Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol

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Abstract
Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.