Effects Of ART On The Expression And Modification Of Imprinted Genes In Human Early Fetus And Its Impact On Newborn Lipid Metabolism And The Mechanism Involved

In-vitro fertilization-embryo transfer (IVF-ET) and intracytoplasm sperm injection (ICSI) as the representative of assisted reproductive technology (ART), have been used widely in the world, there have been millions of children born by ART now. Lots of epidemiological studies have shown that in addition to the relative high rates of low-birth-weight, premature birth and birth defects, ART may also increased the risk of imprinting disorders such as BWS (Beckwith-Wiedemann syndrome) and AS (Angelman syndrome). Animal studies have also indicated that the ART process may impact on the epigenetic modification and expression of imprinted genes such as H19, Ascl2, Peg3, and Xist et al in ART offspring. In humans, limited information is available due to the scarcity of materials for research. The associated studies about human molecular mechanisms are restricted in the ART preimplantation embryos, cord blood, placenta tissues and peripheral blood samples of ART children. From totipotent blastomere or the inner cell mass, three germ layers grown cells differentiated after implantation to various tissues and organs formation, until the birth of the newborn complex changes have taken place in human genomic imprinting from the modification, regulation to the expression levels, few was known about gene expression and modification in human ART intrauterine fetus, which seems to be one of the difficulties for our investigation about the impact of ART process on the offspring genome epigenetic development and progression.Early in1995, Barker brought out the famous hypothesis——"fetal origins of adult disease hypothesis", which indicated that fetus birth weight change resulted from intrauterine growth restriction or overgrowth are closely connected with adult diseases such as obesity, diabetes and hypertension. Several studies have confirmed that the intrauterine nutrition alteration may lead to adult abnormal fat metabolism, insulin resistance, increased arterial blood pressure and renal structural changes. In2010, Motrenko et al further proposed the "embryo-fetal origin of diseases", which suggested that external negative factors exposure during embryo formation period may increase the incidence of adult chronic diseases, the epigenetic modifications change attributed to the adverse environmental exposures of gametes, embryos and fetal tissue is considered to be one of it’s pathogenesis. The interference of ART on gametes mature, sperm-egg binding and embryonic growth process, the birth weight and epigenetic modification alterations in ART child, are similar to the pathogenesis of "embryo-fetal origin of diseases", which indicate that ART may prompt increase the risk of cardiometabolic diseases. Now there is evidence that ART child may have increased blood pressure, fasting glucose and lipid levels. Recently our research team found that there were significantly changes of blood pressure, heart rate, blood lipids, glucose tolerance and insulin levels in old ART mice, which may be correlated with the gene expression and modification alterations of lipid metabolism regulator pathway——insulin-induced gene (INSIG), SREBP cleavage activation protein (SCAP) and sterol regulatory element-binding protein(SREBP). Whereas, few was reported about the mechanisms of lipid metabolism changes in ART child till now, how about the INSIG-SCAP-SREBP pathway gene expression and modification changes in ART fetus and placenta remain unkown.In2007, Kobayashi et al found that spermatozoa from infertile men frequently had abnormal levels of DNA methylation at imprinted loci, and may transmit these epigenetic aberrations to the offsprings, and the poorer sperm quality, the more obvious changes in methylation alterations. Later Kobayashi et al found out that in the ART samples which have abnormal DNA methylation,41%of the identical alterations were present in the parental sperm, this further proved that DNA methylation errors at imprinted loci after assisted conception may originate in the parental sperm. ICSI is increasingly being used to treat the most profoundly infertile couples, especially male infertility. However, because ICSI techniques bypass several barriers in the natural fertilization process, may carry a known or identifiable genetic or epigenetic abnormality to submit to the offspring. Therefore, investigate the epigenetic modification changes in oligoasthenozoospermia sperm for ICSI patients may contribute to the etiology analysis for the epigenetic alterations in human ICSI offspring.Fetus underwent multifetal reduction samples conceived from IVF, ICSI and COS were collected, moreover, we collect the IVF, ICSI and natural conceived singleton term placenta and cord serum to provide fetus basis to reveal the epigenetic changes of ART by detecting the expression and methylation levels of imprinted genes under alternative ART manipulations; examine the lipid level changes in ART neonates and to explore the related mechanism; according to the differentially expressed and/or methylated genes, we detect their methylation status in Oligoasthenozoospermia, in order to reveal the mechanism of epigentic alterations in ART offspring. Part I Expression and methylation status alterations of imprinted genes in ART early fetusObjectiveExamine the expression and methylation status of the imprinted genes IGF2, H19, SNRPN and UBE3A in ART early fetus, provide fetus basis on epigenetic changes in ART offspring.Materials and Methods1IVF, ICSI and control ovarian stimulation (COS) early fetus was collected from ART triplet pregnancy underwent multifetal reduction.2Quantitative real time RT-PCR (qRT-PCR) was used to analyse the imprinted genes IGF2, H19, SNRPN, UBE3A mRNA transcripts in early fetus, compare the expression level differences among three groups.3Pyrosequencing was used to analyse the methylation status of the DMR of these genes, the differences of methylation rates among the three groups were compared.Results1Expression levels of imprinted genes:(1) H19mRNA in IVF was significantly higher than that in COS group, there was no significantly difference between IVF and ICSI group;(2) No significantly difference of IGF2, SNRPN and UBE3A mRNA expression was found among three groups.2Methylation levels of these genes: (1) The methylation levels of IGF2DMR in either IVF or ICSI group were siginificantly higher than those in COS; whereas no significantly difference was found between IVF and ICSI group;(2) H19DMR methlation levels in IVF and ICSI groups were lower than those in COS group, no significantly difference was found in IVF group when compared with ICSI one;(3) The methylation levels of SNRPN DMR in IVF and ICSI groups were significantly higher than COS; whereas there was no significant difference between IVF and ICSI group;(4) UBE3A DMR methlation levels were comparable among three groups.Conclusions1ART procession includes sperm and ovum in vitro treatment, assisted fertilization, in-vitro manipulation and embryo culture etc, may affect the methylation levels of imprinted genes IGF2, H19, SNRPN, whereas the most traumatic ART process—ICSI, didn’t seem to have similar effect.2ART may significantly alter methylation levels of some imprinted genes in early fetus, however, only H19have the gene transcripts alteration, suggesting that there may be other mechanisms involved in epigenetic regulation of imprinted genes which can correct or reduce ART-induced modification changes in early fetus.3Lower methylation level and higher gene transcripts of H19were found in IVF fetus, speculate that IVF may be responsible for the increased risk of low birth weight in ART offspring. Part Ⅱ Effect of ART on the lipid metabolism and the mechanism involvedObjectiveExamine the total cholesterol, triglycerides, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) changes in ART umbilical cord serum; investigate the gene expression and methlation status of lipid metabolism related genes (INSIG-SCAP-SREBP pathway genes) in early fetus and term placenta. Evaluate whether there were lipid metabolism alterations in ART offspring and the mechanism involved.Materials and Methods1IVF, ICSI and COS early fetus samples were collected from ART triplet pregnancy underwent multifetal reduction; term singleton cord blood and placental tissue were collected from IVF, ICSI and natural pregnancy (NC) cesarean deliveries as well as the maternal blood.2Total cholesterol, triglycerides, HDL and LDL were measured in umbilical cord and maternal serum.3Quantitative real time RT-PCR was used to analyse the expression of INSIG1, INSIG2, SCAP, SREBF1, SREBF2mRNA in early fetus and placenta, compare the difference of expression levels among three groups.4According to the different expressed genes, analyse the methylation status of these genes by pyrosequencing.Results 1ICSI umbilical serum triglyceride level was significantly higher than IVF and NC group.2The expression levels of INSIG1mRNA in both fetus and placenta conceived by ICSI were significantly higher than those of IVF and control.3Significantly higher transcripts of SREBF1mRNA were found in the ICSI conceived placenta compared with natural pregnancy.4No significantly difference of INSIG2, SCAP, SREBF2transcripts was found among three groups either in fetus or in placenta.5In the fetus under ICSI, four of five INSIG1CpGs displayed lower DNA methylation rate, whereas, the methylation rate was similar between IVF and COS fetus.6All the five INSIG1CpG sites in IVF and ICSI placenta displayed lower methylation rate than natural pregnancy, moreover, INSIG1was demethylated in ICSI placenta. Otherwise, in all the seven SREBF1CpG sites, two in IVF and three in ICSI placenta displayed lower methylation levels than control.Conclusions1ICSI may significantly affect parts of INSIG-SCAP-SREBP pathway genes mRNA expression in fetus, and the alterations of the corresponding gene promoter CpG sites methylation levels may be involved in these changes.2These gene expression and methylation changes can be enhanced in term placenta, and may be associated with the elevated triglyceride in ICSI newborn cord blood.3IVF can also lead to methylation changes of parts of these genes, but no significantly alteration of gene expression and cord serum triglyceride level was found, indicating that the degree of alteration that IVF brought was relatively minor. Part III DNA methylation alterations of imprinted and non-imprinted genes in oligoasthenozoospermiaObjectiveAccording to the imprinted genes IGF2, H19, SNRPN and non-imprinted genes INSIG1, SREBF1, which we have observed the expression and/or methylation changes in the previous study, investigate their methylation status in different ART sperm.Materials and Methods1Collect the remaining sperm after the completion of the fertilization in IVF/ICSI process, patients were divided into two groups:normozoospermic group (for IVF) and oligoasthenozoospermia group (for ICSI).2Pyrosequencing was used to detect the methylation status of IGF2, H19, SNRPN, INSIG1, SREBF1.Results1The methylation levels of IGF2, SNRPN and INSIG1in oligoasthenozoospermia were comparable with those in normozoospermic.2Five of six H19CpG sites (CpG3,4,5,6) displayed significantly lower methylation rate in oligoasthenozoospermia when compared with those in normozoospermic.3Two of seven SREBF1CpG sites (CpG3, CpG4) displayed significantly higher methylation levels in oligoasthenozoospermia than those in normozoospermic. Conclusions1Methylation status alterations of H19and SREBF1may exist in oligoasthenozoospermia sperm.2No abnormal methylation alteration of IGF2, SNRPN and INSIG1was found in oligoasthenozoospermia sperm.3The methylation alteration trends of imprinted genes and non-imprinted genes in fetus and placenta are not consistent with those in the corresponding sperm, indicating that the epigenetic changes in ICSI treatments are not entirely from the father’s infertility background, the impact of alternative ART manipulations should be concerned.