Schmid T E, Lowe X, Marchetti F, Bishop J, Haseman J, Wyrobek A J
Institut für Säugetiergenetik, GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Neuherberg, Ingolstädter Landstrabetae 1, D-85764, Neuherberg, Germany.
Mutagenesis. 2001 May;16(3):189-95. doi: 10.1093/mutage/16.3.189.
The mouse epididymal sperm aneuploidy (mESA) assay using 3-chromosome fluorescence in situ hybridization (FISH) was recently developed for assessing the aneugenic potential of chemicals on male germ cells. This study was designed to identify the major technical factors that affect inter-scorer and inter-laboratory variability of the mESA assay. Two laboratories participated in this study (GSF and Lawrence Livermore National Laboratory, LLNL). Mice (102/ElxC3H/El) F(1) were exposed in one laboratory (GSF) to vinblastine (VBL; single intraperitoneal injection of 0, 0.5, 1.0 or 2.0 mg/kg), one of the 10 priority compounds of the Commission of the European Communities (CEC) Aneuploidy Program. Twenty-two days later the mESA assay was applied to analyze sperm aneuploidy. In the initial evaluation, small but statistically significant differences were found between the two laboratories in baseline frequencies and there was also disagreement in the determination of a VBL aneuploid effect. Therefore, experiments were conducted to identify the sources of the inter-laboratory differences and technical factors that affected assay reliability and the VBL study was repeated. A harmonization experiment was conducted by bringing the microscope scorers from both laboratories to the same site (LLNL) for a cross-training exercise. Following this exercise, a second group of VBL-treated and control mice were evaluated, and we concluded that VBL is not a sperm aneugen. Our research has identified scoring criteria as the major source of inter-laboratory variation and emphasizes the importance of strict technical controls for the mESA assay, including controlling slide preparations for treatment-induced reductions in sperm count, coding of slides and selection of statistical tests. These considerations are particularly important for the interpretation of small effects (< or =2-fold) on sperm aneuploidy. Our findings suggest that 2-fold differences in frequencies can result from differences among scorers, samples and treatment groups, and are readily within the normal variation for the mESA assay. Such small differences should be viewed with caution until independently confirmed.
最近开发了一种使用3染色体荧光原位杂交（FISH）的小鼠附睾精子非整倍体（mESA）检测方法，用于评估化学物质对雄性生殖细胞的致非整倍体潜能。本研究旨在确定影响mESA检测评分者间和实验室间变异性的主要技术因素。两个实验室参与了本研究（德国环境与健康研究中心和劳伦斯利弗莫尔国家实验室，LLNL）。在一个实验室（德国环境与健康研究中心）中，将（102/ElxC3H/El）F(1)代小鼠腹腔注射长春碱（VBL；剂量分别为0、0.5、1.0或2.0mg/kg），长春碱是欧洲共同体（CEC）非整倍体计划的10种优先化合物之一。22天后，应用mESA检测分析精子非整倍体情况。在初始评估中，发现两个实验室在基线频率上存在微小但具有统计学意义的差异，并且在确定VBL的非整倍体效应方面也存在分歧。因此，进行了实验以确定实验室间差异的来源以及影响检测可靠性的技术因素，并重复了VBL研究。通过将两个实验室的显微镜评分者带到同一地点（LLNL）进行交叉培训，开展了一项协调实验。此次培训后，对第二组经VBL处理的小鼠和对照小鼠进行了评估，我们得出结论，VBL不是精子致非整倍体物质。我们的研究已确定评分标准是实验室间变异的主要来源，并强调了mESA检测严格技术控制的重要性，包括控制因处理导致精子数量减少的载玻片制备、载玻片编码以及统计检验的选择。这些考虑因素对于解释精子非整倍体的微小效应（≤2倍）尤为重要。我们的研究结果表明，频率上2倍的差异可能源于评分者、样本和处理组之间的差异，并且很容易在mESA检测的正常变异范围内。在独立确认之前，应谨慎看待此类微小差异。
