Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

Abstract

Our objective was to optimize the CA technique on mammal embryos.

Materials and methods: 1000 frozen 2-cell embryos from B6CBA mice were used. Based on a literature review, and after checking post-thaw embryo viability, the main outcome measures included: 1) comparison of the embryo recovery rate between 2 CA protocols (2 agarose layers and 3 agarose layers); 2) comparison of DNA damage by the CA on embryos with (ZP+) and without (ZP-) zona pellucida; and 3) comparison of DNA damage in embryos exposed to 2 genotoxic agents (H₂O₂ and simulated sunlight irradiation (SSI)). DNA damage was quantified by the % tail DNA.

Results: 1) The recovery rate was 3,3% (n=5/150) with the 2 agarose layers protocol and 71,3% (n=266/371) with the 3 agarose layers protocol. 2) DNA damage did not differ statistically significantly between ZP- and ZP+ embryos (12.60±2.53% Tail DNA vs 11.04±1.50 (p=0.583) for the control group and 49.23±4.16 vs 41.13±4.31 (p=0.182) for the H₂O₂ group); 3) H₂O₂ and SSI induced a statistically significant increase in DNA damage compared with the control group (41.13±4.31% Tail DNA, 36.33±3.02 and 11.04±1.50 (p<0.0001)). The CA on mammal embryos was optimized by using thawed embryos, by avoiding ZP removal and by the adjunction of a third agarose layer.

Keywords: DNA damage; Single-Cell Gel Electrophoresis; embryo cryopreservation; literature review; technical issue.