1 Department of Gynaecological Endocrinology and Reproductive Medicine, Women's University Hospital, Heidelberg, 2 Department of Dermatology, Division of Andrology and STD, Heidelberg, 3 Department of Internal Medicine II, Klinikum Bad Homburg and 4 Department of Clinical Chemistry, Klinikum Mannheim, Germany
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Abstract |
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Key words: complement/interleukins/leukocytes/male fertility/semen quality
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Introduction |
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Human semen contains a repertoire of cytokines whose effects on semen quality and sperm function, however, are subject to debate (Hill et al., 1987, 1989
; Shimoya et al., 1993
; Comhaire et al., 1994
, 1999
; Rajasekaran et al., 1995
; Huleihel et al., 1996
; Dousset et al., 1997
; Koumantakis et al., 1998
). Inflammatory cytokines are produced by white blood cells (WBC), mainly by macrophages, in response to foreign antigens, pathogens (infection challenge) and also in chronic inflammation (immunological activation). Acute and chronic infections may play a contributory role in male infertility. The clinical relevance of silent infection in asymptomatic patients is, however, not clear. Moreover, the interpretation of the markers commonly used for its diagnosis is controversial; for example the role of seminal leukocytes and clinically significant thresholds (Barratt et al., 1990
; Eggert-Kruse et al., 1992a
; Tomlinson et al., 1992
; Aitken and Baker, 1995
; Kiessling et al., 1995
; Yanushpolsky et al., 1996
). Positive semen cultures, a frequent finding during extended infertility investigation in asymptomatic men, and with bacterial prevalence depending on the extent of the microbial screening, are insufficient to diagnose male genital tract infection (Comhaire et al., 1980
; Eggert-Kruse et al., 1992b
, 1995
).
The reproductive tracts of both men and women contain a myriad of immune response cells. Activation of these cells, for example by microorganisms, stimulates them to secrete lymphokines and monokines. These act, at least in part, locally to regulate immunological reactions, but also affect tissues outside the immune system. Interleukin (IL) -8 is a potent neutrophil chemotactic and activating factor. It is involved in angiogenesis and adhesion processes, and enhances the adherence of neutrophils to endothelial cells and subendothelial matrix proteins. It exerts its biological activities by binding to specific cell surface receptors. IL-8 may be involved, within a network of other cytokines, in intratesticular signal transduction, and may also adversely affect sperm membrane properties (Buch et al., 1994). Few investigations have been made concerning this cytokine with regard to reproduction.
Interleukins are part of the local defence mechanism against infectious diseases, but they are also implicated as mediators of the pathology of these diseases. Recently, it has been suggested that an increase in IL-8 concentration resulting in recruitment of neutrophils may play an important role in viral dissemination throughout the body. IL-8 may increase virus production in endothelial cells by an autocrine mechanism; for example, human cytomegalovirus (CMV) infection up-regulates IL-8 gene expression and stimulates transendothelial neutrophil migration (Craigen et al., 1997; Murayama et al., 1997
; Grundy et al., 1998
).
IL-8 concentrations may fluctuate during certain pathological conditions. There is little information about IL-8 interference with sperm production and semen quality, and whether it increases in parallel to leukocyte counts and other potential markers of subclinical infection/inflammation in the same ejaculates, potentially interrelated with other cytokines, e.g. IL-6. Also unknown is whether potentially pathogenic microorganisms in the semen of patients, who are without symptoms of genital tract infection, are associated with increased pro-inflammatory seminal cytokine concentrations, which might impair sperm functional capacity.
Therefore, in the present prospective study, the concentrations of IL-8, and additionally IL-6, were determined in the seminal plasma of a group of randomly chosen asymptomatic, subfertile males. Cytokine concentrations were related to multiple determinants of semen quality, for example the outcome of microscopical sperm analysis, local antisperm antibodies (Ab) to the evaluation of sperm function using different approaches, to seminal WBC and other potential markers of silent inflammation, and to the results of a broad microbial screening that also included patients' female partners.
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Materials and methods |
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Basic infertility investigation and andrological examination
Standard semen analysis according to World Health Organization criteria (WHO, 1992) included determination of volume, pH, count, progressive motility after liquefaction (at room temperature), after 2 h and 4 h, morphology, viability (eosin testing), and seminal plasma (SP) fructose concentration. Ejaculates were obtained in hospital after at least 5 days of sexual abstinence, and were examined directly after liquefaction. IL concentrations did not differ significantly between males with a longer or shorter duration of abstinence. The mean abstinence period was 7 days, 90% percentile 14 days, 95% percentile 18 days, maximum 24 days. All other parameters of semen or SP which were evaluated in this study [apart from post-coital testing (PCT) results] were determined in aliquots of the same ejaculates.
A detailed medical history was obtained and physical examinations were performed on both partners. Investigation for female infertility factors was carried out as described previously (Eggert-Kruse et al., 1989a, 1997
).
Determination of antisperm antibodies
For screening of antisperm antibodies (Ab) in semen samples, the mixed antiglobulin reaction (MAR) was used (Eggert-Kruse et al., 1991). MAR was performed in parallel with immunoglobulin (Ig) G- and IgA-coated erythrocytes and specific antiserum based on a previously published method (Jager et al., 1978
). Readings were performed in triplicate, and the mean was taken. A percentage of
30% of motile spermatozoa involved in the mixed agglutinates was considered MAR-positive; further analyses were performed with MAR
10% as an additional threshold.
Leukocytes
The number of round cells was counted using a haemocytometer. Round cells were differentiated in leukocytes and in cells of the germ cell line by means of an immunocytochemical method with monoclonal Ab (mAb) and a streptavidinbiotin system as described in detail elsewhere (Eggert-Kruse et al., 1992a). The percentage of leukocytes of the seminal round cells was determined (leukocyte ratio). The mean of triplicate counting was used for analysis. Thresholds were set at 5, 10, 15 and 20% leukocytes. A leukocyte ratio of
15% was considered as `leukocyte-positive', based on previous findings (Eggert-Kruse et al., 1992a
).
With regard to the number of leukocytes, samples with >1x106/ml leukocytes were considered as leukocytospermic, according to WHO definition (WHO, 1992). Additionally, the total leukocyte count per ejaculate was used for analyses, with the median and the 75% percentile used as thresholds. Human peripheral leukocytes, obtained from a healthy donor and separated with a Ficoll technique, were used as positive controls. Positive and negative [phosphate-buffered saline (PBS), pH 7.4] controls were included in all of the test series.
Furthermore, a paper-strip test was used to screen fresh semen samples for increased leukocyte numbers (Cytur®; Roche Diagnostics, Mannheim, Germany).
Complement fraction C3
The complement fraction C3 (C3c) was determined using radial immunodiffusion (Nor-Partigen C3c®; Behring, Marburg, Germany). Briefly, seminal plasma was inoculated into ready-for-use agarose-gel layers containing monospecific antiserum against human C3c. After incubation, the diameter of precipitation was measured and the concentration determined using a standard curve. Negative and positive controls were included in each of the test series. The detection concentration was 0.0085 g/l; threshold values at 0.1 and 0.2 g/l were used to define high and very high C3 concentration in seminal fluid, respectively. If necessary, seminal plasma samples were stored frozen at 70°C until use.
Cytokines
IL-8
For detection of IL-8 in seminal plasma a commercial kit was used (Quantikine®; R & D Systems, Minneapolis MN, USA). Briefly, this was a solid phase enzyme-linked immunosorbent assay (ELISA) with a quantitative sandwich technique using microtitre plates which were pre-coated with mAb specific for IL-8. After reaction, the resulting colour reaction was proportional to the amount of IL-8 bound, and was measured at 450 nm; concentrations were read on a standard curve. Testing was performed strictly according to the manufacturer's instructions. Negative and positive controls were included in each of the test series. All tests were performed in duplicate, and the mean was taken. Thresholds for further analyses were set at 1000, 2000, 3000 and 5000 pg/ml. The intra-assay variation was <7%, and the inter-assay variation <10%. The minimum detectable concentration was 10 pg/ml (manufacturer's information).
IL-6
The concentration of IL-6 in seminal plasma was also determined with a solid phase ELISA, which employed the quantitative sandwich enzyme immunoassay technique (Quantikine HS®). This assay included an amplification system in which the alkaline phosphatase reaction provided a cofactor that activated a redox cycle, leading to the formation of a coloured product. The resulting colour reaction was proportional to the amount of IL-6 bound in the initial step; concentrations were read on a standard curve. Testing was performed strictly according to the manufacturer's instructions, and included negative and positive controls in each of the test series. The mean of duplicate tests was taken for analysis. Seminal plasma concentrations of 10, 15, 30, 50 and 100 pg/ml were used as thresholds. Intra-and inter-assay variations were <10%. The minimum detectable concentration was 0.01 pg/ml (product information).
Microbial screening
To check semen samples for colonizing microorganisms, 10 µl of semen were transferred with a disposable sterile plastic loop into Shepard's medium for identification and differentiation of mycoplasmas; in addition, swabs were obtained and inoculated into a universal transport medium (Port-a-Cul Universal®; BectonDickinson, Heidelberg, Germany). Microbial prevalence was identified with standard methods (Institute of Microbiology, University of Heidelberg) as previously described (Eggert-Kruse et al., 1992b).
For detection of Chlamydia trachomatis, a commercial amplification assay (ligase chain reaction, LCR) (LCX®; Abbott, Wiesbaden, Germany) was used in semen, and in parallel in first void urine (FVU) specimens, according to the manufacturer's instructions. Serological testing for Chlamydia included determination of anti-chlamydial Ab (Chlam Ab) of the IgA and IgG class in seminal plasma, and of Chlam IgA Ab in serum, by means of a commercial recombinant ELISA (rELISA®; Medac, Hamburg, Germany). Chlam IgG Ab in same-day serum were determined with an immunofluorescence assay (Virgo® Chlamydia trachomatis IgG immunofluorescence test; Schiapparelli Biosystems Inc., Columbia, IL, USA). Serology was performed according to the manufacturer's instructions, and included positive and negative controls.
Simultaneously, a microbial screening was performed in patients' female partners. Endocervical material was obtained for detection of C. trachomatis using LCR (performed in parallel in FVU), for culture and differentiation of mycoplasmas, and for bacterial cultures (see above). Serum of female partners, taken at the same time, was checked for Chlam IgG Ab by means of micro-immunofluorescence testing (Virgo®). A serum titre of 1:256 was considered strongly positive (Eggert-Kruse et al., 1997
). Serum samples were stored frozen at 20°C until further processing.
Evaluation of sperm functional capacity
Migration testing
Migration testing was performed as described previously (Biljan et al., 1994). Briefly, semen aliquots (0.1 ml) from the same ejaculates used for IL testing, were carefully layered under the same amount of culture medium (Ham's F-10; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) in a sterile Falcon tube. After 90 min incubation at 37°C in humidified air and 5% CO2, the upper 0.05 ml, into which motile spermatozoa had migrated, were carefully harvested and the concentration of motile spermatozoa was assessed. A concentration of
1x106/ml motile spermatozoa was considered positive, a post-migration count of
3.6x106/ml motile spermatozoa was used as an additional threshold (based on the 75% percentile) and defined as a very good test outcome. Furthermore, the quotient (Q) calculated from the concentration of motile spermatozoa in the initial semen and after migration testing was determined. Two thresholds were used for analyses: the median (Q </
0.067), and the 75% percentile (Q </
0.14). The migration quotient (Q) and the concentration of motile spermatozoa harvested after incubation correlated significantly, with a correlation coefficient (r) of 0.793 (P < 0.0001) (Spearman rank correlation).
Spermcervical mucus penetration test (SCMPT)
The ability of spermatozoa to penetrate the cervical mucus (CM) barrier as a parameter of functional capacity was evaluated by means of the in-vitro spermCM penetration test (SCMPT) performed as previously described (Eggert-Kruse et al., 1989a). Briefly, the penetration of spermatozoa within capillaries, filled with fresh samples of CM, obtained from patients' female partners under hormonally standardized conditions, was observed microscopically after 30 min, 2 h and 6 h incubation. Penetration density, migration distance, quality and duration of motility were examined, graded and summarized in a cumulative score, which served to select two groups with inadequate and adequate sperm penetration ability.
Post-coital test (PCT)
SpermCM interaction in vivo was assessed with the post-coital test (PCT) at 812 h after intercourse. The status of the CM (obtained in the peri-ovulatory period of spontaneous cycles or, if necessary because of endocrine disorders or poor CM quality, after pretreatment with oestrogens; Eggert-Kruse et al., 1989b) was classified according to a previously described scheme (Insler et al., 1972). The number of spermatozoa with forward progression in CM was counted using first the low-power field (LPF, x100) and then the high-power field (HPF, magnification x400), the mean of 20 visual fields being taken. PCT was regarded as adequate when at least two spermatozoa of highly progressive motility/HPF were counted in CM; otherwise it was classified as inadequate. For further evaluation, PCT results were subdivided into four groups: PCT negative (no spermatozoa found in CM/LPF but in vaginal secretions), PCT poor (<2 motile spermatozoa/HPF), PCT good (26 motile spermatozoa/HPF), and PCT excellent (
7 motile spermatozoa/HPF).
Statistical analysis
Data were processed using the statistical analysis system (SAS). Spearman rank correlation, chi-square (2) analysis, Fisher's two-tailed exact test and Wilcoxon rank-sum test were used. The level of significance was set at P < 0.05.
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Results |
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Compared with IL-8, the seminal plasma concentration of IL-6 was much lower, with a median of 15 (range 3.3520) pg/ml (25% percentile 9 pg/ml, 75% percentile 32 pg/ml). A threshold at 30 pg/ml was used to define `high' IL-6 concentrations, which were found in 27% (37/137) of samples. [Very high IL-6 concentrations of
50 pg/ml were measured in 15.3% (21/137), and of
100 pg/ml in 10.9% (15/137) of samples.]
Correlation of IL-8 and IL-6
A high IL-8 concentration was found in 67.6% (25/37) of samples with high IL-6 (30 pg/ml) compared with 10% (10/100) of samples with low IL-6 (P < 0.001). Alternatively, a high IL-6 concentration was found in 71.4% (25/35) of samples with high IL-8 (
2000 pg/ml) compared with 11.8% of the other specimens (12/102) (P < 0.001). Although the seminal plasma concentrations of these cytokines were significantly correlated (r = 0.783; P < 0.0001), results were analysed separately for their potential relationship with parameters of sperm quality.
Relationship with outcome of semen analysis
IL-8
The association of IL-8 concentrations with results of microscopical ejaculate analysis is shown in Table I. Although IL-8 concentration was markedly higher in oligozoospermic men (13.3% of patients), this did not achieve significance. IL-8 concentrations were significantly higher in samples with reduced motility (<20%) (P < 0.05), and were also higher in the group of samples with <40% progressive motility (38.4% of patients), though not to a statistically significant level. This association was confirmed with regard to sperm motility after 2 h and after 4 h. Significant differences were also found when the sperm count per ejaculate or the total number of motile spermatozoa were taken into consideration (P < 0.02). A high IL-8 concentration occurred significantly more frequently in samples with a total motile sperm count below the median (50x106) or below the 75% percentile (100x106/ml) in this population (P < 0.01).
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IL-6
IL-6 concentrations related to the results of semen analysis are shown in Table II. No marked association was found with sperm count per ml or per ejaculate. Although IL-6 concentration was markedly higher in the case of reduced motility, this did not achieve significance, as well as concentrations in samples with total motile sperm counts <50x106/ml (or <100x106/ml) compared with specimens of better quality. Median IL-6 concentration was significantly higher in ejaculates with increased pH (
7.5) (P < 0.04); no relationship was found with standard morphology, viability, and seminal plasma fructose.
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Relationship with seminal leukocytes
The concentration of IL-8 in seminal fluid was significantly correlated with the leukocyte ratio after immunocytochemical differentiation of the round cells (r = 0.514, P < 0.0001) (Spearman rank correlation). The proportion of leukocytes ranged from 0 to 96%. The correlation coefficient with respect to leukocyte numbers per ejaculate and IL-8 was 0.562 (P < 0.0001). Using several threshold values to define samples with increased leukocyte ratios [e.g. `leukocyte-positive' (15%; total 23.5%)], the significant association between leukocytes and IL-8 in semen is shown in Table IV
; high IL-8 was found in 62.5% of the leukocyte-positive group compared with 14.4% of the other ejaculates (P = 0.001). Median IL-8 was four-fold higher in samples with leukocytospermia (P < 0.0001). None of the samples with low IL-8 (<2000 pg/ml) was leukocytospermic. A significant relationship was also found with the total number of leukocytes per ejaculate (Table IV
). [The strong association of the IL-8 concentration with seminal WBC was confirmed when other threshold values were used to define high or very high IL-8 concentrations (
3000 pg/ml or
5000 pg/ml).]
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The close association with WBC in semen was confirmed with regard to IL-6. A strong correlation of the seminal plasma IL-6 concentration with the leukocyte ratio (r = 0.564, P < 0.0001) was noted; with regard to leukocyte numbers per ml, r = 0.645 (P < 0.0001). IL-6 increased significantly with increasing leukocytes ratio (Table V). Leukocytospermic samples had a markedly higher IL-6 concentration (median 152 pg/ml, minimum 32 pg/ml, P < 0.0001). Significant differences were also found with respect to the number of WBC per ejaculate (P < 0.0001), or when other thresholds were used to define samples with high or very high (
50 pg/ml or
100 pg/ml) IL-6 concentration.
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Relationship with complement fraction C3
As an additional parameter of subclinical infection/inflammation, the complement fraction C3 was determined. C3 was detectable in 38.3% (44/115) of ejaculates, and high concentrations (0.02 g/l) were found in 14.8% (17/115). The significant relationship with IL-8 concentrations in seminal plasma aliquots of the same ejaculates is shown in Table IV
(r = 0.443, P < 0.0001).
Seminal fluid IL-6 concentrations were also significantly correlated with this parameter (r = 0.414, P < 0.0001). In the group with high IL-6 concentration, high C3 was found in 33.3% (10/30) compared with 8.2% (7/85) of the other samples (P < 0.0002) (Table V).
Interleukin and sperm functional capacity
The outcome of sperm migration testing, performed in 94 samples (69% of patients), which were also used for interleukin determination in seminal plasma, was very good in 24.5% (23/94) of cases. The median IL-8 concentration was lower in this group (922 pg/ml versus 1332 pg/ml), but this did not reach statistical significance. A high IL-8 concentration was found in 13.0% compared with 28.2% of the other samples (see Table I).
The standardized SCMPT was performed in 132 samples, with inadequate outcome in 43.2% (57/132), and negative result in 15.9% (21/132). SCMPT was not influenced by IL-8 concentrations in SP of the same ejaculates. This could be confirmed when donors' CM was used for the crossed penetration assay instead of CM of the patients' partners [inadequate results in 40.7% (55/135), negative in 11.1% (15/135)].
Spermmucus interaction in vivo was evaluated with the PCT. No marked relationship of IL-8 on results with this screening test was obvious. A high IL-8 concentration was found in the seminal plasma in 26.0% (20/77) of patients with adequate PCT results, and in 24.1% of males of the other couples. However, after exclusion of women with a reduced cervical index (<10), a comparison of median IL-8 concentrations revealed significantly higher concentrations in seminal fluid of men with inadequate PCT results compared with the other patients (median 1470 pg/ml versus 1043 pg/ml, P < 0.03).
As shown in Table II, a high IL-6 concentration occurred significantly more frequently when sperm migration testing revealed reduced functional competence of spermatozoa (P < 0.03). This was found for the number of post-migration spermatozoa (or spermatozoa harvested after the swim-up procedure), as well as for the migration test quotient (with the 75% percentile as threshold).
In addition to IL-8, seminal fluid IL-6 concentration was not significantly associated with the ability of spermatozoa to penetrate the CM of patients' partners or of CM donors in vitro in the standardized SCMPT.
The frequency of samples with high IL-6 concentration was comparable in patients with inadequate or adequate PCT. After exclusion of couples with PCT outcome which indicated poor CM quality (cervical index <10), the median IL-6 concentration was 18 pg/ml in the subgroup with inadequate PCT compared with 12 pg/ml in males of couples with an adequate PCT result (P < 0.03).
Relationship with semen cultures
Semen cultures revealed aerobic growth in 40% of ejaculates (54/135), with potentially pathogenic species in 22 of these specimens (40.7%) (mostly enterococci, group B streptococci, Escherichia coli or Proteus spp.). Positive cultures were not related to IL-8 concentrations in seminal fluid (see Table VI). This lack of association could also be shown with regard to the different species, e.g. E. coli. The detection rate for anaerobes using standard medium and routine transport conditions was low, and was not considered for analysis. There was also no relationship of IL-8 concentration with the colonization of semen with mycoplasmas (Mycoplasma hominis and/or Ureaplasma urealyticum) which were cultured in 11.2% of cases (15/134). Median IL-8 concentrations were lower in this group than in the mycoplasma-negative group. Using amplification methods (LCR) in both semen and urine, C. trachomatis was found in only one patient (1/137) (female partner also positive). This man had a very high IL-8 concentration in his seminal plasma (6336 pg/ml).
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There was also no relationship of IL-8 concentration in the seminal fluid and microbial colonization of the lower genital tract of patients' female partners. Apart from the above-mentioned bacteria, lactobacilli were cultured in 63.4% of the cervical swabs, and Candida albicans in the fornix posterior vaginalis in 4.8%. All male and female samples were free of Trichomonas vaginalis and Neisseria gonorrhoeae. The prevalence of C. trachomatis in women was 1.5% (2/137). The partner of the second woman with LCR-positive endocervical material, who was himself negative for C. trachomatis in semen and FVU, had a low IL-8 concentration (809 pg/ml). IL-8 concentration in the seminal plasma of the males was also not related to anti-chlamydial Ab in their female partners' serum.
The lack of association with the microbial colonization of ejaculates in this asymptomatic group of patients was confirmed with regard to IL-6. This could be shown for standard bacteriology as well as for mycoplasma cultures. The patient with a LCR-proven C. trachomatis infection had a very high IL-6 concentration in seminal plasma (163 pg/ml), but this was not the case in the other patient whose wife only had a chlamydial infection (12 pg/ml). There was no significant association of high seminal fluid IL-6 concentrations and anti-chlamydial Ab in serum or seminal plasma.
As was the case for IL-8, IL-6 was not related to the microbial pattern in the lower genital tract of patients' female partners.
Relationship with general parameters
IL-8 concentrations were not significantly related to the outcome of clinical andrological examination, including testicular volume and consistency; neither were they associated with previous genital trauma, surgery, mumps or varicocoelectomy. More patients of the group with previous genital tract infections (7.6% of patients) had high IL-8 concentrations in seminal plasma [40.0% (4/10) compared with 24.6% (30/122)], but the differences were not statistically significant. There was no association with the duration of infertility, although a high IL-8 concentration was more frequent in men with long-standing infertility (>5 years; total 28.8%). More than half of the men (51.2%) complained of stress because of working conditions. Increased IL-8 was found in 27.3% of this group of patients, and in 23.8% of the other men (not significant). When stress was related to infertility (as indicated by males on a questionnaire; total 17.2%), high IL-8 was found in 36.4% (8/22) compared with 23.6% (25/106, not significant). A high IL-8 concentration in seminal fluid was slightly more frequent in the small group of patients (total 9.2%) who reported occasional sexual dysfunction (mostly erectile dysfunction) (33.3% versus 24.6%, not significant). There was no significant correlation of IL-8 concentration with male age.
IL-6 concentrations were also not significantly related to the outcome of clinical andrological examination, testicular volume and consistency, or to previous genital tract infections. IL-6 in seminal fluid was not significantly associated with stress factors (job-based conditions or stress because of infertility), but was higher in the subgroup with occasional sexual disorders (median IL-6 34 pg/ml compared with 14 pg/ml; P < 0.01). Although median IL-6 concentrations were higher in men with a longer duration of infertility, this did not achieve significance, neither was there any significant relationship of IL-6 concentration with male age.
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Discussion |
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Testing was performed under standardized conditions. All ejaculates were obtained in the hospital, after a period of sexual abstinence of at least 5 days, in the early morning (between 7:00 and 9:00), and were examined directly after liquefaction. None of the men was treated with antibiotics during the time of the study. Functional assays such as sperm migration testing and SCMPT, as well as immunocytochemical round cell differentiation and radial immunodiffusion, were performed by the same individuals (R.B. and M.H.). Neither investigator was aware of the IL results throughout the study; thus, any inter-observer variations could be excluded.
Cytokines rarely act in isolation, but rather in a network of other cytokines. The absolute concentrations of IL-8 in seminal fluid were much higher compared with IL-6, making the use of a highly sensitive assay for the latter parameter necessary. Seminal plasma concentrations of IL-8 were markedly higher than serum concentrations reported elsewhere (Humar et al., 1999). In the present study, IL-6 concentrations in semen were within the range reported for healthy men, but also much higher than in serum (Comhaire et al., 1994
; Maes et al., 1997
), and were within a wide inter-individual range. In this study, the concentrations of IL-8 and IL-6 were significantly interrelated (r = 0.783, P < 0.0001), which has also been shown for other cytokines, such as IL-6 and IL-1 (Comhaire et al., 1999
). Results of this and other studies are limited for the one-point determination of these interleukins, and future investigations will need to show the intra-individual variation over a longer time period and potentially influencing factors. The populations of soluble receptors of the cytokines must also be considered, though previous investigations have failed to show a significant correlation between IL-6 and its soluble receptor in either semen or serum (Comhaire et al., 1994
).
Cytokines are potent polypeptides that are released from inflammatory cells as part of the host response. Many cell types, including monocytes/macrophages, T cells and neutrophils, can produce IL-8 in response to a wide variety of signals frequently initiated by infection or injury. Cytokines play a pivotal role as a mediator of numerous physiological and pathological processes, particularly in the initiation of the immuno-inflammatory cascade (Rees, 1992; Rutanen, 1993
; Fiocchi et al., 1994
; Galley and Webster, 1996
; Grimm et al., 1996
; Henderson et al., 1996
; Sharkey, 1998
). Cytokines are also involved in allograft rejection. An increased production of some pro-inflammatory cytokines has also been reported during immune responses in major depression (Maes et al., 1997
). Genetic factors also substantially influence the production of cytokines (Westendorp et al., 1997
).
In addition to their function in the immune system, cytokines may have a significant role in modulating the neurocrine control of reproduction. They probably play a physiological part as local mediators of the action of sex hormones, and are involved in the paracrine regulation of spermatogenesis (Takao et al., 1990; Bookfor and Schwarz, 1991
; Bookfor et al., 1994
). Another mechanism of interference with sperm quality may be an adverse effect on sperm membrane properties, e.g. lipid peroxidation (Buch et al., 1994
). Increased oxidative stress may additionally modulate the concentration of these cytokines (Rajasekaran et al., 1995
). However, the role of reactive oxygen species (ROS) in human semen is complex (for a review, see Ochsendorf, 1999), and seminal plasma ROS scavenging systems also have to be considered.
When the relationship of sperm with semen quality of the same ejaculates was analysed in the present study, a particular association was noted with the total number of motile spermatozoa. This is considered as a clinically important parameter, notably with regard to fertilization rates in IVF programmes (Acosta et al., 1988; Ben-Chetrit et al., 1995
). It has been suggested that sperm production rates are more suitable for estimating testicular function than standard parameters of semen analysis, for example, sperm count per ml. The relationship of IL-8 with percentage progressive motility or `classical' oligozoospermia (WHO, 1992
) was less obvious, and there was no association with standard morphology. However, in this population which was unselected for infertility factors of the involuntarily childless couples, severe male factor cases were relatively rare. The association might be more pronounced in a group of males selected on the basis of andrological disorders.
The relationship of pro-inflammatory cytokines (e.g. IL-6) with semen quality in other studies is controversial. In landmark studies, others (Hill et al., 1987, 1989
) showed a significant effect of soluble products of activated immune cells and of some lymphokines and tumour necrosis factor (in high concentrations) on sperm motility and on the outcome of the zona-free hamster egg test. This contrasts with other reports which did not show a relationship of, for example, IL-6 with standard parameters of semen analysis (Comhaire et al., 1994
; Huleihel et al., 1996
; Dousset et al., 1997
), possibly due to differing population characteristics and assay methods.
The majority of ejaculates were also examined for sperm migration ability after a swim-up procedure. Migration testing has been suggested as a relatively simple screening test with relevance for IVF success (Biljan et al., 1994). A relationship, particularly of IL-6 concentrations, with results of this method was found in the present investigation. Future studies will have to show if there is also an influence of interleukin concentration in seminal plasma on fertilization rates under in-vitro conditions.
On the other hand, interleukin concentrations were not associated with the outcome of the spermCM penetration test which has been shown to be a reliable indicator of sperm fertilizing capacity under in-vivo conditions of conception (Eggert-Kruse et al., 1989a,b
). This was found when CM of patients' partners obtained under standardized conditions, as well as CM of donors, was used in the present investigation. Differences compared with migration testing might be due to a much closer association of semen quality parameters with the number of motile spermatozoa harvested after the swim-up procedure (e.g. r = 0.851 for sperm count and Q) found here than with the ability of spermatozoa to penetrate the mucus barrier, to adapt to this medium, and to maintain motility in this viscous material for a longer time period (6 h compared with 1.5 h in the migration test). SpermCM interaction testing examines another dimension of sperm functional competence. Furthermore, immunosuppressive properties of CM and potential scavenging systems must be taken into consideration.
There was a certain influence of IL concentrations on PCT outcome which should be interpreted with caution because PCT was the only test performed at a different time (usually some days after cytokine determination), and fluctuations of semen quality must be considered. Furthermore, PCT is influenced by a multiplicity of different factors.
In the present study, a clear association of IL-8 and IL-6 with seminal leukocytes was found. Leukocytes were determined with an immunocytochemical procedure which has several advantages over conventional methods. Not only was the number of leukocytes per ml and per ejaculate determined, but also the leukocyte ratio as a different parameter. Leukocytes play a very important role in successful reproduction, both in the male and female genital tract. However, the role and function of leukocytes in semen, and in particular of the clinically relevant threshold values, are a matter of considerable debate (Barratt et al., 1990; Vogelpoel et al., 1991
; Tomlinson et al., 1992
; Aitken and Baker, 1995
; Kiessling et al., 1995
; Yanushpolsky et al., 1996
; Thomas et al., 1997
). Nevertheless, in the clinical setting, seminal leukocyte counts are widely used as the parameter to diagnose male genital tract infection/inflammation. There is a lack of other markers with clinical significance for this purpose.
In asymptomatic men, silent genital tract infection can only be diagnosed by laboratory parameters. Genital tract infection may impair semen quality by many different mechanisms (Eggert-Kruse et al., 1996; Witkin et al., 1996; Comhaire et al., 1999
; Ochsendorf, 1999
; Weidner et al., 1999
). Treatment of male factor infertility in general is limited. A comprehensive diagnostic work-up and potential subgroup selection is worth considering, as there is a therapeutic potential if semen quality is poor because of infectious or inflammatory reasons. Findings in asymptomatic men of this investigation are in agreement with other studies reporting an increase of IL-6 in seminal fluid of patients exhibiting inflammatory disease of the accessory sex glands (Shimoya et al., 1993
; Comhaire et al., 1994
).
The relationship of interleukins with traditional parameters was confirmed when other biochemical assays, such as the C3c determination with radial immunodiffusion, were used. Factors of the C3 complement component generated by proteolytic cleavage have opsonic power, and are involved in the system of humoral immunity (Clark and Klebanoff, 1976; Katz et al., 1989
; Brandtzaeg et al., 1996
; Heller and Koch, 2000
). C3 measurement is considered to be a sensitive parameter for the identification of inflammatory processes in the prostate and adnexae (Blenk and Hofstetter, 1991
).
The close association of IL-8 with seminal leukocytes suggests that it might be used as sensitive marker for silent infection. It might also provide information about the activation status of local WBC, in addition to cell counts and ratios. It is possible that the determination of IL-8 in SP might offer practical advantages for screening large groups (for research purposes) compared with round cell differentiation in semen. It may also have applications for monitoring patients, and controlling the success of specific therapeutic interventions, though this must be demonstrated in future trials.
Another important finding of this investigation is that the concentrations of the pro-inflammatory cytokines were not related to the microbial colonization of semen samples. In the clinical setting, there is much controversy about the consequences of positive semen cultures in asymptomatic subfertile males. This surely depends on the pathogenicity of the species identified in semen (or urethral swabs). For example, C. trachomatis is very important for subsequent fertility: sexual transmission would have severe sequelae, for example for tubal function in their partners (for a review, see Paavonen and Eggert-Kruse, 1999). These microorganisms were a rare finding in this study, despite the use of sensitive methods for their detection, due to well-known population characteristics. Transmission and prevalence are also markedly influenced by the presence of other sexually transmitted diseases (STD), findings which correspond with the low prevalence of mycoplasmas as well as with the absence of trichomonads and gonorrhoea. It is important to note that the C. trachomatis-positive patient had high interleukin concentrations in seminal plasma, indicative of genital tract infection. More research in large groups of males with actual chlamydial infection is needed. As expected, no marked relationship was found between SP interleukin concentrations and chlamydial serology as a marker of past infection, thus confirming previous findings that used other infection/inflammation markers (Eggert-Kruse et al., 1996b).
With regard to other frequently found bacteria of potentially pathogenic nature (E. coli, enterococci, streptococci, Proteus spp.), there was no relationship with interleukin concentrations. The microbial spectrum was similar to that found in much larger populations (Eggert-Kruse et al., 1992b, 1995
). However, a higher prevalence of anaerobes in semen could be expected in case a more differentiated evaluation for these sensitive bacteria would have been applied. Findings confirm that the mere presence of microorganisms in semen is an inadequate parameter to diagnose male genital tract infection. Cytokine concentrations may more accurately indicate an early phase of infection/inflammation. This is also in agreement with previous studies which did not show an association of seminal bacteria with leukocytes in ejaculates of asymptomatic subfertile patients (Eggert-Kruse et al., 1992a
). However, results might be different in symptomatic men and in patients with evidence of other STD. Future research along this line is needed.
It should also be considered that bacterial infection is not the only factor that might lead to an indirect association between leukocytospermia and infertility. Alternative aetiologies include abnormal spermatozoa, chemical irritants and environmental factors, as well as viral infections. In most studies concerning microbial findings in semen samples, viruses are not considered, but such knowledge is limited and viruses that might have a significant effect on spermatogenesis have still to be defined. In this respect, interleukins (especially IL-8) are of particular interest. The serum concentration of IL-8 was shown to be correlated with activity of CMV infection in transplant recipients (Humar et al., 1999), while cytokine-mediated neutrophil recruitment with subsequent endothelial cellneutrophil interaction may serve to enhance virus dissemination in vivo (Grundy et al., 1998
). These cytokine elevations may represent part of a non-specific acute-phase response, or they may be due to specific interactions between viruses (or other stimuli) and the immune system. The subsequent interaction between virus and inflammatory cytokines could lead to a state of (silent) inflammation (possibly with the generation of ROS) which could induce suppression of adequate spermatogenesis. Cytokine determination in seminal plasma might offer a means to select a subgroup of patients who would benefit from an extended microbiological examination including virus screening, and of obtaining further information on inflammatory causes of semen quality impairment in asymptomatic patients.
In conclusion, although future efforts are necessary to establish the specific mechanisms and the significance of the findings for fertilizing capacity under in-vivo and in-vitro conditions of conception, the results of this study indicate that the seminal plasma interleukin concentration might be a sensitive and useful marker of silent infection/inflammation of the male genital tract, and may also provide information about the activation status of local WBC, in addition to cell counts and ratios.
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Notes |
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5 Present address: Department of Laboratory Medicine, Klinikum Ingolstadt, University of Heidelberg, Germany
6 To whom correspondence should be addressed at: Department of Gynaecological Endocrinology and Reproductive Medicine, Women's Hospital, University of Heidelberg, Voßstr. 9, 69115 Heidelberg, Germany
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Aitken, R.J. and Baker, H.W.G. (1995) Seminal leukocytes: passengers, terrorists or good Samaritans? Hum. Reprod., 10, 17361739.[ISI][Medline]
Barratt, C.L.R., Bolton, A.E. and Cooke, I.D. (1990) Functional significance of white blood cells in the male and female reproductive tract. Hum. Reprod., 5, 639648.[Abstract]
Ben-Chetrit, A., Senoz, S., Greenblatt, E.M. and Casper, R.F. (1995) In vitro fertilization outcome in the presence of severe male factor infertility. Fertil. Steril., 63, 10321037.[ISI][Medline]
Biljan, M.M., Taylor, R.C., Manasse, P.R. et al. (1994) Evaluation of different sperm function tests as screening methods for male fertilization potential the value of the sperm migration test. Fertil. Steril., 62, 591598.[ISI][Medline]
Blenk, H. and Hofstetter, A. (1991) Complement C3, coeruloplasmin and PMN-elastase in the ejaculate in chronic prostato-adnexitis and their diagnostic value. Infection, 19, 138141.[ISI][Medline]
Bookfor, F.R. and Schwarz, L.K. (1991) Effects of interleukin 6, interleukin 2, and tumor necrosis factor on transferrin release from Sertoli cells in culture. Endocrinology, 129, 256262.[Abstract]
Bookfor, F.R., Wang, D., Lin, T. et al. (1994) Interleukin 6 secretion from rat Leydig cells in culture. Endocrinology, 134, 21502155.[Abstract]
Brandtzaeg, P., Hogasen, K., Kierulf, P. and Mollnes, T.E. (1996) The excessive complement activation in fulminant meningococcal sepsis is predominantly caused by alternative pathway activation. J. Infect. Dis., 173, 647655.[ISI][Medline]
Buch, J.P., Kolon, T.F., Maulik, N. et al. (1994) Cytokines stimulate lipid membrane peroxidation of human sperm. Fertil. Steril., 62, 186188.[ISI][Medline]
Clark, R.A. and Klebanoff, S.J. (1976) Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J. Immunol., 117, 13781386.[Abstract]
Comhaire, F., Verschraegen, G. and Vermeulen, L. (1980) Diagnosis of accessory gland infection and its possible role in male infertility. Int. J. Androl., 3, 3245.[ISI][Medline]
Comhaire, F., Bosmans, E., Ombelet, W. et al. (1994) Cytokines in semen of normal men and of patients with andrological diseases. Am. J. Reprod. Immunol., 31, 99103.[ISI][Medline]
Comhaire, F.H., 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, 393398.
Craigen, J.L., Yong, K.L., Jordan, N.J. et al. (1997) Human cytomegalovirus infection upregulates interleukin-8 gene expression and stimulates neutrophil transendothelial migration. Immunology, 92, 138145.[ISI][Medline]
Dousset, B., Hussenet, F., Daudin, M. et al. (1997) Seminal cytokine concentrations (IL-1ß, IL-2, IL-6, sR IL-2, sR IL-6), semen parameters and blood hormonal status in male infertility. Hum. Reprod., 12, 14761479.[Abstract]
Eggert-Kruse, W., Leinhos, G., Gerhard, I. et al. (1989a) Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus. Fertil. Steril., 51, 317323.[ISI][Medline]
Eggert-Kruse, W., Gerhard, I., Tilgen, W. and Runnebaum, B. (1989b) Clinical significance of crossed in vitro sperm-cervical mucus penetration test in infertility investigation. Fertil. Steril., 52, 240.
Eggert-Kruse, W., Hofsäß, A., Haury, E. et al. (1991) Relationship between local antisperm antibodies and sperm-mucus interaction in vitro and in vivo. Hum. Reprod., 6, 267276.[ISI][Medline]
Eggert-Kruse, W., Bellmann, A., Tilgen, W. and Runnebaum, B. (1992a) Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male fertility. Fertil. Steril., 58, 10461055.[ISI][Medline]
Eggert-Kruse, W., Pohl, S., Näher, H. et al. (1992b) Microbial colonization and sperm-mucus interaction: results in 1000 infertile couples. Hum. Reprod., 7, 612620.[Abstract]
Eggert-Kruse, W., Rohr, G., Ströck, W. et al. (1995) Anaerobes in ejaculates of subfertile men. Hum. Reprod. Update, 1, 462478.[Abstract]
Eggert-Kruse, W., Probst, S., Rohr, G. et al. (1996a) Induction of immunoresponse by subclinical male genital tract infection? Fertil. Steril., 65, 12021209.[ISI][Medline]
Eggert-Kruse, W., Buhlinger-Göpfahrt, N., Rohr, G. et al. (1996b) Antibodies to Chlamydia trachomatis in semen and relationship with parameters of male fertility. Hum. Reprod., 11, 14081417.
Eggert-Kruse, W., Rohr, G., Probst, S. et al. (1997) Chlamydial serology in 1303 asymptomatic subfertile couples. Hum. Reprod., 12, 14641475.[Abstract]
Fiocchi, C., Binion, D.G. and Katz, J.A. (1994) Cytokine production in the human gastrointestinal tract during inflammation. Curr. Opin. Gastroenterol., 2, 639644.
Galley, H.F. and Webster, N.R. (1996) The immune-inflammatory cascade. Br. J. Anaesth., 77, 1116.
Grimm, M.C., Elsbury, S.K.O., Pavli, P. and Doe, W.F. (1996) Interleukin 8: cells of origin in inflammatory bowel disease. Gut, 38, 9098.[Abstract]
Grundy, J.F., MacCormac, L.P. and Yong, K.L. (1998) Cytomegalovirus-infected endothelial cells recruit neutrophils by the secretion of C-X-C chemokines and transmit virus by direct neutrophil-endothelial cell contact and during neutrophil transendothelial migration. J. Infect. Dis., 177, 14651474.[ISI][Medline]
Heller, A. and Koch, T. (2000) Das Komplementsystem: Alter Hut oder Ziel neuer Therapieansätze? Anaestsiol. Intensivmed. Notfallmed., 35, 207213.
Henderson, B., Poole, S. and Wilson, M. (1996) Microbial/host interactions in health and disease: who controls the cytokine network? Immunopharmacology, 35, 121.[ISI][Medline]
Hill, J.A., Haimovici, F., Politch, J.A. and Anderson, D.J. (1987) Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil. Steril., 47, 460466.[ISI][Medline]
Hill, J.A., Cohen, J. and Anderson, D.J. (1989) The effects of lymphokines and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test. Am. J. Obstet. Gynecol., 160, 11541159.[ISI][Medline]
Huleihel, M., Lunenfeld, E., Levy, A. et al. (1996) Distinct expression levels of cytokines and soluble cytokine receptors in seminal plasma of fertile and infertile men. Fertil. Steril., 66, 135139.[ISI][Medline]
Humar, A., St Louis, P., Mazzulli, T. et al. (1999) Elevated serum cytokines are associated with cytomegalovirus infection and disease in bone marrow transplant recipients. J. Infect. Dis., 179, 484488.[ISI][Medline]
Insler, V., Melmed, H., Eichenbrenner, I. et al. (1972) The cervical score. A simple semiquantitative method for monitoring of the menstrual cycle. Int. J. Gynaecol. Obstet., 10, 223228.
Jager, S., Kremer, J. and van Schlochteren-Draaisma, T. (1978) A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int. J. Fertil., 23, 1221.[ISI][Medline]
Katz, Y., Revel, M. and Strunk, R.C. (1989) Interleukin 6 stimulates synthesis of complement protein factor B and C3 in human skin fibroblasts. Eur. J. Immunol., 19, 983988.[ISI][Medline]
Kiessling, A.A., Lamparelli, N., Yin, H.Z. et al. (1995) Semen leukocytes: friends or foes? Fertil. Steril., 64, 196198.[ISI][Medline]
Koumantakis, E., Matalliotakis, I., Kyriakou, D. et al. (1998) Increased levels of interleukin 8 in human seminal plasma. Andrologia, 30, 339343.[ISI][Medline]
Maes, M., Bosmans, E., De Jongh, R. et al. (1997) Increased serum IL 6 and IL 1 receptor antagonist concentrations in major depression and treatment resistant depression. Cytokine, 9, 853858.[ISI][Medline]
Murayama, T., Ohara, Y., Obuchi, M. et al. (1997) Human cytomegalovirus induces interleukin 8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1 and NF-K binding sites of the interleukin-8 gene. J. Virol., 71, 56925695.[Abstract]
Ochsendorf, F.R. (1999) Infections in the male genital tract and reactive oxygen species. Hum. Reprod. Update, 5, 399420.
Paavonen, J. and Eggert-Kruse, W. (1999) Chlamydia trachomatis: impact on human reproduction. Hum. Reprod. Update, 5, 433447.
Rajasekaran, M., Hellström, W.J.G., Naz, R.J. and Sikka, S.C. (1995) Oxidative stress and interleukins in seminal plasma during leukocytospermia. Fertil. Steril., 64, 166171.[ISI][Medline]
Rees, R.C. (1992) Cytokines as biological response modifiers. J. Clin. Pathol., 45, 9398.[ISI][Medline]
Rutanen, E.M. (1993) Cytokines in reproduction. Ann. Med., 25, 343347.[ISI][Medline]
Sharkey, A. (1998) Cytokines and implantation. Rev. Reprod., 3, 5261.
Shimoya, K., Matsuzaki, N., Tsutsui, T. et al. (1993) Detection of interleukin-8 (IL-8) in seminal plasma and elevated IL-8 in seminal plasma of infertile patients with leukospermia. Fertil. Steril., 59, 885888.[ISI][Medline]
Takao, T., Mitchell, W.M., Tracey, D.E. and De Souza, E.B. (1990) Identification of interleukin-1 receptor in mouse testis. Endocrinology, 127, 251258.[Abstract]
Thomas, J., Fishel, S.B., Hall, J.A. et al. (1997) Increased polymorphonuclear granulocytes in seminal plasma in relation to sperm morphology. Hum. Reprod., 12, 24182421.[Abstract]
Tomlinson, M.J., White, A., Barratt, C.L.R. et al. (1992) The removal of morphologically abnormal sperm forms by phagocytes: a positive role for seminal leukocytes? Hum. Reprod., 7, 517522.[Abstract]
Vogelpoel, F.R., van Kooij, R.J., te Velde, E.R. and Verhoef, J. (1991) Influence of polymorphonuclear granulocytes on the zona free hamster oocyte assay. Hum. Reprod., 6, 11041107.[Abstract]
Weidner, W., Krause, W. and Ludwig, M. (1999) Relevance of male accessory gland infection for subsequent fertility with special focus on prostatitis. Hum. Reprod. Update, 5, 421432.
Westendorp, R.G.J., Langermans, J.A.M., Huizinga, T.J.W. et al. (1997) Genetic influence on cytokine production and fatal meningococcal disease. Lancet, 349, 170173.[ISI][Medline]
Witkin, S.S., Jeremias, J., Bongiovanni, A.M. and Munoz, M.G. (1996) Immune regulation in the male genital tract. Infect. Dis. Obstet. Gynecol., 4, 131135.
WHO (1992) World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge University Press, Cambridge.
Yanushpolsky, E.H., Politch, J.A., Hill, J.A. and Anderson, D.J. (1996) Is leukocytospermia clinically relevant? Fertil. Steril., 66, 822825.[ISI][Medline]
Submitted on August 17, 2000; accepted on November 13, 2000.