Human Endometrial Stromal Cell Line Research Articles

IL17A Suppresses IGFBP1 in Human Endometrial Stromal Cells

Interleukin (IL) 17A has been implicated in preeclampsia, preterm labor, and miscarriage. IL17A production in non-lymphoid tissues is mainly carried out by unconventional γδ17T cells. Innate lymphoid cells (ILCs) 3, a subgroup of innate lymphocytes, can also be a source of IL17A in the endometrium and are required from implantation to early pregnancy, with their regulation ensuring that pregnancy continues. Herein, we examined the expression of γδ17T cells and ILC3 regulators IL1B, IL23A, and IL17D and IL17A receptors (IL17RA/IL17RC) in human endometrial stromal cells (EnSCs) and cell lines (KC02-44D). Accordingly, quantitative polymerase chain reaction and immunoblotting were employed. IL1B, IL23A, and IL17D were significantly upregulated in decidualized EnSCs and KC02-44D cells. A significant augmentation in IL17RA/IL17RC was also observed in decidualization. IL17A stimulation of KC02-44D cells during decidualization suppressed the decidualization marker IGFBP1. The involvement of transcription factor Forkhead box protein O1 (FOXO1) in this repression was reflected by its translocation from the nucleus into the cytoplasm. A role for IkB kinase alpha in FOXO1 phosphorylation-mediated migration was also suggested. Taken together, our findings indicate that the secretion of IL17A by γδ17T and ILC3 cells in the uterus contributes to EnSCs function and may play critical roles in regulating IGFBP1-mediated implantation and fetal growth.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

CAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization.This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR.Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL.In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

Using a Quantitative High-Throughput Screening Platform to Identify Molecular Targets and Compounds as Repurposing Candidates for Endometriosis

Endometriosis, defined as the growth of hormonally responsive endometrial-like tissue outside of the uterine cavity, is an estrogen-dependent, chronic, pro-inflammatory disease that affects up to 11.4% of women of reproductive age and gender-diverse people with a uterus. At present, there is no long-term cure, and the identification of new therapies that provide a high level of efficacy and favourable long-term safety profiles with rapid clinical access are a priority. In this study, quantitative high-throughput compound screens of 3517 clinically approved compounds were performed on patient-derived immortalized human endometrial stromal cell lines. Following assay optimization and compound criteria selection, a high-throughput screening protocol was developed to enable the identification of compounds that interfered with estrogen-stimulated cell growth. From these screens, 23 novel compounds were identified, in addition to their molecular targets and in silico cell-signalling pathways, which included the neuroactive ligand–receptor interaction pathway, metabolic pathways, and cancer-associated pathways. This study demonstrates for the first time the feasibility of performing large compound screens for the identification of new translatable therapeutics and the improved characterization of endometriosis molecular pathophysiology. Further investigation of the molecular targets identified herein will help uncover new mechanisms involved in the establishment, symptomology, and progression of endometriosis.

Atg9A-mediated mitophagy is required for decidual differentiation of endometrial stromal cells.

Endometrial decidualization is the foundation of a healthy pregnancy. Mitochondrial dysfunction is an independent cause of disease for energy-intensive organs. Mitochondrial homeostasis plays a key role in the differentiation processes of many cell types. We showed increased activation of mitophagy (mitochondrial autophagy) in the decidua compared with proliferative or secretory endometrium. To better comprehend the mechanisms underlying healthy conception, understanding the mechanism of endometrial stromal cell decidualization is of great importance. Here, we artificially induced decidualization of a human endometrial stromal cell line (T HESCs) and characterized subsequent activation of mitophagy using immunofluorescence assay, electron microscopy and Western blot assay. Knockdown of autophagy-related 9A (ATG9A) led to an obvious reduction of mitophagy and deficiencies in decidualization. Our findings demonstrate a key role for proper mitochondrial dynamics in decidual differentiation and identify ATG9A-mediated mitophagy as a novel therapeutic target for repeated implantation failure or recurrent abortion.

Bisphenols A, F, S and AF trigger apoptosis and/or endoplasmic reticulum stress in human endometrial stromal cells

Disruption of non-differentiated endometrial stromal cells could have noxious consequences in female reproduction, impairing endometrial remodelling and implantation. Following the classification of bisphenol A (BPA) as an endocrine disrupting chemical, it started to be gradually withdrawn from the market, being substituted by structural analogues, whose effects in human health are not fully understood. This work used a telomerase-immortalized human endometrial stromal cell line (St-T1b) to study the effects of BPA and its three most commercialized structural analogues (ranked: bisphenols S, F and AF) on endometrial stromal cells to understand their effects on female reproductive function. Bisphenols showed dissimilar effects. All four compounds generated endoplasmic reticulum (ER) stress. In addition, bisphenols A, F and AF induced apoptosis through different mechanisms, with bisphenol AF causing cell cycle arrest at G2/M phase. Bisphenol AF decreased mitochondrial transmembrane potential and bisphenols A, F and AF produced oxidative stress.

The protective effects of Azilsartan against hypoxia in endometrial stromal cells: an implication in endometriosis

IntroductionEndometriosis is a reproductive disorder closely associated with hypoxia stress. Increasing evidences have implied the important roles of angiotensin II (ANG II) receptors in the pathophysiology of endometriosis. Thus, we speculated that Azilsartan (AZL), an ANG II receptor blocker, might have an effective function in controlling endometriosis.Material and methodsThe endometriotic animal model was established in female SD rats (6-8 weeks old, 200-220 g). Rats were divided into sham group and endometriosis (EMS) group. Rats in EMS group were anaesthetized by halothane and a mid-ventral incision was performed to expose the bowels. Human endometrial stromal cell line T-HESC was used for the in vitro assays. The T-HESC cells were cultured in DMEM-F12 mediumwith 10% fetal bovine serum (FBS, Hyclone), and 4 mM L-glutamine, 0.25% HEPES plus necessary antibiotics (Sigma-Aldrich, USA), at 37°C in a humidified atmosphere of 5% CO2.ResultsThe results show that upregulation of ANG II type 1 (AT1) receptor was observed in the endometriotic rat models. Treatment with AZL prevented the development of endometriotic lesions and suppressed the expressions of HIF-1α and cyclooxygenase 2 (COX-2) in endometriotic rats. In vitro assays proved that hypoxia-induced proliferation, migration, and invasion of T-HESC cells were attenuated by AZL. AZL inhibited the expression levels of hypoxia-inducible factor-1α (HIF-1α), COX-2, and prostaglandin E2 (PGE2) production in hypoxia-induced T-HESC cells. Overexpression of HIF-1α blocked the effects of AZL on T-HESC cells in response to hypoxia.ConclusionsAZL showed therapeutic function on endometriosis through inhibiting hypoxia-induced cell proliferation, migration, and invasion of T-HESC cells via HIF-1α/COX-2/PGE2 signaling.

SIRT1 suppresses in vitro decidualization of human endometrial stromal cells through the downregulation of forkhead box O1 expression

SIRT1 regulates survival, DNA repair, and metabolism in human cells and has pleiotropic effects on age-related diseases through either deacetylating target proteins or inhibiting gene transcription. Forkhead box O1 (FOXO1) is one of the most important transcription factors during decidualization. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) are well-known FOXO1-dependent genes in decidualizing cells. To determine whether SIRT1 plays a role in decidualization, we investigated morphological changes in cells following artificially stimulated decidualization and expression levels of PRL, IGFBP1, and FOXO1 in the immortalized non-neoplastic human endometrial stromal cell line T HESCs. SIRT1 expression decreased in the decidualization condition and SIRT1 inhibited morphological changes caused by decidualization of T HESCs. SIRT1 suppressed PRL, IGFBP1, and FOXO1 expression; inhibited FOXO1, PRL, and IGFBP1 promoter activity; and decreased histone protein acetylation of the FOXO1 promoter. We found that FOXO1 expression increased in the secretory phase compared with the proliferative phase, whereas SIRT1 expression decreased in the secretory phase in the human endometrium. We also revealed that SIRT1 may inhibit embryo implantation according to the blastocyst-like spheroid implantation assay. Collectively, these results indicate that SIRT1 suppresses decidualization of human endometrial stromal cells by inhibiting FOXO1 expression.

The uterine natural killer cell, cytotoxic T lymphocyte, and granulysin levels are elevated in the endometrium of women with nonstructural abnormal uterine bleeding

To examine the expression of uterine natural killer (uNK) cells and cytotoxic T lymphocytes (CTLs) in endometrial biopsies from reproductive-age women with and without nonstructural abnormal uterine bleeding (AUB) and evaluate the expression of granulysin within these cell populations and potential modulation of matrix metalloproteinase (MMP) expression. Experimental study, retrospective design. Academic research laboratory. Patients with nonstructural AUB with no other gynecological pathologies and control patients without AUB. None. Immunohistochemical analysis of granulysin, CD56 (uNK cell marker), and CD8 (CTL marker) expression as well as granulysin messenger ribonucleic acid (mRNA) expression levels in endometrial biopsy samples. Assessment of granulysin regulation of human endometrial stromal cell MMP-1 and MMP-3 mRNA expression. The numbers of uNK cells and CTLs were significantly greater in endometrial biopsy tissue from women with AUB than those from controls. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB than in from controls and colocalized to both cell types but not endometrial stromal or epithelialcells. The increased granulysin protein expression was associated with the increased granulysin mRNA expression in adjacent serial sections from these same samples. The treatment of the human endometrial stromal cell line t-HESC with granulysin resulted in a significant increase in MMP-1 and MMP-3 mRNA expression. In the current study, immunohistochemistry showed an increased expression of uNK cells, CTLs, and granulysin among subjects with AUB compared with that of subjects without AUB, leading to conclusions that disturbances in the balance of immune cells and an increase in granulysin expression may have implications in the pathophysiology of AUB and include enhanced MMP-1 and MMP-3 expression.

Dysregulation of X-ray repair cross-complementing 4 expression in the eutopic endometrium of women with endometriosis.

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of dsDNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Toward this, we focused on the X-ray repair cross-complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (P < 0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the Gene Expression Omnibus database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AX and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (hTERT immortalized human endometrial stromal cell line (ThESCs)) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

Peptidome analysis of human intrauterine adhesion tissues and the identification of antifibrotic peptide.

Intrauterine adhesion (IUA) is a common clinical endometrial disease, which can severely damage the fertility and quality of life in women. This study aims to find the differentially expressed endogenous peptides and their possible roles in IUA. Liquid chromatography-mass spectrometry was used to identify the peptidomic profiling of IUA tissues, and the differentially expressed peptides were screened out. Using real-time quantitative PCR, Western blotting, and immunocytochemistry staining, the function of six endogenous peptides was verified in vitro. It was found that peptide 6 (T6) (peptide sequence: TFGGAPGFPLGSPLSSVFPR) could inhibit the expression of TGF-β1-induced cell fibrosis in human endometrial stromal cell line and primary human endometrial stromal cell at a concentration of 50 μmol/L. This study provides new targets for further clarifying the formation and prevention of IUA.

Zinc(II) niflumato complex effects on MMP activity and gene expression in human endometrial cell lines

Endometriosis is a chronic inflammatory disease which increasingly affects young women under 35 years of age and leads to subfertility even infertility. Analysis of the cytotoxic effect of zinc(II) niflumato complex with neocuproine ([Zn(neo)(nif)2] or Zn-Nif) on immortalized human endometriotic cell line (12Z) and on control immortalized human endometrial stromal cell line (hTERT) was performed using xCELLigence technology for approximately 72 h following the treatment with Zn-Nif as well as cell viability Trypan Blue Assay. 12Z cell line proliferated more slowly compared to unaffected cells, whereas hTERT cells did not show similar behavior after treatment. The complex probably reduces the effect of pro-inflammatory pathways due to the effect of NSAID, while presence of zinc might reduce the level of ROS and regulate ER2 levels and MMP activity. The observed effects and high selectivity for rapidly proliferating cells with increased inflammatory activity suggest a good prognosis of successful decrease of endometriosis stage with this complex.

Long non‑coding RNA LINC01224 promotes cell proliferation and inhibits apoptosis by regulating AKT3 expression via targeting miR‑485‑5p in endometrial carcinoma.

Endometrial carcinoma (EC) is the most common cancer in women worldwide, yet little is known about the underlying molecular basis of EC development. LINC01224, a novel long non-coding (lnc)RNA, was recently identified as an oncogene in various types of cancer. However, the function and underlying mechanism of LINC01224 in EC is still unclear. A total of 50 pairs of tumor and adjacent normal tissue from patients with EC, three EC cell lines and one human normal endometrial stromal cell (ESC) line were subjected to reverse transcription-quantitative PCR assay to evaluate the expression levels of LINC01224. Cell Counting Kit-8, colony formation and flow cytometry assays were used to assess cell proliferation and apoptosis. Western blotting was used to measure expression levels of apoptosis- and proliferation-associated proteins and AKT3 protein. A xenograft model of HEC1A cells was established to validate the in vivo function of LINC01224 in EC tumor growth. Starbase 3.0 database prediction and luciferase reporter and RNA pull-down assays were performed to verify the binding sites between LINC01224 and microRNA (miR)-485-5p and miR-485-5p and AKT3. LINC01224 expression was significantly upregulated in both EC tumor tissue and cell lines. The upregulation of LINC01224 was negatively associated with survival of patients with EC. Functionally, LINC01224 promoted proliferation and inhibited apoptosis of EC cells; LINC01224 directly bound to and downregulated miR-485-5p to elevate the expression levels of AKT3, thereby promoting EC progression. LINC01224 depletion in EC cells hindered tumor growth in a xenograft model. The tumor suppressing effect of LINC01224-knockdown on EC progression was partly rescued by treatment with miR-485-5p inhibitor. The present data demonstrated the expression levels, clinical relevance and functional mechanism of LINC01224 in EC. LINC01224 promoted EC development via sponging miR-485-5p to elevate AKT3 expression levels; this may provide a promising therapeutic target pathway for EC treatment.

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

Increasing evidence has revealed a close relationship between non-coding RNAs and recurrent implantation failure (RIF). However, the role of circular RNAs (circRNAs) in RIF pathogenesis remains largely unknown. Microarray analyses were used to identify the differentially expressed circRNA-circSTK40. Functional experiments, including decidualization induction and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, were performed to determine the effects of circSTK40 on human endometrial stromal cells (ESCs). The interactions between circSTK40 and proteins were investigated by RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation (coIP) assays. We observed that circSTK40 expression was upregulated in the RIF midluteal-phase endometrial samples. circSTK40 overexpression in ESCs inhibited the decidualization process but concurrently enhanced cell survival during stress. Mechanistically, circSTK40 directly bound to HSP90 and CLU, thus functioning as a scaffold to block their interactions and hinder the proteasomal degradation of HSP90. The resulting high levels of HSP90 led to the activation of the AKT pathway and downregulation of FOXO1 expression. Inhibitors of AKT (MK-2206) and HSP90 (17AAG) both abolished the effects of circSTK40 overexpression in ESCs and increased the decidualization levels in a dose-dependent manner. Our findings indicate a novel epigenetic mechanism for RIF pathogenesis involving circSTK40 activity and provide a foundation for targeted treatments in patients with low endometrial receptivity.

Antitumor effect of mifepristone on human endometrial stromal cell line

Background/Aim. The main cause for development of endometrial hyperplasia is unopposed effect of estrogen on endometrial cells. The aim of our study was to investigate and compare cytotoxic and apoptotic effects of mifepristone on human endometrial stromal cell line for the first time. Both percentage of cytotoxic and apoptotic cells were determined after 24 h treatment with different doses of mifepristone. Methods. The percentage of cytotoxic cell was evaluated by viability assay while the percentage of apoptotic cells was determined using flow cytometry. Determination of apoptotic effects was confirmed using immunofluorescence method determining expression and localization of active Bax and Bcl-2 proteins. Results. Our results indicated that mifepristone induced cytotoxic and apoptotic effect on human endometrial stromal cell line (ThESC) through changes in expression level of Bcl-2 and active Bax proteins. Conclusion. Cytotoxic and pro-apoptotic effects of mifepristone on human endometrial stromal cell line in vitro was investigated in this study for the first time. It is crucial to point out that mifepristone expressed both cytotoxic and pro-apoptotic effect on ThESC cell line. Our results may contribute to determination of localization and expression level of the crucial proteins involved in apoptosis in ThESC cell line after the treatment with the lowest cytotoxic doses of mifepristone.

Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation.

Does osteoprotegerin (OPG) promote human endometrial stromal decidualization? OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation. OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive. We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs. We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT-PCR (qRT-PCR). Immunofluorescence staining and western blot, and qRT-PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT-PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization. OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P < 0.05) and prolactin (PRL) (P < 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P < 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P < 0.05). N/A. In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration. OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL. This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.

Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells

Autophagy can be dynamically induced in response to stresses and is an essential, ubiquitous intracellular recycling system that impacts the fate of damaged resident cells, thereby influencing wound healing. Endometrial fibrosis is a form of abnormal wound healing that causes intrauterine adhesion (IUA) and infertility. We previously demonstrated that overactivated sonic hedgehog (SHH) signaling exacerbated endometrial fibrosis, but the role of autophagy in this process is still unknown. Here, we report that impaired autophagy participates in SHH pathway-induced endometrial fibrosis. Endometrial stroma-myofibroblast transition accompanied by autophagy dysfunction was present in both endometrial biopsies of IUA patients and Amhr2cre/+R26-SmoM2+/− (AM2) transgenic mouse. Mechanistically, SHH pathway negatively regulated autophagy through pAKT-mTORC1 in a human endometrial stromal cell line (T-HESCs). Furthermore, SHH pathway-mediated fibrosis was partly counteracted by autophagy modulation in both T-HESCs and the murine IUA model. Specifically, the impact of SHH pathway inhibition (GANT61) was reversed by the pharmacological autophagy inhibitor chloroquine (CQ) or RNA interference of autophagy-related gene ATG5 or ATG7. Similar results were obtained from the murine IUA model treated with GANT61 and CQ. Moreover, promoting autophagy with rapamycin reduced fibrosis in the AM2 IUA model to baseline levels. In summary, defective autophagy is involved in SHH pathway-driven endometrial fibrosis, suggesting a potential novel molecular target for IUA treatment.

Decidualization Process Induces Maternal Monocytes to Tolerogenic IL-10-Producing Dendritic Cells (DC-10).

Decidualization is a process that involves phenotypic and functional changes of endometrial stromal cells to sustain endometrial receptivity and the participation of immunoregulatory factors to maintain immune homeostasis. In this context, tolerogenic dendritic cells (DCs) can induce regulatory T cells, which are essential to manage the pro- to anti-inflammatory transition during embryo implantation. Recently, Myeloid Regulatory Cells (MRCs) were proposed as immunosuppressants and tolerance-inducer cells, including the DC-10 subset. This novel and distinctive subset has the ability to produce IL-10 and to induce type 1 regulatory T cells (Tr1) through an HLA-G pathway. Here we focus on the impact of the decidualization process in conditioning peripheral monocytes to MRCs and the DC-10 subset, and their ability to induce regulatory T cells. An in vitro model of decidualization with the human endometrial stromal cell line (HESC), decidualized by medroxyprogesterone and dibutyryl-cAMP was used. Monocytes isolated from peripheral blood mononuclear cells from healthy women were cultured with rhGM-CSF + rhIL-4 and then, the effect of conditioned media from decidualized (Dec-CM) and non-decidualized cells (Non-dec-CM) was tested on monocyte cultures. We found that Dec-CM inhibited the differentiation to the CD1a+CD14– immature DC profile in a concentration-dependent manner. Dec-CM also significantly increased the frequency of CD83+CD86low and HLA-DR+ cells in the monocyte-derived culture. These markers, associated with the increased production of IL-10, are consistent with a MRCs tolerogenic profile. Interestingly, Dec-CM treatment displayed a higher expression of the characteristic markers of the tolerogenic DC-10 subset, HLA-G and ILT2/CD85j; while this modulation was not observed in cultures treated with Non-dec-CM. Moreover, when monocyte cultures with Dec-CM were challenged with LPS, they sustained a higher IL-10 production and prevented the increase of CD83, CD86, IL-12p70, and TNF-α expression. Finally, the DC-10 subset was able to induce a CD4+HLA-G+ regulatory T cells subset. These results suggest that the decidualization process might induce different subsets of MRCs, like DC-10, able to induce regulatory T cells as a novel CD4+HLA-G+ subset which might play an immunoregulatory role in embryo implantation.

Tryptophan and kynurenine stimulate human decidualization via activating Aryl hydrocarbon receptor: Short title: Kynurenine action on human decidualization

Decidualization is essential for successful pregnancy in rodents and primates. Although L-Tryptophan and its metabolites are essential for mammalian pregnancy, the underlying mechanism is poorly defined. We explored effects of tryptophan and kynurenine on human in vitro decidualization in human endometrial stromal cell line and primary endometrial stromal cells. Tryptophan significantly stimulates the expression of prolactin and insulin growth factor binding protein 1, reliable markers for human decidualization. When stromal cells are treated with tryptophan, tryptophan hydroxylase-1 remains unchanged, but indoleamine 2,3-dioxygenase 1 is significantly increased, suggesting tryptophan is mainly metabolized through kynurenine pathway. Kynurenine significantly stimulates insulin growth factor binding protein 1 expression. Aryl hydrocarbon receptor and its target genes (P450 1A1 and P450 1B1) are significantly increased by tryptophan and kynurenine. The induction of tryptophan and kynurenine on insulin growth factor binding protein 1 is abrogated by CH223191, an aryl hydrocarbon receptor inhibitor. Cytochrome P450 1A1 and P450 1B1 catalyze the oxidative metabolism of estradiol to catechol estrogens (2-hydroxy estradiol and 4-hydroxy estradiol), respectively. Insulin growth factor binding protein 1 is up-regulated by 2-hydroxy estradiol and 4-hydroxy estradiol. Interferon-γ significantly induces the expression of indoleamine 2,3-dioxygenase 1, aryl hydrocarbon receptor and insulin growth factor binding protein 1. All the data are also verified in primary human stromal cells. Our data indicate that Interferon-γ-induced kynurenine pathway promotes human decidualization via aryl hydrocarbon receptor signaling.

VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway

Endometrial stromal cells undergo endoplasmic reticulum (ER) stress and unfolded protein response (UPR) during the decidualization linked with the inflammation and angiogenesis processes. Considering VIP (vasoactive intestinal peptide) induces the decidualization program, we studied whether modulates the ER/UPR pathways to condition both processes for embryo implantation.When Human Endometrial Stromal Cell line (HESC) were decidualized by VIP we observed an increased expression of ATF6α, an ER stress-sensor, and UPR markers, associated with an increase in IL-1β production. Moreover, AEBSF (ATF6α -inhibitor pathway) prevented this effect and decreased the expansion index in the in vitro model of implantation. VIP-decidualized cells also favor angiogenesis accompanied by a strong downregulation in thrombospondin-1. Finally, ATF6α, VIP and VPAC2-receptor expression were reduced in endometrial biopsies from women with recurrent implantation failures in comparison with fertile. In conclusion, VIP privileged ATF6α-pathway associated with a sterile inflammatory response and angiogenesis that might condition endometrial receptivity.