Direct Laboratory Assessment of Sperm Oxidative Stress

A full description of the assays used to directly measure sperm oxidative stress is outlined in Chaps. 13 and 14. At present, there are over 30 such assays, but only two are in common usage [6]. One of the oldest and most widely used methods of assessing sperm membrane peroxidation is the measurement of MDA levels in sperm or seminal plasma with the thiobarbituric acid assay (TBARS). MDA levels in sperm are quite low and therefore require the use of sensitive HPLC equipment or the use of iron-based promoters and spectrofluorometry measurement. Seminal plasma levels of MDA are five to tenfold higher than sperm, making measurement on standard spectrophotometers possible [6]. Measurement of MDA appears to be of some clinical relevance, since its concentration within both seminal plasma and sperm is elevated in infertile men with excess ROS production, compared to fertile controls or normozoospermic individuals [6, 157]. Furthermore, in vitro impairment of motility, sperm DNA integrity and sperm-oocyte fusion capacity by ROS is accompanied by an increase in MDA concentration [6] .

Chemiluminescence assays using luminol are probably the most commonly described technique to detect ROS production within semen. This probe is very sensitive and has the advantage of relatively well-established reported ranges for both the fertile and infertile population [3-5, 8, 118, 120]. However, its general uptake by clinical andrology laboratories has been hampered by expensive equipment (luminometer) and difficulties with quality control created by assay confound-ers such as incubation time, leukocyte contamination and presence of seminal plasma contamination [8]. Measurement of total antioxidant capacity (TAC) within seminal plasma can also be measured using luminol, with the ability of seminal plasma to inhibit chemiluminescence elicited by a constant source of ROS (horseradish peroxidase) being the most commonly used technique. The TAC is usually quantified against a vitamin E analogue (Trolox) and expressed as a ROS-TAC score [109]. At the present moment the ROS-TAC score appears to be the best-established method for analysing the balance between free radical production (ROS) and anti-oxidant protection (TAC) of sperm. However, since all assays of sperm oxidative stress are relatively expensive compared to routine semen analysis, many treating physicians will continue to avoid directly testing their patients for oxidative stress and instead offer empirical therapy antioxidant treatment which is relatively inexpensive and without side effects.

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