Transcriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes in the endometrium of patients with polycystic ovary syndrome.
ABSTRACT: CONTEXT: Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by chronic oligo/anovulation, hyperandrogenemia, infertility, and metabolic alterations related to insulin resistance. These abnormalities in PCOS may have complex effects on pathophysiology of the endometrium, contributing to infertility and endometrial disorders. OBJECTIVE: The objective of this study was to examine dysregulated signaling pathways in the endometrium of patients with PCOS (PCOSE) by analyzing expression profiles with a pathway-oriented method. DESIGN: Microarrays, RT-PCR, laser capture microdissection, and immunohistochemistry were performed with endometrial tissues. SETTING: This study was performed at a university hospital laboratory. PATIENTS: This study comprised 12 regularly cycling women and 12 PCOS patients. MAIN OUTCOME MEASURE: Dysregulated signaling pathways in PCOSE were identified as a gene set. RESULTS: Hierarchical clustering revealed distinct expression profiles for PCOSE and the endometrium of normal cycling women. Gene sets associated with androgen signaling were not enriched in PCOSE, although they affect ovarian physiology of PCOS patients. Several biological pathways including cell cycle, apoptosis, glycolysis, and integrin-Rho-cytoskeleton network were aberrantly down-regulated in PCOSE. Expression of genes constituting these gene sets enriched in normal cycling women was systemically down-regulated in PCOSE. Laser capture microdissection-coupled real-time RT-PCR and immunohistochemistry further demonstrated that cell proliferation in the stroma, but not the epithelium, is significantly reduced in PCOSE. CONCLUSIONS: Systemic down-regulation of various signaling pathways in PCOSE with extremely prolonged proliferative phase provides insight into the abnormal phenotypes that reflect pathophysiology of PCOS in the endometrium, possibly leading to increased risks of endometrial disorders.
Project description:Background:Infertility is a critical condition in women with polycystic ovary syndrome (PCOS), caused not only by anovulation but also by endometrial abnormality. Objective:This study aimed to evaluate and compare the hysteroscopic and histological findings of endometrial biopsies in infertile women with PCOS and normal endometrial thickness and women with unexplained infertility (UI). Materials and Methods:This cross-sectional study compared the initial hysteroscopy and endometrial histological findings of 70 infertile women with PCOS and normal endometrial thickness with those of 35 women with UI. The relationship between endometrial histology and clinical parameters such as including luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, testosterone, prolactin, fasting blood sugar, body mass index (BMI), and infertility duration was analyzed. Results:The mean age of women with PCOS was significantly lower than that of women with UI (27.5 ± 4.1 vs. 30 ± 4.5 years, respectively) (p < 0.001). The mean BMI was higher in women with PCOS than in women with UI (28.7 ± 4.4 vs. 25.1 ± 3 kg/m 2 ) (p < 0.001). The hysteroscopic findings of all women with PCOS were normal, whereas 91.4% of women with UI had normal hysteroscopic findings, 2.9% had a polyp, and 5.7% had endometrial thickening. The histological findings of women with PCOS revealed proliferative endometrium in 54.3%, disordered proliferative endometrium in 17.1%, secretory endometrium in 8.6%, and endometrial polyp in 17.1%, whereas these percentages in women with UI were 28.6%, 0%, 54.3%, and 20%, respectively. Conclusion:The hysteroscopic evaluation alone of infertile women might not detect all probable endometrial pathologies in women with PCOS.
Project description:Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, with a prevalence of up to 12% of women of reproductive age, and is in part characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium. A high percentage of PCOS patients suffer from infertility, a condition that appears to be linked to mistimed and incomplete decidualisation critically affecting events surrounding embryo implantation. The aim of this study was to examine the involvement of MAGEA11, and the genome-wide role of AR in PCOS. We determined that elevated androgen levels on PCOS cells had an impact on the delayed and incomplete decidual transformation of endometrial cells. The AR co-regulator MAGEA11, a known enhancer of AR function, was constitutively overexpressed throughout the menstrual cycle of PCOS patients, co-localised in the nucleus of PCOS stromal tissue and cells and formed a molecular complex with AR. Genome-wide AR analysis in PCOS stromal cells revealed that AR targets included genes involved in cell death and apoptosis, as well as genes commonly dysregulated in endometrial cancer. Enhanced MAGEA11 and AR-mediated transcriptional regulation may impact on a correct endometrial decidualisation response, subsequently affecting endometrial receptivity in these infertile women. KEY MESSAGES: MAGEA11 and AR are overexpressed in hyperandrogenic PCOS patients. MAGEA11-AR overexpression in PCOS correlates with delayed decidualisation. AR and MAGEA11 associate in a molecular complex. AR directly regulates a unique set of genes controlling gene differentiation.
Project description:Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium.
Project description:CONTEXT:Polycystic ovary syndrome (PCOS) is a prevalent disorder in reproductive aged women associated with a number of endocrine and metabolic complications, including increased risk of endometrial cancer. OBJECTIVE:To study the effect of the characteristic increased androgen levels in PCOS on the endometrium, a novel scaffold-free multicellular endometrial organoid was established. DESIGN:Human endometrial organoids were constructed using primary endometrial epithelial and stromal cells from endometrial tissues. Organoids were treated for 14 days with physiologic levels of estradiol and testosterone to mimic a normal follicular phase or PCOS hormone profiles. Organoids were harvested for immunostaining and ribonucleic acid sequencing. SETTING:Academic institution. PATIENTS:Endometrial tissues from 10 premenopausal women undergoing hysterectomy for benign pathologies were obtained following written consent. MAIN OUTCOME MEASURES:Organoid architecture, cell specific markers, functional markers, proliferation, and gene expression were measured. RESULTS:A method to generate scaffold-free endometrial organoids containing epithelial and stromal cells was established. These organoids exhibited distinct organization with epithelial cells lining the outer surface and stromal cells in the center of the organoids. Epithelial cells were polarized, organoids expressed cell type specific and functional markers, as well as androgen, estrogen, and progesterone receptors. Treatment with PCOS hormones increased cell proliferation and dysregulated genes in endometrial organoids. CONCLUSIONS:A new multicellular, scaffold-free endometrial organoid system was established that resembled physiology of the native endometrium. Excess androgens in PCOS promoted cell proliferation in endometrial organoids, revealing new mechanisms of PCOS-associated with risk of endometrial neoplasia.
Project description:BACKGROUND:The endometrial luminal epithelium is the first point of attachment of embryos during implantation. Failure of embryos to firmly adhere results in implantation failure and infertility. A receptive endometrial luminal epithelium is achieved through the expression of adhesion molecules in the mid-secretory phase and is a requirement for implantation. Cadherin 6 (CDH6) is an adhesion molecule localizing to the endometrial luminal epithelial cell surface in the mid-secretory/receptive phase and knockdown of CDH6 in the Ishikawa cells (receptive endometrial epithelial cell line) compromises cell integrity. However, there are no studies investigating the role of CDH6 on receptivity and infertility. This study aimed to investigate whether CDH6 is dysregulated in the endometrium of women with infertility during the receptive window and the effect of CDH6 on endometrial adhesion and receptivity. METHODS:The expression and the localization of CDH6 in the human endometrium were determined by immunohistochemistry. Ishikawa cells were used to investigate the functional consequences of CDH6 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids in vitro. CDH6 knockdown was assessed by qPCR and immunoblotting. After CDH6 knockdown, the expression of type II cadherin family members and CDH6 functional partners were assessed by qPCR. Two-tailed unpaired student's t-test or one-way ANOVA as appropriate were used for statistical analysis with a significance threshold of P?<?0.05. RESULTS:A significant reduction of CDH6 immunolocalization was recorded in the luminal and glandular epithelium of endometrium from women with infertility (P?<?0.05) compared to fertile group respective cellular compartments in the mid-secretory phase. Functional analysis using Ishikawa cells demonstrated that knockdown of CDH6 (treated with 50?nM CDH6 siRNA) significantly reduced epithelial adhesive capacity (P?<?0.05) to HTR8/SVneo spheroids compared to control and other type II cadherin family members likely failed to compensate for the loss of CDH6. The expression levels of CDH6 functional partners, catenin family members were not changed after CDH6 knockdown in Ishikawa cells. CONCLUSION:Together, our data revealed that CDH6 was dysregulated in the endometrium from women with infertility and altered Ishikawa cell adhesive capacity. Our study supports a role for CDH6 in regulating endometrial adhesion and implantation.
Project description:Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium: The objective of this study is to evaluate the targets of AR in PCOS patients. Overexpression of AR and altered endometrial signalling pathways in PCOS patients may impact on a correct decidualization response, critically affecting the events surrounding embryo implantation and causing infertility. Overall design: Endometrial stromal cells isolated from 3 PCOS patients were pooled together, grown to confluency and treated with DHT for 72h. One chromatin sample (containing genetic background from 3 different patients) was immunoprecipitated with an anti-AR antibody and subsequently submitted to ChIP-Seq
Project description:Polycystic ovary syndrome (PCOS) is a complex multigenic disorder and women with PCOS suffer from several comorbidities. Although, obesity is a known risk factor for PCOS, the incidence of lean women with PCOS is on the rise. A systematic and comparative study on lean and obese PCOS with respect to genes, pathways and comorbidity analysis has not been attempted so far. Analysis of differentially expressed genes (DEGs) across tissue types for lean and obese PCOS revealed that the majority of them were downregulated for lean and obese PCOS. Ovarian and endometrial tissues shared several commonly dysregulated genes, suggesting shared PCOS pathophysiology mechanisms exist across tissues. Several pathways for cellular homeostasis, such as inflammation and immune response, insulin signaling, steroidogenesis, hormonal and metabolic signaling, regulation of gonadotrophic hormone secretion, cell structure and signaling that are known to be affected in PCOS were found to be enriched in our gene expression analysis of lean and obese PCOS. The gene-disease network is denser for obese PCOS with a higher comorbidity score as compared to lean PCOS.
Project description:Small leucine-rich proteoglycans (SLRPs) play an important role in tissue homeostasis and cell proliferation since these proteoglycans sequester multiple growth factors. However, the content of SLRPs in the endometrium of polycystic ovary syndrome (PCOS) women is unknown. Our purpose was to test the hypothesis that excessive endometrial proliferation in PCOS may be partly related to abnormalities in SLRPs.In a cross section study a total of 20 endometrial samples were collected from 10 patients with PCOS and 10 ovulatory women during their proliferative (pre-ovulatory) phase. The study subjects were matched for age, body mass index and race. The age range was 20 to 35 years. All volunteers were evaluated in reproductive endocrinology clinic, Gynecology Division, Clinics Hospital, University of São Paulo Medical School Profile and concentration of small leucine-rich proteoglycans (decorin, lumican, fibromodulin and biglycan) were determined by immunohistochemical testing and Western blotting.Decorin and lumican demonstrated higher immunoreactivity and relative expression in the endometrium of women with PCOS compared to that of women with regular menstrual cycles.Our data suggests that the endometrium of PCOS women demonstrate a greater content of SLRP than controls; decorin and lumican, in particular, were found in higher concentrations in the endometrium of PCOS women during the proliferative phase. These differences may, in part, explain the excess of endometrial proliferation frequently observed in PCOS. Further studies are warranted.
Project description:Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer.We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial "disease phenotype" affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny.This was a prospective study conducted at an academic medical center.Proliferative-phase endometrium was obtained from 6 overweight/obese PCOS (National Institutes of Health criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting-isolated endometrial epithelial cells (eEPs), endothelial cells, stromal fibroblasts (eSFs), and mesenchymal stem cells (eMSCs). Gene expression data were validated using microfluidic quantitative RT-PCR and immunohistochemistry.The comparison between eEP(PCOS) and eEP(Ctrl) showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSF(PCOS) and eSF(Ctrl) showed up-regulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSC(PCOS) vs eMSC(Ctrl), the most up-regulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). Immunohistochemistry scoring showed increased expression of CCL2 in eEP(PCOS) and eSF(PCOS) compared with eEP(Ctrl) and eSF(Ctrl) and IL-6 in eEP(PCOS) compared with eEP(Ctrl).Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and prooncogenic changes, independent of body mass index, especially in eEP(PCOS) and eMSC(PCOS), compared with controls. The study reveals an endometrial disease phenotype in women with PCOS with potential negative effects on endometrial function and long-term health.
Project description:Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC).To investigate whether the endometrium of women with PCOS possesses gene expression changes similar to those found in EC.Patients with EC, PCOS and control women unaffected by either PCOS or EC were recruited into a cross-sectional study at the Nottingham University Hospital, UK. For RNA sequencing, representative individual endometrial biopsies were obtained from women with EC, PCOS and a woman unaffected by PCOS or EC. Expression of a subset of differentially expressed genes identified by RNA sequencing, including NAD(P)H quinone dehydrogenase 1 (NQO1), was validated by quantitative reverse transcriptase PCR validation (n = 76) and in the cancer genome atlas UCEC (uterine corpus endometrioid carcinoma) RNA sequencing data set (n = 381). The expression of NQO1 was validated by immunohistochemistry in EC samples from a separate cohort (n = 91) comprised of consecutive patients who underwent hysterectomy at St Mary's Hospital, Manchester, between 2011 and 2013. A further 6 postmenopausal women with histologically normal endometrium who underwent hysterectomy for genital prolapse were also included. Informed consent and local ethics approval were obtained for the study.We show for the first that NQO1 expression is significantly increased in the endometrium of women with PCOS and EC. Immunohistochemistry confirms significantly increased NQO1 protein expression in EC relative to nonmalignant endometrial tissue (P < .0001).The results obtained here support a previously unrecognized molecular link between PCOS and EC involving NQO1.