Experimental design

After vitrification-warming, oocytes were incubated in the same medium used for IVM, and a cohort was studied at three different time points to analyze their metabolic status during recuperation: 0 hours (to evaluate oocyte behavior immediately after warming), 3 hours (to determine oocyte metabolic recovery), and 21 hours (to confirm that vitrified-warmed oocytes may survive during the period required for pronuclear formation after fertilization). Fresh matured nonfertilized oocytes were analyzed at the same time points to serve as controls, starting at 44 hours of IVM as 0 hours.

To evaluate oocyte metaphase II recovery, in vitro fertilization (IVF), and intracytoplasmic sperm injection, the control and vitrified-warmed oocytes used came from a different batch of ovaries to synchronize the warming time with the completion of maturation. For the morphological, viability, and biochemical evaluations, control and vitrified-warmed oocytes came from the same batch of ovaries but were processed at different moments because of the extra time needed for the vitrification-warming process.

For both functional and metabolic determinations, three replicates were performed and a total number of 1,040 oocytes were used.