Vitamin D Depletion in Pregnancy Decreases Survival Time, Oxygen Saturation, Lung Weight and Body Weight in Preterm Rat Offspring

Abstract

Animal studies suggest a role of vitamin D in fetal lung development although not studied in preterm animals. We tested the hypothesis that vitamin D depletion aggravates respiratory insufficiency in preterm rat offspring. Furthermore, the effects of vitamin D depletion on growth and lung surfactant were investigated. Female Sprague-Dawley rats were randomly assigned low vitamin D (VDL) or control diet before mating and followed with serum 25-hydroxyvitamin D (s-25(OH)D) determinations. After cesarean section at gestational day 19 (E19) or day 22 (E22), placental weight, birth weight, crown-rump-length (CRL), oxygenation (SaO2) at 30 min and survival time were recorded. The pup lungs were analyzed for phospholipid levels, surfactant protein A-D mRNA and the expression of the vitamin D receptor (VDR). S-25(OH)D was significantly lower in the VDL group at cesarean section (12 vs. 30nmol/L, p<0.0001). Compared to the controls, E19 VDL pups had lower birth weight (2.13 vs. 2.29g, p<0.001), lung weight (0.09 vs. 0.10g, p = 0.002), SaO2 (54% vs. 69%, p = 0.002) as well as reduced survival time (0.50 vs. 1.25h, p<0.0001). At E22, the VDL-induced pulmonary differences were leveled out, but VDL pups had lower CRL (4.0 vs. 4.5cm, p<0.0001). The phospholipid levels and the surfactant protein mRNA expression did not differ between the dietary groups. In conclusion, Vitamin D depletion led to lower oxygenation and reduced survival time in the preterm offspring, associated with reduced lung weight and birth weight. Further studies of vitamin D depletion in respiratory insufficiency in preterm neonates are warranted.

Fig 1. SaO₂ measured in rat pups 30 minutes after birth.

SaO₂ was measured 30 minutes after birth at E19 (VD_L group (n = 13); control group (n = 23)) and E22 (VD_L group (n = 28); control group (n = 16)). Within 2 minutes 3–5 measurements were made and the highest value of SaO₂ was used for analysis. Each measurement was finished within 10–15 seconds to avoid desaturation secondary to prolonged measurement.

Fig 2. Survival-rate in hours after birth.

Kaplan-Meier survival analysis of both A) E19 (VD_L group (n = 58); control group (n = 28)) and B) E22 (VD_L group (n = 38); control group (n = 31)) pups. Comparison of survival curves using log-rank (Mantel-Cox) test.

Fig 3. Effects of vitamin D on mRNA levels of surfactant proteins.

Quantitative real-time PCR analysis of surfactant protein A-D mRNA transcripts in fetal rat lung at E19, VD_L group (n = 15); control group (n = 12) and at E22, VD_L group (n = 15); control group (n = 11). Data were normalized against GAPDH. Results were calculated as mean ± SEM values.

Fig 4. Effects of vitamin D on phospholipid levels.

Phospholipid levels in fetal rat lung at E19, VD_L group (n = 13); control group (n = 12) and at E22, VD_L group (n = 15); control group (n = 9). Phospholipid levels were determined using a modification of the Bartlett method followed by absorbance read at 800nm.

Fig 5. Immunohistochemical analysis of VDR in fetal rat lung.

Immunohistochemistry was performed on lung tissues from each dietary group at both E19 (VD_L group (n = 31); control group (n = 27)) and E22 (VD_L group (n = 35); control group (n = 19)) using the monoclonal D-6 antibody and counterstained with Mayer’s hematoxylin. Representative images of lung tissue from E19 (A) and E22 (B) (Magnification x28). Arrows indicate positive ATII cells. Positive (C) and negative (D) control sections with omission of the monoclonal D-6 antibody in the duodenum (Magnification x5). The expression of VDR in the lung was evaluated as the density of positive ATII cells (cells/μm²) (E).

Fig 6. Survival-rate of pups from mothers stratified by s-25(OH)D <25 nmol/L.

Kaplan-Meier survival analysis of both A) E19 (s-25(OH)D <25 nmol/L (n = 58); ≥25 nmol/L (n = 43)) and B) E22 (s-25(OH)D <25 nmol/L (n = 58); ≥25 nmol/L (n = 28)) pups. Comparison of survival curves using log-rank (Mantel-Cox) test.