Human Reproduction, Vol 13, 376-380, Copyright © 1998 by Oxford University Press
H Newton, J Fisher, JR Arnold, DE Pegg, MJ Faddy and RG Gosden
The recent improvements in the treatment of cancer by chemo- and
radiotherapy have led to a significant increase in the survival rates of
patients with malignant disease, but at the expense of distressing side
effects. One major problem, especially for younger patients, is that
aggressive therapy destroys a significant proportion of the follicular
population, which can result in either temporary or permanent infertility.
Freeze-banking pieces of ovarian cortex prior to treatment is one strategy
for preserving fecundity. When the patient is in remission, fertility
could, theoretically, be restored by autografting the thawed tissue at the
orthotopic site or by growing isolated follicles to maturity in vitro.
Recent studies have found good follicular survival in frozen-thawed human
ovarian tissue but to optimize the process an effective 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 important determinant of post-thaw
cellular survival. In the current study, we have investigated the diffusion
of four cryoprotective agents into human tissue at both 4 degrees C and 37
degrees C. We have also studied the effect of adding different
concentrations of the non penetrating cryoprotective agent, sucrose, to the
freezing media using the release of lactate dehydrogenase as a measure of
its protective effect. At 4 degrees C propylene glycol and glycerol
penetrated the tissue significantly slower than either ethylene glycol or
dimethyl sulphoxide. At the higher temperature of 37 degrees C all four
cryoprotectants penetrated at a faster rate, however concern about enhanced
toxicity prevents 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 at 4 degrees C; this achieved a mean tissue concentration that
was almost 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.
ARTICLES
Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation
Centre for Reproduction, Growth and Development, School of Clinical Medicine, University of Leeds, Leeds General Infirmary, UK.
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