Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes

The accurate determination of the freezing conditions that promote intracel lular ice formation (IIF) is crucial for designing cryopreservation protoco ls far cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, fai led-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope s tage connected to a videomicroscope. Then, with a cooling rate of 0.2 degre es C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thaw ed human oocytes. After adding different cryoprotectants, the median temper ature of IIF (T-MED) was decreased by similar to 23 degrees C in mouse and only by similar to 6.5 degrees C in human oocytes. Using 1.5 M propylene gl ycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the inciden ce of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31 (32 %), 19/34 (56%) and 52/56 (93%) respectively. T he results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large numb er of human oocytes. Undesirable IIF can be prevented and survival rates ma ximized by raising the seeding temperature as close as possible to the melt ing point of the solution, which in our instrument was -4.5 degrees C.