Access chemiluminescent immunoassay

Antimullerian hormone (AMH) was measured via enzyme linked immunuosorbent assay (ELISA, Ansh Labs; <0.023 ng/mL is below the limit of detection). Serum estradiol and follicle stimulating hormone (FSH) levels were measured via Access Chemiluminescent Immunoassay (Beckman Coulter, Fullerton, California) and were not timed to menstrual cycle phase.

Blood samples were obtained by trained research staff in study clinics. For MsFLASH 03, participants were requested to fast for 12 hours prior to morning phlebotomy; for MsFLASH 05, participants were not asked to fast prior to phlebotomy. Serum E₂ and E₁ concentrations were measured by the Brigham Research Assay Core Laboratory (Boston, MA) using a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method certified by the Centers for Disease Control and Prevention (CDC) Hormone Standardization Program. The assay details have been published.[18 (link)] For the E₂ and E₁ assays, the lower limit of quantitation was 1 pg/mL, linear range 1 to 500 pg/mL, the intra-assay coefficient of variation was <5%, and the inter-assay coefficient of variation was <12%. For LC-MS/MS E₂ assay, the mean bias for quality control samples provided by the CDC’s Hormone Standardization Program was 0.81 pg/mL for E₂ concentrations less than or equal to 20mg/mL, and 1.9% for samples with E₂ concentrations >20 pg/mL. SHBG was measured using a two-site directed chemiluminescent immunoassay (Access Chemiluminescent Immunoassay, Beckman Coulter, Fullerton, CA). For the SHBG assay, the lower limit of quantitation was 0.33 nmol/L, linear range 0.33-200 nmol/L, the intra-assay coefficient of variation was 4.5-4.8%, and the inter-assay coefficient of variation was 5.2-5.5%.

Blood specimens were collected after an overnight fast at baseline and three-month follow-up. Complete blood count and high sensitivity CRP concentration were both assessed at Laboratory Corporation of America (Raritan, NJ), using a Sysmex X-series analyzer (Sysmex Corporation, Kobe, Japan) and a particle enhanced turbidimetric assay on a COBAS INTEGRA system (Roche Diagnostics, Mannheim, Germany), respectively. RBC FAs were quantified in the Department of Nutrition at Harvard School of Public Health (Boston, MA), using gas–liquid chromatography on a fused silica capillary cis–trans column SP2560 (Supelco, Bellefonte, PA) with peak retentions analyzed in Agilent Technologies ChemStation A.08.03 software. Individual RBC FA was reported as a proportion of total phospholipid FAs. Serum IL-6 concentration was measured at the Harvard Catalyst Central Laboratory (Boston, MA) using access chemiluminescent immunoassay (Beckman Coulter, Fullerton, CA). The intra-assay coefficients of variation for CRP, IL-6, palmitic, and stearic acids were 1.3%, 1.7–4.6%, 5%, and 19%, respectively.