Decidualization Process Research Articles

Nesfatin-1 expressed in human endometrial stromal cell line (THESC) stimulates decidualization through FAK/PI3K/AKT signaling pathway

This study aimed to investigate the expression and functional role of nesfatin-1, a peptide hormone traditionally associated with appetite regulation, in the human endometrium. Specifically, we examined its presence and regulatory potential in the human endometrial stromal cell line, THESC cells, focusing on the process of endometrial decidualization, which is critical for implantation and pregnancy maintenance. We found that nesfatin-1 and its binding sites were expressed in THESC cells. Furthermore, nesfatin-1 protein expression decreased after treatment with 17β- estradiol but increased upon exposure to progesterone, indicating an influence of sexsteroid hormones on nesfatin-1 expression. Notably, administration of nesfatin-1 protein to THESC cells resulted in significant upregulation of genes associated with decidualization, such as insulin-like growth factor binding protein 1 (IGFBP1) and prolactin. In addition, our research showed that nesfatin-1 promotes decidualization through the activation of the FAK/PI3K/AKT signaling pathway. These findings underscore the central role of nesfatin-1 in endometrial decidualization, and suggest its potential utility in the development of new treatments to improve fertility and pregnancy outcomes.

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.

The landscape of N1-methyladenosine (m1A) modification in mRNA of the decidua in severe preeclampsia.

Recent discoveries in mRNA modification have highlighted N1-methyladenosine (m1A), but its role in preeclampsia (PE) pathogenesis remains unclear. In this study, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify m1A peaks and the expression profile of mRNA in the decidua of humans with early-onset PE (EPE), late-onset PE (LPE), and normal pregnancy (NP). We assessed the m1A modification patterns in preeclamptic decidua using 10 m1A modulators. Our bioinformatic analysis focused on differentially methylated mRNAs (DMGs) and differentially expressed mRNAs (DEGs) in pairwise comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE, as well as m1A-related DEGs. The comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE identified 3110, 2801, and 2818 DMGs, respectively. We discerned three different m1A modification patterns from this data. Further analysis revealed that key PE-related DMGs and m1A-related DEGs predominantly influence signaling pathways critical for decidualization, including cAMP, MAPK, PI3K-Akt, Notch, and TGF-β pathways. Additionally, these modifications impact pathways related to vascular smooth muscle contraction, estrogen signaling, and relaxin signaling, contributing to vascular dysfunction. Our findings demonstrate that preeclamptic decidua exhibits unique mRNA m1A modification patterns and gene expression profiles that significantly alter signaling pathways essential for both decidualization and vascular dysfunction. These differences in m1A modification patterns provide valuable insights into the molecular mechanisms influencing the decidualization process and vascular function in the pathogenesis of PE. These m1A modification regulators could potentially serve as potent biomarkers or therapeutic targets for PE, warranting further investigation.

Clusterin from endometrial glands plays a critical role in decidualization via Trem2

BackgroundDecidualization is a critical step in establishing pregnancy in mammals. Successful decidualization depends on intricate gland-stromal crosstalk. Clusterin (Clu) is a ubiquitously secreted protein in physiological fluids that is involved in numerous physiological functions. However, the role of Clu in decidualization is not fully understood.ResultsIn this study, we examined the expression pattern of Clu during early pregnancy in mice and explored its potential function in decidualization. Our results revealed that Clu was expressed in the uterine glands on Days 1–2 of early pregnancy and on Days 5–8 during decidualization after embryo implantation, as well as in glands at the interimplantation site. Additionally, ovariectomized mice exhibited significant upregulation of Clu expression in the uterine glands 3 h after in vivo estrogen injection. Trem2, a receptor for Clu, was detected in the decidual region of mice on Days 5–8 of early pregnancy, where it mediates Clu to regulate the decidual region. Furthermore, we observed that recombinant CLU protein increased the expression of the decidualization marker molecules insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) in decidual cells. However, this upregulation was not observed when Trem2 expression was inhibited with siRNA.ConclusionsUterine gland-derived Clu, a new paracrine modulator, may participate in early pregnancy by influencing the decidualization process mediated by Trem2 in mice.

Benzo(a)pyrene promotes autophagy to impair endometrial decidualization via inhibiting CXCL12/CXCR4 axis

Benzo(a)pyrene (BaP), a pervasive environmental pollutant with endocrine-disrupting properties, has been associated with detrimental effects on pregnancy. During early pregnancy, the endometrial decidualization process is critical for embryo implantation. Abnormal decidualization can lead to implantation failure, aberrant placental formation, and pregnancy loss. We previously revealed that BaP exposure impaired decidualization and implantation in mice, yet the underlying mechanisms remained elusive. Autophagy, a cellular mechanism pivotal for energy and material recycling, contributes to the decidualization process. The chemokine C-X-C motif chemokine ligand 12 (CXCL12), secreted by endometrium stromal cells (ESCs), is involved in regulating endometrial decidualization and autophagy. Therefore, this study aimed to explore the hypothesis that BaP disrupts the decidualization process by interfering with autophagic pathways via the CXCL12/CXCR4 axis during early pregnancy. We found that BaP inhibited CXCL12/CXCR4 expression, and induced autophagy by promoting autophagosome formation, which in turn impaired the decidualization in early pregnant mice uterus and decidual stromal cells (DSCs). Using autophagy inhibitors 3-methyladenine and chloroquine in combination with BaP to treat DSCs, successfully weakened BaP-induced autophagy, and relieved decidual injury. Additionally, activation of CXCL12/CXCR4 by recombinant protein CXCL12 attenuated BaP-induced autophagy, inhibited the PI3K/AKT signal activation caused by BaP, and partly rescued the expression of decidualization-related genes. In summary, this study demonstrates that BaP induces autophagy in DSCs by inhibiting the CXCL12/CXCR4 axis, leading to damage in endometrial decidualization during early pregnancy. The findings provide a critical chemokine-mediated regulatory mechanism involved in embryo implantation and contribute valuable knowledge to the reproductive toxicology of BaP.

Early pregnancy exposure to Microcystin-LR compromises endometrial decidualization in mice via the PI3K/AKT/FOXO1 signaling pathway

Previous experimental studies have found that exposure to Microcystin-leucine arginine can impact pregnancy outcomes in female mice. The impact of MC-LR on early pregnancy in mammals is not yet well understood. Both mice and humans need to undergo decidualization to maintain pregnancy. In this study, we tried to evaluate whether MC-LR affects decidualization process in mice. Our research showed that MC-LR decreased maternal weight gain, uterine weight, and implantation site weight. These findings suggested that MC-LR exerted adverse effects on decidualization. In mice, we examined decreased number of polyploid decidual cells, but marked proliferation of mouse endometrial stromal cells the expression levels of prolactin (PRL)and insulin-like growth factor binding protein 1 (IGFBP1) were significantly downregulated in the decidual tissue and primary endometrial stromal cells following MC-LR treatment. Furthermore, further in vitro experiments identified that MC-LR promoted endometrial stromal cell division and cycle transition. Lastly, our study demonstrated that MC-LR impaired decidualization through the PI3K/AKT/FOXO1 pathway. Collectively, these data suggested that exposure to MC-LR impaired decidualization during early pregnancy.

Metabolic reprogramming and heterogeneity during the decidualization process of endometrial stromal cells

The human endometrial decidualization is a transformative event in the pregnant uterus that involves the differentiation of stromal cells into decidual cells. While crucial to the establishment of a successful pregnancy, the metabolic characteristics of decidual cells in vivo remain largely unexplored. Here, we integrated the single-cell RNA sequencing (scRNA-seq) datasets on the endometrium of the menstrual cycle and the maternal-fetal interface in the first trimester to comprehensively decrypt the metabolic characteristics of stromal fibroblast cells. Our results revealed that the differentiation of stromal cells into decidual cells is accompanied by increased amino acid and sphingolipid metabolism. Furthermore, metabolic heterogeneity exists in decidual cells with differentiation maturity disparities. Decidual cells with high metabolism exhibit higher cellular activity and show a strong propensity for signaling. In addition, significant metabolic reprogramming in amino acids and lipids also occurs during the transition from non-pregnancy to pregnancy in the uteri of pigs, cattle, and mice. Our analysis provides comprehensive insights into the dynamic landscape of stromal fibroblast cell metabolism, contributing to our understanding of the metabolism at the molecular dynamics underlying the decidualization process in the human endometrium.

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-440 Idiopathic infertility does not increase rates of placental abnormalities among singleton pregnancies conceived with either in-vitro fertilization (IVF) or ovulation induction±intrauterine insemination (OI±IUI)

Abstract Study question Is there an adverse impact of idiopathic infertility diagnosis on placental pathology among singleton pregnancies conceived with fertility treatments? Summary answer Idiopathic infertility does not increase rates of placental abnormalities and exhibits distinctive associations within the inflammatory and vascular spectrum of placental pathology. What is known already Pregnancies resulting from fertility treatments are often considered to be at higher risk for placenta-mediated obstetric complications and subsequent adverse perinatal outcomes. Treatment induced hormonal changes altering the endometrial milieu, along with embryo manipulation and culture conditions, may impact the processes of implantation, decidualization, trophoblast invasion, and placental vascularization. Nevertheless, the impact of infertility diagnoses on placental pathology among singleton pregnancies conceived with fertility treatments has not been elucidated, and whether idiopathic infertility alters the risk of placental abnormalities remains unknown. Study design, size, duration Retrospective review of placental pathology data from 1205 singleton livebirths conceived with fertility treatments (899 IVF, and 306 OI±IUI cycles) between 01/2004 and 04/2022. Placenta pathology was reviewed by one expert pathologist and classified as anatomic, inflammatory, infectious, and vascular [including any features of fetal (FVM) or maternal (MVM) vascular malperfusion], using the Amsterdam Workshop Consensus definitions. Placental abnormalities were compared between idiopathic infertility (IdI, n: 269) and all other, non-idiopathic infertility (non-IdI, n:936) diagnoses. Participants/materials, setting, methods Primary outcomes: anatomic, inflammatory, infectious, and vascular placental abnormalities. Parametric, and non-parametric tests were used as appropriately; odds ratios (OR) with 95% confidence intervals (95%CI) were assessed through logistic regression, adjusting for maternal age, body mass index (BMI, kg/m2), race, parity, gestational age, neonatal gender, treatment type, gestational diabetes, and hypertensive disorders. Analyses were further stratified by OI±IUI vs IVF treatments [the latter further stratified into fresh and frozen embryo transfers (FET)]. Main results and the role of chance Mean age, BMI, and AMH did not differ between groups, and neither did placental weight at term. Overall, unadjusted rates of inflammatory, infectious, and vascular abnormalities did not differ between groups (12.6% vs 11.4%, p 0.568; 22.0% vs 19.1%, p 0.256; 54.5% vs 58.6%, p 0.186, respectively). However, a higher rate of anatomic abnormalities was noted among IdI patients (32.8% vs 27.1%, p 0.044, IdI vs. non-IdI, respectively). When adjusting for potential confounders, no differences were noted between groups regarding inflammatory, infectious, and vascular abnormalities, and the observed difference in anatomic abnormalities lost its significance [adjOR(95%CI); inflammatory: 0.98(0.94-1.03), infectious: 1.03(0.98-1.09), vascular: 0.96(0.90-1.02), and anatomic: 1.02(0.96-1.08), non-IdI: ref]. Similarly, when separating OI±IUI from FET and fresh IVF cycles, adjOR(95%CI) revealed no differences in anatomic, infectious, and vascular abnormalities between groups [OI±IUI: 0.97(0.85-1.1), 0.97(0.87-1.08), 0.99(0.87-1.12); FET: 1.08(0.94-1.24), 1.07(0.94-1.23), 1.04(0.98-1.11) and, fresh IVF: 1.06(0.98-1.15), 1.04(0.96-1.13), 0.96(0.87-1.07), for anatomic, infectious, and vascular, respectively]. However, lower odds of inflammatory abnormalities were noted among fresh IVF but not among FET or OI±IUI cycles in IdI [adjOR (95%CI); fresh IVF: 0.95(0.91-0.99), FET: 1.09(0.98-1.21), OI±IUI: 0.97(0.87-1.08), non-IdI: ref]. Interestingly, among vascular abnormalities studied in the programmed FET cycles, MVM showed a difference between the groups [adjOR(95%CI) 0.90(0.85-0.96), non-IdI: ref]. Limitations, reasons for caution This study is limited by its retrospective design. The reported findings are from an infertile population undergoing treatments and thus not easily generalizable to natural conceptions. Other factors, such as environmental, nutritional, and lifestyle, might alter a patient’s individual pregnancy risk for placental abnormalities. Wider implications of the findings Overall, idiopathic infertility does not appear to increase the risk for placental abnormalities. However, it may impact placental pathology in a unique way, within the vascular and inflammatory spectrum, which might differ from that of other infertility diagnoses, and might be further altered by the treatment protocol. Trial registration number Not Applicable

Embryo Quality Conditions the Secretory Profile of Tolerogenic Dendritic Cell DC-10 During the Peri-Implantation Period.

The decidualization process conditions monocytes to the immunosuppressive and tolerogenic dendritic cell (DC)-10 profile, a DC subset with high IL-10 production. Since the implantation process implies an embryo-endometrium-immune crosstalk, here we focused on the ability of embryonic soluble factors to modify decidual DC conditioning accordingly with its quality. Human endometrial stromal cell line (HESC) decidualized with medroxyprogesterone and dibutyryl-cAMP (Dec) was stimulated with human embryo-conditioned media (ECM), classified as normal (ND) or impaired developed (ID) for 48h (n = 18/group). Monocytes isolated from six healthy women were differentiated to DCs with rhGM-CSF+rhIL-4 in the presence/absence of conditioned media (CM) from decidualized cells stimulated with ECM or nontreated. We found that decidualized cells stimulated with ECM sustain a myeloid regulatory cell profile on monocyte-derived culture with increased frequency of CD1a-CD14+ and CD83+CD86low cells. ND-Dec sustained the higher expression of the DC-10 markers, HLA-G and IL-10 whereas ID-Dec diminished IL-10 production (ID-Dec: 135 ± 37.4vs. Dec: 223.3 ± 49.9 pg/mL, p<0.05). The treatment with ECM-Dec sustained a higher IL-10 production and prevented the increase of CD83/CD86 after LPS challenge regardless of embryo quality. Notably, TNF-α production increased in ID-Dec cultures (ID-Dec: 475.1 ± 134.7vs. Dec: 347.5 ± 98 pg/mL, p<0.05). Although remaining in a tolerogenic profile compatible with DC-10, DCs can differentially respond to decidual secreted factors based on embryo quality, changing their secretome. These results suggest that in the presence of arrested embryo, DCs could differentially shape the immunological microenvironment, contributing to arrested embryo clearance during the menstrual phase.

The downregulation of NSUN5 may contribute to preeclampsia†.

Preeclampsia (PE) is a complication of pregnancy characterized by the new onset of hypertension after 20weeks of gestation. The incidence of PE is steadily rising, posing a significant threat to the lives of both the pregnant woman and the fetus. Most studies on PE pathogenesis currently focus on the placenta, but maternal decidualization forms the foundation for placental growth and development. Recent studies have shown that impaired decidualization is also a cause of PE. Decidualization is a process where endometrial stromal cells gradually transform into secretory decidual cells during early pregnancy. While NSUN5 encodes a member of a conserved family of proteins, its role in pregnancy remains unknown. In this study, we conducted experiments and observed a significant downregulation of NSUN5 expression in severe PE decidual tissues compared to those of normal pregnant women. When inducing decidualization in vitro, we found an increase in NSUN5 expression. However, when we used siRNA to knockdown NSUN5 expression, the process of decidualization was prevented. Moreover, we observed a decrease in ATP content during both cell decidualization and after knockdown of NSUN5. Finally, through immunoprecipitation combined with mass spectrometry, we discovered that the protein ATP5B interacts with NSUN5. Furthermore, after knocking down ATP5B using siRNA, we observed impaired decidualization. Moreover, transfection with siRNA to suppress NSUN5 resulted in a decrease in ATP5B expression. These significant findings provide strong evidence that NSUN5 plays a crucial role in decidualization and is closely associated with the development of PE through its interaction with ATP5B.

Endometrial decidualization status modulates endometrial microvascular complexity and trophoblast outgrowth in gelatin methacryloyl hydrogels

The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown during the menstrual cycle and pregnancy. Decidualization is an endometrial differentiation process driven by steroidal sex hormones that is critical for blastocyst-uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked to decidualization processes. However, much remains unknown regarding the role of decidualization and reciprocal trophoblast-endometrial interactions on endometrial angiogenesis and trophoblast invasion. Here, we report an engineered endometrial microvascular network embedded in gelatin hydrogels that displays morphological and functional patterns of decidualization. Vessel complexity and biomolecule secretion are sensitive to decidualization and affect trophoblast motility, but that signaling between endometrial and trophoblast cells was not bi-directional. Although endometrial microvascular network decidualization status influences trophoblast cells, trophoblast cells did not induce structural changes in the endometrial microvascular networks. These findings add to a growing literature that the endometrium has biological agency at the uterine-trophoblast interface during implantation. Finally, we form a stratified endometrial tri-culture model, combining engineered microvascular networks with epithelial cells. These endometrial microvascular networks provide a well-characterized platform to investigate dynamic changes in angiogenesis in response to pathological and physiological endometrial states.

Decreased thrombospondin-1 impairs endometrial stromal decidualization in unexplained recurrent spontaneous abortion†.

Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications.

Notch1/Akt/Foxo1 Pathway Regulated by Kisspeptin Is Involved in Endometrial Decidualization in Patients With Recurrent Spontaneous Abortion

Kisspeptin, a protein encoded by the KISS1 gene, functions as an essential factor in suppressing tumor growth. The intricate orchestration of cellular processes such as proliferation and differentiation is governed by the Notch1/Akt/Foxo1 signaling pathway, which assumes a central role in maintaining cellular homeostasis. In the specific context of this investigation, the focal point lies in a meticulous exploration of the intricate mechanisms underlying the regulatory effect of kisspeptin on the process of endometrial decidualization. This investigation delves into the interplay between kisspeptin and the Notch1/Akt/Foxo1 signaling pathway, aiming to elucidate its significance in the pathophysiology of recurrent spontaneous abortion (RSA). We enrolled a cohort comprising 45 individuals diagnosed with RSA, who were admitted to the outpatient clinic of the Reproductive Center at the Second Affiliated Hospital of Soochow University between June 2020 and December 2020. On the other hand, an additional group of 50 women undergoing elective abortion at the outpatient clinic of the Family Planning Department during the same timeframe was also included. To comprehensively assess the molecular landscape, Western blot and RT-qPCR were performed to analyze the expression levels of kisspeptin (and its gene KISS1), IGFBP1 (an established marker of decidualization), Notch1, Akt, and Foxo1 within the decidua. Human endometrial stromal cells (hESC) were given targeted interventions, including treatment with siRNA to disrupt KISS1 or exposure to kisspeptin10 (the bioactive fragment of kisspeptin), and were subsequently designated as the siKP group or the KP10 group, respectively. A control group comprised hESC was transfected with blank siRNA, and cell proliferation was meticulously evaluated with CCK8 assay. Following in vitro induction for decidualization across the three experimental groups, immunofluorescence assay was performed to identify differences in Notch1 expression and decidualization morphology between the siKP and the KP10 groups. Furthermore, RT-qPCR and Western blot were performed to gauge the expression levels of IGFBP1, Notch1, Akt, and Foxo1 across the three cell groups. Subsequently, decidualization was induced in hESC by adding inhibitors targeting Notch1, Akt, and Foxo1. The expression profiles of the aforementioned proteins and genes in the four groups were then examined, with hESC induced for decidualization without adding inhibitors serving as the normal control group. To establish murine models of normal pregnancy (NP) and RSA, CBA/J×BALB/c and CBA/J×DBA/2 mice were used. The mice were respectively labeled as the NP model and RSA model. The experimental groups received intraperitoneal injections of kisspeptin10 and kisspeptin234 (acting as a blocker) and were designated as RSA-KP10 and NP-KP234 groups. On the other hand, the control groups received intraperitoneal injections of normal saline (NS) and were referred to as RSA-NS and NP-NS groups. Each group comprised 6 mice, and uterine tissues from embryos at 9.5 days of gestation were meticulously collected for observation of embryo absorption and examination of the expression of the aforementioned proteins and genes. The analysis revealed that the expression levels of kisspeptin, IGFBP1, Notch1, Akt, and Foxo1 were significantly lower in patients diagnosed with RSA compared to those in women with NP (P<0.01 for kisspeptin and P<0.05 for IGFBP1, Notch1, Akt, and Foxo1). After the introduction of kisspeptin10 to hESC, there was an observed enhancement in decidualization capability. Subsequently, the expression levels of Notch1, Akt, and Foxo1 showed an increase, but they decreased after interference with KISS1. Through immunofluorescence analysis, it was observed that proliferative hESC displayed a slender morphology, but they transitioned to a rounder and larger morphology post-decidualization. Concurrently, the expression of Notch1 increased, suggesting enhanced decidualization upon the administration of kisspeptin10, but the expression decreased after interference with KISS1. Further experimentation involved treating hESC with inhibitors specific to Notch1, Akt, and Foxo1 separately, revealing a regulatory sequence of Notch1/Akt/Foxo1 (P<0.05). In comparison to the NS group, NP mice administered with kisspeptin234 exhibited increased fetal absorption rates (P<0.001) and decreased expression of IGFBP1, Notch1, Akt, and Foxo1 (P<0.05). Conversely, RSA mice administered with kisspeptin10 demonstrated decreased fetal absorption rates (P<0.001) and increased expression levels of the aforementioned molecules (P<0.05). It is suggested that kisspeptin might exert its regulatory influence on the process of decidualization through the modulation of the Notch1/Akt/Foxo1 signaling cascade. A down-regulation of the expression levels of kisspeptin could result in suboptimal decidualization, which in turn might contribute to the development or progression of RSA.

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.

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Matrix Metalloproteinase-9 in the Etiopathogenesis of Placenta Accreta Spectrum: A Literature Review

Abstract The recent increase in placenta accreta spectrum has been correlated with a rise in the rate of cesarean sections. A recent study provides evidence that hampered wound healing results in cesarean scar defects that lead to a failure in the normal process of decidualization and deeper adherence of trophoblasts. Matrix metalloproteinase (MMP) is crucial in every step of wound healing as it alters the wound matrix, facilitating cell migration, as well as tissue remodeling. MMP-9 expression is higher in placental and decidual tissue in cases of placenta accreta. Based on these findings, assessment of MMP-9 expression can shed new light on the etiopathology of placenta accreta spectrum disorder and can be a potential diagnostic marker.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

The Multiple Functions of HB-EGF in Female Reproduction and Related Cancer: Molecular Mechanisms and Targeting Strategies.

Heparin-binding growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) ligand family which has a crucial role in women's health. However, there is a lack of comprehensive review to summarize the significance of HB-EGF. Therefore, this work first described the expression patterns of HB-EGF in the endometrium and ovaryof different species and gestational time. Then, the focus was on exploring how it promotes the successful implantation and regulates the process of decidualization and the function of ovarian granulosa cells as an intermediate molecule. Otherwise, we also focused on the clinical and prognostic significance of HB-EGF in female-related cancers (including ovarian cancer, cervical cancer, and endometrial cancer) and breast cancer. Lastly, the article also summarizes the current drugs targeting HB-EGF in the treatment of ovarian cancer and breast cancer. Overall, these studies found that the expression of HB-EGF in the endometrium is spatiotemporal and species-specific. And it mediates the dialogue between the blastocyst and endometrium, promoting synchronous development of the blastocyst and endometrium as an intermediate molecule. HB-EGF may serve as a potentially valuable prognostic clinical indicator in tumors. And the specific inhibitor of HB-EGF (CRM197) has a certain anti-tumor ability, which can exert synergistic anti-tumor effects with conventional chemotherapy drugs. However, it also suggests that more research is needed in the future to elucidate its specific mechanisms and to accommodate clinical studies with a larger sample size to clarify its clinical value.