Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

BACKGROUND Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution. METHODS Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20-30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180 degrees C in the volume of 2 microl and 6 microl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-microl droplets of alpha-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored. RESULTS After the vitrification of 14-day-old preantral follicles using 2 microl or 6 microl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 microl and 6 microl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 microl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group. CONCLUSION Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 microl and 6 microl can be an option.