Key Points

• We still do not have the perfect method to measure ROS generation in human sperm suspensions.

• Methods that target the entire sperm suspension such as chemiluminescence or NBT reduction are sensitive but are only semiquantitative, difficult to calibrate, easily distorted by the activity of cellular oxidoreductases, and too easily dominated by the presence of leukocytes.

• If measures are taken to either quantify the degree of leukocyte contamination or to purify the spermatozoa to the point that leukocytes are no longer present, then these forms of measurement are valuable and of diagnostic significance.

• Flow cytometry may be used to measure the fluorescence of probes that are responsive to the presence of specific ROS. Both DHE and MSR have been validated for the generation of O2-' in different compartments of the spermatozoon, but the ROS responsible for DCFH fluorescence still await resolution.

• The diagnostic application of the DHE and MSR assays requires the simultaneous determination of cell viability.

• The advantage of flow cytometry is that the analysis can be focused directly on the spermatozoa and that acceptably large numbers of cells can be analyzed with ease.

• The major problem with these probes is that they require relatively sophisticated, expensive hardware in the form of flow cytometers and the results do not quantify the target ROS but simply indicate that the percentage of cells are particularly active in their creation. Nevertheless, at present they represent the methods of choice for assessing ROS production by human spermatozoa.

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