Progesterone-mediated gene expression in the human endometrial stromal compartment during the implantation window
ABSTRACT: Progesterone regulated genes in the endometrial stromal compartment were studied in proven fertile women using laser dissection capture microscopy followed by microarray. Endometrial biopsies were obtained from women before (control group, n=9) and after (study group, n=9) treatment with mifepristone. Stromal cells were isolated by Laser-capture microdissection and RNA was extracted. The gene expression was analyzed by microarray and reconfirmed by real-time PCR.
Project description:Progesterone regulated genes in the endometrial stromal compartment were studied in proven fertile women using laser dissection capture microscopy followed by microarray. Endometrial biopsies were obtained from women before (control group, n=9) and after (study group, n=9) treatment with mifepristone. Stromal cells were isolated by Laser-capture microdissection and RNA was extracted. The gene expression was analyzed by microarray and reconfirmed by real-time PCR.
Project description:The control of complement activation in the embryo-maternal environment has been demonstrated to be critical for embryo survival. Complement proteins are expressed in the human endometrium; however, the modulation of this expression by embryo signals has not been explored. To assess the expression of complement proteins in response to human chorionic gonadotropin (hCG), we designed an experimental study using in vivo and in vitro models. Twelve fertile women were treated with hCG or left untreated during the mid-luteal phase, and an endometrial biopsy was performed 24 hours later. The localizations of C3, membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59) were assessed by immunohistochemistry, and the messenger RNA (mRNA) levels of these proteins were quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in cells harvested from endometrial compartments using laser capture microdissection. Endometrial explants were cultured with or without hCG for 24 hours, and the C3 and DAF protein levels were measured by Western blotting. Elevated C3 mRNA levels in stromal cells and elevated DAF levels in epithelial luminal cells were detected after hCG treatment. In the endometrial explant model, the progesterone receptor antagonist RU486 inhibited the increases in the levels of C3 and DAF in response to hCG. The findings of this study indicate that hCG plays a role in embryo-endometrium communication and affects the expression of complement proteins in endometrial compartments during the implantation window.
Project description:Endometrial polyps are common, yet the molecular mechanisms underlying their formation and progression remain unclear. We examined gene mutations possibly related to the pathogenesis of endometrial polyps, as well as to their clinical features. Four premenopausal patients with endometrial polyps, who were not under drug treatment, were recruited. Whole exomes of endometrial polyps and peripheral blood lymphocytes were analyzed by next?generation sequencing, and somatic mutations were derived by subtraction. Then, 35 samples of endometrial polyps and 12 samples of atypical polypoid adenomyoma were newly recruited to validate the identified mutations by polymerase chain reaction?reverse sequence specific oligonucleotide method. The mutations were also analyzed in separate stromal and glandular components of the polyps after laser?capture microdissection. Whole exome sequencing revealed that KRAS mutations were the only type of mutation detectable in multiple cases (2/4). Targeted mutation analysis revealed that 16 of 35 samples (45.7%) of endometrial polyps harbored RAS mutations. Mutation?positive cases exhibited a significantly higher number of endometrial polyps (3.25±2.70 vs. 1.74±0.87, P=0.045). Laser?capture microdissection in NRAS?mutated endometrial polyps revealed that both stromal and glandular components harbored RAS mutations. There was no RAS mutation in 12 samples of atypical polypoid adenomyoma. This is the first report demonstrating that pathogenic RAS mutations are frequent in non?treated endometrial polyps. RAS mutations may have an important role in tumorigenesis and in the formation of multiple endometrial polyps.
Project description:The human endometrial stromal cells (hEndoSCs) could maintain endometrial homeostasis and play a critical role in repairing endometrial injury. Mesenchymal stem cells (MSCs) significantly increase the proliferation of damaged hEndoSCs and protect them from apoptosis. Recent studies indicated that exosomes derived from stem cells could be recruited to damaged tissues for regeneration, which exhibit the potential for stem cell therapy as therapeutic vectors. In this study, we isolated human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) and investigated the effects of hUCMSC-Exos on mifepristone-induced hEndoSC injury. Exosome uptake and cell proliferation as well as cell apoptosis of damaged hEndoSCs treated with hUCMSC-Exos were detected. We also assessed the expression of apoptosis-related proteins and the PTEN/AKT signaling pathway. We found hUCMSC-Exos improved the proliferation of damaged hEndoSCs and protected hEndoSCs from the mifepristone-induced apoptosis. hUCMSC-Exos upregulated Bcl-2 level as well as downregulated Cleaved Caspase-3 level and activated the PTEN/AKT signaling pathway to regulate the proliferation and antiapoptosis. These results indicated hUCMSC-Exos protected hEndoSCs from mifepristone-induced apoptosis and played an active role in repairing the damaged hEndoSCs through the PTEN/AKT signaling pathway in vitro. hUCMSC-Exos may hold great promise in the cell-free therapy of endometrial injury.
Project description:The tumor environment is of vital importance for the incidence and development of colorectal cancer. Increasing evidence in recent years has elaborated the vital role of the tumor environment in cancer subtype classification and patient prognosis, but a comprehensive understanding of the colorectal tumor environment that is purely dependent on the stromal compartment is lacking. To decipher the tumor environment in colorectal cancer and explore the role of its immune context in cancer classification, we performed a gene expression microarray on the stromal compartment of colorectal cancer and adjacent normal tissues. Through the integrated analysis of our data with public gene expression microarray data of stromal and epithelial colorectal cancer tissues processed through laser capture microdissection, we identified four highly connected gene modules representing the biological features of four tissue compartments by applying a weighted gene coexpression network analysis algorithm and classified colorectal cancers into three immune subtypes by adopting a nearest template prediction algorithm. A systematic analysis of the four identified modules mainly reflected the close interplay between the biological changes of intrinsic and extrinsic characteristics at the initiation of colorectal cancer. Colorectal cancers were stratified into three immune subtypes based on gene templates identified from representative gene modules of the stromal compartment: active immune, active stroma, and mixed type. These immune subtypes differed by the immune cell infiltration pattern, expression of immune checkpoint inhibitors, mutation landscape, extent of mutation burden, extent of copy number burden, prognosis and chemotherapeutic sensitivity. Further analysis indicated that activation of the NF-kB signaling pathway was the major mechanism causing the no immune infiltration milieu in the active stroma subtype and that inhibitors of the NF-kB signaling pathway could be candidate drugs for treating patients with an active stroma. Overall, these results suggest that characterizing colorectal cancer by the tumor environment is of vital importance in predicting patients' clinical outcomes and helping guide precision and personalized treatment.
Project description:Endometriosis is a common gynecological disease affecting one in 10 women of reproductive age and is a major cause of pelvic pain and impaired fertility. Endometrial stromal cells of women with endometriosis exhibit a reduced response to in vitro decidualization. NOTCH1 is critical for decidualization of both mouse and human uterine stromal cells.This study aimed to determine whether decidualization failure in women with endometriosis is a consequence of impaired Notch signaling.We investigated expression levels of Notch signaling components in the endometrium of women and baboons with or without endometriosis. We identified NOTCH1-regulated genes during decidualization of human uterine fibroblast (HuF) cells by microarray and quantified their expression levels in in vitro-decidualized endometrial stromal cells isolated from women with or without endometriosis.Notch signaling receptors NOTCH1 and NOTCH4, ligands JAGGED2 and DLL4, as well as direct target genes HES5 and HEY1 were decreased in the eutopic endometrium of women and baboons with endometriosis. Notch signaling was decreased in stromal cells isolated from women with endometriosis, which was associated with impaired in vitro decidualization. Genes that were down-regulated by NOTCH1 silencing in decidualized HuF cells were also decreased in decidualized endometrial stromal cells of women with endometriosis. FOXO1 acts as a downstream target of Notch signaling and endometriosis is associated with decreased expression of NOTCH1-regulated, FOXO1-responsive genes during decidualization.Decreased Notch signaling is associated with endometriosis and contributes to impaired decidualization through the down-regulation of FOXO1.
Project description:The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/?-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3'-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.
Project description:Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription. The resultant PR and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.
Project description: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: Several studies have indicated that human pre-implantation embryo-derived chorionic gonadotropin (hCG) may influence the implantation process by its action on human endometrial epithelial and stromal cells. Despite reports indicating that hCG acts on these cells to affect the production of several cytokines and growth factors (e.g., MIF, IGF-I, VEGF, LIF, IL-11, GMCSF, CXL10 and FGF2), our understanding of the integral influence of hCG on paracrine interactions between endometrial stromal and epithelial cells during implantation is very limited. METHODS: In the present study, we examined the profile of 48 cytokines in the conditioned media of primary cell cultures of human implantation stage endometrium. Endometrial epithelial cells (group 1; n?=?20), stromal cells (group 2; n?=?20), and epithelial plus stromal cells (group 3; n?=?20) obtained from mid-secretory stage endometrial samples (n?=?60) were grown on collagen and exposed to different doses (0, 1, 10 and 100 IU/ml) of rhCG for 24 h in vitro. Immunochemical and qRT-PCR methods were used to determine cytokine profiles. Enrichment and process networks analyses were implemented using a list of cytokines showing differential secretion in response to hCG. RESULTS: Under basal conditions, endometrial epithelial and stromal cells exhibited cell type-specific profiles of secreted cytokines. Administration of hCG (100 IU) resulted in significantly (P?<?0.05) different cytokine secretion profiles indicative of macropinocytic transport (HGF, MCSF) in epithelial cells, signal transduction (CCL4, FGF2, IL-1b, IL-6, IL-17, VEGF) in stromal cells, and epithelial-mesenchymal transition (FGF2, HGF, IL-1b, TNF) in mixed cells. Overall, the administration of hCG affected cytokines involved in the immune response, chemotaxis, inflammatory changes, proliferation, cell adhesion and apoptosis. CONCLUSIONS: CG can influence the function of the endometrium during blastocyst implantation via its differential action on endometrial epithelial and stromal cells. CG may also affect complex paracrine processes in the different endometrial cell types.