Effect Of High Insulin On Endometrial Function In Early Pregnant Mice

Objective: Hormones play a major role in the process of embryo implantation and maintenance of pregnancy.In the past,the study of hormones in pregnancy focused on the relationship between estrogen and progesterone and endometrial function.There is little report on whether insulin is involved in early stage endometrial receptivity and decidualization.Some study found that insulin receptors are present in the endometrium.Clinical women with hyperinsulinemia(such as obese patients)most associated with infertility and early abortion.Therefore,this study intends to explore the effect of hyperinsulin on the endometrial function of early pregnancy mice from the endometrial receptivity and endometrial decidualization in order to understand the regulation role of insulin on pregnancy process,to seek new means in diagnosis and treatment for infertility,spontaneous abortion caused by insulinemia.Methods:1.Establishment of hyperinsulinemia mouse model: Insulin was injected from subcutaneous as a experimental group and the same dose of physiological saline were injected as a conrol group.Blood glucose and body weight are detected.After 23 days,the serum levels of insulin in mice were measured by Elisa method,and it was judged whether the model of high insulin was successfully established.2.Establishment of pregnancy mouse model: After the insulin model was established,the female mice in the insulin treatment group and the control group were respectively mated with the normal male mice.Female and male are combined into a cage(female: male,2: 1)at 8 pm and the vaginal plug was examined at 8 am the next day.Appearance of vaginal plug indicate Day 1 of pregnancy(D1).The pregnant mice were labeled by picric acid.Endometrial tissues were collected on D5,D6,D7 and D8 days.Artificial decidualization model: The female mice of the insulin treatment group and the control group were respectively mated with vasectomized mices,and the appearance of vaginal plug indicate Day 1 of pseudopregnancy(PD1)in the next day.The 100μl corn oil was injected in the one side of uterine horn for inducing decidualization on PD4,the other side of uterine horn as control group.Uterine tissues were collected on PD8.3.Effect of high insulin on endometrial function in early pregnant mice: The levels of serum insulin,progesterone and estradiol were detected in mice by Elis method.The morphology of the uterus was observed.The microstructure of the uterus was observed by HE staining.The expression of endometrial receptivity related markers(such as Esr1,Esr2,Pgr and Hoxa10)were analyzed by Real-time quantitative PCR,Western blotting and immunohistochemistry on D4 and D5 and the expression of endometrial decidualization related markers(such as Pgr、Esr2、Bmp2 and Prl)were analyzed by Real-time quantitative PCR,Western blotting and immunohistochemistry D6,D7 and D8.The expression of mTOR signaling pathway was analyzed by western blot.Finally,we compared with experimental group and control group in the number of days of pregnancy and the number of pups per litter.Results:1.Hyperinsulinemia mouse model and serum hormone analysis: Levels of blood glucose and serum insulin in insulin-treated group mice were significantly higher than those in the control group,it indicated that establishment of the model was successfull.In addition,the blood glucose and HOMA values in the insulin-treated group were significantly higher than those in the control group,suggesting that insulin resistance was observed in the insulin-treated mice.The levels of serum E2 in the insulin-treated group were significantly lower than those in the control group(P <0.05),P4 were significantly higher than those in the control group(P <0.05).2.Effects of hyperinsulin on endometrial receptivity in early pregnancy mice: Compared with the control group,the general morphology and microstructure of D4 and D5 days were not significantly different from those in the control group,and the number of embryo implantation on D5 was not significantly different(P>0.05).However,the expression of Esr1,Pgr and Hoxa10 mRNA of insulin-treated group in endometrial tissue on D4 was significantly higher than that of control group(P<0.05),while the expression of Esr2 mRNA was decreased.The expression of Esr2 and Hoxa10 mRNA of insulin-treated group on D5 was significantly higher than that in control group(P<0.05).The expression of Esr1 mRNA was significantly lower than that of control group(P<0.05).The expression of Esr2 and Hoxa10 proteins was consistent with mRNA expression by western blot.Immunohistochemical results showed that: The expression of Esr1,Esr2,Pgr and Hoxa10 of the uterine epithelial cells and stromal Nuclei in control group on D4 were brown(positive expression).Esr1,Pgr and Hoxa10 in the insulin-treated group were dark brown(strong positive expression),while Esr2 Show brown(positive expression).The expression of Esr2,Pgr and Hoxa10 of the uterine epithelial cells and stromal Nuclei in control group on D5 were brown(positive expression).The expression of Esr2,Pgr and Hoxa10 in the insulin-treated group were dark brown(positive expression),while Esr1 Show brown(positive expression).3.Inhibitory effect of high insulin on endometrial decidualization of pregnant mice: Compared with the control group,there was no significant difference in the uterine shape on D6 in the insulin-treated group.The morphology of embryo implantation point on D7 and D8 in insulin-treatment group was smaller than those in the control group.At D6,D7 and D8,the implantation numbers were significantly reduced(P<0.05),and the microstructure of embryo layer showed partial hypoplasia.In the artificial induced decidualized model,uterine shape was irregular,decidual cells of cytoplasm Vacuolization can be observed.At D6,D7 and D8,The expression of Prl,Bmp2,Pgr and Esr2 mRNA in the decidual tissue of the insulin-treated group were significantly lower than those in the control group(P<0.05)and The expression of Prl,Bmp2,Pgr and Esr2 protein were also significantly lower than that of the control group(P<0.05).The expression of Pgr and Esr2 immunohistochemistry showed that the uterine epithelial cells and stromal nuclei in the control group were brown(positive expression),while the expression of Pgr and Esr2 in the insulin treated group were light yellow(weak positive expression).On the D6 and D7 days,Bmp2 expression of uterine epithelial cells and stromal nuclei were brown in the control group(positive expression),showed dark brown on D8(strong positive expression),but Bmp2 expression in the insulin-treated group showed light yellow(weal positive expression)on D6,D7 and D8.Prl expression was dark brown(strong positive expression)in the uterine epithelial cells and stromal nuclei on D6,D7 and D8 in the control group,while it showed light yellow(weak positive expression)in the insulin-treated group.4.Effect of hyperinsulin on mTOR pathway in endometrium of early pregnancy: By analyzing The expression of mTOR,p-mTOR,p70S6 K and p-p70S6 K proteins in the endometrial tissue of D4,D5 and D6,the expression of p-mTOR and p-p70S6 K protein in the insulin-treated group were Significantly higher than those in the control group(P<0.05).5.The levels of high insulin resulted in an increase in the number of days of pregnancy and a decrease in the number of enbryos: The average number of days of gestation in the insulin treated group(21 ± 0.71 days)was significantly higher than that in the control group(18 ± 0.50 days)(P <0.05),while the average per litter of mice(10 ± 0.50)was significantly lower than that in the control group(13 ± 1.12)(P <0.05).Conclusions:1.Blood glucose and HOMA value in the insulin-treated group was significantly higher than the control group,suggesting that mice develop insulin resistance under the action of high level insulin.E2 was significantly lower than that of the control group and P4 was significantly higher than that of the control group,suggesting that high level insulin can lead to hormonal disorders in pregnant mice.2.Early embryo implantation can be divided into two critical periods: The first period,the blastocyst implant in the uterus to obtain implantation ability and endometrium can accept blastocyst.This period is also known as "the implantation window period".The second period,Blastocysts are completely embedded into the endometrium,induced endometrial decidualization.The number of embryos and the microstructure of the endometrium were not significantly changed in the insulin-treated group,but the expression of receptivity-related genes and proteins Esr1,Esr2,Pgr and Hoxa10 in the endometria were abnormal.The number of embryos and the microstructure of the endometrium were not significantly changed in the insulin-treated group,but the expression of receptivity-related genes and proteins Esr1,Esr2,Pgr and Hoxa10 in the endometrium were abnormal.It is suggesting that the endometrial receptivity of early pregnancy might slightly suffer from damaged under the level of high insulin,although embryo can be implanted.3.During endometrial decidualization,the number of embryos significantly reduced and the shapes of some embryo implantation showed abnormalities in insulin-treated group.The expression of decidualization-related genes and proteins(Prl,Bmp2,Pgr and Esr2)were all abnormal.The results suggested that the level of high insulin might affect the endometrial decidualization and result in embryonic development damage.4.The level of high insulin in the blood of pregnant mice can increased the number of days of pregnancy and decrease number of embryos.5.The expression of p-mTOR and p-p70S6 K protein in the endometrium of pregnant mice with insulin-treated were significantly enhanced,suggesting that mTOR signaling pathway may be involved in the process which induced damage of endometrial receptivity by hyperinsulin,but the specific mechanism is unclear and needs further the study.