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Obstetrics & Gynecology Science, the official journal of six Korean academic societies in the fields of obstetrics and gynecology, will be published in English bimonthly from January 2013. These societies are the Korean Society of Obstetrics and Gynecology, Korean Society of Maternal Fetal Medicine, Korean Society of Gynecologic Endocrinology, Korean Society of Gynecologic Endoscopy and Minimal Invasive Surgery, Korean Society of Ultrasound in Obstetrics and Gynecology, and Korean Society of Contraception and Reproductive Health. Under the initiative of the Korean Society of Obstetrics and Gynecology, the delegates of the six societies have discussed the unification of their representative journals, and concluded that the publication of a unified journal in English is an essential part of the future growth of each society. The primary goal of Obstetrics & Gynecology Science is to become a worldwide journal by publishing highly qualified articles. Concentrating the submission of articles into one journal instead of six separate journals will allow us to reduce the risk of publishing articles of low quality. In addition, we expect that the change will give foreign scholars easier access to Obstetrics & Gynecology Science because it is published in English. Previously, the six journals of the societies were published in Korean, which made it nearly impossible for non-Koreans to read the journals.

ng articles of low quality. In addition, we expect that the change will give foreign scholars easier access to Obstetrics & Gynecology Science because it is published in English. Previously, the six journals of the societies were published in Korean, which made it nearly impossible for non-Koreans to read the journals. Geographical distance is no longer a significant obstacle for international communication. Accordingly, academic exchanges between different countries are so vigorous, fast, and difficult to keep up with, as are exchanges in the medical field. In the meantime, the quality of clinical practice in Korea has gradually increased in proportion to Korea's economic growth. Korean researchers have frequently presented researches at international academic meetings abroad. However, as there is a paucity of Korean journals published in English, they must submit their articles to foreign journals for the purpose of international communication. Moreover, foreign scholars have difficulty in searching Korean journals and citing Korean articles due to language differences. We will make Obstetrics & Gynecology Science an open access journal and assign digital object identifiers to all published articles. We also have a plan to provide a crosscheck service for reviewers to prevent plagiarism.

ve difficulty in searching Korean journals and citing Korean articles due to language differences. We will make Obstetrics & Gynecology Science an open access journal and assign digital object identifiers to all published articles. We also have a plan to provide a crosscheck service for reviewers to prevent plagiarism. Publishing Obstetrics & Gynecology Science is difficult because English is not a native language for Koreans. The burden is shared by the editors and manuscript contributors. Many contributors may hesitate to submit manuscripts to Obstetrics & Gynecology Science, and prefer to submit their work to more world-renowned journals. However, we as editors, have a firm belief that the future of Obstetrics & Gynecology Science is dependent on our continuous self-sacrificing endeavor in terms of submitting articles, peer reviewing, deliberate editing, and providing tools for easy access to this journal. We also believe that the future of Obstetrics & Gynecology Science reflects the future of the six societies. We believe that it is not an unrealistic dream for Obstetrics & Gynecology Science to become an academic journal through which distinguished researchers worldwide freely communicate with each other. We expect that all members of the six societies are eager to contribute to the efforts to reach the goals of Obstetrics & Gynecology Science.

s not an unrealistic dream for Obstetrics & Gynecology Science to become an academic journal through which distinguished researchers worldwide freely communicate with each other. We expect that all members of the six societies are eager to contribute to the efforts to reach the goals of Obstetrics & Gynecology Science. Finally, we would like to extend our appreciation to the presidents of the six societies and the Chairman of the Board of the Korean Society of Obstetrics and Gynecology for their contributions to the publication of Obstetrics & Gynecology Science. We also express our appreciation to the members of the Editorial Board and reviewers of the journal for their willingness to participate in its publication.

Introduction Stage IB cervical cancer has a relatively favorable prognosis but the 20% treatment failure has yet remains. The survival of patients with early stage cervical cancer after radical hysterectomy and pelvic lymphadenectomy depends on the presence or absence of several poor prognostic clinicopathologic risk factors. High-risk factors for recurrent disease are as follow: positive lymph nodes, positive or close surgical margins, parametrial involvement. Intermediate risk factors for recurrent disease are as follow: large tumor size, deep cervical stromal invasion, lymphovascular space invasion. Adjuvant therapy is recommended for both these intermediate and high risk patients to improve survival [1-5]. For high risk tumors, concurrent chemoradiotherapy is now regarded as the standard of treatment [6-8].

ent disease are as follow: large tumor size, deep cervical stromal invasion, lymphovascular space invasion. Adjuvant therapy is recommended for both these intermediate and high risk patients to improve survival [1-5]. For high risk tumors, concurrent chemoradiotherapy is now regarded as the standard of treatment [6-8]. But for intermediate risk cervical cancer patients, method of adjuvant therapy has been debating. Radiotherapy has been used as postoperative adjuvant therapy to reduce recurrence in patients with intermediate risk tumors and the effectiveness of radiotherapy has been widely accepted, based on the results of a randomized studies reported by Sedlis et al. [9]. But radiotherapy have local effects only, so are not effective in distant metastatic diseases, and have high incidence of morbidity and mortality in patients receiving full course radiotherapy following radical surgery [10,11]. Several studies have suggested that chemotherapy alone is promising as postoperative adjuvant therapy for intermediate risk cervical cancer patients. Postoperative adjuvant chemotherapy without radiotherapy is expected to have several advantages including efficacy for control of both microscopic local and distant metastatic diseases, preservation of ovarian function in young female and avoidance of long term serious radiation sequelae. Also the toxicities of chemotherapy were mostly reversible and within acceptable limits [12-14].

ed to have several advantages including efficacy for control of both microscopic local and distant metastatic diseases, preservation of ovarian function in young female and avoidance of long term serious radiation sequelae. Also the toxicities of chemotherapy were mostly reversible and within acceptable limits [12-14]. Cisplatin-based chemotherapy usually has been accepted as effective adjuvant therapy against cervical carcinomas. So we have applied postoperative adjuvant cisplatin based combination chemotherapy to the patients who had at least two of the intermediate risk factors and who had no high risk factors and who refused radiotherapy to avoid serious postoperative radiation complication. Materials and Methods 1. Patients In this retrospective study we reviewed the medical records of stage IB cervical cancer patients who had radical hysterectomy and pelvic lymphadenectomy at the Gospel Hospital, Busan, Korea from January 1993 to December 2007. Among them, 100 intermediate risk tumors were eligible. Of these patients, 22 patients had surgery only and 78 patients had cisplatin based combination chemotherapy postoperatively as adjuvant therapy to improve survival. Patient characteristics are listed in Table 1. Criteria for intermediate risk group are still debating and there are two commonly used criterias for intermediate risk tumors now; Classic vs. gynecological oncology group (GOG) 92 criteria (Table 2).

n chemotherapy postoperatively as adjuvant therapy to improve survival. Patient characteristics are listed in Table 1. Criteria for intermediate risk group are still debating and there are two commonly used criterias for intermediate risk tumors now; Classic vs. gynecological oncology group (GOG) 92 criteria (Table 2). We applied classic criteria for our intermediate risk tumors because of its simplicity and usefulness. Our eligible criteria for intermediate risk tumors are stage IB cervical cancer patients who had type III radical hysterectomy and pelvic lymphadenectomy and who had at least two of the following three intermediate risk factors (deep stromal invasion ≥1/2 or ≥1 cm, lymphovascular space involvement, large tumor size ≥2 cm) and all patients had no high risk factors and no obious any residual tumors.

patients who had type III radical hysterectomy and pelvic lymphadenectomy and who had at least two of the following three intermediate risk factors (deep stromal invasion ≥1/2 or ≥1 cm, lymphovascular space involvement, large tumor size ≥2 cm) and all patients had no high risk factors and no obious any residual tumors. 2. Surgery and chemotherapy All eligible 100 patients had type III radical hysterectomy with pelvic/paraaortic lymph node dissection. Among them, total 78 (78%) patients received postoperative cisplatin based combination adjuvant chemotherapy (1 to 6 cycles/patient, total 300 cycles/78 patients; average 3.8 cycles/patient). The schedules of the cisplatin based combination chemotherapy are as follows. Five-fluorouracilcisplatin combination chemotherapy regimen consisted of cisplatin 50 mg/m2 on day 1 and followed by 5-fluorouracil 1,000 mg/m2/day given intravenously from day 2 to day 5 daily with 3 weekly intervals (total 127 cycles/34 patients) and etoposide-cisplatin combination chemotherapy regimen consisted of cisplatin 30 mg/m2 and etoposide 60 mg/m2 given intravenously daily for 3 days with 3 weekly intervals (total 173 cycles/44 patients). Toxicities were recorded and categorized using the National Cancer Institute Common Toxicity Criteria (ver. 3.0, 2003) and chemotherapy was withheld if any of the following criteria were observed: leukocytes <3,000/mm3, granulocytes <1,500 mm3, platelets <100,000/mm3, serum creatinine level >2.0 mg/dL. In case of severe toxicity, chemotherapy was delayed until the symptoms of toxicities had disappeared.

xicity Criteria (ver. 3.0, 2003) and chemotherapy was withheld if any of the following criteria were observed: leukocytes <3,000/mm3, granulocytes <1,500 mm3, platelets <100,000/mm3, serum creatinine level >2.0 mg/dL. In case of severe toxicity, chemotherapy was delayed until the symptoms of toxicities had disappeared. 3. Follow-up and statistics Median follow-up period is 109 months. Overall survival was offered by the Korean National Stastistical Office. Patient selections between two groups (surgery only and postoperative adjuvant chemotherapy group) were done randomly and Kaplan-Meier survival curves and Cox's proportional-hazards regression model and log-rank test were used for survival analysis and to estimate the impact of prognostic factors on survival and SPSS PASW ver. 18.0 software (IBM, Armonk, NY, USA) was used for the statistical analysis. P-value less than 0.05 was considered significant. Results The mean age of patients at diagnosis was 52 years (range, 28 to 76 years), and 84.0% (84/100) had squamous cell type, and 78.0% (78/100) had stage IB1, and 56.0% (56/100) had stromal invasion ≥1/2 or ≥1 cm but not whole layer, and 36.0% (36/100) had lymphovascular space invasion, and 97.0% (97/100) had tumor size ≥2 cm (Table 1).

of patients at diagnosis was 52 years (range, 28 to 76 years), and 84.0% (84/100) had squamous cell type, and 78.0% (78/100) had stage IB1, and 56.0% (56/100) had stromal invasion ≥1/2 or ≥1 cm but not whole layer, and 36.0% (36/100) had lymphovascular space invasion, and 97.0% (97/100) had tumor size ≥2 cm (Table 1). Comparison of patient characteristics between two groups revealed no significant differences. In overall survival, there are 8 deaths (4 deaths in surgery only group, and 4 deaths in adjuvant chemotherapy group), the overall survival rate of all intermediate tumors are 92% (92/100); surgery only group is 81.8% (18/22) and postoperative adjuvant chemotherapy group is 94.9% (74/78), respectively. Mean survival time from disease diagnosis to death are 26 months in surgery only group and 38 months in postradical adjuvant chemotherapy group. Comparison of survival curves between two groups revealed significant statistical difference in both univariant and multivariant survival analysis (P<0.05) (Tables 3-5; Fig. 1). Toxicities of adjuvant chemotherapy were mostly transient, reversible and within acceptable limits. Grade 3 or 4 myelosuppression was observed in 18%, grade 3 or 4 gastrointestinal toxicity was observed in 5.2%, grade 3 or 4 hepatotoxicity was observed in two cases, and cardiac toxicity was observed in one case in etoposide-cisplatin group. Grade 3 or 4 alopecia was observed in all patients in etoposide-cisplatin group but was not observed in 5-fluorouracil-cisplatin group.

or 4 gastrointestinal toxicity was observed in 5.2%, grade 3 or 4 hepatotoxicity was observed in two cases, and cardiac toxicity was observed in one case in etoposide-cisplatin group. Grade 3 or 4 alopecia was observed in all patients in etoposide-cisplatin group but was not observed in 5-fluorouracil-cisplatin group. Discussion The survival of patients with stage IB cervical cancer after radical hysterectomy and pelvic lymphadenectomy depends on the presence or absence of several poor prognostic clinicopathologic risk factors. The poor risk factors are now classified into two groups: classic high risk and newly developed intermediate risk factors [1-5]. In 1989 and 1990, the GOG published the results of prospective clinicopathologic studies of patients with stage IB cervical carcinoma treated by radical hysterectomy and bilateral pelvic lymphadenectomy. These studies revealed that certain patients with poor prognostic factors related to the primary tumor but without classic high risk factors had a risk of recurrence after radical hysterectomy and pelvic lymphadenectomy. The poor prognostic factors related to the primary tumor are as follow: clinical tumor size, presence or absence of capillary lymphatic space invasion, depth of tumor stromal invasion; the so called newly developed intermediate risk factors [4,5]. Postoperative adjuvant therapy is recommended for these poor risk tumors to reduce recurrence and to prolong survival. Concurrent chemoradiotherapy is now regarded as the standard of treatment for high risk tumors [6-8].

on, depth of tumor stromal invasion; the so called newly developed intermediate risk factors [4,5]. Postoperative adjuvant therapy is recommended for these poor risk tumors to reduce recurrence and to prolong survival. Concurrent chemoradiotherapy is now regarded as the standard of treatment for high risk tumors [6-8]. But adjuvant treatment modalities for intermediate risk tumors have been debating. Use of radiotherapy postoperatively for stage IB, intermediate risk tumors was shown to be beneficial in reducing the risk of recurrence by various studies [9,10]. In 1999, Sedlis et al. [9] showed that adjuvant pelvic radiotherapy reduced recurrence from 28% to 15% at 2 years after treatment and had better 2-year disease free survival than no treatment group in their prospective study for intermediate risk cervical cancer. This result suggested that radiotherapy has a role in adjuvant therapy, and after this report radiotherapy has been used as postoperative adjuvant therapy for stages Ib intermediate risk cervical cancer patients [9]. In 2006, Rotman et al. [11] reported the final results of this study, in which adjuvant pelvic radiotherapy significantly prolonged disease free survival but the improvement in overall survival did not reach statistical significance (Hazard ratio, 0.70; 90% confidence interval [CI], 0.45 to 1.05). Possible reasons of Rotman's results are that radiotherapy have local effects only, so are not effective in distant metastatic disease, and have high incidence of morbidity and mortality in patients receiving full course radiotherapy following radical surgery [11].

io, 0.70; 90% confidence interval [CI], 0.45 to 1.05). Possible reasons of Rotman's results are that radiotherapy have local effects only, so are not effective in distant metastatic disease, and have high incidence of morbidity and mortality in patients receiving full course radiotherapy following radical surgery [11]. The use of cisplatin based combination chemotherapy was found to improve overall survival in women with advanced cervical cancers (large IB2 tumors, high risk early stage diseases and recurrent or metastatic cancer of the cervix) [12-21]. Experiences with adjuvant chemotherapy for poor risk cervical carcinomas suggested that cisplatin-based combination chemotherapy was effective not only in local tumor and may also reduce the incidence of lymph node metastasis and even eradicate microscopic metastasis in distant organs [12-14].

r of the cervix) [12-21]. Experiences with adjuvant chemotherapy for poor risk cervical carcinomas suggested that cisplatin-based combination chemotherapy was effective not only in local tumor and may also reduce the incidence of lymph node metastasis and even eradicate microscopic metastasis in distant organs [12-14]. In 1998, Iwasaka et al. [12] performed postoperative adjuvant chemotherapy only for high risk cervical cancer and compared their patients with those from another hospital where postoperative adjuvant radiotherapy was performed during the same period. All eligible patients had no obvious residual lesions after radical surgery. The results of chemotherapy were similar to those of radiotherapy (83% vs. 81.7% 5-year survival rate). But in the adjuvant chemotherapy group, 5 of 11 (45%) patients with pelvic recurrences survived more than 5 years after retreatment with irradiation to sites of recurrence, which suggests that adjuvant chemotherapy might be preferable because unnecessary irradiation can be avoided and radiotherapy can be reserved for late locoregional recurrence [12]. In 2006, Takeshima et al. [13] have reported a favorable outcome for intermediate and high risk patients treated with adjuvant chemotherapy following radical hysterectomy. They consistently treated their patients with adjuvant chemotherapy alone for poor risk cervical carcinomas postoperatively for several reasons. First, distant metastasis is a major problem when radiotherapy alone is used, and chemotherapy is considered the most powerful means of eradicating subclinical distant metastastic lesions. Second, their chemotherapy regimen yielded good response rate in patients with recurrent cervical cancer with an acceptable incidence of side effects. Third, when chemotherapy alone is used for adjuvant therapy, radiotherapy can be reserved for late locoregional recurrence. Finally, this treatment strategy may provide a better postoperative quality of life by ovarian preservation and precluding radiation related morbidity. Three courses of bleomycin, vincristine, mitomycin, and cisplatin were given for intermediate risk cases (n=30) and 5 courses for high risk cases (n=35). Estimated 5-year disease free survival was 93.3% with intermediate risk tumors, and 85.7% with high risk tumors. The incidence of locoregional recurrence was 3.3% in the intermediate risk group and 8.6% in the high risk group [13].

were given for intermediate risk cases (n=30) and 5 courses for high risk cases (n=35). Estimated 5-year disease free survival was 93.3% with intermediate risk tumors, and 85.7% with high risk tumors. The incidence of locoregional recurrence was 3.3% in the intermediate risk group and 8.6% in the high risk group [13]. These reports suggest that postoperative adjuvant chemotherapy for intermediate risk tumors after radical hysterectomy is promising and intrapelvic recurrence is not a major obstacle when radical operation was performed successfully and all tumor was resected completely [12,13]. In our study, overall survival in adujvant chemotherapy group was 94.9% (74/78), this result was similar to Takeshima's result in intermediate risk group. And the improvement of overall survival in adjuvant chemotherapy group reached statistical significance in both univariant and multivariant survival analysis (odds ratio [OR], 0.173; 95% CI for OR, 0.032 to 0.939; P<0.05) (Tables 4, 5; Fig. 1).

, this result was similar to Takeshima's result in intermediate risk group. And the improvement of overall survival in adjuvant chemotherapy group reached statistical significance in both univariant and multivariant survival analysis (odds ratio [OR], 0.173; 95% CI for OR, 0.032 to 0.939; P<0.05) (Tables 4, 5; Fig. 1). Another issue that must be discussed is complications of these adjuvant therapies following radical surgery. Radiotherapy after radical hysterectomy has been reported to be associated with high incidence of complications and many of these conditions are very tragic and irreversible and result in poor quality of life [22,23]. Barter et al. [23] reported that 30% of patients treated with radiation after radical hysterectomy had serious complications; gastrointestinal and genitourinary complications and leg lymphedema were the major problems of radiation combination therapy. The major toxicities of chemotherapy (etoposide-cisplatin and 5-fluorouracil-cisplatin combination) were bone marrow depepression, mild to moderate gastrointestinal troubles, mild to moderate nephrotoxicities, total alopecia (in etoposide-cisplatin group), relatively low incidence of neurotoxicities and hepatotoxicities, rare incidence of pulmonary and cardiac toxicities and some reported secondary malignancies (acute leukemia and some solid tumors) but rare. And the levels of all toxicities were acceptable and most are reversible and transient. But unexpected severe side effects and idiosyncratic drug reactions can be occurred especially in severe systemic debility, advanced age, poor nutritional status during chemotherapy. Therefore careful monitoring of patients is recommended [17-21,24].

all toxicities were acceptable and most are reversible and transient. But unexpected severe side effects and idiosyncratic drug reactions can be occurred especially in severe systemic debility, advanced age, poor nutritional status during chemotherapy. Therefore careful monitoring of patients is recommended [17-21,24]. In summary, several studies suggest that, when radical operation was performed successfully and all tumor was resected completely, chemotherapy is worth considering as postoperative adjuvant therapy for stage IB intermediate risk cervical cancer from the view of treatment efficacy, morbidity and quality of life. Our study suggests that postoperative cisplatin based combination adjuvant chemotherapy for intermediate risk stage IB cervical cancer after radical hysterectomy is promising with improvement of overall survival (OR, 0.173; 95% CI for OR, 0.032 to 0.939; P<0.05) and with reversible and acceptable toxicity profile. But experiences in postoperative adjuvant chemotherapy for cervical cancer are limited. We think that well balanced prospective studies are needed to confirm the efficacy and safety of the postoperative adjuvant chemotherapy. Acknowledgments This study was funded by the Oncology Center of Kosin University Gospel Hospital. Fig. 1 Overall survival by treatment type. Table 1 Clinicopathologic characteristics of 100 patients with postoperation intermediate risk factors Table 2 Criterias of intermediate risk tumors: Classic vs. GOG criteria GOG, gynecological oncology group; CLS, capillary lymphatic space invasion.

Acknowledgments This study was funded by the Oncology Center of Kosin University Gospel Hospital. Fig. 1 Overall survival by treatment type. Table 1 Clinicopathologic characteristics of 100 patients with postoperation intermediate risk factors Table 2 Criterias of intermediate risk tumors: Classic vs. GOG criteria GOG, gynecological oncology group; CLS, capillary lymphatic space invasion. Table 3 Deaths analysis of 100 patients with intermediate risk factors Values are presented as number (%). EP, etoposide-cisplatin; FP, 5-fluorouracil-cisplatin. Table 4 Survival analysis of 100 patients with intermediate risk factors (Kaplan Meier) Table 5 Cox proportional hazard regression model for survival

Introduction Preeclampsia is one of the leading causes of maternal mortality/morbidity and preterm delivery in the world, affecting 3% to 5% of pregnant women [1,2]. It is a syndrome defined by the onset of hypertension (≥40/≥90 mm Hg) and proteinuria (≥0.3 g/24 hr) after 20 weeks of gestation. Preeclampsia is the leading cause of maternal mortality in developing and developed countries. Delivery of the placenta remains the only known treatment for this disease, suggesting that the placenta is the principal contributor to the pathogenesis of preeclampsia [3]. The pathophysiology of preeclampsia likely involves both maternal and fetal/placental factors. Abnormalities in the development of placental vessels early in pregnancy may result in placental hypoperfusion, hypoxia, or ischemia. However, the molecular mechanisms behind preeclampsia are not clear and the role of angiogenic proteins in early placental vascular development is under investigation.

placental factors. Abnormalities in the development of placental vessels early in pregnancy may result in placental hypoperfusion, hypoxia, or ischemia. However, the molecular mechanisms behind preeclampsia are not clear and the role of angiogenic proteins in early placental vascular development is under investigation. Abnormal Development of the Placenta Because the placenta is central to preeclampsia pathogenesis, this research has focused on the association between abnormal placental vascular development and the development of preeclampsia. In normal pregnancies, extravillous cytotrophoblasts of fetal origin invade the uterine spiral arteries of the decidua and myometrium [3]. These invasive cytotrophoblasts replace the endothelial layer of the maternal spiral arteries, transforming them from small, high-resistance vessels into large-caliber vessels. However, in preeclampsia, this transformation is incomplete [4]. Cytotrophoblast invasion of the spiral arteries is limited to the superficial decidua and does not reach the myometrium (Fig. 1) [4,5].

ayer of the maternal spiral arteries, transforming them from small, high-resistance vessels into large-caliber vessels. However, in preeclampsia, this transformation is incomplete [4]. Cytotrophoblast invasion of the spiral arteries is limited to the superficial decidua and does not reach the myometrium (Fig. 1) [4,5]. Defective differentiation of trophoblasts might be an explanatory mechanism for defective trophoblast invasion of the spiral arteries in preeclampsia. For trophoblast differentiation to occur there must be alterations in the expression of a number of different classes of molecules, including cytokines, adhesion molecules, extracellular matrix molecules, metalloproteinases, and the class IB major histocompatibility complex molecule, histocompatibilty leukocyte antigen-G [6]. During normal pregnancies, invading trophoblasts alter their adhesion molecule expression from one set of endothelial transmembrane receptors (integrin alpha6beta1, integrin alphavbeta5, and E-cadherin) to another (integrin alpha1beta1, integrin alphavbeta3, and VE-cadherin) [7]. Hypoperfusion, hypoxia, and ischemia are critical components in the pathogenesis of preeclampsia because the hypoperfused placenta elaborates many factors into maternal vessels that alter maternal endothelial cell function and lead to the systemic symptoms of preeclampsia [8-10].

grin alphavbeta3, and VE-cadherin) [7]. Hypoperfusion, hypoxia, and ischemia are critical components in the pathogenesis of preeclampsia because the hypoperfused placenta elaborates many factors into maternal vessels that alter maternal endothelial cell function and lead to the systemic symptoms of preeclampsia [8-10]. Altered Angiogenic Balance and Circulating Angiogenic Factors Many angiogenic factors are produced by the human placenta. The most important factors are vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). VEGF is an endothelial-specific mitogen that plays a key role in promoting angiogenesis [11]. VEGF's activities are mediated by its interaction with two high-affinity receptors-tyrosine kinase-kinase-insert domain region (the kinase domain region or vascular endothelial growth factor receptor-2) and fms-like tyrosine kinase 1 (flt-1). PlGF is an angiogenic growth factor that is thought to amplify VEGF signaling by displacing VEGF from the flt-1 receptor and allowing it to bind to the more active kinase-insert domain (KDT) receptor [11]. Increased sFlt-1 during preeclampsia is associated with decreased free VEGF and free PlGF in the blood [5]. Also, administration of sflt-1 to rats resulted in elevated blood pressure and proteinuria, indicating that excessive placenta-derived sflt-1 contributes to preeclampsia [12]. Soluble endoglin is an antiangiogenic protein, which acts together with sflt-1 to induce a severe preeclamptic-like syndrome in pregnant rats. Circulating soluble endoglin levels increased markedly beginning from two to three months before the onset of preeclampsia [11]. An increased level of soluble endoglin was associated with an increased endoglin/sflt-1 ratio [13].

h acts together with sflt-1 to induce a severe preeclamptic-like syndrome in pregnant rats. Circulating soluble endoglin levels increased markedly beginning from two to three months before the onset of preeclampsia [11]. An increased level of soluble endoglin was associated with an increased endoglin/sflt-1 ratio [13]. There is substantial evidence that effective angiogenesis requires the synthesis of bioactive endothelium-derived nitric oxide (NO) [14]. A number of angiogenic factors up-regulate the endothelial expression of NO synthase (NOS) stimulating the release of endothelium-derived NO. NOS is partially regulated by negative feedback from NO, but there are other important inhibitors that are endogenously present in humans, such, the competitive inhibitor asymmetric dimethylarginine (ADMA) [15]. There are some conflicting findings concerning ADMA concentrations in pregnancies complicated by preeclampsia. However, Kim et al. [16] suggested that increased maternal circulating ADMA levels, a higher expression of placental eNOS protein, and a lower expression of placental phospho-eNOS protein contribute to the development of preeclampsia.

g findings concerning ADMA concentrations in pregnancies complicated by preeclampsia. However, Kim et al. [16] suggested that increased maternal circulating ADMA levels, a higher expression of placental eNOS protein, and a lower expression of placental phospho-eNOS protein contribute to the development of preeclampsia. Marinobufagenin, a Bufadienolide Trigger of Preeclampsia Pathogenesis Presently, the key biomarkers of the syndrome associated with preeclampsia pathogenesis are thought to be marinobufagenin (MBG) and angiogenic imbalance [17]. Data from a rat model that mimics human preeclampsia showed that urinary excretion of MBG increased before the onset of hypertension and proteinuria, and that affected animals have increased vascular leakage and blood-brain barrier permeability [17]. The cardenolides and bufadienolides are group of steroid compounds, which belong to a class of circulating substances called "cardiac glycosides" (Fig. 2) [18,19]. Cardiac glycosides have the ability to inhibit the sodium transport enzyme, sodium/potassium adenine triphosphatase [17]. They are also natriuretic and cause vasoconstriction, which can lead to hypertension as an agent of cardiac inotrope [20]. The bufadienolides are more important in diseases such as preeclampsia than the cardenolides [21,22]. The best studied of the bufadienolide compounds is MBG (Fig. 2A), an endogenous vasoconstrictor mammalian cardiotonic bufadienolide [18]. Preeclampsia is thought to result from inadequate placentation, related to a failure of the trophoblasts to adequately remodel the vasculature of the uterus [23]. This leads to hypoperfusion of the placenta, often causing oxidative stress, and endothelial dysfunction, which are related to the development of the preeclampsia syndrome [17]. MBG interferes with proliferation, migration, and the invasive capacity of the cytotrophoblasts [23], and has deleterious effects on human cytotrophoblast cell function, which suggests a role for MBG in the abnormal placentation and altered vascular function of preeclampsia.

velopment of the preeclampsia syndrome [17]. MBG interferes with proliferation, migration, and the invasive capacity of the cytotrophoblasts [23], and has deleterious effects on human cytotrophoblast cell function, which suggests a role for MBG in the abnormal placentation and altered vascular function of preeclampsia. Activation of the Renin-Angiotensin System The renin-angiotensin system (RSA) is involved with regulating blood pressure. Circulating levels of angiotensin II increases as pregnancy advances [24]. In preeclampsia, circulating levels of angiotensin is decreased, despite increased vascular sensitivity to angiotensin with preeclampsia [25]. Uddin et al. [26] propose that MBG can act as a trigger of the RAS alterations observed in preeclampsia cases. Resibufogenin (RBG) is a recently discovered congener of MBG (Fig. 2B) that is postulated to act antagonistically to MBG [17]. Alterations in oxidative stress have been observed in the MBG rat model of preeclampsia [27]. MBG is involved in the alteration of oxidative stress, while RBG attenuates this alteration [17]. Excessive volume expansion in pregnancy causes an increase in the circulating levels of MBG that, in turn, causes an increase in the expression of RAS and the AT1 receptor. These increases produce oxidative stress and endothelial function. MBG also causes angiogenic imbalances, which ultimately lead to endothelial dysfunction (Fig. 3) [17].

sion in pregnancy causes an increase in the circulating levels of MBG that, in turn, causes an increase in the expression of RAS and the AT1 receptor. These increases produce oxidative stress and endothelial function. MBG also causes angiogenic imbalances, which ultimately lead to endothelial dysfunction (Fig. 3) [17]. Epigenetics and Micro-RNAs (miRNAs) in Preeclampsia Recently, there has been a rapid development of new research in the field of epigenetics and fetal and maternal biology [28]. Major areas of epigenetic research include DNA methylation, histone modifications, and genomic imprinting. A promising new concept suggests the possibility that epigenetic dysregulations that occur prior to conception or during pregnancy might increase preeclampsia susceptibility [29]. Chelbi and Vaiman [30] suggest that abnormal methylation patterns may be an important mechanism underlying preeclampsia. In preeclampsia, cytotrophoblasts fail to deeply invade spiral uterine arteries. Shallow invasion of the spiral arteries leads to ischemic lesions and hypoxia. Chelbi and Vaiman [30] confirmed that SERPING1, SERPINE1, and SERPINE2-members of the serine protease inhibitor (SERPIN) gene family-are induced during a hypoxic state and show a modified expression pattern in placentas from preeclamptic pregnancies. Chelbi et al. [31] further found that the promotor of the serine protease inhibitor, SERPINA3, is hypomethylated in preeclamptic placentas compared with normal placentas. In particular, tissue inhibitor of metalloproteinase (TIMP3) showed the greatest degree of hypomethylation in early-onset preeclampsia placentas compared with control placentas, and TIMP3 is highly expressed in the preeclampsia placentas [32]. Increased TIMP3 expression may reduce the invasiveness of the trophoblasts during placental development, leading to placental hypoperfusion in early-onset preeclampsia [29].

thylation in early-onset preeclampsia placentas compared with control placentas, and TIMP3 is highly expressed in the preeclampsia placentas [32]. Increased TIMP3 expression may reduce the invasiveness of the trophoblasts during placental development, leading to placental hypoperfusion in early-onset preeclampsia [29]. Several studies have shown elevated plasma homocysteine concentrations in preeclampsia. Kulkarni et al. [33] found increased homocysteine and global DNA methylation levels in a group of preeclampsia cases compared with a control group. This study also found a positive association between global DNA methylation and systolic/diastolic blood pressure in the term preeclampsia group [33]. In addition to DNA methylation increases and histone modifications in the preeclamptic placentas, the expression patterns of miRNAs are very distinct. A recent study showed that miRNAs could play an important role in controlling DNA methylation and histone modification [34]. The differential expression of specific placental miRNAs (e.g., miR-210) was found to be upregulated in preeclampsia placentas compared with normal placentas [35].

ns of miRNAs are very distinct. A recent study showed that miRNAs could play an important role in controlling DNA methylation and histone modification [34]. The differential expression of specific placental miRNAs (e.g., miR-210) was found to be upregulated in preeclampsia placentas compared with normal placentas [35]. Conclusion To explain the pathogenesis of preeclampsia, there are several hypotheses including altered angiogenic balance, circulating angiogenic factors like marinobufagenin (a bufadienolide trigger), and activation of the renin-angiotensin system, among others. Epigenetically-modified circulating cell-free nucleic acids in plasma and serum could be novel markers with promising non-invasive clinical applications in the diagnosis of preeclampsia. We hope that these promising novel markers may eventually be able to reduce the morbidity and mortality of this severe disorder. Fig. 1 Abnormal placentation in preeclampsia. In normal pregnancies, extravillous cytotrophoblasts of fetal origin invade the uterine spiral arteries of the decidua and myometrium. These invasive cytotrophoblasts replace the endothelial layer of the maternal spiral arteries, transforming them from small, high-resistance vessels into large-caliber vessels (A). However, in preeclampsia, this transformation is incomplete. Cytotrophoblast invasion of the spiral arteries is limited to the superficial decidua and does not reach the myometrium (B) (From Lam et al. [5], with permission from Wolters Kluwer Health).

from small, high-resistance vessels into large-caliber vessels (A). However, in preeclampsia, this transformation is incomplete. Cytotrophoblast invasion of the spiral arteries is limited to the superficial decidua and does not reach the myometrium (B) (From Lam et al. [5], with permission from Wolters Kluwer Health). Fig. 2 (A) Chemical structures of the bufadienolides and the cardenolides. The compounds on the left side are cardenolides and those on the right side are bufadienolides. (B) The chemical structures of marinobufagenin and resibufogenin (From Uddin et al. [17], with permission from Elsevier). Fig. 3 Working model of the role of MBG in preeclampsia pathogenesis and its association with the RAS and oxidative stress (From Uddin et al. [17], with permission from Elsevier). MBG, marinobufagenin; RAS, renin-angiotensin system; RBG, Resibufogenin; sFlt1, soluble fms-like tyrosine kinase 1; sEng, endoglin; VEGF, vascular endothelial growth factor; BP, blood pressure; IUGR, intrauterine growth restriction.

Introduction Ovarian cancer is the leading cause of gynecological cancer death in women in the United States, causing an estimated 13,850 deaths in 2010 [1]. Unfortunately, due to the absence of detectable symptoms in the early stage of the disease and a lack of an effective screening method, approximately 75% of women with ovarian cancer present with stage III and IV [2]. The 5-year relative survival rate of ovary cancer patients is 89.3% and 65.5% for stage I and II, respectively, whereas the rate is only 33.5% and 17.9% for stage III and IV, respectively [3]. In the last two decades, only stage I and II patients showed improvement in cure rate. In contrast, stage III and IV patients showed prolongation in survival time but no improvement in survival rate [4]. Age, performance status, stage of the disease at diagnosis, extent of cytoreductive surgery and residual disease are important independent prognostic factors for survival [5,6]. Many researchers have investigated whether the CA-125 level can be correlated to cancer relapse and survival in various clinical situations. Lower levels at completion of primary therapy [7], lower levels prior to maintenance therapy [8], early changes during maintenance therapy [9] and early normalization during primary chemotherapy [10] could predict positive tumor response and longer survival. In addition, nadir levels of serum CA-125 during primary chemotherapy could predict the risk of tumor recurrence [11,12].

els prior to maintenance therapy [8], early changes during maintenance therapy [9] and early normalization during primary chemotherapy [10] could predict positive tumor response and longer survival. In addition, nadir levels of serum CA-125 during primary chemotherapy could predict the risk of tumor recurrence [11,12]. Markman et al. [13] reported that early changes in CA-125 levels are an independent prognostic factor of survival. In their study, patients who achieved normalization of serum CA-125 levels (<35 U/mL) after the second cycle of platinum-based chemotherapy had significant improvement in median survival compared with those with serum CA-125>35 U/mL. Rocconi et al. [10] compared patients who achieved normalization of CA-125 by the third cycle of chemotherapy with patients who failed to achieve normalization by the third cycle. Patients with early normalization demonstrated improved progression free survival (PFS), overall survival (OS) and platinum sensitivity. Recently, it has been suggested that nadir serum CA-125 levels are independently associated with PFS in patients with complete remission after first-line therapy in ovarian cancer [11,12,14]. The optimal cut-off value of serum CA-125 to predict recurrence varied, ranging from 10 to 25 U/mL.

Markman et al. [13] reported that early changes in CA-125 levels are an independent prognostic factor of survival. In their study, patients who achieved normalization of serum CA-125 levels (<35 U/mL) after the second cycle of platinum-based chemotherapy had significant improvement in median survival compared with those with serum CA-125>35 U/mL. Rocconi et al. [10] compared patients who achieved normalization of CA-125 by the third cycle of chemotherapy with patients who failed to achieve normalization by the third cycle. Patients with early normalization demonstrated improved progression free survival (PFS), overall survival (OS) and platinum sensitivity. Recently, it has been suggested that nadir serum CA-125 levels are independently associated with PFS in patients with complete remission after first-line therapy in ovarian cancer [11,12,14]. The optimal cut-off value of serum CA-125 to predict recurrence varied, ranging from 10 to 25 U/mL. Because there are no reports about the value of serum CA-125 levels in patients with advanced epithelial ovarian cancer with complete remission after chemotherapy with paclitaxel (175 mg/m2) and carboplatin (area under the curve of 5) every 3 weeks for a total of six cycles, we analyzed the value of serum CA-125 levels in this study.

re no reports about the value of serum CA-125 levels in patients with advanced epithelial ovarian cancer with complete remission after chemotherapy with paclitaxel (175 mg/m2) and carboplatin (area under the curve of 5) every 3 weeks for a total of six cycles, we analyzed the value of serum CA-125 levels in this study. Materials and Methods 1. Patient population From 1 January 1998 to 31 December 2005, all patients treated for advanced epithelial ovarian cancer at the Department of Obstetrics and Gynecology of Chonnam National University Hospital (CNUH) were identified from the tumor registry database. All patients were screened retrospectively for their initial serum CA-125 level, age at diagnosis, tumor histology and grade, stage of disease based on the International Federation of Gynecology and Obstetrics (FIGO) staging system, availability of serial serum CA-125 level determinations and timing of recurrence and/or disease status at last follow-up. Patients with epithelial tumors of borderline malignancy, germ cell tumor and gonadal stromal tumor were excluded from this investigation.

ration of Gynecology and Obstetrics (FIGO) staging system, availability of serial serum CA-125 level determinations and timing of recurrence and/or disease status at last follow-up. Patients with epithelial tumors of borderline malignancy, germ cell tumor and gonadal stromal tumor were excluded from this investigation. Following initial eligibility screening, the following inclusion criteria were applied to determine the final study population: 1) elevated (≥ 35 U/mL) serum CA-125 level at the time of diagnosis, 2) histologic documentation of epithelial ovarian cancer, 3) stage of disease III (A, B, and C) and IV treated with primary cytoreductive surgery and postoperative chemotherapy, 4) complete clinical and radiographic response to initial treatment with normalization of CA-125 (<35 U/mL), 5) follow-up serum CA-125 determinations and/or radiographic scans at 1- to 3-month intervals, and 6) clinical and radiographic determination of disease status at the time of last follow-up or recurrence.

erapy, 4) complete clinical and radiographic response to initial treatment with normalization of CA-125 (<35 U/mL), 5) follow-up serum CA-125 determinations and/or radiographic scans at 1- to 3-month intervals, and 6) clinical and radiographic determination of disease status at the time of last follow-up or recurrence. After primary surgical staging procedure, all patients received combination chemotherapy with paclitaxel (175 mg/m2 by 3 hour infusion) and carboplatin (area under the curve of 5) every 3 weeks for a total of six cycles. The treatment response was assessed by a complete history, physical examination, appropriate imaging studies (chest X-ray, computed tomography, magnetic resonance imaging and positron emission tomography) and serum CA-125 level. Serum CA-125 levels were measured preoperatively and within 1 week before each cycle of primary adjuvant chemotherapy. All serum samples were analyzed in the same laboratory at CNUH. Optimal cytoreductive surgery was defined as a following review of the surgery and pathology report <1 cm of residual disease was present (following the recommendations of the Gynecology Oncology Group Protocol 17). Complete response was defined as the absence of all measurable disease for at least 4 weeks and the normalization of the serum CA-125 level.

fined as a following review of the surgery and pathology report <1 cm of residual disease was present (following the recommendations of the Gynecology Oncology Group Protocol 17). Complete response was defined as the absence of all measurable disease for at least 4 weeks and the normalization of the serum CA-125 level. 2. Statistical analyses Overall differences in the serum CA-125 level according to the nadir serum CA-125 level were assessed by Student's t-test and the Wilcoxon rank-sum test. A receiver operating characteristic (ROC) curve was used to determine the most clinically useful serum CA-125 threshold value to predict a recurrence. To estimate linear correlation between nadir CA-125 levels and clinical variables, simple lineal regression analysis was used. A multivariate Cox proportional hazards model for OS and PFS was used to assess differences in outcome on the basis of nadir serum CA-125 levels, including other variables, such as age (>65 years vs. ≤65 years), stage (IV vs. III), tumor grade (3 vs. ≤2), histology (serous vs. non-serous) and residual tumor size. Step-wise regression techniques were used to build multivariate models using a significance level of 0.10 to remain in the model. The differences in OS and PFS by nadir serum CA-125 levels and normalization timing were evaluated using the Kaplan-Meier method with log-rank tests. Statistical analyses were performed using SPSS ver.17.0 (SPSS Inc., Chicago, IL, USA). All P-values reported are two-sided, and a P-value <0.05 was considered statistically significant.

in OS and PFS by nadir serum CA-125 levels and normalization timing were evaluated using the Kaplan-Meier method with log-rank tests. Statistical analyses were performed using SPSS ver.17.0 (SPSS Inc., Chicago, IL, USA). All P-values reported are two-sided, and a P-value <0.05 was considered statistically significant. Results The characteristics of the patients are summarized in Table 1. In the entire study population, the median value of the nadir serum CA-125 level was 10.2 U/mL (range, 0.75 to 34.9 U/mL). The nadir CA-125 level was categorized into two groups: group A, ≤10 U/mL (n=56, 46.7% of total) and group B, 10 to 35 U/mL. The median age, body mass index, tumor stage, tumor histology, tumor grade, distribution of residual tumor size, peritoneal cytology and initial serum CA-125 level were not statistically different between the groups. We evaluated the potential correlation between the residual tumor size and the nadir CA-125 level; non-significant correlation was evident (Table 2). We also tested the correlation between initial serum CA-125 level and the nadir CA-125 level, but it also showed non-significant correlation (P=0.368) (Table 2). The median PFS in group A was 21.6 months (95% confidence interval [CI], 14.0 to 29.2) and in group B was 12.5 months (95% CI, 10.5 to 14.5) (log-rank test, P=0.0047) (Fig. 1). The median OS was 130.2 months in group A (95% CI, 86.2 to 174.3) and 35.3 months (95% CI, 28.4 to 42.5) in group B (log-rank test, P=0.0005) (Fig. 2).

an PFS in group A was 21.6 months (95% confidence interval [CI], 14.0 to 29.2) and in group B was 12.5 months (95% CI, 10.5 to 14.5) (log-rank test, P=0.0047) (Fig. 1). The median OS was 130.2 months in group A (95% CI, 86.2 to 174.3) and 35.3 months (95% CI, 28.4 to 42.5) in group B (log-rank test, P=0.0005) (Fig. 2). In the multivariate Cox regression model, the hazards ratio was adjusted for covariates. PFS was significantly associated with non-serous histology and residual tumor size >1.0 cm (P=0.013 and P=0.03, respectively) (Table 3). Other variables, such as tumor stage, grade and age at the time of diagnosis were not significantly associated with PFS.

gression model, the hazards ratio was adjusted for covariates. PFS was significantly associated with non-serous histology and residual tumor size >1.0 cm (P=0.013 and P=0.03, respectively) (Table 3). Other variables, such as tumor stage, grade and age at the time of diagnosis were not significantly associated with PFS. The serum CA-125 levels after surgery and after each of the three cycles of adjuvant chemotherapy were significantly higher in the recurrent group compared with the non-recurrent group (P=0.037, P<0.001, P=0.003, and P=0.032; respectively). ROC curves after surgery and after each of the three cycles of adjuvant chemotherapy revealed that the most significant factor to predict recurrence was serum CA-125 level following the 1st cycle of chemotherapy (area under curve=0.684, P=0.02) (Table 4). The optimal cut point on the ROC curve of serum CA-125 level after 1st cycle of chemotherapy closest to the left upper corner corresponded to a threshold serum CA-125 level of 35 U/mL. At this cut point, the serum CA-125 level after the 1st cycle of adjuvant chemotherapy was able to predict disease progression with a sensitivity of 79.7% and a specificity of 61.0%. The median PFS was 64.6 months (95% CI, 13.9 to 115.2) and 12.8 months (95% CI, 11.8 to 13.8) in patients with serum CA-125 levels ≤35 U/mL and >35 U/mL after he 1st cycle of adjuvant chemotherapy, respectively (log-rank test, P=0.0001).

redict disease progression with a sensitivity of 79.7% and a specificity of 61.0%. The median PFS was 64.6 months (95% CI, 13.9 to 115.2) and 12.8 months (95% CI, 11.8 to 13.8) in patients with serum CA-125 levels ≤35 U/mL and >35 U/mL after he 1st cycle of adjuvant chemotherapy, respectively (log-rank test, P=0.0001). Median value of preoperative CA-125 in this study group was 636.8 U/mL. The median PFS and OS were not statistically different between the patients with preoperative CA-125 levels ≥636.8 U/mL and patients with values exceeding 636.8 U/mL (log-rank test, P=0.719 and P=0.316; respectively). Discussion The serum CA-125 level is elevated most consistently in advanced epithelial ovarian cancer, and its value has been demonstrated as a marker for patient prognosis, disease progression and response to chemotherapy. The nadir CA-125 is defined as lowest point reached during treatment. Recently, some researchers reported the prognostic value of nadir CA-125 in advanced epithelial ovarian cancer [11,12,14-16]. The optimal cutoff level of the nadir CA-125 to predict PFS varies from 10 to 25 U/mL. There has been no unified consensus about the fixed cut-off nadir value for CA-125.

ached during treatment. Recently, some researchers reported the prognostic value of nadir CA-125 in advanced epithelial ovarian cancer [11,12,14-16]. The optimal cutoff level of the nadir CA-125 to predict PFS varies from 10 to 25 U/mL. There has been no unified consensus about the fixed cut-off nadir value for CA-125. In this study, we analyzed serum CA-125 levels in patients who achieved complete remission after primary adjuvant chemotherapy in advanced epithelial ovarian cancer. The median nadir CA-125 level in our subjects was 10.18 U/mL. PFS and OS showed significant differences between patients with nadir CA-125 ≤10 U/mL and 10 to 35 U/mL. Similar to a recent study [15], our data revealed that serum CA-125 in patients with advanced epithelial ovarian cancer with complete remission after primary adjuvant chemotherapy is an independent prognostic factor.

FS and OS showed significant differences between patients with nadir CA-125 ≤10 U/mL and 10 to 35 U/mL. Similar to a recent study [15], our data revealed that serum CA-125 in patients with advanced epithelial ovarian cancer with complete remission after primary adjuvant chemotherapy is an independent prognostic factor. Previous studies reported the timing of normalization of CA-125 correlates to survival. Markman et al. [13] reported that early changes in the serum CA-125 level 8 weeks after initiation therapy was a significant prognostic factor on OS in advanced ovarian cancer. Rocconi et al. [10] found that the timing of normalization of CA-125 throughout primary treatment confirmed the aforementioned findings and corresponded to survival, especially in the 3rd cycle of chemotherapy as a clinically useful parameter for response to current therapy. But, there has been little consensus concerning the timing and which level of serum CA-125 is more effective to predict the recurrence. In our study, we analyzed comparison of series serum CA-125 between the recurrent and non-recurrent groups during primary adjuvant chemotherapy. Serum CA-125 levels after surgery and after the 1st, 2nd, and 3rd cycles of adjuvant chemotherapy were significantly higher in the recurrent group compared to the non-recurrent group. The ROC curve yield for estimating the prognostic value of each cycle of CA-125 revealed that the serum CA-125 after one cycle of chemotherapy was the most significant prognostic factor.

t, 2nd, and 3rd cycles of adjuvant chemotherapy were significantly higher in the recurrent group compared to the non-recurrent group. The ROC curve yield for estimating the prognostic value of each cycle of CA-125 revealed that the serum CA-125 after one cycle of chemotherapy was the most significant prognostic factor. The role of preoperative CA-l25 levels as an independent prognostic factor in epithelial ovarian cancer has been investigated [17,18]. Cooper et al. [17] reported that, after adjusting for covariates, there was a significant association between CA-125 levels and disease-specific survival. We analyzed the preoperative CA-125 level to estimate the prognostic value of survival. Patients were divided into two groups by median preoperative CA-125 levels (636.8 U/mL). PFS and OS did not differ significantly between the two groups, with the log-rank P-value being were 0.719 and 0.316, respectively. Presently, the serum nadir CA-125 level and serum CA-125 level after the 1st cycle of adjuvant chemotherapy were strong independent prognostic factors for advanced epithelial ovarian cancer after complete response.

The role of preoperative CA-l25 levels as an independent prognostic factor in epithelial ovarian cancer has been investigated [17,18]. Cooper et al. [17] reported that, after adjusting for covariates, there was a significant association between CA-125 levels and disease-specific survival. We analyzed the preoperative CA-125 level to estimate the prognostic value of survival. Patients were divided into two groups by median preoperative CA-125 levels (636.8 U/mL). PFS and OS did not differ significantly between the two groups, with the log-rank P-value being were 0.719 and 0.316, respectively. Presently, the serum nadir CA-125 level and serum CA-125 level after the 1st cycle of adjuvant chemotherapy were strong independent prognostic factors for advanced epithelial ovarian cancer after complete response. Early response to adjuvant chemotherapy and normalized serum CA-125 level <10 U/mL were associated with significantly improved prognosis in patients with advanced epithelial ovarian cancer with complete remission. We recommend that, even for patients with advanced epithelial ovarian cancer achieved complete remission, if serum CA-125 levels after the 1st cycle of adjuvant chemotherapy and before 2nd cycle of chemotherapy exceed 35 U/mL and the nadir serum CA-125 exceeds 10 U/mL, closer follow-up is needed, such as frequent imaging study and short follow-up interval. Fig. 1 Progression free survival according to nadir serum CA-125 levels. Fig. 2 Overall survival according to nadir serum CA-125 levels.

Early response to adjuvant chemotherapy and normalized serum CA-125 level <10 U/mL were associated with significantly improved prognosis in patients with advanced epithelial ovarian cancer with complete remission. We recommend that, even for patients with advanced epithelial ovarian cancer achieved complete remission, if serum CA-125 levels after the 1st cycle of adjuvant chemotherapy and before 2nd cycle of chemotherapy exceed 35 U/mL and the nadir serum CA-125 exceeds 10 U/mL, closer follow-up is needed, such as frequent imaging study and short follow-up interval. Fig. 1 Progression free survival according to nadir serum CA-125 levels. Fig. 2 Overall survival according to nadir serum CA-125 levels. Table 1 Characteristics according to the nadir serum CA-125 value of 120 patients with advanced epithelial ovarian cancer BMI, body mass index. Table 2 Simple regression analysis between the nadir CA-125 levels and clinical variables (residual tumor size, initial CA-125 levels) Table 3 Multivariate analysis of progression free survival Table 4 Characteristic of ROC curve for serum CA-125 level after surgery, after 1 cycles, 2 cycle, and 3 cycle of adjuvant chemotherapy ROC, receiver operating characteristic; CI, confidence interval.

Introduction Menorrhagia is a common symptom, accounting for 20% of all gynecological visits to general practitioners, and is a major clinical problem with significant effects on quality of life [1]. Various types of treatments have been used for treatment of menorrhagia including medical therapy, surgical endometrial ablation and even hysterectomy. However, medical therapy has limited efficacy because of the high incidence of recurrence, the need for prolonged treatment and the presence of adverse side effects [2,3]. For example, a study showed that about 25% of women initially subjected to conservative treatment underwent a hysterectomy within the first year [4]. Recently, women with menorrhagia who prefer less invasive surgical techniques are being treated with thermal balloon endometrial ablation (TBA) or levonorgestrel releasing intrauterine system (LNG-IUS). TBA is a new ablative tool providing a simple and safe alternative to conventional hysteroscopic endometrial ablation and requiring less advanced surgical skills [5,6]. LNG-IUS was originally developed as a contraceptive, and is reported to be effective for the treatment of menorrhagia [7,8]. However, 10% to 30% of women who are managed with TBA or LNG-IUS need additional treatments including hysterectomy. Furthermore, the variables associated with treatment failure have not yet been determined [7,9-13].

ly developed as a contraceptive, and is reported to be effective for the treatment of menorrhagia [7,8]. However, 10% to 30% of women who are managed with TBA or LNG-IUS need additional treatments including hysterectomy. Furthermore, the variables associated with treatment failure have not yet been determined [7,9-13]. Several prospective randomized trials have reported that TBA and LNG-IUS were equally effective in the treatment of menorrhagia [14]. However, the authors hypothesize that there may be subgroups where either TBA or LNG-IUS is more effective than the other treatment type. The objectives of the present study were to identify variables associated with treatment failure in women with menorrhagia who were treated with TBA or LNG-IUS, and to determine if there are subgroups where one treatment type is more effective than the other. Materials and Methods This study identified 170 women with menorrhagia who were treated with TBA or LNG-IUS at the study institute between January 2003 and December 2007. Women with a follow-up period less than 12 months were excluded. The remaining 106 women, all of whom had intramural or submucosal myoma, were included in the study. Menorrhagia was defined as symptomatic excessive menstruation: 1) heavy (more than five fully wet pads) or painful menstrual bleeding for more than seven days, or 2) unable to leave house on heaviest days, or 3) sleep disturbed more than one night. All patients had no history of breast cancer, abnormal cervical cytology, or an ongoing pregnancy.

symptomatic excessive menstruation: 1) heavy (more than five fully wet pads) or painful menstrual bleeding for more than seven days, or 2) unable to leave house on heaviest days, or 3) sleep disturbed more than one night. All patients had no history of breast cancer, abnormal cervical cytology, or an ongoing pregnancy. The data collected by retrospective review of medical records included age, parity, results of transvaginal ultrasonography, and treatment type (TBA vs. LNG-IUS). Every woman had a transvaginal ultrasonographic examination before treatment and the presence, size and location of a uterine myoma were evaluated. Treatment failure was defined as persistent or recurrent menorrhagia within the first year after treatment or a hysterectomy due to intractable recurrent bleeding or pain during the follow-up period. Endometrial curettage was performed prior to treatment to rule out endometrial hyperplasia or any hidden malignancy. The choice of treatment type was decided at the discretion of physicians. The TBA procedure was performed at post-menstrual day 2 or 3 using the Gynecare Thermachoice (Ethicon, Somerville, NJ, USA) under intravenous or spinal anesthesia in an operation theatre. The balloon was inserted into the endometrial cavity and filled with 5% dextrose solution. Balloon pressure was adjusted to 180 mm Hg and fluid temperature was maintained at 87℃ for 8 minutes. LNG-IUS (Mirena, Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ, USA) was inserted into the uterine cavity during the first 7 days of the menstrual cycle according to the manufacturer's instructions.

xtrose solution. Balloon pressure was adjusted to 180 mm Hg and fluid temperature was maintained at 87℃ for 8 minutes. LNG-IUS (Mirena, Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ, USA) was inserted into the uterine cavity during the first 7 days of the menstrual cycle according to the manufacturer's instructions. The analysis involved an intent-to-treat population. In addition, women whose LNG-IUS disappeared, or who received other treatments after TBA or LNG-IUS were also included in the analysis. The relationships between variables and treatment outcome were analyzed using the chi-square or Fisher's exact test. Two-sided P-values <0.05 were considered statistically significant. SPSS for Windows ver. 12.0 (SPSS Inc., Chicago, IL, USA) was used for all data analyses.

phase of the menstrual cycle and administration of gonadotropin-releasing hormone analogs or danazol prior to use of TBA improved both the operating conditions and treatment outcomes [13]. Considering these types of risk factors, physicians tend to favor TBA for the treatment of perimenopausal women with severe anemia. A slow and gradual decrease of menorrhagia is frequently observed in women with menorrhagia treated with LNG-IUS. A literature review of reports on LNG-IUS showed that a considerable number of women had withdrawn from treatment primarily because they felt it was ineffective or they experienced irregular vaginal bleeding [11,21,23]. However, in these studies, most of the patients who used the LNG-IUS for 6 to 24 months were satisfied with the treatment effects and their irregular spotting eventually disappeared. Therefore, prior to insertion of LNG-IUS, the physician should explain the possibility of a delayed effect on menorrhagia and the possibility of irregular vaginal bleeding. Furthermore, additional medication during the several months following insertion could increase the success rate of LNG-IUS by encouraging patients not to withdraw from treatment. Such medication can later be discontinued while treatment with LNG-IUS continues [21].

or LNG-IUS were also included in the analysis. The relationships between variables and treatment outcome were analyzed using the chi-square or Fisher's exact test. Two-sided P-values <0.05 were considered statistically significant. SPSS for Windows ver. 12.0 (SPSS Inc., Chicago, IL, USA) was used for all data analyses. Results The median age was 43 years (range, 26 to 52 years) and most of the subjects were primiparous or multiparous. No endometrial abnormalities were detected at endometrial curettage. Sixty-seven women were treated with TBA and 39 women were managed with LNG-IUS. The median follow-up duration was 36 months (range, 12 to 77 months). During follow-up, treatment failure was observed in 24 women (2 recurrent or persistent menorrhagia and 22 hysterectomies) and the LNG-IUS disappeared or was removed in 8 women (Table 1). Women treated with TBA were of similar age to those treated with LNG-IUS (mean age of 43 years for TBA vs. 42 years for LNG-IUS; P=0.272) and had a similar parity distribution compared to patients who were treated with LNG-IUS (P=0.439) (Table 2). However, in terms of complications, the incidence of postoperative irregular spotting was higher in the TBA group than in the LNG-IUS group. Among the variables evaluated in this study, myoma size was significantly associated with treatment outcome in a univariate analysis. On the other hand, age, parity, and treatment type did not affect treatment outcome. Women who were treated with TBA and LNG-IUS had a 76% and 79% treatment success rate, respectively (Table 1).

e variables evaluated in this study, myoma size was significantly associated with treatment outcome in a univariate analysis. On the other hand, age, parity, and treatment type did not affect treatment outcome. Women who were treated with TBA and LNG-IUS had a 76% and 79% treatment success rate, respectively (Table 1). Because treatment outcome differed according to myoma size, the study population was divided into three subgroups according to myoma size and treatment outcome of TBA and LNG-IUS was analyzed for each subgroup. TBA and LNG-IUS had statistically similar treatment outcomes in all groups. However, in women with a myoma larger than or equal to 5.0 cm, TBA had a higher failure rate than LNG-IUS, although it was not statistically significant (56% for TBA, 25% for LNG-IUS) (Table 3). Discussion Several variables were suggested as risk factors for TBA failure in women with menorrhagia. For example, age, multiple myoma, adenomyosis, dysmenorrheal and large uterine size have been suggested as risk factors for treatment failure in women with menorrhagia [15-17]. However, data regarding factors associated with treatment failure in women with menorrhagia are not consistent [17].

in women with menorrhagia. For example, age, multiple myoma, adenomyosis, dysmenorrheal and large uterine size have been suggested as risk factors for treatment failure in women with menorrhagia [15-17]. However, data regarding factors associated with treatment failure in women with menorrhagia are not consistent [17]. The present study showed that a large myoma was associated with treatment failure in women with menorrhagia who were treated with TBA or LNG-IUS and this is in accordance with other studies [3,18-21]. A large myoma could severely distort the endometrial cavity, thus interfering with contact between the balloon and the endometrium during the TBA procedure. Moreover, a myoma may continue to grow, resulting in increased blood flow to the uterus and an interruption of myometrial contractions during menstruation. This could cause a gradual increase in menstrual blood loss [15,18,19,22]. Results from this study suggest that an endometrium that does not fit into the balloon because of the presence of a large myoma might be free from thermal ablation, resulting in persistent or recurrent menorrhagia in women treated with TBA. However, even if the conformation of the endometrial cavity prevents full contact between the balloon and the entire endometrial wall, TBA could be still effective in menorrhagia with small myomas. Therefore, a thorough evaluation of the severity of endometrial cavity distortion using ultrasonography and/or saline infusion hysterosonography may be helpful in treatment decision-making.

s full contact between the balloon and the entire endometrial wall, TBA could be still effective in menorrhagia with small myomas. Therefore, a thorough evaluation of the severity of endometrial cavity distortion using ultrasonography and/or saline infusion hysterosonography may be helpful in treatment decision-making. The TBA procedure actually destroys the endometrium and the effect may be long-lasting or even permanent. LNG-IUS, by contrast, does not destroy the endometrium. The effect on menorrhagia is mediated by slow-releasing levonorgestrel. TBA could have an immediate effect on menorrhagia in a subject without uterine structural abnormalities. On the other hand, many LNG-IUS patients experience a slow and gradual decrease in menstrual blood loss over 6 to 12 months and irregular spotting disappears with time. The difference in outcome kinetics between the two types of endometrial destruction may influence both the patient's and physician's choice of treatment.

the other hand, many LNG-IUS patients experience a slow and gradual decrease in menstrual blood loss over 6 to 12 months and irregular spotting disappears with time. The difference in outcome kinetics between the two types of endometrial destruction may influence both the patient's and physician's choice of treatment. Although the effects of TBA are dramatic in the early phases, delayed treatment failure was reported in several studies, which was probably caused by endometrial regeneration [22-24]. Ultimately, approximately 10% to 20% of the patients in the cited studies were not cured, and both thick endometrium and young age were risk factors for treatment failure. When young women were treated with TBA, about 20% of the patients required additional treatment after 1 to 2 years. Endometrial thinning in the early proliferative phase of the menstrual cycle and administration of gonadotropin-releasing hormone analogs or danazol prior to use of TBA improved both the operating conditions and treatment outcomes [13]. Considering these types of risk factors, physicians tend to favor TBA for the treatment of perimenopausal women with severe anemia.

ia and the possibility of irregular vaginal bleeding. Furthermore, additional medication during the several months following insertion could increase the success rate of LNG-IUS by encouraging patients not to withdraw from treatment. Such medication can later be discontinued while treatment with LNG-IUS continues [21]. In this study, myoma size was associated with treatment failure of TBA or LNG-IUS in women with menorrhagia. However, this study did not observe a statistically significant difference in treatment outcomes in patients with a large myoma. There should be a thorough physician-patient discussion on the merits and drawbacks of each treatment type prior to the treatment. Table 1 Baseline characteristics and the relationship between treatment outcome and age, parity, myoma size and treatment type in women with menorrhagia Values are presented as number (%). TBA, thermal balloon ablation; LNG-IUS, levonorgestrel-releasing intrauterine system. a)Women with unknown parity were excluded from the analysis when calculating P-values; b)Women without myoma were determined when myoma size was <2.5 cm. Table 2 Comparison of patients in the TBA and LNG-IUS groups Values are presented as number (%). TBA, thermal balloon ablation; LNG-IUS, levonorgestrel-releasing intrauterine system. a)Chi-square test or Fisher's exact test. Table 3 Treatment outcome of TBA and LNG-IUS according to myoma size Values are presented as number (%). TBA, thermal balloon ablation; LNG-IUS, levonorgestrel-releasing intrauterine system.

Introduction Primary hepatoid carcinoma of the ovary (HCO) is a rare alpha-fetoprotein (AFP)-producing ovarian tumor which is histologically similar to a hepatocellular carcinoma (HCC). In 1987, Ishikura and Scully [1] first proposed the conception of a "hepatoid carcinoma" and described HCO which had tumor cells with abundant eosinophilic cytoplasm which satisfies morphologic criteria of HCC and positive staining for AFP by immunohistochemistry. Positive staining for AFP was considered to be an essential feature of this tumor [2]. However, Ishikura and Scully [1] suggested that some ovarian carcinomas with typical histologic features of HCO with negative staining for AFP is also belonged in the HCO category. However, such an ovarian tumor has not been described. Recently, we encountered on ovarian carcinoma appropriate for morphologic criteria of HCC but do not stain positive for AFP.

ed that some ovarian carcinomas with typical histologic features of HCO with negative staining for AFP is also belonged in the HCO category. However, such an ovarian tumor has not been described. Recently, we encountered on ovarian carcinoma appropriate for morphologic criteria of HCC but do not stain positive for AFP. Case Report A 51-year-old woman presented to the department of internal medicine with left lower quadrant pain and hematochezia for a month. Colonoscopy revealed hyperemic mass with spontaneous bleeding at about 20 cm above the anal verge which almost obstructs the lumen. A computed tomography (CT) scan of the abdomen showed 12.5×7.8×11.2 cm sized large heterogeneous pelvic mass invading sigmoid colon and uterus (Fig. 1). There was peritoneal seeding and multiple metastatic lymphadenopathy along the bilateral iliac chain and retroperitoneal space up to level of left renal vein but the liver was normal in size and texture. Laboratory analysis revealed normal renal and liver function. The level of CA-125 was elevated to 37.5 U/mL but carcinoembryonic antigen (CEA) and CA-19-9 was normal. She was transferred to the department of Gynecology to rule out ovarian malignancy and positron emission tomography (PET) scan was done. PET scan showed large mass with heterogeneous FDG uptake in the pelvic cavity and multiple hypermetabolic seeding nodules, suggesting peritoneal seeding. The left supraclavicular lymphadenoapathy was seen and fine needle aspiration biopsy revealed metastatic carcinoma. She underwent an explorative laparotomy with total abdominal hysterectomy, bilateral salpingo-oophorectomy, appendectomy and tumorectomy including sigmoid colon with Hartmann's operation. The white-pink, granular appearance 9×8×6 cm sized cancerous mass with necrosis was found posterior to uterus which invades sigmoid colon. Frozen biopsy of the pelvic mass showed poorly differentiated carcinoma but the cell type was unknown because of severe inflammation. Bilateral pelvic lymph nodes were enlarged on palpation however, dissection was not performed. Histologically, the tumor was composed of solid sheets or aggregates of uniform cells with moderate or abundant eosinophilic cytoplasm, distinct cell borders, and centrally located nuclei with prominent nucleoli (Fig. 2). On immunohistochemical staining, p53, p16 was positive; hepatocyte paraffin-1, caudal type homeobox-2, Wilms Tumor-1, estrogen receptor and AFP was negative (Fig. 3).

of uniform cells with moderate or abundant eosinophilic cytoplasm, distinct cell borders, and centrally located nuclei with prominent nucleoli (Fig. 2). On immunohistochemical staining, p53, p16 was positive; hepatocyte paraffin-1, caudal type homeobox-2, Wilms Tumor-1, estrogen receptor and AFP was negative (Fig. 3). The tumor was relatively homongeneous, with no evidence of conventional germ cell tumor or ovarian surface epithelial tumor morphology. It was consistent with hepatoid carcinoma of the right ovary. After the surgery the CA-125 increased from 37.5 to 43.2 U/mL. But the level of serum AFP was 2.2 ng/mL (normal range, 0.0 to 10.0 ng/mL). Chemotherapy was started with an ovarian cancer regimen of paclitaxel 175 mg/m2 and carboplatin with an area under the curve of 5 every 3 weeks. Serum CA-125 returned to normal range after the first cycle of chemotherapy and remained within normal range (24 to 27 U/mL) during three cycles of chemotherapy. A CT scan after the three cycles of chemotherpy showed progression of disease with 4.3 cm recurrent peritoneal carcinomatosis in pelvic cavity and extensive metastatic lymphadenopathy with necrotic change in retroperitoneum, bilateral iliac chain and retrocrural area. Second line treatment with docetaxel was given three cycles but the disease progressed rapidly and ileus developed due to seeding mass in the abdominal cavity and pulmonary effusion newly developed. The patient expired 6 months after the initial diagnosis.

in retroperitoneum, bilateral iliac chain and retrocrural area. Second line treatment with docetaxel was given three cycles but the disease progressed rapidly and ileus developed due to seeding mass in the abdominal cavity and pulmonary effusion newly developed. The patient expired 6 months after the initial diagnosis. Discussion Primary HCO is a rare AFP-producing undifferentiated epithelial carcinoma that is similar to HCC histologically. HCO has been described in women between the ages of 42 to 78 years, with a mean age at diagnosis of 62 years [2]. Though HCO is usually associated with an elevation in serum AFP, these carcinomas are not associated with germ cell neoplastic components or gonadal dysgenesis [1]. Serum CA-125 is also elevated although less than the AFP. Grossly, the tumor may appear as entirely solid or with cystic areas and there may be multiple foci of hemorrhage and necrosis [3]. Microscopically, HCO resembles hepatocellular carcinoma appearing as tumor cells arranged in sheets, and has abundant eosionophillic cytoplasms with central nuclei and distinct cellular borders [4]. On immunostaining, the tumor is focally positive for AFP and polyclonal CEA and is positive for cytokeratin [4].

. Past surgeries included total abdominal hysterectomy for uterine myoma 14 years previously, and an exploratory laparotomy for abdominal adhesions and a peritoneal inclusion cyst 3 years ago. She had menarche at the age of 12 years, and her family history was unremarkable. She was taking hepatic protective medication. Abdominal examination revealed a fist sized palpable mass, without tenderness or rebound tenderness. On pelvic examination, the uterus was absent due to previous hysterectomy and the right adnexa was not palpable due to the central location of the left adnexal mass. No signs of virilization or hirsutism were observed. Laboratory analysis revealed normal values of blood count, hepatic-renal function, coagulation, and electrolytes. Tumor marker studies showed a slightly increased CA-19-9 of 40.62 U/mL (normal range, 0 to 37 U/mL), a normal carcinoembryonic antigen (CEA) of 3.69 ng/mL, and a normal CA-125 of 8.04 U/mL. Transvaginal ultrasound examination revealed no uterus and a large 10×8×7 cm3 sized well defined cystic mass located at the center of the pelvic cavity, with internal septation and no apparent solid portion (Fig. 1). The right adnexa was not clearly observed and no ascites within the pelvic cavity was found, suggesting a recurrent peritoneal inclusion cyst formed after the previous two surgeries.

sis [3]. Microscopically, HCO resembles hepatocellular carcinoma appearing as tumor cells arranged in sheets, and has abundant eosionophillic cytoplasms with central nuclei and distinct cellular borders [4]. On immunostaining, the tumor is focally positive for AFP and polyclonal CEA and is positive for cytokeratin [4]. The differential diagnoses of HCO include hepatoid yolk sac tumor (HYST), clear-cell carcinoma, lipid cell tumor, endometrial carcinoma and undifferentiated carcinoma [1]. HYST is another AFP producing tumor with hepatoid differentiation which is very similar to HCO. HYST occurs in a younger population between the ages of 7 to 54 years, with a mean age at diagnosis of 22 years and is regarded as a germ-cell tumor and is characterized by uniform-appearing tumor cells. HCO usually occurs in an older population between the ages of 42 to 78 years (mean age, 63 years) and is not associated with germ cell neoplastic components or gonadal dysgenesis and has histological feature of pleomorphic tumor cells with abundant eosinophilic cytoplasm. In case of this patient, HYST was unlikely since HYST occurs at a younger age (30 years), is associated with gonadal dysgenesis, and usually contains other germ cell tumors [5]. HYST is hepatocyte paraffin-1 negative and focally positive for CEA. Our case was also negative for hepatocyte paraffin-1 but gonadal dysgenesis was not seen (Fig. 4).

HYST was unlikely since HYST occurs at a younger age (30 years), is associated with gonadal dysgenesis, and usually contains other germ cell tumors [5]. HYST is hepatocyte paraffin-1 negative and focally positive for CEA. Our case was also negative for hepatocyte paraffin-1 but gonadal dysgenesis was not seen (Fig. 4). It is difficult to distinguish metastatic hepatocellular carcinoma from HCO. Hepatocellular carcinoma rarely metastasizes to the ovary [2]. And elevation of CA-125, a marker for ovarian surface epithelial tumors would support an ovarian origin. In the case of our patient, CA-125 was slightly elevated and AFP level was even within normal range. And there was no evidence of an intrahepatic lesion in a pre-operative abdomino-pelvic CT or during an operative inspection. Therefore, metastatic HCC was excluded.

ace epithelial tumors would support an ovarian origin. In the case of our patient, CA-125 was slightly elevated and AFP level was even within normal range. And there was no evidence of an intrahepatic lesion in a pre-operative abdomino-pelvic CT or during an operative inspection. Therefore, metastatic HCC was excluded. HCO is basically an AFP-producing undifferentiated epithelial carcinoma and the diagnosis of HCO was established on the basis of classic histopathologic findings and immunohistochemical staining pattern. Usually tumor cells of HCO are positive for AFP staining and the presence of focal staining for AFP was considered to be essential when diagnosing AFP. However, not all HCO are associated with AFP positive, AFP is negative in some cases. Undifferentiated carcinoma with abundant eosinophillic cytoplasm which is negative for AFP staining is also considered as hepatoid carcinoma [2]. Two cases of HCO have been reported in Korea, and both of them were immunoreactive for AFP staining and serum AFP was elevated [6,7]. Nishida et al. [2] described a case of ovarian hepatoid carcinoma without staining for AFP in primary site. However, AFP staining was positive in metastatic lesion (uterine corpus). In case of our patient, the tumor of ovary, the primary site, was negative for AFP staining, but histologically the tumor was composed of solid sheets or aggregates of uniform cells with moderate or abundant eosinophilic cytoplasm, distinct cell borders, and centrally located nuclei with prominent nucleoli that is typical histologic feature of hepatoid carcinoma. Ishikura and Scully [1] suggested that some ovarian carcinomas with typical histologic features of a hepatoid carcinoma belonged in hepatoid carcinoma category despite their failure to stain for AFP. AFP is an oncodevelopmental protein, produced by the yolk sac, fetal liver and hepatocellular carcinoma and germ cell tumors composed of yolk sac tumor components in adults. Since the production of AFP is closely coupled to cellular division and the degree of cell differentiation, the capability of AFP production by neoplastic cells is transient. Hence, it is reasonable to suggest that positive staining for AFP is not always essential for the diagnosis of AFP producing tumors [2].

adults. Since the production of AFP is closely coupled to cellular division and the degree of cell differentiation, the capability of AFP production by neoplastic cells is transient. Hence, it is reasonable to suggest that positive staining for AFP is not always essential for the diagnosis of AFP producing tumors [2]. HCO is a rare but highly aggressive tumor of uncertain origin. Usually patients with HCO present with lower abdominal pain and progressive abdominal distension. But it can also be detected as an asymptomatic unilateral ovarian mass. HCO commonly progresses rapidly, with metastases in the abdomen and occasionally to the lungs and the patients died within 2 years [2]. Most patients have been treated with ovarian cancer chemotherapy regimens, such as carboplatin and paclitaxel, with shortlived responses [8]. This was also the case with our patient who despite having an initial normal range of AFP and initial decrease in CA-125 developed progression after three cycles of chemotherapy. After disease progression on first-line regimen, we changed regimen to docetaxel but this drug did not lead to a response in this patient. This suggests that the pathological findings are more important than the level of surface epithelial markers such as AFP or CA-125. Fig. 1 A computed tomography scan of the abdomen showed 12.5×7.8×11.2 cm sized large heterogeneous pelvic mass invading sigmoid colon and uterus.

HCO is a rare but highly aggressive tumor of uncertain origin. Usually patients with HCO present with lower abdominal pain and progressive abdominal distension. But it can also be detected as an asymptomatic unilateral ovarian mass. HCO commonly progresses rapidly, with metastases in the abdomen and occasionally to the lungs and the patients died within 2 years [2]. Most patients have been treated with ovarian cancer chemotherapy regimens, such as carboplatin and paclitaxel, with shortlived responses [8]. This was also the case with our patient who despite having an initial normal range of AFP and initial decrease in CA-125 developed progression after three cycles of chemotherapy. After disease progression on first-line regimen, we changed regimen to docetaxel but this drug did not lead to a response in this patient. This suggests that the pathological findings are more important than the level of surface epithelial markers such as AFP or CA-125. Fig. 1 A computed tomography scan of the abdomen showed 12.5×7.8×11.2 cm sized large heterogeneous pelvic mass invading sigmoid colon and uterus. Fig. 2 The tumor was composed of solid sheets or aggregates of uniform cells with moredate or abundant eosinophilic cytoplasm, distinct cell borders, and centrally located nuclei with prominent nucleoli (H&E, ×200). Fig. 3 Immunohistochemistry for alpha-fetoprotein (AFP). The tumor cells are not immunoreactive for AFP (immunostain for AFP, ×200).

Fig. 2 The tumor was composed of solid sheets or aggregates of uniform cells with moredate or abundant eosinophilic cytoplasm, distinct cell borders, and centrally located nuclei with prominent nucleoli (H&E, ×200). Fig. 3 Immunohistochemistry for alpha-fetoprotein (AFP). The tumor cells are not immunoreactive for AFP (immunostain for AFP, ×200). Fig. 4 Immunohistochemistry for hepatocyte paraffin-1. The tumor cells are not immunoreactive for hepatocyte paraffin-1 and gonadal dysgenesis was not seen (immunostain for hepatocyte paraffin-1, ×200).

Introduction Placenta previa is a major concern in obstetrics because of the high risk of massive hemorrhage. The need for a hysterectomy is significantly enhanced in placenta previa with a relative risk of 30% to 40% [1,2]. Treatment of obstetric hemorrhage by embolization of pelvic vessels has been widely used as an alternative to surgical intervention that can avoid hysterectomy [3]. There have been new attempts to apply this tool for bleeding control during Cesarean sections. [4]. This study discusses two cases of intraoperative uterine artery embolization (UAE) without fetal radiation exposure in patients with placenta previa totalis.

Introduction Placenta previa is a major concern in obstetrics because of the high risk of massive hemorrhage. The need for a hysterectomy is significantly enhanced in placenta previa with a relative risk of 30% to 40% [1,2]. Treatment of obstetric hemorrhage by embolization of pelvic vessels has been widely used as an alternative to surgical intervention that can avoid hysterectomy [3]. There have been new attempts to apply this tool for bleeding control during Cesarean sections. [4]. This study discusses two cases of intraoperative uterine artery embolization (UAE) without fetal radiation exposure in patients with placenta previa totalis. Case Reports 1. Case 1 A 40-year-old woman, gravida 0, was admitted to Seoul National University Bundang Hospital in August 2011 for elective Cesarean section and prophylactic intraoperative UAE at 36 weeks and 6 days gestational age. Two thirds of the placenta was located in the posterior wall of the uterus and the remaining placenta was located in the anterior lower segment of the uterus resulting in complete covering of the internal os of cervix. Under spinal anesthesia, a low transverse uterine incision was made and after gently penetrating part of the placenta directly behind the incision site, a live 2,815 g baby was delivered. The upper and lower cut edges and each lateral angle of the uterine incision were clamped using Allis forceps. The torn placental margin was also clamped using Allis forceps. An interventional radiologist, who had been already stood by, stepped into the operation field and then inserted an angiographic catheter into the right femoral artery and approached both uterine arteries using portable fluoroscopy. Insertion of the catheter took five minutes. During that time, the surgeon had observed the uterus carefully for detection of bleeding. Embolization was performed with gelfoam under fluoroscopic guidance for bilateral enlarged tortuous uterine arteries and it took 20 minutes (Fig. 1). After confirming via angiography that there was markedly decreased blood flow, the surgeon removed the placenta. However, bleeding from the implantation site was profuse even though both uterine arteries had been embolized. Bleeding was ultimately controlled by over-sewing the implantation site with 0-monofilament absorbable suture and the rest of the surgery was uneventful. Estimated blood loss for this surgery was 2,500 mL. The patient received five units of packed red blood cells and three units of fresh frozen plasma during the operation. The next day, the patient's hemoglobin was 10.1 g/dL and the sheaths were removed. Routine postoperative care was performed and the patient was discharged from the hospital four days after surgery with no complications.

ed five units of packed red blood cells and three units of fresh frozen plasma during the operation. The next day, the patient's hemoglobin was 10.1 g/dL and the sheaths were removed. Routine postoperative care was performed and the patient was discharged from the hospital four days after surgery with no complications. 2. Case 2 A 32-year-old woman, gravida 2, para 0, was admitted to Seoul National University Bundang Hospital at 35 weeks and 5 days gestational age with symptoms of vaginal bleeding in October 2011. Trans-vaginal ultrasonography revealed that the placenta completely covered the internal os. Approximately two thirds of the placenta was located in the posterior wall of the uterus and the remaining portion was located in the anterior lower segment of uterus. When the patient and her husband were counseled about the risk of hysterectomy, they strongly requested preservation of the uterus for future fertility. Therefore, a Cesarean section with prophylactic intraoperative UAE was planned. Under spinal anesthesia, a uterine incision was made transversely at the lower uterine segment. The surgeon could not avoid tearing the placenta before the female newborn weighing 2,465 g was delivered. The torn placental margins and the cut edges of the uterus were clamped by Allis forceps. During the interventional radiologist tried to insert an angiographic catheter into the right femoral artery for four minutes, the surgeon found a diffuse hemorrhage from the torn placental margin and cut edges of the uterus. At this point, the surgeon abandoned the plan of a prophylactic UAE. The surgeon removed the placenta manually and profuse hemorrhage from the placental bed sites developed. Although the implantation site was repeatedly over-sewn with 0-monofilament absorbable suture, bleeding was quite severe, thus a Cesarean hysterectomy was indicated. However, it was decided that intraoperative UAE would be attempted once more before the Cesarean hysterectomy since massive transfusion was successful. The interventional radiologist inserted an angiographic catheter into the right femoral artery and approached both uterine arteries. Embolization with gelfoam was performed under fluoroscopic guidance for bilateral enlarged tortuous uterine arteries (Fig. 2A). Fortunately, blood flow decreased on angiography and bleeding through the vagina decreased. At this point it seemed possible to avoid a hysterectomy. The uterine wall was repaired after an additional intraoperative UAE.

as performed under fluoroscopic guidance for bilateral enlarged tortuous uterine arteries (Fig. 2A). Fortunately, blood flow decreased on angiography and bleeding through the vagina decreased. At this point it seemed possible to avoid a hysterectomy. The uterine wall was repaired after an additional intraoperative UAE. Estimated blood loss was 5,000 mL and massive transfusion (9 packs of RBC and 3 packs of FFP) was performed during the operation. Although bleeding decreased markedly after two attempts of intraoperative UAE, the patient was transferred to the angiography room for another attempt at UAE. Embolization of the uterine artery was performed with a coil in the angiography room (Fig. 2B) and then the patient was transferred to the intensive care unit (ICU) for close observation. While in the ICU, the patient received three more packs of RBC and six more packs of FFP as well as cryoprecipitate and platelet. The next day, her hemoglobin level was 10.2 g/dL and vital signs were stable. She was transferred to the general ward for further postoperative care. On the second day after surgery, the patient presented with a fever of 39.1℃ and after a full fever study, her antibiotic regimen was changed. The fever resolved on the fourth day and the patient was discharged from the hospital 10 days after surgery with no other complications.

ral ward for further postoperative care. On the second day after surgery, the patient presented with a fever of 39.1℃ and after a full fever study, her antibiotic regimen was changed. The fever resolved on the fourth day and the patient was discharged from the hospital 10 days after surgery with no other complications. Discussion Abnormal placentation, including placenta previa and placenta accrete, is one of the most common causes of postpartum hemorrhage and the most frequent indication for emergent peripartum hysterectomy [5,6]. Recently, UAE has proven to be very effective and is considered the first choice of treatment for patients with postpartum hemorrhage in well-equipped hospitals with skilled radiologists [3]. When applying UAE to patients with postpartum hemorrhage due to placenta previa, the patient must be transported from the operating room to the angiography room. However, only a small portion of patients are hemodynamically stable enough to be transferred for UAE. Intraoperative embolization of pelvic vessels recently has been introduced as an alternative method to reduce expected hemorrhage and ultimately to avoid hysterectomy [7]. All reported cases placed the catheter before starting surgery. Advancement of a catheter under the fluoroscopy is associated with fetal radiation exposure. It has been reported that during this procedure, the fetus is exposed to approximately 3 to 6 rads of radiation in the uterus. A child has an estimated 0.5% risk of developing a childhood cancer following fetal radiation of doses up to 10 rads [8-11]. To resolve these problems, in the cases presented here the catheter was not placed before surgery but an interventional radiologist was prepared for rapid catheterization and embolization, including the presence of portable fluoroscopy equipment (Fig. 3). In case 1, Catheterization itself only took 5 minutes and embolization took 20 minutes. This case is important because UAE was performed successfully before placenta was removed without fetal radiation exposure. However, the total duration of the operation can affect the amount of bleeding and likelihood of infection, which are important issues that must be taken into serious consideration. In case 1, faster closure of the uterus followed by Bakri balloon insertion or postoperative UAE may have been a better choice. Hence, intraoperative UAE should not be performed on all patients with placenta previa.

g and likelihood of infection, which are important issues that must be taken into serious consideration. In case 1, faster closure of the uterus followed by Bakri balloon insertion or postoperative UAE may have been a better choice. Hence, intraoperative UAE should not be performed on all patients with placenta previa. Unfortunately, in case 2, the first attempt at prophylactic intraoperative embolization of the pelvic vessels before placenta removal failed because of profuse bleeding from the torn placental margin and cut edges of uterus before catheterization. Because bleeding from placental bed site was quite severe, the patients could not be transferred to the Angiography room for UAE. Hence, the authors are strongly convinced that the patient in case 2 would have required a hysterectomy if intraoperative UAE had not been performed. There are many issues which must be addressed for prophylactic intraoperative embolization of pelvic vessels in patients with placenta previa: 1) Indication, which patients with placenta previa need prophylactic intraoperative embolization of the pelvic vessels? 2) Quality of fluoroscopy, a hybrid operation room equipped with high-quality fluoroscopy equipment would be helpful. 3) Method of catheter insertion, preoperative placement of the catheter sheath into the femoral artery under guidance of ultrasound may avoid fetal radiation exposure and reduce the time of angiographic catheterization.

oroscopy, a hybrid operation room equipped with high-quality fluoroscopy equipment would be helpful. 3) Method of catheter insertion, preoperative placement of the catheter sheath into the femoral artery under guidance of ultrasound may avoid fetal radiation exposure and reduce the time of angiographic catheterization. In conclusion, the cases presented here suggest that prophylactic embolization of pelvic vessels under fluoroscopy for patients with placenta previa might help preserve fertility by avoiding a hysterectomy without fetal radiation exposure. However, further studies that establish improved techniques and determine more precise indications for prophylactic embolization are necessary. Fig. 1 Intraoperative left uterine artery angiography and embolization using gelfoam. Fig. 2 (A) Intraoperative uterine artery embolization with gelfoam. (B) Coil embolization at the angioroom. Fig. 3 Intraoperative uterine artery embolization setting including portable fluoroscopy.

Introduction Steroid cell tumors of the ovary are seldom identified and constitute only about 0.1% of ovarian tumors. These tumors have been divided into three subtypes according to their cells of origin: stromal luteoma, Leydig cell tumor, and steroid cell tumor, not otherwise specified (NOS) [1,2]. Of these subtypes, the steroid cell tumors, NOS are grouped by exclusion from the other two subtypes, and account for about 56% of steroid cell tumors [2]. An extensive review of the literature reveals the rarity of these tumors, with barely 100 cases of steroid cell tumors, NOS reported from 1979 to the present.

1,2]. Of these subtypes, the steroid cell tumors, NOS are grouped by exclusion from the other two subtypes, and account for about 56% of steroid cell tumors [2]. An extensive review of the literature reveals the rarity of these tumors, with barely 100 cases of steroid cell tumors, NOS reported from 1979 to the present. Most steroid cell tumors are associated with secretion of steroid hormones, which causes symptoms that lead to diagnosis. Generally, testosterone secretion causes virilization or hirsutism, and only 10% to 15% of patients have no clinical signs or symptoms associated with increased hormone levels [3]. Morphologically, steroid cell tumors, NOS are reported as well-circumscribed masses, yellow in color, and solid in about 89% of cases. Only rarely, in about 1.6% of cases, are the tumors completely cystic [2]. An overall incidence of 3.8% has been reported for ovarian pathologic findings requiring repeat operation after hysterectomy for benign conditions [4]. However, only 3 cases have been described of steroid cell tumor diagnosed after a previous hysterectomy. The present report focuses on a case of a rare steroid cell tumor, NOS, diagnosed in a 52-year-old female. The case was significant due to its lack of clinical symptoms, the unusual tumor morphology that was primarily cystic, with septations, and a small inner solid portion, and the patient's surgical history of a previous hysterectomy and pelvic surgery for peritoneal inclusion cyst.

NOS, diagnosed in a 52-year-old female. The case was significant due to its lack of clinical symptoms, the unusual tumor morphology that was primarily cystic, with septations, and a small inner solid portion, and the patient's surgical history of a previous hysterectomy and pelvic surgery for peritoneal inclusion cyst. Case Report A 52-year-old woman (2-0-1-2) was referred to the gynecology department of our hospital for a left adnexal mass incidentally found by liver computed tomography (CT). She was a hepatitis B carrier diagnosed with liver cirrhosis nine years previously, and the liver CT was part of her routine follow-up. Past surgeries included total abdominal hysterectomy for uterine myoma 14 years previously, and an exploratory laparotomy for abdominal adhesions and a peritoneal inclusion cyst 3 years ago. She had menarche at the age of 12 years, and her family history was unremarkable. She was taking hepatic protective medication.

ized well defined cystic mass located at the center of the pelvic cavity, with internal septation and no apparent solid portion (Fig. 1). The right adnexa was not clearly observed and no ascites within the pelvic cavity was found, suggesting a recurrent peritoneal inclusion cyst formed after the previous two surgeries. An additional CT scan of the abdomen and pelvis showed a 10×8 cm sized round mass at the left adnexa, with internal septations and an enhancing solid portion, indicative of a borderline or malignant epithelial ovarian tumor. There was no lymph node enlargement or ascites, but the liver showed signs of cirrhosis (Fig. 2).

An additional CT scan of the abdomen and pelvis showed a 10×8 cm sized round mass at the left adnexa, with internal septations and an enhancing solid portion, indicative of a borderline or malignant epithelial ovarian tumor. There was no lymph node enlargement or ascites, but the liver showed signs of cirrhosis (Fig. 2). The patient underwent a laparoscopy under general anesthesia. The peritoneum and ileum displayed extensive adhesion from the umbilical trocar site to the previous laparotomy site, and although there was no ascites, the mass was not readily observed due to substantial adhesion. Adhesiolysis was performed on the adhesions to the surrounding peritoneum, small bowel, descending colon and sigmoid colon, revealing several septations of the mass and about 100 mL of dark brown serous fluid. As the adhesions were removed, no uterus was revealed, due to the previous hysterectomy, and adhesiolysis of the bladder revealed a 5×4 cm sized bluish left adnexal mass with a thin wall and serous contents. No abnormal signs were noted in the right adnexa, peritoneum, or omentum. After the left salpingo-oophorectomy, the ileum and sigmoid colon were injured from adhesiolysis by a tendency to tear easily, so laparotomy was performed with the general surgery department. The ileum had a 5 cm linear tear at 80 cm superior to the ileocecal valve and segmentectomy with anastomosis was performed. The sigmoid colon had a 3 cm linear tear, which was sutured. During surgery, gross examination and palpation of the internal abdominal organs yielded no notable abnormalities. The abdominal CT suggested a borderline or malignant epithelial ovarian tumor, but because of the patient's history of surgery 3 years before for abdominal adhesion and peritoneal inclusion cyst, and because extensive adhesion was noted during surgery, a recurrent peritoneal inclusion cyst was suspected and the surgery was concluded without any additional procedures.

ant epithelial ovarian tumor, but because of the patient's history of surgery 3 years before for abdominal adhesion and peritoneal inclusion cyst, and because extensive adhesion was noted during surgery, a recurrent peritoneal inclusion cyst was suspected and the surgery was concluded without any additional procedures. Gross pathological findings showed a 6×6 cm sized mass, mostly cystic. The outer surface had heavy adhesions, and the mass was untidy internally, with a small inner solid portion, but no signs of hemorrhage or necrosis. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization, with no signs of nuclear atypicality and a mitotic count of 5 per 10 HPF (Fig. 3A). Immunohistochemistry revealed a positive reaction to inhibin, calretinin, and vimentin, a moderately positive reaction to CD99, and a negative reaction to pan-CK, S-100, placenta-like alkaline phosphatase, and chromogranin (Fig. 3B, C). The Ki-67 labeling index was low, under 5%. The histopathologic features supported a diagnosis of an ovarian steroid cell tumor, NOS. Postoperatively, the patient's testosterone level was normal, at 0.32 ng/mL (normal range, 0.14 to 0.76 ng/mL). No hormonal assays were done before surgery as there were no clinical manifestations of any hormone secretion. She is on regular follow-up at our outpatient clinic, with no evidence of recurrent disease for 21 months.

tively, the patient's testosterone level was normal, at 0.32 ng/mL (normal range, 0.14 to 0.76 ng/mL). No hormonal assays were done before surgery as there were no clinical manifestations of any hormone secretion. She is on regular follow-up at our outpatient clinic, with no evidence of recurrent disease for 21 months. Discussion Ovarian tumors are rarely diagnosed as steroid cell tumors; these account for less than 0.1% of all ovarian tumors. Steroid cell tumors of the ovary have a diverse cellular origin; they are a group of ovarian neoplasms with morphologic similarities that, in the past, were termed as lipid or lipoid cell tumors. These tumors are made of cells that resemble steroid hormone secreting cells, but some cases show scarce or no lipid components and some tumors have a tendency to secrete various steroid hormones with specific clinical presentations. For these reasons, Hayes and Scully [2] proposed the term steroid cell tumor, which was accepted by World Health Organization in 1999.

roid hormone secreting cells, but some cases show scarce or no lipid components and some tumors have a tendency to secrete various steroid hormones with specific clinical presentations. For these reasons, Hayes and Scully [2] proposed the term steroid cell tumor, which was accepted by World Health Organization in 1999. Steroid cell tumors, NOS are found in almost any age group, from premenarchal girls to postmenopausal women, with an average age at diagnosis of 43 years [2]. The clinical presentation may take many forms, including abdominal pain, distention, and bloating. However, the more noticeable presentations are those associated with hormonal activity. Steroid cell tumors secreting steroid hormones are prevalent, mostly secreting testosterone in 50% of steroid cell tumors, NOS [3]. About 8% to 23% of steroid cell tumors, NOS, show signs of estrogen secretions, and cortisol secretion may be diagnosed in 6% to 10% of cases [2,5]. A minority of 10% to 15% of patients with steroid cell tumor, NOS have no symptoms of increased steroid hormone production [3]. In this case, the apparent absence of androgenic manifestations is an unusual finding.

, show signs of estrogen secretions, and cortisol secretion may be diagnosed in 6% to 10% of cases [2,5]. A minority of 10% to 15% of patients with steroid cell tumor, NOS have no symptoms of increased steroid hormone production [3]. In this case, the apparent absence of androgenic manifestations is an unusual finding. The size of steroid cell tumors, NOS range from under 1 cm to over 45 cm, and the average size at diagnosis is known as 8.4 cm [2]. These tumors are mostly unilateral, with 6% bilateral, and are typically well circumscribed solid tumors in 89% of cases, generally yellow in color, and in some cases lobulated [2,5]. Necrosis and hemorrhage may be found, and only one tumor of the 61 cases studied by Hayes and Scully was described as almost completely cystic [2,5]. An extensive review of case reports in the PubMed database from 1979 until the present revealed only 5 cases that were mainly cystic tumors. No cases have been illustrated, to our knowledge, with inner septa and extensive adhesion to surrounding structures. In our patient, the tumor was a 9 cm sized overall cystic mass with septations and a small solid portion, with tight adhesions to the bladder and bowels (so much so that reparative procedures were necessary after extraction of the mass).

our knowledge, with inner septa and extensive adhesion to surrounding structures. In our patient, the tumor was a 9 cm sized overall cystic mass with septations and a small solid portion, with tight adhesions to the bladder and bowels (so much so that reparative procedures were necessary after extraction of the mass). The majority of steroid cell tumors have benign or low-grade behavior. These tumors are found comparatively early due to endocrine activity, and show low rates of recurrence and metastasis [2,5]. About 25% to 43% are reported clinically malignant [2,5], and in such cases 44% to 83% show symptoms of hormone impairment such as virilization, and 17% are associated with Cushing's syndrome due to cortisol increase [2]. Hayes and Scully [2] detailed five microscopic findings that are highly associated with malignancy: 1) the presence of two or more mitotic figures per 10 HPF in 92%, 2) necrosis in 86%, 3) a diameter of 7 cm or greater in 78%, 4) hemorrhage in 77%, and 5) grade 2 or 3 nuclear atypia in 64% of cases [2]. In our case, as the size of the mass was large (9 cm) and 5 mitotic figures per 10 HPF were observed, malignancy was a possibility, but as no foci of necrosis or hemorrhage, no nuclear atypia, and no signs of invasion or metastasis were evident, we considered the tumor to be benign, and the patient has shown no signs of recurrence during 21 months of follow-up.

rge (9 cm) and 5 mitotic figures per 10 HPF were observed, malignancy was a possibility, but as no foci of necrosis or hemorrhage, no nuclear atypia, and no signs of invasion or metastasis were evident, we considered the tumor to be benign, and the patient has shown no signs of recurrence during 21 months of follow-up. Immunohistochemistry may be utilized in diagnosis, and generally inhibin is used. Inhibin is present in ovarian granulosa and lutein cells, where it suppresses the production of pituitary gonadotropins [6]. Steroid cell tumors, NOS are positive for inhibin and vimentin in 75%, 7% are positive for S-100 protein, and they are negative for chromogranin A [7]. Calretinin, a calcium-binding protein discovered in mesothelial cells, is also present in the ovary and testis, and is expressed by sex-cord stromal tumors [6]. Deavers et al. [6] found that the extent of staining for inhibin ranged from under 5% to over 90%, and 60% to over 90% for calretinin, in 6 cases of steroid cell tumor, NOS; all cases were positive for inhibin and calretinin. CD99 is another marker expressed by sex-cord stromal tumors and it reacts with normal granulosa and Sertoli cells, and it showed membranous staining in one of the previous 6 cases [6]. Our case was positive for inhibin, calretinin, and vimentin, moderately positive for CD99, and negative for S-100 protein and chromogranin A, with a diagnosis of steroid cell tumor, NOS.

l tumors and it reacts with normal granulosa and Sertoli cells, and it showed membranous staining in one of the previous 6 cases [6]. Our case was positive for inhibin, calretinin, and vimentin, moderately positive for CD99, and negative for S-100 protein and chromogranin A, with a diagnosis of steroid cell tumor, NOS. No specific tumor markers or imaging techniques are known for preoperative diagnosis of steroid cell tumors. Tumor markers such as CA-125 and α-fetoprotein, etc., are generally normal and the literature does not indicate if elevated levels signify malignant potential. This case demonstrated a normal level of CA-125, and although CA-19-9 was temporarily mildly elevated, this is considered to result from the patient's underlying liver cirrhosis. Imaging before surgery may be checked for ovarian or adrenal masses, and vaginal or abdominal ultrasound may help to identify ovarian mass characteristics. Wang et al. [8] found preoperative magnetic resonance imaging to be an effective means for staging and gadolinium-diethylene-triamine-pentaacetic acid enhancement to be helpful for locating metastasis within the pelvic cavity. In another study, Wang et al. [9] reported whole-body positron emission tomography with [11C]acetate to aid in the diagnosis of steroid cell tumors that were secreting testosterone. Most recently, Sakamoto et al. [10] proposed chemical shift magnetic resonance imaging as a technique for diagnosis by observing the cytoplasmic lipid content of steroid cell tumors.

hole-body positron emission tomography with [11C]acetate to aid in the diagnosis of steroid cell tumors that were secreting testosterone. Most recently, Sakamoto et al. [10] proposed chemical shift magnetic resonance imaging as a technique for diagnosis by observing the cytoplasmic lipid content of steroid cell tumors. No definitive treatment is established for steroid cell tumors, NOS, due to their rarity, but the tumors are considered to be sex cord-stromal tumors and the primary treatment is surgery. Only 6% are bilateral, so a young woman desiring future childbearing with a low stage tumor may undergo unilateral salpingo-oophorectomy, with regular monitoring of any preoperatively increased hormones. An older woman with a low stage tumor and no desire for parity may have a total hysterectomy with bilateral salpingo-oophorectomy. A high stage tumor should undergo size reduction and adjuvant chemotherapy or radiation therapy should be considered [2]. However, as symptoms lead to early diagnosis and as recurrence or metastasis is rare, the research on adjuvant therapy is insufficient and many are skeptical. If preoperatively increased testosterone does not change after surgery, gonadotropin releasing hormone agonists may be utilized [11]. As steroid cell tumors, NOS are rare, their staging and prognosis is unclear, but Hayes and Scully [2] analyzed 63 cases as 88% stage 1, 6.8% stage 2, 12% stage 3, and 1.7% stage 4 [2]. Cases with a higher stage, larger size, gross necrosis, or hemorrhage were found to have higher malignant potential with worse prognosis [2]. Recurrence or metastasis were also associated with worse prognosis due to insufficient research on additional treatment options.

6.8% stage 2, 12% stage 3, and 1.7% stage 4 [2]. Cases with a higher stage, larger size, gross necrosis, or hemorrhage were found to have higher malignant potential with worse prognosis [2]. Recurrence or metastasis were also associated with worse prognosis due to insufficient research on additional treatment options. Some observations indicate that the incidence of ovarian tumors rises after hysterectomy. The overall incidence of ovarian pathologic findings requiring repeat operation after hysterectomy for benign conditions has been reported as 3.8% [4]. Only 3 cases of steroid cell tumor diagnosed after a previous hysterectomy have been described, and our case is the fourth [12-14].

ian tumors rises after hysterectomy. The overall incidence of ovarian pathologic findings requiring repeat operation after hysterectomy for benign conditions has been reported as 3.8% [4]. Only 3 cases of steroid cell tumor diagnosed after a previous hysterectomy have been described, and our case is the fourth [12-14]. The almost completely cystic characteristic of our case was unusual for a steroid cell tumor, NOS, which are usually solid tumors, and this case shows that even in an apparently benign, mostly cystic, ovarian tumor, a rare steroid cell tumor with undetermined behavior may be diagnosed. Careful clinical assessment should be carried out when evaluating ovarian tumors. In our case, the patient showed no signs of recurrence or metastasis, which are considered to be malignant behavior. The most important factor to be determined in steroid cell tumors of the ovary is whether the tumor has malignant features. Steroid cell tumors, NOS are clinically malignant in 25% to 43% of cases, and pathologically benign steroid cell tumors are known to behave in a clinically malignant fashion. Therefore, careful follow-up after surgery is imperative even in those cases that do not have clinical or pathological evidence of malignancy. We report a patient with a history of hysterectomy, with an incidentally found, asymptomatic, predominantly cystic tumor with internal septa and an inner solid portion, which was diagnosed as steroid cell tumor, NOS. The patient has had no signs of recurrence for 21 months after surgery. We also provide a brief review of the existing literature.

history of hysterectomy, with an incidentally found, asymptomatic, predominantly cystic tumor with internal septa and an inner solid portion, which was diagnosed as steroid cell tumor, NOS. The patient has had no signs of recurrence for 21 months after surgery. We also provide a brief review of the existing literature. Fig. 1 Transvaginal ultrasound indicating a large 10×8×7 cm3 sized cystic mass at the center of the pelvic cavity, with internal septation and no apparent solid portion. Fig. 2 An abdominopelvic computed tomographic scan after enhancement shows a 10×8 cm sized cystic mass in the left ovary, with internal septation (arrow) and a solid portion (arrowhead), which is enhanced by contrast media. Fig. 3 (A) Histological specimen shows large aggregates of tumor cells with abundant eosinophilic to clear cytoplasm, with a granular appearance and vacuolization (H&E, ×200). (B) Immunohistochemical staining shows the tumor cells exhibiting a diffuse positive reaction for inhibin (brown) (×200). (C) Immunohistochemical staining shows a diffuse positive reaction of the tumor cells for calretinin (dark red) (×100).

Introduction The criteria of preeclampsia have remained unchanged for the past several years. This includes: a proteinuria reading of ≥30 mg (≥1+on dipstick) in two random urine samples collected at least 4 to 6 hours apart or 24-hour urine protein excretion ≥300 mg/day with onset at >20 weeks of gestational age, a systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg measured twice. All of these findings should normalize within the 6th postpartum week [1]. The 24-hour urine protein excretion measurement has been the gold standard for quantifying urinary protein, but it is an inconvenient and time-consuming test. In comparison, the urinary dipstick test may be a quicker and simpler method but its drawbacks are its inconsistency and poor correlation with 24-hour urine protein excretion level [2], because of its susceptibility to patients' hydration status. In this respect, an alternative method that is as simple and rapid as the dipstick test but with better accuracy in predicting the amount of secreted urinary protein would be valuable.

tency and poor correlation with 24-hour urine protein excretion level [2], because of its susceptibility to patients' hydration status. In this respect, an alternative method that is as simple and rapid as the dipstick test but with better accuracy in predicting the amount of secreted urinary protein would be valuable. The measurement of a random urine protein-creatinine (P/C) ratio has been tested as a substitute for the 24-hour urine protein excretion test for quantifying protein excretion in patients with renal diseases, such as diabetic nephropathy, lupus nephritis, and transplanted kidneys, with good correlation between the two methods [3-7]. The method relies on calculating the ratio of spot urine protein excretion to creatinine excretion and can normalize the protein excretion to the glomerular filtration rate. Therefore, a random urine P/C ratio is not influenced by variations in hydration status [8]. However, because of the variety of the cutoff values among past studies [9-11], there is no uniform standard so the clinical usefulness of this test is still controversial. The aim of the present study was to evaluate the ability of the random urine P/C ratio to predict significant proteinuria, as well as to introduce a diagnostic test for preeclampsia that would avoid the inconvenience and time consumption of 24-hour urine protein collection.

The measurement of a random urine protein-creatinine (P/C) ratio has been tested as a substitute for the 24-hour urine protein excretion test for quantifying protein excretion in patients with renal diseases, such as diabetic nephropathy, lupus nephritis, and transplanted kidneys, with good correlation between the two methods [3-7]. The method relies on calculating the ratio of spot urine protein excretion to creatinine excretion and can normalize the protein excretion to the glomerular filtration rate. Therefore, a random urine P/C ratio is not influenced by variations in hydration status [8]. However, because of the variety of the cutoff values among past studies [9-11], there is no uniform standard so the clinical usefulness of this test is still controversial. The aim of the present study was to evaluate the ability of the random urine P/C ratio to predict significant proteinuria, as well as to introduce a diagnostic test for preeclampsia that would avoid the inconvenience and time consumption of 24-hour urine protein collection. Materials and Methods 1. Patients We enrolled 140 pregnant women who were admitted to Yonsei University Severance Hospital Obstetrics Department with a suspicion of preeclampsia between January 2006 and June 2011. Women with symptoms of preeclampsia and more than one clinical finding, such as hypertension, edema accompanied by rapid weight gain with or without headache, and new-onset proteinuria on a urinary dipstick test, were included. The only exclusion criterion was a concurrent preexisting renal disease such as immunoglobulin (Ig) A nephropathy.

preeclampsia and more than one clinical finding, such as hypertension, edema accompanied by rapid weight gain with or without headache, and new-onset proteinuria on a urinary dipstick test, were included. The only exclusion criterion was a concurrent preexisting renal disease such as immunoglobulin (Ig) A nephropathy. 2. Laboratory tests Firstly, urine was collected via catheterization for the random urine P/C ratio and the urinary dipstick test. Then, a 24-hour urine was collected via a clean catch. Random urine P/C ratio was determined by a Hitachi 7180 Autoanalyzer (Hitachi, Tokyo, Japan). 3. Statistical analyses Statistical analysis was performed with the SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA) and SAS ver. 9.2 (SAS Inc., Cary, NC, USA). We defined significant proteinuria as 24-hour urine protein excretion ≥300 mg/24 hr. If urine protein excretion was ≥5,000 mg/24 hr, this was considered severe proteinuria, per the guidelines of the International Society for the Study of Hypertension in Pregnancy and the American College of Obstetrics and Gynecology [12,13].

ined significant proteinuria as 24-hour urine protein excretion ≥300 mg/24 hr. If urine protein excretion was ≥5,000 mg/24 hr, this was considered severe proteinuria, per the guidelines of the International Society for the Study of Hypertension in Pregnancy and the American College of Obstetrics and Gynecology [12,13]. The 24-hour urine protein excretion results were used as a gold standard in determining the cutoff points for the significant and severe proteinuria. Sensitivity, specificity, and positive and negative predictive values of random urine P/C ratio were also calculated against this standard. The correlation between random urine P/C ratio and 24-hour urine protein excretion level was analyzed by regression. Receiver operator characteristic (ROC) curves were evaluated to determine the optimal random urine P/C ratio cutoff value that maximized sensitivity and specificity in the detection of significant and severe proteinuria with 24-hour urine protein excretion.

hour urine protein excretion level was analyzed by regression. Receiver operator characteristic (ROC) curves were evaluated to determine the optimal random urine P/C ratio cutoff value that maximized sensitivity and specificity in the detection of significant and severe proteinuria with 24-hour urine protein excretion. Results 1. Study population A total of 140 pregnant women were admitted for the evaluation and management of preeclampsia. Among them, 79 were assigned to random urine P/C ratio or 24-hour urine protein excretion. Of these, 71 (89%) were diagnosed with preeclampsia. In 33 of the 79 (42%), either indicated or spontaneous delivery occurred prior to the completion of a 24-hour urine collection; these patients were excluded from the analysis (Table 1). Both the 24-hour urine and random urine samples were available in 46 (58%) patients and their characteristics were as demonstrated in Table 2. The mean maternal and gestational ages were 33.2±4.8 years and 33.3±3.4 weeks, respectively. The mean systolic and diastolic blood pressure was 157.8±20.7 mm Hg and 97.5±9.5 mm Hg, respectively. None of the patients had a history of chronic hypertension or renal disease. Three patients (7%) had a prior history of preeclampsia. Of the 46 patients, 1 (2%) was diagnosed with mild preeclampsia, 35 (76%) with severe preeclampsia, 8 (17%) with gestational hypertension, and 2 (4%), with superimposed preeclampsia, respectively.

e patients had a history of chronic hypertension or renal disease. Three patients (7%) had a prior history of preeclampsia. Of the 46 patients, 1 (2%) was diagnosed with mild preeclampsia, 35 (76%) with severe preeclampsia, 8 (17%) with gestational hypertension, and 2 (4%), with superimposed preeclampsia, respectively. Urinary protein excretion was 300 to 5,000 mg per 24 hours in 38 patients (83%) and ≥5,000 mg per 24 hours in 6 patients (13%) (Table 3). Among the enrolled patients, 2 urine samples (4%) measured <300 mg in 24-hours, which did not fulfill for the criteria for proteinuria in preeclamptic pregnancy. The mean collected 24-hour urine volume was 1,448.5±868.9 mL and the mean protein excretion in those sample was 2,713±2,930.2 mg/dL. The mean random urine P/C ratio was 4.2±3.7 (Table 4). 2. Correlation statistic The correlation coefficient for random urine P/C ratio to 24-hour urine protein excretion was 0.82 (P<0.01), indicating strong agreement between the two tests.

Urinary protein excretion was 300 to 5,000 mg per 24 hours in 38 patients (83%) and ≥5,000 mg per 24 hours in 6 patients (13%) (Table 3). Among the enrolled patients, 2 urine samples (4%) measured <300 mg in 24-hours, which did not fulfill for the criteria for proteinuria in preeclamptic pregnancy. The mean collected 24-hour urine volume was 1,448.5±868.9 mL and the mean protein excretion in those sample was 2,713±2,930.2 mg/dL. The mean random urine P/C ratio was 4.2±3.7 (Table 4). 2. Correlation statistic The correlation coefficient for random urine P/C ratio to 24-hour urine protein excretion was 0.82 (P<0.01), indicating strong agreement between the two tests. 3. ROC curves The ROC curves for ≥300 mg per 24-hour urine and ≥5,000 mg per 24-hour urine are presented in Fig. 1A, B. The area under the ROC curve of ≥300 mg per 24-hour group was 0.958 (95% confidence interval [CI], 0.903 to 1.000) and for the ≥5,000 mg per 24-hour group was 0.921 (95% CI, 1.074 to 2.002). Analysis of the ROC curve indicated that a random urine P/C ratio of 4.68 was the best cutoff point to detect significant (≥300 mg per 24-hours) proteinuria and to detect severe (≥5,000 mg per 24-hours) proteinuria. The sensitivity, specificity, and positive and negative predictive values were 87.1%, 100%, 100%, and 58.3% for significant preeclampsia and 100%, 85%, 50%, and 100%; for severe preeclampsia. We also compared the area under the ROC curve of the ≥300 mg per 24-hour to that of the urinary dipstick test in Fig. 1C. Each was 0.958 (95% CI, 0.903 to 1.000) and 0.935 (95% CI, 0.883 to 0.899), respectively, demonstrating no significant difference between the two tests. Interestingly, in 4 patients with a negative urine dipstick test but significant proteinuria by 24-hour urine protein excretion, random urine P/C ratio was positive.

those from normal pregnancy (Table 3). When villous tissue explants were cultured for 36 hours with or without interferon-γ, both indoleamine 2,3-dioxygenase activity and the percentage stimulation were still significantly lower in villous tissue from pre-eclampsia than was found for tissue from normal pregnancy [28]. Conclusion Experiments described in this review were conducted in order to investigate the physiological role of the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase in the human placenta on the basis of Munn et al. [4]'s hypothesis in mouse that placental expression of this enzyme is crucial to prevent immunological rejection of the allogeneic fetus. Thus indoleamine 2,3-dioxygenase dependent localised depletion of tryptophan at the site of placentation has been proposed to be the mechanism of the suppression of maternal T cells which attack the conceptus [29].

. Each was 0.958 (95% CI, 0.903 to 1.000) and 0.935 (95% CI, 0.883 to 0.899), respectively, demonstrating no significant difference between the two tests. Interestingly, in 4 patients with a negative urine dipstick test but significant proteinuria by 24-hour urine protein excretion, random urine P/C ratio was positive. Discussion Mild preeclampsia is defined as a systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg measured twice at least 6 hours apart, with proteinuria of ≥1+ on urinary dipstick test or urine protein ≥300 mg/24 hr. Severe preeclampsia is defined as systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥110 mm Hg with urine protein ≥5,000 mg/24 hr, sudden oliguria of <500 mL/day, central nervous system manifestations such as headache, visual disturbance, pulmonary edema or cyanosis, epigastric pain, HELLP syndrome, thrombocytopenia, and fetal intrauterine growth restriction [14]. Of these, measuring proteinuria is mandatory for the diagnosis and evaluation of the severity of the preeclampsia. Some studies have found that maternal and perinatal morbidity and mortality increased with the amount of proteinuria [15].

HELLP syndrome, thrombocytopenia, and fetal intrauterine growth restriction [14]. Of these, measuring proteinuria is mandatory for the diagnosis and evaluation of the severity of the preeclampsia. Some studies have found that maternal and perinatal morbidity and mortality increased with the amount of proteinuria [15]. Several methods are available for measuring proteinuria but 24-hour urine protein excretion has long been regarded as the gold standard. However, this test has some disadvantages such as inconvenience for patients, inaccuracy due to incomplete collection, cost, and delay of diagnosis and management, which makes its wide use difficult for clinicians. For this reason, many investigators have explored simpler and more convenient diagnostic methods to quantify proteinuria. One of the most commonly used is the urinary dipstick test because of its simplicity and low cost. Nevertheless, this method has high false positive and false negative result rates associated with fluctuations throughout the day due to water intake, exercise, diet, posture, or improperly trained laboratory staff [16-18]. The urinary dipstick test values of 3+ to 4+ are also not valid for diagnosis of severe preeclampsia because of their very low positive predictive value, which can be as low as 36% [2]. For this reason, a more rapid and accurate diagnostic test that is capable of predicting 24-hour urine protein excretion would be valuable. Here, we propose the use of random urine P/C ratio to facilitate prompt decision making by clinicians [19,20].

r very low positive predictive value, which can be as low as 36% [2]. For this reason, a more rapid and accurate diagnostic test that is capable of predicting 24-hour urine protein excretion would be valuable. Here, we propose the use of random urine P/C ratio to facilitate prompt decision making by clinicians [19,20]. Our study compared the diagnostic benefits of random urine P/C ratio to the 24-hour urine protein excretion, the gold standard. From all the enrolled patients, 2 24-hour urine samples (4%) showed protein levels <300 mg, although the values were ≥1+ on the urinary dipstick test. This discrepancy probably occurred due to improper 24-hour urine collection. In fact, previous studies have demonstrated inadequate 24-hour urine collection volume in up to 37% of study samples [3].

s, 2 24-hour urine samples (4%) showed protein levels <300 mg, although the values were ≥1+ on the urinary dipstick test. This discrepancy probably occurred due to improper 24-hour urine collection. In fact, previous studies have demonstrated inadequate 24-hour urine collection volume in up to 37% of study samples [3]. The present study demonstrates the value of using the random urine P/C ratio to predict 24-hour urine protein excretion. As shown, a strong correlation was found between the two tests correlation coefficient, 0.82; P<0.01). We also found the optimal cutoff points by analyzing ROC curves for significant (≥300 mg per 24-hours) proteinuria and severe (≥5,000 mg per 24-hours) proteinuria. Also, the cutoff points of 0.63 and 4.68 were found to provide optimal sensitivity and specificity for the detection of significant and severe proteinuria, respectively. This indicates that when a random urine P/C ratio is less than 4.68, it is highly likely that the 24-hour urine protein excretion is less than 5,000 mg (criteria for severe preeclampsia) whereas if random urine P/C ratio is over 0.63, 24-hour urine protein excretion is always over 300 mg. Our results showed no statistically significant correlation between results of random urine P/C ratios and the urinary dipstick tests. However, in the case of 4 patients whose 24-hour urine protein results were more than 300 mg, the urinary dipstick test results were negative, which emphasizes the limitations of the urinary dipstick test method. Meanwhile, in these same patients, the random urine P/C ratios were higher than 0.63, which indicates that it could appropriately diagnose preeclampsia. Therefore, the random urine P/C ratio value could become an effective alternative for making a diagnosis of preeclampsia and can overcome the false negative results of the urinary dipstick test. There are several studies that compare the utility between random urine P/C ratios and the urinary dipstick tests in nephrology units as well as in obstetrics. Gai et al. [21] proposed that the random urine P/C ratio could replace the urinary dipstick test, as they found that the highest regression coefficient was between 24-hour urine protein and random urine P/C ratio (r=0.72), and the lowest between random urine P/C ratio and the urinary dipstick test (r=0.72) in patients with renal disease.

posed that the random urine P/C ratio could replace the urinary dipstick test, as they found that the highest regression coefficient was between 24-hour urine protein and random urine P/C ratio (r=0.72), and the lowest between random urine P/C ratio and the urinary dipstick test (r=0.72) in patients with renal disease. They also revealed that the urinary dipstick test failed to detect a pathological proteinuria in 31.6% of their study population while random urine P/C ratio failed to detect in only 5.4%. Meanwhile, in their study with preeclamptic pregnancy, Eigbefoh et al. [22] showed that a random urine P/C ratio had the highest sensitivity, 92%, while the urinary dipstick test had a sensitivity of 81%. Their results also showed that the urinary dipstick test had the highest false negative rate (19%), while that of the random urine P/C ratio was 8%. Furthermore, the specificity for random urine P/C ratio was 86%, while that for the urinary dipstick test was 47%. Also in their study, the false positive rate was highest with the urinary dipstick test (53%), and only 14% for random urine P/C ratios.

egative rate (19%), while that of the random urine P/C ratio was 8%. Furthermore, the specificity for random urine P/C ratio was 86%, while that for the urinary dipstick test was 47%. Also in their study, the false positive rate was highest with the urinary dipstick test (53%), and only 14% for random urine P/C ratios. Several studies have already established the usefulness of the random urine P/C ratio, including some that have presented evidence of good correlation with acceptable sensitivity in predicting significant proteinuria based on urine collected at different time periods [23-25]. Furthermore, some reports indicate maternal age, gestational age, parity and maternal body size are not confounding factors with regard to random urine P/C ratio [26]. However, some prior studies that evaluated the accuracy of random urine P/C ratio in predicting 24-hour urine protein excretion in preeclampsia reveal some conflicting results for determining an optimal cutoff point for significant proteinuria. This is because of the study-to-study variability in laboratory methods for measuring proteinuria, which precludes valid comparison among the studies. Some reports indicate appropriate cutoff points ranging from 0.15 to 0.5 and sensitivities and specificities from 77.5% to 89.7% and from 72.6% to 80%, respectively [27].

his is because of the study-to-study variability in laboratory methods for measuring proteinuria, which precludes valid comparison among the studies. Some reports indicate appropriate cutoff points ranging from 0.15 to 0.5 and sensitivities and specificities from 77.5% to 89.7% and from 72.6% to 80%, respectively [27]. The strength of our study comes from the fact that the random urine P/C ratio was determined before 24-hour urine collection was completed, thereby reducing the potential for a falsely elevated random urine P/C ratio after completion of 24-hour urine collection because of the progression of preeclampsia. However, our study is limited due to its small sample size. Further studies can overcome this issue incorporating a larger study population that also includes outpatient clinic patients. Based on the results of our study, we conclude that random urine P/C ratios can predict 24-hour urine protein excretion with a high accuracy. This test could be used as a reasonable alternative to 24-hour urine protein excretion, especially in emergent situations, and, it could also complement the urinary dipstick test in preeclamptic pregnancy. Acknowledgments This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2010-0010727).

Based on the results of our study, we conclude that random urine P/C ratios can predict 24-hour urine protein excretion with a high accuracy. This test could be used as a reasonable alternative to 24-hour urine protein excretion, especially in emergent situations, and, it could also complement the urinary dipstick test in preeclamptic pregnancy. Acknowledgments This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2010-0010727). Fig. 1 Receiver operating characteristic (ROC) curves. (A) ROC curves for ≥300 mg per 24-hours urine samples (AUC, 0.958; 95% CI, 0.903 to 1.000). (B) ROC curves for ≥5,000 mg per 24-hours urine samples (AUC, 0.921; 95% CI, 1.074 to 2.002). (C) Comparison of ROC curve of ≥300 mg per 24-hours urine collection to urinary dipstick test (AUC, 0.958; 95% CI, 0.903 to 1.000) and 0.935 (95% CI, 0.883 to 0.899). AUC, area under the curve; UPCR, urine protein-creatinine ratio. Table 1 The causes of incomplete and/or inadequate 24-hour urine collection BP, blood pressure; HELLP SD, hemolytic anemia elevated liver enzyme low platelet syndrome. Table 2 Characteristics of study population (n=46) Values are presented as mean ± standard deviation or number (%). Table 3 Result of 24-hour urine collection Values are presented as number (%). Among all, 2 urine samples (4%) contained <300 mg, which did not suffice for the diagnosis of preeclampsia. Table 4 Mean parameters of the 24-hour urine collections NA, not available.

Introduction It has been recognised as paradoxical that a genetically disparate mammalian conceptus expressing paternal as well as maternal gene products does not succumb to any maternal immune response during pregnancy. Medawar proposed three possible hypotheses to explain the paradox of maternal immunological tolerance to the fetus [1]: anatomical separation of mother and fetus, antigenic immaturity of the fetus and suppression or modification of the maternal immune system. It appears that the first two proposals cannot explain fetal allograft survival. The maternal fetal interface is not a complete barrier and fetal cells can migrate into the maternal circulation or vice versa [2]. The second explanation is also nearly ruled out since both fetal and placental cells express major histocompatibility complex molecules (MHC) [3]. Major interest has therefore been focused on the third mechanism in which the survival of the allogeneic fetus depends on the fetus and placenta actively defending itself from attack by the cells of the maternal immune system. Munn et al. [4] added indoleamine 2,3-dioxygenase as a new candidate to the list of potential immunosuppressive mechanisms in pregnancy.

n the third mechanism in which the survival of the allogeneic fetus depends on the fetus and placenta actively defending itself from attack by the cells of the maternal immune system. Munn et al. [4] added indoleamine 2,3-dioxygenase as a new candidate to the list of potential immunosuppressive mechanisms in pregnancy. The enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42) is a heme-containing protein that catalyses the oxidative cleavage of the indole ring of tryptophan upon the insertion of two oxygen atoms of molecular oxygen [5]. Indoleamine 2,3-dioxygenase is widely expressed in a variety of tissues of mammals such as rabbit, rat, mouse and humans. One tissue with particularly high activity is the human placenta [6]. Although the precise physiological roles of indoleamine 2,3-dioxygenase is still unknown, the enzyme is induced under pathological conditions including virus infection [7] and parasitic infestation [8], resulting in the rapid degradation of tryptophan to kynurenine in the infected cells. Interferon-γ which exerts potent immunomodulatory and antiproliferative effects strongly induces the expression of the gene coding for indoleamine 2,3-dioxygenase [9]. It is possible to speculate that the inhibitory effect of interferon-γ on intracellular pathogens is, at least in part, attributable to the depletion of the essential amino acid, L-tryptophan following induction of indoleamine 2,3-dioxygenase. In addition to defense against pathogens, this enzyme exerts important immunomodulatory effects. Indoleamine 2,3-dioxygenase suppresses T-cell responses by depleting local L-tryptophan, an essential amino acid for T-cell proliferation and function [10]. Indoleamine 2,3-dioxygenase mediated T-cell suppression plays a crucial role in long term allogeneic organ graft function and in the control of graft-versus-host disease after allogeneic bone marrow transplantation and peripheral blood stem cell transplantation [11].

ino acid for T-cell proliferation and function [10]. Indoleamine 2,3-dioxygenase mediated T-cell suppression plays a crucial role in long term allogeneic organ graft function and in the control of graft-versus-host disease after allogeneic bone marrow transplantation and peripheral blood stem cell transplantation [11]. With regard to the role of placental indoleamine 2,3-dioxygenase Munn et al. [4] formulated the hypothesis that in normal pregnancy expression of this enzyme at the maternal-fetal interface is necessary to prevent immunological rejection of the fetal allograft. To test this hypothesis they exposed pregnant mice (carrying syngeneic or allogeneic fetuses) to 1-methyl-tryptophan, a pharmacological agent that inhibits indoleamine 2,3-dioxygenase activity [12]. They found that T cell-induced rejection of all allogeneic (but not syngeneic) concepti occurred rapidly when pregnant mice were treated with the inhibitor. However fetal allograft rejection was not observed when recombination activating gene 1 (RAG-1) deficient mothers that cannot develop lymphocytes were treated with 1-methyl-tryptophan. They also demonstrated that adoptive transfer of splenocytes from T cell receptor transgenic mice harbouring cohorts of CD8 positive T cells specific for a paternally inherited fetal MHC class I alloantigen caused fetal allograft rejection in these mothers lacking lymphocytes. They suggested T cells are inhibited by a mechanism involving indoleamine 2,3-dioxygenase dependent localised depletion of tryptophan at the site of placentation.

CD8 positive T cells specific for a paternally inherited fetal MHC class I alloantigen caused fetal allograft rejection in these mothers lacking lymphocytes. They suggested T cells are inhibited by a mechanism involving indoleamine 2,3-dioxygenase dependent localised depletion of tryptophan at the site of placentation. The maternal syndrome of pre-eclampsia is a major complication of human pregnancy with significant morbidity and mortality. Although the aetiology of pre-eclampsia remains unknown, there is substantial evidence that the clinical disease is secondary to abnormalities of placentation [13], with increased oxidative stress [14] and maternal endothelial cell activation and dysfunction [15]. In addition to endothelial dysfunction, systemic activation of maternal inflammatory cell populations has been observed including granulocytes [16], monocytes [17] and even lymphocytes [18]. Recent studies have shown that pre-eclampsia is indeed associated with an inflammatory response similar to that observed in septic patients and is associated with increased production of intracellular reactive oxygen species. However it seems that normal pregnancy is also associated with an inflammatory response albeit less severe [19]. From Munn et al.'s hypothesis that placental indoleamine 2,3-dioxygenase is responsible for suppressing the maternal immune response against the allogenic fetus by depleting tryptophan at the maternal-fetal interface it is possible to suggest that in pre-eclampsia changes in the activity or levels of indoleamine 2,3-dioxygenase in the placenta may be involved in the pathogenesis of this condition.

onsible for suppressing the maternal immune response against the allogenic fetus by depleting tryptophan at the maternal-fetal interface it is possible to suggest that in pre-eclampsia changes in the activity or levels of indoleamine 2,3-dioxygenase in the placenta may be involved in the pathogenesis of this condition. As mentioned above, Munn et al. [4] made a scientific observation of major biological importance. Their findings raise critical questions for human pregnancy. Specifically, the role of this mechanism (discovered in mouse) in the human, and the extent to which defective activation of this process is responsible for major clinical diseases are unknown. We therefore have studied some key facts about this enzyme expressed in the human placenta in an attempt to test whether Munn et al.'s findings in mouse are met for human pregnancy. In this review, particular focus will be placed on the results of our study on this enzyme in the human placenta. Localisation and characterisation of human placental indoleamine 2,3-dioxygenase In human placental tissue indoleamine 2,3-dioxygenase is detectable immunohistochemically from day 6 human blastocysts and thereafter throughout pregnancy in syncytiotrophoblasts, cytotrophoblasts and macrophages in the villous stroma and in the fetal membranes [20]. The results of cellular fractionation suggest that the placental isoform of indoleamine 2,3-dioxygenase is not expressed in the maternal facing brush border membrane of the syncytiotrophoblast but is a cytoplasmic enzyme [21].

asts, cytotrophoblasts and macrophages in the villous stroma and in the fetal membranes [20]. The results of cellular fractionation suggest that the placental isoform of indoleamine 2,3-dioxygenase is not expressed in the maternal facing brush border membrane of the syncytiotrophoblast but is a cytoplasmic enzyme [21]. 1-Methyl-tryptophan which was used by Munn et al. [4] for inhibiting indoleamine 2,3-dioxygenase activity in the pregnant mouse has been reported to be a competitive inhibitor for the rabbit intestinal isoform of the enzyme. As shown in Fig. 1, 1-methyl-tryptophan is competitive with L-tryptophan for human placental indoleamine 2,3-dioxygenase. The Ki values giving half-maximal inhibition for L-tryptophan was found to be 68 µM for 1-methyl-DL-tryptophan [21]. These results indicate that the hypothesis of Munn et al. [4] that localised placental tryptophan catabolism prevents immunological fetal rejection in mouse is likely to be met for human pregnancy. Regulation of human placental indoleamine 2,3-dioxygenase by interferon-γ In a variety of tissues indoleamine 2,3-dioxygenase activity is known to be modulated by certain cytokines. Thus interferon-γ stimulates its expression in macrophages and monocytes [22]; other cytokines (e.g., interleukin-4) inhibit indoleamine 2,3-dioxygenase expression [23]. In order to clarify physiological significance of human placental indoleamine 2,3-dioxygenase, regulation of its expression by interferon-γ has been investigated using cultured human placental chorionic villi.

and monocytes [22]; other cytokines (e.g., interleukin-4) inhibit indoleamine 2,3-dioxygenase expression [23]. In order to clarify physiological significance of human placental indoleamine 2,3-dioxygenase, regulation of its expression by interferon-γ has been investigated using cultured human placental chorionic villi. Human placental chorionic villi exposed to interferon-γ showed an increased indoleamine 2,3-dioxygenase activity in a time- and concentration-dependent manner (Fig. 2). The significant stimulatory effect of interferon-γ on indoleamine 2,3-dioxygenase activity could be detected after 12 hours of exposure of chorionic villi and was fully expressed 36 hours later, while the enhancement of mRNA expression began after 3 hours stimulation and reached a maximum at 12 hours [24]. Interferon-γ-stimulated indoleamine 2,3-dioxygenase mRNA expression was inhibited by interleukin-4 (Table 1), as was its activity by interleukin-4. Since interleukin-4 alone did not inhibit either mRNA level or enzyme activity, this effect may be due to the suppression of the increased transcription induced by interferon-γ. These data suggest that interleukin-4 may have a role in modulating indoleamine 2,3-dioxygenase availability in the human placenta.

rleukin-4. Since interleukin-4 alone did not inhibit either mRNA level or enzyme activity, this effect may be due to the suppression of the increased transcription induced by interferon-γ. These data suggest that interleukin-4 may have a role in modulating indoleamine 2,3-dioxygenase availability in the human placenta. Concentrations of L-tryptophan and of L-kynurenine, the major indoleamine 2,3-dioxygenase degradation product, in the conditioned medium following in vitro culture of placental villous explants were determined (Fig. 3). The presence of interferon-γ in the culture medium stimulated L-tryptophan degradation markedly, consequently showing a marked increase in L-kynurenine concentration. The indoleamine 2,3-dioxygenase inhibitor 1-methyl-tryptophan showed inhibition of L-tryptophan catabolism in either the presence or absence of interferon-γ; both the decrease in L-tryptophan concentration and the associated increase in L-kynurenine concentration were reduced by the presence of 1-methyl-tryptophan [25].

he indoleamine 2,3-dioxygenase inhibitor 1-methyl-tryptophan showed inhibition of L-tryptophan catabolism in either the presence or absence of interferon-γ; both the decrease in L-tryptophan concentration and the associated increase in L-kynurenine concentration were reduced by the presence of 1-methyl-tryptophan [25]. Regulation of peripheral blood mononuclear cell proliferation by human placental indoleamine 2,3-dioxygenase mediated tryptophan degradation To clarify the physiological potential of indoleamine 2,3-dioxygenase in the human placenta, whether placental indoleamine 2,3-dioxygenase mediated tryptophan degradation regulates peripheral blood mononuclear cell proliferation was examined. For this, peripheral blood mononuclear cells were cultured in the same medium as that previously conditioned by culture of villous explants. Thymidine incorporation into DNA was then determined. The findings are that proliferation is inhibited by conditioned medium, that this inhibition is greater with medium conditioned in the presence of interferon-γ and that these effects are markedly blunted when 1-methyl-tryptophan was present during the previous conditioning with villous explants (Fig. 4). Return of a physiological concentration of L-tryptophan to the conditioned medium, but not of any of the other amino acids tested (including D-tryptophan) returned peripheral blood mononuclear cell proliferation rate to normal [26].

tryptophan was present during the previous conditioning with villous explants (Fig. 4). Return of a physiological concentration of L-tryptophan to the conditioned medium, but not of any of the other amino acids tested (including D-tryptophan) returned peripheral blood mononuclear cell proliferation rate to normal [26]. Tryptophan catabolism by placental indoleamine 2,3-dioxygenase in human pregnancy: a comparison of normal pregnancy and pre-eclampsia It has been reported that plasma tryptophan levels decrease during normal pregnancy. Such a decrease may be related to immune activation phenomena during pregnancy. The maternal syndrome of pre-eclampsia is characterised by an excessive systemic inflammatory response induced by pregnancy [27]. Since the human placenta contains active indoleamine 2,3-dioxygenase, it is possible that in such pregnancies placental indoleamine 2,3-dioxygenase is less effective at controlling local and thus, indirectly, circulating tryptophan concentration. Therefore placental indoleamine 2,3-dioxygenase activity as well as indices of tryptophan catabolism have been studied in pregnant women, with or without pre-eclampsia, and non-pregnant women of reproductive age.

xygenase is less effective at controlling local and thus, indirectly, circulating tryptophan concentration. Therefore placental indoleamine 2,3-dioxygenase activity as well as indices of tryptophan catabolism have been studied in pregnant women, with or without pre-eclampsia, and non-pregnant women of reproductive age. Table 2 summarise the results of high-performance liquid chromatography analysis of tryptophan and kynurenine concentrations in plasma. Tryptophan concentrations in plasma taken from both groups of pregnant women were significantly lower than those from women who were not pregnant. However plasma from women with normal pregnancy also had significantly lower concentrations than those from women with pre-eclampsia. Plasma kynurenine concentrations showed the converse patterns. Hence the ratios of indoleamine 2,3-dioxygenase product (plasma kynurenine) to substrate (plasma tryptophan), an index of tryptophan catabolism, were significantly increased in normal pregnant women compared either with women who were not pregnant or with women with pre-eclampsia [28]. The indoleamine 2,3-dioxygenase activities in fresh placental villous tissue from pre-eclampsia were significantly lower when compared with those from normal pregnancy (Table 3). When villous tissue explants were cultured for 36 hours with or without interferon-γ, both indoleamine 2,3-dioxygenase activity and the percentage stimulation were still significantly lower in villous tissue from pre-eclampsia than was found for tissue from normal pregnancy [28].

lacental expression of this enzyme is crucial to prevent immunological rejection of the allogeneic fetus. Thus indoleamine 2,3-dioxygenase dependent localised depletion of tryptophan at the site of placentation has been proposed to be the mechanism of the suppression of maternal T cells which attack the conceptus [29]. That Munn et al.'s hypothesis in mouse is applicable to human pregnancy is suggested by the following pieces of evidence obtained from experiments in vitro described in this review. The human placenta expresses indoleamine 2,3-dioxygenase from early stages of pregnancy. 1-Methyl-tryptophan, the critical experimental tool used by Munn et al. to inhibit pregnant mouse indoleamine 2,3-dioxygenase, competitively inhibits the human placental enzyme (Fig. 1). Functional indoleamine 2,3-dioxygenase activity is also inhibited by 1-methyl-tryptophan in villous explants; when villous explants were cultured in the presence of 1-methyl-tryptophan, the normal decrease in tryptophan and increase in kynurenine concentrations in the conditioned medium were suppressed (Fig. 3). Indoleamine 2,3-dioxygenase mediated tryptophan degradation can inhibit peripheral blood mononuclear cell proliferation (Fig. 4); peripheral blood mononuclear cell proliferation following culture in the medium previously conditioned by culture of villous explants is markedly suppressed; inhibition of placental indoleamine 2,3-dioxygenase by 1-methyl-tryptophan during conditioning prevents inhibition of peripheral blood mononuclear cell proliferation. In addition to a potential role of indoleamine 2,3-dioxygenase, there are hypotheses suggesting roles for several other molecules or cells to explain maternal tolerance of the fetal allograft [30]. Tolerance of the fetal allograft is the results of the integration of numerous mechanisms of various origins and modes of action.

tion to a potential role of indoleamine 2,3-dioxygenase, there are hypotheses suggesting roles for several other molecules or cells to explain maternal tolerance of the fetal allograft [30]. Tolerance of the fetal allograft is the results of the integration of numerous mechanisms of various origins and modes of action. It has been suggested that the manifestations of the maternal syndrome of pre-eclampsia are not the results of processes distinct from those of normal pregnancy but simply the extreme end of the distribution of maternal intravascular inflammatory responses to normal pregnancy since activation of peripheral blood leukocytes is already well established in normal pregnancy [19,27]. Although the causes of these changes in peripheral blood leukocytes have not been defined, the evidence points to the placenta as the stimulus for pre-eclampsia [31,32]. The changes in the levels of cytokines observed in pre-eclampsia may be causally related to the decreased expression of placental indoleamine 2,3-dioxygenase since indoleamine 2,3-dioxygenase mediated tryptophan depletion can specifically suppress the proliferation of T helper lymphocytes which are a major source of cytokines. The increased circulating pro-inflammatory cytokines can activate maternal leukocytes and thus can develop exaggerated system inflammatory response in this condition. The cause of decreased expression level of indoleamine 2,3-dioxygenase in pre-eclamptic placenta remains to be defined. However, it is likely that increased plasma tryptophan concentration in women with pre-eclampsia and tryptophan dependent proliferation of lymphocytes indicate a causal relationship between suppressed indoleamine 2,3-dioxygenase expression and exaggerated inflammatory response in this maternal syndrome.

to be defined. However, it is likely that increased plasma tryptophan concentration in women with pre-eclampsia and tryptophan dependent proliferation of lymphocytes indicate a causal relationship between suppressed indoleamine 2,3-dioxygenase expression and exaggerated inflammatory response in this maternal syndrome. No potential conflict of interest relevant to this article was reported. Fig. 1 Effect of 1-methyl-tryptophan on indoleamine 2,3-dioxygenase activity [21]. The rate of formation of the product catalysed by indoleamine 2,3-dioxygenase was determined over a 30 minutes period in a medium containing varying concentrations of 1-methyl-DL-tryptophan, 20 mM ascorbic acid, 10 µM methylene blue, 100 units mL-1 catalase and 50 mM potassium phosphate buffer (pH 6.5) in the presence of indicated concentrations of L-tryptophan. 1-Methyl-DL-tryptophan: •, 0 µM; ○, 40 µM; ▼, 200 µM; ▽, 400 µM. Inset is a replot for the apparent Km value (Kmapp) the inhibitor concentrations used to determined the Ki value; the line was determined by least-squares linear regression analysis. Data represent the mean±standard deviation of three separate experiments with triplicate assays.

•, 0 µM; ○, 40 µM; ▼, 200 µM; ▽, 400 µM. Inset is a replot for the apparent Km value (Kmapp) the inhibitor concentrations used to determined the Ki value; the line was determined by least-squares linear regression analysis. Data represent the mean±standard deviation of three separate experiments with triplicate assays. Fig. 2 Effect of interferon-γ (IFN-γ) on the stimulation of indoleamine 2,3-dioxygenase activity in cultured chorionic villi (From Kudo, et al. Mol Hum Reprod 2000;6:369-74, Oxford University Press [24]). Pieces of chorionic villi were cultured for the time indicated with 1,000 unit mL-1 IFN-γ or vehicle (phosphate-buffered saline) (A). Pieces of chorionic villi were cultured for 36 hours with the indicated concentrations of IFN-γ (B). Indoleamine 2,3-dioxygenase activity in tissue extract was determined. Data represent the mean±standard deviation of three separate experiments performed with three placentae. Fig. 3 Effect of interferon (IFN)-γ and 1-methyl-tryptophan on tryptophan catabolism by indoleamine 2,3-dioxygenase in placental explants [25]. Villous explants were cultured with or without 1,000 unit mL-1 IFN-γ and/or 2 mM 1-methyl-tryptophan (1-Met-Trp) or vehicle (Nil) for 36 hours. Concentrations of tryptophan (A) and kynurenine (B) in the conditioned medium were analysed by high-performance liquid chromatography. Values are mean±standard deviation of five separate experiments with triplicate assay from five different samples. Control, values without culture; ND, not detectable. a)Significantly different from Nil; b)Significantly different from IFN-γ.

he conditioned medium were analysed by high-performance liquid chromatography. Values are mean±standard deviation of five separate experiments with triplicate assay from five different samples. Control, values without culture; ND, not detectable. a)Significantly different from Nil; b)Significantly different from IFN-γ. Fig. 4 Peripheral blood mononuclear cell proliferation in medium previously conditioned by culture with villous explants [26]. Peripheral blood mononuclear cells were cultured for 72 hours either in non-conditioned medium or in medium previously conditioned by culture of explants with 1,000 unit mL-1 interferon-γ (IFN) and/or 2 mM 1-methyl-tryptophan (1-Met-Trp) for 36 hours. [3H]thymidine incorporation was then determined. Data represent the standard deviation of five separate experiments with quadruplicate assay, expressed as percentage of control (i.e., values cultured in non-conditioned medium). Control value for [3H]thymidine incorporation into DNA is (5.93±0.34)×104 cpm/well. Nil, vehicle. a)Significantly different from Nil; b)Significantly different from IFN-γ. Table 1 Effect of interleukin-4 on interferon-γ-induced stimulation of indoleamine 2,3-dioxygenase mRNA expression in cultured chorionic villi (From Kudo, et al. Mol Hum Reprod 2000;6:369-74, Oxford University Press [24])

Fig. 4 Peripheral blood mononuclear cell proliferation in medium previously conditioned by culture with villous explants [26]. Peripheral blood mononuclear cells were cultured for 72 hours either in non-conditioned medium or in medium previously conditioned by culture of explants with 1,000 unit mL-1 interferon-γ (IFN) and/or 2 mM 1-methyl-tryptophan (1-Met-Trp) for 36 hours. [3H]thymidine incorporation was then determined. Data represent the standard deviation of five separate experiments with quadruplicate assay, expressed as percentage of control (i.e., values cultured in non-conditioned medium). Control value for [3H]thymidine incorporation into DNA is (5.93±0.34)×104 cpm/well. Nil, vehicle. a)Significantly different from Nil; b)Significantly different from IFN-γ. Table 1 Effect of interleukin-4 on interferon-γ-induced stimulation of indoleamine 2,3-dioxygenase mRNA expression in cultured chorionic villi (From Kudo, et al. Mol Hum Reprod 2000;6:369-74, Oxford University Press [24]) Pieces of chorionic villi were cultured with 1,000 unit mL-1 interferon-γ (IFN-γ) and/or 10 ng mL-1 interleukin-4 or vehicle (control) for 36 hours. The levels of indoleamine 2,3-dioxygenase messenger RNA (mRNA) and glyceraldehyde phosphate 3-dehydrogenase mRNA were analysed by reverse transcription-polymerase chain reaction. The intensity of either the indoleamine 2,3-dioxygenase or the glyceraldehydes phosphate 3-dehydrogenase band was quantitated and the ratio of the two was used as a normalised expression value of the indoleamine 2,3-dioxygenase gene. Values are mean±standard deviation of three separate experiments performed with three placentae, expressed as percentage of control (i.e., values cultured with vehicle alone).

3-dehydrogenase band was quantitated and the ratio of the two was used as a normalised expression value of the indoleamine 2,3-dioxygenase gene. Values are mean±standard deviation of three separate experiments performed with three placentae, expressed as percentage of control (i.e., values cultured with vehicle alone). a)Significantly different from control (P <0.001); b)Significantly different from IFN-γ (P <0.001). Table 2 Tryptophan and kynurenine concentrations and the ratio of kynurenine to tryptophan in plasma from women with pre-eclampsia or normal pregnancies and from non-pregnant women [28] Concentrations of tryptophan and kynurenine in plasma were analysed by high-performance liquid chromatography. Values are given as mean ± standard deviation. a)P <0.001 compared with normal pregnancy (Wilcoxon test); b)P <0.001 compared with pre-eclampsia (Mann-Whitney U test); c)p<0.001 compared with normal pregnancy (Mann-Whitney U test). Table 3 Indoleamine 2,3-dioxygenase activity in placental villous tissue [28] Villous explants were cultured with or without 1,000 unit mL-1 IFN-γ for 36 hours. IDO activity in the tissue extract of fresh or cultured villous explants was determined colorimetrically. Percentage stimulation is given in the parentheses. Values are mean± standard deviation of triplicate assays from indicated number of placentae. IDO, indoleamine 2,3-dioxygenase; IFN-γ, interferon-γ; Nil, vehicle. a)Significantly different from normal pregnancy.

Introduction Fetal echocardiography is an essential tool for screening of the fetal cardiac anatomy. Congenital heart disease is the most common abnormality in the human fetus, occurring in approximately 8-9 per 1,000 live births [1]. Prenatal diagnosis of cardiac defects is important because it allows families to receive appropriate counseling and to properly prepare for the birth of a child with congenital heart disease. Upon diagnosis of a cardiac defect, fetuses should be referred to a tertiary center for proper management. Two-dimensional imaging is still the gold standard and commonly used in fetal echocardiography. Therefore, we present standard views of the normal fetal heart obtained during the second trimester by two-dimensional ultrasound, and color and pulsed wave Doppler. We also present first-trimester fetal echocardiographic findings and a modified myocardial performance index (Mod-MPI), which is a useful tool for evaluating fetal cardiac function.

ard views of the normal fetal heart obtained during the second trimester by two-dimensional ultrasound, and color and pulsed wave Doppler. We also present first-trimester fetal echocardiographic findings and a modified myocardial performance index (Mod-MPI), which is a useful tool for evaluating fetal cardiac function. Determining the abdominal situs Before scanning the fetal heart, it is important to determine whether abdominal situs is normal because congenital heart diseases are frequently associated with abnormal abdominal situs. On the basis of fetal position, several planes can exist. In a fetus with breech presentation, the left side of the fetus should be proximal to the transducer when the fetal occiput is on the left side of the mother (Fig. 1A). When the fetal occiput is on right side of the mother, the left side of the fetus should be distal to the transducer (Fig. 1B). When the fetus is positioned face up, its left side appears on the right side of the screen, and when the fetus is lying face down, its left side appears on the same side of the screen. In a fetus with vertex presentation, determination of abdominal situs is reversed (Fig. 1C, D). While it can be difficult to determine abdominal situs when the fetus is in the transverse position, the right-hand rule of thumb can reliably determine fetal situs (Fig. 2) [2]. With this simple approach, the palm of the right hand corresponds to the fetal abdomen, the dorsal side of the forearm to the fetal back, and the fist to the fetal head. The direction of the thumb always corresponds to the left side of the fetus regardless of the fetal position.

ably determine fetal situs (Fig. 2) [2]. With this simple approach, the palm of the right hand corresponds to the fetal abdomen, the dorsal side of the forearm to the fetal back, and the fist to the fetal head. The direction of the thumb always corresponds to the left side of the fetus regardless of the fetal position. Transverse view of the abdomen A transverse abdominal view is shown in Fig. 3. The stomach and liver are located on the left and right, respectively. The descending aorta (DAo) lies anterior and to the left of the spine, whereas the inferior vena cava (IVC) lies anterior and to the right of the DAo. By moving the transducer cranially, the cardiac apex should be located on the same side as the stomach. Abdominal situs abnormalities, such as those present in heterotaxy syndrome, can be detected with careful observation of this view (Fig. 4). Four-chamber view The four-chamber view (4CV) is not only the easiest view to obtain, but also the most important view of the fetal heart. The 4CV is easily visible by moving the transducer cranially from the transverse abdominal view (Fig. 5A). Approximately 60% of congenital heart disease cases are detected with this view [3]. Examiners should carefully assess heart structure to rule out congenital heart disease, using the following criteria: 1) A normal heart is approximately one-third of the thorax in size, and this proportion (cardiothoracic area, C/T area) is constant throughout gestation [4]. When the C/T area is greater than two standard deviations, cardiomegaly may be suspected [5].

Four-chamber view The four-chamber view (4CV) is not only the easiest view to obtain, but also the most important view of the fetal heart. The 4CV is easily visible by moving the transducer cranially from the transverse abdominal view (Fig. 5A). Approximately 60% of congenital heart disease cases are detected with this view [3]. Examiners should carefully assess heart structure to rule out congenital heart disease, using the following criteria: 1) A normal heart is approximately one-third of the thorax in size, and this proportion (cardiothoracic area, C/T area) is constant throughout gestation [4]. When the C/T area is greater than two standard deviations, cardiomegaly may be suspected [5]. 2) The cardiac axis lies at a 45° angle to the left of the midline [6]. 3) The two atria are equal in size and separated by the atrial septum. The flap of foramen ovale lies in the left atrial cavity.

1) A normal heart is approximately one-third of the thorax in size, and this proportion (cardiothoracic area, C/T area) is constant throughout gestation [4]. When the C/T area is greater than two standard deviations, cardiomegaly may be suspected [5]. 2) The cardiac axis lies at a 45° angle to the left of the midline [6]. 3) The two atria are equal in size and separated by the atrial septum. The flap of foramen ovale lies in the left atrial cavity. 4) The two atrioventricular valves are equally opened and differentially inserted: the septal leaflet of the tricuspid valve is inserted more apically than the mitral valve in the ventricular septum. Blood flow across both valves should be evaluated by color Doppler imaging to ensure that both ventricles fill equally in diastole without regurgitation (Fig. 5B). Doppler waveforms across both atrioventricular valves are shown in Fig. 5C, D. The E wave, which corresponds to the early ventricular filing of diastole, is followed by the A wave, which corresponds to the active ventricular filling of diastole (atrial contraction). The A wave is always higher than E wave in the normal fetus. The peak velocity of both waves is 30-60 cm/sec, and is constant throughout gestation [7]. 5) The crux of the heart, the area of junction of atrial septum, atrioventricular valves, and interventricular septum are intact. 6) The two ventricles are equal in size, although the right side of the heart becomes larger as gestation progresses [8]. 7) The right ventricle contains the moderator band. 8) The ventricular septum is intact.

5) The crux of the heart, the area of junction of atrial septum, atrioventricular valves, and interventricular septum are intact. 6) The two ventricles are equal in size, although the right side of the heart becomes larger as gestation progresses [8]. 7) The right ventricle contains the moderator band. 8) The ventricular septum is intact. 9) Pulmonary veins are connected to the left atrium. 10) The DAo is located anterior and to the left of the spine. Beside the evaluation of 4CV, assessment of great arteries' connections further detects about 90% of serious congenital heart diseases [3]. Left ventricular outflow tract view A slight tilt of the transducer from the lateral 4CV, which is positioned the ventricular septum perpendicular to the ultrasound beam, toward the cardiac apex yields a left ventricular outflow tract (Fig. 6A). This view shows the left ventriculoarterial connection as well as the intact ventricular septum. Blood flow across the aortic valve is laminar flow with no turbulence in systole and no regurgitation in diastole (Fig. 6B). Peak systolic velocity in the aorta increases linearly with advancing gestation, and it ranges from approximately 30 cm/sec at 19 weeks to 100 cm/sec at full term [9,10]. Peak systolic velocity in the aorta is greater than in the pulmonary artery [11].

the patients with PCOS and controls had taken combined oral contraceptives, lipid-lowering agents or insulin sensitizer. The Institute Review Board (IRB) for human research of Seoul National University Hospital approved this project (IRB number: H-0807-031-250) and written informed consent was obtained from each woman. 2. Clinical and biochemical measurements Clinical variables, such as body weight, height, waist circumference, and blood pressure were assessed in all subjects. Using radioimmunoassay (RIA) (Siemens, Los Angeles, CA, USA), serum levels of total testosterone, free testosterone and sex hormone-binding globulin (SHBG) were measured in all patients with PCOS and in a subset of controls (n=14) whose blood samples were taken during the follicular phase of the menstrual cycle. FAI was calculated as total testosterone/SHBG ×100, and the values for testosterone were converted from ng/mL to nmol/L using the following index proposed by the manufacturer: 1 ng/mL = 3.467 nmol/L. The intra-assay and inter-assay coefficients of variation were 4.0% to 11.0% and 5.9% to 12.0% for total testosterone, and 4.0% to 17% and 8.0% to 18.3% for free testosterone, respectively.

o turbulence in systole and no regurgitation in diastole (Fig. 6B). Peak systolic velocity in the aorta increases linearly with advancing gestation, and it ranges from approximately 30 cm/sec at 19 weeks to 100 cm/sec at full term [9,10]. Peak systolic velocity in the aorta is greater than in the pulmonary artery [11]. Right ventricular outflow tract view This view is obtained by sweeping the transducer from left ventricular outflow tract to the fetal head (Fig. 7A). The crossing nature of the great arteries can be confirmed by moving the transducer slightly up and down. This view demonstrates the right ventriculoarterial connection. Using color Doppler imaging, laminar flow across the pulmonary valve is confirmed (Fig. 7B). Peak systolic velocity in the pulmonary artery also increases linearly with advancing gestation, and it reaches 100 cm/sec at full term [10,11]. Three-vessel view This view is obtained by moving the transducer cranially maintaining a transverse position from the 4CV (Fig. 8). The main pulmonary artery, ascending aorta, and superior vena cava (SVC) are arranged in a straight line from the left anterior to the right posterior aspect. The pulmonary artery is the largest in size, followed by the ascending aorta and the superior vena cava. The main pulmonary artery is divided into left and right pulmonary arteries. The thymus is also clearly visible in this view. The three-vessel view (3VV) is useful in diagnosing conotruncal cardiac abnormalities [12].

e pulmonary artery is the largest in size, followed by the ascending aorta and the superior vena cava. The main pulmonary artery is divided into left and right pulmonary arteries. The thymus is also clearly visible in this view. The three-vessel view (3VV) is useful in diagnosing conotruncal cardiac abnormalities [12]. Three-vessel-trachea view (transverse aortic and ductal arch view) Aortic and ductal arches are combined into the DAo, which appear as a V-shaped confluence (Fig. 9A). Both arches are similar in size and located to the left side of the trachea. The thymus is also visible in this view. Color Doppler imaging demonstrates the same direction of blood flow in both arches (Fig. 9B). This view allows comparison of both arches and assessment of aortic arch abnormalities, including aortic arch hypoplasia and coarctation of the aorta [13]. Aortic arch view By rotating the transducer 90 degree either clockwise or counterclockwise from the 3VV, a 'candy cane-like' aortic arch is seen (Fig. 10A). The aortic arch gives rise to the three arterial branches, namely, the brachiocephalic, left common carotid, and left subclavian arteries. All three branches are clearly visible by color Doppler imaging (Fig. 10B). Because the innominate vein is the largest vessel in the mediastinum, it is often observed in front of the brachiocephalic artery [14].

the three arterial branches, namely, the brachiocephalic, left common carotid, and left subclavian arteries. All three branches are clearly visible by color Doppler imaging (Fig. 10B). Because the innominate vein is the largest vessel in the mediastinum, it is often observed in front of the brachiocephalic artery [14]. Ductal arch view By sliding the transducer from the aortic arch view to the left, a 'hockey stick-like' ductal arch is visualized (Fig. 11). This view mainly shows the right ventricle, pulmonary valve, and main pulmonary artery, which is connected to the DAo through the ductus arteriosus. Unlike the aortic arch, the ductal arch does not give rise to any branches. Bicaval view (SVC and IVC view) This view is obtained in a right parasagittal plane (Fig. 12). The SVC and IVC drain into the posterior aspect of the right atrium. The SVC and IVC are similar in size; however, the IVC is widened as it enters the right atrium because of interflow from the ductus venosus and hepatic veins. First-trimester screening of fetal heart Advancements in ultrasonography have paved the way for first-trimester fetal echocardiography since the 1990s [15-17]. Fetal echocardiography performed during the first-trimester is advantageous for at least two reasons. First, early diagnosis of cardiac defects allows families to make well-thought-out plans (i.e., to receive counseling, and/or prepare for the birth of a child with a congenital heart disease). Second, early screening relieves the anxiety of high risk patients who have previous family history of cardiac defects.

easons. First, early diagnosis of cardiac defects allows families to make well-thought-out plans (i.e., to receive counseling, and/or prepare for the birth of a child with a congenital heart disease). Second, early screening relieves the anxiety of high risk patients who have previous family history of cardiac defects. The examination of the fetal heart during early gestation includes the normal situs, cardiac axis, cardiac connections, atrioventricular junction, and septoaortic continuity [18]. If these structures are normal, most structural abnormalities can be excluded. The ultrasonographic findings of a normal heart at 13 weeks of gestation are shown in Fig. 13. Furthermore, increased nuchal translucency and abnormal ductus venosus flow increase the risk of not only chromosomal anomalies, but also congenital heart disease [19,20]. Therefore, these indicators are important for early detection of cardiac defects.

r testosterone were converted from ng/mL to nmol/L using the following index proposed by the manufacturer: 1 ng/mL = 3.467 nmol/L. The intra-assay and inter-assay coefficients of variation were 4.0% to 11.0% and 5.9% to 12.0% for total testosterone, and 4.0% to 17% and 8.0% to 18.3% for free testosterone, respectively. In all subjects, after 12-hour overnight fast, fasting plasma glucose (FPG) (hexokinase method), total cholesterol (cholesterol oxidase-N-[3-sulfopropyl]-3-methoxy-5-methylaniline [HMMPS] method), triglycerides (glycerol-3-Phosphatase oxidase-HMMPS glycerol blanking), high density lipoprotein (HDL)-cholesterol (selective elimination method) and low density lipoprotein (LDL)-cholesterol (selective elimination method) were measured (Wako Pure Chemical Industries Ltd., Osaka, Japan). Circulating highly sensitive C-reactive protein (hs-CRP) was measured using a latex turbidimetric immunoassay with a sensitivity of 0.01 mg/dL (Wako Pure Chemical Industries Ltd., Japan). Fasting insulin levels were measured using RIA (BioSource Europe S.A., Nivelles, Belgium). The homeostatic model for insulin resistance was calculated by glucose (mg/dL) × insulin (µU/mL)/405, and HOMAβcell (%) was calculated as follows: (20×fasting insulin)/(fasting glucose-3.5).

s of a normal heart at 13 weeks of gestation are shown in Fig. 13. Furthermore, increased nuchal translucency and abnormal ductus venosus flow increase the risk of not only chromosomal anomalies, but also congenital heart disease [19,20]. Therefore, these indicators are important for early detection of cardiac defects. Modified myocardial performance index Assessment of fetal cardiac function is now considered as a routine evaluation of fetal status. MPI was originally proposed by Tei et al. [21] for the evaluation of cardiac function in adults. MPI is calculated as the sum of the isovolumetric contraction time and isovolumetric relaxation time divided by the ejection time. Hernandez-Andrade et al. [22] further modified MPI, and this method is now commonly used (Fig. 14). The Mod-MPI is useful tool for evaluating fetal cardiac function in several conditions, such as intrauterine growth retardation, pre-eclampsia, maternal diabetes, and twin-to-twin transfusion syndrome [23-26]. It is also useful in planning for fetal therapy or childbirth. Conclusion Fetal echocardiography is a part of the routine evaluation of normal fetus. The prenatal diagnosis of cardiac defects depends on the knowledge, skill, and experience of practitioners. Therefore, obstetricians should be completely familiar with abovementioned anatomy of the fetal heart in order to detect any abnormalities that might be present. No potential conflict of interest relevant to this article was reported.

Conclusion Fetal echocardiography is a part of the routine evaluation of normal fetus. The prenatal diagnosis of cardiac defects depends on the knowledge, skill, and experience of practitioners. Therefore, obstetricians should be completely familiar with abovementioned anatomy of the fetal heart in order to detect any abnormalities that might be present. No potential conflict of interest relevant to this article was reported. Fig. 1 Schematic images and transverse abdominal views of fetuses with breech and vertex presentations. (A) Breech presentation with fetal occiput on the maternal left side. (B) Breech presentation with fetal occiput on the maternal right side. (C) Vertex presentation with fetal occiput on the maternal left side. (D) Vertex presentation with fetal occiput on the maternal right side. Fig. 2 Right-hand rule of thumb for transabdominal scanning. The palm of the right hand corresponds to the fetal abdomen, the dorsal side of the forearm to the fetal back, and the fist to the fetal head. The direction of the thumb always corresponds to the left side of the fetus. Fig. 3 Transverse abdominal view. The fetal stomach is on the left side and liver is on the right side. The descending aorta is posterior-left located and the IVC is anterior-right located. Lt, left; Rt, right; IVC, inferior vena cava.

Fig. 2 Right-hand rule of thumb for transabdominal scanning. The palm of the right hand corresponds to the fetal abdomen, the dorsal side of the forearm to the fetal back, and the fist to the fetal head. The direction of the thumb always corresponds to the left side of the fetus. Fig. 3 Transverse abdominal view. The fetal stomach is on the left side and liver is on the right side. The descending aorta is posterior-left located and the IVC is anterior-right located. Lt, left; Rt, right; IVC, inferior vena cava. Fig. 4 Transverse abdominal views of both forms of heterotaxy syndrome. (A) Fetus with right atrial isomerism (breech presentation) shows juxtaposition of the aorta and IVC on either left side of the spine. The right-sided stomach and the midline liver are also shown. (B) Fetus with left atrial isomerism (vertex presentation) shows invisible IVC with dilated azygos vein, suggestive of an interrupted IVC with azygos vein continuation. Lt, left; Rt, right; IVC, inferior vena cava.

a and IVC on either left side of the spine. The right-sided stomach and the midline liver are also shown. (B) Fetus with left atrial isomerism (vertex presentation) shows invisible IVC with dilated azygos vein, suggestive of an interrupted IVC with azygos vein continuation. Lt, left; Rt, right; IVC, inferior vena cava. Fig. 5 (A) Apical four-chamber view of the fetal heart. (B) Color Doppler image shows forward flow from both atria to both ventricles. (C) Doppler waveforms of tricuspid and (D) mitral valves show a biphasic pattern. The E wave corresponds to early ventricular filing of the diastole, and the A wave corresponds to active ventricular filling of the diastole (atrial contraction). (D) Because of the continuity between mitral and aortic valves, the Doppler waveform at the mitral valve shows aortic outflow. Lt, left; Rt, right; LA, left atrium; RA, right A atrium; LV, left ventricle; RV, right ventricle; PV, pulmonary vein. Fig. 6 Left ventricular outflow tract view. (A) This view shows the ventriculoarterial connection and intact ventricular septum. (B) Color Doppler across the aortic valve shows laminar flow and no turbulence in systole. Lt, left; Rt, right; LV, left ventricle; RV, right ventricle; Ao, aorta. Fig. 7 Right ventricular outflow tract view. (A) This view shows the right ventriculoarterial connection. (B) Color Doppler image demonstrates laminar flow across the pulmonary valve. Lt, left; Rt, right; RV, right ventricle; PA, pulmonary artery.

Fig. 6 Left ventricular outflow tract view. (A) This view shows the ventriculoarterial connection and intact ventricular septum. (B) Color Doppler across the aortic valve shows laminar flow and no turbulence in systole. Lt, left; Rt, right; LV, left ventricle; RV, right ventricle; Ao, aorta. Fig. 7 Right ventricular outflow tract view. (A) This view shows the right ventriculoarterial connection. (B) Color Doppler image demonstrates laminar flow across the pulmonary valve. Lt, left; Rt, right; RV, right ventricle; PA, pulmonary artery. Fig. 8 Three-vessel view. The main pulmonary artery, ascending aorta, and superior vena cava are arranged in a straight line from the left anterior to the right posterior aspect. The pulmonary artery is the largest in size, followed by the ascending aorta and the superior vena cava. The thymus is clearly visible anterior to the three vessels. Lt, left; Rt, right; MPA, main pulmonary artery; RPA, right pulmonary artery; DA, ductus arteriosus; Ao, aorta; SVC, superior vena cava; DAo, descending aorta. Fig. 9 Three-vessel-trachea view. (A) This view demonstrates a V-shaped confluence of aortic and ductal arches at the descending aorta. (B) The trachea is located to the right side of the aorta. Color Doppler shows the same direction of blood flow in both arches. Lt, left; Rt, right; PA, pulmonary artery; DA, ductus arteriosus; AAo, ascending aorta; DAo, descending aorta; SVC, superior vena cava.

nce of aortic and ductal arches at the descending aorta. (B) The trachea is located to the right side of the aorta. Color Doppler shows the same direction of blood flow in both arches. Lt, left; Rt, right; PA, pulmonary artery; DA, ductus arteriosus; AAo, ascending aorta; DAo, descending aorta; SVC, superior vena cava. Fig. 10 Aortic arch view. (A) A 'candy cane-like' aorta gives rise to the three arterial branches, namely, the brachiocephalic, left common carotid, and left subclavian arteries. (B) These three branches are also visible by color Doppler imaging. LA, left atrium; RPA, right pulmonary artery; AAo, ascending aorta; DAo, descending aorta; BCA, brachiocephalic artery; LCA, left common carotid artery; LSA, left subclavian artery. Fig. 11 Ductal arch view. (A) The ductus arteriosus connects the main pulmonary artery to the DAo, forming hockey stick-shaped arch. (B) Laminar flow across the pulmonary valve is also shown. RV, right ventricle; PA, pulmonary artery; DA, ductus arteriosus; DAo, descending aorta. Fig. 12 Bicaval view. The SVC and IVC drain into the posterior aspect of the right atrium. The IVC is widened as it enters the right atrium because of interflow from the ductus venosus and hepatic veins. RA, right atrium; IVC, inferior vena cava; SVC, superior vena cava.

Fig. 11 Ductal arch view. (A) The ductus arteriosus connects the main pulmonary artery to the DAo, forming hockey stick-shaped arch. (B) Laminar flow across the pulmonary valve is also shown. RV, right ventricle; PA, pulmonary artery; DA, ductus arteriosus; DAo, descending aorta. Fig. 12 Bicaval view. The SVC and IVC drain into the posterior aspect of the right atrium. The IVC is widened as it enters the right atrium because of interflow from the ductus venosus and hepatic veins. RA, right atrium; IVC, inferior vena cava; SVC, superior vena cava. Fig. 13 Ultrasonographic images of fetuses with a normal heart at 13 weeks of gestation. (A) Four-chamber view. (B) Three-vessel view. (C) Left ventricular outflow tract view. Lt, left; Rt, right; LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; MPA, main pulmonary artery; Ao, aorta; SVC, superior vena cava. Fig. 14 Measurement of the fetal modified myocardial performance index. Each cursor should be placed at the beginning of each valve click, and the modified myocardial performance index is calculated as the sum of ICT and IRT divided by ejection time. AV, aortic valve; MV, mitral valve; ICT, isovolumetric contraction time; IRT, isovolumetric relaxation time; ET, ejection time.

Introduction Clinically responsive placental examination, which includes the stages about the involved anatomical region and the grades about the severity in acute histologic chorioamnionitis (acute-HCA), is known to enhance clinico-pathologic correlation [1]. Indeed, previous study demonstrated that increasing histologic stages of inflammation in fetal membranes were associated with an increasing rate of funisitis, perinatal mortality and preterm birth [2]. Moreover, sepsis in the newly born infant more frequently developed with the higher placental inflammatory score [3]. The grading system in acute placental inflammation has been known to play an important role in the assessment for the intensity of inflammation and infection in the fetus and amniotic cavity as following: 1) there was a strong association between the presence and severity of inflammation in each anatomical region of placenta (i.e., chorion-decidua, amnion, chorionic plate and umbilical cord) and the results of amniotic fluid (AF) culture [4]; 2) both the presence and greater severity of acute-HCA were associated with a higher frequency of positive AF culture and an elevated AF white blood cell (WBC) count [5]; 3) the degree of leukocytic infiltration in the fibrin layer under the chorion or in the chorionic membrane of term placentas, correlated positively with the levels of AF cytokines (i.e., tumor necrosis factor, interleukin-1 [IL-1], and IL-6) [6]; and 4) umbilical venous IL-6 levels in preterm and term neonates correlated with the severity of acute placental inflammation [7].

er under the chorion or in the chorionic membrane of term placentas, correlated positively with the levels of AF cytokines (i.e., tumor necrosis factor, interleukin-1 [IL-1], and IL-6) [6]; and 4) umbilical venous IL-6 levels in preterm and term neonates correlated with the severity of acute placental inflammation [7]. Moreover, the identification of involved anatomical region in the placenta and umbilical cord by acute inflammatory changes has been reported to be an essential part for the evaluation of the presence or intensity of intra-amniotic, maternal and fetal inflammatory responses as following: 1) fetal and intra-amniotic inflammatory responses were more intense in patients with funisitis than in those without this lesion [8-10]; 2) the involvement of the amnion in acute-HCA was associated with a more intense fetal and intra-amniotic inflammatory response than chorionitis alone [11]; 3) the intra-amniotic inflammatory response was stronger when inflammation was present in both the chorionic plate and chorio-decidua, than when it was restricted to the chorio-decidua only, which was exposed to the cervical canal in placenta previa [12]; 4) an inflammation restricted to chorio-decidua, an initial stage in ascending intrauterine infection pathway, was associated with a more intense and frequent inflammation in AF, but not fetus and mother, than placenta without any inflammation [13]; and 5) an inflammation in the chorion provided a clinically acceptable minimum threshold for the reporting of pathologic changes [2]. However, there is a paucity of information regarding which is more important for the intensity of intra-amniotic inflammation (IAI) between total grade or involved anatomical region in acute-HCA of preterm gestations. The objective of current study is to examine this issue.

for the reporting of pathologic changes [2]. However, there is a paucity of information regarding which is more important for the intensity of intra-amniotic inflammation (IAI) between total grade or involved anatomical region in acute-HCA of preterm gestations. The objective of current study is to examine this issue. Materials and methods 1. Study design Study population consisted of 225 singleton preterm gestations (<36 weeks) who had acute-HCA including chorio-decidua involvement and delivered within 5 days of amniocentesis at the Seoul National University Hospital between January 1993 and December 2007. This criterion of amniocentesis-to-delivery interval was used to preserve a meaningful temporal relationship between the results of AF studies and the placental pathologic findings at birth. The intensity of IAI was measured by AF WBC count and matrix metalloproteinase-8 (MMP-8) concentration. Patients were divided into 6 groups according to total grade (i.e., 1-6) and the presence or absence of chorio-decidua restriction (i.e., chorio-decidua restriction vs. extension beyond chorio-decidua) of acute-HCA as following: 1) group-1, chorio-decidua restriction with total grade 1; 2) group-2, chorio-decidua restriction with total grade 2; 3) group-3, extension beyond chorio-decidua with total grade 2; 4) group-4, extension beyond chorio-decidua with total grade 3; 5) group-5, extension beyond chorio-decidua with total grade 4; 6) group-6, extension beyond chorio-decidua with grade 5-6. At our institution, trans-abdominal amniocentesis for retrieval of AF was routinely offered to all patients who were admitted with the diagnosis of preterm labor and intact membranes (PTL) or preterm premature rupture of membranes (preterm-PROM). AF was analyzed for microbiologic and inflammatory status, and fetal lung maturity. Amniocentesis was performed to assess fetal lung maturity in patients with maternal-fetal indication such as pre-eclampsia. Moreover, we have routinely recommended and performed placental pathologic examination in all preterm gestations. Written informed consent was obtained from all patients. The Institutional Review Board of Seoul National University Hospital approved the collection and use of these samples and information for research purposes. The Seoul National University has a Federal Wide Assurance with the Office for Human Research Protections of the Department of Health and Human Services of the United States.

tional Review Board of Seoul National University Hospital approved the collection and use of these samples and information for research purposes. The Seoul National University has a Federal Wide Assurance with the Office for Human Research Protections of the Department of Health and Human Services of the United States. Many of patients in this study were included in our previous studies. 2. Clinical characteristics and pregnancy outcomes The demographic and clinical characteristics of the mothers and their neonates were examined through a review of the medical records. We investigated maternal age, parity, the cause of preterm delivery, gestational age (GA) at amniocentesis, GA at delivery and birth weight.

inical characteristics and pregnancy outcomes The demographic and clinical characteristics of the mothers and their neonates were examined through a review of the medical records. We investigated maternal age, parity, the cause of preterm delivery, gestational age (GA) at amniocentesis, GA at delivery and birth weight. 3. Placental pathology Placental tissue samples obtained for pathologic evaluation included chorio-amniotic membrane roll (chorio-decidua and amnion) and chorionic plate. These samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of prepared tissue blocks were stained with hematoxylin and eosin. Pathologists were masked to the clinical information. Acute-HCA was defined in the presence of acute inflammatory changes on examination of chorioamniotic membrane roll and chorionic plate of the placenta. The presence of acute inflammatory change was classified as grade 1 or 2 in each anatomical region of placenta (chorio-decidua, amnion, and chorionic plate) according to previously published criteria [14]. Chorio-decidua restriction in acute-HCA was diagnosed in the presence of at least 1 focus of >5 neutrophils in only a chorio-decidua. Extension beyond chorio-decidua in acute-HCA was defined in the presence of inflammation in amnion or chorionic plate in addition to chorio-decidua: Inflammation of the amnion was diagnosed in the presence of at least 1 focus of >5 neutrophils in the amnion; and inflammation of the chorionic plate was diagnosed in the presence of more than 1 focus of at least 10 neutrophilic collections or diffuse inflammation in subchorionic fibrin, or diffuse and dense inflammation, neutrophilic infiltration into connective tissue of the placental plate, or placental vasculitis, with the use of criteria previously published [14]. Total grade was used to determine the severity of acute-HCA from 1 to 6 according to the criteria previously reported [14].

nic fibrin, or diffuse and dense inflammation, neutrophilic infiltration into connective tissue of the placental plate, or placental vasculitis, with the use of criteria previously published [14]. Total grade was used to determine the severity of acute-HCA from 1 to 6 according to the criteria previously reported [14]. 4. Amniotic fluid AF was cultured for aerobic and anaerobic bacteria and genital mycoplasmas (Ureaplasma urelyticum and Mycoplasma hominis) and analyzed for WBC count according to the methods previously described [15,16]. Of 225 cases, AF WBC count results were available in 208 cases. The remaining fluid was centrifuged and stored in polypropylene tubes at -70℃. MMP-8 concentrations in stored AF were measured with a commercially available enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech Inc., Little Chalfont, Bucks, UK). The sensitivity of the test was <0.3 ng/mL. Both intra- and inter-assay coefficients of variation were <10%. Details about this assay and its performance have been previously described [8]. Of 225 cases, 213 patients were included in the analysis of AF MMP-8, because the test of AF MMP-8 concentration was not performed in 12 cases due to the limited amount of remaining AF.

and inter-assay coefficients of variation were <10%. Details about this assay and its performance have been previously described [8]. Of 225 cases, 213 patients were included in the analysis of AF MMP-8, because the test of AF MMP-8 concentration was not performed in 12 cases due to the limited amount of remaining AF. 5. Statistical analysis Comparisons of continuous variables were performed with Mann-Whitney U test between the two groups and with Kruskal-Wallis test among the three groups and over. The Fisher's exact test or Pearson's chi-square test was used for the comparisons of proportions. Data was analyzed using SPSS Statistics ver. 20.0 (IBM Corp., Somers, NY, USA). Statistical significance was defined as a P<0.05.

U test between the two groups and with Kruskal-Wallis test among the three groups and over. The Fisher's exact test or Pearson's chi-square test was used for the comparisons of proportions. Data was analyzed using SPSS Statistics ver. 20.0 (IBM Corp., Somers, NY, USA). Statistical significance was defined as a P<0.05. Results 1. Clinical characteristics and pregnancy outcomes Table 1 demonstrated clinical characteristics and pregnancy outcomes according to total grade and the presence or absence of chorio-decidua restriction in preterm gestations with acute-HCA including the involvement of chorio-decidua. There was a significant difference in GA at amniocentesis and GA at delivery among the four groups with extension beyond chorio-decidua (group-3 vs. group-4 vs. group-5 vs. group-6, each for P<0.05) (Table 1). However, no difference was found in any clinical characteristics and pregnancy outcomes between the two groups with chorio-decidua restriction (group-1 vs. group-2, each for P>0.05) (Table 1). Moreover, there was no significant difference in any clinical characteristics and pregnancy outcomes between group-2 with chorio-decidua restriction and group-3 with extension beyond chorio-decidua among cases with total grade 2 (each for P>0.05) (Table 1).

restriction (group-1 vs. group-2, each for P>0.05) (Table 1). Moreover, there was no significant difference in any clinical characteristics and pregnancy outcomes between group-2 with chorio-decidua restriction and group-3 with extension beyond chorio-decidua among cases with total grade 2 (each for P>0.05) (Table 1). 2. AF MMP-8 concentrations and WBC counts Figs. 1, 2 show AF MMP-8 concentrations (Fig. 1) and AF WBC counts (Fig. 2) according to total grade and the presence or absence of chorio-decidua restriction in preterm gestations with acute-HCA including the involvement of chorio-decidua. There was no significant difference in a median AF MMP-8 concentration (Fig. 1) and WBC count (Fig. 2) between the two groups (group-1, total grade 1 vs. group-2, total grade 2) in cases with chorio-decidua restriction (each for P>0.05) and among the four groups (group-3, total grade 2 vs. group-4, total grade 3 vs. group-5, total grade 4 vs. group-6, total grade 5-6) in cases with extension beyond chorio-decidua (each for P>0.05). However, group-3 with extension beyond chorio-decidua had a significantly higher median AF MMP-8 concentration (Fig. 1) and WBC count (Fig. 2) than group-2 with chorio-decidua restriction among cases with total grade 2 (each for P<0.05).

total grade 5-6) in cases with extension beyond chorio-decidua (each for P>0.05). However, group-3 with extension beyond chorio-decidua had a significantly higher median AF MMP-8 concentration (Fig. 1) and WBC count (Fig. 2) than group-2 with chorio-decidua restriction among cases with total grade 2 (each for P<0.05). Discussion Principal findings of the study are as following. Firstly, the difference in total grade had little effect on the intensity of IAI in the same context of chorio-decidua restriction and in the same context of extension beyond chorio-decidua among patients with acute-HCA. Secondly, extension beyond chorio-decidua was associated with a significantly higher intensity of IAI than chorio-decidua restriction in the same context of total grade 2 among cases with acute-HCA.

t of chorio-decidua restriction and in the same context of extension beyond chorio-decidua among patients with acute-HCA. Secondly, extension beyond chorio-decidua was associated with a significantly higher intensity of IAI than chorio-decidua restriction in the same context of total grade 2 among cases with acute-HCA. Our results show that there was no significant difference in the intensity of IAI between the two groups of patients with chorio-decidua restriction (Figs. 1, 2). IAI has been defined in the case of an elevated AF WBC count ≥19 cells/mm3 [17] and in the case of an elevated AF MMP-8 concentration ≥23 ng/mL [18] according to the criteria previously published. Considering a median AF MMP-8 concentration and AF WBC count in group-1 or group-2 (AF MMP-8 [ng/mL], 10.1 vs. 107.85; AF WBC count [cells/mm3], 3 vs. 64), no or only a mild inflammation in AF was present in these two groups and ultimately did not cause the difference between the two groups with chorio-decidua restriction (Figs. 1, 2). Indeed, previous studies demonstrated that acute-HCA without amnionitis [11] or chorionic plate inflammation [12] was not associated with an intense IAI.

ld inflammation in AF was present in these two groups and ultimately did not cause the difference between the two groups with chorio-decidua restriction (Figs. 1, 2). Indeed, previous studies demonstrated that acute-HCA without amnionitis [11] or chorionic plate inflammation [12] was not associated with an intense IAI. Our data demonstrated that no significant difference was found in the intensity of IAI according to total grade among cases with extension beyond chorio-decidua in acute-HCA (Figs. 1, 2). An inflammation in amnion or chorionic plate is thought to be the most advanced stage in acute-HCA for the following reason: 1) amnion or chorionic plate, anatomical region beyond chorio-decidua, is located in direct proximity of the amniotic cavity in extra-placental membranes or placental disc; 2) chorionic plate includes large fetal blood vessels that connect to the umbilical cord vessels as a fetal compartment. Therefore, severe intensity of IAI was likely to develop without variation among the four groups according to total grade, because ascending intra-uterine infection had already entered the advanced stages in patients with extension beyond chorio-decidua.

hat connect to the umbilical cord vessels as a fetal compartment. Therefore, severe intensity of IAI was likely to develop without variation among the four groups according to total grade, because ascending intra-uterine infection had already entered the advanced stages in patients with extension beyond chorio-decidua. Patients with total grade 2 had a difference in the involved anatomical region as following: i.e., group-2, chorio-decidua restriction vs. group-3, extension beyond chorio-decidua. Therefore, in view of the previous studies about the significance of involved anatomical region in acute-HCA [11-13], one may expect that extension beyond chorio-decidua was associated with a significantly more intense IAI than chorio-decidua restriction although the two groups had the same total grade. Indeed, patients with extension beyond chorio-decidua had a signficantly higher median AF MMP-8 concentration and WBC count than those with chorio-decidua restriction among cases with total grade 2 in acute-HCA (Figs. 1, 2). Moreover, it should be noted that extension beyond chorio-decidua was associated with a higher rate of positive AF culture than chorio-decidua restriction among cases with total grade 2 (52% vs. 23%) without reaching a statistical significance (Table 1). Our findings demonstrated that patients with the same total grade 2 could be divided into two heterogenous groups (chorio-decidua restriction vs. extension beyond chorio-decidua), which had a different intensity of IAI and a different frequency of intra-amniotic infection.

reaching a statistical significance (Table 1). Our findings demonstrated that patients with the same total grade 2 could be divided into two heterogenous groups (chorio-decidua restriction vs. extension beyond chorio-decidua), which had a different intensity of IAI and a different frequency of intra-amniotic infection. Major strengths of the study are: 1) a large cohort of singleton preterm gestations (n=225) with acute-HCA; 2) it compared a positive culture, MMP-8 concentration and WBC count in AF and therefore, this study examined all potential markers of intra-amniotic infection and inflammation; 3) patients were meticulously divided into as many as 6 groups according to total grade and the presence or absence of chorio-decidua restriction. One potential weakness of the study is that study design was retrospective. To our knowledge, this study is the first study regarding which is more important for the intensity of IAI between total grade or involved anatomical region in acute-HCA of preterm gestations. Although the standardized classification about grading and staging of acute-HCA has not been made yet, our data may suggest that the classification of acute-HCA should attach more importance to the staging about the involved anatomical region of placenta than to the grading about the severity.

CA of preterm gestations. Although the standardized classification about grading and staging of acute-HCA has not been made yet, our data may suggest that the classification of acute-HCA should attach more importance to the staging about the involved anatomical region of placenta than to the grading about the severity. There is a paucity of information about antenatal noninvasive prediction methods for an inflammation in the anatomical region beyond chorio-decidua. Moreover, it should be determined whether the time required for the extension beyond chorio-decidua in acute-HCA is different between patients with PTL and those with preterm-PROM. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2009-0080429). No potential conflict of interest relevant to this article was reported. Fig. 1 Amniotic fluid matrix metalloproteinase-8 (MMP-8) concentrations according to total grade and the presence or absence of chorio-decidua restriction among preterm gestations with acute histologic chorioamnionitis including the involvement of chorio-decidua (median [range], ng/mL; group-1: 10.1 [0.3-4,202.7] vs. group-2: 107.85 [0.3-909.4] vs. group-3: 393.1 [0.3-3,929.0] vs. group-4: 486.15 [0.4-3,392.0] vs. group-5: 408.6 [0.6-6,142.6] vs. group-6: 454.0 [0.5-5,019.5]) (each and all P-value is shown in graph). NS, not significant.

uding the involvement of chorio-decidua (median [range], ng/mL; group-1: 10.1 [0.3-4,202.7] vs. group-2: 107.85 [0.3-909.4] vs. group-3: 393.1 [0.3-3,929.0] vs. group-4: 486.15 [0.4-3,392.0] vs. group-5: 408.6 [0.6-6,142.6] vs. group-6: 454.0 [0.5-5,019.5]) (each and all P-value is shown in graph). NS, not significant. Fig. 2 Amniotic fluid white blood cell (WBC) counts according to total grade and the presence or absence of chorio-decidua restriction among preterm gestations with acute histologic chorioamnionitis including the involvement of chorio-decidua (median [range], cells/mm3; group-1: 3 [0-10000] vs. group-2: 64 [0-2,800] vs. group-3: 773 [0-13,248] vs. group-4: 435 [0-15,000] vs. group-5: 884 [1-19,764] vs. group-6: 380 [0-8,640]) (each and all P-value is shown in graph). NS, not significant. Table 1 Clinical characteristics and pregnancy outcomes according to total grade and the presence or absence of chorio-decidua restriction among 225 preterm gestations (GA at delivery <36 weeks) with acute histologic chorioamnionitis including chorio-decidua involvement GA, gestational age; preterm-PROM, preterm premature rupture of membranes; SD, standard deviation; NS, not significant; AF, amniotic fluid. a)P-value: comparison between group-1 and group-2; b)P-value: comparison between group-2 and group-3; c)P-value: comparison among group-3, group-4, group-5 and group-6; d)P-value: comparison among all study groups; e)Of 225 cases who underwent amniocentesis within 5 days of birth, AF culture results were available in 216 cases.

Introduction Ovarian cancer is the second most common gynecological cancer in Western countries as well as the leading cause of gynecological cancer-related death, with an incidence estimated at 8.8 per 100,000 women-years in USA [1-4]. In Korea, ovarian cancer is also the second most common gynecological cancer, with an incidence of 8.0 per 100,000 women-years in 2010 [4,5]. Though ovarian cancer can be curable in early-diagnosed cases where the disease is limited to the ovary, however most patients are diagnosed when at more advanced stages (International Federation of Obstetrics and Gynecology [FIGO] stage III-IV) [6]. Among gynecologic cancers, the incidence of cervical cancer has been decreasing in Korea [5], which has been attributed to earlier diagnoses secondary to routine pap smears. However, increasing the rate of earlier diagnosis for ovarian cancer has remained difficult due to the relative dearth of associated symptoms and lack of specific serum biomarker. Currently used as a diagnostic marker for ovarian cancer, cancer antigen 125 (CA-125) is elevated in roughly 80% of patients with ovarian cancer and 30% of patients with other primary cancers with extensive intra-abdominal disease. Accordingly, serum CA-125 levels are not only elevated in ovarian malignancies, but also benign ovarian diseases as well as any other inflammatory conditions of the peritonieum, pleura and pericardium [7-10]. Moreover, as CA-125 levels are elevated in less than half of cases of early stage ovarian cancer [11,12], a new biomarker for ovarian cancer is clearly needed.

evated in ovarian malignancies, but also benign ovarian diseases as well as any other inflammatory conditions of the peritonieum, pleura and pericardium [7-10]. Moreover, as CA-125 levels are elevated in less than half of cases of early stage ovarian cancer [11,12], a new biomarker for ovarian cancer is clearly needed. First identified in the epithelium of the distal epididymis, human epididymis protein 4 (HE4) was originally believed to represent a protease inhibitor for sperm maturation and contribute to intrinsic immunity. HE4 is also one of 14 homologous genes on chromosome 20q12-13.1 that encodes proteins with a whey acidic protein-type four disulphide core domain [13-15]. Emerging data now suggests that serum levels of HE4 is elevated in ovarian cancer patients, demonstrating similar sensitivity and increased specificity for ovarian cancer when compared with CA-125 [16]. HE4 is also elevated in lung adenocarcinoma, transitional cell, breast, renal and pancreatic carcinomas [17]. In the current study, we analyzed the serum levels of HE4 and CA-125 among patients with ovarian cancer as well as other benign ovarian tumors in order to assess the possible role of serum HE4 levels as an ovarian cancer biomarker.

First identified in the epithelium of the distal epididymis, human epididymis protein 4 (HE4) was originally believed to represent a protease inhibitor for sperm maturation and contribute to intrinsic immunity. HE4 is also one of 14 homologous genes on chromosome 20q12-13.1 that encodes proteins with a whey acidic protein-type four disulphide core domain [13-15]. Emerging data now suggests that serum levels of HE4 is elevated in ovarian cancer patients, demonstrating similar sensitivity and increased specificity for ovarian cancer when compared with CA-125 [16]. HE4 is also elevated in lung adenocarcinoma, transitional cell, breast, renal and pancreatic carcinomas [17]. In the current study, we analyzed the serum levels of HE4 and CA-125 among patients with ovarian cancer as well as other benign ovarian tumors in order to assess the possible role of serum HE4 levels as an ovarian cancer biomarker. Materials and methods 1. Study population In the current case-controlled 1:2 matching study, patients were recruited from Ewha Woman's University Mokdong Hospital in Seoul, Korea between October 2005 and March 2010. Informed consent was obtained in all cases prior to enrollment. The inclusion criteria were: no other diagnosed gynecologic disease except ovarian mass, the ovarian mass had to be the primary diagnosis availability of complete clinical records, informed consent and agreement to have additional testing for new markers, clinical and histological diagnosis with staging and grading of ovarian cancer, according to the current classification and guidelines. And if any cases were not satisfied in criteria, they were excluded.

ailability of complete clinical records, informed consent and agreement to have additional testing for new markers, clinical and histological diagnosis with staging and grading of ovarian cancer, according to the current classification and guidelines. And if any cases were not satisfied in criteria, they were excluded. During the period, 367 women underwent operation, 47 women received a diagnosis of cancer, and 320 women received a diagnosis of benign ovarian tumor. Based on the inclusion criteria, a total of 94 women were enrolled. The 32 cases of ovarian cancer included 16 serous, 5 clear-cell, 5 mucinous, 4 mixed, 2 endometrioid carcinomas. Of 32 ovarian cancer patients, 6 (18.8%) had stage I disease, 4 (12.5%) had stage II disease, 20 (62.5%) had stage III disease and 2 (6.25%) had stage IV disease as per the International Federation of Gynecology and Obstetrics (FIGO) criteria. Histopathology of 62 patients with benign ovarian tumors were as follows: 23 endometriomas (37.1%), 16 mature cystic teratomas (25.8%), 8 mucinous cystadenomas (12.9%), 8 serous cystadenomas (12.9%), 7 other non-specified neoplasms (11.3%). All enrolled patients underwent laparoscopy or laparotomy, and all diagnoses were histopathologically confirmed by pathologic examination at Ewha Womans University Mokdong Hospital. 2. CA-125 and HE4 levels In all cases, patient sera was obtained on the day prior to the laparotomy/laparoscopy and was stored frozen at -80℃ until analysis.

During the period, 367 women underwent operation, 47 women received a diagnosis of cancer, and 320 women received a diagnosis of benign ovarian tumor. Based on the inclusion criteria, a total of 94 women were enrolled. The 32 cases of ovarian cancer included 16 serous, 5 clear-cell, 5 mucinous, 4 mixed, 2 endometrioid carcinomas. Of 32 ovarian cancer patients, 6 (18.8%) had stage I disease, 4 (12.5%) had stage II disease, 20 (62.5%) had stage III disease and 2 (6.25%) had stage IV disease as per the International Federation of Gynecology and Obstetrics (FIGO) criteria. Histopathology of 62 patients with benign ovarian tumors were as follows: 23 endometriomas (37.1%), 16 mature cystic teratomas (25.8%), 8 mucinous cystadenomas (12.9%), 8 serous cystadenomas (12.9%), 7 other non-specified neoplasms (11.3%). All enrolled patients underwent laparoscopy or laparotomy, and all diagnoses were histopathologically confirmed by pathologic examination at Ewha Womans University Mokdong Hospital. 2. CA-125 and HE4 levels In all cases, patient sera was obtained on the day prior to the laparotomy/laparoscopy and was stored frozen at -80℃ until analysis. Serum HE4 levels were measured by HE4 enzyme immunoassay (Fujirebio Diagnostics Inc., Malvern, PA, USA), which were performed as per the manufacturer's instructions. Specifically, the HE4 assay is a solid-phase immunoassay derived from the direct sandwich technique, which uses biotinylated anti-HE4 monoclonal antibody (MAb), streptavidin coated microstrips, and HRP labeled anti-HE4 MAb. To date, no definitive diagnostic thresholds for HE4 have been reported in Korean women, however previous data from other western countries identified 74.2 pM as a cut-off point, as this value corresponded with the upper 95% among healthy individuals from Verona, Italy [18].

d microstrips, and HRP labeled anti-HE4 MAb. To date, no definitive diagnostic thresholds for HE4 have been reported in Korean women, however previous data from other western countries identified 74.2 pM as a cut-off point, as this value corresponded with the upper 95% among healthy individuals from Verona, Italy [18]. Serum CA-125 levels were determined by Modular analytics E170 module (Roche Diagnostics, Mannheim, Germany), an electrochemiluminescence immunoassay derived from the sandwich principle using two monoclonal antibodies, a biotinylated monoclonal CA-125-specific antibody, and a monoclonal CA-125-specific ruthenium complex-labeled antibody. Notably, this assay is able to measure CA-125 levels between 0.600 to 5,000 U/mL, though the manufacturer's suggested cut-off level is 35 U/mL. 3. Statistical analysis All the data were analyzed using SPSS ver. 21.0 (IBM, Armonk, NY, USA). The median values of the serum HE4 and CA-125 levels were calculated separately for individuals with other benign ovarian tumor and the patients with a diagnosis of ovarian cancer. The relative serum tumor marker levels were compared among the two groups using the Wilcoxon signed-rank test because they did not follow a normal distribution. In all cases, P-values <0.05 were defined as statistically significant. Receiver operating characteristic (ROC) curves were assessed for both serum values of HE4 and CA-125. Values with the best diagnostic performance as per the ROC curve were identified in order to estimate the area under the curve (AUC).

3. Statistical analysis All the data were analyzed using SPSS ver. 21.0 (IBM, Armonk, NY, USA). The median values of the serum HE4 and CA-125 levels were calculated separately for individuals with other benign ovarian tumor and the patients with a diagnosis of ovarian cancer. The relative serum tumor marker levels were compared among the two groups using the Wilcoxon signed-rank test because they did not follow a normal distribution. In all cases, P-values <0.05 were defined as statistically significant. Receiver operating characteristic (ROC) curves were assessed for both serum values of HE4 and CA-125. Values with the best diagnostic performance as per the ROC curve were identified in order to estimate the area under the curve (AUC). Results The clinical characteristics and study groups demographics are presented in Table 1. There were some demographic differences between two groups. The mean age of ovarian cancer group is older than that of benign ovarian tumor group and menopausal patients were more larger in ovarian cancer group. The median serum levels of CA-125 and HE4 were significantly higher among individuals with ovarian cancer when compared with those with other benign ovarian tumors, with the values for each group reaching statistical significance (CA-125, 394.1 U/mL vs. 22.7 U/mL; HE4, 56.7 pM vs. 18.5 pM; P<0.05 in both) (Table 2, Fig. 1).

edian serum levels of CA-125 and HE4 were significantly higher among individuals with ovarian cancer when compared with those with other benign ovarian tumors, with the values for each group reaching statistical significance (CA-125, 394.1 U/mL vs. 22.7 U/mL; HE4, 56.7 pM vs. 18.5 pM; P<0.05 in both) (Table 2, Fig. 1). The patients with benign ovarian tumors were further stratified by known endometriosis as confirmed by a pathologic diagnosis. The median serum CA-125 and HE4 levels were then recalculated for both groups (Table 2) revealing significantly higher serum CA-125 levels in the ovarian endometrioma group when compared with the patients with other benign ovarian tumors (31.95 U/mL vs. 17.9 U/mL, P = 0.03). Conversely, the median serum HE4 levels did not vary significantly between groups (19.0 pM vs. 18.2 pM, P = 0.49). Furthermore, serum CA-125 and HE4 values were compared between patients with ovarian cancer and the ovarian endometrioma subgroup, showing significantly elevated serum levels of both biomarkers among the ovarian cancer group (CA-125, P = 0.004; HE4, P = 0.001). The ROC curve analysis of the diagnostic performance of patients with ovarian cancer revealed a higher AUC with borderline significance for HE4 when compared with CA-125 (0.93 [95% confidence interval, CI: 0.90-0.97] vs. 0.85 [95% CI, 0.77-0.92]) (Fig. 2, Table 3). Additionally, the AUC for the combination of the two serum markers was 0.89 (95% CI, 0.83-0.95), but a significant difference was not found when comparing HE4 and CA-125 alone.

significance for HE4 when compared with CA-125 (0.93 [95% confidence interval, CI: 0.90-0.97] vs. 0.85 [95% CI, 0.77-0.92]) (Fig. 2, Table 3). Additionally, the AUC for the combination of the two serum markers was 0.89 (95% CI, 0.83-0.95), but a significant difference was not found when comparing HE4 and CA-125 alone. Using a serum cut-off level of 76.0 pM for HE4 a sensitivity and specificity of 78.1% and 86.8% was observed. Using a serum cut-off level of 37.45 U/mL for serum CA-125, a sensitivity and specificity of 84.4% and 67.4% was observed. Discussion CA-125 is the most widely used serum biomarker in ovarian cancer screening, as the utility of CA-125 in determining treatment response or monitoring recurrent disease status has been established [19]. Previous data indicates that at a serum level of 35 U/mL CA-125 has a sensitivity of 73.2% and a specificity of 79.2%, which are comparable to other biomarkers in predicting ovarian malignancy [20]. Nevertheless, CA-125 is not only increased in cases of ovarian cancer but also other benign conditions.

established [19]. Previous data indicates that at a serum level of 35 U/mL CA-125 has a sensitivity of 73.2% and a specificity of 79.2%, which are comparable to other biomarkers in predicting ovarian malignancy [20]. Nevertheless, CA-125 is not only increased in cases of ovarian cancer but also other benign conditions. For these reasons, several novel ovarian cancer tumor markers have been assessed for use in screening patients for ovarian cancer, including haptoglobin, osteopontin, HE4, mesothelin (SMRP), B7-H4, prostasin, macrophage colony stimulating factor, vascular endothelial growth factor, several interleukins (IL-6, IL-8), eosinophil-derived neurotoxin, COOH-osteopontin fragments, OVX1, lysophophatidic acid, apolipoprotein A1, and transthyretin [21]. Of these, HE4 has demonstrated high sensitivity and specificity (90% and 77.6%, respectively) in identifying cases of ovarian cancer. In detecting cases of stage I ovarian cancer, HE4 has the highest sensitivity when compared to CA-125, SMRP, CA-72-4, andosteopontin [22]. The results presented here suggest a possible role for serum HE4 as a diagnostic marker for detecting ovarian cancer. Serum HE4 levels were significantly higher in the ovarian cancer group when compared with patients with other benign ovarian tumors (P<0.05), and showed comparable sensitivities in detecting ovarian cancer to CA-125.

esented here suggest a possible role for serum HE4 as a diagnostic marker for detecting ovarian cancer. Serum HE4 levels were significantly higher in the ovarian cancer group when compared with patients with other benign ovarian tumors (P<0.05), and showed comparable sensitivities in detecting ovarian cancer to CA-125. Moreover, HE4 demonstrated a significantly lower false positive rate, especially in cases of other benign ovarian diseases such as endometriosis. Several previous studies suggest that serum levels of HE4 are significantly higher in patients with both endometrial and ovarian malignancies, though not ovarian endometriomas or other types of endometriosis. In comparison, serum CA-125 levels were elevated in patients with ovarian cancer, as well as advanced endometriosis with peritoneal or deep lesions, and ovarian endometriomas, though not in the patients with endometrial cancer [23]. As such, we stratified the other benign ovarian tumor group for ovarian endometriomas, as this common benign tumor has been associated with false elevations in serum CA-125 level. In a comparison between groups of patients with ovarian endometrioma versus other benign ovarian tumors, serum CA-125 but not serum HE4 levels were found to be significantly increased in the setting of endometrioma. Accordingly, serum HE4 levels had a lower false positive rate in the data presented here, as in other recent studies.

on between groups of patients with ovarian endometrioma versus other benign ovarian tumors, serum CA-125 but not serum HE4 levels were found to be significantly increased in the setting of endometrioma. Accordingly, serum HE4 levels had a lower false positive rate in the data presented here, as in other recent studies. We also compared AUC for the ROC analysis for CA-125, HE4, and the combination of the two markers. Specifically, HE4 demonstrated a higher AUC than other two groups in distinguishing benign and malignant pelvic masses. In a previous study, risk of ovarian malignancy, showed excellent diagnostic performance for the detection of epithelial ovarian cancer in post-menopausal women, but just the dual marker combination of HE4 and CA-125 did not exhibit any greater accuracy than HE4 alone [18]. In another cohort of Sweden women, HE4 seems like a CA-125 for diagnostic marker for ovarian mass, although the AUC for the HE4 ROC curve is not greater that CA-125. Nonetheless, the sensitivity for HE4 combined with CA-125 was greater than two other serum HE4 and CA-125 group [24]. Given these findings, HE4 may represent a useful diagnostic marker for excluding ovarian cancer in patients with a known pelvic mass. In the current study, the appropriate cut-off level that yielded a higher sensitivity and specificity was 76.0 pM, a value consistent with data from other studies [18].

We also compared AUC for the ROC analysis for CA-125, HE4, and the combination of the two markers. Specifically, HE4 demonstrated a higher AUC than other two groups in distinguishing benign and malignant pelvic masses. In a previous study, risk of ovarian malignancy, showed excellent diagnostic performance for the detection of epithelial ovarian cancer in post-menopausal women, but just the dual marker combination of HE4 and CA-125 did not exhibit any greater accuracy than HE4 alone [18]. In another cohort of Sweden women, HE4 seems like a CA-125 for diagnostic marker for ovarian mass, although the AUC for the HE4 ROC curve is not greater that CA-125. Nonetheless, the sensitivity for HE4 combined with CA-125 was greater than two other serum HE4 and CA-125 group [24]. Given these findings, HE4 may represent a useful diagnostic marker for excluding ovarian cancer in patients with a known pelvic mass. In the current study, the appropriate cut-off level that yielded a higher sensitivity and specificity was 76.0 pM, a value consistent with data from other studies [18]. Herein, we report early data indicating that serum HE4 levels may represent a new marker for identifying ovarian cancers in Korean women. Until now, many studies for ovarian mass tried to distinguish between ovarian cancer and benign mass in Korean women. Serum CA-125 levels as well as other methods have also been evaluated for ovarian mass screening in Korean women in the past. These previous studies assessed pulsatility index, transvaginal sonographic scoring system and CA-125 preoperatively, and transvaginal Doppler color flow imaging may be useful clinical tools for the differential diagnosis of malignant ovarian tumors [25]. Another study showed that the combination of CA 15-3, Tumor-associated glycoprotein (TAG) 72, and CA-125 may reach an acceptable sensitivity and excellent specificity in differentiating malignant from benign pelvic masses, particularly among patients over 50 years of age. Specifically, these authors collected preoperative serum samples from 78 patients with pelvic masses and measured tumor-associated antigens CA-125, CA-15-3, and TAG 72 by immunoradiometric assay in order to evaluate the efficacy of these markers in differentiating benign and malignant pelvic masses, finding that among patients over 50 years of age the three marker combination was associated with a sensitivity of 79% and a specificity of 100% [26]. Another study of 56 newly-diagnosed epithelial ovarian cancer patients, showed that of five serum biomarkers-leptin, prolactin, osteoponin, insulin-like growth factor-II and CA-125-only the preoperative serum CA-125 level had a significant positive correlation with cancer stage (P<0.01) [27].

specificity of 100% [26]. Another study of 56 newly-diagnosed epithelial ovarian cancer patients, showed that of five serum biomarkers-leptin, prolactin, osteoponin, insulin-like growth factor-II and CA-125-only the preoperative serum CA-125 level had a significant positive correlation with cancer stage (P<0.01) [27]. With specific regard to the association between HE4 and ovarian cancer in Korean women, the initial study recruited 159 women with adnexal masses, including 78 patients with ovarian cancer [28], as well as 224 healthy controls. In this study, serum HE4 and CA-125 levels were found to be significantly elevated in the ovarian cancer patients when compared with those from patients with benign disease or healthy controls (HE4, 80.0 pM; CA-125, 216.8 U/mL; P<0.0001 in both). But no definitive diagnostic threshold for these tests was ever determined in Korean women. So we need to perform more studies about serum HE4 level in Korean healthy women, then we may determinate the proper definitive diagnostic threshold. A larger case-control study of Korean females was done in 2011. The population of that study comprised 2,182 healthy women, 72 pregnant women, 66 women with ovarian cancers, and 257 women with benign gynecologic disease. The authors suggested an HE4 cut-off level of 33.2 pmol/L for 97% upper reference limits. Using this value as a cutoff point, the sensitivity and specificity for diagnosing ovarian cancer as differentiated from benign gynecologic disease were 90.9% and 94.1%, respectively. The cut-off HE4 level was different from that in our study due to the use of different machines and methods [29].

pper reference limits. Using this value as a cutoff point, the sensitivity and specificity for diagnosing ovarian cancer as differentiated from benign gynecologic disease were 90.9% and 94.1%, respectively. The cut-off HE4 level was different from that in our study due to the use of different machines and methods [29]. The primary limitation of the present study is the relatively small size of sample number. As such, no statistical analysis was performed according to the ovarian cancer histological subtype. Furthermore, the serum HE4 and CA-125 levels were also not compared among the healthy controls. A second limitation is that age and menstruation status were not included in the analysis, both of which may have influenced the serum HE4 level. In one prior study, the serum HE4 level increased with increasing age, while serum CA-125 levels were lower in older subjects. However, the upper limits of serum HE4 levels did not vary significantly in the individuals without ovarian cancer regardless of menopausal status [29]. Given these findings, the differences in age between the two groups may have influenced another demographic factor, and a larger scale age-matched case-control study is needed to better characterize this relationship.

not vary significantly in the individuals without ovarian cancer regardless of menopausal status [29]. Given these findings, the differences in age between the two groups may have influenced another demographic factor, and a larger scale age-matched case-control study is needed to better characterize this relationship. In conclusion, serum HE4 likely represents a useful tumor marker for ovarian cancer in Korean women. Assessing serum HE4 levels has the potential to increase the accuracy of ovarian cancer screening and provide better information in differentiating ovarian cancer from other benign ovarian tumors. We did this study as a pilot study and larger, more extended studies are needed to confirm the accuracy of serum HE4 as a tumor marker for the early diagnosis of ovarian cancer in patient with ovarian masses. Acknowledgments This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120071). No potential conflict of interest relevant to this article was reported. Fig. 1 Comparison of (A) the serum CA-125 and (B) human epididymis-specific protein E4 (HE4) levels between ovarian cancer and benign ovarian tumor. Fig. 2 Comparison of the area under the curve from the receiver operating characteristics (ROC) curve analysis for serum CA-125 and human epididymis-specific protein E4 (HE4) levels. Table 1 The demographic and clinical characteristics of the study group Values are presented as median (range) or number (%).

Fig. 1 Comparison of (A) the serum CA-125 and (B) human epididymis-specific protein E4 (HE4) levels between ovarian cancer and benign ovarian tumor. Fig. 2 Comparison of the area under the curve from the receiver operating characteristics (ROC) curve analysis for serum CA-125 and human epididymis-specific protein E4 (HE4) levels. Table 1 The demographic and clinical characteristics of the study group Values are presented as median (range) or number (%). Table 2 Comparison of the serum human epididymis-specific protein E4 (HE4) and CA-125 levels among patients with ovarian cancer versus other benign ovarian tumors a)P-value, calculated using the Wilcoxon signed-rank test. Table 3 Analysis of the receiver operating characteristics (ROC) curve analysis for the serum CA-125 and HE4 levels HE4, human epididymis-specific protein E4; ROC, receiver operating characteristics; AUC, area under the curve.

Introduction Dysmenorrhea is characterized as a periodic menstrual cramping pain occurring in the center of the lower abdominal region. Dysmenorrhea is a common gynecological problem in women of childbearing age; in general, it begins with the first ovulation cycle, which occurs about two years after menarche. The prevalence is 60% to 90% in adolescent women, and it gradually decreases with age [1-6]. The condition can be classified into primary dysmenorrhea and secondary dysmenorrhea depending on the cause. Primary dysmenorrhea is due to the contraction of the myometrium without ovarian or cervical lesions; it appears either immediately before or after menstruation and continues for one to three days. This primary type is closely related to ovulatory menstruation; dysmenorrhea rarely occurs during the temporary anovulatory menstrual periods of menarche. Secondary dysmenorrhea is caused by a gynecological disorder and may also occur during anovulatory menstrual cycles. The known risk factors of primary dysmenorrhea are a body mass index (BMI) of 20 kg/m2 or less, early menarche before 12 years old, long menstrual intervals or long menstrual periods, massive menstrual volume, and smoking. Delivery experience is known to decrease dysmenorrhea, but having a miscarriage does not affect symptoms [6-12]. On the other hand, socioeconomic factors are associated with dysmenorrhea.

less, early menarche before 12 years old, long menstrual intervals or long menstrual periods, massive menstrual volume, and smoking. Delivery experience is known to decrease dysmenorrhea, but having a miscarriage does not affect symptoms [6-12]. On the other hand, socioeconomic factors are associated with dysmenorrhea. Recently, Korean society has resolved some socioeconomic problems, such as low fertility and late marriage, through international marriage and accepting immigrant workers. As a result, a rapid change to a multicultural society is occurring. International marriages between Korean men and Vietnamese women accounted for about 40% of the total international marriages; more than 13,000 Vietnamese women move to Korea to live. Because they are an important part of the Korean socioeconomy and they have significant roles in family composition, domestic Vietnamese marriage immigrants were investigated and compared to Vietnamese women living in the southern and northern regions; frequency of dysmenorrhea and factors affecting dysmenorrhea were evaluated, including socioeconomic factors.

Korean socioeconomy and they have significant roles in family composition, domestic Vietnamese marriage immigrants were investigated and compared to Vietnamese women living in the southern and northern regions; frequency of dysmenorrhea and factors affecting dysmenorrhea were evaluated, including socioeconomic factors. Materials and methods From March 2010 to March 2011, 3,017 Vietnamese women aged 17 to 42 years were investigated. The cohort was composed of 993 domestic marriage immigrants, 1,026 women living in the northern region of Vietnam (Bavi) and 998 women living in southern Vietnam (Can Tho). In the Korean provinces of Jinju, Okcheon, Gongju, Pohang, Gyeongju, Pusan, Masan, and Sangju, where many Vietnam marriage immigrants live, participants in this cohort study were recruited using advertising through the Marriage Immigrant Family Support Center and the Korean School for Marriage Immigrants. Information regarding the contents and items of examination as well as the procedure and introduction was mailed, then questionnaires were given and examinations were conducted on the day of examination. Participants received questionnaires written in Vietnamese, and an interpreter explained the contents if required before the women completed the questionnaires. Basic items such as age, height and weight, as well as the history of menstruation and pregnancy and delivery were investigated. In addition, life habits, such as drinking history, smoking history and exercise frequency, and socioeconomic factors, such as resident region, education, religion, and average gross income, were also assessed. This study was approved by the Institutional Review Board of Ewha University Mokdong Hospital. This study results were analyzed using the SPSS ver. 19.0 (IBM, Armonk, NY, USA), and continuous variables were expressed as mean ± standard deviation for the statistical analysis. Continuous variables for the target comparison were tested with independent t-test, while categorized variables used chi-square; each factor's dysmenorrheal risk factors were analyzed using logistic regression. When the P-value was less than 0.05 for all items, the result was considered to be statistically significant.

Continuous variables for the target comparison were tested with independent t-test, while categorized variables used chi-square; each factor's dysmenorrheal risk factors were analyzed using logistic regression. When the P-value was less than 0.05 for all items, the result was considered to be statistically significant. Results The average age of participants was 25.5 ± 4.4 years; the average age at menarche was 14.8 years. Women with dysmenorrhea made up 1,774 of the participants (58.8%), and the average BMI was 20.2 kg/m2. Most women (91.3%) did not drink alcohol, and all women who participated in the survey were non-smokers. The average menstrual period length was 4.6 ±1.6 days. The most frequently reported amount of menstrual volume was common (65.9%), followed by too much (23.3%) and a very large amount (2.0%) (Table 1).

s 20.2 kg/m2. Most women (91.3%) did not drink alcohol, and all women who participated in the survey were non-smokers. The average menstrual period length was 4.6 ±1.6 days. The most frequently reported amount of menstrual volume was common (65.9%), followed by too much (23.3%) and a very large amount (2.0%) (Table 1). The average age of women without dysmenorrhea (24.9 ± 4.3 years) was significantly lower than the average age of women with dysmenorrhea (26.3 ± 4.4 years), and women without dysmenorrhea had a significantly lower age at menarche (14.7 ±1.8 years) than the age at menarche of women with dysmenorrhea (14.9 ±1.6 years); a younger overall age and a younger age at menarche were associated with higher amounts of dysmenorrhea. Women's menstrual intervals did not differ significantly according to the presence or absence of dysmenorrhea; women with dysmenorrhea had menstrual periods of 4.73 ±1.6 days, which was longer than those of women without dysmenorrhea (4.31±1.6 days). However, menstrual cycle and body mass index were not significantly associated with dysmenorrhea (Table 2). In women with highly massive menstrual volumes, the dysmenorrhea prevalence was significantly higher (30.0%) than in women without dysmenorrhea (18.6%) (Table 3).

se of women without dysmenorrhea (4.31±1.6 days). However, menstrual cycle and body mass index were not significantly associated with dysmenorrhea (Table 2). In women with highly massive menstrual volumes, the dysmenorrhea prevalence was significantly higher (30.0%) than in women without dysmenorrhea (18.6%) (Table 3). Our investigation of the participants' delivery history and its association with dysmenorrhea revealed that experiencing pregnancy and breastfeeding produced a lower incidence of dysmenorrhea. We found that 72.1% of women without pregnancy experience showed dysmenorrhea, while only 54.1% of women who had been pregnant reported dysmenorrhea. The frequency of dysmenorrhea was also influenced by breastfeeding history; 52.2% of women who had breastfed a child showed dysmenorrhea compared to 68.8% of those who had not (Table 3).

.1% of women without pregnancy experience showed dysmenorrhea, while only 54.1% of women who had been pregnant reported dysmenorrhea. The frequency of dysmenorrhea was also influenced by breastfeeding history; 52.2% of women who had breastfed a child showed dysmenorrhea compared to 68.8% of those who had not (Table 3). The incidence of dysmenorrhea in northern Vietnamese residents at 43.4% was significantly lower than that reported by domestic marriage immigrants (68.3%) and residents of southern Vietnam (65.2%, P<0.001). When considering education, the high school graduation group had the highest frequency of dysmenorrhea (64.4%). A significantly low incidence was reported in the middle school graduation or high school dropout group (51.9%). The presence or absence of religion and religion-specific incidence of dysmenorrhea was also reviewed; women who were religious showed a higher incidence of dysmenorrhea than women who practiced no religion (55.0%, P<0.001); there was no difference among the type of religion practiced (Table 3). In contrast, the average monthly gross income and living period in Korea had no significant associations with dysmenorrhea.

d; women who were religious showed a higher incidence of dysmenorrhea than women who practiced no religion (55.0%, P<0.001); there was no difference among the type of religion practiced (Table 3). In contrast, the average monthly gross income and living period in Korea had no significant associations with dysmenorrhea. Age, menstrual volume, pregnancy experience, resident region, education, and age at menarche were used for logistic regression analysis; the result was that dysmenorrhea was 1.847 times higher in women with massive menstrual volumes, and there was a significant correlation between massive menstrual volume and dysmenorrhea. Pregnancy lowered the risk of dysmenorrhea by about half (odds ratio [OR], 0.550; 95% CI, 1.349-2.501). Korean residents had a higher risk of dysmenorrhea than Vietnam residents overall, while southern Vietnam residents (OR, 0.590; 95% CI, 0.459-0.758) reported more dysmenorrhea than northern Vietnam residents (OR, 0.411; 95% CI, 0.338-0.499) (Table 4).

norrhea by about half (odds ratio [OR], 0.550; 95% CI, 1.349-2.501). Korean residents had a higher risk of dysmenorrhea than Vietnam residents overall, while southern Vietnam residents (OR, 0.590; 95% CI, 0.459-0.758) reported more dysmenorrhea than northern Vietnam residents (OR, 0.411; 95% CI, 0.338-0.499) (Table 4). Discussion This study was to conducted investigate the incidence of dysmenorrhea and socioeconomic factors affecting the dysmenorrhea occurrence in Vietnam women of childbearing age. Various factors were found to be significantly related with the incidence of dysmenorrhea. Age, age at menarche, menstrual period, and menstrual volume were correlated with dysmenorrhea, and a history of obstetric factors, such as pregnancy experience, full-term delivery and breastfeeding, as well as socioeconomic factors including resident region, education and religion, were significantly correlated with dysmenorrhea. BMI, menstrual period, average monthly gross income, and residence period in Korea did not show a clear association.

s, such as pregnancy experience, full-term delivery and breastfeeding, as well as socioeconomic factors including resident region, education and religion, were significantly correlated with dysmenorrhea. BMI, menstrual period, average monthly gross income, and residence period in Korea did not show a clear association. Various studies have investigated the current prevalence of dysmenorrhea. In a study of adolescents between 13 and 18 years, Wong discovered that one out of six experienced dysmenorrhea [13]. Banikarim et al. [14] reported an 85% prevalence of dysmenorrhea in Latin American women, and Sundell et al. [7] found an incidence of 72% in 19-year-old and 67% in 24-year-old Swedish women. Jamieson and Steege [15] reported 90% in 18-year-old to 45-year-old American women, and Lu [16] reported 51.3% in 15-year-old to 54-year-old Taiwan women [8]. This study was the first to determine the prevalence of dysmenorrhea in domestic Vietnamese marriage immigrants and Vietnamese resident women; the dysmenorrhea prevalence of Vietnamese women was similar (58.8%) with Turkish women of the same age (55.5% to 58.2%) [12,17].

in 15-year-old to 54-year-old Taiwan women [8]. This study was the first to determine the prevalence of dysmenorrhea in domestic Vietnamese marriage immigrants and Vietnamese resident women; the dysmenorrhea prevalence of Vietnamese women was similar (58.8%) with Turkish women of the same age (55.5% to 58.2%) [12,17]. In 1988, the dysmenorrhea prevalence amongst Korean high school girls was 77.8%, and it had fallen to 73.9% in 1998 [18]. This incidence was still higher than this study's finding of 58.8%, which might have been caused by the age difference. This study's participants had an average age of 25.5 ± 4.4 years. When we reanalyzed our data to include only 17-year-old and 18-year-old women in this study, the prevalence of dysmenorrhea was 78.3%, which confirmed that there was no difference between same-aged Korean high school girls and this study's result (data not shown).

's participants had an average age of 25.5 ± 4.4 years. When we reanalyzed our data to include only 17-year-old and 18-year-old women in this study, the prevalence of dysmenorrhea was 78.3%, which confirmed that there was no difference between same-aged Korean high school girls and this study's result (data not shown). The average age of women who experienced dysmenorrhea was lower compared to women without dysmenorrhea. When logistic regression analysis was done, the result was consistent that the risk of dysmenorrhea was 0.97 times lower as age increased (P<0.006). Therefore, it may be worthwhile to review the relationship between age at menarche and dysmenorrhea. In this study, the average age at menarche of the group who experienced dysmenorrhea was 14.7 ± 1.8 years old, which was significantly lower than the group who had no dysmenorrhea (14.9 ± 1.6 years old) (P=0.001). It might be inferred that early menarche produces more prevalent dysmenorrhea, which matches with the study of Andersch and Milsom [8] with Swedish women and Tangchai et al. [19] study with Thai adolescents. Another study reported that delayed menarche showed a high dysmenorrhea occurrence [20]. The association with early age at menarche and dysmenorrhea was thought to result from the fact that women who matured earlier showed similar hormone patterns with adults, and slower-maturing women had only a half ovulation efficiency.

her study reported that delayed menarche showed a high dysmenorrhea occurrence [20]. The association with early age at menarche and dysmenorrhea was thought to result from the fact that women who matured earlier showed similar hormone patterns with adults, and slower-maturing women had only a half ovulation efficiency. The menstrual period was significantly longer in the dysmenorrhea group (P<0.05), but the correlation of menstrual cycle length and dysmenorrhea was not significant. This finding agrees with the study that showed no correlation between menstrual period regularity and dysmenorrhea [21], but it did not match up with the study that found women with irregular menstrual cycles, massive menstrual volumes and long menstrual cycles showed high incidences of dysmenorrhea [22]. The result was consistent with an existing report that revealed high menstrual volumes were significantly correlated with dysmenorrhea [8]. The correlation between weight and dysmenorrhea was somewhat contradictory; women who were underweight showed a high incidence of dysmenorrhea in our study, and another study found that dysmenorrhea of sub-standard weight women was 1.5 times higher than overweight or obese women [19,23]. However, a different study reported higher complaints of dysmenorrhea in obese participants [8]. BMI and dysmenorrhea had no significant correlation in this study, which was consistent with local study results among adolescents [21].

sub-standard weight women was 1.5 times higher than overweight or obese women [19,23]. However, a different study reported higher complaints of dysmenorrhea in obese participants [8]. BMI and dysmenorrhea had no significant correlation in this study, which was consistent with local study results among adolescents [21]. Pregnancy and breastfeeding experience was associated with a low frequency of dysmenorrhea. Our findings matched with other reports that showed a significantly lower incidence of dysmenorrhea in women who had a delivery history [7,8]. Our results also disagreed with another report that found miscarriages did not decrease the frequency of dysmenorrhea [8]. No report has investigated the correlation between breastfeeding and dysmenorrhea. Therefore, to uncover the existence of an association among obstetrics history, menstrual history and dysmenorrhea frequency, further research is required for miscarriage experience, delivery method, breastfeeding period, etc.

rrhea [8]. No report has investigated the correlation between breastfeeding and dysmenorrhea. Therefore, to uncover the existence of an association among obstetrics history, menstrual history and dysmenorrhea frequency, further research is required for miscarriage experience, delivery method, breastfeeding period, etc. Socioeconomic factors, resident region and dysmenorrhea frequency were associated, but there was no correlation with the average monthly gross income. Place of residence also affected the frequency of dysmenorrhea; 68.3% of domestic marriage immigrants reported dysmenorrhea, while 65.2% of southern Vietnam residents and 43.3% of northern Vietnamese residents complained of the same symptoms. Similarly, our regression analysis result showed a lower risk of dysmenorrhea in northern and southern residents (0.411 and 0.459, respectively) compared to the Vietnamese domestic marriage immigrants; Vietnamese northern residents were at the lowest risk. The average monthly gross income was highest in Korea, and residents of the southern Vietnam region earned more income than those in the northern region. Therefore, these results could imply that socioeconomic status in each residence as well as resident region were correlated. However, the incidence of dysmenorrhea was not correlated with average monthly gross income (P=0.298), which agreed with the existing report [6] that showed no correlation between the incidence of dysmenorrhea and income but disagreed with the report [18] that indicated women with higher monthly gross incomes had a higher dysmenorrhea incidence than those making a lower monthly gross income. The association between dysmenorrhea and economic factors will need further research.

correlation between the incidence of dysmenorrhea and income but disagreed with the report [18] that indicated women with higher monthly gross incomes had a higher dysmenorrhea incidence than those making a lower monthly gross income. The association between dysmenorrhea and economic factors will need further research. The limitations of this study were that we did not distinguish between primary dysmenorrhea and secondary dysmenorrhea, and mental types of socioeconomic factors, such as depression, stress, social isolation, etc., were also not considered. In addition, as the problem of co-linearity among various socioeconomic factors could not be ruled out, independent factors affecting the incidence of dysmenorrhea should be revealed through further analysis. Since the participants filled out the questionnaires independently, the possibility of recall bias and difficulty making an objective dysmenorrhea assessment cannot be ruled out completely. In further research on dysmenorrhea in different races, the dysmenorrhea prevalence could be surveyed with Korean women living in the same area compared to this study's Vietnamese marriage immigrant women. In conclusion, this study showed that the incidence of dysmenorrhea was significantly correlated with age, menstrual history and obstetric history as well as socioeconomic factors, such as resident region, education and religion. No potential conflict of interest relevant to this article was reported. Table 1 Baseline characteristics of women in the study group Values are presented as mean±standard deviation or %.

In conclusion, this study showed that the incidence of dysmenorrhea was significantly correlated with age, menstrual history and obstetric history as well as socioeconomic factors, such as resident region, education and religion. No potential conflict of interest relevant to this article was reported. Table 1 Baseline characteristics of women in the study group Values are presented as mean±standard deviation or %. Table 2 Clinical characteristics of women stratified by presence of dysmenorrhea Values are presented as mean±standard deviation. NS, non-significant. Table 3 Factors related with dysmenorrhea in Vietnamese women Values are presented as n (%). Table 4 Logistic regression analysis of significant variables related to dysmenorrhea The menarche age and educational status was also taken into account other than age, menstrual volume, pregnancy experience, and place of residence.

Introduction Polycystic ovary syndrome (PCOS) is one of the most common causes of endocrine dysfunction in women of reproductive age with a prevalence that ranges from 4% to 7% [1,2]. Metabolic disturbances such as visceral obesity, hypertension, dyslipidemia, insulin resistance, and glucose intolerance are well-recognized clinical features of this syndrome. These factors, which are cluster in patients with PCOS, are also closely related to atherosclerosis. Carotid intima-media thickness (CIMT) has been widely used as a surrogate marker of atherosclerosis and cardiovascular disease (CVD) events [3-8]. The association between PCOS and CIMT has been investigated in many studies [9-13], but there has been only one report in the Korean population: in 24 women with PCOS and 16 matched controls, mean CIMT was significantly higher in PCOS group than controls (0.57 ± 0.12 mm vs. 0.49 ± 0.11 mm, respectively, P = 0.004) [14]. The aim of the present study was to compare the presence of premature atherosclerosis in young untreated Korean women with PCOS and age matched controls, specifically by measuring their CIMT.

ean CIMT was significantly higher in PCOS group than controls (0.57 ± 0.12 mm vs. 0.49 ± 0.11 mm, respectively, P = 0.004) [14]. The aim of the present study was to compare the presence of premature atherosclerosis in young untreated Korean women with PCOS and age matched controls, specifically by measuring their CIMT. Materials and methods 1. Subjects Fifty-six women with PCOS (range, 18 to 40 years) were recruited using the Rotterdam criteria [15]. Clinical hyperandrogenism (HA) was defined as a modified Ferriman and Gallwey score (mF-G score) of 6 or greater and biochemical HA was defined as follows: total testosterone>0.68 ng/mL, free testosterone >1.72 pg/mL, and free androgen index (FAI) >5.36 [16,17]. To determine the distribution of the different PCOS phenotypes, patients with PCOS were divided into two subgroups according to the presence of HA. All women with PCOS were screened to exclude hyperprolactinemia and thyroid dysfunction. Serum 17-hydroxyprogesterone (OHP) was also measured, and if the serum 17-OHP level was over 2 ng/mL, a repeat test was performed during the early morning follicular phase. The patients who showed continuous elevation of 17-OHP were excluded from the study group.

d to exclude hyperprolactinemia and thyroid dysfunction. Serum 17-hydroxyprogesterone (OHP) was also measured, and if the serum 17-OHP level was over 2 ng/mL, a repeat test was performed during the early morning follicular phase. The patients who showed continuous elevation of 17-OHP were excluded from the study group. A total of 56 age-matched (±1 year) premenopausal women served as controls, and the match ratio was 1 to 1. Control women visited Seoul National University Hospital as part of a group check-up for work and lacked specific health problems. All controls had regular (21 to 35 day) menstrual cycles, a mF-G score <6, and all received a transvaginal or transrectal pelvic ultrasound examination to evaluate ovarian morphology and were excluded if PCOS morphology was identified. None of the patients with PCOS and controls had taken combined oral contraceptives, lipid-lowering agents or insulin sensitizer. The Institute Review Board (IRB) for human research of Seoul National University Hospital approved this project (IRB number: H-0807-031-250) and written informed consent was obtained from each woman.

Pure Chemical Industries Ltd., Japan). Fasting insulin levels were measured using RIA (BioSource Europe S.A., Nivelles, Belgium). The homeostatic model for insulin resistance was calculated by glucose (mg/dL) × insulin (µU/mL)/405, and HOMAβcell (%) was calculated as follows: (20×fasting insulin)/(fasting glucose-3.5). Although transducer frequency is best between 8 to 12 MHz [4], CIMT measurement was conducted using a high-resolution 7.5-MHz phased-array transducer (Vivid 7 Cardiovascular Ultrasound, GE Healthcare, Milwaukee, WI, USA) by one radiologist (K.J.H.) who was blinded to the patients' clinical profiles. Depth and gain were optimized to reduce noise, and to get best image, the operator manipulated transducer for ultrasound beam is perpendicular to the intima-media structure. Both common carotid arteries were explored in B-mode and intra-assay variation was <10%. The posterior carotid wall at 1 cm of the common carotid bulb was imaged and CIMT was estimated by visual assessment of the distance between the lumen/intima and intima/adventicia interphases in longitudinal frame. Each left and right carotid artery IMT was calculated as the average of three recordings, and the mean CIMT, which was calculated from the bilateral CIMT values, was used as the outcome variable.

mated by visual assessment of the distance between the lumen/intima and intima/adventicia interphases in longitudinal frame. Each left and right carotid artery IMT was calculated as the average of three recordings, and the mean CIMT, which was calculated from the bilateral CIMT values, was used as the outcome variable. 3. Statistical analysis Deviation of the data from a normal distribution was examined through visual inspection of quantile-normal plots and/or the Shapiro-Wilk test of normality. The data are shown as the mean ± standard deviation or median value with the range. If Gaussian distribution was achieved by natural logarithmic or square root transformation, the data are shown as geometric means and 95% confidence intervals (95% CI). Continuous parameters were compared using Student's t or Mann-Whitney U test. Univariate regression analyses were conducted with CIMT as a dependent variable and traditional CVD risk factors and serum androgens as independent variables. All data analyses were performed using the Statistical Package for the Social Sciences software ver. 19.0 (IBM SPSS, Somers, NY, USA), and statistical significance was set at two-sided P-values <0.05. Power calculations were performed using the G-power ver. 3.1.5 software (http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3). Given the specified sample size (56 PCOS patients and 56 controls), the power to detect a mean CIMT difference 0.5 mm (an α value of 0.05) was 0.75.

3. Statistical analysis Deviation of the data from a normal distribution was examined through visual inspection of quantile-normal plots and/or the Shapiro-Wilk test of normality. The data are shown as the mean ± standard deviation or median value with the range. If Gaussian distribution was achieved by natural logarithmic or square root transformation, the data are shown as geometric means and 95% confidence intervals (95% CI). Continuous parameters were compared using Student's t or Mann-Whitney U test. Univariate regression analyses were conducted with CIMT as a dependent variable and traditional CVD risk factors and serum androgens as independent variables. All data analyses were performed using the Statistical Package for the Social Sciences software ver. 19.0 (IBM SPSS, Somers, NY, USA), and statistical significance was set at two-sided P-values <0.05. Power calculations were performed using the G-power ver. 3.1.5 software (http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3). Given the specified sample size (56 PCOS patients and 56 controls), the power to detect a mean CIMT difference 0.5 mm (an α value of 0.05) was 0.75. Results Clinical and biochemical characteristics of the subjects are shown in Table 1. Women with PCOS and controls were same in age (30.9 vs. 30.8, P = 0.904). Although mean BMI of the PCOS patients was significantly higher than that of the controls (21.2 kg/m2 vs. 19.8 kg/m2, respectively, P = 0.004), most (87.5%) of the women with PCOS in the present study were not obese. The PCOS group had significantly higher blood pressure, HOMA-IR as well as lower SHBG levels than the controls, but there were no significant differences in serum androgen, lipid profiles, fasting glucose and A1C levels between the two groups.

4), most (87.5%) of the women with PCOS in the present study were not obese. The PCOS group had significantly higher blood pressure, HOMA-IR as well as lower SHBG levels than the controls, but there were no significant differences in serum androgen, lipid profiles, fasting glucose and A1C levels between the two groups. The CIMT ranged from 0.30 to 0.70 mm in women with PCOS and from 0.24 to 0.82 mm in controls. Despite the significant differences in some vascular risk factors between women with PCOS and controls, the mean CIMT was not different between the two groups (0.49 ± 0.09 mm in PCOS patients vs. 0.50 ± 0.11 mm in controls, respectively, P = 0.562). When the CIMT in the control group was compared with hyperandrogenic and non-hyperandrogenic PCOS groups, there were also no significant differences between the groups (Table 2). The univariate linear correlations between CIMT and common atherogenic factors or androgenic parameters are presented in Table 3. CIMT was not correlated with any of the above parameters in all subjects as well as the subset of PCOS patients. Discussion We have presented a case-control study of the CIMT, a marker of subclinical atherosclerosis, in women with PCOS and age-matched controls. Although young Korean PCOS patients were more obese and had higher blood pressure and insulin resistance index than the age-matched controls, carotid artery intima-media thickness measurements as a pre-indicator of CVD was not found to be different between the patients and controls.

with PCOS and age-matched controls. Although young Korean PCOS patients were more obese and had higher blood pressure and insulin resistance index than the age-matched controls, carotid artery intima-media thickness measurements as a pre-indicator of CVD was not found to be different between the patients and controls. Numerous studies have investigated whether CIMT is elevated in women with PCOS or not [9-13]. However, these studies have not shown consistent results, and it may stem from the quality of the CIMT measurement, small sample sizes and young age of the subjects. Recently, systematic review and meta-analysis has been reported [18]. Eight studies were included in the systematic review and 19 studies were included in the meta-analysis (total n = 1,123 women with PCOS, n = 923 controls). In this analysis, the mean difference in CIMT among women with PCOS compared with controls was 0.072 mm (95% CI, 0.040-0.105; P < 0.0001) for highest quality studies, and 0.084 mm (95% CI, 0.042-0.126; P < 0.0001) for good quality studies, and the authors concluded that women with PCOS demonstrated higher CIMT than controls. However, in our current study, there were no significant differences in CIMT between the PCOS group and controls. Additionally, there were also no significant associations between CIMT and serum androgen and metabolic markers.

udies, and the authors concluded that women with PCOS demonstrated higher CIMT than controls. However, in our current study, there were no significant differences in CIMT between the PCOS group and controls. Additionally, there were also no significant associations between CIMT and serum androgen and metabolic markers. One outstanding issue is the role of androgens in cardiovascular risk, and in the current study, PCOS cases with HA did not show higher CIMT than the controls. Although there have been studies reporting similar occurrence of metabolic risk across PCOS subgroups [19,20], patients with no evidence of HA may have milder metabolic profile compared with the other phenotypes [21,22]. In current study, mean serum androgen levels of the whole PCOS patients were not different compared to those of the controls, and almost half (41.2%) of the PCOS subjects had non-hyperandrogenic phenotype. Thus, no difference in CMIT between PCOS patients and controls may stem from the presence of this mild phenotype.

In current study, mean serum androgen levels of the whole PCOS patients were not different compared to those of the controls, and almost half (41.2%) of the PCOS subjects had non-hyperandrogenic phenotype. Thus, no difference in CMIT between PCOS patients and controls may stem from the presence of this mild phenotype. Although our study could not find any difference in CIMT between PCOS patients and controls, the present study has some limitations that need to be discussed. First, mean age of the patients was 30.9 (±4.6) years old, and mean BMI was 21.2 (±2.8) kg/m2, suggesting that the subjects mainly consisted of young and non-obese women. The prevalence of atherogenic CIMT pattern increases with age and BMI [3,4], thus the absence of CIMT difference between PCOS patients and controls may be, in part, due to the relatively young age of our subjects. However, previous studies, which found that the PCOS patients presented with an increased CIMT, were also performed using patients under 35 years of age [11,12,23], suggesting that the premature atherosclerosis may be found even in these young PCOS patients. Second, as mentioned above, no difference in CIMT between patients and controls also might stem from non-obese state of the subjects. Although mean BMI of the PCOS patients was significantly higher than that of the controls (21.2 ± 2.8 kg/m2 vs. 19.8 ± 2.1 kg/m2, respectively, P = 0.004), women with PCOS in the present study were definitely thin. Thus, the degree of metabolic dysfunction in this study was modest such that serum insulin, lipid, hs-CRP levels of the patients were not different compared to the controls. This would be the most important reason why there were no differences in CIMT. Data on obese subjects could offer complementary findings about the possible relationship between the magnitude of obesity and CIMT. Third, given the specified sample size (56 PCOS patients and 56 controls), the power to detect a mean CIMT difference 0.5 mm (an α value of 0.05) was 0.75. Since this study has limited power for the small CIMT difference, no difference between PCOS patients and controls could be due to insufficient power. Finally, we cannot exclude the possibility of other potentially confounding factors, such as differences in diet and/or exercise patterns.

(an α value of 0.05) was 0.75. Since this study has limited power for the small CIMT difference, no difference between PCOS patients and controls could be due to insufficient power. Finally, we cannot exclude the possibility of other potentially confounding factors, such as differences in diet and/or exercise patterns. In conclusion, despite the significant differences in some vascular risk factors between women with PCOS and controls, PCOS patients did not have a significantly higher CIMT (even in the hyperandrogenic subgroups). Although our study did not show the increased risk of subclinical atherosclerosis in PCOS patients, the role of CIMT continues to be investigated considering the importance of screening and monitoring CVD risk factors in women with PCOS. No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by Supported by grant no. 04-2008-0900 from the Seoul National University Hospital Research Fund. Table 1 Clinical features of the patients with PCOS and matched controls

In conclusion, despite the significant differences in some vascular risk factors between women with PCOS and controls, PCOS patients did not have a significantly higher CIMT (even in the hyperandrogenic subgroups). Although our study did not show the increased risk of subclinical atherosclerosis in PCOS patients, the role of CIMT continues to be investigated considering the importance of screening and monitoring CVD risk factors in women with PCOS. No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by Supported by grant no. 04-2008-0900 from the Seoul National University Hospital Research Fund. Table 1 Clinical features of the patients with PCOS and matched controls PCOS, polycystic ovary syndrome; BMI, body mass index; WC, waist circumference; T, testosterone; SHBG, sex-hormone binding globulin; FAI, free androgen index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostatic model assessment for insulin resistance; HOMA-BC, homeostatic model assessment for beta cell capacity; hs-CRP, high-sensitivity C-reactive protein; A1C, hemoglobin A1c; C, cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; CIMT, carotid intima media thickness. a)P-values were analysed by Student's t test or Mann-Whitney U test except the prevalence of obesity (Fisher's exact test); b)Data are shown as means ± standard deviations; c)Medians (ranges); d)Geometric means with 95% confidence intervals.

-C, low-density lipoprotein cholesterol; CIMT, carotid intima media thickness. a)P-values were analysed by Student's t test or Mann-Whitney U test except the prevalence of obesity (Fisher's exact test); b)Data are shown as means ± standard deviations; c)Medians (ranges); d)Geometric means with 95% confidence intervals. Table 2 Comparison of CIMT between the control group and hyperandrogenic and non-hyperandrogenic PCOS patients CIMT, carotid intima media thickness; PCOS, polycystic ovary syndrome. a)P-values were analysed by Student's t test or Mann-Whitney U test. Table 3 Univariate linear regression analysis with CIMT as the dependent variable CIMT, carotid intima media thickness; PCOS, polycystic ovary syndrome; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HOMA-IR, homeostatic model assessment for insulin resistance; A1C, hemoglobin A1C; FAI, free androgen index.

Introduction Postoperative physical inactivity is a risk factor for various adverse outcomes. For example, a retrospective study reported that postoperative prolonged bed rest is a risk factor for pulmonary embolism following cardiac surgery [1]. A systematic review showed that immobilization defined as confinement to bed and/or armchair was associated with 6 fold increase in deep vein thrombosis [2]. A randomized trial including patients who underwent a coronary artery bypass surgery revealed that systematic turning activity in bed decreased the risk of postoperative fever and length of stay (LOS) in intensive care unit [3]. A retrospective study found that delayed ambulation after hip fracture surgery was related to delirium, pneumonia and increased LOS [4]. LOS is one of the most important metrics for cost-effective care [5]. In addition, adequate LOS was associated with quality of care in several settings. For example, a nationwide population-based cohort study showed that higher volume hospitals significantly reduced LOS and in-hospital mortality for elderly patients with acute biliary diseases [6]. Another study reported that patients admitted to high volume stroke units received a higher quality care and spent fewer days in the hospital [7].

e population-based cohort study showed that higher volume hospitals significantly reduced LOS and in-hospital mortality for elderly patients with acute biliary diseases [6]. Another study reported that patients admitted to high volume stroke units received a higher quality care and spent fewer days in the hospital [7]. The association of physical activity during hospitalization with LOS was mainly investigated in elderly populations. For example, an observational cohort study examined the total number of steps in elderly patients hospitalized with acute medical illness and reported that patients with shorter LOS tended to have higher physical activity [8]. A possible association of physical activity with LOS following surgery can be deduced from trials testing the benefit of fast-track approach. Specifically, the fast-track approach including early ambulation after surgery was known to reduce LOS in various settings [9-11]. However, in trials testing the fast-track approach, the physical activity of patients was not measured. Therefore, to our knowledge, the association of physical activity during hospitalization with LOS was not thoroughly investigated in patients following surgery. The objective of this study was to examine the association of postoperative physical activity measured by nursing-documented ambulation with LOS following an elective total laparoscopic hysterectomy for benign gynecologic diseases.

The association of physical activity during hospitalization with LOS was mainly investigated in elderly populations. For example, an observational cohort study examined the total number of steps in elderly patients hospitalized with acute medical illness and reported that patients with shorter LOS tended to have higher physical activity [8]. A possible association of physical activity with LOS following surgery can be deduced from trials testing the benefit of fast-track approach. Specifically, the fast-track approach including early ambulation after surgery was known to reduce LOS in various settings [9-11]. However, in trials testing the fast-track approach, the physical activity of patients was not measured. Therefore, to our knowledge, the association of physical activity during hospitalization with LOS was not thoroughly investigated in patients following surgery. The objective of this study was to examine the association of postoperative physical activity measured by nursing-documented ambulation with LOS following an elective total laparoscopic hysterectomy for benign gynecologic diseases. Materials and methods 1. Population From 2009 to 2012, 745 patients with benign gynecologic diseases entered a critical pathway for elective total laparoscopic hysterectomy in our institute. All patients received same perioperative management according to a prespecified protocol. Patients were admitted the day before operation day and received a bowel preparation using bisacodyl rectally and laxatives orally and rectally. On operation day, prophylactic antibiotics were intravenously given 30 minutes before skin incision and no postoperative antibiotics were administered. On postoperative first day, diet was tried and ambulation was encouraged. On postoperative second day, patients were discharged. Postoperative pain was managed with ketorolac tromethamine intravenously administered via patient-controlled analgesia device.

kin incision and no postoperative antibiotics were administered. On postoperative first day, diet was tried and ambulation was encouraged. On postoperative second day, patients were discharged. Postoperative pain was managed with ketorolac tromethamine intravenously administered via patient-controlled analgesia device. After the approval of Institutional Review Board, the characteristics of 745 patients were abstracted from medical records. One hundred twenty-five patients with intraoperative severe complications, concurrent surgeries or unexpected cancer diagnosis were excluded. For the remaining 620 patients, age, body mass index, endometriosis, systemic disease, previous abdominal surgery and current medication were abstracted from medical records. Seventy patients who were discharged on postoperative third day or later comprised the case group and the other 550 patients were the initial control group. The Institutional Review Board reviewed this protocol in a brief review track and exempted this study from further review. 2. Matching Propensity score calculation and matching were performed using Matchlt package in R ver. 2.15.0 (R Foundation for Statistical Computing, http://www.R-project.org) [12]. Year, age, body mass index, endometriosis, systemic disease, previous abdominal surgery and current medication were used to calculate the propensity score. The ratio for case and control was 1:3 and the nearest matching was performed. Of 550 patients in initial control group, 210 patients were matched and composed a matched control group.

index, endometriosis, systemic disease, previous abdominal surgery and current medication were used to calculate the propensity score. The ratio for case and control was 1:3 and the nearest matching was performed. Of 550 patients in initial control group, 210 patients were matched and composed a matched control group. 3. Measurement of physical activity In our institution, nurses described the status of patients using the predetermined sentences in electronic medical record. There were nine sentences describing the ambulation event of patients. For example, the sentence of "The patient is ambulating" is used to describe the ambulation event of patients. We examined whether the sentences describing the ambulation event was present in nursing record of each patient from operation to 9 AM of postoperative second day. When the sentences describing the ambulation event was present one or more times, the patient was determined to have a nursing-documented ambulation.

ients. We examined whether the sentences describing the ambulation event was present in nursing record of each patient from operation to 9 AM of postoperative second day. When the sentences describing the ambulation event was present one or more times, the patient was determined to have a nursing-documented ambulation. 4. Analysis Characteristics of the case group were compared with those of the initial and matched control group. Chi-square or Fisher's exact test was used for categorical variables. Student's t-test was used for continuous variables. If there is a significant difference in any variable between case and matched control groups, ratio for matching was planned to be reduced. When the difference was not corrected despite reduction of ratio, conditional logistic regression analysis including the variable with difference was planned. Propensity score calculation and matching were performed using Matchlt package in R ver. 2.15.0 and the other analyses were performed using SPSS ver. 20.0.0 (SPSS Inc., IBM Co., Somers, NY, USA). Null hypotheses of no difference were rejected if P-values were less than 0.05.

ng the variable with difference was planned. Propensity score calculation and matching were performed using Matchlt package in R ver. 2.15.0 and the other analyses were performed using SPSS ver. 20.0.0 (SPSS Inc., IBM Co., Somers, NY, USA). Null hypotheses of no difference were rejected if P-values were less than 0.05. Results 1. Baseline characteristics The characteristics of case, matched control, and initial control groups are summarized in Table 1. Over 70% of patients received an operation in 2010 and 2011. The mean age of the cases was 48 years, of the matched controls 48 and the initial control group 47. The mean body mass index was 24 kg/m2 in all groups. The prevalence of endometriosis, systemic disease, previous abdominal surgery and current medication was not different among groups, and all variables were well-balanced between case and matched control groups. 2. Physical activity The number of patients with nursing-documented ambulation in case group (19%) was not different from that in matched-control group (11%) (Table 2).

Results 1. Baseline characteristics The characteristics of case, matched control, and initial control groups are summarized in Table 1. Over 70% of patients received an operation in 2010 and 2011. The mean age of the cases was 48 years, of the matched controls 48 and the initial control group 47. The mean body mass index was 24 kg/m2 in all groups. The prevalence of endometriosis, systemic disease, previous abdominal surgery and current medication was not different among groups, and all variables were well-balanced between case and matched control groups. 2. Physical activity The number of patients with nursing-documented ambulation in case group (19%) was not different from that in matched-control group (11%) (Table 2). Discussion 1. Our finding In this study, nursing-documented ambulation was not associated with LOS. Several explanations are possible. First, patients who underwent total laparoscopic hysterectomy for benign gynecologic diseases might not be an ideal group to test the hypothesis of 'physical activity of patients with longer LOS would be less than that of patients with shorter LOS.' In other words, most patients who underwent total laparoscopic hysterectomy would not experience significant decline of function resulting in reduced physical activity and longer LOS. Second, nursing-documented ambulation might not be a reliable method to measure physical activity. Nursing-documented ambulation could be affected by various factors such as walking time, walking area and nurse's attitude to document the ambulation. Recently, several studies used more accurate methods such as accelerometer or pedometer to measure the physical activity [13,14].

le method to measure physical activity. Nursing-documented ambulation could be affected by various factors such as walking time, walking area and nurse's attitude to document the ambulation. Recently, several studies used more accurate methods such as accelerometer or pedometer to measure the physical activity [13,14]. 2. Low level of physical activity during hospitalization In this study, only 36 of 280 patients (13%) did a nursing-documented ambulation. The low level of physical activity during hospitalization was reported in previous studies. For example, in a study examining the frequency of hallway ambulation in hospitalized patients aged ≥55, only 318 of 19,363 (2%) patient-minutes were used in hallway walking [15]. Another study measuring the number of steps in hospitalized older adults reported that the average number of steps per day was only one tenth of community-living older adults [8]. 3. Electronic nursing record using predetermined sentences We can measure the physical activity of hospitalized patients by counting the number of predetermined sentences describing the ambulation event. We think that electronic nursing record using predetermined sentences could be a powerful tool in evaluating the status of patients in a group. For example, we can measure the quality of postoperative pain control by counting the number of sentences describing the pain experience. Hypothetically, the increased number could trigger us to improve the quality of postoperative pain control.

could be a powerful tool in evaluating the status of patients in a group. For example, we can measure the quality of postoperative pain control by counting the number of sentences describing the pain experience. Hypothetically, the increased number could trigger us to improve the quality of postoperative pain control. 4. Future studies Although we did not find any association of physical activity with LOS in this study, we obtained valuable lessons for future studies. First, to detect the effect of physical activity to LOS, the study population should be a group with diseases causing significant decline of function. Second, physical activity should be measured prospectively through reliable methods such as accelerometer or pedometer. In spite of the negative result of this study, we still believe that physical activity is an essential metric to evaluate the function and health status of patients. Therefore, we hold that accurate measurement of physical activity can help predict the prognosis of patients in various settings. Furthermore, we believe that patient prognosis could be improved by increasing the physical activity of patients. Further studies investigating the association of physical activity with function and health status are warranted. No potential conflict of interest relevant to this article was reported. Acknowledgments The authors thank the Medical Research Collaborating Center at Seoul National University Bundang Hospital for statistical analyses. Table 1 The characteristics of case, matched control, and initial control groups

In spite of the negative result of this study, we still believe that physical activity is an essential metric to evaluate the function and health status of patients. Therefore, we hold that accurate measurement of physical activity can help predict the prognosis of patients in various settings. Furthermore, we believe that patient prognosis could be improved by increasing the physical activity of patients. Further studies investigating the association of physical activity with function and health status are warranted. No potential conflict of interest relevant to this article was reported. Acknowledgments The authors thank the Medical Research Collaborating Center at Seoul National University Bundang Hospital for statistical analyses. Table 1 The characteristics of case, matched control, and initial control groups Values are presented as number (%) or mean ± standard deviation. Table 2 Association of nursing-documented ambulation with delay of discharge Values are presented as or number (%). P>0.05.

Introduction Postpartum visual disturbance is often associated with pre-eclampsia or eclampsia, and these symptoms are generally transient [1,2]. Blindness in pre-eclampsia is very rare and may develop from brain damage or retinal lesions [3]. Blindness from retinal lesions is known as Purtscher-like retinopathy, and is caused by either retinal ischemia or infarction [3]. It has been estimated to develop in 0.24 cases per million in the UK [4]. Apart from the treatment of the precipitating conditions, there are no specific treatment guidelines at present [4,5]. Here, we report a rare case of Purtscher-like retinopathy after pre-eclampsia combined with acute pancreatitis, which resulted in persistent visual disturbance. Case Report A 33-year-old primigravida was transferred to the Kyung Hee University Medical Center for preterm labor and pre-eclampsia at 36+4 weeks gestation. She had a twin pregnancy after in vitro fertilization-embryo transfer. Her blood pressure measured 140/100 mm Hg. Her urine protein was 4+ and 24-hour urinary protein excretion was 2,333 mg/day. She did not have headache, visual disturbances, or pitting edema. She had regular uterine contractions and a cervix dilated about 2 cm. An emergency cesarean section was performed uneventfully, and healthy twin babies were born.

100 mm Hg. Her urine protein was 4+ and 24-hour urinary protein excretion was 2,333 mg/day. She did not have headache, visual disturbances, or pitting edema. She had regular uterine contractions and a cervix dilated about 2 cm. An emergency cesarean section was performed uneventfully, and healthy twin babies were born. On the second postoperative day, the patient had abdominal distension, steady epigastric and whole abdominal pain. An abdominal X-ray examination showed paralytic ileus. Laboratory examination showed white blood cell (WBC) count of 11,890, low Ca+ of 6.4 mg/dL, elevated blood urea nitrogen (BUN) of 64 mg/dL, serum creatinine of 1.5 mg/dL, aspartate transaminase (AST) of 52 IU/L, alanine aminotransferase (ALT) of 21 IU/L, elevated C-reactive protein (CRP) of 20.74 mg/dL, triglyceride 1,080 mg/dL and otherwise non-specific. At that time, she started complaining of blurry vision. Because pre-eclampsia-related visual disturbances are common, we did not perform a formal ophthalmic examination. We decided on conservative management including nasogastric drainage, parenteral nutrition, non per os, pain control, and avoidance of toxic renal medications. On the fifth postoperative day, the patient had flank pain, diarrhea, tachycardia (pulse 112 per minutes) and fever (38℃). Laboratory examination showed WBC count of 14,900, low Ca+ of 5.6 mg/dL, AST of 61 IU/L, ALT of 33 IU/L, elevated CRP of 8.56 mg/dL, and normalized BUN and serum creatinine. Bacterial cultures were taken from blood and urine, and antibiotics were started. Her visual loss was equivocal.

112 per minutes) and fever (38℃). Laboratory examination showed WBC count of 14,900, low Ca+ of 5.6 mg/dL, AST of 61 IU/L, ALT of 33 IU/L, elevated CRP of 8.56 mg/dL, and normalized BUN and serum creatinine. Bacterial cultures were taken from blood and urine, and antibiotics were started. Her visual loss was equivocal. The patient still had epigastric pain, a fever (38.3℃) and visual loss in both eyes until eight days after operation. At this point, we therefore decided to do an abdominal computed tomography (CT) scan and an ophthalmology consultation. The abdominal CT scan showed enlargement of pancreas with fat infiltration, fluid collection in the pancreatic area and no other abnormal findings. Serum amylase and lipase levels were elevated at 149 U/L (normal range, 25 to 125 U/L), and 128 U/L (normal range, 5.5 to 58 U/L) respectively, on the day of CT scan. The patient was diagnosed as having acute pancreatitis based on her steady epigastric pain and abdominal CT scan. She recovered fully upon resolution of pancreatic necrosis with conservative management and drainage of the underlying fluid collection.

/L (normal range, 5.5 to 58 U/L) respectively, on the day of CT scan. The patient was diagnosed as having acute pancreatitis based on her steady epigastric pain and abdominal CT scan. She recovered fully upon resolution of pancreatic necrosis with conservative management and drainage of the underlying fluid collection. On the same day, the patient underwent an ophthalmologic examination, and her visual acuity test was 0.02 (right) and finger count 30 cm (left). There were multiple discrete retinal changes, and macular yellow patches, known as Purtscher flecken, in both eyes. A retinal splinter hemorrhage was seen in the right eye upon retinal photography (Fig. 1). A fluorescein angiogram showed arteriolar occlusion, and retinal ischemia, typical signs of Purtscher-like retinopathy. She was diagnosed with Purtscher-like retinopathy based on her symptoms and the results of the ophthalmologic examinations. Because there are no definite guidelines for the treatment of this condition, her visual acuity was closely monitored with conservative management. We also performed a brain magnetic resonance imaging (MRI) to rule out cortical blindness. The brain MRI showed unremarkable findings (Fig. 2). Two months after onset of visual loss, her visual acuity test was 0.08 (right) and 0.01 (left) and the multiple yellowish patches had decreased in both eyes on retinal photographs. At a recent follow-up (two years after the onset of her visual symptoms), her corrected visual acuity was 0.2 (right) and 0.1 (left). There was gradual improvement in the visual functions of both eyes, but she still had visual impairment.

the multiple yellowish patches had decreased in both eyes on retinal photographs. At a recent follow-up (two years after the onset of her visual symptoms), her corrected visual acuity was 0.2 (right) and 0.1 (left). There was gradual improvement in the visual functions of both eyes, but she still had visual impairment. Discussion Pre-eclampsia affects microvascular circulation and may involve cerebral, placental, hepatic, renal and ocular function [1]. Visual symptoms have been reported to occur in approximately 25% of patients with pre-eclampsia and 19% to 45% of patients with eclampsia, and include blurry vision, diplopia, amaurosis fugax, and photopsia [1,3]. Fortunately, pre-eclampsia-related visual acuity loss is usually reversible within weeks to months after the onset of symptoms [1-3]. In this case, because visual symptoms are relatively common with pre-eclampsia, serious ophthalmic diseases were not suspected when the patient complained visual problems at the first time. However, her visual symptoms had persisted until the eighth postoperative day, so we decided to perform a formal ophthalmologic examination. Purtscher-like retinopathy was confirmed according to the results of ophthalmologic examinations.

were not suspected when the patient complained visual problems at the first time. However, her visual symptoms had persisted until the eighth postoperative day, so we decided to perform a formal ophthalmologic examination. Purtscher-like retinopathy was confirmed according to the results of ophthalmologic examinations. According to the recent study, the incidence of Purtscher's retinopathy estimate 0.24 case per million population per year based on the UK population of 58.8 million [4]. Purtscher's retinopathy is a rare condition, usually associated with severe head injury and blunt thoracic trauma, but also seen in patients with acute pancreatitis, fat embolism, renal failure, pre-eclampsia, childbirth and connective tissue disorders with vasculitis [4-8]. Especially Purtscher-like retinopathy is used to describe the retinopathy seen in conditions other than trauma [5]. Various systemic conditions mentioned above are attributed to the occlusion of retinal arterioles by aggregated granulocytes or platelets following complement activation, fat microemboli, or fibrin clots [4-6]. Most cases resolve spontaneously 1 to 3 months after systemic disease resolution [4-6]. Visual prognosis is variable, and is poor if optic disc swelling or leakage, choroidal hypoperfusion and involvement of the outer retina are seen upon intravenous fluorescein angiography [4,5]. At present no definite guidelines exist for the treatment of Purtscher-like retinopathy [4,5]. Although there are some case reports of successful treatment using high-dose steroids in IV form, the major treatment modality is expectancy [4,5]. The patient in this case had a gradual recovery of visual functions without treatments. However, she still has acuity impairments, and ophthalmologic follow up is ongoing.

Although there are some case reports of successful treatment using high-dose steroids in IV form, the major treatment modality is expectancy [4,5]. The patient in this case had a gradual recovery of visual functions without treatments. However, she still has acuity impairments, and ophthalmologic follow up is ongoing. Acute pancreatitis during pregnancy and postpartum is uncommon with an incidence of 1 case per 1,000 to 3,000 pregnancies [9]. Nearly 70% of cases of acute pancreatitis during pregnancy are secondary to biliary stones or sludge, followed by hyperlipidemia or alcohol abuse in approximately 20% of cases [9]. Pregnancy increases serum cholesterol and triglyceride levels, bile stasis, and may induce gallstone formation [9,10]. However, hyperlipidemia may directly induce acute pancreatitis [9,10]. The symptoms include the classic colicky or stabbing pain in the right upper quadrant or epigastric area, which can radiate to the right flank, scapula, and shoulder [10]. Diagnostic criteria for acute pancreatitis are presentation with at least two of the following three manifestations: 1) the typical symptoms mentioned above; 2) elevated levels of pancreatic enzyme (over three times of normal limit) in the blood, urine, or ascitic fluid; and 3) abnormal imaging findings in the pancreas associated with acute pancreatitis [9]. The general treatment of mild acute pancreatitis in pregnancy is conservative and supportive, while severe acute pancreatitis requires hospitalization in the intensive care unit and endoscopic or surgical interventions [9-11]. This patient had typical epigastric pain which was different from that normally seen after cesarean section, and abdominal CT findings compatible with acute pancreatitis. Because acute pancreatitis was not suspected initially, serum amylase and lipase were tested on eight days after symptom onset therefore the level were low for diagnosis of acute pancreatitis. The prolonged postoperative course with ileus, vague upper abdominal discomfort with mild tenderness, and hypocalcemia were likely secondary to acute pancreatitis. The disease resolved with protracted drainage of the fluid collected around the pancreas, broad-spectrum antibiotics and nutritional support.

pancreatitis. The prolonged postoperative course with ileus, vague upper abdominal discomfort with mild tenderness, and hypocalcemia were likely secondary to acute pancreatitis. The disease resolved with protracted drainage of the fluid collected around the pancreas, broad-spectrum antibiotics and nutritional support. To our knowledge, this is the first case report of Purtscher-like retinopathy developed after pre-eclampsia combined with acute pancreatitis in Korea. It is very meaningful that this case let us know there are several conditions that cause permanent visual disturbances after childbirth related to pre-eclampsia. Purtscher-like retinopathy is rare in women with pre-eclampsia, but some cases with permanent visual loss have been reported [4,5]. Therefore, when patients complain of ongoing visual disturbance with pre-eclampsia, other ophthalmologic causes such as Purtscher retinopathy should be considered. No potential conflict of interest relevant to this article was reported. Fig. 1 The retinal photography shows characteristic multiple cotton-wool exudates, known as Purtscher flecken, in both eyes and a retinal splinter hemorrhage in the right eye. Fig. 2 Brain magnetic resonance imaging shows normal findings.

Introduction Prune-belly syndrome is a rare disease that occurs about 1 per 50,000 live births and more than 90% of the affected patients are male [1]. Early diagnosis and appropriate management before renal compromise is essential for better prognosis. There are few literatures concerning the diagnosis of prune belly syndrome in the first trimester [2-4]. We present a case of an early diagnosed prune belly syndrome and the usefulness of vesicocentesis instead of vesicoamniotic shunt. Case report A 36-year-old woman (gravida 1, para 0) at gestational age of 11+6 weeks was transferred to our hospital for further evaluation of the suspected fetal abdominal cyst. She had been diagnosed with diabetes mellitus 2 years before and had regular insulin injections. Otherwise, she had neither familial nor past medical history. Ultrasonography at our hospital confirmed enlarged fetal bladder with the size of 2.7×2.5 cm (Fig. 1) but the classic 'key hole sign' was absent. Other fetal biometric values were appropriate for the gestational age; crown rump length (CRL) 45 mm, neural tube (NT) 0.7 mm.

had neither familial nor past medical history. Ultrasonography at our hospital confirmed enlarged fetal bladder with the size of 2.7×2.5 cm (Fig. 1) but the classic 'key hole sign' was absent. Other fetal biometric values were appropriate for the gestational age; crown rump length (CRL) 45 mm, neural tube (NT) 0.7 mm. Chorionic villus sampling resulted in 46, XY karyotype. Based upon informed consent, vesicocentesis with 22-gauge spinal needle was performed under ultrasonographic guidance (Voluson E8, GE Medical Systems Kretztechnik GmbH & Co. OHG, Zipf, Austria). The aspirated fetal urine was analyzed. As fetal bladder was not enlarged again until 1 week after vesicocentesis, the procedure seemed to be effective. However, the pregnancy was interrupted because the patient and her husband changed their mind and strongly requested termination of pregnancy. Postmortem gross examination confirmed prenatal ultrasooographic diagnosis. Besides, undiagnosed anomalies were identified; bilateral cleft lip, cleft palate, bilateral club feet, and polydactyly (Fig. 2).

nterrupted because the patient and her husband changed their mind and strongly requested termination of pregnancy. Postmortem gross examination confirmed prenatal ultrasooographic diagnosis. Besides, undiagnosed anomalies were identified; bilateral cleft lip, cleft palate, bilateral club feet, and polydactyly (Fig. 2). Discussion Prune-belly syndrome, also called Eagle-Barrett syndrome is a rare congenital malformation. The clinical manifestations are [1] deficiency of abdominal wall muscles [2], cryptorchidism in male fetus [3] genitourinary malformations. The pathogenesis of prune belly syndrome is not yet fully understood. Some theories have been suggested; urinary tract obstruction, maldevelopment of the mesoderm, genetic deficit, and abnormalities of allantois [4]. Prune belly syndrome is also associated with pulmonary, cardiovascular, gastrointestinal and musculoskeletal abnormalities [5]. In our case, postmortem examination revealed cleft lip and palate, club feet and polydactyly. Although cryptorchidism was not so evident because of early geatational age, the key hole sign was not found. Therefore it would be diagnosed as prune belly syndrome with the exclusion of other urinary tract anomalies.

es [5]. In our case, postmortem examination revealed cleft lip and palate, club feet and polydactyly. Although cryptorchidism was not so evident because of early geatational age, the key hole sign was not found. Therefore it would be diagnosed as prune belly syndrome with the exclusion of other urinary tract anomalies. Prune belly syndrome is classified into 3 groups by antenatal and anatomical features [5,6]. Among them, category I is characterized by pulmonary hypoplasia, pneumothorax, oligohydramnios, renal dysplasia, urethral obstruction patent urachus or club feet. The prognosis is worst in the type I group. Although pulmonary hypoplasia was not clearly identified in the very early gestational age, the fetus with club feet would belong to the category I. Fetal urine production begins between 8 and 10 gestational weeks and fetal bladder can be identified by ultrasound at least from 11 weeks. Therefore the detection of prune belly syndrome by ultrasound in the very early gestational period is uncommonly reported [2-4]. It is noteworthy that, in our case, very large bladder was identified and the fetus was diagnosed as prune belly syndrome by prenatal abdominal ultrasound before 12 weeks of gestational age.

Therefore the detection of prune belly syndrome by ultrasound in the very early gestational period is uncommonly reported [2-4]. It is noteworthy that, in our case, very large bladder was identified and the fetus was diagnosed as prune belly syndrome by prenatal abdominal ultrasound before 12 weeks of gestational age. Hypotonic urine is associated with a better prognosis [7] and kidney functions normally when the urinary concentration of sodium is lower than 50 mmol/L [8]. Early onset of bladder distension and hypertonic urine result in worse prognosis. In our case, the urine analysis revealed hypertonic urine and the concentration of sodium was over 120 mmol/L. This implies that renal function was deteriorated in the very early gestational age. However urine analysis before 20 weeks of gestation may be inaccurate. As renal function compromise is a factor that determines the prognosis, prenatal intervention is needed to preserve renal function and to provide the aqueous environment for lung maturation [9,10]. Various management modalities can be applied; regular sonographic monitoring of the urinary tract and amniotic fluid volume in utero, fetal, vesicoamniotic shunting, termination of pregnancy, or early delivery so long as the neonatal viability is achieved.

to provide the aqueous environment for lung maturation [9,10]. Various management modalities can be applied; regular sonographic monitoring of the urinary tract and amniotic fluid volume in utero, fetal, vesicoamniotic shunting, termination of pregnancy, or early delivery so long as the neonatal viability is achieved. Vesicoamniotic shunt for urinary obstruction can prevent pulmonary hypoplasia and renal dysplasia or even may improve postnatal quality of life. Early intervention can lead to better results [11,12]. However, vesicoamniotic shunt is not possible in the very early gestational age as in our case because the fetus is too small for the shunt instrument. In addition, the relatively wide location of placenta and frequent fetal position can be obstacles to vesicoamniotic shunt [13]. However, early intervention would be crucial before deterioration of renal function, especially for early complete urethral obstruction just as our case. Vesicocentesis for the treatment of fetal megacystis and prune belly syndrome have been reported [14]. We performed vesicocentesis by spinal needle under ultrasound guidance. It was so effective as to reduce the bladder volume without severe complications.

Vesicoamniotic shunt for urinary obstruction can prevent pulmonary hypoplasia and renal dysplasia or even may improve postnatal quality of life. Early intervention can lead to better results [11,12]. However, vesicoamniotic shunt is not possible in the very early gestational age as in our case because the fetus is too small for the shunt instrument. In addition, the relatively wide location of placenta and frequent fetal position can be obstacles to vesicoamniotic shunt [13]. However, early intervention would be crucial before deterioration of renal function, especially for early complete urethral obstruction just as our case. Vesicocentesis for the treatment of fetal megacystis and prune belly syndrome have been reported [14]. We performed vesicocentesis by spinal needle under ultrasound guidance. It was so effective as to reduce the bladder volume without severe complications. Although the pregnancy in this case was terminated upon parental request, this study suggests the importance of the early diagnosis of the disease and the prompt intervention to improve prognosis. Our case is one of the earliest diagnoses and managements of prune-belly syndrome. Furthermore, the vesicocentesis with finer needle may be used as an effective treatment modality for the lower urinary tract obstruction developed in the first trimester. This case was previously reported as a view only poster presentation (P34.06) at the 20th World Congress on Ultrasound in Obstetrics and Gynecology, 10-14 October 2010, Prague, Czech Republic. No potential conflict of interest relevant to this article was reported.

Although the pregnancy in this case was terminated upon parental request, this study suggests the importance of the early diagnosis of the disease and the prompt intervention to improve prognosis. Our case is one of the earliest diagnoses and managements of prune-belly syndrome. Furthermore, the vesicocentesis with finer needle may be used as an effective treatment modality for the lower urinary tract obstruction developed in the first trimester. This case was previously reported as a view only poster presentation (P34.06) at the 20th World Congress on Ultrasound in Obstetrics and Gynecology, 10-14 October 2010, Prague, Czech Republic. No potential conflict of interest relevant to this article was reported. Fig. 1 Fetal bladder (A) dilated fetal bladder (BL) around which umbilical arteries are shown (color Doppler), bladder size 2.7×2.5 cm. (B) Collapsed fetal bladder (arrow head) after vesicocentesis. Fig. 2 Postmortem appearance. (A) Cleft lip (arrow) and palate (arrow head). (B) Polydactyly (arrow).

Introduction Malignant mixed müllerian tumor (MMMT) arising from female internal genitalia is rare with the uterine corpus being the most prevalently affected site. Ovarian MMMT contains malignant epithelial and stromal elements comprising less than 1% of ovarian malignancies [1]. Uterine adenosarcoma is a rare variant of uterine mixed müllerian tumor the prevalence of which is only 8% of uterine sarcoma [2]. It is characterized by a mixture of benign glandular epithelium and a malignant sarcomatous stroma resembling that of endometrial stromal sarcoma or mixed müllerian tumors, which are regarded as low-grade sarcoma [3]. We describe a case of synchronized occurrence of MMMT on both ovary and uterus with review of literature.

Introduction Malignant mixed müllerian tumor (MMMT) arising from female internal genitalia is rare with the uterine corpus being the most prevalently affected site. Ovarian MMMT contains malignant epithelial and stromal elements comprising less than 1% of ovarian malignancies [1]. Uterine adenosarcoma is a rare variant of uterine mixed müllerian tumor the prevalence of which is only 8% of uterine sarcoma [2]. It is characterized by a mixture of benign glandular epithelium and a malignant sarcomatous stroma resembling that of endometrial stromal sarcoma or mixed müllerian tumors, which are regarded as low-grade sarcoma [3]. We describe a case of synchronized occurrence of MMMT on both ovary and uterus with review of literature. Case Report A 57-year-old woman was admitted on January 2011 because of irregular vaginal bleeding for the last 10 months and recently aggravating in amount. She experienced the menopause on her age of 50. She had visited other clinic on June 2010 and was advised to follow up for the uterine tumor. Her family history was not contributory. Vital signs were within normal range and consciousness was clear. On physical examination, no abdominal tenderness or rebound tenderness was identified. Necrotic tissue covered with blood clot was noticed on cervix observed through vaginal speculum. The results of complete blood count, urinalysis, serologic tests, electrocardiogram, and chest X-ray were within normal range. Serum CA-125 and CA-19-9 levels were 12 U/mL and 2 U/mL, respectively. On abdominopelvic computed tomography (CT) examination, a mass of 8 cm in diameter arising from uterine corpus and 5.5 cm sized cystic and solid mass on right ovary were identified (Fig. 1).

ectrocardiogram, and chest X-ray were within normal range. Serum CA-125 and CA-19-9 levels were 12 U/mL and 2 U/mL, respectively. On abdominopelvic computed tomography (CT) examination, a mass of 8 cm in diameter arising from uterine corpus and 5.5 cm sized cystic and solid mass on right ovary were identified (Fig. 1). Explo-laparotomy was performed on February 2011 under the impression of endometrial cancer with ovarian metastasis. Low midline incision was placed under general anesthesia. Right ovarian mass was fixed on the posterior aspect of uterus and rectal wall by dense adhesive band and surrounded by necrotic tissue debris and blood clot. Extrafascial hysterectomy and bilateral adnexectomy was performed. Right ovarian mass was ruptured during the manipulation. Poorly differentiated sarcoma was suspected from frozen biopsy of both uterus and right ovary. Left adnexa was grossly free of tumor. However, multiple metastases of diameter 0.5 to 2 cm were identified on greater omentum. Partial omentectomy and appendectomy was performed. Small metastatic nodules were identified and all removed from right pelvic wall and mesentery of small intestine. No gross residual tumor was left.

nexa was grossly free of tumor. However, multiple metastases of diameter 0.5 to 2 cm were identified on greater omentum. Partial omentectomy and appendectomy was performed. Small metastatic nodules were identified and all removed from right pelvic wall and mesentery of small intestine. No gross residual tumor was left. On gross examination, huge polypoid mass of 8×4 cm was identified in uterine cavity and two myomatous nodule in myometrium. Cervix and both fallopian tubes were free of tumor (Fig. 2A). On microscopic examination, endometrial tumor was composed of mostly invasive cells with large and polymorphic nucleus with pale and distinct plasmid. Sarcomatous component was overwhelming and included small area of adenomatous component which was attributed as adenosarcoma of MMMT (Fig. 2B). Immunohistochemical staining with CD10 of both ovary and uterus was positive. The tumor arising from ovary was mostly composed of sarcoma while the individual cells looked different from those of uterine tumor and did not include adenomatous component (Fig. 2C). The metastatic nodules showed the histologic feature that are similar to that of the ovarian tumor suggesting the metastases originated from the ovary. This case was diagnosed as primary MMMT arising from uterus and from ovary as well. Oral intake was initiated on postoperative day 3 and no significant complication was noticed. She was discharged on day 9 and three cycles of chemotherapy with paclitaxel 175 mg/m2 plus carboplatin with area under the curve (AUC) 5. The follow-up was lost because she refused further treatment.

On gross examination, huge polypoid mass of 8×4 cm was identified in uterine cavity and two myomatous nodule in myometrium. Cervix and both fallopian tubes were free of tumor (Fig. 2A). On microscopic examination, endometrial tumor was composed of mostly invasive cells with large and polymorphic nucleus with pale and distinct plasmid. Sarcomatous component was overwhelming and included small area of adenomatous component which was attributed as adenosarcoma of MMMT (Fig. 2B). Immunohistochemical staining with CD10 of both ovary and uterus was positive. The tumor arising from ovary was mostly composed of sarcoma while the individual cells looked different from those of uterine tumor and did not include adenomatous component (Fig. 2C). The metastatic nodules showed the histologic feature that are similar to that of the ovarian tumor suggesting the metastases originated from the ovary. This case was diagnosed as primary MMMT arising from uterus and from ovary as well. Oral intake was initiated on postoperative day 3 and no significant complication was noticed. She was discharged on day 9 and three cycles of chemotherapy with paclitaxel 175 mg/m2 plus carboplatin with area under the curve (AUC) 5. The follow-up was lost because she refused further treatment. Discussion MMMT arising from female genital organ is a very rare tumor and affects mostly uterine corpus followed by cervix, vagina, ovary, and fallopian tube [2]. MMMT comprises 10% of ovarian tumors and 5% of uterine tumors and it is even more rare to be found in both uterus and ovary [4]. In 1985, Ulbright and Roth [5] firstly suggested the pathological criteria to classify the independent malignancies arising concurrently from individual sites. The relationship between the tumors in individual site is not always clear. However, it is evident that the tumors are independent such as this case when the histologic findings of the tumors are different.

ed the pathological criteria to classify the independent malignancies arising concurrently from individual sites. The relationship between the tumors in individual site is not always clear. However, it is evident that the tumors are independent such as this case when the histologic findings of the tumors are different. Uterine adenosarcoma develops mostly in postmenopausal women. However, one third affects premenopausal women including the teens [3]. Eighty percent of ovarian MMMT also affects postmenopausal women majority of which lies on 50 to 70 of age [1]. Abnormal uterine bleeding is the most frequent clinical presentation followed by low abdominal pain, cervical mass, and increase in uterine size. Cervical MMMT typically resembles polyp or submucosal myoma filling uterine cavity and extends into the cervix [3]. MMMT is usually diagnosed at early stage that stage III or IV comprises only 11% of all cases [6]. However, the recurrence and death rate is reported to be 30% to 40% and 20% to 55%, respectively. The death rate of uterine adenosarcoma is 65% and tends to diagnosed in earlier stage than carcinosarcoma [6]. The symptoms of ovarian MMMT is identical to those of advanced epithelial ovarian cancer such as abdominal distention, pelvic mass, and belching [1]. Peritoneal seeding is a common finding in ovarian MMMT accompanying ascites in 67% to 100% [6,7]. Ovarian MMMT is diagnosed as stage III or IV in 75% and metastasize to extragonadal sites in over 90% [1,7,8].

cal to those of advanced epithelial ovarian cancer such as abdominal distention, pelvic mass, and belching [1]. Peritoneal seeding is a common finding in ovarian MMMT accompanying ascites in 67% to 100% [6,7]. Ovarian MMMT is diagnosed as stage III or IV in 75% and metastasize to extragonadal sites in over 90% [1,7,8]. The prognostic factors of uterine adenosarcoma include stage, myometrial invasion, mitotic index, presence of heterotopic tissue, and tissue necrosis [9]. Deep myometrial invasion and extrauterine metastasis affects more adversely and deep myometrial invasion is the only prognostic factor for the recurrence of uterine adenosarcoma. Tumor is limited to endometrium in most cases and myometrial invasion is observed in 15% while deep myometrial invasion is reported only in 4%. Extrauterine metastasis occurs in less than 50% [3]. The prognostic factors of ovarian MMMT are reported to be the age at presentation, insufficient surgical removal, and the stage. The recurrence rate is 50% in stage I and up to 90% to 100% in stage II or greater [7].

The prognostic factors of uterine adenosarcoma include stage, myometrial invasion, mitotic index, presence of heterotopic tissue, and tissue necrosis [9]. Deep myometrial invasion and extrauterine metastasis affects more adversely and deep myometrial invasion is the only prognostic factor for the recurrence of uterine adenosarcoma. Tumor is limited to endometrium in most cases and myometrial invasion is observed in 15% while deep myometrial invasion is reported only in 4%. Extrauterine metastasis occurs in less than 50% [3]. The prognostic factors of ovarian MMMT are reported to be the age at presentation, insufficient surgical removal, and the stage. The recurrence rate is 50% in stage I and up to 90% to 100% in stage II or greater [7]. Preoperative diagnosis is possible for uterine adenosarcoma by endometrial biopsy. However, ovarian MMMT is not diagnosed before surgery and the incidence is extremely rare. Thus the treatment strategy for ovarian MMMT is not established to date and maximal surgical resection and adjuvant chemotherapy is generally performed [1,10]. Optimal cytoreduction resulted in longer recurrence-free interval and median survival [1]. Duska et al. [11] reported that 14 out of 24 who received optimal cytoreduction were associated with increased time to recurrence. Silasi et al. [12] reported that median survival was 46 months in optimally debulked patients and 27 months in suboptimally debulked. The treatment is not established for uterine adenosarcoma either but surgical resection and adjuvant chemotherapy is generally performed as well. Radiotherapy for uterine adenosarcoma may effectively control local disease without significant increase in survival rate [6].

atients and 27 months in suboptimally debulked. The treatment is not established for uterine adenosarcoma either but surgical resection and adjuvant chemotherapy is generally performed as well. Radiotherapy for uterine adenosarcoma may effectively control local disease without significant increase in survival rate [6]. Three to six cycles with doxorubicin, dacarbazine, and vincristine was suggested for adjuvant chemotherapy for uterine adenosarcoma but the data are limited to determine efficacy [13]. Platinum based monotherapy or combination chemotherapy with platinum plus paclitaxel or ifosphamide was reported for ovarian MMMT [1,14]. Tate Thigpen et al. [14] reported that complete response was reached in 1 out of 44 who received cisplatin monotherapy while partial response in 8, stable disease in 10, and progression in 25 was observed. Overall response was 20%, which is identical to the repose rate of uterine carcinosarcoma. Progression-free survival was 5.2 months and median survival was 11.7 months. Duska et al. [11] reported the experience of doublet with paclitaxel and carboplatin. Complete response was reached in 16 out of 28 patients while 6 patients showed partial response and 5 patients progressed. The overall response rate was 72% and median survival was 27.1 months. Of the 16 patients of complete response, 10 patients received optimal cytoreduction and had significantly longer recurrence-free survival [11]. The doublet with ifosfamide and cisplatin showed median survival of 23 months, which is improved over paclitaxel and carboplatin doublet. However, digestive complications such as nausea and vomiting and hematopoietic complications such as neutropenia and anemia were more severe in patients who treated with ifosfamide+cisplatin doublet [15]. Chemotherapies with vincristine+adriamycin+cyclophosphamide (VAC), ifosfamide+mesna, and adriamycin were reported, however, the response rate was not comparable [1,11].

omiting and hematopoietic complications such as neutropenia and anemia were more severe in patients who treated with ifosfamide+cisplatin doublet [15]. Chemotherapies with vincristine+adriamycin+cyclophosphamide (VAC), ifosfamide+mesna, and adriamycin were reported, however, the response rate was not comparable [1,11]. We performed optimal cytoreduction and adjuvant chemotherapy with paclitaxel 175 mg/m2 and carboplatin with AUC 5. However the follow-up was lost after three cycles of chemotherapy because the patient refused further treatment. The uterine adenosarcoma and ovarian MMMT are both rare tumors and treatment strategy is not settled to date. Proper management might be established when more cases are cumulated in the future. No potential conflict of interest relevant to this article was reported. Fig. 1 Computed tomography showing a huge sized mass in uterine cavity. And 5.5-cm sized cystic and solid mass in right adnexa (white arrows). Fig. 2 Gross and microscopic findings. (A) Gross findings after surgery: 8×5-cm sized polypoid mass in uterine cavity. (B) The uterine mass shows mainly diffuse sheets of anaplastic sarcomatous components with scattered benign looking glandular components (H&E, ×200). (C) The ovarian mass shows diffuse sheets of mixed adenocarcinoma and anaplastic sarcomatous component (H&E, ×200).

Introduction Combined oral contraceptives (COCs) are a common method of contraception, but they carry a risk of venous and arterial thrombosis. The association between estrogen-containing oral contraceptives (OCs) and venous thromboembolism (VTE) is well established [1]. Until 1995, it was generally thought that the progestogen component of COCs did not contribute to the risk of VTE. However, a number of studies published in late 1995 and early 1996 reported an increased risk of VTE for users of the so-called third-generation COCs containing the progestogens, desogestrel or gestodene, compared with second-generation COCs, containing levonorgestrel [2]. These findings were unexpected and led to debate over possible bias and confounding. New studies, re-analyses of the original studies and a meta-analysis have since been published [3]. VTE is rare event in young women and the risk associated with a new COC. But, many studies indicate that the well-known risk of VTE related to the use of COC needs to be considered as a cause of pulmonary embolism (PE) even among very young females [4]. So, prevention of these life-threatening conditions in patients without a family history of VTE consists of timely examination of their thrombotic profile and selection of appropriate medication. We have experienced a patient, 24-year-old young woman with PE and deep vein thrombosis (DVT) without inherited risk factors four months after taking Yasmin. We report this case with brief review of literatures.

Introduction Combined oral contraceptives (COCs) are a common method of contraception, but they carry a risk of venous and arterial thrombosis. The association between estrogen-containing oral contraceptives (OCs) and venous thromboembolism (VTE) is well established [1]. Until 1995, it was generally thought that the progestogen component of COCs did not contribute to the risk of VTE. However, a number of studies published in late 1995 and early 1996 reported an increased risk of VTE for users of the so-called third-generation COCs containing the progestogens, desogestrel or gestodene, compared with second-generation COCs, containing levonorgestrel [2]. These findings were unexpected and led to debate over possible bias and confounding. New studies, re-analyses of the original studies and a meta-analysis have since been published [3]. VTE is rare event in young women and the risk associated with a new COC. But, many studies indicate that the well-known risk of VTE related to the use of COC needs to be considered as a cause of pulmonary embolism (PE) even among very young females [4]. So, prevention of these life-threatening conditions in patients without a family history of VTE consists of timely examination of their thrombotic profile and selection of appropriate medication. We have experienced a patient, 24-year-old young woman with PE and deep vein thrombosis (DVT) without inherited risk factors four months after taking Yasmin. We report this case with brief review of literatures. Case report A 24-year-old woman (gravida 0) visited our clinic due to left inguinal pain on every step and edema of left leg four months after taking Yasmin (Bayer HealthCare, New York, NY, USA). This pain and edema were started a week ago and aggravated three days ago. Her weight was 69 kg, and height 170 cm. She was a non-smoker. Her job was a company worker. The patient and family members had not suffered prior VTE. She received pelviscopic ovarian cystectomy of 5 cm sized left ovarian endometrioma at another clinic a year ago. Two weeks after operation, she was injected gonadotropin releasing hormone (GnRH) per four weeks for six months continuously. A month after sixth GnRH injection, she had started taking Yasmin 21-tablet continuously without seven-day discontinuation period for four months to prevent recurrence of endometriosis. During taking the fifth pack of Yasmin, she felt left inguinal pain on ambulation, and edema of left leg. So, she admitted our clinic. Her vital signs were as stable. Body temperature 120/80 mm Hg, pulse rate 72/min, respiratory rate 20/min, body temperature 36.5℃. She had no chest discomfort, breathing difficulties and skin color change of left leg. The circumference of her left thigh was proximal 64 cm, distal 48 cm, and right proximal 57 cm, distal 43 cm, respectively. Her left radial artery and dorsalis pedis artery were palpable well. Laboratory findings were below. Complete blood count, chemistry profile, and urine analysis were all normal range. Prothrombin time 11.6 sec (normal, 9.8 to 12.4 sec), partial thromboplastin time 96.6% (normal, 80% to 135%). Anti-cardiolipin antibody IgG/IgM, Lupus anticoagulant, Factor V Leiden (FV, Q506) were all negative. Protein C activity was 109% (normal, 70% to 130%), and protein S activity 99% (normal, 65% to 140%). D-dimer was elevated to 2.68 µg/mL (normal, 0 to 0.5 µg/mL). Chest posterial-anterial and electrocardiogram were normal findings.

ipin antibody IgG/IgM, Lupus anticoagulant, Factor V Leiden (FV, Q506) were all negative. Protein C activity was 109% (normal, 70% to 130%), and protein S activity 99% (normal, 65% to 140%). D-dimer was elevated to 2.68 µg/mL (normal, 0 to 0.5 µg/mL). Chest posterial-anterial and electrocardiogram were normal findings. Chest pulmonary angiography computed tomography (CT) (contrast enhanced) showed pulmonary thromboembolism in BLLs' basal pulmonary arteries and 32-mm sized consolidation in right lower lobe posterior segment as considering pulmonary infarction (Fig. 1). Lower extremity venogram CT (contrast enhanced) showed thrombosis in inferior vena cava (IVC) and diffuse DVT below the level of left external iliac vein (Fig. 2). We injected low molecular weight heparin (LMWH) 60 mg per 12 hours subcutaneously for seven days and overlapped heparin with 4 mg warfarin five days. After five days of combined therapy, we changed to 4 mg warfarin per oral. We determined the proper dose of warfarin by a blood test reported as the international normalized ratio. She discharged with warfarin. She had taken warfarin for six months thereafter. She had normal menstrual cycles and no recurrence of PE and DVT during 12-month follow-up.

rapy, we changed to 4 mg warfarin per oral. We determined the proper dose of warfarin by a blood test reported as the international normalized ratio. She discharged with warfarin. She had taken warfarin for six months thereafter. She had normal menstrual cycles and no recurrence of PE and DVT during 12-month follow-up. Discussion VTE poses a public health threat with an estimated incidence in the United States of 250,000 to 2 million cases per year. Predisposition to VTE arises from acquired conditions, inherited disorders, or both. Acquired risk factors include obesity, cigarette smoking, hypertension, immobilization, surgery, trauma, oral contraceptives, pregnancy, and hormone replacement therapy. Chronic medical illnesses such as congestive heart failure, chronic obstructive pulmonary disease, and cancer also predispose to PE. Heredity plays an important role in a patient's susceptibility to PE. So, we do genetic tests such as factor V Leiden and the prothrombin gene mutation that can identify those who are predisposed. The presence of these risk factors is sometimes called a prothrombotic or thrombophilic state. DVT most often originates in the calf, with a persistent cramping or "charley horse" that intensifies over several days. Leg swelling and discoloration may accompany the increase in discomfort. Upper-extremity DVT may cause otherwise unexplained upper arm or neck swelling. The most frequently used diagnostic imaging test is the noninvasive venous ultrasound examination. To establish the diagnosis of PE, the most frequently used noninvasive imaging test is the rapid-speed chest CT scan. If D-dimer is normal, PE is extremely unlikely. Chest CT scan, which directly images blood clots causing blockages in the pulmonary arteries. Lung scan, which indirectly identifies areas of decreased blood flow in the lung tissue as a consequence of PE.

ly used noninvasive imaging test is the rapid-speed chest CT scan. If D-dimer is normal, PE is extremely unlikely. Chest CT scan, which directly images blood clots causing blockages in the pulmonary arteries. Lung scan, which indirectly identifies areas of decreased blood flow in the lung tissue as a consequence of PE. Anticoagulation begins with a combination of heparin and warfarin. The traditional unfractionated form ordinarily requires intravenous administration. More recently, LMWHs have begun replacing unfractionated heparin. In patients who cannot tolerate anticoagulation or those for whom anticoagulation fails, a permanent metal filter is inserted into the IVC, the largest vein below the heart, to prevent large blood clots from reaching the pulmonary arteries and causing PE. Unfortunately, the filter devices do not halt the clotting process. Their presence predisposes to future venous clots on or below the filter. The most controversial area in VTE therapy is the optimal duration of warfarin anticoagulation. The current recommendation is usually at least 6 months of anticoagulation, but it can sometimes be longer, according to individual patient circumstances [5].

esence predisposes to future venous clots on or below the filter. The most controversial area in VTE therapy is the optimal duration of warfarin anticoagulation. The current recommendation is usually at least 6 months of anticoagulation, but it can sometimes be longer, according to individual patient circumstances [5]. Genetic risk factors were found in about 5% to 10% of cases. Activated protein C resistance caused by factor V Leiden mutation is the most common inherited cause of an underlying predisposition to PE and DVT. Factor V Leiden mutation is an important risk factor for PE or DVT during pregnancy, after pregnancy, or during oral contraceptive use [6]. The phenotypic expression of resistance to activated protein C is characterized by a poor response to the anticoagulant activity of activated protein C, a key enzyme in the down-regulation of blood coagulation, which causes a disposition for a hypercoagulable state. At least 90% of the cases with resistance to activated protein C are explained by a point mutation in the gene for coagulation factor V, resulting in replacement of an Arg to Gln at position 506 (factor V, Q506, often denoted factor V Leiden), one of the three activated protein C cleavage sites in activated factor V. Thrombotic events are often triggered through the presence of a combination of inherited and circumstantial risk factors. The high prevalence of activated protein C resistance raises the issue whether it would be cost-beneficial to screen for this trait in connection with surgery, pregnancy, and OCs [7].

ated factor V. Thrombotic events are often triggered through the presence of a combination of inherited and circumstantial risk factors. The high prevalence of activated protein C resistance raises the issue whether it would be cost-beneficial to screen for this trait in connection with surgery, pregnancy, and OCs [7]. The use of drospirenone-containing OCs was associated with an increased risk of DVT and PE, but not transient ischemic attack or cerebrovascular attack, relative to second- and third-generation COCs [8]. The risk of non-fatal VTE among users of OCs containing drospirenone seems to be around twice that of users of OCs containing levonorgestrel [9,10]. The risk of VTE in current users of COCs decreases with duration of use and decreasing estrogen dose. For the same dose of estrogen and the same length of use, OCs with desogestrel, gestodene, or drospirenone were associated with a significantly higher risk of VTE than COCs with levonorgestrel. Progestogen only pills and hormone releasing intrauterine devices were not associated with any increased risk of VTE [11]. The American College of Obstetricians and Gynecologists Committee said that when prescribing any oral contraceptive, clinicians should consider a woman's risk factors for VTE and decisions regarding choice of oral contraceptive should be left to their patients, taking into account the possible minimally increased risk of VTE, patient preference, and available alternatives [12].

ittee said that when prescribing any oral contraceptive, clinicians should consider a woman's risk factors for VTE and decisions regarding choice of oral contraceptive should be left to their patients, taking into account the possible minimally increased risk of VTE, patient preference, and available alternatives [12]. Several specific potential risk factors for a fatal outcome from a COC-induced PE were identified. Recognition of these in combination with a high suspicion of VTE in COC users may reduce the risk of a fatal outcome. We have experienced a patient, 24-year-old young woman with PE and DVT without inherited risk factors four months after taking Yasmin. So, we report this rare case with brief review of literatures. Acknowledgments This work was supported in part by the Soonchunhyang University Research Fund. No potential conflict of interest relevant to this article was reported. Fig. 1 Chest pulmonary angiography computed tomography (contrast enhanced) showed positron emission tomography in both lower lobes basal pulmonary arteries and 32-mm sized extent consolidation (arrow) in right lower lobe posterior segment considering pulmonary infarction. Fig. 2 Lower extremity venogram computed tomography (contrast enhanced) showing diffuse deep vein thrombosis (arrow) below the level of left external iliac vein.

Introduction Allogenic peripheral blood stem cell transplantation (Allo-PBSCT) is being used to treat hematological malignancies with increasing frequency. Allo-PBSCT has many complications, such as infections, veno-occlusive disease of the liver, drug reactions, and graft-versus-host disease (GvHD). GvHD is a complex complication with acute and chronic stages that are categorized based on whether symptoms developed within 100 days after the transplant or later. Currently, the number of days after the transplant is not sufficient to differentiate acute from chronic GvHD. The pathogenesis of GvHD is believed to be a complex, primarily T-cell mediated, immune response in which the grafted donor cells react against histocompatibility antigens in the host [1]. Manifestations of GvHD involving the skin, gastrointestinal tract, lungs, and liver are well described [2-4]. However, involvement of the vagina has not been well characterized. Therefore, GvHD may be an unfamiliar topic to gynecologists. However, if the possible impact of GvHD on the vulva or vagina is not underestimated, diagnosis and management would not be difficult. This case provides a chance to review the gynecologic complications of GvHD.

of the vagina has not been well characterized. Therefore, GvHD may be an unfamiliar topic to gynecologists. However, if the possible impact of GvHD on the vulva or vagina is not underestimated, diagnosis and management would not be difficult. This case provides a chance to review the gynecologic complications of GvHD. Case report A 43-year-old, gravida 4, para 2 woman came to the gynecology clinic for counseling about premature menopause. She had been diagnosed with acute myeloid leukemia 2 years earlier and treated with induction and consolidation chemotherapy using idarubicin and cytosine arabinoside. After developing complete remission, physician recommended patient to have gynecological examination. At the gynecology clinic office, gynecologist conducted pelvic examination with Papanicolaou (Pap) test. The uterine cervix revealed cooper intrauterine device (IUD) tail string which got stuck into exocervix, but otherwise non-specific finding (Fig. 1A). Of course the IUD was immediately removed. The result of Pap test was reactive cellular changes. Eleven days after gynecologic consultation, patient underwent Allo-PBSCT and the donor was her younger brother. There was no evidence of residual disease on a bone marrow biopsy performed 10 months after transplantation.

). Of course the IUD was immediately removed. The result of Pap test was reactive cellular changes. Eleven days after gynecologic consultation, patient underwent Allo-PBSCT and the donor was her younger brother. There was no evidence of residual disease on a bone marrow biopsy performed 10 months after transplantation. Twenty-six months after Allo-PBSCT, she revisited our gynecology clinic and it was found that her follicle-stimulating hormone level was greater than 100 IU/mL and estradiol was 4.14 pg/mL. By reviewing the patient medical record and taking a medical history, it was found that when she started chemotherapy, her menstrual cycle completely disappeared. About one year after chemotherapy, the patient tried coitus with husband, but vaginal dryness and dyspareunia were very severe, so could not have sexual intercourse. However, patient had no other menopausal symptoms like hot flush, sleep disturbance, mood swing and so on. To introduce sequential hormone replacement therapy (HRT), we performed clinical examination. The clinical examination revealed extensive vulvar atrophy with flattening but otherwise non-specific appearance including uterine cervix. After that, the patient started sequential HRT.

leep disturbance, mood swing and so on. To introduce sequential hormone replacement therapy (HRT), we performed clinical examination. The clinical examination revealed extensive vulvar atrophy with flattening but otherwise non-specific appearance including uterine cervix. After that, the patient started sequential HRT. Fourteen months after initiation of HRT, it was discovered that her upper vaginal canal was completely obstructed (Fig. 1B). The lower vagina had a normal appearance. The human papillomavirus (HPV) deoxyribonucleic acid (DNA) chip test and Pap test were conducted in vagina not in uterine cervix because of obstruction of upper vaginal canal. The HPV DNA chip test was positive for HPV (other type) in the vagina. The Pap test was negative. Trans-rectal ultrasonographic findings were hematocolpos and atrophic uterus (Fig. 1C). We explained the results to the patient and provided reassurance. The patient is currently being treated with tibolone and we recommended non-hormonal moisturizing vaginal gel and vaginal dryness and dyspareunia were much improved. Here, we present the case of a woman who after Allo-PBSCT, showed obstruction of the upper vagina as a chronic complication of GvHD.

Fourteen months after initiation of HRT, it was discovered that her upper vaginal canal was completely obstructed (Fig. 1B). The lower vagina had a normal appearance. The human papillomavirus (HPV) deoxyribonucleic acid (DNA) chip test and Pap test were conducted in vagina not in uterine cervix because of obstruction of upper vaginal canal. The HPV DNA chip test was positive for HPV (other type) in the vagina. The Pap test was negative. Trans-rectal ultrasonographic findings were hematocolpos and atrophic uterus (Fig. 1C). We explained the results to the patient and provided reassurance. The patient is currently being treated with tibolone and we recommended non-hormonal moisturizing vaginal gel and vaginal dryness and dyspareunia were much improved. Here, we present the case of a woman who after Allo-PBSCT, showed obstruction of the upper vagina as a chronic complication of GvHD. Discussion GvHD is a systemic syndrome. The commonly involved organs include the skin, mouth, liver, eyes, esophagus, and upper respiratory tract. Clinical features are similar to those of autoimmune diseases, including scleroderma, Sjögren's syndrome [5], lichen planus, and primary biliary cirrhosis [6]. However, gynecologic manifestations of GvHD in patients treated with Allo-PBSCT are rare and probably underestimated. Vaginal inflammation, dryness, stricture and stenosis have been reported, together with desquamative web formation [7].

roderma, Sjögren's syndrome [5], lichen planus, and primary biliary cirrhosis [6]. However, gynecologic manifestations of GvHD in patients treated with Allo-PBSCT are rare and probably underestimated. Vaginal inflammation, dryness, stricture and stenosis have been reported, together with desquamative web formation [7]. For 80% of patients with chronic GvHD, drying of the vagina would be expected [8]. It is hypothesized that inflamed and atrophic vaginal surfaces might cause obstruction of the vaginal canal. It should be recognized that the severity of vaginal and vulvar symptoms does not correlate with the severity of GvHD found in other organ systems. That is, some women with severe vaginal stenosis have only mild chronic GvHD.

ized that inflamed and atrophic vaginal surfaces might cause obstruction of the vaginal canal. It should be recognized that the severity of vaginal and vulvar symptoms does not correlate with the severity of GvHD found in other organ systems. That is, some women with severe vaginal stenosis have only mild chronic GvHD. There have been some reports describing similar cases [9-13]. Corson et al. [9] first reported vaginal involvement in five women with sclerosing vaginitis and stricture formation in 1982. Since then, several cases have been reported with complete vaginal obliteration [10-13]. Vaginal obstruction limits the ability to perform a routine Pap test and prevents sexual intercourse. However, most cases show mild chronic GvHD of the vulva or vagina that may be asymptomatic and detected only on examination. Because of this, careful gynecologic approach is needed in Allo-PBSCT [14]. Management of vulvovaginal lesions is based on the onset of therapy at early stages of the disorder and maintenance of sexual activity. HRT is recommended when menopause is confirmed, but its local beneficial effects are limited in advanced disease. Sequential HRT may contribute to the formation of hematocolpometra in the presence of vaginal synechiae. It has been reported that HRT does not influence the severity or activity of GvHD and can be safely used as a prophylactic measure to treat ovarian failure [15].

l beneficial effects are limited in advanced disease. Sequential HRT may contribute to the formation of hematocolpometra in the presence of vaginal synechiae. It has been reported that HRT does not influence the severity or activity of GvHD and can be safely used as a prophylactic measure to treat ovarian failure [15]. Management of the early stages of anatomical distortion of the external and internal genitalia includes vaginal dilatation, local corticotherapy, and estrogen therapy to help prevent stenosis. Surgery is indicated in advanced cases to restore normal anatomy. Progression or recurrence of lesions may be observed, despite treatment, in cases of extensive GvHD. Vulvar and vaginal GvHD seems to be a discrete entity in the continuum of chronic GvHD. As more studies are performed, women who experience these complications can promptly and effectively be treated. We report a rare case of partial vaginal obstruction as a complication of chronic GvHD and review the literature. We expect that this case report provides an opportunity to remind clinician of the gynecologic complications of GvHD. Acknowledgements This work was supported in part by the Soonchunhyang University Research Fund. No potential conflict of interest relevant to this article was reported.

We report a rare case of partial vaginal obstruction as a complication of chronic GvHD and review the literature. We expect that this case report provides an opportunity to remind clinician of the gynecologic complications of GvHD. Acknowledgements This work was supported in part by the Soonchunhyang University Research Fund. No potential conflict of interest relevant to this article was reported. Fig. 1 (A) Before allogenic peripheral blood stem cell transplantation, photograph shows normal uterine cervix with intra-uterine device tail string which got stuck into exocervix. (B) Twenty-six months after allogenic peripheral blood stem cell transplantation, photograph shows almost complete obstruction of the upper vaginal canal. A lower vagina had normal appearance. (C) Twenty-six months after allogenic peripheral blood stem cell transplantation, trans-rectal ultrasonography shows hematocolpos (arrow). asterisk, uterine cervix; star, vagina.

Introduction Endometriosis is a chronic progressive disease inducing various symptoms such as infertility, pelvic pain, and menstrual pain due to the presence of endometrial tissues outside of the uterus. Even though endometriosis occurs in approximately 6% to 10% of women, the incidence increases to up to 35% to 50% for women with pelvic pain or infertility [1]. The causes of endometriosis are not clearly understood in spite of many research efforts. However, the transplantation theory by Sampson is generally accepted, suggesting that retrogradely menstruated endometrial cells implant in the pelvic peritoneum and thereby cause endometriosis. However, approximately 76% to 90% of women have shown the backflow of menstruation in laparoscopic examinations, while the incidence of endometriosis is much lower [2]. Thus, genetic factors, environmental factors, and changes in immunological as well as endocrinologic functions are thought to be involved in the occurrence of endometriosis. Additionally, some studies have demonstrated that the eutopic endometrial tissues in patients with endometriosis are considerably different from those in patients without endometriosis; therefore, such abnormal findings are also anticipated to play important roles in the occurrence of endometriosis [3]. The mechanism of the adherence of endometrial cells to the peritoneum has not yet been elucidated. Nevertheless, Lucidi et al. [4] found that endometrial stromal cells (ESCs) adhere to peritoneal mesothelial cells (PMCs) via experimental models, and they further suggested that the source of endometrium, rather than the source of PMCs, has the greatest effects on ESCs binding to PMCs.

eum has not yet been elucidated. Nevertheless, Lucidi et al. [4] found that endometrial stromal cells (ESCs) adhere to peritoneal mesothelial cells (PMCs) via experimental models, and they further suggested that the source of endometrium, rather than the source of PMCs, has the greatest effects on ESCs binding to PMCs. Lately, several researchers have demonstrated that CD44, the main receptor of hyaluronic acid (HA), was associated with the peritoneal adherence of gastric and ovarian carcinoma cells [5,6]. CD44, a transmembrane glycoprotein, is involved in intercellular interactions as well as in interactions between cells and the extracellular matrix [7]. As the genomic locus for CD44 includes 20 exons, several isoforms are present. The smallest CD44 isoforms lack all variable regions and are called "standard" CD44 (CD44s), and the CD44 isoforms with variable regions consisting of 10 exons via alternative splicing are called v1-v10. To the best of our knowledge, this is the first systematic investigation of the expression of CD44 isoforms in the endometrium. Although the expression of CD44 was determined through reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining in several studies, the results were not consistent [8-12]. Therefore, the objectives of this study were to compare the expression of CD44 in ESCs between patients with endometriosis and controls and to investigate the roles of CD44 in the adherence of ESCs to PMCs.

Lately, several researchers have demonstrated that CD44, the main receptor of hyaluronic acid (HA), was associated with the peritoneal adherence of gastric and ovarian carcinoma cells [5,6]. CD44, a transmembrane glycoprotein, is involved in intercellular interactions as well as in interactions between cells and the extracellular matrix [7]. As the genomic locus for CD44 includes 20 exons, several isoforms are present. The smallest CD44 isoforms lack all variable regions and are called "standard" CD44 (CD44s), and the CD44 isoforms with variable regions consisting of 10 exons via alternative splicing are called v1-v10. To the best of our knowledge, this is the first systematic investigation of the expression of CD44 isoforms in the endometrium. Although the expression of CD44 was determined through reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining in several studies, the results were not consistent [8-12]. Therefore, the objectives of this study were to compare the expression of CD44 in ESCs between patients with endometriosis and controls and to investigate the roles of CD44 in the adherence of ESCs to PMCs. Materials and Methods 1. Subjects For the endometriosis group, we recruited 13 patients who were confirmed to have endometriosis based on pathologic examinations after conservative surgery in the department of obstetrics and gynecology at Pusan National University Yangsan Hospital. The recruited patients had no history of medications such as gonadotropin releasing hormone agonist or steroids within the previous six months. The patients were not pregnant and had normal menstrual cycles. For the control group, we recruited 13 females with normal menstrual cycles who underwent hysterectomy because of benign gynecologic diseases or carcinoma in situ of the cervix. There were no gross lesions to consider the presence of endometriosis during surgery of the control group.

nt and had normal menstrual cycles. For the control group, we recruited 13 females with normal menstrual cycles who underwent hysterectomy because of benign gynecologic diseases or carcinoma in situ of the cervix. There were no gross lesions to consider the presence of endometriosis during surgery of the control group. 2. Methods This study was approved by the Institutional Review Board at Pusan National University. Sufficient explanation regarding the study was provided to all subjects, and informed consent was obtained. 1) Collection of endometrial tissues Endometrial aspiration biopsy using a Novak curette was performed during conservative surgery in the endometriosis group, whereas the endometrial tissues were obtained from the resected uterus after hysterectomy in the control group. The entire process of collecting tissues was conducted under aseptic conditions. The collected tissues were placed in conical tubes containing Dulbecco's modified Eagle's media F12 (DMEM:F12, Gibco, Grand Island, NY, USA) with 1% antibiotics/antimycotics (Gibco), and then the samples were transferred from the operation room to laboratory. The rest of the blood was removed by washing several times with phosphate-buffered saline (PBS, Gibco).

l tubes containing Dulbecco's modified Eagle's media F12 (DMEM:F12, Gibco, Grand Island, NY, USA) with 1% antibiotics/antimycotics (Gibco), and then the samples were transferred from the operation room to laboratory. The rest of the blood was removed by washing several times with phosphate-buffered saline (PBS, Gibco). 2) Endometrial stromal cell culture The endometrial tissues were placed in culture dishes with DMEM:F12 and then minced to 1-2 mm in size using sterile scissors. Minced tissues in the solution were transferred to conical tubes and treated with 0.1% collagenase type 1 (Gibco) and 0.05% DNAse (Boehringer Mannheim, Mannheim, Germany) followed by incubation in a waterbath at 37℃ for 1 hour 30 minutes while shaking [13]. After that, the media containing 10% fetal bovine serum (FBS, Gibco, USA) was added to stop the enzyme reaction, the samples were filtered through a 100 µm nylon membrane (BD Falcon, Bedford, MA, USA) and the supernatant was removed through centrifugation.

in a waterbath at 37℃ for 1 hour 30 minutes while shaking [13]. After that, the media containing 10% fetal bovine serum (FBS, Gibco, USA) was added to stop the enzyme reaction, the samples were filtered through a 100 µm nylon membrane (BD Falcon, Bedford, MA, USA) and the supernatant was removed through centrifugation. The precipitated cells were reconstituted in media and filtered through a 40 µm nylon membrane (BD Falcon, Bedford, MA, USA) [14]. After centrifugation of the precipitates, the supernatant was removed and the precipitated cells were resuspended in DMEM:F12 (1:1) media with 10% FBS, 1% antibiotics/antimycotics and 5 µg/mL of insulin (Sigma, St. Louis, MO, USA). Then, the cells were aliquoted into 100 mm culture dishes (BD Falcon) and incubated at room temperature for 20 minutes. The cells that did not adhere to the culture dishes were discarded, and new media was added to the stromal cells adhering to the culture dishes. Then, the cells were placed in an incubator at 37℃ and 5% CO2 for 24 hour with replacement of the media once every two days [15]. Immunohistochemical staining against Vimentin and Cytokeratin (Novocastra Laboratories Ltd., Newcastle, UK) was performed on the cultured cells. Based upon the positive and negative results, only ESCs with greater than 97% purity were utilized in the study.

4 hour with replacement of the media once every two days [15]. Immunohistochemical staining against Vimentin and Cytokeratin (Novocastra Laboratories Ltd., Newcastle, UK) was performed on the cultured cells. Based upon the positive and negative results, only ESCs with greater than 97% purity were utilized in the study. 3) Cell line culture The LP9 human peritoneal cell line was purchased from NIA Aging Cell Culture Repository (USA). The cell line was maintained in MCDB131/M199 (1:1) media containing 20 ng/mL of epithelial growth factor (Sigma), 2 mM L-glutamin (Gibco), HEPES buffer (Sigma), 0.05 µg/mL of hydrocortisone (Sigma), 1% antibiotics/antimycotics, and 10% FBS [4]. SK-OV-3 human ovarian carcinoma cells were obtained from the Korean Cell Bank and utilized as the control group for adherence assays. SK-OV-3 cells were cultured in RPMI (Gibco) media with HEPES buffer, 10% FBS, and 1% antibiotics/antimycotics. Each cell line was cultured in an incubator at 37℃ and 5% CO2 with replacement of the media once every two to three days.

ained from the Korean Cell Bank and utilized as the control group for adherence assays. SK-OV-3 cells were cultured in RPMI (Gibco) media with HEPES buffer, 10% FBS, and 1% antibiotics/antimycotics. Each cell line was cultured in an incubator at 37℃ and 5% CO2 with replacement of the media once every two to three days. 4) Adherence assay of peritoneal cells To perform an in vivo assay with adherence conditions similar to those of endometrial and peritoneal cells in the body, LP9 cells were attached to the bottom of the culture dishes, and adherence was determined by attaching fluorescent materials on the primarily cultured ESCs. The cultured LP9 cells (1×104 cells/well) were aliquoted in 96-well plates and then utilized in the study after culturing for three days. The cultured ESCs were detached from the culture dishes using non-enzymatic cell dissociation media (Sigma) and centrifuged. The precipitated cells were reconstituted in media and attached to Calcein AM, a fluorescent material. The precipitated fluorescent cells (100 µL/well, 2×104 cells/100 µL) were aliquoted in the 96-well plates in which LP9 cells had been placed. After 1 hour of incubation, absorbance was measured at 485 to 538 nm utilizing a fluorometer (FIUOstar OPTIMA, BMG Labtech, Offenburg, Germany). After the measurement, the substances in the 96-well plates were discarded and then cultured upside down in an incubator containing PBS with calcium and magnesium at 37℃ and 5% CO2 while shaking at 20 rpm for 20 minutes. The precipitated fluorescent ESCs that are not attached to LP9 cell layer will be removed, and then ESCs attached to LP9 cells are left in the 96-well plates. Then, the 96-well plates were subjected to absorbance measurements. The adherence was calculated using the equation (fluorescent values after washing/fluorescent values before washing ×100) in each well and then expressed as a percentage [4]. The assays were performed in triplicate, and the averages were used in the results.

the 96-well plates were subjected to absorbance measurements. The adherence was calculated using the equation (fluorescent values after washing/fluorescent values before washing ×100) in each well and then expressed as a percentage [4]. The assays were performed in triplicate, and the averages were used in the results. 5) Collection and treatment of peritoneal fluids The peritoneal fluids were centrifuged at 800 g for 10 minutes within 30 minutes after collection in the operating room, and then the supernatants were tightly sealed and stored in a deep freezer at -70℃. The mixture of the peritoneal fluids from the endometriosis group and the control group was used for the treatment of cells. The endometrial cells (1×106 cells) were aliquoted in 100 mm dishes. After incubation for 48 hours, the peritoneal fluids mixed with 10% of the culture media were added, and the cells were treated for 24 hours. The cells were then collected for further analysis. 6) Anti-CD44 antibody treatment The endometrial cells collected for the adherence assay with peritoneal cells (1×106 cells/mL) were treated with 10 µg/mL of anti-CD44 antibody (Lab vision corp., USA) at 37℃ for 30 minutes, and then the cells were collected for further analysis.

5) Collection and treatment of peritoneal fluids The peritoneal fluids were centrifuged at 800 g for 10 minutes within 30 minutes after collection in the operating room, and then the supernatants were tightly sealed and stored in a deep freezer at -70℃. The mixture of the peritoneal fluids from the endometriosis group and the control group was used for the treatment of cells. The endometrial cells (1×106 cells) were aliquoted in 100 mm dishes. After incubation for 48 hours, the peritoneal fluids mixed with 10% of the culture media were added, and the cells were treated for 24 hours. The cells were then collected for further analysis. 6) Anti-CD44 antibody treatment The endometrial cells collected for the adherence assay with peritoneal cells (1×106 cells/mL) were treated with 10 µg/mL of anti-CD44 antibody (Lab vision corp., USA) at 37℃ for 30 minutes, and then the cells were collected for further analysis. 7) RNA separation and RT-PCR For RNA separation, the cells were dissolved by adding Trizol (Gibco, BRL) to the tissues, and chloroform (Sigma), 10% of total volume, was added prior to centrifugation to obtain supernatant. Then, the same amount of isopropanol (Sigma) was mixed to precipitate RNA. After washing the RNA precipitates with 75% ethanol, the supernatant was removed through centrifugation, and the precipitates were air-dried. The dried RNA was dissolved in diethylpyrocarbonate (DEPC, Sigma) and then subjected to absorbance and purity measurements at 260 and 280 nm, respectively. The dried RNA was stored in at -70℃ until needed for further analysis.

nol, the supernatant was removed through centrifugation, and the precipitates were air-dried. The dried RNA was dissolved in diethylpyrocarbonate (DEPC, Sigma) and then subjected to absorbance and purity measurements at 260 and 280 nm, respectively. The dried RNA was stored in at -70℃ until needed for further analysis. A total of 2 µg of RNA was added to the reaction solution containing 5×RNA PCR buffer, dNTP (2.5 mM), MgCl2 (25 mM), oligo dT primer, RNase inhibitor, and MMLV reverse transcriptase (Promega) and reacted at 42℃ for 60 minutes and at 95℃ for 5 minutes to synthesize cDNA. PCR was performed using the synthesized cDNA as templates. PCR was carried out utilizing CD44 primer (forward primer, 5' TCC CAG TAT GAC ACA TAT TGC 3'; reverse primer, 5' CAC CTT CTT CGA CTG TTG AC 3') [16]. The expression of mRNA was standardized by GAPDH (forward primer, 5' CGG AGT CAA CGG GAT ATG GA 3'; reverse primer, 5' AGC CTT CTC CAT GGT GGT GAA GAC 3') in each sample. The results of the PCR products were confirmed by gel electrophoresis with 2% agarose gel. 8) Western blot analysis Proteins were extracted from the cultured cells using cell extraction buffer (Pierce, Rockford, IL, USA) and then quantified utilizing Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA).

A total of 2 µg of RNA was added to the reaction solution containing 5×RNA PCR buffer, dNTP (2.5 mM), MgCl2 (25 mM), oligo dT primer, RNase inhibitor, and MMLV reverse transcriptase (Promega) and reacted at 42℃ for 60 minutes and at 95℃ for 5 minutes to synthesize cDNA. PCR was performed using the synthesized cDNA as templates. PCR was carried out utilizing CD44 primer (forward primer, 5' TCC CAG TAT GAC ACA TAT TGC 3'; reverse primer, 5' CAC CTT CTT CGA CTG TTG AC 3') [16]. The expression of mRNA was standardized by GAPDH (forward primer, 5' CGG AGT CAA CGG GAT ATG GA 3'; reverse primer, 5' AGC CTT CTC CAT GGT GGT GAA GAC 3') in each sample. The results of the PCR products were confirmed by gel electrophoresis with 2% agarose gel. 8) Western blot analysis Proteins were extracted from the cultured cells using cell extraction buffer (Pierce, Rockford, IL, USA) and then quantified utilizing Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA). The protein extracts, 20 µg of each experimental group, were loaded on 10% SDS-polyacrylamide gel to separate the proteins via gel electrophoresis, and the acrylamide gel with separated proteins was transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated in TBS with 0.1% TBS-T containing 5% non-fat dry milk for 1 hour in order to block non-specific binding. Then, the membrane was reacted with 1:1000 anti-CD44H (R&D Systems, Minneapolis, MN, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4℃ overnight, followed by six ten-minute washes with TBS-T. Then, the membrane was reacted with 1:5,000 horseradish peroxidase (HRP)-conjugated anti-mouse goat Ig G antibody (Santa Cruz, San Diego, CA, USA) diluted by TBS-T at room temperature for 1 hour and washed six times for 5 minutes each. Lastly, the proteins were developed using an enhanced chemiluminescence (ECL) kit (Western blotting detection reagents, Amersham-Biosciences), and the X-ray film was exposed to light to determine protein expression.

Diego, CA, USA) diluted by TBS-T at room temperature for 1 hour and washed six times for 5 minutes each. Lastly, the proteins were developed using an enhanced chemiluminescence (ECL) kit (Western blotting detection reagents, Amersham-Biosciences), and the X-ray film was exposed to light to determine protein expression. 9) Statistical analysis All statistical analysis was performed using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). Mann-Whitney U-test was applied to compare the adherence of ESCs to the peritoneal cells between the two groups, while the Kruskall-Wallis test was utilized in the comparison of the three groups after the treatment with peritoneal fluids. A P-value <0.05 was considered to be statistically significant. Results 1. Adherence of ESCs to peritoneal cells The adherence of ESCs to peritoneal cells is shown in Fig. 1. Slightly higher adherence was observed in the endometriosis group (42.8±7.4%) compared to the control group (38.9±6.4%), but no statistically significant differences were found (P=0.204). 2. CD44 mRNA expression in endometrial stromal cells RT-PCR was carried out using a primer from the study by Cannistra et al. [5] in order to identify expression of CD44 standard (CD44s) type and variant type in ESCs (Fig. 2). CD44s isoforms were expressed in both the endometriosis group and the control group, whereas the expression of variant CD44 isoforms was not observed in either group. Statistically significant differences were not found in the expression of CD44 types between the endometriosis group and the control group.

SCs (Fig. 2). CD44s isoforms were expressed in both the endometriosis group and the control group, whereas the expression of variant CD44 isoforms was not observed in either group. Statistically significant differences were not found in the expression of CD44 types between the endometriosis group and the control group. 3. CD44 protein expression in endometrial stromal cells Fig. 3 demonstrates the expression of CD44s proteins via Western blot analysis. The CD44 protein expression in the endometriosis group and the control group showed differences between individuals, but expression was not consistent in each group. In addition, the CD44 protein expression in ESCs of each individual did not show a significant correlation with the adherence to peritoneal cells. 4. CD44 protein expression of the ESCs treated with anti-CD44 antibody and the adherence to peritoneal cells Fig. 4 describes the results of anti-CD44 antibody treatment of ESCs to evaluate the effects of CD44 expression on the adherence to the peritoneal cells. Anti-CD44 antibody induced remarkable reductions in CD44 protein expression in the endometriosis group as well as the control group, but the reductions were not significantly different between the two groups (Fig. 4A). However, despite the decrease of CD44 protein expression in ESCs, the adherence to peritoneal cells was not decreased in either the endometriosis group or the control group (Fig. 4B).

endometriosis group as well as the control group, but the reductions were not significantly different between the two groups (Fig. 4A). However, despite the decrease of CD44 protein expression in ESCs, the adherence to peritoneal cells was not decreased in either the endometriosis group or the control group (Fig. 4B). 5. Adherence of ESCs treated with peritoneal fluids to the peritoneal cells The adherence of the ESCs to peritoneal cells after treatment with peritoneal fluids was not found to be significantly different among the control group, non-peritoneal fluid-treated group, normal peritoneal fluid-treated group, and endometriosis peritoneal fluid-treated group (Fig. 5A). In the endometriosis group, on the other hand, the adherence tended to be the same or slightly increased in the normal peritoneal fluid-treated group compared to the non-peritoneal fluid-treated group. In particular, when treating with endometriosis peritoneal fluids, there was a statistically significant increase in the adherence of ESCs to the peritoneal cells (Fig. 5B).

rence tended to be the same or slightly increased in the normal peritoneal fluid-treated group compared to the non-peritoneal fluid-treated group. In particular, when treating with endometriosis peritoneal fluids, there was a statistically significant increase in the adherence of ESCs to the peritoneal cells (Fig. 5B). Discussion The attachment of endometrial cells to the peritoneum is an essential step in the development of endometriosis. Lucidi et al. [4] demonstrated that ESCs cultured in experimental models adhere to PMCs. Recently, a study performed by Griffith et al. [17] compared endometriosis patients with normal women using menstrual endometrial cells. For the first time, they reported that the adherence to PMCs was increased in endometriosis patients and further suggested that the menstrual endometrium of endometriosis patients was different from that in normal women. In the present study, the ESCs of the endometriosis group and normal group were cultured, and the adherence to PMCs was tested. We found that the adherence tended to increase in the endometriosis group, but statistically significant differences were not exhibited. However, it seems to be very difficult to draw definitive conclusions because the endometrial tissues collected during surgery were not classified based upon menstrual cycles in this study, and the study by Griffith was focused on the endometrial tissues during menstruation assuming that the pathogenesis of endometriosis would be the backflow of menstruation.

draw definitive conclusions because the endometrial tissues collected during surgery were not classified based upon menstrual cycles in this study, and the study by Griffith was focused on the endometrial tissues during menstruation assuming that the pathogenesis of endometriosis would be the backflow of menstruation. In the analysis of the CD44 expression of ESCs in this study, the CD44 mRNA and protein were expressed, but the variant type of CD44 was not, without notable differences between the endometriosis group and the control group. As mentioned earlier, studies have investigated CD44 isoform expression in the endometrium, but the results were inconsistent. For normal endometrium, CD44 expression was increased in the secretory phase rather than proliferative phase [8,10], and CD44 was not always expressed in the proliferative phase [12,18]. Moreover, studies found that all CD44s were expressed in the secretory phase, while consistent patterns were not exhibited in the expression of variant type [8-12,18]. As shown in the present study, CD44 expression in endometria from endometriosis patients did not show any difference from normal endometrial tissues [9], or the expression of some variant types was increased in endometriosis patients [17]. Such differences may be due to differences in experimental methods. In particular, in the earlier studies, CD44 expression was mostly measured via immunohistochemical methods, while protein expression was determined through Western blot analysis after culture of the endometrial tissues. Furthermore, the small number of subjects, about 20 subjects in most studies, may have an effect on the results. In this study, the finding that variant type CD44 was not expressed could be due to the insufficient number of subjects as well as the different experimental methods. In addition, the timing of endometrial tissue collection seems to be important. According to the studies reported so far, CD44 expression is increased in the secretory phase and during menstruation and is more likely for the standard type than the variant type. In the present study, endometrial tissues were obtained during surgery. Considering that the surgeries were mostly performed within two weeks after menstruation, more endometrial tissues would be present in the proliferative phase than in the secretory phase, so that the variant type was not expressed, even though the obtained endometrial tissues were not classified by menstrual cycle.

ry. Considering that the surgeries were mostly performed within two weeks after menstruation, more endometrial tissues would be present in the proliferative phase than in the secretory phase, so that the variant type was not expressed, even though the obtained endometrial tissues were not classified by menstrual cycle. Anti-CD44 antibody treatment of ESCs showed a notable decrease in the expression of CD44 proteins in endometriosis patients and the control group, while the adherence of peritoneal cells was not affected. In contrast, the adherence to peritoneal cells was remarkably increased when ESCs of the endometriosis patients were treated with endometriosis peritoneal fluids. CD44 adheres to extracellular matrix HA and induces cell-to-cell attachments and cell-to-matrix attachments. It is known that CD44 is particularly involved in the adherence of gastric carcinoma and ovarian carcinoma cells to peritoneal mesothelium [5,6]. Many studies have shown that the adherence to peritoneal cells was reduced when ovarian carcinoma cells were treated with anti-CD44 antibody or hyaluronidase [5,6,19]. However, CD44 expression in the present study was not significantly different between the endometriosis group and the control group, and the adherence to peritoneal cells was not influenced by the treatment with anti-CD44 antibody. Therefore, it seems that CD44 does not play an important role in peritoneal adherence of endometrial cells for endometriosis. The remarkable increase in adherence when ESCs of endometriosis patients were treated with endometriosis peritoneal fluids is in agreement with the results from previous studies indicating that cytokines and adhesion molecules in peritoneal fluids are associated with the peritoneal attachments of the endometrial tissues [20,21]. Additionally, considering that the adherence to peritoneal cells was not affected even with treatment with endometriosis peritoneal fluids in the ESCs of the control group, abnormalities in endometrial tissues of endometriosis patients are suggested to be considerably responsible for the occurrence of endometriosis [3].

dditionally, considering that the adherence to peritoneal cells was not affected even with treatment with endometriosis peritoneal fluids in the ESCs of the control group, abnormalities in endometrial tissues of endometriosis patients are suggested to be considerably responsible for the occurrence of endometriosis [3]. In conclusion, CD44 expression in ESCs did not differ between the endometriosis group and the control group. Although a decrease of CD44 protein expression was observed in the endometriosis group and the control group after the CD44 antibody treatment, there were no differences in the attachment of ESCs to PMCs between the two groups even after the decrease in CD44 protein. Thus, CD44 expression does not seem to be directly associated with the attachment of ESCs to PMCs. However, since our study did not classify the ESCs based upon menstrual cycle, further studies are necessary to clarify if similar results are obtained when using endometrial cells in the secretory phase and during menstruation, where CD44 expression is known to be increased. Acknowledgments This study was supported by the Medical Research Institute Grant (2005-21), Pusan National University Hospital. Fig. 1 Endometrial stromal cell adherence to peritoneal mesothelial cells in the normal group and the endometriosis group, as measured by peritoneal mesothelial cells adherence assay. Fig. 2 Expression of CD44 mRNA in the control group (A) and the endometriosis group (B), as measured by reverse transcription-polymerase chain reaction.

Fig. 1 Endometrial stromal cell adherence to peritoneal mesothelial cells in the normal group and the endometriosis group, as measured by peritoneal mesothelial cells adherence assay. Fig. 2 Expression of CD44 mRNA in the control group (A) and the endometriosis group (B), as measured by reverse transcription-polymerase chain reaction. Fig. 3 Expression of CD44 protein in the control group (A) and the endometriosis group (B), as measured by Western blot analysis. Fig. 4 Expression of CD44 protein after anti-CD44 antibody treatment in the control group and the endometriosis group, as measured by Western blot analysis (A). Peritoneal mesothelial cells adherence assay after anti-CD44 antibody treatment in the control group and the endometriosis group (B). Fig. 5 Peritoneal mesothelial cells adherence assay after peritoneal fluid treatment in the control group (A) and the endometriosis group (B). Con, control; Ep, endometriosis peritoneal fluid; Np, normal peritoneal fluid. Significantly different *P < 0.05, **P < 0.01.

Introduction Based on the causal relationship of persistent high-risk human papillomavirus (HPV) infection in cervical carcinogenesis [1], HPV tests have been introduced as part of cervical cancer screening. Since receiving U.S. Food and Drug Administration (FDA) approval, the HPV test has been used to triage equivocal cervical cytology and as a co-test for women aged 30 years and older [2]. The HPV test is more reliable and more sensitive but less specific than routinely performed cervical cytology for detecting cervical intraepithelial neoplasia [3].

Drug Administration (FDA) approval, the HPV test has been used to triage equivocal cervical cytology and as a co-test for women aged 30 years and older [2]. The HPV test is more reliable and more sensitive but less specific than routinely performed cervical cytology for detecting cervical intraepithelial neoplasia [3]. Because HPV16 and HPV18 are the most commonly observed genotypes of cervical cancer, a few HPV genotyping tests and Cervista HPV16/18, the first FDA-approved HPV genotyping test, have been validated in clinical trials for assessing persistent HPV infection and detecting cervical precancerous lesions and cancer [4,5]. However, HPV16 and HPV18 are more commonly detected in invasive cancer than in high-grade precancerous lesions, although their rankings appear to be similar [6-8]. In fact, Clifford et al. [9] demonstrated the different squamous cell carcinoma (SCC)/low-grade squamous intraepithelial lesions (LSIL) prevalence ratios of each high-risk HPV genotype, highlighting the importance of individual HPV genotypes for the risk of progression from LSIL to malignancy. Even in in situ cervical cancer, the proportions and rankings of individual high-risk HPV genotypes significantly differed from those of invasive cervical cancer [10]. According to previous Korean studies, aside from HPV16, HPV33, HPV58, and HPV31 were the most prevalent HPV types in cervical cancer, whereas HPV52, HPV58, and HPV51 were commonly detected in precancerous lesions [11-13].

vidual high-risk HPV genotypes significantly differed from those of invasive cervical cancer [10]. According to previous Korean studies, aside from HPV16, HPV33, HPV58, and HPV31 were the most prevalent HPV types in cervical cancer, whereas HPV52, HPV58, and HPV51 were commonly detected in precancerous lesions [11-13]. Many data from many countries have been published regarding the type-specific prevalence of high-risk HPV [8,11,13,14], but the results of HPV genotyping surveillance combined with cervical cytology has been rarely reported in the well-organized screening programs for the healthy Korean population [15,16]. Therefore, we conducted a retrospective study to investigate the type-specific risk of high-risk HPV in the development and progression of precancerous or invasive cervical diseases, and to present the foundations for a follow-up guideline for high-risk HPV-positive normal cytology subjects based on the data from routine health care screenings. It is our hope that this information supports the understanding of which HPV types future vaccines should target. As baseline data in this cross-sectional study, we evaluated the type-specific prevalence of high-risk HPV types and their distribution by cervical cytology severity and age.

routine health care screenings. It is our hope that this information supports the understanding of which HPV types future vaccines should target. As baseline data in this cross-sectional study, we evaluated the type-specific prevalence of high-risk HPV types and their distribution by cervical cytology severity and age. Materials and Methods 1. Study population A total of 16,600 women visited Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Korea for a routine health check-up between December 2008 and October 2010. Among the women who were screened, 7,014 consecutive subjects who underwent both a liquid-based cervical cytology (SurePath LBC, Beckton Dickinson, Franklin Lakes, NJ, USA) and an HPV genotyping test by HPV DNA chip (MyHPV Chip, Biomedlab Co., Seoul, Korea) for cervical cancer screening were analyzed in this cross-sectional study. All of the participants were asked to complete systemic questionnaires regarding demographic characteristics, past medical histories, history of surgical diseases, and present medical condition/medication. Information regarding menstruation/pregnancy history, history of contraceptive methods and hormone use, menopausal status, lifestyle characteristics (such as smoking history) and clinical infection regarding gynecologic surgeries, including hysterectomy and/or ovarian surgery, was obtained via direct interview by three expert gynecologists. Based on the responses to the questionnaires and the medical interview, we excluded the following subjects: those with a history of invasive cervical cancer or precancerous lesions, those who had received a hysterectomy and/or bilateral salpingo-oophorectomy and those who had undergone any procedures for treating cervical intraepithelial neoplasm, such as loop excisional electrosurgical procedure, within the previous 5 years. Postmenopausal status was defined as the cessation of menses for at least 1 year or the presence of 40 international units (IU)/L or more of serum follicle-stimulating hormone (FSH). The women were grouped according to age at 10-year intervals from 20 to 60 years. The women aged 60 years or older were grouped in a single comparably sized group.

defined as the cessation of menses for at least 1 year or the presence of 40 international units (IU)/L or more of serum follicle-stimulating hormone (FSH). The women were grouped according to age at 10-year intervals from 20 to 60 years. The women aged 60 years or older were grouped in a single comparably sized group. 2. HPV DNA test DNA isolation was sequentially performed on the remaining suspension of the SurePath Pap test vial. For the HPV genotyping, we used a commercially available HPV DNA chip, which is a PCR-based DNA microarray system. The HPV DNA chip contains 24 type-specific probes for 12 types of high-risk HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), four probable high-risk HPV types (types 53, 54, 66, and 68) and eight types of low-risk HPV (types 6, 11, 34, 40, 42, 43, 44, and 70) [17].

h is a PCR-based DNA microarray system. The HPV DNA chip contains 24 type-specific probes for 12 types of high-risk HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), four probable high-risk HPV types (types 53, 54, 66, and 68) and eight types of low-risk HPV (types 6, 11, 34, 40, 42, 43, 44, and 70) [17]. Twenty-four type-specific 30-meroligonucleotide probes containing an amine group at the 5'teminus were immobilized onto a chip slide of glass. Each slide had eight chambers, and each chamber was used for a test. Briefly, the target HPV DNA was amplified by polymerase chain reaction (PCR) using the primers (HPV and β-globin) and conditions provided by the Biomedlab Company. The amplified DNA was then labeled with a single dye, indocardocyanine-dUTP (MEM Life Science Products Inc., Boston, MA, USA). The PCR product size of the HPV DNA was 150 base pairs (bp) on the gel electrophoresis. Next, the PCR product was hybridized onto the chip. Hybridization was performed at 43℃ for 90 minutes; the product was then washed with 3×saline-sodium phosphate-ethylenediamine tetra-acetic acid (SSPE) for 5 minutes and 1×SSPE for 5 minutes and dried at room temperature. The hybridized signals were visualized with a DNA chip scanner (Scanarray lite, GSI Lumonics, Ottawa, Ontario, Canada). The samples that showed a positive band of 150 bp on the gel electrophoresis of the HPV-PCR but a negative hybridized signal on the DNA chip scanner were designated as HPV-other samples [18]. None of the negative controls (without DNA) revealed HPV positivity.

er (Scanarray lite, GSI Lumonics, Ottawa, Ontario, Canada). The samples that showed a positive band of 150 bp on the gel electrophoresis of the HPV-PCR but a negative hybridized signal on the DNA chip scanner were designated as HPV-other samples [18]. None of the negative controls (without DNA) revealed HPV positivity. Initially, we used an HPV DNA chip that covered 24 genotypes. In December 2009, our institution introduced an HPV DNA chip that identified 19 genotypes covering 11 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58) and eight low-risk HPV types (6, 11, 34, 40, 42, 43, 44, and 54); thereafter, HPV59, one of the 12 high-risk HPV genotypes, was omitted from genotyping. High-risk HPV was ultimately defined by Munoz et al. [19] as the presence of one or more of the following 11 HPV types: types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58. Type-specific high-risk HPV prevalence is expressed as the proportion of all high-risk HPV-positive subjects, including all subjects who were positive for either a single infection or a co-infection with HPV genotypes other than the corresponding HPV genotype.

ypes: types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58. Type-specific high-risk HPV prevalence is expressed as the proportion of all high-risk HPV-positive subjects, including all subjects who were positive for either a single infection or a co-infection with HPV genotypes other than the corresponding HPV genotype. 3. Cervical cytology Independent of the HPV testing result, all of the cytologies on the slides were read according to the routine laboratory protocol and reported a cytological classification based on the Bethesda nomenclature system [20]: squamous cervical carcinoma/adenocarcinoma, high-grade squamous intraepithelial lesions (HSIL), atypical squamous cells, cannot exclude HSIL (ASC-H), atypical glandular cells (AGC), LSIL, atypical squamous cells of undetermined significance (ASCUS) and negative. A panel of cytopathologists was masked to the HPV DNA chip test results. To analyze the trends of individual HR-HPV genotypes distribution, all cervical cytologies were grouped as follows: negative, ASCUS/LSIL, and HSIL or worse. Because of their substantial likelihood of developing cervical cancer [21], 11 subjects with ASC-H were categorized as HSIL or worse; two subjects with AGC were excluded from the study population. 4. Statistical analysis The baseline characteristics of continuous variables were compared using Student's t-test. For categorical variables, the chi-squared (χ2) test or Fisher's exact test was used. The data were analyzed with SPSS ver. 19.0 (IBM, Armonk, NY, USA), and two-sided P-values <0.05 were considered statistically significant.

ysis The baseline characteristics of continuous variables were compared using Student's t-test. For categorical variables, the chi-squared (χ2) test or Fisher's exact test was used. The data were analyzed with SPSS ver. 19.0 (IBM, Armonk, NY, USA), and two-sided P-values <0.05 were considered statistically significant. Written informed consent was obtained from all participants. This retrospective cross-sectional study was approved by the Institutional Review Board of Seoul National University Hospital (IRB-No. H-1012-095-344). Results 1. Baseline characteristics A total of 7,014 women with both cervical cytology and HPV test results were enrolled. The mean age of the participants was 48.2±9.2 years, and 81.5% were aged 40 years or older. The clinical characteristics of the subjects are shown in Table 1. The overall prevalence of abnormal cervical cytologies (ASCUS or worse) was 4.7%, showing a U-shaped trend according to age groups: the highest prevalence was in the youngest group, the lowest was in the 30- to 39-year-old group, and there was a slight increase in the oldest group (13.2%, 2.7%, and 3.9%, respectively) (Fig. 1). Squamous cervical carcinoma in the cervical cytology was noted only in two subjects.

shaped trend according to age groups: the highest prevalence was in the youngest group, the lowest was in the 30- to 39-year-old group, and there was a slight increase in the oldest group (13.2%, 2.7%, and 3.9%, respectively) (Fig. 1). Squamous cervical carcinoma in the cervical cytology was noted only in two subjects. The overall positivity for any HPV and high-risk HPV was 19.7% and 8.4%, respectively. When the HPV prevalence was reexamined with regard to the HPV DNA chip probes, no significant difference in the overall prevalence of high-risk HPV was observed between the two probes of the DNA chip (9.3% for the 24-probe chip and 8.3% for 19-probe chip, P=0.131). In comparison, the overall prevalence of any HPV was significantly higher with the 24-probe chip than with the 19-probe chip (21.9% vs. 18.2%, P<0.001), whereas the prevalence of HPV-other was significantly lower with the 24-probe chip than with the 19-probe chip (33.8% vs. 44.2%, P<0.001; data not shown). 2. Prevalence and distribution of high-risk HPV types according to age groups The overall prevalence of any HPV and high-risk HPV declined markedly with age (P for trend<0.001 for each) (Table 2). The youngest group had the highest positivity for high-risk HPV, and the oldest had the lowest positivity, showing an age-dependent decrease (29.0% and 5.5%, respectively; P for trend<0.001). In contrast, the proportion of HPV-other showed a significant age-specific increase (P for trend<0.001). There was no age-specific trend for low-risk HPV prevalence.

sitivity for high-risk HPV, and the oldest had the lowest positivity, showing an age-dependent decrease (29.0% and 5.5%, respectively; P for trend<0.001). In contrast, the proportion of HPV-other showed a significant age-specific increase (P for trend<0.001). There was no age-specific trend for low-risk HPV prevalence. We then calculated the proportion of individual high-risk HPV types among high-risk HPV positive-subjects. In total, the most prevalent HPV types were HPV58 (23.9%), HPV16 (21.8%), HPV52 (16.6%), HPV18 (11.7%), HPV33 (9.0%), and HPV35 (9.0%), in order of frequency. Regarding the age-specific prevalence trend of individual high-risk HPV types, there was a marked decline in the proportion of HPV52 with age, ranging from 21.1% in the youngest group to 11.4% in the oldest group (P for trend=0.014). Among women aged 30 years or older, the proportion of HPV39 also progressively decreased with increasing age (P for trend=0.051, data not shown). The proportion of the other high-risk HPV types, including HPV16, did not vary significantly according to age group. Co-infection with several HPV types was detected in 19.0% (262 out of 1,380) of the HPV-positive women and was markedly prevalent in women aged 30 years or younger, but showed no significant age-specific trend.

portion of the other high-risk HPV types, including HPV16, did not vary significantly according to age group. Co-infection with several HPV types was detected in 19.0% (262 out of 1,380) of the HPV-positive women and was markedly prevalent in women aged 30 years or younger, but showed no significant age-specific trend. 3. Prevalence and distribution of high-risk HPV types according to cervical cytology The prevalence of any HPV, high-risk HPV and the proportion of individual high-risk HPV genotypes according to cervical cytology are shown in Table 3. As a whole, the prevalence of any HPV and all high-risk HPV types increased progressively with increasing severity of cervical cytology (P for trend<0.001, respectively). For low-risk HPV, no age-specific trend was observed. In terms of individual HPV genotypes, only HPV56 showed a strong increase in proportion with cervical cytology severity (P for trend=0.041), ranging from 7.2% of subjects with normal cytology to 11.7% and 12.5% of subjects with ASCUS/LSIL and HSIL or worse. Among women aged 30 years or older, the positivity of HPV52 and HPV56 was marginally related to abnormal cervical cytology (P=0.055 and P=0.058 for each, data not shown). For the other high-risk HPV genotypes, including HPV16 and HPV18, no significant type-specific trends were observed according to the cervical cytology severity.

aged 30 years or older, the positivity of HPV52 and HPV56 was marginally related to abnormal cervical cytology (P=0.055 and P=0.058 for each, data not shown). For the other high-risk HPV genotypes, including HPV16 and HPV18, no significant type-specific trends were observed according to the cervical cytology severity. Co-infection with several HPV types also significantly increased with the severity of cervical cytology (P for trend=0.029). Among the high-risk HPV-positive women who had abnormal cervical cytology (≥ASCUS), 45.4% (54 out of 119) tested positive for more than one HPV type. The positivity of HPV-other significantly increased with the increasing severity of cervical cytology (P for trend=0.026). Discussion The principal finding of our study was that the most prevalent HPV types were HPV58, HPV16, HPV52, and HPV18, all of which accounted for 73.9% of the 591 high-risk HPV infections detected. In terms of individual HPV types by cervical cytology and age, there was a significantly increased prevalence of HPV56 with cervical cytology of increasing severity. The prevalence of HPV 52 had a marked age-specific decline but a marginal incease with cervical cytology severity among women aged 30 years or older. Additionally, an age-specific decline in the overall high-risk HPV prevalence was reaffirmed independent of cervical cytology, except for HSIL or worse (Fig. 2).

everity. The prevalence of HPV 52 had a marked age-specific decline but a marginal incease with cervical cytology severity among women aged 30 years or older. Additionally, an age-specific decline in the overall high-risk HPV prevalence was reaffirmed independent of cervical cytology, except for HSIL or worse (Fig. 2). Among women aged 30 years or younger, the prevalence of any HPV and high-risk HPV reached peak; however, there was no discrete trend in type-specific HPV prevalence by cervical cytology. The overall prevalence of abnormal cervical cytology (ASCUS or worse) also peaked in the youngest age group (Fig. 1). In the youngest group, however, no subjects had severe abnormal cervical cytology (HSIL or worse), whereas all of the subjects with SCC were in the oldest group (data not shown). These findings correspond well to the background of the revised American Society for Colposcopy and Cervical Pathology (ASCCP) guideline [22] and can be supported by the study by Trottier et al. [23] on incident HPV infection and the timing of cervical lesions in young women. They reported that women who did not experience an HPV infection had a very small risk of developing a lesion, but that a transient high-risk HPV infection as short as 3 months was associated with the risk of cervical abnormality, and persistent cervical high-risk HPV infection, specifically with HPV16/18, was a much stronger predictor of cervical lesions.

xperience an HPV infection had a very small risk of developing a lesion, but that a transient high-risk HPV infection as short as 3 months was associated with the risk of cervical abnormality, and persistent cervical high-risk HPV infection, specifically with HPV16/18, was a much stronger predictor of cervical lesions. Of all of the HPV-tested subjects, HPV58 was the most common type in normal or mildly abnormal cervical cytology (ASCUS/LSIL), consistent with other data on type-specific HPV prevalence in either Korean or Asian populations. However, in our study, the prevalence of HPV58 did not have a significant association with the severity of cervical cytology, unlike other studies in which there was the high prevalence of HPV58 in both HSIL and SCC [24,25].

nsistent with other data on type-specific HPV prevalence in either Korean or Asian populations. However, in our study, the prevalence of HPV58 did not have a significant association with the severity of cervical cytology, unlike other studies in which there was the high prevalence of HPV58 in both HSIL and SCC [24,25]. A significant type-specific trend toward cervical cytology severity in our study was observed only for HPV56. Among women aged 30 years or older, the prevalences of HPV52 and HPV56 were marginally associated with abnormal cervical cytology. Despite the small number of subjects with abnormal cervical cytology (including only one HPV56-positive subject who had HSIL or worse), the prevalence of HPV56 was relatively high, specifically in abnormal cervical cytology, compared with the prevalence reported in other studies [13,26]. In the study by Choi et al. [26], HPV56 had a relatively low prevalence, but it was more commonly detected in low-grade cervical intraepithelial neoplasia (CIN) than were HPV16, HPV58, and HPV52. HPV52 has been often noted as one of the prevalent high-risk HPV types in precancerous lesions in the Korean population, followed by HPV16, HPV18, and HPV58 [27,28]. In comparison, the prevalence of HPV16 in our study was considerably low (1.8% of the women overall and 21.8% of the high-risk HPV-positive women), with only a 12.5% prevalence among the 22 subjects who had HSIL or worse. All of these findings are inconsistent with the findings of other studies [24,25,29-30]. Most studies have reported that HPV16 was the most prevalent type [6,10,29,30], reaching an overall prevalence of 3.3% and a range from 1.5% in normal cytology to 45.6% in HSIL+ and 9.7% at age 20 to 29 years to 0.7% at age 50 to 64 years [29].

findings are inconsistent with the findings of other studies [24,25,29-30]. Most studies have reported that HPV16 was the most prevalent type [6,10,29,30], reaching an overall prevalence of 3.3% and a range from 1.5% in normal cytology to 45.6% in HSIL+ and 9.7% at age 20 to 29 years to 0.7% at age 50 to 64 years [29]. One possible explanation for our findings' inconsistency with other studies is that the proportion of young women (<30 years old) was very low (only 1.9%) in our study population. The predominance of HPV16 in young women has been already noted across the cervical disease categories [10,31,32]. Some authors have demonstrated that the age at diagnosis of HPV16- and HPV18-related invasive cancers was 5 years earlier than the age of diagnosis for cancers caused by high-risk HPV types other than HPV16/18, suggesting that the age of first cervical cancer screening could be delayed in the HPV-vaccinated population [10].

authors have demonstrated that the age at diagnosis of HPV16- and HPV18-related invasive cancers was 5 years earlier than the age of diagnosis for cancers caused by high-risk HPV types other than HPV16/18, suggesting that the age of first cervical cancer screening could be delayed in the HPV-vaccinated population [10]. Another possible explanation is that our study population consists of apparently healthy people who voluntarily seek health check-ups and thus have a substantially low prevalence of abnormal cervical cytology (less than 5%). In a recent population-based study, HPV16 was the most commonly encountered HPV type even in normal cervical cytology, but its prevalence was substantially low among all tested women [33]. It has also been noted that the performance of each of high-risk HPV detection methods differs when applied to the detection of genital HPV DNA, especially in cervical swabs with low grades of dysplasia or normal cells [34]. Presumably, the relatively much lower detection rate of HPV DNA in our study population is related to the lower levels of HPV DNA in these specimens [34,35].

sk HPV detection methods differs when applied to the detection of genital HPV DNA, especially in cervical swabs with low grades of dysplasia or normal cells [34]. Presumably, the relatively much lower detection rate of HPV DNA in our study population is related to the lower levels of HPV DNA in these specimens [34,35]. Finally, we assume that individual HPV types have different potentials to develop abnormal cytology from normal uterine cervixes, similar to those involved in the progression from LSIL to SCCA suggested by Clifford et al. [9]. Despite the fact that HPV16 and HPV18 are the most commonly detected high-risk HPV types, the prevalence and distribution of the high-risk HPV types that are prevalent in precancerous lesions differ greatly from those of cervical cancers, depending on the subjects' race and geography [29,36,37]. In addition, it can be also supported by the fact that the efficacy of the HPV vaccine has been attributed to its protection against lesions associated with non-vaccine HPV types, which are shown to be higher against CIN3+ than against CIN2+, thereby indicating the greater contribution of HPV16 and HPV18 to precancerous lesions of increasing severity [38].

by the fact that the efficacy of the HPV vaccine has been attributed to its protection against lesions associated with non-vaccine HPV types, which are shown to be higher against CIN3+ than against CIN2+, thereby indicating the greater contribution of HPV16 and HPV18 to precancerous lesions of increasing severity [38]. Regarding co-infection with several HPV types, the proportion of HPV16 in our study was inconsistent with that reported in other studies. According to ARTISTIC trial data, the proportion of women positive for HPV16 as a single infection increased with increasing severity of cervical cytology, thus suggesting that HPV16 may compete with less virulent types in the progression to neoplasia rather than in normal infective processes [29].

ed in other studies. According to ARTISTIC trial data, the proportion of women positive for HPV16 as a single infection increased with increasing severity of cervical cytology, thus suggesting that HPV16 may compete with less virulent types in the progression to neoplasia rather than in normal infective processes [29]. In our study, only 30.9% (103 out of 333) of the subjects who had high-risk HPV-positive ASCUS or LSIL+cytology had available histologic confirmation data. Among the 15 of these 103 subjects who had CIN2 lesions or worse (CIN2+), only 13.3% were positive for HPV16 (95% CI, 1.7% to 40.5%); in all cases, HPV16 was present a as co-infection with other high-risk HPV types. In contrast, the positivity of high-risk HPV types other than HPV16 (i.e., HPV52 and HPV58) was to some extent higher than that of HPV16 (20.0%, 95% CI 4.3% to 48.1% for each). As single infections, the proportions of HPV52 and HPV58 were much higher than that of HPV16 (for HPV52, 13.3%, 95% CI 1.7% to 40.5%; for HPV58, 6.7%, 95% CI 0.17% to 32.0%; for HPV16, 0.0%, 95% CI 0.0% to 21.8%). Considering the prevalence and distribution of HPV52, HPV56 and HPV58 by cervical cytology and age, our study's findings may support the hypothesis that by showing different potentials, high-risk HPV types other than HPV16/18 may play great roles the development of abnormal cervical cytology or precancerous lesions in the normal cervix in the general population. These findings are also consistent with the high-risk HPV types detected in the progression to cervical cancer [24-26,29,38].

ent potentials, high-risk HPV types other than HPV16/18 may play great roles the development of abnormal cervical cytology or precancerous lesions in the normal cervix in the general population. These findings are also consistent with the high-risk HPV types detected in the progression to cervical cancer [24-26,29,38]. Our study has some other limitations. First, because of its cross-sectional design based only on cervical cytology, the attributable type-specific risk of high-risk HPV for cervical precancerous lesions and invasive cancer could not be assessed. However, a further study with follow-up data is being planned with the hope of finding evidence of the role of individual high-risk HPV types in the risk of progression to precancerous lesions and invasive cancer. A recent study by Kjaer et al. [30] reported a good correlation of prevalent high-risk HPV type distributions between subjects with abnormal cervical cytology and those with cervical disease, such as histologically confirmed CIN; based on that study's findings, we can anticipate that individual high-risk HPV types that gradually increase in prevalence with increasing severity of cervical cytology, appear to correspond to the HPV types commonly detected in high-grade CIN.

ogy and those with cervical disease, such as histologically confirmed CIN; based on that study's findings, we can anticipate that individual high-risk HPV types that gradually increase in prevalence with increasing severity of cervical cytology, appear to correspond to the HPV types commonly detected in high-grade CIN. Second, the prevalence of the individual high-risk HPV types in our study is determined by a mathematical sum of both single infection and co-infection with several HPV types other than the corresponding HPV type; therefore, the totals are often greater than 100%. Still, there are no established methods or analyses for addressing the complexity of co-infections with several HPV types, although some authors have developed mathematical models or HPV type assignment algorithms to estimate the contribution of each high-risk HPV type to the development or progression of precancerous lesions and invasive cancer when several HPV types are detected [39,40]. Finally, our study population consisted of healthy subjects who lived primarily in a metropolis and were interested in their health, as they voluntarily participated in the private screening program. These subject characteristics may have led to the very low prevalence of abnormal cervical cytology and CIN, consequently requiring cautious interpretation of our results.

subjects who lived primarily in a metropolis and were interested in their health, as they voluntarily participated in the private screening program. These subject characteristics may have led to the very low prevalence of abnormal cervical cytology and CIN, consequently requiring cautious interpretation of our results. Despite these potential limitations, our study has several advantages. First, to our knowledge, it presents the largest Korean study population to date that includes a wide age range, and combined cervical cytology and HPV genotyping test using an HPV DNA chip for primary cervical cancer screening [11-13]. Second, our study population consists of apparently healthy people who sought health check-ups, which we hope will help to elucidate the natural history of HPV infection and its relationship with the development of cervical diseases.

V genotyping test using an HPV DNA chip for primary cervical cancer screening [11-13]. Second, our study population consists of apparently healthy people who sought health check-ups, which we hope will help to elucidate the natural history of HPV infection and its relationship with the development of cervical diseases. In conclusion, our finding is that the prevalence and distribution of specific HPV types by cervical cytology and age in the apparently healthy population differs significantly from the HPV type distribution and prevalence detected in patients with cervical cancer and precancerous lesions. Given important role of HPV in the pathogenesis of cervical cancer, the development of precancerous lesions and the progression to invasive cancer, we can hypothesize that similar to the progression from LSIL to malignancy reported by Clifford et al. [9], high risk-HPV types other than HPV16 might play a role in the progression from a normal cervix to mild cervical diseases [8]. This hypothesis implies that by identifying the specific high-risk HPV types with different carcinogenic potentials, HPV genotyping test shows clinical relevance in cervical cancer screening programs for the follow-up of type-specific persistency and triage for women aged 30 years or older. Poster presentation at the 26th International Papillomavirus Conference & Clinical Workshop on July 3-8, 2010 in Montréal, Canada.

In conclusion, our finding is that the prevalence and distribution of specific HPV types by cervical cytology and age in the apparently healthy population differs significantly from the HPV type distribution and prevalence detected in patients with cervical cancer and precancerous lesions. Given important role of HPV in the pathogenesis of cervical cancer, the development of precancerous lesions and the progression to invasive cancer, we can hypothesize that similar to the progression from LSIL to malignancy reported by Clifford et al. [9], high risk-HPV types other than HPV16 might play a role in the progression from a normal cervix to mild cervical diseases [8]. This hypothesis implies that by identifying the specific high-risk HPV types with different carcinogenic potentials, HPV genotyping test shows clinical relevance in cervical cancer screening programs for the follow-up of type-specific persistency and triage for women aged 30 years or older. Poster presentation at the 26th International Papillomavirus Conference & Clinical Workshop on July 3-8, 2010 in Montréal, Canada. Fig. 1 The prevalence of abnormal cervical cytology by age. ASCUS, atypical squamous cells with undetermined significance; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; ASC-H, atypical squamous cells, cannot exclude HSIL. *Ptrend<0.001.

Poster presentation at the 26th International Papillomavirus Conference & Clinical Workshop on July 3-8, 2010 in Montréal, Canada. Fig. 1 The prevalence of abnormal cervical cytology by age. ASCUS, atypical squamous cells with undetermined significance; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; ASC-H, atypical squamous cells, cannot exclude HSIL. *Ptrend<0.001. Fig. 2 The prevalence of high-risk human papillomavirus by age and cervical cytology. ASCUS, atypical squamous cells with undetermined significance; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; ASC-H, atypical squamous cells, cannot exclude HSIL. Table 1 Characteristics of study population Values are presented as mean±standard deviation or number (%). HRT, hormone replacement therapy; WNL, negative for malignancy; ASCUS, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial neoplasia; ASC-H, atypical squamous cells; HSIL, high-grade squamous intraepithelial neoplasia; SCCA, squamous cell carcinoma; HR-HPV, high risk-human papillomavirus; LR-HPV, low risk-human papillomavirus; HPV-Other, unspecified HPV genotypes. Table 2 Distribution of individual HR-HPV genotype according to age groups Values are presented as number of cases (%). HR-HPV, high risk-human papillomavirus; HPV-other, unspecified HPV genotype. a)Data are number of cases and % representing the proportion of individual HPV genotypes among all high risk-HPV positive women unless otherwise specified.

Table 2 Distribution of individual HR-HPV genotype according to age groups Values are presented as number of cases (%). HR-HPV, high risk-human papillomavirus; HPV-other, unspecified HPV genotype. a)Data are number of cases and % representing the proportion of individual HPV genotypes among all high risk-HPV positive women unless otherwise specified. Table 3 Distribution of individual HR-HPV genotype according to cervical cytology Values are presented as number of cases (%). HR-HPV, high risk human papillomavirus; WNL, negative for malignancy; ASCUS, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial neoplasia; HSIL, high-grade squamous intraepithelial neoplasia; SCCA, squamous cell carcinoma; HPV-other, unspecified HPV genotype. a)Data are number of cases and % representing the proportion of individual HPV genotypes among all high risk-HPV positive women unless otherwise specified.

Introduction The incidence of adnexal masses during pregnancy is 1% to 9% [1]. Mature cystic teratomas (MCT) are common during pregnancy with the most dreadful complication being malignant transformation which occurs in approximately 1% to 3% of MCTs [2]. In comparison with benign MCT, malignant transformation occurs in the older population with a mean age of 45 to 60 years [3]. The prevalent malignant evolution is a squamous cell carcinoma (SCC) accounting for 63.7% to 88.9% of all malignancies originating from dermoid tumours [2]. There are no established standard preoperative diagnostic, surgical or postoperative procedures due to the low incidence of cases [4,5]. A case of SCC which developed from an MCT in a 30-year-old woman during pregnancy is presented and issues regarding the diagnosis and management of this rare complication are discussed. Case Report A 30-year nulliparous woman was referred to our clinic for a tumor in the left lower quadrant at 8+5 weeks of gestation. Ultrasonography revealed a tumour of the left ovary (about 18 cm) (Fig. 1A, B).

A case of SCC which developed from an MCT in a 30-year-old woman during pregnancy is presented and issues regarding the diagnosis and management of this rare complication are discussed. Case Report A 30-year nulliparous woman was referred to our clinic for a tumor in the left lower quadrant at 8+5 weeks of gestation. Ultrasonography revealed a tumour of the left ovary (about 18 cm) (Fig. 1A, B). An 18 cm solid and cystic left ovarian mass with a smooth surface and two small right ovarian cysts were detected resulting in a laparotomy at 13 weeks of gestation and left salpingo-oophorectomy and right ovarian cystectomy (Fig. 1C). The report of the frozen section from both tissues revealed MCT. The permanent histology report showed a 17×13×10 cm, 1,029 g MCT. In addition, a well-differentiated invasive SCC was detected arising from the MCT of the left ovary and confined within the MCT of the ovary which did not involve the surface of the ovary (Fig. 2).

rt of the frozen section from both tissues revealed MCT. The permanent histology report showed a 17×13×10 cm, 1,029 g MCT. In addition, a well-differentiated invasive SCC was detected arising from the MCT of the left ovary and confined within the MCT of the ovary which did not involve the surface of the ovary (Fig. 2). SCC antigen levels were assayed following the report of SCC and were 0.37 ng/mL. There was no evidence of metastasis to the brain, chest, abdomen and pelvis via MRI. The patient opted for continuation of the pregnancy and delivered a healthy female 3.3 kg at term of gestation through vaginal route 6 months later following the operation. After delivery, positron emission tomography-computed tomography (PET-CT) was performed revealing no significant hypermetabolic lesions suggestive of a lack of recurrence or metastasis. However, there was diffuse increased fluorodeoxyglucose (FDG) uptake in both breasts during breast feeding. One year following the operation, the patient presented with complaints of headache but a brain angio-CT was normal. Two years after her first delivery, the patient underwent Cesarean section for a twin delivery: 2 females weighing 2.74 kg each. The tissue sampled during the Cesarean section showed no pathologic finding. Two and a half years following the first operation, there was an abnormal hypermetabolic lesion in the right ovary on PET-CT suggesting a physiologic rather than pathologic uptake. However, there was no demonstration of any recurred mass or abnormal lymphadenopathy in the pelvis on follow-up magnetic resonance imaging (MRI). The PET-CT performed one month later revealed no significant hypermetabolic lesions to suggest recurrence or metastasis. However, four years after the initial operation, there was an abnormal hypermetabolic lesion in the right ovary and endometrium. Follow-up MRI revealed a small sized uterine myoma with multiple follicular cysts in the right ovary. During the follow-up period, every cyst as viewed on sonography had faded out. During the five years postoperation, 10 measurements of the SCC level had been performed and these values were within normal limits. The patient was taking care of her 3 kids following the initial operation for more than 60 months without any issues.

e follow-up period, every cyst as viewed on sonography had faded out. During the five years postoperation, 10 measurements of the SCC level had been performed and these values were within normal limits. The patient was taking care of her 3 kids following the initial operation for more than 60 months without any issues. Discussion The management of persistent adnexal masses during pregnancy is controversial. Following cervical cancer, ovarian cancer is the second most frequent gynaecological neoplasm complicating pregnancy [6]. Leiserowitz et al. [7] reported that ovarian cancers diagnosed during pregnancy were mostly borderline lesions or germ cell tumours and the overall prognosis is highly favorable.

y is controversial. Following cervical cancer, ovarian cancer is the second most frequent gynaecological neoplasm complicating pregnancy [6]. Leiserowitz et al. [7] reported that ovarian cancers diagnosed during pregnancy were mostly borderline lesions or germ cell tumours and the overall prognosis is highly favorable. SCC is rarely diagnosed pre- or intraoperatively due to its rare incidence and generally occurs after 40 years of age with the mean age ranging from 45 to 60 years. Preoperative distinction between MCT and SCC is extremely difficult due to the rarity of malignant transformation and the complex contents of MCT. Studies examining the clinical viability of tumour markers such as CA-125, CEA, CA-19-9, and SCC antigen have proven inconclusive [8]. Though MCT presents with a wide range of size, malignant transformation correlates with increasing dimensions. It has been recommended that a tumour size greater than 10 cm should prompt suspicion [9]. In our case, the size of the tumor was about 17 cm. Surgical intervention should ideally be performed after 12 to 14 weeks of gestation. In our case, the operation was performed at 13 weeks of gestation. An exploratory laparotomy was performed as indicated in case of suspected malignancy since the risk of spillage of dermoid content increases with laparoscopic management [10]. Analogous to any ovarian cancer, optimal cytoreduction is associated with significantly improved survival. Tseng et al. [11] reported a disease-free survival for 2 years in 100% of patients with the International Federation of Gynecology and Obstetrics (FIGO) stage I disease. While para-aortic and pelvic lymph node dissection is controversial since the tumor spreads by direct extension and/or peritoneal seeding, metastatic lymph nodes have been described in the literature [11]. In our case, lymph node dissection was not performed as the report from frozen section revealed MCT. Due to the rarity of this disease, there is no sufficient data supporting the safety of fertility-sparing surgery even for early stage disease. Thus, complete surgical staging as performed in case of ovarian cancer (i.e., total hysterectomy, bilateral salpingo-oophorectomy, omentectomy and lymphonodectomy) is opted as the safest approach. There is no consensus regarding adjuvant treatment and effectiveness of chemotherapy or radiotherapy for ovarian SCC. Tseng et al. [11] reported a disease-free survival of 100% in four 1A patients without adjuvant treatment.

ateral salpingo-oophorectomy, omentectomy and lymphonodectomy) is opted as the safest approach. There is no consensus regarding adjuvant treatment and effectiveness of chemotherapy or radiotherapy for ovarian SCC. Tseng et al. [11] reported a disease-free survival of 100% in four 1A patients without adjuvant treatment. Patients with IIB-IIIC disease were treated with cisplatinum-based chemotherapy with or without sequential radiotherapy. In our case, only a left salpingo-oophorectomy and right ovarian cystectomy was performed without adjuvant treatment. Dos Santos et al. [12] proposed whole-pelvis radiation and platinum-based chemotherapy for patients with stage I-II disease since SCC is a radiosensitive tumor. The prognosis of this tumor heavily depends on the FIGO stage. Other prognostic factors include tumor grade, growth pattern, capsular rupture and vascular invasion [13]. In premenopausal women, focal FDG uptake is often identified in the normal ovaries and could cause misinterpretation of the FDG PET-CT scan [14]. Kim et al. [15] retrospectively reviewed FDG PET-CT findings in 449 women patients with breast cancer, cervical cancer and endometrial cancer or as part of a regular checkup. Their results revealed focal ovarian uptake in 19 cases, 15 of which did not have FDG uptake on short-term follow-up PET-CT. For some cases, the disappearance of the uptake on a follow-up scan following subsequent menstruation could confirm benign uptake of the ovaries in the earlier scan [15]. In our case, every focal uptake of the ovary disappeared on a follow-up scan.

es, 15 of which did not have FDG uptake on short-term follow-up PET-CT. For some cases, the disappearance of the uptake on a follow-up scan following subsequent menstruation could confirm benign uptake of the ovaries in the earlier scan [15]. In our case, every focal uptake of the ovary disappeared on a follow-up scan. In summary, our patient developed SCC from an MCT during pregnancy, was treated conservatively without adjuvant chemotherapy and exhibited no evidence of disease for more than 60 months following the initial surgery. Acknowledgments This work was supported by INHA UNIVERSITY Research Grant. Fig. 1 (A) Sonography shows intrauterine pregnancy at 8+5 weeks of gestation. (B) Sonography shows about 18 cm solid and cystic left ovarian mass. (C) The specimen obtained shows huge cystic and solid left ovary with smooth surface and two small right ovarian cysts. Fig. 2 (A) The ovarian cyst filled with sebaceous material shows a protuberance covered with hair tuffs (arrowhead). Cut surface of the protuberance discloses a cauliflower-like solid mass (arrow) protruding into the cyst. (B) A typical squamous cell carcinoma arises in the epidermal component (H&E, ×12.5). (C) Transition from benign epidermal tissue to squamous cell carcinoma with invasive growth is evident (H&E, ×40). (D) Mature glial tissue is partly invaded by squamous cell carcinoma (arrow) (H&E, ×200).

Introduction Multicystic benign mesothelioma (MBM) of the peritoneum is a very rare disease. Since the first description of MBM in 1979, approximately 100 cases have been reported. The preoperative diagnosis is difficult and covers the whole range of cystic and papillary lesions of the peritoneum and the ovaries [1,2]. This is a case report of MBM of the pelvic peritoneum presenting as acute abdominal pain in a young woman. Case Report A 28-year-old woman, gravida 2, para 0, abortion 2, had been experiencing progressive lower abdominal pain for almost 7 months. The pain worsened a week prior to her admittance and remained constant. She denied fever or chills. Her appetite, bowel and urinary function were normal and she had no recent weight loss. She experienced regular menstrual cycles and a long-standing history of dysmenorrhea and dyspareunia. Past medical history was unremarkable and there was no history of previous abdominal surgery or gynecologic disorders. Family history was not notable. On physical examination, the abdomen was mildly rigid and moderately distended. She had a tender mass behind the uterus but the cervix and genital tract appeared normal. Complete blood screening profile, urinalysis and serum CA-125 assay were within normal limits. Ultrasonography and computed tomography of the pelvis revealed a 10 × 7 cm multicystic mass with irregular borders occupying the rectouterine pouch (Fig. 1).

behind the uterus but the cervix and genital tract appeared normal. Complete blood screening profile, urinalysis and serum CA-125 assay were within normal limits. Ultrasonography and computed tomography of the pelvis revealed a 10 × 7 cm multicystic mass with irregular borders occupying the rectouterine pouch (Fig. 1). We decided to perform laparoscopic surgery. Laparoscopic trochars were placed below the umbilicus and lower abdomen (×3, 12 mm, 5 mm, 5 mm). During the laparoscopy, we observed multiple grapelike clusters of cysts within the rectouterine pouch. The cysts appeared to originate from the peritoneum of the rectouterine pouch and attached to the uterus and left adnexa. Additional small cysts were found on the greater omentum and serosal surface of the right fallopian tube. Careful exploration of the whole abdomen, including appendix, liver and diaphragm, revealed no other abnormalities. Intraoperative frozen section histology of the multiple grapelike clusters of cysts were suspicious for serous type benign cysts. A decision was made to excise all macroscopically visible cysts. Excision of multiple clusters of cysts in the rectouterine pouch via laparotomy, resection of the peritoneum of the rectouterine pouch, partial greater omentectomy and electro-fulguration (argon plasma coagulator) of small cysts on the serosal surface of the right fallopian tube was performed.

scopically visible cysts. Excision of multiple clusters of cysts in the rectouterine pouch via laparotomy, resection of the peritoneum of the rectouterine pouch, partial greater omentectomy and electro-fulguration (argon plasma coagulator) of small cysts on the serosal surface of the right fallopian tube was performed. Grossly, multiple cysts were present measuring up to 2 cm. Microscopically, the cysts were lined by flattened or cuboidal cells. The lining cells were reactive immunohistochemically for Pan-CK, calretinin, D2-40 and CK5/6 but negative for CD34 (Fig. 2). Histological examination of the excised specimens were consistent with a MBM of the pelvic peritoneum. The postoperative course was uneventful. The patient is well at 3-month follow-up with no clinical evidence of disease recurrence. Discussion MBM of the peritoneum is a rare tumor that occurs predominantly in women of reproductive age (median age, 28 years; range, 18 to 54 years) with only 17% of patients being men [2,3]. One case has been reported in a 9 month old infant [2]. Clinically, there is no specific symptomatology and MBM may even be asymptomatic; the most frequent complaint is chronic pain [3]. MBM, due to compressive effects, presents with abdominal pain and distention and other mass symptoms such as pelvic peritoneum and has a predilection for serosal surfaces of the pelvic viscera. In 1928, Plaut first described the condition as cysts of the pelvis incidentally discovered during surgery for uterine leiomyomas.

due to compressive effects, presents with abdominal pain and distention and other mass symptoms such as pelvic peritoneum and has a predilection for serosal surfaces of the pelvic viscera. In 1928, Plaut first described the condition as cysts of the pelvis incidentally discovered during surgery for uterine leiomyomas. The etiology of MBM is controversial but is probably either neoplastic or reactive because of the strong association in women to surgical intervention, inflammatory disease or endometriosis. In general, there is no association with asbestos exposure. These findings along with the predominant benign behavior of MBM support the reactive nature of lesions as opposed to a neoplastic origin [3]. However, recurrence of MBM is as high as 75% and may occur from 1 month to 16 years [4-6]. No risk factors including size, tumor bulk or adequacy of initial resection have been helpful in predicting which tumors will recur [4]. Malignant degeneration is rare but has been reported in 2 of 130 adults [7].

tic origin [3]. However, recurrence of MBM is as high as 75% and may occur from 1 month to 16 years [4-6]. No risk factors including size, tumor bulk or adequacy of initial resection have been helpful in predicting which tumors will recur [4]. Malignant degeneration is rare but has been reported in 2 of 130 adults [7]. On radiologic examination, MBM appears as intra-abdominal lesions [1,2]. Preoperatively, solitary or multilocular, thin-walled cysts containing watery secretions and multiseptated anechoic cysts, on ultrasonographic examination, point to the presence of MBM [1]. Computed tomography and magnetic resonance imaging confirm ultrasonographic findings but do not permit differentiation from other cystic lesions [1]. The clinical, radiological and morphologic differential diagnosis of MBMs covers the whole range of cystic diagnoses as imaging is not specific. The most reliable method of preoperative diagnosis appears to be cellular analysis of peritoneal washings which has been recently reported to be diagnostic for MBM when abundant mesothelial cells are demonstrated [8]. This analysis should be performed either preoperatively on aspirated peritoneal fluid or on surgically obtained cystic fluid as an adjunct for diagnosis. The cysts are translucent and arranged in grapelike clusters separated by fibrous tissue. The tumor is multicystic with the cysts ranging in size from several millimeters to as much as 20 cm [1].

On radiologic examination, MBM appears as intra-abdominal lesions [1,2]. Preoperatively, solitary or multilocular, thin-walled cysts containing watery secretions and multiseptated anechoic cysts, on ultrasonographic examination, point to the presence of MBM [1]. Computed tomography and magnetic resonance imaging confirm ultrasonographic findings but do not permit differentiation from other cystic lesions [1]. The clinical, radiological and morphologic differential diagnosis of MBMs covers the whole range of cystic diagnoses as imaging is not specific. The most reliable method of preoperative diagnosis appears to be cellular analysis of peritoneal washings which has been recently reported to be diagnostic for MBM when abundant mesothelial cells are demonstrated [8]. This analysis should be performed either preoperatively on aspirated peritoneal fluid or on surgically obtained cystic fluid as an adjunct for diagnosis. The cysts are translucent and arranged in grapelike clusters separated by fibrous tissue. The tumor is multicystic with the cysts ranging in size from several millimeters to as much as 20 cm [1]. Although MBM has not been shown to have malignant potential, operation with complete surgical resection is the recommended treatment. Advantages of resection include confirmation of the diagnosis, ruling out the possibility of malignancy and amelioration of symptoms. Therefore, the treatment of choice, including recurrences, is complete surgical resection. In the present case, laparoscopic approach enabled not only histologic diagnosis but also surgical treatment. Other modalities have been attempted for treating recurrences. Based on the predominance of MBM in women, successful treatment with tamoxifen has been reported in single cases leading to a reduction of the cyst volume and cyst growth [9]. Unfortunately, only about one-fifth of the MBMs are immunoreactive for steroid hormone receptor analysis [3]. One adult patient was treated with intra-abdominal tetracycline through a closed drain following several surgical resections with no recurrence in a 2-year follow-up [10]. Adjuvant chemotherapy and radiation is not warranted because the tumor has a prevailing benign behavior [7]. Rare cases of malignant transformation may be treated with optimal cytoreductive surgery and adjuvant hyperthermic intra-peritoneal administration of cisplatin and doxorubicin [11]. Due to the relatively high frequency of recurrence and possible malignant transition, we recommend a close clinical follow-up of affected patients. Lifelong follow-up is planned with serial physical examinations and imaging using ultrasound for documentation and computed tomography for preoperative localization.

[11]. Due to the relatively high frequency of recurrence and possible malignant transition, we recommend a close clinical follow-up of affected patients. Lifelong follow-up is planned with serial physical examinations and imaging using ultrasound for documentation and computed tomography for preoperative localization. In summary, we wish to alert gynecologists of the diagnostic and therapeutic approach to MBM which can be accomplished by laparoscopy. Fig. 1 (A) Large multilocular hypoechogenic cysts with irregular border on vaginal ultrasonography. (B) Contrast enhanced computed tomography with a hypodense, multicystic mass with contact to the uterus without infiltrative growth, ascites or lymph node involvement. Fig. 2 (A) Multiple transparent cysts. (B) Multiple cysts resembling a lymphangioma (H&E, ×40). (C) The flat shape of the mesothelioma lining the cyst (H&E, ×400). (D) The lining cells are diffuse positive for calretinin (×400).

Introduction Homonymous hemianopsia is a specific visual disturbance induced by the lesions of the retrochiasmal visual pathways, including the optic tract, lateral geniculate body, parietotemporal radiation, and occipital cortex [1]. The most frequent cause is apoplexy, followed by trauma, neoplasm, brain surgery, and other less common mechanisms [1-3]. We report on an occurrence of temporary homonymous hemianopsia after spinal anesthesia and epidural blood patch. One common side effect of spinal anesthesia, which is often performed during operations in the lower abdomen and pelvis, is a headache due to a cerebrospinal fluid leak from a dural puncture. An epidural blood patch (EBP) procedure using autologous blood is commonly performed for post-dural puncture headache (PDPH). Pneumocephalus may occur because of the presence of within the dural space during spinal puncture or EBP procedures, and subsequent nerve-related symptoms may appear. The authors report on a unique case of temporary homonymous hemaniopsia after EBP due to pneumocephalus and briefly review the literature.

Introduction Homonymous hemianopsia is a specific visual disturbance induced by the lesions of the retrochiasmal visual pathways, including the optic tract, lateral geniculate body, parietotemporal radiation, and occipital cortex [1]. The most frequent cause is apoplexy, followed by trauma, neoplasm, brain surgery, and other less common mechanisms [1-3]. We report on an occurrence of temporary homonymous hemianopsia after spinal anesthesia and epidural blood patch. One common side effect of spinal anesthesia, which is often performed during operations in the lower abdomen and pelvis, is a headache due to a cerebrospinal fluid leak from a dural puncture. An epidural blood patch (EBP) procedure using autologous blood is commonly performed for post-dural puncture headache (PDPH). Pneumocephalus may occur because of the presence of within the dural space during spinal puncture or EBP procedures, and subsequent nerve-related symptoms may appear. The authors report on a unique case of temporary homonymous hemaniopsia after EBP due to pneumocephalus and briefly review the literature. Case Report A 33-year-old pregnant woman had been transferred to our outpatient clinic with the concern for preterm labor at 23 weeks gestation but was ultimately sent home. After a triplet pregnancy achieved through an in vitro fertilization embryo transfer in a private clinic, the patient underwent a McDonald operation at 16 weeks gestation due to the risk of preterm birth in multiple gestations. The patient then presented to the emergency department of our hospital at 30 weeks and 2 days gestation due to premature rupture of membranes. She was prepared for an urgent Caesarean section at 31 weeks and 3 days gestation due to sudden contractions. In a seated position, the patient underwent a midline approach to the L3-4 interspinous space using an 18 G Tuohy needle. Cerebrospinal fluid leaked while identifying the epidural space using the loss of resistance (LOR) technique with 2.5 mL of air. Dural puncture was suspected, and the needle was removed. As a result, the initially scheduled epidural anesthesia was changed to spinal anesthesia. Subsequently, we successfully initiated spinal anesthesia using a 26 G Quincke needle in the same location. The patient was move to a supine position after administering 10 mg of bupivacaine. The Caesarean section was initiated after confirmation of adequate anesthesia up to the fourth thoracic segment. All three babies and the placenta were delivered. All vital signs were stable before and after the anesthetic injection, and no abnormal findings were observed at the time of the procedure. The patient was advised postoperative bed rest and began early ambulation on the first postoperative day. Since the patient complained of mild tinnitus during ambulation without any other associated symptoms, closer follow-up was initiated.

injection, and no abnormal findings were observed at the time of the procedure. The patient was advised postoperative bed rest and began early ambulation on the first postoperative day. Since the patient complained of mild tinnitus during ambulation without any other associated symptoms, closer follow-up was initiated. On the second postoperative day, she complained of headache, nausea, and other symptoms, and she began complaining of a stiff neck and vomiting on the third postoperative day. These symptoms were alleviated in the supine position but recurred when the patient assumed standing or sitting positions. No other focal neurologic symptoms were identified at that time, and vital signs were stable. Despite conservative management including bed rest, the administration of fluid, analgesics and anti-nauseants, her symptoms persisted. The patient was sent to the Department of Neurology on the fourth postoperative day for suspected PDPH. She was encouraged to undergo epidural blood patch. We were initially planning to perform EBP after the confirmation of PDPH based on computed tomography (CT) scans; however, the scanning was delayed after the procedure due to limited availability. Subsequently, the patient was placed in the left lateral position, and the epidural space was identified using about 2.5 mL of air with the LOR technique at the L4-5 spinal level. No leakage of cerebrospinal fluid was detected at that time. Fifteen milliliters of the patient's own blood were injected using an aseptic technique. No specific physical findings were identified at the time of the procedure. The patient stayed on bed rest in the supine position and slept in the same posture. The CT scan taken immediately after the EBP procedure demonstrated pneumocephalus in the prepontine and premedullar cisterns (Fig. 1A). After waking on the fifth postoperative day, the first postprocedure day following the EBP, she started to complain of an abrupt vision abnormality. Her vital signs were stable and no neurological symptoms were detected other than her visual disturbance, apart from her persistent mild headache. Visual field and fundal examinations were conducted in the Department of Ophthalmology. A right homonymous hemianopia was detected in the Humphrey visual field test (Fig. 2), but no abnormal findings were identified on fundal examination.

ected other than her visual disturbance, apart from her persistent mild headache. Visual field and fundal examinations were conducted in the Department of Ophthalmology. A right homonymous hemianopia was detected in the Humphrey visual field test (Fig. 2), but no abnormal findings were identified on fundal examination. Magnetic resonance imaging (MRI) was performed due to the clinical suspicion of homonymous hemianopsia, and demonstrated air bubble movement toward the left retrochiasmal visual pathways (Fig. 1B). After pneumocephalus was identified as the cause of her visual disturbance and headache, we administered prophylactic antibiotics and highly concentrated oxygen. The patient was then placed on bed rest. Her symptoms of visual disturbance improved considerably over the course of the day, and a follow-up CT scan was performed. No further air density was observed. Although her mild headache remained, the visual disturbance completely disappeared by the end of the fifth postoperative day. The patient was discharged on the sixth postoperative day and was asymptomatic at her outpatient follow-up visit one week later.

ow-up CT scan was performed. No further air density was observed. Although her mild headache remained, the visual disturbance completely disappeared by the end of the fifth postoperative day. The patient was discharged on the sixth postoperative day and was asymptomatic at her outpatient follow-up visit one week later. Discussion A case of temporary homonymous hemianopsia due to pneumocephalus after EBP has not been previously reported. Pneumocephalus is one of the potential complications in procedures that may result in dural puncture and is often reported after unexpected dural puncture, spinal anesthesia, or medication injection into the spinal canal during epidural anesthesia. Although it is very rare, a few cases of pneumocephalus have been reported after EBP for PDPH treatment, similar to this case report. Silvia et al. reported a case of hemipupillary dilatation associated with PDPH with evidence of pneumocephalus on CT scan. However, no previous studies have reported the unique constellations of symptoms including homonymous hemianopsia [4-8].

ave been reported after EBP for PDPH treatment, similar to this case report. Silvia et al. reported a case of hemipupillary dilatation associated with PDPH with evidence of pneumocephalus on CT scan. However, no previous studies have reported the unique constellations of symptoms including homonymous hemianopsia [4-8]. The LOR technique is commonly applied to identify the epidural space using either air or a physiological saline solution. The LOR technique with air can better detect the LOR than that using saline solution and enables more accurate and convenient verification of cerebrospinal fluid in cases of dural puncture. In addition, when saline is used, the identification of cerebrospinal fluid is more complicated, and local anesthetic agents may be diluted in the case of dural puncture. However, the use of air in this procedure brings with it the risks of pneumocephalus, aeroembolism, subcutaneous emphysema, and an uneven degree of sensory deprivation. These complications are nonetheless very rare, and air is more widely used because of the superior performance and convenience [4]. Pneumocephalus after the EBP procedure is assumed to occur due to the movement of air bubbles from the epidural space to subdural and subarachnoid spaces through the defect formed during the dural puncture [4]. Peumocephalus may also be generated by the movement of the remaining air in epidural space during dural puncture while performing the LOR technique or to remaining air in the subarachnoid space during dural puncture with additional air inflow during the EBP procedure [9]. Negative pressure induced by the inhalation of the patient during the procedure could also reduce cerebrospinal fluid pressure lower than atmospheric pressure. This is thought to be another potential mechanism of pneumocephalus [6,7,10].

ace during dural puncture with additional air inflow during the EBP procedure [9]. Negative pressure induced by the inhalation of the patient during the procedure could also reduce cerebrospinal fluid pressure lower than atmospheric pressure. This is thought to be another potential mechanism of pneumocephalus [6,7,10]. Although not always simple, pneumocephalus and PDPH following a dural puncture can be distinguished by differences in clinical presentation. PDPH usually appears within one to four days of the dural puncture and persists for an average of four days; it is characterized by alleviation of headache in the supine position [4]. In comparison, the headache associated with pneumocephalus commonly appears immediately after the surgical procedure. Since the headache is generated by cerebral stimulation by the air present in the skull, the condition deteriorates despite any postural changes and continues even in the supine position [5].

ition [4]. In comparison, the headache associated with pneumocephalus commonly appears immediately after the surgical procedure. Since the headache is generated by cerebral stimulation by the air present in the skull, the condition deteriorates despite any postural changes and continues even in the supine position [5]. In this case, the patient had symptoms consistent with PDPH, but the possibility of preoperative pneumocephalus could not be completely excluded. The comparison of pre- and post-operative images were unattainable since CT scanning was performed after the EBP procedure in this case report. However, symptoms prior to the EBP included only the typical postural headache without any vision abnormality. On the other hand, abrupt visual disturbance did occur immediately after the surgical procedure with postural change from supine position to sitting position. In addition, the pneumocephalus detected within the cistern on the CT scan taken before the onset of the homonymous hemianopsia moved to behind the optic chiasm in the MRI scan taken the next day, once the visual changes had been noted. This clinical course is consistent with the absence of pneumocephalus before the EBP procedure.

the pneumocephalus detected within the cistern on the CT scan taken before the onset of the homonymous hemianopsia moved to behind the optic chiasm in the MRI scan taken the next day, once the visual changes had been noted. This clinical course is consistent with the absence of pneumocephalus before the EBP procedure. Our hospital frequently performs EBP without CT scanning in the presence of symptoms suspicious for PDPH. Through the experience of this unusual case, the authors realized that leakage of cerebrospinal fluid and possibilities for other illnesses should be considered, and CT scanning should be performed even in the presence of the typical history and symptoms of PDPH. In addition, the use of physiological saline is much safer than air for identifying the epidural space using the LOR technique, and the use of air needs to be carefully considered, especially after an unexpected dural puncture. In cases of air use according to the preference of anesthesiologist, the amount of air and the durations of syringe and guide needle use need to be reduced to minimize the air inflow to the epidural space. Acknowledgments This work was supported by research grant of Chungbuk National University in 2012.

Our hospital frequently performs EBP without CT scanning in the presence of symptoms suspicious for PDPH. Through the experience of this unusual case, the authors realized that leakage of cerebrospinal fluid and possibilities for other illnesses should be considered, and CT scanning should be performed even in the presence of the typical history and symptoms of PDPH. In addition, the use of physiological saline is much safer than air for identifying the epidural space using the LOR technique, and the use of air needs to be carefully considered, especially after an unexpected dural puncture. In cases of air use according to the preference of anesthesiologist, the amount of air and the durations of syringe and guide needle use need to be reduced to minimize the air inflow to the epidural space. Acknowledgments This work was supported by research grant of Chungbuk National University in 2012. Fig. 1 (A) Computed tomography just after the epidural blood patch procedure demonstrates a small amount of pneumocephalus at the prepontine and premedullar cisterns (arrows). Visual disturbance had not yet developed. (B) T2-weighted fast field echo imaging demonstrates a small amount of pneumocephalus in the left retrochiasmal region (arrow) while the patient was complaining of visual disturbance. Fig. 2 Humphrey visual field test shows a complete right homonymous hemianopsia.

Introduction Prenatal testing is an integral component of obstetric practice, and is commonly performed in professional medical organizations. The primary aim of prenatal testing is the diagnosis of fetal aneuploidies, such as trisomy 21 (T21, Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome), as well as aneuploidies related to the X and Y chromosomes [1]. Although the majority of fetuses with aneuploidy result in termination during the development of the fetus, T21 has the highest survival rate, which affects 1 in 800 births [2]. Therefore, the prenatal detection of T21 is considered the most common and important aspect of prenatal genetic testing.

nd Y chromosomes [1]. Although the majority of fetuses with aneuploidy result in termination during the development of the fetus, T21 has the highest survival rate, which affects 1 in 800 births [2]. Therefore, the prenatal detection of T21 is considered the most common and important aspect of prenatal genetic testing. Prenatal testing of T21 falls into 'screening' and 'diagnosis' category. Current prenatal screening tests have greatly improved by using a combination of maternal serum markers and fetal sonographic markers such as nuchal translucency [3-6]. The best performing screening tests are able to identify more than 90% of T21 cases, with a 5% rate of false positives. However, positive screening results require confirmation with diagnostic testing, such as amniocentesis or chorionic villus sampling (CVS). The accuracy of these diagnostic methods is estimated to be 98% to 99% [7]. However, both sampling procedures are invasive, and are associated with significant risks to the fetus and mother, including the potential loss of a healthy fetus [7,8]. For this reason invasive prenatal diagnosis tests are currently preformed only in high-risk pregnancies or in pregnancies with increased maternal age and/or family history of having a child with an inherited disease. Therefore, developing a reliable method for non-invasive prenatal diagnosis (NIPD) for fetal T21 is of critical importance in prenatal care.

gnosis tests are currently preformed only in high-risk pregnancies or in pregnancies with increased maternal age and/or family history of having a child with an inherited disease. Therefore, developing a reliable method for non-invasive prenatal diagnosis (NIPD) for fetal T21 is of critical importance in prenatal care. To perform NIPD, a source of fetal genetic material that could be sampled without harm to the fetus would be needed. Since the 1970s, researchers have isolated intact fetal cells in maternal circulation [9]. However, fetal cells in maternal blood are rare in quantity and tend to remain in the mother's body for years [10]. Hence, this method is unsuitable for NIPD [11]. In 1997, Lo et al. [12] discovered the existence of cell-free fetal DNA (cff-DNA) in maternal circulation. Compared to fetal cells, cff-DNA is relatively more abundant in maternal blood and thus has been regarded as a promising new material for NIPD. It constitutes approximately 10% of the total DNA in maternal plasma and is rapidly cleared from maternal blood, within two hours of delivery [13,14]. Moreover, it has recently been found that the entire fetal genome, in the form of cff-DNA, is present in maternal blood [15]. Therefore, cff-DNA has become the focus of research for the development of NIPD.

he total DNA in maternal plasma and is rapidly cleared from maternal blood, within two hours of delivery [13,14]. Moreover, it has recently been found that the entire fetal genome, in the form of cff-DNA, is present in maternal blood [15]. Therefore, cff-DNA has become the focus of research for the development of NIPD. Currently, the clinical potential of cff-DNA has been demonstrated. In particular, the determination of fetal sex and fetal Rhesus D status using cff-DNA is already applied as routine tests in Denmark, Sweden, and the Netherlands [16-18]. However, the application of cff-DNA for the NIPD of fetal T21 has been considered to be technically challenging. One aspect of the challenge relates to the presence of the large amount of maternal DNA which interferes with the analysis of the fetal DNA in maternal plasma [13]. Another challenge is related to the characteristics of the cff-DNA that pose a difficulty in determining the chromosome dosage of the fetus. Recently, various methods have been applied to overcome these challenges in the NIPD of fetal T21 using cff-DNA and promising results have been reported. In this review, we discuss the most recent technologies for the NIPD of fetal T21 using cff-DNA, and their use in clinical practice.

e chromosome dosage of the fetus. Recently, various methods have been applied to overcome these challenges in the NIPD of fetal T21 using cff-DNA and promising results have been reported. In this review, we discuss the most recent technologies for the NIPD of fetal T21 using cff-DNA, and their use in clinical practice. NIPD of Fetal T21 Using Single-molecule Counting Methods of Chromosome Sequences The need for reliable methods for NIPD of T21 has created a strong interest in the field of rapid and accurate single-molecule counting methods (e.g., digital polymerase chain reaction [PCR] and massively parallel DNA sequencing [MPS]), which could be used in routine clinical diagnosis in the form of automated platforms [19]. The single molecule counting techniques can detect fetal aneuploidy without the analysis of fetal-specific DNA in maternal plasma [20]. These methods are based on the detection of the extra copy of chromosome 21 to distinguish normal cases from T21 cases. For example, in cases where a woman is carrying a fetus with T21, the number of copies of chromosome 21 in the maternal blood is expected to be slightly higher in comparison with other autosomes. Currently, rapidly developing MPS technologies, which provide a vast amount of data across the entire genome, appear to be suitable for counting genome representation and determining the over-represented chromosomes 21 in the affected fetus. Moreover, these techniques can simultaneously detect all trisomies in a single step.

tly, rapidly developing MPS technologies, which provide a vast amount of data across the entire genome, appear to be suitable for counting genome representation and determining the over-represented chromosomes 21 in the affected fetus. Moreover, these techniques can simultaneously detect all trisomies in a single step. Digital PCR Digital PCR is a single molecule counting technique that allows the quantification of DNA by counting one molecule at a time. It has superior analytical precision compared with conventional PCR methods. Thus, it can precisely quantify small increments within the total (maternal+fetal) amount of DNA molecules derived from chromosome 21 for T21, when compared with euploid pregnancies. Lo et al. [21] reported on the application of digital PCR for the NIPD of T21. They used an approach called relative chromosome dosage where the amount of plasma DNA molecules from chromosome 21 was compared with that of a reference chromosome, that is, a chromosome expected to have a normal dosage in the fetus [21]. The relative chromosome dosage of chromosome 21 to the reference chromosome was elevated in maternal plasma of women with T21 fetus and the degree of increments was dependent on the fetal DNA concentration. However, in the application of digital PCR for the NIPD of fetal T21, the analytical platform of digital PCR needs to be quantitatively more precise to reliably determine the small expected increment. Quantitative precision can be improved by increasing the number of PCR analyses performed. A previous study has shown that the accurate detection of fetal T21 in a maternal plasma sample containing 25% fetal DNA requires approximately 8,000 digital PCRs [21]. Therefore, the clinical setting for the NIPD of fetal T21 using digital PCR may require the use of automated platforms.

mber of PCR analyses performed. A previous study has shown that the accurate detection of fetal T21 in a maternal plasma sample containing 25% fetal DNA requires approximately 8,000 digital PCRs [21]. Therefore, the clinical setting for the NIPD of fetal T21 using digital PCR may require the use of automated platforms. Next-generation DNA Sequencing New next-generation DNA sequencing (NGS) technologies permit the simultaneous sequencing of extremely large quantities of DNA molecules. NGS produces millions or billions of short sequence reads per instrument run. NGS of cff-DNA from maternal blood has enormous potential, not only for increasing our understanding of the causes of prenatal genetic disorders in the fetus but also for designing non-invasive clinical diagnostic tests [15]. The possibility of using NGS to detect non-invasive fetal trisomy from maternal blood has been demonstrated [22-24], and this finding has been confirmed in other recent studies (Table 1) [25-30]. An alternative approach to sequencing whole genomes for the non-invasive detection of fetal abnormalities is to enrich only interest regions prior to sequencing [29-31]. Moreover, NGS technologies show remarkable potential for detecting the most common aneuploidies, including T21, T18, and T13. Currently, these discoveries have been translated into clinical tests, resulting in major benefits for NIPD.

f fetal abnormalities is to enrich only interest regions prior to sequencing [29-31]. Moreover, NGS technologies show remarkable potential for detecting the most common aneuploidies, including T21, T18, and T13. Currently, these discoveries have been translated into clinical tests, resulting in major benefits for NIPD. Generally, the NIPD of fetal T21 using NGS is done through the following process. First, a short region at one end of each DNA molecule of maternal plasma is sequenced using synthesis technology and mapped against the reference human genome to determine the chromosomal origin of each sequence. Next, the density of the sequenced tags from the chromosome 21 of interest from a T21 fetus is compared with cases of trisomy and euploid pregnancies. Consequently, NGS can clearly identify samples from women carrying aneuploid fetuses by comparing them with samples taken from women with known euploid fetuses. Previous studies demonstrated that NGS was highly accurate in the direct detection of fetal T21 from maternal plasma (Table 1) [22-30]. The accuracy of NGS for the NIPD of T21 has already been validated by large-scale clinical studies. However, sequence information of NGS is obtained for the various chromosomes proportional to their sizes.

amount of time to develop invasive cancers through a progress of dysplasia and intraepithelial carcinoma, and early detection of precancerous lesions is readily available due to periodic screening and the development of cervical cancer examination methods utilizing colpos copy and human papillomavirus (HPV) tests [2]. Pap smears are the most frequently utilized method for cervical cancer screening. The Pap smear technique was first developed by Papanicolaou and Taut [3], in 1941, and although it is a convenient, inexpensive, and safe method for cervical cancer screening, the rate of false negative errors is between 6% and 55%. In order to improve upon this error rate, liquid-based Pap [4], colposcopy, and human papillomavirus tests have been suggested as alternative screening methods [5]. Likewise, 'The Bethesda System' (TBS) was established by the National Cancer Institute meeting, held in Bethesda, USA in 1988, to address problems regarding diagnostic classification of Pap smear results. Currently, a variety of classification methods are utilized around the world, especially in the US. Specifically, atypical squamous cells of undetermined significance (ASCUS) and atypical glandular cells of undetermined significance (AGUS) are among the most important characteristics of TBS. According to TBS, ASCUS is defined as foremost by abnormal cells observed are worse than either reparative or reactive cells, and secondly as cells that do not satisfy the quantitative and qualitative criteria of squamous intraepithelial lesions [6], where AGUS are defined as those cells that satisfy the range of positive reactive changes but are not enough sufficient to be diagnosed as invasive adenocarcinomas [7]. In 2001, the Bethesda III classification, the terminology of both ASCUS and AGUS, were updated as ASC and atypical glandular cell (AGC), respectively; where ASC is subdivided into ASC-US and ASC-H while AGUS is subdivided into AGC-not otherwise specified (NOS) and AGC-favor neoplastic, respectively, and are now utilized in clinical diagnoses [8].

nstrated that NGS was highly accurate in the direct detection of fetal T21 from maternal plasma (Table 1) [22-30]. The accuracy of NGS for the NIPD of T21 has already been validated by large-scale clinical studies. However, sequence information of NGS is obtained for the various chromosomes proportional to their sizes. Therefore, chromosome 21, being the smallest autosome, would only be represented by a relatively small percentage of the sequence reads. As a result, the throughput of NGS for NIPD of fetal T21 is too low. To overcome the limitations of NGS, several targeted sequencing approaches were developed based on the a priori selection of DNA regions for analysis. Compared to sequencing and counting all reads from chromosomes, limiting the number of DNA regions greatly reduces the effort required to assess the dosage of a chromosome. Moreover, the careful selection of the DNA regions to quantify can potentially reduce the confounding variation in the number of reads per locus by taking into account only the loci with similar properties (e.g., GC content or the number of repeats of a particular sequence in the genome) [29,30]. Sparks et al. [30] described a method for detecting chromosome aneuploidy using NGS combined with an amplificationbased enrichment assay. The assay is comprised of three oligos per analyzed locus. Of the 298 samples, including 39 trisomy 21 samples and seven trisomy 18 samples, all aneuploidy samples were correctly distinguished from the controls, and as such the authors concluded the assay to have 100% sensitivity and specificity. The level of sequencing, covering only 420,000 reads per sample, was nevertheless sufficient to detect trisomy 21 and trisomy 18 reliably (z statistics exceeded 3.6 in all samples). This level corresponds to a <5% of the level required by non-targeted approaches. Moreover, this method enables multiplexing (96 samples were processed simultaneously), thus greatly reducing the cost of the analysis. The recent single nucleotide polymorphism (SNP)-based targeted NGS method was developed for the NIPD of fetal aneuploidies [29,30] and seems to be highly efficient. The key feature of this method is that it takes the mixture of maternal and fetal DNA obtained from blood plasma into account, separately from the DNA from one or both parents.

leotide polymorphism (SNP)-based targeted NGS method was developed for the NIPD of fetal aneuploidies [29,30] and seems to be highly efficient. The key feature of this method is that it takes the mixture of maternal and fetal DNA obtained from blood plasma into account, separately from the DNA from one or both parents. Along with T21, T18, T13, and sex chromosome aneuploidies (e.g., X0, XXY, XXX, XYY) can also be detected, which is an important advantage of this method in light of the high occurrence of these abnormalities. A clinical trial of the prenatal non-invasive aneuploidy testing using SNPs, supported by the National Institutes of Health, is currently underway [32]. As it is SNP-based, the method may need to be tested on patients from different populations. Nevertheless, as targeted DNA sequencing can be performed on a sequencing machine with a lower price per run and lower throughput (e.g., PGM, Ion Torrent [Life Technologies, San Francisco, CA, USA], or MiSeq [Illumina Inc., San Diego, CA, USA]), these methods are preferred, especially for average-sized clinics.

ations. Nevertheless, as targeted DNA sequencing can be performed on a sequencing machine with a lower price per run and lower throughput (e.g., PGM, Ion Torrent [Life Technologies, San Francisco, CA, USA], or MiSeq [Illumina Inc., San Diego, CA, USA]), these methods are preferred, especially for average-sized clinics. Epigenetic Approaches for the NIPD of Fetal T21 The major challenge for the development of NIPD using cff-DNA is that cff-DNA only constitutes around 10% of the total DNA in the maternal circulation [13]. To differentiate the fetal-derived sequences from that of the mother, the most intuitive targets for the detection of fetal DNA were based on absolute discriminative genetic markers, such as Y-chromosome-specific loci or paternally-inherited polymorphic loci that are either absent or different in the maternal genome [33-35]. However, these types of fetal markers were associated with certain limitations in practice. Firstly, diagnostic tests developed based on Y-specific targets could only be applied to pregnancies involving male fetuses. Secondly, the detection of a paternally inherited polymorphism requires prior knowledge of the polymorphic status of the parents, and could only apply to a subset of individuals who possessed that particular polymorphism. Therefore, it would be desirable to develop a type of marker that allows for a confident differentiation of the fetus from the mother, and yet is independent of the gender or polymorphic status of the fetuses. Recently, epigenetic modifications as fetal-specific signatures to detect cff-DNA from circulating maternal DNA have been investigated.

sirable to develop a type of marker that allows for a confident differentiation of the fetus from the mother, and yet is independent of the gender or polymorphic status of the fetuses. Recently, epigenetic modifications as fetal-specific signatures to detect cff-DNA from circulating maternal DNA have been investigated. Fetal-specific Epigenetic Makers for NIPD Epigenetic modifications refer to inheritable molecular processes that affect gene expression without changing the DNA sequence or content, and the most widely studied epigenetic process is DNA methylation. The possibility of DNA methylation as a non-invasive biomarker was first demonstrated in the plasma of patients with cancer [36-38]. Soon after such discoveries, various attempts have been made to identify fetal-specific epigenetic markers based on differential methylation patterns between the fetus and the mother [39-41]. Fetal-specific methylation pattern is divided to parent origin-specific methylation pattern and placenta specific methylation pattern.

fter such discoveries, various attempts have been made to identify fetal-specific epigenetic markers based on differential methylation patterns between the fetus and the mother [39-41]. Fetal-specific methylation pattern is divided to parent origin-specific methylation pattern and placenta specific methylation pattern. First, parent origin-specific methylation pattern is based on genomic imprinting in humans [42,43]. Fetal epigenetic markers are developed with an imprinted region, in which the DNA methylation patterns are inherited in a parent origin-specific manner [44]. For example, if a pregnant woman has inherited the methylated copy of an imprinted region from her father, an imprinted region in her fetus would become unmethylated because she passed. The methylation status of this region is distinguishable between the fetus and the mother in an allele-specific manner. In 2002, the imprinted region between the IGF2 and H19 genes was investigated to detect fetal-specific methylation from maternal plasma [39] and was confirmed by genotyping a biallelic polymorphism within the differentially methylated regions [39]. However, this method would be relatively complicated to use as a routine fetal marker, because this marker was based on an imprinted locus. Next, placenta specific methylation pattern is based on the human placenta with a specific DNA methylation pattern that is different with somatic tissues [45-47]. The majority of cff-DNA in the maternal plasma was derived from the placenta, while the maternal cell free DNA in the maternal plasma was predominantly derived from the maternal hematopoietic cells [48-50]. Therefore, genomic regions that are differentially methylated between the placenta and the maternal blood cells have been considered as fetal-specific epigenetic makers in maternal plasma. In 2005, a region on the maspin (SERPINB5) gene promoter was firstly found to be hypomethylated in the placenta, while hypermethylated in the maternal blood cells [40], and the hypomethylated sequences of the SERPINB5 gene were detectable in maternal plasma throughout the course of pregnancy, and its level dropped significantly after delivery. Therefore, this was reported as the first universal fetal marker that can be used in all pregnancies, regardless of fetal gender and genotype.

and the hypomethylated sequences of the SERPINB5 gene were detectable in maternal plasma throughout the course of pregnancy, and its level dropped significantly after delivery. Therefore, this was reported as the first universal fetal marker that can be used in all pregnancies, regardless of fetal gender and genotype. After this discovery, various attempts were made to identify a number of genomic regions that are differentially methylated between the placental tissue and the maternal peripheral blood cells according to the principle of NIPD. This feature allows for the development of a single, simple test to determine the presence of cff-DNA in the maternal plasma with greater simplicity and coverage. The approaches used for the detection of these markers are variable, depending on whether the placental-derived sequences are hypermethylated or hypomethylated compared with the maternal blood cells. Detection Method of Fetal Epigenetic Markers To detect fetal epigenetic markers in maternal plasma, the first step is to differentiate methylated and unmethylated sequences. Various methods, such as a bisulfite modification of the template DNA, differential cleavage by restriction enzymes and antibody-mediated enrichment of methylated fragments by methylated DNA immunoprecipitation (MeDIP), are applied. The next step is to quantify a fetal-specific methylation pattern. In general, PCR-based methods, such as quantitative methylation-specific PCR and quantitative real-time PCR, are used.

striction enzymes and antibody-mediated enrichment of methylated fragments by methylated DNA immunoprecipitation (MeDIP), are applied. The next step is to quantify a fetal-specific methylation pattern. In general, PCR-based methods, such as quantitative methylation-specific PCR and quantitative real-time PCR, are used. Briefly, the process of bisulfite conversion changes unmethylated cytosine residues into uracil, leaving methylated cytosine unchanged [51]. The bisulfite-converted DNA is differentially amplified by PCR-based methods, depending on the methylation status of the regions where the primers bind [52]. However, bisulfite DNA conversion results in the degradation of >90% of the template DNA [53]. Therefore, this technique is undesirable for the detection of cff-DNA, which is present at a lower abundance in maternal plasma, particularly during early gestation. Methylation sensitive restriction enzymes, such as BstU I or Hpa II, can also be distinguished to differentiate between methylation patterns in DNA sequences. These restriction enzymes sensitively digest ummethylated cytosine bases in their recognition sequence, such as CGCG or CCGG. To quantify cff-DNA in maternal blood using methylation-sensitive restriction enzymes, cell-free maternal DNA should be unmethylated. This unmethylated maternal DNA is removed in cell-free total plasma DNA by the treatment of such enzymes, and then can be quantified the digestion-resistant (methylated) cff-DNA by quantitative methods, including real-time PCR or digital PCR [41,54]. Compared with bisulfite conversion, this digestion-based method introduces less damage to the plasma DNA. However, the enzyme cleavage effectiveness, depending on the duration of digestion or the amount of enzymes used, can affect the quantification of cff-DNA [55]. Recently, MeDIP, which captures DNA containing methylcytosine, has been applied to quantify cff-DNA. This method can capture only methylated DNA fragments using a monoclonal antibody specific for methylcytosine and provides up to a 90-fold enrichment of methylated DNA. Generally, the unmethylated or methylated DNA sequences can be quantitatively measured by a methylation-specific PCR (MSP) using a fluorescence probe. The copy number is calculated directly from the amplification curves of the fluorescence signal by a series of calibration standards.

-fold enrichment of methylated DNA. Generally, the unmethylated or methylated DNA sequences can be quantitatively measured by a methylation-specific PCR (MSP) using a fluorescence probe. The copy number is calculated directly from the amplification curves of the fluorescence signal by a series of calibration standards. This method has been widely used to identify methylation patterns of cff-DNA in maternal plasma [56,57] and applied to develop effective epigenetic tests for the NIPD of fetal T21. Potential of Fetal-specific Epigenetic Marker in NIPD of Fetal T21 Analysis of differences in the DNA methylation patterns between the maternal and fetal circulating DNA molecules has been proposed as an alternative strategy to the analysis of cff-DNA sequences in the NIPD of fetal T21. Such epigenetic markers could be useful either via the analysis of the epigenetic allelic ratios or directly compared with a placenta-derived DNA methylation marker on a reference chromosome.

lating DNA molecules has been proposed as an alternative strategy to the analysis of cff-DNA sequences in the NIPD of fetal T21. Such epigenetic markers could be useful either via the analysis of the epigenetic allelic ratios or directly compared with a placenta-derived DNA methylation marker on a reference chromosome. The fetal-specific epigenetic markers require:1) the detection of a number of DNA sequences that are differentially methylated between maternal and fetal DNA and 2) quantification of these fetal-specific DNA sequences by methods such as quantitative MSP or quantitative real-time PCR. Previous studies described that PDE9A on 21q22.3, which were hypomethylated in the placental tissues while completely methylated in the maternal peripheral blood cells, can be used for the NIPD of T21 [58,59]. The putative promoter regions of HLCS on 21q22.13, which are hypermethylated in the placental tissue compared with the maternal blood cells, are also applied for the NIPD of fetal T21 and have reported promising results [60]. Theoretically, the allelic ratio of a fetal-specific epigenetic marker may present equal signal intensity for unaffected fetuses and an increased signal intensity of chromosome 21 for T21 fetuses. Using this approach, fetal T21 can be detected non-invasive even during the first trimester [42,60]. The enrichment of sequences which are specifically methylated in the placenta and/or the analysis of multiple informative markers on the chromosome 21 have been applied to detect fetal T21 with high sensitivity and specificity. Recently, various methylation-specific techniques, such as antibody-mediated enrichment of methylated fragments by MeDIP and differential amplification of methylated fragments via Hpa II tiny fragment enrichment by ligation-mediated PCR (HELP), were used for the NIPD of fetal T21 using fetal epigenetic markers [61-64]. The correct diagnosis in the NIPD for fetal T21 using fetal epigenetic markers is based on the ratio of a subset of fetal-specific methylated regions located on chromosome 21 compared with normal cases. This new platform is calculated with further statistical analysis of multiple markers and has exhibited excellent clinical performance (both the sensitivity and specificity were 100%) [65]. This methodology seems to be easily reproducible and can be readily performed by equipment currently present in most diagnostic laboratories without sophisticated analytical platforms.

al analysis of multiple markers and has exhibited excellent clinical performance (both the sensitivity and specificity were 100%) [65]. This methodology seems to be easily reproducible and can be readily performed by equipment currently present in most diagnostic laboratories without sophisticated analytical platforms. Moreover, this approach can be simultaneously detected in all known aneuploidies, if regions exist where the fetal DNA is hypermethylated compared to the maternal peripheral blood DNA are provided. Therefore, this technique seems to have the right properties to become a NIPD technique for T21 and would provide a cost-effective alternative. However, such an approach is limited in the practical applicability of NIPD for fetal T21 because of the low number of copies of cff-DNA in maternal blood and the variability in the levels of DNA methylation of individual fetal-derived epigenetic markers can affect the results and its clinical value remains to be proven in large-scale clinical studies.

ed in the practical applicability of NIPD for fetal T21 because of the low number of copies of cff-DNA in maternal blood and the variability in the levels of DNA methylation of individual fetal-derived epigenetic markers can affect the results and its clinical value remains to be proven in large-scale clinical studies. Conclusion The development of an NIPD technique for fetal T21 that would provide true genetic information without carrying risk for the progress of the pregnancy will continue to be an actively researched area in prenatal diagnosis. Trials performed so far highlight the medical and commercial potential of NIPD, but the proposed techniques warrant further validation in clinical practice. Throughout the last decade, considerable achievement has been made regarding the technical possibilities for the NIPD of T21. In the previous years, male-specific signals or paternally inherited polymorphisms have been proposed as targeted fetal DNA markers, but research interest has now evolved to the detection of fetal-specific patterns or epigenetic signatures with a unique methylation pattern that will allow the application of NIPD in all pregnancies. In parallel, novel sequencing methods with high diagnostic accuracy have already been applied in the clinical setting as an effective breakthrough for the NIPD using cff-DNA. Yet, population-based, double-blind, large-scale clinical trials are required to verify the diagnostic potential of these methods and their cost-effectiveness compared with the conventional screening tests before their introduction into the clinical practice of fetal medicine. In particular, the fact that NIPD using cff-DNA requires a small sample of maternal blood may create numerous ethical, social and legal implications, owing to the ease with which the test can be performed. Therefore, the use of this method should be carefully considered in clinical situations. Nevertheless, in the near future, the NIPD of fetal T21 using cff-DNA will be applied in the clinical setting as an effective choice for all pregnant women who opt for safer prenatal diagnostic testing. Eventually, the availability of a reliable non-invasive test to determine fetal T21 would reduce unintended fetal losses and would presumably be welcomed by pregnant women.

using cff-DNA will be applied in the clinical setting as an effective choice for all pregnant women who opt for safer prenatal diagnostic testing. Eventually, the availability of a reliable non-invasive test to determine fetal T21 would reduce unintended fetal losses and would presumably be welcomed by pregnant women. Acknowledgments This article was presented at 17th Seoul International Symposium, Korean Society of Obstetrics and Gynecology, October 4, 2012, and supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111550). Table 1 Diagnostic accuracy for fetal trisomy 21 of next generation sequencing using cell-free DNA Gestational age is range in the trisomy 21 cases and the number in parentheses indicates mean. In sensitivity and specificity, parentheses indicate the 95% confidence interval.

Introduction The levonorgestrel-releasing intrauterine system (LNG-IUS), designed initially in the mid-1970s, provides highly effective, safe, and long-term reversible contraception. More than 120 countries, including Korea, have approved it for use [1]. The approved Korean applications include contraception, treatment of heavy menstrual bleeding, treatment of dysmenorrhea, and endometrial protection during estrogen replacement therapy in postmenopausal women. LNG-IUS is a T-shaped device that releases 20 µg/day of LNG into the uterine cavity over a 5-year period [2]. LNG-IUS provides, by contrast with the relatively low serum levels, locally high concentrations of LNG in the endometrium and adjacent tissues. This leads to decidualization of the stroma, mucosal thinning, and eventually, by suppression of endometrial growth, an inactive endometrium [3]. It has been demonstrated that LNG-IUS, additionally to its high contraceptive efficacy, benefits women also in the treatment of gynecologic diseases related to heavy menstrual bleeding and dysmenorrheal, which include endometriosis, leiomyoma, adenomyosis, endometrial hyperplasia, and early-stage endometrial cancer [4,5]. In this article, we summarize the current clinical applications status of LNG-IUS as relates to gynecologic diseases.

ent of gynecologic diseases related to heavy menstrual bleeding and dysmenorrheal, which include endometriosis, leiomyoma, adenomyosis, endometrial hyperplasia, and early-stage endometrial cancer [4,5]. In this article, we summarize the current clinical applications status of LNG-IUS as relates to gynecologic diseases. Heavy Menstrual Bleeding Heavy menstrual bleeding (HMB), clinically defined as greater than 80 mL of blood loss per menstrual cycle, is a common health problem in women. Hysterectomy is an often-employed treatment option, though various alternative approaches, such as tranexamic acid, nonsteroidal anti-inflammatory drugs, danazol, combined oral pills, progestins, and LNG-IUS, also can be successful.

ater than 80 mL of blood loss per menstrual cycle, is a common health problem in women. Hysterectomy is an often-employed treatment option, though various alternative approaches, such as tranexamic acid, nonsteroidal anti-inflammatory drugs, danazol, combined oral pills, progestins, and LNG-IUS, also can be successful. Hurskainen et al. [6] conducted a 5-year randomized comparison of clinical outcomes and costs associated with the use of LNG-IUS and hysterectomy for treatment of HMB. In the results, patient' satisfaction and quality of life were similar, but costs were 40% lower in the LNG-IUS group. Lethaby et al. [7] published a review of ten randomized controlled trials with reproductive-aged women treated with LNG-IUS versus medical (cyclic progestins) or surgical therapy (hysteroscopic endometrial resection, thermal ablation, or hysterectomy). LNG-IUS was more effective than cyclic progestins; and whereas side effects were more common, the LNG-IUS patients were more satisfied with their results. Endometrial ablation was more effective than LNG-IUS for reduction of menstrual blood loss, and yet there was no difference in patient' satisfaction between the groups. Once again, women treated with LNG-IUS experienced more drug-induced side effects, but there was no significant difference in their perceived quality of life. Comparing LNG-IUS with hysterectomy, the former was more cost effective, and there were no significant differences in the quality of life measures [7]. Gupta et al. [8] conducted a multicenter, randomized controlled trial involving 571 women with HMB who were treated with LNG-IUS or the usual medical therapy (tranexamic acid, mefenamic acid, combined estrogen-progestogen, or progestogen alone). In both groups, the patient-reported scores on the menorrhagia multi-attributes scale (MMAS) improved from the baseline to six months, though the LNG-IUS group showed significantly better improvement sustainment over a 2-year period (P<0.001). Moreover, all of the MMAS domains showed significantly superior improvements for the LNG-IUS group. Also, at 2 years, the LNG-IUS group had a higher continuation rate than the usual-medical-treatment group (64% vs. 38%, P<0.001), with no significant differences in the rates of hysterectomy, endometrial ablation or sexual activity scores [8]. The LNG-IUS is a good alternative to surgical management such as hysterectomy and endometrial ablation in heavy menstrual bleeding.

uation rate than the usual-medical-treatment group (64% vs. 38%, P<0.001), with no significant differences in the rates of hysterectomy, endometrial ablation or sexual activity scores [8]. The LNG-IUS is a good alternative to surgical management such as hysterectomy and endometrial ablation in heavy menstrual bleeding. In ESHRE Capri Workshop Group [9], they recommended that the LNG-IUS or other medical treatments firstly adopted in treatment of HMB. Overall, LNG-IUS was proved to be highly effective in reducing menstrual blood loss, was well tolerated, boasted a high user-satisfaction rate, and was cost effective [9]. Currently, LNG-IUS is considered to be the first-line treatment for HMB [10].

G-IUS or other medical treatments firstly adopted in treatment of HMB. Overall, LNG-IUS was proved to be highly effective in reducing menstrual blood loss, was well tolerated, boasted a high user-satisfaction rate, and was cost effective [9]. Currently, LNG-IUS is considered to be the first-line treatment for HMB [10]. Endometriosis Endometriosis is associated with dysmenorrhea, dyspareunia, non-cyclic pelvic pain, and subfertility. For women with dysmenorrhea, reported endometriosis incidences have been as high as 40% to 60% [11,12]. Endometriosis is a chronic disease that has a recurrence rate of approximately 10% to 15% one year after conservative surgical treatment alone, and fully 40% to 50% at 5 years' follow-up [13,14]. Cheong et al. [15], having conducted a retrospective study, reported re-operation rate as high as 51% for a 10-year period. Recurrence is a important issue indeed, as repeated surgery can significantly impact upon the patient's quality of life and endanger her future fertility [16]. In order to prolong symptom-free interval and prevent recurrence, postoperative adjunctive hormonal therapy usually is prescribed. Gonadotropin releasing hormone (GnRH) agonist, danazol, combined oral contraceptives, and progestins are the common hormonal methods employed for the management of endometriosis-related pain. GnRH agonist is the gold standard for adjunctive treatment of endometriosis [17]. Such treatment often needs to be continued many years or until pregnancy is desired. Although effective, the hypoestrogenism induced by the GnRH agonist is associated with systemic side effects, which can affect patient' compliance and preclude long-term use. Thus, new therapeutic options, including the continuous use of oral pills or LNG-IUS, are being explored [18-21].

or until pregnancy is desired. Although effective, the hypoestrogenism induced by the GnRH agonist is associated with systemic side effects, which can affect patient' compliance and preclude long-term use. Thus, new therapeutic options, including the continuous use of oral pills or LNG-IUS, are being explored [18-21]. Several hypotheses have been formulated to explain the mechanism of action of LNG-IUS in endometriosis-related pain. One is a local effect on the ectopic endometrium resulting from depletion of the estrogen and progesterone receptors though inhibition of synthesis and expression of estrogen and progestin receptors [22,23]. Other possibilities are a direct effect on the eutopic endometrium by inhibition of endometrial production of estrogen-induced growth factors or growth factor-binding protein, as resulting in an anti-proliferative effect, glandular atrophy and decidualization [24]. Or, the LNG-IUS effect might be a function of a reduction of local vascular angiogenesis, a reduction in pelvic-vessel congestion and an increase in apoptosis, a reduction in peritoneal fluid macrophage activity and a modification in the production of cytokines responsible for maintenance of lesions and pain [25-28].

. Or, the LNG-IUS effect might be a function of a reduction of local vascular angiogenesis, a reduction in pelvic-vessel congestion and an increase in apoptosis, a reduction in peritoneal fluid macrophage activity and a modification in the production of cytokines responsible for maintenance of lesions and pain [25-28]. LNG-IUS was first used for endometriosis-related dysmenorrhea by Vercellini et al. [29]. They reported that it greatly reduced menstrual pain and was highly rated in terms of patient satisfaction. As a follow-up to this pilot study, Vercellini et al. [30] thoroughly investigated the application of LNG-IUS to endometriosis in a randomized controlled trial, comparing it with expectant management after laparoscopic conservative surgery. According to a post-12-month evaluation, and an intention-to-treat analysis, postoperative recurrence of dysmenorrhea was significantly decreased in the LNG-IUS group (10% vs. 45%, P=0.03). Tanmahasamut et al. [31], similarly, conducted a double-blind randomized controlled trial with 55 post-conservative-surgery patients. At 12 months, the LNG-IUS group relative to an expectant group, showed a greater reduction in dysmenorrhea visual analogue scale (VAS) (-81 mm vs. -50 mm, P=0.06) and pelvic pain VAS (-48.5 mm vs. -22 mm, P=0.038). Recurrent dysmenorrhea within one year also was reduced in the LNG-IUS group (7.4% vs. 39.1%, P=0.014) [31]. Petta et al. [32] conducted a multicenter randomized controlled trial to compare LNG-IUS (n=39) with a GnRH agonist (n=43), finding no statistical differences in VAS pain score or quality-of-life improvement. However, Bayoglu et al. [33] reported different results for a 12-month prospective randomized study involving 40 severe endometriosis patients. They reported that the total endometriosis severity profile (TESP) score decreased in the LNG-IUS group over the initial 6 months, but that by 12 months of follow-up, the TESP scores had risen back to values similar to those at pretreatment. At the end of the study, the LNG-IUS group relative to the GnRH subjects showed a significant increase in VAS and TESP scores, and recorded lower levels of satisfaction [33]. Even so, given the additional advantages of LNG-IUS, namely the facts that it is not associated with hypoestrogenism and the possibility of long-term (5-year) use, it may yet be used for chronic pelvic pain-associated endometriosis in women who do not wish to conceive.

s, and recorded lower levels of satisfaction [33]. Even so, given the additional advantages of LNG-IUS, namely the facts that it is not associated with hypoestrogenism and the possibility of long-term (5-year) use, it may yet be used for chronic pelvic pain-associated endometriosis in women who do not wish to conceive. Leiomyoma Leiomyoma is the most common benign gynecologic tumor in reproductive-aged women, producing symptoms including HMB, dysmenorrhea, pelvic pressure and pain, and reproductive dysfunction, though many patients remain asymptomatic [34,35]. In many studies, LNG-IUS use by leiomyoma patients ameliorated leiomyoma-related menorrhagia. This treatment modality is utilized primarily in cases of leiomyoma-related HMB, though treatment is not as effective as for idiopathtic HMB [36-41]. Sivin and Stern [42] reported a multicenter prospective 7-year randomized study, the chief finding of thich was that long-term use of LNG-IUS reduced the incidence of newly developed myoma and myoma-related surgery in comparison with copper T. However, there is no coherence to changes of uterine volume or leiomyoma volume in users of LNG-IUS [36,41,43].

ticenter prospective 7-year randomized study, the chief finding of thich was that long-term use of LNG-IUS reduced the incidence of newly developed myoma and myoma-related surgery in comparison with copper T. However, there is no coherence to changes of uterine volume or leiomyoma volume in users of LNG-IUS [36,41,43]. There are some limitations on the suitability of LNG-IUS for women with leiomyoma, including leiomyomas causing distortion of the uterine cavity or cases of submucosal myoma [44]. The reported LNG-IUS expulsion rates among women with uterine leiomyomas range between 0% and 20%, and are higher than those without uterine leiomyoma (0% to 3%). Also, significantly higher rates of expulsion have been noted among women with greater uterine volumes (a possible proxy for fibroid size) than among those with smaller ones [36-39,45,46]. Adenomyosis Adenomyosis characterized by the presence of heterotopic endometrial glands and stroma in the myometrium, is a common cause of menorrhagia and dysmenorrhea. The definitive treatment is hysterectomy, at least traditionally. However, alternative management, including oral pills, danazol, GnRH agonist, LNG-IUS, endometrial ablation/resection, uterine artery embolization, and magnetic resonance guided focused ultrasound also can be considered [47-55].

rrhagia and dysmenorrhea. The definitive treatment is hysterectomy, at least traditionally. However, alternative management, including oral pills, danazol, GnRH agonist, LNG-IUS, endometrial ablation/resection, uterine artery embolization, and magnetic resonance guided focused ultrasound also can be considered [47-55]. The use of LNG-IUS for adenomyosis was first reported by Fedele et al. [50], in 1997. They evaluated the efficacy of LNG-IUS in 25 patients suffering from adenomyosis-associated menorrhagia, and found that 92% of them showed decreases on the pictorial blood loss assessment chart (PBAC) and diminished dysmenorrheal symptoms, along with significant increases in hemoglobin, hematocrit, and serum ferritin levels [50]. Bragheto et al. [56] reported on the employment of LNG-IUS in the treatment of 29 adenomyosis patients diagnosed and monitored by magnetic resonance imaging. After 6 months, significant reductions of junctional zone thickness and VAS pain scores were observed, though there was no significant change in uterine volume. Cho et al. [57] and Sheng et al. [58] reported 3-year follow-up data on the application of LNG-IUS for the treatment of adenomyosis, in which indicated significantly decreased menorrhagia and VAS pain scores and high patient' satisfaction. Additionally to these observational study, Ozdegirmenci et al. [59] compared LNG-IUS with hysterectomy in a prospective randomized trial, the results of which showed that the LNG-IUS group enjoyed significant and comparable improvements in hemoglobin levels and along with superior health-related quality of life improvements during the first year. Conclusively, LNG-IUS is an effective treatment option for management of dysmenorrhea and menorrhagia in patients with clinically diagnosed adenomyosis. As such, it offers patients a practical alternative to hysterectomy.

levels and along with superior health-related quality of life improvements during the first year. Conclusively, LNG-IUS is an effective treatment option for management of dysmenorrhea and menorrhagia in patients with clinically diagnosed adenomyosis. As such, it offers patients a practical alternative to hysterectomy. Endometrial Hyperplasia Endometrial hyperplasia is defined as a morphologic and biologic alteration of the endometrium as a result of continuous estrogenic stimulation unopposed by adequate levels of progesterone. Among reproductive-aged women, chronic anovulation, commonly seen in those diagnosed with polycystic ovarian syndrome, is the most common cause of endogenous unopposed estrogen [60].

and biologic alteration of the endometrium as a result of continuous estrogenic stimulation unopposed by adequate levels of progesterone. Among reproductive-aged women, chronic anovulation, commonly seen in those diagnosed with polycystic ovarian syndrome, is the most common cause of endogenous unopposed estrogen [60]. Hormonal therapy is regarded as the standard management plan for endometrial hyperplasia without atypia or benign endometrial hyperplasia, owing to the facts that the malignany potential is low, the spontaneous resolution rate is high, and the response to hormonal therapy, moreover, also is high [61,62]. In the case of atypical endometrial hyperplasia, total hysterectomy with or without bilateral salpingooophorectomy is the current standard treatment option [63]. However, hormonal therapy can be selected in atypical endometrial hyperplasia patients who desire to preserve their fertility or in patients who are poor surgical candidates due to severe medical comorbidities. The hormonal classes with potential therapeutic options include progestins, selective estrogen receptor modulators, aromatase inhibitors, and gonadotropin-releasing hormone agonists. Among these, progestin is most commonly used as the safe, uterus-preserving alternative to hysterectomy. Nonetheless, systemic side effects and poor compliance reportedly are associated with oral progesterone; clinical trials of progestin therapies for atypical endometrial hyperplasia, furthermore, have not yet established a standard regimen [64-66].

ommonly used as the safe, uterus-preserving alternative to hysterectomy. Nonetheless, systemic side effects and poor compliance reportedly are associated with oral progesterone; clinical trials of progestin therapies for atypical endometrial hyperplasia, furthermore, have not yet established a standard regimen [64-66]. Compared with oral progestin, LNG-IUS in many studies has been found to have less severe systemic side effects and higher efficacy as a treatment for endometrial hyperplasia [66-71]. Gallos et al. [72] recently published a systematic review and meta-analysis of 24 studies that had compared endometrial hyperplasia regression rates between oral progestin and LNG-IUS for a total of 1,001 patients. In cases of simple hyperplasia, 213 women (9 studies) treated with oral progestin showed a pooled regression rate of 89%, versus the 96% rate for 72 LNG-IUS patients (6 studies). Meta-regression confirmed that these rates were not statistically significant (P=0.41). In cases of complex hyperplasia, 389 patients (9 studies) administered oral progestin showed a pooled regression rate of 66%, versus the 92% rate for 102 LNG-IUS patients (4 studies). Overall, the treatment outcomes for LNG-IUS were statistically more significant than those for oral progestin (P<0.01). In atypical hyperplasia, 189 women (14 studies) treated with oral progestin showed a pooled regression rate of 69%, versus the 90% rate for 36 LNG-IUS patients (7 studies) (P=0.03) [72].

(4 studies). Overall, the treatment outcomes for LNG-IUS were statistically more significant than those for oral progestin (P<0.01). In atypical hyperplasia, 189 women (14 studies) treated with oral progestin showed a pooled regression rate of 69%, versus the 90% rate for 36 LNG-IUS patients (7 studies) (P=0.03) [72]. Lee et al. [73] reported on the effectiveness of LNG-IUS in 12 patients (4 simple, 7 complex, 1 atypical complex hyperplasia) evaluated at our institution in Korea. In all of the cases, complete regression of endometrial hyperplasia was achieved. The mean duration to regression was 4.5 months (66% achieved compete regression within 3 months), and all of the patients had achieved regression within 9 months. Additionally, a prospective multicenter trial on Korean women (planned number of patients: 80), this one by the Korean Gynecologic Oncology Group (KGOG2006), has been ongoing since 2006 [74].

s 4.5 months (66% achieved compete regression within 3 months), and all of the patients had achieved regression within 9 months. Additionally, a prospective multicenter trial on Korean women (planned number of patients: 80), this one by the Korean Gynecologic Oncology Group (KGOG2006), has been ongoing since 2006 [74]. Early-stage Endometrial Cancer Endometrial carcinoma is the most common gynecologic malignancy in developed countries [75,76]. The majority of cases are diagnosed in postmenopausal women, and up to 14% of patients are premenopausal, among whom 3% to 5% are aged under 40 years, 70% of those 3% to 5% being nulliparous at the time of diagnosis [77,78]. On histologic examination, 84% of all endometrial cancers are endometroid adenocarcinomas, which typically have a good prognosis [79]. Endometrial cancer in younger women usually is most commonly of the early clinical stage, well-differentiated and endometrioid type, which also carries a good prognosis [80]. The current standard treatment is total hysterectomy and bilateral salpingo-oophorectomy with or without surgical staging [81-83]. However, the data from multiple studies suggest that for select patients with early clinical stage carcinoma and a strong desire to maintain fertility, hormonal therapy is an attractive and effective alternative [84-88].

is total hysterectomy and bilateral salpingo-oophorectomy with or without surgical staging [81-83]. However, the data from multiple studies suggest that for select patients with early clinical stage carcinoma and a strong desire to maintain fertility, hormonal therapy is an attractive and effective alternative [84-88]. Progestins are the first medical treatment option for endometrial cancer. Progestins effect secretory differentiation of endometrial glands, inhibit estrogen receptor function and endometrial cell mitosis, and promote apoptosis; additionally, some progestins have an anti-angiogenic effect [89]. According to a meta-analysis by Gallos et al. [90], a total 408 women (32 studies) who underwent fertility-sparing treatment by various methods, including progestin, LNG-IUS, GnRH agonist, aromatase inhibitor, and hysteroscopic resection, showed a pooled regression rate of 76.2%, a relapse rate of 40.6%, and a live birth rate of 28%.

meta-analysis by Gallos et al. [90], a total 408 women (32 studies) who underwent fertility-sparing treatment by various methods, including progestin, LNG-IUS, GnRH agonist, aromatase inhibitor, and hysteroscopic resection, showed a pooled regression rate of 76.2%, a relapse rate of 40.6%, and a live birth rate of 28%. Montz et al. [91] reported the results of LNG-IUS-based treatment of the International Federation of Gynecology and Obstetrics (FIGO) stage IA, grade 1 endometrioid cancer patients at high risk for perioperative complications. Among 12 patients, the biopsy results were negative for 64% at 6 months and 75% at 12 months. Cade et al. [92] reported on 16 patients who had been treated with MPA (4 patients), LNG-IUS (3 patients), or both (9 patients). Ten of the patients responded to treatment (MPA only, 2; LNG-IUS only, 1; both, 7), and the mean time to response was 5.5 months. The results of 5 young patients who had been treated, at our institution, with a daily 500 mg dose of MPA plus LNG-IUS insertion were published in 2010. Complete remission was achieved for 4 patients, and another one showed partial remission. During the mean 10.2 months follow-up period, there was no recurrence of disease [93]. Also in Korea, a prospective multicenter study (KGOG2009), initiated in 2009, has been treating patients with 500 mg of MPA plus LNG-IUS for early-stage endometrial cancer in young women [94].

nd another one showed partial remission. During the mean 10.2 months follow-up period, there was no recurrence of disease [93]. Also in Korea, a prospective multicenter study (KGOG2009), initiated in 2009, has been treating patients with 500 mg of MPA plus LNG-IUS for early-stage endometrial cancer in young women [94]. Hormonal management with LNG-IUS and supplemental oral progestin appears to be a safe and moderately effective option for early-stage endometrial cancer patients who want to retain their reproductive potential. However, given that the relapse rate is higher than 40% [90], post-childbirth women should consider hysterectomy, which remains the standard of care. Conclusion The non-contraceptive benefits of LNG-IUS, particularly the effects on heavy menstrual bleeding and dysmenorrhea, as well as the option for 5-year use, add to its utility and efficacy as an alternative to long-term contraception. LNG-IUS in fact has proved to be an effective treatment modality for a great variety of gynecologic conditions: idiopathic, myoma- or adenomyosis-related HMB, endometriosis- or adenomyosis-related pelvic pain, as well as endometrial hyperplasia and early-stage endometrial cancer. However, further large-scale randomized study and comparion with conventional treatment methods, along with long-term follow-up data, are needed.

Introduction Among malignant tumors in Korean women, cervical cancers have been reported as the fifth most common form of cancer after breast, stomach, colorectal, and thyroid cancers [1]. Recently, however, the occurrence of cervical cancer has decreased as it takes considerable amount of time to develop invasive cancers through a progress of dysplasia and intraepithelial carcinoma, and early detection of precancerous lesions is readily available due to periodic screening and the development of cervical cancer examination methods utilizing colpos copy and human papillomavirus (HPV) tests [2].

assification, the terminology of both ASCUS and AGUS, were updated as ASC and atypical glandular cell (AGC), respectively; where ASC is subdivided into ASC-US and ASC-H while AGUS is subdivided into AGC-not otherwise specified (NOS) and AGC-favor neoplastic, respectively, and are now utilized in clinical diagnoses [8]. ASCUS findings accounts for 3% to 5% of Pap smear results, although this figure has been reported to vary between 10% to 20% and 3% to 5% of the diagnosed patients possessed the risk of cervical intraepithelial neoplasia (CIN)1 and CIN2 or CIN3, respectively. A significant proportion of ASCUS patients with CIN1 are accompanied by positive HPV infection, the lesions of which have been reported to spontaneously disappear in more than 60% of cases. Thus, follow-up analysis via an outpatient clinic after confirming whether lesions of grade CIN2 or CIN3 are present in the patients who were diagnosed ASCUS appears to be an important research avenue [9-13]. Indeed, the prevalence of AGC accounts for 0.08% to 5.96% of known cases, and it has been shown that 8% of diagnosed patients are associated with malignant lesions, thereby requiring more attention and additional histological examinations and outpatient clinic follow-up for the patients clinically diagnosed with AGC [14]. Thus, the objective of this study was to investigate the clinical significance of AGC (AGC-NOS, AGC-favor neoplastic) by analyzing the final diagnosis results obtained from biopsies of female patients diagnosed with AGC based on the TBS classification method according to Pap smear.

ASCUS findings accounts for 3% to 5% of Pap smear results, although this figure has been reported to vary between 10% to 20% and 3% to 5% of the diagnosed patients possessed the risk of cervical intraepithelial neoplasia (CIN)1 and CIN2 or CIN3, respectively. A significant proportion of ASCUS patients with CIN1 are accompanied by positive HPV infection, the lesions of which have been reported to spontaneously disappear in more than 60% of cases. Thus, follow-up analysis via an outpatient clinic after confirming whether lesions of grade CIN2 or CIN3 are present in the patients who were diagnosed ASCUS appears to be an important research avenue [9-13]. Indeed, the prevalence of AGC accounts for 0.08% to 5.96% of known cases, and it has been shown that 8% of diagnosed patients are associated with malignant lesions, thereby requiring more attention and additional histological examinations and outpatient clinic follow-up for the patients clinically diagnosed with AGC [14]. Thus, the objective of this study was to investigate the clinical significance of AGC (AGC-NOS, AGC-favor neoplastic) by analyzing the final diagnosis results obtained from biopsies of female patients diagnosed with AGC based on the TBS classification method according to Pap smear. Materials and Methods 1. Subjects The medical records of 83 patients who were diagnosed AGC (AGC-NOS, 55 subjects; AGC-favor neoplastic, 28 subjects) were analyzed retrospectively among 54,160 subjects who underwent Pap smear analysis in our hospital between January 1998 and March 2006 (Table 1). The average age of the patients was 49.2 ± 8.89 with a range of 33 to 74 years old. The average gravidity and parity were 3.91± 2.3 times and 2.01±1.1, respectively (Table 2).

e analyzed retrospectively among 54,160 subjects who underwent Pap smear analysis in our hospital between January 1998 and March 2006 (Table 1). The average age of the patients was 49.2 ± 8.89 with a range of 33 to 74 years old. The average gravidity and parity were 3.91± 2.3 times and 2.01±1.1, respectively (Table 2). 2. Methods 1) Pap smear and biopsy Slides of patients with AGUS and AGC were reviewed by a pathology specialist based upon the 2001 revision of TBS III classification system (Fig. 1). Results of punch biopsy and cervical curettage in AGC patients were analyzed retrospectively. 2) Correlation with clinicopathologic examination results Age, presence or absence of HPV infection, and CA-125 values during diagnosis were analyzed in patients with AGC-NOS and AGC-favor neoplastic AGC patients (Table 2). 3) Histologic correlation of AGC-NOS and AGC-favor neoplastic Punch biopsy and hysterotrachelectasia were performed in patients with AGC. Depending upon indications, several examinations including conization, gastrointestinal examinations, and laparotomy were performed. The cases were classified into AGC-NOS and AGC-favor neoplastic cases, and the histologic results of the patient groups were compared (Tables 3, 4). 3. Results and statistical analysis Statistical analyses were performed using SPSS ver. 10.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was determined by Pearson chi-square test. Differences of P<0.05 were considered to be statistically significant different.

3) Histologic correlation of AGC-NOS and AGC-favor neoplastic Punch biopsy and hysterotrachelectasia were performed in patients with AGC. Depending upon indications, several examinations including conization, gastrointestinal examinations, and laparotomy were performed. The cases were classified into AGC-NOS and AGC-favor neoplastic cases, and the histologic results of the patient groups were compared (Tables 3, 4). 3. Results and statistical analysis Statistical analyses were performed using SPSS ver. 10.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was determined by Pearson chi-square test. Differences of P<0.05 were considered to be statistically significant different. Results 1. Prevalence of AGC Among 54,160 subjects who received a Pap smear, the number of patients with AGC, AGC-NOS, and AGC-favor neoplastic cells was 83 (0.15%), 55 (0.10%), and 28 (0.05%), respectively. Of the examination performed, conventional Pap smear and liquid-based Pap (ThinPrep) were carried out in 51,510 subjects (95.1%) and 2,650 subjects (4.9%), respectively. In conventional Pap smear, AGC, AGC-NOS, and AGC-favor neoplastic were present in 64 (0.12%), 41 (0.08%), and 23 subjects (0.04%), respectively; in liquid-based Pap (ThinPrep), AGC, AGC-NOS, and AGC-favor neoplastic were observed in 19 (0.72%), 14 (0.53%), and 5 subjects (0.19%), respectively (Table 1).

, respectively. In conventional Pap smear, AGC, AGC-NOS, and AGC-favor neoplastic were present in 64 (0.12%), 41 (0.08%), and 23 subjects (0.04%), respectively; in liquid-based Pap (ThinPrep), AGC, AGC-NOS, and AGC-favor neoplastic were observed in 19 (0.72%), 14 (0.53%), and 5 subjects (0.19%), respectively (Table 1). 2. Characteristics and clinicopathologic correlation of the patients with AGC-NOS and AGC-favor neoplastic The average age of the female patients was 47.3 ± 7.8 years old for AGC-NOS, 51.9 ±10.1 years old for AGC-favor neoplastic, and 49.2 ± 8.89 years old for AGC. AGC-NOS represented 3.35 ±1.9 times the average gravidity and 1.79 ±1.0 times the average parity; AGC-favor neoplastic represented 5.15 ± 2.4 times the average gravidity and 2.51 ±1.2 times the average parity; and AGC represented 3.91± 2.3 times the average gravidity and 2.01±1.1 times the average parity. For AGC-NOS cases, HPV examination was performed in 16 subjects out of 55 cases; where 5/16 (31.2%) subjects (type 33, 1 subject; type 52, 1 subject; and type 53, 2 subjects) were deemed at high risk of infection and 1/16 (6.3%) subjects (type 6, 1 subject) were deemed at low risk of infection. In the case of AGC-favor neoplastic, evidence of a high risk of infection was found in 5/10 (50.0%) subjects (type 18, 1 subject; type 33, 2 subjects; type 35, 1 subject; type 53, 1 subject). In patients with AGC, high risk and low risk of HPV infection was present in 9/26 (34.6%) subjects and 1/26 (3.8%) subjects, respectively. During diagnosis, the CA-125 (U/mL) average was 24.4 ± 36.5 (U/mL) in 31 AGC-NOS cases, 97.6 ±155.4 (U/mL) in 19 AGC-favor neoplastic cases, and 50.8 ±101.6 for AGC cases (Table 2).

In patients with AGC, high risk and low risk of HPV infection was present in 9/26 (34.6%) subjects and 1/26 (3.8%) subjects, respectively. During diagnosis, the CA-125 (U/mL) average was 24.4 ± 36.5 (U/mL) in 31 AGC-NOS cases, 97.6 ±155.4 (U/mL) in 19 AGC-favor neoplastic cases, and 50.8 ±101.6 for AGC cases (Table 2). 3. Histologic correlation of AGC-NOS and AGC-favor neoplastic Histologic results and clinical findings diagnosed during follow-up in the patients with AGC-NOS and AGC-favor neoplastic were described as follows. For AGC-NOS cases, benign pathology indicated by normal histologic findings or benign disease was observed in 34/55 (61.8%) subjects; low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion, carcinoma in situ, adenocarcinoma in situ, and endometrial hyperplasia, which corresponded to pre-malignancy, were found in 13/55 (23.6%) subjects; and malignant pathology was observed in 8/55 (14.6%) subjects. As primary sites of the malignancy, cervical adenocarcinomas, endometrial cancer, breast cancer, and ovarian cancer were observed in 3/55 (5.4%), 2/55 (3.6%), 1/55 (1.8%), and 2/55 (3.6%) subjects, respectively. For AGC-favor neoplastic cases, benign pathology, pre-malignant, and malignant pathology were observed in 7/28 (25.0%), 5/28 (17.9%), and 16/28 (57.1%) subjects, respectively. As primary sites of the malignant, cervical adenocarcinomas, endometrial cancers, breast cancers, ovarian cancers, and stomach cancers were represented in 5/28 (17.8%) subjects, 4/28 (14.2%) subjects, 2/28 (7.1%) subjects, 2/28 (7.1%) subjects, and 3/28 (10.7%) subjects, respectively (Table 3, Fig. 2).

s, respectively. As primary sites of the malignant, cervical adenocarcinomas, endometrial cancers, breast cancers, ovarian cancers, and stomach cancers were represented in 5/28 (17.8%) subjects, 4/28 (14.2%) subjects, 2/28 (7.1%) subjects, 2/28 (7.1%) subjects, and 3/28 (10.7%) subjects, respectively (Table 3, Fig. 2). 4. Relative distribution of malignant diseases in AGC-NOS and AGC-favor neoplastic The relative distribution of malignant diseases in AGC-NOS and AGC-favor neoplastic was as follows. When patients were diagnosed with AGC-NOS, the observed distribution of patients was 8/55(14.6%) with malignant disease, 3/8 (37.5%) with cervical adenocarcinoma, 2/8 (25%) with endometrial cancer, 2/8 (25%) with ovarian cancer, and 1/8 (12.5%) with breast cancers. When patients were diagnosed with AGC-favor neoplastic, the observed distribution of patients was 16/28 (57.1%) with malignant disease, 5/16 (31.2%) with cervical adenocarcinoma, 4/16 (25%) with endometrial cancer, 3/16 (18.7%) with stomach cancer, 2/16 (12.5%) with ovarian cancer, and 2/16 (12.5%) with breast cancer (Table 3, Fig. 3).

e diagnosed with AGC-favor neoplastic, the observed distribution of patients was 16/28 (57.1%) with malignant disease, 5/16 (31.2%) with cervical adenocarcinoma, 4/16 (25%) with endometrial cancer, 3/16 (18.7%) with stomach cancer, 2/16 (12.5%) with ovarian cancer, and 2/16 (12.5%) with breast cancer (Table 3, Fig. 3). 5. Relative distribution of extrauterine malignancy in AGC-NOS and AGC-favor neoplastic Of AGCs, the relative distribution of AGC-NOS and AGC-favor neoplastic in the 10 cases with malignant disease found in extrauterine tissues at the time of diagnosis was as follows: ovarian cancer (2/10 subjects, 20%) and breast cancer (1/10 subjects, 10%) were observed in AGC-NOS, while ovarian cancer (2/10 subjects, 20%) and stomach cancer (2/10 subjects, 20%) were observed in AGC-favor neoplastic. As a result of the follow-up during the study period, malignancy was not observed in AGC-NOS, whereas malignancy was found in AGC-favor neoplastic including 1/10 subjects (10%) with stomach cancer and 2/10 subjects (20%) with breast cancers (Table 4).

h cancer (2/10 subjects, 20%) were observed in AGC-favor neoplastic. As a result of the follow-up during the study period, malignancy was not observed in AGC-NOS, whereas malignancy was found in AGC-favor neoplastic including 1/10 subjects (10%) with stomach cancer and 2/10 subjects (20%) with breast cancers (Table 4). Discussion Pap smears are a meaningful diagnostic tool for identifying early stages of precancerous lesions of cervical cancers, thereby reducing cancer mortality. Thus, when examination results indicate the presence of cell abnormalities accurate interpretation and subsequent follow-up is critical. In the present study, we aimed to analyze the meaning of Pap smear results in females who diagnosed as AGC (NOS, favor-neoplastic) among the females who had abnormal Pap smear results during regular check-ups. A total of 83 of 54,160 subjects (0.15%) were diagnosed with AGC, which was in agreement with a previously reported value of 0.08% to 5.96% [14], indicating that AGC diagnosis our hospital can be considered relatively well managed.

oplastic) among the females who had abnormal Pap smear results during regular check-ups. A total of 83 of 54,160 subjects (0.15%) were diagnosed with AGC, which was in agreement with a previously reported value of 0.08% to 5.96% [14], indicating that AGC diagnosis our hospital can be considered relatively well managed. A total of 64/51,510 subjects (0.12%) and 19/2,650 subjects (4.9%) were diagnosed via conventional Pap and liquid-based Pap (ThinPrep), respectively. AGC-NOS was present in 41/51,510 subjects (0.08%) for conventional Pap and 14/2,650 subjects (0.53%) for liquid-based Pap. In addition, AGC-favor neoplastic were observed in 23/51,510 subjects (0.04%) for conventional Pap and 5/2,650 subjects (0.19%) for liquid-based Pap. Given these results, diagnosis of AGC (NOS, favor-neoplastic) was relatively more frequent in liquid-based Pap than that of conventional Pap (P=0.011), and thus AGC diagnosis utilizing liquid-based Pap appeared to increase the frequency of diagnosis. However, as liquid-based Pap accounted for only 2,650/54,160 subjects (4.9%), it is somewhat unreasonable to determine an overall diagnosis ratio, and more results are needed to support whether this observation is clinically relevant.

diagnosis utilizing liquid-based Pap appeared to increase the frequency of diagnosis. However, as liquid-based Pap accounted for only 2,650/54,160 subjects (4.9%), it is somewhat unreasonable to determine an overall diagnosis ratio, and more results are needed to support whether this observation is clinically relevant. Since it has been reported that HPV18 is highly associated with cervical adenocarcinoma [15], we investigated the correlation of HPV infection. In AGC, HPV infection was present in 10/26 subjects (38.4%) including 1 case of low risk infection and 9 cases of high risk of infection. In cases of high risk HPV infection, 5/10 subjects (50%) and 4/16 subjects (25%) exhibited AGC-favor neoplastic and AGC-NOS, respectively, indicating that the high risk infection was significantly higher in AGC-favor neoplastic compared with AGC-NOS. Of the cases of HPV observed, type 18 accounted for 1 case, while type 33 accounted for 3 cases; these represent the most common types of HPV infections. Even though these results represent different patterns within cervical adenocarcinoma, evaluation of more subjects is required to determine whether there is a direct correlation, due to the small number of patients in this study. The average CA-125 of patients with AGC was 50.8 ±101.6 (U/mL), whereas the average CA-125 in AGC-NOS and AGC-favor neoplastic was 24.4 ± 36.5 (U/mL) and 97.6 ±155.4 (U/mL), respectively. Although the average CA-125 was higher in AGC-favor neoplastic, it was difficult to establish whether these values were significant due to the large standard deviation.

AGC was 50.8 ±101.6 (U/mL), whereas the average CA-125 in AGC-NOS and AGC-favor neoplastic was 24.4 ± 36.5 (U/mL) and 97.6 ±155.4 (U/mL), respectively. Although the average CA-125 was higher in AGC-favor neoplastic, it was difficult to establish whether these values were significant due to the large standard deviation. Chhieng et al. [16], reported that AGUS (AGC) is highly correlated to malignant diseases (15.45%). Moreover, Seok et al. [17], reported that invasive diseases were found in 50 subjects (57.4%) during histologic examination, among 87 subjects who were diagnosed with AGUS by Pap smear. In the present study, 24/83 subjects (28.9%) were diagnosed with malignancy in the histologic examination results of the patients with AGC, indicating high correlation. Particularly, the frequency of AGC-favor neoplastic (16/28 subjects, 57.1%) was significantly higher than that of AGC-NOS (8/55 subjects, 14.6%), indicating that AGC-favor neoplastic was relatively more correlated to malignant diseases (P=0.000). In patients with AGC, cervical adenocarcinoma (8/83 subjects, 9.6%) was the most frequently observed malignant disease, followed by, in decreasing order, endometrial cancer (6/83 subjects, 7.2%), ovarian cancer (4/83 subjects, 4.8%), breast cancer (3/83 subjects, 3.6%), and stomach cancer (3/83 subjects, 3.6%). Recently, Schnatz et al. [18,19], and Sharpless et al. [20], also found that malignancy in extrauterine organs, primarily the large intestine and breast, can be observed in histologic examination results of patients with AGUS.

bjects, 4.8%), breast cancer (3/83 subjects, 3.6%), and stomach cancer (3/83 subjects, 3.6%). Recently, Schnatz et al. [18,19], and Sharpless et al. [20], also found that malignancy in extrauterine organs, primarily the large intestine and breast, can be observed in histologic examination results of patients with AGUS. Based upon the results above, liquid-based Pap smear, colposcopy and biopsy, human papillomavirus tests, and cervical curettage are needed in patients with AGC to obtain appropriate examination results; cervical curettage may be especially important for patients who aged 35 years and older, or who possess risk factors for endometrial cancer. Moreover, examination of abnormalities within the pelvis, including pelvic examination and pelvic sonography, is also necessary. If no abnormalities are observed according the examinations described above, additional evaluation of extrauterine adenocarcinoma such as breast examination, gastroscopy, and colonoscopy is recommended, with consideration of the patient's age. Regardless of such comprehensive examinations, a 4.7% false negative error rate has been reported [18]. Thus, it is suggested that follow-up be carried out with Pap smear every 4 to 6 months, even if patients appear to be normal according to the examinations listed above.

ended, with consideration of the patient's age. Regardless of such comprehensive examinations, a 4.7% false negative error rate has been reported [18]. Thus, it is suggested that follow-up be carried out with Pap smear every 4 to 6 months, even if patients appear to be normal according to the examinations listed above. Given the results of the study, we can speculate that AGC, especially in cases of AGC-favor neoplastic, may be associated with adenocarcinoma (e.g., ovarian cancers, breast cancers, stomach cancers) or certain modifications found before or after AGC formation. Large-scale investigations with more subjects and investigation of the molecular basis for these results are warranted. Acknowledgments This work was supported for two years by Pusan National University Research Grant.

Given the results of the study, we can speculate that AGC, especially in cases of AGC-favor neoplastic, may be associated with adenocarcinoma (e.g., ovarian cancers, breast cancers, stomach cancers) or certain modifications found before or after AGC formation. Large-scale investigations with more subjects and investigation of the molecular basis for these results are warranted. Acknowledgments This work was supported for two years by Pusan National University Research Grant. Fig. 1 (A) Atypical endocervical cells, not otherwise specified (cervical smears by Pap stain, 34-year-old woman). Groups of cells show round to oval nuclei with nuclear enlargement, small nucleoli, and smooth nuclear membrane. Follow-up revealed chronic cervicitis (×400). (B) Atypical endocervical cells, favor neoplastic (cervical smears by Pap stain, 67-year-old woman). Sheet of cells show enlarged nuclei with hyperchromasia, some variation in nuclear size, small nucleoli, and feathering. Follow-up revealed endocervical adenocarcinoma (×400). (C) Atypical endometrial cells, favor neoplastic (cervical smears by Pap stain, 53-year-old woman). A small group of cells have mildly hyperchromatic nuclei, small nucleoli, and a vacuolated cytoplasm. Histologic diagnosis was endometrial adenocarcinoma (×400). Fig. 2 Relative distribution of benign pathology, pre-malignant diseases, and malignant diseases after clinical follow-up in AGC-NOS (A) and AGC-favor neoplastic (B). AGC, atypical glandular cell; NOS, not otherwise specified.

Fig. 1 (A) Atypical endocervical cells, not otherwise specified (cervical smears by Pap stain, 34-year-old woman). Groups of cells show round to oval nuclei with nuclear enlargement, small nucleoli, and smooth nuclear membrane. Follow-up revealed chronic cervicitis (×400). (B) Atypical endocervical cells, favor neoplastic (cervical smears by Pap stain, 67-year-old woman). Sheet of cells show enlarged nuclei with hyperchromasia, some variation in nuclear size, small nucleoli, and feathering. Follow-up revealed endocervical adenocarcinoma (×400). (C) Atypical endometrial cells, favor neoplastic (cervical smears by Pap stain, 53-year-old woman). A small group of cells have mildly hyperchromatic nuclei, small nucleoli, and a vacuolated cytoplasm. Histologic diagnosis was endometrial adenocarcinoma (×400). Fig. 2 Relative distribution of benign pathology, pre-malignant diseases, and malignant diseases after clinical follow-up in AGC-NOS (A) and AGC-favor neoplastic (B). AGC, atypical glandular cell; NOS, not otherwise specified. Fig. 3 Relative distribution of malignancies after clinical follow-up in AGC-NOS (A) and AGC-favor neoplastic (B). AGC, atypical glandular cell; NOS, not otherwise specified. Table 1 Prevalence of AGC using conventional Pap and liquid-based Pap (ThinPrep) Pearson-chi square analysis. Values are presented as number (%). AGC, atypical glandular cell; NOS, not otherwise specified. Table 2 Clinicopathological relevance of AGC-NOS and AGC-favor neoplastic Values are presented as number (%) or mean ± standard deviation. Data evaluated by unpaired t test.

Table 1 Prevalence of AGC using conventional Pap and liquid-based Pap (ThinPrep) Pearson-chi square analysis. Values are presented as number (%). AGC, atypical glandular cell; NOS, not otherwise specified. Table 2 Clinicopathological relevance of AGC-NOS and AGC-favor neoplastic Values are presented as number (%) or mean ± standard deviation. Data evaluated by unpaired t test. AGC, atypical glandular cell; NOS, not otherwise specified; HPV, human papillomavirus (DNA-chip method results are presented and polymerase chain reaction results are excluded). Table 3 Histological outcome of AGC-NOS and AGC-favor neoplastic on follow-up Values are presented as number (%) AGC, atypical glandular cell; NOS, not otherwise specified; LSIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion. a)Benign pathology includes polyps, cervicitis, metaplasia, and normal. Table 4 Extrauterine malignancy of AGC-NOS and AGC-favor neoplastic Values are presented as number (%) AGC, atypical glandular cell; NOS, not otherwise specified.

Introduction The autophagy maintains the homeostasis by binding the intracellular organelles and protein to lysosome and thereby degrading them when the nutrient supply to the cells is blocked. The major functions of autophagy include self-control, response to the stress and the alternative measures for energy and survival. But if this is persistently present, this would lead to a complete disruption of the cellular morphology. These phenomena are referred to as type II programmed cell death or intracytoplasmic cell death. It is known that the cancer cells supply the nutrients through the autophagy but this leads to the apoptosis when exposed to the long-standing stimuli [1,2]. One of the anti-cancer drugs that are commonly used to treat patients with gynecological cancer, paclitaxel has an anti-cancer effect are based on the apoptosis [3]. But most of the patients with progressive or recurrent cancer have a lower rate of treatment response to these anti-cancer drugs. To overcome this, efforts have been made to raise the treatment response by administering concomitant anti-cancer drugs rather than single one and introducing new anti-cancer drugs. To date, these efforts have revealed the limitations to the applicability to a clinical setting.

vel therapeutic approach to PCOS. In addition to improvement of lipid profiles, PCOS women receiving statins showed a significant decrease of testosterone, free androgen index, C-reactive protein, and insulin resistance [42-44]. However, the use of statins in pregnancy is contraindicated, and contraception is required. Conclusion Although prevalence of obesity in Korean women with PCOS is low, even young and non-obese Korean women with PCOS have substantially increased prevalence of dyslipidemia. Dyslipidemia in women with PCOS may be consistent with those found in the insulin resistant state: decreased levels of HDL-C and ApoA-I, increased levels of TG. Women with PCOS have both quantitative and qualitative changes in LDL-C profile: not only increased LDL-C level but also increased proportion of atherogenic small dense LDL. However, in a recent Korean study, non-obese Korean women with PCOS had no such abnormalities. In women with PCOS, ApoA-I, ApoC-I, and lipoprotein (a) abnormalities were also reported. Women with PCOS should receive a complete lipid test, and lifestyle modification is the first line therapy for all women with PCOS and is particularly important for those with dyslipidemia. Acknowledgments This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A100624).

ment response to these anti-cancer drugs. To overcome this, efforts have been made to raise the treatment response by administering concomitant anti-cancer drugs rather than single one and introducing new anti-cancer drugs. To date, these efforts have revealed the limitations to the applicability to a clinical setting. On the other hand, it is known that the phosphatidylinositol 3 phosphate kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway as well as the apoptosis is involved in the mechanisms by which the autophagy occurs. As described here, if there are any chances that these two involved mechanisms for apoptosis might be regulated, this would further lead to the programmed cell death. In this study, we examined whether there is a possibility to maximize the treatment effect using rapamycin, an inducer of the autophagy, concomitantly with conventional types of apoptosis-based anti-cancer drugs in a model of uterine cervical cancer. Materials and Methods 1. Culture of HeLa cell lines and the treatment of them with drugs The HeLa uterine cervical cancer cell lines were cultured in a Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) in conditions of 37℃ and 5% CO2. 1×104/mL HeLa cell lines were cultured on a 96-well plate (Nunc, Rochester, NY, USA) for 48 hours. This was followed by the treatment with an anti-cancer drug, paclitaxel (Sigma-Aldrich, St. Louis, MO, USA), and rapamycin (Sigma-Aldrich), an inducer of autophagy.

etal bovine serum (FBS) in conditions of 37℃ and 5% CO2. 1×104/mL HeLa cell lines were cultured on a 96-well plate (Nunc, Rochester, NY, USA) for 48 hours. This was followed by the treatment with an anti-cancer drug, paclitaxel (Sigma-Aldrich, St. Louis, MO, USA), and rapamycin (Sigma-Aldrich), an inducer of autophagy. Following the culture of HeLa uterine cervical cancer cell lines, they were treated with paclitaxel at varying concentrations of 0, 5, 10, 15, and 20 nM for 24 hours. This was followed by the examination of their inhibitory effects on cell survival. The cell survival was examined 24 hours after the treatment with rapamycin at varying concentrations of 0, 5, 10, 15, and 20 nM. On the other hand, to examine the synergistic effects of two drugs in increasing the survival of cells, we administered paclitaxel at varying concentrations of 0, 5, 10, and 20 nM to the HeLa cells that had been pre-treated with rapamycin 10 nM for three hours. Then, the cell viability was examined. The assessment was done with the methods for measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Colorimetric assay kit, Chemicon Inc., Temecula, CA, USA) which had been modified from tetrazolium based colorimetric assay. Following the addition of MTT samples 20 mg/mL, the culture was done for four hours. After the removal of culture medium, followed by the addition of dimenthylsulfoxide (DMSO), an analysis was done at a wave-length of 450 nm.

on Inc., Temecula, CA, USA) which had been modified from tetrazolium based colorimetric assay. Following the addition of MTT samples 20 mg/mL, the culture was done for four hours. After the removal of culture medium, followed by the addition of dimenthylsulfoxide (DMSO), an analysis was done at a wave-length of 450 nm. 2. The quantitative analysis of the apoptosis and autophagy due to paclitaxel in HeLa uterine cervical cancer cell lines 1-5×105 HeLa uterine cervical cancer cells were cultured for a day and then treated with 5 nM paclitaxel. Twenty-four hours after the treatment with paclitaxel, both paclitaxel-treated cells and their non-treated controls were administered trypsin. Then, the cells were placed in phosphate buffered saline (PBS) containing 1% paraformaldehyde. This was followed by the fixation with 70% cool ethanol. Annexin V propidium iodide (PI) staining were done with ApoDETECT Annexin V-FITC kit (Invitrogen, Calsbad, CA, USA). According to the manufacturer's instructions, changes in the apoptosis were examined. The stained cells were subjected to a quantitative apoptosis assay using a flow cytometry (FACS Vantage SE, BD Bioscience, San Jose, CA, USA). Thus, the cells undergoing apoptosis were counted.

FITC kit (Invitrogen, Calsbad, CA, USA). According to the manufacturer's instructions, changes in the apoptosis were examined. The stained cells were subjected to a quantitative apoptosis assay using a flow cytometry (FACS Vantage SE, BD Bioscience, San Jose, CA, USA). Thus, the cells undergoing apoptosis were counted. For the quantitative analysis of autophagy, changes in the development of acidic vesicular organelles were examined. That is, 5×105 HeLa cells were cultured overnight. This was followed by a 24-hour treatment with 5 nM paclitaxel. Paclitaxel-treated cells and their non-treated controls were stained with acridine orange (Sigma-Aldrich) at a concentration of 0.5 ug/mL at 37℃ for 15 minutes with the sunlight blocked. The cells were rinsed with PBS twice and then treated with trypsin. Then, the cells were suspended on PBS containing 1% FBS, whose number was counted using a flow cytometry. This was followed by the analysis using CellQuest 7.0 software (Beckman Coulter Co., Brea, CA, USA). 3. An assay of the proteins undergoing apoptosis and autophagy 1×106/mL cells were cultured on a 100 mm2 dish for 48 hours and then harvested. This was followed by a 30-minute centrifugation at a temperature of 4℃ and at 12,000 rpm. The extracted cells were reacted in a 100 µL lysis buffer at 4℃ for 30 minutes and then dissolved. The cell lysate was centrifuged at a temperature of 4℃ and at 12,000 rpm. Thus, the protein was harvested. This was followed by the measurement of the concentration of protein using Bio-Rad Protein Assay (Bio-Rad, Philadelphia, PA, USA).

Introduction During pregnancy and lactation, a woman's breasts face several physiological changes. These changes can be attributed to various hormones, which may also cause vascular hyperplasia and hyperplastic lobules [1]. Such changes may hinder the interpretation of physical and medical imaging examinations of the breasts. It is important to note that most breast lesions that are diagnosed during pregnancy and lactation are benign; however, the different diagnosis of breast cancer is challenging during these periods. Therefore, the aim of this article was to review the changes occurring in the breast, which are related to pregnancy and lactation, and to identify methods for the different diagnosis and treatment of breast disease.

lls were reacted in a 100 µL lysis buffer at 4℃ for 30 minutes and then dissolved. The cell lysate was centrifuged at a temperature of 4℃ and at 12,000 rpm. Thus, the protein was harvested. This was followed by the measurement of the concentration of protein using Bio-Rad Protein Assay (Bio-Rad, Philadelphia, PA, USA). The protein 50 µg was boiled for 5 minutes and then subjected to a 2-hour electrophoresis using a 10% sodium dodecyl sulfate/polyacrylamide gel at 100 V. Then, the protein was transferred to a nitrocellulose paper at 360 mA for an hour. This was followed by the blockage of unnecessary factors from 5% skim milk and 0.05% tris buffered saline with Tween 20 (TBS-T) solution on the membrane for an hour. Using the primary antibodies against caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and light chain (LC) 3 (Santa Cruz Biotechnology), the membrane was reacted overnight at 4℃. The membrane was rinsed with the tris-buffered saline and TBS-T, three times. Each membrane was reacted with the secondary antibody (anti-mouse IgG, Santa Cruz Biotechnology) at room temperature for an hour. Then, the membrane was rinsed with TBS-T three times. The specific protein bands were obtained using the ECL Western blotting system (Amersham, Piscataway, NJ, USA). 4. LC3 transfection For the quantitative analysis of autophagy, the cells were labeled with green fluorescent protein (GFP)-LC3. Then, changes in the number of spots were examined using a confocal microscope (quantitative autophagic assay).

Using the primary antibodies against caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and light chain (LC) 3 (Santa Cruz Biotechnology), the membrane was reacted overnight at 4℃. The membrane was rinsed with the tris-buffered saline and TBS-T, three times. Each membrane was reacted with the secondary antibody (anti-mouse IgG, Santa Cruz Biotechnology) at room temperature for an hour. Then, the membrane was rinsed with TBS-T three times. The specific protein bands were obtained using the ECL Western blotting system (Amersham, Piscataway, NJ, USA). 4. LC3 transfection For the quantitative analysis of autophagy, the cells were labeled with green fluorescent protein (GFP)-LC3. Then, changes in the number of spots were examined using a confocal microscope (quantitative autophagic assay). HeLa cells were transiently transfected into pCMV-GFP-LC3 expression vector (Cell Biolab, San Diego, CA, USA). Then, following the treatment with 10 nM rapamycin and 5 nM paclitaxel for 24 hours, the cells were fixed with 4% formaldehyde. The cells were rinsed with PBS and then examined using a polarized microscope (Olympus FluoView 1000, Olympus, Melville, NY, USA). At a magnification of ×200, we counted and then compared the number of cells where more than ten GFP-LC3 spots were stained. 5. Statistical analysis All the experimental procedures were performed independently at least three times in triplicate. All the data was expressed as mean±standard deviation. Statistical analysis was done using a two-sided Student's t-test. Statistical significance was accepted as P<0.05.

HeLa cells were transiently transfected into pCMV-GFP-LC3 expression vector (Cell Biolab, San Diego, CA, USA). Then, following the treatment with 10 nM rapamycin and 5 nM paclitaxel for 24 hours, the cells were fixed with 4% formaldehyde. The cells were rinsed with PBS and then examined using a polarized microscope (Olympus FluoView 1000, Olympus, Melville, NY, USA). At a magnification of ×200, we counted and then compared the number of cells where more than ten GFP-LC3 spots were stained. 5. Statistical analysis All the experimental procedures were performed independently at least three times in triplicate. All the data was expressed as mean±standard deviation. Statistical analysis was done using a two-sided Student's t-test. Statistical significance was accepted as P<0.05. Results 1. Anti-cancer effects of paclitaxel (an anti-cancer drug) and rapamycin (a regulator of the autophagy) on the HeLa cells HeLa uterine cervical cancer cell lines were treated with paclitaxel at varying concentrations. Then, we determined the 50% inhibition concentration (IC50) where the cytotoxicity reached the maximum level at 24 hours. The cell survival was inhibited in a concentration-dependent manner following a 24-hour treatment with paclitaxel at varying concentrations of 5, 10, 15, and 20 nM. In addition, the IC50 of paclitaxel, at which the growth of HeLa cells is decreased by 50%, ranged between 5 nM and 10 nM (Fig. 1A).

mum level at 24 hours. The cell survival was inhibited in a concentration-dependent manner following a 24-hour treatment with paclitaxel at varying concentrations of 5, 10, 15, and 20 nM. In addition, the IC50 of paclitaxel, at which the growth of HeLa cells is decreased by 50%, ranged between 5 nM and 10 nM (Fig. 1A). The HeLa cells were treated with rapamycin (a regulator of the autophagy) at varying concentrations of 5, 10, 15, and 20 nM for 24 hours. But there were no concentrations of rapamycin at which the cytotoxicity appeared (Fig. 1B).

mum level at 24 hours. The cell survival was inhibited in a concentration-dependent manner following a 24-hour treatment with paclitaxel at varying concentrations of 5, 10, 15, and 20 nM. In addition, the IC50 of paclitaxel, at which the growth of HeLa cells is decreased by 50%, ranged between 5 nM and 10 nM (Fig. 1A). The HeLa cells were treated with rapamycin (a regulator of the autophagy) at varying concentrations of 5, 10, 15, and 20 nM for 24 hours. But there were no concentrations of rapamycin at which the cytotoxicity appeared (Fig. 1B). In addition, we also examined the effects of concomitant treatment with paclitaxel and rapamycin at varying concentrations in inhibiting the cell survival. Following a 3-hour pre-treatment with 10 nM rapamycin, the cells were then treated with paclitaxel at concentrations of 0, 5, 10, and 20 nM. Then, 24 hours later, it was examined whether there were inhibitory effects on the survival of HeLa uterine cervical cancer cells. This showed that montherapy with rapamycin had no effects on the cell survival. In addition, following the pre-treatment with 10 nM rapamycin, the cells were treated with paclitaxel at concentrations of 10 nM and 20 nM. This showed that there were no significant differences in the inhibitory effects on the cell survival as compared with a lack of the pre-treatment with rapamycin. With the pre-treatment with rapamycin followed by the treatment with 5 nM paclitaxel, the degree of inhibitory effects on the cells survival was higher as compared with a lack of the pre-treatment with rapamycin. This difference reached a statistical significance (P<0.05) (Fig. 1C).

of the pre-treatment with rapamycin. With the pre-treatment with rapamycin followed by the treatment with 5 nM paclitaxel, the degree of inhibitory effects on the cells survival was higher as compared with a lack of the pre-treatment with rapamycin. This difference reached a statistical significance (P<0.05) (Fig. 1C). The difference in the cytotoxicity, described herein, can be explained by the synergistic effects of rapamycin (an inducer of the autophagy) at a certain concentration of paclitaxel. 2. The quantitative analysis of apoptosis and autophagy It was examined whether paclitaxel-induced inhibitory effects on the cell survival originate from the apoptosis and autophagy. To examine the mechanisms by which the cell survival is inhibited at 24 hours following the treatment of HeLa cells with 10 nM paclitaxel, the cells were stained with annexin V. The staining property was examined using a flow cytometry (BD FACS). Thus, it was confirmed that the apoptosis was involved in the mechanisms (Fig. 2A). It was also shown that the proportion of cells undergoing apoptosis was increased from 2.74% to 23.26% as compared with prior to the treatment with paclitaxel.

exin V. The staining property was examined using a flow cytometry (BD FACS). Thus, it was confirmed that the apoptosis was involved in the mechanisms (Fig. 2A). It was also shown that the proportion of cells undergoing apoptosis was increased from 2.74% to 23.26% as compared with prior to the treatment with paclitaxel. Moreover, to examine the quantitative changes in the autophagy, the cells were treated with 10 nM paclitaxel and then stained with acridine orange within 24 hours thereafter. Then, the number of acridine orange-positive cells was counted using a flow cytometry. This showed that the proportion of cells undergoing the autophagy was increased from 0.87% to 1.89% as compared with prior to the treatment with paclitaxel. It was also shown that the paclitaxel monotherapy had no effects on the autophagy (Fig. 2B). To identify the proteins that are involved in the apoptosis and autophagy, HeLa cells were treated with paclitaxel at varying concentrations of 0, 10, 20, and 50 nM for 24 hours. Following the treatment with 10 nM paclitaxel for 24 hours and 48 hours, the degree of the expressions of caspase 3 and LC3 was measured using a western blot analysis. Cleaved caspase 3, involved in the apoptosis, showed an increased degree of expression as the concentration of paclitaxel was elevated. But there were no changes in LC3 protein that is involved in the autophagy (Fig. 2C).

rs, the degree of the expressions of caspase 3 and LC3 was measured using a western blot analysis. Cleaved caspase 3, involved in the apoptosis, showed an increased degree of expression as the concentration of paclitaxel was elevated. But there were no changes in LC3 protein that is involved in the autophagy (Fig. 2C). 3. LC3 transfection After the LC3 transfection into the HeLa cells, followed by the treatment with paclitaxel and rapamycin, changes in the cells were examined using a light microscopy. Once the autophagy occurs, LC3 binds to the autophagosome. This leads to the formation of spots, which is a key marker of the autophagy (Fig. 3A). There was no increase in the number of cells where more than ten LC3 spots appeared following the treatment with 5 nM paclitaxel. Following the treatment with 10 nM rapamycin, however, it was increased. With the concomitant administration of two drugs, it was further increased (P<0.05) (Fig. 3B, C). Discussion In cases of nutrient deficiency, the tumor cells are dependent on the autophagy for their survival in the early stage of tumorigenesis. If this is continued, it would lead to the apoptosis. But this remains obscure at the present. In addition, it also remains unclear whether the autophagy directly causes the apoptosis or it is simply a phenomenon that occurs secondarily to the apoptosis [1,2].

agy for their survival in the early stage of tumorigenesis. If this is continued, it would lead to the apoptosis. But this remains obscure at the present. In addition, it also remains unclear whether the autophagy directly causes the apoptosis or it is simply a phenomenon that occurs secondarily to the apoptosis [1,2]. As the proteins involved in the autophagy, beclin 1 was first identified as a substance that binds to Bcl-2 (oncogene) based on the yeast two hybrid methods. It plays a role in triggering the occurrence of autophagy. Earlier studies have reported that the expression of beclin 1 led to the occurrence of autophagy and this eventually inhibited the tumor occurrence [4,5]. It has been shown that the inhibition of beclin 1 expression promoted the development of several types of tumor. Thus, it was confirmed that beclin 1 is a tumor suppressor gene. Moreover, it has also been shown that the autophagy is involved in the suppression of tumor occurrence [6,7]. Recent studies have also shown that some tumor suppressor genes such as the phosphatase and tensin homologue (PTEN) and TSC1/2, as well as beclin 1, are also involved in triggering the occurrence of autophagy [8,9]. The proposed mechanisms by which the autophagy is involved in the suppression of tumor occurrence are as follows: 1) The degradation of the key protein and cell organelles that are essential for suppressing the cell growth. 2) The production of reactive oxygen species and the removal of damaged cell organelles causing the genotoxic stress. 3) The induction of the occurrence of autophagy, another programmed cell death than the apoptosis, leading to the occurrence of apoptosis [10].

nelles that are essential for suppressing the cell growth. 2) The production of reactive oxygen species and the removal of damaged cell organelles causing the genotoxic stress. 3) The induction of the occurrence of autophagy, another programmed cell death than the apoptosis, leading to the occurrence of apoptosis [10]. It is widely known that the PI3K/Akt/mTOR signaling pathway is involved in the molecular biological mechanisms by which the autophagy occurs [8]. In the presence of the stimulation of growth factors, the growth factor receptors activate type I PI3K on the plasma membrane and thereby inhibits the transformation of autophagy. PI3K stimulates the Akt, the downstream target, and activates mTOR. Thus, it suppresses the p70S6 kinase-induced autophagy. On the other hand, if the PTEN gene is over-expressed, this would cause an autophagy with the antagonistic effects of type I PI3K. Ras two effects. If it stimulates the PI3K, this leads to the suppression of autophagy. If it selectively stimulates the RAF1-mitogen activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK), this triggers the occurrence of autophagy. The complex between rapamycin (mTOR inhibitor), type III PI3K and beclin 1 stimulates the autophagy. The differential features of autophagy-induced type II programmed cell death from type I one are not only that the apoptosis is morphologically characterized by changes in chromatin density, the formation of DNA laddering or fragmentation but also that the autophagy is characterized by an abundant presence of the intracellular autophagic vacuoles, the degradation of Golgi apparatus, ribosome and endoplasmic reticulum. In addition, it has also been known that the autophagy leads to a programmed cell death that occurs in a non caspase-dependent manner. But both events are involved in the PI3K/Atk/mTOR signaling pathway that is essential for the tumorigenesis. It is therefore presumed that the molecular biological mechanisms of programmed cell death might be inter-related.

at the autophagy leads to a programmed cell death that occurs in a non caspase-dependent manner. But both events are involved in the PI3K/Atk/mTOR signaling pathway that is essential for the tumorigenesis. It is therefore presumed that the molecular biological mechanisms of programmed cell death might be inter-related. There are some reports confirming that z-VAD (caspase inhibitor) triggers the occurrence of autophagy rather than apoptosis [11]. In addition, it has also been reported that the apoptosis is inhibited following the treatment with rapamycin (an inducer of the autophagy) [12]. Still, however, it remains unclear whether the autophagy would be helpful for the apoptosis during the anti-cancer treatment or it would have an antagonistic effect. In Korea, the uterine cervical cancer is one of the most common gynecological cancers, most of which are treated with surgery and radiotherapy. It is known as a carcinoma which is sensitive to platinum anti-cancer drugs such as cisplatin or taxanes such as paclitaxel [13]. Recently, there is a decreasing tendency in the incidence of uterine cervical cancer with the help of the Korean Uterine Cervical Cancer Screening Program. It is also known that the treatment outcomes are so excellent that the rate of complete cure is relatively higher with the early detection of it.

s paclitaxel [13]. Recently, there is a decreasing tendency in the incidence of uterine cervical cancer with the help of the Korean Uterine Cervical Cancer Screening Program. It is also known that the treatment outcomes are so excellent that the rate of complete cure is relatively higher with the early detection of it. Paclitaxel is one of the representative taxane anti-cancer drugs. It is known to stabilize the microtubules during the DNA synthesis and thereby to suppress the mitosis of the cancer cells. It has been tested on several clinical trials during the 1980's. In the 1990's, its indications for various types of cancer have been approved by the Food and Drug Administration. Recent studies have reported its excellent efficacy on not only uterine cervical cancer but also breast cancer, ovarian cancer, lung cancer, head-and-neck cancer and Kaposi's sarcoma. In cases of progressive cancer or recurrent uterine cervical cancer, however, it shows a lower degree of treatment effect. A poor prognosis is therefore unavoidable in these cancers. For this reason, the compound regimens are used to raise the efficacy of anti-cancer drugs. But side effects prevalently occur without markedly increasing the survival. This indicates that other treatment regimens than the anti-cancer drugs are necessary [14].

A poor prognosis is therefore unavoidable in these cancers. For this reason, the compound regimens are used to raise the efficacy of anti-cancer drugs. But side effects prevalently occur without markedly increasing the survival. This indicates that other treatment regimens than the anti-cancer drugs are necessary [14]. Recent studies have disclosed the mode of action of biological agents. Meanwhile, novel target treatment agents based on the inhibitors of monoclonal antibodies such as vascular endothelial growth factor and epidermal growth factor receptor have been actively introduced for the treatment of other cancers than uterine cervical cancer [15]. An inducer of the autophagy, rapamycin is also an inhibitor of mTOR; its efficacy on renal cancers has been demonstrated on several clinical studies conducted in cancer patients [16]. But rapamycin itself has an inhibitory effect on the cell growth to a greater extent than its cytotoxic effect. Therefore, its efficacy should be controlled with concomitant regimens with other drugs with a different mode of action rather than the monotherapy. In addition, the autophagy may induce the secondary apoptosis, but this is somewhat reversible and time-consuming. Presumably, it would therefore be recommended to use the adjuvant use rather than the direct anti-cancer effect of rapamycin [17].

h other drugs with a different mode of action rather than the monotherapy. In addition, the autophagy may induce the secondary apoptosis, but this is somewhat reversible and time-consuming. Presumably, it would therefore be recommended to use the adjuvant use rather than the direct anti-cancer effect of rapamycin [17]. Our results showed that rapamycin (an inducer of the autophagy) was effective in significantly increasing the autophagy in HeLa uterine cervical cancer cells. It was also shown that its degree of inhibitory effect was relatively higher as compared with the paclitaxel monotherapy. Besides, we also transfected LC3, an intermediate substance from autophagy using green fluorescent protein and then examined the quantitative changes. This showed that LC3 spots were increased at a lower concentration of paclitaxel. In addition, they were further increased when the autophagy was induced prior to the paclitaxel treatment. Therefore, our results showed that the use of an inducer of the autophagy could enhance the primary treatment response of conventional types of anti-cancer drugs in a model of gynecological cancer. In addition, it was also shown that the use of an inducer of the autophagy promoted the programmed cell death and this led to the increased anti-cancer effect.

e use of an inducer of the autophagy could enhance the primary treatment response of conventional types of anti-cancer drugs in a model of gynecological cancer. In addition, it was also shown that the use of an inducer of the autophagy promoted the programmed cell death and this led to the increased anti-cancer effect. At a certain concentration of paclitaxel, its concomitant use with rapamycin (a regulator of the autophagy) further increased an inhibitory effect on the cell survival (Fig. 1C). With the administration of 10 nM and 20 nM paclitaxel, there was a progression of the apoptosis. Therefore, we failed to confirm whether there are additional effects in inhibiting the cell survival when the survival of cells were suppressed at a proportion of 70% to 80%. With the administration of 5 nM paclitaxel at which the survival of cells was inhibited at a proportion of 50%, however, rapamycin additonally had an inhibitory effect on the cell survival at a proportion of approximately 15%. These results suggest that the autophagy would be helpful for the anti-cancer effects when the anti-cancer drugs such as paclitaxel are used in an in vivo setting if there are any chances that they could not sufficiently reach the target sites because the size of tumor is relatively larger or the blood vessels are not well developed.

uggest that the autophagy would be helpful for the anti-cancer effects when the anti-cancer drugs such as paclitaxel are used in an in vivo setting if there are any chances that they could not sufficiently reach the target sites because the size of tumor is relatively larger or the blood vessels are not well developed. Sun et al. [18] reported that the degree of cytotoxic effect of anti-cancer drugs was increased with the over-expression of beclin 1 in CaSki uterine cervical cancer cells. According to these authors, the toxicity of anti-cancer drugs such as cisplatin and paclitaxel increased and then amplified the autophagy of beclin 1 as well as apoptosis. Then, they noted that the autophagy play a key role in regulating the anti-cancer effects. According to Zou et al. [19], ARHI (a tumor suppressor gene) induced the autophagy and this led to a further increase in the inhibitory effects of paclitaxel in breast cancer cells. Recent reports have shown that the inhibitory effect of paclitaxel was increased following the use of siRNA transcripts of 3-methyladenine or beclin 1, both of which are inhibitors of the autophagy [20]. This implies that a regulator of the autophagy would be used as an adjuvant treatment strategy to enhance the efficacy of anti-cancer drugs.

n that the inhibitory effect of paclitaxel was increased following the use of siRNA transcripts of 3-methyladenine or beclin 1, both of which are inhibitors of the autophagy [20]. This implies that a regulator of the autophagy would be used as an adjuvant treatment strategy to enhance the efficacy of anti-cancer drugs. In the current study, we attempted to raise the degree of treatment response in patients with uterine cervical cancer and to identify novel treatment modalities that can overcome the limitations of conventional types of anti-cancer drugs. To put this in another way, it has been hypothesized that the future treatment modalities will be effective in enhancing the early efficacy of the pharmacological treatments, preventing the recurrence of cancer and treating it by concomitantly using the target treatment agents or immune boosters based on other biological agents with a different mode of action from conventional types of standard anti-cancer therapies. In conclusion, our results indicate that an inducer of the autophagy was effective in increasing the efficacy of conventional types of anti-cancer drugs causing an apoptosis in uterine cancer cells. Thus, our results provided an evidence demonstrating that an inducer of the autophagy might be used an adjuvant modality during the anti-cancer pharmacological treatments. Acknowledgments The authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation made in the program years of 2009 and 2010.

In conclusion, our results indicate that an inducer of the autophagy was effective in increasing the efficacy of conventional types of anti-cancer drugs causing an apoptosis in uterine cancer cells. Thus, our results provided an evidence demonstrating that an inducer of the autophagy might be used an adjuvant modality during the anti-cancer pharmacological treatments. Acknowledgments The authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation made in the program years of 2009 and 2010. Fig. 1 (A, B) Cell viability after treatment of paclitaxel and rapamycin in HeLa cervical cancer cells. (C) Cell viability after treatment with various doses of paclitaxel with or without 10 nM of rapamycin pretreatment. a)Rapamycin pretreatment followed by 5 nM of paclitaxel showed significant suppression of cellular growth (P<0.05). Fig. 2 (A, B) Apoptotic cells stained by Anenxin V and autophagic cells stained by acridine orange counted at 24 hour after treatment with 5 nM of paclitaxel (detected by flow cytometry). (C) Paclitaxel exposure induced increased dose- and time-dependent caspase-3 expression. Fig. 3 (A) Increased GFP-LC3-treated cells (arrows) in the process of autohpagy (×40). (B, C) Autophagic vacuoles in LC3-transfected cells increased in number after addition of 10 nM of rapamycin and a combination of 10 nM of rapamycin and 5 nM of paclitaxel for 24-hour to HeLa cells (×10).

Introduction Surgery is a major stressor that induces secretion of various substances such as prostaglandin, serotonin, and histamine as a reaction to localized tissue damage. In laparotomies with larger incisions, intra-abdominal surgical incision site pain is the most significant cause of acute postoperative pain. Extended lower midline laparotomies have relatively long incisions, and thus effective pain control of the surgical incision site is particularly important. Pain at the intra-abdominal incision site which is not effectively controlled interferes with the deep breathing necessary for early ambulation and recovery from atelectasis. Uncontrolled pain can also affect the respiratory, cardiovascular, digestive, urinary and musculoskeletal system, thereby making it difficult to recover quickly from surgery, as well as affecting the overall success of the surgery. Various methods including epidural anesthesia, intraspinal anesthesia, intrapleural anesthesia, and intravenous patient controlled analgesia are used to control postoperative pain, and parenteral narcotics are commonly used as anesthesia. Even though parenteral narcotics play a key role in the reduction of postoperative pain, they include many side effects such as nausea, vomiting, an itching sensation, palpitation, low blood pressure, weakening of muscles, and dizziness. Other serious side effects include difficult urinating, paralytic ileus, miosis, increased intracranial pressure and respiratory suppression. There have been many attempts to reduce these side effects, such as combining parenteral narcotics with other drugs or adjusting the dose of narcotic analgesia, but some side effects still persist.

effects include difficult urinating, paralytic ileus, miosis, increased intracranial pressure and respiratory suppression. There have been many attempts to reduce these side effects, such as combining parenteral narcotics with other drugs or adjusting the dose of narcotic analgesia, but some side effects still persist. Continuous injection of local analgesia into the surgical incision site has been effectively used to reduce the side effects of narcotics [1-16]. The ON-Q pain management system (ON-Q PainBuster, referred to as ON-Q pump) created by the I Flow Corp. (Lake Forest, CA, USA) is a device that continuously administers local analgesia directly into the intra-abdominal surgical wound site. Studies comparing the use of this device to a placebo have been carried out over the past several years in numerous surgical departments [4,17-19]. However, no study has evaluated postoperative surgical incision site pain control using the ON-Q pump in gynecologic cancer patients who underwent oncologic surgeries with an extended lower midline incision. This study randomly selected gynecologic cancer patients who had undergone oncologic surgeries with an extended lower midline incision, and compared postoperative intra-abdominal surgical incision site pain between a group that administered local analgesia directly into the incision site wound by ON-Q infusion pump, and a group that administered parenteral narcotics via intravenous patient-controlled analgesia (referred to as IV PCA).

midline incision, and compared postoperative intra-abdominal surgical incision site pain between a group that administered local analgesia directly into the incision site wound by ON-Q infusion pump, and a group that administered parenteral narcotics via intravenous patient-controlled analgesia (referred to as IV PCA). Materials And Methods Gynecology oncologic patients who completed baseline studies with a confirmed diagnosis of carcinoma between November 2011 and April 2012 at the Department of Obstetrics and Gynecology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea were selected as study subjects. Twenty patients consented to use a pain control device following gynecology oncologic surgery and were randomly divided into two groups of ten. Postoperative intra-abdominal surgical incision site pain was compared between the group that administered local analgesia through an ON-Q pump (ON-Q pain management system, I Flow Corp.) inserted into the surgical incision site, and the group that administered parenteral narcotics via IV PCA. The groups consisted of patients who underwent laparotomy through a lower midline incision that extended 6 to 7 cm above the umbilicus from the pubic bone. Surgeries were limited to the staging operation, including total abdominal hysterectomy (or radical abdominal hysterectomy) and bilateral pelvic lymph node dissection, but could be modified by each diagnosis. Patients who received an American Society of Anesthesiologist grade of more than IV; had an allergic reaction to ropivacaine hydrochloride, fentanyl citrate or ketorolac tromethamine; had a history of drug or alcohol addiction in the last six months; or had serum creatinine levels greater than 2 mg/dL were excluded from this study.

received an American Society of Anesthesiologist grade of more than IV; had an allergic reaction to ropivacaine hydrochloride, fentanyl citrate or ketorolac tromethamine; had a history of drug or alcohol addiction in the last six months; or had serum creatinine levels greater than 2 mg/dL were excluded from this study. A multifilament synthetic absorbable suture was used for closure from the peritoneum to the subcutaneous layer, and a disposable skin stapler with a width of 9.9 mm was used to suture the skin. All surgeries and insertion of the ON-Q pump catheter were performed by the same surgeon and surgical assistants using standard surgical procedures.

tic absorbable suture was used for closure from the peritoneum to the subcutaneous layer, and a disposable skin stapler with a width of 9.9 mm was used to suture the skin. All surgeries and insertion of the ON-Q pump catheter were performed by the same surgeon and surgical assistants using standard surgical procedures. Age, body mass index (BMI), duration of surgery, length of incision, duration of hospitalization following surgery, final diagnosis, old scar revisions, name of operation, postoperative surgical incision site pain score at each point, and the number of times postoperative analgesia was administered were all noted in electronic medical records. The visual analogue scale (VAS) was used to assess pain (Fig. 1). VAS provides a simple, minimally intrusive measure of pain intensity that has been widely utilized in research settings that require a quick and quantitative pain index. VAS consists of a 10-cm horizontal line with two endpoints labeled as "no pain" and "the worst possible pain." The distance (in centimeters) from the low end point of VAS to the patient's mark is considered a numerical index of pain intensity. The patients used the VAS to report a postoperative pain score at the intra-abdominal surgical incision site immediately following surgery, as well as 6, 24, 48, 72, and 96 hours after surgery. During surgery, both groups received 20 µg/mL of remifentanil at an average rate of 10 mL/hr. For the ON-Q group, 50 µg/mL of fentanyl citrate was intravenously injected once at the end of surgery. For the IV PCA administered group, no additional analgesia was provided at the end of the surgery. Both groups controlled additional pain only with an intravenous injection of ketorolac tromethamine (30 mg/mL) at the patients' request. When the patients started ingesting soft diet, oral nonsteroidal anti-inflammatory drugs (NSAIDs) were given three times daily.

ional analgesia was provided at the end of the surgery. Both groups controlled additional pain only with an intravenous injection of ketorolac tromethamine (30 mg/mL) at the patients' request. When the patients started ingesting soft diet, oral nonsteroidal anti-inflammatory drugs (NSAIDs) were given three times daily. 1. ON-Q pain management system The ON-Q pain management system includes an elastomeric pump filled with local analgesia solution (total volume of 300 mL), which is a mixture of 400 mg of ropivacaine hydrochloride (Nacaine [Huons] 40 mg/20 mL) and 100 mL of 0.9% normal saline. This elastomeric pump is connected to a catheter inserted into the intra-abdominal surgical incision site, and is designed to inject 300 mL of 0.5% ropivacaine solution through two strands of soaker catheter at the rate of 2 mL/hr each for 72 hours with consistent pressure (10 psi) (Fig. 2).

0 mL of 0.9% normal saline. This elastomeric pump is connected to a catheter inserted into the intra-abdominal surgical incision site, and is designed to inject 300 mL of 0.5% ropivacaine solution through two strands of soaker catheter at the rate of 2 mL/hr each for 72 hours with consistent pressure (10 psi) (Fig. 2). Soaker catheters were placed by inserting 20-gauge guiding needles into the subfascial supraperitoneal layer of the surgical incision after having sutured up to the peritoneum. The guiding needles were placed about 3 cm from the midline incision at the upper and lower position of the incision line. Once the introducer was fixed in the desired location, the needle was removed and a length of catheter approximately 6 to 10 cm was embedded in the incisional wound prior to suturing the skin. When both upper and lower catheters were fixed securely, a bolus of ropivacaine (10 mg/5 mL) was injected through both strands of soaker catheters and the ON-Q infusion pump was connected immediately. The pump contents were then continuously infused into the surgical incision site at a flow rate of 4 mL/hr (2 mL/hr per catheter) for 72 hours. The catheters located in the subfascial supraperitoneal layer were so thin that no subcutaneous suture was necessary, although fixed dressing of catheters was necessary at the injection site. An extra 20 mg/10 mL of ropivacaine was injected directly into the subcutaneous layer prior to suturing the skin (Fig. 3).

ours. The catheters located in the subfascial supraperitoneal layer were so thin that no subcutaneous suture was necessary, although fixed dressing of catheters was necessary at the injection site. An extra 20 mg/10 mL of ropivacaine was injected directly into the subcutaneous layer prior to suturing the skin (Fig. 3). 2. Intravenous patient-controlled analgesia Before surgery was completed, IV PCA was initiated via intravenous infusion (Accufuser Plus) with a total of 100 mL of mixed solution of fentanyl citrate (20 mg/mL per kg) and ondansetron hydrochloride (16 mg/8 mL) in 0.9% normal saline, which is the standard solution at the study institution. Although IV PCA was infused continuously at the basic flow rate of 2 mL/hr, 0.5 mL of bolus can be injected if the patients press the button once upon experiencing pain. The lock-out time for each bolus injection was 15 minutes. For statistical analysis, a descriptive analysis was carried out first. The Mann-Whitney U test was used to examine differences in continuous variables between the groups, and Fisher's exact chi-square test was utilized to examine differences in nominal variables between groups. P-values less than 0.05 was considered statistically significant. Statistical analyses were performed with PASW statistics ver. 18.0 for Windows (SPSS Inc., Chicago, IL, USA).

inuous variables between the groups, and Fisher's exact chi-square test was utilized to examine differences in nominal variables between groups. P-values less than 0.05 was considered statistically significant. Statistical analyses were performed with PASW statistics ver. 18.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Among the 20 patients that participated in this study, ten were in the ON-Q group and ten were in the IV PCA group. No patients stopped taking medication due to side effects or for other reasons, although two patients in the IV PCA administered group had to temporarily stop medication due to nausea and dizziness in the middle of administration on the day of surgery. In these cases, medication was resumed within several hours once the side effects were controlled by allopathic medication, and ultimately the complete dosage of IV PCA was dispensed.

administered group had to temporarily stop medication due to nausea and dizziness in the middle of administration on the day of surgery. In these cases, medication was resumed within several hours once the side effects were controlled by allopathic medication, and ultimately the complete dosage of IV PCA was dispensed. Table 1 summarizes the basic characteristics of each analgesia group including age, BMI, duration of surgery, length of incision, duration of hospitalization, final diagnosis, whether or not old scars were revised, and name of operation. The average age of the ON-Q and IV PCA groups were 56.2 ±8.2 years and 50.8 ±8.2 years, respectively. The average length of incision for the ON-Q group was longer than that of the IV PCA group (25.3±1.2 cm vs. 24.7±1.1 cm). Differences in age and incision length were not statistically significant. There was also no statistically significant difference between the ON-Q group and the IV PCA group in terms of BMI, duration of surgery, duration of hospitalization, and whether or not old scar revision was performed. Two patients in the ON-Q group and three patients in the IV PCA group underwent surgery following a diagnosis of uterine cervical cancer, and two patients in the ON-Q group and three patients in the IV PCA group underwent surgery following a diagnosis of endometrial cancer. One patient in the ON-Q group was diagnosed with recurrent ovarian cancer and underwent a debulking operation including total abdominal hysterectomy and lymphadenectomy at this study institution, followed by bilateral salpingo-oophorectomy at other institution. There were no statistically significant differences between the groups in terms of final diagnosis and surgeries that were performed (Table 1).

nt a debulking operation including total abdominal hysterectomy and lymphadenectomy at this study institution, followed by bilateral salpingo-oophorectomy at other institution. There were no statistically significant differences between the groups in terms of final diagnosis and surgeries that were performed (Table 1). The average postoperative pain score at the surgical incision site in the ON-Q group was lower than the IV PCA group immediately after surgery, as well as 24, 48, and 72 hours after surgery (Table 2), although the differences were statistically significant only at 24 hours and 48 hours (24 hours, P=0.023; 48 hours, P<0.001). This suggests that the intra-abdominal surgical incision site was less painful in the ON-Q group during the postoperative acute phase. On the other hand, the average pain score 96 hours after surgery was significantly higher in the ON-Q group (2.9±0.9) than the IV PCA group (2.1±0.6) (P=0.035) (Fig. 4). The number of times painkiller administered additional postoperative analgesia was higher in the ON-Q group (5.1±3.4) than that in the IV PCA group (4.3±3.3), although this difference was not statistically significant (P=0.481).

ntly higher in the ON-Q group (2.9±0.9) than the IV PCA group (2.1±0.6) (P=0.035) (Fig. 4). The number of times painkiller administered additional postoperative analgesia was higher in the ON-Q group (5.1±3.4) than that in the IV PCA group (4.3±3.3), although this difference was not statistically significant (P=0.481). Discussion Local analgesia is a relatively safe and inexpensive painkiller that controls pain by blocking the transmission of pain from peripheral nerves at the damaged site. However, the use of local analgesia is limited and frequent re-administration is necessary due to the relatively short analgesic effect, which is 2 to 6 hours on average for single dose. The ON-Q pump created by I Flow resolves the issue of repetitive administration and efficiently blocks the transmission of pain from the surgical incision site to the spinal cord via a continuous infusion system [20-25]. Wound perfusion with local anesthetic solution by an indwelling irrigation unit was first designed by Capelle [26] in the 1930's. In the 1950's, Blades and Ford [27] refined this technique for thoracotomy incisions with the insertion of a fine catheter before closure, and noticed an associated decrease in opioid requirements. Additional research on upper abdominal wounds confirmed a decrease in opioid requirements. Nevertheless, this technique did not achieve popularity at that time. It did, however, begin to receive attention as a 'new method of pain relief' when re-discovered by Samarji [28] in 1972 [29,30]. The ON-Q pump, which provides a continuous infusion of local analgesia into the surgical incision, has proven to be an effective method of managing postoperative pain and reducing the use of opioids in a wide range of medical fields including general surgery, urology, and thoracic surgery [31-39]. Fredman et al. [17] and Givens et al. [18] evaluated the effect of the ON-Q pump on pain reduction and opioid use in obstetric patients after caesarian section. They demonstrated the importance of maintaining blood levels of unbound ropivacaine below the toxic threshold by comparing a group using local anesthetics with a placebo group using normal saline. Gupta et al. [19] showed that application of the ON-Q pump provides more effective postoperative pain relief for total abdominal hysterectomy than a placebo, while Zimberg et al. [40] revealed that the ON-Q pump can reduce the duration of hospitalization following total abdominal hysterectomy, thereby cutting hospital costs up to 30%.

] showed that application of the ON-Q pump provides more effective postoperative pain relief for total abdominal hysterectomy than a placebo, while Zimberg et al. [40] revealed that the ON-Q pump can reduce the duration of hospitalization following total abdominal hysterectomy, thereby cutting hospital costs up to 30%. IV PCA is the most commonly used method in postoperative pain control for patients undergoing gynecology oncologic surgery with an extended midline incision. However, this study found that the ON-Q pump was more helpful in pain control at the surgical incision site during the postoperative acute phase, and was significantly more effective at 24 hours and 48 hours after surgery (P=0.023, P<0.001, respectively). Pain scores at the surgical incision site decreased as time passed after surgery in both the ON-Q group and the IV PCA group, whereas the pain score increased at 96 hours after surgery in both groups. This finding at 96 hours after surgery suggests less meaningful because both ON-Q and IV PCA had already been dispensed at this point. In terms of the number of times a patient administered additional analgesia, on average the ON-Q group administered analgesia more frequently than the IV PCA group (5.1±3.4 vs. 4.3 ±3.3, respectively). The autonomic nerve system including the inferior hypogastric nerve plexus can be damaged by a wide transection of the uterosacral ligament, particularly in radical abdominal hysterectomy, and is known as one of the main factors in immediate postoperative pain [41,42]. This study only considered postoperative pain at the surgical incision site and excluded visceral pain such as the pain described above, or musculoskeletal pain due to the long operating time. Considering the difference in the number of times of analgesic was administered between the two groups was less than 1.0, pain score errors associated with the relief of visceral or musculoskeletal pain should be negligibly small. The average period of hospitalization was longer in the ON-Q group (9.9 ±3.7 days) than in the IV PCA group (9.2 ±3.0 days) (P=0.796), but the median value for duration of hospitalization in the ON-Q group was 8.0 days, which was shorter than the 8.5 days for the IV PCA group. The average duration of hospitalization in the ON-Q group seems to be skewed by one patient in the ON-Q group who was discharged on the day 19 after finishing the first postoperative adjuvant chemotherapy on day 14 following the staging operation.

was 8.0 days, which was shorter than the 8.5 days for the IV PCA group. The average duration of hospitalization in the ON-Q group seems to be skewed by one patient in the ON-Q group who was discharged on the day 19 after finishing the first postoperative adjuvant chemotherapy on day 14 following the staging operation. While two patients in the IV PCA group temporarily stopped administration of IV PCA due to nausea and dizziness, the ON-Q group did not have any side effects or any cessation during administration. Thus the ON-Q pump was able to substantially reduce the need for additional drugs such as anti-emetics. This study differs from previous ON-Q pump studies in that it compares the effect of the ON-Q pump with IV PCA instead of a placebo. IV PCA is generally administered in gynecology oncologic laparotomies and has limited use during the postoperative acute phase due to various side effects of narcotics. However, if there are minimal side effects, IV PCA can be used in accordance with additional medication such as antiemetics.

PCA instead of a placebo. IV PCA is generally administered in gynecology oncologic laparotomies and has limited use during the postoperative acute phase due to various side effects of narcotics. However, if there are minimal side effects, IV PCA can be used in accordance with additional medication such as antiemetics. Hypersensitivity is a known side effect of the ON-Q pump and is caused by the ester series of anesthetics, but it is very rare and occurs in less than 1% of patients using the ON-Q pump [43]. Because ON-Q pump catheters are embedded in the incisional wound during the operation, it is possible to reduce unnecessary medication without generalized side effects or additional pain. Moreover, by relieving pain in the surgical wound area, which is the main cause of postoperative pain in the acute phase, the ON-Q pump helps prevent pulmonary complications such as atelectasis and aids in early ambulation and early recovery of bowel movements in gynecology oncologic surgeries with a long incision, all without the use of IV PCA. Consequently, the ON-Q pump is highly beneficial to cancer patients who desire a quick recovery from surgery for postoperative adjuvant anti-cancer therapy.

electasis and aids in early ambulation and early recovery of bowel movements in gynecology oncologic surgeries with a long incision, all without the use of IV PCA. Consequently, the ON-Q pump is highly beneficial to cancer patients who desire a quick recovery from surgery for postoperative adjuvant anti-cancer therapy. This study shows that acute postoperative surgical incision site pain is well controlled using only the ON-Q pump and additional intravenous NSAIDs, without opioid-based IV PCA, after laparotomy of gynecologic cancer with an extended lower midline incision. However, this study had a small sample size which made it difficult to use a linear regression model to determine whether the use of additional pain relievers or the average period of hospitalization was affected by parameters other than the method of postoperative pain control. Therefore, future studies should use a larger sample size and should evaluate the effect of the ON-Q pump on generalized pain such as visceral and musculoskeletal pain. In addition, future studies should specify indices of early recovery and establish a multi-regimen protocol for postoperative pain control. Fig. 1 Visual analogue scale (VAS): The VAS provides a simple and efficient measure of pain intensity that has been used widely and consists of a 10 cm horizontal line with the two endpoints labeled as "no pain" and "worst pain." The distance (centimeters) between the low end of the VAS and the patient's mark is used as a numerical index of pain intensity.

e VAS provides a simple and efficient measure of pain intensity that has been used widely and consists of a 10 cm horizontal line with the two endpoints labeled as "no pain" and "worst pain." The distance (centimeters) between the low end of the VAS and the patient's mark is used as a numerical index of pain intensity. Fig. 2 Elastometric pump filled with local anesthetic. The protective cap is first removed from the pump, a syringe filled with local anesthetic is attached to the fill port, and the fluid is injected into the pump. Fig. 3 Placement of infusion catheters. Inserting the Soaker catheters into the subfacial supraperitoneal layer (A). The introducer needle is placed approximately 3 cm from the midline incision (B). Fig. 4 Mean change in postoperative surgical site pain. VAS, visual analogue scale; post op, postoperative; IV PCA, intravenous patient-controlled analgesia. Table 1 Patient characteristics (n=20) Values are presented as mean±standard deviation or number (%). IV PCA, intravenous patient-controlled analgesia; BMI, body mass index; TAH, total abdominal hysterectomy; BSO, bilateral salpingo-oophorectomy; BPLD, bilateral pelvic lymph node dissection; PALD, paraaortic lymph node dissection; Om, total omentectomy; Ia, incidental appendectomy; RAH, radical abdominal hysterectomy. Table 2 Comparison On-Q group and intravenous patient-controlled analgesia (IV PCA) group Values are presented as mean (standard deviation).

Advanced maternal age (AMA) is usually defined as a mother who is 35 years of age or older at delivery. AMA is related to aneuploidies by nondisjunction of the chromosomes during maternal oogenesis (meiosis) and is therefore an important risk factor for fetuses with chromosomal abnormalities. AMA has also been used as a sole indicator for undergoing cytogenetic amniocentesis. As such, pregnant women with AMA have been offered invasive diagnostic testing, such as amniocentesis or chorionic villus sampling (CVS), as well as genetic counseling. In Korea, the proportion of pregnant women with AMA is continuously increasing due to increases in the average age of marriage and the social participation of females, and has more than doubled over the past 10 years according to source of the National Statistical Office (from 7.4% in 2001 to 18.0% in 2011). It is important to monitor various indicators in pregnant women with AMA in order to predict the risk of fetal chromosomal abnormalities that depend on the mother's age. However, the validity of AMA as a potential indicator relating to the risk of fetal chromosomal abnormalities is still being studied and its usefulness is still under debate.

or various indicators in pregnant women with AMA in order to predict the risk of fetal chromosomal abnormalities that depend on the mother's age. However, the validity of AMA as a potential indicator relating to the risk of fetal chromosomal abnormalities is still being studied and its usefulness is still under debate. In 2007, the American Congress of Obstetricians and Gynecologists (ACOG) recommended that patients should have the option to take a diagnostic test such as amniocentesis or CVS regardless of maternal age [1]. In previous studies, Forabosco et al. [2] and Boyd et al. [3] reported that the decision to undergo amniocentesis should not be based solely on maternal age. These changes in the guidelines of genetic counseling for pregnant women with AMA appear to be based on the high detection rate of prenatal screening tool such as integrated test. Due to improvements in the diagnostic accuracy of prenatal screening, doctors' individual risk assessments show a tendency to prefer the easier screening algorithms rather than offering amniocentesis to all pregnant women with AMA. Recently, Kwon et al. [4] reported that the quadruple screening test is a better choice than direct amniocentesis for pregnancies complicated by AMA under conditions in which first trimester screening test is not available.

e easier screening algorithms rather than offering amniocentesis to all pregnant women with AMA. Recently, Kwon et al. [4] reported that the quadruple screening test is a better choice than direct amniocentesis for pregnancies complicated by AMA under conditions in which first trimester screening test is not available. However, quite a few obstetricians still use a maternal age of 35 years as a cutoff for offering diagnostic testing. Park et al. [5] determined that AMA was a strong risk factor for chromosomal abnormalities. Bornstein et al. [6] suggested that the benefit of undergoing genetic amniocentesis based only on AMA far outweighs the potential amniocentesis-related fetal loss rate and Henry et al. [7] reported that the rate of Down syndrome births for women with AMA increased considerably, while the same rate for pregnant women <35 years of age remained stable. This demonstrates that especially for women who do not undergo combined screening and who want definitive information on the chromosomal status of their fetus, AMA is a reasonable indicator for invasive prenatal diagnosis.

increased considerably, while the same rate for pregnant women <35 years of age remained stable. This demonstrates that especially for women who do not undergo combined screening and who want definitive information on the chromosomal status of their fetus, AMA is a reasonable indicator for invasive prenatal diagnosis. In this controversial situation regarding the use of AMA as a sole indicator for invasive diagnostic testing, all pregnant women should have the option to choose invasive diagnostic testing such as amniocentesis or CVS, regardless of maternal age, and women should be counseled regarding the differences between prenatal screening and invasive diagnostic testing, according to ACOG guidelines [1]. Obstetricians should guide pregnant women through genetic counseling that is focused on the importance of individual choice, based on the risk that the fetus will have a chromosomal abnormality and the risk of pregnancy loss from an invasive procedure, as well as the morals and viewpoints of the individual, couple, and/or family.

Introduction Polycystic ovary syndrome (PCOS) is one of the most common causes of endocrine dysfunction in women of reproductive age with a prevalence that ranges from 4% to 7% [1,2]. Metabolic disturbances are well-recognized clinical features of this syndrome. Especially, dyslipidemia is a very common metabolic abnormality in women with PCOS, with a prevalence of up to 70% [3]. Insulin resistance is a key pathophysiology of PCOS, and dyslipidemia in women with PCOS may therefore be consistent with that found in the insulin resistant state: decreased levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein (Apo) A-I, and increased levels of triglycerides (TG), ApoB and very low-density lipoprotein [4-7]. The aim of the present review was to address the detailed profile of dyslipidemia in PCOS. In addition, obesity can alter lipoprotein lipid profiles, but most of the Korean women with PCOS are not obese [8]. Thus, we also reviewed the unique characteristics in dyslipidemia profile in Korean women with PCOS.

Introduction Polycystic ovary syndrome (PCOS) is one of the most common causes of endocrine dysfunction in women of reproductive age with a prevalence that ranges from 4% to 7% [1,2]. Metabolic disturbances are well-recognized clinical features of this syndrome. Especially, dyslipidemia is a very common metabolic abnormality in women with PCOS, with a prevalence of up to 70% [3]. Insulin resistance is a key pathophysiology of PCOS, and dyslipidemia in women with PCOS may therefore be consistent with that found in the insulin resistant state: decreased levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein (Apo) A-I, and increased levels of triglycerides (TG), ApoB and very low-density lipoprotein [4-7]. The aim of the present review was to address the detailed profile of dyslipidemia in PCOS. In addition, obesity can alter lipoprotein lipid profiles, but most of the Korean women with PCOS are not obese [8]. Thus, we also reviewed the unique characteristics in dyslipidemia profile in Korean women with PCOS. Serum TG, HDL cholesterol, and LDL cholesterol Decrease in HDL-C and increase in TG levels are well known lipid profile characteristics in women with PCOS [3,9-16]. Recently, Wild et al. [17] reported meta-analysis of lipid levels in world-wide cross-sectional studies in women with PCOS (mainly performed in European and American women). In this analysis, TG levels were 26 mg/dL (95% confidence interval [CI], 17 to 35) higher and HDL-C concentrations were 6 mg/dL (95% CI, 4 to 9) lower in women with PCOS than those of controls. Also, low-density lipoprotein cholesterol (LDL-C) and non HDL-C concentrations were 12 mg/dL (95% CI, 10 to 16) and 19 mg/dL (95% CI, 16 to 22) higher, respectively. With body mass index (BMI) matching, LDL-C and non HDL-C were still higher in PCOS subjects: by 9 mg/dL (95% CI, 6 to 12) and 16 mg/dL (95% CI, 14 to 19), respectively [17].

ipoprotein cholesterol (LDL-C) and non HDL-C concentrations were 12 mg/dL (95% CI, 10 to 16) and 19 mg/dL (95% CI, 16 to 22) higher, respectively. With body mass index (BMI) matching, LDL-C and non HDL-C were still higher in PCOS subjects: by 9 mg/dL (95% CI, 6 to 12) and 16 mg/dL (95% CI, 14 to 19), respectively [17]. HDL encompasses several different classes of lipoproteins (HDL2 and HDL3) according to its density, metabolism and properties [18]. HDL subclasses are known to differ in their capacity to confer cardio-protection, and HDL2 has been reported as the most anti-atherogenic HDL subtype. Thus decreased levels of HDL2 have been strongly associated with coronary heart disease, and HDL subclass profile has been investigated in women with PCOS. Talbott et al. [19] recruited a total of 206 women with PCOS and 206 age-matched controls, and total HDL and HDL2 levels were significantly lower in women with PCOS than controls even after controlling for both age and BMI. Conway et al. [20] also found that even lean women with PCOS had reduced serum HDL and HDL2 concentrations compared to controls. These findings suggest that women with PCOS not only have low serum HLD-C levels, but also show alterations in HDL quality.

ith PCOS than controls even after controlling for both age and BMI. Conway et al. [20] also found that even lean women with PCOS had reduced serum HDL and HDL2 concentrations compared to controls. These findings suggest that women with PCOS not only have low serum HLD-C levels, but also show alterations in HDL quality. Many studies have reported that LDL-C is increased in women with PCOS [3,21], which is usually not noted in insulin resistant states. The reason why LDL-C in also increased in women with PCOS in not clear yet, but increased LDL-C levels in women with PCOS may be related to hyperandrogenism or genetic factor. Recabarren et al. [22] investigated the metabolic profiles in sons of women with PCOS during different stages of life. In this study, 80 sons of women with PCOS and 56 sons of control women were enrolled since early infancy, and during adulthood, sons of women with PCOS exhibited significantly higher LDL-C levels than those of controls (106.8 mg/dL [range, 52.6 to 224.3] versus 94.0 mg/dL [range, 60.4 to 142.5], respectively, P = 0.022) [22]. Sam et al. [23] also investigated the metabolic phenotypes in 215 non-Hispanic white mothers of women with PCOS and 62 control women. In the results, mothers had higher LDL-C levels (3.58 ± 0.97 mmol/L versus 3.11 ± 0.66 mmol/L, respectively, P = 0.007), whereas HDL-C and TG levels did not differ compared with control women. The only predictors of LDL-C level in the mothers were their daughters' LDL-C (r2=0.11, P < 0.001) and their own unbound testosterone levels (r2=0.04, P = 0.03) [23]. Sam et al. [24] also have found that LDL-C levels are increased in sisters of women with PCOS compared to control women.

fer compared with control women. The only predictors of LDL-C level in the mothers were their daughters' LDL-C (r2=0.11, P < 0.001) and their own unbound testosterone levels (r2=0.04, P = 0.03) [23]. Sam et al. [24] also have found that LDL-C levels are increased in sisters of women with PCOS compared to control women. As stated above, LDL-C is increased in women with PCOS; however, recently, alterations in LDL quality also have been reported in women with PCOS [14,25-31]. LDL comprises different subclasses according to size, density and atherogenicity. Small dense LDL particles are more atherogenic than large buoyant ones, and are strongly associated with coronary artery disease [32]. Women with PCOS have an increased proportion of atherogenic small dense LDL or decreased mean LDL particle size [14,25-31], and such alterations may be associated with increased cardiovascular risk. However, all of these studies have been based primarily on obese or over-weight PCOS women, but prevalence of obesity in Korean women with PCOS is low [8]. Thus, we investigated whether altered LDL particle profiles are also observed in non-obese women with PCOS. Complete lipid and lipoprotein profiles were obtained in 64 non-obese PCOS patients and 64 age- and BMI-matched controls. The results showed no differences in the absolute level of LDL-C, mean LDL diameter or percentage of atherogenic small dense LDL between PCOS patients and controls. Our findings suggest that non-obese Korean women with PCOS have no significant quantitative or qualitative changes in LDL-C profile [33].

matched controls. The results showed no differences in the absolute level of LDL-C, mean LDL diameter or percentage of atherogenic small dense LDL between PCOS patients and controls. Our findings suggest that non-obese Korean women with PCOS have no significant quantitative or qualitative changes in LDL-C profile [33]. ApoA-I, ApoB, ApoC-1, and lipoprotein (a) and lipoprotein (a) Lipids are transported through the circulation via lipoproteins. Apolipoproteins are located on the surface of lipoproteins, and regulate lipoprotein metabolism and lipid transport. Among the 13 known apolipoproteins, ApoA-I is carried in HDL and has cardioprotective properties, and ApoB is a potential atherogenic risk factor [32]. Some studies have investigated ApoA-I and ApoB measurement in women with PCOS. Valkenburg et al. [21] enrolled 557 women with PCOS and 295 controls, and found that across the entire range of BMI values, ApoA-I levels were significantly lower in women with PCOS. However, there were no differences in ApoB levels between PCOS patients and controls. They also analyzed whether hyperandrogenism and obesity were independent predictors for the presence of a more atherogenic lipid profile in women with PCOS and found that free androgen index and BMI were independent predictors for serum ApoA-I levels in women with PCOS. They concluded that ApoA-I levels were significantly lower in women with PCOS without any difference in ApoB levels, and both obesity and hyperandrogenism contribute to these changes [21].

women with PCOS and found that free androgen index and BMI were independent predictors for serum ApoA-I levels in women with PCOS. They concluded that ApoA-I levels were significantly lower in women with PCOS without any difference in ApoB levels, and both obesity and hyperandrogenism contribute to these changes [21]. Among human apolipoproteins, ApoC-I inhibits the uptake of TG-rich lipoproteins via hepatic receptors, and has been reported to increase postprandial serum lipid level as is common in coronary artery disease patients [34,35]. In women with PCOS, Huang et al. [36] evaluated the role of ApoC-I level and assessed relationships between ApoC-I and clinical features of PCOS. The serum levels of ApoC-I in women with PCOS were significantly higher compared with those of controls (1.23 ± 0.29 mg/mL versus 1.01 ± 0.24 mg/mL, respectively, P < 0.05). In PCOS patients without any abnormal serum lipid index, ApoC-I levels were still higher than in controls (1.39 ± 0.37 mg/mL versus 1.03 ± 0.22 mg/mL, respectively, P < 0.05), and even lean PCOS women had higher ApoC-I levels than controls. Thus, the authors concluded that ApoC-I may be the earliest variation of lipid metabolic abnormality in women with PCOS [36].

pid index, ApoC-I levels were still higher than in controls (1.39 ± 0.37 mg/mL versus 1.03 ± 0.22 mg/mL, respectively, P < 0.05), and even lean PCOS women had higher ApoC-I levels than controls. Thus, the authors concluded that ApoC-I may be the earliest variation of lipid metabolic abnormality in women with PCOS [36]. Among the human lipoproteins, lipoprotein (a) has been identified as an independent risk factor for coronary heart disease in large-scale meta-analyses [37-39]. Lipoprotein (a) concentration in women with PCOS has been reported in some studies. Recently, we compared serum lipoprotein (a) concentration in 64 lean women with PCOS and 64 age- and BMI-matched controls. In this study, non-obese women with PCOS presented a significantly higher level of lipoprotein (a) than matched controls (15.3 mg/dL [95% CI, 12.8 to 17.8] versus 9.1 mg/dL [95% CI, 7.2 to 11.0], P = 0.002), and one-third (29.7%) of the PCOS patients had elevated (≥30 mg/dL) lipoprotein (a) levels [33]. Berneis et al. [40] also reported that lipoprotein (a) abnormalities may be found in one-third of Mediterranean women with PCOS who have a normal lipid pattern, which was similar with our previous results. Rizzo et al. [29] investigated the prevalence of dyslipidemia in different PCOS phenotypes and also reported that levels of lipoprotein (a) were significantly increased in anovulatory women with PCOS than ovulatory women. Thus, they concluded that measurement of lipoprotein (a) in women with different PCOS phenotypes may potentially help to assess cardiovascular risk.

yslipidemia in different PCOS phenotypes and also reported that levels of lipoprotein (a) were significantly increased in anovulatory women with PCOS than ovulatory women. Thus, they concluded that measurement of lipoprotein (a) in women with different PCOS phenotypes may potentially help to assess cardiovascular risk. Prevalence of dyslipidemia in Korean women with PCOS Chae et al. [41] reported the clinical and biochemical characteristics of PCOS in Korean women. In 166 women with PCOS and 277 controls, prevalence of elevated TG (≥150 mg/dL) was 26.7%, whereas that of controls was 1.0% (P < 0.001); prevalence of low HDL-C (<50 mg/dL) was 30.0%, whereas that of controls was 3.0% (P = 0.004) [41]. Recently, we investigated complete phenotypic and metabolic profiles in a large cohort of untreated Korean women with PCOS. From May 2010 to December 2012, consecutive women with PCOS were recruited from 13 medical centers; three were infertility clinics, and the remaining ten were tertiary university hospitals. The mean age of the patients was 24.9 (±6.0) years, the mean BMI was 22.4 (±4.1) kg/m2, and the prevalence of dyslipidemia was 35.7% in 865 consecutive patients (unpublished). These findings also suggest that even young and non-obese Korean women with PCOS also have increased prevalence of dyslipidemia.

sity hospitals. The mean age of the patients was 24.9 (±6.0) years, the mean BMI was 22.4 (±4.1) kg/m2, and the prevalence of dyslipidemia was 35.7% in 865 consecutive patients (unpublished). These findings also suggest that even young and non-obese Korean women with PCOS also have increased prevalence of dyslipidemia. Implication for treatment The Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society has recommended that women with PCOS receive a complete lipid test (total cholesterol, LDL-C, non-HDL-C, HDL-C, and TG). The primary target goal is LDL-C, with level of non-HDL-C as the secondary target. Lipid target values are categorized according to cardiovascular disease risk factors. In women with PCOS without additional cardiovascular disease risk factors, LDL-C levels should be less than 130 mg/dL and target serum non-HDL-C levels should be 30 mg/dL higher than the designated LDL-C goal. Serum TG levels should be less than 150 mg/dL. Lifestyle modification, including diet and exercise, is the first line therapy for all women with PCOS and is particularly important for those with dyslipidemia (individuals with serum LDL-C levels greater than 160 mg/dL and/or non-HDL-C levels of at least 190 mg/dL according to the AE-PCOS Society). Statin drugs are used when lifestyle modifications are not enough, and have emerged as a novel therapeutic approach to PCOS. In addition to improvement of lipid profiles, PCOS women receiving statins showed a significant decrease of testosterone, free androgen index, C-reactive protein, and insulin resistance [42-44]. However, the use of statins in pregnancy is contraindicated, and contraception is required.

tion are benign; however, the different diagnosis of breast cancer is challenging during these periods. Therefore, the aim of this article was to review the changes occurring in the breast, which are related to pregnancy and lactation, and to identify methods for the different diagnosis and treatment of breast disease. Breast changes during pregnancy and lactation Breast begins to change under the influence of estrogen, progesterone, and prolactin from the mid-term in the first trimester of pregnancy. Particularly, by the influence of estrogen the blood vessels show remarkable growth, and lobules are proliferated. For the meantime, fibrolipoma substrate decreases, blood flow increases, and infiltration of mononuclear cell are often accompanied. During the second and the third period of pregnancy, the proliferation of lobules and the decrease in the substrate become more apparent. Normally, due to progesterone, cell proliferation in the unit of lobule appears apparently, and due to estrogen, the ductal proliferation comes to be much intense. Under the influence of the hormones prolactin and oxytoxin (secreted by the posterior pituitary gland), during late pregnancy, the alveolar cells produce early breast milk called colostrum by taking up nutrients from the blood. Foremilk and hindmilk, whose compositions are different from that of colostrum, are subsequently produced with the help of the hormone progesterone (lactogenesis I). In a period of lactation, due to drastic reduction of progesterone, prolactin levels increase. Shape of myoepithelial cell becomes much thinner and flatter, and along with insulin, thyroid hormone and other metabolic hormones, the myoepithelial cell synthesizes the basic nutrients of breast milk-fat, lactose, and protein (lactogenesis II). During a period of lactation, breast displays the lobular expansion as well as the accumulation of ductal secretion (Fig. 1). The breast milk is secreted by oxytocin and neuroendocrine interactions. To producing of breastfeeding constantly being generated during a period of lactation, a certain amount of oxytocin from the posterior pituitary needs to secrete by stimulating that comes from sucking (lactogenesis III) [2-6].

secretion (Fig. 1). The breast milk is secreted by oxytocin and neuroendocrine interactions. To producing of breastfeeding constantly being generated during a period of lactation, a certain amount of oxytocin from the posterior pituitary needs to secrete by stimulating that comes from sucking (lactogenesis III) [2-6]. It takes three months after discontinuation of breast-feeding in order to pre-pregnancy state, and during this process pronounced atrophy of lobules is detected [3]. Imaging and biopsy during pregnancy and lactation In this period, it is very difficult to distinguish between tumor and normal breast by clinical examination or imaging test. Since the breasts continue to grow and feel firm and nodular during pregnancy, it is possible that a tumor mass may appear as normal tissue during this period. Furthermore, as the breast increases its size, the tumor mass may be located deeper, making it more difficult to identify via palpation.

nation or imaging test. Since the breasts continue to grow and feel firm and nodular during pregnancy, it is possible that a tumor mass may appear as normal tissue during this period. Furthermore, as the breast increases its size, the tumor mass may be located deeper, making it more difficult to identify via palpation. 1. Mammography The physiological changes as stated above proliferates the breast parenchyma so that it increases the size, and increases the density of cells, blood vessels, or amount of moisture, therefore, the mammographic parenchymal density increase and diffuse (Fig. 2). Especially, during the lactation period, breast parenchymal density is further increased so that expansion of the lactic duct seems to be nodular and hyperdensity appearance by breast milk filling nipples. Therefore, in pregnant and lactating women, it is difficult to diagnose a tumor by mammography because of the architectural distortion and asymmetric density of the tumor. In such cases, ultrasonography is a more suitable diagnostic procedure than mammography. If a biopsy is indicated by ultrasonography, further mammography must be performed to identify the range of lesions, including microcalcification lesions [7-12]. It is thought that fetal abnormalities, which may occur due to radiation during pregnancy, would appear in case of more than 0.05 Gy. Yet, the radiation that fetus can receive through the mammography introduced in 4 times, is very small as 0.004 Gy, even if one does not wear lead aprons [13-15]. However, except special cases, it is recommended not conduct radiation tests during the first quarter of pregnancy.

ould appear in case of more than 0.05 Gy. Yet, the radiation that fetus can receive through the mammography introduced in 4 times, is very small as 0.004 Gy, even if one does not wear lead aprons [13-15]. However, except special cases, it is recommended not conduct radiation tests during the first quarter of pregnancy. 2. Breast ultrasonography One of the characteristics of breast ultrasonography conducted during pregnancy is that nonfatty fibroglandular tissue with diffuse feature becomes larger, as showing hypoechogenicity (Fig. 3). The other is that during a period of lactation, the nonfatty fibroglandular tissue displays hyperechogenicity, and both vascular tissue and vascular characteristic increase (Fig. 4) [7-11]. Ultrasonic waves can be used as the most dependable medical imaging examination to diagnose pregnancy and breast diseases that may appear during lactation period. In case of malignant breast lumps, ultrasonic represents its higher sensitivity against the lumps, rather than mammography [8,9]. It is known that pregnancy-associated breast cancer (PABC) occupies approximately 3% of all breast cancers. About 90% of PABC show lumps that are possible to be easily diagnosed by ultrasonic.

case of malignant breast lumps, ultrasonic represents its higher sensitivity against the lumps, rather than mammography [8,9]. It is known that pregnancy-associated breast cancer (PABC) occupies approximately 3% of all breast cancers. About 90% of PABC show lumps that are possible to be easily diagnosed by ultrasonic. 3. Magnetic resonance imaging It is not recommended to use magnetic resonance imaging (MRI) as a general inspection for the diagnosis and treatment of breast disease during pregnancy. According to American College of Radiology, in terms of diagnosis of breast diseases during pregnancy, MRI should only be used in cases that it would be regarded to have obvious benefits. It is also said that except special cases, contrast media should not be used [16]. However, in recent studies, it was reported that contrast media would not be closely related to foetal abnormalities [17,18]. European Committee on Radiation Risk reported that gadolinium based contrast could be safely utilized during pregnancy, because it would be less absorbed into placenta and also, it would be rapidly excreted to kidney [19]. For MRI using gadolinium based contrast during a period of lactation makes less excretion to breast milk, it is known to be safe relatively [18,19].

European Committee on Radiation Risk reported that gadolinium based contrast could be safely utilized during pregnancy, because it would be less absorbed into placenta and also, it would be rapidly excreted to kidney [19]. For MRI using gadolinium based contrast during a period of lactation makes less excretion to breast milk, it is known to be safe relatively [18,19]. 4. Cytologic examination Since the mammary epithelial cells undergo several cellular morphological changes during pregnancy and lactation, a false-positive result may be obtained during the diagnosis of breast cancer. Therefore, the results of these diagnostic procedures must be carefully interpreted. A core biopsy examination of the suspected malignant lesion must be conducted and interpreted by a skilled pathologist [20,21]. 5. Core biopsy Core biopsy, most appropriate method of tissue diagnosis in pregnancy and lactation period, is safe and economical (cost-effective). The process of breast milk production is characterized by increased blood flow to the breast, which increases the risk of bleeding, and expansion of the milk ducts increases the risk of infection, which can cause a milk fistula because breast milk is generated in this stage. Milk fistula is known to occur more in central than in peripheral. Thus feeding temporarily suspend before aseptic surgery and a biopsy, biopsy is in the correct location and after the procedure, hemostasis using compresses or ice packs will help [22].

ause a milk fistula because breast milk is generated in this stage. Milk fistula is known to occur more in central than in peripheral. Thus feeding temporarily suspend before aseptic surgery and a biopsy, biopsy is in the correct location and after the procedure, hemostasis using compresses or ice packs will help [22]. Breast disease related to pregnancy, lactation period 1. Benign breast disease associated with physiological change 1) Gestational and secretory hyperplasia Microcalcification can be the result due to gestational hyperplasia associated with pregnancy and secretory hyperplasia associated with breast-feeding in mammography. This calcification looks mostly round and focal or diffuse distribution, but rarely shows a irregular, linear distribution, branching pattern. Round and small punctate calcifications represent lobular acinar proliferation and linear distribution represents the growth of the cannulated (Fig. 5) [23-25].

in mammography. This calcification looks mostly round and focal or diffuse distribution, but rarely shows a irregular, linear distribution, branching pattern. Round and small punctate calcifications represent lobular acinar proliferation and linear distribution represents the growth of the cannulated (Fig. 5) [23-25]. 2) Spontaneous bloody nipple discharge Bloody nipple discharge does not occur commonly in pregnancy or in a period of lactation. However, it can be cause by even small stimuli because the blood flow in breast increases rapidly and the change of epithelial cells gets even worst in 3rd trimester of pregnancy. Slight bloody nipple discharge can be seen from approximately 20% of nipple discharge during pregnancy and 15% of a period of lactation [26]. This phenomenon mostly stops as the lactate feeding begins, but also has a possibility of a severe case that may persist throughout the whole lactation period. Diagnostic cytology tests must be performed for the wounds of nipples by feeding. If the result, physical examination and ultrasound are normal, the follow up test should be recommended in a timely manner. If it seems to be abnormal in cytology, galactography should be performed especially when the bloody discharge is observed from one duct. However, it is important to help the patient to be relieved because this kind of bloody discharge is a rare symptom of PABC (Figs. 6, 7) [26-28].

2) Spontaneous bloody nipple discharge Bloody nipple discharge does not occur commonly in pregnancy or in a period of lactation. However, it can be cause by even small stimuli because the blood flow in breast increases rapidly and the change of epithelial cells gets even worst in 3rd trimester of pregnancy. Slight bloody nipple discharge can be seen from approximately 20% of nipple discharge during pregnancy and 15% of a period of lactation [26]. This phenomenon mostly stops as the lactate feeding begins, but also has a possibility of a severe case that may persist throughout the whole lactation period. Diagnostic cytology tests must be performed for the wounds of nipples by feeding. If the result, physical examination and ultrasound are normal, the follow up test should be recommended in a timely manner. If it seems to be abnormal in cytology, galactography should be performed especially when the bloody discharge is observed from one duct. However, it is important to help the patient to be relieved because this kind of bloody discharge is a rare symptom of PABC (Figs. 6, 7) [26-28]. 3) Galactocele Galactocele is a benign lesion, which is mostly detected after a few weeks or months from the time the patient stopped breast feeding, or during lactation or during the 3rd trimester of pregnancy [11,29]. This means it is surrounded by the epithelial cells and myoepithelial cells and contains liquid that is similar to lactate in cystic changed terminal duct and ductules. Most of the galactoceles are found as painless palpable mass which contains protein, fat, lactose in aspiration analysis and are often accompanied by inflammation and necrosis. Therefore, it is considered that the galactocele is formed by the wall fibrosis due to the inflammatory response of extension of lactate ducts. When the contents leak within the breast, chronic inflammation and fat necrosis are observed [30]. Needle aspiration biopsy is helpful for diagnosis and treatment, however, milk-like liquid during lactation and sticky milky contents after lactation, are found in aspiration [29,31]. If it recurrs even after several aspirations, imaging test is needed. Depending on the proportion of fat and protein in breast milk, mammographic findings of galactoceles may vary, which can be seen as follows.

milk-like liquid during lactation and sticky milky contents after lactation, are found in aspiration [29,31]. If it recurrs even after several aspirations, imaging test is needed. Depending on the proportion of fat and protein in breast milk, mammographic findings of galactoceles may vary, which can be seen as follows. (1) Pseudolipoma: When a higher fat content, seems to be completely radiolucent mass. (2) Cystic mass with fat-fluid level: Low concentration of fat content rises up and the liquid sinks to the bottom. It is a diagnostic finding that is seen in the mediolateral oblique view of Mammography. (3) Pseudohamartoma: Galactoceles does not separate lipid from liquid and it is seen radiologic findings which is simliar to hamartoma that contains high viscosity of breast milk [31].

(2) Cystic mass with fat-fluid level: Low concentration of fat content rises up and the liquid sinks to the bottom. It is a diagnostic finding that is seen in the mediolateral oblique view of Mammography. (3) Pseudohamartoma: Galactoceles does not separate lipid from liquid and it is seen radiologic findings which is simliar to hamartoma that contains high viscosity of breast milk [31]. Sonographic findings of galactoceles may also vary depending on the time and site of lesion. In the acute phase, it may appear as an anechoic unilocular simple cyst or a multilocular cyst with thin septation. It is common multilocular cyst that occurs in peripheral region and it is common unilocular or bilocular cyst that occurs in central region. The intensity of hypoechoic echo increases gradually due to the interface between the fat and water components. In this case, Doppler examination performed by the blood vessels in the center to make sure that no particles moving when you press the transducer lesions may be helpful in the diagnosis. In ultrasound, galactoceles only breast milk composition consists of benign solid masses as shown boundaries if and posterior acoustic enhancement seems clear. A long time later, several ingredients are mixed and if you look inside looks mixed echogenic heterogeneous mass (Figs. 8-10) [11,32-34]. Galactoceles is rich in nutrients, and sometimes it can be infected. This finding is well observed in the ultrasound. Aspiration here, you can see that the purulent fluid and breast milk ingredients are mixed in (Fig. 11). Most natural decay takes over several weeks, but occasionally a more long-lasting.

0) [11,32-34]. Galactoceles is rich in nutrients, and sometimes it can be infected. This finding is well observed in the ultrasound. Aspiration here, you can see that the purulent fluid and breast milk ingredients are mixed in (Fig. 11). Most natural decay takes over several weeks, but occasionally a more long-lasting. 2. Inflammatory and infectious diseases 1) Postpartum mastitis (puerperal mastitis) It's not common to see infections during pregnancy, but it is often observed in lactation. Staphylococcus aureus and Streptococcus, which are common pathogens present in the nose and throat of newborn babies, may infect the breast via the damaged epithelial cells of the nipple-areola complex during breast feeding. Nipple cracks or abrasions of the skin can be often discovered on the patient's past history. If breast milk is stagnant, it would be a good culture medium and cause symptoms [24,29,30]. S. aureus infections, being topical, result in severe clinical symptoms right from the beginning, whereas infections caused by streptococci are diffuse and cause local abscess only in the advanced stage of infection (Figs. 12,13). It is treated well by antibiotics, especially amoxicillin-clavulanate. It is quite rare, but puerperal mastitis by methicillin-resistant S. aureus can be very fatal [35-37]. Mammography should not be performed, unless there are suspicious malignant findings. The reason is that it mostly shows thickening of the skin and fibrous tissue even though it is very severe mastitis. Moreover, it is rare to discover other severe abnormalities. If there is abscess formation suspected, the ultrasound examination is first required for the diagnosis and treatment. Irregular boudaries, hypoechoic or anechoic mass, thick, irregular walls, posterior acoustic enhancement, and liquid debris (fluid-debris) shades can be observed in the abscess. Sometimes the air in abscess can cause bright reflection. These hyperechoic dots floating point is the differentiation of malignant. Mastitis in lactation abscess developed in the subacute form, periarthritis [38,39]. Abscess is easily treated by incision drainage. If the abscess lesion size is less than 3 cm, it can be treated with 14 to 21 gauge needle aspiration. If the abscess lesion size is 3 cm or more, it can be aspirated with the 6 to 8 Fr catheter drainage or surgical drainage. Before doing this, it is important to dry up the abscess first.

treated by incision drainage. If the abscess lesion size is less than 3 cm, it can be treated with 14 to 21 gauge needle aspiration. If the abscess lesion size is 3 cm or more, it can be aspirated with the 6 to 8 Fr catheter drainage or surgical drainage. Before doing this, it is important to dry up the abscess first. It is necessary that examiner should irrigate the cavity with normal saline under local anesthesia after the drainage to prevent recurrence [38-40]. After treatment, continuing breast feeding helps improve inflammation and promote drainage. Also, breast feeding can be safely performed during antibiotics therapy [29,35]. If there is no improvement despite these treatments, examiner should suspect tumor and perform cytology test and biopsy.

revent recurrence [38-40]. After treatment, continuing breast feeding helps improve inflammation and promote drainage. Also, breast feeding can be safely performed during antibiotics therapy [29,35]. If there is no improvement despite these treatments, examiner should suspect tumor and perform cytology test and biopsy. 2) Granulomatous mastitis Granulomatous mastitis occurs mainly in young women, and it is an uncommon type which mainly occurs within five years after pregnancy [41-43]. It exhibits specific histological findings characterized by non-caseating (noncaseating), non-vascular (nonvasculitic) granulomatous inflammatory changes, which can be noted in the center of the lobules. Mammographic findings are diverse from normal to benign with focally asymmetric shadow and malignant. Sonographic findings helpful in diagnosis are multiple appearances, which often show hypoechoic and sometimes large hypoechoic mass in adjacent lactiferous gland (Fig. 14) [43]. However, it requires special attention in diagnosis because it is not well differentiated from malignancy, and reactive lymphadenopathy is observed in about 15%. Local excision can be a treatment [40-46], but sometimes it disappears naturally. Therefore close follow-up is necessary [47].

ctiferous gland (Fig. 14) [43]. However, it requires special attention in diagnosis because it is not well differentiated from malignancy, and reactive lymphadenopathy is observed in about 15%. Local excision can be a treatment [40-46], but sometimes it disappears naturally. Therefore close follow-up is necessary [47]. 3) Juvenile papillomatosis of the breast Benign proliferative lesion is a common type because of the influence of hormones during pregnancy and lactation. Juvenile papillomatosis of the breast rarely occurs in young women, and it is characterized by ductal hyperplasia and papillary proliferation of the wall surrounding its sac [48]. It is seen as a hypoechoic mass with an unclear margin in an ultrasonogram, and no specific findings, except an asymmetric shadow, is seen in a mammogram; however, sometimes microcalcification can be observed. The treatment is a surgical resection. It is important to check if the resection margin is completely negative when it comes to preventing recurrence.

unclear margin in an ultrasonogram, and no specific findings, except an asymmetric shadow, is seen in a mammogram; however, sometimes microcalcification can be observed. The treatment is a surgical resection. It is important to check if the resection margin is completely negative when it comes to preventing recurrence. 3. Benign tumor 1) Tumors related to pregnancy and lactation Lactating adenoma is a benign tumor mainly caused by the physiological changes that occur during pregnancy and lactation. This typically occurs during feeding or the third trimester of pregnancy. Lactating adenoma is sometimes interpreted as a variant of fibroadenoma, tubular adenoma, or lobular hyperplasia, which are also caused by physiological changes [49-51]. Fibroadenoma is mixed with stromal and epithelial components. Lactating adenomas, however, only consists of epithelial component. Grossly, it seems to be a certain boundary and elastic mass. Histologically, it is densely packed with lobules, which are separated from each other by a thin connective tissue. Lactating adenoma is composed of acinars and mature thin tubes and filled with secretion is softer than a fibroadenoma The physiological changes that occurs in the surrounding normal parenchyma takes place in the lesions of the internal secretion and secretory hyperplasia appears [49-51]. Lactating adenoma can naturally disappear at the end of pregnancy or lactation. It may also disappear by necrosis, like a fibroadenoma [52]. Most of Lactating adenoma does not relapse after complete resection. Lactating adenoma accompanying malignant lesions is very rare. And it does not increase the risk of breast cancer. It is difficult to distinguish between a lactating adenoma and a fibroadenoma by imaging. A radiolucent or hyperechoic area, which indicates fat content of breast milk, according to lactation hyperplasia, can be seen on mammography or ultrasonography and is useful in diagnosis (Fig. 15). It appears to be a small hypoechoic mass, which is difficult to distinguish from the surrounding tissue on ultrasonography. Moreover, because of its posterior acoustic enhancement and hormonal changes, it appears hypervascular in color or output Doppler. Occasionally, the differential diagnosis of malignant lesions is difficult mainly due to unclear boundaries, microlobulated periphery, reducing posterior acoustic effect, structural heterogeneity. And it occurred by the necrosis of lactating adenoma [11,53-55].

nges, it appears hypervascular in color or output Doppler. Occasionally, the differential diagnosis of malignant lesions is difficult mainly due to unclear boundaries, microlobulated periphery, reducing posterior acoustic effect, structural heterogeneity. And it occurred by the necrosis of lactating adenoma [11,53-55]. 2) Morphologic and physiologic changes in fibroadenomas secondary to pregnancy and lactation (1) Growing fibroadenoma Fibroadenoma is the most frequent lesion found during pregnancy and the period of lactation. It can be formed before pregnancy, however, there are many instances wherein it is undetected because it is not palpable or breast screening inspection is not performed on young women. Fibroadenoma increases in size during pregnancy because of its sensitivity to hormone levels. Imaging findings of fibroadenoma is not very different during pregnancy, however, findings such as dilation of lactiferous ducts and increase in blood of flow is very similar to complex fibroadenoma (Fig. 16) [29,56,57]. When performing fine needle aspiration for diagnosing palpable fibroadenoma, you must consider the physiological changes of cells during pregnancy [58]. Central biopsy is also appropriate and must be carefully done to avoid hemorrhage, lacto fistula and infection.

y similar to complex fibroadenoma (Fig. 16) [29,56,57]. When performing fine needle aspiration for diagnosing palpable fibroadenoma, you must consider the physiological changes of cells during pregnancy [58]. Central biopsy is also appropriate and must be carefully done to avoid hemorrhage, lacto fistula and infection. (2) Fibroadenoma with infarction Spontaneous necrosis of fibroadenoma is very rare, but it is sometimes observed during pregnancy or lactation. It can be presumed when there is sudden pain during the third trimester or during birth. The cause is embolism within the vessels and it shows more borderlines of lobules and mixes echos and shows posterior shadow depending on the degree of necrosis. If necrosis is more extensive, central biopsy must be performed to differentiate from a malignant lesion [30,59-61]. (3) Fibroadenoma with secretory hyperplasia or lactational change During pregnancy, hormone-sensitive breast parenchyma of fibroadenoma is stimulated by pregnancy hormones leading to secretory hyperplasia. This is very similar to the phenomena seen in lactating adenoma, so it is very difficult to distinguish between both. When fibroadenoma undergoes changes during lactiferous phase, there can be lactation during sucking and it is noted that this phenomena is not seclusive to breast cysts. Similar to complex fibroadenoma, it is diagnosed based on the findings of secretory hyperplasia, which appears as mixed echo, and dilated lactiferous ducts and cysts. Furthermore, calcification may sometimes be detected during breast imaging.

sucking and it is noted that this phenomena is not seclusive to breast cysts. Similar to complex fibroadenoma, it is diagnosed based on the findings of secretory hyperplasia, which appears as mixed echo, and dilated lactiferous ducts and cysts. Furthermore, calcification may sometimes be detected during breast imaging. 4. Malignant tumor (PABC) 1) Introduction PABC is defined as breast cancer that occurred during pregnancy and after delivery within one year, represents up to approximately 3% of all breast cancers, occurring in one incidence per 3,000 to 10,000 pregnancy. Delay of childbirth age shows increased prevalence and advanced stage at the time of diagnosis and poor prognosis and seems that more than 50% are high-grade tumors [29,62,63]. And large number of them are form of inflammatory tumor and shows in more than 50% lymph node metastasis, negative at hormone receptor and positive at HER2/neu [62,63]. Late diagnosis of tumors owing to the biological effects of pregnancy results in poor prognosis and rapid tumor growth; recurrence is common within 2 to 3 years of the diagnosis [29,62,63]. Mostly palpable lesions and clinical symptoms are visible swelling, redness, and diffuse mastosis at locally advanced carcinoma [29,62,63]. The imaging findings of breast cancer in non-pregnant women are not significantly different from those in pregnant women due to the increase in glandular density. Even in non-pregnant women, mammography has low sensitivity, and ultrasound is considered a better diagnostic tool [7-12].

lly advanced carcinoma [29,62,63]. The imaging findings of breast cancer in non-pregnant women are not significantly different from those in pregnant women due to the increase in glandular density. Even in non-pregnant women, mammography has low sensitivity, and ultrasound is considered a better diagnostic tool [7-12]. 2) Cause of delayed diagnosis The mammary gland undergoes clinical and physiological changes during pregnancy, making imaging diagnosis difficult. The majority of patients with advanced stage looks so delayed diagnosis is as follows [64,65]. Young women who are not a screening test. Self-examination is difficult and is not performed frequently. The clinical examination is difficult because of increased blood flow and dense mammary tissues. Until delivery they are not good at follow up test. Breast care at the department of obstetrics and gynecology does not enforce. Reluctant to biopsy during pregnancy. During pregnancy is known to be delayed an average of 5 to 7 months of breast cancer diagnosis, whenever be delayed one month diagnosis shows increased by approximately 0.9% to 1.8% of axillary lymph node metastasis [66]. 3) Symptoms In most cases, it appears as a breast mass with no associated pain, and sometimes, it can be found on breast examination in the absence of breast feeding [66]. During the third trimester of pregnancy, hemoid secretion is often seen due to loss of epithelial cells alike papilloma in lactiferous drifts and this is a common symptom. Cytological examination will be carried out when hemoid secretion is continued more than 2 months after delivering.

3) Symptoms In most cases, it appears as a breast mass with no associated pain, and sometimes, it can be found on breast examination in the absence of breast feeding [66]. During the third trimester of pregnancy, hemoid secretion is often seen due to loss of epithelial cells alike papilloma in lactiferous drifts and this is a common symptom. Cytological examination will be carried out when hemoid secretion is continued more than 2 months after delivering. 4) Diagnosis Although mammography is not effective in pregnancy because physiological changes decreased sensitivity and specificity, it is performed suspected malignancy. 68% to 74% of PABC has abnormal findings with mammography and 90% of PABC has Breast Imaging Reporting and Data System (BIRADS) 3 to 4 score. During pregnancy, ultrasound seems the most effective imaging studies in the diagnosis of breast disease. And it's sensitivity to the breast disease is 93% to 98% (Figs. 17, 18). Complex echo pattern, posterior acoustic enhancement is a common finding in the PABC compared with non-pregnancy breast cancer. Necrosis or cystic change is seen in the carcinoma present non-echo pattern and posterior acoustic enhancement because tumor grows up quickly by stimulation of the hormonal changes in pregnancy.

mplex echo pattern, posterior acoustic enhancement is a common finding in the PABC compared with non-pregnancy breast cancer. Necrosis or cystic change is seen in the carcinoma present non-echo pattern and posterior acoustic enhancement because tumor grows up quickly by stimulation of the hormonal changes in pregnancy. 5) PABC in carrier with BRCA mutation Carriers with breast cancer (BRCA)1 or BRCA2 mutation are at high risk for breast cancer. Therefore carriers with BRCA1 or BRCA2 mutation certainly needs to undergo strict clinical and image examination before and after pregnancy. This strict examination uses ultrasonography and MRI, but ultrasonography is more useful than MRI because the latter is difficult to find during the period of lactation (Fig. 19). According to former studies, the PABC prevalence of carrier with BRCA1 mutation is higher than BRCA2 mutation [67]. Carriers of mutations in the BRCA1 or BRCA2 genes who have parity have an increased risk of PABC than those who do not have parity before the age of 40 years, and pregnancy at an early age does not decrease the prevalence [68]. But it was recently reported by International BRCA1/2 Carrier Cohort Study (IBCCS) that carriers with BRCA1, BRCA2 malignant and control group has decreased the risk of being malignant after the age of 40 years [69].

parity before the age of 40 years, and pregnancy at an early age does not decrease the prevalence [68]. But it was recently reported by International BRCA1/2 Carrier Cohort Study (IBCCS) that carriers with BRCA1, BRCA2 malignant and control group has decreased the risk of being malignant after the age of 40 years [69]. 6) Treatment (1) Pregnancy interruption In the past, due to the adverse affects of tumor growth and proliferation due to increasing hormone levels during pregnancy, therapeutic abortion was encouraged. But according to several studies, the therapeutic abortion does not improve the prognosis. The reason is that the mammary gland of most of PABC is hormone receptor negative and imcomplete growth and division occur in the point at which the pregnancy ends. For this reason therapeutic abortion brings about growth of tumor. So termination of pregnancy should be considered in case of a very aggressive tumor or receptor positive. (2) Staging and histologic types According to stage by Tumor-Node-Metastasis (TNM) system of American Joint Committee on Cancer, the most common histologic type is infiltrating ductal adenocarcinoma for 80%.

6) Treatment (1) Pregnancy interruption In the past, due to the adverse affects of tumor growth and proliferation due to increasing hormone levels during pregnancy, therapeutic abortion was encouraged. But according to several studies, the therapeutic abortion does not improve the prognosis. The reason is that the mammary gland of most of PABC is hormone receptor negative and imcomplete growth and division occur in the point at which the pregnancy ends. For this reason therapeutic abortion brings about growth of tumor. So termination of pregnancy should be considered in case of a very aggressive tumor or receptor positive. (2) Staging and histologic types According to stage by Tumor-Node-Metastasis (TNM) system of American Joint Committee on Cancer, the most common histologic type is infiltrating ductal adenocarcinoma for 80%. (3) Surgical treatment During pregnancy axillary disection can be performed safely with modified radical mastectomy and lympectomy. Breast parenchyma can be preserved in the following 2 ways during pregnancy. First, conserving surgery can be performed in the second trimester of pregnancy, and radiation therapy, after delivery. Second, conservation therapy and neoadjuvant chemotherapy can be performed in the second trimester, and radiation therapy, after delivery.

ma can be preserved in the following 2 ways during pregnancy. First, conserving surgery can be performed in the second trimester of pregnancy, and radiation therapy, after delivery. Second, conservation therapy and neoadjuvant chemotherapy can be performed in the second trimester, and radiation therapy, after delivery. (4) Chemotheraphy The majority of anticancer drugs that are administered to a PABC patient can cause teratogenesis and mutation that are related to intrauterine growth retardation (IUGR), deformity, still birth. Teratogenesis increases 20% especially in the first trimester of pregnancy. Furthermore, due to the physiological changes during pregnancy, i.e., increased renal blood flow, glomerular transmittance, and creatinine, the pharmacokinetics of a cytotoxic drug are altered. Moreover, the drug may be accumulated in the amniotic fluid, which may interrupt its excretion. In general, the cytotoxic drug crosses the placenta, but do not know how to pass suspension. However, because teratogenic in first trimester, 5 to 10 weeks attention and 13 weeks after the safety. Methotrexate is prohibited because of the high risk of contraindication for teratogenesis and miscarriage. Drugs belonging to Food and Drug Administration category D, such as cyclophosphamide, fluorouracil, and cisplatinum, affect teratogenesis in the first trimester, but rarely have an adverse effect during the second and third trimesters. In general, taxanes and trastuzumab are considered safe during pregnancy; however, more studies are needed to confirm their safety.

category D, such as cyclophosphamide, fluorouracil, and cisplatinum, affect teratogenesis in the first trimester, but rarely have an adverse effect during the second and third trimesters. In general, taxanes and trastuzumab are considered safe during pregnancy; however, more studies are needed to confirm their safety. (5) Hormone therapy Most PABC are hormone receptor negative, and in these patients, it is necessary to administer tamoxifen. However, this medicine is prohibited during pregnancy, because it is related to colporrhagia, natural abortion, stillbirth, and malformation. If necessary, it would be recommended to use after the delivery [70]. (6) Radiotherapy Even a little radiation, which can be hardly detected, could be fatal to the infant. Pregnant women are most sensitive to radiation during the first 10 to 38 days of pregnancy. During this period, radiation can cause fetal abnormalities such as microencephaly, anencephaly, ocular damage, and clubfoot. According to the pregnancy period, Mother's body surface is increasing. Therefore, the quantity of the radiation which the infant absorbs depends on the physiological changes from Mother. For example, if radiation of 5,000 cGy is measured in Mother's breast, the infant is exposed to the radiation of 10 to 15 cGy in the first-quarter and to the radiation of more than a hundred in the third-quarter. Consequently, it is recommended that radiotherapy should folllow after giving birth (Table 1).

from Mother. For example, if radiation of 5,000 cGy is measured in Mother's breast, the infant is exposed to the radiation of 10 to 15 cGy in the first-quarter and to the radiation of more than a hundred in the third-quarter. Consequently, it is recommended that radiotherapy should folllow after giving birth (Table 1). Conclusion Breast lesions that are detected during pregnancy or nursing are not very different from those detected in non-pregnant women. But, it is difficult to diagnose these lesions in pregnant women due to the hormone-induced physiological changes occurring in the breast. However given enough understanding and knowledge about special clinical and imaging features in pregnancy and having aggressive approach or action toward these symptoms accordingly it will be helpful to treat and diagnose. Acknowledgments This research was supported by research funds from Chosun University, 2008. Fig. 1 Changes of breast tissue during lactation. (A) Terminal duct-lobular unit in non-pregnancy (H&E, ×200). (B) Dilated lobular acini with vacuoles and secretions can be seen during lactation (H&E, ×200). Fig. 2 Mammographic changes during lactation. (A) Type 2 American College of Radiology classification shows before pregnancy. (B) Mammogram during lactation shows a marked diffuse increase in density. Fig. 3 Typical ultrasonographic feature during pregnancy shows diffuse enlargement of the non-fatty glandular component and global hypoechogenicity.

Fig. 2 Mammographic changes during lactation. (A) Type 2 American College of Radiology classification shows before pregnancy. (B) Mammogram during lactation shows a marked diffuse increase in density. Fig. 3 Typical ultrasonographic feature during pregnancy shows diffuse enlargement of the non-fatty glandular component and global hypoechogenicity. Fig. 4 Ultrasonographic changes during lactation. (A) Ultrasound (US) image shows irregular margined, hypoechoic dilated duct (black arrowhead). (B) US image reveals diffuse enlargement of the glandular component with diffuse hyperechogenicity. (C) Color Doppler US image reveals increased vascularity. Fig. 5 Microcalcifications during lactation. (A) Image shows new cluster of indeterminate asymmetric microcalcification. (B) Craniocaudal spot-compression magnification mammograms: several clusters of heterogeneous and granular calcifications. Some clusters display linear distribution. (C) Photomicrograph of histopathologic specimen: a Coarse microcalcification group is seen in the dilated duct. The relatively bigger microcalcification (thick arrow) shows in the single duct with homogeneous and eosiophillic feature. The smaller microcalcification (thin arrow) is seen in the lobule (H&E, ×50).

bution. (C) Photomicrograph of histopathologic specimen: a Coarse microcalcification group is seen in the dilated duct. The relatively bigger microcalcification (thick arrow) shows in the single duct with homogeneous and eosiophillic feature. The smaller microcalcification (thin arrow) is seen in the lobule (H&E, ×50). Fig. 6 A woman who presented with palpable mass and bloody nipple discharge at 24 weeks of pregnancy. (A) Magnification view mammogram of left breast in craniocaudal projection: extensive pleomorphic microcalcifications (arrows). (B) Corresponding longitudinal ultrasound image: irregular solid hypoechoic mass (long arrows) with internal calcifications (short arrows) corresponding to mammographic finding. (C) A mammogram of 30 weeks of pregnant woman presented with bloody nipple discharge: Multilobulated filling defect (long arrows), which focally expands duct. Proximal duct is dilated (short arrows). Fig. 7 (A) The galactogram in a lactating woman who presented with bloody nipple discharge: lobular filling defect is seen which is expected to be intraductal papilloma. (B) The galactograms in patients with papillomatosis showing intra-ductal growth in a separate duct. Fig. 8 Pseudolipoma type galactocele. (A) Mammography shows a 1.5 cm oval circumscribed mass (arrow) at the subareolar region. (B) Sonography shows a 1.5 cm oval circumscribed hypoechoic nodule with posterior shadowing. An echogenic rim (arrow) can be seen at the anterior margin.

Fig. 7 (A) The galactogram in a lactating woman who presented with bloody nipple discharge: lobular filling defect is seen which is expected to be intraductal papilloma. (B) The galactograms in patients with papillomatosis showing intra-ductal growth in a separate duct. Fig. 8 Pseudolipoma type galactocele. (A) Mammography shows a 1.5 cm oval circumscribed mass (arrow) at the subareolar region. (B) Sonography shows a 1.5 cm oval circumscribed hypoechoic nodule with posterior shadowing. An echogenic rim (arrow) can be seen at the anterior margin. Fig. 9 Galactocele with fat-fluid level. (A) An oval circumscribed cystic mass (arrows) with fat-fluid level is imaged in mammogram. (B) Ultrasound image shows the fat-fluid level with high echogenicity of lipid component and low echogenicity of fluid in the same patient. Fig. 10 Galactocele 6 months post delivery which shows various features. (A) Ultrasound (US) image reveals 2 cystic mass: typical galactocele with homogenous anechoic, acoustic attenuation and lateral edge shadowing in bigger cyst. (B) US image shows the lobulated, fat-fluid (arrows) galactocele. Fig. 11 Infected galactocele: lactating woman who presented with reddish skin changes in the breast. (A) Ultrasound image shows a heterogeneous echoic, irregular margined collection which was suspicious of abscess. (B) Fine-needle aspiration and culture was performed.

Fig. 10 Galactocele 6 months post delivery which shows various features. (A) Ultrasound (US) image reveals 2 cystic mass: typical galactocele with homogenous anechoic, acoustic attenuation and lateral edge shadowing in bigger cyst. (B) US image shows the lobulated, fat-fluid (arrows) galactocele. Fig. 11 Infected galactocele: lactating woman who presented with reddish skin changes in the breast. (A) Ultrasound image shows a heterogeneous echoic, irregular margined collection which was suspicious of abscess. (B) Fine-needle aspiration and culture was performed. Fig. 12 Puerperal mastitis with abscess formation. (A) Lactational abscess grossly apparent secondary to flaming redness, hemorrhagic area, swelling, and peeling skin. (B) Ultrasound image shows large mass and purulent material was obtained by fine-needle aspiration. Fig. 13 Ultrasound findings in puerperal mastitis. Early stage mastitis shows various features that is presented with thickness of skin and subcutaneous layer, and irregular border between subcutaneous layer and parenchyme. (A) US shows irregular margin and hypoechoic lesion. (B) If abscess is develop, hypoechoic or anechoic fluid collections can be seen. Irregular margin and echoic lesion can be also seen along with acoustic enhancement. Fig. 14 Granulomatous mastitis after pregnancy. (A) Photomicrograph (enlarged, H&E, ×10) shows epitheloid and giant cell granulomas (arrows) in polymorphous inflammatory infiltrate. (B) Mammogram shows irregular enhanced mass almost filled the right breast.

Fig. 13 Ultrasound findings in puerperal mastitis. Early stage mastitis shows various features that is presented with thickness of skin and subcutaneous layer, and irregular border between subcutaneous layer and parenchyme. (A) US shows irregular margin and hypoechoic lesion. (B) If abscess is develop, hypoechoic or anechoic fluid collections can be seen. Irregular margin and echoic lesion can be also seen along with acoustic enhancement. Fig. 14 Granulomatous mastitis after pregnancy. (A) Photomicrograph (enlarged, H&E, ×10) shows epitheloid and giant cell granulomas (arrows) in polymorphous inflammatory infiltrate. (B) Mammogram shows irregular enhanced mass almost filled the right breast. Fig. 15 Lactating adenoma. (A) Ultrasound image demonstrates oval, well defined, regular margined mass. (B) Mammogram shows an oval circumscribed mass in the left lower breast. (C) The lobules are lined by actively secreting epithelial cells with vacuolated cytoplasm. Secretions may accumulate in the glands. The cells have basophilic cytoplasm, hyperchromatic nuclei with prominent nucleoli, and inconspicuous myoepithelial cell layer (H&E, ×400). Fig. 16 Fibroadenoma. (A) Aside from cystic lesion, fibroadenoma shows internal echogenicity and fibroadenoma can not be distinguished from malignant lesion perfectly. (B) Color Doppler Ultrasound image shows a fibroadenoma with increased vascularity and lobulated hypoechoic mass.

Fig. 15 Lactating adenoma. (A) Ultrasound image demonstrates oval, well defined, regular margined mass. (B) Mammogram shows an oval circumscribed mass in the left lower breast. (C) The lobules are lined by actively secreting epithelial cells with vacuolated cytoplasm. Secretions may accumulate in the glands. The cells have basophilic cytoplasm, hyperchromatic nuclei with prominent nucleoli, and inconspicuous myoepithelial cell layer (H&E, ×400). Fig. 16 Fibroadenoma. (A) Aside from cystic lesion, fibroadenoma shows internal echogenicity and fibroadenoma can not be distinguished from malignant lesion perfectly. (B) Color Doppler Ultrasound image shows a fibroadenoma with increased vascularity and lobulated hypoechoic mass. Fig. 17 Inflammatory carcinoma during pregnancy. (A) Craniocaudal view. (B) Mediolateral-oblique view: mammogram shows a mark diffuse increase in parenchymal density with skin thickening. (C) Subtraction 1 minute after bolus injection-the diffuse enhancement infiltrating the skin and the pectoralis muscle (continuous arrows). (D) T2-weighted image-edema in a cutaneous/subcutaneous, diffuse and prepectoral localization (discontinuous arrows).

diffuse increase in parenchymal density with skin thickening. (C) Subtraction 1 minute after bolus injection-the diffuse enhancement infiltrating the skin and the pectoralis muscle (continuous arrows). (D) T2-weighted image-edema in a cutaneous/subcutaneous, diffuse and prepectoral localization (discontinuous arrows). Fig. 18 Pregnancy-associated breast cancer in a lactating woman who is presented with paeau d'orange skin. (A) Bilateral craniocaudal mammogram: severe dense breast with hypertrophic skin. (B) Mediolateral-oblique view: multiple mass (thick and thin arrows). (C) Ultrasound image in a lactating woman presented with palpable mass 9 months post delivery: it shows taller than wide mixed echogeic lesion revealed with invasive ductal carcinoma. Fig. 19 Atypical medullary carcinoma of the breast with cartilaginous metaplasia. (A) Ultrasound image reveals lobulated hypoechoic lesion in BRCA1 germline muation patient. (B) Higher magnification view of the previous slide shows the highly anaplastic tumor cells in a background of lymphoplasmacytic infiltrate (H&E, ×200). Table 1 Treatment based on pregnancy trimester during which breast cancer is diagnosed

Introduction Pregnancy in advanced age is defined as childbirth at the age of 35 years or order regardless of whether the childbirth is the first or not. Recently, the percentages of pregnancies in advanced age have shown a gradual increase as women have entered the workforce in increasing numbers. In South Korea, the percentages of pregnancies in women of advanced age have more than doubled, increasing from 6.2% in 1999 to 15.4% in 2009 [1]. Fetal chromosomal abnormalities may be caused by a nondisjunction phenomenon that occurs in the period of meiosis during maternal oogenesis, which has been reported to have a direct association with maternal age. Therefore, pregnancy in advanced age is a critical risk factor for fetal chromosomal abnormalities [2-4]. Currently, fetal chromosomal abnormalities due to maternal age have been reported to include trisomy 21, trisomy 18, trisomy 13, triple X syndrome, and Klinefelter's syndrome [5]. As the percentages of pregnancies in women of advanced age have increased, there has been increasing demand for prenatal genetic counseling that helps to predict the risk of fetal chromosomal abnormalities depending on age. Most of the data were obtained from studies of women in North America or Europe. However, rare data from study of fetal chromosomal abnormalities depending on age targeting Korean women have been reported [6].

renatal genetic counseling that helps to predict the risk of fetal chromosomal abnormalities depending on age. Most of the data were obtained from studies of women in North America or Europe. However, rare data from study of fetal chromosomal abnormalities depending on age targeting Korean women have been reported [6]. Against this background, the intention of this study was to examine the incidence rate of fetal chromosomal abnormalities depending on maternal age and to investigate correlation between fetal chromosomal abnormalities and maternal age, which targeted Korean pregnant women who underwent amniocentesis or chorionic villous sampling (CVS). In addition, this study examined if there was any meaningful difference in the incidence rate of fetal chromosomal abnormalities between cases where only advanced age of pregnant women was considered as an indication for amniocentesis or CVS and cases where other indications were considered for such examinations.

CVS). In addition, this study examined if there was any meaningful difference in the incidence rate of fetal chromosomal abnormalities between cases where only advanced age of pregnant women was considered as an indication for amniocentesis or CVS and cases where other indications were considered for such examinations. Materials and methods This study targeted 15,454 pregnant women who underwent amniocentesis or CVS in Gangnam and Bundang CHA Medical Centers during the period from January 2001 to February 2012 and was approved by Institutional Review Board in CHA Medical Center. This study was conducted as a retrospective study based on genetic information and inpatient and outpatient charts. However, 71 persons whose age could not be determined and 2 persons whose indication for amniocentesis or CVS could not be confirmed were excluded from this study. As a result, the cases of 15,381 pregnant women were included. Age of the cases was the age at the time of delivery. Fetal chromosomal abnormalities were classified according to numeric and structural chromosomal abnormalities. The numeric chromosomal abnormalities were divided again according to autosomal and sex chromosomal abnormalities. In regard to structural chromosomal abnormalities, normal variations such as heterochromatin, satellite chromosomes and pericentric inversion of chromosome 9 were excluded from the abnormal category.

ties. The numeric chromosomal abnormalities were divided again according to autosomal and sex chromosomal abnormalities. In regard to structural chromosomal abnormalities, normal variations such as heterochromatin, satellite chromosomes and pericentric inversion of chromosome 9 were excluded from the abnormal category. For analysis of maternal age-specific rates of fetal chromosomal abnormalities in pregnant women of advanced maternal age (AMA), maternal age beyond 35 years was segmented with a one-year interval. In addition to, among women ≥35 years of age, when only AMA was considered as indication of amniocentesis or CVS was performed, analysis was also performed to determine whether there was any difference in incidence rate of fetal chromosomal abnormalities compared to cases where other indications were considered. Logistic regression analysis was performed in order to investigate correlation between increase in maternal age and fetal chromosomal abnormalities. In addition, SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the study results. Significance probability of less than 0.05 was considered to show statistical significance. Results Among pregnant women who underwent amniocentesis or CVS during the study period 15,381 persons were selected for inclusion in this study. Age of the cases ranged from 19 years to 52 years. Average age was 34.0 years (±4.3); age distribution of the entire group of study subjects is shown in Fig. 1.

For analysis of maternal age-specific rates of fetal chromosomal abnormalities in pregnant women of advanced maternal age (AMA), maternal age beyond 35 years was segmented with a one-year interval. In addition to, among women ≥35 years of age, when only AMA was considered as indication of amniocentesis or CVS was performed, analysis was also performed to determine whether there was any difference in incidence rate of fetal chromosomal abnormalities compared to cases where other indications were considered. Logistic regression analysis was performed in order to investigate correlation between increase in maternal age and fetal chromosomal abnormalities. In addition, SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the study results. Significance probability of less than 0.05 was considered to show statistical significance. Results Among pregnant women who underwent amniocentesis or CVS during the study period 15,381 persons were selected for inclusion in this study. Age of the cases ranged from 19 years to 52 years. Average age was 34.0 years (±4.3); age distribution of the entire group of study subjects is shown in Fig. 1. According to the results of fetal chromosomal examination, 14,818 cases (96.34%) were found to have normal fetus chromosomes while 563 cases (3.66%) were found to have abnormal fetus chromosomes. Among the abnormal cases (3.66%), 357 cases (2.32%) had numeric chromosomal abnormalities, while 206 cases (1.34%) had structural chromosomal abnormalities. With respect to numeric chromosomal abnormalities, trisomy 21 was found in 181 cases (1.18%), which was the highest frequency. Then, trisomy 18 was found in 72 cases (0.47%), Turner's syndrome in 41 cases (0.27%), Klinefelter's syndrome in 28 cases (0.18%), trisomy 13 and triple X syndrome in 14 cases for each (0.09%), and XYY syndrome in 7 cases (0.05%), in this order.

risomy 21 was found in 181 cases (1.18%), which was the highest frequency. Then, trisomy 18 was found in 72 cases (0.47%), Turner's syndrome in 41 cases (0.27%), Klinefelter's syndrome in 28 cases (0.18%), trisomy 13 and triple X syndrome in 14 cases for each (0.09%), and XYY syndrome in 7 cases (0.05%), in this order. Tables 1, 2 show the analyses of the incidence rate of numeric and structural chromosomal abnormalities when age was increased by a one-year interval among women older than 35 years at the time of delivery. Trisomy 21 showed an incidence rate of 11.34 out of 1,000 cases at the age of 35 years, 15.41 cases at the age of 40, and 37.04 cases at the age of 45. Trisomy 18 showed an incidence rate of 1.89 out of 1,000 cases at the age of 35 years, 5.14 cases at the age of 40, and 37.04 cases at the age of 45, which is a sharp increase. According to the results of logistic regression analysis, trisomy 21 and trisomy 18 showed close correlation with age. In other words, as age increased by one year, odds ratio of trisomy 21 tended to increase by 1.177 times (Table 3, Fig. 2). According to increase in age by one year, odds ratio of trisomy 18 also tended to increase by 1.182 times (Table 3, Fig. 3). In addition, in regard to all of the fetal aneuploidies, odds ratio also tended to increase by 1.160 times as age increased by one year (Table 3, Fig. 4).

crease by 1.177 times (Table 3, Fig. 2). According to increase in age by one year, odds ratio of trisomy 18 also tended to increase by 1.182 times (Table 3, Fig. 3). In addition, in regard to all of the fetal aneuploidies, odds ratio also tended to increase by 1.160 times as age increased by one year (Table 3, Fig. 4). According to the results of this study, trisomy 13, Turner's syndrome, triple X syndrome, Klinefelter's syndrome, and structural chromosomal abnormalities did not show any significant correlation with maternal age (Table 3). XYY syndrome was found in two of the study subjects. Therefore, due to the very small number, analysis of the correlation between fetal chromosomal abnormalities and age was not possible. When only advanced age of women aged 35 years or older was considered as indication of amniocentesis or CVS were performed, the incidence rate of fetal chromosomal abnormalities was 1.80%. When other indications, including AMA were considered, the incidence rate of fetal chromosomal abnormalities was 6.45%. As a result, incidence of fetal chromosomal abnormalities was found to be significantly low when only advanced age was considered as an indication. However, both the AMA only group and other indication groups showed an increasing tendency in the expected rate of fetal chromosomal abnormalities with the increase in age (Table 4).

t, incidence of fetal chromosomal abnormalities was found to be significantly low when only advanced age was considered as an indication. However, both the AMA only group and other indication groups showed an increasing tendency in the expected rate of fetal chromosomal abnormalities with the increase in age (Table 4). Discussion This study targeted pregnant women who underwent amniocentesis or CVS in South Korea in order to analyze correlation between fetal chromosomal abnormalities and maternal age. As the percentages of pregnancies in advanced age have increased in Korea, demand for prediction of risk of fetal chromosomal abnormalities depending on age in prenatal genetic counseling has also increased. However, data from studies targeting Korean women have rarely been reported. Consequently, this study has significant meaning in that it targeted Korean women and it utilized a large quantity of data that had accumulated over the past 10 years. Because fetal chromosomal abnormalities were examined among women in high-risk groups who underwent amniocentesis or CVS, incidence rate of fetal chromosomal abnormalities was relatively higher than incidence rate in the normal group.

and it utilized a large quantity of data that had accumulated over the past 10 years. Because fetal chromosomal abnormalities were examined among women in high-risk groups who underwent amniocentesis or CVS, incidence rate of fetal chromosomal abnormalities was relatively higher than incidence rate in the normal group. A number of studies have reported that fetal chromosomal abnormalities showing a close association with maternal age included trisomy 21, trisomy 18, trisomy 13, triple X syndrome, and XYY syndrome. In 2010, Park et al. [6] published a research paper on a study of fetal chromosomal abnormalities depending on maternal age among Korean women. In a study targeting 2,032 women who underwent amniocentesis due to the sole indication of AMA, they analyzed correlation between fetal chromosomal abnormalities and maternal age. They reported that risk of trisomy 21, trisomy 18, triple X syndrome, and all aneuploidies showed a significant increase according to increase in maternal age. In this study, risk of trisomy 21, trisomy 18, and all of the fetal aneuploidies was found to show a significant increase according to increase in maternal age. In other words, as maternal age increased by one year, odds ratio of trisomy 21 tended to increase by 1.177 times, odds ratio of trisomy 18 tended to increase by 1.182 times, and odds ratio of all of the fetal aneuploidies tended to increase by 1.160 times. In addition, a sharp increase was found in incidence rate per 1,000 cases of trisomy 21 and trisomy 18 at the age of 45. However, because the number of cases at the age of 46 or older was very small, analysis of correlation between fetal chromosomal abnormalities and extreme maternal age was not possible. No significant correlation with maternal age was found in trisomy 13, Turner's syndrome, triple X syndrome, XYY syndrome, and structural chromosomal abnormalities. In particular, according to the statistics, trisomy 13, Turner's syndrome, and triple X syndrome were not found to show any significant correlation with maternal age. However, there was limitation in that the number of study subjects with each type of abnormality was too small to determine whether the resulting value was significant.

according to the statistics, trisomy 13, Turner's syndrome, and triple X syndrome were not found to show any significant correlation with maternal age. However, there was limitation in that the number of study subjects with each type of abnormality was too small to determine whether the resulting value was significant. Amniocentesis is usually performed after 16 weeks, while CVS is generally performed within 10 to 12 weeks. If spontaneous abortion occurs before 12 weeks, more than 50% of cases are likely to be attributable to fetal chromosomal abnormalities. According to the results of studies on analysis of abnormal karyotype frequency depending on gestational age, the frequency was higher as the gestational age was lower [7]. In this study, a separate adjustment was not made for cases where fetal chromosomal abnormalities caused spontaneous abortion before amniocentesis or CVS was performed. Therefore, it is considered that there may be a slight difference from the actual incidence rate of fetal chromosomal abnormalities.

age was lower [7]. In this study, a separate adjustment was not made for cases where fetal chromosomal abnormalities caused spontaneous abortion before amniocentesis or CVS was performed. Therefore, it is considered that there may be a slight difference from the actual incidence rate of fetal chromosomal abnormalities. In addition, this study compared incidence rate of fetal chromosomal abnormalities between when only pregnancy in advanced age was considered as an indication for performance of amniocentesis or CVS and when other indications as well as AMA were considered among women older than 35 years. The results of comparison demonstrated that the incidence rate of fetal chromosomal abnormalities was significantly high when other indications, including pregnancy in advanced age were considered for performance of genetic testing. Recently, remarkable development has been achieved not only in ultrasonography but also various prenatal screening tests. According to Malone et al. [8] in 2005, when prenatal screening test was performed using the combined method (i.e., sequential or integrated test) in a medical institution where experienced medical staff worked, detection rate of fetal chromosomal abnormalities was reported to be 90% or higher. Therefore, these results support the opinion that advanced age of a pregnant woman cannot be considered as a sole indication for amniocentesis, as shown in a Practice Bulletin published by the American College of Obstetrics and Gynecology [9]. However, genetic testing has been recommended for pregnant woman aged 35 years or older worldwide for the past 35 years. Despite the recently published guidelines, quite a few obstetricians still consider the age of 35 years as a cutoff for genetic testing [10]. According to recent studies conducted in Colorado of the US, birth rate of children with Down's syndrome increased when frequency of amniocentesis was reduced among women aged 35 years or older [11,12]. Consequently, maternal age is a factor that cannot be completely excluded from examination of fetal chromosomal abnormalities. In particular, advanced age of 35 years or older can be used as indication for conduct of prenatal genetic testing for pregnant women who did not undergo a separate screening test previously or could not provide exact information.

r that cannot be completely excluded from examination of fetal chromosomal abnormalities. In particular, advanced age of 35 years or older can be used as indication for conduct of prenatal genetic testing for pregnant women who did not undergo a separate screening test previously or could not provide exact information. Currently, thanks to development of not only a serum screening test but also precision ultrasound, the possibility of detection of fetal chromosomal abnormalities before childbirth has increased. Therefore, for more precise analysis of fetal chromosomal abnormalities, prenatal genetic counseling along with serum screening test, ultrasound screening, past history of fetal chromosomal abnormalities, or obstetrical history of pregnant women, rather than a simple reference to maternal age is needed. It is believed that the results of this study can be used as basic data that are suitable for domestic circumstances when prenatal genetic counseling is provided for investigation of risk of fetal chromosomal abnormalities depending on maternal age. In addition, conduct of future study of association with fetal chromosomal abnormalities targeting Koreans and persons with a broader range of age will be needed. Fig. 1 Age distribution in the study group. Fig. 2 Observed rates per 1,000 of trisomy 21 based on maternal age. Fig. 3 Observed rates per 1,000 of trisomy 18 based on maternal age. Fig. 4 Observed rates per 1,000 of all aneuploidies based on maternal age. Table 1 Incidences of numeric chromosomal abnormalities found in amniocentesis or chorionic villus sampling

Fig. 1 Age distribution in the study group. Fig. 2 Observed rates per 1,000 of trisomy 21 based on maternal age. Fig. 3 Observed rates per 1,000 of trisomy 18 based on maternal age. Fig. 4 Observed rates per 1,000 of all aneuploidies based on maternal age. Table 1 Incidences of numeric chromosomal abnormalities found in amniocentesis or chorionic villus sampling Values are presented as number (incidences per 1,000). a)Maternal age at the time of delivery. Table 2 Incidences of structural chromosomal abnormalities found in amniocentesis or chorionic villus sampling Values are presented as number (incidences per 1,000). a)Maternal age at the time of delivery. Table 3 Parameters and standard errors of logistic regression equations for fetal chromosomal abnormalities diagnosed at amniocentesis or chorionic villus sampling Table 4 Comparison with incidences of fetal chromosomal abnormalities between the advanced maternal age (AMA) only group and the others groups as an indication for amniocentesis or chorionic villus sampling among women older than 35 years Values are presented as number (incidences per 1,000). a)Maternal age at the time of delivery.

Introduction Matrix metalloproteinase-8 (MMP-8), human neutrophil collagenase, is released from the secondary or specific granules of polymorphonuclear leukocytes stimulated by chemotactic cytokines [1], and has the potent capabilities for degrading extracellular matrix, in particular collagen type I [2,3]. The degradation of extracellular matrix of fetal membranes is an important biochemical process in chorio-decidua activation, which is a part of the common terminal pathway of human parturition [4]. Indeed, MMP-8 has been documented in the amniotic cavity, fetal membranes and lower uterine segment in the context of preterm or term parturition [5-7].

ar matrix of fetal membranes is an important biochemical process in chorio-decidua activation, which is a part of the common terminal pathway of human parturition [4]. Indeed, MMP-8 has been documented in the amniotic cavity, fetal membranes and lower uterine segment in the context of preterm or term parturition [5-7]. Intra-amniotic inflammation (IAI) is associated with impending preterm delivery, acute histologic chorioamnionitis (acute-HCA), positive amniotic fluid (AF) culture and significant neonatal morbidity [8-19]. AF white blood cell (WBC) count has been generally used as a method for the rapid analysis of IAI, and a low AF WBC count has been thought to reflect the amniotic cavity without inflammation. However, AF MMP-8 has recently emerged as a reliable indicator of IAI [20-28]. We have previously observed that some patients with preterm labor and intact membranes (PTL) had low AF WBC counts but high AF MMP-8 concentrations. However, there is a paucity of data about the frequency and clinical significance of IAI defined as an elevated AF MMP-8 concentration in patients with PTL and low AF WBC counts. We hypothesized that only a low AF WBC count could not guarantee the continuation of pregnancy after 5 days of amniocentesis, placenta without any inflammation and a negative AF culture when AF MMP-8 concentration was elevated in patients with PTL. The objective of the study is to examine this hypothesis.

F WBC counts. We hypothesized that only a low AF WBC count could not guarantee the continuation of pregnancy after 5 days of amniocentesis, placenta without any inflammation and a negative AF culture when AF MMP-8 concentration was elevated in patients with PTL. The objective of the study is to examine this hypothesis. Materials and methods 1. Study design Study population consisted of 220 singleton gestations who underwent amniocentesis due to PTL (gestational age [GA] <35.7 weeks) and had low AF WBC counts (defined as AF WBC count<19 cells/mm3) at Seoul National University Hospital between January 1993 and December 2007. Adverse pregnancy outcomes were compared according to the presence or absence of IAI. Adverse pregnancy outcomes included preterm birth within 5 days of amniocentesis, acute-HCA and a positive AF culture. Of adverse pregnancy outcomes, the relationship between IAI and acute-HCA was examined in 110 cases delivered within 7 days of amniocentesis. This criterion was used to preserve a meaningful temporal relationship between the results of AF studies and the placental pathologic findings at birth. At our institution, transabdominal amniocentesis for retrieval of AF and placental pathologic examination after delivery were routinely offered to all patients who were admitted with the diagnosis of PTL. AF was analyzed for microbiologic and inflammatory status and fetal lung maturity. Written informed consent was obtained from all these patients. The Institutional Review Board of Seoul National University Hospital approved the collection and use of these samples and information for research purposes. The Seoul National University has a Federal Wide Assurance with the Office for Human Research Protections of the Department of Health and Human Services of the United States. Many of patients in this study were included in our previous studies.

collection and use of these samples and information for research purposes. The Seoul National University has a Federal Wide Assurance with the Office for Human Research Protections of the Department of Health and Human Services of the United States. Many of patients in this study were included in our previous studies. 2. Clinical characteristics and pregnancy outcomes The demographic and clinical characteristics of the mothers and their neonates were examined through a review of the medical records. We investigated parity (≥1), maternal age, antenatal use of corticosteroid, GA at amniocentesis, gender of newborn, route of delivery, GA at delivery, birth weight, 1 minute Apgar score and 5 minutes Apgar score. 3. Clinical chorioamnionitis Clinical chorioamnionitis was diagnosed when maternal body temperature was elevated to 37.8℃ and ≥2 of the following criteria were present according to the definitions previously described in detail [29]: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/mm3), maternal tachycardia (>100 beats/min) and fetal tachycardia (>160 beats/min).

ody temperature was elevated to 37.8℃ and ≥2 of the following criteria were present according to the definitions previously described in detail [29]: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/mm3), maternal tachycardia (>100 beats/min) and fetal tachycardia (>160 beats/min). 4. Placental pathology Placental tissue samples obtained for pathologic evaluation included the chorio-decidua, amnion, chorionic plate and umbilical cord. These samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of prepared tissue blocks were stained with hematoxylin and eosin. Pathologists were masked to the clinical information. Acute-HCA was defined in the presence of acute inflammatory changes on examination of a membrane roll and chorionic plate of the placenta; inflammation of the chorio-decidua or amnion was diagnosed in the presence of at least 1 focus of >5 neutrophils in the chorio-decidua or amnion; inflammation of the chorionic plate was diagnosed in the presence of more than 1 focus of at least 10 neutrophilic collections or diffuse inflammation in subchorionic fibrin, or diffuse and dense inflammation, neutrophilic infiltration into connective tissue of the placental plate, or placental vasculitis; funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton's jelly with the use of criteria previously published [30]. The presence of acute inflammation was classified as grade 1 or 2 in each anatomical region of placenta (amnion, chorio-decidua, chorionic plate and umbilical cord), and total grade was used to determine the severity of acute-HCA from 1 to 8 with the use of criteria previously published [30].

previously published [30]. The presence of acute inflammation was classified as grade 1 or 2 in each anatomical region of placenta (amnion, chorio-decidua, chorionic plate and umbilical cord), and total grade was used to determine the severity of acute-HCA from 1 to 8 with the use of criteria previously published [30]. 5. Amniotic fluid AF was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas (Ureaplasma urelyticum and Mycoplasma hominis) and analyzed for WBC count according to the methods previously described [31,32]. The remaining fluid was centrifuged and stored in polypropylene tubes at -70℃. MMP-8 concentrations in stored AF were measured with a commercially available enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech Inc., Little Chalfont, Bucks, UK). The sensitivity of the test was <0.3 ng/mL. Both intra- and inter-assay coefficients of variation were <10%. Details about this assay and its performance have been previously described [24]. IAI was defined as an elevated AF MMP-8 concentration (>23 ng/mL) as previously reported [25].