Untreated genital Chlamydia trachomatis infection (“chlamydia”) may ascend from the lower genitourinary tract to the upper genitourinary tract, leading to pelvic inflammatory disease (PID)(1). The subsequent inflammation can lead to scarring, fallopian tube obstruction, and tubal factor infertility (TFI), with repeated PID episodes and more severe PID increasing TFI risk(2). US women of black race have higher rates of reported chlamydial infections than women of other races(3); however, little is known about ascending chlamydial infection that may ultimately result in chlamydia-associated TFI in women of different racial groups.

Chlamydia serological assays can identify women who have been infected with chlamydia. Seropositivity with these assays has been associated with reproductive chlamydial complications, including TFI(4, 5). Consequently, these assays have been used to estimate TFI population attributable fraction (PAF) related to prior chlamydia(6, 7), although rarely stratified by race. Unfortunately, to date most serological assays have poor sensitivity for measuring prior chlamydia in women(8) regardless of race, resulting in estimates that may not reflect actual chlamydia-related TFI. We previously investigated a relationship between different chlamydia serological assays and hysterosalpingogram (HSG)-diagnosed TFI stratified by race using a reference standard of positivity by the Medac IgG plus assay or a research elementary body ELISA assay; however, we did not find an independent association between chlamydia seropositivity and TFI in black women nor in non-black women(9).

C. trachomatis Pgp3 (plasmid gene product 3) serological assays have significantly higher sensitivity (74–83%) (10–12) than commercially available major outer membrane protein (MOMP) peptide-based commercial assays (46–60%) for detecting serum IgG antibodies to C. trachomatis. The specificity of Pgp3 assays is comparable to other MOMP peptide-based serological assays(11, 12). Additionally, Pgp3 antigen has been associated with upper tract infection in mice, suggesting possible utility of Pgp3 serology for upper tract disease in humans(13).

Both the Pgp3 indirect ELISA and Pgp3 double antigen (enhanced) ELISA assays have been used to evaluate for prior chlamydia using nationally representative data from the United Kingdom(14); however the enhanced assay demonstrates longer serum antibody detection than the indirect assay(8, 12, 15). In this analysis, we used data from our original case-control study which assessed chlamydia seropositivity in women with and without TFI, to determine whether chlamydia seropositivity measured with the enhanced Pgp3 assay is associated with TFI. We also estimated the chlamydia TFI PAF among women with infertility, and examined the data stratified by race to determine the proportion of TFI that might be averted with chlamydia prevention and prompt diagnosis and treatment.

In these women with TFI and non-TFI infertility, we measured prior chlamydia using the enhanced Pgp3 ELISA — a chlamydia serological assay with better sensitivity and comparable specificity to other MOMP-based serological assays — and estimated the PAF of TFI due to chlamydia. While other studies have characterized chlamydia seropositivity in women with TFI(5, 17), our study is notable because we found a significant association of Pgp3 seropositivity among some women in our stratified analysis by racial group. Among non-black women without endometriosis, Pgp3 seropositivity was independently associated with the odds of TFI and in these women, 20% of TFI was attributable to chlamydia.

In the context of the imperfect sensitivity and specificity of many chlamydia serological assays for detecting prior chlamydia(8), with a more sensitive serological assay, we found that Pgp3 seropositivity was associated with the unadjusted odds of TFI among all women combined, and also among non-black women. We also found an overall chlamydia PAF of 20% in non-black women without endometriosis which is higher than our prior study evaluating the unadjusted PAF of HSG-diagnosed TFI due to chlamydia, where 11% [95% CI −3 to 23%] of TFI was attributed to chlamydia when chlamydia seropositivity in non-black women was assessed with a reference of positivity on a combination of the commercial Medac IgG MOMP ELISA or the research-based EB ELISA assays(9), although this was not a statistically significant association. Pgp3’s higher sensitivity compared to the Medac MOMP assay may have resulted in the association we observed in this study compared to the lack of association with our previous reference assay(8). Using prior serologic data and modeling to account for imperfect sensitivity and specificity of chlamydia serological assays, UK investigators estimated the PAF of TFI from chlamydia in a Dutch population from 1992–2003 to be 45% [28–62%] (6). Price et al. applied multi-parameter evidence synthesis of available non-serologically based data sources and estimated, using modelling, that the TFI chlamydia PAF in England in the early 2000s was 29% (95% CI 9%–56%) and pooled modeled estimates from observational case-control studies found the TFI chlamydia PAF ranged from 28–60%(7). Another study using modeled distributions of women in the UK between 1985 and 1995 with differing whole-cell immunofluorescence chlamydia antibody titers estimated that the minimum PAF of TFI from chlamydia was 28% (95% CI 7% to 50%) and maximum PAF was 46.8% (95% CI 23% to 64%)(18). Our 20% estimate using Pgp3 is higher than our previous estimate using different assays but lower than the previously published modeled estimates. Taken together, the estimates suggest that a minority of TFI is caused by chlamydia, the most common reportable STI in the US and worldwide(3, 19). While gonorrhea has also been implicated as a cause of TFI, gonorrhea is generally less common than chlamydia(3). When both of these STIs are considered together, they are estimated to be responsible for less than half of all PID, and presumably also less than half of TFI cases(20).

Interestingly, we found the association of Pgp3 chlamydia serology with TFI varied by the presence or absence of endometriosis. Endometriosis has been associated with infertility and obstruction of the fallopian tubes possibly due to anatomical distortions due to adhesions and fibrosis(21, 22). Although chlamydial infection has been linked with endometriosis in one paper, the data for this association are poor and the mechanism for this association is not clear(23, 24). Because we did not see a significant association between Pgp3 serology and TFI among women with endometriosis, we might speculate that among these women, the association between chlamydia and TFI may be obscured by an association between endometriosis and TFI.

As with our previous study(9), there was no observed association between chlamydia seropositivity and HSG-diagnosed TFI among black women. Among these women, we did interestingly observe a negative, although non-significant, association of Pgp3 seropositivity and TFI, noting that women with TFI had lower absolute numeric Pgp3 seropositivity than controls. However, for black women in whom we might most expect to see chlamydia seropositivity (women with a history of chlamydia, women with PID history, and women with both), we did see positive, although non-significant, associations between chlamydia serology and TFI. We may not have seen a positive association between seropositivity and TFI outcome among black women because of possible higher rates of prior chlamydia in black women in the control group through mechanisms including increased frequency of uncomplicated chlamydial infections and repeated undetected chlamydial infections or undetected PID infections. It is possible that subsetting our analysis to women whom we presumed to be at higher risk for chlamydia infection than the general study population, such as those with a prior STI or PID history, may have excluded women with positive serology because of uncomplicated STIs. This exclusion may have resulted in a non-statistically significantly positive association in black women where it had previously been non-significantly negative.

An additional perspective on the seropositivity of these black women and possible limitation of our study is that black control women may have been misclassified and may have actually had TFI that was not evident on HSG, a less sensitive TFI test compared to laparoscopy(25). One study that found differing sensitivities of HSG for tubal disease comparing women without risk factors for tubal disease to women with risk factors suggested that an HSG reading could be affected by a woman’s medical history and may not be simply based on blinded test results(26). In addition, there is evidence that chlamydia may also impair fertility through mechanisms other than overt tubal damage. Steiner et al. observed that infertile women with patent tubes were less likely to conceive if chlamydia seropositive(4). Thus, our study may have been improved by the inclusion of fertile controls to address the potential misclassification bias of subclinical TFI or non-TFI chlamydia-related infertility in women considered to be controls. However, the higher proportion of chronic pelvic pain and suggestion of higher PID among black case women compared to black control women in our study argues against misclassification. Higher background chlamydia seropositivity in black controls, which is in accordance with higher chlamydia prevalence in black women(3), may be the underlying reason for a lack of association of chlamydia seropositivity with TFI in black women.

Our findings suggest that the Pgp3 assay might be a more reliable approach to differentiate prior chlamydia from a lack of prior chlamydia in non-black women with TFI compared to the commercial and research assays used in our previous analysis. It is important to note that while the enhanced Pgp3 ELISA has better or equivalent sensitivity than existing serologic assays(11, 12), any serologic assay for chlamydia has inherent challenges. Chlamydial antibodies can wane over time. Although chlamydial Pgp3 antibodies detected with the enhanced Pgp3 assay have been documented to remain positive in the majority of, but not all, women for 12 years independent of reported reinfection(12), other studies found that detection of chlamydial antibodies using the Medac MOMP(27) and indirect Pgp3 ELISA(8) declines within the first few years after initial infection. Additionally, not all women even mount an antibody response after an initial chlamydial infection(27). An additional limitation in our study is that there is no gold standard test for past infection that we could have assessed as a reference standard. This is particularly notable because we were unable to detect all reported chlamydial infections with Pgp3, resulting in possible misclassification.

Even in the context of the limitations of the enhanced Pgp3 assay, we estimate that 20% of TFI among non-black women without endometriosis in these clinics is attributable to chlamydia. Given the limited sensitivity of Pgp3 and chlamydia serologies in general, this figure may be an underestimate. With the differential association we observed by race, our data suggest that stratified analyses of TFI in the US by race should be done when assessing the contribution of chlamydia and other STIs to this pathology. With the lack of association of Pgp3 seropositivity with TFI among black women, additional research should be done to understand chlamydia risk in TFI for these women and whether it may cause infertility through other mechanisms. Although serologic tests for previous chlamydia can provide some estimate of previous disease and are needed to identify outcomes of chlamydial disease(28), they are imperfect and there is need for more accurate biomarkers that can predict ascension of chlamydia and other STIs into the upper genital tract predisposing to PID and TFI in all women.