ANNEXIN-V LABELING AND TERMINAL TRANSFERASE-MEDIATED DNA END LABELING(TUNEL) ASSAY IN HUMAN ARRESTED EMBRYOS

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilizat ion, Observation of the cellular actin cortex and chromatin showed a h igh frequency of embryos with blastomeres exhibiting two or more nucle i, while others had nuclei displaying chromatin condensation and fragm entation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigat ed using two detection methods for cells undergoing apoptosis. Detecti on of phosphatidylserine exposure was performed using annexin V;the ch romosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which conti nued to develop normally after thawing. The TUNEL assay was positive i n 30% (15/50) of arrested embryos, all of which had cytoplasmic fragme nts. In contrast, embryos showing regular size blastomeres without fra gments were TUNEL negative.