PERMEATION OF HUMAN OVARIAN TISSUE WITH CRYOPROTECTIVE AGENTS IN PREPARATION FOR CRYOPRESERVATION

The recent improvements in the treatment of cancer by chemo-and radiot herapy have led to a significant increase in the survival rates of pat ients with malignant disease, but at the expense of distressing side e ffects, One major problem, especially for younger patients, is that ag gressive therapy destroys a significant proportion of the follicular p opulation, which can result in either temporary or permanent infertili ty, Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity, When the patient is in remission, f ertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to matu rity in vitro. Recent studies have found good follicular survival in f rozen-thawed human ovarian tissue but to optimize the process an effec tive cryopreservation method needs to be developed, An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an imp ortant determinant of post-thaw cellular survival, In the current stud y, we have investigated the diffusion of four cryoprotective agents in to human tissue at both 4 degrees C and 37 degrees C, We have also stu died the effect of adding different concentrations of the non-penetrat ing cryoprotective agent, sucrose, to the freezing media using the rel ease of lactate dehydrogenase as a measure of its protective effect. A t 4 degrees C propylene glycol and glycerol penetrated the tissue sign ificantly slower than either ethylene glycol or dimethyl sulphoxide, A t the higher temperature of 37 degrees C all four cryoprotectants pene trated at a faster rate, however concern about enhanced toxicity preve nts the use of these conditions in practice, Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide a t 4 degrees C; this achieved a mean tissue concentration that was almo st 80% that of the bathing solution, We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.