PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jargspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
 
J Assist Reprod Genet. 2009 August; 26(8): 427–432.
Published online 2009 September 19. doi:  10.1007/s10815-009-9343-5
PMCID: PMC2767489

Antioxidants and sperm DNA damage: a clinical perspective

Abstract

Purpose

Infertile men possess substantially more sperm DNA damage than do fertile men, damage that may impact negatively on reproductive outcomes. In this era of assisted reproductive technologies there is mounting concern regarding the safety of utilizing DNA-damaged spermatozoa in this setting. Therefore, it is important to identify strategies that may reduce sperm DNA damage. The purpose of this review is to discuss the rationale for antioxidant therapy in men with sperm DNA damage and to evaluate the data on the efficacy of dietary and in vitro antioxidant preparations on sperm DNA damage.

Methods

We reviewed the literature on antioxidants and sperm DNA damage.

Results

To date, the data suggest that dietary antioxidants may be beneficial in reducing sperm DNA damage, particularly, in men with high levels of DNA fragmentation. However, the mechanism of action of dietary antioxidants has not been established and most of the clinical studies are small. A beneficial effect of in vitro antioxidant supplements in protecting sperm DNA from exogenous oxidants has been demonstrated, however, the effect of these antioxidants in protecting sperm from endogenous ROS, gentle sperm processing and cryopreservation has not been established.

Keywords: Sperm DNA fragmentation, Oxidative stress, Vitamins, Sperm washing, Male infertility

Introduction

There is evidence to show that infertile men possess substantially more sperm DNA damage than do fertile men and that this DNA damage may adversely affect reproductive outcomes [14]. This is particularly relevant in an era where advanced forms of assisted reproductive technologies (ARTs) are commonly utilized (ARTs often bypass the barriers to natural selection), as there is some uncertainty and mounting concern regarding the safety of utilizing DNA-damaged spermatozoa in this setting [5]. Therefore, it is important to identify strategies that may reduce sperm DNA damage. The proposed strategies include eliminating testicular gonadotoxins or hyperthermia, treatment of semen or genital tract infections, correction of varicoceles and the use of antioxidants [68]. The purpose of this review is to explore the rationale for antioxidant therapy in men with sperm DNA damage and to present data on the efficacy of dietary and in vitro antioxidant preparations on sperm DNA damage.

Etiology of sperm DNA damage

The etiology of sperm DNA damage is multi-factorial and may be due to primary testicular or secondary (e.g. environmental) factors. Sperm DNA damage is believed to be the result of aberrant protamine expression, excessive ROS (reactive oxygen species) generation and abortive apoptosis during spermatogenesis [912].

Relationship between ROS (reactive oxygen species) and sperm DNA damage

The association between sperm DNA damage and semen ROS is the basis for the use of antioxidants in the treatment of sperm DNA damage. High levels of ROS have been detected in the semen of 25% of infertile men [13, 14]. The levels of sperm-derived ROS (measured in sperm preparations having minimal leukocyte contamination) have been associated with sperm DNA damage, although no ROS threshold level above which sperm DNA damage is detected has been established [1517]. Moreover, the levels of sperm DNA oxidation are higher in infertile compared to fertile men [18, 19]. Semen ROS are generated by spermatozoa (especially, defective or immature) and semen leukocytes [16, 2023]. While the controlled release of low levels of ROS is necessary for normal sperm function, high levels of ROS can cause sperm dysfunction [23]. The susceptibility of human spermatozoa to oxidative stress stems primarily from the abundance of unsaturated fatty acids in the sperm plasma membrane. These unsaturated fatty acids provide fluidity that is necessary for membrane fusion events (e.g. the acrosome reaction and sperm-egg interaction) and for sperm motility. However, the unsaturated nature of these molecules predisposes them to free radical attack and ongoing lipid peroxidation throughout the sperm plasma membrane. Once this process has been initiated, accumulation of lipid peroxides occurs on the sperm surface and oxidative damage to DNA can ensue [6, 24, 25]. Studies have demonstrated that exogenous and endogenous ROS can induce sperm DNA damage in vitro, confirming that ROS may play a role in the etiology of sperm DNA damage in infertile men [2527].

Semen antioxidants and sperm DNA damage

Seminal fluid is an important source of antioxidants (ROS scavengers) and is key in protecting spermatozoa from oxidative injury [14, 28, 29]. This is particularly important because spermatozoa have little cytoplasmic fluid (antioxidant enzymes are generally intracellular), virtually no capacity for protein synthesis and little antioxidant capacity [14]. The endogenous free radical scavenging enzymes in the male reproductive tract include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) [14, 3033]. These same antioxidant enzymes (SOD, catalase and GPX) are found in semen [34]. Moreover, several non-enzymatic antioxidants (e.g. vitamins C and E, hypotaurine, taurine, L-carnitine, lycopene) are also found in semen and this non-enzymatic component accounts for much of the total seminal antioxidant activity [14, 35].

Several clinical studies have evaluated the relationship between semen antioxidant levels and sperm DNA damage and have reported conflicting results. Some studies have shown that a deficiency in semen antioxidants is associated with sperm DNA damage, whereas, other studies have not observed such a relationship [6, 3639]. Similarly, some studies have found that seminal antioxidant activity is reduced in infertile men with high levels of seminal ROS (relative to those with normal levels of ROS) whereas others have not shown this [14, 4042]. To date, there are no studies to indicate a relationship between systemic antioxidant or vitamin deficiency and male infertility. Silver et al, 2005 evaluated a cohort of fertile men and did not identify any relationships between dietary antioxidant intake (vitamins C, E or ß-carotene) and sperm DNA damage [43]. Nonetheless, it is possible that a subset of infertile men may be at risk for antioxidant deficiency, particularly, vitamin C deficiency [44]. Moreover, infertile men with specific lifestyles may also be at risk for antioxidant or vitamin deficiency (e.g. smoking, increased alcohol intake, dieting)[45, 46].

Dietary antioxidant supplements and sperm DNA damage

In order to be active, a dietary antioxidant should be effectively absorbed and concentrated in reproductive tract organs. The antioxidant should also replete a deficiency (in the testis, epididymis or semen) and play a role in reproductive function. The antioxidant must either improve spermatogenesis and/or epididymal function, ultimately, resulting in improved sperm function and chromatin compaction and integrity. Alternatively, the antioxidant should enhance semen antioxidant capacity in order to reduce oxidative DNA damage.

There are a small number of reports on the effects of dietary antioxidant supplementation on sperm DNA integrity. In general, these are small studies that do not evaluate the mechanism of action of antioxidants: the only endpoint that is measured is the integrity of the sperm DNA or pregnancy rate. Moreover, most studies evaluate the effects of a short treatment course (with no long-term follow-up), are not randomized and fail to include a placebo-control group. Most of these clinical studies have evaluated men with high levels of sperm DNA damage. In these men, treatment with antioxidant supplements is generally associated with reduced levels of sperm DNA integrity and/or improved fertility potential (Table 1) [19, 4752].

Table 1
Effect of dietary antioxidant supplements on sperm DNA integrity

In 1991, Fraga et al. demonstrated that dietary vitamin C increases semen vitamin C levels and improves sperm DNA integrity (lowers DNA oxidation levels) in men with a vitamin C deficiency (on a vitamin C depleted diet). More recent studies of infertile men with high levels of sperm DNA damage (2 randomized controlled and 3 uncontrolled trials) have shown that antioxidant therapy is effective in improving sperm DNA integrity or pregnancy rates (Table 1). In men with unselected infertility, the effect of dietary antioxidants on sperm DNA integrity is equivocal with one of two controlled trials showing a benefit of antioxidants on sperm DNA integrity (Table 1).

In vitro antioxidants and sperm DNA damage

Several studies have examined the role of in vitro antioxidant supplementation in protecting the sperm DNA from oxidative damage. This is clinically relevant as sperm washing is routinely performed prior to ARTs (e.g. intrauterine insemination and in vitro fertilization) and the process may result in injury to the sperm DNA, particularly, as spermatozoa are now vulnerable to oxidative stress because seminal plasma (rich in antioxidants) has been removed in the process [53]. However, it is important to note that subpopulations of spermatozoa will exhibit variable susceptibility to oxidative stress: the DNA of normal spermatozoa is reportedly less susceptible to gentle processing techniques than is the DNA of abnormal or immature spermatozoa [21, 54].

The studies on in vitro antioxidant supplementation have looked at the role of antioxidants in protecting sperm from exogenous and endogenous ROS, and, from the effects of semen processing and cryopreservation. Antioxidants (e.g. vitamins C and E, catalase, glutathione) have been shown quite clearly to protect sperm DNA from the effects of exogenous ROS (see Table 2) [5558]. This is of clinical relevance as many of the semen samples contain leukocytes and these cells have the potential to generate exogenous ROS. In contrast, antioxidants appear to be of limited value in protecting the DNA of normal spermatozoa from endogenous ROS production (e.g. NADPH-induced or centrifugation-induced) (see Table 3) [25, 5961]. In samples with poor morphology and poor sperm chromatin compaction, antioxidants may protect the sperm DNA from endogenous ROS production, as these samples are more vulnerable to oxidative stress [21, 54]. In general, antioxidants appear to be of limited value in protecting sperm DNA from gentle semen processing (e.g. incubation or density-gradient centrifugation) (see Table 4) [6265]. In some cases, antioxidants supplementation in vitro (e.g. combination of vitamins C and E) may cause sperm DNA damage [63, 64]. The one study evaluating the effects of sperm cryopreservation suggests that antioxidants (vitamin E) do not protect sperm DNA in this setting [66].

Table 2
Role of in vitro antioxidant supplements in protecting sperm DNA from exogenous ROS
Table 3
Role of in vitro antioxidant supplements in protecting sperm DNA from stimulated endogenous reactive oxygen species (ROS) generation
Table 4
Role of in vitro antioxidant supplements in protecting sperm DNA from semen processing

In summary, the data suggest that ROS appear to play an important role in the generation of sperm DNA damage. Although in vitro studies have demonstrated a beneficial effect of antioxidant supplements in protecting sperm DNA from exogenous oxidants, the effect of these antioxidants in protecting sperm from endogenous ROS, gentle sperm processing and cryopreservation has not been established. The data suggest that dietary antioxidants may be beneficial in reducing sperm DNA damage, particularly, in men with high levels of DNA fragmentation. However, the mechanism of action of dietary antioxidants has not been established and most of the clinical studies are small.

Footnotes

Capsule Dietary and in vitro antioxidant preparations may help reduce sperm DNA damage, however, larger, well-designed studies are needed to firmly establish the utility of antioxidants in this setting.

References

1. 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;23:2663–8. [PubMed]
2. 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;22:174–9. [PubMed]
3. Sigman M, Zini A. Semen analysis and sperm function assays: what do they mean? Semin Reprod Med. 2009;27:115–23. [PubMed]
4. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril. 2008;89:823–31. [PubMed]
5. 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;78:761–72. [PubMed]
6. Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ, Jacob RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci U S A. 1991;88:11003–6. [PubMed]
7. Zini A, Blumenfeld A, Libman J, Willis J. Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity. Hum Reprod. 2005;20:1018–21. [PubMed]
8. Ochsendorf FR. Infections in the male genital tract and reactive oxygen species. Hum Reprod Update. 1999;5:399–420. [PubMed]
9. 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;7:428–32. [PubMed]
10. Aitken RJ, De Iuliis GN, McLachlan RI. Biological and clinical significance of DNA damage in the male germ line. Int J Androl. 2009;32:46–56. [PubMed]
11. Carrell DT, Liu L. Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. J Androl. 2001;22:604–10. [PubMed]
12. de Yebra L, Ballesca JL, Vanrell JA, Bassas L, Oliva R. Complete selective absence of protamine P2 in humans. J Biol Chem. 1993;268:10553–7. [PubMed]
13. Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril. 1992;57:409–16. [PubMed]
14. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl. 1993;16:183–8. [PubMed]
15. Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21:33–44. [PubMed]
16. 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. [PubMed]
17. Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ Jr. Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertil Steril. 2003;80:1431–6. [PubMed]
18. Shen HM, Chia SE, Ong CN. Evaluation of oxidative DNA damage in human sperm and its association with male infertility. J Androl. 1999;20:718–23. [PubMed]
19. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril. 1997;68:519–24. [PubMed]
20. Sati L, Ovari L, Bennett D, Simon SD, Demir R, Huszar G. Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation. Reprod Biomed Online. 2008;16:570–9. [PubMed]
21. Muratori M, Piomboni P, Baldi E, Filimberti E, Pecchioli P, Moretti E, et al. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 2000;21:903–12. [PubMed]
22. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl. 1996;17:276–87. [PubMed]
23. Aitken J, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays. 1994;16:259–67. [PubMed]
24. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl. 1987;8:338–48. [PubMed]
25. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum Reprod. 1998;13:1429–36. [PubMed]
26. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod. 1998;59:1037–46. [PubMed]
27. Sawyer DE, Mercer BG, Wiklendt AM, Aitken RJ. Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat Res. 2003;529:21–34. [PubMed]
28. Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res. 1989;24:185–96. [PubMed]
29. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann N Y Acad Sci. 1991;637:436–44. [PubMed]
30. Zini A, Schlegel PN. Catalase mRNA expression in the male rat reproductive tract. J Androl. 1996;17:473–80. [PubMed]
31. Zini A, Schlegel PN. Expression of glutathione peroxidases in the adult male rat reproductive tract. Fertil Steril. 1997;68:689–95. [PubMed]
32. Jow WW, Schlegel PN, Cichon Z, Phillips D, Goldstein M, Bardin CW. Identification and localization of copper-zinc superoxide dismutase gene expression in rat testicular development. J Androl. 1993;14:439–47. [PubMed]
33. Zini A, Schlegel PN. Identification and characterization of antioxidant enzyme mRNAs in the rat epididymis. Int J Androl. 1997;20:86–91. [PubMed]
34. Sanocka D, Miesel R, Jedrzejczak P, Chelmonska-Soyta AC, Kurpisz M. Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility. Int J Androl. 1997;20:255–64. [PubMed]
35. Holmes RP, Goodman HO, Shihabi ZK, Jarow JP. The taurine and hypotaurine content of human semen. J Androl. 1992;13:289–92. [PubMed]
36. Song GJ, Lewis V. Mitochondrial DNA integrity and copy number in sperm from infertile men. Fertil Steril. 2008;90:2238–44. [PubMed]
37. Appasamy M, Muttukrishna S, Pizzey AR, Ozturk O, Groome NP, Serhal P, et al. Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility. Reprod Biomed Online. 2007;14:159–65. [PubMed]
38. Verit FF, Verit A, Kocyigit A, Ciftci H, Celik H, Koksal M. No increase in sperm DNA damage and seminal oxidative stress in patients with idiopathic infertility. Arch Gynecol Obstet. 2006;274:339–44. [PubMed]
39. Xu DX, Shen HM, Zhu QX, Chua L, Wang QN, Chia SE, et al. The associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma. Mutat Res. 2003;534:155–63. [PubMed]
40. Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma is different in fertile and infertile men. Fertil Steril. 1995;64:868–70. [PubMed]
41. Smith R, Vantman D, Ponce J, Escobar J, Lissi E. Total antioxidant capacity of human seminal plasma. Hum Reprod. 1996;11:1655–60. [PubMed]
42. Sanocka D, Miesel R, Jedrzejczak P, Kurpisz MK. Oxidative stress and male infertility. J Androl. 1996;17:449–54. [PubMed]
43. Silver EW, Eskenazi B, Evenson DP, Block G, Young S, Wyrobek AJ. Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men. J Androl. 2005;26:550–6. [PubMed]
44. Hampl JS, Taylor CA, Johnston CS. Vitamin C deficiency and depletion in the United States: the Third National Health and Nutrition Examination Survey, 1988 to 1994. Am J Public Health. 2004;94:870–5. [PubMed]
45. Jacob RA. Assessment of human vitamin C status. J Nutr. 1990;120(Suppl 11):1480–5. [PubMed]
46. Ryle PR, Thomson AD. Nutrition and vitamins in alcoholism. Contemp Issues Clin Biochem. 1984;1:188–224. [PubMed]
47. Greco E, Romano S, Iacobelli M, Ferrero S, Baroni E, Minasi MG, et al. ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment. Hum Reprod. 2005;20:2590–4. [PubMed]
48. 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. [PubMed]
49. Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J, Cohen-Bacrie P, et al. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online. 2007;14:418–21. [PubMed]
50. Tremellen K, Miari G, Froiland D, Thompson J. A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment. Aust N Z J Obstet Gynaecol. 2007;47:216–21. [PubMed]
51. Gil-Villa AM, Cardona-Maya W, Agarwal A, Sharma R, Cadavid A. Role of male factor in early recurrent embryo loss: do antioxidants have any effect? Fertil Steril 2008. [PubMed]
52. Piomboni P, Gambera L, Serafini F, Campanella G, Morgante G, De Leo V. Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia. Asian J Androl. 2008;10:201–6. [PubMed]
53. Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod. 1998;4:439–45. [PubMed]
54. Said TM, Agarwal A, Sharma RK, Thomas AJ Jr, Sikka SC. Impact of sperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucleotide phosphate. Fertil Steril. 2005;83:95–103. [PubMed]
55. Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod. 1998;13:896–900. [PubMed]
56. Potts RJ, Notarianni LJ, Jefferies TM. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutat Res. 2000;447:249–56. [PubMed]
57. Sierens J, Hartley JA, Campbell MJ, Leathem AJ, Woodside JV. In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm. Teratog Carcinog Mutagen. 2002;22:227–34. [PubMed]
58. Russo A, Troncoso N, Sanchez F, Garbarino JA, Vanella A. Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species. Life Sci. 2006;78:1401–6. [PubMed]
59. Cemeli E, Schmid TE, Anderson D. Modulation by flavonoids of DNA damage induced by estrogen-like compounds. Environ Mol Mutagen. 2004;44:420–6. [PubMed]
60. Dobrzynska MM, Baumgartner A, Anderson D. Antioxidants modulate thyroid hormone- and noradrenaline-induced DNA damage in human sperm. Mutagenesis. 2004;19:325–30. [PubMed]
61. Anderson D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM. Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay). Mutat Res. 2003;544:173–8. [PubMed]
62. Chi HJ, Kim JH, Ryu CS, Lee JY, Park JS, Chung DY, et al. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum Reprod. 2008;23:1023–8. [PubMed]
63. Donnelly ET, McClure N, Lewis SE. The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis. 1999;14:505–12. [PubMed]
64. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod. 1998;13:1240–7. [PubMed]
65. Donnelly ET, McClure N, Lewis SE. Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagenesis. 2000;15:61–8. [PubMed]
66. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online. 2009;18:184–9. [PubMed]

Articles from Journal of Assisted Reproduction and Genetics are provided here courtesy of Springer Science+Business Media, LLC