Vitamin D promotes human extravillous trophoblast invasion in vitro

Introduction: Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. Methods: This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D-3 (1,25-D-3), and its precursor, 25-hydroxyvitamin D-3 (25-D-3), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D-3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D-3 (0-10 nM) or 25-D-3 (0-100 nM) for 48-60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MIT. Results: EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25D(3) treatment. EVT could respond to 1,25-D3 and 25-D-3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D-3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p <0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and proMMP9 secretion. The invasion was independent of cell viability, which remained unchanged. Discussion: These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia. (C) 2015 Elsevier Ltd. All rights reserved.