Comparison of day 3 embryo cryo-survival and morphology after slow-rate freeze/thaw or vitrification/warming

Abstract

OBJECTIVE: Slow-rate freezing/thawing (SRF) and vitrification/warming (V) differ in concentration of cryoprotectants used, times of cryoprotectant exposure, rate of temperature decrease and increase, and presence or absence of intracellular and extracellular ice crystal formation. We aimed to compare cleavage-stage embryo SRF/thawing to V/warming in relation to number of degenerated embryos, blastomere loss and change in fragmentation grade after cryopreservation. DESIGN: Retrospective study. MATERIALS AND METHODS: Supernumerary embryos not transferred during a fresh IVF cycle were cryopreserved by either SRF or V between 01/04 to 02/08. Embryo cryopreservation by SRF was performed with propanediol and sucrose, with a programmable freezer. Rapid thawing was performed with stepwise dilutions of sucrose. Vitrification and warming were performed according to manufacturer's instructions (Vitrification Freeze and Thaw Kit, Irvine Scientific). Within 1 hour of thawing/warming embryos were microscopically assessed for survival (degenerate: < 50% of original blastomeres present), number of blastomeres, and percentage of fragmentation. Fragmentation grades were assigned: 1: ≤10%, 2: 11-20%, 3: 21-50%, 4: >50%. Results were represented as mean±standard error of the mean. Statistical comparisons were performed with Student's t-test and considered significant when p<0.05. RESULTS: Embryos thawed following SRF numbered 145 from 30 cycles, equating to 5±2 embryos thawed per cycle. Embryos warmed following V numbered 508 from 126 cycles, yielding 4.0±2 embryos warmed per cycle. The number of degenerated embryos was significantly higher after SRF/thawing (1.5±0.1) in comparison to V/warming (0.5±0.1), giving survival percentages of 69% (SRF) and 88% (V). Of embryos that survived there was no significant difference in numbers of blastomeres lost following thawing or warming. However, there was an increase in fragmentation following SRF/thawing compared to V/warming which resulted in a significant increase in fragmentation grade of embryos after thawing (0.3±0.05) compared to warming (0.1±0.01). CONCLUSIONS: Cryopreservation of cleavage-stage embryos by vitrification, followed by warming, results in enhanced embryo survival rates and better embryo morphology in comparison to SRF/thawing. Future prospective studies are required to determine which procedure is superior in yielding pregnancy rates, live-birth rates, and healthy offspring.