A total of 368 patients attending the gynecology out patient department, Safdarjung Hospital, New Delhi, India for gynecological complaints (cervical discharge, cervicitis and infertility) were enrolled in the study. The study received approval by the hospital's ethics review committee. Thirty-nine healthy age-matched controls attending the family-planning department for birth-control measures and with no previous history of any sexually transmitted disease (STD) were also enrolled. At recruitment, a detailed clinical questionnaire was administered to each patient for collecting information on reasons for referral, gynecology history including menstruation, symptoms of genital and urinary tract infection, obstetric and medical histories. Patients with positive urine pregnancy test, recent antibiotic therapy and history of recently treated sexually transmitted infection (STI) and genital tuberculosis were excluded from the study.

The vulva was examined for lesions and vaginal/cervical discharge. The cervix was inspected for ulcers, warts, ectopy, erythema, discharge or any other abnormalities. After cleaning the endocervix with a cotton swab (Hi Media, Mumbai, India), endocervical swabs were collected from patients for diagnosis of C. trachomatis and other STI pathogens.

The cervical canal was wiped clean, and a cytobrush was placed within the endocervical canal so that cells from the endocervical region and the zone between the endocervical and ectocervical regions (transformation zone) could be obtained. The cytobrush was then held in a sterile centrifuge tube containing phosphate buffered saline (PBS) (pH 7.2) supplemented with 100 U penicillin/ml, 100 μg streptomycin/ml, and 100 μg glutamine/ml. All cytobrush samples had negative results for blood contamination.

Spots were made on glass slides from cervical swab samples. These were stained with fluorescein isothiocyanate conjugated monoclonal antibodies to C. trachomatis major outer membrane protein (MOMP) using Chlamydia trachomatis Direct Specimen Test kit (Microtrak, Syva Corporation, Palo Alto, CA, USA) according to the manufacturer's instructions. A sample was considered to be positive when at least 10 elementary bodies were detected. Samples with greater than one and less than 10 EBs were confirmed for positivity by polymerase chain reaction (PCR) analysis using a primer specific for 200 base pair (bp) plasmid of C. trachomatis 28. Diagnosis for other STD pathogens were done by culture for Neisseria gonorrhoeae, Mycoplasma hominis, Ureaplasma urealyticum and by microscopy on gram stained smears for Candida sps., bacterial vaginosis, Trichomonas vaginalis as mentioned earlier 27.

Full-length cHSP60 gene (GenBank accession no. M58027) was amplified using unique primer set (Forward primer – 5'-TCCCcccgggATGGTCGCTAAAAACATTAAA-3' and Reverse primer 5'-ACGCgtcgacTTAATAGTCCATTCCTGCGCC-3' with restriction endonuclease sites, XmaI and SalI respectively at 5'-end). Oligonucleotides used as primers were synthesized by Microsynth, Balgach, Switzerland. PCR products were initially cloned into pGEM-T Easy vector (Promega, Madison, Wisconsin) and checked for their correct reading frame by sequencing. The cloned inserts were then excised by digestion with restriction enzymes XmaI and SalI (New England Biolabs) and ligated to the compatible sites of the expression vector pQE-60 (Qiagen Inc., Chatsworth, California) in frame with a His6 affinity tag-coding sequence at the 3' terminus. The resulting plasmids were introduced into Escherichia coli M15 cells and routinely grown in the presence of ampicillin (100 μg/ml) and kanamycin (25 μg/ml) (Sigma).

Cloning of cHSP10 and large-scale expression and purification of chlamydial recombinant proteins was performed as mentioned earlier 29. Proteins under native conditions were purified by nickel chelate affinity chromatography (Qiagen Ni-NTA Superflow resin). The protein concentration was determined with the Bradford assay (Sigma). We controlled for the non-specific effects of LPS contamination by treating recombinant proteins with polymyxin B (Sigma-Aldrich). Purified proteins were subsequently characterized by SDS-PAGE and immunoblotting using monoclonal antibody against chlamydia (Alexis Biochemicals, Lausen, Switzerland), separated into aliquots and frozen at -80°C.

Cervical specimens were vortexed before the removal of cytobrush. They were filtered through a 70-μm nylon cell strainer (Becton Dickinson, San Diego, CA, USA) and centrifuged at 200 g for 10 min and the cell pellet was resuspended in PBS. Mononuclear cells were separated by Ficoll-Paque density gradient centrifugation. The mononuclear cells were counted on haemocytometer and samples containing less than one million cells/ml were excluded. The mononuclear cells were washed three times with PBS and suspended in RPMI-1640 (Sigma-Aldrich) containing 5% fetal calf serum (FCS) (PAA, Austria). Briefly, endocervical mononuclear cells were cultured in round-bottomed 96-well plates (5 × 10⁴ cells/well) in a total volume of 200 μl and subsequently stimulated with cHSP60 (2 μg/ml) and cHSP10 (3 μg/ml) in triplicate. Phytoheamagglutinin (PHA 2 μg/ml) (Sigma) was used as a positive control mitogen in each experiment. Optimum concentrations of antigen and mitogen were determined in preliminary experiments as optimum concentrations giving maximal proliferation post stimulation.

Cervical washes of patients and controls were assayed for presence of IgG antibodies to cHSP60 and cHSP10. Briefly, the proteins were bound to the wells of a microtitre plate (1 μg/well) in carbonate buffer (14.2 mM Na₂CO₃, 34.9 mM NaHCO₃, 3.1 mM NaN₃, pH 9.5) and were incubated overnight at 4°C. After washing, wells were blocked with PBS-0.5% bovine serum albumin (BSA) at 37°C for 60 min; 100 μl of cervical washes were then added, and after incubation at 37°C for 120 min 100 μl of 1: 10 000 dilution of peroxidase-conjugated goat antibody to human IgG (Jackson Immunoresearch, Baltimore, MD, USA) was added to each well. After further incubation of 60 min at 37°C, the peroxidase substrate tetramethylene benzidine was added. The reaction was stopped with 0.5 M H₂SO₄ and the plates were read at 450 nm. Known positive and negative controls were always assayed in parallel to test samples. A positive sample was defined as one yielding an OD value that was at least 2 standard deviations (SD) above the mean value of known negative samples as mentioned earlier 30.

Quantification of IFN-γ, IL-10, TNF-α, IL-13 and IL-4 (ebiosciences, San Diego, CA, USA) in the supernatant of cervical mononuclear cell cultures of samples after 72 h stimulation with proteins was performed by commercially available ELISA kits, in accordance with the manufacturer's instructions. The minimum detectable cytokine concentrations for these assays were IFN-γ-(4 pg/ml), IL-10-(2 pg/ml), TNF-α-(4 pg/ml), IL-13-(4 pg/ml) and IL-4-(2 pg/ml).

Since the distributions in cytokine production were not normal, differences between two groups were evaluated using Mann-Whitney U test. A comparison between two groups was made only when the Kruskal-Wallis test yielded a statistically significant result. Categorical variables were compared using the χ² test. Correlation was tested with Spearman's correlation coefficient.

Cervical C. trachomatis infection was diagnosed by direct fluorescent assay (DFA)/PCR in 174 patients. Thirty-one of these patients were found to have bacterial vaginosis, or to be co-infected with either Candida sp., T. vaginalis, M. hominis, U. urealyticum or N. gonorrhoeae in the cervix and were thus excluded from the study. Ten Chlamydia positive patients were excluded, as the count of mononuclear cells in the cervical cells was less than 1 million cells/ml and epithelial cells were present. Based on diagnosis the women were divided into three groups. Group I (n = 39) comprised of uninfected healthy controls with no infertility problem; Group II (n = 63) comprised of Chlamydia positive women with no infertility problem; Group III (n = 70) comprised of Chlamydia positive women with infertility and who had laparoscopic or hysterosalpingographic evidence of tubal damage. Candidates were considered infertile if they had regular unprotected intercourse for at least 2 years without conception. The median ages of women in each group were comparable (Table 1).

ELISA results showed that the prevalence was significantly higher for IgG antibodies to both cHSP60 (P = 0.04 & P < 0.0001) and cHSP10 (P = 0.03 & P < 0.0001) in Group II and III respectively as compared to Group I. In Group III the prevalence was significantly higher for IgG antibodies to both cHSP60 and cHSP10 as compared to Group II (P = 0.002 & P = 0.04 respectively) (Table 1). Correlation between cHSP60 and cHSP10 specific IgG antibodies were significant (P = 0.04, r = 0.26 & P < 0.0001, r = 0.6) in both Group II and Group III respectively (Figure 1a &1b).

IFN-γ levels were significantly higher after stimulation with both cHSP60 and cHSP10 in Group II (P = 0.04 & 0.02 respectively) and in Group III (P < 0.0001) as compared to Group I. Significantly higher levels of IFN-γ were observed after stimulation with both cHSP60 (P = 0.006) and cHSP10 (P = 0.04) in Group III when compared with Group II (Figure 2a). Similarly when IL-10 levels were compared significant difference was observed after stimulation with both cHSP60 and cHSP10 between Group II (P = 0.03 & 0.04 respectively) and Group III (P = 0.0005 & 0.0007 respectively) as compared to Group I and IL-10 was significantly higher (P = 0.04) in Group III as compared to Group II after cHSP60 and cHSP10 stimulation (Figure 2b).

TNF-α levels were significantly higher after stimulation with cHSP60 and cHSP10 (P < 0.0001 & P = 0.02 respectively) in Group III as compared to Group I whereas there was no significant difference (P = 0.07 & 0.25 respectively) in Group II as compared to Group I. The TNF-α levels were significantly higher (P = 0.0008) in Group III when stimulated with cHSP60 as compared to Group II. The TNF-α levels were higher although not significant (P = 0.1) in Group III when stimulated with cHSP10 as compared to Group II (Figure 2c).

IL-13 levels were low (close to the minimum detectable cytokine concentrations) and no significant difference was observed after stimulation with both cHSP60 and cHSP10 in any group. There was no detectable IL-4 production in any culture supernatant after stimulation with both cHSP60 and cHSP10 (Data not shown).

IFN-γ and IL-10 levels after stimulation with both cHSP60 and cHSP10 were significantly correlated in Group III (P < 0.0001, r = 0.54 and P = 0.004, r = 0.33 respectively)

Median and range for cytokine concentration in all the groups after cHSP60 and cHSP10 stimulation are mentioned in Table 2.

Median and range for cytokine concentration after PHA stimulation were: IFN-γ [Group I: 22 (0–276), Group II: 87 (5–643), Group III: 120 (11–1049)]; IL-10: [Group I: 20 (0–366), Group II: 69 (7–823), Group III: 104 (5–669)]; TNF-α [Group I: 17 (0–114), Group II: 72 (0–445), Group III: 92 (0–382)]; IL-4 [Group I: 0 (0–2), Group II: 7 (0–25), Group III: 8 (0–18)]; IL-13 [Group I: 0.8 (0–5), Group II: 24 (0–86), Group III: 18 (0–63)].

There was high standard error for cytokines as one or two samples showed very high concentration of cytokines. However, these high values did not affect the median value as well as the significance of the results when data was analyzed excluding these values.