Mitochondrial DNA (mtDNA) mutations cause inherited diseases and are implicated in the pathogenesis of common late-onset disorders, but how they arise is not clear1,2. Here we show that mtDNA mutations are present in primordial germ cells (PGCs) within healthy female human embryos. Isolated PGCs have a profound reduction in mtDNA content, with discrete mitochondria containing ~5 mtDNA molecules. Single-cell deep mtDNA sequencing of in vivo human female PGCs showed rare variants reaching higher heteroplasmy levels in late PGCs, consistent with the observed genetic bottleneck. We also saw the signature of selection against non-synonymous protein-coding, tRNA gene and D-loop variants, concomitant with a progressive upregulation of genes involving mtDNA replication and transcription, and linked to a transition from glycolytic to oxidative metabolism. The associated metabolic shift would expose deleterious mutations to selection during early germ cell development, preventing the relentless accumulation of mtDNA mutations in the human population predicted by Muller’s ratchet. Mutations escaping this mechanism will show shifts in heteroplasmy levels within one human generation, explaining the extreme phenotypic variation seen in human pedigrees with inherited mtDNA disorders.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

  • 19 April 2018

    In the version of this Letter originally published, an author error led to the affiliations for Brendan Payne, Jonathan Coxhead and Gavin Hudson being incorrect. The correct affiliations are: Brendan Payne: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. 6Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 6 and subsequent existing affiliations have been renumbered. Jonathan Coxhead: 11Genomic Core Facility, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 11 and subsequent existing affiliations have been renumbered. Gavin Hudson: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. In addition, in Fig. 2d, the numbers on the x-axis of the left plot were incorrectly labelled as negative; they should have been positive. These errors have now been corrected in all online versions of the Letter.


  1. 1.

    Vafai, S. B. & Mootha, V. K. Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374–383 (2012).

  2. 2.

    Stewart, J. B. & Chinnery, P. F. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat. Rev. Genet. 16, 530–542 (2015).

  3. 3.

    Wallace, D. C. Colloquium paper: bioenergetics, the origins of complexity, and the ascent of man. Proc. Natl. Acad. Sci. USA 107, 8947–8953 (2011).

  4. 4.

    Payne, B. A. et al. Universal heteroplasmy of human mitochondrial DNA. Hum. Mol. Genet. 22, 384–390 (2013).

  5. 5.

    Hanley, N. A. et al. SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. Mech. Dev. 91, 403–407 (2000).

  6. 6.

    Anderson, R. A., Fulton, N., Cowan, G., Coutts, S. & Saunders, P. T. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 7, 136 (2007).

  7. 7.

    Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).

  8. 8.

    Dayama, G., Emery, S. B., Kidd, J. M. & Mills, R. E. The genomic landscape of polymorphic human nuclear mitochondrial insertions. Nucleic Acids Res. 42, 12640–12649 (2014).

  9. 9.

    Kennedy, S. R., Salk, J. J., Schmitt, M. W. & Loeb, L. A. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. PLoS. Genet. 9, e1003794 (2013).

  10. 10.

    Copeland, W. C. & Longley, M. J. Mitochondrial genome maintenance in health and disease. DNA Repair 19, 190–198 (2014).

  11. 11.

    Stewart, J. B., Freyer, C., Elson, J. L. & Larsson, N. G. Purifying selection of mtDNA and its implications for understanding evolution and mitochondrial disease. Nat. Rev. Genet. 9, 657–662 (2008).

  12. 12.

    Freyer, C. et al. Variation in germline mtDNA heteroplasmy is determined prenatally but modified during subsequent transmission. Nat. Genet. 44, 1282–1285 (2012).

  13. 13.

    Falkenberg, M., Larsson, N. G. & Gustafsson, C. M. DNA replication and transcription in mammalian mitochondria. Annu. Rev. Biochem. 76, 679–699 (2007).

  14. 14.

    Pyle, A. et al. Extreme-depth re-sequencing of mitochondrial DNA finds no evidence of paternal transmission in humans. PLoS. Genet. 11, e1005040 (2015).

  15. 15.

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

  16. 16.

    Cree, L. M. et al. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nat. Genet. 40, 249–254 (2008).

  17. 17.

    Ohinata, Y., Sano, M., Shigeta, M., Yamanaka, K. & Saitou, M. A. Comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter. Reproduction 136, 503–514 (2008).

  18. 18.

    Wai, T., Teoli, D. & Shoubridge, E. A. The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat. Genet. 40, 1484–1488 (2008).

  19. 19.

    Cao, L. et al. The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat. Genet. 39, 386–390 (2007).

  20. 20.

    Wonnapinij, P., Chinnery, P. F. & Samuels, D. C. Previous estimates of mitochondrial DNA mutation level variance did not account for sampling error: comparing the mtDNA genetic bottleneck in mice and humans. Am. J. Hum. Genet. 86, 540–550 (2010).

  21. 21.

    Kobayashi, T. et al. Principles of early human development and germ cell program from conserved model systems. Nature 546, 416–420 (2017).

  22. 22.

    Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016).

  23. 23.

    Stewart, J. B. et al. Strong purifying selection in transmission of mammalian mitochondrial DNA. PLoS. Biol. 6, e10 (2008).

  24. 24.

    Ma, H., Xu, H. & O’Farrell, P. H. Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster. Nat. Genet. 46, 393–397 (2014).

  25. 25.

    Hill, J. H., Chen, Z. & Xu, H. Selective propagation of functional mitochondrial DNA during oogenesis restricts the transmission of a deleterious mitochondrial variant. Nat. Genet. 46, 389–392 (2014).

  26. 26.

    Chen, Y. & Dorn, G. W. 2nd PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria. Science 340, 471–475 (2013).

  27. 27.

    Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS. Biol. 8, e1000298 (2010).

  28. 28.

    Fukuoh, A. et al. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase. Mol. Syst. Biol. 10, 734 (2014).

  29. 29.

    Muller, H. J. The relation of recombination to mutational advance. Mutat. Res. 1, 2–9 (1964).

  30. 30.

    Upholt, W. B. & Dawid, I. B. Mapping of mitochondrial DNA of individual sheep and goats: rapid evolution in the D loop region. Cell 11, 571–583 (1977).

  31. 31.

    Chinnery, P. F. et al. The inheritance of mtDNA heteroplasmy: random drift, selection or both? Trends Genet. 16, 500–505 (2000).

  32. 32.

    Keogh, M. & Chinnery, P. F. Hereditary mtDNA heteroplasmy: a baseline for aging? Cell. Metab. 18, 463–464 (2013).

  33. 40.

    Funkuda, T. Ultrastructure of primordial germ cells in human embryo. Virchows Arch. B Cell. Pathol. 20, 85–89 (1976).

  34. 33.

    Villesen, P. & Fredsted, T. Fast and non-invasive PCR sexing of primates: apes, Old World monkeys, New World monkeys and Strepsirrhines. BMC Ecol. 6, 8 (2006).

  35. 34.

    Noli, L., Capalbo, A., Ogilvie, C., Khalaf, Y. & Ilic, D. Discordant growth of monozygotic twins starts at the blastocyst stage: a case study. Stem Cell. Rep. 5, 946–953 (2015).

  36. 35.

    Sterio, D. C. The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc. 134, 127–136 (1984).

  37. 36.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  38. 37.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  39. 38.

    Koboldt, D. C. et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics 25, 2283–2285 (2009).

  40. 39.

    Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

Download references


P.F.C. is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UP_1501/2), the Medical Research Council (UK) Centre for Translational Muscle Disease research (G0601943) and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. W.W.C.T. is supported by a Croucher Foundation studentship, and M.A.S. by a Wellcome Investigator Award.

Author information

Author notes

  1. Vasileios I. Floros and Angela Pyle contributed equally to this work.


  1. MRC-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    • Vasileios I. Floros
    • , Wei Wei
    •  & Patrick F. Chinnery
  2. Department of Clinical Neurosciences, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK

    • Vasileios I. Floros
    • , Wei Wei
    •  & Patrick F. Chinnery
  3. Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK

    • Angela Pyle
    • , Brendan Payne
    •  & Gavin Hudson
  4. Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK

    • Sabine Dietmann
  5. Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    • Walfred C. W. Tang
    • , Naoko Irie
    •  & M. Azim Surani
  6. Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK

    • Brendan Payne
  7. GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy

    • Antonio Capalbo
  8. GENETYX, Reproductive Genetics Laboratory, Marostica, Italy

    • Antonio Capalbo
  9. Division of Women’s Health, Faculty of Life Sciences and Medicine, King’s College London, London, UK

    • Laila Noli
    • , Yacoub Khalaf
    •  & Dusko Ilic
  10. Assisted Conception Unit, Guy’s Hospital, London, UK

    • Laila Noli
    • , Yacoub Khalaf
    •  & Dusko Ilic
  11. Genomic Core Facility, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK

    • Jonathan Coxhead
  12. Human Developmental Biology Resource, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK

    • Moira Crosier
  13. Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

    • Henrik Strahl
  14. Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    • Mitinori Saitou
  15. JST, ERATO, Kyoto, Japan

    • Mitinori Saitou


  1. Search for Vasileios I. Floros in:

  2. Search for Angela Pyle in:

  3. Search for Sabine Dietmann in:

  4. Search for Wei Wei in:

  5. Search for Walfred C. W. Tang in:

  6. Search for Naoko Irie in:

  7. Search for Brendan Payne in:

  8. Search for Antonio Capalbo in:

  9. Search for Laila Noli in:

  10. Search for Jonathan Coxhead in:

  11. Search for Gavin Hudson in:

  12. Search for Moira Crosier in:

  13. Search for Henrik Strahl in:

  14. Search for Yacoub Khalaf in:

  15. Search for Mitinori Saitou in:

  16. Search for Dusko Ilic in:

  17. Search for M. Azim Surani in:

  18. Search for Patrick F. Chinnery in:


V.I.F. developed methods and isolated the in vivo human and mouse PGCs, and performed the microscopy; S.D. performed RNA-seq bioinformatic analysis; A.P. and W.W.C.T. carried out the real-time PCR assays; W.W.C.T. performed the RNA-seq experiments and N.I. derived and isolated the hESCs, hPGCLCs and in vitro somatic cells; W.W. performed additional informatic and statistical analysis; A.C. and L.N. isolated the human inner cell mass and trophectoderm cells, overseen by D.I. and Y.K. J.C. carried out the library preparation and deep sequencing; M.C. helped with the human tissue dissection; H.S. helped with the super-resolution microscopy; B.P. performed the technical validation of the deep sequencing protocol; M.S. provided the BVSC mouse; G.H. advised on the NGS data analysis; M.A.S. supervised the RNA-seq experiments and real-time PCR expression assays and advised on the project; P.F.C. supervised the project, designed experiments, analysed the data and wrote the paper. All authors contributed to the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Patrick F. Chinnery.

Supplementary information

  1. Supplementary Information

    Supplementary legends and Supplementary Figures 1–5

  2. Life Sciences Reporting Summary

    Reporting Summary and Flow Cytometry Reporting Summary

  3. Supplementary Table

    Supplementary Table 1

  4. Supplementary Table

    Supplementary Table 2

  5. Supplementary Table

    Supplementary Table 3

  6. Supplementary Table

    Supplementary Table 4

  7. Supplementary Table

    Supplementary Table 5

  8. Supplementary Table

    Supplementary Table 6


  1. Supplementary Movie

    Supplementary Movie 1

  2. Supplementary Movie

    Supplementary Movie 2

About this article

Publication history






Further reading