Abstract
As a natural by-product of mitochondrial respiration, reactive oxygen species (ROS) in sperm play a role in promoting fertilization, by intervening in a series of events. Nevertheless, an abnormal and uncounteracted increase in ROS production leads to oxidative stress (OS) which can, ultimately, culminate in cell death. An established relationship between OS and male infertility highlights the importance of an accurate detection method for ROS content that can be easily implemented and reproduced in any andrology lab. More recently, reactive nitrogen species (RNS) production and subsequent nitrosative stress have also been described. Here we describe the use of fluorescent probes, including some that targeted to the mitochondria due to the coupling of a cation (TPP+), in order to assess the levels of different ROS and RNS in human sperm using flow cytometry and/or fluorescent microscopy. This methodology is user friendly and accurate and can be safely applied in research- and/or clinical-based contexts.