title: Detection of DNA damage in cumulus cells using a chromatin dispersion assay.
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.
authors: a Clínica EUGIN , Barcelona , Spain .
authors: Pablo
authors: P
authors: Barcena
authors: b Departamento de Biología , Universidad Autónoma de Madrid , Madrid , Spain .
authors: Carmen
authors: C
authors: López-Fernández
authors: c CEFIVA , Oviedo , Spain , and.
authors: Carlos
authors: C
authors: García-Ochoa
authors: a Clínica EUGIN , Barcelona , Spain .
authors: Albert
authors: A
authors: Obradors
authors: a Clínica EUGIN , Barcelona , Spain .|d Fundación Privada EUGIN , Barcelona , Spain.
authors: Valerie
authors: V
authors: Vernaeve
authors: b Departamento de Biología , Universidad Autónoma de Madrid , Madrid , Spain .
authors: Jaime
authors: J
authors: Gosálvez
authors: a Clínica EUGIN , Barcelona , Spain .
authors: Rita
authors: R
authors: Vassena
mesh_terms: D000328:Adult; D002843:Chromatin / Q000737:chemistry; D020552:Comet Assay / Q000379:methods; D054885:Cumulus Cells; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D005260:Female; D006801:Humans; D055815:Young Adult
chemical_list: D002843:Chromatin
keywords: Assisted reproduction; DNA fragmentation; chromatin dispersion test; cumulus cells