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Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia
ABSTRACT
Chlamydia trachomatis infections are prevalent worldwide, but current research, screening, and treatment are focused on females, with the burden of disease and infertility sequelae considered to be a predominantly female problem. The prevalence of chlamydial infection, however, is similar in males and females. Furthermore, a role for this pathogen in the development of male urethritis, epididymitis, and orchitis is widely accepted. The role of Chlamydia in the development of prostatitis is controversial, but we suggest that Chlamydia is an etiological agent, with incidences of up to 39.5% reported in patients with prostatitis. Infection of the testis and prostate is implicated in a deterioration of sperm, possibly affecting fertility. Chlamydia infections also may affect male fertility by directly damaging the sperm, because sperm parameters, proportion of DNA fragmentation, and acrosome reaction capacity are impaired with chlamydial infection. Furthermore, the proportion of male partners of infertile couples with evidence of a Chlamydia infection is greater than that documented in the general population. An effect of male chlamydial infection on the fertility of the female partner also has been reported. Thus, the need for a vaccine to protect both males and females is proposed. The difficulty arises because the male reproductive tract is an immune-privileged site that can be disrupted, potentially affecting spermatogenesis, if inappropriate inflammatory responses are provoked. Examination of responses to infection in humans and in experimental animal models suggest that an immunoglobulin A-inducing vaccine will be able to target the male reproductive tract effectively while avoiding harmful inflammatory responses that may impair fertility.
Chlamydia, male fertility, male reproductive tract, male sexual function, prostate, prostatis, sperm
ARE CHLAMYDIA INFECTIONS A MALE PROBLEM?
Chlamydia trachomatis (CT) is the most prevalent bacterial cause of sexually transmitted infections and can result in severe genital and ocular disease. The World Health Organization estimates that 92 million CT infections occurred worldwide in 1999 [1]. Currently, World Health Organization guidelines recommend that screening strategies be aimed at women. In addition, research efforts are focused mainly on females, and the burden of disease sequelae is considered to be primarily a female problem. Thus, the importance of this pathogen in male genital tract infection often is underrated. In the present review, we aim to comprehensively examine the role of Chlamydia infections in men, both in terms of disease sequelae and potential to cause infertility, and look at current advances in screening and vaccine development that may aid in the eradication of this pathogen from both men and women.
The reported incidence rates of genital chlamydial infections in the population likely are an underestimate because of the highly asymptomatic nature of the pathogen. Furthermore, as more cross-sectional population surveys are being conducted, it is becoming increasingly apparent that the prevalence rates of genital CT infection are very similar for heterosexual men and women (for review, see [2, 3]). The results of recent large surveys reiterate this trend and are presented in Table 1 [4–7]; Chlamydia positivity was determined by nucleic acid amplification techniques (NAAT) in those studies.
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The reason for the higher prevalence of positivity in the study by LaMontagne et al. [5] is uncertain. The prevalence in females is greater in health care settings of any kind than in population-based studies [3], however, and this also may be the case with males. Before the reports presented in Table 1, a systematic review of prevalence studies in the United Kingdom was performed. Only 11% of the reported prevalence estimates were from males, with estimates ranging from 0% to 4.8% (in 25- to 29-yr age group) [3]. Clearly, there is paucity in the literature of information regarding male prevalence, but variation does exist among studies. The similar rates of infection between males and females is consistent, however, and highlights the importance of this pathogen in genital tract infection of both men and women.
UNDERDIAGNOSIS AND TREATMENT OF CT INFECTIONS IN MEN
Approximately 75% of CT infections in women and up to 50% of those in men are asymptomatic (for review, see [8, 9]). In comparison to the high prevalence rates reported in Table 1 from screened populations, the most recent data from the Centers for Disease Control and Prevention documented CT infection rates of only 0.161% and 0.497% in the entire male and female populations of the United States, respectively [10]. Thus, a large proportion of CT infections will go undetected unless targeted screening programs are put in place.
Asymptomatic men who are infected with CT are younger than their symptomatic counterparts [11]. This also emphasizes the need for better screening or prevention practices, because although up to 13.3% of young men may have a genital chlamydial infection [5], only half of these will present with any symptoms—and even fewer are likely to pursue treatment.
The deficiency in screening/treatment of men is further highlighted by a U.K. study that found the testing rate for CT infection in men reached a maximum of only 5% of the testing rates in women. In 2004, only 5.1% of the diagnosed infections in men were treated, compared with 25% in women [12]. Thus, a very high proportion of infections in men remain undetected or untreated, enabling further spread of the bacterium within the population. Screening of young men reduces cases of inflammatory disease [13], and cost-effectiveness could be improved by prescreening for Chlamydia-associated seminal markers, such as antibodies, and then using NAAT [14].
The Centers for Disease Control and Prevention guidelines for screening proposed in 2007 suggested that programs should have a primary emphasis on screening women [10]. We believe, however, that because male infection is as prevalent as female infection and likely plays a role in infertility, screening should be focussed on both males and females. NAAT on first-void urine with internal PCR controls should be used, because it is more amenable than urethral swab collection and has comparable sensitivity [15].
Following detection, standard treatment for genital infection with Chlamydia consists of a single oral dose of 1 g of azithromycin or 100 mg of doxycycline orally twice daily for 7 days (for review, see [16]). A recent study, however, has shown that early identification and treatment of infected patients within a population can actually interfere with long-term immunity and result in eventual higher incidence rates related to an increase in reinfections [17]. Reinfection is a significant problem, with a median probability of reinfection in men of 11.3%, which is comparable to reinfection rates in women [18]. Hence, with the current deficiencies in detection, uncertain long-term success of treatment when provided, and high reinfection rates, a vaccine appears to be the only viable option for eradication of Chlamydia-associated genital disease. This will be discussed in greater detail later.
DURATION OF INFECTION AND BACTERIAL LOAD IN MALES
Serious difficulties are involved in determining the duration of infection in humans, because the onset of infection generally is unknown, reexposure is common, and clearance is rarely followed up [19]. To a large degree, we have to rely on information provided from animal studies for our information. In male rodent models of Chlamydia muridarum infection, the bacterium has been found to persist in upper reproductive tract tissues beyond 7 wk [20, 21]. Primates, however, tend to experience more prolonged infections than mice or guinea pigs and, thus, are more likely to reflect the progression of disease in humans. Persistence of CT infection has been observed for up to 9 wk [22], and resultant chronic lymphocytic response was seen for up to 3 mo in primate studies [23]. It is interesting to note also that animals often are antibody [24] and culture negative but can still have chlamydial DNA in their tissues [19].
A limited number of studies have demonstrated that chlamydial positivity can, indeed, persist for a number of weeks in humans (for review, see [19]), as it does in primates. To our knowledge, however, no study has consistently followed up infection in men for greater than 4 wk; thus, the frequency of prolonged infections is unknown. For a comparison, most evidence in women suggests that infection persists for more than 60 days and even up to years in the upper female reproductive tract [19, 25], and interestingly, it has been suggested that a CT infection can maintain itself for up to 4 yr within a couple [26]. This may have implications for fertility, which will be discussed later in this review.
Few studies have examined the infectious chlamydial load in the reproductive tracts of males and females. The median inclusion-forming unit count is 72 for urethral swabs from men and 450 for cervical swabs from women [27], with other investigators finding similar results [28]. Organism load in first-void urine samples and in urethral swabs correlates with clinical signs or symptoms of disease in men [15, 28]. Urine is a suitable alternative diagnostic specimen to urethral swabs, because they both have similar infectious loads [15]. One obvious deficiency in the literature, however, is the organism load in ejaculated semen. This information would be invaluable in determining the transmission dose from men to women.
PATHOLOGY OF THE MALE REPRODUCTIVE TRACT RESULTING FROM CHLAMYDIAL INFECTIONS
The role of CT infection in the development of urethritis and epididymitis is now well accepted in the literature, but a role for this pathogen in the development of prostatitis remains controversial and will be discussed in detail. Another complication arising from chlamydial infections in men can include proctitis resulting from infection with the Lymphogranuloma venereum (LGV) strain. Large clusters of LGV infections in homosexual men have been found in Europe, the United States, and Australia since 2003, making it an increasing health problem (for review, see [29]).
The primary site of infection in males is the penile urethra. Undeniably, CT infection is a major cause of urethritis in men. Estimates using NAAT for diagnosis attribute up to 30% of urethritis cases to a CT infection, compared to only 4% in healthy control subjects [30], whereas up to 42% of nongonococcal urethritis cases may be caused by CT [31]. One study even suggested the presence of CT infection in as many as 56.6% of male patients with urethritis using enzyme immunoassay on urethral swabs [32]. CT infection appears to be equally prevalent in symptomatic and asymptomatic urethral disease [33, 34], again reiterating the highly asymptomatic nature of this pathogen.
The role of CT as an etiological agent for the development of epididymitis also is widely accepted (for review, see [35]). There also are animal models in which this phenomenon can be examined [21, 36, 37]. Direct immunofluorescence on urethral swabs of patients with epididymitis found that 30.8% had a CT infection, whereas 51.3% presented with serum immunoglobulin (Ig) A antibodies against CT [38], indicating a past or current infection. Chlamydial antigen also has been detected in tissues from patients with acute or chronic epididymitis by immunoperoxidase staining, indicating that the bacterium ascends the male reproductive tract [39]. The development of CT epididymitis is most predominant in younger men, with a number of studies demonstrating significantly higher rates in men under the age of 35 yr [38, 40].
Ascending chlamydial infection to the testes has been observed experimentally in rats [21], mice [20], and monkeys [24]. Furthermore, natural infection of bulls with Chlamydia psittaci commonly results in the development of orchitis [41]. In humans, chlamydial antigen has been detected in urethral or urine samples from 11% to 35% of men presenting with epididymo-orchitis [42–45]. A causative link between CT infection and epididymo-orchitis in men is now accepted [46].
Decreased sperm counts and decreased motility often are demonstrated in cases of acute epididymo-orchitis of nonspecific etiology [47], and this pathology also is consistently associated with high rates of infertility [46]. Ascending urethral infection to the sites of spermatogenesis provides a plausible means by which Chlamydia can interact with and impair sperm function and, thus, affect fertility.
Ascending C. muridarum infection to the prostate has been observed in experimental mouse models [20] (K.A. Cunningham, unpublished data), and a male koala has been documented that presented naturally with chlamydial organisms in the prostate [48]. Controversy exists in the literature, however, as to whether CT is a cause of prostatitis in humans, despite a large number of studies examining this relationship. This largely is a result of the difficulties in accurately and conclusively diagnosing prostatic infection with Chlamydia. These can include variance in the method of detection and difficulties in obtaining pure prostatic samples. Semen and prostatic secretion samples may be contaminated via passage through an infected urethra, making diagnosis of upper male reproductive tract infection difficult [49]. It has been suggested that care also be taken in the interpretation of prostatic biopsy findings when determining chlamydial presence, because some biopsy samples may contain prostatic-urethral material [50].
The prevalence of CT infection in patients with chronic prostatitis when detected by NAAT on secretions from the upper male reproductive tract has ranged from 8.3% to 18.8% in studies performed since 2000 (Table 2) [51–55]. A meta-analysis to determine the magnitude of this association conclusively could not be performed because of the great differences among diagnostic methods, population demographics, and lack of control groups. One case-controlled study by Weidner et al. [56] using culturing methods for detection found that 14.9% of patients with prostatitis had positive cultures from postprostatic massage urethral swabs; by comparison, only 5% of healthy controls were positive.
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Doubt has been raised concerning the reliability of these samples for accurate diagnosis, but a number of studies indicate that seminal fluid/expressed prostatic secretion often is positive for Chlamydia in patients with negative urethral swabs [54, 57, 58]. In fact, in one study, 39.5% of patients with chronic prostatitis and negative urethral swabs were positive for CT infection by McCoy cell culture on expressed prostatic secretion/postprostatic massage urine [59]. Antigen and DNA detection techniques have indicated the presence of CT in approximately 30% of biopsy specimens [60, 61], and pure prostatic biopsy samples also can demonstrate chlamydial positivity in the absence of urethral infection [52, 62]. These examples warn against negative diagnosis of upper reproductive tract infection using specimens such as urethral swabs, and they highlight the importance of relevant testing for determination of chlamydial etiology. They also suggest that contamination of prostatic samples, be they fluid or biopsy, via passage through the urethra may not be as much of a problem for diagnosis of prostatic infection as has been postulated.
We believe that findings from studies employing sensitive detection methods on suitable samples provide compelling evidence of an association between CT infection and the development of prostatitis. A recent comparative study examining sperm parameters of patients with chronic pelvic pain syndrome/prostatitis and healthy controls found that those with prostatitis had poorer sperm morphology and concentration and a lower inducibility of the acrosome reaction [63]. Thus, Chlamydia-induced prostatic disease could have clinical implications for male infertility. In the future, NAAT and direct antigen detection should be conducted on transperineal/transrectal biopsy specimens when possible to clinically prove a role for CT in prostatitis. In terms of screening, when symptoms are present or urine NAAT is positive for CT, expressed prostatic secretion could be used as a supplementary diagnostic sample to test for upper reproductive tract infection.
DOES CHLAMYDIA PLAY A ROLE IN INFERTILITY?
The potential mechanisms for the effect of CT on fertility may be a direct, negative effect of Chlamydia on spermatozoa or indirect effect, via infection leading to inflammatory obstruction of the tubules and/or epithelial damage that results in impaired spermatogenesis. These possibilities will be reviewed in detail in this section.
Does Chlamydia Interact with and Impair Sperm Function?
A number of electron microscopy studies have demonstrated an interaction of Chlamydia and sperm. This has been shown both in biopsy samples of the testis and epididymis and in semen samples [64, 65]. In fact, transmission-electron microscopy has shown that CT serovars D, H, and I all attach to human spermatozoa in vitro [66], and CT has been observed attached to sperm in the peritoneal fluid of women with salpingitis [67]. An interaction, however, does not provide indication of a negative effect of Chlamydia on sperm function.
Conclusive evidence for a direct effect of Chlamydia on spermatozoa is shown by the tyrosine phosphorylation of sperm proteins following interaction [68]. Furthermore, elementary bodies of CT serovar E and LGV can lead to apoptosis of human sperm in vitro involving caspases [69–71]. This can be induced by chlamydial lipopolysaccharide, which has a 550-fold greater spermicidal activity than that of Escherichia coli lipopolysaccharide [72, 73]. Eley et al. [74] proposed that chlamydial lipopolysaccharide interacts with CD14 on the sperm surface (and, possibly, Toll-like receptors if present), leading to increased production of reactive oxygen species, resulting in caspase-mediated apoptosis. Excessive generation of reactive oxygen species is related to an increase in sperm defects both in vitro [75] and in infertile men [76]. An association of the bacterium with sperm has been demonstrated, and this potential for Chlamydia to affect sperm function adversely has great implications for fertility.
The literature contains conflicting evidence concerning the relationship between chlamydial infection and sperm function. A large number of studies have suggested that positive markers for CT infection are not associated with altered sperm parameters [53, 77–83]. Others, however, have found that CT infection correlates with reduced sperm motility [84–87]; increased proportion of sperm abnormalities [88]; significant reductions in semen density, sperm morphology, and viability [89]; and increased likelihood of leukocytospermia [87, 90].
In a large study of 627 semen samples, 136 of which had evidence of CT infection, the presence of Chlamydia reduced normal sperm morphology by 14.4%, volume by 6.4%, concentration by 8.3%, motility by 7.8%, and velocity by 9.3% [91]. In addition to these frequently examined sperm parameters, a recent study has, to our knowledge, been the first to demonstrate that coinfection with Chlamydia and Mycoplasma results in 3.2-fold more sperm cells with fragmented DNA than in uninfected controls [87]. Chlamydia infection affected DNA fragmentation more strongly than it did motility or structural parameters [87]. Antibiotic therapy was able to reduce the number of patients with fragmented DNA and to improve the pregnancy outcomes of their partners [87]. Of further relevance to fertilization, men with high levels of antichlamydial antibodies in seminal plasma had a decreased acrosome reaction capacity of spermatozoa [92] and a higher level of sperm lipid peroxidation [93].
Almost all studies examining differences in sperm parameters between Chlamydia infected and noninfected males are performed on male partners of infertile couples (MPIC). One study, however, demonstrated that 45% of subfertile men can have reduced semen quality [83], whereas another found that 74% of MPIC had reduced viability and 70% had reduced sperm motility [85], making it potentially difficult to determine an effect of chlamydial infection in the MPIC population. The most reliable studies examining this phenomenon will be those comparing sperm of Chlamydia-infected versus noninfected men regardless of fertility status [46].
Chlamydia and Male Factor Infertility
A role for CT in male factor infertility is not yet proven [8, 94]. A direct interaction between sperm and Chlamydia has been shown [67], however, as has a subsequent increase in DNA damage [87], but whether this has a detrimental effect on male fertility is uncertain. A number of studies have been performed examining the rates of Chlamydia infection in men from infertile couples. Proving an association between infection and infertility, however, has been difficult because of the variance among the studies, with differences in patient demographics, method of Chlamydia diagnosis, and samples examined.
Conclusively proving a link also is difficult because of the scarcity of strong, case-controlled, prospective studies comparing chlamydial incidence rates in infertile men versus fertile men using suitable diagnostic means. Mosli et al. [95] examined chlamydial incidence by direct immunofluorescence and culture on urethral swabs in age-matched MPIC and fertile controls, and those authors found rates of 25% and 4%, respectively, strongly supporting a role for Chlamydia infection in male infertility.
To investigate the relationship between Chlamydia infection and male infertility, we have compared reports of rates of infection in MPIC to what is known about general prevalence rates in males of similar demographics from population-based studies. In virtually all studies concerning MPIC, the mean or median age is in the mid-thirties. Infection rates in males in the general population aged 30–44 yr were reported as being 1.1% [4], and rates in those aged 18–45 yr were reported as 1.9% [96] as determined by NAAT on urine samples. A number of studies examining the incidence from NAAT on semen of asymptomatic MPIC are reported in Table 3 [58, 77, 78, 80, 83, 86, 90, 97–99]. Positive NAAT results are considered to be indicative of a current active infection, and urine and semen NAAT findings have high concordance [77].
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No clear methodological explanations are available for the wide variance in semen sample findings, with reported rates ranging from 0.4% [78] to 42.3% [77]. On the other hand, documented rates of infection determined from urine samples of MPIC range from 0.3% to 7.1% [100, 101]. Irrespective of the varied findings, the majority of these studies report a higher rate of incidence of chlamydial infection than that in the general population.
Accurate determination of an active infection is important for defining incidence rates, but it is plausible that determination of a previous infection (antibody presence) in men may be more relevant for delineating a link to subsequent development of sequelae, such as infertility. We have tabulated a number of studies since 1995 that report levels of antichlamydial IgA in samples from the reproductive tract of MPIC (Table 4) [80, 85, 102–106].
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With the exception of the study performed by Ochsendorf et al. [80], the reported positivity rates are approximately 20%. In infertile couples, a male factor is implicated only in approximately 60% of cases [78]. If the incidence of Chlamydia infection had been examined in the approximately 60% subset of MPIC that were proven to be infertile, a link between infection and infertility might have proven to be even stronger. Also of interest, antisperm antibodies on motile sperm are found in 16.3% of asymptomatic MPIC, mostly associated with the presence of antichlamydial IgA in semen [106]; thus, previous chlamydial infection appears to be associated with the development of antisperm antibodies, potentially affecting male fertility.
Male Chlamydia Infection and Female Factor Infertility
The evidence presented strongly supports a link between CT infection and male factor infertility, but male chlamydial infection also is of great importance in the fertility of couples. Because of the observed interaction of spermatozoa and Chlamydia, it is postulated that CT infections in men serve as reservoirs for transmission to females [35, 79]. It also is possible that Chlamydia sp. may hitch a ride in leukocytes in semen, such as neutrophils and macrophages, which are cells that may be more prevalent during an infection [74, 107, 108]. Transmission between infected men and their sexual partners has been established [62, 85], and it is interesting to note that partners of men with symptomatic urethral infection are more likely to be infected than are partners of men with asymptomatic urethral infection [109], a probable corollary of transmission dose.
The presence of antichlamydial antibodies in males is related to the presence of infertility factors in female partners and subsequent reductions in pregnancy rates [78, 110, 111]. Furthermore, antibiotic treatment of MPIC with high levels of sperm DNA fragmentation and CT infection was able to increase assisted pregnancy rates significantly [87]. Given the high proportion of MPIC that have antibodies to Chlamydia (Table 4), these findings have huge implications for assisted reproduction. Furthermore, the cryopreservation process used in assisted reproduction techniques is not able to affect the presence of infectious Chlamydia in semen [112], indicating that prescreening of these subjects is of the utmost importance. These studies highlight the need to abrogate infection in both males and females to alleviate successfully the problems of Chlamydia-associated infertility.
DEVELOPMENT OF A CHLAMYDIA VACCINE IS ESSENTIAL
Efforts in Vaccine Development
Given the high rate of asymptomatic infections and the potential of early antibiotic intervention to interfere with the development of immunity within the population as a whole [17], the need for a vaccine against Chlamydia is undeniable. A computer modeling simulation examining the efficacy of vaccination strategies against CT found that even a 50% efficacious vaccine would result in a significant reduction in the prevalence of disease [113]. Given the structural differences between the male and female reproductive tracts, however, and the differences in innate and adaptive immune induction capacity, development of a vaccine that will prevent infection in both males and females will need to account for these differences.
Sperm production begins at puberty, after immune tolerance to self is well established. Because of the need to avoid development of antibodies against the immunogenic sperm, the male reproductive tract is an immune-privileged site, which results from the blood-testis barrier formed by the Sertoli cells and the blood-epididymal barrier formed by the epididymal epithelial cells (for review, see [114]). Inflammatory processes, such as the activation of complement, are undesirable in the male reproductive tract, because they may negatively affect sperm development via disruption of this barrier. For example, mice with a disruption in the CD59B complement regulatory protein have decreased sperm production and increased infertility [115]. Furthermore, an interferon-
response is considered to be necessary for clearance of a Chlamydia infection in females (for review, see [116]), but such a response is likely to affect the immune barrier of the male reproductive tract and, potentially, contribute to infertility [114]. Disruption of the barrier in experimental animals by an infection results in autoimmune orchitis and impaired spermatogenesis because of infiltration of Th1 CD4+ T cells [117] and interferon- [114]. Therefore, the induction of these responses by an experimental vaccine should be avoided.
We would like to direct the reader to a comprehensive review of immunity within the male reproductive tract for further information [118]. We require a good understanding of the immune induction capacity of the male reproductive tract to target suitable protective immunity to the correct site without inducing immunopathologic inflammatory responses.
Innate Immunity of the Male Reproductive Tract
Adaptive immunity in the male reproductive tract is tightly regulated to avoid inflammatory processes; thus, immune cells, such as tolerigenic antigen-presenting cells and regulatory T cells, within the tract are functionally modified under the regulation of androgens [118]. Furthermore, the immune-privileged nature of the male reproductive tract poses a barrier to the entry of serum Igs, particularly IgG [119, 120]. Therefore, we see an enhanced capacity for innate immunity at this site.
To illustrate this concept, incubation with semen results in a dose-dependent reduction in chlamydial infectivity on McCoy cells [121]. In a guinea pig model of Chlamydia caviae (formerly guinea pig inclusion conjunctivitis), female guinea pigs infected by mating had a shorter duration of infection compared to those inoculated experimentally with the determined transmission dose (102 inclusion-forming units), suggesting that semen may have innate stimulators or antimicrobial activity [122]. Zhao et al. [123] found that human beta-defensin 1 transcripts were present in prostate and testis epithelial cell lines. Human neutrophilic proteins 1–3 (alpha-defensins) also were present in the prostate cell line. Furthermore, zinc salts inhibit the growth of Chlamydia in McCoy, HeLa, and primary human prostate epithelial cell cultures. The high levels of zinc in human prostatic secretions may inhibit Chlamydia from infecting the prostate in vivo [124]. Another potential mediator is surfactant protein D, which inhibits Chlamydia infection of epithelial cells by binding to the bacteria [125].
Antimicrobials (e.g., defensins) in semen and urine may serve to protect the lower reproductive tract from infection [125, 126], but infection of rat prostate epithelial cells in vitro also increases the secretion of innate immune mediators and upregulates expression of Toll-like receptors [127, 128]. Whereas these mediators may aid in immune cell recruitment to the site of infection, they have the potential to induce inflammation. Despite the wide array of innate immune mediators that can be induced in response to Chlamydia, in many cases this immunity clearly is insufficient to block infection and transmission to partners, and the potential of innate immune responses to negatively affect spermatogenesis via inflammation is not well characterized.
Adaptive Immunity of the Male Reproductive Tract
We can glean some information from animal models about the time course of immune induction following chlamydial infection. The induction of an adaptive immune response to infection has been described most comprehensively by Pal et al. [20] following intrapenile inoculation of C3H/HeN(H2-K1) mice with C. muridarum. They demonstrated a very strong cell-mediated immune bias, with IgG2a levels 16-fold greater than IgG1, and high levels of interferon- and tumor necrosis factor-
. Unfortunately, the effect of such an inflammatory response on sperm function and fertility was not examined in their study. Serum antibodies peaked from 4 to 7 wk in mice and rats, but antibody induction local to the genital tract was not examined in these models [20, 21].
An infection in men results in significant inflammation, as indicated by high interleukin 8 levels [129], a finding supported by in vitro work demonstrating that infected prostate epithelial cells secrete interleukins 6 and 8 [130]. It is anticipated, however, that these proinflammatory responses have the potential to disrupt the immune-privileged nature of the site; thus, the induction of these responses may need to be avoided in a male vaccine so as not to affect fertility.
The induction of local IgA appears to be of great significance in male Chlamydia infection, because whereas the prevalence of antichlamydial antibodies of the IgG isotype in the male genital tract is only one eighth that of IgG levels in serum, the levels of seminal plasma IgA are two thirds those of serum IgA levels [82]. In contrast, IgM is barely detected in the genital tract secretions of men with bacterial prostatitis [131] or urethral swabs of CT-infected men [129], suggesting that this Ig isotype is not required for protection against chlamydial infection.
The groups of antigens that are recognized by serum antibodies in infected male mice are similar to those observed in a female salpingitis model [20, 132], including Major Outer Membrane Protein/Outer Membrane Protein A, the 60-kDa cysteine-rich protein, chlamydial heat shock protein 60, and chlamydial lipopolysaccharide. Determining antigens common to male and female infection may be useful in the design of an efficacious vaccine. Vaccine-induced immunity will need to be better than that elicited by a natural response to infection; thus, a multiantigen vaccine with strong, rapid IgA antibody-inducing mucosal-targeting adjuvants is likely to be the most successful approach in males.
Lessons from Male Animal Models of Vaccination
Vaccination studies to date have focused primarily on female animal models. A few studies, however, have examined the induction of protective immunity against Chlamydia infection in male animal models. Urethral infection with C. caviae in male guinea pigs resulted in subsequent protection from a second infection, while ultraviolet-irradiated elementary bodies administered parenterally also were able to reduce the infection [133, 134]. Similarly, Digiacomo et al. [135] found a reduced secondary infection when male baboons were inoculated first with CT serovar D. To highlight the importance of these studies, Patterson and Rank [133] performed urethral inoculation of both male and female guinea pigs with C. caviae and, on secondary challenge, found that males were more resistant to reinfection than females. Thus, they determined that the vaccination of males should be a priority.
Intranasal immunization with the major outer membrane protein of C. muridarum in combination with cholera toxin was able to elicit high levels of locally produced, neutralizing IgA in the prostatic fluid [136]. This induction of IgA (and the transepithelial transport involving the poly-Ig receptor) was able to reduce levels of C. muridarum in all regions of the male reproductive tract; however, sterilizing immunity was not observed with the high challenge dose that was used (K.A. Cunningham, unpublished data). Because IgA is a natural response to chlamydial infection [82] and, in high levels, may even serve to prevent the activation of complement [137], a strong antichlamydial IgA response could suit the criteria for a suitable male vaccine. Studies in females support this: Levels of major outer membrane protein/Chlamydia-specific IgA correlated with reduced shedding of Chlamydia in mice [138, 139] and in women [140].
Further studies will need to be undertaken in an attempt to optimally target the male and female reproductive tracts and to determine the antigen or combination of antigens that will best afford protective immunity against infection, without the potential for immunopathology. Thus, vaccine development efforts could best be directed toward a combination of microbicides and adaptive immune effectors, such as IgA, that will not disrupt the immune-privileged nature of the male reproductive tract.
Figure 1 summarizes the role of Chlamydia in the development of male reproductive tract pathology and the immune effectors that are present throughout the tract. Some key questions have been raised that should direct future research in this area:
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The present review highlights the necessity for a vaccine to protect against infection in both men and women, despite current screening and treatment efforts targeting females. We provide evidence of similar levels of prevalence in both sexes and a role for Chlamydia infection in degenerative male reproductive tract disease. A strong potential exists for an effect on male fertility, but more well-designed studies are required to prove conclusively a causal role of Chlamydia infection. A role for male chlamydial infection and female infertility via transmission, however, has been demonstrated. A vaccine therefore will be optimal only if it can protect against infection in both males and females. This will require either a separate design for each of the sexes or one that is able to induce protective immunity in the reproductive tracts of both sexes despite their obvious structural differences. The difficulties of targeting immunity to the male reproductive tract are the barrier to serum Igs and the greater potential for inflammatory processes to affect this barrier and, thus, allow the development of antisperm antibodies or immunopathology, each of which may potentially affect the fertility of males. We have found that the male reproductive tract can produce local neutralizing IgA following intranasal immunization, and this may provide a basis for further rational design of vaccination strategies in males.
FOOTNOTES
1Supported by the University of Newcastle Research Management Committee. 
Correspondence: 2Kenneth W. Beagley, Institute of Health and Biomedical Innovation, Queensland University of Technology, Cnr Musk Ave and Blamey Street, Kelvin Grove, QLD 4059, Australia. FAX: 61 7 3138 6030; e-mail: k2.beagley{at}qut.edu.au
Received: 17 January 2008.
First decision: 19 February 2008.
Accepted: 2 April 2008.
REFERENCES
