Chlamydia trachomatis infection in women with secondary infertility

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1 Chlamydia trachomatis infection in women with secondary infertility Abida Malik, M.D., a Suchitra Jain, M.D., a Meher Rizvi, M.D., a Indu Shukla, M.D., a and Seema Hakim, M.D. b a Department of Microbiology, and b Department of Obstetrics and Gynaecology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India Objective: To assess the role of Chlamydia in secondary infertility in a prospective study. Design: Forty women with secondary infertility and 30 healthy term pregnant women of similar age composition were studied for past and present Chlamydia trachomatis infection. Setting: Women attending the outpatient Department of Obstetrics and Gynaecology with complaint of secondary infertility were enrolled as patients in the study. Patient(s): Forty women with secondary infertility formed the study group, and 30 healthy women served as the controls. Intervention(s): Chlamydia IgG was detected by ELISA; titers of 1:320 or more were considered positive. Endocervical swabs were collected for culture on cycloheximide-treated McCoy cell lines, and ELISA was used to detect Chlamydia antigen. Hysterosalpingography was performed to assess tubal patency. Main Outcome Measure(s): A difference was expected between the prevalence of C. trachomatis infection in the infertile study subjects and fertile control group. Result(s): Immunoglobulin G antibodies were present in 22 (55%) women with secondary infertility, whereas positivity was seen among 2 (5.5%) controls. Tubal occlusion occurred in 16 (63.6%) cases positive for chlamydial antibody. Sensitivity of chlamydial IgG antibody as a diagnostic marker for infertility was 72.7%, and specificity was 44.4%. The majority of Chlamydia IgG antibody positive cases, 17 (77.2%), were symptomatic. Unfavorable obstetric history was found in 16 (72.7%) cases. Active infection was found in 12 (30%) cases with one (3.3%) case of current infection occurring in the controls. Conclusion(s): Prevalence of past chlamydial infection is strongly statistically significant in women with secondary infertility. Current infection was also found statistically significantly in these women. Immunoglobulin G antibody detection is an effective and noninvasive tool for detection of Chlamydia and a more viable option than HSG in a developing country such as India. Screening of women with secondary infertility for C. trachomatis is strongly recommended to allow early therapeutic interventions. (Fertil Steril Ò 2009;91:91 5. Ó2009 by American Society for Reproductive Medicine.) Key Words: Chlamydia trachomatis, secondary infertility, IgG antibodies, Chlamydia antigen, culture Chlamydia trachomatis infections are one of the most prevalent sexually transmitted pathogens throughout the world (1). C. trachomatis is a common cause of urethritis, pelvic inflammatory disease (PID), ectopic pregnancy, and tubal factor infertility. The sharp worldwide increase in the incidence of PID during the last two to three decades has led to the secondary epidemics of primary and secondary tubal factor infertility (2). Chlamydial PID is the most important preventable cause of infertility and adverse pregnancy outcomes. Studies pertaining to infertility in general and secondary infertility in particular are extremely limited in India. The present study was carried out to evaluate chlamydial infection in women with secondary infertility in North India. Received December 24, 2006; revised May 29, 2007; accepted May 31, Presented at the 12th International Congress of Infectious Diseases, Lisbon, Portugal, June 15 18, Reprint requests: Abida Malik, M.D., Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Medical Road, Aligarh, Uttar Pradesh , India ( abidamalik2k@ yahoo.com). Past infection was investigated by detection of IgG antibodies by ELISA, and present infection was assessed by a twopronged approach: isolation of C. trachomatis on McCoy cell line and antigen detection by ELISA. Thus we attempted to assess the role of both past and present Chlamydia infection in causation of secondary infertility. MATERIALS AND METHODS The study group was recruited from consecutive women of reproductive age complaining of secondary infertility who came to the obstetrics and gynecology outpatient department of Jawaharlal Nehru Medical College, Aligarh, India. They had normal results on ovulatory tests, normal Mantoux test results, normal chest x-ray examination results, no specific findings on endometrial biopsy, and husbands who had normal results on a semenogram. Because tuberculosis is endemic in India this criterion was used to rule out tuberculosis as a possible cause of infertility. Thirty healthy term pregnant women of similar age composition and free of all signs and symptoms attending the /09/$36.00 Fertility and Sterility â Vol. 91, No. 1, January doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 antenatal clinic constituted the control group. The study was conducted for a period of 1 year from May 2003 to June 2004 in the Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh. A written informed consent was obtained from each participant. Secondary infertility is defined as inability of a couple to conceive after a year or two of unprotected and appropriately timed intercourse when one or both partners have previously conceived children. Detailed history and clinical features were recorded, and all relevant investigations were performed. Hysterosalpingography (HSG) was done in all cases subsequent to enrollment. If the patient had symptoms, that is, had evidence of PID or mucopurulent discharge or had tender adnexa she was given antibiotic treatment, and HSG was performed only after the symptoms had abated. Tubal infertility was said to be present if bilateral tubal occlusion with or without hydrosalpinx was seen on HSG. A total of 96 women were seen with secondary infertility at the obstetrics and gynecology outpatient department during the study period. After screening, 40 women were recruited for the study. Twenty-five (26%) women were excluded because of evidence of tubercular infection, and 17 (17.7%) were not considered because of history of antibiotic treatment in the last 2 months. Nine (9.3%) women were excluded because of abnormal results on ovulatory function tests, and in 5 (5.2%) cases their husbands had abnormal semenogram findings. The study group thus comprised 40 patients with secondary infertility after excluding the cases mentioned above. They were further categorized on the basis of whether they presented with symptoms suggestive of urogenital tract infection (30 patients) or had no symptoms (10 cases). The term bad obstetrics history was used when the woman had a history of recurrent miscarriages, habitual abortion, recurrent spontaneous abortions, or repetitive pregnancy loss. Determination of IgG Antibodies to C. Trachomatis Serum samples were collected in clean dry vials and stored at 20 C. The IgG antibodies to C. trachomatis were detected by VIR-ELISA Antichlamydia IgG Kit (VIR Immune Labor Diagnostica, Oberursel, Germany). According to the manufacturer s documentation, titers of more than 1:320 were considered significant. Specimen Collection for Cell Culture and Antigen Detection The endocervix was first cleaned with a sterile cotton swab to remove mucus and exudate, after which endocervical specimens were collected in triplicate from all the women. For cell culture Two Dacron swabs on a plastic shaft were used. The samples were collected by inserting the swab into the cervical canal up to a depth of 1 to 2 cm close to the endocervix and rotated through an angle of 15 to 30 to collect the specimen from the squamocolumnar junction. Care was taken to prevent the swab from touching the vaginal mucosa. They were transported to the laboratory immediately in 2-sucrose-phosphate (2SP) transport medium. The swabs were processed and cultured immediately. For antigen detection Chlamydia swab collection kit (Bio- Rad Laboratories, Hercules, CA) was used for sample collection. One small Dacron swab on a stainless steel/plastic shaft for collecting the specimen and one tube containing phosphate-buffered saline solution for transporting the specimen to the laboratory were provided with the kit. Specimens, after collection, were stored at 20 C till use. Tissue culture technique McCoy cell line was used for the isolation of C. trachomatis obtained from the National Centre for Cell Culture and Science, Pune, India. The cell line was maintained in the laboratory according to standard technique (3). C. trachomatis was cultured on cycloheximide-treated McCoy cell lines as follows. A 1-mL suspension of 10 5 McCoy cells per milliliter of growth medium was seeded in Leighton tubes containing coverslips. The tubes were incubated at 37 C in a stationary position for 2 to 3 days for adequate growth to appear, after which minimum essential medium (MEM, Mumbai, India) was aspirated from the vials and 0.1 ml from each 2SP specimen extract was inoculated into two tubes, one for iodine staining and one for Giemsa staining (Bio-lab, Bangalore, India). Tubes were centrifuged at 2,500g to 3,000g for 1 hour. After addition of 1 ml of MEM containing 1 mg/ ml cycloheximide, the tubes were incubated at 37 C for 48 to 72 hours. Inclusion bodies were detected by Giemsa and iodine staining (3, 4) The inclusion-forming units were graded from 1 to 4 according to the number of inclusions seen. The grading was as follows: in grade 1, 5 to 9 inclusion-forming units per coverslip; in grade 2, 10 to 20 inclusion-forming units per coverslip; in grade 3, 1 to 10 inclusion-forming units per high-power field, and in grade 4, >10 inclusion-forming units per high-power field (5). Antigen detection C. trachomatis antigen was detected by ELISA (BioRad). Chlamydia Microplate EIA is a monoclonal antibody test based on qualitative enzyme immunoassay for the direct detection of chlamydial organisms in adult urogenital specimens. The samples were processed weekly. The same kit was used throughout the period of study. The Chlamydia Microplate EIA was performed and interpreted as per the manufacturer s instructions. Ethical clearance The study was approved by the Institutional Ethics Committee of the Jawaharlal Nehru Medical College. A written informed consent was obtained from each patient. Statistical Methods The data were analyzed with use of statistical software SPSS for Windows, version 11.0 (SPSS, Inc., Chicago, IL). The c 2 92 Malik et al. Chlamydia infection in secondary infertility Vol. 91, No. 1, January 2009

3 test, Fisher s exact test, and McNemar s c 2 test were used for significance. RESULTS The mean age of the 40 women with secondary infertility enrolled in this study was years, and the mean age of the control group was years. Among the infertile cases, 16 (40%) were in the 26- to 30-year age group, followed by 10 (25%) in the 31- to 35-year group and 7 in the 36- to 40- year group. Of the 7 remaining women, one was in the 18- to 20-year bracket and 6 were in the 21- to 25-year age group. In the control group, 14 (46.66%) were in the 26- to 30-year bracket, 6 (20%) were in the 36- to 40-year age group, and 3(10%) were in the 21- to 25-year age group. Twenty-nine (72.5%) women in the study group had one live issue, 7 (17.5%) had two live issues, and 4 (10%) had no live issues. Eighteen (45%) of these women had a bad obstetrics history. Five (27.7%) had a history of three abortions in the first trimester, and 8 (44.4%) had a history of two abortions in the second trimester. Four women (22.2%) had a history of one abortion each in both the first and second trimester. In three (16.6%) cases there was a history of unexplained intrauterine death. Seven (17.5%) had a history of miscarriages, and 3 (7.5%) had a history of intrauterine death. Immunoglobulin G antibodies were present in 22 (55%; 95% confidence interval ) women with secondary infertility, whereas 2 (6.7%; 95% confidence interval women in the control group had positive results for IgG antibodies, P<.001. Among these 22 cases, in 16 (72.7%) there was evidence by HSG of tubal occlusion. Considering tubal occlusion as a good predictor of infertility, sensitivity of chlamydial IgG antibodies as a diagnostic marker for infertility was 72.7% and specificity was 77.7% (Table 1). Ten (45.4%) women among the 22 IgG-positive cases also had evidence of active infection (Table 2).Taking culture of Chlamydia as gold standard, sensitivity of ELISA for antigen detection was 80%, specificity was 90%, positive predictive value was 72.7%, and negative predictive value was 93.1%. Past chlamydial infection occurred most often in the 26- to 35-year age groups, 17 (77%), as did current infection, 7 (70%) cases, followed by 3 (13.6%) cases of past infection occurring in women of 36 years of age or more and 2 (9%) cases in the 18- to 25-year age group. Two (20%) and 1 (10%) cases of current infection also occurred in these respective age groups. The solitary case in the control group that was positive for Chlamydia IgG antibodies also belonged to this age group. The duration of infertility in a majority, 16 (72.7%), cases positive for C. trachomatis was 4 to 6 years. The majority of women with symptoms, 17 (77.2%), with past chlamydial infection had significantly raised IgG levels, which appears to indicate a correlation between raised IgG titers and development of overt symptoms. Presenting symptoms of women with secondary infertility in relation to Chlamydia status are given in Table 3. Pelvic inflammatory disease and mucopurulent discharge were the most common presentations, P<.001. Bad obstetrics history was present in 16 (94.1%) of these cases. Seven (43.7%) had a history of recurrent abortions in the first trimester, 6 (37.5%) had a history of two abortions in the second trimester, and 3 (18.75%) had a history of intrauterine death. Among the 18 IgG antibody negative cases, 3 (16.6%) patients had active infection (Table 2). Hysterosalpingography results were positive in 4 (22.2%) of these 18 cases but not in those with active infection. DISCUSSION Secondary infertility is becoming an increasingly significant health problem in many countries of the world including India (6). The increase appears to coincide with the growing role played by C. trachomatis as a sexually transmitted disease (2). In our study in women with secondary infertility, past C. trachomatis infection was found in 55% and current infection was observed in 45.4% of cases (P<.001), a rate that is quite high. A surprisingly high 72.7% of the patients with positive results for IgG antibodies had tubal infertility, P<.001. This finding suggests that in a resource-poor country such as India ELISA for chlamydial IgG antibodies can easily be substituted for HSG for detection of tubal occlusion. Other studies have reported similar results (7, 8). Dabekausen et al. reported that C. trachomatis antibody testing is more accurate than HSG in predicting tubal factor infertility (9). Our serologic data show a higher prevalence of chlamydial antibodies in secondary infertility as compared with the control, P<.001, which is consistent with other reports (10). The incidence of C. trachomatis infection appears to be more common in the secondarily infertile women than was found in studies in women with primary infertility (11). Other TABLE 1 Comparison of Chlamydia IgG antibodies and HSG as markers of tubal occlusion. Chlamydia Chlamydia IgG-positive IgG-negative cases cases Total HSG-positive 16 (80) 4 (20) 20 cases, n (%) HSG-negative 6 (30) 14 (70) 20 cases, n (%) Total (n) Note: Sensitivity ¼ 72.7%, positive predictive value ¼ 80%; specificity ¼ 77.7%, negative predictive value ¼ 70%. Fertility and Sterility â 93

4 TABLE 2 Detection of active C. trachomatis infection in patients with IgG antibodies. IgG status Total patients with active infection, n (%) Both cell culture and antigen, n (%) Chlamydia detected by Cell culture alone, n(%) Antigen alone, n (%) IgG-positive cases in study group, n ¼ 22 (55%) IgG-negative cases in study group, n ¼ 18 (45%) 10 (45.4) 7 (70) 2 (20) 1 (10) 3 (16.6) 1 (33.3) 1 (33.3) 1 (33.3) studies report similar association of secondary infertility and C. trachomatis infection (12, 13). This increased susceptibility could be due to the longer period of active sexual life in secondarily infertile women, thus enhancing their risk of exposure to chlamydial infection. A significant number of these women had PID and bad obstetric history pointing to prior exposure to Chlamydia. Chlamydia has been implicated in causing bad obstetric history in other studies too (14, 15). Prevalence of current infection in a significant number of women with evidence of past chlamydial infection, P<.01, implies that recurrent infections occur often. Such repeated infections rather than solitary episodes of chlamydial infection appear to lead to salpingitis causing tubal occlusion and subsequently secondary infertility. Such a relationship has also been mentioned in another study (16). Our study suggests that women with secondary infertility should be screened for both past and present C. trachomatis infection because the likelihood of Chlamydia being the incriminating factor appears to increase with evidence of recurrent infection. We conclude that IgG antibody detection by ELISA for past infection is an effective, rapid, noninvasive, and indirect tool for detection of tubal obstruction. A similar approach has been suggested by other studies also (8, 9). It appears to be a more viable option in a developing country such as India for predicting tubal infertility where facilities for performing HSG may not be available in many areas. In the absence of requisite infrastructure and skills for culture and for direct fluorescent assay, ELISA for antigen detection can play a significant role in screening for current C. trachomatis infection in secondarily infertile women because the results of both are comparable and should be used for the workup of these patients. Chlamydial infection should also be investigated in women with bad obstetrics history because appropriate treatment will help the woman to have a successful pregnancy. There does appear to be a minor shortcoming in the Chlamydia antigen and antibody detection methods, wherein three separate cases of active infection were missed by both tests, but if used together they supplement one another, missing only one patient. TABLE 3 Clinical profile of women with symptoms with secondary infertility in relation to Chlamydia positivity. Clinical presentation No. of symptomatic cases (n [ 35) H/o Chlamydia exposure (or infection) (n [ 17) No h/o Chlamydia exposure (or infection) (n [ 18) Bleeding per vaginum 2 (5.7) 2 (11.7) 0 Mucopurulent discharge 3 (48.5) 1 (5.8) 2 (11.1) PID 17 (48.5) 15 (88.2) 2 (11.1) a Bad obstetrics history 18 (51.4) 16 (94) 2 (11.1) a Chronic cervicitis 1 (2.8) 1 (5.8) 0 Burning micturition 3 (8.5) 1 (5.8) 2 (11.1) Menorrhagia 1 (2.8) 0 1 (5.5) Note: H/o ¼ History of. Figures in parenthesis indicate percentage. a P<.001 compared with history of Chlamydia exposure. 94 Malik et al. Chlamydia infection in secondary infertility Vol. 91, No. 1, January 2009

5 We maintain that emphasis should be laid on detection of both IgG antibodies and Chlamydia antigen rather than treating all women with secondary infertility in toto or all patients with positive results for IgG. In a world where rational use of antibiotics is gaining ever-increasing momentum, treatment should ideally be begun subsequent to testing for Chlamydia infection. However, in resource-poor countries treatment of IgG-positive patients may be considered without resorting to detection of Chlamydia antigen. In situations where performance of either of these tests is not possible because of a lack of facilities as in rural India, treatment of all women with secondary infertility appears to be the answer. Where facilities are available we suggest that patients who are antibody positive but negative for Chlamydia antigen by ELISA should be referred to a higher referral center for treatment. All chlamydial antigen positive patients irrespective of antibody status should be treated with the appropriate antibiotics and asked to try to conceive over a period of 6 months. If this fails they too should be referred to a higher center where necessary therapeutic interventions (tubal surgery or IVF) could be initiated. Furthermore, we suggest that, as the results of both are comparable, ELISA for IgG antibodies should be preferred to HSG because ELISA is noninvasive and causes minimal inconvenience to the patients. If C. trachomatis IgG antibody test results are negative then recourse can be taken to not HSG but laparoscopy, which is more sensitive. We conclude that ELISA for antigen and antibody detection for chlamydial infection is easy to perform, cost-effective, and not labor intensive thus allowing early therapeutic intervention to be instituted enabling the patient to conceive naturally. However, the relative merits of the strategies clearly depend on the prevalence of infection in the given population and the relative cost of antibiotics versus laboratory tests. Studies with a larger sample size will further elucidate the extent of the problem of secondary infertility caused by C. trachomatis in India. REFERENCES 1. Joyee AG, Thyagarajan SP, Sowmya B, Venkatesan C, Ganapathy M. Need for specific & routine strategy for the diagnosis of genital chlamydial infection among patients with sexually transmitted diseases in India. Indian J Med Res 2003;118: Paavonen J, Eggert-Kruse W. Chlamydia trachomatis: impact on human reproduction. Hum Reprod Update 1999;5: Ripa KT, Mardh PA. Cultivation of Chlamydia trachomatis in cycloximide treated McCoy cells. J Clin Microbiol 1977;6: Thomas BJ, Evans RT, Hutchison GR, Taylor Robinson D. Early detection of chlamydia inclusions combining the use of cycloheximide-treated McCoy cells and immunofluorescence staining. J Clin Microbiol 1977;6: Reichart CA, Gaydos CA, Bredy WE, Quinn TC, Hook EW. Evaluation of Abbott Testpack Chlamydia for detection of chlamydia trachomatis in patients attending Sexually Transmitted Diseases Clinics. Sex Transm Dis 1990;17: Nachtigall R. International disparities in access to infertility services. Fertil Steril 2006;85: Veenemans LM, van der Linden PJ. The value of chlamydia trachomatis antibody testing in predicting tubal factor infertility. Hum Reprod 2002;17: Perquin DA, Beersma MF, de Craen AJ, Helmerhorst FM. The value of chlamydia trachomatis specific IgG antibody testing and hysterosalpingography for predicting tubal pathology and occurrence of pregnancy. Fertil Steril 2007;88: Dabekausen YA, Evers JL, Land JA, Stals FS. Chlamydia trachomatis antibody testing is more accurate than hysterosalpingography in predicting tubal factor infertility. Fertil Steril 1994;61: Kosseim M, Brunham RC. Fallopian tube obstruction as a sequelae to chlamydia trachomatis infection. Eur J Clin Microbiol 1986;5: Malik A, Jain S, Hakim S, Shukla I, Rizvi M. Chlamydia trachomatis infection in females and infertility. Indian J Med Res 2006;123: Shi XB, Liu FY, Zhang HW. Study of Chlamydia trachomatis infection on cervical secretion of women with early pregnancy and secondary infertility. Hunan Yi Ke Da Xue Xue Bao 2001;26: Cengiz L, Kiyan M, Cengiz AT, Aksoy AM, Kara F, Seekin L, et al. Chlamydia trachomatis antigens in endocervical samples and serum IgG antibodies in sterile infertile women using ELISA. Mikrobiyol Bul 1992;26: Sharma K, Aggarwal A, Arora U. Seroprevalence of Chlamydia trachomatis in women with bad obstetric history and infertility. Indian J Med Sci 2002;56: Gogate A, Deodhar LP, Shah PK, Vaidya P. Detection of Chlamydia trachomatis antigen & Toxoplasma gondii (IgM) & Mycoplasma hominis (IgG) antibodies by ELISA in women with bad obstetric history. Indian J Med Res 1994;100: Westrom L. Influence of sexually transmitted diseases on sterility and ectopic pregnancy. Acta Eur Fertil 1985;16:21 4. Fertility and Sterility â 95