Cord blood vitamin D is correlated with problem solving domain in early childhood neurodevelopment: A Cohort Study on Vitamin D Status and Its Impact during Pregnancy and Childhood in Indonesia

Fetal vitamin D level is known to in�uence brain development and subsequent postnatal neurodevelopment, although several studies stated that this correlation remains controversial. We aimed to determine the correlation between cord blood vitamin D level and neurodevelopmental status in the �rst 1000 days of life. This was a cohort study, located in Sukabumi and Waled districts of West Java, Indonesia, from 2016–2019. Subjects were term infants with no congenital abnormalities, born from pregnant women involved in a previously conducted cohort study. The cord blood sample collected at delivery was used to measure 25 (OH)-vitamin D level. Neurodevelopmental status was examined after the infants reached 6, 12, and 24 months of age using Ages and Stages Questionnaire-3 (ASQ-3). Statistical analysis was performed using rank Spearman’s correlation. Blood samples were collected from 116 subjects. The mean value of vitamin D levels was 16.2 ng/mL (8.0–35.4 ng/mL). As many as 12.9%, 65.5% and 21.6% of newborns had vitamin D de�ciency, insu�ciency and normal vitamin D levels. The correlation was found between cordblood vitamin D levels and the problem-solving domain at the age of 12 and 24 months old; r = 0.217, and r = 0.414 respectively. Multiple linier regression analysis result showed a decrease problem solving domain score of 0.641 was associated with decreased of vitamin D. Cordblood vitamin D level correlates with infant neurodevelopmental status. Screening of vitamin D level is crucial during pregnancy and early childhood to improve neurodevelopmental outcome.


Background
Vitamin D is a pro-hormone secosteroid with active metabolites of calcidiol/25(OH)D and calcitriol/1,25-(OH)2-D. 1,2 Vitamin D classically participates in calcium and phosphorus metabolism, thus contributes to bone growth and reorganization.The non-classical role of vitamin D found signi cantly on extra skeletal sites, including in reducing oxidative stress, antimicrobial defense, immunoregulation, anti-in ammatory, anticancer, neuroprotective, and child neurodevelopment roles. 1,3,4e emerging awareness of hypovitaminosis D, including both de ciency and insu ciency conditions, is a major health problem across countries and regions among all age groups. 5According to the South East Asian Nutrition Survey (SEANUT), as serum 25-hydroxy-vitamin D (25 (OH) D) < 25 nmol / L (< 10 ng / mL), and 25 nmol / L to < 50 nmol / L (< 20 ng / mL) are de ned as vitamin D de ciency and insu ciency, respectively. 5ere are numerous study investigating the correlation between maternal vitamin D levels in various gestational ages and cord blood / or newborn vitamin D levels.A previous study stated that a positive correlation was observed, while this positive correlation was absent in another study. 6,7The presence of this correlation was assumed to be a result of vitamin D diffusing across the placenta from mother to fetus, therefore, fetuses are utterly dependent on their mother's vitamin D status. 6me observational studies had been conducted to analyze the relationship between newborn vitamin D levels taken from cord blood and neurodevelopmental status, with various results.Two previous studies conducted in the United State 8 and Australia 9 discovered a positive relationship between cord blood vitamin D levels and early childhood development, including neurocognitive and language aspects.However, another study in Shanghai observed there was no association between cord blood vitamin D levels and all developmental aspects in children aged 2 years old. 10e rst 1000 days of life was considered as a critical period, in which rapid central nervous system (CNS) development occurred.Disturbance in that period, one of which is hypovitaminosis D, could create a huge impact on the neurodevelopmental process.Therefore, researchers were interested in investigating the correlation of vitamin D levels with the developmental status of children at this critical period. 11

Design and study population
This was a cohort study and a part of larger study on 'Vitamin D status and its impact on pregnancy and childhood in Indonesia'.The study was conducted at Sukabumi and Waled district, West Java, Indonesia, from 2016 to 2019.The ethical approval had been obtained by the Research Ethics Committee, Universitas Padjadjaran.
The study subjects included infants born from pregnant women in the previously mentioned study.The pregnant women were informed about the research by our team in respective locations.Written consent was obtained from women who had agreed to all research procedures.Cord blood samples were taken immediately after the delivery process, then stored and examined for 25-(OH) vitamin D levels.The development status of their child was assessed in the 6 th , 12 th and 24 th month.
The inclusion criteria in this study were babies born at term (37-42 weeks gestation) and appropriate for gestational age.Exclusion criteria were infants with congenital abnormalities, history of bilirubin encephalopathy, seizures, and asphyxia.

Laboratory examination
Vitamin D samples were taken from biological material withdrawn from cord blood and stored in the temperature of -70 to -80˚C.Measurement of vitamin D levels was done using the Enzyme Linked Fluorescent Assay (ELFA) technique, with the VIDAS® 25 OH Vitamin D TOTAL (VITD) measuring device. 12This method was chosen because it has a wide measurement range and has a good correlation with the standard vitamin D test using liquid chromatography-tandem mass spectroscopy (LC-MS/MS).This method had the ability to detect 25(OH) vitamin D levels as low as 8.1 ng/mL. 12Vitamin D levels are categorized as being de cient if serum levels were less than 10 ng/mL or 25 nmol/L. 5ild developmental tools Child developmental assessments were carried out in the study site at Sukabumi and Waled regional health centres.Mothers recruited to the study were contacted by the research team to schedule follow-up of the infants at the ages of 6 (V1), 12 (V2) and 24 (V3) months at the regional primary health centres for assessment of their neurodevelopment status using the Ages and Stages Questionnaire-3(ASQ-3).The ASQ-3 was translated into Bahasa Indonesia and it had been validated.This tool consisted of 30 questions assessing five development domains: gross motor, fine motor, communication, problemsolving and personal-social domains.The answers to each question were scored as follows: 'yes' obtained a score of 10, 'sometimes' had a score of 5 and 'no' had a score of 0; each domain was then summed.Higher scores reflected better developmental state.These assessments were conducted by researchers and research assistants who had received standardized training.13

Statistical analysis
Mean, median, range, standard deviations, frequencies, and percentages were used to describe numerical or categorical data in descriptive analysis.To determine the differences between the development status of children from various aspects based on subject characteristics, the Mann-Whitney test or the Kruskal-Wallis test was used.The correlation between newborn vitamin D levels and the scores for each development domains was calculated using Spearman's Rank correlation.Categorical differences between vitamin D levels and each developmental domain were tested using the ANOVA test.Multiple linear regression analysis was performed to adjust all variables tightly correlated with vitamin D level and external variables.Result with a p-value less than 0.05 was considered significant.Data were managed and analyzed using the Statistical Product and Service Solution (SPSS version 20.0) program.

Covariate
The covariates in this study were mother's age, parity status, mother's education status, and occupation.Characteristics of infants were sex and exclusive breastfeeding.

Results
Of the 141 babies born from previous cohort studies, 4 babies were born premature, 10 babies were intrauterine growth restriction (IUGR), and 11 babies did not come for follow-up until the end of the observation, so they were excluded.( gure 1) Therefore, the total subjects in this study were 116.The minimum sample had been ful lled, with the power of a test of 95%.
Data on the subject's characteristics were presented in table 1; the majority of mothers were housewives (86.2%), primipara (72.4%) and had a low educational level (72.43%).The majority of infants received exclusive breastfeeding (90.5%).The mean ASQ development score for each domains of 3 different times visit can be seen on table 3.
There is a signi cant difference in the ASQ development score in the ve development domains at each different visit (V1, V2, V3).
The correlation was found between newborn vitamin D levels and the developmental problem-solving domain (table 4) at the age of 12 and 24 months old; r = 0.217, and r = 0.414 respectively; in other words, the higher the newborn vitamin D levels, the higher the problem-solving scores.This showed that the strength of correlation had increased in the 24 th month of observation; which according to Guilford correlation criteria, it had a moderate correlation.Based on the ANOVA test (table 5), the results show that vitamin D levels have a signi cant relationship to the problem-solving domain at the last two visits (V2, V3).To further prove the relationship between vitamin D levels and the personal-social domain, a multivariate logistic regression analysis was carried out (table 6).The result shows that a decrease in the problem-solving domain score of 0.641 is associated with a decrease in vitamin D levels.

Discussion
Hypovitaminosis D in pregnant women and neonates is a current major concern in obstetrics and neonatology eld. 6This study, as a part of the rst cohort study in Indonesia about Vitamin D status and its impact on pregnancy and childhood in Indonesia. 7The rst evidence related to vitamin D role on brain function was reported two decades ago through the discovery of vitamin D receptors (VDR) autoradiography in experimental animal brains and the discovery of 1,25-(OH)2-D/calcitriol in cerebrospinal uid. 14In addition to that, VDRs are also widely distributed in mammalian brain and rst expressed in brain development during the critical period of cell proliferation.These receptors could be found in certain brain regions, such as temporal lobes, cingulate, thalamus, cerebellum, amygdala and hippocampus areas. 14,15Calcitriol also works by affecting the production of cytokines and affecting neurotransmitters and synaptic plasticity, which play important roles in the learning process and neurocognitive development. 15,16tal brain development starts during early period of pregnancy.Some parts of the brain will develop rapidly in the last trimester, and the process of differentiation and synaptogenesis will develop up to postnatal period with the critical time in the rst 1000 days of life. 16Thus, disruption in that period such as low maternal vitamin D level during pregnancy could lead to impaired fetal brain structure formation, such as brain ventricular enlargement and neocortex region thinning. 4,17Another concept suggests that vitamin D de ciency may weaken the integrity of perineuronal nets (PNNs), so that neural-circuit function will be disturbed and cognitive processes such as learning and memory will be impeded. 18he mean value of newborn vitamin D level from 116 subjects in this study was 16.2 ng/mL (8.0-35.4ng/mL).As many as 12.9% of infants have vitamin D de ciency and 65.5% have insu ciency condition. 5A previous study on vitamin D level in the rst trimester of pregnancy, which is a part of this cohort study, showed that the mean value of maternal 25 (OH) vitamin D level was 17.52 ng/mL. 7This result is similar to other previous studies 6,8 which showed that newborn vitamin D level is lower (75-90%) than maternal vitamin D level.This could happen because the mother had to ful ll her own vitamin D requirement beside her fetal. 19evious studies conducted to determine the correlation of vitamin D levels during early 4 , mid 20 , and late pregnancy, and/or cord blood at birth 8 with child development had been carried out in several countries with various results. 9The previous part of this cohort study in Indonesia about associations between maternal vitamin D levels in early pregnancy (10-14 weeks gestation) with child development in the rst year ages of life (3 months, 6 months, and 12 months), showed that ASQ scores in gross motor domains were signi cantly lower at 3 months of age, and did not have a signi cant difference for all developmental aspects at older ages (6 and 12 months). 7,19 this study, we examined cord blood vitamin D samples because maternal vitamin D could pass through the placenta and enter the fetal bloodstream, with the half-life around 2 months.Therefore, it could represent vitamin D level in the newborns. 21,22This study showed correlations between cord blood vitamin D levels and the developmental problem-solving domain at 12 and 24 months of age (r = 0.217 and r = 0.414), but no correlation with gross motor, ne motor, communication and personal social domains.Multiple linear regression analysis reinforces the state that a decrease in problem-solving scores is associated with a decrease in vitamin D levels.This could be explained by the extraskeletal effect of vitamin D on neuroplasticity 15,16 , which determined neurocognition aspect.Problem-solving domain can be a representative of neurocognition aspect.Adverse effects occuring in early life, such as hypovitaminosis D and inadequate stimulations, could cause neurocognition disturbance that can persist until later life.This was consistent with a recent meta-analysis study, regarding the association of maternal or newborn vitamin D levels with neurodevelopmental outcomes, which found that prenatal vitamin D levels had borderline positive associations with the infant cognitive development but no association with infant motor development. 23cohort study in the United States assessing the relationship between cord blood vitamin D and developmental and cognitive achievement scores, showed that an increase in 5 nmol/L of vitamin D levels in the cord blood was associated with a very small increase in the Wechsler Intelligence Scale for Children (WISC) score at the age of seven years old. 8,24Other study in Australia stated that cord blood vitamin D level had a positive association with language development in children aged 18 months and 4 years old, although the association was weak. 9Thus, it could be concluded that vitamin D has an effect on child development, despite the weak association found on the currently available evidence.
The effort to increase infant vitamin D levels can be done in several ways.The provision of vitamin D supplementation in Indonesia is still di cult as a standard due to the high price.As an alternative, exposing 18.59% of body surface area to sunlight for 37.5 minutes per day especially between 10.00−13.00 is proven to be an effective method to meet daily vitamin D requirements, as reported by Judistiani et al. 25 The limitation of this study was not report, several factors that could have affected vitamin D levels and neurodevelopmental status at the age of two year old, such as parenting style and complementary food given after the age of 6 months old.Future long-term cohort studies taking these factors into consideration or randomized clinical trial giving vitamin D supplementation is required.

Conclusion
Vitamin D level correlates with infant neurodevelopmental status.Screening of vitamin D level is crucial to be done during early infancy, for early detection and intervention, as well.

Declarations
Ethics approval and consent to participate An approval was issued by the Health Research Ethics Committee Faculty of Medicine, Universitas Padjadjaran 335/UN6.KEP/EC/2018.Written informed consent was obtained from the parents SS, MD contributed equally to this work, MD, DS, SS designed the research with contributions from all authors, SS, LG searched the literature and extracted the data, RTD, BS contributed to the critical revision of the manuscript before publication

Table 1 .
General characteristic of infants and mothers (n = 116)One hundred and sixteen serum newborns were examined for vitamin D levels.The mean value of newborn vitamin D levels in this study was 16.27 ng/mL.As many as 12.9% of infants have vitamin D de ciency, 65.5% have insu ciency condition, and 21.6% have normal vitamin D levels.(table2)

Table 2 .
Cord blood vitamin D in newborn

Table 4 .
Correlation between newborn vitamin D levels and infant development

Table 5 .
Comparison of infant development status in three different time based on level of vitamin D levels