Risk And Mechanism Of Neurodegenerative Disorders Involved In The Mice Generated After Blastomere Biopsy

Preimplantation genetic diagnosis (PGD) has been one of the main clinicalcomponents of assisted reproductive technology (ART) and it has been widelyapplied in the field of ART for twenty years. Blastomere biopsy is involved in PGDbut there has generally been little investigation into the long-term health implicationsof this procedure on the resulting offspring. In our previous study, through spatiallearning assay, a high-risk of defective memory function was shown in an adultmouse model generated after the blastomere biopsy. It suggested a potential high-riskof neurodegenerative diseases in aged offspring. However, little is known about themolecular mechanism underlying the neural disorders.In this study, the risk evaluation was conducted in aged mice generated afterblastomere biopsy. Morphologic analysis of brain tissue indicated more seriousmitochondrial degeneration, increase of lipofuscin and hypomyelination in the brainsof these aged offspring. The results of western blot and immunohistochemistryshowed a decrease expression of MBP, a myelination-related protein.Two-dimensional gel electrophoresis proteomic analysis identified several differentlyexpressed proteins including SOD1, NEFL, DPYSL2, ALDH2, which revealedneurodegenerative-like disorders in biopsied mice.Moreover, a methylated DNA immunoprecipitation (MeDIP) assay suggestedthat epigenetic alterations in the brain were associated with neurodegenerativedisorders in the biopsied mice. Bisulfite sequencing showed hypomethylation in theregion of chd4promoter compared to the control group, and the results of real-timePCR analysis showed an increase in chd4mRNA expression in the biopsied groupcompared with the control group. In addition, it was found that the level of PLP,which is necessary for myelination, was decreased markedly in some biopsied mice from both the adult and aged groups compared with the control mice.To study whether the different methylation in mice brain was substantially due tothe baltomere biopsy before birth, we profiled the global methylation level in E7.5early embryo. The results showed a hypomethylation status in biopsied groupcompared to the control group, and it suggested blastomere biopsy was an obstacle tode novo methylation during early embryo development.Finally, we profiled the differently expressed genes between blastocysts afterblastomere biopsy and control blastocysts. The results of bioinformatic analysissuggested58genes associated with a serious of biology events including DNAmethylation, chromatin remodeling, cell cycle, oxidative stress, embryo developmentand cell differentiation. The results of real-time PCR showed a lower expression levelof some important transcripts which participated in de novo methylation and cellulardifferentiation in biopsied group, including ehmt2, cul3, ncor2and phgdh.In conclusion, this study indicated the potential risk of neurodegenerativedisorders in the mice generated from biopsied embryos. The epigenetic brainalterations demonstrated in this study may be related to the impairment of nervoussystem function. Thus, this study suggested that more studies should be performed toaddress the possible adverse effects of blastomere biopsy on the development ofoffspring and the overall safety of PGD technology should be subjected to furtherrigorous assessment.