Primary Endometrial Stromal Cells Research Articles

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.

M6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

P-453 Non-autologous human platelet lysate as a therapy for recurrent implantation failure: lessons from in vitro models

Abstract Study question Which biological pathways are modulated by primary human endometrial cells in response to in vitro treatment with non-autologous human platelet lysate (HPL)? Summary answer HPL treatment stimulates endometrial growth and trophoblast attachment by activating cell proliferation, and modulating cell-cell signaling and extracellular matrix organization. What is known already Inadequate endometrial receptivity and a thin endometrium (TE) are contributing factors to recurrent implantation failure (RIF), which occurs in 10% of in vitro fertilization cycles. Studies have reported that autologous platelet-rich plasma stimulates endometrial growth and enhances receptivity, thereby improving implantation rate in RIF patients. We previously showed that non-autologous human platelet lysate (HPL) promotes in vitro proliferation of primary endometrial epithelial (EECs) and stromal cells (ESCs). Our data suggest that HPL may standardize and improve future clinical treatments for a TE and endometrial origins of RIF. Study design, size, duration Endometrial tissue was collected from five proliferative phase control and eighteen RIF patients at the CReATe Fertility Centre, Toronto, Canada. The RIF patients were separated into three groups (N = 6 each): 1) proliferative phase RIF, 2) secretory phase RIF, and 3) secretory phase RIF+TE. Primary EECs and ESCs were enzymatically isolated, cultured separately, and treated with either serum-free media (SFM), or SFM + 1% HPL (EECs), or 10% HPL (ESCs). Participants/materials, setting, methods Cell proliferation was assessed using PrestoBlueTM reagent and immunocytochemistry for Ki67. Pooled RNA libraries were prepared and sequenced for 150 cycles paired-end. Differential expression was performed using DESeq2 and pathway analysis with Enrichr. Using an in vitro model of embryo attachment, attachment between HTR-8/SVneo trophoblast spheroids and EECs was assessed by fluorescent microscopy and ImageJ™ software. Main results and the role of chance For all groups, 48-hour treatment with non-autologous HPL stimulates a significant increase (1.24-1.49-fold, P<0.05) in EEC proliferation. Whereas ESCs experienced a significant 1.29-fold and 1.92-fold increase (P<0.05) in proliferation for proliferative phase RIF and secretory phase RIF+TE respectively. HPL-treated EECs also exhibited significantly increased attachment to HTR-8 spheroids from 57.52% to 86.5% (P<0.01) in RIF+TE cultures and 42.58% to 68.90% (P<0.01) in RIF only cultures. Comparing SFM vs. HPL treatment identified 45 significantly upregulated (log2FoldChange >2 and padj <0.05) genes, including MMP1 and MMP9, and 30 significantly downregulated genes including KL and ADRA2A. Whereas ESCs had 429 significantly upregulated genes, including BUB1 and PLK1, and 378 significantly downregulated genes, including PTGIS and ADAMTS16. Upregulated pathways included extracellular matrix organization and mitotic cell cycle. Downregulated pathways included NOTCH1 signaling and synthesis of prostaglandins. Limitations, reasons for caution Our data suggests in vitro HPL treatment significantly stimulates endometrial cell proliferation, trophoblast attachment, and modulates receptivity. However, the small sample size per group (N = 6) underscores the need for larger samples and randomized controlled trials to establish the clinical efficacy of HPL as a treatment for endometrial origins of RIF. Wider implications of the findings HPL treatment for RIF and TE is gaining global acceptance by fertility clinics. Our study establishes the molecular groundwork to understand how HPL impacts endometrial growth and receptivity, offering mechanistic insights into this therapeutic approach and informing future treatments aimed at enhancing the success of embryo transfers. Trial registration number not applicable

P-309 Injectable “homing-like” bioactive short-fibers for endometrial repair and efficient live births

Abstract Study question To develop effective therapeutic interventions to promote endometrial growth and facilitate the restoration of the damaged endometrium of rats with intrauterine adhesion (IUA). Summary answer We have developed injectable “homing-like” bioactive decellularized extracellular matrix short-fibers which can achieve efficient endometrial repair and live births of rats with IUA. What is known already The prevalence of infertility caused by endometrial defects has been steadily increasing, posing a significant challenge to women’s reproductive health. Various factors, such as infection and intrauterine surgery, can lead to damage to the basal layer of the endometrium, which hinders functional endometrial repair and results in the pathological development of IUA. However, there is currently no effective clinical treatment strategy for IUA. Therefore, it is imperative to develop effective therapeutic interventions to promote endometrial growth and receptivity, ameliorate endometrial fibrosis, and facilitate the restoration of the damaged endometrium that ultimately reverses the poor pregnancy outcomes of patients with IUA. Study design, size, duration We have developed an innovative method for creating injectable acellular extracellular matrix short fibers (DEFs) from porcine skin. We investigated the effects of DEFs by in vitro and in vivo experiments over a period of two years. Participants/materials, setting, methods In vitro, we investigated the pro-proliferative, anti-fibrosis and angiogenesis effects of DEFs in human primary endometrial stromal cells (HESCs) and human umbilical vein endothelial cells (HUVECs). In vivo, we constructed a rat IUA model to investigate the promotion of endometrial repair and fertility remodeling by DFEs. Main results and the role of chance The DEFs can effectively attract endometrial cells, enabling cell adhesion, spreading, and proliferation on their surface. DEFs are rich in bioactive growth factors, which creates a nourishing microenvironment for endometrial recovery while attracting and recruiting endogenous endometrial cells for in situ regeneration. Initially, DEFs formed stable bonds with the surface of endometrial cells via electrostatic interactions after injection into the uterine cavity, which acted as a physical barrier to prevent intrauterine adhesions. Subsequently, DEFs effectively facilitated the proliferation and angiogenesis of HESCs and HUVECs, and inhibited fibrosis of pretreated HESCs. Next, DEFs could decrease endometrial collagen deposition and promote endometrial repair and endometrial angiogenesis by releasing growth factors. Therefore, bioactive DEFs could efficiently improve endometrial receptivity, promote endometrial repair, and achieve efficient live births in endometrium-injured rats. Limitations, reasons for caution The composition of DEFs is complex. The mechanism by which DEFs promotes endometrial repair remains unclear. In our future endeavors, our primary aim is to conduct a proteomic analysis of the DEFs to pinpoint the vital proteins responsible for their ability to induce tissue growth. Wider implications of the findings The DEFs can achieve efficient live births and the preparation method is suitable for mass production. In addition, the injectability of DEFs can achieve non-invasive intrauterine injection, which can reduce the risk of endometrial re-injury. Therefore, the DEFs system provides a new promising clinical strategy for endometrial factor infertility. Trial registration number enter ‘not applicable

PDLIM3 knockdown promotes ferroptosis in endometriosis progression via inducing Gli1 degradation and blocking Hedgehog signaling pathway.

Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS. PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs. Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions. Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future.

Long-Term Maintenance of Viable Human Endometrial Epithelial Cells to Analyze Estrogen and Progestin Effects.

There are fewer investigations conducted on human primary endometrial epithelial cells (HPEECs) compared to human primary endometrial stromal cells (HPESCs). One of the main reasons is the scarcity of protocols enabling prolonged epithelial cell culture. Even though it is possible to culture HPEECs in 3D over a longer period of time, it is technically demanding. In this study, we successfully established a highly pure, stable, and long-term viable human conditionally reprogrammed endometrial epithelial cell line, designated as eCRC560. These cells stained positive for epithelial markers, estrogen and progesterone receptors, and epithelial cell-cell contacts but negative for stromal and endothelial cell markers. Estradiol (ES) reduced the abundance of ZO-1 in a time- and dose-dependent manner, in contrast to the dose-dependent increase with the progestin dienogest (DNG) when co-cultured with HPESCs. Moreover, ES significantly increased cell viability, cell migration, and invasion of the eCRC560 cells; all these effects were inhibited by pretreatment with DNG. DNG withdrawal led to a significantly disrupted monolayer of eCRC560 cells in co-culture with HPESCs, yet it markedly increased the adhesion of eCRC560 to the human mesothelial MeT-5A cells. The long-term viable eCRC560 cells are suitable for in vitro analysis of HPEECs to study the epithelial compartment of the human endometrium and endometrial pathologies.

Enhancing endometrial receptivity: the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells

Inadequate endometrial receptivity often results in embryo implantation failure and miscarriage. Human chorionic gonadotropin (hCG) is a key signaling molecule secreted during early embryonic development, which regulates embryonic maternal interface signaling and promotes embryo implantation. This study aimed to examine the impact of hCG on endometrial receptivity and its underlying mechanisms. An exploratory study was designed, and endometrial samples were obtained from women diagnosed with simple tubal infertility or male factor infertile (n = 12) and recurrent implantation failure (RIF, n = 10). Using reverse transcription-quantitative PCR and western blotting, luteinizing hormone (LH)/hCG receptor (LHCGR) levels and autophagy were detected in the endometrial tissues. Subsequently, primary endometrial stromal cells (ESCs) were isolated from these control groups and treated with hCG to examine the presence of LHCGR and markers of endometrial receptivity (HOXA10, ITGB3, FOXO1, LIF, and L-selectin ligand) and autophagy-related factors (Beclin1, LC3, and P62). The findings revealed that the expressions of receptivity factors, LHCGR, and LC3 were reduced in the endometrial tissues of women with RIF compared with the control group, whereas the expression of P62 was elevated. The administration of hCG to ESCs specifically activated LHCGR, stimulating an increase in the endometrial production of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligands. Furthermore, when ESCs were exposed to 0.1 IU/mL hCG for 72 h, the autophagy factors Beclin1 and LC3 increased within the cells and P62 decreased. Moreover, the apoptotic factor Bax increased and Bcl-2 declined. However, when small interfering RNA was used to knock down LHCGR, hCG was less capable of controlling endometrial receptivity and autophagy molecules in ESCs. In addition, hCG stimulation enhanced the phosphorylation of ERK1/2 and mTOR proteins. These results suggest that women with RIF exhibit lower levels of LHCGR and compromised autophagy function in their endometrial tissues. Thus, hCG/LHCGR could potentially improve endometrial receptivity by modulating autophagy and apoptosis.

Injectable "Homing-Like" Bioactive Short-Fibers for Endometrial Repair and Efficient Live Births.

The prevalence of infertility caused by endometrial defects is steadily increasing, posing a significant challenge to women's reproductive health. In this study, injectable "homing-like" bioactive decellularized extracellular matrix short-fibers (DEFs) of porcine skin origin are innovatively designed for endometrial and fertility restoration. The DEFs can effectively bind to endometrial cells through noncovalent dipole interactions and release bioactive growth factors in situ. In vitro, the DEFs effectively attracted endometrial cells through the "homing-like" effect, enabling cell adhesion, spreading, and proliferation on their surface. Furthermore, the DEFs effectively facilitated the proliferation and angiogenesis of human primary endometrial stromal cells (HESCs) and human umbilical vein endothelial cells (HUVECs), and inhibited fibrosis of pretreated HESCs. In vivo, the DEFs significantly accelerated endometrial restoration, angiogenesis, and receptivity. Notably, the deposition of endometrial collagen decreased from 41.19 ± 2.16% to 14.15 ± 1.70% with DEFs treatment. Most importantly, in endometrium-injured rats, the use of DEFs increased the live birth rate from 30% to an impressive 90%, and the number and development of live births close to normal rats. The injectable "homing-like" bioactive DEFs system can achieve efficient live births and intrauterine injection of DEFs provides a new promising clinical strategy for endometrial factor infertility.

Novel Diagnostic Biomarker BST2 Identified by Integrated Transcriptomics Promotes the Development of Endometriosis via the TNF-α/NF-κB Signaling Pathway.

Endometriosis (EMS) is a common gynecological condition with apparent heterogeneity, lack of diagnostic markers, and unclear pathogenesis. A series of bioinformatics methods were employed to explore EMS's pathological mechanisms and potential biomarkers by analyzing the combined datasets of EMS (GSE7305, GSE7307, GSE58198, E-MTAB-694), which included 34 normal, 127 eutopic, and 46 ectopic endometrium samples. Then, wet-laboratory experiments (including Western blot, qRT-PCR, and Immunohistochemistry, Immunofluorescence, CCK-8, EdU, Wound healing, Transwell, and Adhesion assays) were applied to examine the biomarkers' expression and function in primary endometrial stromal cells. Bioinformatic analysis indicated that the core pathogenesis of EMS was dysregulated immune-inflammation and tissue remolding processes. Among the upregulated DEGs, BST2 was screened as a potential diagnostic biomarker in EMS, which associated with the revised American Fertility Society (r-AFS) stage and immune-inflammation processes of EMS. Moreover, BST2's overexpression was affirmed in the RNA and protein levels in EMS tissues. In vitro experiments demonstrated that TNF-α promoted the expression of BST2 in ESCs. And BST2 knockdown inhibited migration, invasion, adhesion, and inflammation except for the proliferation of ESCs, probably via the TNF-α/NF-κB pathway. Through a combination of wet and dry studies, we concluded that the core pathogenesis of endometriosis was dysregulated immune-inflammation and tissue remolding, and BST2 might be a potential diagnostic and therapeutic target in endometriosis.

Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 μl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). Raw data have been uploaded to GEO (accession number GSE234338). The findings of the study will require validation utilizing a second cohort of EV samples. The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. The study was supported by a Marie Skłodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

Galectin-3 promotes fibrosis in ovarian endometriosis.

This study aimed to investigate the potential role of galectin-3 (Gal-3) in the pathogenesis of fibrotic alterations in ovarian endometriosis (OVE). In this study, we collected the ectopic endometrial tissues and eutopic endometrial tissues from 31 OVE patients treated by laparoscopy, and the eutopic endometrial tissues from 23 non-OVE patients with leiomyoma or other benign diseases were used as control. Hematoxylin and eosin (H&E) and Masson's trichrome staining were utilized for histopathological assessment. The primary normal endometrial stromal cells (NESC), ectopic endometrial stromal cells (ECSC), and eutopic endometrial stromal cells (EUSC) were isolated. Gal-3 overexpression plasmids (Gal-OE) and short hairpin RNA targeting Gal-3 (Gal-3-shRNA) were transfected into the immortalized human endometriotic cell line 12Z, respectively. RT-qPCR, Western blot analysis, and immunohistochemistry were used to detect the mRNA and protein expression levels of Gal-3, type I collagen (COL-1), connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA), respectively. H&E and Masson staining showed that ovarian ectopic endometrium exhibited glandular hyperplasia, high columnar glandular epithelium, apical plasma secretion, more subnuclear vacuoles, and obvious fibrosis, compared with normal endometrium. The mRNA and protein levels of Gal-3 , CTGF, α-SMA, and COL-1 were all upregulated in the ectopic endometrial tissues of OVE patients compared to the eutopic endometrial tissues from OVE patients and non-OVE patients. Moreover, ECSC expressed higher levels of Gal-3, CTGF, α-SMA, and COL-1 than EUSC and NESC. Follow-up investigations demonstrated that the Gal-3 overexpression substantially increased fibrosis-related markers including CTGF, α-SMA, and COL-1 within the 12Z cell line. Conversely, Gal-3 knockdown showed the opposite effects. Gal-3 promotes fibrosis in OVE, positioning it as a prospective therapeutic target for mitigating fibrosis in endometriosis.

Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

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.

Effect of Aurora kinase B on polyploidy and decidualization in mouse uterus.

Decidualization is critical to the establishment of mouse normal pregnancy. The fibroblast-like stromal cells in the process form polyploid multinucleated cells. Aurora kinase B (Aurora B) has previously been shown to regulate polyploidy in various cells. However, whether Aurora B regulates the formation of decidual cell polyploidization and its regulatory mechanisms remain poorly understood. Establish decidualization model of mouse primary endometrial stromal cells in vitro. Construct pseudopregnancy mouse models and delayed-activation mouse models. Detect Aurora B and polyploidization related genes in mouse uteri treated by Aurora B specific inhibitor Barasertib and CPT. In this study, we found that Aurora B was strongly expressed in endometrial stromal cells after implantation. Additionally, Aurora B was remarkably up regulated in the stromal cells of oil-induced deciduomoa and in vitro decidualization. As an Aurora B specific inhibitor, Barasertib significantly inhibits the mRNA expression of Prl8a2, a marker of mouse decidualization. Furthermore, the protein levels of p-Plk1, Survivin and p-Cdk1 were inhibited by Barasertib. CPT-induced DNA damage suppressed Aurkb (encodes Aurora B) expression, thus resulting in polyploidization. Our data shows that Aurora B is expressed in decidual stromal cells of implantation sites and plays a key role for mouse decidualization. The protein of Plk1, Survivn, and Cdk1 may participate in formation of decidual cell polyploidization during mouse decidualization.

Inhibitory effect of telocyte-induced M1 macrophages on endometriosis: Targeting angiogenesis and invasion

PurposeTelocytes (TCs), a novel type of stromal cells found in tissues, induce macrophage differentiation into classically activated macrophages (M1) types and enhance their phagocytic function. The purpose of this study was to investigate the inhibitory effects of TC-induced M1 macrophages on endometriosis (EMs). Methodsmouse uterine primary TCs and endometrial stromal cells (ESCs) were isolated and identified using double immunofluorescence staining. For the in vitro study, ESCs were treated with TC-induced M1 macrophages, and the vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP9), and nuclear factor kappa B (NF-κb) genes were identified by quantitative real-time PCR (qRT-PCR) or western blotting (WB). For the in vivo study, an EMs mouse model received TC-conditioned medium (TCM) via abdominal administration, and characterized the inhibitory effects on growth (lesion weight, volume, and pathology), tissue-resident macrophages differentiation by immunostaining, angiogenic capacity (CD31 and VEGF), invasive capacity (MMP9), and NF-κb expression within EMs lesions. Resultsimmunofluorescent staining showed that uterine TCs expressed CD34+ and vimentin+, whereas ESCs expressed vimentin+ and cytokeratin-. At the cellular level, TC-induced M1 macrophages can significantly inhibit the expression of VEGF and MMP9 in ESCs through WB or qRT-PCR, possibly by suppressing the NF-κb pathway. The in vivo study showed that macrophages switch from the alternatively activated macrophages (M2) in untreated EMs lesions to the M1 subtype after TCM exposure. Thereby, TC-induced M1 macrophages contributed to the inhibition of EMs lesions. More importantly, this effect may be achieved by suppressing the expression of NF-κb to inhibit angiogenesis (CD31 and VEGF) and invasion (MMP9) in the tissue. ConclusionTC-induced M1 macrophages play a prevailing role in suppressing EMs by inhibiting angiogenic and invasive capacity through the NF-κb pathway, which provides a promising therapeutic approach for EMs.

LAMB3 Promotes Myofibrogenesis and Cytoskeletal Reorganization in Endometrial Stromal Cells via the RhoA/ROCK1/MYL9 Pathway

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman’s syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-β-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS.

Long non coding RNA, C8orf49, a novel diagnostic and prognostic biomarker, enhances PTEN/FZD4-mediated cell growth and metastasis by sponging miR-1323 in endometriosis

Lack of sensitive biomarkers in the early stages of endometriosis (EMs) results in delayed diagnosis and intervention. Long non-coding RNAs (lncRNAs) have prognostic and diagnostic values in various diseases. However, the prognostic and diagnostic effects of lncRNAs on EMs have rarely been discussed in EMs. In this study, we found that lncRNA C8orf49 was stably overexpressed in EMs tissues/plasma, and its expression greatly influenced dysmenorrhea (p = 2.2605E-9) and the revised American Society for Reproductive Medicine stage (p = 0.040765) of EMs. Multivariate logistic regression results revealed that C8orf49 expression was an independent risk factor for EMs [p = 6.4997E-17, 95% confidence interval (CI) = 0.000559–0.023853]. In primary endometrial stromal cells (ESCs), inhibition of C8orf49 could impede the proliferation and metastasis of ESCs. C8orf49 influenced the expression of PTEN/FZD4 by absorbing miR-1323, thus controlling ESCs activity. The results of a subcutaneous endometriosis animal model showed that the inhibition of C8orf49 restrained endometrial growth. Overall, C8orf49 functioned as an activator of EMs pathogenesis via the C8orf49/miR-1323/PTEN/FZD4 axis.

Paradoxical expression of NRP1 in decidual stromal and immune cells reveals a novel inflammation balancing mechanism during early pregnancy.

To investigate the balancing mechanisms between decidualization-associated inflammation and pregnancy-related immunotolerance. Decidual samples from women with normal pregnancy (n = 58) or unexplained spontaneous miscarriage (n = 13), peripheral blood from normal pregnancy and endometria from non-pregnancy (n = 10) were collected. Primary endometrial stromal cells (ESCs), decidual stromal cells (DSCs), decidual immune cells (DICs) and peripheral blood mononuclear cells (PBMCs) were isolated. The plasmid carrying neuropilin-1 (NRP1) gene was transfected into ESC for overexpression. To induce decidualization in vitro, ESCs were treated with a combination of 10nM estradiol, 100nM progesterone and 0.5mM cAMP. Anti-Sema3a and anti-NRP1 neutralizing antibodies were applied to block the ligand-receptor interactions. RNA-seq analysis was performed to identify differentially expressed genes in DSCs and DICs, and NRP1 expression was verified by Western blotting and flow cytometry. The secretion of inflammatory mediators was measured using a multifactor cytometric bead array. The effects of Sema3a-NRP1 pathway on DICs were determined by flow cytometry. Statistical differences between groups were compared using the T test and one way or two-way ANOVA. Combined with five RNA-seq datasets, NRP1 was the only immune checkpoint changing oppositely between DSCs and DICs. The decreased expression of NRP1 in DSCs allowed intrinsic inflammatory responses required for decidualization, while its increased expression in DICs enhanced tolerant phenotypes beneficial to pregnancy maintenance. DSC-secreted Sema3a promoted immunosuppression in DICs via NRP1 binding. In women with miscarriage, NRP1 was abnormally elevated in DSCs but diminished in decidual macrophages and NK cells. NRP1 is a multifunctional controller that balances the inflammatory states of DSCs and DICs in gravid uterus. Abnormal expression of NRP1 is implicated in miscarriage.

LincRNA RP24-315D19.10 promotes endometrial decidualization via upregulation of hnRNPA2B1

Decidualization is a critical process for successful pregnancy. Disorders in this process are tightly associated with adverse pregnancy outcomes including spontaneous abortion. However, the potential molecular mechanisms of lncRNAs underlying this process are yet to be fully elucidated. In this study, we utilized RNA sequencing (RNA-seq) to identify differentially expressed lncRNAs during endometrial decidualization with a pregnant mouse model. Based on RNA-seq analysis, weighted gene co-expression network analysis (WGCNA) was performed to construct the lncRNA-mRNA co-expression network and to identify decidualization-associated hub lncRNAs. Through comprehensive screening and validation, we identified a novel lncRNA, RP24-315D19.10 and studied its function in primary mouse endometrial stromal cells (mESCs). lncRNA RP24-315D19.10 was highly expressed during decidualization. Knockdown of RP24-315D19.10 significantly inhibited mESCs decidualization in vitro. Mechanistically, RNA pull-down and RNA immunoprecipitation assays indicated that cytoplasmic RP24-315D19.10 could bind to hnRNPA2B1, thereby upregulating hnRNPA2B1 expression. Site-directed mutagenesis followed by biolayer interferometry analysis additionally illustrated that hnRNPA2B1 protein specifically bound to the ~-142ccccc~-167 region of the RP24-315D19.10 sequence. hnRPA2B1 deficiency impairs mESCs decidualization in vitro and we found that the inhibition in decidualization caused by RP24-315D19.10 knockdown was rescued by hnRNPA2B1 overexpression. Moreover, the expression of hnRNPA2B1 in spontaneous abortion women with deficient decidualization was significantly lower than that in healthy individuals, suggesting that hnRNPA2B1 may be involved in the development and progression of spontaneous abortion caused by decidualization failure. Collectively, our study indicates RP24-315D19.10 is a critical regulator for endometrial decidualization and RP24-315D19.10-regulated hnRNPA2B1 might be a new mark of decidualization-related spontaneous abortion.

Mitochondrial energy metabolism is downregulated in repeated implantation failure patients related to alteration of PGC-1α acetylation level.

Herein we aimed at exploring mitochondrial energy metabolism status in patients with repeated implantation failure (RIF) and whether key regulatory factor PGC-1α of energy metabolism is involved in the decidualization of endometrial stromal cells. Mitochondrial oxidative phosphorylation level and ATP synthesis were compared in primary endometrial stromal cells from RIF and control group. At the same time, as one of key transcription regulators of mitochondrial energy metabolism, the expression level and acetylation level of PGC-1α were compared with two groups. Then, we downregulated the acetylation levels of PGC-1α, and the expression of decidual markers (PRL and IGFBP1) was observed further. Mitochondrial energy metabolism, showing by mitochondrial oxidative phosphorylation level and ATP synthesis, was decreased in the endometrial stromal cells of the RIF group (RIF-hEnSCs). Meanwhile, PGC-1α acetylation levels were significantly higher in RIF-hEnSCs. When we reduced the acetylation levels of PGC-1α in RIF-hEnSCs, the basal O2 consumption rate and maximal respiration were increased, and also the PRL and IGFBP1. Overall, our data indicated that the endometrial stromal cells of the RIF patients had low level of mitochondrial energy metabolism. Reducing acetylation level of key energy metabolism regulator PGC-1α can increase the decidualization level of RIF-hEnSCs. These findings may inspire new ideas about the treatment of RIF.