The first trimester placental stroma becomes populated by extravascular RBCs that enucleate. (A) Localization and enucleation status of erythroid cells in the placenta was determined by H&E staining. At 3 weeks of developmental age (i), placental blood vessels had started to form and were devoid of RBCs (white arrow). By 4 weeks (ii), nucleated RBCs had appeared in the placental vasculature (arrowhead). Between 5-7 weeks of developmental age, RBCs were also found in the villous stroma (arrows) with increasing frequency (iii-iv), and many were enucleated, whereas most circulating RBCs still retained their nuclei (arrowheads). Toward the end of first trimester, RBCs were found mainly in circulation (arrowheads in v-vi). All images shown were acquired at 400× original magnification. (B) Calculation of ERs (number of enucleated RBCs divided by the total number of RBCs counted, see “Histologic analysis of erythroblast maturation”) for the placental RBC pool for each week of development revealed a marked change in their maturation state between 4 and 8 weeks. Bars represent median for each age. (C) Analysis of enucleation ratios of RBCs in circulation (inside blood vessels) or extravascular (in placental stroma) revealed an enrichment of enucleated RBCs in the extravascular stroma during 5-7 weeks of development. Dotted line at 1 indicates equal representation of enucleated cells in both compartments, and bars represent the median at each age.