High Estradiol Exposure Alters CDKN1C And IGF2 Expression In Human Trophoblast Cells Through DNA Methylation

IntroductionControlled ovarian stimulation(COS),one of assisted reproductive technology(ART),has been shown to induce maternal supra physiological estradiol(E2)levels which might influence oocyte,embryo and fetus development.Our previous study indicated that the maternal high-E2 levels in the first trimester were correlated with increased risks of low birth weight(LBW)and small-for-gestational-age(SGA)birth.However,little is known about the exact underlying mechanisms.Estrogen exposure can regulate relative gene expression through epigenetic mechanisms.There are many imprinted genes located in placenta regulating fetal growth.Their expressions are controlled by the DNA methylation of their imprinting control regions.Our previous study also indicated that the mRNA expression of CDKN1C and IGF2 were significantly up-regulated and the DNA methylation was significantly changed in their differential methylation regions(DMRs)in ART conceived placentas.Whether these changes are derived from the maternal high E2 exposure is also unknown.In the present study,we compared the birth weights between ART conceived singletons and the naturally conceived(NC)ones,and investigated the relationship between the birth weights and the maternal E2 levels on the day of human chorionic gonadotropin(hCG)administration.We treated human trophoblast cells(HTR8)with E2 and analyzed the mRNA expression of CDKN1C and IGF2 genes and the DNA methylation of their differential methylation regions(DMRs),in order to illuminate the mechanisms how maternal high E2 exposure induced by ART influences the birth weight of offspring.Materials and Methods1.One thousand and twenty-three(1023)singletons conceived after COS and fresh embryo transfer were included as the ART group.One thousand two hundred and twenty-six(1226)NC singletons with the gestational and the maternal ages matched to the fresh ET singletons served as the control group.The birth weights of the two groups and the birth weights between groups divided according to the median value of maternal E2 levels on the day of hCG administration were compared.2.The human first trimester trophoblast cell line HTR8 was treated with 17-?-estradiol at the concentrations of 0(control),10-9,10-7 and 10-5 mol/L for 24 hours(h)and 48 h respectively to simulate the physiological and high E2 exposure in vivo.3.The reverse transcription(RT)and Real time PCR was used to analyze the mRNA expression of CDKN1C and IGF2 in the cells treated with E2.4.The bisulfite sequencing was used to investigate the DNA methylation status in the DMR of CDKN1C(KvDMR1)and the DMR of IGF2(H19 DMR).Results1.Perinatal outcomes of singletons born after ART and NCThere was no significant difference in birth weight between ART and NC group(p>0.05).However,when dividing the ART singletons into two groups according to the median value of maternal E2 levels(9,974 pmol/L)on the day of hCG administration,we found that the mean birth weight of the high-E2 group(E2>9,974 pmol/L)was significantly lower than that of the low-E2 group(E2?9,974 pmol/L)(p<0.05)and the NC group,respectively(p<0.05).2.The mRNA expression of CDKN1C and IGF2 genes in the HTR8 cells treated with E2The dose-dependent and time-dependent experiments in the human first trimester trophoblast cell line HTR8 cells showed that the treatment with the E2 concentration of 10-5 mol/L for 24h or the E2 concentrations ?10-9 mol/L for 48h could significantly up-regulate the mRNA expression of CDKN1C(p<0.01).The mRNA expression of IGF2 was not altered by the treatment of E2 for 24h,but up-regulated by the treatment with 10-5 mol/L E2 for 48h(p<0.05).3.DNA methylation patterns of KvDMR1 and H19 DMR in the HTR8 cells treated with E2The in vitro treatment of the HTR8 cells with E2 showed that both the treatment with 10-5 mol/L E2 for 24h and the E2 concentrations>10-7 mol/L for 48h could significantly down-regulate the DNA methylation percentage of KvDMRl(p<0.01).Although the treatment of E2 for 24h could not change the DNA methylation percentage of H19 DMR,the treatment with 10-5 mol/L E2 for 48h significantly up-regulated the DNA methylation percentage of H19 DMR(p<0.05).ConclusionThe high maternal E2 levels on the day of hCG administration(>9,974 pmol/L)were associated with the lower birth weight of offspring conceived after fresh embryo transfer.The supra physiological E2 treatment could significantly upregulate CDKN1C and IGF2 mRNA expression and changed the DNA methylation in their DMRs in human trophoblast cells.The supra physiological E2 levels of women after ART could upregulate the expression of imprinted genes CDKN1C and IGF2 via altering the DNA methylation in their DMRs,which might affect the growth potential of the offspring,leading to lower birth weight.