A rationale for glutathione therapy

Is antioxidant therapy a promising strategy to improve human reproduction? Perry, A.C.F., Jones, R., Niang, L.S.P. et al. (1992) Genetic-evidence for an androgen-regulated epididymal secretory glutathione-peroxidase whose transcript does not contain a selenocysteine codon. Biochem. J., 285, 863–870. Perry, A.C.F., Jones, R. and Hall, L. (1993) Isolation and characterization of a rat cdna clone encoding a secreted superoxide-dismutase reveals the epididymis to be a major site of its expression. Biochem. J., 293, 21–25. Rao, B., Soufir, J.-C., Martin, M. and David, G. (1989) Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res., 24, 127–134. Selley, M., Lacey, M.J., Bartlett, M.R. et al. (1991) Content of significant amounts of a cytotoxic end-product of lipid peroxidation in human semen. J. Reprod. Fertil., 92, 291–298. Storey, B.T. (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol. Hum. Reprod., 3, 203–213. Sukcharoen, N., Keith, J., Irvine, D.S. and Aitken, R.J. (1995) Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination. Fertil. Steril., 63, 1293–1300. Suleiman, S.A., Ali, M.E., Zaki, Z.M.S. et al. (1996) Lipid-peroxidation and human sperm motility – protective role of vitamin E. J. Androl., 17, 530–537. Therond, P., Auger, J., Legrand, A. and Jouannet, P. (1996) α-Tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes. Mol. Hum. Reprod., 2, 739–744. Thiele, J.J., Freisleben, H.J., Fuchs, J. and Ochsendorf, F.R. (1995) Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen. Hum. Reprod., 10, 110–115. Tomlinson, M.J., Barratt, C.L.R. and Cooke, I.D. (1993) Prospective-study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male-infertility. Fertil. Steril., 60, 1069–1075. Whittington, K. (1997) Origin and Effects of Reactive Oxygen Species in Sperm Suspensions. Ph.D. Dissertation, University of Bristol. 311 pp. Wolff, H. (1995) The biologic significance of white blood-cells in semen. Fertil. Steril., 63, 1143–1157. Wolff, H., Politch, J.A., Martinez, A. et al. (1990) Leukocytospermia is associated with poor semen quality. Fertil. Steril., 53, 528–536.

A rationale for glutathione therapy A.Lenzi1,3, L.Gandini1 and M.Picardo2 1Laboratory

of Seminology and Immunology of Reproduction, Department of Medical Pathophysiology, University of Rome ‘La Sapienza’, 00161 Rome, and 2Laboratory of Physiopathology, IRCSS ‘S.Gallicano’, Rome, Italy 3To

whom correspondence should be addressed

This Debate was previously published on Webtrack on Web 10, on March 31, 1998 Cellular homeostasis is regulated in part by the membrane levels of peroxidable substances, such as polyunsaturated fatty acids (PUFA) and by the effectiveness of the free radical scavenger systems. Therefore, oxidative stress can be defined as any disturbance in the balance between pro-oxidants and anti-oxidants in which the former prevail. In mature spermatozoa, a high concentration of membrane unsaturated lipids is associated with a relative paucity of oxyradical scavenger enzymes, such as superoxide dismutase (SOD) catalase and glutathione peroxidase (Aitken, 1991). The deficiency is probably due to the virtual absence of cytoplasm in mature

sperm cells. This, however, is compensated by the powerful antioxidant system in seminal plasma. Several studies have demonstrated that, in contrast to other biological fluids, seminal plasma contains significant levels of superoxide dismutase, xanthine oxidase, nitric oxide, catalase and glutathione peroxidase together with significant concentrations of chemical antioxidants such as ascorbic acid (vitamin C) and α-tocopherol (vitamin E) (Daunter et al., 1981; Sanoka et al., 1996; The`rond et al., 1996). Of the antioxidant systems, a special attribute of seminal plasma is the relatively high concentration of reduced glutathione (GSH). The harmful impact of oxyradicals and toxic compounds on sperm function has been well studied with particular emphasis on the negative effects of reactive oxygen species (ROS) on sperm function (Aitken and Clarkson, 1987; Alvarez et al., 1987; Aitken et al., 1989; D’Agata et al., 1990). ROS are be toxic for spermatozoa and can significantly affect sperm motility (Alvarez and Storey, 1982, 1989). However, some reports have shown that ROS in vitro can also trigger physiological sperm functions, such as capacitation and hyperactivated motility (de Lamirande and Gagnon, 1993; Griveau and Le Lannou, 1994; Griveau et al., 1994). Significant ROS production by spermatozoa has been shown during the capacitation process (de Lamirande and Gagnon, 1995). Experimental data on mouse and bovine spermatozoa demonstrate that ROS may be beneficial to gamete function under specific peroxidative conditions that increase lipoperoxidation without modifying free sulphydryl groups (Kodama et al., 1996; Blondin et al., 1997). These positive/negative effects are strictly related to the equilibrium between ROS and the scavenger systems. Also in assisted reproduction great differences have been observed in the results of sperm selection techniques, if the semen characteristics are not taken into account and if the sperm preparation methods and composition of culture media are not chosen case by case (Mortimer, 1991; Gavella and Lipovac, 1994). The main sources of ROS in the genital tract are the spermatozoa themselves (especially if damaged, with excess residual cytoplasm and morphologically abnormal spermatozoa), leukocytes during inflammatory processes, and ischaemia/ hypoxia produced during vascular diseases such as varicocele (Aitken et al., 1989; Gomez et al., 1996). Based on the above, the idea of using antioxidant-scavenger therapies to treat some forms of dyspermia have recently been proposed. Treatments, based on different rationales, have varied over the years and have involved the use of carnitine, phosphatidilcholine, kallicreine, penthossiphylline and vitamins A, E and C. Glutathione antioxidant-scavenger therapy Given the interesting possibilities of these antioxidant therapies our group established at the beginning of the 1990s a research programme using GSH in selected forms of sperm pathology. One of the most important reasons for selecting GSH as a therapy is its physiologically significant presence in seminal plasma. Even though it cannot cross cell membranes, the concentration of this antioxidant can be increased in biological fluids by systemic administration. It is able to reach the seminal plasma and concentrate there, thus exerting its physiological 1419

A.Lenzi et al.

and therapeutic role. Glutathione, through its thiolic group, can react directly with hydrogen peroxide, superoxide anion and hydroxyl radicals and its sulphydryl group can react with alkoxyl radicals and hydroperoxides, producing alcohols. Moreover, GSH is the substrate of the selenium-containing enzyme glutathione-peroxidase (the main enzyme involved in removing hydrogen peroxides) and of glutathione transferase (an enzyme which catalyses covalent reactions of GSH with electrophile substances such as quinones). We carried out a 2 month pilot study on the therapeutic use of glutathione (600 mg/day injected i.m.) and found a statistically significant effect on sperm motility patterns and sperm morphology (Lenzi et al., 1992). After this, we carried out a placebo controlled double-blind cross-over trial in a selected group of infertile patients suffering only from unilateral varicocele or germ-free genital tract inflammation. The patients were randomly and blindly assigned to treatment with one i.m. injection every other day of either glutathione 600 mg or an equal volume of a placebo preparation (for a detailed description of selection criteria and the cross-over statistical analysis results see Lenzi et al., 1993). All the selected patients showed an increase in sperm concentration and a highly statistically significant improvement in sperm motility, sperm kinetic parameters and sperm morphology. These results were confirmed by other groups using orally administered vitamin E both a double-blind cross-over trial (resulting in an increase in fertilization potential) (Kessopoulou et al., 1995) and in a open study on patients with low fertilization rate in in-vitro fertilization (IVF) programmes (resulting in an improvement of IVF rate) (Geva et al., 1996). Finally, we studied the in vitro therapeutic effect of GSH. We selected semen samples from sperm bank donors and infertile patients, with and without leukospermia and we used swim-up with and without GSH in the medium. The only statistically significant difference in sperm forward motility was found in leukospermic samples treated with pellet swim up. In these samples, an increase was seen when the migration media contained GSH (Gandini et al., 1993). These results indicate that glutathione also protects sperm motility in vitro during pelleting when there can be contact between seminal ROS, produced by leukocytes or damaged spermatozoa, and normal spermatozoa. Recently, a paper confirmed, in part, the above results and showed invitro enhancement of sperm motility using a migration salt solution containing glucose and glutathione as an antioxidant (Parinaud et al., 1997). Seeking a rationale With these results in hand, we tried to find a rationale for the use of glutathione antoxidant-scaveger therapy. We strictly selected another group of infertile patients with unilateral varicocele or germ free genital tract inflammation and studied the modifications produced by glutathione therapy on: (i) the risk of lipoperoxidation of the sperm membrane, as evaluated by thiobarbituric acid (TBA) assay (Barber and Bernheim, 1967; Alvarez and Storey, 1982; Aitken et al., 1989); and (ii) on the pattern of PUFA of red blood cell membranes. Red blood cells (RBC) are widely considered to be a representative 1420

model of the constitution of general cell membranes and we used these as it was practically impossible to evaluate the PUFA pattern of sperm membrane phospholipids in patients with severe oligozoospermia. An improvement in sperm concentration, motility, morphology and kinetic variables was observed also in this series of patients. These improvements were associated with a decrease in the levels of lipoperoxides in the spermatozoa (TBA assay) and with an increase of the RBC levels of PUFA of membrane phospholipids. The arachidonic acid/linoleic acid ratio (a valuable indicator of the polyunsaturated fatty acid metabolism) in both RBC and serum phospholipids before therapy, was lower in the infertile patients than in fertile subjects. After therapy, together with the reduction of sperm lipid peroxidation, the levels of the precursor di-omo-γ-linolenic acid and of the arachidonic acid increased significantly in both red blood cells and serum phospholipids (for more detailed data see Lenzi et al., 1994). This indicated that, at least in part, the therapeutic action of glutathione is due to its general protective effect on the lipid components of the cell membrane. Furthermore, from the above results, we postulated that the patients studied had an impairment of the desaturase enzymes, possibly due to genetic factors or acquired pathologies leading to chronic systemic damage of all cell membranes. This constitutional alteration in cell membranes may increase the harmful effect of the oxyradicals generated in the epididymis following vascular (varicocele) or inflammatory (germ free genital tract inflammation) processes, facilitating dyspermia. It seems likely that subjects with systemic lipid membrane disturbances associated with strictly selected andrological pathologies express this membrane damage in spermatozoa and are prone to develop dyspermia. In this context, it is likely that GSH therapy acts by modifying the biochemical lipid membrane constitution. Another possible rationale for the gluthatione therapeutic effect in selected cases of dyspermia, is an improvement in the epididymal microenvironment and a subsequent higher unsaturation of the PUFA of the sperm plasma membrane. It is probable that GSH also acts as a free radical scavenger in the epididymis, thus reducing the lipoperoxidative process generated by the above vascular or inflammatory pathologies. The significant reduction in the lipoperoxide level in seminal plasma as detected by the TBA test supports this hypothesis. To support this last hypothesis we studied PUFA of the sperm plasma membrane in various sperm populations with different morpho–functional characteristics. In fact, sperm plasma membrane plays a fundamental role in sperm fertilization. Current theories of membrane fusion suggest that membrane fluidity is a prerequisite for normal cell functions and that the fluidity and flexibility of cell membranes are mainly dependent on their lipid constitution. In particular, PUFA are known to contribute to membrane fluidity and flexibility (Israelachvili et al., 1980; Fleming and Yanagimachi, 1981; Meizel and Turner, 1983). Analyses of the fatty acid pattern of membrane phospholipids of human spermatozoa have demonstrated significant levels of polyunsaturated acids. The definitive lipid pattern of ejaculated spermatozoa is reached only during the sperm passage through the epididymis and after maturation, as shown in rams (Wolf et al., 1986), guinea pigs (Myles and

Is antioxidant therapy a promising strategy to improve human reproduction?

Primakoff, 1984; Cowan et al., 1986) and rats (Gaunt et al., 1983; Hall et al., 1991). Biomembrane fluidity in humans can increase as the degree of unsaturation increases when spermatozoa pass from the caput to the cauda of the epididymis and this would indicate an active sperm lipid metabolism. Furthermore, as stated above, PUFA of phospholipids play a major role in membrane constitution and function and are one of the main targets of the lipoperoxidative process. Their degree of unsaturation is therefore an essential parameter in the ability of spermatozoa to maintain equilibrium in an oxidative environment. To study this, the fatty acid pattern of the membranes in human ejaculated spermatozoa was studied by our group using combined gas chromatography–mass spectrometry (Lenzi et al., 1994). In a first experiment spermatozoa were analysed after washing (whole spermatozoa) and following sperm selection on Percoll gradients (Percoll selected spermatozoa). Percoll gradients were used to eliminate all interference from cells other than spermatozoa. Results showed significant differences in samples obtained by the two methods with regard to the percentage of PUFA. In whole spermatozoa, the percentage of PUFA was 36–39%, whereas in Percoll-selected spermatozoa, PUFA represented 48–52% of the total fatty acids. In particular, higher concentrations of C20:4 n6 (arachidonic acid) and C22:6 n3 (docosaesaenoic acid) were found in Percoll-selected sperm cells, indicating that this pattern is characteristic of morphologically normal sperm cells (Lenzi et al., 1996). The PUFA pattern differences between whole and Percoll selected spermatozoa suggest that the presence of cells other than normal spermatozoa modify the percentage of each fatty acid. In a second experiment, after sperm selection on Percoll gradients (40:50:70:80:90:100%), the highest percentage of PUFA was detected in the 80:90:100% fractions, which showed spermatozoa with the best morpho–functional characteristics. The spermatozoa collected from the 80, 90 and 100% layer showed a significant difference in concentration of C20:4 n6 and C22:6 n3 compared with spermatozoa from the 40, 50 and 70% layers. In fact, mean 6 SD of C20:4 n6 and C22:6 n3 was 5.2 6 1.1% and 38.2 6 2.2% respectively in the 80, 90 and 100% layers whereas the concentrations were significantly reduced in the 40, 50 and 70% layers, 3.4 6 1.6% and 11.5 6 5.6% respectively (unpublished data). These results show that morpho–functionally normal spermatozoa have a higher percentage of the most representative PUFA (C22:6 n3) than those detected in other cell membranes. This seems to confirm the postulated active fatty acid metabolism and desaturation either during spermatogenesis or during epididymal sperm maturation. These experimental data suggest that in andrological pathologies the sperm membrane becomes more fragile as a consequence of modifications in the PUFA composition. This would indicate that, in vivo, epididymal fluid acts as a nutritiveprotective medium for sperm cells during maturation, but that it can become a hostile medium when andrological pathologies alter its anti/pro-oxidant equilibrium and/or when the sperm membrane is more fragile. The higher epididymal concentration of glutathione obtained by therapy can thus play a protective role, helping to avoid ROS induced damage by bringing

unsaturation levels of sperm membrane PUFA near to those of the morphologically better spermatozoa, selected by Percoll gradients. It is now necessary to demonstrate that GSH therapy can increase the capacity of sperm cells to desaturate essential fatty acids during the epididymal passage and to show whether the high percentage of n-3 PUFA in the sperm membrane is essential for in-vivo and in-vitro fertilizing capacity. References Aitken, R.J. and Clarkson, J.S. (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil., 81, 459–469. Aitken, R.J., Clarkson, J.S. and Fishel, S. (1989) Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod., 40, 183–187. Aitken, R.J. (1991) Reactive oxygen species and human sperm function. In Bacetti, B. (ed.), Comparative Spermatology 20 years After. Serono series, Vol. 75. Raven Press, New York, USA, pp 787–792. Alvarez, J.G. and Storey, B.T. (1982) Spontaneous lipid peroxidation in rabbit epididymal spermatozoa its effect on sperm motility. Biol. Reprod., 27, 1102–1108. Alvarez, J.G. and Storey, B.T. (1989) Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res., 23, 77–90. Alvarez, J.G., Touchtone, J.C., Blasco, L. and Storey, B.T. (1987) Spontaneous lipid peroxidation and production of superoxide and hydrogen peroxide in human spermatozoa: superoxide dismutase as major protectant against oxygen toxicity. J. Androl., 8, 338–348. Barber, A.A. and Bernheim, F. (1967) Lipid peroxidation: its measurement, occurence, and significance in animal tissues. Adv. Gerontol. Res., 2, 355–403. Blondin, P., Coenen, K. and Sirard, M.A. (1997) The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte matuartion. J. Androl., 18, 454–460. Cowan, A.E., Primakoff, P. and Myles, D.G. (1986) Sperm exocytosis increases the amount of Ph-20 antigen on the surface of guinea-pig sperm. J. Cell. Biol., 103, 1289–1297. D’Agata, R., Vicari, E., Moncada, M.L. et al. (1990) Generation of reactive oxygen species in subgroups of infertile men. Int. J. Androl., 13, 344–351. Daunter, B., Hill, R., Hennessey, J. and Mackay, E.V. (1981) Preliminary report: a possible mechanism for the liquefaction of human seminal plasma and its relationship to spermatozoal motility. Andrologia, 13, 131–141. De Lamirande, E. and Gagnon, C. (1993) Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma. Fertil. Steril., 59, 1291–1295. De Lamirande, E. and Gagnon, C. (1995) Capacitation-associated production of superoxide ion by human spermatozoa. Free Rad. Biol. Med., 18, 487–495. Fleming, A.D. and Yanagimachi, R. (1981) Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipid in the acrosome reaction. Gamete Res., 4, 253–273. Gandini, L., Lenzi, A., Lombardo, F. and Dondero, F. (1993) Glutathione: in vitro effects on human spermatozoa. [Abstr.] In Proceedings of the International Meeting on the Neuroendocrine and Intragonadal regulation of testicular function, Modena. Italy. p. 165. Gaunt, S.J., Brown, C.R. and Jones, R. (1983) Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies. Exp. Cell. Res., 144, 275–284. Gavella, M. and Lipovac, V. (1994) Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa. Int. J. Androl., 17, 308–313. Geva, E., Bartoov, B., Zabludovsky, N. et al. (1996) The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program. Fertil. Steril., 66, 430–434. Gomez, E., Buckingam, D.W., Brindle, J. et al. (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. J. Androl., 17, 276–287

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A.Lenzi et al. Griveau, J.F. and Le Lannou, D. (1994) Effects of antioxidants on human sperm preparation tecniques. Int. J. Androl., 17, 225–231. Griveau, J. F., Renard, P. and Le Lannou, D. (1994) An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int. J. Androl., 17, 300–307. Hall, J.C., Hadley, J. and Doman, T. (1991) Correlation between changes in rat sperm membrane lipids, protein, and the membrane physical state during epididymal maturation. J. Androl., 12, 76–87. Israelachvili, J.N., Marcelja, S. and Horn, R.G. (1980) Physical principles of membrane organization. Q. Rev. Biophys., 13, 121–200. Kessopoulou, E., Powers, H.J., Sharma, K.K. et al. (1995) A double-blind randomized placebo cross-over trial using the antioxidant vitamin E to treat reactive oxygen species associated male infertility. Fertil. Steril., 64, 825–831. Kodama, H., Kuribayashi, Y. and Gagnon, C. (1996) Effect of sperm lipid peroxidation on fertilization. J. Androl., 17, 151–157. Lenzi, A., Lombardo, F., Gandini, L. et al. (1992) Glutathione therapy for male infertility. Arch. Androl., 29, 65–68. Lenzi, A., Culasso, F., Gandini, L. et al. (1993) Placebo controlled, double blind, cross-over trial of glutathione therapy in male infertility. Hum. Reprod., 8, 1657–1662. Lenzi, A., Picardo, M., Gandini, L. et al. (1994) Glutathione treatment of dyspermia: effect on the lipoperoxidation process. Hum. Reprod., 9, 2044–2050. Lenzi, A., Picardo, M., Gandini, L. and Dondero, F. (1996) Lipids of the sperm plasma membrane: from polyunsaturated fatty acids considered as markers of sperm function to possible scavenger therapy. Hum. Reprod. Update, 2, 246–256. Meizel, S. and Turner, K.O. (1983) Stimulation of an exocytotic event, the hamster sperm acrosome reaction by cis-unsaturated fatty acids. FEBS Letts., 161, 315–318. Mortimer, D. (1991) Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum. Reprod., 2, 173–176. Myles, D.G. and Primakoff, P. (1984) Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J. Cell Biol., 99, 1634–1641. Parinaud, J., Le Lannou, D., Viertez, G. et al. (1997) Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit®) following ejaculation. Hum. Reprod., 12, 2434–2436. Sanoka, D., Miesel, R., Jedrzejczak, P. and Kurpisz, M. (1996) Oxidative stress and male fertility. J. Androl., 17, 449–454. Te`rond, P., Auger, J., Legrand, A. and Jouannet, P. (1996) α-tocopherol in human spermatozoa and seminal plasma: relationship with motility, antioxidant enzymes and leukocytes. Hum. Reprod. Update, 2, 739–744. Wolf, D.E., Hagopian, S.S., Lewis, R.G. et al. (1986) Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane. J. Cell. Biol., 102, 1826–1831.

Free radicals, antioxidants and human spermatozoa: clinical implications Eli Geva, Joseph B.Lessing, Liat Lerner-Geva and Ami Amit1 Sara Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, 6 Weixmann Street, Tel Aviv 64239, Israel 1To

whom correspondence should be addressed

This Debate was previously published on Webtrack on Web 10, on March 31, 1998 In recent years, interest has grown regarding the role of oxygen toxicity and free radical reactions in association with fertility 1422

potential, such as superoxide radicals (O2–), hydrogen peroxide (H2O2) and the hydroxyl radical (OH–), can result in peroxidative damage to the cell lipid membranes. Polyunsaturated fatty acids are most susceptible to lipid peroxidation, presumably because of the presence of carbon–carbon double bonds that weaken the carbon–hydrogen bond on the adjacent hydrogen atom, making it susceptible to cleavage. Lipid peroxidation triggers the loss of membrane integrity, causing increased cell permeability to electrolytes. The inward leakage of calcium, and sodium ions in particular, can affect the cell’s energyforming mechanism by causing the cell to become ATPdepleted. An increase in intracellular calcium ions also activates proteases and phospholipases which can lead to further damage to proteins and lipids. This free radical-mediated process may also cause enzyme inactivation with structural damage to DNA, which produces cell death (Halliwell, 1994; Cummins et al., 1994). Mammalian spermatozoa are known to be highly sensitive to injuries caused by high oxygen concentration (Aitken and Clarkson, 1987). Since polyunsaturated fatty acids in the phospholipids of the human spermatozoa are highly susceptible to peroxidation, oxygen free radicals generated by spermatozoa may be involved in the production of spermicidal cytotoxic end products (Selly et al., 1991). The production of abnormal levels of reactive oxygen species (ROS) is now thought to be engaged in many aspects of human male infertility, where spermatozoa are rendered dysfunctional by lipid peroxidation and altered membrane function, together with impaired metabolism, morphology, motility, and fertility (Cummins et al., 1994). A controversy has evolved as to whether the source of ROS in semen of subfertile men originates in the spermatozoa themselves, or in the infiltrating leukocytes (Aitken and West, 1984; Aitken and Clarkson, 1987). Sperm dysfunction may originate in the process of sperm release from the Sertoli cell, and imperfect spermiation (Russell, 1991). Evidently, it is imperative for spermatozoa to eliminate excess cytoplasm. The presence of retained cytoplasmic droplets has been associated with reduced fertility. There may be a link between ROS generation and residual cytoplasm in human spermatozoa through cytoplasmic glucose-6-phosphate dehydrogenase (G6PDH) producing nicotinamide adenine dinucleotide phosphate (NADPH), that in turn serves as a source of ROS (Cummins et al., 1994). The concentration of creatine kinase reflects the degree of cytoplasmic extrusion during the last phase of spermatogenesis (Huszar and Vigue, 1994). Huszar and Vigue (1994) found a close correlation between the rates of sperm creatine kinase activity and lipid peroxidation in the semen. However, a limitation to this model is only circumstantial evidence that spermatozoa contain NADPH oxidase (Ford, 1990). Leukocytes are a potent source of ROS in the semen (Zalata et al., 1995; Parinaud et al., 1997 Parinaud et al., 1997). Following phagocytosis, neutrophils, monocytes, and macrophages produce a burst of oxygen consumption. There is an increase in glucose catabolism caused by activation of the pentose phosphate shunt. This event is termed the ‘respiratory burst’ and is associated with the production and release of ROS (Babior et al., 1976). In addition to spermatozoa, the