| Part Ⅰ Alterations in the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technologyObjective:To investigate the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology (ART) when compare with the frequency of these mutations in control offspring conceived from spontaneous pregnancies, to illuminate the effects of IVF/ICSI on the genome stablility.Materials and methods:A prospective clinical observational study was performed on246families recruited from an in vitro fertilisation (IVF) centre at a tertiary-care, university-affiliated teaching hospital from2008to2012. The study included147ART families [75IVF and72intracytoplasmic sperm injection (ICSI)] in the study group and99natural-conception families in the control group.Parental, umbilical cord and infant peripheral blood samples were collected, and the trinucleotide repeats of the ATN1, AR, ATXN1, ATXN3, Huntington, DMPK and FMR-1genes were investigated between the generations; these genes were chosen due to their ability to undergo dynamic mutation. The frequencies and sizes of the mutational repeats, as well as the intergenerational instability, were measured. Result (s):In2466transmissions identified in the ART offspring,2.11%(n=52/2466) of the alleles were unstable upon transmission, while in the control group offspring, the frequency of dynamic mutation was0.77%(n=10/1300); this difference was statistically significant. The unstable transmission alleles were detected in32(2.48%) of the1288alleles fromthe IVF offspring and in20(1.70%) of the1178alleles from the ICSI offspring; both of these frequencies were significantly different from that of naturally conceived offspring (0.77%). However, there were no significant differences in the sizes of the mutational repeats or in the rates of expansion or contraction among the three groups. The repeat copy numbers of the examined genes were found to be within the normal ranges in all parents and infants.We divided the infertile couples into several categories, such as female factor (tubal infertility, endometriosis and anovulatory), male factor (oligozoospermia, asthenozoospermia, teratozoospermia and obstructive azoospermia), male and female factor, unexplained infertility. Unstable transmission appears to occur more frequently in the families who required ART because of female only or both male and female infertility, and this difference was significant in the ICSI group.Conclusion (s):1. There is a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring.2. In this study, we report an association between ART and the frequency of dynamic mutation. The instability could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation and/or the in vitro culture conditions. Part Ⅱ Effects of ART on DNA damage repair associated genes in ART offspringObjective:To investigate the expression and the promoter methylation levels of DNA damage repair associated genes in ART offspring, to illuminate the root cause of genome instability in ARToffspring.Materials and methods:We collected52ART conceived, term and singleton placentas (32IVF and20ICSI) and32comparative spontaneously conceived placentas. The birth weights of babies, materal age and gestational weeks were recorded.Based on the results of the Part Ⅰ, a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring. The DNA damage repair associated genes were analyzed in the placenta, including the exapression and DNA methylation.(1) mRNA expression of DNA damage repair associated genes Gene expressions of OGG1, MSH6, MSH2, MSH3, MLH1, MLH3, PMS2, PMS1, XPA, APEX1and RPA1were analyzed by real-time quantitative PCR.(2) DNA methylation status of DNA damage repair associated genes The methylation status of CpG inslands of OGG1, RPA1,PMS2, MSH6and XPA was determined by pyrosequencing.(3) Protein expression of DNA damage repair associated genesThe protein expressions of MLH1, OGG1,RPA1, PMS2, MSH2, MSH6and XPA were detected by western blotting.Result (s): (1)Gene expressions of PMS2, RPA1, XPA, MSH2, MSH6were significantly higher in the IVF and ICSI groups than in the control group. In addition, gene expression of MLH1was significantly higher in IVF group when compared with the ICSI and control groups. The gene expression of MSH2, XPA,OGG1and RPA1was significantly higher in the ICSI group when compared with the IVF group.(2) Both IVF and ICSI groups showed significant differences in the DNA methylation rate of OGG1, RPA1, PMS2, MSH6and XPA..(3)The protein expression of PMS2, MSH6and MLH1were significantly higher in the ICSI group as compared with the IVF and control groups.Conclusion (s):1. The alterations in gene expression of DNA damage repair associated genes could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation, ICSI and/or the in vitro culture conditions.2. ART procedures and the background of infertility could affect the epigenetic modification such as DNA methylation.3. The inconsistence expression between mRNA and protein may be the result of the post-transcript regulation. Part Ⅲ Effects of ART on DNA oxidative stress and neurodegenerative changes in elder miceObjective:To investigate the expression of ART on DNA oxidative stress and the neurodegenerative disease associated genes in elder mice, to determine the risk of neurodegenerative changes by ART.Materials and methods:Mice of the C57BL/6J were used as oocyte and sperm donors. Superovulated mouse oocytes were fertilized in vivo constituted the control group. The ART groups were established containing IVF and ICSI conceived mice. In total,8controls,8IVF-conceived,8ICSI-conceived mice were examined at1.5years of age.8-OHdG was used as the biomark of DNA oxidative damage; the DNA repair associated genes, neurodegenerative disease associated genes and miRNA were analyzed in the brain of elder mice conceived by ART.(1) The level of8-OHdG in elder mice The level of8-OHdG was detected by ELIS A in elder mice(2) mRNA expression of DNA repair associated genes in elder miceGene expressions of Msh2, Apex1, Msh6, Ogg1, Pcna, Xpa and Mlhl were analyzed by real-time quantitative PCR.(3) mRNA expression of neurodegenerative disease associated genes and miRNA in elder miceGene expressions of Mapt, Bacel, Psen1,Psen2, App, Apoe,Igf-1, Igf-1r, Insr, Irs-1, Irs-2, miR-34and miR-101were analyzed by real-time quantitative PCR.(4) DNA methylation status of DNA repair associated genes in elder miceThe methylation status of CpG inslands of DNA repair associated genes were determined by pyrosequencing.Result (s): (1)8-OHdG:The level of8-OHdG was4.26±1.54pg/ml in the control group,9.59±4.27pg/ml in the IVF group and8.57±3.90pg/ml in the ICSI group. It was significantly higher in IVF and ICSI groups than in the control group. However, there was no significantly difference between the IVF group and ICSI group.(2) Gene expressions of Oggl, Apex1and Pcna were significantly higher in the IVF and ICSI groups than in the control group. In addition, the expression of Pcna was significantly higher inthe ICSI group when compared with the IVF groups. However, Gene expressions of Msh6, Msh2and Mlh1were significantly lower in the IVF and ICSI groups than in the control group.(3) Gene expression of Psenlwere significantly hifher in the ICSI groups than in the control group. Both IVF and ICSI groups showed significant differences in the expressions of Igf-1, Igf-1r, Insr, Irs-1, when compared with the control group. Both IVF and ICSI groups showed significant differences in the expressions of miR-34when compared with the control group, the expressions of miR-101in the ICSI group was significantly lower than in the IVF and control groups.(4) Both IVF and ICSI groups showed significant differences in the DNA methylation rates of Oggl and Mlhl.Conclusion (s):1. IVF/ICSI may induce higher risk of oxidative damage in the elder mice.2. IVF/ICSI procedure may alter the progress of neurodegenerative changes in old age, further investigation are needed. |
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