Chalmydia trachomatis infection among asymptomatic males in an infertility clinic

Introduction

Chlamydia trachomatis (C. trachomatis) infection is now considered as an important cause of non-specific urethritis and epididymitis in men, and cervicitis and pelvic inflammatory disease in women, leading to infertility.[1] Several studies suggest that one or more infectious agents in the genito-urinary tract may be associated with male infertility.[2],[3],[4],[5] Many of these organisms have been considered commensal flora but some organisms like C. trachomatis in the semen are significantly associated with tubal disease in female partners. Salpingitic tubal damage is a common cause of acquired subfertility in women, which is preventable. A considerable group of subfertile men have experienced chlamydial infection as indicated by the presence of chlamydial antibodies and provide further evidence for the sexually transmitted nature of C. trachomatis infection with potential severe complications in their partners, threatening the couples fertility.[8] At the Institutes Infertility Clinic, females were screened for

C. trachomatis infection. The prevalence of this infection among the infertile females enrolled in the Clinic was 34% (unpublished data). This was determined by endocervical swabs stained by FITC method. In the present study, the objective is to find out the infection rate among the asymptomatic male partners of these infected females.

Subjects and Methods

Fifteen male partners of infertile couples where the female partners were infected with C. trachomatis were selected for the study. They were not taking any antibiotic in the recent past. Their history and genital examination did not show any symptoms or signs of infection and were therefore considered as asymptomatic. Consent was taken for collection of samples for diagnosis of C. trachomatis infection.

Samples

For antigen detection, first voided morning urine (15-20ml) and semen sample were collected from each individual. Briefly, 15-20ml of urine collected in a sterile container was centrifuged at 3000 g for 20 minutes and the sediment was collected for further analysis. Similarly semen sample was obtained from each of these individuals after an abstinence of 3-4 days and kept in a beaker for liquefication. The semen sample after liquefication was processed for routine analysis and the rest of the sample was vortexed and sonicated. Both the urine sediment and the semen sample were kept at -20°C for further processing within seven days. For antibody detection 1 ml of blood was collected from each individual using sterile disposable syringe and needle. Serum was separated and refrigerated for further analysis.

Antigenic detection

The ′chlamydiazyme′ kit from Abbott′s diagnostic was used for antigen detection. Each urine sediment was dissolved in 400 µl of sample dilution buffer while 200 µl of semen sample was diluted in 1 ml of sample dilution buffer. These samples were then vortexed at high speed to get a homogenous suspension. Simultaneously positive and negative controls were used as per the instruction manual. Vortexed specimens or freshly prepared controls each of 200 µl were pipetted into wells of the reaction tray. Each sample testing was done in duplicate. One bead from the kit was added to each well containing the specimen or control. The wells were sealed, gently tapped and incubated at 37° C for 1 hr. After incubation, the liquid was aspirated and the beads were washed properly in distilled water. Anti-C. trachomatis antibody (200 pl) was added to each well, sealed and kept in the incubator for 1 hr. Again the beads were washed and enzyme conjugate (IgG-HRP, 200 µl) was added to each well containing the beads and incubated for 1 hr. After incubation, the beads were washed and transferred to properly identified assay tubes. OPD (o-phyenylenediamine) substrate solution of 300 µl was freshly prepared and added to each tube containing a bead and kept at room temperature for 30 minutes. One ml of IN H2SO4 was added to each tube, agitated to mix and the reading was taken at 492 nm. The cut off value was determined by calculating the mean of negative controls. Individual specimens whose value was more than this cut off value, was taken as positive and the other with an O.D. (optical density) reading less than it, was considered negative.

Antibody detection

Antibody (IgG) to C. trachomatis was determined in the serum sample of each individual using the kit ′IMMUNOCOMB′ from Orgenics Ltd., Israel. As per the instruction, using the comb supplied, samples were processed through rows of wells (A to F), containing diluent (A), wash solution (B), conjugate (C), two wash solutions (D,E) and chromogen solution (F). At a time 10 specimens, one positive and one negative control could be tested. The sample which developed a colour on the respective comb was considered as positive.

Routine semen analysis

Semen analysis such as volume, concentration, viscosity, motility and WBC cell count (pus cell) was also carried out in each of these cases.[9]

Results

The average age (±S.D.) of the 15 males selected for the study was 31.3±3.92 years (range:25-38 years). Of these 15 cases, 5 (33.3%) were positive for C. trachomatis antigen in their urine sample. Among these 5 cases, two were also positive for this infection in their semen samples. In others, this infection was not detectable either in urine or in semen.

C. trachomatis antibody (IgG) was detected in 7 (46.7%) serum samples. Among these cases 3 had antigen in their urine samples, indicating that 3 (20%) cases had both antigen and antibody. Considering the presence of antigen or antibody or both, 9 (60%) cases were with the C. trachomatis infection.

The routine semen analysis result showed that 6 (40%) individuals had a semen volume less than the average normal volume (1.5-4.5 ml). Sperm count was low (< 20 mill/ml) in 6 (40%) cases. Of these 6 cases, 3 had low semen volume. Sperm motility was low in 4 (26.7%) cases while the others had normal rapid linear progressive motility (>25%). Average pus cell count was 4-5/hpf (high power field). Semen viscosity was normal in 6 (40%) cases.

When the routine semen parameters were correlated with the C. trachomatis infection, it was observed that among the infected cases (n=9), 5 (55.6%) had semen volume less than the normal volume. Sperm count, motility and viscosity were abnormal in 66.7% (n=6), 33.3% (n=3) and 55.6% (n=5) of cases respectively. Among the infected cases, the average pus cell count was 5-7 cells/hpf whereas in non-infected cases, it was 2-3 cells/hpf Table 1.

Discussion

Asymptomatic male partners of fifteen infertile couples whose female partners were infected with C. trachomatis were screened for this infection in the present study. It was observed that 60% of these males had this infection in their urine and / or in blood. Of the 9 infected cases, 2 had chlamydia antigen present in both urine and semen whereas in another 3 cases antigen was only present in their urine sample. This suggests the probable site of chlamydia infection may be the urethra. Previous reports also suggest that C. trachomatis is a common cause of urethritis.[10],[11],[12] Presence of only chlamycdia antibody in 4 cases, revealed a past infection, i.e. they might have already infected their spouse or received infection from them. It was also reported earlier that C. trachomatis infection of the male genital tract was transmitted to female partners with a frequency of 40%.[13] The mechanism that results in infertility through infection is not clear. It is assumed that bacterial infections of the genital tract, in particular with C. trachomatis, may stimulate the immune system, perhaps via vasoepididymitis with unilateral obstruction or exposure of the spermatozoa to immunologically competent cells in inflammatory conditions.[14],[15],[16],[17] This infection may cause occlusion in the canalicular system of male genital tract, may damage the epithelial cells involved in the spermatogenesis.[18] This might be the cause in the present observation where 40% (n=6) of these cases had a low sperm count and among which 3 had also low semen volume. Of the 9 infected cases 3 (33.3%) had impaired sperm motility suggesting a probable association with the infertility of the couple. Among the infected cases the average pus cell count was higher than the uninfected cases. Cs ccachomat\s has a direct effect on sperm cells which may also cause infertility.[19] It has the ability to attack the sperm membrane which has an important role in the transportation of the infected agent into the upper genital system leading to infertility.[20] Spermatozoa with adhering chlamydia have been demonstrated in the peritoneal fluid of women with salpingitis at laparoscopy.[21] The observation in rest of the males was in agreement with studies that demonstrated no relationship of chlamydia antibody and semen quality.[22],[23],[24],[25] They remained asymptomatic and were at risk of transmitting the infection. Hence it is important to screen and treat such asymptomatic cases with obligatory partner therapy in order to prevent sexually transmitted disease leading to infertility.

Acknowledgement

We are thankful to Dr. H.S. Juneja, the Director of our Institute for introducing this subject of research with constant encouragement. We also acknowledge the support and guidance of Dr. U.M. Donde, Head of the Department of Immunodiagnostics to carry out the study.