Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols

Objective: To study the thermal properties of a cryoprotectant solution, called VS4, and of VS4-impregnated whole sheep ovaries with pedicle. Design: Physical and experimental animal study. Setting: Academic research environment. Animal(s): Five- to 6-month-old ewes. Intervention(s): Thermal properties on cooling of a cryoprotectant solution called VS5 were measured by differential-scanning calorimetry. VS4 contains 2.75 mol/L dimethyl sulfoxide, 2.76 mol/L formamide, and 1.97 mol/L propylene glycol. Whole sheep ovaries were collected at the slaughterhouse and prepared for a vitrification procedure. Cortex and vessels underwent histologic examination before and after vitrification, and the thermal properties of VS4-impregnated ovarian cortex were then studied. Main Outcome Measure(s): Critical cooling rates (V-ccr), vitreous transition temperature (T-g), end-of-melting temperature (T-m), follicle viability assessment by trypan blue test, and histologic examination of ovary and vessel structure. Result(s): The critical cooling rate (V-ccr) of VS4 solution was estimated to be 14.3 +/- 1.1 degrees C/min. Its vitreous transition temperature (T-g) was -125.2 +/- 0.2 degrees C, and its end-of-melting temperature (T-m) -34.3 +/- 0.1 degrees C. Following our vitrification procedure procedure, immediate follicle viability was 61.4% +/- 2.2%. The percentage if normal primordial follicles remained after vitrification was 48% +/- 3.8%. The V-ccr of VS4-impregnated cortex could not be determined because of the quantity of ice forming as of the top programmed cooling rate (-300 degrees C min). The mean ovarian cortex cooling rate actually attained experimentally during vitrification was -342.9 +/- 49.6 degrees C/min. Conclusion(s): Vitrification of entire organs, such as ovaries, is a great challenge in cryobiology and reproductive medicine. Physical studies seem indispensible for progress with this technique. Thus, our ovarian perfusion procedure needs improving to enhance ovarian cortex impregnation and bring down the V-ccr rate in both tissue and VS4 solution.