Maternal Vitamin D in the Late First and Second Trimester is Beneficial for Healthy Development of Fetal Head Circumference

Background: We previously showed that vitamin D supplementation might ameliorate typical clinical symptoms in children with autism spectrum disorder. In this study we examined the effects of maternal vitamin D status during pregnancy on the development of the fetus. Methods: Seventy-eight healthy Japanese women with uncomplicated pregnancies were enrolled in the study. Urine 25-hydroxy vitamin D (25OHD) and creatinine were measured. 25OHD concentrations were normalized for creatinine (ng/mg creatinine). Newborn anthropometry (height (cm), head circumference (cm), chest circumference (cm) and birth weight (kg)) of babies delivered naturally were measured and the relationship with the urinary vitamin D concentration of the mother at less than 90 days (third trimester) to delivery and more than 90 days to delivery (late first to second trimester) was examined. PC12 cells with various neurobiological processes were cultured and differentiation induced by 25OHD enhanced nerve growth factor was assessed. Results: Maternal and newborn characteristics were not significantly different between the late first and third trimesters. The associations between newborn length, chest circumference, birth weight and the urinary 25OHD concentration of mother in the late first and second trimester and the third trimester were not significant. As lightly positive association between urinary 25OHD and head circumference was observed in the late first and second trimester (r= 0.309), but not in the third trimester. Six nmol/L of 25OHD (=1/5 of serum level) enhanced nerve growth factor induced differentiation in PC12 cells. Conclusion: These findings show that vitamin D supplementation in the late first and second trimester beneficial for the healthy development of the fetal head circumference and neural system.


Introduction
Vitamin D is a secosteroid associated with peripheral calcium homeostasis and nervous system function [1]. Significant changes in maternal vitamin D and calcium metabolism occur during pregnancy to provide calcium for fetal bone mineral accretion [2]. It is important that 25OHD readily crosses the placental tissue to the fetus [3,4]. Low 25OHD levels in the maternal and cord blood have been found to be significantly associated with decreased birth weight of infants [5]. Vitamin D deficiency is common in preterm infants [6] and vitamin D supplementation in women at high risk of vitamin D deficiency leads to improved neonatal handling of Ca [2]. Many studies have shown that maternal vitamin D level might affect fetal growth.
Autism spectrum disorders are developmental disorders associated with a high individual and social burden, but their aetiology is poorly understood. We previously reported that vitamin D supplementation for 9 months might ameliorate typical clinical symptoms in children with autism spectrum disorder [7]. Vitamin D may have an important role in the development of the brain.
In comparison with serum 25OHD concentration in non-pregnant women or women with uncomplicated pregnancies, half of urinary 25OHD was excreted in uncomplicated pregnant women at 15 weeks' gestation [8]. Given this, serum vitamin D state was quantified from maternal urinary vitamin D.
PC12 cells represent a useful in vitro model of neuronal differentiation with various neurobiological properties [9]. Exposure of nerve growth factor (NGF) cause PC12 cells to differentiate into sympathetic neuron-like cells that exhibit increased neurite outgrowth

Serum 25OHD and creatinine
Urinary 25OHD and creatinine were analyzed using a commercially available ELISA kit (Immundiagnostik AG, Germany) and colorimetric reaction kit using Jaffe reagent (QuantiChrom TM, CA, USA), respectively. The 25OHD concentrations were normalized for urinary creatinine (ng/mg creatinine).

Newborn anthropometry
Newborn anthropometry (height (cm), head circumference (cm), chest circumference (cm) and birth weight (kg)) of babies delivered naturally were measured by skilled midwives.

Cell culture and assay
PC12 cells obtained from the Riken Cell Bank (Ibaraki, Japan) were maintained in DMEM supplemented with 10% fetal bovine and horse serum (Gibco, Life Technologies) and 1% penicillin-streptomycin (Sigma). The cells were then incubated at 37°C in an atmosphere of 5% CO2/95% air. PC12 cells were seeded at a density of 1.5 x 10 5 cells [9]. Following 24 h of incubation, PC12 cells were treated with 0 ng/mL NGF with or without 6 nmol/L of 25OHD. The ACE activity was measured for an index of the differentiation of the PC12 cells and expressed as absorbance mIU of mg protein. AchE activity and protein were analyzed using a commercially available kit (Amplite TM Colorimetric kit, AAT Bioquest, USA) and Pierce TM BCA protein assay kit (Thermofisher Scientific, USA), respectively.

Statistical analysis
The differences between urinary 25OHD and newborn anthropometry were evaluated using the t-test. AChE activity was expressed as mean ±SD and evaluated using the t-test. A p-value of < 0.05 was considered to be statistically significant. Analyses were carried out using SPSS 21 for Windows (IBM, Japan).

Study subjects
The characteristics of the study subjects are shown in Table 1 Page of two groups classified by the times of urine specimen collection. The height and the weight at the time of the birth were a little bigger than the mean values shown by the Ministry of Health, Labor and Welfare. These data showed that the deliveries in this study were typical Japanese deliveries.

Newborn anthropometry outcomes and urinary 25OHD concentration in pregnancy
The associations between newborn length, chest circumference and birth weight and the urinary 25OHD concentration of mothers in the late first and second trimester and the third trimester were not significant. A slightly positive association between urinary 25OHD and birth head circumference was observed in the late first and second trimester (r= 0.358), but not in the third trimester (Figure 1).
The vitamin D receptor is found in multiple brain regions of the neonatal central nervous system [10,11]. Maternal vitamin D supplementation from the 2nd half of pregnancy until birth does not influence newborn growth [12]. Our study showed a slightly positive association between birth head circumference and urinary 25OHD concentration for the late first and second trimester. These results suggest that vitamin D levels in early pregnancy (1st trimester) are associated with newborn head size.

Enhancement of NGF-induced neuronal differentiation in PC12 cells by 25OHD
Six nmol/L of 25 OHD (=1/5 of serum level) enhanced nerve growth factor induced differentiation in PC12 cells (Figure 2).
Vitamin D plays a role in brain developmental mechanisms [13]. Lower maternal serum 25 OHD in the first trimester is associated with a higher autism risk [14]. These results suggest that preventing vitamin D deficiency in early pregnancy is essential to decrease the risk of autism. Vitamin D deficiency is highly prevalent in the winter months, seen in 64% of pregnant women [15]. Instruction with advice on how to supplement vitamin D by sunbathing and vitamin D-rich food is necessary. This is a preliminary study with a very small number of subjects. Further study with larger numbers of subjects is warranted, and could