Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- andradiotherapy have led to a significant increase in the survival rates ofpatients with malignant disease, but at the expense of distressing sideeffects. One major problem, especially for younger patients, is thataggressive therapy destroys a significant proportion of the follicularpopulation, which can result in either temporary or permanent infertility.Freeze-banking pieces of ovarian cortex prior to treatment is one strategyfor preserving fecundity. When the patient is in remission, fertilitycould, theoretically, be restored by autografting the thawed tissue at theorthotopic site or by growing isolated follicles to maturity in vitro.Recent studies have found good follicular survival in frozen-thawed humanovarian tissue but to optimize the process an effective cryopreservationmethod needs to be developed. An essential part of such a technique is topermeate the tissue with a cryoprotectant to minimize ice formation and theextent of this equilibration is an important determinant of post-thawcellular survival. In the current study, we have investigated the diffusionof four cryoprotective agents into human tissue at both 4 degrees C and 37degrees C. We have also studied the effect of adding differentconcentrations of the non penetrating cryoprotective agent, sucrose, to thefreezing media using the release of lactate dehydrogenase as a measure ofits protective effect. At 4 degrees C propylene glycol and glycerolpenetrated the tissue significantly slower than either ethylene glycol ordimethyl sulphoxide. At the higher temperature of 37 degrees C all fourcryoprotectants penetrated at a faster rate, however concern about enhancedtoxicity prevents the use of these conditions in practice. Thus, theresults suggest that the best method of preparing tissue for freezing isexposure for 30 min to 1.5 M solutions of ethylene glycol or dimethylsulphoxide at 4 degrees C; this achieved a mean tissue concentration thatwas almost 80% that of the bathing solution. We also report that theaddition of low concentrations of sucrose to the freezing medium does nothave a significant protective effect against freezing injury.