title: PP065. dNK and dNK-CM mediated alterations of DNA methylation in extravillous cytotrophoblasts (EVTS).
abstract: BACKGROUND
  Placental DNA methylation is thought to be influenced by environmental exposure. Decidual natural killer cells (dNKs) directly contact with cytotrophoblasts in the early stage of pregnancy. dNKs may affect DNA methylation of extravillous cytotrophoblasts (EVTs) directly or indirectly through their secreted soluble factors.
  
  
  OBJECTIVE
  Previously, we showed that EVT outgrowth and migration on collagen gel were restricted by exposure to dNK or dNK-derived conditioned medium (dNK-CM) (ref [1]). The aim of this study was to determine if EVT DNA methylation was altered by treating with dNK or dNK-CM.
  
  
  METHODS
  Placental explants collected from 6 first-trimester healthy pregnancy terminations were cultured on a rat collagen gel model. Outgrowth EVTs from each subject were treated with medium or concordant dNK (trapped inside of hollow fibers) or dNK-CM. EVTs were harvested after 96-hour co-culturing and underwent DNA extraction. DNA methylation was quantified using the Infinium Human Methylation 450 BeadChip, which targets over 450,000 CpG sites in the human genome. Differential methylation was defined by having p<0.05 (Student's t-test) and average DNA methylation change >10% before and after treatments. Functional enrichment was assessed by gene ontology analysis with False Discovery Rate <10% defined as significantly enriched.
  
  
  RESULTS
  Increased DNA methylation was observed for 360 loci and 572 loci by dNK or dNK-CM respectively, and decreased DNA methylation was shown for 62 loci and 188 loci by dNK or dNK-CM respectively. DNA methylation at 44 loci was altered by both dNK and dNK-CM. The common loci were overrepresented for associations with EVT differentiation, adhesion and migration. Examples of the relevant overlapped loci with increased DNA methylations were MYO15A and PRDM16 (PR domain zinc finger protein 16); and the overlapped loci with reduced DNA methylation were CDH9 and USP29 (ubiquitin specific protein 29). dNK but not dNK-CM reduced IL18 methylation and increased methylation on ITGAL (integrin, alpha L) and ITGB7. dNK-CM but not dNK reduced methylation of ITGAD and PCDH8 (protocadherin 8) and increased methylation of CDH4 and CDH6.
  
  
  CONCLUSIONS
  DNA methylation of EVT was altered by exposure to surrounding dNK and their secreted soluble molecules. These results serve as a basis for further investigations on whether DNA methylation can mediate the changes in protein expression that influence EVT differentiation, adhesion and migration.
authors.affiliation: Obstetrics & Gynaecology, Child and Family Research Institute, Vancouver, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Medical Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Yuen
authors.affiliation: Obstetrics & Gynaecology, Child and Family Research Institute, Vancouver, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Eastabrook
authors.affiliation: Dermatology and Skin Science, Vancouver, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Dutz
authors.affiliation: Pathology, , Vancouver, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Tan
authors.affiliation: Obstetrics & Gynaecology, Child and Family Research Institute, Vancouver, Canada.
authors.forename: P
authors.initials: P
authors.lastname: von Dadelszen
authors.affiliation: Medical Genetics, Child and Family Research Institute, Vancouver, Canada.
authors.forename: W
authors.initials: W
authors.lastname: Robinson