Table 2.

Pros and cons of direct tests used to measure oxidative stress levels in the seminal plasma.

Assay	Description	Pros	Cons	Reference
Chemiluminescence assay	The emission of electromagnetic radiation brought on by a chemical process that results in the production of light is known as chemiluminescence (CL). Chemiluminescence immunoassay (CLIA) is a test that combines immunochemical responses with the chemiluminescence method. CLIA uses chemical probes that can label the antibody by a chemical reaction, much as other labeled immunoassays (RIA, FIA, and ELISA).	Extremely specific and responsive	Constrained in time Large, high-maintenance, and expensive machinery. Requires a significant sample volume. Does not allow for the measurement of reactive oxygen species in samples that are frozen, gelatinous, or azoospermia. Changes in pH, centrifugation, and the presence of NADPH, cysteine, ascorbic acid, or uric acid may affect the results.	Agarwal et al. (2015), Li et al. (2016)
Cytochrome c reduction test	This test gauges how much cytochrome c is reduced by NADPH-cytochrome c reductase when NADPH is present. Cytochrome C’s oxidation/reduction status affects its absorption spectrum. At 550 nm, a significant absorption peak is seen after reduction.	It is effective for measuring a high level of reactive oxygen production and can quantify the amount of oxygen released by neutrophils during their respiratory burst.	Ferricytochrome c can be reduced by electron transfer from enzymes and other molecules The change in absorbance for O2• is unclear. In circumstances where the enzyme activity is limited, it is impossible to identify the presence of NADPH oxidase activity. As a result of the enzyme’s lack of access to intracellular spaces, only the quantity of reactive oxygen species present outside of the cell can be determined	Melendez-Ferro et al. (2013)
Nitroblue tetrazolium (NBT)	The nitroblue tetrazolium measures intracellular ROS level as well as give insight to the potential source of OS with the aid of a light microscope. NBT is converted into a blue pigment (diformazin) following its interaction with the superoxide released from the leukocytes or spermatozoa. The concentration of diformazin is positively correlated with the concentration of intracellular ROS.	Always on the market, less expensive, and very effective Give specifics regarding the distinct roles that damaged spermatozoa and leukocytes play in the production of reactive oxygen species. Identifies intracellular ROS in a diverse population of cells	The levels of NBT reduction are influenced by variations in the cellular material of various oxidation–reduction Due to the assay’s focus on the reduction of NBT, there are discrepancies in the detection of reactive oxygen species.	Tvrdá et al. (2011), Agarwal & Majzoub (2017)
Oxidation–reduction potential (ORP)	The interaction between oxidants and antioxidants is measured by ORP, which offers a thorough assessment of oxidative stress. Higher ORP readings distinguish the level of oxidative stress caused by an imbalance in the activity of oxidants and antioxidants. reproductive issues caused by men	Simple method that takes less time. Favors both fresh and frozen samples without being altered or treated in any way. Results remain stable for up to 2 h. It can isolate sperm samples based on seminal parameters or fertility status. It can quantify the number of antioxidants and oxidants in a sample.	Viscous samples may affect the reading. Azoospermia samples are not supported.	Agarwal et al. (2017)
Electron spin resonance	A spectroscopic method called electron spin resonance (ESR) uses a static magnetic field to detect transitions between the energy levels of electron spins caused by electromagnetic radiation.	Oxidation of hydroxyl spin probes in hydroxylamine spin results in the formation of extremely stable nitroxide with a half-life of several hours. Spin trap distinguishes between various oxidative molecules.	Basic analyses in hydroxylamine spin are partially inactive at biological pH. In both methods, the scavenging action of antioxidants may hinder the formation of adducts.	Kopáni et al. (2006)