Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.

Meenakshi Choudhary,Doug M Turnbull ,Nilendran Prathalingam,Sissy E Wamaitha,Louise Hyslop ,Alison Murdoch ,Hannah O’Keefe,Norah M E Fogarty,Dimitrios Kalleas,Dagan Wells,Jessica Richardson,Paul Blakeley,Qi Zhang,Mahdi Lamb,Elpida Fragouli,Lyndsey Craven,Mary Herbert,Yuko Takeda,Lucia Arizzi,Kathy K Niakan,Helen A.l Tuppen ,S Alfarawati ,Laura Irving

doi:10.1038/nature18303

Abstract

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.

Highlights

Progression through female meiosis offers a number of opportunities for transplanting nuclear DNA
Control experiments in which PN were replaced in the same zygote indicated that blastocyst quality was compromised by the manipulations (Fig. 2a and Extended Data Fig. 2f)
Heterologous pronuclear transfer (PNT) resulted in reduced blastocyst formation (Fig. 2b), possibly due to an effect of vitrification at the meiosis II (MII) stage[13], which was not ameliorated by delaying vitrification until after exit from MII (Extended Data Fig. 3a-f)

Summary

Introduction

Progression through female meiosis offers a number of opportunities for transplanting nuclear DNA. While the increased survival of ePNT zygotes (Series I) resulted in improved blastocyst formation compared with LtPNT, both approaches produced few good quality blastocysts (Extended Data Fig. 2e,f).

Results

Conclusion