68 VITRIFICATION OF CLEAVAGE-STAGE MOUSE EMBRYOS BY THE VITROLOOP^™ (CRYOLOOP) PROCEDURE

Abstract

By decreasing the volume of the cryoprotective solution, we were able to dramatically increase the freezing speed and decrease the toxicity and osmotic side effects of the cryoprotectants (CP). Several carriers have been developed successfully (Vajta G et al. 1998 Mol. Reprod. Dev. 51, 53–58; Liebermann J et al. 2002 Reprod. Biomed. Online 4, 146–150; Park SP et al. 1999 Hum. Reprod. 15, 1787–1790; Chung HM et al. 2000 Fertil. Steril. 73, 545–551; Kuwayama M and Kato O 2000 Fertil. Steril. 74(Suppl. 3), S49 O-127; Matsumoto H et al. 2001 Cryobiology 42, 139–144; Lane M et al. 1999 Fertil. Steril. 72, 1073–1078; Dinnyés A et al. 2000 Biol. Reprod. 63, 513–518). The objective of our study was to vitrify Day 3 cleavage stage mouse embryos with the Vitroloop^™ (Vitrolife, Kungsbacka, Sweden) cryopreservation technology. Vitrification was carried out in RapidVit^™ Cleave (Vitrolife) solutions (holding, equilibration, and vitrification medium). Embryos were exposed to a 2-step loading of CP, ethylene glycol (EG), and propylene glycol (PG), before being placed in a small loop attached to the lid of a cryo-vial and rapidly submerged into liquid nitrogen (LN). First, the embryos were transferred from the G-MOPS holding medium to the equilibration medium containing 8% EG for 2 min. Then, embryos were transferred into a 20-μL drop of vitrification medium containing 16% EG, 16% PG, 10 mg mL^–1 Ficoll 400, and 0.65 m sucrose for 30 s. After that, the embryos (maximum of 2 at a time) were transferred onto the loop, which was quickly sealed in a cryo-vial of LN and stored. After storage in LN, embryos were warmed by a 3-step dilution of the CP with sucrose (RapidWarmCleave^™, Vitrolife) carried out at 37°C. First, the loop with the embryos was quickly immersed in 37°C warming medium 1 (0.65 m sucrose) for 20 s. Then, the embryos were transferred into warming medium 2 (0.25 m sucrose) for 1 min, then into warming medium 3 (0.125 m sucrose) for 2 min, and finally into warming medium 4 (G-MOPS medium) for 5 min. Following warming, embryos were cultured in G1 medium (Vitrolife) at 37°C with 6.5% CO₂ and maximum humidity in air. Embryo viability was assessed by 48 h in vitro culture; the survival of embryos was based on morphological appearance in vitro after thawing and continued development to expanded blastocysts upon subsequent culture. The control embryos were treated likewise except that they were not vitrified. A total of 229 cleavage-stage embryos were vitrified and warmed; out of these, 11 were lost (11/229; 4.8%). Of the remaining 218 embryo, 202 survived vitrification (202/218; 92.7%) and 180 developed further to expanded blastocysts during in vitro culture (180/202; 82.6%). In the control group, 91.4% of the embryos developed to expanded blastocysts (75/82) indicating that the solutions used were not toxic. Our data show that a high percentage of cleavage-stage mouse embryos survived vitrification in the mixture of EG and PG combined with the use of cryoloop and developed normally in vitro after thawing. To our knowledge, this is the first report of the successful use of the Vitroloop^™ vitrification procedure with cleavage-stage mouse embryos.

The authors thank Vitrolife Ltd. (Kungsbacka, Sweden) and FertiCad Ltd. (Budapest, Hungary) for providing the solutions. The 3-month fellowship for Phillip Klambauer was provided by CEEPUS.