Decidualization Of Endometrial Stromal Cells Research Articles

#123 : Caveolin-1 Function and Regulation in Mouse Uterus During Early Pregnancy and Under Human In Vitro Decidualization

Background and Aims: Endometrial decidualization is a step in establishing and maintaining pregnancy. Senescence was considered to accompany with decidualization. Failure to clear senescence during decidualization could cause pregnancy abnormalities. Caveolin-1, as a scaffold protein, play a role in senescence, but the expression, regulation and function were not very clear in uterine endometrium. Here, we investigated the profile of Caveolin-1 in mouse and human decidualization to tease out the potential relationship among the Caveolin-1, senescence and decidualization. Method: ICR strain mice and immortalized human endometrial stromal cells (ESCs) were used. Immunofluorescence, Western-Blot, Real-time PCR were used to detect the levels of molecules. Results: In endometrium of mice during early pregnancy, the expression of Caveolin-1 gradually decreased in stroma and epithelium before embryo implantation. However, the level of Caveolin-1 converts in stromal cell after implantation. Estrogen could stimulate expression of Caveolin-1 in epithelium and progesterone could up-regulate level of Caveolin-1 in endometrial stromal cells. In vitro, expression of Caveolin-1 was up-regulated during decidualization in mESCs, while the level of Caveolin-1 was reduced during decidualization in hESCs. siRNA of Caveolin-1 could promote the expression of IGFBP-1 (decidualization biomarker) and senescence in hESCs, while over-expression of Caveolin-1 had the opposite results. In mESCs, Caveolin-1 seems to play a different role in decidualization, over-expression of Caveolin-1 could promote decidualization and senescence in mESCs, but siRNA depressed it. Blastocysts-derived TNF and hCG stimulated Caveolin-1 in mice and depressed it in human decidual cells, respectively. Caveolin-1 levels are also regulated by high glucose and insulin. Conclusion: Caveolin-1 play a role in ESCs decidualization, but the mode of action was different between species. A low level of Caveolin-1 will benefit for human decidualization, disorder of Caveolin-1 could impair decidualization.

EHD1 impaired decidualization of endometrial stromal cells in recurrent implantation failure: role of SENP1 in modulating progesterone receptor signalling†.

Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness and exposure to progesterone, which is an intractable clinical problem. However, the associated molecular mechanisms remain elusive. We found that EH domain containing 1 (EHD1) expression was abnormally elevated in RIF and linked to aberrant endometrial decidualization. Here we show that EHD1 overexpressed in human endometrial stromal cells significantly inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness to progesterone. No significant changes were observed in PGR mRNA levels, while a significant decrease in progesterone receptor B (PRB) protein level. Indeed, EHD1 binds to the PRB protein, with the K388 site crucial for this interaction. Overexpression of EHD1 promotes the SUMOylation and ubiquitination of PRB, leading to the degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. To establish a functional link between EHD1 and the PGR signalling pathway, sg-EHD1 were utilized to suppress EHD1 expression in HESCs from RIF patients. A significant increase in the expression of prolactin and insulin-like growth factor-binding protein 1 was detected by interfering with the EHD1. In conclusion, we demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF.

ATOH8 Expression Is Regulated by BMP2 and Plays a Key Role in Human Endometrial Stromal Cell Decidualization.

During the secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to transdifferentiate into polygonal epithelioid-like (decidual) cells. This decidualization process continues more broadly during early pregnancy, and the resulting decidual tissue supports successful embryo implantation and placental development. This study was carried out to determine if atonal basic helix-loop-helix transcription factor 8 (ATOH8) plays a role in human endometrial stromal fibroblast (ESF) decidualization. ATOH8 messenger RNA and protein expression levels significantly increased in human ESF cells undergoing in vitro decidualization, with the protein primarily localized to the nucleus. When ATOH8 expression was silenced, the ability of the cells to undergo decidualization was significantly diminished. Overexpression of ATOH8 enhanced the expression of many decidualization markers. Silencing the expression of ATOH8 reduced the expression of FZD4, FOXO1, and several known FOXO1-downstream targets during human ESF cell decidualization. Therefore, ATOH8 may be a major upstream regulator of the WNT/FZD-FOXO1 pathway, previously shown to be critical for human endometrial decidualization. Finally, we explored possible regulators of ATOH8 expression during human ESF decidualization. BMP2 significantly enhanced ATOH8 expression when cells were stimulated to undergo decidualization, while an ALK2/3 inhibitor reduced ATOH8 expression. Finally, although the steroids progesterone plus estradiol did not affect ATOH8 expression, the addition of cyclic adenosine monophosphate (cAMP) analogue alone represented the major effect of ATOH8 expression when cells were stimulated to undergo decidualization. Our results suggest that ATOH8 plays a crucial role in human ESF decidualization and that BMP2 plus cAMP are major regulators of ATOH8 expression.

DHT and Insulin Upregulate Secretion of the Soluble Decoy Receptor of IL-33 From Decidualized Endometrial Stromal Cells.

Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling.

Steroid receptor coactivator-2 drives epithelial reprogramming that enables murine embryo implantation.

Although we have shown that steroid receptor coactivator-2 (SRC-2), a member of the p160/SRC family of transcriptional coregulators, is essential for decidualization of both human and murine endometrial stromal cells, SRC-2's role in the earlier stages of the implantation process have not been adequately addressed. Using a conditional SRC-2 knockout mouse (SRC-2d/d ) in timed natural pregnancy studies, we show that endometrial SRC-2 is required for embryo attachment and adherence to the luminal epithelium. Implantation failure is associated with the persistent expression of Mucin 1 and E-cadherin on the apical surface and basolateral adherens junctions of the SRC-2d/d luminal epithelium, respectively. These findings indicate that the SRC-2d/d luminal epithelium fails to exhibit a plasma membrane transformation (PMT) state known to be required for the development of uterine receptivity. Transcriptomics demonstrated that the expression of genes involved in steroid hormone control of uterine receptivity were significantly disrupted in the SRC-2d/d endometrium as well as genes that control epithelial tight junctional biology and the emergence of the epithelial mesenchymal transition state, with the latter sharing similar biological properties with PMT. Collectively, these findings uncover a new role for endometrial SRC-2 in the induction of the luminal epithelial PMT state, which is a prerequisite for the development of uterine receptivity and early pregnancy establishment.

METTL3-regulated m6A modification impairs the decidualization of endometrial stromal cells by regulating YTHDF2-mediated degradation of FOXO1 mRNA in endometriosis-related infertility

BackgroundEndometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing research, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduction. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires further investigation.MethodsWe examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken from normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) or from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary endometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophosphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed the expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) or women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, and decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, we investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Furthermore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore the mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A.ResultsThe expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demonstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual treatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, whereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers and embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted embryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation of FOXO1 mRNA through YTHDF2, a m6A binding protein.ConclusionsMETTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular decidualization and embryo implantation. These findings provide novel insights into the development of therapies for women with endometriosis-related infertility.

USP7 promotes decidualization of ESCs by STAT3/PR axis during early pregnancy

Decidualization is an essential process for embryo implantation during early pregnancy. Defective decidualization is a critical leading cause of early pregnancy loss (EPL). Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that is involved in uterine function. This study aimed to explore the underlying mechanism by which USP7 regulates decidualization in EPL. We found that USP7 was downregulated in the decidual tissue of EPL patients. Upregulation of USP7 enhanced decidualization in endometrial stromal cells (ESCs), with increased decidualized biomarkers IGFBP1 and PRL and progesterone receptor A/B (PR-A/B) expression. Moreover, we found that signal transducer and activator of transcription 3 (STAT3) is a direct target of USP7 in ESCs. USP7 bound to STAT3 by deubiquitination and increased STAT3 levels in ESCs. Suppression of STAT3 impeded the USP7-promoted cell viability, decidualization, and PR-A/B expression of ESCs. USP7 promoted the decidualization of ESCs through the STAT3/PR signaling pathway during early pregnancy, which provides new insight into the pathological mechanism of EPL and may contribute to the clinical treatment of EPL.

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Elevated high-mannose N-glycans hamper endometrial decidualization

Decidualization of endometrial stromal cells is a hallmark of endometrial receptivity for embryo implantation, and dysfunctional decidualization is associated with pregnancy failure. Protein glycosylation is an important posttranslational modification that affects the structure and function of glycoproteins. Our results showed that high-mannose epitopes were elevated in the decidual tissues of miscarriage patients compared with early pregnant women by Lectin microarray. Furthermore, the level of mannosyl-oligosaccharide α-1,2 mannosidase IA (MAN1A1), a key enzyme for high-mannose glycan biosynthesis, was decreased in the decidual tissues of miscarriage patients. Screening of lncRNAs showed that lncNEAT1 level was increased in the serum and decidua of miscarriage patients, and negatively correlated with MAN1A1 expression. The results also revealed that specific binding of lncNEAT1 with nucleophosmin (NPM1)-SP1 transcription complex inhibited MAN1A1 expression and hampered endometrial decidualization and embryo implantation potential. The study suggests the new insights into the function of high-mannose glycans/MAN1A1 modification during endometrial decidualization.

OR09-05 GPBAR1- Understanding Its Function In The Secretory Endometrium

Abstract Disclosure: M. Saad-Naguib: None. P. Timmins: None. L. George: None. C. Samuels: None. N. Douglas: None. A.V. Babwah: None. Every successful pregnancy relies on implantation of an embryo into a receptive uterine endometrium. While implantation failure accounts for 50% of pregnancy failures, it remains the least understood aspect of pregnancy. In the mid-secretory (MS) phase of the menstrual cycle, under the influence of progesterone and cAMP, fibroblasts decidualize, a critical requirement for implantation. Decidualization involves the differentiation of stromal fibroblasts into secretory epithelioid cells that contribute to the formation of the decidua, the support of the pregnancy prior to placentation. A defective decidua can lead to implantation failure, but if implantation occurs, it could compromise placenta formation and function. Several studies have demonstrated the importance of G protein-coupled receptors (GPCRs) in mouse endometrial receptivity and decidualization. GPCRs are transmembrane signaling molecules that are ubiquitously expressed in the body and regulate almost every cellular and physiological process. They are major pharmaceutical targets, with 35% of all the drugs approved by the US FDA targeting GPCRs. To identify novel GPCR regulators of embryo implantation, we conducted bulk RNA sequencing of the human endometrium at the periovulatory (PO) and mid-secretory (MS) phase in natural menstrual cycles in healthy women of reproductive age and observed that genes encoding 11 GPCRs, not previously reported to regulate implantation, were differentially expressed between these two time points. Among these was the gene encoding the G protein-coupled bile acid receptor 1 (GPBAR1), and this was upregulated 4.6-fold in the MS vs PO phase. Single cell RNA sequence analysis showed that within the human endometrium, GPBAR1 mRNA expression is mostly and strongly localized to stromal fibroblasts. Upon activation by secondary bile acids, GPBAR1 couples to Gαs leading to an increase in intracellular cAMP levels. We hypothesize that in the MS endometrium, upon ligand binding, GPBAR1 produces cAMP and induces human endometrial stromal cell (HESC) decidualization. This hypothesis is being tested in vitro using primary HESCs. Interestingly, in the pregnant mouse, GPBAR1 mRNA is barely expressed at the implantation site on E7.5, and therefore unlikely plays a role in stromal cell decidualization in the mouse. Whether Gpbar1 is expressed in the pregnant mouse endometrium prior to implantation and plays a role in regulating endometrial receptivity is being investigated. Overall, our studies have uncovered a GPCR, that to our knowledge, has not been previously described in the human endometrium. Our data show that the expression of this GPCR is upregulated at the time of endometrial stromal cell decidualization, and therefore it might regulate decidualization and/or be required for decidual function in women. These possibilities are being examined. Presentation: Friday, June 16, 2023

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

THU633 Atonal BHLH Transcription Factor 8 (ATOH8) Functions In Decidualization

Abstract Disclosure: R. Thapa: None. B. Bany: None. ATOH8 is a member of the vast basic helix-loop-helix (bHLH) protein family that is widely known to play a role in various physiological and pathological processes. However, the functional significance of ATOH8 expression in the endometrium during implantation and in the first trimester of pregnancy in women is still lacking. In the mid to late secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to trans-differentiate into polygonal epithelioid-like (decidual) cells. During pregnancy, this process continues more broadly and supports successful embryo implantation and early placental development. We employed an established in vitro model using uterine fibroblast (HuF) cells derived from the decidua parietalis of term patients to investigate the function of ATOH8 during in vitro decidualization. All experiments were carried out using cells isolated from at least six independent patients (N=6). We measured the expression of classical decidualization marker genes and the characteristic shape change from small fibroblast-like cells to large epitheloid-like decidual cells to assess decidualization. Treating cells with steroids plus a membrane-permeable cAMP analog (EPC) for 48h caused in vitro decidualization of these cells, while treatment with vehicle alone had no effect. We discovered that ATOH8 expression was dramatically upregulated during this decidualization at both the mRNA and protein levels, with the protein primarily localized to the nucleus. The functional role of ATOH8 during decidualization was investigated using lentivirus-mediated shRNA knockdown or overexpression of ATOH8. When ATOH8 expression was silenced, we discovered that the ability of the cells to undergo decidualization was greatly and significantly diminished. On the other hand, overexpression of ATOH8 enhanced some aspects of decidualization. The addition of recombinant BMP2 alone had little effect on ATOH8 expression. However, in the presence of EPC it significantly enhanced ATOH8 expression. This effect of BMP2 on ATOH8 expression was inhibited by preincubating the cells with 500 nM DMH-1 (an ALK2/3 inhibitor) for one hr prior to inducing decidualization. Steroids alone did not affect ATOH8 or BMP2 expression. However, the addition of cAMP alone represented the major effect of EPC on ATOH8 expression while having a slight impact on BMP2 expression. A robust increase in BMP2 expression required the presence of both steroids and cAMP together. Our results indicate that ATOH8 plays a crucial role in human endometrial stromal cell decidualization. Further, our results suggest that ALK2/3 receptors are most likely involved in mediating the effect of BMP2 on increasing ATOH8 expression. Keywords: ATOH8, Decidualization, Embryo Implantation Presentation: Thursday, June 15, 2023

The effect of obesity on uterine receptivity is mediated by endometrial extracellular vesicles that control human endometrial stromal cell decidualization and trophoblast invasion

AbstractThe objectives of the present study were to determine whether obesity impacts human decidualization and the endometrial control of trophoblast invasion (both of which are required for embryo implantation) and evaluate the potential involvement of endometrial extracellular vesicles (EVs) in the regulation of these physiological processes. Using primary human cell cultures, we first demonstrated that obesity is associated with significantly lower in vitro decidualization of endometrial stromal cells (ESCs). We then showed that a trophoblastic cell line's invasive ability was greater in the presence of conditioned media from cultures of ESCs from obese women. The results of functional assays indicated that supplementation of the culture medium with EVs from nonobese women can rescue (at least in part) the defect in in vitro decidualization described in ESCs from obese women. Furthermore, exposure to endometrial EVs from obese women (vs. nonobese women) was associated with significantly greater invasive activity by HTR‐8/SVneo cells. Using mass‐spectrometry‐based quantitative proteomics, we found that EVs isolated from uterine supernatants of biopsies from obese women (vs. nonobese women) presented a molecular signature focused on cell remodelling and angiogenesis. The proteomics analysis revealed two differentially expressed proteins (fibronectin and angiotensin‐converting enzyme) that might be involved specifically in the rescue of the decidualization capacity in ESCs from obese women; both of these proteins are abundantly present in endometrial EVs from nonobese women, and both are involved in the decidualization process. In conclusion, our results provided new insights into the endometrial EVs’ pivotal role in the poor uterine receptivity observed in obese women.

P-313 Dysregulation of endometrial stromal serotonin homeostasis impairs decidualization in patients with recurrent implantation failure via phosphatidylcholine metabolism

Abstract Study question Does abnormal serotonin homeostasis contribute to impaired endometrial decidualization in patients with recurrent implantation failure (RIF)? Summary answer Abnormal serotonin homeostasis in patients with RIF, which is accompanied by decreased expression of monoamine oxidase (MAO), affects decidualization of endometrial stromal cells. What is known already Previous studies have found 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 the mechanism remains unclear. Study design, size, duration Endometrial samples from 31 patients with RIF and 31 fertile patients were used to investigate the expression levels of MAOA, 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 mechanism using RNA-seq and LC/MS analyses. Participants/materials, setting, methods The levels of serotonin in the endometrium of patients with RIF were detected by ELISA and immunohistofluorescence, and the key genes of abnormal serotonin metabolism were analyzed combined with single-cell sequencing data. The effects of MAO on the decidualization of stromal cells were investigated using human endometrial stromal cells in vitro induced decidualization model and mouse artificially induced decidualization model. The potential mechanisms of MAO regulating decidualization were explored by RNA-seq and LC/MS analysis. Main results and the role of chance 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. Attenuated MAO caused 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 MAO-deficiency induced defective decidualization. Furthermore, decidualization defects were rescued by phosphatidylcholine supplementation. Limitations, reasons for caution This study found that impaired serotonin metabolic homeostasis and abnormally reduced MAO expression were one of the reasons for repeated implantation failure. However, the source and other potential function of serotonin in the endometrium remain to be further explored. Wider implications of the findings This study shows new insights into the mechanisms of serotonin homeostasis in human endometrial decidualization and provides new biomarkers or targets for the treatment of patients with RIF. Trial registration number not applicable

P-313 Dysregulation of endometrial stromal serotonin homeostasis impairs decidualization in patients with recurrent implantation failure via phosphatidylcholine metabolism

Abstract Study question Does abnormal serotonin homeostasis contribute to impaired endometrial decidualization in patients with recurrent implantation failure (RIF)? Summary answer Abnormal serotonin homeostasis in patients with RIF, which is accompanied by decreased expression of monoamine oxidase (MAO), affects decidualization of endometrial stromal cells. What is known already Previous studies have found 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 the mechanism remains unclear. Study design, size, duration Endometrial samples from 31 patients with RIF and 31 fertile patients were used to investigate the expression levels of MAOA, 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 mechanism using RNA-seq and LC/MS analyses. Participants/materials, setting, methods The levels of serotonin in the endometrium of patients with RIF were detected by ELISA and immunohistofluorescence, and the key genes of abnormal serotonin metabolism were analyzed combined with single-cell sequencing data. The effects of MAO on the decidualization of stromal cells were investigated using human endometrial stromal cells in vitro induced decidualization model and mouse artificially induced decidualization model. The potential mechanisms of MAO regulating decidualization were explored by RNA-seq and LC/MS analysis. Main results and the role of chance 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. Attenuated MAO caused 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 MAO-deficiency induced defective decidualization. Furthermore, decidualization defects were rescued by phosphatidylcholine supplementation. Limitations, reasons for caution This study found that impaired serotonin metabolic homeostasis and abnormally reduced MAO expression were one of the reasons for repeated implantation failure. However, the source and other potential function of serotonin in the endometrium remain to be further explored. Wider implications of the findings This study shows new insights into the mechanisms of serotonin homeostasis in human endometrial decidualization and provides new biomarkers or targets for the treatment of patients with RIF. Trial registration number not applicable Study funding No Funding source Funding by national/international organization(s)

P-515 EHD1 impaired endometrial decidualization linked to recurrent implantation failure through SENP1-mediated progesterone resistance

Abstract Study question Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness to progesterone, an intractable clinical problem with elusive mechanisms. Summary answer EHD1 overexpression promoted the SUMOylation and ubiquitinated degradation of the PRB protein leading to decreased transcriptional activity and responsiveness of endometrial stromal cells to progesterone. What is known already Abnormal decidualization is a significant cause of infertility in RIF patients. A previous study has demonstrated that EH homeodomain 1 (EHD1) is significantly elevated in the endometrium of RIF patients and plays an essential role in the decidualization of human endometrial stromal cells (HESCs). However, far less is known about the function of EHD1 in the endometrium during embryo implantation. Study design, size, duration After approval from the Ethics Committee of Nanjing Drum Tower Hospital (2013-081-01), endometrial specimens were collected from women who received treatment at the Reproductive Center of Nanjing Drum Tower Hospital from January 2020 to December 2021. Twelve RIF women and Twelve fertile normal control women were enrolled in the present study. Endometrium in the middle and late stages of secretion was obtained by aspiration and curettage 5-7 days after ovulation. Participants/materials, setting, methods The expression and location of EH homeodomain 1 (EHD1) in endometrium were evaluated by IHC, qRT-PCR and Western blotting. Transcriptomic analysis was performed to predict the mechanism by which EHD1 is mediated in human endometrial stromal cells (HESCs) decidualization. Luciferase reporter assays and Western blotting were used to detect the protein activity and stability of progesterone receptor B (PRB) regulated by EHD1. The potential mechanisms were further confirmed through immunoprecipitation, nucleocytoplasmic separation and immunofluorescence experiments. Main results and the role of chance EHD1 was abnormally elevated in RIF patients and linked to aberrant endometrial decidualization. EHD1 overexpression inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness of HESCs to progesterone. Indeed, EHD1 overexpression promoted the SUMOylation of PRB, leading to ubiquitinated degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. In addition, the expression of PRL and IGFBP1 was rescued by interfering with the expression of EHD1 in HESCs from RIF patients. Limitations, reasons for caution In our study, we provided novel insight into the molecular scaffold of EHD1 to mediate the ubiquitination of PRB for degradation. However, our data do not account for the specific mechanisms by which EHD1 promotes the ubiquitination of PRB, and The specific enzymes involved remain to be elucidated. Wider implications of the findings We demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF. Trial registration number not applicable

P-517 Downregulated INHBB in endometrial tissue of recurrent implantation failure patients impeded decidualization through the ADCY1/cAMP signalling pathway

Abstract Study question To identify the mechanism of Inhibin Subunit Beta B (INHBB) in the regulation of human endometrial stromal cells (HESCs) decidualization in recurrent implantation failure (RIF). Summary answer INHBB was decreased in endometrial stromal cells of women with RIF, which attenuated decidualization in vitro by suppressing ADCY1-induced cAMP production and cAMP-mediated signalling. What is known already INHBB encodes a preprotein subunit of inhibin/ activin, functional cytokines belonging to the transforming growth factor-β (TGF-β) family. INHBB mRNAs were expressed in human decidualized endometrium from the first trimester of pregnancy and increased as the gestation progressed. Furthermore, INHBB expression increases in the mouse uterus in areas undergoing decidualization. These studies indicated that INHBB might play an essential role in endometrial decidualization. Study design, size, duration The Medical Ethics Committee of Nanjing Drum Tower Hospital approved this study (No. 2013-081-01). Written informed consent was obtained from each participant. Between January 2020 and November 2021, 28 infertile and 18 fertile control women aged 20-40 years with normal and regular menstrual cycles (25-35 days) and no history of steroid hormone medication in the last 3 months were included in the study. Participants/materials, setting, methods RNA-seq was conducted to identify the differentially expressed genes in the endometria from control and RIF patients. RT-qPCR, WB, and immunohistochemistry were performed to analyse the expression levels of INHBB in decidualised HESCs. RT-qPCR and immunofluorescence were used to detect changes in decidualization after knockdown INHBB. Then, RNA-seq was used to dig out the mechanism of INHBB regulating decidualisation. Main results and the role of chance Our results showed significantly reduced expression of INHBB in endometrial stromal cells of women with RIF. In addition, INHBB was increased in the endometrium of the secretory phase and significantly induced in in-vitro decidualization of HESCs. Notably, with RNA-seq and siRNA-mediated knockdown approaches, we demonstrated that the INHBB-ADCY1-mediated cAMP signalling pathway regulates the reduction of decidualization. We found a positive association between the expression of INHBB and ADCY1 in endometria with RIF (R2= 0.3785, P = 0.0005). Limitations, reasons for caution Our results indicated that INHBB regulates cAMP level by inducing the expression of ADCY1, while the regulation mechanism between INHBB and ADCY1 in endometrial decidualization requires further exploration. Wider implications of the findings The decline of INHBB in HESCs suppressed ADCY1-induced cAMP production and cAMP-mediated signalling, which attenuated decidualization in RIF patients, indicating that INHBB is an essential component in the decidualization process. Trial registration number not applicable

P-114 Abdominal obesity in Polycystic ovary syndrome patients can increase endometrial stromal cells decidualization

Abstract Study question Whether abdominal obesity can change the decidualization induction potential in PCOS patients? Summary answer We hypothesized that considering the inflammatory origin of abdominal obesity, it may affect decidualization potential of endometrial stromal cells (ESCs). What is known already Polycystic ovary syndrome (PCOS) as a chronic inflammatory disease is the most common hormonal disorder in women of reproductive age, which is often associated with subfertility or infertility due to ovulation disorders and subsequently low pregnancy rate, live birth rate and a high miscarriage rate. Inflammatory Abdominal obesity (AO) is the most common phenotype in PCOS patients, which has a high association with PCOS pathogenesis. Embryo-endometrium cross-talk has a key role in successful embryo implantation, which contains inflammatory phase (decidualization induction). Followed by inflammatory phase for pregnancy support. Study design, size, duration Our study population were consisting of 53 infertile women including PCOS diagnosed according to Rotterdam criteria (n = 25) and normal oogenesis women (male factor infertility) (n = 28) with in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) and frozen embryo transfer (FET) indication. All patients were aged 25-35 referred to Royan Institute between 1st May until 1st December in 2021 for infertility treatments. Participants/materials, setting, methods The embryos were subdivided into four groups: PCOS with AO, PCOS without AO, nonPCOS with AO, and nonPCOS without AO patients. The embryos from each group were single cultured up to the blastocyst stage. Their embryo condition media (ECM) were pooled and added to the culture media of healthy ESCs monolayer to investigate their effects on decidualization potential via gene and protein expression analysis and ESCs migration assay. Main results and the role of chance Results showed that ECM generally can improved decidualization capacity in ESCs. Morphological analysis, migration assay, protein and gene expressional analysis showed PCOS with AO had the highest decidualization potential and revealed by higher expression of decidualization markers (P ≤ 0.05). NonPCOS individuals without AO had the lowest level of both gene and protein decidualization markers (P ≤ 0.05). Limitations, reasons for caution Due to small sample population and the ECM volume limitation, the present study remained focused on the some main decidualization markers rather than extended study. Wider implications of the findings Inflammation may increase endometrial receptivity, allowing more suboptimal embryos to implant; this could explain the high rate of abortion in these cases. Controlling the inflammation in PCOS patients with AO may correct the decidualization profile of these patients. Trial registration number Not applicable

O-Fucosylation of BMP1 promotes endometrial decidualization by activating BMP/Smad signaling pathway.

Endometrial decidualization is critical to successful uterine receptivity and embryo implantation. Dysfunction of decidualization is associated with some pregnancy-related disorders, including miscarriage. Protein glycosylation is involved in many physiological and pathological processes. Protein O-fucosyltransferase 1 (poFUT1) is a key enzyme responsible for O-fucosylation biosynthesis on glycoproteins. Bone morphogenetic protein 1 (BMP1) is an essential glycoprotein in reproduction. However, the role and molecular mechanism of fucosylated BMP1 in endometrial stromal cell decidualization are still unknown. In the current study, we found that BMP1 contains a potential O-fucosylation site. Moreover, poFUT1 and BMP1 levels in the secretory phase are higher than those in the proliferative phase, and the highest level was observed in the human uterine tissues of early pregnancy, while a decrease of poFUT1 and BMP1 in the decidua was observed in miscarriage patients. Using human endometrial stromal cells (hESCs), we demonstrated that O-fucosylation of BMP1 was elevated after induced decidualization. Moreover, the increase of BMP1 O-fucosylation by poFUT1 promoted BMP1 secretion to the extracellular matrix, and more actively binds to CHRD. The binding of BMP1 and CHRD further released BMP4 originally bound to CHRD, and activated BMP/Smad signaling pathway, thereby accelerating the decidualization of human endometrial stromal cells. In summary, these results suggest that BMP1 O-fucosylation by poFUT1 could be a potential diagnostic and therapeutic target to predict miscarriage in early pregnancy examinations.

Stromal cell decidualization and embryo implantation: a vulnerable step leading to successful pregnancy

Endometrial stromal cell decidualization is a crucial step in endometrial remodeling during pregnancy. Decidualization is controlled by orchestrated ovarian hormones, followed by the activation of various downstream signaling pathways. Accumulating evidence has shown multiple functions of decidualized endometrial stromal cells during embryo implantation, including tissue remodeling, antioxidative stress, angiogenesis, and immune tolerance. The distinct secretomes of decidualized stromal cells also reveal their intensive interactions with epithelial, endothelial, and immune cells. However, aberrant decidualization leads to pregnancy failures, such as recurrent pregnancy loss and repeated implantation failure. This review aimed to provide an overview of the molecular mechanisms underlying the divergent functions of decidualized endometrial stromal cells and their potential clinical applications. Moreover, the use of single-cell RNA sequencing data further enhances our understanding of these biological processes. This review discusses decidualization-related signaling pathways that serve as potential therapeutic targets for treating implantation failure in in vitro fertilization and provides novel approaches to investigate the underlying causes of female infertility.