Evaluation of Human Sperm Quality In Vitro—Purification of Motile Sperm and Subsequent Assessment of Potential Apoptotic Signs Beyond DNA Fragmentation

In our previous studies, OptiPrep and Percoll density gradients separated human motile sperm without DNA fragmentation from immotile sperm with DNA damage. Even in normospermia, over half of the sperm were already immotile, and angle-modulated two-dimensional single-cell pulsed-field gel electrophoresis showed that these were at the end stage of fragmentation. We developed sperm-specific dye- and lectin-exclusion assays to evaluate plasma and acrosomal membranes, mitochondrial endogenous reactive oxygen species, and vacuole negative staining. Comprehensive analyses suggested that they corresponded to sperm that had not yet undergone apoptosis and to those that had undergone apoptotic denaturation. In ICSI, injectable motile sperm that fully meet criteria have an oval-shaped head, intact membranes on both the head and tail, and normal oxidative phosphorylation in cylindrical mitochondria, and they lack vacuoles and DNA damage. Conversely, sperm exhibiting apoptotic signs, such as immotility, plasma membrane damage, and DNA fragmentation, are not injectable. We must establish threshold criteria for injectable sperm; multiple impairments in sperm hinder the study of these issues. The topic of functional impairments in human sperm is too extensive to cover in a single review; for the full scope of the issue, technical guidance for DNA fragmentation analyses is presented in our previous review.