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Introduction The umbilical cord is an important lifeline for a fetus, and abnormalities of the umbilical cord sometimes cause fetal mortality or morbidity [1]. One of the abnormalities is its length. A short cord increases the probability of non-reassuring fetal status, emergent cesarean delivery, and neonatal asphyxia [1]. A long cord is associated with entanglement, knots, prolapse, and a thrombus of the cord. A long cord also increases the probability of neonatal asphyxia [2,3]. Though little is known about the factors determining umbilical cord length, fetal movement is considered by many to be one of the promoting factors. There have been several animal experiments suggesting just that. Moessinger et al. [4] reported that tensile forces on the cord, secondary to fetal movements, were an important determinant of cord length in rats. Baron S et al. exposed cocaine [5] and alcohol [6] to fetal rats to reduce fetal movement and demonstrated that these exposures made umbilical cords shorter. Similarly, Katz V et al. [7] exposed beta-blockers to fetal rabbits to reduce fetal movement and concluded that a decrease in fetal movement led to a shorter cord.

linical experience also demonstrated a significant difference between the two groups with 98% of obstetricians having 10 or more years of experience compared to 30% of nurses and midwives.Table 1 Background demographic data and prior understanding of obstetric violence in the survey responders. (chi square * p < 0.05). Table 1Survey question Obstetricians (n=50) % Obstetric nurse/midwifery staff (n=167) % Please select your gender male 58% 0%* female 42% 100% How many years have you been working in the field of Obstetrics? less than 5 years 0% 37%* 5 to 10 years 4% 34% more than 10 years 96% 29% In what country/region did you complete your primary clinical training? North American/Europe 56% 30%* Other 44% 70% Have you heard of the term Obstetric violence before? Yes 58% 50% No or unsure 42% 50% From the option list, what do you think the term obstetric violence means? correct 60% 44% incorrect 40% 56% Fifty two percent of those who responded had previously heard of the term obstetric violence, and 48% of responders understood what the term obstetric violence meant. There were no significant differences in these responses between the two groups.

aron S et al. exposed cocaine [5] and alcohol [6] to fetal rats to reduce fetal movement and demonstrated that these exposures made umbilical cords shorter. Similarly, Katz V et al. [7] exposed beta-blockers to fetal rabbits to reduce fetal movement and concluded that a decrease in fetal movement led to a shorter cord. On the other hand, several clinical studies reported an indirect relationship between cord length and fetal movements in human beings. Miller et al. [8] reported that a short umbilical cord was found in newborns with gross structural or functional limb defects that would limit intrauterine movement. Kivistö J et al. [9] studied pregnant women taking selective serotonin reuptake inhibitors and found that new-borns exposed to the medicine had longer umbilical cords. They said that the increased cord length could be explained by the increase of fetal movement caused by the medicine. Wright D and Chan GM [10] studied the relationship between the bone mass of infants and their umbilical cord lengths. They found that infants with a short cord length had less bone strength and that the finding was likely due to decreased fetal movement. Therefore, fetal movement seems to promote cord length. On the other hand, a longer cord may give a baby more freedom to move. However, there have been no reports that demonstrate a direct correlation between fetal movement and cord length in human beings. That is because there has never been a practical way to count fetal movements in many fetuses over long periods of time.

n the other hand, a longer cord may give a baby more freedom to move. However, there have been no reports that demonstrate a direct correlation between fetal movement and cord length in human beings. That is because there has never been a practical way to count fetal movements in many fetuses over long periods of time. We have developed a fetal movement acceleration measurement recorder (FMAM recorder, http://e-mother.co-site,jp) that was designed for home use [11,12]. The recorder records gross fetal movements by detecting oscillations of the mother’s abdominal wall caused by fetal movements. In a previous study, we simultaneously observed gross fetal movements and maternal abdominal wall oscillations respectively by ultrasonography and the FMAM recorder to examine the agreements between the two. The agreements expressed by prevalence-adjusted bias-adjusted kappa were 0.82-0.83 which meant almost perfect values [11]. Furthermore, using the FMAM recorder, we recently made normal reference values for gross fetal movement counts and demonstrated that the values are similar to those made using ultrasonography [12]. The FMAM recorder has made it possible to count gross fetal movements over many hours. The purpose of the current study was to examine the relationship between umbilical cord length and fetal movements as counted by the FMAM recorder.

We have developed a fetal movement acceleration measurement recorder (FMAM recorder, http://e-mother.co-site,jp) that was designed for home use [11,12]. The recorder records gross fetal movements by detecting oscillations of the mother’s abdominal wall caused by fetal movements. In a previous study, we simultaneously observed gross fetal movements and maternal abdominal wall oscillations respectively by ultrasonography and the FMAM recorder to examine the agreements between the two. The agreements expressed by prevalence-adjusted bias-adjusted kappa were 0.82-0.83 which meant almost perfect values [11]. Furthermore, using the FMAM recorder, we recently made normal reference values for gross fetal movement counts and demonstrated that the values are similar to those made using ultrasonography [12]. The FMAM recorder has made it possible to count gross fetal movements over many hours. The purpose of the current study was to examine the relationship between umbilical cord length and fetal movements as counted by the FMAM recorder. Materials and methods Counting fetal movements The FMAM recorder was described in detail in our past studies [12,13]. It weighs 290 g and can be used at home. It has two acceleration sensors: one is a fetal movement sensor (FM sensor), which attaches to the mother’s abdomen, and the other is a mother’s movement sensor (MM sensor), which attaches to her thigh. The sensitivity of the FM and MM sensors is 700 mV/0.1 G and 120 mV/0.1 G, respectively. The FM sensor detects oscillations of the mother’s abdominal wall caused by gross fetal movements. The recorder is unsuitable when the mother moves frequently because the mother’s body movements themselves also cause oscillations. That is why the recorder is used during her sleep. However, the mother does move occasionally even when she is asleep. In principle, when the MM sensor detects no maternal leg movement and the FM sensor detects oscillations of her abdominal wall, gross fetal movements are judged to have occurred.

so cause oscillations. That is why the recorder is used during her sleep. However, the mother does move occasionally even when she is asleep. In principle, when the MM sensor detects no maternal leg movement and the FM sensor detects oscillations of her abdominal wall, gross fetal movements are judged to have occurred. The mothers were asked to record fetal movements during their sleep weekly or biweekly after 28 weeks, because the accuracy of the FMAM recorder is limited before 28 weeks [11]. The data were uploaded to a PC. We accepted records only when data could be obtained for more than 4 h per night. The recording was divided into 10-s time intervals (epochs), meaning 360 epochs per hour. All epochs were reviewed to determine whether any fetal movements occurred within each epoch. An epoch with any fetal movements was judged to be one positive epoch. The decision was made using a custom-made software system (Version 1.04 A, NoruPro Light Systems, Inc. Tokyo Japan), which was developed for the FMAM recorder [12,14]. The ratio of positive epochs to all epochs during one night was calculated as the movement index.

y fetal movements was judged to be one positive epoch. The decision was made using a custom-made software system (Version 1.04 A, NoruPro Light Systems, Inc. Tokyo Japan), which was developed for the FMAM recorder [12,14]. The ratio of positive epochs to all epochs during one night was calculated as the movement index. Subjects There were a total of 62 pregnant women who could record fetal movement for more than 4 h per night and delivered a baby at term between February 2010 and the end of 2016 at Teikyo University Hospital. None of the mothers had any medical complications, and none of the babies had any anomalies or neurological problems. The characteristics of the mothers and babies are shown in Table 1.Table 1 Characteristics of all women and three groups depending on the cord length. Table 1 total groups short middle long p n 62 20 22 20 mother’s age 32.9 32.5 33.4 32.8 0.798 (20.0, 44.0) 　(20.0, 44.0) 　(25.0, 41.0) 　(24.0, 41.0) BMI 21.1 21.4 20.4 21.5 0.327 (16.8, 28.0) 　(17.6, 28.0) (16.8, 24.7) 　(17.8, 26.8) Para / non-para 20 / 42 8 / 12 9 / 13 3 / 17 0.138 anterior located placenta(+ / -) 18 / 44 4 / 16 8 / 14 6 / 14 0.516 delivery weeks 39.2 39.0 39.1 39.4 0.649 (37.0, 41.1) 　(37.3, 40.9) 　(37.0, 41.1) 　(37.1, 41.0) newborn weight (g) 2990.6 2853.6 3126.8 2977.9 0.067 (2340, 3790) (2340, 3404) (2396, 3790) 　(2400, 3576) male / female 30 / 32 8 / 12 10 / 12 12 / 8 0.436 placental weight (g) 573 525 594.3 595.3 0.070-1.000 (400., 790) (400, 730) (420, 785) (425, 790) Data are presented as number or mean　(range).

41.1) 　(37.1, 41.0) newborn weight (g) 2990.6 2853.6 3126.8 2977.9 0.067 (2340, 3790) (2340, 3404) (2396, 3790) 　(2400, 3576) male / female 30 / 32 8 / 12 10 / 12 12 / 8 0.436 placental weight (g) 573 525 594.3 595.3 0.070-1.000 (400., 790) (400, 730) (420, 785) (425, 790) Data are presented as number or mean　(range). Comparisons among the three groups were conducted with ANOVA or Tukey-Kramer. Methods After all delivery, the length of the umbilical cord was measured after separated from the baby. The cord segment attached to the baby was left routinely 3 cm in length. A trained midwife checked cord abnormalities and measured cord length using a ruler. The length was determined as the nearest centimeters and added 3 cm in order to get the total length. First, the correlation between the cord length and the movement index was examined. In a previous study, we reported that the movement index decreases as pregnancy progresses, with the decrease being relatively rapid around 32–35 weeks [12]. Therefore, the recordings were divided into three groups based on gestational weeks, i.e. 28–31, 32–35, and 36–39 weeks, and a mean value of the movement index was calculated for each period per one woman. Multiple linear regression analyses were then conducted with six explanatory variables (primipara / multipara, anterior / posterior located placenta, placental weight, and the mean movement index value of 28–31, 32–35, and 36–39 week) and a response valuable (umbilical cord length).

lated for each period per one woman. Multiple linear regression analyses were then conducted with six explanatory variables (primipara / multipara, anterior / posterior located placenta, placental weight, and the mean movement index value of 28–31, 32–35, and 36–39 week) and a response valuable (umbilical cord length). Next, all women were divided into three groups depending on the cord length. The average umbilical cord length has been reported to be around 55 cm [2]; therefore, when the cord length was 50–60 cm, the women were classified in the middle cord group. The shorter or longer cord length groups were less than 50 cm and more than 60 cm, respectively. Table 1 shows the characteristics of the three groups. Based on SD = 10% and an expected 10% movement difference, we calculated a sample size of 16 women for each group with α = 0.05 and 80% power. The numbers of women in each group were between 20 and 22. The index of movement was then compared among the three groups by ANOVA for the three gestational periods, i.e. 28–31, 32–35, and 36–39 weeks. All data was analyzed with JMP Pro 12.0.1. The statistically significant difference was set at p value of less than 0.05. This study was approved by the ethics committee of Teikyo University. All women gave written informed consent before participating in the study.

Next, all women were divided into three groups depending on the cord length. The average umbilical cord length has been reported to be around 55 cm [2]; therefore, when the cord length was 50–60 cm, the women were classified in the middle cord group. The shorter or longer cord length groups were less than 50 cm and more than 60 cm, respectively. Table 1 shows the characteristics of the three groups. Based on SD = 10% and an expected 10% movement difference, we calculated a sample size of 16 women for each group with α = 0.05 and 80% power. The numbers of women in each group were between 20 and 22. The index of movement was then compared among the three groups by ANOVA for the three gestational periods, i.e. 28–31, 32–35, and 36–39 weeks. All data was analyzed with JMP Pro 12.0.1. The statistically significant difference was set at p value of less than 0.05. This study was approved by the ethics committee of Teikyo University. All women gave written informed consent before participating in the study. Results A total of 2355.6 h from 368 night records were available for this study. Umbilical cord length varied from 35 to 100 cm. The mean length (10–90%) was 54.8 (39.6–69.7) cm. There were no umbilical cord complications, such as a single artery, excessive torsion, no torsion, or velamentous insertion.

This study was approved by the ethics committee of Teikyo University. All women gave written informed consent before participating in the study. Results A total of 2355.6 h from 368 night records were available for this study. Umbilical cord length varied from 35 to 100 cm. The mean length (10–90%) was 54.8 (39.6–69.7) cm. There were no umbilical cord complications, such as a single artery, excessive torsion, no torsion, or velamentous insertion. Primipara and anterior located placenta were valuables to elongate cord length (p = 0.013, and 0.015, respectively), and the placental weight also had positive relationship with cord length (p = 0.005); however, there were no relationships between the cord length and the movement index of 28–31, 32–35, and 35–39 weeks (p = 0.090, 0.235, 0.129, respectively).

were valuables to elongate cord length (p = 0.013, and 0.015, respectively), and the placental weight also had positive relationship with cord length (p = 0.005); however, there were no relationships between the cord length and the movement index of 28–31, 32–35, and 35–39 weeks (p = 0.090, 0.235, 0.129, respectively). Fig. 1 shows the changes in the index of movements in the shorter, middle, and longer cord groups depending on gestational weeks. In a previous study, we reported normal reference values where the index of movement decreases as pregnancy progresses, with the decrease being relatively rapid around 32–35 weeks [12]. Similar changes are shown in the shorter and middle cord groups; however, the decrease seems to be slight in the longer cord group. Statistical comparisons also demonstrated that there were no differences in the index of movement among the three groups at 28–31 and 32–35 gestational weeks (p = 0.096, and 0.465, respectively); however, the longer cord group had a greater index of movement than the other two groups at 36–39 weeks (p = 0.0008).Fig. 1 Changes in the median index of fetal movement in shorter (< 50 cm), middle (50–60 cm), and longer (> 60 cm) umbilical cord groups. There were no differences at 28–31 and 32–35 gestational weeks (p = 0.096, and 0.465, respectively). The longer cord group showed more movement than the other two at 36–39 weeks (p = 0.0008). Fig. 1

Fig. 1 shows the changes in the index of movements in the shorter, middle, and longer cord groups depending on gestational weeks. In a previous study, we reported normal reference values where the index of movement decreases as pregnancy progresses, with the decrease being relatively rapid around 32–35 weeks [12]. Similar changes are shown in the shorter and middle cord groups; however, the decrease seems to be slight in the longer cord group. Statistical comparisons also demonstrated that there were no differences in the index of movement among the three groups at 28–31 and 32–35 gestational weeks (p = 0.096, and 0.465, respectively); however, the longer cord group had a greater index of movement than the other two groups at 36–39 weeks (p = 0.0008).Fig. 1 Changes in the median index of fetal movement in shorter (< 50 cm), middle (50–60 cm), and longer (> 60 cm) umbilical cord groups. There were no differences at 28–31 and 32–35 gestational weeks (p = 0.096, and 0.465, respectively). The longer cord group showed more movement than the other two at 36–39 weeks (p = 0.0008). Fig. 1 Discussion The factors determining umbilical cord length are largely unknown. Marpas P [15] reported that little correspondence was found between cord length and the fetal or placental weight. On the contrary, Georgiadis L et al. [16] reported that cord length was associated with birth weight, placental weight, and gestational age. Georgiadia et al. also reported that girls had shorter cords.

n. Marpas P [15] reported that little correspondence was found between cord length and the fetal or placental weight. On the contrary, Georgiadis L et al. [16] reported that cord length was associated with birth weight, placental weight, and gestational age. Georgiadia et al. also reported that girls had shorter cords. The results of this study demonstrated that there were no correlations found between the cord length at birth and fetal movement counts after 28 weeks; however, the longer cord group had higher fetal movement counts than the other two groups after 36 weeks. What did these results mean? Positive correlations include both the cord becomes shorter and longer depending fetal movement. Simple explanation could be that decreased fetal movement did not shorten the cord length but increased one elongated that. Actually, there were no differences in the movement index between the shorter and middle cord groups in the results. In a study by Richard LN [17], the authors noted that umbilical cords progressively grew longer toward term, and the rate of growth gradually slowed. In another study, Georgiadis L et al. [16] also found that umbilical cords grew progressively longer toward term; however, the growth rate was greatest between 31 and 39 weeks, and the maximum growth was at around 35–36 weeks. Both studies were consistent that the umbilical cord continued to grow until term. Continuing grow of the cord seemed to relate our study’s results that the group with longer cords had higher fetal movement counts after 36 weeks than the other.

between 31 and 39 weeks, and the maximum growth was at around 35–36 weeks. Both studies were consistent that the umbilical cord continued to grow until term. Continuing grow of the cord seemed to relate our study’s results that the group with longer cords had higher fetal movement counts after 36 weeks than the other. We made normal reference curves for fetal movements in our previous study [12] and demonstrated that fetal movements decreased at around 32–35 weeks; however, the results of this study showed that the decreased fetal movements after that period could be an important factor in finally determining whether or not the umbilical cord becomes longer. Looking at the changes in fetal movements based on the gestational weeks shown in Fig. 1, we see that fetal movement counts for the three groups were not so different until around 32 weeks. After that, decreases in movement in the longer cord group seemed smaller than those in the other two groups. In the longer cord group, a smaller decrease in fetal movements near term might make the umbilical cord longer and longer as pregnancy progresses, which might eventually make a significant difference in cord length between the longer cord group and the other two.

group seemed smaller than those in the other two groups. In the longer cord group, a smaller decrease in fetal movements near term might make the umbilical cord longer and longer as pregnancy progresses, which might eventually make a significant difference in cord length between the longer cord group and the other two. However, even if this is true, it is still difficult to understand why the shorter cord group did not demonstrate less movement compared with the middle cord group. There are several limitations of this study. One limitation of studies about cord length is that we can measure only final cord length but not the process of cord elongation during pregnancy. Another limitation is that we can count fetal movement only after 28 weeks when the cord length already becomes 2-third of full length.

re several limitations of this study. One limitation of studies about cord length is that we can measure only final cord length but not the process of cord elongation during pregnancy. Another limitation is that we can count fetal movement only after 28 weeks when the cord length already becomes 2-third of full length. On top of that, an important problem of this study was that the accuracy of the FMAM recorder has not been fully confirmed. We have discussed about the accuracy of the FMAM recorder in several previous studies [[11], [12], [13], [14]]. Overall, the recorder has a good record of counting fetal movements: however, additional clinical studies are still needed to fully confirm its accuracy. If this study had demonstrated no positive results about relation between fetal movements and cord length, it would have been unclear whether the FMAM recorder was at fault or whether there was truly no relationship between the two. However, this study showed the positive and reasonable result that the group with longer umbilical cords had higher fetal movement counts than the other groups. We feel that this study is one more that supports the accuracy of the FMAM recorder. Georgiadis L et al. [16] reported that girls seemed to have shorter cords. If true, that raises the question of whether female fetuses move less compared with male fetuses. In a previous study, we compared fetal movement counts between boys and girls and found no difference between the two [12].

On top of that, an important problem of this study was that the accuracy of the FMAM recorder has not been fully confirmed. We have discussed about the accuracy of the FMAM recorder in several previous studies [[11], [12], [13], [14]]. Overall, the recorder has a good record of counting fetal movements: however, additional clinical studies are still needed to fully confirm its accuracy. If this study had demonstrated no positive results about relation between fetal movements and cord length, it would have been unclear whether the FMAM recorder was at fault or whether there was truly no relationship between the two. However, this study showed the positive and reasonable result that the group with longer umbilical cords had higher fetal movement counts than the other groups. We feel that this study is one more that supports the accuracy of the FMAM recorder. Georgiadis L et al. [16] reported that girls seemed to have shorter cords. If true, that raises the question of whether female fetuses move less compared with male fetuses. In a previous study, we compared fetal movement counts between boys and girls and found no difference between the two [12]. In conclusion, this study suggested that fetal movement near term is an important factor in determining whether cord length becomes relatively longer in normal pregnancies. Funding This work was supported by the Japan Society for the Promotion of Science (Grant Number JP 16K10109). Declarations of interest None. Conflict of interest The authors have no conflicts of interest to declare.

In conclusion, this study suggested that fetal movement near term is an important factor in determining whether cord length becomes relatively longer in normal pregnancies. Funding This work was supported by the Japan Society for the Promotion of Science (Grant Number JP 16K10109). Declarations of interest None. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgements We thank Professor Takuya Ayabe and Mrs. Mieko Fuse for their support, and we appreciate language help by Mr. Howard Stacey. We also appreciate the cooperation of the women who participated in this study.

Dear Editor, A 33 year old primiparous patient presented to our Obstetric triage at 24 + 3 weeks gestation with pyrexia and left iliac fossa pain. Significant medical history included a background of diverticular disease, gastric ulcer and previous cholecystectomy for bile acid malabsorption. Routine investigations on admission revealed elevated inflammatory markers, a swinging pyrexia and positive urine dip. A renal ultrasound reported the presence of renal calculi with no evidence of hydronephrosis. The patient was treated with intravenous antibiotics for a differential diagnosis of pyelonephritis. One day following admission she complained of lower pelvic pains and examination revealed a dilated cervix of 2 cm with bulging membranes. She completed a course of steroids and magnesium sulphate in view of threatened pre term labour for fetal lung maturation and neuroprotection. She rapidly developed septicaemia and proceeded to deliver a live female baby. Post-delivery she continued to have swinging temperatures despite broad spectrum antibiotics and developed septic shock. An urgent CT pelvic scan reported a possible left tubo- ovarian abscess. She was taken to theatre for an urgent laparoscopy and was noted to have a large diverticular abscess on the sigmoid colon fistulating into the left abdominal wall, involving the left ovary and salpinx. Due to significant inflammation and necrotic tissue she underwent a defunctioning- loop ileostomy and pelvic washout. She continues to make good progress with a future plan for ileostomy reversal surgery.

icular abscess on the sigmoid colon fistulating into the left abdominal wall, involving the left ovary and salpinx. Due to significant inflammation and necrotic tissue she underwent a defunctioning- loop ileostomy and pelvic washout. She continues to make good progress with a future plan for ileostomy reversal surgery. Diverticular disease in pregnancy is extremely rare. Only one study reviewing a 20 year period of pregnancies within an obstetric department reported an incidence of 1 in 6000 pregnancies [1]. It commonly presents with uncomplicated diverticulitis in which bowel rest and antibiotic therapy are the mainstay treatment. The disease itself is often one associated with an elderly population. A prevalence of 65% has been reported in over 85 year olds compared to 5% among patients less than 40 years old [1]. Recent literature however, reports increasing incidence among younger patients between 18–44 years old presenting with more aggressive forms of the disease [2]. A review of patients by Weizman at al reported a five -fold increase in risk of complications such as fistula formation, peri-diverticular abscess, perforation, strictures and obstruction when compared with older patients [2]. Given the presumption of a low incidence of the disease within younger patients, it is not surprising to find a high percentage (50%) are often misdiagnosed at presentation- most commonly with appendicitis [2].

on, peri-diverticular abscess, perforation, strictures and obstruction when compared with older patients [2]. Given the presumption of a low incidence of the disease within younger patients, it is not surprising to find a high percentage (50%) are often misdiagnosed at presentation- most commonly with appendicitis [2]. It has been reported that 30% of cases of pre term labour are secondary to infection [3]. Elevated concentrations of inflammatory cytokines such as Interleukin- 1 and tumour necrosis factor within the amniotic fluid produced by the maternal decidua and fetal membranes, have been found in patients presenting with pre term labour <30 weeks gestation [4]. Such cytokine mediated inflammation is thought to trigger pre term contractions, cervical ripening and rupture of membranes [3]. Therefore any infectious stimuli such as intra- abdominal sepsis in pregnancy, can potentially initiate cytokine release and the onset of pre term labour.

term labour <30 weeks gestation [4]. Such cytokine mediated inflammation is thought to trigger pre term contractions, cervical ripening and rupture of membranes [3]. Therefore any infectious stimuli such as intra- abdominal sepsis in pregnancy, can potentially initiate cytokine release and the onset of pre term labour. It is important to consider diverticular abscess as a differential diagnosis of swinging pyrexia in pregnancy, as evidently it can be associated with significant maternal and fetal morbidity. In view of recent evidence of an increasing prevalence among a younger population in the Western population, it is certainly a condition Obstetricians may expect to see increase as more women delay their pregnancies till later in life. Early imaging and antibiotics may improve overall maternal and fetal outcomes. If operative intervention is required, the decision for tocolysis or early delivery prior to surgery should be discussed with a multidisciplinary team. Conflict of interest None.

Introduction Determination of whether a suspicious adnexal mass is an ovarian cancer is currently made only following invasive surgery and pathological analysis of excised tissue. Since only a minority of these women will in fact have a malignancy [1], the discovery of a biomarker to noninvasively determine whether or not an adnexal mass is malignant could greatly reduce the incidence of unnecessary surgery. Currently, serum CA125 is the most commonly used biomarker to detect ovarian cancer. However, about 25% of women with stage 1 ovarian cancer will have a normal CA125 and multiple benign conditions may cause CA125 elevations [2]. To date, CA125 has been shown to be most useful in predicting the response to treatment and recurrence rather than detection of early stage malignancy [3]. Recent findings indicate that some ovarian cancers originate as premalignant intraepithelial lesions in the distal fallopian tube and that transformed cells subsequently migrate to the ovary [4,5]. Women who have undergone a tubal ligation have a decreased risk of developing ovarian cancer, especially endometrioid and serous types [6]. Thus, in women with ovarian cancer compounds associated with development of this malignancy may be present in the upper genital tract, migrate to the lower genital tract and accumulate in the vagina.

rgone a tubal ligation have a decreased risk of developing ovarian cancer, especially endometrioid and serous types [6]. Thus, in women with ovarian cancer compounds associated with development of this malignancy may be present in the upper genital tract, migrate to the lower genital tract and accumulate in the vagina. A scarcity of studies have evaluated vaginal fluid as a potential source of cancer-associated biomarkers. An investigation in 1997 determined that CA125 as well as carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC) antigen were detectable in vaginal fluid and that the levels of all three compounds were highest in 20 women with benign gynecological diseases as compared to 10 with squamous cell carcinoma, 5 with endometrial cancer, 3 with vulva carcinoma, 8 healthy pre-menopausal and 7 healthy post-menopausal women [7]. A later study reported that the level of CA125 in the vagina and cervix was highest in women with endometrial hyperplasia and endometrial cancer [8]. A comparative evaluation of three tumors biomarkers – CA125, CEA and CA19-9 – in vaginal wash samples from 30 women in each group with either advanced primary ovarian cancer or benign ovarian cysts revealed highly elevated levels of all 3 compounds in the malignant group [9]. In the most recent study women with precancerous endometrial intraepithelial neoplasia or endometrial adenocarcinoma had higher and similar vaginal concentrations of CA125 than did women with non-endometrioma ovarian cysts, endometrial hyperplasia or dysfunctional uterine bleeding [10].

unds in the malignant group [9]. In the most recent study women with precancerous endometrial intraepithelial neoplasia or endometrial adenocarcinoma had higher and similar vaginal concentrations of CA125 than did women with non-endometrioma ovarian cysts, endometrial hyperplasia or dysfunctional uterine bleeding [10]. Lysophosphatidic acid (LPA, 1-acyl-2hydroxy-sn-glycero-3-phosphate) is a phospholipid produced by malignantly transformed ovarian epithelial cells. It has been shown to stimulate cell proliferation and invasion, increase expression of factors involved in angiogenesis and levels are elevated in sera and ascites fluid from women with ovarian cancer [[11], [12], [13], [14]]. LPA has also been shown to inhibit autophagy in a prostate tumor cell line [15]. We previously reported that vaginal fluid from women with a malignant adnexal mass inhibited autophagy in peripheral blood mononuclear cells to a greater extent than did vaginal fluid from women with a benign mass [16]. As a component of our ongoing investigation of possible predictive biomarkers of a malignant adnexal mass in vaginal fluid, and based on prior associations between circulating LPA levels and ovarian cancer, we evaluated whether the LPA concentration in vaginal fluid would be elevated in women subsequently diagnosed with a malignant adnexal mass.

ng investigation of possible predictive biomarkers of a malignant adnexal mass in vaginal fluid, and based on prior associations between circulating LPA levels and ovarian cancer, we evaluated whether the LPA concentration in vaginal fluid would be elevated in women subsequently diagnosed with a malignant adnexal mass. Material and methods Subjects The subjects in this prospective study were 100 women referred to the Division of Gynecologic Oncology at Weill Cornell Medicine for evaluation of a suspicious adnexal mass. All subjects were referred for evaluation due to a pelvic mass of presumed gynecologic origin noted on pelvic sonography or magnetic resonance imaging. Inclusion criteria were post-menopausal status, the ability to collect vaginal samples that could be sent to the laboratory for processing within a 3 h time period and ability to provide written informed consent. Exclusion criteria were women with prior hysterectomy, history of malignancy, presence of vaginal bleeding, signs or symptoms of infection or no plan for undergoing surgical evaluation of their mass at Weill Cornell. Subsequent to vaginal sample collection all women underwent surgical exploration by laparoscopy or laparotomy and surgical resection of the adnexal mass, as described previously [16]. Briefly, the adnexal masses were resected and intraoperative pathology consultation was utilized. When an invasive malignancy was present, further surgical staging procedures including total hysterectomy, bilateral salpingo-oopherectomy, pelvic and paraarortic lymph node sampling and oomentectomy were performed when clinically indicated. The final pathological diagnosis was determined by an attending gynecologic pathologist utilizing techniques and tissue samples standard at our hospital. The study was approved by the Institutional Review Board of Weill Cornell Medicine and all subjects signed informed written consent.

formed when clinically indicated. The final pathological diagnosis was determined by an attending gynecologic pathologist utilizing techniques and tissue samples standard at our hospital. The study was approved by the Institutional Review Board of Weill Cornell Medicine and all subjects signed informed written consent. Samples Vaginal fluid was obtained during a speculum-based examination by scraping a cotton swab against the posterior vaginal wall and shaking the contents into a tube containing one ml sterile phosphate-buffered saline. The tube was centrifuged and the supernatant stored in aliquots at −80 °C until assayed. Samples with visible blood contamination were discarded. Assays Thawed aliquots of vaginal secretions were assayed for LPA by a commercial ELISA kit (Echelon Biosciences, Salt Lake City, Utah). LPA was detected with a monoclonal antibody that did not exhibit reactivity with LPA analogs. Values were converted to μM by reference to a standard curve generated in parallel to each assay. The lower limit of sensitivity was 0.064 μM. Intra-and inter-assay variability was <10%. Peripheral blood serum was assayed for CA125 in the Weill Cornell clinical laboratory by a standard chemiluminescent immunoassay. The laboratory staff was blinded to all clinical data including CA125 results that were obtained by chart review only after completion of the lab analyses.

inter-assay variability was <10%. Peripheral blood serum was assayed for CA125 in the Weill Cornell clinical laboratory by a standard chemiluminescent immunoassay. The laboratory staff was blinded to all clinical data including CA125 results that were obtained by chart review only after completion of the lab analyses. Statistics Differences in LPA and CA125 levels between women with a benign or malignant mass were analyzed by the non-parametric Mann-Whitney test since values were not normally distributed. A serum CA125 concentration of 35 U/ml is typically used as the cutoff for malignancy [3], and we utilized this value in our study. An LPA concentration >7.25 μM was operationally designated as the cutoff for detection of a malignant lesion, based on the observation that >90% of women with a benign diagnosis had a vaginal LPA level below this value. Differences between positive values for CA125 and LPA for each type of malignancy were assessed by Fisher’s exact test. A two sided p value <0.05 was considered significant. GRAPH PAD INSTAT (Graph Pad Software, San Diego, CA) was utilized for the analysis.

h a benign diagnosis had a vaginal LPA level below this value. Differences between positive values for CA125 and LPA for each type of malignancy were assessed by Fisher’s exact test. A two sided p value <0.05 was considered significant. GRAPH PAD INSTAT (Graph Pad Software, San Diego, CA) was utilized for the analysis. Results Characteristics of the study population are detailed in Table 1. A malignant adnexal mass was detected in 28 women, four had a borderline tumor while 68 had a benign lesion. Women in the three groups were of a similar mean age, had a comparable mean age at menarche and menopause and were of similar gravidity, parity and body mass index. The majority of women in each group were White. Analysis of these multiple variables negated their possible confounding influence on vaginal LPA levels. There was no difference in the use of hormone replacement therapy (HRT) between groups. The median CA125 level was 52.2 U/ml in women with a malignant mass, 14.6 U/ml in those with a borderline tumor and 11.0 U/ml in those whose mass was benign (p < 0.0001 malignant vs. the other two groups).Table 1 Characteristics of post-menopausal women evaluated for an adnexal mass.

ement therapy (HRT) between groups. The median CA125 level was 52.2 U/ml in women with a malignant mass, 14.6 U/ml in those with a borderline tumor and 11.0 U/ml in those whose mass was benign (p < 0.0001 malignant vs. the other two groups).Table 1 Characteristics of post-menopausal women evaluated for an adnexal mass. Table 1Characteristic Benign Borderline Malignant N = 68 N = 4 N = 28 Mean age in years (SD) 63.2 (9.4) 63.0(11.7) 66.2(10.2) Using HRT 12 (18.2%) 0 9 (26.5%) Mean age at menarche (SD) 12.5(1.2) 13.0(0) 13.3 (1.9) Mean age at menopause (SD) 49.1(5.8) 47.5(3.3) 50.7(4.1) Median gravida (range) 2 (0–10) 3 (0–4) 2 (0–9) Median parity (range) 2 (0–9) 2(0–3) 2 (0–5) Mean body mass index (SD]b 26.4(5.8) 27.1(5.2) 25.3 (5.0) Per cent White race 70.7% 75.0% 85.2% Median CA125 (range) 11.0(2.8–75) 14.6(9.3–117) 52.2 (5.7–7778)a HRT, hormone replacement therapy. a p < 0.0001 vs. benign and borderline. b kg/m2. Among women with a malignant ovarian mass, 11 (39.3%) had endometrioid ovarian cancer with or without Clear cell tumor components, six (21.4%) had high grade serous cancer, three (10.7%) had a mucinous tumor, two each (7.1%) had a malignant mixed mesodermal or a granulosa tumor and one of four women (3.6%) had a Clear cell tumor, a mixed cell tumor, leimyosarcoma or metastatic adrenal tumor. The majority of women with a benign diagnosis had either a cystadenoma (44.1%) or another type of cyst (33.8%).

had a mucinous tumor, two each (7.1%) had a malignant mixed mesodermal or a granulosa tumor and one of four women (3.6%) had a Clear cell tumor, a mixed cell tumor, leimyosarcoma or metastatic adrenal tumor. The majority of women with a benign diagnosis had either a cystadenoma (44.1%) or another type of cyst (33.8%). The concentration of LPA in vaginal fluid from women with each of the different ovarian malignancies as well as the individual benign conditions is shown in Table 2. Individual values for women with a malignant or benign mass are presented in Fig. 1. The median vaginal LPA concentration was highest in the six women with serous ovarian cancer (9.5 μM), one woman with a granulosa tumor (9.1 μM) and eleven with an endometrioid malignancy (7.9 μM). However, most likely due to the low number of subjects evaluated only in women with an endometrioid ovarian malignancy was the LPA level significantly elevated (p = 0.0137) compared to the median level in women with benign diagnoses (1.5 μM). None of the women with a malignant mass and 6 women with a benign mass had an undetectable LPA level in their vaginal sample. Only four women with benign conditions – two with a serous cystadenoma and one each with a mucinous cystadenoma and a mesothelial cyst - had a vaginal LPA concentration >7.25 μM. HRT administration had no effect on the vaginal LPA level (data not shown).Table 2 Association between lysophosphatidic acid concentration in vaginal secretions and differential pathological diagnosis in women with a benign or malignant adnexal mass.

ma and a mesothelial cyst - had a vaginal LPA concentration >7.25 μM. HRT administration had no effect on the vaginal LPA level (data not shown).Table 2 Association between lysophosphatidic acid concentration in vaginal secretions and differential pathological diagnosis in women with a benign or malignant adnexal mass. Table 2Diagnosis No. women LPA (μM) Malignant Endometroid +/- Clear cell 11 7.9 (1.9–24.3)a Serous 6 9.5 (1.0–16.1)b Mucinous 3 2.6 (1.2–15.2) MMMT 2 1.1 (0.9–1.4) Granulosa 2 9.1 (4.2–14.1) Other 2 4.4 (1.6–7.3) Clear cell 1 1.6 Mixed 1 0.2 Borderline Serous 2 Mucinous 2 Benign Cystadenoma 30 1.2 (0.4–6.6) Other cysts 23 2.7 (1.2–21.8) Teratoma 6 1.1 (0.5–2.3) Fibroma 6 2.0 (0.5–4.4) Endometriosis 3 0.3 (0–7.9) Total benign 68 1.5 (0.5–4.3) Values are median (25%–75%) μM; Endometrioid +/− Clear cell, endometrioid tumor with and without clear cell components; MMMT, malignant mixed mesoderm tumor (carcinosarcoma); Other, metastatic adrenal, leiomyosarcoma. ap = 0.0137 vs. benign; bp = 0.1267 vs. benign; cp = 0.1208 vs. benign. Fig. 1 LPA in vaginal fluid from women with a benign or malignant adnexal mass. Vaginal fluid was collected from women being screened for an adnexal mass and tested for LPA by ELISA. The type of malignancy was subsequently determined by a gynecologic pathologist. MMMT, malignant mixed mesoderm tumor (carcinosarcoma). Fig. 1

ap = 0.0137 vs. benign; bp = 0.1267 vs. benign; cp = 0.1208 vs. benign. Fig. 1 LPA in vaginal fluid from women with a benign or malignant adnexal mass. Vaginal fluid was collected from women being screened for an adnexal mass and tested for LPA by ELISA. The type of malignancy was subsequently determined by a gynecologic pathologist. MMMT, malignant mixed mesoderm tumor (carcinosarcoma). Fig. 1 Values for serum CA125 were available for 23 of the 28 women with a malignant adnexal mass. The relative ability of vaginal LPA and serum CA125 to detect the various adnexal mass-related malignancies is shown in Table 3. Of six women with endometrioid ovarian cancer five (83.3%) were positive only for vaginal LPA while one (16.6%) was only CA125 positive. None were positive for both LPA and CA125. Of six women with a serous carcinoma five were CA125 positive (three only CA125 positive and two CA125 and LPA positive) and three were LPA positive (one LPA only and two LPA and CA125 positive). The median CA125 level was higher in women with a serous carcinoma (71 U/ml) than in those with endometrioid cancer (20 U/ml). Among the other malignancies only one, a woman with a granulosa tumor, was LPA positive and CA125 negative.Table 3 Comparison of women with different ovarian malignancies positive for vaginal lysophosphatidic acid and CA125.

er in women with a serous carcinoma (71 U/ml) than in those with endometrioid cancer (20 U/ml). Among the other malignancies only one, a woman with a granulosa tumor, was LPA positive and CA125 negative.Table 3 Comparison of women with different ovarian malignancies positive for vaginal lysophosphatidic acid and CA125. Table 3Diagnosis No. tested No. (%) positive CA125 only LPA only Both Neither Endometrioid+/− Clear cell 6 1 (16.6) 5 (83.3) 0 0 Serous 6 3 (50.0) 1(16.7) 2(33.3) 0 Mucinous 3 1(33.3) 0 1(33.3) 1(33.3) MMMT 2 2(100) 0 0 0 Granulosa 2 0 1(50.0) 0 1 (50.0) Other 2 0 0 1(50.0) 1 (50.0) Clear cell 1 0 0 0 1(100) Mixed 1 1 (100%) 0 0 0 Endometrioid +/− Clear cell, endometrioid tumor with and without clear cell components; MMMT, malignant mixed mesoderm tumor (carcinosarcoma); Other, metastatic adrenal, leiomyosarcoma.

.3) MMMT 2 2(100) 0 0 0 Granulosa 2 0 1(50.0) 0 1 (50.0) Other 2 0 0 1(50.0) 1 (50.0) Clear cell 1 0 0 0 1(100) Mixed 1 1 (100%) 0 0 0 Endometrioid +/− Clear cell, endometrioid tumor with and without clear cell components; MMMT, malignant mixed mesoderm tumor (carcinosarcoma); Other, metastatic adrenal, leiomyosarcoma. Discussion LPA was detected in vaginal fluid from all women with a malignant adnexal mass as well as in 91% of those whose mass was benign. The vaginal LPA concentration only in women with endometrioid ovarian cancer was significantly higher than the level present in vaginal fluid from women with various benign diagnoses. Furthermore, LPA predicted the detection of an endometrioid ovarian cancer in 83.3% of women with this diagnosis while CA125 was positive in only 16.6% of these women. In contrast, the vaginal LPA assay was inferior to serum CA125 determination for detection of serous ovarian carcinoma. Serum CA125 and vaginal LPA were positive in 83% and 50% of women with this diagnosis, respectively. Among women with a malignant adnexal mass the serum concentration of CA125 was lower in those with an endometrioid ovarian tumor than in those with serous ovarian cancer. This further suggests that CA125 is superior for detection of serous carcinoma than for endmetrioid covarian cancer. The tentative conclusion from these observations is that determination of the vaginal LPA concentration might be a more sensitive measurement than serum CA125 to noninvasively detect the presence of endometrioid ovarian cancer, but not serous carcinoma, in women with a suspicious adnexal mass.

metrioid covarian cancer. The tentative conclusion from these observations is that determination of the vaginal LPA concentration might be a more sensitive measurement than serum CA125 to noninvasively detect the presence of endometrioid ovarian cancer, but not serous carcinoma, in women with a suspicious adnexal mass. Endometrioid ovarian cancer is classified as a type 1 low grade malignancy. It has been estimated to represent about 10% of ovarian cancers and is often associated with prior endometriosis. Prognosis is usually favorable, especially if confined to the ovary [17,18]. Two studies have reported that vaginal levels of CA125 were higher in women with endometrial adenocarcinoma than in women with benign ovarian cysts, endometrial hyperplasia or uterine bleeding [7,8]. In addition, the incidence of endometriod ovarian cancer is lowered in women who have undergone a tubal ligation [6]. These observations suggest the involvement of genital tract components in development of this malignancy. The finding that vaginal LPA is higher in women with this diagnosis is consistent with this suggestion. Elevated levels of LPA in the vagina might be due to its transduction from the systemic circulation and/or by its local production within the genital tract and subsequent migration and collection in the vagina. The association of LPA with only one type of ovarian malignancy, endometrioid cancer, argues against transduction being a major contributor to vaginal LPA levels.

its transduction from the systemic circulation and/or by its local production within the genital tract and subsequent migration and collection in the vagina. The association of LPA with only one type of ovarian malignancy, endometrioid cancer, argues against transduction being a major contributor to vaginal LPA levels. The source(s) of LPA in vaginal fluid remain to be determined. Platelet activation has been shown to be a source of LPA release into the circulation [19]. In addition, transformed epithelial cells from women with gynecologic malignancies produce LPA [10]. Thus, both a pro—inflammatory immune response that stimulates platelet activation and local production by transformed cells may likely contribute to the elevation in vaginal LPA in women with endometrioid ovarian cancer. Limitations of the study must be acknowledged. Due to the low number of subjects analyzed the study must be designated as hypothesis testing and is subject to confirmation. In addition, studies on pre-menopausal women with an adnexal mass, as well as pre- and post-menopausal women without an adnexal mass, also remain to be conducted. The use of more sophisticated measurements of vaginal LPA, for example by mass spectrometry-based methods, are needed to provide additional information on fatty acid chain length and saturation. Advantages of the study are that it was prospective, all procedures and diagnoses were from a single institution and samples for CA125 and for LPA were obtained at the same time.

LPA, for example by mass spectrometry-based methods, are needed to provide additional information on fatty acid chain length and saturation. Advantages of the study are that it was prospective, all procedures and diagnoses were from a single institution and samples for CA125 and for LPA were obtained at the same time. In conclusion, detection of an association between vaginal LPA levels and an ovarian malignancy in this pilot study suggests that analysis of vaginal components as biomarkers for this cancer is biologically plausible and deserves further investigation. The LPA assay may especially be of value for the pre-surgical detection of endometrioid ovarian cancer in post-menopausal women with a suspicious adnexal mass. Further analysis on a larger sample size and among different populations is needed to verify the tentative conclusions and assess the potential clinical utility of vaginal LPA determination. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgement Supported by a grant from The Macy Foundation.

Introduction Respectful and dignified healthcare provision is a fundamental right for every pregnant woman, leading to a positive birth experience delivered by compassionate, skilled providers. The care provided to women in childbirth varies across the globe and in many settings there are examples of non-dignified and sometimes even abusive patterns of care being provided to pregnant women. Reporting on the scale and types of these deficiencies in maternity care is increasing [1,2]. Whilst this behavior is by no means restricted to low socio-economic countries, it is often seen in cultures where empowerment of women and accountability of providers is not the normal standard of care. Recent evidence suggests that exposure to a disrespectful, abusive or coercive service by skilled maternity providers, which results in actual or perceived poor quality of care, is both directly and indirectly associated with both adverse maternal and neonatal outcomes [[3], [4], [5], [6], [7]]. The term ‘Obstetric Violence’ was coined to reflect the ‘professional’ deficiencies in healthcare provision to pregnant women. Obstetric Violence is defined as ‘the appropriation of the body and reproductive processes of women by health personnel, which is expressed as dehumanised treatment, an abuse of medications and to convert the natural processes into pathological ones, bringing with it loss of autonomy and the ability to decide freely about their bodies and sexuality, negatively impacting the quality of life of women [8].

sses of women by health personnel, which is expressed as dehumanised treatment, an abuse of medications and to convert the natural processes into pathological ones, bringing with it loss of autonomy and the ability to decide freely about their bodies and sexuality, negatively impacting the quality of life of women [8]. In parts of Latin America, over the past 10 years, the term “Obstetric Violence’ has become part of the legal framework, with specific laws against obstetric violence being introduced [[9], [10], [11], [12]]. To highlight the problem Amnesty International produced a short video (Available at https://youtu.be/glwP60-g77A) depicting an example of Obstetric Violence in order to raise awareness of the problem seen in many birthing units in Latin America. The video is two minutes and forty seconds in length, presented in Spanish, with English subtitles. The video depicts a woman going into labour, she is on her own without a birthing partner and her confinement is attended, at various points, by two midwives/nurses, a doctor and two medical students. The video is filmed from the patient’s perspective and depicts the woman in distress, asking for help. The medical team are not compassionate or caring and do little to either communicate with the woman or to help her as the labour progresses. They undertake siting of intravenous access, giving of uterotonics and vaginal examination without effective communication or informed consent, resulting in a harrowing, traumatic and lonely birthing experience filmed from the patient’s perspective.

er communicate with the woman or to help her as the labour progresses. They undertake siting of intravenous access, giving of uterotonics and vaginal examination without effective communication or informed consent, resulting in a harrowing, traumatic and lonely birthing experience filmed from the patient’s perspective. Sidra Medicine (Doha, Qatar) opened its private inpatient maternity services In February 2018. As with most healthcare workforces in Qatar, the staff are multinational; with around 80 nationalities represented within the Obstetric unit. The aim of this study was to determine the multinational staff awareness of the term obstetric violence and their opinion of the conduct of the professional caregivers depicted in the Amnesty International video by means of an anonymous questionnaire. Materials and methods The project was reviewed by the Sidra Research Department and as this was a survey of clinicians, formal ethical approval for the study was not required. No funding was received for this study. In July 2018 an online electronic survey was sent to the hospital email accounts of all midwifery, nursing and medical staff within the Obstetrics division of Sidra Medicine (n = 640). The survey was live for a period of four weeks, during which time a weekly reminder was sent to all potential participants by email.

Materials and methods The project was reviewed by the Sidra Research Department and as this was a survey of clinicians, formal ethical approval for the study was not required. No funding was received for this study. In July 2018 an online electronic survey was sent to the hospital email accounts of all midwifery, nursing and medical staff within the Obstetrics division of Sidra Medicine (n = 640). The survey was live for a period of four weeks, during which time a weekly reminder was sent to all potential participants by email. The email explained the purpose of the survey and confirmed that participation was entirely voluntary and the results anonymous. It included a link to the Amnesty International video followed by 21 questions. The first questions assessed the participant’s demographics and asked whether staff had heard of the term obstetric violence and what was meant by the term obstetric violence. The following eight questions assessed the participant’s perceptions on the video they were reviewing. These questions utilised a visual analogue scale and were each scored from 0-100. Higher scores indicate a greater approval/positive response to the question. The final six questions assessed participant’s perceptions on their own practice after reflecting on the content of the video and one of these also utilized the visual analogue scale. Staff were asked to watch the short video and then to complete the questionnaire. Comparisons of the survey responses were made between both doctors and nursing/midwifery staff members.

The email explained the purpose of the survey and confirmed that participation was entirely voluntary and the results anonymous. It included a link to the Amnesty International video followed by 21 questions. The first questions assessed the participant’s demographics and asked whether staff had heard of the term obstetric violence and what was meant by the term obstetric violence. The following eight questions assessed the participant’s perceptions on the video they were reviewing. These questions utilised a visual analogue scale and were each scored from 0-100. Higher scores indicate a greater approval/positive response to the question. The final six questions assessed participant’s perceptions on their own practice after reflecting on the content of the video and one of these also utilized the visual analogue scale. Staff were asked to watch the short video and then to complete the questionnaire. Comparisons of the survey responses were made between both doctors and nursing/midwifery staff members. The results were analysed using SPSS Version 23.0. Comparisons between results for doctors and midwifery/nursing staff were made using Student’s t test or chi square test as appropriate. Results Two hundred and seventeen staff members completed the survey fully after confirming they had watched the short video in its entirety. These included 50 obstetricians and 167 obstetric nursing/midwifery staff representing a response rate of 60% for obstetricians and 30% for nursing and midwifery staff.

The results were analysed using SPSS Version 23.0. Comparisons between results for doctors and midwifery/nursing staff were made using Student’s t test or chi square test as appropriate. Results Two hundred and seventeen staff members completed the survey fully after confirming they had watched the short video in its entirety. These included 50 obstetricians and 167 obstetric nursing/midwifery staff representing a response rate of 60% for obstetricians and 30% for nursing and midwifery staff. Table 1 shows the background demographic data and prior understanding of the term obstetric violence. There was a significant difference in gender between the obstetricians and nursing/midwifery staff with 40% of obstetricians being female compared to 100% of nurses and midwifes. There was also a significant difference in nationality with proportionately more obstetricians originating from the United States of America or Northern Europe, compared to nursing and midwifery staff (56% v 30%). Duration of clinical experience also demonstrated a significant difference between the two groups with 98% of obstetricians having 10 or more years of experience compared to 30% of nurses and midwives.Table 1 Background demographic data and prior understanding of obstetric violence in the survey responders. (chi square * p < 0.05).

Table 1Survey question Obstetricians (n=50) % Obstetric nurse/midwifery staff (n=167) % Please select your gender male 58% 0%* female 42% 100% How many years have you been working in the field of Obstetrics? less than 5 years 0% 37%* 5 to 10 years 4% 34% more than 10 years 96% 29% In what country/region did you complete your primary clinical training? North American/Europe 56% 30%* Other 44% 70% Have you heard of the term Obstetric violence before? Yes 58% 50% No or unsure 42% 50% From the option list, what do you think the term obstetric violence means? correct 60% 44% incorrect 40% 56% Fifty two percent of those who responded had previously heard of the term obstetric violence, and 48% of responders understood what the term obstetric violence meant. There were no significant differences in these responses between the two groups. Table 2 demonstrates the mean scores (+1SD) given by the responders to questions specifically around the professional behaviours seen in the video, the t statistic, 95% confidence interval and p value for each are shown. The t value indicates the size of the variation or difference in responses between the two groups. The p value indicates the statistical significance, with values less than or equal to p < 0.05 being considered statistically significant in this study.Table 2 Comparison between professional groups of visual analogue scale mean scores for questions related to the professional conduct demonstrated in the video and future perceptions on this (* = p < 0.05).

significance, with values less than or equal to p < 0.05 being considered statistically significant in this study.Table 2 Comparison between professional groups of visual analogue scale mean scores for questions related to the professional conduct demonstrated in the video and future perceptions on this (* = p < 0.05). Table 2Survey question Obstetric nursing/midwifery staff (mean + 1SD) Obstetricians (mean + 1SD) t statistic 95% Confidence interval of the t statistic Significance level (* denotes statistical significance: p<0.05) Having watched the video, how well did you feel the Obstetric team worked together? 13.0 + 21.7 17.3 + 21.5 −1.30 −11.4156 to 2.3556 p = 0.1961 How well do you think the Obstetric team did in their manner of communication with the patient, given the clinical circumstances? 8.6 + 20.0 11.2 + 20.4 −0.80 −9.0234 to 3.8034 p = 0.4233 How well do you think the doctor did in giving good quality information to the patient and gaining informed consent? 6.8 + 18.8 7.8 + 15.1 −0.36 −6.7223 to 4.6823 p = 0.7247 How well do you think the team as a whole were mindful of the patient's dignity and privacy made an effort to preserve these as far as possible in the clinical situation? 6.6 + 19.7 7.8 + 17.4 −0.38 −7.2548 to 4.9348 p = 0.7079 How well did the team involve the patient and put the patient at the centre of their efforts to provide care and in planning care? 7.0 + 19.1 8.3 + 15.1 −0.47 −7.1889 to 4.4129 p = 0.6377 In your opinion, how professionally did the doctor behave in this video? 3.7 + 11.2 8.9 + 17.5 −2.55 −9.3738 to -1.1924 p = 0.0116* In your opinion, how well did the medical trainees behave professionally in this scenario? 3.9 + 11.6 7.9 + 18.6 −1.84 −8.3078 to 0.2795 p = 0.0667 In your opinion, how professionally did the midwife/nurses behave professionally in this video? 4.7 + 12.6 11.2 + 17.5 −2.90 −10.8801 to -2.0784 p = 0.0041* How much did watching this video change your perception of how a medical team should act/ behave when caring for a patient? 75.4 + 36.4 29.6 + 35.9 7.71 33.7096 to 56.8636 p < 0.0001*

professionally did the midwife/nurses behave professionally in this video? 4.7 + 12.6 11.2 + 17.5 −2.90 −10.8801 to -2.0784 p = 0.0041* How much did watching this video change your perception of how a medical team should act/ behave when caring for a patient? 75.4 + 36.4 29.6 + 35.9 7.71 33.7096 to 56.8636 p < 0.0001* Significant differences were seen when each clinical group (obstetricians or midwives/obstetric nurses) were asked to report on the behavior of the opposite clinical team members in the video, with the nurses and midwives being more significantly critical of the medical team in the video (p = 0.01) compared to their own professional group, and the medical team also significantly more critical of the nursing and midwifery team in the video (p < 0.0001). Obstetricians completing the survey were also more critical of the doctors in training than the midwifery/nursing staff, however this was not significant. (p = 0.06).

pared to their own professional group, and the medical team also significantly more critical of the nursing and midwifery team in the video (p < 0.0001). Obstetricians completing the survey were also more critical of the doctors in training than the midwifery/nursing staff, however this was not significant. (p = 0.06). The final part of the survey questioned the responder’s personal response to the video in terms of whether the video had highlighted a need for self-reflection and which areas they would reflect, prior obstetric exposure to similar situations in their career and whether they had received training on professional behaviours (Table 3). Both the obstetricians and nursing/midwifery responders identified patient dignity and privacy, and patient centred care as the leading areas of professional deficiencies seen in the video. There was a significant difference (p < 0.001) in how the responders felt the medical team should interact after watching the video. Doctors felt that the video changed their perceptions of how a care team should interact with a patient significantly less than midwifery/nursing staff (mean score on visual analogue scale 30/100 versus 75/100, p < 0.0001). Forty six percent of doctors felt the video highlighted a need for them to reflect on their professional behavior compared to 86% of nursing and midwifery staff. Communication, team working and patient dignity and privacy were selected as areas all responders felt would be areas to reflect on their own practice.Table 3 Impact of the video on self-reflection and prior exposure to obstetric violence scenarios in the workplace. Comparison of means with visual analogue scales using student’s t test.

m working and patient dignity and privacy were selected as areas all responders felt would be areas to reflect on their own practice.Table 3 Impact of the video on self-reflection and prior exposure to obstetric violence scenarios in the workplace. Comparison of means with visual analogue scales using student’s t test. Table 3Survey question Obstetricians (n=50) Obstetric nurse/midwifery staff (n=167) How much did watching this video change your perception of how a medical team should act/ behave when caring for a patient? Mean 29.6 Mean 75.4 (p<0.0001) (Scored 0 to 100 on visual analogue scale) Has viewing this video prompted you to reflect on your own practice and possibly identify areas of change? yes 46% 86% no or unsure 54% 14% If you have been prompted to change, what area/areas are you planning to change most? teamwork 28% 51% communication 26% 70% patient dignity and privacy 23% 58% patient respect 14% 54% patient centred care approach 23% 60% informed consent 12% 49% professionalism 19% 48% During your career, have you seen real life situations scenario depicted in this video? yes 74% 59% no or unsure 26% 41% If you have seen such a scenario take place in real life, how often have you seen it? never witnessed 12% 34% rarely (annually) 57% 42% occasionally (monthly) 29% 20% frequently (weekly) 2% 4% Have you personally received training in professional conduct as part of your clinical training? yes 76% 84% no 24% 16%

26% 41% If you have seen such a scenario take place in real life, how often have you seen it? never witnessed 12% 34% rarely (annually) 57% 42% occasionally (monthly) 29% 20% frequently (weekly) 2% 4% Have you personally received training in professional conduct as part of your clinical training? yes 76% 84% no 24% 16% In terms of prior exposure to episodes of obstetric violence, 136 (63%) staff members said they had witnessed similar situations as depicted in the video at some point through their career (37 obstetricians (74%) versus 99 nursing/midwifery staff (60%)). Twenty five percent of all staff groups had previously witnessed some elements of this kind of behavior as frequently as monthly, despite around 75–80% of all staff groups having received training in professional conduct in maternity care in their previous or current maternity unit. Comment This is the first study to describe a survey regarding ‘obstetric violence’ completed by obstetricians and nursing/midwifery staff working in a high resource setting. The main findings are that only around 50% of doctors, midwives and obstetric nurses in this cohort had previously heard of obstetric violence and were able to accurately define it. This is despite 63% of staff witnessing such behavior previously, in many cases on a frequent basis. Doctors and nursing/midwifery staff were both significantly more critical of the other professional group (p < 0.05) and doctors were significantly less likely to change their perception of how patients in these settings should be cared for as a result of watching the video (p < 0.05)

in many cases on a frequent basis. Doctors and nursing/midwifery staff were both significantly more critical of the other professional group (p < 0.05) and doctors were significantly less likely to change their perception of how patients in these settings should be cared for as a result of watching the video (p < 0.05) The range in incidence of obstetric violence has previously been reported as 15–97% [[13], [14], [15], [16], [17], [18], [19]] with the risk of being exposed to obstetric violence being influenced by socioeconomic and educational status, particularly in societies where empowerment of women is low [19]. Similarly, facilities which are overwhelmed by workforce shortages, inadequate provider training and supervision, where a lack of accountability exists, promotes an environment for unprofessional behaviours to develop and grow; impacting negatively on obstetric outcomes and childbirth complications [3,13,[20], [21], [22]]. Such behaviours have been clearly identified and described in public enquiries into recent healthcare scandals in the United Kingdom [23,24] and it is clear that such behaviors and scenarios are by no means confined to low resource settings. Steps to address obstetric violence must focus on mandatory practical health provider training in how to respect women’s rights and facilitate shared decision making, facilitating improvement in interpersonal skills. Institutional policies on respectful maternity care must also be developed, implemented and enforced.

. Steps to address obstetric violence must focus on mandatory practical health provider training in how to respect women’s rights and facilitate shared decision making, facilitating improvement in interpersonal skills. Institutional policies on respectful maternity care must also be developed, implemented and enforced. The definition of obstetric violence covers a broad range of deficient behaviours which include physical abuse, non-consented care, non-confidential care, non-dignified care, discrimination based on specific patient attributes, abandonment of care and finally patient detention [25]. As would be anticipated non-dignified care from providers with poor negative unfriendly attitudes predominate, with physical abuse and detention least likely to occur [19]. The Amnesty International video demonstrates graphically in just over two minutes the more common elements of obstetric violence reported and how they fail to provide the patient with the respect and dignity she should expect to receive during her confinement. The Women’s unit of Sidra Medicine is staffed by a team of around 600 clinical staff from around 80 Nationalities with different clinical backgrounds and possibly different attitudes to patient care in childbirth. Given the disparity in terms of experience, ethnicity, cultural and training background we aimed to determine, in this small study, whether there was a knowledge gap around professional behaviours on the labour ward and, more importantly, that staff recognised the unacceptability of the type of patient care depicted in the training video.

in terms of experience, ethnicity, cultural and training background we aimed to determine, in this small study, whether there was a knowledge gap around professional behaviours on the labour ward and, more importantly, that staff recognised the unacceptability of the type of patient care depicted in the training video. Around 50% of the cohort in this study were aware of the term ‘obstetric violence’ and around half were able to correctly identify what is meant the term obstetric violence. Reassuringly, it appears that both medical and nursing/midwifery staff groups recognised the unprofessional level of care provided by the obstetric team in the training video with no significant differences observed between the medical and nursing and midwifery scores when evaluating the behaviors of the healthcare team in the video. Where doctors and nursing and midwifery did disagree is in using the video as a tool to reflect on their own professional behaviours. The nursing and midwifery responders were significantly more likely to personally reflect (86% versus 46%) than the doctors. This may simply be due to seniority of the doctors and unfamiliarity with reflective practice, however one cannot help but wonder whether the level of unprofessional behavior ‘hit a raw nerve’ with the nursing staff who already act as advocates for women in their care. This is supported by the fact both clinical groups were significantly more likely to be critical of the other groups behavior in the video (p < 0.05).

er one cannot help but wonder whether the level of unprofessional behavior ‘hit a raw nerve’ with the nursing staff who already act as advocates for women in their care. This is supported by the fact both clinical groups were significantly more likely to be critical of the other groups behavior in the video (p < 0.05). In our cohort, sixty percent of clinical staff claimed to have witnessed some elements of the behaviours depicted in the video during their career, with 25% of responders claiming that they had witnessed it at least as frequently as monthly. Obstetricians were more likely to report having seen obstetric violence (74%) compared nursing and midwifery staff (60%), which may be explained by the doctors having a better understanding of the term ‘obstetric violence’ and being more senior in terms of how long they have been in the specialty. Despite the high levels of reported episodes of obstetric violence the staff in this cohort have witnessed previously, this study demonstrates that both nursing/midwifery and medical staff in this cohort have an awareness of what constitutes unprofessional behavior and an ability to self-reflect on their own standards of care. There is however a need to further improve this understanding and ensure that high professional standards continue to be met through education and multidisciplinary simulation and training [26].

have an awareness of what constitutes unprofessional behavior and an ability to self-reflect on their own standards of care. There is however a need to further improve this understanding and ensure that high professional standards continue to be met through education and multidisciplinary simulation and training [26]. In conclusion, obstetric violence becoming increasingly understood as an important issue to understand and address in maternity care. The results of this survey have demonstrated that the majority of staff in this cohort have witnessed examples of obstetric violence, but were able to identify the negative behaviours in the video they viewed and reflect on how this impacts the care they provide as individuals. Further studies are needed to identify ways in which obstetric violence can be identified and prevented in both low resource and high resource settings. Conflict of interest The authors have no conflicts of interest to declare.

Introduction First trimester combined screening (FTCS) has reported detection rates for trisomy 21 (T21) of 85–90% with a false positive rate of 3–5% [1,2] and its influence in women’s prenatal choices has already been demonstrated [[3], [4], [5]]. Pregnant women with a low risk at FTCS usually decline an invasive test because of the risk of miscarriage that is quoted in 0.1–1% [6,7]. Cell-free fetal DNA (cf-DNA) test in maternal blood for T21, trisomy 18 (T18) and trisomy 13 (T13) offers an alternative option with a high specificity and sensitivity for all pregnant women but especially as a contingent screening model in women at risk for fetal common aneuploidies. T21, T18 and T13 represented approximately 70% of aneuploidies prenatally detected [8,9], but there are other chromosomal abnormalities that must be evaluate before the implementation of the cf-DNA test in order to avoid their undiagnosis [8,[10], [11], [12]]. The aim of this study is to assess the results of the FTCS to design a prenatal protocol in our hospital, with the introduction of the cf-DNA test as a contingent screening model to achieve a better selection of pregnant women at risk for fetal aneuploidies and to avoid the invasive test. We present the analysis of a population of pregnant women with their results at birth and propose a cut-off risk for the introduction of the cf-DNA test for T21, T18 and T13 as well as the inclusion of other criteria for the cf-DNA test.

n of pregnant women at risk for fetal aneuploidies and to avoid the invasive test. We present the analysis of a population of pregnant women with their results at birth and propose a cut-off risk for the introduction of the cf-DNA test for T21, T18 and T13 as well as the inclusion of other criteria for the cf-DNA test. Methods We performed an observational retrospective cohort study, to evaluate the results of the FTCS and the prenatal and postnatal cytogenetic studies, during 2009–2014, on pregnant women who came to our hospital. The inclusion criteria were all singleton pregnancies who were undergone to FTCS and the exclusion criteria those with no FTCS, twin pregnancies or miscarriages.

t study, to evaluate the results of the FTCS and the prenatal and postnatal cytogenetic studies, during 2009–2014, on pregnant women who came to our hospital. The inclusion criteria were all singleton pregnancies who were undergone to FTCS and the exclusion criteria those with no FTCS, twin pregnancies or miscarriages. All pregnant women underwent an ultrasound scan between 10 and 14 weeks’ gestational for measuring crown-rump length and nuchal translucency (NT) following by a biochemical analysis of the pregnancy-associated plasma protein-A (PAPP-A) and β fraction of free human chorionic gonadotrophin at the same day. NT percentile was estimated with the scale of Borrell et al [13]. FTCS was performed and provided a risk assessment for T21 and T18 (SsdwLab V. 5.0. © SBP Software CB. Spain). Pregnancies with a FTCS risk greater than 1/300 for T21 or T18 were considered at risk for fetal aneuploidies. Pregnant women with advanced maternal age (AMA, ≥ 35 years old), abnormal ultrasound, FTCS at risk, family or personal history of aneuploidies or maternal anxiety were referred for prenatal counselling. An invasive prenatal testing by chorionic villus sampling or amniocentesis was offered to them and a rapid test by quantitative fluorescence-polymerase chain reaction (QF-PCR, Devyser Compact v3) for the most common aneuploidies and a karyotype study were performed. Pregnancies with risk at FTCS ≥ 1/50 for T21 or T18 were considered at high risk, with a FTCS risk between 1/51–1/300 for T21 or T18 were considered at intermediate risk and with a FTCS risk <1/300 for T21 and T18 were considered at low risk. In pregnancies with high risk or an abnormal ultrasound scan, subtelomeric and microdeletion syndromes were ruled out by multiplex ligation dependent probe amplification techniques (MLPA, Salsa P036, P070, P245, MRC Holland) or array comparative genomic hybridization (array-CGH, KaryoNIM 60K®prenatal), as long as the results of QF-PCR and karyotypes were normal. Birth outcome data were compiled and postnatal karyotypes were performed if newborn´s physical examination suggested a chromosomal syndrome.

A, Salsa P036, P070, P245, MRC Holland) or array comparative genomic hybridization (array-CGH, KaryoNIM 60K®prenatal), as long as the results of QF-PCR and karyotypes were normal. Birth outcome data were compiled and postnatal karyotypes were performed if newborn´s physical examination suggested a chromosomal syndrome. In order to decide the FTCS risk cut-off in which pregnant women could benefit from cf-DNA test for T21, T18 and T13 as an alternative option to an invasive procedure, we analyzed the prenatal and postnatal unbalanced karyotypes classifying them in three groups: high, intermediate and low risk just for T21, just for T18 and for both trisomies (T21-T18) at FTCS. Other risk cut-offs for T21 and T18 at FTCS have also been evaluated. False negative cases for T21 and NT percentile in our risk groups were evaluated. Other chromosomal abnormalities with relevant clinical significance which could not be detected by cf-DNA test were included into the analysis. This study was approved by the research ethics committee of our hospital. Results During the period 2009–2014 a total of 12,327 singleton pregnancies came to our hospital for fetal screening of common aneuploidies by FTCS. Table 1 shows demographic and clinical data of our population.Table 1 Demographic and clinical data in 12,327 pregnant women.

This study was approved by the research ethics committee of our hospital. Results During the period 2009–2014 a total of 12,327 singleton pregnancies came to our hospital for fetal screening of common aneuploidies by FTCS. Table 1 shows demographic and clinical data of our population.Table 1 Demographic and clinical data in 12,327 pregnant women. Table 1Population data n = 12,327 Risk ≥1/50 n = 147 Risk 1/51–1/300 n = 386 Risk <1/300 n = 11,794 Maternal age 36.3 ± 4.7 36.9 ± 4.4 31.1 ± 5.4 Weight 65.9 ± 12.2 71.6 ± 15.2 62.8 ± 11.1 Size 161.6 ± 6.6 163.3 ± 6.5 162 ± 6.8 Smoker 24 (16.3) 71 (18.4) 1,432 (12.2) Origin Caucasian 108 (73.5) 275 (71.2) 7,587 (64.3) Afro-Caribbean 2 (1.4) 5 (1.3) 516 (4.4) Asian 1 (0.7) 9 (2.3) 410 (3.5) Arabs 4 (2.7) 9 (2.3) 304 (2.6) South-American 30 (20.4) 79 (20.5) 2,769 (23.5) Others 2 (1.4) 9 (2.3) 208 (1.8) Nuchal translucency (mm) 3.5 ± 2 1.5 ± 0.6 1.3 ± 0.3 Crown-rump length (mm) 57.6 ± 8.8 61.6 ± 8.3 60.4 ± 8.1 aFree-βHCG (MoM) 1.4 ± 1.3 1.3 ± 1.2 1.2 ± 0.9 bPAPP-A (MoM) 0.69 ± 0.5 0.63 ± 0.5 1.3 ± 0.7 a Free-βHCG: β fraction of free human chorionic gonadotrophin. b PAPP-A: Pregnancy-associated plasma protein-A. Data are given as frequencies and percentages (%) for qualitative variables and as mean and standard deviation for quantitative variables with symmetric distribution.

Table 1Population data n = 12,327 Risk ≥1/50 n = 147 Risk 1/51–1/300 n = 386 Risk <1/300 n = 11,794 Maternal age 36.3 ± 4.7 36.9 ± 4.4 31.1 ± 5.4 Weight 65.9 ± 12.2 71.6 ± 15.2 62.8 ± 11.1 Size 161.6 ± 6.6 163.3 ± 6.5 162 ± 6.8 Smoker 24 (16.3) 71 (18.4) 1,432 (12.2) Origin Caucasian 108 (73.5) 275 (71.2) 7,587 (64.3) Afro-Caribbean 2 (1.4) 5 (1.3) 516 (4.4) Asian 1 (0.7) 9 (2.3) 410 (3.5) Arabs 4 (2.7) 9 (2.3) 304 (2.6) South-American 30 (20.4) 79 (20.5) 2,769 (23.5) Others 2 (1.4) 9 (2.3) 208 (1.8) Nuchal translucency (mm) 3.5 ± 2 1.5 ± 0.6 1.3 ± 0.3 Crown-rump length (mm) 57.6 ± 8.8 61.6 ± 8.3 60.4 ± 8.1 aFree-βHCG (MoM) 1.4 ± 1.3 1.3 ± 1.2 1.2 ± 0.9 bPAPP-A (MoM) 0.69 ± 0.5 0.63 ± 0.5 1.3 ± 0.7 a Free-βHCG: β fraction of free human chorionic gonadotrophin. b PAPP-A: Pregnancy-associated plasma protein-A. Data are given as frequencies and percentages (%) for qualitative variables and as mean and standard deviation for quantitative variables with symmetric distribution. Fig. 1 shows a schematic distribution of pregnant women. In 954 cases an invasive prenatal test was performed for fetal karyotype, which was normal or balanced in 878 cases (92%). There were 76 unbalanced prenatal karyotypes and 5 unbalanced postnatal karyotypes. The prevalence of chromosomal abnormalities in our cohort was 0.7%.Fig. 1 Distribution of pregnant women referred for prenatal counselling. Fig. 1

Fig. 1 shows a schematic distribution of pregnant women. In 954 cases an invasive prenatal test was performed for fetal karyotype, which was normal or balanced in 878 cases (92%). There were 76 unbalanced prenatal karyotypes and 5 unbalanced postnatal karyotypes. The prevalence of chromosomal abnormalities in our cohort was 0.7%.Fig. 1 Distribution of pregnant women referred for prenatal counselling. Fig. 1 FTCS and unbalanced karyotypes There were 533 (4.3% of 12,327) of all pregnant women with FTCS risk above 1/300. Only 12.9% of these pregnancies with FTCS at risk had an unbalanced karyotype. The results of FTCS and karyotypes studies in our risk groups are shown in Table 2. The detection rate of FTCS for T21 and T18 was 88.5% with a 4% of false positives. The detection rate only for T21 was 83.3% with a 2.3% of false positives. The positive predictive values were 10.1% and 10.9% for both T21-T18 and only for T21, respectively.Table 2 Results of FTCS and karyotype studies performed in pregnant women by risk group during 2009–2014.

was 88.5% with a 4% of false positives. The detection rate only for T21 was 83.3% with a 2.3% of false positives. The positive predictive values were 10.1% and 10.9% for both T21-T18 and only for T21, respectively.Table 2 Results of FTCS and karyotype studies performed in pregnant women by risk group during 2009–2014. Table 2 †FTCS Risk for T21-T18 Risk just for T21 Risk just for T18 Low risk FTCS Risk groups ‡HR-T21 §IR-T21 HR-T21 IR-T21 ¶LR-T21 LR-T21 ††HR-T18 ‡‡IR-T18 HR-T18 IR-T18 §§LR-T18 HR-T18 IR-T18 LR-T18 Total Pregnant women on each risk group 6 20 2 28 92 173 27 185 11,794 12,327 Invasive test 6 20 1 19 81 108 18 103 598 954 Normal or balanced karyotype 0 5 1 18 49 105 12 99 589 878 Unbalanced karyotype 6 15 0 1 33 3 6 5 12 81 Trisomy 21 6 1 a26 2 g7 42 Trisomy 18 6 6 6 e1 19 Trisomy 13 3 1 4 Monosomy X 2 2 Triploidy 1 1 2 Sexual Chromosomal Abnormalities b1 d1 f2 h1 5 Uncommon Chromosomal Abnormalities c3 i4 7 Ratio between unbalanced karyotype and pregnant women on each risk group 1:1 1:1.2 0:2 1:28 1:3 1:58 1:4.5 1:37 1:983 1:152 † FTCS: First Trimester Combined Screening. ‡ HR-T21: High Risk for T21 (1/2-1/50). § IR-T21: Intermediate Risk for T21 (1/51-1/300). ¶ LR-T21: Low Risk for T21(<1/300). †† HR-T18: High Risk for T18 (1/2-1/50). ‡‡ IR-T18: Intermediate Risk for T18 (1/51-1/300). §§ LR-T18: Low Risk for T18(<1/300). a 25 Prenatal and 1 postnatal karyotypes. b 1 mosaic XXY/XY Trisomy. c 1 Mosaic Trisomy 22, 1 Partial Duplication 5q and 1 Partial Trisomy 14q with Partial Monosomy 17q. d XXX Trisomy. e 1 Postnatal Karyotype. f 1 XYY Trisomy and 1 Mosaic Monosomy X.

‡‡ IR-T18: Intermediate Risk for T18 (1/51-1/300). §§ LR-T18: Low Risk for T18(<1/300). a 25 Prenatal and 1 postnatal karyotypes. b 1 mosaic XXY/XY Trisomy. c 1 Mosaic Trisomy 22, 1 Partial Duplication 5q and 1 Partial Trisomy 14q with Partial Monosomy 17q. d XXX Trisomy. e 1 Postnatal Karyotype. f 1 XYY Trisomy and 1 Mosaic Monosomy X. g 4 Prenatal and 3 postnatal karyotype. h XXY Trisomy. i 1 Mosaic Tetraploidy, 1 Mosaic Trisomy 20, 1 Mosaic 13q Deletion and 1 Supernumerary marker chromosome 15. T21, T18 and T13 represented 80.2% of unbalanced karyotypes in our cohort. All T18 and T13 had a FTCS at risk. There were 39 cases of T21 prenatally detected, 35 by a FTCS at risk (R ≥ 1/300), 3 by abnormal ultrasound and 1 by AMA. In the remaining abnormal group, 8.65% of the cases were sex chromosomal aneuploidies (SCAs), 8.65% other uncommon chromosomal abnormalities and 2.5% were triploidy. In pregnancies with high risk (≥1/50) in the FTCS were detected 43% of the uncommon chromosomal abnormalities, and 57% were detected in the low risk group in the FTCS, all of them by karyotype. There were no cases in pregnancies with intermediate risk in the FTCS. All the complementary studies by MLPA or array-CGH in pregnancies with high risk (R ≥ 1/50) in the FTCS or with abnormal ultrasound scan, as long as they had normal QF-PCR and karyotypes, were normal.

T21, T18 and T13 represented 80.2% of unbalanced karyotypes in our cohort. All T18 and T13 had a FTCS at risk. There were 39 cases of T21 prenatally detected, 35 by a FTCS at risk (R ≥ 1/300), 3 by abnormal ultrasound and 1 by AMA. In the remaining abnormal group, 8.65% of the cases were sex chromosomal aneuploidies (SCAs), 8.65% other uncommon chromosomal abnormalities and 2.5% were triploidy. In pregnancies with high risk (≥1/50) in the FTCS were detected 43% of the uncommon chromosomal abnormalities, and 57% were detected in the low risk group in the FTCS, all of them by karyotype. There were no cases in pregnancies with intermediate risk in the FTCS. All the complementary studies by MLPA or array-CGH in pregnancies with high risk (R ≥ 1/50) in the FTCS or with abnormal ultrasound scan, as long as they had normal QF-PCR and karyotypes, were normal. FTCS risk cut-offs The Table 3 shows the distribution of several risk cut-offs at FTCS according to T21, T18 and others unbalanced karyotypes. Of the 81 unbalanced chromosomal abnormalities, 60% (49/81) were detected when the risk was greater than 1/10 and 74% (60/81) when it was greater than 1/50. The 97.7% of the pregnancies in the intermediate risk group (1/51-1/300) had a normal outcome. In this intermediate risk group there were 9 unbalanced karyotypes (9/386): three T21, one T18, one T13, one triploidy and three SCAs cases. In the low risk group (<1/300) there were 25% (5/20) of the chromosomal abnormalities others than T21 and T18. These 5 unbalanced karyotypes are shown in Table 2, and only the partial deletion of 13q chromosome mosaicism detected by a Dandy-Walker malformation on second trimester ultrasound had clinical relevance.Table 3 Distribution of the FTCS risk cut-offs according to T21, T18 and others unbalanced karyotypes.

T21 and T18. These 5 unbalanced karyotypes are shown in Table 2, and only the partial deletion of 13q chromosome mosaicism detected by a Dandy-Walker malformation on second trimester ultrasound had clinical relevance.Table 3 Distribution of the FTCS risk cut-offs according to T21, T18 and others unbalanced karyotypes. Table 3Risk cutt offs at †FTCS Trisomy 21 cases (%) n = 42 Trisomy 18 cases (%) n = 19 Others unbalanced karyotypes cases (%) n = 20 Unbalanced / total cases R ≥ 1/10 27 (64.3) 13 (68.4) 9 (45) 49/86 R ≥ 1/50 32 (76.2) 18 (94.7) 10 (50) 60/147 R 1/11-1/50 5 (11.9) 5 (26.3) 1 (5) 11/150 R 1/11-1/300 8 (19) 6 (31.6) 6 (30) 20/447 R 1/51-1/300 3 (7.1) 1 (5.3) 5 (25) 9/386 R 1/301-1/1000 2 (4.8) _ 2 (10) 4/1048 R 1/1001-1/2000 3 (7.1) _ 1 (5) 4/1215 R 1/2001-1/3000 1 (2.4) _ _ 1/926 R 1/3001-1/6000 1 (2.4) _ 1 (5) 2/2130 R < 1/6001 _ _ 1 (5) 1/6475 † FTCS: First Trimester Combined Screening. When there were high or intermediate risks for both T21-T18, the highest risk of the two was considered to stratify the results of the karyotypes. Dates are given as frequencies and percentages. False negative cases for T21 The seven false negatives cases of the FTCS for T21 are shown in Table 4. Two of them (numbers 1 and 5) had a FTCS risk between 1/301-1/1000 and both had a NT in 97.5 percentile (p97.5th).Table 4 False negatives cases at FTCS for T21.

Table 3Risk cutt offs at †FTCS Trisomy 21 cases (%) n = 42 Trisomy 18 cases (%) n = 19 Others unbalanced karyotypes cases (%) n = 20 Unbalanced / total cases R ≥ 1/10 27 (64.3) 13 (68.4) 9 (45) 49/86 R ≥ 1/50 32 (76.2) 18 (94.7) 10 (50) 60/147 R 1/11-1/50 5 (11.9) 5 (26.3) 1 (5) 11/150 R 1/11-1/300 8 (19) 6 (31.6) 6 (30) 20/447 R 1/51-1/300 3 (7.1) 1 (5.3) 5 (25) 9/386 R 1/301-1/1000 2 (4.8) _ 2 (10) 4/1048 R 1/1001-1/2000 3 (7.1) _ 1 (5) 4/1215 R 1/2001-1/3000 1 (2.4) _ _ 1/926 R 1/3001-1/6000 1 (2.4) _ 1 (5) 2/2130 R < 1/6001 _ _ 1 (5) 1/6475 † FTCS: First Trimester Combined Screening. When there were high or intermediate risks for both T21-T18, the highest risk of the two was considered to stratify the results of the karyotypes. Dates are given as frequencies and percentages. False negative cases for T21 The seven false negatives cases of the FTCS for T21 are shown in Table 4. Two of them (numbers 1 and 5) had a FTCS risk between 1/301-1/1000 and both had a NT in 97.5 percentile (p97.5th).Table 4 False negatives cases at FTCS for T21. Table 4Case number Trisomy 21 risk at ¶FTCS Maternal age at term †Nuchal Translucency mm (Percentile) Indication for prenatal counselling 1 1/445 28 3.1 (p97.5th) NT>3 mm 2 1/1167 39 1.5 (p5th) §FHD 3 1/1273 28 1.8 (p75th) §FHD 4 1/1626 38 1 (p10th) ‡AMA 5 1/882 30 2.9 (p97.5th) 6 1/2687 24 1.9 (p75th) 7 1/5116 30 1 (p10th) ¶ FTCS: First trimester combined screening. † NT millimeters (percentile) depending on CRL [13]. § FHD: Fetal heart defect. ‡ AMA: Advanced maternal age.

Table 4Case number Trisomy 21 risk at ¶FTCS Maternal age at term †Nuchal Translucency mm (Percentile) Indication for prenatal counselling 1 1/445 28 3.1 (p97.5th) NT>3 mm 2 1/1167 39 1.5 (p5th) §FHD 3 1/1273 28 1.8 (p75th) §FHD 4 1/1626 38 1 (p10th) ‡AMA 5 1/882 30 2.9 (p97.5th) 6 1/2687 24 1.9 (p75th) 7 1/5116 30 1 (p10th) ¶ FTCS: First trimester combined screening. † NT millimeters (percentile) depending on CRL [13]. § FHD: Fetal heart defect. ‡ AMA: Advanced maternal age. NT and unbalanced karyotypes There were a total amount of 38 cases with NT higher than p97.5th and low risk for T21 at FTCS (21 cases, Group A + 17 cases, Group B, Table 5), and 33 of these pregnant women were under 35 years old. There were 35 cases with a NT between p97.5th-p99th and only T21 cases have been detected. The two false negatives of the FTCS for T21 were in this group (Group A, Table 5). Of the 142 fetuses with a NT ≥ p99th (Group B, Table 5), 17 cases presented low risk for T21 and normal outcome.Table 5 Karyotype and Nuchal Traslucency ≥97.5th percentile [13]. Table 5 Group A: p97.5th≤ †NT < p99th (35 cases) Group B: NT ≥ p99th (142 cases) ‡FTCS §HR-T21 ¶IR-T21 ††LR-T21 HR-T21 IR-T21 LR-T21 Age (years) < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 Total Trisomy 21 – 1 – – a2 – 8 21 1 – – – 33 Other Unbalanced Fetal Karyotype – – – – – – 4 17 – – – – 21 Normal Karyotype – – 5 8 14 5 27 25 12 10 b17 – 123 Total 1 13 21 102 23 17 177 † NT: Nuchal translucency. ‡ FTCS: Combined First Trimester Screening test. § HR-T21: High Risk for T21 (1/2-1/50). ¶ IR-T21: Intermediate Risk for T21 (1/51-1/300).

Table 5 Group A: p97.5th≤ †NT < p99th (35 cases) Group B: NT ≥ p99th (142 cases) ‡FTCS §HR-T21 ¶IR-T21 ††LR-T21 HR-T21 IR-T21 LR-T21 Age (years) < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 < 35 ≥ 35 Total Trisomy 21 – 1 – – a2 – 8 21 1 – – – 33 Other Unbalanced Fetal Karyotype – – – – – – 4 17 – – – – 21 Normal Karyotype – – 5 8 14 5 27 25 12 10 b17 – 123 Total 1 13 21 102 23 17 177 † NT: Nuchal translucency. ‡ FTCS: Combined First Trimester Screening test. § HR-T21: High Risk for T21 (1/2-1/50). ¶ IR-T21: Intermediate Risk for T21 (1/51-1/300). †† LR-T21: Low Risk for T21 (<1/300). a false negatives cases for T21. b 14 cases with NT < 3,5 mm. cf-DNA as contingent screening model The introduction of the cf-DNA test for T21, T18 and T13 will be recommended in our hospital as first choice in pregnant women with an intermediate risk at FTCS (1/51-1/300) and in those with a NT ≥ p97.5th and low risk at FTCS (<1/300). An invasive test will be recommended in pregnant women with a risk at combined test ≥1/50.

oduction of the cf-DNA test for T21, T18 and T13 will be recommended in our hospital as first choice in pregnant women with an intermediate risk at FTCS (1/51-1/300) and in those with a NT ≥ p97.5th and low risk at FTCS (<1/300). An invasive test will be recommended in pregnant women with a risk at combined test ≥1/50. Discussion In our series, 74% (60/81) of chromosomal unbalanced abnormalities with serious effect on fetus phenotype, including uncommon aneuploidies, had a high risk (R ≥ 1/50) at the FTCS. At this risk cut-off we can detect the highest percentage of T21 (32/42) and T18 (18/19) cases as well as 50% (10/20) of others unbalanced karyotypes: monosomy X, T13, triploidy and the uncommon chromosomal abnormalities associated with adverse outcome. Our results are according with previous reports [11,14]. Also, it seems that the ratio between unbalanced karyotype and pregnancies on each risk groups support the recommendation of an invasive test (between 1/1 and 1/4.5, Table 2). We have considered as intermediate risk the pregnancies which have a FTCS between 1/51-1/300 because unbalanced karyotypes represent only 2.3% (9/386) of the cases. In this intermediate risk group the ratio between chromosomal abnormalities and pregnancies on each risk groups suggests a cf-DNA test (between 1/28 and 1/58, Table 2). Other authors raise the possibility of offering cf-DNA test at a risk cut-off of 1/11 to 1/500 or 1/1000 [15]. In our series there are 11.9% of T21 and 26.3% of T18 between the risk cut-off 1/11-1/50 which makes us consider an invasive test in this group.

groups suggests a cf-DNA test (between 1/28 and 1/58, Table 2). Other authors raise the possibility of offering cf-DNA test at a risk cut-off of 1/11 to 1/500 or 1/1000 [15]. In our series there are 11.9% of T21 and 26.3% of T18 between the risk cut-off 1/11-1/50 which makes us consider an invasive test in this group. There are reports which analyze the distribution of risk from FTCS according to T21, T18, T13 and others chromosomal abnormalities outcome. The authors propose different policies for the introduction of cf-DNA test. With a cut-off risk at 1/1000, their detection rate for T21 was 98% [15,16]. In our series the detection rate at this cut-off would be 88% but between 1/301–1000 there were 1048 pregnant women and only two cases of T21. So this option would increase the cost of cf-DNA test implementation. Besides we could detect this two T21 cases through the NT percentile because these two cases had a NT percentile between p97.5th–p99th and low risk at FTCS in a <35 years old women. The scope of maternal age on the calculation of risks in FTCS is probably modifying the risk despite the NT measurement. We could detect more T21 cases if we offered a cf-DNA test to all pregnancies with NT ≥ p97.5th when FTCS risk is low and it suppose a small increase in cost because there are 21 pregnant women that meet these criteria (Group A, Table 5).

ation of risks in FTCS is probably modifying the risk despite the NT measurement. We could detect more T21 cases if we offered a cf-DNA test to all pregnancies with NT ≥ p97.5th when FTCS risk is low and it suppose a small increase in cost because there are 21 pregnant women that meet these criteria (Group A, Table 5). An increased NT (p99th or ≥3.5 mm) is associated with a high risk of chromosomal abnormalities [11], being monosomy X, T21 and T18 cases the more frequent unbalanced karyotypes found in these cases [14,17,18]. NT in p99th is an indication for invasive test but we think that it is necessary to get the result of FTCS in these pregnancies because it provides information that can help women to make a decision about all their options, mainly when the NT is <3.5 mm. In our series, there was only one abnormal karyotype (T21) with intermediate risk for T21 and none with low risk for T21 despite of the NT in p99th. Although other authors report different percentages of uncommon chromosomal abnormalities in pregnancies with a NT in p99th depending on the technique used for its detection [19,20] in our series all the complementary molecular studies were normal and had a normal outcome. So we could offer cf-DNA test to all pregnancies with NT in ≥ p99th and low risk for T21.

centages of uncommon chromosomal abnormalities in pregnancies with a NT in p99th depending on the technique used for its detection [19,20] in our series all the complementary molecular studies were normal and had a normal outcome. So we could offer cf-DNA test to all pregnancies with NT in ≥ p99th and low risk for T21. Evidence data about the performance of cf-DNA test in a general population show a positive predictive value range for T21 from 45.5% [21] to 94.4% [22] and show high sensitivity and specificity in low risk population [23]. Because the positive predictive value of FTCS is less than in cf-DNA test and the FTCS false negative cases for T21 are more frequent in women under the age of 35, it would be necessary to perform the cf-DNA test into general obstetric population [24]. Currently this is not possible in our public health service due to the cost of the test.

predictive value of FTCS is less than in cf-DNA test and the FTCS false negative cases for T21 are more frequent in women under the age of 35, it would be necessary to perform the cf-DNA test into general obstetric population [24]. Currently this is not possible in our public health service due to the cost of the test. There are several studies that have shown that contingent model can be cost-effective because it implies a reduction in the rate of the invasive tests and a lower procedure-related loss rate [25,26]. Although the cost effectiveness studies should be done with caution due to the large number of topics that must be evaluated, in our cohort we have 7 false negatives cases for T21 that would mean an additional cost between 594,000–790,000 € for the health and social care per case [27,28], and therefore that would imply an additional cost between 337–448 € for each pregnant woman. With this theoretical saving we could introduce the cf-DNA to all pregnant women, but maintaining the FTCS that allows us to have additional information on PAPP-A levels to assess the risk of preeclampsia or preterm delivery. Besides, both FTCS and TN allow us to identify other chromosomal abnormalities other than T21 which are not estimated by the cf-DNA. In addition, cf-DNA also has false negative and positive results, although there are very few. Detecting pathological cases does not always imply cost reduction since we ought always to respect the autonomy of the pregnant women. In the near future a more accurate study about economic feasibility should be done.

f-DNA. In addition, cf-DNA also has false negative and positive results, although there are very few. Detecting pathological cases does not always imply cost reduction since we ought always to respect the autonomy of the pregnant women. In the near future a more accurate study about economic feasibility should be done. Available data indicate that cf-DNA test has less accuracy for SCAs than for T21 and T18, mainly due to a confined placental or maternal mosaicism that can increase the false positive rates [[29], [30], [31]]. We assume that we will not diagnose SCAs different than non mosaic monosomy X if we do not analyze the fetal sex by cf-DNA test. These SCAs have a mild phenotype and their diagnosis is usually fortuitous when invasive test are performed by AMA or FTCS at risk to rule out a T21 fetus [32]. In addition, X chromosome aneuploidies have been associated to AMA and these cases probably remain undiagnosed at this moment because in our cohort, 88% (3,022/3,423) of pregnant women with AMA and low risk at FTCS declined an invasive test. It is necessary to remind the important role of ultrasound scan screening that could detect fetus at risk for any chromosomal abnormalities, including uncommon abnormalities, triploidies and false negative cases of cf-DNA test or FTCS. In our series, three of the FTCS false negatives, one triploidy and a partial deletion of 13q chromosome mosaicism were detected by an abnormal ultrasound scan and they could be missed by cf-DNA test or FTCS.

l abnormalities, including uncommon abnormalities, triploidies and false negative cases of cf-DNA test or FTCS. In our series, three of the FTCS false negatives, one triploidy and a partial deletion of 13q chromosome mosaicism were detected by an abnormal ultrasound scan and they could be missed by cf-DNA test or FTCS. Regardless of our recommendations, all pregnant women must be informed about the chromosomal abnormalities that couldn´t be detected by cf-DNA test or by second trimester ultrasound as well as about all prenatal diagnosis options. One of the limitations of this study is that the cohort belongs only to our center so this proposed policy cannot be applied to another population. Another limitation could be the lack of MLPA or array-CGH results that detect unusual cryptic anomalies in all invasive tests of pregnant women at intermediate risk and that could have a future clinical impact on newborn, although the data at birth were normal. The low prevalence of uncommon chromosome abnormalities could affect the validation of our new policy.

or array-CGH results that detect unusual cryptic anomalies in all invasive tests of pregnant women at intermediate risk and that could have a future clinical impact on newborn, although the data at birth were normal. The low prevalence of uncommon chromosome abnormalities could affect the validation of our new policy. Here we present a contingent screening model for the introduction of cf-DNA test in our hospital in two pregnancies group: i) those with an intermediate risk at FTCS and ii) those with a low risk at FTCS and NT ≥ p97.5th. We consider that those two assumptions could allow us to detect the most common aneuploidies, and to decrease the number of invasive test and consequently, the fetal losses. Our data suggest that the others uncommon chromosomal abnormalities with severe effect in the fetal phenotype would not be underdiagnosed since all of them had a high risk at FTCS or an abnormal ultrasound scan in which it was recommended to perform an invasive test. The new proposed policy has been implemented in our hospital since October 2015 and future analyses of those data with the previous policy will allow us to verify and to improve our prenatal protocol. Contribution to authorship Carmen Cotarelo-Pérez, Raluca Oancea-Ionescu and María Fenollar-Cortés contributed equally to this work. Disclosure statement The authors declare no conflict of interest. Funding sources None. Acknowledgements We appreciate the collaboration for the analysis of the data to predoctoral student Miss Irene Serrano García, Mathematics of the Research Unit, Department of Epidemiology.

Introduction Preeclampsia is characterized by maternal hypertension and proteinuria, and is a leading cause of maternal mortality worldwide [1]. Preeclampsia is also associated with complications such as fetal growth restriction and fetal death [2,3]. In pregnancies with preeclampsia, the prevalence of fetal growth restriction is increased [4,5], and it is well known that fetal growth restriction is associated with increased risk of fetal death [6,7]. Thus, if fetal growth restriction is diagnosed in a preeclamptic pregnancy, the offspring may be delivered to prevent intrauterine death. Most infants born to preeclamptic mothers have birthweight appropriate for gestational age, and birthweight may also be increased in pregnancies with preeclampsia [8,9]. Reliable knowledge about the risk of fetal death in normal, and in large for gestational age offspring in pregnancies with preeclampsia is important for clinical decisions about intervention in such pregnancies. During the last decades, the fetal death rate has declined significantly in the Western world [10], and the decline has been more prominent in preeclamptic as compared to non-preeclamptic pregnancies [11,12]. It is not known whether the decline in fetal death rate in preeclamptic pregnancies has differed by offspring birthweight. Therefore, we studied the temporal changes in fetal death risk in pregnancies with and in pregnancies without preeclampsia within categories of offspring birthweight. We included all singleton pregnancies in Norway during the years 1967–2014.

During the last decades, the fetal death rate has declined significantly in the Western world [10], and the decline has been more prominent in preeclamptic as compared to non-preeclamptic pregnancies [11,12]. It is not known whether the decline in fetal death rate in preeclamptic pregnancies has differed by offspring birthweight. Therefore, we studied the temporal changes in fetal death risk in pregnancies with and in pregnancies without preeclampsia within categories of offspring birthweight. We included all singleton pregnancies in Norway during the years 1967–2014. Materials and methods We used data from the Medical Birth Registry of Norway. This registry includes information about all births in Norway from 16+0 weeks of gestation and beyond since 1967 [13]. The reporting of births to the Medical Birth Registry is compulsory by law and is performed by the midwife or the doctor attending the delivery. Study population We aimed at including all singleton pregnancies in Norway during the years 1967–2014 (n = 2 747 844) (Fig. 1). We excluded pregnancies with missing information about gestational age of the offspring at birth and pregnancies with gestational age at birth less than 23+0 weeks. We also excluded pregnancies with missing offspring birthweight, offspring sex or maternal age. Of the remaining, we excluded pregnancies with outlying values on gestational age at birth (≥46+0 weeks) or offspring birthweight (<250 g or >6500 g). A total of 2 607 199 pregnancies could be included in our data analyses.Fig. 1 Flow chart of the study sample. Fig. 1

Study population We aimed at including all singleton pregnancies in Norway during the years 1967–2014 (n = 2 747 844) (Fig. 1). We excluded pregnancies with missing information about gestational age of the offspring at birth and pregnancies with gestational age at birth less than 23+0 weeks. We also excluded pregnancies with missing offspring birthweight, offspring sex or maternal age. Of the remaining, we excluded pregnancies with outlying values on gestational age at birth (≥46+0 weeks) or offspring birthweight (<250 g or >6500 g). A total of 2 607 199 pregnancies could be included in our data analyses.Fig. 1 Flow chart of the study sample. Fig. 1 Study factors Our main outcome measure was fetal death (yes/no). Fetal death was defined as no sign of life at birth in offspring born in pregnancy week 23+0 or beyond. A total of 65.5% of the fetal deaths were reported to have occurred antepartum, 11.4% intrapartum, and for 23.1% the time of death was not reported. Induced abortions are not performed in pregnancy week 23+0 or beyond, according to the Norwegian Act on Induced Abortion (https://lovdata.no/dokument/NL/lov/1975-06-13-50). Preeclampsia (yes/no) included pregnancies with preeclampsia and/or eclampsia. Preeclampsia was defined as blood pressure ≥140/90 mmHg and proteinuria with dip-stick ≥1, whereas eclampsia was defined as preeclampsia with maternal seizures. This definition of preeclampsia has been used in Norway throughout our study period.

clampsia (yes/no) included pregnancies with preeclampsia and/or eclampsia. Preeclampsia was defined as blood pressure ≥140/90 mmHg and proteinuria with dip-stick ≥1, whereas eclampsia was defined as preeclampsia with maternal seizures. This definition of preeclampsia has been used in Norway throughout our study period. Birthweight was reported in grams. Since offspring birthweight varies by gestational age, and gestational age at birth may vary by maternal preeclampsia status, we made adjustment for gestational age at birth by using birthweight z-scores in our data analyses [14]. We calculated z-scores by using means and standard deviations of birthweight by gestational week in the study sample as a whole [15]. Z-scores were calculated separately for male and female offspring. The distribution of z-scores was grouped into percentiles as follows: <1, 1–2.5, 2.5–10, 10–90, 90–97.5, 97.5–99 and >99 percentile of birthweight. During the years 1967–1999, gestational age at birth was calculated from the date of the last menstrual period. After 1999, the gestational age at birth estimate was based on fetal size at routine fetal ultrasonographic examination in pregnancy week 17-19. Such examination was performed for 97.3% of all pregnancies after 1999.

he years 1967–1999, gestational age at birth was calculated from the date of the last menstrual period. After 1999, the gestational age at birth estimate was based on fetal size at routine fetal ultrasonographic examination in pregnancy week 17-19. Such examination was performed for 97.3% of all pregnancies after 1999. Statistical analyses We calculated the absolute risk of fetal death (in percent) in pregnancies with and in pregnancies without preeclampsia. The absolute risk was calculated for the study sample as a whole, and within categories of offspring birthweight (categories described above). The relative risk of fetal death in pregnancies with preeclampsia compared to pregnancies without preeclampsia was estimated as crude and adjusted odds ratios (OR) with 95% confidence intervals (CI) in the sample as a whole, and within categories of offspring birthweight. Non-preeclamptic pregnancies were used as the reference group within each birthweight category. We made adjustments for maternal age (in years at the time of delivery), parity (previous deliveries in pregnancy week 16+0 or beyond, coded 0 or ≥1) and maternal diabetes (type 1 or type 2 diabetes mellitus, gestational diabetes, or use of anti-diabetic medication during pregnancy, coded yes or no).

thweight category. We made adjustments for maternal age (in years at the time of delivery), parity (previous deliveries in pregnancy week 16+0 or beyond, coded 0 or ≥1) and maternal diabetes (type 1 or type 2 diabetes mellitus, gestational diabetes, or use of anti-diabetic medication during pregnancy, coded yes or no). We repeated the above analyses for births during the years: 1967–1983 and 1984–2014, and in supplementary analyses, we further sub-grouped the year of birth into: 1967–1973, 1974–1983, 1984–1993, 1994–2003 and 2004-2014. To test for consistency of our findings, we studied pregnancies with delivery in pregnancy week 28+0 or beyond separately. In these analyses, only fetal deaths that were reported to have occurred antepartum were used as outcome. All statistical analyses were conducted by using IBM SPSS Statistics for Windows, Version 21.0 (Armonk, NY, USA). Ethical approval Use of the Medical Birth Registry of Norway for research is approved by the Norwegian Data Inspectorate. The advisory committee for the Medical Birth Registry has recommended this study (Reference number 07/944/236).

All statistical analyses were conducted by using IBM SPSS Statistics for Windows, Version 21.0 (Armonk, NY, USA). Ethical approval Use of the Medical Birth Registry of Norway for research is approved by the Norwegian Data Inspectorate. The advisory committee for the Medical Birth Registry has recommended this study (Reference number 07/944/236). Results A total of 16 105 fetal deaths occurred during the years 1967–2014, representing 0.6% of all births from 23+0 weeks of gestation and beyond (Table 1). Preeclampsia occurred in 76 066 pregnancies (2.9% of all pregnancies). Mean birthweight was 3154.2 g in pregnancies with preeclampsia, and 3537.7 g in pregnancies without preeclampsia (Student’s t-test, P <  0.001). Preeclamptic pregnancies were on average 9.8 days shorter than non-preeclamptic pregnancies (Student’s t-test, P <  0.001).Table 1 Characteristics of the study sample, 2 607 199 singleton pregnancies in Norway during the period 1967–2014. Table 1 Preeclampsia Fetal death Characteristics Yes No Yes No Total Maternal age (years, mean) 27.7 27.8 27.8 27.8 27.8 Number of previous deliveries (mean) 0.6 0.9 1.1 0.9 0.9 Gestational age of offspring at birth (days, mean) 270.8 280.6 240.1 280.5 280.3 Offspring birthweight (grams, mean) 3154.2 3537.7 2040.4 3535.7 3526.5 Offspring birthweight z-score (mean) −0.262 0.008 −0.794 0.005 0.000 Total number of pregnancies (% of total) 76 066 (2.9) 2 531 133 (97.1) 16 105 (0.6) 2 591 094 (99.4) 2 607 199 (100)

f offspring at birth (days, mean) 270.8 280.6 240.1 280.5 280.3 Offspring birthweight (grams, mean) 3154.2 3537.7 2040.4 3535.7 3526.5 Offspring birthweight z-score (mean) −0.262 0.008 −0.794 0.005 0.000 Total number of pregnancies (% of total) 76 066 (2.9) 2 531 133 (97.1) 16 105 (0.6) 2 591 094 (99.4) 2 607 199 (100) During the study period as a whole, the absolute risk of fetal death was higher in preeclamptic compared to non-preeclamptic pregnancies (1.6% vs 0.6%) (Table 2). The crude OR for fetal death associated with preeclampsia was 2.73 (95% CI 2.57–2.89).Table 2 Risk of fetal death according to presence of preeclampsia within percentiles of birthweight. Risks are presented as absolute risks (percent) and crude and adjusted odds ratios with 95% confidence intervals. Non-preeclamptic pregnancies represent the reference group. Results are presented for all singleton pregnancies in Norway during the total study period 1967–2014, during the years 1967-83 and during the years 1984–2014.

sented as absolute risks (percent) and crude and adjusted odds ratios with 95% confidence intervals. Non-preeclamptic pregnancies represent the reference group. Results are presented for all singleton pregnancies in Norway during the total study period 1967–2014, during the years 1967-83 and during the years 1984–2014. Table 2 Preeclampsia Yes No Birthweight percentiles Total (n) Fetal death % (n) Live birth % (n) Fetal death % (n) Live birth % (n) cOR (95% CI) aOR* (95% CI) During the total study period, 1967–2014 <1 26 085 7.0 (182) 93.0 (2425) 6.3 (1472) 93.7 (22 006) 1.12 (0.96-1.32) 1.13 (0.96-1.32) 1–2.5 39 280 4.0 (105) 96.0 (2547) 1.9 (679) 98.1 (135 949) 2.18 (1.77-2.69) 2.22 (1.80-2.75) 2.5–10 194 965 2.5 (230) 97.5 (8959) 1.1 (2063) 98.9 (183 713) 2.29 (1.99-2.62) 2.27 (1.97-2.61) 10–90 2 086 067 1.2 (658) 98.8 (53 641) 0.5 (10 127) 99.5 (2 021 641) 2.45 (2.26-2.65) 2.40 (2.22-2.60) 90–97.5 195 468 0.3 (13) 99.7 (4974) 0.2 (347) 99.8 (190 134) 1.43 (0.82-2.49) 1.15 (0.66-2.02) 97.5–99 39 277 0.6 (8) 99.4 (1266) 0.2 (91) 99.8 (37 912) 2.63 (1.28-5.44) 2.13 (1.01-4.47) >99 26 057 1.2 (13) 98.8 (1045) 0.5 (117) 99.5 (24 882) 2.65 (1.49-4.71) 2.05 (1.13-3.70) Total 2 607 199 1.6 (1209) 98.4 (74 857) 0.6 (14 896) 99.4 (2 516 237) 2.73 (2.57-2.89) 2.63 (2.48-2.79)

1.43 (0.82-2.49) 1.15 (0.66-2.02) 97.5–99 39 277 0.6 (8) 99.4 (1266) 0.2 (91) 99.8 (37 912) 2.63 (1.28-5.44) 2.13 (1.01-4.47) >99 26 057 1.2 (13) 98.8 (1045) 0.5 (117) 99.5 (24 882) 2.65 (1.49-4.71) 2.05 (1.13-3.70) Total 2 607 199 1.6 (1209) 98.4 (74 857) 0.6 (14 896) 99.4 (2 516 237) 2.73 (2.57-2.89) 2.63 (2.48-2.79) During 1967–1983 <1 13 739 12.5 (172) 87.5 (1199) 8.7 (1071) 91.3 (11 297) 1.51 (1.27-1.80) 1.50 (1.26-1.79) 1–2.5 17 623 8.7 (90) 91.3 (950) 2.6 (434) 97.4 (16 149) 3.53 (2.78-4.47) 3.45 (2.71-4.39) 2.5–10 81 466 6.6 (187) 93.4 (2653) 1.5 (1171) 98.5 (77 455) 4.66 (3.98-5.47) 4.28 (3.64-5.03) 10–90 733 127 2.8 (383) 97.2 (13 200) 0.7 (5150) 99.3 (714 394) 4.03 (3.62-4.47) 3.74 (3.36-4.16) 90–97.5 61 053 0.4 (5) 99.6 (1242) 0.3 (173) 99.7 (59 633) 1.39 (0.57-3.38) 1.15 (0.47-2.82) 97.5–99 11 812 1.3 (4) 98.7 (306) 0.4 (46) 99.6 (11 456) 3.26 (1.17-9.10) 2.65 (0.92-7.63) >99 7 653 3.2 (8) 96.8 (239) 0.7 (50) 99.3 (7356) 4.93 (2.31-10.50) 4.31 (1.98-9.39) Total 926 473 4.1 (849) 95.9 (19 789) 0.9 (8095) 99.1 (897 740) 4.76 (4.43-5.11) 4.34 (4.04-4.67)

9.7 (59 633) 1.39 (0.57-3.38) 1.15 (0.47-2.82) 97.5–99 11 812 1.3 (4) 98.7 (306) 0.4 (46) 99.6 (11 456) 3.26 (1.17-9.10) 2.65 (0.92-7.63) >99 7 653 3.2 (8) 96.8 (239) 0.7 (50) 99.3 (7356) 4.93 (2.31-10.50) 4.31 (1.98-9.39) Total 926 473 4.1 (849) 95.9 (19 789) 0.9 (8095) 99.1 (897 740) 4.76 (4.43-5.11) 4.34 (4.04-4.67) During 1984–2014 <1 12 346 0.8 (10) 99.2 (1226) 3.6 (401) 96.4 (10 709) 0.22 (0.12-0.41) 0.22 (0.12-0.41) 1–2.5 21 657 0.9 (15) 99.1 (1597) 1.2 (245) 98.8 (19 800) 0.76 (0.45-1.28) 0.76 (0.45-1.28) 2.5–10 113 499 0.7 (43) 99.3 (6306) 0.8 (892) 99.2 (106 258) 0.81 (0.60-1.10) 0.81 (0.60-1.11) 10–90 1 352 940 0.7 (275) 99.3 (40 441) 0.4 (4977) 99.6 (1 307 247) 1.79 (1.58-2.02) 1.74 (1.54-1.97) 90–97.5 134 415 0.2 (8) 99.8 (3732) 0.1 (174) 99.9 (130 501) 1.61 (0.79-3.27) 1.28 (0.62-2.63) 97.5–99 27 465 0.4 (4) 99.6 (960) 0.2 (45) 99.8 (26 456) 2.45 (0.88-6.83) 1.92 (0.67-5.48) >99 18 404 0.6 (5) 99.4 (806) 0.4 (67) 99.6 (17 526) 1.62 (0.65-4.04) 1.29 (0.51-3.28) Total 1 680 726 0.6 (360) 99.4 (55 068) 0.4 (6801) 99.6 (1 618 497) 1.56 (1.40-1.73) 1.49 (1.34-1.66) cOR, crude odds ratio; aOR, adjusted odds ratio; CI, confidence interval. * Adjusted for gestational age at birth, offspring sex, maternal age, parity and maternal diabetes.

During 1984–2014 <1 12 346 0.8 (10) 99.2 (1226) 3.6 (401) 96.4 (10 709) 0.22 (0.12-0.41) 0.22 (0.12-0.41) 1–2.5 21 657 0.9 (15) 99.1 (1597) 1.2 (245) 98.8 (19 800) 0.76 (0.45-1.28) 0.76 (0.45-1.28) 2.5–10 113 499 0.7 (43) 99.3 (6306) 0.8 (892) 99.2 (106 258) 0.81 (0.60-1.10) 0.81 (0.60-1.11) 10–90 1 352 940 0.7 (275) 99.3 (40 441) 0.4 (4977) 99.6 (1 307 247) 1.79 (1.58-2.02) 1.74 (1.54-1.97) 90–97.5 134 415 0.2 (8) 99.8 (3732) 0.1 (174) 99.9 (130 501) 1.61 (0.79-3.27) 1.28 (0.62-2.63) 97.5–99 27 465 0.4 (4) 99.6 (960) 0.2 (45) 99.8 (26 456) 2.45 (0.88-6.83) 1.92 (0.67-5.48) >99 18 404 0.6 (5) 99.4 (806) 0.4 (67) 99.6 (17 526) 1.62 (0.65-4.04) 1.29 (0.51-3.28) Total 1 680 726 0.6 (360) 99.4 (55 068) 0.4 (6801) 99.6 (1 618 497) 1.56 (1.40-1.73) 1.49 (1.34-1.66) cOR, crude odds ratio; aOR, adjusted odds ratio; CI, confidence interval. * Adjusted for gestational age at birth, offspring sex, maternal age, parity and maternal diabetes. The absolute risk of fetal death was highest in pregnancies with the lowest offspring birthweight (<1 percentile) (Table 2, Fig. 2), and in these pregnancies there was little difference in fetal death risk in pregnancies with preeclampsia compared to pregnancies without preeclampsia (7.0% vs 6.3%, crude OR 1.12, 95% CI 0.96–1.32). In pregnancies with offspring birthweight within the 10–90 percentiles, fetal death occurred in 1.2% of pregnancies with preeclampsia, and in 0.5% of pregnancies without preeclampsia (crude OR 2.45, 95% CI 2.26–2.65). In pregnancies with the highest offspring birthweight (>99 percentile, fetal death occurred in 1.2% of pregnancies with preeclampsia, and in 0.6% of non-preeclamptic pregnancies (crude OR 2.65, 95% CI 1.49–4.71).Fig. 2 Prevalence (%) of fetal death in pregnancies with and in pregnancies without preeclampsia within birthweight percentile categories in Norway during the years.

9 percentile, fetal death occurred in 1.2% of pregnancies with preeclampsia, and in 0.6% of non-preeclamptic pregnancies (crude OR 2.65, 95% CI 1.49–4.71).Fig. 2 Prevalence (%) of fetal death in pregnancies with and in pregnancies without preeclampsia within birthweight percentile categories in Norway during the years. a) 1967–2014, b) 1967–1983 and c) 1984–2014. Fig. 2 Changes over time During the years 1967–1983, the prevalence of preeclampsia was 2.2% (Table 2). Fetal death occurred in 4.1% of all pregnancies with preeclampsia, and in 0.9% of non-preeclamptic pregnancies (crude OR 4.76, 95% CI 4.43–5.11). In pregnancies with the lowest offspring birthweight (<1 percentile), fetal death occurred in 12.5% of pregnancies with preeclampsia, and in 8.7% of non-preeclamptic pregnancies (crude OR 1.51, 95% CI 1.27–1.80) (Table 2, Fig. 2). In pregnancies with offspring birthweight within the 10–90 percentiles, fetal death occurred in 2.8% of pregnancies with preeclampsia, and in 0.7% of pregnancies without preeclampsia (crude OR 4.03, 95% CI 3.62–4.47). Similar figures were seen in pregnancies with the highest offspring birthweight (>99 percentile), (3.2% vs 0.7%, crude OR 4.93, 95% CI 2.31–10.50).

hin the 10–90 percentiles, fetal death occurred in 2.8% of pregnancies with preeclampsia, and in 0.7% of pregnancies without preeclampsia (crude OR 4.03, 95% CI 3.62–4.47). Similar figures were seen in pregnancies with the highest offspring birthweight (>99 percentile), (3.2% vs 0.7%, crude OR 4.93, 95% CI 2.31–10.50). During the years 1984–2014, the absolute risk of fetal death was greatly reduced compared to the years 1967–1983, particularly in preeclamptic pregnancies (Table 2). Thus, during the years 1984–2014, fetal death occurred in 0.6% of pregnancies with preeclampsia, and in 0.4% of pregnancies without preeclampsia (crude OR 1.56, 95% CI 1.40–1.73). The prevalence of preeclampsia during these years was 3.3%. The reduction in fetal death risk was most prominent in pregnancies with the lowest offspring birthweight (<1 percentile), and in these pregnancies, the risk of fetal death during the years 1984–2014 was lower in preeclamptic pregnancies compared to non-preeclamptic pregnancies (0.8% vs 3.6%, crude OR 0.22, 95% CI 0.12-0.41) (Table 2, Fig. 2). Only in pregnancies with offspring birthweight within the 10–90 percentiles, the risk of fetal death was significantly higher in pregnancies with preeclampsia compared to non-preeclamptic pregnancies (0.7% vs 0.4%, crude OR 1.79, 95% CI 1.58–2.02). In pregnancies with the highest offspring birthweight (>99 percentile), fetal death occurred in 0.6% of pregnancies with preeclampsia, and in 0.4% of non-preeclamptic pregnancies (crude OR 1.62, 95% CI 0.65–4.04).

ith preeclampsia compared to non-preeclamptic pregnancies (0.7% vs 0.4%, crude OR 1.79, 95% CI 1.58–2.02). In pregnancies with the highest offspring birthweight (>99 percentile), fetal death occurred in 0.6% of pregnancies with preeclampsia, and in 0.4% of non-preeclamptic pregnancies (crude OR 1.62, 95% CI 0.65–4.04). The overall decline in fetal death risk occurred gradually during our study period (Supplementary material, Table S1). However, in pregnancies with preeclampsia and offspring birthweight <1 percentile, there was a distinct decline from 1974–1983 to 1984–1993. In 1974–1983, fetal death occurred in 8.3% of pregnancies with preeclampsia, and in 7.5% of pregnancies without preeclampsia (crude OR 1.13, 95% CI 0.84–1.52). In the next decade, 1984–1993, the corresponding figures were 0.7% and 3.9% (crude OR 0.17, 95% CI 0.06-0.45). After 1994, the point OR estimate for fetal death has been lower in pregnancies with preeclampsia compared to pregnancies without preeclampsia in pregnancies with offspring birthweight < 10 percentile. We repeated our main analyses among pregnancies with delivery in pregnancy week 28+0 and beyond, and we used antepartum fetal death as outcome measure (54.6% of all fetal deaths in our main analyses). The results remained essentially unchanged (Supplementary material, Table S2). Discussion Main findings In this population study of more than 2.5 million singleton pregnancies in Norway, the risk of fetal death associated with preeclampsia declined during the years 1967–2014, particularly in pregnancies with very small for gestational age offspring.

We repeated our main analyses among pregnancies with delivery in pregnancy week 28+0 and beyond, and we used antepartum fetal death as outcome measure (54.6% of all fetal deaths in our main analyses). The results remained essentially unchanged (Supplementary material, Table S2). Discussion Main findings In this population study of more than 2.5 million singleton pregnancies in Norway, the risk of fetal death associated with preeclampsia declined during the years 1967–2014, particularly in pregnancies with very small for gestational age offspring. Strengths and limitations The major strength of our study is the large sample size, which has provided statistical power to study temporal changes in fetal death risk in preeclamptic and non-preeclamptic pregnancies within categories of birthweight. We included all singleton pregnancies in Norway. It is therefore unlikely that a skewed selection of study participants has biased our results. The definition of fetal death has remained unchanged over time. Also, the definition of preeclampsia has not changed notably in Norway during the study period [16]. Thus, it is unlikely that changes in the definitions of our main study factors have influenced our estimates.

Strengths and limitations The major strength of our study is the large sample size, which has provided statistical power to study temporal changes in fetal death risk in preeclamptic and non-preeclamptic pregnancies within categories of birthweight. We included all singleton pregnancies in Norway. It is therefore unlikely that a skewed selection of study participants has biased our results. The definition of fetal death has remained unchanged over time. Also, the definition of preeclampsia has not changed notably in Norway during the study period [16]. Thus, it is unlikely that changes in the definitions of our main study factors have influenced our estimates. The likelihood of reporting fetal deaths and preeclampsia to the Medical Birth Registry may have changed during our study period. For instance, in the first part of our study period, it is possible that preeclampsia was more likely to be reported if fetal death had occurred. However, we found little difference in the prevalence of preeclampsia according to offspring vital status in pregnancies with very small offspring in the first part of our study period among pregnancies with very small offspring. This observation does not support differential reporting or diagnosing of preeclampsia. We made supplementary analyses of pregnancies with delivery in pregnancy week 28+0 or beyond. In these analyses, we excluded pregnancies where fetal death had occurred intrapartum or the time of fetal death was not reported, since fetal death during labor may be unrelated to preeclampsia. The results remained essentially the same.

lementary analyses of pregnancies with delivery in pregnancy week 28+0 or beyond. In these analyses, we excluded pregnancies where fetal death had occurred intrapartum or the time of fetal death was not reported, since fetal death during labor may be unrelated to preeclampsia. The results remained essentially the same. We made adjustments for risk factors of preeclampsia that have increased in prevalence over time, such as high maternal age, being a first time mother and maternal diabetes [2], but the estimated associations of preeclampsia with fetal death remained essentially unchanged. We also made adjustment for differences in gestational age at birth by using birthweight z-score. The occurrence of fetal death could possibly influence birthweight [17,18]. Nevertheless, it is unlikely that such changes are differential by maternal preeclampsia status, birthweight or year of birth. Interpretation During the first part of our study period (1967–1983), we found that preeclampsia increased the risk of fetal death within all categories of offspring birthweight. Although the absolute risk of fetal death was highest in very small for gestational age offspring, the relative risk of fetal death associated with preeclampsia was highest in pregnancies with normal or high offspring birthweight. This finding suggests that there are other factors than fetal growth restriction that cause fetal death in preeclamptic pregnancies.

as highest in very small for gestational age offspring, the relative risk of fetal death associated with preeclampsia was highest in pregnancies with normal or high offspring birthweight. This finding suggests that there are other factors than fetal growth restriction that cause fetal death in preeclamptic pregnancies. There has been an overall decline in fetal death rate during the past decades [21], and this decline has been most prominent in pregnancies with preeclampsia [11,12]. Interestingly, we found that the temporal decline in fetal death rate was particularly pronounced in preeclamptic pregnancies with small for gestational age offspring. In fact, the risk of fetal death in small for gestational age offspring was lower in preeclamptic than in than non-preeclamptic pregnancies in the last part of our study period. Only in pregnancies with offspring birthweight within the 10–90 percentiles, we found a persistent increased risk of fetal death in pregnancies with preeclampsia throughout the study period.

stational age offspring was lower in preeclamptic than in than non-preeclamptic pregnancies in the last part of our study period. Only in pregnancies with offspring birthweight within the 10–90 percentiles, we found a persistent increased risk of fetal death in pregnancies with preeclampsia throughout the study period. Identification of high risk pregnancies and timely intervention are preconditions for prevention of fetal death. Preeclampsia is a well-known risk factor for fetal death, and in 1984, a public screening program for preeclampsia was implemented in primary antenatal health care in Norway. At least ten routine clinical examinations were recommended [22], and antenatal and obstetric health care is free of charge. It is assumed that virtually all pregnant women who live in Norway follow the program [23,24]. Women with clinical signs of preeclampsia are referred to hospitals with specialized obstetric health care for further clinical examinations.

s were recommended [22], and antenatal and obstetric health care is free of charge. It is assumed that virtually all pregnant women who live in Norway follow the program [23,24]. Women with clinical signs of preeclampsia are referred to hospitals with specialized obstetric health care for further clinical examinations. Both small and large for gestational age offspring are known to be at increased risk of fetal death [19,20], and ultrasonography has made it possible to identify pregnancies with abnormal fetal growth. Fetal ultrasonography was gradually introduced in Norway after 1975, and by 1985, almost all deliveries in the country (96%) took place in maternity wards where ultrasonographic examinations could be performed [25]. Since women with preeclampsia are routinely referred to obstetric health care, abnormal fetal growth is more likely to be diagnosed in these pregnancies than in non-preeclamptic pregnancies. In non-preeclamptic pregnancies, fetal ultrasonographic examinations are not routinely performed, except for in pregnancy week 17–19, which is prior to the occurrence of preeclampsia. Interestingly, after 1984, we found a marked decrease in fetal death risk in preeclamptic pregnancies with a small for gestational age offspring. This marked decrease strongly suggests that the introduction of screening for preeclampsia, in combination with fetal ultrasonographic examinations, have been important for preventing fetal death in small for gestational age offspring.

l death risk in preeclamptic pregnancies with a small for gestational age offspring. This marked decrease strongly suggests that the introduction of screening for preeclampsia, in combination with fetal ultrasonographic examinations, have been important for preventing fetal death in small for gestational age offspring. Conclusion In this study of all pregnancies in Norway, we found that the risk of fetal death associated with preeclampsia was greatly reduced during the years 1967–2014, particularly in pregnancies with a small for gestational age offspring. In pregnancies with a normal weight offspring, the risk of fetal death in pregnancies with preeclampsia is still higher than in non-preeclamptic pregnancies. This increased risk may not be sufficiently acknowledged, and it is likely that there still is a potential for further prevention of fetal death in preeclamptic pregnancies with normal offspring birthweight. Conflict of interest The authors have no conflicts of interest to declare. Appendix A Supplementary data The following are Supplementary data to this article: Acknowledgements We thank the South-Eastern Regional Health Authority in Norway for funding this study, grant number 2014008. This study has used data from the Medical Birth Registry of Norway. The interpretation and reporting of these data is the sole responsibility of the authors, and no endorsement by the Medical Birth Registry of Norway is intended nor should be inferred. Appendix A Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.eurox.2019.100009.

Introduction Female genital mutilation (FGM) is a public health problem which is a violation of the reproductive right of women [1]. It includes all procedure that intentionally alters the female external genitalia for non-medical reasons [2]. Globally, it is estimated that 100–140 million women and girls have experienced the procedure with 3 million girls at risk each year [3,4]. It is a harmful traditional practice that is deep-rooted in sub-Saharan Africa. The prevalence of female genital mutilation varies and a prevalence rate of > 70% have been documented in Burkina Faso, Djibouti, Egypt, Eritrea, Ethiopia, Sudan, and Somalia [5]. In Nigeria and Ghana, a prevalence rate of 19% and 5% respectively have been noted in a World Health Organization (WHO) collaborative prospective study [6]. Nigeria has the highest absolute number of genitally mutilated women throughout the world [7] accounting for about one-quarter of the estimated 115–130 million circumcised women worldwide [8]. There is a geographical variation on the prevalence of FGM in Nigeria, the highest burden is found in southern part of Nigeria [9].

ria has the highest absolute number of genitally mutilated women throughout the world [7] accounting for about one-quarter of the estimated 115–130 million circumcised women worldwide [8]. There is a geographical variation on the prevalence of FGM in Nigeria, the highest burden is found in southern part of Nigeria [9]. It is classified into four types according to the WHO classification of 1995 [10] (Type I, II, III and IV). Various reasons have been advanced by different societies for the perpetuation of the practice but none is good enough for its rationalization. It is often described as a means to safeguard against the premarital sexual activity and as such prevent female promiscuity and preserve virginity [5]. Female genital mutilation is mainly practiced by traditional/local healers although, in some countries, medical personnel including doctors, nurses and certified midwives perform the procedure [5]. Highest rates of medicalization of female genital mutilation are found in Egypt (61%), Kenya (34%) and Sudan (36%) while in Nigeria and Guinea the rates are 13% and 9% respectively [5]. It is performed mainly on girls aged 4–12 years and in some cultures as early as a few days after birth or before marriage.

edure [5]. Highest rates of medicalization of female genital mutilation are found in Egypt (61%), Kenya (34%) and Sudan (36%) while in Nigeria and Guinea the rates are 13% and 9% respectively [5]. It is performed mainly on girls aged 4–12 years and in some cultures as early as a few days after birth or before marriage. It has no health benefit only harm. Some of the complications of the procedure include shock, haemorrhage, infection, sepsis, chronic pain, cheloids formation, infertility, and psychological problem [[11], [12], [13]]. Female genital mutilation has a myriad of the adverse obstetric outcome on the maternal and neonatal well-being [6]. The current study will help to add to the body of knowledge on the impacts of FGM on the obstetric outcome and helps also to understand the role of skilled health care in addressing possible health impacts of FGM. This project, therefore, intends to evaluate the effects of different types of Female genital mutilation on a range of maternal and neonatal outcomes during and immediately after delivery. This will help in policy formulation in the study area on the eradication of female genital mutilation. It will also assist in the education of our women as an earlier study has shown that some women still advocate for the perpetuation of the act [14]. We hypothesized that parturient with Female genital mutilation is not associated with adverse maternal and neonatal obstetric outcome.

cation of female genital mutilation. It will also assist in the education of our women as an earlier study has shown that some women still advocate for the perpetuation of the act [14]. We hypothesized that parturient with Female genital mutilation is not associated with adverse maternal and neonatal obstetric outcome. Materials and methods This cross-sectional comparative study was carried out in Obstetrics and Gynaecology department of Federal Teaching Hospital Abakaliki (FETHA), Ebonyi. Ebonyi state was created in 1996 with 13 local government areas, one urban, one semi-urban, and the rest rural. It has a population of 2.4 million according to 2006 Nigerian national population commission census and it occupies a land mass of 5932 km. About 75% of the population dwells in the rural areas with farming as their major occupation. FETHA is a tertiary institution which is the only referral center in the state. It was the product of a merger in December 2011 between the former Federal Medical Centre Abakaliki and Ebonyi State University Teaching Hospital, Abakaliki. Apart from FETHA, there are private, primary/secondary health facilities and two Catholic mission hospitals (St Patrick hospital, Abakaliki and St Vincent hospital Ndubia) in the study area. They provide maternal and child health services to numerous patients within and outside Ebonyi state.

aching Hospital, Abakaliki. Apart from FETHA, there are private, primary/secondary health facilities and two Catholic mission hospitals (St Patrick hospital, Abakaliki and St Vincent hospital Ndubia) in the study area. They provide maternal and child health services to numerous patients within and outside Ebonyi state. The Department of Obstetrics and Gynaecology of the hospital (FETHA) ran an antenatal clinic that is managed by consultants and resident doctors with trained nurses. The antenatal clients who are booked on Wednesday are distributed to the five (5) teams in the department with 30 consultants. The choice for a place for antenatal and labour care by the women is a personal decision but they are encouraged to access care in a center with a skilled birth attendant like FETHA. The average delivery rate is 150 deliveries per month. The ethical committee of the hospital granted approval for the study.

consultants. The choice for a place for antenatal and labour care by the women is a personal decision but they are encouraged to access care in a center with a skilled birth attendant like FETHA. The average delivery rate is 150 deliveries per month. The ethical committee of the hospital granted approval for the study. Study participants The study population was recruited from 1st January 2012 and December 2012 at the labour ward of the hospital. A total of 520 women were recruited (260 on each arm) for study at 95% confidence interval, exposed prevalence of 19% [6] and at a precision of 5%. Ten percent (10%) attrition rate was added. They were recruited by the chief researcher with the help of five trained senior registrar of each team. The study population was healthy consenting women at term with singleton foetus who was in the first stage of labour. High-risk patients, non-consenting women and those in the advanced 1st stage of labour were excluded. They were recruited using the ballot method of the simple random sample. After counseling and obtainment of an informed consent from the parturients, they were asked to pick a card from an opaque bag containing white and blue cards with replacement. The white signified inclusion while blue is for exclusion. The women recruited from above method were examined early in labour to ascertain whether or not they have FGM. The type of FGM was determined using WHO classification, thus:1 No FGM: No evidence of any genital mutilation 2 FGM I: Excision of the prepuce, with or without excision of part or all of the clitoris

Study participants The study population was recruited from 1st January 2012 and December 2012 at the labour ward of the hospital. A total of 520 women were recruited (260 on each arm) for study at 95% confidence interval, exposed prevalence of 19% [6] and at a precision of 5%. Ten percent (10%) attrition rate was added. They were recruited by the chief researcher with the help of five trained senior registrar of each team. The study population was healthy consenting women at term with singleton foetus who was in the first stage of labour. High-risk patients, non-consenting women and those in the advanced 1st stage of labour were excluded. They were recruited using the ballot method of the simple random sample. After counseling and obtainment of an informed consent from the parturients, they were asked to pick a card from an opaque bag containing white and blue cards with replacement. The white signified inclusion while blue is for exclusion. The women recruited from above method were examined early in labour to ascertain whether or not they have FGM. The type of FGM was determined using WHO classification, thus:1 No FGM: No evidence of any genital mutilation 2 FGM I: Excision of the prepuce, with or without excision of part or all of the clitoris 3 FGM II: Excision of the clitoris with the partial or total removal of the labia minora 4 FGM III: Excision of part or all of the external genitalia and stitching or narrowing of the vaginal opening (infibulation).

Study participants The study population was recruited from 1st January 2012 and December 2012 at the labour ward of the hospital. A total of 520 women were recruited (260 on each arm) for study at 95% confidence interval, exposed prevalence of 19% [6] and at a precision of 5%. Ten percent (10%) attrition rate was added. They were recruited by the chief researcher with the help of five trained senior registrar of each team. The study population was healthy consenting women at term with singleton foetus who was in the first stage of labour. High-risk patients, non-consenting women and those in the advanced 1st stage of labour were excluded. They were recruited using the ballot method of the simple random sample. After counseling and obtainment of an informed consent from the parturients, they were asked to pick a card from an opaque bag containing white and blue cards with replacement. The white signified inclusion while blue is for exclusion. The women recruited from above method were examined early in labour to ascertain whether or not they have FGM. The type of FGM was determined using WHO classification, thus:1 No FGM: No evidence of any genital mutilation 2 FGM I: Excision of the prepuce, with or without excision of part or all of the clitoris 3 FGM II: Excision of the clitoris with the partial or total removal of the labia minora 4 FGM III: Excision of part or all of the external genitalia and stitching or narrowing of the vaginal opening (infibulation). The participants with FGM were the cases and the control were consenting women at term without FGM who delivered in our facility within 24 h of selection of a case. The participants were interviewed using a structured questionnaire to obtain information about social and demographic characteristics and obstetric history. The social class was classified using the education of the woman and her husband’s occupation according to the work of Olusanya et al. [15] the labour and deliveries were done according to the departmental protocol. An individualized partograph was opened for each parturient when in active phase labour (cervical Os = 4 cm) and episiotomy was only given when the perineum threatened to tear. Following admission of a parturient into labour routine investigation done include pack cell volume, urinalysis, blood grouping and typing of blood. Other investigations requested are based on the clinical finding.

ive phase labour (cervical Os = 4 cm) and episiotomy was only given when the perineum threatened to tear. Following admission of a parturient into labour routine investigation done include pack cell volume, urinalysis, blood grouping and typing of blood. Other investigations requested are based on the clinical finding. Exclusion criteria include refusal to give consent, those booked for the elective caesarean section, unbooked status, women undergoing VBAC, multiple gestations, non-cephalic presentation and pregnancy complicated by medical disease. Pregnancy whose labour’s were too advanced to allow vaginal examination and including those with intrauterine growth restriction (IUGR) were also excluded. Following delivery, the obstetric and neonatal outcome variables collected included: duration of the second stage of labour, mode of delivery, episiotomy, perineal tear, blood loss, Apgar score, and neonatal weight. The degree of the perineal tear was classified at the time of delivery using 9th International Classification of Disease. A first-degree vaginal tear is defined as damage to the superficial vaginal epithelium, a second-degree tear as involving the vaginal epithelium and deeper muscle but excluding the anal sphincter. A third perineal is defined as a partial or complete anal sphincter rupture without the involvement of anal mucosa and a fourth-degree tear as a rupture of the anal sphincter and mucosa. Women and their infants were then followed up until maternal discharge from hospital for details of delivery and health status.

cter. A third perineal is defined as a partial or complete anal sphincter rupture without the involvement of anal mucosa and a fourth-degree tear as a rupture of the anal sphincter and mucosa. Women and their infants were then followed up until maternal discharge from hospital for details of delivery and health status. Data analysis The data were analyzed using IBM SPSS statistic version 20 software (IBM Corp. Armonk, NY, USA). A categorical variable was analysed using simple percentages, Odd ratio, and Chi-square. Mean and Student t-test was used for a continuous variable where appropriate. P values less than 0.05 was considered significant

lysis The data were analyzed using IBM SPSS statistic version 20 software (IBM Corp. Armonk, NY, USA). A categorical variable was analysed using simple percentages, Odd ratio, and Chi-square. Mean and Student t-test was used for a continuous variable where appropriate. P values less than 0.05 was considered significant Out of 520 women that were recruited into the study, only 496 women had complete data for analysis. From Table 1, the mean age of the study population was 27.9 ± 4.8 years with a range of 40 years. Majority of the women were within the age bracket of 25–29 years (209, 42%). About one-third of the women were obese and the mean body mass index (BMI) was 28.3 ± 4.7 kg/m2. An independent sample t-test was conducted to compare the maternal age and weight between the two groups of women studied (FGM and no FGM). There was no significant difference in the age (M = 27.6 SD = 4.8) of group with FGM and those without FGM (M = 28.4 SD = 4.9) t (494), p = 0.153; for the weight a significance difference exist in the weight (M = 70.7 SD = 11.6) of the group with FGM and those without (M = 75.6 SD = 13.6) t (494) p = 0.001. The odds of a woman having FGM in the cohort of women residing in rural communities is 66% more than in the group in urban communities with the true population effect that is between 46% and 94%. This result was statistically significant (p = 0.012).Table 1 Social demographic characteristics of the women.

4) p = 0.001. The odds of a woman having FGM in the cohort of women residing in rural communities is 66% more than in the group in urban communities with the true population effect that is between 46% and 94%. This result was statistically significant (p = 0.012).Table 1 Social demographic characteristics of the women. Table 1 Female genital mutilation Variables Yes (%) No (%) X2 / t test P value Age (years) <20 10(4.0) 13(5.2) 20-24 61(24.6) 32(12.9) 9.27 0.15* 25-29 99(39.9) 110(44.4) 30-34 53(21.4) 73(29.4) ≥35 25(10.1) 20(8.1) BMI(kg/m2) <18.5 1(0.4) 0(0.0) 18.5-24.9 67(27.0) 44(17.4) 12.73 0.001* 25.0 -29.9 123(49.6) 112(45.2) 30.0-34.9 39(15.7) 63(25.4) 35.0-39.9 17(6.8) 21(8.5) ≥40.0 1(0.4) 8(3.2) Residence Rural 112(45.2) 138(55.6) 5.45 0.012† Urban 136(54.8) 110(44.4) Ethnicity Igbo 246(99.2) 242(97.6) 2.13 0.28‡ Others 2(0.8) 6(2.4) Social class 1 17(6.8) 31(12.5) 2 32(12.9) 72(29.0) 38.20 0.001† 3 66(26.6) 72(29.0) 4 95(38.3) 47(18.9) 5 38(15.3) 26(10.5) * Independent sample t-test. ‡ Likelihood ratio. † Chi-square. Table 2 Female genital mutilation and obstetric characteristics of the study population. Table 2 Type of female genital mutilation (n, %) Variables Nil 1 2 3 X2 P value GA(weeks) <37 35(14.1) 10(4.0) 23(9.3) 3(1.2) 37-39 132(53.2) 23(9.3) 101(40.7) 2(0.8) 7.29 0.27* >39 81(32.7) 17(6.9) 68(27.4) 1(0.4) Parity 0 84(33.9) 22(8.9) 92(37.1) 4(1.6) 1-4 130(52.4) 23(9.3) 75(30.2) 2(0.8) 11.33 0.06* >4 34(13.7) 5(2.0) 25(10.1) 0(0.0)

Table 2 Type of female genital mutilation (n, %) Variables Nil 1 2 3 X2 P value GA(weeks) <37 35(14.1) 10(4.0) 23(9.3) 3(1.2) 37-39 132(53.2) 23(9.3) 101(40.7) 2(0.8) 7.29 0.27* >39 81(32.7) 17(6.9) 68(27.4) 1(0.4) Parity 0 84(33.9) 22(8.9) 92(37.1) 4(1.6) 1-4 130(52.4) 23(9.3) 75(30.2) 2(0.8) 11.33 0.06* >4 34(13.7) 5(2.0) 25(10.1) 0(0.0) Neonatal weight (kg) <2.5 5(2.0) 13(5.2) 11(4.4) 0(0.0) 2.5-3.5 197(79.4) 31(12.5) 161(64.9) 4(1.6) 6.21 0.35* >3.5 47(18.9) 6(2.4) 20(8.1) 2(0.8) In Table 2, the majority of the women that have FGM where classified as type II (192, 77.4%). * Fisher’s Exact Test. Type III FGM was seen in six (6) women accounting for less than three percent of the different type of FGM that was seen. The mean gestational age at delivery was 38.9 ± 1.5 weeks with half of the women delivering at a gestational age of 37–39 weeks.

Neonatal weight (kg) <2.5 5(2.0) 13(5.2) 11(4.4) 0(0.0) 2.5-3.5 197(79.4) 31(12.5) 161(64.9) 4(1.6) 6.21 0.35* >3.5 47(18.9) 6(2.4) 20(8.1) 2(0.8) In Table 2, the majority of the women that have FGM where classified as type II (192, 77.4%). * Fisher’s Exact Test. Type III FGM was seen in six (6) women accounting for less than three percent of the different type of FGM that was seen. The mean gestational age at delivery was 38.9 ± 1.5 weeks with half of the women delivering at a gestational age of 37–39 weeks. Table 3 shows that majority of the women studied achieved vaginal delivery (477, 96.2%) with more women on the FGM arm having abdominal delivery. The second stage of labour was prolonged in 13% (65) of women with 60% (37) of it occurring in FGM group. Cohort of women with FGM has increased odd of delayed labour which is significant (OR = 0.78 95%CI 0.64-0.97). Our study also showed that the presence or absence of FGM was not associated with neonatal Apgar score (X2 (2) = 9.30, P = 0.46). Among the study population, 36% (177) had episiotomy; 58% occurred in women that had genital mutilation. Women with FGM had increased odds of being given episiotomy during delivery (OR = 1.69 95% CI 1.17–2.45) though not significant and significantly not having an intact perineum (OR = 0.76 95% CI 0.63 - 0.91). A chi-square test of independence was performed to examine the relationship between the presence or absence of FGM and duration of the second stage of labour, the risk of episiotomy, intactness of the perineum and volume of blood lost while controlling for possible confounders. The result showed a significant association between presence or absence of FGM and duration of the 2nd stage only among women with BMI of 25.9–29.9 kg/m2 (P = 0.009) and ≥ 40.0 kg/m2 (P = 0.003). A significant association was also seen only among women in social class 2 and 4. Even though there was a significant association between the presence or absence of FGM and episiotomy (X2 (1) = 7.85, P = 0.005); this association remained significant across all the social class and was only significant among women with BMI of 18.5–24.9 and 25.9–29.9 kg/m2 (P = 0.001). A significant association was seen (X2 (1) 9.34, P = 0.002) between the presence or absence of FGM and intactness of the perineum. Only women with BMI of 18.5–24.9 kg/m2 and social class 2 and 3 were significant contributors. The study also showed that being in social class 5 and in FGM arm is the only significant contributor to excessive blood loss (P = 0.023).Table 3 Cross-tabulation of the obstetric outcome of the women with the different types of Female genital mutilation.

.9 kg/m2 and social class 2 and 3 were significant contributors. The study also showed that being in social class 5 and in FGM arm is the only significant contributor to excessive blood loss (P = 0.023).Table 3 Cross-tabulation of the obstetric outcome of the women with the different types of Female genital mutilation. Table 3 Type of Female genital mutilation (n, %) Variables Nil 1 2 3 X2 P value Mode of delivery SVD 241(97.2) 48(19.4) 182(73.4) 6(2.4) C/S 5(2.0) 2(0.8) 6(2.4) 0(0.0) 4.17 0.64* Vacuum 2(0.8) 0(0.0) 4(1.6) 0(0.0) Duration of 2nd stage labour ≤ 2 hours 220(88.7) 44(17.7) 161(64.9) 0(0.0) 39.48 0.001 >2 hours 28(11.3) 6(2.4) 31(12.5) 6(2.4) Episiotomy Yes 75(30.2) 14(5.6) 85(34.3) 6(2.4) 20.58 0.001* No 173(69.8) 36(14.5) 107(43.1) 0(0.0) Intact perineum Yes 135(54.4) 30(12.1) 71(28.6) 0(0.0) 21.95 0.001 No 113(45.6) 20(8.1) 121(48.8) 6(2.4) Blood loss(ml) ≤500 224(90.3) 48(19.4) 170() 2(0.8) 22.78 0.001 >500 24(9.7) 2(0.8) 22(8.9) 4(1.6) Neonatal weight (kg) <2.5 5(2.0) 3(1.2) 10(4.0) 0(0.0) 6.21 0.35 2.5-3.5 197(79.4) 41(16.5) 150(60.5) 6(2.4) >3.5 46(18.5) 6(2.4) 32(12.9) 0(0.0) Apgar 0 2(0.8) 0(0.0) 5(2.0) 0(0.0) 9.38 0.46* ≤3 2(0.8) 0(0.0) 0(0.0) 0(0.0) 4-7 12(4.8) 2(0.8) 17(6.9) 0(0.0) ≥8 232(93.5) 48(19.4) 170(68.5) 6(2.4) * Fisher’s Exact Test.

Blood loss(ml) ≤500 224(90.3) 48(19.4) 170() 2(0.8) 22.78 0.001 >500 24(9.7) 2(0.8) 22(8.9) 4(1.6) Neonatal weight (kg) <2.5 5(2.0) 3(1.2) 10(4.0) 0(0.0) 6.21 0.35 2.5-3.5 197(79.4) 41(16.5) 150(60.5) 6(2.4) >3.5 46(18.5) 6(2.4) 32(12.9) 0(0.0) Apgar 0 2(0.8) 0(0.0) 5(2.0) 0(0.0) 9.38 0.46* ≤3 2(0.8) 0(0.0) 0(0.0) 0(0.0) 4-7 12(4.8) 2(0.8) 17(6.9) 0(0.0) ≥8 232(93.5) 48(19.4) 170(68.5) 6(2.4) * Fisher’s Exact Test. Discussion Female genital mutilation is one of the social vices against women which are associated with obstetrics complication [1,6]. This study compares obstetrics outcome between women with and those without Female genital mutilation. The prevalence of Female genital mutilation is high in the study area [16] which is higher than the National prevalence rate [17]. The prevalence of the different type of Female genital mutilation that was seen among the study population was: type I FGM (20.2%), type II FGM (77.4%) and type III FGM (2.4%). Majority of the women in our study had type I or II genital mutilation which agrees with the earlier finding in Nigeria [14,18]. The results also agree with Morison’s survey, in which more than 99% of the cohort of women in their survey had type II FGM.19 It is however different from the work of Kaplan et al. in Gambia; they reported a higher rate of type I and type III FGM [3]. The difference from our study could be ascribed to a difference in study design and population. Type III FGM of 2.4% seen in our survey is, however, higher than the WHO collaborative finding in Nigeria [6]. It is a worrisome finding (type III FGM) because of increase complication associated with this type of mutilation. Our finding also suggests that the prevalence of type III FGM might be high in the study area and concerted effort is needed to eradicate the obnoxious procedure.

WHO collaborative finding in Nigeria [6]. It is a worrisome finding (type III FGM) because of increase complication associated with this type of mutilation. Our finding also suggests that the prevalence of type III FGM might be high in the study area and concerted effort is needed to eradicate the obnoxious procedure. Amongst the obstetric outcome that was evaluated in our study, only the rate of episiotomy and whether the perineum was intact or not were significantly associated with the presence of FGM. Our study suggests that the presence of FGM increases the odd of these occurring in parturient in labour. The mode of delivery and the neonatal outcome measures were not significantly associated with the presence or absence of genital mutilation. It is however evident from our study that a majority of the parturient on the FGM arm had delayed labour, assisted delivery and abnormal Apgar score. A causal effect, however, cannot be established as this is subject to variegate of labour. From our study, parturient that are overweight or obese are significantly affected by delayed labour and astute labour care is needed to circumvent it. This could be effected by second stage augmentation of labour, as this group is prone to dysfunctional labour or by assisted vaginal delivery. Some studies gave credence to some of our findings. A collaborative prospective study by WHO summarises that women with FGM are significantly more likely than those without FGM to have adverse obstetric outcomes [6]. The episiotomies rate and perineal tear were significantly more on the group with FGM in their work which is similar to our findings. It is apparent from our study that women who are overweight or obese are more likely to suffer this which might result from challenges of conducting delivery in this class of parturient. Support for increased perineal tear among women with FGM was reported by Varol et al. in Australia [20].

is similar to our findings. It is apparent from our study that women who are overweight or obese are more likely to suffer this which might result from challenges of conducting delivery in this class of parturient. Support for increased perineal tear among women with FGM was reported by Varol et al. in Australia [20]. Previous studies had reported a significant association between FGM and stillbirth [6,21] which was not seen in our study. The difference in the study population and design might be a plausible reason for this difference. In one of the studies mentioned above, the study population was recruited from probably primary, secondary and tertiary health centre with different accoucheurship which might affect labour care and neonatal outcome. This differs from our study, as only booked patients in our hospital were evaluated and labour managed only by qualified personnel. Post-partum haemorrhage is a major contributor to maternal mortality in developing world like Nigeria [22]. Even though uterine atony is the commonest cause; genital laceration contributes significantly to it in our environment. FGM is associated with excessive blood loss via bleeding from episiotomy site, genital tract laceration and amongst other. In our study, women with FGM had average blood loss of 497 ml 95% CI 446.8–547.6 ml which is not significantly different from the cohort of women with no FGM although they are at increased risk of excessive blood loss (OR = 0.91 95% CI 0.70–1.20). Our finding did not agree with earlier report [6] and could be attributed to the level of skill birth attendant that attended to these women. Cohort of women in our study had Active management of the third stage of labour with prompt repair of their episiotomies and genital lacerations to avert primary post-partum haemorrhage. In this study, the mode of delivery is not associated with the presence of FGM and those that were delivered by caesarean section were for other obstetrics reason and not because of FGM. This finding is in tandem with the finding in Australia [19].

omies and genital lacerations to avert primary post-partum haemorrhage. In this study, the mode of delivery is not associated with the presence of FGM and those that were delivered by caesarean section were for other obstetrics reason and not because of FGM. This finding is in tandem with the finding in Australia [19]. In conclusion, the current study has highlighted the obstetric complications of female genital mutilation. It shows increased odd of these women been given episiotomies and having a genital laceration. It calls for astute labour care to help prevent the possible complication of PPH, major perineal tear with its late possible squealer. We recommend continued awareness creation to stop FGM. The finding from this study will help in advocacy and the continued fight on the eradication of this act. Declarations Authors’ contributions CCA & BCO: Study design, data collection/analysis, and interpretation of finding and drafting of manuscript. JAO: participated in the interpretation of findings and data analysis. BNE & NJO: interpretation of findings and drafting of the manuscript. LOA & FAO: participated in the interpretation of findings, data collection, and drafting of the manuscript. All participated in the review of the final manuscript. All the authors approved the manuscript. Funding Funding is borne by the authors. Ethics approval and consent to participate Ethical approval for the study was obtained from the Research and Ethics committee of the hospital and only consented mothers were recruited for the study. Consent for publication Not applicable.

Declarations Authors’ contributions CCA & BCO: Study design, data collection/analysis, and interpretation of finding and drafting of manuscript. JAO: participated in the interpretation of findings and data analysis. BNE & NJO: interpretation of findings and drafting of the manuscript. LOA & FAO: participated in the interpretation of findings, data collection, and drafting of the manuscript. All participated in the review of the final manuscript. All the authors approved the manuscript. Funding Funding is borne by the authors. Ethics approval and consent to participate Ethical approval for the study was obtained from the Research and Ethics committee of the hospital and only consented mothers were recruited for the study. Consent for publication Not applicable. Availability of data and material All data generated or analysed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgments Not applicable.

Introduction Urinary incontinence (UI), defined as the complaint of any involuntary leakage of urine, affects nearly 40% of women; stress urinary incontinence (SUI) accounts for approximately half of all UI [1]. UI significantly impacts on quality of life, affecting the woman’s physical, mental, social and sexual well-being and leading to avoidance of intimacy, depression and social isolation [[2], [3], [4]]. In addition, the economic impact of UI was estimated to be $710 million in 1998 and was projected to be $1.6 billion by 2009 in Australia [5]. Surgical options for SUI include trans-vaginal insertion of a mid-urethral sling (MUS) and the more invasive, traditional gold-standard Burch colposuspension procedure, requiring an abdominal approach (laparotomic or laparoscopic). With similar success rates of 74–90% and similar longevity, the choice of one procedure over the other often depends on surgical training, experience, and preference [6]. A systematic review suggests MUS to be a superior surgical treatment, which has rapidly become the procedure of choice for SUI due to shorter operating time and quicker patient recovery; there are nevertheless inherent surgical risks applicable to both procedures, including bleeding, infection, bladder and urethral injury, voiding dysfunction and pain [7].

a superior surgical treatment, which has rapidly become the procedure of choice for SUI due to shorter operating time and quicker patient recovery; there are nevertheless inherent surgical risks applicable to both procedures, including bleeding, infection, bladder and urethral injury, voiding dysfunction and pain [7]. In 2016, the US Food and Drug Administration (FDA) reclassified the use of mesh kits in urogynaecology as Class III medical devices requiring pre-market approval [8]. The resultant media attention and class actions against manufacturers of these devices has resulted in the withdrawal of most pelvic organ prolapse (POP) mesh devices from the market [9]. Thus, there is strong public interest in and a clinical need for a minimally-invasive, non-hormonal, effective treatment for SUI. Fractional micro-ablative laser therapy has been shown to be a potential non-surgical treatment alternative for SUI [10,11]. The subclinical thermal tissue effect from the laser beam induces human dermal fibroblasts to initiate an inflammatory healing cascade, stimulating de novo collagen and elastin synthesis resulting in a thicker vaginal epithelium with larger diameter, glycogen-rich epithelial cells [[12], [13], [14]]. The aim of this study is to evaluate the change in SUI symptoms after trans-vaginal fractional micro-ablative CO2 laser in women at baseline, compared to follow-up at 3 months and 12–24 months post-treatment.

Fractional micro-ablative laser therapy has been shown to be a potential non-surgical treatment alternative for SUI [10,11]. The subclinical thermal tissue effect from the laser beam induces human dermal fibroblasts to initiate an inflammatory healing cascade, stimulating de novo collagen and elastin synthesis resulting in a thicker vaginal epithelium with larger diameter, glycogen-rich epithelial cells [[12], [13], [14]]. The aim of this study is to evaluate the change in SUI symptoms after trans-vaginal fractional micro-ablative CO2 laser in women at baseline, compared to follow-up at 3 months and 12–24 months post-treatment. Materials and methods This is a prospective observational study on women with SUI symptoms who were treated with trans-vaginal fractional CO2 laser. The study received Human Research Ethics approval from Bellberry Limited (Application ID: 2016-04-293). All participants provided informed, written consent; participants were not compensated for their participation.

observational study on women with SUI symptoms who were treated with trans-vaginal fractional CO2 laser. The study received Human Research Ethics approval from Bellberry Limited (Application ID: 2016-04-293). All participants provided informed, written consent; participants were not compensated for their participation. Study population During 2014–2017, all women aged 18 years or more being treated by a single gynaecology consultant at FBW Gynaecology Plus were invited to participate in the study. Inclusion criteria were no/unsatisfactory response to conservative treatments and a preference for non-surgical management of bothersome SUI symptoms. The women also demonstrated a positive cough test and urethral hypermobility on ultrasound. All women that participated were offered urodynamic studies and encouraged to continue with topical oestrogen therapy and pelvic floor muscle exercises. Women with ≥stage II pelvic organ prolapse quantification system (POPQ) score, acute or recurrent urinary tract infections, pregnancy, current malignancy, known cervical dysplasia or undiagnosed abnormal uterine bleeding were excluded. All participants were asked to complete the bladder function section of the Australian Pelvic Floor Questionnaire (APFQ) (Appendix A) at baseline (T1), 3 months after third treatment (T2), and at 12–24 months’ follow-up (T3). The APFQ is a validated self-administered pelvic floor questionnaire utilised for quantification of clinical and research outcomes [15]. The primary outcome of this study is to describe the change in self-reported SUI symptoms based on question 6 of APFQ.

after third treatment (T2), and at 12–24 months’ follow-up (T3). The APFQ is a validated self-administered pelvic floor questionnaire utilised for quantification of clinical and research outcomes [15]. The primary outcome of this study is to describe the change in self-reported SUI symptoms based on question 6 of APFQ. The secondary outcomes were bladder function, urgency, urge incontinence, pad usage, quality of life, degree of bothersome bladder score as assessed by APFQ. Improvement of SUI was calculated based on severity scoring 0-3. Bladder function score was calculated by adding all 15 questions in the bladder subsection of APFQ, with maximum score of 45. Scores 0–11.25 was normal bladder function, 11.26–22.5 was mild bladder dysfunction, 22.6–33.75 was moderate bladder dysfunction, and 33.76–45 was severe bladder dysfunction. Questionnaires were distributed to participants and collated by practice staff; the identity of individual respondents remained blinded to the investigators.

25 was normal bladder function, 11.26–22.5 was mild bladder dysfunction, 22.6–33.75 was moderate bladder dysfunction, and 33.76–45 was severe bladder dysfunction. Questionnaires were distributed to participants and collated by practice staff; the identity of individual respondents remained blinded to the investigators. Intervention The intervention was carried out at the FBW Gynaecology Plus office. Participants were pre-treated with topical anaesthetic cream at the level of vestibulum. After 10 min, they were treated with trans-vaginal fractional micro-ablative CO2 laser system (MonaLisa Touch, SmartXide2 V2LR, DEKA, Italy) using the following settings: power 40 W, dwell time 1000 μs, DOT spacing 700 μm, SmartStak parameter 3 and D-pulse mode. The laser beam was emitted from a 90° vaginal probe gently inserted up to the level of the bladder neck, then rotated and withdrawn in order to provide treatment of the anterior lower one third of the vagina and external urethral meatus. Each patient also received three total vaginal length laser treatments with a 360-degree probe as per Salvatore et al. [15]. Three treatments were delivered at intervals of 4 to 6 weeks. Patients were advised to abstain from vaginal intercourse for 5 days after laser application and avoid heavy lifting (>1 kg) for 6 weeks. Participants with a past history of genital herpes were given antiviral prophylaxis 2 h prior to laser treatment.

Intervention The intervention was carried out at the FBW Gynaecology Plus office. Participants were pre-treated with topical anaesthetic cream at the level of vestibulum. After 10 min, they were treated with trans-vaginal fractional micro-ablative CO2 laser system (MonaLisa Touch, SmartXide2 V2LR, DEKA, Italy) using the following settings: power 40 W, dwell time 1000 μs, DOT spacing 700 μm, SmartStak parameter 3 and D-pulse mode. The laser beam was emitted from a 90° vaginal probe gently inserted up to the level of the bladder neck, then rotated and withdrawn in order to provide treatment of the anterior lower one third of the vagina and external urethral meatus. Each patient also received three total vaginal length laser treatments with a 360-degree probe as per Salvatore et al. [15]. Three treatments were delivered at intervals of 4 to 6 weeks. Patients were advised to abstain from vaginal intercourse for 5 days after laser application and avoid heavy lifting (>1 kg) for 6 weeks. Participants with a past history of genital herpes were given antiviral prophylaxis 2 h prior to laser treatment. Statistical analysis Power analysis showed that a sample size of 29 participants would be required to achieve 5% significance, 80% power. Wilcoxon signed-rank tests were used to detect differences for the primary and all secondary outcomes. The threshold for statistical significance was set at 0.05. Data were analysed using IBM SPSS software, version 21.0 (IBM, Chicago, Illinois)

of 29 participants would be required to achieve 5% significance, 80% power. Wilcoxon signed-rank tests were used to detect differences for the primary and all secondary outcomes. The threshold for statistical significance was set at 0.05. Data were analysed using IBM SPSS software, version 21.0 (IBM, Chicago, Illinois) Results Fifty-eight women were recruited to the study with an average age of 57.4 ± 11.4 years (30–85 years); 45 (77.6%) were postmenopausal, 44 (75.9%) women were on vaginal oestrogen and 33 (56.9%) women underwent urodynamic studies, which confirmed SUI. There were 54 women who were followed-up at 3 months (T2) and 36 at 12–24 months (T3). In relation to the primary outcome (question 6 of APFQ), Fig. 1 illustrates the reduction in SUI symptoms reported by women at follow-up (T2 and T3) compared to baseline (T1). At T1, all 58 participants reported frequent or daily SUI symptoms. At T2, 80% (44 of 55) reported an improvement in SUI symptoms, which included 45.5% (25 of 55) participants reporting no SUI symptoms, 16.4% (9 of 55) participants reported frequent symptoms, 3.6% (2 of 55) reported daily symptoms. These changes were also reflected in the median score reduction for Q6 (p < 0.01).Fig. 1 Changes in stress incontinence symptoms in women who underwent fractional CO2 laser at pre-treatment (T1; n = 58), 3 months post-treatment (T2; n = 55), and 12–24-month post-treatment (T3; n = 36). Fig. 1

In relation to the primary outcome (question 6 of APFQ), Fig. 1 illustrates the reduction in SUI symptoms reported by women at follow-up (T2 and T3) compared to baseline (T1). At T1, all 58 participants reported frequent or daily SUI symptoms. At T2, 80% (44 of 55) reported an improvement in SUI symptoms, which included 45.5% (25 of 55) participants reporting no SUI symptoms, 16.4% (9 of 55) participants reported frequent symptoms, 3.6% (2 of 55) reported daily symptoms. These changes were also reflected in the median score reduction for Q6 (p < 0.01).Fig. 1 Changes in stress incontinence symptoms in women who underwent fractional CO2 laser at pre-treatment (T1; n = 58), 3 months post-treatment (T2; n = 55), and 12–24-month post-treatment (T3; n = 36). Fig. 1 At T2 (3 months); 27 of the 36 women (75%) that returned reported SUI symptoms ‘not more than occasionally,’ including 10 of the 36 (27.8%) (T3) who reported no symptoms. These women also had a negative cough test at the time of clinical examination. The remaining 9 of the 36 (25%) women experienced a return of occasional SUI symptoms.

27 of the 36 women (75%) that returned reported SUI symptoms ‘not more than occasionally,’ including 10 of the 36 (27.8%) (T3) who reported no symptoms. These women also had a negative cough test at the time of clinical examination. The remaining 9 of the 36 (25%) women experienced a return of occasional SUI symptoms. Similar results were demonstrated for secondary outcomes of bladder function (APFQ questions 1–15), urge incontinence (APFQ question 5) and bothersome bladder (APFQ question 15). Normal bladder function increased from 31% (18 of 58) at T1 to 72.2% (39 of 54) at T2. This trend decreased to 69.4% (25 of 36) at T3. There was an overall improvement in participants’ urge incontinence scores from T1 to T2, with an increase in the number of patients reporting “never” leaking urine when they rush to the toilet from T1 to T2 (19% to 60%, p < 0.01); this trend decreased slightly to at T3 (44.4%, p < 0.01). There was an improvement in the participants’ degree of bothersome bladder from T1 to T2; more women reported “not at all (bothersome)” from T1 to T2 (3.4% to 50%, p < 0.01); this trend reduced at T3 (36%, p = 0.01). The results are summarised in Table 1.Table 1 Outcomes for women who underwent fractional CO2 laser at pre-treatment (T1) compared to 3 months after treatment (T2) and 12–24 months after treatment (T3). Table 1 APFQ Improvement % (n) Median T1 Median T2 p-value Improvement % (n) Median T1 Median T3 p-value Primary outcome Stress incontinence 6 80 (55) 2 1 <0.01 75 (36) 2 1 <0.01

Similar results were demonstrated for secondary outcomes of bladder function (APFQ questions 1–15), urge incontinence (APFQ question 5) and bothersome bladder (APFQ question 15). Normal bladder function increased from 31% (18 of 58) at T1 to 72.2% (39 of 54) at T2. This trend decreased to 69.4% (25 of 36) at T3. There was an overall improvement in participants’ urge incontinence scores from T1 to T2, with an increase in the number of patients reporting “never” leaking urine when they rush to the toilet from T1 to T2 (19% to 60%, p < 0.01); this trend decreased slightly to at T3 (44.4%, p < 0.01). There was an improvement in the participants’ degree of bothersome bladder from T1 to T2; more women reported “not at all (bothersome)” from T1 to T2 (3.4% to 50%, p < 0.01); this trend reduced at T3 (36%, p = 0.01). The results are summarised in Table 1.Table 1 Outcomes for women who underwent fractional CO2 laser at pre-treatment (T1) compared to 3 months after treatment (T2) and 12–24 months after treatment (T3). Table 1 APFQ Improvement % (n) Median T1 Median T2 p-value Improvement % (n) Median T1 Median T3 p-value Primary outcome Stress incontinence 6 80 (55) 2 1 <0.01 75 (36) 2 1 <0.01 Secondary outcomes Bladder Function 1-15 67 (54) 1 0 <0.01 87.5 (36) 1 0 <0.01 Urge incontinence 5 79 (55) 2 0 <0.01 63 (36) 2 1 <0.01 Urgency 4 65 (55) 2 1 <0.01 45 (36) 2 1 <0.01 Wearing pads 10 54 (55) 2 0 0.01 46 (36) 2 0 0.01 Impact of urinary leakage on QOL 14 63 (55) 1 0 <0.01 44 (36) 1 0 0.01 Degree of bothersome bladder 15 56 (54) 2 1 <0.01 45 (36) 2 1 <0.01 APFQ = Australian Pelvic Floor Questionnaire.

e 5 79 (55) 2 0 <0.01 63 (36) 2 1 <0.01 Urgency 4 65 (55) 2 1 <0.01 45 (36) 2 1 <0.01 Wearing pads 10 54 (55) 2 0 0.01 46 (36) 2 0 0.01 Impact of urinary leakage on QOL 14 63 (55) 1 0 <0.01 44 (36) 1 0 0.01 Degree of bothersome bladder 15 56 (54) 2 1 <0.01 45 (36) 2 1 <0.01 APFQ = Australian Pelvic Floor Questionnaire. STD = Standard deviation. p < 0.05 as statistically significant. Differences were assessed by Wilcoxon sign-rank testing. Women lost to follow-up were contacted to offer review. Upon phoning 22 of the 58 patients to arrange the 12-month follow-up, 5 participants reported no further SUI symptoms (cured) and 3 participants reported 50–60% improvement of their SUI symptoms which they deemed manageable. There were another 14 women who did not improve after 3 sessions of CO2 laser treatment, 4 of whom opted for MUS surgery and some women desired to avoid surgery by undergoing autologous cell therapy, such as platelet-rich plasma (RegenPRP®). There were no serious adverse events as a result of the treatment protocol. Out of 58 participants, 3 (5.4%) participants noticed a change in vaginal discharge diagnosed as thrush, which resolved with treatment; 2 (3.4%) participants reported symptoms of urinary tract infections (both of these patients had previous history of post-coital UTIs) and were treated with appropriate antibiotics; 1 (1.7%) participant developed a recurrence of genital herpes and required antiviral therapy.

sed as thrush, which resolved with treatment; 2 (3.4%) participants reported symptoms of urinary tract infections (both of these patients had previous history of post-coital UTIs) and were treated with appropriate antibiotics; 1 (1.7%) participant developed a recurrence of genital herpes and required antiviral therapy. Comments This study describes the change in prevalence of SUI symptoms before and after fractionated CO2 laser to treat both pre- and post-menopausal women with SUI. The study showed that following 3 treatments at 4–6-week intervals, SUI symptoms improved in 80% of participants at 3 months (p < 0.01) and that these benefits persisted in 75% of participants at 12 months (p < 0.01). SUI is a significant condition affecting women with their physical, mental and social well-being [1,2]. Women who seek non-surgical treatment are presented with a limited range of low-risk treatment options, such as pelvic floor muscle strengthening and vaginal pessaries [17]. For women who are sexually active, vaginal pessaries can pose coital problems. In addition, pessaries can be problematic for women with severe vaginal atrophy and mobility issues, as the need for regular examinations can be traumatising, painful and requiring concomitant topical oestrogen therapy.

ginal pessaries [17]. For women who are sexually active, vaginal pessaries can pose coital problems. In addition, pessaries can be problematic for women with severe vaginal atrophy and mobility issues, as the need for regular examinations can be traumatising, painful and requiring concomitant topical oestrogen therapy. Topical oestrogen therapy has been shown to provide modest treatment benefits for women experiencing UI, mostly improving urinary urgency and frequency related symptoms [18]. Recent literature suggests that topical oestrogen does not increase incidence of oestrogen-dependent malignancies, cardiovascular or thromboembolic complications [19]. However, women with a personal history of these conditions, especially breast cancer, are often advised to avoid any hormonal treatment and many others still decline, leaving them with few alternatives for management of their SUI. Furthermore, topical oestrogen benefits only last while the product is being applied, which requires patient compliance.

history of these conditions, especially breast cancer, are often advised to avoid any hormonal treatment and many others still decline, leaving them with few alternatives for management of their SUI. Furthermore, topical oestrogen benefits only last while the product is being applied, which requires patient compliance. Since the 1990s, surgical management of SUI has shifted from the more invasive traditional Burch colposuspension to increasing use of the MUS, due to shorter operating time and fewer complications (except for bladder injury) with improved outcomes [7]. Furthermore, recent FDA statements, TGA withdrawal of mesh products, class actions, and media focus on mesh in urogynaecological procedures has seen a strong public reaction against mesh. There is a 2.4–9.8% risk of mesh erosion after MUS insertion with higher erosion rates after use of transvaginal mesh in POP surgery [[20], [21], [22], [23]].

atements, TGA withdrawal of mesh products, class actions, and media focus on mesh in urogynaecological procedures has seen a strong public reaction against mesh. There is a 2.4–9.8% risk of mesh erosion after MUS insertion with higher erosion rates after use of transvaginal mesh in POP surgery [[20], [21], [22], [23]]. Trans-vaginal fractionated CO2 laser treatment has been shown to improve vaginal tissue health in women with vaginal atrophy [12]. Since 2014, a growing number of studies have been published exploring the use of trans-vaginal laser treatment for gynaecogical conditions such as SUI, mixed UI (MUI) and genitourinary symptoms of menopause (GSM) [16,25]. Salvatore et al. first published a pilot study of 50 women with GSM who were dissatisfied with topical oestrogen therapy. Following three treatments with fractional CO2 laser, the outcomes were assessed with the Vaginal Health Index Score (VHIS) and Short Form-12 Quality of Life (SF-12 QOL) survey. They found significant improvements in the symptoms and impact of GSM on QOL by all measures assessed at 12 weeks [15]. Behnia-Willison et al. showed in a study treating 102 women with GSM with fractional CO2 laser having improvements up to 24 months by colposcopic examinations and responses to APFQ [24].

QOL) survey. They found significant improvements in the symptoms and impact of GSM on QOL by all measures assessed at 12 weeks [15]. Behnia-Willison et al. showed in a study treating 102 women with GSM with fractional CO2 laser having improvements up to 24 months by colposcopic examinations and responses to APFQ [24]. Regarding use of laser for UI, Ogrinc et al. recruited 175 women with symptoms of SUI or MUI. Their treatment protocol included an erbium-doped yttrium-aluminium-garnet (Er:YAG) laser to deliver both non-ablative full circumferential vaginal and fractionated anterior vaginal wall treatment; this significantly improved symptoms in 77% of women with SUI and 34% of women with MUI [26]. Two other prospective cohort studies using Er:YAG laser to treat SUI showed similar positive results [26,27]. This study builds on data from other prospective cohort studies showing that Er:YAG vaginal laser treatment was able to significantly improve symptoms of SUI short-term (up to 12 months) as assessed by the International Consultation on Incontinence Questionnaire (ICIQ) and the Incontinence Severity Index (ISI) [27]. Similarly, Isaza et al. demonstrated long-term benefits up to 36 months after CO2 laser for women with mild SUI and GSM [28]. Hence, the treatment poses minimal risks and this study also adds to data demonstrating a good safety profile with trans-vaginal use.

naire (ICIQ) and the Incontinence Severity Index (ISI) [27]. Similarly, Isaza et al. demonstrated long-term benefits up to 36 months after CO2 laser for women with mild SUI and GSM [28]. Hence, the treatment poses minimal risks and this study also adds to data demonstrating a good safety profile with trans-vaginal use. The purpose of this study was to include women who were still symptomatic after first line conservative treatment, including pelvic floor exercises and vaginal oestrogen therapy, and desired to have an alternative non-surgical therapy. Whilst a blinded RCT would be ideal, data from this observational study suggests that fractionated CO2 laser treatment of the vagina results was able to produce comparable outcomes to MUS procedures, with ongoing improvement of SUI symptoms at 12–24 months. It is most likely that maintenance CO2 laser therapy is required. Due to individual ageing pattern and menopause effect and uncertain treatment intervals after T3, further studies are required.

a results was able to produce comparable outcomes to MUS procedures, with ongoing improvement of SUI symptoms at 12–24 months. It is most likely that maintenance CO2 laser therapy is required. Due to individual ageing pattern and menopause effect and uncertain treatment intervals after T3, further studies are required. Limitations of the study include the study design and attrition rate. We experienced a high attrition rate for responses from T1 (n = 58) to T3 (n = 36), which introduces a possibility of attrition bias and can weaken the strengths of our findings. At T2, all patients attended follow-up but 3 patients did not complete the data. At T3, there was 62% of women who attended follow-up. Another limitation was our primary outcome being based on a subjective measure reported by the participants and objective cough test and bladder neck funnelling by the treating specialist; only 33 participants agreed to undergo urodynamic studies. The remaining participants declined this test due to invasiveness and social circumstances, such as cost and insurance cover. There were 4 participants who underwent urodynamics and surgical management which had resolved their SUI symptoms. Another limitation is that this is an uncontrolled study and subjected to the usual biases. In addition there are well known placebo effects with the use of new medical devices. However, the strength of this study is the potential to develop a another non-surgical treatment option for symptomatic SUI. The first line treatment includes oestrogen cream for vaginal atrophy, pelvic floor muscle training, and lifestyle optimisation. The second line treatment includes CO2 laser treatment +/- platelet-rich plasma (PRP). Then third line treatment would be surgery.

a another non-surgical treatment option for symptomatic SUI. The first line treatment includes oestrogen cream for vaginal atrophy, pelvic floor muscle training, and lifestyle optimisation. The second line treatment includes CO2 laser treatment +/- platelet-rich plasma (PRP). Then third line treatment would be surgery. In summary, micro-ablative fractional CO2 laser treatment appears to be a promising, non-surgical, non-hormonal, minimally invasive, durable, low risk treatment option for women with SUI. The safety this treatment modality and the reduced prevalence as per self-reported SUI symptom reduction from baseline suggests a possible alternative for women with SUI who are unwilling to accept the inherent risks of MUS and Burch colposuspension, or whose medical comorbidities exclude surgical treatment. Further research should compare the use of fractionated CO2 laser with placebo and/or established treatments, as well as determine whether booster treatment is required to sustain improvements in SUI symptoms longer term. Disclosures The authors have no conflict of interest. FBW is a preceptor for MonaLisa Touch and runs laser workshops for the company. FBW is paid to run these teaching workshops but received no support, financial or otherwise, to run this study. Funding There were no sources of funding for this study. Conflict of interest The authors have no conflicts of interest to declare.

Disclosures The authors have no conflict of interest. FBW is a preceptor for MonaLisa Touch and runs laser workshops for the company. FBW is paid to run these teaching workshops but received no support, financial or otherwise, to run this study. Funding There were no sources of funding for this study. Conflict of interest The authors have no conflicts of interest to declare. Appendix A Australian Pelvic Floor Questionnaire – Bladder Function Items Questionnaire item Response options APFQ1. How many times do you pass urine in the day? (0) Up to 7 (1) Between 8-10 (2) Between 11-15 (3) More than 15 APFQ2. How many times do you get up at night to pass urine? (0) 0–1 (1) 2 times (2) 3 times (3) More than 3 times APFQ3. Do you wet the bed before you wake up at night? (0) Never (1) Occasionally–less than once per week (2) Frequently–once or more than per week (3) Always–every night APFQ4. Do you need to rush or hurry to pass urine when you get the urge? (0) Never (1) Occasionally (2) Frequently (3) Daily APFQ5. Does urine leak when you rush or hurry to the toilet? i.e. You can’t make it in time? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ6. Do you leak urine with coughing, sneezing, laughing or exercising? (0) Not at all (1) Occasionally (2) Frequently (3) Daily APFQ7. Is your urinary stream (urine flow) weak, prolonged or slow? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ8. Do you have a feeling of incomplete bladder emptying? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ9. Do you need to strain to empty your bladder? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ10. Do you wear pads because of urinary leakage? (0) None-never (1) As a precaution (2) When exercising/during a cold (3) Always APFQ11. Do you limit your fluid intake to decrease leakage? (0) Never (1) Before going out/socially (2) Moderately (3) Daily APFQ12. Do you have frequent bladder infections? (0) No (1) 1–3 per year (2) 4–12 per year (3) More than once per month APFQ13. Do you have pain in your bladder or urethra when you empty your bladder? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ14. Does the urine leakage affect your routine activities like recreation, socialising, sleeping, shopping, etc.? (0) Not at all (1) Slightly (2) Moderately (3) Greatly APFQ15.

r urethra when you empty your bladder? (0) Never (1) Occasionally– less than once per week (2) Frequently–more than once per week (3) Daily APFQ14. Does the urine leakage affect your routine activities like recreation, socialising, sleeping, shopping, etc.? (0) Not at all (1) Slightly (2) Moderately (3) Greatly APFQ15. How much does the bladder problem bother you (0) Not at all (1) Slightly (2) Moderately (3) Greatly Acknowledgements We wish to acknowledge the staff of FBW Gynaecology Plus in assisting with the running and follow-up of participants of the study. We wish to thank Aidan Norbury for his contribution to the paper.

Introduction Abdominal sacrocolpopexy has been shown to be an effective treatment for pelvic organ prolapse. However, in a 7 year follow-up study of over 200 cases, failure rates increased with time and the overall risk of mesh extrusion was 10.5% [1]. The laparoscopic approach to sacrocolpopexy has also been shown to be effective in terms of anatomical and functional success at long term follow-up and mesh extrusion was seen in 6% cases [2]. Mesh complications can vary from a small thread of mesh protruding through the vaginal epithelium to infection of the whole mesh, requiring complete removal. Increasing concern about the problem of mesh extrusion and exposure has led to the development of meshes with a different structure and weight. However there is no evidence to determine whether the reduction in density has an impact on efficacy or risk of extrusion. Materials and methods This study is a retrospective review of the outcome of a consecutive series of women treated with a laparoscopic sacrocolpopexy (LSCP) since 2005 using the same techniques we published in a previous study [2], but employing an ultra-lightweight polypropylene mesh (Restorelle®, Coloplast A/S, Denmark, acquired from Mpathy Medical Devices in 2010). The objectives of the study were to explore the functional and anatomical outcomes of women following LSCP using an ultra-lightweight polypropylene mesh including subjective and objective recurrence of prolapse and mesh adverse events.

(Restorelle®, Coloplast A/S, Denmark, acquired from Mpathy Medical Devices in 2010). The objectives of the study were to explore the functional and anatomical outcomes of women following LSCP using an ultra-lightweight polypropylene mesh including subjective and objective recurrence of prolapse and mesh adverse events. Restorelle is a Type I monofilament polypropylene mesh with a density of 19 g/m [3]. It is therefore considered an ultra-lightweight mesh [4]. Restorelle has been used exclusively at our institution for LSCP since 2005. Prior to this Prolene© (Ethicon, Johnson & Johnson Medical Limited, USA) was used, which is classified as a Standard mesh, with a density of 82.5 g/m2 [5]. LSCP has been the preferred method for surgical treatment of vaginal vault prolapse in the department since 1993. Patients are not excluded on account of obesity or previous surgery (unless complicated) and several patients have had a previous open sacrocolpopexy. The cohort included all women who underwent LSCP using Restorelle mesh at either St Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester (n = 189) or BMI The Alexandra Hospital, Manchester (n = 49) between March 2005 and January 2013. These women were subsequently invited to attend a follow-up examination and complete a number of self-report questionnaires between August 2012 and August 2013.

chester University Hospitals NHS Foundation Trust, Manchester (n = 189) or BMI The Alexandra Hospital, Manchester (n = 49) between March 2005 and January 2013. These women were subsequently invited to attend a follow-up examination and complete a number of self-report questionnaires between August 2012 and August 2013. Women willing to participate were invited to attend an appointment during which a pelvic examination was performed to assess vaginal support using the POPQ assessment and mesh palpability. The pelvic examination was performed by a member of the research team rather than a member of the surgical team who had performed the LSCP in an attempt to reduce the potential for information bias. The women were asked to complete the following questionnaires; the Patient Global Impression of Improvement questionnaire for urogenital prolapse (PGI-I)-a validated questionnaire which asks the participant to describe how their post-operative condition is now compared with prior to their surgery; The Electronic Personal Assessment Questionnaire (EPAQ)-a validated patient-focused symptom and quality of life questionnaire exploring the frequency, severity and impact of symptoms related to bladder, bowel, vaginal and sexual domains; The Pelvic Floor Distress Inventory (PFDI-20)-a validated questionnaire in which the participant is asked to consider bowel, bladder and pelvic symptoms over the past 3 months and if present to report how bothersome the symptom is for them.

y and impact of symptoms related to bladder, bowel, vaginal and sexual domains; The Pelvic Floor Distress Inventory (PFDI-20)-a validated questionnaire in which the participant is asked to consider bowel, bladder and pelvic symptoms over the past 3 months and if present to report how bothersome the symptom is for them. Women unable or unwilling to attend a follow-up appointment were invited to complete the study questionnaires at home and give written permission for the study team to collect relevant data from their medical records. 7 patients had died since surgery, 6 of these deaths were unrelated to the procedure, therefore approval was obtained to allow access to the deceased patients’ medical records wherever possible. The surgical technique used Restorelle as a flat mesh which was attached to the posterior and anterior surface of the vagina. It is sutured to the vagina with 6–12 vicryl sutures and secured to the anterior aspect of the 5th lumbar vertebrae with titanium helical screws.

Women unable or unwilling to attend a follow-up appointment were invited to complete the study questionnaires at home and give written permission for the study team to collect relevant data from their medical records. 7 patients had died since surgery, 6 of these deaths were unrelated to the procedure, therefore approval was obtained to allow access to the deceased patients’ medical records wherever possible. The surgical technique used Restorelle as a flat mesh which was attached to the posterior and anterior surface of the vagina. It is sutured to the vagina with 6–12 vicryl sutures and secured to the anterior aspect of the 5th lumbar vertebrae with titanium helical screws. Results 231 women were identified; however 75 women declined to participate or failed to respond, leaving the remaining sample of 156 women. 101 women (65%) attended a follow-up appointment and 147 (94%) completed study questionnaires. Median follow-up time was 34 months post-operatively (range 1–94 months). 25 (16%) study participants had their surgery more than 5 years prior to follow-up. At the time of surgery the mean age was 61 (range: 38–85 SD: 10.3), mean BMI was 27 (range: 19–38 SD: 3.9) and the median parity was 2 (range 0–7). 87 (58%) of the sample had previously had a laparotomy. The number of participants with comorbidities at the time of surgery was low with 6 (4%) having diabetes, 19 (13%) receiving cardiovascular treatment (primarily for hypertension) and 2 (1%) receiving long-term anti-coagulation therapy.

an parity was 2 (range 0–7). 87 (58%) of the sample had previously had a laparotomy. The number of participants with comorbidities at the time of surgery was low with 6 (4%) having diabetes, 19 (13%) receiving cardiovascular treatment (primarily for hypertension) and 2 (1%) receiving long-term anti-coagulation therapy. 28 (18%) of the women had a history of previous vault support surgery. All women presented with vault prolapse; none of the LCSP were performed at the same time as hysterectomy. The route of previous hysterectomy was evenly distributed between the women, with 77 (51%) having had an abdominal hysterectomy and 75 (49%) a vaginal hysterectomy. The number of previous prolapse operations amongst the sample ranged between 0–5 (median = 1 SD: 0.9). Further details of participants’ history of prolapse surgery are documented within Table 1.Table 1 Previous gynaecological surgery. Table 1 n= % LSCP 6 4% Open SCP 9 6% SSF 13 8% Vaginal repair 90 58% TVT 6 4% TOT 5 3.2% Colposuspension 14 9% Abdominal hysterectomy 77 50% Vaginal hysterectomy 75 49% Adverse events during or immediately following surgery are detailed in Table 2. There was one death 16 days following surgery due to port site hernia complications (patient included within bowel injury data).Table 2 Adverse Events. Table 2Adverse event n = Percentage Bladder injury 2 1.3% Ureter injury 1 0.6% Vaginal injury 2 1.3% Rectal injury 1 0.6% Bowel injury 2 1.3% Blood transfusion 2 1.3% Death 1 0.6%

Adverse events during or immediately following surgery are detailed in Table 2. There was one death 16 days following surgery due to port site hernia complications (patient included within bowel injury data).Table 2 Adverse Events. Table 2Adverse event n = Percentage Bladder injury 2 1.3% Ureter injury 1 0.6% Vaginal injury 2 1.3% Rectal injury 1 0.6% Bowel injury 2 1.3% Blood transfusion 2 1.3% Death 1 0.6% Global outcome 147 women completed the PGI-I; 75% of participants reported that they felt their post-operative condition was “very much better"” or “much better”. 7% reported that their condition was now “worse”. The patients who felt that their post-operative condition had worsened ranged between 1–57 months post-operatively (Median: 28 SD: 17.1). Further analysis of the anatomical outcome of the 10 women who felt that their post-operative condition had worsened, revealed that of the 7 who had been re-examined as part of the study, only one woman (14%) had a failure of apical support (defined as stage 2 or more) with point C at +5 (Stage 3). This same degree of recurrent apical prolapse was also noted at the woman’s routine 6 month post-operative follow-up appointment 51 months previously. Of the remaining women who felt that their condition had deterioriated, 71% (n = 5) had an apical stage of 0 and 14% (n = 1) an apical stage of 1. When comparing pre and post-operative EPAQ scores for this group of women, an improvement was seen in the mean scores for bladder, bowel, vaginal and sexual symptoms.

ously. Of the remaining women who felt that their condition had deterioriated, 71% (n = 5) had an apical stage of 0 and 14% (n = 1) an apical stage of 1. When comparing pre and post-operative EPAQ scores for this group of women, an improvement was seen in the mean scores for bladder, bowel, vaginal and sexual symptoms. Anatomical outcome As detailed in Table 3, the median position of C changed from −3 pre-op (IQR = −4 to 1) to −7 post-operatively (IQR = −8 to −6). Similar improvements were noted in the other points. 2 women (2%) had a failure of apical support, both being beyond the hymen.Table 3 - Comparison of Pre and Post Surgery POPQ. Table 3 Pre POPQ C Post POPQ C n= 132 100 Range −6 to +10 −12 to +8 Median −3 −7 Stdev 4 2.5 Pre POPQ Aa Post POPQ Aa n= 128 100 Range −1 to +3 −3 to +3 Median 0 −2 Stdev 1.9 1.6 Pre POPQ Ba Post POPQ Ba n= 128 100 Range −3 to +10 −3 to +4 Median +1 −2 Stdev 3 1.7 Pre POPQ Ap Post POPQ Ap n= 127 100 Range −3 to +3 −3 to +3 Median −1 −2 Stdev 2.1 1.6 Pre POPQ Bp Post POPQ Bp n= 127 100 Range −3 to +10 −3 to +3 Median 0 −1 Stdev 3 1.4 Pre POPQ GH Post POPQ GH n= 73 100 Range 2–8 2–7 Median 5 4 Stdev 1.1 0.8 Pre POPQ PB Post POPQ PB n= 73 100 Range 2–7 3–6 Median 4 4 Stdev 0.9 0.6 Pre POPQ TVL Post POPQ TVL n= 86 100 Range 5–11 6–13 Median 8 8 Stdev 1.3 1.2

v 2.1 1.6 Pre POPQ Bp Post POPQ Bp n= 127 100 Range −3 to +10 −3 to +3 Median 0 −1 Stdev 3 1.4 Pre POPQ GH Post POPQ GH n= 73 100 Range 2–8 2–7 Median 5 4 Stdev 1.1 0.8 Pre POPQ PB Post POPQ PB n= 73 100 Range 2–7 3–6 Median 4 4 Stdev 0.9 0.6 Pre POPQ TVL Post POPQ TVL n= 86 100 Range 5–11 6–13 Median 8 8 Stdev 1.3 1.2 Functional outcome It is acknowledged that a patient’s functional outcome may be affected by concomitant surgery, which was performed for 4.6% (n = 8) of participants. The most frequent concomitant surgery was rectopexy in 5 women (3%), followed by TVT in 2 women (1%) and laparoscopic colposuspension for 1 women (0.6%). Data from EPAQ was analysed to assess functional outcome post. EPAQ provides scores from 0 to 100 with higher scores representing more severe symptoms. Therefore a patient reporting an improvement in symptoms would have a lower symptom score post-operatively. Persistent bowel and bladder symptoms following LSCP were reported as detailed in Table 4. Bowel symptoms were both the most prevalent and bothersome symptoms at follow-up. Despite experiencing on-going symptoms, the prevalence and bothersomeness of both bowel and bladder symptoms had significantly improved following surgery.Table 4 -Comparison of Pre and Post Surgery Functional Outcome.

detailed in Table 4. Bowel symptoms were both the most prevalent and bothersome symptoms at follow-up. Despite experiencing on-going symptoms, the prevalence and bothersomeness of both bowel and bladder symptoms had significantly improved following surgery.Table 4 -Comparison of Pre and Post Surgery Functional Outcome. Table 4 Pre Urinary Quality of Life Post Urinary Quality of Life Significance n= 77 72 Symptomatic patients n= 66 35 Percentage of symptomatic patients 86% 49% p = .01 Mean 37.7 16.5 Stdev 28.1 25.6 Pre Bowel Quality of Life Post Bowel Quality of Life Significance n= 72 71 Symptomatic patients n= 38 35 Percentage of symptomatic patients 53% 49% Mean 18.5 13.1 p = .01 Stdev 24.1 21.3 Pre Vaginal Quality of Life Post Vaginal Quality of Life Significance n= 73 132 Symptomatic patients n= 72 29 Percentage of symptomatic patients 99% 22% Mean 50.3 10.2 p = .01 Stdev 32.9 19.6 Responses to EPAQ questions within the vaginal domain demonstrated a significant improvement in prolapse symptoms reported post-operatively as detailed in Table 4. 76 (76%) of participants reported no awareness of vaginal bulge at study follow up. Women’s perception of sexual function improved from a mean of 42 pre-operatively to 25 post-operatively. However the percentage of women reporting that they were sexually active did not change pre (53% n = 31) and post-operatively (52% n = 60).

of participants reported no awareness of vaginal bulge at study follow up. Women’s perception of sexual function improved from a mean of 42 pre-operatively to 25 post-operatively. However the percentage of women reporting that they were sexually active did not change pre (53% n = 31) and post-operatively (52% n = 60). Mesh extrusion No mesh complications were identified during the course of the study. However, one patient required a posterior vaginal repair 12 months post LSCP which was performed without mesh. When this patient was seen as part of the research study, 28 months following LSCP, there was no evidence of mesh extrusion. However, 12 months later the patient was diagnosed with upper posterior vaginal wall extrusion of mesh requiring surgical management. The mesh extrusion was noted along the path of the posterior repair wound.

ient was seen as part of the research study, 28 months following LSCP, there was no evidence of mesh extrusion. However, 12 months later the patient was diagnosed with upper posterior vaginal wall extrusion of mesh requiring surgical management. The mesh extrusion was noted along the path of the posterior repair wound. Mesh palpability It had been noted that mesh was palpable in many of the women examined vaginally after LSCP with standard weight mesh. In this study mesh palpability was assessed using a non-validated palpability assessment (Appendix A). Mesh was palpable during examination in only 3 women (3%). The palpable mesh was identified in the three cases during study participation at 19, 21 and 34 months post-operatively. Since the examination was only performed on one occasion in each case, it is not known whether this is a progressive phenomenon. All three women reported dyspareunia when completing study questionnaires but had no other symptoms. When their medical records were reviewed it was noted that all three women reported dyspareunia pre-operatively, with one woman’s dyspareunia worsening since her surgery. No further treatment was requested for any of the women with palpable mesh. Dyspareunia was also an issue for women who did not have palpable mesh. 54% (n = 47) reported this as a symptom post-operatively. When comparing dysparaeunia pre and post operatively, 6.5% (n = 2) of participants experienced de novo dysparaeunia, mesh was not palpable upon examination for either patient.

palpable mesh. Dyspareunia was also an issue for women who did not have palpable mesh. 54% (n = 47) reported this as a symptom post-operatively. When comparing dysparaeunia pre and post operatively, 6.5% (n = 2) of participants experienced de novo dysparaeunia, mesh was not palpable upon examination for either patient. Anatomical and functional outcome and mesh extrusion over time All patients were invited to participate regardless of the period of time since surgery. The outcomes have been divided between short-term (less than 12 months since surgery), medium-term (12 months to 5 years since surgery) and long-term (more than 5 years since surgery) Table 5.Table 5 -Study outcomes for short, medium and long-term follow-up participants. Table 5 PFDI20 <1 year PFDI20 1 year-5 year PFDI20 >5years n= 16 102 25 Mean 16.5 22 24.1 Stdev 19.7 5.5 20.2 PGI <1 year PGI 1 year-5 year PGI >5years n= 16 101 25 Mean 1.8 2 1.7 Stdev 1.3 1.6 0.7 POPQ C <1 year POPQ C 1 year-5 year POPQ C >5years n= 11 69 16 Median −8 −7 −7 IQR −8.5 to −7 −8 to −6 −7.5 to −6.5 Mesh palpability <1 year Mesh palpability 1 year-5 year Mesh palpability >5years n= 11 70 16 Palpable mesh 0 3 0 % 0 4% 0% Patients in the long term follow-up group had higher PFDI scores than those in the shortest follow-up group. The global impression of improvement did not differ between groups of different follow-up length.

Table 5 PFDI20 <1 year PFDI20 1 year-5 year PFDI20 >5years n= 16 102 25 Mean 16.5 22 24.1 Stdev 19.7 5.5 20.2 PGI <1 year PGI 1 year-5 year PGI >5years n= 16 101 25 Mean 1.8 2 1.7 Stdev 1.3 1.6 0.7 POPQ C <1 year POPQ C 1 year-5 year POPQ C >5years n= 11 69 16 Median −8 −7 −7 IQR −8.5 to −7 −8 to −6 −7.5 to −6.5 Mesh palpability <1 year Mesh palpability 1 year-5 year Mesh palpability >5years n= 11 70 16 Palpable mesh 0 3 0 % 0 4% 0% Patients in the long term follow-up group had higher PFDI scores than those in the shortest follow-up group. The global impression of improvement did not differ between groups of different follow-up length. The median position of the POPQ measurement ‘C’ was similar at all time periods demonstrating the durability of anatomical outcome. The likelihood of mesh palpability does not appear to increase over time with all 3 cases being reported in the medium-term follow-up group. Comment This is the first study to report a cohort of patients undergoing LSCP using an ultra-light weight polypropylene mesh 19 g/m2. The study has demonstrated good, durable anatomical and patient reported outcomes.

The median position of the POPQ measurement ‘C’ was similar at all time periods demonstrating the durability of anatomical outcome. The likelihood of mesh palpability does not appear to increase over time with all 3 cases being reported in the medium-term follow-up group. Comment This is the first study to report a cohort of patients undergoing LSCP using an ultra-light weight polypropylene mesh 19 g/m2. The study has demonstrated good, durable anatomical and patient reported outcomes. The reason for using a lighter weight mesh is the theoretical reduction in mesh extrusion and dyspareunia. Brown et al and Liang et al demonstrated that this ultra-lightweight mesh (19 g/m2) generated a reduced inflammatory response and better tissue remodelling when compared to other meshes [5,6]. The outcomes in this current study can be compared to a previous published cohort from the same institution [2], using a heavier polypropylene mesh (Ethicon, Johnson & Johnson Medical Limited, USA) with a density of 82.5 g/m2. Whilst it is acknowledged that there are limitations when making comparisons between two cohorts, at different times, the surgical technique of LSCP has not changed significantly over the time period and the study population is a comparable group of patients; the only significant change at our institution has been the use of the new ultra-lightweight mesh. A comparison of the two cohorts, in Table 6, suggests that use of an ultra-lightweight mesh does not appear to have any negative effect on the vaginal support. There appears to be a lower incidence of mesh extrusion with the ultra-lightweight mesh, although the 2005 study had a longer follow-up interval. The median follow-up in the 2005 cohort was 66 months compared to 28 months for the current cohort. The only mesh extrusion within the ultra-lightweight mesh cohort was in a woman who had a posterior repair 12 months following LSCP; it is possible that opening the posterior vaginal wall during this procedure may have increased the potential for mesh extrusion particularly since the extrusion occurred along the posterior repair wound. It is reported in the literature that when hysterectomy is performed concomitantly with LSCP the risk of mesh extrusion increases significantly [7]. Possible explanations for this include devascularisation of the vaginal cuff and bacterial infection [7]. In the department’s previous experience, mesh extrusion has developed up to 8 years after surgery. It is not known whether this is related to the mesh type. The current cohort only contained 25 women who had undergone surgery more than 5 years earlier.

devascularisation of the vaginal cuff and bacterial infection [7]. In the department’s previous experience, mesh extrusion has developed up to 8 years after surgery. It is not known whether this is related to the mesh type. The current cohort only contained 25 women who had undergone surgery more than 5 years earlier. Future research exploring the long-term outcome of LSCP performed with Restorelle mesh in a large cohort of women who are more than 5 years post-surgery is required to answer this question.Table 6 Comparison of study outcome data with 2005 study [2]. Table 6After surgery 2005 [2] Current study Failure of apical support 8% 2% Awareness of a lump/bulge 38% 24% Urinary symptoms improved 28% 63% Urinary symptoms no change 49% 26% Urinary symptoms worsened 23% 11% Bowel symptoms improved 44% 34% Bowel symptoms no change 48% 47% Bowel symptoms worsened 8% 18% Ten women (7%) perceived that their condition had worsened post-operatively despite only one of these women having anatomical prolapse and an overall improvement in all EPAQ scores. Due to the long-term nature of the study, this may reflect participant reporting of new symptoms which have occurred since surgery but are not related to the LSCP. However, data from both studies (Table 6) demonstrates that many women experience persistent bowel and bladder symptoms following LSCP regardless of the mesh used and the support gained. Hence patients should be advised that although surgery may correct anatomical defects it is difficult to predict the functional outcome.

ever, data from both studies (Table 6) demonstrates that many women experience persistent bowel and bladder symptoms following LSCP regardless of the mesh used and the support gained. Hence patients should be advised that although surgery may correct anatomical defects it is difficult to predict the functional outcome. The use of different outcome measures between this and our earlier study prevents direct comparison of study findings. In 2005 participants were simply asked whether they had experienced an improvement, no change or a worsening of their bladder and bowel symptoms. Therefore although the findings facilitate some comparison between the studies, limitations with this comparison is acknowledged. The percentage of women who were sexually active before and after surgery did not change which suggests that prolapse surgery and a subsequent improvement in prolapse symptoms, does not increase the likelihood that a woman will be sexually active. For many women there may be reasons other than prolapse which impact upon their sexual activity. The study evaluated sexual function using EPAQ. Within EPAQ dyspareunia is a collection of symptoms of discomfort and pain, such as vaginal dryness, lack of sensation, tightness and obstruction. Some women reported an improvement in dyspareunia but for over half of the sexually active women the problem persisted. Therefore although LSCP may improve sexual function for women whose cause of dyspareunia is obstruction due to prolapse, dyspareunia related to other issues may remain unchanged.

ness and obstruction. Some women reported an improvement in dyspareunia but for over half of the sexually active women the problem persisted. Therefore although LSCP may improve sexual function for women whose cause of dyspareunia is obstruction due to prolapse, dyspareunia related to other issues may remain unchanged. We hypothesised that mesh palpability would be associated with dyspareunia. However mesh was only palpable in three women, all of whom reported pre-operative dyspareunia with one lady’s symptoms worsening following surgery. Dyspareunia was also an issue for over half of women who did not have palpable mesh, therefore the relationship between mesh palpability and dyspareunia is not clear. It is possible that post-operative infections were under reported in this study as the incidence of these events was collected from hospital records. Reduced length of hospital stay means that minor post-operative complications are more likely to be reported after discharge from hospital. It is possible that women who were least satisfied with their surgical outcome were less motivated to participate in the study. Conversely, those who were least satisfied with their surgical outcome may also have been increasingly motivated to participate in the study to access further treatment and assessment. The influence of selection bias amongst the sample is unknown. It is acknowledged that the generalisability of the findings may be restricted as the sample is limited to women who had surgery performed by one group of surgeons.

It is possible that women who were least satisfied with their surgical outcome were less motivated to participate in the study. Conversely, those who were least satisfied with their surgical outcome may also have been increasingly motivated to participate in the study to access further treatment and assessment. The influence of selection bias amongst the sample is unknown. It is acknowledged that the generalisability of the findings may be restricted as the sample is limited to women who had surgery performed by one group of surgeons. This study demonstrates that LSCP performed with an ultra-lightweight polypropylene mesh improves patient’s functional and anatomical outcome. The diagnosis of a single mesh extrusion within this patient cohort suggests that ultra-lightweight mesh is associated with a lower risk of a mesh extrusion than LSCP performed with heavier mesh. The use of the lighter mesh does not appear to reduce the strength of the support provided. Conflict of interest The authors declare that they have no conflict of interest. Funding The study was Investigator initiated and financially supported by Coloplast Corp and facilitated by the Greater Manchester Local Clinical Research Network. Coloplast Corp had no role in the collection, analysis or interpretation of data or writing of the manuscript. Ethical approval This research study has been reviewed by the National Research Ethics Service Committee North West - Greater Manchester Central. Reference: 12/NW/0277.

Funding The study was Investigator initiated and financially supported by Coloplast Corp and facilitated by the Greater Manchester Local Clinical Research Network. Coloplast Corp had no role in the collection, analysis or interpretation of data or writing of the manuscript. Ethical approval This research study has been reviewed by the National Research Ethics Service Committee North West - Greater Manchester Central. Reference: 12/NW/0277. ISRCTN reference: 19907894 http://www.isrctn.com/ISRCTN19907894?q=laparoscopic%20sacrocolpopexyrelease&filters=&sort=&offset=1&totalResults=1&page=1&pageSize=10&searchType=basic-search. Contribution to authorship All authors made substantial contributions to this study and article. LD, WK, KW, FR and ARBS participated in the design and/or execution of the study, the interpretation of the data, and the drafting and/or revision of the article. All authors approve this version to be published. Appendix A Palpability Assessment Acknowledgements The study team would like to acknowledge and thank the women who have made this research possible by participating in the study. ☆ The preliminary findings of this study were presented as a poster at The 34th Annual Scientific Meeting of The American Urogynecology Society (AUGS), Las Vegas, Nevada, October 16–19, 2013.

Introduction Endometriosis is a condition in which endometrial tissue (uterine gland and stroma) is present outside the endometrium of the uterus. These ectopic endometrial cells express receptors for various sex hormones, depending on the patient’s menstrual cycle. Lesions are characterized by localized bleeding and inflammation, resulting in fibrosis and adhesions and causing pain and infertility. Ectopic endometrial cells can also invade other tissues. Although endometriosis is not malignant, its ability to infiltrate into and invade distant tissues shows the same pattern as metastases of malignant tumors. The etiology and pathogenesis of endometriosis have not yet been clarified, although various hypotheses have been suggested [1,2].

s can also invade other tissues. Although endometriosis is not malignant, its ability to infiltrate into and invade distant tissues shows the same pattern as metastases of malignant tumors. The etiology and pathogenesis of endometriosis have not yet been clarified, although various hypotheses have been suggested [1,2]. Since autophagy-related genes (Atg) were first detected in yeast, more than 30 Atg genes have been identified [3,4]. Autophagy is a major catabolic process, in which intracellular constituents that are unnecessary or malfunctioning are degraded by lysosomes [5,6]. Autophagy regulates organ development, differentiation and tissue regeneration in various organs by regulating the balance between the synthesis and degradation of cell organs and proteins, i.e., the conversion rate [7]. Autophagy may be induced by deficiencies in intracellular glucose and amino acids, as well as by hypoxia, oxidative stress, and treatment with chemotherapeutic agents. In addition, autophagy has been shown to interact with reactive oxygen species to regulate cellular signaling and protein damage, and to act as a mediator of various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as various pathological phenomena such as cancer, diabetes, cardiovascular disease, and inflammatory reactions [8]. Studies on the mechanisms of action and roles of Atgs in various diseases have shown that autophagy plays important roles not only in the degradation of clumps of protein, but in the removal of damaged intracellular organelles and external pathogens. Dysregulation of autophagy is a major cause of diseases such as cancer, metabolic diseases, pathogen infection, otitis media, lung disease, Cohn's disease, degenerative brain disease, and heart disease, and even aging [[9], [10], [11]]. Autophagic processes are elaborately controlled by the proteins they produce, and can be classified into various steps, consisting of initiation, vesicle nucleation, vesicle elongation, fusion, degradation, and termination. The present study analyzed the expression of agonists and antagonists of autophagy. These mTOR is involved in induction and initiation P13KC3 and Beclin-1 are involved in nucleation and BCL-2 acts an antagonist of Beclin-1. LC3 II is involved in autophagosome biogenesis. FLIP is involved in vesicle elongation and Atg antagonistic activity.

lyzed the expression of agonists and antagonists of autophagy. These mTOR is involved in induction and initiation P13KC3 and Beclin-1 are involved in nucleation and BCL-2 acts an antagonist of Beclin-1. LC3 II is involved in autophagosome biogenesis. FLIP is involved in vesicle elongation and Atg antagonistic activity. Rubicon is involved in the inhibition of maturation and BIRC2 & BIRC5 act as antagonists of Rubicon [[9], [10], [11]]. To assess the role of autophagy in the development of endometriosis and gynecologic cancers, this study evaluated the expression of autophagy-associated mRNAs in the peritoneal fluid of patients with endometriosis; benign, non-endometriosis tumors; and gynecologic (ovarian, uterine, and cervical) cancers.

[10], [11]]. To assess the role of autophagy in the development of endometriosis and gynecologic cancers, this study evaluated the expression of autophagy-associated mRNAs in the peritoneal fluid of patients with endometriosis; benign, non-endometriosis tumors; and gynecologic (ovarian, uterine, and cervical) cancers. Subjects & methods Subjects Intraperitoneal fluid samples were obtained from 112 patients who visited Obstetrics and gynecology department of St. Vincent's Hospital, the Catholic University of Korea and underwent surgery. During laparoscopy, peritoneal fluid was collected aseptically from the Douglas pouch, taking care to avoid bleeding. Patients with inflammatory diseases, hormone producing condition, including pregnancy, and blood contaminated peritoneal fluid or without peritoneal fluid were excluded from the study. The samples were centrifuged at 1800 x g for 10 min; the supernatants were stored at −80 °C in 1.5 ml aliquots; and the cell pellets were stored at −80 °C in 1.5 ml aliquots after adding RNase inhibitor. Findings in the three groups were compared. We excluded patients treated prior to surgery in the gynecologic cancer group in order to eliminate situations that could affect the expression of autophagy by preoperative treatment The study protocol was approved by the institutional review boards (IRBs) of Vincent’s Hospital, The Catholic University of Korea and informed consent was obtained from each patient (VC16TISI0148).

Subjects & methods Subjects Intraperitoneal fluid samples were obtained from 112 patients who visited Obstetrics and gynecology department of St. Vincent's Hospital, the Catholic University of Korea and underwent surgery. During laparoscopy, peritoneal fluid was collected aseptically from the Douglas pouch, taking care to avoid bleeding. Patients with inflammatory diseases, hormone producing condition, including pregnancy, and blood contaminated peritoneal fluid or without peritoneal fluid were excluded from the study. The samples were centrifuged at 1800 x g for 10 min; the supernatants were stored at −80 °C in 1.5 ml aliquots; and the cell pellets were stored at −80 °C in 1.5 ml aliquots after adding RNase inhibitor. Findings in the three groups were compared. We excluded patients treated prior to surgery in the gynecologic cancer group in order to eliminate situations that could affect the expression of autophagy by preoperative treatment The study protocol was approved by the institutional review boards (IRBs) of Vincent’s Hospital, The Catholic University of Korea and informed consent was obtained from each patient (VC16TISI0148). RNA extraction and real-time PCR and CA125 measurment Total RNA was purified from peritoneal fluid using TRIzol solution following the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed with 1 μg of total RNA, which was transcribed to cDNA using a reverse transcription system with random hexamers (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The primer sequences are shown in Table 1. Real-time PCR was performed on a StepOnePlus real-time PCR system with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCR was performed with 1 μl of cDNA in a 20-μl reaction mixture containing 10 μl of Power SYBR Green PCR Master Mix, 2 μl of primers, and 7 μl of PCR-grade water. The reaction conditions were denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The crossing points of the target genes with β-actin were calculated using the formula 2–(target gene–β−actin), and relative amounts were quantified.Table 1 Primers for real-time RT-PCR.

de water. The reaction conditions were denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The crossing points of the target genes with β-actin were calculated using the formula 2–(target gene–β−actin), and relative amounts were quantified.Table 1 Primers for real-time RT-PCR. Table 1Name Sequences annealing temperature Size (bp) mTOR 5’-CCTGCCACTGAGAGATGACA-3’ 5’-TCCGGCTGCTGTAGCTTATT-3’ 60 168 P13KC3 5’-GGAACACCGACCTCACAGTT-3’ 5’-CACAGCACCTCCTCTGTGAA-3’ 60 128 Beclin-1 5’-AGGTTGAGAAAGGCGAGACA-3’ 5’-AATTGTGAGGACACCCAAGC-3’ 60 112 Bcl-2 5’-ATGTGTGTGGAGAGCGTCAA-3’ 5’-ACAGTTCCACAAAGGCATCC-3’ 60 124 LC3 II 5’-AGCAGCATCCAACCAAAATC-3’ 5’-CTGTGTCCGTTCACCAACAG-3’ 60 187 FLIP 5’-GCAGAGTTTCTGCCAAGGAG-3’ 5’-CTCCCAAAGTGCTGGGATTA-3’ 60 123 Rubicon 5’-CAGATTCTGCTGCCTCTTCC-3’ 5’-AGTGTCTGCCCCTCTGAGAA-3’ 60 105 BIRC2 5’-CCAGGTCCCTCGTATCAAAA-3’ 5’-GCACGACAAGACTCCTTTC-3’ 60 179 BIRC5 5’-GCCCAGTGTTTCTTCTGCTT-3’ 5’-TCTCCGCAGTTTCCTCAAAT-3’ 60 104 β-Actin 5′-GCGAGAAGATGACCCAGATC-3′ 5′-GGATAGCACAGCCTGGATAG-3′ 60 77 Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction; mTOR, mechanistic target of rapamycin; P13KC3, class III phosphatidylinositol 3-kinase;Bcl-2, B cell lymphoma 2; LC3 II, light chain 3-II; FLIP, FLICE-inhibitory protein; FLICE, FADD-like IL-1β-converting enzyme; BIRC, baculoviral IAP repeat-containing protein.

: RT-PCR, reverse transcriptase-polymerase chain reaction; mTOR, mechanistic target of rapamycin; P13KC3, class III phosphatidylinositol 3-kinase;Bcl-2, B cell lymphoma 2; LC3 II, light chain 3-II; FLIP, FLICE-inhibitory protein; FLICE, FADD-like IL-1β-converting enzyme; BIRC, baculoviral IAP repeat-containing protein. We measured the CA 125 test in the patient's serum. We take the patient's blood before the operation and measured the CA125 using an immunoradiometric assay kit (IRMA-Count OM-MA) from DPC (Los Angeles, USA) and the normal cut off was 35 U / ml. Statistical analysis The Kolmogorov-Smirnov test was used to assess normality and Levene’s test was used to assess the equality of variances between groups. Differences among groups were analyzed by the Kruskal-Wallis test, with the Bonferroni adjusted Mann-Whitney U test used for post hoc analysis. We showed mean +/- standard deviation in graph. All statistical analyses were performed using SPSS version 13, with a p-value less than 0.05 considered statistically significant.

fferences among groups were analyzed by the Kruskal-Wallis test, with the Bonferroni adjusted Mann-Whitney U test used for post hoc analysis. We showed mean +/- standard deviation in graph. All statistical analyses were performed using SPSS version 13, with a p-value less than 0.05 considered statistically significant. Results Characteristics of patients in the control and experimental groups (Table 2) Peritoneal fluid was obtained from 27 control patients with benign non-endometriosis tumors (mean age, 42.5 ± 12.6 years), 42 patients with endometriosis (mean age, 36.20 ± 13.5 years), and 43 patients with gynecologic (ovarian, uterine, and cervical) cancers (mean age, 49.2 ± 14.1 years).The concentration of cancer antigen 125 (CA-125) was significantly higher in the cancer group than in the other two groups and significantly higher in the endometriosis than in the control group (p < 0.05 each).Table 2 Demographic data. Table 2 control endometriosis cancer (n = 27) (n = 42) (n = 43) Age (years) 42.5 ± 12.6 36.20 ± 13.5 49.2 ± 14.1 Diabetes mellitus 1 (3.7%) 0 (0%) 3 (6.9%) Hypertension 2 (7.4%) 3 (7.1%) 7 (16.3%) Menopause 2 (7.4%) 1 (2.3%) 8 (18.6%) CA-125 (U/ml)* 26.8 ± 18.4 73.9 ± 45.1 205.6 ± 339.1 * p < 0.05.

Results Characteristics of patients in the control and experimental groups (Table 2) Peritoneal fluid was obtained from 27 control patients with benign non-endometriosis tumors (mean age, 42.5 ± 12.6 years), 42 patients with endometriosis (mean age, 36.20 ± 13.5 years), and 43 patients with gynecologic (ovarian, uterine, and cervical) cancers (mean age, 49.2 ± 14.1 years).The concentration of cancer antigen 125 (CA-125) was significantly higher in the cancer group than in the other two groups and significantly higher in the endometriosis than in the control group (p < 0.05 each).Table 2 Demographic data. Table 2 control endometriosis cancer (n = 27) (n = 42) (n = 43) Age (years) 42.5 ± 12.6 36.20 ± 13.5 49.2 ± 14.1 Diabetes mellitus 1 (3.7%) 0 (0%) 3 (6.9%) Hypertension 2 (7.4%) 3 (7.1%) 7 (16.3%) Menopause 2 (7.4%) 1 (2.3%) 8 (18.6%) CA-125 (U/ml)* 26.8 ± 18.4 73.9 ± 45.1 205.6 ± 339.1 * p < 0.05. Expression of autophagy mRNAs in peritoneal fluid (Fig. 1) The levels of PI3K, FLIP, and Rubicon mRNAs were significantly higher in the endometriosis than in the control group (p < 0.05 each). The levels of LC3II and FLIP mRNA were significantly lower, and the levels of Beclin-1 and Rubicon mRNAs significantly higher in the endometriosis than in the gynecologic cancer group (p < 0.05 each). The levels of expression of PI3K and FLIP mRNAs were significantly higher in peritoneal fluid samples from the endometriosis and gynecologic cancer groups than from the control group (p < 0.05 each). The expression of mTOR mRNA was similar in all three groups (p > 0.05).Fig. 1 Comparison of autophagy expression associated mRNAs in control, endometriosis and cancer. Differences among groups were analyzed by the Kruskal-Wallis test, with the Bonferroni adjusted Mann-Whitney U test used for post hoc analysis.

5 each). The expression of mTOR mRNA was similar in all three groups (p > 0.05).Fig. 1 Comparison of autophagy expression associated mRNAs in control, endometriosis and cancer. Differences among groups were analyzed by the Kruskal-Wallis test, with the Bonferroni adjusted Mann-Whitney U test used for post hoc analysis. Fig. 1 Discussion Endometriosis is a common gynecologic disease affecting 5–15% of women of reproductive age and 20–50% of all infertile women [12,13]. Endometriosis is the most frequent cause of chronic pelvic pain and of dysmenorrhea and infertility in women with chronic pelvic pain [14].

5 each). The expression of mTOR mRNA was similar in all three groups (p > 0.05).Fig. 1 Comparison of autophagy expression associated mRNAs in control, endometriosis and cancer. Differences among groups were analyzed by the Kruskal-Wallis test, with the Bonferroni adjusted Mann-Whitney U test used for post hoc analysis. Fig. 1 Discussion Endometriosis is a common gynecologic disease affecting 5–15% of women of reproductive age and 20–50% of all infertile women [12,13]. Endometriosis is the most frequent cause of chronic pelvic pain and of dysmenorrhea and infertility in women with chronic pelvic pain [14]. Although the polymeric glycoprotein CA-125 was first identified in the early 1980s as a tumor marker, its role in the body has not yet been accurately determined. The expression of CA-125 is increased in gynecological tumors, such as ovarian and endometrial cancer, as well as in other types of malignant tumors, including pancreatic, lung, breast, colorectal, and gastrointestinal cancer.CA-125 expression is also increased in benign diseases, such as liver cirrhosis (70%), benign ovarian cyst, and pelvic inflammatory disease, as well as during menstruation and the first trimester of pregnancy. Therefore, CA-125 is not appropriate as a diagnostic test for specific diseases. Studies have therefore sought to identify other markers of early endometriosis in serum, peritoneal fluid, and various tissues [15,16]. In agreement with findings showing that CA-125 is expressed in benign tumors, the present study showed that its expression was significantly higher in the endometriosis group than in the control group. Moreover, its expression was significantly higher in the tumor group than in the other two groups, with CA-125 expression correlating with disease severity.

t CA-125 is expressed in benign tumors, the present study showed that its expression was significantly higher in the endometriosis group than in the control group. Moreover, its expression was significantly higher in the tumor group than in the other two groups, with CA-125 expression correlating with disease severity. Pathologically, autophagy can have both beneficial and harmful effects. Autophagic processes in neurodegeneration allow for the removal of aggregated proteins before they are toxic and induce the death of neurons that accumulate aggregated proteins. In diabetes, there is a need for maintenance of the structure, mass, and function of β-cells, with an age-associated decline in autophagic activity affecting insulin sensitivity and impairing glucose tolerance. In cardiovascular diseases, increased autophagy can compensate for defects in lysosome function, with defects in the completion of autophagy resulting in the accumulation of autophagosomes that impair cellular function. In immunity, autophagy affects cellular defenses against invasion by bacteria and virus, and may allow replication of nucleic acids by microbial pathogens as well as supplying nutrients for their growth. Autophagy in cancer can act as a tumor suppressor, promoting cell death, or as an oncogene, preventing cell death.

. In immunity, autophagy affects cellular defenses against invasion by bacteria and virus, and may allow replication of nucleic acids by microbial pathogens as well as supplying nutrients for their growth. Autophagy in cancer can act as a tumor suppressor, promoting cell death, or as an oncogene, preventing cell death. The present study assessed the expression of autophagy-associated mRNAs in groups of patients with benign tumors, endometriosis, and gynecologic malignancies. We were unable to determine whether the expression of these autophagy mRNAs was normal in any of these groups because, for ethical reasons, we could not obtain peritoneal fluid from normal, disease-free individuals. However, autophagy mRNAs were present in all peritoneal fluid samples from the three groups of patients. Autophagy was found to be involved in intraperitoneal tumor immunity. In particular, levels of PI3K, FLIP, and Rubicon mRNAs were significantly higher in the endometriosis than in the control group, suggesting that these autophagy mRNAs were associated with intraperitoneal endometriosis formation. Endometriosis is a chronic inflammatory condition in a pelvic environment that changes to a complex disease characterized by disparate morphological, histological and biochemical properties. Although endometriosis is not a malignant tumor, it is clinically invasive, infiltrating or metastatic, similar to malignant tumors.

ometriosis is a chronic inflammatory condition in a pelvic environment that changes to a complex disease characterized by disparate morphological, histological and biochemical properties. Although endometriosis is not a malignant tumor, it is clinically invasive, infiltrating or metastatic, similar to malignant tumors. The present study found that the expression of PI3K and FLIP mRNA in peritoneal fluid was similar in the gynecologic malignancy and endometriosis groups and was significantly higher in both groups than in the control group. In contrast, the levels of LC3II and FLIP mRNAs were significantly lower, and the levels of Beclin-1 and Rubicon mRNAs significantly higher, in the endometriosis than in the gynecologic cancer group. These findings indicate that different autophagy genes are involved in different diseases. Mechanistically, however, this study could not explain why mTOR mRNA expression were similar in the three groups, and why genes associated with the induction and initiation processes of autophagy did not participate in endometriosis and gynecologic cancer. Moreover, PI3K, which is involved in nucleation in endometriosis and gynecologic cancers, was higher in these two groups than in the control group; and FLIP, which is antagonistic to autophagy-related gene 3 and not an autophagy agonist, was higher in the endometriosis than in the control group. Further studies are needed to determine whether autophagy genes are involved in different stages of endometriosis and gynecologic cancer.

groups than in the control group; and FLIP, which is antagonistic to autophagy-related gene 3 and not an autophagy agonist, was higher in the endometriosis than in the control group. Further studies are needed to determine whether autophagy genes are involved in different stages of endometriosis and gynecologic cancer. Autophagy plays two roles in tumorigenesis. The first is the induction of apoptosis as a tumor suppressor, and the second is the inhibition of apoptosis during tumorigenesis. In the latter, autophagy can promote cancer cell survival by removing damaged proteins and cellular organelles during conditions of nutrient limitation and hypoxia and following exposure to radioactivity [[17], [18], [19]]. During early tumor stages, inhibition of autophagy can enhance the survival of cancer cells and promotes tumor growth [20,21], whereas, during later stages, autophagy induces cancer cells in the inner part of the tumor, due to the restriction of nutrients, allowing these cells to live in extreme situations; alternatively, these cells may be damaged by therapy to rapidly remove the endoplasmic reticulum. This study found that all autophagy-associated mRNAs, except for Beclin-1 mRNA, were increased in cancer patients. Our finding, that Beclin-1 mRNA was reduced in these patients, was consistent with the results of previous studies [22,23]. The disappearance of Beclin-1, which is related to the formation of autophagosome, has also been observed in breast, uterine, and prostate cancers. Little Beclin-1 protein was detected in the breast cancer cell line MCF7, with MCF7 cells expressing Beclin-1 showing increased autophagic activity and suppression of tumor activity [24]. In addition, beclin-1 +/- mice showed a high rate of tumor formation and beclin-1 - / - mouse stem cells showed a reduced number of autophagy vacuoles [25]. These results suggest that autophagy induces tumor cell death and tumor suppression through growth inhibition.

gic activity and suppression of tumor activity [24]. In addition, beclin-1 +/- mice showed a high rate of tumor formation and beclin-1 - / - mouse stem cells showed a reduced number of autophagy vacuoles [25]. These results suggest that autophagy induces tumor cell death and tumor suppression through growth inhibition. This study had several limitations. Because we could not obtain peritoneal fluid from healthy subjects due to ethical reasons, we used peritoneal fluid from patients with benign tumors without endometriosis as a control. Second, autophagy mRNAs were assessed in peritoneal fluid rather than lesion tissue. Third, the present study measured mRNAs, not proteins. Fourth, only some autophagy-associated mRNAs were measured in these samples. Fifth, although studies have investigated the association between immune response and the severity of endometriosis, the present study analyzed the association between autophagy mRNAs and the presence or absence of endometriosis.

not proteins. Fourth, only some autophagy-associated mRNAs were measured in these samples. Fifth, although studies have investigated the association between immune response and the severity of endometriosis, the present study analyzed the association between autophagy mRNAs and the presence or absence of endometriosis. Conclusions Autophagy-associated mRNAs were detected in the intraperitoneal fluids of all patients with benign tumors, endometriosis, and gynecologic malignancies, suggesting that these mRNAs may be involved in the pathogenesis of gynecologic diseases.PI3K, FLIP, and Rubicon mRNAs are closely associated with the pathogenesis of endometriosis. The increased expression of PI3K and FLIP mRNAs in the peritoneal fluid of the endometriosis and gynecologic cancer groups suggests that endometriosis and gynecologic cancers have similar autophagic characteristics. The level of Beclin-1 mRNA was lower in the gynecologic malignancy group than in the control and endometriosis groups, suggesting that the decrease in Beclin-1 mRNA expression is associated with the development of gynecologic tumors. Conflicts of interest No potential conflict of interest relevant to this article was reported. Financial disclosures None of the authors reported financial disclosures. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF 2017R1D1A1B3030021).

Introduction Maternal intrapartum pyrexia ≥38 °C has been associated with a risk of neonatal early-onset Group B Streptococcus (GBS) disease of 5.3 per 1000, compared to a background risk of 0.6 per 1000. [1] In an effort to avert these infections, guidelines in the US [2], UK [3], and Ireland [4] instruct healthcare providers to consider isolated maternal intrapartum fever of ≥38 °C as a sign of infective chorioamnionitis and an indication for antibiotic therapy. While maternal pyrexia during labour may be an indicator of chorioamnionitis, it is difficult to determine clinically owing to the range of other causes of pyrexia such as epidural use [[5], [6], [7]], direct physiological effects of labour, [8] dehydration [9], elevated ambient temperature [9], and use of misoprostol [10].

maternal pyrexia during labour may be an indicator of chorioamnionitis, it is difficult to determine clinically owing to the range of other causes of pyrexia such as epidural use [[5], [6], [7]], direct physiological effects of labour, [8] dehydration [9], elevated ambient temperature [9], and use of misoprostol [10]. Diagnosing chorioamnionitis is an ongoing clinical challenge: signs and symptoms may be subtle and non-specific, while microbiological cultures and histology of the placenta are not available at the time of clinical suspicion. Hence, a clinical diagnosis of chorioamnionitis may be made when a combination (or even one) of the following are noted: maternal fever ≥38 °C, maternal tachycardia, fetal tachycardia, elevated white cell count, uterine tenderness, or purulent discharge from the cervical os. [4,9] In 2016, an expert panel of the National Institute of Child Health and Human Development (NICHD) contended that isolated maternal fever is not synonymous with chorioamnionitis. Furthermore, the panel argued that clinical use of the term chorioamnionitis is outdated as it implies presence of infection for what is in reality a heterogenous array of conditions characterized by infection or inflammation or both.[9]

D) contended that isolated maternal fever is not synonymous with chorioamnionitis. Furthermore, the panel argued that clinical use of the term chorioamnionitis is outdated as it implies presence of infection for what is in reality a heterogenous array of conditions characterized by infection or inflammation or both.[9] Maternal intrapartum pyrexia leading to a clinical diagnosis of chorioamnionitis has significant implications for management of mothers and newborns, usually leading to intravenous antibiotic therapy and increased medical interventions. Since maternal intrapartum pyrexia is not always of infectious origin, treating all fevers with antibiotics results in overuse and may contribute to increased antimicrobial resistance. Furthermore, alterations in the neonatal microbiome caused by antibiotics have been linked to adverse effects later in childhood including obesity, diabetes and allergy. [[11], [12], [13]]

nfectious origin, treating all fevers with antibiotics results in overuse and may contribute to increased antimicrobial resistance. Furthermore, alterations in the neonatal microbiome caused by antibiotics have been linked to adverse effects later in childhood including obesity, diabetes and allergy. [[11], [12], [13]] In Ireland, maternal temperature ≥38 °C forms part of the obstetric systemic inflammatory response (SIRS) criteria utilised to identify women at risk of sepsis, along with temperature <36 °C, maternal heart rate (HR) ≥100 bpm, fetal heart rate >160 bpm, respiratory rate (RR) ≥20/min, white cell count (WCC) >16.9 or <4.0 × 109/L, bedside glucose >7.7 mmol/L (in the absence of diabetes) or acutely altered mental status. [14] The presence of two or more of these SIRS criteria triggers escalation to medical review frequently leading to a septic workup (SWU). However, maternal vital signs (VS) undergo changes during pregnancy and labour [8]. In 2016 the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) task force considered the use of two or more SIRS criteria to identify sepsis to be unhelpful, as their presence may reflect the host response to inflammation, infection or other insults [15]. These obstetric SIRS criteria are in need of validation as markers of acute clinical deterioration around the time of labour. The aims of this study were:1 To determine the infection rate related to peripartum maternal pyrexia.

In Ireland, maternal temperature ≥38 °C forms part of the obstetric systemic inflammatory response (SIRS) criteria utilised to identify women at risk of sepsis, along with temperature <36 °C, maternal heart rate (HR) ≥100 bpm, fetal heart rate >160 bpm, respiratory rate (RR) ≥20/min, white cell count (WCC) >16.9 or <4.0 × 109/L, bedside glucose >7.7 mmol/L (in the absence of diabetes) or acutely altered mental status. [14] The presence of two or more of these SIRS criteria triggers escalation to medical review frequently leading to a septic workup (SWU). However, maternal vital signs (VS) undergo changes during pregnancy and labour [8]. In 2016 the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) task force considered the use of two or more SIRS criteria to identify sepsis to be unhelpful, as their presence may reflect the host response to inflammation, infection or other insults [15]. These obstetric SIRS criteria are in need of validation as markers of acute clinical deterioration around the time of labour. The aims of this study were:1 To determine the infection rate related to peripartum maternal pyrexia. 2 To evaluate the diagnostic accuracy of the obstetric SIRS criteria and cardiotocography (CTG) for identification of infection related to peripartum maternal pyrexia.

In Ireland, maternal temperature ≥38 °C forms part of the obstetric systemic inflammatory response (SIRS) criteria utilised to identify women at risk of sepsis, along with temperature <36 °C, maternal heart rate (HR) ≥100 bpm, fetal heart rate >160 bpm, respiratory rate (RR) ≥20/min, white cell count (WCC) >16.9 or <4.0 × 109/L, bedside glucose >7.7 mmol/L (in the absence of diabetes) or acutely altered mental status. [14] The presence of two or more of these SIRS criteria triggers escalation to medical review frequently leading to a septic workup (SWU). However, maternal vital signs (VS) undergo changes during pregnancy and labour [8]. In 2016 the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) task force considered the use of two or more SIRS criteria to identify sepsis to be unhelpful, as their presence may reflect the host response to inflammation, infection or other insults [15]. These obstetric SIRS criteria are in need of validation as markers of acute clinical deterioration around the time of labour. The aims of this study were:1 To determine the infection rate related to peripartum maternal pyrexia. 2 To evaluate the diagnostic accuracy of the obstetric SIRS criteria and cardiotocography (CTG) for identification of infection related to peripartum maternal pyrexia. Materials and Methods Study population and setting Research ethics approval was granted by the National Maternity Hospital (NMH) Dublin Ethics Committee in September 2015. The study was conducted in a tertiary referral centre in the Republic of Ireland that had an average of 9247 births per annum between 2007 and 2016. This study aimed to investigate the clinical profile of women with peripartum pyrexia, which was defined as pyrexia that developed anytime after onset of labour until four hours after delivery. Pyrexia that developed immediately after delivery was included as it is likely to relate to infection or inflammation that was evolving during labour. A convenience sample size (n = 198) was chosen whereby data was collected on all women with peripartum pyrexia who delivered in the NMH over a four month period. Patients were excluded if they had any evidence of infection at the time of labour onset. Preterm births were more likely to have symptoms of infection prior to labour and therefore many, but not all, were excluded. Hence, a cohort of 175 mother-newborn pairs who presented with peripartum maternal temperature ≥38 °C on at least 1 occasion was included for prospective analysis from November 2015 to February 2016 (Fig. 1). There were 3006 deliveries (≥500 g) during the study period. The prevalence of peripartum pyrexia that met the inclusion criteria was 5.8% (175/3006).Fig. 1 Inclusion and Exclusion Criteria.

nal temperature ≥38 °C on at least 1 occasion was included for prospective analysis from November 2015 to February 2016 (Fig. 1). There were 3006 deliveries (≥500 g) during the study period. The prevalence of peripartum pyrexia that met the inclusion criteria was 5.8% (175/3006).Fig. 1 Inclusion and Exclusion Criteria. Fig. 1 Data Collection Information on maternal demographics, labour and delivery outcomes, maternal VS, white cell count and CTG, were collected from patient medical records, shortly after delivery during the postnatal inpatient stage. The laboratory information system was accessed prospectively for maternal and neonatal microbiological results and placental histology reports. Maternal VS were recorded at maximum intervals of every two hours during labour and every four hours after delivery. Peripartum VS were selected at the same time that the highest temperature reading was observed. All VS recordings were performed by trained delivery ward staff.

l results and placental histology reports. Maternal VS were recorded at maximum intervals of every two hours during labour and every four hours after delivery. Peripartum VS were selected at the same time that the highest temperature reading was observed. All VS recordings were performed by trained delivery ward staff. Protocol for Determination of Infection Status A SWU was obtained from women with fever ≥38 °C on at least one occasion. The maternal SWU included blood cultures, urine culture and vaginal swab. An incomplete SWU was defined as omission of one or more of the above tests. Placental swabs were taken where the obstetrician was suspicious of chorioamnionitis. Specimens from other sites were obtained if clinically indicated. A SWU was performed on all neonates born to mothers who had peripartum pyrexia. The standard neonatal SWU included a full blood count and blood culture. GBS polymerase chain reaction (PCR) on EDTA was performed if the neonatal blood culture was negative at 36 hours and the baby remained symptomatic with clinical evidence of early-onset sepsis. A lumbar puncture (LP) was performed in any baby with a clinical suspicion of meningitis, neurological findings associated with sepsis, a positive blood culture, a positive EDTA PCR, a maternal positive blood culture in labour, or failure to respond to treatment. Cases were microbiologically categorised as “confirmed infection” if any of the following criteria were met:• Sterile site infection in mother or neonate • Maternal UTI defined as a pure or predominant growth of a single organism >100,000 organisms/ml

Protocol for Determination of Infection Status A SWU was obtained from women with fever ≥38 °C on at least one occasion. The maternal SWU included blood cultures, urine culture and vaginal swab. An incomplete SWU was defined as omission of one or more of the above tests. Placental swabs were taken where the obstetrician was suspicious of chorioamnionitis. Specimens from other sites were obtained if clinically indicated. A SWU was performed on all neonates born to mothers who had peripartum pyrexia. The standard neonatal SWU included a full blood count and blood culture. GBS polymerase chain reaction (PCR) on EDTA was performed if the neonatal blood culture was negative at 36 hours and the baby remained symptomatic with clinical evidence of early-onset sepsis. A lumbar puncture (LP) was performed in any baby with a clinical suspicion of meningitis, neurological findings associated with sepsis, a positive blood culture, a positive EDTA PCR, a maternal positive blood culture in labour, or failure to respond to treatment. Cases were microbiologically categorised as “confirmed infection” if any of the following criteria were met:• Sterile site infection in mother or neonate • Maternal UTI defined as a pure or predominant growth of a single organism >100,000 organisms/ml • A significant perinatal pathogen isolated from a placental swab i.e. pure or predominant growth of Group B Streptococcus (GBS), Group A Streptococcus (GAS), Escherichia coli (E.coli), Listeria monocytogenes or Haemophilus influenzae.

• Maternal UTI defined as a pure or predominant growth of a single organism >100,000 organisms/ml • A significant perinatal pathogen isolated from a placental swab i.e. pure or predominant growth of Group B Streptococcus (GBS), Group A Streptococcus (GAS), Escherichia coli (E.coli), Listeria monocytogenes or Haemophilus influenzae. Mother-newborn pairs that had a full SWU were categorised as “no infection” in the absence of microbiological evidence of potential pathogens from any site. Placentas were examined histologically if there was clinical chorioamnionitis, preterm birth <34 weeks gestation, preterm prelabour rupture of membranes or low APGAR score. Other placentas from women with PIL were examined only after consultant histopathologist review or clinical request. In non-sterile sites such as urine, vaginal, placental or rectal swabs, it was not always possible to distinguish between growth of potentially pathogenic micro-organisms and colonisation with normal microbiota; in addition, a small proportion of SWUs were incomplete. Examination of placentas was sought 47 times to determine infection status in these cases. All placentas were reported by placental pathologists who were blinded to microbiological results. Chorioamnionitis was reported using Society for Paediatric Pathology nomenclature. [16] Participants with no histological evidence of chorioamnionitis or subchorionitis were categorised as “no infection”.

in these cases. All placentas were reported by placental pathologists who were blinded to microbiological results. Chorioamnionitis was reported using Society for Paediatric Pathology nomenclature. [16] Participants with no histological evidence of chorioamnionitis or subchorionitis were categorised as “no infection”. Statistical Analysis Nominal data were reported as frequencies and proportions. Where appropriate, continuous demographic and labour outcome data were collapsed into clinically relevant nominal categories. Normally and non-normally distributed continuous data were reported as the mean ± standard deviation (SD) and the median ± interquartile range, respectively. Study objectives were analysed as follows:1 Infection outcomes were reported as frequencies and proportions. 2 SIRS criteria in patients with confirmed infection versus those without evidence of infection were compared using independent samples t-test for continuous data and pearson chi-square test for nominal data. Diagnostic accuracy of obstetric SIRS criteria as markers of infection related to peripartum pyrexia were evaluated using receiver operating characteristic (ROC) curves. Accuracy of SIRS criteria was reported as the area under the ROC curve (AUC), with the 95% confidence interval (CI) and significance level.

inal data. Diagnostic accuracy of obstetric SIRS criteria as markers of infection related to peripartum pyrexia were evaluated using receiver operating characteristic (ROC) curves. Accuracy of SIRS criteria was reported as the area under the ROC curve (AUC), with the 95% confidence interval (CI) and significance level. Statistical analysis was carried out using IBM SPSS Statistics version 22. A bonferroni correction was performed in order to reduce the risk of Type I error. Since twelve statistical tests were carried out on SIRS criteria and CTG as predictors of infection, a P-value threshold of <0.004 was chosen to claim statistical significance.

alysis was carried out using IBM SPSS Statistics version 22. A bonferroni correction was performed in order to reduce the risk of Type I error. Since twelve statistical tests were carried out on SIRS criteria and CTG as predictors of infection, a P-value threshold of <0.004 was chosen to claim statistical significance. Results Demographics In this pyrexia cohort 79.4% (139/175) were nulliparous, and 99.4% (174/175) delivered at term (≥37 weeks gestation). The median duration of rupture of membranes (ROM) was 12.3 hours (IQR: 7.8 – 21.7), while 33.7% (59/175) of women had prolonged rupture of membranes greater than 18 hours. Pyrexia began during labour in 57.1% (100/175) of women and within four hours after delivery in 42.9% (75/175). There were no maternal admissions to critical care for infective reasons and there were no cases of maternal sepsis. APGAR scores were less than 7 at 1 minute in 9.1% (16/176) of neonates, and less than 7 at 5 minutes in 0.6% (1/176). A comparison of maternal demographics and delivery outcomes between those with confirmed infection and those with no evidence of infection are summarised in Table 1. The impact of antibiotic administration on neonatal management is summarised in Table 2.Table 1 Demographic Data, Labour and Delivery Outcomes in “Confirmed Infections” versus “Colonisation / No Infections”.

tcomes between those with confirmed infection and those with no evidence of infection are summarised in Table 1. The impact of antibiotic administration on neonatal management is summarised in Table 2.Table 1 Demographic Data, Labour and Delivery Outcomes in “Confirmed Infections” versus “Colonisation / No Infections”. Table 1 Confirmed Infections (n = Confirmed Infections (mean, SD) (n = 30) Colonisation / Colonisation / No Infection (mean, SD) (n = 145) Statistical Statistical Significance Maternal Age (years) 31.5, 4.7 32.9, 4.2 0.49 Gestation at Delivery (weeks) 39.6, 1.7 39.6, 1.9 0.76 Confirmed Infections (n = 30) Colonisation / No Infection (n = 145) Statistical Significance Post-partum Haemorrhage (ml): Normal (<500) 21 (70%) 95 (65.5%) 0.38 Minor (500 – 1000) 8 (26.7%) 42 (29%) Major (1001 – 2000) 0 (0%) 7 (4.8%) Severe (> 2000) 1 (3.3%) 1 (0.7%) Sepsis (life-threatening organ dysfunction caused by a dysregulated host response to infection) 0 (0%) 0 (0%) -- Spontaneous Vaginal Delivery Instrumental Delivery Caesarean Section in Labour 10 (33.3%) 10 (33.3%) 10 (33.3%) 57 (39.3%) 49 (33.8%) 39 (26.9%) 0.74 Confirmed Infections Delivered by SVD or Instrumental (n = 20) Colonisation / No Infection Delivered by SVD or Instrumental (n = 106) Statistical Significance Episiotomy 13 (65%) 68 (64.2%) 0.94 3rd / 4th Degree Tear 1 (5%) 2 (1.9%) 0.40 Manual Removal of Placenta 0 (0%) 3 (2.8%) 0.45

Confirmed Infections (n = 30) Colonisation / No Infection (n = 145) Statistical Significance Post-partum Haemorrhage (ml): Normal (<500) 21 (70%) 95 (65.5%) 0.38 Minor (500 – 1000) 8 (26.7%) 42 (29%) Major (1001 – 2000) 0 (0%) 7 (4.8%) Severe (> 2000) 1 (3.3%) 1 (0.7%) Sepsis (life-threatening organ dysfunction caused by a dysregulated host response to infection) 0 (0%) 0 (0%) -- Spontaneous Vaginal Delivery Instrumental Delivery Caesarean Section in Labour 10 (33.3%) 10 (33.3%) 10 (33.3%) 57 (39.3%) 49 (33.8%) 39 (26.9%) 0.74 Confirmed Infections Delivered by SVD or Instrumental (n = 20) Colonisation / No Infection Delivered by SVD or Instrumental (n = 106) Statistical Significance Episiotomy 13 (65%) 68 (64.2%) 0.94 3rd / 4th Degree Tear 1 (5%) 2 (1.9%) 0.40 Manual Removal of Placenta 0 (0%) 3 (2.8%) 0.45 Neonatal Outcomes: Neonates Born in Confirmed Infection Group (n = 30) Neonates Born in Colonisation / No Infection Group (n = 146) Statistical Significance Live Births 30 (100%) 146 (100%) -- Immediate NICU / SCBU admission 5 (16.7%) 20 (13.8%) 0.68 NOTE: all continuous data was normally distributed Table 2 Impact of Antibiotic Administration on Neonatal Management Table 2Neonatal Investigations performed Number Blood Cultures 175 PCR on EDTA 1 Lumbar Punctures 5 WCCs 170 Mean (SD) WCCa 19.2 × 109/L (5.2) a data was normally distributed.

Neonatal Outcomes: Neonates Born in Confirmed Infection Group (n = 30) Neonates Born in Colonisation / No Infection Group (n = 146) Statistical Significance Live Births 30 (100%) 146 (100%) -- Immediate NICU / SCBU admission 5 (16.7%) 20 (13.8%) 0.68 NOTE: all continuous data was normally distributed Table 2 Impact of Antibiotic Administration on Neonatal Management Table 2Neonatal Investigations performed Number Blood Cultures 175 PCR on EDTA 1 Lumbar Punctures 5 WCCs 170 Mean (SD) WCCa 19.2 × 109/L (5.2) a data was normally distributed. Infection Outcomes The infection rate for women with peripartum pyrexia was 17.1% (30/175). Subgroup analysis found that pyrexia in labour was associated with an infection rate of 22% (22/100) while pyrexia occurring within four hours after delivery was associated with a rate of 10.7% (8/75). Of the 175 women with pyrexia, 13.2% (23/175) had chorioamnionitis, the majority of which were diagnosed histologically (10.9%, 19/175) as opposed to microbiologically (2.3%, 4/175). The rate of chorioamnionitis was 16% (16/100) among women with intrapartum pyrexia and 9.3% (7/75) among women who developed pyrexia within four hours after delivery. Blood stream infection (BSI) occurred in 2.3% of the cohort (4/175). These consisted of 3 maternal blood stream infections and one neonatal infection identified by PCR in blood. UTIs occurred in 1.7% of women (3/175). A further 14.3% (25/175) had microbial colonisation at a non-sterile genito-urinary site but were not deemed to have chorioamnionitis histologically. There was no evidence of infection or colonisation in 68.6% (120/175) of the cohort (Fig. 2).Fig. 2 Infection Outcomes in Pyrexia Cohort.

curred in 1.7% of women (3/175). A further 14.3% (25/175) had microbial colonisation at a non-sterile genito-urinary site but were not deemed to have chorioamnionitis histologically. There was no evidence of infection or colonisation in 68.6% (120/175) of the cohort (Fig. 2).Fig. 2 Infection Outcomes in Pyrexia Cohort. †One Maternal blood stream infection was associated with a UTI. ‡One Neonatal blood stream infection was associated with histologically confirmed chorioamnionitis. Fig. 2 GBS was the most prevalent pathogen, accounting for 43.3% (13/30) of infections, the majority of which were chorioamnionitis. E. coli accounted for 16.7% (5/30) of infections, anaerobic bacteria 10% (3/30), Enterococcus faecalis 6.7% (2/30) and one each by Proteus mirabilis and Peptostreptococcus asaccharolyticus. No pathogen was isolated in 16.6% (5/30) of histologically confirmed infections. Diagnostic Accuracy of Obstetric SIRS Criteria and CTG Two or more obstetric SIRS criteria consisting of pyrexia ≥ 38 °C plus at least one of maternal heart rate ≥ 100bpm, respiratory rate ≥ 20 breaths/min, or WCC > 16.9 × 109/L were observed in 70.9% (124/175) of mothers. The rate of infection in this group was 16.1% (20/124). No significant differences were observed in mean temperature, heart rate, respiratory rate, systolic blood pressure or WCC between the groups with “confirmed infection” and “no evidence of infection”(Table 3).Table 3 Comparison of SIRS criteria and CTG between “Confirmed Infections” versus “Colonisation / No Infection”.

o significant differences were observed in mean temperature, heart rate, respiratory rate, systolic blood pressure or WCC between the groups with “confirmed infection” and “no evidence of infection”(Table 3).Table 3 Comparison of SIRS criteria and CTG between “Confirmed Infections” versus “Colonisation / No Infection”. Table 3Maternal Vital Sign Confirmed Infections (mean, SD) Colonisation / No Infections (mean, SD) Statistical Significance Temperature (°C) 38.30, 0.24 38.30, 0.29 0.53 Heart Rate (bpm) 96, 15 93, 16 0.51 Respiratory Rate (breaths per min) 17, 1 17, 2 0.62 Systolic Blood Pressure (mmHg) 118, 13 123, 13 0.99 White Cell Count (WCC) 18, 5 18, 4 0.32 Maternal Vital Sign / CTG Confirmed Infections (%) n = 30 Colonisation / No Infections (%) n = 145 Statistical Significance WCC > 16.9 × 10 [9]/L 16 (53.3%) 86 (59.4%) 0.54 WCC < 4.0 × 10 [9]/L 0 (0%) 0 (0%) -- Non-reassuring CTG 14 (46.7%) 63 (43.4%) 0.75 SIRS (systemic inflammatory response syndrome); CTG (cardiotocography); bpm (beats per minute); WCC (white cell count). NOTE: all continuous data was normally distributed.

Maternal Vital Sign / CTG Confirmed Infections (%) n = 30 Colonisation / No Infections (%) n = 145 Statistical Significance WCC > 16.9 × 10 [9]/L 16 (53.3%) 86 (59.4%) 0.54 WCC < 4.0 × 10 [9]/L 0 (0%) 0 (0%) -- Non-reassuring CTG 14 (46.7%) 63 (43.4%) 0.75 SIRS (systemic inflammatory response syndrome); CTG (cardiotocography); bpm (beats per minute); WCC (white cell count). NOTE: all continuous data was normally distributed. In the ROC curve analysis (Fig. 3), the 95% confidence intervals for all peripartum maternal pyrexia related clinical observations (HR, RR, SBP, WCC) included an AUC of 0.5 (Table 4), indicating that these variables have poor predictive value as markers of infection. A non-reassuring cardiotocograph (NRCTG) was associated with an infection rate of 18.2% (14/77); for normal CTGs the infection rate was 16.4% (16/98). For NRCTGs there was no significant difference between those with infections and those with no evidence of infection (Table 3).Fig. 3 ROC Curves for maternal RR,HR,SBP,and WCC as predictors of infection in cases with peripartum pyrexia. Fig. 3Table 4 Receiver Operator Characteristic (ROC) Curve Analysis. Table 4Area under the ROC Curve Peripartum Pyrexia Related Maternal Vital Sign AUC 95% Confidence Interval Statistical Significance Elevated Heart Rate 0.542 0.43 to 0.65 P = 0.48 Elevated Respiratory Rate 0.515 0.40 to 0.63 P = 0.81 Decreased Systolic Blood Pressure 0.399 0.29 to 0.51 P = 0.09 Elevated White Cell Count 0.505 0.39 to 0.62 P = 0.93 Null hypothesis: true area = 0.5 ROC (receiver operator characteristic curve).

tatistical Significance Elevated Heart Rate 0.542 0.43 to 0.65 P = 0.48 Elevated Respiratory Rate 0.515 0.40 to 0.63 P = 0.81 Decreased Systolic Blood Pressure 0.399 0.29 to 0.51 P = 0.09 Elevated White Cell Count 0.505 0.39 to 0.62 P = 0.93 Null hypothesis: true area = 0.5 ROC (receiver operator characteristic curve). Comment Utilising a broad range of diagnostic information from SWUs and histological examination of placentas, we found a low rate of infection among mother-newborns pairs where maternal pyrexia ≥ 38 °C on at least one occasion occurred after onset of labour or within four hours after delivery, at term gestation, with or without two or more obstetric SIRS criteria. Infection was more commonly diagnosed when pyrexia occurred during labour compared to pyrexia that occurred shortly after delivery. The most commonly identified infection was histologically confirmed chorioamnionitis (10.9%; 19/175). [16] Our findings call into question the view that peripartum maternal pyrexia, especially in the presence of two or more obstetric SIRS criteria, is a sign of infection. The low rate of infection (17.1%; 30/175) associated with peripartum pyrexia confirms that aetiologies other than infection were the cause in the majority of cases. These findings support the assertion of the expert panel of the NICHD that maternal fever alone should not automatically lead to a diagnosis of infective chorioamnionitis. The panel propose to replace the use of the intrapartum term chorioamnionitis with the term “intrauterine inflammation or infection or both” or “Triple I”. [9]

ngs support the assertion of the expert panel of the NICHD that maternal fever alone should not automatically lead to a diagnosis of infective chorioamnionitis. The panel propose to replace the use of the intrapartum term chorioamnionitis with the term “intrauterine inflammation or infection or both” or “Triple I”. [9] We found that the combination of two obstetric SIRS criteria consisting of temperature ≥38 °C plus any one of HR ≥100bpm, RR ≥ 20 breaths/minute, systolic blood pressure <100 mmHg, or WCC > 16.9 × 109/L were poor predictors of infection in women with peripartum pyrexia. At a time when doubts have been expressed about the diagnostic accuracy of SIRS criteria for the identification of sepsis [15], our findings highlight the inadequacy of the approach to escalate to medical review and SWU based solely on the presence of two or more obstetric SIRS criteria around the time of labour. In addition, our findings suggest that CTG may not be useful in identifying infection related to peripartum pyrexia.

of sepsis [15], our findings highlight the inadequacy of the approach to escalate to medical review and SWU based solely on the presence of two or more obstetric SIRS criteria around the time of labour. In addition, our findings suggest that CTG may not be useful in identifying infection related to peripartum pyrexia. The neonatal “sepsis calculator” of Puopolo et al [17] can be used to guide decisions on whether or not antibiotics should be used in late preterm and term neonates born to mothers who had intrapartum pyrexia. In the UK, NICE provide guidance on the duration of such courses of antibiotics for neonates, suggesting discontinuation at 36 hours if the blood culture is negative [18]. No such tools or guidance exist to aid decision-making in the initiation or de-escalation of antibiotic therapy for women with intrapartum pyrexia. There is a need for future research to accurately identify infection in women during term labour, making use of biomarkers and prediction models similar to those utilised for neonates. This could greatly reduce exposure to antibiotics among women in labour and their newborns as well as reduce interventional delivery.

. There is a need for future research to accurately identify infection in women during term labour, making use of biomarkers and prediction models similar to those utilised for neonates. This could greatly reduce exposure to antibiotics among women in labour and their newborns as well as reduce interventional delivery. While there may be a low rate of infection among women with peripartum pyrexia at term, the presence of bloodstream infections in 2.3% (4/175) of mother-newborn pairs means that it may still be prudent to initiate SWUs in these patients, until such time as we can accurately diagnose infection at the time of peripartum pyrexia. However the findings of this study provide evidence to support the safe discontinuation of antibiotics in mothers with peripartum pyrexia ≥ 38 °C on at least one occasion, with or without two or more obstetric SIRS criteria, at term, without signs of infection prior to labour, provided the 48 hour blood culture is negative and the patient remains well in the postnatal period. Disclosure of Interests No conflicts of interest declared. No funding was sought for this study. Acknowledgements Research was carried out as part fulfilment of an MSc in Clinical Pharmacy through Queen’s University Belfast (QUB), Northern Ireland. NMH: Ricardo Segurado for statistical advice. Mary Hunter and all scientific staff in the histology department. Martina Cronin and all midwifery staff on the Labour ward. QUB: Dr Carole Parsons for review and guidance.

fulfilment of an MSc in Clinical Pharmacy through Queen’s University Belfast (QUB), Northern Ireland. NMH: Ricardo Segurado for statistical advice. Mary Hunter and all scientific staff in the histology department. Martina Cronin and all midwifery staff on the Labour ward. QUB: Dr Carole Parsons for review and guidance. ☆ Abstracts of this research have been presented at the following conferences: 3rd National Sepsis Summit, Dublin Castle, September 2016; Irish Society of Clinical Microbiologists Spring Meeting, Dublin, March 2017; Hospital Pharmacists’ Association of Ireland Annual Conference, Dublin, April 2017; European Congress of Clinical Microbiology and Infectious Diseases, Vienna, April 2017.

Introduction Intra-uterine inseminations (IUI) with fresh husband sperm is one of the first line treatments in case of subfertility. They can be used in mild male abnormality [1,2], ovulation disorders [3] and unexplained infertility (UI) [4]. Numerous factors have been reported to have a great influence on the success rate of IUI. The number of inseminated motile spermatozoa, as well as the number of mature follicles and the use of GnRH antagonist have been widely reported to be significantly linked to the chance to obtain a pregnancy after IUI [[5], [6], [7], [8]]. Conflicting results have been reported concerning female age. Indeed, if some authors have shown that advanced age was associated with a lower pregnancy rate [3,[8], [9], [10]] or with an increased miscarriage rate [11], others found no correlation [12,13]. The influence of the ovarian reserve parameters has been studied by different authors with major discrepancies since some have found that high levels of ovarian reserve as measured by anti-Mullerian hormone (AMH) and/or antral follicle count (AFC) are good predictors of the pregnancy rate [11,[14], [15], [16], [17], [18]] while other have found that AMH was not a useful tool to predict IUI outcome [19,20]. These differences can be explained by the fact that ovarian reserve is closely correlated to age [21] making difficult to differentiate the respective part of age and of ovarian reserve on the ability of motherhood.

[16], [17], [18]] while other have found that AMH was not a useful tool to predict IUI outcome [19,20]. These differences can be explained by the fact that ovarian reserve is closely correlated to age [21] making difficult to differentiate the respective part of age and of ovarian reserve on the ability of motherhood. To try to answer this question, the present study aimed to evaluate the ability of the age-specific AMH levels [22,23] to predict the cumulative live birth rate after 4 IUI. Materials and methods Patients Five hundred and nine couples who underwent their first IUI between January 2011 and July 2017 in the Toulouse University Hospital entered the study. The indications of IUI were: unexplained infertility (252; 49.5%), ovulation disorder (151; 29.7%), moderate oligo-asthenospermia allowing to inseminate at least 106 motile spermatozoa (59; 11.6%), stage 1 endometriosis (32; 6.3%), moderate oligo-asthenospermia associated to ovulation disorders or to stage 1 endometriosis (15; 2.9%). The means female age was 33.4 ± 4.2 and 385 (75.6%) women had a primary infertility. All patients had an evaluation of tubal permeability using hysterosalpingography completed by laparoscopy in case of suspected abnormality before the beginning of IUI. Couples were excluded when the female partner had no two patent tubes [24] or if less than 106 motile spermatozoa were obtained after semen preparation [25].

All patients had an evaluation of tubal permeability using hysterosalpingography completed by laparoscopy in case of suspected abnormality before the beginning of IUI. Couples were excluded when the female partner had no two patent tubes [24] or if less than 106 motile spermatozoa were obtained after semen preparation [25]. Semen preparation Sperm were prepared according to WHO 2010 using discontinuous density gradient centrifugation (three layers: 60%, 80%, 90%) (Puresperm®, Nidacon, Mölndal, Sweeden). After preparation spermatozoa were incubated in 400 μl universal IVF medium (Origio, Versailles, France) at 37 °C in a 6% CO2 atmosphere. The number of recovered spermatozoa and their progressive motility were assessed in the medium to allow the measurement of the number of recovered motile spermatozoa. IUI procedures Ovarian stimulation used a combination of recombinant FSH (Gonal F, Merck, Lyon, France or Puregon, MSD, Paris, France) and GnRH antagonist (Cetrotide 0.25 mg, Merck, Lyon, France or Orgalutran, MSD, Paris, France). The initial dose of FSH was chosen according the female age and the score described by Chalumeau et al. [26]. Ovulation was triggered with recombinant hCG (Ovitrelle, Merck, Lyon, France) when at least one follicle ≥ 18 mm was obtained. Insemination was performed 36 h after hCG injection. A luteal support of 400 mg per day of intra-vaginal progesterone was administrated during 15 days, starting on the day of insemination.

26]. Ovulation was triggered with recombinant hCG (Ovitrelle, Merck, Lyon, France) when at least one follicle ≥ 18 mm was obtained. Insemination was performed 36 h after hCG injection. A luteal support of 400 mg per day of intra-vaginal progesterone was administrated during 15 days, starting on the day of insemination. Clinical pregnancy was defined as the presence of a fetal heartbeat evaluated during the transvaginal ultrasonographic examination seven weeks after insemination. Live birth was defined as the delivery of at least one live born infant after a 22 weeks or more pregnancy [27].

26]. Ovulation was triggered with recombinant hCG (Ovitrelle, Merck, Lyon, France) when at least one follicle ≥ 18 mm was obtained. Insemination was performed 36 h after hCG injection. A luteal support of 400 mg per day of intra-vaginal progesterone was administrated during 15 days, starting on the day of insemination. Clinical pregnancy was defined as the presence of a fetal heartbeat evaluated during the transvaginal ultrasonographic examination seven weeks after insemination. Live birth was defined as the delivery of at least one live born infant after a 22 weeks or more pregnancy [27]. Evaluation of the ovarian reserve Evaluations were done in the year before the first IUI. The hormonal measurements (FSH, LH, AMH and E2) were performed between cycle days 2 and 5 in the biochemistry laboratory of the Toulouse University Hospital with the same kits and the antral follicle count (AFC) at the same time, through 2D transvaginal ultrasonography by several different physicians in the department of obstetrics of the Toulouse University Hospital. Every follicle within 2–9 mm mean diameter (after 2 measures on orthogonal plans) was count. Serum LH, FSH, and E2 levels were assayed by an automated electrochemiluminescent based-assay (Elecsys® e602 Roche Diagnostics, Meylan, France) The interassay coefficient of variation (CV) were respectively 3.4% (around 11 UI/L) for LH, 2.2% (around 17 UI/L) for FSH and 3.4% (around 95 pg/mL) for E2. AMH was measured by ELISA GENII Beckman essay (Beckman Coulter Inc, Brea, CA) with an interassay CV of 4.4% (around 3.8 ng/mL) and a 0.08 ng/mL low limit of detection.

nterassay coefficient of variation (CV) were respectively 3.4% (around 11 UI/L) for LH, 2.2% (around 17 UI/L) for FSH and 3.4% (around 95 pg/mL) for E2. AMH was measured by ELISA GENII Beckman essay (Beckman Coulter Inc, Brea, CA) with an interassay CV of 4.4% (around 3.8 ng/mL) and a 0.08 ng/mL low limit of detection. Statistical analysis Data were extracted from the Gynelog clinical database used in our department. This database is approved by the French National Commission for Information Technology and Civil Liberties (CNIL) to be used for clinical research. According to French law (2012−300), patients are aware that their data can be used for anonymous clinical studies unless they specifically state otherwise. This information is detailed in posters in the rooms of the centre, and patients can inform the centre through a letter if they do not want to participate in clinical studies. The measured primary outcome was the cumulative live birth rate (CLBR) after a maximum of 4 attempts. Pregnancy loss was defined as the outcome of any pregnancy that does not result in at least one live birth [27]. Statistical analyses were performed using StatView software (Abacus Concepts Inc., Berkeley, CA). Data are means ± SD or median (range) according to the normality of the data. Percentages were compared by the χ2 test. Means were compared using the Student’s t-test and medians using the Mann-Whitney test according to the normality of data distribution.

ed using StatView software (Abacus Concepts Inc., Berkeley, CA). Data are means ± SD or median (range) according to the normality of the data. Percentages were compared by the χ2 test. Means were compared using the Student’s t-test and medians using the Mann-Whitney test according to the normality of data distribution. Groups of age were defined by the 25th, 50th 75th percentiles. Groups of age-specific AMH were defined by the 25th and 75th percentiles of AHM inside each group of ageThe age-specific AMH was called “low” when it was lower than the 25th percentile, “normal” between the 25th and the 75th percentile and “high” when it was higher than the 75th percentile, to allow easy reading. The demographic data of de different groups are described in Table1.Table 1 Demographic data of the different groups of age-specific AMH levels.

Groups of age were defined by the 25th, 50th 75th percentiles. Groups of age-specific AMH were defined by the 25th and 75th percentiles of AHM inside each group of ageThe age-specific AMH was called “low” when it was lower than the 25th percentile, “normal” between the 25th and the 75th percentile and “high” when it was higher than the 75th percentile, to allow easy reading. The demographic data of de different groups are described in Table1.Table 1 Demographic data of the different groups of age-specific AMH levels. Table 1Age ≤ 30 30 – 33 33 – 37 ≥ 37 AMH (ng/ml) ≤ 1.6 1.6 – 4.6 ≥ 4,6 ≤ 1.2 1.2 to 3.8 ≥ 3.8 ≤ 1.1 1.1 – 3.8 ≥ 3.8 ≤ 1.0 1.0 – 2.6 ≥ 2.6 n 34 67 43 33 58 32 33 63 32 35 59 29 Origin of infertilty (%) Ovulatory 17 (50) 16 (28) 14 (33) 16 (48) 12 (21) 13 (41) 18 (55) 8 (13) 8 (25) 10 (29) 9 (15) 10 (34) Endometriosis 0 3 (5) 9 (21) 3 (9) 6 (10) 1 (3) 2 (6) 4 (6) 1 (3) 1 (3) 2 (3) 0 Male 3 (9) 8 (14) 4 (9) 3 (9) 3 (5) 1 (3) 3 (9) 12 (19) 3 (9) 5 (14) 12 (20) 2 (7) Male and female 0 2 (3) 2 (5) 0 3 (5) 2 (6) 0 0 4 (13) 0 2 (3) 0 Unexplained 14 (41) 29 (50) 14 (33) 11 (33) 34 (59) 15 (47) 10 (30) 39 (62) 16 (62) 19 (54) 34 (58) 17 (58) Primary infertility (%) 30 (88) 50 (86) 33 (77) 29 (88) 50 (86) 19 (59) 25 (76) (44 (70) 24 (75) 26 (74) 34 (57) 21 (72) Basal FSH (mIU/ml) 7.9 ± 3.0 7.2 ± 1.5 6.5 ± 1.5 8.3 ± 2.2 7.1 ± 1.8 6.8 ± 1.7 7.9 ± 1.6 7.5 ± 2.2 6.7 ± 1.5 8.0 ± 2.1 7.1 ± 2.1 6.6 ± 1.1 Basal LH (mIU/ml) 4.8 ± 1.6 5.2 ± 2.0 6.7 ± 3.0 5.3 ± 1.9 6.2 ± 4.7 6.6 ± 2.9 4.6 ± 1.5 5.5 ± 2.1 6.9 ± 2.9 4.8 ± 2.0 5.2 ± 2.0 6.6 ± 4.5 Antral follicle count 15 ± 6 25 ± 9 33 ± 13 13 ± 6 19 ± 8 32 ± 14 11 ± 5 21 ± 8 34 ± 13 11 ± 4 17 ± 6 29 ± 15 Groups of age were defined as the 25th, 50th and 75th percentile.

± 1.1 Basal LH (mIU/ml) 4.8 ± 1.6 5.2 ± 2.0 6.7 ± 3.0 5.3 ± 1.9 6.2 ± 4.7 6.6 ± 2.9 4.6 ± 1.5 5.5 ± 2.1 6.9 ± 2.9 4.8 ± 2.0 5.2 ± 2.0 6.6 ± 4.5 Antral follicle count 15 ± 6 25 ± 9 33 ± 13 13 ± 6 19 ± 8 32 ± 14 11 ± 5 21 ± 8 34 ± 13 11 ± 4 17 ± 6 29 ± 15 Groups of age were defined as the 25th, 50th and 75th percentile. Groups of AMH were defined as the 25th and the 75th percentile in each group of age. Results Table 2 shows the results of IUI as a function of age-specific AMH. There was a trend, but not statistically significant, for a lower live birth rate in all low age-specific AMH groups. This was also true after considering age-specific AMH groups (low, normal and high) whatever the age (Table 3), the CLBR rate was not significantly different but showed a trend for a decrease in the low group. It must be pointed out that patients in the low AMH group had significantly higher initial FSH administrated doses and more mature follicles. Therefore, we have calculated the ratio of the number of newborns to the cumulative number of mature follicles. This ratio appeared significantly different among the groups of age-specific AMH: it appeared that, to obtain a newborn, twice more mature follicles were needed in the low than in the high AMH group (Table 3). There was a non-significant trend for a higher miscarriage rate per pregnancy in the low AMH group.Table 2 Results of IUI as a function of age and age-specific AMH levels.

of age-specific AMH: it appeared that, to obtain a newborn, twice more mature follicles were needed in the low than in the high AMH group (Table 3). There was a non-significant trend for a higher miscarriage rate per pregnancy in the low AMH group.Table 2 Results of IUI as a function of age and age-specific AMH levels. Table 2Age ≤ 30 30 – 33 33 – 37 ≥ 37 AMH (ng/ml) ≤ 1.6 1.6 – 4.6 ≥ 4,6 ≤ 1.2 1.2 – 3.8 ≥ 3.8 ≤ 1.1 1.1 – 3.8 ≥ 3.8 ≤ 1.0 1.0 – 2.6 ≥ 2.6 n 34 58 43 33 58 32 33 63 32 35 59 29 N IUI 2.5 ± 1.3 2.4 ± 1.3 2.4 ± 1.2 2.9 ± 1.3 2.3 ± 1.3 2.6 ± 1.2 2.8 ± 1.2 2.5 ± 1.2 2.7 ± 1.3 2.6 ± 1.2 2.9 ± 1.2 2.5 ± 1.2 Initial FSH dose 73 ± 29 58 ± 13 62 ± 24 76 ± 17 71 ± 14 60 ± 15 86 ± 22 72 ± 15 64 ± 14 97 ± 24 82 ± 16 75 ± 23 N follicles ≥ 15 mm 1.5 ± 0.6 1.3 ± 0.4 1.3 ± 0.5 1.7 ± 0.8 1.5 ± 0.5 1.2 ± 0.5 1.6 ± 0.7 1.5 ± 0.6 1.3 ± 0.5 1.8 ± 0.7 1.6 ± 0.8 1.6 ± 0.7 N inseminated motile spermatozoa (106) 24.3 ± 28.0 19.4 ± 28.5 22.0 ± 30.6 22.9 ± 28.2 28.2 ± 25.7 18.6 ± 15.9 23.3 ± 25.4 21.1 ± 28.6 18.2 ± 19.0 19.4 ± 23.5 26.0 ± 32.2 23.2 ± 29.3 Cumulative live birth after 4 IUI (%) 12 (35) 25 (43) 19 (44) 11 (33) 26 (45) 14 (44) 9 (27) 26 (41) 12 (38) 10 (29) 14 (24) 12 (41) Cumulative pregnancy losses after 4IUI (%) 5/17 (29) 4/29 (14) 7/23 (30) 2/13 (15) 3/29 (10) 2/16 (12) 3/12 (25) 8/34 (24) 5/17 (29) 6/16 (37) 10/24 (42) 3/15 (20) Ratio of the number of newborns to the cumulative number of follicles ≥ 15 mm (%) 13/129 (10) 28/192 (15) 19/137 (14) 12/165 (7) 27/192 (14) 18/100 (18) 11/160 (7) 30/242 (12) 14/121 (12) 10/161 (6) 15/293 (5) 15/124 (12) Groups of age were defined as the 25th, 50th and 75th percentile.

) 6/16 (37) 10/24 (42) 3/15 (20) Ratio of the number of newborns to the cumulative number of follicles ≥ 15 mm (%) 13/129 (10) 28/192 (15) 19/137 (14) 12/165 (7) 27/192 (14) 18/100 (18) 11/160 (7) 30/242 (12) 14/121 (12) 10/161 (6) 15/293 (5) 15/124 (12) Groups of age were defined as the 25th, 50th and 75th percentile. Groups of AMH were defined as the 25th and the 75th percentile in each group of age. Table 3 Results of IUI when regrouping age specific AMH into low, normal and high groups. Table 3 Age specific AMH Low Normal High Statistical comparison n 135 238 136 Initial FSH administrated dose (UI) 83 ± 25 71 ± 17 65 ± 21 P  < 0.0001 Number of follicles ≥ 15 mm 1.7 ± 0.7 1.5 ± 0.6 1.3 ± 0.5 P  < 0.01 Cumulative live births after 4 IUI (%) 45 (31) 91 (38) 57 (42) P = 0.344 Ratio of the number of newborns to the cumulative number of mature follicles 0.075 (46/615) 0.109 (100/919) 0.137 (66/482) P = 0.0035 Miscarriages (%) 12/57 (26) 22/113 (19) 9/66 (14) P = 0.209 Discussion Even if low age-specific AMH tend to be associated with a lower live birth rate and with a higher risk of miscarriage, its predictive value remained poor. The impact of AMH levels on the chances of success in ART highly varies among the studies.

Table 3 Age specific AMH Low Normal High Statistical comparison n 135 238 136 Initial FSH administrated dose (UI) 83 ± 25 71 ± 17 65 ± 21 P  < 0.0001 Number of follicles ≥ 15 mm 1.7 ± 0.7 1.5 ± 0.6 1.3 ± 0.5 P  < 0.01 Cumulative live births after 4 IUI (%) 45 (31) 91 (38) 57 (42) P = 0.344 Ratio of the number of newborns to the cumulative number of mature follicles 0.075 (46/615) 0.109 (100/919) 0.137 (66/482) P = 0.0035 Miscarriages (%) 12/57 (26) 22/113 (19) 9/66 (14) P = 0.209 Discussion Even if low age-specific AMH tend to be associated with a lower live birth rate and with a higher risk of miscarriage, its predictive value remained poor. The impact of AMH levels on the chances of success in ART highly varies among the studies. The ability of AMH to predict the ovarian response to stimulation has been widely reported and numerous studies have shown good correlations between AMH and the ovarian stimulation index [26] or the number of collected oocytes in IVF [28] and thus has a good ability to diagnose high and poor responders [29,30]. In IUI, Freiesleben et al., using the same FSH dose (75UI) for all patients, have shown that the number of recruited mature follicles (18 mm) was dependent on the AMH levels [19]. We found opposite data but this can be explained by the fact that we have adapted FSH dose to age-specific AMH in order to compensate the defect in ovarian responsiveness of patients with low AMH, as attested by the significantly higher follicles≥ 15 mm obtained when age-specific AMH is low.

ent on the AMH levels [19]. We found opposite data but this can be explained by the fact that we have adapted FSH dose to age-specific AMH in order to compensate the defect in ovarian responsiveness of patients with low AMH, as attested by the significantly higher follicles≥ 15 mm obtained when age-specific AMH is low. Concerning the relations between AMH and pregnancy rate in IUI, several authors have reported a significantly higher AMH in patients who achieved a clinical pregnancy [15,17,18]. In the same way, Moro et al. found a threshold of 2.3 ng/ml allowing to discriminate women according their chances of success [16] and Li et al. found a similar threshold at 1.8 ng/ml [14]. However, other authors found a modest [14] even a non-significant [20] correlation between the AHM level and the chances of ongoing pregnancy. Similar discrepancies have been reported in IVF with a significant impact on the results for some authors [[31], [32], [33], [34], [35]] and no predictive value for others [18,[36], [37], [38]]. These discrepancies could be due to the fact that these studies were focused on the sole AMH level. While AMH and age are closely linked, wide variations exist inside a year of age [39], thus the use of age-specific AMH allows to better discriminate the effect of age and of diminished ovarian reserve [40].

Concerning the relations between AMH and pregnancy rate in IUI, several authors have reported a significantly higher AMH in patients who achieved a clinical pregnancy [15,17,18]. In the same way, Moro et al. found a threshold of 2.3 ng/ml allowing to discriminate women according their chances of success [16] and Li et al. found a similar threshold at 1.8 ng/ml [14]. However, other authors found a modest [14] even a non-significant [20] correlation between the AHM level and the chances of ongoing pregnancy. Similar discrepancies have been reported in IVF with a significant impact on the results for some authors [[31], [32], [33], [34], [35]] and no predictive value for others [18,[36], [37], [38]]. These discrepancies could be due to the fact that these studies were focused on the sole AMH level. While AMH and age are closely linked, wide variations exist inside a year of age [39], thus the use of age-specific AMH allows to better discriminate the effect of age and of diminished ovarian reserve [40]. In our study, there were a non-significantly higher miscarriage rate when age-specific AMH was low. Low AMH has been shown to be linked, independently of age, to increased pregnancy loss as well in naturally conceived pregnancies [41] as after IVF [42], probably due to a higher embryonic aneuploidy [43,44] These data suggest that the diminution of the ovarian reserve; notably when unexplained, may not only be a quantitative problem but also a qualitative one. Our observation of a significant decrease of the efficiency of the stimulation, which we have estimated through the ratio of the number of newborns to the cumulative number of follicles ≥ 15 mm, with the age-specific AMH levels, is in line with this hypothesis. For example, the mechanisms by which some molecules, such as environmental pollutants, can alter the pool of follicles, can also impair oocyte quality [45].

mated through the ratio of the number of newborns to the cumulative number of follicles ≥ 15 mm, with the age-specific AMH levels, is in line with this hypothesis. For example, the mechanisms by which some molecules, such as environmental pollutants, can alter the pool of follicles, can also impair oocyte quality [45]. The main limitation of this study is the relatively low number of subjects in each group of age and AMH, which decrease the statistical power of the analyses. In conclusion, these data show that correct live birth rates can be obtained by IUI in case of low age-specific AMH if higher doses of FSH are used. Indeed, our results have shown that, to obtain a newborn, patients with low AMH required 1.5 more follicles than those with normal AMH and twice more than those with high AMH. Conflict of interest The authors have no conflicts of interest to declare.

Introduction Postpartum urinary retention is a frequent complication after childbirth [1]. Although the exact pathophysiology remains unclear, post void residual (PVR) is assumed to be a multifactorial process based on physiological, neurological and mechanical conditions. [2]. Risk factors for PVR after delivery are instrumental delivery, perineal trauma, epidural anesthesia, higher birth weight and nulliparity [3,4]. High PVR is usually a temporary condition. However, unrecognised retention can lead to with considerable morbidity due to bladder over distention, detrusor atony and long term voiding dysfunction [2,5]. Therefore it is important to adequately diagnose significant PVR. Common used methods for PVR measurements are catheterization and (three-dimensional bladder) ultrasonography. Although catheterization is mentioned as the golden standard, it is an invasive procedure which can lead to an increased risk of urinary tract infections and trauma [6]. Therefore, preferably a non-invasive ultrasonography is used. Previous research has shown that three-dimensional bladder ultrasonography (BladderScan®) is a reliable instrument to determine PVR in men and non-pregnant women [7,8]. The three-dimensional bladder ultrasonography (BladderScan® (US, using the BVI 3000®, Verathon, WA, USA) is most commonly used method in the Netherlands for determination of PVR in postoperative patients. However, the reliability of the BladderScan® in the postpartum period are conflicting [1,[9], [10], [11], [12], [13], [14]].

-dimensional bladder ultrasonography (BladderScan® (US, using the BVI 3000®, Verathon, WA, USA) is most commonly used method in the Netherlands for determination of PVR in postoperative patients. However, the reliability of the BladderScan® in the postpartum period are conflicting [1,[9], [10], [11], [12], [13], [14]]. In our clinic we noticed an overestimation of PVR’s measured with the BladderScan® in comparison with catheterization in women one week after delivery. With this study, we intend to ascertain whether the BladderScan® is a reliable instrument for measurement of PVR in patients with postpartum urinary retention who conducted a micturition trial one week after delivery. Materials and methods We included 25 women in this prospective pilot study. These women had a urinary retention over 1000 ml within 4–5 h postpartum. Conform our local protocol, an indwelling catheter was inserted for one week. After removal of the indwelling catheter, a micturition trial was conducted. In our protocol, a catheterization is a normal procedure for evaluating PVR, but the BladderScan® is used as well. In this study, PVR was measured with the BladderScan® (US, using the BVI 3000®, Verathon, WA, USA). This evaluation was directly followed by clean intermittent catheterization (CIC) to collect and determine the real volume of urinary retention, as catheterization is the golden standard to assess PVR. We defined PVR above 200 ml as significant.

asured with the BladderScan® (US, using the BVI 3000®, Verathon, WA, USA). This evaluation was directly followed by clean intermittent catheterization (CIC) to collect and determine the real volume of urinary retention, as catheterization is the golden standard to assess PVR. We defined PVR above 200 ml as significant. The measurement of PVR was conducted by a continence nurse specialist who was trained in adequate measurement by firm Verathon; developers of the BladderScan®. All measurements were conducted according to the instructions of the BladderScan®. Statistical analysis was performed using IBM SPSS Statistics version 20.0. A two-sided p-value <0.05 was considered to indicate statistical significance. The Wilcoxon test was used to determine difference between PVR data of the BladderScan® and CIC. Spearman rank test was used for correlation between PVR data of the BladderScan® and CIC. For categorical characteristics frequencies were analysed in contingency tables with X2 statistics. Continuous variables were depicted as median with interquartile range. We calculated the sensitivity, specificity and positive predictive value of our PVR measurements with BladderScan® in comparison to CIC. Ethical approval Ethical approval was not applicable, because this was an evaluation of standard care. The patients in this evaluation were informed and had no objection to the use of their information. Results The median interquartile range of PVR of the BladderScan® and CIC are respectively 403 ml (314–637 ml) and 95 ml (35–95 ml).

Ethical approval Ethical approval was not applicable, because this was an evaluation of standard care. The patients in this evaluation were informed and had no objection to the use of their information. Results The median interquartile range of PVR of the BladderScan® and CIC are respectively 403 ml (314–637 ml) and 95 ml (35–95 ml). According to our PVR definition, only 3 patients measured a residu <200 ml by the BladderScan®. In 20 patients the BladderScan® measured PVR ≥200 ml. However, in 13 of these patients, CIC showed a PVR <200 ml (table S1). There was a significant mean difference in PVR measurements with the BladderScan® and CIC of 312 ml (95% CI 220–404 ml) (p < 0.001). There was a positive correlation between PVR data of the BladderScan® and CIC (r = 0.60; p < 0.05) According to our PVR definition of 200 ml the sensitivity and specificity of the BladderScan® was respectively 100% and 17.6%. The positive predictive value was 36% (Table 1).Table 1 2x2 contingency table between three-dimensional bladder ultrasonography (Bladderscan®BVI 3000) versus clean intermittent catheterization. Table 1 CIC (≥200 ml) CIC (<200 ml) Bladderscan® (≥200 ml) 8 14 Bladderscan® (<200 ml) 0 3 Results: Sensitivity 100% specificity 17.6% positive predictive value 36%; CIC = clean intermittent catheterization. Comment According to our study, the BladderScan® (BVI 3000) is a non-reliable instrument to measure adequate PVR one week postpartum.

Table 1 CIC (≥200 ml) CIC (<200 ml) Bladderscan® (≥200 ml) 8 14 Bladderscan® (<200 ml) 0 3 Results: Sensitivity 100% specificity 17.6% positive predictive value 36%; CIC = clean intermittent catheterization. Comment According to our study, the BladderScan® (BVI 3000) is a non-reliable instrument to measure adequate PVR one week postpartum. To our knowledge we are the first to investigate the reliability of the BladderScan® one week postpartum. Our findings are in line with Pallis et al. [12] who measured the PVR one day postpartum. This is in contrast with all other previous studies [1,[9], [10], [11]] showed that the BladderScan® is a reliable instrument to measure PVR’s (cut-off 300 ml and 400 ml) after vaginal delivery. However, they all measured PVR directly postpartum and use a higher cut of for significant PVR. Sensitivity and specificity vary with different cut-off values. Lukasse et al showed an decrease in specificity from 96% to 65%, but increase in sensitivity from 76% to 100% when the cut-off value changed from 400 ml until 300 ml [9]. This implicates, that small PVR leads to an overestimation and large PVR to a possible underestimation, which is in line with our findings.

f values. Lukasse et al showed an decrease in specificity from 96% to 65%, but increase in sensitivity from 76% to 100% when the cut-off value changed from 400 ml until 300 ml [9]. This implicates, that small PVR leads to an overestimation and large PVR to a possible underestimation, which is in line with our findings. In all studies a similar type of the BladderScan® (BVI 3000 or 6100) was used. The different outcome of the studies might be based on population size, the variety time of measurement (directly - <24 h) and heterogeneity of definitions of PVR (150–500 ml). However, the major difference might be related to the difficulty for the Bladderscan® to differentiate the bladder from the uterus postpartum. Directly postpartum, the uterus is about 18 week gestation, while after one week it is on the same level of the bladder. Thus, this may lead to an overestimation in measured bladder volume, as the Bladderscan® may be unable to differentiate in volume of the bladder and uterus. Therefore, the volume of the uterus might be counted in the measurement of the bladder volume. Regarding the latest prospective study of Mulder et all, the reliability of measuring PVR with the BladderScan® (BVI 9400) is promising, according to a sensitivity of 85.4% and sensitivity of 85.6% with a cut-of range for PVR >500 ml directly postpartum [14]. Even though it is a methodically wise strong and large study, it also is the first which used the newer BladderScan® (BVI 9400). And therefore, their measurements may be more precise/accurate compared to the older models (BVI 3000, BVI 6100).

tivity of 85.6% with a cut-of range for PVR >500 ml directly postpartum [14]. Even though it is a methodically wise strong and large study, it also is the first which used the newer BladderScan® (BVI 9400). And therefore, their measurements may be more precise/accurate compared to the older models (BVI 3000, BVI 6100). Strength of our study is that CIC and measurements with the BladderScan® was performed by one investigator who was trained adequately. Therefore, observational bias was minimalized. A limitation of our study is the small study population. But the difference in this small population is already clinically significant. In practise, we prefer to use the BladderScan®, to maintain the benefits of non-invasive measurement. However, the Bladderscan® (BVI 9400) is only validated for PVR >500 ml. Also, this is the newest type of scan which is very expensive and therefore not (directly) available in all hospitals. Therefore, we advise further research in order to develop a formula for the BladderScan® BVI 3000 for the postpartum period. When postmictal urinary retention occurs, we advise the use of CIC above an indwelling catheter as described in Mulder et al. [16]. Conflict of interest All authors report no conflict of interest. Role of the funding source No specific funding was obtained. Acknowledgements Not applicable.

Introduction Persistent infection with high-risk human papillomavirus (HPV) is a prerequisite for the development of cervical cancer [1]. Persistence of HPV is consistently and strongly associated with the risk of developing high-grade cervical intraepithelial neoplasia (CIN2+) [2] which in turn include an elevated risk to progress to cervical cancer [3]. HPV testing has greater sensitivity in revealing CIN2+ than cytology [4], and is presently recommended as the primary screening method for cervical cancer in Europe [5]. HPV-positive women are recommended cytological triage, the less sensitive method for detection of CIN2 + . This will result in a group of HPV-positive women with normal cytology and the optimal clinical handling of these women is not known [6].

ommended as the primary screening method for cervical cancer in Europe [5]. HPV-positive women are recommended cytological triage, the less sensitive method for detection of CIN2 + . This will result in a group of HPV-positive women with normal cytology and the optimal clinical handling of these women is not known [6]. Although some persistent infections clear spontaneously, women with normal cytology who are positive for HPV have a much higher risk of developing CIN3 than HPV-negative women [7]. Katki et al. have found a 7.4% 5-year risk of CIN3+ in women with persistent HPV infection and normal cytology [8]. Kjaer et al did a population based prospective cohort study on HPV positive women <30 years with normal Pap smear at baseline showing risks of developing CIN3 in 12 years in different HPV genotypes as following: HPV16 26,7%, HPV18 19,1% and HPV31/33 over 14% [9]. The Swedescreen study, involving women aged 32–38 years, described that among women with a normal Pap smear attending organized screening, the positive predictive value of HPV persistence as regards detection of biopsy-confirmed CIN2+ was 29% [10]. Long-term follow-up of this study pointed out that all the HPV-positive women with initially normal cytology either become HPV-negative or developed CIN2+ within seven years [11]. Mittal et al additionally describe highest incidence rate of CIN2+ developing from persistent HPV infection in women over 50 years [12].

0]. Long-term follow-up of this study pointed out that all the HPV-positive women with initially normal cytology either become HPV-negative or developed CIN2+ within seven years [11]. Mittal et al additionally describe highest incidence rate of CIN2+ developing from persistent HPV infection in women over 50 years [12]. Cytological screening is less sensitive in women of 50 years of age or more compared with younger women [13]. Colposcopy have moderate sensitivity for detection of CIN2+ in premenopausal women, and the sensitivity decreases further in postmenopausal women [14]. In colposcopic examination the type of the transformation zone (TZ) is crucial for interpretation of the examination. In types 1 and 2 the TZ is fully visible. In TZ type 3 the upper limit is not visible and this is a common finding in the postmenopausal period when the TZ often retracts into the endocervix [15]. Performance of biopsies lacking the TZ is insufficient [16]. It is also more difficult to obtain adequate amounts of tissue from the endocervix and the sensitivity of cytobrush sampling in detecting dysplasia varies between 44-93%, and is even lower with endocervical curettage for histopathological samples [17,18]. In addition, the positive predictive rate of colposcopic examination is better as regards high-grade cervical lesions and less accurate for low-grade cervical lesions [19]. Petry et al have described a higher colposcopy failure rate in the HPV-positive/Pap-normal group than in the HPV-positive/Pap-abnormal group [16]. The most reliable method to obtain representative samples is to excise the whole TZ surgically, for example by using a loop electrosurgical excision procedure (LEEP), for histological analysis. This procedure is already recommended as clinical practice for women with low-grade colposcopic changes or TZ3 in colposcopy associated with high-grade cytological changes [20].

amples is to excise the whole TZ surgically, for example by using a loop electrosurgical excision procedure (LEEP), for histological analysis. This procedure is already recommended as clinical practice for women with low-grade colposcopic changes or TZ3 in colposcopy associated with high-grade cytological changes [20]. The management of women with persistent HPV infection and no cytological or colposcopic evidence of CIN represents an unsolved clinical problem. We therefore performed LEEP on a group of women over 40 years with HPV persistency but normal cytology, to determine the prevalence of histologically confirmed CIN2+.

amples is to excise the whole TZ surgically, for example by using a loop electrosurgical excision procedure (LEEP), for histological analysis. This procedure is already recommended as clinical practice for women with low-grade colposcopic changes or TZ3 in colposcopy associated with high-grade cytological changes [20]. The management of women with persistent HPV infection and no cytological or colposcopic evidence of CIN represents an unsolved clinical problem. We therefore performed LEEP on a group of women over 40 years with HPV persistency but normal cytology, to determine the prevalence of histologically confirmed CIN2+. Materials and methods This prospective study was performed from April 2013 until March 2016 and carried out at the gynecological out-patient clinic, Uppsala University Hospital, Sweden. We recruited 91 women with persistent HPV infection but without abnormalities in cytology by sending them an invitation letter (Fig. 1). We excluded women who had plans for future pregnancies, who could not understand the information in Swedish, and where LEEP was regarded as being technically difficult to perform, since we wished to carry it out using local anesthesia. Forty-four women were interested in taking part in the study and these women were contacted by telephone for more information and to schedule a study visit. In four women LEEP could not be performed for anatomical reasons. All postmenopausal women were treated with local estradiol for at least two weeks before the visit to optimize the vaginal mucosa and minimize the possible risk of postoperative cervical stenosis. A signed informed consent document was obtained at the visit. The examination was performed by an experienced colposcopist (RA) and included a) a cytobrush sample for HPV analysis, b) a conventional Pap smear, c) a separate endocervical cytobrush sample on a glass slide, and d) colposcopic evaluation with application of 5% acetic acid and iodine solution, identification of squamocolumnar junction and transformation zone and punch biopsy sampling. The tissues with the most pathological appearance were biopsied and in the absence of abnormality a random biopsy sample was taken. Finally, diagnostic LEEP was performed in local anesthesia for histological analysis. All women that were HPV-positive at the study visit underwent follow-up 6–12 months after LEEP, with a Pap smear and an HPV test. Women that did not participate in the study were followed up with annual Pap smears and HPV tests.Fig. 1 Diagram of all the invited women including key results.

ia for histological analysis. All women that were HPV-positive at the study visit underwent follow-up 6–12 months after LEEP, with a Pap smear and an HPV test. Women that did not participate in the study were followed up with annual Pap smears and HPV tests.Fig. 1 Diagram of all the invited women including key results. Fig. 1

omyosis of the inner myometrium or serrated JZ and linear JZ at 6 months follow-up, with the most pronounced improvement among patients with linear JZ. At 18 months, when patients undergoing reintervention surgery were removed from analyses, there was no longer a statistically significant difference between the groups. Presence and intensity of pelvic pain at baseline, and 6 and 18 months after TCRE is shown in Table 4. There were no statistically significant differences in the distribution of patients with pain or the pain intensity between the groups at baseline. Excluding patients with concomitant pelvic pathology did not change the results significantly (S2). At 18 months follow-up, when patients with reintervention surgery were excluded, the number of patients with pelvic pain was considerably reduced and there was no significant difference in pain reduction according to histopathological findings.Table 4 Pelvic pain at baseline and follow-up after transcervical resection of the endometrium.

ia for histological analysis. All women that were HPV-positive at the study visit underwent follow-up 6–12 months after LEEP, with a Pap smear and an HPV test. Women that did not participate in the study were followed up with annual Pap smears and HPV tests.Fig. 1 Diagram of all the invited women including key results. Fig. 1 All cytology and histology was performed at the Clinic of Pathology and Cytology, Uppsala University Hospital, Uppsala. The highest histological grade found in each patient was used for interpretation of the results. In HPV-testing cervical cytobrush samples were applied to an indicating FTA elute micro-cardTM (GE Healthcare, United Kingdom, art. no WB129308). The FTA cards were processed using a dedicated automated laboratory system (easyPunch STARlet, Hamilton Robotics, USA) which collects each card, takes a photograph of the sample collection area, identifies the parts of the sampling deposition area with the highest amount of cellular material using a machine learning software, and then takes 4 punches with a 3-mm diameter knife from the area containing most material and deposits the punches in a single well in a 96-well microtiter plate. DNA extraction from punches was performed as described earlier [21]. Testing for HPV was performed using a multiplex real-time PCR assay (hpVIR), which detects the following high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 (18 and 45 are detected together, and 33, 52 and 58 as one group) and also measures a human single copy gene (HMBS), which serves as a control for that the samples contain sufficient amounts of cellular material for the test to be informative. The limit of detection (LOD) for the nuclear single copy gene HMBS and HPV was both set to 10 copies per PCR. The FTA cards were analyzed at the HPV laboratory, Uppsala University.

gene (HMBS), which serves as a control for that the samples contain sufficient amounts of cellular material for the test to be informative. The limit of detection (LOD) for the nuclear single copy gene HMBS and HPV was both set to 10 copies per PCR. The FTA cards were analyzed at the HPV laboratory, Uppsala University. The endpoint of the study was histologically identified CIN2+ in LEEP samples from women with persistent HPV infection but without evident CIN in either cytology or colposcopy. The study was approved by the Regional Ethics Committee in Uppsala (Dnr 2012/460).

gene (HMBS), which serves as a control for that the samples contain sufficient amounts of cellular material for the test to be informative. The limit of detection (LOD) for the nuclear single copy gene HMBS and HPV was both set to 10 copies per PCR. The FTA cards were analyzed at the HPV laboratory, Uppsala University. The endpoint of the study was histologically identified CIN2+ in LEEP samples from women with persistent HPV infection but without evident CIN in either cytology or colposcopy. The study was approved by the Regional Ethics Committee in Uppsala (Dnr 2012/460). Results In the 40 women who underwent complete examination including LEEP, the mean duration of known HPV persistence was 20 months (median 12, range 4–93). The mean age of the women was 58 years (median 59, range 41–77) and 33/40 women were postmenopausal. Before the study, 25/40 women had taken 1 Pap smear, 13/40 had taken 2 Pap smears, 1/40 had taken 3 Pap smears and 1/40 had taken 7 Pap smears. They were all normal. All the cytological analysis of Pap smears and endocervical samples obtained at the study visit were normal. Only 3/40 Pap smears were reported lacking columnar cells which can be interpreted that in 37/40 cases the junction was reached. None of the biopsy samples confirmed CIN2+, but six showed CIN1. Five cases lack a biopsy sample because it was difficult to obtain due to technical reasons. Notably, for 28/40 (70%) of the women had a TZ type 3. The mean depth of excision was 12.3 mm (median 12 mm, range 8–18 mm) and 34/40 histological samples included the whole TZ. No complications were reported. Histological evaluation of the LEEP samples showed that 20 women had no CIN, 14 women had CIN1 and only 3 of these samples did not include the whole TZ. Further two women had CIN2 and four women had CIN3. None of the LEEP samples displayed invasive disease and five out of six CIN2+ excisions had free endocervical margins. Four out of six women with CIN2+ had TZ type 3 but two women with CIN2+ had a fully visible normal TZ. HPV analysis revealed that 21/40 women had cleared their HPV infection, while 19/40 women still had a persistent HPV infection at the study visit. The most common HPV types that persisted were HPV16 (n = 5) and the HPV33/52/58 group (n = 5). All the women with CIN2+ were HPV-positive at the study visit with different HPV types. The known duration of HPV persistence among the women with CIN2+ was between 7 and 20 months (mean 13 months) and the known duration of HPV persistence among the women showing no CIN was between 7 and 93 months (mean 26 months). None of the 21 women who were HPV-negative at the study visit showed CIN2+ in histology.

s. The known duration of HPV persistence among the women with CIN2+ was between 7 and 20 months (mean 13 months) and the known duration of HPV persistence among the women showing no CIN was between 7 and 93 months (mean 26 months). None of the 21 women who were HPV-negative at the study visit showed CIN2+ in histology. All results from women that underwent LEEP are presented in Table 1.Table 1 Individual data on all the women who underwent LEEP presented in order of CIN findings with highest grade first.

s. The known duration of HPV persistence among the women with CIN2+ was between 7 and 20 months (mean 13 months) and the known duration of HPV persistence among the women showing no CIN was between 7 and 93 months (mean 26 months). None of the 21 women who were HPV-negative at the study visit showed CIN2+ in histology. All results from women that underwent LEEP are presented in Table 1.Table 1 Individual data on all the women who underwent LEEP presented in order of CIN findings with highest grade first. Table 1Baseline data at invitation Test result at study visit Follow up 6 months after study visit Age Former excision for CIN Smoking Persistent HPV type Persistency (months) HPV PAP-smear Endocervical cytology TZ Histology in biopsy Histology in excision Margin status cervical/vaginal Excision height (mm) HPV PAP-smear 59 0 0 16 20 16 normal normal 3 normal CIN3 neg/neg 13 neg normal 56 0 0 31 7 31 normal normal 3 normal CIN3 neg/neg 15 neg normal 41 0 1 39 10 39 normal normal 3 0 CIN3 neg/neg 15 neg normal 59 0 0 33/52/58 10 33/52/58 normal normal 1 or 2 CIN1 CIN3 pos/pos 13 neg normal 60 0 ? 56 19 56 normal normal 3 0 CIN2 neg/neg 14 neg normal 54 0 1 59 11 59 normal normal 1 or 2 0 CIN2 neg/neg 12 neg normal 59 0 1 16 7 16 normal normal 1 or 2 CIN1 CIN1 neg/neg 12 neg normal 45 0 0 18/45 57 18/45 normal normal 3 normal CIN1 neg/neg 11 35 0 45 0 0 18/45 11 18/45 normal normal 3 CIN1 CIN1 neg/neg 13 neg normal 61 0 0 33/52/58 29 33/52/58 normal normal 3 CIN1 CIN1 pos/pos 10 neg normal 71 1 0 33/52/58 18 33/52/58 normal normal 1 or 2 normal CIN1 neg/neg 9 33/52/58 ASCUS 62 0 0 59 33 neg normal normal 1 or 2 CIN1 CIN1 neg/neg 12 0 0 67 1 0 16 8 neg normal normal 3 CIN1 CIN1 neg/pos 12 neg normal 60 0 0 51 12 neg normal normal 1 or 2 normal CIN1 neg/neg 18 0 0 62 0 0 16 4 neg normal 0 3 normal CIN1 neg/neg 12 0 0 60 0 1 39 12 neg normal normal 3 0 CIN1 neg/neg 11 neg normal 58 1 0 56 10 neg normal normal 3 normal CIN1 neg/neg 14 0 0 59 0 ?

ospital discharge forms from 2001 to 2012. Their study showed that the risk of PACs was 122.9 per 100,000 pregnancies with the most common cancers being breast, thyroid and blood cancers. Furthermore, the incidence increased significantly with age but it did not show any increase over time in the considered decade [3]. The aim of the present study was to investigate the incidence of pregnancy related cancers in Apulia, a region with 4 million inhabitants in the southern Italian peninsula from 2003 to 2015, and to compare the Apulian data with those reported from other geographical areas. Materials and methods We evaluated data from the National electronic database containing all the hospital discharge forms, in Italian “Scheda di Dimissione Ospedaliera (SDO)” regarding hospitals in Apulia from January 2003 to December 2015. In each SDO the patient is identified by a nationwide unique reference anonymous code and a series of clinical information is reported. More precisely, each SDO contains personal and demographic data (e.g. date of birth, nationality, qualification, job), patients’ main complaint, a comprehensive provision of medical services during hospitalization, history of the present illness, remote pathological anamnesis, review of systems with principal vital parameters, regular and acute medications, allergies, discharge dates. The main diagnosis and 5 secondary diagnoses are coded according to the International Classification of Disease, Ninth Revision (ICD-9) while up to 5 interventions and hospitalization- related costs are encoded according to the national diagnosis-related group (DRG) system. The first step was to select all those SDOs reporting DRG codes 370-375 and 380-381 (regarding deliveries and abortions respectively), and to check for any possible lapse including only the SDOs reporting diagnoses or delivery/abortion-related interventions.

g 12 0 0 67 1 0 16 8 neg normal normal 3 CIN1 CIN1 neg/pos 12 neg normal 60 0 0 51 12 neg normal normal 1 or 2 normal CIN1 neg/neg 18 0 0 62 0 0 16 4 neg normal 0 3 normal CIN1 neg/neg 12 0 0 60 0 1 39 12 neg normal normal 3 0 CIN1 neg/neg 11 neg normal 58 1 0 56 10 neg normal normal 3 normal CIN1 neg/neg 14 0 0 59 0 ? 56 12 neg normal normal 3 normal CIN1 neg/neg 15 neg normal 47 1 0 33/52/58 12 neg normal normal 1 or 2 normal CIN1 neg/neg 12 0 normal 59 0 0 18/45 12 neg normal normal 1 or 2 normal CIN1 neg/neg 13 neg normal 59 0 0 16 45 16 normal normal 3 normal normal 12 16 normal 68 0 0 16 33 16 normal normal 1 or 2 normal normal 15 16 normal 46 1 0 16 7 16 normal normal 3 normal normal 15 16 normal 56 0 0 39 12 39 normal normal 3 normal normal 8 neg normal 69 0 0 56 29 56 normal normal 3 normal normal 14 56 ASCUS 53 0 1 56 10 56 normal not possible 3 normal normal 11 neg normal 54 0 0 33/52/58 25 33/52/58 normal normal 3 0 normal 9 33/52/58 normal 62 0 0 33/52/58 93 33/52/58 normal not possible 3 normal normal 9 33/52/58 ASCUS 61 0 0 16 44 neg normal normal 3 normal normal 14 0 0 62 0 0 16 30 neg normal normal 1 or 2 normal normal 10 0 0 73 0 0 16 18 neg normal normal 3 normal normal 11 0 0 64 0 0 18/45 28 neg normal normal 3 normal normal 11 0 0 58 0 1 16 24 neg normal normal 1 or 2 normal normal 12 0 0 41 1 0 51 12 neg normal 0 1 or 2 normal normal 9 0 0 62 0 0 33/52/58 7 neg normal normal 3 normal normal 10 0 0 77 1 0 33/52/58 16 neg normal not possible 3 normal normal 10 0 0 56 0 0 31 10 neg normal normal 3 normal normal 15 neg normal 43 0 ? 16 10 neg normal normal 3 normal normal 15 0 0 53 0 0 56 10 neg normal normal 3 normal normal 14 0 0 59 0 0 35 12 neg normal normal 3 normal normal 13 0 0

l normal 3 normal normal 10 0 0 77 1 0 33/52/58 16 neg normal not possible 3 normal normal 10 0 0 56 0 0 31 10 neg normal normal 3 normal normal 15 neg normal 43 0 ? 16 10 neg normal normal 3 normal normal 15 0 0 53 0 0 56 10 neg normal normal 3 normal normal 14 0 0 59 0 0 35 12 neg normal normal 3 normal normal 13 0 0 At follow-up 6–12 months after LEEP, all six women with CIN2+ had become HPV-negative and showed normal cytology. Among eight HPV-positive women with no dysplasia in the LEEP sample six women still had a persistent HPV-infection at follow-up (Fig. 1).

l normal 3 normal normal 10 0 0 77 1 0 33/52/58 16 neg normal not possible 3 normal normal 10 0 0 56 0 0 31 10 neg normal normal 3 normal normal 15 neg normal 43 0 ? 16 10 neg normal normal 3 normal normal 15 0 0 53 0 0 56 10 neg normal normal 3 normal normal 14 0 0 59 0 0 35 12 neg normal normal 3 normal normal 13 0 0 At follow-up 6–12 months after LEEP, all six women with CIN2+ had become HPV-negative and showed normal cytology. Among eight HPV-positive women with no dysplasia in the LEEP sample six women still had a persistent HPV-infection at follow-up (Fig. 1). Discussion In our study 6/40 (15%) women with persistent HPV infection and no detectable abnormalities in cytology had CIN2+ verified by histological examination of the LEEP sample. Two thirds of these women had a TZ3. HPV genotyping showed different high-risk types in all six of these women, a finding also noticed in an earlier study from our group [22], which indicates that in order to cover the HPV types associated with persistence, it is necessary to genotype for more than HPV16/18 in this older age group. An important notice is that only 19 women in this cohort still tested HPV-positive at the study visit and all the CIN2+ women were in this group. By contrast, none of the 21 women with cleared HPV infection had CIN2+, a finding that was also noted in follow-up in the Swedescreen study [11]. However, a recent Dutch post-hoc analysis describes higher risk for CIN3+ in five years for women that were HPV positive in the first screening round and then turned HPV negative at follow-up [23]. A continued surveillance of these women might thus be needed. Our results further highlight the importance of carrying out follow-up of women with persistent HPV infection and a normal cytology, since a moderate proportion have CIN2+ and thus are at risk of developing cervical cancer.

ed HPV negative at follow-up [23]. A continued surveillance of these women might thus be needed. Our results further highlight the importance of carrying out follow-up of women with persistent HPV infection and a normal cytology, since a moderate proportion have CIN2+ and thus are at risk of developing cervical cancer. The optimal management of women with histological CIN1 is surveillance, since at least 70% of these lesions will resolve spontaneously and only few will progress [24]. While CIN2+ is considered as a true precursor of cervical cancer and is associated with oncogenic types of HPV, CIN1 is merely an insensitive histopathological sign of HPV infection [25] that can be caused even by low-risk HPV types [26]. Even if LEEP performed because of CIN tends to eliminate HPV infection [27], treatment of CIN1 is associated with a higher HPV-positive rate at follow-up when compared with treatment of CIN2+ [28]. In our study also, LEEP performed in connection with CIN2+ tended to clear the HPV infection earlier than in women with CIN1. In addition, it does not seem possible to treat a persistent HPV infection without CIN by means of LEEP, since most of these women still tested HPV-positive at follow-up. In women with CIN1 or normal histology in the excision the future risk of development of CIN or cancer remains unclear, but excision samples including the whole TZ (29/34 women) exclude an existing CIN2+.

stent HPV infection without CIN by means of LEEP, since most of these women still tested HPV-positive at follow-up. In women with CIN1 or normal histology in the excision the future risk of development of CIN or cancer remains unclear, but excision samples including the whole TZ (29/34 women) exclude an existing CIN2+. The number of study subjects is rather small since only 44/91 (48%) of the invited women chose to participate. We do not find this unexpected since the study visit included an invasive treatment demanding local anesthesia. Also the usually self-healing nature of the HPV-infection and a trust to a normal Pap smear could explain the low participation rate. We do however believe that the obtained data gives a quite fair estimate of the CIN2+ prevalence in this group of women.

cording to the national diagnosis-related group (DRG) system. The first step was to select all those SDOs reporting DRG codes 370-375 and 380-381 (regarding deliveries and abortions respectively), and to check for any possible lapse including only the SDOs reporting diagnoses or delivery/abortion-related interventions. In this report we consider PAC as the diagnosis of malignancy occurring 9 or 3 months before delivery or abortion respectively, or within 12 months after the date of pregnancy outcome considered as the discharge date. Among those patients, we selected all SDOs reporting ICD codes 140.-208., which mean a diagnosis of malignant cancer among the main or secondary diagnoses. We excluded SDOs in which cancer was recorded as secondary diagnosis or if a previous SDO reported cancer as the main diagnosis because our aim was to obtain only incident neoplasms.

visit included an invasive treatment demanding local anesthesia. Also the usually self-healing nature of the HPV-infection and a trust to a normal Pap smear could explain the low participation rate. We do however believe that the obtained data gives a quite fair estimate of the CIN2+ prevalence in this group of women. The present cohort represents a highly selected group with previously identified persistent HPV infection and no former evidence of CIN. Currently it is not clear how to manage these women and even though a persistent HPV infection is a risk factor of cervical cancer, very few HPV-positive women will develop the disease. About half or more of HPV infections will clear within a year [29] as they also did in this study. It is important to consider the balance between overtreatment of women with transient infections against the risk of not detecting those women who may develop cervical cancer. Unnecessary treatments are not only uncomfortable but have short-term risks such as infection or bleeding and can in the long-term also cause preterm labor [30] or cervical stenosis [31]. Knowledge of having a potentially dangerous HPV infection can also cause anxiety [32]. However, the remaining HPV infections may persist longer and the risk of progression from CIN to cancer is considerable [33] and especially in case of TZ3 the LEEP must kept in mind.

ause preterm labor [30] or cervical stenosis [31]. Knowledge of having a potentially dangerous HPV infection can also cause anxiety [32]. However, the remaining HPV infections may persist longer and the risk of progression from CIN to cancer is considerable [33] and especially in case of TZ3 the LEEP must kept in mind. There is still no consensus concerning the definition of persistency of an HPV infection. A Columbian prospective study on HPV persistence proposed the definition that persistent infections are those lasting more than the median duration, which was for example 9,5 months for HPV16 in women >30 years [34]. In a large meta-analysis, Koshiol et al. [2] remarked that even testing intervals of ≤ six months produce strong summative relative risks as regards the association between HPV persistence and CIN2+. It is therefore suggested that repeat HPV testing at six months is a valuable way to identify women at increased risk of cervical precancer and cancer. In our study the women with CIN2+ had a detected HPV persistence for seven to 20 months. Follow-up in the Swedescreen study pointed out that all initially cytology-negative women with persistent HPV infection developed colposcopically verified CIN2+ within seven years [11]. The women in that study were between 32 and 38 years of age at entry, in contrast to our study where the majority were postmenopausal. There is a need for further studies on different age groups to form a firm basis for future decisions on how to handle these patients.

scopically verified CIN2+ within seven years [11]. The women in that study were between 32 and 38 years of age at entry, in contrast to our study where the majority were postmenopausal. There is a need for further studies on different age groups to form a firm basis for future decisions on how to handle these patients. Conclusion A persistent HPV infection needs to be monitored despite normal Pap smears, since 6/40 (15%) women older than 40 years, was revealed to have an undiagnosed CIN2+ when LEEP was performed. Counseling women regarding the risk of cervical cancer and the expected effect of an eventual LEEP can help them to make an optimal informed choice. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement Lions Cancer Foundation, Uppsala, Sweden. Grant ID 1050023.

Introduction Pregnancy associated cancer (PAC), although fairly uncommon, raises puzzling ethical, social, familial and religious issues. This situation also affects patients' physical and psychosocial health while threatening foetal integrity [1]. The incidence of cancer during or immediately after pregnancy has generally been reported to range between 0.09 and 0.14% [[2], [3], [4], [5], [6], [7], [8]]. However, despite a quite large number of papers in literature, the current incidence of PACs still remains uncertain. Indeed, available data are poorly comparable due to different inclusion criteria (invasive and non invasive disease), incoherent time intervals (12 or 18 months) after delivery and finally dissimilar population references. Moreover, the incidence of different types of cancer in women shows a wide variation worldwide with many genetic and epigenetic influences. This implies that data from a certain geographical area cannot be generalized and as a matter of fact are nearly useless for public health purposes in cancer screening policies. In 2017, Parazzini et al. reported the incidence of pregnancy associated cancers (PACs) in Lombardy (a region in northern Italy), based on data from regional hospital discharge forms from 2001 to 2012. Their study showed that the risk of PACs was 122.9 per 100,000 pregnancies with the most common cancers being breast, thyroid and blood cancers. Furthermore, the incidence increased significantly with age but it did not show any increase over time in the considered decade [3].

ospital discharge forms from 2001 to 2012. Their study showed that the risk of PACs was 122.9 per 100,000 pregnancies with the most common cancers being breast, thyroid and blood cancers. Furthermore, the incidence increased significantly with age but it did not show any increase over time in the considered decade [3]. The aim of the present study was to investigate the incidence of pregnancy related cancers in Apulia, a region with 4 million inhabitants in the southern Italian peninsula from 2003 to 2015, and to compare the Apulian data with those reported from other geographical areas.

ts, we selected all SDOs reporting ICD codes 140.-208., which mean a diagnosis of malignant cancer among the main or secondary diagnoses. We excluded SDOs in which cancer was recorded as secondary diagnosis or if a previous SDO reported cancer as the main diagnosis because our aim was to obtain only incident neoplasms. The date of admission was tabbed as the date of cancer diagnosis and for each woman we collected only the first discharge form reporting an oncologic diagnosis. We divided tumours by principal anatomic sites using the aforementioned ICD-9 codes: breast (174.), thyroid (193.), skin except melanoma (173.), lymphoma (200.-202.), melanoma (172.), cervix (180.), nervous system (191.-192.), leukaemia (204.-208.), colorectum (153.-154.), ovary (183.), head and neck (140.-149., 160.), skeletal or connective tissue (170.-171.), kidney (189.), urinary tract (188., 189.1-189.4, 189.8-189.9), lung (162.), other gastrointestinal (150., 152., 156., 158.-159.), stomach (151.), pancreas (157.), endometrium (182.), placenta (181.), other gynaecologic tract (184.), multiple myeloma (203.), other (164., 190., 194.-199.). The risk of PAC is the ratio between the whole number of PACs and all pregnancies occurring during the interval between January 1 st, 2003 and December 31 st, 2015.

mach (151.), pancreas (157.), endometrium (182.), placenta (181.), other gynaecologic tract (184.), multiple myeloma (203.), other (164., 190., 194.-199.). The risk of PAC is the ratio between the whole number of PACs and all pregnancies occurring during the interval between January 1 st, 2003 and December 31 st, 2015. As secondary endpoints we also expressed the odds ratio (OR) for some potential risk predictors using a logistic model and so we stratified the incidence rate of PACs by year, age, nationality, pregnancy outcome. Women were divided by age into 4 groups (<30, 30–34, 35–39, >=40) and by nationality between Italian women or foreign nationals. Each pregnancy had two possible outcomes: delivery or abortion. The effect of the above items as potential risk predictors was estimated as were the odds ratio (OR). Results From January 2003 to December 2015 we recorded a total number of 682,173 pregnancies in women residing in Apulia; as raw data, we obtained 1008 women with pregnancy associated cancer within 9 or 3 months before the date of the pregnancy outcome (delivery or abortion respectively) and within 12 months after the same outcome, but we must exclude several cases from the SDO database to achieve our aim.

ies in women residing in Apulia; as raw data, we obtained 1008 women with pregnancy associated cancer within 9 or 3 months before the date of the pregnancy outcome (delivery or abortion respectively) and within 12 months after the same outcome, but we must exclude several cases from the SDO database to achieve our aim. We identified 876 women with cancer as main diagnosis and 132 with cancer as secondary diagnosis according to ICD-9 codes. Among the latter, we rejected 81 patients who had a non-cancer primary diagnosis and other 15 cases with cancer diagnosis before pregnancy, with a total of only 36 women with incident cancer as a secondary item in the SDO form. Thus the number of pregnancy associated cancers was 912 cases; a further 50 cases were excluded because of unclear cancer site or metastasis of unspecified origin.

diagnosis and other 15 cases with cancer diagnosis before pregnancy, with a total of only 36 women with incident cancer as a secondary item in the SDO form. Thus the number of pregnancy associated cancers was 912 cases; a further 50 cases were excluded because of unclear cancer site or metastasis of unspecified origin. Overall we achieved a final cohort of 867 women with incident cancer in pregnancy: thus, the risk for a PAC in our population was calculated as 127.1 per 100,000 pregnancies. Table 1 shows the incidence of cancer by anatomical region. Breast cancer was the most common pregnancy associated cancer (257 cases) with an incidence of 37.7 cancers per 100,000 pregnancies: 229 women were diagnosed in post-pregnancy while 28 during pregnancy. Thyroid cancers followed breast by incidence (22.3 per 100,000 pregnancies) with 133 malignancies in post-pregnancy (19.5 per 100,000) and 19 cancer in pregnancy (2.8 per 100,000 cases). The ranking goes as follows: skin except melanoma (89 cancers and 13.0 per 100,000 pregnancies), lymphoma (77 cases and 11.3 per 100,000 pregnancies), melanoma (6.2 per 100,000), cervical cancer and central nervous system (3.8 per 100,000), leukaemia (3.7 per 100,000).Table 1 Classification of pregnancy-associated cancer by site.

goes as follows: skin except melanoma (89 cancers and 13.0 per 100,000 pregnancies), lymphoma (77 cases and 11.3 per 100,000 pregnancies), melanoma (6.2 per 100,000), cervical cancer and central nervous system (3.8 per 100,000), leukaemia (3.7 per 100,000).Table 1 Classification of pregnancy-associated cancer by site. Table 1 Pregnancy Post-pregnancy All Cancer site No Risk No Risk No Risk Breast 28 4,1 229 33,6 257 37,7 Thyroid 19 2,8 133 19,5 152 22,3 Skin excluding melanoma 15 2,2 74 10,8 89 13,0 Lymphoma 19 2,8 58 8,5 77 11,3 Melanoma 6 0.9 36 5,3 42 6,2 Cervix 3 0.4 23 3,4 26 3,8 Nervous system 2 0.3 24 3,5 26 3,8 Leukemia 6 0.9 19 2,8 25 3,7 Colorectum 4 0,6 20 2,9 24 3,5 Ovary 4 0.6 18 2,6 22 3,2 Head and neck 6 0.9 15 2,2 21 3,1 Other or ill defined 2 0,3 19 2,8 21 3,1 Connective tissue 6 0,9 11 1,6 17 2,5 Kidney 5 0.7 9 1,3 14 2,1 Urinary tract 3 0.4 8 1,2 11 1,6 Lung 1 0,1 7 1,0 8 1,2 Other gastrointestinal tract 0 0,0 8 1,2 8 1,2 Stomach 0 0,0 7 1,0 7 1,0 Pancreas 0 0.0 6 0,9 6 0,9 Endometrium 0 0.0 4 0,6 4 0,6 Uterus 0 0.0 3 0,4 3 0,4 Placenta 0 0.0 3 0,4 3 0,4 Other Gynecological 1 0.1 1 0,1 2 0,3 Multiple myeloma 1 0,1 1 0,1 2 0,3 All cancers 131 19,2 736 107,9 867 127,1

6 Lung 1 0,1 7 1,0 8 1,2 Other gastrointestinal tract 0 0,0 8 1,2 8 1,2 Stomach 0 0,0 7 1,0 7 1,0 Pancreas 0 0.0 6 0,9 6 0,9 Endometrium 0 0.0 4 0,6 4 0,6 Uterus 0 0.0 3 0,4 3 0,4 Placenta 0 0.0 3 0,4 3 0,4 Other Gynecological 1 0.1 1 0,1 2 0,3 Multiple myeloma 1 0,1 1 0,1 2 0,3 All cancers 131 19,2 736 107,9 867 127,1 According to the data 19.2 and 107.9 per 100,000 pregnancies were diagnosed with cancer during pregnancy and in post-pregnancy respectively. Table 2 shows distribution of pregnancies stratified by four items or potential risk predictors. Women younger than 30 years were about 1/5 (20.07%) of our cohort with the lowest risk for PACs (64.5 per 100,000, OR = 1) while one third (33.10%) of our cohort was made up of women ranging from 35 to 39 years and the highest risk for PACs was for women aged >/ = 40 years (296.3 cancers per 100,000 pregnancies, OR = 4.29, p < 0.05).Table 2 Distribution, risks and OR for pregnancy-associated cancer and their relative p-value.

,000, OR = 1) while one third (33.10%) of our cohort was made up of women ranging from 35 to 39 years and the highest risk for PACs was for women aged >/ = 40 years (296.3 cancers per 100,000 pregnancies, OR = 4.29, p < 0.05).Table 2 Distribution, risks and OR for pregnancy-associated cancer and their relative p-value. Table 2 Pregnancies Pregnancy-associated cancer Frequency (n.) Frequency (n.) % Risk OR p-value Age <30 269820 174 20,07 64,5 1 30-34 215169 259 29,87 120,4 1,88 <0,05 35-39 147567 287 33,10 194,5 2,96 <0,05 > = 40 49617 147 16,96 296,3 4,29 <0,05 Nationality Foreign 111507 108 12,46 96,9 1 Italian 570666 759 87,54 133,0 1,33 <0,05 Outcome Delivery 212924 334 38,52 156,9 1 Abortion 469249 533 61,48 113,6 1,26 <0,05 Year of pregnancy 2003 58901 60 6,92 101,9 1 2004 59877 70 8,07 116,9 1,12 0,51 2005 56959 56 6,46 98,3 1,22 0,387 2006 56545 84 9,69 148,6 1,39 0,05 2007 55518 52 6,00 93,7 0,87 0,46 2008 54254 77 8,88 141,9 1,31 0,12 2009 54213 84 9,69 154,9 1,41 <0,05 2010 54100 57 6,57 105,4 0,95 0,78 2011 52180 78 9,00 149,5 1,34 0,09 2012 49546 76 8,77 153,4 1,32 0,11 2013 46790 71 8,19 151,7 1,33 0,1 2014 44510 58 6,69 130,3 1,13 0,49 2015 38780 44 5,07 113,5 0,99 0,97

9 0,05 2007 55518 52 6,00 93,7 0,87 0,46 2008 54254 77 8,88 141,9 1,31 0,12 2009 54213 84 9,69 154,9 1,41 <0,05 2010 54100 57 6,57 105,4 0,95 0,78 2011 52180 78 9,00 149,5 1,34 0,09 2012 49546 76 8,77 153,4 1,32 0,11 2013 46790 71 8,19 151,7 1,33 0,1 2014 44510 58 6,69 130,3 1,13 0,49 2015 38780 44 5,07 113,5 0,99 0,97 Eighty-seven percent of women were Italian with 759 cancers versus 108 cancers (12.46%) in foreign pregnant women: being a foreigner did not increase the risk for PACs while being Italian or born in Italy meant higher association with cancer during pregnancy. More than 500 pregnancies out of 867 ended with abortion so we can conclude that the incidence rate of developing a pregnancy-associated cancer was higher for pregnancies resulting in miscarriage. Considering calendar years, we observed an increased OR from 2006 to 2009 (OR = 1,39 and OR = 1,41 respectively). Comments Benign neoplasms, such as leiomyomas or adnexal cysts are commonly found in pregnant women while data regarding malignancies are scarce [1,9,10]. Knowing the exact incidence and distribution of cancer in pregnancy in each geographical area represents a key point in the field of obstetrics and gynaecology.

Eighty-seven percent of women were Italian with 759 cancers versus 108 cancers (12.46%) in foreign pregnant women: being a foreigner did not increase the risk for PACs while being Italian or born in Italy meant higher association with cancer during pregnancy. More than 500 pregnancies out of 867 ended with abortion so we can conclude that the incidence rate of developing a pregnancy-associated cancer was higher for pregnancies resulting in miscarriage. Considering calendar years, we observed an increased OR from 2006 to 2009 (OR = 1,39 and OR = 1,41 respectively). Comments Benign neoplasms, such as leiomyomas or adnexal cysts are commonly found in pregnant women while data regarding malignancies are scarce [1,9,10]. Knowing the exact incidence and distribution of cancer in pregnancy in each geographical area represents a key point in the field of obstetrics and gynaecology. In 2016, Parazzini et al. reported the incidence of pregnancy associated cancer in Lombardy (a region in northern Italy), based on data from regional hospital discharge forms and we decided to use the same system to collect data [3]. In the current study a raw incidence of 127.1 cancers per 100,000 pregnancies was similar to that reported in Lombardy (122.9 per 100,000 maternities), and in other geographical areas such as Australia (137 per 100,000 pregnancies), California (94 per 100,000 live births), Washington DC, Germany and other single centre and multicentric experiences from nearby or faraway Countries [[2], [3], [4], [5], [6], [7], [8]]. So we can assume that there is no critical geographical difference.

ographical areas such as Australia (137 per 100,000 pregnancies), California (94 per 100,000 live births), Washington DC, Germany and other single centre and multicentric experiences from nearby or faraway Countries [[2], [3], [4], [5], [6], [7], [8]]. So we can assume that there is no critical geographical difference. However, there are noticeable differences regarding each cancer in different regions: melanoma is the most common in Australia (45.7 per 100,000 maternities) [11] while it is the fifth cancer by frequency in our experience (6.2 per 100,000 maternities). In our study the ranking of each tumour during pregnancy reflects more or less its demographics in reproductive age females in the so-called high-resource countries [15]: breast cancer is the most frequent PAC [[12], [13], [14]] and as for any other cancer, it is expected to become more frequent since first births in older women continue to rise [16]. The second most frequent cancer in our series is thyroid cancer: this result is specific for Italian population which shows an important increase in incidence for this neoplasm in both sexes compared to other countries worldwide [17]: it is the second neoplasm by incidence in women younger than 49 years old while it is the 18th by number of deaths from cancer in women. That means that the increasing incidence just relates to the milder histotypes and this goes along with the improving 5 years overall survival rate (2005–2009, 95%; 1990–1994, 86%) [18].

is the second neoplasm by incidence in women younger than 49 years old while it is the 18th by number of deaths from cancer in women. That means that the increasing incidence just relates to the milder histotypes and this goes along with the improving 5 years overall survival rate (2005–2009, 95%; 1990–1994, 86%) [18]. Notably, the incidence of pregnancy-associated thyroid cancer in our study was higher than that recorded in Lombardy. In fact there is a statistically significant discrepancy between its incidence in northern and southern Italy: contrary to almost all cancers, the incidence of thyroid cancer is higher in southern Italy (27.5 × 100,000 inhabitants vs 23.5 in northern Italy) with a greater (+17%; ×100,000 inhabitants) incidence rate standardized for geographical area and sex [18].

discrepancy between its incidence in northern and southern Italy: contrary to almost all cancers, the incidence of thyroid cancer is higher in southern Italy (27.5 × 100,000 inhabitants vs 23.5 in northern Italy) with a greater (+17%; ×100,000 inhabitants) incidence rate standardized for geographical area and sex [18]. We also detected 77 cases per 100,000 women of lymphoma, which represents the most common haematological malignant disorder in pregnancy, followed by acute leukaemia. Approximately 3 every 100 women [19] with HL (Hodgkin lymphoma) receive the diagnosis during pregnancy, usually at the same stage as in non-pregnant counterparts. Melanoma is the fifth most frequent cancer in our series with special concerns about this PAC in literature: one of the features of melanoma is the risk for trans-placental metastases, with newborns developing clinical evidence of metastases having a poor prognosis [20]. Cervical cancer is the most frequent gynaecologic cancer in both our and other series. As previously mentioned, it is reasonable to expect a rising incidence of PACs but, surprisingly, the trend in our series diverges from this expected corollary with a peak in 2009 and unexplained statistically noticeable spikes in 2006 and 2011 and 2012 without increase.

ynaecologic cancer in both our and other series. As previously mentioned, it is reasonable to expect a rising incidence of PACs but, surprisingly, the trend in our series diverges from this expected corollary with a peak in 2009 and unexplained statistically noticeable spikes in 2006 and 2011 and 2012 without increase. Italian women seem to have an increased risk for PACs (OR = 1,29) compared to other countries and in Apulia we find a greater proportion for miscarriage/abortion (200.4 every 100,000 pregnancies in our report) compared to our counterpart in Lombardy (116 per 100,000 pregnancies). Probably the main weaknesses of our study is inherent to the specific nature of our database which prevents us from obtaining any more data than those considered, thus we had no information on the gestational age at birth or on neonatal outcomes.

t) compared to our counterpart in Lombardy (116 per 100,000 pregnancies). Probably the main weaknesses of our study is inherent to the specific nature of our database which prevents us from obtaining any more data than those considered, thus we had no information on the gestational age at birth or on neonatal outcomes. Finally, as expected, the incidence of tumours by age showed an increasing trend in older women compared to younger ones. In fact, in agreement with data from either far-away countries like Australia or nearby regions (e.g. Lombardy), age seems to be a major factor in the incidence of PACs: in fact being older than 40 years during childbearing increases the risk for oncologic diagnosis more than four-fold. This finding is of great clinical and social importance as in developed countries women tend to postpone childbearing because of socio-economic reasons. This study does not provide any information on clinical management and follow up of cancer during pregnancy. However, we believe that improved knowledge of the exact incidence of this rare condition might make a significant contribution for a better management and treatment.

Introduction Chronic pelvic pain (CPP) has a prevalence of 3.8% among women between 15- and 73-years-old. In women of reproductive age, CPP varies from 14% to 34%, having a direct impact on their conjugal, social, and professional lives [1]. The intensity of pain is the most assessed dimension and can be measured with different instruments. The visual analogue scale (VAS), numeric rating scale (NRS), and verbal descriptor scales are the most commonly used self-reported pain perception scales [2]. Nevertheless, pain provocation tests with the use of an algometer can also measure the intensity of pain through the pressure trigger points for pain [3]. Different studies have debated which method is the best for assessing pain [4,5]. Although the one-dimensional scales are a good way to assess pain, they cannot evaluate the continuous spectrum of pain [5]. Therefore, this study assessed the intensity of pain in women with CPP with two different methods in the hope of understanding the pain better and to evaluate the possibility of treating patients with better care once the pain intensity was evaluated on different dimensions. Materials and methods Patients Forty-seven women who had been referred to the gynaecology and obstetrics outpatient clinic of the Hospital de Clínicas de Porto Alegre from May 2012 to August 2013 participated in this cross-sectional study. The participants were divided into two groups, with endometriosis (n = 20) and with other gynaecological causes (n = 27) based on the laparoscopy diagnosis.

e gynaecology and obstetrics outpatient clinic of the Hospital de Clínicas de Porto Alegre from May 2012 to August 2013 participated in this cross-sectional study. The participants were divided into two groups, with endometriosis (n = 20) and with other gynaecological causes (n = 27) based on the laparoscopy diagnosis. The eligibility criteria were as follows: female patients, between 18- to 45-years-old, diagnosed with CPP and signed an informed consent form. The exclusion criteria were as follows: patients who underwent abdominal surgery, acute lumbar pain (less than six weeks), obesity defined as a body mass index (BMI) equal to or greater than 30 kg/m2, inflammatory disease, gastrointestinal or urinary disorders, menopausal women, women menstruating on the day of the assessment, hysterectomy, pregnant women and the use of analgesic medication in the past six months. The inclusion of patients with endometriosis was confirmed (or excluded) by laparoscopy. Therefore, all patients suffering chronic pelvic pain were investigated by laparoscopy and biopsy of the suspected areas of endometriosis. All included patients with endometriosis had the peritoneal form of this disease. Deep or ovarian endometriosis was excluded by laparoscopy, physical exam and pelvic ultrasound. In addition, we only included subjects in this research protocol who underwent laparoscopy by our medical team during the study protocol. The local ethics committee approved the study and all of the experiments under the number: 08–650, and all of the participants provided their informed, written consent.

The inclusion of patients with endometriosis was confirmed (or excluded) by laparoscopy. Therefore, all patients suffering chronic pelvic pain were investigated by laparoscopy and biopsy of the suspected areas of endometriosis. All included patients with endometriosis had the peritoneal form of this disease. Deep or ovarian endometriosis was excluded by laparoscopy, physical exam and pelvic ultrasound. In addition, we only included subjects in this research protocol who underwent laparoscopy by our medical team during the study protocol. The local ethics committee approved the study and all of the experiments under the number: 08–650, and all of the participants provided their informed, written consent. Protocol and assessment CPP was defined as a painful syndrome originating in one or more pelvic organs [6] that was not associated with menstruation; therefore, the pain was non-cyclic, lasted at least six months, and presented as continuous or intermittent pain that was intense enough to interfere with the patient’s usual activities and required clinical or surgical treatment [7]. The participants’ social and gynaecological histories, BMI, and information on CPP, including diagnosis, treatment, symptoms, location, and intensity of pain, were collected during a visit to the gynaecology and obstetrics outpatient clinic at Hospital de Clínicas de Porto Alegre (HCPA). The pain scores were determined based on pain intensity through the use of self-reported pain scales [2].

n on CPP, including diagnosis, treatment, symptoms, location, and intensity of pain, were collected during a visit to the gynaecology and obstetrics outpatient clinic at Hospital de Clínicas de Porto Alegre (HCPA). The pain scores were determined based on pain intensity through the use of self-reported pain scales [2]. The two self-reported scales have verbal anchors at the beginning and end with “no pain” and “worst pain imaginable”, respectively. Patients were asked to indicate their pain intensity on a 10 cm line when using the VAS [4] and the number that indicates the pain intensity when using the NRS [4]. The participants’ pressure pain thresholds were assessed using an electronic pressure algometer (model Kratos Equipamentos Industriais LTDA; DDK 10 kg, São Paulo, Brazil). The manufacturer calibrated the compression gauge of the device, and the readings were expressed as kgf/cm2. The same examiner, a physical therapist trained to identify and locate the investigated trigger points, assessed all of the participants.

odel Kratos Equipamentos Industriais LTDA; DDK 10 kg, São Paulo, Brazil). The manufacturer calibrated the compression gauge of the device, and the readings were expressed as kgf/cm2. The same examiner, a physical therapist trained to identify and locate the investigated trigger points, assessed all of the participants. For the pressure algometry assessment, the participants were requested to lay in the supine position. The examiner located all of the trigger points in the abdominal, pubic, pelvic and lumbar areas by manual palpation following the positional release technique assessment protocol [8]. The trigger points assessed were as follows: anterior 1 st lumbar points (A1Ls), which are located medially to the anterior-superior iliac spine (ASIS); anterior 2nd lumbar points (A2Ls), which are located on the medial surface of the anterior-inferior iliac spine; abdominal 2nd lumbar points (Ab2Ls), which are located on the abdominal surface, approximately 5 cm laterally and somewhat caudally to the umbilicus, corresponding to the lateral margin of the rectus abdominis muscle; iliac points (ILs), which are located approximately 3 cm medially to the ASIS and deep into the iliac fossa; superior pubic points (SPs), which are located on the superior surface of the pubic area, approximately 2 cm laterally to the pubic symphysis; and posterior lumbar points (P1L-P5L), which are located on the lateral surfaces of the spinous processes, paraspinal sulci or the posterior surface of the transverse processes. All of the trigger points were assessed on the right and left sides.

e of the pubic area, approximately 2 cm laterally to the pubic symphysis; and posterior lumbar points (P1L-P5L), which are located on the lateral surfaces of the spinous processes, paraspinal sulci or the posterior surface of the transverse processes. All of the trigger points were assessed on the right and left sides. The algometer was placed perpendicularly to the body surface at a constantly increasing pressure rate (1 kg/s) until the participants reported pain at the palpated site. Between the assessment of one point and the next, there was a 5-second interval. The measurements were performed twice, with a 20-minute interval between each series. The average of the measurements for each point was used for the statistical analysis. Statistical analysis Continuous variables are described as the average and standard deviation or as the median and interquartile range, according to their distribution. Categorical variables are expressed as the absolute and relative frequencies. The results of the right- and left-side algometric measurements and the NRS and VAS results were compared by Wilcoxon’s test. The agreement between the scales was assessed by the intraclass correlation coefficient (ICC). Moreover, Bland-Altman’s method was used to assess the agreement for pain, dysmenorrhea, and dyspareunia between the two one-dimensional scales.

etric measurements and the NRS and VAS results were compared by Wilcoxon’s test. The agreement between the scales was assessed by the intraclass correlation coefficient (ICC). Moreover, Bland-Altman’s method was used to assess the agreement for pain, dysmenorrhea, and dyspareunia between the two one-dimensional scales. The comparison of the scales and algometry results according to the cause of pain was determined by Mann-Whitney or Student’s t-test for continuous variables, according to the distribution, and for categorical variables, Pearson’s chi-square test was used. The correlation between the scales and algometry results was assessed using Spearman’s correlation coefficient. The comparisons among the algometry results for the trigger points used Friedman’s test, complemented by Wilcoxon’s test. The level of significance was established at 5% (p < 0.05), and the analyses were performed using SPSS version 18.0 (IBM, Armonk, NY, USA). The post hoc power calculation showed a pβ > 80%, considering our sample size (47) [9]. Funding None of the funding institutions participated in the collection, analysis and interpretation of the data.

The comparison of the scales and algometry results according to the cause of pain was determined by Mann-Whitney or Student’s t-test for continuous variables, according to the distribution, and for categorical variables, Pearson’s chi-square test was used. The correlation between the scales and algometry results was assessed using Spearman’s correlation coefficient. The comparisons among the algometry results for the trigger points used Friedman’s test, complemented by Wilcoxon’s test. The level of significance was established at 5% (p < 0.05), and the analyses were performed using SPSS version 18.0 (IBM, Armonk, NY, USA). The post hoc power calculation showed a pβ > 80%, considering our sample size (47) [9]. Funding None of the funding institutions participated in the collection, analysis and interpretation of the data. Results The patients had a median age of 40.00 [36.06–40.54]-years-old (median [25th-75th percentile]), a median weight of 65.00 [65.14–71.33] kg, a median height of 1.64 [1.61–1.64] m and an average BMI of 25.82 ± 3.50 kg/m2 (average ± standard deviation ‘SD’). A total of 76.6% of the participants were white, and 68.1% were in a relationship. The patients’ educational level had an average of 8.72 ± 2.6 years of formal schooling. Other data referring to the sample characteristics that were collected during the study are shown in Table 1.Table 1 Characterization of the study patients.

of 76.6% of the participants were white, and 68.1% were in a relationship. The patients’ educational level had an average of 8.72 ± 2.6 years of formal schooling. Other data referring to the sample characteristics that were collected during the study are shown in Table 1.Table 1 Characterization of the study patients. Table 1Variables n (n%) Ethnicity White 36.6 (76.6) Black 11 (23.4) Marital status Single 7 (14.9) Married 32 (68.1) Divorced 8 (17.0) Physical activity Yes 8 (17.0) No 39 (83.0) Paid job Yes 25 (53.2) No 22 (46.8) Smoking Never 31 (66) Smoker 15 (31.9) Ex-smoker 1 (2.1) Constipation Yes 25 (53.2) No 22 (46.8) Children Yes 40 (85.1) No 7 (14.9) No. of births 1 (1 – 3) Contraceptive method Yes 23 (48.9) No 24 (51.1) Treatment Yes 33 (70.2) No 14 (29.8) Continuous treatment Yes 27 (57.4) No 20 (42.6) Delivery type (n = 40) Vaginal 21 (52.5) Caesarean section 17 (42.5) Vaginal + caesarean section 2 (5.0) Legend: Continuous variables that characterize the study patients (n = 47). Variables are presented as the absolute and relative frequencies, “n” and “n%”, respectively.

ntinuous treatment Yes 27 (57.4) No 20 (42.6) Delivery type (n = 40) Vaginal 21 (52.5) Caesarean section 17 (42.5) Vaginal + caesarean section 2 (5.0) Legend: Continuous variables that characterize the study patients (n = 47). Variables are presented as the absolute and relative frequencies, “n” and “n%”, respectively. Considering the characteristics of CPP, the duration of pain was 73 [36–180] months (median [25th – 75th percentiles]). Table 2 presents the data from all patients (n = 47) regarding pain site, symptoms and duration. The average scores on the scales were as follows: pain, 8.02 ± 1.39 on the NRS and 7.96 ± 1.4 on the VAS; dysmenorrhea, 8 [5–10] on the NRS and 7 [5–10] on the VAS; and dyspareunia, 7 [6–10] on the NRS and 7 [6–10] on the VAS.Table 2 Characterization of the sample of patients with chronic pelvic pain. Table 2Variables Sample (n = 47) Pelvic pain Right side 7 (14.9) Left side 9 (19.1%) Both sides 31 (66.0%) Lumbar pain Right side 3 (6.4%) Left side 2 (4.3%) Both sides 26 (55.3%) No pain 16 (34%) Dyspareunia 40 (85.1) Dysmenorrhea 37 (78.7%) Activities of daily living 26 (55.3%) Continuous pain 25 (53.2%) Intermittent pain 22 (46.8%) Legend: Analysis of patients who participated in the study. Variables evaluated pain site, symptoms, and duration. Data are presented as the absolute and relative frequency, n (n%), respectively. Statistical analysis was performed by chi-square testing.

26 (55.3%) Continuous pain 25 (53.2%) Intermittent pain 22 (46.8%) Legend: Analysis of patients who participated in the study. Variables evaluated pain site, symptoms, and duration. Data are presented as the absolute and relative frequency, n (n%), respectively. Statistical analysis was performed by chi-square testing. The causes of CPP were categorized as endometriosis (n = 20) and other causes (n = 27). In the comparison regarding the cause of pain, the duration of the pain was 132 [39–240] months in women with endometriosis (42.55%) and 60 [24–120] months in the group with other causes (57.45%); that difference was statistically significant (p = 0.026) (Table 3).Table 3 Comparison of variables regarding pain location and measurements according to one-dimensional scales.

ation of the pain was 132 [39–240] months in women with endometriosis (42.55%) and 60 [24–120] months in the group with other causes (57.45%); that difference was statistically significant (p = 0.026) (Table 3).Table 3 Comparison of variables regarding pain location and measurements according to one-dimensional scales. Table 3Variables Endometriosis (n = 20) Other causes (n = 27) p-value Duration of pain (months) 132 [39-240] 60 [24-120] 0.026* Site of pelvic pain 0.819 Right 3 (15.0%) 4 (14.8%) Left 3 (15.0%) 6 (22.2%) Bilateral 14 (70.0%) 17 (63.0%) Site of lumbar pain 0.504 Right 1 (5.0%) 2 (7.4%) Left 0 (0.0%) 2 (7.4%) Bilateral 13 (65.0%) 13 (48.1%) No pain 6 (30.0%) 10 (37.0%) Symptoms Dyspareunia 17 (85.0%) 23 (85.2%) 1.000 Dysmenorrhea 14 (70.0%) 23 (85.2%) 0.286 During activities of daily living 10 (50.0%) 16 (59.3%) 0.738 Continuous pain 10 (50.0%) 15 (55.6%) 0.935 Scales NRS for pain 8.0 ± 1.4 8.0 ± 1.4 0.929 VAS for pain 8.0 ± 1.4 7.9 ± 1.4 0.861 NRS for dysmenorrhea 8[0-9.8] 7 [[6], [7], [8], [9]] 0.991 VAS for dysmenorrhea 8[0-9.8] 7 [[6], [7], [8], [9]] 0.965 NRS for dyspareunia 7[5-8.8] 8 [[6], [7], [8], [9]] 0.310 VAS for dyspareunia 7[5.3-8.8] 8 [[6], [7], [8], [9]] 0.406 Legend: Comparison of variables regarding pain location and measurements according to two different one-dimensional scales; NRS stands for numerical rating scale, and VAS stands for visual analogue scale. Continuous variables are described as the mean ± SD or median [25th – 75th percentile]; Student’s t-test and Mann-Whitney’s test, respectively, were used, according to variables’ distribution. Categorical variables are expressed as the absolute and relative frequencies, n (n%), respectively.

for visual analogue scale. Continuous variables are described as the mean ± SD or median [25th – 75th percentile]; Student’s t-test and Mann-Whitney’s test, respectively, were used, according to variables’ distribution. Categorical variables are expressed as the absolute and relative frequencies, n (n%), respectively. The intraclass correlation coefficient (ICC) between the scales was 0.992 for pain, 1.00 for dysmenorrhea and 0.996 for dyspareunia, which denotes very good agreement (p < 0.01). The agreement between the NRS and VAS was 93.6%, 97.9%, and 91.5% for pain, dysmenorrhea, and dyspareunia, respectively. Fig. 1 presents the Bland-Altman method of agreement for CPP symptoms based on the one-dimensional scales.Fig. 1 Bland-Altman for the NRS and VAS scales regarding pain, dysmenorrhea, and dyspareunia.

The agreement between the NRS and VAS was 93.6%, 97.9%, and 91.5% for pain, dysmenorrhea, and dyspareunia, respectively. Fig. 1 presents the Bland-Altman method of agreement for CPP symptoms based on the one-dimensional scales.Fig. 1 Bland-Altman for the NRS and VAS scales regarding pain, dysmenorrhea, and dyspareunia. Legend: Bland-Altman’s method of agreement for pain, dysmenorrhea, and dyspareunia between two one-dimensional scales. (a) Bland-Altman’s method for pain regarding the NRS and VAS agreement. The lower (-0.43) and upper (0.55) limits of agreement are represented as dotted lines, and the formula used to obtain the limits is found on the right side of the image. SD stands for standard deviation (0.25), and the mean difference (0.06) is represented as a continuous line. Statistical analysis was performed using Bland-Altman’s method (p >  0.05; paired t-test). (b) Bland-Altman’s method for dysmenorrhea regarding the NRS and VAS agreement. The lower (-0.26) and upper (0.31) limits of agreements are represented as dotted lines, and the formula to obtain the limits is found on the right side of the image. SD stands for standard deviation (0.15), and the mean difference (0.02) is represented as a continuous line. Statistical analysis was performed using Bland-Altman’s method (p >  0.05; paired t-test). (c) Bland-Altman’s method for pain regarding the NRS and VAS agreement. The lower (-0.69) and upper (0.82) limits of agreements are represented as dotted lines, and the formula to obtain the limits is found on the right side of the image. SD stands for standard deviation (0.38), and the mean difference (0.06) is represented as a continuous line. Statistical analysis was performed using Bland-Altman’s method (p >  0.05; paired t-test).

greements are represented as dotted lines, and the formula to obtain the limits is found on the right side of the image. SD stands for standard deviation (0.38), and the mean difference (0.06) is represented as a continuous line. Statistical analysis was performed using Bland-Altman’s method (p >  0.05; paired t-test). Fig. 1 The pressure pain thresholds measured by algometry represent the average value of two measurements at each trigger point. The comparison of the thresholds between the right and left sides showed a significant difference only for the anterior 2nd lumbar point (A2L) with 1.03 [0.55–1.73] and 0.90 [0.62–1.45] kgf/cm2 on the right and left sides, respectively, indicating a lower pressure pain threshold on the left side than on the right side (p = 0.026; Student’s t-test). The differences in the pressure pain thresholds between the right and left sides at the remaining trigger points assessed were not significant: anterior 1st lumbar point (A1L) (p = 0.664), abdominal 2nd lumbar point (Ab2L) (p = 0.907), superior pubic point (SP) (p = 0.057), pelvic 1st lumbar point (P1L) (p = 0.882), pelvic 2nd lumbar point (P2L) (p = 0.861), pelvic 3rd lumbar point (P3L) (p = 0.941), pelvic 4th lumbar point (P4L) (p = 0.421), pelvic 5th lumbar point (P5L) (p = 0.070) and Iliac point (IL) (p = 0.495).

lumbar point (Ab2L) (p = 0.907), superior pubic point (SP) (p = 0.057), pelvic 1st lumbar point (P1L) (p = 0.882), pelvic 2nd lumbar point (P2L) (p = 0.861), pelvic 3rd lumbar point (P3L) (p = 0.941), pelvic 4th lumbar point (P4L) (p = 0.421), pelvic 5th lumbar point (P5L) (p = 0.070) and Iliac point (IL) (p = 0.495). The comparison of the pressure pain thresholds among the investigated trigger points found a significant difference (p = 0.001; Student’s t-test). The trigger points with the lowest pressure pain thresholds were the Ab2L, SP, and IL, in that order, and the trigger points with the highest thresholds were the P1L and P2L, in that order. The analysis of the algometry measurements regarding the causes of CPP showed that the pressure pain thresholds were lower at the following trigger points in the participants with endometriosis: the left A1L, 0.86 [0.62–1.18] kgf/cm2 (p = 0.039; Student’s t-test); left A2L, 0.72 [0.44 – 0.98] kgf/cm2 (p = 0.020); and right SP, 0.54 [0.34–1.14] kgf/cm2 (p = 0.014). Table 4 describes the comparison of the trigger points assessed by algometry relative to the causes of pain.Table 4 Comparison of algometry measures regarding the pain location and causes.

p = 0.039; Student’s t-test); left A2L, 0.72 [0.44 – 0.98] kgf/cm2 (p = 0.020); and right SP, 0.54 [0.34–1.14] kgf/cm2 (p = 0.014). Table 4 describes the comparison of the trigger points assessed by algometry relative to the causes of pain.Table 4 Comparison of algometry measures regarding the pain location and causes. Table 4Variables Endometriosis (n = 20) Other causes (n = 27) p-value Anterior 1st Lumbar point (A1L) Right 1.02 [0.46-1.86] 1.09 [0.74-1.54] 0.451 Left 0.86 [0.62-1.18] 1.19 [0.74-2.03] 0.039* Anterior 2nd Lumbar point (A2L) Right 0.71 [0.49-1.46] 1.27 [0.60-2.02] 0.057 Left 0.72 [0.44-0.98] 1.07 [0.71-1.56] 0.020* Abdominal 2nd Lumbar point (Ab2L) Right 0.49 [0.29-0.71] 059 [0.39-1.03] 0.333 Left 0.49 [0.32-0.91] 0.52 [0.34-1.15] 0.796 Superior Pubic point (SP) Right 0.54 [0.34-1.14] 1.04 [0.65-1.45] 0.014* Left 0.55 [0.28-1.11] 0.95 [0.43-1.50] 0.083 Pelvic 1st Lumbar point (P1L) Right 2.68 [1.21-4.16] 2.86 [1.86-3.99] 0.491 Left 2.63 [1.25-3.95] 2.87 [1.51-4.35] 0.439 Pelvic 2nd Lumbar point (P2L) Right 1.99 [1.03-3.79] 2.16 [1.47-3.80] 0.651 Left 1.82 [1.11-3.70] 2.24 [1.61-3.54] 0.426 Pelvic 3rd Lumbar point (P3L) Right 1.75 [0.92-2.93] 1.81 [1.19-2.54] 0.780 Left 1.54 [0.80-2.88] 1.74 [1.32-3.10] 0.407 Pelvic 4th Lumbar point (P4L) Right 1.41 [0.75-2.34] 1.73 [0.92-2.37] 0.606 Left 1.34 [0.73-2.81] 1.74 [0.81-2.34] 0.813 Pelvic 5th Lumbar point (P5L) Right 1.49 [0.71-2.35] 1.62 [0.73-2.36] 0.576 Left 1.37 [0.70-2.09] 1.64 [0.77-2.51] 0.302 Iliac point (IL) Right 0.62 [0.31-1.11] 0.84 [0.39-1.25] 0.259 Left 0.60 [0.32-0.77] 0.62 [0.31-1.11] 0.109 Legend: Comparison of algometry measures regarding location and causes of pain. The results were expressed as the median [25th-75th percentile] according to trigger points assessed by the physical therapist responsible for the measurements. p-Values with the (*) symbol had values ≤0.05, indicating a significant difference; Mann-Whitney’s test.

ometry measures regarding location and causes of pain. The results were expressed as the median [25th-75th percentile] according to trigger points assessed by the physical therapist responsible for the measurements. p-Values with the (*) symbol had values ≤0.05, indicating a significant difference; Mann-Whitney’s test. The correlation between algometry and the pain scales is shown in Table 5. As the scores on the NRS and VAS regarding dyspareunia increased, the algometry thresholds decreased, except for the trigger points A1L and A2L on both sides; this association was moderate, inverse and statistically significant. There were inverse and significant correlations between the NRS and VAS scores regarding dysmenorrhea and the threshold of the right Ab2L trigger point and between the VAS score for pain and the right SP trigger point.Table 5 Correlation of the algometry pressure points and self-reported scales based on Spearman’s correlation coefficient.

nd significant correlations between the NRS and VAS scores regarding dysmenorrhea and the threshold of the right Ab2L trigger point and between the VAS score for pain and the right SP trigger point.Table 5 Correlation of the algometry pressure points and self-reported scales based on Spearman’s correlation coefficient. Table 5Variables NRSpain VASpain NRS dysm VAS dysm NRS dysp VAS dysp Anterior 1st Lumbar point (A1L) Right 0.0001 −0.024 −0.137 −0.145 −0.125 −0.125 Left −0.043 −0.085 −0.145 −0.149 −0.134 −0.144 Anterior 2nd Lumbar point (A2L) Right −0.037 −0.063 −0.074 −0.080 −0.158 −0.155 Left 0.117 0.085 −.0086 −0.092 −0.141 −0.150 Abdominal 2nd Lumbar point (Ab2L) Right −0.161 −0.190 −0.305* −0.311* −0.174 −0.171 Left −0.153 −0.191 −0.188 −0.192 −0.288* −0.271 Superior Pubic point (SP) Right −0.259 −0.301* −0.216 −0.223 −0.258 −0.258 Left −0.163 −0.199 −0.164 −0.172 −0.363* −0.364* Pelvic 1st Lumbar point (P1L) Right −0.175 −0.192 −0.094 −0.096 −0.344* −0.318* Left −0.199 −0.219 −0.058 −0.054 −0.399** −0.373** Pelvic 2nd Lumbar point (P2L) Right −0.120 −0.127 −0.093 −0.092 −0.315* −0.305* Left −0.107 −0.120 0.011 0.014 −0.371** −0.362* Pelvic 3rd Lumbar point (P3L) Right 0.002 −0.010 0.071 0.071 −0.223 −0.196 Left −0.127 −0.141 0.046 0.052 −0.412** −0.392** Pelvic 4th Lumbar point (P4L) Right 0.027 0.003 0.106 0.110 −0.249 −0.230 Left −0.114 −0.137 0.064 0.066 −0.333* −0.317* Pelvic 5th Lumbar point (P5L) Right −0.039 −0.062 0.051 0.049 −0.274 −0.259 Left −0.170 −0.200 0.053 0.054 −0.362* −0.343* Iliac point (IL) Right 0.009 −0.019 −0.091 −0.098 −0.256 −0.251 Left −0.020 −0.055 −0.079 −0.076 −0.343* −0;315* Legend: Correlation of algometry trigger point measurements and one-dimensional scales regarding pain assessment and its symptoms based on Spearman’s correlation coefficient. The table’s first column shows the one-dimensional scale for pain assessment and its symptoms: NRSpain - numerical rating scale for pain; VASpain - visual analogue scale for pain; NRSdysm - numerical rating scale for dysmenorrhea; VASdysm - visual analogue scale for dysmenorrhea; NRSdysp - numerical rating scale for dyspareunia; VASdysp - visual analogue scale for dyspareunia. Measurements with (*) had a statistically significant difference with p ≤ 0.05, and those with (**) had a statistically significant difference with p < 0.01; Spearman’s correlation coefficient.

gue scale for dysmenorrhea; NRSdysp - numerical rating scale for dyspareunia; VASdysp - visual analogue scale for dyspareunia. Measurements with (*) had a statistically significant difference with p ≤ 0.05, and those with (**) had a statistically significant difference with p < 0.01; Spearman’s correlation coefficient. Comment In the present study, we investigated the correlation between self-reported pain perception instruments, the NRS and the VAS, as well as the correlation between those scales and a pain provocation test with pressure algometry in women with CPP. We used one-dimensional instruments that have been recommended by several studies [5,9,10]. However, other authors have disagreed with that approach, as they consider one-dimensional instruments to be less effective because they do not reflect the full complexity of the painful experience [12,13]. A study conducted in Brazil found that the average time from the onset of pain to a diagnosis of endometriosis is 7.0 years [14]. In the present study, that interval was 11 years (132 months), which agrees with the results of a comparative study that found that the diagnostic delay was 12 years in the United States versus 8 years in the United Kingdom [15].

the average time from the onset of pain to a diagnosis of endometriosis is 7.0 years [14]. In the present study, that interval was 11 years (132 months), which agrees with the results of a comparative study that found that the diagnostic delay was 12 years in the United States versus 8 years in the United Kingdom [15]. A previous comparison between women with CPP due to endometriosis or other gynaecological causes did not find any difference in the levels of pain [16]. The results of our study were similar; the assessment of pain by NRS and VAS did not differ between the women with CPP due to endometriosis and women with pelvic pain from other gynaecological or idiopathic causes. However, in the algometry assessment, the participants with endometriosis exhibited lower pressure pain thresholds compared with women with CPP from other gynaecological causes. The reason for this finding might be that endometriosis is an inflammatory disease, while none of the other gynaecological or idiopathic causes of CPP are characterized by inflammation. ICC assessed the correlation between the self-reported instruments for pain assessment, the NRS, and the VAS, regarding pain, dysmenorrhea, and dyspareunia, and the results showed excellent agreement between the two scales. Our results are supported by the findings of other studies, which indicate a strong positive correlation between the NRS and VAS, suggesting that both can be considered equally efficient for pain assessment [14,[17], [18], [19]].

rrhea, and dyspareunia, and the results showed excellent agreement between the two scales. Our results are supported by the findings of other studies, which indicate a strong positive correlation between the NRS and VAS, suggesting that both can be considered equally efficient for pain assessment [14,[17], [18], [19]]. The results of the present study agree with the findings reported in the meta-analysis by Hjermstad et al., who concluded that the numeric, verbal and visual analogue scales exhibit satisfactory agreement [5] and could be recommended for the assessment of the intensity of pain. In particular, the NRSs are the most widely recommended scales, as a function of their better response capacity and ease of use, which make them more widely applicable compared with the VASs and verbal rating scales [5]. The complexity of the experience of pain requires multidimensional assessment that combines pain intensity scores and other measurements of the various domains of pain. It is worthwhile to stress the need to standardize the assessment of the subjective experience of pain to improve its management and promote research [5]. One weak point of our study is related to the fact that we investigated the correlation between one-dimensional measures only and did not include multidimensional instruments that are able to encompass the complexity of pain and its multiple dimensions.

rience of pain to improve its management and promote research [5]. One weak point of our study is related to the fact that we investigated the correlation between one-dimensional measures only and did not include multidimensional instruments that are able to encompass the complexity of pain and its multiple dimensions. The crucial feature in the assessment of pain is not the choice of the scale to be used but the conditions of its use, which includes the following: the standardized anchor descriptors, methods of application, time intervals, interpretation of the clinical meaning, level of cognitive development, age, educational level, and patient’s preferences [20]. Upon the comparison of the algometry test and the NRS and VAS instruments regarding pain, dysmenorrhea, and dyspareunia, the correlations that were found had moderate strength and were statistically significant. These results agreed with previous reports in the literature, which also found moderate [21] or good [22] correlation between these measures. However, it should be noted that other studies have found only weak correlations between pressure algometry and the pain analogue scales [20]. According to some authors, the correlation between psychophysical indices, such as pressure algometry, and the subjective reports of pain, such as with scales, is usually poor [22]. That may be due to the different nature of both types of instruments, because although they measure the same feature, they do so in different manners [10].

authors, the correlation between psychophysical indices, such as pressure algometry, and the subjective reports of pain, such as with scales, is usually poor [22]. That may be due to the different nature of both types of instruments, because although they measure the same feature, they do so in different manners [10]. None of the currently available instruments is able to provide a global and unbiased assessment of pain. Our results suggest that it is advisable to combine a self-reported instrument, such as the scales, and provocation tests, such as algometry, when selecting methods for pain assessment to obtain a more thorough picture of pain in women with CPP; therefore, patients might be able to better locate their pain and be treated in a better way. Therefore, the main clinical application of this research is to introduce a new instrument to quantify and better understand the pain quantity and even the pain mechanism in the clinical setting. The new forms of treatments (drugs that act on the central or peripheral nervous system) and clinical presentations (pain centralization) in those patients with chronic pain were not properly evaluated and validated. Thus, in the assessment of chronic pelvic pain, the use of this cheap and easy-to-use instrument (algometry) could be essential to better characterize this common and important condition (chronic pain).

al presentations (pain centralization) in those patients with chronic pain were not properly evaluated and validated. Thus, in the assessment of chronic pelvic pain, the use of this cheap and easy-to-use instrument (algometry) could be essential to better characterize this common and important condition (chronic pain). Furthermore, we recommend that future studies achieve the following goals: investigate the correlation of the various causes of CPP with algometry and self-reported scales (the VAS and NRS); perform subgroup analyses of the women with endometriosis and CPP, correlating the disease stage and localization with the results of algometry and self-reported scales; investigate the correlation of the serum interleukin (e.g., IL-1 and IL-6) levels with the results of algometry and the self-reported scales; and investigate the correlation between the algometry results of women with CPP and asymptomatic women. Authors' contributions MMF, MMCM, and JSCF were responsible for the statistical analysis and the draft of the manuscript. RC and CABS were responsible for obtaining patient data, organizing databases and patient recruitment. VKG was responsible for analysing the data, patient recruitment and the draft of the manuscript. All authors agreed with the final version. Conflicts of interests The authors declare that they have no conflicts of interest.

Authors' contributions MMF, MMCM, and JSCF were responsible for the statistical analysis and the draft of the manuscript. RC and CABS were responsible for obtaining patient data, organizing databases and patient recruitment. VKG was responsible for analysing the data, patient recruitment and the draft of the manuscript. All authors agreed with the final version. Conflicts of interests The authors declare that they have no conflicts of interest. Ethics approval and consent to participate The bioethics committee of the Hospital de Clínicas de Porto Alegre (IRB) approved this research project. All forty-seven participants signed an informed consent form to participate in the study. Consent for publication Not applicable. Availability of data and material The datasets used and analysed during the study are available from the corresponding author upon reasonable request. Funding This study was funded by the following institutions: CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FIPE (Fundo de Incentivo à Pesquisa e Eventos) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). Acknowledgements We would like to acknowledge CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FIPE (Fundo de Incentivo à Pesquisa e Eventos) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for funding this study.

Introduction Abnormal uterine bleeding can severely affect quality of life [1,2] and is a leading reason for outpatient contacts [3]. Adenomyosis is a common cause of abnormal uterine bleeding [4] and is known as a heterogeneous disease of the junctional zone (JZ) with ectopic endometrium infiltrating varying degrees of the myometrium. A hysterectomy may be the last resort to treat adenomyosis, but attempts with hormonal and minimally invasive treatment such as transcervical endometrial resection (TCRE) are often preferred [[5], [6], [7], [8]]. Adenomyosis is often found in patients who have a hysterectomy following treatment failure of TCRE [[9], [10], [11], [12], [13], [14], [15]]. However, of all studies assessing the effect of TCRE none have a preoperative diagnosis of adenomyosis verified by histopathology [[9], [10], [11], [12], [13], [14], [15]]. Thus, it is not clear whether adenomyosis is induced by TCRE or whether it is the cause of treatment failure. The number of patients with adenomyosis and a good response to the procedure is unknown due to the lack of a preoperative diagnosis of adenomyosis. The degree of JZ changes before TCRE in relation to symptom relief is of particular interest [14]. It is likely that symptoms may persist after TCRE depending on the degree of JZ changes. We hypothesized that women with more extensive down growth of the endometrium may have no or less symptom relief after TCRE compared with women with no or minimal down growth of the endometrium. Therefore, the aim of this study was to investigate the association between outcome of TCRE (symptom severity, health-related quality of life, pelvic pain and reintervention rate) and histopathological findings of the JZ.

s symptom relief after TCRE compared with women with no or minimal down growth of the endometrium. Therefore, the aim of this study was to investigate the association between outcome of TCRE (symptom severity, health-related quality of life, pelvic pain and reintervention rate) and histopathological findings of the JZ. Materials and methods Study population This prospective study was approved by The Central Denmark Region Committee of Health Research Ethics, journal number: 20010175 and The Danish Data Protection Agency, journal number: 2007-58-0010. Written informed consent was obtained from all study participants before study entry. From October 2011 to January 2015, a consecutive sample of premenopausal women undergoing TCRE due to benign abnormal uterine bleeding and/or pelvic pain was recruited at the Department of Gynecology and Obstetrics, Aarhus University Hospital, Denmark. The exclusion criteria were non-elective surgery, malignancy, mental illness, coagulopathy, lack of Danish language skills or presence of five or more leiomyomas / uterine volume > 300 ml due to leiomyomas. Women were only able to participate once in the study project, thus repeat TCRE were excluded (Fig. 1).Fig. 1 Flowchart. 1 No adenomyosis of the inner myometrium and no reintervention surgery 2 Five with a histopathological diagnosis of adenomyosis of the inner myometrium and no reinterventions surgery. Two with serrated junctional zone had reintervention surgery (“no adenomyosis” verified by hysterectomy)

Materials and methods Study population This prospective study was approved by The Central Denmark Region Committee of Health Research Ethics, journal number: 20010175 and The Danish Data Protection Agency, journal number: 2007-58-0010. Written informed consent was obtained from all study participants before study entry. From October 2011 to January 2015, a consecutive sample of premenopausal women undergoing TCRE due to benign abnormal uterine bleeding and/or pelvic pain was recruited at the Department of Gynecology and Obstetrics, Aarhus University Hospital, Denmark. The exclusion criteria were non-elective surgery, malignancy, mental illness, coagulopathy, lack of Danish language skills or presence of five or more leiomyomas / uterine volume > 300 ml due to leiomyomas. Women were only able to participate once in the study project, thus repeat TCRE were excluded (Fig. 1).Fig. 1 Flowchart. 1 No adenomyosis of the inner myometrium and no reintervention surgery 2 Five with a histopathological diagnosis of adenomyosis of the inner myometrium and no reinterventions surgery. Two with serrated junctional zone had reintervention surgery (“no adenomyosis” verified by hysterectomy) Patients with at least one follow-up questionnaire (6 or 18 months) were kept for the questionnaire analyses. Reintervention surgery was recorded for all patients who completed baseline questionnaire (n = 126). Fig. 1

2 Five with a histopathological diagnosis of adenomyosis of the inner myometrium and no reinterventions surgery. Two with serrated junctional zone had reintervention surgery (“no adenomyosis” verified by hysterectomy) Patients with at least one follow-up questionnaire (6 or 18 months) were kept for the questionnaire analyses. Reintervention surgery was recorded for all patients who completed baseline questionnaire (n = 126). Fig. 1 Questionnaire Right before surgery, all study participants were asked to fill out a comprehensive questionnaire to obtain information on baseline characteristics, symptom severity, health-related quality of life (HR-QoL) and pelvic pain. The questionnaire was in Danish and consisted of the validated fibroid symptom and quality of life questionnaire (UFS-QOL) [[16], [17], [18]] including eight questions related to symptom severity (menstrual bleeding characteristics, pelvic and urinary discomfort, and fatigue) and 29 questions related to HR-QoL (concern, activities, energy/mood, control, self-consciousness, and sexual function). Each question was scored on a scale between 1 and 5. A total score was calculated and transformed into a symptom severity score (SSS) from 0 to 100 (higher score = more symptoms) and an HR-QoL score ranging from 0 to 100 (higher score = better HR-QoL). The UFS-QoL has been used for patients with adenomyosis before [[19], [20], [21], [22]]. Pelvic pain was assessed using the visual analogue scale for pain (VAS pain) [23], which consists of a 100-mm line with the endpoints "no pain" and "worst pain". Study participants were asked to mark the point along the line that expressed their average pain intensity. Identical questionnaires for follow-up were sent by email or post 6 and 18 months after surgery. The changes between baseline and follow-up scores (at 6 and 18 months) were calculated for SSS, HR-QoL, and VAS pain intensity. Women with an SSS < 20, an overall HR-QoL > 80, and no pelvic pain at 18 months follow-up were considered asymptomatic.

for follow-up were sent by email or post 6 and 18 months after surgery. The changes between baseline and follow-up scores (at 6 and 18 months) were calculated for SSS, HR-QoL, and VAS pain intensity. Women with an SSS < 20, an overall HR-QoL > 80, and no pelvic pain at 18 months follow-up were considered asymptomatic. In addition to the questionnaires, the rate of reintervention surgery was recorded on January 2018 from the national database Patoweb. This is a database where all surgical pathology tests are registered.

for follow-up were sent by email or post 6 and 18 months after surgery. The changes between baseline and follow-up scores (at 6 and 18 months) were calculated for SSS, HR-QoL, and VAS pain intensity. Women with an SSS < 20, an overall HR-QoL > 80, and no pelvic pain at 18 months follow-up were considered asymptomatic. In addition to the questionnaires, the rate of reintervention surgery was recorded on January 2018 from the national database Patoweb. This is a database where all surgical pathology tests are registered. Histopathology The procedure for obtaining deep endomyometrial biopsies during TCRE was standardized and applied in the same manner to all study participants. The surgical procedure took place in an outpatient setting with patients in local or brief general anesthesia. TCRE was performed with standardized electro current settings to avoid changes by electrocoagulation. The surgeon used a Storz resectoscope (26 F 12 oblique optic) (Karl Storz, Holte, Denmark) with a large monopolar cutting loop. To ensure that the resection could penetrate deep into the myometrium, careful suction curettage was performed immediately before resection, if the endometrium was not in the early proliferative phase. Furthermore, a firm pressure was applied to the resectoscope. Parallel slices representing the entire endometrium were obtained. Each biopsy was removed separately after each pass of the resectoscope, stretched and immediately fastened to a surface with the endometrium pointing downwards to avoid twisting. A needle was placed at the end, representing the beginning of the biopsy and the upper part of the uterus. Each biopsy was numbered according to its origin and then fixed in formalin prior to histopathological analysis.

hed and immediately fastened to a surface with the endometrium pointing downwards to avoid twisting. A needle was placed at the end, representing the beginning of the biopsy and the upper part of the uterus. Each biopsy was numbered according to its origin and then fixed in formalin prior to histopathological analysis. The endometrial-myometrial JZ was defined by histopathology of endomyometrial biopsies from TCRE and defined as: Adenomyosis of the inner myometrium, ≥ 2 mm myometrial invasion without contact to the basal endometrium, serrated JZ, > 3 mm myometrial invasion with contact to the basal endometrium, and linear JZ (no or marginal myometrial invasion ≤ 3 mm with contact to the basal endometrium) (Fig. 2).Fig. 2 Left: Linear junctional zone, no or marginal myometrial invasion ≤ 3 mm with contact to the basal endometrium. Middle: Serrated junctional zone, > 3 mm myometrial invasion with contact to the basal endometrium. Right: Adenomyosis of the inner myometrium (intrinsic adenomyosis), ≥ 2 mm myometrial invasion without contact to the basal endometrium. Fig. 2 For an accurate evaluation of the JZ, this method required thick endomyometrial biopsies with a depth of ≥ 5 mm into the myometrium. The histopathological evaluation took place within 1 to 3 weeks after surgery. The pathologist documented the findings in a standardized form designed for this study. Histopathology was inconclusive if the biopsies were either twisted or did not contain myometrium of sufficient depth (< 5 mm). Inconclusive histopathology samples were excluded from the study.

1 6 months follow-up questionnaire missing in four patients (these patients did not have surgical reintervention), three patients excluded due to reintervention surgery within the follow-up period. 2 18 months follow-up questionnaire missing in six patients (one of the patients had reintervention), ten patients exclude due to reintervention surgery within the follow-up period. No statistically significant difference in baseline SSS and HR-QoL between the groups (p-value > 0.05). *Statistically significant difference in 6 months follow-up SSS and HR-QoL between patients with either serrated junctional zone or adenomyosis of the inner myometrium and linear junctional zone (p-value ≤ .05). Fig. 3 Health-related quality of life on a scale from 0 to 100 at baseline, 6 and 18 months follow-up. Fig. 3 There was a statically significant difference in mean improvement of SSS and HR-QoL following TCRE between patients with either adenomyosis of the inner myometrium or serrated JZ and linear JZ at 6 months follow-up, with the most pronounced improvement among patients with linear JZ. At 18 months, when patients undergoing reintervention surgery were removed from analyses, there was no longer a statistically significant difference between the groups.

ace within 1 to 3 weeks after surgery. The pathologist documented the findings in a standardized form designed for this study. Histopathology was inconclusive if the biopsies were either twisted or did not contain myometrium of sufficient depth (< 5 mm). Inconclusive histopathology samples were excluded from the study. All statistical analyses were made using STATA 13.1. The sample size calculation was based on the assumption of at least 30% reintervention among patients with adenomyosis of the inner myometrium and 5% reintervention among patients with normal JZ (linear JZ), with standard errors of 5%. The estimated required sample size was then at least n = 72. The three groups according to histopathology of the JZ (adenomyosis of the inner myometrium, serrated JZ or linear JZ) were compared using the Kruskal-Wallis one-way analysis of variance for data that were not normally distributed and one-way ANOVA for data that were normally distributed. In addition, we used the Wilcoxon signed-rank test (not normally distributed data) and student's t-test (normally distributed data) to compare patients with either adenomyosis of the inner myometrium or serrated JZ to patients with linear JZ. P-values of < .05 were considered statistically significant. Patients who had reintervention surgery within the follow-up period (6 or 18 months) were excluded from the respective analyses. Missing items from the questionnaires were handled as follows: if < 50% of the scale items were missing, the scale would be retained, with the mean scale score of the items present used to impute a score for the missing items (subscale analysis). If ≥ 50% of the items were missing, no scale score was calculated, and the subscale score was considered missing [16].

ndled as follows: if < 50% of the scale items were missing, the scale would be retained, with the mean scale score of the items present used to impute a score for the missing items (subscale analysis). If ≥ 50% of the items were missing, no scale score was calculated, and the subscale score was considered missing [16]. Results In total, 112 patients filled out the baseline questionnaire. At 6 and 18 months, follow-up questionnaires were completed by 96.4% and 94,6% of the patients, respectively. Among patients who did not respond to the questionnaire, only two underwent reintervention surgery. Baseline characteristics are shown in Table 1. The presence of intrauterine device (IUD) did not affect the histopathological results. Based on histopathology twenty-four patients (21%) had adenomyosis of the inner myometrium, 31 (28%) had serrated JZ and 57 patients (51%) had linear JZ. There was no patients with a histopathological diagnosis of adenomyomas.Table 1 Baseline characteristics of patients scheduled for transcervical resection of the endometrium.

histopathology twenty-four patients (21%) had adenomyosis of the inner myometrium, 31 (28%) had serrated JZ and 57 patients (51%) had linear JZ. There was no patients with a histopathological diagnosis of adenomyomas.Table 1 Baseline characteristics of patients scheduled for transcervical resection of the endometrium. Table 1 All (n = 112) N 95% CI 75% PI% Adenomyosis of the inner myometrium (n = 24) N 95% CI 75% PI % Serrated junctional zone (n = 31) N 95% CI 75% PI % Linear junctional zone (n = 57) N 95% CI 75% PI % Mean age 45.2 44-46 45.5 44-47 45.1 43-47 45.1 43-47 Median BMI1 26.1 23-29 27.1 24-28 26.5 23-29 24.7 22-29 Mean number of pregnancies 3 3-3 4 3-4 3 3-4 3 2-3 Mean number of labors 2 2-2 2 2-2 2 2-3 2 2-2 Previous hysteroscopic surgery or myomectomy2 18 16% 4 17% 6 19% 8 14% Previous evacuation 23 21% 7 29% 6 19% 10 18% Previous caesarean section 16 14% 3 13% 10 52% 3 5% IUD or hormonal therapy before TCRE3 43 38% 4 17% 16 52% 23 40% IUD or hormonal therapy after TCRE4 34 30% 6 25% 10 32% 18 32% Ultrasound diagnosis of leiomyomas (all types) 27 24% 4 17% 9 29% 14 25% Ultrasound diagnosis of leiomyomas (intramural >3 cm in diameter or type 0-3) 16 24% 2 8% 5 16% 9 16% Ultrasound diagnosis of polyps 10 9% 3 13% 0 0% 7 12% Mean thickness of endomyometrial biopsies 5.3 5-6 5.4 5-6 5.4 5-6 5.2 5-5 CI: Confidence interval. PI: Percentile interval. BMI: Body Mass Index. IUD: Intrauterine device, TCRE: Transcervical resection of the endometrium.

iameter or type 0-3) 16 24% 2 8% 5 16% 9 16% Ultrasound diagnosis of polyps 10 9% 3 13% 0 0% 7 12% Mean thickness of endomyometrial biopsies 5.3 5-6 5.4 5-6 5.4 5-6 5.2 5-5 CI: Confidence interval. PI: Percentile interval. BMI: Body Mass Index. IUD: Intrauterine device, TCRE: Transcervical resection of the endometrium. There were no statistically significant differences (p-value <.05) between the groups (adenomyosis of the inner myometrium, serrated JZ and linear JZ) in baseline characteristics, except for mean number of pregnancies. 1One missing. 216 had resection of polyps, one had fibroid resection and one had myomectomy. 3Three months or more prior to operation. 4Five missing.

There were no statistically significant differences (p-value <.05) between the groups (adenomyosis of the inner myometrium, serrated JZ and linear JZ) in baseline characteristics, except for mean number of pregnancies. 1One missing. 216 had resection of polyps, one had fibroid resection and one had myomectomy. 3Three months or more prior to operation. 4Five missing. Reintervention surgery reported in the follow-up questionnaires is shown in Table 2. In total, twelve patients (80%) underwent reintervention surgery within 18 months after TCRE and an additional three patients (20%) underwent reintervention surgery after the 18-month follow-up period and before January 2018. Reintervention surgery was performed in 33% (95% CI: 16–55) patients with adenomyosis of the inner myometrium compared to 13% (95% CI: 4–30) and 5% (95% CI: 1–15) with serrated or linear JZ respectively (p-value < .05). Indications for reintervention surgery were either heavy menstrual bleeding and/or pelvic pain. All patients who had a reintervention surgery had a hysterectomy, but three (20%) had undergone repeated TCRE before hysterectomy. Adenomyosis of the inner myometrium was verified by histopathological examination of hysterectomy specimens in all but one woman. This woman had repeat TCRE prior to hysterectomy, thus it is likely that adenomyosis was removed before hysterectomy.Table 2 Surgical reintervention during follow-up according to histopathology and clinical symptoms.

yometrium was verified by histopathological examination of hysterectomy specimens in all but one woman. This woman had repeat TCRE prior to hysterectomy, thus it is likely that adenomyosis was removed before hysterectomy.Table 2 Surgical reintervention during follow-up according to histopathology and clinical symptoms. Table 2 Baseline symptoms Symptoms at 6 months follow-up Use of analgesics after TCREα Use of IUD after TCRE Surgical reintervention Histopathology SS HR-QoL Pelvic pain (VAS intensity %) SS HR-QoL Pelvic pain (VAS intensity %) #1 Adenomyosis of the inner myometrium 37.5 56.0 During (39) and between menstruation (30) Hysterectomy #2 Adenomyosis of the inner myometrium 59.4 57.8 No Repeat TCRE* #3 Serrated junctional zone 53.1 40.5 During (30) and between menstruation (15) Hysterectomy #41 Linear junctional zone 56.3 50.0 During (30) and between menstruation (88) 21.9 93.7 Between menstruation (87) Yes Yes Hysterectomy #5 Adenomyosis of the inner myometrium 62.5 41.4 During (87) and between menstruation (62) 56.3 46.6 During (90) and between menstruation (70) Yes Yes Repeat TCRE* #6 Adenomyosis of the inner myometrium 53.1 75.9 No 18.8 89.7 During menstruation (22) Yes No Repeat TCRE* #7 Adenomyosis of the inner myometrium 78.1 42.2 No 43.8 81.0 No No No Hysterectomy #8 Adenomyosis of the inner myometrium 62.5 42.2 During (71) and between menstruation (65) 15.6 90.5 Between menstruation (14) Yes No Hysterectomy #92 Linear junctional zone 56.3 75.0 During (77) and between menstruation (71) 53.1 82.8 During (56) and between menstruation (58) Yes Yes Hysterectomy #10 Serrated junctional zone 12.5 80.2 No 9.4 69.8 Between menstruation (23) No Yes Hysterectomy #11 Serrated junctional zone 50.0 44.8 During menstruation (78) Hysterectomy #12 Serrated junctional zone 18.8 81.9 During (85) and between menstruation (80) Hysterectomy #13 Adenomyosis of the inner myometrium 71.9 63.8 During (65) and between menstruation (23) 6.3 90.5 No No No Hysterectomy #14 Adenomyosis of the inner myometrium 62.5 49.1 During menstruation (72) 53.1 61.2 During menstruation (54) Yes No Hysterectomy #15 Linear junctional zone 34.4 30.2 No 65.6 7.8 During (63) and between menstruation (60) Yes No Hysterectomy TCRE: Transcervical resection of the endometrium. SSS: Symptom severity. HR-QoL: Health-related quality of life. VAS: Visual analogue scale. IUD: Intrauterine device.

ring menstruation (54) Yes No Hysterectomy #15 Linear junctional zone 34.4 30.2 No 65.6 7.8 During (63) and between menstruation (60) Yes No Hysterectomy TCRE: Transcervical resection of the endometrium. SSS: Symptom severity. HR-QoL: Health-related quality of life. VAS: Visual analogue scale. IUD: Intrauterine device. #1-3 had surgical reintervention < 6 months after TCRE, #4-12 had surgical reintervention 6–18 months after TCRE and #13-15 had surgical reintervention > 18 months after TCRE (before January 2018). Follow-up questionnaire missing in #11 and #12. * All three patients had subsequently hysterectomy due to persisting symptoms. Presence or absence of adenomyosis of the inner myometrium was verified in all hysterectomy specimens except for patient #2. This patient had one foci of adenomyosis of the inner myometrium, and it is likely that this foci may have been removed by repeat TCRE prior to hysterectomy. α Reported use at 6 months follow-up, including paracetamol, aspirin, non-steroidal anti-inflammatory drugs. 1 Concomitant uterine polyps. 2 Concomitant leiomyomas.

Presence or absence of adenomyosis of the inner myometrium was verified in all hysterectomy specimens except for patient #2. This patient had one foci of adenomyosis of the inner myometrium, and it is likely that this foci may have been removed by repeat TCRE prior to hysterectomy. α Reported use at 6 months follow-up, including paracetamol, aspirin, non-steroidal anti-inflammatory drugs. 1 Concomitant uterine polyps. 2 Concomitant leiomyomas. SSS and HR-QoL results at baseline, at 6 months follow-up (median (75% PI), 7.0 months (6–9)), 18 months follow-up (median (75% PI) and 18.4 months (18–20)) after TCRE are shown in Table 3. In addition, Fig. 3 illustrates the HR-QoL at baseline, 6 and 18 months follow-up. There was no statistically significant difference in the baseline SSS and HR-QoL between patients with adenomyosis of the inner myometrium, serrated JZ or linear JZ. When patients with concomitant pelvic pathology (intramural leiomyomas > 3 cm in diameter or type 0–3 leiomyomas, endometriosis and uterine polyps) were excluded from analyses, there was still no statistically significant difference between the groups (S1).Table 3 Symptom severity and health-related quality of life at baseline and follow-up after transcervical resection of the endometrium.

3 cm in diameter or type 0–3 leiomyomas, endometriosis and uterine polyps) were excluded from analyses, there was still no statistically significant difference between the groups (S1).Table 3 Symptom severity and health-related quality of life at baseline and follow-up after transcervical resection of the endometrium. Table 3 Baseline (n = 112) 6 months follow-up (n = 105)1 6 months improvement 18 months follow-up (n = 96)2 18 months improvement N Median 75% PI N Median 75% PI Mean 95% CI Difference N Median 75% PI Mean 95% CI Difference SSS Adenomyosis of the inner myometrium 24 59.4 48-66 22 18.8 9-44 34.4 25-44 p-value >.05* 15 18.8 6-19 39.8 30-50 p-value > .05 Serrated junctional zone 31 50.0 34-63 29 9.4 6-25 33.4 25-41 28 10.9 3-19 35.0 28-42 Linear junctional zone 57 56.3 44-72 54 7.8 0-25 41.6 35-48 53 9.4 3-22 42.3 36-48 HR-QoL total Adenomyosis of the inner myometrium 24 56.5 46-63 22 85.8 69-91 22.9 17-29 p-value ≤ .05 15 90.5 76-95 28.6 21-37 p-value > .05 Serrated junctional zone 31 56.0 42-66 29 93.1 83-97 31.0 22-40 28 93.1 80-97 30.7 22-39 Linear junctional zone 57 49.1 40-63 54 94.0 84-98 36.3 31-41 53 92.2 86-98 35.4 29-42 SSS: Symptom severity score. HR-QoL: Health-Related Quality of Life. PI: Percentile interval, CI: Confidence interval. No subscale score was considered missing. 1 6 months follow-up questionnaire missing in four patients (these patients did not have surgical reintervention), three patients excluded due to reintervention surgery within the follow-up period.

Table 3 Baseline (n = 112) 6 months follow-up (n = 105)1 6 months improvement 18 months follow-up (n = 96)2 18 months improvement N Median 75% PI N Median 75% PI Mean 95% CI Difference N Median 75% PI Mean 95% CI Difference SSS Adenomyosis of the inner myometrium 24 59.4 48-66 22 18.8 9-44 34.4 25-44 p-value >.05* 15 18.8 6-19 39.8 30-50 p-value > .05 Serrated junctional zone 31 50.0 34-63 29 9.4 6-25 33.4 25-41 28 10.9 3-19 35.0 28-42 Linear junctional zone 57 56.3 44-72 54 7.8 0-25 41.6 35-48 53 9.4 3-22 42.3 36-48 HR-QoL total Adenomyosis of the inner myometrium 24 56.5 46-63 22 85.8 69-91 22.9 17-29 p-value ≤ .05 15 90.5 76-95 28.6 21-37 p-value > .05 Serrated junctional zone 31 56.0 42-66 29 93.1 83-97 31.0 22-40 28 93.1 80-97 30.7 22-39 Linear junctional zone 57 49.1 40-63 54 94.0 84-98 36.3 31-41 53 92.2 86-98 35.4 29-42 SSS: Symptom severity score. HR-QoL: Health-Related Quality of Life. PI: Percentile interval, CI: Confidence interval. No subscale score was considered missing. 1 6 months follow-up questionnaire missing in four patients (these patients did not have surgical reintervention), three patients excluded due to reintervention surgery within the follow-up period. 2 18 months follow-up questionnaire missing in six patients (one of the patients had reintervention), ten patients exclude due to reintervention surgery within the follow-up period. No statistically significant difference in baseline SSS and HR-QoL between the groups (p-value > 0.05).

-up, when patients with reintervention surgery were excluded, the number of patients with pelvic pain was considerably reduced and there was no significant difference in pain reduction according to histopathological findings.Table 4 Pelvic pain at baseline and follow-up after transcervical resection of the endometrium. Table 4 Baseline (n = 112) 6 months follow-up (n = 105)1 18 months follow-up (n = 96)2 N With pain N (%) VAS* Median % 75% PI N With pain N (%) VAS* Median % 75% PI N With pain N (%) VAS* Median % 75% PI Pelvic pain during menstruation Adenomyosis of the inner myometrium 24 18 (75) 67 51-73 22 7 (32) 52 22-67 15 2 (13) 26 17-35 Serrated junctional zone 31 21 (68) 60 38-68 29 5 (17) 35 32-57 28 7 (25) 59 50-70 Linear junctional zone 57 45 (79) 57 36-70 54 15 (28) 56 11-63 53 11 (21) 31 20-39 Pelvic pain between menstruations Adenomyosis of the inner myometrium 24 11 (46) 30 17-62 22 5 (23) 42 19-56 15 0 0 Serrated junctional zone 31 14 (45) 40 24-50 29 8 (28) 25 21-39 28 3 (11) 35 15-53 Linear junctional zone 57 29 (51) 39 25-63 54 15 (28) 37 5-60 53 11 (21) 40 12-47 VAS: Visual Analogue Scale. PI: Percentile interval, CI: Confidence interval. *Average pain. 1 6 months follow-up questionnaire missing in four patients (these patients did not have reintervention surgery), three patients excluded due to reintervention surgery within the follow-up period. 2 18 months follow-up questionnaire missing in six patients (one of the patients had reintervention), ten patients exclude due to reintervention surgery within the follow-up period.

1 6 months follow-up questionnaire missing in four patients (these patients did not have reintervention surgery), three patients excluded due to reintervention surgery within the follow-up period. 2 18 months follow-up questionnaire missing in six patients (one of the patients had reintervention), ten patients exclude due to reintervention surgery within the follow-up period. There was no statistically significant difference in baseline or follow-up pelvic pain and VAS intensity between groups (p-value > 0.05). Before surgery more than 50% in each of the three groups used analgesic drugs; this was reduced to less than 20% at 18 months follow-up. In total, 31 patients had IUD’s inserted after TCRE (six with adenomyosis of the inner myometrium, eight with serrated JZ and 17 with linear JZ). There was no statistically significant difference in SSS, HR-QoL and pelvic pain between the groups who had an IUD or between those with and without IUD at 18 months follow-up. Finally, at 18 months follow-up, nine patients with adenomyosis of the inner myometrium had no pelvic pain, a symptom severity score < 20 and an HR-QoL > 80 and were therefore considered asymptomatic.

HR-QoL and pelvic pain between the groups who had an IUD or between those with and without IUD at 18 months follow-up. Finally, at 18 months follow-up, nine patients with adenomyosis of the inner myometrium had no pelvic pain, a symptom severity score < 20 and an HR-QoL > 80 and were therefore considered asymptomatic. Comment Surgical reintervention after TCRE was required among 12% of the study participants, and more commonly among patients who had adenomyosis of the inner myometrium before initial TCRE than those who did not (p-value < .05). However, TCRE completely resolved symptoms in 1/3 of all the patients with adenomyosis of the inner myometrium. A serrated JZ was rarely associated with treatment failure, although it was associated with less symptom improvement after surgery compared to linear JZ. However, the majority of all patients including those with adenomyosis of the inner myometrium and serrated JZ who did not require reintervention surgery experienced a considerable improvement in SSS and HR-QoL as well as a reduction in pelvic pain.

ted with less symptom improvement after surgery compared to linear JZ. However, the majority of all patients including those with adenomyosis of the inner myometrium and serrated JZ who did not require reintervention surgery experienced a considerable improvement in SSS and HR-QoL as well as a reduction in pelvic pain. Previous studies have shown that TCRE is an effective treatment for abnormal uterine bleeding, particularly for heavy menstrual bleeding [6,7], but 20% of patients might require surgical reintervention [24]. This may be due to the presence of adenomyosis [[9], [10], [11], [12], [13], [14], [15]]. However, to the best of our knowledge, no previous studies have evaluated the effect of TCRE according to a histopathologically proven preoperative diagnosis of adenomyosis of the inner myometrium. Moreover, outcome in relation to serrated JZ and linear JZ has not been reported. Our results suggest that TCRE does not cause adenomyosis of the inner myometrium but in fact, it seems to be an acceptable treatment option for some patients with adenomyosis of the inner myometrium and for most patients with serrated JZ or linear JZ. The cutting loop was able to penetrate at least 5 mm into the myometrium and destroy additional 1–2 mm of the myometrium due to electro-currency [14], thus we may assume that TCRE is an effective treatment option in patients with 0–7 mm endometrial penetration.

trium and for most patients with serrated JZ or linear JZ. The cutting loop was able to penetrate at least 5 mm into the myometrium and destroy additional 1–2 mm of the myometrium due to electro-currency [14], thus we may assume that TCRE is an effective treatment option in patients with 0–7 mm endometrial penetration. The presence of concomitant pelvic pathology did not affect our results as demonstrated in the supplementary tables (S1 + S2). This may be explained by the selection of patients without large or multiple leiomyomas. In patients with both adenomyosis and small leiomyomas, adenomyosis may be the main contributor to the clinical symptoms [25]. Furthermore, there was no difference in baseline symptoms between patients with either adenomyosis of the inner myometrium or serrated JZ and linear JZ. Again, this may be explained by the selection of only premenopausal women scheduled for surgery; anovulatory bleeding may also contribute to heavy menstrual bleeding [26] no matter the presence of adenomyosis of the inner myometrium. We were surprised not to find any difference in baseline pelvic pain between the groups. We expected that especially patients with adenomyosis of the inner myometrium and serrated JZ would have more menstrual pain because JZ changes may contribute to dysperistalsis or hyperperistalsis [27].

sis of the inner myometrium. We were surprised not to find any difference in baseline pelvic pain between the groups. We expected that especially patients with adenomyosis of the inner myometrium and serrated JZ would have more menstrual pain because JZ changes may contribute to dysperistalsis or hyperperistalsis [27]. There are some limitations to this study. The study population was small and we did not perform a systematic preoperative imaging to determine the JZ appearance prior to surgery. A recent study suggests that preoperative two- and three-dimensional transvaginal ultrasound may differentiate between adenomyosis of the inner myometrium, serrated JZ and linear JZ [28]. Unfortunately, there is still no consistent definition of how deep the endometrial penetration must be to be diagnosed as adenomyosis of the inner myometrium. Based on previously published textbooks, we chose a definition of > 2 mm with or without surrounding smooth muscle hyperplasia/hypertrophia [29,30]. To determine the effect of TCRE, we assessed several subjective parameters (questionnaire) but only one objective parameter (surgical reintervention). Additional objective parameters such as the amount of menstrual blood loss may have been assessed as well. Finally, the number of patients with IUDs was very limited which may explain why we did not find a difference in symptom improvement between patients with and without IUDs.

ive parameter (surgical reintervention). Additional objective parameters such as the amount of menstrual blood loss may have been assessed as well. Finally, the number of patients with IUDs was very limited which may explain why we did not find a difference in symptom improvement between patients with and without IUDs. A major strength of the study is the prospective study design of consecutively enrolled study participants undergoing the same standardized procedure for obtaining deep endomyometrial biopsies. This unique method allowed for a detailed histopathological evaluation of the entire JZ circumference. We focused not only on adenomyosis of the inner myometrium but also included serrated JZ as we hypothesized that this finding may also be associated with inferior outcome. Patients had a long-term follow-up and the response rate was high. Furthermore, we were able to gain access to information from Patoweb regarding surgical reintervention, even for the patients who did not complete the questionnaire.

d JZ as we hypothesized that this finding may also be associated with inferior outcome. Patients had a long-term follow-up and the response rate was high. Furthermore, we were able to gain access to information from Patoweb regarding surgical reintervention, even for the patients who did not complete the questionnaire. For successful management of women suffering from chronic abnormal uterine bleeding with or without pelvic pain, it is important to consider a preoperative diagnosis of adenomyosis, as this may negatively affect the outcome of TCRE. Nevertheless, in the present study, most women with adenomyosis of the inner myometrium experienced improved clinical symptoms after TCRE. Thus, it seems possible to treat adenomyosis of the inner myometrium with a minimally invasive approach. Future studies should include a large population of women with a preoperative transvaginal ultrasound measure of endometrial penetration before undergoing TCRE. Ultrasound-guided resection [31] and routine IUD insertion after resection may further improve the outcome after endometrial ablation [32,33]. Women with intrinsic adenomyosis are more likely to undergo reintervention surgery after transcervical endometrial resection due to persisting pelvic pain or heavy menstrual bleeding. On the other hand, approximately 1/3 of women with intrinsic adenomyosis may be asymptomatic 18 months after surgery. Overall, women with serrated and linear JZ responded well to TCRE though the highest improvement in symptoms, HR-QoL and pelvic pain was observed among women with linear JZ.

or heavy menstrual bleeding. On the other hand, approximately 1/3 of women with intrinsic adenomyosis may be asymptomatic 18 months after surgery. Overall, women with serrated and linear JZ responded well to TCRE though the highest improvement in symptoms, HR-QoL and pelvic pain was observed among women with linear JZ. Disclosure The authors have no conflicts of interest to declare. Appendix A Supplementary data The following are Supplementary data to this article: Acknowledgments This study was supported by Aarhus University, Denmark and by funding from A.P. Moeller, Helga and Peter Korning, The Family Hede Nielsen and Manager Jacob Madsen and his wife. Appendix A Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.eurox.2019.100029.

Introduction Pelvic organ prolapse (POP) is defined as the displacement, slippage or descent of the uterus and / or different vaginal compartments and its neighboring organs, such as the bladder, rectum, or intestine. It is, therefore, the anatomical change of the pelvic organs. [1] It affects approximately 40% of women after menopause. Its etiology is multifactorial, and includes age, hypoestrogenism, genetic, racial factors, those associated with increased intra-abdominal pressure, parity and other obstetric risk factors, vaginal hysterectomy and connective tissue diseases [2]. Collagen is one of the main constituents of the pelvic floor, with types I and III being the most prevalent. The fibers of type I collagen are well structured and are part of the ligaments. The fibers of type III collagen, however, make up the adventitia of the vaginal wall and involve the pelvic organs [3]. Recent studies have shown that the content, components and structure of collagen are modified in patients with POP. [4,5],

fibers of type I collagen are well structured and are part of the ligaments. The fibers of type III collagen, however, make up the adventitia of the vaginal wall and involve the pelvic organs [3]. Recent studies have shown that the content, components and structure of collagen are modified in patients with POP. [4,5], In the human species, metalloproteinases (MMPs) are a family of at least 20 proteolytic enzymes that are fundamental for extracellular matrix (ECM) turnover and are capable of regulating collagen metabolism, besides having an important role in the catabolism of most matrix components. [4,6], MMP-3 is capable of activating other MMPs and degrading proteoglycans, fibronectin, laminin, gelatin, elastin and collagens type II, III, IV and IX. [7,8], On the other hand, tissue inhibitors of metalloproteinases combine with inactive forms (pre-enzymes) or active forms to inhibit their production and activation, thus interfering with collagen degradation [4].

d degrading proteoglycans, fibronectin, laminin, gelatin, elastin and collagens type II, III, IV and IX. [7,8], On the other hand, tissue inhibitors of metalloproteinases combine with inactive forms (pre-enzymes) or active forms to inhibit their production and activation, thus interfering with collagen degradation [4]. The gene expression of these proteins depends on several factors, mainly the different polymorphisms of the promoter. A single nucleotide polymorphism (SNP) is a site in the DNA where a single base pair varies from person to person in a given population. Most of the polymorphisms do not have clinical repercussions, but another part is related to differences in transcription and expression of proteins, which confers phenotypic differences and individual susceptibility or resistance to the development of diseases. [8,9], According to a survey in the public database of SNPs (dbSNP), more than one thousand polymorphisms of the MMP-3 gene are described in the human species. The polymorphism rs3025058 results in the insertion of the adenosine (A) base in the promoter of the MMP-3 gene at position -1612/-1617 and creates a polymonomeric run of six adenosines, while the other variant has five adenosines. The presence of the 6A allele downregulates the expression of the MMP-3 gene. Thus, in 5A/5A the degradation of collagen and ECM may be accelerated. [6]

sine (A) base in the promoter of the MMP-3 gene at position -1612/-1617 and creates a polymonomeric run of six adenosines, while the other variant has five adenosines. The presence of the 6A allele downregulates the expression of the MMP-3 gene. Thus, in 5A/5A the degradation of collagen and ECM may be accelerated. [6] The interrelationship of epidemiologic, environmental, and genetic risk factors for POP constitutes the genetic epidemiology of prolapse. The motivation for our study is that with improved understanding of these relationships, there may be a role for individual risk assessment in future. Perhaps, women at high-risk for prolapse may choose her treatment. Maybe, in the future, this information may be incorporated into patient counseling and treatment decisions. Given this, we hypothesized that variations in the MMP-3 gene may alter gene expression and increase the risk of POP. In the present study we evaluated the polymorphism -1171 5A / 6A rs3025058 of the MMP-3 gene in a small sample of Brazilian postmenopausal women. We also assessed the associated clinical features and the risk for prolapse. Materials and methods The present study was reviewed and approved by the Research Ethics Committee of the Faculdade de Medicina do ABC (FMABC), Santo André, São Paulo, Brazil under number 554.670 / 2014. Participants were informed about the study protocol and procedures and written informed consent forms were obtained.

Materials and methods The present study was reviewed and approved by the Research Ethics Committee of the Faculdade de Medicina do ABC (FMABC), Santo André, São Paulo, Brazil under number 554.670 / 2014. Participants were informed about the study protocol and procedures and written informed consent forms were obtained. This is a cohort study, and all patients attended the Urogynecology and Vaginal Surgery Section of the FMABC, from 2014 to 2016 and they were randomly recruited by the researchers at the first medical appointment. All the women were subjected to history and physical examination. The POP-Q (Pelvic Organ Prolapse Quantification) classification proposed by Bump et al in 1996, a consensus statement from ICS- International Continence Society, AUGS -American Urogynecologic Society and SGS- Society of Gynecologic Surgeons, was used. [10] The group (A) consisted of 112 patients with POPs in stages III and IV. The control group (B) consisted of 180 patients with stages 0 and I. In both groups, patients were in the postmenopausal period. Those who did not have POP had urinary incontinence, hypertrophy of labia minora, Bartholin’s gland cyst and Skene’s gland cyst, desire for contraception by vaginal ligature and Müllerian duct anomalies. Patients who refused blood collection after they were provided with clarification about the study and those with previous vaginal surgery were excluded.

ry incontinence, hypertrophy of labia minora, Bartholin’s gland cyst and Skene’s gland cyst, desire for contraception by vaginal ligature and Müllerian duct anomalies. Patients who refused blood collection after they were provided with clarification about the study and those with previous vaginal surgery were excluded. In the clinical history, the following data were collected: age, ethnicity, body mass index (BMI), parity, place of birth and delivery, obstetric interventions (analgesia and episiotomy), weight of newborns, previous diseases, hypertension, dyslipidemia, chronic cough and constipation), life habits (physical activity with physical exertion and smoking) and previous hysterectomy. In terms of hormone replacement therapy (HRT), those patients who regularly used vaginal or systemic hormones for at least six months were considered as users. The frequencies of polymorphism genotypes at the MMP-3 gene site -1171 5A / 6A were compared between cases and controls. Molecular analysis of the -1171 5A / 6A polymorphism of the MMP3 gene Genomic DNA was extracted from 5 ml of venous blood from each patient collected in an EDTA anticoagulant bottle. DNA extraction was conducted from the leukocyte phase using the commercial kit, Illustra blood genomicPrep mini spin (GE Healthcare). The obtained DNA was stored at −20 °C until use. Amplification of the DNA by polymerase chain reaction (PCR) was performed in a volume of 10 μL with PCR Master Mix reagent (Promega) and 1 μM of each primer.

Molecular analysis of the -1171 5A / 6A polymorphism of the MMP3 gene Genomic DNA was extracted from 5 ml of venous blood from each patient collected in an EDTA anticoagulant bottle. DNA extraction was conducted from the leukocyte phase using the commercial kit, Illustra blood genomicPrep mini spin (GE Healthcare). The obtained DNA was stored at −20 °C until use. Amplification of the DNA by polymerase chain reaction (PCR) was performed in a volume of 10 μL with PCR Master Mix reagent (Promega) and 1 μM of each primer. The primers used were described by Gnasso et al (2000): 5′-GGTTCTCCATTCCTTTGATGGGGGGAAAGA-3′(sense); 5′-CTTCCTGGAATTCACATCACTGCCACCACT-3′(antisense). [7] The reaction in the thermal cycler was programmed as follows: one cycle at 94 °C with a duration of 5 min; 45 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 1 min; 1 cycle of 72 °C with a duration of 10 min and finally, the products were kept at 10 °C. The PCR products were subjected to digestion with 2 U of the restriction enzyme, Psyl (Thermo Scientific), under the conditions described by the supplier for 16 h. For homozygous patients, 6A / 6A, a 129 bp DNA band was expected; for the 5A / 5A homozygous patients, two bands of 97 and 32 bp were expected, while for the heterozygous patients the combination of bands of both alleles (129, 97 and 32 bp) was expected. Restriction fragment length polymorphism (RFLP) analysis was performed by 2.5% agarose gel electrophoresis plus ethidium bromide for the development of DNA bands.

ents, two bands of 97 and 32 bp were expected, while for the heterozygous patients the combination of bands of both alleles (129, 97 and 32 bp) was expected. Restriction fragment length polymorphism (RFLP) analysis was performed by 2.5% agarose gel electrophoresis plus ethidium bromide for the development of DNA bands. Statistical analysis The continuous quantitative variables are presented as the means. Qualitative variables are presented as numbers or percentages. Chi-squared test was used to compare the frequencies of polymorphisms between the groups of cases and controls. For those characteristics with statistical relevance, the crude odds ratio (OR) and its respective 95% confidence intervals were calculated; and, by logistic regression, were adjusted for each of the other characteristics, obtaining the adjusted OR. Hardy-Weinberg gene balance was determined using Pearson's Chi-squared test. Values of p < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism version 7.0 and SPSS version 23. Results The clinical characteristics of the patients are presented in Table 1. The patients in the study group were older than the patients in the control group. In addition, they also had on average a greater number of pregnancies and births, and among those, the vaginal route was the prevalent one. Home delivery contributed significantly to the occurrence of pelvic organ prolapse in the studied group.Table 1 Analysis of the clinical characteristics of women with and without pelvic organ prolapse.

o had on average a greater number of pregnancies and births, and among those, the vaginal route was the prevalent one. Home delivery contributed significantly to the occurrence of pelvic organ prolapse in the studied group.Table 1 Analysis of the clinical characteristics of women with and without pelvic organ prolapse. Table 1 Group A (n = 112) Mean or % Group B (n = 180) Mean or % p* Age, mean (years) 68.4 57.8 < 0.0001a White 69.90% 64.80% 0.422b Non-White 30.10% 35.20% Body mass index (kg/m²) 28.8 28.9 0.874a Age of menopause (years) 48.8 46.6 0.07a Hormonal therapy 10.70% 18.10% 0.09b Smoking 13.10% 20.10% 0.15b Arterial hypertension 57.80% 49.40% 0.186b Diabetes mellitus 24.50% 23.70% 0.888b Dyslipidemia 25.40% 24.70% 0.889b Chronic cough 1.80% 6.80% 0.08b Constipation 14.30% 10.40% 0.35b Pregnancy 5.6 3.5 < 0.0001a Parity 4.8 2.9 < 0.0001a Vaginal delivery 4.1 2.3 < 0.0001a C-section 0.08 0.12 0.377a Heaviest birth weight (g) 3516 3059 0.147a Episiotomy 8.30% 9.20% > 0.99b Labor analgesia 3.70% 4.80% 0.768b Home birth 25.90% 3.05% < 0.0001b Hysterectomy 15.20% 15.60% > 0.99b a Unpaired t-test. b Fisher’s exact-test. The logistic regression of factors significantly associated with genital prolapse adjusted for each of the other factors showed that age (adjusted OR = 11.89, 95% CI, 3.53–40) and home birth (adjusted OR = 9.645; 95% CI, 3.35–27.7) remained risk factors for the occurrence of genital prolapse in the studied samples (Table 2).Table 2 Logistic regression of factors associated with risk of pelvic organ prolapse.

for each of the other factors showed that age (adjusted OR = 11.89, 95% CI, 3.53–40) and home birth (adjusted OR = 9.645; 95% CI, 3.35–27.7) remained risk factors for the occurrence of genital prolapse in the studied samples (Table 2).Table 2 Logistic regression of factors associated with risk of pelvic organ prolapse. Table 2Factors Crude OR CI 95% p Adjusted OR CI 95% Age ≥ 51 15.57 (4.73 – 51.2) < 0.0001 11.89 (3.53 – 40) Pregnancy ≥ 3 2.02 (1.24 – 3.28) 0.004 0.656 (0.283 – 1.51) Vaginal delivery ≥ 3 3.12 (1.86 – 5.23) < 0.0001 1.91 (0.7 – 5.22) Parity ≥ 3 2.64 (1.62 – 4.31) < 0.0001 2.01 (0.665 – 6.1) Home birth 11.1 (4.14 – 29.7) < 0.0001 9.645 (3.35 – 27.7) OR: Odds ratio. CI: Confidence Interval. The frequencies of 5A / 5A, 5A / 6A and 6A / 6A genotypes were in Hardy-Weinberg equilibrium. Table 3 shows the frequency of MMP-3 genotypes in the study and control groups. There was no statistically significant difference between the groups in the distribution of genotypes, even after calculating the contribution of the 5A recessive allele in the aggregated genotypes (5A / 5A + 5A / 6A).Table 3 Distribution of the frequencies of MMP-3 genotypes between cases and controls. Table 3MMP3 genotypes Group A (n = 106) Group B (n = 169) p 6A/6A 55 (51.9%) 83 (49.1%) 0.895a 5A/6A 38 (35.9%) 65 (38.4%) 5A/5A 13 (12.2%) 21 (12.5%) MMP3 aggregated genotypes 6A/6A 55 (51.9%) 83 (49.1%) 0.7105b 5A/6A + 5A/5A 51 (48.1%) 86 (50.9%) a Chi-square test. b Fisher’s exact-test.

The frequencies of 5A / 5A, 5A / 6A and 6A / 6A genotypes were in Hardy-Weinberg equilibrium. Table 3 shows the frequency of MMP-3 genotypes in the study and control groups. There was no statistically significant difference between the groups in the distribution of genotypes, even after calculating the contribution of the 5A recessive allele in the aggregated genotypes (5A / 5A + 5A / 6A).Table 3 Distribution of the frequencies of MMP-3 genotypes between cases and controls. Table 3MMP3 genotypes Group A (n = 106) Group B (n = 169) p 6A/6A 55 (51.9%) 83 (49.1%) 0.895a 5A/6A 38 (35.9%) 65 (38.4%) 5A/5A 13 (12.2%) 21 (12.5%) MMP3 aggregated genotypes 6A/6A 55 (51.9%) 83 (49.1%) 0.7105b 5A/6A + 5A/5A 51 (48.1%) 86 (50.9%) a Chi-square test. b Fisher’s exact-test. Discussion POP negatively affects the quality of life of women, especially after the age of 50, and among these, approximately 10% will require surgery until the age of 80. Its etiology is multifactorial, and lately, genetic factors have been extensively studied. [11,12], SNPs at specific sites of genes that encode the synthesis and protein degradation of extracellular matrix components are a possibility that seek to explain this genetic origin for POP and other diseases. The current information suggests that changes in ECM turnover play an important role in the pathogenesis of genital prolapse. Studies have already evaluated the synthesis, degradation, types and amount of collagen in patients with POPs. [5,13,14],

SNPs at specific sites of genes that encode the synthesis and protein degradation of extracellular matrix components are a possibility that seek to explain this genetic origin for POP and other diseases. The current information suggests that changes in ECM turnover play an important role in the pathogenesis of genital prolapse. Studies have already evaluated the synthesis, degradation, types and amount of collagen in patients with POPs. [5,13,14], The polymorphism -1171 5A / 6A rs3025058 of the MMP-3 gene has already been related to certain diseases, mainly cancer and cardiovascular diseases. It is associated with an increased risk of development and metastasis of esophageal, lung and colorectal cancer. MMPs stimulate the proteolysis of ECM and basement membrane components, thus facilitating tumor dissemination. [15,16], This polymorphism was also associated with increased risk of atherosclerosis and restenosis in 6A / 6A homozygous individuals, probably due to failure of tissue remodeling [17]. In another study, the 5A allele was related to increased risk of deep vein thrombosis [18]. Some studies were conducted based on the various genomic polymorphisms of MMPs in women with genital prolapse and obtained contradictory results. According to the systematic reviews by Ward et al (2014) and Cartwright et al (2015), only two previous studies specifically addressed the rs3025038 polymorphism of MMP-3 in women with genital prolapse. [2,6,8,19],

various genomic polymorphisms of MMPs in women with genital prolapse and obtained contradictory results. According to the systematic reviews by Ward et al (2014) and Cartwright et al (2015), only two previous studies specifically addressed the rs3025038 polymorphism of MMP-3 in women with genital prolapse. [2,6,8,19], In the first systematic review, 21 articles pertained to the genetic epidemiology of POP. Ten candidate genes were studied. All of the case-control studies defined the control as POP-Q stage 0 or 0-I. Only two studies looked at Brazilian population (9.5%, 2/21). Age was similar between cases and controls for nine studies. Two studies preferentially recruited controls from an older population; all other studies with an age discrepancy had controls that were younger than the prolapse cases. The metaanalysis suggests that COL3A1 rs1800255 genotype AA is associated with POP (OR, 4.79; 95% confidence interval [CI], 1.91–11.98; P < .001); compared with the reference genotype GG in the same populations. The similar finding in both Asian and Dutch populations increases the likelihood that this is a true association. [2]

nalysis suggests that COL3A1 rs1800255 genotype AA is associated with POP (OR, 4.79; 95% confidence interval [CI], 1.91–11.98; P < .001); compared with the reference genotype GG in the same populations. The similar finding in both Asian and Dutch populations increases the likelihood that this is a true association. [2] Cartwright et al (2015) evaluated 34 studies that provided data on polymorphisms in or near 32 different genes. In pooled analysis, the rs4994 polymorphism of the ADRB3 gene was associated with overactive bladder (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.7–3.6; n = 419). The rs1800012 polymorphism of the COL1A1 gene was associated with prolapse (OR, 1.3; 95% CI, 1.0–1.7; n = 838) and stress urinary incontinence (OR, 2.1; 95% CI, 1.4–3.2; n = 190). Other metaanalyses, including those for polymorphisms of COL3A1, LAMC1, MMP-1, MMP-3, and MMP-9 did not show significant effects. In this systematic review and meta-analysis, other epidemiological characteristics of the populations studied were not addressed. [19] When we deal specifically with the polymorphism in question, we have two articles in the literature that address its relationship with POP.

Cartwright et al (2015) evaluated 34 studies that provided data on polymorphisms in or near 32 different genes. In pooled analysis, the rs4994 polymorphism of the ADRB3 gene was associated with overactive bladder (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.7–3.6; n = 419). The rs1800012 polymorphism of the COL1A1 gene was associated with prolapse (OR, 1.3; 95% CI, 1.0–1.7; n = 838) and stress urinary incontinence (OR, 2.1; 95% CI, 1.4–3.2; n = 190). Other metaanalyses, including those for polymorphisms of COL3A1, LAMC1, MMP-1, MMP-3, and MMP-9 did not show significant effects. In this systematic review and meta-analysis, other epidemiological characteristics of the populations studied were not addressed. [19] When we deal specifically with the polymorphism in question, we have two articles in the literature that address its relationship with POP. Ferrari et al (2012) studied 137 women with stage II pelvic organ prolapse or more, while controls (96) were those defined as having stage 0–1. Controls were matched for POP risk factors such as age, BMI, smoking habits, parity and rate of instrumental deliveries. Previous hysterectomy and/or surgery for POP or urinary incontinence as well as a past history of malignancy were exclusion criteria. Family history showed that 35% of patients with POP had a first-degree relative affected by the same disease compared with a prevalence of 16% in the control group (p = 0.002; OR: 2.9, 95% CI 1.5–5.7). They concluded that MMP-3 polymorphism -1171 5A / 6A did not increase the risk for prolapse in the group of cases compared to controls, both including Italian women. The groups were homogeneous for all the clinical characteristics studied. They also found that positive first-degree family history for POP constituted a factor significantly associated with risk of prolapse. [8]

ot increase the risk for prolapse in the group of cases compared to controls, both including Italian women. The groups were homogeneous for all the clinical characteristics studied. They also found that positive first-degree family history for POP constituted a factor significantly associated with risk of prolapse. [8] Shorupski et al (2013) studied 133 women with stage II pelvic organ prolapse or more. The patients were subjected to pelvic floor repair procedures with mesh. The control group consisted of 132 women without significant POP. The vast majority of these patients were admitted with uterine myomas and subsequently underwent total abdominal hysterectomy or supracervical abdominal hysterectomy. The rest of the control group consisted of patients with dysfunctional uterine bleeding. The patients in the control group were younger than their counterparts in the study group (p < 0.05). Not surprisingly, fewer patients in the control group had entered the menopause (p < 0.05). Besides that, they found overexpression of MMP-1 1 G / 2 G 1 G / 2 G SNPs and MMP-3 -1612 / -1617 5A / 6A SNPs in Polish women with genital prolapse, but when analyzed in isolation, both polymorphisms were not associated with increased risk for POP. [6]

atients in the control group had entered the menopause (p < 0.05). Besides that, they found overexpression of MMP-1 1 G / 2 G 1 G / 2 G SNPs and MMP-3 -1612 / -1617 5A / 6A SNPs in Polish women with genital prolapse, but when analyzed in isolation, both polymorphisms were not associated with increased risk for POP. [6] Thus, our study matches the findings of previous studies that investigated the same MMP-3 polymorphism associated with risk of genital prolapse. This is the first study developed with Brazilian postmenopausal women that seeks to relate the occurrence of genital prolapse with the polymorphism rs3025038 of the MMP-3 gene. We also evaluated a greater number of clinical characteristics of the sample than the previously mentioned studies. Our samples were not fully comparable in some clinical characteristics, probably because the occurrence of more advanced degrees of prolapse occurs in older women with more gestations and deliveries, and in this case, in those of the study group. The relatively young age of the controls increases the risk for misclassification of women who have not yet manifested prolapse. Horst et al (2016) also found that age greater than 35 years, normal delivery and fetal weight greater than 4 kg are risk factors for genital prolapse in Brazilian women. [20] Vergeldt et al (2015) concluded that parity, vaginal delivery, age, and BMI are factors significantly associated with disease risk [21].

olapse. Horst et al (2016) also found that age greater than 35 years, normal delivery and fetal weight greater than 4 kg are risk factors for genital prolapse in Brazilian women. [20] Vergeldt et al (2015) concluded that parity, vaginal delivery, age, and BMI are factors significantly associated with disease risk [21]. As a limitation, our study evaluated only one polymorphism and one gene alone. Shorupski et al found a correlation between polymorphisms of two different genes (MMP-1 and MMP-3) in patients with genital prolapse, further suggesting the complexity of genetic factors, such as etiology for the disease. In addition, history collection may present a significant recall bias. [6] In conclusion, the polymorphism rs3025058 of the MMP-3 gene was not associated with the risk for POP in this sample. Age and home delivery were significantly associated with increased risk for the disease. The negative findings of the study stimulate new research that analyzes polymorphisms of other genes related to the synthesis of collagen and other constituents of the ECM. The identification of patients at increased risk for developing POP would revolutionize the prevention of this change; guiding the choice of the birth route for example. In addition, it would assist in the identification of patients with greater risk of recurrence of POP, allowing a better evaluation of patients who are candidates for vaginal surgeries. Conflict of interest The authors have no conflicts of interest to declare.

The negative findings of the study stimulate new research that analyzes polymorphisms of other genes related to the synthesis of collagen and other constituents of the ECM. The identification of patients at increased risk for developing POP would revolutionize the prevention of this change; guiding the choice of the birth route for example. In addition, it would assist in the identification of patients with greater risk of recurrence of POP, allowing a better evaluation of patients who are candidates for vaginal surgeries. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgement This study was funded by Foundation for Research Support of the State of São Paulo (FAPESP) (grant number 2014 / 01107-6).

Background Obstetric cholestasis (OC), otherwise known as intrahepatic cholestasis of pregnancy is a condition specific to pregnancy The prevalence of OC is influenced by genetic and environmental factors and varies between 0.7 and 5% in different populations around the world [1]. Evidence also suggests a risk of recurrence of OC in up to two-third of subsequent pregnancies [2]. OC has been associated with an increased risk of perinatal morbidity and mortality [[3], [4], [5]], particularly with regards to stillbirths [6,7]. OC is commonly investigated as one potential cause in unexplained stillbirth [1]. Stillbirth is defined as ‘a baby delivered at or after 24 + 0 weeks gestational age showing no signs of life, irrespective of when the death occurred’ [8]. Stillbirth is common in pregnancy with about 1 in 200 babies born dead according to the Confidential Enquiry into Maternal and Child Health (CEMACH) report in 2009 [9] which is similar to the latest Mothers and Babies: Reducing Risk through Audits and Confidential Enquiries in the UK (MBRRACE-UK) report of 4.16 stillbirths per 1000 total births in 2016 [8]. In a large prospective cohort study by United Kingdom Obstetric Surveillance System (UKOSS) looking at severe cases of OC, stillbirth rates in severe OC were reported to be increased (adjusted OR 2.58,95% CI 1.03–6.49) compared to controls [10]. A number of observational studies showing varied rates of stillbirth and associated perinatal outcomes with OC have been published. However, the available evidence has not been systematically reviewed and is widely debated.

OC were reported to be increased (adjusted OR 2.58,95% CI 1.03–6.49) compared to controls [10]. A number of observational studies showing varied rates of stillbirth and associated perinatal outcomes with OC have been published. However, the available evidence has not been systematically reviewed and is widely debated. The hypothesis is the question “Is OC associated with stillbirth” led to the aim of this study to provide an overview of stillbirth and other outcomes in OC to inform clinical and health policy decisions. The objective was to systematically review the available evidence as outlined in the PRISMA statement [11] and critically appraisal it to produce a summary outcome of results to give an insight into the epidemiological outcomes of OC available globally. Methods Protocol and registration The study had a priori protocol designed and registered. (PROSPERO CRD42016052682). Search strategy and selection criteria The following electronic databases and registries were searched from inception up to 03 Nov 2018. Databases (for published data): PubMed, Embase (OVID), CENTRAL (Cochrane central Register of Controlled Trials), Cumulative Index to Nursing and Allied Health (CINAHL) and Literatura Latino Americana em Ciências da Saúde (LILACS) and Cochrane Library’s Database of Abstracts of Reviews of Effects (DARE).

Search strategy and selection criteria The following electronic databases and registries were searched from inception up to 03 Nov 2018. Databases (for published data): PubMed, Embase (OVID), CENTRAL (Cochrane central Register of Controlled Trials), Cumulative Index to Nursing and Allied Health (CINAHL) and Literatura Latino Americana em Ciências da Saúde (LILACS) and Cochrane Library’s Database of Abstracts of Reviews of Effects (DARE). Registries (for published and unpublished data): Clinicaltrials.gov, International Standard Randomized Controlled Trial Number registry and World Health Organization International Trials Registry Platform. (Appendix S1 in Supplementary material: Search sample for PubMed and Embase) In addition to the above, grey literature was also searched for in Open Grey (www.opengrey.eu). Hand searches methodology was done where relevant literature was considered to be available for inclusion into the studies and when not possible to be obtained from the above defined search strategy. No language restriction was used and articles identified in Chinese were excluded as per eligibility criteria. A search strategy was defined, agreed and independently carried by authors MM and AA. The searches were undertaken using index terms and key words relating to obstetric cholestasis (or) intrahepatic cholestasis of pregnancy, stillbirth (or) stillborn, neonatal (or) perinatal mortality (or) morbidity, premature (or) preterm birth, postpartum hemorrhage, meconium, cesarean section and induce (or) induced (or) induction of labo*.

aken using index terms and key words relating to obstetric cholestasis (or) intrahepatic cholestasis of pregnancy, stillbirth (or) stillborn, neonatal (or) perinatal mortality (or) morbidity, premature (or) preterm birth, postpartum hemorrhage, meconium, cesarean section and induce (or) induced (or) induction of labo*. Search results obtained was imported to Mendeley reference manager and duplicates were removed. Following this, authors MM and AA independently identified articles by screening titles. The authors then evaluated all full text abstracts of potentially relevant articles for their eligibility using criteria listed in Table 1. Any discrepancies in study selection were resolved through discussions at a consensus meeting. We had obtained all data electronically from databases and therefore no effort was made to contact authors of any included studies.Table 1 Study eligibility criteria. Table 1A study was considered eligible if it: • was a case-control or cohort study AND • published either as an original full length article or letter in a peer reviewed journal AND • included pregnant women with either singleton or multiple pregnancies with a diagnosis of OC (or) intrahepatic cholestasis of pregnancy (ICP) as per:- RCOG green top guidelines1 OR - WHO International classification of disease (ICD) codes AND • reported stillbirth (as a primary outcomes) AND • compared the outcomes in women diagnosed with OC or ICP with a control group (women without a diagnosed with OC or ICP).

• included pregnant women with either singleton or multiple pregnancies with a diagnosis of OC (or) intrahepatic cholestasis of pregnancy (ICP) as per:- RCOG green top guidelines1 OR - WHO International classification of disease (ICD) codes AND • reported stillbirth (as a primary outcomes) AND • compared the outcomes in women diagnosed with OC or ICP with a control group (women without a diagnosed with OC or ICP). Data extraction and assessment of risk of bias Details on study design, participants and clinical outcomes (stillbirths, cesarean section, induction of labor, preterm birth, meconium events, postpartum hemorrhage and admission to neonatal unit) were extracted. The Newcastle - Ottawa Scale (NOS), a tool designed to assess the quality of non-randomized studies included in a systematic review and/or meta-analyses, was used to appraise studies [12]. The NOS star rating system was used to evaluate eight items grouped into three categories: the selection of the study groups; the comparability of the groups; and the ascertainment of either the exposure or outcome of interest for case-control or cohort studies respectively. As per the tool, a study could be given a maximum of one star for each numbered item within the selection and outcome categories and a maximum of two stars within the comparability category – Table 2. Studies with 0–3 stars (red color), 4–6 stars (yellow color) and 7–9 stars (green color) are classified as studies with high, moderate or low risk of bias respectively.Table 2 Newcastle - Ottawa Scale categories and items.

on and outcome categories and a maximum of two stars within the comparability category – Table 2. Studies with 0–3 stars (red color), 4–6 stars (yellow color) and 7–9 stars (green color) are classified as studies with high, moderate or low risk of bias respectively.Table 2 Newcastle - Ottawa Scale categories and items. Table 2Category Items Selection 1 Representativeness of the exposed cohort 2 Selection of the non-exposed cohort 3 Ascertainment of exposure 4 Demonstration that outcome of interest was not present at start of study Defined as a robust approach in selecting obstetric patients (max. 4 stars) Comparability 1 Comparability of cohorts on the basis of the design or analysis controlled for confounders Defined as a study design that utilized associate controls and/or additional controls to compare with the obstetric cholestasis cohort (max. 2 stars) Outcome 3 Assessment of outcome 4 Was follow-up long enough for outcomes to occur 5 Adequacy of follow-up of cohorts Defined as identification and quantification of data on stillbirth max. (3 stars) Data extraction and assessment of risk of bias was carried out by authors MM and AA independently. Any discrepancies in data extraction and risk assessment were resolved through discussions.

4 Was follow-up long enough for outcomes to occur 5 Adequacy of follow-up of cohorts Defined as identification and quantification of data on stillbirth max. (3 stars) Data extraction and assessment of risk of bias was carried out by authors MM and AA independently. Any discrepancies in data extraction and risk assessment were resolved through discussions. Data synthesis Characteristics of included studies and the results of risk of bias assessment were tabulated. Where suitable data was available, meta-analyses of odds ratios (for binary outcomes) and were carried out in Review Manager 5.3 using a random effects model. In addition to the PRISMA-P checklist (Appendix S2 in Supplementary material), studies were cross checked using the meta-analysis of observational studies in epidemiology (MOOSE) (Appendix S3 in Supplementary material) approach to assess their suitability for meta-analysis. [12] Statistical heterogeneity between studies was evaluated by visual inspection of forest plots and the I2 statistic. A funnel plot was developed to undertake assessment of potential publication bias. Subgroup analysis was performed for the stillbirth outcome and also for the secondary outcomes including cesarean section and induction of labor. A subgroup analysis of cohort and case-control study was performed and results provided below. Multiple sensitivity analysis on the primary outcome with expected heterogeneity of included observational studies and described below. Ethics approval This review did not require any ethics committee approval or informed patient consent as it entirely relies on published data.

A subgroup analysis of cohort and case-control study was performed and results provided below. Multiple sensitivity analysis on the primary outcome with expected heterogeneity of included observational studies and described below. Ethics approval This review did not require any ethics committee approval or informed patient consent as it entirely relies on published data. Results A total of 13 studies [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]] met the inclusion criteria Fig. 1. A list of excluded studies [[26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60]] with reasons for exclusion can be found in supplementary file (Appendix S4 in Supplementary material).Fig. 1 PRISMA 2009 Flow Diagram. Fig. 1 Characteristics of included studies Five case-control [13,14,18,19,25] and eight cohort studies [[15], [16], [17],[20], [21], [22], [23], [24]]. The included studies were published between 1994 to 2016 and originated from different parts of the world including; Australia, China, Finland, India, Mexico, Saudi Arabia, Sweden, Switzerland, United Kingdom and The United States of America. Table3 shows the summary characteristics of the included studies.Table 3 Summary characteristics of included studies.

een 1994 to 2016 and originated from different parts of the world including; Australia, China, Finland, India, Mexico, Saudi Arabia, Sweden, Switzerland, United Kingdom and The United States of America. Table3 shows the summary characteristics of the included studies.Table 3 Summary characteristics of included studies. Table 3Study author and year Type of study Enrolment period Country of study Women with OC Women without OC Reported outcomes: Primary Reported outcome: Secondary Rioseco 1994 Case-control 1988-1990 United States of America 320 320 Stillbirth None Heinonen 1999 Cohort 1990-1996 Finland 91 16818 Stillbirth Preterm birth; Cesarean section; Meconium; Neonatal admission Yoong 2008 Cohort 2002-2005 United Kingdom 144 144 Stillbirth Cesarean section; Induction of labor; Meconium; Postpartum hemorrhage Sosa 2009 Cohort 1999-2007 Mexico 50 51 Stillbirth Meconium Padmaja 2010 Case control 2003-2006 India 45 90 Stillbirth Preterm birth; Cesarean section; Meconium; Postpartum haemorrhage; Neonatal admission Turunen 2010 Cohort 1969-1988 Finland 687 1374 Stillbirth Cesarean section; Induction of labor; Al Shobaili 2010 Cohort 2008-2010 Saudi Arabia 76 200 Stillbirth Preterm birth; Cesarean section; Meconium; Postpartum hemorrhage; Neonatal admission Shemer 2013 Cohort 1997-2009 Sweden 5477 1208191 Stillbirth Preterm birth; Cesarean section; Induction of labor; Geenes 2014 Case control 2010-2011 United Kingdom 713 2205 Stillbirth Preterm birth; Cesarean section; Neonatal admission Martineau 2014 Case-Control 2005-2011 United States of America 143 57581 Stillbirth Preterm birth; Cesarean section; Induction of labor; Bannister-T 2014 Cohort 2001-2011 Australia 1868 972898 Stillbirth Preterm birth; Cesarean section; Induction of labor; Postpartum hemorrhage Liu 2016 Cohort 2006-2014 China 1319 92876 Stillbirth Preterm birth; Meconium Furrer 2016 Case control 2004-2014 Switzerland 345 1725 Stillbirth Cesarean section; Induction of labor; Meconium; Neonatal admission

Australia 1868 972898 Stillbirth Preterm birth; Cesarean section; Induction of labor; Postpartum hemorrhage Liu 2016 Cohort 2006-2014 China 1319 92876 Stillbirth Preterm birth; Meconium Furrer 2016 Case control 2004-2014 Switzerland 345 1725 Stillbirth Cesarean section; Induction of labor; Meconium; Neonatal admission Assessment of risk of bias The risk of bias of studies as per the NOS tool assessment was considered to be low for 8 studies and moderate for 5 studies – Table 4. Publication bias was considered because of the stillbirth reporting variance in different countries across the world. However a funnel plot to assess of potential publication bias was symmetrical suggesting a possible absence of publication bias Fig. 2.Table 4 NOS SCORING. Table 4 Fig. 2 Funnel plot of stillbirth in patients with OC compared with patients without OC. Fig. 2 Outcomes Outcomes from the observational studies were reported in different formats and were of various completeness. Narrative and tabulated summaries including diagnostic criteria of included studies are provided (Table 3 and supplementary file - Appendix S5 in Supplementary material). Meta-analyses were performed for four outcomes - stillbirth, cesarean section, induction of labor and preterm labor. Primary outcome Stillbirth This outcome was reported in all 13 included studies. The included studies were of two designs i.e. case-control and cohort designs, we therefore subjected the meta-analysis as described below and presented in the

Outcomes Outcomes from the observational studies were reported in different formats and were of various completeness. Narrative and tabulated summaries including diagnostic criteria of included studies are provided (Table 3 and supplementary file - Appendix S5 in Supplementary material). Meta-analyses were performed for four outcomes - stillbirth, cesarean section, induction of labor and preterm labor. Primary outcome Stillbirth This outcome was reported in all 13 included studies. The included studies were of two designs i.e. case-control and cohort designs, we therefore subjected the meta-analysis as described below and presented in the The pooled estimates for OC showed that the stillbirth rates did not have a significant difference in outcome compared to stillbirth rates when the population did not have obstetric cholestasis OR 1.22 (0.73–2.04), with a subgroup analysis of cohort study showed OR 0.91 (0.66–1.25) with an I2 = 7%. Therefore the cohort subgroup analysis showed that OC was associated with a stillbirth rate of 5.25 per 1000 births compared to the reference population without obstetric cholestasis (non-OC), with a stillbirth rate of 4.91 per 1000 births and this was not statistically significant.

OR 0.91 (0.66–1.25) with an I2 = 7%. Therefore the cohort subgroup analysis showed that OC was associated with a stillbirth rate of 5.25 per 1000 births compared to the reference population without obstetric cholestasis (non-OC), with a stillbirth rate of 4.91 per 1000 births and this was not statistically significant. A sensitivity analysis for stillbirth events was performed after the removal of the studies where there was no stillbirth [17,20,23] in either group (OR 1.13,0.65–1.95), alternatively studies with wider confidence intervals [14,17,20,22,23] were removed (OR1.45,0.82–2.55) and finally data from the studies published before year 2010 [13,17,20,23] were removed (OR 1.12,0.62–2.01). This suggests that as heterogeneity was assumed with the included observational studies, however, with various sensitivity analyses, the primary outcome showed similar results as described above and therefore the reliability of the primary outcome results is more robust. The Forest plot is provided in Fig. 3Fig. 3 Results of meta-analysis of stillbirth events in patients with OC compared with patients without OC. Fig. 3 Secondary outcomes Cesarean section This outcome was reported in 10 studies [[14], [15], [16], [17], [18], [19],[22], [23], [24], [25]]. All of the 10 studies have presented the cesarean section as a similar outcome, with a variance in comparability of elective versus emergency cesarean section, however when both elective and emergency was combined together they were considered as a comparable group and therefore we analyzed as below.

[23], [24], [25]]. All of the 10 studies have presented the cesarean section as a similar outcome, with a variance in comparability of elective versus emergency cesarean section, however when both elective and emergency was combined together they were considered as a comparable group and therefore we analyzed as below. The pooled estimates for cesarean section in the OC population when compared to the non-OC group showed an OR 1.28 (1.15–1.42). The subgroup analysis with cohort study showed a similar OR 1.34 (1.27–1.41). However the subgroup of case-control studies showed a paradox of no difference in cesarean section with an OR 1.11(0.87–1.41). The Forest plot is provided in Fig. 4.Fig. 4 Results of meta-analysis of Cesarean section in patients with OC compared with patients without OC. Fig. 4 Induction of labor Induction of labor was reported by 6 studies [15,16,18,[23], [24], [25]] and all studies that reported on induction of labor also reported cesarean section. Pooled estimates of induction of labor in women with OC compared to women without OC showed an OR of 3.03(1.38–6.68) – Fig. 4. To understand the paradox of the cesarean section results, a subgroup analysis by study design were undertaken. The results showed cohort studies had an OR of 3.34 (1.38–8.07) while results for case-control studies showed an OR of 2.56 (0.41–15.87).

compared to women without OC showed an OR of 3.03(1.38–6.68) – Fig. 4. To understand the paradox of the cesarean section results, a subgroup analysis by study design were undertaken. The results showed cohort studies had an OR of 3.34 (1.38–8.07) while results for case-control studies showed an OR of 2.56 (0.41–15.87). Induction of labor with obstetric cholestasis compared to the control had a pooled estimate of OR 3.03(1.38–6.68). All the studies reporting induction of labor had also reported cesarean section. When we split the cohort and case-control to understand the paradox of cesarean section this showed the cohort studies had an OR of 3.34 (1.38–8.07), while the case-control studies showed an OR of 2.56 (0.41–15.87). This might explain the cesarean paradox of no difference does exist even with the induction group. The Forest plot is provided in Fig. 5.Fig. 5 Results of meta-analysis of induction of labor in patients with OC compared with patients without OC. Fig. 5 Preterm birth This outcome was reported in 8 studies [[14], [15], [16], [17], [18], [19],21,22]. All of the 8 studies used a delivery gestational age below 37 weeks gestation and therefore it was considered to be a group useful for meta-analysis. The pooled estimates for preterm birth rates in the OC population when compared to the regular (non-OC) group showed an OR 3.60 (2.61–4.96). The Forest plot is provided in Fig. 6.Fig. 6 Results of meta-analysis of preterm birth in patients with OC compared with patients without OC. Fig. 6

Preterm birth This outcome was reported in 8 studies [[14], [15], [16], [17], [18], [19],21,22]. All of the 8 studies used a delivery gestational age below 37 weeks gestation and therefore it was considered to be a group useful for meta-analysis. The pooled estimates for preterm birth rates in the OC population when compared to the regular (non-OC) group showed an OR 3.60 (2.61–4.96). The Forest plot is provided in Fig. 6.Fig. 6 Results of meta-analysis of preterm birth in patients with OC compared with patients without OC. Fig. 6 Other outcomes Meconium intrapartum events with obstetric cholestasis showed an OR 2.29 (1.35–3.88) when compared with the control (non-OC). Postpartum hemorrhage in OC compared to the non-OC group showed an OR 2.33(0.75–7.16) and the need for neonatal admission in the OC compared to the regular population was OR1.74 (1.03–2.96).

Other outcomes Meconium intrapartum events with obstetric cholestasis showed an OR 2.29 (1.35–3.88) when compared with the control (non-OC). Postpartum hemorrhage in OC compared to the non-OC group showed an OR 2.33(0.75–7.16) and the need for neonatal admission in the OC compared to the regular population was OR1.74 (1.03–2.96). Discussion This is the first study that systematically reviews current evidence and undertakes a meta-analysis of outcomes associated OC. comprehensive literature search was performed, which identified 8 cohort and 5 case-control studies that met the inclusion criteria. Results suggest stillbirth rates in women with OC are not significantly different when compared to women without OC. However overall pooled estimates and a subgroup analysis of cohort studies showed as increase of cesarean section and induction of labour in women with OC compared with women without OC. However no significant difference was seen for the outcomes in case control studies. An increase in preterm births, meconium intrapartum events and need for neonatal admission in women with OC compared with women without OC was also noted but with no statistical difference in post-partum hemorrhage. The overall risk of bias in included studies was considered low to moderate according to the NOS scoring.

n increase in preterm births, meconium intrapartum events and need for neonatal admission in women with OC compared with women without OC was also noted but with no statistical difference in post-partum hemorrhage. The overall risk of bias in included studies was considered low to moderate according to the NOS scoring. Comparison with previous studies Incidence of stillbirth rates of approximately 2% were reported in women with OC [7,61]. In a previous study, a delayed diagnosis of OC has been associated with an increased risk of stillbirths [62] and early delivery to reduce the risk [[62], [63], [64]], However, findings from the meta-analysis suggest no increase in risk of stillbirths. Similar results of stillbirth are shown in the recent systematic review specifically looking at biomarkers used in OC [65]. However for symptomatic OC the treatments benefits exists without much of a difference in stillbirths [66] and perinatal outcomes unless they were classified as severe OC [67].

isk of stillbirths. Similar results of stillbirth are shown in the recent systematic review specifically looking at biomarkers used in OC [65]. However for symptomatic OC the treatments benefits exists without much of a difference in stillbirths [66] and perinatal outcomes unless they were classified as severe OC [67]. Previous studies have found an increased rate of cesarean section in women with OC of up to 36% [68], These results are in line with findings of this study. However, the assumed cesarean section rate showed a paradox of higher rates in the cohort studies and no difference in the case control studies. - This may be attributable due to multiple variables including, the increased preterm birth rates, possible higher intrapartum meconium events (approximately 3 fold) and also other variables not included in the study such as increased interventions including abnormal cardiotocographic / electronic fetal monitoring abnormalities. Induction of labour in gestational weeks 37–39 is commonly performed with the perspective to avoid the complication of stillbirth [68]. Therefore, the paradox of cesarean section may be a represented with the induction of labor outcome as it also showed the same paradox as the cesarean section. Therefore it appears the paradox may be true. As matter of fact, it may be the case that there is no significant difference in stillbirth rates. Therefore, it is likely the cesarean section and induction rates are possible non-significant or may represent the reason for keeping the stillbirth rates non-significant.

on. Therefore it appears the paradox may be true. As matter of fact, it may be the case that there is no significant difference in stillbirth rates. Therefore, it is likely the cesarean section and induction rates are possible non-significant or may represent the reason for keeping the stillbirth rates non-significant. Preterm births in previous studies have shown to occur in 44% of women with OC [7,61]. The findings of the meta-analysis showed as increased preterm birth rate. These results are a direct representation of the comparison groups showing an increased rate with the OC population; this included both spontaneous preterm birth and iatrogenic preterm birth. The difference between the spontaneous and iatrogenic preterm birth were not studied, however, some of the included studies [16,19,21] showed significant rates of iatrogenic preterm birth as a likely contributor. This could possibly be due to the intervention associated with OC. Differences in post-partum hemorrhage rates were not statistically significant; but the neonatal admission rates were 74% higher in the OC compared to the reference population. Possibly this was secondary to the larger prevalence of preterm birth however, if this was directly as a representation of the preterm group, was not possible to assess this outcome from our study design.

cally significant; but the neonatal admission rates were 74% higher in the OC compared to the reference population. Possibly this was secondary to the larger prevalence of preterm birth however, if this was directly as a representation of the preterm group, was not possible to assess this outcome from our study design. Strengths and limitations This study has some limitations. Firstly, while a comprehensive search was undertaken to retrieve unpublished data, none was identifies and therefore the review findings are based upon published data only. Secondly, the findings of this study are based on observational studies, most of which had small sample sizes. Thirdly, the findings may have been influenced due to the different ways in which outcomes were selected and reported in individual studies. Implications for clinical practice and policy The study was not able to demonstrate that the actual stillbirth rates are reduced secondary to current practice as advised by international bodies such as the RCOG [1]. These guidelines may lead to intervention in pregnancies affected with OC such that the stillbirth rate is reduced. However, it may be that clinicians are intervening the obstetric population when there is not much evidence to show reduction in stillbirth rates as previously presumed, but instead contributing to an increase in relevant obstetric outcomes such as iatrogenic preterm births which are all likely due to increased intervention without statistically significant added benefits.

obstetric population when there is not much evidence to show reduction in stillbirth rates as previously presumed, but instead contributing to an increase in relevant obstetric outcomes such as iatrogenic preterm births which are all likely due to increased intervention without statistically significant added benefits. However as some of the included studies suggest a significant increased rates of stillbirth with the specific group of severe obstetric cholestasis [19,67], we recommend a definite need for intervention in this specific group as per the RCOG recommendation [1] “women should be informed that the case for intervention (after 37 weeks of gestation) maybe stronger in those with more severe biochemical abnormality (transaminase and bile acids)”. However we still lack the evidence when there is no severe obstetric cholestasis diagnosed and possibly associated with unwanted increased risks. We should therefore include these findings when we proceed for early intervention as we currently lack evidence for supporting our intervention for early delivery when there is no diagnosis of severe obstetric cholestasis. Even though the paradox of cesarean section and induction of labor may suggest that we need a much more robust testing before we conclude that either of the outcomes is different in the OC population and we may not be able to include these information’s when we use for patient counselling until higher evidence arises.

We should therefore include these findings when we proceed for early intervention as we currently lack evidence for supporting our intervention for early delivery when there is no diagnosis of severe obstetric cholestasis. Even though the paradox of cesarean section and induction of labor may suggest that we need a much more robust testing before we conclude that either of the outcomes is different in the OC population and we may not be able to include these information’s when we use for patient counselling until higher evidence arises. To avoid these stillbirths, there has been international acceptance of active management of OC-affected pregnancies with the goal of delivering the infant at <39 weeks. The general agreement suggests that delivery should not be delayed after 37–38 wks of gestation in patients with OC but not all obstetric professionals accept the association between OC and stillbirth or agree with the concept of active management in OC. Although American College of Obstetricians and Gynecologists and the Society for Maternal Fetal Medicine endorsed active management of OC-affected pregnancies, RCOG reported that active management of OC should be replaced by individual management decisions according to the evidence concerning the known perinatal risk of early term birth vs the small but unknown risk of OC-associated term still birth

aternal Fetal Medicine endorsed active management of OC-affected pregnancies, RCOG reported that active management of OC should be replaced by individual management decisions according to the evidence concerning the known perinatal risk of early term birth vs the small but unknown risk of OC-associated term still birth Conclusions This study provides an overview of risks associated with obstetric cholestasis based on currently available literature for the management of OC in pregnancy. The currently available evidence on risk factors of OC is still limited in quantity and remains inconclusive given the lack of robustly conducted studies. In light of this, there is a need for large prospective studies to determine the impact of OC on perinatal outcomes which are appropriately selected Systematic review registration PROSPERO CRD42016052682 Funding No funding obtained for this study Conflict of interest The authors have no conflicts of interest to declare. Ethics approval No ethical consideration as published data used for the systematic review. Disclosure All authors disclose with no financial, personal, political, intellectual or religious interest in development of this systematic review. Contribution to authorship All authors contributed significantly to be included as authors of the study.

Ethics approval No ethical consideration as published data used for the systematic review. Disclosure All authors disclose with no financial, personal, political, intellectual or religious interest in development of this systematic review. Contribution to authorship All authors contributed significantly to be included as authors of the study. MM conceptualized the idea to perform the study and designed the initial protocol and registration process. MM along with AA participated as the two independent reviewers for searches, identifying study subjects and collecting data independently, when discrepancies arouse SL contributed with finalizing the included studies. MM did the statistical analysis with support from MA and AAk. JK contributed in overall assessment and revision at each stage of the study. Appendix A Supplementary data The following are Supplementary data to this article: Appendix A Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.eurox.2019.100026.

Introduction Circulating levels of the anti-angiogenic factors fms-like tyrosine kinase (sFlt-1) and soluble endoglin are elevated in preeclampsia and fetal growth restriction [[1], [2], [3], [4]]. Studies to date have focused on placental trophoblast expression of Flt-1 and endoglin [[5], [6], [7]]. A contribution to the pathogenesis of preeclampsia and fetal growth restriction by Flt-1 and endoglin production by other cell types such as endothelial cells, monocytes and macrophages in the placenta and maternal peripheral circulation, has not been well explored. Monocytes are a functionally heterogeneous cell type with plasticity in subset types and polarization. The contribution of monocyte subsets to the angiogenic milieu in the pathogenesis of preeclampsia and fetal growth restriction is not established.

centa and maternal peripheral circulation, has not been well explored. Monocytes are a functionally heterogeneous cell type with plasticity in subset types and polarization. The contribution of monocyte subsets to the angiogenic milieu in the pathogenesis of preeclampsia and fetal growth restriction is not established. Circulating monocytes [8,9] express Flt-1 as a cell surface molecule, through which they bind VEGF and PlGF [10]. Both VEGF and PlGF stimulate tissue factor production, chemotaxis and trigger of further release of VEGF [11]. Flt-1 is also a mediator of pro-coagulant activity [10]. Both PlGF and VEGF activate the Flt-1–dependent signaling pathways of PI-3 K, p38 kinase, Akt, and ERK1/2 in primary human monocytes, leading to the activation of several intracellular signaling pathways that are critically involved in primary monocyte chemotaxis [12]. These data suggest that Flt-1 is a cell surface marker as well as a biologically functional molecule for monocyte-macrophage lineages. Any expression of sFlt-1 from these cells could be an additional, extra-placental, source of sFlt-1 that contributes to the pathogenesis of preeclampsia while surface expression of Flt-1 may play a pro-angiogenic role. In addition, the strong placental expression of PlGF could contribute to the marked angiogenesis seen in the growing placenta in part by its chemo-attraction of monocytes [13,14]. While there is some data available on monocytes in preeclampsia, there are no published studies exploring peripheral monocyte-derived angiogenic factors in fetal growth restriction or in the fetal / placental circulation.

ed angiogenesis seen in the growing placenta in part by its chemo-attraction of monocytes [13,14]. While there is some data available on monocytes in preeclampsia, there are no published studies exploring peripheral monocyte-derived angiogenic factors in fetal growth restriction or in the fetal / placental circulation. Endoglin (CD105) is a component of the TGF-β receptor system and acts as a co-receptor for TGF- β1 and TGF- β3 with high affinity [[15], [16], [17]], playing a critical role in maintaining cardiovascular homeostasis [18]. While endoglin is primarily expressed on endothelial cells [19] and induces pro-angiogenic proliferation and migration of these cells, it has also been demonstrated on macrophages [20], erythroid precursors [21], syncytiotrophoblast [22], monocytes [20], and stromal cells [23]. Soluble endoglin (sEng) inhibits TGFβ1 binding to its receptor, disordering signaling and preventing stimulation of endothelial nitric oxide synthase and vessel dilatation and is known to be elevated in preeclampsia, fetal growth restriction [7], and severe placental disease [24,25]. While monocytes can express endoglin, whether they do so in preeclampsia and fetal growth restriction has not been demonstrated.

venting stimulation of endothelial nitric oxide synthase and vessel dilatation and is known to be elevated in preeclampsia, fetal growth restriction [7], and severe placental disease [24,25]. While monocytes can express endoglin, whether they do so in preeclampsia and fetal growth restriction has not been demonstrated. Little is known about the pro and anti-angiogenic factor expression by different subsets of monocytes. Monocytes are subdivided into 3 subsets based on the expression of CD14 and the CD16 receptors, classical monocytes that lack CD16 (CD14++CD16−), and two subsets that express CD16 intermediate (CD14++CD16+), and the non-classical (CD14+CD16++) [26]. The intermediate and non-classical subsets are considered inflammatory monocytes [26]. The surface markers KDR and endoglin are suggested to be expressed on intermediate monocytes [27], at a low threshold. Monocyte chemotaxis towards the angiogenic ligands VEGF and PlGF was reduced in CD16+ intermediate and non-classical monocytes compared to CD16- classical monocytes [28]. Currently there is no data available on the relative levels of endoglin expression by different monocyte subsets.

monocytes [27], at a low threshold. Monocyte chemotaxis towards the angiogenic ligands VEGF and PlGF was reduced in CD16+ intermediate and non-classical monocytes compared to CD16- classical monocytes [28]. Currently there is no data available on the relative levels of endoglin expression by different monocyte subsets. Monocytes and macrophages can be phenotypically polarized by the environment to mount specific functional programs. Polarized activation of cells of the monocyte-macrophage lineage into classically activated inflammatory (M1) and alternatively activated healing (M2), cells is an operationally useful, simplified descriptor of the functional plasticity of these cells [29]. A transition from pro-inflammatory M1 to anti-inflammatory M2 phenotype is characterized by changes in cell surface marker expression including increases in CD163, CD206, and CD11b, which are distinctive of M2 monocytes and macrophages [30]. CD86 is a cell surface protein strongly expressed by M1-type macrophages / monocytes [[31], [32], [33]]. The monocyte expression of anti-inflammatory molecule, CD163 and the CD86/CD163 ratio can be used as a marker of M1 and M2 phenotypes [34]. M2 polarized macrophages play a role in resolution of inflammation, accompanied by reduced pro-inflammatory cytokine secretion [35]. Differentiation of monocytes into M1 and M2 macrophages play a central role in wound healing [36]. The association of monocyte polarization into M1/M2 phenotypes and correlation with angiogenic factor expression has not been described in the literature.

, accompanied by reduced pro-inflammatory cytokine secretion [35]. Differentiation of monocytes into M1 and M2 macrophages play a central role in wound healing [36]. The association of monocyte polarization into M1/M2 phenotypes and correlation with angiogenic factor expression has not been described in the literature. The current study explores the associations of peripheral monocyte expression of angiogenic factors (Flt-1 and endoglin), with clinical groups (normal, preeclampsia, fetal growth restriction and preeclampsia + fetal growth restriction), monocyte subsets (classical, non-classical and intermediate), polarization (by ratio of CD86/CD163), as well as by gestational age. We examined both maternal and fetal monocytes. Materials and methods A prospective cross-sectional study was conducted. Pregnant women between 24–40 weeks of gestation dated by first trimester ultrasound, who satisfied the selection criteria, confining at an Australian tertiary hospital during 2013–2014 were recruited from ultrasound service, antenatal ward, and antenatal clinics into four clinical groups of normal pregnancy, preeclampsia, fetal growth restriction and preeclampsia + fetal growth restriction. The normal pregnancy samples were collected at the routine antenatal clinics for gestations less than 37 weeks and prior to elective repeat caesarean section at over 37 weeks. The study was conducted with the approval of the institutional Human Research Ethics committee. Written consent was obtained from all participants in the study.

ancy samples were collected at the routine antenatal clinics for gestations less than 37 weeks and prior to elective repeat caesarean section at over 37 weeks. The study was conducted with the approval of the institutional Human Research Ethics committee. Written consent was obtained from all participants in the study. All patients classified as preeclampsia in this study satisfied the ISSHP 2014 criteria for preeclampsia [37]. Fetal growth restriction was defined as birth weight less than 10th centile with elevated umbilical artery Doppler systolic / diastolic ratio or Resistance Index > 95th centile for gestation. All patients with preeclampsia, preeclampsia + fetal growth restriction or fetal growth restriction underwent antenatal ultrasound examination after 24 weeks of gestation and within 7 days of delivery. Patients with pre-existing hypertension, renal disease, pre-existing diabetes, gestational diabetes and multiple pregnancies were excluded from the study.

clampsia + fetal growth restriction or fetal growth restriction underwent antenatal ultrasound examination after 24 weeks of gestation and within 7 days of delivery. Patients with pre-existing hypertension, renal disease, pre-existing diabetes, gestational diabetes and multiple pregnancies were excluded from the study. Flow cytometry Maternal venous blood samples were collected prior to delivery in EDTA tubes. Blood counts were performed. Cells were stained at room temperature using antibodies against CD14 (BD 560349, Biosciences, USA), CD16 (Ab 140477, Abcam, USA), Flt-1 (FAB321 A, R&D), endoglin (BD561443), CD86 (BD 555658 Biosciences, USA) and CD163 (BD 556018, Biosciences, USA). Incubation with cell surface antibodies was followed by Optilyse C for lysing red blood cells and fixation. Monocyte phenotype assessment and cell marker profile was assessed by flow cytometry using a BD Canto II flow cytometer (BD Biosciences, USA) and Flow Jo software version 10.6 (Tree star, Inc., Ashland, OR, USA). A fluorescence minus one control and a three-color panel were used for data acquisition. The median fluorescence intensity (MFI) was calculated for each marker in each clinical group, Flt-1 and endoglin expression were examined and correlated with clinical group, monocyte subset and CD86/CD163 as an indicator of the degree of monocyte M1/M2 polarization.

a three-color panel were used for data acquisition. The median fluorescence intensity (MFI) was calculated for each marker in each clinical group, Flt-1 and endoglin expression were examined and correlated with clinical group, monocyte subset and CD86/CD163 as an indicator of the degree of monocyte M1/M2 polarization. Statistics The statistical software packages SPSS for windows Version 21 and SPLUS version 8 were used. The patient numbers used in this study were considered adequate from previous work on similar exploratory studies. Similar work has been published on sample numbers ranging from 5 to 10 in each group. ANOVA for multiple comparisons was used to assess the association between tested variables. Kruskal Wallis non-parametric analysis of variance was used to test for homogeneity across the four clinical groups for each of the variables; percentage of monocytes, percentage of monocyte subsets, median fluorescence intensity of CD86, CD163, Flt-1 and endoglin. Where heterogeneity was identified, post hoc Mann-Whitney tests were used for pairwise comparisons between normal pregnancy and each of the clinical groups as well as between each of the pathological groups. The Spearman's rank correlation was used to quantify the extent of the association between monocyte surface Flt-1, endoglin expression and gestational age in normal pregnancies. Data were also analyzed for any association between Flt-1, endoglin expression and monocyte subtype as well as monocyte polarization and pro-inflammatory status as defined by CD86/CD163 ratio.

fy the extent of the association between monocyte surface Flt-1, endoglin expression and gestational age in normal pregnancies. Data were also analyzed for any association between Flt-1, endoglin expression and monocyte subtype as well as monocyte polarization and pro-inflammatory status as defined by CD86/CD163 ratio. Results Maternal and neonatal demographic data, clinical characteristics and results are presented for 54 maternal and 27 fetal samples for pregnancies from 24 to 40 weeks of gestation. There were no statistically significant differences noted between the pathological (preeclampsia, preeclampsia + fetal growth restriction and fetal growth restriction) groups in gestational age at sample collection. Gestational age and weight at delivery were lower in study groups compared to controls (Table 1). There was a 71% caesarean section rate in the normal pregnancy controls due to the inclusion of elective caesarean deliveries at term.Table 1 Maternal and fetal demographic data and clinical characteristics of the study population. Results are presented as mean ± SD for each continuous variable unless otherwise specified. * Significantly different from normal pregnancy. † Significantly different to PE. # Significantly different to FGR. p < 0.05. LSCS Lower segment caesarean section.

aphic data and clinical characteristics of the study population. Results are presented as mean ± SD for each continuous variable unless otherwise specified. * Significantly different from normal pregnancy. † Significantly different to PE. # Significantly different to FGR. p < 0.05. LSCS Lower segment caesarean section. Table 1Experimental Groups Normal PE FGR PE + FGR Number of maternal samples 24 9 12 9 Number of cord blood / fetal samples 8 4 9 6 Maternal age (years) 29.4(3.6) 27.9(6.1) 28.3(4.9) 33.0(7.1)* † BMI 27.3(5.3) 29.6(8.1) 24.2(5.3) † 28.2(6.3) Gestation at sample collection (weeks) 34.8(4.2) 35.7(3.3) 34.8(4.2) 32.3(2.3) Gestation at delivery (weeks) 39.2(0.8) 36.2(3.5)* 35.5(3.6)* 32.68(2.5)* † # Birth weight (g) 3316(505) 2749(821)* 1879(670)* † 1509(540)* † Primiparous (%) 37.5 77.8* 58.3 88.9* Antihypertensive treatment (%) 0% 88% 0% 44% Smoking 8.3% 0.0% 8.3% 7.4% Mode of delivery (Rate of LSCS) 71% 78% 100% 100%

.8(4.2) 32.3(2.3) Gestation at delivery (weeks) 39.2(0.8) 36.2(3.5)* 35.5(3.6)* 32.68(2.5)* † # Birth weight (g) 3316(505) 2749(821)* 1879(670)* † 1509(540)* † Primiparous (%) 37.5 77.8* 58.3 88.9* Antihypertensive treatment (%) 0% 88% 0% 44% Smoking 8.3% 0.0% 8.3% 7.4% Mode of delivery (Rate of LSCS) 71% 78% 100% 100% Maternal monocyte Flt-1 and endoglin expression Circulating maternal monocytes expressed Flt-1 and endoglin as surface markers (Table 2). Although the median fluorescence intensity of Flt-1 expression of total monocytes was not different across the clinical groups, differences were observed in the monocyte subsets with Flt-1 mainly expressed by classical and intermediate monocytes (Fig. 1A).Table 2 Maternal monocyte expression of Flt-1 and Endoglin presented for total monocytes and different monocyte subsets. Results are presented as median ± interquartile range for each continuous variable. * Statistically significantly different from normal pregnancy p < 0.05. † Statistically significantly different from PE p < 0.05.

al monocyte expression of Flt-1 and Endoglin presented for total monocytes and different monocyte subsets. Results are presented as median ± interquartile range for each continuous variable. * Statistically significantly different from normal pregnancy p < 0.05. † Statistically significantly different from PE p < 0.05. Table 2 Clinical Group Normal PE only FGR only PE + FGR Median (Percentile 25, 75) Median (Percentile 25, 75) Median (Percentile 25, 75) Median (Percentile 25, 75) Percentage of total monocytes expressing Flt-1 42.0 (33.4, 51.0) 37.6 (30.2, 54.9) 48.2 (25.1, 70.6) 42.4 (37.7, 44.5) Total monocytes Flt-1 MFI 80.0 (57.0, 126.0) 49.0 (19.0, 71.0) 104.0 (33.5, 194.5) 73.0 (51.0, 102.0) Classical monocytes Flt-1 MFI 140.0 (81.0, 181.0) 100.0 (65.0, 167.0) 185.5 (96.0, 278.0) 147.0 (102.0, 178.0) Intermediate monocytes Flt-1 MFI 167.0 (99.0, 238.5) 128.0 (90.0, 204.0) 191.5 (99.5, 337.5) 140.0 (119.0, 212.0) Non Classical Flt-1 MFI 52.5 (30.5, 98.0) 88.0 (48.0, 93.0) 84.0 (54.5, 103.0) 97.0 (73.0, 160.0) Percentage of monocytes expressing Endoglin 62.7 (57.1, 76.2) 63.3 (58.8, 73.3) 74.5 (63.0, 82.1) 54.4*† (43.4, 63.6) Total monocytes Endoglin MFI 330.0 (291.0, 446.5) 300.0 (267.0, 403.0) 362.5 (299.5, 575.5) 264.0* (75.0, 296.0) Classical monocytes Endoglin MFI 342.0 (302.5, 403.5) 320.0 (308.0, 330.0) 454.5 (314.0, 644.0) 236.0* (216.0, 351.0) Intermediate monocytes Endoglin MFI 653.0 (523.0, 1034.5) 483.0 (444.0, 896.0) 692.0 (468.0, 937.0) 499.0 (395.0, 595.0) Non Classical monocytes Endoglin MFI 582.0 (526.0, 811.0) 458.0 (332.0, 678.0) 658.0 (293.0, 725.5) 464.0 (203.0, 641.0) Fig. 1 Analysis of maternal monocyte surface (A) Flt-1 and (B) endoglin expression (MFI) according to the subtype and clinical group. Results are presented as median and interquartile range. * Statistically significant difference between groups p < 0.05.

0) 458.0 (332.0, 678.0) 658.0 (293.0, 725.5) 464.0 (203.0, 641.0) Fig. 1 Analysis of maternal monocyte surface (A) Flt-1 and (B) endoglin expression (MFI) according to the subtype and clinical group. Results are presented as median and interquartile range. * Statistically significant difference between groups p < 0.05. CM = Classical monocytes, IM = Intermediate monocytes, NCM = Non classical monocytes. Fig. 1 The expression of endoglin MFI appears to be significantly decreased in preeclampsia + fetal growth restriction for total monocytes and classical monocytes, as compared to the normal pregnancies (Table 2). In all clinical groups, the expression of endoglin was significantly higher in the intermediate monocytes compared to classical monocyte subtype (Fig. 1B). Fetal monocyte Flt-1 and endoglin expression The results for fetal monocyte expression of Flt-1 and endoglin are presented in Fig. 2 and Table 3. The percentage and MFI of fetal monocytes Flt-1 expression did not show a statistically significant differences between the clinical groups. The results did not vary for the different subsets. The endoglin expression however was increased in the fetal growth restriction group for total monocytes as well as all the subtypes.Fig. 2 Fetal monocytes expression of Endoglin. A: Total monocytes B: Classical monocytes. C: Intermediate monocytes. D: Non-classical monocytes. The results are presented as median ± interquartile range. * Statistically significant differences between clinical groups p < 0.05.

otal monocytes as well as all the subtypes.Fig. 2 Fetal monocytes expression of Endoglin. A: Total monocytes B: Classical monocytes. C: Intermediate monocytes. D: Non-classical monocytes. The results are presented as median ± interquartile range. * Statistically significant differences between clinical groups p < 0.05. Fig. 2Table 3 Fetal monocyte expression of Flt-1 and Endoglin as markers of anti-angiogenic activity. *Significantly different from normal pregnancy. # Significantly different from PE. †Significantly different from FGR. Results are presented as median ± interquartile range for each continuous variable. For PE, 3 out of 4 MFI values for Flt-1 were not higher than the isotype control, resulting in a median value of 0.

y. *Significantly different from normal pregnancy. # Significantly different from PE. †Significantly different from FGR. Results are presented as median ± interquartile range for each continuous variable. For PE, 3 out of 4 MFI values for Flt-1 were not higher than the isotype control, resulting in a median value of 0. Table 3 Clinical Group Normal PE only FGR only PE + FGR Median (Percentile 25, 75) Median (Percentile 25, 75) Median (Percentile 25, 75) Median (Percentile 25, 75) Percentage of monocytes expressing Flt-1 10.0 (6.6, 28.9) 32.3 (12.6, 49.0) 17.6 (12.7, 43.0) 30.4 (12.3, 37.7) Total monocytes Flt-1 MFI 6.0 (0.0, 49.0) 43.5 (8.5, 87.5) 30.0 (15.0, 64.0) 34.0 (16.0, 87.0) Classical monocytes Flt-1 MFI 58.0 (43.5, 98.0) 0.0 (0.0, 55.5) 42.0 (26.0, 137.0) 68.0 (34.0, 142.0) Intermediate monocytes Flt-1 MFI 107.0 (68.0, 182.5) 41.5 (13.5, 128.0) 50.0 (43.0, 218.0) 157.5 (77.0, 223.0) Non Classical Flt-1 MFI 72.0 (67.5, 140.5) 61.5 (0.0, 168.5) 48.0 (4.0, 80.0) 107.0 (71.0, 152.0) Percentage of monocytes expressing Endoglin 49.7 (36.3, 59.3) 52.5 (39.9, 58.7) 77.9 *# (74.5, 81.9) 59.5 † (46.8, 73.5) Total monocytes Endoglin MFI 181.0 (76.0, 331.0) 222.5 (108.5, 272.0) 469.0 *# (426.0, 490.0) 219.0 † (111.0, 280.0) Classical monocytes Endoglin MFI 330.0 (207.0, 511.5) 235.5 (204.0, 270.5) 549.0*# (496.0, 642.0) 333.5 (259.0, 362.0) Intermediate monocytes Endoglin MFI 375.0 (228.5, 548.0) 335.0 (229.0, 403.5) 745.0 *# (616.0, 869.0) 417.0 (269.0, 426.0) Non Classical monocytes Endoglin MFI 279.0 (184.5, 496.0) 198.5 (76.0, 307.5) 537.0*# (457.0, 752.0) 224.0 (203.0, 332.0)

.0, 511.5) 235.5 (204.0, 270.5) 549.0*# (496.0, 642.0) 333.5 (259.0, 362.0) Intermediate monocytes Endoglin MFI 375.0 (228.5, 548.0) 335.0 (229.0, 403.5) 745.0 *# (616.0, 869.0) 417.0 (269.0, 426.0) Non Classical monocytes Endoglin MFI 279.0 (184.5, 496.0) 198.5 (76.0, 307.5) 537.0*# (457.0, 752.0) 224.0 (203.0, 332.0) Correlation of Flt-1 and endoglin expression with gestation Differential distribution of maternal monocyte Flt-1 and endoglin expression with increasing gestation in third trimester of pregnancy was assessed in 24 pregnancies from 26 weeks to 40 weeks of gestation. Spearman's rank correlation between gestational age in normal third trimester pregnancy and the markers of interest Flt-1 and endoglin showed that the percentage of monocytes expressing Flt-1 and endoglin did not change with gestation but the monocyte Flt-1 MFI (correlation coefficient 0.402, p = 0.049) and endoglin MFI (correlation coefficient 0.457, p = 0.025) showed a statistically significant increase with gestation (Fig. 3).Fig. 3 Mean fluorescent intensity of Flt-1 and Endoglin with increased gestation in normal pregnancies. There was a moderate and statistically significant correlation seen between gestational age and MFI of both Flt-1 and Endoglin. r = Correlation coefficient, Spearman rank correlation. Significance p < 0.05. Fig. 3

Correlation of Flt-1 and endoglin expression with gestation Differential distribution of maternal monocyte Flt-1 and endoglin expression with increasing gestation in third trimester of pregnancy was assessed in 24 pregnancies from 26 weeks to 40 weeks of gestation. Spearman's rank correlation between gestational age in normal third trimester pregnancy and the markers of interest Flt-1 and endoglin showed that the percentage of monocytes expressing Flt-1 and endoglin did not change with gestation but the monocyte Flt-1 MFI (correlation coefficient 0.402, p = 0.049) and endoglin MFI (correlation coefficient 0.457, p = 0.025) showed a statistically significant increase with gestation (Fig. 3).Fig. 3 Mean fluorescent intensity of Flt-1 and Endoglin with increased gestation in normal pregnancies. There was a moderate and statistically significant correlation seen between gestational age and MFI of both Flt-1 and Endoglin. r = Correlation coefficient, Spearman rank correlation. Significance p < 0.05. Fig. 3 Correlation of anti-angiogenic factor Flt-1 and endoglin expression with polarisation of monocytes into M1/M2 inflammatory phenotypes Spearman's rank correlations were performed to investigate the relationship between total monocyte Flt-1 and endoglin expression and M1/M2 polarization of monocytes (Table 4). A strongly positive correlation was noted between Flt-1 and CD86 MFI (correlation coefficient 0.643, p = 0.001). The correlation between Flt-1 and CD163 MFI (correlation coefficient 0.274, p = 0.045) was low but statistically significant. Positive correlations were noted between Flt-1 and endoglin (correlation coefficient 0.547, p = 0.001), Flt-1 and CD86/CD163 expression (correlation coefficient 0.412, p = 0.002), and between endoglin and CD86/CD163 expression (correlation coefficient 0.45, p = 0.001) (Fig. 4A and B).Table 4 Spearman rank correlations for relationships between total monocyte Flt-1 MFI, Endoglin MFI, CD86 MFI, CD163 MFI and CD86/CD163 MFI ratio. *Correlation is significant at p < 0.05 level (2-tailed).

= 0.002), and between endoglin and CD86/CD163 expression (correlation coefficient 0.45, p = 0.001) (Fig. 4A and B).Table 4 Spearman rank correlations for relationships between total monocyte Flt-1 MFI, Endoglin MFI, CD86 MFI, CD163 MFI and CD86/CD163 MFI ratio. *Correlation is significant at p < 0.05 level (2-tailed). Table 4Variable 1 Variable 2 Correlation coefficient Significance (2 tailed) Interpretation Total monocyte Flt-1 MFI Total monocyte Endoglin MFI 0.547 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86 MFI 0.643 p < 0.001* Strong positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD163 MFI 0.274 P = 0.045* Low positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86/CD163 MFI ratio 0.412 P = 0.002* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD86 MFI 0.571 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD163 MFI 0.145 P = 0.297 No correlation Total monocyte Endoglin MFI Total monocyte CD86/CD163 MFI ratio 0.45 P = 0.001* Moderate positive correlation Statistically significant Fig. 4 Spearman's rank correlations for relationships between total monocyte Flt-1 MFI (A), Endoglin MFI (B) and markers of monocyte polarisation, CD86/CD163 MFI. r = Spearman's rank correlation. Correlation is significant at p < 0.05 level (2-tailed). Fig. 4

Table 4Variable 1 Variable 2 Correlation coefficient Significance (2 tailed) Interpretation Total monocyte Flt-1 MFI Total monocyte Endoglin MFI 0.547 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86 MFI 0.643 p < 0.001* Strong positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD163 MFI 0.274 P = 0.045* Low positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86/CD163 MFI ratio 0.412 P = 0.002* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD86 MFI 0.571 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD163 MFI 0.145 P = 0.297 No correlation Total monocyte Endoglin MFI Total monocyte CD86/CD163 MFI ratio 0.45 P = 0.001* Moderate positive correlation Statistically significant Fig. 4 Spearman's rank correlations for relationships between total monocyte Flt-1 MFI (A), Endoglin MFI (B) and markers of monocyte polarisation, CD86/CD163 MFI. r = Spearman's rank correlation. Correlation is significant at p < 0.05 level (2-tailed). Fig. 4 Discussion The current study has contributed to the literature by documentation of Flt-1 and endoglin expression by maternal and fetal monocytes in normal and complicated pregnancies as well as exploration of these markers by monocyte subtype and polarization.

Table 4Variable 1 Variable 2 Correlation coefficient Significance (2 tailed) Interpretation Total monocyte Flt-1 MFI Total monocyte Endoglin MFI 0.547 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86 MFI 0.643 p < 0.001* Strong positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD163 MFI 0.274 P = 0.045* Low positive correlation Statistically significant Total monocyte Flt-1 MFI Total monocyte CD86/CD163 MFI ratio 0.412 P = 0.002* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD86 MFI 0.571 p < 0.001* Moderate positive correlation Statistically significant Total monocyte Endoglin MFI Total monocyte CD163 MFI 0.145 P = 0.297 No correlation Total monocyte Endoglin MFI Total monocyte CD86/CD163 MFI ratio 0.45 P = 0.001* Moderate positive correlation Statistically significant Fig. 4 Spearman's rank correlations for relationships between total monocyte Flt-1 MFI (A), Endoglin MFI (B) and markers of monocyte polarisation, CD86/CD163 MFI. r = Spearman's rank correlation. Correlation is significant at p < 0.05 level (2-tailed). Fig. 4 Discussion The current study has contributed to the literature by documentation of Flt-1 and endoglin expression by maternal and fetal monocytes in normal and complicated pregnancies as well as exploration of these markers by monocyte subtype and polarization. While it is established that soluble Flt-1 and soluble endoglin play a significant role in the pathogenesis of preeclampsia [7,24,38], this is the first study to document maternal and fetal monocyte expression of Flt-1 and endoglin in preeclampsia and fetal growth restriction. This study has clearly shown that circulating monocytes from maternal circulation express Flt-1 and endoglin as surface markers and that gestational variations in the monocyte Flt-1 and endoglin expression are evident in normal third trimester of pregnancy. While the percentage of monocytes expressing Flt-1 did not show any difference with gestation, the demonstrated gestational increase in intensity of Flt-1 and endoglin expression (MFI) may reflect the increased inflammatory nature of late gestation [39].

in expression are evident in normal third trimester of pregnancy. While the percentage of monocytes expressing Flt-1 did not show any difference with gestation, the demonstrated gestational increase in intensity of Flt-1 and endoglin expression (MFI) may reflect the increased inflammatory nature of late gestation [39]. Lack of variation in maternal monocyte surface expression of Flt-1 between clinical groups suggests that Flt-1 is unlikely to be a significant contributor to the circulatory anti-angiogenic factors in preeclampsia and fetal growth restriction. The monocyte Flt-1 has been described to have a significant role in the chemotaxis of peripheral monocytes and tissue macrophages [10]. It is possible that surface expression of Flt-1 on monocytes in preeclampsia, particularly by non-classical monocytes, may have a functional role in chemotaxis and autocrine expression.

he monocyte Flt-1 has been described to have a significant role in the chemotaxis of peripheral monocytes and tissue macrophages [10]. It is possible that surface expression of Flt-1 on monocytes in preeclampsia, particularly by non-classical monocytes, may have a functional role in chemotaxis and autocrine expression. This study further explored the circulating monocyte Flt-1 and endoglin expression by subset and clinical groups. Variable expression of Flt-1 and endoglin was noted with the different monocyte subset populations, suggesting different functional roles for the different monocyte subsets in the angiogenic contribution. Flt-1 was mainly expressed on classical and intermediate cells. This finding is consistent with previous literature suggesting that monocyte chemotaxis towards the angiogenic ligands VEGF and PlGF is reduced in CD16+ intermediate and non-classical monocytes compared to CD16- classical monocytes [28]. The differences in chemotactic function may relate to their lower level of Flt-1 protein expression. Such differences may predict different functional roles of monocyte subsets in vascular repair, arteriogenesis and atherogenesis [28]. The membrane bound and circulating forms of Flt-1 and endoglin have different functions, with pro-angiogenic activity of the membrane bound forms and anti-angiogenic activity of the soluble forms. They may be controlled by different regulatory mechanisms and may reflect opposing regulatory mechanisms of the angiogenesis milieu.

ane bound and circulating forms of Flt-1 and endoglin have different functions, with pro-angiogenic activity of the membrane bound forms and anti-angiogenic activity of the soluble forms. They may be controlled by different regulatory mechanisms and may reflect opposing regulatory mechanisms of the angiogenesis milieu. The association of monocyte polarization into M1/M2 phenotypes in vivo and correlation with angiogenic factors Flt-1 and endoglin expression as presented has not been previously described in the literature. This study has demonstrated a moderate association between M1 polarization and surface expression of endoglin and Flt-1 on monocytes. While Flt-1 weakly correlated with CD163, the findings are consistent with Flt-1 related M1 upregulation of macrophages demonstrated in preeclamptic decidua [40] and may reflect monocyte activity such as chemotaxis. The inflammatory intermediate monocyte subset has been shown to be increased in preterm preeclampsia and fetal growth restriction as well as term pregnancy [34]. The current study findings of a gestational increase in Flt-1 and endoglin as well as with M1 polarization may represent an increase in the Flt-1 and endoglin expression by more inflammatory monocytes. With Flt-1 associated with monocyte recruitment, then this may lead to an increased influx of inflammatory monocytes into the placenta.

findings of a gestational increase in Flt-1 and endoglin as well as with M1 polarization may represent an increase in the Flt-1 and endoglin expression by more inflammatory monocytes. With Flt-1 associated with monocyte recruitment, then this may lead to an increased influx of inflammatory monocytes into the placenta. Interestingly, M1 polarization has also been shown to be atherogenic [41]. The long term increased vascular risk associated with preeclampsia is now well established [42]. The role of endoglin in cardiovascular homeostasis has been established. Increased expression of endoglin is associated with angiogenesis and myocardial fibrosis after infarction leading to increased risk of heart failure [18]. Soluble endoglin post myocardial infarction is an indicator of poor prognosis [43]. It is interesting to speculate, and investigate in the future, whether the M1 monocyte /macrophage related plaque formation and endoglin plays a role in adult as well as placental vascular disease. Up-regulation of endoglin in M1 monocytes as well as by the trophoblast may play a role in the pathogenesis of increased long-term cardiovascular risk associated with preeclampsia. The findings of this study are exploratory. The data and the interpretations are limited by small patient numbers and the cross-sectional design of the study. Further study on monocyte expression of Flt-1 and endoglin and correlation with soluble forms may help to shed further light on the monocyte contribution to the pathogenesis of preeclampsia and fetal growth restriction.

he interpretations are limited by small patient numbers and the cross-sectional design of the study. Further study on monocyte expression of Flt-1 and endoglin and correlation with soluble forms may help to shed further light on the monocyte contribution to the pathogenesis of preeclampsia and fetal growth restriction. We conclude that circulating monocytes from maternal and fetal circulation express Flt-1 and endoglin as surface markers and that their expression increase with gestational age and M1 polarization suggesting their upregulation with inflammatory changes in monocytes. Increased fetal monocyte expression of endoglin expression may be a response to placental injury. Conflict of interest The authors declare no competing interests. Acknowledgments This work was supported by Ella Macnight Research Scholarship, Royal Australian and New Zealand College of Obstetrics and Gynecology (RANZCOG) Research Foundation. We acknowledge the contribution by Karen Byth, senior hospital statistician to the statistical analysis.

Introduction Cesarean delivery (CD) is an obstetrical surgical procedure that is used to save lives of mothers and fetuses [1]. During the last decades, a global dramatic increase in the incidence of both primary and repeated CDs was recorded [2]. Nevertheless, the impact of this rise on maternal health or on neonatal morbidity and mortality still needs to be proved [3].

surgical procedure that is used to save lives of mothers and fetuses [1]. During the last decades, a global dramatic increase in the incidence of both primary and repeated CDs was recorded [2]. Nevertheless, the impact of this rise on maternal health or on neonatal morbidity and mortality still needs to be proved [3]. Twin pregnancies account for 2–3% of all births and the rate of CDs for twin pregnancy increased as well in the United States, from 53.4% to 75.3% during 1995 and 2009, respectively. This increase was seen for both vertex and breech presenting twins [[4], [5], [6], [7]]. Several factors contributed to the increase in CD rate among twin pregnancies: advanced maternal age seen in these women, the high rate of accompanied comorbidities, increase in proportion of women who conceived as a result of assisted reproduction techniques, and women’s demand. Each factor represents an independent reason for the CD rate increase in twin pregnancies [8,9]. Additionally, the optimal mode of delivery is a point of debate, particularly in regard to its effect on neonatal outcomes. Large population-based retrospective cohort studies suggested that planned CD may improve perinatal outcome of the second twin [10,11]. Nevertheless, prospective studies suggest otherwise [[12], [13], [14]]. Although most health care providers support planned vaginal delivery in cases of vertex–vertex twins, fewer are comfortable with breech vaginal delivery of the second twin [4]. Despite this rise in CD rate in twin pregnancies, the impact on maternal health or on neonatal outcome still needs to be proved. The objective of the current study was to determine the trend of overall CD rate among twin pregnancies at a single university teaching hospital during the last two decades, and to explore the parallel trend in maternal and early neonatal morbidities during this period.

or on neonatal outcome still needs to be proved. The objective of the current study was to determine the trend of overall CD rate among twin pregnancies at a single university teaching hospital during the last two decades, and to explore the parallel trend in maternal and early neonatal morbidities during this period. Materials and methods A population-based cohort study was conducted at a single university medical center in Israel using data from January 1995 to December 2015. The study was approved in June, 2016 (IRB no: 0073-16-EMC) by the local institutional review board, Emek Medical Center, Afula, Israel. For this type of study signed consent was not required. All pregnant women with twin gestation who delivered at a gestational age beyond 24 weeks were included. Mono-amniotic twins and twins with major fetal malformations or death of one or both twins were excluded. Women were detected through International Classification of Diseases-9 codes, electronic labor charts and medical records at admission and discharge. All medical files that were detected were then checked manually for confirmation.

ns and twins with major fetal malformations or death of one or both twins were excluded. Women were detected through International Classification of Diseases-9 codes, electronic labor charts and medical records at admission and discharge. All medical files that were detected were then checked manually for confirmation. The policy of twin delivery in our institution, throughout the study period, was to support a woman’s choice for a trial of vaginal delivery in cases of cephalic presenting first twin. CD was recommended for non-cephalic first twins and for other obstetrical reasons as in the case of placenta previa. Trial of labor after one low transverse CD was not an indication for repeat CD. When induction of labor was required it was accomplished by a balloon catheter for cervical ripening and amniotomy with or without intravenous oxytocin infusion. Continuous fetal heart rate monitoring was employed in all twin deliveries. Labor was supervised by midwives and a senior obstetrician. Women were encouraged to receive epidural analgesia throughout labor, but it was not mandatory and was performed upon request. In cases of second twin presented in vertex or breech presentation, expectant management was employed after delivery of the first twin, as long as the fetal heart rate monitoring was reassuring. Interventions such as oxytocin infusion and amniotomy were employed as a decision of the senior physician during labor. In these cases, spontaneous vaginal delivery was intended given that maternal pushing efforts were preserved and continuous fetal descent was noticed. Vacuum extraction was the only instrumental delivery used and was employed in cases of prolonged second stage, maternal exhaustion, or a non-reassuring fetal monitoring. Internal podalic version followed by breech extraction or cesarean delivery were performed if the obstetrician presumed that the fetal status required an immediate delivery and the fetal head station did not permit a safe vacuum delivery. Assisted breech delivery was the preferred method for delivering the breech second twin. Total breech extraction was done in cases of non-reassuring fetal status or in incomplete breech presentation. For a transverse or oblique lie of the second fetus with reassuring fetal tracing, external cephalic version or internal podalic versions were attempted, according to the obstetrician’s judgment. Delivery of a second twin in a non-cephalic presentation was carried out in the operating theatre.

lete breech presentation. For a transverse or oblique lie of the second fetus with reassuring fetal tracing, external cephalic version or internal podalic versions were attempted, according to the obstetrician’s judgment. Delivery of a second twin in a non-cephalic presentation was carried out in the operating theatre. Data collected and examined for each year included demographic and obstetric parameters, mode of deliveries, Apgar score < 7 at 5 min and cord artery pH < 7.1 of each twin, neonatal death not attributed to major malformations, early postpartum hemorrhage (PPH), blood transfusion, and intrapartum fever, were extracted, plotted, and trends analyzed. Besides hypertension and diabetes, other maternal diseases that included thrombophilia, thyroid disorders, asthma, cardiac disease, and epilepsy were also recorded. The primary outcome was delivery mode, which was categorized as either vaginal or cesarean. The main predictor variable of interest was birth year. CD was categorized as laboring, i.e., performed after a trial of labor, for one or both twins, and non-laboring, i.e., performed without a trial of labor. Women admitted in labor but who were delivered by an indicated cesarean, as in the case of non-vertex first twin, were categorized as having a non-laboring cesarean.

CD was categorized as laboring, i.e., performed after a trial of labor, for one or both twins, and non-laboring, i.e., performed without a trial of labor. Women admitted in labor but who were delivered by an indicated cesarean, as in the case of non-vertex first twin, were categorized as having a non-laboring cesarean. Statistical analysis The Cochran-Armitage Trend Test was used to identify trends in the categorical data and linear regression was used to test linear trend in the continuous variables of maternal age and BMI. Logistic regression was used to adjust the incidence of cesarean sections and significant outcome variables (such as blood transfusion) for significant demographic and obstetric variables. Significance was considered when p-value < 0.05. The statistical analysis was performed using XLSTAT (MS Excel, Addinsoft) and SPSS version 21 (IBM Corp, 2012).

to adjust the incidence of cesarean sections and significant outcome variables (such as blood transfusion) for significant demographic and obstetric variables. Significance was considered when p-value < 0.05. The statistical analysis was performed using XLSTAT (MS Excel, Addinsoft) and SPSS version 21 (IBM Corp, 2012). Results Of all 88,145 deliveries that took place during this period, 1955 (2.2%) were twin gestations. Of these, 53 were ineligible and were excluded from the final analysis. Demographic and obstetric characteristics are presented in Table 1. During the study period there was a statistically significant trend (increase) in twin deliveries over time (χ2 = 8.45, p <  0.004) from 1.9% in 1996 to a maximum of 2.6% in 2013 and 2014. Linear regression analysis revealed a statistically significant increase in maternal age over time (b=0.085, se=0.021, p =  0.001) from a minimum average of 29.1 years in 2001 to a maximum average of 32.2 years in 2015. There was no linear trend for body mass index (b=-0.0308, se=0.023, p = 0.10).Table 1 Demographic and Obstetric Characteristics.

evealed a statistically significant increase in maternal age over time (b=0.085, se=0.021, p =  0.001) from a minimum average of 29.1 years in 2001 to a maximum average of 32.2 years in 2015. There was no linear trend for body mass index (b=-0.0308, se=0.023, p = 0.10).Table 1 Demographic and Obstetric Characteristics. Table 1Year All deliveries Twin deliveries Maternal age Body mass index, kg/m2 Dichorionic diamniotic Fertility treatment Any Diabetes Any Hypertension Other maternal diseases* Induction of labor Vertex first twin First vertex, second non-vertex 1995 3854 82 (2.13) 30.02 23.78 68 (89.5) 15 (18.3) 1 (1.2) 6 (7.3) 0 (0.0) 1 (1.2) 59 (72.0) 29 (50.0) 1996 3861 74 (1.92) 30.34 25.22 59 (90.0) 23 (31.1) 3 (4.1) 9 (12.2) 2 (2.7) 5 (6.8) 53 (71.6) 28 (53.8) 1997 4023 90 (2.24) 29.89 26.49 66 (81.5) 46 (51.1) 5 (5.6) 15 (16.7) 2 (2.2) 1 (1.1) 60 (66.7) 27 (45.0) 1998 4131 92 (2.23) 29.83 24.72 64 (85.3) 38 (41.3) 11(12.0) 7 (7.6) 0 (0.0) 3(3.3) 68 (73.9) 24 (35.3) 1999 4246 85 (2.00) 30.95 24.64 62 (92.5) 32 (37.6) 5 (5.9) 19 (22.4) 2 (2.4) 0 (0.0) 60 (70.6) 20 (33.3) 2000 4301 78 (1.81) 31.13 27.13 66 (90.4) 35 (44.9) 7 (9.0) 12 (15.4) 2 (2.6) 2 (2.6) 54 (69.6) 28 (51.9) 2001 4418 102 (2.31) 29.11 25.58 71(75.5) 40 (39.2) 5 (4.9) 12 (11.8) 0 (0.0) 6(5.9) 73 (72.6) 37 (50.7) 2002 4477 92 (2.05) 29.76 25.84 74 (87.1) 36 (39.1) 7 (7.6) 7 (7.6) 2 (2.2) 6 (6.5) 64 (69.2) 27 (42.2) 2003 4457 73 (1.64) 29.58 24.96 55 (80.9) 29 (39.7) 2 (2.7) 8 (11.0) 5 (6.8) 1 (1.4) 53 (62.4) 23 (43.4) 2004 4273 78 (1.83) 31.41 25.20 67 (87.0) 34 (43.6) 4 (5.1) 6 (7.7) 1 (1.3) 2 (2.6) 54 (77.1) 21 (38.9) 2005 4232 93 (2.20) 29.58 24.39 78 (86.7) 36 (38.7) 4 (4.3) 16 (17.2) 0 (0.0) 2 (2.2) 58 (70.7) 29 (50.0) 2006 4215 96 (2.28) 30.74 24.92 78 (84.0) 41 (42.7) 10(10.4) 18 (18.8) 3 (3.1) 7 (7.3) 74 (73.0) 36 (49.3) 2007 4135 82 (1.98) 30.17 25.54 66 (81.5) 29 (35.4) 8 (9.8) 9 (11.0) 2 (2.4) 0 (0.0) 58 (71.4) 18 (31.0) 2008 4222 74 (1.75) 31.99 25.33 68 (93.2) 37 (50.0) 6 (8.1) 12 (16.2) 2 (2.7) 1 (1.4) 54 (73.0) 17 (31.5) 2009 4171 91 (2.18) 30.51 24.66 70 (78.7) 34(37.4) 2 (2.2) 14(15.4) 2 (2.2) 9 (9.9) 65 (71.4) 24 (36.9) 2010 4285 97 (2.26) 30.86 25.31 84 (86.6) 44 (45.4) 6 (6.2) 16 (16.5) 4 (4.1) 4 (4.1) 61 (62.9) 26 (42.6) 2011 4289 112 (2.61) 31.07 25.08 92 (82.9) 46 (41.1) 11 (9.8) 16 (14.3) 7 (6.3) 2 (1.8) 84 (75.0) 32 (38.1) 2012 4228 99 (2.34) 31.17 25.14 91 (91.9) 49 (49.5) 5 (5.1) 13 (13.1) 6 (6.1) 4 (4.0) 58 (58.6) 19 (32.8) 2013 4057 107 (2.64) 30.87 23.77 90 (85.7) 55 (51.4) 6 (5

2) 16 (16.5) 4 (4.1) 4 (4.1) 61 (62.9) 26 (42.6) 2011 4289 112 (2.61) 31.07 25.08 92 (82.9) 46 (41.1) 11 (9.8) 16 (14.3) 7 (6.3) 2 (1.8) 84 (75.0) 32 (38.1) 2012 4228 99 (2.34) 31.17 25.14 91 (91.9) 49 (49.5) 5 (5.1) 13 (13.1) 6 (6.1) 4 (4.0) 58 (58.6) 19 (32.8) 2013 4057 107 (2.64) 30.87 23.77 90 (85.7) 55 (51.4) 6 (5 .6) 10 (9.3) 7 (6.5) 11 (10.3) 77 (72.0) 26 (33.8) 2014 4103 108 (2.63) 31.60 24.48 98 (90.7) 61 (56.5) 6 (5.6) 14 (13.0) 3 (2.8) 15 (13.9) 73 (67.6) 33 (45.2) 2015 4167 97 (2.33) 32.18 25.15 82 (86.3) 38 (39.2) 6 (6.2) 10 (10.3) 1 (1.0) 14 (14.4) 71 (73.2) 34 (47.9) χ2 -- 8.45 -- -- 0.11 13.60 0.14 0.04 7.69 24.14 0.35 2.86 p -- <0.01 <0.01 0.10 0.74 0.03 0.71 0.86 <0.01 <0.01 0.56 0.10 Data are mean or N (%). * Include Thrombophilia, Thyroid disorders, Asthma, Cardiac disease, and Epilepsy. There was a significant trend for increased Jewish ethnicity over time (χ2 = 8.45, p =  0.01). In addition, there was a significant increasing trend for use of fertility treatments (χ2 = 13.60, p =  0.03) from 18.3% in 1995 to 56.5% in 2015, an increase in the use of labor induction (χ2 = 24.14, p =  0.001) from a minimum of 1.2% in 1995 to a maximum of 14.4% in 2015. While there was no change in incidences of diabetes or hypertension over time there was an increase in other maternal diseases (χ2 = 7.69, p =  0.006). The presentations of the first and second twins at the start of delivery did not vary over time (Table 1).

There was a significant trend for increased Jewish ethnicity over time (χ2 = 8.45, p =  0.01). In addition, there was a significant increasing trend for use of fertility treatments (χ2 = 13.60, p =  0.03) from 18.3% in 1995 to 56.5% in 2015, an increase in the use of labor induction (χ2 = 24.14, p =  0.001) from a minimum of 1.2% in 1995 to a maximum of 14.4% in 2015. While there was no change in incidences of diabetes or hypertension over time there was an increase in other maternal diseases (χ2 = 7.69, p =  0.006). The presentations of the first and second twins at the start of delivery did not vary over time (Table 1). As can be seen in Table 2, CD rate increased significantly from 43.4% in 1995 to 66.0% in 2015 (χ2 = 31.32, p =  0.001). This increase was observed only among non-laboring cesareans (χ2 = 63.92, p =  0.001) (Fig. 1). There was a significant decrease in the incidence of laboring cesareans (χ2 = 24.06, p =  0.001). During this period significant decreases in the incidences of neonatal death (χ2 = 13.05, p =  0.001) and blood transfusions (χ2 = 7.38, p =  0.007), and an increase in the incidence of cord artery pH < 7.1 (χ2 = 4.11, p =  0.04) of any twin were observed. There was no significant change in the rates of Apgar score < 7 at 5 min and/or early postpartum hemorrhage or in intrapartum fever. There was no linear trend for birth weight (twin 1: b=1.742, se=2.161, p =  0.42; twin 2: b=2.384, se=2.182, p =  0.28).Table 2 Annual twin births, cesarean deliveries, and unadjusted neonatal outcomes.

gnificant change in the rates of Apgar score < 7 at 5 min and/or early postpartum hemorrhage or in intrapartum fever. There was no linear trend for birth weight (twin 1: b=1.742, se=2.161, p =  0.42; twin 2: b=2.384, se=2.182, p =  0.28).Table 2 Annual twin births, cesarean deliveries, and unadjusted neonatal outcomes. Table 2Year All Cesareans Non-laboring Cesarean Laboring Cesarean Male first twin Male second twin First twin weight, gr Second twin weight, gr pH <7.1 Apgar <7 at 5 minutes Neonatal death 1995 36 (43.4) 26 (31.7) 10 (12.2) 42 (51.2) 39 (47.6) 2348 ± 618 2297 ± 614 2 (2.44) 2 (2.44) 2 (2.44) 1996 37 (50.0) 26 (35.1) 11 (14.9) 40 (54.1) 32 (43.2) 2272 ± 631 2232 ± 580 2 (2.70) 3 (4.05) 3 (4.05) 1997 55 (61.1) 40 (44.4) 15 (16.7) 47 (52.2) 50 (55.6) 2415 ± 607 2332 ± 627 1 (1.11) 0 (0.00) 0 (0.00) 1998 48 (51.6) 37 (40.0) 11 (12.0) 46 (50.0) 42 (45.7) 2335 ± 561 2223 ± 552 3 (3.26) 1 (1.09) 2 (2.17) 1999 41 (47.7) 28 (32.9) 13 (15.3) 41 (48.2) 44 (51.8) 2469 ± 495 2462 ± 497 5 (5.88) 1 (1.18) 0 (0.00) 2000 50 (63.3) 41 (52.6) 9 (11.5) 38 (48.7) 41 (52.6) 2392 ± 518 2362 ± 497 2 (2.56) 0 (0.00) 0 (0.00) 2001 68 (66.0) 59 (57.8) 9 (8.8) 46 (45.1) 47 (46.1) 2241 ± 618 2232 ± 629 3 (2.94) 4 (3.92) 0 (0.00) 2002 56 (60.2) 48 (52.2) 8 (8.7) 49 (52.7) 42 (45.7) 2326 ± 598 2238 ± 578 2 (2.17) 2 (2.17) 2 (2.17) 2003 43 (57.3) 38 (52.1) 5 (6.8) 44 (53.1) 39 (53.4) 2294 ± 630 2355 ± 611 1 (1.37) 1 (1.37) 1 (1.37) 2004 57 (73.1) 50 (64.1) 7 (9.0) 39 (50.0) 40 (51.3) 2415 ± 760 2305 ± 654 1 (1.28) 1 (1.28) 2 (2.56) 2005 70 (74.5) 65 (69.9) 5 (5.4) 49 (52.7) 53 (57.0) 2392 ± 517 2323 ± 505 0 (0.00) 0 (0.00) 0 (0.00) 2006 71 (73.2) 59 (61.5) 12 (12.5) 51 (53.1) 47 (49.0) 2357 ± 590 2294 ± 540 0 (0.00) 0 (0.00) 0 (0.00) 2007 58 (70.7) 52 (63.4) 6 (7.3) 39 (47.6) 36 (43.9) 2287 ± 532 2256 ± 555 0 (0.00) 1 (1.22) 0 (0.00) 2008 51 (68.9) 45 (60.8) 6 (8.1) 42 (56.8) 39 (52.7) 2301 ± 544 2300 ± 560 1 (1.35) 1 (1.35) 0 (0.00) 2009 66 (71.7) 58 (63.7) 8 (8.8) 48 (52.7) 46 (50.0) 2389 ± 541 2325 ± 600 1 (1.10) 1 (1.10) 0 (0.00) 2010 70 (72.2) 67 (69.1) 3 (3.1) 47 (48.5) 49 (50.0) 2368 ± 560 2304 ± 545 2 (2.06) 0 (0.00) 0 (0.00) 2011 84 (75.0) 77 (68.8) 7 (6.3) 51 (45.5) 53 (47.3) 2360 ± 498 2279 ± 535 3 (2.68) 0 (0.00) 0 (0.00) 2012 70 (70.7) 69 (69.7) 1 (1.0) 46 (46.5) 49 (49.5) 2337 ± 541 2324 ± 588 5 (5.05) 1 (1.01) 0 (0.00) 2013 67 (62.6) 60 (56.1) 7 (6.5) 47 (43.9) 52 (48.6) 2335 ± 638 2297 ± 645 3 (2.80) 1 (0.93) 0 (0.00) 2014 73 (67.6) 70(64.8) 3 (2.8) 60 (55.6) 58 (53.7) 2404 ± 6

(47.3) 2360 ± 498 2279 ± 535 3 (2.68) 0 (0.00) 0 (0.00) 2012 70 (70.7) 69 (69.7) 1 (1.0) 46 (46.5) 49 (49.5) 2337 ± 541 2324 ± 588 5 (5.05) 1 (1.01) 0 (0.00) 2013 67 (62.6) 60 (56.1) 7 (6.5) 47 (43.9) 52 (48.6) 2335 ± 638 2297 ± 645 3 (2.80) 1 (0.93) 0 (0.00) 2014 73 (67.6) 70(64.8) 3 (2.8) 60 (55.6) 58 (53.7) 2404 ± 6 03 2369 ± 658 5 (4.63) 5 (4.63) 0 (0.00) 2015 64 (66.0) 56 (57.7) 8 (8.2) 49 (50.0) 58 (59.8) 2451 ± 470 2406 ± 529 10 (10.31) 1 (1.03) 0 (0.00) χ2 31.32 63.92 24.06 0.35 1.28 -- -- 4.11 0.32 13.05 p <0.01 <0.01 <0.01 0.57 0.26 0.42 0.28 0.04* 0.57 <0.01** Data are mean ± standard deviation or N (%). * p = 0.07 after adjustment for maternal age, ethnicity, maternal diseases, use of fertility treatment, and labor induction. ** Adjustment could not be performed due to the small number of cases. Fig. 1 Trends in cesarean delivery rate among twin pregnancies (1995–2015). Fig. 1 Multivariate logistic regression analysis was used to estimate the odds for CD during the study period, adjusting for maternal age, ethnicity, maternal diseases, use of fertility treatments, and labor induction. For every additional year from 1995 to 2015, the odds of CD rate increased by 5.1% (OR: 1.051, 95% CI: 1.034–1.069). Among preterm births (<37 weeks) the odds of CD rate increased by 4.1% (OR: 1.044, 95% CI: 1.020–1.068) and among term births (>37 weeks) the odds of CD rate increased by 6.2% for every additional year from 1995 to 2015 (OR: 1.062, 95% CI: 1.037–1.088).

dds of CD rate increased by 5.1% (OR: 1.051, 95% CI: 1.034–1.069). Among preterm births (<37 weeks) the odds of CD rate increased by 4.1% (OR: 1.044, 95% CI: 1.020–1.068) and among term births (>37 weeks) the odds of CD rate increased by 6.2% for every additional year from 1995 to 2015 (OR: 1.062, 95% CI: 1.037–1.088). Repetition of the analysis for non-laboring and laboring CDs revealed that for every additional year from 1995 to 2015, a mean increase of 7.8% in the incidence of non-laboring CDs was observed (OR: 1.078, 95% CI: 1.061–1.096). The increase was statistically significant for both term (OR: 1.094, 95% CI: 1.068–1.121) and preterm (OR: 1.064, 95% CI: 1.040–1.088) births. On the other hand, the incidence of laboring CDs declined during this period by 7.5% (OR: 0.925, 95% CI: 0.899-0.951). This decline was evident for both term (OR: 0.901, 95% CI: 0.862-0.942) and preterm (OR: 0.944, 95% CI: 0.910-0.980) births.

4, 95% CI: 1.068–1.121) and preterm (OR: 1.064, 95% CI: 1.040–1.088) births. On the other hand, the incidence of laboring CDs declined during this period by 7.5% (OR: 0.925, 95% CI: 0.899-0.951). This decline was evident for both term (OR: 0.901, 95% CI: 0.862-0.942) and preterm (OR: 0.944, 95% CI: 0.910-0.980) births. Multivariate logistic regression analysis was then used to estimate the odds for blood transfusions and pH less < 7.1 during the study period adjusting for maternal age, ethnicity, maternal diseases, use of fertility treatment, and labor induction. After adjustment there was no difference in the incidences of blood transfusions (OR: 0.965, 95% CI: 0.909–1.024, p = 0.16) or pH < 7.1 (OR: 1.045, 95% CI: 0.997–1.095, p = 0.07) over time. During the study period there were 12 cases of neonatal death, of them 10 were born < 28 weeks, one at 30.5 weeks and one at term (37.5 weeks). Due to the small number of cases (n=12), the multivariate model could not be adjusted for induction or other maternal diseases.

1.045, 95% CI: 0.997–1.095, p = 0.07) over time. During the study period there were 12 cases of neonatal death, of them 10 were born < 28 weeks, one at 30.5 weeks and one at term (37.5 weeks). Due to the small number of cases (n=12), the multivariate model could not be adjusted for induction or other maternal diseases. Comment In the current study we observed a statistically significant increase in the rate of CD for twin pregnancies from 43.4% in 1995 to 66.0% in 2015. The increase was simply due to increase in the rate of non-laboring CD while the rate of laboring CD decreased. Overall, for every additional year from 1995 to 2015, a mean increase of 5.1% in the incidence of CD was noticed, similar to the annual increase in the United States reported by Lee et al., [4]. The trend in maternal morbidity and short-term neonatal outcomes in terms of Apgar score and cord pH did not change over the study period despite the increase in non-laboring CD. The increase in preterm CD rate was associated with decrease in the incidence of neonatal deaths among preterm deliveries. Nevertheless, the number of cases was small and adjustment could not be performed. For that reason, the results regarding the effect of the increase in CD rate on neonatal deaths should be interpreted with caution.

reterm CD rate was associated with decrease in the incidence of neonatal deaths among preterm deliveries. Nevertheless, the number of cases was small and adjustment could not be performed. For that reason, the results regarding the effect of the increase in CD rate on neonatal deaths should be interpreted with caution. Several factors may have influenced the change in CD rate during the study period. The increase in maternal age and rate of twin pregnancies conceived following fertility treatments found during the study period may explain this increase to some extent. Twin pregnancies conceived following fertility treatment, particularly among women of advanced age, are at higher risk of obstetric complications and adverse neonatal outcome in comparison with naturally conceived twin gestations [[15], [16], [17]]. Both advanced maternal age and fertility treatments in general are predictable reasons for increase in planned CD but not in laboring CD [17]. Other main factors may have contributed to the increase in CD rate in twins but were not examined in the current study. These are the increase in the less-skilled providers that manage vaginal delivery of twin pregnancies and the prejudice of the involved infertility providers that may affect women’s decisions regarding mode of delivery [18,19]. Additionally, the change that occurred in medico-legal milieu during the last two decades is probably a concern [[20], [21], [22]]. Both factors are possibly related as well.

of twin pregnancies and the prejudice of the involved infertility providers that may affect women’s decisions regarding mode of delivery [18,19]. Additionally, the change that occurred in medico-legal milieu during the last two decades is probably a concern [[20], [21], [22]]. Both factors are possibly related as well. As compared to the study of Lee et al., [4] who examined trend in CD rate in the United States, we were able to determine presentations at delivery. Presentation combinations of both fetuses during the study period did not change significantly. Additionally, it is not possible in the current study (strength of single institution study) that some of the increase in twin CD rate was attributed to change in policy regarding managing non-vertex first twin since during the whole study period same policy was considered, i.e., delivering by CD. For these reasons the increase in CD rate was attributed only to change in mode of delivery of vertex-first twin pregnancies.

f the increase in twin CD rate was attributed to change in policy regarding managing non-vertex first twin since during the whole study period same policy was considered, i.e., delivering by CD. For these reasons the increase in CD rate was attributed only to change in mode of delivery of vertex-first twin pregnancies. In the current study, we observed a trend of increase in induction of labor during the study period similar to the trend found by Lee et al., [4] In contrast to singleton gestations where recent randomized trials showed that induction of labor was not associated with increase in CD rate [23,24], data regarding induction in twin gestation is limited. Previous studies reported an increase in CD rate with induction of labor in twin gestation [4,25]. Nevertheless this change does not explain the increase noticed in the last decades since induced women are designated to have a planned vaginal delivery while the increase was found only in non-laboring CDs.

n is limited. Previous studies reported an increase in CD rate with induction of labor in twin gestation [4,25]. Nevertheless this change does not explain the increase noticed in the last decades since induced women are designated to have a planned vaginal delivery while the increase was found only in non-laboring CDs. CD is an invasive intervention used to avoid or treat life-threatening maternal or fetal complications [26]. Nevertheless, CD may increase both short-term and long-term maternal morbidity, and for that reason justifications for the increased use in terms of potential maternal and perinatal benefit are warranted [26]. In this study we were unable to demonstrate significant change in short neonatal outcomes, similar to the results of Schmitz et al., [27]. Additionally, it has been suggested that planned CD could potentially reduce maternal morbidity by decreasing the need for unplanned cesarean deliveries [27]. Nevertheless, maternal morbidities including fever and bleeding were also not affected during the study period.

, similar to the results of Schmitz et al., [27]. Additionally, it has been suggested that planned CD could potentially reduce maternal morbidity by decreasing the need for unplanned cesarean deliveries [27]. Nevertheless, maternal morbidities including fever and bleeding were also not affected during the study period. The current study has limitations including a possibility of bias due to the use of a retrospective database. Furthermore, the study lacks information regarding long-term maternal and neonatal outcomes that were not explored in this study and for that reason the impact of the increase in CD rate cannot be explored entirely. In addition, the fact that the study was conducted at a single hospital may limit its generalizability. Nevertheless, a distinctive advantage related to a single center study is the use of similar protocol in managing twin pregnancies. Differences, even minor, in intrapartum management that may occasionally be found between institutions may affect results of multicenter studies. In conclusion, the results of the current study combined with results of other retrospective and randomized trials [4,13,28] are unable to justify the increase in CD rate among twin pregnancies. Training young physicians in delivering twin pregnancies before total "disappearance" of the skilled obstetricians is essential to block or at least to slow the increase in CD rate among twins before it's too late. Conflict of interest The authors report no conflict of interest.

In conclusion, the results of the current study combined with results of other retrospective and randomized trials [4,13,28] are unable to justify the increase in CD rate among twin pregnancies. Training young physicians in delivering twin pregnancies before total "disappearance" of the skilled obstetricians is essential to block or at least to slow the increase in CD rate among twins before it's too late. Conflict of interest The authors report no conflict of interest. Acknowledgment The authors thank Paula S. Herer, biostatistician, MSc., MPH, for statistical guidance and assistance.

1 Introduction Aging process is complex due to its interaction among cultural, social, physiological and economic factors, so it would be inaccurate to restrict it just to age. In Spain, life expectancy presents an ascendant pattern that nowadays is greater in women than in men, a mean of seven years, establishing an aging population profile characterized not only by low birth but also low mortality rates, so there is a reduced natural growth of the population. It should be emphasized that in Spain there are approximately six million women aged over 50 years old [1]. About 40% of them will exhibit symptoms due to menopause that will significantly affect their quality of life. So, a therapeutic approach could be a public health priority in the next years, with a great medical, social and economic impact due to the increasing number of women who will live a post-menopausal period. Some aspect such diet and lifestyle should be take into account in order to improve the quality of women`s life during this period of their life [2]. Hence the importance of adult female population in the context of health cares.

nomic impact due to the increasing number of women who will live a post-menopausal period. Some aspect such diet and lifestyle should be take into account in order to improve the quality of women`s life during this period of their life [2]. Hence the importance of adult female population in the context of health cares. Menopause is defined as the permanent cessation of menstruation which induces physiological changes due to the decline in estrogen secretion with loss of follicular function [3]. Generally, it tends to appear at the age of 45–55 years. The overall mean onset is 50 years old, with differences between industrialized and developing countries. Spanish Society of Gynecology and Obstetrics (SEGO) established that the onset age in this cohort is 51.4 years old, ranging from 48 to 54 years [4]. Hormonal changes experience by women in physiological and non-physiological situation (i.e. hormone replacement therapy) produce significant changes in the periodontium, especially in plaque-induced gingivitis [5]. Gingiva is a target tissue for steroid hormones action. During hormonal fluctuations periods, clinical modifications have been identified in the periodontal tissue [6]. In particular, estrogens may influence cytodifferentiation of the stratified squamous epithelium and also in the synthesis and maintenance of collagen fibers [7]. It has been demonstrated that estrogen receptors are present in osteoblasts related to bone metabolism [8], in fibroblasts from periosteum, in lamina propria [9] and, in periodontal ligament [10].

cytodifferentiation of the stratified squamous epithelium and also in the synthesis and maintenance of collagen fibers [7]. It has been demonstrated that estrogen receptors are present in osteoblasts related to bone metabolism [8], in fibroblasts from periosteum, in lamina propria [9] and, in periodontal ligament [10]. Estrogen-progesterone interaction with inflammation mediators may explain the higher inflammation observed during hormonal fluctuation periods. Thus, a reduction in the levels of interleukin 1β (IL-1β) and interleukin 6 (IL-6) induced by progesterone may lead reductions of the tissue inhibitor of metalloproteinases and increase the activity of proteolytic enzymes and high levels of tumor necrosis factor (TNF), resulting in inflammation and clinical manifestations [5,11]. Therefore, normal estrogen levels in plasma may be necessary for periodontal protection. In fact, the amount of circulating estradiol appears to be inversely correlated with the prevalence of periodontal diseases [12,13]. In vitro studies suggest that reduced levels of female sex hormones (estradiol and progesterone) alter the local and systemic production of IL-1β and IL-6 and, postulating that these changes may be responsible for an increased bone loss associated to menopause. [14,15].

h the prevalence of periodontal diseases [12,13]. In vitro studies suggest that reduced levels of female sex hormones (estradiol and progesterone) alter the local and systemic production of IL-1β and IL-6 and, postulating that these changes may be responsible for an increased bone loss associated to menopause. [14,15]. Currently there are no clinical studies assessing the effect of menopausal hormone therapy (MHT) in post-menopausal women with periodontitis. The specific hypothesis of this investigation was that moderate chronic periodontitis women under MHT would present differences in immunological patient-based variables compared to those without MHT. Therefore, the purpose of the present study was to compare the effect of MHT on clinical and immunological variables in moderate chronic periodontitis patients with and without MHT. 2 Material and methods In this cross-sectional, pilot study, a consecutive sample of 83 menopausal women with moderate chronic periodontitis from the Menopause Program of the Gynecology and Obstetric Department at Madrid Centro Médico was screened for participation in the study for their posterior evaluation in the Graduate Periodontology Clinic of the Complutense University of Madrid (Spain). The protocol was submitted and approved by the Research Ethics Committee of the Hospital Clínico San Carlos (Madrid).

etric Department at Madrid Centro Médico was screened for participation in the study for their posterior evaluation in the Graduate Periodontology Clinic of the Complutense University of Madrid (Spain). The protocol was submitted and approved by the Research Ethics Committee of the Hospital Clínico San Carlos (Madrid). The following inclusion criteria were considered: systemically healthy women aged between 48–64 years with physiologic menopause and established amenorrhea for more than 12 months, presenting at least 16 teeth and with a diagnosis of moderate to advance chronic periodontitis, characterized with clinical attachment loss (CAL) ≥5 mm in ≥4 sites and probing pocket depth (PPD) ≥6 mm in ≥8 sites in, at least, two different quadrants [16]. Patients were excluded if they had taken antibiotics in the previous 3 months or those presenting systemic conditions or medications that may affect the periodontal status. After signing an IRB-approved informed consent and having explained the study aims, procedures and the voluntary character of their participation, medical and socio-demographic data were collected. Socio-demographic data included the following: age, gender, tobacco usage, employment status, marital status, schooling, systemic diseases, allergies and medications. Subsequently, all patients were examined by one calibrated examiner, for periodontal parameters, microbiological and gingival crevicular fluid (GCF) sampling.

-demographic data included the following: age, gender, tobacco usage, employment status, marital status, schooling, systemic diseases, allergies and medications. Subsequently, all patients were examined by one calibrated examiner, for periodontal parameters, microbiological and gingival crevicular fluid (GCF) sampling. 2.1 Clinical evaluation One calibrated examiner assessed plaque index [17] (PI), gingival index [18] (GI), PPD, gingival recession (GR) and CAL, recorded at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual and mesiolingual), at all teeth excluding third molars. PPD, GR and CAL were recorded using a periodontal probe (North Caroline Probe, Hu-Friedy, Leinmen, Germany). A calibration trial for periodontal clinical parameters was performed with the examiner involved in 10 randomly selected patients with chronic periodontitis, not related with the study, where CAL and PPD full-mouth measures were assessed in a duplicate manner within 2 weeks.

aroline Probe, Hu-Friedy, Leinmen, Germany). A calibration trial for periodontal clinical parameters was performed with the examiner involved in 10 randomly selected patients with chronic periodontitis, not related with the study, where CAL and PPD full-mouth measures were assessed in a duplicate manner within 2 weeks. 2.2 Microbiological evaluation Four sites, one per quadrant, being the most accessible site with the deepest pocket and bleeding, were selected. Clinical variables were specifically recorded at these sites (presence of plaque, bleeding on probing [BOP], PPD and GR). Samples were taken with two consecutive medium size paper points (Paper Point, Maillefer, Ballaigues, Switzerland) in each site. All paper points were pooled in a vial with reduced transport fluid, and transported to the laboratory, where they were processed within the next 2 h. At the laboratory, the samples were vortexed (30 s), serially diluted and plated in different media: blood agar medium (No. 2 of Oxoid; Oxoid Ltd, Basingstoke, UK), with 5% horse blood, and haemin (5 mg/l) and menadione (1 mg/l); and a selective medium for Aggregatibacter actinomycetemcomitans. The blood agar plates were studied after 7 and 14 days of anaerobic incubation (80% N2; 10% H2; 10% CO2 at 37 °C), and the selective plates after 3–5 days of 37 °C incubation in air with 5% CO2. Plates were carefully examined for the identification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Tannerella forsythia, Parvimonas micra, Capnocytophaga spp., Eikenella corrodens and Fusobacterium spp., based on the morphology of the colony and using different standard biochemical tests to confirm the initial identification (RapID ANA II, Thermo Scientific, Waltham, MA, USA). Other relevant colonies (those representing an important proportion of the microbiota) were also isolated for further characterization. The total number of colonies, as well as the number of each bacterial species, was counted in a representative plate (containing between 30 and 300 colonies). Counts of A. actinomycetemcomitans were performed on the selective plates, based on its typical colony morphology, a catalase reaction and a set of specific enzymes.

total number of colonies, as well as the number of each bacterial species, was counted in a representative plate (containing between 30 and 300 colonies). Counts of A. actinomycetemcomitans were performed on the selective plates, based on its typical colony morphology, a catalase reaction and a set of specific enzymes. Total counts of anaerobes in colony forming units (CFU) per sample and the frequency of detection, counts and proportions of periodontal pathogens were calculated. 2.3 Gingival crevicular fluid samples Gingival crevicular fluid was collected from the mesiobuccal sulcus of upper first molars by means of filter paper strips (Periopaper, Harco, Irvine, CA, USA). All samples were measured for gingival fluid volume with a calibrated gingival fluid meter (Periotrom 8000, Harco, Irvine, CA, USA) and placed in a sterile eppendorf tube [11,19]. GCF was used to measured IL-1β and IL-6 using enzyme-linked immunosorbent assays (BLK Diagnostic International, Badalona, Barcelona, Spain). Analyses were performed according to the manufacter’s protocol. Results were calculated using the standard curves created. Concentrations were corrected for GCF volume and defined as nanograms per milliliter. The total amount of IL-1β and IL-6 was expressed in picograms. 2.4 Data analysis Two groups of patients were determined at baseline examination: a) the MHT users group, defined as subjects with menopausal hormone therapy (MHT); b) the non-MHT users group, defined as those without menopausal hormone therapy.

2.3 Gingival crevicular fluid samples Gingival crevicular fluid was collected from the mesiobuccal sulcus of upper first molars by means of filter paper strips (Periopaper, Harco, Irvine, CA, USA). All samples were measured for gingival fluid volume with a calibrated gingival fluid meter (Periotrom 8000, Harco, Irvine, CA, USA) and placed in a sterile eppendorf tube [11,19]. GCF was used to measured IL-1β and IL-6 using enzyme-linked immunosorbent assays (BLK Diagnostic International, Badalona, Barcelona, Spain). Analyses were performed according to the manufacter’s protocol. Results were calculated using the standard curves created. Concentrations were corrected for GCF volume and defined as nanograms per milliliter. The total amount of IL-1β and IL-6 was expressed in picograms. 2.4 Data analysis Two groups of patients were determined at baseline examination: a) the MHT users group, defined as subjects with menopausal hormone therapy (MHT); b) the non-MHT users group, defined as those without menopausal hormone therapy. 2.5 Statistical analysis The patient was considered the unit of the analysis. Socio-demographic data were computed for each participant from data provided and compared between groups by means of χ2 tests. Clinical parameters were registered and averaged, first for each individual patient, and then for each group. Clinical variables were expressed as mean and standard deviation (SD), including the mean percentage of sites in each PPD category: 1–3 mm (shallow), 4–6 mm (moderate) and >6 mm (deep). Clinical variables of plaque index and gingival index were transformed into quantitative variables. Inter-group differences were evaluated using non-paired Student t-tests. All variables were first evaluated to confirm a normal distribution, by means of Kolmogorov–Smirnov tests.

ow), 4–6 mm (moderate) and >6 mm (deep). Clinical variables of plaque index and gingival index were transformed into quantitative variables. Inter-group differences were evaluated using non-paired Student t-tests. All variables were first evaluated to confirm a normal distribution, by means of Kolmogorov–Smirnov tests. Data were analyzed (Statistical Package for Social Sciences for MAC, SPSS Inc., Chicago, IL, USA) and the statistical significance level was set at 5% (p ≤ 0.05) for all analysis. 3 Results Out of the 83-screened subjects, 30 postmenopausal women (aged 50–64 years) were included in the study. The average age of menopause onset was established in 52.33 years. Forty-one subjects did not fulfill the inclusion-exclusion criteria, and twelve refused to participate in the study (Fig. 1).Fig. 1 Flow-chart of the studt, with number of patients. MHT = Menopausal hormone therapy. Fig. 1 3.1 Socio-demographic data The socio-demographic data of all subjects are summarized in Table 1 and it shows the population distribution according to the socio-demographic data. No statistically significant differences (p > 0.05) were detected between groups for any of the variables collected.Table 1 Socio-demographic characteristics of participants. Table 1Variable MHT users (n = 15) Non-MHT users (n = 15) p Age Mean ± standard deviation 58.2 ± 4.1 58.2 ± 3.7 0.772 Menopause age onset Mean ± standard deviation 52.8 ± 3.8 51.86 ± 2.7 0.541 Marital status Unmarried 4 3 0.648 Married 9 11 Divorced 0 0 Widowed 2 1 Tobacco Smokers 8 9 0.068 Non-smokers 7 6 Drugs Yes 2 1 0.271 No 13 14

Table 1Variable MHT users (n = 15) Non-MHT users (n = 15) p Age Mean ± standard deviation 58.2 ± 4.1 58.2 ± 3.7 0.772 Menopause age onset Mean ± standard deviation 52.8 ± 3.8 51.86 ± 2.7 0.541 Marital status Unmarried 4 3 0.648 Married 9 11 Divorced 0 0 Widowed 2 1 Tobacco Smokers 8 9 0.068 Non-smokers 7 6 Drugs Yes 2 1 0.271 No 13 14 Systemic disease Yes 1 0 0.585 No 14 15 Allergies Familiar 1 2 0.749 No 14 13 Schooling Middle school 1 3 0.164 High school 10 7 Professional training 0 0 Degree 4 5 Employment Status Hired hand 2 2 0.978 Self-employment 4 3 Student 0 0 Housewife 7 5 Retired 1 1 Unemployed 1 4 Other 0 0 MHT = Menopausal hormone therapy. 3.2 Clinical parameters 3.2.1 Plaque and gingival index There were no statistically significant differences between groups, being the average plaque value of 1.06 ± 0.44. The highest value corresponded to proximal sites (1.20 ± 0.46) while the minimum value to free surfaces (0.78 ± 0.42) (Table 2).Table 2 Mean percentages (±standard deviation) of periodontal clinical parameters in the two groups. Table 2Variables MHT users Non-MHT users p Plaque Index 1.04 ± 0.38 10.8 ± 0.51 0.813 Gingival Index 0.60 ± 0.29 0.80 ± 0.38 0.353 CAL (mm) 3.7 ± 0.46 4.6 ± 0.39 0.002 PPD (mm) 2.93 ± 0.14 3.71 ± 0.16 0.001 REC (mm) 0.77 ± 0.78 0.87 ± 0.63 0.699 MHT = Menopausal hormone therapy; CAL = clinical attachment level; PPD = probing pocket depth; REC = recession.

ers p Plaque Index 1.04 ± 0.38 10.8 ± 0.51 0.813 Gingival Index 0.60 ± 0.29 0.80 ± 0.38 0.353 CAL (mm) 3.7 ± 0.46 4.6 ± 0.39 0.002 PPD (mm) 2.93 ± 0.14 3.71 ± 0.16 0.001 REC (mm) 0.77 ± 0.78 0.87 ± 0.63 0.699 MHT = Menopausal hormone therapy; CAL = clinical attachment level; PPD = probing pocket depth; REC = recession. 3.2.2 Gingival recession Mean gingival recession was 0.82 ± 0.10 mm and there were no statistically significant differences between groups (p > 0.05). There were also no differences when data was analyzed based on the type of site: proximal and buccal/lingual/palatal sites (p > 0.05) 3.2.3 Probing pocket depth Mean PPD of the whole sample was 3.32 ± 0.70 mm. In the subgroup analysis, the non-MHT users group showed statistically significant higher values than the MHT users group: 3.71 ± 0.16 mm vs 2.93 ± 0.14 mm (p < 0.05). Deeper PPD were localized at the proximal sites (3.70 ± 0.80 mm) and shallower PPD were localized at buccal and lingual/palatal sites (2.55 ± 0.59 mm), there were statistically significant differences between groups for PPD in both type of sites, showing deeper values, approximately 1 mm, in the MHT users group. 3.2.4 Clinical attachment level There were statistically significant differences between groups for CAL, showing grater levels in the MHT users group (4.6 ± 0.20 mm).

Deeper PPD were localized at the proximal sites (3.70 ± 0.80 mm) and shallower PPD were localized at buccal and lingual/palatal sites (2.55 ± 0.59 mm), there were statistically significant differences between groups for PPD in both type of sites, showing deeper values, approximately 1 mm, in the MHT users group. 3.2.4 Clinical attachment level There were statistically significant differences between groups for CAL, showing grater levels in the MHT users group (4.6 ± 0.20 mm). 3.3 Immunological variables Mean GCF volume obtained from the patients was 0.67 ± 0.21 μL. There were statistically significant differences between groups: more CGF volume was obtained from patients without menopausal hormone therapy (0.81 μL) than patients with the therapy (0.53 μL) (p = 0.026). The whole sample mean concentration of Il-1ß was 53.24 ± 30.22 pg/mL. For this variable, there were statistically significant differences between groups (p < 0.001), being the group without menopausal hormone therapy the one showing higher levels. For the IL-6, whole sample mean concentration was 51.37 ± 30.13 pg/ml and there were also statistically significant differences between groups (p < 0.001), being the group without menopausal hormone therapy the one showing higher levels (80.16 ± 9.44 pg/mL) (Table 3).Table 3 Mean percentages (±standard deviation) of immunological parameters in the two groups.

tion was 51.37 ± 30.13 pg/ml and there were also statistically significant differences between groups (p < 0.001), being the group without menopausal hormone therapy the one showing higher levels (80.16 ± 9.44 pg/mL) (Table 3).Table 3 Mean percentages (±standard deviation) of immunological parameters in the two groups. Table 3Variables MHT users Non-MHT users p GCF volume (μL) 0.53 ± 0.38 0.81 ± 0.51 0.026 IL-1β concentration (pg/mL) 25.24 ± 8.55 81.25 ± 11.74 <0.001 IL-6 concentration (pg/mL) 22.58 ± 6.19 80.16 ± 9.44 <0.001 MHT = Menopausal hormone therapy; GCF = gingival crevicular fluid; IL-1β=interleukin 1 beta; IL6=interleukin 6. 3.4 Correlation analysis between immunological and periodontal clinical parameters IL-1ß GCF levels showed to be statistically related to the study group (p < 0.001), the smoking habit (p = 0.013), mean PPD (p = 0.005), PPD in posterior teeth (p < 0.001), PPD at proximal sites (p = 0.003) and Il-6 levels (p < 0.001). Both the study group and Il-6 levels obtained the highest correlation with the Il-1ß concentration (r = 0.943 and r = 0.940 respectively). PPD showed also high correlation while the smoking habit showed moderate correlation (r = 0.448). Then the smoking habit, deeper PPD and higher Il-6 levels in non-MHT users group, tend to increase the Il-1ß GCF levels.

obtained the highest correlation with the Il-1ß concentration (r = 0.943 and r = 0.940 respectively). PPD showed also high correlation while the smoking habit showed moderate correlation (r = 0.448). Then the smoking habit, deeper PPD and higher Il-6 levels in non-MHT users group, tend to increase the Il-1ß GCF levels. Il-6 GCF levels showed to be statistically related to the study group (p < 0.001), mean PPD (p < 0.001) at both tooth level: anterior teeth (p = 0.160) and posterior teeth (p < 0.001), and site level: proximal sites (p < 0.001) and non-proximal (p = 0.010); and Il-1ß levels (p < 0.001). Both the study group and Il-1ß levels obtained the highest correlation with the Il-6 (r = 0.966 and r = 0.940 respectively). High correlation was obtained for PPD at posterior teeth and proximal sites. Then the increase of the value of any of these variables would tend to increase also Il-6 GCF levels (Table 4).Table 4 Correlation analysis between immunological and periodontal clinical parameters. Table 4Variables IL-1β concentration (pg/mL) IL-6 concentration (pg/mL) Group 0.943 0.966 Tobacco 0.448 0.284 Mean PPD 0.501 0.601 Mean anterior PPD 0.353 0.436 Mean posterior PPD 0.556 0.660 Mean interproximal PPD 0.524 0.616 Mean free PPD 0.355 0.461 IL-1β concentration (pg/mL) 1 0.940 IL-6 concentration (pg/mL) 0.940 1 PPD = probing pocket depth; IL-1β=interleukin 1 beta; IL-6=interleukin 6.

0.943 0.966 Tobacco 0.448 0.284 Mean PPD 0.501 0.601 Mean anterior PPD 0.353 0.436 Mean posterior PPD 0.556 0.660 Mean interproximal PPD 0.524 0.616 Mean free PPD 0.355 0.461 IL-1β concentration (pg/mL) 1 0.940 IL-6 concentration (pg/mL) 0.940 1 PPD = probing pocket depth; IL-1β=interleukin 1 beta; IL-6=interleukin 6. 3.5 Microbiological variables The percentage of sites positive for Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Prevotella intermediate, Parvimonas micra, Fusobacterium nucleatum and Campylobacter rectus over the course of the study is shown in Table 5. There were no statistically significant differences between groups.Table 5 Percentages of sites positive for each bacterial species in the two groups. Table 5Variables MHT users Non-MHT users Aa 4.6 4.7 Pg 49.56 53.4 Tf 8.16 6.65 Pi 19.3 23.5 Pm 30.4 25.0 Fn 25.5 30.6 Cr 2.9 1.0 MHT = Menopausal hormone therapy; A.a. = Aggregatibacter actinomycetemcomitans; P.g. = Porphyromonas gingivalis; T.f. = Tannerella forsythia; P.i. = Prevotella intermediate; P.m. = Parvimonas micra; F.n. = Fusobacterium nucleatum; C.r. = Campylobacter rectus. 4 Comment The results of the present pilot study show that the patients undergoing menopausal hormone therapy (MHT), test group, had shallower PPD compared to the control group, those who didn’t receive the therapy. This difference was statistically significant (p < 0.001). For the rest of the clinical variables, the control group showed greater values but these differences didn’t reach a statistically significance (p > 0.05).

(MHT), test group, had shallower PPD compared to the control group, those who didn’t receive the therapy. This difference was statistically significant (p < 0.001). For the rest of the clinical variables, the control group showed greater values but these differences didn’t reach a statistically significance (p > 0.05). Risks associated to the MHT generate controversy [20,21] although it is being prescribed to avoid hot flashes and systemic menopause consequences as osseous fractures due to osteoporosis. Prospective studies [22,23] showed a 50% reduction in hip fractures in women undergoing MHT compared to those who didn’t received the therapy. This effect of the MHT in bone metabolism has been proposed to influence alveolar bone loss [12,13] and periodontal status.

onsequences as osseous fractures due to osteoporosis. Prospective studies [22,23] showed a 50% reduction in hip fractures in women undergoing MHT compared to those who didn’t received the therapy. This effect of the MHT in bone metabolism has been proposed to influence alveolar bone loss [12,13] and periodontal status. Previous research has shown that during menopause the risk of tooth loss is reduced in women receiving MHT [13,23]. Plasmatic estrogen levels influence alveolar bone density and lower levels have shown to be related to a more advanced periodontal affection [24]. In this pilot study women in the test group showed presented better periodontal situation after receiving MHT. These results are correlated to those obtained in a study with 190 Spanish patients that showed better CAL in those receiving MHT for at least one year [25]. However, a randomized clinical trial by Pilgram and coworkers [26] didn’t find statistically significant differences for CAL in 135 women under MHT. For gingival recession, the results of the present study showed a tendency for more recession in women without MHT (p = 0.669), these results are in agreement with those obtained by Ronderos and coworkers [27] who did get statistically significant differences for this parameter. Reindhart and coworkers [5] reported that women under MHT had less gingival inflammation and that having serum levels of estradiol >40 pg/ml was associated to less loss of attachment. In the current study blood samples were not taken to report hormonal serum levels, being one of the study’s limitation.

Previous research has shown that during menopause the risk of tooth loss is reduced in women receiving MHT [13,23]. Plasmatic estrogen levels influence alveolar bone density and lower levels have shown to be related to a more advanced periodontal affection [24]. In this pilot study women in the test group showed presented better periodontal situation after receiving MHT. These results are correlated to those obtained in a study with 190 Spanish patients that showed better CAL in those receiving MHT for at least one year [25]. However, a randomized clinical trial by Pilgram and coworkers [26] didn’t find statistically significant differences for CAL in 135 women under MHT. For gingival recession, the results of the present study showed a tendency for more recession in women without MHT (p = 0.669), these results are in agreement with those obtained by Ronderos and coworkers [27] who did get statistically significant differences for this parameter. Reindhart and coworkers [5] reported that women under MHT had less gingival inflammation and that having serum levels of estradiol >40 pg/ml was associated to less loss of attachment. In the current study blood samples were not taken to report hormonal serum levels, being one of the study’s limitation. When analyzing other studies, the effect of the MHT on the periodontal status is similar between them. Studies in Brazilian and German patients established that in those aged between 50–69 years old the prevalence of periodontal disease is higher in menopause women who are not under MHT compared to those who are this statement is in agreement to the results of the present study [28,29]. However, the American population analyzed in the NHANES III study observed a lower prevalence in patients without MHT [30] and for the Japanese woman differences between both study groups could not be found [31].

r MHT compared to those who are this statement is in agreement to the results of the present study [28,29]. However, the American population analyzed in the NHANES III study observed a lower prevalence in patients without MHT [30] and for the Japanese woman differences between both study groups could not be found [31]. Sexual hormones have shown to influence periodontal tissues and periodontal disease progression [[32], [33], [34]] then it could explain the results obtained in the present study that seem to justify that MHT plays a roll in chronic periodontitis. However there is little information about the effect of feminine sexual hormones on oral microbiota in menopause women. In the present study it was observed that patients of the non-MHT users group showed more oral manifestations than the MHT users group but didn’t reach statistically significant differences, this has been also observed in previous similar studies [25,35].

Sexual hormones have shown to influence periodontal tissues and periodontal disease progression [[32], [33], [34]] then it could explain the results obtained in the present study that seem to justify that MHT plays a roll in chronic periodontitis. However there is little information about the effect of feminine sexual hormones on oral microbiota in menopause women. In the present study it was observed that patients of the non-MHT users group showed more oral manifestations than the MHT users group but didn’t reach statistically significant differences, this has been also observed in previous similar studies [25,35]. Reindhart and coworkers [36] showed that GCF levels of IL-1ß y IL-6 were increased in menopause patients with refractory periodontitis who didn’t received MHT compared to those who received it, so local inflammation would not be the only cause for this increase but also the level of estrogens. Another study performed by the same authors determined that patients under MHT showed lower levels of IL-1ß compared to patients without MHT; this data suggests that low levels of estrogens are associated to gingival production of IL-1ß influencing the progression of periodontal disease. In the present study the results are in agreement with previous data: test group showed lower levels of inflammatory mediators and a better periodontal status. The level of estrogens and PPD explains these differences. According to the variable PPD, when it increases 0.2 mm, there is an increment of 8.01 pg/ml of IL-1ß and 7.73 pg/ml of IL-6.

results are in agreement with previous data: test group showed lower levels of inflammatory mediators and a better periodontal status. The level of estrogens and PPD explains these differences. According to the variable PPD, when it increases 0.2 mm, there is an increment of 8.01 pg/ml of IL-1ß and 7.73 pg/ml of IL-6. To our knowledge, the present study is the first to analyze in detail the association of the immunological, microbiological and periodontal status in menopause women comparing two groups based on the presence of MHT. However, one of the main limitations of this study is that there is no information about the estrogens levels to established future detection thresholds and relate them with other variables. The preliminary results of this pilot study should be interpreted with caution due to the small sample size and the cross-sectional design. In this type of study it can be established an association between variables but not a temporary consistency or casualty due to the lack of a follow-up of the patients. The type of MHT was not assessed in the present study, and despite there are no systematic reviews that evaluate the effect of different type of MHT on the microbiological, immunological and periodontal status, it should be considered as an important factor affecting the estrogen levels. Finally, another limitation of the present study is the lack of stratification by estrogen levels, PPD or number of cigarettes in smoker patients.

e effect of different type of MHT on the microbiological, immunological and periodontal status, it should be considered as an important factor affecting the estrogen levels. Finally, another limitation of the present study is the lack of stratification by estrogen levels, PPD or number of cigarettes in smoker patients. Within the limitations of the present study, the hypothesis that moderate chronic periodontitis women under MHT would present differences in immunological variables, based on patient, compared to those without MHT was supported. Conflict of interest The authors declare that they have no conflicts of interest in this study. The study was designed, conducted and analyzed by researchers belonging to the Host Response in Oral Pathology (HROP) Research Group (Complutense University, Madrid, Spain). Role of the funding source No external funding, apart from the support of the author’s institution, was available for this study. Acknowledgments Authors would like to thank Madrid Centro Médico and researchers for their help with this clinical research.

Introduction Endometrial cancer is the most frequent malignant gynecologic neoplasm in developed countries, with 70% of cases diagnosed in the initial stages, with good prognosis and a five-year survival rate of 80–85% [[1], [2], [3]]. Standard surgical staging considers the extent of the disease, particularly the presence of myometrial infiltration (MI), histological type, histological differentiation grade (G) and regional (lymph node) or distant metastasis. Detection of locoregional metastasis is preferably performed by retroperitoneal lymphadenectomy of the pelvic and/or para-aortic region. The presence of compromised lymph nodes is related to worse prognoses and decreased five-year survival rates (44–52%) [4,5], signaling the need for adjuvant therapy [6]. Despite the importance of lymphadenectomy in staging, the procedure is associated with significant morbidity and it could be omitted depending on the extent of the disease and risk factors [2,7,8]. From previous published studies, the American College of Obstetrics and Gynecology (ACOG) advises that lymphadenectomy may be dispensed in early-stages low-risk patients for lymph node metastasis [9,10].

with significant morbidity and it could be omitted depending on the extent of the disease and risk factors [2,7,8]. From previous published studies, the American College of Obstetrics and Gynecology (ACOG) advises that lymphadenectomy may be dispensed in early-stages low-risk patients for lymph node metastasis [9,10]. There are three distinct risk groups for lymph node metastasis, defined with some variations depending on the published study: (1) low risk (27% of the cases, with disease in an initial stage and up to 50% MI and G1), with lymph node metastasis found in <5%. In these cases, the staging surgery may be restricted to total hysterectomy and bilateral salpingo-oophorectomy (BSO) [6,[11], [12], [13]]; (2) the opposite group, at high-risk (24% of the cases, with >50% MI and G3), with lymph node metastasis found in 25%–40%. This group has indication for systematic lymphadenectomy (SL) of the pelvic and para-aortic region, for the correct staging of the neoplasm [[13], [14], [15]]; (3) the third group, at intermediate risk (at least 50% of all cases, when considered endometrioid endometrial carcinoma (EEC) with up to 50% MI and G2 or G3 (FIGO Stage IAG2 or IAG3), and tumors with deep MI (>50%) and G1 or G2 (FIGO Stage IBG1 or IBG2) [13,16], with lymph node metastasis rate between 5% and 25%. Uncertainty persists for this third group in respect of the advantages and disadvantages of SL in the accuracy of definitive staging, as regards possible therapeutic outcome.

IAG2 or IAG3), and tumors with deep MI (>50%) and G1 or G2 (FIGO Stage IBG1 or IBG2) [13,16], with lymph node metastasis rate between 5% and 25%. Uncertainty persists for this third group in respect of the advantages and disadvantages of SL in the accuracy of definitive staging, as regards possible therapeutic outcome. This study aims to evaluate the indication and performance of SL carried out over time in women with stage I EEC at intermediate risk and its impact on recurrence, disease free survival (DFS) and cancer survival.

IAG2 or IAG3), and tumors with deep MI (>50%) and G1 or G2 (FIGO Stage IBG1 or IBG2) [13,16], with lymph node metastasis rate between 5% and 25%. Uncertainty persists for this third group in respect of the advantages and disadvantages of SL in the accuracy of definitive staging, as regards possible therapeutic outcome. This study aims to evaluate the indication and performance of SL carried out over time in women with stage I EEC at intermediate risk and its impact on recurrence, disease free survival (DFS) and cancer survival. Materials and methods After obtaining approval from the Research Ethics Committee at the State University of Campinas (UNICAMP) in Campinas (SP), Brazil, we identified from institutional surgical database 1068 women with EC treated with hysterectomy in the period 1990 to 2014 and with no history of other neoplasms, previous chemotherapy or radiotherapy. From this cohort, 291 were under 75 years old, had preoperative Stage I, and were submitted for surgery by laparotomy as first treatment, and exhibited histopathological evaluation with endometrioid-type histology, with MI, and no lymph-vascular space invasion detected. Final Stage was reclassified according to FIGO-2014 staging system [17]. There were 152 women treated with hysterectomy and BSO, and the final Stage were IAG1 for 50, Stage II (cervical invasion) for three, and 99 were at intermediate risk (IAG2/G3, IBG1/G2 - ‘non-SL’ group) [16,17]. Other 139 women were treated with hysterectomy and BSO plus pelvic and para-aortic lymphadenectomy, and 22 had final Stage IAG1, 15 Stage II and three Stage III (lymph node metastasis), with remaining 95 at intermediate risk (‘SL’ group). In all cases, the hysterectomy was performed according to Paver 1 (Querleu A) [18,19], and the pelvic lymphadenectomy consisted of the removal of adipose tissue around the iliac vessels (common, internal and external) bilaterally and in obturator fossa until visualization of the obturator nerve, while the para-aortic lymphadenectomy comprised the peri aortal and pericaval region below the renal vessels. Surgical procedures were performed or supervised by surgeons specialized in gynecological cancer treatment. Until 1998, the recommendations guided complete surgical staging (with SL) in all cases that clinical conditions permitted it. After 1998, the selection of patients to undergo SL started taking into consideration the risk of lymph node disease and more careful evaluation of clinical and/or technical restrictions, as due to obesity. All lymph nodes removed were processed, counted, and sectioned in their half, with both sections were microscopically analyzed.

e selection of patients to undergo SL started taking into consideration the risk of lymph node disease and more careful evaluation of clinical and/or technical restrictions, as due to obesity. All lymph nodes removed were processed, counted, and sectioned in their half, with both sections were microscopically analyzed. All pathological evaluations were performed by pathologists specialized in gynecological malignancies. Adjuvant therapy was determined according to woman performance and tumor pattern and stage and guided by Brazilian National Cancer Institute (INCA). The options were no further treatment, or treatment with brachytherapy or brachytherapy plus teletherapy. Adjuvant pelvic radiation therapy usually started within 4 to 6 weeks after surgery using a conventional four-field technique, with dose ranged from 40 Gy in 23 daily fractions to 50.4 Gy in 28 daily fractions. Brachytherapy in vaginal cuff was performed weekly in four fractions and total dose of 28 Gy. At follow-up visits, all patients received a complete physical and gynecological examination every four months for the first year and every six months for the next five years. Chest radiography, pelvic and abdominal ultrasound exam, CT scan and biopsy were performed when clinical abnormalities were found. Recurrence was defined as histological presence of tumor cells or enlarged lymph nodes or detection of pelvic tumor or distant metastasis.

year and every six months for the next five years. Chest radiography, pelvic and abdominal ultrasound exam, CT scan and biopsy were performed when clinical abnormalities were found. Recurrence was defined as histological presence of tumor cells or enlarged lymph nodes or detection of pelvic tumor or distant metastasis. Information were obtained from medical files of hospital records regarding diagnosis period (1990–1998, 1999–2008 and 2009–2014), age-group (<50, 50–59, 60–69 and >70 years), body mass index-BMI (<35.0 kg/m2 and ≥35.0 kg/m2), presence of comorbidities (diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease), surgical and pathological information, final staging, use of adjuvant radiotherapy, relapse and death in the follow-up and considered until 2016. Statistical analysis Data were entered into a computerized database and statistical analysis was performed using the StatsDirect statistical software 3.0 (England, www.statsdirect.com), and p values < 0.05 were considered statistically significant.

Information were obtained from medical files of hospital records regarding diagnosis period (1990–1998, 1999–2008 and 2009–2014), age-group (<50, 50–59, 60–69 and >70 years), body mass index-BMI (<35.0 kg/m2 and ≥35.0 kg/m2), presence of comorbidities (diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease), surgical and pathological information, final staging, use of adjuvant radiotherapy, relapse and death in the follow-up and considered until 2016. Statistical analysis Data were entered into a computerized database and statistical analysis was performed using the StatsDirect statistical software 3.0 (England, www.statsdirect.com), and p values < 0.05 were considered statistically significant. Categorical variables between groups were analyzed by Chi-Square or Fisher's exact tests. Kaplan-Meier survival curves were assembled and analyzed using the log-rank test, considering DFS - the interval between the end of the treatment and the relapse and cause-specific survival - the interval between the date of surgery and death, and only deaths due to illness or related to treatment were considered. Follow-up was censored at 120 months. Factors possibly associated with recurrence and DFS (p <  0.20) were selected for Cox regression analyses (univariate and multivariate).

e-specific survival - the interval between the date of surgery and death, and only deaths due to illness or related to treatment were considered. Follow-up was censored at 120 months. Factors possibly associated with recurrence and DFS (p <  0.20) were selected for Cox regression analyses (univariate and multivariate). Results The comparison between the groups studied is shown in Table 1. SL was performed in 93% (41/44) of women managed before 1998 and decreasing the proportion after that (p < 0.001). Lymphadenectomy was also more frequent if BMI under 35.0 kg/m2 (p < 0.001) and in women without comorbidities (p = 0.017). There was no difference in age-group distribution (p = 0.128), cancer stage (p = 0.174) and adjuvant radiotherapy (p = 0.156) regarding lymphadenectomy done or not. The mean number of lymph nodes removed in SL group was 12 for the pelvic region and 5 for the para-aortic region (data not shown).Table 1 Comparison of women with endometrioid-type endometrial carcinoma at intermediate risk (IAG2G3 and IBG1G2) managed by hysterectomy with or without lymphadenectomy, and according to diagnosis period, the women’s characteristics, cancer stage and adjuvant radiotherapy.

5 for the para-aortic region (data not shown).Table 1 Comparison of women with endometrioid-type endometrial carcinoma at intermediate risk (IAG2G3 and IBG1G2) managed by hysterectomy with or without lymphadenectomy, and according to diagnosis period, the women’s characteristics, cancer stage and adjuvant radiotherapy. Table 1Characteristics Lymphadenectomy No (n = 99) Yes (n = 95) p* n % n % Period of diagnosis 1990–1998 3 3.0 41 43.2 1999–2008 44 44.4 35 36.8 2009–2014 52 52.6 19 20.0 < 0.001 Age-group (in years) <50 4 4.0 8 8.4 50–59 26 26.3 33 34.8 60–69 46 46.5 42 44.2 ≥70 23 23.2 12 12.6 0.128 BMI (kg/m2) <35.0 52 58.4 79 83.2 ≥35.0 37 41.6 16 16.8 < 0.001 Missing information 10 0 Comorbidities§ Yes 73 77.7 58 61.7 No 21 22.3 36 38.3 0.017 Missing information 5 1 Stage (FIGO) IA G2 57 57.6 46 48.4 IA G3 5 5.0 11 11.6 IB G1 18 18.2 13 13.7 IB G2 19 19.2 25 26.3 0.174 Radiotherapy EBRT (±brachytherapy) 41 41.4 49 51.6 Brachytherapy/None 58 58.6 46 48.4 0.156 FIGO: International Federation of Gynecology and Obstetrics. EBRT: external beam radiotherapy. BMI: body mass index. * Chi-square test or Fisher's exact test. § Diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease.

Table 1Characteristics Lymphadenectomy No (n = 99) Yes (n = 95) p* n % n % Period of diagnosis 1990–1998 3 3.0 41 43.2 1999–2008 44 44.4 35 36.8 2009–2014 52 52.6 19 20.0 < 0.001 Age-group (in years) <50 4 4.0 8 8.4 50–59 26 26.3 33 34.8 60–69 46 46.5 42 44.2 ≥70 23 23.2 12 12.6 0.128 BMI (kg/m2) <35.0 52 58.4 79 83.2 ≥35.0 37 41.6 16 16.8 < 0.001 Missing information 10 0 Comorbidities§ Yes 73 77.7 58 61.7 No 21 22.3 36 38.3 0.017 Missing information 5 1 Stage (FIGO) IA G2 57 57.6 46 48.4 IA G3 5 5.0 11 11.6 IB G1 18 18.2 13 13.7 IB G2 19 19.2 25 26.3 0.174 Radiotherapy EBRT (±brachytherapy) 41 41.4 49 51.6 Brachytherapy/None 58 58.6 46 48.4 0.156 FIGO: International Federation of Gynecology and Obstetrics. EBRT: external beam radiotherapy. BMI: body mass index. * Chi-square test or Fisher's exact test. § Diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease. Four cases (one from the SL group) did not return after performing vaginal cuff brachytherapy and were not followed up. There was one intraoperative death due to uncontrollable bleeding in the non-SL group. Follow-up of the remaining 189 cases, exhibited two deaths (one in each group) caused by bowel complications, four to six months after the end of the radiotherapy and six deaths not related to the disease or treatment (two from the SL group). There were 14 (7.4%) recurrences, nine of which were pelvic (including peritoneal cavity), and five as distant metastases (2-lung, 2-CNS and one neck lymph node, all cases in the SL group; Table 2). All 14 recurred women died, 10 had short post-relapse survival up to 12 months (Table 2).Table 2 Cases with recurrence of endometrioid-type endometrial carcinoma at intermediate risk, according to some patient characteristics, management, and recurrence pattern.

one neck lymph node, all cases in the SL group; Table 2). All 14 recurred women died, 10 had short post-relapse survival up to 12 months (Table 2).Table 2 Cases with recurrence of endometrioid-type endometrial carcinoma at intermediate risk, according to some patient characteristics, management, and recurrence pattern. Table 2Case (Age) First treatment Recurrence Survival post-relapse§ (months) Year Lymph-adenectomy Stage* Adjuvant radiotherapy Site Time after treatment (months) 1 (75y) 1996 No IBG2 EBRT Pelvic 50 4 2 (73y) 2014 No IAG3 EBRT Upper abdomen 24 1 3 (61y) 1990 Yes IAG2 VBT Upper abdomen 31 18 4 (57y) 1991 Yes IAG2 EBRT Neck limph node 59 9 5 (57y) 1992 Yes IBG2 EBRT Lung 6 23 6 (64y) 1993 Yes IAG2 EBRT Pelvic 26 10 7 (67y) 1993 Yes IAG3 VBT CNS 5 1 8 (68y) 1993 Yes IAG3 EBRT Pelvic 48 2 9 (61y) 1995 Yes IBG1 EBRT Upper abdomen 20 5 10 (70y) 1996 Yes IAG3 EBRT Lung 6 3 11 (47y) 2004 Yes IBG2 EBRT Para-aortic 64 34 12 (65y) 2008 Yes IAG3 EBRT CNS 20 7 13 (62y) 2009 Yes IAG2 VBT Vagina 11 7 14 (64y) 2010 Yes IAG2 VBT Para-aortic 185 24 EBRT: external beam radiotherapy; VBT: vaginal brachytherapy; CNS: central nervous system; NA: not available. * FIGO Stage/2014 [16]: A=myometrial invasion <50% deep; B=≥50% deep; G=histological grade. § Survival post-relapse: time after recurrence and until death.

Table 2Case (Age) First treatment Recurrence Survival post-relapse§ (months) Year Lymph-adenectomy Stage* Adjuvant radiotherapy Site Time after treatment (months) 1 (75y) 1996 No IBG2 EBRT Pelvic 50 4 2 (73y) 2014 No IAG3 EBRT Upper abdomen 24 1 3 (61y) 1990 Yes IAG2 VBT Upper abdomen 31 18 4 (57y) 1991 Yes IAG2 EBRT Neck limph node 59 9 5 (57y) 1992 Yes IBG2 EBRT Lung 6 23 6 (64y) 1993 Yes IAG2 EBRT Pelvic 26 10 7 (67y) 1993 Yes IAG3 VBT CNS 5 1 8 (68y) 1993 Yes IAG3 EBRT Pelvic 48 2 9 (61y) 1995 Yes IBG1 EBRT Upper abdomen 20 5 10 (70y) 1996 Yes IAG3 EBRT Lung 6 3 11 (47y) 2004 Yes IBG2 EBRT Para-aortic 64 34 12 (65y) 2008 Yes IAG3 EBRT CNS 20 7 13 (62y) 2009 Yes IAG2 VBT Vagina 11 7 14 (64y) 2010 Yes IAG2 VBT Para-aortic 185 24 EBRT: external beam radiotherapy; VBT: vaginal brachytherapy; CNS: central nervous system; NA: not available. * FIGO Stage/2014 [16]: A=myometrial invasion <50% deep; B=≥50% deep; G=histological grade. § Survival post-relapse: time after recurrence and until death. Recurrences were associated with FIGO Stage IAG3 (35.7%, p = 0.009) and concentrated in the SL group (12/14 recurrences; Table 3). Age-group, BMI, comorbidities, and adjuvant radiotherapy were not associated with relapses (Table 3). Survival and DFS time were short when recurrence happened. Cox regression analyses considered the stage, radiotherapy, and lymphadenectomy. Only Stage IAG3 (Hazard Ratio 6.48, IC95% 1.88–22.39, p = 0.003) was associated with less DFS in univariate analysis. In the longitudinal evaluation, DFS of women with intermediate-risk EEC exhibited 97% 5-year survival rate for non-SL group and 85% for SL group (p = 0.004, Fig. 1). Cancer-specific survival curves present significant differences according to whether SL was performed. After five years of follow-up, the survival rate was 95% for the non-SL group vs. 88% for the SL group (p = 0.039, Fig. 1). Comparing cancer stage and tumor grade, stage IAG3 had a worse survival time rate (75% at 5-year vs. 89–97% for other stage, p = 0.013, Fig. 2).Table 3 Recurrence rate in women with endometrioid-type endometrial carcinoma at intermediate risk (IAG2G3 and IBG1G2) according to diagnosis period, the women's characteristics, cancer stage, adjuvant radiotherapy and surgery staging, with or without lymphadenectomy.

year vs. 89–97% for other stage, p = 0.013, Fig. 2).Table 3 Recurrence rate in women with endometrioid-type endometrial carcinoma at intermediate risk (IAG2G3 and IBG1G2) according to diagnosis period, the women's characteristics, cancer stage, adjuvant radiotherapy and surgery staging, with or without lymphadenectomy. Table 3Characteristics Recurrence No (n = 175) Yes (n = 14) p* n % n % Period of diagnosis 1990–1998 34 19.4 9 64.3 1999–2008 76 43.4 3 21.4 2009–2014 65 37.2 2 14.3 0.002 Age-group (in years) <50 11 6.3 1 7.1 50–59 57 32.6 2 14.3 60–69 77 44.0 8 57.2 ≥70 30 17.1 3 21.4 0.465 BMI (kg/m2) <35.0 119 72.1 11 78.6 ≥35.0 46 27.9 3 21.4 0.454 Missing information 10 Comorbidities§ Yes 116 68.6 10 71.4 No 53 31.4 4 28.6 0.546 Missing information 6 Stage (FIGO) IA G2 95 54.3 5 35.7 IA G3 11 6.3 5 35.7 IB G1 30 17.1 1 7.1 IB G2 39 22.3 3 21.4 0.009 Radiotherapy EBRT (±brachytherapy) 79 45.1 10 71.4 Brachytherapy/none 96 54.9 4 28.6 0.052 Lymphadenectomy Yes 82 46.9 12 85.7 No 93 53.1 2 14.3 0.004 FIGO: International Federation of Gynecology and Obstetrics. EBRT: external beam radiotherapy. BMI: body mass index. * Chi-square test or Fisher’s exact test. § Diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease. Fig. 1 Disease-free survival (DFS) and cancer-specific survival curves of women with endometrioid-type endometrial carcinoma at intermediate-risk according to whether they underwent systematic lymphadenectomy (SL).

* Chi-square test or Fisher’s exact test. § Diabetes mellitus and/or systemic arterial hypertension and/or cardiovascular disease. Fig. 1 Disease-free survival (DFS) and cancer-specific survival curves of women with endometrioid-type endometrial carcinoma at intermediate-risk according to whether they underwent systematic lymphadenectomy (SL). Fig. 1Fig. 2 Cancer-specific survival curve of women with endometrioid-type endometrial carcinoma at intermediate-risk according to cancer stage and tumor grade. Fig. 2 Comment SL in EEC of intermediate risk (IAG2G3, IBG1G2) did not bring advantages regarding less relapses or increasing survival over time as compared with women who underwent surgery without lymphadenectomy. By contrast, according to the possible therapeutic outcome of SL, the SL group exhibited DFS, and cancer-specific survival curves always below the curves of the group treated with hysterectomy and BSO without lymphadenectomy, even though some of these latter cases may have been down-staged. This worse result observed in SL group can be related to more liberation to perform complete surgical staging in the first period (93.2% of the cases up to 1998 vs. 26.8% after 2008), following the guidelines at that time, which advised complete staging surgery for all candidate patients. Maybe, in that first period, we were less selective and managed cases with worse prognosis, such as those with larger tumors. Tumor size is considered associated with prognosis [13,20,21], but we did not have access to consistent information about it, as in preoperative exams as in pathologic reports.

ndidate patients. Maybe, in that first period, we were less selective and managed cases with worse prognosis, such as those with larger tumors. Tumor size is considered associated with prognosis [13,20,21], but we did not have access to consistent information about it, as in preoperative exams as in pathologic reports. Additionally, another independent risk factor for lymph node involvement or worse prognosis is histologic grade 3 [13,21] and the cases G3 studied (all Stage IA with MI) were twice more frequent at SL group than non-SL group. Regarding all 14 women with recurrence, five were Stage IAG3 and four performed SL (three with distant metastasis). Although the SL group may have worse cases selected, such as in stage IAG3, even with lymphadenectomy performed, they presented lower survival rate, with no evidence of benefit of complete staging surgery. Currently, several studies have diverged from the idea of performing SL to obtain more accurate staging. From our 139 women submitted to SL, only three (2.2%, two G3) had lymph node positive and final Stage III. Vargas et al. (2014) adopting some specific risk strata, analyzed almost 20 thousand cases of SEER data from 1988 to 2010 and observed 1.4% of lymph node positive, if low-risk EC, and 6.4% if high-risk [21].

ging. From our 139 women submitted to SL, only three (2.2%, two G3) had lymph node positive and final Stage III. Vargas et al. (2014) adopting some specific risk strata, analyzed almost 20 thousand cases of SEER data from 1988 to 2010 and observed 1.4% of lymph node positive, if low-risk EC, and 6.4% if high-risk [21]. Two meta-analyses have shown that the therapeutic value of SL remains controversial as regards survival rate [3,22] and another recent study found no differences in the reduction of recurrences or mortality related to the performance of SL in stage I endometrial cancer [23]. Two randomized clinical trials with stage I cancer found that performing SL did not improve DFS or cancer-specific survival and the patients could have been spared the procedure and its possible complications [5,6]. While studies tend to point to a lack of benefits of SL in cases of type 1 cancer restricted to the uterine body [[22], [23], [24], [25], [26], [27], [28]], there is no consensus about how to proceed when the EC had an intermediate risk, as the women evaluated here. Longitudinal evaluation of our data exhibited high 5-year survival rate for both groups, although significantly worse for women performed complete surgical staging, including DFS.

[24], [25], [26], [27], [28]], there is no consensus about how to proceed when the EC had an intermediate risk, as the women evaluated here. Longitudinal evaluation of our data exhibited high 5-year survival rate for both groups, although significantly worse for women performed complete surgical staging, including DFS. The absence of positive impact over SL on cancer-specific survival observed in this study in women with EEC at intermediate risk agrees with some other published studies [3,6,[23], [24], [25]]. Besides the lymph node status, these studies showed that survival is mainly related to stage and risk factors such as myometrial invasion, tumor grade, and histological type. Our results reinforcing what already been put forward in the literature aimed to suppress SL in surgical staging in EEC at intermediate risk [4,8,9,13,23].

e lymph node status, these studies showed that survival is mainly related to stage and risk factors such as myometrial invasion, tumor grade, and histological type. Our results reinforcing what already been put forward in the literature aimed to suppress SL in surgical staging in EEC at intermediate risk [4,8,9,13,23]. Another result worth highlighting was the maintenance of the use of adjuvant radiotherapy even in women who underwent SL, which is currently less indicated in cases of negative lymphadenectomy and without associated risk factors, as guided by more recent studies [13,[26], [27], [28], [29], [30], [31]]. In this study, even in the SL group and considering only women without lymph node involvement, 52% received pelvic radiotherapy, although these were performed before 2010. Recurrences occurred even in women submitted to external beam radiotherapy (EBRT), and this approach did not provide any benefits concerning DFS and cancer-specific survival rates, since the outcomes were no different between the groups. Furthermore, the association between SL and EBRT can increase complications in the follow-up.

occurred even in women submitted to external beam radiotherapy (EBRT), and this approach did not provide any benefits concerning DFS and cancer-specific survival rates, since the outcomes were no different between the groups. Furthermore, the association between SL and EBRT can increase complications in the follow-up. Stage I EC presents a good prognosis and the treatment based on surgery and/or radiotherapy is usually associated with non-negligible morbidity and mortality. To these negative events add other prevalent in the pattern of the women with EC, i.e., resulting of older age, obesity and frequent comorbidities. Morbidity and mortality may be partially controlled by reducing the extent of staging surgery and individualize the treatment. Therefore, it is necessary to consider whether SL adds information about disease extension, especially if performed by laparotomy, as evaluated in this study, and how much is used to avoid radiotherapy in cases of negative lymph node involvement. As expected, our cases had less indicated SL in older women, with higher BMI and comorbidities. Limitations of this study were its retrospective pattern and not considered some relevant risk factors, as lymph-vascular space involvement and tumor size. Strengths of this study were its relatively uniform management, surgery by laparotomy, number of lymph nodes removed, pathological evaluation by pathologists specialized in gynecological malignancies, long-term follow up with high adhesion, and applying established algorithms in a real world.

nvolvement and tumor size. Strengths of this study were its relatively uniform management, surgery by laparotomy, number of lymph nodes removed, pathological evaluation by pathologists specialized in gynecological malignancies, long-term follow up with high adhesion, and applying established algorithms in a real world. Relapses were predominated in the first two years of follow up and generally found after symptoms had been reported, similar of the pattern described by Salani et al. in 2011 [32]. Three cases with distant metastasis had G3 primary tumor and happened in the SL group. It is striking to note the poor prognosis of relapses in most of the cases, even treated, which resulted in death after a few months. Although additional studies in the future can define parameters to select women with Stage I EEC at intermediate risk candidate to perform complete surgical staging, image exams, biomolecular markers, and minimally invasive surgery seems to be a reasonable alternative to control operative complications and costs. Tumors G3 need to be managed more carefully and indication of adjuvant therapeutic, even systemic, can be evaluated.

e risk candidate to perform complete surgical staging, image exams, biomolecular markers, and minimally invasive surgery seems to be a reasonable alternative to control operative complications and costs. Tumors G3 need to be managed more carefully and indication of adjuvant therapeutic, even systemic, can be evaluated. In conclusion, systematic lymphadenectomy in the surgical staging of EEC patients at intermediate risk presented no benefits regarding recurrence, disease-free survival, cancer-specific survival or mortality when compared to patients who did not undergo complete surgical staging. Our results provide further evidence to support the current trend to avoid systematic lymphadenectomy in the surgical approach to selected women, particularly those with intermediate risk neoplasms. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgement None [The authors provided the financial support for this study].

Introduction Endometriosis is a gynecological disorder defined by the histological presence of endometrial glands and stroma outside the uterus [1]. It is estimated to affect 10–15 % of women of reproductive age [2,3]. Three types of endometriosis have been identified although they are often associated: peritoneal, ovarian (also called endometrioma), and deep endometriosis (DE). Based on the relation between the depth of infiltration and intensity of pain, DE has been defined as endometriosis infiltrating beneath the peritoneum over 5 mm [4,5]. However, in accordance with previous reports, DE should be defined by the infiltration of anatomical structures and organs regardless of the depth of penetration [6,7]. The most common locations of pelvic DE are the uterosacral ligaments, vagina, rectovaginal septum, colorectum junction and bladder [[8], [9], [10]].

]. However, in accordance with previous reports, DE should be defined by the infiltration of anatomical structures and organs regardless of the depth of penetration [6,7]. The most common locations of pelvic DE are the uterosacral ligaments, vagina, rectovaginal septum, colorectum junction and bladder [[8], [9], [10]]. First intention treatment of DE is based on hormonal contraception, progestins, and GnRH analogues. Surgery is restricted to patients who do not respond to medical treatment. This strategy is supported by previous studies as well as national and international guidelines [[11], [12], [13]] highlighting the risk of severe complications after DE resection. However, in contrast to colorectal endometriosis, few data have focused on surgical morbidity using the standardized Clavien-Dindo classification in patients with pelvic DE without bowel involvement [14,15]. Moreover, few data are available to determine whether the Enzian and ASRM (American Society for Reproductive Medicine) classifications are useful to predict postoperative complications [16,17]. Hence, the objectives of this study were to evaluate the incidence of complications after surgery for DE without bowel involvement, and to develop a nomogram for predicting their occurrence. Materials and methods Patients We conducted a retrospective analysis of our prospective database using information from women with DE without bowel involvement who underwent surgery from January 2006 to December 2014 at Tenon University Hospital in Paris, France.

Hence, the objectives of this study were to evaluate the incidence of complications after surgery for DE without bowel involvement, and to develop a nomogram for predicting their occurrence. Materials and methods Patients We conducted a retrospective analysis of our prospective database using information from women with DE without bowel involvement who underwent surgery from January 2006 to December 2014 at Tenon University Hospital in Paris, France. For each woman, the following parameters were recorded: age at surgery, body mass index (BMI), smoking status, presence of endometrioma and adenomyosis on magnetic resonance imaging (MRI), fertility before surgery, symptoms, previous surgery for endometriosis, type of surgery, surgical route (laparoscopy or laparotomy), Enzian and ASRM scores calculated during surgery. All patients gave their consent to participate in the study. The study was approved by the Ethics Committee (CEROG) of the College National des Gynécologues et Obstétriciens Français (CNGOF) (reference number: CEROG 2012-GYN-10-03). Preoperative diagnosis and surgery All the patients underwent transvaginal sonography and MRI to evaluate the locations of DE using previously published criteria, and the presence of endometrioma and adenomyosis [5,18]. Indication for surgery was based on symptoms, failure of medical treatment, and associated infertility.

Preoperative diagnosis and surgery All the patients underwent transvaginal sonography and MRI to evaluate the locations of DE using previously published criteria, and the presence of endometrioma and adenomyosis [5,18]. Indication for surgery was based on symptoms, failure of medical treatment, and associated infertility. Endometriosis surgery was performed by laparoscopy by three experienced surgeons. The first step of the surgery consisted of exploring the abdominopelvic cavity to exhaustively assess the endometriotic lesions and calculate the ASRM and Enzian scores. Adhesiolysis and salpingo-ovariolysis were performed if necessary. The endometrioma were treated by cystectomy, PlasmaJet® vaporization or expectant management depending on lesion size and preoperative ovarian reserve evaluation. The ureters were systematically identified before dissection in the case of major infiltration, and uni- or bilateral ureterolysis was performed when required. Once the external lateral surface of the uterosacral ligament had been fully liberated, the rectovaginal space and the ipsilateral pararectal fossa were opened. The uterosacral ligaments and torus were removed if infiltrated. When the vaginal wall was involved, an en bloc resection including the uterosacral ligaments and a partial colpectomy was performed. All patients underwent a first postoperative visit 4–6 weeks after surgery. When data were not available in the hospital medical records, the patients were contacted by phone or by e-mail.

hen the vaginal wall was involved, an en bloc resection including the uterosacral ligaments and a partial colpectomy was performed. All patients underwent a first postoperative visit 4–6 weeks after surgery. When data were not available in the hospital medical records, the patients were contacted by phone or by e-mail. Outcome measures All intra- and postoperative complications were recorded. In accordance with the Clavien-Dindo classification, complications were classified as minor when of grade I-II (deviation from the normal postoperative course without the need for surgical, endoscopic or radiological interventions) and major when of grade IIIa (requiring surgical, endoscopic or radiological intervention without general anesthesia), IIIb (requiring surgical, endoscopic or radiological intervention under general anesthesia), IV (life-threatening complication, including central nervous system complications or requiring intermediate or intensive care unit management) and V (death). In addition, de novo voiding dysfunction requiring self-catheterization lasting more than 1 month was considered a major complication. For statistical analysis, the primary composite endpoint was the occurrence of any Clavien-Dindo complication or voiding dysfunction lasting more than 1 month.

Outcome measures All intra- and postoperative complications were recorded. In accordance with the Clavien-Dindo classification, complications were classified as minor when of grade I-II (deviation from the normal postoperative course without the need for surgical, endoscopic or radiological interventions) and major when of grade IIIa (requiring surgical, endoscopic or radiological intervention without general anesthesia), IIIb (requiring surgical, endoscopic or radiological intervention under general anesthesia), IV (life-threatening complication, including central nervous system complications or requiring intermediate or intensive care unit management) and V (death). In addition, de novo voiding dysfunction requiring self-catheterization lasting more than 1 month was considered a major complication. For statistical analysis, the primary composite endpoint was the occurrence of any Clavien-Dindo complication or voiding dysfunction lasting more than 1 month. Statistical analysis Development and predictive accuracy of the model A nomogram was developed for predicting the likelihood of complications after surgery for DE without bowel involvement. We considered three groups at risk of postoperative complications according to the Enzian classification (Table 1); (i) a low-risk group with only location A0, A1, B1 and C0, (ii) an intermediate risk group with A2 and/or B2 location, and (iii) a high-risk group with at least A3, B3 or C1 location.Table 1 Distribution of surgical complications according to Enzian risk groups.

cations according to the Enzian classification (Table 1); (i) a low-risk group with only location A0, A1, B1 and C0, (ii) an intermediate risk group with A2 and/or B2 location, and (iii) a high-risk group with at least A3, B3 or C1 location.Table 1 Distribution of surgical complications according to Enzian risk groups. Table 1ENZIAN classification (N = 220) N (%) Low risk 53 (24.1) - Grade I-II Clavien-Dindo complications 4 (7.5) - Grade III Clavien-Dindo complications 2 (3.8) - Voiding dysfunction 1 (1.9) Intermediate risk 87 (39.5) - Grade I-II Clavien-Dindo complications 7 (8) - Grade III Clavien-Dindo complications 2 (2.3) - Voiding dysfunction 4 (4.6) High risk 80 (36.4) - Grade I-II Clavien-Dindo complications 20 (25) - Grade III Clavien-Dindo complications 7 (8.75) - Voiding dysfunction 6 (7.5)

dysfunction 1 (1.9) Intermediate risk 87 (39.5) - Grade I-II Clavien-Dindo complications 7 (8) - Grade III Clavien-Dindo complications 2 (2.3) - Voiding dysfunction 4 (4.6) High risk 80 (36.4) - Grade I-II Clavien-Dindo complications 20 (25) - Grade III Clavien-Dindo complications 7 (8.75) - Voiding dysfunction 6 (7.5) A multivariate analysis was performed using the logistic regression model and including all the factors that were statistically significant on univariate analysis or clinically relevant from the literature [19]. The complexity of the model was controlled using the Akaike information criterion [20]. A P-value of 0.05 was considered significant. The final model equation was then organized as a nomogram designed to calculate patient-specific probabilities of complications after surgery for DE without bowel involvement. Values for each of the model covariates were mapped to points on a scale ranging from 0 to 100, with total points obtained for each model covariate mapped to the probability of a live birth associated with the area under the receiver-operating characteristic curve (AUC) to measure the model’s discriminatory power. It is generally accepted that an AUC of 1.0 indicates perfect accuracy between cases with or without a live birth, an AUC of 0.7–0.8 indicates satisfactory discrimination, values of 0.8 represent good discrimination whereas an AUC of 0.5 indicates no relationship [21]. Calibration was assessed using plots that overlap the prediction model.

ted that an AUC of 1.0 indicates perfect accuracy between cases with or without a live birth, an AUC of 0.7–0.8 indicates satisfactory discrimination, values of 0.8 represent good discrimination whereas an AUC of 0.5 indicates no relationship [21]. Calibration was assessed using plots that overlap the prediction model. A bootstrapping technique to obtain relatively unbiased estimates (200 repetitions) was used for internal validation. The bootstrapping method is based on resampling obtained by randomly drawing data and replacing them with samples from the original dataset. It provides an estimate of the average optimism of the AUC of the receiver-operating characteristics (AUC-ROC) [22]. Calibration was assessed using plots that overlapped the prediction model. Additional statistical tests The categorical and numerical variables were analyzed using the chi2 test and the Student t test, respectively. Differences were considered significant at a P-value of 0.05. All analyses were performed using the R package with the Design, Hmisc, Presence/absence (http:// lib.stat.cmu.edu/R/CRAN).

onal statistical tests The categorical and numerical variables were analyzed using the chi2 test and the Student t test, respectively. Differences were considered significant at a P-value of 0.05. All analyses were performed using the R package with the Design, Hmisc, Presence/absence (http:// lib.stat.cmu.edu/R/CRAN). Results Description of the study population During the study period, 370 women with DE without bowel involvement underwent a resection for DE. One hundred and fifty women for whom it was impossible to evaluate the ASRM or Enzian scores were excluded resulting in a study population of 220 women. The median age of the patients was 32 years (range 19–53 years). The BMI was 22.5 kg/m2 (range: 14.1–35.8). The majority of the patients were nulliparous (73%). Epidemiological and clinical characteristics of the population are summarized in Table 2 and Table 3.Table 2 Characteristics of the 220 patients with deep endometriosis.

age of the patients was 32 years (range 19–53 years). The BMI was 22.5 kg/m2 (range: 14.1–35.8). The majority of the patients were nulliparous (73%). Epidemiological and clinical characteristics of the population are summarized in Table 2 and Table 3.Table 2 Characteristics of the 220 patients with deep endometriosis. Table 2Characteristics (N = 220) Items Age (years) median (range) 32 (19-53) BMI (Kg/m2) median (range) 22.5 (14.1-35.8) Smoking N (%) 55 (25) Parity median (range) 0.48 (0-5) - 0 N (%) 159 (72.3) - 1 N (%) 27 (12.3) - ≥ 2 N (%) 34 (15.5) Previous surgery for endometriosis - No N (%) 149 (67.7) - Yes N (%) 71 (32.3) 1 48 ≥ 2 23 Preoperative symptoms N (%) - Gynecologic dysmenorrhea 180 (81.8) dyspareunia 149 (67.7) chronic pelvic pain 99 (45) - Digestive dyschezia 83 (37.7) - Urinary mvoiding dysfunction 19 (8.6) urinary infection 23 (10.5) Clinical lesion N (%) - Vagina 35 (15.9) - Uterosacral ligaments 167 (75.9) - Torus uterinum 141 (64.1) - Parametrium 15 (6.8) - Rectum 24 (10.9) Table 3 Location of deep endometriosis by MRI of the 220 patients. Table 3Lesion location (N = 220) N (%) Vagina 41 (18.6) Torus Uterinum 186 (84.5) Utero-sacral ligaments 206 (92.3) - right 43 - left 27 - bilateral 136 Rectum 15 (6.8) Other digestive lesion 2 (0.9) Bladder 13 (5.9) Parametrium 19 (8.6) - right 5 - left 14 Ureterohydronephrosis 9 (4.1) - right 2 - left 7 Endometrioma 80 (36.4) - right 26 - left 29 - bilateral 25 Associated adenomyosis 50 (22.7)

.5) Utero-sacral ligaments 206 (92.3) - right 43 - left 27 - bilateral 136 Rectum 15 (6.8) Other digestive lesion 2 (0.9) Bladder 13 (5.9) Parametrium 19 (8.6) - right 5 - left 14 Ureterohydronephrosis 9 (4.1) - right 2 - left 7 Endometrioma 80 (36.4) - right 26 - left 29 - bilateral 25 Associated adenomyosis 50 (22.7) Surgical procedures (Table 4) The main indication for surgical management was pain (155 patients, 70%), followed by the association of pain and infertility (60 patients, 27%), and infertility (five patients, 3%). Nearly all the patients (97%) underwent laparoscopic management. Only one conversion to laparotomy (0.5%) was required due to extensive abdominopelvic adhesions. The remaining 2.5% of the patients underwent a laparotomy due to the association of DE with uterine fibroids requiring a multiple myomectomy.Table 4 Surgical procedures for DE resection for the 220 patients.

pic management. Only one conversion to laparotomy (0.5%) was required due to extensive abdominopelvic adhesions. The remaining 2.5% of the patients underwent a laparotomy due to the association of DE with uterine fibroids requiring a multiple myomectomy.Table 4 Surgical procedures for DE resection for the 220 patients. Table 4Surgical characteristics (N = 220) N (%) Indication for resection - Pain 155 (70.4) - Infertility 5 (2.3) - Pain and infertility 60 (27.3) Surgical approach - Laparotomy 5 (2.3) - Laparoscopy 214 (97.3) - Laparoconversion 1 (0.5) Operating time (min) median (range) 125 (40-320) Resection of Gynecologic lesions - Ovarian fenestration 9 (4.1) - Ovarian cystectomy 54 (23.2) right 25 left 21 bilateral 8 - Salpingectomy 30 (13.6) right 16 left 9 bilateral 5 - Adnexectomy 18 (8.2) right 6 left 6 bilateral 6 - Hysterectomy 36 (16.4) - Torus uterinum resection 170 (77.3) - Uterosacral ligaments resection 201 (91.4) right 30 left 28 bilateral 143 - Partial colpectomy 37 (16.8) Resection of urinary lesions - Partial bladder resection 9 (4.1) - Ureterolysis 155 (70.5) right 22 left 43 bilateral 90 - Ureterolysis with parametrectomy 41 (18.6) right 10 left 19 bilateral 12 - Ureteroneocystostomy 4 (1.8) right 1 left 3 Resection of bowel lesions - Superficial rectal shaving 56 (25.5) - Appendectomy 4 (1.8) The median operating time was 125 min (range: 40–320 minutes). No intraoperative transfusions were required. Complications (Supplementary data 1) The mean hospital stay was 3.7 days (range: 1–19).

Table 4Surgical characteristics (N = 220) N (%) Indication for resection - Pain 155 (70.4) - Infertility 5 (2.3) - Pain and infertility 60 (27.3) Surgical approach - Laparotomy 5 (2.3) - Laparoscopy 214 (97.3) - Laparoconversion 1 (0.5) Operating time (min) median (range) 125 (40-320) Resection of Gynecologic lesions - Ovarian fenestration 9 (4.1) - Ovarian cystectomy 54 (23.2) right 25 left 21 bilateral 8 - Salpingectomy 30 (13.6) right 16 left 9 bilateral 5 - Adnexectomy 18 (8.2) right 6 left 6 bilateral 6 - Hysterectomy 36 (16.4) - Torus uterinum resection 170 (77.3) - Uterosacral ligaments resection 201 (91.4) right 30 left 28 bilateral 143 - Partial colpectomy 37 (16.8) Resection of urinary lesions - Partial bladder resection 9 (4.1) - Ureterolysis 155 (70.5) right 22 left 43 bilateral 90 - Ureterolysis with parametrectomy 41 (18.6) right 10 left 19 bilateral 12 - Ureteroneocystostomy 4 (1.8) right 1 left 3 Resection of bowel lesions - Superficial rectal shaving 56 (25.5) - Appendectomy 4 (1.8) The median operating time was 125 min (range: 40–320 minutes). No intraoperative transfusions were required. Complications (Supplementary data 1) The mean hospital stay was 3.7 days (range: 1–19). Intraoperative complications Two hundred twelve patients (96.4%) did not experience any intraoperative complications. The intraoperative complications observed in the remaining eight patients included: three cases of digestive injury requiring laparoscopic suture; two of vaginal injury; one bladder injury; one ureteral injury requiring a suture with JJ stent; and one intra-abdominal hemorrhage with abdominal wall hematoma subsequent to epigastric vessel injury and treated by transparietal suture.

t patients included: three cases of digestive injury requiring laparoscopic suture; two of vaginal injury; one bladder injury; one ureteral injury requiring a suture with JJ stent; and one intra-abdominal hemorrhage with abdominal wall hematoma subsequent to epigastric vessel injury and treated by transparietal suture. Postoperative complications One hundred sixty-seven patients (76%) did not experience any postoperative complications. Among the 53 patients (24%) presenting at least one postoperative complication: 31 patients had a Clavien-Dindo grade I-II complication (minor); 11 had a grade III complication (major); and 11 patients had voiding dysfunction. No grade IV-V complications were observed. Three of the 31 minor complications were of Clavien-Dindo grade I: two cases of seizure episodes and one case of pelvic hematoma. The remaining 28 patients had a grade II complication: 10 cases of urinary infection; nine cases of pyelonephritis; four of pelvic abscess; four cases of fever of unknown cause and treated by antibiotics; and one case of deep venous thrombosis.

ndo grade I: two cases of seizure episodes and one case of pelvic hematoma. The remaining 28 patients had a grade II complication: 10 cases of urinary infection; nine cases of pyelonephritis; four of pelvic abscess; four cases of fever of unknown cause and treated by antibiotics; and one case of deep venous thrombosis. Two of the 11 major complications were of Clavien-Dindo grade IIIa: one pelvic abscess treated by radiological drainage; and one uretero-hydronephrosis treated by nephrostomy followed by ureteral reimplantation. The remaining nine patients had a grade IIIb complication: three cases of pelvic peritonitis requiring an ileostomy (one case subsequent to ileal injury treated by segmental small bowel resection and ileostomy, one ileal injury treated by simple ileostomy, and one case of rectovaginal fistula); three cases of vaginal bleeding due to leakage and treated by simple suture; one case of uretero-vaginal fistula treated by JJ stent; one laparoscopic drainage of pelvic hematoma; and one case of abdominal wall hematoma treated by drainage. Six of the 11 patients experiencing voiding dysfunction lasting more than 1 month required self-catheterization for less than 6 months. The remaining five required self-catheterization for more than 6 months. In addition, three patients had late complications corresponding to two cases of vaginal granuloma treated by silver nitrate and one case of incisional hernia requiring a second surgery.

Six of the 11 patients experiencing voiding dysfunction lasting more than 1 month required self-catheterization for less than 6 months. The remaining five required self-catheterization for more than 6 months. In addition, three patients had late complications corresponding to two cases of vaginal granuloma treated by silver nitrate and one case of incisional hernia requiring a second surgery. Relation between ASRM and Enzian classifications and the occurrence of postoperative complication The distribution of postoperative complications according to the Enzian classification is given in Supplementary data 2. Based on the three Enzian classification risk groups: 53 (24.1%) patients were at low risk; 87 (39.5%) at intermediate risk; and 80 (36.4%) at high risk. A relation was observed between the Enzian risk groups and the occurrence of complications (Table 5).Table 5 Risk factors associated with occurrence of postoperative complications: univariate and multivariate analysis. Table 5H Univariate analysis Multivariate analysis OR (95 % CI) p OR (95 % CI) p Age 1.036 (0.982 – 1.093) 0.446 1.022 (0.968 – 1.079) 0.447 Previous surgical procedure for endometriosis - Non 0.33 (0.155 – 0.702) 0.007 0.343 (0.157 – 0.751) 0.007 - Oui 1 1 Risk group of Enzian classification - Low risk 1 1 - Intermediate risk 1.413 (0.413 – 4.84) 0.6308 1.359 (0.389 – 4.745) - High risk 4.083 (1.309 – 12.741) 0.0209 3.918 (1.229 – 12.484) 0.011

.079) 0.447 Previous surgical procedure for endometriosis - Non 0.33 (0.155 – 0.702) 0.007 0.343 (0.157 – 0.751) 0.007 - Oui 1 1 Risk group of Enzian classification - Low risk 1 1 - Intermediate risk 1.413 (0.413 – 4.84) 0.6308 1.359 (0.389 – 4.745) - High risk 4.083 (1.309 – 12.741) 0.0209 3.918 (1.229 – 12.484) 0.011 According to the ASRM classification, 15 patients had stage I, 86 stage II, 50 stage III, and 69 stage IV. Among the patients with stage I disease, one patient had a grade I-II complication and one had a grade III complication. Among the patients with stage II disease, 10 patients had a grade I-II complication, two had a grade III complication and three had voiding dysfunction. Among the patients with stage III disease, 10 patients had a grade I-II complication, two had a grade III complication and seven had voiding dysfunction. Among the patients with stage IV disease, 10 patients had a grade I-II complication, six patients had a grade III complication and one voiding dysfunction. No statistical relation was observed between the ASRM classification and the occurrence of complications. Moreover, no differences in the complication rate was observed between ASRM stages I and II or between ASRM stages III and IV. Model to predict complications after surgery for DE without bowel involvement In multivariate analysis (Table 5), a p-value below 0.20 was considered significant. Age, Enzian risk group, and previous surgery for endometriosis were significantly associated with postoperative complication after surgery and were included in the logistic regression model.

ter surgery for DE without bowel involvement In multivariate analysis (Table 5), a p-value below 0.20 was considered significant. Age, Enzian risk group, and previous surgery for endometriosis were significantly associated with postoperative complication after surgery and were included in the logistic regression model. The predictive model (Fig. 1) had an AUC of 0.72 (95% CI, 0.70–0.74) before the 200 repetitions of bootstrap sample corrections and 0.70 (95% CI, 0.68–72) afterwards (Fig. 2). No significant difference was observed between the predicted probability obtained from the bootstrap correction and the actual probabilities of postoperative complications (p = 0.19), implying that the nomogram was well calibrated. The average difference and the maximal difference in predicted and calibrated probabilities of recurrence were 0.023 and 0.089%, respectively (Supplementary data 3)Fig. 1 Predictive model of complications after surgery for DE without bowel involvement. The probability of complications is calculated by drawing a line to the point on the axis for each of the following variables: age, previous surgery for endometriosis and risk group of ENZIAN classification. The points for each variable are summed and located on the total point line. Next, a vertical line is projected from the total point line to the predicted probability bottom scale to obtain the individual probability of complications. Fig. 1Fig. 2 ROC curve. Fig. 2

The probability of complications is calculated by drawing a line to the point on the axis for each of the following variables: age, previous surgery for endometriosis and risk group of ENZIAN classification. The points for each variable are summed and located on the total point line. Next, a vertical line is projected from the total point line to the predicted probability bottom scale to obtain the individual probability of complications. Fig. 1Fig. 2 ROC curve. Fig. 2 Discussion The present retrospective study of complications following surgery for women with DE without bowel involvement, allowed us to develop a nomogram to predict postoperative complications based on three simple criteria: the patient’s age, previous surgery for DE and the Enzian classification. We found a postoperative complication rate of 24% during the first postoperative month. Most of these complications corresponded to Clavien-Dindo grade I-II complications were classified as minor according to the Clavien-Dindo classification. Among these, 90% were grade II mainly related to urinary tract infection and treated by antibiotics. Eleven patients (5%) experienced a major complication (two grade IIIa requiring a radiological intervention and nine grade IIIb requiring a second surgery) while no grade IV or V complications were observed. A further 11 patients required bladder self-catheterization.

urinary tract infection and treated by antibiotics. Eleven patients (5%) experienced a major complication (two grade IIIa requiring a radiological intervention and nine grade IIIb requiring a second surgery) while no grade IV or V complications were observed. A further 11 patients required bladder self-catheterization. In contrast to DE with bowel involvement [14,[23], [24], [25]], relatively few data focusing on complications of DE without bowel endometriosis are available. In a series of 568 patients with DE, Kondo et al [26] reported an overall complication rate of 13.9% (with 9.5% minor and 4.6% major complications) but did not describe these complications. In contrast, De La Hera-Lazaro observed a complication rate of 30.4% [27]. However, it is difficult to compare our series with previous studies because the reports do not systematically distinguish DE resection with and without bowel involvement. Moreover, most of the studies did not use the Clavien-Dindo classification to report the complication rates. In a prospective series of 203 patients with moderate to severe endometriosis according to the ASRM classification, Meuleman et al reported 1% of Clavien-Dindo grade I—II and 2% of grade ≥ III complications in the 127 patients without bowel involvement [14]. This apparent discrepancy in complication rates could be explained by several factors such as the inclusion in the group of patients with severe endometriosis those exhibiting endometrioma of more than 3 cm in diameter or with extensive adhesions without true DE. Moreover, the rate of voiding dysfunction was not reported while this complication represented 5% of our postoperative complications. The difficulties to compare morbidity of DE resection according to series impose the use a consensual scoring system such as the Clavien-Dindo classification but adapted to patients with endometriosis to take into account the specific risk of voiding dysfunction. Finally, although inclusion criteria excluded patients with colorectal endometriosis, it is important to note that some patients required a rectal shaving probably linked to an underestimation of serosal rectal involvement. However, none of our patients required a discoid or segmental colorectal resection. Moreover, in accordance with previous studies [28], appendicular endometriosis was ignored by preoperative IRM in four patients.

ome patients required a rectal shaving probably linked to an underestimation of serosal rectal involvement. However, none of our patients required a discoid or segmental colorectal resection. Moreover, in accordance with previous studies [28], appendicular endometriosis was ignored by preoperative IRM in four patients. Few data exist on urinary dysfunction after surgery of DE without bowel resection. Dubernard et al [29], comparing the incidence of voiding dysfunction according to uni- or bilateral uterosacral ligament resection, demonstrated an increased risk correlated with the extent of the resection. In a literature review on urinary dysfunction, despite the absence of a consensual definition of bladder voiding dysfunction, Bonneau et al [30] reported that DE surgery was associated with a risk of urinary dysfunction, mainly corresponding to de novo voiding dysfunction, in 1.4%–29.2% of cases with a mean value of 4.8%. This is in agreement with our results.

ion, despite the absence of a consensual definition of bladder voiding dysfunction, Bonneau et al [30] reported that DE surgery was associated with a risk of urinary dysfunction, mainly corresponding to de novo voiding dysfunction, in 1.4%–29.2% of cases with a mean value of 4.8%. This is in agreement with our results. However, complications should be evaluated not only by the percentage but also according to the extent of the DE lesions. In the present study, both ASRM and Enzian classifications were used to evaluate the extent of the DE lesions. Although a trend for a relation was observed between ASRM classification and the occurrence of postoperative complications, it is interesting to note that this difference was not significant between stages I and II or between stages III and IV implying that it cannot be used as a predictor of postoperative complications. In contrast, using uni- and multivariate analysis, a relation was found between the Enzian score and Enzian risk groups and the occurrence of postoperative complications. Indeed, all Clavien-Dindo grade III complications occurred in patients with Enzian grade A3 or B3. Moreover, using three simple items – the age of the patient, previous surgery for DE and the Enzian classification – it was possible to build a nomogram to predict the occurrence of severe postoperative complications. The usefulness of the Enzian score in predicting the risk of surgical complications associated with DE resection is supported by a recent study by Di Paola et al [31] who demonstrated a high correlation between the preoperative MRI Enzian score and the intraoperative Enzian score.

of severe postoperative complications. The usefulness of the Enzian score in predicting the risk of surgical complications associated with DE resection is supported by a recent study by Di Paola et al [31] who demonstrated a high correlation between the preoperative MRI Enzian score and the intraoperative Enzian score. Some limits of the present study deserve to be mentioned. First, the retrospective nature of the study cannot exclude the risk of biases. Second, the long study period and the exclusion of patients with incomplete data on the exact location of DE on MRI and due to the lack of ASRM or Enzian values from the initial population is another limit of the present study. Third, we included voiding dysfunction requiring self-catheterization for more than 1 month as a severe complication although this complication cannot be clearly categorized according to Clavien-Dindo classification. Fourth, the Enzian score calculation was based on intraoperative evaluation of DE but not on MRI. While all DE lesions were clearly distinguished on MRI, the lesion size was not systematically measured. Fifth, we classified the Enzian score into three risk groups. This approach is not subject to consensus but reflects the extent of the disease and the requirement for multiple surgical procedures. Finally, although the calibration of the model was good, the ROC curve was only 0.72. Taking into account these limitations, further studies are required to externally validate and assess the robustness the present nomogram.

us but reflects the extent of the disease and the requirement for multiple surgical procedures. Finally, although the calibration of the model was good, the ROC curve was only 0.72. Taking into account these limitations, further studies are required to externally validate and assess the robustness the present nomogram. Conclusion The nomogram developed by this study based on three simple criteria – the age of the patient, previous surgery for DE and the Enzian classification – could be used to evaluate the risk of severe postoperative complications associated with the resection of DE without bowel involvement. This is important in a context where clinicians are increasingly interested in supporting women to make an informed decision based on individual criteria. Conflict of interest None.

Introduction Hyperthyroidism complicates approximately 0.1–0.4% of pregnancies, with 85–90% of cases due to Graves’ disease (GD), an autoimmune disorder caused by an immunoglobulin G antibody that stimulates the thyroid-stimulating hormone (TSH) receptor [1]. If not properly managed, it can result in severe maternal, fetal, and neonatal morbidity and mortality. Maternal complications include abortion, pre-eclampsia, preterm labor, placental abruption, congestive heart failure, and thyroid storm [2]. The fetus of a pregnant woman with GD is at risk of thyroid dysfunction from the transplacental transfer of the TSH receptor antibody. A pregnant woman with GD is usually treated with antithyroid drugs (ATDs), but ATDs can pass through the placenta, and high doses can cause fetal hypothyroidism [3]. Fetal goiter is an abnormal enlargement of the fetal thyroid gland, and it indicates thyroid dysfunction [4]. Untreated fetal goiters are often associated with perinatal and postnatal complications caused by the goiters themselves and the attendant thyroid dysfunction. Prenatal management can prevent these adverse outcomes, but systematic verifications of these interventions are limited, and the risk-to-benefit ratio is controversial. This review summarizes the current knowledge about fetal goiter associated with maternal GD and evaluates the most significant new findings regarding its in utero and peripartum management.

adverse outcomes, but systematic verifications of these interventions are limited, and the risk-to-benefit ratio is controversial. This review summarizes the current knowledge about fetal goiter associated with maternal GD and evaluates the most significant new findings regarding its in utero and peripartum management. Fetal goiter associated with maternal Graves’ disease Fetal goiter is found in 19% of the fetuses carried by mothers with GD [5]. Fetal goiter associated with maternal GD can result from either fetal hyperthyroidism or hypothyroidism. Fetal goiter is a very rare phenomenon; indeed, the incidence of goitrous hypothyroidism is approximately one per 40,000 live births, and goiter is found in only 10%–15% of all cases of congenital hypothyroidism [6,7]. The incidence of goitrous hyperthyroidism is unknown.

either fetal hyperthyroidism or hypothyroidism. Fetal goiter is a very rare phenomenon; indeed, the incidence of goitrous hypothyroidism is approximately one per 40,000 live births, and goiter is found in only 10%–15% of all cases of congenital hypothyroidism [6,7]. The incidence of goitrous hyperthyroidism is unknown. After 20 weeks of gestation, the fetal TSH receptors become responsive to TSH. Maternal thyroid-stimulating antibodies (TSAbs) cross the placenta and can overstimulate the fetal thyroid, causing hyperthyroidism and goiter [8]. Fetal goitrous hypothyroidism may occur in the presence of thyroid-blocking antibodies (TBAbs) or when high-dose ATDs are administered to the mother. As fetal hypothyroidism caused by TBAbs is extremely rare [8], fetal hypothyroidism accompanied by maternal GD is commonly caused by the excessive placental transfer of maternal ATDs [3]. Before the onset of fetal thyroid function during midgestation, the fetus relies on the transplacental passage of maternal thyroid hormone [8]; therefore, there is no need to consider any direct effects of ATDs. After midgestation, the maternal thyroxine (T4) supply remains extremely important to the fetus, but the transfer of maternal T4 may not be able to fully compensate for the blockage of fetal thyroid hormone synthesis by maternal ATDs. Consequently, the thyroid lobes enlarge, which is likely caused by the hyperstimulation of the tissue by the increased levels of fetal TSH in response to fetal thyroid insufficiency. Fetal goiter can cause complications that are attributable either to the goiter itself, for example, the mechanical effects of the neck mass, or to the hormonal abnormalities. In utero, a very large goiter may cause esophageal compression with secondary polyhydramnios or tracheal compression, both of which are compounded by dystocia caused by neck hyperextension [9]. At birth, tracheal compression caused by the enlarged thyroid gland can lead to respiratory distress and death [9].

rmalities. In utero, a very large goiter may cause esophageal compression with secondary polyhydramnios or tracheal compression, both of which are compounded by dystocia caused by neck hyperextension [9]. At birth, tracheal compression caused by the enlarged thyroid gland can lead to respiratory distress and death [9]. Fetal hyperthyroidism can cause fetal growth restriction (FGR) which is accompanied by accelerated bone maturation, goiter development, and craniosynostosis. Thyrotoxicosis, comprising tachycardia, cardiac failure, and hydrops, can occur, which may cause premature birth, intrauterine or perinatal death, or serious neurological sequelae [10]. Fetal hypothyroidism is associated with retarded skeletal development, mental retardation, hearing defects, poor visuomotor abilities, delayed speech and language development, and poor attention and memory skills [11]. Despite immediate postnatal evaluations and thyroid hormone replacement, some neurological abnormalities may persist. The degree of neurological impairment has been associated with the severity of the hypothyroidism and the age at diagnosis. The treatment of fetal hypothyroidism without a goiter remains controversial, because it is difficult to diagnose, and it is unclear whether the condition affects a child’s prognosis [3,9]. Since the presence of a goiter clearly indicates serious fetal thyroid dysfunction, prenatal identification and treatment of goiter are recommended [12].

hypothyroidism without a goiter remains controversial, because it is difficult to diagnose, and it is unclear whether the condition affects a child’s prognosis [3,9]. Since the presence of a goiter clearly indicates serious fetal thyroid dysfunction, prenatal identification and treatment of goiter are recommended [12]. Diagnosis of fetal goiter and thyroid status The early detection of fetal goiter and appropriate evaluation of fetal thyroid dysfunction are crucial to prevent adverse outcomes in fetuses and neonates. Fetal ultrasonography As the fetal thyroid gland can be accurately assessed using serial ultrasonography (US) at 20–36 weeks of gestation, a fetal goiter can be diagnosed using US during the second or third trimester. On US, a fetal goiter appears as a homogeneous, symmetrical, and hyperechogenic mass in the anterior neck region, and it is defined as a fetal goiter when the thyroid gland’s width and circumference exceed the 95th percentile for the gestational age [4]. When a goiter is detected, it is important to document its size and vascularity, and more importantly, the size of the trachea and the presence of fetal swallowing should be noted.

nd it is defined as a fetal goiter when the thyroid gland’s width and circumference exceed the 95th percentile for the gestational age [4]. When a goiter is detected, it is important to document its size and vascularity, and more importantly, the size of the trachea and the presence of fetal swallowing should be noted. In fetuses with goiters, determining whether the cause is fetal hypothyroidism or hyperthyroidism is the main clinical challenge [3]. On US, goiter vascularization, the fetal heart rate, bone maturation, and fetal movement differ appreciably according to the presence of fetal hyperthyroidism or hypothyroidism. For example, using the Doppler technique, an increased blood flow throughout the gland is linked to fetal hyperthyroidism, whereas an increased blood flow at the periphery of the gland has been associated with fetal hypothyroidism [12]. Huel et al. proposed an ultrasound scoring system, based on the color Doppler pattern (peripheral/central vascularization), fetal heart rate (tachycardia/normal heart rate), bone maturation (early/late), and fetal movements (intense/normal), to predict fetal thyroid function in cases of fetal goiter [5]. Although the researchers correctly distinguished 36 cases of fetal hypothyroidism from hyperthyroidism using this scoring system, some inconsistency remains, and its practical applicability is poor.

ation (early/late), and fetal movements (intense/normal), to predict fetal thyroid function in cases of fetal goiter [5]. Although the researchers correctly distinguished 36 cases of fetal hypothyroidism from hyperthyroidism using this scoring system, some inconsistency remains, and its practical applicability is poor. Fetal magnetic resonance imaging Fetal magnetic resonance imaging (MRI) complements US examinations by helping to differentiate goiters from cervical tumors, delineate a goiter’s full extent [13], and to assess the levels by which a goiter obstructs the trachea and esophagus in the presence of massive polyhydramnios [14]. Fetal MRI also helps differentiate the status of the thyroid by analyzing the T1 and T2 signals. A fetal thyroid gland can be identified as a hyperintense structure in the neck on T1-weighted images, which can differentiate goiters from other neck masses more readily than US [15]. Moreover, the intensity of the thyroid T2 signals varies with the functional status, because it correlates with the iodine concentration; hence, a thyroid gland with a high iodine concentration has a low signal intensity, and a gland with a low iodine concentration has a high signal intensity [16]. As a normal thyroid gland’s signal intensity is as low as that of muscle on T2-weighted images, the presence of a signal that is higher than that associated with muscle signifies a hypofunctioning thyroid gland [16]. Table 1 summarizes our review of previous reports that describe MRI findings from fetal goiters between 2000 and 2018 [13,15,[17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]]. Without a maternal thyroid gland abnormality, fetal hypothyroidism is characterized by a fetal thyroid gland that shows a hyperintense signal on T2-weighted images. In contrast, in the presence of maternal GD, there might be no relationship between the signal intensity and fetal thyroid abnormalities on T2-weighted images. Therefore, MRI may not correspond to expectations regarding the discrimination of fetal hyperthyroidism and hypothyroidism in the presence of maternal GD.Table 1 Intrauterine magnetic resonance imaging for fetuses with goiters, 2000–2018.

tween the signal intensity and fetal thyroid abnormalities on T2-weighted images. Therefore, MRI may not correspond to expectations regarding the discrimination of fetal hyperthyroidism and hypothyroidism in the presence of maternal GD.Table 1 Intrauterine magnetic resonance imaging for fetuses with goiters, 2000–2018. Table 1Reference Maternal thyroid disease Antithyroid drugs Maternal thyroid status GA at MRI (weeks) Signal intensity Fetal thyroid status Comments T1-weighted image T2-weighted image High Iso Miyata, et al. [17] GD Yes Hyper 36 N/A High Hypo Harreld, et al. [18] GD Yes N/A 34 High Low N/A Matsumoto, et al. [19] GD Yes Euthyroid 34 N/A High Hyper Kanai, et al. [20] GD Yes Euthyroid 36 High Low Hypo Oguma, et al. [21] GD Yes Hyper 32 High Low Hypo Overcash, et al. [22] Hypo No Hypo 29 N/A High N/A Matsumoto, et al. [23] No No Euthyroid 29 High N/A Hypo Kondoh, et al. [15] No No N/A 32 High High Hypo Ohira, et al. [24] No No Hypo 28 N/A High Hypo Maternal TSBAb Miyamoto, et al. [25] No No Euthyroid 37 High High Hypo Maternal iodine excess Inoue, et al. [26] No No Euthyroid 29 High High Hypo Neto, et al. [27] No No Euthyroid 34 N/A High N/A GA: gestational age; MRI: magnetic resonance imaging; GD: Graves’ disease; N/A: not available; Hypo: hypothyroidism; Hyper: hyperthyroidism; Iso: isointense; TSBAb: thyroid stimulation-blocking antibody.

iodine excess Inoue, et al. [26] No No Euthyroid 29 High High Hypo Neto, et al. [27] No No Euthyroid 34 N/A High N/A GA: gestational age; MRI: magnetic resonance imaging; GD: Graves’ disease; N/A: not available; Hypo: hypothyroidism; Hyper: hyperthyroidism; Iso: isointense; TSBAb: thyroid stimulation-blocking antibody. Amniotic fluid sampling Amniocentesis to evaluate the TSH levels in the amniotic fluid may be useful to check fetal thyroid metabolism, and it is less technically demanding and safer than cordocentesis. Previous reports indicate that the amniotic fluid TSH levels reflect fetal rather than maternal thyroid function, because TSH does not cross the placenta [28]. Moreover, reference ranges have been established for thyroid hormone levels in the amniotic fluid [29]. These data may support the use of amniotic fluid analysis to diagnose fetal hypothyroidism; however, the value of amniotic fluid analysis alone in the presence of fetal thyroid dysfunction is controversial. Discrepancies between the intra-amniotic and neonatal blood TSH levels have been described, and the relative maternal and fetal contributions to the hormone levels in the amniotic fluid are unclear [28]. Indirect quantification based on the thyroid hormone levels in the amniotic fluid does not accurately reflect the function of the fetal thyroid gland, and it only enables the assessment of dynamic changes. Hence, the TSH levels in the amniotic fluid may have some diagnostic value for fetuses with severe hypothyroidism, especially when cordocentesis is not available.

mone levels in the amniotic fluid does not accurately reflect the function of the fetal thyroid gland, and it only enables the assessment of dynamic changes. Hence, the TSH levels in the amniotic fluid may have some diagnostic value for fetuses with severe hypothyroidism, especially when cordocentesis is not available. Fetal blood sampling Cordocentesis, which is the gold standard diagnostic method [9], enables direct measurements of the fetal thyroid hormone levels and accurate determinations of the fetal thyroid status because normal reference values for fetal thyroid function have been established [8,30]. However, the minor and major complications associated with this invasive procedure should be stressed; these include fetal bleeding, infections, bradycardia, premature rupture of membranes, and death, which occur in 0.5–9.0% of patients [28]. Cordocentesis may not be required for all patients, and it should only be undertaken in specialist centers. The European Society for Paediatric Endocrinology consensus guidelines on the screening, diagnosis, and management of congenital hypothyroidism recommend using cordocentesis rather than amniocentesis to assess fetal thyroid function [31]. Intrauterine treatment of fetal goiter Regardless of whether hyperthyroidism or hypothyroidism is present, the treatment of a large fetal goiter is essential, because tracheal obstruction could cause neonatal death and fetal neck hyperextension could cause mechanical issues during delivery.

Fetal blood sampling Cordocentesis, which is the gold standard diagnostic method [9], enables direct measurements of the fetal thyroid hormone levels and accurate determinations of the fetal thyroid status because normal reference values for fetal thyroid function have been established [8,30]. However, the minor and major complications associated with this invasive procedure should be stressed; these include fetal bleeding, infections, bradycardia, premature rupture of membranes, and death, which occur in 0.5–9.0% of patients [28]. Cordocentesis may not be required for all patients, and it should only be undertaken in specialist centers. The European Society for Paediatric Endocrinology consensus guidelines on the screening, diagnosis, and management of congenital hypothyroidism recommend using cordocentesis rather than amniocentesis to assess fetal thyroid function [31]. Intrauterine treatment of fetal goiter Regardless of whether hyperthyroidism or hypothyroidism is present, the treatment of a large fetal goiter is essential, because tracheal obstruction could cause neonatal death and fetal neck hyperextension could cause mechanical issues during delivery. Treatment of fetal goiter caused by hyperthyroidism A fetus that has hyperthyroidism benefits directly from the administration of ATDs to the mother, because ATDs cross the placenta and act on the fetal thyroid gland. A mother with hyperthyroidism who receives ATDs will usually require an increased ATD dose. If a euthyroid mother who is not receiving medication is carrying a fetus with a goiter, it is usually possible to treat the mother with ATDs. Although propylthiouracil (PTU) and methimazole (MMI) control hyperthyroidism similarly, PTU is a first-line drug for the treatment of fetal hyperthyroidism, and its use as the initial treatment is preferred, because MMI causes a series of malformations when it is used during the first trimester [32]. The resolution of fetal tachycardia has been used to indicate the successful intrauterine medical treatment of fetal hyperthyroidism. Nonetheless, ATDs may expose a fetus to the risk of hypothyroidism; therefore, ATDs should be administered at low doses that maintain the fetal heart rate at 140 beats/min [8]. If the mother develops hypothyroidism during this treatment, levothyroxine (L-T4) is added, because very little T4 is transferred across the placenta.

less, ATDs may expose a fetus to the risk of hypothyroidism; therefore, ATDs should be administered at low doses that maintain the fetal heart rate at 140 beats/min [8]. If the mother develops hypothyroidism during this treatment, levothyroxine (L-T4) is added, because very little T4 is transferred across the placenta. A hyperthyroid fetus that required urgent treatment and was administered potassium iodide in combination with ATDs has been described with favorable results [19]. Potassium iodide acutely inhibits hormonal secretions within hours of its administration to hyperthyroid patients, and there are few side-effects. However, additional fetal monitoring is advised, because administering iodine to mothers may cause fetal and neonatal hypothyroidism and goiters [32]. Beta-adrenergic blocking agents, including propranolol, can be used in severe cases. As most symptoms of fetal thyrotoxicosis, including tachycardia, are mediated by increased adrenergic responses, beta-blockers can ameliorate these symptoms within a few hours of their administration; however, their use requires caution because of their adverse effects, including FGR, prolongation of labor, neonatal bradycardia, hypotension, hypoglycemia, and prolonged jaundice [32].

re mediated by increased adrenergic responses, beta-blockers can ameliorate these symptoms within a few hours of their administration; however, their use requires caution because of their adverse effects, including FGR, prolongation of labor, neonatal bradycardia, hypotension, hypoglycemia, and prolonged jaundice [32]. Treatment of fetal goitrous hypothyroidism The treatment of fetal goitrous hypothyroidism requires optimization of the maternal thyroid status, which includes reducing or discontinuing treatment with ATDs [3], followed by conservative management involving neonatal administration of l-T4 [9,14]. In contrast to fetal hyperthyroidism, fetal hypothyroidism cannot be treated by administering l-T4 to the mother because the placental transfer of maternal T4 is minimal [9]. Hence, fetal hypothyroidism can only be treated by directly administering thyroid hormone to the fetal blood vessels or muscles, or to the amniotic fluid. Injecting the fetus itself is associated with the risk of injury and may require several puncture attempts. Injecting l-T4 intra-amniotically represents a safer and simpler management option compared with other methods, and it has a long treatment interval [33]; the fetus swallows and ingests the l-T4, and the fetal thyroid function normalizes as a consequence of the higher serum l-T4 levels and reduced TSH levels, which may be beneficial to the developing fetal brain [9]. The simultaneous decrease in the size of the goiter should reduce the obstetric risks at delivery [33]. To our knowledge, the first case of intra-amniotic l-T4 injections associated with maternal ATD overtreatment was reported in 1980 [34]. Since then, several intra-amniotic l-T4 injections have been performed; however, most data that describe the prenatal treatment of fetuses with goitrous hypothyroidism are from anecdotal case reports. The findings from one retrospective cohort study of 12 patients with fetal goitrous hypothyroidism support the benefits associated with reducing goiter sizes [35]; however, only two of the patients in this study had normal serum T4 levels at birth. The findings from a recent systematic review showed that six of 22 neonates (27%) had hypothyroidism after prenatal maternal ATD dose reductions and intra-amniotic l-T4 injections [3]. Hence, this effective treatment does not always prevent hypothyroidism after birth.

in this study had normal serum T4 levels at birth. The findings from a recent systematic review showed that six of 22 neonates (27%) had hypothyroidism after prenatal maternal ATD dose reductions and intra-amniotic l-T4 injections [3]. Hence, this effective treatment does not always prevent hypothyroidism after birth. Unanswered questions remain that include when treatment should begin, l-T4 dosing, and the interval and frequency of administration. As few pharmacokinetic data portray the fetal uptake and absorption of l-T4 from the amniotic fluid, different treatment regimens have been described. Table 2 summarizes 22 case reports that describe intra-amniotic l-T4 injections for fetal goitrous hypothyroidism in maternal GD [6,9,17,34,[36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]]. The median (interquartile range) dose, frequency, interval, and gestational age at the initiation of therapy were 250 (128–350) μg, 3 (2–4) times, 1 (1–2) weeks, and 32 (29–34) weeks of gestation, respectively. The critical gestational age at which therapy must be initiated should be determined clinically by balancing the risk of not treating the condition against the risk of prematurity at that gestational age. While some clinicians believe that the hormonal deficiency should be corrected quickly to prevent negative effects on the fetal brain and have initiated treatment at as early as 20 weeks of gestation, other clinicians have initiated treatment at as late as 37 weeks of gestation [9,33]. Regarding the fetal outcomes, intra-amniotic l-T4 injections reversed the laboratory evidence that indicated hypothyroidism, resolved the fetal goiters, and prevented asphyxia and respiratory distress in all except one of the patients. Moreover, all except one of the patients grew and developed normally after birth, but the follow-up period was no more than 4 years. Whether prenatal l-T4 treatment improves neurodevelopmental outcomes remains a matter of debate. In this series, most of the patients did not experience any adverse events, but two babies were born prematurely and chorioamnionitis occurred in one patient. Another publication describes preterm labor, FGR, chorioamnionitis, and fetal death following intra-amniotic l-T4 injections [14]. Moreover, a T4 overdose may cause fetal thyrotoxicosis.Table 2 . Previous cases of fetal goiter in maternal Graves’ disease treated with intraamniotic thyroxine administration.

one patient. Another publication describes preterm labor, FGR, chorioamnionitis, and fetal death following intra-amniotic l-T4 injections [14]. Moreover, a T4 overdose may cause fetal thyrotoxicosis.Table 2 . Previous cases of fetal goiter in maternal Graves’ disease treated with intraamniotic thyroxine administration. Table 2 Treatment Reference Maternal ATD and dose (mg/day) GA at diagnosis of fetal goiter (WGA) Fetal blood thyroid function at treatment completion ATDs reduction Intraamniotic LT4 administration Goiter reduction Complication GA at birth (WGA) and delivery mode Birth weight (g) Birth asphyxia Respiratory distress Neonatal blood thyroid function at birth Developmental delay TSH (mIU/L) Free T4 (pmol/L) Total T4 (nmol/L) GA at initiation (WGA) Dose (μg/dose) Frequency Interval (week) TSH (mIU/L) Free T4 (pmol/L) Total T4 (nmol/L) Weiner, et al. [34] PTU, 400 30 N/A N/A N/A Yes 34 200 1 – Yes No 36, CS 2891 No Yes (TTN) 17.9 N/A 100 N/A Davidson, et al. [9] PTU, 450 28 25 N/A 41 Yes 35 250 3 1 Yes No 38, VD 2960 No No 15.8 N/A 81 No Noia, et al. [36] PTU, 200 26 17 N/A 62 N/A 36 250 1 – Yes No Term, VD 3639 No No 2.8 24.4 188 No Van Loon, et al. [37] PTU, 600 33 >50 3.5 N/A Yes 33 250 4 1 Yes No 39, VD 3090 No No 15 8.8 N/A No Hadi, et al. [38] PTU, 600 31 32 N/A 45 Yes 33 250 3 1 Yes No 37, VD 2980 No Yes 15 N/A 116 No Hadi, et al. [38] PTU, 300 27 25 N/A 37 Yes 29 230 4 1 Yes Preterm birth 33, VD 2210 No No 17 N/A 103 No Nicolini, et al. [39] PTU, 300 24 1640 1 N/A Yes 25 600 5 1–6 Yes No 38, VD 1830 No No 96.5 N/A 145 No Bruner, et al. [40] PTU, 100 28 378 3.2 <16 Yes 29 250–500 5 1 Yes Preterm birth 35, VD 2413 No No Normal Normal Normal No Bruner, et al. [40] PTU, 150 31 324 N/A <16 Yes 32 250 1 – Yes Preterm birth 32, CS 1723 No No N/A N/A N/A No Maragliano, et al. [41] PTU, 250 31 15,74 6 N/A Yes 34 250–500 2 2 Yes No 39, CS 2650 No No <0.1 11.8 N/A No Okumura, et al. [42] PTU, 300 25 98.65 11.6 N/A Yes 32 250 2 3 Yes No 39, VD 2630 No No Normal Normal Normal No Yanai, et al. [43] PTU, 200 29 38 9.8 N/A Yes 29 200 3 1 Yes No 36, VD 3000 No No Normal Normal Normal No Nath, et al. [44] PTU, 100 23 23.61 N/A N/A Yes 30 500 2 2 Yes No 36, CS N/A No No Normal Normal Normal No Miyata, et al. [17] PTU, 300 36 99 3.7 N/A No 37 300 2 1 Yes No 38, CS 3042 No No 33 18 N/A No Lassen, et al. [45] MMI, 30 31 29.9 10.7 N/A Yes 32 70 μg/kg/EFW 4 1 Yes No 36, CS 2880 No Yes (RDS) 8.3 14.2 N/A No Corral, et al.

[44] PTU, 100 23 23.61 N/A N/A Yes 30 500 2 2 Yes No 36, CS N/A No No Normal Normal Normal No Miyata, et al. [17] PTU, 300 36 99 3.7 N/A No 37 300 2 1 Yes No 38, CS 3042 No No 33 18 N/A No Lassen, et al. [45] MMI, 30 31 29.9 10.7 N/A Yes 32 70 μg/kg/EFW 4 1 Yes No 36, CS 2880 No Yes (RDS) 8.3 14.2 N/A No Corral, et al. [6] PTU, 300 24 480 1.8 18.5 Yes 34 500 4 1 Yes No 37, CS 3500 No No 26 18 149 Yes Koyuncu, et al. [46] PTU, 200 30 69.9 5.9 N/A No 30 250 1 – No Preterm birth 30, CS 1200 Yes Yes (RDS) 39.2 10.7 N/A No Bliddal, et al. [47] PTU, 200 23 >200 3.4 N/A Yes 23 50–100 6 1 Yes No 40, VD 4100 No No 5.4 N/A N/A No Bliddal, et al. [47] MMI, 20 21 34.5 13.8 N/A Yes 25 55–150 2 1 Yes No 40, CS 3630 No No 0.7 40.4 N/A No Munoz, et al. [48] PTU, 600 23 N/A N/A N/A No 29 200–400 2 4 Yes Preterm birth FGR 35, CS 1880 No No N/A N/A N/A No Kim, et al. [49] Radioactive 30 390 6.7 N/A – 30 200–400 2 1–4 Yes No 38, VD 2495 No No 11.8 17.9 N/A No Kobayashi, et al. [50] PTU, 150 32 97.8 6.1 N/A Yes 35 300 2 1 Yes No 37, CS 3224 No No 42.5 17.9 N/A No ATD: antithyroid drug; GA: gestational age; WGA: weeks of gestation; TSH: thyroid-stimulating hormone; T4: thyroxine; LT4: levothyroxine; PTU: propylthiouracil; MMI: methimazol; N/A: not available; EFW: estimated fetal weight; FGR: fetal growth restriction; CS: cesarean delivery; VD: vaginal delivery; TTN: transient tachypnea of the newborn; RDS: respiratory distress syndrome. Reference ranges of fetal thyroid function according to Thorpe-Beeston et al. are: TSH: 2–12  mIU/L; Free T4: 5.1–27 pmol/L; T4: 15–125 nmol/L [24]. Reference ranges of neonatal thyroid function according to Fisher are: TSH: 1–39 mIU/L; free T4: 28–68 pmol/L; T4: 142–277 nmol/L [10].

RDS: respiratory distress syndrome. Reference ranges of fetal thyroid function according to Thorpe-Beeston et al. are: TSH: 2–12  mIU/L; Free T4: 5.1–27 pmol/L; T4: 15–125 nmol/L [24]. Reference ranges of neonatal thyroid function according to Fisher are: TSH: 1–39 mIU/L; free T4: 28–68 pmol/L; T4: 142–277 nmol/L [10]. As a possible alternative, intra-amniotic l-tri-iodothyronine (L-T3) injections given either alone or in combination with l-T4, have been administered successfully [33,51]. As the effects of l-T3 on the fetus begin within 4–8 h of its administration, intra-amniotic l-T3 injections may reduce the size of the goitrous mass faster and at a lower dose than l-T4 injections. On the other hand, more frequent intra-amniotic l-T3 injections may be required to maintain a consistent effect because the half-lives of l-T3 and l-T4 are 1–2 days and 6–7 days, respectively [33]. The optimal dosing regimen for l-T3 remains unclear. Limited literature reviews have evaluated the different administration routes for l-T4, including umbilical cord injections or fetal intramuscular injections [6,52]. These methods should be considered for a patient whose ability to swallow is critically impaired, because the goitrous mass is causing extreme pressure on the esophagus. The wide variety of approaches to treatment exemplifies the lack of guidelines, and no systematic studies have been undertaken to date.

ctions [6,52]. These methods should be considered for a patient whose ability to swallow is critically impaired, because the goitrous mass is causing extreme pressure on the esophagus. The wide variety of approaches to treatment exemplifies the lack of guidelines, and no systematic studies have been undertaken to date. Peripartum management of fetal goiter The major problem regarding fetuses with goiters is how to manage their delivery when intrauterine treatment fails; however, there has been no generally accepted strategy for the management of childbirth. As the peripartum prognosis is mainly related to the risk of dystocia at delivery as a consequence of cervical deflection in such cases, cesarean delivery is highly recommended [48]. In all cases, the pediatric team must be informed of the goiter diagnosis to optimize the prevention of potential serious perinatal mechanical complications.

ognosis is mainly related to the risk of dystocia at delivery as a consequence of cervical deflection in such cases, cesarean delivery is highly recommended [48]. In all cases, the pediatric team must be informed of the goiter diagnosis to optimize the prevention of potential serious perinatal mechanical complications. For a patient in whom a fetal goiter may be causing airway obstruction, a cesarean delivery with ex utero intrapartum treatment (EXIT) should be considered. This procedure permits access to the fetal airway via intubation, laryngoscopy, or bronchoscopy, and, eventually, tracheostomy while the fetus remains under placental support, and can prevent respiratory insufficiency after birth, and it has significantly reduced the mortality of patients with large neck masses [53]. However, to the best of our knowledge, only eight cases involving EXIT for fetal goiter have been reported to date, although the detailed clinical course in each case was not described (Table 3) [[54], [55], [56], [57], [58], [59], [60], [61]]. Meanwhile, EXIT is accompanied by difficulties associated with preserving an adequate blood flow through the umbilical cord, protecting the placenta, and avoiding uterine contractions to ensure that there is sufficient time to establish an airway. Although there have been few fetal complications directly related to EXIT, the partially delivered fetus should be directly monitored with pulse oximetry and continuous echocardiography to detect fetal bradycardia and decreased myocardial contractility [53]. Regarding maternal complications, an important concern is bleeding, which is usually associated with uterine atony, related to the administration of high-dose tocolytic drugs or excess time spent establishing the fetal airway during the procedure [62]. Therefore, careful coordination among a multidisciplinary team that includes obstetricians (usually perinatologists), neonatologists, pediatric surgeons, otolaryngologists, and anesthesiologists is required to achieve successful outcomes from EXIT [53].Table 3 . Reported cases in which the ex utero intrapartum treatment procedure was performed in fetus with goiter.

sciplinary team that includes obstetricians (usually perinatologists), neonatologists, pediatric surgeons, otolaryngologists, and anesthesiologists is required to achieve successful outcomes from EXIT [53].Table 3 . Reported cases in which the ex utero intrapartum treatment procedure was performed in fetus with goiter. Table 3Reference Maternal thyroid disease Cause of fetal goiter Intrauterine treatment Gestational age at delivery (weeks) EXIT procedure Time to airway Apgar score (1 min/5 min) Maternal or neonatal complication Neonatal outcome Klee, et al. [54] Graves’ disease Hypothyroidism Intraamniotic T4 injection 38 Endotracheal intubation N/A 8 / 9 No Alive Fink, et al. [55] Graves’ disease N/A No 36 Endotracheal intubation 9 min 3 / 8 N/A Alive Harreld, et al. [56] Graves’ disease Hypothyroidism No 36 N/A N/A 3 / 8 Stridor in the neonate Alive Sriram, et al. [57] Unknown Hypothyroidism No 37 Endotracheal intubation N/A N/A / 8 No Alive Niiya, et al. [58] Unknown Hypothyroidism No 35 Endotracheal intubation 6 min N/A No Alive Whited, et al. [59] N/A N/A N/A 36 Endotracheal intubation 9 min N/A No Alive Scott, et al. [60] N/A N/A N/A 34 N/A N/A N/A No Alive Kornacki, et al. [61] Unknown Hypothyroidism Intraamniotic T4 injection 37 Endotracheal intubation N/A 9 / 9 No Alive EXIT: ex utero intrapartum treatment; N/A: not available; T4: thyroxine.

ted, et al. [59] N/A N/A N/A 36 Endotracheal intubation 9 min N/A No Alive Scott, et al. [60] N/A N/A N/A 34 N/A N/A N/A No Alive Kornacki, et al. [61] Unknown Hypothyroidism Intraamniotic T4 injection 37 Endotracheal intubation N/A 9 / 9 No Alive EXIT: ex utero intrapartum treatment; N/A: not available; T4: thyroxine. An innovative alternative to EXIT is fetal endoscopic tracheal intubation. The procedure involves fetal tracheoscopy and the subsequent insertion of an intrauterine orotracheal cannula under ultrasound guidance. This procedure ensures fetal tracheal permeability before delivery. However, to date, only two publications describe this new procedure that was performed on patients with large neck masses but not goiters [63,64].

acheoscopy and the subsequent insertion of an intrauterine orotracheal cannula under ultrasound guidance. This procedure ensures fetal tracheal permeability before delivery. However, to date, only two publications describe this new procedure that was performed on patients with large neck masses but not goiters [63,64]. Summary and recommendations The potential morbidity and mortality associated with a fetal goiter warrants a complete evaluation of the fetal thyroid status. This can be achieved through a combination of imaging methods, such as ultrasound examinations, which include color Doppler imaging, and MRI, and cordocentesis or amniocentesis to measure the fetal thyroid hormone levels. However, the thyroid hormone levels in the amniotic fluid may not accurately reflect fetal thyroid function. Regarding the treatment of fetal goitrous hypothyroidism, administering thyroid hormone intra-amniotically shrinks goiters and results in good developmental outcomes. In addition, when intrauterine treatment fails and a fetal goiter causes airway obstruction, intrapartum management, including EXIT, is required; however, no guidelines are available currently. A large-scale, well-designed study is necessary for monitoring therapeutic efficacy and long-term prognosis. For now, intrauterine and intrapartum treatments should be reserved for selected patients who are at a high risk of complications, and they should only be conducted by physicians with experience in intrauterine and peripartum care and related procedures.

sary for monitoring therapeutic efficacy and long-term prognosis. For now, intrauterine and intrapartum treatments should be reserved for selected patients who are at a high risk of complications, and they should only be conducted by physicians with experience in intrauterine and peripartum care and related procedures. Disclosure statement The author has no conflicts of interest to declare.

Introduction Functional hypothalamic amenorrhoea (FHA) is characterised by reversible menses cessation in women without any organic disorders, as a result of gonadotropin-releasing hormone (GnRH) pulsatile secretion suppression below a critical range. There is a complex interplay of excessive exercise, energy deficit, abnormal eating attitudes (AEA) in addition to external and intrapersonal psychological stressors on hypothalamic-pituitary-gonadal (HPG) axis desynchronization [1]. There is cumulative evidence suggesting that FHA is attributable to cognitive, behavioural, emotional and psychological stressors triggering a stress-arousal response [[2], [3], [4]]. Previous studies underscored the psychological FHA correlates; notwithstanding, the pivotal role of SleD has not been elucidated thus far. Given that the anxiety levels (AL) and sleep disorders (SleD) are often interrelated, we hypothesised that FHA women suffer from SleD. We aimed to revisit the AL role on FHA pathophysiology and to identify possible associations with other known FHA predisposing factors. Materials and methods Study approval We conducted a prospective case-controlled study at a tertiary referral clinic for gynaecological endocrinology and paediatric and adolescent gynaecology, spanning the period January 2016 to April 2018. The study received approval by the Ethics Committee of the University of Athens prior to enrolment (Protocol No 33/131 2016). Study participants We recruited a total of 127 women following written informed consent. Anonymity and confidentiality was secured for all data collected.

Materials and methods Study approval We conducted a prospective case-controlled study at a tertiary referral clinic for gynaecological endocrinology and paediatric and adolescent gynaecology, spanning the period January 2016 to April 2018. The study received approval by the Ethics Committee of the University of Athens prior to enrolment (Protocol No 33/131 2016). Study participants We recruited a total of 127 women following written informed consent. Anonymity and confidentiality was secured for all data collected. Patients with FHA Forty-one women met the diagnostic criteria of FHA: [1] history of secondary amenorrhoea for at least six months; [2] low serum luteinising hormone (<1 IU/Lit); [3] negative progesterone withdrawal test, where norethisterone was administered at a dose of 5 mg twice a day for five days; [4] a normal prolactin levels; [5] at least one of the following predisposing factors: excessive exercise (> 5 h per week) and more than 15% body weight loss. None of these women met the criteria for anorexia nervosa [5]. All women with headache, nausea, or visual disturbances underwent Magnetic Resonance Imaging (MRI) of the pituitary and brain to rule out central nervous system pathology. They were all high school or university students. Controls We recruited 86 High School and University students, who had normal menstrual cycles (27–32 days) for the previous year and normal body-mass index (BMI) for their age. We sought to relatively match the two groups concerning the participants’ age.