Many studies have examined the impact of genital tract infections and bacterial semen contamination in male fertility; however, the putative detrimental effect of bacteria on the sperm quality is still controversial [17
]. Microorganisms can affect the male reproductive function directly, causing the agglutination of motile sperm, reducing the ability of acrosome reaction and causing alterations in cell morphology—and indirectly, through the production of reactive oxygen species generated by the inflammatory response to the infection [18
]. However, there is not complete agreement on the detrimental role of the presence of bacteria in the semen. Sanocka–Maciejewska et al. [4
] have reported that the bacteria most frequently isolated from the genitourinary tracts of men have no effect on semen quality in normozoospermic males; however, in infertile patients with pathological semen parameters bacteria usually determined a diminished antioxidant capacity of sperm. These findings have in part been confirmed in the present study, i.e.
, the bacterial contamination of semen samples of fertile individuals did not compromise the sperm quality, while in infertile men, it is possible that bacteria further deteriorated the whole quality of the seminal plasma.
The presence of leukocytes in the semen often complicates the interpretation of results of sperm analyses and alterations of sperm parameters. In this study, in order to avoid the additional factors that could increase sperm damage, such as the cellular inflammatory response [19
], we enrolled only asymptomatic individuals who were referred to this centre for sperm analysis. Nevertheless, 15% of our patients had leukocytospermia. The prevalent opinion is that leukocytospermia is always concomitant with bacteriospermia; however the lack of leukocytospermia does not preclude the development of genitourinary symptoms of disease [20
]. The fact that in the present study the prevalence of leukocytospermia was equally distributed in all groups analysed and was independent of bacterial species isolated from the samples reinforces our interpretation that the presence of white blood cells in the semen only had a negligible pathogenic importance.
Our findings show that the simple presence of bacteria might alter the sperm quality. The mean sperm concentration in the group of individuals positive for bacteria was significantly lower than that observed in controls; however, it was always > 20
sperm/ml, value considered normal for WHO [13
The negative influence of bacteria on sperm motility is well known [4
]. In our study, motility was significantly reduced in all groups except for in those with S. agalactiae
and S. anginosus
. This suggests that the bacterial flagella and pili, i.e.
constant accessory structures of E. coli
and M. morganii
, could be an important determinant of pathogenicity. Recent studies have hypothesized that the mechanisms of sperm damage caused by bacteria passes through the expression of the adhesive properties of the flagella and pili to mannose receptors [21
]. The fact that receptors to mannose have been demonstrated also at the surface of human spermatozoa [22
] suggests that flagella and pili could play a considerable causative role in sperm damage. As far as gram positive organisms are concerned, the production of pili is not a characteristic present in all clones of the same species. Some studies [23
] have underlined the pathogenetic importance of such structures also in gram positive organisms, as they have shown that bacteria isolated from the site of infection are more likely to be piliated, while when they are simple bystanders they are not; this is the case of bacteria colonizing the urethral tract of human beings. These observations suggest that pili could possibly constitute a putative determinant of pathogenicity in gram positive cocci, too.
Sperm morphology is generally evaluated by light microscopy, although a deep analysis of subcellular sperm anomalies can only be performed by TEM. Applying the statistical mathematical formula to TEM data [14
], FI, an indicator of sperm quality, was determined together with the percentage of sperm pathologies such as immaturity, apoptosis and necrosis. FI score was significantly reduced in the studied groups compared to controls; however, as observed for sperm concentration, the values of standard deviations indicated a wide variability in each group. To better clarify this issue we divided the patients into fertile and infertile following the FI score. Despite fertile individuals distributed in each group (20.2% in E. faecalis
30% in E. coli
, 51.5% in S. agalactiae
, 13.8% in U. urealyticum
, 33.3% in S. epidermidis
, 30.4% in S. anginosus
, and 12.5% in M. morganii
group), the statistical calculation showed that the number of infertile patients in the groups with U. urealyticum
and E. faecalis
contamination was significantly higher than that with S. agalactiae
. Similarly, M. morganii
seems to damage the spermatozoa more than S. epidermidis
and S. anginosus
, even though the number of the cases was very small.
The characteristics of the contaminated semen samples from fertile patients were similar to those from men of proven fertility (controls), except for sperm necrosis in E. coli group. The observation that the presence of bacteria in semen samples did not compromise the sperm quality of fertile men could be explained by the fact that the bacterial colonization was possibly recent and the contact of bacteria with spermatozoa had not been long enough to result in damage. Alternatively, we can suppose that the semen of fertile individuals is capable of hampering the potential mechanisms by which bacteria may damage spermatozoa.
Regarding sperm pathologies, only necrosis and apoptosis were significantly increased in the groups with semen bacterial colonization compared to controls, whereas the frequency of immaturity, a pathology generally related to the presence of varicocele [25
], was equally dispersed in the various groups. As previously reported, apoptosis and necrosis seem to have an important role in spermatogenetic damage associated with semen bacterial colonization [26
]. In an in vitro
study performed by Villegas et al. [21
], a single incubation with E. fecalis
, E. coli
and S. aureus
induced apoptosis in human sperm with two possible, putative mechanisms: a direct cytotoxic activity of bacterial toxins and the contact with pili and flagella. It has also been demonstrated that E. coli
can start the apoptotic process by activating several caspases, proteases responsible for mitochondrial changes, alterations in membrane symmetry, and DNA fragmentation [21
In the present study, E. faecalis
, E. coli, U. urealyticum
and M. morganii
have been shown to exert a negative influence on the sperm quality of infertile males as a whole; similar findings have also been reported by other researchers [2
]. The possible mechanisms of sperm damage, in addition to adhesion by flagella and pili, include the production of toxins and metabolic products originating from bacterial proliferation. Some researches reported different pathogenetic mechanisms exerted by U. urealyticum
upon sperm, such as the production of H2
a source of hydroxide anion, and phospholipases A and C which may injure sperm membranes. In addition, the presence of U. urealyticum
in semen samples may decrease the amount of microelements such as zinc and selenium, which are important to maintain the antioxidative defensive properties of such biological fluids [11
In conclusion, our data suggest that the presence of bacteria in semen samples may influence the sperm quality, mainly by the induction of apoptosis and necrosis, which may in part be responsible for the observed reduction of sperm motility. However, another possible explanation may reside in the putative existence of an antigenic mimicry between some constituents of sperm flagella such as tubulin, one of the major component of axoneme, and bacterial proteins. Molecular mimicry of host structures with proteins encoded by microorganisms can have pathogenetic consequences; infection may induce antibodies and T cells to react against bacterial cell constituents, which can also recognize self components and immunomediated damage may follow. To verify such a possibility, we blasted the aminoacidic sequence of tubulin with proteins of E. coli (strain UTI89), M. morganii, U. urealyticum and E. faecalis and we found significant linear homologies with proteins expressed by all these species (Fig.1). Human spermatozoa may therefore share epitopes with antigens of the most frequent species colonizing the genitourinary tract of man. In case of infection or contamination, these antigens may induce an antibody response, which could cross-react with the flagella of spermatozoa, thus reducing their motility.
In conclusion, we have shown that contamination of sperm samples by some species is more closely associated with infertility. However, this is only part of the problem. In this study, we only dealt with aerobic and facultative anaerobic bacteria. The clinical significance of strict anaerobes in sperm samples is a subject of dispute. Anaerobic bacteria are not routinely sought in sperm samples, because they are fastidious to cultivate and may be damaged by the contact with oxygen for the duration of transportation. In fact, the studies concerning the presence of anaerobes in semen samples are neither numerous nor conclusive; however, already in 1995, Eggert–Kruse et al. [28
] reported that almost all the ejaculates they examined were positive for anaerobic microorganisms and potentially pathogenic species were found in 71% of men. It is noteworthy that, in a recent study, Kiessling et al. [29
] detected and identified bacteria, including anaerobes, in the semen of men undergoing fertility evaluation, by sequencing polymerase chain reaction amplified rRNA gene. By this method they found a significant contamination (≥ 2
bacteria/mL) with strict anaerobic microorganisms in a discrete proportion of cases. It is not clear, yet, whether the presence of anaerobes in the semen may influence fertility.
Future studies, also based on this molecular approach, should be addressed to this topic in order to provide new insights into the microorganisms coming into contact with sperm, helping to monitor the health of male genitourinary organs. In any case, a re-evaluation of sperm characteristics after patients have been treated, could confirm the role, if any, of bacterial presence in the determination of the sperm abnormalities observed.