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Journal of Assisted Reproduction and Genetics

, Volume 36, Issue 10, pp 2079–2086 | Cite as

Removal of DNA-fragmented spermatozoa using flow cytometry and sorting does not improve the outcome of intracytoplasmic sperm injection

  • Christian De GeyterEmail author
  • Ursula Gobrecht-Keller
  • Astrid Ahler
  • Manuel Fischer
Assisted Reproduction Technologies

Abstract

Purpose

The DNA fragmentation in sperm is associated with reduced outcome in assisted reproduction. Using YoPro1 as the staining dye and flow cytometry and sorting (FACS), the number of spermatozoa with DNA fragmentation can be lowered to 5%. Can the cumulative outcome of ICSI be improved using FACS?

Methods

A prospective, randomized, double-blind clinical trial was conducted in 104 infertile couples with male infertility based on abnormal conventional semen analysis results. Cumulative ongoing pregnancy rate was the primary outcome parameter. In 52 cases, semen was processed for ICSI using swim-up. In another 52 cases, spermatozoa with fragmented DNA were removed with FACS.

Results

The cumulative pregnancy rate at 12 weeks of gestation (51.9% versus 46.2%) and live birth rate (42.3% versus 34.6%) were higher and the miscarriage rate was lower (27.8% versus 35.3%) after FACS-sorting as compared with swim-up. An interim analysis scheduled before initiation of the study after 100 cases demonstrated that the aim of a 20% gain in pregnancy rate could not be achieved. For that reason, the prospective study was stopped prematurely.

Conclusions

A trend towards consistently better results was achieved by removing spermatozoa with fragmented DNA. The fragmentation of the DNA in sperm is the end stage of apoptosis. Sorting of spermatozoa may be improved by selecting parameters of processes active more upstream of apoptosis, such as chromatin decondensation.

Trial registration

NCT02166567. June 14, 2014.

Keywords

DNA fragmentation  Apoptosis  Flow cytometry  ICSI  Chromatin remodeling 

Notes

Acknowledgments

We are grateful to the work of the monitoring group of the Clinical Trial Unit of the Basler University Hospital (www.dkf.unibas.ch) and to Ms. Hanna Flükiger for coordinating all activities related to this study.

Funding information

This study was funded by the Repronatal Foundation, Basel, Switzerland.

Compliance with ethical standards

The study was presented to and approved by the local ethics committee (EKBB178/12) and monitored by the clinical trial unit (CTU) of the University Hospital of Basel.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Bonde JP, Ernst E, Jensen TK, Hjollund NH, Kolstad H, Henriksen TB, et al. Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet. 1998;352:1172–7.CrossRefGoogle Scholar
  2. 2.
    Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16:231–45.CrossRefGoogle Scholar
  3. 3.
    De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T, et al. European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2018;33:1586–601.CrossRefGoogle Scholar
  4. 4.
    Muratori M, De Geyter C. Chromatin condensation, fragmentation of DNA and differences in the epigenetic signature of infertile men. Best Pract Res Clin Endocrinol Metab. 2018;33(1):117–26.CrossRefGoogle Scholar
  5. 5.
    Osman A, Alsomait H, Seshadri S, El-Toukhy T, Khalaf Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis. Reprod BioMed Online. 2015;30:120–7.CrossRefGoogle Scholar
  6. 6.
    Simon L, Emery BR, Carrell DT. Review: diagnosis and impact of sperm DNA alterations in assisted reproduction. Best Pract Res Clin Obstet Gynaecol. 2017;44:38–56.CrossRefGoogle Scholar
  7. 7.
    Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23:2663–8.CrossRefGoogle Scholar
  8. 8.
    Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum Reprod. 2012;27:2908–17.CrossRefGoogle Scholar
  9. 9.
    Taha EA, Ez-Aldin AM, Sayed SK, Ghandour NM, Mostafa T. Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men. Urology. 2012;80:822–5.CrossRefGoogle Scholar
  10. 10.
    Dupont C, Faure C, Sermondade N, Boubaya M, Eustache F, Clément P, et al. Obesity leads to higher risk of sperm DNA damage in infertile patients. Asian J Androl. 2013;15:622–5.CrossRefGoogle Scholar
  11. 11.
    Roessner C, Paasch U, Kratzsch J, Glander HJ, Grunewald S. Sperm apoptosis signalling in diabetic men. Reprod BioMed Online. 2012;25:292–9.CrossRefGoogle Scholar
  12. 12.
    Said TM, Agarwal A, Zborowski M. Utility of magnetic cell separation as a molecular sperm preparation technique. J Androl. 2008;29:134–42.CrossRefGoogle Scholar
  13. 13.
    Ye H, Huang GN, Gao Y, Liu DY. Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization. Hum Reprod. 2006;21:1545–50.CrossRefGoogle Scholar
  14. 14.
    Gil M, Sar-Shalom V, Melendez Sivira Y, Carreras R, Checa MA. Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic review and meta-analysis. J Assist Reprod Genet. 2013;30:479–85.CrossRefGoogle Scholar
  15. 15.
    McDowell S, Kroon B, Ford E, Hook Y, Glujovsky D, Yazdani A. Advanced sperm selection techniques for assisted reproduction. Cochrane Database Syst Rev. 2014;CD010461.Google Scholar
  16. 16.
    Ribeiro SC, Sartorius G, Pletscher F, de Geyter M, Zhang H, de Geyter C. Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting. Fertil Steril. 2013;100:686–94.CrossRefGoogle Scholar
  17. 17.
    De Geyter C, Bals-Pratsch M, Doeren M, Yeung CH, Grunert JH, Bordt J, et al. Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization. Hum Reprod. 1988;3:948–54.CrossRefGoogle Scholar
  18. 18.
    De Geyter C. Assisted reproductive medicine in Switzerland. Swiss Med Wkly. 2012;142:w13569.PubMedGoogle Scholar
  19. 19.
    Raat H, Landgraf JM, Oostenbrink R, Moll HA, Essink-Bot ML. Reliability and validity of the Infant and Toddler Quality of Life Questionnaire (ITQOL) in a general population and respiratory disease sample. Qual Life Res. 2007;16:445–60.CrossRefGoogle Scholar
  20. 20.
    De Geyter C, Sterthaus O, Miny P, Wenzel F, Lapaire O, De Geyter M, et al. First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa. Swiss Med Wkly. 2013;143:w13718.PubMedGoogle Scholar
  21. 21.
    Aitken RJ, De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod. 2010;16:3–13.CrossRefGoogle Scholar
  22. 22.
    Muratori M, Marchiani S, Tamburrino L, Cambi M, Lotti F, Natali I, et al. DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters. Fertil Steril. 2015;104:582–90.CrossRefGoogle Scholar
  23. 23.
    Ribeiro SC, Muratori M, De Geyter M, De Geyter C. TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation. PLoS One. 2017;12:e0181802.CrossRefGoogle Scholar
  24. 24.
    Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature. 2009;460:473–8.CrossRefGoogle Scholar
  25. 25.
    Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, Roloff TC, et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol. 2010;17:679–87.CrossRefGoogle Scholar
  26. 26.
    Erkek S, Hisano M, Liang C-Y, Gill M, Murr R, Dieker J, et al. Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa. Nat Struct Mol Biol. 2013;20:868–75.CrossRefGoogle Scholar
  27. 27.
    Hammoud SS, Nix DA, Hammoud AO, Gibson M, Cairns BR, Carrell DT. Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men. Hum Reprod. 2011;26:2558–69.CrossRefGoogle Scholar
  28. 28.
    Azpiazu R, Amaral A, Castillo J, Estanyo JM, Guimera’ M, Ballesca’ JL, et al. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction. Hum Reprod. 2014;29:1225–37.CrossRefGoogle Scholar
  29. 29.
    Ni K, Spiess AN, Schuppe HC, Steger K. The impact of sperm protamine deficiency and sperm DNA damage on human male fertility: a systematic review and meta-analysis. Andrology. 2016;4:789–99.CrossRefGoogle Scholar
  30. 30.
    Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, et al. Investigation on the origin of sperm DNA fragmentation: role of apoptosis. Immaturity and Oxidative Stress Mol Med. 2015;21:109–22.PubMedGoogle Scholar
  31. 31.
    Manochantr S, Chiamchanya C, Sobhon P. Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men. Andrologia. 2012;44:187–99.CrossRefGoogle Scholar
  32. 32.
    Sakkas D, Urner F, Bizzaro D, Manicardi G, Bianchi PG, Shoukir Y, et al. Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Hum Reprod. 1998;13(Suppl 4):11–9.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Reproductive Medicine and Gynecological Endocrinology (RME), University HospitalUniversity of BaselBaselSwitzerland
  2. 2.Reproductive Medicine and Gyn. Endocrinology (RME)BaselSwitzerland

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