Proinflammatory Cytokines as an Intermediate ... - Wiley Online Library

Journal of Andrology, Vol. 29, No. 1, January/February 2008 Copyright E American Society of Andrology

Proinflammatory Cytokines as an Intermediate Factor Enhancing Lipid Sperm Membrane Peroxidation in In Vitro Conditions MONIKA FRACZEK, DOROTA SANOCKA, MARZENA KAMIENICZNA, AND MACIEJ KURPISZ From the Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT: We have examined the effect of white blood cells (WBCs), various proinflammatory cytokines, or a combination of the two on the peroxidation of human sperm membrane lipids in in vitro conditions. Six recombinant cytokines, such as interleukin-1b (IL-1b), IL-6, IL-8, IL-12, IL-18, and tumor necrosis factor alpha (TNF-a), used singly or in combinations, were analyzed. WBCs were isolated from the whole heparinized blood using a density gradient technique (Histopaque 1.077). Spermatozoa were isolated from semen samples with normal sperm parameters by both the swim-up technique (swim-up fraction) and by a discontinuous Percoll gradient centrifugation (90% and 47% Percoll fractions). Peroxidative damage to sperm membrane lipids was assessed by determining the concentration of malondialdehyde (MDA) in lysates of spermatozoa using high-performance liquid chromatography (HPLC). There were no statistically significant differences in MDA concentrations be-

tween sperm fractions incubated with cytokines and respective controls (spermatozoa alone). In spermatozoa isolated by the swimup technique, the MDA level was significantly higher only after incubation with IL-6 and IL-8 plus WBCs when compared to sperm incubated with leukocytes alone (0.62 6 0.21 mmol/L and 0.42 6 0.22 mmol/L, respectively; P , .05). In spermatozoa recovered from the 47% Percoll, only a combination of IL-12 and IL-18 used together with WBCs was linked with a significant increase in MDA concentration (from 0.41 6 0.13 mmol/L to 0.65 6 0.19 mmol/L; P , .05). The results obtained suggest that cytokines produced during the inflammatory process intensify the level of oxidative stress caused by leukocytes, which may have serious consequences for sperm membrane integrity. Key words: Semen inflammation, peroxidative damage. J Androl 2008;29:85–92

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Proinflammatory cytokines are the natural components of seminal plasma (Maegawa et al, 2002). In numerous reports, proinflammatory cytokines were found to regulate a physiologic function of the male gonad and to act as testicular immunomodulatory elements (Hales et al, 1999; Soder et al, 2000; Rozwadowska et al, 2005). Most of them are also involved in the fertilization process (Naz and Evans, 1998; Diemer et al, 2003; Huleihel and Lunenfeld, 2004). On the other hand, the same cytokines have been linked with a decrease in semen quality of infertile men, particularly those with urogenital tract infections (Naz and Kaplan, 1994; Gruschwitz et al, 1996; Eggert-Kruse et al, 2001; Kocak et al, 2002; Friebe et al, 2003; Paulis et al, 2003). In our earlier study, we demonstrated that cytokines deepen oxidative stress, not only by the intensification of the inflammatory process but by a direct influence on pro-oxidative and antioxidative system components as well (Sanocka et al, 2003). The oxidative stress with its consequences for sperm membranes can lead to a damage of sperm biologic function, in spite of the fact that semen consists of spermatozoa subpopulations with different fertilizing potential. As clinical significance of selected proinflammatory cytokines and their influence on male fertility reduction is poorly documented, we decided to assess an in vitro

n a number of studies performed thus far, the inflammatory process within the male genitourinary tract and accompanying oxidative stress were found to reduce fertilizing potential of mature spermatozoa (de Lamirande and Gagnon, 1995; Griveau and Le Lannou, 1997; Aitken, 1999). In response to an inflammatory factor, leukocytes migrate to the site of inflammation, where they mobilize the host defense system. The activated macrophages and neutrophils release reactive oxygen intermediates (ROIs) and secrete proinflammatory cytokines, both of which can affect spermatozoa (Aitken and Fisher, 1994; Wolff, 1995). Proinflammatory cytokines are one of the mediators of the host response to inflammatory reaction. Generated by both the cells of the immune system as well as those of surrounding tissues in response to external stimuli, inflicted injury, or other cytokines, they modulate inflammatory reactions, acting as the regulatory factors in different biologic processes, such as cell activation, proliferation, growth, differentiation, and mobility (Feldmann and Saklatvala, 2001). Correspondence to: Dr Maciej Kurpisz, Professor, Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479 Poznan, Poland (e-mail: [email protected]). Received for publication May 11, 2007; accepted for publication August 15, 2007. DOI: 10.2164/jandrol.107.003319

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effect of various cytokines (pathologic concentrations) taking part in the inflammatory process on peroxidation of sperm membrane lipids. Six recombinant proinflammatory cytokines, such as interleukin-1b (IL-1b), IL-6, IL-8, IL-12, IL-18, and tumor necrosis factor alpha (TNF-a), were chosen for the study. They were used singly or in combinations according to natural interactions taking place during the inflammatory process, which some investigators have already observed in semen (Depuydt et al, 1996; Kocak et al, 2002; Sanocka et al, 2003).

Materials and Methods Human recombinant cytokines were applied in the study. IL1b was purchased from R&D Systems Inc. (Minneapolis, MN). IL-6, IL-8, IL-12, and TNF-a were obtained from Sigma (St. Louis, MO). IL-18 was purchased from MBL Co. Ltd. (Nagoya, Japan).

Preparation of White Blood Cell (WBC) Suspensions Specimens of heparinized venous blood were collected from 10 healthy adults donating to the Regional Blood Center, Poznan´, Poland. The WBCs were isolated by a density gradient centrifugation technique using Histopaque 1.077 (Sigma). The gradients were centrifuged at 400 6 g for 20 minutes at room temperature. The WBCs were harvested from buffy coats. After two washes in Hanks’ balanced salt solution (HBSS) supplemented with 0.5% lactoalbumin hydrolyzate and sodium bicarbonate (Biokom, Lublin, Poland), the cell pellets were resuspended in 0.83% NH4Cl-Tris, pH 7.4, and incubated for 15 minutes at room temperature in order to lyse the contaminating erythrocytes. The cell suspensions were centrifuged at 250 6 g for 10 minutes and washed twice in phosphate-buffered saline (PBS), pH 7.4. Cells were counted and checked for the viability by 1% trypan blue staining. The WBC suspensions were then adjusted with PBS to a concentration of 1 6 107 cells/ml.

Semen Preparation Semen samples were obtained from 10 healthy volunteers with no sperm abnormalities attending the Outpatient Clinic for Andrology (Poznan, Poland) after 4 days of sexual abstinence. The specimens were allowed to liquefy for 30 minutes at room temperature. An aliquot of each semen specimen was subjected to routine semen analysis in accordance with the guidelines published by the World Health Organization (WHO, 1999). Data recorded included semen volume, viscosity, pH, sperm count, motility, morphology, and viability. Sperm concentration and motility were determined using a Makler counting chamber. Sperm viability was assessed using 1% eosin Y solution. Sperm morphology was evaluated after Papanicolaou staining and was scored according to Kruger’s strict criteria (Kruger et al, 1986). Each semen specimen was tested for the presence of leukocytes by the Endtz test (Endtz, 1974). All

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tested samples were subjected to microbiologic analysis. In addition, semen samples were checked for the presence of antisperm antibodies using direct immunobead test (DIBT; Irvine Scientific, Santa Ana, Calif ). Only semen samples from healthy volunteers with sperm concentrations .20 6 106/mL of semen, sperm progressive motility (A + B category) .50%, sperm morphology .14%, absence of antisperm antibodies, leukocyte concentrations ,1 6 106/mL of semen, and bacteria concentration ,1 6 103/mL of semen were used for further experiments. Semen samples were then divided into two aliquots. The first aliquot was used for spermatozoa separation by swim-up technique incubating of fresh semen samples for 1 hour at 37uC in F10 (Ham’s) solution supplemented with L-glutamine and sodium bicarbonate (Biokom). From the second aliquot, spermatozoa were isolated by a double-step discontinuous Percoll gradient (47% and 90%; Sigma), centrifuging at 450 6 g for 30 minutes at room temperature. The seminal plasma was then discarded, and spermatozoa were harvested from the 90% Percoll and 47% Percoll fractions. The cells from all three sperm fractions were then washed twice in PBS and finally adjusted to a density of 1 6 107 spermatozoa/mL in PBS. Samples containing 1 6 106 spermatozoa (the pool of two volunteers’ semen samples) from each fraction were incubated with recombinant cytokines and/or WBCs for 1 hour at 37uC. WBCs (the pool of two donors’ blood samples) were added at a concentration of 1 6 106 cells per milliliter of co-incubated mixture. Cytokines were added at pathologic concentrations of 50 pg, 200 pg, 500 pg, 50 pg, 500 pg, and 50 pg per milliliter of co-incubated cell suspension, respectively, for IL-1b, IL-6, IL-8, IL-12, IL-18, and TNF-a. The concentrations of IL-1b, IL-6, IL-8, and TNF-a were chosen according to our earlier observations regarding infertile patients with genital tract infection/inflammation (Sanocka et al, 2003; Sanocka et al, 2004). As for the concentrations of IL-12 and IL-18, they were chosen according to the other relevant reports (Nakanishi et al, 2001; Matalliotakis et al, 2006). These co-incubated cell suspensions were next selectively depleted to obtain pure sperm samples for membrane lipid peroxidation assay.

Depletion of WBCs WBCs were removed from the reaction mixture using a Dynal MPC-1 immunomagnetic cell isolation system (Dynal, Oslo, Norway). During 20 minutes of incubation at 4uC, CD45+ cells were adsorbed onto magnetic M-450 Dynabeads (Dynal, Oslo, Norway). Spermatozoa then were eluted in a magnetic field at 4uC. The eluates were collected, washed, and resuspended in PBS for a final concentration of 1 6 106 sperm/mL. Sperm pellets were lysed with isotonic 10 mmol/L potassium buffer phosphate, pH 7.2, and stored at 220uC until they were used for malondialdehyde (MDA) measurement.

Determination of MDA The lipid peroxidation was measured on the basis of MDA amounts produced in different sperm fractions incubated together with cytokines and/or WBCs. MDA levels were determined by a high-performance liquid chromatography (HPLC) technique in a Waters HPLC system (Waters,

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Cytokines and Sperm Lipid Peroxidation

Milford, MA; Nielsen et al, 1997). Each 1 ml of sample contained: 100 ml sperm lysate; water (for a blank sample) or the known MDA concentration; 700 ml of 1% orthophosphoric acid; and 200 ml of 42 mmol/L 2-thiobarbituric acid (TBA) in 0.1 mol HCl. The samples were heated in a water bath for 1 hour at 100uC. After cooling, the samples were mixed with 2 mol/L NaOH/methanol (1:12, v/v) and centrifuged for 3 minutes at 13 000 6 g. Fifty-microliter aliquots of supernatant were injected onto the LiChroCART 250-4 column packed with 5 mm LiChrospher 100 RP-18 (Merck, Darmstadt, Germany). Each sample was run in duplicate. Water served as a blank sample. The samples were eluted for 8 minutes at a flow rate of 0.5 ml/min using a 3:2 (v/v) mixture of 10 mol/L KH2PO4 (pH 6.8) and methanol. The absorbance of the eluate at 532 nm was determined using an ultraviolet detector. MDA concentration was calculated from the area of the peak at 6.8 minutes using a standard curve prepared with triplicate serial dilutions of freshly hydrolyzed 0.1 mmol/L 1,1,3,3-tetraethoxypropane (TEP; Sigma). The TEP was hydrolyzed for 1 hour at 50uC.

Statistical Analysis All statistical calculations were performed using the STATISTICA software package, 6.0 version (StatSoft, Tulsa, OK). The significant differences were assessed using nonparametric (Friedman and Mann-Whitney U) tests and were presented as median 6 average deviation (AD). P , .05 was considered significant, P , .01 very significant, and P , .001 most significant.

Results Effect of Cytokines on MDA Concentrations in Sperm Fractions The results for MDA concentrations in the three sperm preparations incubated with cytokines, leukocytes, or both are presented in the Figure. There were no statistical differences in MDA concentrations in sperm incubated with cytokines versus sperm incubated alone. This was true for all sperm fractions used in the study (Figure, panel A). In general, the addition of leukocytes resulted in elevated sperm MDA, regardless of proinflammatory cytokine applied (Figure, panel B). Incubation of spermatozoa isolated by the swim-up technique with IL-6 and IL-8 plus WBCs significantly increased the MDA content in comparison with sperm incubated with leukocytes alone (0.62 6 0.21 mmol/L and 0.42 6 0.22 mmol/L, respectively; P , .05). An increase in MDA concentration was also significant in spermatozoa recovered from the 47% Percoll after their incubation with a combination of IL-12 and IL-18 together with WBCs (from 0.41 6 0.13 mmol/L to 0.65 6 0.19 mmol/L); P , .05. In a situation where spermatozoa from 90% Percoll fraction were applied,

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none of the cytokines used together with WBCs exerted a significant effect on sperm lipid peroxidation compared with sperm incubated with leukocytes alone (Figure, panel B).

Effect of WBCs on MDA Concentration in Sperm Fractions The Mann-Whitney test discriminated the WBC effect on MDA content depending on the proinflammatory cytokines and sperm fractions used in the study (Table 1). Only in cases of spermatozoa obtained from the 90% Percoll fraction did WBCs themselves induce the lipid peroxidation in the absence of cytokines, and the effect was close to a significant levels. In the swim-up sperm fraction, the presence of WBCs had a positive influence on the level of MDA when IL-12 or IL-18 (used individually) or when IL-6 combined with IL-8 or TNF-a was applied. In most cases, leukocytes and cytokines used together caused a significant increase of membrane lipid peroxidation in spermatozoa from the 90% Percoll fraction in comparison to sperm incubated only with respective cytokines. As for spermatozoa from 47% Percoll fraction, only in cases of IL-18 and TNFa used individually or together with other cytokines did WBCs have a statistically significant influence on MDA content.

Discussion The activation of leukocytes in a site of the inflammatory process is strictly connected with both the oxidative burst and the initiation of an immune response directed against infectious agents. The secretion of cytokines is one of the first signals from the innate host defense to combat inflammation. The in vitro model of semen inflammation created in this study was designed to determine the effect of two main mediators of the inflammatory process, namely, proinflammatory cytokines and WBCs, on sperm lipid peroxidation as measured by the MDA levels. In the present study we did not observe a direct in vitro effect (no statistical differences when compared to sperm controls) of proinflammatory cytokines on MDA content in sperm membranes (Figure, panel A). This observation is a proof that proinflammatory cytokines do not act separately but in association with the other mediators of the inflammatory process, which was previously suggested (Das, 1991; Rajasekaran et al, 1995; Depuydt et al, 1996). Our results are not entirely in agreement with another recent report published by Martinez et al (2007) in which the authors showed that some cytokines in physiologic concentrations can act directly on sperma-

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Figure. Influence of selected proinflammatory cytokines on malondialdehyde (MDA) concentration in different spermatozoal fractions: (A) without white blood cells (WBCs); (B) after incubation with WBCs. The results are expressed as median 6 AD. Significantly different when compared with sperm alone or sperm with WBCs only ([A] and [B], respectively); *P , .05; calculated using Friedman test.

tozoa enhancing the level of their lipid membrane peroxidation. In our study, we have also analyzed the wider set of proinflammatory cytokines, mostly at pathologic concentrations. Demonstration of lipid peroxidation increase in the presence of cytokines at physiologic concentrations seems somehow controversial, although the longer incubation time and different statistical evaluation could have some influence on final interpretation of obtained by Martinez et al (2007). A characteristic feature of proinflammatory cytokines is their synergistic, additive, or antagonistic activity, due to which they can influence the function of the target cells in vivo in a number of ways. This suggestion was also included in the final conclusions of the paper of Martinez and coworkers, although it was not experimentally verified (Martinez et al, 2007). In our study, in which we have used various combinations of cytokines, this particular conclusion was extended an empirical level, thus adding to the understanding of cytokines’

nature during the inflammatory process in male genitourinary tract. The levels of IL-6 and IL-8 in seminal plasma have been often demonstrated as the factors linked with a decrease in quality of seminologic parameters (Gruschwitz et al, 1996; Eggert-Kruse et al, 2001; Furuya et al, 2003; Sanocka et al, 2003; Kopa et al, 2005). The significant increase in the MDA level in spermatozoa with the best seminologic parameters (swim-up sperm fraction) after their in vitro incubation with IL-6, IL-8, and WBCs (Figure, panel B) may probably be related to observed infertility in vivo. This was already suggested by several authors who presented positive correlations between IL-6 or IL-8 levels and MDA concentration in seminal plasma of infertile men (Camejo et al, 2001; Liu et al, 2003). Moreover, the application of IL-6 together with IL-8 exerted a synergistic effect in respect to their harmful influence on the sperm membranes (Figure, panel B). Probably, the prolonged presence of high levels of IL-6 and IL-8 in

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Table. Comparison of malondialdehyde (MDA) concentrations in spermatozoal fractions without leukocytes and following incubation with leukocytes applying selected proinflammatory cytokines (n 5 5)* ‘‘Swim-Up’’ Sperm Fraction

No cytokines vs WBCs (control) IL-1b vs IL-1b + WBCs IL-6 vs IL-6 + WBCs IL-8 vs IL-8 + WBCs IL-1b + IL-8 vs IL-1b + IL-8 + WBCs IL-6 + IL-8 vs IL-6 + IL-8 + WBCs IL-12 vs IL-12 + WBCs IL-18 vs IL-18 + WBCs IL-12 + IL-18 vs IL-12 + IL-18 + WBCs TNF-a vs TNF-a + WBCs IL-1b + TNF-a vs IL-1b + TNF-a + WBCs IL-6 + TNF-a vs IL-6 + TNF-a + WBCs IL-8 + TNF-a vs IL-8 + TNF-a + WBCs

90% Percoll Isolated Sperm Fraction

47% Percoll Isolated Sperm Fraction

Z

P

Z

P

Z

P

NS NS NS NS NS 22.498 22.876 23.142 NS NS NS 22.624 NS

NS NS NS NS NS 0.012 0.004 0.0017 NS NS NS 0.009 NS

21.925 23.689 24.760 22.225 23.334 23.393 22.890 23.216 NS 22.779 22.580 25.448 23.519

0.046 0.0002 0.000002 0.026 0.0008 0.0007 0.0038 0.0013 NS 0.0054 0.01 0.000000 0.0004

NS NS NS NS NS NS NS 22.033 23.223 22.210 22.839 22.720 23.208

NS NS NS NS NS NS NS 0.042 0.0013 0.027 0.0045 0.0065 0.0013

* Z indicates Mann-Whitney test values; WBCs, white blood cells; NS, statistically nonsignificant; IL-1b, interleukin-1b; and TNF-a, tumor necrosis factor alpha.

semen during the genitourinary tract inflammation can lead to persistent sperm damage resulting from the peroxidative process. The assessment of the proinflammatory cytokine levels in semen can be supplementary to the evaluation of the male genital tract inflammation in vivo. Our data once again support the need for the examination of these two cytokines as the excellent markers that can be used to identify an early phase of the inflammatory process in the male genitourinary system (Depuydt et al, 1996; Eggert-Kruse et al, 2001; Sanocka et al, 2003). Many authors have recently focused on a new member of the IL-1 family, namely, IL-18 (Dinarello, 1999; Nakanishi et al, 2001). Most of them postulated a rather pathologic role for this cytokine in many diseases (Fassbender et al, 1999; Tsutsui et al, 2000; Matsui et al, 2003; Ogata et al, 2004; Malaguarnera et al, 2006). Matalliotakis et al (2006) have also emphasized the detrimental influence of the elevated IL-18 levels on semen parameters and suggested the presence of IL-18 as another marker for male genital tract inflammation. In our research, IL-18 applied in combination with IL-12 was more effective in exerting a deleterious effect of oxidative stress caused by leukocytes on sperm membranes than either of cytokines used singly (Figure, panel B). Thus, our results confirmed a synergism between these two cytokines that has been previously postulated (Munder et al, 2001). However, views on the relationship between high levels of IL-12 and male infertility are not coherent, and this cytokine is also known for taking part in the normal functioning of male reproductive system (Naz and Evans, 1998). It is conceivable that IL-18 is mainly

a mediator that may affect sperm membranes through the induced changes in the antioxidative system of male gametes (Wereszczynska-Siemiatkowska et al, 2004). Taking into consideration that some cytokines, such as IL-1b, IL-18, or TNF-a, participate in inflammatory reaction through the induction of cell apoptosis, we cannot preclude a fact that membrane peroxidation is not the only mechanism by which some proinflammatory cytokines affect spermatozoa during the male reproductive tract infection/inflammation. Many authors have observed correlations between the levels of proinflammatory cytokines and the number of leukocytes in semen (Shimoya et al, 1993; Rajasekaran et al, 1995; Eggert-Kruse et al, 2001; Liu et al, 2003; Jedrzejczak et al, 2005; Kopa et al, 2005). However, there have been also reports demonstrating the elevated levels of proinflammatory cytokines in semen, regardless of the presence or absence of leukocytes (Alexander et al, 1998; Naz and Evans, 1998). The intensification of sperm cells’ membrane peroxidation after their exposure to both cytokines and leukocytes observed in this study reconfirmed proof for the involvement of proinflammatory cytokines in the deepening of harmful activity of oxidative stress to spermatozoa. Moreover, during the inflammatory process, ROIs produced by leukocytes and due to proinflammatory cytokines cooperate with each other in provoking the structural sperm disorder. This is most probably connected with the changes in the activity of both enzymatic and nonenzymatic members of the semen antioxidative system. Our previous reports demonstrated a clear relationship between some proinflammatory cytokines and the pro-oxidative and antioxidative components of seminal plasma in patients with

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genital tract inflammation (Sanocka et al, 2003). Other authors also showed inducing properties of IL-6 toward mitochondrial SOD (Isoherranen et al, 1997). It may be hypothesized that high activities of proinflammatory cytokines in semen influence the intensity of oxidative stress, which may then have dangerous consequences for spermatozoa, indirectly affecting their redox profile as well as their close environment. The present study is a continuation of our previous observations regarding inflammatory mediators in human semen, in which we used three preparations of spermatozoa differing structurally and functionally (Fraczek et al, 2004; Fraczek et al, 2007). In general, MDA concentrations observed in this study were lower in sperm from the 90% Percoll pellet when compared to the other fractions (Figure). These results are in agreement with the data presented in the other papers showing that differences in the rates of lipid peroxidation are related to sperm maturity and, presumably, sperm membrane structure (Aitken et al, 1994; Huszar and Vigue, 1994; Zalata et al, 1998) that can be also affected by gradient procedure itself (Kobayashi et al, 1991; Alvarez et al, 1993). The mediating role of leukocytes with respect to the harmful effects of secreted proinflammatory cytokines toward sperm membranes depended on the type of applied spermatozoal fraction and was the highest in spermatozoa from the 90% Percoll fraction, although in most observed cases the levels of MDA still remained lower when compared to sperm from the swim-up as well as the 47% Percoll fraction (Table 1). On the other hand, the subpopulation of spermatozoa recovered from the 90% Percoll gradient turned out to be the most susceptible to lipid peroxidative process in the presence of cytokines combined with leukocytes. These results are in agreement with our earlier report, in which we demonstrated differences in quantities of peroxidative products between sperm separated by the swim-up technique and those obtained from 90% Percoll gradient in the presence of bacterial strains (Fraczek et al, 2007). In our view, lower MDA levels observed in spermatozoa from the 90% Percoll fraction compared with the swim-up sperm fraction may originate from the gradient procedure and number of centrifugation used that can initiate shedding of peroxidative products from membranes. Thus, such manipulations cannot be a recommendation to use of any gradients for the separation of gametes aimed for assisted reproductive applications. To conclude, the results obtained in this study clearly demonstrated that proinflammatory cytokines per se, even in pathologic concentrations, are unable to cause oxidative stress in semen to the level of membrane peroxidative damage. The assessment of sperm DNA integrity rather than membrane peroxidation in sper-


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matozoa could provide further useful information in explaining the pathologic role of cytokines toward male gametes during male reproductive tract infection/inflammation. Because the harmful effect of cytokines on spermatozoa is closely connected to the accompanying leukocytospermia, we may also conclude that the evaluation of leukocyte concentration in semen still remains the important but insufficient approach to the diagnosis and treatment of male genital tract infection/ inflammation and its particular stage or kinetics.

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