Human Reproduction, Vol. 14, No. 6, 1569-1577,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes
1 Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, 2 Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, 51 Blossom Street, Boston, MA 02114 and 3 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, USA
The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-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°C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2°C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen–thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by ~23°C in mouse and only by ~6.5°C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5°C, the incidence 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. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6°C and -8°C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5°C.
Key words: cryobiology/freezing/human/intracellular ice formation/oocyte
4 Present address: Trad Hospital, 53 Mexico Street, Beirut, Lebanon
5 To whom correspondence should be addressed
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