Ness, Roberta B. MD, MPH*; Soper, David E. MD†; Richter, Holly E. PhD, MD‡; Randall, Hugh MD§; Peipert, Jeffrey F. MD, MPH∥; Nelson, Deborah B. PhD¶; Schubeck, Diane MD#; McNeeley, S Gene MD**; Trout, Wayne MD††; Bass, Debra C. MS*; Hutchison, Katherine PhD*; Kip, Kevin PhD*; Brunham, Robert C. MD‡‡
LONG-TERM SEQUELAE FROM pelvic inflammatory disease (PID) include infertility, ectopic pregnancy, recurrent episodes of PID, and chronic pelvic pain.1–4 Among the microorganisms associated with PID, Chlamydia trachomatis has been the best studied and is the organism most likely causally linked to tubal infertility, based on retrospective epidemiologic studies, limited prospective data, and animal experiments. In numerous case-control studies, women with tubal infertility were substantially more likely than women without infertility to have serologic evidence of past chlamydia infections.5–7 In a prospective cohort study from Lund, Sweden, one-fifth of women with salpingitis experienced tubal infertility. Between 20% and 25% had further PID episodes and of those with 3 or more episodes of PID, over half subsequently became infertile.8–10 Experimentally, although single upper genital tract chlamydial infections in macaque monkeys are usually self-limiting, repeated episodes of chlamydial salpingitis often produce tubal scarring.11
A provocative hypothesis about how chlamydia induces post-PID morbidity is that immune reactions to the chlamydia heat shock protein 60 (Chsp60) may incite an autoimmune response and the chronic inflammation that manifests as infertility, recurrence, and the other outcomes12 or that antibody responses to Chsp60 may signal chronic persistent infection, which incites chronic inflammation that causes the adverse reproductive outcomes.13 However, limited human data support either of these hypotheses; no previous prospective analysis has evaluated antibody responses to Chsp60 and PID sequelae.
We assessed IgG antibodies to serovar D of C. trachomatis elementary bodies (EB), the extracellular form of chlamydia, and antibodies to Chsp60 among women with mild to moderate PID enrolled in the PID Evaluation and Clinical Health (PEACH) Study and followed over an average of 7 years. Our aim was to test whether high serologic titers were associated with the PID sequelae of lack of pregnancy and recurrent PID.
Materials and Methods
Methods of subject selection and recruitment, data collection, and follow-up for the PEACH Study have been described in detail elsewhere.14,15 Briefly, between March 1996 and February 1999, women aged 14 to 37 were recruited from emergency departments, clinics, and sexually transmitted disease units at 7 major (over 90% of enrollment) and 6 minor clinical sites in the eastern, southern, and central regions of the United States. Human subject use approval was obtained at each participating institution, and all participants provided informed consent. Eligibility was based on clinically generalizable criteria that included: (a) a history of pelvic discomfort for a period of 30 days or less, (b) findings of pelvic organ tenderness (uterine or adnexal) on bimanual examination, and (c) leukorrhea or mucopurulent cervicitis or both and/or untreated but documented gonococcal or chlamydial cervicitis. Leukorrhea was defined as white blood cells in excess of epithelial cells viewed microscopically and mucopurulent cervicitis was defined by the presence of grossly yellow/green exudate on a cervical swab.
Subjects were selected from 2941 women screened. Excluded were 346 (11.9%) women who did not meet the inclusion criteria. An additional 1080 (36.7%) women were excluded on the basis of a priori criteria, including 141 (4.8%) because of pregnancy; 246 (8.4%) who had taken antimicrobial agents in the preceding 7 days; 248 (8.4%) with a previous hysterectomy or bilateral salpingectomy; 51 (1.7%) with an abortion, delivery, or gynecologic surgery in the preceding 14 days; 191 (6.5%) with suspected tubo-ovarian abscess or other condition necessitating surgery; 163 (5.5%) with an allergy to the study medication; 29 (1.0%) who were homeless, and 11 (0.4%) who vomited after a trial of antiemetic treatment. There were 1515 women eligible for the study. Of these, 651 refused participation and 831 were enrolled and contacted at least once after randomization. Blood samples were a supplemental (elective) component of the initial protocol and so not collected on all women; however, in 443 women, we obtained blood samples from which we were able to measure chlamydia EB and Chsp60 antibodies. Women who did have chlamydia versus who did not have chlamydia EB and Chsp60 values were not statistically significantly different about age (P = 0.51), race (P = 0.74), education (P = 0.77), history of PID (P = 0.84), gonococcal/chlamydial cervicitis on baseline examination (P = 0.14), endometritis (0.72), or bacterial vaginosis (BV) by Gram stain (P = 0.89).
Participants were monitored with in-person visits at 5 and 30 days after treatment. Subsequent telephone follow-ups were conducted every 3 months during the first year after enrollment and then every 4 months until June 2004, at which point we were in contact with and obtained self-reported follow-up information for 69.1% of the cohort, representing a mean follow-up time of 84 months. Because inpatient and outpatient treatment were no different in terms of any of the outcomes measured, the 2 groups are combined into a single cohort in this analysis.15
Baseline data on demographic descriptors, gynecologic and reproductive history, lifestyle habits, and clinical aspects of the current illness were obtained by a standardized 20-minute interview conducted by study nurses at each center. Subsequent follow-ups elicited self-reported information about pelvic pain, pregnancy and births, signs/symptoms of PID, sexually transmitted infections, contraceptive use, pattern of sexual intercourse, and health care utilization.
Gynecologic examinations were conducted at 5 and 30 days and included tenderness assessment using the 36-point scale developed by McCormack et al.16, ascertainment of cervical swabs for N. gonorrhoeae cultures and C. trachomatis polymerase chain reaction detection, collection of vaginal swabs for Gram stain detection of BV, and aspiration of the endometrium for detection of gonorrhea and chlamydia. A central reference laboratory performed the polymerase chain reactions and Gram stains. Interpreted according to the Nugent criteria, a Gram stain score of 7 to 10 categorized BV.17
The primary outcomes included time-to-pregnancy and time-to-recurrent PID. Recurrent PID was self-reported and verified whenever medical records were available. As we previously reported, confirmation of recurrent PID was found in 76% of medical records that could be obtained and rates of PID by self-report and medical record review were similar.15 Ectopic pregnancy and chronic pelvic pain have been examined in other PEACH analyses; however, we do not report them here.
Bloods were collected whenever possible at baseline and also within the final 2 years of follow-up; however, in reality all but 53 women had a single measurement (n = 202 at baseline and n = 188 late in follow-up). Serologic testing for IgG antibodies to serovar D of C. trachomatis EB, as well as Chsp60 were conducted in the reference laboratory of one of the authors (RB) using a research enzyme-linked immunosorbent serologic assay (ELISA) technique. C. trachomatis serovar D was cultured in HeLa cells in Eagles MEM containing 10% fetal bovine serum and 2 mmol/L glutamine. EB preparations were purified by step gradient centrifugation using 40%, 44%, and 50% Renografin (Amershan Health). Purified EBs were collected from the interface between the 44% and 50% density gradient layers. The EB stock was titrated on HeLa cells and after heat inactivation was used to coat ELISA plate cells of the equivalent of 3 × 105 IFU per well. The Chsp60 gene was cloned from serovar D genomic DNA into pET Vector (NOVagen). The histidine tagged recombinant protein was expressed in Escherichia coli BL 21 and purified using Ni-NTA agarose column (QIAGEN, Valencia, CA). Flat-bottomed (96-well), polystyrene, microtiter plates (Corning Science Products, Corning, NY) were coated with 100 μL of recombinant Chsp60 (0.1 μg/well) diluted in 0.1 mol/L carbonate buffer (pH 9.6) in each well. Plates were incubated overnight at 4°C and then washed 3 times with phosphate-buffered saline (PBS) with 0.05% Tween 20. Nonspecific binding sites were blocked by adding 150 μL of 3% BSA in PBS to each well for 2 hours at 37°C. After 2 washings with PBS with Tween 20, 100 μL of human sera diluted to 1:250 in PBS with 0.5% BSA were added in duplicate and incubated at 4°C overnight. After 4 washings with PBS-Tween 20, 100 μL of 1:2000 diluted peroxidase conjugated-antihuman IgG secondary antibody was added and incubated for 2 hours at 37°C. After 4 washings with PBS-Tween 20, 100 μL of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate was added and allowed to develop at room temperature in the dark for between 5 minutes and 2 hours until the positive control wells on each plate reached the optical density value of 1.0 at OD405 on microplate reader. The Bio-Rad microplate reader model 680 (Bio-Rad Laboratories, Inc.; Hercules, CA) has a dedicated function to monitor the OD reading of the positive well, which was routinely incorporated on each plate. The positive well used 2 separate sera that were known to contain high titers of EB or Chsp60 antibodies, respectively. When the OD value of the positive well reached 1.0 units, the reader then began readings of each of the experimental wells. Typically it took between 10 and 15 minutes for the control well to reach this OD value when the substrate was kept at 4°C, before addition to the plate.
Final readings are based on a mean of duplicate runs. The same stock of EB and Chsp60 was used throughout the study, which took 3 months to complete. All sera as been collected and stored before the ELISA assay.
All assays were conducted by personnel masked to case/control status. The intra-assay coefficient of variation for chlamydia EB antibodies was 0.06 and for Chsp60 antibodies was 0.09, both representing excellent intra-assay replication.
Baseline differences between groups were analyzed with the t test for normally distributed continuous variables and the 32 χ2 test for categorical variables. Time-to-pregnancy and time-to-recurrence were analyzed using Kaplan-Meier life-table analyses. Cox proportional hazards model were employed to examine these outcomes after adjusting for age, history of PID, history of chlamydia, history of gonorrhea, parity, race, and education.
Each of the antibody levels tested was measured in optical density (OD) units (range 0.0–>2.0). Because the distributions of antibody OD units were highly skewed, we examined tertiles of OD units. Nonparametric correlation tests used were Spearman's ρ.
For analyses in which early and late blood samples were combined into overall estimates, imputed values were based on the 53 women who had both a baseline and follow-up blood sample. We used a linear regression model to best predict the baseline value from the follow-up values of women who had no baseline value. We imputed baseline (rather than follow-up) values to concord with the presumption that infection precedes outcome. The most predictive model was:
Equation (Uncited)Image Tools
Having predicted the baseline value for the 53 women, this predicted value was then compared with the actual baseline value. That being the case, the antilog of values obtained from the algorithm were used.
Each model was run for each of the main dependent variables of chlamydial antibodies and Chsp60 antibodies. Interactions between EB and Chsp60 as well as EB or Chsp60 and covariates were explored. Finally, we examined a Cox proportional hazards model including both EB and Chsp60 so as to separate the independent effects of these 2 exposures. Statistical significance was set at an α (2-tailed) <0.05.
At baseline, women enrolled in the PEACH study were predominately black (75%) and aged less than 25 (65%) (Table 1). Approximately one-third of participants reported a previous history of PID and showed evidence of N. gonorrhoeae and/or C. trachomatis at baseline. Women in the highest EB tertile were more likely to be black; to have a previous history of PID and a history of infertility; and on the baseline examination to have chlamydia, gonorrhea, or both, upper genital tract infection (UGTI) or endometritis, and BV. Women in the highest tertile for Chsp60 had the same characteristics, although some of these were not statistically significant.
Correlations Among Measures
Among the 53 women with repeated measures, first and second EB serologies were substantially correlated (r = 0.67; P <0.001) although not identical. The proportion of women whose titers remained stable over time was high. Of women in the lowest tertile at baseline, 11 of 16 remained in the lowest tertile, 4 of 16 rose to the midtertile, and 1 of 16 rose to the highest tertile. Of women in the highest tertile at baseline, 12 of 16 remained in the highest tertile, 3 of 16 fell to the midtertile, and 1 of 16 fell to the lowest tertile.
EB and Chsp60, were also strongly correlated (r = 0.73; P<0.001), although not identical. Of 146 women with a low EB tertile, 92 (63%) were in the lowest Chsp60 tertile. Among 148 women in the highest EB tertile, 106 (72%) were in the highest Chsp60 tertile.
Associations Among EB, Chsp60, and PID Sequelae
After the first year of follow-up, rates of pregnancy were lowest in women in the highest chlamydia EB antibody titer tertile (25.6% lowest; 25.1% mid; 17.6% highest) (Fig. 1). Pregnancy rates continued to be differential by EB tertile after 7 years of follow-up (72.6% lowest; 64.3% mid; 58.6% highest). Similarly, pregnancy rates were lowest among women in the highest Chsp60 tertile throughout follow-up and after the seventh year were 72.0% in the lowest; 65.9% in the mid; and 59.6% in the highest tertile.
Rates of recurrent PID were also elevated in the highest EB and Chsp60 tertile (Fig. 2). By the seventh year of follow-up, these PID recurrence rates were 14.7% EB lowest; 22.5% EB mid; 29.3% EB highest and 16.9% Chsp60 lowest; 24.9% Chsp60 mid; 31.7% Chsp60 highest.
After adjustment for the possible confounding factors age, history of PID, history of chlamydia, history of gonorrhea, parity, race, and education, presence of an EB titer in the highest tertile was associated with a reduced overall occurrence of pregnancy [adj. hazard ratio (HR) 0.71, 95% confidence interval (CI) 0.50–1.02] (Table 2). This effect was significant for antibody tests on samples collected in the final year of follow-up (adj. HR 0.47, 95% CI 0.28–0.79) but not on samples collected at baseline. Presence of a high EB titer was associated with an increased adjusted rate of PID recurrence (adj. HR 1.79, 95% CI 0.93–3.43). Again, this effect was only significant when antibody tests were collected near the end of follow-up (adj. HR 2.48, 95% CI 1.00–6.27).
After adjustment for possible confounding, women with Chsp60 antibodies in the highest tertile had only modestly reduced adjusted rates of pregnancy (adj. HR 0.78, 95% CI 0.56–1.08), an effect that remained nonsignificant in testing bloods collected near the end of the study (adj. HR 0.67, 95% CI 0.41–1.12) (Table 2). Similarly, the highest tertile for Chsp60 was not significantly associated with recurrent PID either overall (adj. HR 1.24, 95% CI 0.70–2.20) or when collected late in the final year of the study (adj. HR 1.06, 95% CI 0.47–2.39).
We tested a number of interactions. In multivariable models, the interactions between EB and Chsp60 in relation to pregnancy were not significant (0.86 for early samples and 0.95 for late samples); similarly, EB and Chsp60 did not result in significant interactions in relation to PID recurrence (0.89 for early samples and 1.20 for late samples). We also found no relevant interactions about either pregnancy or PID recurrence for EB and age; Chsp60 and age; EB and race; Chsp60 and race; EB and history of PID; Chsp60 and history of PID; EB and baseline endometritis; Chsp60 and baseline endometritis.
We also tested quartiles of EB and Chsp60, rather than tertiles. The results were essentially the same, with the highest quartile being significantly different from the lowest; however, the cell sizes were smaller and thus estimates were less stable.
Among women who had endometritis or chlamydial/gonococcal UGTI, i.e., pathologic or microbiologic evidence of PID, those with EB antibody titers in the highest tertile had a reduced adjusted rate of pregnancy (adj. HR 0.59, 95% CI 0.56–1.08) and a markedly reduced rate when the serum collection occurred late in the study (adj. HR 0.34, 95% CI 0.15–0.74). Similarly, the highest tertile for EB antibodies was associated with an elevated rate of recurrent PID overall (adj. HR 2.69, 95% CI 0.86–8.38). Sample size for this endpoint within the subset was insufficient to analyze baseline and late blood collections separately. Among women with endometritis/UGTI, Chsp60 was not significantly associated with pregnancy or recurrent PID.
In models including both EB and Chsp60, a high EB titer continued to be significantly related to reduced rates of pregnancy, particularly for antibodies detected in the late blood collection (adj. HR 0.49, 95% CI 0.26–0.91) and to increased rates of recurrent PID (adj. HR 3.01, 95% CI 1.10–8.23) (Table 3). In contrast, associations between Chsp60 and pregnancy or PID recurrence were all nonsignificant and close to null.
After an initial episode of mild to moderate PID, rates of pregnancy were lower and PID recurrence higher among women whose antibody titers to chlamydia EB were in the highest tertile remote from baseline. Surprisingly, Chsp60 antibody titers were not significantly associated with PID-related sequelae. When considered simultaneously, only high tertile chlamydia EB antibodies (and not Chsp60 antibodies) remained independently associated with reduced pregnancy and increased PID recurrence rates. Moreover, the interaction between EB and Chsp60 antibodies was not significant for either the pregnancy or recurrence outcome, providing little support for the hypothesis that Chsp60 amplifies the likelihood of PID sequelae among women with previous chlamydial exposure. These data suggest that chlamydia EB antibodies measured some years remote from baseline are a significant predictor of reduced pregnancy and elevated PID recurrence.
In retrospective studies, Chsp60 has been found in 16% to 25% of fertile women with positive serology for chlamydia, 36% to 44% of women with C. trachomatis cervicitis, 48% to 60% of women with chlamydial PID, and 81% to 90% of women with chlamydia-associated tubal infertility.9,18–23 A prospective study of female sex workers in Nairobi confirmed the Chsp60 and PID relationship, but post-PID sequelae were not studied.24 In a macaque monkey model, introduction of Chsp60 into chlamydial sensitized animals was associated with a delayed hypersensitivity immune response with mononuclear infiltration of salpingeal tissue, supporting the suggestion that Chsp60 is an immunologic mediator of chlamydial-related infertility.25 Yet, the paucity of prospective human data make the notion that Chsp60 specific antibodies as mediating or being a marker for PID sequelae an as yet untested hypothesis.
Our study is the first to our knowledge to prospectively examine the independent and joint roles for Chsp60 and chlamydia EB antibodies in the etiology of post-PID sequelae. Previous studies have been retrospective, conducted in nonhuman primates, or studying an outcome other than PID-related sequelae. Nonetheless, the discordance between our result and that of previous studies begs explanation. Previous results could have been false positives, first because Chsp60 measures may have been surrogates for prior chlamdyial exposure rather than an additional etiologic factor. Second, selection and diagnostic biases may have resulted in overestimates of effect sizes. However, we must also consider that our study may have missed a real relationship. First, fallopian tube pathology in women with tubal infertility may vary by chlamydial serovar, and we did not examine chlamydia serovar-specific correlates of virulence.26 Second, a comparison of paired follicular fluid and sera from women undergoing in vitro fertilization demonstrated the import of measuring mucosal chlamydial antibodies; IgA antibodies to chlamydia in follicular fluid (as opposed to IgG antibodies in follicular fluid or serum) were associated with failure to become pregnant after embryo transfer.27 We also did not measure mucosal immunity in this study. Finally, a larger study with more data points may have uncovered an association that we missed. Moreover, the collinearity between EB and Chsp60 adds complexity to the interpretation of the joint model.
The greatest potential limitation of our analysis was the incomplete serologic measurement. Only about half of all women had bloods drawn for analysis. Yet, women with and without antibodies had similar demographic and sexually transmitted disease-related characteristics. Moreover, about half of the women with serum available had blood collected near the end of follow-up and the other half had blood collected early in follow-up. Very few women had 2 blood collections (n = 53). Thus, the estimates for early and late antibodies were in different women on the whole. On the other hand, the availability of 2 time-periods of blood collection provided the important insight that baseline serology, reflecting chlamydial exposures before or at the time of the index episode of PID, was not strongly related to later pregnancy or recurrence.22 However, later serology, reflecting the incident PID episode and subsequent episodes, was significantly related to these outcomes. Are the higher titers associated with morbidity an indication of further exposure to the microorganism and possibly recurrent or persistent UGTI episodes? It appears that, as shown in animal models, second exposures to chlamydia and the associated memory immune response portends to sequelae after PID.
Other limitations include the self-reported documentation of outcomes, which, despite our attempts to validate endpoints, remains a caveat to interpretation of results. Additionally, the cohort largely involved low-income black women, who represent only one component of all women with PID. Nonetheless, our recruitment strategy was designed to include a clinically generalizable group of women with PID-related signs and symptoms, not all of whom had endometritis/UGTI. Among women with endometritis/UGTI, the association between chlamydia EB antibodies and adverse reproductive outcomes was as strong or stronger than in the cohort as a whole, demonstrating the robustness of our results.
Our current findings support independent associations among chlamydia EB antibodies, reduced pregnancy, and increased PID recurrence in women after mild to moderate PID. Our data thus support a link between infection with C. trachomatis and PID sequelae, whether or not there is an independent or interactive role for Chsp60 antibodies in the etiology of PID sequelae remains unclear.
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