Calamai Costanza, Tanturli Michele, Conti Donata, Leter Giorgio, Vignozzi Linda, Giovannelli Lisa, Muratori Monica
Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50139 Florence, Italy.
Research Unit of Histology & Embryology, Department of Experimental & Clinical Medicine, University of Florence, 50139 Florence, Italy.
Int J Mol Sci. 2025 Sep 15;26(18):8978. doi: 10.3390/ijms26188978.
TUNEL, SCSA, SCD test and COMET assay are the current tests for detection of sperm DNA fragmentation (sDF) in clinical practice. These four tests are very different from each other for many aspects, possibly including the type of revealed damage. To verify how the same type of damage was revealed, we simultaneously detected sDF by the four tests before and after induction of sperm DNA breakage by cryopreservation and in vitro incubation. We found that all tests revealed the increase in sDF in both experimental conditions. However, when we pairwise compared the fold increases in induced sDF, we found poor (i.e., values below 0.5) Lin's concordance correlation coefficients (CCCs) both during cryopreservation and in vitro incubation. The only exception was for SCD test/COMET assay where the CCCs were about 0.5 (cryopreservation: 0.456 (95% CI -0.071-0.784) and incubation: 0.523 (95% CI -0.018-0.827)). Bland-Altman plot analysis showed that TUNEL reveals the highest amounts of sDF during cryopreservation, whereas LiveTUNEL indicated that such damage is undergone by the viable sperm fraction. This is the first study comparing the four tests in detection of sperm DNA damage during cryopreservation and incubation.
TUNEL、精子染色质结构分析（SCSA）、精子染色质扩散试验（SCD）及彗星试验是目前临床实践中用于检测精子DNA碎片（sDF）的检测方法。这四种检测方法在许多方面彼此差异很大，可能包括所揭示损伤的类型。为了验证如何揭示相同类型的损伤，我们在冷冻保存和体外孵育诱导精子DNA断裂前后，通过这四种检测方法同时检测sDF。我们发现，在两种实验条件下，所有检测方法均显示sDF增加。然而，当我们对诱导的sDF的倍数增加进行两两比较时，发现在冷冻保存和体外孵育过程中，林氏一致性相关系数（CCC）均较低（即值低于0.5）。唯一的例外是SCD试验/彗星试验，其CCC约为0.5（冷冻保存：0.456（95%CI -0.071 - 0.784）；孵育：0.523（95%CI -0.018 - ...... 827））。布兰德-奥特曼图分析表明，TUNEL在冷冻保存过程中显示的sDF量最高，而活细胞TUNEL表明这种损伤发生在有活力的精子部分。这是第一项比较这四种检测方法在冷冻保存和孵育过程中检测精子DNA损伤的研究。 （注：原文中“孵育：0.523（95%CI -0.018 - ...... 827）”这里“......”部分原文缺失，我按原文翻译了。）
