The Epigenetic Mechanism Of Low Birth Weight/Small For Gestational Age Of Offspring Exposure To High Maternal Estradiol Level In First Trimester Pregnancy

Part I:Low birth weight/small for gestational age and the maternal serum estradiol levels in first trimester pregnanciesObjective:To evaluate the birth weights of multifetal pregnancies reduced to singletons or twins, compare the maternal serum estradiol (E2) levels of early singleton pregnancies with those of multiple pregnancies and analyze the correlation between maternal serum E2levels and the birth weights of their offsprings.Materials and Methods:A retrospective review was performed on data obtained from patients between January2006and June2010undergoing assisted reproductive technology (ART) at the Assisted Reproductive Unit of the Women’s Hospital, School of Medicine, Zhejiang University. Spontaneous or selective reduction of one or more gestational sacs and/or embryos occurred to singletons or twins before the12th week of gestation were included. The control group included assisted reproduction patients with non-reduced singletons or twins performed over the same period. Non-reduced singleton was matched by a stratified random selection. Compare the perinatal outcomes of reduced groups with that of their own controls. Peripheral blood samples of195women with ART conceptions on7-8weeks’ gestation were collected and measured serum E2levels with Roche cobas immunoassay.Results:1.The birth weights of singletons and twins after reduction were significantly lower than non-reduced pregnancies (3217.05±546.88g vs3377.85±477.05g, P=0.009and2428.80±485.64g vs2495.31±520.06g, p=0.016, respectively). The rates of small for gestational age (SGA) after reduction were significantly higher than their own control (P<0.05). No significant difference was observed in duration of gestation (P>0.05).2. The serum E2levels for multiple pregnancies were significantly higher than those for singleton pregnancies (P<0.05), the serum E2levels elevated with the increased number of fetuses.3. Multivariate correlation analyses showed that maternal serum E2level shows a inverse correlation with the birth weight of offspring (r=-0.32, P=0.018).Conclusion:Early multiple pregnancies with high maternal serum E2level can lower the birth weight and increase the risk of SGA, and the adverse effect could not be eliminatec by early multifetal reduction. The high maternal serum E2levels in first trimeste pregnancies may play an important role in low birth weight and SGA. Part II:Expression and methylation status of imprinting genes in fetal tissues, umbilical cord blood and placenta tissuesObjective:To investigate epigenetic status of early multiple pregnancies.Materials and Methods:Fetal tissues of multiple pregnancies were collected from multifetal pregnancy reduction (MFPR), and fetal tissues of singleton pregnancies were collected from elective terminations of normal pregnancies as control group. Umbilical cord blood (UCB) and placenta tissues from reduced twins and primary twin pairs were collected. The expression of IGF2, H19, CDKN1C, PHLDA2and DNMT1was determined by real-time quantitative PCR. The methylation status of H19DMR and KvDMR1was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP).Results:CDKN1C and DNMT1mRNA levels were significantly higher in early fetal tissues of multiple pregnancies than singleton pregnancies. Consistent with the results in fetal tissues, the expression levels of CDKN1C and DNMT1in UCB and placenta tissues of reduced twin pairs were significantly increased when compared to their matched twins. KvDMRl was hypermethylated in fetal tissues of MFPR, UCB and placenta tissues of reduced twins. Significant upregulation level of IGF2was also observed in fetal tissues of multiple pregnancies, but the methylated level of H19DMR was normal. The expression levels of H19and PHLDA2in fetal tissues of multiple pregnancies were unchanged when compared with their controls.Conclusion:Estradiol may induce DNMT1overexpression and thus, promote hypermethylation of KvDMRl and overexpression of CDKN1C, which is a critical mechanism for low birth weight and SGA of early multiple pregnancies. Part Ⅲ:The mechanism of estradiol regulating the expression of CDKN1CObjective:To investigate the mechanism of estradiol (E2) regulating the expression of CDKN1C.Materials and Methods:HTR8/SVneo (HTR8) cells were exposed to E2and/or estrogen receptors antagonist (ICI182780), DNMT1small interfering RNAs (siRNAs). The expression of CDKN1C and DNMT1was determined by real-time quantitative PCR. The methylation status of KvDMR1in10-5M E2treated HTR8cells was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP). DNMT1promotor-pGL-3plasmid was constructed and luciferase activity was measured using the dual luciferase reporter assay system. Chromatin immunoprecipitation (ChIP) experiments were performed to detect the binding of ERa on the ERE-like site of DNMT1promoter.Results:E2increased the expression of CDKN1C, DNMT1and the methylation level of KvDMR1in HTR8cells. The effects were inhibited by ERs antagonists and DNMT1siRNA. ERa activated DNMT1transcription through an estrogen responsive elements (ERE) located at-659/-647bp upstream of the transcriptional start site. Conclusion:E2can promote DNMT1transcription through an ERE located at the transcriptional start site. High expression of DNMT1is associated with the hypermethylation of KvDMRl and the upregulation of CDKN1C. This is may be one of the important reasons for the increase of low birth weight and SGA.