Study subjects are a subset of a cohort of women enrolled in a study of outcomes in children exposed to large amounts of alcohol in utero
. Detailed methods for identifying the full cohort of study participants are described elsewhere (Aros et al., 2006
). Briefly, women (n = 9,628) presenting for prenatal care at the Maipú Clinic in Santiago, Chile, between August 1995 and July 2000, were questioned about alcohol use. There were 887 women who gave answers suggestive of risky alcohol use (see Aros et al. for details) who we further evaluated with home visits. Using information from the home visits, we identified 101 pregnant women who were consuming ≥48g alcohol/day on average during pregnancy. We used the same method to identify a control group of non-drinking pregnant women (n = 101) matched for age (± 2 years) and parity (0 or ≥1). None of the women included in this study had a history of chronic hypertension, pregestational or gestational diabetes, gestational hypertension, or preeclampsia.
The larger study described above was underway when sample collection for the current investigation began. Random urine specimens were collected during pregnancy from 26 of the matched drinking and non-drinking pairs. In addition, unpaired urine specimens were collected from 3 drinking gravidas and 13 non-drinking gravidas. Thus, samples were available for a total of 29 drinkers and 39 non-drinkers. Urine samples were stored at −70° C until analysis.
Subjects were classified as FAS if they demonstrated evidence of growth deficiency, characteristic facial dysmorphology, and neurodevelopmental abnormalities as described by Jones in 1973 (Jones et al., 1973
). Growth deficiency was defined as weight or length ≤ 10th
percentile at any one or more assessments in infancy or childhood. Facial dysmorphology was determined by serial examinations by a geneticist. Specific features required for diagnosis were short palpebral fissure length, thin vermilion border, and flat philtrum. Neurodevelopmental impairment was based on evidence of microcephaly, results of developmental and cognitive testing (Bayley Scales of Infant Development II, Wechsler Preschool and Primary Scale of Intelligence, and Wechsler Intelligence Scale for Children), serial examinations by pediatric neurologists, and evaluation by a pediatric psychologist. We considered a child to have partial FAS if he exhibited abnormalities in two of the above three domains.
Free 8-isoprostane F2α (8-iso-P), 2,3-dinor-6-keto-prostaglandin F1α (2,3-dinor), and 11-dehydro-thromboxane B2 (11-dehydro-TxB2) were quantified as previously described using highly accurate and precise mass spectrometric methods (Daniel et al., 1994
; Morrow and Minton, 1993
; Morrow and Roberts, 1999
). Precision of each of the three assays was +/− 4%, +/− 5%, and +/− 7%, respectively. Intra- and inter-day variabilities were < 10%. All results were corrected for intersubject differences in renal function and are expressed in ng/mg urinary creatinine. All analyses were conducted by personnel blinded to the alcohol exposure status of the subjects.
Visual inspection of the data demonstrated an outlying (>3.2 standard deviations above the mean) 8-iso-P value in a control subject; this value was deleted from our final analyses.
Baseline characteristics of the drinking and non-drinking subjects were compared using the Wilcoxon Rank Sum Test for continuous variables, and Fisher’s Exact Test for categorical variables. Multiple linear regression was used to compare transformed values of eicosanoid levels after adjusting for maternal age, marital status, number of cigarettes smoked per day, and gestational age at sampling. To express the result in more meaningful units, the eicosanoid values were first log-transformed until approximately normally distributed, and then standardized by the control group mean and standard deviation. Regression analyses were conducted on both the set of matched pairs, and on the larger, unmatched study sample. Because the results of the matched and unmatched analyses did not differ substantially, only the unmatched analyses are presented. All data were analyzed using SAS v. 9.0 (SAS System, Cary, NC). A P value of < 0.05 was considered statistically significant.
Written informed consent was obtained from all study participants. This study was approved by the Institutional Review Boards of both the National Institute of Child Health and Human Development and the San Borja Arriaran Hospital, which is affiliated with the University of Chile School of Medicine.