WHO laboratory manual for the examination and processing of human semen

Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the bodies own natural antioxidant defenses, resulting in cellular damage. Oxidative stress is a common pathology seen in approximately half of all infertile men. ROS, defined as including oxygen ions, free radicals and peroxides are generated by sperm and seminal leukocytes within semen and produce infertility by two key mechanisms. First, they damage the sperm membrane, decreasing sperm motility and its ability to fuse with the oocyte. Second, ROS can alter the sperm DNA, resulting in the passage of defective paternal DNA on to the conceptus. This review will provide an overview of oxidative biochemistry related to sperm health and will identify which men are most at risk of oxidative infertility. Finally, the review will outline methods available for diagnosing oxidative stress and the various treatments available.

/pdf/oxidative-stress-and-male-infertility-a-clinical-perspective-2nu3kdi0jn.pdf

Oxidative stress and male infertility—a clinical perspective

Recent studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species and that this activity is significantly accelerated in cases of defective sperm function. In view of the pivotal role played by lipid peroxidation in mediating free radical damage to cells, we have examined the relationships between reactive oxygen species production, lipid peroxidation, and the functional competence of human spermatozoa. Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion, which could be reversed by the inclusion of a chain-breaking antioxidant, alpha-tocopherol. Low levels of lipid peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to ionophore, without influencing the steady-state activity. At higher levels of lipid peroxidation, both the basal level of reactive oxygen species production and the response to A23187 were significantly diminished. In contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by alpha-tocopherol. These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the reactive oxygen species generated by human spermatozoa in mediating sperm-zona interaction.

Generation of reactive oxygen species, lipid peroxidation, and human sperm function.

Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.

/pdf/cellular-basis-of-defective-sperm-function-and-its-zab3dedi9x.pdf

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.

Spontaneous lipid peroxidation in washed human spermatozoa was induced by aerobic incubation at 32 C and measured by malonaldehyde production; loss of motility during the incubation was determined simultaneously. Malonaldehyde production at the point of complete loss of motility, defined as the lipoperoxidative lethal endpoint (LLE), was 0.10 +/- 0.03 nmol/10(8) cells (mean +/- SD, n = 40), and was independent of the time to complete loss of motility. Human spermatozoa produced both H2O2 and O2-. during aerobic incubation. Inhibition of superoxide dismutase in these cells with KCN showed that all the H2O2 production is due to action of the dismutase. The superoxide dismutase activity of individual human sperm samples varied between 1 and 10 U/10(8) cells, variations between samples from a single donor being nearly as great as those between different donors. The time to complete motility loss (tL) showed equal variation of 1 to 10 hours among samples. The rate of spontaneous lipid peroxidation, calculated as LLE/tL, for a given sperm sample and the superoxide dismutase activity of the same sample, determined prior to aerobic incubation, gave a good linear correlation (r = 0.97). Glutathione reductase, glutathione peroxidase, and glutathione were found to be present in human spermatozoa, but showed little variation among samples. These results suggest that superoxide dismutase plays the major role in protecting human spermatozoa against lipid peroxidation. In addition, the superoxide dismutase activity of a fresh sperm sample appears to be a good predictor of the lifetime (up to the complete loss of motility) of that particular sample, and so may prove useful in semen analysis.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1987.tb00973.x

Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.

Peroxidative Breakdown of Phospholipids in Human Spermatozoa, Spermicidal Properties of Fatty Acid Peroxides, and Protective Action of Seminal Plasma

Peroxidation of the sperm cells' own phospholipids, closely bound up with declining motility, has been demonstrated in human spermatozoa; the rate of peroxidation was determined quantitatively by the reaction with thiobarbituric acid. Immotile or poorly motile spermatozoa from necrospermic or oligospermic semen exhibited a higher rate of peroxidation than highly motile spermatozoa from normal ejaculates. Peroxidized unsaturated fatty acids added to washed sperm suspensions immobilized the spermatozoa rapidly and permanently. Human seminal plasma was found to contain a non-ultrafiltrable factor which effectively prevented, but did not reverse, the toxic effect upon spermatozoa of either endogenous or exogenous lipid peroxides.

Adverse effects of peroxidized lipid on human spermatozoa

Anaerobic fructolysis was studied in human spermatozoa from 1) normal, 2) oligospermic, and 3) necrospermic semen, as well as 4) in normal spermatozoa irreversibly immobilized with a spermicidal agent (lipid peroxide); the rate of fructolysis was determined by measuring the amount of L-(+)-lactic acid produced during anaerobic incubation in fructose-supplemented sperm suspensions with identical concentrations of spermatozoa and fructose. Motile spermatozoa from normal semen produced lactic acid at a steady rate for at least 2 hours at 37 C, but when immobilized with peroxidized linoleic acid they lost rapidly and irreversibly all fructolytic ability. There was no substantial difference in the rates of anaerobic fructolysis between sperm suspensions prepared 1) from six individual specimens of normal semen and 2) from pooled ejaculates of ten oligospermic patients. In three of a group of four infertile men diagnosed as necrospermic, the immotile spermatozoa failed to produce lactic acid from fructose. In the fourth individual, the spermatozoa, although immotile at the time of testing, were able to convert fructose to lactic acid, but at a reduced rate; this patient's semen has been examined periodically over the last three years and has contained mostly immotile spermatozoa, but a few times motility was definitely observed, especially after treatment with caffeine. The authors conclude from their results that necrospermia may be associated with diverse metabolic defects, one of them being loss of fructolytic ability by human spermatozoa.

Fructolysis in Human Spermatozoa Under Normal and Pathological Conditions

In agreement with earlier studies, we have found that the human seminal plasma yields considerably higher values for cyclic nucleotides when compared with spermatozoa. However, we have also shown that these values vary markedly both between different individuals and in specimens from the same individual at different times. Levels recorded for seminal plasma were 25 to 18, 937 nM of cyclic AMP and 0.54 to 3.57 nM of cyclic GMP. These levels were unrelated to those of either fructose (from the seminal vesicles) or zinc and citric acid (from the prostate). We have demonstrated also that in most specimens of seminal plasma the level of cyclic AMP remained stable during a 1-hour incubation at 37 C or during prolonged storage at −40 C. Washed suspensions contained 13.2 to 27.2 pmol cyclic AMP/100 million spermatozoa. Adenylate cyclase activity of human semen was confined to the spermatozoa; it was several times higher in the presence of Mn2+ than Mg2+ and was unresponsive to sodium fluoride. The latter findings are consistent with enzyme activity derived from a dissociated catalytic component of adenylate cyclase.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1981.tb00624.x

Roy Jones

Papers

Peroxidative Breakdown of Phospholipids in Human Spermatozoa, Spermicidal Properties of Fatty Acid Peroxides, and Protective Action of Seminal Plasma

Adverse effects of peroxidized lipid on human spermatozoa

Fructolysis in Human Spermatozoa Under Normal and Pathological Conditions

Observations on Cyclic Nucleotides in Human Semen