Maternal plasma folate, vitamin B12 levels and multivitamin supplementation during pregnancy and risk of Autism Spectrum Disorder in the Boston Birth Cohort
Authors: Raghavan Ramkripa , Fallin M. Daniele , Wang Xiaobin 
Published: November 2023

BackgroundThe role of preconception/prenatal nutrition in the development of Autism Spectrum Disorder (ASD) is under‐studied. Maternal adequate folate intake is important in protecting against neural tube defects in offspring, but uncertainty remains on its role in ASD.ObjectiveTo understand the relationship between maternal multivitamin supplementation during pregnancy and maternal plasma biomarkers of folate and vitamin B12 measured 24–72 hours after delivery and risk of later Autism Spectrum Disorder in children.MethodsData are from the Boston Birth Cohort, an ongoing longitudinal prospective birth cohort study that recruited low‐income urban, primarily minority mother‐offspring pairs (n=1,391) at the Boston Medical Center and followed them from birth through childhood between 1998–2013. Based on electronic medical records, children were diagnosed as having ASD (n=107) or categorized as ‘typical’ (n=1284). Prenatal multivitamin supplement intake data and maternal blood samples were collected 24–72 hours post‐delivery. Plasma folate, vitamin B12 and homocysteine levels were later analyzed and maternal MTHFR genotype status was also determined. Cox proportional hazard regression was used to account for differential follow‐up time and pertinent covariates were adjusted.ResultsThere was a considerable variability in the frequency of multivitamin use and plasma levels among the study mothers. Maternal multivitamin supplementation (3–5 times/wk) was associated with significantly lower risk of ASD in offspring across all trimesters (adjusted hazard ratio (HR): 0.33, 0.38 and 0.43 for 1st, 2nd and 3rd trimesters respectively) (Table 1). However, when maternal plasma folate and vitamin B12 levels were analyzed as exposure variables, high levels of maternal vitamin B12 (>600 pmol/L) were associated with significantly increased risk of ASD (HR: 3.01; 95% CI: 1.64 – 5.52; p value: 0.001). High maternal folate levels (>59 nmol/L) were also associated with increased risk of ASD (HR: 2.27; 95% CI: 1.26 – 4.09; p value: 0.007). The risk was greatest for those children whose mothers had both high plasma folate (>59 nmol/L) and vitamin B12 (>600 pmol/L) (HR: 17.59; p value: <0.001) (Table 2). The risk of ASD in children did not differ based on maternal MTHFR genotypes. Maternal homocysteine levels did not predict offspring's ASD risk (HR: 0.99; 95% CI: 0.91 −1.07; p value: 0.86).ConclusionIn this urban low‐income minority birth cohort, we observed an elevated risk of ASD associated with high maternal plasma folate levels (>59 nmol/L), which far exceeds the excess cutoff suggested by the WHO (>45.3 nmol/L); however reported maternal vitamin supplementation was protective. Excess maternal vitamin B12 (>600 pmol/L) in pregnancy was also shown to be associated with greater ASD risk in offspring. The risk of ASD was highest if mothers had both excess prenatal folate and vitamin B12 levels. Our findings warrant additional investigation and highlight the need to identify optimum prenatal folate and vitamin B12 levels that maximize health benefits, at the same time minimize the risk of excess and its associated adverse consequences such as ASD.Support or Funding InformationThis study is funded in part by Wendy Klag Center for Autism and Developmental Disorders, JHBSPH. The parent study was supported in part by the March of Dimes PERI grants (20‐FY02‐56); NIEHS (R21 ES011666); and NICHD (2R01 HD041702). The follow‐up study is supported in part by the National Institute of Allergy and Infectious Diseases (U01AI090727, and R21AI079872); and Maternal and Child Health Bureau (R40MC27443).
Maternal multivitamin supplementation during preconception, first, second and third trimesters and risk of ASD in children

Maternal Supplementation
aHR*
95% CI
P value

Preconception
0.72
0.22 – 2.33
0.58

First Trimester

No Supplements
Ref

1–2 times/wk
0.61
0.21 – 1.78
0.37

3–5 times/wk
0.33
0.17 – 0.65
0.001

Almost everyday
0.61
0.34 – 1.10
0.1

Second Trimester

No Supplements
Ref

1–2 times/wk
0.95
0.35 – 2.62
0.93

3–5 times/wk
0.38
0.19 – 0.78
0.008

Almost everyday
0.67
0.35 – 1.27
0.22

Third Trimester

No Supplements
Ref

1–2 times/wk
1.18
0.44 – 3.15
0.75

3–5 times/wk
0.43
0.21 – 0.86
0.017

Almost everyday
0.77
0.41 – 1.43
0.41

Maternal plasma folate, Vitamin B12 levels measured during third trimester and risk of ASD in their children

Risk of ASD by WHO categories of plasma folate and vitamin B12 levels of deficient, normal and excess

Prenatal plasma folate (nmol/L)
aHR*
95% CI
P value

<13.5 nmol/L (Possibly Deficient)
0.95
0.40 – 2.28
0.91

>=13.5 to <=45.3 nmol/L (Normal)
Ref

>45.3 nmol/L (Excess)
1.31
0.78 – 2.19
0.31

Prenatal plasma vitamin B12 (pmol/L)

<200 pmol/L (Deficient)
1.68
0.60 – 4.72
0.32

>=200 & <=600 pmol/L (Normal)
Ref

>600 pmol/L (Excess)
3
1.64 – 5.49
<0.001

Using cut‐offs used in the literature

Prenatal plasma folate (nmol/L)

<=59 nmol/L (Deficient/Normal)
Ref

>59 nmol/L (Excess)
2.27
1.26 – 4.09
0.007

Prenatal plasma vitamin B12 (pmol/L)

<=600 pmol/L (Deficient/Normal)
Ref

>600 pmol/L (Excess)
3.01
1.64 – 5.52
0.001

Joint effects of maternal folate and vitamin B12

Neither elevated (<=59 nmol/L & <=600 pmol/L)
Ref

Either elevated (>59 nmol/L | >600 pmol/L)
1.2
0.63 – 2.29
0.59

Both elevated (>59 nmol/L & >600 pmol/L)
17.59
8.03 – 38.54
<0.001

aHR ‐ Adjusted Hazard Ratio