Ten healthy women were recruited in 2000 for a pilot feasibility study on early pregnancy loss, at which time a serum sample was collected and archived, and a health and lifestyle questionnaire was completed. Newly married women in Chapaevsk were invited to join, and 10 enrolled in the pilot project. To be included in the original study women needed to be married, age 20-34 years, and trying to conceive. To investigate temporal trends in PCDD/PCDF/PCB exposure, participants were re-contacted in 2009, and a second serum sample and lifestyle questionnaire was collected from eight of the ten women (of the two others, one had left Chapaevsk, and one declined to participate).
The research protocol was approved by the Human Studies Institutional Review Boards of the Chapaevsk Medical Association, UMass Medical School, and Harvard School of Public Health. All participants gave their consent before participating in each of the two assessments.
Measurement of dioxin, furan, and PCB serum concentrations
Samples were stored at -20 degrees C until analysis. All 16 serum samples (10-15 ml each) were analyzed in 2009 using isotope dilution gas chromatography-high-resolution mass spectrometry (GC-HRMS-ID) at the Laboratory of Analytical Toxicology of the Severtsov Institute of Ecology and Evolution in Moscow, Russia. The list of dioxin-like congeners (called dioxins in this manuscript) included 7 PCDDs, 10 PCDFs, 4 co-planar PCBs, and 6 mono-ortho PCBs. The list of non-dioxin like PCBs (NDL-PCBs) included congeners 18, 28, 44, 49, 52, 66, 74, 87, 99, 101, 110, 128, 138, 146, 149, 151, 153, 170, 172, 177, 178, 180, 183, 187, 190, 194, 195, 196, 199, 203, 206, 209. Congeners are identified according to the International Union for Pure and Applied Chemistry (IUPAC) nomenclature.
Prior to analysis of study samples the laboratory carefully cleaned and checked all reagents, solvents, and glassware, and analyzed a method blank to confirm that it was free from interference. Then each set of four samples was extracted and analyzed with one method blank, which was treated identically to the serum samples.
For the analysis of dioxins, furans and co-planar PCBs the serum samples were spiked with a mixture of 13C12-labeled PCDDs/PCDFs and PCBs as internal standards. Methods for sample preparation, clean up and analysis follow US Environmental Protection Agency (EPA) methods 1613  and 1668 . Analysis was performed using a Thermo Finnigan MAT 95XP. The analytes were separated on a capillary column (SGE-BPX5, 30 meters, 0.25 mm, 0.25 microns) and quantified using selected-ion-monitoring high resolution (10,000 resolving power) mass spectrometry (HRGC-ID/HRMS). Quantification was by isotope dilution mass spectrometry using calibration standards containing 13C labeled and unlabeled analytes. Mono-ortho (M-PCBs) and non-dioxin-like PCBs were separated on a capillary column (SGE-HT8, 30 meters, 0.25 mm, 0.25 microns).
LODs were calculated as three times chromatographic noise. The LODs for dioxins and furans ranged from 0.01 to 0.04 pg/g, and for C-PCBs from 0.1 to 0.6 pg/g serum. The levels of target analytes in 4 blanks analyzed with study samples were below the LODs. The mean (SD) recoveries were 90% (5.5) for TCDD and 84% (3.1) for HxCDD.
Serum total cholesterol and triglycerides were measured with an enzymatic colorimetric assay using the COBAS INTEGRA 400 plus system, and the serum total lipid content was calculated using the Phillips equation . All PCDD/PCDF/PCB serum concentrations were lipid-adjusted. Individual congener concentrations below the limit of detection (LOD) were assigned a value equal to the LOD divided by the square root of 2 [19, 20].
The serum concentrations of PCDD/PCDF/PCBs were summarized with two measures: the total sum of toxic equivalencies (i.e. total TEQ ) for PCDDs, PCDFs, co-planar PCBs (C-PCBs) and mono-ortho PCBs (M-PCBs); and the sum of all 36 assayed non-coplanar PCB concentrations (i.e. ∑PCBs).