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Chapter 19 - DNA Damage: TdT-Mediated dUTP Nick-End-Labelling Assay

Published online by Cambridge University Press:  05 April 2021

By
Rakesh Sharma ,
Concetta Iovine  and
Ashok Agarwal
Edited by
Ashok Agarwal ,
Ralf Henkel  and
Ahmad Majzoub
Ashok Agarwal
Affiliation:
The Cleveland Clinic Foundation, Cleveland, OH
Ralf Henkel
Affiliation:
University of the Western Cape, South Africa
Ahmad Majzoub
Affiliation:
Hamad Medical Corporation, Doha
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Summary

Male infertility affects men worldwide with about 20 percent of couples having male factor infertility [1]. The routine semen analysis is the first step in the assessment of male infertility. However, conventional semen analysis does not provide a complete understanding of fertility potential, especially in patients with idiopathic infertility [2]. In this scenario, DNA integrity is the most important feature to ensure normal fertilization, implantation, pregnancy and embryonic development. Sperm DNA fragmentation (SDF) can be due to several intrinsic factors such as varicocele, oxidative stress, apoptosis, and chromatin packaging defects. Further, SDF can be caused by extrinsic factors such as lifestyle alterations, infections, exposure to xenobiotics, etc. [3–7].

Type
Chapter
Information
Manual of Sperm Function Testing in Human Assisted Reproduction , pp. 163 - 191
Publisher: Cambridge University Press
Print publication year: 2021

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References

Agarwal, A, Gupta, S, Sharma, R. (2016) Measurement of DNA fragmentation in spermatozoa by TUNEL assay using bench top flow cytometer. In Agarwal, A, Gupta, S, Sharma, R, eds., Andrological Evaluation of Male Infertility: A Laboratory Guide. Geneva: Springer International Publishing, pp. 181–203.CrossRefGoogle Scholar
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. Trans Androl Urol 2016; 5(6): 935.Google Scholar
Tremellen, K. Oxidative stress and male infertility – a clinical perspective. Hum Reprod Update 2008; 14(3): 243–58.CrossRefGoogle ScholarPubMed
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: 126.CrossRefGoogle ScholarPubMed
Sakkas, D, Seli, E, Tarozzi, Bizzaro D Manicardi, N GC. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online 2003; 7: 428–32.Google Scholar
Lewis, SE, Aitken, RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005; 322(1): 33–41.Google Scholar
Aitken, RJ, Smith, TB, Jobling, MS, Baker, MA, De Iuliis, GN. Oxidative stress and male reproductive health. Asian J Androl 2014; 16(1): 31–8.Google Scholar
Aitken, RJ, De Iuliis, GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod 2010; 16: 3–13.Google Scholar
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: 1912–21.Google Scholar
Aitken, RJ, Sawyer, D. The human spermatozoon-not waving but drowning. Adv Exp Med Biol 2003; 518: 85–98.CrossRefGoogle ScholarPubMed
Saleh, RA, Agarwal, A, Nada, EA, El-Tonsy, MH, Sharma, RK, Meyer, A, Nelson, DR, Thomas, AJ, Jr. 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: 1597–1605.Google Scholar
De Iuliis, GN, Thomson, LK, Mitchell, LA, Finnie, JM, Koppers, AJ, Hedges, A, Nixon, B, Aitken, RJ. 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: 517–24.CrossRefGoogle ScholarPubMed
Sakkas, D, Mariethoz, E, Manicardi, G, et al. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4: 31–7.Google Scholar
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: 910–18.Google Scholar
Weng, SL, Taylor, SL, Morshedi, M, Schuffner, A, Duran, EH, Beebe, S, Oehninger, S. Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod 2002; 8: 984–91.Google Scholar
Matsuda, Y, Tobari, I, Maemori, M, Seki, N. Mechanism of chromosome aberration induction in the mouse egg fertilized with sperm recovered from postmeiotic germ cells treated with methyl methanesulfonate. Mutat Res 1989; 214: 165–80.CrossRefGoogle ScholarPubMed
Aitken, RJ. Founders’ Lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev 2004; 16: 655–64.Google Scholar
Steele, EK, Lewis, SE, McClure, N. Science versus clinical adventurism in treatment of azoospermia. Lancet 1999; 13(353): 516–17.Google Scholar
O’Connell, M, McClure, N, Powell, LA, Steele, EK, Lewis, SE. Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril 2003; 79: 754–62.Google Scholar
Lewis, SE, O’Connell, M, Stevenson, M, Thompson-Cree, L, McClure, N. An algorithm to predict pregnancy in assisted reproduction. Hum Reprod 2004; 19: 1385–94.Google Scholar
Suganuma, R, Yanagimachi, R, Meistrich, ML. Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI. Hum Reprod 2005; 20: 3101–8.Google Scholar
Sergerie, M, Laforest, G, Bujan, L, Bissonnette, F, Bleau, G. Sperm DNA fragmentation: threshold value in male fertility. Hum Reprod 2005; 20(12): 3446–51.Google Scholar
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.Google Scholar
Ribas-Maynou, J, Garcia-Peiro, A, Fernandez-Encinas, A, Abad, C, Amengual, MJ, Prada, E, Navarro, J, Benet, J. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay. Andrology 2013; 1(5): 715–22.Google Scholar
Feijo, 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.Google Scholar
Majzoub, A, Esteves, SC, Gosálvez, J, Agarwal, A. Specialized sperm function tests in varicocele and the future of andrology laboratory. Asian J Androl 2016; 18(2): 205–12.Google Scholar
Ni, K, Steger, K, Yang, H, Wang, H, Hu, K, Zhang, T, Chen, B. A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele. Andrology 2016; 4(5): 816–24.Google Scholar
Spanò, M, Bonde, JP, Hjøllund, HI, Kolstad, HA, Cordelli, E, Leter, G. The Danish First Pregnancy Planner Study Team. Sperm chromatin damage impairs human fertility. Fertil Steril 2000; 73: 43–50.Google Scholar
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.Google Scholar
Cho, CL, Agarwal, A. Role of sperm DNA fragmentation in male factor infertility: a systematic review. Arab J Urol 2017; 16(1): 21–34.Google Scholar
Rai, R, Regan, L. Recurrent miscarriage. Lancet 2006; 368(9535): 601–11.CrossRefGoogle ScholarPubMed
Kirkman-Brown, JC, De Jonge, C. Sperm DNA fragmentation in miscarriage: a promising diagnostic, or a test too far? Reprod Biomed Online 2017; 34(1): 3–4.Google Scholar
Practice Committee of the American Society for Reproductive Medicine. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril 2013; 99: 673–7.Google Scholar
Practice Committee of the American Society for Reproductive Medicine. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril 2012; 98(5): 1103–11.Google Scholar
Zini, A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med 2011; 57(1–2): 78–85.Google Scholar
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.Google Scholar
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.Google Scholar
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: 1–16.Google Scholar
Anzar, M, He, L, Buhr, MM, Kroetsch, TG, Pauls, KP. Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol Reprod 2002; 66(2): 354–60.CrossRefGoogle ScholarPubMed
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(8): e0181802.CrossRefGoogle ScholarPubMed
Domínguez-Fandos, D, Camejo, MI, Ballescà, JL, Oliva, R. Human sperm DNA fragmentation: correlation of TUNEL results as assessed by flow cytometry and optical microscopy. Cytometry 2007; 71: 1011–18.Google Scholar
Muratori, M, Piomboni, P, Baldi, E, Filimberti, E, Pecchioli, P, Moretti, E, Gambera, L, Baccetti, B, Biagiotti, R, Forti, G, Maggi, M. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl 2000; 21: 903–12.Google Scholar
Muratori, M, Maggi, M, Spinelli, S, Filimberti, E, Forti, G, Baldi, E. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 2003; 24: 253–62.Google Scholar
Lopes, S, Jurisicova, A, Sun, J-G, Casper, RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998; 13(4): 896–900.Google Scholar
Barroso, G, Morshedi, M, Oehninger, S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000; 15: 1338–44.Google Scholar
Donnelly, ET, O’Connell, M, McClure, N, Lewis, SE. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod 2000; 15: 1552–61.Google Scholar
Gandini, L, Lombardo, F, Paoli, D, Caponecchia, L, Familiari, G, Verlengia, C, Dondero, F, Lenzi, A. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 2000; 15: 830–9.CrossRefGoogle ScholarPubMed
Oosterhuis, GJ, Mulder, AB, Kalsbeek-Batenburg, E, Lambalk, CB, Schoemaker, J, Sergerie, M, Ouhilal, S, Bissonnette, F, Brodeur, J, Bleau, G. Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men. Hum Reprod 2000; 15: 1314–21.Google Scholar
Sergerie, M, Ouhilal, S, Bissonnette, F, Brodeur, J, Bleau, G. Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men. Hum Reprod 2000; 15: 1314–21.Google Scholar
Ramos, L, Wetzels, AM. Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay. Hum Reprod 2001; 16: 1703–7.Google Scholar
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: 674–7.Google Scholar
Duran, EH, Morshedi, M, Taylor, S, Oehninger, S. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 2002; 17: 3122–8.Google Scholar
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.Google Scholar
Shen, HM, Dai, J, Chia, SE, Lim, A, Ong, CN. Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality. Hum Reprod 2002; 17: 1266–73.CrossRefGoogle ScholarPubMed
Benchaib, M, Braun, V, Lornage, J, Hadj, S, Salle, B, Lejeune, H, Guérin, JF. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003; 18: 1023–8.Google Scholar
Carrell, DT, Liu, L, Peterson, CM, Jones, KP, Hatasaka, HH, Erickson, L, Campbell, B. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003; 49: 49–55.Google Scholar
Erenpreisa, J, Erenpreiss, J, Freivalds, T, Slaidina, M, Krampe, R, Butikova, J, Ivanov, A, Pjanova, D. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry 2003; 52: 19–27.Google Scholar
Erenpreiss, J, Jepson, K, Giwercman, A, Tsarev, I, Erenpreisa, J, Spano, M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod 2004; 19: 2277–82.Google Scholar
Lachaud, C, Tesarik, J, Cañadas, ML, Mendoza, C. Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod 2004; 19: 607–10.CrossRefGoogle ScholarPubMed
Tesarik, J, Greco, E, Mendoza, C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod 2004; 19: 611–15.Google Scholar
Greco, E, Iacobelli, M, Rienzi, L, Ubaldi, F, Ferrero, S, Tesarik, J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl 2005; 26: 349–53.Google Scholar
Stahl, O, Eberhard, J, Jepson, K, Spano, M, Cwikiel, M, Cavallin Stahl, E, Giwercman, A. The impact of testicular carcinoma and its treatment on sperm DNA integrity. Cancer 2004; 100: 1137–44.Google Scholar
Sépaniak, S, Forges, T, Monnier-Barbarino, P. Cigarette smoking and fertility in women and men. Gynecol Obstet Fertil 2006; 34: 945–9.Google Scholar
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: 53–9.Google Scholar
de Paula, TS, Bertolla, RP, Spaine, DM, Cunha, MA, Schor, N, Cedenho, AP. Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia. Fertil Steril 2006; 86: 597–600.CrossRefGoogle ScholarPubMed
Aoki, VW, Emery, BR, Liu, L, Carrell, DT. Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and DNA integrity. J Androl 2006; 27: 890–8.Google Scholar
Spermon, JR, Ramos, L, Wetzels, AM, Sweep, CG, Braat, DD, Kiemeney, LA, Witjes, JA. Sperm integrity pre- and post-chemotherapy in men with testicular germ cell cancer. Hum Reprod 2006; 21: 1781–6.Google Scholar
Sakamoto, Y, Ishikawa, T, Kondo, Y, Yamaguchi, K, Fujisawa, M. The assessment of oxidative stress in infertile patients with varicocele. BJU Int 2008; 101: 1547–52.Google Scholar
Muratori, M, Forti, G, Baldi, E. Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labeling. Cytometry A 2008; 73(9): 785–7.Google ScholarPubMed
Sharma, R, Gupta, S, Henkel, R, Agarwal, A. Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation: an appeal for performance verification. Asian J Androl 2019; 21(5): 438–44.Google Scholar
Cissen, M, Wely, MV, Scholten, I, Mansell, S, Bruin, JP, Mol, BW, Braat, D, Repping, S, Hamer, G. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis. PLOS One 2016; 11: 165125.CrossRefGoogle ScholarPubMed
Mitchell, LA, De Iuliis, GN, Aitken, RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl 2011; 34(1): 2–13.CrossRefGoogle ScholarPubMed
Muratori, M, Tamburrino, L, Marchiani, S, Cambi, M, Olivito, B, Azzari, C, Forti, G, Baldi, E. Investigation on the origin of sperm DNA fragmentation: role of apoptosis, immaturity and oxidative stres. Mol Med 2015; 21(1): 109–22.Google Scholar
Oleszczuk, K, Augustinsson, L, Bayat, N, et al. Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples. Androl 2013; 1: 357–60.CrossRefGoogle ScholarPubMed
Saleh, RA, Agarwal, A, Nada, EA, 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.Google Scholar

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