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

Clinical significance of asymptomatic urogenital Mycoplasma hominis and Ureaplasma urealyticum in relation to seminal fluid parameters among infertile Jordanian males Academic research paper on "Health sciences"

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Abstract of research paper on Health sciences, author of scientific article — Hala I. Al-Daghistani, Muna Abdel-Dayem

Abstract Objectives To investigate Mycoplasma prevalence rate among infertile Jordanian patients and determine the possible role of asymptomatic Mycoplasma hominis and Ureaplasma urealyticum as an infectious factor that might affect semen quality in infertility, mainly varicose-related one. Setting Medical Hussein City Hospital in Jordan. Materials and methods Seminal fluids obtained from 99 infertile patients were tested for the presence of M. hominis and U. urealyticum by polymerase chain reaction (PCR) and analyzed for motility, counts and viscosity. Cases involved were 33(27.5%) infertile with varicocele, 8(24.2%) with normal seminal fluid parameters, 25(75.8%) with abnormal, and 66(55%) infertile patients (all showed a decrease in sperm concentration and motility). Twenty-one fertile males (17.5%) were used as control. DNA primer pairs specific for 16S ribosomal RNA gene of M. hominis and urease gene of U. urealyticum were utilized for PCR. 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.

Academic research paper on topic "Clinical significance of asymptomatic urogenital Mycoplasma hominis and Ureaplasma urealyticum in relation to seminal fluid parameters among infertile Jordanian males"

Middle East Fertility Society Journal (2010) 15, 29-34

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.com www.sciencedirect.com

RESEARCH ARTICLE

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

Hala I. Al-Daghistani a *, Muna Abdel-Dayem b

a Department of Medical Allied Sciences, Al-Salt University College, Al-Balqa Applied University, Al-Salt 19117, Jordan b Medical Hussein City, Medical Laboratories, Amman, Jordan

Received 18 June 2009; accepted 29 October 2009 Available online 28 March 2010

KEYWORDS

Mycoplasma hominis; Ureaplasma urealyticum; Seminal fluid; Varicocele

Abstract Objectives: To investigate Mycoplasma prevalence rate among infertile Jordanian patients and determine the possible role of asymptomatic Mycoplasma hominis and Ureaplasma urealyticum as an infectious factor that might affect semen quality in infertility, mainly varicose-related one. Setting: Medical Hussein City Hospital in Jordan.

Materials and methods: Seminal fluids obtained from 99 infertile patients were tested for the presence of M. hominis and U. urealyticum by polymerase chain reaction (PCR) and analyzed for motility, counts and viscosity. Cases involved were 33(27.5%) infertile with varicocele, 8(24.2%) with normal seminal fluid parameters, 25(75.8%) with abnormal, and 66(55%) infertile patients (all showed a decrease in sperm concentration and motility). Twenty-one fertile males (17.5%) were used as control. DNA primer pairs specific for 16S ribosomal RNA gene of M. hominis and urease gene of U. urealyticum were utilized for PCR.

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

Corresponding author. Fax: +962 6 5856636. E-mail address: hala2002dagh@yahoo.com (H.I. Al-Daghistani).

1110-5690 © 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved. Peer-review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2010.03.003

(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. ure-alyticum 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.

© 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

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). Vari-cocele 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 motil-ity 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 Myco-plasmas 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 asympto-

matic 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 Mycoplas-mas exert an effect on male fertility and semen quality. Myco-plasma 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 Mycoplas-mas 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 varico-cele 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 (x106/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 ig/ml proteinase K (29). To lyse the cells and inactivate proteinase K, the samples were microcen-trifuged, overlaid with mineral oil, and incubated in a Minicy-cler (MJ Research, USA) for 60 min at 56 0C, and then at 95 0C for 10 min (30). Aliquots of 25 il were microcentrifuged, heated at 94 0C 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 P1, potassium chloride 50 mmol P1, and 200 imol P1 dNTP, 1 imol P1 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 imol P1 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 P1, pH 8.3. The mixtures were subjected to 40 cycles composed of sequential incubations at 95 0C for 1 min for DNA denaturation, 55 0C for 1 min for annealing primers to the template, and 72 0C 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 H2O 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. hom-inis 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

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 Myco-found to be statistically significant (Table 2). Regarding sperm plasmas can occur during birth and the carriers are asymp-

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 tomatic, but under certain circumstances the organisms could be opportunistic pathogens (33). Because asymptomatic infections may remain undetected, screening programs

Table 1 Prevalences of M. hominis and U. urealyticum among infertile-varicose male, infertile, and fertile male.

Study groups M. hominis U. urealyticum

Absent no. (%) Present no. (%) Absent no. (%) Present no. (%)

Infertile-varicose patients a. With normal SF (no. = 8) b. With abnormal SF (no. = 25) Infertile without varicocele (no. = 66) Fertile male (no. = 21) Total: 120 (100%) 7(87.5) 17(б8) 54(81.8) 18(85.7) 9б(80) 1(12.5) 8(32) 12(18.2) з(14.З) 24(20) 7(87.5) 22(88) 55(83.3) 21(100) 105(87.5) 1(12.5) 3(12) 11(1б.7) 0(0) 15(12.5)

SF: seminal fluid.

Table 2 Mean percentage of sperm motility in relation to M. hominis and U. urealyticum in the seminal fluid.

Motility grade M. hominis U. urealyticum

PCR positive (mean%) n = 24 PCR negative (mean%) n = 9б PCR positive (mean%) n =15 PCR negative (mean%) n = 105

Grade a (fast progressive) 0.б7 0.б7 0.47 0.б9

Grade b (slow progressive) 2б.21 2б.09 22.53 2б.б2

Grade c (non progressive) 31.92 2б.88 30.53 27.51

Grade d (immotile) 40.79 43.07 46.47 42.07

Table 3 Mean sperm count in relation to M. hominis and U. urealyticum obtained from infertile patients (with and without varicocele) and healthy males.

Sperm count (1 x 106/ml) M. hominis U. urealyticum

PCR positive Mean i SD PCR negative Mean i SD PCR positive Mean ± SD PCR negative Mean i SD

Infertile with varicocele a. with normal SF (no. = 8) b. with abnormal SF (no. = 25) Infertile without varicocele (no. = 66) Fertile male (no. = 21) 30.0 i 0.0 72.50 i 33.49 34.04 i 34.40 145.0 i 124.9 93.43 i 84.04 29.0б i 52.29 57.70 i 41.05 97.55 i 78.09 180.5 ± 0.0 61.66 ± 27.24 45.04 ± 45.7 0 72.00 i 43.83 б1.0 i 41.б2 47.б2 i 39.8б 104.0 i 92.29

1 0 9 S 7 б 5 4 3 2 1 S

Figure 1 Detection of Mycoplasmas by PCR in the seminal fluids. M. hominis was tested using primer pairs specific for a 324 base pair region of 16 rRNA genes. U. urealyticum was tested using primer base specific for 224 base pair region of urease gene. Lane S, base pair slandered; lanes 1-3 positive samples for U. urealyticum; lanes 4-6, 8-10 positive samples for M. hominis; lane 7, positive for both.

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 1040% in the seminal fluids (35).

Despite intensive investigations, the pathogenesis of varico-cele-related infertility remains controversial. In a study done by Gattuccio et al., a real correlation was found between var-icocele 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 hydrody-namic drag on the infested sperm which causes loss of its motility (41). Some researchers report that infection with Ure-aplasma 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 anti-sperm 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 hypervis-cosity 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.

In conclusion, the prevalence of Mycoplasma was higher than that of Ureaplasma, with the absence of the latter from fertile male. A significant association was found between U. urea-lyticum and infertile patients, but this has no detectable impacts on poor semen parameters among infertility, mainly varicose-related one. Further studies were needed to clarify the potential role of Mycoplasmas in the pathophysiology of varicocele.

Acknowledgments

The author wants to acknowledge Al-Balqa Applied University for the grant offered to support this study, and also a lot of appreciation to Fatema Sousarbi for her technical laboratory helps.

References

(1) McLachlan RI, de Kretser DM. Male infertility: the case for continued research. Med J Aust 2001;174:116-7.

(2) Kursh ED. What is the incidence of varicocele in a fertile population? Fertil Steril 1987;48:510-1.

(3) World Health Organization. The influence of varicocele on parameters of fertility in a large group of men presenting to infertility clinics. Fertil Steril 1992;57:1289-93.

(4) Moein MR, Soleimani M, Tabibnejad N. Reactive oxygen species (ROS) production in seminal fluid correlates with the severity of varicocele in infertile men. Ind J Rep Med 2008;6(2):65-9.

(5) Ficarra V, Porcaro AB, Righetti R, Cerruto MA, Pilloni S, Cavalleri S, et al. Antegrade scrotal sclerotherapy in the treatment of varicocele: a prospective study. BJU Int 2002;89:264-8.

(6) Zucchi A, Mearini L, Mearini E, Fioretti F, Bini V, Porena M. Varicocele and infertility: relationship between testicular volume and seminal parameters before and after treatment. J Androl 2006;27(4):548-51.

(7) Zargooshi J. Sperm count and sperm motility in incidental highgrade varicocele. Fertil Steril 2007;88(5):1470-3.

(8) Golomb J, Vardinon N, Homonnai ZT, Braf Z, Yust I. Demonstration of antispermatozoal antibodies in varicocele-related infertility with an enzyme-linked immunosorbent assay (ELISA). Fertil Steril 1986;45(3):397-402.

(9) Pasqualotto FF, Sundaram A, Sharma RK, Borges Jr E, Pasqualotto EB, Agarwal A. Semen quality and oxidative stress scores in fertile and fertile patients with varicocele. Fertil Steril 2008;89(3):602-7.

(10) Giamarellou H, Tympanidis K, Bitos NA, Leonidas E, Daikos GK. Infertility and chronic prostatitis. Andrologia 1984;16:417-22.

(11) Weidner W, Jantos C, Schiefer HG, Haidl G, Friedrich HJ. Semen parameters in men with and without proven chronic prostatitis. Arch Androl 1991;26:173-83.

(12) Diemer T, Huwe P, Ludwig M, Hauck EW, Weidner W. Urogenital infection and sperm motility. Andrologia 2003;35:283-7.

(13) Bornman MS, Mahomed MF, Boomker D, Schulenburge GW, Reif S, Crewe-Brown HH. Microbial flora in semen of infertile African men at Garankuwa hospital. Andrologia 1990;22(2): 118-21.

(14) Mandar R, Raukas E, Turk S, Korrovits P, Punab M. Mycoplasmas in semen of chronic prostatitis patients. Scand J Urol Nephrol 2005;39(6):479-82.

(15) Fernandez C, Alvarez K, Muy L, Martinez M. Detection using molecular biology techniques of Mycoplasma hominis and Ureaplasma urealyticum in urogenital samples. Rev Argent Microbiol 1998;30(2):53-8.

(16) Dieterle S. Urogenital infections in reproductive medicine. Andrologia 2008;40(2):117-9.

(17) Andrade-Rocha FT. Ureaplasma urealyticum and Mycoplasma hominis in men attending for routine semen analysis. Prevalence, incidence by age and clinical setting, influence on sperm characteristics, relationship with leukocyte count and clinical value. Urol Int 2003;71(4):377-81.

(18) Unskula A, Kohl PK. Genital mycoplasmas, including Myco-plasma genitalium as sexually transmitted agents. Int J STD AIDS 2002;13:79-85.

(19) Potts JM, Sharma R, Pasqualotto F, Nelson D, Hall G, Agrwal A. Association of Ureaplasma urealyticum with abnormal reactive oxygen species levels and absence of leukocytospermia. J Urol 2002;163:1775-8.

(20) Li H, Guo Y, Wang Y, Sun X. Genital Ureaplasma urealyticum infection in varicocele related infertility. Clin Med J 1997;110(11):865-8.

(21) Han XD, Wang Y, Chen JX. A comparative study on the interrelations among microelements, infection of Ureaplasma urealyticum, and male infertility. Arch Androl 2003;49:265-9.

(22) Xu C, Sun GF, Zhu YF, Wang YF. The correlation of Ureaplasma urealyticum infection with fertility. Andrologia 1997;29(4):219-26.

(23) Busolo F, Zanchetta R. Effects of Mycoplasma hominis and Ureaplasma urealyticum on hamster egg in vitro penetration by human spermatozoa. Fertil Steril 1985;43(1):110-4.

(24) Abdurazzak AA, Bakr SS. Role of mycoplasma in male infertility. East Med Health J 2000;6(1):1-6.

(25) de-Jong Z, Pontonnier F, Plante P, Perie N, Talazac N, Mansat A, et al. Comparison of the incidence of Ureaplasma urealyticum in fertile men and in donors of semen. Eur Urol 1990;18(2): 127-31.

(26) Weidner W, Krause W, Schiefer HG, Brunner H, Friedrich HJ. Ureaplasma infections of the male urogenital tract, in particular prostatitis and semen quality. Urol Int 1985;40(1):5-9.

(27) World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge: Cambridge University Press; 1999.

(28) Comhaire F, Vermeulen L. Human semen analysis. Hum Reprod Update 1995;1(4):343-62.

(29) Blancgard A, Yanez A, Dybvig K, Watson HL, Griffiths G, Cassell GH. Evaluation of interspecies variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction. J Clin Microbiol 1993;31:1358-61.

(30) Witkin SS, Jeremias J, Toth M, Ledger WJ. Detection of Chlamydia trachomatis by the polymerase chain reaction in the cervices of women with acute salpingitis. Am J Obstet Gynecol 1993;168:1438-42.

(31) Lee AH, Ramanujam T, Ware P, Edelstein PH, Brooks JJ, Freundlich B, et al. Molecular diagnosis of Ureaplasma urealyt-icum septic arthritis in a patient with hypogammaglobulinemia. Arthritis Rheum 1992;354:443-8.

(32) Koch A, Bilina A, Teodorowicz L, Stary A. Mycoplasma hominis and Ureaplasma urealyticum in patients with sexually transmitted diseases. Wlen Klin Wochenschr 1997;114(15):584-9.

(33) Klein JO, Buckland D, Finland M. Colonization of newborn infants by mycoplasmas. New Engl J Med 1969;280:1025-30.

(34) Daxboeck F, Zitta S, Stadler M, Iro E, Krause R. Mycoplasma hominis and Ureaplasma urealyticum in patients with sterile pyuria. J Infect 2005;51(1):54-8.

(35) Andrade-Rocha FT. Ureaplasma urealyticum and Mycoplasma hominis in men attending for routine semen analysis. Prevalence, incidence by age and clinical setting, influence on sperm characteristics, relationship with a leukocytes count and clinical value. Urol Int 2003;71:377-81.

(36) Gattuccio F, Di Trapani D, Romano C, Turtulici AS, Milici M, Pavone C, et al. Urogenital inflammations: etiology, diagnosis and their correlation with varicocele and male infertility. Acta Eur Fertil 1988;19(4):201-8.

(37) Vespasiani G, Virgili G, Giurioli A, Di Stasi SM Torelli F, Valitutti M. Echography in prostatitis. Arch Ital Urol Androl 1994;66(Suppl. 4):37-40.

(38) Soffer Y, Ron-El R, Golan A, Herman A, Caspi E, Samra Z. Male genital mycoplasma and Chlamydia trachomatis culture: its relationship with accessory gland function, sperm quality, and autoimmunity. Fertil Steril 1990;53(2):331-6.

(39) Corradi G, Molnar G, Panovics J. A genitalis mycoplasma kandrologiai jelentosege (Andrologic significance of genital mycoplasma). Orvosi Hetilap 1992;133(48):3085-8.

(40) Stalheim OH, Gallagher JE. Ureaplasma epithelial lesions related to ammonia. Inf Immunol 1977;15:995-6.

(41) O'Leary WM. Ureaplasmas and human disease. Crit Rev Microbiol 1990;17(3):161-8.

(42) Han XD, Wang Y, Chen JX. A comparative study on interrelations among microelements, infection of Ureaplasma Urealty-icum, and male infertility. Arch Androl 2003;49:265-9.

(43) Huang PZ, Li YH. Male infertility. Beijing: Scientific and Technical Literature; 1996, p. 262-4.

(44) Mehraban D, Ansari M, Arab D. Comparison of nitric oxide concentration in seminal fluid between infertile patients with and without varicocele and normal fertile male. Urol J 2005;2(2):106-10.

(45) Diaz-Garcia FJ, Herrera-Mendoza AP, Giono-Cerezo S, Guerra-Infante FM. Mycoplasma hominis attaches to and locates intra-cellularly in human spermatozoa. Hum Reprod 2006;21(6): 1591-8.

(46) Gonzales-Jimenez AM, Villanueva-Diaz CA. Epididymal stereo-cilia in semen of infertile men: evidence of chronic epididymitis. Andrology 2006;38(1):26-30.

(47) Munuce MJ. Semen culture, leukocytospermia, and the presence of sperm antibodies in seminal hyperviscosity. Arch Androl 1999;42(1):21-8.