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. O ur aim was to determine whether apoptosis is also a feature. A method was t herefore required to differentiate unequivocally necrosis and apoptosis, In itially, terminal deoxynucleotide transferase-mediated dUTP nick-end labell ing was used to visualize apoptotic cells. However, non-specific labelling, probably due to free DNA released by necrotic cells, was excessive; thus, in-situ DNA ligation was employed. In this technique, two DNA fragments wit h single-base 3' overhangs and blunt-ends were labelled with a fluorochrome and then ligated to the DNA breaks on the sections. Immunolabelling for cy tokeratin or leukocyte common antigen was performed to determine the phenot ype of apoptotic cells identified by the in-situ DMA ligation technique. A proportion of the dying cells was confirmed to, be trophoblasts. No co-loca lization with leukocyte common antigen was found in this region, suggesting that maternal macrophages and natural killer cells (CD56(+)) were not dyin g by apoptosis in significant numbers. In conclusion, insitu DNA ligation i n association with immunocytochemistry can readily distinguish apoptosis fr om necrosis in the placental bed. The results suggest that a proportion of invading trophoblast cells are eliminated by apoptosis in early pregnancy.