Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice.

Successful mouse embryo implantation requires a receptive uterus and an activated blastocyst. A large number of genes, cytokines, and other factors are involved in the process. MicroRNAs (miRNAs) regulate the expression of many genes, and previous studies have investigated the relationship between miRNA expression and embryo implantation. In this study, we show that mmumicroRNA-200a (mmu-miR-200a) is expressed in a spatiatemporal manner during implantation in mouse uterus and found that phosphatase and tensin homolog (PTEN), SON, and programmed cell death 4 (Pdcd4) are the target genes of mmu-miR-200a by bioinformatics analysis. In vitro gain and loss of function experiments confirm that PTEN, a critical gene for cell proliferation and apoptosis, is the target gene of mmu-miR-200a. Our experiments also show that injection of the uterine horn with mmu-miR-200a lentivirus leads to a decreased implantation rate. Collectively, our results suggest that mmu-miR-200a affects embryo implantation by regulating PTEN protein expression. Thus, clarifying the physiological functions of uterine miRNAs will help to elucidate the embryo implantation process and may even contribute to curing infertility and inventing new contraceptives.

Rapamycin (sirolimus) is an antiproliferative drug that has been widely used in the clinic as an immunosuppressant and a potential anticancer agent. Certain reports have indicated that rapamycin may induce male infertility through impairing sperm quality. The present study investigated the mechanism of male infertility caused by rapamycin and examined whether withdrawal of rapamycin could recover the number of sperm in rats. Male Sprague-Dawley rats (n=100) were divided randomly into 5 groups: 3 rapamycin-treated groups (2, 4 and 6 mg/kg) and 2 control groups [Blank and dimethyl sulfoxide (DMSO)]. Organ coefficients of the testes, number of sperm and hematoxylin-eosin staining analyses demonstrated that rapamycin treatment markedly damaged the structure of the seminiferous tubule and reduced the number of sperm. Immunohistochemistry of mechanistic target of rapamycin (mTOR) and Ki67 in testes tissue, and western blotting of phosphorylated-p70S6K and p70S6K, supported the hypothesis that rapamycin causes sperm reduction through inhibiting proliferation of spermatogonia. Unfortunately, 24 weeks after cessation of rapamycin treatment, only the number of sperm in 2 mg/kg group was restored back to the normal level. In addition, to the best of our knowledge, the present study was the first to demonstrate that low doses rapamycin leads to activation of autophagy in rat testes. This may be a self-protective mechanism of the cell in response to external stress. Thus, spermatogenesis can be recovered in the testes from rats in the low dose group. High doses of rapamycin resulted in excessive consumption of autophagy proteins, and the damage could not be compensated. In addition, it was revealed that cell apoptosis increased after treatment with rapamycin. In conclusion, the present study demonstrated that rapamycin inhibits spermatogenesis through suppressing phosphorylation of p70S6K and changing the autophagy status, ultimately reducing the number of sperm. These findings provide important guidance for the clinical application of rapamycin.

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.