Journal of Assisted Reproduction and Genetics

, Volume 32, Issue 1, pp 147–154 | Cite as

DNA fragmentation in human sperm after magnetic-activated cell sorting

  • Sara Bucar
  • Ana Gonçalves
  • Eduardo Rocha
  • Alberto Barros
  • Mário Sousa
  • Rosália SáEmail author
Gamete Biology



As fertilization with unselected apoptotic spermatozoa may contribute to failures in assisted reproductive techniques, it has become essential to remove this type of sperm in order to increase the success rates. Magnetic-activated cell sorting (MACS) is a sperm preparation technique that isolates non-apoptotic spermatozoa based on the expression of phosphatidylserine in the membrane of apoptotic sperm. Therefore, we aimed to evaluate whether there was a significant decrease in sperm DNA fragmentation (sDNAfrag) and verify which protocol was the most efficient.


Hundred semen samples were allocated into five distinct groups and processed according to a combination of MACS with density gradient centrifugation (DGC) and swim-up (SU) techniques. Sperm DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.


Groups DGC-SU (73.4 %), DGC-MACS-SU (78.9 %), DGC-SU-MACS (53.8 %) and MACS-SU (73.5 %) presented a significant decrease in sDNAfrag but the highest reduction rate was obtained with MACS-DGC-SU (83.3 %). The later was also negatively correlated with sperm vitality, membrane integrity and progressive motility. Additionally, teratozoospermic patients presented a tendency to have lower sDNAfrag reduction rates than asthenozoospermic and asthenoteratozoospermic patients.


Based on the results, MACS showed potential to optimize the sDNAfrag reduction rate, when applied to raw semen, before DGC and SU, especially in samples with low values of progressive motility, vitality and hypoosmotic swelling test.


Apoptosis DNA fragmentation Sperm TUNEL MACS 



We would like to acknowledge Ângela Alves, Technical assistant for teaching and research (ICBAS-UP) in laboratorial assistance, Claúdia Osório, Bsc, Biologist (CGR-ABarros) in spermiogram analysis assistance and Gabriela Rodrigues, PhD, Assistant Professor (University of Lisbon) for reviewing the manuscript.

Multidisciplinary Unit for Biomedical Research (UMIB) is funded by National Funds through FCT-Foundation for Science and Technology, under the Pest-OE/SAU/UI0215/2014.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    World Health Organization (WHO). WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization; 2010.Google Scholar
  2. 2.
    Nallella KP, Sharma RK, Aziz N, Agarwal A. Significance of sperm characteristics in the evaluation of male infertility. Fertil Steril. 2006;85(3):629–34. doi: 10.1016/j.fertnstert.2005.08.024.PubMedCrossRefGoogle Scholar
  3. 3.
    ASRM. Effectiveness and treatment for unexplained infertility. Fertil Steril. 2006;86(5 Suppl 1):S111–4. doi: 10.1016/j.fertnstert.2006.07.1475.Google Scholar
  4. 4.
    Zinaman MJ, Brown CC, Selevan SG, Clegg ED. Semen quality and human fertility: a prospective study with healthy couples. J Androl. 2000;21(1):145–53. doi: 10.1002/j.1939-4640.2000.tb03284.x.PubMedGoogle Scholar
  5. 5.
    Aitken RJ. Sperm function tests and fertility. Int J Androl. 2006;29(1):69–75. doi: 10.1111/j.1365-2605.2005.00630.x. discussion 105–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl. 2006;27(1):53–9. doi: 10.2164/jandrol.05068.PubMedCrossRefGoogle Scholar
  7. 7.
    Delbes G, Hales BF, Robaire B. Toxicants and human sperm chromatin integrity. Mol Hum Reprod. 2010;16(1):14–22. doi: 10.1093/molehr/gap087.PubMedCrossRefGoogle Scholar
  8. 8.
    Simon L, Proutski I, Stevenson M, Jennings D, McManus J, Lutton D, et al. Sperm DNA damage has a negative association with live-birth rates after IVF. Reprod Biomed Online. 2013;26(1):68–78. doi: 10.1016/j.rbmo.2012.09.019.PubMedCrossRefGoogle Scholar
  9. 9.
    Henkel R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, et al. DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online. 2003;7(4):477–84.PubMedCrossRefGoogle Scholar
  10. 10.
    Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, Francois GJ. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril. 2007;87(1):93–100. doi: 10.1016/j.fertnstert.2006.05.057.PubMedCrossRefGoogle Scholar
  11. 11.
    Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81(5):1289–95. doi: 10.1016/j.fertnstert.2003.09.063.PubMedCrossRefGoogle Scholar
  12. 12.
    Lin MH, Kuo-Kuang Lee R, Li SH, Lu CH, Sun FJ, Hwu YM. Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertil Steril. 2008;90(2):352–9. doi: 10.1016/j.fertnstert.2007.06.018.PubMedCrossRefGoogle Scholar
  13. 13.
    Tavalaee M, Razavi S, Nasr-Esfahani MH. Influence of sperm chromatin anomalies on assisted reproductive technology outcome. Fertil Steril. 2009;91(4):1119–26. doi: 10.1016/j.fertnstert.2008.01.063.PubMedCrossRefGoogle Scholar
  14. 14.
    Henkel R. Sperm preparation: state-of-the-art–physiological aspects and application of advanced sperm preparation methods. Asian J Androl. 2012;14(2):260–9. doi: 10.1038/aja.2011.133.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Boitrelle F, Guthauser B, Alter L, Bailly M, Bergere M, Wainer R, et al. High-magnification selection of spermatozoa prior to oocyte injection: confirmed and potential indications. Reprod Biomed Online. 2014;28(1):6–13. doi: 10.1016/j.rbmo.2013.09.019.PubMedCrossRefGoogle Scholar
  16. 16.
    Kheirollahi-Kouhestani M, Razavi S, Tavalaee M, Deemeh MR, Mardani M, Moshtaghian J, et al. Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome. Hum Reprod. 2009;24(10):2409–16. doi: 10.1093/humrep/dep088.PubMedCrossRefGoogle Scholar
  17. 17.
    Fleming SD, Ilad RS, Griffin AM, Wu Y, Ong KJ, Smith HC, et al. Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation. Hum Reprod. 2008;23(12):2646–51. doi: 10.1093/humrep/den330.PubMedCrossRefGoogle Scholar
  18. 18.
    Tarozzi N, Nadalini M, Bizzaro D, Serrao L, Fava L, Scaravelli G, et al. Sperm-hyaluronan-binding assay: clinical value in conventional IVF under Italian law. Reprod Biomed Online. 2009;19 Suppl 3:35–43.PubMedCrossRefGoogle Scholar
  19. 19.
    Almeida C, Sousa M, Barros A. Phosphatidylserine translocation in human spermatozoa from impaired spermatogenesis. Reprod Biomed Online. 2009;19(6):770–7.PubMedCrossRefGoogle Scholar
  20. 20.
    Grunewald S, Paasch U. Sperm selection for ICSI using annexin V. Methods Mol Biol. 2013;927:257–62. doi: 10.1007/978-1-62703-038-0_23.PubMedCrossRefGoogle Scholar
  21. 21.
    Said TM, Grunewald S, Paasch U, Glander HJ, Baumann T, Kriegel C, et al. Advantage of combining magnetic cell separation with sperm preparation techniques. Reprod Biomed Online. 2005;10(6):740–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Delbes G, Herrero MB, Troeung ET, Chan PT. The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting. Andrology. 2013;1(5):698–706. doi: 10.1111/j.2047-2927.2013.00106.x.PubMedCrossRefGoogle Scholar
  23. 23.
    Vendrell X, Ferrer M, Garcia-Mengual E, Munoz P, Trivino JC, Calatayud C, et al. Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients. Reprod Biomed Online. 2014;28(4):492–502. doi: 10.1016/j.rbmo.2013.12.001.PubMedCrossRefGoogle Scholar
  24. 24.
    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(4):479–85. doi: 10.1007/s10815-013-9962-8.PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Sheikhi A, Jalali M, Gholamian M, Jafarzadeh A, Jannati S, Mousavifar N. Elimination of apoptotic spermatozoa by magnetic-activated cell sorting improves the fertilization rate of couples treated with ICSI procedure. Androl. 2013;1(6):845–9. doi: 10.1111/j.2047-2927.2013.00140.x.CrossRefGoogle Scholar
  26. 26.
    Rawe VY, Boudri HU, Alvarez Sedo C, Carro M, Papier S, Nodar F. Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI. Reprod Biomed Online. 2010;20(3):320–3. doi: 10.1016/j.rbmo.2009.12.004.PubMedCrossRefGoogle Scholar
  27. 27.
    Polakde Fried E, Denaday F. Single and twin ongoing pregnancies in two cases of previous ART failure after ICSI performed with sperm sorted using annexin V microbeads. Fertil Steril. 2010;94(1):351.e15–8. doi: 10.1016/j.fertnstert.2009.12.037.Google Scholar
  28. 28.
    Herrero MB, Delbes G, Chung JT, Son WY, Holzer H, Buckett W, et al. Case report: the use of annexin V coupled with magnetic activated cell sorting in cryopreserved spermatozoa from a male cancer survivor: healthy twin newborns after two previous ICSI failures. J Assist Reprod Genet. 2013;30(11):1415–9. doi: 10.1007/s10815-013-0086-y.PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Cohen J. Statistical power analysis for the behavioral sciences. 2nd ed. NewJersey: Lawrence Erlbaum Associates; 1988.Google Scholar
  30. 30.
    Lee TH, Liu CH, Shih YT, Tsao HM, Huang CC, Chen HH, et al. Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility. Hum Reprod. 2010;25(4):839–46. doi: 10.1093/humrep/deq009.PubMedCrossRefGoogle Scholar
  31. 31.
    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(3):530–7. doi: 10.1095/biolreprod.105.046607.PubMedCrossRefGoogle Scholar
  32. 32.
    Tavalaee M, Deemeh MR, Arbabian M, Nasr-Esfahani MH. Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection? J Assist Reprod Genet. 2012;29(1):31–8. doi: 10.1007/s10815-011-9686-6.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Oehninger S, Acosta R, Morshedi M, Philput C, Swanson RJ, Acosta AA. Relationship between morphology and motion characteristics of human spermatozoa in semen and in the swim-up sperm fractions. J Androl. 1990;11(5):446–52. doi: 10.1002/j.1939-4640.1990.tb00174.x.PubMedGoogle Scholar
  34. 34.
    Lachaud C, Tesarik J, Canadas ML, Mendoza C. Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod. 2004;19(3):607–10. doi: 10.1093/humrep/deh130.PubMedCrossRefGoogle Scholar
  35. 35.
    de Vantery AC, Lucas H, Chardonnens D, de Agostini A. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential. Reprod Biol Endocrinol. 2009;7:1. doi: 10.1186/1477-7827-7-1.CrossRefGoogle Scholar
  36. 36.
    Zahedi A, Tavalaee M, Deemeh MR, Azadi L, Fazilati M, Nasr-Esfahani MH. Zeta potential vs apoptotic marker: which is more suitable for ICSI sperm selection? J Assist Reprod Genet. 2013;30(9):1181–6. doi: 10.1007/s10815-013-0022-1.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sara Bucar
    • 1
    • 2
  • Ana Gonçalves
    • 3
  • Eduardo Rocha
    • 4
  • Alberto Barros
    • 3
    • 5
  • Mário Sousa
    • 1
  • Rosália Sá
    • 1
    Email author
  1. 1.Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS)University of Porto (UP), Multidisciplinary Unit for Biomedical Research (UMIB)PortoPortugal
  2. 2.Department of Animal Biology, Faculty of SciencesUniversity of LisbonLisbonPortugal
  3. 3.Centre for Reproductive Genetics Prof. Alberto Barros (CGR-ABarros)PortoPortugal
  4. 4.Department of Microscopy, Laboratory of Histology and Embryology, ICBAS-UPPortoPortugal
  5. 5.Department of Genetics, Faculty of Medicine, UPPortoPortugal

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