[Frontiers in Bioscience 1, e78-86, August 1,1996]


Suresh C. Sikka, Ph.D., HCLD

Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana, USA

Received 05/30/96; Accepted 07/02/96; On-line 08/01/96


In many complex biological systems including semen, the true ROS status leading to oxidative stress reflects a relative balance between the ROS-generated and ROS-scavenged. The measurement of the rate of ROS generation by luminol induced chemiluminescence has been the most common method for quantitating ROS. Although this rate measurement is dynamic, it may not accurately reflect the status of potential sperm damaging ROS. For such evaluations, the amount of ROS-detected, rather than the ROS-generated will represent a more physiological assessment of oxidative stress (5). The methods commonly used for measuring ROS can be categorized into: (a) reactions involving nitroblue tetrazolium (NBT) or cytochrome c-Fe3+ complexes which measure ROS on the cell membrane surface, (b) reactions that measure ROS (generated inside or outside the cell) utilizing luminol-dependent chemiluminescence, and (c) the electron spin resonance methods which are more sensitive and can identify the type of ROS generated inside the cell but require skillful operation, accurate interpretations, and expensive instrumentation.

To further study their mode of action on human spermatozoa, ROS can be artificially generated under defined experimental conditions. The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O2-). anion and hydrogen peroxide (H2O2), respectively. In the presence of ferric ions, these radicals further generate the highly reactive hydroxyl radical (OH.) which is especially deleterious to spermatozoa.

xanthine oxidase
Xanthine-hypoxanthine-------------------------------->uric acid + O2.- + H2O2
O2.- + H2O2-------------------------------->OH. + OH- + O2

Electrolysis of physiological buffer under defined conditions also generates ROS which can damage sperm motion (41). Selective modifications of these defined conditions can identify: (a) the free radicals involved, (b) their mode of action on spermatozoa, and (c) the evaluation of selective protective mechanisms.

9.1. Oxidative stress status (OSS) evaluation

The balance of ROS can be termed as the "balance of creation and destruction". Under normal circumstances, there is an appropriate balance between pro-oxidants and anti-oxidants. A shift in the levels of ROS towards pro-oxidants in semen and vaginal secretions can induce an oxidative stress on spermatozoa. Concomitantly, a decrease in antioxidant activities (e.g., SOD, catalase, glutathione peroxidase and reductase, GSH) in semen correlates with idiopathic infertility (8). It is possible that an increased rate of ROS production (suggesting high oxidative stress) may inhibit the action of these antioxidant enzymes, or alternatively the inherent decreased expression of these antioxidant enzymes may cause increased oxidative stress (5). This will result in increased LPO, decreased sperm motility, viability and function, and ultimately leads to infertility (Fig 1).

Assessment of the rate of ROS production/generation using luminol as a probe can be a dynamic measure of oxidative stress (9). However, clinically the evaluation of this ROS generation is limited by a very short half life of these free radicals (12). The potential methods that can be developed for evaluation of OSS may utilize measurement of an oxidized component that remains in the body fluids (e.g., TBA reactive substances; GSH/GSSG balance; the levels of unaltered tocopherol or ascorbate). Although there have been concerns about the specificity, interference, and reliability of measuring TBA-MDA activity as an indicator of LPO, this test remains one of the most efficacious methods for assessing the oxidative damage to sperm (18). Eventually, this TBA-MDA measurement will need to be combined with other assays which would be able to measure the rate of ROS production and antioxidant protection for the overall assessment of OSS in infertility. Measurement of IL-8, for example, when combined with assessment of SOD or other antioxidants in infertile patients with leukocytospermia will indicate a positive OSS in this population and can be treated accordingly (16). Thus, it would be important to assess OSS either in the semen in the male or the vaginal fluids in the female before, during, and after any clinical studies. This would be indicative that an individual with low OSS does not contribute to the infertility. If a positive correlation is observed between OSS and the outcome of the trial, a predictive value could be determined.

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