Evaluation of the impact of leucocytospermia on semen oxidative status by chemiluminescence technique in infertile men


The presence of high reactive oxygen species (ROS) levels in semen is a major factor involved in the decline of male fertility. In seminal plasma, ROS are mainly produced by activated leucocytes. Spermatozoa were the first cell type reported to show a potential susceptibility to oxidative damage. The aim of our study was to evaluate the impact of leucocytospermia on basal and FMLP (Formyl-Methionyl-Leucyl-Phenylalanine) induced oxidative status in semen of infertile men. We also analyzed the correlations of the spermatic parameters with amounts of ROS in semen. Our study included 50 semen samples of infertile men. Sperm analysis was performed using WHO standardized method. Seminal leucocytes were quantified using peroxidase technique. The measurement of ROS levels in semen was made by chemiluminescence assay. We measure respectively ROS amounts in neat semen and in washed sperm cells suspension from the same ejaculate. We also applied the test of provocation of leucocytes by FMLP on neat and washed samples to assess the spermatic oxidative status after leucocyte stimulation. Our results showed significant correlations between ROS levels in neat semen and many sperm parameters: motility, sperm concentration, leucocytes concentration and the rate of sperm cytoplasmic droplets. The studied samples were divided into 2 groups: (G1) composed of 36 samples without leucocytospermia and (G2) composed of 14 leucospermic samples. ROS levels were significantly lower in G1 than in G2 (p = 0.002). ROS production was significantly increased after application of FMLP in washed leucospermic samples (p = 0.001). The measurement of ROS in neat semen is a considerable contribution to explore the impairment of semen quality in infertile men. ROS levels in washed semen reflect the oxidative status generated by sperm preparation techniques used in assisted reproductive procedures. Levels of ROS are highly influenced by the presence of leucocytes and associated with decreased seminal parameters.

Share and Cite:

Sellami, A. , Chakroun, N. , Rtaib, Y. , Hdhili, H. , Mansour, R. , Dolira, L. , Chaabene, K. , Keskes, L. , Lassoued, S. and Rebai, T. (2013) Evaluation of the impact of leucocytospermia on semen oxidative status by chemiluminescence technique in infertile men. Advances in Bioscience and Biotechnology, 4, 30-40. doi: 10.4236/abb.2013.411A2005.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Rahman, K. (2007) Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging, 2, 219-236.
[2] Brieger, K., Schiavone, S., Miller Jr., F.J. and Krause, K.H. (2102) Reactive oxygen species: From health to disease. Swiss Medical Weekly, 142, w13659.
[3] Agarwal, A. and Saleh, R.A. (2002) Role of oxidants in male infertility: Rationale, significance, and treatment. Urologic Clinics of North America, 29, 817-827.
[4] Cocuzza, M., Sikka, S.C., Athayde, K.S. and Agarwal, A. (2007) Clinical relevance of oxidative stress and sperm chromatin damage in male infertility: An evidence based analysis. International braz j urol, 33, 603-621.
[5] Agarwal, A., Makker, K. and Sharma, R. (2008) Clinical relevance of oxidative stress in male factor infertility: An update. American Journal of Reproductive Immunology, 59, 2-11.
[6] Gharagozloo, P. and Aitken, R.J. (2011) The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Human Reproduction, 26, 1628-1640. http://dx.doi.org/10.1093/humrep/der132
[7] Agarwal, A. and Said, T.M. (2005) Oxidative stress, DNA damage and apoptosis in male infertility: A clinical approach. BJU International, 95, 503-507.
[8] Saalu, L.C. (2010) The incriminating role of reactive oxygen species in idiopathic male infertility: An evidence based evaluation. Pakistan Journal of Biological Sciences, 13, 413-422. http://dx.doi.org/10.3923/pjbs.2010.413.422
[9] Tremellen, K. (2008) Oxidative stress and male infertileity—A clinical perspective. Human Reproduction Update, 14, 243-258. http://dx.doi.org/10.1093/humupd/dmn004
[10] Aitken, R.J. and Koppers, A.J. (2011) Apoptosis and DNA damage in human spermatozoa. Asian Journal of Andrology, 13, 36-42.
[11] Kessopoulou, E., Tomlinson, M.J., Barratt, C.L., Bolton, A.E. and Cooke, I.D. (1992) Origin of reactive oxygen species in human semen: Spermatozoa or leucocytes? The Journal of the Society for Reproduction and Fertility, 94, 463-470. http://dx.doi.org/10.1530/jrf.0.0940463
[12] Sharma, R.K. and Agarwal, A. (1996) Role of reactive oxygen species in male infertility. Urology, 48, 835-850.
[13] Anderson, D.J. and Politch, J.A. (1996) Evaluation and treatment of the infertile male. In: Centola, G.M. and Ginsburg, K.A., Eds., Cambridge University Press, New York, 263-277.
[14] Depuydt, C.E., Bosmans, E., Zalata, A., Schoonjans, F. and Comhaire, F.H. (1996) The relation between reactive oxygen species and cytokines in andrological patients with or without male accessory gland infection. Journal of Andrology, 17, 699-707.
[15] Keck, C., Gerber-Schäfer, C., Clad, A., Wilhelm, C. and Breckwoldt, M. (1998) Seminal tract infections: Impact on male fertility and treatment options. Human Reproduction Update, 4, 891-903.
[16] World Health Organization (1999) Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press, Cambridge.
[17] World Health Organization (2010) Laboratory manual for the examination and processing of human semen. WHO Press, Geneva.
[18] Kashou, A.H., Sharma, R. and Agarwal, A. (2013) Assessment of oxidative stress in sperm and semen. In: Carrell, D.T. and Aston, K.I., Eds., Spermatogenesis: Methods and protocols, Methods in Molecular Biology, Humana Press, New York, 351-361.
[19] Krausz, C., Mills, C., Rogers, S., Tan, S.L. and Aitken, R.J. (1994) Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: Relationships with motility and fertilization in vitro. Fertility and Sterility, 62, 599-605.
[20] Storey, B.T. (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Molecular Human Reproduction, 3, 203-213.
[21] Tomlinson, M.J., Barratt, C.L. and Cooke, I.D. (1993) Prospective study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male infertility. Fertility and Sterility, 60, 1069-1075.
[22] Aitken, R.J., Irvine, D.S. and Wu, F.C. (1991) Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. American Journal of Obstetrics & Gynecology, 164, 542-551. http://dx.doi.org/10.1016/S0002-9378(11)80017-7
[23] du Plessis, S.S., McAllister, D.A., Luu, A., Savia, J., Agarwal, A. and Lampiao, F. (2010) Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels. Andrologia, 42, 206-210. http://dx.doi.org/10.1111/j.1439-0272.2009.00980.x
[24] Sharma, R.K., Pasqualotto, A.E., Nelson, D.R., Thomas Jr., A.J. and Agarwal A. (2001) Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. Journal of Andrology, 22, 575-583.
[25] Barraud-Lange, V., Pont, J.C., Ziyyat, A., Pocate, K., Sifer, C., Cedrin-Durnerin, I., Fechtali, B., Ducot, B. and Wolf, J.P. (2011) Seminal leukocytes are good samaritans for spermatozoa. Fertility and Sterility, 96, 1315-1319.
[26] Zini, A. and Sigman, M. (2009) Evaluation of sperm function. In: Lipshultz, L.I., Howards, S.S. and Niederberger, C.S., Eds., Infertility in the Male, Cambridge University Press, New York, 177-198.
[27] Agarwal, A. and Prabakaran, S.A. (2005) Oxidative stress and antioxidants in male infertility: A difficult balance. Iranian Journal of Reproductive Medicine, 3, 1-8.
[28] Agarwal, A., Sharma, R.K., Nallella, K.P., Thomas Jr., A.J., Alvarez, J.G. and Sikka, S.C. (2006) Reactive oxygen species as an independent marker of male factor infertility. Fertility and Sterility, 86, 878-885.
[29] Doshi, S.B., Khullar, K., Sharma, R.K. and Agarwal, A. (2012) Role of reactive nitrogen species in male infertileity. Reproductive Biology and Endocrinology, 10, 109.
[30] Sikka, S.C. (2004) Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. Journal of Andrology, 25, 5-18.
[31] Ochsendorf, F.R., Thiele, J., Fuchs, J., Schuttau, H., Freisleben, H.J., Buslau, M. and Milbradt, R. (1994) Chemiluminescence in semen of infertile men. Andrologia, 26, 289-293.
[32] Williams, A.C. and Ford, W.C. (2005) Relationship between reactive oxygen species production and lipid peroxidation in human sperm suspensions and their association with sperm function. Fertility and Sterility, 83, 929-936. http://dx.doi.org/10.1016/j.fertnstert.2004.11.031
[33] Athayde, K.S., Cocuzza, M., Agarwal, A., Krajcir, N., Lucon, A.M., Srougi, M. and Hallak, J. (2007) Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. Journal of Andrology, 28, 613-620.
[34] Plante, M., de Lamirande, E. and Gagnon, C. (1994) Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertility and Sterility, 62, 387-393.
[35] Griveau, J.F. and Le Lannou, D. (1997) Reactive oxygen species and human spermatozoa: Physiology and pathology. International Journal of Andrology, 20, 61-69.
[36] Armstrong, J.S., Rajasekaran, M., Chamulitrat, W., Gatti, P., Hellstrom, W.J. and Sikka, S.C. (1999) Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radical Biology and Medicine, 26, 869-880.
[37] Baumber, J., Ball, B.A., Gravance, C.G., Medina, V. and Davies-Morel, M.C. (2000) The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Journal of Andrology, 21, 895-902.
[38] Wang X., Sharma, R.K., Gupta, A., George, V., Thomas, A.J., Falcone, T. and Agarwal, A. (2003) Alterations in mitochondria membrane potential and oxidative stress in infertile men: A prospective observational study. Fertility and Sterility, 80, 844-850.
[39] Agarwal, A., Saleh, R.A. and Bedaiwy, M.A. (2003) Role of reactive oxygen species in the pathophysiology of human reproduction. Fertility and Sterility, 79, 829-843.
[40] Aitken, R.J., Wingate, J.K., De Iuliis, G.N., Koppers, A.J. and McLaughlin, E.A. (2006) Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. The Journal of Clinical Endocrinology & Metabolism, 91, 4154-4163.
[41] de Lamirande, E. and Gagnon, C. (1992) Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. Journal of Andrology, 13, 368-378.
[42] Tavilani, H., Doosti, M. and Saeidi, H. (2005) Malondialdehyde levels in sperm and seminal plasma of asthenozoospermic and its relationship with semen parameters. Clinica Chimica Acta, 356, 199-203.
[43] Kothari, S., Thompson, A., Agarwal, A. and du Plessis, S.S. (2010) Free radicals: Their beneficial and detrimental effects on sperm function. Indian Journal of Experimental Biology, 48, 425-435.
[44] Aitken, R.J., Buckingham, D., West, K.M., Wu, F.C., Zikopoulos, K. and Richardson, D.W. (1992) Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. The Journal of the Society for Reproduction and Fertility, 94, 451-462.
[45] Zalata, A., Hafez, T. and Comhaire, F. (1995) Evaluation of the role of reactive oxygen species in male infertility. Human Reproduction, 10, 1444-1451.
[46] Pasqualotto, F.F., Sharma, R.K., Nelson, D.R., Thomas, A.J. and Agarwal, A. (2000) Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing infertility investigation. Fertility and Sterility, 73, 459-464.
[47] Boonstra, J. and Post, J.A. (2004) Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene, 337, 1-13.
[48] Tvrdá, E., Kňazická, Z., Bárdos, L., Massányi, P. and Lukác, N. (2011) Impact of oxidative stress on male fertility—A review. Acta Veterinaria Hungarica, 59, 465-484. http://dx.doi.org/10.1556/AVet.2011.034
[49] Ochsendorf, F.R. (1999) Infections in the male genital tract and reactive oxygen species. Human Reproduction Update, 5, 399-420.
[50] Fraczek, M. and Kurpisz, M. (2007) Inflammatory mediators exert toxic effects of oxidative stress on human spermatozoa. Journal of Andrology, 28, 325-333.
[51] Villegas, J., Schulz, M., Soto, L., Iglesias, T., Miska, W. and Sánchez, R. (2005) Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis in mature human spermatozoa. Fertility and Sterility, 83, 808-810.
[52] Wang, A., Fanning, L., Anderson, D.J. and Loughlin, K.R. (1997) Generation of reactive oxygen species by leukocytes and sperm following exposure to urogenital tract infection. Archives of Andrology, 39, 11-17.
[53] Saleh, R.A., Agarwal, A., Kandirali, E., Sharma, R.K., Thomas, A.J., Nada, E.A., Evenson, D.P. and Alvarez, J.G. (2002) Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa. Fertility and Sterility, 78, 1215-1224.
[54] Krausz, C., West, K., Buckingham, D. and Aitken, R.J. (1992) Development of a technique for monitoring the contamination of human semen samples with leukocytes. Fertility and Sterility, 57, 1317-1325.
[55] Sukcharoen, N., Keith, J., Irvine, D.S. and Aitken, R.J. (1996) Prediction of the in-vitro fertilization (IVF) potential of human spermatozoa using sperm function tests: The effect of the delay between testing and IVF. Human Reproduction, 11, 1030-1034.
[56] Gomez, E., Buckingham, D.W., Brindle, J., Lanzafame, F., Irvine, D.S. and Aitken, R.J. (1996) Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: Correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. Journal of Andrology, 17, 276-287.
[57] Said, T.M., Agarwal, A., Sharma, R.K., Thomas Jr., A.J. and Sikka, S.C. (2005) Impact of sperm morphology on DNA damage caused by oxidative stress induced by betanicotinamide adenine dinucleotide phosphate. Fertility and Sterility, 83, 95-103.
[58] Fisher, H.M. and Aitken, R.J. (1997) Comparative analysis of the ability of precursor germ cells and epididymal spermatozoa to generate reactive oxygen metabolites. Journal of Experimental Zoology, 277, 390-400.
[59] Said, T.M., Agarwal, A., Sharma, R.K., Mascha, E., Sikka, S.C. and Thomas Jr., A.J. (2004) Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility. Fertility and Sterility, 82, 871-877.
[60] Pasqualotto, F.F., Sharma, R.K., Kobayashi, H., Nelson, D.R., Thomas Jr., A.J. and Agarwal, A. (2001) Oxidative stress in normospermic men undergoing infertility evaluation. Journal of Andrology, 22, 316-322.
[61] Agarwal, A., Ikemoto, I. and Loughlin, K.R. (1994) Effect of sperm washing on levels of reactive oxygen species in semen. Archives of Andrology, 33, 157-162.
[62] Fingerova, H., Oborna, I., Novotny, J., Svobodova, M., Brezinova, J. and Radova, L. (2009) The measurement of reactive oxygen species in human neat semen and in suspended spermatozoa: A comparison. Reproductive Biology and Endocrinology, 7, 118.
[63] Venkatesh, S., Shamsi, M.B., Dudeja, S., Kumar, R. and Dada, R. (2011) Reactive oxygen species measurement in neat and washed semen: Comparative analysis and its significance in male infertility assessment. Archives of Gynecology and Obstetrics, 283, 121-126.
[64] Hughes, C.M., Lewis, S.E., McKelvey-Martin V.J. and Thompson, W. (1998) The effects of antioxidant supplementation during percoll preparation on human sperm DNA integrity. Human Reproduction, 13, 1240-1247.
[65] Donnelly, E.T., McClure, N. and Lewis, S.E. (1999) The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis, 14, 505-512. http://dx.doi.org/10.1093/mutage/14.5.505
[66] Chi, H.J., Kim, J.H., Ryu, C.S., Lee, J.Y., Park, J.S., Chung, D.Y., Choi, S.Y., Kim, M.H., Chun, E.K. and Roh, S.I. (2008) Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Human Reproduction, 23, 1023-1028. http://dx.doi.org/10.1093/humrep/den060
[67] Donnelly, E.T., McClure, N. and Lewis, S.E. (2000) Glutathione and hypotaurine in vitro: Effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagenesis, 15, 61-68.
[68] Baumber, J., Ball, B.A. and Linfor, J.J. (2005) Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants. American Journal of Veterinary Research, 66, 772-779.
[69] Mahfouz, R., Sharma, R., Thiyagarajan, A., Kale, V., Gupta, S., Sabanegh, E. and Agarwal, A. (2010) Semen characterristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertility and Sterility, 94, 2141-2146.
[70] Aitken, R.J. and Krausz, C. (2001) Oxidative stress, DNA damage and the Y chromosome. Reproduction, 122, 497-506. http://dx.doi.org/10.1530/rep.0.1220497
[71] Aitken, R.J. and De Iuliis, G.N. (2007) Origins and consequences of DNA damage in male germ cells. Reproductive BioMedicine Online, 14, 727-733.
[72] Alvarez, J.G., Sharma, R.K., Ollero, M., Saleh, R.A., Lopez, M.C., Thomas, A.J. Jr., Evenson, D.P. and Agarwal, A. (2002) Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay. Fertility and Sterility, 78, 319-329.
[73] Erenpreiss, J., Hlevicka, S., Zalkalns, J. and Erenpreisa J. (2002) Effect of leukocytospermia on sperm DNA integrity: A negative effect in abnormal semen samples. Journal of Andrology, 23, 717-723.
[74] Zribi, N., Chakroun, N.F., Elleuch, H., Abdallah, F.B., Ben Hamida, A.S., Gargouri, J., Fakhfakh, F. and Keskes, L.A. (2011) Sperm DNA fragmentation and oxidation are independent of malondialdheyde. Reproductive Biology and Endocrinology, 9, 47-54.
[75] Makker, K., Agarwal, A. and Sharma, R. (2009) Oxidative stress and male infertility. Indian Journal of Medical Research, 129, 357-367.
[76] Aitken, R.J., De Iuliis, G.N., Finnie, J.M., Hedges, A. and McLachlan, R.I. (2010) Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: Development of diagnostic criteria. Human Reproduction, 25, 2415-2426.
[77] Muriel, L., Garrido, N., Fernández, J.L., Remohí, J., Pellicer, A., de los Santos, M.J. and Meseguer, M. (2006) Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertility and Sterility, 85, 371-383.
[78] Avendaño, C., Franchi, A., Duran, H. and Oehninger S. (2010) DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome. Fertility and Sterility, 94, 549-557.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.