Detection of fractalkine in human seminal plasma and its role in infertile patients

Qing Zhang, Koichiro Shimoya,1, Yukinibu Ohta, Rika Chin, Kumiko Tenma, Shigeyuki Isaka, Hitomi Nakamura, Masayasu Koyama, Chihiro Azuma and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita city, Osaka 565-0871, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Fractalkine is a relatively newly discovered CX3C chemokine, which is a chemoattractant for T cells, monocytes and natural killer cells. Several reports have demonstrated the association between chemokine levels in seminal plasma and semen quality. The fractalkine levels in ejaculates from normal donors and infertile male patients with or without asthenozoospermia, were examined and correlated with sperm motility and morphology. METHODS AND RESULTS: Western blot analysis showed fractalkine protein to be present in the seminal plasma. Fractalkine titres in the seminal plasma of infertile men with asthenozoospermia (0.64 ± 0.04 µg/ml; n = 58) were lower than those in patients without asthenozoospermia (0.94 ± 0.10 µg/ml; n = 22, P < 0.01) and fertile donors (1.04 ± 0.07 µg/ml; n = 10, P < 0.001). There was no significant difference between fractalkine levels in patients with and without leukospermia. No significant correlation was found between fractalkine and interleukin-8 levels in seminal plasma. Sperm motility was positively correlated (R2 = 0.14, P < 0.001) with fractalkine concentration. The existence of CX3CR-positive leukocytes in semen was confirmed using specific primers for CX3CR. CONCLUSIONS: These results suggest that fractalkine is a chemokine associated with sperm motility and the migration of CX3CR-positive leukocytes into semen.

Key words: asthenozoospermia/fractalkine/seminal plasma


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemokines are small proteins that stimulate the migration of leukocytes and mediate inflammation. These proteins are classified into subgroups according to characteristic cysteine (Cys) signature motifs (Baggiolini, 1998Go). Cys-X-Cys (CXC) molecules target neutrophils and, to some degree, lymphocytes; Cys-Cys (CC) molecules target monocytes, lymphocytes, basophils and eosinophils with variable selectivity; and the Cys (C)-chemokine seems to act only on lymphocytes (Rollins, 1997Go). Recently, a chemokine bearing a new Cys-X-X-X-Cys (CX3C) cysteine motif has been cloned. In contrast to other chemokines, this chemokine—which is named fractalkine—displays potent chemoattractant activity for T cells, natural killer (NK) cells and monocytes (but not neutrophils), and is of non-haematopoietic origin. The extracellular domain of fractalkine has been shown to be released into the supernatants of transfected cells as a 95 kDa glycoprotein, possibly by proteolysis at the dibasic cleavage site proximal to the membrane, to generate soluble fractalkine (Bazan et al., 1997Go; Pan et al., 1997Go). Fractalkine is produced by endothelial cells and neurones and occurs as a cell surface-bound as well as a cleaved protein (Harrison et al., 1999Go; Papadopoulos et al., 1999Go; Muehlhoefer et al., 2000Go). Human fractalkine mRNA expression is most abundant in the brain and heart (Bazan, et al., 1997Go). The expression of fractalkine in microglia, endothelial cells and fibroblastic cells has been reported to be up-regulated by inflammatory signals (Schall, 1997Go). Fractalkine may be responsible for the accumulation of lymphocytes in inflammatory regions. Recently, the orphan receptors V28 and RBS11 have been characterized as the human and rat receptors for fractalkine respectively, and have been renamed CX3CR1. The fractalkine receptor is a seven transmembrane-spanning G protein-coupled receptor expressed in leukocytes (Imai et al., 1997Go; Jiang et al., 1998Go). The surface expression of CX3CR1 has been demonstrated in NK cells, monocytes and CD8+ T cells, and its signal transduction presumably plays a role in their migration and adhesion (Imai et al., 1997Go; Jiang et al., 1998Go).

Human semen contains sperm and leukocytes, as well as various types of proteins such as enzymes, hormones and those with unknown functions. Large numbers of leukocytes have been detected in some semen samples associated with male genital tract infection and infertility (Caldamone et al., 1980Go; Berger et al., 1982Go). Indeed, it has been reported that an elevated concentration of leukocytes impairs the fertilizing ability of sperm (Maruyama et al., 1985Go; Thomas, et al., 1997Go). There is also increasing evidence that chemokines are responsible for the elevation of lymphocytes in inflammatory regions in the reproductive tracts. Seminal plasma also contains various chemokines, such as interleukin (IL)-8, and monocyte chemotactic and activating factor (MCAF). The levels of cytokines produced in seminal plasma are markedly elevated in genital infections such as leukospermia (Shimoya et al., 1993Go, 1995Go; Comhaire et al., 1994Go; Zalata et al., 1995Go; Depuydt et al., 1996Go; Eggert-Kruse et al., 2001Go). These cytokines may accumulate and activate leukocytes in the male genital tract, where activated leukocytes produce large amounts of elastase (Jochum et al., 1986Go; Wolff and Anderson, 1988Go; Micic et al., 1989Go). It has also been shown that polymorphonuclear (PMN) elastase is an inhibitor of sperm motion (Satoh et al., 1990Go; Wolff et al., 1990Go), and that secretory leukocyte protease inhibitor (a potent inhibitor of leukocyte elastase) in seminal plasma reduces the extent of motility inhibition caused by elastase (Moriyama et al., 1998Go).

In the present study, fractalkine was quantified in ejaculates from normal donors and infertile patients, and the relationship between fractalkine and seminal parameters was examined. The existence of CX3CR-positive leukocytes in semen samples was also examined in order to determine their possible migration into semen.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reagents
Goat anti-fractalkine polyclonal antibodies and recombinant (r-) fractalkine were purchased from R&D systems (Minneapolis, MN, USA).

Semen collection
Semen was obtained by masturbation after 5 days of abstinence. Samples were collected in a sterile container and examined within 1 h after ejaculation (World Health Organization, 1992Go, 1999Go). Semen samples were obtained from 80 infertile and 10 proven fertile men. The fertile men had fathered at least one child and had no recent history of venereal infection. Informed consent to use seminal plasma was obtained from all patients in this study.

Separation of lymphocytes from semen
Semen was layered onto a four-step discontinuous Percoll®(Pharmacia, Uppsala, Sweden) gradient (the components of each density fraction are listed in Table IGo), and centrifuged at 800xg in a swing-out rotor for 25 min at room temperature. Enriched lymphocyte fractions were washed three times with phosphate-buffered saline (PBS).


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Table I. Composition of solutions used for Percoll gradient centrifugation
 
Semen analysis
Semen analysis, including the determination of motility, morphological features, number of sperm cells and viscosity of the ejaculate was performed following published guidelines (World Health Organization, 1992Go, 1999Go); peroxidase staining was performed as described previously (Shibata et al., 1985Go). Leukospermia was diagnosed as >106 white blood cells/ml semen, while asthenozoospermia was diagnosed as <25% grade (a) motility or <50% grade (a + b) motile sperm (World Health Organization, 1992Go, 1999Go). The sperm concentrations of the infertile group and donors were 7.8 ± 0.5 x107/ml and 8.8 ± 1.2x107/ml respectively. Sperm motilities [grade (a + b)] of the infertile group and donors were 34 ± 3 and 67 ± 4% respectively.

Preparation of seminal plasma
At 30 min after collection, liquefied semen samples were first centrifuged at 1000xg for 10 min, after which the supernatants were re-centrifuged at 10 000xg for 15 min to remove cellular elements and debris (Shimoya et al., 1993Go). After centrifugation, the clear seminal plasma was collected and stored at –80°C until determination of fractalkine and IL-8 concentrations (Shimoya et al., 1993Go).

Western blot analysis
In order to determine fractalkine protein levels in the seminal plasma, Western blotting analysis was performed using an anti-human fractalkine polyclonal antibody. Seminal plasma (5 µl) was electrophoresed on a 7.5 % SDS–polyacrylamide gel and transferred onto nitrocellulose membranes (0.45 µm; Schleicher and Schuell, Dassel, Germany). The membrane was incubated with 5% dried milk protein followed by anti-human fractalkine polyclonal antibody. The primary antibody was used at a final concentration of 1.0 µg/l. Fractalkine immunoreactivity was visualized using an ECL Western blotting analysis system (Amersham, Aylesbury, UK).

Determination of IL-8 in seminal plasma
Seminal plasma levels of IL-8 were monitored using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA). Dilution of the seminal plasma caused no adverse effect on IL-8 measurements using this kit. Seminal plasma levels of IL-8 detected were >31.2 pg/ml, and no cross-reactivity was identified with either cytokines (e.g. IL-1, IL-6), chemokines (e.g. MCAF) or growth factors (e.g. epidermal growth factor; EGF). The intra-assay variability of the IL-8 kit was 6.0–9.2%; inter-assay variability was 7.2–9.6%.

Fractalkine titre in seminal plasma
Fractalkine titres in seminal plasma were measured by densitometric analysis of the Western blots. The expression of fractalkine protein was quantified and analysed using a NIH image software program [developed and provided by the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH)]. Intra-assay and inter-assay variabilities of the fractalkine titres were 6.0–9.8 and 6.9–9.7% respectively.

RNA extraction
RNA was extracted from lymphocytes in the semen using a guanidine thiocyanate-phenol-chloroform extraction technique (Chomczynski and Sacchi, 1987Go).

RT-PCR amplification
RT-PCR was performed using an RT-PCR high kit (Toyobo Co., Tokyo, Japan). The reaction was carried out in the presence of M-MLVRTase and 1 ml RNA sample in a 5x RTase buffer, random primers and dNTP mix for 40 min at 42°C. PCR amplification was performed using an RT mixture (10 µl), with sequence-specific primers against human CX3CR (5'-TTGAGTACGATGATTTGGCTGA-3'/5'-GGCTTTGGCTTTCTTGTGG-3') (Gene Bank accession number U28934). PCR was carried out for 35 cycles using a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT, USA). Each cycle consisted of denaturation at 94°C (40 s), annealing at 52°C (40 s) and extension at 72°C (40 s). The amplification yielded a 653-bp DNA product according to the published sequence of the fractalkine gene (Muehlhoefer et al., 2000Go). RT was performed with total RNA without reverse transcriptase (a mock RT sample). PCR of a mock RT sample was carried out to detect any possible contamination in the RNA samples by genomic DNA. An aliquot (20 µl) of a 50 µl PCR mixture was electrophoresed on 1% agarose gel and stained using ethidium bromide; the amplified products were visualized using ultraviolet illumination. Molecular sizes were estimated using a 100-bp DNA ladder. All primers were obtained from Becks (Tokyo, Japan).

Statistical analysis
All values were presented as mean ± SEM. Statistical analysis of fractalkine and IL-8 levels in seminal plasma was conducted using Welch's t-test, and a P-value < 0.05 was considered significant. The correlation was analysed by simple linear regression.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Western blot analysis confirmed the existence of fractalkine protein in seminal plasma as a 95 kDa band (Figure 1Go). Fractalkine concentrations in seminal plasma of infertile men with (0.64 ± 0.04 µg/ml; n = 58) and without asthenozoospermia (0.94 ± 0.10 µg/ml; n = 22, P < 0.01) and fertile donors (1.04 ± 0.07 µg/ml; n = 10, P < 0.01) are shown in Figure 2Go. There was no significant difference between the fractalkine titre of infertile patients without asthenozoospermia and that of fertile donors. In contrast to fractalkine, there was no significant difference between IL-8 levels in seminal plasma among the three groups (Figure 3Go).



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Figure 1. Western blotting of fractalkine protein in seminal plasma. Seminal plasma of infertile patients was electrophoresed on a 15% SDS–polyacrylamide gel. The fractalkine signal was detected as described in the text. Lanes 1–10 = seminal plasma of infertile patients; lane pc = positive control (10 ng r-fractalkine).

 


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Figure 2. Fractalkine concentration in seminal plasma of infertile patients with or without asthenozoospermia, and in controls.

 


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Figure 3. Interleukin-8 concentration in seminal plasma of infertile patients with or without asthenozoospermia, and in controls. There was no significant difference among these three groups.

 
With regard to the effect of fractalkine on other sperm parameters, no correlation was found between sperm concentration, leukocyte count or sperm morphology such as head defects, neck and midpiece defects, tail defects and cytoplasmic droplet. The correlation between fractalkine levels in seminal plasma and sperm motility is shown in Figure 4Go (R2 = 0.14, P < 0.001); no correlation was found between fractalkine titre and IL-8 level in the seminal plasma (R2= 1.5x10–3, P = 0.71) (Figure 5Go).



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Figure 4. Correlation of fractalkine concentration in seminal plasma and sperm motility. The correlation was analysed by simple linear regression (R2 = 0.14, P < 0.001).

 


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Figure 5. Correlation of fractalkine concentrations and IL-8 levels in seminal plasma. The correlation was analysed by simple linear regression (R2 = 1.5x10–3, P = 0.71).

 
RT-PCR using specific primers for CX3CR confirmed the presence of CX3CR mRNA expression in semen leukocytes (Figure 6Go).



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Figure 6. CX3CR mRNA expression in lymphocytes of the semen samples from infertile patients. Agarose gel electrophoresis of PCR-amplified DNA of CX3CR. Lane M = DNA size marker: 100 bp ladder. Lane 1 = cDNA from leukocytes of semen, patient 1; lane 2 = cDNA from mock RT sample of leukocytes from semen, patient 1; lane 3 = cDNA from leukocytes from semen, patient 2; lane 4 = cDNA from mock RT sample of leukocytes from semen, patient 2; lane 5 = cDNA from leukocytes of semen, patient 3; lane 6 = cDNA from mock RT sample of lymphocytes from semen, patient 3.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fractalkine, which has a unique CX3C cysteine motif, is one of several chemokines that have recently been identified through bioinformatics. In the present study it was shown that limited amounts of fractalkine are contained in human semen, and that the fractalkine titre in the seminal plasma correlates positively with sperm motility. In recent years, several reports have been made regarding the correlation between cytokine levels and asthenozoospermia (Comhaire et al., 1994Go; Zalata et al., 1995Go; Depuydt et al., 1996Go), though none of these has demonstrated any significant correlation between cytokine levels and sperm motility. Although one group (Shimonovitz et al., 1994Go) demonstrated a high concentration of soluble IL-2 receptors in ejaculates with low sperm motility, the results of the present study suggested that fractalkine might represent a good marker for asthenozoospermia.

IL-8 in seminal plasma has been associated with genital tract infection and is a good marker for leukospermia (Shimoya et al., 1993Go). It has also been reported (Depuydt et al., 1996Go) that certain cytokines, including IL-8, IL-6, IL-1 receptor antagonist, and IL-1 {alpha} were associated with genital tract infection, though no correlation was found between IL-8 and sperm motility in the present study. Several inflammatory mediators are known to enhance fractalkine mRNA. The up-regulation of fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage (Yoneda et al., 2000Go). However, in contrast to other chemokines such as IL-8, elevated fractalkine levels could not be detected in the seminal plasma of infertile patients with leukospermia. These results suggest that male genital tract infections, such as leukospermia, do not affect the production of fractalkine and that fractalkine is produced in the male genital tract by a different pathway to that used to produce IL-8. Taken together, the combination of IL-8 and fractalkine in seminal plasma might provide more useful information about male infertility.

A large number of leukocytes in the semen is associated with male genital tract infection and infertility (Caldamone et al., 1980Go; Berger et al., 1982Go), and the value of flow cytometry in detecting leukocytes in human semen has been reported (Ricci et al., 2000Go). In the present study, the presence of CX3CR mRNA in leukocytes in human semen was demonstrated. Seminal plasma constitutively contains a certain amount of fractalkine. Hence, these results show that fractalkine contained in the seminal plasma might induce the migration of CX3CR-positive cells into the male genital tract, where these lymphocytes might contribute to the immune defence system. However, further studies are necessary to investigate the mechanism by which fractalkine improves sperm motility.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported in part by Grants-in-Aid for Scientific Research (Nos. 13671712, 13671713, 13877273 and 12671596) from the Ministry and Education, Science and Culture of Japan.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita city, Osaka 565-0871, Japan. E-mail: shimoya{at}gyne.med.osaka-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature, 392, 565–568.[ISI][Medline]

Bazan, J.F., Bacon, K.B., Hardiman, G., Wang, W.S.K., Rossi, D., Greaves, D.R., Zlotnik, A. and Schall, T.J. (1997) A new class of membrane-bound chemokine with a CX3C motif. Nature, 385, 640–644.[ISI][Medline]

Berger, R.E., Karp, L.E., Williamson, R.A., Koehler, J., Moore, D.E. and Holmes, K.K. (1982) The relationship of pyospermia and seminal fluid bacteriology to sperm function as reflected in the sperm penetration assay. Fertil. Steril., 37, 557–564.[ISI][Medline]

Caldamone, A.A., Emilson, L.B.V., Al-Juburi, A. and Cockett, A.T. (1980) Prostatitis: prostatic secretory dysfunction affecting fertility. Fertil. Steril., 34, 602–603.[ISI][Medline]

Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Ann. Biochem., 162, 156.

Comhaire, F., Bosmans, E., Ombelet, W., Punjabi, U. and Schoonjans, F. (1994) Cytokines in semen of normal men and of patients with andrological diseases. Am. J. Reprod. Immunol., 31, 99–103.[ISI][Medline]

Depuydt, C.E., Bosmans, E., Zalata, A., Schoonjans, F. and Comhaire, F.H. (1996) The relation between reactive oxygen species and cytokines in andrological patients with or without male accessory gland infection. J. Androl., 17, 699–707.[Abstract/Free Full Text]

Eggert-Kruse, W., Boit, R., Rohr, G., Aufenanger, J., Hund, M. and Strowitzki, T. (2001) Relationship of seminal plasma interleukin (IL)-8 and IL-6 with semen quality. Hum. Reprod., 16, 517–528.[Abstract/Free Full Text]

Harrison, J.K., Jiang, Y., Wees, E.A., Salafranca, M.N., Liang, H.X., Feng, L. and Belardinelli, L. (1999) Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart. J. Leukocyte Biol., 66, 937–944.[Abstract]

Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T.J. et al. (1997) Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell, 91, 521–530.[ISI][Medline]

Jiang, Y., Salafranca, M.N. and Adhikari, S. (1998) Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis. J. Neuroimmunol., 86, 1–12.[ISI][Medline]

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.[ISI][Medline]

Maruyama, D.K., Jr, Hale, P.W. and Rogers, B.J. (1985) Effects of white blood cells on the in vitro penetration of zona-free hamster eggs by human spermatozoa. J. Androl., 6, 127–135.[Abstract/Free Full Text]

Micic, S., Macura, M., Lalic, N. and Dotlic, R. (1989) Elastase as an indicator of silent genital tract infection in infertile men. Int. J. Androl., 12, 423–429.[ISI][Medline]

Moriyama, A., Shimoya, K., Kawamoto, A., Hashimoto, K., Ogata, I., Kunishige, I., Ohashi, K., Azuma, C., Saji, F. and Murata, Y. (1998) Secretory leukocyte protease inhibitor (SLPI) levels in seminal plasma: SLPI restores sperm motility reduced by elastase. Mol. Hum. Reprod., 4, 946–950.[Abstract]

Muehlhoefer, A., Saubermann, L.J., Gu, X., Luedtke-Heckenkamp, K., Xavier, R., Blumberg, R.S., Podolsky, D.K., MacDermott, R.P. and Reinecker, H.C. (2000) Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J. Immunol., 164, 3368–3376.[Abstract/Free Full Text]

Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, J.A., Vath, J., Gosselin, M., Ma, J., Dussault, B. et al. (1997) Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature, 387, 611–617.[ISI][Medline]

Papadopoulos, E.J., Sassetti, C., Saeki, H., Yamada, N., Kawamura, T., Fitzhugh, D.J., Saraf, M.A., Schall, T., Blauvelt, A., Rosen, S.D. et al. (1999) Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is upregulated upon dendritic cell maturation. Eur. J. Immunol., 29, 2551–2559.[ISI][Medline]

Ricci, G., Presani, G., Guaschino, S., Simeone, R. and Perticarari, S. (2000) Leukocyte detection in human semen using flow cytometry. Hum. Reprod., 15, 1329–1337.[Abstract/Free Full Text]

Rollins, B.J. (1997) Chemokines. Blood, 90, 909–928.[Free Full Text]

Satoh, S., Satoh, K., Orikasa, S., Maehara, I., Takahashi, M. and Hiramatsu, M. (1990) [Studies on pyospermia in male infertility]. Nippon Hinyokika Gakkai Zasshi, 81, 170–177.[Medline]

Schall, T. (1997) Fractalkine – a strange attractor in the chemokine landscape. Immunology Today, 18, 147.[ISI][Medline]

Shibata, A., Bennett, J.M., Castoldi, G.L., Catovsky, D., Flandrin, G., Jaffe, E.S., Katayama, I., Nanba, K., Schmalzl, F. and Yam, L.T. (1985) Recommended methods cytological procedures in haematology. International Committee for Standardization in Haematology (ICSH). Clin. Lab. Haematol., 7, 55–74.[ISI][Medline]

Shimonovitz, S., Barak, V., Zacut, D., Ever-Hadani, P., Ben Chetrit, A. and Ron, M. (1994) High concentrations of soluble interleukin-2 receptors in ejaculate with low sperm motility. Hum. Reprod., 9, 653–655.[Abstract]

Shimoya, K., Matsuzaki, N., Tsutsui, T., Taniguchi, T., Saji, F. and Tanizawa, O. (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, 885–888.[ISI][Medline]

Shimoya, K., Matsuzaki, N., Ida, N., Okada, T., Taniguchi, T., Sawai, K., Itoh, S., Ohashi, K., Saji, F. and Tanizawa, O. (1995) Detection of monocyte chemotactic and activating factor (MCAF) and interleukin (IL)-6 in human seminal plasma and effect of leukospermia on these cytokine levels. Am. J. Reprod. Immunol., 34, 311–316.[ISI][Medline]

Thomas, J., Fishel, S.B., Hall, J.A., Green, S., Newton, T.A. and Thornton, S.J. (1997) Increased polymorphonuclear granulocytes in seminal plasma in relation to sperm morphology. Hum. Reprod., 12, 2418–2421.[Abstract]

Wolff, H. and Anderson, D.J. (1988) Evaluation of granulocyte elastase as a seminal plasma marker for leukocytospermia. Fertil. Steril., 50, 129–132.[ISI][Medline]

Wolff, H., Politch, J.A., Martinez, A., Hartinez, A., Haimovici, F., Hill, J.A. and Anderson, D.J. (1990) Leukocytospermia is associated with poor semen quality. Fertil. Steril., 53, 528–536.[ISI][Medline]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, Cambridge.

World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, Cambridge.

Yoneda, O., Imai, T., Goda, S., Inoue, H., Yamauchi, A., Okazaki, T., Imai, H., Yoshie, O., Bloom, E.T., Domae, N. et al. (2000) Fractalkine-mediated endothelial cell injury by NK cells. J. Immunol., 164, 4055–4062.[Abstract/Free Full Text]

Zalata, A., Hafez, T., Hoecke, M.J.V. and Comhaire, F. (1995) Evaluation of ß-endorphin and interleukin-6 in seminal plasma of patients with certain andrological diseases. Hum. Reprod., 10, 3161–3165.[Abstract]

Submitted on September 10, 2001; resubmitted on November 26, 2001; accepted on February 22, 2002.