Chlamydia trachomatis and chlamydial heat shock protein 60-specific antibody and cell-mediated responses predict tubal factor infertility

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1 Human Reproduction Page 1 of 6 Hum. Reprod. Advance Access published February 14, 2006 doi: /humrep/del014 Chlamydia trachomatis and chlamydial heat shock protein 60-specific antibody and cell-mediated responses predict tubal factor infertility A.Tiitinen 1, H.-M.Surcel 2, M.Halttunen 1, S.Birkelund 3, A.Bloigu 2, G.Christiansen 3, P.Koskela 2, S.G.Morrison 4, R.P.Morrison 4 and J.Paavonen 1,5 1 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, 2 National Public Health Institute, Oulu, Finland, 3 Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark and 4 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 5 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, PO Box 140, HUS, Helsinki, Finland. aila.tiitinen@hus.fi BACKGROUND: To evaluate the role of Chlamydia trachomatis-induced humoral and cell-mediated immune (CMI) responses in predicting tubal factor infertility (TFI). METHODS: Blood samples were taken from 88 women with TFI and 163 control women. C. trachomatis and chlamydial heat shock protein 60 (CHSP60)-specific immunoglobulin G (IgG) antibodies were analysed using enzyme-linked immunosorbent assay (ELISA) kits. Proliferative reactivity of peripheral blood mononuclear cells was studied in vitro against Chlamydia elementary body (EB) and recombinant CHSP60 antigens. RESULTS: C. trachomatis-specific IgG antibodies were found more frequently (43.2 versus 13.5%), and the antibody levels were higher in the TFI cases than in the controls (P < 0.001). C. trachomatis EB-induced lymphocyte responses were positive in 81.8% of the TFI cases and 58.9% of the controls (P < 0.001). Similarly, CHSP60- induced lymphocyte responses were found in 45.5% of the TFI cases and 30.7% of the controls (P < 0.001). CHSP60 antibody test was the best single test predicting TFI. Compared to cases with all four markers negative, the estimated risk for TFI was 4.1 (95% CI ) among those with one positive marker and 19.9 (95% CI ) among those with three to four positive markers. CONCLUSION: Our results show that TFI prediction model can be improved by combining tests for humoral and CMI response to chlamydial antigens. Key words: C. trachomatis/immune response to Chlamydia/tubal factor infertility Introduction Subfertility is a major health problem requiring extensive and costly evaluation and management. It would be helpful to have simple screening tests that could reliably predict those positive with tubal factor infertility (TFI) to simplify the work-up. After an association between TFI and past Chlamydia trachomatis infection was discovered (Punnonen et al., 1979), many studies have confirmed the impact of C. trachomatis infection on human reproductive health (Paavonen and Eggert-Kruse, 1999) and focused on the role of Chlamydia antibody test (CAT) in the infertility work-up (Land et al., 1998; Gijsen et al., 2002). Although serological screening for past C. trachomatis infection lacks sensitivity and accuracy (Mol et al., 1997), it may be useful as a screening test for probable tubal damage among infertile women and may facilitate further management (Thomas et al., 2000; Akande et al., 2003). Sensitivity may be higher with CAT tests using chlamydial lipopolysaccharide (LPS) or other broadly reacting antigens (Land and Evers, 2002), although false-positive results because of cross-reactions with other Chlamydia species are difficult to rule out (Mannion et al., 1991; Gijsen et al., 2001; Jones et al., 2003; Land et al., 2003). This is especially true for chlamydial heat shock protein 60 (CHSP60), which is a highly conserved protein sharing antigenic epitopes not only between Chlamydia species but also between other bacteria and eucaryotic cells. Development of C. trachomatis peptide-based enzyme-linked immunosorbent assay (ELISA) tests has improved specificity and established the role of serology in screening for chlamydial TFI among subfertile women (Minassian and Wu, 1992; Verkooyen et al., 2002; Land et al., 2003). Predictive value may be improved by increasing the threshold levels for positive results (Land et al., 1998; Thomas et al., 2000; Akande et al., 2003) or by combining serological results with transvaginal ultrasound findings (Dabekausen et al., 1994; Logan et al., 2003; Hubacher et al., 2004). However, the overall accuracy of serology in predicting TFI remains far from optimal. The development of TFI may be due to enhanced immune response to C. trachomatis (Witkin et al., 2000; Debattista The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 1 For Permissions, please journals.permissions@oxfordjournals.org

2 A.Tiitinen et al. et al., 2003), especially to chlamydial CHSP60. CHSP60 antibodies seem to perform well in predicting TFI (Toye et al., 1993; Claman et al., 1997; Peeling et al., 1997; Ault et al., 1998; Persson et al., 1999; LaVerda et al., 2000; Bax et al., 2004; den Hartog et al., 2005). Cell-mediated immune (CMI) response to CHSP60 can be demonstrated in women with pelvic inflammatory disease (PID) or TFI (Witkin et al., 1994; Kinnunen et al., 2003). Thus, immunopathogenesis of TFI also involves cell-mediated mechanisms (Witkin et al., 2000; Debattista et al., 2003), and CHSP60 seems to be the key antigen in this process (Kinnunen et al., 2002). The purpose of the current study was to determine whether combining markers for C. trachomatis-specific humoral and CMI responses would better predict TFI. Subjects and methods Study population The study population consisted of 88 women (mean ± SD, 34.6 ± 7.8 years) with proven TFI attending the IVF Unit, Department of Obstetrics and Gynecology, Helsinki University Hospital. Clinical evaluation consisted of hormonal evaluation, vaginal ultrasound examination and semen analysis. Tubal patency was tested during laparoscopy. Patients with endometriosis were excluded. Ultrasound and laparoscopy findings of the cases are summarized in Table I. The control group consisted of 163 women (mean ± SD, 33.6 ± 4.0 years), of whom 34 were partners of couples with male factor infertility, 37 had other female infertility factors (endometriosis, anovulation and unexplained infertility) and 92 were healthy female blood donors. All infertility patients had laparoscopy performed during the infertility evaluation. Heparinized blood samples were obtained from all subjects and transported to the laboratory at room temperature. Plasma was separated and stored at 20 C for antibody analysis. The study protocol was approved by the Ethics Committee of the Helsinki University Hospital. Table I. Ultrasound (US) or laparoscopy (LAP) findings among 88 women with tubal factor infertility Tubal pathology n Sactosalpinx (US) None 52 (59.1) Unilateral 15 (17.0) Bilateral 12 (13.6) Sactosalpinx (LAP) a None 26 (29.5) Unilateral 19 (21.6) Bilateral 33 (11.4) Site of tubal occlusion (LAP) b None 8 (9.1) Proximal 5 (5.7) Distal 58 (65.9) Both 4 (4.5) Adhesions present (LAP) c None 8 (9.1) Minimal 15 (17.0) Moderate 32 (36.4) Severe 26 (30.7) a No data in 10 cases. b No data in 13 cases. c No data in seven cases. 2 Antibody analysis C. trachomatis and CHSP60-specific immunoglobulin G (IgG) plasma antibodies were analysed by ELISA kits (Medac Diagnostika, Hamburg, Germany). The peptide-based C. trachomatis ELISA kit is considered as species specific with a minimal cross-reactivity with Chlamydia pneumoniae antibodies (Land et al., 2003). High structural homology between chlamydial and other HSP60 antigens may cause cross-reactivity, which has an impact on the anti-chsp60 antibody results. Specific IgG antibodies in the plasma were attached to C. trachomatis peptide or recombinant CHSP60 protein bound to polystyrene surface of the ELISA plates and detected using horse-radish peroxidase-conjugated anti-human IgG and tetramethylbenzidine substrate. Results were obtained as a mean absorbance of duplicated samples at 450 nm. Less than 10% variation was seen in doublets (optical density [OD] > 0.2). Threshold for a positive antibody level (mean OD value of the negative control (0.350) was OD > 0.4. Lymphocyte proliferation assay Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Cells were washed three times with Hanks balanced salt solution (Sigma, St Louis, MO, USA) and suspended in Roswell Park Memorial Institute 1640 medium (RPMI, Sigma) containing 10% heat-inactivated human AB plasma (Finnish Red Cross, Helsinki, Finland) for the PBMC proliferative assay. PBMC reactivity was studied in vitro by culture stimulation in roundbottomed 96-well plates ( cells/well) with or without antigen in a total volume of 200 μl. C. trachomatis elementary body (EB; serovar E and F, total protein concentration 3 μg/ml) and recombinant CHSP60 (2.5 μg/ml) (LaVerda et al., 2000) were used as the lymphocytestimulating antigens. To evaluate an antigen-specific character of lymphocyte responses, C. pneumoniae EB (3 μg/ml) was included as the control Chlamydia antigen. Optimum concentrations of antigens were determined in preliminary experiments as the minimum concentrations giving the maximal PBMC proliferation. The cultures were incubated in a humidified 5% CO 2 atmosphere at 37 C for 6 days, and [methyl- 3 H]thymidine (0.2 μci/well; Amersham Life Science, Buckinghamshire, UK) was added to the cultures for the last 18 h of incubation (Surcel et al., 1993; Kinnunen et al., 2003). The PBMC proliferative responses were measured as counts per minute (cpm) of incorporated [methyl- 3 H]thymidine with a liquid scintillation counter (Wallac, Turku, Finland), and the results were expressed as stimulation indices (SI = mean cpm in the presence of antigen divided by mean cpm in its absence) for triplicate cultures. SI > 5 was considered a positive response to chlamydial EB and SI > 2.5 was considered a positive response to CHSP60 antigen. The viability and reactivity of the cultured lymphocytes were controlled in each experiment by requiring SI > 10 in response to pokeweed mitogen (PWM, Gibco, Paisley, UK; 12.5 μg/ml). Statistical analysis Chi-squared test was used to compare categorical variables between the study groups. Continuous variables were compared by Mann Whitney U-test. Spearman s correlation coefficient was used for an analysis of correlation. Odds ratios (OR) with 95% confidence interval (CI) were estimated by logistic regression. The statistical analyses were performed with SPSS for Windows 11.5 software (SPSS, Chicago, IL, USA). Results Humoral immune response C. trachomatis-specific IgG antibodies (CTA) were more common (43.2 versus 13.5%, P > 0.001), and the antibody levels

3 C. trachomatis-specific immune response in TFI 3,0 A 125 B 2, ,0 75 OD 1,5 SI 1,0 50,5 25 0,0 N pos % Controls Prox/None Distal anti-ctr anti-chsp60 Figure 1. Chlamydia trachomatis (CTR) (grey box) and CHSP60 (clear box)-specific antibodies (A) and lymphocyte proliferation responses (B) in control women and in women with TFI and either proximal/no occlusion or distal occlusion. were higher in the TFI cases than in the controls (median OD 0.284; interquartile range [IQR] versus 0.135; , P < 0.001) (Figure 1A). CHSP60 antibodies were also more common (59.1 versus 22.1%, P < 0.001), and the antibody levels were higher in the TFI cases than in the controls (median 0.561; IQR versus 0.157; , P < 0.001). CTA and CHSP60 antibody levels correlated both among cases (R = 0.616, P < 0.001) and controls (R = 0.532, P < 0.001). Altogether, 28 (31.8%) of the TFI cases and 118 (72.4%) of the controls were negative by both tests (P = 0.001). Of the 28 TFI cases, 19 (67.9%) had moderateto-strong pelvic adhesions and 17 (60.1%) had distal tubal occlusion. We then evaluated the relationship of C. trachomatis antibody prevalence and the severity of tubal damage. Distal tubal occlusion (53 cases were available for anti-chsp60 analysis) was associated with higher CHSP60 antibody levels compared to the cases (16 cases) with proximal or no visible occlusion (median OD 0.62; IQR versus 0.28; , P = 0.053). On the other hand, CTA levels did not differ between the cases with distal versus proximal or no tubal occlusion (median 0.26; versus 0.19; , P = 0.754), although highest antibody levels (OD > 1.0) were found in cases with distal occlusion (Figure 1A). C. trachomatis-specific CMI response As shown in Figure 1B, PBMC proliferative responses to C. trachomatis EB antigens were significantly higher in the 0 N pos % Controls Prox/None Distal SI_E SI_CHSP TFI group than in the control group (median 17.0; IQR versus 6.5; , P < 0.001). PBMC response to C. trachomatis EB was slightly stronger than that to CHSP60 antigen (median 1.8; IQR versus 1.4; , P = 0.171) in both groups. PBMC responses varied remarkably, especially in cases with distal tubal occlusion (Figure 1B). Strongest proliferative responses against C. trachomatis EB antigen (SI > 90) were found in such cases. Positive responses to C. trachomatis EB antigen were found in 82% of the TFI cases and in 59% of the controls (P < 0.001). Positive PBMC responses to CHSP60 antigen were found in 46% of the cases and 31% of the controls (P < 0.05). A positive correlation was found between the PBMC responses to C. trachomatis EB and CHSP60 both in TFI cases (R = 0.613, P < 0.001) and in controls (R = 0.619, P < 0.001). Fourteen (16%) of the cases and 61 (37%) of the controls were negative for both antigens (P < 0.001). C. pneumoniae induced similar lymphocyte responses in TFI cases and controls (median 8.9; IQR versus 12.2; ). In the TFI cases, C. pneumoniae-induced responses were lower than C. trachomatisinduced responses, although the difference was not statistically significant. Predictive value of combined immune response markers Of the humoral and CMI response markers, no single marker differentiated those with distal tubal occlusion, bilateral sactosalpinx or moderate-to-strong adhesions (Figure 1). However, CHSP60 antibody test and PBMC proliferation test 3

4 A.Tiitinen et al. (sensitivity 81.8, specificity 41.4) to C. trachomatis EB picked up most cases with any tubal pathology. Table II summarizes the performance of each of the markers and combination of all four markers in the diagnosis of TFI. PBMC proliferative response to C. trachomatis EB was a highly sensitive (81.8%) marker, but the specificity was lower than that by CTA. Although CHSP60 antibody test was the best single test for TFI (area under curve 0.74; 95% CI , P < 0.001) (Figure 2), the specificity and the positive predictive value improved when all four immunologic markers were used. Comparing subjects with all the four markers negative, the estimated risk for TFI was 4.1 (95% CI ) among Table II. Performance of one or more immunological markers in the diagnosis of tubal factor infertility 4 Sensitivity Specificity PPV NPV Accuracy Humoral immunity CHSP60 antibody C. trachomatis antibody Cell-mediated immunity CHSP C. trachomatis EB Combination All four positive PPV, positive predictive value; NPV, negative predictive value; CHSP60, chlamydial heat shock protein 60. Sensitivity 1,0,8,5,3 0,0 0,0 1 2,3 1 - Specificity Figure 2. Comparison of the area under receiver operating characteristic curves for Chlamydia trachomatis and CHSP60-specific immune markers in the prediction of TFI (1, C. trachomatis (CTA) (immunoglobulin G antibodies); 2, anti-chsp60; 3, lymphocyte proliferation response to C. trachomatis EB; 4, lymphocyte proliferation response to CHSP60).,5, ,0 Table III. Rates of immune response markers in tubal factor infertility (TFI) cases, in cases with other infertility factors and in cases with male factor infertility TFI (n = 88) Male factor (n = 34) All negative One positive Two positive Three to four positive Other causes (n = 37) those with one positive marker, 5.3 (95% CI ) among those with two positive markers and 19.9 (95% CI ) among those with three or four positive markers with any test combination. To test the validity of combining the tests, we compared the prevalence of positive markers of past C. trachomatis infection in 88 laparoscopically verified TFI cases, in 37 cases with other infertility factors and in 34 cases with male factor infertility. As summarized in Table III, the discriminatory value of the four negative markers was excellent and differentiated TFI from other causes of infertility. Three or four positive markers were found in 41 (46.6%) cases, 26 of whom (63.4%) had distal occlusion and four (9.8%) had proximal or no occlusion. Three or four positive markers were found in 27 (63%) of 43 cases with moderate-to-strong tubal adhesions and in six (14%) of 43 cases with thin adhesions. Discussion Our results show that the TFI prediction model which has been based on an antibody analysis (Dabekausen et al., 1994; Claman et al., 1997; Mol et al., 1997; Land et al., 1998; Gijsen et al., 2002; Akande et al., 2003; den Hartog et al., 2004) can be improved by combining tests for humoral and CMI response to C. trachomatis. The presence of CHSP60 antibodies seemed to best predict TFI, but the CMI response to C. trachomatis EB antigen picked up more cases with TFI. When testing the impact of combined analysis of humoral and cellular responses, we found that the presence of three to four positive test results from any test combination increased the risk for TFI 20-fold. This was clearly more than by combining several serological markers to different Chlamydia species (den Hartog et al., 2004) or chlamydial antigens such as LPS or CHSP60 (Claman et al., 1997; Persson et al., 1999; LaVerda et al., 2000). Our favourable results may be explained by the clinical setting and by the fact that we did not use CTA test results as selection criteria for the study. However, using serological and cell-mediated tests in clinical practice may not be easy. Furthermore, tests for cell-mediated responses are not commercially available. Low sensitivity of CTA in the diagnosis of TFI (Mol et al., 1997) cannot be explained by poor technique only. Rather, it emphasizes the fact that the host response to Chlamydia infection varies more than appreciated (Witkin et al., 2000). Lack of antibodies is not a definitive marker of negative history for C. trachomatis infection (LaVerda et al., 2000).

5 C. trachomatis-specific immune response in TFI Although many TFI cases are because of past C. trachomatis infection (Punnonen et al., 1979; Paavonen and Eggert-Kruse, 1999), the multifactor aetiology of TFI (Kodaman et al., 2004) is often ignored when evaluating the validity of CTA or other markers of past C. trachomatis infection in TFI. In this study, only 46.6% of TFI cases were found with three of four markers of C. trachomatis infection, suggesting that no more than 50% of TFI is caused by C. trachomatis infection. Also, not all women with C. trachomatis infection and positive CTA develop tubal damage (van Valkengoed et al., 2004). This reduces the predictive value when a more sensitive test is used. C. trachomatis is an obligate intracellular bacterium which induces antigen-specific CMI response (Brunham et al., 1981; Mabey et al., 1991). Diagnostic tests for CMI response have not been introduced into subfertility work-up so far. We have already demonstrated in our earlier studies PBMC proliferation against recalled chlamydial antigen, indicating species-specific immune activation in a species-specific manner (Surcel et al., 1993). Although cross-reactivity between Chlamydia species also seen in this study is not excluded, we found positive PBMC proliferation as an analogous test to whole EB antigenbased antibody assays and therefore took this test as being valid in studying the role of Chlamydia in chronic diseases (Surcel et al., 1993; Halme et al., 1997; Kinnunen et al., 2002). Antibody responses generally overlap with PBMC responses but do not necessarily correlate (Brunham et al., 1981; Mabey et al., 1991), suggesting that C. trachomatis-induced immune responses vary between individuals. In this study, 16% of the TFI cases had serological markers only, whereas 30% had no serum antibodies although the PBMC proliferative responses were positive. Thus, measuring only one arm of the immune response may not always reveal past C. trachomatis infection. The severity of tubal damage correlates with C. trachomatis antibody levels resulting from microimmunofluorescence assay (Thomas et al., 2000; Akande et al., 2003). Similarly, we showed high prevalence and levels of CHSP60 antibodies in women with distal tubal occlusion, which is in line with earlier observations. The fact that antibody levels correlate with the severity of damage might facilitate individual decisions on management of women with subfertility. In our study, CTA prevalence rates and difference between cases and controls were comparable with earlier reports where serological analysis to chlamydial antigens has been used in screening for TFI (Mol et al., 1997). Distal tubal occlusion was associated with increased antibody levels to C. trachomatis, which is in agreement with Land et al. (1998) who suggested that a higher threshold level should be used to improve specificity of CTA test. However, CHSP60 is considered a key antigen in the immunopathogenesis of TFI (Kinnunen et al., 2002, 2003). Immune response to CHSP60 reflects chronic C. trachomatis infection better than that to C. trachomatis EB or major outer membrane protein (MOMP) antigen (Witkin et al., 1994, 2000). Accordingly, anti-chsp60 test showed higher positive likelihood ratio than any other single marker of C. trachomatis infection in our study, confirming the role of CHSP60-induced immune responses as an indicator of TFI. Diagnosis and management of TFI is important in clinical practice. Accurate tests are needed. Laparoscopy is the gold standard for detecting tubal occlusion, pelvic adhesions or endometriosis. However, laparoscopy is costly, is invasive and carries risk for complications. Hysterosalpingosonography (HSSG) is less costly and less risky but performs poorly in the diagnosis of peritubal adhesions. Furthermore, false-positive results because of tubal spasm or false-negative results are not uncommon. Medical history alone is highly unreliable in the evaluation of women with TFI (Hubacher et al., 2004). Costeffectiveness analyses suggest that the diagnostic work-up to detect tubal pathology should start with CAT and then proceed to HSSG, providing that the antibody levels are indicative for C. trachomatis infection (Mol et al., 2001). However, patients with positive immune response markers are likely to show pelvic pathology justifying laparoscopy (Khalaf, 2003). In conclusion, we have shown that prediction of TFI can be improved by combining tests for humoral and CMI response to C. trachomatis. We are now implementing these results into a busy clinical practice. 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