Maternal and Cord Blood Manganese Concentrations and Early Childhood Neurodevelopment among Residents near a Mining-Impacted Superfund Site

Background: Environmental manganese exposure has been associated with adverse neurodevelopmental outcomes among school-aged children; yet, few studies have evaluated prenatal exposure. Objectives: Our study examines associations between prenatal manganese concentrations and placental transfer of manganese with neurodevelopment in 224 2-y-old children residing near the Tar Creek Superfund Site. Methods: We collected maternal and cord blood at delivery, measured manganese using inductively coupled plasma mass spectrometry, and assessed neurodevelopment using the Bayley Scales of Infant Development-II. Associations between manganese and mental (MDI) and psychomotor (PDI) development indices were estimated in multivariable models. Placental transfer, approximated by cord/maternal manganese ratio, cord/total manganese ratio (total=maternal+cord), and by joint classification according to high or low (above or below median) maternal and cord manganese, was evaluated as a predictor of neurodevelopment. Results: Median levels [interquartile ranges (IQR)] of manganese in maternal and cord blood, respectively, were 24.0 (19.5–29.7) and 43.1 (33.5–52.1) μg/L. Adjusting for lead, arsenic, and other potential confounders, an IQR increase in maternal manganese was associated with −3.0 (95% CI: −5.3, −0.7) points on MDI and −2.3 (95% CI: −4.1, −0.4) points on PDI. Cord manganese concentrations were not associated with neurodevelopment scores. Cord/maternal and cord/total manganese ratios were positively associated with MDI [cord/maternal: β=2.6 (95% Cl: −0.04, 5.3); cord/total: β=22.0 (95% Cl: 3.2, 40.7)] and PDI (cord/maternal: β=1.7 (95% Cl: −0.5, 3.9); cord/total: β=15.6 (95% Cl: 0.3, 20.9)). Compared to mother–child pairs with low maternal and cord manganese, associations with neurodevelopment scores were negative for pairs with either high maternal, high cord, or high maternal and cord manganese. Conclusions: Maternal blood manganese concentrations were negatively associated with early childhood neurodevelopment scores in our study. Findings highlight the importance of understanding maternal exposures during pregnancy and factors influencing placental transfer. https://doi.org/10.1289/EHP925


Introduction
Manganese (Mn) is a trace essential element, necessary for physiologic processes such as neuronal function (Prohaska 1987;Sloot and Gramsbergen 1994), protein and energy metabolism, bone growth (Aschner and Aschner 2005;Hurley 1981), and enzyme activation (Erikson and Aschner 2003). During fetal and neonatal development, there is an increased need for manganese due to its critical role in brain function and skeletal development (Hurley 1981). Manganese crosses the placenta via active transport (Yoon et al. 2009), likely reflecting fetal nutrient demand. However, excess or accumulated manganese exposure can be neurotoxic and has been associated with deficits in cognition and motor function (Sanders et al. 2015;Zoni and Lucchini 2013). Little is known about how manganese transfer from the mother is regulated. The way in which manganese is partitioned in the maternal/fetal unit may be an important factor in fetal development (Kopp et al. 2012).
Concerns about heightened potential sensitivity to manganese neurotoxicity during fetal and early life compared to adulthood have recently been raised. Research is complicated by the increased fetal demand for manganese during development, as well as the unique physiology of the fetus and infant in which rapid growth makes it susceptible to nutrient deficiency. Increasing maternal blood manganese levels during pregnancy may by a physiologic response to this fetal demand, but the optimal range of manganese levels has not been determined and it remains unclear at what level maternal blood manganese may become harmful to the fetus. Manganese crosses the blood-brain barrier in the fetus at a higher rate than in adults, based on animal experimental data (Cahill et al. 1980;Kostial et al. 1978;Takeda et al. 1999). Because of the developing brain's high oxygen and energy consumption, it is sensitive to oxidative stress and damage from free radicals that can result from elevated manganese exposure (Blomgren and Hagberg 2006;Buonocore et al. 2001;Ikonomidou and Kaindl 2011). Given that neurodevelopment occurs as a cascade of well-timed, regulated events, exposure to toxic insults can cause damage at any stage, which may impair subsequent processes and result in developmental disability (Nowakowski and Hayes 1999).
Environmental manganese exposure has been associated with various neurodevelopmental outcomes among school-age children (Bouchard et al. 2011;Khan et al. 2012;Oulhote et al. 2014a;Wasserman et al. 2006;Wright et al. 2006). Relatively few studies, however, have evaluated neurotoxic effects of prenatal manganese exposure. An umbilical cord serum manganese concentration greater than 5:0 lg=L was associated with poorer performance on neurobehavioral tests in a population-based study of 933 3-d-old neonates in China (Yu et al. 2014). An inverted U-shaped association was reported between maternal blood manganese at delivery and mental and psychomotor development scores among 232 6-mo-old Korean infants with no known exposure source (Chung et al. 2015). Prenatal exposure estimated in deciduous teeth was not associated with mental and psychomotor development scores among 197 six-to 12-mo-old Mexican-American children living in an agricultural area of California where use of Mn-containing fungicides is common (Gunier et al. 2015). In the same cohort, prenatal tooth manganese was associated with poorer behavioral scores in 248 school-age children, but positively associated with scores on tests of memory and cognition . High (>75th percentile) cord blood manganese concentrations were associated with worse cognitive, language, and overall neurodevelopment scores among 230 2-y old Taiwanese children with no known exposure source (Lin et al. 2013). Cord blood manganese levels were inversely associated with performance on psychomotor tests among 126 3-y-olds in France, whereas no association was observed with maternal blood manganese (Takser et al. 2003). Most prior studies, except Takser et al. (2003), relied on a single biomarker of prenatal manganese exposure, typically cord blood or serum.
Our study examines associations between prenatal manganese exposure and neurodevelopment among young children living near a former mining area in rural Oklahoma. The objectives were a) to estimate associations of manganese, measured in paired maternal-infant blood samples, with neurodevelopment at 2 y of age while adjusting for exposures to other metals; and b) to explore the role of placental transfer of manganese as a predictor of neurodevelopment.

Study Participants
Subjects were participants in a prospective birth cohort study of biologic markers of fetal and early childhood exposure to metals, maternal psychosocial stress, and their impact on neurodevelopment. This research was conducted in the area of the Tar Creek Superfund site in Ottawa County, Oklahoma. This Superfund site, a former lead and zinc mining area, contains numerous piles of mine waste enriched in metals that are dispersed throughout the region (ATSDR 2004;Schaider et al. 2007). Study location and objectives have been described elsewhere Zota et al. 2009). Briefly, pregnant women were recruited during prenatal visits or at delivery from the Integris Baptist Medical Center in Miami, Oklahoma. Mothers and offspring were followed until children were 7 y of age. Eligibility criteria included a) giving birth at Integris Hospital; b) intention to live within the study area for the next 2 y; c) not being currently enrolled in the study with another child; and d) having Englishlanguage proficiency sufficient to participate in the informed consent process. Eligible mothers received a detailed explanation of study procedures before consenting to participate. The research protocol was approved by the human subjects committees of Integris Baptist Medical Center and Harvard T.H. Chan School of Public Health (HSPH).
The original cohort included 713 mother-infant pairs, who were enrolled between 2002 and 2011. Biomarkers of prenatal manganese exposure data were available for 637 mother-infant pairs (5 pairs missing blood samples; 71 pairs excluded to avoid batch effects inherent in different instruments due to our laboratory's purchase of a new ICP-MS instrument near the end of the study). Neurodevelopment test scores at 2 y of age were available for 225 of these 637 pairs. One subject was excluded from this analysis due to scores that were more than 3 standard deviations below the expected means for both mental and psychomotor development, necessitating referral for intervention. A total of 224 mother-infant pairs were included in this analysis.

Prenatal Manganese Exposure Assessment
Prenatal exposure to manganese was estimated by measuring manganese concentrations in maternal blood and umbilical cord blood samples collected at the time of birth ( ± 12 hr). Blood collection procedures have been detailed elsewhere ). Briefly, venous whole blood from mothers and umbilical cord blood from the umbilical vein were collected in trace element-free tubes [BD Vacutainer royal blue top, with K2EDTA #368381 (Becton Dickinson)] following routine clinical procedures by delivery room staff and shipped frozen to the Trace Metals Laboratory at HSPH (Boston, MA). One milliliter of blood was digested with concentrated HNO 3 acid, followed by the addition of hydrogen peroxide and dilution with deionized water. We measured total manganese concentrations in blood with a dynamic reaction cell-inductively coupled plasma mass spectrometer (DRC-ICP-MS; Elan 6,100; PerkinElmer, Norwalk, CT) using previously published methods and quality control measures (Chen et al. 1999;Ettinger et al. 2009). Average recovery of quality control standards for manganese (NIST 1643e, 1 ppb CV, human hair GBW 07601) was 96-104%. Lead and arsenic concentrations were also measured in blood samples and considered as covariates in all analyses given their co-occurrence in environmental media at this site (Zota et al. 2011). The limit of detection (LOD) was 0:02 lg=dL for manganese, lead, and arsenic. All manganese and arsenic measurements were above the LOD. Three (1.3%) lead measurements in cord blood were below the LOD and were assigned a value of one-half the LOD.

Child Neurodevelopment Assessment
Child neurodevelopment was assessed at 2 y of age using the Bayley Scales of Infant Development-II (Bayley 1993). Ageadjusted scores from the Mental Development Index (MDI) and the Psychomotor Development Index (PDI) were used as the primary outcomes. Two trained study personnel administered the test using a standardized protocol and were overseen by a licensed psychologist (D.C.B.) and a graduate student clinical developmental psychologist. All study testers were blinded to the participants' exposure information.

Covariate Assessment
We used interviewer-administered questionnaires at the time of enrollment to collect information on sociodemographic characteristics, including maternal education, race/ethnicity, and smoking and alcohol consumption during pregnancy, as well as potential sources of metals exposure. Information on child's birth weight, head circumference, and gestational age at birth was abstracted from medical records. Gestational age at birth was based on clinical assessment using data from the last menstrual period, the first accurate ultrasound examination during the first trimester, and clinical examination (ACOG 2014). Hemoglobin and hematocrit concentrations were measured in a maternal blood sample collected within 12 hr of admission to labor and delivery according to routine clinical procedures (as well as an extra tube collected at this time for measurement of blood manganese). Maternal IQ was assessed using the Kaufman Brief Intelligence Test (KBIT) at 6-mo postpartum (Kaufman and Kaufman 1990).

Statistical Analysis
Distributional plots were examined and descriptive statistics were calculated for all variables. Bivariate associations were calculated between all exposures, outcomes, and covariates. The correlation between maternal and cord blood manganese was estimated using Spearman's r correlation coefficient. Characteristics of motherinfant pairs included in all analyses were compared to those excluded from analyses using t-tests for continuous variables and chi-square tests for categorical variables.
We estimated associations between prenatal manganese concentrations and neurodevelopment using multivariable regression. We examined potential nonlinear associations between manganese and neurodevelopment using generalized additive models with penalized splines (constrained to 4 knots). We used a likelihood ratio test comparing models with a smoothed manganese term to models with a linear manganese term to assess linearity of the manganese-neurodevelopment association. To address skewness, we used natural logarithmic-transformed metals concentrations in exposure-neurodevelopment models. We modeled manganese concentrations as continuous log e -transformed concentrations and compared the 25th to 75th percentile [interquartile range (IQR)]. Neurodevelopment test scores were normally distributed and analyzed as continuous variables.
Potential confounders were selected a priori based on previous literature and on established or plausible associations with neurodevelopment (Grandjean and Landrigan 2006;Lanphear et al. 2000;Tong et al. 2007). We included child sex, maternal education (≥12th grade vs: <12th grade), maternal IQ, maternal hemoglobin at time of delivery (g/dL), and concentrations of maternal or cord blood lead and arsenic, centered at the mean of the log e distribution and modeled as penalized splines (constrained to 4 knots). Maternal hemoglobin was used as a proxy for iron status, which is a potential confounder given that iron deficiency in the neonate can adversely impact neurodevelopment (Georgieff 2008) and that iron status may influence manganese levels (Gunshin et al. 1997). In sensitivity analyses, we considered the following additional potential confounders because they were associated with log e -transformed blood manganese levels or neurodevelopment scores in bivariate models (p < 0:10): maternal smoking during pregnancy (yes/no), gestational age at birth (weeks), maternal age at time of delivery (years), first-born child (yes/no), annual household income ($20,000-40,000, >$40,000 vs: <$20,000), maternal marital status (married/living with partner vs. never married/separated/divorced), and use of prenatal vitamins. Given the recent emphasis on sex-specific metal effects (Llop et al. 2013) and possible sex-related metabolic differences in manganese regulation (Oulhote et al. 2014b), we explored sex differences in the association between manganese and neurodevelopment by including an interaction term (child sexÃ manganese) in regression models.
Some participants were missing data on one or more key potential confounders (i.e., maternal IQ, maternal hemoglobin, maternal and cord arsenic levels). We used multiple imputation to impute missing values using chained equations with the MI procedure in SAS (SAS Institute Inc., Cary, NC, USA) (van Buuren 2007; White et al. 2011). We assumed data were missing at random, that is, that the missingness did not depend on the unobserved data. We generated 40 imputed data sets. In the imputation process, we included all exposure and outcome variables and covariates thought to be related to the process causing the missing data (see Table S1, for a list of variables). We combined results from models fit with the multiply imputed data sets by applying Rubin's formula (Rubin 2004) in R (R Foundation for Statistical Computing).
We explored the hypothesis that the efficiency with which manganese is transferred from mother to fetus is an important determinant of neurodevelopment, as has been posited previously (Kopp et al. 2012). We evaluated cord/maternal blood manganese ratio as well as cord/total blood manganese ratio (total = maternal + cord manganese) as predictors of neurodevelopment. Ratios were calculated using untransformed manganese concentrations. We also clustered mother-infant pairs into four groups, based on blood manganese concentrations dichotomized at the medians of the two distributions: a) low maternal/low cord, representing concordant low exposures; b) low maternal/high cord; c) high maternal/low cord; and d) high maternal/high cord, representing concordant high exposures. Using multivariable regression models, we estimated the associations between this categorical variable, a crude representation of placental transfer, and neurodevelopment. Models with ratios and with clustered mother-infant pairs were adjusted for the same set of a priori covariates as manganese-neurodevelopment models (i.e., child sex, maternal education, maternal IQ, maternal hemoglobin at time of delivery, and concentrations of maternal or cord blood lead and arsenic).
We conducted sensitivity analyses to evaluate the robustness of our findings. a) We ran models using complete cases only and compared results with those using the imputed datasets. b) We examined the influence of extreme values of prenatal manganese concentrations by fitting models with and without outliers identified using the generalized extreme Studentized deviate (ESD) many-outlier procedure, set to identify up to 10 outliers (Rosner 1983). c) We examined the potential confounding effects of additional sociodemographic characteristics (listed above) by rerunning models including these variables. For all statistical tests, the significance level was set at 5%. We conducted statistical analyses using SAS (version 9.4; SAS Institute Inc.) and R (version 3.1.2; R Foundation for Statistical Computing). Table 1 shows characteristics of mother-infant pairs included in all analyses, as well as those excluded from analyses due to missing exposure and/or neurodevelopment data. Included pairs (n = 224) differed from excluded pairs (n = 489) on several characteristics: mothers included were older at time of delivery, had higher household incomes, were more likely to be married/ living with partner and have at least a high school education, had higher blood arsenic levels, and were less likely to have smoked or have had smokers in the household during pregnancy. For study participants, the average age ± SD at time of neurodevelopment assessment was 2:1 ± 0:1 y. MDI scores ranged from 54 to 128 (mean ± SD = 98:8 ± 15:9); PDI scores ranged from 57 to 133 (mean ± SD = 105:3 ± 12:2). Among included pairs, characteristics of participants with complete data on all variables included in the imputation process (n = 122) were similar to those of participants with incomplete data (n = 102; see Table S2).

Results
Median (25-75th percentile) manganese concentrations in maternal and cord blood were, respectively, 24:0 ð19:5-29:7Þ and 43:1 ð33:5-52:1Þ lg=L. The median manganese concentration in cord blood was nearly twice the median concentration in maternal blood, and the median (25-75th percentile) cord/maternal manganese ratio was 1.8 (1:4-2:3). Manganese levels were in the range of levels reported in other studies of mother-infant pairs (Figure 1). Maternal and cord blood manganese levels were correlated (Spearman rho = 0:39, p < 0:0001, n = 224), though in a nonlinear manner (Figure 2). At maternal manganese less than 40 lg=L, cord manganese level increased by an estimated 1:1 ½95% confidence interval ðCIÞ: 0:8, 1:4 lg=L for each 1-lg=L increase in maternal manganese. At maternal manganese levels greater than 40 lg=L, however, a negative association was apparent, although the estimated curve is imprecise in this range because it is based on a limited number of observations (n = 18).
There was a lack of evidence for a departure from linearity in the associations between log e -transformed maternal manganese levels and neurodevelopment scores, based on likelihood ratio tests and visual inspection ( Figure 3A,B). Maternal manganese was significantly negatively associated with both mental and psychomotor development (Table 2): an interquartile range increase in maternal manganese (10:1 lg=L) was associated with decreases of 3.0 (95% CI: −5:3, −0:7) and 2.3 (95% CI: −4:1, −0:4) points in MDI and PDI scores, respectively.
Visual inspection of smoothed plots of log e -transformed cord blood manganese with neurodevelopment suggest an increase in MDI and PDI scores at lower concentrations and a leveling off at mid-range concentrations ( Figure 3C,D). However, there was a lack of evidence for a departure from linearity based on likelihood ratio tests comparing models with penalized spline terms to models with linear terms (MDI: p = 0:15; PDI: p = 0:12). There were no significant associations between log e -transformed cord blood manganese and MDI or PDI scores (Table 2). Results from exploratory analyses of sex-specific manganese associations, in which we included an interaction term (child sex Ã manganese) in regression models, were inconclusive (see Table S3).
a Numbers may not sum to total sample size (n = 224) for some characteristics due to missing data: first-born child n = 1, delivery type n = 9, household smokers n = 47, race/ethnicity n = 2, income n = 66, anemia n = 4. Percentages are based on observations with known values only.
b Numbers may not sum to total sample size (n = 489) for some characteristics due to missing data: sex n = 2, first-born child n = 1, marital status n = 16, education n = 3, prenatal smoking n = 1, household smokers n = 234, race/ethnicity n = 18, income n = 176, anemia n = 8. Percentages are based on observations with known values only. Participants excluded from analyses due to missing blood samples (n = 5), new ICP-MS instrument error (n = 71), missing outcome data (n = 412), and neurodevelopment scores more than 3 SD below expected mean (n = 1). Anemia at delivery defined as maternal hemoglobin <11:0 g=dL at delivery, which is based on the definition from the Centers for Disease Control and Prevention (during 3rd trimester), the World Health Organization (during pregnancy), and the American Congress of Obstetricians and Gynecologists (in 1st and 3rd trimesters; ACOG 2014).
lower among pairs in all three groups, compared to the low concordant exposure group.

Sensitivity Analyses
Sensitivity analyses produced three main findings. a) Results from models of maternal and cord manganese with neurodevelopment using complete cases only were similar to results of analyses that included imputed data (Table 2). Adjusted associations between neurodevelopment and joint classification of maternalinfant groups according to high or low (above or below median) maternal and cord manganese were also similar when based only on complete cases (Table 3). b) Three subjects were identified with outlying blood manganese levels (two maternal: 73.2, 117:4 lg=L; one cord: 139:1 lg=L; Figure 2) using the generalized ESD many-outlier procedure. When these subjects were excluded, associations between manganese and neurodevelopment remained similar (Table 2), indicating that the few outlying manganese levels did not drive the associations we observed. Further, the correlation between maternal and cord manganese was unchanged (Spearman rho = 0:40, p < 0:0001, n = 221 vs. for all observations: Spearman rho = 0:39, p < 0:0001, n = 224) and a nonlinear pattern remained (Figure 2). c) With adjustment for additional sociodemographic variables, conclusions were unchanged (Table 2).

Discussion
We estimated inverse linear associations between log etransformed maternal blood manganese and early childhood mental and psychomotor development scores. Our findings are generally consistent with other studies that have estimated associations between biomarkers of prenatal manganese and neurodevelopmental outcomes (Lin et al. 2013;Takser et al. 2003;Yu et al. 2014). Our study is unique in that it is among the first to evaluate neurodevelopment outcomes in association with both maternal and infant biomarkers of manganese exposure, and with proxy measures of manganese placental transfer. To our knowledge, only one other study has estimated both maternal and cord blood effects: Takser et al. (Takser et al. 2003) observed inverse associations between cord, but not maternal, blood manganese and  performance on psychomotor subscales of the McCarthy Scales of Children's Abilities among 126 3-y-olds with prenatal biomarker levels similar in magnitude to our study [geometric mean (range) 20:4 lg=L ð6:3-151:2Þ and 38:5 lg=L ð14:9-92:9Þ in maternal and cord blood samples, respectively]. In contrast, we found that maternal, but not cord, blood manganese was associated with neurodevelopment, as indicated by lower mean MDI and PDI scores, in our study population. Other studies have also reported evidence of adverse effects of cord blood manganese (Lin et al. 2013;Yu et al. 2014), although maternal biomarkers were not assessed.
Median blood manganese levels in our U.S.-based cohort were lower than median values reported for children in China (Chen et al. 2014), but higher than median or mean values reported for study cohorts in Germany, France, South Africa, and Canada (Abdelouahab et al. 2010;Chen et al. 2014;Kopp et al. 2012;Rudge et al. 2009;Takser et al. 2004). In this cohort, the routes of exposure likely include ingestion of manganese in dusts. We have previously reported positive associations between child hair manganese levels and house dust, but not indoor air, yard soil or tap water, in a subset of this cohort (Zota et al. 2015). Our observation that the median manganese level in cord blood was about twice as high as in maternal blood likely reflects active transport of manganese across the placenta (Krachler et al. 1999;Nandakumaran et al. 2016). Our finding is similar to previous reports in whole blood from 62 mother-infant pairs in South Africa (median 16:8 lg=L and 34:9 lg=L in maternal and cord blood samples, respectively) (Rudge et al. 2009) and in  Additionally adjusted for maternal smoking during pregnancy, gestational age at birth, maternal age at time of delivery, first-born child, annual household income, maternal marital status, and prenatal vitamin use.
serum from 202 mother-infant pairs in China (median 2:8 lg=L and 4:0 lg=L in maternal and cord serum samples, respectively) (Yu et al. 2013). The Spearman correlation coefficient we observed between maternal and cord blood manganese was in the range of correlation coefficients reported by other studies (Abdelouahab et al. 2010;Chen et al. 2014;Gunier et al. 2014;Takser et al. 2004;Yu et al. 2013), though few studies appear to have allowed for possible nonlinearity (Chen et al. 2014;Gunier et al. 2014).
Our observation that the concentration of manganese in maternal blood, despite generally being lower than the concentration in cord blood, was a stronger predictor of neurodevelopment was unexpected, and the explanation is not apparent. We hypothesized that cord blood would more proximally represent fetal exposure, and therefore be more strongly associated with neurodevelopment. However, given that manganese is a nutrient, relationships are difficult to predict.
It is possible that high maternal manganese levels produce adverse effects on the placenta that are subsequently responsible for poorer neurodevelopment. This might explain differences in results compared to cord blood manganese. The fetus may be protected from direct adverse effects of excess maternal manganese by accumulating manganese in the placenta, but it may be indirectly affected by maternal manganese via placental factors that regulate neurodevelopment. An in vitro human placenta study has demonstrated that during normal steady-state exposure, the placenta efficiently transfers manganese to the fetus, but at highdose maternal exposure the placenta limits transfer to the fetal circulation and accumulates manganese (Miller et al. 1987). This is consistent with the nonlinear relationship we observed between maternal and cord blood manganese levels, whereby levels were positively associated up to approximately 40 lg=L maternal blood, and cord blood levels subsequently leveled off or declined. There was, however, a relatively small number of observations in this higher exposure range. Alternative explanations may include mechanisms that do not involve a causal effect of maternal manganese, and findings should be confirmed in other studies.
Several biological mechanisms for the neurotoxic effects of prenatal manganese exposure have been proposed. Animal studies have reported that maternal developmental exposure in mice affects neurogenesis in the offspring, altering the number of immature granule cells in the hippocampal dentate gyrus and causing neuronal mismigration Wang et al. 2012). In an experimental study, in utero exposure altered epigenetic gene regulation in offspring, which may affect programing of cells involved in neurogenesis (Wang et al. 2013). Manganese neurotoxicity appears to involve oxidative stress and dopaminergic dysfunction (Racette et al. 2012), and a link to adverse neurodevelopmental outcomes through disruption of thyroid homeostasis following alterations to dopamine activity has also been proposed (Soldin and Aschner 2007).
In our exploration of placental transfer, we found that MDI and PDI scores increased as the cord/maternal or cord= ðmaternal + cordÞ manganese ratios increased, suggesting that increasing cord manganese levels relative to maternal levels or relative to total (i.e., cord + maternal) levels might be beneficial. We also found that both MDI and PDI scores were lower among mother-infant pairs with either high maternal levels or high cord levels, compared to mother-infant pairs with both low maternal and low cord manganese. Overall, these results are consistent with our finding of a negative association between maternal manganese levels and scores for cognition and psychomotor function at 2 y of age. Further study is necessary, but these preliminary findings suggest that not only are concentrations of manganese in maternal blood predictors of neurodevelopment, but the relative amount of manganese transferred from maternal to fetal circulation may also be important. Factors that influence manganese transfer should be investigated more deeply.
Our study has several limitations. Prenatal maternal blood manganese may not best represent manganese total body burden given that it is a nutrient with a steady-state normal concentration range that is regulated in the body, rather than a xenobiotic that undergoes metabolism and excretion. Physiologic factors such as genetic variability or variability in metabolic function or liver function may contribute to differences in manganese levels (Claus Henn et al. 2011;Zerón et al. 2011). In addition, little is known about how maternal blood manganese levels vary during labor and delivery; therefore, timing of blood sample collection may influence blood manganese levels. However, blood manganese levels also increase in the setting of chronic exposure. For example, blood levels have been correlated with airborne manganese levels (Smith et al. 2007), with an indicator of take-home occupational pesticide exposure in a region where manganesecontaining pesticides are commonly applied (Gunier et al. 2014), and with MRI intensity index in the globus pallidus among manganese-exposed children (Kafritsa et al. 1998). Our modest sample size limits the precision in our effect estimates. However, by multiply imputing missing covariate data, an approach that is valid if data are missing at random, we maximized the use of available exposure and outcome data. Finally, there is a possibility of selection bias from loss to follow up if factor(s) related to participation are associated with both manganese levels and neurodevelopment. This is among the first studies to examine both maternal and infant biomarkers of prenatal manganese exposure in relation to neurodevelopment, which allows for a comparison of biomarkers as well as an exploration of placental transfer. The prospective design of our study strengthens the growing literature on manganese and neurodevelopment, which remains dominated by crosssectional studies that cannot establish temporality. We collected data on a large number of potential confounders, including coexposures to lead and arsenic, and results were robust to additional confounder adjustment.

Conclusions
In our U.S. study population, the concentration of manganese in maternal blood at or near the time of delivery was associated with lower neurodevelopment scores at 2 y of age. In addition, we found preliminary evidence suggesting that placental factors may influence associations between prenatal manganese exposure Table 3. Adjusted associations between neurodevelopment and joint classification of maternal-infant groups according to high or low (above or below median) maternal and cord manganese.