Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes

Nature Protocols volume 11, pages 1229–1243 (2016)Cite this article

Subjects

Abstract

We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping of crossovers and analysis of chromosome segregation patterns. The protocol takes a minimum of 3–5 d and requires a clinical embryologist with micromanipulation experience and a molecular biologist with basic bioinformatic skills. It has several advantages over previous methods; importantly, the use of artificial oocyte activation avoids the creation of embryos for research purposes. In addition, compared with next-generation sequencing, targeted SNP genotyping is cost-effective and it simplifies the bioinformatic analysis, as only one haploid reference sample is required to establish phase for maternal haplotyping. Finally, meiomapping is more informative than copy-number analysis alone for analysis of chromosome segregation patterns. Using this protocol, we have provided new insights that may lead to improvements in assisted reproduction for the treatment of infertility.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic showing an adult oocyte completing the first and second meiotic divisions.
Figure 2: Images showing oocyte activation and sequential biopsy of first and second polar bodies.
Figure 3: A representation of phasing of maternal SNPs in oocyte–PB trios and an illustration of some of the technical limitations that accompany SNP genotyping.
Figure 4: Screenshot of SNP patterns in a meiomap showing a region of a single chromosome across an oocyte–PB trio.
Figure 5: Informative SNPs are phased using a reference chromatid to reveal positions of haplotype switches (crossovers).
Figure 6: Anticipated results of meiomapping.

Similar content being viewed by others

References

  1. Hassold, T. & Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2, 280–291 (2001).

    Article  CAS  Google Scholar 

  2. Nagaoka, S.I., Hassold, T.J. & Hunt, P.A. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat. Rev. Genet. 13, 493–504 (2012).

    Article  CAS  Google Scholar 

  3. Angell, R. First-meiotic-division nondisjunction in human oocytes. Am. J. Hum. Genet. 61, 23–32 (1997).

    Article  CAS  Google Scholar 

  4. Handyside, A.H. et al. Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation. Eur. J. Hum. Genet. 20, 742–747 (2012).

    Article  Google Scholar 

  5. Handyside, A.H. et al. Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes. J. Med. Genet. 47, 651–658 (2010).

    Article  Google Scholar 

  6. Natesan, S.A. et al. Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro. Genet. Med. 16, 838–845 (2014).

    Article  CAS  Google Scholar 

  7. Ottolini, C.S. et al. Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates. Nat. Genet. 47, 727–735 (2015).

    Article  CAS  Google Scholar 

  8. Hou, Y. et al. Genome analyses of single human oocytes. Cell 155, 1492–1506 (2013).

    Article  CAS  Google Scholar 

  9. Fulton, B.P. & Whittingham, D.G. Activation of mammalian oocytes by intracellular injection of calcium. Nature 273, 149–151 (1978).

    Article  CAS  Google Scholar 

  10. Winston, N., Johnson, M., Pickering, S. & Braude, P. Parthenogenetic activation and development of fresh and aged human oocytes. Fertil. Steril. 56, 904–912 (1991).

    Article  CAS  Google Scholar 

  11. Taylor, A.S. & Braude, P.R. The early development and DNA content of activated human oocytes and parthenogenetic human embryos. Hum. Reprod. 9, 2389–2397 (1994).

    Article  CAS  Google Scholar 

  12. Liu, Y. et al. Three-day-old human unfertilized oocytes after in vitro fertilization/intracytoplasmic sperm injection can be activated by calcium ionophore a23187 or strontium chloride and develop to blastocysts. Cell. Reprogram. 16, 276–280 (2014).

    Article  CAS  Google Scholar 

  13. de Fried, E.P. et al. Human parthenogenetic blastocysts derived from noninseminated cryopreserved human oocytes. Fertil. Steril. 89, 943–947 (2008).

    Article  Google Scholar 

  14. Escrich, L. et al. Spontaneous in vitro maturation and artificial activation of human germinal vesicle oocytes recovered from stimulated cycles. J. Assist. Reprod. Genet. 28, 111–117 (2011).

    Article  Google Scholar 

  15. Montag, M., Koster, M., van der Ven, K., Bohlen, U. & van der Ven, H. The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle. Reprod. Biomed. Online 24, 521–526 (2012).

    Article  Google Scholar 

  16. Kuentz, P. et al. Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status. Hum. Reprod. 28, 1054–1061 (2013).

    Article  CAS  Google Scholar 

  17. Heindryckx, B., Van der Elst, J., De Sutter, P. & Dhont, M. Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum. Reprod. 20, 2237–2241 (2005).

    Article  CAS  Google Scholar 

  18. Heindryckx, B., De Gheselle, S., Gerris, J., Dhont, M. & De Sutter, P. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection. Reprod. Biomed. Online 17, 662–668 (2008).

    Article  Google Scholar 

  19. Ebner, T. et al. Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study. Hum. Reprod. 30, 97–102 (2015).

    Article  CAS  Google Scholar 

  20. Hassold, T., Merrill, M., Adkins, K., Freeman, S. & Sherman, S. Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16. Am. J. Hum. Genet. 57, 867–874 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Oliver, T.R. et al. New insights into human nondisjunction of chromosome 21 in oocytes. PLoS Genet. 4, e1000033 (2008).

    Article  Google Scholar 

  22. Middlebrooks, C.D. et al. Evidence for dysregulation of genome-wide recombination in oocytes with nondisjoined chromosomes 21. Hum. Mol. Genet. 2, 408–417 (2013).

    Google Scholar 

  23. Kong, A. et al. Recombination rate and reproductive success in humans. Nat. Genet. 36, 1203–1206 (2004).

    Article  CAS  Google Scholar 

  24. Gruhn, J.R., Rubio, C., Broman, K.W., Hunt, P.A. & Hassold, T. Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks. PLoS One 8, e85075 (2013).

    Article  Google Scholar 

  25. Tease, C., Hartshorne, G.M. & Hulten, M.A. Patterns of meiotic recombination in human fetal oocytes. Am. J. Hum. Genet. 70, 1469–1479 (2002).

    Article  CAS  Google Scholar 

  26. Holloway, J.K., Booth, J., Edelmann, W., McGowan, C.H. & Cohen, P.E. MUS81 generates a subset of MLH1-MLH3-independent crossovers in mammalian meiosis. PLoS Genet. 4, e1000186 (2008).

    Article  Google Scholar 

  27. Zaragoza, M.V. et al. Nondisjunction of human acrocentric chromosomes: studies of 432 trisomic fetuses and liveborns. Hum. Genet. 94, 411–417 (1994).

    Article  CAS  Google Scholar 

  28. Capalbo, A. et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum. Reprod. 28, 509–518 (2013).

    Article  CAS  Google Scholar 

  29. Christopikou, D. et al. Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Hum. Reprod. 28, 1426–1434 (2013).

    Article  CAS  Google Scholar 

  30. Palermo, G.D., Cohen, J., Alikani, M., Adler, A. & Rosenwaks, Z. Development and implementation of intracytoplasmic sperm injection (ICSI). Reprod. Fertil. Dev. 7, 211–217 (1995).

    Article  CAS  Google Scholar 

  31. Findlay, I., Ray, P., Quirke, P., Rutherford, A. & Lilford, R. Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis. Hum. Reprod. 10, 1609–1618 (1995).

    Article  CAS  Google Scholar 

  32. Bonetti, A. et al. Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices. Fertil. Steril. 95, 928–935 (2011).

    Article  Google Scholar 

  33. Kuwayama, M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 67, 73–80 (2007).

    Article  CAS  Google Scholar 

  34. Alexander, M. & Kusleika, D. Excel 2016 Power Programming with VBA. (Chichester, UK: John Wiley & Sons, 2016).

Download references

Author information

Author notes

  1. Christian S Ottolini, Antonio Capalbo and Louise Newnham: These authors contributed equally to this work.

Authors and Affiliations

  1. The Bridge Centre, London, UK

    Christian S Ottolini & Alan H Handyside

  2. School of Biosciences, University of Kent, Canterbury, UK

    Christian S Ottolini & Alan H Handyside

  3. GENERA, Centers for Reproductive Medicine, Rome, Italy

    Antonio Capalbo, Danilo Cimadomo, Filippo M Ubaldi & Laura Rienzi

  4. GENETYX, Molecular Genetics Laboratory, Marostica, Italy

    Antonio Capalbo, Danilo Cimadomo, Filippo M Ubaldi & Laura Rienzi

  5. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK

    Louise Newnham & Eva R Hoffmann

  6. Department of Cellular and Molecular Medicine, DNRF Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark

    Eva R Hoffmann

  7. Illumina, Cambridge, UK

    Senthilkumar A Natesan & Alan H Handyside

Authors
  1. Christian S Ottolini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonio Capalbo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Louise Newnham
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danilo Cimadomo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Senthilkumar A Natesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eva R Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Filippo M Ubaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Laura Rienzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alan H Handyside
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.S.O., A.C., D.C., L.R. and F.M.U. were responsible for donor consent, oocyte collection and oocyte activation. L.R., F.M.U. and A.H.H. oversaw ethical and legal regulations in Italy and the UK. A.C., C.S.O., S.A.N. and D.C. carried out amplification, SNP array and array CGH experiments. A.H.H., L.N. and C.S.O. analyzed the SNP data. A.H.H., C.S.O. and L.N. generated the figures. A.H.H., L.N. and C.S.O. wrote the manuscript. A.H.H., E.R.H. and L.N. edited the manuscript. All authors proofread and accepted the manuscript. A.H.H. developed the meiomapping algorithm.

Corresponding author

Correspondence to Alan H Handyside.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Meiomap SNP data (XLSM 1464 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ottolini, C., Capalbo, A., Newnham, L. et al. Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes. Nat Protoc 11, 1229–1243 (2016). https://doi.org/10.1038/nprot.2016.075

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.075

This article is cited by

Comments

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.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing