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.

中文翻译：

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使用染色质分散测定法检测卵丘细胞中的DNA损伤。

卵丘细胞（CCs）中的DNA损伤可能与封闭卵母细胞的发育能力有关，但是，缺少可靠的结论性研究，部分原因是缺乏可靠，便宜，快速和可重复的DNA损伤试验。我们报道了染色质分散测试的发展，该测试可以快速评估CC中双链DNA（ds-DNA）的损伤。整个实验是使用来自103个卵母细胞回收周期的CC进行的，评估原型D3-MAX能力（基于染色质分散的检测）以检测针对原位缺口翻译（ISNT）的DNA断裂和双尾彗星检测（TT-comet）。从年龄小于35岁的女性身上采集样品，对刺激反应良好。使用了MII卵母细胞的汇集卵丘细胞。染色质分散测定结果与通过TT彗星测定法评估的双链DNA断裂值相关（Spearman Rho = 0.624; p = 0.003;），而与单链DNA（ss-DNA）相比，相关性较差在TT-彗星试验中也观察到断裂（Spearman Rho = -0.141; p = 0.554）。ISNT在同一细胞中显示出酶促修饰核苷酸的掺入与染色质分散体的光晕之间的对应关系。我们得出的结论是，D3-Max测试主要检测卵丘细胞中的ds-DNA断裂，并且一旦确定了对卵母细胞质量的影响，它便是一种可靠，快速且易于重现的测定方法，适用于常规临床实践。与TT-comet分析相比，与单链DNA（ss-DNA）断裂相比，相关性较差（Spearman Rho = -0.141; p = 0.554）。ISNT在同一细胞中显示出酶促修饰核苷酸的掺入与染色质分散体的光晕之间的对应关系。我们得出的结论是，D3-Max测试主要检测卵丘细胞中的ds-DNA断裂，并且一旦确定了对卵母细胞质量的影响，它便是一种可靠，快速且易于重现的测定方法，适用于常规临床实践。与TT-comet分析相比，与单链DNA（ss-DNA）断裂相比，相关性较差（Spearman Rho = -0.141; p = 0.554）。ISNT在同一细胞中显示出酶促修饰核苷酸的掺入与染色质分散体的光晕之间的对应关系。我们得出的结论是，D3-Max测试主要检测卵丘细胞中的ds-DNA断裂，并且一旦确定了对卵母细胞质量的影响，它便是一种可靠，快速且易于重现的测定方法，适用于常规临床实践。