1 Department of Clinical Microbiology and 2 Gynecology and Obstetrics, Malmö University Hospital, S-205 02 Malmö, Sweden, 3 Department of Medical Microbiology and Immunology, University of Aarhus, Denmark and 4 Division of Medical and Biochemical Microbiology, Forschungszentrum, Borstel, Germany
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Abstract |
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Key words: Chlamydia trachomatis/Chlamydia pneumoniae/hsp60/hsp10/infertility
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Introduction |
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Tubal occlusion after pelvic inflammatory disease is reminiscent of the situation in trachoma where relapsing or chronic chlamydial infection results in tarsal scarring. Animal studies have been helpful to uncover the pathogenic processes which are likely to be involved. An extract of Chlamydia after treatment by the detergent, Triton X-100, produced a follicular response in monkeys previously immunized by a serovar B of C. trachomatis but had no effect in immunologically naive animals (Taylor et al., 1987). Triton extracts from either C. trachomatis or Chlamydia psittaci were active while chlamydial lipopolysaccharide or major outer membrane preparations from C. trachomatis were not.
Similar scarring reactions in the conjunctivae were produced in guinea pigs by a Triton X-100 extract of C. trachomatis or of the GPIC strain of C. psittaci suggesting a genus-specific component (Watkins et al., 1986). When the primary infection was induced at another mucosal site delayed type hypersensitivity (DTH) reactions could still be elicited in the conjunctiva. Later it was shown that a 60 kDa heat shock protein similar to GroEL from E. coli was the active component (Morrison et al., 1989a
, b
). In a monkey model of genital chlamydial infection repeated inoculations produced tubal scarring probably due to a delayed hypersensitivity reaction (Patton et al., 1990
, 1994
).
Chlamydial infections then seem to be able to sensitize individuals for DTH-like reactions at various mucosal surfaces. The scarring reaction observed in animals was genus-related rather than species-specific. It is therefore conceivable that C. pneumoniae infections might prime an immune response in humans which may influence the development of tubal infertility in response to a subsequent genital infection by C. trachomatis.
Antibodies to heat shock proteins (hsp) have been shown to be more common in infertile women compared to different control groups (Wager et al., 1990; Brunham et al., 1992
; Toye et al., 1993
; Arno et al., 1995
; Dieterle and Wollenhaupt, 1996
; Eckert et al., 1997
; Claman et al., 1997
). Chlamydial hsp antibodies are also more common in patients with pelvic inflammatory disease or in cases with ectopic pregnancies (Peeling et al., 1997
; Eckert et al., 1997
; Sziller et al., 1998
). Some of the studies have demonstrated that the presence of hsp60 antibodies is a predictor of tubal occlusion independent of other chlamydial antibodies.
We (Osser et al., 1989) have previously confirmed the association between C. trachomatis antibodies and tubal factor infertility reported by several different groups. In this study we re-examined the material with regard to the antibody response to the hsp60 and hsp10 of C. trachomatis. The correlation between such antibodies and species-specific antibodies to structural antigens of C. trachomatis and C. pneumoniae was analysed to be able to detect any joint effect between these two infections with regard to hsp antibodies. Antibodies to the lipopolysaccharide chlamydial antigen, which is shared by the different species, were also analysed and the association to hsp antibodies examined.
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Materials and methods |
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Chlamydial antibodies by micro-immunofluorescence (MIF)
The sera have been examined for antibodies to C. trachomatis previously. At that time C. pneumoniae had not been recognized as a common human pathogen. The sera were therefore re-tested for antibodies to different species of Chlamydia. The micro-immunofluorescence test according to Wang (Wang and Grayston, 1970) was used and performed in the following way.
Prototype strains were grown in yolk sacs of embryonated hens' eggs. Suspensions of Chlamydia from infected yolk sacs were treated with 0.1% formalin in PBS and then used as antigens. All antigens were titrated using specific monoclonal antibodies to the different species of Chlamydia to obtain optimal reactivity. The C. pneumoniae, IOL-207 strain, and C. psittaci, 6BC strain, were used. A pool of C. trachomatis serovars D through K was also included. One dot of each antigen was placed in a cluster on microscopic slides. Each slide had 12 such antigen clusters in two rows. Serum of different dilutions was placed on the antigen clusters. End-point titrations of the sera were performed. Geometric mean titres (GMT) were calculated for positive sera. Cut-off titres for positive sera were 1:64 for both C. trachomatis and C. pneumoniae.
Antibodies to the chlamydial lipopolysaccharide antigen
Antibodies to the lipopolysaccharide (LPS) antigen of Chlamydia were measured by a commercial kit (Medac, Hamburg, Germany). The test employs a recombinant antigen of the chlamydial LPS in an enzyme-linked immunosorbent assay (ELISA) format. IgG and IgA antibodies were measured. Titres were calculated according to the manufacturer's suggestions. Positive titres for IgG were >150 and for IgA >75.
Antibodies to heat shock proteins 60 and 10
The C. trachomatis genes for GroEL (Hsp60) and GroES (Hsp10) were cloned into the expression vector pGEX3X, as previously described (Cerrone et al., 1991; Larsen et al., 1994
). The heat shock protein was expressed as a fusion protein with glutathione-S-transferase and affinity-purified on glutathione-sepharose columns. Likewise glutathione-S-transferase itself was also purified and used as control protein.
An ELISA test was developed where StarWell MaxiSorp® 96-well plastic plates (Nunc, Roskilde, Denmark) were coated with the hsp fusion proteins or the control protein. An appropriate dilution of the hsp60 was determined in preliminary tests using a monoclonal antibody (Sigma, St Louis, MO, USA, H-3524 that cross-reacts with hsp60 from both procaryotic and eucaryotic cells). Human sera diluted 1/100 were tested in microtitre plates coated with approximately 0.2 µg of the fusion hsp60 protein. An anti-human-IgG serum conjugated with alkaline phosphatase was used together with a sodium p-nitrophenylphosphate substrate to detect human antibodies. The optical densities of the plates were measured in a spectrophotometer at 405 nm. ELISA for hsp10 was performed in a similar way. A positive serum for hsp60 and another for hsp10 were included in all test runs and used as reference sera. The positive control serum was assigned a relative titre of 100. Negative control sera were also included in each run. The titre was calculated as the difference between the ODs for a serum between positive and negative antigens in relation to that of the reference positive serum with a titre of 100. Sera with a titre value of >10 were considered antibody positive and sera with titre values of <10 were considered negative.
Statistical methods
Student's t-test was used to compare group mean values, but for some variables, where severe skewness could not be eliminated even after transformation, the MannWhitney rank sum test was used. The 2 test was used to compare proportions, and Spearman's rank correlation coefficient was used to investigate correlations among variables. Logistic regression analysis was used to identify associations between tubal factor infertility and different chlamydial antibodies.
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Results |
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As the LPS-IgA antibody response is generally considered more transient than that of LPS-IgG the ratio between IgA and IgG antibodies to chlamydial LPS was calculated. In the patient group this ratio was 0.91 and in the controls it was 0.96, not statistically different (2 test). The GMT for LPS-IgG among patients who had such antibodies was 1142 and 645 among controls, which was a statistically significant difference (P < 0.001, Student's t-test). The corresponding LPS-IgA mean titres in patients and controls were 205 and 172 respectively, a difference not statistically significant.
Antibodies to chlamydial hsp60 and hsp10
The prevalence of antibodies to chlamydial hsp60 and hsp10 were higher in patients than in controls and with higher titres in patients. The individual association of C. trachomatis and C. pneumoniae antibodies to hsp antibodies was analysed by a logistic regression model. The model included MIF antibodies to C. trachomatis and C. pneumoniae and IgG and IgA antibodies to the chlamydial LPS. The only factor that independently predicted the presence of antibodies to hsp60 or hsp10 was MIF IgG antibodies to C. trachomatis. This was the case in both patients and controls. These results are summarized in Table II.
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The correlations between titres of hsp60 and the various antibodies to Chlamydia were also examined. Antibodies to hsp60 were correlated to C. trachomatis in patients (rs = 0.46, P < 0.001) and in controls (rs = 0.52, P < 0.001) but not to antibodies to C. pneumoniae (rs = 0.17/0.18, P = 0.03/0.02) for patients and controls (Table III). The correlation between antibodies to hsp60 and chlamydial LPS antibodies was as good (rs = 0.430.54, P < 0.001) as the correlation between hsp60 and C. trachomatis antibodies (Table III
).
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Predictors of tubal factor infertility
The prevalence of hsp antibodies and MIF antibodies as well as LPS IgG and IgA antibodies was higher in patients with tubal factor infertility than in fertile control women. The frequency of antibodies to C. pneumoniae did not differ between these groups. The individual association between the different antibodies and tubal factor infertility was tested in a logistic regression model where patients and controls were grouped together. Only hsp60 antibodies and MIF antibodies to C. trachomatis came out as independent predictors of tubal factor infertility while LPS IgG and IgA did not. The odds ratio for hsp60 was 3.54 (CI 1.925.86) and for C. trachomatis antibodies 1.36 (CI 1.101.69).
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Discussion |
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The results of our study, however, tend to argue against a synergistic effect of C. pneumoniae and C. trachomatis infections for the development of tubal damage causing infertility. Had such an effect existed a higher frequency of C. pneumoniae antibodies would have been expected among the patients with tubal infertility as prior C. pneumoniae infection would have been a risk factor for tubal damage at subsequent C. trachomatis infection.
The relation between antibodies to C. pneumoniae and the antibody response to hsp60 was also examined. The hsp60 has been associated with tarsal scarring in animal models of trachoma and antibodies to the hsp60 have been found more often in patients with tubal factor infertility, ectopic pregnancy and pelvic inflammatory disease than in control women (Wager et al., 1990; Brunham et al., 1992
; Toye et al., 1993
; Arno et al., 1995
; Dieterle and Wollenhaupt, 1996
; Claman et al., 1997
; Peeling et al., 1997
; Eckert et al., 1997
; Sziller et al., 1998
). Our results are in agreement with these previous studies as we detected a higher prevalence and a higher mean titre of antibodies to hsp60 in patients than in controls. The relation between hsp60 antibodies and MIF and LPS antibodies was examined in a logistic regression model. Only MIF antibodies to C. trachomatis were associated with hsp 60 antibodies in patients and controls while antibodies to C. pneumoniae and chlamydial LPS were not. This association suggests that infection by C. trachomatis but not by C. pneumoniae is important for the hsp60 antibody response. We also examined how the different antibodies predicted tubal factor infertility. Only MIF antibodies to C. trachomatis and hsp60 antibodies independently predicted tubal factor infertility where hsp60 was the stronger predictor. This was true even when only individuals who were antibody positive for C. trachomatis were included in the analysis. These findings agree with what have been reported by others as well (Wager et al., 1990
; Toye et al., 1993
; Claman et al., 1997
; Peeling et al., 1997
).
In most studies MIF antibodies to C. trachomatis have been measured in addition to hsp60 antibodies. In this study we also tested for IgG and IgA antibodies to the common chlamydial LPS. Both C. trachomatis and C. pneumoniae can elicit such antibodies. LPS antibodies were more prevalent among patients than controls and correlated with antibodies to hsp60 and C. trachomatis but not with antibodies to C. pneumoniae. An association between LPS antibodies and antibody reactivity to hsp60 has been reported in one other study (Domeika et al., 1998). Thus the LPS antibody response in our patients seems to be mainly due to C. trachomatis.
Tubal occlusion might occur after a long-standing chronic Chlamydia infection of the Fallopian tubes or after relapsing or repeated infections, as would seem to be the case in advanced trachoma. Weström reported, however, that the severity of acute salpingitis as noted at laparoscopic examination could predict the fertility outcome (Weström, 1980). Tubal damage with fertility implications may therefore occur during the acute phase of chlamydial salpingitis whether or not chronic infection will follow. IgA antibodies are of shorter duration than IgG antibodies. This is the case particularly with LPS-IgA antibodies. The presence of such antibodies might therefore indicate continuing active infection. Although the prevalence of IgA antibodies to chlamydial LPS was higher in patients than in controls this was not the case after adjustment for different exposure rates in the two groups. Thus the ratios of IgA and IgG LPS antibodies were similar in patients and controls and with similar mean titres. These serological results did not suggest persistent infection.
C. trachomatis infection of the Fallopian tubes leading to tubal occlusion and infertility elicit a strong antibody response to several different chlamydial antigens. In agreement with similar studies we found that hsp60 and MIF antibodies to C. trachomatis independently predicted the presence of tubal factor infertility. Although animal models of trachoma have suggested that the immune response leading to scarring is a genus specific reactivity and not species specific we could find no evidence of C. pneumoniae being involved in the development of tubal factor infertility.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 17, 1998; accepted on April 21, 1999.