Normandie Univ, UNIROUEN, EA 4308 "Gametogenesis and Gamete Quality", Rouen University Hospital, Department of Reproductive Biology - CECOS, F 76000, Rouen, France; Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
OmicX | Seine Innopolis, Le-Petit-Quevilly, France.
Reprod Biol. 2021 Jun;21(2):100507. doi: 10.1016/j.repbio.2021.100507. Epub 2021 Apr 24.
Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.
免疫组织化学分析是男性生育力临床和研究的常规程序。然而，大多数解释仍然是主观的，耗时的，存在内在的观察者间和观察者内变异性。鉴于睾丸评估的预后和研究意义，需要一种更客观、耗时更少的方法。在本研究中，我们使用体外成熟的未成年小鼠睾丸作为模型。主要目的是开发一种负担得起的自动化数字免疫组织化学图像分析工具，用于对睾丸组织切片进行公正和定量的评估。睾丸外植体被固定、切割并染色用于特定的生殖细胞标记物。评估了经典的手动计数程序。在明场图像上降低了背景和噪声。将显微照片拼接（Background_Elimination_Stitching）以创建高质量的图像。评估了两种程序（IHC_Tool 和染色核区域）；然后评估了一种允许在培养后去除观察到的坏死区域的程序（Necrotic_Area_Elimination）。最后，提取未改变组织区域中的染色核数量。以 ×200 放大倍数保存为 TIFF 的自动 IHC_Tool 程序允许最严格的细胞定量。为睾丸样本分析开发的 IHC_Tool 可用于各种类型的组织。我们预计这种方法将最大限度地减少实验室之间的观察者间变异性，并有助于临床试验和转化计划。
