Journal of Assisted Reproduction and Genetics

, Volume 30, Issue 11, pp 1519–1524 | Cite as

Sperm DNA and chromatin integrity in semen samples used for intrauterine insemination

  • Abdullah Alkhayal
  • Maria San Gabriel
  • Krista Zeidan
  • Khalid Alrabeeah
  • Diana Noel
  • Rachelle McGraw
  • Francois Bissonnette
  • Isaac Jacques Kadoch
  • Armand ZiniEmail author
Gamete Biology



Sperm DNA damage is associated with male infertility but whether normozoospermic infertile men also have DNA damage is unknown.


To evaluate sperm DNA and chromatin integrity in men with mild male factor infertility.

Design, setting and participants

Prospective study of 102 consecutive men (78 normozoospermic, 15 asthenozoospermic, 9 oligozoospermic) enrolled for intrauterine insemination (IUI) and 15 fertile controls.

Outcome measurements and statistical analysis

Standard semen parameters and sperm chromatin and DNA integrity were assessed and compared between groups. Sperm chromatin quality was assessed by (1) aniline blue staining (AB is specific to histone lysines), (2) iodoacetamide fluorescein fluorescence (IAF targets free protamine sulfhydryl groups) and (3) sperm chromatin structure assay (SCSA) with the results expressed as % DNA fragmentation index (%DFI).

Results and limitations

The mean (±SD) percentage of spermatozoa with positive IAF fluorescence was significantly higher in the IUI population compared to fertile controls (17 % ± 10 % vs. 8 % ± 6 %, P = 0.0011) and also in the normozoospermic subset (n = 78) compared to controls (16 % ± 9 % vs. 8 % ± 6 %, P < 0.0001, ANOVA). We also observed a trend toward lower %progressive motility, and higher %AB staining and %DFI in the IUI group compared to controls. We observed significant relationships between sperm %DFI and progressive motility (r = −0.40, P < 0.0001) and between positive AB staining and IAF fluorescence (r = 0.58, P < 0.0001).


The data indicate that sperm chromatin integrity may be abnormal in men enrolled in IUI treatment cycles, despite the fact that most of these men are normozoospermic.


Sperm DNA Intrauterine insemination Male infertility Normozoospermia Semen Sperm chromatin 


  1. 1.
    Brandes M, Hamilton CJ, de Bruin JP, Nelen WL, Kremer JA. The relative contribution of IVF to the total ongoing pregnancy rate in a subfertile cohort. Hum Reprod. 2010;25(1):118–26.PubMedCrossRefGoogle Scholar
  2. 2.
    Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999;4(1):31–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Marcon L, Boissonneault G. Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling. Biol Reprod. 2004;70(4):910–8.PubMedCrossRefGoogle Scholar
  4. 4.
    McPherson S, Longo FJ. Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur J Histochem. 1993;37(2):109–28.PubMedGoogle Scholar
  5. 5.
    Sakkas D, Manicardi G, Bianchi PG, Bizzaro D, Bianchi U. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod. 1995;52(5):1149–55.PubMedCrossRefGoogle Scholar
  6. 6.
    Aitken J, Krausz C, Buckingham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod Dev. 1994;39(3):268–79.PubMedCrossRefGoogle Scholar
  7. 7.
    Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod. 2005;20(1):226–30.PubMedCrossRefGoogle Scholar
  8. 8.
    Moskovtsev SI, Jarvi K, Mullen JB, Cadesky KI, Hannam T, Lo KC. Testicular spermatozoa have statistically significantly lower DNA damage compared with ejaculated spermatozoa in patients with unsuccessful oral antioxidant treatment. Fertil Steril. 2010;93(4):1142–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Barratt CL, Aitken RJ, Bjorndahl L, Carrell DT, de Boer P, Kvist U, et al. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications—a position report. Hum Reprod. 2010;25(4):824–38.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Moskovtsev SI, Willis J, White J, Mullen JB. Sperm DNA damage: correlation to severity of semen abnormalities. Urology. 2009;74(4):789–93.PubMedCrossRefGoogle Scholar
  12. 12.
    Zini A, Fischer MA, Sharir S, Shayegan B, Phang D, Jarvi K. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology. 2002;60(6):1069–72.PubMedCrossRefGoogle Scholar
  13. 13.
    WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: UCUP; 1999.Google Scholar
  14. 14.
    Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 1999;14(4):1039–49.PubMedCrossRefGoogle Scholar
  15. 15.
    Zini A, Azhar R, Baazeem A, Gabriel MS. Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial. Int J Androl. 2011;34(1):14–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril. 2001;75(4):674–7.PubMedCrossRefGoogle Scholar
  17. 17.
    de Lamirande E, San Gabriel MC, Zini A. Human sperm chromatin undergoes physiological remodeling during in vitro capacitation and acrosome reaction. J Androl. 2012;33(5):1025–35.PubMedCrossRefGoogle Scholar
  18. 18.
    Chatterjee S, de Lamirande E, Gagnon C. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol Reprod Dev. 2001;60(4):498–506.PubMedCrossRefGoogle Scholar
  19. 19.
    Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas Jr AJ. Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertil Steril. 2003;80(6):1431–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril. 2003;79 Suppl 3:1597–605.PubMedCrossRefGoogle Scholar
  21. 21.
    Chi HJ, Chung DY, Choi SY, Kim JH, Kim GY, Lee JS, et al. Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program. Clin Exp Reprod Med. 2011;38(1):10–7.PubMedCrossRefGoogle Scholar
  22. 22.
    Oliveira JB, Massaro FC, Baruffi RL, Mauri AL, Petersen CG, Silva LF, et al. Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage. Fertil Steril. 2010;94(5):1937–40.PubMedCrossRefGoogle Scholar
  23. 23.
    Varghese AC, Bragais FM, Mukhopadhyay D, Kundu S, Pal M, Bhattacharyya AK, et al. Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics. Andrologia. 2009;41(4):207–15.PubMedCrossRefGoogle Scholar
  24. 24.
    Cohen-Bacrie P, Belloc S, Menezo YJ, Clement P, Hamidi J, Benkhalifa M. Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients. Fertil Steril. 2009;91(5):1801–5.PubMedCrossRefGoogle Scholar
  25. 25.
    De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, et al. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biol Reprod. 2009;81(3):517–24.PubMedCrossRefGoogle Scholar
  26. 26.
    Leduc F, Nkoma GB, Boissonneault G. Spermiogenesis and DNA repair: a possible etiology of human infertility and genetic disorders. Syst Biol Reprod Med. 2008;54(1):3–10.PubMedCrossRefGoogle Scholar
  27. 27.
    Sakkas D, Seli E, Bizzaro D, Tarozzi N, Manicardi GC. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online. 2003;7(4):428–32.PubMedCrossRefGoogle Scholar
  28. 28.
    Darzynkiewicz Z. Critical aspects in analysis of cellular DNA content. Curr Protoc Cytom. 2011;Chapter 7:Unit 7 2.Google Scholar
  29. 29.
    Gregoire MC, Massonneau J, Simard O, Gouraud A, Brazeau MA, Arguin M, et al. Male-driven de novo mutations in haploid germ cells. Mol Hum Reprod. 2013;19(8):495–9.PubMedCrossRefGoogle Scholar
  30. 30.
    Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, et al. Rate of de novo mutations and the importance of father’s age to disease risk. Nature. 2012;488(7412):471–5.PubMedCrossRefGoogle Scholar
  31. 31.
    The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril. 2013;99:673–7.Google Scholar
  32. 32.
    Giwercman A, Lindstedt L, Larsson M, Bungum M, Spano M, Levine RJ, et al. Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case–control study. Int J Androl. 2010;33:e221–7.Google Scholar
  33. 33.
    Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril. 2000;73(1):43–50.PubMedCrossRefGoogle Scholar
  34. 34.
    Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod. 2007;22(1):174–9.PubMedCrossRefGoogle Scholar
  35. 35.
    Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril. 2008;89(4):823–31.PubMedCrossRefGoogle Scholar
  36. 36.
    Zini A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med. 2011;57(1–2):78–85.PubMedCrossRefGoogle Scholar
  37. 37.
    Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl. 2009;30(3):219–29.PubMedCrossRefGoogle Scholar
  38. 38.
    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(10):2908–17.PubMedCrossRefGoogle Scholar
  39. 39.
    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(12):2663–8.PubMedCrossRefGoogle Scholar
  40. 40.
    Bungum M, Bungum L, Lynch KF, Wedlund L, Humaidan P, Giwercman A. Spermatozoa DNA damage measured by sperm chromatin structure assay (SCSA) and birth characteristics in children conceived by IVF and ICSI. Int J Androl. 2012;35(4):485–90.PubMedCrossRefGoogle Scholar
  41. 41.
    Fernandez-Gonzalez R, Moreira PN, Perez-Crespo M, Sanchez-Martin 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(4):761–72.PubMedCrossRefGoogle Scholar
  42. 42.
    Wyrobek AJ, Eskenazi B, Young S, Arnheim N, Tiemann-Boege I, Jabs EW, et al. Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm. Proc Natl Acad Sci U S A. 2006;103(25):9601–6.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Abdullah Alkhayal
    • 1
  • Maria San Gabriel
    • 1
  • Krista Zeidan
    • 1
  • Khalid Alrabeeah
    • 1
  • Diana Noel
    • 2
  • Rachelle McGraw
    • 2
  • Francois Bissonnette
    • 2
    • 3
  • Isaac Jacques Kadoch
    • 2
    • 3
  • Armand Zini
    • 1
    • 4
    Email author
  1. 1.Division of Urology, Department of SurgeryMcGill UniversityMontrealCanada
  2. 2.OVO Fertility ClinicMontrealCanada
  3. 3.Centre Hospitalier de l’Universite de MontrealMontrealCanada
  4. 4.St. Mary’s HospitalMontrealCanada

Personalised recommendations