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

L. Rolland 1, 2 B. Courbiere 1, 2 V. Tassistro 2 A. Sansoni 3 T. Orsiere 2 W. Liu 4 C. Di Giorgio 2 J. Perrin 2, 5
1 Service de Gynécologie et Obstétrique [Marseille]
2 IMBE - Institut méditerranéen de biodiversité et d'écologie marine et continentale
3 CIPHE - Centre d'Immunophénomique
4 CEREGE - Centre européen de recherche et d'enseignement de géosciences de l'environnement
5 Centre Clinico-Biologique d’Assistance Médicale à la Procréation - CECOS [Hôpital de la Conception - APHM]
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 (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.
Keywords : technical issue literature review DNA damage embryo cryopreservation Single-Cell Gel Electrophoresis
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L. Rolland, B. Courbiere, V. Tassistro, A. Sansoni, T. Orsiere, et al.. Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos. Toxicology in Vitro, Elsevier, 2017, 44, pp.266-272. ⟨10.1016/j.tiv.2017.07.010⟩. ⟨hal-01785372v2⟩

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