2.4. Survey and Biospecimen Collection, Storage, and Analyses
BLOOD
Data collection included surveys (including sociodemographic and behavioral information as well as the ASA-24 described above), maternal blood, urine, and stool collection at three time points (24- and 36-weeks gestation [pre- and 4-weeks post-intervention initiation, respectively]; and at 6 weeks postpartum), and infant stool at age 6 weeks. Blood and urine specimens were obtained in the prenatal clinic and immediately aliquoted and stored at –80 °C. Fecal samples were self-collected at home and sent to the laboratory by mail. Fecal aliquots were stored at –80 °C upon reaching the lab. The average time between home sample collection and laboratory receipt was 3.8 ± 1.9 days (median = 3.5 days). We utilized the laboratory services provided by the National Institutes of Health Children’s Health Exposure Analysis Resource (CHEAR) Pilot and Feasibility (P&F) Program to measure plasma carotenoids and lipids, as well as urinary heavy metals and urinary metabolites. Trace elements reported in this manuscript were assessed from urine samples collected at 36-weeks gestation and were measured using an Agilent 8800 Triple Quadrupole ICP-MS (Santa Clara, CA, USA). These elements were analyzed in part to determine if metals serve as a marker for the presence of environmental contaminants, but they are reported here to show the feasibility of outcome assessment. Stool sample DNA extraction and amplification were performed in the laboratory of one of the study principal investigators (S Comstock). 16SrRNA gene sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core. All stool microbiome methods used this previously described protocol [