Exposure To High Estrogen During Early Pregnancy Affects Lipid Metabolism And Epigenetic Mechanism Involved In Offspring

Part Ⅰ Follow-up study of offspring exposure to high estrogen during early pregnancyObjective:To study effect of high estrogen environment exposure during early pregnancy on lipid metabolism in offspring, analyze correlation between the mother’s estrogen levels during pregnancy and serum lipids in offspring, and determine the risk of intrauterine high estrogen environment induced hyperlipemia.Materials and Methods:In this study we followed up401single offspring born from assisted reproductive technology during January2002to July2010at the Obstetrics and Gynecology Hospital, School of Medicine, Zhejiang University, including142cases of natural pregnancy,153cases of fresh embryo transfer, and106cases of frozen embryo transfer. Follow-up indicators included the basic situation of offspring, the situation of its mother during pregnancy, and blood pressure, heart rate, fasting serum lipids of offspring. The peripheral blood was collected from patients undergone assisted reproductive therapy as well as the natural mother in the same period of pregnancy. Serum estradiol levels on the embryo transfer day and about8th week of pregnancy (singleton pregnancy) were detected. Correlation analysis between serum estrogen levels of mothers on embryo transfer day and lipid levels of offspring was carried out.Results:After exclusion of birth weight, birth length, gestational week, gender, age, BMI and maternal gestational age, there was no significant difference of blood routine, blood pressure, heart rate among offspring from natural pregnancy group, fresh embryo transfer group and frozen embryo transfer group, but serum lipid levels in offspring appeared to change. Total cholesterol and LDL cholesterol levels in fresh embryo transfer group were significantly higher than the other two groups, but there was no significant difference in frozen transfer group and control. There was no statistically significance of high-density lipoprotein cholesterol and triglycerides among the three groups. Patients undergone fresh embryo transfer group have taken ovulation induction treatment during pregnancy, and the estrogen levels on the embryo transfer day and8th week of pregnancy were significantly higher than the corresponding natural pregnancy group. Meanwhile, incidence of hypercholesterolemia (10.53%) in their offspring was significantly higher than control (3.29%). There was a significantly positive correlation between total cholesterol and low-density lipoprotein cholesterol levels of offspring and estrogen levels on embryo transfer day of mothers, but the high-density lipoprotein cholesterol and triglyceride levels of offspring and estrogen levels on embryo transfer day in mothers were not significantly correlated.Conclusion:Ovulation induction treatment of pregnant women in early pregnancy induces a super-physiological range of high estrogen state, and is the preferred model for the study of intrauterine exposure to high estrogen. The high estrogen state of the mother during pregnancy tends to cause hyperlipemia in offspring, and embryo cryopreservation technology has no effect on serum lipid levels in offspring. Intrauterine exposure to high estrogen increases risk of hypercholesterolemia of offspring. A positive correlation between high levels of total cholesterol and LDL cholesterol levels in offspring who have undergone estrogen exposure in utero and estrogen levels on embryo transfer day of mothers. Part Ⅱ Regulation of estrogen on lipid metabolism in hepatocytesObjective:To study effect of estrogen on lipid metabolism in vitro and its mechanism, specifically to ensure the role of estrogen in regulation of lipid metabolism.Materials and Methods:Human hepatoma cell line HepG2was cultured in vitro experiments. Different concentrations of estrogen and (or) estrogen receptor blocker were used to treat cells, and cholesterol content in cell supernatants were detected by ELISA; quantitative PCR technique was used to detect expression of HMGCR, CYP7A1, LCAT, LPL; Chromatin immunoprecipitation and luciferase activity were measured to detect region of estrogen response element binding with estrogen receptors in HMGCR promoter. High concentration of estrogen was used to treat primary mouse fetal liver cells, quantitative PCR technique was used to detect expression of HMGCR.Results:Estrogen can significantly upregulate the secretion of total cholesterol and low density lipoprotein in HepG2cells supernatant, and can increase the gene expression of HMGCR, but this upregulation will be completely blocked by estrogen receptor antagonist.17β-estradiol has no effect on the expression of CYP7A1, LCAT, LPL. There exists estrogen response element structure in the transcription initiation site-3081/-3069bp of HMGCR in HepG2cells, and estrogen can regulate the expression of HMGCR by estrogen response elements. High concentrations of17β-estradiol significantly upregulates the expressing of HMGCR in primary mouse fetal liver cells.Conclusion:High concentrations of estrogen can upregulate the expression of HMGCR in human hepatoma cell lines and primary fetal liver cells. estrogen increases the expression of HMGCR through estrogen response element structure in the transcription initiation site-3081/-3069bp, thus increases the cholesterol synthesis in the liver cells and plays the role in regulating lipid metabolism. Part III Establishment of intrauterine high estrogen mouse model and detection of methylation status of HMGCR in offspringObjective:To study the effect of intrauterine high estrogen on growth and lipid metabolism in mouse offspring, as well as the methylation status on the HMGCR DMR.Materials and Methods:We establish intrauterine high estrogen during pregnancy in a mouse model by ovulation induction technology, monitoring serum estrogen levels in pregnant mouse at different stages of pregnancy. Weight and liver proportion were detected in3-week-old and8-week-old offspring; Serum lipid levels were assayed by ELISA in3-week-old and8-week-old offspring. For12.5-day-old and3-week-old progeny liver, expression of HMGCR were detected by real-time quantitative PCR. Bisulfite sequencing method were taken to detect the methylation status of HMGCR DMR in the liver of the12.5-day-old and3-week-old offspring.Results:Ovulation induction technology can create a mouse model of intrauterine high estrogen status. There was no difference of weight in3-week-old and8-week-old offspring of the two groups, while the proportion of the liver was significantly higher in3-week-old offspring and no difference in8-week-old offspring. Serum TC, LDL-C, and TG levels were significantly higher in childhood, while there was no difference of HDL-C level between the two groups. Till adulthood, lipid levels in offspring from intrauterine high estrogen exposure were reduced, but the LDL-C was still significantly higher than control. Expression of HMGCR was significantly increased in12.5-day-old and3-week-old offspring from intrauterine high estrogen exposure, while the overall methylation level of HMGCR DMR in the liver of these two periods is significantly lower than control.Conclusion:Offspring born from intrauterine high estrogen exposure appeared abnormal phenotypes such as significantly higher lipid levels in childhood and adult stage. Expression of HMGCR was significantly higher, and methylation levels of HMGCR DMR were significantly reduced in the fetal period and childhood. Part IV LncRNA expression profiling of fetal liver exposure to high estrogen and Gm5196involved in regulation of cholesterol generationObjective:To systematically understand the influence of exposure to high estrogen on the long non-coding RNA (lncRNA) expression profiles in offspring. LncRNA associated with high cholesterol was screened, so as to verify its biological function and to explore underlying molecular mechanisms.Materials and Methods:Ten12.5days of ICR mouse livers are selected and using lncRNAs expression microarray analysis, five cases are from high estrogen intrauterine pregnancy while the other five are from normal pregnancy. Using real-time quantitative PCR validation of eight lncRNA which is significantly different in25cases of fetal liver tissue. Bioinformatics analysis of the results of the chip screening target lncRNA, and using real-time quantitative PCR method to verify the changes of expression in HepG2with estrogen treatment. After using specific RNA interference to reduce the expression of Gm5196, detect changes of Osbpl5and TC in cell supernatants.Results:The number of differentially expressed lncRNAs in intrauterine fetal liver tissue exposed to high estrogen is633, of which the number of up regulating the expression is316, while down regulating the expression is317. We verify eight significant differences lncRNA in the fetal liver, consistent with the results of the chip. CNC network diagram hints Gm5196may affect lipid metabolism.HepG2cell experiments have shown that estrogen can downregulate the expression of Gm5196and upregulate the expression of Osbpl5, and interference Gm5196can upregulate the expression of Osbp15and promote the secretion of total cholesterol.Conclusion:Intrauterine exposure to high estrogen environment changes the expression of lncRNAs spectrum in offspring, estrogen decreases the expression of lncRNA Gm5196, increases the expression of lncRNA Osbp15. Interference Gm5196can upregulate the expression of Osbpl5and promote the secretion of cholesterol. LncRNA Gm5196may be involved in epigenetic regulation on intrauterine exposure to high estrogen-induced high blood offspring.