Transcript
Human Reproduction vol.15 no.9 pp.1978–1984, 2000
Semen granulocyte elastase: its relevance for the diagnosis and prognosis of silent genital tract inflammation
B.Zorn1, I.Virant-Klun and H.Meden-Vrtovec Andrology Centre, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia 1To
whom correspondence should be addressed at: Andrology Centre, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, S˘lajmerjeva 3, SI-1000 Ljubljana, Slovenia. E-mail:
[email protected]
Elastase–inhibitor complex was assessed by immunoassay in the seminal plasma of 312 men attending the outpatient infertility clinic. Using receiver operating characteristic (ROC) curve analysis, elastase at the cut-off value of ≥290 ng/ml was shown to be efficient (sensitivity 79.5%, specificity 74.4%) in the detection of genital tract inflammation as defined by leukocytospermia (>1⍥106 leukocytes/ml). The prevalence of increased elastase in 292 infertile men was significantly higher (34%) as compared with that (5%) observed in 20 fertile men (P ⍧ 0.02). Moreover, high elastase concentration (≥290 ng/ml) was observed in 66 of the 264 men (25%) without leukocytospermia. A significant positive correlation was found between elastase concentration and patient age (r ⍧ 0.202, P < 0.0001) and the number of leukocytes (r ⍧ 0.330, P < 0.0001). A negative correlation was found between elastase concentration and semen volume (r ⍧ –0.146, P ⍧ 0.01) and the percentage of spermatozoa with singlestranded DNA (r ⍧ –0.194, P ⍧ 0.024), but there was no correlation between elastase and sperm reactive oxygen species production. A higher seminal elastase concentration was significantly associated with tubal damage in female partners (P < 0.001). After norfloxacine antibiotic therapy, decrease in elastase concentration was observed in 15 (25%) of the 60 treated patients. Tubal damage in the partner negatively affected the response to antibiotic therapy. In conclusion, granulocyte elastase is a reliable screening test for silent genital tract inflammation of the couple. The elastase–inhibitor complex may have a protective effect in reducing sperm DNA denaturation. Key words: diagnosis/prognosis/seminal elastase/silent inflammation/sperm quality
Introduction 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 often, the main difficulty resides first in the diagnosis of inflammation and second, in demonstration of the causal link between inflammation and male infertility (Auger, 1998). 1978
Several tests have been proposed as markers of inflammation in men, including either markers of specific infection known for a deleterious effect on male reproductive function [for example the determination of antibodies specific for Chlamydia trachomatis in seminal plasma; (Keck et al., 1998)], or nonspecific markers of inflammation such as leukocytospermia (Wolff, 1998). According to World Health Organization criteria (WHO, 1992), leukocytospermia, defined as the presence of ⬎1⫻106 white blood cells (WBC)/ml of semen, is considered as a possible indicator of ongoing male genital tract infection. Besides leukocytospermia, organ-specific markers such as fructose, α-glucosidase, citric acid (Zalata et al., 1996), and organ non-specific markers such as albumin, C-reactive protein, different cytokines, mainly interleukins 6 and 8 (Shimoya et al., 1993; Eggert-Kruse et al., 1995; Comhaire et al., 1999), and complement C3 (Ludwig et al., 1998) have been evaluated in semen. Others (Purvis and Christiansen, 1993) found rectal ultrasound to be important in the detection of non-symptomatic deep pelvic infections. One of the main changes during the inflammatory process is the discharge by polymorphonuclear granulocytes (PMN) of large amounts of proteases such as elastase. As granulocytes are the main constituents of the WBC population in semen, the evolution of elastase in seminal plasma is of clinical importance in detecting inflammation. The enzyme elastase, particularly the elastase-α1–protease inhibitor complex (Ela/ α1–PI) has been suggested as a sensitive and quantitative marker of genital tract inflammation in general (Jochum et al., 1986; Schill et al., 1995) and in particular of chronic prostatitis (Ludwig et al., 1998), as determined by clinical and bacteriological features. Apart from its beneficial anti-inflammatory effects, elastase per se provokes cell deterioration with the synthesis of reactive oxygen species (ROS) which may lead to cell death (Hautamaki et al., 1997). The aims of the present study were to assess the prevalence of high elastase concentration in the seminal plasma of infertile men in comparison with that in fertile men, to study the reliability of seminal elastase in detection of silent male genital tract inflammation, and to verify the hypothetical deleterious effect of elastase on sperm classical characteristics and sperm functional activity evaluated by ROS production and the presence of single-stranded DNA.
Materials and methods Study population The study population consisted of 312 men attending the infertility outpatient clinic at the Andrology Centre of the Department of © European Society of Human Reproduction and Embryology
Seminal elastase and silent genital tract inflammation
Obstetrics and Gynecology of Ljubljana between January 1996 and June 1998. The infertile population (n ⫽ 292) consisted of 224 men with oligoasthenoteratozoospermia (OAT) according to WHO criteria (WHO, 1992), 40 normozoospermic men from infertile couples, and 28 normozoospermic men with one or more unsuccessful conventional IVF–embryo transfer attempts. The control group consisted of 20 fertile men who fathered children spontaneously within the study period. None of the patients had clinical signs or symptoms of a genital tract infection. None had been treated by antibiotics within 3 months before enrolment into the study. All patients had given their informed consent for participation. Clinical examination Male partners were interviewed about their andrological history, with a particular emphasis on clinical or biological events possibly related to a previous genital tract infection; this was followed by a standard clinical examination. Rectal examination of the prostate was performed in cases of leukocytospermia or high elastase concentration. When the rectal examination was abnormal, it was completed by a prostatovesicular ultrasound scan according to WHO recommendations (Rowe et al., 1993). Abacterial chronic prostatitis was suspected in the presence of glandular asymmetry, hypoechogenicity or hyperechogenicity associated with areas of calcification (Vicari, 1999). Additionally, data from the female partner’s history (hysterosalpingography, endometrial histology and laparoscopy) as well as histological endometritis, endometriosis and tubal damage found at laparoscopy, were taken into consideration. Tubal damage was defined as the presence of one or both tubes closed at hysterosalpingography, and/or one or both tubes closed with adhesions (slight and/or severe) at laparoscopy. Analysis of semen samples In all 312 men, the semen was assessed according to WHO (1992) guidelines with regard to volume, pH, sperm count, rapid progressive 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/α1–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 (5amino-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
and minimize ROS production by leukocytes. The sperm ROS production detected by luminescence was recorded in the integration mode for 10 s with constant stirring of the analysed sample. Readings were taken every 5 min for 30 min, with a peak of luminescence observed between 5 and 15 min. After subtracting the appropriate blanks, the peak luminescence was considered detectable when the luminescence was 艌0.05 mV/s. Peak luminescence observed at 5– 15 min after the addition of luminol was expressed in mV/s per 109 spermatozoa. Sperm single-stranded DNA assessment by acridine orange staining After semen preparation on discontinuous PureSperm gradient and washing, single-stranded DNA was detected by acridine orange (AO) staining according to a published method (Liu and Baker, 1994). Airdried sperm smears were fixed in Carnoy’s solution overnight, after which they were rinsed in phosphate-buffered saline and air-dried. Sperm smears were stained for 5 min with 1% AO solution, prepared as follows: 10 ml of 1% AO in distilled water was added to a mixture of 40 ml of 0.1 mol/l citric acid and 2.5 ml of 0.3 mol/l Na2HPO4·7H2O. After AO staining, the slides were rinsed and mounted in distilled water, and then observed by fluorescence microscopy (Axioskop; Carl Zeiss Jena, Germany) at ⫻400 final magnification. AO stain intercalates in sperm single-stranded DNA as a polymer, providing red, orange, or yellow fluorescence of the sperm head, whereas in normal, double-stranded DNA, it intercalates as a monomer, providing green fluorescence. At least 100 spermatozoa were assessed on each slide. From that count and the count of red, orange or yellow spermatozoa, the percentage of sperm containing single-stranded DNA was calculated. Antibiotic therapy Sixty patients with an elastase concentration 艌290 ng/ml were treated with antibiotic therapy, whereas no female partner received antibiotics. Antibiotic treatment was performed with norfloxacine (Nolicin, Krka, 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
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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 patients (n ⫽ 292) Age (years) 34.4 ⫾ 5.6a* Duration of infertility 6.6 ⫾ 4.9 (years) Abstinence (days) 3.6 ⫾ 0.9 Semen volume (ml) 3.3 ⫾ 1.6 Rapid progressive 18.7 ⫾ 14.2 motility (%) Vitality (%) 55.9 ⫾ 13.1 Sperm count (⫻106 90 ⫾ 149 spermatozoa) Normal sperm 15.7 ⫾ 15.1 morphology (%) Semen leukocyte count 0.8 ⫾ 0.5 (⫻106/ml) No. of men with 54 (17) leukocytospermia (%)
Fertile men (n ⫽ 20) OAT patients Normozoospermic Normozoospermic (n ⫽ 224) men (n ⫽ 40) men with unsuccessful IVF attempt (n ⫽ 28) 34.1 ⫾ 5.5b* 6.7 ⫾ 5.0
33.7 ⫾ 4.3c* 4.5 ⫾ 3.5
38.4 ⫾ 6.9d* 9.1 ⫾ 4.6
32.8 ⫾ 4.6e* 3.7 ⫾ 3.7
3.6 ⫾ 0.9 3.3 ⫾ 1.6 14.7 ⫾ 12.0
3.8 ⫾ 0.8 3.5 ⫾ 1.5 35.9 ⫾ 8.2
3.3 ⫾ 0.7 3.3 ⫾ 1.6 29.1 ⫾ 12.8
3.6 ⫾ 0.6 3.8 ⫾ 1.4 32 ⫾ 14
51.6 ⫾ 13.0 44 ⫾ 70
64.0 ⫾ 6.7 298 ⫾ 252
57.6 ⫾ 13.9 172 ⫾ 134
64.0 ⫾ 7.7 134 ⫾ 103
10.4 ⫾ 10.0
37.6 ⫾ 9.3
30.4 ⫾ 18.4
24 ⫾ 17
0.8 ⫾ 1.4
0.7 ⫾ 1.5
0.6 ⫾ 1.2
0.5 ⫾ 0.7
6 (15)
8 (28)
36 (15)
2 (10)
*Values with superscripts a and d, b and d, c and d, and e and d are statistically different by means of χ2test (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 (艌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
Fertile men (n ⫽ 20)
All infertile patients OAT patients (n ⫽ 292) (n ⫽ 224)
ROS production (mV/s per 109 spermatozoa) Spermatozoa with single-stranded DNA (%) Elastase conc. (ng/ml) No. of men with elastase conc. 艌290 ng/ml (%)
28.8 ⫾ 50.1 61.7 ⫾ 26.9a,* 341.7 ⫾ 748.9 102 (35)a,**
Normozoospermic men (n ⫽ 40)
32.1 ⫾ 53.9 8.5 ⫾ 3.4 66.1 ⫾ 23.9b,* 36.9 ⫾ 31.5c,* 332.1 ⫾ 782.8 492.7 ⫾ 837.1 75 (34)b,** 16 (40)c,**
Normozoospermic men with unsuccessful IVF attempt (n ⫽ 28) 30.1 ⫾ 45.8 58.8 ⫾ 26.8d,* 241.0 ⫾ 230.8 10 (36)d,**
8.3 ⫾ 4.7 49.3 ⫾ 20.9 113.0 ⫾ 105.0 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 χ2-test (P ⬍ 0.05).
transformation of these parameters was performed to reduce the degree of skew. The data were back-transformed for presentation in Tables I and II. Statistical analysis was performed using the statistical package SPSS for Windows (SPSS Inc., version 9.0, Chicago, IL, USA). Pearson’s test was used to identify correlations between elastase 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 semen bacteriology, men with abacterial prostatitis, and men whose female partner was affected by tubal damage in comparison with men without these pathologies, were calculated by the Mann– Whitney test. The difference in elastase concentration between infertile and fertile men and the influences of age, positive semen bacteriology, abnormal
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ultrasound scan indicative of chronic prostatitis, and female partner tubal impairment on the changes in elastase concentrations after antibiotic therapy were checked using a χ2-test. An analysis of variance was performed to analyse the differences in ROS production and the percentage of spermatozoa with single-stranded DNA. Statistical significance was set at P ⬍ 0.05.
Results Elastase as a marker for male genital inflammation Compared with leukocytospermia, the elastase at the cut-off value of 艌290 ng/ml had a sensitivity of 79.5% and a specificity of 74.4% in detecting genital inflammation (Figure 1). The positive predictive value was 63.3 and negative
Seminal elastase and silent genital tract inflammation
Figure 2. Correlation between elastase concentration and percentage of spermatozoa with single-stranded DNA (r ⫽ –0.194, P ⫽ 0.024). 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 (⬎1⫻106 white blood cells/ml).
Table III. Correlation analysis between elastase concentration and patient age, semen volume, leukocytes and percentage of spermatozoa with singlestranded DNA Seminal elastase concentration
predictive value 87.2 at a 35% estimated incidence of high elastase concentration (艌290 ng/ml). Study populations, classical sperm characteristics, and sperm functional activity Age, duration of infertility, duration of sexual abstinence, sperm characteristics (volume, rapid sperm motility, vitality, sperm count, normal sperm morphology, number of leukocytes), sperm ROS production, sperm single-stranded DNA and elastase concentrations for each infertile and fertile population 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 (10%), though the difference was not significant (Table I). The percentages of men with high elastase concentration were significantly different in the four groups (P ⬍ 0.05) (Table II). Normozoospermic men in infertile couples had normal ROS production and normal percentage of spermatozoa with single-stranded DNA. A higher percentage of spermatozoa with single-stranded DNA was observed in men with OAT and in normozoospermic men with an unsuccessful IVF attempt in comparison with normozoospermic men (Table II). Correlations between elastase concentration and studied sperm characteristics A positive correlation was found between elastase concentration and patient age (r ⫽ 0.202, P ⬍ 0.0001) (Table III). There was also a positive correlation between elastase concentration and number of leukocytes, but a negative correlation between elastase concentration and both semen volume and sperm single-stranded DNA. Six of the 62 semen samples evaluated for antisperm antibodies by the MAR test were positive. No significant relationship was found between elastase concentration and the presence of antisperm antibodies (Table III).
Study characteristics
Correlation coefficient (r)
P
Age of male partner Duration of infertility Semen volume Seminal leukocytes Sperm single-stranded DNA Semen pH, sperm count, rapid progressive motility, vitality, morphology, and MAR test
0.202 0.074 –0.146 0.330 –0.194
⬍ 0.0001 NS 0.01 ⬍ 0.0001 0.024 NS
NS ⫽ not statistically related by means of Pearson’s test. MAR ⫽ mixed antiglobulin reaction.
Elastase concentration, ROS production and singlestranded DNA No correlation was found between elastase concentration and ROS production, which in turn was not correlated with the number of leukocytes. ROS production and single-stranded DNA were positively correlated (r ⫽ 0.236, P ⫽ 0.003); the higher the percentage of spermatozoa with single-stranded DNA, the higher the ROS production. Moreover, there was a negative correlation between elastase concentration and the percentage of spermatozoa with single-stranded DNA (r ⫽ –0.194, P ⫽ 0.024) (Figure 2). Seminal elastase concentration and female partner characteristics Significantly (P ⬍ 0.001) higher elastase concentrations were observed in men whose female partner had tubal damage, as compared with those whose partner was without this pathology (Figure 3). On the other hand, no correlation was found between elastase concentration and the occurrence of endometritis or endometriosis. Elastase concentration and semen bacteriology Bacteriological examination of semen samples from 31 patients with leukocytospermia gave positive results in eight men. Ten 1981
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Table IV. Variations in, and factors affecting, elastase concentration after antibiotic therapy in patients (n ⫽ 50)
Tubal impairment Abnormal prostate ultrasound scan (chronic prostatitis) Semen infection Men aged 艌35 years
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 艌105 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 (艌290 ng/ml) was observed in 66 (25%) of the 264 men with ⬍106 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 (539.0 ⫾ 113.5 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
Elastase concentration reduced (⬍290 ng/ml) (n ⫽ 15)
Elastase Statistical concentration not significance of reduced (艌290 differencea ng/ml) (n ⫽ 35)
3/15 (20) 0/7
22/35 (63) 2/18 (11)
P ⫽ 0.01 NS
0/5 6/15 (40)
4/20 (20) 18/35 (51)
NS NS
Values in parentheses are percentages. aχ2 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 antibodies (Eggert-Kruse et al., 1996a,b, 1998). No apparent negative impact was found between classical sperm characteristics 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-
Seminal elastase and silent genital tract inflammation
ant role of elastase determination for screening and preventing infectious disease in the couple. Similarly, a relationship was found between high C. trachomatis seminal serology in men and the occurrence of tubal damage in the female partner (Eggert-Kruse et al., 1996b). Leukocytospermia as a selection criterion failed to detect all cases of inflammation. In non-leukocytospermic men with high elastase concentration, we found a significantly higher proportion of men whose partner had tubal damage. In this group, we found that all patients but one were suspected of having abacterial prostatitis. For all these reasons, we may conclude that elastase is a reliable marker of male silent inflammation. Although leukocytes are the main producers of ROS (Aitken and West, 1990; Wang et al., 1997), ROS are also produced by spermatozoa (Whittington and Ford, 1999), mainly of poor quality (Iwasaki and Gagnon, 1992). At present, there are many unresolved questions concerning the exact role of ROS during infection of the male genital tract because of the difficulty in assessing the site and the origin of ROS production (Ochsendorf, 1999). To eliminate leukocytes as ROS producers as completely as possible, we used sperm density gradient centrifugation as proposed previously (Aitken and West, 1990). Under these conditions, we did not observe any relationship between leukocytes and ROS production. Moreover, the elastase concentration was not associated with ROS produced by spermatozoa. However ROS production was related positively to the percentage of spermatozoa with single-stranded DNA. These negative effects of ROS on sperm DNA integrity have also been reported by others (Lopes et al., 1998; Twigg et al., 1998). Although in our experience ROS are generated at low rates by poor-quality spermatozoa, they are harmful to sperm DNA integrity. Acridine orange staining is used to evaluate the chromatin integrity, as the test distinguishes between the normal doublestranded and abnormal single-stranded DNA (Gopalkrishnan et al., 1999). Spermatozoa with single-stranded DNA, when used in an IVF–embryo transfer attempt, are functionally worse in terms of fertilization ability (Hoshi et al., 1996) and embryo development (Virant-Klun et al., 1998). We found that the elastase–inhibitor complex was related to sperm single-stranded DNA: in the presence of high elastase–inhibitor complex concentrations DNA denaturation appeared to be lower. Our finding suggests a protective role for the elastase–inhibitor complex towards sperm DNA integrity. In the presence of genital tract inflammation, elastase is not correlated with negative changes of sperm characteristics (Eggert-Kruse et al., 1997; Henkel and Schill, 1998; Gopalkrishnan et al., 1999), possibly due to some positive role of the elastase–inhibitor complex? By using different experimental conditions, a positive role of elastase inhibitor was found (Lee et al., 1998): serine elastase inhibitor reduced inflammation and fibrosis, and preserved cardiac function after experimentally induced murine myocarditis. Only antibiotic therapy has proved to be really efficacious in eradicating microorganisms and reducing cellular and humoral inflammatory parameters (Weidner, 1999). Others (Micic et al., 1989; Reinhardt et al., 1997) have reported that elastase
concentrations decreased after antibiotic therapy and antiinflammatory drug administration. Elastase concentration has been reported to be a useful tool in following patients after antibiotic therapy; an amelioration of sperm parameters was observed in 67% of men in whom elastase concentration was reduced (Micic et al., 1989). In this study elastase concentrations declined after antibiotic therapy in 25% of patients, but not in the remaining 75%. Moreover, after antibiotic therapy we did not observe any improvement in classical sperm characteristics. We are aware that elastase concentration as well as other current diagnostic criteria may be insufficient to decide which men with signs of inflammation should be treated, and whether they will benefit with regard to fertility (Krause, 1999). The female partner’s tubal damage appeared to be discriminatory, elastase concentration being reduced most infrequently in the presence of tubal damage. Because of its relationship with the partner’s tubal damage, we found that elastase activity has a prognostic value. When the elastase concentration does not decrease after antibiotic therapy, one must consider the couple, knowing in most cases that the partner’s tubal damage may be involved and should be treated medically or surgically. Elastase is probably involved in sexually transmitted diseases where non-bacterial microorganisms are implicated. In conclusion, seminal elastase is related to the partner’s tubal damage, and it is most likely that any antibiotic therapy would be of low efficacy until such tubal damage were corrected. The elastase–inhibitor complex is not related to reduced semen quality; however, its relationship with reduced sperm DNA denaturation is suggestive of a positive role for the complex during infection.
Acknowledgements The authors would like to thank Ms A.Bris˘ki-Ses˘ek, Institute of Clinical Chemistry and Clinical Biochemistry, University Medical Centre Ljubljana for elastase concentration evaluation, Ms M.Kolbezen-Simoniti, Laboratory of Andrology, Department of Obstetrics and Gynecology, for semen analyses, Mr I.Verdenik, Research Unit, for statistical analyses, and Ms M.Pirc, for revising the manuscript.
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