Table 3.

The advantages and disadvantages of indirect tests used to measure oxidative stress levels in the seminal plasma.

Assay	Description	Advantages	Disadvantages	Reference
Myeloperoxidase	Myeloperoxidase, which is found in the leukocyte granules, converts substrates from an insoluble blue/brown derivative to a colorless form in the presence of hydrogen peroxide. As substrates, benzidine, 3,3′-diaminobenzidine, or p-phenylenediamine dihydrochloride are used.	Quick, simple, and inexpensive to operate Suggested by WHO for the purpose of examining leukocytospermia in sperm samples >1 × 106 peroxidase positive WBC/mL of semen (leukocytospermia)	PMNs and macrophages, both peroxidase-positive leukocytes, make up between 50 and 60% of all seminal leukocytes. Unable to determine whether spermatozoa produce ROS.	Shekarriz et al. (1995)
Total antioxidant capacity	Antioxidant capacity (AC) provides an integrated metric rather than the mere total of measured antioxidants by taking into account the cumulative activity of all antioxidants present in plasma and bodily fluids.	Automatically measures the total antioxidant content of seminal plasma. Recommended cut off ≥1950 µM Trolox significative of good antioxidant reserves	Makes use of a costly assay kit and microplate reader	Robert et al. (2021)
HNE-HIS adduct ELISA	The protein-HNE adduct ELISA is a technique for detecting HNE bound to proteins, which is thought to be the type of HNE occurrence in biological systems	Fast	Cross-reactivity	Agarwal et al. (2017)
Malondialdehyde assay	This assay is based on the formation of the MDA-TBA2 adduct, a strong absorber at 532 nm, by the interaction of malondialdehyde (MDA) with thiobarbituric acid (TBA). The most widely used technique for calculating MDA in biological samples is this reaction.	Easy to use, Capable of determining lipid peroxidation, Able to identify the MDA-TBA adduct using either fluoroscopy or colorimetry	Expensive materials Thorough controls required Generic for MDA	Khoubnasabjafari et al. (2015)
DNA fragmentation	TUNEL is a technique for identifying apoptotic DNA fragmentation that is frequently used to recognize and measure apoptotic cells or to find cells that have excessively broken DNA. The test depends on the enzyme terminal deoxynucleotidyl transferase (TdT), which is used to attach deoxynucleotides that have been dyed or otherwise marked to the 3'-hydroxyl termini of DNA double-strand breaks. Additionally, it may identify cells whose DNA has been damaged in ways beyond apoptosis.	Different methods are available (TUNEL, SCSA, Comet, SCD, and 8-OHdG) Comet assay is easy to carry out, flexible, sensitive, and rapid assay and has shown some similarities with other assays like the SCSA and TUNEL SCSA and TUNEL using a flow cytometry is robust and sensitive assay TUNEL assay is very direct in terms of measuring single- and double-strand DNA breakage	Lack of standardized reference value The 8-OHdG method may itself trigger DNA oxidation tempering with basal level Cost is a major concern in TUNEL and SCSA assays	Homa et al. (2019)
Protein alterations ASSA	Determine the quantity or concentration of a particular protein or a variety of distinct proteins in a sample using a protein test. Many clinical and scientific procedures include the isolation and detection of proteins.	Incredibly precise and sensitive Western blot, ELISA, or immunochemistry can be used to verify certain proteins. It is possible to identify changes in sperm protein that are specific to male infertility conditions.	It takes a long time and requires expensive instruments.	Agarwal et al. (2017)

8-OHdG, 8-hydroxy-2-deoxyguanosine; ELISA, enzyme linked immunosorbent assay; MDA, malondialdehyde; OS, oxidative stress; PMN, polymorphonuclear neutrophils; ROS, reactive oxygen species; SCD, sperm chromatin dispersion; SCSA, sperm chromatin structure assay; TBA, thiobarbituric acid; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WBC, white blood cell; WHO, World Health Organization.