title: Vitamin D promotes human extravillous trophoblast invasion in vitro.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centre 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.
authors.forename: S Y
authors.initials: SY
authors.lastname: Chan
authors.affiliation: Centre 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.
authors.forename: R
authors.initials: R
authors.lastname: Susarla
authors.affiliation: Centre 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.
authors.forename: D
authors.initials: D
authors.lastname: Canovas
authors.affiliation: Centre 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.
authors.forename: E
authors.initials: E
authors.lastname: Vasilopoulou
authors.affiliation: Women, Children and Sexual Health Directorate, Walsall Hospitals NHS Trust, Walsall, WS2 9PS, UK.
authors.forename: O
authors.initials: O
authors.lastname: Ohizua
authors.affiliation: Centre for Endocrinology, Diabetes and Metabolism, and the School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: McCabe
authors.affiliation: Centre for Endocrinology, Diabetes and Metabolism, and the School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: M
authors.initials: M
authors.lastname: Hewison
authors.affiliation: Centre 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; Fetal Medicine Centre, Birmingham Women's NHS Foundation Trust, Edgbaston, Birmingham, B15 2TG, UK. Electronic address: m.d.kilby@bham.ac.uk.
authors.forename: M D
authors.initials: MD
authors.lastname: Kilby
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000378:metabolism*; D054804:Cathelicidins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010929:Placentation; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism*; D015854:Up-Regulation; D065668:Vitamin D3 24-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D054804:Cathelicidins; D018167:Receptors, Calcitriol; C579457:VDR protein, human; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; C000600378:CYP27B1 protein, human; D053505:Matrix Metalloproteinases, Secreted; D002117:Calcitriol; D002112:Calcifediol
keywords: Cell invasion; Extravillous trophoblast; Placenta; Pre-eclampsia; Vitamin D