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Background/objectives: A high percentage of women having polycystic ovarian syndrome (PCOS) exhibit hyperinsulinemia and obesity. Transforming necrosis factor-α (TNF-α) is an adipokine that increases in obesity and negatively affects insulin action in several tissues, including the endometrium. In fact, it has been reported that insulin signaling is altered in the endometrium of PCOS women, affecting its reproductive function. The aim of this study was to determine the proinflammatory environment and TNF-α signaling in endometrium from obese women with PCOS, and also to evaluate the effect of TNF-α on endometrial cell energy homeostasis., Methods: Serum and endometrial tissues were obtained from four study groups: normal-weight, normal-weight-PCOS, obese and obese-PCOS (hyperandrogenemia/hyperinsulinemia) (n=7 per group). Serum TNF-α level was assayed by enzyme-linked immunosorbent assay (ELISA); endometrial TNF-α level and its receptors (TNFR1/TNFR2) as well as nuclear factor (NF)-κB content were determined by immunohistochemistry. Finally, we evaluated TNF-α effect on glucose uptake in cultured human endometrial stromal cells (T-HESC) treated or not with testosterone/insulin resembling partially the PCOS condition., Results: TNF-α plasma levels were similar between groups, whereas cytokine levels and macrophage number increased in endometrium from obese-PCOS women (P<0.001). Both receptor types were higher in obese vs normal-weight women, particularly TNFR2 content in the obese-PCOS group (P<0.001). Furthermore, an increased NF-κB nuclear content in endometrium from obese-PCOS was observed (P<0.001). Finally, TNF-α treatment of T-HESC cultures exhibited a decrease of glucose uptake (P<0.05), although similar to cells treated with testosterone or testosterone/insulin/TNF-α., Conclusions: These results suggest that the PCOS condition induces an inflammatory state exacerbated when obesity is present, where a higher TNF-α signaling is observed, all of which could affect glucose uptake in the tissue and may cause fertility failures in these women.

Background: Women with polycystic ovary syndrome (PCOS) exhibit a low fertility by chronic hyperandrogenemia. Different evidence have shown that androgens could regulate the endoplasmic reticulum (ER) homeostasis and glucose metabolism. However, it is unclear whether androgens can exert these effects on human endometrial stromal cells. Our goal was to study the protein content of GRP78 (an ER homeostasis marker) in endometria from women with PCOS and healthy women and to assess the GRP78 protein levels and its relationship with glucose uptake on a human endometrial stromal cell line stimulated with testosterone., Methods: Immunohistochemistry assays for GRP78 were performed on endometrial samples obtained from women with PCOS (n = 8) and control women subjected to hysterectomy (n = 8). Western blot analysis for GRP78 and glucose uptake was assessed in a telomerase-immortalized human endometrial stromal cell line (T-HESC) exposed to testosterone for 24 or 48 hours and challenged to an insulin short-term stimulation. Tukey test was performed for human samples comparison. Student t test or ANOVA-Bonferroni test was carried out according to the in vitro experiment. P < .05 was considered as significant., Results: GRP78 stromal immunostaining was reduced in PCOS endometria compared to controls (P < .05). The T-HESC shows a testosterone-dependent downregulation of GRP78 protein content (P < .05), concomitant with half-reduction in glucose uptake compared to controls (P < .05). Moreover, enhanced small interfering RNA against GRP78 messenger RNA leads to a decrease in glucose uptake (P < .05). Such effects were reverted by hydroxyflutamide, an inhibitor of androgen receptor., Conclusion: These results suggest that hyperandrogenemic PCOS environment could compromise the endometrial homeostasis confirmed by the decrease in glucose uptake induced by testosterone and exhibited by stromal cells., (© The Author(s) 2015.)

Polycystic ovarian syndrome (PCOS) has been associated with endometrial hyperplasia and cancer. The aim of this study was to establish whether the expression of proliferation regulatory proteins in the endometria of patients having PCOS, with or without hyperplasia, differs from control women. Control endometria (CE), patients having PCOS without and with endometrial hyperplasia (PCOSE and HPCOSE, respectively), and that of women with endometrial hyperplasia (HE) were used. The phosphorylated estrogen receptor form (pERα), similar to mother against decapentaplegic (SMAD) 2, SMAD3, and SMAD4, vascular epithelial growth factor (VEGF), and phosphorylated SMAD (pSMAD) 2 and pSMAD3 were detected by immunohistochemistry or Western blot. The results show higher levels of pERα in HE versus CE (P < .05), while higher VEGF levels were found in PCOSE and HE (P < .05) compared to CE; SMAD2 diminished in HE (P < .05) versus CE. Consequently, the higher levels of VEGF and pERα in PCOSE could represent early changes in the progression of PCOSE toward hyperplasia and cancer, whereas changes observed in SMAD proteins support the differential origin of the pathologies of HPCOSE and HE., (© The Author(s) 2015.)

Background: Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine/ metabolic disorder characterized by hyperandrogenemia and in most cases, by hyperinsulinemia in addition to obesity. Besides ovarian dysfunction, endometrial physiology is also disrupted since this tissue is highly dependent on the action of steroids; in case of conception cycles, high percentage of abortion is observed. Because of the endocrine/metabolic alterations, PCOS-women present high probability to develop hyperplasia and endometrial cancer, where an imbalance of cell proliferation/apoptosis processes is detected. Additionally, insulin pathway and the endometrial energetic homeostasis are also compromised., Methods: The aim of this review was to report molecular alterations related to insulinresistance and/or obesity in PCOS-women endometria that could drive to infertility. For this, several methods were employed: immunohistocytochemistry, qPCR, western-blot, glucoseuptake, cell cultures, among others., Results: Diminished levels and activity of several insulin signaling pathway molecules, like IRS-1/AS160/PKCζ, were detected. Concomitantly, a defect in the synthesis and GLUT4 translocation to cell surface is induced. Oral administration of metformin (insulin sensitizer) to PCOS-patients increases GLUT4 endometrial levels, improving fertility of those patients. Another relevant feature is the high percentage of obesity in PCOS-women; adiponectin is an obesity marker and elicits an insulin-sensitizer action, being diminished in plasma of obese PCOSwomen similar to its endometrial level, adiponectin-receptors and APPL1, an adapter molecule of adiponectin pathway. Moreover, obesity and PCOS can induce a pro-inflammatory environment, exaggerating the alterations in insulin pathway., Conclusion: The evidences obtained in PCOS-endometria clearly indicate that these molecular defects could partially explain the reproductive failures of these patients.

Intracrinology mechanism involves the metabolism of steroids in peripheral tissues, such as DHEA, to molecules with estrogenic or androgenic activity. Proliferation rate of endometria from Polycystic Ovary Syndrome women (PCOS) is increased, favoring hyperplasia development. Besides, in endometria from PCOS-women the synthesis of androst-5-ene-3β,17β-diol (androstenediol), an estrogenic molecule, is enhanced concomitantly to increased cellular proliferation. DHEA, the major intracrinological precursor, circulates mainly in its sulfated form and requires transporters for cell intake, that belong to the families of organic anion transporting polypeptides (OATP) and organic anion transporters (OAT). The aim of this study was to determine protein levels and activity of sulfated steroid transporters OATP2B1, OATP3A1, OATP4A1 and OAT4 in endometria from control and PCOS-women and to evaluate the activity of the enzyme 3β-HSD. Levels of transporters were done by RT-PCR (OAT4 only) and Western-blot (WB). Additionally, in primary culture cells stimulated with steroids, protein levels by WB and uptake of tritiated DHEAS, were evaluated; 3β-HSD activity was assessed using radiolabel substrate. PCOS-endometrium had higher levels of OATP2B1 and OATP4A1 than CE (p<0.05); decreased OATP4A1 levels were found in androstenediol or testosterone-stimulated cells. Accordingly, the entry of DHEAS to cells was lower in cells stimulated with testosterone (p<0.05); 3β-HSD-activity was similar in control and PCOS-endometria. Therefore, this study describes that steroids can modulate the expression and activity of transporters of OATPs-family in human endometria and that some transporter levels are increased in PCOS-endometria, suggesting a potential role in the pathogenesis of endometrial hyperplasia of these patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Study Question: Does treatment with the insulin sensitizer metformin modify the levels and activation of proteins related to the expression of the insulin-dependent glucose transporter (GLUT4), such as adenosine monophosphate-activated protein kinase (AMPK) and myocyte enhancer factor 2A (MEF2A), in endometria from hyperinsulinemic hyperandrogenemic polycystic ovary syndrome (PCOS h-Ins) patients?, Summary Answer: In PCOS h-Ins patients, metformin increases endometrial levels of GLUT4 mRNA and protein levels by normalizing the quantity and activation of molecules that regulate GLUT4 expression to healthy values. These changes could improve endometrial metabolic function., What Is Already Known: PCOS is an endocrine-metabolic disorders closely associated with insulin resistance. In particular, the insulin signaling pathway is impaired in endometria from these patients and the concentration of GLUT4, as well as the molecules involved in its translocation to the cell surface, is decreased. However, there are limited data about the mechanisms that regulate the GLUT4 expression in the endometria and the effect of metformin on them., Study Design, Size and Duration: This is a case-control study in the setting of a research unit, approved by the Ethical Committees of our institution. The groups whose endometria were studied were PCOS h-Ins (n = 8); PCOS patients with hyperandrogenemia hyperinsulinemia taking only metformin for at least 3 months (PCOS-MTF, n = 8) and healthy fertile women at the time of hysterectomy because of benign pathology as controls (CE, n = 8)., Participants/materials, Setting, Methods: Steroids and sex hormone-binding globulin were measured and glucose and insulin levels were evaluated during an oral glucose tolerance test. Protein levels for αAMPK (catalytic subunit of AMPK), phosphorylated (p)-AMPKαThr(172) (activating phosphorylation site), MEF2A, p-MEF2AThr312 (activating phosphorylation site) and GLUT4 were assessed by western blot and immunohistochemistry. In addition, GLUT4 gene expression was evaluated by RT-PCR., Main Results and the Role of Chance: We found significantly lower levels of MEF2A and p-MEF2AThr312 in PCOS h-Ins compared with CE endometria (P < 0.05). Also, we detected lower levels of p-AMPKαThr(172) in PCOS h-Ins endometria compared with the PCOS-MTF group (P < 0.05). The ratios of phospho-AMPK/total AMPK and phospho-MEF2A/total MEF2A were significantly increased in the PCOS-MTF compared with the PCOS h-Ins group (P < 0.05). The RT-PCR experiments showed lower levels of GLUT4 mRNA transcripts in PCOS h-Ins compared with PCOS-MTF-treated group (P < 0.05), the protein levels of GLUT4 were decreased in a similar way., Limitations, Reasons for Caution: The limited number of patients included in this study who presented large clinical variability. Therefore, it would be necessary to recruit a greater number of patients to minimize our data dispersion in order to prove the clinical benefits of metformin described by others., Wider Implications of the Findings: Since the insulin sensitizer metformin increases the expression of the GLUT4, it may improve endometrial physiology in PCOS patients and, therefore, promote better reproductive outcomes. These results suggest that in PCOS patients, metformin may act directly at the endometrial level and decrease insulin resistance condition by increasing the expression of GLUT4 and, in this way, indirectly restore endometrial function., Study Funding/competing Interest(s): This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number 1095127 to M.V.). None of the authors has any conflict of interest to declare.

Endometrial tissue of patients with polycystic ovary syndrome (PCOS) shows an impaired expression of insulin signaling molecules. Tyrosine phosphorylation of the insulin receptor (IR) by insulin promotes glucose uptake by activating the PI3K/Akt pathway. IR stability and function depend on the presence of the protein caveolin-1. Activation of IR increases phosphorylation of Y¹⁴caveolin-1. Since the endometrium of PCOS patients is proposed to be insulin resistant, we evaluated the phosphorylation of IR and caveolin-1 in endometria of patients with insulin resistance (PCOSE-IR) compared to controls (CE). To explore the mechanism associated with this condition, cultured endometrial cells (T-HESC) were exposed to high glucose (25 mM, 24 h), an experimental condition that leads to insulin resistance in other cell types. Endometrial protein levels of phospho-Y⁹⁷²IR, phospho-Y¹⁴caveolin-1 and caveolin-1 were determined by Western blotting. In cultured cells, protein levels of caveolin-1, IR, and Akt were evaluated by Western blotting. After acute insulin stimulation, phospho-S⁴⁷³Akt, phospho-Y¹⁴caveolin-1, and 2-deoxyglucose (2-DOG) uptake were determined. PCOSE-IR samples showed high protein levels of caveolin-1, but reduced phospho-Y¹⁴caveolin-1 compared to CE. No differences were observed for phospho-Y⁹⁷²IR between both groups. Cells pretreated with glucose showed a reduction in protein levels of IR and caveolin-1 and were unable to increase 2-DOG uptake, phospho-S⁴⁷³Akt and phospho-Y¹⁴caveolin-1 after insulin stimulation. In conclusion, in PCOSE-IR the impaired phosphorylation of IR downstream molecules such as phospho-Y¹⁴caveolin-1 suggests a diminished insulin sensitivity in endometria, condition that could be supported in vitro by the ability of T-HESCs to become insulin resistant when they are exposed to high glucose., (© Georg Thieme Verlag KG Stuttgart · New York.)

Enhanced endometrial proliferation correlates obesity to type-I (estrogen-dependent) endometrial cancer (EC). Our aim was to distinguish obese women (without EC) with differing endometrial proliferation. Endometrial and blood samples were obtained from normal-weight and obese women without EC. Type-I EC samples were obtained from obese patients. On measuring endometrial proliferation (Ki67 and phosphorylated histone H3 (p-H3)), two groups of obese women without EC were identified: obese(High Proliferating) (O(HP)) and obese(Low Proliferating) (O(LP)). Increased Ki67 (88.5%, P<0.001), p-H3 (62.6%, P<0.01), 17β-estradiol/progesterone ratio (46.3%, P<0.01) and endometrial estrogen receptor alpha (ERα) (82.2%, P<0.001) were observed in O(HP) compared with O(LP) patients. ECs possessed similar ERα and enhanced proliferation as O(HP), suggesting that O(HP) women are at higher risk of type-I EC. O(LP) women were indistinguishable from normal-weight women regarding these determinants of endometrial proliferation, ERα and 17β-estradiol/progesterone ratio. Our data may further define the obesity phenotype in regards to type-I EC risk and may help identify obese women more susceptible to develop type-I EC, allowing early intervention and a potential reduction in mortality.

Background: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder commonly associated with insulin resistance (IR). Previous studies indicate about the expression of molecules involved in the insulin pathway in endometria of women with PCOS-IR. Therefore, the aim of the present study was to evaluate the effect of insulin and testosterone in the expression of these proteins in the endometria and immortal endometrial stromal cell line (T-HESCs)., Methods: We examined the protein levels of Munc18c, PKC zeta, phospho-PKC Zeta, and Syntaxin-4. Protein levels were assessed by Western Blot and/or immunohistochemistry in proliferative endometria (NPE = 6) and in PCOS endometria with insulin resistance (PCOSE-IR = 6). We also evaluated whether high concentrations of insulin (100 nM) and/or testosterone (100 nM), during a 24 h stimulatory period, affected the expression of these proteins in an immortal endometrial stromal cell line (T-HESCs). Once stimulated, proteins were extracted from cells and were assessed by Western Blot analysis. Immunocytochemistry was performed to detect AR in T-HESC cells., Results: Western Blot data showed decreased expression (p < 0,05) of Munc18c and phospho-PKC Zeta in PCOS-IR endometria (PCOSE-IR) with respect to the control (NPE). In the in vitro study, Western Blot analysis showed decreased levels of Munc18c, PKC Zeta and phospho-PKC Zeta with the different hormonal treatments when compared to the control condition (no hormonal stimulation) (p < 0,05). The AR was present in the endometrial stromal cell line (T-HESC)., Conclusion: The conditions of hyperinsulinism and hyperandrogenism present in PCOS-IR patients modulate the expression and/or phosphorylation of the proteins involved in the insulin pathway at the endometrial level. These data extend to the T-HESCs cells results, where insulin and testosterone exert an effect on both the expression and phosphorylation of proteins present in the pathway.

The changes in endometrial homeostasis found in women with polycystic ovarian syndrome (PCOS) could be associated with alterations in the intracrine metabolism of steroid hormones. The uptake of dehydroepiandrosterone-sulphate (DHEA-S), precursor of the intracrine pathway, is achieved by transporters, such as organic anion transporter polypeptides (OATPs), and molecules with oestrogenic activity, such as androst-5-ene-3beta,17beta-diol (androstenediol), can be generated. We aimed to determine androstenediol generation and the expression of OATPs in human endometria throughout the menstrual cycle and in endometria from PCOS women. Endometrial samples were obtained from control women in the proliferative phase (control endometria (CEp), n=7), secretory phase (CEs, n=7), and from PCOS patients (PCOSEp, n=7). The mRNA levels of OATP-B, OATP-D and OATP-E were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels of OATP-E by immunofluorescence; 3beta-hydroxysteroid dehydrogenase (HSD) by immunohistochemistry/Western blot; the metabolism of DHEA to androstenediol was evaluated by thin layer chromatography-high-performance liquid chromatography (TLC-HPLC). Lower levels of OATP-E transcript were obtained in PCOSEp (p<0.05) compared with CEp, while OATP-E protein levels (p<0.05) and DHEA conversion to androstenediol (p<0.01) were higher in PCOSEp. Lower 3beta-(hydroxysteroid dehydrogenase) HSD protein levels were found in PCOSEp (p<0.05) (Western blot, immunohistochemistry). These results reveal a higher capacity of the endometria from PCOS women to metabolise DHEA to androstenediol, which, coupled with the high oestrogen sensitivity previously found in these endometria, may account for the increase in cell proliferation in PCOSEp already reported., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder highly associated with insulin resistance and compensatory hyperinsulinemia. It is known that the insulin signaling pathway is impaired in endometria from PCOS hyperinsulinemic women, but no information is available about molecules associated with cell surface GLUT4 translocation. We therefore evaluated the protein levels of AS160 target molecules, Rab8A and Rab10, and the WAVE family proteins involved in the cortical-actin remodeling, Neural Wiskott-Aldrich Syndrome Protein (N-WASP) and WASP, in endometria from hyperinsulinemic PCOS women and controls., Methods: Protein levels were assessed by western blot, immunohistochemistry and immunofluorescence in proliferative (PE = 7) and secretory (SE = 7) phase endometria from control women and in endometria from hyperinsulinemic PCOS women (PCOS h-INS = 7)., Results: Similar levels were detected for Rab10 in the three studied groups; however, Rab8A levels decreased in SE (P < 0.05) while higher levels were obtained in PCOSE h-INS compared with PE (P < 0.05). In the normal menstrual cycle, Neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP levels were increased in SE versus PE (P < 0.05), but in PCOSE h-INS, the levels were diminished compared with PE (P < 0.05)., Conclusions: SE is characterized by protein expression changes associated with glucose uptake. In endometria from PCOS women with hyperinsulinemia, reduced levels of WAVE family proteins could compromise the cell surface GLUT4 exposure and the consequent glucose uptake in this tissue.

Fifty to seventy percent of patients with polycystic ovary syndrome (PCOS) present hyperinsulinemia. On the other hand, reports indicate that forkhead box class O 1 (FOXO1) and peroxisome proliferator-activated receptor-gamma (PPARG) are involved in the insulin signaling pathway, regulating the gene expression of SLC2A4 (GLUT4). The negative effect of FOXO1 over PPARG transcription disappears when FOXO1 is phosphorylated (p-FOXO1) and excluded from the nucleus, whereas PPARG can suppress gene expression of SLC2A4. Scarce knowledge is available in endometrium of women with PCOS and hyperinsulinemia (PCOSE h-Ins) about the role of these factors. We aimed to evaluate whether the endocrine and metabolic status of PCOS modify the levels of gene and protein expression of FOXO1, PPARG, and SLC2A4 in the endometria from hyperinsulinemic PCOS women compared with controls. In endometria from control (CE, n=7) or PCOSE h-Ins (n=7), we determined the subcellular location and protein levels of p-FOXO1Ser319 and FOXO1/FOXO4 by immunohistochemistry and western blot respectively; gene and/or protein levels of PPARG and SLC2A4 were evaluated by RT-PCR and/or western blot. Cytoplasm location for FOXO1 and p-FOXO1Ser319 was immunodetected in both groups of endometria, showing significantly higher staining in PCOSE h-Ins for these proteins (P<0.05). In PCOSE h-Ins, gene and protein levels of PPARG were significantly higher than in CE, whereas SLC2A4 mRNA was decreased (P<0.05). In conclusion, the derepression of PPARG transcription by the high levels of p-FOXO1Ser319 could partially account for the lower levels of SLC2A4 found in PCOSE h-Ins, suggesting an alteration of the endometrial function in these patients.

Objective: To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women., Methods: Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subject's body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays., Results: In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subject's BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology., Conclusion: These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women., (Copyright 2009 Elsevier Inc. All rights reserved.)

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder associated with insulin resistance and compensatory hyperinsulinemia. Scarce information is available on the expression of molecules involved in the insulin pathway in endometria from women with PCOS. Therefore, we examined the protein levels of insulin-signaling molecules, like insulin receptor, insulin-receptor substrate (IRS)-1, pIRS-1Y612, Akt, AS160, pAS160T642 and GLUT4 in endometria from PCOS women with or without hyperinsulinemia. Protein levels were assessed by Western blot and immunohistochemistry in 21 proliferative-phase endometria from control women (CE = 7), normoinssulinemic PCOS women (PCOSE-NI = 7) and hyperinsulinemic PCOS women (PCOSE-HI = 7). The data show no differences in the expression of insulin receptor between all groups as assessed by Western blot; however, IRS-1 and pIRS-1Y612 were lower in PCOSE-HI than controls and PCOSE-NI (P < 0.05). AS160 was detected in all analyzed tissues with similar expression levels between groups. Importantly, PCOSE-HI exhibited lower levels of pAS160T642 (P < 0.05) and of GLUT4 (P < 0.05) compared with CE. The immunohistochemistry for insulin receptor, IRS-1, Akt, AS160 and GLUT4 showed epithelial and stromal localization; IRS-1 staining was lower in PCOSE-HI (P < 0.05). In conclusion, human endometrium has the machinery for glucose uptake mediated by insulin. The diminished expression of GLUT4, as well as the lower level of pIRS-1Y612 and pAS160T642 exhibited by PCOSE-HI, suggests a disruption in the translocation of vesicles with GLUT4 to the cell surface in these patients.

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE (p<0.05, p<0.01, respectively) and of EST for HPCOSE and EH compared to control (p<0.05). However, higher levels for EST mRNA were obtained in PCOSE (p<0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE (p<0.05) versus CE. The ratio between the mRNA from 17beta-HSD type 1/type 2 was higher in PCOSE (p<0.05), whereas, a diminution in the 17beta-HSD type 2 activity was observed in PCOSE (p<0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.

Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic pathology related with infertility and recurrent miscarriage. We have previously shown that the endometrium of these patients can exhibit a potentially higher sensitivity to estrogen action, being estrogens important regulators of the cell cycle and tissue homeostasis. The effect of estrogens on tissues depends on their in situ availability, which is in part regulated by the activity of steroid metabolic enzymes within the tissues. Therefore, the objective of the present study was to analyze if the activity and/or expression of steroid metabolic enzymes in endometria from women with PCOS differ from controls. For this purpose, the activity of the enzymes was determined by using radiometric assays and the mRNA levels measured by semi-quantitative RT-PCR. Both assays were assessed in endometria obtained during mid secretory phase from control (CE, n=12) and PCOS women (PCOSE, n=11). For the statistical analyses, Mann-Whitney and Student's t-tests were used to compare CE and PCOSE, considering a p value <0.05 significantly different. The results showed an increase in the sulfatase activity in PCOS respect to control endometria (200+/-28pmol/mg vs. 115+/-13pmol/mgproth; p<0.05), in agreement with the higher mRNA levels found for the enzyme in PCOSE. In addition, a PCOSE exhibited lower activity of sulfotransferase respect to the control group (50+/-21pmol/mg vs. 124+/-10pmol/mgproth; p<0.05), whereas a higher level of 17beta-hydroxysteroid dehydrogenase type 1mRNA was found in PCOSE compared with the control tissues (p<0.05). The activity of 17beta-hydroxysteroid dehydrogenase type 2 and the mRNA levels of sulfotransferase were similar in both groups; meanwhile, the expression of aromatase was undetectable. These data indicate that the sulfatase pathway could play an important role in the local production of estrogens in PCOSE from secretory phase. This potentially higher bioavailability of estrogens in endometria from PCOS women could influence the deregulation of tissue homeostasis that we have previously reported, and could partially explain the poor reproductive performance observed in this group of patients.

Objective: To evaluate the protein and messenger RNA expression of sex hormone-binding globulin (SHBG) in endometria from women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Thirty-three women with PCOS, and 17 fertile, healthy women of similar age to those with PCOS., Intervention(s): Endometrial and blood samples were obtained from women with PCOS (PCOSEs) and from control women (CEs) during the proliferative phase of the menstrual cycle., Main Outcome Measure(s): Expression studies for SHBG (immunohistochemistry and reverse transcription-polymerase chain reaction). Hormonal studies for determining sex steroids (T, P, and E(2)) and SHBG concentration. Insulin sensitivity was assessed by composite insulin sensitivity index (ISI(composite))., Result(s): In stroma, the protein expression of SHBG was lower in PCOSEs than in CEs. Epithelial cells had a similar expression of SHBG protein in both groups. Messenger RNA of variant 548 base pairs (wild-type) tended to be lower in PCOSEs compared to CEs. When PCOSEs were classified by insulin resistance, the PCOSEs with normal insulin sensitivity showed an expression of stromal SHBG similar to that observed in CEs., Conclusion(s): The low SHBG expression in the stromal compartment of endometria from women with PCOS with insulin resistance may contribute to generate an abnormal steroid milieu in the endometria of these women.

Objective: To study the proteins involved in endometrial homeostasis in PCOS women., Methods: Protein expression of Ki67, Bcl-2, Bax, Pro-Caspase-3 and Caspase-3 by immunohistochemistry and/or Western blot, and DNA fragmentation using in situ 3'-end labeling of apoptotic cells, was measured in 9 samples of normal endometrium (NE), 12 PCOS endometria without treatment (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE) and 9 endometria from patients with endometrial hyperplasia (HE)., Results: Cell proliferation was higher in epithelium from PCOSE (P<0.05), HPCOSE and HE vs NE. A higher Bcl-2/Bax relative ratio in PCOSE and HPCOSE was observed, in absence of active Caspase-3 and scarce DNA fragmentation in the four groups of endometria studied., Conclusion: As the apoptosis was scarce in all of the groups studied, endometrial homeostasis deregulation in PCOS could be a result of increased proliferation. Therefore, the onset of endometrial hyperplasia in PCOS endometrium could be linked to inadequate cell proliferation, and concomitantly to inadequate cell survival.

Background: The hormonal alterations observed in women with polycystic ovary syndrome (PCOS) may promote implantation failure as well as disruption of their endometrial homeostasis. To evaluate cell survival of mid-secretory endometrium from untreated women with PCOS, we measured the expression of apoptosis and proliferation-related proteins., Methods: A case-control study of 11 patients with PCOS and 11 fertile women in the Hospital Research Unit was performed. Endometrial samples were obtained from PCOS women (PCOSE) and fertile healthy women (CE) during the mid-secretory phase of the menstrual cycle. Protein expressions for Akt, p-AktSer473 and p-AktThr308, Bad, p-BadSer136, Bcl-2, Bax and pro-caspase-3/caspase-3, were assessed by western blot, and Ki67 and p-histone-3 (p-H3) by immunohistochemistry., Results: In CE and PCOSE, a predominance of p-AktThr308 over p-AktSer473 is observed; p-BadSer136 expression is higher in PCOSE than in CE (P < 0.05). Also, Bcl-2 protein is overexpressed in PCOSE (P < 0.05), with no changes in Bax expression among the two groups, resulting in a significantly higher Bcl-2/Bax ratio in PCOSE than in CE (P < 0.05). No changes in the expression of caspase-3 are obtained between both groups of endometria. Furthermore, cell proliferation detected by the expression of Ki67 and p-H3 proteins is higher in the epithelia than the stroma of PCOSE versus CE (P < 0.05)., Conclusion: The abnormal tissue homeostasis exhibited by the secretory endometrium from PCOS patients with spontaneous ovulation may interfere with their endometrial receptivity.

Objective: To evaluate gene and protein expression of steroid receptors, nuclear receptor coregulators, and uterine receptivity markers in midsecretory phase endometria from untreated women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Eight patients with PCOS and eight fertile women of similar age to those with PCOS., Intervention(s): Endometrial samples were obtained from women with PCOS (PCOSE) and normal (NE) women during the midsecretory phase of the menstrual cycle., Main Outcome Measure(s): Expression studies (immunohistochemistry, reverse transcription-polymerase chain reaction [RT-PCR] and Western blot)., Result(s): Endometria from PCOS exhibit higher levels of messenger RNA (mRNA) and protein for estrogen receptor alpha and coactivators than NE. Epithelial cells had a greater expression of progesterone receptor in PCOSE, whereas, no differences were observed in gene and protein expression of the nuclear corepressor (NcoR) and the antiadhesion molecule mucin type-1 (MUC-1) between PCOSE and NE. Immunodetection for the coactivator ARA70 was higher in PCOSE than in NE; in contrast, expression of beta3-integrin in epithelia was lower in PCOSE than in control endometria., Conclusion(s): The higher response to steroid hormones of endometria from untreated PCOS-women induces diminished expression of beta3 integrin, which partially explain implantation failure in PCOS patients.

Objective: To assess the effect of clomiphene citrate (CC) on endometrial epithelial integrins and P receptors (PR) during the window of implantation., Design: Controlled, prospective, clinical study., Setting: Teaching hospital and university research laboratory., Patient(s): Thirty-one fertile, normo-ovulatory women participated in this trial. Thirteen women exhibited a CC-stimulated cycle with 50 mg on days 5-9, and 18 women with spontaneous menstrual cycles served as controls., Intervention(s): Endometrial biopsies in the midluteal phase., Main Outcome Measure(s): Immunohistochemical determination and endometrial cellular localization of alpha1, alpha v, beta3, and alpha4 epithelial integrins and PR during the window of implantation. The staining intensity was assessed by a semiquantitative index (HSCORE) and compared by nonparametric Mann-Whitney test., Result(s): Higher plasma levels of P and E2 and delayed histologic dating of the endometrium (38%) were features of CC-treated women. In addition, a low epithelial beta3 integrin expression and persistent PR were observed in glandular epithelial cells of "out-of-phase" endometrial biopsies from CC-treated women. In contrast, in "in-phase" biopsies, neither epithelial PR nor beta3 integrin were different from spontaneous control cycles. There was no difference in the expression of alpha1, alpha v, and alpha4 between the groups studied., Conclusion(s): The administration of clomiphene produces aberrant endometrial beta3 integrin expression in conjunction with a failure in the down-regulation of PR during the window of implantation in a significant number of normo-ovulatory women, notwithstanding the higher plasma P levels. Therefore, CC might affect the expression of endometrial receptivity markers.

Objective: To evaluate the clinical response and endometrial morphology during the implantation window on ovarian hyperstimulation with the aromatase inhibitor letrozole in infertile ovulatory women., Design: Prospective trial in infertile patients., Setting: Tertiary care hospital., Patient(s): Eight ovulatory infertile patient candidates for ovarian superovulation., Intervention(s): Subjects were monitored in one control cycle. In the next cycle, they received letrozole 5.0 mg daily on days 3 through 7 after menses., Main Outcome Measure(s): Number of ovulatory follicles; dominant follicle diameter; endometrial thickness; hormonal profile of FSH, LH, E(2), A, T, and P; endometrial histological dating; and pinopode formation assessed by scanning electron microscopy., Result(s): Cycles stimulated with letrozole resulted in more ovulatory follicles than did natural cycles (mean +/- SD 2.0 +/- 0.9 vs. 1.0 +/- 0.0), which attained a greater preovulatory diameter (mean +/- SD 23.8 +/- 2.7 vs. 19.3 +/- 2.1 mm), with similar endometrial thickness at midcycle compared with spontaneous cycles. Endocrine profile of medicated cycles was characterized on day 7 by increased levels of LH (5.9 +/- 0.8 vs. 3.5 +/- 0.4 IU/mL), reduced E(2) (98.4 +/- 11.4 vs. 161.5 +/- 14.7 pmol/L), and elevated androgens. Preovulatory and midsecretory E(2) were similar to spontaneous cycle, and P levels during midluteal phase were significantly elevated (44.2 +/- 4.6 vs. 27.7 +/- 4.6 pmol/L). Endometrial morphology during the implantation window in letrozole-stimulated cycles was characterized by in-phase histological dating and pinopode expression on scanning electron microscopy., Conclusion(s): Letrozole induces moderate ovarian hyperstimulation in ovulatory infertile patients with E(2) levels similar to spontaneous cycles and higher midluteal P, leading to both a normal endometrial histology and development of pinopodes, considered to be relevant markers of endometrial receptivity.

Objective: To evaluate the expression of steroid hormone receptors, proteins related to apoptosis, and cell proliferation in endometria from women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Eight women with PCOS and 12 fertile healthy women of similar age to those with PCOS., Intervention(s): Endometrial samples were obtained from women with PCOS (PCOSE) and normal (NE) women during the proliferative phase of the menstrual cycle., Main Outcome Measure(s): Expression studies (immunohistochemistry and reverse transcription-polymerase chain reaction) and DNA fragmentation [TdT-mediated dUTP nick end labeling (TUNEL)]., Result(s): In stroma, protein expression of estrogen receptor alpha, Bcl-2, and Bax was higher in PCOSE than in NE; epithelial cells had a greater expression of androgen receptor in the nucleus and a lower expression in the cytoplasm of PCOSE. Cell proliferation was higher in the epithelia of NE, while the expression of caspase-3 and DNA fragmentation was similar in both groups. The bax mRNA expression was higher in PCOSE, and bcl-2 mRNA expression was similar between groups. A higher bcl-2/bax relative ratio in PCOSE was observed., Conclusion(s): An alternating expression of proteins related to cell survival in endometria from PCOSE may potentially be associated with the disruption of their endometrial cell cycle.

Objective: To investigate the role of nitric oxide (NO) and death regulatory genes, bcl-2 and bax, in human endometria apoptosis., Design: Expression of bcl-2, bax, NO synthases (NOS), and the apoptotic effect of L-arginine on endometrial explants in vitro., Setting: Prospective study., Patient(s): Thirty-seven eumenorrheic women., Intervention(s): Endometrial samples were obtained with Pipelle suction curette after women signed institutional informed consent forms., Main Outcome Measure(s): DNA fragmentation (TUNEL), immunohistochemistry, and reverse transcription polymerase chain reaction., Result(s): Apoptosis was detected in mid and late secretory endometria. L-arginine induced an increase in apoptosis in stroma (threefold), glands (eightfold), and surface epithelia (fourfold) in proliferative but not secretory endometria explants. Immunostaining of Bcl-2 was almost absent in the secretory endometria, whereas Bax increased in the stroma at the end of the menstrual cycle, coincident to the decrease in the bcl-2/bax mRNA relative ratio (P<.05) observed in secretory endometria., Conclusion(s): The induction of DNA fragmentation by L-arginine on proliferative endometria suggests that NO may be involved in the endometrial apoptotic process, whose control may be related predominantly to the changes of Bcl-2 expression.

Unlabelled: Determine quantitative expression of endometrial integrins that reflect receptivity during implantation window in fertile women treated with clomiphene citrate (CC), and in intrauterine device users (IUD) as compared to fertile controls. Comparative study of the quantitative expression of a1, a4, av and b3 integrins in epithelial and stromal cells in mid-secretory endometrium of CC treated fertile women, IUD users and controls. All subjects included in this study had regular and ovulatory menstrual cycles., Subjects: Ten women treated with a daily dose of 50 mg of CC. Six women T-Cu device users and nine fertile controls. Age ranges for all groups were similar, 29-41 years old (mean 36.3). Tissue samples were taken at the mid-secretory phase or implantation window. A histological dating of the endometrial biopsies was assessed according to Noyes criteria. Ovulation was assessed by repeated transvaginal ultrasonography. The expression of a1, a4, aV and b3 integrins in dispersions of epithelial (EEC) and stromal (ESC) cells isolated from endometrial biopsies was quantitatively determined by flow cytometry using specific monoclonal antibodies. Immunohistochemistry was also used to detect integrin expression. Biopsies from CC-treated women had a high incidence of out-of-phase endometria. Interestingly, CC-treated women over-expressed a1, aV and b3-ESC integrins and under-expressed b3-EEC subunit (P<0.05). IUD users over-expressed the a1-EEC and under-expressed a4-ESC (P<0.05) at the time of the implantation window. CC treatment in fertile women provokes a high frequency of out-of-phase endometrium and desynchronises the expression of endometrial integrins at the implantation window. The epithelial b3 integrin was under-expressed in all CC-treated patients. The T-Cu intrauterine device alters endometrial receptivity by a different mechanism independent of the expression of the epithelial b3 integrin. However, both CC and IUD use alter the expression of some epithelial and stromal integrins during the implantation window.