In-vitro growth of murine pre-antral follicles after isolation from cryopreserved ovarian tissue.

The low temperature storage of ovarian tissue allows patients at risk of premature menopause to preserve their fecundity. One strategy for harvesting mature oocytes may be to isolate small follicles from the stroma for in-vitro culture. The aim of this study was to assess the survival and growth of murine follicles during in-vitro culture after isolation from tissue frozen-thawed in various cryoprotective agents. The effect of different seeding and thawing temperatures was also investigated. Pre-antral follicles 100--135 microm in diameter were isolated from fresh and frozen-thawed tissue and cultured in vitro for 8 days. In the fresh control group 79 +/- 3% of follicles survived in-vitro culture and grew to antral stages. Fewer follicles survived after isolation from tissue cryopreserved in dimethyl sulphoxide (43 +/- 5%) or propylene glycol (24 +/- 2%) and none survived freeze-thawing in glycerol. Lowering the seeding temperature from -5 degrees to -7 degrees or -9 degrees C reduced follicle survival rates from 49 +/- 4% to 26 +/- 1% and 28 +/- 3% respectively. If thawing was carried out at 27 degrees C follicle survival rate was higher (38 +/- 7%) than at 37 degrees C (26 +/- 2%) or 47 degrees C (20 +/- 6%). Follicles surviving 8 days of in-vitro culture were stimulated with human chorionic gonadotrophin. The number of mature oocytes released did not differ between any experimental group. The results indicate that follicles isolated from frozen-thawed tissue can be grown to antral sizes and produce mature oocytes. The in-vitro culture system also proved a sensitive method for testing variations in the freeze-thaw protocol.