Clinical significance of asymptomatic urogenital Mycoplasma hominis and Ureaplasma urealyticum in relation to seminal fluid parameters among infertile Jordanian males

Results M. hominis and U. urealyticum were present in 9(27.3%), 4(12.1%) of seminal fluids of infertile-varicose patients, 12(18.2%), 11(16.7%) of infertile and only in 3(14.3%), and 0(0%) fertile male, respectively. The presence of the two species among infertility cases was significantly correlated ( p = 0.039). A high percentage of M. hominis was recorded in varicose-infertile males. U. urealyticum was significantly associated with infertility cases (without varicocele) in comparison with control ( p = 0.046). It exerts a minor effect on the mean values of sperm motility by decreasing a and b grades’ motility. Conclusion The differences in the occurrence of M. hominis were statistically insignificant among infertility and control groups, but it was significant for U. urealyticum ( p = 0.046). M. hominis occurs more frequently in the semen of infertile-varicose male and normal seminal fluid quality. It seems to have no adverse effects on sperm motility but it might decline the fertility potential in such cases. U. urealyticum on the other hand have no clear significant impacts on sperm motility. The mean values for sperm motility, concentrations, and viscosity were not affected by the presence of the two species. Despite the significant presence of Ureaplasma among infertility, further studies were needed to clarify their potential effect on semen quality and infertility status. Keywords PCR Mycoplasma hominis Ureaplasma urealyticum Seminal fluid Varicocele 1 Introduction Male factor accounts for up to half of all cases of infertility and affects one man in 20 in the general population (1) . Varicocele was considered as one of male infertility causes present in 2–22% of the adult male population (2) . In men with abnormal semen analysis, the prevalence of varicocele was about 25% (3) . This condition has been linked to a series of biochemical changes in the epididymal fluid and sperm cells, and has a role in affecting sperm motility and morphology which lead to sperm dysfunction (4) . It is characterized by the stasis of the internal spermatic vein, leading to elevated scrotal temperature, testicular hypoxia and retrograde blood flow of adrenal and renal metabolites (5) . A reduction in the volume of the affected testicle has been detected mostly in such cases which could be restored after surgical treatment with an increase in sperm count and motility rate (6) . However, varicocele, especially the high grade one, is not a progressive condition and some patients retain normal semen quality, after certain time (7) . Immunologic factors and urogenital Infections appeared to have certain role in varicocele-related infertility (8) . Data about their influence on seminal fluid parameters are contradictory, since males with varicocele showed infertility with variable semen finding. Moreover, some varicose male appeared fertile, but their fertility potential might decline gradually (9) . Infections of the male reproductive tract have been recognized to cause chronic damage to the organs where they are lodged and have been considered as one of the causes of male infertility (10,11) . Different species of bacteria known to have an impact on spermatogenesis and participating in male infertility were identified. Beside their influence on spermatozoa, infectious mediators that appear to be responsible for specific molecular process in infections particularly affected the motility of the sperm (12) . Mycoplasmas ( Mycoplasma hominis and Ureaplasma urealyticum ) are the smallest free-living, unusual, bacteria that possess a very small genome, and characterized by their strict dependence on the host for their nutrients. They have the capability to attach to spermatozoa and influence their motility in a negative fashion (13) . Mycoplasmas are either commensal with a detectable percentage of 1/25 in healthy control (14) or benign pathogens associated with mild and chronic infections (15) . This leads to the suspicion that chronic asymptomatic genital tract colonization with Mycoplasmas might contribute to human infertility. Dieterle, in 2008, found that sperm motility and viability were impaired by symptomatic urogenital infections, while no clear evidence have been reached concerning the adverse effect of asymptomatic urogenital infection on male infertility (16) . However, generations of subclinical genital infection or non gonococcal urethritis have been detected in 25% of infertile men. The most widespread species in the genital tract of both sexes was U. urealyticum (17) . Its reported prevalence varied from 10% to 40% in the male seminal fluids (18,19) and showed to have a role in varicose-infertile male with higher rate in asthenozoospermia (20) . The presence of U. urealyticum in the seminal fluid has a direct effect on sperm motility, density and morphology (21,22) . Preincubation of spermatozoa with the supernatant of U. urealyticum culture decreases the human sperm-hamster egg penetration rate which suggests the presence of toxic factor that impairs sperm function. The extent of penetration inhibition varied considerably among ureaplasma serotypes (23) . The importance of Mycoplasmas is obscured by the presence of many asymptomatic persons from whom M. hominis and U. urealyticum can be isolated from urogenital specimens. However, there is no clear evidence that asymptomatic urogenital infections with Mycoplasmas have an adverse effect on seminal fluid quality. The amount of data which has been collected to support this concept failed to explain their influence on sperm quality. Studies in the field were hampered by the frequent isolation of ureaplasma from fertile groups (24) . De-Jong et al., failed to find a significant difference between ureaplasma isolation rates from the semen of infertile and fertile men (25) . It has been proposed that ureaplasma titer of 103 colony-forming units/ml of semen is significant, whereas lower titers are due to contamination by normal urethral colonization (26) . Varicocele and seminal fluid colonization with Mycoplasmas exert an effect on male fertility and semen quality. Mycoplasma infections are known to cause some reproductive problems which mean that chronic asymptomatic genital colonization might have an association with male infertility. In this study, we investigated the prevalence rate of M. hominis and U. urealyticum among infertile Jordanian patients (with or without varicocele) to determine if there is any role for Mycoplasmas on the semen quality in such cases. 2 Materials and methods 2.1 Study groups Ninety nine infertile patients (33 with varicocele and 66 without) were enrolled in the study with mean duration of infertility of 3.0167 ± 0.2687 year. The patients were attending infertility department at Medical Hussein City Hospital in Jordan, during February 2006 to May 2007 with complete medical, clinical histories. Any patient with a history of genital tract infections was excluded from the study. Varicocele was diagnosed after physical examination, duplex, and Color Doppler Ultrasonography. All cases of varicocele were classified as grade I (when spermatic veins were palpable only with Valsalva). Regarding seminal fluid analysis, some varicose-infertile patients showed normal semen parameters (8 cases) and others (25) with abnormal semen parameters (abnormal sperm count and/or motility). Infertile males without varicocele showed abnormal seminal fluid parameters. All patients never achieved pregnancy more than one year of unprotected intercourses. Control group consist of 21(17.5%) fertile married male without varicocele and normal seminal fluids. They are clinically asymptomatic males who were attending for routine check-up. Informed consent was obtained from them. All subjects enrolled in the study were non-smokers. The mean age of the study group was 30.4 ± 0.47138 years. Study protocol has been approved by the scientific committee at Al-Balqa Applied University. The patients who had received antibiotics during the previous month were excluded from the study. The study protocol has been approved by the ethics committee. 2.2 Seminal fluid samples Semen samples were obtained by masturbation after 3–5 days of sexual abstinence and kept in sterile nontoxic recipients. Written and verbal advices were given to the patients to follow the procedure. Each patient provided at least two samples with in one month. Semen samples were put in the incubator directly for liquefaction and analyzed by the same person for sperm concentration, viscosity and motility as indicated by the WHO manual for semen analysis (27) . Viscosity of the liquefied sample was estimated by introducing a glass rod into the sample and observing the thread that forms on withdrawal of the rod. The threads obtained from normal samples should not exceed 2 cm in length (28) . Sperm motility was calculated by multiplying sperm concentration (×10 6 /ml) and semen volume (ml). Motility is graded from a to d and sperm of grade c and d was considered poor. The presence of 50% of sperms or more with categories a and b or 25% or more with category a within 60 min of ejaculation was considered as normal results. The results were averaged for the two samples and a single value was used for each parameter. 2.3 Molecular investigation of Mycoplasmas All PCR reagents and enzymes were purchased from Promega (Promega, Co., Madison, WI, USA). Seminal fluid samples were centrifuge and the pellets were resuspended in 0.25 ml of Tris–hydrochloric acid 10 mmol/l pH 8.3, containing potassium chloride 50 mmol/l, magnesium chloride 2.5 mmol/l, 1% Brij detergent, and 200 μg/ml proteinase K (29) . To lyse the cells and inactivate proteinase K, the samples were microcentrifuged, overlaid with mineral oil, and incubated in a Minicycler (MJ Research, USA) for 60 min at 56 °C, and then at 95 °C for 10 min (30) . Aliquots of 25 μl were microcentrifuged, heated at 94 °C for 10 min, and immediately plunged into ice water to prevent reannealing of DNA. An equal volume of reaction mixture was then added to yield a final concentration of 1.25 units of heat stable Taq DNA polymerase, magnesium chloride 2 mmol l −1 , potassium chloride 50 mmol l −1 , and 200 μmol l −1 dNTP, 1 μmol l −1 of each of the two oligonucleotide DNA primer pairs specific for M. hominis (324 base pair regions of the 16S ribosomal RNA gene) and 1 μmol l −1 of each of the two oligonucleotide DNA primer pairs specific for U. urealyticum (224 base pair regions of the urease gene) (Alpha DNA, Montreal, Quebec HA4 IW3), and Tris–hydrochloric acid 10 mmol l −1 , pH 8.3. The mixtures were subjected to 40 cycles composed of sequential incubations at 95 °C for 1 min for DNA denaturation, 55 °C for 1 min for annealing primers to the template, and 72 °C for 2 min for chain extension. PCR products were resolved electrophoretically through agarose gel containing ethedium bromide (Promega, CO., Madison, WI, USA). The bands were visualized with UV transilluminator, photographed with the gel documentation system (Doc Print DP-001-FDC, Vilber Loumate, France). Mycoplasma positive control (Clonit S.r.I., Milano-Italy) was always processed in parallel to the test samples and H 2 O blank was used as a negative control (31) . 2.4 Statistical analysis SPSS software version 13.0 was used for data analysis. The results were presented as mean values with deviations (±SDs). Significance of the differences was performed using t-test for equality of means, ANOVA correlation, Descriptive, Frequency and chi-squared test. A p-value of <0.05 was considered. 3 Results Electrophoretic analysis of PCR products for M. hominis and U. urealyticum revealed their presence in 21(28.6%) and 15(12.5%) of the seminal fluids of infertile patients and in 3(8.33%) and 0(0%) of fertile ones, respectively ( Fig. 1 ). The presence of the two species of Mycoplasmas was significantly correlated ( p = 0.03) and Six of semen samples showed the occurrence of both species. An increase in the prevalence of M. hominis among cases with varicocele was observed in comparison with other infertility cases and fertile male; 9(27.3%), 12(18.2%), 3(14.3%), respectively, but this does not appear to be significant. Despite the finding of only one patient (12.5%) harboring M. hominis showed normal semen parameters compared to 8(32%) with abnormal semen parameters, there was no significant relation between M. hominis and poor semen quality in varicose-related infertility ( Table 1 ). On the other hand, a significant increase in the percentage of U. urealyticum was observed among infertility patients {11(16.7%) without varicocele and 4(12.1%) varicose one} in comparison with fertile male 0(0%) ( p = 0.05). The relation appeared more significant when comparing a group of infertile patients without varicocele and control ( p = 0.04). Sperm motility and PCR positive for M. hominis and U. urealyticum were compared for their relation. No significant differences were observed among samples positive for M. hominis and sperm motility. The samples with positive results for U. urealyticum showed a minor decrease in grades a and b, and a minor increase in grades c and d in comparison with those that showed negative results. The differences were not found to be statistically significant ( Table 2 ). Regarding sperm concentration, no association was found between M. hominis , U. urealyticum and sperm count ( Table 3 ). Sperm count among infertile patients (with and without varicocele) with positive PCR results for Mycoplasmas was observed to be lower than those with PCR negative, but this was not significant. The data concerning sperm morphology were incomplete, so this parameter was excluded from the study results. Viscosity of seminal fluid was also recorded, in which 8(6.7%) semen samples with PCR positive for M. hominis showed hyperviscosity in comparison with 16(13.3%) without viscosity, and only 5(4.2%) semen samples diagnosed as U. urealyticum positive revealed hyperviscosity as compared to 10(8.3%) that showed normal viscosity. These results were not significantly different. 4 Discussion Acute or chronic infections have been shown to compromise spermatogenesis resulting in a detectable reduction in the fertilizing potential of spermatozoa (12) . M. hominis and U. urealyticum are species that are closely related to urogenital diseases such as pyelonephritis, nongonococcal urethritis, epididymitis and infertility (32) . Colonization with Mycoplasmas can occur during birth and the carriers are asymptomatic, but under certain circumstances the organisms could be opportunistic pathogens (33) . Because asymptomatic infections may remain undetected, screening programs in men should be used to reduce the carrier rate of urogenital infections. In this study, Mycoplasmas have been found to be widespread among infertile patients. Their prevalences correspond to the expected prevalences present in other studies (13,20,34) . Although the overall prevalence of M. hominis was higher than that for U. urealyticum among our study cases, the latter was significantly associated with infertility (with or without varicocele). In some studies U. urealyticum has been shown to be the most detectable species in male genital tract with a reported prevalence of 10–40% in the seminal fluids (35) . Despite intensive investigations, the pathogenesis of varicocele-related infertility remains controversial. In a study done by Gattuccio et al., a real correlation was found between varicocele and genital inflammations mainly Chlamydia and Mycoplasmas (36) . Inflammation does not appear to be the only cause of infertility, but it frequently reduces the probability of male fertility (37) . Several mechanisms have been reported to be associated with seminal fluid alterations in infertile male with varicocele including microbial one (5) . Li et al., find a significant quantitative difference in the incidence of genital U. urealyticum between varicose men and other infertile without (14) . Some studies have correlated the incidence of M. hominis and U. urealyticum with a decrease in semen count, morphology, motility, and volume (3) , but others were unable to find such relations (17,38) . We questioned if Mycoplasmas might contribute to abnormality in seminal fluids associated with varicose patients. A detectable increase in M. hominis among infertile-varicose patients appeared, but this does not seem to have a direct effect on sperm parameters. Sperm count, motility and PCR positive for M. hominis among infertile-varicose patients showed no significant differences when compared to those without varicocele and healthy control. It seems logical that the presence of M. hominis was not considered as one of the factors that affect varicocele status since no negative effect has been detected on semen quality. On the other hand, U. urealyticum appeared to have a minor impact on infertile male and its effect concentrated on sperm motility rather than count or viscosity. In a number of studies, ureaplasma appears to be significantly associated with poor semen quality and male infertility (22,39) . It produces a toxic factor that impairs sperm function after adsorption to the surface of mammalian cells and replication. Because of its urealytic activity and subsequent release of ammonium ions, U. urealyticum induces cytotoxicity in a variety of established cell lines (40) . The toxic effects appeared to be dose dependent, and a titer of 103 colony-forming units/ml of ureaplasma in the semen is significant in affecting sperm motility (26) . U. urealyticum can attach massively to sperm, especially at the midpiece, thus producing marked hydrodynamic drag on the infested sperm which causes loss of its motility (41) . Some researchers report that infection with Ureaplasma leads to a decrease in the contents of Zn and Se in the seminal fluids which causes a significant decline in the sperm quality (42) . Beside that, the presence of Ureaplasma brings some changes in prostate function and disrupts its secretion, thus causing a decline in certain microelements secretion in the semen which reduces its quality (43) . Some investigator noticed that certain immunologic mediators that appeared to be responsible for specific molecular process in infections seem to particularly affect the motility of the sperm (12) . Seminal antisperm antibody activity significantly increased in cases with positive Mycoplasma culture. By this increase, Mycoplasma may indirectly reduce sperm motility and egg penetration ability participating in a state of infertility (38) . Furthermore, it was mentioned that the abnormal sperm motility and function among infertile-varicose male appeared to be associated with an increase in nitric oxide concentration in the seminal fluids which exert some toxic effect on sperms and ignore the participation of microbial factors (44) . Diaz-Garcia et al., showed that M. hominis locates intracellularly in human spermatozoa with fourfold higher interaction to sperm head or tail than to midpiece. This provides specific evidence of Mycoplasma attachment and invasiveness of sperm cells (45) . Although sperm damage showed to be non–apparent, it might have an implication on male infertility. It seems logical that a short-term Mycoplasma interaction with spermatozoa results in non-apparent or subtle damage, but their effects might have implications on long-term male infertility (46) . These results allowed us to assume that although ureaplasma appeared to be significantly associated with infertility, its presence is not significantly correlated to sperm abnormality in cases without varicocele or with varicocele-associated infertility. Excessive bacterial concentrations and contact time might be needed to produce a desirable effect on sperm motility. Seminal hyperviscosity is generally thought to reveal genitourinary infection. No association was found between seminal hyperviscosity and positive samples for Mycoplasmas. This was supported by the results of others (47) . However, more studies should be done to consider the etiology of hyperviscosity. 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