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.

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Oxidative stress and male infertility—a clinical perspective

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2000.tb03273.x

DNA Integrity in Human Spermatozoa: Relationships With Semen Quality

Evolutionary theory is firmly grounded on the existence of trade-offs between life-history traits, and recent interest has centred on the physiological mechanisms underlying such trade-offs. Several branches of evolutionary biology, particularly those focusing on ageing, immunological and sexual selection theory, have implicated reactive oxygen species (ROS) as profound evolutionary players. ROS are a highly reactive group of oxygen-containing molecules, generated as common by-products of vital oxidative enzyme complexes. Both animals and plants appear to intentionally harness ROS for use as molecular messengers to fulfil a wide range of essential biological processes. However, at high levels, ROS are known to exert very damaging effects through oxidative stress. For these reasons, ROS have been suggested to be important mediators of the cost of reproduction, and of trade-offs between metabolic rate and lifespan, and between immunity, sexual ornamentation and sperm quality. In this review, we integrate the above suggestions into one life-history framework, and review the evidence in support of the contention that ROS production will constitute a primary and universal constraint in life-history evolution.

https://royalsocietypublishing.org/doi/pdf/10.1098/rspb.2008.1791

Reactive oxygen species as universal constraints in life-history evolution

Extensive research in our center at the Cleveland Clinic indicates that the seminal oxidative stress test has diagnostic and prognostic capabilities beyond those of conventional tests of sperm quality or functions. An oxidative stress test can accurately discriminate between fertile and infertile men and identify patients with a clinical diagnosis of male-factor infertility who are likely to initiate a pregnancy if they are followed over a period of time. In addition, the test can help select subgroups of patients with infertility in which oxidative stress is a significant factor, and who may benefit from antioxidant supplementation. Incorporation of such a test into routine andrology laboratory practice may be of particular importance to the future management of male infertility. In recent years, the generation of reactive oxygen species (ROS) in the male reproductive tract has become a real concern because of their potential toxic effects at high levels on sperm quality and function. ROS are highly reactive oxidizing agents belonging to the class of free radicals (Aitken, 1994). A free radical is defined as ‘‘any atom or molecule that possesses one or more unpaired electrons’’ (Warren et al, 1987). Recent reports have indicated that high levels of ROS are detected in semen samples of 25% to 40% of infertile men (de Lamirande et al, 1995; Padron et al, 1997). However, a strong body of evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities (Aitken, 1999). Spermatozoa, like all cells living in aerobic conditions, constantly face the oxygen (O2) paradox: O2 is required to support life, but its metabolites such as ROS can modify cell functions, endanger cell survival, or both (de Lamirande and Gagnon, 1995). Hence, ROS must be continuously inactivated to keep only a small

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02324.x

Oxidative stress and male infertility: from research bench to clinical practice.

Although the importance of seminal plasma in the protection of spermatozoa against reactive oxygen species is well known, only a few studies have investigated its antioxidative properties and the possible relationship between infertility and plasmatic antioxidant defences. The aim of the present study was to assess the status of the total non-enzymatic antioxidant defences of human seminal plasma. Semen samples were obtained from 101 patients consulting for infertility and 15 fertile donors. A total reactive antioxidant potential (TRAP) in seminal plasma was determined by luminol-chemiluminescence. The relationship of seminal TRAP values to lipid peroxidation was also evaluated. Our results show that semen samples from fertile controls show total antioxidant capacity at higher frequency and value than equivalent samples from suspected subfertile men. This fact as well as the inverse relationship observed between antioxidant capacity and lipid peroxidation potential strongly suggest that impaired antioxidant defences may play a role in infertile disorders.

J. Escobar

Papers

Total antioxidant capacity of human seminal plasma.