Infections with Chlamydia trachomatis, genital ureaplasmas

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1 Journal of Andrology, Vol. 29, No. 2, March/April 2008 Copyright E American Society of Andrology Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium in Semen and First Void Urine Specimens of Asymptomatic Male Partners of Infertile Couples R. GDOURA,* W. KCHAOU,* L. AMMAR-KESKES,{ N. CHAKROUN,{ A. SELLEMI,{ A. ZNAZEN,* T. REBAI,{ AND A. HAMMAMI* From the *Department of Microbiology and Research Laboratory Microorganismes et Pathologie Humaine, Habib Bourguiba Hospital, Sfax, Tunisia; Àand the Laboratory of Histology-Embryology and Biology of Reproduction, Medical School, Sfax, Tunisia. ABSTRACT: The purpose of this study was threefold: to compare semen and first void urine (FVU) specimens from asymptomatic infertile men for the detection of Chlamydia trachomatis, genital ureaplasma, and genital mycoplasma infections using in-house inhibitor-controlled polymerase chain reaction (PCR) microtiter plate hybridization assay; to determine the prevalence of those organisms in infertile men in Tunisia; and to study the relationship between these bacteria and male infertility. Paired urine and semen specimens from 104 patients were examined by in-house PCR for the presence of DNA of Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and genital mycoplasmas (Mycoplasma hominis and Mycoplasma genitalium). Semen analysis was assessed according to the guidelines of the World Health Organization. Nominal scale variables, the Mann-Whitney test, and the Kruskal-Wallis nonparametric analysis of variance test were used for statistical analysis. There was a very high concordance (.95%) and a very good agreement (k. 0.9) between the detection of Chlamydia trachomatis, genital ureaplasmas, and Mycoplasma hominis in semen and corresponding FVU specimens. Our findings also show a high concordance (81.1%) and a good agreement (k ) between the detection of Mycoplasma genitalium in both specimens. C trachomatis, genital mycoplasmas, and genital ureaplasmas were found to be widespread among infertile male patients in Tunisia, as shown by their respective prevalences of 43.3%, 18.3%, and 14.4%. The mean values of seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte count were not significantly related either to the detection of C trachomatis DNA or to that of genital ureaplasma or mycoplasma DNA in semen specimens. Using our in-house PCR, both semen and FVU were found to be sensitive diagnostic specimens for the detection of C trachomatis, ureaplasmas, and mycoplasmas. The FVU, a less invasive and self-collected specimen, can serve as a marker for the presence of these organisms in the genital tract and can be used as a reliable way of detecting asymptomatic carriers of infection. Key words: Male infertility, sperm quality. J Androl 2008;29: Infections with Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and genital mycoplasmas (Mycoplasma hominis and Mycoplasma genitalium) have been recognized as being common sexually transmitted diseases in industrial countries (Tully et al, 1981; Gerbase et al, 1998; Yoshida et al, 2002). Chlamydial infection in the male urethra can be complicated by inflammation of the epididymis (Stamm et al, 1984) and the prostate gland (Dan et al, 1991), but the role that C trachomatis infection plays in male infertility is controversial (Soffer Correspondence to: Dr R. Gdoura or Prof A. Hammami, Laboratory of Microbiology, Faculté de Médecine de Sfax, Avenue Magida Boulila, 3029, Sfax, Tunisia ( gdourar@yahoo.com) (Dr Gdoura) ( Adnene.hammami@rns.tn) (Prof Hammami). Received for publication June 9, 2007; accepted for publication November 27, DOI: /jandrol et al, 1990; Wolff et al, 1991; Witkin et al, 1995; Eggert- Kruse et al, 1996; Cengiz et al, 1997; Gdoura et al, 2001b). Genital ureaplasmas and genital mycoplasmas are natural inhabitants of the male urethra contaminating the semen during ejaculation. However, these microorganisms, particularly U urealyticum, are potentially pathogenic species playing an etiologic role in both genital infections and male infertility (Upadhyaya et al, 1984; De Jong et al, 1990; Andrade-Rocha, 2003; Wang et al, 2006). The impact of M hominis colonization on semen parameters and male fertility remains unclear. Hitherto, M genitalium and U parvum have seldom been investigated in the semen of infertile men. The demonstration of the relationship between C trachomatis, genital ureaplasmas, and genital mycoplasmas and male infertility would have important public health consequences. Various studies conducted in developed countries have demonstrated that C trachomatis, genital urea- 198

2 Gdoura et al N Bacterial Infections and Semen Quality 199 plasma, and genital mycoplasma infections can lead to sterility (Upadhyaya et al, 1984; Paavonen and Wolner-Hassen, 1989; Vigil et al, 2002; Askienazy- Elbhar, 2005; Wang et al, 2006). In developing countries, however, infections caused by Chlamydia and Mycoplasma have not been well studied, and the prevalence of C trachomatis, genital ureaplasmas, and genital mycoplasmas among infertile couples has not been determined. Asymptomatic infections of the male urogenital tract are difficult to detect. Screening with classical cell culture requires specimens from endourethral swabs, which are unacceptable to many asymptomatic men. For Chlamydia, the cell culture method cannot be used for semen and urine samples because of their cytotoxicity (Mardh et al, 1980), and is not sufficiently sensitive to rule out infections of accessory glands (Berger et al, 1978). Other routine tests for Chlamydia, such as enzyme immunoassays, may have a reduced sensitivity in asymptomatic individuals. Genital mycoplasma and ureaplasma colonizations are commonly diagnosed by culture. Cultures, however, are time-consuming as they require 2 5 days for Ureaplasma spp. and M hominis and up to 8 weeks for M genitalium, whereas nucleic acid amplification techniques can detect infectious agents in less than 8 hours. A great deal of research has led to the development and evaluation of sensitive and specific diagnostic tests based on nucleic acid amplification tests for C trachomatis, M hominis, M genitalium, U urealyticum, and U parvum. The important aspect of these assays is their capacity to be used on less-invasive specimens, such as first void urine (FVU), which can be selfcollected. Studies have shown that nucleic acid amplification tests performed on these less-invasive samples are able to detect as many or more infected patients than traditional swabs from the urethra or cervix and are more suitable methods for screening and diagnosing chlamydial and genital Mycoplasmataceae infections (Chernesky et al, 1994; Consentino et al, 2003; Gaydos et al, 2004; Jensen et al, 2004). However, one major problem with them is the presence of potential inhibitors in clinical specimens that can lead to false negative results. The purposes of our study were: first, to compare semen specimens and FVU specimens from asymptomatic male members of infertile couples for the detection of C trachomatis, U urealyticum, U parvum, M hominis, and M genitalium infections using an in-house inhibitorcontrolled polymerase chain reaction (PCR) microtiter plate hybridization assay; and second, to determine the prevalence of those organisms in infertile men in Tunisia. We also investigated the relationship between those bacteria and semen quality. Patients and Methods Patients A total of 104 male patients attending obstetrics and gynecology clinics in Sfax (in the south of Tunisia) for infertility were included in this study. All men were asked for sexual history and past history of infections and were screened for chronic prostatitis or urethritis. All 104 patients did not present any clinical symptoms of infection of the lower genital tract, and, apart from their infertility problem, were healthy individuals. The mean duration of infertility was 4.3 years (range, 1 19). The mean age of the patients was 37 years (range, 26 58). All subjects were assessed by clinical examination and semen analysis. Specimens Semen and corresponding FVU samples of the 104 patients were collected after approval of our institutional review board and each patient s informed consent. In the morning, when patients had not had oral intake for at least 8 hours, FVU was collected in a sterile plastic container and stored at 220uC. After FVU had been collected, patients were instructed to wash the penis, scrotum, and hands with soap and water, and then semen specimens were obtained by masturbation and collected into standard containers that had previously been shown not to have any cytotoxic effects on human spermatozoa. All male partners had at least 3 days of sexual abstinence before donation of semen. Semen Analysis Semen analysis was performed according to the methods outlined by the World Health Organization (WHO, 1999) to determine the following variables: seminal volume, sperm concentration, viability, total progressive motility (category [a + b]), rapid progressive motility (category [a]), and normal morphology, as well as leukocyte count. Azoospermia was defined by the absence of spermatozoa after at least 2 consecutive spermiograms, oligospermia by a sperm concentration of, /ml, asthenospermia by sperm motility (category [a + b]),50%, necrospermia by sperm viability,50%, and teratospermia by normal morphology,30%. Peroxidase staining, being a practical and reliable method recommended by the WHO for determining leukocytes in the semen, was employed to count and differentiate leukocytes from immature germ cells (1999). Leukocytospermia was defined by a concentration of leukocytes $10 6 /ml. Detection of C trachomatis, Genital Mycoplasmas, and Genital Ureaplasmas in Semen Specimens by In-House PCR Microtiter Plate Hybridization Assay For each male patient, 200 ml of semen specimens and 1 ml of corresponding FVU samples were used for DNA detection of C trachomatis, genital mycoplasmas (M hominis and M genitalium), and genital ureaplasmas (U urealyticum and U parvum) by an amplification-based method according to the following protocol.

3 200 Journal of Andrology N March ÙApril 2008 Extraction of DNA by Cetyltrimethylammonium Bromide Phenol-Chloroform/Isoamyl Alcohol Method The precipitates from each 200 ml of semen specimens and 1 ml of corresponding FVU samples were harvested by centrifugation at g for 20 minutes. The precipitates were then treated with 5 ml of proteinase K (20 mg/ml) at 55uC for 2 hours in 600 ml of digestion buffer (30 ml of10% sodium dodecyl sulfate and 570 ml of TE buffer [10 mm Tris-HC1 (ph 8), 1 mm EDTA]). After homogenization, the samples were incubated in a solution of cetyltrimethylammonium bromide (CTAB)-NaCl (100 ml of 5 M NaCl and 80 ml of 10% CTAB) for 10 minutes at 65uC, and then mixed with 750 ml of chloroform isoamyl alcohol (24:1 [vol/vol]) and centrifuged for 15 minutes at g in an Eppendorf centrifuge. The aqueous phase was separated, mixed with 750 ml of phenolchloroform isoamyl alcohol (25:24:1 [vol/vol/vol]), and centrifuged for 15 minutes at g in an Eppendorf centrifuge. The obtained aqueous phase was reseparated and mixed with an equal volume of isopropanol. The samples were left at 280uC for 1 hour and then centrifuged for 15 minutes at g. The DNA pellet was washed up once with 70% ethanol, air-dried, and dissolved in a final volume of 100 ml of TE buffer. In-house PCR Assay Initially, the extracted DNA was tested for human b-globin gene to check that there were no PCR inhibitors in the samples. Primers b-gpco (59-ACACA- ACTGTGTTCACTAGC-39) and b-gpcpo (59-GAAACC- CAAGAGTCTTCTCT-39) were used to amplify a 209-bp fragment of the human b-globin gene (Vogels et al, 1993). Samples found to be b-globin negative by PCR were retested after dilution 10-fold in distilled water. Samples shown to be b- globin positive were then examined for the presence of C trachomatis, U urealyticum, U parvum, M hominis, and M genitalium. A forward primer, T1 (59-GGACAAATCGTATTTCGG-39), and a reverse primer, T2 (59-GAAACCAACTCTACGCTG-39), were used to amplify an approximately 517-bp region of the cryptic plasmid of C trachomatis (Claas et al, 1990). A forward primer, My-ins (59-GTAATACATAGGTCG- CAAGCGTTATC-39), and 2 reverse primers, MGSO-2-Bi (59- CACCATCTGTCACTCTGTTAACCTC-39) and UGSO-Bi (59-CACCACCTGTCATATTGTTAACCTC-39), were used to amplify an approximately 520-bp region of the 16S rrna gene of mycoplasmas and ureaplasmas (Yoshida et al, 2003). The PCR mixture, which was made up to 50 ml with sterile water, contained 16 PCR buffer (50 mm Tris-HCl [ph 8.3], 10 mm KCl, 5.0 mm (NH 4 )2SO 4, and 2.0 mm MgCl 2 ); 0.5 mm each My-ins and MGSO-2-Bi for detection of genital mycoplasmas, 0.5 mm each My-ins and UGSO-Bi for detection of genital ureaplasmas, or 0.5 mm each T1 and T2 for detection of C trachomatis; 0.2 mm each datp, dctp, and dgtp; 0.6 mm dutp; 1.25 U of Go Taq DNA polymerase (Promega, Lyon, France); and 10 ml of prepared DNA solution. PCR was performed using the Gene-Amp PCR System 9700 (PerkinElmer, Waltham, Mass) under the following conditions: an initial cycle at 95uC for 5 minutes, followed by 40 cycles of denaturation at 94uC for 30 seconds, annealing at 50uC (T1/T2) or at 55uC (My-ins/MGSO-2-Bi or My-ins/UGSO-Bi) for 30 seconds, and elongation at 72uC for 45 seconds, with a final cycle at 72uC for 7 minutes. Each run PCR included a positive control (M hominis PG21, U urealyticum, or C trachomatis genotype E) and 2 negative controls (previously negatively tested samples and distilled water). The PCR products were then subjected to hybridization assays. Twenty semen specimens from infertile men were used as controls and were blindly re-examined. Twelve semen specimens were used as positive controls; 5 of these were previously shown to be C trachomatis positive by Cobas Amplicor CT/ NG PCR (Gdoura et al, 2001b), and 4 and 3 were previously shown to be U urealyticum positive and M hominis positive, respectively, by culture (Mycoplasma IST; biomérieux, Lyon, France). Eight semen specimens negative for these 3 bacteria were used as negative controls. Hybridization Amplified products of 517 bp of C trachomatis and of 520 bp of U urealyticum, U parvum, M hominis, and M genitalium were detected by using a molecular hybridization technique in a liquid phase assay (Argene, Varilhes, France) with species-specific internal biotinylated probes (Claas et al, 1990; Yoshida et al, 2003): T3 (59-biotin- CGCAGCGCTAGAGGCCGGTCTATT- TATGAT-39) specific for C trachomatis, Uure-P4-Am (59- biotin-ggctcgaacgagtcggtgt-39) specific for U urealyticum, Upar-P6-Am (59-biotin-GTCTGCCTGAAT- GGGTCGGT-39) specific for U parvum, Mhom-P10-Am (59- biotin - GACACTAGCAAACTAGAGTTAG-39) specific for M hominis, Mgen-P3-Am (59-biotin-TCGGAGCGATCCC- TTCGGT-39) specific for M genitalium. The amplified products were denatured in a denaturing solution for 10 minutes and then immobilized to a microtiter plate (DNA-BIND strip well plates; Argene) as described in the manufacturer s instructions. Species-specific capture probe diluted in hybridization solution was added to the wells of each microtiter plate immobilized by the denatured amplicons, and hybridization was carried out at 37uC for 30 minutes. Following hybridization, the wells were washed 4 times (soaking for 30 seconds) with a washing solution. One hundred microliters of ready-to-use conjugate were added per well and incubated for 15 minutes at room temperature. Wells were washed as described above. One hundred microliters of substrate (tetramethylbenzidine) were added per well and incubated for 30 minutes at room temperature in the darkness. The reaction was stopped by adding 100 ml of stop solution to each well, and the optical density (OD) at 450 nm was measured. Samples with an OD value of greater than the cutoff value (OD of negative control ) + 10% of cutoff value were considered positive, as suggested by the manufacturer. Statistical Analysis All data were collected using standardized forms and were analyzed using SPSS 13.0 software (SPSS Inc, Chicago, Ill). To assess the agreement between the detection of C trachomatis, U urealyticum, U parvum, M hominis, and M genitalium in semen and corresponding FVU specimens, we

4 Gdoura et al N Bacterial Infections and Semen Quality 201 Table 1. Results of the detection of C. trachomatis, U urealyticum, U parvum, M hominis, and M genitalium DNA in semen and FVU samples of 104 infertile male patients by in house-pcr microtiter plate hybridization Species Semen samples, n (%) FVU samples, n (%) Semen/FVU samples +/+ 2/2 +/2 2/+ Semen-FVU Concordance, % C trachomatis 44 (42.3) 41 (39.4) U urealyticum 16 (15.4) 16 (15.4) U parvum 3 (2.9) 3 (2.9) M hominis 10 (9.6) 11 (10.6) M genitalium 5 (4.8) 5 (4.8) Abbreviations: FVU indicates first void urine; PCR, polymerase chain reaction; k, nominal scale variables. k used k (nominal scale variables) as proposed by Landis and Koch (1977). Guidelines for the interpretation of k were as follows: k, 0.20, poor agreement; k , fair agreement; k , moderate agreement; k , good agreement; k , very good agreement. Age, seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte counts are presented as mean values with standard deviation (SD). Semen characteristics were compared among C trachomatis semen-positive and semen-negative groups, ureaplasma semenpositive and semen-negative groups and mycoplasma semenpositive and semen-negative groups. The statistical significance of the differences between groups regarding age, seminal volume, sperm concentration, percentage of viability, percentage of progressive motility, percentage of rapid progressive motility, percentage of normal morphology, and leukocyte counts was assessed using the Mann- Whitney test and the Kruskal-Wallis nonparametric analysis of variance test. Fisher s exact test was used to test the statistical significance of the differences between groups regarding proportions of oligospermia, necrospermia, asthenospermia, teratospermia, and leukocytospermia. Results Detection of C trachomatis DNA in Semen and Corresponding FVU Specimens by PCR A total of 45 (43.3%) of the 104 patients were positive for C trachomatis in semen or FVU specimens. C trachomatis positive men had a median age of 37.2 years, not statistically different from C trachomatis negative men, who had a median age of 36.9 years (P 5.8). C trachomatis DNA was detected in 44 (42.3%) of semen specimens and in 41 (39.4%) of corresponding FVU samples (Table 1). Forty men were positive in both FVU and semen specimens, 4 were positive in the semen specimen only, and 1 was positive in the FVU specimen only (Table 1). Thus, the concordance between the detection of C trachomatis DNA in semen specimens and corresponding FVU specimens of infertile men was 95.2%. A very good agreement between the 2 specimens was found (k ). Detection of Genital Ureaplasma DNA in Semen and Corresponding FVU Specimens by PCR Genital ureaplasma DNA was detected in 19 (18.3%) of the semen or FVU specimens. Genital ureaplasma positive men had a median age of 39.1 years; this was not statistically different from the median age of the negative men (36.6 years; P 5.1). U urealyticum DNA and U parvum DNA were detected in 16 (15.4%) and in 3 (2.9%) of semen and corresponding FVU specimens respectively (Table 1). Thus, the concordance between the detection of genital ureaplasma DNA in semen specimens and corresponding FVU specimens of infertile men was 100%. A very good agreement between the 2 specimens was found (k 5 1). Detection of Genital Mycoplasma DNA in Semen and Corresponding FVU Specimens by PCR A total of 15 (14.4%) of the 104 patients were positive for genital mycoplasmas in semen or FVU specimens. There was no significant difference between the median age of men with or without genital mycoplasma infections (37.4 years vs 37 years; P 5.8). M hominis DNA was detected in 10 (9.6%) of semen specimens and in 11 (10.6%) of corresponding FVU samples (Table 1). Ten patients were positive in both semen and FVU specimens and 1 was positive in the FVU specimen only (Table 1). Thus, the concordance between the detection of C trachomatis DNA in semen specimens and the corresponding FVU of infertile men was 99%. A very good agreement between the 2 specimens was found (k ). M genitalium DNA were detected in 5 (4.8%) of both semen and corresponding FVU specimens (Table 1). Four patients were positive in both FVU and semen specimens, whereas 1 was positive in the semen specimen only, and 1 was positive in the FVU specimen only (Table 1). The concordance between the detection of M genitalium DNA in semen specimens and the corresponding FVU of infertile men was 81.1%. Therefore, a good agreement between the 2 specimens was found (k ).

5 202 Journal of Andrology N March ÙApril 2008 C trachomatis, Genital Ureaplasma, and Genital Mycoplasma Infections and Semen Quality A standard semen analysis showed that 10.6% (11/104) of specimens were azoospermic. The remaining were asthenospermic (96.8%), teratospermic (91.4%), oligospermic (32.3%), necrospermic (20.4%), and leukocytospermic (17.2%). The prevalence of M genitalium DNA in semen samples was significantly higher in azoospermic patients than in nonazoospermic patients (27.3% vs 2.1% respectively, P 5.008; Table 2). A summary of abnormalities in seminal fluid of nonazoospermic patients with C trachomatis, genital ureaplasma, or genital mycoplasma DNA in semen samples is shown in Table 2. Oligospermia, necrospermia, asthenospermia, teratospermia, and leukocytospermia were not significantly related to the presence of C trachomatis, genital ureaplasma, or genital mycoplasma DNA in semen samples. The mean values of seminal volume, sperm concentration, sperm viability, total progressive sperm motility, rapid progressive sperm motility, sperm morphology, and leukocyte counts were significantly related neither to the detection of C trachomatis DNA nor to that of genital ureaplasma or mycoplasma DNA in semen specimens (Table 3). Discussion It is estimated that 15% of male infertility is related to genital tract infection (Keck et al, 1998). C trachomatis and U urealyticum are among the most prevalent sexually transmitted pathogens. Thus, it is important to determine the presence of these pathogens (Gerbase et al, 1998; Gonzales et al, 2004; Wang et al, 2006). The majority of patients with C trachomatis, genital ureaplasma, and genital mycoplasma infections are not aware of their infection because they do not have symptoms. Because asymptomatically infected individuals may shed fewer organisms (Witkin, 2002), and in order to make tests easier and with a higher specificity, nucleic acid amplification tests such as PCR may be the techniques of choice for C trachomatis, genital ureaplasma, and genital mycoplasma assessment in asymptomatic male partners. The main purpose of the current study was to evaluate the relative performance of FVU compared with semen specimens for the detection of C trachomatis, U urealyticum, U parvum, M hominis, and M genitalium in asymptomatic male members of infertile couples. Thus, we used in-house PCR microtiter plate hybridization assays that can facilitate the detection of C trachomatis Table 2. Association between the detection of C trachomatis, U urealyticum, U parvum, M hominis, and M genitalium DNA in semen specimens of infertile male patients and sperm quality Abnormalities in seminal fluid of nonazoospermic patients (n 5 93) Sperm concentration Sperm motility Sperm viability Sperm morphology Sperm count Azoospermic vs nonazoospermic patients N.Leuk (n 5 77) Leuk (n 5 16) N.Ter (n 5 8) Ter (n 5 85) N.Necr (n 5 74) Necr (n 5 19) N.Asth (n 5 3) Asth (n 5 90) N.Oligo (n 5 63) Oligo (n 5 30) N.Azoo (n 5 93) Azoo (n 5 11) Pathogens C. trachomatis positive 7 (36.4%) 40 (43.0%) 13 (43.1%) 17 (42.9%) 39 (43.3%) 1 (33.3%) 7 (36.8%) 33 (44.6%) 35 (41.1%) 5 (62.5%) 7 (43.8%) 33 (42.9%) Genital ureaplasma positive 3 (27.3%) 16 (17.2%) 3 (10%) 13 (20.6%) 13 (14.4%) 0 2 (10.5%) 14 (18.9%) 15 (17.6%) 1 (12.5%) 2 (12.5%) 14 (18.2%) U urealyticum positive 3 (27.3%) 13 (14.0%) 3 (10%) 10 (15.9%) 16 (17.8%) 0 1 (5.3%) 12 (16.2%) 13 (15.3%) 0 2 (12.5%) 11 (14.3%) U parvum positive 0 3 (3.2%) 0 3 (4.8%) 3 (3.3%) 0 1 (5.3%) 2 (2.7%) 2 (2.4%) 1 (12.5%) 0 3 (3.9%) Genital mycoplasma positive 5 (45.5%) 8 (8.6%) a 4 (13.3%) 4 (6.3%) 8 (8.9%) 0 4 (21.1%) 4 (5.4%) 7 (8.2%) 1 (12.5%) 0 8 (10.4%) M hominis positive 3 (27.3%) 7 (7.5%) 4 (13.3%) 3 (4.8%) 7 (7.8%) 0 3 (15.8%) 4 (5.4%) 6 (7.1%) 1 (12.5%) 0 7 (9.1%) M genitalium positive 3 (27.3%) 2 (2.1%) b 0 3 (4.8%) 3 (3.3%) 0 1 (5.3%) 2 (2.7%) 3 (3.5%) (3.9%) Abbreviations: Azoo indicates azoospermia; N.Azoo, nonazoospermia; Oligo, oligospermia; N.Oligo, nonoligospermia; Asth, asthenospermia; N.Asth, nonasthenospermia; Necr, necrospermia; N.Necr, nonnecrospermia; Ter, teratospermia; N.Ter, nonteratospermia; Leuk, leukocytospermia; N.Leuk, nonleukocytospermia. a P 5.04, azoospermic patients vs nonazoospermic patients. b P 5.08, azoospermic patients vs nonazoospermic patients.

6 Gdoura et al N Bacterial Infections and Semen Quality 203 Table 3. Semen characteristics of 93 semen samples of nonazoospermic infertile men patients with and without C trachomatis, U urealyticum, U parvum, M hominis, or M genitalium DNA in semen specimens Pathogens C trachomatis Volume, ml Sperm Concentration, /ml Viability, % Total Progressive Motility (category [a + b]), % Rapid Progressive Motility (category [a]), % Normal Morphology, % Leukocyte Count, /ml Positive (n 5 40) Negative (n 5 53) Genital ureaplasmas Positive (n 5 16) Negative (n 5 77) U urealyticum Positive (n 5 13) Negative (n 5 80) U parvum Positive (n 5 3) Negative (n 5 90) Genital mycoplasmas Positive (n 5 8) Negative (n 5 85) M hominis Positive (n 5 7) Negative (n 5 86) M genitalium Positive (n 5 2) Negative (n 5 91) and the identification of U urealyticum, U parvum, M hominis, and M genitalium in semen and FVU specimens. Initially, we tested extracted DNA of each semen and FVU samples for human b-globin gene to check that there were no PCR inhibitors in the samples. In a routine diagnostic setting, the internal processing control is important for providing a high sensitivity and specificity (Mahony et al, 1992), which is particularly important when dealing with sexually transmitted infections. In our study, known positive and negative control specimens were also blindly re-examined to confirm the sensitivity and specificity of the assays. In this study, we found a very high concordance (.95%) and a very good agreement (k. 0.8) between the detection of C trachomatis DNA, genital ureaplasma DNA, and M hominis DNA in semen and corresponding FVU specimens. A high concordance (81.1%) and a good agreement (k ) were also found between the detection of M genitalium DNA in both specimens. Using our in-house PCR and sample preparation methods, FVU was found to be a sensitive diagnostic specimen for these pathogens. Several studies have shown that nucleic acid amplification tests performed on FVU samples are able to detect as many or more infected patients than traditional swabs from the urethra or cervix or from semen (Chernesky et al, 1994; Pannekoek et al, 2003; Gaydos et al, 2004; Hamdad-Daoudi et al, 2004; Jensen et al, 2004). In some cases, we found discrepancies between the detection of C trachomatis DNA and genital mycoplasma DNA in semen and corresponding FVU specimens. The presence of C trachomatis DNA and genital mycoplasma DNA in FVU samples and its absence in semen specimens may indicate an asymptomatic urethral infection. The detection of C trachomatis in 4 patients and M genitalium in 1 patient only in semen may indicate that these organisms are harboured in the epididymis or seminal vesicles. Various studies conducted in industrialized countries proved the high prevalence of C trachomatis, genital ureaplasmas, and genital mycoplasmas among male partners of infertile couples and its important role in some cases in infertility (Upadhyaya et al, 1984; Paavonen and Wolner-Hassen, 1989; Vigil et al, 2002; Askienazy-Elbhar, 2005; Wang et al, 2006). Yet, in developing countries, the situation is not always clear. Our study demonstrated that C trachomatis seems to be widespread among male partners of infertile couples in Tunisia, as shown by its high prevalence (43.3%). Our findings confirm our previous reports (Gdoura et al, 2001a,b) and are generally similar to those reported in studies conducted in developed countries (Vigil et al,

7 204 Journal of Andrology N March ÙApril ; Askienazy-Elbhar, 2005). Our results revealed also that genital mycoplasmas and genital ureaplasmas seem to affect a large number of infertile male patients, as disclosed by their rates of 18.3% and 14.4% respectively. These data are comparable with those reported in previous studies (Tully et al, 1981; Andrade-Rocha, 2003; Knox et al, 2003). U urealyticum was the most prevalent species detected (15.4%) in the current study. The prevalence of U urealyticum in the semen samples of male infertile patients in the literature varies from 5% to 42% (De Jong et al, 1990; Andrade-Rocha, 2003; Knox et al, 2003; Rosemond et al, 2006; Wang et al, 2006). This wide range might be explained by the diversity of detection methods used for characterizing the studied populations. Most of the previous reported studies have discussed the role of ureaplasmas in male infertility without discriminating between U urealyticum and U parvum (Naessens et al, 1986; Bornman et al, 1990; De Jong et al, 1990). In our study, we used the PCR microtiter plate hybridization assay for facilitating the identification of U urealyticum, U parvum, M hominis, and M genitalium in semen specimens. By this method, U parvum was detected in 2.9% of semen samples. The prevalence of this species in our study was lower than that reported by Knox et al (2003) (2.9% vs 19.2%). In the literature, M hominis has been associated with bacterial vaginosis, pelvic inflammatory disease, postpartum fever, and postabortal fever, as well as a number of gynecologic infections (Soffer et al, 1990; Yoshida et al, 2003). However, its role in nongonoccocal urethritis and in infertility is rarely investigated (Pannekoek et al, 2000). The prevalence of M hominis in our study was comparable to that reported by Andrade-Rocha (2003) but higher than that found by Rosemond et al (2006). M genitalium is gaining increasing interest as a pathogen in male urethritis. Several studies (Björnelius et al, 2000; Totten et al, 2001) have shown a significant association between the presence of M genitalium DNA and symptoms and/or signs of urethritis (recently reviewed by Jensen et al, 2004). Hitherto, M genitalium has seldom been investigated in semen of infertile men. In our study, the prevalence of M genitalium in male partners of infertile couples was higher than that reported by Kjaergaard et al (1997) (4.8% vs 0.9%). This difference might be explained by the use of different methods for the detection of this bacterium. We have used PCR, which is more sensitive than a culture and facilitates the detection of M genitalium in clinical samples (Yoshida et al, 2003). In addition to the determination of the prevalence of C trachomatis, genital mycoplasmas, and genital ureaplasmas in the male partners of infertile couples, we tried to evaluate the relationship between the detection of these pathogens in semen specimens and sperm quality. Several lines of evidence suggest a role of C trachomatis in the etiology of male infertility. However, no direct link has been demonstrated (Witkin et al, 1993; Dieterle et al, 1995; Bollmann et al, 1998). In the current study, no relationship has been found between the detection of C trachomatis DNA in semen specimens and sperm abnormalities (concentration, sperm motility, sperm viability, sperm morphology, and leukocyte count). This result was in accordance with several studies (Gregoriou et al, 1989; Soffer et al, 1990; Dieterle et al, 1995; Eggert-Kruse et al, 1996, 1997; Weidner et al, 1996; Habermann and Krause, 1999; Gdoura et al, 2001b). However, some other studies have shown that infection is associated with poorer semen quality (Custo et al, 1989; Wolff et al, 1991; Witkin et al, 1995; Cengiz et al, 1997). Reports are controversial about the effects of genital mycoplasma and ureaplasma infections or colonizations on semen andrology variables (Potts et al, 2000; Knox et al, 2003; Sanocka-Maciejewska et al, 2005; Wang et al, 2006). Previous studies have established that the presence of mycoplasma and ureaplasma in sperm specimens has no real effect on the semen quality (Soffer et al, 1990; Kjaergaard et al, 1997; Andrade- Rocha, 2003; Knox et al, 2003). Conversely, a relationship between U urealyticum and semen characteristics has been documented in literature (Potts et al, 2000; Sanocka-Maciejewska et al, 2005; Wang et al, 2006). In the present study, we failed to demonstrate any correlation between the abnormalities in seminal fluid and the detection of genital ureaplasma DNA or the detection of genital mycoplasma DNA in semen specimens. We found only a significantly higher prevalence of M genitalium in azoospermic patients than in nonazoospermic patients. Recent investigations seem to show that the presence of mycoplasmas reflects a silent colonization rather than an infection in infertile patients (Pannekoek et al, 2000), although the attachment and invasiveness of mycoplasmas towards human sperm cells has been demonstrated in vitro (Diaz-Garcia et al, 2006; Baczynska et al, 2007). These findings suggest that M genitalium may have an effect on the genital tract, leading to an inflammatory obstructive process, rather than on functional characteristics of semen. In conclusion, using our in-house PCR and sample preparation methods, both semen and FVU were found to be sensitive diagnostic specimens for the detection of C trachomatis, genital ureaplasma, and genital mycoplasma DNA. The FVU, a less invasive and self-collected specimen, can be used as a marker for the presence of these organisms in the genital tract and as a reliable way of detecting asymptomatic carriers of infection.

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