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Behavioral Ecology Vol. 14 No. 1: 40–47 Do bacterial infections cause reduced ejaculate quality? A meta-analysis of antibiotic treatment of male infertility Philip A. Skau and Ivar Folstad Department of Evolution and Ecology, IB, University of Tromsø, 9037 Tromsø, Norway Ejaculate quality may limit male reproductive success, and consequently, sperm quality is of importance. Spermatozoa are perceived as ‘‘non-self’’ by the immune system and are exposed to immunological attacks in the male reproductive tract. To reduce immunological reactions against their own sperm, males are dependent on the testis being an immunoprivileged site. Immunoprivilege is obtained by the blood-testis barrier and by local immunosuppression by androgens. Despite this testicular immunosuppression, an influx of leukocytes may occur in testes. The condition in which males have a heightened level of leukocytes in semen is called leukocytospermia, and it is associated with reduced fertility. As the ability of immunosuppression by androgens may depend on current intensities of infectious organisms in the extratesticular soma, only males with high parasite resistance may be able to bear the cost of immunosuppression and consequently produce high quality ejaculates. This issue is addressed by a meta-analysis on the effects of broad-spectrum antibiotic treatment of male leukocytospermia-associated infertility. The analysis showed that antibiotic treatment of leukocytospermic men, without diagnosed genital tract infections, resulted in a significant improvement of ejaculate quality, that is, an increase in ejaculate volume, sperm concentration, number of motile spermatozoa, and number of spermatozoa with normal morphology. Moreover, the amount of leukocytes in semen was also reduced. This suggests that broad-spectrum treatment targeted toward bacterial infections reduces the density of leukocytes in semen and, at the same time, improves the quality of ejaculates produced. Our results emphasize the importance of parasitic resistance and immunity as factors that cause variations in ejaculate quality. Key words: antibiotic treatment, ejaculate quality, immunity, leukocytospermia, male infertility, meta-analysis, parasite resistance, sperm. [Behav Ecol 14:40–47 (2003)] W hen assessing the relative importance of pre- and postcopulatory mechanisms for fitness, the former have previously been considered to be the most important determinant of male reproductive success (Trivers, 1985; Williams, 1975). Postcopulatory mechanisms, such as male fertilization potential and fertilizing capacity, have therefore acquired less attention (Ginsberg and Huck, 1989). Male fertilization potential is, however, sometimes limited by ejaculate quality, and sperm density in ejaculates is an important determinant of fertilization success (Bonde et al., 1998; Gibson and Jewell, 1982; Marconato and Shapiro, 1996; Warner et al., 1995). Males show reduced sperm density in successive ejaculations (e.g., Freund, 1963; Synnot et al., 1981), and in humans (Homo sapiens), sperm counts begin to decrease at ejaculation rates greater than 3.5 times per week (Freund, 1963). This suggests that the rate of sperm production or sperm maturation may be limited in some species (Marson et al., 1989; Synnot et al., 1981). In species in which males experience sperm competition, which is the competition between the ejaculates of different males for the fertilization of an ova (Parker, 1970), males allocate more resources to sperm production. That is, they have larger testes and release more sperm per ejaculate relative to males in species with low risk of sperm competition, (Hosken and Ward, 2001; Møller, 1988, 1989; Møller and Briskie, 1995). This suggests that the importance of high sperm numbers is greater under sperm competition (Birkhead and Møller, 1992; Møller, 1988; Møller and Briskie, 1995; Parker, 1998). Address correspondence to P.A. Skau. E-mail: [email protected] uit.no. Received 4 April 2001; revised 7 February 2002; accepted 24 April 2002. 2003 International Society for Behavioral Ecology In species in which females may risk not having their eggs fertilized owing to poor ejaculate quality among males, females choosing males with good ejaculates should be favored by selection. In addition, in such species, females should allow sperm competition as fertility insurance. Sperm competition exists in a variety of species, and it has generally been considered more common among birds and insects than among mammalian species (Birkhead and Møller, 1998). However, even if female mammals do not possess proper sperm-storage organs and if mammalian spermatozoa are relatively short-lived, recent studies have documented that sperm competition in mammals is more common than previously assumed (Gomendio and Roldan, 1993; Gomendio et al. 1998). The rate of sperm production in humans is significantly lower than that of other mammals (Amann and Howard, 1980), and human semen is unique among mammals because of its large number of morphologically abnormal cells. The number of spermatozoa in a normal ejaculate is also low in humans (Birkhead, 1999a). These findings suggest that sperm competition may not be particularly intense in humans. The human testis size is, on the other hand, close to that expected from body mass, indicating intermediate sperm competition (Birkhead, 1999b). Sperm competition may occur in situations in which females engage in extrapair copulations. The extent to which production of extrapair offspring occurs is currently subject to controversy, but the most commonly cited estimates are around 10% (Birkhead, 1999b), which is a relatively small figure compared with that of other mammals (Birkhead and Appleton, 1998). However, 10% can still be considered enough to increase the variation in male reproductive success and consequently contribute to an important component of sexual selection in humans. The frequency of extrapair copulation is, however, probably higher, as not all extrapair Skau and Folstad • Meta-analysis of antibiotic treatment of male infertility copulations lead to extrapair offspring. Even though the extent of extrapair copulation in humans is at present quite small, it may have been of larger importance in earlier stages of human evolution. Human females have been suggested to prefer testosteronerelated facial characteristics in mates when conception is most likely (Penton-Voak and Perrett, 2000). Testosterone-related facial traits are also preferred when women engage in extrapair copulations or short-term relationships (PentonVoak et al., 1999; Perrett et al., 1998). Many sexual selected characters and behaviors in male vertebrates are testosterone dependent, and testosterone-related traits may honestly advertise immunocompetence. That is, testosterone is immunosuppressive, and the conditional development of testosterone-dependent characters reduces individual immunocompetence toward infectious organisms. Testosterone-dependent sex-trait development may thus be a reliable signal of individual parasite resistance (Folstad and Karter, 1992). In 1997 both Hillgarth et al. and Folstad and Skarstein suggested that the adaptive significance of immunosuppression by testosterone was related to the antigenicity of sperm. Because spermatozoa are differentiated at puberty, after the immune system has acquired a tolerance to all ‘‘self,’’ they are perceived as ‘‘non-self.’’ Males are consequently exposed to new antigens when spermatogonia mature, and the immune activity involved in the male reproductive system may lead to autoimmunity (Turek and Lipshultz, 1994). This is a condition in which the immune system produces antibodies and sensitized lymphocytes against the body’s own sperm (Stites et al., 1987). Such autoimmunity does, however, not normally occur (Turek and Lipshultz, 1994), as the testis is an immunologically privileged site. This is partly caused by the blood-testes barrier (Weienbauer et al., 1997), which isolates the antigenic germ cells from the somatic immune system. Among seasonal breeders, the blood-testes barrier is redeveloped before each breeding season and reabsorbed after breeding (Setchell, 1997), which indicates the importance of this barrier for development of germ cells. In addition, immune activity within the testis is also thought to be inhibited by locally produced hormones such as testosterone (Lehmann, 1986; Lehmann and Emmons, 1989). The blood-testes barrier and its associated immunosuppressive mechanisms, however, do not seem to hinder all immunological activity within the testes (Turek and Lipshultz, 1994), and both sperm antibodies and white blood cells are able to enter the interior of the testes and the epididymis. The normal cell population of the testis also includes macrophages, and human testis macrophages represent about 25% of all interstitial cells in adults (Weienbauer et al., 1997). Macrophages participate in normal immunological surveillance by presenting the antigen for cell-mediated immunity and are capable of phagocytosis (Janeway and Travers, 1994). The macrophages in testes are also capable of engulfing numerous spermatozoa (Hughes et al., 1981), and such phagocytosis could represent a process for removal of ageing spermatozoa (Tomlinson et al., 1992). However, it has also been suggested that there could be need for immunological control over ‘‘outlaw’’ haploid spermatozoa, as conflicts may arise when diploid and haploid self-interest differ, for example, as in some forms of meiotic drive (Folstad and Skarstein, 1997). Even though it has been suggested that testicular immune activity may be adaptive, current evidence indicates that it can also be pathological (Bronson, 1999; Turek and Lipshultz, 1994). The presence of white blood cells in semen has been shown to play an important role in male infertility, and there 41 are numerous reports on the negative effects of white blood cells on sperm function (Branigan and Muller, 1994; Wolff, 1995; Wolff et al., 1990). The negative effects described are, for example, decreased sperm number and decreased sperm motility (Wolff et al., 1990). Individuals with high densities of white blood cells in semen are referred to as leukocytospermic (World Health Organization, 1993), and the frequency of leukocytospermia is documented to be from 10–20% among male infertility patients (Wolff, 1995). Infectious organisms may interfere with immunosuppression during reproduction and may consequently influence ejaculate production. Folstad and Skarstein (1997) suggested that the relationship between immunosuppression and development of secondary sex traits might be a derived characteristic of the testicular immunosuppression conducted by sex hormones. As a consequence of such association, females could be able to evaluate both the ejaculate qualities of a male and his resistance toward infectious organisms from his sex-trait development. Males with high intensities of parasites should display an increased level of systemic immune activity and consequently have a heightened level of testicular immunity. Thus, high parasite intensities could result in a reduction of ejaculate quality and fertility (Folstad and Skarstein, 1997). Infections in the reproductive tract of infertile men have been acknowledged for decades (Nikkanen et al., 1979). Until recently, the condition of leukocytospermia has been used as an indicator of genital tract infection, and it has been presumed that high densities of leukocytes and antisperm antibodies have their origin in infections in the genital tract (Behre et al., 1997). However, a relatively large number of men who attend fertility clinics exhibit leukocytospermia without symptoms of genital infections, indicating that there is not a necessary relationship between infections in the genital tract and the amount of leukocytes or antisperm antibodies in semen (Eggert-Kruse et al., 1998; Micic et al., 1990; Trum et al., 1998; Wolff, 1995). Leukocytospermic men are consequently of interest as they may show symptoms of heightened systemic immune activity that is not caused by genital tract infections (Anderson, 1995; Purvis and Christiansen, 1993). Infections outside the genital tract may be asymptomatic but could still contribute to an increase in somatic immune activity and increased influx of leukocytes to the genital tract. The most commonly used group of antibiotics in the treatment of leukocytospermia is broad-spectrum antibiotics, which are not targeted toward specific bacterial infections and are used for treatment for a number of nongonadal infections, for example, pneumonia and gastrointestinal infections (Dollery, 1999). Yet, even if it has not been diagnosed as an inflammation in the genital tract or isolated as a pathological organism in leukocytospermic patients, leukocytospermia has routinely been treated with antibiotics (Anderson, 1995; Purvis and Christiansen, 1993). Today there exists, however, no clear consensus on the effect of such treatment (Anderson, 1995) or on the importance of leukocytospermia in general (Kaleli et al., 2000), and several studies have been conducted with differing results. The hypothesis tested here is that a general reduction in the level of bacterial infections after broad-spectrum antibiotic treatment of leukocytospermic men will reduce the somatic immune activity and lead to a reduction in the level of leukocytes in testes and epididymis. This will subsequently lead to an improved ejaculate quality. This hypothesis is addressed with a meta-analysis of the results from studies conducted to examine the effects of antibiotic treatment of leukocytospermic infertile men without diagnosed infections in the reproductive organs. Behavioral Ecology Vol. 14 No. 1 42 METHODS Identification and classification of outcome variables To identify clinical trials, a computer search of the databases Medline, EmBase, and Cochrane Collaboration was performed. This was conducted to identify all published articles in any languages published in the period from 1966–2000. These sources of information should provide a near complete coverage of published data on the subject. The following keywords were used: leukocytospermia, male infertility, antibiotic treatment of male infertility, immunological infertility, male genital tract infection, antisperm antibodies, asthenozoospermia (i.e., loss or reduction of sperm motility), and oligozoospermia (i.e., low numbers of spermatozoa in ejaculates). To reduce the risk of missing studies, a search of the Science Citation Index was also performed by using the name of the first author of papers obtained from the databases. A search on the references (and references of references) cited in the primary sources was also performed. Only published papers were included in the analysis. The definition of normal semen values is given by the World Health Organization (1993). Semen volume is the total volume of an ejaculate and is classified as normal above 2.0 ml. Sperm concentration is the number of spermatozoa per milliliter semen and is classified as normal above 20 million sperm per milliliter. A semen sample has normal motility if more than 50% of the spermatozoa show forward progression. The evaluation of sperm motility is often subjective, and various methods are in use. The morphology of a sperm cell is defined by its shape and form, for example, whether the head is round or tapered, or whether a different tail or a neck or mid-piece defect is present. A semen sample with more than 15% cells with normal morphology is classified as normal. According to the World Health Organization, leukocytospermia is defined as the presence of more than 106 white blood cells per milliliter semen. Identified trials A total of 23 clinical studies of antibiotic treatment of male infertility were identified. To be included in the analysis, each study had to report original data, a quantitative measure of effect size, or p values. Moreover, we excluded studies in which leukocytospermia was assumed to be caused by local infections causing pathology (e.g., in cases of prostatitis). Studies on leukocytospermic patients showing bacteriospermia were, however, included in the analysis because bacteriospermia was most likely not caused by a local infection in testes or epididymis but rather by infections situated elsewhere or by contamination in the urethra (Bieniek and Riedel 1993; Busolo et al., 1984; Purvis and Christiansen 1995). Following these selection criteria, we were left with 12 studies. In 11 of these, the effect size was converted from p values. Often researchers only stated whether their results were significant or not, and consequently we could only extract p values of .05, even if the original results may have been significant at higher levels. This procedure will result in conservative effect-size estimates. The most commonly applied antibiotics were doxycycline, erythromycin, and trimethoprim in combination with sulfamethoxazole, which are all considered broad-spectrum antibiotics. The trials were classified according to their different outcome measurements: sperm counts, motility, morphology, ejaculate volume, and the release of leukocytospermia. Pregnancy rates are the best measure of fertility, but the lack of sufficient data in the identified studies did not allow fertility as an outcome variable to be included in the meta-analysis. Some authors have reported that poor studies tend to exaggerate the overall estimate of treatment effect and thus lead to incorrect inference (Khan et al., 1996), whereas others have failed to find that the inclusiveness of poor studies affect the overall estimate in meta-analysis (e.g., Greenwald and Russell, 1991). It might consequently be difficult to find exact rationales for inclusiveness of studies in meta-analysis (Greenwald and Russell, 1991). Because the total number of available studies is rather low in the present analysis, no attempt was made to exclude studies because of poor methodological quality. Studies included in the analysis are given in Table 1. Measures of effect size were transformed into Pearson’s product-moment correlation coefficients by using the formulas given by Rosenthal (1991: 19). The sampling distribution of r is usually skewed, and in order to meet the assumption of normality, we transformed r values into zr values. The relationship between r and zr is given by Sokal and Rohlf (1995: 575). The results from studies that include small samples are more subject to chance than those of larger studies and should therefore be given less weight. In this study we used a weighted average of the results, in which the larger trials are given more influence than that of the smaller ones. The overall effect size was consequently calculated from the mean effect size adjusted for sample size (after zr transformation) by using the equation provided by Rosenthal (1991: 73–74). If the result of the studies differ greatly, it may not be appropriate to combine the results into one overall measure of effect. It is, however, unclear how to assert whether or not this is appropriate. One approach is to statistically examine the degree of similarity between the outcomes of the studies, that is, to test for heterogeneity across studies. Such tests examine whether the results of a study reflect a single underlying effect rather than a distribution of effects. Statistically significant heterogeneity may be caused by methodological variation between trials; for example, study designs, method of randomization, and different methods of measuring sperm quality or quantity. If the test shows homogenous results, then the differences between studies are assumed to be a result of sampling variation alone. Heterogeneity was calculated by using the formula provided by Rosenthal (1991: 73–74). The ‘‘file-drawer problem’’ is one aspect of the more general problem of publication bias (Rosenthal, 1979). There are reports suggesting that the set of available studies does not reflect the total set of studies ever conducted, because some reports are not published owing to the lack of statistically significant results (Begg and Berlin, 1988; Rosenthal, 1991). Rosenthal (1991) suggested calculating the fail-safe number, which is an estimate of the number of studies with zero effect needed to nullify an overall effect. This procedure was conducted by using the equation provided by Rosenthal (1991: 104). Moreover, the lack of consensus regarding the effect of antibiotic treatment of leukocytospermic men may have reduced publication bias in this particular field of science. RESULTS Effect of treatment The meta-analysis revealed that there was a significant positive effect of antibiotic treatment for the following sperm parameters: sperm volume, sperm concentration, sperm motility, and sperm morphology (Table 2). Antibiotic treatment also significantly reduced the number of leukocytes in ejaculates of male infertility patients. Thus, in general, males treated with antibiotics were relieved from leukocytospermia and produced ejaculates of higher quality. Skau and Folstad • Meta-analysis of antibiotic treatment of male infertility 43 Table 1 Treatment period (in days of treatment) and the total number of patients enrolled (n) in each of the 12 studies included in the meta-analysis Treatment and study design Treatment of leukocytospermia with doxycycline and trimethoprim. The treatment group was compared with a control group of untreated patients. Treatment of bacteriospermia with trimethoprim/sulfamethoxazole. The treatment group was compared with a control group of patients given placebo drugs. Treatment of leukocytospermia with doxycycline. The treatment group was compared with a control group of patients given placebo drugs. Treatment of leukocytospermia with ciprofloxacin; evaluation of treatment group before and after medical treatment, and treatment with trimethoprim and sulfamethoxazole; evaluation of treatment group before and after medical treatment. Treatment of leukocytospermia with doxycycline. The treatment group was compared with a control group of untreated patients. Treatment of seminal infection with doxycycline. The treatment group was compared with a control group of untreated patients. Treatment of bacteriospermia with doxycycline. Evaluation of treatment group before and after medical treatment. Treatment of oligoasthenozoospermia (small amounts of spermatozoa) with doxycycline and erythromycin. Evaluation of treatment group before and after medical treatment. Treatment of asthenospermia (loss or reduction of motility) with erythromycin. The treatment group was compared with a control group of patients given placebo drugs. Treatment of accessory gland infection with doxycycline. The treatment group was compared with a control group of patients given placebo drugs. Treatment of leukocytospermia with doxycycline. The treatment group was compared with a control group of untreated patients. Treatment of leukocytospermia with doxycycline. The treatment group was compared with a control group of untreated patients. Treatment period n Reference 14 41 Yanushpolsky et al., 1995 30 20 Merino and Carranza-Lira, 1995 10 25 Erel et al., 1997 14 7 10 76 Keck et al., 1998 30 24 Omu et al., 1998 30 24 Cardoso et al., 1998 90 56 Micic, 1988 30 65 Baker et al., 1984 30 33 Comhaire et al., 1986 30 50 Branigan and Muller, 1994 28 53 Branigan et al., 1995 Carransa-Lira et al., 1998 The drugs doxycycline, erythromycin, trimethoprim/sulfamethoxazole, cephalexin, and ciprofloxacin are all considered broad-spectrum antibiotics. Studies with patients showing bacteriospermia are included, as bacteriospermia in these cases most likely was not caused by infections situated in reproductive tissue. Fail-safe number and heterogeneity The fail-safe number for the different parameters ranged from six to 68 (Table 2). No firm guidelines can be given as to what constitutes an unlikely number of unretrieved and unpublished studies, but a robust result has been suggested to be five times the number of studies included in an analysis plus 10 (Rosenthal, 1991: 106). Consequently, one of the present results, those on morphology, had a fail-safe number higher than this rule of thumb. Sperm morphology showed the highest heterogeneity of the different sperm parameters, but none of the included parameters showed significant heterogeneity among studies. This is interesting, as several of the included studies show low sample size. DISCUSSION Treatment of male infertility with broad-spectrum antibiotics significantly reduces the density of leukocytes in ejaculates. In addition, treatment also significantly increases ejaculate quality. That is, treatment increases ejaculate volume, sperm concentration, the number of motile spermatozoa, and the number of spermatozoa with normal morphology. Leukocytospermia has a heterogeneous etiology, including infections, inflammations, and autoimmunity (Barratt et al., 1990), making the immediate cause for this condition quite complex and unclear. Most cases of leukocytes in semen are presumed to originate from some form of infection (Behre et al., 1997). However, the majority of men with leukocytospermia have semen cultures that fail to show genital tract infections (Anderson, 1995; Barratt, 1988; Hillier et al., 1990). A heightened level of seminal leukocytes in individuals in whom no infection in the genital tract is identified may have its cause in infections situated outside the genital tract (Bieniek and Riedel 1993; Busolo et al., 1984; Purvis and Christiansen 1995). Thus, a wide variety of infections may cause leukocytospermia. We have only included studies of antibiotic treatment of male infertility, and this may have contributed to our low effect sizes, as only bacteriological infections should be affected by treatment. Other infectious organisms of potential importance for a male’s ability to down-regulate the immune response could be viral, protozoan, or metazoan infections. HIV-infected men are, for example, shown to have low seminal quality (Anderson et al., 1990), and ejaculate quality decreases with the progression of the infection. Moreover, treatment of HIV-infected men with antiviral drugs reduces seminal levels of leukocytes and also improves ejaculate quality (Politch et al., 1994). It can, however, not be excluded that these effects could be caused by the presence of HIV in testes tissue (Fan et al., 2000). Yet, negative effects on host sperm quality, sperm quantity, and fertility have also been observed from nongonadal infections (Baker, 1991; El-Gamal et al., 1991; Liljedal et al., 1999; MacLeod, 1951; Sheriff, 1987; Wang et al., 1987; see also Måsvær M, Liljedal S, Folstad I, in preparation). Treatment of leukocytospermia with antibiotics resulted in a significant reduction in the level of leukocytes in semen. Behavioral Ecology Vol. 14 No. 1 44 Table 2 Results from the meta-analysis of male infertility with broad-spectrum antibiotics Parameters No. of Studies n Weighted effect size Unweighted effect size Heterogeneity test Fail safe number Sperm volume Sperm concentration Motility Morphology Leukocytospermia 4 8 9 9 5 182 327 360 333 258 0.20* 0.16* 0.20** 0.22*** 0.23** 0.19 0.19 0.24 0.35 0.25 0.98 0.14 0.18 0.08 0.17 6 25 48 68 26 Weighted mean effect size and unweighted effect size of the outcome variables sperm volume, sperm concentration, sperm motility, and sperm morphology. The number of studies, sample size (n), and effect sizes (mean zr values) are given. The heterogeneity test provides information about the difference between correlation coefficients (p values are reported), and the fail-safe number is the number of studies with zero effect needed to nullify the mean effect size. * p , .01, ** p , .001, *** p , .0001. Leukocyte density was also the parameter that showed the largest response to treatment. Current research suggests that some leukocytes may be adapted to remove defect and immature sperm cells in the testicular environment (Tomlinson et al., 1992), and it is documented that leukocytes are present in testes as early as the seventh week of gestation (Weienbauer et al., 1997). This suggests that the presence of leukocytes in testes is not only of a pathological nature. However, densities of leukocytes in semen are positively associated with levels of antisperm antibodies (Wolff, 1995), and antisperm antibodies are able to bind to the surface of sperm cells and reduce the acrosome reaction (Bandoh et al., 1992). This will, in turn, reduce the number of sperm cells in the ejaculate capable of fertilizing an ovum. A reduced level of leukocytes in semen should consequently be associated with improved ejaculate quality and improved fertility. Antibiotic treatment resulted in an increased ejaculate volume. The volume of ejaculates show large differences both between and within individuals, and is strongly related to ejaculation frequency (Freund, 1963). Ejaculate volume may, however, also be influenced by infections outside the urogenital tract that reduce the elasticity in prostate and seminal vesicles (Behre et al., 1997). In humans, ejaculate volume is not a commonly used measure of ejaculate quality, and volumes of ejaculates are not positively related to pregnancy rates, except at exceptionally low volumes (Bonde et al., 1998; World Health Organization, 1993). Because ejaculate volume, for a large part, is determined by the production of seminal fluid in the prostate and the seminal vesicles, it should not be affected by immune activity in testes or epididymis. It could consequently not be expected that a reduced immunological activity in testes resulting from antibiotic treatment would have a strong effect on ejaculate volume, and volume was also one of the outcome parameters that showed low response to treatment. Antibiotic treatment lead to an increased sperm concentration. This was the parameter showing the lowest response to treatment. Sperm numbers appear to be related to the number of sperm surviving during the passage through the female tract (Gomendio et al., 1998), and sperm concentration is also strongly associated with the probability of pregnancy (Bonde et al., 1998). It is, however, not obvious how antibiotic treatment should affect sperm concentration, because sperm cells of the treated individuals are produced before medication. One explanation may be that the number of macrophages in testes has been reduced. In general, macrophages in testes have been considered to be of negative influence on sperm cell production (Rossi and Aitken, 1997; Wang and Fanning, 1997), and as macrophages are shown to remove sperm cells through phagocytosis (Kurpisz and Fernandez, 1995), a reduction in macrophage density in the testes may lead to an increased number of sperm cells. Another, not mutually exclusive, explanation of the increased sperm concentration after treatment may be that the number of immunological cells with a cytotoxic effect in testes, such as cytotoxic T-lymphocytes, may have been reduced. Cytotoxic cells have been shown to inflict oxidative stress on spermatozoa (Aitken et al., 1994), which leads to a reduction in sperm motility and altered morphology of sperm cells (Rossi and Aitken, 1997). If treatment results in a lowered oxidative stress because of the reduced level of cytotoxic cells in testes, fewer sperm cells are likely to be abnormal and therefore are less likely to be engulfed by macrophages. Although this may result in differences in the numbers of sperm cells ejaculated between individuals, variation in sperm numbers in the ejaculate appears not to provide a full explanation of differential fertilizing capacity. Even when an approximately equivalent number of sperm from two or more males have been inseminated in females, systematic differences in reproductive outcome between males have been found (Martin and Dzuik, 1977). This suggests that differences in fertilizing capacity between ejaculates may also be related to the quality of the ejaculated sperm cells. Antibiotic treatment resulted in a significant improvement of sperm motility. Motility is considered to be an important factor in male fertility (Drobnis and Overstreet, 1992; World Health Organization, 1993; for an alternative view, see Ben Chitrit et al., 1995; Bonde et al., 1998). In mice, it was shown that subordinate individuals produce sperm cells with lower motility compared with that of dominant individuals (Koyama and Kamimura, 1999), and this lower motility may be caused by low levels of androgens in subordinate mice (Bronson and Desjardins, 1971; Gandelman, 1980). Androgens, which may be immunosuppressive, could influence the level of white blood cells and antisperm antibodies in semen. Antisperm antibodies present in semen may bind to the surface of spermatozoa (Bronson, 1999), and such binding is shown to considerably reduce sperm motility (Barratt et al., 1990; Purvis and Christiansen, 1995; Turek and Lipshultz, 1994). A reduction in the intratesticular immune activity may therefore reduce the amount of antisperm antibodies and consequently lead to improved motility. The level of morphologically normal sperm cells increased as a result of antibiotic treatment. Large numbers of cytotoxic cells present in the testes have been shown to negatively affect sperm morphology (Aitken et al., 1994), and the formation of Skau and Folstad • Meta-analysis of antibiotic treatment of male infertility normal spermatozoa is dependent on low levels of testicular immune activity. Therefore, a reduction in immune activity in the testes should lead to a higher number of spermatozoa with normal morphology. Moreover, sperm morphology seems to be an important determinant for spermatozoa motility, as spermatozoa with odd morphology usually show dysfunctional movement. Thus, morphology and motility are closely associated variables (Katz et al., 1982). It has also been suggested that the proportion of sperm with normal morphology is strongly related to the likelihood of pregnancy, independent of both sperm concentration and ejaculate volume (Bonde et al., 1998; Liu and Baker, 1994). It has clearly been illustrated in the case of the stomach ulcer–producing bacteria Helicobacter pylori that infectious organisms of relatively low pathogenicity may play an important role in human illness (Bourke et al., 1996). In accordance with this view, antibiotic treatment of infertility patients, without clear symptoms of genital tract infections, leads to a significant decrease of leukocyte numbers in semen and an increase in ejaculate quality. This suggests that a reduction in the level of parasites may lead to a reduction in the somatic immune activity, a lower influx of immunocompetent cells in the testes, and a higher ejaculate quality. Thus, increased pathogenicity from infectious organisms associated with immunosuppression may be an unrecognized cost of ejaculate production. We are currently conducting a follow-up study on the published effects of corticosteroid treatment on sperm quality in humans. We thank Monica Martinussen for guidance in meta-analysis, Yngve Figenschau for providing information about andrology, and Mathilde Berg, Henning Klausen Ståle Liljedal, Marthe Måsvær, Ingebrigt Uglem, and two anonymous referees for commenting on earlier versions of the manuscript. REFERENCES Aitken RJ, West K, Buckingham D, 1994. Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress, and sperm function. J Androl 15:343–352. Amann RP, Howard SS, 1980. 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