The diagnosis of OS, DNA damage, and apoptosis in the context of male infertility can be made by focusing on the clinical and laboratory presentations that were proven to be associated with these conditions. Additionally, there are specific tests that can be used to identify whether OS, DNA damage, and apoptosis do actually play a role in a specific case of male infertility . There has been no recommendation to implement these tests in routine clinical practice to date. However, there are some specific cases that will definitely benefit from such testing .
Testing for OS should include the assessment of ROS and the total antioxidant capacity (TAC) of seminal plasma since it is the end result of an imbalance between the two. The chemiluminescence assay with luminol and lucigenin has been consistently used to detect ROS levels . This method is the most standardized to date for the assessment of ROS and provides a clear cut-off point as threshold for normality . Flow cytometry has been recently successfully used for the detection of intracellular ROS in samples with low sperm concentration . The seminal TAC can be evaluated using the enhanced chemiluminescence assay. This method is accurate, but colorimetry has been identified as less cumbersome .
Apoptosis and apoptosis-like manifestations can be evaluated in ejaculated spermatozoa using fluorescent staining methods. Using this approach, several markers such as externalization of phosphatidylserine, mitochondrial membrane potential, and caspase activation have been identified in human spermatozoa . Assessment of sperm DNA fragmentation has been an issue of controversy. Several methods are currently available; however, the lack of standardization and cut-off point between normal levels in the average fertile population and the minimal levels of sperm DNA integrity required for achieving pregnancy deprives most of these methods of clinical significance . Currently, there are two methods recognized to be of a clinical benefit. The sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) [21, 119]. Both assays correlate well with fertility as well as with IVF outcomes . Although results from both assays correlate very well, each has a different range for normal values. In addition, these different techniques determine different aspects of sperm DNA damage .
The clinical application of DNA fragmentation testing includes the use of tes-ticular sperm in patients with high levels of sperm DNA fragmentation in semen. In these cases, the use of surgically retrieved testicular sperm in ART may be recommended. In addition to the use of testicular sperm, another strategy that could be used in patients with high levels of sperm DNA fragmentation is the selection of spermatozoa with low levels of DNA damage .
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