Muratori M, Marchiani S, Tamburrino L, Tocci V, Failli P, Forti G, Baldi E
Department of Clinical Physiopathology, Andrology Unit, Center for Research, Transfer and High Education DeNothe, University of Florence, Viale Pieraccini, 6, I-50139 Firenze, Italy.
Hum Reprod. 2008 May;23(5):1035-43. doi: 10.1093/humrep/den058. Epub 2008 Mar 6.
Sperm DNA fragmentation is a possible predictive parameter for male fertility status. The occurrence of M540 bodies in semen of subfertile subjects affects flow cytometric investigations in sperm. We set up a new method to evaluate DNA fragmentation excluding M540 bodies.
DNA fragmentation was evaluated by flow cytometry in semen of 75 subjects both by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL, traditional method) and by double staining with TUNEL and propidium iodide (PI, new method).
The use of the new method revealed that TUNEL underestimates sperm DNA fragmentation in flow cytometry and showed two sperm populations stained with low (PI(dim)) and high (PI(br)) avidity for PI. The PI(dim) population is entirely composed of DNA fragmented sperm and its incidence shows highly significant negative correlations with morphology, motility, sperm count and concentration (respectively, r = -0.51, -0.52, -0.46 and -0.32, n = 75). DNA fragmentation in the PI(br) sperm population is independent from semen quality.
The correlations between sperm DNA breakage and semen quality previously reported are mainly driven by the occurrence of the PI(dim) population. DNA fragmented sperm in this population are more likely to have poorer morphology, reduced motility and thus a reduced chance to fertilize an oocyte than DNA damaged sperm in PI(br) population. Distinguishing between the two types of sperm DNA fragmentation appears to be important in clinical investigations.
精子DNA碎片化是男性生育状况的一个可能的预测参数。不育男性精液中M540小体的出现影响了精子的流式细胞术检测。我们建立了一种新方法来评估不包括M540小体的DNA碎片化情况。
通过末端脱氧核苷酸转移酶介导的荧光素-dUTP缺口末端标记法（TUNEL，传统方法）以及TUNEL与碘化丙啶双重染色法（PI，新方法），采用流式细胞术对75名受试者的精液中的DNA碎片化情况进行评估。
新方法的应用表明，TUNEL法在流式细胞术中低估了精子DNA碎片化情况，并且显示出对PI有低亲和力（PI（dim））和高亲和力（PI（br））两种染色的精子群体。PI（dim）群体完全由DNA碎片化精子组成，其发生率与形态、活力、精子计数和浓度呈高度显著负相关（分别为r = -0.51、-0.52、-0.46和-0.32，n = 75）。PI（br）精子群体中的DNA碎片化与精液质量无关。
先前报道的精子DNA断裂与精液质量之间的相关性主要由PI（dim）群体的出现所驱动。与PI（br）群体中DNA受损的精子相比，该群体中DNA碎片化的精子更有可能具有较差的形态、降低的活力，从而使卵子受精的机会减少。区分这两种类型的精子DNA碎片化在临床研究中似乎很重要。
