Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro cultureenvironment differs from in-vivo conditions in that the oxygenconcentration is higher, and in such conditions the mouse embryos show ahigher concentration of reactive oxygen species (ROS) in simple culturemedia. ROS are believed to cause damage to cell membranes and DNAfragmentation in somatic cells. This study was conducted to ascertain thelevel of H2O2 concentration within embryos and the morphological featuresof cell damage induced by H2O2. A total of 62 human oocytes and embryos (31fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) wasobtained from the IVF-embryo transfer programme. The relative intensity ofH2O2 concentrations within embryos was measured using2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescenceimaging and DNA fragmentation was observed with transmission electronmicroscopy and an in-situ apoptosis detection kit. The H2O2 concentrationswere significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/-SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) andunfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observedonly in fragmented embryos, and was absent in non-fragmented embryos.Electron microscopic findings confirmed apoptotic bodies and cytoplasmiccondensation in the fragmented blastomeres. We conclude that there is adirect relationship between increased H2O2 concentration and apoptosis, andthat further studies should be undertaken to confirm these findings.