Advertisement

Journal of Assisted Reproduction and Genetics

, Volume 35, Issue 12, pp 2215–2221 | Cite as

Magnetic-activated cell sorting is not completely effective at reducing sperm DNA fragmentation

  • Mercedes González Martínez
  • Pascual Sánchez-Martín
  • Mónica Dorado-Silva
  • José Luís Fernández
  • Estibaliz Girones
  • Stephen D. JohnstonEmail author
  • Jaime Gosálvez
Gamete Biology

Abstract

Purpose

To determine whether there is a homogeneous reduction of sperm DNA fragmentation (SDF) in sperm samples recovered from the MACS procedure, compared to spermatozoa in the initial ejaculate (NEAT) and those retained in the column.

Methods

This study investigated the relative change in sperm DNA quality (SDF) of neat ejaculates (10 idiopathic infertile and 10 normozoospermic patients) to subpopulations of spermatozoa that had passed through the column (MACS−) and those retained (MACS+) by the annexin-V conjugated microbeads.

Results

While the MACS protocol was capable of reducing the mean proportion of SDF (59.2%; P = 0.000) and sperm with highly degraded DNA (SDD; 65.7%, P = 0.000) in all patients, the reduction was not homogeneous across the patient cohort. A significant positive correlation (r = 0.772, P = 0.000) was apparent between the level of SDF in the NEAT ejaculate and the efficacy of SDF reduction observed in the MACS− fraction.

Conclusion

MACS is capable of reducing the proportion of SDF, especially spermatozoa with a highly degraded DNA molecule. However, this reduction did not preclude the presence of a small subpopulation of spermatozoa with damaged DNA in the MACS− fraction. The MACS protocol was two- to threefold more efficient when the SDF in NEAT ejaculate was equal to or greater than 30%. In 4 of 20 individuals, the level of SDF after MACS resulted in semen for ICSI with a higher or non-significant reduction when compared to SDF observed in the NEAT ejaculate.

Keywords

Male factor MACS Sperm DNA fragmentation 

Notes

Acknowledgments

The authors want to acknowledge Francisca Arroyo for technical assistance.

Author’s contribution

PS-M, MD-S, JLF, SDJ and JG were involved in the experimental design. MD-S, JLF, and EG processed samples. EG, SDJ, and KG drafted the manuscript. SDJ revised the manuscript. JG conducted the statistical analysis.

Funding

This project was partially funded by the Spanish Ministry of Science and Innovation (BFU-2013- 44290-R). The funding body had no involvement in the study.

Compliance with ethical standards

This study was approved by the GINEMED Ethics Committee (Protocol Version v-4).

References

  1. 1.
    Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health. 2014;31:1–17.CrossRefGoogle Scholar
  2. 2.
    Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev. 2010;4:118–26.CrossRefGoogle Scholar
  3. 3.
    Scandalios JG. Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res. 2005;38:955–1014.CrossRefGoogle Scholar
  4. 4.
    Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ. 2001;8:551–63.CrossRefGoogle Scholar
  5. 5.
    Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995;184:39–51.CrossRefGoogle Scholar
  6. 6.
    van Genderen HO, Kenis H, Hofstra L, Narula J, Reutelingsperger CPM. Extracellular annexin A5: functions of phosphatidylserine-binding and two-dimensional crystallization. Biochim Biophys Acta Mol Cell Res. 2008;1783:953–63.CrossRefGoogle Scholar
  7. 7.
    Said T, Agarwal A, Grunewald S, Rasch M, Baumann T, Kriegel C, et al. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biol Reprod. 2006;74:530–7.CrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C. Semen preparation techniques for intrauterine insemination. Cochrane Database Syst Rev. 2007;3:CD004507.Google Scholar
  10. 10.
    Bartke A. Apoptosis of male germ cells, a generalized or a cell type- specific phenomenon? Endocrinology. 1995;136:3–4.CrossRefGoogle Scholar
  11. 11.
    Sakkas D, Mariethoz E, St John JC. Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the fas-mediated pathway. Exp Cell Res. 1999;251:350–5.CrossRefGoogle Scholar
  12. 12.
    Sánchez-Martín P, Dorado-Silva M, Sánchez-Martín F, González-Martínez M, Johnston SD, Gosálvez J. Magnetic cell sorting of semen containing spermatozoa with high DNA fragmentation in ICSI cycles decreases miscarriage rate. Reprod BioMed Online. 2017;34:506–12.CrossRefGoogle Scholar
  13. 13.
    Fernández JL, Muriel L, Goyanes V, Segrelles E, Gosálvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 2005;84:833–42.CrossRefGoogle Scholar
  14. 14.
    Gosálvez J, Rodríguez-Predreira M, Mosquera A, López-Fernández C, Esteves SC, Agarwal A, et al. Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test. Andrologia. 2014;46:602–9.CrossRefGoogle Scholar
  15. 15.
    Almeida C, Sousa M, Barros A. Phosphatidylserine translocation in human spermatozoa from impaired spermatogenesis. Reprod BioMed Online. 2009;19:770–7.CrossRefGoogle Scholar
  16. 16.
    Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod. 2002;66:1061–7.CrossRefGoogle Scholar
  17. 17.
    Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol. 2004;5:897–907.CrossRefGoogle Scholar
  18. 18.
    Slee EA, Adrain C, Martin SJ. Executioner Caspase-3, −6, and −7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem. 2001;276:7320–6.CrossRefGoogle Scholar
  19. 19.
    Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998;391:96–9.CrossRefGoogle Scholar
  20. 20.
    Collins JA, Schandl CA, Young KK, Vesely J, Willingham MC. Major DNA fragmentation is a late event in apoptosis. J Histochem Cytochem. 1997;45:923–34.CrossRefGoogle Scholar
  21. 21.
    Gjertsen BT, Cressey LI, Ruchaud S, Houge G, Lanotte M, Døskeland SO. Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line. J Cell Sci. 1994;107:3363–77.PubMedGoogle Scholar
  22. 22.
    Marchetti C, Marchetti P. Detection of apoptotic markers in human ejaculated spermatozoa as new methods in human reproductive biology. Gynécol Obs Fertil. 2005;33:669–77.CrossRefGoogle Scholar
  23. 23.
    Martí E, Pérez-Pé R, Colás C, Muiño-Blanco T, Cebrián-Pérez JA. Study of apoptosis-related markers in ram spermatozoa. Anim Reprod Sci. 2008;106:113–32.CrossRefGoogle Scholar
  24. 24.
    Fraczek M, Hryhorowicz M, Gaczarzewicz D, Szumala-Kakol A, Kolanowski TJ, Beutin L, et al. Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection? J Assist Reprod Genet. 2015;32:711–9.CrossRefGoogle Scholar
  25. 25.
    Didenko VV, Ngo H, Baskin DS. Early necrotic DNA degradation: presence of blunt-ended DNA breaks, 3′ and 5′ overhangs in apoptosis, but only 5′ overhangs in early necrosis. Am J Pathol. 2004;162:1571–8.CrossRefGoogle Scholar
  26. 26.
    Fernández-Gonzalez R, Moreira PN, Pérez-Crespo M, Sánchez-Martín M, Ramirez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod. 2008;78:761–72.CrossRefGoogle Scholar
  27. 27.
    Marchetti F, Wyrobeck AJ. Mechanisms and consequences of paternally-transmitted chromosomal abnormalities. Birth Defects Res C. 2005;75:112–29.CrossRefGoogle Scholar
  28. 28.
    Marchetti F, Essers J, Kanaar R, Wyrobeck AJ. Disruption of maternal DNA repair increases sperm-derived chromosomal aberrations. PNAS. 2007;104:17725–9.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mercedes González Martínez
    • 1
  • Pascual Sánchez-Martín
    • 1
  • Mónica Dorado-Silva
    • 1
  • José Luís Fernández
    • 2
  • Estibaliz Girones
    • 3
  • Stephen D. Johnston
    • 4
    • 5
    Email author
  • Jaime Gosálvez
    • 3
  1. 1.GinemedSevillaSpain
  2. 2.Genetics UnitINIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC)A CoruñaSpain
  3. 3.Unit of Genetics, Department of BiologyUniversidad Autónoma de MadridMadridSpain
  4. 4.School of Agriculture and Food ScienceThe University of QueenslandGattonAustralia
  5. 5.School of Agriculture and Food ScienceThe University of QueenslandGattonAustralia

Personalised recommendations