OD units <0.10 0.10–0.199 0.20–0.399 ≥0.40 EB quartiles <0.06 0.06–0.11 0.11–0.24 ≥0.25 Chlamydia EB Case subjects 248 138 73 62 Chlamydia EB Case subjects 133 133 146 109 Control subjects 342 173 113 138 Control subjects 189 189 176 212 OR (95% CI) 1.0 1.1 (0.8–1.5) 0.9 (0.6–1.2) 0.6 (0.4–0.9) OR (95% CI) 1.0 1.1 (0.8–1.5) 1.2 (0.9–1.7) 0.7 (0.5–1.1) Roberta B. Ness et al. Table 2: Frequencies and adjusted odds ratios (ORs) and 95% conﬁdence intervals (CIs) for chlamydial elementary bodies optical density (OD) units, in cases and controls, categorized by previously deﬁned cut points. (ORs were adjusted for age, education, family history of ovarian cancer, tubal ligation, nulliparity/any parity, and years of oral-contraceptive use). Table 3: Frequencies and adjusted odds ratios (ORs) and 95% conﬁdence intervals (CIs) for chlamydial elementary bodies, cat- egorized by quartiles, in case and control subjects. (ORs were adjusted for age, education, family history of ovarian cancer, tubal ligation, nulliparity/any parity, and years of oral-contraceptive use.) 2.1. Serologic testing Serologic testing for IgG antibodies to serovar D of C. trachomatis elementary bodies (EBs), the extracellular form of the chlamydia bacteria, was conducted in the reference laboratory of one of the authors (RB) using an ELISA technique. Final readings are based on a mean of duplicate runs. All assays were conducted by personnel masked to case/control status. The intra-assay coeﬃcient of variation for chlamydia antibodies was 0.06, representing excellent intra-assay replication. Among masked replicates admixed into the test set, Pearson correlation coeﬃcients were 0.90 for chlamydia, again representing excellent interassay variability. 2.2. Statistical analysis Each of the antibody levels tested was measured in optical density (OD) units (range 0.0–0.4+). We log transformed all OD units to reduce skewing when considering these as continuous measures and categorized OD units into neat whole number categories when considering these as discrete measures. These cut points corresponded to those in our published pilot study [10]. Odds ratios, with corresponding 95 percent conﬁdence intervals, were calculated as the primary measure of eﬀect size. Odd ratios were adjusted in unconditional logistic regression models for any residual eﬀect of age and for family history of ovarian cancer in 3 any ﬁrst degree relative (yes/no), tubal ligation (yes/no), nulliparity versus any parity, years of oral contraception (continuous), and education (<high school/high school or equivalent/>high school). In secondary analyses, we divided serologic titers by quartiles; we also performed secondary analysis limiting our evaluation to only women with invasive ovarian cancer. We also stratiﬁed by age to assess cohort and age eﬀects. Chlamydial infections typically occur in younger women. Older women may have time-related diminished antibody titers. 3. RESULTS About one quarter of women were younger than the age of 50 and half were age 50 to 65 (see Table 1). Only about 1% of women reported having PID and 4% reported having gon- orrhea or chlamydia. The well-established protection against ovarian cancer aﬀorded by oral contraception, pregnancies, and tubal ligation, and the risk from a family history of ovarian cancer were demonstrated here. After adjustment for possible confounding factors and based on our previous antibody titer cut points [10], women with ovarian cancer were less likely than controls to have high chlamydia EB OD unit titers (≥0.40 versus <0.10) (OR 0.6, 95% CI 0.4–0.9; test for trend P = .001) (see Table 2). We then recategorized chlamydia EB OD units as quartiles based on the distribution of antibody values among controls (see Table 3). After adjustment for covariates, the highest quartile of chlamydia EB antibodies was inversely nonsigniﬁcantly associated with ovarian cancer (OR 0.7, 95% conﬁdence interval 0.5–1.1). In age-stratiﬁed analyses, we continued to ﬁnd the chlamydia antibodies that reduced ovarian cancer risk (see Table 4). Results were also similar in analyses limited to invasive ovarian cancer; the risk for ovarian cancer among women in the highest quartile of chlamydia EB antibodies versus the lowest was 0.6 (0.4–0.9, test for trend P = .021). 4. DISCUSSION We found that women with ovarian cancer were less likely to have high levels of IgG to C. trachomatis serovar D EBs. Our data are inconsistent with our previously published pilot results [10] which showed higher serologic titers for C. trachomatis among ovarian cancer patients. Our current ﬁnding is the reverse of the link between chlamydia and ovarian cancer in our previous analysis [10]. This is diﬃcult to explain. The same laboratory conducted serologic analyses using similar methods for both studies. A population-based case-control method was used to recruit women into both ovarian cancer case-control studies. Explanations for a lack of association include the long period between exposure to chlamydia (in the reproductive period) and blood collection (generally after the age of 50) in our population. If titers fall signiﬁcantly over time, they may have become undetectable in some women. Data from a Dutch study suggested that 18% of women had a decline of twofold in chlamydia titers over four years [11]. However in our own data, over 5–7 years of followup, women with the