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

Thin slices of human ovarian cortex were evaluated following cryopreservati on in 1,2-propanediol (PROH)/sucrose under various conditions, Following ra pid thawing, 1 mu m sections were assessed by light microscopy and oocyte a bnormalities were further examined by electron microscopy. Follicles (n = 5 03) 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 signi ficantly reduced following cooling at three different rates with the highes t levels of intactness (55 and 85% respectively) being achieved with slow c ooling. The frequency of oocyte abnormalities [loss of organelles (mitochon dria), organelle-free areas, and/or cytoplasmic vacuolation] was significan tly increased at all cooling rates with slow cooling resulting in the highe st proportion (56%) of normal oocytes, With slow cooling, increasing dehydr ation time increased the proportions of intact pre-granulosa cells and oocy tes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cel ls intact (44%) was observed, as was the highest proportion of 'normal' ooc ytes (85%), In this study, single step dehydration in PROH/sucrose for 90 m in and slow cooling/rapid thawing results in the highest proportion of inta ct human primordial and primary follicles.