[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


"Oxidative stress" is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen-derived oxidants commonly known as reactive oxygen species (5). Reactive oxygen species (ROS ) have been implicated in over a hundred of disease states which range from arthritis and connective tissue disorders to carcinogenesis, aging, toxin exposure, physical injury, infection, and acquired immunodeficiency syndrome (6). The role of oxidative stress in infertility and methods for counteracting its impact on reproductive tissues with antioxidants is still in its infancy.

4.1. Reactive oxygen species and oxidative stress

ROS are highly reactive oxidizing agents belonging to the class of free-radicals. A free radical is any compound (not necessarily derived from oxygen) which contains one or more unpaired electrons. The most common ROS that have potential implications in reproductive biology include superoxide (O2-). anion, hydrogen peroxide (H2O2), peroxyl (ROO-). radicals, and the very reactive hydroxyl (OH-). radicals. The nitrogen-derived free radical nitric oxide (NO.) and peroxynitrite anion (ONOO-) also appear to play a significant role in the reproduction and fertilization. The ultimate effects of (NO.)depend upon its concentration and interactions with hydrogen peroxide. Peroxynitrite (oxoperoxonitrate) anion may be formed in vivo from superoxide and nitric oxide and actively reacts with glutathione, cysteine, deoxyribose, and other thiols/thioethers (7). This can form a strongly nitrating species in the presence of metal ions or complexes.

The assumption that free radicals can influence male fertility has received substantial scientific support (8). The proposed mechanism for loss of sperm function upon oxidative stress has been shown to involve excessive generation of ROS (9). The H2O2 has both beneficial and damaging effects on sperm and thus can influence the fertilization process. Hence, free radicals and ROS are associated with oxidative stress and are likely to play a number of significant and diverse roles in reproduction. Basic and clinical research on the involvement of ROS and antioxidants in maintaining normal sperm function is very much warranted.

4.2. Spermatozoa and leukocytes as sites of ROS production

Presence of leukocytes (predominantly granulocytes) in semen has been associated with severe male factor infertility cases (10,11). There has been much speculation as to whether the origin of ROS in semen is from spermatozoa or from infiltrating leukocytes (12,13). Iwasaki and Gagnon reported that the leukocyte free percoll fractions of semen samples obtained from non-azoospermic infertile men generate detectable levels of ROS when compared to the semen of normal and azoospermic men suggesting that damaged spermatozoa are likely to be the source of ROS (14). Also, higher levels of ROS were correlated with a decreased number of motile sperm; conversely, greater sperm motility was observed in samples with lesser amounts of detectable ROS (14). It is important for the clinician to recognize that assisted reproductive techniques (percoll gradients/sperm washing/centrifugation) may induce damage to spermatozoa by either inadvertently removing the scavenging capability of seminal plasma or by increasing ROS generation by spermatozoa (9).

4.3. Leukocytospermia and oxidative stress

The exact site of origin of these leukocytes in semen, their mode of action, and the role that bacteria, viruses and subsequent genitourinary-inflammation might have on sperm function are not clear. Experimentally, ROS production by human spermatozoa and contaminating leukocytes can be stimulated by phorbol esters and certain formyl peptides with deleterious effects on sperm motility and fertilization (13). Although the presence of leukocytes in semen did not diminish the in vitro fertilizing capacity of spermatozoa, the introduction of leukocytes into washed sperm preparations did reduce sperm function by the production of ROS (15). This finding seems paradoxic but does indicate that seminal plasma has significant antioxidant or ROS scavenging capacity which may prevent sperm damage by leukocytes.

An association between leukocytospermia and ROS has been recently found to correlate with increased chemokine (IL-8), and decreased SOD activity of the semen (16). This demonstrates that increased oxidative stress during leukocytospermia is caused by a defective ROS scavenging system which, in turn, can be modulated by certain proinflammatory cytokines. A significant shift towards increased production of proinflammatory chemokine (GRO-alpha) compared to anti-inflammatory cytokine (IL-10) during leukocytospermia suggests an active chemotactic pro-inflammatory response (17). This shift may be responsible for a significant oxidative stress to spermatozoa due to leukocytes in the semen of the infertile patient (5). Based upon these observations, it may be useful to assess the oxidative stress status (OSS) of semen in infertile or subfertile patients, particularly those with chronic genitourinary inflammation.

4.4 Oxidative stress and sperm function

Theoretically, cellular damage in the semen is the result of an improper balance between ROS generation and scavenging activities. The scavenging potential in gonads and seminal fluid is normally maintained by adequate levels of antioxidants superoxide dismutase (SOD), catalase, and probably glutathione (GSH) peroxidase and reductase (5). This balance can be referred to as oxidative stress status (OSS) and its assessment may play a critical role in monitoring sperm damage and infertility (Fig 1).

Figure 1: Scheme suggesting interacting mechanisms in the role of oxidative stress and antioxidants affecting sperm function and fertility. (The key words are in bold and are italicised. See text for further details).

A situation in which there is a shift in this ROS balance towards pro-oxidants, because of either excess ROS or diminished anti-oxidants, can be classified in terms of positive oxidative stress status (OSS). At present, there is no true ROS detection method available which will evaluate this balance. However, assessment of OSS, or a similar paradigm when monitored more objectively, would be a good indicator of sperm damage caused by oxidative stress (5). Chronic asymptomatic genitourinary inflammation can be regarded as a condition with positive OSS, which may be the real cause of idiopathic infertility in such patients. Superoxide dismutase (SOD) may directly act as antioxidant enzymes involved in the inhibition of sperm LPO (18). A high GSH/GSSG ratio will help spermatozoa to combat oxidative insult (19). It seems that the role of these biological antioxidants and their associated mechanisms is an important area for further investigation in the treatment of infertility. Though the therapeutic use of antioxidants appears attractive, until proper multicenter clinical trials have been completed, clinicians need to be aware of exaggerated antifertility claims in various commercial antioxidants.

Nitric oxide radical (NO.) and reactive nitrogen species (RNS) have recently been found to have biological roles in inflammation and in mediating many cytotoxic and pathological events (20). Synthesis of NO. in response to infection and inflammation could contribute to poor sperm motility and function and may lead to infertility (21). RNS (e.g., NO.) like ROS, may normally be useful for maintaining sperm motility but can be toxic in excess (22). Other RNS such as nitrogen dioxide (NO2.) radical and peroxynitrite (ONOO-) anion are considered to be damaging. The primary mechanism of nitric oxide-induced sperm damage is likely to be inhibition of mitochondrial respiration and DNA synthesis (23). Nitric oxide-induced toxicity is also mediated indirectly through its interaction with superoxide anions and formation of peroxynitrite anion, which when protonated, decomposes to form OH- and NO2, both of which are cytotoxic agents (24).

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