Prenatal exposure to methyl mercury from fish consumption and polyunsaturated fatty acids: associations with child development at 20 mo of age in an observational study in the Republic of Seychelles

ABSTRACT

Background: Fish is a rich source of n–3 polyunsaturated fatty acids (PUFAs) but also contains the neurotoxicant methyl mercury (MeHg). PUFAs may modify the relation between prenatal MeHg exposure and child development either directly by enhancing neurodevelopment or indirectly through the inflammatory milieu.

Objective: The objective was to investigate the associations of prenatal MeHg exposure and maternal PUFA status with child development at 20 mo of age.

Design: The Seychelles Child Development Study Nutrition Cohort 2 is an observational study in the Republic of Seychelles, a high-fish-eating population. Mothers were enrolled during pregnancy and their children evaluated at 20 mo of age by using the Bayley Scales of Infant Development II (BSID-II), the MacArthur Bates Communicative Development Inventories (CDI), and the Infant Behavior Questionnaire–Revised. There were 1265 mother-child pairs with complete data.

Results: Prenatal MeHg exposure had no direct associations with neurodevelopmental outcomes. Significant interactions were found between MeHg and PUFAs on the Psychomotor Developmental Index (PDI) of the BSID-II. Increasing MeHg was associated with lower PDI but only in children of mothers with higher n–6/n–3. Among mothers with higher n–3 PUFAs, increasing MeHg was associated with improved PDI. Higher maternal docosahexaenoic acid (DHA) was associated with improved CDI total gestures (language development) but was significantly adversely associated with the Mental Development Index (MDI), both with and without MeHg adjustment. Higher n–6:n–3 ratios were associated with poorer scores on all 3 CDI outcomes.

Conclusions: We found no overall adverse association between prenatal MeHg exposure and neurodevelopmental outcomes. However, maternal PUFA status as a putative marker of the inflammatory milieu appeared to modify the associations of prenatal MeHg exposure with the PDI. Increasing DHA status was positively associated with language development yet negatively associated with the MDI. These findings may indicate the existence of an optimal DHA balance with respect to arachidonic acid for different aspects of neurodevelopment.

child development, maternal fish consumption, methyl mercury, polyunsaturated fatty acids, docosahexaenoic acid, arachidonic acid, n–6:n–3 ratio, Mental Developmental Index, Psychomotor Developmental Index, language development

INTRODUCTION

The risks and benefits of fish consumption during pregnancy have received much attention in recent years (1–4). Studies have predominantly focused on methyl mercury (MeHg)⁵ exposure as a potential risk from fish consumption and on the n–3 PUFA, DHA, as a potential benefit. Although prenatal MeHg is a well-documented neurotoxicant at high doses, the extent to which low-level exposure from fish consumption affects child development remains controversial. Results from prospective mother-child cohorts in the Republic of Seychelles have consistently shown no adverse associations between prenatal MeHg exposure and children’s subsequent development (5–11). Other large studies in the United Kingdom and Spain have reported similar findings (12, 13), whereas studies from New Zealand, the Faroe Islands, and the United States have reported adverse developmental influences of prenatal MeHg exposure (14–17). These inconsistencies may be related to variability in study designs, populations, genetic susceptibility, biomarkers of exposure or coexposures, or other factors. Alternatively, the benefits of fish consumption may outweigh or mask any potential adverse effects of MeHg on developmental outcomes (8).

In a smaller previous cohort [Nutrition Cohort 1 (NC1)] we reported improved psychomotor performance with better prenatal status of n–3 PUFAs at 9 and 30 mo (8, 9, 18) and improved verbal skills and comprehension at 5 y of age (11). In that cohort, children exposed to higher prenatal n–6 PUFAs had poorer outcomes. However, in that study, there was no evidence to support the hypothesis that higher prenatal MeHg exposure was associated with adverse outcomes over 5 y of follow-up (11). On the basis of those findings, we hypothesized that the n–3 PUFA and other nutritional components associated with fish consumption might mask an association with prenatal MeHg exposure or that the nutritional benefits might exceed possible neurotoxicity. The toxic effects of MeHg on the developing brain are considered to be mediated by oxidative damage, which in turn causes inflammation (19). As both n–6 and n–3 PUFAs compete for the same enzymes in biosynthetic pathways and incorporation into cell membranes, the relative amounts of these PUFAs available in the diet are important for determining the physiologic n–6/n–3 balance. Furthermore, a high n–6:n–3 ratio in favor of n–6 PUFAs, which are more proinflammatory than n–3 PUFAs, may augment any possible inflammatory insults that might result from MeHg exposure in the brain (20).

Accordingly, we enrolled a larger cohort (Nutrition Cohort 2; NC2) to confirm our earlier findings of no adverse effects of prenatal MeHg exposure and to clarify the role of prenatal PUFA status in either enhancing neurodevelopment and/or modifying any association with MeHg.

METHODS

Study design and characteristics

NC2 is part of the Seychelles Child Development Study, a multicohort observational study with the overall aim of investigating associations between prenatal MeHg exposure and child development. NC2 was conducted between 2008 and 2011 on Mahé, the main island of the Republic of Seychelles. Mothers were recruited during their first antenatal visit (from 14 wk of gestation) at 8 health centers between January 2008 and January 2011. Power calculations were based on the magnitudes of associations observed for the Bayley Scales of Infant Development II (BSID-II) Psychomotor Developmental Index (PDI) at 9 and 30 mo in our earlier NC1 cohort (8, 18) by using a 2-sided significance level of α = 0.05; power for interaction models dichotomized PUFAs at the median. We determined that a cohort of 1200 mother-child pairs would be sufficient to detect an interaction between n–6 PUFAs and MeHg on the 30-mo PDI with 81% power, between n–3 PUFAs and MeHg on the 9-mo PDI with 99% power, and between the n–6:n–3 ratio and MeHg on the 9-mo PDI with 99% power. A cohort size of 1200 would also be sufficient to detect main effects of n–3 and the n–6:n–3 ratio on both the 9-mo and the 30-mo PDI with 99% power. Inclusion criteria for NC2 included being native Seychellois, being ≥16 y of age, having a singleton pregnancy, and having no obvious health concerns. The study was reviewed and approved by the Seychelles Ethics Board and the Research Subjects Review Board at the University of Rochester. We used quality control procedures at all collaborating study sites to ensure the integrity of the data and included independent data verification, data management, and statistical analysis. All investigators and staff other than those measuring MeHg exposure or directly preparing for and carrying out statistical analyses were blinded to MeHg data. Investigators measuring MeHg exposure and PUFA status were blinded to developmental outcome data. Statistical analysis plans based on biological hypotheses were developed a priori and are described in the statistical analysis section.

Blood sampling and PUFA analysis

At 28 wk of gestation, nonfasting maternal blood samples were collected. Samples were processed promptly and stored at −80°C until analyzed, as previously described (8). Blood samples were shipped at −80°C to the University of Ulster for serum total PUFA analysis, which was undertaken according to an adaptation of the method by Folch et al. (21). Fatty acid methyl esters were detected and quantified by using the gold-standard technique of gas chromatography–mass spectrometry (7890A-5975C; Agilent) using heptadecanoic acid (C17:0) as the internal standard, as previously described (8). All analytic standards were of ≥99% purity and purchased from Sigma-Aldrich. Total serum PUFA status was chosen as a biomarker to encompass recent PUFA concentrations of the triacylglycerol fraction, to which the majority of circulating PUFAs are bound during pregnancy and circulate to the fetus (22). We measured the individual PUFAs—linoleic acid, arachidonic acid (AA), α-linolenic acid, EPA, and DHA—and results were presented as milligrams per milliliter to indicate physiologic quantities.

Methyl mercury exposure

At delivery, maternal hair samples were collected to determine prenatal MeHg exposure. Total mercury was measured by the standard technique of atomic absorption spectroscopy at the University of Rochester in the longest hair segment available to reflect exposure throughout pregnancy. Hair was assumed to grow at a rate of 1.1 cm/mo (7). Mercury deposited in hair is ∼80% MeHg and is known to correlate with mercury deposited in the infant brain (7). Therefore, we refer to this measurement as the prenatal MeHg exposure.

Developmental assessment

When infants were about 20 mo of age (range: 15.9–28.4 mo), they completed the BSID-II, a well-established measure of cognition and development previously administered to previous Seychelles Child Development Study cohorts. The BSID-II Mental Developmental Index (MDI) and PDI are scaled scores obtained by direct examination of the child. Testing was implemented by specially trained nurses at the Child Development Centre, Mahé. All study forms were shipped to the University of Rochester and double entered. Data from the 2 BSID-II endpoints were scaled according to child age at testing. Interobserver reliability for the BSID-II was determined for about 10% of the cohort by comparing the independent test scoring of 2 nurses. The median agreement on scored MDI and PDI items was 100%.

At testing, mothers completed questionnaires on the Infant Behavior Record–Revised (IBQ-R), a measure of infant and toddler social and play behaviors, and the MacArthur-Bates Communicative Development Inventories (CDI), a measure of social communication and early language development. Children were older than the normative group for the IBQ-R, but some were too young for the toddler version of this instrument, so we administered the IBQ-R and checked each outcome for ceiling effects. Similarly, many children were older than the normative group for the CDI when tested, so we analyzed only the 3 outcomes that did not show ceiling effects: total gestures, vocabulary produced, and vocabulary understood. We used raw scores for the 3 IBQ-R subscales: surgency, negative affect, and effortful control. We also used raw scores for CDI analysis as have other authors (23). For vocabulary produced and vocabulary understood, raw scores were square root transformed to meet regression model assumptions.

Statistical analysis

We calculated measures of central tendency and variability to describe demographic, exposure, nutritional, and developmental characteristics of mothers and children. We computed Pearson correlations between prenatal MeHg exposure and PUFA status and carried out linear regression to evaluate the main and interactive effects of MeHg and PUFAs on outcomes. In main effects models, we evaluated DHA and AA because these PUFAs are considered to have a direct influence on brain development (20). In the MeHg by PUFA interaction models, we used total n–3, total n–6, and the n–6:n–3 ratio because the balance of these PUFAs can influence the inflammatory response to MeHg toxicity in the developing brain (24), and in turn might modify MeHg toxicity. We evaluated the n–6:n–3 ratio in models both with and without an interaction with MeHg. All models were fit by using the statistical package R version 3.0.2 (www.r-project.org).

We examined the main effects of MeHg and PUFAs on developmental outcomes with and without adjustment for each other. We then examined interactions between MeHg and tertiles of total n–3 and total n–6 PUFAs and, in a separate model, interactions between MeHg and tertiles of the n–6:n–3 ratio. The main effects models used PUFAs as continuous variables, whereas we used PUFA tertiles in the interaction model for interpretability, particularly to compare MeHg slopes among subjects with low, medium, or high PUFA concentrations. Model assumptions were checked by using standard methods (25), which included checking for linearity and constant variance and normality of the residuals. All models were adjusted for covariates known to be associated with child development (7)—namely, maternal age, child age at testing, child sex, Hollingshead socioeconomic status, and number of parents living with the child (family status). In secondary regression models, we also adjusted for mother’s cognitive ability [Kaufman Brief Intelligence Test (KBIT)] and the child’s home environment [Pediatric Review of Children’s Environmental Support and Stimulation (PROCESS)]. These data were available on only a subset of mothers (n = 1155 for KBIT and n = 1070 for PROCESS). To evaluate whether differences in MeHg and PUFA effects between the primary and secondary models resulted from adjustment for KBIT and PROCESS or from the different sample sizes, we also fit models by using the smaller set of observations without adjusting for KBIT and PROCESS. We used 2-sided α = 0.05 to determine statistical significance. The analyses were specified in an a priori analysis plan, developed before model fitting.

RESULTS

We recruited a total of 1535 mother-child pairs and conducted primary analyses on 1265 with complete covariate data after exclusions and a measure of at least one outcome (Figure 1). Summary statistics for selected maternal characteristics, prenatal MeHg and PUFA status, child outcomes, and model covariates in the cohort analyzed (n = 1265) are presented in Table 1. Mothers reported consuming an average of 8.5 fish meals per week during pregnancy, as assessed by a Fish Use Questionnaire. Pearson correlation analysis showed prenatal MeHg exposure to be positively correlated with serum concentrations of several n–3 PUFAs: α-linolenic acid (r = 0.07, P = 0.01), EPA (r = 0.08, P < 0.01), and DHA (r = 0.11, P < 0.01). The main effects associations between MeHg and PUFAs with BSID-II outcomes are presented in Table 2, and those with CDI and IBQ-R outcomes are shown in Table 3. Results from interaction models are presented in Table 4.

FIGURE 1

Description of exclusions and missing data for the current analysis. BSID-II, Bayley Scales of Infant Development II; CDI, Communicative Development Inventories; IBQ-R, Infant Behavior Questionnaire–Revised; MeHg, methyl mercury.

TABLE 1

Summary statistics for selected maternal characteristics, infant cognitive outcomes at 20 mo of age, and model covariates¹

Variable	n	Mean ± SD	Minimum	Maximum
Mothers
Maternal age at enrollment, y	1265	26.85 ± 6.33	16.03	46.56
Estimated weekly fish meals	1208	8.52 ± 4.56	0.00	37.00
Hair MeHg, ppm	1265	3.92 ± 3.46	0.00	31.66
Serum PUFAs, mg/mL
Linoleic acid	1265	0.90 ± 0.25	0.31	2.34
AA	1265	0.20 ± 0.08	0.04	0.38
α-Linolenic acid	1265	0.04 ± 0.01	0.00	0.11
EPA	1265	0.05 ± 0.01	0.05	0.12
DHA	1265	0.19 ± 0.09	0.04	0.52
Total n–3 PUFAs	1265	0.27 ± 0.09	0.12	0.64
Total n–6 PUFAs	1265	1.11 ± 0.30	0.43	2.71
n–6:n–3 ratio	1265	4.36 ± 1.63	1.56	15.81
AA:DHA ratio	1265	1.26 ± 0.77	0.13	7.93
Developmental outcomes
BSID-II MDI, scaled score	1243	87.88 ± 10.62	49.00	118.00
BSID-II PDI, scaled score	1241	96.90 ± 10.40	49.00	125.00
CDI total gestures	1265	46.46 ± 8.36	2.00	63.00
CDI vocabulary produced	1265	123.60 ± 87.60	0.00	388.00
CDI vocabulary understood	1265	233.39 ± 90.93	0.00	396.00
IBQ-R surgency	1264	5.38 ± 0.75	2.15	7.00
IBQ negative affect	1264	4.12 ± 1.00	1.00	7.00
IBQ effortful control	1264	5.18 ± 0.74	1.00	7.00
Covariates
Child age at testing, mo	1265	20.71 ± 1.34	15.90	28.39
Hollingshead SES	1265	32.01 ± 10.31	11.00	63.00
Family status	1265	0.74 ± 0.44	0.00	1.00
KBIT scaled	1155	87.08 ± 17.03	40.00	126.00
PROCESS	1070	156.41 ± 16.47	96.00	200.00

1

Total n–3 PUFAs = sum of α-linolenic acid + EPA + DHA; total n–6 PUFAs = sum of linoleic acid + AA. AA, arachidonic acid; BSID-II, Bayley Scales of Infant Development II; CDI, Communicative Development Inventories; IBQ, Infant Behavior Questionnaire; IBQ-R, Infant Behavior Questionnaire–Revised; KBIT, Kaufman Brief Intelligence Test; MDI, Mental Developmental Index; MeHg, methyl mercury; PDI, Psychomotor Developmental Index; ppm, parts per million; PROCESS, Pediatric Review of Children’s Environmental Support and Stimulation; SES, socioeconomic status.

TABLE 2

Main effects models for prenatal MeHg exposure and PUFA status variables and the BSID-II MDI and PDI at 20 mo of age¹

	20-mo MDI									20-mo PDI
	MeHg + PUFAs (n = 1243)			PUFAs only (n = 1243)			MeHg only (n = 1243)			MeHg + PUFAs (n = 1241)			PUFAs only (n = 1241)			MeHg only (n = 1241)
	β	SE	P	β	SE	P	β	SE	P	β	SE	P	β	SE	P	β	SE	P
DHA + AA
MeHg	−0.06	0.08	0.46				−0.08	0.08	0.31	0.02	0.09	0.80				0.03	0.09	0.68
DHA	−9.73	4.33	0.02*	−10.11	4.30	0.02*				5.54	4.4	0.21	5.67	4.37	0.19
AA	8.13	4.47	0.07	8.34	4.46	0.06				−2.4	4.54	0.60	−2.48	4.53	0.58
Maternal age	−0.07	0.05	0.17	−0.07	0.05	0.15	−0.08	0.05	0.10	−0.12	0.05	0.01*	−0.12	0.05	0.02*	−0.11	0.05	0.02*
Hollingshead SES	0.12	0.03	<0.01*	0.12	0.03	<0.01*	0.13	0.03	<0.01*	0.04	0.03	0.19	0.04	0.03	0.19	0.03	0.03	0.24
Child sex (girl)	2.98	0.58	<0.01*	2.99	0.58	<0.01*	2.95	0.58	<0.01*	1.67	0.59	<0.01*	1.67	0.59	<0.01*	1.69	0.59	<0.01*
Child age	−1.57	0.23	<0.01*	−1.56	0.22	<0.01*	−1.59	0.22	<0.01*	−0.49	0.23	0.03*	−0.49	0.23	0.03*	−0.45	0.22	0.04*
Family status	0.76	0.68	0.26	0.77	0.68	0.26	0.81	0.68	0.23	−1.4	0.69	0.04*	−1.4	0.69	0.04*	−1.43	0.69	0.04*
n–6:n–3 ratio
MeHg	−0.07	0.08	0.39				−0.08	0.08	0.31	0.03	0.09	0.70				0.03	0.09	0.68
n–6:n–3 ratio	0.38	0.18	0.04*	0.39	0.18	0.03*				−0.07	0.18	0.72	−0.07	0.18	0.70
Maternal age	−0.07	0.05	0.13	−0.08	0.05	0.11	−0.08	0.05	0.10	−0.11	0.05	0.02*	−0.11	0.05	0.02*	−0.11	0.05	0.02*
Hollingshead SES	0.12	0.03	<0.01*	0.12	0.03	<0.01*	0.13	0.03	<0.01*	0.04	0.03	0.23	0.04	0.03	0.23	0.03	0.03	0.24
Child sex (girl)	2.97	0.58	<0.01*	2.98	0.58	<0.01*	2.95	0.58	<0.01*	1.69	0.59	<0.01*	1.68	0.59	<0.01*	1.69	0.59	<0.01*
Child age	−1.53	0.22	<0.01*	−1.51	0.22	<0.01*	−1.59	0.22	<0.01*	−0.46	0.22	0.04*	−0.47	0.22	0.04*	−0.45	0.22	0.04*
Family status	0.74	0.68	0.27	0.75	0.68	0.27	0.81	0.68	0.23	−1.42	0.69	0.04*	−1.42	0.69	0.04*	−1.43	0.69	0.04*

1

Estimated regression coefficients and P values are shown. *Significant associations, P < 0.05. AA, arachidonic acid; BSID-II, Bayley Scales of Infant Development II; MDI, Mental Developmental Index; MeHg, methyl mercury; PDI, Psychomotor Developmental Index; SES, socioeconomic status.

TABLE 3

Main effects models for prenatal MeHg exposure and PUFA status variables and the MacArthur-Bates CDI and IBQ-R at 20 mo of age¹

	MacArthur-Bates CDI									IBQ-R
	Sqrt (vocabulary produced) (n = 1265)			Sqrt (vocabulary understood) (n = 1265)			Total gestures (n = 1265)			Surgency (n = 1264)			Negative affect (n = 1264)			Effortful control (n = 1264)
	β	SE	P	β	SE	P	β	SE	P	β	SE	P	β	SE	P	β	SE	P value
DHA + AA
MeHg	−0.03	0.03	0.37	−0.01	0.03	0.82	−0.07	0.07	0.27	−0.00	0.01	0.93	−0.01	0.01	0.35	−0.00	0.01	0.99
DHA	−0.53	1.76	0.76	1.78	1.31	0.17	10.26	3.37	<0.01*	0.15	0.31	0.63	−0.03	0.42	0.94	0.15	0.31	0.62
AA	2.38	1.81	0.19	2.07	1.35	0.13	0.65	3.48	0.85	0.46	0.32	0.16	0.17	0.44	0.70	0.07	0.32	0.82
Maternal age	−0.07	0.02	<0.01*	−0.04	0.01	<0.01*	−0.06	0.04	0.10	−0.00	0.00	0.33	−0.01	0.00	0.06	0.01	0.00	0.07
Hollingshead SES	0.02	0.01	0.09	−0.02	0.01	0.07	0.1	0.02	<0.01*	0.01	0.00	<0.01*	0.01	0.00	0.01*	0.01	0.00	<0.01*
Child sex (girl)	1.49	0.23	<0.01*	0.70	0.17	<0.01*	3.22	0.45	<0.01*	−0.04	0.04	0.37	0.04	0.06	0.46	0.08	0.04	0.06
Child age	0.76	0.09	<0.01*	0.36	0.07	<0.01*	0.99	0.17	<0.01*	0.01	0.02	0.38	−0.04	0.02	0.08	0.01	0.02	0.54
Family status	0.54	0.27	0.05	0.23	0.2	0.26	0.49	0.53	0.35	0.05	0.05	0.28	−0.05	0.07	0.46	0.07	0.05	0.14
n–6:n–3 ratio
MeHg	−0.04	0.03	0.28	−0.01	0.03	0.82	−0.06	0.07	0.33	0.00	0.01	1.00	−0.01	0.01	0.36	−0.00	0.01	0.99
n–6:n–3 ratio	−0.17	0.07	0.02*	−0.16	0.05	<0.01*	−0.54	0.14	<0.01*	0.00	0.01	0.93	0.01	0.02	0.73	−0.01	0.01	0.31
Maternal age	−0.07	0.02	<0.01*	−0.03	0.01	0.02*	−0.04	0.04	0.24	−0.00	0.00	0.50	−0.01	0.00	0.06	0.01	0.00	0.05
Hollingshead SES	0.02	0.01	0.05*	−0.01	0.01	0.10	0.1	0.02	<0.01*	0.01	0.00	<0.01*	0.01	0.00	0.01*	0.01	0.00	<0.01*
Child sex (girl)	1.48	0.23	<0.01*	0.70	0.17	<0.01*	3.23	0.45	<0.01*	−0.04	0.04	0.38	0.04	0.06	0.46	0.08	0.04	0.06
Child age	0.76	0.09	<0.01*	0.39	0.07	<0.01*	1.05	0.17	<0.01*	0.02	0.02	0.15	−0.04	0.02	0.09	0.01	0.02	0.50
Family status	0.56	0.27	0.04*	0.24	0.2	0.24	0.52	0.53	0.32	0.05	0.05	0.30	−0.05	0.07	0.45	0.07	0.05	0.13

1

Estimated regression coefficients and P values are shown. *Significant associations, P < 0.05. AA, arachidonic acid; CDI, Communicative Development Inventories; IBQ-R, Infant Behavior Questionnaire–Revised; MeHg, methyl mercury; SES, socioeconomic status; Sqrt, square root transformation.

TABLE 4

Interaction models for prenatal MeHg exposure against BSID-II MDI and PDI at 20 mo of age with PUFA status¹

	20-mo MDI (n = 1243)			20-mo PDI (n = 1241)
	MeHg β	SE	P	MeHg β	SE	P
Interaction with n–3 PUFAs
n–3 PUFA lowest tertile	0.09	0.16	0.59	−0.23	0.16	0.15
n–3 PUFA middle tertile	−0.17	0.14	0.24	−0.11	0.14	0.42
n–3 PUFA highest tertile	−0.11	0.14	0.43	0.37	0.14	<0.01*
Hg by n–3 interaction P value			0.47			<0.01*
Interaction with n–6/n–3 ratio
n–6/n–3 PUFA lowest tertile	−0.04	0.13	0.78	0.19	0.13	0.15
n–6/n–3 PUFA middle tertile	−0.06	0.15	0.70	0.14	0.15	0.36
n–6/n–3 PUFA highest tertile	−0.12	0.17	0.48	−0.37	0.17	0.03*
Hg by n–6/n–3 ratio interaction P value			0.93			0.02*

1

Estimated regression coefficients and P values are shown. *Significant associations, P < 0.05. Models shown were adjusted for maternal age, child age, child sex, Hollingshead socioeconomic status, and family status. High n–3 PUFAs, >0.308 mg/mL; medium n–3 PUFAs, 0.228–0.308 mg/mL; low n–3 PUFAs, <0.228 mg/mL. Serum concentrations for the tertiles were as follows: high n–6/n–3, >4.496; medium n–6/n–3, 3.523–4.496; low n–6/n–3, <3.523. BSID-II, Bayley Scales of Infant Development II; MDI, Mental Developmental Index; MeHg, methyl mercury; PDI, Psychomotor Developmental Index.

MeHg associations

Prenatal MeHg exposure both with and without adjustment for PUFAs was not associated with any test score (Tables 2 and 3). In models that included total n–3 and total n–6 and did not adjust for KBIT and PROCESS, there were no statistically significant interactions between MeHg and n–6 PUFA tertiles for any outcome. Therefore, we report the results from these models with MeHg and n–3 PUFA tertile interactions only (Table 4). For the MDI, the interactions between MeHg and n–3 PUFAs were not significant (P = 0.47; 2 df test). However, for the PDI, there was a significant MeHg by n–3 interaction (P < 0.01), indicating that the MeHg effect differed across tertiles of n–3 PUFAs (Figure 2A). For subjects in the low and medium n–3 PUFA tertiles, increased MeHg exposure was not significantly associated with lower PDI scores. However, among subjects in the highest tertile of n–3 PUFAs, the estimated MeHg slope showed a significant improvement in PDI scores with increasing MeHg exposure (P < 0.01). The MeHg by n–3 PUFA tertile interactions were also significant (P = 0.05) in secondary models adjusted for KBIT and PROCESS with similar MeHg slopes (data not shown).

FIGURE 2

Interactions between maternal hair mercury and tertiles of maternal serum n–3 PUFAs (mg/mL) (A) and maternal n–6:n–3 ratio (B) on the BSID-II PDI score, with superimposed mercury slopes and tertile-specific P values from the covariate-adjusted regression (n = 1241). The maternal hair mercury slopes within tertiles of maternal serum n–3 PUFA (A) and within tertiles of n–6/n–3 (B) were significantly different from each other (P < 0.01 and P = 0.02, respectively, for the 2 df tests). Serum concentrations for the tertiles were as follows: high n–3 PUFA, >0.308 mg/mL; medium n–3 PUFA, 0.228–0.308 mg/mL; low n–3 PUFA, <0.228 mg/mL. High n–6/n–3, >4.496; medium n–6/n–3, 3.523–4.496; low n–6/n–3, <3.523. BSID-II, Bayley Scales of Infant Development II.

In primary interaction models of n–6:n–3 ratio tertiles, the interactions between MeHg and n–6/n–3 were not significant for the MDI (P = 0.93; 2 df test) (Table 4). For the PDI, however, there were significant MeHg by n–6/n–3 interactions (P = 0.02), indicating that the MeHg direct association differed across n–6:n–3 ratio tertiles (Figure 2B). However, increased MeHg exposure was significantly associated with lower scores among subjects in the highest n–6:n–3 ratio tertile only (P = 0.03), indicating an adverse MeHg association. The estimated MeHg slopes for MDI and PDI within n–6:n–3 ratio tertiles were similar in secondary models adjusted for KBIT and PROCESS (data not shown).

There were no significant direct associations between MeHg and any of the CDI outcomes (Table 3), including total gestures (P = 0.27) (Figure 3A), nor were there any significant interactions between MeHg and PUFAs for any of the CDI and IBQ-R outcomes (data not shown).

FIGURE 3

Associations between the 20-mo McArthur-Bates CDI total gestures score and maternal hair mercury (slope = −0.07, SE = 0.07, P = 0.27) (A), maternal DHA status (mg/mL serum) (slope = 10.26, SE = 3.37, P < 0.01) (B), and maternal n–6:n–3 ratio (slope = −0.54, SE = 0.14, P < 0.01) (C), with superimposed slopes from the covariate-adjusted regression (n = 1265). CDI, Communicative Development Inventories.

PUFA associations

In main effect models, DHA was significantly adversely associated with the MDI score both with and without MeHg adjustment (Table 2). In the primary model adjusting for MeHg, with each 0.1-mg/mL serum increase in DHA, the MDI score was estimated to decline by 0.97 points. The n–6:n–3 ratio was significantly associated with an improved MDI score with or without adjustment for MeHg (Table 2). No significant associations between PUFAs and the PDI score were observed. Higher DHA was associated with improved total gestures (P < 0.01) (Figure 3B). Higher n–6:n–3 ratios were associated with poorer scores on all 3 CDI outcomes of vocabulary produced (P = 0.02), vocabulary understood (P < 0.01), and total gestures (P < 0.01) (Table 3, Figure 3C). IBQ-R scores were not significantly associated with PUFA status (Table 3).

Covariate associations

Child sex and age and Hollingshead socioeconomic status were associated with MDI scores, and maternal age, child sex and age, and family status were associated with PDI scores (Table 2).

DISCUSSION

The primary finding from this longitudinal observational study in the Seychelles was the absence of an overall association between MeHg exposure and child developmental outcomes at 20 mo of age. This finding did not change after adjusting for maternal PUFA status and confirms our findings from the NC1 and main cohorts, in which MeHg exposure had no consistent direct influence on cognitive outcomes during 5 and 19 y of follow-up, respectively (8, 26). Some studies have shown conflicting adverse neurotoxic effects of prenatal MeHg exposure (14–17), but such findings cannot be directly compared with the current study because of wide variability in study design, population, and choice of biomarkers of exposure or coexposures.

The current cohort was large enough to test the extent to which MeHg associations with outcomes are modified by PUFAs. There were 2 significant associations with the PDI: an adverse association of MeHg on the PDI at a high serum n–6:n–3 ratio and a beneficial association with MeHg at high n–3 status. These findings suggest the n–6 to n–3 PUFA balance is important to consider when studying MeHg associations at these levels of exposure and may reflect the capability of n–6 PUFAs or n–3 PUFAs, at higher concentrations, to augment or counteract, respectively, MeHg-induced inflammation.

The n–6:n–3 ratio can be regarded as an indirect measure of inflammation, reflecting the potential for greater production of n–6 PUFA–derived eicosanoids, which are more proinflammatory than those derived from n–3 PUFAs. The physiologic effects of a higher n–6:n–3 ratio have been associated with increased systemic inflammation and increased risk of disease (24). Other prospective studies have reported adverse associations of a high maternal n–6:n–3 ratio (high n–6 PUFAs) with child development, including attention problems at age 5–6 y (27) and language at age 2 y (28). Another cross-sectional study of children aged 7–9 y reported that associations between intake of n–3 PUFAs and performance on some outcomes of the Cambridge Neuropsychological Test Assessment Battery varied by n–6:n–3 ratio (29). Data from these studies, using dietary estimates of PUFAs (28, 29), agree with results of our study, in which biological measures of PUFA status were used. Biological measures of PUFA status could be described as more robust than dietary data and differ in that they reflect various physiologic factors that are known to influence PUFA status, such as mobilization of maternal adipose tissue stores during pregnancy (30). There is no currently recommended biological n–6/n–3 ratio, but ratios in the current cohort (4.36; range: 1.56–15.81) are lower than most Western populations as a result of a high dietary intake of n–3 PUFAs from fish (24). Nevertheless, our results suggest that if there are adverse developmental associations with the effects of prenatal MeHg exposure at these levels, they may depend on the physiologic balance of n–6 to n–3 PUFAs. If true, the mechanisms of this association deserve further study.

This study also suggests that PUFA status, independent of MeHg, may be associated with improved communication. Increased maternal DHA concentrations were associated with an improved CDI vocabulary understood score. In contrast, increased maternal n–6/n–3 ratios were associated with poorer scores on all 3 CDI scores. These results suggest a higher maternal DHA status, and a lower n–6/n–3 ratio may be beneficial for child language and communication skills. These data support our earlier finding in the NC1 cohort at age 5 y in which, on another test of language development, the Preschool Language Scale, scores improved with increasing DHA (11). Other studies of children at a similar age and that used the CDI reported that language skills improved with higher n–3 PUFAs (12, 31) and maternal fish intake (12). We did not predict lower test scores on the MDI with increasing maternal DHA, and this finding conflicts with findings from the earlier NC1 cohort in which we found no significant effects of PUFAs on the MDI (8, 9, 11). One explanation might be that AA becomes the limiting nutrient for some aspects of fetal development when DHA concentrations are high, such as in high fish-eating populations, whereas DHA is the limiting nutrient in populations consuming low quantities of fish. This hypothesis was proposed following studies in Tanzania, where tribes with differing levels of fish consumption were studied. These studies, which assessed erythrocyte PUFAs during pregnancy and postpartum, reported a synergistic relation between DHA and AA with low DHA status and an antagonistic relation with high DHA status (32–34).

Our study has a number of strengths. The cohort was large and enrolled specifically to address the complex relationships of fish consumption, MeHg exposure, and nutritional status. Mothers were early in pregnancy and followed prospectively. Mothers consumed large quantities of fish, resulting in mean MeHg exposures about 10 times higher than in US (35) or UK women (36). Robust physiologic measures of both MeHg exposure and PUFA status were used as previously described, and the cohort size permitted investigation of interactions between maternal MeHg exposure and PUFA status. The study also has limitations. It was an observational study and thus cannot determine causation, as unmeasured covariates might have confounded some relationships. Missing covariates reduced the size of the cohort available for analysis, although there was no meaningful difference in demographic and exposure characteristics between those who were and were not included.

We found no adverse associations between prenatal MeHg exposure from fish consumption and child development through 20 mo of age in this study, even though the mothers consumed large quantities of fish and had exposures about 10 times those of Western nations. These results confirm our past MeHg findings in multiple cohorts from this population, support the importance of maternal PUFA status, and suggest that the developmental consequences of exposure to MeHg from fish consumption and maternal PUFA status are more complex than previously thought.

We thank Jean Reeves and Joanne Janciuras from the University of Rochester for their assistance with database management.

The authors’ responsibilities were as follows—JJS: had full access to all data in the study, with the exception of mercury data, and took responsibility for data integrity and the accuracy of data analysis, as well as the decision to submit for publication; JJS, EvW, EMM, GEW, THS, CFS, GJM, and PWD: were involved in study concept, design, and funding acquisition; CFS, JH, KY, and EvW: were involved in fieldwork and acquisition of the data; EvW, SWT, TML, and DH: conducted the statistical analysis and interpretation of data; EvW, AJY, SWT, MSM, GEW, and DH: conducted quality control assessment and were responsible for analysis and interpretation of data; JJS and AJY: drafted the manuscript; and EvW, SWT, MSM, EMM, GEW, TML, THS, KY, DH, CFS, JH, GJM, and PWD: contributed to critical revision of the manuscript. All authors read and approved the final manuscript. All authors declared no conflicts of interest related to this study.

FOOTNOTES

2

" Supported by the NIH (grants R01-ES010219 and P30-ES01247) and inkind support from the government of Seychelles.

3

" The study sponsors had no role in the design, collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

ABBREVIATIONS

 
• AA

  arachidonic acid

 
• BSID-II

  Bayley Scales of Infant Development II

 
• CDI

  Communicative Development Inventory

 
• IBQ-R

  Infant Behavior Questionnaire–Revised

 
• KBIT

  Kaufman Brief Intelligence Test

 
• MDI

  Mental Developmental Index

 
• MeHg

  methyl mercury

 
• NC1

  Nutrition Cohort 1

 
• NC2

  Nutrition Cohort 2

 
• PDI

  Psychomotor Developmental Index

 
• PROCESS

  Pediatric Review of Children’s Environmental Support and Stimulation