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.


METHODS
Physical and experimental animal study.


METHODS
Academic research environment.


METHODS
Five- to 6-month-old ewes.


METHODS
Thermal properties on cooling of a cryoprotectant solution called VS4 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.


METHODS
Critical cooling rates (V(ccr)), vitreous transition temperature (Tg), end-of-melting temperature (Tm), follicle viability assessment by trypan blue test, and histologic examination of ovary and vessel structure.


RESULTS
The critical cooling rate (V(ccr)) of VS4 solution was estimated to be 14.3 +/- 1.1 degrees C/min. Its vitreous transition temperature (Tg) was -125.2 +/- 0.2 degrees C, and its end-of-melting temperature (Tm) -34.3 +/- 0.1 degrees C. Following our vitrification procedure, immediate follicle viability was 61.4% +/- 2.2%. The percentage of normal primordial follicles remaining 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.


CONCLUSIONS
Vitrification of entire organs, such as ovaries, is a great challenge in cryobiology and reproductive medicine. Physical studies seem indispensable 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.
Courbiere
Blandine
B
Unité INSERM 418, Hôpital Debrousse, Lyon, France.
Odagescu
Valentina
V
Baudot
Anne
A
Massardier
Jérôme
J
Mazoyer
Claire
C
Salle
Bruno
B
Lornage
Jacqueline
J
D000818:Animals; D001808:Blood Vessels / Q000187:drug effects; D002152:Calorimetry, Differential Scanning; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D007552:Isotonic Solutions / Q000494:pharmacology; D006080:Ovarian Follicle / Q000187:drug effects / Q000473:pathology; D010053:Ovary / Q000098:blood supply; D012756:Sheep; D013997:Time Factors; D014022:Tissue Survival
D003451:Cryoprotective Agents; D007552:Isotonic Solutions