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

This article has been updated


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


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.


  1. 1.

    , & Human mitochondrial DNA: roles of inherited and somatic mutations. Nature Rev. Genet. 13, 878–890 (2012)

  2. 2.

    et al. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85 (2010)

  3. 3.

    & Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring Harb. Perspect. Biol. 5, a021220 (2013)

  4. 4.

    et al. Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation. Nature Genet. 30, 145–146 (2002)

  5. 5.

    et al. Data from artificial models of mitochondrial DNA disorders are not always applicable to humans. Cell Reports 7, 933–934 (2014)

  6. 6.

    et al. Concise reviews: assisted reproductive technologies to prevent transmission of mitochondrial DNA disease. Stem Cells 33, 639–645 (2015)

  7. 7.

    et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature 493, 627–631 (2013)

  8. 8.

    et al. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632–637 (2013)

  9. 9.

    & Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220, 1300–1302 (1983)

  10. 10.

    , , & Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. Hum. Reprod. 17, 407–412 (2002)

  11. 11.

    & The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol. Hum. Reprod. 15, 531–538 (2009)

  12. 12.

    , , , & Human pre-implantation embryo development. Development 139, 829–841 (2012)

  13. 13.

    et al. Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting. Fertil. Steril. 98, 644–649 (2012)

  14. 14.

    et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165 (2015)

  15. 15.

    & Visualizing high-dimensional data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605 (2008)

  16. 16.

    et al. PGD and heteroplasmic mitochondrial DNA point mutations: a systematic review estimating the chance of healthy offspring. Hum. Reprod. Update 18, 341–349 (2012)

  17. 17.

    , & Preventing the transmission of pathogenic mitochondrial DNA mutations: can we achieve long-term benefits from germ-line gene transfer? Hum. Reprod. 28, 554–559 (2013)

  18. 18.

    & The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation. Stem Cell Rev. 5, 140–158 (2009)

  19. 19.

    , & Mitochondrial DNA disease and developmental implications for reproductive strategies. Mol. Hum. Reprod. 21, 11–22 (2015)

  20. 20.

    et al. Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model. Proc. Natl Acad. Sci. USA 110, E3622–E3630 (2013)

  21. 21.

    et al. Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck. Cell Reports 1, 506–515 (2012)

  22. 22.

    et al. Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder. Fertil. Steril. 98, 1236–1240 (2012)

  23. 23.

    , , & Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases. Cell Reports 7, 935–937 (2014)

  24. 24.

    et al. Preimplantation genetic diagnosis in mitochondrial DNA disorders: challenge and success. J. Med. Genet. 50, 125–132 (2013)

  25. 25.

    et al. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis. J. Med. Genet. 43, 244–247 (2006)

  26. 26.

    , , , & Meiosis and maternal aging: insights from aneuploid oocytes and trisomy births. Cold Spring Harb. Perspect. Biol. 7, a017970 (2015)

  27. 27.

    et al. Egg sharing for research: a successful outcome for patients and researchers. Cell Stem Cell 10, 239–240 (2012)

  28. 28.

    HFEA Guidance on Payments for Donors.HFEA Code of Practice Section 13 (Human Fertilisation and Embryology Authority, 2009)

  29. 29.

    et al. A novel isolator-based system promotes viability of human embryos during laboratory processing. PLoS ONE 7, e31010 (2012)

  30. 30.

    , , , & Elective single embryo transfer: guidelines for practice British Fertility Society and Association of Clinical Embryologists. Hum. Fertil. (Camb.) 11, 131–146 (2008)

  31. 31.

    , & International community consensus standard for reporting derivation of human embryonic stem cell lines. Regen. Med. 2, 349–362 (2007)

  32. 32.

    et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J. Med. Genet. 51, 553–562 (2014)

  33. 33.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  34. 34.

    , & HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  35. 35.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  36. 36.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

  37. 37.

    , , & The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations. Nucleic Acids Res. 29, e74 (2001)

  38. 38.

    et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nature Genet. 23, 147 (1999)

  39. 39.

    et al. Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing. PLoS Genet. 10, e1004620 (2014)

  40. 40.

    et al. Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells. Genes Dev. 29, 1239–1255 (2015)

  41. 41.

    , , , & Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 5, 621–628 (2008)

  42. 42.

    et al. Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing. Genet. Test. 9, 190–199 (2005)

Download references


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.).


  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


  1. Search for Louise A. Hyslop in:

  2. Search for Paul Blakeley in:

  3. Search for Lyndsey Craven in:

  4. Search for Jessica Richardson in:

  5. Search for Norah M. E. Fogarty in:

  6. Search for Elpida Fragouli in:

  7. Search for Mahdi Lamb in:

  8. Search for Sissy E. Wamaitha in:

  9. Search for Nilendran Prathalingam in:

  10. Search for Qi Zhang in:

  11. Search for Hannah O’Keefe in:

  12. Search for Yuko Takeda in:

  13. Search for Lucia Arizzi in:

  14. Search for Samer Alfarawati in:

  15. Search for Helen A. Tuppen in:

  16. Search for Laura Irving in:

  17. Search for Dimitrios Kalleas in:

  18. Search for Meenakshi Choudhary in:

  19. Search for Dagan Wells in:

  20. Search for Alison P. Murdoch in:

  21. Search for Douglass M. Turnbull in:

  22. Search for Kathy K. Niakan in:

  23. Search for Mary Herbert in:


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


  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.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.