1 Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
2 Division of Epidemiology, Statistics, and Prevention Research, National Institute of Child Health and Human Development, Rockville, MD.
3 Chemical Exposure and Molecular Biology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
4 National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA.
5 Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
Received for publication February 7, 2002; accepted for publication October 1, 2002.
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ABSTRACT |
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infant; mental status schedule; motor skills; polychlorinated biphenyls; prenatal exposure delayed effects
Abbreviations: Abbreviations: DDE, dichlorodiphenyldichloroethylene; MDI, Mental Development Index; PCB, polychlorinated biphenyl; PDI, Psychomotor Development Index.
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INTRODUCTION |
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Studies of low-level exposure to PCBs in utero in relation to neurodevelopment have produced various results. Among infants, the Bayley Scales of Infant Development have been among the most frequently used instruments for assessing mental and psychomotor development. Low-level exposure to PCBs in utero has been modestly associated with a decreased Bayley Psychomotor Development Index (PDI) among infants in studies from the United States and Europe (911). In the Netherlands, however, the inverse association between PCBs and the PDI observed among children at 3 months of age had attenuated by 7 months (11); and in Michigan, no association was observed among children 5 months of age (12). The Mental Development Index (MDI), measured by the Bayley Scales, has been inversely associated with PCBs in only one cohort in Germany (13). Delays in other aspects of development, such as infants visual recognition memory measured by the Fagan Test of Infant Intelligence and intelligence quotient measured among older children, have been detected in relation to PCB exposure in some studies (12, 14, 15) but not confirmed in others (16). Inconsistencies among results have been attributed to variation in the age at examination, examination protocol, variation in the total concentration or composition of PCB mixtures, the inability to distinguish acute high-level exposure from chronic low-level exposure in total PCB measures (which may present different associated risks), and limited control for social or environmental influences (14, 17).
We evaluated the relation between PCB exposure and mental and psychomotor development at 8 months of age, using the Bayley Scales of Infant Development in a cohort of children born to women participating in the Collaborative Perinatal Project. Participants from the 12 study centers offered a large demographically diverse population whose PCB levels varied within the background range of 40 years ago, and for whom the Bayley assessment instrument was administered to offspring in a standardized manner.
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MATERIALS AND METHODS |
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Children were eligible for the present study if they were a liveborn singleton, had a 3-ml third trimester maternal serum sample available, and had completed the Bayley Scales of Infant Development at the age of 8 months. From the 43,628 eligible children, 1,065 were selected at random. An additional 194 children were selected at random from among the children scoring more than 1 standard deviation below or above the mean Bayley Psychomotor Development Index in the total study population. If some or all subjects are selected using outcome-dependent sampling and the data are analyzed using a newly available statistical procedure, then greater statistical power can be obtained than if the total sample were selected using simple random sampling (19). In this report, however, we used standard, more straightforward, methods to account for the sampling, because additional power would not have altered our main conclusions.
Exposure assessment
PCB levels have been shown to be highly correlated between maternal and cord serum at birth (r > 0.7) (20). Maternal nonfasting blood was collected every 8 weeks during pregnancy, at delivery, and 6 weeks postpartum. Sera were stored in glass at 20°C, with no recorded thaws. Because third trimester samples were available for the greatest number of mothers, these samples were analyzed for PCBs. The correlations for lipid-adjusted PCBs among serial measures, such as first and third trimester specimens, were high (Pearsons r 0.77) (21). Eleven PCB congeners (International Union of Pure and Applied Chemistry designations: PCBs 28, 52, 74, 105, 118, 138, 153, 170, 180, 194, 203) were analyzed in 19971999 using solid-phase extraction followed by dual-column gas chromatography with electron capture detection at the Centers for Disease Control and Prevention (22). Total PCB exposure represented the sum of the 11 measured congeners, expressed as µg/liter of serum ("wet weight"). Specimens were analyzed in batches of 10, each including an aliquot from a single large pool of serum to assess interbatch variation (19 percent at a total PCB concentration of 3.49 µg/liter). Serum cholesterol and tri-glyceride levels, which affect serum PCB levels, were analyzed using standard enzymatic assays.
Outcome measurement
The Bayley Scales of Infant Development were used to assess the infants mental and psychomotor development at 8 months of age. The Bayley MDI assesses age-appropriate cognitive, language, and social development. The Bayley PDI assesses fine and gross motor development (23). Testing procedures were standardized across centers and conducted by specially trained personnel. Dr. Bayley made regular site visits to monitor testing procedures (24). Tests were edited on site and again after the forms were received by the National Institutes of Health in Bethesda, Maryland. Raw scores were converted to age-standardized scores for these analyses (23). Most infants (93 percent) were tested between 7.5 and 9 months of age. Seven children (0.6 percent) were tested before 7.5 months; the remaining 6.4 percent were tested between 9 and 10 months of age.
Data analysis
The relations between maternal third trimester PCB levels and the childs standardized MDI and PDI were evaluated using linear regression analysis with PCBs modeled as a continuous variable. The data met the assumptions necessary to support the use of linear models. The PCB-neurodevelopment relation was estimated for children from all centers combined and by each study center separately.
Total PCBs were also categorized by concentration intervals of 1.25 µg/liter to produce five groups. The interval for the highest exposure category was wider, however, in order to achieve ample representation for analysis. Adjusted mean developmental scores (MDI and PDI) were calculated for each category of maternal PCB using generalized linear models. Trend significance was also evaluated by linear regression of an ordinal variable that represented the five levels of PCB exposure. All primary analyses were weighted by the inverse sampling probability to account for the sampling scheme (25).
Covariates were evaluated as potential confounders in this analysis if they were important in the design of the study, were related to both PCBs and neurodevelopment in these data, or had been reported as confounders in other comparable studies. The covariates evaluated included study center; maternal race (White, Black, other); education (less than high school, high school, greater than high school); socioeconomic index (continuous scale); intelligence quotient (continuous); marital status (married, single, divorced, widowed); prenatal smoking (none, <10 cigarettes per day, 10 cigarettes per day); prepregnancy body mass index (underweight, normal, overweight, obese); third trimester serum triglyceride, total cholesterol, and dichlorodiphenyldichloroethylene (DDE) levels (continuous); the childs birth order (first born compared with others); gestational age (continuous); and whether the child ever breastfed (yes/no). Only the design variable (center) and covariates that changed the effect estimate for the PCB-neurodevelopment relation by more than 10 percent in these data were included as confounders in the final models presented.
To assess heterogeneity in the PCB-neurodevelopment relation among centers, we added product terms for PCB (continuous) by each study center to the primary linear regression models. Estimates summarizing the PCB effects over all centers were calculated using both traditional linear regression and multilevel models (i.e., random-effects models). The random-effects models adjusted the covariance structure for the clustering of PCB levels and covariates within centers and were run in SAS software (SAS Institute, Inc., Cary, North Carolina). In addition, we fit a multilevel model that assessed the individual- and center-level influences of race, breastfeeding, and PCB congener ratios using BUGS software (26, 27). To determine whether the results were sensitive to the sampling scheme, we also conducted all analyses on the random sample only.
The ratio of PCB 118 to PCB 153 has been shown to vary considerably across the studies that have measured multiple PCB congeners (28). To determine whether the PCB-Bayley relation depended on congener mixture, we evaluated the interaction of total PCBs with the ratio of PCB 118 to PCB 153, and at the center level, we examined whether the ratio was related to center-specific differences in the PCB-Bayley relation. We also evaluated each of the 11 measured PCB congeners in separate models for the total study population. Individual assessment of all congeners in the same model was not possible because seven were highly correlated (Spearmans correlation coefficient > 0.6 for PCBs 74, 105, 118, 138, 153, 170, and 180); however, we created one variable that reflected the sum of the concentration of the highly correlated congeners and ran a model that included the PCB-combination variable as well as the remaining four congeners (PCBs 28, 52, 194, and 203) separately to evaluate the independent effects of those congeners.
Our analysis included 1,207 children. Those excluded were missing information on maternal serum PCB concentration (n = 37) or cholesterol (n = 1), maternal education (n = 12), or parity (n = 2). Those missing PCB levels appeared demographically similar to those with measured levels.
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RESULTS |
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We did not detect an association between maternal serum PCB level and the childs MDI (ß = 0.1, p = 0.71) or PDI (ß = 0.5, p = 0.14) (table 2). The mean PDI scores appeared to increase slightly with increasing PCB levels (p = 0.09). Estimates of the relation between PCBs and Bayley scores were adjusted for the mothers triglycerides, cholesterol, and education and for the childs birth order (MDI crude ß = 0.1, p = 0.62; or PDI crude ß = 0.5, p = 0.13). Inclusion in the model of the other covariates evaluated, including maternal serum DDE, race, prepregnancy body mass index, and breastfeeding, did not affect the associations (data not shown). Inclusion of gestational age in the multivariate model strengthened the direct association between PCB levels and MDI (ß = 0.2, p = 0.41) and PDI (ß = 0.6, p = 0.06) (data not shown), but this was not considered further because it could act as an intermediate on the causal pathway. Results were similar when restricted to nonbreastfed children (n = 950; MDI (ß = 0.2, p = 0.60) and PDI (ß = 0.4, p = 0.21)), which excluded those with postnatal exposure through breast milk. Transforming the continuous PCB variable by log (dose + 1) did not alter results. The summary estimates from the multilevel models, which adjusted the covariance structure within and between centers, were similar to those from the fixed effects models. Results were also similar when restricted to the random sample, which excluded children sampled specifically because of their very low or very high Bayley scores.
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DISCUSSION |
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The baseline developmental scores, PCB levels, and the change in developmental score due to PCBs differed with respect to study center. Although maternal serum PCB levels were not related to Bayley scores among most centers, increased PCBs were associated with a decreased PDI in New Orleans and Baltimore and, contrary to expectation, an increased PDI in Richmond and Providence.
The statistical significance of the heterogeneity was not robust, with detection depending on the analytic method. Differences among centers were most pronounced for the PDI when all children, from the random sample and the outcome-dependent sample, were included. Although the overall results and interpretation did not differ on the basis of the source of the sample, the outcome-dependent sample increased the power to detect heterogeneity among centers. We did not anticipate heterogeneity when designing this study. By selecting subjects in our outcome-dependent sample according to their placement on the PDI distribution of the total study population, we found that these subjects tended to be from centers with high or low mean PDIs. Had we used center-specific sampling probabilities to select these and the simple random sample subjects, the sample could have been better balanced and more statistically efficient. Our sampling approach, however, combined with a weighted analysis caused no bias.
There was considerable demographic heterogeneity across centers. Both race and breastfeeding rates varied by center, but there was no pattern of variation in the relation between PCBs and neurodevelopment explained by individual- or center-specific race or breastfeeding rates in the multilevel models examined. In addition, results were similar when restricted to nonbreastfed children, who were the majority. Although the center effectively served as a proxy for characteristics such as race and breastfeeding, which did not prove to be important factors in this analysis, the center may have also served as a proxy for important unmeasured characteristics, such as diet, mercury, or lead exposure. The two sites with the most striking adverse associations were New Orleans and Baltimore. It is possible that women in these coastal cities consumed more fish, which if contaminated with mercury could have had a synergistic relation with PCBs in adversely affecting development. Yet, because no inverse relation was observed in Boston, Providence, or New York, which are also coastal cities where fish is consumed, such an explanation is not supported. Results from the multilevel models with random terms for PCBs reflected the variation in results by center but were not markedly different from the results from the more standard fixed effects models. In addition, adjusting for center as a random effect variable to account for clustering of the individuals characteristics within centers had little effect on results.
Exposure variability is another potential explanation for variation in results across centers. The sites showing adverse associations, however, were not those with the highest total PCB concentrations (table 3). Although unlikely, variation in the composition of congeners comprising the mixture could have affected our results. Our ability to evaluate the congener-specific effects was limited by the relatively small sample size for each center and the correlation among congener levels. Although the ratio of PCB 118 to PCB 153 ranged from 0.7 to 1.2 across centers, variation in the relation between PCB and neurodevelopment by the ratio of these two congeners was not statistically significant at the individual level or site level.
Variability in testing protocol and examiners among study sites could have also affected the PCB-neurodevelopment results; however, Bayley testing procedures were carefully standardized across study sites and regularly monitored by the test developer and other experts. The Bayley Scales performed as expected in relation to other characteristics known to influence childrens development, such as socioeconomic indicators, preterm birth, and birth weight (2931). We suspect that any differences in testing procedures across study sites were minimal and would not vary by PCB level within a site nor account for important differences in the PCB-Bayley relation across study sites.
The difference in the PCB-PDI relation among study sites remains unexplained. It is possible that the unknown factors responsible for heterogeneity among sites in this study may also be responsible for differences between our overall results and those of studies showing an inverse relation (911).
The PCB-Bayley relations reported in previous studies have varied by the timing of exposure (pre- vs. postnatal), the timing of the childs examination, and the outcome associated with PCBs (usually PDI, not MDI). In the Netherlands, prenatal PCB exposure was associated with a decreased PDI at 3 months of age, but not at 7 or 18 months, yet postnatal PCB exposure was associated with a decreased PDI at 7 months of age (11). A larger cohort from North Carolina reported a subtle but consistent inverse association between prenatal PCB exposure and the PDI at 6, 12, 18, and 24 months of age but no association between postnatal PCB exposure and MDI scores (9, 10). Other studies have shown relations between PCBs and neurodevelopment among very young infants and among older children using assessments such as the Fagan Test of Infant Intelligence or McCarthy Scales (12, 14, 17); these instruments, compared with the Bayley Scales, may be more sensitive for detecting subtle neurodevelopmental effects that may be related to PCBs.
Most studies have accounted for socioeconomic influences, but the factors measured and methods used to adjust analyses have varied. Some have suggested that associations with PCBs may be more difficult to detect among children from lower socioeconomic households because such factors as poor parenting, nutrition, or lead exposure may have greater impact on neurodevelopment relative to PCBs (14, 17). Likewise, positive factors, such as stimulating home environment and breastfeeding, could counter any negative effects of PCBs in homes of higher socioeconomic status. Our study included children from a variety of socioeconomic backgrounds, and our analyses accounted for relevant differences by social factors, but the overall PCB-Bayley relation did not differ by the socioeconomic indicators in this study.
Other potential explanations for the differences among studies include the type and timing of the exposure and the biologic sample from which PCBs were measured. Breastfeeding confers postnatal PCB exposure. We did not have information on PCB levels in breast milk or breastfeeding duration. Yet, only 15 percent of the women in this study population ever breastfed their infant, limiting the proportion of children exposed to PCBs postnatally through breast milk, and results were similar when restricted to the nonbreastfed children. Some suggest breastfeeding as the most important source of exposure to PCBs among children (12); however, the critical time period for PCB activity in relation to a childs neurodevelopment remains controversial (17).
The ratio of PCB 118 to PCB 153 has varied across similar studies and geographic regions. In this study, the PCB 118:PCB 153 ratios from all the centers were generally higher than those reported by other studies of PCBs. Although PCB levels have dropped in the United States since these data were collected, the levels in the Collaborative Perinatal Project from 40 years ago appear similar to those in some European countries today (28). For example, the median level of PCB 153 measured from a larger sample of this study population around 1960 was 140 ng/g of lipid. In the early 1990s, PCB 153 levels measured in a New York population were much lower (40 ng/g of lipid), but levels measured in Germany (140 ng/g of lipid) were similar (28). Thus, we did not find a relation between PCBs and neurodevelopment at PCB levels comparable with those of studies that reported adverse effects (911).
In summary, prenatal exposure was indicated by PCB levels in maternal third trimester sera. Despite standardization across study centers of 1) the timing of and procedures related to the childs Bayley examination and 2) the analytic methods for quantifying PCBs, which are two important factors suspected to be responsible for variation among studies, we found variability in the association between the PCB and PDI across study centers. Thus, the variation in results across studies may not be attributable to differences in timing or measurement of the exposure or outcome. A more plausible explanation is that study populations vary with respect to other factors, such as nutrition or other environmental toxicants, which might confound or modify the effects of PCBs. Given the heterogeneity in results across the centers within this study and among other distinct studies, the relation between low-level prenatal PCB exposure and the childs early mental and motor development remains unclear.
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NOTES |
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REFERENCES |
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