Ribeiro S, Sharma R, Gupta S, Cakar Z, De Geyter C, Agarwal A
Clinic of Gynecological Endocrinology and Reproductive Medicine, University Hospital, University of Basel, Basel, Switzerland.
American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
Andrology. 2017 May;5(3):477-485. doi: 10.1111/andr.12334. Epub 2017 Feb 28.
One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting.
精子DNA片段化检测结果面临的挑战之一是不同技术所获结果的不一致性和巨大变异性。末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）检测通过用TdT标记3'-OH末端来量化荧光素化dUTP掺入单链和双链DNA断裂处的情况。本研究的目的是通过方法标准化以及比较瑞士巴塞尔和美国俄亥俄州克利夫兰诊所这两个参考实验室的数据，优化用于评估精子DNA片段化的TUNEL方案。将31名受试者的精液样本分为三个队列。由两名经验丰富的操作人员在两个不同实验室使用相同的精液样本、检测试剂盒、方案和采集设置，并使用相同的流式细胞仪（BD Accuri C6）来测量精子DNA片段化数据。在进行的任何实验中，重复测量之间均未观察到显著差异。通过在多聚甲醛固定后增加一个洗涤步骤，两个实验室之间呈现出高度相关性（r = 0.94）。平均精子DNA片段化率之间观察到强正相关（r = 0.719）。每个实验室测量的平均精子DNA片段化情况相似。两台流式细胞仪的设置和性能相同。这项实验室间和实验室内部的研究表明，当采用标准化染色方案和流式细胞仪采集设置时，TUNEL检测具有可重复性。TUNEL的标准化和共识性指南验证了该检测方法，并确立了TUNEL作为一种可靠的检测方法，用于测量精子DNA片段化，尤其是在多中心环境中。
