Another intensively studied pathogen is C. trachomatis. Chlamydia is the most commonly detected bacterial sexually transmitted infection (STI) worldwide, and the numbers of infections keep increasing [13, 23, 98]. Mostly asymptomatic [14], chlamydia infection is a major cause of infertility [98], mostly among females. Although no significant differences between infertile and fertile men in terms of the prevalence of C. trachomatis have been reported [52], large epidemiological data suggest an association of a past C. trachomatis infection with infertility in men [41]. Chlamydia has been directly associated with decreased sperm motility as well as abnormal sperm concentration and morphology [10, 24]. The pathogen has been reported to be able to attach to human spermatozoa, which results in their C. trachomatis-induced death in vitro [38]. Albeit the proven biological significance of chlamydia infection in male infertility has only been accepted for acute epididymitis and subsequent azoospermia [78], the pathogen has been associated with decreased sperm motility and abnormal sperm concentration and morphology in young prostatitis patients [59].

Nevertheless, the role of chlamydia infection in the pathogenesis of prostatitis remains inconclusive and controversial. C. trachomatis has been detected in semen samples of male partners in infertile couples [50], in the fluid of dilated seminal vesicles [30] and EPS [87], and in prostate tissue removed by transurethral resection or by open surgery [17], or obtained via perineal approach [45], but the discrimination from possible urethral contamination of the obtained material remains impossible to exclude.

Interestingly, chlamydia infection has been associated with increased leukocyte counts and pain severity in men with CPPS [73], and the role of immune system activation in the pathophysiology of chronic prostatitis has been proposed [60]. Some studies suggested anti-C. trachomatis immunoglobulin A (IgA) as a more sensitive marker for detection of C. trachomatis prostatitis than currently used amplification or culture techniques. Interestingly, a significant increase in mucosal IgA has been detected in the ejaculate of men with chronic prostatitis as compared to controls, and, also, significant correlations between IL-8 and mucosal IgA and between IL-8 levels and prostate symptom score results were found [59, 60].

Among genital mycoplasmas, Mycoplasma genitalium has also been considered as an STI [77]. However, their role in chronic prostatitis and related male subfertility is even more controversial that in the case of chlamydia infection. In fact, M. genitalium has been detected by amplification techniques in semen from some men with chronic abacterial inflammatory prostatitis [55] and, also, in a small number of prostatic biopsy specimens [46]. On the other hand, an earlier study failed to detect mycoplasmas in all biopsy specimens [20].

No significant differences have been reported between infertile and fertile men in terms of the prevalence of U. urealyticum and M. hominis [52]. Importantly, being untreated or successfully treated, the ascending urethral infection caused by genital mycoplasmas might trigger the immune response that would involve the prostate. In fact, an early infection might set in motion immunological processes that culminate in chronic prostatitis [90], which may potentially affect male fertility.

Further studies, employing novel diagnostic approaches, are required to investigate the clinical significance of the infective pathogens and potential mechanisms of action in male infertility. Culture tests and microscopy have been considered as the gold standard for diagnosing urinary tract infections (UTIs), with an additional amplification system used if sexually transmitted pathogens are suspected [32, 51]. However, the current evaluation of UTIs, under the umbrella of “significant bacteriuria” first introduced in 1960 [42], was based on analyses of a cultured single bacterial isolate assembly representing the dominant pathogen. A significant limitation in the current widely used method is the detection of only culturable bacteria. The method is also limited in the detection of multiple pathogens or the strain diversity. Thus, caution is required when considering studied human-ejaculated sperm or EPS as “sterile” based on the lack of bacteria detected by classical methods alone. The wide use of amplification methods in detection of urogenital pathogens, in particular STIs [23], is a result of the methods’ good specificity and sufficient sensitivity. Noteworthily, only good laboratory practice and monitoring reference reagents should be employed [92], to be able to differentiate between infection and contamination. These detection methods are rapid and some of them allow detection of several pathogens during the same experiment. For instance, a novel bead-based multiplex assay is using a multiplex PCR followed by Luminex bead-based hybridization and has shown its effectiveness in assessing 18 urogenital infections in the same experiment [82]. However, the abovementioned tests are able to detect already known pathogens. The novel metagenomic sequencing (MGS) approach allows comprehensive high-throughput analyses in a sample without prior cloning, and it has become instrumental in detecting a broad range of non-culturable bacteria in “sterile” (based on “classical” diagnostic practices) EPS. The recent first-in-principle study on MGS of EPS found several bacterial reads in men with chronic prostatic inflammation. Most of the obtained bacterial sequences belonged to the family Proteobacteria, which includes such main causative agents of bacterial prostatitis as E. coli, Klebsiella, Shigella, Proteus, Enterobacter, etc. [86]. These findings raise the question of whether MGS could be useful for microbiological diagnosis in some cases, when conventional diagnostics may have failed to identify an appropriate treatment [6]. MGS studies would be also beneficial in exploring the prostate microbiome and in discovery of the role of potential non-culturable bacterial or viral cofactors in particular. For instance, in a population of prostatitis-related symptoms attributable to chlamydia infection, coinfection with human papillomavirus (HPV) was found to have a significant role in decreasing male fertility, in particular with regard to sperm motility and morphology [11]. A highly statistically significant correlation between pregnancy loss rate after the assisted reproductive technologies and positive HPV DNA testing in semen samples has been reported [75], providing an additional support that HPV coinfection may be related to impaired sperm motility [58], as well as other potential bacterial and viral coinfections. For cost reasons it will take time before novel assays become clinically applicable, but while the MGS methods remain expensive, the sequencing-related costs have decreased rapidly. This might significantly change the current viewpoint on the pathogenesis of prostatic disorders where bacteria may not play a role, such as chronic abacterial prostatitis.

The reduced volume of EPS may result in decreased semen volume. This can be a result of both secretory and ejaculatory dysfunctions. The role of male sex gland infections in diminishing the volume of EPS was observed in 1999 [95], but the mechanism of dysfunction in prostatitis remains unclear. The prostate infection may decrease the production of enzymes or secretion of citric acid, alpha-glucosidase, fructose, and zinc [56] or increase semen viscosity [25, 48], which adversely affects sperm motility. A complete lack of sperm occurs in about 10–15 % of infertile men. In fact, the most common problems in such cases are related to the testicles, hormone imbalances, or blockages in the male reproductive organs. However, chronic prostatitis may result in premature ejaculation [104] and ejaculatory and erectile dysfunctions [48]. The lack of sexual desire can be an additional component of sexual dysfunctions. These situations may lead to a decreased number of sexual intercourses. In order to optimize the likelihood of conception, a frequency of sexual intercourse from 2 to 3 times weekly was recommended [89].

Inflammation in the prostate gland has been speculated to promote an autoimmune response, which might be leading to deleterious effects on semen quality and function [28, 65]. Moreover, anti-inflammatory treatment has been reported to have a positive impact on sperm parameters [93], although the exact details remain unclear. The originally suggested [39] role of prostatitis-induced antisperm antibodies against human sperm has not been supported in later studies [37, 56]. Instead, poor semen quality has been linked with the potential role of multiple cytokines, such as seminal interleukins 6, 8, 10, 12, and 18 and TNF-alpha [57, 65], in which detection might also be dependent on the presence of leukocytes or bacterial pathogens [57]. Although the leukocyte (or white blood cell (WBC)) counts may not correlate with the severity of symptoms [80], they are known to produce seminal reactive oxygen species (ROS) in response to infection or inflammation stimuli [74]. An abnormal ROS level is associated with human infertility in several ways, including some STIs, in particular C. trachomatis [10]. Increased levels of ROS are associated with leukocytospermia [85] and may lead to damage of cell membranes, intracellular proteins, organelles, [69] and sperm DNA [1, 69], with subsequent impairment of sperm motility [5] and decreased seminal total antioxidant capacity [74]. Interestingly, while increased seminal WBCs in the ejaculate of patients with CPPS were not found to affect semen parameters [53], elevated seminal leukocytes alone were associated with poor semen parameters [22]. An increased amount of ROS in semen is also linked to high DNA fragmentation index (DFI), a measure of the proportion of sperms with chromosome breaks and a predictor of male fertility independent of standard sperm parameters. Urogenital infections have been reported to imply increase in DFI [88]. However, although the role of oxidative stress has been associated with prostatitis and male infertility [47], the true pathways remain controversial, requiring further studies.