Genital Infection in Males with Idiopathic Infertility

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1 Genital Infection in Males with Idiopathic Infertility Hisham A. Mosli, MBBCh, FRCSC, FACS; Faten S.B. Gazzaz, MBBCh, MSc; Hasan M.A. Farsi, MBBCh, FRCSC, FACS; Hassan S.O. AbdulJabar, MD, frcsc From the Departments of Urology (Drs. Mosli, Farsi), Virology (Dr. Gazzaz) and Gynecology (Dr. Abduljabar), King Abdulaziz University Hospital, Jeddah. Address reprint requests and correspondence to Dr. Mosli: Consultant and Associate Professor of Urology, Department of Urology, King Abdulaziz University Hospital, P.O. Box 6615, Jeddah 21452, Saudi Arabia. Accepted for publication 2 July Received 24 January We investigated a group of male patients with idiopathic infertility to determine the presence of genital infection and to identify the pattern of this infection using a specially designed protocol. A prospective study was carried out on 63 patients and 23 controls. We cultured the first voided urine, semen and swabs taken from the anterior urethra of these patients and controls for bacteria, chlamydia, Ureaplasma urealyticum and Mycoplasma hominis. Two techniques were used for chlamydial isolation and identification. These involved the use of chlamydial culture on McCoy cells with culture confirmation test and the direct fluorescent identification of Chlamydia trachomatis. The all-liquid media (MYCOFAST ALL-IN) kits were used for the identification of mycoplasma. Our results indicate that there is a significantly higher incidence of genital infection among male patients with idiopathic infertility than in normal fertile controls (P=0.0004). Extensive microbial investigations are indicated when genital infection is suspected to be the cause of the infertile state or cannot be ruled out as a possible cause in case of idiopathic infertility. Ann Saudi Med 1996;16(1): HA Mosli, FSB Gazzaz, HMA Farsi, HSO AbdulJabar, Genital Infection in Males with Idiopathic Infertility. 1996; 16(1): More information about the role of infection in male infertility is needed. This information could be utilized in constructing the plan for the investigations and subsequent disease-specific successful treatment. 1-3 There is enough evidence to believe that infection can alter semen quality, 1-4,20,21 leading to azoospermia, oligospermia, asthenospermia or oligoasthenospermia, pyospermia, increased volume of seminal plasma, a change in ph towards the alkaline side and hemospermia. 4,8 The possible underlying mechanisms of infection are epididymovasal ductal obstruction secondary to postinflammatory edema and fibrosis, epididymal dysfunction, and prostatic and seminal vesicular dysfunction. The direct toxic effect of the infection process affects sperm maturation, transport, motility and the sperm function as a whole. 4,6,8,20,21 When a male patient presents with infertility associated with such abnormal semen parameters and a history of urethritis, then urogenital infection is highly suspected, especially in the absence of other medical and surgical causes of infertility, such as testicular failure and varicocele. Symptoms of prostatitis and burning on ejaculation are also important. Yet the organisms can be present in the genital tract of many persons who have no obvious problem or complaints. 2,12 On physical examination, one would focus on detecting any pathological conditions of the urethra, prostate (and seminal vesicles) and epididymis. The investigation of the patient with infertility for urogenital infection should target several organisms which are commonly implicated in inducing infertility via the above-mentioned mechanisms. 3 These are chronic gonorrhea, nonspecific nongonococcal bacteria (both gram positives and gram negatives), Mycoplasma hominis, 1,6,9 Ureaplasma urealyticum and Chlamydia trachomatis. 1-3,6,9,20 Each of these requires a special method of isolation and identification. A special method for specimen collection and transport is also required. The aim of this current prospective study is to explore the role and identify the pattern of urogenital infection in a selected cohort of men with idiopathic infertility. Materials and Methods Sixty-three patients were selected for this study from others seen in the infertility clinic. The criteria for selection were the presence of abnormal semen parameters and the exclusion of possible causes of infertility other than urogenital infection. Therefore, these 63 were patients presenting with idiopathic infertility and azoo-, oligo-, astheno- or oligoasthenospermia with or without pyospermia. Complete histories and physical examinations were done and the data were recorded on a special protocol sheet designed for this study. All patients admitted had normal testicular volume and consistency, absence of varicocele (clinically and by scrotal ultrasound) and normal

2 serum androgens and gonadotropins. Patients with positive antisperm antibodies were excluded from the study. The following investigations were then carried out for each patient: urinalysis, urine bacterial culture and sensitivity, urine mycoplasmal culture and sensitivity, semen analysis, semen bacterial culture and sensitivity, semen mycoplasmal culture and sensitivity, urethral swabs for bacteriological, chlamydial and mycoplasmal studies. These investigations proceeded in the following manner: 1) Urinalysis: A detailed urinalysis was performed on all the patients using the automated analyzer machine, the sample spun at 5000 rpm for 10 minutes and then the deposit was examined for casts, pus cells, ova, trichomonas and yeast cells. Gram's stain was done on those samples showing evidence of bacteria. 2) Urine bacteriological culture: The first voided samples were cultured on CLED (cystine-lactose-electrolyte deficient agar plate) medium and more identification was performed as required, using the A.P.I, kits (Analytab Products Inc., Plainview, N.Y.). 3) Urine culture for Ureaplasma urealyticum and Mycoplasma hominis (U.u. and M.h.) using the Mycofast All-in kits (international Mycofast, B.P 705, Toulon cedex 9, France ). This all-liquid media kit is used to detect, identify and enumerate these organisms and test resistance to three different antibiotics, namely: minocin (minocycline), tetracycline and ciprofloxacin, within 24 hours. The technique depends on a shift in the ph of the liquid media towards the alkaline side with subsequent change in the color of the media. The colonies were identified as color-changing units (CCUs/mL). A7 and A8 agar cultures are the classically accepted methods for isolation of genital mycoplasma, but several reports validated the sensitivity and specificity of the Mycofast kits with two added advantages: lower rate of contamination and much faster results ) Semen analysis: The specimen was obtained in the laboratory bathroom by hand masturbation. After ejaculation, the samples were placed in the incubator at 37 C for approximately 10 to 20 minutes to allow for liquefaction. Semen analysis was done by the standard method. The diagnosis of abnormal semen quality was confirmed by performing three analyses on three different occasions one month from each other, each after three days of abstinence. Once the patient was admitted to the study, a full seminal analysis was performed by the fully automated analyzer machine (Hamilton-Thron) ) Semen bacterial culture: The procedure was explained to each patient and each was instructed to wash his genitalia before giving the specimen. All of our patients are of the Moslem religion and therefore all are circumcised. A loopful each of the samples was cultured on blood agar, McConkey, gonococcal (GC) selective medium and Saboraud plates. 6) Semen culture for U.u. and M.h.: This was performed by using the Mycofast All-in kits. Again, it is worth mentioning here that all of the study subjects were circumcised Moslem men. Genital mycoplasmas can be found under the foreskin of uncircumcised men. 6 7) Urethral swabs: Four specimens were taken to study chlamydial (two techniques used), bacterial and mycoplasmal presence. The swabs were taken in the Andrology Clinic by one of the two urologists participating in the study (HAM and HMAF). The fine sterile cotton swab was introduced into the anterior urethra for 1 to 2 cm under no anesthesia and this was repeated twice under strict sterile conditions. The first urethral swab was mounted on a preautoclaved slide and used for the direct detection of the chlamydial extracellular elementary 15,16 and reticulate 17 bodies by the immunofluorescent technique utilizing the Syva MicroTrak (Syva Co., Palo Alto, Calif.) direct immunofluorescence test The same swab was placed in a transport media and sent for chlamydial culture. McCoy cells were used for culturing Chlamydia trachomatis. Fluorescein-labeled monoclonal antibody for C. trachomatis was used for culture confirmation (Microtrak culture confirmation reagent, Syva Co., Palo Alto, Calif.). 15 The second urethral swab was detached from the wooden part and cut in two halves using sterile scissors. Each half of the swab was placed in a transport media and sent for bacterial and mycoplasmal culture respectively. The specimen was inoculated on CLED, blood and chocolate agar for the bacteriological studies and Saboraud medium to detect fungi. The same Mycoplasma All-in kits were used to culture Ureaplasma urealyticum and Mycoplasma hominis. Twenty-three normal males with recent parity volunteered to contribute to this study. They underwent the same set of investigations mentioned above. The statistical analysis was done using the chi-squared method. Results The average age of the patients was 34 years and the average duration of infertility was eight years. Forty-eight patients had primary infertility and 15 patients had secondary infertility. Patients were classified according to semen quality into four groups: oligospermics, 24 patients; asthenospermics, 22 patients; pyospermics, 13 patients; and azoospermics, four patients. In general, there were 40 infertile subjects with documented infection in one or more of the tests done. This constitutes 64% of the total patients studied (Table 1). Table 2 shows the total number of organisms identified in relation to specimen source. The number of infections in specimens obtained from urethral swabs is higher than the number of infections per patient because we could isolate more than one organism from the same patient (mixed colonization or infection). Ureaplasma urealyticum

3 was isolated from 29 patients out of the 40 infected (72.5%), Mycoplasma hominis from 11 patients (27.5%), chlamydia from 10 (25%) and bacteria from nine patients only (22.5%). Urethral swabs yielded the highest isolation rate. Table 3 shows the difference in numbers of the total microbial agents isolated from patients and controls. In anterior urethral swabs, a microbial agent was isolated in 45 specimens from 40 patients and only once from controls, P value = , which is significant. For mycoplasma alone (Ureaplasma urealyticum and Mycoplasma hominis) it was isolated in 28 specimens from patients and was isolated from two controls, P value = In this population, mycoplasma may be of importance in male infertility as a frequent cause of genital infection, as it was the most frequently isolated organism and it may deserve further investigation. Chlamydia was isolated from 10 patients using the cell culture technique and in seven of those it was also detected using the direct immunofluorescence test (DIF, Table 2). Chlamydia was isolated from the anterior urethra in one fertile control subject only, P value = , insignificant. The same insignificant result is detected from male swabs to detect bacteriological growth. The bacteriology obtained in this study and the site of isolation of each bacterial species is presented in Table 4. Bacteria was grown in only nine patients, P value = We were unable to grow bacteria in urine specimens on repeated occasions, even when we could isolate bacteria from a patient's urethral swab or semen. On no occasion in any of the 258 cultures (3 sites: urine, semen and swab for 63 patients and 23 controls, i.e., 3x86=258) were we able to isolate staphylococci or Escherichia coli. Table 4 illustrates the microbiology obtained from urine specimens. The highest yield was for Ureaplasma urealyticum. Since chlamydia are primarily obligatory intracellular organisms 19 and since the bladder cells are not targeted in this study, contrary to others, 10 no attempts were made to isolate chlamydia from the urine, but attention was given instead to studying chlamydia on the anterior urethral swabs. 11 Discussion In the absence of historic or clinical evidence of genital tract infection, there are little diagnostic or therapeutic rewards from extensive microbial investigations or by blind administration of antimicrobial agents in male infertility. 3 On the other hand, the role of infection in male infertility is of enormous practical importance to the clinician, since many of the infections of the male genital tract can be cured with antimicrobial therapy with subsequent improvement in the fertility state if the offending organisms were known. 3,9,20 Therefore, the extensive investigations for genital tract infection are strongly indicated in a selected group of patients when this infection is suspected and if a disease-specific therapy is to be given. Table 1. Relation of infection to male patients and controls. Patients (63) Controls (23) N % N % Infected* Not infected* Total Chi square (Yates)= 12.55; P=0.0004; *=significant. Table 2. Relation of different organisms to male specimen sites. Urethral swabs Urine Semen Pts Ctrls Pts Ctrls Pts Ctrls Bacteria Mycoplasma Ureaplasma C. Trachoma Culture 10 1 N/A N/A N/A N/A DIF 7 0 N/A N/A N/A N/A N/A=not applicable; DIF=direct immunofluorescence test. The importance of Ureaplasma urealyticum in genital infection and its relationship to infertility has been stressed before. 1-3,20 Treatment of these specific types of microorganisms resulted in improvement in semen quality. 20 The results of treatment of the patients with genital infection in this series will be covered in an upcoming

4 report. Our data confirms the high association between mycoplasma and idiopathic infertility in the male patient, although there are reports in the literature which dispute this association. 13,14 De Louvois et al. 13 found no significant difference in the frequency of mycoplasma in fertile and infertile couples. However, their criteria for admitting the infertile patient to the study were not clear. Also, they stated that "even if doxycycline does improve infertility, it may have a beneficial effect other than the eradication of mycoplasma." 13 To our minds the disease-treatmentoutcome relationship is clear. If other known possible endocrinological and surgical causes of infertility are excluded, mycoplasmal genital infection will be identified and documented and then successfully eradicated by adequate treatment. If the patients (in statistically acceptable numbers) have reverted to the normal fertile capability, then it will be very hard to dispute this relationship. The work of Fowler and Mariano 4,5 suggested that subclinical E. coli infections of the male reproductive tract are not commonly associated with infertility, and that expressed prostatic secretions and seminal fluid are more susceptible to contamination by urethral bacteria than urine, and that this phenomenon, rather than infection of the prostate, was the cause of culture results suggesting prostatic infection. We did not include prostatic expressate cultures in this study and took the utmost of precautions in interpreting the data of cultures of the seminal fluid. As was the case with the above-mentioned work, we could not isolate E. coli in any of the specimens studied. We recommend taking a urethral swab under sterile conditions and culturing it for bacteria when there is bacterial growth in the seminal fluid as a confirmatory test to check for contamination. Table 3. The number of positive specimens for different organisms isolated from urethral swabs and urine in patients and controls. Urethral swabs Urine Organism isolated Pts Ctrls Pts Ctrls Bacteria Mycoplasma Ureaplasma Chlamydia Total Table 4. Relation of different kinds of bacteria and the site of isolation. Name of bacteria No. of patients Site Hemophilusparainfluenza 1 Urethral swab Streptococci group F 1 Swab & semen Streptococci group B 3 Swab & semen Streptococci group D 1 Swab & semen Strept., B-hemolytic 2 Urethral swab Klebsiella pneumonia 1 Semen Our data show that in this particular population, genitourinary infection deserves thorough investigation in male patients with idiopathic infertility in order to provide disease-specific treatment. We conclude that there is a statistically significant difference between the occurrence of genital infection in males with idiopathic infertility and normal fertile controls. There was no control subject infected or colonized with multiple agents (mixed infection) in any of the sites studied. The two microbial agents isolated from the control subjects were Ureaplasma urealyticum in three subjects (in urethral swab and seminal plasma) and Chlamydia trachomatis in the other (in urethral swab by cell culture technique). We notice that the male anterior urethral swab yielded the highest rate of isolation of organisms and is the best specimen site to detect any kind of infection in the male genital tract. We notice that the highest frequency of infection with Mycoplasma hominis is in the urethral swabs and that no control circumcised fertile subject harbored it in any of the specimens; therefore, when it is isolated it is more likely to be pathogenic in our population. We conclude that in none of the cases studied has there been a documented isolation of bacteria from the urine of infertile patients and that unless there was a definite history of urinary tract infection (UTI) or symptoms of UTI, a urine culture and sensitivity is useless in the investigation of male infertility. Mycoplasma was the most prevalent disease encountered in this series of infertile men, followed by chlamydia, and bacteria was the least prevalent.

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