NHS population

The NHS began in 1976 when 121,700 female US registered nurses between the ages of 30-55 years completed a self-administered questionnaire on their medical histories and baseline health-related exposures. Information regarding endometrial cancer risk factors was obtained from biennial questionnaires as well as a questionnaire completed at the time of blood collection. These questionnaires include data on reproductive variables, oral contraceptive and postmenopausal hormone use, cigarette smoking, and (since 1980) dietary intake. From 1989 through 1990, blood samples were collected from 32,826 women; details regarding the blood collection methods have been published previously. Briefly, each woman arranged to have her blood drawn and shipped, via overnight courier with an icepack, where it was processed; 97% of the samples were returned within 26 hours of blood draw. Upon receipt, the samples were centrifuged; aliquoted into plasma, red blood cells, and buffy coat fractions; and stored in liquid nitrogen freezers. Subsequent compliance with follow-up has been >98% for this subcohort of NHS participants who have given blood.

Case-control study

For this study, eligible endometrial cancer cases consisted of women with pathologically confirmed invasive endometrioid endometrial adenocarcinoma that had been diagnosed at any time point after blood collection (1989-1990) and up to June 1, 2004, with no history of cancer except for nonmelanoma skin cancer. Controls were randomly selected participants who had given a blood sample, had not had a hysterectomy, and were free of diagnosed cancer (except nonmelanoma skin cancer) up to and including the interval in which the case was diagnosed. Controls were matched to cases according to year of birth, menopausal status at blood draw and at the time of cancer diagnosis, and use of hormone replacement therapy at time of blood draw (current vs not current users). Controls were also matched to cases by time of day of blood collection, month of blood return, and fasting status at blood draw because of planned plasma analyses. A secondary analysis including cases diagnosed with any invasive epithelial endometrial cancer (not just endometrioid) during this time was also performed. The study protocol was approved by the Committee on Use of Human Subjects of the Brigham and Women’s Hospital, Boston, MA. The institutional review board at University of Texas, M. D. Anderson Cancer Center, approved this study with an exemption for informed consent, as all of the received samples and clinical information were deidentified.

Measurement of adiponectin

Both case and control samples were run on the same microplates and the investigators were blinded to study group. Serum adiponectin levels were measured using a commercially available quantitative sandwich enzyme-linked immunoassay with a sensitivity of 0.246 ng/mL (R&D Systems, Minneapolis, MN). The coefficients of variation from masked quality control samples (embedded with the case-control samples) for intraassay and interassay precision were 4.5-8.5%.

The enzyme-linked immunoassays were performed at room temperature, according to the instructions from the manufacturer. Recombinant adiponectin was used as the standard. Study samples were diluted 100-fold with calibrator diluent and placed into 96-well plates that were precoated with mouse monoclonal antibody to adiponectin. All samples were run in triplicate. Following a 2-hour incubation period, the microplates were washed thoroughly with buffer. Adiponectin conjugate was then added to each well for an additional 2-hour incubation. Stabilized chromogen (tetramethylbenzidine) was used for color development. Absorbance of the solution in the wells was measured immediately by spectrometer at a wavelength of 450 nm, with the correction wavelength set at 540 nm. Serum adiponectin concentrations were then determined by comparison to the standard curve generated for each individual microplate. We previously reported a correlation of 0.97 between adiponectin levels from blood samples processed immediately vs after 24-48 hours suggesting our blood collection methods did not adversely influence adiponectin levels.

Statistical analysis

Descriptive statistics were used to report demographic characteristics, clinical and pathologic variables, and serum adiponectin levels for all cases and controls. A χ ² test was used to assess the significance of differences in categorical variables between the 2 groups. Continuous variables were compared using the Student t test. In logistic regression models, plasma adiponectin levels were categorized into tertiles based on the distribution of values in the control group (lowest tertile <10.0 mg/mL, intermediate tertile 10.00-14.99 mg/mL, highest tertile ≥15.00 mg/mL). In multivariable models, BMI was included as a continuous variable while diabetes, parity, and age at last birth were included as categorical variables. Using the lowest tertile of adiponectin level as the reference group, crude and adjusted odds ratio (OR) and 95% confidence interval (CI) were calculated using conditional logistic regression for the intermediate and highest tertiles. A 2-sided P value of < .05 was considered statistically significant. Tests for trend were assessed by modeling the median value of each category as a single ordinal variable. Tests for interaction were calculated using the product of the median of each category and a binary stratification factor.

Analyses were also stratified by menopausal status. Women were defined as postmenopausal at the time of blood collection if they reported a natural menopause defined as no menstrual cycle within 12 months of the blood draw. Although many women were perimenopausal, they were categorized as premenopausal if they had a period within the previous year. In multivariable models, menopausal status and postmenopausal hormone use were updated until the date of diagnosis for endometrial cancer cases and matched controls.