It is estimated that 15% of male infertility is related to genital tract infection . Among infectious microorganisms, U. urealyticum is one of the most common species [11, 22]. Since 1967, the ureaplasmas have been shown as an etiology of male infertility , and especially when Friberg and Gnarpe  first demonstrated a higher frequency of ureaplasmas in the semen of men with unexplained infertility (76%) compared with fertile men (19%). Previously, U. urealyticum had been differentiated, into biovars 1 and 2. biovar 1 is composed of serovars 1, 3, 6, and 14, and biovar 2 is composed of serovars 2, 4, 5, and 7 to 13 [2, 25]. In 1999, U. urealyticum biovars 1 and 2 were classified into U. parvum and U. urealyticum, respectively . Most of the previous reported studies have discussed the role of ureaplasmas in male infertility without discriminating between U. parvum and U. urealyticum [9, 14, 15, 23]. In our study, we have used the PCR-microtiter plate hybridization assay that can facilitate the identification of U. urealyticum, U. parvum, M. hominis and M. genitalium in semen specimens. Our results demonstrated that genital mycoplasmas and ureaplasmas seem to be widespread among infertile male patients, as shown respectively by the frequency of 19.2% and 15.8%. These data are comparable with those reported in previous studies [1, 10, 27]. U. urealyticum was the most prevalent species detected (15%) in this study. The frequency of U. urealyticum in the semen samples of male infertile patients in the literature varies from 5 to 42% [9–11, 27, 28]. This wide range might be explained by the diversity of detection methods used for characterizing the studied populations.
In our study, U. parvum was detected in 4.2% of semen samples. The frequency of this species was lower than that reported by Knox et al.  (4.2% vs 19.2%).
M. hominis has been associated with bacterial vaginosis, pelvic inflammatory disease, postpartum fever, and postabortal fever, as well as a number of gynaecological infections [20, 29]. However, its role in non-gonoccocal urethritis (NGU) and in infertility is rarely investigated . The frequency of M. hominis, in our study, was comparable to that reported by Andra-Rocha et al.  but higher than that reported by Rosemond et al. .
M. genitalium was first isolated in urethral cultures from two men with NGU in 1981 . Although M. genitalium has been suggested as a cause of human NGU, the precise role of this mycoplasma in the etiology of NGU remains not established because of the immense difficulty in isolating it from clinical samples. More recently, however, PCR-based assays have facilitated the detection of M. genitalium in clinical samples  and a significant association has been demonstrated between M. genitalium and NGU [5, 32]. Hitherto, M. genitalium has seldom been investigated in semen of infertile men. In our study, the frequency of M. genitalium was higher than that reported by Kjaergaard et al.  (5% vs 0.9%). This difference might be explained by the use of different methods for the detection of this bacterium. We have used PCR that is more sensitive than culture and that can facilitate the detection of M. genitalium in clinical samples .
In the present study, the frequency of the U. urealyticum was higher than that of M. hominis. U. urealyticum was also detected more often than U. parvum and M. genitalium. These findings were consistent with other studies [10, 27, 33].
In the literature, mixed species (U.urealyticum + M. hominis) have been found in 7–14% of semen samples of infertile men [27, 29]. In our study, although M. genitalium was not separately isolated among patients, it was detected together with U. urealyticum + M. Hominis in one patient, with M. hominis in one patient, with U. parvum in two patients and with U. urealyticum in two patients.U. urealyticum and M. hominis were observed together in two patients. Thus, eight patients (6.7%) had mixed species and our results are similar to those of previously reported studies [27, 29]. These results show that the hybridization-based microtiter plate assay can be a useful method to detect mixed infection when multiple species of mycoplasmas or ureaplasmas were present in semen specimens.
Previous studies have reported that the presence of mycoplasmas and ureaplasmas in sperm specimens has no real effect on the semen quality, nor on the leukocyte count [10, 29]. Recent investigations seem to show that the presence of mycoplasmas reflects a silent infection rather than infection in infertile patients , even though when the attachment and invasiveness towards human sperm cell has been demonstrated in vitro [34, 35]. Reports are controversial about the effects of genital mycoplasmas and ureaplasmas infections or infection on sperm seminological variables [11, 27, 36, 37]. We have compared semen and first void urine specimens from the 120 infertile men for the detection of genital ureaplasmas and mycoplasmas infections using in-house PCR (unpublished data). We have found a very high concordance (> 95%) and a very good agreement (K > 0.8) between the detection of genital mycoplasmas and ureaplasmas DNA in semen and corresponding first void urine specimens. Several studies have shown that nucleic acid amplification tests performed on first void urine samples are able to detect as many or more infected patients than traditional swabs from the urethra or cervix or semen [38–42]. In some cases, we have found discrepancies between the detection of genital mycoplasmas and ureplasmas DNA in semen and corresponding first void urine specimens. The presence of genital mycoplasmas and ureaplasmas DNA in first void urine samples and its absence in semen specimens may indicate an asymptomatic urethral infection. The detections of genital mycoplasmas and ureaplasmas DNA only in semen may indicate that these organisms are harboured in the epididymis or seminal vesicles.
In the present study, the comparison of the sperm seminological variables of U. urealyticum-positive and U. urealyticum-negative infertile men demonstrated no significant differences in sperm seminological variables, which confirms previous findings [27, 33]. Conversely, a relationship between U. urealyticum and semen characteristics was observed in some literature [11, 36, 37]. The influence or the lack of influence of mycoplasmas and ureaplasmas on seminology may come from the capability of bacterial species to attach to spermatozoa and to affect directly via cellular interactions their vitality, motility, morphology, cellular integrity and their molecular structure or the development of protective immunity to genital infection by the host (population sensitivity to microbial agents) or other host factors.
In our study, a positive correlation was found between sperm morphology and the detection of M. hominis in semen samples of infertile men. Yet, a correlation was reversely observed between sperm concentration and the detection of this organism in semen samples. The sperm concentration (14.14 × 106/mL) was lower than the normal reference of WHO manual (≥ 20 × 106/ml) in semen of M. hominis-positive infertile men and higher (52.63 × 106/mL) in semen of M. hominis-negative infertile men. The present data show that M. hominis may affect sperm concentration and sperm morphology of infertile men. A negative correlation was also found between sperm concentration and the detection of M. genitalium in semen samples of infertile men. Although, the sperm count with the presence of M. genitalium was within a normal range, a decrease in sperm concentration was significant. However, we have failed to demonstrate a correlation between sperm concentration and sperm morphology and the detection of genital ureaplasmas in semen samples. The comparison of the sperm seminological variables between semen with mixed infections and semen without mixed infections or without infections showed that only the sperm concentration in the male partners of infertile couples with mixed infection in semen specimens were significantly lower than that of the male partners without infections. Our findings show that the mixed infections have no additional effect on seminology.
Semen with M. hominis presented a higher mean of leukocytes than semen with negative M. hominis; this difference not was statistically significant. The detected mean (1.153 × 106 leukocytes/mL) was higher than the reference value of the WHO manual (≥ 1 × 106 leukocytes/mL). In contrast, the means of leukocyte count of the positive DNA in semen samples for U. urealyticum, U. parvum and M. genitalium were smaller than the reference value of the WHO manual. In addition, no significant difference was detected among our studied patients. These findings indicate that the presence of mycoplasmas and ureaplasmas in semen is not necessary associated with leukocytospermia, and thus, in spite of potentially pathogenic species. Our results are consistent with previous reports [10, 36]. The unreliability of leukocytospermia levels to predict the presence of genital mycoplasmas and ureaplasmas, when evaluating subfertile men and the absence of leukocytospermia, does not exclude the presence of genital mycoplasmas and ureaplasmas [43, 44].