Table 3.

Indirect assays of oxidative stress

Test	Method of measurement	Function	Advantages	Disadvantages
Myeloperoxidase or Endtz test	Peroxidase positivity is assessed through staining using benzidine as a buffer	Detection of granulocytes in semen	Specifically distinguishes WBC’s especially producing granulocytes from immature germ cells in semen	Cannot be used to detect ROS production in spermatozoa
Lipid peroxidation levels	Detection of MDA and toxic 4-HNE through colorimetric and thiobarbituric acid assays	Identification of by-products of lipid peroxidation	MDA is a colored substance that can be measured by fluorometry or spectrophotometry. Low sperm concentration of MDA can be measured through sensitive HPLC equipment or spectrofluorometric measurement of iron-based promoters	Not a widely-used test in clinic practice
MiOXSYs	Assessment of electron transfer in millivolts from a reducing agent to the oxidant using a galvanostat-based system	Measurement of oxidation-reduction potential	Easy to employ in a clinical setting Can be used in patients with low semen volume	Larger cohort studies to establish the reference value are needed
Total antioxidant capacity	Evaluates the reductive ability of the antioxidants within the semen against an oxidative agent such as hydrogen peroxide and measures the effect on the substrate	Assesses the cumulative effect of antioxidants within the semen	Rapid colorimetric method Total antioxidants in seminal plasma can be measured	Does not measure individual or enzymatic antioxidants. Requires expensive assay kit and microplate reader
Gpx activity	The activity of Gpx is measured by the decrease in GSH content after incubating the sample in the presence of H2O2 and NaN3	Based on the principle that Gpx catalyzes the reaction between hydrogen peroxide and reduced GSH	Gpx protects the sperm from lipid peroxidation and the DNA damage can be significant if the Gpx levels are lower	Some studies show that Gpx activity does not correlate with sperm motility or concentration
Comet assay	Allows DNA migration in an agarose gel under an electric field. The loose DNA forms a pattern of migration that resembles a comet	Single-cell gel electrophoresis assay to assess DNA damage	Can detect extent of DNA damage equivalent to 50-single strand breaks per cell Can be employed in men with low sperm concentrations (requires only 100 cells for analysis)	No consensus reached on the standardization protocol

GSH=Glutathione, GPx=GSH peroxidase, 4-HNE=4-hydorxynonenal, MDA=Malondialdehyde, WBC’s=White blood cells, ROS=Reactive oxygen species, HPLC=High-performance liquid chromatography