ABSTRACT
DNA damage in cumulus cells (CCs) might be related with the developmental competence of the enclosed oocytes, however, conclusive studies are missing, partially due to the lack of a reliable, cheap, fast, and reproducible DNA damage test. We report the development of a chromatin dispersion test that allows for a fast evaluation of double strand DNA (ds-DNA) damage in CCs. The whole experiment was performed using CCs from 103 oocyte retrieval cycles evaluating the prototype D3-MAX ability (a chromatin dispersion based assay) to detect DNA breaks against in situ nick translation (ISNT) and a two tailed comet assay (TT-comet). Samples were collected from women younger than 35 years of age with a good response to stimulation. Pooled cumulus cells of MII oocytes were used. The chromatin dispersion assay results correlate with the double strand-DNA breaks values assessed by the TT-comet assay (Spearman Rho = 0.624; p = 0.003;), while the correlation was poor when compared to the single strand DNA (ss-DNA) breaks observed also with the TT-comet assay (Spearman Rho = -0.141; p = 0.554). ISNT showed a correspondence in the same cells between enzymatic incorporation of modified nucleotides and halos of chromatin dispersion. We conclude that D3-Max test detects mainly ds-DNA breaks in cumulus cells and is a reliable, fast, and easy reproducible assay suitable for routine clinical practices once the influence on oocyte quality has been established.
