High stretch cycling inhibits the morphological and biological decidual process in human endometrial stromal cells.

PURPOSE: Subendometrial myometrium exerts wave-like activity throughout the menstrual cycle, and uterine peristalsis is markedly reduced during the implantation phase. We hypothesized that abnormal uterine peristalsis has an adverse effect on the endometrial decidualization process. We conducted an in vitro culture experiment to investigate the effect of cyclic stretch on the morphological and biological endometrial decidual process. METHODS: Primary human endometrial stromal cells (HESCs) were isolated from hysterectomy specimens and incubated with or without 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) and medroxyprogesterone acetate (MPA) for 3 days. After decidualization, cultures were continued for 24 hours with or without cyclic stretch using a computer-operated cell tension system. RESULTS: Cyclic stretch significantly repressed expression of decidual markers including insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), forkhead box O1 (FOXO1), and WNT4 on decidualized HESCs. In addition, cyclic stretch of decidualized HESCs affected the decidual morphological phenotype to an elongated shape. The alternation of F-actin localization in decidualized HESCs was not observed in response to cyclic stretch. CONCLUSIONS: These data suggest that cyclic stretch inhibits the morphological and biological decidual process of HESCs. Our findings imply that uterine abnormal contractions during the implantation period impair endometrial decidualization and contribute to infertility.

Endometrial microRNAs and their aberrant expression patterns.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They play fundamental roles in several biological processes, including cell differentiation and proliferation, embryo development, organ development, and organ metabolism. Besides regulating the physiological processes, miRNAs regulate various pathological conditions such as tumors, metastases, metabolic diseases, and osteoporosis. Although several studies have been performed on miRNAs, only few studies have described the miRNA expression and functions in human reproductive tract tissues. During menstruation, the human endometrium undergoes extensive cyclic morphological and biochemical modifications before embryo implantation. In addition to the ovarian steroid hormones (estrogen and progesterone), endometrial autocrine or paracrine factors and embryo-derived signals play a significant role in endometrial functions. miRNAs are considered key regulators of gene expression in the human endometrium and implantation process, and their aberrant expression levels are associated with the development of various disorders, including tumorigenesis. In this review, we summarize the studies that show the role of miRNAs in regulating the physiological conditions of the endometrium and the implantation process and discuss the aberrant expression of miRNAs in ectopic pregnancy, endometriosis, and endometrial cancer.

Heparin prevents oxidative stress-induced apoptosis in human decidualized endometrial stromal cells.

Clinical trials have shown that administering heparin during the luteal phase has beneficial effects on implantation and live birth rates. Heparin exerts direct effects on decidual human endometrial stromal cells (HESCs), which are independent of its anticoagulant effect. However, the accurate effects of heparin on the decidualization process remain unidentified. Here, we demonstrate that HESCs become dramatically resistant to oxidative stress upon decidualization, and we hypothesize a possible direct action of heparin on the decidualization of HESCs, which would lead to improved implantation. To test this hypothesis, we established primary HESC cultures and propagated them, and then we decidualized confluent cultures with 8-bromo-cAMP, with medroxyprogesterone acetate, and with or without heparin. We treated the cells with hydrogen peroxide (H2O2) as a source of reactive oxygen species (ROS). Adding heparin to decidualized HESCs induced prolactin secretion. Decidualized HESCs treated with heparin were prevented from undergoing apoptosis induced by oxidative stress. Heparin induced nuclear accumulation of the forkhead transcription factor FOXO1 and expression of its downstream target, the ROS scavenger superoxide dismutase 2. These results demonstrate that heparin-treated decidualized HESCs acquired further resistance to oxidative stress, suggesting that heparin may improve the implantation environment.

Loss of miR-542-3p enhances IGFBP-1 expression in decidualizing human endometrial stromal cells.

Endometrial decidualization represents an essential step for the successful implantation of the embryo; however, the molecular mechanism behind this differentiation process remains unclear. This study aimed to identify novel microRNAs (miRNAs) involved in the regulation of decidual gene expression in human endometrial stromal cells (HESCs). An in vitro analysis of primary undifferentiated and decidualizing HESCs was conducted. HESCs were isolated from hysterectomy specimens from normally cycling premenopausal women with uterine fibroids, who were not on hormonal treatment at the time of surgery. Primary HESCs were expanded in culture and decidualized with 8-bromo-cyclic adenosine monophosphate and medroxyprogesterone acetate. Microarray analysis identified six miRNAs differentially expressed in response to decidualization of HESCs. All but one miRNA were downregulated upon decidualization, including miR-542-3p. We demonstrated that miR-542-3p overexpression inhibits the induction of major decidual marker genes, including IGFBP1, WNT4 and PRL. In addition, miR-542-3p overexpression inhibited the morphological transformation of HESCs in response to deciduogenic cues. A luciferase reporter assay confirmed that the 3'-untranslated region of IGFBP1 mRNA is targeted by miR-542-3p. The results suggest that miR-542-3p plays an important role in endometrial decidualization by regulating the expression of major decidual marker genes.

MicroRNA-135b suppresses extravillous trophoblast-derived HTR-8/SVneo cell invasion by directly down regulating CXCL12 under low oxygen conditions.

The expression of numerous microRNAs (miRNAs) in the trophoblasts changes under low oxygen conditions. However, little is known regarding the regulation of the trophoblast invasion by miRNAs under low oxygen conditions. The aim of this study was to identify those miRNAs and their target genes associated with the trophoblast invasion under low oxygen conditions. Culturing the extravillous trophoblast (EVT) cell line, HTR-8/SVneo, at 2% oxygen as compared to 20% oxygen suppressed trophoblast invasion that correlated with increased expression of microRNA-135b (miR-135b) and decreased expression of the its predicted target gene CXCL12. Overexpression of miR-135b suppressed CXCL12 mRNA expression and invasion of HTR-8/SVneo cells. Adding a neutralizing antibody against CXCL12 to the culture medium suppressed HTR-8/SVneo cell invasion. Reporter assays showed that the 3'UTR sequence of CXCL12 was directly targeted by miR-135b. Our results suggest that miR-135b and CXCL12 play important roles in modulating the EVT invasion under low oxygen conditions.

Androgens modulate the morphological characteristics of human endometrial stromal cells decidualized in vitro.

The activated androgen receptor (AR) in decidualizing human endometrial stromal cells (HESCs) regulates genes involved in cytoskeletal organization, cell motility, and cell cycle progression. Androgens also enhance the secretion of prolactin, a widely used marker of decidualized HESCs. The purpose of the present study was to investigate the direct effects of androgens on the ultrastructural changes associated with decidual transformation of HESCs. Primary HESC cultures were established and propagated, and confluent cultures were decidualized for 6 days with 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) and progesterone (P4) in the presence or absence of dihydrotestosterone (DHT). Phase-contrast image analysis demonstrated that DHT increases the shape index of decidualizing cells, which was reversed upon cotreatment with the AR antagonist flutamide. Electron microscopy demonstrated that DHT enhances many of the ultrastructural changes induced by 8-br-cAMP and P4 in HESCs. Decidualizing cells are characterized by an abundant cytoplasm, multiple cell surface projections and, unlike undifferentiated HESCs, form 2 or more cell layers. The DHT further stimulated cytoplasmic expansion, lipid droplet formation, the production of an abundant extracellular matrix, and gap junction formation in decidualized HESCs. The present study demonstrates that androgen signaling has an impact on the morphological and ultrastructural changes associated with the decidual process. Our findings show that androgens promote the development and expansion of cytoplasmic organelles and gap junctions in decidualizing HESCs. These results suggest that androgens in early pregnancy play an important role in promoting the cellular transformation associated with decidualization.

Androgen signaling in decidualizing human endometrial stromal cells enhances resistance to oxidative stress.

OBJECTIVE: To investigate the effect of androgens on the expression of genes involved in oxidative stress resistance in decidualized human endometrial stromal cells (HESCs). DESIGN: In vitro experiment. SETTING: University hospital. PATIENT(S): Premenopausal women undergoing hysterectomy for uterine fibroids. INTERVENTION(S): Human endometrial stromal cells isolated from hysterectomy specimens were decidualized with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) and P in the presence or absence of dihydrotestosterone (DHT) at various concentrations. Hydrogen peroxide was used as a source of reactive oxygen species. MAIN OUTCOME MEASURE(S): Prolactin secretion, apoptosis, FOXO1, and the free radical scavengers superoxide dismutase 2 (SOD2) and SOD1 protein expression. RESULT(S): Prolactin production was induced in HESCs in response to 8-br-cAMP and P. Dihydrotestosterone further enhanced the secretion of PRL in cells treated with 8-br-cAMP plus P. The effect of DHT was blocked by the antiandrogen flutamide. Dihydrotestosterone enhanced resistance to oxidative stress-induced apoptosis on decidualized HESCs. Moreover, DHT enhanced FOXO1 expression in parallel with increased SOD2 protein but not with SOD1. CONCLUSION(S): Androgens might play a critical role in the decidualization process at the time of embryo implantation and trophoblast invasion by promoting resistance to oxidative stress.