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

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 (H 2 O 2 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 H 2 O 2 group); 3) H 2 O 2 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.