Age and Sex as Determinants of Acute Domoic Acid Toxicity in a Mouse Model.

The excitatory neurotoxin domoic acid (DA) consistently contaminates food webs in coastal regions around the world. Acute exposure to the toxin causes Amnesic Shellfish Poisoning, a potentially lethal syndrome of gastrointestinal- and seizure-related outcomes. Both advanced age and male sex have been suggested to contribute to interindividual DA susceptibility. To test this, we administered DA doses between 0.5 and 2.5 mg/kg body weight to female and male C57Bl/6 mice at adult (7-9-month-old) and aged (25-28-month-old) life stages and observed seizure-related activity for 90 min, at which point we euthanized the mice and collected serum, cortical, and kidney samples. We observed severe clonic-tonic convulsions in some aged individuals, but not in younger adults. We also saw an association between advanced age and the incidence of a moderately severe seizure-related outcome, hindlimb tremors, and between advanced age and overall symptom severity and persistence. Surprisingly, we additionally report that female mice, particularly aged female mice, demonstrated more severe neurotoxic symptoms following acute exposure to DA than males. Both age and sex patterns were reflected in tissue DA concentrations as well: aged mice and females had generally higher concentrations of DA in their tissues at 90 min post-exposure. This study contributes to the body of work that can inform intelligent, evidence-based public health protections for communities threatened by more frequent and extensive DA-producing algal blooms.

The Role of Nonhuman Primates in Neurotoxicology Research: Preclinical Models and Experimental Methods.

Although noteworthy progress has been made in developing alternatives to animal testing, nonhuman primates still play a critical role in advancing biomedical research and will likely do so for many years. Core similarities between monkeys and humans in genetics, physiology, reproduction, development, and behavior make them excellent models for translational studies relevant to human health. This unit is designed to specifically address the role of nonhuman primates in neurotoxicology research and outlines the specialized assessments that can be used to measure exposure-related changes at the structural, chemical, cellular, molecular, and functional levels. © 2023 Wiley Periodicals LLC.

Prolonged, Low-Level Exposure to the Marine Toxin, Domoic Acid, and Measures of Neurotoxicity in Nonhuman Primates.

BACKGROUND: The excitotoxic molecule, domoic acid (DA), is a marine algal toxin known to induce overt hippocampal neurotoxicity. Recent experimental and epidemiological studies suggest adverse neurological effects at exposure levels near the current regulatory limit (20 ppm, ∼0.075-0.1mg/kg). At these levels, cognitive effects occur in the absence of acute symptoms or evidence of neuronal death. OBJECTIVES: This study aimed to identify adverse effects on the nervous system from prolonged, dietary DA exposure in adult, female Macaca fascicularis monkeys. METHODS: Monkeys were orally exposed to 0, 0.075, and 0.15mg/kg per day for an average of 14 months. Clinical blood counts, chemistry, and cytokine levels were analyzed in the blood. In-life magnetic resonance (MR) imaging assessed volumetric and tractography differences in and between the hippocampus and thalamus. Histology of neurons and glia in the fornix, fimbria, internal capsule, thalamus, and hippocampus was evaluated. Hippocampal RNA sequencing was used to identify differentially expressed genes. Enrichment of gene networks for neuronal health, excitotoxicity, inflammation/glia, and myelin were assessed with Gene Set Enrichment Analysis. RESULTS: Clinical blood counts, chemistry, and cytokine levels were not altered with DA exposure in nonhuman primates. Transcriptome analysis of the hippocampus yielded 748 differentially expressed genes (fold change≥1.5; p≤0.05), reflecting differences in a broad molecular profile of intermediate early genes (e.g., FOS, EGR) and genes related to myelin networks in DA animals. Between exposed and control animals, MR imaging showed comparable connectivity of the hippocampus and thalamus and histology showed no evidence of hypomyelination. Histological examination of the thalamus showed a larger microglia soma size and an extension of cell processes, but suggestions of a GFAP+astrocyte response showed no indication of astrocyte hypertrophy. DISCUSSION: In the absence of overt hippocampal excitotoxicity, chronic exposure of Macaca fascicularis monkeys to environmentally relevant levels of DA suggested a subtle shift in the molecular profile of the hippocampus and the microglia phenotype in the thalamus that was possibly reflective of an adaptive response due to prolonged DA exposure. https://doi.org/10.1289/EHP10923.

Ice seals as sentinels for algal toxin presence in the Pacific Arctic and subarctic marine ecosystems.

Domoic acid (DA) and saxitoxin (STX)-producing algae are present in Alaskan seas, presenting exposure risks to marine mammals that may be increasing due to climate change. To investigate potential increases in exposure risks to four pagophilic ice seal species (Erignathus barbatus, bearded seals; Pusa hispida, ringed seals; Phoca largha, spotted seals; and Histriophoca fasciata, ribbon seals), this study analyzed samples from 998 seals harvested for subsistence purposes in western and northern Alaska during 2005-2019 for DA and STX. Both toxins were detected in bearded, ringed, and spotted seals, though no clinical signs of acute neurotoxicity were reported in harvested seals. Bearded seals had the highest prevalence of each toxin, followed by ringed seals. Bearded seal stomach content samples from the Bering Sea showed a significant increase in DA prevalence with time (logistic regression, p = .004). These findings are consistent with predicted northward expansion of DA-producing algae. A comparison of paired samples taken from the stomachs and colons of 15 seals found that colon content consistently had higher concentrations of both toxins. Collectively, these results suggest that ice seals, particularly bearded seals (benthic foraging specialists), are suitable sentinels for monitoring HAB prevalence in the Pacific Arctic and subarctic.

Public health risks associated with chronic, low-level domoic acid exposure: A review of the evidence.

Domoic acid (DA), the causative agent for the human syndrome Amnesic Shellfish Poisoning (ASP), is a potent, naturally occurring neurotoxin produced by common marine algae. DA accumulates in seafood, and humans and wildlife alike can subsequently be exposed when consuming DA-contaminated shellfish or finfish. While strong regulatory limits protect people from the acute effects associated with ASP, DA is an increasingly significant public health concern, particularly for coastal dwelling populations, and there is a growing body of evidence suggesting that there are significant health consequences following repeated exposures to levels of the toxin below current safety guidelines. However, gaps in scientific knowledge make it difficult to precisely determine the risks of contemporary low-level exposure scenarios. The present review characterizes the toxicokinetics and neurotoxicology of DA, discussing results from clinical and preclinical studies after both adult and developmental DA exposure. The review also highlights crucial areas for future DA research and makes the case that DA safety limits need to be reassessed to best protect public health from deleterious effects of this widespread marine toxin.

Cytokine and chemokine responses to injury and treatment in a nonhuman primate model of hypoxic-ischemic encephalopathy treated with hypothermia and erythropoietin.

Predicting long-term outcome in infants with hypoxic-ischemic encephalopathy (HIE) remains an ongoing clinical challenge. We investigated plasma biomarkers and their association with 6-month outcomes in a nonhuman primate model of HIE with or without therapeutic hypothermia (TH) and erythropoietin (Epo). Twenty-nine Macaca nemestrina were randomized to control cesarean section (n = 7) or 20 min of umbilical cord occlusion (UCO, n = 22) with either no treatment (n = 11) or TH/Epo (n = 11). Initial injury severity was scored using 30-min arterial pH, base deficit, and 10-min Apgar score. Twenty-four plasma cytokines, chemokines, and growth factors were measured 3, 6, 24, 72, and 96 h after UCO. Interleukin 17 (IL-17) and macrophage-derived chemokine (MDC) differentiated the normal/mild from moderate/severe injury groups. Treatment with TH/Epo was associated with increased monocyte chemotactic protein-4 (MCP-4) at 3 h-6h, and significantly lower MCP-4 and MDC at 24 h-72h, respectively. IL-12p40 was lower at 24 h-72h in animals with death/cerebral palsy (CP) compared to survivors without CP. Baseline injury severity was the single best predictor of death/CP, and predictions did not improve with the addition of biomarker data. Circulating chemokines associated with the peripheral monocyte cell lineage are associated with severity of injury and response to therapy, but do not improve ability to predict outcomes.

Maternal-fetal disposition of domoic acid following repeated oral dosing during pregnancy in nonhuman primate.

Domoic acid (DA) is a marine algal toxin that causes acute and chronic neurotoxicity in animals and humans. Prenatal exposure to DA has been associated with neuronal damage and cognitive and behavioral deficits in juvenile California sea lions, cynomolgus monkeys and rodents. Yet, the toxicokinetics (TK) of DA during pregnancy and the maternal-fetal disposition of DA have not been fully elucidated. In this study, we investigated the TK before, during, and after pregnancy and the maternal-fetal disposition of DA in 22 cynomolgus monkeys following daily oral doses of 0.075 or 0.15 mg/kg/day of DA. The AUC0-τ of DA was not changed while the renal clearance of DA was increased by 30-90% during and after pregnancy when compared to the pre-pregnancy values. DA was detected in the infant plasma and in the amniotic fluid at delivery. The infant plasma concentrations correlated positively with both the maternal plasma and the amniotic fluid concentrations. The paired infant-to-maternal plasma DA concentration ratios ranged from 0.3 to 0.6 and increased as a function of time which suggests placental efflux and longer apparent fetal half-life than the maternal half-life. The paired amniotic fluid-to-infant plasma DA concentration ratios ranged from 4.5 to 7.5 which indicates significant accumulation of DA in the amniotic fluid. A maternal-fetal TK model was developed to explore the processes that give the observed maternal-fetal disposition of DA. The final model suggests that placental transport and recirculation of DA between the fetus and amniotic fluid are major determining factors of the maternal-fetal TK of DA.

Chronic, low-level oral exposure to marine toxin, domoic acid, alters whole brain morphometry in nonhuman primates.

Domoic acid (DA) is an excitatory neurotoxin produced by marine algae and responsible for Amnesiac Shellfish Poisoning in humans. Current regulatory limits (˜0.075-0.1 mg/kg/day) protect against acute toxicity, but recent studies suggest that the chronic consumption of DA below the regulatory limit may produce subtle neurotoxicity in adults, including decrements in memory. As DA-algal blooms are increasing in both severity and frequency, we sought to better understand the effects of chronic DA exposure on reproductive and neurobehavioral endpoints in a preclinical nonhuman primate model. To this end, we initiated a long-term study using adult, female Macaca fascicularis monkeys exposed to daily, oral doses of 0.075 or 0.15 mg/kg of DA for a range of 321-381, and 346-554 days, respectively. This time period included a pre-pregnancy, pregnancy, and postpartum period. Throughout these times, trained data collectors observed intentional tremors in some exposed animals during biweekly clinical examinations. The present study explores the basis of this neurobehavioral finding with in vivo imaging techniques, including diffusion tensor magnetic resonance imaging and spectroscopy. Diffusion tensor analyses revealed that, while DA exposed macaques did not significantly differ from controls, increases in DA-related tremors were negatively correlated with fractional anisotropy, a measure of structural integrity, in the internal capsule, fornix, pons, and corpus callosum. Brain concentrations of lactate, a neurochemical closely linked with astrocytes, were also weakly, but positively associated with tremors. These findings are the first documented results suggesting that chronic oral exposure to DA at concentrations near the current human regulatory limit are related to structural and chemical changes in the adult primate brain.

Preclinical modeling of exposure to a global marine bio-contaminant: Effects of in utero Domoic acid exposure on neonatal behavior and infant memory.

Domoic Acid (DA) is a naturally-occurring marine neurotoxin that is increasingly recognized as an important public health issue. Prenatal DA exposure occurs through the maternal consumption of contaminated shellfish/finfish. To better understand the fetal risks associated with DA, we initiated a longitudinal, preclinical study focused on the reproductive and developmental effects of chronic, low-dose oral DA exposure. To this end, 32 adult female Macaca fascicularis monkeys were orally dosed with 0, 0.075 or 0.15 mg/kg/day DA on a daily basis prior to breeding and throughout breeding and pregnancy. The doses included the proposed human Tolerable Daily Intake (TDI) (0.075 mg/kg/day) for DA. Adult females were bred to nonexposed males. To evaluate development during early infancy, offspring were administered a Neonatal Assessment modeled after the human Neonatal Behavior Assessment Scale and a series of Visual Recognition Memory problems using the novelty paradigm. Results indicated that prenatal DA exposure did not impact early survival reflexes or responsivity to the environment. Findings from the recognition memory assessment, given between 1 and 2 months of age, showed that exposed and control infants demonstrated robust novelty scores when test problems were relatively easy to solve. Performance was not diminished by the introduction of delay periods. However, when more difficult recognition problems were introduced, the looking behavior of the 0.15 mg/kg DA group was random and infants failed to show differential visual attention to novel test stimuli. This finding suggests subtle but significant impairment in recognition memory and demonstrates that chronic fetal exposure to DA may impact developing cognitive processes.

Effects of oral domoic acid exposure on maternal reproduction and infant birth characteristics in a preclinical nonhuman primate model.

Domoic Acid (DA) is a naturally-occurring excitotoxin, produced by marine algae, which can bioaccumulate in shellfish and finfish. The consumption of seafood contaminated with DA is associated with gastrointestinal illness that, in the case of high DA exposure, can evolve into a spectrum of responses ranging from agitation to hallucinations, memory loss, seizures and coma. Because algal blooms that produce DA are becoming more widespread and very little is known about the dangers of chronic, low-dose exposure, we initiated a preclinical study focused on the reproductive and developmental effects of DA in a nonhuman primate model. To this end, 32 adult female Macaca fascicularis monkeys were orally exposed to 0, 0.075 or 0.15 mg/kg/day DA on a daily basis, prior to and during pregnancy. Females were bred to non-exposed males and infants were evaluated at birth. Results from this study provided no evidence of changes in DA plasma concentrations with chronic exposure. DA exposure was not associated with reproductive toxicity or adverse changes in the physical characteristics of newborns. However, in an unanticipated finding, our clinical observations revealed the presence of subtle neurological effects in the form of intentional tremors in the exposed adult females. While females in both dose groups displayed increased tremoring, the effect was dose-dependent and observed at a higher rate in females exposed to 0.15 mg/kg/day. These results demonstrate that chronic, low-level exposure to DA is associated with injury to the adult CNS and suggest that current regulatory guidelines designed to protect human health may not be adequate for high-frequency shellfish consumers.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Validated HPLC-MS/MS Method To Quantify Low Levels of Domoic Acid in Plasma and Urine after Subacute Exposure.

Domoic acid (DA) is a marine neurotoxin produced by several species of Pseudo-nitzschia. DA causes severe neurological toxicity in humans and animals. To address the current analytical need to quantify low levels of DA in human and animal body fluids, a sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed to measure DA in plasma and urine. This method was fully validated to accurately and precisely quantify DA between 0.31 and 16 ng/mL in plasma and between 7.8 and 1000 ng/mL in urine. Our group introduced the use of a novel internal standard, tetrahydrodomoic acid to control for matrix effects and other sources of variability. This validated method will be useful to assess DA concentrations in biological samples of human or animal origin after suspected DA exposure from contaminated food. It will also be applicable to sentinel programs and research studies to analyze body fluids with low levels of DA.

Cannabis use during pregnancy: Pharmacokinetics and effects on child development.

The broad-based legalization of cannabis use has created a strong need to understand its impact on human health and behavior. The risks that may be associated with cannabis use, particularly for sensitive subgroups such as pregnant women, are difficult to define because of a paucity of dose-response data and the recent increase in cannabis potency. Although there is a large body of evidence detailing the mode of action of Δ9-tetrahydrocannabinol (THC) in adults, little work has focused on understanding how cannabis use during pregnancy may impact the development of the fetal nervous system and whether additional plant-derived cannabinoids might participate. This manuscript presents an overview of the historical and contemporary literature focused on the mode of action of THC in the developing brain, comparative pharmacokinetics in both pregnant and nonpregnant model systems and neurodevelopmental outcomes in exposed offspring. Despite growing public health significance, pharmacokinetic studies of THC have focused on nonpregnant adult subjects and there are few published reports on disposition parameters during pregnancy. Data from preclinical species show that THC readily crosses the placenta although fetal exposures appear lower than maternal exposures. The neurodevelopmental data in humans and animals suggest that prenatal exposure to THC may lead to subtle, persistent changes in targeted aspects of higher-level cognition and psychological well-being. There is an urgent need for well-controlled studies in humans and preclinical models on THC as a developmental neurotoxicant. Until more information is available, pregnant women should not assume that using cannabis during pregnancy is safe.

Toxicokinetics and Physiologically Based Pharmacokinetic Modeling of the Shellfish Toxin Domoic Acid in Nonhuman Primates.

Domoic acid (DA), a neurotoxin, is produced by marine algae and has caused toxications worldwide in animals and humans. However, the toxicokinetics of DA have not been fully evaluated, and information is missing on the disposition of DA following oral exposures at doses that are considered safe for human consumption. In this study, toxicokinetics of DA were investigated in cynomolgus monkeys following single doses of 5 µg/kg DA intravenously, 0.075 mg/kg DA orally, and 0.15 mg/kg DA orally. After intravenous dosing, DA had a systemic clearance of 124 ± 71 (ml/h)/kg, volume of distribution at steady state of 131 ± 71 ml/kg and elimination half-life of 1.2 ± 1.1 hours. However, following oral dosing, the average terminal half-life of DA was 11.3 ± 2.4 hours, indicating that DA disposition follows flip-flop kinetics with slow, rate-limiting absorption. The absorption of DA was low after oral dosing with absolute bioavailability of 6% ± 4%. The renal clearance of DA was variable [21-152 (ml/h)/kg] with 42% ± 11% of the intravenous DA dose recovered in urine. A physiologically based pharmacokinetic model was developed for DA in monkeys and humans that replicated the flip-flop kinetics observed after oral administration and allowed simulation of urinary excretion and brain and kidney distribution of DA following intravenous and oral dosing. This study is the first to characterize DA disposition at exposure levels close to the current estimated tolerable daily intake and to mechanistically model DA disposition in a model species, providing important information of the toxicokinetics of DA for human safety assessment.

Effectiveness of community outreach and engagement in recruitment success for a prebirth cohort.

INTRODUCTION: We describe the effectiveness of community outreach and engagement in supporting recruitment for the US National Children's Vanguard Study between 2009 and 2012. METHODS: Thirty-seven study locations used 1 of 4 strategies to recruit 18-49-year-old pregnant or trying to conceive women: (1) Initial Vanguard Study used household-based recruitment; (2) Direct Outreach emphasized self-referral; (3) Enhanced Household-Based Recruitment enhanced Initial Vanguard Study strategies; and (4) Provider-Based Recruitment recruited through healthcare providers. Outreach and engagement included advance letters, interactions with healthcare providers, participation in community events, contacts with community organizations, and media outreach. RESULTS: After 1-2 years, 41%-74% of 9844 study-eligible women had heard about the National Children's Vanguard Study when first approached. Women who heard were 1.5-3 times more likely to consent. Hearing via word-of-mouth or the media most frequently predicted consent. The more sources women heard from the higher the odds of consent. CONCLUSIONS: We conclude that tailored outreach and engagement facilitate recruitment in cohort studies.

Neurobehavioral assessment of mice following repeated oral exposures to domoic acid during prenatal development.

Domoic acid (DA) is an algal toxin which has been associated with significant neurotoxicity in humans, non-human primates, rodents, and marine mammals. Developmental exposure to DA is believed to result in neurotoxicity that may persist into adulthood. DA is produced by harmful algal blooms of Pseudo-nitzschia, raising concerns about the consumption of contaminated seafood. We evaluated oral exposures to DA during pregnancy in mice. Doses of 0 (vehicle), 1 or 3mg/kg/d of DA were administered by gavage to C57BL/6J mice on gestational days 10 to 17. The offspring were tested for persistent neurobehavioral consequences during early development, adolescence and adulthood. Neurobehavioral tests revealed both dose- and gender-related differences in several neurobehavioral measures, including motor coordination in the rotarod test, behavior in the elevated plus maze, circadian patterns of activity in Phenotyper cages, gait as assessed in the Catwalk, and exploratory activity in the Morris water maze. This study demonstrated significant gender-specific and persistent neurobehavioral effects of repeated prenatal oral exposures to DA at low-dose levels that did not induce toxicity in dams.

A longitudinal study of hair cortisol concentrations in Macaca nemestrina mothers and infants.

Cortisol is a well-known glucocorticoid that can be used as a biomarker of hypothalamic-pituitary-adrenocortical activity. To explore basal cortisol physiology during pregnancy and infancy in Macaca nemestrina monkeys, hair was collected from a convenience sample of 22 healthy mother-infant dyads. Adult females were housed in pairs as part of a small breeding colony at the Washington National Primate Research Center and infants were reared in a specialized nursery. Maternal samples were collected from females during a pregnancy-detection ultrasound and immediately following labor and delivery. Infant samples were collected at birth, 20 days, 4, 6, 8, and 10 months of age. Hair cortisol concentrations (HCCs) were determined using an enzyme immunoassay in washed and ground hair samples. Like human mothers, macaque HCCs rose during pregnancy (paired t = 5.8, df = 16, P < 0.001). Maternal HCCs at pregnancy-detection (114.2 ± 12.07 picogram/milligram [pg/mg]) were highly predictive of maternal HCCs at delivery (144.8 ± 13.60 pg/mg), suggesting a trait-like quality (r = 0.90, P < 0.001). When maternal HCCs were viewed on a continuum, the absolute rise in cortisol over the course of pregnancy was significantly related to newborn HCCs (r = 0.55, P = 0.02). Infant birth HCCs (1,027.43 ± 97.95 pg/mg) were seven times higher than maternal HCCs at delivery (paired t = 19.1, df = 16, P < 0.001). Higher birth HCCs were strongly associated with larger decreases in infant hair cortisol until 6 months of postnatal age when infant HCCs converged on values indistinguishable from adults. Overall, study results demonstrate a marked degree of fetal cortisol exposure during the latter part of gestation and suggest that the rise in maternal cortisol over pregnancy may play an influential role on HCCs in the newborn.

Periconceptional seafood intake and pregnancy complications.

OBJECTIVE: To investigate associations of maternal periconceptional shellfish, lean fish and fatty fish intake with risk of pregnancy complications. DESIGN: In this prospective cohort study, we collected information on intake of seafood subtypes using FFQ. We categorized seafood intake into frequencies of 1 servings/week. We ascertained gestational hypertension, pre-eclampsia, gestational diabetes and preterm birth diagnoses from medical records. Using generalized linear models with a log link, the Poisson family and robust standard errors, we estimated risk ratios and 95 % confidence intervals across seafood intake categories. SETTING: The Omega study, a study of risk factors for pregnancy complications among women recruited from prenatal clinics in Washington State, USA, 1996-2008. SUBJECTS: The current study included 3279 participants from the Omega study. RESULTS: Median (interquartile range) shellfish, lean fish and fatty fish intake was 0·3 (0-0·9), 0·5 (0-1·0) and 0·5 (0·1-1·0) servings/week, respectively. Lean fish intake of >1 servings/week (v. <0·2 servings/month) was associated with a 1·55-fold higher risk of preterm birth (95 % CI 1·04, 2·30) and was not associated with the other pregnancy complications. Higher intake of seafood (total or other subtypes) was not associated with pregnancy complications (separately or combined). CONCLUSIONS: Higher intake of lean fish, but not fatty fish or shellfish, was associated with a higher risk of preterm birth; these findings may have significance for preterm birth prevention. Studies of mechanisms and potential contributing factors (including seafood preparation and nutrient/contaminant content) are warranted.

Periconceptional Seafood Intake and Fetal Growth.

BACKGROUND: Previous reports of associations of maternal seafood intake with fetal growth were inconsistent. Further, little is known whether associations differ across seafood subtypes or fetal growth indices. METHODS: Among 3141 participants of the Omega study, a pregnancy cohort study, we investigated associations of periconceptional shell, lean, and fatty fish intake with fetal growth indices. We categorised food frequency questionnaire reported seafood intake into frequencies of: <0.2 servings/month, 0.2 servings/month -<0.5 servings/week, 0.5-1 servings/week, and >1 servings/week. We abstracted birthweight, birth length, and head circumference from medical records. Using generalised linear models with a log link, the Poisson family, and robust standard errors, we estimated relative risks and 95% confidence intervals (CI) for low birthweight (LBW, <2500 g) and linear regression models to estimate mean differences for continuous fetal growth indices across seafood intake categories. RESULTS: Medians (interquartile range) of shell, lean, and fatty fish intake were 0.3 (0-0.9), 0.5 (0-1.0), and 0.5 (0.1-1.0) servings/week, respectively. Lean fish intake of >1 servings/week (vs. <0.2 servings/month) was associated with a 2.2-fold higher risk of LBW (95% CI 1.2, 4.1). Shellfish intake of >1 servings/week (vs. <0.2 servings/month) was associated with a 0.6 kg/m(3) higher mean ponderal index (95% CI 0.0, 1.2 kg/m(3) ). There was no evidence for associations of total seafood or seafood subtype intake with other fetal growth indices. CONCLUSIONS: Higher intakes of lean fish and shellfish were associated with a higher risk of LBW and higher mean ponderal index, respectively. Findings highlight the importance of considerations of seafood subtype in similar investigations.