Association of cord blood vitamin D with child neurodevelopment at 7 years of age

Aim: The role of fetal vitamin D [25-hydroxyvitamin D (25(OH)D)], one of the nuclear steroid transcription regulators, and brain development is unclear. We previously found a weak but persistent association between cord blood 25(OH)D and child language abilities at 18 months and 4 years of age, but no association with cognition or behaviour. The aim of this study was to investigate the association between cord blood 25(OH)D and a range of neurodevelopmental outcomes in these same children at 7 years of age. Methods: Cord blood samples from 250 Australian mother – child pairs were analysed for 25(OH)D by mass spectroscopy. Children underwent tests of cognition, language, academic abilities and executive functions with a trained assessor at 7 years of age. Caregivers completed questionnaires to rate their child ’ s behaviour and executive functioning in the home environment. Associations between standardised 25(OH)D and outcomes were assessed using regression models, taking into account possible social and demographic confounders. Results: Standardised 25(OH)D in cord blood was not associated with any test or parent-rated scores. Nor was there any association with the risk of having a poor test or parent-rated score. Likewise, cord blood 25(OH)D categorised as <25, 25 – 50 and >50 nmol/L was not associated with test scores or parent-rated scores. Conclusions: There was no evidence that cord blood vitamin D concentration or de ﬁ ciency was associated with cognition, language, academic abilities, executive functioning or behaviour at 7 years of age.

5][6] The implications of nutritional deficiencies during this period for normal child development are serious [1][2][3][4] and later supplementation often does not restore development after an early deficit. 5,6For example, early vitamin D deficiency leads to rickets. 7Vitamin D is also thought to have a role in neurodevelopment as the main form of circulating Vitamin D [25-hydroxyvitamin D (25(OH)D)] 8 is a member of the superfamily of nuclear steroid transcription regulators 9 with receptors widely distributed throughout the human brain. 10Concerns about increasing prevalence of low vitamin D status among pregnant women have prompted the Australian Government Pregnancy Care Guidelines to recommend that pregnant women may need vitamin D supplements if 25(OH)D status is <50 nmol/L. 114][25][26][27] Two reviews of trials of vitamin D supplementation for children with attention-deficit/hyperactivity disorder also found a small decrease in symptoms, although authors noted the quality of evidence was low. 28,29[16][30][31][32][33] One review concluded that there was a beneficial association between child cognition and language and vitamin D exposure that is grouped across pregnancy, infancy and childhood, but mixed results for associations with behavioural symptoms and motor abilities. 15A more recent narrative review of vitamin D exposure in pregnancy and childhood concluded that maintaining adequate prenatal vitamin D may protect against poor neurocognitive outcomes; however, the authors did not discuss the quality of the evidence base or whether there were differences for specific domains. 30A review of vitamin D trials and neurodevelopment did not include prenatal supplementation but reported interventions after birth had mixed, largely null effects on child neurodevelopment. 20Similarly, a review of trials in preterm and low birthweight infants found no benefits to neurodevelopmental outcomes. 16However, reviews focused on prenatal vitamin D, 13 or prenatal and neonatal vitamin D 14 judged there to be some evidence supporting an association between low vitamin D status and early language development, although no evidence of associations with other neurological domains. 13,14Differing conclusions are likely due to variations in inclusion and exclusion criteria (although there was overlap in the original studies included in the reviews), and heterogeneity of included studies, as well as their appraisal and synthesis.All reviews of vitamin D in pregnancy highlighted that language appears to be neurodevelopmental domain most sensitive to vitamin D exposure, although there was a general lack of strong evidence, and all reviews emphasised the need for studies of high quality with neurodevelopmental assessments, particularly targeting language, beyond early childhood when language abilities are emerging. 13,14,31r own previous exploration of the association between cord blood 25(OH)D concentration and child development at 18 months and at 4 years of age 34 in 300 Australian motherinfant pairs found no association between cognition or motor skills.However, we identified a small consistent positive relationship between cord blood 25(OH)D and language at 18 months and at 4 years. 34We subsequently conducted a battery of neurodevelopmental assessments, including language, on these children when they reached 7 years of age. 35,36Based on our previous finding of an association between 25(OH)D and early language skills, 34 combined with the calls for language assessments beyond early childhood, [13][14][15] we now explore the association between cord blood vitamin D status and 7-year cognition, language, academic abilities, behaviour and executive functioning.

Methods
This is an exploratory analysis of data collected for the multicentre randomised controlled trial (registered at www.anzctr.org.au as ACTRN1260500056906) evaluating the effect of maternal omega-3 supplementation during the second half of pregnancy. 37other-child pairs enrolled at two Adelaide sites (Adelaide latitude 34 S55 0 S) between 2005 and 2008 and were followed up at 18 months, 37 4 years, 38 and 7 years 35,36 so that child neurodevelopment could be assessed.6][37][38] To summarise, women with a singleton pregnancy less than 21 weeks' gestation were randomised to receive daily capsules of either omega-3-rich fish oil or vegetable oil (control group) from enrolment until birth.Capsules did not contain vitamin D.

Exposure assessment
The major circulating form of vitamin D is 25(OH)D, which is widely used to indicate vitamin D status. 8,39Samples of umbilical cord blood were collected at delivery, centrifuged and the plasma immediately stored at À20 C, as detailed previously. 34,40Cord blood samples were shipped on dry ice to the Steroid and Immunobiochemistry Laboratory in Christchurch (New Zealand) for 25(OH)D analysis by liquid chromatography-tandem mass spectroscopy. 41 To explore the consequences of vitamin D deficiency, we categorised our cord blood sample as <25, 25-50 and >50 nmol/L, according to Australian definitions of vitamin D 'deficiency', 'likely insufficiency' and 'sufficiency' for blood 25(OH)D at the time of sample collection. 8,42

Sample characteristics and confounder assessment
Information on possible confounding factors such as maternal age, race, education, smoking status and current use of vitamin or mineral supplements at mid-pregnancy were collected by trained staff who interviewed pregnant women at enrolment into the trial (mid-pregnancy).At each follow-up appointment, parents were interviewed about a range of home and child health characteristics.

Outcome assessments
A neurodevelopmental assessment battery was administered by a trained psychologist or technician when children reached 7 years of age. 36Parents completed a series of questionnaires while the children were being assessed.General cognition was assessed with the Full-scale Intelligence Quotient (FSIQ) score of the Wechsler Abbreviated Scale of Intelligence, Second Edition (WASI-II). 43The WASI-II Verbal Comprehension Index measuring verbal intelligence and the Perceptual Reasoning Index measuring non-verbal intelligence were included. 43General language ability was tested with the Core Language Score of the Clinical Evaluation of Language Fundamentals, Fourth Edition (CELF-4). 44Children's reading, spelling and mathematical abilities were measured with the Word Reading, Spelling and Math Computation subtests of the Wide Range Achievement Test, Fourth Edition (WRAT-4). 45The WASI-II, CELF-4 and WRAT-4 have scores that are age-standardised to a mean of 100 (SD = 15), and corrected age was used to standardise the performance of children that were born preterm.Children with WASI-II FSIQ or CELF-4 Core Language scores that were more than 1 SD below the mean (<85) were categorised as having a mild impairment.
Executive functions were assessed 36 with the Number Repetition subtest of the CELF-4, 44 the Fruit Stroop, 46 and the Sky Search, Score!, Creature Counting and Sky Search Dual Task of the Test of Everyday Attention for Children (TEACh). 47TEACh scores are standardised to a mean of 10, 47 with the exception of the Sky Search Dual Task composite score. 48Parents completed the Behaviour Rating Inventory of Executive Function (BRIEF), measuring behavioural manifestations of executive dysfunction, 49 along with the behavioural-rating questionnaires, the Strengths and Difficulties Questionnaire (SDQ) 50 and the Conners 3rd Edition Attention Deficit Hyperactivity Disorder Index (Conners 3-AI). 51The Conners 3-AI and BRIEF were standardised to a mean of 50 (SD = 10).Children with high scores were categorised as having a possible behavioural problem (Conners 3-AI ≥60, BRIEF Global Executive Composite T Score >65 and SDQ Total Difficulties Score >14).

Statistical analyses
To account for seasonal variation in 25(OH)D levels, we standardised 25(OH)D values by regressing 25(OH)D on the month of cord blood collection and adding residuals to the overall mean 25(OH)D, weighted by month to account for the differing number of cord blood samples collected each month. 34,40,52e previously explored standardising 25(OH)D using periodic regression, 53 although this model resulted in a slightly worse fit to the 25(OH)D values. 34e combined randomisation groups as we previously demonstrated that the intervention did not affect 25(OH)D concentration or its associations with child development. 34,40nalyses were performed using linear regression models for continuous outcomes and log-binomial regression models for binary outcomes to estimate the effect (difference in means or relative risk, respectively) of a 10 nmol/L increase in continuous 25(OH)D (rather than a 1 nmol/L increase, which is very small), and to compare infants whose 25(OH)D was 25-50 or >50 nmol/L compared with <25 nmol/L. 8,39,42For analyses comparing categorised 25(OH)D, the overall P value from the global test for any difference between groups is presented in addition to two pairwise comparisons (25-50 nmol/L vs. <25 nmol/L and >50 nmol/L vs. <25 nmol/L).
Child neurodevelopment results were adjusted for a priori determined characteristics randomised treatment group (omega-3 or vegetable oil) from the original trial, centre, parity, infant sex, maternal secondary education, maternal further education and smoking status, in line with our previous analyses. 34No adjustment was made for any post-birth characteristics known to influence neurodevelopment, as these may lie on the causal pathway.Since the effect of vitamin D on outcomes may be mediated by preterm birth, 54 we conducted sensitivity analyses involving only the term-born children, similar to previous robust observational studies. 34,55,56All analyses were performed using R version 4.1.3. 57

Results
There were 250 children who underwent neurodevelopmental assessments at 7 years of age who had cord blood 25(OH)D status available (46% of the 543 mother-child pairs who participated in the 7-year follow-up, see Fig. 1).Families in the 7-year follow-up with available cord blood samples did not differ in characteristics from the overall trial sample.Nor were the characteristics of mother-child pairs who did not provide data for all outcomes (data not shown).The mean (SD) standardised cord blood 25(OH)D concentration was 55.6 (24.5) nmol/L (Table 1).Over half (56.0%) of the mothers in the sample had cord blood 25(OH)D status >50 nmol/L, 37.2% were in the 25-50 nmol/L range, and 6.8% had 25(OH)D <25 nmol/L.Characteristics of participants appeared proportionally distributed among the three 25(OH)D statuses, although the distribution of participants across the three status groups was not balanced (Table 1).
Average test scores and parent-rated scores were all within the normal range of development.None of the test scores or parentrated scores were associated with cord blood 25(OH)D at delivery once confounding was accounted for (Table 2).Likewise, there was no association between cord blood 25(OH)D and risk of an impaired FSIQ or Core Language Score, or a possible behavioural problem.
None of the test scores from the WASI-II, CELF-4, WRAT-4, TEACh or Fruit Stroop were associated with categorised 25(OH)D status (Table 3).Individual pairwise comparisons (25-50 nmol/L vs. <25 nmol/L and >50 nmol/L vs. <25 nmol/L) showed no significant difference between groups, possibly due to the low numbers in the deficient category, though significant differences were observed between the 25-50 and >50 nmol/L groups (data not shown).After controlling for potential confounders, there were generally null associations between categorised cord blood 25(OH)D and parent-reported scores (Table 3).
Adjusted analyses were not conducted for cord blood 25(OH)D <25, 25-50 and >50 nmol/L with impaired FSIQ or Core Language Score, or indicators of behavioural problems due to the small sample size and limited numbers in these categories (Table 3).There was no indication of association between categorised 25(OH)D and the unadjusted risk of scores indicative of a possible problem.
Results of the sensitivity analyses restricted to the n = 233 termborn children were largely consistent with null findings in the main analyses (data not shown), with the exception that a 10 nmol/L increase in cord blood 25(OH)D was associated with a decrease (benefit) of 0.38 points (95% confidence interval = À0.68 to À0.09, P = 0.01) on the SDQ total difficulties score in adjusted analyses.

Discussion
We explored the association between cord blood 25(OH)D and a comprehensive range of robust neurodevelopmental assessments at 7 years of age in a cohort of 250 Australian mother-child pairs.As with our assessments of these children at 18 months and at 4 years of age, 34 there were no associations between cord blood 25(OH)D and cognition.Null associations were also observed between cord blood 25(OH)D and child academic abilities, executive functions and parent-rated behaviour.The consistent positive association between language at 18 months and 4 years of age and cord blood 25(OH)D 34 was not evident in the 7-year results.It may be that the association between vitamin D and language diminishes with age, or is overtaken by more prominent environmental drivers of language abilities.Exploration of vitamin D deficiency (cord blood 25(OH)D <25 nmol/L) was not associated with any aspect of neurodevelopment.Our findings indicate that cord blood vitamin D may not have a significant influence on long-term child neurodevelopment in generally healthy, well-nourished children.
][16]20,[30][31][32][33] Two notable reviews focused on prenatal vitamin D exposure noted limited evidence to support an association between low vitamin D exposure and adverse early language development, 13,14 and null associations in the handful of assessments in older children. 14The only review to include a meta-analysis of observational vitamin D studies found null associations with language or any other neurodevelopmental outcome. 15 handful of small randomised controlled trials of vitamin D supplementation with child neurodevelopment assessments are emerging, [58][59][60][61][62][63][64][65][66][67][68][69][70][71] although only two have intervened during pregnancy. 33,72Over 600 pregnant Danish women were randomised to either 2800 IU or 400 IU vitamin D 3 per day and a mixture of parent-reported and psychologist assessments was conducted at regular intervals between 1 and 6 years of age. 72The authors reported no effect of vitamin D on language skills assessed at 1, 2.5 or 3 years of age, and poorer language skills at 2 years of age in the high vitamin D group. 72There were no group differences in motor, cognitive or behavioural assessments at 1-6 years of age. 72An American trial randomised 506 pregnant women to either 400 IU, 2000 IU or 4000 IU of vitamin D 3 per day and assessed child language, motor and academic abilities summed into one score at 3, 4 and 5 years. 33Children whose mothers were randomised to 2000 IU vitamin D per day were reported to have higher scores. 33Other trials conducted in preterm neonates 58,71 or older children [59][60][61][62][63][65][66][67][68][69][70]73 have similarly showed largely null results. Evidence rom observational studies and randomised controlled trials of vitamin D are limited due to the lack of large, robust studies conducted in deficient or insufficient samples.In addition, there is uncertainty around the definition of vitamin D insufficiency or deficiency, 74 particularly for neurodevelopment, 14 as well as lack of clearly defined underlying mechanisms through which vitamin D influences neurodevelopment.[12][13][14][15][16][17][18][19][20][21][22] Even the most recent Cochrane review of vitamin D in pregnancy identified 30 trials in over 7000 women (without reporting child neurodevelopment) and found the quality of evidence to be moderate to very low.75 Future studies should focus on sufficiently large (powered) samples of vitamin D-deficient populations to verify that low vitamin D in pregnancy does not pose a threat to child neurodevelopment.
Our study is one of the few to explore the association between fetal vitamin D exposure and neurodevelopmental assessments beyond early childhood.Most noteworthy is that long-term language assessments are largely absent from the evidence base.We administered a comprehensive range of robust neurodevelopment assessments and had the benefit of both overall associations of cord blood 25(OH)D as well as deficiency, using 25(OH)D status based on Australian definitions of vitamin D inadequacy 8,42,76 and were still unable to detect associations between them.We had the added advantage that we were able to explore 4 the association between vitamin D and language assessed beyond early childhood. 13Our strong methodology was similar to our earlier report, 34 which had the highest quality ranking in a review of observational studies of vitamin D in pregnancy and subsequent neurodevelopment. 13We used the gold standard methodology for determining 25(OH)D due to high sensitivity and specificity. 77Our use of cord blood 25(OH)D is likely to reflect fetal exposure, and cord blood concentrations are slightly lower although strongly correlated with maternal levels. 8,78Given possible associations between preterm birth and lower vitamin D status, we conducted additional sensitivity analyses excluding children born preterm, which gave similar results.Although we did not measure child 25(OH)D status, we have now explored associations between 25(OH)D exposure and child development at 18 months, 4 years, 34 and 7 years in the same cohort.4][15] Attrition increased across the follow-ups which further decreased the sample available for inclusion in the present analyses.Further, the sample is drawn from a trial of omega-3 supplementation and was not specifically targeting the effect of vitamin D exposure and we may have missed collecting potentially relevant information about the mothers or children that may be relevant for the present analyses.For example, maternal vitamin D supplementation after birth, or supplementation of the infants or children was not captured.As with any observational analyses, there is the possibility of residual confounding. 79

Conclusion
We observed no associations between cord blood 25(OH)D and childhood cognition, language, academic abilities, executive functioning or behaviour at 7 years of age, even when vitamin D status was categorised as deficient.
Standard Reference Material 972 from the National Institute of Standards and Technology was used to determine the accuracy of the methods.In levels 1, 2 and 3 of the Standard Reference Material 972, we obtained 25(OH)D concentrations of 60, 30 and 47 nmol/L, respectively, compared with the National Institute of Standards and Technology Certified Concentration Values of 60, 31 and 46 nmol/L.Method precision (6-7%) was established by repeat (n = 16) measurements of three control serum samples, which had concentrations of 23, 59 and 107 nmol/L 25(OH)D.

Had cord blood available for inclusion in vitamin D side-study Total n = 337 Fig. 1
Fig. 1 Participant flow from enrolment during pregnancy to assessments of children at 7 years of age.

Table 1
Characteristics of mother-child pairs included in the 7-year follow-up at baseline and 7 years by standardised cord blood vitamin D (25(OH)D)

Table 2
Effect of a 10 nmol/L increase in standardised cord blood vitamin D (25(OH)D) on neurodevelopment assessments at 7 years of age † Values are difference in means (95% CI) for a 10 nmol/L increase in standardised 25(OH)D unless otherwise indicated.‡Adjustedanalysis includes one fewer child than unadjusted analysis due to missing data for adjustment covariates.§Adjustedanalyses were adjusted for treatment group, centre, parity, infant sex, mother's secondary education, mother's further education and smoking status.¶Values are n/N (%) and relative risk (95% CI) for a 10 nmol/L increase in standardised 25(OH)D.25(OH)D,Vitamin D; BRIEF, Behaviour Rating Inventory of Executive Function; Conners 3-AI, Conners 3rd Edition Attention Deficit Hyperactivity Disorder Index; CELF-4, Clinical Evaluation of Language Fundamentals, Fourth Edition; FSIQ, Full Scale Intelligence Quotient; SDQ, Strengths and Difficulties Questionnaire; TEACh, Test of Everyday Attention for Children; WASI-II, Wechsler Abbreviated Scale of Intelligence, Second Edition; WRAT-4, Wide Range Achievement Test, Fourth Edition.

Table 3
Effect of standardised vitamin D (25(OH)D) status at delivery categorised as <25, 25-50 and >50 nmol/L on neurodevelopment outcomes at 7 years of age