Apoptosis in the early human placental bed and its discrimination from necrosis using the in-situ DNA ligation technique

Extensive areas of necrosis are present in the early human placental bed.Our aim was to determine whether apoptosis is also a feature. A method wastherefore required to differentiate unequivocally necrosis and apoptosis.Initially, terminal deoxynucleotide transferase-mediated dUTP nick-endlabelling was used to visualize apoptotic cells. However, non-specificlabelling, probably due to free DNA released by necrotic cells, wasexcessive; thus, in-situ DNA ligation was employed. In this technique, twoDNA fragments with single-base 3' overhangs and blunt- ends were labelledwith a fluorochrome and then ligated to the DNA breaks on the sections.Immunolabelling for cytokeratin or leukocyte common antigen was performedto determine the phenotype of apoptotic cells identified by the in-situ DNAligation technique. A proportion of the dying cells was confirmed to betrophoblasts. No co-localization with leukocyte common antigen was found inthis region, suggesting that maternal macrophages and natural killer cells(CD56+) were not dying by apoptosis in significant numbers. In conclusion,in-situ DNA ligation in association with immunocytochemistry can readilydistinguish apoptosis from necrosis in the placental bed. The resultssuggest that a proportion of invading trophoblast cells are eliminated byapoptosis in early pregnancy.