• A Corrigendum to this article was published on 27 July 2016

This article has been updated

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. 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.

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Change history

  • 15 June 2016

    The reviewer information statement did not display correctly online when this paper was first published; this has been corrected and the statement is now available.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Raw RNA-seq data and reads per kilobase per million mapped reads (RPKM) table have been deposited in the Gene Expression Omnibus under accession number GSE76284.

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Acknowledgements

We are very grateful to those who donated gametes for this research and we thank M. Nesbitt and K. Lennox for obtaining their consent. We also thank P. Chinnery and V. Floros for helpful discussion. The work was funded by the Wellcome Trust (096919) and by grants from the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre and the Barbour Foundation. K.K.N. and co-workers are supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council (MC_UP_1202/9) and the Wellcome Trust, and by the March of Dimes Foundation (FY11-436). D.W. and co-workers are funded by the NIHR Oxford Biomedical Research Centre.

Author information

Author notes

    • Qi Zhang
    • , Laura Irving
    •  & Dimitrios Kalleas

    Present addresses: Department of Cell Biology, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing 100850, China (Q.Z.); Gateshead Fertility Unit, Gateshead Health NHS Trust, Queen Elizabeth Hospital, Sheriff Hill, Gateshead NE9 6SX, UK (L.I.); St Mary’s Hospital, Department of Reproductive Medicine, Old Saint Mary’s Hospital, Oxford Road, Manchester M13 9WL, UK (D.K.).

Affiliations

  1. Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Biomedicine West Wing, Centre for Life, Times Square, Newcastle upon Tyne NE1 3BZ, UK

    • Louise A. Hyslop
    • , Jessica Richardson
    • , Mahdi Lamb
    • , Nilendran Prathalingam
    • , Qi Zhang
    • , Hannah O’Keefe
    • , Yuko Takeda
    • , Lucia Arizzi
    • , Laura Irving
    • , Dimitrios Kalleas
    •  & Mary Herbert
  2. Newcastle Fertility Centre, Biomedicine West Wing, Centre for Life, Times Square, Newcastle upon Tyne NE1 4EP, UK

    • Louise A. Hyslop
    • , Nilendran Prathalingam
    • , Lucia Arizzi
    • , Meenakshi Choudhary
    • , Alison P. Murdoch
    •  & Mary Herbert
  3. The Francis Crick Institute, Human Embryo and Stem Cell Laboratory, Mill Hill Laboratory, Mill Hill, London NW7 1AA, UK

    • Paul Blakeley
    • , Norah M. E. Fogarty
    • , Sissy E. Wamaitha
    •  & Kathy K. Niakan
  4. Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK

    • Lyndsey Craven
    • , Helen A. Tuppen
    •  & Douglass M. Turnbull
  5. Reprogenetics UK, Institute of Reproductive Sciences, Oxford Business Park North, Oxford OX4 2HW, UK

    • Elpida Fragouli
    •  & Samer Alfarawati
  6. University of Oxford, Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford OX3 9DU, UK

    • Dagan Wells

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Contributions

M.H. and L.A.H. conceived and designed the PNT experiments. L.A.H., L.I., L.C. and L.A. performed PNT experiments and embryo manipulations. J.R., D.K. and Q.Z. performed cell counts. D.W., E.F. and S.A. performed whole-genome amplification and array-CGH. K.K.N., P.B. and N.M.E.F. performed RNA-seq experiments. L.C., H.A.T. and D.M.T. measured mtDNA carryover and performed mtDNA haplogroup analysis. N.P., K.K.N., N.M.E.F., S.E.W., Y.T. and H.O’K. derived, cultured and characterized ES cell lines. K.K.N., P.B., M.L., J.R., L.A.H., L.C., Y.T., P.B. and M.H. analysed data. A.P.M. and M.C. coordinated the oocyte donation program. M.H. wrote the manuscript with input from D.M.T., D.W., K.K.N., J.R. and M.L.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mary Herbert.

Reviewer Information: Nature thanks J. Carroll, G. Manfredi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

Videos

  1. 1.

    The ePNT procedure in human zygotes

    Video showing pronuclei being extracted in separate karyoplasts from a human zygote before being placed underneath the zona pellucida of a previously enucleated zygote.

  2. 2.

    Removal of excess cytoplasm from the karyoplast during ePNT in human zygotes

    Video showing karyoplast removal and shearing of excess cytoplasm in preparation for fusion with a previously enucleated zygote.

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DOI

https://doi.org/10.1038/nature18303

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