Effects Of Intracytoplasmic Sperm Injection (ICSI)on Imprinted Genes Expression And Methylation Status In Mouse Kidney

Since the birth of Louise Brown in1978, in vitro fertilization and intracytoplasmic sperm injection (ICSI), as widely accepted assisted reproductive technologies (ART), have become widely-accepted medical treatments for human infertility. The safety of ART offspring, especially the safety of ICSI offspring, has become a hot topic in the field of ART.ICSI was presented as a successful method of treatment for certain types of male infertility. The ICSI procedure involves the isolation of one particular spermatozoon that is subsequently injected directly into the oocyte with a micropipette. Several concerns have therefore been raised. Theoretically in the absence of natural selection of the fertilizing sperm, any structural damage inflicted by the operation procedure or even the transfer of heterologous substances, such as polyvinylpyrrolidone (PVP), that normally would not be passed on to a child, could increase the risk of health problems of the children.The risk of birth defects is among these concerns, but the reason why ICSI increased the risk of birth defect is still unclear. Experimental evidence in mice suggests that genomic imprints in the parental gametes are erased upon every reproductive cycle, reestablished in the immature germ cells of the developing embryo according to their fate either as male or female gametes, and maintained through both the preimplantation period (when the rest of the genome is demethylated) and postimplantation development. As such, imprints are dynamically changing during both germ cell and embryo developments. ICSI treatment, which was operated in the timing of in vitro culture of germ cell and fertilization and embryo development, may interfere the acquisition and maintaining of genomic imprints.Previous research reported kidney disease related genes, such as MEST, H19, IGF2, PEGS, SNRPN, CDKNLC, SOCS-3, SFRP1and RASAL1, but seldom focus on the mechanism of the effect of ICSI on urinary birth defects. In the present study, to exclude the effect of gonadotropin stimulation and transplantation, we established ICSI mice model (experimental group) and2cell embryo transplantation mice model (control group). We focused on paternally imprinted gene H19, and maternally imprinted genes Igf2, Snrpn, Mest, and Peg3, and detected the expression of these genes and the DNA methylation patterns of their differentially methylated region (DMR). In this research, we elucidated the underlying epigenetic mechanisms for abnormal expression of imprinted genes and provided a theoretical basis for the safety of ICSI.Part Ⅰ The expression of imprinted genes H19, Igf2, Mest, Peg3and Snrpn changed in ICSI mice kidneyObjective:To establish models of ICSI and2cell embryo transplantation, to investigate the expression differences of H19, Igf2, Mest, Peg3and Snrpn between ICSI and2cell embryo transplantation conceived kidney.Methods:To establish models of ICSI and2cell embryo transplantation by using C57BL/6J mice. The2cell embryo transplantation group was used as the control group. The kidney tissue was obtained after the offspring of ICSI and control group were at their adulthood (10-week) and old age (1.5-year). We compared the weights of adult and old mice and weights of their kidney, used reverse transcription (RT) Real time PCR to analyze the mRNA expression of the target genes. Results:In adult offspring, the expressions of H19, Mest, Peg3and Snrpn were significantly up-regulated in kidney of ICSI mice compared with control group, and there was no difference in Igf2mRNA expression between two groups. In old offspring, the expression of Snrpn were significantly down-regulated, and no differences were found in the expressions of the other four genes between two groups.Conclusion:The expression of H19, Mest, Peg3and Snrpn were changed in ICSI conceived kidney, which may influence the risk of kidney diseases. Part Ⅱ DNA methylation analysis of differentially methylated region (DMR) of imprinted genes H19,Mest, Peg3and Snrpn in mice kidney derived from ICSIObjective:To investigate the change of DNA methylation patterns in differentially methylated region of H19, Mest, Peg3and Snrpn between ICSI and2cell embryo transplantation kidney, and elucidate the relationship between aberrant DNA methylation and abnormal gene expression.Methods:Kidneys of adult and old mice both in ICSI and control group were subjected to bisulfite sequencing (BSP) and/or pyrosequencing. DNA methylation patterns in CpG islands of H19DMR, MestDMR, Peg3DMR and SnrpnDMR were analyzed.Results:In adult offspring, significantly hypomethylated in H19DMR, MestDMR and Peg3DMR were found in ICSI group compared with control group, hypomethylation of SnrpnDMR. were also found, and the difference between two group was marginally significant. In old mice offspring, there was a significant increase in methylation of Snrpn in ICSI group.Conclusion:The aberrant DNA methylation status in the differentially methylated region contribute to the abnormal expression of imprinted genes in ICSI kidney. ICSI treatment may interfere the dynamical changing of genomic imprints. Reprogramming of methylation status in SnrpnDMR may occurred in ICSI kidney after adulthood.