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

STUDY QUESTION: 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? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LARGE-SCALE DATA: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

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.

Progesterone suppresses podocalyxin partly by up-regulating miR-145 and miR-199 in human endometrial epithelial cells to enhance receptivity in in vitro models.

Establishment of endometrial surface receptivity is crucial for the initiation of embryo implantation yet the molecular mechanisms are not well understood, especially in humans. We have recently discovered that podocalyxin (PODXL) is a critical negative regulator of human endometrial surface receptivity. PODXL is highly expressed in all epithelial and endothelial cells in the non-receptive endometrium, but down-regulated specifically in the luminal epithelium at receptivity. We have further shown that PODXL inhibits embryo implantation, and that PODXL down-regulation is essential for endometrial surface receptivity. Our previous study also indicated that progesterone down-regulates PODXL; however, the exact molecular regulations are unknown. Here, we investigated whether progesterone suppresses PODXL via microRNAs (miRNAs). We first bioinformatically predicted 13 miRNAs that may potentially target human PODXL, then experimentally determined whether any of these 13 miRNAs are altered in primary human endometrial epithelial cells (HEECs) by progesterone, and whether the identified miRNAs can affect PODXL expression in Ishikawa cells without progesterone and alter receptivity to embryo implantation. Progesterone significantly up-regulated miR-145 and miR-199 while suppressing PODXL in HEECs. When these two miRNAs were transfected into Ishikawa cells, both significantly down-regulated PODXL mRNA and protein in the absence of progesterone. Moreover, both miR-145 and miR-199 significantly enhanced receptivity of the Ishikawa monolayer to embryo implantation in in vitro models. This study thus provides in vitro evidence that PODXL is down-regulated by progesterone partly via miR-145 and miR-199 during the development of human endometrial epithelial receptivity. These results also reveal the likely importance of hormonal regulation of miRNAs for embryo implantation.

Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids.

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells.

BACKGROUND: Successful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells' ability to invade the endometrium. We hypothesized that PIF's effects on the endometrium counteract its pro-invasive effects. METHODS: We tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay. RESULTS: Firstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line's invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF's anti-invasive action was associated with a specifically decrease in MMP-9 activity. CONCLUSION: Taken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Label-free proteomics uncovers SMC1A expression is Down-regulated in AUB-E.

BACKGROUND: While heavy menstrual bleeding (HMB) is a prevalent symptom among women with abnormal uterine bleeding caused by endometrial disorder (AUB-E) seeking gynecologic care, the primary endometrial disorder remains poorly understood. METHODS: Five human endometrial samples from women with AUB-E and the age-matched healthy women were selected, respectively. Proteins from the samples were analyzed by a linear ion trap (LTQ)-Orbitrap Elite mass spectrometer based label-free proteomic approach. The purpose protein was validated by western blot and immunohistochemistry staining. RESULTS: A total of 2353 protein groups were quantified under highly stringent criteria with a false discovery rate of < 1% for protein groups, and 291 differentially expressed proteins were significantly changed between the two groups. The results showed that the down-regulation of structural maintenance of chromosomes protein 1A (SMC1A) in AUB-E patients. Next, this change in the glandular epithelial cells was validated by immunohistochemistry. CONCLUSION: The results indicated a novel mechanism for the cause of AUB-E, as down-expression SMC1A potentially regulated the cell cycle progression in endometrial glandular epithelium further led to bleeding.

Decreased number of p16-positive senescent cells in human endometrium as a marker of miscarriage.

PURPOSE: The aim of this study was to evaluate whether the number of p16-positive cells in the functional layer of the endometrium could be a useful biomarker to identify women with recurrent implantation failure (RIF) undergoing in vitro fertilization (IVF) at risk of miscarriage. METHODS: Immunohistochemical staining was performed in 311 endometrial biopsies taken during mid-luteal phase using antibody against p16INK4A. The percentage of p16-positive cells was determined in luminal, glandular and stromal endometrial cells. After embryo transfer, women were divided into the following groups: unsuccessful embryo implantation (n = 151), miscarriage (n = 66) and live birth (n = 94). The percentage of p16-positive cells in all endometrial compartments was compared among these groups. RESULTS: We found that the percentages of p16-positive glandular and luminal epithelial endometrial cells were significantly higher in patients with live births compared to women with miscarriage (9.3% vs. 2.9%, P < 0.001; and 35.2% vs. 11.7%, P = 0.001, respectively). This tendency was not confirmed in thе stroma. The cut-off values with p16-positive luminal cells lower than 12.5% and p16-positive glandular cells lower than 3.2% could be predictive factors for miscarriage (AUC 0.80 and 0.79; sensitivity 71.3% and 74.5%; specificity 74.2% and 71.2%, respectively). CONCLUSION: A decreased number of senescent p16-positive cells could be involved in the implantation failures and aetiology of recurrent miscarriage. Women with history of RIF with reduced populations of p16-positive cells in the endometrial glandular and luminal epithelium may be at greater risk for unsuccessful implantation and miscarriage. The percentage of p16-positive luminal epithelial cells may be clinically useful as a biomarker of miscarriage.

Women with polycystic ovary syndrome present with altered endometrial expression of stanniocalcin-1†.

Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium.

Varying levels of serum estradiol do not alter the timing of the early endometrial secretory transformation.

STUDY QUESTION: Do supraphysiologic estradiol (E2) levels in the ranges attained during normal and high response superovulation cycles modify the onset of endometrial secretory transformation? SUMMARY ANSWER: Highly supraphysiologic levels of E2 do not alter the ability of physiologic levels of progesterone (P4) to induce secretory transformation. WHAT IS KNOWN ALREADY: Previous studies have demonstrated that premature P4 elevations during IVF cycles are associated with a decrement in clinical pregnancy rates after fresh embryo transfer due to shifts in the window of implantation (WOI). However, alterations in the onset of secretory transformation may not apply uniformly to all patients. High responders with supraphysiologic E2 levels accompanied by similar subtle increases in P4 have not been shown to have decreased sustained implantation rates. This prospective investigation in which whole-genome transcriptomic and methylomic analysis of the endometrium is performed for individual patients under a range of E2 concentrations brings clarity to a long-debated issue. STUDY DESIGN, SIZE, DURATION: A randomized, prospective and paired trial was conducted in which 10 participants were enrolled and randomized to the order in which they completed three distinct uterine stimulation cycles, each at a specific E2 concentration: physiologic (∼180 pg/ml), moderately supraphysiologic (600-800 pg/ml) or supraphysiologic (2000 pg/ml). Target E2 ranges were selected to mimic those seen in natural, controlled ovarian stimulation and IVF cycles. E2 valerate was administered in order to maintain stable E2 levels for 12 days followed by intramuscular P4 in oil 10 mg/day for two doses, after which an endometrial biopsy was performed. A total of 30 endometrial biopsies were included in a whole-genome transcriptomic and methylomic analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthy volunteers without a history of infertility were included in this study at a single large infertility center. DNA was isolated from the endometrial biopsy specimens and bisulfite sequencing was performed to construct a methylation array. Differential methylation analysis was conducted based on differences in M-values of individuals across treatment groups for each probe as well as carrying out t-tests. RNA was isolated for RNA-Seq analysis and gene expression values were compared using DESeq2. All analyses were performed in a pairwise fashion to compare among the three stimulation cycles within individuals and secondarily to compare all participants in each of the cycles. MAIN RESULTS AND THE ROLE OF CHANCE: The mean peak E2 and P4 levels were 275 pg/ml and 4.17 ng/ml in the physiologic group, 910 pg/ml and 2.69 ng/ml in the moderate group was, and 2043 pg/ml and 2.64 ng/ml in the supraphysiologic group, respectively. Principal component analysis of 834 913 CpG sites was performed on M-values of individuals within the low, moderate and supraphysiologic conditions in a paired approach. There were no differences in genome-wide methylation within participants across E2 groups. A paired analysis revealed that gene expression profiles did not differ within the same individual at each of the three E2 levels. No significant alterations in gene expression as related to endometrial physiology were identified between the low, moderate and supraphysiologic groups in an inter-participant analysis. LIMITATIONS, REASONS FOR CAUTION: Although each participant completed a physiologic cycle in which E2 levels were maintained in a range that would simulate a natural cycle, our findings are limited by lack of an unmedicated control to assess if there was a potential effect from E2V. Additionally, our results were obtained in fertile individuals, who may have a different endometrial response compared to an infertile population. Despite the whole genomic endometrial assessment and rigorous, paired study design, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS: Given that the endometrial response to P4 is unaffected by E2 levels in the supraphysiologic range, diminutions in implantation seen in stimulated cycles may result from embryonic-endometrial dyssynchrony following early P4 elevations or slowly blastulating embryos, which occur independently of the magnitude of the E2 rise. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. Dr E.S. reports consultancy work for The Foundation for Embryonic Competence, Basking Ridge, NJ, USA. The other authors declare no conflict of interests related to this topic. TRIAL REGISTRATION NUMBER: NCT02458404.

Insulin regulation of solute carrier family 2 member 1 (glucose transporter 1) expression and glucose uptake in decidualizing human endometrial stromal cells: an in vitro study.

BACKGROUND: Solute carrier family 2 member 1 (SLC2A1; previously known as glucose transporter 1), is the most abundant glucose transporter in human endometrium and is up-regulated during decidualization, whereas high insulin may have a negative impact on this process. The present study aimed to investigate the effect of insulin on the expression of SLC2A1 and glucose uptake in decidualizing human endometrial stromal cells. METHODS: We induced in vitro decidualization of endometrial stromal cells obtained from regularly menstruating healthy non-obese women. The cells were treated with increasing concentrations of insulin, and the involvement of the transcription factor forkhead box O1 (FOXO1) was evaluated using a FOXO1 inhibitor. SLC2A1 mRNA levels were measured by Real-Time PCR and protein levels were evaluated by immunocytochemistry. Glucose uptake was estimated by an assay quantifying the cellular uptake of radioactive glucose. One-way ANOVA, Dunnett's multiple comparisons test and paired t-test were used to determine the statistical significance of the results. RESULTS: We found that insulin dose-dependently decreased SLC2A1 mRNA levels and decreased protein levels of SLC2A1 in decidualizing human endometrial stromal cells. Transcriptional inactivation of FOXO1 seems to explain at least partly the down-regulation of SLC2A1 by insulin. Glucose uptake increased upon decidualization, whereas insulin treatment resulted in a slight inhibition of the glucose uptake, although not significant for all insulin concentrations. CONCLUSIONS: These results indicate an impairment of decidualization by high concentrations of insulin. Future studies will determine the clinical significance of our results for endometrial function and decidualization in women with insulin resistance and hyperinsulinemia.

PI3K/AKT pathway is altered in the endometriosis patient's endometrium and presents differences according to severity stage.

Based on the inflammatory nature and hormone-dependency of endometriosis, PI3K/AKT signaling appears to influence its progression. Could the endometriosis stages be linked to differential changes in PI3K/AKT pathway regulation? The objective is to evaluate the expression of PI3K, PTEN, AKT and p-AKT in endometrial human biopsies, according to the presence or absence of the disease, and to assess the underlying differences regarding the endometriosis stages. Biopsy specimens of the ectopic and eutopic endometrium were obtained from twenty women with untreated peritoneal endometriosis as well as endometrium biopsies from nine controls. Our study revealed an increased expression of PI3K in eutopic and ectopic endometrium from patients with endometriosis, and a reduced expression of PTEN and increased levels of AKT phosphorylation, compared to control endometrium. Both eutopic and ectopic endometrium from patients with minimal-mild endometriosis expressed a significant reduced PTEN level compared to the respective endometrium from patients with moderate-severe endometriosis. The ratio p-AKT/total AKT showed higher levels of AKT phosphorylation in endometriotic tissue from patients with minimal-mild endometriosis. This study has firmly confirmed the alteration in PI3K/AKT pathway regulation and demonstrated clear differences between the stages of endometriosis, emphasizing the importance of this pathway in the first stage of the disease.

Recurrent pregnancy loss is associated to leaky gut: a novel pathogenic model of endometrium inflammation?

BACKGROUND: Recurrent pregnancy loss (RPL) occurs in 3-5% in about 30% of cases no cause can be found. Women with RPL show higher prevalence of undiagnosed gut disorders. Furthermore, in endometrial tissues of RPL women, higher expression of pro-inflammatory cytokines and Nalp-3 inflammasome has been observed. Aim of this study was to investigate whether an abnormal gut permeability might occur in RPL women and allow passage into systemic circulation of pro-inflammatory molecules able to induce endometrial inflammation. METHODS: 70 women with idiopathic RPL and 30 healthy women were recruited at the Recurrent Pregnancy Loss Outpatient Unit of the Gemelli Hospital of Rome from March 2013 to February 2017. Enrolled women underwent 51Cr-ethylene-diamine-tetraacetic acid absorption test to evaluate intestinal permeability. Sera obtained from enrolled women were analysed for lipopolysaccharide (LPS) by ELISA. Anxiety and depression state were evaluated by administering STAI-Y and Zung-SDS tests, respectively. Of all recruited individuals, 35 women with idiopathic RPL and 20 healthy controls accepted to undergo diagnostic hysteroscopy and endometrial biopsy. Endometrial lysates were investigated for inflammasome Nalp-3 by Western blot analysis, and caspase-1, IL-1ß and IL-18 by ELISA, respectively. RESULTS: Higher prevalence of abnormal intestinal permeability (P < 0.0001), increased circulating levels of LPS (P < 0.05), anxiety (P < 0.05) and depression (P < 0.05) were observed in RLP women compared to controls. Endometrial expression of Nalp-3, caspase-1 and IL-1ß was significantly increased in RPL group (P < 0.0001; P < 0.05 and P < 0.001, respectively). IL-18 endometrial levels were not found to be higher in RPL cases. Statistically significant association between higher intestinal permeability and abnormally increased expression of endometrial Nalp-3, was observed in RPL (P < 0.01). Furthermore, higher LPS serum levels, a bacterial-derived activator of Nalp-3 complex, was shown to be statistically associated to abnormal endometrial expression of Nalp-3 inflammasome (P < 0.01) in RPL women. CONCLUSIONS: In women with RLP, leaky gut might occur and allow passage into circulation of immune triggers, potentially able to elicit endometrial innate immune response and, thus, to contribute to miscarriage pathogenesis. Diagnosis and treatment of intestinal disorders underlying leaky gut might improve endometrial environment and pregnancy outcome.

The genetic regulation of transcription in human endometrial tissue.

Study question: Do genetic effects regulate gene expression in human endometrium? Summary answer: This study demonstrated strong genetic effects on endometrial gene expression and some evidence for genetic regulation of gene expression in a menstrual cycle stage-specific manner. What is known already: Genetic effects on expression levels for many genes are tissue specific. Endometrial gene expression varies across menstrual cycle stages and between individuals, but there are limited data on genetic control of expression in endometrium. Study design, size, duration: We analysed genome-wide genotype and gene expression data to map cis expression quantitative trait loci (eQTL) in endometrium. Participants/materials, setting, methods: We recruited 123 women of European ancestry. DNA samples from blood were genotyped on Illumina HumanCoreExome chips. Total RNA was extracted from endometrial tissues. Whole-transcriptome profiles were characterized using Illumina Human HT-12 v4.0 Expression Beadchips. We performed eQTL mapping with ~8 000 000 genotyped and imputed single nucleotide polymorphisms (SNPs) and 12 329 genes. Main results and the role of chance: We identified a total of 18 595 cis SNP-probe associations at a study-wide level of significance (P < 1 × 10-7), which correspond to independent eQTLs for 198 unique genes. The eQTLs with the largest effect in endometrial tissue were rs4902335 for CHURC1 (P = 1.05 × 10-32) and rs147253019 for ZP3 (P = 8.22 × 10-30). We further performed a context-specific eQTL analysis to investigate if genetic effects on gene expression regulation act in a menstrual cycle-specific manner. Interestingly, five cis-eQTLs were identified with a significant stage-by-genotype interaction. The strongest stage interaction was the eQTL for C10ORF33 (PYROXD2) with SNP rs2296438 (P = 2.0 × 10-4), where we observe a 2-fold difference in the average expression levels of heterozygous samples depending on the stage of the menstrual cycle. Large scale data: The summary eQTL results are publicly available to browse or download. Limitations, reasons for caution: A limitation of the present study was the relatively modest sample size. It was not powered to identify trans-eQTLs and larger sample sizes will also be needed to provide better power to detect cis-eQTLs and cycle stage-specific effects, given the substantial changes in expression across the menstrual cycle for many genes. Wider implications of the findings: Identification of endometrial eQTLs provides a platform for better understanding genetic effects on endometriosis risk and other endometrial-related pathologies. Study funding/competing interest(s): Funding for this work was provided by NHMRC Project Grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321 and APP1083405. There are no competing interests.

Establishment of a novel human embryonic stem cell-derived trophoblastic spheroid implantation model.

STUDY QUESTION: Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation? SUMMARY ANSWER: We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation. WHAT IS KNOWN ALREADY: Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research. STUDY DESIGN, SIZE, DURATION: Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-ß inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells. MAIN RESULTS AND THE ROLE OF CHANCE: After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture. LIMITATIONS, REASONS FOR CAUTION: The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles. WIDER IMPLICATIONS OF THE FINDINGS: BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

STUDY QUESTION: Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? SUMMARY ANSWER: The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. WHAT IS KNOWN ALREADY: Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. STUDY DESIGN, SIZE, DURATION: Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. LIMITATIONS, REASONS FOR CAUTION: Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. WIDER IMPLICATIONS OF THE FINDINGS: Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. STUDY FUNDING/ COMPETING INTERESTS: This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Metal status in human endometrium: relation to cigarette smoking and histological lesions.

Human endometrium is a thick, blood vessel-rich, glandular tissue which undergoes cyclic changes and is potentially sensitive to the various endogenous and exogenous compounds supplied via the hematogenous route. As recently indicated, several metals including Cd, Pb, Cr and Ni represent an emerging class of potential metalloestrogens and can be implicated in alterations of the female reproductive system including endometriosis and cancer. In the present study, we investigated the content of five metals: Cd, Cr, Ni, Pb and Zn in 25 samples of human endometrium collected from Polish females undergoing diagnostic or therapeutic curettage of the uterine cavity. The overall mean metal concentration (analyzed using microwave induced plasma atomic emission spectrometry MIP-OES) decreased in the following order: Cr>Pb>Zn>Ni>Cd. For the first time it was demonstrated that cigarette smoking significantly increases the endometrial content of Cd and Pb. Concentration of these metals was also positively correlated with years of smoking and the number of smoked cigarettes. Tissue samples with recognized histologic lesions (simple hyperplasia, polyposis and atrophy) were characterized by a 2-fold higher Cd level. No relation between the age of the women and metal content was found. Our study shows that human endometrium can be a potential target of metal accumulation within the human body. Quantitative analyses of endometrial metal content could serve as an additional indicator of potential impairments of the menstrual cycle and fertility.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) as a putative human endometrial stem cell marker.

The endometrium is recognized for its remarkable regenerative and remodeling capacity. Every month this hormonally regulated organ undergoes cycles of growth (from 0.5-2 to 7 mm), regression and shedding of two-third of the tissue, leading to its monthly renewal that occurs ∼400 times in a woman's reproductive lifetime. Several groups have suggested the existence of a human endometrial somatic stem cell (SSC) population located around the spiral arterioles of the basalis. Different groups have isolated, identified and characterized putative endometrial SSC populations in human endometrium based on the general features of undifferentiated cells, such as slow cycling detected using the 5-bromo-2-deoxyuridine technique or identification of a side population using the Hoechst efflux dye technique. Nevertheless, specific markers to isolate these endometrial SSC have not yet been consistently elucidated. Accumulated evidence based on lineage tracing studies indicates that a surface protein named Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a marker that can identify SSC in several tissues such as small intestine mucosa (endodermal origin), hair follicles (ectodermal origin) or mature kidney nephrons (mesodermal origin). This protein plays a crucial role in the Wnt/ß-catenin signaling system by acting on the self-renewal and maintenance of the SSC population. In this work, we present novel data suggestive of Lgr5 as a putative human endometrial SSC marker, and since this is a mesoderm-derived tissue, these findings reinforce the concept that Lgr5 can be considered a universal SSC marker.

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

The 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.

Decreased CD56+CD16-CD94+uNK cells in the mid-luteal phase in women with recurrent implantation failure are associated with IL-15 deficiency.

PROBLEM: Whether the abnormal development of uterine natural killer (uNK) cells contributes to women with recurrent implantation failure (RIF) remains unclear. METHOD OF STUDY: We characterized the development of uNK cells and peripheral blood NK cells (pbNK) in the mid-luteal phase in women with RIF (n = 31) and controls (n = 14) by flow cytometry. Endometrial IL-15 mRNA expression was studied by quantitative reverse transcription-PCR. The GSE58144 dataset was used to validate the correlation results. RESULTS: We found decreased proportions of stage 4 CD56+CD16-CD94+ uNK cells (median: 9.56% vs. 17.78%, P .014) and increased proportions of stage 6 CD56+CD16+CD57+ uNK cells (median: 1.54% vs. 0.74%, P = .020) in the mid-luteal endometrium of women with RIF compared to fertile women. We also found that there was no quantitative correlation between uNK cells and the corresponding pbNK cell subpopulations (P > .05). In addition, IL-15 mRNA levels in the mid-luteal endometrium were positively correlated with the proportion of CD56+ uNK cells (r = .392, P = .008), especially with stage 4 uNK cell populations (r = .408, P = .005). CONCLUSIONS: We showed that the proportion of stage 4 uNK cells decreased in the RIF group compared to controls, and the decrease in stage 4 uNK cells correlated positively with low IL-15 mRNA expression. We suggest that the reduced stage 4 uNK cells in women with RIF are associated with IL-15 deficiency.

The single-cell atlas of cultured human endometrial stromal cells.

OBJECTIVE: To systematically analyze the cell composition and transcriptome of primary human endometrial stromal cells (HESCs) and transformed human endometrial stromal cells (THESCs). DESIGN: The primary HESCs from 3 different donors and 1 immortalized THESC were collected from the human endometrium at the midsecretory phase and cultured in vitro. SETTING: Academic research laboratory. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Single-cell ribonucleic acid sequencing analysis. RESULT(S): We found the individual differences among the primary HESCs and bigger changes between the primary HESCs and THESCs. Cell clustering with or without integration identified cell clusters belonging to mature, proliferative, and active fibroblasts that were conserved across all samples at different stages of the cell cycles with intensive cell communication signals. All primary HESCs and THESCs can be correlated with some subpopulations of fibroblasts in the human endometrium. CONCLUSION(S): Our study indicated that the primary HESCs and THESCs displayed conserved cell characters and distinct cell clusters. Mature, proliferative, and active fibroblasts at different stages or cell cycles were detected across all samples and presented with a complex cell communication network. The cultured HESCs and THESCs retained the features of some subpopulations within the human endometrium.