Persistent Impact of Antenatal Maternal Anaemia on Child Brain Structure at 6–7 Years of Age: A South African Child Health Study

Background The study aim was to determine whether associations of antenatal maternal anaemia with smaller corpus callosum, putamen, and caudate nucleus volumes previously described in children at age 2–3 years persist to age 6–7 years in the Drakenstein Child Health Study (DCHS). Methods This neuroimaging sub-study was nested within the DCHS, a South African population-based birth cohort. Pregnant women were enrolled (2012–2015) and mother-child dyads were followed prospectively. A sub-group of children had magnetic resonance imaging at 6–7 years of age (2018–2022). Mothers had haemoglobin measurements during pregnancy and a proportion of children were tested postnatally. Maternal anaemia (haemoglobin<11g/dL) and child anaemia were classified using WHO and local guidelines. Linear modeling was used to investigate associations between antenatal maternal anaemia status, maternal haemoglobin concentrations, and regional child brain volumes. Models included potential confounders and were conducted with and without child anaemia to assess the relative roles of antenatal versus postnatal anaemia. Results Overall, 157 children (Mean [SD] age of 75.54 [4.77] months; 84 [53.50%] male) were born to mothers with antenatal haemoglobin data. The prevalence of maternal anaemia during pregnancy was 31.85% (50/157). In adjusted models, maternal anaemia status was associated with smaller volumes of the total corpus callosum (adjusted percentage difference, −6.77%; p=0.003), left caudate nucleus (adjusted percentage difference, −5.98%, p=0.005), and right caudate nucleus (adjusted percentage difference, −6.12%; p=0.003). Continuous maternal haemoglobin was positively associated with total corpus callosum (β=0.239 [CI: 0.10 to 0.38]; p<0.001) and caudate nucleus (β=0.165 [CI: 0.02 to 0.31]; p=0.027) volumes. In a sub-group (n=89) with child haemoglobin data (Mean [SD] age of 76.06[4.84]), the prevalence of antenatal maternal anaemia and postnatal child anaemia was 38.20% (34/89) and 47.19% (42/89), respectively. There was no association between maternal and child anaemia (c2 = 0.799; p=0.372), and child anaemia did not contribute to regional brain volume differences associated with maternal anaemia. Conclusions Associations between maternal anaemia and regional child brain volumes previously reported at 2–3 years of age were consistent and persisted to 6–7 years of age. Findings support the importance of optimizing antenatal maternal health and reinforce these brain regions as a future research focus on intervention outcomes.


INTRODUCTION
From conception through early postnatal life, the developing child brain is extremely sensitive to environmental in uences [1][2][3].While the importance of this rst 1000-day window is well-known, recent research is providing increasing evidence to support a renewed focus on optimising antenatal maternal well-being for improved neurodevelopmental outcomes in children [1].In recognizing the foetal origins of brain health and the persistent impact of early risk, maternal anaemia during pregnancy is an important health priority for targeted prevention and intervention strategies [4].
Anaemia, indicated by low serum haemoglobin, affects one quarter of the world's population [5].Women and children bear the greatest burden with 30% of women of reproductive age and 36% of pregnant women estimated to be anaemic [6].In considering possible aetiologies for anaemia, chronic iron de ciency has consistently been identi ed as the most common underlying direct cause [5], accounting for approximately 50% of cases in this group [6].Although anaemia is a global health priority, it is most prevalent in Low-and Middle-Income Countries (LMICs), particularly in Africa and South Asia [6].This is due to multiple risk factors including malnutrition, food insecurity, and infectious disease [7][8][9], all of which are highly prevalent in these regions [10][11][12][13].Given that progress in reducing anaemia has stagnated between 2000 and 2019 [6], it has been identi ed by the World Health Organization (WHO) as a Sustainable Development Goal (SDG) for accelerated action [5].
The risk of anaemia rises during pregnancy due to increased blood volume to support the haemoglobin-facilitated supply of oxygen to the foetus, higher metabolic demand of the developing brain, and foetal iron loading [2,14].In turn, antenatal maternal anaemia and iron de ciency have consistently been associated with a greater probability of poor maternal and infant health outcomes including placental abruption, maternal shock, ICU admission, maternal mortality, post-partum haemorrhage, foetal growth restriction, stillbirth, prematurity, and low birthweight [15][16][17].Additionally, antenatal maternal anaemia and iron status are well-known risk factors for poorer general paediatric neurocognitive outcomes in multiple settings [18][19][20], including South Africa [1].Similarly, an association has been identi ed between maternal anaemia during early pregnancy and increased risk of a range of neurodevelopmental conditions including Autism Spectrum Disorder (ASD), Attention-De cit Hyperactivity Disorder (ADHD), and Intellectual Disability (ID) [21].
Non-invasive approaches such as neuroimaging provide an opportunity to objectively explore the potential neurobiological impact of anaemia on the developing brain comparably across cultures and settings [22,23].However, while recent neuroimaging research using magnetic resonance imaging (MRI) suggests a striking neural vulnerability to iron de ciency and anaemia [24][25][26][27][28][29], this remains an emergent eld with varying research foci.For example, existing studies on brain imaging outcomes associated with postnatal foetal (cord blood) or child (venous blood) iron and anaemia status do not address the role of maternal anaemia [26,27], and emphasize severe pathological presentations due to haemorrhage [26].Other neuroimaging research has been designed with brain imaging measures as the exposure variable to assess postnatal brain iron concentration as a risk factor for poor cognitive outcomes [28] or developmental disorders in childhood [29].The few studies that have investigated maternal iron de ciency and anaemia exposure have targeted particular brain structures [24], used different MRI modalities (structural [24] versus diffusion tensor imaging [25]), and were limited by the use of self-report indicators of maternal iron based on dietary intake [25].They also largely focused on neonatal brain outcomes, resulting in very little being known about the relative effects of antenatal versus postnatal anaemia and the persistence of effects in early and middle childhood [24,25].
Given the scarcity of MRI studies in LMICs [30,31] and the particularly high prevalence of anaemia in Africa [6], a neuroimaging sub-study of the Drakenstein Child health Study (DCHS; South African birth cohort) [32,33] investigated the associations between antenatal maternal anaemia, postnatal child anaemia, and child brain structure at 2-3 years of age [4].In this cohort, maternal anaemia during pregnancy was associated with smaller volumes of the bilateral caudate, left putamen, and total corpus callosum in the child brain.While this study highlights a critical need to optimise the timing of anaemia interventions, further research is necessary to corroborate results and to determine whether these novel neuroimaging ndings persist with age.This may aid in understanding the longitudinal impact of maternal risk on patterns of child brain development by identifying whether structural brain changes are long-lasting or indicative of a temporary delay in growth.The aim of this study was to determine whether the association between antenatal maternal anaemia and lower corpus callosum and basal ganglia volumes described in toddlers (aged 2-3 years) from the DCHS birth cohort persisted in this cohort at school-age (6-7 years of age).

Study design and setting
The DCHS is an observational population-based birth cohort located in the peri-urban Drakenstein district of Cape Town, South Africa [32,33].This community is characterised by low socio-economic status (SES) and multiple health and psychosocial adversities with a high prevalence of risk factors such as maternal HIV, food insecurity, and malnutrition.However, more than 90% of the population have access to public health services, with TC Newman and Mbekweni Clinics being the two primary healthcare centres for this study.Pregnant women were recruited for the DCHS while attending antenatal clinic visits, and well-characterised mother-child dyads have been followed prospectively.

Participants
Between 2012 and 2015, 1125 pregnant women were enrolled in the DCHS and 1143 live births were included with good retention in postnatal follow-up care.In a nested neuroimaging study [34], a sub-group of children were invited for MRI as neonates (2012-2016), at 2-3 years (2015-2018), and at 6-7 years (2018-2022).Exclusion criteria included (1) medical comorbidity (genetic syndrome, neurological disorder, or congenital abnormality); (2) gestation less than 36 weeks; (3) low Apgar score (less than 7 at 5 minutes); (4) neonatal intensive care admission; (5) maternal use of illicit drugs; (6) MRI contraindications; and (7) child HIV infection.Of the eligible children, those who were scanned at birth were followed-up at subsequent imaging sessions alongside additional children who were selected at the 2-3 year timepoint based on known neurodevelopmental risk factors (maternal HIV and alcohol exposure) and a randomly selected control group.Previous DCHS research has demonstrated comparability between the nested neuroimaging sub-study sample and the full cohort [34].
Findings regarding the association between antenatal maternal anaemia and child brain structure at 2-3 years of age in this cohort have been published [4].The current study is focused on the 6-7 year timepoint, at which 157 children had both useable structural neuroimaging data (see neuroimaging measures section below) and maternal haemoglobin data for inclusion in analysis.A study owchart is available as supplementary information (Additional File 1, Figure S1).

Measures
Contextual measures.Demographic, environmental, psychosocial, and clinical data for mother-child dyads were collected antenatally and postnatally for descriptive purposes and for consideration as covariates.All mothers underwent repeat testing for HIV during pregnancy and infants were tested postnatally as per national guidelines.Birth details were obtained by study staff at delivery and child gestational age was calculated using ultrasonography in the 2 nd trimester of pregnancy or, where this was unavailable, symphysis-fundal height or maternal report of the most recent menstrual cycle.Child anthropometry measures were observed and classi ed according to WHO guidelines [35] at routine study visits as well as neuroimaging sessions.Maternal tobacco smoking in pregnancy was self-reported and a dichotomous classi cation of antenatal alcohol use was retrospectively assessed using the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST) [36].
Anaemia and iron de ciency.Antenatal maternal and postnatal child anaemia was assessed based on serum haemoglobin measurements in pregnancy and childhood, respectively.Maternal haemoglobin measurements were acquired using rapid tests at antenatal clinic visits as per standard-of-care policy, and iron and folic acid supplementation was recommended as per national guidelines.This data was abstracted from clinical records by trained DCHS staff at study enrolment.Based on WHO guidelines [37], haemoglobin levels of <11g/dL during pregnancy were used to classify pregnant women as anaemic.Further classi cations into mild (haemoglobin 10.0 -10.9g/dL), moderate (haemoglobin 7.0 -9.9g/dL), and severe anaemia (haemoglobin <7.0g/dL) were determined.Child haemoglobin was only available for children who presented at hospital with pneumonia between birth and the MRI scan, as part of a full blood count.Child anaemia was classi ed based on age-speci c cut-offs using WHO guidelines for all measurements in children aged over 6 months and local guidelines (Groote Schuur Hospital/University of Cape Town Pathology Laboratory) for children under 6 months (Additional File 1, Table S1).A dichotomous variable for child anaemia status was created based on meeting anaemia criteria at least once between birth and 6-7 years of age.
Neuroimaging outcomes.Based on earlier ndings [4], structural MRI was chosen as the most relevant imaging measure.Brain volume was acquired on a 3T Siemens Skyra MRI system at the Cape Universities Body Imaging Centre (CUBIC) using a 32 channel head-coil.All children were scanned awake while watching a movie inside the MRI machine.The neuroimaging protocol and MRI speci cations are available as supplementary material (Additional File 1, Text S1).
All structural brain scans were processed with FreeSurfer Version 7.1.1using an automated process of cortical reconstruction and volumetric segmentation [38,39].Regional brain volumes were extracted for analysis using the Desikan-Killiany atlas [40] and an inbuilt probabilistic atlas [38] for cortical parcellation and subcortical segmentation, respectively [40].Given that maternal anaemia was associated with smaller caudate nucleus, putamen, and corpus callosum volumes in the DCHS analysis at 2-3 years of age [4], these brain regions were chosen as key apriori regions of interest (ROIs) for a targeted analysis at 6-7 years of age.However, based on broader literature [24][25][26]28], other potentially vulnerable subcortical regions including the hippocampus, amygdala, thalamus, nucleus accumbens, and pallidum were identi ed for exploratory analyses.All subcortical structures were segmented into left and right hemispheres.The corpus callosum was segmented into posterior, mid-posterior, central, mid-anterior, and anterior regions.The total corpus callosum volume was computed by summing all individual subregions, and the body was de ned as the sum of the mid-posterior, central, and mid-anterior volumes.Intracranial volume (ICV) was included as a covariate in analyses to account for normal interindividual variability in brain size.
All regional segmented output (n=204) was subject to a standardised quality control check using the ENIGMA Cortical Quality Control Protocol 2.0 [41].This was conducted independently by two senior research staff with experience in neuroimaging processing and analysis.Subjects with consistent failures across all brain regions on internal and external quality control (QC) were excluded (n=36).Further decisions on inclusion in the dataset were made by identifying participants that emerged as statistical outliers in SPSS (using Tukey's method) [42] for subcortical (n=0) and corpus callosum (n=1) ROIs.Overall, a sample of 167 participants passed the visual and statistical QC testing, of which 157 had maternal haemoglobin data (Additional File 1, Figure S1).

Statistical analysis
Sample characteristics.Demographic data and clinical characteristics were presented as means and standard deviations for continuous variables and frequencies for categorical variables.Sociodemographic and clinical (e.g., maternal exposures) group differences between children with antenatal maternal anaemia exposure and children without antenatal maternal anaemia exposure were calculated using unpaired t-tests for continuous data and chi-squared tests or Fisher's exact tests for categorical data.
Maternal anaemia status.The exposure variable was antenatal maternal anaemia status (dichotomized as anaemic versus non-anaemic based on WHO haemoglobin cut-offs for pregnancy) and the outcomes were regional child brain volumes selected apriori.Between-group differences were investigated using multivariate analysis of variance (MANOVA) general linear models.Given that this analysis aimed to determine whether previously identi ed ndings in 2-3 year-old children persist with age at 6-7 years in the same cohort, a similar statistical approach was conducted.This included a targeted analysis for key apriori ROIs, namely the corpus callosum, putamen, and caudate nucleus.However, a separate MANOVA for an exploratory analysis of other subcortical regions was run to ensure no emerging ndings were missed.
A separate set of MANOVA models were performed for grey (left and right hemispheric volumes of subcortical structures) and white (individual corpus callosum regions) matter ROIs.Models were built using a hierarchical stepwise approach with 1) an unadjusted model assessing group differences without the inclusion of covariates, 2) a partially adjusted model including age at scan, sex, SES (represented by maternal education and total household income), and ICV as covariates known to affect brain volume apriori [1,43], and 3) a fully adjusted model including maternal exposures with demonstrated group differences, placing a particular focus on antenatal alcohol exposure which has consistently been associated with smaller corpus callosum volumes in the broader literature [44] and is known to interact with iron metabolism at a physiological level [45,46].A series of fully adjusted post-hoc univariate analyses (ANOVAs) were additionally performed for each individual ROI, correcting for multiple comparisons using the False Discovery Rate (FDR) method [47].
Separate ANOVAs were conducted to assess the association between antenatal maternal anaemia status and overall summed volumes for the body and total corpus callosum.
In comparing volumes based on maternal anaemia status, adjusted mean differences were calculated using pairwise comparisons of estimated marginal means based on the fully adjusted MANOVA and ANOVA models.Percentage differences were calculated using the adjusted mean difference relative to the unadjusted mean volume in the control group (no maternal anaemia) for each brain structure.
Maternal haemoglobin concentration.In regions where an association between maternal anaemia status and child brain volumes was observed (p < .05),we explored hierarchical multivariable linear regression models using standardised regression coe cients for continuous maternal haemoglobin concentrations.This allowed us to assess the relationship between maternal anaemia severity and regional child brain volumes.
Child anaemia sub-analysis.To explore the relative role of postnatal child anaemia on regional child brain volumes in a sub-analysis (children with both maternal and child haemoglobin data), child anaemia status was included as an additional covariate for consideration in the established fully adjusted models for maternal anaemia status described above.
Sensitivity analyses and statistical considerations.Sensitivity analyses were conducted to consider the potential role of a broader range of factors, including timing.We adjusted for trimester of pregnancy given the anticipated increase in maternal blood volume and haemoglobin with gestational time [37].Secondly, other relevant clinical maternal exposures such as HIV and smoking, both of which are prevalent in this community, were adjusted for in the models to account for any unmeasured confounding [48].
All analyses were conducted using SPSS.A two-sided signi cance level of p<0.05 was used throughout.Collinearity and biological plausibility was considered in the establishment of all models and checks for assumptions including normality of residuals and homogeneity of variance were conducted throughout.
There were no group differences in maternal or child sociodemographic characteristics and anthropometric measures.While antenatal maternal smoking and maternal HIV infection were prevalent across the whole neuroimaging sample, there were no group differences in these exposures.However, antenatal maternal alcohol use was signi cantly more prevalent in mothers with anaemia during pregnancy (22/50 [44%]) than mothers without anaemia during pregnancy (26/107 [24%]).
Antenatal maternal anaemia status and child brain structure Corpus callosum.In a partially adjusted MANOVA model, antenatal maternal anaemia had a main effect on the corpus callosum regions overall, F(5,146)=0.022.This effect was no longer signi cant in the fully adjusted model which included antenatal alcohol exposure, F(5,145)=2.195, p=0.058.However, fully adjusted post-hoc univariate analyses (see Table 2) revealed that children born to mothers who were anaemic during pregnancy had signi cantly smaller volumes of the posterior (p=0.017),mid-posterior (p=0.009), and central (p=0.018),regions than children born to mothers who were not anaemic during pregnancy, after multiple comparisons correction.
Following on from these ndings, in fully adjusted ANOVAs, the volumes of the body and the total corpus callosum were found to be smaller in children born to mothers with maternal anaemia during pregnancy (Body: M=1221.09mmRight: M=3944.82mm 3 , SD=531.17), p=0.005 and p=0.003, respectively.In considering adjusted mean differences, this corresponds with smaller volumes of approximately 5.98% and 6.12%, respectively.This association between antenatal maternal anaemia and the caudate nucleus (see Figure 1) demonstrated a medium effect size for both the left (partial η 2 = 0.052) and right (partial η 2 = 0.057) hemispheres.
It is noted that the sequential adjustment for antenatal alcohol exposure in the fully adjusted model did not change the effects of antenatal maternal anaemia on the caudate nucleus.There was no signi cant effect of antenatal maternal anaemia on the left or right putamen in this age group.In an exploratory analysis based on the fully adjusted model, there were no other subcortical regions associated with antenatal maternal anaemia (Additional File 1, Table S2).The adjusted mean difference was calculated from the fully adjusted MANOVA models via post-hoc pairwise comparison using estimated marginal means.A negative mean difference and the corresponding percentage difference represent a smaller volume in children born to mothers with anaemia during pregnancy.

Antenatal maternal haemoglobin concentration and child brain structure
Given the demonstrated associations between antenatal maternal anaemia status and child brain volumes of the corpus callosum and bilateral caudate nucleus, these brain regions were explored further using multivariable linear regression for continuous haemoglobin concentrations (see Figure 2).male) who also had imaging data and maternal haemoglobin data.Overall, 216 measurements (Additional File 1, Table S1) were observed due to the inclusion of multiple measurements for the same child at different timepoints where relevant (median age [IQR] at haemoglobin measurement across all measurements: 10.50 [3.76-22.80]).Of the 89 children, 42 (47.19%)were classi ed as anaemic at least once in the postnatal period using age-speci c thresholds (Additional File 1, Table S1, Table S3).In this sub-group, 34/89 (38.20%) of the mothers were found to be anaemic during pregnancy, of which 16/34 (47%) had mild anaemia and 18/34 (53%) had moderate anaemia.
There were no group differences in sample characteristics between children with haemoglobin measurements and without across the sample (n=157; Additional File 1, Table S4), or between children with and without anaemia in the sub-group (n=89; Additional File 1, Table S5).In line with the full sample, there were no differences in sample characteristics in this sub-group between children born to mothers with and without anaemia during pregnancy (n=89; Additional File 1, Table S6), other than antenatal alcohol exposure which was more prevalent in anaemic mothers as noted previously.
Overall, there was no association between antenatal maternal anaemia and postnatal child anaemia in this group (n=89; Additional File 1, Table S3), c 2 = 0.799, p=0.372.However, to investigate whether postnatal child anaemia was contributing to group differences in regional child brain volumes, it was included in previously established models as a covariate.In this sub-analysis (n=89), the fully adjusted models conducted for the main analyses on maternal anaemia were replicated with the addition of postnatal child anaemia (see Table 3).Overall, the previously identi ed effects of antenatal maternal anaemia on child brain volumes remained robust with very similar effect sizes when adjusting for postnatal child anaemia.Furthermore, in this model, child anaemia status was not found to be associated with corpus callosum, caudate nucleus, or putamen volumes.

Sensitivity analyses
All identi ed associations between antenatal maternal anaemia and regional brain volumes (corpus callosum and caudate nucleus) were robust, remaining signi cant in sensitivity analyses before and after adjusting for trimester of pregnancy, antenatal smoking exposure, and maternal HIV status.Survived FDR correction for multiple comparisons.For the body and total corpus callosum where univariate analyses (ANOVAs) were run, multiple comparisons was not applicable.
f The adjusted mean difference was calculated from the fully adjusted MANOVA models via post-hoc pairwise comparison using estimated marginal means.A negative mean difference and the corresponding percentage difference represent a smaller volume in children born to mothers with anaemia during pregnancy.

Principal ndings and implications
In this neuroimaging sub-study, antenatal maternal anaemia was associated with smaller child brain volumes of the corpus callosum and caudate nucleus at 6-7 years of age.In contrasting the results from this DCHS research on school-age children (6-7 years old) with previous work from the same birth cohort on toddlers (2-3 years old) [4], the adjusted volume differences for ROIs were found to be comparable between timepoints.This was evident with consistently smaller volumes of the corpus callosum (7% at 6-7 years versus 8% at 2-3 years) and caudate nucleus (6% at 6-7 years versus 5% at 2-3 years).Furthermore, the nature and the strength of the relationship between maternal haemoglobin concentrations and child volumes for the total corpus callosum (standardised coe cient of 0.24 at both timepoints) and caudate nucleus (standardised coe cient of 0.17 at 6-7 years versus 0.15 at 2-3 years) remained similar.However, in exploratory analyses, no other emerging subcortical brain regions were associated with antenatal maternal anaemia.Similarly, as seen at 2-3 years of age, postnatal child anaemia was still not associated with regional brain volumes of the caudate nucleus, putamen, or corpus callosum at 6-7 years of age.Overall, these results indicate that the effects of antenatal maternal anaemia on child brain structure persist with age and remain regionally consistent over time.
Given the magnitude of the volume difference in affected regions, and the known role of the corpus callosum caudate nucleus in neuropsychological functioning [49][50][51], these ndings are likely to be clinically important.This is corroborated by recent systematic reviews suggesting a direct relationship between maternal anaemia and iron status during pregnancy, and poorer offspring performance across domains of cognition, motor function, language, memory, and behaviour on standardized testing [19,20].In parallel to the neuroimaging ndings linking maternal anaemia to altered child brain structure in a sub-group of DCHS toddlers [4], previous research from the broader cohort has also demonstrated a strong link between maternal anaemia and poor developmental outcomes at the same age [1].Therefore, the persistent effects of anaemia on volumes of key brain volumes in this group of children at 6-7 years of age in this study suggests an increased risk of lasting cognitive di culties that may potentially emerge at school age.
In this nested neuroimaging cohort, the prevalence of antenatal maternal anaemia was 32%.This is consistent with WHO estimates for African countries [6] and reports of stagnated progress in global efforts to reduce anaemia in LMICs [5].In addition to suggesting the antenatal period as important for the timing of anaemia interventions, this study highlights the necessity of a multifactorial approach that addresses its complex aetiology.In the South African context, various other risk factors become relevant in considering the physiological mechanisms for iron de ciency anaemia.For example, HIV infection may have a negative impact on iron bioavailability due to increased sequestration within the context of in ammation [52][53][54][55].Similarity, antenatal alcohol use may contribute to iron de ciency directly by limiting the intestinal absorption of iron and disrupting iron homeostasis [45,46], and indirectly by negatively impacting nutritional choices [56].In turn, antenatal tobacco exposure, which is highly overlapping with alcohol consumption during pregnancy [57,58], is known to increase haemoglobin levels [37] resulting in the underestimation of functional anaemia in people who smoke.While all of these risk factors were prevalent in this cohort across groups, antenatal alcohol exposure was signi cantly more common in women who were anaemic during pregnancy suggesting that this interaction may be particularly important.
In addition to potential interactions between iron de ciency anaemia and risk factors such as maternal HIV and alcohol exposure, there is a well-known overlapping association with the corpus callosum as an associated outcome of interest.For example, this structure has also been widely implicated in research on antenatal alcohol exposure [44,59] as well as HIV infection [60, 61].After statistically accounting for these covariates in analyses, the association of maternal anaemia in pregnancy with smaller corpus callosum volumes was found to be robust.However, the inclusion of alcohol consumption as a covariate did weaken its effect.Given that in utero exposure to HIV, alcohol, and tobacco are highly prevalent and often comorbid in this cohort and other high-risk LMIC communities, an integrated approach to understanding and preventing anaemia is necessary.This is an important consideration in ongoing work to accelerate anaemia reduction using prevention and treatment strategies [5].
While the prevention of anaemia includes addressing food insecurity, managing disease, and mitigating psychosocial risk, current treatments are via simple interventions such as iron supplementation [5].Given the growing body of research suggesting that postnatal iron supplementation may be less effective in improving long-term cognitive outcomes in children [62-64], these results re-emphasize the importance of considering the antenatal period as an important opportunity.According to WHO policy [65], iron and folic acid in pregnancy are recommended as per standard antenatal care practice but may be insu cient to combat the current burden of maternal anaemia in LMICs.This is due to a myriad of challenges including unclear aetiologies and imprecise strategies for intervention, dosage insu ciency, late presentation to clinics for antenatal care, limited public knowledge around the importance of nutrition and iron supplementation, unaffordability of nutritious iron-rich foods, and unclear directions for optimised supplementation use [52,53,66].Future work should be focused on increased community engagement for understanding barriers to screening for anaemia and iron de ciency, and opportunities for context-speci c strategizing for optimisation of anaemia interventions.The consistency of ndings in the same key brain structures across early childhood in this study highlight these regions as a potential focus for outcomes in future research on intervention.

Limitations
While this study is the rst to demonstrate the persistent effects of antenatal maternal anaemia on child brain structure, it has limitations to consider.Firstly, given that this is an observational birth cohort study, causality can certainly be queried.However, the groups were comparable, covariates were included in models, and analyses were robust to sensitivity analyses.Furthermore, the ndings remained consistent with previous results at an earlier age, a temporal association was demonstrated, and there was evidence for a biological gradient using continuous haemoglobin concentrations.Secondly, the neuroimaging subgroup was embedded within a high-risk community with multiple potential confounders including HIV and alcohol use.Although this contributes to complicated interactions which deserve further exploration, it is an ecologically valid representation of many LMICs and should not limit the generalisability of ndings.Thirdly, this study is limited in its ability to determine the exact nature and cause of maternal anaemia due to the reliance on point-of-care testing for haemoglobin.Fourthly, this study is restricted in its ability to assess the role of postnatal child anaemia due to power limitations and selection bias.This is because child haemoglobin data was only available for half the sample in a subgroup of children who had blood tests while ill with pneumonia.Furthermore, the child anaemia variable was a dichotomous indication of whether a child had ever been diagnosed with anaemia which prevented the exploration of severity, duration, and age-speci c associations.Lastly, due to power limitations, this study was unable to assess whether altered child brain structure in key affected ROIs mediated the relationship between antenatal maternal anaemia and school readiness.Future work could bene t from more comprehensive anaemia data and iron metrics, as well as the addition of cognitive outcomes with a su cient sample size.

Conclusion
This nested neuroimaging cohort study demonstrated that associations of maternal anaemia in pregnancy with child brain volumes are regionally consistent and persist from age 2-3 years through to age 6-7 years.Overall, antenatal maternal anaemia was associated with smaller volumes of the corpus callosum and caudate nucleus in school-age children, with comparable adjusted volume differences and coe cients to ndings in toddlers.The persistent associations of antenatal maternal anaemia with structural child brain ndings in regions underlying important cognitive functions emphasizes the need for optimised anaemia interventions in women of reproductive age before and during pregnancy for improved child neurodevelopmental outcomes.Given the high prevalence of comorbid antenatal alcohol consumption in anaemic women from this cohort, this risk factor is likely to play a key causal role via a physiological interaction with iron and nutritional behaviours.Therefore, prevention and intervention strategies for maternal anaemia should be multifaceted to account for overlapping risk factors such as malnutrition and alcohol use in pregnancy.Given the consistency of ndings in key brain structures across early childhood, the importance of these regions is emphasized as a focus for future research, particularly on intervention outcomes.

Supplementary Files
This is a of supplementary les associated with this preprint.Click to download. AdditionalFile1.pdf

FDR
ADHD Attention-De cit Hyperactivity Disorder ANOVA Analysis of Variance ASD Autism Spectrum Disorder ASSIST Alcohol, Smoking, and Substance Involvement Screening Test BMI Body Mass Index BMIZ Body Mass Index-for-Age CI Con dence Interval CUBIC Cape Universities Body Imaging Centre DCHS Drakenstein Child Health Study , and MTL had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.Concept and design: JER, KAD, CJW, HJZ, DJS.Acquisition, analysis, or interpretation of data: JER, KAD, CJW, CH, SCRW, HJZ, DJS, SHJ, SS, LEB, CNN, SRW.Drafting of the manuscript: JER, KAD, CJW, CH.Critical revision of the manuscript for important intellectual content: All authors.Statistical analysis: JER, CH.Obtained funding: JER, KAD, HJZ.

Figures
Figures

Figure 1 Regional
Figure 1

Figure 1
Figure1Footnote.Glass brain image of regional child brain volumes visualized on a cortical surface from lateral views (right and left hemispheres of the brain) and a coronal view.In children aged 6-7 years, the brain structures associated with antenatal maternal anaemia were the total corpus callosum (blue) and total caudate nucleus (green).

Table 1 .
Sample Characteristics of Children Born to Mothers with and without Anaemia During Pregnancy Values for continuous variables are presented as: mean ± standard deviation (range).Values for categorical variables are presented as: number (%).
a b Maternal anaemia during pregnancy was classi ed according to the WHO threshold of Hb<11g/dL.Severity classi cations were de ned as mild (10.0-10.9g/dL),moderate(7.0-9.9g/dL), and severe (<7.0g/dL).cTrimester of pregnancy de ned as rst (0-12 weeks), second (13-27 weeks), and third (28 weeks onwards).dThebirth anthropometric measurements were conducted by trained labour staff in the ward.Infant length was measures in cm to the nearest completed 0.5cm and weight was measured in kgs.Child weight and length measurements were converted to z-scores based on age and sex using Anthro software for WAZ, HAZ, and HCZ.Children were classi ed as underweight, stunted, or having microcephaly if they had z-scores of less than -2 SDs. e Levene's test was signi cant.T-test results were interpreted based on equal variance not assumed.fFisher's exact test result interpreted due to one or more cells having an expected count of less than 5.g

Table 2 .
Effects of Maternal Anaemia on Regional Child Brain Volumes of Interest with and without adjusting for covariates (n=150) Corpus callosum body: Sum of mid-posterior, central, and mid-anterior regions.Total Corpus callosum: Sum of posterior, midposterior, central, mid-anterior, and anterior regions.Fully adjusted ANOVA models run separately on the body and total corpus callosum summed volumes.
1Unadjusted model including only antenatal maternal anaemia status.a,bFully adjusted MANOVA models run separately for the corpus callosum and caudate nucleus regions.c e Survived FDR correction for multiple comparisons.For the body and total corpus callosum where univariate analyses (ANOVAs) were run, multiple comparisons was not applicable.f

Table 3 .
Effects of Maternal Anaemia on Regional Child Brain Volumes of Interest in Sub-Analysis Adjusting for the Role of Child Anaemia (n=89) Partially adjusted model including antenatal maternal anaemia status, ICV, child age and sex at scan, SES (indicated by maternal education and household income), and antenatal alcohol exposure p values are adjusted for covariates but are not corrected for multiple comparisons.*p is signi cant at <0.05, ** p is signi cant at <0.01, ***p is signi cant at <0.001.
1 2Fully adjusted model including antenatal maternal anaemia status, ICV, child age and sex at scan, SES (indicated by maternal education and household income), antenatal alcohol exposure, and postnatal child anaemia a,b Fully adjusted MANOVA models run separately for the corpus callosum and caudate nucleus regions.cFully adjusted ANOVA models run separately on the body and total corpus callosum summed volumes.d e