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Bisphenol A (BPA) is a well-known endocrine disruptor, widely used in various consumer products and ubiquitously found in air, water, food, dust, and sewage leachates. Recently, several countries have restricted the use of BPA and replaced them with bisphenol S (BPS) and bisphenol F (BPF), which have a similar chemical structure to BPA. Compared to BPA, both BPS and BPF have weaker estrogenic effects, but their effects on human reproductive function including endometrial receptivity and embryo implantation still remain largely unknown. We used an in vitro spheroid (blastocyst surrogate) co-culture assay to investigate the effects of BPA, BPS, and BPF on spheroid attachment on human endometrial epithelial cells, and further delineated their role on steroid hormone receptor expression. We also used transcriptomics to investigate the effects of BPA, BPS, and BPF on the transcriptome of human endometrial cells. We found that bisphenol treatment in human endometrial Ishikawa cells altered estrogen receptor alpha (ERα) signaling and upregulated progesterone receptors (PR). Bisphenols suppressed spheroid attachment onto Ishikawa cells, which was reversed by the downregulation of PR through PR siRNA. Overall, we found that bisphenol compounds can affect human endometrial epithelial cell receptivity through the modulation of steroid hormone receptor function leading to impaired embryo implantation.

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin β3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 μg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 μg/mL and ETU at 300 μg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 μg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor β and integrin β3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor β and integrin β3 expression of endometrial epithelial cells., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro., Design: Experimental study., Setting: Reproductive biology laboratory., Patient(s): Healthy nonpregnant women., Intervention(s): No patient interventions., Main Outcome Measure(s): Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay., Result(s): Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression., Conclusion(s): Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Epidemiological studies have shown that cigarette smoking is a major risk factor for tubal ectopic pregnancy but the reason for this remains unclear. Here, we set out to determine the effect of smoking on Fallopian tube gene expression. An oviductal epithelial cell line (OE-E6/E7) and explants of human Fallopian tubes from non-pregnant women (n = 6) were exposed to physiologically relevant concentrations of cotinine, the principle metabolite of nicotine, and changes in gene expression analyzed using the Illumina Human HT-12 array. Cotinine sensitive genes identified through this process were then localized and quantified in Fallopian tube biopsies from non-pregnant smokers (n = 10) and non-smokers (n = 11) using immunohistochemistry and TaqMan RT-PCR. The principle cotinine induced change in gene expression detected by the array analysis in both explants and the cell line was significant down regulation (P<0.05) of the pro-apoptotic gene BAD. We therefore assessed the effect of smoking on cell turnover in retrospectively collected human samples. Consistent with the array data, smoking was associated with decreased levels of BAD transcript (P<0.01) and increased levels of BCL2 transcript (P<0.05) in Fallopian tube biopsies. BAD and BCL2 specific immunolabelling was localized to Fallopian tube epithelium. Although no other significant differences in levels of apoptosis or cell cycle associated proteins were observed, smoking was associated with significant changes in the morphology of the Fallopian tube epithelium (P<0.05). These results suggest that smoking may alter tubal epithelial cell turnover and is associated with structural, as well as functional, changes that may contribute to the development of ectopic pregnancy.

Exposure of animals to perfluorooctanoic acid (PFOA), a surfactant used in emulsion polymerization processes causes early pregnancy loss, delayed growth and development of fetuses. The mechanisms of action are largely unknown. We studied the effect of PFOA on implantation using an in vitro spheroid-endometrial cell co-culture model. PFOA (10-100μM) significantly reduced Jeg-3 spheroid attachment on RL95-2 endometrial cells. PFOA also suppressed β-catenin expression in Jeg-3 cells. The Wnt agonist Wnt3a stimulated β-catenin expression in Jeg-3 cells and reversed the PFOA suppression of the spheroid attachment. The putative PFOA receptors (PPARα, β, γ) present in both cell lines were not affected by PFOA (0.01-100μM). The PPARα antagonist MK886 restored the β-catenin and E-cadherin expression levels in Jeg-3 cells and reversed the suppression of the spheroid attachment caused by PFOA. Taken together, PFOA suppresses spheroid attachment through PPARα and Wnt signaling pathways via down-regulation of β-catenin and E-cadherin expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The female reproductive system is a complex system. Epithelia of the female reproductive system including the ovaries, the oviduct, and the uterus are important sites for follicular development, ovulation, fertilization, implantation, and embryo development. They are also able to synthesize and secrete various hormones, growth factors, and cytokines, which are essential to women's health, sexuality, and reproduction. Conversely, their dysfunction has been implicated in disorders such as infertility, endometriosis, and many other gynecological diseases, as well as cancer. In this chapter, we describe detailed procedures for establishing and maintaining primary cultures of human ovarian surface epithelium, oviductal epithelium, and endometrium. We also provide protocols for cell immortalization, clonal isolation, and in coculture with stromal cells. These cultures can be useful models for investigating the molecular and cellular functions of these epithelia in both normal and pathological states.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects embryo development, implantation and fertility in humans. The underlying molecular mechanism by which TCDD suppresses implantation remains largely unknown. We used the trophoblastic spheroids (embryo surrogate)-endometrial cells co-culture assay to study the attachment of trophoblastic spheroids (BeWo and Jeg-3) onto the endometrial epithelial (RL95-2 and Ishikawa) cells. TCDD dose-dependently induced cytochrome P450 1A1 (Cyp1A1) expression in trophoblastic and endometrial epithelial cells. Moreover, TCDD at 1 and 10 nM suppressed β-catenin (a Wnt-signaling molecule) and E-cadherin expression, as well as spheroids attachment onto endometrial cells. Interestingly, activation of the canonical Wnt-signaling pathway via Wnt3a or lithium chloride reverted the suppressive effect of TCDD on β-catenin and E-cadherin expressions in the BeWo and RL95-2 cells, and restored the spheroids attachment rate to be comparable to the untreated controls. Taken together, TCDD induces Cyp1A1 expression, modulates the Wnt-signaling pathway and suppresses spheroids attachment onto endometrial cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To determine whether OE-E6/E7, an immortalized human oviductal epithelial cell line, expresses oviductin messenger RNA (mRNA) and its translated protein., Design: Transmission electron microscopy was employed to characterize the morphology of OE-E6/E7 cells followed by reverse-transcription polymerase chain reaction (PCR) analysis of oviductin mRNA and sequencing of the nested-PCR product. Confocal microscopy was used, using a polyclonal antibody against human oviductin and Con A as a marker for mannose residues, to reveal the colocalization of human oviduct-specific glycoprotein with the endoplasmic reticulum and Golgi compartments., Setting: University-based anatomy and cell biology department., Patient(s): Women undergoing laparoscopy for tubal ligation or hysterectomy due to uterine fibroma., Intervention(s): An immortalized OE-E6/E7 cell line was previously established using human oviductal epithelial cells. Electron microscopy, RT-PCR, sequencing, immunohistochemistry and confocal microscopy were performed., Main Outcome Measure(s): The presence of human oviductin mRNA and protein in OE-E6/E7 cells., Result(s): OE-E6/E7 cells retain morphological features characteristic of secretory cells and express human oviductin mRNA and its translated protein., Conclusion(s): OE-E6/E7 cells were characterized for the first time by electron microscopy and shown to exhibit histological features typical of secretory cells. Reverse-transcription PCR with sequencing and confocal microscopy showed, respectively, that human oviductin mRNA and protein are expressed in OE-E6/E7 cells. Our results suggest that OE-E6/E7 could be a useful tool for future studies of the function of human oviductin.

The decidualization of endometrial stromal cells (ESCs) is an essential process facilitating embryo implantation. However, the roles of non-decidualized and decidualized ESCs in regulating the microenvironment of a receptive endometrium remain unclear. We investigated single-cell transcriptomic changes in the uterus of a CD-1 mouse model at the post-implantation stage. The implantation and inter-implantation sites of the uteruses of pregnant mice at 4.5 and 5.5 days post-coitum were dissected for single-cell RNA sequencing. We identified eight cell types: epithelial cells, stromal cells, endothelial cells, mesothelial cells, lymphocytes, myocytes, myeloids, and pericytes. The ESC transcriptome suggests that the four ESC subtypes are involved in the extracellular remodeling during implantation. The trajectory plot of ESC subtypes indicates embryo implantation that involves a differentiation pathway from undifferentiated ESCs (ESC 1) to decidualized ESCs (DEC ESCs), with distinct signaling pathways between the ESC subtypes. Furthermore, the ligand-receptor analysis suggests that ESCs communicate with epithelial cells and immune cells through nectin and ICAM signaling. Collectively, both decidualized and non-decidualized ESCs may regulate the endometrial microenvironment for optimal endometrial receptivity and immune tolerance. This study provides insights on the molecular and cellular characteristics of mouse ESCs in modulating the epithelial and lymphocyte functions during early embryo implantation. [ABSTRACT FROM AUTHOR]

Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not β-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception. [ABSTRACT FROM AUTHOR]

BACKGROUND Olfactomedin (Olfm) is a member of a diverse group of extracellular matrix proteins important for neuronal growth. Recent microarray studies identified Olfm as one of the down-regulated transcripts in receptive endometrium at the time of embryo attachment and implantation. However, the underlying molecular mechanisms that govern Olfm expression and its effect on embryo attachment and implantation remain unknown. METHODS The expression of Olfm in the human endometrium was investigated by real-time PCR, western blotting and immunohistochemistry on human endometrial biopsies from natural and ovarian stimulated cycles. To investigate the function of Olfm in trophoblast–endometrial cell attachment, an in vitro spheroid-endometrial cell co-culture study was performed. RESULTS Human endometrial Olfactomedin-1 and -2(Olfm-1 and -2) transcripts decreased significantly from the proliferative to the secretory phases of the menstrual cycle. Olfm protein was strongly expressed in the luminal and glandular epithelium and moderately in the stromal cells of human endometria. Ovarian stimulation significantly decreased (P < 0.05) the expression of endometrial Olfm-1 and -2 transcripts in patients receiving IVF treatment when compared with those in the natural cycle. Importantly, recombinant Olfm-1 suppressed JAr spheroid attachment onto Ishikawa cells and this was not associated with changes of β-catenin and E-cadherin expression in trophoblast and endometrial cells. CONCLUSIONS Decreased expression of Olfm during the receptive phase of the endometrium may allow successful trophoblast attachment for implantation. [ABSTRACT FROM AUTHOR]

Abstract: Members of the claudin family play important roles in the formation of tight junctions in the kidneys, liver and intestine. Claudin-19 (Cldn19), a newly identified member of this family, is highly expressed in the kidney of the mouse. To have a better understanding on mouse claudin-19 gene expression, a 0.9-kb DNA fragment containing the 5′-flanking region of the Cldn19 gene was isolated. DNA sequence comparison between the mouse and human Cldn19 promoter regions exhibited little homology. One transcription initiation site was located at 104 nucleotides upstream of the start codon (ATG) of the Cldn19 gene. The mouse claudin-19 promoter lacked typical CAAT or GC-box. Deletion constructs of the 0.9-kb DNA fragment were generated and fused to a promoterless luciferase (Luc) reporter plasmid. Transfection studies using various kidney cell lines (MDCK, mIMCD3 and HEK293) revealed that the minimal promoter fragment resided in the −39 to −108 region, which contained a number of binding sites for transcription factors including Sp1. Site-directed mutagenesis using specific oligo probes confirmed that Sp1 was crucial for Cldn19 transactivation in the three cell lines studied. Electromobility shift assay confirmed that the nuclear extracts of these cells bound to the Sp1 oligo derived from Cldn19 promoter, but not to the mutated Sp1 oligo probe. Moreover, this DNA–protein complex would be recognized by Sp1 antibody, indicating specific Sp1 binding. Collectively, our data suggest that Sp1 binds to the claudin-19 promoter region and is responsible for its expression in the kidney cell lines in vitro. [Copyright &y& Elsevier]

To evaluate the attachment rate of a human embryonic stem cell–derived trophoblastic spheroid onto endometrial epithelial cells in predicting the cumulative live birth rate of an in vitro fertilization (IVF) cycle. A prospective observational study. University hospital and research laboratory. A total of 240 infertile women from 2017–2021. Infertile women with regular cycles attending for IVF were recruited. An endometrial aspirate was collected from a natural cycle 1 month before IVF to determine the BAP-EB attachment rate. Cumulative live birth rates from a stimulated cycle and its derived frozen embryo transfer cycles within 6 months of ovarian stimulation were obtained. The BAP-EB attachment rate in women who attained a cumulative live birth was similar to that in those who did not. When women were stratified by age into <35 years and ≥35 years, the BAP-EB attachment rate was significantly higher only in women aged ≥35 years having a live birth when compared with those in the same age group without a live birth. Receiver operating characteristic curve analysis of BAP-EB attachment rate in predicting cumulative live birth showed the areas under the curve of 0.559 (95% confidence interval [CI], 0.479–0.639), 0.448 (95% CI, 0.310–0.585), and 0.613 (95% CI, 0.517–0.710) for all ages, an age of <35 years, and an age of ≥35 years, respectively. The BAP-EB attachment rate offers only a very modest prediction of the cumulative live birth rate in women aged ≥35 years undergoing IVF. NCT02713854 (https://clinicaltrials.gov/ct2/show/NCT02713854 ; Date of registration, March 21, 2016; date of enrollment of the first subject, August 1, 2017). [ABSTRACT FROM AUTHOR]

Objective: To study the use of human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) as human blastocyst surrogates for studying early implantation and trophoblast development.Design: Laboratory study.Setting: University research laboratory.Patient(s): Infertile in vitro fertilization patients donating endometrial aspirates and human embryonic stem cells (hESCs: VAL3 and H9/WA09).Intervention(s): In BAP-EB derived from hESC, transcriptomes analyzed by next-generation RNA sequencing, effects of Hippo signaling pathway studied by a YAP inhibitor, comparison of attachment of BAP-EB onto primary endometrial epithelial cells (EEC) collected at prereceptive and receptive phases, and antibody blocking assay used to study the molecule(s) involved in BAP-EB attachment.Main Outcome Measure(s): Gene expression profiles and endometrial cell attachment rates.Result(s): The BAP-EB differentiation protocol for VAL3 could be used to induce trophoblast differentiation in another hESC line, H9. Transcriptomic analysis showed that the epiblast signature gene expression was reduced while that of the trophoblast was induced during BAP-EB differentiation. Specifically, trophectoderm signature genes were induced in BAP-EB at 48 hours and 72 hours after induction of differentiation. The Hippo signaling pathway was one of the pathways induced during BAP-EB differentiation, and YAP1 inhibitor statistically significantly reduced attachment, outgrowth, and trophoblast gene expressions of BAP-EB. A statistically significantly higher number of BAP-EB derived from both VAL3 and H9 attached onto receptive EEC than prereceptive EEC. The antibody blocking assay demonstrated that endometrial E-cadherin might be critical in early implantation.Conclusion(s): The data suggest that BAP-EB possesses a trophectoderm-like signature, which supports the use of BAP-EB as a blastocyst surrogate for the study of trophoblast development and endometrial receptivity. [ABSTRACT FROM AUTHOR]