Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

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

  • Sine Lykkedegn,

    Affiliations Hans Christian Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark, Clinical Institute, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

    ⨯
  • Grith Lykke Sorensen,

    Affiliation Institute of Molecular Medicine, Department of Cancer and Inflammation, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

    ⨯
  • Signe Sparre Beck-Nielsen,

    Affiliations Hans Christian Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark, Clinical Institute, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

    ⨯
  • Bartosz Pilecki,

    Affiliation Institute of Molecular Medicine, Department of Cancer and Inflammation, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

    ⨯
  • Lars Duelund,

    Affiliation MEMPHYS, University of Southern Denmark, Odense, Denmark

    ⨯
  • Niels Marcussen,

    Affiliation Institute of Pathology, Odense University Hospital, Odense, Denmark

    ⨯
  • Henrik Thybo Christesen

    henrik.christesen@rsyd.dk

    Affiliations Hans Christian Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark, Clinical Institute, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

    ⨯

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.

Citation: Lykkedegn S, Sorensen GL, Beck-Nielsen SS, Pilecki B, Duelund L, Marcussen N, et al. (2016) Vitamin D Depletion in Pregnancy Decreases Survival Time, Oxygen Saturation, Lung Weight and Body Weight in Preterm Rat Offspring. PLoS ONE 11(8): e0155203. https://doi.org/10.1371/journal.pone.0155203

Editor: Colette Kanellopoulos-Langevin, Xavier Bichat Medical School, INSERM-CNRS - Université Paris Diderot, FRANCE

Received: December 22, 2015; Accepted: July 28, 2016; Published: August 29, 2016

Copyright: © 2016 Lykkedegn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are presented within the paper thus fully available. Additional data may be requested by contacting the corresponding author (henrik.christesen@rsyd.dk).

Funding: All studies were financially supported by: Dagmar Marshalls foundation, Aase & Ejner Danielsens foundation, Takeda Pharma, The A.P. Moeller Foundation for the Advancement of Medical Science and Odense University Hospital. The funding sources had no involvement in collection, analysis and interpretation of data, writing of the report, or decision to submit the article for publication.

Competing interests: The authors confirm that the financial support by Takeda Pharma does not alter the authors' adherence to PLOS ONE policies on sharing data and material.

Abbreviations: CRL, crown-rump length; LW/BW ratio, lung weight/birth weight ratio; PW/BW ratio, placental weight/birth weight ratio; S-25(OH)D, serum 25-hydroxyvitamin D; SaO2, oxygen saturation; VDL, low vitamin D diet; VDR, vitamin D receptor

Introduction

At birth, adequate lung development and growth is the key to successful adaptation to extrauterine life [1]. Thus, appropriate lung function depends on both proper development of the lung structure as well as sufficient surfactant synthesis and secretion [1, 2]. In preterm neonates inadequate lung maturation leads to respiratory distress syndrome (RDS) characterized by structural immaturity and inadequate synthesis and secretion of surfactant [2]. Antenatal corticosteroids [3, 4], intratracheal surfactant [5] and nasal continuous positive airway pressure (nCPAP) or mechanical ventilation [6, 7] are important in modern treatment of RDS. Yet, respiratory insufficiency due to lung immaturity is still a major cause of mortality in extremely preterm neonates [6], and research in other treatment modalities are needed.

Placenta plays a vital role in normal fetal development, and fetal or neonatal disorders may be a result of placental insufficiency. Hypovitaminosis D, most commonly defined as s-25(OH)D levels below 50 nmol/L, is frequent in both pregnant women and preterm neonates [8–13]. Some human studies have shown an association between low s-25(OH)D levels during pregnancy and reduced placental development and weight [14–16], while others have not [17, 18]. Both placental weight and placental weight/birth weight (PW/BW) ratio have been used to describe the growth conditions for the fetus [19–22]. In humans, a high PW/BW ratio has been associated with RDS, low apgar scores and increased risk of admission to the neonatal intensive care unit [21, 23]. Only one human study [17] has approached the association between vitamin D and PW/BW ratio, however, without positive findings.

The impact of calcitriol (1,25(OH)2D), the active metabolite of vitamin D) on early lung development and maturation as well as on development of lung diseases in early life is an emerging field of research. A recent human study found an association between s-25(OH)D levels below 30 nmol/L and increased oxygenation requirement and greater need of assisted ventilation in preterm neonates [24]. Yet, the human evidence on the impact of 1,25(OH)2D on lung development and maturation remains sparse. In contrary, both animal and laboratory studies, primary based on vitamin D enrichment, have provided detailed insights into the mechanisms through which 1,25(OH)2D stimulates the development and maturation of the lung [3]. In vivo studies in mice [25] and rats [26, 27] have shown that vitamin D deficiency during pregnancy causes alterations in lung growth and structure in term offspring. In vitro studies of cells from fetal rat lung explants have shown an impact of vitamin D on the embryogenesis and cellular growth and differentiation, including surfactant synthesis and secretion [28–35]. Laboratory studies on human pulmonary adenocarcinoma-derived cell lines support these findings [34, 36, 37]. However, in vivo studies of the role of vitamin D in lung development in preterm offspring have not been performed.

We therefore tested the hypothesis that vitamin D depletion in pregnancy does not aggravate respiratory insufficiency in the preterm rat offspring.

Materials and Methods

Animals

Sprague-Dawley rat dams were purchased from Taconic Biosciences (Denmark) and housed in a facility without windows and light without UV-B radiation. All fluorescence lamps were shatterproof and had built-in UV-filters (Phillips MASTER TL-D Secura 58W/830 (150cm)). Room air was kept at 21–24 degrees Celsius and the animals were exposed to day-night cycles alternating every 12 hours. The animals (n = 34) were randomly assigned to two different dietary groups; a low vitamin D (VDL) diet group (n = 18) (<5 IU/kg cholecalciferol, purified vitamin D3 deficient diet, Art No E15312-24) and a control diet group (n = 16) (1500 IU/kg cholecalciferol, purified control diet, Art No E15000-04). Both diets were designed for maintenance, were identical in terms of all other components including calcium and phosphorus and were obtained from Ssniff, Soest, Germany. Adult ten-week-old females were fed ad libitum for five weeks with the assigned diet before mating with adult males and continued throughout the pregnancy. The males were maintained on a standard rat chow (600 IU/kg cholecalciferol, Art No #1320, Altromin Spezialfutter, Lage, Germany). The pregnant females from each dietary group were randomly assigned to either the E19 or E22 subgroup immediately after the appearance of vaginal plug (gestational day 0 (E0)). Blood samples were collected from the tail vein before mating and after cesarean section before euthanizing. To observe the welfare, the weight of the pregnant rats was followed throughout the study. In a supplementary study, a whole-body dual-energy x-ray absorptiometry (DXA) scan of some of the pups from each dietary group was performed to describe the bone mineral content. The Danish National Animal Experiments Inspectorate approved the study (permit number: 2012-15-2934-00243).

Cesarean section and survival

On gestational day 19 (E19) or 22 (E22)—roughly equivalent to human gestational week 24 and term, pregnant mothers were anesthetized with isoflurane inhalation and the pups were recovered by cesarean section. Maternal oxygen saturation (SaO2) was monitored during the procedure and all efforts were made to minimize suffering. After removal of the pups, serum samples from the mothers were collected by intracardiac puncture followed by euthanization with pentobarbital sodium. Newborn pups were immediately wiped dry with a cloth and placed on a heating pad. After the cesarean section, the placenta and pups were weighed and the pup crown-rump length (CRL) was assessed within 30 minutes after birth. SaO2 was measured at 30 minutes after birth, and the pups were randomly, selected to be sacrificed immediately hereafter or kept with foster mother rats. The lungs from the euthanized pups were weighed and dissected for qPCR (quantitative real-time PCR) analysis, immunohistochemical analysis and phospholipid analysis. An investigator blinded to the dietary group monitored the survival of the non-sacrificed pups with a frequency of 15 minutes for the first two hours. The pups were euthanized if no movements, low heart rate and undetectable low SaO2 were observed. To observe the welfare and describe the survival time of the E22 pups, the blinded investigator registered daily weight, the ability to suckle and the activity level the first seven days after birth. Our human endpoints were observed distress and/or affected welfare described as weight loss > 20% within the first two days of life, inactivity and/or ruffled fur. In case of doubt, a veterinarian examined the pups.

Oxygen saturation

SaO2 was measured by using Nonin Pulse Oximeter® on the rats and pups in room air. The sensor was attached to the precordial site, and the animals were placed in a prone position to maintain a good attachment of the sensor. During the measurements, the pups were kept on a heating pad to avoid hypothermia. The readings of SaO2 were accepted as valid when the simultaneously monitored heart rate was stable. Each measurement was finished within 10–15 seconds to avoid desaturation secondary to prolonged measurement. For each animal, three to five measurements were taken within 2 minutes and the highest value of SaO2 was used for analysis.

Serum 25(OH)D, calcium and phosphorus levels

Serum samples were centrifuged and kept frozen at -20°C until analysis. All analyses were made after the study was finished. While the levels of s-25(OH)D were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [38], serum phosphorus and calcium levels were measured using a Roche Cobas 8000 Autoanalyzer Spectrophotometric (Roche Diagnostics®).

qPCR analysis

To quantify mRNA expression of the surfactant protein A-D genes (sftpa, sftpb, sftpc, sftpd), total RNA was extracted from homogenized lung tissue using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Two μg RNA were used for cDNA production. Reverse transcription was performed using M-MLV Reverse Transcriptase (Sigma), and the reaction product was diluted to a final concentration of 10 ng/μl. Real-time PCR was performed in duplicates using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays specific for the given gene. The assay kits used were as follows: Gapdh, Rn01775763_g1; Sftpa1, Rn00824545_m1; Sftpb, Rn00684778_m1; Sftpc, Rn00569225_m1; Sftpd, Rn00563557_m1. The results were calculated using the 2ΔΔCt method.

Phospholipid levels

Isolated lungs were snap-frozen in liquid nitrogen and stored at -80°C until analysis. Lipid extraction was performed using a MTBE/methanol/ammonium acetate protocol [39]. Afterwards, the phospholipid concentration was determined in duplicates using a modification of the Bartlett method [40]. Briefly, 0.65 ml 70% perchloric acid was added to the dried samples followed by heating at 190°C for 60 minutes. Thereafter, the cooled samples were mixed with 3.3 ml water, 0.5 ml 2.5% ammonium molybdate and 0.5 ml 10% ascorbic acid followed by incubation at 100°C for 10 minutes. Finally, 250 μl from each cooled sample was transferred to a 96-well microtiter plate and absorbance was read at 800nm.

Immunohistochemical detection of VDR

Isolated lungs were immersed in 4% paraformaldehyde at room temperature for 24 hours fixation, processed, embedded in paraffin wax and cut into sections of 1.5 μm thickness. Before nuclear staining with Mayer’s hematoxylin, the sections were pretreated with MBO/TEG for 15 minutes followed by D-6 monoclonal antibody against VDR (dilution 1:500, Santa Cruz Biotechnologies cat. no. sc-13133). The sections were scanned, and microscopic analysis was performed using newCAST software (Visiopharm, Hoersholm, Denmark). Each section was analyzed according to previously described procedures [41] by an investigator blinded to the dietary group as well as the gestational age of the pups. The expression of VDR in the lung was estimated as the density of VDR-positive alveolar type II cells (cells/μm2 lung tissue).

Statistics

Shapiro-Wilks test revealed non-normal distribution of s-25(OH)D, maternal SaO2, CRL and lung weight. Between-group comparisons were made using two-way ANOVA and two-tailed unpaired t-tests, or non-parametric Mann-Whitney U test when appropriate. Survival analysis was performed using log-rank Mantel-Cox test. Normally distributed data were presented as mean ± SEM and non-normally distributed data as median and range. Spearman’s correlation was used to study within group correlations between SaO2 and LW, BW, LW/BW ratio and PW/BW ratio.

A power calculation assuming an E19 SaO2 SD of 10, two-sided alpha 0.05 and beta 0.20, showed a need of 34 pups to detect a true difference in SaO2 of 10 between groups. As we only expected to be able to measure SaO2 in one pup per litter, mother rat n = 34 were chosen.

Due to a substantial overlap in s-25(OH)D levels between the two dietary groups, we further performed a post hoc analysis. In this analysis associations between maternel s-25(OH)D at cesarean section and the measured phenotypical outcomes (SaO2, PW, BW, LW, LW/BW ratio, PW/BW ratio and CRL) were analyzed using linear uni- and multivariate regression models disregarding of initial dietary group. Moreover, we also performed a post hoc survival analysis comparing pups of mothers with s-25(OH)D <25nmol/L to pups of mothers with s-25(OH)D ≥25 nmol/L disregarding of initial dietary group.

A p-value <0.05 was considered statistically significant; p = 0.05–0.10 was considered a trend. Data were analyzed using the Prism software package (version 6.0, GraphPad) and STATA software, version 13.1 (StataCorp, College Station, TX, USA).

Results

Maternal data

Before mating, the median s-25(OH)D in the VDL group was lower compared to the controls, but no significant differences in s-total calcium or s-phosphorus were found between the two dietary groups, Table 1. However, the finding of lower s-25(OH)D values in the VDL group only reached significance in the E22 subgroup, S1 Table.

thumbnail
Download:
Table 1. Maternal data.

https://doi.org/10.1371/journal.pone.0155203.t001

At cesarean section, both s-25(OH)D levels and s-phosphorus levels had decreased in both dietary groups and s-total calcium levels increased, Table 1. At time of cesarean section, s-25(OH)D was significantly lower in the VDL animals compared to controls both at E19 and at E22, S1 Table. In the E19 control subgroup, the s-25(OH)D had a median of 25 nmol/L, e.g. an unintended moderate s-25(OH)D deficiency level, whereas the median in the E22 subgroup was 53 nmol/L, e.g. a normal s-25(OH)D level. S-total calcium and s-phosphorus became significantly lower in the VDL animals compared to controls at cesarean section in the E19 subgroup, but not at E22, S1 Table. Both s-total calcium and s-phosphorus were within the laboratory reference range for adult rodents at all times.

All mother rats achieved a weight gain during the experiment with no significant difference in gestational weight gain between the groups. At the cesarean section, no differences were observed in maternal SaO2, the duration of maternal anesthesia or in the number of pups in each litter.

Offspring data

Half of the animals from each dietary group had cesarean section at E19, n = 65 (VDL) vs. n = 56 (controls): the other half at E22, n = 75 (VDL) vs. n = 49 (controls). When comparing preterm and term offspring in general, a significant difference was observed in all measurements (lung, birth and placental weights and CRL) as the results of maturation, Table 2.

thumbnail
Download:
Table 2. Offspring data.

https://doi.org/10.1371/journal.pone.0155203.t002

When comparing the two dietary groups at E19 or E22, respectively, there was no difference between the weights of placenta, but a significant difference in PW/BW ratio was observed at E19, giving a higher ratio in the VDL pups compared to control pups due to lower body weight. At E19, birth weight, lung weight and lung weight/birth weight ratio (LW/BW ratio) were significantly lower in the pups born of VDL mothers than in the controls. While only insignificant difference in CRL was observed between the two dietary groups at E19, pups born at E22 of VDL mothers were significantly shorter than controls. Potential alterations in bone mineral content were not detectable using whole-body DXA scans (data not shown). All E22 weight measurements showed no difference between the dietary groups.

Pups born at E19 had significantly (p < 0.001) lower 30 min SaO2 values than pups born at E22 (Fig 1). At E19, the pups of VDL mothers further had significantly (p = 0.002) lower SaO2 values than controls, whereas no significant difference was observed between the dietary groups at E22.

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

SaO2 was measured 30 minutes after birth at E19 (VDL group (n = 13); control group (n = 23)) and E22 (VDL 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.

https://doi.org/10.1371/journal.pone.0155203.g001

At E19, the survival-rate was significantly (p < 0.0001) reduced in the VDL pups compared to controls and none of the pups survived more than 1.5 hours after birth (Fig 2). During the first 24 hours the majority of the E22 pups survived, leading to a 24h survival time of 74% in the VDL group and 84% in the control group. Further 3% of the pups in the VDL group died within day 2, after which all pups survived, leading to no significant difference in the long-term survival time between the groups born at E22 (Fig 2).

thumbnail
Download:
Fig 2. Survival-rate in hours after birth.

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

https://doi.org/10.1371/journal.pone.0155203.g002

Results from correlation analysis are presented in Table 3. In the E19 VDL pups, we found a significant positive correlation between SaO2 and birth weight and a trend towards a negative correlation between PW/BW ratio and SaO2, but no correlation between lung weight and SaO2. In E19 control pups, a significant negative correlation between LW/BW ratio and SaO2 was observed.

thumbnail
Download:
Table 3. Correlations between saturation and lung weight, birth weight, LW/BW ratio and PW/BW ratio.

https://doi.org/10.1371/journal.pone.0155203.t003

Pulmonary surfactant protein expression, phospholipids and VDR levels

The qPCR analysis showed a significantly lower surfactant protein A-D mRNA levels (Fig 3) in lung tissue obtained from pups born at E19 compared to E22. The concentration of pulmonary phospholipids was also significantly lower in the E19 pups compared to the E22 pups (Fig 4). No significant differences in surfactant protein mRNA expression or phospholipid concentrations were observed when comparing the two dietary groups at either E19 or E22.

thumbnail
Download:
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, VDL group (n = 15); control group (n = 12) and at E22, VDL group (n = 15); control group (n = 11). Data were normalized against GAPDH. Results were calculated as mean ± SEM values.

https://doi.org/10.1371/journal.pone.0155203.g003

thumbnail
Download:
Fig 4. Effects of vitamin D on phospholipid levels.

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

https://doi.org/10.1371/journal.pone.0155203.g004

Immunohistochemical analysis showed that VDR was expressed in the ATII cells during the last days of gestation in both dietary groups. The density of VDR-positive ATII cells in lung tissue in both E19 dietary groups was significantly (p < 0.001) higher compared to E22. At E19, a trend (p = 0.068, Fig 5) towards lower levels of VDR was observed in the VDL group compared to controls. The expression of VDR in both E19 groups was significantly higher compared to E22 whereas no difference in VDR expression was found between the dietary groups at E22.

thumbnail
Download:
Fig 5. Immunohistochemical analysis of VDR in fetal rat lung.

Immunohistochemistry was performed on lung tissues from each dietary group at both E19 (VDL group (n = 31); control group (n = 27)) and E22 (VDL 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).

https://doi.org/10.1371/journal.pone.0155203.g005

S-25(OH)D associations with pulmonary outcomes and survival

Due to a substantial overlap in maternal s-25(OH)D between the two dietary groups at cesarean section, post hoc analyses on the association between s-25(OH)D and outcomes was performed for all animals disregarding initial dietary group, Table 4.

thumbnail
Download:
Table 4. Uni- and multivariate linear regression models of the association between maternal s-25(OH)D at cesarean section and SaO2, PW, BW, LW, LW/BW ratio, PW/BW ratio and CRL.

https://doi.org/10.1371/journal.pone.0155203.t004

At E19, s-25(OH)D was positively associated with SaO2, birth weight, lung weight and LW/BW ratio, and negatively associated with PW/BW ratio.

At E22, maternal s-25(OH)D was positively associated with CRL, birth weight, placental weight and PW/BW ratio and negatively associated with LW/BW ratio.

Adjustment for maternal weight and littersize did not change the associations at E19. But at E22 only birth weight, placental weight and CRL remained positive associated with s-25(OH)

Moreover, a survival analysis comparing offspring of mothers with s-25(OH)D levels <25nmol/L at cesarean section to offspring of mothers with s-25(OH)D levels ≥25 nmol/L was performed (Fig 6). While the survival-rate was significantly (p = 0.0001) lower in the E19 pups of mothers with s-25(OH)D <25 nmol/L compared to s-25(OH)D ≥25 nmol/L, there was no significant difference between the pups of mothers with s-25(OH)D <25 nmol/L compared to s-25(OH)D ≥25 nmol/L at E22.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0155203.g006

Discussion

We found that premature rat offspring from mothers with severe vitamin D deficiency had reduced oxygenation and survival-rate compared to offspring of mothers with moderate vitamin D deficiency. Potential explanatory findings were significantly lower lung weight, birth weight, LW/BW ratio higher PW/BW ratio and lower s-total calcium in the premature VDL pups, whereas no reduction in the expression of surfactant protein A-D mRNA, pulmonary phospholipid concentration, or VDR expression, was found. Furthermore, birth weight and PW/BW ratio correlated significantly to SaO2 levels at 30 min in E19 VDL pups. The VDL-induced pulmonary outcomes were only detectable in the premature pups and not in fully matured pups, but were supported by direct multivariate associations between s-25(OH)D levels and lung weight and SaO2 and decreased survival with s-25(OH)D deficiency in the premature.

Our obtained results support that 25(OH)D is implicated in early fetal growth but are in contrast to the previously described positive impact of vitamin D on the synthesis and secretion of surfactant proteins and phospholipids [3]. We used an animal model of vitamin D deficiency during pregnancy, while most others either studied the in vitro synthesis of surfactant by incubating ATII cells with 1,25(OH)2D [29, 30, 33], or in vivo by vitamin D enrichment before sacrifice [26, 27]. Our model was designed to resemble the clinical setting of hypovitaminosis D in humans and we achieved a s-25(OH)D level of 12 nmol/L in the VDL mothers at cesarean section, which corresponds to severe vitamin D deficiency. In normal pregnant rats, levels of s-25(OH)D gradually decrease during the last period of gestation [42], as did in our control animals, which explains the substantial overlap in low s-25(OH)D between dietary groups. Our negative results on VDL-mediated surfactant protein A-D mRNA expression and pulmonary phospholipid levels may be attributed to failure of remaining s-25(OH)D concentrations >50 nmol/L in the E19 control group. Moreover, pups for lung studies were randomly chosen within the dietary groups, which had overlapping maternal s-25(OH)D concentration ranges.

In rats, the synthesis of surfactant evolves in the saccular phase only between the 19th gestational day and term (day 21–22) [43]. In accordance, our observed surfactant protein A-D mRNA expression and phospholipid levels at E19 were very low, which may have induced a detection error of a true difference. However, at E22, we still found no difference between VDL pups and control pups. The observed lack of impact of vitamin D on these outcomes does not rule out that at higher s-25(OH)D concentrations in the saccular phase can have effects as observed by others [30, 33].

We further described the immunohistochemical localization and relative expression of VDR in the preterm pup and term pup lung. Previous studies have shown that the widely used antibody 9A7γ [32–34] not only binds to VDR, but also possesses non-specific interactions with yet unidentified proteins [44, 45]. Based on a parallel comparison of a large selection of VDR antibodies [44], we chose to use the mouse monoclonal antibody D-6, which possesses the highest specificity, sensitivity and versatility. Our study brings further evidence that the VDR is present in the ATII cells during the last period of gestation, but with unaffected expression by s-25(OH)D in the studied concentration range. Furthermore, our study showed a significantly higher expression of VDR in the E19 pups compared to E22 confirming previous observations of reduced VDR expression in the last days before term in rat offspring [34, 36].

Despite these null-findings for the surfactant synthesis and VDR expression, premature pups with the lowest s-25(OH)D levels had decreased oxygenation and survival time. In humans, the postmortem diagnosis of pulmonary hypoplasia is based on a low LW/BW ratio [46], and we found a strong negative correlation between LW/BW ratio and oxygenation in the premature pups. The premature VDL pups had both significantly reduced lung weight and LW/BW ratio, supporting that pulmonary hypoplasia was likely to be responsible for the decreased survival. The significant positive correlation between birth weight and oxygenation may further indicate a reduced muscle mass leading to earlier muscular fatigue. Moreover, muscular weakness associated to the well-known vitamin D deficiency-associated myopathy in both humans and rodents [47] and lower levels of s-total calcium may have been a contributing factor. We were, however, not able to observe differential signs of muscular fatigue, or increased respiratory distress with increased respiratory muscular work between the pup groups. E19 pups hardly moved and showed only very little respiratory effort at all and E22 pups showed no visual signs of muscular fatigue or respiratory distress. Decreased mineralization and hence more soft ribs as a further explanatory factor was not supported by our whole-body DXA-scans, which showed unchanged bone mineral content between vitamin D depleted offspring and controls at both E19 and E22 (data not shown). This finding is in accordance with findings by Zosky et al. [25].

The significantly reduced BW in the premature VDL pups may indicate a more general affection of organ development in utero as a result of the severe vitamin D deficiency as previously shown [3, 13, 48, 49]. In humans, a high PW/BW ratio has been associated with RDS, low Apgar scores and increased risk of admission to the neonatal intensive care unit [21, 23]. We showed that the reduced birth weight was not secondary to a reduced placental weight, which was unchanged. Although there are conflicting results in both animal and human data assessing the role of vitamin D in somatic growth [25, 48, 50, 51], there are several biological mechanisms possibly connecting maternal vitamin D to fetal growth.

The vitamin D conversion enzyme gene, CYP27B1, and VDR are expressed in both placenta and lungs [50, 52]. In placenta, 1,25(OH)2D stimulates the secretion of placental hormones, which supports fetal growth and energy needs through delivery of calcium as well as glucose and fatty acids [17, 50]. In the lungs, 1,25(OH)2D stimulates pulmonary artery endothelial cell (PAEC) growth in the same manner as vascular endothelial growth factor (VEGF) [52]. Furthermore, 1,25(OH)2D regulates transforming growth factor β1 (TGF-β1), a known mediator of airway remodeling [53, 54].

The observed lack of differences in pulmonary and survival outcomes between dietary groups in mature pups may have several explanations: The VDR expression in the lungs was lower at E22 compared to E19 suggesting that pulmonary vitamin D effects are of minor importance at term compared to the preterm period. Moreover, term offspring do not suffer from respiratory insufficiency, why an effect on oxygenation and survival-rate should not be suspected in the mature lung.

We observed reduced CRL in the VDL pups compared to control pups at E22, but not at E19. Direct association analyses across dietary groups between maternal s-25(OH)D and CRL at E22, but not at E19, supported these findings. Vitamin D deficiency per se, as well as VDR mutations and VDR knock-out, has shown not to affect serum mineral concentrations, bone mineralization and fetal longitudinal bone growth [55, 56]. Moreover, animal studies in severely vitamin D deficient rats [57–60] and VDR-null mice [61, 62] showed a normal increase in intestinal calcium absorption during pregnancy and normal mineral content of the term offspring. Thus, a low calcium effect on bone growth could not readily explain the significant VDL dependent shortening of CRL at E22. However, CRL is a measure of skull and spine growth rather than longitudinal bone growth and bone mineralization and our association analyses showed not only increased CRL, but also BW and PW at E22 with higher s-25(OH)D, suggesting a general growth-promoting effect of vitamin D at term.

Strengths of the present study include the randomized, blinded design, the obtained severe deficiency level of s-25(OH)D in the VDL group, the between-group similarity in maternal weight gain, duration of anesthesia and maternal SaO2 during cesarean section. The difference in s-25-(OH)D between the dietary groups, was further designed to mimic differences in vitamin D status in the clinical setting rather than extreme vitamin D depletion versus pharmacological supplementation.

Limitations included the large with-in group variation resulting in overlapping s-25(OH)D concentrations between the dietary groups. Yet, s-25(OH)D was associated with pup anthropometrics, SaO2 and survival time in our post hoc analyses, which supported the findings between the dietary groups. The females were ten weeks old and may not have been housed under conditions similar to ours, in terms of lighting and diet, before purchase potentially causing variation in s-25(OH)D which have a long half-life of 2–3 weeks [63, 64]. Variations in appetite may also have contributed to the relatively high within-group variation. To reduce the within group variations and ensure a larger difference between the dietary groups, future studies should assign the diets to young females immediately after the weaning from their mothers. We did not measure pup s-25(OH)D, because of technical volume limitations. However, maternal s-25(OH)D correlates strongly to offspring s-25(OH)D concentrations [65]. Lastly, the fragility of the lung tissue, especially at E19, did not allow in situ fixation of the premature lungs under physiological pressure enabling comparison of lung morphology between groups.

The perspectives of our study include the emphasis of whole body animal models to study the effects of vitamin D on the lungs. Our results indicate that aggravation of respiratory failure may occur due to reduced lung and birth weight as the result of severe vitamin D depletion. However, we were unable to show a direct effect of vitamin D deficiency on surfactant measures or VDR expression. Future studies should pursue to evaluate our results using an optimized version of our model with animals assigned to the diets immediately after weaning.

In conclusion, vitamin D depletion during pregnancy led to a lower SaO2 and shorter survival-rate in premature rat offspring despite no reduction in lung surfactant constituents. Explanatory factors include reduced lung weight, which may imply a reduced total lung diffusion area, and decreased birth weight, which may indicate a reduced muscle mass leading to earlier muscular fatigue. In support of the hypothesis, vitamin D alleviates respiratory insufficiency at preterm birth. Studies of vitamin D effects in human preterm neonates regarding respiratory insufficiency are warranted.

Acknowledgments

The authors wish to thank animal technician Anne Mette Durand and medical student Kathrine Work Havelund for very helpful assistance.

Author Contributions

  1. Conceptualization: SL GLS SSBN HTC.
  2. Data curation: SL.
  3. Formal analysis: SL GLS SSBN HTC.
  4. Funding acquisition: SL HTC.
  5. Investigation: SL.
  6. Methodology: SL GLS SSBN HTC.
  7. Project administration: SL GLS HTC.
  8. Resources: SL GLS BP LD NM.
  9. Supervision: GLS SSBN HTC.
  10. Validation: SL NM LD BP.
  11. Visualization: SL HTC.
  12. Writing – original draft: SL.
  13. Writing – review & editing: SL GLS SSBN LD BP NM HTC.

References

  1. 1. Vannucchi C, Silva L, Lúcio C, Regazzi F, Veiga G, Angrimani D. Prenatal and Neonatal Adaptations with a Focus on the Respiratory System. Reproduction in Domestic Animals. 2012;47:177–81.
  2. 2. Hermansen CL, Lorah KN. Respiratory Distress in the Newborn. American Family Physician. 2007;76:987–94. pmid:17956068
  3. 3. Lykkedegn S, Sorensen GL, Beck-Nielsen SS, Christesen HT. The impact of vitamin D on fetal and neonatal lung maturation. A systematic review. American journal of physiology Lung cellular and molecular physiology. 2015;308:L587–L602. pmid:25595644
  4. 4. Ballard PL, Ballard RA. Scientific basis and therapeutic regimens for use of antenatal glucocorticoids. Am J Obstet Gynecol. 1995;173(1):254–62. pmid:7631700
  5. 5. Halliday HL. Surfactants: past, present and future. Journal of perinatology: official journal of the California Perinatal Association. 2008;28 Suppl 1:S47–56. Epub 2008/06/18. pmid:18446178.
  6. 6. Cools F, Offringa M, Askie LM. Elective high frequency oscillatory ventilation versus conventional ventilation for acute pulmonary dysfunction in preterm infants (Review). Cochrane database Syst Rev. 2015;(3).
  7. 7. Verder H, Bohlin K, Kamper J, Lindwall R, Josson B. Nasal CPAP and surfactant for treatment of respiratory distress syndrome and prevention of bronchopulmonary dysplasia. Acta paediatrica. 2009;98:1400–8. pmid:19572989
  8. 8. Andersen LB, Abrahamsen B, Dalgard C, Kyhl HB, Beck-Nielsen SS, Frost-Nielsen M, et al. Parity and tanned white skin as novel predictors of vitamin D status in early pregnancy: a population-based cohort study. Clinical endocrinology. 2013;79(3):333–41. Epub 2013/01/12. pmid:23305099.
  9. 9. Dawodu A, Nath R. High prevalence of moderately severe vitamin D deficiency in preterm infants. Pediatrics International. 2011;53:207–10. pmid:20667028
  10. 10. Kaushal M, Magon N. Vitamin D in pregnancy: A metabolic outlook. Indian journal of endocrinology and metabolism. 2013;17(1):76–82. Epub 2013/06/19. pmid:23776856; PubMed Central PMCID: PMC3659910.
  11. 11. Kazemi A, Sharifi F, Jafari N, Mousavinasab N. High Prevalence of Vitamin D Deficiency among Pregnant Women and their Newborns in an Iranian Population. Journal of Womens Health. 2009;18(6):835–9.
  12. 12. Vandevijvere S, Amsalkhir S, Van Oyen H, Moreno-Reyes R. High Prevalence of Vitamin D Deficiency in Pregnant Women: A National Cross-Sectional Survey. PloS one. 2012;7(8):e43868. Epub 2012/09/01. pmid:22937114; PubMed Central PMCID: PMC3427250.
  13. 13. Hart PH, Lucas RM, Walsh JP, Zosky GR, Whitehouse AJO, Zhu K, et al. Vitamin D in Fetal Development: Findings From a Birth Cohort Study. Pediatrics. 2015;135(1):e167–e73. pmid:25511121
  14. 14. Bodnar LM, Klebanoff MA, Gernand AD, Platt RW, Parks WT, Catov JM, et al. Maternal Vitamin D Status and Spontaneous Preterm Birth by Placental Histology in the US Collaborative Perinatal Project. American Journal of Epidemiology. 2013:1–9.
  15. 15. Evans KN, Bulmer JN, Kilby MD, Hewison M. Vitamin D and Placental-Decidual Function. Journal of the Society for Gynecologic Investigation. 2004;11:263–71. 10.1016/j~jsgi.2004.02.002. pmid:15219879
  16. 16. Evans KN, Nguyen L, Chan J, Innes BA, Bulmer JN, Kilby MD, et al. Effects of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 on Cytokine Production by Human Decidual Cells. Biology of reproduction. 2006;75(6):816–22. Epub 2006/09/08. pmid:16957024.
  17. 17. Gernand AD, Simhan HN, Klebnoff MA, Bodnar LM. Maternal Serum 25-Hydroxyvitamin D and Measures of Newborn and Placenta Weight in a U.S. Multicenter Cohort Study. J Clin Endocrinol Metab. 2013;98(1):398–404. pmid:23162094
  18. 18. Javaid MK, Crozier SR, Harvey NC, Gale CR, Dennison EM, Boucher BJ, et al. Maternal vitamin D status during pregnancy and childhood bone mass at age 9 years: a longitudinal study. Lancet. 2006;367(9504):36–43. Epub 2006/01/10. pmid:16399151.
  19. 19. Godfrey K. The Role of the Placenta in Fetal Programming—A Review. Placenta. 2002;23 (suppl A):s20–7. pmid:11978056
  20. 20. Salafia C, Charles A, Maas E. Placenta and Fetal Growth Restriction. Clinical obstetrics and gynecology 2006;49(2):236–56. pmid:16721104
  21. 21. Shehata F, Levin I, Shrim A, Ata B, Weisz B, Gamzu R, et al. Placenta/birthweight ratio and perinatal outcome: a retrospective cohort analysis. BJOG. 2011;118:741–7. pmid:21332633
  22. 22. Myatt L. Placental adaptive responses and fetal programming. J Physiol. 2006;572.1:25–30.
  23. 23. Madkar C, Musale J, Deshpande H, Shitole R. A Study of placental weight and birth weight ratio (PW/BW) and it's effects on perinatal outcome. Indian Journal of Obstetrics and Gynaecology. 2015;2(1):1–6.
  24. 24. Onwuneme C, Martin F, McCarthy R, Carroll A, Segurado R, Murphy J, et al. The Association of Vitamin D Status with Acute Respiratory Morbidity in Preterm Infants. The Journal of Pediatrics. 2015;166(5):1175–80.e1. pmid:25919726
  25. 25. Zosky GR, Berry LJ, Elliot JG, James AL, Gorman S, Hart PH. Vitamin D Deficiency Causes Deficits in Lung Function and Alters Lung Structure. American journal of respiratory and critical care medicine. 2011;183:1336–43. pmid:21297070
  26. 26. Mandell E, Seedorf G, Gien J, Abman SH. Vitamin D Treatment Improves Survival and Infant Lung Structure After Intra-Amniotic Endotoxin Exposure in Rats: Potential Role for the Prevention of Bronchopulmonary. American journal of physiology Lung cellular and molecular physiology. 2014;306(5):L420–8. Epub 2014/01/15. pmid:24414254; PubMed Central PMCID: PMC3949057.
  27. 27. Yurt M, Liu J, Sakurai R, Gong M, Husain SM, Siddiqui MA, et al. Vitamin D Supplementation Blocks Pulmonary Structural and Functional Changes in a Rat Model of Perinatal Vitamin D deficiency. Am j Physiol lung Cell Mol Physiol 2014;307:L859–L67. pmid:25305247
  28. 28. Edelson JD, Chan S, Jassal D, Post M, Tanswell KA. Vitamin D stimulates DNA synthesis in alveolar type-II cells. Biochimica et Biophysica Acta. 1993;1221:159–66.
  29. 29. Marin L, Dufour ME, Nguyen TM, Tordet C, Garabedian M. Maturational changes induced by 1 alpha,25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants. American Journal of Physiology. 1993;265:L45–L52. pmid:8338181
  30. 30. Marin L, Dufour ME, Tordet C, Nguyen TM. 1,25(OH)2D3 stimulates Phospholipid Biosynthesis and Surfactant Release in Fetal Rat Lung Explants. Biol Neonate. 1990;57:257–60. pmid:2322608
  31. 31. Nguyen M, Guillozo H, Garabedian M, Balsan S. Lung as a Possible Additional Target Organ for Vitamin D during Fetal Life in the Rat. Biol Neonate. 1987;52:232–40. pmid:2823916
  32. 32. Nguyen TM, Guillozo H, Marin L, Dufour ME, Tordet C, Pike JW, et al. 1,25-Dihydroxyvitamin D3 Receptors in Rat Lung during the Perinatal Period: Regulation and Immunohistochemical Localization. Endocrinology. 1990;127:1755–62. pmid:2169401
  33. 33. Nguyen TM, Guillozo H, Marin L, Tordet C, Koite S, Garabedian M. Evidence for a vitamin D paracrine system regulating maturation of developing rat lung epithelium. American Journal of Physiology-Lung Cellular and Molecular Physiology. 1996;15:L392–L9.
  34. 34. Nguyen M, Trubert CL, Rizk-Rabin M, Rehan VK, Besancon F, Cayre YE, et al. 1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase. The Journal of steroid biochemistry and molecular biology. 2004;89-90(1–5):93–7. Epub 2004/07/01. pmid:15225753.
  35. 35. Sakurai R, Shin E, Fonseca S, Sakurai T, Litonjua AA, Weiss ST, et al. 1α,25(OH)2D3 and its 3-epimer promote rat lung alveolar epithelial-mesenchymal interactions and inhibit lipofibroblast apoptosis. American journal of physiology Lung cellular and molecular physiology. 2009;297(3):L496–505. Epub 2009/07/04. pmid:19574420; PubMed Central PMCID: PMC2739775.
  36. 36. Phokela SS, Peleg S, Moya FR, Alcorn JL. Regulation of human pulmonary surfactant protein gene expression by 1 α,25-dihydroxyvitamin D3. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2005;289:617–26.
  37. 37. Rehan VK, Torday JS, Peleg S, Gennaro L, Vouros P, Padbury J, et al. 1a,25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1a,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells. Molecular Genetics and Metabolism. 2002;76:46–56. pmid:12175780
  38. 38. Vogeser M, Parhofer KG. Liquid Chromatography Tandem-mass Spectrometry (LC-MS/MS)–Technique and Applications in Endocrinology. Exp Clin Endocrinol Diabetes. 2007;115:559–70. pmid:17943689
  39. 39. Abbott SK, Jenner AM, Mitchell TW, Brown SH, Halliday GM. An Improved High-Throughput Lipid Extraction Method for the Analysis of Human Brain Lipids. Lipids. 2013;48(3):307–18. pmid:23355308
  40. 40. Bartlett GR. Phosphorus Assay in Column Chromatography. J Biol chem. 1959;234:466–8. pmid:13641241
  41. 41. Ochs M, Mühlfeld C. Quantitative microscopy of the lung: a problem-based approach. Part 1: basic principles of lung stereology American journal of physiology Lung cellular and molecular physiology 2013;305:L 15–22.
  42. 42. Clements MR, Fraser DR. Vitamin D Supply to the Rat Fetus and Neonate. J Clin Invest. 1988;81:1768–73. pmid:2838521
  43. 43. Samtani MN, Pyszczynski NA, Dubois DC, Almon RR, Jusko WJ. Modeling Glucocorticoid-Mediated Fetal Lung Maturation: I. Temporal Patterns of Corticosteroids in Rat Pregnancy. The Journal of pharmacology and experimental therapeutics. 2005;317(1):117–26. Epub 2005/12/24. pmid:16371449.
  44. 44. Wang Y, Becklund BR, DeLuca HF. Identification of a highly specific and versatile vitamin D receptor antibody. Arch Biochem Biophys. 2010;494(2):166–77. Epub 2009/12/03. pmid:19951695.
  45. 45. Wang Y, Zhu J, DeLuca HF. Where is the vitamin D receptor? Arch Biochem Biophys. 2012;523(1):123–33. Epub 2012/04/17. pmid:22503810.
  46. 46. De Paepe M, Friedman RM, Gundogan F, Pinar H. Postmortem Lung Weight/Body Weight Standards for Term and Preterm Infants. Pediatric Pulmononology. 2005;45:445–8.
  47. 47. Polly P, Tan TC. The role of vitamin D in skeletal and cardiac muscle function. Frontiers in Physiology. 2014;5:1–6.
  48. 48. O'Loan J, Eyles DW, Kesby J, Ko P, McGrath JJ, Burne THJ. Vitamin D deficiency during various stages of pregnancy in the rat; its impact on development and behaviour in adult offspring. Psychoneuroendocrinology. 2007;32:277–34.
  49. 49. Eyles DW, Burne THJ, McGrath JJ. Vitamin D, effects on brain development, adult brain function and the links between low levels of vitamin D and neuropsychiatric disease. Frontiers in Neuroendocrinology. 2013;34:47–64. pmid:22796576
  50. 50. Lapillonne A. Vitamin D deficiency during pregnancy may impair maternal and fetal outcomes. Medical Hypotheses. 2010;74:71–5. pmid:19692182
  51. 51. Galthen-Sørensen M, Andersen L, Sperling L, Christesen H. Maternal 25-hydroxyvitamin D level and fetal bone growth assessed by ultrasound: a systematic review. Ultrasound Obstet Gynesol. 2014;44:633–40.
  52. 52. Mandell E, Seedorf GJ, Ryan S, Gien J, Cramer SD, Abman SH. Antenatal Endotoxin Disrupts Lung Vitamin D Receptor and 25-hydroxyvitamin D 1-alpha Hydroxylase in the Developing Rat. American journal of physiology Lung cellular and molecular physiology. 2015.
  53. 53. Chen G, Khalil N. TGF-beta1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases. Respiratory Research. 2006;7(2):1–10.
  54. 54. Foong RE, Shaw NC, Berry LJ, Hart PH, Gorman S, Zosky GR. Vitamin D deficiency causes airway hyperresponsiveness, increases airway smooth muscle mass, and reduces TGF-B expression in the lungs of female BALB/c mice. Physiological Reports. 2014;2(3):1–12.
  55. 55. Kovacs C. The Role of Vitamin D in Pregnancy and Lactation: Insights from Animal Models and Clinical Studies.. The annual review of nutrition. 2012;(32):9.1–9.27.
  56. 56. Kovacs CS. Bone Development and Mineral Homeostasis in the Fetus and Neonate: Roles of the Calciotropic and Phosphotropic Hormones. Physiol Rev. 2014;94:1143–218. pmid:25287862
  57. 57. Halloran BP, De Luca HF. Effect of vitamin D deficiency on skeletal development during early growth in the rat. Arch Biochem Biophys. 1981;209(1):7–14. Epub 1981/06/01. pmid:7283447.
  58. 58. Brommage R, DeLuca HF. Placental transport of calcium and phosphorus is not regulated by vitamin D. The American journal of physiology. 1984;246(4 Pt 2):F526–9. Epub 1984/04/01. pmid:6720905.
  59. 59. Miller SC, Halloran BP, DeLuca HF, Jee WS. Studies on the role of vitamin D in early skeletal development, mineralization, and growth in rats. Calcified tissue international. 1983;35(4–5):455–60. Epub 1983/07/01. pmid:6616315.
  60. 60. Glazier JD, Mawer EB, Sibley CP. Calbindin-D9K gene expression in rat chorioallantoic placenta is not regulated by 1,25-dihydroxyvitamin D3. Pediatr Res. 1995;37(6):720–5. Epub 1995/06/01. pmid:7651755.
  61. 61. Lieben L, Stockmans I, Moermans K, Carmeliet G. Maternal hypervitaminosis D reduces fetal bone mass and mineral acquisition and leads to neonatal lethality. Bone. 2013;57(1):123–31. Epub 2013/07/31. pmid:23895994.
  62. 62. Kovacs CS, Woodland ML, Fudge NJ, Friel JK. The vitamin D receptor is not required for fetal mineral homeostasis or for the regulation of placental calcium transfer in mice. American journal of physiology Endocrinology and metabolism. 2005;289(1):E133–44. Epub 2005/03/03. pmid:15741244.
  63. 63. Kovacs C. Maternal vitmain D deficiency: Fetal and neonatal implications. Seminars in Fetal and Neonatal Medicine. 2013;18:129–35.
  64. 64. Zerwekh JE. Blood biomarkers of vitamin D status. Am j Clin Nutr. 2008;87 (suppl):1087S–91S.
  65. 65. Kovacs C. Maternal Mineral and Bone Metabolism During Pregnancy, Lactation, and Post-Weaning Recovery. Physiol Rev. 2016;96:449–547. pmid:26887676