de Lima Rosa Josiane, de Paula Freitas Dell'Aqua Camila, de Souza Fabiana Ferreira, Missassi Gabriela, Kempinas Wilma De Grava
Institute of Biosciences, Department of Structural and Functional Biology, Laboratory of Reproductive and Developmental Biology and Toxicology, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
School of Veterinary Medicine and Animal Science, Department of Veterinary Surgery and Animal Reproduction, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Reprod Toxicol. 2023 Apr;117:108353. doi: 10.1016/j.reprotox.2023.108353. Epub 2023 Feb 18.
Multiparametric analysis by flow cytometry solves one of the major problems in sperm evaluation, the inability to test multiple attributes simultaneously in a single cell, which would increase the precision to predict fertility potential since several sperm parameters are tested. The association of fluorochromes and compounds conjugated to fluorochromes in multiparametric sperm analysis is well-established in microscopy techniques. However, these techniques are subjective and limit the assessment in small cell numbers, thereby harming analytic accuracy. Therefore, the current study aimed to present new possibilities for assessing the integrity and stability of the sperm plasma membrane, acrosome status, mitochondrial potential, and superoxide anion production in the mitochondrial matrix in only 2 cytometric assays using cytometers equipped with 2 and 3 lasers. For this, human semen samples collected by masturbation and selected by the swim-up technique were divided into 3 treatments: T0 (flash-frozen semen), T50 (flash-frozen semen + fresh semen, V: V), and T100 (fresh semen) for the validation of the multiparametric protocols by flow cytometry. For both protocols, sperm percentage with positive stain for all fluorophores differed significantly between treatments. The determination coefficients presented values close to 1, which validated objective, sensitive, rapid, and reproducible methodologies. Therefore, we concluded that the results reflect the status of analyzed structure, enabling a more accurate diagnosis of male infertility that has become an increasingly prevalent worldwide setback due to exposure to a variety of environmental toxicants.
流式细胞术的多参数分析解决了精子评估中的一个主要问题，即无法在单个细胞中同时检测多个属性，而若能检测多个精子参数，将提高预测生育潜力的精度。在显微镜技术中，多参数精子分析中荧光染料及与荧光染料偶联的化合物之间的关联已得到充分确立。然而，这些技术具有主观性，且在小细胞数量情况下会限制评估，从而损害分析准确性。因此，本研究旨在利用配备2激光和3激光的细胞仪，仅通过2种细胞计数测定法，为评估精子质膜的完整性和稳定性、顶体状态、线粒体电位以及线粒体基质中超氧阴离子的产生提供新的可能性。为此，通过手淫采集并经上浮技术筛选的人类精液样本被分为3种处理组：T0（速冻精液）、T50（速冻精液 + 新鲜精液，体积比1:1）和T100（新鲜精液），用于通过流式细胞术验证多参数方案。对于这两种方案，所有荧光团染色阳性的精子百分比在各处理组之间存在显著差异。测定系数的值接近1，这验证了客观、灵敏、快速且可重复的方法。因此，我们得出结论，这些结果反映了所分析结构的状态，能够对男性不育症进行更准确的诊断，由于接触多种环境毒物，男性不育症在全球范围内已成为日益普遍的挫折。
