Skip to main content
SpringerLink
Log in

Routine cryopreservation of spermatozoa is safe — Evidence from the DNA methylation pattern of nine spermatozoa genes

  • Gamete Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Assess short- and mid-term impact of cryopreservation on DNA methylation status of different genes in spermatozoa.

Methods

Semen samples from 10 healthy normozoospermic men were collected at the Department of Clinical Andrology of the Centre of Reproductive Medicine and Andrology (Muenster, Germany). Each was divided into four equal aliquots: 1) untreated, 2) diluted in cryoprotectant, 3) short term (2 days) cryopreserved and 4) mid term (4 weeks) cryopreserved. Samples were “swim-up” purified prior to analysis. DNA fragmentation was measured using comet assay and Flow cytometric evaluation with Acridine Orange (FCEAO). The degree of methylation of nine genes was determined by bisulfite pyrosequencing of genomic DNA.

Result(s)

Analysis of three maternally imprinted genes (LIT1, SNRPN, MEST), two paternally imprinted genes (MEG3, H19), two repetitive elements (ALU, LINE1), one spermatogenesis-specific gene (VASA) and one gene associated with male infertility (MTHFR) in semen samples demonstrated no alteration in methylation pattern regardless of duration of cryopreservation.

Conclusion(s)

The lack of any changes in the sub-fraction of the genome examined in our study, implies that sperm DNA methylation is unaffected by cryopreservation and suggests that this daily clinical routine is safe in terms of DNA methylation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Allen C, Reardon W. Assisted reproduction technology and defects of genomic imprinting. BJOG. 2005;112:1589–94.

    Article  PubMed  Google Scholar 

  2. Biermann K, Steger K. Epigenetics in male germ cells. J Androl. 2007;28:466–80.

    Article  PubMed  CAS  Google Scholar 

  3. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 2003;72:156–60.

    Article  PubMed  CAS  Google Scholar 

  4. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod. 2001;16:1191–9.

    Article  PubMed  CAS  Google Scholar 

  5. El Hajj N, Zechner U, Schneider E, Tresch A, Gromoll J, Hahn T, et al. Methylation status of imprinted genes and repetitive elements in sperm DNA from infertile males. Sex Dev. 2011;5:60–9.

    Article  PubMed  CAS  Google Scholar 

  6. Evenson D, Jost L (2001) Sperm chromatin structure assay for fertility assessment. Curr Protoc Cytom Chapter 7:Unit 7.13

    Google Scholar 

  7. Feldschuh J, Brassel J, Durso N, Levine A. Successful sperm storage for 28 years. Fertil Steril. 2005;84:1017.

    Article  PubMed  Google Scholar 

  8. Gandini L, Lombardo F, Lenzi A, Spano M, Dondero F. Cryopreservation and sperm DNA integrity. Cell Tissue Bank. 2006;7:91–8.

    Article  PubMed  CAS  Google Scholar 

  9. Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet. 2003;72:1338–41.

    Article  PubMed  CAS  Google Scholar 

  10. Guo X, Gui YT, Tang AF, Lu LH, Gao X, Cai ZM. Differential expression of VASA gene in ejaculated spermatozoa from normozoospermic men and patients with oligozoospermia. Asian J Androl. 2007;9:339–44.

    Article  PubMed  CAS  Google Scholar 

  11. Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, Reik W, et al. Epigenetic reprogramming in mouse primordial germ cells. Mech Dev. 2002;117:15–23.

    Article  PubMed  CAS  Google Scholar 

  12. Hammadeh ME, Askari AS, Georg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl. 1999;22:155–62.

    Article  PubMed  CAS  Google Scholar 

  13. Hammoud SS, Purwar J, Pflueger C, Cairns BR, Carrell DT. Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility. Fertil Steril. 2010;94:1728–33.

    Article  PubMed  CAS  Google Scholar 

  14. Horne G, Atkinson AD, Pease EH, Logue JP, Brison DR, Lieberman BA. Live birth with sperm cryopreserved for 21 years prior to cancer treatment: case report. Hum Reprod. 2004;19:1448–9.

    Article  PubMed  CAS  Google Scholar 

  15. Horsthemke B, Ludwig M. Assisted reproduction: the epigenetic perspective. Hum Reprod Update. 2005;11:473–82.

    Article  PubMed  Google Scholar 

  16. Hu J, Zhu W, Liu W, Fan L. Factors affecting fecundity among sperm donors: a multivariate analysis. Andrologia. 2011;43:155–62.

    Article  PubMed  CAS  Google Scholar 

  17. Kelly TL, Li E, Trasler JM. 5-Aza-2′-deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome. J Androl. 2003;24:822–30.

    PubMed  CAS  Google Scholar 

  18. Kerjean A, Dupont JM, Vasseur C, Le Tessier D, Cuisset L, Paldi A, et al. Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol Genet. 2000;9:2183–7.

    Article  PubMed  CAS  Google Scholar 

  19. Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet. 2009;17:1582–91.

    Article  PubMed  CAS  Google Scholar 

  20. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet. 2007;16:2542–51.

    Article  PubMed  CAS  Google Scholar 

  21. Le Lannou D, Blanchard Y. Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure. J Reprod Fertil. 1988;84:551–6.

    Article  PubMed  Google Scholar 

  22. Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol. 2006;24:2917–31.

    Article  PubMed  Google Scholar 

  23. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril. 1998;69:528–32.

    Article  PubMed  CAS  Google Scholar 

  24. Maher ER (2005) Imprinting and assisted reproductive technology. Hum Mol Genet 14 Spec No 1:R133-8

    Google Scholar 

  25. Manipalviratn S, DeCherney A, Segars J. Imprinting disorders and assisted reproductive technology. Fertil Steril. 2009;91:305–15.

    Article  PubMed  CAS  Google Scholar 

  26. Marques CJ, Joao Pinho M, Carvalho F, Bieche I, Barros A, Sousa M (2011) DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages. Epigenetics 6

  27. Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, et al. Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia. Mol Hum Reprod. 2008;14:67–74.

    Article  PubMed  CAS  Google Scholar 

  28. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the zygotic paternal genome. Nature. 2000;403:501–2.

    Article  PubMed  CAS  Google Scholar 

  29. Niu ZH, Shi HJ, Zhang HQ, Zhang AJ, Sun YJ, Feng Y. Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF. Asian J Androl. 2011;13:862–6.

    Article  PubMed  Google Scholar 

  30. Oakes CC, Kelly TL, Robaire B, Trasler JM. Adverse effects of 5-aza-2′-deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation. J Pharmacol Exp Ther. 2007;322:1171–80.

    Article  PubMed  CAS  Google Scholar 

  31. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Dev Biol. 2007;307:368–79.

    Article  PubMed  CAS  Google Scholar 

  32. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun. 1984;123:291–8.

    Article  PubMed  CAS  Google Scholar 

  33. Pacey AA. Fertility issues in survivors from adolescent cancers. Cancer Treat Rev. 2007;33:646–55.

    Article  PubMed  CAS  Google Scholar 

  34. Patrizio P, Sanguineti F, Sakkas D. Modern andrology: from semen analysis to postgenomic studies of the male gametes. Ann N Y Acad Sci. 2008;1127:59–63.

    Article  PubMed  Google Scholar 

  35. Poplinski A, Tuttelmann F, Kanber D, Horsthemke B, Gromoll J. Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1. Int J Androl. 2010;33:642–9.

    PubMed  CAS  Google Scholar 

  36. Practice Committee of American Society for Reproductive Medicine, Practice Committee of Society for Assisted Reproductive Technology. 2008 Guidelines for gamete and embryo donation: a Practice Committee report. Fertil Steril. 2008;90:S30–44.

    Google Scholar 

  37. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089–93.

    Article  PubMed  CAS  Google Scholar 

  38. Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril. 2010;93:1027–36.

    Article  PubMed  CAS  Google Scholar 

  39. Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod. 1996;11:837–43.

    Article  PubMed  CAS  Google Scholar 

  40. Spano M, Cordelli E, Leter G, Lombardo F, Lenzi A, Gandini L. Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. Mol Hum Reprod. 1999;5:29–37.

    Article  PubMed  CAS  Google Scholar 

  41. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod. 1997;56:602–7.

    Article  PubMed  CAS  Google Scholar 

  42. Thomson LK, Fleming SD, Barone K, Zieschang JA, Clark AM. The effect of repeated freezing and thawing on human sperm DNA fragmentation. Fertil Steril. 2010;93:1147–56.

    Article  PubMed  CAS  Google Scholar 

  43. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24:2061–70.

    Article  PubMed  CAS  Google Scholar 

  44. Trasler JM. Epigenetics in spermatogenesis. Mol Cell Endocrinol. 2009;306:33–6.

    Article  PubMed  CAS  Google Scholar 

  45. Tunc O, Tremellen K. Oxidative DNA damage impairs global sperm DNA methylation in infertile men. J Assist Reprod Genet. 2009;26:537–44.

    Article  PubMed  Google Scholar 

  46. Wang JX, Norman RJ, Wilcox AJ. Incidence of spontaneous abortion among pregnancies produced by assisted reproductive technology. Hum Reprod. 2004;19:272–7.

    Article  PubMed  CAS  Google Scholar 

  47. World Health Organization. WHO laboratory manual for the examination and processing of human semen. Geneva: World Health Organization; 2010.

    Google Scholar 

  48. Wu W, Shen O, Qin Y, Niu X, Lu C, Xia Y, et al. Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR). PLoS One. 2010;5:e13884.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Raphaele Kürten, Daniela Hanke, Jolonta Körber, Sabine Strüwing and Barbara Hellenkemper for excellent technical assistance and Prof. Thomas Haaf from the University of Wuerzburg for information concerning pyrosequencing assays. We also thank Victoria Sánchez for help with Fenton’s reactions and FCEAO.

Financial Support: This study was supported by Graduate Program Cell Dynamics and Disease (CEDAD) and the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM) and by German Research Foundation (Research Unit “Germ cell potential”, FOR 1041).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Centre of Reproductive Medicine and Andrology, University Clinics Muenster, Albert-Schweitzer-Campus 1, Building D11, 48149, Muenster, Germany

    Ruth Kläver, Andreas Bleiziffer, Klaus Redmann, Con Mallidis, Sabine Kliesch & Jörg Gromoll

Authors
  1. Ruth Kläver
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Bleiziffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klaus Redmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Con Mallidis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sabine Kliesch
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jörg Gromoll
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jörg Gromoll.

Additional information

Capsule

Cryopreservation of spermatozoa in daily clinical routine can be considered safe with regard to DNA methylation, since neither short- nor mid-term cryostorage alters methylation patterns of spermatozoa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 31 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kläver, R., Bleiziffer, A., Redmann, K. et al. Routine cryopreservation of spermatozoa is safe — Evidence from the DNA methylation pattern of nine spermatozoa genes. J Assist Reprod Genet 29, 943–950 (2012). https://doi.org/10.1007/s10815-012-9813-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-012-9813-z

Keywords

Search

Navigation