Effects Of The Manipulation After Controlled Ovarian Stimulation In ART On The Expression And Modification Of Imprinted Genes H19,IGF2 And SNRPN In Early Pregnancy Fetuses

Since the world's first case of in vitro fertilization(IVF)infant Louise Brown's birth in 1978,assisted reproductive technology(ART)had developed rapidly and was becoming more and more mature.And more and more infants were born through it.With the increase of the proportion of ART offspring in the population,more and more scholars paid attention to the safety of ART then carried out the relevant research.In recent years,the research of assisted reproductive technology has been focused on ART and epigenetic research.Epigenetic modifications are ubiquitous in life phenomena and are a special kind of gene regulation method that is crucial to maintain the normal life activities of mammals.Epigenetic modification mainly includes the following: DNA methylation,histone acetylation and histone methylation modification,which usually coordinate the gene expression and are easily influenced by many environmental factors.Abnormalities in epigenetic modification during embryogenesis may lead to the development of multiple diseases in embryos and even in adulthood.In mammals,there are some special genes,they only express a single parent gene,this epigenetic modification is known as genomic imprinting.The gene is called the maternally imprinted gene when a imprinted gene is silenced from the maternal parent and activated from the paternal parent.When a imprinted gene is silenced from the paternal parent and activated from the maternal parent,the gene is called the paternally imprinted gene.DNA methylation is an important epigenetic modification,the main site of modification occurs on the CpG island of DNA.The role of imprinted genes has always been throughout the embryo's development.During gametogenesis and embryo development,imprinted genes undergo three stages of removal,establishment and maintenance of imprinted genes.However,the various operations involved in assisted reproductive technologies are at a crucial stage of it.Will they lead to abnormal epigenetic modification of the embryo? And whether they increase the risk of defects? It remains to be further studied.ObjectiveTo examine the effects of IVF,ICSI and FET,as well as in vitro culture,on the safety of offspring,this study was conducted from the perspective of genetic imprinting to investigate whether assisted reproductive technology would influence the parental and maternal imprinting genes.Materials and Methods1.Materials:The study protocol was approved by the Third Affiliated Hospital of Zhengzhou University Institutional Ethics Committee,and informed consent was obtained from all patients.Between January 2014 and December 2016,18 multifoetal reduction foetuses were obtained from 18 patients respectively for this study.The inclusion criteria for this study were single gestational sacs and 6-9 weeks of pregnancy.The preoperative B ultrasounds showed intrauterine live births.2.Methods: We collected 18 fetuses from multifetal reduction and divided them into 6 groups:multifetal reduction after IVF fresh transferred D3 embryos(n=3),multifetal reduction after IVF frozen transferred D3 embryos(n=3),multifetal reduction after IVF frozen transferred D5 embryos(n=3),multifetal reduction after ICSI fresh transferred D3 embryos(n=3),multifetal reduction after ICSI frozen transferred D3 embryos(n=3),multifetal reduction after controlled ovarian stimulation(COS)(n=3).We chose the imprinted genes H19,IGF2 and SNRPN to analyze.The expression and DNA methylation at some CpG sites of H19,IGF2,SNRPN were examined using real-time quantitative polymerase chain reaction(PCR)and pyrosequencing.3.Statistical methods: All statistics were performed with IBM SPSS Statistics 23.0.Quantitative data were calculated as the mean±SD.The Kruskal-Wallis H test was used to evaluate the results among the six groups.A difference was considered significant when P?0.05.Results1.Patient CharacteristicsA total of 18 fetuses from multifetal reduction were included in this study.The specific grouping was as described above.There were no significant difference in the maternal age between all groups(P=0.791);and there were also no significant difference in gestational age between groups(P=0.068).2.Changes in the expression levels of genesWe conducted real-time PCR to determine mRNA expression levels of the imprinted genes H19,IGF2 and SNRPN among the six groups.The result showed that,no significant differences in expression were detected for H19,IGF2 and SNRPN genes(H19:P=0.688;IGF2:P=0.527;SNRPN: P=0.295).3.DNA methylation status of H19,IGF2 and SNRPNThe mean percentage of H19 methylation(eight CpG sites),IGF2 methylation(five CpG sites)and SNRPN methylation(nine CpG sites)were not significantly different in all groups(H19:P=0.169;IGF2:P=0.058;SNRPN:P=0.748).ConclusionOur results suggest that ART after controlled ovarian stimulation(IVF,ICSI,cryopreservation and duration of in vitro culture)may not increase the risk of abnormal expression and DNA methylation at some CpG sites of H19,IGF2 and SNRPN on fetuses.