Lack of cyclical fluctuations of endometrial GLUT4 expression in women with polycystic ovary syndrome: Evidence for direct regulation of GLUT4 by steroid hormones.
ABSTRACT: Background Determination of the role of steroid hormones in expression and regulation of endometrial glucose transport 4 (GLUT4) in humans is important for understanding endometrial disorders such as polycystic ovary syndrome (PCOS), a common hormone-imbalance disease. Methods Endometrial biopsy samples were collected from non-PCOS patients with regular menstrual cycles or with hyperplasia and from PCOS patients with or without hyperplasia. In addition, endometrial tissues from postmenopausal women were incubated with human chorionic gonadotropin (hCG, 10 IU/ml), 17?-estradiol (E2, 10 nM), progesterone (P4, 100 nM), or a combination of E2 and P4 for 24 h. The expression of GLUT4 was measured at the mRNA level using quantitative real-time polymerase chain reaction (qRT-PCR) and at the protein level using Western blot analysis and immunohistochemistry. Results A cyclical change in GLUT4 expression pattern was observed in non-PCOS patients, and a high level of GLUT4 expression was seen in the proliferative phase compared to the secretory phase. Low levels of GLUT4 expression were found in PCOS patients compared to menstrual cycle phase-matched non-PCOS patients, and there was no significant change in GLUT4 expression in PCOS patients during the menstrual cycle. GLUT4 was localized in both epithelial and stromal cells, with notable changes in epithelial cells. We postulate that decreased GLUT4 expression might be regulated by steroid hormones. In support of this, we showed that in cultured endometrial tissues hCG and E2 alone had no effect on GLUT4 expression. However, P4 alone and P4 in combination with E2 decreased GLUT4 expression. Compared with non-PCOS controls, PCOS patients with endometrial hyperplasia exhibited decreased GLUT4 expression in particular in the epithelial cells. Conclusion We conclude that P4 can induce changes in endometrial GLUT4 expression during the menstrual cycle and that abnormal hormonal conditions such as PCOS disrupt normal patterns of GLUT4 expression in endometrial cells.
Project description:Conflicting results have been reported regarding whether or not insulin-regulated glucose transporter 4 (GLUT4) is expressed in human and rodent endometria. There is an inverse relationship between androgen levels and insulin-dependent glucose metabolism in women. Hyperandrogenemia, hyperinsulinemia, and insulin resistance are believed to contribute to endometrial abnormalities in women with polycystic ovary syndrome (PCOS). However, it has been unclear in previous studies if endometrial GLUT4 expression is regulated by androgen-dependent androgen receptors (ARs) and/or the insulin receptor/Akt/mTOR signaling network. In this study, we demonstrate that GLUT4 is expressed in normal endometrial cells (mainly in the epithelial cells) and is down-regulated under conditions of hyperandrogenemia in tissues from PCOS patients and in a 5α-dihydrotestosterone-induced PCOS-like rat model. Western blot analysis revealed reduced endometrial GLUT4 expression and increased AR expression in PCOS patients. However, the reduced GLUT4 level was not always associated with an increase in AR in PCOS patients when comparing non-hyperplasia with hyperplasia. Using a human tissue culture system, we investigated the molecular basis by which GLUT4 regulation in endometrial hyperplasia tissues is affected by metformin in PCOS patients. We show that specific endogenous organic cation transporter isoforms are regulated by metformin, and this suggests a direct effect of metformin on endometrial hyperplasia. Moreover, we demonstrate that metformin induces GLUT4 expression and inhibits AR expression and blocks insulin receptor/PI3K/Akt/mTOR signaling in the same hyperplasia human tissues. These findings indicate that changes in endometrial GLUT4 expression in PCOS patients involve the androgen-dependent alteration of AR expression and changes in the insulin receptor/PI3K/Akt/mTOR signaling network.
Project description:The failure of reproductive success in polycystic ovary syndrome (PCOS) patients could be in part due to endometrial dysfunction. However, no studies have investigated any causality between androgen, androgen receptor (AR) expression, and adenosine monophosphate activated protein kinase (AMPK) activation in the endometrium under physiological and pathological conditions. In the present study, we show that 1) endometrial AR expression levels fluctuate in non-PCOS and PCOS patients during the menstrual cycle; 2) the menstrual phase-dependent alteration of p-AMPKα expression occurs in non-PCOS patients but not in PCOS patients; 3) AR expression is higher in PCOS patients than non-PCOS patients during hyperplasia while AMPKα activation (indicated by the ratio of p-AMPKα to AMPKα); and 4) co-localization of AR and Ki-67 in epithelial cell nuclei is observed in endometrial hyperplasia. Importantly, using in vitro human tissue culture and an in vivo 5α-dihydrotestosterone-treated rat model, we show that the action of androgen on AMPKα activation is likely mediated through nuclear AR, especially in epithelial cells. Collectively, we present evidence that AR expression and AMPKα activation depend on menstrual cycle phase and the presence of PCOS, and the data suggest that AR-mediated regulation of AMPKα activation might play a role in the development of endometrial hyperplasia.
Project description:OBJECTIVE:The aim of the study was to evaluate the effect of a single-capsule 17?-estradiol/progesterone (E2/P4), TX-001HR, on endometrial safety, to report on amenorrhea and bleeding patterns of users, and to identify predictors of amenorrhea. METHODS:The REPLENISH trial (NCT01942668) evaluated use of TX-001HR in menopausal women (40-65 y) with vasomotor symptoms (VMS) and a uterus. Women were randomized to daily E2/P4 (mg/mg: 1/100, 0.5/100, 0.5/50, or 0.25/50), or placebo for 12 months. Incidence rate of endometrial hyperplasia was calculated from endometrial biopsies conducted at screening and study completion. Women reported bleeding and spotting in daily diaries. The number of bleeding and/or spotting days and the proportion of women with no bleeding or amenorrhea were compared between treatment and placebo using the Fisher exact test. Predictors of cumulative amenorrhea were assessed by univariate analyses. RESULTS:Women (n?=?1,835) who took at least one study dose comprised the safety population; 1,255 had baseline and 12-month biopsies and comprised the endometrial safety population. Incidence of endometrial hyperplasia was ?0.36% with any dose of TX-001HR after 1 year of use (one-sided upper 95% confidence interval ?4%). Cumulative amenorrhea (no bleeding/spotting) rates increased over time and were relatively high from cycle 1 to 13 with TX-001HR (56%-73%; placebo 79%; P?<?0.05 except with 0.25/50 dose). Few vaginal bleeding adverse events (1.0%-4.6% TX-001HR vs 0.7% placebo) were reported and discontinuations due to bleeding were low (0.4%-1.4% vs 0%). Cumulative amenorrhea was significantly more frequent in older women, those further from their last menstrual period, and those with lower baseline E2 concentrations (all; P?<?0.01). CONCLUSIONS:All doses of TX-001HR provided endometrial protection and were associated with an improved bleeding profile over time; older age, further last menstrual period, or lower baseline E2 may predict amenorrhea with TX-001HR.
Project description:The underlying mechanisms of polycystic ovarian syndrome (PCOS)-induced endometrial dysfunction are not fully understood, and although accumulating evidence shows that the use of metformin has beneficial effects in PCOS patients, the precise regulatory mechanisms of metformin on endometrial function under PCOS conditions have only been partially explored. To address these clinical challenges, this study aimed to assess the protein expression patterns of glycolytic enzymes, estrogen receptor (ER), and androgen receptor (AR) along with differences in mitochondria-dependent apoptosis in PCOS patients with and without endometrial hyperplasia in vivo and to investigate the effects of metformin in PCOS patients with endometrial hyperplasia in vitro. Here, we showed that compared to non-PCOS patients and PCOS patients without hyperplasia, the endometria from PCOS patients with hyperplasia had a distinct protein expression pattern of glycolytic enzymes, including pyruvate kinase isozyme M2 isoform (PKM2) and pyruvate dehydrogenase (PDH), and mitochondrial transcription factor A (TFAM). In PCOS patients with endometrial hyperplasia, increased glandular epithelial cell secretion and infiltrated stromal cells in the glands were associated with decreased PDH immunoreactivity in the epithelial cells. Using endometrial tissues from PCOS patients with hyperplasia, we found that in response to metformin treatment in vitro, hexokinase 2 (HK2) expression was decreased, whereas phosphofructokinase (PFK), PKM2, and lactate dehydrogenase A (LDHA) expression was increased compared to controls. Although there was no change in PDH expression, metformin treatment increased the expression of TFAM and cleaved caspase-3. Moreover, our in vivo study showed that while endometrial ER? expression was no different between non-PCOS and PCOS patients regardless of whether or not hyperplasia was present, ER? and AR protein expression was gradually increased in women with PCOS following the onset of endometrial hyperplasia. Our in vitro study showed that treatment with metformin inhibited ER? expression without affecting ER? expression. Our findings suggest that decreased glycolysis and increased mitochondrial activity might contribute to the onset of ER?-dependent endometrial hyperplasia and that metformin might directly reverse impaired glycolysis and normalize mitochondrial function in PCOS patients with endometrial hyperplasia.
Project description:STUDY QUESTION:How does aquaporin-3 (AQP3) affect endometrial receptivity? SUMMARY ANSWER:AQP3, which is regulated by the combination and estrogen (E2) and progesterone (P4), induces epithelial-mesenchymal transition (EMT) of endometrial epithelial cells. WHAT IS KNOWN ALREADY:Embryo implantation is an extremely complex process, and endometrial receptivity is essential for successful embryo implantation. Estrogen and progesterone regulate endometrial receptivity. AQP3, which is regulated by estrogen (E2), increases cell migration and invasion ability by regulating the expression of EMT-related factors and influencing the reorganization of the actin cytoskeleton. STUDY DESIGN, SIZE, DURATION:This study investigated the pathophysiological significance of AQP3 in human endometrial function during different phases of the menstrual cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS:AQP3 expression levels during different phases of the menstrual cycle were measured using immunohistochemical assays. In cells of different receptivity (high-receptive RL95-2 cells and low-receptive HEC-1A cells), the expression of AQP3 was measured using western blotting, qRT-PCR and immunofluorescence assays. Activities of AQP3, and its regulation by E2 and P4, were studied through in-vitro experiments using RL95-2 cells. MAIN RESULTS AND THE ROLE OF CHANCE:AQP3 expression in the mid- and late-secretory phases of the human endometrium is significantly higher than in other phases. Since AQP3 expression levels were higher in RL95-2 cells than in HEC-1A cells, mechanisms of AQP3 regulation by E2 and P4 were studied using RL95-2 cells. We provided the first report that P4 up-regulates AQP3 by directly targeting the promoter of the AQP3 gene. The up-regulation of AQP3 expression by a combination of E2 and P4 is significantly higher than that caused by either E2 or P4 alone. Together E2 and P4 promote RL95-2 cell migration and invasion by inducing EMT through AQP3. We also found that AQP3 co-localizes with ezrin and affects the formation of filopodia and lamellipodia during the E2 and P4-induced EMT process but has no effect on the expression of ezrin and F-actin. LARGE SCALE DATA:N/A. LIMITATIONS, REASONS FOR CAUTION:It is still unclear whether AQP3 is a main regulator of endometrial receptivity or one of several factors influencing the process. WIDER IMPLICATIONS OF THE FINDINGS:Further investigation on AQP3 may contribute to a greater understanding of endometrial receptivity. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Natural Scientific Grants of China (No. 31570798), the Program for Liaoning Excellent Talents in University (LR2017042), the Doctoral Scientific Research Foundation of Liaoning province (201601236), and the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences. There are no conflicts of interest.
Project description:The optimal protocol for endometrial preparation in patients with infertility remains unclear. Due to this, the current study retrospectively analyzed 1,589 patients with infertility and regular menstrual cycles to assess reproductive outcomes per embryo transferred and per embryo transfer (ET) cycle following the transfer of frozen-thawed embryos (FET) in a modified natural cycle (mNC) or hormone therapy cycle (HT) with or without gonadotropin-releasing hormone agonist (GnRHa)-induced pituitary suppression. The molecular mechanisms involved were also studied using tissues from endometrial biopsies. Patients who underwent FET were assigned to 5 groups as follows: Group A underwent a mNC (n=276); group B (n=338) received estradiol (E2) and progesterone (P4); group C received 1 cycle of GnRHa, E2 and P4 (n=323); group D received 2 cycles of GnRHa, E2 and P4 (n=329); and group E received 3 cycles of GnRHa, E2 and P4 (n=323). Tissues from endometrial biopsies of 91 patients performed on the day of ET were tested for endometrial receptivity marker mRNA expression and microRNA (miR)-223-3p mRNA. Furthermore, endometrial stromal cells (ESCs) were used for an in-depth study of the molecular mechanisms involved. Among the 5 groups of patients, implantation rates, clinical pregnancy rates and live birth rates were not significantly different. However, endometrial receptivity was enhanced in group E when compared with groups A-D, which was associated with endometrial leukemia inhibitory factor (LIF), osteopontin, vascular endothelial growth factor, integrin ?3 and homeobox gene 10 and 11 mRNA upregulation, and miR-223-3p miRNA downregulation. Transfection of ESCs with an miR-223-3p mimic significantly reduced levels of LIF mRNA and protein. In addition, pre-treating ESCs with GnRHa upregulated mRNA and protein expression of the decidualization markers prolactin and insulin-like growth factor binding protein-1 in a time-dependent manner. In conclusion, these results indicated that HT with GnRHa may be a potential endometrial preparation protocol for FET.
Project description:Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal endometrial proliferation and, ultimately, endometrial adenocarcinoma, the fourth most common cancer in women. We have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of progesterone receptor (PR) and steroid receptor coactivator (SRC-1) action in the uterus. Here, we demonstrate that absence of Mig-6 in mice results in the inability of P4 to inhibit E2-induced uterine weight gain and E2-responsive target genes expression. At 5 months of age, the absence of Mig-6 results in endometrial hyperplasia. Ovariectomized Mig-6(d/d) mice exhibit this hyperplastic phenotype in the presence of E2 and P4 but not without ovarian hormone. Ovariectomized Mig-6(d/d) mice treated with E2 developed invasive endometrioid-type endometrial adenocarcinoma. Importantly, the observation that endometrial carcinomas from women have a significant reduction in MIG-6 expression provides compelling support for an important growth regulatory role for Mig-6 in the uterus of both humans and mice. This demonstrates the Mig-6 is a critical regulator of the response of the endometrium to E2 in regulating tissue homeostasis. Since Mig-6 is regulated by both PR and SRC-1, this identifies a PR, SRC-1, Mig-6 regulatory pathway that is critical in the suppression of endometrial cancer.
Project description:Approximately 70% of women with polycystic ovary syndrome (PCOS) have intrinsic insulin resistance (IR) above and beyond that associated with body mass, including dysfunctional glucose metabolism in adipose tissue (AT). In AT, analysis of the IRS/PI3-K/AKT pathway signaling components identified only GLUT4 expression to be significantly lower in PCOS patients and in control subjects with IR. We examined the role of miRNAs, particularly in the regulation of GLUT4, the insulin-sensitive glucose transporter, in the AT of PCOS and matched control subjects. PCOS AT was determined to have a differentially expressed miRNA profile, including upregulated miR-93, -133, and -223. GLUT4 is a highly predicted target for miR-93, while miR-133 and miR-223 have been demonstrated to regulate GLUT4 expression in cardiomyocytes. Expression of miR-93 revealed a strong correlation between the homeostasis model assessment of IR in vivo values and GLUT4 and miR-93 but not miR-133 and -223 expression in human AT. Overexpression of miR-93 resulted in downregulation of GLUT4 gene expression in adipocytes through direct targeting of the GLUT4 3'UTR, while inhibition of miR-93 activity led to increased GLUT4 expression. These results point to a novel mechanism for regulating insulin-stimulated glucose uptake via miR-93 and demonstrate upregulated miR-93 expression in all PCOS, and in non-PCOS women with IR, possibly accounting for the IR of the syndrome. In contrast, miR-133 and miR-223 may have a different, although yet to be defined, role in the IR of PCOS.
Project description:To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria.The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay.CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS.The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.
Project description:The ovarian steroid hormones, estrogen (E2) and progesterone (P4), are essential regulators of uterine biology. The imbalance of these ovarian steroid hormones leads to uterine diseases such as endometrial cancer, endometriosis, and infertility. Mitogen-inducible gene 6 (MIG-6) is an adaptor protein. MIG-6 mediates P4 signaling and acts as a tumor suppressor during endometrial tumorigenesis in both humans and mice. In previous studies, we developed the conditional knockout of Mig-6 in all uterine compartments (Pgrcre/+Mig-6f/f; Mig-6KO) and endometrial epithelial cell-specific Mig-6 knockout (Sprr2fcre/+Mig-6f/f; Mig-6Ep-KO) mice. Both mouse models developed endometrial hyperplasia and E2-dependent endometrial cancer. P4 treatment significantly decreases aberrant epithelial proliferation and AKT signaling in Mig-6Ep-KO mice but not in Mig-6KO mice. In the present study, we identified a calcium-dependent phospholipase A2 (cPla2) as one of the genes down-regulated by Mig-6 in the uterus. We performed immunohistochemistry and Western Blot analysis to investigate the regulation of cPLA2 by MIG-6 as well as determine the expression patterns of cPLA2 in the uterus. While the expression of cPLA2 was stronger at the uterine epithelial cells of Mig-6KO and Mig-6Ep-KO mice compared to control mice, P4 suppressed the expression of cPLA2 in Mig-6Ep-KO mice but not in Mig-6KO mice. To determine the ovarian steroid hormone regulation of cPLA2, we examined the expression of cPLA2 in ovariectomized control, Mig-6KO, Mig-6Ep-KO, and PRKO mice treated with P4 or E2. After P4 treatment, cPLA2 expression was remarkably reduced in Mig-6Ep-KO mice but not in Mig-6KO mice. However, the expression of cPLA2 was not changed in PRKO mice. Our results identified cPLA2 as a novel target of MIG-6 in the murine uterus and identified its important role during endometrial tumorigenesis.