Mouse ovarian follicle cryopreservation using vitrification or slow programmed cooling: assessment of in vitro development, maturation, ultra-structure and meiotic spindle organization.
OBJECTIVE
To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and to evaluate different cryo-devices for vitrification of isolated follicles.


METHODS
Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slow freezing. A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolated follicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n=100), nylon mesh (n=96), electron microscopy grid (n=102), and micro-capillary tips (n=116). Subsequently, a total of 979 follicles were randomly assigned to three different groups: CT fresh (n=256), vitrification (n=399) and slow freezing (n=324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily for development. Final maturation was triggered with human chorionic gonadotrophin and rates of oocyte maturation were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electron microscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosight imaging system.


RESULTS
Micro-capillary tips resulted in poor immediate post-warming survival but no differences were observed in the subsequent in vitro development characteristics between different cryo-devices. Nylon mesh proved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared to vitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the culture period (P<0.0001). However all other outcome measures were comparable between both techniques.


CONCLUSIONS
Isolated follicles were more vulnerable to cryodamage after slow freezing as compared to vitrification.
Desai
Nina
N
Department of OB-GYN, Cleveland Clinic Foundation, Cleveland, Ohio, USA. desain@ccf.org
AbdelHafez
Faten
F
Ali
Mansour Y
MY
Sayed
Ezzat H
EH
Abu-Alhassan
Ahmed M
AM
Falcone
Tomasso
T
Goldfarb
James
J
D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D005260:Female; D005615:Freezing / Q000009:adverse effects; D008540:Meiosis; D051379:Mice; D009866:Oogenesis; D006080:Ovarian Follicle / Q000502:physiology* / Q000648:ultrastructure*; D058989:Vitrification