Effect Of ART On Epigenetic Modification Mechanisms In Pre-implantation Mouse Embryos

Objective:1.To explore the difference of genome-wide DNA methylation level in vivo fertilization(IVO)embryos(control group)?superovulation embryos?in vitro fertilization(IVF)embryos?in vitro maturation(IVM)embryos at the blastocyst stage2.To explore the effect of ART on imprinted genes Igf2 and Grb10 expression in all the above four groups embryos.3.To explore the effect of ART on CGI1 methylation level in the imprinted gene Grb10 at the blastocyst stage4.To explore the application security of ART from epigenetic perspective,which helps to evaluate the ART's influence on the offspring and application securityMethods: The experiment include in vivo fertilization(IVO)embryos(control group),superovulation embryos group,in vitro fertilization(IVF)embryos group,in vitro maturation(IVM)embryos group.Observe the changes in the genome-wide DNA methylation level of pre-implantation embryos at the blastocyst stage by using immunofluorescence technique;Selecting the suitable “relative standard preparation”,serial diluting and constructing relative standard curve,then detecting the expression of imprinted genes Igf2 and Grb10 in a single blastocyst by real-time fluorescent quantitation PCR;Using bisulphite genomic sequencing to detect the methylation of Grb10 CGI1 in all the above four groups.The experimental data are collected,the use the mean ± standard deviation?ANOVA?Dunnett t test to analysis the data.Results: Immunofluorescence showed that genome-wide DNA methylation level of IVF embryos at blastocyst stage was higher than IVO and IVM embryos,however,no significant differences between IVF and superovulation group,DNA methylation level among IVO ? superovulation ? IVM group were no significant difference;Real-Time Quantitative PCR analysis showed that the expression of Igf2 in superovulation and IVM group are higher then the IVO and IVF group,however,there was no significant difference between superovulation and IVM,aslo in IVO and IVF group;the expression of Grb10 in superovulation group and the IVF group are lower then IVO group,however,there was no significant difference between superovulation and IVF.The expression of Grb10 are no significant difference among superovulation group ? IVF group ? IVM group.The bisulfite modification based on the sequencing results showed the methylation of Grb10 CGI1 in IVF group of are higher then IVO and IVM group,while the methylation of Grb10 CGI1 in IVF and superovulation group were no significant difference.The methylation of Grb10 CGI1 among IVO?superovulation?IVM group were no significant difference.CONCLUSION: ART cause genome-wide DNA methylation level hypermethylated was more obvious in IVF group,which may be associated with the exogenous hormone HCG used during ART procedure;Superovulation and IVF play opposite effect on the expression of Igf2.The expression levels of Grb10 was lower in IVF and superovulation group,which was associated with ART caused the methylation level of CGI1 increased;ART can cause epigenetic abnormalities.