Vitamin C supplementation improves placental function and alters placental gene expression in smokers

Abstract

Maternal smoking during pregnancy (MSDP), driven by nicotine crossing the placenta, causes lifelong decreases in offspring pulmonary function and vitamin C supplementation during pregnancy prevents some of those changes. We have also shown in animal models of prenatal nicotine exposure that vitamin C supplementation during pregnancy improves placental function. In this study we examined whether vitamin C supplementation mitigates the effects of MSDP on placental structure, function, and gene expression in pregnant human smokers. Doppler ultrasound was performed in a subset of 55 pregnant smokers participating in the "Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function" (VCSIP) randomized clinical trial (NCT01723696) and in 33 pregnant nonsmokers. Doppler ultrasound measurements showed decreased umbilical vein Doppler velocity (Vmax) in placebo-treated smokers that was significantly improved in smokers randomized to vitamin C, restoring to levels comparable to nonsmokers. RNA-sequencing demonstrated that vitamin C supplementation to pregnant smokers was associated with changes in mRNA expression in genes highly relevant to vascular and cardiac development, suggesting a potential mechanism for vitamin C supplementation in pregnant smokers to improve some aspects of offspring health.

Fig. 1

Consort diagram and study design. Pregnant smokers were randomized to vitamin C or placebo at less than 23 weeks of gestation as part of a clinical trial to evaluate impact on offspring lung function. Non-smoking subjects were recruited as part of an ancillary study to perform doppler ultrasound. Placentas were collected at delivery from 210 subjects for histology and gene expression analysis. Samples with pregnancy-related complications and poor-quality control metrics were excluded from molecular analysis.

Fig. 2

Comparison of Doppler ultrasound measures. Boxplots show effects by treatment group on (A) Umbilical vein Doppler velocity; (B) Umbilical vein volume blood flow; (C) Umbilical artery pulsatility index. Plots show median and interquartile ranges. * p < 0.05 compared to nonsmokers and vitamin-C treated groups, † p < 0.09 compared to vitamin-C treated group by F-test using mixed linear model per measurements adjusted for gestational age at ultrasound.

Fig. 3

Fetal/placental weight ratio by group and correlation with pulmonary function at 5 years of age. (A) Boxplot shows the distribution by treatment group. (B) Scatterplot shows the Pearson correlation between fetal/placental weight ratio and FEF_{25 − 75} measured at 5-year follow-up visit for each treatment group. The correlation was significant in the vitamin C treated smokers (orange) with a similar trend in nonsmokers (green), however this relationship was not observed in placebo treated smokers (purple).

Fig. 4

RNA-sequencing results of placentas. Volcano plots show difference in log fold change (log FC) versus –log10 p-value in vitamin C vs. placebo (A) and placebo vs. nonsmokers (B) Lines at y = 1.3 are equivalent to nominal p = 0.05. (C) Venn-diagram shows overlap of nominally significant changes in expression between groups. (D) Heatmap for overlapping set of 164 genes shown in C with nominally significant differential expression in placentas from smokers randomized to placebo vs. nonsmokers and in placentas from placebo vs. vitamin C-treated smokers. Columns represent individual subjects and rows represent individual genes. The mean direction of effect in 160/164 overlapping genes was reversed with vitamin C.

Fig. 5

Top differentially expressed genes validated by qPCR. (A–C) Boxplots for top 3 genes with significant differential expression in placentas from smokers randomized to placebo vs. nonsmokers and in placentas from placebo- or vitamin C-treated smokers validated by qPCR. (D) IPA analysis of nominally significant overlap identified enrichment of canonical signaling related to vasculogenesis. All genes shown were significantly upregulated in vitamin C supplemented smokers versus placebo and in never smokers vs. placebo.