Previous research on the role of insulin has focused on metabolism. This study investigated the effect of insulin on angiogenesis in endometrial decidualization. High insulin-treated mouse model was constructed by subcutaneous injection of insulin. Venous blood glucose, serum insulin, P4, E2, FSH and LH levels in the pregnant mice were detected by ELISA. Decidual markers, angiogenesis factors and decidual vascular network were detected during decidualization in the pregnant mouse model and an artificially induced decidualization mouse model. Tube formation ability and angiogenesis factors expression were also detected in high insulin-treated HUVECS cells. To confirm whether autophagy participates in hyperinsulinemia-impaired decidual angiogenesis, autophagy was detected in vivo and in vitro. During decidualization, in the condition of high insulin, serum insulin and blood glucose were significantly higher, while ovarian steroid hormones were also disordered (P < 0.05), decidual markers BMP2 and PRL were significantly lower (P < 0.05). Uterine CD34 staining showed that the size of the vascular sinus was significantly smaller than that in control. Endometrial VEGFA was significantly decreased after treatment with high insulin in vivo and in vitro (P < 0.05), whereas ANG-1 and TIE2 expression was significantly increased (P < 0.05). In addition, aberrant expression of autophagy markers revealed that autophagy participates in endometrial angiogenesis during decidualization (P < 0.05). After treatment with the autophagy inhibitor 3-MA in HUVEC, the originally damaged cell tube formation ability and VEGFA expression were repaired. This study suggests that endometrial angiogenesis during decidualization was impaired by hyperinsulinemia in early pregnant mice.
Supplementary Figure 1. The expression of angiogenesis-related factors in the endometrium was disturbed by high levels of insulin. The expression of ANG-1 (A), TIE2 (B), VEGFA (C) and VEGFR2 (D) in the endometrium of pregnant mice on D6, D7 and D8 was detected by real-time PCR. Real-time PCR results showed that the expression of ANG-1, TIE2, VEGFA and VEGFR2 was disordered in the endometrium of mice with artificially induced decidualization (induced side) (E). n=3. * p<0.05. CON: control group; IN: high insulin group.