Morphological and phenotypic analyses of the human placenta using whole mount immunofluorescence

The human placenta performs multiple essential functions required for successful pregnancy. Alterations in the placental vasculature have been implicated in severe complications of pregnancy. Despite the importance of placental vascular function during pregnancy, there are gaps in our knowledge regarding the molecular pathways that control vessel development. Furthermore, there are limited tools available to simultaneously examine the morphology, phenotype, and spatial arrangement of cells within intact placental structures. To overcome these limitations, we developed whole mount immunofluorescence (WMIF) of the human placenta. Morphological analyses using WMIF revealed that blood vessel structures were consistent with an immature, angiogenic morphology in first-trimester placentas and mature, remodeled endothelium at term. To investigate placental expression of factors that control blood vessel development, we utilized WMIF to examine gestation age-specific expression of 1) the receptors for vascular endothelial growth factor (VEGFR-1, VEGFR-2, and VEGFR-3), which are required for placental vascular development in mice, and 2) activated, tyrosine phosphorylated STAT3 (pSTAT3), a transcription factor that mediates VEGFR2 signaling. We detected high levels of VEGFR2, VEGFR3, and pSTAT3 expression in early placental blood vessels that were significantly diminished by term. VEGFR1 was expressed primarily in trophoblast and Hofbauer cells throughout gestation. Based on our collective results, we propose that VEGFR2, VEGFR3, and STAT3 play essential roles in the development of the human placental vasculature. In addition, we anticipate that WMIF will provide a powerful approach for comparing placental morphology and protein expression in normal versus pathological pregnancies and for investigating the effects of environmental factors on placental function.