Male infertility is an increasing health problem. Studies show that in infertile couples, the malefactor contribution is 40% (1). One of the leading causes of male sterility is decreased sperm function (2). Nitrosative stress or nitrosylation of redox‐sensitive thiols by reactive nitrogen species is often associated with male infertility and induces mitochondrial damage in human spermatozoa. While paternal mitochondria are generally not passed on to the next generation, they are critical for sperm swimming speed and force to propel through the female reproductive tract to meet and penetrate the egg for fertilization. Uribe and coworkers established a four‐parameter functional test to evaluate sperm quality using flow cytometry (3) (Fig. 1). Propidium iodide was used as a viability dye combined with three cell‐permeable tracers: boronate, monobromobimane, and tetramethylrhodamine methyl ester perchlorate. Boronate is a commonly used probe to identify cell‐derived peroxynitrite which is a potent mediator of nitrosative stress (4). Pinacol boronic ester covalently bound to fluorescein allows flow cytometric quantification of the reaction between peroxynitrite and boronate. Monobromobimane is another fluorometric probe that, upon linking to non‐oxidized thiols, forms a fluorescent thiol‐monobromobimane adduct (excitation 380 nm; emission 478–480 nm) (5). Tetramethylrhodamine methyl ester perchlorate is a rhodamine dye that has a transmembrane potential‐dependent accumulation in mitochondria and is broadly used to analyze mitochondrial health by flow cytometry (6). The authors showed that this panel of dyes offers a useful and easy tool to evaluate both fresh and frozen–thawed human semen quality. In a first approach, the “swim‐up” method, a common technique in the clinic based on the self‐migration of the gametes, was utilized to purify spermatocytes with the highest motility. These migratory spermatozoa exhibited high mitochondrial transmembrane potential and non‐oxidized thiol levels and low amounts of peroxynitrite, compared with native semen samples. In a second approach, swim‐up spermatozoa were exposed to a peroxynitrite‐inducing chemical compound 3‐morpholinosydnonimine or sin‐1 before labeling with the dyes. The generation of peroxynitrite did not affect the viability, but lowered the mitochondrial transmembrane potential and increased thiol oxidation. This promising work further strengthened the use of flow cytometry as a valuable and quantitative tool in the repertoire of sperm evaluation techniques. Indeed flow cytometry has been used to study several aspects of cellular function in semen and potential selection of a baby's gender (7, 8). The current publication opens new venues to apply flow cytometry in andrology research. For example, by expanding the panel with additional mitochondria function probes. It would be interesting to know how the retention of the dyes utilized in this article relates to mitochondrial biomass, which can be readily accessed by flow cytometry using MitoTracker (9, 10). Quantification of nicotinamide adenine dinucleotide (NADH) fluorescence is another flow cytometric measure which provides insight into the mitochondrial complex I function (11, 12). Generally, NADH is considered as a significant contributor to cellular autofluorescence. Still, these energy‐rich intermediates also serve as one of the electron sources of the mitochondrial electron transport chain that maintains the transmembrane potential. Variations in mitochondrial mass and other mitochondria functions may be additional essential parameters in the evaluation of spermatocytes (13). The newly described assay combining four probes needs further validation in a clinical trial to study its efficacy in the selection of a more functional sperm subpopulation than the classic swim‐up method. Expression of the fluorescence values in absolute units rather than arbitrary mean fluorescence intensity values may also facilitate standardization of the assay and, ultimately, its routine use in clinical medicine. The impact of the current work is beyond andrology. Nitrosative stress is involved in many other diseases such as cancer, atherosclerosis, inflammation, and neurodegenerative diseases. Utilization of the flow cytometry technique described here may thus also benefit the research in these areas. As the mitochondrion gains increase the interest for its role in health and disease, so do the tools to study this organelle function and flow cytometric application interrogating mitochondrial biology continues to be a hot topic in the cytometry world.

男性不育是一个日益严重的健康问题。研究表明，在不育夫妇中，男性因素的贡献为 40% ( 1 )。男性不育的主要原因之一是精子功能下降 ( 2 )。活性氮物种对氧化还原敏感硫醇的亚硝化应激或亚硝基化通常与男性不育有关，并诱导人类精子的线粒体损伤。虽然父本线粒体通常不会传递给下一代，但它们对于精子游动速度和推动通过雌性生殖道与卵子相遇并穿透卵子进行受精至关重要。Uribe 及其同事建立了一项四参数功能测试，以使用流式细胞术评估精子质量 ( 3)（图 1）。碘化丙啶被用作活性染料，结合三种细胞渗透性示踪剂：硼酸盐、一溴二苯胺和四甲基罗丹明甲酯高氯酸盐。硼酸盐是一种常用的探针，用于鉴定细胞来源的过亚硝酸盐，它是亚硝化应激的有效介质 ( 4 )。频哪醇硼酸酯与荧光素共价结合，允许对过亚硝酸盐和硼酸盐之间的反应进行流式细胞仪定量。Monobromobimane 是另一种荧光探针，在与未氧化的硫醇连接后，形成荧光硫醇-单溴二烷加合物（激发 380 nm；发射 478–480 nm）（5）。四甲基罗丹明甲酯高氯酸盐是一种罗丹明染料，在线粒体中具有跨膜电位依赖性积累，广泛用于通过流式细胞术分析线粒体健康（6）。作者表明，这组染料提供了一种有用且简单的工具来评估新鲜和冷冻解冻的人类精液质量。在第一种方法中，“游泳”方法是一种基于配子自迁移的临床常用技术，用于纯化具有最高运动能力的精母细胞。与天然精液样本相比，这些迁移精子表现出高线粒体跨膜电位和非氧化硫醇水平以及低含量的过氧亚硝酸盐。在第二种方法中，在用染料标记之前，将游泳的精子暴露于诱导过氧亚硝酸盐的化合物 3-吗啉基二亚胺或 sin-1。过氧亚硝酸盐的产生不影响细胞活力，但降低了线粒体跨膜电位并增加了硫醇氧化。这项有前途的工作进一步加强了流式细胞术作为精子评估技术库中一种有价值的定量工具的使用。事实上，流式细胞术已被用于研究精液中细胞功能的多个方面以及婴儿性别的潜在选择。7、8）。目前的出版物为流式细胞术在男科研究中的应用开辟了新的途径。例如，通过使用额外的线粒体功能探针扩展面板。了解本文中使用的染料的保留如何与线粒体生物量相关会很有趣，线粒体生物量可以通过使用 MitoTracker 的流式细胞术轻松访问 ( 9, 10 )。烟酰胺腺嘌呤二核苷酸 (NADH) 荧光的定量是另一种流式细胞术测量方法，可提供对线粒体复合体 I 功能的洞察 ( 11, 12）。通常，NADH 被认为是细胞自发荧光的重要贡献者。尽管如此，这些富含能量的中间体还是线粒体电子传递链的电子源之一，可维持跨膜电位。线粒体质量和其他线粒体功能的变化可能是评估精母细胞的额外重要参数（13）。新描述的结合四种探针的测定需要在临床试验中进一步验证，以研究其在选择比经典游泳方法更具功能性的精子亚群方面的功效。以绝对单位而不是任意平均荧光强度值表示荧光值也可以促进测定的标准化，并最终促进其在临床医学中的常规使用。当前工作的影响超出了男科学。亚硝化应激与许多其他疾病有关，例如癌症、动脉粥样硬化、炎症和神经退行性疾病。因此，这里描述的流式细胞术技术的利用也可能有利于这些领域的研究。随着线粒体获得增加对其在健康和疾病中的作用的兴趣，