Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients

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1 Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients Ralf Henkel, Ph.D., a,b Eva Kierspel, M.Sc., a Thomas Stalf, Ph.D., c Claas Mehnert, M.Sc., c Roelof Menkveld, Ph.D., d Hans-Rudolf Tinneberg, M.D., c Wolf-Bernhard Schill, M.D., a and Thinus F. Kruger, M.D. d a Center for Dermatology and Andrology and c Center for In-Vitro Fertilization, Justus Liebig University, Giessen; b Urological Clinic, Friedrich Schiller University, Jena, Germany; and d Reproductive Biology Unit, Tygerberg Hospital and University of Stellenbosch, Tygerberg, South Africa Objective: To investigate whether there is an impact of different sources of reactive oxygen species (ROS) on sperm functions. Design: Prospective study. Setting: Patients at the Center for Dermatology and Andrology, Giessen, Germany. Patient(s): Semen collected from 63 randomly collected patients attending the IVF unit of the University of Giessen, Germany. Intervention(s): Only patients with nonleukocytospermia were included in this study. Main Outcome Measure(s): Sperm count and motility before and after sperm separation by swim-up, morphology, DNA fragmentation, and extrinsic (by leukocytes) and intrinsic ROS production (by spermatozoa) were evaluated. Result(s): Leukocytes correlated significantly with extrinsic ROS production (r 0.576), but markedly less with intrinsic ROS production (r 0.296). Sperm count, morphology, and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. The DNA fragmentation was strongly positively correlated with intrinsic ROS production, whereas this correlation was weaker for extrinsic ROS production. No correlation was found between DNA fragmentation and the number of leukocytes, whereas the correlations with motility in the ejaculate and the motile sperm count after swim-up were highly significant. Moreover, significant differences were observed for extrinsic and intrinsic ROS production between groups of patients having a high ( /ml) and a low number ( /ml) of leukocytes in the ejaculate. Conclusion(s): The origin of ROS seems to have an influence on the site of the damage. Because leukocyte counts /ml caused a significant decrease of motility and DNA integrity, the threshold given by the World Health Organization (WHO) should be re-evaluated. (Fertil Steril 2005;83: by American Society for Reproductive Medicine.) Key Words: Reactive oxygen species, sperm functions, DNA fragmentation, leukocytospermia, human spermatozoa Received April 15, 2004; revised and accepted August 7, Supported by Research Grant No. I/ of the Volkswagen Stiftung, Hannover, Germany. Reprint requests: Prof. Ralf Henkel, Ph.D., Department of Urology, Friedrich Schiller University of Jena, Lessingstr. 1, D Jena, Germany (FAX: ; ralf.henkel@med.uni-jena. de). Fertilization and pregnancy are dependent on a series of functional sperm parameters, which are reportedly affected by reactive oxygen species (ROS) like hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ), or hydroxyl radical ( OH) (1 5). These highly reactive substances, which exhibit halflife times in the nanosecond ( OH) to the millisecond range (O 2 ), are very strong oxidants and physiologically produced in any living cell during respiration. Because of the extraordinary high content of polyunsaturated fatty acids in the plasma membrane, spermatozoa are highly susceptible to oxidative stress (1), which has repeatedly been shown to be a cause of impaired sperm function and thus male infertility (6 8). This is probably due to the very low content of protective systems and the absence of catalase. On the other hand, it was shown that ROS also have a key position in the control of sperm function by redox regulation of tyrosine phosphorylation (9 11). In the male reproductive system, ROS can derive from leukocytes or from the sperm cells themselves. In the ejaculate, spermatozoa have been repeatedly shown to be only a minor source of ROS production, whereas infiltrating or contaminating leukocytes (peroxidase-positive cells) are the predominant source of these oxidants (7, 12, 13). Leukocytes are present in almost any ejaculate (14) and produce at least 1,000 times more ROS than spermatozoa (15) within the scope of their normal physiological function, immunosurveillance, and thus immunologic defense with elimination of pathogenic germs /05/$30.00 Fertility and Sterility Vol. 83, No. 3, March 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 635

2 TABLE 1 Summary statistics of the 63 non-leukocytospermic patients analyzed in the study. Parameter Mean SD Median Age of patients (y) Sperm count in the ejaculate (10 6 /ml) Motility in the ejaculate (%) Progressive motility (%) Normal morphology (%) Sperm count after swim-up (10 6 /ml) Motility after swim-up (%) Number of peroxidase-positive cells (10 6 /ml) ROS production in the ejaculate (counts/10 7 live sperm) 296, ,043, Percentage of ROS-producing sperm (%) Percentage of TUNEL-positive sperm (%) The World Health Organization (WHO) has defined seminal leukocyte counts of 10 6 leukocytes/ml as leukocytospermia (16). A large number of patients (about 10% 20% of infertile men) (14) show, according to the WHO criteria, an increased number of leukocytes in the ejaculate. Although the determination of leukocytes in the ejaculate is one of the corner stones in andrological diagnosis the clinical relevance of leukocytospermia has been questioned repeatedly. Although some investigators found that ROS levels and seminal leukocyte counts appear to have only little or even no prognostic value for reproduction (17, 18), other researchers attributed even favorable effects on sperm morphology or on some sperm functions (19, 20). On the other hand, there are also reports showing its importance for the deterioration of semen quality and male factor infertility (21, 22) and it was suggested recently that the cutoff value of 10 6 leukocytes/ml for leukocytospermia might be too high (23, 24). In this context, the question raises whether the influence of ROS produced by leukocytes or the sperm cells themselves affect sperm functions in non-leukocytospermic patients. Moreover, it is of clinical interest for assisted reproduction programs to know whether or not these two sources of ROS act differently on male germ cells. Therefore, this study aimed at investigating the impact of extrinsic ROS produced in the ejaculate by leukocytes and defective spermatozoa, and intrinsic ROS produced by the spermatozoa themselves on motility and DNA fragmentation. MATERIALS AND METHODS To investigate the influence of intrinsic ROS, produced by the sperm cells themselves, and extrinsic ROS, mainly produced by leukocytes in the ejaculate, on sperm functions, ejaculates from 63 randomly selected nonleukocytospermic (leukocyte count /ml semen) patients attending the Centre for IVF at the University of Giessen, Germany, were examined. Sperm count, sperm motility, and the number of peroxidase-positive cells (leukocytes) were analyzed according to WHO guidelines (16), whereas normal sperm morphology was performed after Shorr stain according to the Düsseldorf classification and is correlated with the results obtained with strict criteria (25, 26). In addition, motile sperm count before and after conventional swim-up procedure was determined. In brief, semen aliquots were diluted 1:5 with human tubular fluid medium according to Quinn et al. (27) containing 1% serum albumin (Centeon Pharma, Marburg, Germany) (HTF-HSA), centrifuged for 10 minutes at 400 g. The supernatant was discarded and the remaining pellet was overlaid with 1 ml of fresh HTF-HSA and incubated at 37 C for 1 hour. Afterwards, the medium containing the motile spermatozoa was collected. For the determination of sperm DNA fragmentation, a detection kit (Apoptosis Detection System Fluorescein; Promega, Mannheim, Germany) was used and the test was performed as described previously (28). After the final rinses, excess water was drained off, a drop of Anti-Fade solution (Molecular Probes, Eugene, OR) was added, coverslip applied, and 300 randomly selected spermatozoa were immediately analyzed with an epifluorescence microscope (Zeiss, Oberkochen, Germany) at a 1,000 magnification. The percentage of sperm showing green fluorescence (TUNEL-positive) was determined. Negative controls without the TdT enzyme were prepared for each batch of analyzed slides. To determine the intrinsic ROS production within the spermatozoa, 100 L of native ejaculate were diluted 1:2 with phosphate-buffered saline (PBS) (Oxoid, Hampshire, UK) and centrifuged at 300 g for 10 minutes. The supernatant was discarded and the remaining pellet resuspended to a final sperm count of /ml. Twenty microliters ofa20 M dihydroethidine solution (Molecular Probes) was added to 180 L of the cell suspension and incubated for 15 minutes at 37 C. This uncharged, cell-permeant compound, 636 Henkel et al. Intrinsic and extrinsic ROS Vol. 83, No. 3, March 2005

3 TABLE 2 Correlation of the number of leukocytes in the ejaculate, extrinsic and intrinsic ROS production with different sperm parameters from the ejaculate. Number of leukocytes in the ejaculate Extrinsic ROS (ROS production by the leukocytes in the ejaculate) Intrinsic ROS (percentage of ROS-producing sperm) Sperm concentration in the ejaculate Motility in the ejaculate Motile sperm count in the ejaculate Percent normal sperm morphology in the ejaculate r 0.149; P.2473 r 0.465; P.0003 r 0.249; P.0496 r 0.375; P.0057 r 0.418; P.0011 r 0.477; P.0002 r 0.247; P.0552 r 0.516; P.0001 r 0.397; P.0018 r 0.136; P.2918 r 0.345; P.0065 r 0.322; P.0112 Note: The number of leukocytes in the ejaculate shows only a significant negative relationship to sperm motility. By comparing the ROS production, the relation between the respective parameters and the level of significance are markedly stronger; n 63. which has been shown to be specific for the detection of early oxygen metabolites like superoxide (29), is enzymatically dehydrogenated in viable cells to form the cationic ethidium, which is membrane impermeant. It intercalates with the DNA and was visualized under the epifluorescence microscope. Immediately after the labeling of the spermatozoa with the fluorescent probe, a droplet of the sperm suspension was smeared onto a microslide and the percentage of red orange fluorescing sperm was calculated. Considering that leukocytes show an about 1,000 times higher ROS production than spermatozoa and the release of ROS from spermatozoa in the surrounding medium is only about one-third of the total sperm ROS production (7), the sperm cell s contribution to the ROS production in the ejaculate can be neglected and the ROS activity in the ejaculate was regarded as extrinsic ROS. The ROS production in the ejaculate was measured using an MTP reader (Hamamatsu Photronics, Herrsching, Germany) as described before (13) using 0.2 mm luminol (Serva, Heidelberg, Germany) (final concentration) as a nonspecific probe. All statistical calculations (mean, median, Spearman s rank correlation, and Wilcoxon test) were performed after checking normal distribution of the data by means of the Kolmogorov-Smirnov test using the MedCalc program (version , MedCalc Software, Mariakerke, Belgium). A value of P.05 was considered as significantly different. RESULTS The summary statistics of the parameters analyzed are depicted in Table 1. The number of leukocytes in the ejaculate correlated significantly positively with the extrinsic ROS production (ROS produced by leukocytes) (r 0.576; P.0001), but markedly less with the intrinsic ROS production (percentage of spermatozoa producing ROS themselves) (r 0.296; P.0218). There was also a significant positive correlation between the extrinsic and intrinsic ROS production (r 0.324; P.0107). The correlation between extrinsic and intrinsic ROS production with sperm count, morphology and motile sperm count in the ejaculate revealed in every case negative correlations with a higher level of significance for the ROS production in the ejaculate, whereas correlation coefficient and level of significance was rather similar for the motility in the ejaculate (Table 2). However, although the match sperm concentration in the ejaculate and intrinsic ROS correlated significantly (P.0496), it can only be regarded as a tendency. In contrast to the significant matches for extrinsic ROS production, there was only a significantly negative correlation between the number of leukocytes and sperm motility in the ejaculate (r 0.375; P.0057). After sperm separation by means of a conventional swim-up procedure, the correlation between motility and the percentage of ROS-producing sperm (intrinsic ROS) was significant, whereas the respective correlation with extrinsic ROS production as well as the number of leukocytes were not (Table 3). A comparison of the correlation coefficients and the levels of significance of the motility in the ejaculate and after sperm separation revealed markedly lower values after swim-up. Although no correlation was found between sperm concentration after swim-up and intrinsic ROS and the number of leukocytes, the match for extrinsic ROS was significantly negative (Table 3). On the contrary, the levels Fertility and Sterility 637

4 TABLE 3 Correlation of the number of leukocytes in the ejaculate, extrinsic and intrinsic ROS production with different sperm parameters after sperm separation by means of conventional swim-up. Number of leukocytes in the ejaculate Extrinsic ROS (ROS production by the leukocytes in the ejaculate) Intrinsic ROS (percentage of ROS-producing sperm) Sperm concentration r 0.252; P.0569 r 0.333; P.0113 r 0.220; P.0944 after swim-up Motility after swim-up r 0.002; P.9865 r 0.134; P.3088 r 0.315; P.0164 Motile sperm count after swim-up r 0.269; P.0426 r 0.366; P.0053 r 0.376; P.0041 Note: The number of leukocytes in the ejaculate does not or only hardly correlates with sperm concentration, motility, and motile sperm count after swim-up. The comparison of extrinsic and intrinsic ROS production reveals a negative influence of extrinsic ROS on sperm concentration and motile sperm count, whereas intrinsic ROS is negatively correlated with motility and motile sperm count; n 63. of significance for the motile sperm count after swim-up was similar for the number of leukocytes as well as for extrinsic and intrinsic ROS. The correlation between the number of leukocytes and the percentage of spermatozoa showing DNA fragmentations in the TUNEL assay was not significant (r 0.237; P.0716), whereas extrinsic ROS production correlated significantly positively with the TUNEL assay (r 0.576; P.0001). Likewise, the matches between the TUNEL assay with the amount or externally produced ROS (r 0.400; P.0019) and the percentage of ROS-producing sperm (r 0.504; P.0001), respectively, also correlated significantly positive with a stronger correlation for internally by the spermatozoaproduced ROS. Moreover, there were highly significant negative correlations between the percentage of TUNEL-positive sperm in the ejaculate and motility (r 0.523; P.0001), motile sperm count in the ejaculate (r 0.598; P.0001), and the motile sperm count after sperm separation (r 0.539; P.0001), respectively. In contrast, the motility after swim-up revealed no correlation with the TUNEL assay (r 0.104; P.4357). Although 50.4% of the spermatozoa showed intrinsic ROS production in patients having a high number of leukocytes in the ejaculate ( /ml), only 38.8% of the spermatozoa produced ROS in patients with a low number of leukocytes in the ejaculate ( /ml). The difference between both these groups was significant (P.0047) (Fig. 1). The level of significance decreased if this cutoff was set to a higher value ( /ml; P.2811). For the extrinsic ROS production by leukocytes a similar result was observed ( /ml: mean ROS production: 433,547.8 counts/10 7 sperm; /ml: mean ROS production: 5,709.2 counts/10 7 sperm). Although the difference between these two groups is significant (P.0003), the level of significance decreases to P.0549 if this cutoff is set to more than leukocytes/ml. For the percentage of DNA fragmented spermatozoa (Fig. 2) and for sperm motility in the ejaculate (Fig. 3) similar results were found. Compared with patients with a high number of leukocytes in the ejaculate, these exhibited a significantly higher percentage of DNA fragmented sperm (P.0125; mean: 19.2%) and lower motility (P.0013; mean: 44.7% motile sperm) in the ejaculate of those patients with a low number of leukocytes showed a lower percentage of DNA fragmented sperm (mean: 12.7%) and a higher motility (61.6%). In addition, the level of significance decreased if this cutoff was set to a higher value (DNA fragmentation: /ml, P.9783; motility: /ml, P.8747). DISCUSSION The clinical value of leukocytospermia as defined by the WHO (more than 10 6 leukocytes/ml ejaculate) and the influence of leukocytes or leukocyte-derived oxidants have been debated frequently and a final common agreement that leads to clinical guidelines other than that given by the WHO has not been concluded yet. Although Yanushpolsky et al. (30) observed statistically and clinically significant differences at a threshold of more than leukocytes/ml, Tomlinson et al. (17) and Aitken et al. (9) provided data suggesting that leukocyte contaminations in the ejaculate in the range regarded as pathological by the WHO do not affect the fertility status of the subjects. On the contrary, recent work by Sharma et al. (22) and Punab et al. (24) questioned whether the WHO threshold of leukocytospermia is too high. Reasons for these obvious discrepancies can be found in differing study designs, including different methods of de- 638 Henkel et al. Intrinsic and extrinsic ROS Vol. 83, No. 3, March 2005

5 FIGURE 1 Comparison of the percentage of reactive oxygen species (ROS)-producing spermatozoa (intrinsic ROS production) between patients showing a low ( /ml) and high ( /ml) number of leukocytes in the ejaculate. The intrinsic sperm ROS production in patients with a high number of leukocytes in the ejaculate is significantly (P.0047) higher (mean 50.4% ROSproducing spermatozoa) than in those patients showing only a small number of leukocytes in the ejaculate (mean 38.8% ROS-producing spermatozoa). termining leukocytes (31) or in the presence of powerful antioxidants in the seminal plasma (32). However, it is without doubt that leukocytes are the main source of ROS in the ejaculate (12) and that excessive ROS are extremely detrimental to motility, membrane function, and sperm penetration in the sperm penetration assay (6, 8) and can also cause the death of male germ cells. Recently, Alvarez et al. (33) showed that even sperm DNA integrity was impaired in leukocytospermic semen samples. At present, this finding is of particular importance as sperm DNA fragmentation is a cause for fertilization and pregnancy failure in IUI, IVF, and intracytoplasmic sperm injection (ICSI) (13, 28, 34 37). Because Lopes et al. (5) and Irvine et al. (38) showed that sperm DNA fragmentation can also be induced by ROS, produced either by the sperm cells themselves or by leukocytes, the aim of this study was to investigate the impact of ROS produced by leukocytes on sperm functions in nonleukocytospermic patients. In addition, these effects should be differentiated from those caused by the sperm cell s own ROS production. Previous findings revealed that leukocyte-derived extrinsic ROS production is positively correlated with the sperm cell s own intrinsic ROS production (13, 39) and could clearly be confirmed in this study. However, the markedly lower correlation coefficient and level of significance for the correlation between the number of leukocytes in the ejaculate and intrinsic ROS production as compared to the correlation between the number of leukocytes in the ejaculate and extrinsic ROS production is indicative for a different action of ROS from a different origin. This assumption is further supported by the observation that the correlation coefficients and the levels of significance are rather lower for the percentage of ROS-producing spermatozoa if compared with extrinsic parameters like motility in the ejaculate. On the contrary, for intrinsic parameters like sperm DNA fragmentation, a higher level of significance was observed. In addition, the correlation of intrinsic ROS production is much stronger for the intrinsic parameter DNA fragmentation than for motility, even after sperm separation. Thus, the site of ROS production, either inside the spermatozoa themselves or outside by leukocytes, appears to play a role for sperm function. This effect can be explained by the fact that among the oxidants produced by leukocytes, hydrogen peroxide (H 2 O 2 ) is persistent and can even penetrate plasma membranes, whereas other ROS like superoxide (O 2 ) or the hydroxyl radical ( OH) are nonmembrane permeable. Thus, externally by leukocytes produced nonmembrane penetrating ROS will oxidize the phospholipids in the sperm plasma membrane and cause lipid peroxidation, which would affect sperm function or morphology. For the latter, however, it remains questionable as to where leukocytes affect spermatozoa after ejaculation, during epididymal maturation, or during spermatogenesis. It should be noted that leukocytemediated sperm damage gains importance when spermatozoa are separated in vitro and when the seminal plasma, which contains scavengers for ROS (40), is being eliminated. During spermatogenesis, Sertoli cell function can be affected, thus resulting in poor morphogenesis of the sperm. It is well known that poor morphology, especially excess residual cytoplasm, significantly affects sperm fertilizing potential (41). There are also significant cell-to-cell differences in ROS production in subsets of spermatozoa at different maturational stages (42). Spermatozoa that have cytoplasmic residues have a higher content of cytoplasmic enzymes (43, 44), that is, glucose-6-phosphate dehydrogenase (45), which is thought to stimulate the generation of ROS in the spermatozoa themselves (45, 46). The clinical importance of the sperm s own ROS production was underlined by considerably stronger correlations of the percentage of ROSproducing spermatozoa with the different parameters measured compared to the ROS production in the ejaculate (13). This is particularly important as excessively ROS-producing sperm seem to damage themselves. Fertility and Sterility 639

6 FIGURE 2 Comparison of the percentage of spermatozoa with DNA fragmentations as determined by the TUNEL assay between patients showing a low ( /ml) and high ( /ml) number of leukocytes in the ejaculate. Sperm DNA fragmentation in patients with a high number of leukocytes in the ejaculate is significantly (P.0125) higher (mean 19.2% TUNEL-positive spermatozoa) than in those patients showing only a small number of leukocytes in the ejaculate (mean 12.7% TUNEL-positive spermatozoa). On the other hand, hydrogen peroxide, however, can penetrate the plasma membrane and therefore directly damage DNA integrity or induce intrinsic ROS production and thus act in an indirect way on sperm DNA. Our findings confirm the observations by Ramos and Wetzels (47) who found DNA fragmentation in human sperm after the addition of hydrogen peroxide to a sperm suspension. They, as well as Giwercman et al. (48), emphasized the correlation between sperm motility and DNA integrity. In the light of the membrane permeability of H 2 O 2, it is obvious that the external production or addition of this oxidant must have a negative effect on both sperm motility and DNA integrity. In this study and in a previous report (13), we showed that extrinsic ROS produced by leukocytes significantly affect both sperm motility and DNA integrity. However, intrinsic ROS affected the intrinsic parameter of DNA fragmentation. This is consistent with the recent findings of Alvarez et al. (33) and Erenpreiss et al. (49) that leukocytospermia negatively affects sperm DNA integrity. In this report, we show that even in non-leukocytospermic patients seminal leukocytes significantly impair sperm DNA integrity and motility. This is reflected by significantly higher percentages of DNAfragmented sperm and lower motility, respectively, in patients showing leukocytes/ml compared to those patients with a leukocyte count below this threshold. If the threshold was increased, no difference could be observed anymore. The very same criterion was applied to the influence of seminal leukocytes on sperm ROS production. Thus, we can confirm the observation of Sharma et al. (22) that oxidative stress that reduces sperm fertilizing capacity can even occur in patients with seminal leukocyte counts much less than /ml. Plante et al. (7) showed that leukocytes, even at leukocytes/ml, did not affect sperm motility, unless they were activated. On the other hand, Aitken et al. (32) demonstrated that leukocyte contaminations of more than leukocytes/ml have detrimental effects on Percoll-separated spermatozoa. Consequently, the question arises whether the WHO threshold for leukocytospermia ( /ml) is still justified, at least if the seminal fluid that normally provides a massive protection against oxidative stress is removed by sperm preparation for assisted reproduction. Therefore, it appears to be of paramount importance if spermatozoa and leukocytes are in suspension in ejaculate or medium (which does or does not contain scavengers for ROS) and thus being far away from each other or if they are in close cell-to-cell contact as it is the case in sperm separation for assisted reproduction. This might explain the discrepancies between different reports. Because even low amounts of ROS are harmful to sperm DNA integrity (50), a causality between leukocytes in the ejaculate and DNA fragmentation should not be neglected. Therefore, to maintain sperm fertilizing capacity for assisted reproduction purposes there is a necessity to reduce the seminal ROS levels as well as leukocyte concentrations, even below /ml, by antibiotic and antiphlogistic treatments. Considering this enormous detrimental effect of ROS on sperm functions and DNA fragmentation with its possibility of failed pregnancy (13, 28) and an increased risk of childhood cancer in the offspring (51), which is due to the vulnerability of human sperm DNA during late stages of spermatogenesis and epididymal maturation (52), careful sperm separation techniques play a crucial role. This would not only include the application of such sperm separation methods that separate leukocytes from the spermatozoa first and only in a second step the seminal fluid (for review, see Henkel and Schill, 53), but also the addition of antioxidants to sperm separation media as suggested by Baker et al. (54). This would also require the determination of an excessive production of ROS or a lack of antioxidant capacity in the sperm environment (22, 55). In conclusion, this study shows that the origin of ROS either from leukocytes or from the sperm cells themselves seems to have an influence on the site of the damage. In addition, because even leukocyte counts much less than the normal value as defined by the WHO contributed to a sig- 640 Henkel et al. Intrinsic and extrinsic ROS Vol. 83, No. 3, March 2005

7 FIGURE 3 Comparison of sperm motility in the ejaculate between patients showing a low ( /ml) and high ( /ml) number of leukocytes in the ejaculate. Sperm motility in patients with a high number of leukocytes in the ejaculate is significantly (P.0013) lower (mean 44.7% motile spermatozoa in the ejaculate) than in those patients showing only a small number of leukocytes in the ejaculate (mean 61.6% motile spermatozoa in the ejaculate). nificant decrease of motility and DNA integrity, this definition given by the WHO should be re-evaluated. Perhaps, the evaluation of a leukocyte contamination should be done in more detail by especially differentiating macrophages as suggested by Haidl (56) or more reliable and accurate detection methods should be applied (31). Finally, the pathophysiology of ROS and the impact of leukocytes on spermatozoa and DNA integrity should be better understood. 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