PBDEs and thyroid hormones in pregnant women Materials and Methods Participant recruitment. Participants were recruited from within an ongoing observational prospective cohort study assessing the joint effect of social, environmental, and host factors on pregnancy outcomes [the Healthy Pregnancy, Healthy Baby (HPHB) Study] (Burgette and Reiter 2010; Miranda et al. 2010; Swamy et al. 2011). The HPHB study enrolls preg- nant women from the Duke Obstetrics Clinic and the Durham County Health Department Prenatal Clinic. Women receiving prenatal care at these sites were eligible to participate if they were at least 18 years of age, English liter- ate, between 18 and 28 weeks of gestation at study enrollment, lived in Durham County, were planning on delivering at Duke University Medical Center, and did not have a multiple gestation or any known fetal genetic or con- genital abnormalities. All patients > 34 weeks of gestation enrolled in the HPHB study and who were routine patients at the Durham County Health Department’s Prenatal Clinic at the Lincoln Community Health Center (Durham, NC) between September 2008 and June 2010 were eligible to participate in the present study. The choice of this clinic ensured a predomi- nantly African-American study population. Women were approached during their routine third trimester laboratory visit (35–36 weeks) for participation in this study. Of all women approached for this study, > 97% agreed to par- ticipate. After women gave informed consent, samples were collected during the third trimes- ter of pregnancy (> 34 weeks of gestation). All aspects of this study were carried out in accor- dance with a human subjects research protocol approved by the Duke University Institutional Review Board. Sample collection. Consenting women filled out a short questionnaire that documented information regarding their personal, diet, and lifestyle characteristics. Two tubes of blood were collected during the clinic visit: one 4.5-mL tube (BD Vacutainer plasma separation with lithium heparin) and one 10-mL tube (BD Vacutainer serum separator). The tubes were allowed to sit for several hours in a refrigerator and were later centrifuged (within 24 hr) at 3,500 RMP for 5 min to isolate the plasma and serum, respectively. The 10-mL tubes (~ 4–5 g serum) were stored at –20°C until analysis. The smaller tubes were sent to the clinical laboratory at Duke University Hospital for thyroid hor- mone analysis within 8 hr of centrifugation. hTyroid hormone analysis and lipid meas - urement. Plasma samples were analyzed by the clinical laboratory at Duke University Hospital (Durham, NC). TSH was measured using the Access HYPERsensitive hTSH assay; free T4 (FT4) was measured using the Access Free T4 assay; total T4 (TT4) was measured using the Access Total T4 assay; free triiodothyronine (FT3) was measured using the Access Free T3 assay; and total T3 (TT3) was measured using the Access Total T3 assay. All Access assays were paramagnetic particle, chemiluminescent immunoassays manufactured by Beckman Coulter Inc. (Fullerton, CA). Lipid content of the serum was determined using an enzymatic method based on measurements of serum cho- lesterol and triglycerides (Covaci et al. 2006). Chemicals. Information on the specific internal, recovery, and quantitative stan- dards used can be found in Supplemental Material, p. 2 (http://dx.doi.org/10.1289/ ehp.1003235). Sample extraction. Serum samples (approxi- mately 3–5 g) were extracted using a previously published method (Stapleton et al. 2008). More information regarding sample extraction can be found in Supplemental Material, p. 2 (http:// dx.doi.org/10.1289/ehp.1003235). Sample analysis. Extracts were analyzed for 27 PBDE congeners using a method reported by Stapleton et al. (2008). A subset of 57 serum extracts was spiked with 10 ng 13C 6-OH-BDE 47 and 13C α-HBCD, then blown to dryness under pure nitrogen gas. Dried extracts were reconstituted in 100 µL methanol and analyzed using liquid chromatography tandem mass spectrom- etry (LC/MS-MS) for phenolic metabolites including 2,4,6-tribromophenol (246 TBP), 4´-hydroxy-2,2´,4,5´-tetrabromodiphenyl ether (4´-OH-BDE-49), 6´-hydroxy-2,2´,4,5´- tetrabromodiphenyl ether (6´-OH-BDE-49), 6-hydroxy-2,2´,4,4´-tetrabromodiphenyl ether (6-OH-BDE-47), 6-hydroxy-2,2´,4,4´,5- pentabromodiphenyl ether (6-OH-BDE-99), and alpha-, beta- and gamma hexabromo- cyclododecane (HBCD). More information on the LC/MS-MS method can be found in Supplemental Material, pp. 2–3 (http:// dx.doi.org/10.1289/ehp.1003235). Quality control. Bovine serum (Invitrogen, Carlsbad, CA) and deionized water were both used as laboratory blanks, and SRM 1958 [Fortified Human Serum; NIST (National Institute of Standards and Technology), Gaithersburg, MD] was used for quality assur- ance to test performance of the extraction method for PBDEs and HBCD. Further details on the calculation of method detection limits (MDLs) and internal standard and SRM recov- eries can be found in Supplemental Material (http://dx.doi.org/10.1289/ehp.1003235). Statistical analysis. Statistical analyses were performed using Stata 11 (StataCorp, LP, College Station, TX). Values below MDL were assigned a value equal to half the detection limit for statistical analyses. Summary statistics were computed for thyroid hormones, individual and total PBDEs, and phenolic metabolites, using only those compounds with detection fre- quencies ≥ 50%. Correlations among PBDEs, metabolites, and thyroid hormones were exam- ined using Spearman rank-order correlation. Environmental Health Perspectives • volume 119 | number 10 | October 2011 The distributions of thyroid hormone, PBDEs, and metabolites were assessed for normality (Shapiro–Wilkes) and log-transformed if they were log-normally distributed. We imple- mented multiple linear regression analysis to assess the association between thyroid hormone levels and PBDE/metabolite levels, controlling for maternal characteristics known to influ- ence thyroid hormone levels, including smok- ing status (two categories of nonsmoker and ever-smoked before 28 weeks of gestation), race (non-Hispanic black or other), age (three cate- gories: 18–19, 20–24, and 25–39 years), ges- tational age at blood draw (weeks), and parity (operationalized as whether or not the infant was firstborn). In addition, we used logistic regression (adjusted for the same covariates) to examine whether PBDE or PBDE metabolite levels were associated with TT3 levels above the normal range in the general population (80–178 ng/dL) or FT4 levels below the normal range (0.52–1.21 ng/dL) and ordered logistic regression to estimate associations of PBDEs/ metabolites with low, normal, or high hormone TT4 levels (normal range 5.5–10.8 µg/dL). For TSH, the normal range in the general popula- tion was defined as 0.34–5.66 µIU/mL. Few observations fell outside the normal range for FT3 (2.2–3.8 pg/mL), precluding further analy- sis. Significance levels were set at p < 0.05. Results P o p u l a t i o n c h a r a c t e r i s t i c s . B e t w e e n September 2008 and June 2010, 140 preg- nant women enrolled in this study. Women were all residents of Durham County, North Carolina. Characteristics of the 137 partici- pants are presented in Table 1. Eighty percent Table 1. Cohort characteristics (n = 137). Characteristic n (%) Maternal race Non-Hispanic white 12 (9) Non-Hispanic black 110 (80) Hispanic 12 (9) Other 3 (2) Maternal age (years) 18–19 32 (23) 20–24 69 (50) 25–39 36 (26) Parity First birth 70 (51) Second birth 38 (28) Third birth 19 (14) Fourth birth 6 (4) Five or more 4 (3) Male infant sexa 62 (46) Maternal education Less than high school 19 (14) High school diploma 72 (53) More than high school 46 (34) Not married 127 (93) Smoking Nonsmoker 90 (66) Smoked before 28 weeks of gestation 47 (34) aTotal n = 133 because of missing values. Percentages may not sum to 100 because of rounding. 1455