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, S̆lajmerjeva 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
1979
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
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
1980
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
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)
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.Bris̆ki-Ses̆ek, 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.
References
Aitken, R.J. and Gordon-Baker, H.W. (1995) Seminal leukocytes: passengers,
terrorists or good Samaritans? Hum. Reprod., 10, 1736–1739.
Aitken, R.J. and West, K.M. (1990) Analysis of the relationship between
reactive oxygen species production and leukocyte infiltration in fractions
of human semen separated on Percoll gradients. Int. J. Androl., 13, 433–451.
Auger, J. (1998) Derives actifs de l’oxygene et dysfonctions spermatiques:
role de l’infection du tractus genital de l’homme. Andrologie, 8, 234–244.
Comhaire, F., Mahmoud, A.M.A., Depuydt, C.E. et al. (1999) Mechanisms
and effects of male genital tract infection on sperm quality and fertilizing
potential: the andrologist’s viewpoint. Hum. Reprod. Update, 5, 393–398.
Cumming, J.A., Dawes, J. and Hargreave, T.B. (1990) Granulocyte elastase
levels do not correlate with anaerobic and aerobic bacterial growth in
seminal plasma from infertile men. Int. J. Androl., 13, 273–277.
Eggert-Kruse, W., Probst, S., Rohr, G. et al. (1995) Screening for subclinical
inflammation in ejaculates. Fertil. Steril., 64, 1012–1022.
Eggert-Kruse, W., Probst, S., Rohr, G. et al. (1996a) Induction of
immunoresponse by subclinical male tract infection. Fertil. Steril., 65,
1202–1209.
Eggert-Kruse, W., Buhlinger-Gopfarth, N., Rohr, G. et al. (1996b) Antibodies
1983
B.Zorn, I.Virant-Klun and H.Meden-Vrtovec
to Chlamydia trachomatis in semen and relationship with parameters of
male fertility. Hum. Reprod., 11, 1408–1417.
Eggert-Kruse, W., Beck, R., Rohr, G. et al. (1997) Cytokines in seminal
plasma: relationship with semen quality. Hum. Reprod., 12 (Abstract book
1), 16–17.
Eggert-Kruse, W., Rohr, G., Probst, S. et al. (1998) Antisperm antibodies and
microorganisms in genital secretions – a clinically significant relationship?
Andrologia, 30 (Suppl. I), 61–71.
Gopalkrishnan, K., Hurkadli, K., Padwal, V. et al. (1999) Use of acridine
orange to evaluate chromatin integrity of human spermatozoa in different
groups of infertile men. Andrologia, 31, 277–282.
Hautamaki, R.D., Kobayashi, D.K., Senior, R.M. et al. (1997) Requirement
for macrophage elastase for cigarette smoke-induced emphysema in mice.
Science, 277, 2002–2004.
Henkel, R. and Schill, W.B. (1998) Sperm preparation in patients with
urogenital infections. Andrologia, 30 (Suppl. I), 91–97.
Hoshi, K., Katayose, H., Yanagida, K. et al. (1996) The relationship between
acridine orange fluorescence of sperm nuclei and the fertilizing ability of
human sperm. Fertil. Steril., 66, 634–639.
Iwasaki, A. and Gagnon, C. (1992) Formation of reactive oxygen species in
spermatozoa of infertile patients. Fertil. Steril., 57, 409–417.
Jochum, M., Pabst, W. and Schill, W.B. (1986) Granulocyte elastase as a
sensitive diagnostic parameter of silent male genital tract inflammation.
Andrologia, 18, 413–419.
Keck, C., Gerber-Schafer, C., Clad, A. et al. (1998) Seminal tract infections:
impact on male fertility and treatment options. Hum. Reprod. Update, 4,
891–903.
Kiessling, A.A., Lamparelli, N., Yin, H.Z. et al. (1995) Semen leukocytes:
friends or foes? Fertil. Steril., 64, 196–198.
Krause, W. (1999) Which efforts towards conservative treatment of male
infertility will be successful? Antiphlogistics and glucocorticoids.
Andrologia, 31, 301–303.
Lee, J.K., Zaidi, S.H.E., Liu, P. et al. (1998) A serine elastase inhibitor
reduces inflammation and fibrosis and preserves cardiac function after
experimentally-induced murine myocarditis. Nature Med., 4, 1383–1391.
Liu, D.Y. and Baker, H.W.G. (1994) A new test for the assessment of spermzona pellucida penetration: relationship with results of other tests and
fertilization in vitro. Hum. Reprod., 9, 489–496.
Lopes, S., Jurisicova, A. and Sun, J.G. (1998) Reactive oxygen species:
potential cause for DNA fragmentation in human spermatozoa. Hum.
Reprod., 13, 896–900.
Ludwig, M., Kummel, C., Schroeder-Printzen, I. et al. (1998) Evaluation
of seminal plasma parameters in patients with chronic prostatitis or
leukocytospermia. Andrologia, 30 (Suppl. 1), 41–47.
Micic, S., Macura, M., Lalic, N. et al. (1989) Elastase as an indicator of silent
genital tract infection in infertile men. Int. J. Androl., 12, 423–429.
Ness, R.B., Markovic, N., Carlson, C.L. et al. (1997) Do men become infertile
after having sexually transmitted urethritis? An epidemiologic examination.
Fertil. Steril., 68, 205–213.
Neumann, S., Gunzer, G., Hennrich, N. et al. (1984) ‘PMN-Elastase assay’:
enzyme immunoassay for human polymorphonuclear elastase complexed
with µ1- proteinase inhibitor. J. Clin. Chem. Clin. Biochem., 22, 693–697.
Ochsendorf, F.R. (1999) Infections in the male genital tract and reactive
oxygen species. Hum. Reprod. Update, 5, 399–420.
Purvis, K. and Christiansen, E. (1993) Infection in the male reproductive tract.
Impact, diagnosis and treatment in relation with male infertility. Int. J.
Androl., 16, 1–13.
Rajasekaran, M., Hellstrom, W.J.G., Naz, R.K. et al. (1995) Oxidative stress
and interleukins in seminal plasma during leukocytospermia. Fertil. Steril.,
64, 166–171.
Reinhardt, A., Haidl, G. and Schill, W.B. (1997) Granulocyte elastase indicates
silent male genital tract inflammation and appropriate anti-inflammatory
treatment. Andrologia, 29, 187–192.
Rowe, P.J., Comhaire, F.H., Hargreave, T.B. et al. (1993) World Health
Organization Manual for the Standardized Investigation and Diagnosis of
the Infertile Couple. Cambridge University Press, Cambridge, UK, pp. 6–39.
Schill, W.H.B., Kohn, F.H., Henkel, R. et al. (1995) Biochemical aspects of
semen analysis. In Hedon, B., Bringer, J. and Mares, P. (eds), Fertility and
Sterility: A Current Overview. Proceedings of the 15th World Congress on
Fertility and Sterility Montpellier, France, 17–22 September 1995. The
Parthenon Publishing Group, New York London, pp. 303–309.
Shimoya, K., Matsuzaki, N., Tsutsui, T. et al. (1993) Detection of interleukin8 (IL-8) in seminal plasma of infertile patients with leukospermia. Fertil.
Steril., 59, 885–888.
1984
Thonneau, P., Quesnot, S., Ducot, B. et al. (1992) Risk factors for female and
male infertility: results of a case-control study. Hum. Reprod., 7, 55–58.
Tomlinson, M.J., Barratt, C.L.R. and Cooke, I.D. (1993) Prospective study of
leukocytes and leukocyte subpopulations in semen suggests they are not a
cause of male infertility. Fertil. Steril., 60, 1069–1075.
Twigg, J., Fulton, N., Gomez, E. et al. (1998) Analysis of the impact of
intracellular reactive oxygen species generation on the structural and
functional integrity of human spermatozoa: lipid peroxidation, DNA
fragmentation and effectiveness of antioxidants. Hum. Reprod., 13, 1429–
1436.
Vicari, E. (1999) Seminal leukocyte concentration and related specific reactive
oxygen species production in patients with male accessory gland infections.
Hum. Reprod., 14, 2025–2030.
Virant-Klun, I., Zorn, B., Tomazevic, T. et al. (1998) Influence de la
denaturation de l’ADN spermatique sur le potentiel de developpement
cellulaire embryonnaire apres injection intracytoplasmique de spermatozoide
(ICSI). Andrologie, 8, 90–91.
Wang, A., Fanning, L., Anderson, D.J. et al. (1997) Generation of reactive
oxygen species by leukocytes and sperm following exposure to urogenital
tract infection. Arch. Androl., 39, 11–17.
Weidner, W. (1999) Which efforts towards conservative treatment of male
infertility will be successful? Antibiotic therapy. Andrologia, 31, 297.
Whittington, K. and Ford, W.C.L. (1999) Relative contribution of leukocytes
and of spermatozoa to reactive oxygen species production in human sperm
suspensions. Int. J. Androl., 22, 229–235.
Wolff, H. (1995) The biological significance of white blood cells in semen.
Fertil. Steril., 63, 1143–1157.
Wolff, H. (1998) Methods for the detection of male genital tract inflammation.
Andrologia, 30 (Suppl. 1), 35–39.
Wolff, H. and Anderson, D.J. (1988) Evaluation of granulocyte elastase as a
seminal plasma marker for leukocytospermia. Fertil. Steril., 50, 129–132.
Wolff, H., Politch, J.A., Martinez, A. et al. (1990) Leukocytospermia is
associated with poor semen quality. Fertil. Steril., 53, 528–536.
World Health Organization (1992) WHO Laboratory Manual for the
Examination of Human Semen and Semen-Cervical Mucus Interaction. 3rd
edn, Cambridge University Press, Cambridge, UK.
Yanushpolsky, E.H., Politch, J.A., Hill, J.A. et al. (1996) Is leukocytospermia
clinically relevant? Fertil. Steril., 66, 822–825.
Zalata, A., Comhaire, F.H., Vermeulen, L. et al. (1996) Detection of a causal
factor of male infertility. In Comhaire F.H. (ed.), Male Infertility: Clinical
Investigation, Cause Evaluation and Treatment. Chapman & Hall Medical,
Oxford, UK, pp. 173–175.
Zorn, B., Virant-Klun, I., Verdenik, I. et al. (1999) Semen quality changes
among Slovenian healthy men included in the IVF-ET programme during
1983–1996. Int. J. Androl., 22, 178–183.
Received on February 21, 2000; accepted on June 7, 2000