Decidualization Of Endometrial Stromal Cells Research Articles

YAP1 and WWTR1 are required for murine pregnancy initiation.

Endometrial stromal cell decidualization is required for pregnancy success. Although this process is integral to fertility, many of the intricate molecular mechanisms contributing to decidualization remain undefined. One pathway that has been implicated in endometrial stromal cell decidualization in humans in vitro, is the Hippo signaling pathway. Two previously conducted studies showed that the effectors of the Hippo signaling pathway, YAP1 and WWTR1, are required for decidualization of primary endometrial stromal cells in vitro. To investigate the in vivo role of YAP1 and WWTR1 in decidualization and pregnancy initiation, we generated Progesterone receptor Cre mediated mutation of a combination of Yap1 and Wwtr1 alleles. Female Yap1 and Wwtr1 triple allele mutants exhibited subfertility, a compromised decidualization response, decreased endometrial receptivity, delayed embryonic development, and a unique transcriptional profile at 7.5 days post coitus. Bulk mRNA sequencing revealed aberrant maternal remodeling evidenced by significant alterations in extracellular matrix encoding genes at 7.5 days post-coitus in mutant dams and enrichment for terms associated with fertility-compromising diseases like pre-eclampsia and endometriosis. In addition, differentially expressed genes overlapped directionally with Estrogen receptor and Epidermal growth factor receptor regulated genes as identified by microarray. Our results indicate that Yap1 and Wwtr1 are necessary for successful mammalian pregnancy initiation.

Identification of potential hub genes associated with recurrent miscarriage through combined transcriptomic and proteomic analysis.

Recurrent miscarriage (RM) is currently difficult to prevent and treat due to a lack of comprehensive understanding of its molecular mechanisms. The aim of this study was to identify genes potentially involved in the pathogenesis of RM and to observe their expression in the decidual tissues of RM patients. A total of 1823 differentially expressed genes (DEGs) and 148 differentially expressed proteins (DEPs) in decidual tissues between RM and control groups were identified. Subsequently, DCN, DPT, LUM, MFAP4, and ISG15 were identified from the DEGs/DEPs as RM-related hub genes through systematic bioinformatics analysis. Bioinformatics analysis of the single-cell dataset GSE214607 revealed that the expression of these five hub genes in the decidual stromal cells of RM patients appeared to be upregulated, while the RT-qPCR assay showed that their decidual expression levels were significantly increased in RM patients. Uterine Isg15expression was significantly increased, whereas the uterine expression of Dcn, Dpt, Lum, and Mfap4 was decreased in LPS-induced early pregnancy loss mice. MiR-16-5p, -21-3p, -27a-3p, and -941 were identified as potentially involved in the regulation of these five hub genes, and their decidual expression levels were significantly decreased in RM patients. The abnormally increased ISG15 expression in the decidual tissues of RM patients and uterine tissues of LPS-induced mice was validated by WB analysis. ISG15 expression was significantly reduced during the in vitro decidualization of human endometrial stromal cells (hESCs). Collectively, DCN, DPT, LUM, MFAP4, and ISG15 were identified as RM-related hub genes, and their expression in the decidual tissues of RM patients was significantly increased. The decidualization of hESCs was accompanied by reduced ISG15 expression, suggesting that increased decidual ISG15 expression might lead to early pregnancy loss by disrupting the decidualization process.

7954 Vitamin D Deficiency Underlies Defective Endometrial Decidualization In Vivo and In Vitro

Abstract Disclosure: M. Yi: None. T. Wang: None. A. Jukic: None. F.J. DeMayo: None. Several epidemiologic studies have reported associations between vitamin D and both menstrual cycles and fertility. Vitamin D signaling occurs most often through the interaction of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) with its cognate receptor, the transcription factor vitamin D receptor (VDR). This study aims to define the role of vitamin D and VDR on female reproduction and identify vitamin D-mediated signaling. In our studies, preliminary results demonstrate mice on a vitamin D-deficient diet showed a significantly impaired uterine decidualization reaction in response to exogenous hormones. To knockdown VDR expression, human endometrial stroma cells (THESCs) were transfected with either non-targeting control (siNT) or VDR siRNA (siVDR) for 48 h. And, to perform the in vitro decidualization, the cell culture media was replaced to differentiate the cells by treatment with 17β-estradiol (E), medroxyprogesterone acetate (P), and db-cAMP (C) for 72 h. At the 48 h exposure of EPC, the decidual media was replaced with containing 1,25(OH)2D3 or vehicle and cultured for 24 h additionally. Treatment of 1,25(OH)2D3 (ligand) and knockdown of VDR (receptor) in THESCs resulted in an increased expression of the decidualization markers, PRL and IGFBP1. Transcriptomic analysis was then conducted to identify the biological networks regulated by the ligand and receptor in THESCs. THESCs were transfected with either siNT or siVDR for 48 h, and the culture media was replaced with either treatment of 2 nM of 1,25(OH)2D3 or vehicle for an additional 24 h. Differentially expressed genes (DEGs) were affected by the ligand and receptor (N = 626 and 1499, respectively) based on a three-way ANOVA filtered with false discovery rate (FDR)-adjusted p-value<0.05 and fold-change (FC)≥|1.4|. Pathway analyses and predicted upstream regulators were analyzed by Ingenuity Pathways Analysis (IPA), and among the predicted upstream regulators of the DEGs, β-estradiol was predicted as an activator for both. Thus, to test the role of vitamin D in estrogen signaling, THESCs were treated with either EPC or PC for 72 h in total, and at the 48 h exposure, the decidual media was replaced with 1,25(OH)2D3 or vehicle and cultured for 24 h additionally. Removal of estrogen (PC) caused loss of the enhanced expression of decidual markers by 1,25(OH)2D3 that was observed in control EPC-treated cells. In conclusion, Vitamin D signaling modulates human and mouse uterine stromal cell differentiation by modulating the ability of estrogen to stimulate decidualization in endometrial stromal cells, a process critical to establishing pregnancy. Presentation: 6/2/2024

In-utero transfer of decidualized endometrial stromal cells increases the frequency of regulatory T cells and normalizes the abortion rate in the CBA/J × DBA/2 abortion model.

Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization. Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking. Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model.

Effects of butyrate on endometrial stromal cell decidualization and glandular epithelial cell maturation

Effects of butyrate on endometrial stromal cell decidualization and glandular epithelial cell maturation

Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

Polycystic ovary syndrome (PCOS) is a complex common endocrine disorder affecting women of reproductive age. Ovulatory dysfunction is recognized as a primary infertile factor, however, even when ovulation is medically induced and restored, PCOS patients continue to experience reduced cumulative pregnancy rates and a higher spontaneous miscarriage rate. Hyperandrogenism, a hallmark feature of PCOS, affects ovarian folliculogenesis, endometrial receptivity, and the establishment and maintenance of pregnancy. Decidualization denotes the transformation that the stromal compart of the endometrium must undergo to accommodate pregnancy, driven by the rising progesterone levels and local cAMP production. However, studies on the impact of hyperandrogenism on decidualization are limited. In this study, we observed that primary endometrial stromal cells from women with PCOS exhibit abnormal responses to progesterone during in vitro decidualization. A high concentration of testosterone inhibits human endometrial stromal cells (HESCs) decidualization. RNA-Seq analysis demonstrated that pyruvate dehydrogenase kinase 4 (PDK4) expression was significantly lower in the endometrium of PCOS patients with hyperandrogenism compared to those without hyperandrogenism. We also characterized that the expression of PDK4 is elevated in the endometrium stroma at the mid-secretory phase. Artificial decidualization could enhance PDK4 expression, while downregulation of PDK4 leads to abnormal decidualization both in vivo and in vitro. Mechanistically, testosterone excess inhibits IGFBP1 and PRL expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. Based on co-immunoprecipitation analysis, we observed an interaction between SIRT1 and PDK4, promoting glycolysis to facilitate decidualization. Restrain of AR activation resumes the AMPK/SIRT1/PDK4 pathway suppressed by testosterone excess, indicating that testosterone primarily acts on decidualization through AR stimulation. Androgen excess in the endometrium inhibits decidualization by disrupting the AMPK/SIRT1/PDK4 signaling pathway. These data demonstrate the critical roles of endometrial PDK4 in regulating decidualization and provide valuable information for understanding the underlying mechanism during decidualization.

Nuclear actin assembly is an integral part of decidualization in human endometrial stromal cells

Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs’ morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPβ), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy.

P-411 CD55 deficiency hinders decidualization in unexplained early miscarriage through the Src/ERK signaling pathway

Abstract Study question What is the role of CD55 in the process of human endometrial stromal cells (HESCs) decidualization in unexplained early miscarriage? Summary answer CD55, responsive to cAMP signaling, is deficient in decidua of patients with unexplained early miscarriage, which hinders decidualization in vitro through perturbing Src/ERK signaling pathway. What is known already Decidualization is a process where HESCs differentiate into decidual stromal cells and present morphological changes and acquisition of secretory function, which is essential for embryo implantation and maintenance of pregnancy while abnormal decidualization contributed to several pregnancy disorders like a miscarriage. Reprogramming of several signaling pathways and transcription factors are involved in this process and cAMP is a critical mediator. Our previous bioinformatic analysis revealed that CD55 was upregulated over five times in the window of implantation, responding to estrogen and progesterone. We speculated that CD55, regulated by cAMP signaling pathway, might play an important role in decidualization. Study design, size, duration Decidua tissues were collected from 8 patients with unexplained early miscarriage and 10 control women with normal early pregnancy undergoing artificial abortion between June 2020 and September 2020. Immortalized HESCs line T-hESCs (ATCC CRL-4003) were cultured for up to 6 days to establish in-vitro decidualization model. Each experiment was conducted in triplicate and replicated at least three times independently. Participants/materials, setting, methods CD55 expression in control and miscarriage decidua was compared by immunohistochemistry and RT-qPCR. T-hESCs were induced into decidualization by medroxyprogesterone acetate (MPA) and 8-Br-cAMP, with prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) as decidualization biomarkers. The changes of genes and signaling pathways were detected by RT-qPCR and western blot during decidualization after CD55 knockdown by lentivirus-mediated RNA interference. Signaling pathway disruptors were added to verify the involvement of signaling pathways. Main results and the role of chance The decidua of women with unexplained early miscarriage exhibited decreased expression of CD55, accompanied by a compromised level of decidualization. During in-vitro decidualization, CD55 was significantly upregulated by cAMP in a time- and dose-dependent manner, which was attenuated by a PKA inhibitor H89. Knocking down CD55 expression diminished the expression of decidualization markers PRL and IGFBP1 through inhibition of Src, aberrant activation of ERK and frustrated expression of decidualization-related genes like FOXO1, EGFR, and STAT3. Limitations, reasons for caution The mechanisms revealed in this study were based on in-vitro cell models. It is necessary to verify the significance of CD55 in decidualization with in-vivo models to directly explain the link between CD55, decidualization, and pregnancy loss. Wider implications of the findings CD55 deficiency hinders decidualization through perturbing Src/ERK signaling pathway and expression of decidualization-related genes, potentially contributing to the pathogenesis of spontaneous miscarriage. Progestin supplementation or other factors stimulating cAMP accumulation can restore CD55 expression and improve decidualization, providing a basis for the therapy of spontaneous miscarriage. Trial registration number not applicable

USP22 is required for human endometrial stromal cell proliferation and decidualization by deubiquitinating FoxM1

Despite significant advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) still occurs in some patients. Poor endometrial receptivity and abnormal human endometrial stromal cell (HESC) proliferation and decidualization have been identified as the major causes. Ubiquitin-specific protease 22 (USP22) has been reported to participate in the decidualization of endometrial stromal cells in mice. However, the role of USP22 in HESC function and RIF development remains unknown. In this study, clinical endometrial tissue samples were gathered to investigate the involvement of USP22 in RIF, and HESCs were utilized to examine the molecular mechanisms of USP22 and Forkhead box M1 (FoxM1). The findings indicated a high expression of USP22 in the secretory phase of the endometrium. Knockdown of USP22 led to a notable reduction in the proliferation and decidualization of HESCs, along with a decrease in FoxM1 expression, while overexpression of USP22 yielded opposite results. Furthermore, USP22 was found to deubiquitinate FoxM1 in HESCs. Moreover, both USP22 and FoxM1 were downregulated in the endometria of patients with RIF. In conclusion, these results suggest that USP22 may have a significant impact on HESCs proliferation and decidualization through its interaction with FoxM1, potentially contributing to the underlying mechanisms of RIF pathogenesis.

Interleukin-1β induces and accelerates human endometrial stromal cell senescence and impairs decidualization via the c-Jun N-terminal kinase pathway

As the mean age of first-time mothers increases in the industrialized world, inquiries into causes of human reproductive senescence have followed. Rates of ovulatory dysfunction and oocyte aneuploidy parallel chronological age, but poor reproductive outcomes in women older than 35 years are also attributed to endometrial senescence. The current studies, using primary human endometrial stromal cell (ESC) cultures as an in vitro model for endometrial aging, characterize the proinflammatory cytokine, IL-1β-mediated and passage number-dependent effects on ESC phenotype. ESC senescence was accelerated by incubation with IL-1β, which was monitored by RNA sequencing, ELISA, immunocytochemistry and Western blotting. Senescence associated secreted phenotype (SASP) proteins, IL-1β, IL-6, IL-8, TNF-α, MMP3, CCL2, CCL5, and other senescence-associated biomarkers of DNA damage (p16, p21, HMGB1, phospho-γ-histone 2 A.X) were noted to increase directly in response to 0.1 nM IL-1β stimulation. Production of the corresponding SASP proteins increased further following extended cell passage. Using enzyme inhibitors and siRNA interference, these effects of IL-1β were found to be mediated via the c-Jun N-terminal kinase (JNK) signaling pathway. Hormone-induced ESC decidualization, classical morphological and biochemical endocrine responses to estradiol, progesterone and cAMP stimulation (prolactin, IGFBP-1, IL-11 and VEGF), were attenuated pari passu with prolonged ESC passaging. The kinetics of differentiation responses varied in a biomarker-specific manner, with IGFBP-1 and VEGF secretion showing the largest and smallest reductions, with respect to cell passage number. ESC hormone responsiveness was most robust when limited to the first six cell passages. Hence, investigation of ESC cultures as a decidualization model should respect this limitation of cell aging. The results support the hypotheses that “inflammaging” contributes to endometrial senescence, disruption of decidualization and impairment of fecundity. IL-1β and the JNK signaling pathway are pathogenetic targets amenable to pharmacological correction or mitigation with the potential to reduce endometrial stromal senescence and enhance uterine receptivity.

Expression of Long Non-Coding RNA H19 in the Endometrium of Mice During Peri-Implantation and Its Regulation on Embryo Implantation

In this study, we utilized magnetic nanobeads for the extraction of nucleic acids from tissues to investigate the expression levels and correlation between lncRNA H19, miR-612, and their target gene HOXA10 in peri-implantation endometrium of mice. Furthermore, we conducted overexpression or gene knockout experiments on lncRNA H19 to observe its impact on the expression of miR-612 and HOXA10. The targeted binding relationship between lncRNA H19, miR-612, and HOXA10 was detected by dual luciferase reporter assay. The regulatory relationship between lncRNA H19, miR-612, and HOXA10 was verified through silencing or overexpression of these genes. Intrauterine transfection was used to modulate the expression of lncRNA H19 in endometria during pregnancy, followed by the detection of the expression levels of miR-612 and HOXA10 as well as ITGB3 and IGFBP-1 proteins. Compared with non-pregnant mice, we observed a significant upregulation of both lncRNA H19 and HOXA10 in the endometria of pregnant mice, while miR-612 was found to be downregulated (P < 0.05). Further analysis revealed that the expression levels of lncRNA H19 and HOXA10 increased progressively with gestational days, peaking on Day 4 (P < 0.05). Moreover, Through database analysis, we identified binding sites for lncRNA H19-miR-612 as well as HOXA10-miR-612 interactions. The dual-luciferase reporter assay further supported our conjecture that lncRNA H19 could specifically bind the miR-612, which in turn targets HOXA10 to regulate its expression (P < 0.05). In conclusion, regulations of lncRNA H19 and HOXA10 expression contribute to enhancing endometrial receptivity and facilitating decidualization of endometrial stromal cells, ultimately promoting successful embryo implantation.

Dysregulation of endometrial stromal serotonin homeostasis leading to abnormal phosphatidylcholine metabolism impairs decidualization in patients with recurrent implantation failure.

Does abnormal serotonin homeostasis contribute to impaired endometrial decidualization in patients with recurrent implantation failure (RIF)? Abnormal serotonin homeostasis in patients with RIF, which is accompanied by decreased monoamine oxidase (MAO) expression, affects the decidualization of endometrial stromal cells and leads to embryo implantation failure. Previous studies have indicated that the expression of MAO, which metabolizes serotonin, is reduced in the endometrium of patients with RIF, and serotonin can induce disruption of implantation in rats. However, whether abnormal serotonin homeostasis leads to impaired decidualization in patients with RIF and, if so, the mechanism involved, remains unclear. Endometrial samples from 25 patients with RIF and 25 fertile patients were used to investigate the expression levels of monoamine oxidase A (MAOA), monoamine oxidase B (MAOB), and serotonin. We isolated human endometrial stromal cells to investigate the role of MAOA, MAOB, and serotonin in inducing decidualization in vitro and further explored the underlying mechanism using RNA-sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC/MS) analyses. The levels of serotonin in the endometrium of patients with RIF were detected by ELISA and immunohistofluorescence, and the key genes involved in abnormal serotonin metabolism were analyzed via combination with single-cell sequencing data. The effects of MAOA or MAOB on the decidualization of stromal cells were investigated using an in vitro human endometrial stromal cell-induced decidualization model and a mouse artificially induced decidualization model. The potential mechanisms by which MAOA and MAOB regulate decidualization were explored by RNA-seq and LC/MS analysis. We found that women with RIF have abnormal serotonin metabolism in the endometrium and attenuated MAO in endometrial stromal cells. Endometrial decidualization was accompanied by increased MAO in vivo and in vitro. However attenuated MAO caused an increased local serotonin content in the endometrium, impairing stromal cell decidualization. RNA-seq and LC/MS analyses showed that abnormal lipid metabolism, especially phosphatidylcholine metabolism, was involved in the defective decidualization caused by MAO deficiency. Furthermore, decidualization defects were rescued by phosphatidylcholine supplementation. RNA-seq information and raw data can be found at NCBI Bioproject number PRJNA892255. This study revealed that impaired serotonin metabolic homeostasis and abnormally reduced MAO expression were among the reasons for RIF. However, the source and other potential functions of serotonin in the endometrium remain to be further explored. This study provides new insights into the mechanisms of serotonin homeostasis in human endometrial decidualization and new biomarkers or targets for the treatment of patients with RIF. X. Sheng is supported by grants from the National Natural Science Foundation of China (82001629), the Wenzhou Basic Public Welfare Research Project (Y20240030), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116), and Jiangsu Province Postdoctoral Research Funding (2021K277B). H.S. is supported by grants from the National Natural Science Foundation of China (82030040). G.Y. is supported by grants from the National Natural Science Foundation of China (82171653). The authors declare no conflicts of interest.

METTL3-dependent m6A modification facilitates decreased endometrial decidualization via attenuation of MET in endometriosis.

Failure to induce mesenchymal-epithelial transition (MET) during stromal cell decidualization can lead to consequences such as impaired fertility in patients with endometriosis. METTL3-mediated m6A modification plays an important role in attenuating MET and defective decidualization of endometrial stromal cells and contributes to the development of reduced endometrial receptivity in endometriosis. Mesenchymal-epithelial transition (MET)-mediated endometrial decidualization is pivotal for achieving endometrial receptivity and successful pregnancy. We observed blockade of MET in the eutopic secretory endometrium of patients with endometriosis, but the underlying mechanism is unknown. In this study, real-time PCR was used to detect PRL and IGFBP1 expression, whereas western blotting was used to detect the expression of MET markers and METTL3. Phalloidin staining was used to identify changes in cell morphology. M6A levels were quantified using a colorimetric method and m6A dot blots, and functional analysis was performed using spheroid adhesion assays. We first found that increased E-cadherin expression was accompanied by decreased vimentin and Slug expression in the eutopic secretory endometrium of individuals with endometriosis. We also detected a significant increase in both the m6A level and the expression of the related methyltransferase METTL3. Finally, METTL3 expression was negatively correlated with PRL, IGFBP1, and MET markers expression. Collectively, our findings suggest that METTL3 mediates m6A modification, thereby inhibiting MET formation within the eutopic secretory endometrium of patients with endometriosis. Increased METTL3-mediated m6A modification plays a crucial role in attenuating MET formation and decidualization impairment in endometrial stromal cells, ultimately contributing to compromised endometrial receptivity in individuals with endometriosis. These insights could lead to the identification of potential therapeutic targets for improving both endometrial receptivity and pregnancy rate among individuals affected by endometriosis.

Yes Associated Transcriptional Regulator 1 (YAP1) and WW Domain Containing Transcription Regulator (WWTR1) are required for murine pregnancy initiation.

Endometrial stromal cell decidualization is required for pregnancy success. Although this process is integral to fertility, many of the intricate molecular mechanisms contributing to decidualization remain undefined. One pathway that has been implicated in endometrial stromal cell decidualization in humans in vitro is the Hippo signaling pathway. Two previously conducted studies showed that the effectors of the Hippo signaling pathway, YAP1 and WWTR1, were required for decidualization of primary stromal cells in culture. To investigate the in vivo role of YAP1 and WWTR1 in decidualization and pregnancy initiation, we generated a Progesterone Cre mediated partial double knockout (pdKO) of Yap1 and Wwtr1. Female pdKOs exhibited subfertility, a compromised decidualization response, partial interruption in embryo transport, blunted endometrial receptivity, delayed implantation and subsequent embryonic development, and a unique transcriptional profile. Bulk mRNA sequencing revealed aberrant maternal remodeling evidenced by significant alterations in extracellular matrix proteins at 7.5 days post-coitus in pdKO dams and enrichment for terms associated with fertility-compromising diseases like pre-eclampsia and endometriosis. Our results indicate a required role for YAP1 and WWTR1 for successful mammalian uterine function and pregnancy success.

Lysosome dynamics during human endometrial stromal cells decidualization: effect of para-nonylphenol.

During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the pre-implantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers Connexin-43, insulin like growth factor binding protein 1 (IGFBP1) and Prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on ECM remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of Prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy.

Long noncoding RNA BMPR1B-AS1 stability regulated by IGF2BP2 affects the decidualization in endometriosis patients through the SMAD1/5/9 pathway.

Endometriosis (EMs)-related infertility commonly has decreased endometrial receptivity and normal decidualization is the basis for establishing and maintaining endometrial receptivity. However, the potential molecular regulatory mechanisms of impaired endometrial decidualization in patients with EMs have not been fully clarified. We confirmed the existence of reduced endometrial receptivity in patients with EMs by scanning electron microscopy and quantitative real-time PCR. Here we identified an lncRNA, named BMPR1B-AS1, which is significantly downregulated in eutopic endometrium in EMs patients and plays an essential role in decidual formation. Furthermore, RNA pull-down, mass spectrometry, RNA immunoprecipitation, and rescue analyses revealed that BMPR1B-AS1 positively regulates decidual formation through interaction with the RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Downregulation of IGF2BP2 led to a decreased stability of BMPR1B-AS1 and inhibition of activation of the SMAD1/5/9 pathway, an inhibitory effect which diminished decidualization in human endometrial stromal cells (hESCs) decidualization. In conclusion, our identified a novel regulatory mechanism in which the IGF2BP2-BMPR1B-AS1-SMAD1/5/9 axis plays a key role in the regulation of decidualization, providing insights into the potential link between abnormal decidualization and infertility in patients with EMs, which will be of clinical significance for the management and treatment of infertility in patients with EMs.

MiR-135a-5p regulates window of implantation by suppressing pinopodes development and decidualization of endometrial stromal cells.

The window of implantation (WOI) is a brief period during which the endometrium is receptive to embryo implantation. This study investigated the relationship between miR-135a-5p and endometrial receptivity. Peripheral blood was collected on the day of ovulation and the 5th day after ovulation for high-throughput sequencing from women who achieved clinical pregnancy through natural cycle frozen embryo transfer. RT-qPCR assessed miR-135a-5p expression in the endometrium tissue or cells during the mouse implantation window or decidualization. Scanning electron microscopy was utilized to observe pinopode morphology and quantity in mice overexpressing miR-135a-5p during the WOI. Human endometrial stromal cells (HESC) and artificial induction of mouse uterine decidualization were used to explore whether miR-135a-5p overexpression inhibits decidualization by regulating HOXA10 and BMPR2. Furthermore, the impact of miR-135a-5p on HESC proliferation and HTR8/SVneo invasion was explored. A total of 54 women were enrolled in the study. bioinformatics analysis and animal models demonstrated that miR-135a-5p was significantly downregulated during the WOI, and its high expression can lead to abnormal pregnancy outcomes. Overexpression of miR-135a-5p resulted in the absence of pinopode in mouse endometrial tissue during the WOI. High miR-135a-5p levels were found to potentially inhibit endometrial tissue decidualization by downregulating HOXA10 and BMPR2 expression. Finally, CEBPD was identified as a potential regulator of miR-135a-5p, which would explain the decreased miR-135a-5p expression during the WOI. MiR-135a-5p expression is significantly downregulated during the WOI. High miR-135a-5p levels suppress pinopode development and endometrial tissue decidualization through HOXA10 and BMPR2, contributing to inadequate endometrial receptivity.

Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor.

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-β estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy.

Dysregulation of CREB5 Impairs Decidualization and Maternal-Fetal Interactions by Inhibiting Autophagy in Recurrent Spontaneous Abortion.

Clinically, recurrent spontaneous abortion (RSA) is a pregnancy illness that is difficult to treat. Impaired decidualization is a documented cause of RSA, but the etiology and mechanism are still unknown. cAMP-responsive element binding protein 5 (CREB5) is a member of the ATF/CREB family. CREB5 has been reported to be related to pathological pregnancy, but there are few related studies on this topic in patients with RSA, and the underlying mechanism is unclear. We collected decidual tissues from RSA patients and healthy pregnant women to measure the expression level of CREB5, PRL, IGFBP1, ATG5, LC3B, and SQSTM/p62. Then, the changes in CREB5 expression and autophagy levels were measured in human endometrial stromal cells (hESCs) during decidualization. The expression levels of PRL and IGFBP1 were tested in sh-CREB5/ov-CREB5 hESCs after decidualization induction, and the autophagy level in sh-CREB5/ov-CREB5 hESCs was measured without decidualization induction. The decidualization ability of sh-CREB5 and ov-CREB5 hESCs treated with an autophagy inducer or inhibitor was measured. To investigate the effect of CREB5 in hESCs on the invasion and migration of HTR8/SVneo cells, we performed a coculture experiment. Finally, we examined the expression of CREB5 and autophagy key proteins in mouse decidual tissues by constructing an abortion mouse model. In our study, we found that the expression of CREB5 was unusually elevated in the uterine decidua of RSA patients, but the expression of PRL, IGFBP1, and autophagy were decreased. During the decidualization of hESCs, the expression of CREB5 gradually decreases in a time-dependent manner with increasing autophagy. Moreover, by knocking down or overexpressing CREB5 in hESCs, it was found that CREB5 can impair decidualization and reduce autophagy in hESCs. Furthermore, the damage caused by CREB5 in terms of decidualization can be reversed by the addition of an autophagy inducer (rapamycin). In addition, CREB5 can increase the secretion of proteins (IL-1β and TGF-β1) in hESCs to inhibit trophoblast invasion and migration. Our data support the supposition that CREB5 disturbs the decidualization of endometrial stromal cells and interactions at the maternal-fetal interface by inhibiting autophagy and that its abnormal upregulation and dysfunction may lead to RSA. It may function as a diagnostic and therapeutic target for RSA. Similarly, we found that in the spontaneous abortion mouse model, the expression of CREB5 in the decidual tissue of the abortion group was significantly increased, and autophagy was decreased.

The involvement of RNA N6-methyladenosine and histone methylation modification in decidualization and endometriosis-associated infertility.

Defective decidualization of endometrial stromal cells (ESCs) in endometriosis (EM) patients leads to inadequate endometrial receptivity and EM-associated infertility. Hypoxia is an inevitable pathological process of EM and participates in deficient decidualization of the eutopic secretory endometrium. Enhancer of zeste homology 2 (EZH2) is a methyltransferase which catalyses H3K27Me3, leading to decreased expression levels of target genes. Although EZH2 expression is low under normal decidualization, it is abundantly increased in the eutopic secretory endometrium of EM and is induced by hypoxia. Chromatin immunoprecipitation-PCR results revealed that decidua marker IGFBP1 is a direct target of EZH2, partially explaining the increased levels of histone methylation modification in defected decidualization of EM. To mechanism controlling this, we examined the effects of hypoxia on EZH2 and decidualization. EZH2 mRNA showed decreased m6 A modification and increased expression levels under hypoxia and decidualization combined treatment. Increased EZH2 expression was due to the increased expression of m6 A demethylase ALKBH5 and decreased expression of the m6 A reader protein YTHDF2. YTHDF2 directly bind to the m6 A modification site of EZH2 to promote EZH2 mRNA degradation in ESCs. Moreover, selective Ezh2 depletion in mouse ESCs increased endometrial receptivity and improved mouse fertility by up-regulating decidua marker IGFBP1 expression. This is the first report showing that YTHDF2 can act as a m6 A reader to promote decidualization by decreasing the stability of EZH2 mRNA and further increasing the expression of IGFBP1 in ESCs. Taken together, our findings highlight the critical role of EZH2/H3K27Me3 in decidualization and reveal a novel epigenetic mechanism by which hypoxia can suppress EM decidualization by decreasing the m6 A modification of EZH2 mRNA.