To implant in the uterus, mammalian embryos form blastocysts comprising trophectodermsurrounding an inner cell mass, confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered trophectoderm until they implant in the characteristic thick deciduum, whereas humanblastocysts attach via polar trophectoderm directly to the uterine wall. Usingimmunofluorescence of rapidly isolated inner cell masses, blockade of RNA and protein synthesis in whole embryos, or 3D visualisation of immunostained embryos we provide evidence of multi-layering in human polar trophectoderm before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiateformation of the placenta. Using sequential fluorescent labelling, we demonstrate that the majority of inner trophectoderm in human blastocysts arises from existing outer cells with no evidence of conversion from the inner cell mass in the context of the intact embry
