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Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. This provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg-1 ·day -1 BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.

Circular RNAs (circRNAs), a class of newly discovered endogenous noncoding RNAs, have been found to play important roles in regulating gene expression and participate in several biological processes. However, their specific roles in embryo implantation remain unclear. This study aims to investigate the roles of circRNAs in embryo implantation. In the current study, we screened the expression profiles of circRNAs between implantation sites and interimplantation sites in the endometrial tissue of early pregnant mice on Day 5 using microarray assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of four dysregulated circRNAs was performed to validate the microarray results. Bioinformatics analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and circRNA-microRNA (miRNA)-messenger RNA (mRNA) negative correlation network analyses were performed. The microarray results showed 101 upregulated and 75 downregulated circRNAs (fold change [FC] ≥ 1.5 and p value < 0.05) at implantation sites compared with interimplantation sites. Four randomly selected circRNAs were successfully verified by qRT-PCR. The GO and KEGG analyses revealed some meaningful terms. Moreover, based on circRNAs microarray data and the miRNA and mRNA high-throughput data previously published by our groups, differently expressed circRNAs, miRNAs, and mRNAs were performed to conjoint analysis. Then circRNA-miRNA-mRNA negative correlation networks were constructed and 21 downregulated circRNAs, 14 upregulated miRNAs, and 79 downregulated mRNAs were involved in the networks. The findings of this study may provide a novel perspective on circRNAs and lay a foundation for future research into the potential roles of circRNAs in embryo implantation.

Embryo implantation is essential for normal pregnancy, and the process of decidualization is critical for embryo implantation. However, the mechanism of decidualization during early pregnancy is still unknown. Forkhead box O3a (FOXO3a) is the most important functional transcription factor of the forkhead box family and is a highly conserved transcription factor of apoptosis-related genes. In the mouse uterus, FOXO3a was found to be expressed regularly from Days 1-7 of early pregnancy. Upon further exploration, it was found that FOXO3a was expressed at significantly higher levels at the implantation site than at the interimplantation site on Days 5-7 of pregnancy. Under artificial decidualization, FOXO3a was highly expressed in the first and second decidual zones. After decidualization, the expression of FOXO3a was significantly increased both in vivo and vitro. In primary stromal cells, apoptosis was reduced by decreased expression of FOXO3a after inducing decidualization. Moreover, when FOXO3a-small interfering RNA was transfected into the uteri of mice, the expression of decidualization- and apoptosis-related factors was impaired. Thus, FOXO3a might play an important role in decidualization during early pregnancy, and cell apoptosis might be one of pathways for FOXO3a-regulated decidualization.

Folate deficiency results in abnormal embryonic development, but the underlying mechanisms remain to be comprehensively investigated. Mutation of Vangl genes belonging to the planar cell polarity (PCP) pathway is associated with abnormal embryonic development, but the effect of folate deficiency on the PCP pathway is unclear. In this study, we found that folate deficiency inhibited Vangl gene expression and Vangl protein binding to the ligand Dvl. As a methyl donor, folate can chemically alter the DNA methylation levels of genomic sequences. Here, reduced representation bisulfite sequencing (RRBS) was employed to detect the methylation profiles of mouse embryos. The results confirmed that folate deficiency affected the genomic methylation levels of mouse embryos, which resulted in down-regulation of key genes involved in embryonic development. Gene ontology (GO) analysis suggested that the genes located in the differentially methylated regions (DMRs) are primarily involved in biological regulation, cellular processes, development, metabolism, and signaling pathways. The data revealed that folate deficiency inhibits the PCP pathway and alters genomic methylation profiles, which may be the underlying mechanisms through which folate deficiency impairs embryonic development.