Primary Cultures Of Endometrial Stromal Cells Research Articles

P-297 Regulation and functions of PGRMC1 in the human endometrium and in endometriosis

Abstract Study question What is the role of PGRMC1 in the human endometrium (especially in tissue remodeling) and its potential contribution to the development of endometrial diseases? Summary answer In primary endometrial stromal cell culture, PGRMC1 downregulation modifies expression of genes implicated in extracellular matrix remodeling as well as in cell migration and invasion. What is known already It has been suggested that Progesterone Receptor Membrane Component-1 (PGRMC1) is involved in gynecological pathologies. In particular, Pgrmc1 conditional KO in the mouse female reproductive tract induced subfertility and spontaneous development of endometrial cysts. Moreover, siRNA-mediated downregulation of PGRMC1 expression influenced TNF-α-induced activity of matrix metalloproteinase-9 (MMP-9) in cytotrophoblast cells, suggesting that PGRMC1 is a regulator of MMPs. In addition to playing a major role in the physiological breakdown of the human endometrium at menstruation, MMPs are strongly suspected to favor the pathogenesis of endometriosis lesions. Study design, size, duration Not applicable Participants/materials, setting, methods We compared transcriptomes obtained by RNA-sequencing from distinct primary endometrial stromal cell (ESC) cultures transfected with one of two specific siRNA targeting PGRMC1 mRNA or with a control siRNA. Expression changes for selected genes were confirmed by RT-qPCR. Migration and invasion capacities of the cells were also studied using Transwell assay. Finally, immunocytochemistry was performed on these primary cultures to localize the presence of PGRMC1 as well as the canonical progesterone receptor (PR). Main results and the role of chance RNA-sequencing data converged to show that the reduction of PGRMC1 expression by the specific siRNA significantly increased (up to 3-fold) the expression of several genes involved in the extracellular matrix (ECM) remodeling, as well as in the processes of migration and invasion. These changes were reproduced by RT-qPCR on other distinct primary endometrial stromal cultures isolated from other patients. In parallel, Transwell assays showed no significant changes in the migration and invasion capacities of the primary endometrial stromal cell cultures previously invalidated for PGRMC1 by transfection of the cells with a specific siRNA by comparison with a control siRNA. However, immunocytochemistry studies on these primary endometrial stromal cells showed that progesterone receptor (PR) labeling was restricted to cell nuclei, suggesting a constitutively activated PR in these cultures. Limitations, reasons for caution The absence of changes in migration and invasion properties of primary ESC cultures observed by Transwell assay could be due to the fact that the progesterone receptor seems to be constitutively activated in these cultures potentially causing a repressive effect on the migration and invasion capacity of the cells. Wider implications of the findings The modification of expression of genes implicated in ECM remodeling, and cell migration and invasion after PGRMC1 downregulation in primary ESC cultures is interesting in the context of endometriosis in which reduction of PGRMC1 expression could improve the migration and invasion capacities of lesions to implant into the host tissue. Trial registration number Not applicable

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200 µM of H2O2 and evaluated the cytoprotective effects of Vit E (5 µM) and folate (0.01 µM) in H2O2-treated cells for 24 h. Following the exposure of endometrial cells to H2O2 alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H2O2 treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H2O2 toxicity. An increasing number of alive cells was showed in the cells exposed to H2O2 (50 µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.

Decidualization Mediated by Steroid Hormones Modulates the Innate Immunity in Response to Group B Streptococcal Infection in vitro

Background: Decidual cells play a role in the modulation of the innate immune response to protect pregnancy against infection. Steroid hormones regulate the innate immune response in different tissues, and they are involved in several biological processes like decidualization. The aim of this study was to assess if steroid hormones modulate the innate immunity in endometrial stromal cells (ESCs) and decidual stromal cells (DSCs) in response to group B streptococcus (GBS) infection in vitro. Methods: Primary cultures of ESC were differentiated into DSC using 36 n<smlcap>M</smlcap> estradiol + 300 n<smlcap>M</smlcap> progesterone, and both were infected with GBS overnight. Concentrations of pro- and anti-inflammatory mediators (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, IL-10, and TGF-β), chemokines (IL-8 and GCP-2), and human β-defensins (HBD-1, HBD-2, and HBD-3) were measured in the culture supernatants. Results: DSCs showed a significant increase in IL-6 (p < 0.05), TNF-α (p < 0.05), IL-10 (p < 0.01), and TGF-β (p < 0.05) secretion after GBS infection, while these changes were not observed in infected ESCs. IL-8 and GCP-2 increased after GBS infection, regardless of decidualization. β-Defensins 1-3 decreased (p < 0.05) in ESCs after GBS infection, and hormone decidualization preserved the secretion of these antimicrobial peptides. Conclusions: Decidualization mediated by steroid hormones balance the pro- and anti-inflammatory response at the maternal-fetal interface under infection conditions.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Interferon-gamma modulates prolactin and tissue factor expression in differentiating human endometrial stromal cells.

Cytokines such as interferon-gamma (IFNgamma) released by resident uterine immune cells are thought to influence the expression of differentiated function in the human endometrium. Decidualization of the stromal cell compartment is confined to the superficial endometrial layer in the nonpregnant uterus. To explore the molecular mechanism underlying the spatial expression of the decidual phenotype, the effect of IFNgamma on the induction of two well characterized markers of endometrial stromal (ES) cell differentiation, PRL and tissue factor (TF), has been investigated. IFNgamma antagonizes cAMP-mediated PRL protein and messenger RNA expression in primary ES cell cultures through inhibition of decidual PRL promoter activity. In parallel, IFNgamma stimulates Stat-1 (signal transducer and activator of transcription-1) expression, phosphorylation, and translocation to the nucleus. Exogenously expressed Stat-1 potently represses decidual PRL promoter activation, indicating the potential for the inhibitory effects of IFNgamma to be mediated by Stat-1. We demonstrate that although the coactivator CREB-binding protein/p300 is essential for decidual PRL transcription, this coactivator does not appear to be the target for IFNgamma-mediated repression. By contrast, IFNgamma has little effect on cAMP-mediated TF expression, but induces TF in ES cells not exposed to a decidualizing stimulus. This suggested that in vivo TF expression may not be restricted to decidualizing cells of the superficial layer and was confirmed by immunohistochemical analysis demonstrating intense TF staining in the basal stromal compartment during the regeneration phase of the cycle. The differential sensitivity of decidualization-associated genes to IFNgamma illustrates its potential role as a selective biological response modifier that influences regional function within the endometrium.

Effect of peritoneal fluid from patients with mild and severe endometriosis on endometrial stromal cell proliferation.

The aim of this study was to evaluate and compare the mitogenic effect of peritoneal fluid (PF) from women with mild and severe endometriosis on the endometrial stromal cell proliferation. Increasing concentrations of PF from women with and without mild or severe endometriosis were added to primary endometrial stromal cell cultures and 3H-thymidine incorporation was used to assess DNA synthesis in these cultures. PF from women with mild endometriosis induced a statistically significant dose-dependent increase in stromal cell thymidine uptake ranged from 5.8 to 14.5 fold, whereas PF from women with severe endometriosis produced an average 51% inhibition of stromal cell proliferation of compared with cells exposed to non-endometriosis PF or exposed to nutrient medium supplemented with 2.5% calf serum alone. PF samples from patients with stage I endometriosis induced a statistically dose-dependent increase in stromal cell proliferation, whereas PF from patients with stage IV endometriosis caused a significant inhibition.