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PRECIS: This study identifies, higher levels of leptin expressions in umbilical cord from preeclampsia placenta, suggesting that provocation of interleukin-8 secretion by leptin in endothelial cells may contribute to preeclampsia-related inflammation. INTRODUCTION Preeclampsia (PE) is a multisystem disorder. Hypertension, proteinuria, and pathologic edema in pregnancy are the classic clinical manifestations. Worldwide, approximately 5-7% of primigravid women develop PE in pregnancy(1). Many studies implied that the fundamental pathophysiologic abnormality is endothelial dysfunction associated with exaggerated inflammation and immunologic reaction(2,3,4). In the pathogenesis of PE, activation of neutrophils, monocytes, and natural killer cells initiate inflammation, resulting in endothelial destruction in the pathogenesis of PE(4). The damaged endothelium produces various chemokines, one of which is interleukin (IL)-8(5). Leptin was initially defined as an adipocyte hormone that controls energy balance, reproductive functions, and immune reactions in the body(6). Mounting data imply that leptin is a novel proinflammatory adipocyte-originated element that manages the cytokine pathway by connecting the immune and inflammatory system(7,8). The plasma leptin concentrations in patients with PE are considerably higher than those in normal patients(9). Moreover, recent reports revealed that leptin showed vital roles in diverse physiologic processes including angiogenesis and arterial blood pressure regulation(9).

ting the immune and inflammatory system(7,8). The plasma leptin concentrations in patients with PE are considerably higher than those in normal patients(9). Moreover, recent reports revealed that leptin showed vital roles in diverse physiologic processes including angiogenesis and arterial blood pressure regulation(9). IL-8 has been reported to activate chemotactic migration, proliferation, and survival of vascular endothelial cells; induce the expressions of vascular endothelial growth factor (VEGF) and VEGF receptor(10), and regulate pathologic angiogenesis(11). Was first purified as a potent neutrophil chemotactic factor(12). IL-8 is also characterized as a proinflammatory cytokine. Increased IL-8 levels have been described in various inflammatory diseases(13). Many inflammatory cells, such as monocytes, lymphocytes, and mast cells, release IL-8 that is accumulated inside endothelial cells. IL-8 and its receptors C-X-C chemokine receptor 1-2 have been detected in endothelial cells(14). Moreover, human placental tissue produces IL-8 during pregnancy. Previous studies showed a robust correlation between IL-8 levels and the severity of PE(5,15). Neutrophil activation results in progressive damage to endothelial cells(16). The present study aimed to investigate umbilical cord (UC) leptin receptor (LEPR) levels in PE and to examine the direct effect of leptin on IL-8 production by human endometrial endothelial cells (HEECs) and human umbilical vein endothelial cells (HUVECs), thus to describe the function of leptin in PE.

elial cells(16). The present study aimed to investigate umbilical cord (UC) leptin receptor (LEPR) levels in PE and to examine the direct effect of leptin on IL-8 production by human endometrial endothelial cells (HEECs) and human umbilical vein endothelial cells (HUVECs), thus to describe the function of leptin in PE. MATERIALS AND METHODS Tissue collection Serial paraffin sections of human placental UC specimens were obtained from the University of South Florida under the protocol approved by the Ethics and Human Investigation Committees of the University of South Florida (approvel number: 00015578). Written and verbal informed consents were obtained from each patient. All of the samples were grouped according to clinical diagnosis: UC control (n=12) or PE (n=7). For in vitro studies, previously frozen HEECs (n=2) and HUVEC (n=1) from normal women undergoing hysterectomy (laparoscopy or laparotomy) or normal delivery were thawed and grown to confluence, as previously described(17).

. All of the samples were grouped according to clinical diagnosis: UC control (n=12) or PE (n=7). For in vitro studies, previously frozen HEECs (n=2) and HUVEC (n=1) from normal women undergoing hysterectomy (laparoscopy or laparotomy) or normal delivery were thawed and grown to confluence, as previously described(17). Immunohistochemistry Collected PE and normal patient UC paraffin blocks were cut into 5 µm sections that were then put into a heater to incubate overnight at 56 °C. The slides were deparaffinized in xylene (x3) for 20 min., followed by 100, 90, 80, and 70% alcohol x1 for 10 min. per gradient. Following deparaffinization, the slides were heated in 10 mM citrate buffer (pH 6.0) for 3x5 min in a microwave oven for antigen retrieval. The slides were then immersed in 3% hydrogen peroxide (in 1:1 v/v methanol/distilled water) for 12 min. to quench endogenous peroxidase activity. After washing with tris-buffered saline (TBS); (pH: 7.4) (x3) for 5 min., the slides were incubated in a humidified chamber with 5% blocking normal goat serum (Vector Labs, Burlingame, CA) for 30 min at room temperature (RT) in TBS. Excess serum was emptied, and then the slides were incubated overnight with a primary rabbit polyclonal anti-LEPR antibody (1:60; Santa Cruz Biotechnology, Dallas, TX) in 1% normal goat serum at 4 °C. Normal rabbit immunoglobulin G (IgG) (Vector Labs) isotypes were used for negative controls at the equal primary antibody concentrations. The slides were rinsed (x3) for 5 min. with TBS, and then biotinylated anti-rabbit IgG (Vector Labs) was used at a 1:400 dilution for 30 min. at RT. The antigen-antibody complex was identified using an avidin-biotin-peroxidase kit (Vector Labs) for 30 min. at RT. 3.3´-Diaminobenzidine tetrahydrochloride dihydrate (Vector Labs) was added as the chromogen to visualize immunoreactivity for 90 seconds. The slides were then counterstained with hematoxylin and mounted. Immunoreactive LEPR levels were semi-quantitatively assessed using the subsequent intensity categories: 0, no staining; 1+, weak but visible staining; 2+, moderate or distinct staining; and 3+, strong staining.

visualize immunoreactivity for 90 seconds. The slides were then counterstained with hematoxylin and mounted. Immunoreactive LEPR levels were semi-quantitatively assessed using the subsequent intensity categories: 0, no staining; 1+, weak but visible staining; 2+, moderate or distinct staining; and 3+, strong staining. As described previously(18), for each tissue, a histologic score (HSCORE) was derived by adding the percentages of cells that were stained at each intensity category and then multiplying that value by the weighted intensity of the staining using the formula HSCORE=Σ Pi (i + l), where i represents the intensity scores, and Pi is the corresponding percentage of the cells. In each slide, three randomly selected areas were assessed under a light microscope (x40 magnification), and the percentage of the cells at each intensity within these regions was evaluated at different times by two blinded researchers. The average HSCORE of the two examiners was used. Human endometrial endothelial cell and human umbilical vein endothelial cell isolation and experimental treatment with leptin. Frozen primary HEECs (n=2) and HUVECs (n=1) were derived from banked samples. The samples had been isolated and categorized as previously described(17) from endometrial specimens obtained from reproductive-age women undergoing hysterectomy (laparoscopy or laparotomy) and UC vein specimens obtained from the delivery of a normal pregnant woman. Written informed consent for sample retrieval was obtained from Yale University Faculty of Medicine, Human Investigation Committee and approved by the University of South Florida. Aliquots of frozen primary HEECs and HUVECs were thawed and grown to confluence in basal medium (BM), and a phenol red-free 1:1 v/v Dulbecco’s Modified Eagle Medium/Ham’s F-12 (Gibco, Grand Island, NY) mixture, containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL Fungizone complex (Gibco) supplemented with 10% charcoal-stripped calf serum (Gibco). At ~80% confluence, HEECs and HUVECs were transferred to 6-well plates at a density of 150x103 cells/well, for the corresponding treatments, as designated by each experimental condition. Confluent HEECs and HUVECs were incubated in parallel in BM with 0.1% ethanol (vehicle control) or leptin (0.1, 1.0, 10, 100 and 1000 ng/mL leptin, respectively).

HUVECs were transferred to 6-well plates at a density of 150x103 cells/well, for the corresponding treatments, as designated by each experimental condition. Confluent HEECs and HUVECs were incubated in parallel in BM with 0.1% ethanol (vehicle control) or leptin (0.1, 1.0, 10, 100 and 1000 ng/mL leptin, respectively). After incubation for 24 h, HEECs and HUVECs were rinsed with ice-cold 1x phosphate buffered saline and stored at -80 °C until used for immunoblotting analysis to measure IL-8 total protein levels.

HUVECs were transferred to 6-well plates at a density of 150x103 cells/well, for the corresponding treatments, as designated by each experimental condition. Confluent HEECs and HUVECs were incubated in parallel in BM with 0.1% ethanol (vehicle control) or leptin (0.1, 1.0, 10, 100 and 1000 ng/mL leptin, respectively). After incubation for 24 h, HEECs and HUVECs were rinsed with ice-cold 1x phosphate buffered saline and stored at -80 °C until used for immunoblotting analysis to measure IL-8 total protein levels. Immunoblot analysis Total protein was extracted using a cell extraction buffer (BioSource International, Camarillo, CA) containing 3 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The protein level was determined using a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Samples (40 µg) were loaded on 10% Tris-hydrochloric acid-ready gels (Bio-Rad), electrophoretically separated, and electroblotted onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk powder in TBS containing 0.1% Tween 20 (TBS-T) for 1 h to reduce any non-specific antibody binding. Subsequently, the membrane was incubated overnight with a monoclonal mouse IgG1 clone primary antibody against IL-8 (1:800 R&D Systems, Inc., Minneapolis, MN) in 5% non-fat milk powder in TBS-T. The membrane was then rinsed several times with 1x TBS-T for 1 h and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Vector Labs) in TBS-T. Following several washes, IL-8 was visualized through light emission from the film (Denville Scientific, Holliston, MA) with enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL). Band intensities were quantified using computer densitometry analysis (Image J, National Institutes of Health, Bethesda, MD).

llowing several washes, IL-8 was visualized through light emission from the film (Denville Scientific, Holliston, MA) with enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL). Band intensities were quantified using computer densitometry analysis (Image J, National Institutes of Health, Bethesda, MD). Statistical Analysis Data from immunohistochemistry and Western blot analysis that were normally distributed, according to the Kolmogorov-Smirnov test, were compared using Student’s t-test or one-way analysis of variance, followed by the post hoc Holm-Sidak test. Immunohistochemistry and Western blot analysis data that were not normally distributed were analyzed using the Kruskal-Wallis nonparametric ANOVA-by-Ranks test, followed by the post hoc Student-Newman-Keuls test. Statistical calculations were performed using Sigmaplot 13 for Windows (Jandel Scientific Corp., San Rafael, CA). Statistical significance was considered as p<0.05.

were not normally distributed were analyzed using the Kruskal-Wallis nonparametric ANOVA-by-Ranks test, followed by the post hoc Student-Newman-Keuls test. Statistical calculations were performed using Sigmaplot 13 for Windows (Jandel Scientific Corp., San Rafael, CA). Statistical significance was considered as p<0.05. RESULTS Immunohistochemistry of human placental umbilical cord specimens evaluating leptin receptor immunostaining in the control vs. preeclamptic groups LEPR immunostaining was detected in the cytoplasm and nucleus of the UC artery and vein endothelial cells. LEPR HSCOREs were significantly different between the control vs. PE specimens in the sectioned UC arterial endothelial cells [mean ± standard error of the mean (SEM): 67.9±8.868 vs. 127.6±23.1; p=0.011, respectively] (Figure 1) and UC vein endothelial cells (mean ± SEM: 55.4±8.043 vs. 93.7±17.15; p=0.035, respectively). LEPR immunostaining was moderate in the PE specimens (n=7) (Figure 1a-d), whereas the control UC endothelial cells (n=12) displayed weak immunostaining (Figure 1e-h). There were no statistically significant differences in maternal age, gestational age (GA), body mass index (BMI) at delivery, and baby weight between the control and PE groups (Table 1).

in the PE specimens (n=7) (Figure 1a-d), whereas the control UC endothelial cells (n=12) displayed weak immunostaining (Figure 1e-h). There were no statistically significant differences in maternal age, gestational age (GA), body mass index (BMI) at delivery, and baby weight between the control and PE groups (Table 1). Effect of leptin on interleukin-8 protein expression in cultured human endometrial endothelial cells and human umbilical vein endothelial cells Experimental incubations were followed by immunoblotting of the cell extracts to establish the functional regulation of leptin on IL-8 protein expression in the primary cultures of HEECs and HUVECs. Representative immunoblotting (Figure 2a) and the accompanying graphs (Figure 2b) indicated that IL-8 protein level was significantly increased by 1000 ng/mL leptin and 100 ng/mL compared with the control (p=0.003). Conversely, 0.1, 1, and 10.0 ng/mL leptin had not statistically significant effect on IL-8 levels when added to the culture medium (p>0.05) (Figure 2a,b). Incubation with 100 and 1000 ng/mL leptin induced greater IL-8 protein level vs. the control, 0.1 and 1 ng/mL leptin (Figure 2a). Compared with the control, 0.1, 1, and 10 ng/mL leptin showed no significant change in the basal IL-8 protein expression in HUVECs and HEECs.

ed to the culture medium (p>0.05) (Figure 2a,b). Incubation with 100 and 1000 ng/mL leptin induced greater IL-8 protein level vs. the control, 0.1 and 1 ng/mL leptin (Figure 2a). Compared with the control, 0.1, 1, and 10 ng/mL leptin showed no significant change in the basal IL-8 protein expression in HUVECs and HEECs. DISCUSSION Several studies illustrated the potential role of cytokines, chemokines, and their receptors in the development and progression of PE(19,20,21). Pregnancy complications associated with PE are major causes of materno-fetal morbidity and mortality, but their pathogenesis remains unclear(22). Despite significant development in the management of PE, it still constitutes an unsolved health problem in pregnancy(23). The current study examined the molecular mechanism of leptin-mediated inflammation in PE. The data in this study imply that leptin/LEPR interaction stimulated IL-8 production in endothelial cells, consequently promoting neutrophil chemotaxis and PE progression. Therefore, a view was instigated to connect leptin concentrations and IL-8 level in endothelial cells. The structure and function of endothelial cells are crucial to the preservation of arterial and vein vessel wall homeostasis, as well as immune cells migration(24). Damaged endothelial cells, increasing inflammatory cell recruitment, and high concentrations of inflammatory agents in the plasma of pregnant patients are responsible for the pathogenesis of PE(13,24). Impaired placental function and placental vascular disorders result in the occurrence of poor perinatal outcome(25). Leptin is produced by cytotrophoblasts and syncytiotrophoblasts in the human placenta and adipose tissue, which is then secreted into the circulation, where it exerts its effects via interaction with the LEPR. The effect of leptin on endothelial cells has attracted particular attention(20,26). LEPRs are expressed in many normal tissues, but also in pathologic tissues generally associated with obesity and abnormal energy balance(27). Immunohistochemically, endothelial cells, syncytiotrophoblasts, and cytotrophoblasts were stained with leptin(28). The present study demonstrated that the LEPR expression in UC artery and vein endothelial cells was augmented in PE and localized to the cytoplasm and nuclei of endothelial cells. Significant increases in the concentration and amounts of LEPR-positive endothelial cells, as implied by HSCORES in PE vs.

with leptin(28). The present study demonstrated that the LEPR expression in UC artery and vein endothelial cells was augmented in PE and localized to the cytoplasm and nuclei of endothelial cells. Significant increases in the concentration and amounts of LEPR-positive endothelial cells, as implied by HSCORES in PE vs. normal pregnancy suggests a function for LEPR in the inflammatory pathway of PE. A previous study demonstrated a high level of the leptin gene expression in microarray investigations in PE(29). In this regard, the current study was designed to elucidate the possible correlation between UC artery and vein LEPR levels and PE and its functional effects on endothelial cells. The study data can also be considered as indirect evidence for LEPR and its contribution to PE, and the functional results of leptin on endothelial cells. The data of the study can also be considered as indirect evidence for the contribution of LEPR to PE. Leptin possibly has an effect on the regulation of arterial blood pressure in pregnant women, as indicated by the direct relationship between plasma leptin concentrations and mean arterial blood pressure(30). Furthermore, dysfunctions of leptin metabolism or regulation in the placental unit and plasma are enhanced in pregnancies complicated with various abnormalities such as intrauterine growth restriction, gestational diabetes mellitus, and PE. Leptin synthesis and secretion have been shown as positively correlated with BMI and GA(31,32,33). In contrast, in this study, BMI, GA, maternal age, and birth weight of the baby were not significantly different between the control and PE groups. Mise et al.(34) stated that leptin messenger RNA (mRNA) levels in severe PE were significantly higher than in those with mild PE, also that placental leptin mRNA levels were generally similar to plasma leptin concentrations in women with PE compared with GA-matched healthy pregnant women. Higher concentrations of UC plasma leptin have been revealed in infants of mothers with PE than in a GA-, sex-, and infant ponderal index-matched control group(35). Compromised placental blood distribution causes chronic disturbance of nutrient resource and ultimately results in intrauterine growth restriction(36). Impaired placental perfusion also creates a depressed oxygen source at the placental level, which subsequently enhances leptin gene expression in the placental unit(21).

ompromised placental blood distribution causes chronic disturbance of nutrient resource and ultimately results in intrauterine growth restriction(36). Impaired placental perfusion also creates a depressed oxygen source at the placental level, which subsequently enhances leptin gene expression in the placental unit(21). It is likely that the high leptin concentrations in maternal plasma may augment hypertension because leptin provokes endothelial dysfunction and hypertension via aldosterone-related mechanisms and milieu in gestations complicated by intrauterine growth restriction(37). PE is concomitant with shallow trophoblastic invasion into the endometrial layer, which leads to poor placental perfusion and augmented fetal and maternal plasma leptin levels that are considerably increased over the concentration of leptin specific to human gestation(38). This exaggerated hyperleptinemia may be linked to a compensatory response to augment nutrient supply to the growing fetus(39). The present study showed that although not significant, the LEPR level was slightly higher in the UC artery than the paired UC vein, especially in the preeclamptic group, suggesting that UC artery LEPR may be more functional than UC vein LEPR in PE. In a previous report, leptin levels were found to be higher in the UC vein than the UC artery(40). One other study showed that the leptin concentrations were considerably higher in UC artery and UC vein than those in paired maternal plasma, implying that leptin is produced in placental trophoblastic cells and is secreted into the maternal blood circulation. Furthermore, plasma leptin concentrations in the UC vein were notably higher than those in the paired UC artery, suggesting that leptin is released from placental trophoblastic cells into the fetal blood circulation(41).

n is produced in placental trophoblastic cells and is secreted into the maternal blood circulation. Furthermore, plasma leptin concentrations in the UC vein were notably higher than those in the paired UC artery, suggesting that leptin is released from placental trophoblastic cells into the fetal blood circulation(41). The increase in the proinflammatory chemoattractant cytokine levels in PE suggests an inflammatory basis for this disease. The proinflammatory and regulatory cytokine IL-8 has been discovered in endothelial cells. Prior studies demonstrated upregulation of IL-8 protein levels in PE. IL-8 is regulated by neutrophil and monocyte chemotaxis regarding the inflammation site and stimulated inflammatory response(42). IL-8 is secreted by some cell types, including endothelial cells, monocytes, macrophages. Neutrophils, and fibroblasts. This study investigated UC LEPR levels in PE and the relationship of IL-8 expression in HEECs and HUVECs with leptin at different concentrations in an attempt to explain the significant association between leptin and IL-8 in patients with PE. Increasing placental endothelial IL-8 production may contribute to the improvement of placental endothelial pathology in PE. There was a dose-dependent progressive connection between increasing leptin level and endothelial IL-8 protein expression. Exposing endothelial cells to a high leptin concentration plays a role in leukocyte migration into the placental area and in the management of the tissue-specific modifications related with the leukocyte extravasation. A prior study showed that circulating plasma IL-8 concentrations were elevated in women with PE compared with normal pregnant women(5,15). These data indicate that IL-8 may have a critical role in endothelial cell proliferation and differentiation and regulating endothelial function. Human placental tissues constitutively produce IL-8 in pregnancy, and IL-8 secretion increases with progressing GA. IL-8 is critical for leukocyte recruitment(43). Namely, the vascular endothelial cell layer acts as the gatekeeper for maternal immune rejection and immune cells. Similarly, increased IL-8 plasma levels in PE have been documented (15,44). Moreover, leptin induced the production of IL-8 in human cartilage, fibroblasts, and M2 macrophages(45,46). These previous data also support the role of leptin-induced IL-8 secretion in endothelial cells(46).

immune rejection and immune cells. Similarly, increased IL-8 plasma levels in PE have been documented (15,44). Moreover, leptin induced the production of IL-8 in human cartilage, fibroblasts, and M2 macrophages(45,46). These previous data also support the role of leptin-induced IL-8 secretion in endothelial cells(46). Additionally, the current study is the first to describe the association between leptin and IL-8 in HUVECs and HEECs. IL-8 influences early vascular remodeling by recruiting circulating neutrophil cells to the endothelial cells(24). Understanding the mechanism for the increased IL-8 expression in HEECs and HUVECs of women in pregnancy may contribute to explaining the pathophysiology and development of PE. Speculatively, increased leptin production may exaggerate cytokine-induced destruction of endothelial cells in PE or overweight patients. Neutrophil-endothelial contact is a hallmark of vascular inflammation that results in endothelial injury/dysfunction(47). Increasing IL-8 levels in endothelial cells probably contribute to enhanced neutrophil recruitment and cytokine production. Furthermore, neutrophil stimulation is amplified in inflammatory reactions in the maternal artery and vein blood circulation in PE. These observations reveal that there is a high rate of IL-8 production in PE, consistent with other studies(44). However, further studies are required to investigate the role of leptin and LEPR in other inflammatory-related cellular mechanisms.

ammatory reactions in the maternal artery and vein blood circulation in PE. These observations reveal that there is a high rate of IL-8 production in PE, consistent with other studies(44). However, further studies are required to investigate the role of leptin and LEPR in other inflammatory-related cellular mechanisms. Collectively, these in vivo and in vitro results indicate that endothelial cells contribute to increased IL-8 concentrations in maternal and fetal circulation, as well as neutrophil recruitment. Leptin-associated augmented IL-8 secretion in endothelial cells is probably related with the development of PE. Thus, the increase in IL-8 level may potentiate leukocyte activation into the placental tissue under the effects of leptin. Consequently, the source and physiologic importance of IL-8 in the maternal and fetal circulation are noteworthy objectives of potential investigation. However, whether this phenomenon is a compensatory effect or amplified reaction to the severity of the PE remains enigmatic(48).

tal tissue under the effects of leptin. Consequently, the source and physiologic importance of IL-8 in the maternal and fetal circulation are noteworthy objectives of potential investigation. However, whether this phenomenon is a compensatory effect or amplified reaction to the severity of the PE remains enigmatic(48). CONCLUSION These observations provide direct evidence of the stimulation of IL-8 gene expression in endothelial cells in vitro by high leptin concentrations. In this manner, the increased leptin and LEPR may cause or contribute to increased IL-8 production, leading to increased neutrophil recruitment and endothelial destruction and consequently, increase cytokine expression in PE. This study may provide an in vivo basis for the application of an anti-human IL-8 antibody for the treatment of PE. Further studies researching the possible role of LEPR in normal and PE UC endothelial cells are needed to explore these possibilities and to support new insight into our understanding of the pathogenesis of PE. The authors would like to thank the lab members, Lockwood C.J. and Magnes R., Ümit A Kayışlı, and Dr. John Tsibris for their assistance. Ethics Ethics Committee Approval: Serial paraffin sections of human placental UC specimens were obtained from the University of South Florida under the protocol approved by the Ethics and Human Investigation Committees of the University of South Florida (approval number: 00015578). Informed Consent: Written and verbal informed consents were obtained from each patient. Peer-review: External and internal peer-reviewed.

Ethics Committee Approval: Serial paraffin sections of human placental UC specimens were obtained from the University of South Florida under the protocol approved by the Ethics and Human Investigation Committees of the University of South Florida (approval number: 00015578). Informed Consent: Written and verbal informed consents were obtained from each patient. Peer-review: External and internal peer-reviewed. Conflict of Interest: No conflict of interest was declared by the author. Financial Disclosure: The author declared that this study received no financial support. Figure 1 Leptin receptor immunoreactivity in umbilical cord arterial and venous sections from gestational age-matched preeclamptic and normal pregnancies. Representative micrographs of immunohistochemical staining for leptin receptor in preeclampsia (n=7) (A, B, C, D) and normal pregnancy (n=12) (E, F, G, H). Graphs represent the histologic score analysis of leptin receptor immunostaining in umbilical cord vein (I) and umbilical cord artery (J) endothelial cells expressed as mean ± standard error of the mean *p=0.035; endothelial cells of the umbilical vein, preeclampsia vs. normal pregnancy **p=0.011; endothelial cells of the umbilical artery, preeclampsia vs. normal pregnancy

Figure 1 Leptin receptor immunoreactivity in umbilical cord arterial and venous sections from gestational age-matched preeclamptic and normal pregnancies. Representative micrographs of immunohistochemical staining for leptin receptor in preeclampsia (n=7) (A, B, C, D) and normal pregnancy (n=12) (E, F, G, H). Graphs represent the histologic score analysis of leptin receptor immunostaining in umbilical cord vein (I) and umbilical cord artery (J) endothelial cells expressed as mean ± standard error of the mean *p=0.035; endothelial cells of the umbilical vein, preeclampsia vs. normal pregnancy **p=0.011; endothelial cells of the umbilical artery, preeclampsia vs. normal pregnancy PE: Preeclampsia Figure 2 Leptin stimulates interleukin-8 expression in endothelial cells. Representative immunoblotting in human endometrial endothelial cells (n=2) and human umbilical vein endothelial cells (n=1) cultures treated with leptin for 24 hours. Western blot analysis demonstrating the effect of leptin on interleukin-8 levels in human endometrial endothelial cells and human umbilical vein endothelial cells. Confluent human endometrial endothelial cells and human umbilical vein endothelial cells cultures were treated with vehicle (control), 0.1 1, 10, 100 and l000 ng/mL leptin, respectively, for 24 hours, to evaluate the effect of leptin. Immunoblot bands for interleukin-8 were quantified using Image J. Bars represent mean ± standard error of the mean (n=3) *p<0.05; both for 100 and 1000 ng/mL leptin vs. control. Data are representative of three independent experiments

PE: Preeclampsia Figure 2 Leptin stimulates interleukin-8 expression in endothelial cells. Representative immunoblotting in human endometrial endothelial cells (n=2) and human umbilical vein endothelial cells (n=1) cultures treated with leptin for 24 hours. Western blot analysis demonstrating the effect of leptin on interleukin-8 levels in human endometrial endothelial cells and human umbilical vein endothelial cells. Confluent human endometrial endothelial cells and human umbilical vein endothelial cells cultures were treated with vehicle (control), 0.1 1, 10, 100 and l000 ng/mL leptin, respectively, for 24 hours, to evaluate the effect of leptin. Immunoblot bands for interleukin-8 were quantified using Image J. Bars represent mean ± standard error of the mean (n=3) *p<0.05; both for 100 and 1000 ng/mL leptin vs. control. Data are representative of three independent experiments IL: Interleukin Table 1 Demographic data of the control and preeclamptic groups from which umbilical cord specimens were obtained

PRECIS: We have assessed that the estradiol-stimulated regulation of inhibitory kappa Bα and subsequent decrease in nucleor factor faktör kappa B affects inflammatory reactions in human endometrial cells.

PRECIS: We have assessed that the estradiol-stimulated regulation of inhibitory kappa Bα and subsequent decrease in nucleor factor faktör kappa B affects inflammatory reactions in human endometrial cells. Introduction Immunologic-endocrine interactions mediate and participate in complex physiologic processes that occur within the uterus throughout the menstrual cycle and pregnancy, and are also important to the pathophysiology of endometriosis(1,2,3). One of the molecular signaling pathways that may be regulated by the endocrine system, which also participates in the regulation of inflammation, is the nuclear factor kappa B (NF-kB) signaling cascade(4,5,6). NF-kB is a transcription factor that is kept in an inactive state in the cytosol while bound to the inhibitory kappa B (IkB) protein(7,8). First described in B cells, NF-kB was subsequently recognized as a nuclear and cytoplasmic protein that is found in multiple cell types(9). In many cells, NF-kB positively regulates the expression of a number of genes including those of cytokines, cell adhesion molecules, complement factors, anti-apoptotic factors, and immunoreactions(10,11,12). The IkB protein family is composed of 35-70 kDa proteins that are localized in the cytoplasm and inhibit the activation of NF-kB. This protein family includes IkBa, IkBb, IkBg, IkB-R, B-cell leukemia-3, p105/p50, p100/52 and the Drosophila melanogaster proteins Cactus and Relish. IkBa and IkBb preferentially interact with NF-κB dimers composed of proteins p65 and p50, and regulate NF-kB function by converting the heterodimer structure to a trimer that is incapable of binding DNA(13,14,15). Tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1 induce the phosphorylation and subsequent degradation of IkBα. This, in turn, results in the activation and relocation of NF-kB to the nucleus, leading to NF-kB-mediated transcription of responsive genes(16,17). Ligand binding to most, if not all, of the inflammatory cytokine receptors activates intracellular signaling molecules that engender the activation of NF-kB. Activation of such signaling molecules results in a transient activation of IkB kinase (IKK) and a transient phosphorylation of IkBα (phospho-IkBα). Often, phospho-IkBα peaks 2-15 min after stimulation with the cytokine, and is followed by a rapid acceleration of IkBα degradation.

that engender the activation of NF-kB. Activation of such signaling molecules results in a transient activation of IkB kinase (IKK) and a transient phosphorylation of IkBα (phospho-IkBα). Often, phospho-IkBα peaks 2-15 min after stimulation with the cytokine, and is followed by a rapid acceleration of IkBα degradation. Often, IkBα levels may subsequently increase in the cytosol over the following 2-6 h, in response to NF-kB-mediated upregulation of the IkB promotor(11,13,18). Several proteins and molecules that activate NF-kB signaling have been described. IL-1 and TNF-α are two principal cytokines that promote IkBα degradation and NF-kB activation. Although these cytokines bind to specific receptors to activate different intracellular second messengers, downstream signals merge with the activation of the same target, namely IKK(19,20,21). Estrogen influences the growth, differentiation, and function of many target cells by genomic and non-genomic pathways. Although the genomic effects of estrogen are mediated via estrogen receptors (ERs) and occur over a period of hours or days, the non-genomic effects occur within minutes(22,23,24). Previous studies have shown that estrogen down-regulates the expression of many cytokines such as IL-1, TNF-α, IL-6 and regulated-upon activation, normal T-cell-expressed and secreted (RANTES), which are regulated by NF-kB in various cell types(25,26,27). Previously, we have shown that estrogen inhibits monocyte chemotactic protein-1 expression in human endometrial stromal cells (ESCs)(28). Moreover, in response to estrogen, chemokine-mediated regulation of endometrial cells obtained from women with endometriosis is distinct from that observed in normal endometrial cells(29,30,31). An estrogen-dependent disease, endometriosis develops outside of the uterus and is characterized by a proinflammatory peritoneal environment(32,33).Thus, there may be differential regulation of NFkB signaling by estrogen and by cytokines such as TNF-α and IL-l in endometriotic cells as compared with normal endometrial cells. In endometriotic cells, there appears to be synergy between the effects of E2 and IL-1/TNF-α, whereas these molecules appear to function antagonistically in normal endometrial cells. We hypothesized that estrogen might regulate IkBα phosphorylation and degradation in vivo and in vitro in normal endometrium and in eutopic and ectopic endometrium of women with endometriosis, thus influencing NFkB-dependent gene expression.

ese molecules appear to function antagonistically in normal endometrial cells. We hypothesized that estrogen might regulate IkBα phosphorylation and degradation in vivo and in vitro in normal endometrium and in eutopic and ectopic endometrium of women with endometriosis, thus influencing NFkB-dependent gene expression. First, we investigated the in vivo expression of IkBα in normal endometrium and in eutopic and ectopic endometrium of women with endometriosis. We then investigated the modulation of IkBα by E2 in TNF-α- and IL-1α-treated endometrial stromal and glandular cells, in vitro, using Western blot analysis and immunocytochemistry. Materials and Methods Tissue collection Endometrial tissues were obtained from human uteri after hysterectomy conducted for benign diseases excluding endometrial disease, and from endometrial biopsies. Approval for this study was granted by the Human Investigation Committee of Yale University (HIC#22334) and written informed consent was obtained from each patient prior to surgery. The mean age of the patients was 36 years (range, 30-45 years).

ign diseases excluding endometrial disease, and from endometrial biopsies. Approval for this study was granted by the Human Investigation Committee of Yale University (HIC#22334) and written informed consent was obtained from each patient prior to surgery. The mean age of the patients was 36 years (range, 30-45 years). For immunohistochemistry, normal cyclic endometrium (n=12) of women without endometriosis, and eutopic and ectopic endometrium pairs of women with endometriosis (n=6) were collected, and paraffin blocks were routinely prepared and cut at 5-7 mm. For the endometrial cells used in culture, the diagnoses of the patients were leiomyomata uteri or voluntary sterilization by tubal ligation (n=5). The day of the menstrual cycle was established from the patient’s menstrual history and was verified through histologic examination of the endometrium. The tissues were placed in Hank’s balanced salt solution and transported to the laboratory for separation and culture of endometrial stromal and glandular cells. Each experimental setup was repeated on at least three occasions using cells obtained from different patients.

ough histologic examination of the endometrium. The tissues were placed in Hank’s balanced salt solution and transported to the laboratory for separation and culture of endometrial stromal and glandular cells. Each experimental setup was repeated on at least three occasions using cells obtained from different patients. Isolation and culture of human endometrial stromal and glandular cells Endometrial tissues were separated and conserved in a monolayer culture, as described previously(34). The isolated endometrial cells were separated by filtration through a wire sieve (73 mm diameter pore, Sigma). The endometrial glands (largely undispersed) were retained by the sieve, whereas the dispersed stromal cells passed through the sieve into the filtrate. The stromal cells were plated in plastic flasks (75 cm2, Falcon, Franklin Lakes, NJ), maintained at 37 °C in a humidified atmosphere (5% CO2 in air), and allowed to replicate to confluence. Thereafter, the stromal cells were passed by standard methods of trypsinization, plated in culture dishes (100 mm diameter), and allowed to replicate to confluence. ESCs after the first passage were characterized as described previously(34) and were found to contain 0-7% epithelial cells, no detectable endothelial cells, and 0.2% macrophages. Experiments were commenced 1-3 days after the cells reached confluence. The confluent cells were treated with serum-free, phenol red-free media for 24 h before treatment with test agents. Stromal cells reached confluence in 7-10 days.

contain 0-7% epithelial cells, no detectable endothelial cells, and 0.2% macrophages. Experiments were commenced 1-3 days after the cells reached confluence. The confluent cells were treated with serum-free, phenol red-free media for 24 h before treatment with test agents. Stromal cells reached confluence in 7-10 days. Experiments with glandular cells were performed using a well-differentiated endometrial adenocarcinomacell line (Ishikawa cell) provided to us by Dr. R. Hochberg (Departmentof Obstetrics and Gynecology, Yale University, New Haven, CT)from a frozen stock. Thawed cells were maintained in T75 flasks(BD Biosciences, Franklin Lakes, NJ) until passage.The cells were treated with serum-free phenol red-free media for 24 h before treatment with test agents. Cells were treated with E2 (Sigma) for 3-90 min and immunocytochemistry and Western blot analysis were performed as described.

ed cells were maintained in T75 flasks(BD Biosciences, Franklin Lakes, NJ) until passage.The cells were treated with serum-free phenol red-free media for 24 h before treatment with test agents. Cells were treated with E2 (Sigma) for 3-90 min and immunocytochemistry and Western blot analysis were performed as described. Immunohistochemistry and immunocytochemistry Endometrial tissue sections from normal, eutopic, and ectopic endometrium were deparaffinized and washed with phosphate buffered saline (PBS). Thereafter, sections were twice microwaved in citric acid buffer (0.1 M, pH: 6) and thoroughly rinsed in PBS. The same steps used for immunocytochemistry (described below) were followed. ESCs were grown to pre-confluence on four-chamber slides. Following treatment, the chamber slides were fixed in 4% paraformaldehyde for 20 min. After several washes with distilled water and then with PBS (pH 7.4) (three times 10 min each), endogenous peroxidase activity was quenched by 3% H2O2 (0.6 mL H2O2 and 5.4 mL methanol) for 10 min and the slides were then rinsed in PBS-tween. Slides were then incubated with rabbit anti-IkBa polyclonal antibody (Cell signaling Technology, Beverly, MA) for 60 min at room temperature. In negative control slides, normal rabbit immunoglobulin G (IgG) was used as a control instead of primary antibody. After several rinses in PBS, goat biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) was applied for 30 min. After several rinses with PBS, the slides were incubated with streptavidin-peroxidase complex for 30 min (Vector Laboratories). The slides were then rinsed several times in PBS and incubated with 3-amino-9-ethyl-carbazole (Vector Laboratories) for 10 min. The slides were lightly counterstained with hematoxylin prior to permanent mounting. Immunocytochemical staining intensity was ranked between 0 (absent) to 3 (most intense). For each slide, an HSCORE value was derived by summing the percentages of cell staining at each intensity multiplied by the weighted intensity of the staining [HSCORE=Σ Pi(i+1), where i is the intensity scores and Pi is the corresponding percentage of the cells]. In each slide, five randomly selected areas were assessed microscopically using 50× magnification. Two investigators who were blinded to the treatments analyzed each slide for intensity. The averages for the scores of both investigators are presented.

tensity scores and Pi is the corresponding percentage of the cells]. In each slide, five randomly selected areas were assessed microscopically using 50× magnification. Two investigators who were blinded to the treatments analyzed each slide for intensity. The averages for the scores of both investigators are presented. IkBa and phospho-IkBa Western blot analysis Total protein from endometrial cells was extracted in a lysis buffer composed of 50 mM hydroxyethyl piperazineethanesulfonic, pH: 7.4; 150 mM NaCl; 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2-6H2O; 1 mM EGTA; 100 mM NaF, 10 mM sodium pyrophosphate and protease inhibitors, 1 mM Na3VO4, 10 mg/mL leupeptin, 10 mg/mL aprotinin; and 4 mM phenylmethylsulfonyl fluoride. The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA). Protein lysates (20 μg) were loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% Tris-Hydrogen chloride Ready Gels (Bio-Rad Laboratories) and electroblotted onto nitrocellulose membrane (Bio-Rad Laboratories). Equal loading of proteins in each lane was confirmed by staining the membrane with Ponceau 2S (Sigma). The membrane was incubated with 5% nonfat dry milk in tris-buffered saline-tween (TBS-T) buffer (0.05% tween-20 in PBS, pH 7.4) for 1 h to reduce nonspecific binding of antibody. The membrane was probed with rabbit anti-IkBα and rabbit anti-phospho-IkBα (Ser32) antibodies (Cell Signaling Technology) overnight to quantitate total and phospho-IκBα forms. After washing with TBS-T, blots were incubated for 1 h with peroxidase labeled anti-rabbit IgG (Vector Laboratories) diluted at 1:10000. Membranes were washed with TBS-T and the immunoblots were developed using chemiluminescent kit following the manufacturer’s instructions. (NEN Life Science, Boston, MA).  The signal was normalized by dividing the arbitrary densitometry units for phospho-IkBα to the amount of total IkBα for each band. The signals were quantified by using a laser densitometer (Molecular Dynamics, Sunnyvale, CA) to analyze the autoradiographic bands.

acturer’s instructions. (NEN Life Science, Boston, MA).  The signal was normalized by dividing the arbitrary densitometry units for phospho-IkBα to the amount of total IkBα for each band. The signals were quantified by using a laser densitometer (Molecular Dynamics, Sunnyvale, CA) to analyze the autoradiographic bands. Preparation of nuclear extracts and the active-NF-κB assay To quantify the amount of active NF-κB, which binds to NFkB response element sites on gene promotors, an enzyme-linked immunosorbent (ELISA) plate covered with NF-κB binding consensus sequence oligonucleotide (5’-GGGACTTTCC-3’) was used in combination with nuclear extracts from our cultured cells. Two different primary antibodies against NF-κB each recognize either an epitope on p65 or on p50 that is accessible only after dissociation of IκB from NF-κB, indicating the activation of cytoplasmic NF-κB.  An horseradish peroxidase-conjugated secondary antibody provides a colorimetric readout that is quantitated using spectrophotometry (450 nm). As a positive control for activated NF-κB, nuclear extracts from HeLA cells were used. To monitor the specificity of the assay, both wild type and mutated consensus oligonucleotides were employed in each reaction. Nuclear extracts from endometrial cells grown to confluence in 60 mm plates were obtained using a nuclear extraction kit (Active Motif, Carlsbad, CA). Briefly, cells were washed with ice-cold PBS and protease/phosphatase inhibitors, removed from the dish by scraping with a cell lifter and transferred to pre-chilled tubes. Cell suspensions were centrifuged at 4 °C for 5 min at 500 rpm. Pellets were resuspended in hypotonic buffer and incubated for 15 min on ice, detergent was added, and the cells were centrifuged at 4 °C for 30 seconds at 14.000 ×g. The pellet was resuspended in a lysis buffer and incubated for 30 min on ice on a rocking platform. The suspension was centrifuged at 4 °C for 10 min at 14.000 ×g and the supernatant (nuclear fraction) was aliquoted and frozen at -80 °C. Nuclear fractions were quantitated using a Coomassie protein assay (Pierce; Rockford, IL) as per the manufacturer’s protocol. Four micrograms of nuclear extract sample were loaded into each well and assayed according to the manufacturer’s directions (Active Motif) using a microplate reader. Quantification of the NF-κB p50 subunit was expressed as mean absorbance (λ) per sample.

assay (Pierce; Rockford, IL) as per the manufacturer’s protocol. Four micrograms of nuclear extract sample were loaded into each well and assayed according to the manufacturer’s directions (Active Motif) using a microplate reader. Quantification of the NF-κB p50 subunit was expressed as mean absorbance (λ) per sample. Statistical Analysis IkBα immunocytochemistry scores and Western blot results were normally distributed as assessed using the Kolmogorov-Smirnov test. Analysis of variance (ANOVA) and post hoc Tukey test for pair-wise comparisons were used in statistical analysis. p<0.05 was considered to be significant. Statistical calculations were performed using Sigma stat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA).

using the Kolmogorov-Smirnov test. Analysis of variance (ANOVA) and post hoc Tukey test for pair-wise comparisons were used in statistical analysis. p<0.05 was considered to be significant. Statistical calculations were performed using Sigma stat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA). Results Expression of IкBα in normal endometrium, and in eutopic and ectopic endometrium from women with endometriosis Eutopic endometrial stromal and glandular cells from women without endometriosis express immunoreactive IkBα (Figure 1). The antibody used for immunohistochemistry recognizes both phosphorylated and unphosphorylated forms of IkBα. In normal endometrium, glandular cells reveal stronger immunoreactivity for IkBα compared with stromal cells throughout the menstrual cycle. Stronger immunoreactivity was detected in samples of mid-late proliferative endometrium compared with late secretory and early proliferative phase samples (p<0.05) (Figure 1, Table 1). When proliferative phase and secretory phase immunoreactivity for IkB were compared, the proliferative phase showed a trend for stronger immunoreactivity although this difference did not reach statistical significance. Eutopic and ectopic endometrium from women with endometriosis also revealed immunoreactivity for IkBα. When the eutopic endometrium from women with endometriosis was compared with the endometrium of women without endometriosis, no significant difference was observed in staining intensity, although eutopic endometrial cells of women with endometriosis showed a trend towards decreased immunoreactivity for IkBα (p=0.1) (Figure 1, Table 2). On the other hand, when compared with eutopic endometrium, homologous ectopic endometrium revealed significantly less immunoreactivity for IkBα (p<0.05) (Figure 1, Table 2).

ough eutopic endometrial cells of women with endometriosis showed a trend towards decreased immunoreactivity for IkBα (p=0.1) (Figure 1, Table 2). On the other hand, when compared with eutopic endometrium, homologous ectopic endometrium revealed significantly less immunoreactivity for IkBα (p<0.05) (Figure 1, Table 2). Estradiol-regulated expression of IкB in endometrial cells as assessed using immunocytochemistry ESCs grown on four-chamber slides were placed in serum-free, phenol red-free media for 24 h, and were then treated for 15 min with fresh serum-free, phenol red-free media as control, with TNF-α (2 ng/mL),  or estradiol (10-8 M) combined with TNF-α (2 ng/mL) for 15 min. Slides were stained with rabbit anti-IkBα antibody. Cells treated with TNF-α alone showed a very weak immunoreactivity for IkBα when compared with the control (Figure 2a, b). On the other hand, cells treated with TNF-α combined with E2 displayed a stronger IkBα immunoreactivity than those treated with TNF-α alone (p<0.05) (Figure 2b, c).  We also compared cells maintained for 24 h in serum-free phenol red-free media for 24 h, with or without E2 (10-8 M), followed by TNF-α (2 ng/mL) treatment for an additional 15 min. TNF-α–stimulated IkBα immunoreactivity was stronger in cells pre-treated with E2 compared with those pre-treated with serum-free media alone (p<0.05) (Figure 2d-f).

ntained for 24 h in serum-free phenol red-free media for 24 h, with or without E2 (10-8 M), followed by TNF-α (2 ng/mL) treatment for an additional 15 min. TNF-α–stimulated IkBα immunoreactivity was stronger in cells pre-treated with E2 compared with those pre-treated with serum-free media alone (p<0.05) (Figure 2d-f). Regulation of IкBα expression and phosphorylation in endometrial cells as assessed using Western blot analysis We sought to understand whether the increased IkBα immunoreactivity observed in cells treated with both TNF-α and E2 was associated with a phosphorylation and subsequent degradation of IkBα. After  24 h of incubation with serum-free, phenol red-free media, ESCs were treated with media alone (control), E2 10-8 M alone, TNF-α 2 ng/mL alone, or with E2 10-8 M combined with TNF-α 2 ng/mL for 3, 6, 12, 30, and 60 min. Total protein was extracted and levels of total IкBa and phospho-IкBa were measured using Western blot analysis. Control and E2-treated cells showed similar levels of IkBα throughout the treatment period. On the other hand, treatment with TNF-α resulted in a time-dependent decrease in IkBα levels compared with the control. Moreover, this treatment caused a time-dependent increase in phospho-IkBα levels with a peak between 6 and 12 min of treatment. Meanwhile, E2 combined with TNF-α treatment showed markedly higher levels of IkBα when compared with TNF-α alone (Figure 3). When groups were compared in terms of phospho-IkBα levels, control and E2-treated cells revealed the lowest levels of phospho-IkBα throughout the treatment periods. However, in cells treated with TNF-α, co-treatment with E2 induced higher IkBα levels and lower phospho-IkBα levels during the first 12 minutes of treatments (p<0.05) (Figure 3). Following 60 min of treatment, IkBα levels were still higher in cells co-treated with E2 compared with cells treated with TNF-α alone (Figure 4a). Interestingly, in glandular cells, longer treatment with E2 with TNF-α (90 min) resulted in a significantly higher level of IkBα compared with other treatments, including the control group (Figure 4b). Glandular cells treated with E2 plus TNF-α demonstrated higher phospho-IkBα levels when compared with cells treated with TNF-α alone (p<0.05).

lar cells, longer treatment with E2 with TNF-α (90 min) resulted in a significantly higher level of IkBα compared with other treatments, including the control group (Figure 4b). Glandular cells treated with E2 plus TNF-α demonstrated higher phospho-IkBα levels when compared with cells treated with TNF-α alone (p<0.05). As observed using immunoblotting, the effect of E2 on IkBα was more pronounced when glandular cells were pre-treated with E2 for 24 h prior to TNF-α treatment (Figure 5). To determine whether the effect of E2 on IkBα phosphorylation was specific to the TNF-α signaling cascade, we also explored the effect of estrogen on IL-1α-mediated activation of NF-kB. Cells were treated with E2 (10-8 M), IL-1α (2 ng/mL), E2 plus IL-1α, or vehicle alone (control). E2 induced lower phospho-IkBα and higher IkBα levels in IL-1α-treated cells as compared with cells treated with IL-1α alone (Figure 6). Regulation of TNF-α– and IL-1α-induced activation of NF-кB by E2 as assessed using an NF-кB binding assay To understand whether the TNF-α– and IL-1α-induced IkBα levels in E2-treated cells was associated with a decrease in free NF-kB, ESCs were treated with serum-free, phenol red-free media as control, and with E2 (10-8 M) alone, TNF-α (2 ng/mL) alone, IL-1α (2 ng/mL) alone, E2 combined with TNF-α or IL-1α for 15 min. Free NF-kB levels in control cells and E2-treated cells were lower than those in TNF-α– and IL-1α-treated cells. On the other hand, E2 decreased the TNF-α– and IL-1α–induced free NF-kB levels as compared with cells treated with TNF-α alone or IL-lα alone (Figure 7).

L) alone, E2 combined with TNF-α or IL-1α for 15 min. Free NF-kB levels in control cells and E2-treated cells were lower than those in TNF-α– and IL-1α-treated cells. On the other hand, E2 decreased the TNF-α– and IL-1α–induced free NF-kB levels as compared with cells treated with TNF-α alone or IL-lα alone (Figure 7). Discussion Steroid hormones classically bind to cognate nuclear receptors to regulate target gene expression(35). Estrogen takes part in cell and tissue regulation at many stages of human life. In addition to the reproductive tract of women, other systems such as the skeletal and nervous systems are important targets for estrogen action(36,37). Estrogen mainly affects cells through the genomic pathway(38). Estrogen actions may also result from non-genomic activity, possibly related to the cell type, receptor type, and the presence of intracellular co-factors that may interact with typical or atypical ERs. Non-genomic effects occur within minutes and appear to include cell membrane-dependent signaling mechanisms such as the nitric oxide cascade, stimulation of p38-mitogen-activated protein kinase, or phosphorylation of protein kinase B, among others(39,40,41,42). In contrast, long-term effects of estrogen, namely genomic effects, arise over hours or longer and are directed in part by DNA estrogen response elements(43). Some biologic processes can also play a role in both genomic and nongenomic pathways. A previous study showed that the lipopolysaccharide-stimulated activation of NF-kB was reduced by cell-impermeable E2–bovine serum albumin in mouse bone marrow-derived macrophage cultures in both genomic and nongenomic pathways(44). Eutopic and ectopic endometrium undergoes cycle-dependent changes predominantly controlled by estrogen and progesterone in their implantation site(45,46,47). The present study is focused on the anti-inflammatory effects of estrogen, assessing IkBα phosphorylation and NF-kB activation in endometrial and endometriotic cells. In vitro and in vivo studies indicate that NF-kB–mediated gene transcription stimulates inflammation, invasion, angiogenesis, and cell proliferation, and reduces apoptosis of endometriotic cells. Excessive activation of NF-kB has been confirmed in endometriotic implants and peritoneal macrophages of patients with endometriosis(48,49).

vivo studies indicate that NF-kB–mediated gene transcription stimulates inflammation, invasion, angiogenesis, and cell proliferation, and reduces apoptosis of endometriotic cells. Excessive activation of NF-kB has been confirmed in endometriotic implants and peritoneal macrophages of patients with endometriosis(48,49). In inflammatory tissue, an increase in TNF-α is often the first step in the cascade, followed by increases in the expression of various chemokines and the recruitment of leukocytes(27,50,51,52,53). Previous studies have shown that, when bound to their receptors, TNF-α and IL-1 increase IkBα phosphorylation, degradation, and eventually NF-kB activation, which results in increased inflammatory cells and expression of several inflammatory cytokines and chemokines(27,54,55). Our findings suggest that E2 may reduce phospho-IkBα and therefore decrease its degradation in endometrial cells. In this way, estrogen may block NF-kB transport into the nucleus and attenuate the inflammatory response. To our knowledge, this is the first study to report IkBα regulation by estrogen in endometrial stromal and glandular cells. It is possible that this increase arises from effects on the transcriptional or translational machinery, because a previous study has shown that E2 has a down-regulatory effect on IkBα at the mRNA level in phorbol ester-induced HeLa cells(56). Alternatively, a previous study performed using MCF-7 cells suggested that this increase was related to the increase of p105 protein level(57). On the other hand, another research group showed that estrogen treatment decreased liver IkB mRNA and protein expression and also increased ethanol-induced liver NF-kB levels and TNF-α expression(58). These disparate findings are likely to be related to the cell-specific effects of estrogen and merit further analysis. Several cytokines participate in NF-kB activation. In addition to TNF-α, IL-1α also regulates IkBα levels in the cytosol. The similar effects on IkBα levels by E2 co-treatment with TNF-α and with IL-1α, compared with treatments with TNF-α or IL-1α alone, indicate that the effect of E2 is not specific for the TNF-α signaling cascade. IL-1α initiates an alternate cascade for IkBα-related NF-kB activation to that of TNF-α. Furthermore, because both signaling pathways merge on IKK activation, the effect of estrogen may be on IKK activation or on subsequent steps.

1α alone, indicate that the effect of E2 is not specific for the TNF-α signaling cascade. IL-1α initiates an alternate cascade for IkBα-related NF-kB activation to that of TNF-α. Furthermore, because both signaling pathways merge on IKK activation, the effect of estrogen may be on IKK activation or on subsequent steps. Bulun et al.(59) studied NF-kBα and IkBα expression in human fetal membranes and decidua at preterm and term gestation. The authors observed a marked increase in the nuclear localization of p65 and in the IkBα immunoreactivity in tissues obtained at term compared with tissues delivered preterm, suggesting a role for p65 in the regulation of parturition-related gene transcription in the decidua(59). Our in vivo results show an increase in IkBα levels from early proliferative to the late proliferative phase, and suggest direct or indirect estrogenic regulation of IkBα in human endometrial cells. On the other hand, persistently low levels of IkBα immunoreactivity in ectopic endometrial cells are likely to be related to the increased local inflammation observed in endometriosis and may contribute to the increased inflammatory cytokine levels in the peritoneal cavity of women with endometriosis(60,61). Endometriosis is an estrogen-dependent disease and implants of endometriosis have sufficient enzymes for the local production of estrogen(54,59,62,63,64). The low levels of IkBα in ectopic endometrial cells suggest that the signaling effects of estrogen on IkBα may function similarly to those observed in eutopic endometrium. It seems that there is a lack of the inhibitory effect of E2 on cytokine-induced IkBα phosphorylation in ectopic endometrium. Supporting this hypothesis, a recent study has shown that E2 increases phospho-IkB levels, and more interestingly, induces higher IL-8 levels in endometriotic cells when compared with eutopic endometrium(65). Similarly, Akoum et al.(66)  showed that E2 and IL-1b had synergistic effects on the expression of RANTES, revealing that E2 enhanced the mRNA stability of RANTES, and IL-1b increased its transcription. A recent study reported the expressions of IkBα, IkBβ, and p50 in human endometrial cells throughout the menstrual cycle(67). Expression of these inhibitory proteins decreased significantly during the mid-secretory phase of the cycle. The study detected maximal immunoreactivity for IkBα during the late proliferative phase, consistent with our findings.

sions of IkBα, IkBβ, and p50 in human endometrial cells throughout the menstrual cycle(67). Expression of these inhibitory proteins decreased significantly during the mid-secretory phase of the cycle. The study detected maximal immunoreactivity for IkBα during the late proliferative phase, consistent with our findings. Another study showed an increase in IkBα mRNA levels in the pre-menstrual endometrium, suggesting activation of NF-kB during this phase or alternate regulation of IkBα expression(68). Our results support the findings of this study because activation of NF-kB requires IkBα phosphorylation and degradation, low levels of IkBα protein would stimulate high level of IkBα mRNA during the pre-menstrual phase to replenish degraded IkB protein. One reason for the inhibitory effect of estrogen on chemokine expression may be related to decreased IkBα degradation. As a consequence, estrogen may decrease the amount of free-NF-kB in the cytosol, and therefore decrease the level of activation. Recently, we showed that the presence of ligand ERs suppressed free-NF-kB subunits (both p65 and p50) binding to NF-kB response element,(26) suggesting a second mechanism for estrogen-dependent inhibition of NF-kB-mediated gene activation. ERs in ESCs inhibited DNA binding of p50 and p65 subunits of NF-kB. Also, NF-kB activation significantly reduced estrogen responsiveness of ER-alpha–transfected ESCs, but p50 did not impair ER-alpha DNA binding, suggesting possible indirect mechanisms for this type of interaction(26).

of NF-kB-mediated gene activation. ERs in ESCs inhibited DNA binding of p50 and p65 subunits of NF-kB. Also, NF-kB activation significantly reduced estrogen responsiveness of ER-alpha–transfected ESCs, but p50 did not impair ER-alpha DNA binding, suggesting possible indirect mechanisms for this type of interaction(26). Study Limitations There were some limitations in the present study. This study presented a limitation with regard to experimental circumstances. These results also need to be assessed under in vivo conditions. Conclusion Our results support the hypothesis that E2 inhibits NFkB activation through the down-regulation of IkBα phosphorylation and consequent reduction of free NF-kB in the cytosol. These results demonstrate that the regulation of IkBα by E2 may regulate the inflammatory response in eutopic and ectopic endometrial cells. Our in vivo and in vitro findings suggest that this effect of estrogen on IkBα may not be optimal in ectopic endometrium, which may be an important factor in the pathogenesis of endometriosis. The authors appreciate, Özlem Guzeloglu-Kayisli PhD (Department of Obstetrics and Gynecology, College of Medicine, University of South Florida) for their scientific supporting as well as language editing of the manuscript. Ethics Ethics Committee Approval: The study was approved by the Human Investigation Committee of Yale University Local Ethics Committee (approval number: HIC#22334). Informed Consent: Consent form was filled out by all participants. Peer-review: External and internal peer-reviewed. Authorship Contributions

The authors appreciate, Özlem Guzeloglu-Kayisli PhD (Department of Obstetrics and Gynecology, College of Medicine, University of South Florida) for their scientific supporting as well as language editing of the manuscript. Ethics Ethics Committee Approval: The study was approved by the Human Investigation Committee of Yale University Local Ethics Committee (approval number: HIC#22334). Informed Consent: Consent form was filled out by all participants. Peer-review: External and internal peer-reviewed. Authorship Contributions Surgical and Medical Practices: A.A., Concept:  A.A., Design:  A.A., Ü.A.K., Data Collection or Processing: Ü.A.K., Analysis or Interpretation: S.A., Literature Search: S.A., Writing: Ü.A.K., S.A., A.A. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: The authors declared that this study received no financial support.

Surgical and Medical Practices: A.A., Concept:  A.A., Design:  A.A., Ü.A.K., Data Collection or Processing: Ü.A.K., Analysis or Interpretation: S.A., Literature Search: S.A., Writing: Ü.A.K., S.A., A.A. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: The authors declared that this study received no financial support. Table 1 Inhibitory kappa B immunoreactivity in various cell types of human endometrium throughout the cycle. Early proliferative (n=2), late proliferative (n=4), early secretory (n=4) and late secretory (n=2) Table 2 Inhibitory kappa B immunoreactivity in various cell types of normal, eutopic and ectopic endometrium. Menstrual cycle matched normal endometrium (n=6), eutopic and ectopic pairs of endometriotic endometrium samples (n=6) Figure 1 Inhibitory kappa B (IкBα) immunoreactivity in human normal (a, d), eutopic (b, e) and ectopic (c, f) endometrial tissues. IкBα immunoreactivity in proliferative (a, c) and secretory phase (d, f) tissue samples are seen. Stronger immunoreactivity in endometrial glands and stromal cells in normal endometrium are observed when compared with ectopic endometrial and stromal cells. (a-f x40) Figure 2 Inhibitory kappa B (IкBα) immunoreactivity in endometrial stromal cells treated with estradiol and tumor necrosis factor-alpha (TNF-α.) Endometrial stromal cells were treated for 12 min with vehicle (control) (a), TNF-α (2 ng/mL) (b), or estradiol (10-8 M) combined with TNF-α (c), and were immunostained for IкBα. Cells treated with estradiol combined with TNF-α showed stronger immunoreactivity for IкBα than cells treated with TNF-α alone. Endometrial stromal cells were pretreated with vehicle (control) (d, e) or estradiol (f) for 24 h prior to stimulation with TNF-α (e, f) for 15 min. Following stimulation with TNF-α cells pretreated with estradiol for 24 h (f) showed stronger immunoreactivity for IкBα than cells that were not pretreated with estradiol (e) Figure 3 Regulation of inhibitory kappa B (IкBα) in endometrial stromal cells by estradiol and tumor necrosis factor-alpha (TNF-α). Endometrial stromal cells treated with estradiol (E2; 10-8 M), TNF-α (mg/mL) alone, or estradiol with TNF-α (E2+T), or vehicle (C; control) were analyzed for IкBα and its phosphorylated form following 3-12 min treatment. Estradiol treatment suppressed partially the TNF-α-induced IкBα degradation at 6 and 12 min. (+: positive control from TNF-α -induced HeLa cell extracts)

10-8 M), TNF-α (mg/mL) alone, or estradiol with TNF-α (E2+T), or vehicle (C; control) were analyzed for IкBα and its phosphorylated form following 3-12 min treatment. Estradiol treatment suppressed partially the TNF-α-induced IкBα degradation at 6 and 12 min. (+: positive control from TNF-α -induced HeLa cell extracts) Phosphorylation of IкBα: Inhibitory kappa B alpha Figure 4 Regulation of inhibitory kappa B-alpha (IкBα) in endometrial stromal cells by estradiol and tumor necrosis factor-alpha (TNF-α). Endometrial stromal cells were treated with estradiol (E2); 10-8 M, TNF-α (T; 1 mg/mL), estradiol in addition to TNF-α (E2+T), or vehicle (C; control) for 30-60 min. Estradiol has a partial opposing effect on TNF-α–induced IкBα phosphorylation and degradation at both time points (a). Endometrial glandular cells were treated in a similar manner for 90 min, and similar effects were observed (b) Phospho-IкBα: Phosphorylation of inhibitory kappa B-alpha Figure 5 Regulation of inhibitory kappa B alpha (IкBα) in endometrial glandular cells by estradiol and tumor necrosis factor-alpha (TNF-α). Endometrial glandular cells were pre-treated with estradiol (E2); 10-8 M, or vehicle (C; control) for 24 h prior to treatment with TNF-α (1 mg/mL) for 4-12 min. E2 pre-treatment inhibited IкBα degradation compared with control

a (IкBα) in endometrial glandular cells by estradiol and tumor necrosis factor-alpha (TNF-α). Endometrial glandular cells were pre-treated with estradiol (E2); 10-8 M, or vehicle (C; control) for 24 h prior to treatment with TNF-α (1 mg/mL) for 4-12 min. E2 pre-treatment inhibited IкBα degradation compared with control Phospho-IкBα: Phosphorylation of inhibitory kappa B-alpha, TNF: Tumor necrosis factor-alpha Figure 6 Regulation of inhibitory kappa B (IкBα) in endometrial stromal cells by estradiol and interleukin (IL)-1α. Endometrial stromal cells were treated for 6 and 12 min with estradiol (E2); 10-8 M, IL-1α (IL; E2 ng/mL), estradiol with IL-1к (E2+IL), or vehicle (C; control) and were analyzed for phospho-IкBα. Estradiol treatment suppressed IL-1α-induced IкBα degradation at 6 and 12 min Phospho-IкBα: Phosphorylation of inhibitory kappa B-alpha, IL: Interleukin Figure 7 Regulation of active nuclear factor kappa B level in endometrial stromal cells by estradiol. The amount of activated NF-кB in endometrial stromal cells after 15 min of treatment with estradiol (E2); 10-8 M, interleukin (IL)-1a (IL-1; 2 ng/mL) and E2+IL-1 (10-8 M and 2 ng/mL), tumor necrosis factor-alpha (TNF-a) (TNF; 2 ng/mL) and E2+TNF (10-8 M and 2 ng/mL) were compared with control cells. Experiments were repeated on three occasions with similar results and a representative graph from one experiment is presented NF-кB: Nuclear factor kappa B, IL: Interleukin, TNF-α: Tumor necrosis factor-alpha

PRECIS: No difference was identified in terms of right and left USL thicknesses of the OAB and control groups. This was a preliminary study, and further research with larger sample sizes is required to reach a conclusion.

PRECIS: No difference was identified in terms of right and left USL thicknesses of the OAB and control groups. This was a preliminary study, and further research with larger sample sizes is required to reach a conclusion. Introduction Overactive bladder (OAB) is a significant health problem that can negatively affect quality of life(1). “OAB” is a term that describes a syndrome of urinary urgency with or without incontinence, which is often accompanied by nocturia and urinary frequency(2,3). The terms “urgency incontinence” and “OAB with incontinence” are often used interchangeably. Significant risk factors for urinary incontinence are primarily known as age, obesity, births, menopause, hysterectomy, and cigarette smoking(4). It is thought to result from detrusor overactivity, leading to uninhibited detrusor muscle contractions during bladder filling(3). In the etiology, neurologic disorders (e.g., spinal cord injury), bladder abnormalities, increased or altered bladder microbiome may be other reasons, or this may be idiopathic(5). The integral theory describes the pathophysiology of urinary incontinence. The integral theory indicates that pelvic organ prolapses and abnormal pelvic symptoms such as urge, frequency, nocturia, and pelvic pain are usually caused by connective tissue laxity in the vagina or its supporting ligaments(6). In the theory, the pelvic floor muscle forces the vaginal membrane to stretch against the suspensory ligaments to stimulate the micturition stretch receptors. Laxity in the membrane or suspensory ligaments may activate stretch receptors, which are perceived by the cortex as urgency, frequency, and nocturia untimely. The cortex then perceives this stimulation as urgency, frequency, and nocturia(7). Imaging methods are playing an increasingly important part in the diagnosis of pelvic floor disorders. In many studies, pelvic floor disorders have been evaluated with magnetic resonance imaging (MRI)(8). Measurements of the uterosacral ligaments (USL), which are strong ligaments of the uterus, have previously been made with ultrasound in cadaver studies and in patienst with endometriosis(9,10,11). The aim of the current study was to investigate the role of USL anatomy in stretching the vaginal membrane in patients with OAB.

rements of the uterosacral ligaments (USL), which are strong ligaments of the uterus, have previously been made with ultrasound in cadaver studies and in patienst with endometriosis(9,10,11). The aim of the current study was to investigate the role of USL anatomy in stretching the vaginal membrane in patients with OAB. Materials and Methods Approval for this controlled clinical study was granted by the Adana Numune Training and Research Hospital Ethics Committee (approval number: 122/03.11.2015) and written informed consent was obtained from all participants. The study included a total of 27 patients who had been diagnosed as having OAB in our clinic between January 2013 and December 2015. Patients were excluded from the study if they were determined to have concomitant stress urinary incontinence or a malignant pathology. The control group comprised 27 healthy women. The diagnosis of OAB was made from the anamnesis and physical examination. On the first presentation, the patients were questioned in respect of the times that urine leakage occurred, the amount of urine leakage, the reason for the leakage, and what increased or decreased the leakage. Questions were also asked regarding the presence of additional diseases that could cause urine leakage. A physical and pelvic examination was performed to all patients. The stress test was applied. The patients kept a urine diary and this was examined. Patients with OAB who met the study criteria were included in the evaluation. For all the patients included in the study, a record was made of age, height, weight, body mass index (BMI), parity, type of births, and the Pelvic Organ Prolapse Quantification system (POP-Q) was used, which was first published in 1996, in an article by Bump et al.(12) The hymen acts as the set point of indication throughout the POP-Q staging. There are six described points for quantity in the POP-Q system. Anterior: Aa, Ba, C, posterior: Ap, Bp, D. Three others milestones: genital hiatus, the vaginal length (TVL), and perineal body. Each is measured in centimeters above or proximal to the hymen (negative number) or centimeters below or distal to the hymen (positive number) with the plane of the hymen being defined as zero (0).

or: Aa, Ba, C, posterior: Ap, Bp, D. Three others milestones: genital hiatus, the vaginal length (TVL), and perineal body. Each is measured in centimeters above or proximal to the hymen (negative number) or centimeters below or distal to the hymen (positive number) with the plane of the hymen being defined as zero (0). Stage 0: no prolapse; Stage I: the most distal portion of prolapse is >1 cm above level of hymen; Stage II: the most distal part of prolapse is <1 cm proximal to or distal to the plane of hymen; Stage III: the most distal portion of the prolapse protrudes more than 1 cm below the hymen but no farther than 2 cm less than the total TVL (for example, not all of the vagina has prolapsed); Stage IV: complete vaginal eversion is needed, full urine test and urine culture. Lower abdominal MRI was taken using a 1.5 Tesla MRI system (Siemens Magnetom Avanto, Philadelphia, USA). The same protocol was applied to all patients and the control group. Axial T1 and T2 sequences and coronal and sagittal T2 sequences were used. The parameters for the sequences used in the study were as follows: T1-weighted axial repetition time (TR): 370 ms, echo time (TE): 7.11 ms, slice thickness: 3 mm, field of view (FOV): 34x34 mm; T2-weighted axial TR: 3185 ms, TE: 105 ms, slice thickness: 3 mm, FOV: 33x33 mm; T2-weighted turbo spin echo (TSE) sagittal TR: 5117 ms, TE: 120 ms, slice thickness: 3 mm, FOV: 30x30 mm; T2-weighted TSE coronal TR: 5484 ms, TE: 120 ms, slice thickness: 3 mm, FOV: 32x32 mm. The images were evaluated on a separate workstation by a radiology specialist with 10 years’ experience who was blinded to the study. All images were investigated in respect of pelvic pathology, then the USLs on both sides were identified and an evaluation was made concerning their thickness and nodularity. The USL thickness was measured using MRI at the closest points to the cervix and the sacrum and at the mid point between those two points, and the results of the OAB group were compared with those of a control group. Any patients with nodularity that was found to be significant for endometriosis were excluded from the study. A measurement was made of the transverse thickness of the area observed as hypointense on T1 and T2-weighted sequences from the closest points to the cervix and the sacrum and from the midpoint of those two points (Figure 1). The mean of the three measurements was then calculated for the right and left USL.

the study. A measurement was made of the transverse thickness of the area observed as hypointense on T1 and T2-weighted sequences from the closest points to the cervix and the sacrum and from the midpoint of those two points (Figure 1). The mean of the three measurements was then calculated for the right and left USL. Statistical Analysis The statistical analysis of the study data was made using Statistical Package for Social Sciences (SPSS) v. Twenty-one software (SPSS , Chicago, IL, USA). Comparisons were made between the OAB patient group and the control group in respect of the above-mentioned clinical parameters and the USL thickness measured on MRI. Categorical variables were stated as number and percentage (%) and numerical variables as mean (minimum-maximum) ± standard deviation (SD). Conformity of the data to normal distribution was assessed using the the Kolmogorov-Smirnov test. In the comparisons of categorical data between the groups, the chi-square test or Fisher’s exact test was used. In the comparison of numerical data, Student’s t-test was used when there was conformity to normal distribution and the Mann-Whitney U test where there was non-normal distribution. A value of p<0.05 was accepted as statistically significant.

data between the groups, the chi-square test or Fisher’s exact test was used. In the comparison of numerical data, Student’s t-test was used when there was conformity to normal distribution and the Mann-Whitney U test where there was non-normal distribution. A value of p<0.05 was accepted as statistically significant. Results The study included a total of 33 patients with OAB and 30 control subjects who met the study criteria within the specified period. A total of 6 patients with OAB and 3 control group subjects were excluded from the final evaluation because the MRIs were not clear. The mean age was 43.88±9.36 years in the OAB group and 39.92±5.36 years in the control group. The mean BMI value was 29.77±4.82 kg/m2 in the OAB group and 27.49±3.44 kg/m2 in the control group. The mean parity was 3.37±1.59 in the OAB group and 2.7±1.68 in the control group (Table 1). In the OAB group, previous births were performed with vaginal delivery in 59.3%, caesarean section in 25.9%, and with vaginal delivery and cesarean in 14.8%. In the control group, previous births were performed with vaginal delivery in 55.6%, with cesarean section in 37%, and with vaginal delivey and cesarean in 7.4% (p>0.05) (Table 2). No statistically significant difference was determined between the groups in respect of episiotomy (p=0.08). In the comparison of the POP-Q stages between the groups, no statistically significant relationship was determined (p>0.05) (Table 3). The mean (± SD) thickness of the right USL and left USL of both the OAB and control groups was 2.10±0.4 mm and 2.06±0.51 mm, respectively. In the OAB group, the mean thickness of the right USL was 2.04±0.34 mm and the mean thickness of the left USL was 2.04±0.52 mm. In the control group, the mean thickness the right USL was 2.17±0.47 mm and the mean thickness of the left USL was 2.09±0.51 mm. No statistically significant difference was found between the groups in respect of the thickness of the right USL (p=0.71) and the thickness of the left USL (p=0.206) (Table 4).

2.04±0.52 mm. In the control group, the mean thickness the right USL was 2.17±0.47 mm and the mean thickness of the left USL was 2.09±0.51 mm. No statistically significant difference was found between the groups in respect of the thickness of the right USL (p=0.71) and the thickness of the left USL (p=0.206) (Table 4). Discussion In current study, we aimed to evaluate thickness of the USL using MRI to find anatomic disorders of USL that may cause laxness of the vaginal membrane. We hypothesized that the thickness of the USL might correlate with OAB. The role of imaging methods in the evaluation of pelvic floor dysfunctions has been questioned in many studies. MRI in particular has become increasingly useful in the diagnosis of pelvic organ prolapse and pelvic floor disorders(12,13). In previous studies, a correlation has been determined between pelvic floor measurements made with MRI and POP clinical staging(14). However, as study data are limited and cannot be compared, there is no standardized method as yet for MRI measurements(15). In a study by Tan et al.(8) pelvic MRI was performed in young healthy females and cadavers, clearly showing the pelvic and urogenital diaphragm and the previously defined uterus-supporting tissues, and the periuretal and parauretal ligaments were able to be seen anatomically. Stoker et al.(16) examined the whole pelvic floor aiming to find a solution to both urinary and anal dysfunction. The integral theory of female urinary incontinence states that stress and urge symptoms both derive from the same anatomical defect, a lax vagina(17). According to the integral theory, urge, frequency, and nocturia are neurogenic symptoms and can happen with minimal prolapse(6). The integral theory suggests sustentation of the mid-urethra (the anterior vaginal wall along the arcus tendineous, and the vaginal cuff along the uterosacral “neoligament”) will prevent a lax vaginal membrane, which will cure OAB and/or urge incontinence symptoms. This is based on the presence of hypothesized stretch receptors at the proximal urethra and bladder neck(6). On the other hand, hysterectomy has been implicated as a risk factor for the development of urinary incontinence(18). Urinary incontinence after hysterectomy can be the result of a lasting injury to the pelvic plexus at the time of uterosacral/cardinal ligament complex transection, bladder flap formation, and possibly disruption of the anatomic support to the bladder neck and urethra(19).

r the development of urinary incontinence(18). Urinary incontinence after hysterectomy can be the result of a lasting injury to the pelvic plexus at the time of uterosacral/cardinal ligament complex transection, bladder flap formation, and possibly disruption of the anatomic support to the bladder neck and urethra(19). The USL is an important ligament that supports the pelvic floor. No systematic mapping has been performed to define the length and thickness of the USL(10) and there are very few studies in the literature with any information related to USL thickness. In a cadaver dissection study by Vu et al.(9), the USL thickness was reported to be a mean 5-20 mm in the cervical region, 5 mm in the central region, and 5 mm in the sacral region. In addition to cadaver studies, USL thickness has been evaluated in endometriosis. In a study by Bazot et al.(20) MRI findings showed USL thickness as >9 mm in patients with endometriosis. In our study, we were also able to identify the POP-Q stages between the control and OAB groups. However, a statistically significant relationship was not determined between the two groups. It is possible that laxness of the USL may be correlated with both thickness of the ligament and the distribution and function of the collagen types within the tissue(21,22,23). In the whole group of the current study, including the OAB patients and the control group, the mean thickness of the left USL was 2.06±0.51 mm, and the right was 2.10±0.40 mm. None of the current study patients had endometriosis or any pelvic pain. In the OAB group, the mean thickness of the right USL was determined as 2.04±0.34 mm, and the left as 2.04±0.52 mm. In the control group, the mean thickness of the right USL was 2.17±0.46 mm, and the left was 2.09±0.51 mm (Table 4). According to the integral theory, vagina and bladder base defects are displayed as “OAB” symptoms. A previous study by Petros and Ulmsten(7) reported that repair of ligament stretch and tension restored anatomy and function. The USLs are major insertion points for the directional vectors that stretch the vaginal membrane to block premature activation of the micturition reflex. All different appearances of a prematurely activated micturition reflex such as urge incontinence depend on the link between loose ligaments and diminished striated muscle force.

major insertion points for the directional vectors that stretch the vaginal membrane to block premature activation of the micturition reflex. All different appearances of a prematurely activated micturition reflex such as urge incontinence depend on the link between loose ligaments and diminished striated muscle force. The Testicular Feminisation syndrome creates a strong suspension structure to restore muscle contractility and to prevent urge incontinence, as well as urge symptoms(6,23,24,25). Study Limitations This study provides useful preliminary data of the relationship between USL thickness and incontinences. However, the present study has some limitations. First, the sample size was small. Due to the low number of patients, this report should be regarded as a preliminary study. A larger number of patients may be more likely to elucidate the relationship of USL thickness with OAB and the development of urinary problems. Second, a urodynamic test was not used to determine causes of incontinence. Conclusion In conclusion, no statistically significant difference was found between the two groups examined in respect of both left and right USL thickness. There is a need for further studies including a larger number of patients. Ethics Ethics Committee Approval: The study was approved by the Adana Numune Training and Research Hospital Local Ethics Committee (approval number: 122/03.11.2015). Informed Consent: Consent form was filled out by all participants. Peer-review: Externally peer-reviewed. Authorship Contributions

Conclusion In conclusion, no statistically significant difference was found between the two groups examined in respect of both left and right USL thickness. There is a need for further studies including a larger number of patients. Ethics Ethics Committee Approval: The study was approved by the Adana Numune Training and Research Hospital Local Ethics Committee (approval number: 122/03.11.2015). Informed Consent: Consent form was filled out by all participants. Peer-review: Externally peer-reviewed. Authorship Contributions Surgical and Medical Practices: C.A., Concept: C.A., Design: C.A., A.S., Data Collection or Processing: C.A., S.A., S.S., G.S., G.U., Analysis or Interpretation: C.A., A.S., D.Y, Literature Search: C.A., S.A., O.Y., Critical Revision: A.S., E.S.S.Y., Writing: C.A., S.A. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: The authors declared that this study received no financial support. Table 1 Demographic characteristics of overactive bladder and control groups Table 2 Obstetric history of overactive bladder and control groups Table 3 Pelvic Organ Prolapse Quantification system staging of overactive bladder and control groups Table 4 Uterosacral ligament thickness of overactive bladder and control groups Figure 1 Measurement points of the uterosacral ligament on magnetic resonance imaging