Sperm chromatin structure assay (SCSA
Sperm chromatin structure assay (SCSA
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DOI：
10.1007/978-1-62703-038-0_14
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发表时间：
2011
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影响因子：
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通讯作者：
D. Evenson
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文献类型：
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作者：
D. Evenson
SCSA®是通过吖啶橙子（AO）染色精子的流式细胞术检测受损精子DNA和精子核中改变的蛋白质的开创性检测方法。SCSA®被认为是最精确和可重复的测试，在1，024 × 1，024通道规模上提供非常独特的双参数数据（红色与绿色荧光），不仅针对DNA片段化，而且针对以缺乏组蛋白与鱼精蛋白的正常交换为特征的异常精子。原始精液/精子等分试样或纯化的精子可以快速冷冻，置于装有干冰的盒子中，并通过隔夜快递运送到经验丰富的SCSA®实验室。在10分钟内分别解冻、制备和分析样本。重要的是，在绿色（天然DNA）和红色（断裂DNA）荧光的1，024 × 1，024点图上记录了5，000个精子的数据。重复测量具有几乎相同的点图模式，表明在断裂位点打开DNA链的低pH处理和吖啶橙子（AO）染色具有高度精确性和可重复性（CV为1-3%），并且新鲜和冷冻样品之间相同。SCSAsoft®软件将X-Y数据转换为总DNA荧光与红/红+绿色荧光（DFI），提供更准确的% DFI测定以及更灵敏的DFI标准偏差值（SD DFI），如动物生育力和剂量反应毒理学研究所证明的。目前建立的临床阈值为25% DFI，用于将男性置于以下统计概率中：（a）自然妊娠时间较长，（B）IUI妊娠几率较低，（c）流产较多，或（d）未妊娠。生活方式的改变以及医疗干预可以降低DFI %，以增加自然妊娠的可能性。建议DFI>25%的男性夫妇尝试ICSI，当DFI>50%时，可以考虑TESE/ICSI。SCSA®同时测定与未成熟精子一致的与保留的核组蛋白相关的高DNA含量（%HDS）精子的百分比;高HDS值预示着妊娠失败。SCSA®被认为是技术人员最友好、时间和成本效益最高、精确和可重复的DNA片段化测定，具有最多的数据和唯一的具有可接受的临床阈值的片段化测定，用于将男性置于不孕症的风险中。SCSA®数据比经典精液分析更能预测男性因素不育。
The SCSA®is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA®is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA®lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1–3%) and the same between fresh and frozen samples. SCSAsoft®software transforms theX-Ydata to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose–response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA®simultaneously determines the % of sperm with high DNA stainability (%HDS) related to retained nuclear histones consistent with immature sperm; high HDS values are predictive of pregnancy failure.The SCSA®is considered to be the most technician friendly, time- and cost-efficient, precise and repeatable DNA fragmentation assay, with the most data and the only fragmentation assay with an accepted clinical threshold for placing a man at risk for infertility. SCSA®data are more predictive of male factor infertility than classical semen analyses.