Mice endometrium receptivity in early pregnancy is impaired by maternal hyperinsulinemia

Abstract

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.

Figures

Figure 1.

Number of implantation sites at D5 in insulin-treated and control mice. (A) Trypan blue was injected on D5 and the number of implantation sites as indicated by distinct blue bands was recorded. (B) Morphology of the uteri of pregnant mice at D5. Left image are the uteri from control mice and the right image are the uteri from insulin-treated mice. D5, day 5 of pregnancy.

Figure 2.

Implantation sites on D5 of pregnancy from control and insulin-treated mice were stained with hematoxylin and eosin (scale bar, 200 µm). No obvious morphological alterations between the two groups were identified.

Figure 3.

Expression of genes associated with endometrial receptivity during the embryo implantation window. Reverse transcription-quantitative polymerase chain reaction was performed to compare the mRNA expression levels of Esr1, Esr2, Pgr and Hoxa10 in the endometrium between control and insulin-treated groups at (A) D4, (B) D5 and (C) D6. Data are presented as the mean ± standard error (n=6). *P

Figure 4.

Endometrial mTOR signaling was altered during the embryo implantation window by maternal hyperinsulinemia. (A) An increase in the phosphorylation of mTOR and p70S6K following insulin treatment was determined using western blotting. (B) Quantification of protein expression levels. Data are presented as the mean ± standard error (n=3). *P