ACE inhibitors on ACE1, ACE2, and TMPRSS2 expression and spheroid attachment on human endometrial Ishikawa cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells via receptor angiotensin-converting enzyme 2 (ACE2) and co-receptor transmembrane serine protease 2 (TMPRSS2). However, patients with SARS-CoV-2 infection receiving ACE1 inhibitors had higher ACE2 expression and were prone to poorer prognostic outcomes. Until now, information on the expression of ACE1, ACE2, and TMPRSS2 in human endometrial tissues, and the effects of ACE inhibitors on embryo implantation are limited. We found human endometria expressed ACE1, ACE2, and TMPRSS2 transcripts and proteins. Lower ACE1, but higher ACE2 transcripts were found at the secretory than in the proliferative endometria. ACE1 proteins were weakly expressed in endometrial epithelial and stromal cells, whereas ACE2 and TMPRSS2 proteins were highly expressed in luminal and glandular epithelial cells. However, ACE1 and TMPRSS4 were highly expressed in receptive human endometrial epithelial (Ishikawa and RL95-2) cells, but not in non-receptive AN3CA and HEC1-B cells. Treatment of human endometrial epithelial cells with ACE1 (Captopril, Enalaprilat, and Zofenopril) or ACE2 (DX600) inhibitors did not significantly alter the expression of ACE1, ACE2 and TMPRSS2 transcripts and spheroid (blastocyst surrogate) attachment onto Ishikawa cells in vitro. Taken together, our data suggest that higher ACE2 expression was found in mid-secretory endometrium and the use of ACE inhibitors did not alter endometrial receptivity for embryo implantation.

High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation.

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 ß-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.

Bisphenol A Analogues Suppress Spheroid Attachment on Human Endometrial Epithelial Cells through Modulation of Steroid Hormone Receptors Signaling Pathway.

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.

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for 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 ß3 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.

Differential expression of protein disulfide isomerase (PDI) in regulating endometrial receptivity in humans.

Estrogen and progesterone regulate the expression of endometrial proteins that determine endometrial receptivity for embryo implantation. The protein disulfide isomerase (PDI) family of proteins play a diverse role in regulating protein modification and redox function. Although the role of PDIs in cancer progression has been widely studied, their role in endometrial receptivity is largely unknown. We have focused on the expressions of PDIA1, PDIA2, PDIA3, PDIA4, PDIA5, and PDIA6 isoforms in endometrial epithelium under the influence of estrogen and progesterone and investigated their functional role in regulating endometrial receptivity. We found PDIA1-6 transcripts were expressed in endometrial epithelial Ishikawa, RL95-2, AN3CA, and HEC1-B cell lines. The expression of PDIA1 was low and PDIA5 was high in HEC1-B cells, whereas PDIA2 was high in both AN3CA and HEC1-B cells. In Ishikawa cells, estrogen (10 and 100 nM) upregulated PDIA1 and PDIA6, whereas estrogen (100 nM) downregulated PDIA4 and PDIA5; and progesterone (0.1 and 1 µM) downregulated transcript expressions of PDIA1-6. In human endometrial samples, significantly lowered transcript expressions of PDIA2 and PDIA5 were observed in the secretory phase compared with the proliferative phase, whereas no change was observed in the other studied transcripts throughout the cycle. Inhibition of PDI by PDI antibody (5 and 10 µg/mL) and PDI inhibitor bacitracin (1 and 5 mM) significantly increased the attachment of Jeg-3 spheroids onto AN3CA cells. Taken together, our study suggests a role of PDI in regulating endometrial receptivity and the possibility of using PDI inhibitors to enhance endometrial receptivity.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.