Evaluation of Maturation Status of Oocytes
Three days after IVF, all noncleaved oocytes were
separated from embryos, and sperm was removed by
repeated pipetting with a micropipette of 150-mdiameter.
Naked oocytes were then fixed in methanol, followed
by staining with 4′,6-diamidino-2-phenylindole
dihydrochloride (Molecular Probes, Eugene, OR) and
evaluation of the maturation status under epifluorescent
microscopy. Oocytes in the germinal vesicle stage,
containing diplotene chromatin, were considered to be
immature. Oocytes in metaphase II demonstrated extrusion
of the first polar body and were considered to
be mature (Luther et al., 2005; Pant et al., 2005).