Chlamydia trachomatis remains the most common sexually transmitted infection, with an estimated 2.8 million cases annually in the United States and 100 million cases worldwide.1,2 Up to 84% of women with lower genital tract infections are asymptomatic and therefore may remain undiagnosed.3 C. trachomatis is the most common infectious etiology for pelvic inflammatory disease (PID) and whether symptomatic or asymptomatic can spread to upper genital tract and persist. Although PID is usually polymicrobial, C. trachomatis is one of the most common inciting organisms. The risk for developing PID after a lower genital tract infection is as high as 30%, and the risk of developing tubal blockage after PID is at least 20%.4,5 The resultant tubal pathology accounts for 30% to 35%% of all causes of infertility.6
Numerous studies have documented that a positive C. trachomatis serology result is associated with tubal infertility on hysterosalpingography (HSG) and laparoscopy.4–18 Serologic testing for C. trachomatis has generally used type-specific microimmunofluorescence assays (MIF); in which elevated immunoglobulin G (IgG) titers are highly predictive of either recent or past exposure to C. trachomatis. In addition, increasing chlamydia antibody titers seem to predict a worsening severity of tubal damage.19,20 Chlamydia antibody testing (CAT) is often used in combination with, or in lieu of, HSG to screen for tubal infertility.4 Laparoscopy, however, has remained the gold standard for the definitive diagnosis of tubal infertility.
In previous studies from our institution and from several European centers, elevated chlamydia antibody titers were detected in up to 40% of infertile patients.19,21,22 A Finnish Study documented the association between general population–positive chlamydia serology and a reduction in spontaneous pregnancies.23 However, there are no prior studies with infertile participants that assessed the non-IVF pregnancy rates in chlamydia serology–positive versus chlamydia serology–negative women. Previous studies have, however, noted no significant difference in IVF pregnancy rates based on chlamydia serology status.24–30 The purpose of this study was to determine whether elevated C. trachomatis IgG titers at initial infertility visit predicts a reduced cumulative non-IVF pregnancy rate. We also assessed any impact of a positive CAT on IVF outcomes.
MATERIALS AND METHODS
A prospective observational study was conducted at a university-affiliated reproductive center. After obtaining approval from our institutional review board, all new patients seen between January 2007 and December 2009 underwent serum C. trachomatis IgG screening. Results from Quest Diagnostics Laboratories (Teterboro, NJ) were available for 1279 participants. All women coming to Continuum reproductive center were screened for C. trachomatis with urine DNA-based polymerase chain reaction testing and were all found to have negative results. The chlamydia antibody test was preformed with MIFs, using in vitro growth culture of elementary bodies to Chlamydia pneumoniae, C. trachomatis, and C. psittaci. The elementary bodies were used as antigen dots attached to the slide, and the patient’s serum was added to the slides. If the patient’s serum contained antibodies to chlamydia, it remained fixed on the slide and detected by a fluorescent dye–labeled antibody on human IgG. The results were detected by fluorescent microscopy. After a positive reaction, serial dilutions were made and CAT results were considered positive at an IgG titer cutoff of 1:64. The patient’s serum was also tested for C. pneumoniae and C. psittaci because of the cross-reactivity of antibodies among different chlamydia species. We were able to more accurately diagnose positivity and reduce false positives using the titer results of the other species because infection by C. trachomatis, if real, shows a higher titer of IgG and then those seen by C. psittaci or C. pneumoniae. Therefore, CAT was only considered positive if MIF titers of C. trachomatis were at least 1/64 and titers of the other chlamydia organisms C. psittaci and C. pneumoniae were lower than the C. trachomatis titer.
All 1279 participants were followed up for a range of 2 to 30 months, and their records were reviewed in detail. The results of all hysterosalpingograms and any laparoscopies and operative reports were recorded. Ultrasound evidence of clinical intrauterine pregnancies including any spontaneous or non-IVF cycles, as well as the outcomes of all IVF cycles, was also collected for all participants.
All statistical analyses were performed using Systat Version 13 and SAS Version 9.2 (SAS Institute, Inc, Cary, NC). All hypothesis tests were conducted at the 0.05 level of significance. Proportions of pregnancy outcome variables were compared between CAT-positive and CAT-negative groups using the Pearson χ2 test (or Fisher exact test when the cell count was less than 5). Probabilities of spontaneous pregnancy over time were estimated using the Kaplan-Meier product limit method with comparison between CAT groups evaluated by the log-rank test statistic. The Cox proportional hazards model was used to estimate hazard ratios for spontaneous pregnancy and 95% confidence intervals. All patients were followed up from their initial chlamydia serology evaluation until clinical pregnancy or, if no clinical pregnancy, then until their last date evaluated in the office.
Seventy (5.5%) of 1279 participants were found to have a positive C. trachomatis IgG serology result, and 700 (54.7%) of 1279 participants had an HSG from our institution on file. One hundred fifty-six (12.2%) of 1279 underwent a laparoscopy during their infertility workup. There were no differences in age, follicle stimulating hormone, or anti-mullerian hormone in the serology-positive and serology-negative participants. There were no differences in follow-up time, non-IVF treatment cycles (intrauterine insemination), and nontubal factor infertility diagnosis between CAT-positive and CAT-negative participants. Patients with positive chlamydia serology result were significantly more likely to have abnormal tubal findings on HSG and laparoscopy. However, Patients with a positive CAT result had a significantly lower rate of endometriosis at laparoscopy (Table 1).
Participants who were CAT positive and underwent a laparoscopic tuboplasty were significantly more likely to get pregnant without IVF (37.5% vs. 6.5%, P = 0.028) than CAT-positive participants who did not undergo laparoscopy with tuboplasty. Likewise, participants with an abnormal HSG result who underwent laparoscopic tuboplasty were significantly more likely to achieve a non-IVF clinical pregnancy (23.8% vs. 5.6%, P = 0.042). Per-cycle IVF pregnancy rates and cumulative live birth rates were not significantly different in chlamydia serology–positive and chlamydia serology–negative participants (Table 1).
Regarding the primary outcome variable, CAT-positive participants were significantly less likely to achieve an intrauterine pregnancy when excluding treatment with IVF (Table 1). Likewise, an abnormal HSG result was also associated with lower non-IVF pregnancy rates (11.0% vs. 26.5%, P = 0.002). There were no spontaneous pregnancies in patients that were CAT positive with abnormal HSG result. Kaplan-Meier curves for non-IVF clinical pregnancy were significantly different between the CAT-positive and CAT-negative groups (log-rank P = 0.025; Fig. 1). The time to pregnancy rate of non-IVF clinical pregnancy among CAT-positive patients was 57% less than the rate of pregnancy among CAT-negative patients (hazard ratio, 0.43; 95% confidence interval, 0.20–0.92).
Currently, the most reliable laboratory assay in CAT is C. trachomatis type-specific micro-immunofluorescence. However, there are laboratories that use the inferior test enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay uses synthetic peptides, for example, from major outer membrane protein–containing species and uses highly specific peptides that may be so specific that they are not able to detect antibodies in all cases, resulting in false-negative CAT results.
In this cohort, only 5.5% of the 1279 participants were CAT positive, far below the 40% positive rate found in our previous study from a decade ago.19 This rate is also well below the rates noted in larger European studies such as Land et al.,22 who showed a CAT-positive rate of 40% to 64%, and Mol et al.,21 who found a serology-positive rate of 20% to 73%. Possible reasons for this marked decrease in positive serology result include more type-specific testing in the newer MIF assays that exclude false positives from prior C. pneumoniae infections and a higher titer cutoff for a positive test result, which increased at Quest Laboratories from 1:32 to 1:64. An increase in the titer cutoff will increase the false-negative serology rate, but only among participants likely to have milder tubal damage.19 In addition, our participant population may have shifted to a lower-risk population from our more clinic-based participant population of a decade ago.
As noted in our prior study and nearly all reports in the world literature, CAT-positive participants had a significantly higher rate of tubal damage and obstruction on HSG and laparoscopy.4–22 This reconfirms the high positive predictive value of CAT screening for tubal infertility. Because fewer patients with infertility are now undergoing laparoscopy (only 156/1279 in our cohort), it is interesting but not surprising to find that endometriosis was associated with being CAT negative among those undergoing laparoscopy for suspected anatomical infertility. Our finding that laparoscopic tuboplasty increased the non-IVF pregnancy rate among serology-positive patients is intriguing but requires a prospective randomized trial for confirmation. This cohort also corroborated the general consensus in the literature that chlamydia serology has no impact on IVF outcome.24–30
This is the first large cohort study to report that infertile patients with a positive chlamydia serology result are less likely than seronegative patients to conceive without IVF, and those with CAT-positive result have a longer time to non-IVF pregnancy than do CAT-negative patients. We also found that an HSG showing at least unilateral tubal obstruction predicted a lower rate of non-IVF intrauterine pregnancy. Because it is less invasive, our data support CAT being used for initial screening. Hysterosalpingography could then be used for CAT-negative participants to rule out other causes of tubal infertility such as gonorrhea, endometriosis, or adhesions from prior surgery.
In many European countries, chlamydia serology is used as a screening test for tubal factor infertility and if positive patients proceed to diagnostic laparoscopy. In practice, however, the combination of CAT and HSG at initial workup is quite useful. We recommend a combined approach using both chlamydia serology and HSG, and if either gives abnormal result, we recommend proceeding to diagnostic laparoscopy. Patients with a negative CAT and negative HSG results can proceed to less invasive therapies than laparoscopy or IVF.31 These less invasive options include intrauterine insemination with or without ovulation induction agents. We found that HSG is 78% sensitive in diagnosing tubal disease; however, an abnormal HSG and positive chlamydia serology result combined increased the sensitivity for finding tubal disease to 97%.19 Our results show that there were no spontaneous pregnancies in patients who were CAT positive with abnormal HSG result, with the highest number of pregnancies achieved with the combination of negative CAT and normal HSG result. These findings support the combination of CAT and HSG as a valuable tool in predicting a spontaneous pregnancy. The finding from Finland that CAT positivity is associated with subfertility in the general population is consistent with our findings in the infertile population but does not provide justification for screening the general population.18
The limitations of this study are derived from its observational design and the wide ranging period of follow-up after initial screening. For example, a number of our patients were lost to follow-up after the initial chlamydia serology screen and did not obtain HSG. However, we minimized follow-up bias by performing a Kaplan-Meier study to analyze the probability of spontaneous pregnancy over time. Although this study design will accurately assess the association of CAT with other diagnostic findings and pregnancy outcomes, it is less reliable for establishing management algorithms. To determine the best route of management for infertile patients who screen positive with CAT, prospective randomized trials are required.
In conclusion, positive C. trachomatis serology screening result is highly predictive of tubal disease and seropositive participants are less likely than seronegative participants to conceive without IVF. Although chlamydia serology–positive participants are far more likely to need IVF because of tubal damage, they were no more or less likely to conceive or achieve a live birth after IVF than seronegative participants.
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