Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease

Journal name:
Nature
Volume:
465,
Pages:
82–85
Date published:
DOI:
doi:10.1038/nature08958
Received
Accepted
Published online

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births1, 2, 3 and at least 1 in 10,000 adults in the UK are affected by mtDNA disease4. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease5, 6. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 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 the potential to prevent the transmission of mtDNA disease in humans.

At a glance

Figures

  1. Pronuclear transfer using abnormally fertilized human zygotes.
    Figure 1: Pronuclear transfer using abnormally fertilized human zygotes.

    ag, Transfer of two pronuclei between human zygotes. a, Recipient zygote (one pronucleus removed) and donor zygote (three pronuclei, two of which are removed and fused with the recipient zygote). b, Recipient zygote containing a single pronucleus (marked with arrow), which is removed by a biopsy pipette to leave an enucleated zygote (d). c, Donor zygote with three pronuclei (marked with arrows), two of which are removed as karyoplasts (e). f, Enucleated recipient zygote with two pronuclear karyoplasts from the donor zygote (arrows) before fusion. g, Recipient zygote 20min after transfer, already showing fusion of the karyoplast membranes (arrow). h, Development of unmanipulated abnormally fertilized zygotes (n = 76; black bars), embryos receiving one transferred pronucleus (n = 44; grey bars) and embryos receiving two transferred pronuclei (n = 36; white bars). i, j, Hatching blastocyst on day 6 (i) and hatched blastocyst on day 7 (j) containing two donor pronuclei. Scale bars, 50µm.

  2. Mitochondrial DNA analysis of pronuclear-transfer embryos.
    Figure 2: Mitochondrial DNA analysis of pronuclear-transfer embryos.

    a, The potential transfer of donor zygote mtDNA to the recipient zygote. b, Sequence electropherograms of mtDNA non-coding control region in donor and recipient zygotes with the sequence variant used for the last hot cycle PCR RFLP assay highlighted. c, Scheme of RFLP designed using the sequence variant. d, Last hot cycle PCR RFLP analysis of donor mtDNA carry-over detected in embryos receiving two transferred pronuclei with products separated by 12% non-denaturing polyacrylamide gel electrophoresis. U, undigested; C1 and C2, controls (C1, donor embryo for E3, recipient embryo for E1 and E2; C2, donor embryo for E1 and E2, recipient embryo for E3). bp, base pairs. e, Mitochondrial DNA copy number in human mature oocytes.

  3. Mitochondrial DNA analysis of individual blastomeres disaggregated from pronuclear-transfer embryos.
    Figure 3: Mitochondrial DNA analysis of individual blastomeres disaggregated from pronuclear-transfer embryos.

    a, Last hot cycle PCR RFLP of individual blastomeres from a pronuclear-transfer embryo showing variable levels of mtDNA donor genotype in individual blastomeres. The arrow indicates the band representing carry-over mtDNA. b, Levels of donor mtDNA carry-over in individual blastomeres from eight embryos before modifications to minimize levels of donor mtDNA in pronuclear karyoplasts. In some embryos not all blastomeres could be collected. Figures represent the percentage mtDNA carry-over in individual blastomeres after pronuclear transfer. n.d., Non-detectable. c, Pronuclear karyoplasts after additional manipulation showing minimal amount of donor cytoplasm compared with Fig. 1e. Scale bar, 25µm. d, Last hot cycle PCR RFLP of individual blastomeres from a pronuclear-transfer embryo showing no detectable levels of mtDNA donor genotype in individual blastomeres. The arrow indicates the band representing carry-over mtDNA. e, Levels of donor mtDNA carry-over in individual blastomeres from nine embryos after improvements to pronuclear karyoplast removal. In some embryos, not all blastomeres could be collected. Figures represent the percentage of mtDNA carry-over in individual blastomeres after pronuclear transfer. n.d., Non-detectable.

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Author information

Affiliations

  1. Mitochondrial Research Group, Institute for Ageing and Health,

    • Lyndsey Craven,
    • Helen A. Tuppen,
    • Julie L. Murphy,
    • Lynsey M. Cree,
    • Patrick F. Chinnery,
    • Robert W. Taylor,
    • Robert N. Lightowlers &
    • Douglass M. Turnbull
  2. Newcastle University Centre for Brain Ageing and Vitality, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

    • Douglass M. Turnbull
  3. Newcastle Fertility Centre, International Centre for Life,

    • Gareth D. Greggains,
    • Stephen J. Harbottle,
    • Alison P. Murdoch &
    • Mary Herbert
  4. Institute for Ageing and Health, International Centre for Life,

    • Gareth D. Greggains &
    • Mary Herbert
  5. North East England Stem Cell Institute (NESCI), Bioscience Centre, International Centre for Life, Newcastle University, Newcastle upon Tyne NE1 4EP, UK

    • Alison P. Murdoch,
    • Mary Herbert &
    • Douglass M. Turnbull

Contributions

M.H., A.P.M., R.N.L. and D.M.T. conceived the project and designed the experiments. L.C., H.A.T., S.J.H., G.D.G., J.L.M., L.M.C., P.F.C. and R.W.T. performed experiments and analysed data. L.C., M.H., H.A.T. and D.M.T. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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