Detection of interleukin-8 (IL-8) in seminal plasma and elevated IL-8 in seminal plasma of infertile patients with leukospermia

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1 6 Detection of interleukin-8 (IL-8) in seminal plasma and elevated IL-8 in seminal plasma of infertile patients with leukospermia Koichiro Shimoya, 2)Takeshi Taniguchi, Takayoshi Okada, Kaoru Yamanaka, Reiko Neki, 1)Noboru Matsuzaki, Kazutomo Ohashi, Tatsuhiro Tomiyama, Fumitaka Saji, Osamu Tanizawa. Department of Obstetrics and Gynecology, Osaka Unuversity Medical School, 1-1-5, Fukushima, Fukushimaku, Osaka 553, Japan Osaka Medical Center and Institute for Maternal and Child Health Department of Obstetrics and Gynecology, Osaka Rosai Hospital INTRODUCTION Large numbers of leukocytes have been detected in some semen samples associated with male genital tract infection and infertility (1, 2). It is shown that large numbers of granulocytes impair the fertilizing ability of sperm in the zona free hamster-egg penetration test, suggesting a close linkage between infection and infertility (3). Various spermiogram parameters, such as elevated ph-values of semen, changes in color and viscosity of semen, increased leukocyte counts, positive bacterial culture, and reduced sperm motility, have been used for diagnosis of silent genital tract inflammation. Several reports document that, among these parameters, the quantification of PMN elastase in seminal plasma makes possible a rapid and accurate diagnosis of silent male genital tract inflammation (4, 5). Interleukin-8 (IL-8), an 8.4 kda cytokine, possesses specific chemotactic activity to accumulate and activate neutrophils at an inflammatory site (6). In this study, we quantified IL-8 in ejaculates from normal donars and infertile patients with or without leukospermia and examined the correlations of IL-8 with spermiogram parameters and PMN elastase level in seminal plasma. MATERIALS AND METHODS Semen collection Semen was obtained by masturbation after a period of 3 days abstinence. Samples were collected in a sterile container and examined in the laboratory within one hour following ejaculation. Semen samples were obtained from 44 infertile men and 14 proven -112-

2 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. Semen analysis Semen analysis including the determination of motility, morphologic features, the number of sperm cells and viscosity of the ejaculate, was performed as previopsly described (1). Diagnosis of leukospermia (>106 WBC/ml semen) was made by morphologic technique. Preparation of seminal plasma Liquefied semen was centrifuged at 1000xg for 10 minutes. The seminal plasma was aspirated, filtered, and frozen at -80 C until titration of IL-8 and PMN elastase. IL-8 assay enzyme immunoassay technique. The kit consistently detects IL-8 concentrations of more than 18.1 ng/l, but is incapable of detecting other cytokines such as IL-1, TNF-ƒ, or IL-6. Intra-assay and inter-assay variabilities of the method were % and %, respectively. PMN elastase assay PMN elastase in cell-free seminal plasma was determined by the method of Neumann and Jochum by enzyme-linked immunosorbent assay (7) (commercially available from E. Merck, Darmstadt). The kit consistently detects PMN elastase concentrations of more than 1.0 ng/l. Intra-assay and inter-assay variabilities of the method were % and %, respectively. Statistical analysis Statistical analysis was performed by X 2 test or by Student's t test, P<0.05 being considered statistically significant. Values were expressed as mean } SEM. The correlation between IL-8 and other markers was analyzed by simple linear regression. RESULTS The IL-8 titer of seminal plasma with (n=10) and without leukospermia (n=34) was analyzed (Figure 1). The IL-8 titer of seminal plasma with leukospermia } 0.82 ƒêg/l) was significantly higher than that of seminal plasma without leukospermia (2.35 } 0.34 ; P<0.0001). There was no significant differencd

3 between the IL-8 titer of infertile patients without leukospermia and that of fertile donors (1.64 } 0.29 n=14). The PMN elastase level of seminal plasma with leukospermia ( Đg/l) was significant higher than that of seminal plasma without leukospermia (47.7 } 5.1 Đg/l; P<0.0001). No statistical difference in the PMN elastase titer could be detected among the infertile men without leukospermia and fertile donors (32.4 } 2.1 Đg/l)(figire 2). Figure 3 shows a high degree of correlation between PMN elastase and IL-8 levels in seminal plasma P<0.001, y=0.03x+0.96). No correlation could be found between the IL-8 level of the seminal plasma and other spermiogram parameters such as ejaculate volume, sperm concentration, total sperm number, percent sperm motility, sperm velocity and ph of seminal plasma. DISCUSSION The present study shows that a certain amount of IL-8 is constitutively detectable in human seminal plasma. Since IL-8 exerts chemotactic and activating capacity on neutrophils to eliminate invading bacteria (6), the male genital organs produce IL-8 to stimulate neutrophils to accumulate in the genital tract and make them competent against bacterial invasion into the genital tract. Thompson reported a significantly larger number of leukocytes from cervical mucous samples of postinsemination compared with preinsemination (8). They found these leukocytes to be predominantly composed of neutrophils. Thus possibily, IL-8 in seminal plasma after being ejaculated into the vagina induces neutrophil accumulation in the cervical mucous and functions to bring about phagocytic clearance of non-fertilized population of sperm. Wolff reported a significant different distribution patterns in total cell numbers of leukocytes, granulocytes, samples from and fertile monocytes/macrophages and infertile men between semen (9). Since IL- 8 is a chemotactic factor for neutrophils, present study showing higher IL-8 level in seminal plasma in infertile men than fertile men might explain the reason for the increased cell number of granulocytes in infertile semen. Bacteria-derived endotoxin is a strong enhancer of IL-8 production (10). Several authors including Naessens (11) reported high bacterial isolation rate -114-

4 of semen. A significantly high IL-8 level in seminal plasma with leukospermia may be produced from various cells in the genital tract including immunocompetent cells, fibroblasts and endotherial cells activated by invading bacteria. Since neutrophils release PMN elastase from azuropholic granules during phagocytosis, degranulation, or cell death, the protease can be used as an objective indicator for granulocyte activity in semen. IL-8 is essential for activating neutrophils to release granules (12) and PMN elastase, and thus increased IL-8 level in seminal plasma may be associated with a high level of PMN elastase in seminal plasma of infertile men with leukospermia. Our results support this possibility by the high degree of correlation between IL-8 and PMN elastase levels in seminal plasma. REFERENCES 1. Fertil Steril 1980; 34: Fertil Steril 1982; 37: J Androl 1985; 6: Andrologia 1986;18(4): Fertil Steril 1988; 50: J Immunol 1987; 139: Methods of Enzymatic Analysis, 3rd edn. Weinheim: Verlag Chemie, 1984; Am J Reprod Immunol 1991; 26: Fertil Steril 1988; 49: J Immunol 1989; 142: Fertil Steril 1986; 45: J Immunol 1987; 139:

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