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Updated: Apr 18, 2026
In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines
Published on: March 5, 2019
Vitamin D promotes human extravillous trophoblast invasion in vitro
S Y Chan1, R Susarla1, D Canovas1
1Centre for Women's & Children's Health and the School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
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 D3 (1,25-D3), and its precursor, 25-hydroxyvitamin D3 (25-D3), 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-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0-10 nM) or 25-D3 (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 MTT.
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 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (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 pro-MMP9 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.
