Effects Of Assisted Reproductive Technologies On DNA Damage Repair Genes And Mechanisms Involved

Part I Expression and methylation status of DNA damage repair genes in ART early fetusObjective:To investigate the expression and methylation status of DNA damage repair genes in ART early fetus,to illuminate the effect of ART process on DNA damage repair genes in early fetus and related mechanisms.Materials and methods:1.We collected IVF,ICSI and COS early fetus samples from ART triplet pregnancy underwent multifetal reduction during 2012 and 2015 in Obstetrics&Gynecology,School of Medicine,Zhejiang University,including 32 IVF,20 ICSI and 32 COS early fetuses.2.Real-time quantitative PCR was used to analyze the expressions of MSH2,MSH3,MSH6,MLH1,PMS2,OGG1,APEX1,XPA and RPA1 among three groups in early fetuses.3.According to the different expressed genes,pyrosequencing was used to analyze the methylation rates of MSH2,MSH3,MSH6,MLH1 and APEX1 among three groups in early fetuses.4.Western blotting was used to detect the protein expressions of MSH2,MSH3,MSH6,MLH1 and APEX1.Results:1.Gene expressions of MSH2,MSH3,MSH6,MLH1 and APEX1 were significantly lower in both IVF and ICSI groups compared to COS group.In addition,the expressions of PMS2 and XPA in ICSI group was significantly lower compared to COS group,and the expression of XPA in ICSI group was significantly lower compared to IVF group.2.Both IVF and ICSI groups showed significant differences in the DNA methylation rates of MSH2,MSH3,MSH6,MLH1 and APEX1 compared to COS group.Moreover,no obvious differences were detected in the DNA methylation rates between IVF and ICSI groups.3.No significant difference in protein expression of MSH2,MSH3,MSH6,MLH1 or APEX1 was detected among IVF,ICSI and COS groups.Conclusion:1.The alterations of gene expressions of some DNA damage repair genes in IVF/ICSI early fetuses could be explained by altered methylation rates of promoter region CpG islands.2.The similar alterations of gene expressions and methylation rates of some DNA damage repair genes in IVF/ICSI early fetuses indicated that this kind of alteration could be caused by the the common procedure of IVF and ICSI-in vitro operation and embryo culture.3.The inconsistent expressions between mRNA and protein levels of DNA damage repair genes may be the result of the post-transcript regulations.Part ? The effect of IVF on the expression and methylation status of DNA damage repair genes in the brainObjective:To investigate the expression and methylation status of DNA damage repair genes in the brain of IVF and IVO mouse model,and to illuminate the short-term,medium-term and long-term effects of IVF and mechanism involved.Materials and methods:1.The IVF and IVO mouse model were established by using C57BL/6J mice,containing three representative ages of mice groups.The IVO group was used as the control group.Left frontal lobe brain tissues from 6 IVF and 6 IVO mice at 3 wk,10 wk and 1.5 yr were examined.2.Real-time quantitative PCR was used to analyze the effects of IVF on gene expressions of MSH2,MSH3,MSH6,MLHl,PMS2,OGG1,APEX1,XPA and RPA1 at 3 wk,10 wkand 1.5 yr.3.Pyrosequencing was used to analyze the methylation rates of the different expressed genes at 3 wk,10 wk and 1.5 yr.4.Western blotting was used to detect the protein expressions of the different expressed genes at 3 wk,10 wk and 1.5 yr.Results:1.At 3 wk,gene expressions of MSH2,MSH6 and MLH1 were significantly lower in IVF group compared to IVO group.However,only MSH2 and MSH6 were significantly lower in IVF group compared to IVO group at 10 wk.By the age of 1.5 yr,gene expressions of MSH2,MSH6,MLH1,OGGI and APEX1 showed significantly differences between two groups2.At 3 wk,IVF group showed significant differences in the DNA methylation rates of MSH2,MSH6 and MLH1 compared to IVO group.At 10 wk,DNA methylation rate of MSH6 showed a significant difference,however,MSH2 showed no difference between two groups.By the age of 1.5 yr,there were significant differences in the DNA methylation rates of MSH2,MSH6,MLH1,OGGI and APEX]between two groups.3.At 3 wk,the expression of MSH6 protein was significantly lower in IVF group compared to IVO group.However,no significant difference of any protein was detected between IVF and IVO groups at 10 wk.By the age of 1.5 yr,the expressions of MSH6 and OGGI were significantly different between IVF and IVO groups.Conclusion:1.There existed alterations of gene expressions and methylation rates of some DNA damage repair genes in the brain of IVF mouse.2.Although the alterations of gene expressions and methylation rates of some DNA damage repair genes in the brain at 3 wk could be partially reversed at 10 wk,this kind of genes showed more significant modification at the age of 1.5 yr.Part ? The effect of in vitro operation and culture condition on DNA damage repair genes of mouse embryo and mouse embryonic stem cellObjective:To investigate the effect of in vitro operation and culture condition on the expressions of DNA damage repair genes of mouse embryos and mouse embryonic stem cells,and to illuminate the original main etiological factor that how the alterations of DNA damage repair genes happen.Materials and methods:Mice of the C57BL/6J were used as oocyte and sperm donors to establish IVF-EC and IVO embryo model.Using mouse 2-cell embryos and blastocysts cultured in different concentrations of oxygen and estradiol environment,real-time quantitative PCR was used to analyze the effects of different treatments on gene expressions of MSH2,MSH3,MSH6,MLH1,PMS2,OGG1,APEX1,XPA,RPA1 and DNMT1.Western blot and immunofluorescence were used to detect the protein expressions of the different expressed genes in mouse blastocysts.Meanwhile,the gene expressions and methylation rates of DNA damage genes in mouse embryonic stem cell cultured in different concentrations of oxygen and estradiol environment were detected.Results:1.The effect of in vitro operation and culture on the expressions of DNA damage repair genes and DNMT1 of mouse 2-cell embryosThere were no significant differences of the expressions of DNA damage repair genes and DNMT1of 2-cell embryos among IVO,20%O2IVF-EC and 5%O2IVF-EC groups.2.The effect of in vitro operation and culture on the expressions of DNA damage repair genes and DNMT1 of mouse blastocysts(1)Gene expressions of MSH2,MSH3,MSH6,OGG1,APEX1 and RPA1 were significantly lower in 20%O2 IVF-EC group compared to IVO group;DNMT1 was higher in 20%O2IVF-EC group compared to IVO group.And in 5%O2IVF-EC group,only expression of MSH2,MSH3,MSH6 and RPA1 were significantly lower compared to IVO group.Meanwhile,in 5%O2 IVF-EC group,the expressions of MSH2,MSH3 and MSH6 were significantly higher compared to 20%O2 IVF-EC group,closer to IVO group.(2)No significant difference of the expression of any protein was detected among three groups.3.The effect of estradiol on the expressions of DNA damage repair genes and DNMT1 of mouse blastocystsThere were no significant differences of the expressions of DNA damage repair genes among different concentrations of estradiol groups.4.The effect of oxygen on the expressions and methylation rates of DNA damage repair genes of mouse embryonic stem cellGene expressions of MSH2 and MSH6 were significantly higher in 5%O2 group compared to 20%O2 group;meanwhile,DNA methylation rate of MSH6 showed a significant difference.5.The effect of estradiol on the expressions and methylation rates of DNA damage repair genes of mouse embryonic stem cellThere were no significant differences of the expressions and methylation rates of DNA damage repair genes among different concentrations of estradiol groups.Conclusion:1.There existed alterations of gene expressions of some DNA damage repair genes in IVF-EC blastocysts.2.The alterations of gene expressions of some DNA damage repair genes in IVF-EC blastocysts could be related to exposure of high oxygen concentration,not to estradiol exposure.3.The high oxygen concentration of embryo culture environment could lead to the up-regulation of DNMT1 gene expression and result in different methylation statuses and gene expressions of DNA damage repair genes,which could be the possible mechanism of the alterations of gene expressions of some DNA damage repair genes in IVF-EC blastocysts.4.The high oxygen concentration of embryo culture environment could be the important cause of the alterations of gene expressions of some DNA damage repair genes in ART offspring.Promoting the use of low oxygen embryo culture system could be beneficial to reduce the safety risk caused by the alterations of gene expressions and methylation statuses of DNA damage repair genes.