Epithelial-mesenchymal transition in the differentiation of cyto- to extravillous trophoblast

The transformation of cytotrophoblast (CTB) to extravillous trophoblast (EVT) is an essential process for placental implantation. EVT generated at the tips of the anchoring villi migrate away from the placenta and invade the endometrium and maternal spiral arteries, where they modulate maternal immune responses and remodel the arteries into high-volume conduits to facilitate uteroplacental blood flow. The process of EVT differentiation has several factors in common with the epithelial-to-mesenchymal transition (EMT) observed in embryonic development, wound healing and cancer metastasis. We hypothesized that the generation of invasive EVT from CTB was a form of EMT. We isolated paired CTB and EVT from first trimester placentae by flow cytometry for integrin beta 4 (CTB) and HLA-G (EVT). We compared their gene expression using a PCR array comprising probes for genes involved in EMT. Out of 84 genes, 24 were down-regulated in EVT compared to CTB, including epithelial markers such as E-cadherin (-11-fold) and occludin (-75-fold). Another 30 genes were up-regulated in EVT compared to CTB including mesenchymal markers such as vimentin (235-fold) and fibronectin (107-fold) as well as the matrix metalloproteinases, MMP2 and MMP9 (357-fold, 129-fold). These alterations also included major increases in the ZEB2 (zinc finger E-box binding homeobox 2, 198-fold) and TCF4 (transcription factor 4, 18-fold) transcription factors, recognized as EMT master regulators, suggesting possible stimulatory mechanisms. There was substantial up-regulation of the genes encoding TGFb1 and TGFb2 (48-fold, 115-fold), which may contribute to the maintenance of the mesenchymal-like phenotype. We conclude that transformation of CTB to EVT is consistent with an EMT, although the differences with other types of EMT suggest this may be a unique form Paired sets of CTB and EVT obtained from single placenta by fluorescence activated cell sorting (integrin beta4, CTB; HLA-G, EVT). RNA extracted from paired sets obtained from 6 separate placentae ranging from 6-10 weeks gestational age and reverse-transcribed, pre-amplified and analyzed on a PCR Array containing probes for 84 genes associated with the EMT (SABiociences, RT2 Profiler PCR Array Cat# PAHS-090Z). Relative gene expression determined for EVT compared to CTB.