The mechanism of birth defects induced by folate deficiency was focused on mainly in fetal development. Little is known about the effect of folate deficiency on the maternal uterus, especially on decidual angiogenesis after implantation which establishes vessel networks to support embryo development. The aim of this study was to investigate the effects of folate deficiency on decidual angiogenesis. Serum folate levels were measured by electrochemiluminescence. The status of decidual angiogenesis was examined by cluster designation 34 (CD34) immunohistochemistry and the expression of angiogenic factors, including vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), and VEGF receptor 2 (VEGFR2) were also tested. Serum levels of homocysteine (Hcy), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), progesterone (P4), and estradiol (E2) were detected by Enzyme-linked immunosorbent assay. The folate-deficient mice had a lower folate level and a higher Hcy level. Folate deficiency restrained decidual angiogenesis with significant abnormalities in vascular density and the enlargement and elongation of the vascular sinus. It also showed a reduction in the expressions of VEGFA, VEGFR2, and PLGF. In addition, the serum levels of P4, E2, LH, and PRL were reduced in folate-deficient mice, and the expression of progesterone receptor (PR) and estrogen receptor α (ERα) were abnormal. These results indicated that folate deficiency could impaire decidual angiogenesis and it may be related to the vasculotoxic properties of Hcy and the imbalance of the reproductive hormone.

Previous studies have investigated the lower embryo implantation rates in women with polycystic ovary syndrome, obesity and type 2 diabetes, and specifically the association between the abnormal oocyte and embryo and hyperinsulinemia. The importance of hyperinsulinemia on maternal endometrium receptivity remains to be elucidated. The present study used a hyperinsulinemic mouse model to determine whether hyperinsulinemia may affect endometrial receptivity. An insulin intervention mouse model was first established. The serum levels of insulin, progesterone and estradiol were subsequently detected by ELISA assay analysis. The number of implantation sites was recorded using Trypan blue dye and the morphology of mice uteri was investigated using hematoxylin and eosin staining. The expression levels of molecular markers associated with endometrial receptivity were detected by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry analyses. Finally, the importance of mechanistic target of rapamycin (mTOR) expression following insulin treatment was determined. Mice treated with insulin developed insulin resistance and hyperinsulinemia. The number of implantation sites following insulin treatment did not differ between the control and insulin‑treated groups. Additionally, no significant morphological alterations in mice uteri between control and insulin‑treated groups were observed. However, the expression levels of estrogen receptor (Esr) 1, Esr2, progesterone receptor and homeobox A10 associated with endometrial receptivity, were imbalanced during endometrium receptivity when maternal hyperinsulinemia was induced. Western blot analysis revealed that expression levels of endometrial phosphorylated (p)‑mTOR and p‑ribosomal protein S6 kinase β‑1 were significantly greater in the insulin‑treated group. These results demonstrated that although an embryo may implant into endometrium, mice endometrium receptivity in early pregnancy may be impaired by maternal hyperinsulinemia. In addition, mTOR signaling may be involved in this process. The present study provides preliminary results demonstrating that female reproduction may be compromised during hyperinsulinemia, which requires further investigation in future studies.

It is well known that maternal folate deficiency results in adverse pregnancy outcomes. In addition to aspects in embryonic development, maternal uterine receptivity and the decidualization of stromal cells is also very important for a successful pregnancy. In this study, we focused on endometrium decidualization and investigated whether apoptosis, which is essential for decidualization, was impaired. Flow cytometry and TUNEL detection revealed that apoptosis of mouse endometrium decidual cells was suppressed in the dietary folate-deficient group on Days 7 and 8 of pregnancy (Day 1 = vaginal plug) when decidua regression is initiated. The endometrium decidual tissue of the folate deficiency group expressed less Bax compared to the normal diet group while they had nearly equal expression of Bcl2 protein. Further examination revealed that the mitochondrial transmembrane potential (ΔΨm) decreased, and the fluorescence of diffuse cytoplasmic cytochrome c protein was detected using laser confocal microscopy in normal decidual cells. However, no corresponding changes were observed in the folate-deficient group. Western blotting analyses confirmed that more cytochrome c was released from mitochondria in normal decidual cells. Taken together, these results demonstrated that folate deficiency could inhibit apoptosis of decidual cells via the mitochondrial apoptosis pathway, thereby restraining decidualization of the endometrium and further impairing pregnancy.