Mitochondrial DNA (mtDNA) mutations are a common cause of genetic disease with pathogenic mtDNA mutations being detected in approximately 1 in 250 live births1-3 and at least 1 in 10,000 adults in the UK affected by mtDNA disease4. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. MtDNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach to prevent the transmission of human mtDNA disease5,6. Here we show that transfer of pronuclei between abnormally fertilised human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimising the procedure we found the average level of carry-over following transfer of two pronuclei is <2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has potential to prevent the transmission of mtDNA disease in humans.

The homeobox transcription factor Nanog has been proposed to play a crucial role in the maintenance of the undifferentiated state of murine embryonic stem cells. A human counterpart, NANOG, has been identified, but its function and localization have not hitherto been described. We have used a combination of RNA interference and quantitative realtime polymerase chain reaction to study NANOG in human embryonic stem and embryonic carcinoma cells. Transfection of NANOG-specific small interfering RNAs reduced levels of NANOG transcript and protein and induced activation of the extraembryonic endoderm-associated genes GATA4, GATA6, LAMININ B1, and AFP as well as upregulation of trophectoderm-associated genes CDX2, GATA2, hCG-alpha, and hCGbeta. Immunostaining of preimplantation human embryos showed that NANOG was expressed in the inner cell mass of expanded blastocysts but not in earlier-stage embryos, consistent with a role in the maintenance of pluripotency. Taken together, our findings suggest that NANOG acts as a gatekeeper of pluripotency in human embryonic stem and carcinoma cells by preventing their differentiation to extraembryonic endoderm and trophectoderm lineages. Stem Cells

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. Following optimisation, 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.