The metabolism of vitamin D and its effect on the epigenetic regulation of gene expression within the human placenta

Abstract

Poor placental function impairs fetal growth and development. Suboptimal fetal growth is associated with an increased risk of disease in later life. Women of childbearing age have a high prevalence of 25-hydroxyvitamin D3 (25(OH)D3) deficiency, which is associated with reduced fetal growth and may impair placental delivery of vitamin D to the fetus. In this thesis I investigate which metabolites of vitamin D are passed from the mother to the fetus via the placenta, and how vitamin D directly affects placental tissue.

Placental 25(OH)D3 transfer and metabolism was investigated in ex vivo perfused placental cotyledons. Following perfusion of 13C-25(OH)D3 in the maternal circulation, vitamin D metabolites were measured in the maternal and fetal perfusate using liquid chromatography mass spectroscopy and enzyme-linked immunosorbent assay. Placental metabolism of 13C25(OH)D3 was identified, and the metabolites 13C-25(OH)D3, 13C-24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were transferred from the placenta to the fetal circulation. This study suggests that the placenta produces 1,25(OH)2D3, and releases it to both the mother and the fetus.

The effects of short term 25(OH)D3 treatment on the placenta were determined in villous fragments and cultured syncytiotrophoblast cells. 25(OH)D3 altered DNA methylation in villous fragments when measured by Illumina 850K DNA methylation array, demonstrating alterations to placental epigenetics with 25(OH)D3. In addition 25(OH)D3 treatment of villous fragments resulted in altered RNA and protein expression, measured by RNA sequencing and liquid chromatography mass spectroscopy. Altered RNA expression was also observed in 25(OH)D3 treated cultured syncytiotrophoblast, measured by quantitative real time polymerase chain reaction. Altered DNA methylation, RNA and protein expression mapped to biological pathways of transcriptional regulation, histone protein activity, immune function and inorganic ion transport. Components of the altered biological pathways may alter placental function in ways that could impact upon fetal growth.

Long term vitamin D (cholecalciferol) treatment was investigated in placentas from the Maternal Vitamin D and Osteoporosis Study (MAVIDOS) and the Southampton Pregnancy Intervention for the Next Generation (SPRING) studies. Pyrosequencing identified increased DNA methylation at one CpG site of the RXRα gene in MAVIDOS with 1000 IU cholecalciferol daily treatment, but no other changes in DNA methylation with treatment in either MAVIDOS or SPRING.

Data presented in this thesis suggest that the metabolites 25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3 are transferred across the placenta to the fetal circulation, were they could act directly on the fetus. 25(OH)D3 is metabolised within the placenta, and this metabolism potentially alters levels of vitamin D metabolites in both the fetal and maternal circulation. 25(OH)D3 may also indirectly impact fetal growth and development via its actions on the placenta, which alter placental functions important for fetal growth. The data presented here helps explain the relationships underlying the observed association between maternal 25(OH)D3 concentration and fetal growth.

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Identifiers

ORCID for Jane Cleal: orcid.org/0000-0001-7978-4327

ORCID for Rohan Lewis: orcid.org/0000-0003-4044-9104

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