OUR FIRST EXPERIENCE WITH THE COMET ASSAY IN THE STUDY OF SPERM DNA INTEGRITY
Authors:
Božena Novotná 1; Lucie Svobodová 2; Marcela Čechová 3; Ladislav Jarolím 3; Matúš Chocholatý 3; Yana Bagryantseva 1; Sr. Milan Macek 2; Milan Macek jr. 2
Authors‘ workplace:
Ústav experimentální medicíny v. v. i., AV ČR, Praha
1; Ústav biologie a lékařské genetiky 2. LF UK a FN Motol, Praha
2; Urologická klinika 2. LF UK a FN Motol, Praha
3
Published in:
Ces Urol 2016; 20(4): 317-325
Category:
Original Articles
Overview
Major statement:
Based on published data, the authors highlight the importance of assessment of sperm DNA integrity in the investigation of causes of infertility. Information is complemented by our own experimental results obtained by comet assay and commercial kit Halosperm.
Aim:
Abnormalities in chromatin arrangement and/or structure of the sperm DNA can significantly reduce the fertilizing potential of individuals, even in cases with normal results of conventional sperm analysis. Therefore, evaluation of sperm DNA integrity is currently emphasized in identifying the causes of male infertility. For this purpose, we decided to use comet assay and a commercially available test Halosperm with the aim to compare the two methods in the investigation of control subjects and patients with fertility disorders.
Patients and Methods:
Conventional semen analysis was performed in 32 control subjects, 24 men with infertility and 26 men with vascular disorders of male genital organs, and then the levels of DNA fragmentation in the sperm were examined using the comet assay and Halosperm.
Results:
Correlation analysis confirmed good agreement between the results of both methods (Pearson coeficient r = 0.241; p < 0.05); unlike the comet assay, the results of Halosperm correlated well with sperm motility (r = 0.4365; p < 0.001). While comet assay showed significantly higher sperm DNA damage in both groups of patients than in the controls (t-test, both p < 0.001), Halosperm demonstrated increased levels of fragmented DNA over control values only in the group of men with infertility (p < 0.001). From the results of comet assay in the control group, 32% of fragmented DNA per sperm was determined as the upper limit of the tolerance interval indicating a reduced fertilizing potential.
Conclusion:
The comet assay has proven to be a useful complementary method to the standard examination of sperm quality.
Key words:
Comet assay, DNA fragmentation, Halosperm, infertility, sperm.
Sources
1. Saleh RA, Agarwal A, Nelson DR, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002; 78: 313–318.
2. Nallella KP, Sharma RK, Aziz N, et al. Significance of sperm characteristics in the evaluation of male infertility. Fertil Steril 2006; 85: 629–634.
3. Agarwal A, Allamaneni SS. Sperm DNA damage assessment: a test whose time has come. Fertil Steril 2005; 84: 850–853.
4. Lewis SE, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med 2008; 54: 111–125.
5. Agarwal A, Said M. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003; 9: 331–345.
6. Evgeni E, Charalabopoulos K, Asimakopoulos B. Human sperm DNA fragmentation and its correlation with conventional semen parameters. J Reprod Infertil 2014; 15: 2–14.
7. Lin MH, Kuo-Kuang Lee R, Li SH, et al. 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: 352–359.
8. Muriel L, Garrido N, Fernández JL, et al. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2006; 85: 371–383.
9. Singh A, Agarwal A. The role of sperm chromatin integrity and DNA damage on male infertility. Open Reprod Sci J 2011; 3: 65–71.
10. Coccuza M, Sikka SC, Athayade KS, et al. Clinical relevance of oxidative stress and sperm chromatin damage in male infertility: An evidence based analysis. Int Braz J Urol 2007; 33: 603–621.
11. Dvořák M, Matejovičová M. Principy a využití kometové analýzy při detekci poškození DNA. Chem Listy 2008; 102: 977–983.
12. Schmid TE, Eskenazi B, Baumgartner A, et al. The effects of male age on sperm DNA damage in healthy nonsmokers. Hum Reprod 2007: 22: 180–187.
13. Hughes CM, Lewis SEM, McKelvey-Martin V, et al. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using modified comet assay. Mol Hum Reprod 1996; 2: 613–619.
14. 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: 25–43.
15. Fernández JL, Muriel L, Rivero MT, et al. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003; 24: 59–66.
16. Peluso G, Palmieri A, Cozza PP, et al. The study of spermatic DNA fragmentation and sperm motility in infertile subjects. Arch Ital Urol Androl 2013; 85: 1–13.
17. World Health Organization (1999). WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, 4th edn. Cambridge University Press, Cambridge, UK, ISBN 9780521645997.
18. World Health Organization (2010). WHO laboratory manual for the examination and processing of human semen, 5th edn. World Health Organization, Geneva, Switzerland, ISBN 978 92 4 154778 9.
19. Rubes J, Rybar R, Prinosilova P, et al. Genetic polymorphisms influence the susceptibiliy of men to sperm DNA damage associated with exposure to air pollution. Mutat Res 2010; 683: 9–15.
20. Novotná B, Topinka J, Solanský I, et al. Impact of air pollution and genotype variability on DNA damage in Prague policemen. Toxicol Lett 2007; 172: 34–47.
21. Owen DB. Handbook of Statistical Tables, Addison-Wesley publishing Company, Massachusetts Palo Alto London, 1962.
22. Irvine DS, Twigg JP, Gordon EL, et al. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000; 21: 33–44.
23. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008; 59: 2–11.
24. Singh NP, Muller ChH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril 2003; 80: 1420–1430.
25. Talebi AR, Vahidi S, Aflatoonian A, et al. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia 2012; 44: 462–470.
Labels
Paediatric urologist Nephrology UrologyArticle was published in
Czech Urology
2016 Issue 4
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