Semen granulocyte elastase: its relevance for the diagnosis and prognosis of silent genital tract inflammation

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1 Human Reproduction vol.15 no.9 pp , 2000 Semen granulocyte elastase: its relevance for the diagnosis and prognosis of silent genital tract inflammation B.Zorn 1, I.Virant-Klun and H.Meden-Vrtovec Several tests have been proposed as markers of inflammation in men, including either markers of specific infection known Andrology Centre, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia for a deleterious effect on male reproductive function [for example the determination of antibodies specific for Chlamydia To whom correspondence should be addressed at: Andrology trachomatis in seminal plasma; (Keck et al., 1998)], or non- Centre, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, S lajmerjeva 3, SI-1000 Ljubljana, specific markers of inflammation such as leukocytospermia Slovenia. milica.trenkic@guest.arnes.si (Wolff, 1998). According to World Health Organization criteria (WHO, 1992), leukocytospermia, defined as the presence of Elastase inhibitor complex was assessed by immunoassay white blood cells (WBC)/ml of semen, is considered in the seminal plasma of 312 men attending the outpatient as a possible indicator of ongoing male genital tract infection. infertility clinic. Using receiver operating characteristic Besides leukocytospermia, organ-specific markers such as (ROC) curve analysis, elastase at the cut-off value of fructose, α-glucosidase, citric acid (Zalata et al., 1996), and 290 ng/ml was shown to be efficient (sensitivity 79.5%, organ non-specific markers such as albumin, C-reactive protein, specificity 74.4%) in the detection of genital tract different cytokines, mainly interleukins 6 and 8 (Shimoya inflammation as defined by leukocytospermia (> et al., 1993; Eggert-Kruse et al., 1995; Comhaire et al., 1999), leukocytes/ml). The prevalence of increased elastase in 292 and complement C3 (Ludwig et al., 1998) have been evaluated infertile men was significantly higher (34%) as compared in semen. Others (Purvis and Christiansen, 1993) found rectal with that (5%) observed in 20 fertile men (P 0.02). ultrasound to be important in the detection of non-symptomatic Moreover, high elastase concentration ( 290 ng/ml) was deep pelvic infections. observed in 66 of the 264 men (25%) without leukocyto- One of the main changes during the inflammatory process spermia. A significant positive correlation was found is the discharge by polymorphonuclear granulocytes (PMN) between elastase concentration and patient age (r 0.202, of large amounts of proteases such as elastase. As granulocytes P < ) and the number of leukocytes (r 0.330, are the main constituents of the WBC population in semen, P < ). A negative correlation was found between the evolution of elastase in seminal plasma is of clinical elastase concentration and semen volume (r 0.146, importance in detecting inflammation. The enzyme elastase, P 0.01) and the percentage of spermatozoa with singleparticularly the elastase-α 1 protease inhibitor complex (Ela/ stranded DNA (r 0.194, P 0.024), but there was no correlation between elastase and sperm reactive oxygen α 1 PI) has been suggested as a sensitive and quantitative species production. A higher seminal elastase concentration marker of genital tract inflammation in general (Jochum et al., was significantly associated with tubal damage in female 1986; Schill et al., 1995) and in particular of chronic prostatitis partners (P < 0.001). After norfloxacine antibiotic therapy, (Ludwig et al., 1998), as determined by clinical and bacterio- decrease in elastase concentration was observed in 15 logical features. Apart from its beneficial anti-inflammatory (25%) of the 60 treated patients. Tubal damage in the effects, elastase per se provokes cell deterioration with the partner negatively affected the response to antibiotic synthesis of reactive oxygen species (ROS) which may lead therapy. In conclusion, granulocyte elastase is a reliable to cell death (Hautamaki et al., 1997). screening test for silent genital tract inflammation of The aims of the present study were to assess the prevalence the couple. The elastase inhibitor complex may have a of high elastase concentration in the seminal plasma of infertile protective effect in reducing sperm DNA denaturation. men in comparison with that in fertile men, to study the Key words: diagnosis/prognosis/seminal elastase/silent inflamtract inflammation, and to verify the hypothetical deleterious reliability of seminal elastase in detection of silent male genital mation/sperm quality effect of elastase on sperm classical characteristics and sperm functional activity evaluated by ROS production and the Introduction presence of single-stranded DNA. Inflammation of the genital tract is alleged to be responsible for between 4% and 10% of male infertility (Thonneau et al., 1992), but this is not easily proven (Ness et al., 1997). Most Materials and methods often, the main difficulty resides first in the diagnosis of Study population inflammation and second, in demonstration of the causal link The study population consisted of 312 men attending the infertility between inflammation and male infertility (Auger, 1998). outpatient clinic at the Andrology Centre of the Department of 1978 European Society of Human Reproduction and Embryology

2 Seminal elastase and silent genital tract inflammation Obstetrics and Gynecology of Ljubljana between January 1996 and and minimize ROS production by leukocytes. The sperm ROS June production detected by luminescence was recorded in the integration The infertile population (n 292) consisted of 224 men with mode for 10 s with constant stirring of the analysed sample. Readings oligoasthenoteratozoospermia (OAT) according to WHO criteria were taken every 5 min for 30 min, with a peak of luminescence (WHO, 1992), 40 normozoospermic men from infertile couples, and observed between 5 and 15 min. After subtracting the appropriate 28 normozoospermic men with one or more unsuccessful conventional blanks, the peak luminescence was considered detectable when the IVF embryo transfer attempts. The control group consisted of 20 luminescence was 0.05 mv/s. Peak luminescence observed at 5 fertile men who fathered children spontaneously within the study 15 min after the addition of luminol was expressed in mv/s per 10 9 period. None of the patients had clinical signs or symptoms of a spermatozoa. genital tract infection. None had been treated by antibiotics within 3 months before enrolment into the study. All patients had given their Sperm single-stranded DNA assessment by acridine orange staining informed consent for participation. After semen preparation on discontinuous PureSperm gradient and washing, single-stranded DNA was detected by acridine orange (AO) Clinical examination staining according to a published method (Liu and Baker, 1994). Airdried Male partners were interviewed about their andrological history, with sperm smears were fixed in Carnoy s solution overnight, after a particular emphasis on clinical or biological events possibly related which they were rinsed in phosphate-buffered saline and air-dried. to a previous genital tract infection; this was followed by a standard Sperm smears were stained for 5 min with 1% AO solution, prepared clinical examination. Rectal examination of the prostate was performed as follows: 10 ml of 1% AO in distilled water was added to a mixture in cases of leukocytospermia or high elastase concentration. When of 40 ml of 0.1 mol/l citric acid and 2.5 ml of 0.3 mol/l Na 2 HPO 4 7H 2 O. the rectal examination was abnormal, it was completed by a prostato- After AO staining, the slides were rinsed and mounted in distilled vesicular ultrasound scan according to WHO recommendations (Rowe water, and then observed by fluorescence microscopy (Axioskop; et al., 1993). Abacterial chronic prostatitis was suspected in the Carl Zeiss Jena, Germany) at 400 final magnification. AO stain presence of glandular asymmetry, hypoechogenicity or hyperechogenicity intercalates in sperm single-stranded DNA as a polymer, providing associated with areas of calcification (Vicari, 1999). Additionally, red, orange, or yellow fluorescence of the sperm head, whereas in data from the female partner s history (hysterosalpingography, endometrial normal, double-stranded DNA, it intercalates as a monomer, providing histology and laparoscopy) as well as histological endometritis, green fluorescence. At least 100 spermatozoa were assessed on each endometriosis and tubal damage found at laparoscopy, were taken slide. From that count and the count of red, orange or yellow into consideration. Tubal damage was defined as the presence of one spermatozoa, the percentage of sperm containing single-stranded or both tubes closed at hysterosalpingography, and/or one or both DNA was calculated. tubes closed with adhesions (slight and/or severe) at laparoscopy. Antibiotic therapy Analysis of semen samples Sixty patients with an elastase concentration 290 ng/ml were treated In all 312 men, the semen was assessed according to WHO (1992) with antibiotic therapy, whereas no female partner received antibiotics. guidelines with regard to volume, ph, sperm count, rapid progressive Antibiotic treatment was performed with norfloxacine (Nolicin, Krka, motility, vitality and normal morphology by means of techniques described elsewhere (Zorn et al., 1999). Leukocytes were determined using the peroxidase test. In addition, antisperm antibodies were determined by means of mixed antiglobulin reaction (MAR) test. PMN elastase activity in seminal plasma Elastase concentration was measured in seminal plasma according to a previously described method (Neumann et al., 1984). Each semen sample was first centrifuged at 300 g for 10 min and the supernatant removed and frozen. Granulocyte elastase in the form of its complex with α1-protease inhibitor Ela/α 1 PI was determined in frozen thawed seminal plasma, prepared in physiological solution, using an immunoassay (IMAC PMN elastase; Merck, Darmstadt, Germany). The method was applied 25 min after centrifugation. The elastase concentration was determined spectrophotometrically using a standard curve, and expressed in ng/ml. The sensitivity of the assay was 4 ng/ ml; intra- and inter-assay coefficients of variation were 6.2% and 6.7% respectively. Sperm ROS production Measurement of ROS was performed using a LKB Wallac 1250 Luminometer (LKB-Wallac, Turku, Finland). Luminescence was recorded at room temperature after the addition of luminol (5- amino-2,3 dehydro-1,4-phthalazinedione; Fluka Chemie AG, Buchs, Switzerland) at 0.2 mmol/l final concentration to 500 µl of prepared semen. Semen was layered on a discontinuous PureSperm (Nidacon International AB, Gothenburg, Sweden) concentration gradient (90% and 40%) and then washed with sperm preparation medium (MediCult, Copenhagen, Denmark) in order to avoid the presence of leukocytes Slovenia) 400 mg twice daily for at least 20 days. The semen of each patient was controlled for elastase concentration and sperm classical characteristics within 3 weeks after the end of therapy. Bacteriological examination Before antibiotic therapy, semen samples of 31 men with leukocytospermia were cultured aerobically and anaerobically. Standard bacteriological methods were used to quantify and identify the microorganisms. After antibiotic therapy, additional tests to detect C. trachomatis were carried out in 12 patients because of persistent elevated elastase concentration. C. trachomatis was assayed in urine samples using an immunohistochemical method (Chlamydia Easy-Card; Sentinel, Milan, Italy). In those 12 men, Ureaplasma urealyticum was further diagnosed by using a mycoscreen test (International Mycoplasma S.A., Toulon, France). Statistical analysis To determine the predictive value of elastase concentration in the detection of male genital tract inflammation as defined by leukocytospermia, the ROC (receiver operating characteristic) test was used. In this test, the greater the discriminating power of the parameter, the more the curve will deviate from the diagonal to the upper left corner. The calculated elastase concentration, located at the greatest distance from the diagonal, is that which allows the best differentiation between the patients with and those without inflammation. This criterion was used to establish the cut-off value of elastase. Because the distributions of elastase concentrations, ROS chemiluminescence data and the sperm count were not normal, a log 1979

3 B.Zorn, I.Virant-Klun and H.Meden-Vrtovec Table I. Age, duration of infertility, duration of sexual abstinence, classical sperm characteristics (mean SD) and incidence of leukocytospermia in infertile men [men with oligoasthenoteratozoospermia (OAT), normozoospermic men in infertile couples, normozoospermic men with more than one unsuccessful IVF embryo transfer attempt] and fertile men (controls) Infertile men All infertile OAT patients Normozoospermic Normozoospermic patients (n 224) men (n 40) men with (n 292) unsuccessful IVF attempt (n 28) Fertile men (n 20) Age (years) a * b * c * d * e * Duration of infertility (years) Abstinence (days) Semen volume (ml) Rapid progressive motility (%) Vitality (%) Sperm count ( spermatozoa) Normal sperm morphology (%) Semen leukocyte count ( 10 6 /ml) No. of men with 54 (17) 36 (15) 6 (15) 8 (28) 2 (10) leukocytospermia (%) *Values with superscripts a and d, b and d, c and d, and e and d are statistically different by means of χ 2 - test (P 0.05). Table II. Mean SD sperm reactive oxygen species (ROS) production, percentages of spermatozoa with single-stranded DNA, elastase concentrations and numbers of men with high elastase concentration ( 290 ng/ml) in infertile groups (men with oligoasthenoteratozoospermia (OAT), normozoospermic men in infertile couples, normozoospermic men with more than one unsuccessful IVF embryo transfer attempt) and fertile control group Infertile men All infertile patients OAT patients Normozoospermic Normozoospermic men with (n 292) (n 224) men (n 40) unsuccessful IVF attempt (n 28) Fertile men (n 20) ROS production (mv/s per 10 9 spermatozoa) Spermatozoa with single-stranded DNA (%) a, * b, * c, * d, * Elastase conc. (ng/ml) No. of men with elastase conc. 290 ng/ml 102 (35) a, ** 75 (34) b, ** 16 (40) c, ** 10 (36) d, ** 1 (5) e, ** (%) **Values with superscripts a and c, b and c, and d and c are statistically different by analysis of variance. *Values with superscripts a and e, b and e, c and e, and d and e are statistically different by means of χ 2 -test (P 0.05). transformation of these parameters was performed to reduce the ultrasound scan indicative of chronic prostatitis, and female partner degree of skew. The data were back-transformed for presentation in tubal impairment on the changes in elastase concentrations after Tables I and II. antibiotic therapy were checked using a χ 2 -test. An analysis of Statistical analysis was performed using the statistical package variance was performed to analyse the differences in ROS production SPSS for Windows (SPSS Inc., version 9.0, Chicago, IL, USA). and the percentage of spermatozoa with single-stranded DNA. Statist- Pearson s test was used to identify correlations between elastase ical significance was set at P concentration and patient age, different semen parameters including semen volume and the number of leukocytes, sperm ROS production and sperm single-stranded DNA. The differences in elastase concentration in men with positive Results semen bacteriology, men with abacterial prostatitis, and men whose Elastase as a marker for male genital inflammation female partner was affected by tubal damage in comparison with men without these pathologies, were calculated by the Mann Compared with leukocytospermia, the elastase at the cut-off Whitney test. value of 290 ng/ml had a sensitivity of 79.5% and a The difference in elastase concentration between infertile and fertile specificity of 74.4% in detecting genital inflammation (Figure men and the influences of age, positive semen bacteriology, abnormal 1). The positive predictive value was 63.3 and negative 1980

4 Seminal elastase and silent genital tract inflammation Figure 1. The receiver operating characteristic (ROC) test to determine the sensitivity and specificity of elastase concentration to detect male genital inflammation as defined by leukocytospermia ( white blood cells/ml). Figure 2. Correlation between elastase concentration and percentage of spermatozoa with single-stranded DNA (r 0.194, P 0.024). Table III. Correlation analysis between elastase concentration and patient age, semen volume, leukocytes and percentage of spermatozoa with singlestranded DNA predictive value 87.2 at a 35% estimated incidence of high elastase concentration ( 290 ng/ml). Seminal elastase concentration Study characteristics Correlation coefficient (r) P Age of male partner Study populations, classical sperm characteristics, and sperm Duration of infertility NS Semen volume functional activity Seminal leukocytes Age, duration of infertility, duration of sexual abstinence, Sperm single-stranded DNA Semen ph, sperm count, rapid NS sperm characteristics (volume, rapid sperm motility, vitality, progressive motility, vitality, sperm count, normal sperm morphology, number of leuko- morphology, and MAR test cytes), sperm ROS production, sperm single-stranded DNA and elastase concentrations for each infertile and fertile popula- NS not statistically related by means of Pearson s test. MAR mixed antiglobulin reaction. tion are expressed as mean SD, and are presented in Tables I and II. In infertile men, the incidence of leukocytospermia ranged from 15% to 28%, and was higher than in fertile men Elastase concentration, (10%), though the difference was not significant (Table I). The stranded DNA ROS production and single- percentages of men with high elastase concentration were No correlation was found between elastase concentration and significantly different in the four groups (P 0.05) (Table ROS production, which in turn was not correlated with the II). Normozoospermic men in infertile couples had normal number of leukocytes. ROS production and single-stranded ROS production and normal percentage of spermatozoa with DNA were positively correlated (r 0.236, P 0.003); the single-stranded DNA. A higher percentage of spermatozoa higher the percentage of spermatozoa with single-stranded with single-stranded DNA was observed in men with OAT DNA, the higher the ROS production. Moreover, there was a and in normozoospermic men with an unsuccessful IVF attempt negative correlation between elastase concentration and the in comparison with normozoospermic men (Table II). percentage of spermatozoa with single-stranded DNA (r 0.194, P 0.024) (Figure 2). Correlations between elastase concentration and studied Seminal elastase concentration and female partner charactersperm characteristics istics A positive correlation was found between elastase concentra- Significantly (P 0.001) higher elastase concentrations were tion and patient age (r 0.202, P ) (Table III). observed in men whose female partner had tubal damage, as There was also a positive correlation between elastase concen- compared with those whose partner was without this pathology tration and number of leukocytes, but a negative correlation (Figure 3). On the other hand, no correlation was found between between elastase concentration and both semen volume and elastase concentration and the occurrence of endometritis or sperm single-stranded DNA. Six of the 62 semen samples endometriosis. evaluated for antisperm antibodies by the MAR test were positive. No significant relationship was found between elastase Elastase concentration and semen bacteriology concentration and the presence of antisperm antibodies Bacteriological examination of semen samples from 31 patients (Table III). with leukocytospermia gave positive results in eight men. Ten 1981

5 B.Zorn, I.Virant-Klun and H.Meden-Vrtovec Table IV. Variations in, and factors affecting, elastase concentration after antibiotic therapy in patients (n 50) Elastase Elastase Statistical concentration concentration not significance of reduced ( 290 reduced ( 290 difference a ng/ml) (n 15) ng/ml) (n 35) Tubal impairment 3/15 (20) 22/35 (63) P 0.01 Abnormal prostate 0/7 2/18 (11) NS ultrasound scan (chronic prostatitis) Semen infection 0/5 4/20 (20) NS Men aged 35 years 6/15 (40) 18/35 (51) NS Figure 3. Seminal elastase concentrations in men whose partner was affected by tubal damage, and men whose partner had no tubal pathology; semi-logarithmic presentation (median values, and 25th, 75th, 5th and 95th percentiles). different types of microorganisms were isolated with 10 5 colony-forming units (CFU). Among seven patients there were four types of Gram-positive bacteria, including Enterococcus faecalis (n 4), Streptococcus agalactiae (n 1), coagulasenegative Staphylococcus (n 1) and Staphylococcus aureus (n 1). A Gram-negative bacterium (Escherichia coli) was found in two patients. U. urealyticum was isolated in one patient with a persistently high elastase concentration after antibiotic therapy. All urine assessments for C. trachomatis were negative; neither was any correlation found between elastase concentration and the presence of bacteria in semen. Elastase concentration in non-leukocytospermic men A high elastase concentration ( 290 ng/ml) was observed in 66 (25%) of the 264 men with 10 6 leukocytes/ml. Among this group, ultrasound identified five men with abacterial prostatitis of the total of six found among the whole group of infertile patients (n 292). These men were characterized by very high elastase concentration ( ng/ml). Moreover, among non-leukocytospermic men with a high elastase concentration we found a significantly higher (P 0.05) percentage whose partner had tubal damage (21/27; 78%) than in the overall population (74/187; 40%). Follow-up after antibiotic therapy Among 60 men who received antibiotic therapy, a decrease in elastase concentration was seen in 15 (25%) cases. In 45 (75%) men, no decrease below 290 ng/ml was observed. Information on partners tubal damage, however, was available for only 50 of the 60 men receiving antibiotics, and 35 of these 50 showed no decrease in elastase concentration below 290 ng/ml (Table IV). Most female partners of those 35 men had tubal damage (63%), compared with 20% of the female partners of the men who showed a decrease in elastase concentration (Table IV). However, antibiotic therapy did not affect the sperm characteristics. Discussion Leukocytospermia has been considered as an indicator of male genital tract inflammation. We confirmed that elastase 1982 Values in parentheses are percentages. a χ 2 test. NS not statistically different. concentration is strongly correlated with leukocytospermia. On the basis of leukocytospermia, we found the cut-off elastase value of at least 290 ng/ml to be discriminative for the detection of inflammation. This discriminatory level is similar to that of 250 ng/ml proposed earlier (Jochum et al., 1986) for infertile men, but lower than the 600 ng/ml value observed in patients with prostatitis (Reinhardt et al., 1997). In our infertile men the incidence of leukocytospermia was similar to that (10 20%) reported previously (Wolff, 1995). Leukocytospermia has been related to poor semen parameters (Wolff et al., 1990; Aitken and Gordon-Baker, 1995; Rajasekaran et al., 1995; Yanushpolsky et al., 1996). Others argued that leukocytospermia is not a cause of male infertility (Tomlinson et al., 1993), or it might play a positive role in semen (Kiessling et al., 1995). We did not observe any negative effect of leukocytes on classical sperm characteristics. In infertile men, inflammation detected by high elastase concentration is a frequent occurrence (Wolff and Anderson, 1988; Reinhardt et al., 1997), whatever the semen quality. Significantly higher concentrations of elastase were observed in the semen of infertile patients (Rajasekaran et al., 1995). A lower incidence of high elastase concentration was also reported in men of proven fertility (Jochum et al., 1986). Our data confirm a lower incidence of high elastase concentration in fertile men when compared with infertile men. The relationship between elastase concentration and male age is an indirect indication of the role of elastase as a marker of inflammation (older men generally have greater exposure to infection and inflammation). We found that elastase concentration was not correlated with the presence of bacteria usually assessed in the semen, as was shown previously (Cumming et al., 1990). Similarly, we confirmed that elastase concentration was not correlated with the presence of antisperm anti- bodies (Eggert-Kruse et al., 1996a,b, 1998). No apparent negative impact was found between classical sperm character- istics and elastase concentration. The only adverse change we observed was a reduction in semen volume, which may be related to infection of male accessory glands such as the prostate or seminal vesicles. The finding that elastase concentration is correlated with tubal impairment in the female partner indicates a new import-

6 Seminal elastase and silent genital tract inflammation ant role of elastase determination for screening and preventing concentrations decreased after antibiotic therapy and antiinflammatory infectious disease in the couple. Similarly, a relationship was drug administration. Elastase concentration has found between high C. trachomatis seminal serology in men been reported to be a useful tool in following patients after and the occurrence of tubal damage in the female partner antibiotic therapy; an amelioration of sperm parameters was (Eggert-Kruse et al., 1996b). observed in 67% of men in whom elastase concentration Leukocytospermia as a selection criterion failed to detect was reduced (Micic et al., 1989). In this study elastase all cases of inflammation. In non-leukocytospermic men with concentrations declined after antibiotic therapy in 25% of high elastase concentration, we found a significantly higher patients, but not in the remaining 75%. Moreover, after proportion of men whose partner had tubal damage. In this antibiotic therapy we did not observe any improvement in group, we found that all patients but one were suspected of classical sperm characteristics. We are aware that elastase having abacterial prostatitis. For all these reasons, we may concentration as well as other current diagnostic criteria may conclude that elastase is a reliable marker of male silent be insufficient to decide which men with signs of inflammation inflammation. should be treated, and whether they will benefit with regard Although leukocytes are the main producers of ROS (Aitken to fertility (Krause, 1999). The female partner s tubal damage and West, 1990; Wang et al., 1997), ROS are also produced appeared to be discriminatory, elastase concentration being by spermatozoa (Whittington and Ford, 1999), mainly of poor reduced most infrequently in the presence of tubal damage. quality (Iwasaki and Gagnon, 1992). At present, there are Because of its relationship with the partner s tubal damage, many unresolved questions concerning the exact role of ROS we found that elastase activity has a prognostic value. When during infection of the male genital tract because of the the elastase concentration does not decrease after antibiotic difficulty in assessing the site and the origin of ROS production therapy, one must consider the couple, knowing in most cases (Ochsendorf, 1999). To eliminate leukocytes as ROS producers that the partner s tubal damage may be involved and should as completely as possible, we used sperm density gradient be treated medically or surgically. Elastase is probably involved centrifugation as proposed previously (Aitken and West, 1990). in sexually transmitted diseases where non-bacterial microorganisms Under these conditions, we did not observe any relationship are implicated. between leukocytes and ROS production. Moreover, the elas- In conclusion, seminal elastase is related to the partner s tase concentration was not associated with ROS produced by tubal damage, and it is most likely that any antibiotic therapy spermatozoa. However ROS production was related positively would be of low efficacy until such tubal damage were to the percentage of spermatozoa with single-stranded DNA. corrected. The elastase inhibitor complex is not related to These negative effects of ROS on sperm DNA integrity have reduced semen quality; however, its relationship with reduced also been reported by others (Lopes et al., 1998; Twigg et al., sperm DNA denaturation is suggestive of a positive role for 1998). Although in our experience ROS are generated at low the complex during infection. rates by poor-quality spermatozoa, they are harmful to sperm DNA integrity. Acridine orange staining is used to evaluate the chromatin Acknowledgements integrity, as the test distinguishes between the normal double- The authors would like to thank Ms A.Bris ki-ses ek, Institute of stranded and abnormal single-stranded DNA (Gopalkrishnan Clinical Chemistry and Clinical Biochemistry, University Medical et al., 1999). Spermatozoa with single-stranded DNA, when Centre Ljubljana for elastase concentration evaluation, Ms M.Kolbezen-Simoniti, Laboratory of Andrology, Department of used in an IVF embryo transfer attempt, are functionally worse Obstetrics and Gynecology, for semen analyses, Mr I.Verdenik, in terms of fertilization ability (Hoshi et al., 1996) and embryo Research Unit, for statistical analyses, and Ms M.Pirc, for revising development (Virant-Klun et al., 1998). 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