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Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases1. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes2 were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.
Predicting the risk of serious disease in children of women who carry mtDNA mutations is complicated by a number of factors. Mutations in mtDNA can be either homoplasmic (all copies of mtDNA are mutated) or heteroplasmic (mixture of mutated and wild-type mtDNA). In the case of heteroplasmy, women produce oocytes with widely varying mutation loads3. While pathogenicity is generally proportional to the ratio of mutated to wild-type mtDNA, the severity of disease for a given mutation load can vary, even among homoplasmic individuals4. The resulting unpredictability in the risk of transmitting disease raises profoundly difficult reproductive decisions for women from affected families. While preimplantation genetic diagnosis (PGD) can be used to reduce the risk of mtDNA disease by identifying embryos with low mutation loads5, it is not useful for women who are homoplasmic for pathogenic mtDNA mutations6. In such cases, it may be possible to reduce the risk of transmission by transplanting the oocyte nuclear DNA to an enucleated donor oocyte free of pathogenic mtDNA mutations.
Progression through female meiosis offers a number of opportunities for transplanting nuclear DNA. Proof-of-concept studies7,8 indicate that transplantation of the nuclear...