Learning Aspects in Implementing Vitrification Program Using Mouse Blastocysts

Oocyte vitrification is important for fertility preservation. There are debates, however, surrounding the components of the procedure itself.

before starting a vitrification program, we decided to (1) determine if blastocysts derived from previously frozen mouse embryos would be suitable to practice vitrification and (2) to analyze the factors that contributed to improving our "learning curve".

58 expanded blastocysts cultured from commercially available frozen 1-cell mouse embryos (B6D2F1 x B6C3F1) were used. Embryos were vitrified, during 2 separate attempts, in a closed device (Cryopette) and vitrified and warmed using Ethylene Glycol-cryoprotectant vitrification kit (Vitrification Attempt 1 and 2 - VA1 and 2; Warming Attempt 1 and 2 - WA1 and 2). Differences in attempts by more focusing on the reaction of the embryo itself. The survival rate (SR) of embryos was evaluated immediately after warming (T0), then rinsed and cultured for 24 hours in an HTF-like medium (Cleavage Medium - Sage; Cooper Surgical; U.S.A.). Additional evaluations were done at 2 and 24 hours of culture (T2 and T24, respectively). RESULTS: The combined SR for both attempts were 70.6, 60.3 and 34.4% at T0, T2 and T24, respectively. Embryo loss was significantly higher in WA1 compared to WA2 (30.8 versus 10.1%; p=.05). The SR at T0 after VA1 and VA2 were similar (90.9 versus 80.9%) and the SR after VA1+WA1 versus VA1+WA2 was 55.6 vs. 76.9% at T2 and 22.2 vs. 61.5% at T24, respectively (NS).

Commercially frozen mouse embryos can provide essential information for initiating a Vitrification Program applicable to human embryos and oocytes. Practice is critical, particularly Warming, which can provide the confidence needed to adapt and optimize these protocols before vitrifying limited human tissues or gametes.