Diagnostic application of total antioxidant capacity in seminal plasma to assess oxidative stress in male factor infertility

  • Shubhadeep Roychoudhury
  • Rakesh Sharma
  • Suresh Sikka
  • Ashok AgarwalEmail author
Reproductive Physiology and Disease



This study was undertaken in order to establish a new reference value for the total antioxidant capacity (TAC) in seminal plasma as a predictor of fertility. This study also aims to propose a detailed protocol for the TAC assay including calculation of assay results and assessment of sensitivity and specificity over possible cutoff values in infertile men and controls with proven and unproven fertility.


Seminal plasma from 279 infertile patients and 46 normal healthy men referred to a male infertility testing laboratory were tested to measure TAC by a colorimetric assay kit. Receiver operating characteristics (ROC) curves were generated to establish cutoff values, sensitivity, and specificity, and the distribution of cutoff values in controls and infertile patients was calculated.


Infertile patients showed significantly lower levels (mean ± SEM) of total antioxidants (micromolar Trolox equivalents) in their seminal plasma (1863.84 ± 27.16 μM) compared to those from fertile men (2013 ± 56.04 μM, P = 0.019). A preferred cutoff TAC value of 1947 μM could facilitate better diagnosis of oxidative stress (OS) in men with male factor infertility. At this threshold, the specificity of TAC assay was 63.0 % and the sensitivity 59.5 % with a positive predictive value of 90.7 % and a negative predictive value of 20.4 %.


Our results establish a new diagnostic cutoff TAC value of 1947 μM in seminal plasma to distinguish prevalence of OS in infertile patients compared to healthy men. This study provides a robust reference value of seminal plasma TAC that may provide an important diagnostic tool to the physicians for managing OS and male factor infertility in such patients.


Male infertility Oxidative stress Seminal plasma Total antioxidant capacity Diagnostic value Specificity and sensitivity 



The study was supported by funds from the American Center for Reproductive Medicine. Dr. Shubhadeep Roychoudhury was supported by a fellowship from the Department of Biotechnology, Government of India. The authors are grateful to the Andrology Center technologists for scheduling the study subjects and Jeff Hammel, senior biostatistician, for his contribution to data analysis.


  1. 1.
    Gangel EK. AUA and ASRM produce recommendations for male infertility. Am Fam Physician. 2002;65(12):2589–90.PubMedGoogle Scholar
  2. 2.
    Tremellen K. Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update. 2008;14(3):243–58.CrossRefPubMedGoogle Scholar
  3. 3.
    Chen H, Zhao HX, Huang XF, Chen GW, Yang ZX, Sun WJ, et al. Does high load of oxidants in human semen contribute to male factor infertility? Antioxid Redox Signal. 2012;16(8):754–59.CrossRefPubMedGoogle Scholar
  4. 4.
    Aitken RJ, De Iuliis GN, Finnie JM, Hedges A, McLachlan RI. Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria. Hum Reprod. 2010;25(10):2415–26.CrossRefPubMedGoogle Scholar
  5. 5.
    Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. Adv Exp Med Biol. 2008;636:154–71.CrossRefPubMedGoogle Scholar
  6. 6.
    Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril. 1992;57(2):409–16.CrossRefPubMedGoogle Scholar
  7. 7.
    Khosrowbeygi A, Zarghami N. Levels of oxidative stress biomarkers in seminal plasma and their relationship with seminal parameters. BMC Clin Pathol. 2007;7:6.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    du Plessis SS, Makker K, Desai NR, Agarwal A. Impact of oxidative stress on IVF. Expet Rev Obstet Gynecol. 2008;3(4):539–54.CrossRefGoogle Scholar
  9. 9.
    Agarwal A, Allamaneni SS, Said TM. Chemiluminescence technique for measuring reactive oxygen species. Reprod Biomed Online. 2004;9(4):466–68.CrossRefPubMedGoogle Scholar
  10. 10.
    de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radic Biol Med. 1993;14(2):157–66.CrossRefPubMedGoogle Scholar
  11. 11.
    Rengan AK, Agarwal A, van der Linde M, du Plessis SS. An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet. Reprod Biol Endocrinol. 2012;10:92.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kessopoulou E, Tomlinson MJ, Barratt CL, Bolton AE, Cooke ID. Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J Reprod Fertil. 1992;94(2):463–70.CrossRefPubMedGoogle Scholar
  13. 13.
    Whittington K, Ford WC. Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl. 1999;22(4):229–35.CrossRefPubMedGoogle Scholar
  14. 14.
    Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989;41(1):183–97.CrossRefPubMedGoogle Scholar
  15. 15.
    Aitken RJ. The Amoroso Lecture. The human spermatozoon—a cell in crisis? J Reprod Fertil. 1999;115(1):1–7.CrossRefPubMedGoogle Scholar
  16. 16.
    Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update. 2001;7(2):175–89.CrossRefPubMedGoogle Scholar
  17. 17.
    Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod Biomed Online. 2004;8(6):616–27.CrossRefPubMedGoogle Scholar
  18. 18.
    Sikka SC, Rajasekaran M, Hellstrom WJ. Role of oxidative stress and antioxidants in male infertility. J Androl. 1995;16(6):464–68.PubMedGoogle Scholar
  19. 19.
    Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996;48(6):835–50.CrossRefPubMedGoogle Scholar
  20. 20.
    Sikka SC. Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 2001;8(7):851–62.CrossRefPubMedGoogle Scholar
  21. 21.
    Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res. 2005;322(1):33–41.CrossRefPubMedGoogle Scholar
  22. 22.
    Pasqualotto FF, Sharma RK, Potts JM, Nelson DR, Thomas AJ, Agarwal A. Seminal oxidative stress in patients with chronic prostatitis. Urology. 2000;55(6):881–85.CrossRefPubMedGoogle Scholar
  23. 23.
    Shiraishi K, Matsuyama H, Takihara H. Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology. Int J Urol. 2012;19(6):538–50.CrossRefPubMedGoogle Scholar
  24. 24.
    Doshi SB, Khullar K, Sharma RK, Agarwal A. Role of reactive nitrogen species in male infertility. Reprod Biol Endocrinol. 2012;10:109.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Agarwal A, Ahmad G, Sharma R. Reference values of reactive oxygen species in seminal ejaculates using chemiluminescence assay. J Assist Reprod Genet. 2015;32(12):1721–29.CrossRefPubMedGoogle Scholar
  26. 26.
    Homa ST, Vessey W, Perez-Miranda A, Riyait T, Agarwal A. Reactive oxygen species (ROS) in human semen: determination of a reference range. J Assist Reprod Genet. 2015;32(5):757–64.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    WHO: World Health Organization laboratory manual for the examination and processing of human semen. 5th edition. Geneva, Switzerland, 2010.Google Scholar
  28. 28.
    Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl. 1987;8(5):338–48.CrossRefPubMedGoogle Scholar
  29. 29.
    Wang XL, Rainwater DL, VandeBerg JF, Mitchell BD, Mahaney MC. Genetic contributions to plasma total antioxidant activity. Arterioscler Thromb Vasc Biol. 2001;21(7):1190–95.CrossRefPubMedGoogle Scholar
  30. 30.
    Cocuzza M, Athayde KS, Agarwal A, Sharma R, Pagani R, Lucon AM, et al. Age-related increase of reactive oxygen species in neat semen in healthy fertile men. Urology. 2008;71(3):490–94.CrossRefPubMedGoogle Scholar
  31. 31.
    Alshahrani S, Agarwal A, Assidi M, Abuzenadah AM, Durairajanayagam D, Ayaz A, et al. Infertile men older than 40 years are at higher risk of sperm DNA damage. Reprod Biol Endocrinol. 2014;12:103.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Shekarriz M, DeWire DM, Thomas Jr AJ, Agarwal A. A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol. 1995;28(1):31–5.PubMedGoogle Scholar
  33. 33.
    Bani Hani SA. Semen quality and chemical oxidative stress: quantification and remediation. PhD thesis in Analytical Biochemistry from Cleveland State University, Cleveland, Ohio, USA 2011, 153 p.Google Scholar
  34. 34.
    Ko EY, Sabanegh ES, Agarwal A. Male infertility testing: reactive oxygen species and antioxidant capacity. Fertil Steril. 2014;102(6):1518–27.CrossRefPubMedGoogle Scholar
  35. 35.
    Agarwal A, Prabakaran S, Allamaneni SS. Relationship between oxidative stress, varicocele and infertility: a meta-analysis. Reprod Biomed Online. 2006;12(5):630–33.CrossRefPubMedGoogle Scholar
  36. 36.
    Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male infertility. Reprod Biomed Online. 2014;29(1):32–58.CrossRefPubMedGoogle Scholar
  37. 37.
    Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. Oxidative stress in an assisted reproductive techniques setting. Fertil Steril. 2006;86(3):503–12.CrossRefPubMedGoogle Scholar
  38. 38.
    Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health. 2014;32(1):1–17.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Muller CH, Lee TK, Montano MA. Improved chemiluminescence assay for measuring antioxidant capacity of seminal plasma. Methods Mol Biol. 2013;927:363–76.CrossRefPubMedGoogle Scholar
  40. 40.
    Mahfouz R, Sharma R, Sharma D, Sabanegh E, Agarwal A. Diagnostic value of the total antioxidant capacity (TAC) in human seminal plasma. Fertil Steril. 2009;91(3):805–11.CrossRefPubMedGoogle Scholar
  41. 41.
    Hosseinzadeh Colagar A, Karimi F, Jorsaraei SG. Correlation of sperm parameters with semen lipid peroxidation and total antioxidants levels in astheno- and oligoasheno-teratospermic men. Iran Red Crescent Med J. 2013;15(9):780–85.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Pahune PP, Choudhari AR, Muley PA. The total antioxidant power of semen and its correlation with the fertility potential of human male subjects. J Clin Diagn Res. 2013;7(6):991–95.PubMedPubMedCentralGoogle Scholar
  43. 43.
    Mancini A, Festa R, Silvestrini A, Nicolotti N, Di Donna V, La Torre G, et al. Hormonal regulation of total antioxidant capacity in seminal plasma. J Androl. 2009;30(5):534–40.CrossRefPubMedGoogle Scholar
  44. 44.
    Ozturk U, Ozdemir E, Buyukkagnici U, Dede O, Sucak A, Celen S, et al. Effect of spermatic vein ligation on seminal total antioxidant capacity in terms of varicocele grading. Andrologia. 2012;44 Suppl 1:199–204.CrossRefPubMedGoogle Scholar
  45. 45.
    Marshburn PB, Giddings A, Causby S, Matthews ML, Usadi RS, Steuerwald N, et al. Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation. Fertil Steril. 2014;102(3):705–10.CrossRefPubMedGoogle Scholar
  46. 46.
    Eroglu M, Sahin S, Durukan B, Ozakpinar OB, Erdinc N, Turkgeldi L, et al. Blood serum and seminal plasma selenium, total antioxidant capacity and coenzyme q10 levels in relation to semen parameters in men with idiopathic infertility. Biol Trace Elem Res. 2014;159(1–3):46–51.CrossRefPubMedGoogle Scholar
  47. 47.
    Kumar D, Salian SR, Kalthur G, Uppangala S, Kumari S, Challapalli S, et al. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation. Environ Res. 2014;132:297–304.CrossRefPubMedGoogle Scholar
  48. 48.
    Kasperczyk A, Dobrakowski M, Czuba ZP, Horak S, Kasperczyk S. Environmental exposure to lead induces oxidative stress and modulates the function of the antioxidant defense system and the immune system in the semen of males with normal semen profile. Toxicol Appl Pharmacol. 2015;284(3):339–44.CrossRefPubMedGoogle Scholar
  49. 49.
    Haghighian HK, Haidari F, Mohammadi-Asl J, Dadfar M. Randomized, triple-blind, placebo-controlled clinical trial examining the effects of alpha-lipoic acid supplement on the spermatogram and seminal oxidative stress in infertile men. Fertil Steril. 2015;104(2):318–24.CrossRefPubMedGoogle Scholar
  50. 50.
    Raigani M, Yaghmaei B, Amirjannti N, Lakpour N, Akhondi MM, Zeraati H, et al. The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men. Andrologia. 2014;46(9):956–62.CrossRefPubMedGoogle Scholar
  51. 51.
    Guo L, Jing J, Feng YM, Yao B. Tamoxifen is a potent antioxidant modulator for sperm quality in patients with idiopathic oligoasthenospermia. Int Urol Nephrol. 2015;47(9):1463–69.CrossRefPubMedGoogle Scholar
  52. 52.
    Layali I, Tahmasbpour E, Joulaei M, Jorsaraei SG, Farzanegi P. Total antioxidant capacity and lipid peroxidation in semen of patient with hyperviscosity. Cell J. 2015;16(4):554–59.PubMedPubMedCentralGoogle Scholar
  53. 53.
    Siciliano L, Tarantino P, Longobardi F, Rago V, De Stefano C, Carpino A. Impaired seminal antioxidant capacity in human semen with hyperviscosity or oligoasthenozoospermia. J Androl. 2001;22(5):798–803.PubMedGoogle Scholar
  54. 54.
    Kratz EM, Piwowar A, Zeman M, Stebelova K, Thalhammer T. Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility. Reprod Fertil Dev. 2016. doi:10.1071/RD14165.Google Scholar
  55. 55.
    Kratz EM, Kaluza A, Ferens-Sieczkowska M, Olejnik B, Fiutek R, Zimmer M. et al. Gelatinases and their tissue inhibitors are associated with oxidative stress: a potential set of markers connected with male infertility. Reprod Fertil Dev. 2016. doi:10.1071/RD14268.Google Scholar
  56. 56.
    Yousefniapasha Y, Jorsaraei G, Gholinezhadchari M, Mahjoub S, Hajiahmadi M, Farsi M. Nitric oxide levels and total antioxidant capacity in the seminal plasma of infertile smoking men. Cell J. 2015;17(1):129–36.PubMedPubMedCentralGoogle Scholar
  57. 57.
    Pasqualotto FF, Sundaram A, Sharma RK, Borges Jr E, Pasqualotto EB, Agarwal A. Semen quality and oxidative stress scores in fertile and infertile patients with varicocele. Fertil Steril. 2008;89(3):602–7.CrossRefPubMedGoogle Scholar
  58. 58.
    Sharma RK, Pasqualotto FF, Nelson DR, Thomas Jr AJ, Agarwal A. The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999;14(11):2801–07.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Shubhadeep Roychoudhury
    • 1
    • 2
  • Rakesh Sharma
    • 1
  • Suresh Sikka
    • 3
  • Ashok Agarwal
    • 1
    Email author
  1. 1.American Center for Reproductive MedicineCleveland ClinicClevelandUSA
  2. 2.Department of Life Science and BioinformaticsAssam UniversitySilcharIndia
  3. 3.Tulane University Health Sciences CenterNew OrleansUSA

Personalised recommendations