Fertility and Sterility
Volume 92, Issue 2 , Pages 819-827 , August 2009

Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa

  • Reda Mahfouz, M.D.

      Affiliations

    • Center for Reproductive Medicine, Glickman Urological and Kidney Institute, and Ob-Gyn and Woman's Health Institute, Cleveland Clinic, Cleveland, Ohio
  • ,
  • Rakesh Sharma, Ph.D.

      Affiliations

    • Center for Reproductive Medicine, Glickman Urological and Kidney Institute, and Ob-Gyn and Woman's Health Institute, Cleveland Clinic, Cleveland, Ohio
  • ,
  • Jakob Lackner, M.D.

      Affiliations

    • Department of Urology, Medical University of Vienna, Austria
  • ,
  • Nabil Aziz, M.D.

      Affiliations

    • Liverpool Women's Hospital, Liverpool, United Kingdom
  • ,
  • Ashok Agarwal, Ph.D., H.C.L.D.

      Affiliations

    • Center for Reproductive Medicine, Glickman Urological and Kidney Institute, and Ob-Gyn and Woman's Health Institute, Cleveland Clinic, Cleveland, Ohio
    • Corresponding Author InformationReprint requests: Ashok Agarwal, Ph.D., H.C.L.D., Professor and Director, Center for Reproductive Medicine, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1 Cleveland, Ohio 44195 (FAX: 216-636-3118).

Received 25 March 2008 ,Revised 12 May 2008 ,Accepted 27 May 2008.

References 

  1. Agarwal A, Gupta S, Sikka S. The role of free radicals and antioxidants in reproduction. Current Opin Obstet Gynecol. 2006;18:325–332
  2. Aitken RJ. Sperm function tests and fertility. Int J Androl. 2006;29:69–75discussion, 105–8
  3. Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003;79:829–843
  4. Agarwal A, Prabakaran S, Allamaneni S. What an andrologist/urologist should know about free radicals and why. Urology. 2006;67:2–8
  5. Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction (Camb). 2001;122:497–506
  6. de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod. 1995;10(Suppl 1):15–21
  7. Saleh RA, Agarwal A, Kandirali E, Sharma RK, Thomas AJ, Nada EA, et al. Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa. Fertil Steril. 2002;78:1215–1224
  8. Alvarez JG, Sharma RK, Ollero M, Saleh RA, Lopez MC, Thomas AJ, et al. Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay. Fertil Steril. 2002;78:319–329
  9. Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril. 2002;78:313–318
  10. Erenpreiss J, Hlevicka S, Zalkalns J, Erenpreisa J. Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples. J Androl. 2002;23:717–723
  11. Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod. 2002;17:1257–1265
  12. Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. J Androl. 2001;22:575–583
  13. Aitken RJ, De Iuliis GN. Origins and consequences of DNA damage in male germ cells. Reprod Biomed Online. 2007;14:727–733
  14. Agarwal A, Allamaneni SS, Said TM. Chemiluminescence technique for measuring reactive oxygen species. Reprod Biomed Online. 2004;9:466–468
  15. Kobayashi H, Gil-Guzman E, Mahran AM, Sharma R, Nelson DR, Thomas AJ, et al. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Androl. 2001;22:568–574
  16. Aitken RJ, Baker MA, O'Bryan M. Shedding light on chemiluminescence: the application of chemiluminescence in diagnostic andrology. J Androl. 2004;25:455–465
  17. Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM, Srougi M, et al. Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J Androl. 2007;28:613–620
  18. Shai S, Roudebush W, Powers D, Dirnfeld M, Lamb DJ. A multicenter study evaluating the flow cytometric-based kit for semen analysis. Fertil Steril. 2005;83:1034–1038
  19. Guthrie HD, Welch GR. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci. 2006;84:2089–2100
  20. Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S, et al. A new multiparameter flow cytometric method for human semen analysis. Hum Reprod. 2007;22:485–494
  21. Soh N. Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal Bioanal Chem. 2006;386:532–543
  22. Fridovich I. Editorial commentary on “Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide” by H. Zhao, et al. Free Rad Biol Med. 2003;34:1357–1358
  23. Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Rad Biol Med. 2003;34:1359–1368
  24. Bonini MG, Rota C, Tomasi A, Mason RP. The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species: a self-fulfilling prophesy?. Free Rad Biol Med. 2006;40:968–975
  25. World Health Organization WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed.. Cambridge: Cambridge University Press; 1999;
  26. Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16:1912–1921
  27. In:  Team RDC editors. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2006;
  28. Eustache F, Jouannet P, Auger J. Evaluation of flow cytometric methods to measure human sperm concentration. J Androl. 2001;22:558–567
  29. Rinaldi M, Moroni P, Paape MJ, Bannerman DD. Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species. Vet Immunol Immunopathol. 2007;115:107–125
  30. Robinson JP, Bruner LH, Bassoe CF, Hudson JL, Ward PA, Phan SH. Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism. J Leuk Biol. 1988;43:304–310
  31. De Iuliis GN, Wingate JK, Koppers AJ, McLaughlin EA, Aitken RJ. Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa. J Clin Endocrinol Metab. 2006;91:1968–1975
  32. Agarwal A, Saleh R. Role of oxidants in male infertility: rationale, significance, and treatment. Urol Clin N Am. 2002;29:817–827
  33. Aitken RJ, Buckingham DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol. 1992;151:466–477
  34. Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl. 1997;20:61–69
  35. Saleh R, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl. 2002;23:737–752
  36. Sikka S. Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl. 2004;25:5–18
  37. Lackner J, Schatzl G, Waldhor T, Resch K, Kratzik C, Marberger M. Constant decline in sperm concentration in infertile males in an urban population: experience over 18 years. Fertil Steril. 2005;84:1657–1661
  38. Keel BA. How reliable are results from the semen analysis?. Fertil Steril. 2004;82:41–44
  39. Kobayashi H, Gil-Guzman E, Mahran A, Rakesh R, Nelson D, Thomas AJ, et al. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Androl. 2001;22:568–574
  40. Peris SI, Bilodeau JF, Dufour M, Bailey JL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol Reprod Devel. 2007;74:878–892

 R.M. has nothing to disclose. R.S. has nothing to disclose. J.L. has nothing to disclose. N.A. has nothing to disclose. A.A. has nothing to disclose.

 Presented at the 63rd Annual Meeting of the American Society of Reproductive Medicine, which was held in Washington, D.C., on October 13–19, 2007.

 Supported by a research grant from the Research Programs Committee, Cleveland Clinic (RPC no. 07549).

PII: S0015-0282(08)01246-6

doi: 10.1016/j.fertnstert.2008.05.087

Fertility and Sterility
Volume 92, Issue 2 , Pages 819-827 , August 2009