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Assessment of Sperm Chromatin Damage by TUNEL Method Using Benchtop Flow Cytometer

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In Vitro Fertilization

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Abstract

Routine semen analysis is the basic step in the laboratory evaluation of male fertility; however, it cannot explain why men are infertile even when the semen parameters are normal. Many studies evaluating sperm DNA integrity have demonstrated an inverse association between DNA fragmentation and fertilization and pregnancy rates. Advance molecular techniques allow the measurement of DNA fragmentation. A brief description of each technique is provided in this chapter. As these techniques are becoming more accessible to the clinical and research laboratories, the number of studies trying to relate DNA fragmentation with pregnancy outcome and fertilization rate is increasing every day. In this chapter we will describe the causes of sperm DNA fragmentation, highlight various techniques that are available to measure DNA fragmentation, and describe the measurement of DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by flow cytometry and explain why TUNEL should be the method of choice in evaluating DNA fragmentation.

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References

  1. Björndahl L, Kvist U. Structure of chromatin in spermatozoa. In: Genetic damage in human spermatozoa. New York: Springer; 2014. p. 1–11.

    Google Scholar 

  2. Fuentes-Mascorro G, Serrano H, Rosado A. Sperm chromatin. Arch Androl. 2000;45(3):215–25.

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  4. Esteves SC. Clinical relevance of routine semen analysis and controversies surrounding the 2010 World Health Organization criteria for semen examination. Int Braz J Urol. 2014;40(4):433–53.

    Article  Google Scholar 

  5. Hamada A, Esteves SC, Nizza M, Agarwal A. Unexplained male infertility: diagnosis and management. Int Braz J Urol. 2012;38(5):576–94.

    Article  PubMed  Google Scholar 

  6. Gosálvez J, Lopez-Fernandez C, Fernandez J, Esteves S, Johnston S. Unpacking the mysteries of sperm DNA fragmentation: ten frequently asked questions. J Reprod Biotech Fertil. 2015;4 https://doi.org/10.1177/2058915815594454.

    Article  Google Scholar 

  7. Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, Thomas AJ, Sharma RK. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril. 2002;78(2):313–8.

    Article  PubMed  Google Scholar 

  8. Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril. 2010;93(4):1027–36.

    Article  CAS  PubMed  Google Scholar 

  9. Evenson D, Jost L, Marshall D, Zinaman M, Clegg E, Purvis K, De Angelis P, Claussen O. 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.

    Article  CAS  PubMed  Google Scholar 

  10. Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril. 2003;80(4):895–902.

    Article  PubMed  Google Scholar 

  11. Spanò M, Bonde JP, Hjøllund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. Fertil Steril. 2000;73(1):43–50.

    Article  PubMed  Google Scholar 

  12. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR, Thomas AJ. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril. 2003;79:1597–605.

    Article  PubMed  Google Scholar 

  13. 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.

    Article  PubMed  Google Scholar 

  14. Lin M-H, Lee RK-K, Li S-H, Lu C-H, Sun F-J, Hwu Y-M. 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.

    Article  PubMed  Google Scholar 

  15. 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.

    Article  CAS  PubMed  Google Scholar 

  16. Giwercman A, Lindstedt L, Larsson M, Bungum M, Spano M, Levine RJ, Rylander L. Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case–control study. Int J Androl. 2010;33(1):e221.

    Article  PubMed  Google Scholar 

  17. Wells D, Bermudez M, Steuerwald N, Thornhill A, Walker D, Malter H, Delhanty J, Cohen J. Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development. Hum Reprod. 2005;20(5):1339–48.

    Article  CAS  PubMed  Google Scholar 

  18. Gasca S, Pellestor F, Assou S, Loup V, Anahory T, Dechaud H, De Vos J, Hamamah S. Identifying new human oocyte marker genes: a microarray approach. Reprod Biomed Online. 2007;14(2):175–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gledhill BL, Gledhill MP, Rigler RJ, Ringertz NR. Changes in deoxyribonucleoprotein during spermiogenesis in the bull. Exp Cell Res. 1966;41(3):652–65.

    Article  CAS  PubMed  Google Scholar 

  20. Evenson D, Darzynkiewicz Z, Melamed M. Relation of mammalian sperm chromatin heterogeneity to fertility. Science. 1980;210(4474):1131–3.

    Article  CAS  PubMed  Google Scholar 

  21. Aravindan G, Bjordahl J, Jost L, Evenson D. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA Strand breaks identified by single-cell electrophoresis. Exp Cell Res. 1997;236(1):231–7.

    Article  CAS  PubMed  Google Scholar 

  22. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res. 1993;207(1):202–5.

    Article  CAS  PubMed  Google Scholar 

  23. Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl. 2003;24(1):59–66.

    CAS  PubMed  Google Scholar 

  24. Pienta KJ, Coffey DS. A structural analysis of the role of the nuclear matrix and DNA loops in the organization of the nucleus and chromosome. J Cell Sci. 1984;1984(Supplement 1):123–35.

    Article  Google Scholar 

  25. Ioannou D, Miller D, Griffin DK, Tempest HG. Impact of sperm DNA chromatin in the clinic. J Assist Reprod Genet. 2016;33(2):157–66.

    Article  PubMed  Google Scholar 

  26. Ward WS, Coffey D. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod. 1991;44(4):569–74.

    Article  CAS  PubMed  Google Scholar 

  27. Ward WS, Coffey DS. Specific organization of genes in relation to the sperm nuclear matrix. Biochem Biophys Res Commun. 1990;173(1):20–5.

    Article  CAS  PubMed  Google Scholar 

  28. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003;9(4):331–45.

    Article  CAS  PubMed  Google Scholar 

  29. Poccia D. Remodeling of nucleoproteins during gametogenesis, fertilization, and early development. In: International review of cytology, vol. 105. Burlington: Elsevier; 1986. p. 1–65.

    Google Scholar 

  30. Rey RA. Commentary on sperm DNA fragmentation testing clinical guideline. Transl Androl Urol. 2017;6(Suppl 4):S522.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Aitken R, Bronson R, Smith T, De Iuliis G. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies. Mol Hum Reprod. 2013;19(8):475–85.

    Article  CAS  PubMed  Google Scholar 

  32. Bennetts LE, Aitken RJ. A comparative study of oxidative DNA damage in mammalian spermatozoa. Mol Reprod Dev. 2005;71(1):77–87.

    Article  CAS  PubMed  Google Scholar 

  33. Zini A, Albert O, Robaire B. Assessing sperm chromatin and DNA damage: clinical importance and development of standards. Andrology. 2014;2(3):322–5.

    Article  CAS  PubMed  Google Scholar 

  34. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod. 1998;59(5):1037–46.

    Article  CAS  PubMed  Google Scholar 

  35. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl. 2002;23(1):25–43.

    Article  PubMed  Google Scholar 

  36. Fernández JL, de la Calle JF, Tamayo M, Cajigal D, Agarwal A, Gosálvez J. Sperm DNA integrity and male infertility: current perspectives. Arch Med Sci. 2009;2009(1):62.

    Google Scholar 

  37. 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(4):1061–7.

    Article  CAS  PubMed  Google Scholar 

  38. Carrell DT, Emery BR, Hammoud S. Altered protamine expression and diminished spermatogenesis: what is the link? Hum Reprod Update. 2007;13(3):313–27.

    Article  CAS  PubMed  Google Scholar 

  39. McPherson S, Longo F. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev. 1992;31(4):268–79.

    Article  CAS  PubMed  Google Scholar 

  40. McPherson S, Longo F. Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur J Histochem. 1993;37(2):190–28.

    Google Scholar 

  41. Sharma R, Harlev A, Agarwal A, Esteves SC. Cigarette smoking and semen quality: a new meta-analysis examining the effect of the 2010 World Health Organization laboratory methods for the examination of human semen. Eur Urol. 2016;70(4):635–45.

    Article  PubMed  Google Scholar 

  42. Lalinde-Acevedo PC, Mayorga-Torres BJM, Agarwal A, du Plessis SS, Ahmad G, Cadavid ÁP, Maya WDC. Physically active men show better semen parameters than their sedentary counterparts. Int J Fertil Steril. 2017;11(3):156.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Sharma R, Agarwal A, Harlev A, Esteves S. A meta analysis to study the effects of body mass index on sperm DNA fragmentation index in reproductive age men. Fertil Steril. 2017;108(3):e138–9.

    Article  Google Scholar 

  44. Harlev A, Agarwal A, Gunes SO, Shetty A, du Plessis SS. Smoking and male infertility: an evidence-based review. World J Mens Health. 2015;33(3):143–60.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Sharma R, Agarwal A, Rohra VK, Assidi M, Abu-Elmagd M, Turki RF. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring. Reprod Biol Endocrinol. 2015;13(1):35.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Alshahrani S, Agarwal A, Assidi M, Abuzenadah AM, Durairajanayagam D, Ayaz A, Sharma R, Sabanegh E. Infertile men older than 40 years are at higher risk of sperm DNA damage. Reprod Biol Endocrinol. 2014;12(1):103.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Kaarouch I, Bouamoud N, Madkour A, Louanjli N, Saadani B, Assou S, Aboulmaouhib S, Amzazi S, Copin H, Benkhalifa M. Paternal age: negative impact on sperm genome decays and IVF outcomes after 40 years. Mol Reprod Dev. 2018;85:271.

    Article  CAS  PubMed  Google Scholar 

  48. Mathur PP, Huang L, Kashou A, Vaithinathan S, Agarwal A. Environmental toxicants and testicular apoptosis. Open Reprod Sci J. 2011;3:114–24.

    Article  CAS  Google Scholar 

  49. Martenies SE, Perry MJ. Environmental and occupational pesticide exposure and human sperm parameters: a systematic review. Toxicology. 2013;307:66–73.

    Article  CAS  PubMed  Google Scholar 

  50. Cho C-L, Esteves SC, Agarwal A. Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation. Asian J Androl. 2016;18(2):186.

    Article  CAS  PubMed  Google Scholar 

  51. Agarwal A, Mulgund A, Alshahrani S, Assidi M, Abuzenadah AM, Sharma R, Sabanegh E. Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia. Reprod Biol Endocrinol. 2014;12(1):126.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Pourmasumi S, Sabeti P, Rahiminia T, Mangoli E, Tabibnejad N, Talebi AR. The etiologies of DNA abnormalities in male infertility: an assessment and review. Int J Reprod Biomed (Yazd). 2017;15(6):331.

    Article  CAS  Google Scholar 

  53. Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, Thomas AJ Jr, Alvarez JG. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16(9):1912–21.

    Article  CAS  PubMed  Google Scholar 

  54. Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Franco G, Anniballo N, Mendoza C, Tesarik J. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod. 2005;20(1):226–30.

    Article  PubMed  Google Scholar 

  55. Laberge R-M, Boissonneault G. On the nature and origin of DNA strand breaks in elongating spermatids. Biol Reprod. 2005;73(2):289–96.

    Article  CAS  PubMed  Google Scholar 

  56. 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.

    Article  CAS  PubMed  Google Scholar 

  57. Muratori M, Marchiani S, Maggi M, Forti G, Baldi E. Origin and biological significance of DNA fragmentation in human spermatozoa. Front Biosci. 2006;11:1491–9.

    Article  CAS  PubMed  Google Scholar 

  58. Egozcue S, Blanco J, Vendrell J, Garcia F, Veiga A, Aran B, Barri P, Vidal F, Egozcue J. Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum Reprod Update. 2000;6(1):93–105.

    Article  CAS  PubMed  Google Scholar 

  59. Piomboni P, Stendardi A, Gambera L. Chromosomal aberrations and aneuploidies of spermatozoa. In: Genetic damage in human spermatozoa. New York: Springer; 2014. p. 27–52.

    Chapter  Google Scholar 

  60. Sloter E, Nath J, Eskenazi B, Wyrobek AJ. Effects of male age on the frequencies of germinal and heritable chromosomal abnormalities in humans and rodents. Fertil Steril. 2004;81(4):925–43.

    Article  PubMed  Google Scholar 

  61. Ford JH, Schultz CJ, Correll AT. Chromosome elimination in micronuclei: a common cause of hypoploidy. Am J Hum Genet. 1988;43(5):733.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 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.

    Article  CAS  PubMed  Google Scholar 

  63. 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(2):350–5.

    Article  CAS  PubMed  Google Scholar 

  64. Tease C, Hulten MA. Inter-sex variation in synaptonemal complex lengths largely determine the different recombination rates in male and female germ cells. Cytogenet Genome Res. 2004;107(3–4):208–15.

    Article  CAS  PubMed  Google Scholar 

  65. Hann MC, Lau PE, Tempest HG. Meiotic recombination and male infertility: from basic science to clinical reality? Asian J Androl. 2011;13(2):212–8.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2001;2(4):280–91.

    Article  CAS  PubMed  Google Scholar 

  67. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell. 1993;75(6):1169–78.

    Article  CAS  PubMed  Google Scholar 

  68. Sakkas D, Mariethoz E, John JCS. Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res. 1999;251(2):350–5.

    Article  CAS  PubMed  Google Scholar 

  69. McVicar CM, McClure N, Williamson K, Dalzell LH, Lewis SE. Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm. Fertil Steril. 2004;81:767–74.

    Article  CAS  PubMed  Google Scholar 

  70. Burrello N, Arcidiacono G, Vicari E, Asero P, Di Benedetto D, De Palma A, Romeo R, D’agata R, Calogero AE. Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate. Hum Reprod. 2004;19(10):2298–302.

    Article  PubMed  Google Scholar 

  71. Simon L, Emery BR, Carrell DT. Impact of sperm DNA damage in assisted reproduction. Best Pract Res Clin Obstet Gynaecol. 2017;44:38–56.

    Article  PubMed  Google Scholar 

  72. Tomlinson M, White A, Barratt C, Bolton A, Cooke I. The removal of morphologically abnormal sperm forms by phagocytes: a positive role for seminal leukocytes? Hum Reprod. 1992;7(4):517–22.

    Article  CAS  PubMed  Google Scholar 

  73. Pudney J, Anderson D. Organization of immunocompetent cells and their function in the male reproductive tract. In: Local immunity in reproductive tract tissues. Oxford: Oxford University Press; 1993. p. 131.

    Google Scholar 

  74. Kiessling AA, Lamparelli N, Yin H-Z, Seibel MM, Eyre RC. Semen leukocytes: friends or foes? Fertil Steril. 1995;64(1):196–8.

    Article  CAS  PubMed  Google Scholar 

  75. Barraud-Lange V, Pont J-C, Ziyyat A, Pocate K, Sifer C, Cedrin-Durnerin I, Fechtali B, Ducot B, Wolf JP. Seminal leukocytes are good Samaritans for spermatozoa. Fertil Steril. 2011;96(6):1315–9.

    Article  PubMed  Google Scholar 

  76. Aitken R, Baker HG. Seminal leukocytes: passengers, terrorists or good samaritans? Hum Reprod. 1995;10:1736.

    Article  CAS  PubMed  Google Scholar 

  77. Hsu PC, Chen IY, Pan CH, Wu KY, Pan MH, Chen JR, Chen CJ, Chang-Chien GP, Hsu CH, Liu CS, Wu MT. Sperm DNA damage correlates with polycyclic aromatic hydrocarbons biomarker in coke-oven workers. Int Arch Occup Environ Health. 2006;79(5):349–56.

    Article  CAS  PubMed  Google Scholar 

  78. Oh E, Lee E, Im H, Kang H-S, Jung W-W, Won NH, Kim E-M, Sul D. Evaluation of immuno- and reproductive toxicities and association between immunotoxicological and genotoxicological parameters in waste incineration workers. Toxicology. 2005;210(1):65–80.

    Article  CAS  PubMed  Google Scholar 

  79. Migliore L, Naccarati A, Zanello A, Scarpato R, Bramanti L, Mariani M. Assessment of sperm DNA integrity in workers exposed to styrene. Hum Reprod. 2002;17(11):2912–8.

    Article  CAS  PubMed  Google Scholar 

  80. Morris ID. Sperm DNA damage and cancer treatment. Int J Androl. 2002;25(5):255–61.

    Article  CAS  PubMed  Google Scholar 

  81. Tvrda E, Agarwal A, Alkuhaimi N. Male reproductive cancers and infertility: a mutual relationship. Int J Mol Sci. 2015;16(4):7230–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Esquerré-Lamare C, Isus F, Moinard N, Bujan L. Sperm DNA fragmentation after radioiodine treatment for differentiated thyroid cancer. Basic Clin Androl. 2015;25(1):8.

    PubMed  PubMed Central  Google Scholar 

  83. Sharma R, Biedenharn KR, Fedor JM, Agarwal A. Lifestyle factors and reproductive health: taking control of your fertility. Reprod Biol Endocrinol. 2013;11(1):66.

    Article  PubMed  PubMed Central  Google Scholar 

  84. Esteves SC, Sánchez-Martín F, Sánchez-Martín P, Schneider DT, Gosálvez J. Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm. Fertil Steril. 2015;104(6):1398–405.

    Article  PubMed  Google Scholar 

  85. Oleszczuk K, Augustinsson L, Bayat N, Giwercman A, Bungum M. Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples. Andrology. 2013;1(3):357–60.

    Article  CAS  PubMed  Google Scholar 

  86. Duru NK, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steril. 2000;74(6):1200–7.

    Article  Google Scholar 

  87. 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.

    Article  PubMed  Google Scholar 

  88. Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, Zini A. Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios. Transl Androl Urol. 2016;5(6):935.

    Article  PubMed  PubMed Central  Google Scholar 

  89. Evenson D, Jost L. Sperm chromatin structure assay: DNA denaturability. Methods Cell Biol. 1994;42:159–76.

    Article  CAS  PubMed  Google Scholar 

  90. Oleszczuk K, Giwercman A, Bungum M. Sperm chromatin structure assay in prediction of in vitro fertilization outcome. Andrology. 2016;4(2):290–6.

    Article  CAS  PubMed  Google Scholar 

  91. Bisht S, Faiq M, Tolahunase M, Dada R. Oxidative stress and male infertility. Nat Rev Urol. 2017;14(8):470–85.

    Article  CAS  PubMed  Google Scholar 

  92. Kumar K, Deka D, Singh A, Mitra D, Vanitha B, Dada R. Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception. J Assist Reprod Genet. 2012;29(9):861–7.

    Article  PubMed  PubMed Central  Google Scholar 

  93. Gosálvez J, López-Fernández C, Fernández JL. Sperm chromatin dispersion test: technical aspects and clinical applications. In: Sperm Chromatin. Biological and Clinical Applications in Male Infertility and Assisted Reproduction. A. Zini and A. Agarwal (eds). Springer Science + Business Media, LLC. Springer Nature, New York, p. 151–70.

    Chapter  Google Scholar 

  94. Agarwal A, Cho C-L, Majzoub A, Esteves SC. The Society for Translational Medicine: clinical practice guidelines for sperm DNA fragmentation testing in male infertility. Transl Androl Urol. 2017;6(Suppl 4):S720.

    Article  PubMed  PubMed Central  Google Scholar 

  95. Feijó CM, Esteves SC. Diagnostic accuracy of sperm chromatin dispersion test to evaluate sperm deoxyribonucleic acid damage in men with unexplained infertility. Fertil Steril. 2014;101(1):58–63.e53.

    Article  PubMed  CAS  Google Scholar 

  96. Sharma R, Ahmad G, Esteves SC, Agarwal A. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control. J Assist Reprod Genet. 2016;33(2):291–300.

    Article  PubMed  PubMed Central  Google Scholar 

  97. Agarwal A, Gupta S, Sharma R. Measurement of DNA fragmentation in spermatozoa by TUNEL assay using bench top flow cytometer. In: Andrological evaluation of male infertility. Cham: Springer; 2016. p. 181–203.

    Chapter  Google Scholar 

  98. Shamsi M, Kumar R, Dada R. Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction. Indian J Med Res. 2008;127(2):115.

    CAS  PubMed  Google Scholar 

  99. Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken R. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21(1):33–44.

    CAS  PubMed  Google Scholar 

  100. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175(1):184–91.

    Article  CAS  PubMed  Google Scholar 

  101. Fernández JL, Muriel L, Goyanes V, Segrelles E, Gosálvez J, Enciso M, LaFromboise M, De Jonge C. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 2005;84(4):833–42.

    Article  PubMed  CAS  Google Scholar 

  102. Gekas J, Thepot F, Turleau C, Siffroi J, Dadoune J, Briault S, Rio M, Bourouillou G, Carre-Pigeon F, Wasels R. Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum Reprod. 2001;16(1):82–90.

    Article  CAS  PubMed  Google Scholar 

  103. Sharma RK, Sabanegh E, Mahfouz R, Gupta S, Thiyagarajan A, Agarwal A. TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology. 2010;76(6):1380–6.

    Article  PubMed  Google Scholar 

  104. Bareh GM, Jacoby E, Binkley P, Schenken RS, Robinson RD. Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: a prospective study. Fertil Steril. 2016;105(2):329–36.e1.

    Article  CAS  PubMed  Google Scholar 

  105. Agarwal A, Cho C-L, Esteves SC. Should we evaluate and treat sperm DNA fragmentation? Curr Opin Obstet Gynecol. 2016;28(3):164–71.

    Article  PubMed  Google Scholar 

  106. Zidi-Jrah I, Hajlaoui A, Mougou-Zerelli S, Kammoun M, Meniaoui I, Sallem A, Brahem S, Fekih M, Bibi M, Saad A. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss. Fertil Steril. 2016;105(1):58–64.

    Article  CAS  PubMed  Google Scholar 

  107. Henkel R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Schill W-B, Kruger TF. DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online. 2003;7(4):477–84.

    Article  PubMed  Google Scholar 

  108. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, Schill W-B, Kruger TF. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril. 2004;81(4):965–72.

    Article  CAS  PubMed  Google Scholar 

  109. Duran EH, Morshedi M, Taylor S, Oehninger S. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod. 2002;17(12):3122–8.

    Article  CAS  PubMed  Google Scholar 

  110. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, Giwercman A. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod. 2006;22(1):174–9.

    Article  PubMed  CAS  Google Scholar 

  111. Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl. 2009;30(3):219–29.

    Article  CAS  PubMed  Google Scholar 

  112. Høst E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand. 2000;79(7):559–63.

    Article  PubMed  Google Scholar 

  113. Huang C-C, Lin DP-C, Tsao H-M, Cheng T-C, Liu C-H, Lee M-S. Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. Fertil Steril. 2005;84(1):130–40.

    Article  PubMed  Google Scholar 

  114. Borini A, Tarozzi N, Bizzaro D, Bonu M, Fava L, Flamigni C, Coticchio G. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod. 2006;21(11):2876–81.

    Article  CAS  PubMed  Google Scholar 

  115. Agarwal A, Gupta S, Sharma R. Andrological evaluation of male infertility: a laboratory guide. Cham: Springer; 2016.

    Book  Google Scholar 

  116. Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. Clinics. 2011;66(4):691–700.

    Article  PubMed  PubMed Central  Google Scholar 

  117. Cho C-L, Agarwal A, Majzoub A, Esteves SC. Clinical utility of sperm DNA fragmentation testing: concise practice recommendations. Transl Androl Urol. 2017;6(Suppl 4):S366.

    Article  PubMed  PubMed Central  Google Scholar 

  118. Medicine PCASR. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril. 2013;99(3):673–7.

    Article  CAS  Google Scholar 

  119. Esteves SC, Gosálvez J, López-Fernández C, Núñez-Calonge R, Caballero P, Agarwal A, Fernández JL. Diagnostic accuracy of sperm DNA degradation index (DDSi) as a potential noninvasive biomarker to identify men with varicocele-associated infertility. Int Urol Nephrol. 2015;47(9):1471–7.

    Article  CAS  PubMed  Google Scholar 

  120. Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ. Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertil Steril. 2003;80(6):1431–6.

    Article  PubMed  Google Scholar 

  121. Lewis SE, Aitken RJ, Conner SJ, De Iuliis G, Evenson DP, Henkel R, Giwercman A, Gharagozloo P. The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment. Reprod Biomed Online. 2013;27(4):325–37.

    Article  CAS  PubMed  Google Scholar 

  122. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 1993;53(8):1945–51.

    CAS  PubMed  Google Scholar 

  123. Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl. 1995;16(1):80–7.

    CAS  PubMed  Google Scholar 

  124. Ribeiro S, Sharma R, Gupta S, Cakar Z, De Geyter C, Agarwal A. Inter-and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation. Andrology. 2017;5(3):477–85.

    Article  CAS  PubMed  Google Scholar 

  125. Gupta S, Sharma R, Agarwal A. Inter-and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation. Curr Protoc Toxicol. 2017;74:16.11.11–22.

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Urology and American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA

    Ana D. Martins, Rakesh Sharma & Ashok Agarwal

  2. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine, Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto, Porto, Portugal

    Ana D. Martins

Authors
  1. Ana D. Martins
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  2. Rakesh Sharma
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  3. Ashok Agarwal
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Corresponding author

Correspondence to Ashok Agarwal .

Editor information

Editors and Affiliations

  1. Reproductive Biology Associates – Prelude, Atlanta, GA, USA

    Zsolt Peter Nagy

  2. Astra Fertility Group, Mississauga, ON, Canada

    Alex C. Varghese

  3. Cleveland Clinic, Cleveland, OH, USA

    Ashok Agarwal

Review Questions

Review Questions

  1. 1.

    In sperm DNA, during chromatin packaging the majority of the histones are replaced with:

    1. (a)

      Protamines

    2. (b)

      DNA topoisomerases

    3. (c)

      Disulfide bonds

    4. (d)

      None of the above

  2. 2.

    Which of these causes can be responsible for DNA fragmentation:

    1. (a)

      Aging

    2. (b)

      Lifestyle

    3. (c)

      Medical history

    4. (d)

      All of the above

  3. 3.

    In the TUNEL assay the samples can be evaluated by:

    1. (a)

      Flow cytometry

    2. (b)

      Standard fluorescence microscope

    3. (c)

      Polymerase chain reaction

    4. (d)

      (a) and (b)

  4. 4.

    In the TUNEL assay:

    1. (a)

      The intensity of labeling is not proportional to the number of DNA strand break sites.

    2. (b)

      The intensity of labeling is proportional to the number of DNA strand break sites.

    3. (c)

      There is no relationship between the intensity labeling and the number of DNA strand break sites.

    4. (d)

      None of the above.

  5. 5.

    What is the most important parameter in the sperm chromatin structure assay:

    1. (a)

      DNA fragmentation index

    2. (b)

      Number of DNA strand breaks

    3. (c)

      Sperm chromatin packing index

    4. (d)

      Protamine concentration

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Martins, A.D., Sharma, R., Agarwal, A. (2019). Assessment of Sperm Chromatin Damage by TUNEL Method Using Benchtop Flow Cytometer. In: Nagy, Z., Varghese, A., Agarwal, A. (eds) In Vitro Fertilization. Springer, Cham. https://doi.org/10.1007/978-3-319-43011-9_24

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  • DOI: https://doi.org/10.1007/978-3-319-43011-9_24

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  • Publisher Name: Springer, Cham

  • Print ISBN: 978-3-319-43010-2

  • Online ISBN: 978-3-319-43011-9

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