AJMB

Sperm Chromatin Integrity: Etiologies and Mechanisms of Abnormality, Assays, Clinical Importance, Preventing and Repairing Damage

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Volume 1, Issue 3, October-December , Page 147 to 160
Sunday, November 8, 2009 :Received , Tuesday, December 1, 2009 :Accepted


  • - Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR , Tehran, Iran
    - Department of Human Nutrition, Faculty of Nutrition & Food Industry, Shaheed Beheshti University of Medical Sciences , Tehran, Iran

  • - Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR , Tehran, Iran
  • Corresponding author Ph.D., Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, P.O. Box: 19615-1177, Tel: +98 21 22432020, Fax: +98 21 22432021, E-mail: Sadeghi@avicenna.ac.ir
    - Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR , Tehran, Iran

Abstract:

The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples? infertility. One of the most important parameters of sperm in its fertilizing potential is "Sperm chromatin integrity" that has direct positive correlation with Assisted Reproductive Techniques (ART) outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DNA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons under-standing the sperm chromatin structure, etiology of sperm chromatin abnor-mality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples? infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DNA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it.


 

 

Introduction :
Infertility is a major problem in 15-20% of couples trying to conceive in the reproductive ages (1). Male infertility may contribute in half of all couples who refer to infertility clinics (2).
The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and vital-ity. However, it is well-known that normal results of semen analysis can not exclude men from causes of couples? infertility (3).

Today, it is well known that the quality and integrity of sperm chromatin is very important in the reproductive potential of men. Sperm DNA is known to contribute to half of the genomic material of the embryo. Sperm chro-matin is much more compact in somatic cells, with the aim of protecting the paternal genetic materials against damaging factors during its passage from testis as production site to fallopian tubes as final destination (4).
It is a fact that normal sperm genomic

material is required for a normal fertilization, pregnancy, live birth, and postnatal child well being. Therefore, abnormal sperm chromatin may result in male sub-fertility or even infer-tility, recurrent abortion, increased congenital anomalies and testicular cancer in offsprings (5). Thus, more knowledge on sperm chro-matin damage including: etiology, mechan-ism, detection methods, clinical outcomes and treatment may provide better diagnostic and prognostic capabilities than standard sperm parameters for both in vivo and in vitro fertil-izing ability of human spermatozoa (6).

Etiology of sperm chromatin damage
Many factors are involved in inducing sperm DNA damage, which may result in the male infertility. One of these factors is Leuko-cytospermia (7), which is the increase of leuko-cytes in semen due to genital infection and/or inflammation (8).
Leukocytospermia could result in the pro-duction of inflammatory cytokines followed by the overproduction of Reactive Oxygen Species (ROS) leading to increase in sperm DNA damage (9,10). Cigarette smokes also in-duce leukocytospermia and ROS overproduc-tion (11-13). It seems that leukocytes can gener-ate high levels of ROS in semen, which may overwhelm the antioxidant capacity of semen and resultant Oxidative Stress (OS) could dis-turb sperm functions (14, 15).
Also, some of the sperm preparation and cryopreservation protocols could decrease sperm chromatin integrity (16,17). It has been shown that rapid and ultra rapid freezing (flash-freezing in liquid nitrogen) induce the least damage to sperm during cryopreserva-tion (18). Malignancies such as leukemia, Hodgkin’s disease, and testicular malign-nancies could affect sperm chromatin integ-rity by themselves or following their treat-ment with cytotoxic drugs and/ or radiation therapy (19-22).
Many drugs might also affect the semen quality and increase sperm DNA damage
(23-25). Even some types of herbal remedies may induce sperm DNA damage. For ex-ample high dose administration of Gingko biloba, St. John's wort and Echinacae purpura were associated with increased damage of the reproductive cells (26).
Finally, a growing body of evidences shows that environmental and occupational exposures to chemical agents, heat, and agri-cultural toxins could play a role in sperm DNA damage (10). In a recent study, it has been shown that increased scrotal heat not only reduces the quality of semen parameters, but also compromises sperm chromatin integ-rity (27).

Mechanism of Sperm DNA Damage
While the exact causes of sperm DNA damage have not yet been fully elucidated, several interrelated mechanisms have been suggested (28-30). These mechanisms are cat-egorized as abnormal chromatin packaging, apoptosis and oxidative stress.

Abnormal chromatin packaging
The sperm chromatin is an extremely compact and stable structure which must be organized in a specific manner to achieve this unique condensed state (31). This DNA organ-ization provides mor

 

Discussion :
It has recently been accepted that sperm chromatin integrity is an independent index of sperm quality and has better diagnostic and prognostic capacities in association with routine semen analysis results for both in vivo and in vitro fertility. Although there are nu-merous methods to evaluate sperm chromatin integrity, each procedure needs to be studied more and standardized for routine use in diag-nostic andrology lab. However, all of these methods destroy sperm during the evaluation process. Introducing methods with some ef-fect on whole sperm integrity will improve outcomes of ARTs using spermatozoa with approved chromatin integrity. The results may help the physicians to counsel infertile couples referred for ART in a better manner. In addition, sperm preparation techniques and in vitro culture of spermatozoa in special conditions, may develop its quality in men having high percentage of DFI.

 


References :
  1. Kidd SA, Eskenazi B, Wyrobek AJ. Effects of male age on semen quality and fertility: a review of the literature. Fertil Steril 2001;75(2):237-248.
  2. Schoor RA. Prostatitis and male infertility: evi-dence and links. Curr Urol Rep 2002;3(4):324-329.
  3. Lewis SEM, Agbaje I, Alvarez J. Sperm DNA tests as useful Adjuncts to semen analysis. Syst Biol Reprod Med 2008;(3)54:111-125.
  4. Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 1999;60(3):615-620.
  5. Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol 2003;518:85-98.
  6. Alvarez JG. DNA fragmentation in human sperm-atozoa: significance in the diagnosis and treatment of infertility. Minerva Ginecol 2003;55(3):233-239.
  7. Tomlinson MJ, White A, Barratt CLR, Bolton AE, Cooke ID. The removal of morphologically abnor-mal sperm forms by phagocytes: a positive role for seminal leukocytes. Hum Reprod 1992;7(4):517-522.
  8. Sigman M, Lopes L. The correlation between round cells and white blood cells in the semen. J Urol 1993;149(5):1338-1340.
  9. Cohen PE, Pollard JW. Cytokines and growth factors in reproduction. In: Bronson R, (eds). Re-productive Immunology. Cambridge, MA: Black-well Science; 1996,52-102.
  10. Reichart M, Kahane I, Bartoov B. In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted Urea-plasma urealyticum infection. Biol Reprod 2000; 63(4):1041-1048.
  11. Kunzle R, Mueller MD, Hanggi W, Birkahuser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril 2003;79(2):287-291.
  12. Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jef-feries TM. Sperm chromatin damage associated with male smoking. Mutat Res 1999;423(1-2):103-111.
  13. Comhaire FH, Mahmoud AM, Depuydt CE, Zalata AA, Christofe AB. Mechanism and effects of male genital tract infection on sperm quality and fertiliz-ing potential: the andrologist's view point. Hum Reprod Update 1999;5(5):393-398.
  14. Aitken RJ, Buckingham D, Brindle J, Gomez E, Baker G, Irvine, S. Analysis of sperm movement in relation to the oxidative stress created by leuko-cytes in washed sperm preparations and seminal plasma. Hum Reprod 1995;10(8):2061-2071.
  15. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res 1996;351(2):199-203.
  16. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Re-prod 2001;16(6):1191-1199.
  17. Donnelly ET, McClure N, Lewis SE. Cryopreser-vation of human semen and prepared sperm: ef-fects on motility parameters and DNA integrity. Fertil Steril 2001;76(5):892-900.
  18. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Hunag T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  19. Giwercman A, Petersen PM. Cancer and male infertility. Baillieres Best Pract Res Clin Endo-crinol Metab 2000;14:453-471.
  20. Richter MA., Haning RV, Jr Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 1984;41(2): 277-280.
  21. Revel A, Haimov-Kochman R, Porat A, Lewin A, Simon A, Laufer N, et al. In vitro fertilization-intracytoplasmic sperm injection success rates with cryopreserved sperm from patients with malignant disease. Fertil Steril 2005;84(1):118-122.
  22. Kobayashi H, Larson K, Sharma RK, Nelson DR, Evenson DP, Toma H, et al. DNA damage in cancer patients before treatment as measured by the sperm chromatin structure assay. Fertil Steril 2001;75(3):469-475.
  23. Chatterjee R, Haines GA., Perera DM, Goldstone A, Morris ID. Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukemia. Hum Reprod 2000;15(4): 762-766.
  24. Cai L, Hales BF, Robaire B. Induction of apoptosis on the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 1997;56(6): 1490-1497.
  25. Li H, Jian Y, Rajpurkar R, Dunbar, JC, Dhabu-wala CB. Cocaine induced apoptosis in rat testes. J Urol 1999;162(1):213-216.
  26. Ondrizek RR, Chan PJ, Patton WC, King A. An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid. Fertil Steril 1999;71(3):517-522.
  27. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev 2008;75(1):40-47.
  28. Aitken RJ, De Iulis GN. Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online 2007;14(6):727-733.
  29. Tesarik J, Mendoza TR, Mendoza C. Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment. Reprod Biomed Online 2006;12(6):715-721.
  30. Ozmen B, Koutlaki N, Youssry M, Diedrich K, Al Hasani S. DNA damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts, and safety. Reprod Biomed Online 2007;14(3): 384-395.
  31. Carrell DT, Emery BR, Hammoud S. The etiology of sperm protamine abnormalities and their poten-tial impact on the sperm epigenome. Int J Andro-logy 2008;31(6):537-545.
  32. Evenson DP, Larson KL, Jost LK. Sperm chro-matin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons. J Andrology 2002;23(1):25-43.
  33. Ward WS. Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod 1993;48(6):1193-1201.
  34. D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relation-ship to male fertility and embryonic survival. Animal Reprod Sci 2007;101(1-2):1-17.
  35. Balhorn R, Cosman M, Thornton K, Krishnan VV, Corzett M, Bench E, Kramer C, et al. Protamine mediated condensation of DNA in mammalian sperm. In: Gagnon C. The male gamete: from basic knowledge to clinical applications. Illinois: Cache River Press;1999,55-70.
  36. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4:31-37.
  37. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, et al. Influ-ence of deoxyribonucleic acid damage on fertil-ization and pregnancy. Fertil Steril 2004;81(4): 965-972.
  38. Lewis SEM, Aitken RJ. DNA damage to sperm-atozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322(1):33-41.
  39. Vogt PH. Molecular genetics of human male infer-tility: from genes to new therapeutic perspectives. Curr Pharm Design 2004;10(5):471-500.
  40. Carrell DT, Emery BR, Hammoud S. Altered protamine expression and diminished spermato-genesis: what is the link? Hum Reprod Update 2007;13(3):313-327.
  41. Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson M, Peterson CM, et al. Sperm protamine1/ protamine 2 ratios are related to in vitro fertil-ization pregnancy rates and predictive of fertile-ization ability. Fertil Steril 2006;86(5):1408-1415.
  42. Tseden K, Topaloglu O, Meinhardt A, Dev A, Adham I, Muller C, et al. Premature translation of transition protein 2 mRNA causes sperm abnor-malities and male infertility. Mol Reprod Dev 2007;74(3):273-279.
  43. Torregrosa N, Dominguez-Fandos D, Camejo MI, Shirley CR, Meistrich ML, Ballesca JL, et al. Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Human Reprod 2006;21(8):2084-2089.
  44. Dadoune JP. Expression of mammalian spermato-zoal nucleoproteins. Microsc Res Tech 2003;61 (1):56-75.
  45. Brewer L, Corzett M, Balhorn R. Condensation of DNA by spermatid basic nuclear proteins. J Biol Chem 2002;41:38895-38900.
  46. Brewer L, Corzett M, Lau EY, Balhorn R. Dy-namics of protamine 1 binding to single DNA molecules. J Biol Chem 2003;278:42403-42408.
  47. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331-345.
  48. Kocak I, Dundar M, Hekimgil M, Okyay P. As-sessment of germ cell apoptosis in cryptorchid rats. Asian J Androl 2002;4(3):183-186.
  49. Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999;4:38-47.
  50. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 1997;138(5): 2081-2088.
  51. Suda T, Takahashi T, Goldstein P , Nagata S. Mol-ecular cloning and expression of Fas ligand, a novel member of tumor necrosis factor family. Cell 1993;75(6):1169-1178.
  52. Rodriguez I, Ody C, Araki K, Garcia I, Vassali P. An early and massive wave of germinal cell ap-optosis is required for the development of functional spermatogenesis. EMBO J 1997;16(9): 2262-2270.
  53. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenic maturation in men: relationship among plasma membrane beat 1,4 galacto-syltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol Reprod 1997;56(4):1020-1024.
  54. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281(5381):1312-1316.
  55. Kim JM, Ghosh SR, Weil ACP, Zirkin BR. Cas-pase-3 and caspase-activated deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced intratesticular testosterone. Endocrinology 2001;142(9):3809-3816.
  56. Shafik A, Shafik AA, Shafik I, El Sibai O. Sperm DNA fragmentation. Arch Androl 2006;52:197-208.
  57. 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(3):519-524.
  58. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  59. Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma mem-brane integrity of human spermatozoa. Fertil Steril 2000;74(6):1200-1207.
  60. Aitken RJ, Krausz C. Oxidative stress, DNA dam-age and the Y chromosome. Reproduction 2001; 122:497-506.
  61. Spiropoulos J, Turnbull DM, Chinnery PF. Can mitochondrial DNA mutations cause sperm dys-function? Mol Hum Reprod 2002;8(8):719-721.
  62. Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 2004;6:139-148.
  63. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001;65(4):1102-1113.
  64. Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and signifi-cance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 2005;3:67-75.
  65. Balercia G, Moretti S, Vignini A, Magagnini M, Mantero F, Boscaro M, et al. Role of nitric oxide concentrations on human sperm motility. J Androl 2004;25(2):245-249.
  66. Agarwal A, Makker K, Sharma R. Clinical rele-vance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008;59:2-11.
  67. 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 struc-ture and TUNEL assays. Hum Reprod 2004;19 (10):2277-2282.
  68. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR, King A. A simple comet assay for arch-ived sperm correlates DNA fragmentation to re-duced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001;75(1):186-192.
  69. Hodjat M, Akhondi MA, Al-Hasani S, Mobaraki M, Sadeghi MR. Increased sperm ubiquitination correlates with abnormal chromatin integrity. Re-prod Biomed Online 2008;17(3):324-330.
  70. Hodjat M, Akhondi MA, Amirjanati N, Savadi Shirazi E, Sadeghi MR. The comparison of four different sperm chromatin assays and their correl-ation with semen parameters. TUMJ 2008;65 (Suppl 3):33-40.
  71. Hammadeh M, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 2001;46(2):99-104.
  72. Foresta C, Zorzi M, Rossato M, Varotto A. Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. lnt J Androl 1992;15(4):330-337.
  73. Erenpreiss A, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22(1):45-53.
  74. Ashok Agarwal, Tamer M Said. Sperm chromatin assessment. In: Gardner DK, Weismann A (eds). Textbook of Assisted Reproductive Techniques. United Kingdom: InForma Health Care; 2009,93-106.
  75. Manicardi G, Bianchi P, Pantano S, Azzoni P, D. Bizzaro D, Bianchi U. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessi-bility. Biol Reprod 1995;52:864-867.
  76. Esterhuizen A, Franken D, Laurens JGH, Prinsloo e, van Rooyen LH. Sperm chromatin packaging as an indicator of in vitro fertilization rates. Hum Reprod 2000;15(3):657-661.
  77. Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosál-vez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82(3-4):251-256.
  78. Fernandez J, Vazquez-Gundin F, Delgado A, Goy-anes J, Ramiro-Diaz J, de la Torre J, et al. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453(1):77-82.
  79. Gopalkrishnan K, Hurkadli K, Padwal V, Balaiah D. Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andralogia 1999;31(5):277-282.
  80. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  81. Variant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI derived embryos. J Assist Reprod Genet 2002;19(7):319-328.
  82. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluores-cence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79:670-676.
  83. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giweroman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495-2505.
  84. Singh N, Danner DB, Tice R, Mccoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  85. Fernandez J, Muriel L, Rivero MT, Goyanes V, Vazque R, Alvarez JG. The sperm chromatin dis-persion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24 (1):59-66.
  86. Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damages on indi-vidual cells. Biochem Biophys Res Commun 1984; 123(1):291-298.
  87. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(1):184-191.
  88. Singh NP, Danner DB, Tice RR, Mccoy MT, Col-lins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  89. Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995;336(2):123-131.
  90. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Huang T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  91. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm as-sessed by single cell gel electrophoresis (comet assay) and its relationship to fertilization. Hum Reprod 2002;17(4):990-998.
  92. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17(7):1856-1862.
  93. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single cell electro-phoresis. Exp Cell Res 1997;236(1):231-237.
  94. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  95. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 2009;18(6):761-768.
  96. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  97. Shen HM, Ong CN. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000;28(4):529-536.
  98. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased deoxyribonucleic acid dam-age in the spermatozoa of infertile male patients. Fertil Steril 1997;68(3):519-524.
  99. Loft S, Kold-Jensen T, Hjollund N, Giweroman A, Geyllemborg J, Ernst E, et al. Oxidative DNA damage in human sperm influences time to preg-nancy. Hum Reprod 2003;18(6):1265-1272.
  100. 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):1597-1605.
  101. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of sperm chro-matin structure assay as a diagnostic and prog-nostic tool in the human fertility clinic. Hum Reprod 1999;14(4):1039-1049.
  102. Duran EH, Morshedi M, Taylor S, Oehringer S. Sperm DNA quality predicts intrauterine insemina-tion, outcome: a prospective cohort study. Hum Reprod 2002;17(12):3122-3128.
  103. Host 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):559-563.
  104. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracyto-plasmic sperm injection. Fertil Steril 1998;69(3): 528-32.
  105. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602-607.
  106. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation de-creases the pregnancy rate in an assisted reproduct-ive technique. Hum Reprod 2003;18(5):1023-1028.
  107. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rates after intracytoplasmic sperm injec-tion with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10(1):148-152.
  108. Cummins JM, Jequire AM, Kan R. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol Reprod Dev 1994;37(3):345-362.
  109. Larson KL, DeJonge CJ, BarnesAM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy fol-lowing assisted reproductive techniques. Hum Reprod 2000;15(8):1717-1722.
  110. Edwards RG, Beard HK. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum Reprod 1999;14(1):1-6.
  111. Matsuda Y, Tobari I. Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes. Mutat Res 1989;210(1):35-47.
  112. Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  113. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999; 284(6): 696-704.
  114. Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod 1999;14(9): 2279-2285.
  115. Genesca A, Caballin MR, Miro R, Benet J, Germ JR, Egoscue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  116. 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(7): 1552-1561.
  117. Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol Hum Reprod 1997;3:47-54.
  118. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA dam-age before and after density gradient centrifuga-tion: implications for assisted conception. Hum Reprod 2001;16(10):2160-2165.
  119. Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up technique and DNA fragmentation. Hum Reprod 2001;16(9):1950-1953.
  120. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Verdoodt M, Englert Y. Increased spermatozoa motility after in vitro culture of testicular biopsies from obstructive azoospermic patients results in better post thaw recovery rate. Hum Reprod 2000;15(11):2371-2374.
  121. Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril 1999;72(5):809-813.
  122. Hughes CM, Lewis SEM, McKelvey-Martin VJ. Thompson, W. The effects of antioxidant supple-mentation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240-1247.
  123. 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(3): 227-234.
  124. 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(4):550-556.
  125. 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(5):870-875.
  126. Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, et al. Coenzyme Q10 treat-ment in infertile men with idiopathic asthenozoo-spermia: a placebo-controlled, double-blind ran-domized trial. Fertil Steril 2009;91(5):1785-1792.
  127. Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril 2002;77(3):491-498.
  128. Lenzi A, Lombardo F, Sgro P, Salacone P, Capon-ecchia L, Dondero F, et al. Use of carnitine ther-apy in selected cases of male factor infertility: a double-blind crossover trial. Fertil Steril 2003;79 (2):292-300.
  129. Schoor RA. Prostatitis and male infertility: evi-dence and links. Curr Urol Rep 2002;3(4):324-329.
  130. Lewis SEM, Agbaje I, Alvarez J. Sperm DNA tests as useful Adjuncts to semen analysis. Syst Biol Reprod Med 2008;(3)54:111-125.
  131. Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 1999;60(3):615-620.
  132. Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol 2003;518:85-98.
  133. Alvarez JG. DNA fragmentation in human sperm-atozoa: significance in the diagnosis and treatment of infertility. Minerva Ginecol 2003;55(3):233-239.
  134. Tomlinson MJ, White A, Barratt CLR, Bolton AE, Cooke ID. The removal of morphologically abnor-mal sperm forms by phagocytes: a positive role for seminal leukocytes. Hum Reprod 1992;7(4):517-522.
  135. Sigman M, Lopes L. The correlation between round cells and white blood cells in the semen. J Urol 1993;149(5):1338-1340.
  136. Cohen PE, Pollard JW. Cytokines and growth factors in reproduction. In: Bronson R, (eds). Re-productive Immunology. Cambridge, MA: Black-well Science; 1996,52-102.
  137. Reichart M, Kahane I, Bartoov B. In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted Urea-plasma urealyticum infection. Biol Reprod 2000; 63(4):1041-1048.
  138. Kunzle R, Mueller MD, Hanggi W, Birkahuser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril 2003;79(2):287-291.
  139. Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jef-feries TM. Sperm chromatin damage associated with male smoking. Mutat Res 1999;423(1-2):103-111.
  140. Comhaire FH, Mahmoud AM, Depuydt CE, Zalata AA, Christofe AB. Mechanism and effects of male genital tract infection on sperm quality and fertiliz-ing potential: the andrologist's view point. Hum Reprod Update 1999;5(5):393-398.
  141. Aitken RJ, Buckingham D, Brindle J, Gomez E, Baker G, Irvine, S. Analysis of sperm movement in relation to the oxidative stress created by leuko-cytes in washed sperm preparations and seminal plasma. Hum Reprod 1995;10(8):2061-2071.
  142. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res 1996;351(2):199-203.
  143. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Re-prod 2001;16(6):1191-1199.
  144. Donnelly ET, McClure N, Lewis SE. Cryopreser-vation of human semen and prepared sperm: ef-fects on motility parameters and DNA integrity. Fertil Steril 2001;76(5):892-900.
  145. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Hunag T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  146. Giwercman A, Petersen PM. Cancer and male infertility. Baillieres Best Pract Res Clin Endo-crinol Metab 2000;14:453-471.
  147. Richter MA., Haning RV, Jr Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 1984;41(2): 277-280.
  148. Revel A, Haimov-Kochman R, Porat A, Lewin A, Simon A, Laufer N, et al. In vitro fertilization-intracytoplasmic sperm injection success rates with cryopreserved sperm from patients with malignant disease. Fertil Steril 2005;84(1):118-122.
  149. Kobayashi H, Larson K, Sharma RK, Nelson DR, Evenson DP, Toma H, et al. DNA damage in cancer patients before treatment as measured by the sperm chromatin structure assay. Fertil Steril 2001;75(3):469-475.
  150. Chatterjee R, Haines GA., Perera DM, Goldstone A, Morris ID. Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukemia. Hum Reprod 2000;15(4): 762-766.
  151. Cai L, Hales BF, Robaire B. Induction of apoptosis on the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 1997;56(6): 1490-1497.
  152. Li H, Jian Y, Rajpurkar R, Dunbar, JC, Dhabu-wala CB. Cocaine induced apoptosis in rat testes. J Urol 1999;162(1):213-216.
  153. Ondrizek RR, Chan PJ, Patton WC, King A. An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid. Fertil Steril 1999;71(3):517-522.
  154. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev 2008;75(1):40-47.
  155. Aitken RJ, De Iulis GN. Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online 2007;14(6):727-733.
  156. Tesarik J, Mendoza TR, Mendoza C. Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment. Reprod Biomed Online 2006;12(6):715-721.
  157. Ozmen B, Koutlaki N, Youssry M, Diedrich K, Al Hasani S. DNA damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts, and safety. Reprod Biomed Online 2007;14(3): 384-395.
  158. Carrell DT, Emery BR, Hammoud S. The etiology of sperm protamine abnormalities and their poten-tial impact on the sperm epigenome. Int J Andro-logy 2008;31(6):537-545.
  159. Evenson DP, Larson KL, Jost LK. Sperm chro-matin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons. J Andrology 2002;23(1):25-43.
  160. Ward WS. Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod 1993;48(6):1193-1201.
  161. D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relation-ship to male fertility and embryonic survival. Animal Reprod Sci 2007;101(1-2):1-17.
  162. Balhorn R, Cosman M, Thornton K, Krishnan VV, Corzett M, Bench E, Kramer C, et al. Protamine mediated condensation of DNA in mammalian sperm. In: Gagnon C. The male gamete: from basic knowledge to clinical applications. Illinois: Cache River Press;1999,55-70.
  163. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4:31-37.
  164. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, et al. Influ-ence of deoxyribonucleic acid damage on fertil-ization and pregnancy. Fertil Steril 2004;81(4): 965-972.
  165. Lewis SEM, Aitken RJ. DNA damage to sperm-atozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322(1):33-41.
  166. Vogt PH. Molecular genetics of human male infer-tility: from genes to new therapeutic perspectives. Curr Pharm Design 2004;10(5):471-500.
  167. Carrell DT, Emery BR, Hammoud S. Altered protamine expression and diminished spermato-genesis: what is the link? Hum Reprod Update 2007;13(3):313-327.
  168. Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson M, Peterson CM, et al. Sperm protamine1/ protamine 2 ratios are related to in vitro fertil-ization pregnancy rates and predictive of fertile-ization ability. Fertil Steril 2006;86(5):1408-1415.
  169. Tseden K, Topaloglu O, Meinhardt A, Dev A, Adham I, Muller C, et al. Premature translation of transition protein 2 mRNA causes sperm abnor-malities and male infertility. Mol Reprod Dev 2007;74(3):273-279.
  170. Torregrosa N, Dominguez-Fandos D, Camejo MI, Shirley CR, Meistrich ML, Ballesca JL, et al. Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Human Reprod 2006;21(8):2084-2089.
  171. Dadoune JP. Expression of mammalian spermato-zoal nucleoproteins. Microsc Res Tech 2003;61 (1):56-75.
  172. Brewer L, Corzett M, Balhorn R. Condensation of DNA by spermatid basic nuclear proteins. J Biol Chem 2002;41:38895-38900.
  173. Brewer L, Corzett M, Lau EY, Balhorn R. Dy-namics of protamine 1 binding to single DNA molecules. J Biol Chem 2003;278:42403-42408.
  174. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331-345.
  175. Kocak I, Dundar M, Hekimgil M, Okyay P. As-sessment of germ cell apoptosis in cryptorchid rats. Asian J Androl 2002;4(3):183-186.
  176. Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999;4:38-47.
  177. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 1997;138(5): 2081-2088.
  178. Suda T, Takahashi T, Goldstein P , Nagata S. Mol-ecular cloning and expression of Fas ligand, a novel member of tumor necrosis factor family. Cell 1993;75(6):1169-1178.
  179. Rodriguez I, Ody C, Araki K, Garcia I, Vassali P. An early and massive wave of germinal cell ap-optosis is required for the development of functional spermatogenesis. EMBO J 1997;16(9): 2262-2270.
  180. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenic maturation in men: relationship among plasma membrane beat 1,4 galacto-syltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol Reprod 1997;56(4):1020-1024.
  181. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281(5381):1312-1316.
  182. Kim JM, Ghosh SR, Weil ACP, Zirkin BR. Cas-pase-3 and caspase-activated deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced intratesticular testosterone. Endocrinology 2001;142(9):3809-3816.
  183. Shafik A, Shafik AA, Shafik I, El Sibai O. Sperm DNA fragmentation. Arch Androl 2006;52:197-208.
  184. 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(3):519-524.
  185. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  186. Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma mem-brane integrity of human spermatozoa. Fertil Steril 2000;74(6):1200-1207.
  187. Aitken RJ, Krausz C. Oxidative stress, DNA dam-age and the Y chromosome. Reproduction 2001; 122:497-506.
  188. Spiropoulos J, Turnbull DM, Chinnery PF. Can mitochondrial DNA mutations cause sperm dys-function? Mol Hum Reprod 2002;8(8):719-721.
  189. Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 2004;6:139-148.
  190. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001;65(4):1102-1113.
  191. Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and signifi-cance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 2005;3:67-75.
  192. Balercia G, Moretti S, Vignini A, Magagnini M, Mantero F, Boscaro M, et al. Role of nitric oxide concentrations on human sperm motility. J Androl 2004;25(2):245-249.
  193. Agarwal A, Makker K, Sharma R. Clinical rele-vance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008;59:2-11.
  194. 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 struc-ture and TUNEL assays. Hum Reprod 2004;19 (10):2277-2282.
  195. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR, King A. A simple comet assay for arch-ived sperm correlates DNA fragmentation to re-duced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001;75(1):186-192.
  196. Hodjat M, Akhondi MA, Al-Hasani S, Mobaraki M, Sadeghi MR. Increased sperm ubiquitination correlates with abnormal chromatin integrity. Re-prod Biomed Online 2008;17(3):324-330.
  197. Hodjat M, Akhondi MA, Amirjanati N, Savadi Shirazi E, Sadeghi MR. The comparison of four different sperm chromatin assays and their correl-ation with semen parameters. TUMJ 2008;65 (Suppl 3):33-40.
  198. Hammadeh M, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 2001;46(2):99-104.
  199. Foresta C, Zorzi M, Rossato M, Varotto A. Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. lnt J Androl 1992;15(4):330-337.
  200. Erenpreiss A, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22(1):45-53.
  201. Ashok Agarwal, Tamer M Said. Sperm chromatin assessment. In: Gardner DK, Weismann A (eds). Textbook of Assisted Reproductive Techniques. United Kingdom: InForma Health Care; 2009,93-106.
  202. Manicardi G, Bianchi P, Pantano S, Azzoni P, D. Bizzaro D, Bianchi U. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessi-bility. Biol Reprod 1995;52:864-867.
  203. Esterhuizen A, Franken D, Laurens JGH, Prinsloo e, van Rooyen LH. Sperm chromatin packaging as an indicator of in vitro fertilization rates. Hum Reprod 2000;15(3):657-661.
  204. Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosál-vez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82(3-4):251-256.
  205. Fernandez J, Vazquez-Gundin F, Delgado A, Goy-anes J, Ramiro-Diaz J, de la Torre J, et al. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453(1):77-82.
  206. Gopalkrishnan K, Hurkadli K, Padwal V, Balaiah D. Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andralogia 1999;31(5):277-282.
  207. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  208. Variant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI derived embryos. J Assist Reprod Genet 2002;19(7):319-328.
  209. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluores-cence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79:670-676.
  210. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giweroman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495-2505.
  211. Singh N, Danner DB, Tice R, Mccoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  212. Fernandez J, Muriel L, Rivero MT, Goyanes V, Vazque R, Alvarez JG. The sperm chromatin dis-persion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24 (1):59-66.
  213. Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damages on indi-vidual cells. Biochem Biophys Res Commun 1984; 123(1):291-298.
  214. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(1):184-191.
  215. Singh NP, Danner DB, Tice RR, Mccoy MT, Col-lins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  216. Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995;336(2):123-131.
  217. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Huang T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  218. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm as-sessed by single cell gel electrophoresis (comet assay) and its relationship to fertilization. Hum Reprod 2002;17(4):990-998.
  219. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17(7):1856-1862.
  220. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single cell electro-phoresis. Exp Cell Res 1997;236(1):231-237.
  221. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  222. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 2009;18(6):761-768.
  223. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  224. Shen HM, Ong CN. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000;28(4):529-536.
  225. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased deoxyribonucleic acid dam-age in the spermatozoa of infertile male patients. Fertil Steril 1997;68(3):519-524.
  226. Loft S, Kold-Jensen T, Hjollund N, Giweroman A, Geyllemborg J, Ernst E, et al. Oxidative DNA damage in human sperm influences time to preg-nancy. Hum Reprod 2003;18(6):1265-1272.
  227. 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):1597-1605.
  228. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of sperm chro-matin structure assay as a diagnostic and prog-nostic tool in the human fertility clinic. Hum Reprod 1999;14(4):1039-1049.
  229. Duran EH, Morshedi M, Taylor S, Oehringer S. Sperm DNA quality predicts intrauterine insemina-tion, outcome: a prospective cohort study. Hum Reprod 2002;17(12):3122-3128.
  230. Host 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):559-563.
  231. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracyto-plasmic sperm injection. Fertil Steril 1998;69(3): 528-32.
  232. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602-607.
  233. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation de-creases the pregnancy rate in an assisted reproduct-ive technique. Hum Reprod 2003;18(5):1023-1028.
  234. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rates after intracytoplasmic sperm injec-tion with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10(1):148-152.
  235. Cummins JM, Jequire AM, Kan R. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol Reprod Dev 1994;37(3):345-362.
  236. Larson KL, DeJonge CJ, BarnesAM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy fol-lowing assisted reproductive techniques. Hum Reprod 2000;15(8):1717-1722.
  237. Edwards RG, Beard HK. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum Reprod 1999;14(1):1-6.
  238. Matsuda Y, Tobari I. Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes. Mutat Res 1989;210(1):35-47.
  239. Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  240. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999; 284(6): 696-704.
  241. Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod 1999;14(9): 2279-2285.
  242. Genesca A, Caballin MR, Miro R, Benet J, Germ JR, Egoscue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  243. 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(7): 1552-1561.
  244. Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol Hum Reprod 1997;3:47-54.
  245. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA dam-age before and after density gradient centrifuga-tion: implications for assisted conception. Hum Reprod 2001;16(10):2160-2165.
  246. Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up technique and DNA fragmentation. Hum Reprod 2001;16(9):1950-1953.
  247. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Verdoodt M, Englert Y. Increased spermatozoa motility after in vitro culture of testicular biopsies from obstructive azoospermic patients results in better post thaw recovery rate. Hum Reprod 2000;15(11):2371-2374.
  248. Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril 1999;72(5):809-813.
  249. Hughes CM, Lewis SEM, McKelvey-Martin VJ. Thompson, W. The effects of antioxidant supple-mentation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240-1247.
  250. 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(3): 227-234.
  251. 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(4):550-556.
  252. 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(5):870-875.
  253. Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, et al. Coenzyme Q10 treat-ment in infertile men with idiopathic asthenozoo-spermia: a placebo-controlled, double-blind ran-domized trial. Fertil Steril 2009;91(5):1785-1792.
  254. Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril 2002;77(3):491-498.
  255. Lenzi A, Lombardo F, Sgro P, Salacone P, Capon-ecchia L, Dondero F, et al. Use of carnitine ther-apy in selected cases of male factor infertility: a double-blind crossover trial. Fertil Steril 2003;79 (2):292-300.
  256. Schoor RA. Prostatitis and male infertility: evi-dence and links. Curr Urol Rep 2002;3(4):324-329.
  257. Lewis SEM, Agbaje I, Alvarez J. Sperm DNA tests as useful Adjuncts to semen analysis. Syst Biol Reprod Med 2008;(3)54:111-125.
  258. Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 1999;60(3):615-620.
  259. Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol 2003;518:85-98.
  260. Alvarez JG. DNA fragmentation in human sperm-atozoa: significance in the diagnosis and treatment of infertility. Minerva Ginecol 2003;55(3):233-239.
  261. Tomlinson MJ, White A, Barratt CLR, Bolton AE, Cooke ID. The removal of morphologically abnor-mal sperm forms by phagocytes: a positive role for seminal leukocytes. Hum Reprod 1992;7(4):517-522.
  262. Sigman M, Lopes L. The correlation between round cells and white blood cells in the semen. J Urol 1993;149(5):1338-1340.
  263. Cohen PE, Pollard JW. Cytokines and growth factors in reproduction. In: Bronson R, (eds). Re-productive Immunology. Cambridge, MA: Black-well Science; 1996,52-102.
  264. Reichart M, Kahane I, Bartoov B. In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted Urea-plasma urealyticum infection. Biol Reprod 2000; 63(4):1041-1048.
  265. Kunzle R, Mueller MD, Hanggi W, Birkahuser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril 2003;79(2):287-291.
  266. Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jef-feries TM. Sperm chromatin damage associated with male smoking. Mutat Res 1999;423(1-2):103-111.
  267. Comhaire FH, Mahmoud AM, Depuydt CE, Zalata AA, Christofe AB. Mechanism and effects of male genital tract infection on sperm quality and fertiliz-ing potential: the andrologist's view point. Hum Reprod Update 1999;5(5):393-398.
  268. Aitken RJ, Buckingham D, Brindle J, Gomez E, Baker G, Irvine, S. Analysis of sperm movement in relation to the oxidative stress created by leuko-cytes in washed sperm preparations and seminal plasma. Hum Reprod 1995;10(8):2061-2071.
  269. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res 1996;351(2):199-203.
  270. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Re-prod 2001;16(6):1191-1199.
  271. Donnelly ET, McClure N, Lewis SE. Cryopreser-vation of human semen and prepared sperm: ef-fects on motility parameters and DNA integrity. Fertil Steril 2001;76(5):892-900.
  272. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Hunag T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  273. Giwercman A, Petersen PM. Cancer and male infertility. Baillieres Best Pract Res Clin Endo-crinol Metab 2000;14:453-471.
  274. Richter MA., Haning RV, Jr Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 1984;41(2): 277-280.
  275. Revel A, Haimov-Kochman R, Porat A, Lewin A, Simon A, Laufer N, et al. In vitro fertilization-intracytoplasmic sperm injection success rates with cryopreserved sperm from patients with malignant disease. Fertil Steril 2005;84(1):118-122.
  276. Kobayashi H, Larson K, Sharma RK, Nelson DR, Evenson DP, Toma H, et al. DNA damage in cancer patients before treatment as measured by the sperm chromatin structure assay. Fertil Steril 2001;75(3):469-475.
  277. Chatterjee R, Haines GA., Perera DM, Goldstone A, Morris ID. Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukemia. Hum Reprod 2000;15(4): 762-766.
  278. Cai L, Hales BF, Robaire B. Induction of apoptosis on the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 1997;56(6): 1490-1497.
  279. Li H, Jian Y, Rajpurkar R, Dunbar, JC, Dhabu-wala CB. Cocaine induced apoptosis in rat testes. J Urol 1999;162(1):213-216.
  280. Ondrizek RR, Chan PJ, Patton WC, King A. An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid. Fertil Steril 1999;71(3):517-522.
  281. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev 2008;75(1):40-47.
  282. Aitken RJ, De Iulis GN. Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online 2007;14(6):727-733.
  283. Tesarik J, Mendoza TR, Mendoza C. Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment. Reprod Biomed Online 2006;12(6):715-721.
  284. Ozmen B, Koutlaki N, Youssry M, Diedrich K, Al Hasani S. DNA damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts, and safety. Reprod Biomed Online 2007;14(3): 384-395.
  285. Carrell DT, Emery BR, Hammoud S. The etiology of sperm protamine abnormalities and their poten-tial impact on the sperm epigenome. Int J Andro-logy 2008;31(6):537-545.
  286. Evenson DP, Larson KL, Jost LK. Sperm chro-matin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons. J Andrology 2002;23(1):25-43.
  287. Ward WS. Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod 1993;48(6):1193-1201.
  288. D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relation-ship to male fertility and embryonic survival. Animal Reprod Sci 2007;101(1-2):1-17.
  289. Balhorn R, Cosman M, Thornton K, Krishnan VV, Corzett M, Bench E, Kramer C, et al. Protamine mediated condensation of DNA in mammalian sperm. In: Gagnon C. The male gamete: from basic knowledge to clinical applications. Illinois: Cache River Press;1999,55-70.
  290. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4:31-37.
  291. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, et al. Influ-ence of deoxyribonucleic acid damage on fertil-ization and pregnancy. Fertil Steril 2004;81(4): 965-972.
  292. Lewis SEM, Aitken RJ. DNA damage to sperm-atozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322(1):33-41.
  293. Vogt PH. Molecular genetics of human male infer-tility: from genes to new therapeutic perspectives. Curr Pharm Design 2004;10(5):471-500.
  294. Carrell DT, Emery BR, Hammoud S. Altered protamine expression and diminished spermato-genesis: what is the link? Hum Reprod Update 2007;13(3):313-327.
  295. Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson M, Peterson CM, et al. Sperm protamine1/ protamine 2 ratios are related to in vitro fertil-ization pregnancy rates and predictive of fertile-ization ability. Fertil Steril 2006;86(5):1408-1415.
  296. Tseden K, Topaloglu O, Meinhardt A, Dev A, Adham I, Muller C, et al. Premature translation of transition protein 2 mRNA causes sperm abnor-malities and male infertility. Mol Reprod Dev 2007;74(3):273-279.
  297. Torregrosa N, Dominguez-Fandos D, Camejo MI, Shirley CR, Meistrich ML, Ballesca JL, et al. Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Human Reprod 2006;21(8):2084-2089.
  298. Dadoune JP. Expression of mammalian spermato-zoal nucleoproteins. Microsc Res Tech 2003;61 (1):56-75.
  299. Brewer L, Corzett M, Balhorn R. Condensation of DNA by spermatid basic nuclear proteins. J Biol Chem 2002;41:38895-38900.
  300. Brewer L, Corzett M, Lau EY, Balhorn R. Dy-namics of protamine 1 binding to single DNA molecules. J Biol Chem 2003;278:42403-42408.
  301. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331-345.
  302. Kocak I, Dundar M, Hekimgil M, Okyay P. As-sessment of germ cell apoptosis in cryptorchid rats. Asian J Androl 2002;4(3):183-186.
  303. Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999;4:38-47.
  304. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 1997;138(5): 2081-2088.
  305. Suda T, Takahashi T, Goldstein P , Nagata S. Mol-ecular cloning and expression of Fas ligand, a novel member of tumor necrosis factor family. Cell 1993;75(6):1169-1178.
  306. Rodriguez I, Ody C, Araki K, Garcia I, Vassali P. An early and massive wave of germinal cell ap-optosis is required for the development of functional spermatogenesis. EMBO J 1997;16(9): 2262-2270.
  307. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenic maturation in men: relationship among plasma membrane beat 1,4 galacto-syltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol Reprod 1997;56(4):1020-1024.
  308. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281(5381):1312-1316.
  309. Kim JM, Ghosh SR, Weil ACP, Zirkin BR. Cas-pase-3 and caspase-activated deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced intratesticular testosterone. Endocrinology 2001;142(9):3809-3816.
  310. Shafik A, Shafik AA, Shafik I, El Sibai O. Sperm DNA fragmentation. Arch Androl 2006;52:197-208.
  311. 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(3):519-524.
  312. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  313. Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma mem-brane integrity of human spermatozoa. Fertil Steril 2000;74(6):1200-1207.
  314. Aitken RJ, Krausz C. Oxidative stress, DNA dam-age and the Y chromosome. Reproduction 2001; 122:497-506.
  315. Spiropoulos J, Turnbull DM, Chinnery PF. Can mitochondrial DNA mutations cause sperm dys-function? Mol Hum Reprod 2002;8(8):719-721.
  316. Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 2004;6:139-148.
  317. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001;65(4):1102-1113.
  318. Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and signifi-cance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 2005;3:67-75.
  319. Balercia G, Moretti S, Vignini A, Magagnini M, Mantero F, Boscaro M, et al. Role of nitric oxide concentrations on human sperm motility. J Androl 2004;25(2):245-249.
  320. Agarwal A, Makker K, Sharma R. Clinical rele-vance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008;59:2-11.
  321. 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 struc-ture and TUNEL assays. Hum Reprod 2004;19 (10):2277-2282.
  322. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR, King A. A simple comet assay for arch-ived sperm correlates DNA fragmentation to re-duced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001;75(1):186-192.
  323. Hodjat M, Akhondi MA, Al-Hasani S, Mobaraki M, Sadeghi MR. Increased sperm ubiquitination correlates with abnormal chromatin integrity. Re-prod Biomed Online 2008;17(3):324-330.
  324. Hodjat M, Akhondi MA, Amirjanati N, Savadi Shirazi E, Sadeghi MR. The comparison of four different sperm chromatin assays and their correl-ation with semen parameters. TUMJ 2008;65 (Suppl 3):33-40.
  325. Hammadeh M, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 2001;46(2):99-104.
  326. Foresta C, Zorzi M, Rossato M, Varotto A. Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. lnt J Androl 1992;15(4):330-337.
  327. Erenpreiss A, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22(1):45-53.
  328. Ashok Agarwal, Tamer M Said. Sperm chromatin assessment. In: Gardner DK, Weismann A (eds). Textbook of Assisted Reproductive Techniques. United Kingdom: InForma Health Care; 2009,93-106.
  329. Manicardi G, Bianchi P, Pantano S, Azzoni P, D. Bizzaro D, Bianchi U. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessi-bility. Biol Reprod 1995;52:864-867.
  330. Esterhuizen A, Franken D, Laurens JGH, Prinsloo e, van Rooyen LH. Sperm chromatin packaging as an indicator of in vitro fertilization rates. Hum Reprod 2000;15(3):657-661.
  331. Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosál-vez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82(3-4):251-256.
  332. Fernandez J, Vazquez-Gundin F, Delgado A, Goy-anes J, Ramiro-Diaz J, de la Torre J, et al. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453(1):77-82.
  333. Gopalkrishnan K, Hurkadli K, Padwal V, Balaiah D. Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andralogia 1999;31(5):277-282.
  334. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  335. Variant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI derived embryos. J Assist Reprod Genet 2002;19(7):319-328.
  336. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluores-cence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79:670-676.
  337. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giweroman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495-2505.
  338. Singh N, Danner DB, Tice R, Mccoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  339. Fernandez J, Muriel L, Rivero MT, Goyanes V, Vazque R, Alvarez JG. The sperm chromatin dis-persion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24 (1):59-66.
  340. Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damages on indi-vidual cells. Biochem Biophys Res Commun 1984; 123(1):291-298.
  341. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(1):184-191.
  342. Singh NP, Danner DB, Tice RR, Mccoy MT, Col-lins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  343. Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995;336(2):123-131.
  344. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Huang T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  345. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm as-sessed by single cell gel electrophoresis (comet assay) and its relationship to fertilization. Hum Reprod 2002;17(4):990-998.
  346. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17(7):1856-1862.
  347. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single cell electro-phoresis. Exp Cell Res 1997;236(1):231-237.
  348. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  349. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 2009;18(6):761-768.
  350. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  351. Shen HM, Ong CN. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000;28(4):529-536.
  352. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased deoxyribonucleic acid dam-age in the spermatozoa of infertile male patients. Fertil Steril 1997;68(3):519-524.
  353. Loft S, Kold-Jensen T, Hjollund N, Giweroman A, Geyllemborg J, Ernst E, et al. Oxidative DNA damage in human sperm influences time to preg-nancy. Hum Reprod 2003;18(6):1265-1272.
  354. 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):1597-1605.
  355. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of sperm chro-matin structure assay as a diagnostic and prog-nostic tool in the human fertility clinic. Hum Reprod 1999;14(4):1039-1049.
  356. Duran EH, Morshedi M, Taylor S, Oehringer S. Sperm DNA quality predicts intrauterine insemina-tion, outcome: a prospective cohort study. Hum Reprod 2002;17(12):3122-3128.
  357. Host 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):559-563.
  358. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracyto-plasmic sperm injection. Fertil Steril 1998;69(3): 528-32.
  359. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602-607.
  360. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation de-creases the pregnancy rate in an assisted reproduct-ive technique. Hum Reprod 2003;18(5):1023-1028.
  361. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rates after intracytoplasmic sperm injec-tion with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10(1):148-152.
  362. Cummins JM, Jequire AM, Kan R. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol Reprod Dev 1994;37(3):345-362.
  363. Larson KL, DeJonge CJ, BarnesAM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy fol-lowing assisted reproductive techniques. Hum Reprod 2000;15(8):1717-1722.
  364. Edwards RG, Beard HK. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum Reprod 1999;14(1):1-6.
  365. Matsuda Y, Tobari I. Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes. Mutat Res 1989;210(1):35-47.
  366. Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  367. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999; 284(6): 696-704.
  368. Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod 1999;14(9): 2279-2285.
  369. Genesca A, Caballin MR, Miro R, Benet J, Germ JR, Egoscue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  370. 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(7): 1552-1561.
  371. Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol Hum Reprod 1997;3:47-54.
  372. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA dam-age before and after density gradient centrifuga-tion: implications for assisted conception. Hum Reprod 2001;16(10):2160-2165.
  373. Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up technique and DNA fragmentation. Hum Reprod 2001;16(9):1950-1953.
  374. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Verdoodt M, Englert Y. Increased spermatozoa motility after in vitro culture of testicular biopsies from obstructive azoospermic patients results in better post thaw recovery rate. Hum Reprod 2000;15(11):2371-2374.
  375. Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril 1999;72(5):809-813.
  376. Hughes CM, Lewis SEM, McKelvey-Martin VJ. Thompson, W. The effects of antioxidant supple-mentation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240-1247.
  377. 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(3): 227-234.
  378. 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(4):550-556.
  379. 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(5):870-875.
  380. Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, et al. Coenzyme Q10 treat-ment in infertile men with idiopathic asthenozoo-spermia: a placebo-controlled, double-blind ran-domized trial. Fertil Steril 2009;91(5):1785-1792.
  381. Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril 2002;77(3):491-498.
  382. Lenzi A, Lombardo F, Sgro P, Salacone P, Capon-ecchia L, Dondero F, et al. Use of carnitine ther-apy in selected cases of male factor infertility: a double-blind crossover trial. Fertil Steril 2003;79 (2):292-300.
  383. Schoor RA. Prostatitis and male infertility: evi-dence and links. Curr Urol Rep 2002;3(4):324-329.
  384. Lewis SEM, Agbaje I, Alvarez J. Sperm DNA tests as useful Adjuncts to semen analysis. Syst Biol Reprod Med 2008;(3)54:111-125.
  385. Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 1999;60(3):615-620.
  386. Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol 2003;518:85-98.
  387. Alvarez JG. DNA fragmentation in human sperm-atozoa: significance in the diagnosis and treatment of infertility. Minerva Ginecol 2003;55(3):233-239.
  388. Tomlinson MJ, White A, Barratt CLR, Bolton AE, Cooke ID. The removal of morphologically abnor-mal sperm forms by phagocytes: a positive role for seminal leukocytes. Hum Reprod 1992;7(4):517-522.
  389. Sigman M, Lopes L. The correlation between round cells and white blood cells in the semen. J Urol 1993;149(5):1338-1340.
  390. Cohen PE, Pollard JW. Cytokines and growth factors in reproduction. In: Bronson R, (eds). Re-productive Immunology. Cambridge, MA: Black-well Science; 1996,52-102.
  391. Reichart M, Kahane I, Bartoov B. In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted Urea-plasma urealyticum infection. Biol Reprod 2000; 63(4):1041-1048.
  392. Kunzle R, Mueller MD, Hanggi W, Birkahuser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril 2003;79(2):287-291.
  393. Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jef-feries TM. Sperm chromatin damage associated with male smoking. Mutat Res 1999;423(1-2):103-111.
  394. Comhaire FH, Mahmoud AM, Depuydt CE, Zalata AA, Christofe AB. Mechanism and effects of male genital tract infection on sperm quality and fertiliz-ing potential: the andrologist's view point. Hum Reprod Update 1999;5(5):393-398.
  395. Aitken RJ, Buckingham D, Brindle J, Gomez E, Baker G, Irvine, S. Analysis of sperm movement in relation to the oxidative stress created by leuko-cytes in washed sperm preparations and seminal plasma. Hum Reprod 1995;10(8):2061-2071.
  396. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res 1996;351(2):199-203.
  397. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Re-prod 2001;16(6):1191-1199.
  398. Donnelly ET, McClure N, Lewis SE. Cryopreser-vation of human semen and prepared sperm: ef-fects on motility parameters and DNA integrity. Fertil Steril 2001;76(5):892-900.
  399. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Hunag T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  400. Giwercman A, Petersen PM. Cancer and male infertility. Baillieres Best Pract Res Clin Endo-crinol Metab 2000;14:453-471.
  401. Richter MA., Haning RV, Jr Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 1984;41(2): 277-280.
  402. Revel A, Haimov-Kochman R, Porat A, Lewin A, Simon A, Laufer N, et al. In vitro fertilization-intracytoplasmic sperm injection success rates with cryopreserved sperm from patients with malignant disease. Fertil Steril 2005;84(1):118-122.
  403. Kobayashi H, Larson K, Sharma RK, Nelson DR, Evenson DP, Toma H, et al. DNA damage in cancer patients before treatment as measured by the sperm chromatin structure assay. Fertil Steril 2001;75(3):469-475.
  404. Chatterjee R, Haines GA., Perera DM, Goldstone A, Morris ID. Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukemia. Hum Reprod 2000;15(4): 762-766.
  405. Cai L, Hales BF, Robaire B. Induction of apoptosis on the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 1997;56(6): 1490-1497.
  406. Li H, Jian Y, Rajpurkar R, Dunbar, JC, Dhabu-wala CB. Cocaine induced apoptosis in rat testes. J Urol 1999;162(1):213-216.
  407. Ondrizek RR, Chan PJ, Patton WC, King A. An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid. Fertil Steril 1999;71(3):517-522.
  408. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev 2008;75(1):40-47.
  409. Aitken RJ, De Iulis GN. Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online 2007;14(6):727-733.
  410. Tesarik J, Mendoza TR, Mendoza C. Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment. Reprod Biomed Online 2006;12(6):715-721.
  411. Ozmen B, Koutlaki N, Youssry M, Diedrich K, Al Hasani S. DNA damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts, and safety. Reprod Biomed Online 2007;14(3): 384-395.
  412. Carrell DT, Emery BR, Hammoud S. The etiology of sperm protamine abnormalities and their poten-tial impact on the sperm epigenome. Int J Andro-logy 2008;31(6):537-545.
  413. Evenson DP, Larson KL, Jost LK. Sperm chro-matin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons. J Andrology 2002;23(1):25-43.
  414. Ward WS. Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod 1993;48(6):1193-1201.
  415. D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relation-ship to male fertility and embryonic survival. Animal Reprod Sci 2007;101(1-2):1-17.
  416. Balhorn R, Cosman M, Thornton K, Krishnan VV, Corzett M, Bench E, Kramer C, et al. Protamine mediated condensation of DNA in mammalian sperm. In: Gagnon C. The male gamete: from basic knowledge to clinical applications. Illinois: Cache River Press;1999,55-70.
  417. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4:31-37.
  418. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, et al. Influ-ence of deoxyribonucleic acid damage on fertil-ization and pregnancy. Fertil Steril 2004;81(4): 965-972.
  419. Lewis SEM, Aitken RJ. DNA damage to sperm-atozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322(1):33-41.
  420. Vogt PH. Molecular genetics of human male infer-tility: from genes to new therapeutic perspectives. Curr Pharm Design 2004;10(5):471-500.
  421. Carrell DT, Emery BR, Hammoud S. Altered protamine expression and diminished spermato-genesis: what is the link? Hum Reprod Update 2007;13(3):313-327.
  422. Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson M, Peterson CM, et al. Sperm protamine1/ protamine 2 ratios are related to in vitro fertil-ization pregnancy rates and predictive of fertile-ization ability. Fertil Steril 2006;86(5):1408-1415.
  423. Tseden K, Topaloglu O, Meinhardt A, Dev A, Adham I, Muller C, et al. Premature translation of transition protein 2 mRNA causes sperm abnor-malities and male infertility. Mol Reprod Dev 2007;74(3):273-279.
  424. Torregrosa N, Dominguez-Fandos D, Camejo MI, Shirley CR, Meistrich ML, Ballesca JL, et al. Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Human Reprod 2006;21(8):2084-2089.
  425. Dadoune JP. Expression of mammalian spermato-zoal nucleoproteins. Microsc Res Tech 2003;61 (1):56-75.
  426. Brewer L, Corzett M, Balhorn R. Condensation of DNA by spermatid basic nuclear proteins. J Biol Chem 2002;41:38895-38900.
  427. Brewer L, Corzett M, Lau EY, Balhorn R. Dy-namics of protamine 1 binding to single DNA molecules. J Biol Chem 2003;278:42403-42408.
  428. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331-345.
  429. Kocak I, Dundar M, Hekimgil M, Okyay P. As-sessment of germ cell apoptosis in cryptorchid rats. Asian J Androl 2002;4(3):183-186.
  430. Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999;4:38-47.
  431. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 1997;138(5): 2081-2088.
  432. Suda T, Takahashi T, Goldstein P , Nagata S. Mol-ecular cloning and expression of Fas ligand, a novel member of tumor necrosis factor family. Cell 1993;75(6):1169-1178.
  433. Rodriguez I, Ody C, Araki K, Garcia I, Vassali P. An early and massive wave of germinal cell ap-optosis is required for the development of functional spermatogenesis. EMBO J 1997;16(9): 2262-2270.
  434. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenic maturation in men: relationship among plasma membrane beat 1,4 galacto-syltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol Reprod 1997;56(4):1020-1024.
  435. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281(5381):1312-1316.
  436. Kim JM, Ghosh SR, Weil ACP, Zirkin BR. Cas-pase-3 and caspase-activated deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced intratesticular testosterone. Endocrinology 2001;142(9):3809-3816.
  437. Shafik A, Shafik AA, Shafik I, El Sibai O. Sperm DNA fragmentation. Arch Androl 2006;52:197-208.
  438. 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(3):519-524.
  439. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  440. Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma mem-brane integrity of human spermatozoa. Fertil Steril 2000;74(6):1200-1207.
  441. Aitken RJ, Krausz C. Oxidative stress, DNA dam-age and the Y chromosome. Reproduction 2001; 122:497-506.
  442. Spiropoulos J, Turnbull DM, Chinnery PF. Can mitochondrial DNA mutations cause sperm dys-function? Mol Hum Reprod 2002;8(8):719-721.
  443. Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 2004;6:139-148.
  444. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001;65(4):1102-1113.
  445. Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and signifi-cance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 2005;3:67-75.
  446. Balercia G, Moretti S, Vignini A, Magagnini M, Mantero F, Boscaro M, et al. Role of nitric oxide concentrations on human sperm motility. J Androl 2004;25(2):245-249.
  447. Agarwal A, Makker K, Sharma R. Clinical rele-vance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 2008;59:2-11.
  448. 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 struc-ture and TUNEL assays. Hum Reprod 2004;19 (10):2277-2282.
  449. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR, King A. A simple comet assay for arch-ived sperm correlates DNA fragmentation to re-duced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001;75(1):186-192.
  450. Hodjat M, Akhondi MA, Al-Hasani S, Mobaraki M, Sadeghi MR. Increased sperm ubiquitination correlates with abnormal chromatin integrity. Re-prod Biomed Online 2008;17(3):324-330.
  451. Hodjat M, Akhondi MA, Amirjanati N, Savadi Shirazi E, Sadeghi MR. The comparison of four different sperm chromatin assays and their correl-ation with semen parameters. TUMJ 2008;65 (Suppl 3):33-40.
  452. Hammadeh M, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 2001;46(2):99-104.
  453. Foresta C, Zorzi M, Rossato M, Varotto A. Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. lnt J Androl 1992;15(4):330-337.
  454. Erenpreiss A, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22(1):45-53.
  455. Ashok Agarwal, Tamer M Said. Sperm chromatin assessment. In: Gardner DK, Weismann A (eds). Textbook of Assisted Reproductive Techniques. United Kingdom: InForma Health Care; 2009,93-106.
  456. Manicardi G, Bianchi P, Pantano S, Azzoni P, D. Bizzaro D, Bianchi U. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessi-bility. Biol Reprod 1995;52:864-867.
  457. Esterhuizen A, Franken D, Laurens JGH, Prinsloo e, van Rooyen LH. Sperm chromatin packaging as an indicator of in vitro fertilization rates. Hum Reprod 2000;15(3):657-661.
  458. Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosál-vez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82(3-4):251-256.
  459. Fernandez J, Vazquez-Gundin F, Delgado A, Goy-anes J, Ramiro-Diaz J, de la Torre J, et al. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453(1):77-82.
  460. Gopalkrishnan K, Hurkadli K, Padwal V, Balaiah D. Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andralogia 1999;31(5):277-282.
  461. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  462. Variant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI derived embryos. J Assist Reprod Genet 2002;19(7):319-328.
  463. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluores-cence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79:670-676.
  464. Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giweroman A, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998;13:2495-2505.
  465. Singh N, Danner DB, Tice R, Mccoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  466. Fernandez J, Muriel L, Rivero MT, Goyanes V, Vazque R, Alvarez JG. The sperm chromatin dis-persion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24 (1):59-66.
  467. Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damages on indi-vidual cells. Biochem Biophys Res Commun 1984; 123(1):291-298.
  468. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(1):184-191.
  469. Singh NP, Danner DB, Tice RR, Mccoy MT, Col-lins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184(2):461-470.
  470. Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995;336(2):123-131.
  471. Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Huang T, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17(5):1274-1280.
  472. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm as-sessed by single cell gel electrophoresis (comet assay) and its relationship to fertilization. Hum Reprod 2002;17(4):990-998.
  473. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17(7):1856-1862.
  474. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single cell electro-phoresis. Exp Cell Res 1997;236(1):231-237.
  475. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans-ferase and nick translation assays. Cancer Res 1993;53:1945-1951.
  476. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 2009;18(6):761-768.
  477. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane trans-location of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15(6): 1338-1344.
  478. Shen HM, Ong CN. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000;28(4):529-536.
  479. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased deoxyribonucleic acid dam-age in the spermatozoa of infertile male patients. Fertil Steril 1997;68(3):519-524.
  480. Loft S, Kold-Jensen T, Hjollund N, Giweroman A, Geyllemborg J, Ernst E, et al. Oxidative DNA damage in human sperm influences time to preg-nancy. Hum Reprod 2003;18(6):1265-1272.
  481. 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):1597-1605.
  482. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of sperm chro-matin structure assay as a diagnostic and prog-nostic tool in the human fertility clinic. Hum Reprod 1999;14(4):1039-1049.
  483. Duran EH, Morshedi M, Taylor S, Oehringer S. Sperm DNA quality predicts intrauterine insemina-tion, outcome: a prospective cohort study. Hum Reprod 2002;17(12):3122-3128.
  484. Host 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):559-563.
  485. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracyto-plasmic sperm injection. Fertil Steril 1998;69(3): 528-32.
  486. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602-607.
  487. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation de-creases the pregnancy rate in an assisted reproduct-ive technique. Hum Reprod 2003;18(5):1023-1028.
  488. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rates after intracytoplasmic sperm injec-tion with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10(1):148-152.
  489. Cummins JM, Jequire AM, Kan R. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol Reprod Dev 1994;37(3):345-362.
  490. Larson KL, DeJonge CJ, BarnesAM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy fol-lowing assisted reproductive techniques. Hum Reprod 2000;15(8):1717-1722.
  491. Edwards RG, Beard HK. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum Reprod 1999;14(1):1-6.
  492. Matsuda Y, Tobari I. Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes. Mutat Res 1989;210(1):35-47.
  493. Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  494. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999; 284(6): 696-704.
  495. Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod 1999;14(9): 2279-2285.
  496. Genesca A, Caballin MR, Miro R, Benet J, Germ JR, Egoscue J. Repair of human sperm chromo-some aberrations in the hamster egg. Hum Genet 1992;89(2):181-186.
  497. 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(7): 1552-1561.
  498. Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol Hum Reprod 1997;3:47-54.
  499. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA dam-age before and after density gradient centrifuga-tion: implications for assisted conception. Hum Reprod 2001;16(10):2160-2165.
  500. Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up technique and DNA fragmentation. Hum Reprod 2001;16(9):1950-1953.
  501. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Verdoodt M, Englert Y. Increased spermatozoa motility after in vitro culture of testicular biopsies from obstructive azoospermic patients results in better post thaw recovery rate. Hum Reprod 2000;15(11):2371-2374.
  502. Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril 1999;72(5):809-813.
  503. Hughes CM, Lewis SEM, McKelvey-Martin VJ. Thompson, W. The effects of antioxidant supple-mentation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240-1247.
  504. 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(3): 227-234.
  505. 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(4):550-556.
  506. 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(5):870-875.
  507. Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, et al. Coenzyme Q10 treat-ment in infertile men with idiopathic asthenozoo-spermia: a placebo-controlled, double-blind ran-domized trial. Fertil Steril 2009;91(5):1785-1792.
  508. Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril 2002;77(3):491-498.
  509. Lenzi A, Lombardo F, Sgro P, Salacone P, Capon-ecchia L, Dondero F, et al. Use of carnitine ther-apy in selected cases of male factor infertility: a double-blind crossover trial. Fertil Steril 2003;79 (2):292-300.
  510. Patel SR, Sigman M. Antioxidant therapy in male infertility. Urol Clin North Am 2008;35(2):319-330.