464

Journal of Andrology, Vol. 16, No. 6, November/December 1995Copyright © American Society of Andrology

Role of Oxidative Stress andAntioxidants in Male Infertility

SURESH C. SIKKA,MAHADEVAN RAJASEKARAN,AND WAYNE J. 0. HELLSTROM

From the Department of Urology, Tulane University

School of Medicine, New Orleans, Louisiana.

“Oxidative stress” is a condition associated with an in-creased rate of cellular damage induced by oxygen andoxygen-derived oxidants commonly known as reactive

oxygen species (ROS). All living aerobic cells are normallyexposed to a background level of oxidative stress. Variousstress-related conditions, such as chronic disease states,aging, toxin exposure, physical injury, and exposure tomany types of foods, can enhance this oxidative processand cause cell damage. Recently, a plethora of knowledgeon free-radical biology has provoked our collective re-search interests, with emphasis on clinical conditions suchas aging, dietary intake, health awareness, cosmetic use,the pathobiology of disease, and the perceived beneficialeffects of antioxidants. ROS have been implicated in overa hundred disease states, ranging from arthritis and con-nective tissue disorders to carcinogenesis, infection, andacquired immunodeficiency syndrome. In the area of re-production, the role of oxidative stress and its therapywith antioxidants remain in their initial stages and therebywarrant additional exploration. The aim of this minire-view is to further stimulate interest in basic and clinicalresearch on the roles of ROS and antioxidants in repro-duction, particularly the development of newer modes ofdiagnosis and therapy for the infertile couple.

What are ROS and How Do TheyRelate to Ox/dative Stress?

ROS are highly reactive oxidizing agents belonging to theclass of free radicals. A free radical is any compound (notnecessarily derived from oxygen) containing one or moreunpaired electrons. The most common ROS that havepotential implications in reproductive biology include su-peroxide (02 -) anion, hydrogen peroxide (H202), peroxyl(R00) radicals, and the very reactive hydroxyl (OW)radicals.

Correspondence to: Dr. Suresh C. Sikka, Urology Research Laboratory,SL42, Tulane University School of Medicine, 1430 Tulane Avenue, New

Orleans, Louisiana 70112-2699.Received for publication August 9, 1995; accepted for publication

September 1, 1995.

Minireview

The assumption that free radicals can influence malefertility has received substantial scientific support (seeGagnon et al, 1991, for review). The proposed mechanismfor loss of sperm function due to oxidative stress has beenshown to involve excessive generation of ROS (Aitkenand Clarkson, 1987). H202 has both beneficial and dam-aging effects on sperm and thus can influence the fertil-ization process. The nitrogen-derived free radical nitricoxide (NO-) also appears to play a significant role in re-production and fertilization. The ultimate effects of NOdepend upon its concentration and interactions with H202.Hence, free radicals and ROS are associated with oxi-dative stress and likely play a number of significant anddiverse roles in reproduction.

Spermatozoa and ROS

There had been much speculation as to whether the originof ROS in semen is from spermatozoa or from infiltratingleukocytes (Kessopoulou et al, 1992; Krausz et al, 1994).Iwasaki and Gagnon (1992) reported that the leukocyte-free Percoll fractions of semen samples obtained fromnon-azoospermic infertile men generate detectable levelsof ROS when compared to the semen of normal andazoospermic men, suggesting that damaged spermatozoaare likely to be the source of ROS. Also, higher levels ofROS were correlated with a decreased number of motilesperm; conversely, greater sperm motility was observedin samples with lesser amounts of detectable ROS.

It is important for the clinician to recognize that assistedreproductive techniques (Percoll gradients/sperm wash-ing/centrifugation) may induce damage to spermatozoaby either inadvertently removing the scavenging capabil-ity of seminal plasma or by increasing ROS generationby spermatozoa (Aitken and Clarkson, 1987). Cellulardamage is theoretically the result of an improper balancebetween ROS generation and scavenging activities. Thescavenging potential of spermatozoa is normally main-tained by adequate levels of superoxide dismutase (SOD),catalase, and probably glutathione (GSH) peroxidase andreductase in the seminal plasma.

Leukocytospermia and ROS

The presence of leukocytes (predominantly granulocytes)in semen has been associated with severe male factorinfertility cases. The exact site of origin of these leukocytesin semen, their mode of action, and the roles that bacteria,viruses, and subsequent genitourinary inflammation might

Sikka et al . Oxidative Stress and Infertility 465

FIG. 1. Scheme suggesting, interacting mechanisms In the role ofoxidative stress and antioxidants in male infertility.

have on sperm function are not clear. Experimentally,ROS production by human spermatozoa and contami-nating leukocytes can be stimulated by phorbol esters andcertain formyl peptides with deleterious effects on spermmotility and fertilization (Krausz et al, 1994). Althoughthe presence of leukocytes in semen did not diminish thein vitro fertilizing capacity of spermatozoa, the introduc-tion of leukocytes into washed sperm preparations didreduce sperm function by the production of ROS (Aitkenet al, 1994). This finding seems paradoxical but does in-dicate that seminal plasma has significant antioxidant orROS scavenging capacity that may prevent sperm damageby leukocytes.

An association between leukocytospermia and ROS hasbeen recently found to correlate with increased chemokine(interleukin 8; IL-8), and decreased SOD activity (Raja-sekaran et al, 1995). This demonstrates that increasedoxidative stress during leukocytospermia is caused by adefective ROS scavenging system that can in turn be mod-ulated by certain proinflammatory cytokines. Based uponthese observations, it may be useful to assess the oxidativestress status (OSS) in infertile or subfertile patients, par-ticularly those with chronic genitourinary inflammation.In this context it would also be worthwhile to exploreROS production and the presence of potential scavengersin the vaginal secretions and cervical mucus of the femalepartner of infertile couples.

Detection of ROS

In many complex biological systems, including semen,the true ROS status reflects a relative balance betweenROS generated and ROS scavenged. The measurementof the rate of ROS generation by luminol-induced che-miluminescence has been the most common method toquantitate ROS. Although this rate measurement is a dy-namic system, it may not accurately represent the poten-tial sperm-damaging ROS status. For such evaluations,the amount of ROS detected instead of ROS generatedwill represent a more physiological assessment of oxida-tive stress (Gagnon, 1992). The methods commonly usedfpr measuring ROS can be categorized into 1) reactionsinvolving nitroblue tetrazolium (NBT) or cytochromec-Fe3 complexes that measure ROS on the cell mem-brane surface; 2) those that measure ROS (generated in-side or outside the cell) utilizing luminol-dependent che-miluminescence; and 3) electron spin resonance methods,which are more sensitive and can identify the type of ROSgenerated inside the cell but require skillful operation,accurate interpretations, and expensive instrumentation.

Artificially Induced ROS andSperm Function

ROS can be artificially generated under defined experi-mental conditions in order to further study their mode of

action on human spermatozoa. The reaction between xan-thine and xanthine oxidase results in the univalent anddivalent reduction of dioxygen to generate superoxide(O2) and hydrogen peroxide (H202), respectively. In thepresence of ferric ions, these radicals further generate thehighly reactive hydroxyl radical (OH-), which is especiallydeleterious to spermatozoa.

xanthinco,,idasc

xanthine-hypoxanthine -p uric acid + O2 + H202

Fe3

O2 + H2O2-’OH- + 0H + 02.

Electrolysis of physiological buffer under defined condi-tions also generates ROS, which can damage sperm mo-tion (Rajasekaran et al, 1994). Selective modifications ofthese defined conditions can identify 1) the free radicalsinvolved, 2) their mode of action on spermatozoa, and 3)the evaluation of selective protective mechanisms.

Mode of Action of ROS

Mammalian spermatozoa are rich in polyunsaturatedfatty acids and are thus very susceptible to ROS attack,resulting in a decrease in sperm motility, presumably bya rapid loss of intracellular ATP, causing axonemal dam-age (de Lamirande and Gagnon, 1992), a decrease in spermviability, and an increase in midpiece morphology defects,with deleterious effects on sperm capacitation and theacrosome reaction. Lipid peroxidation of sperm mem-brane is considered to be the key mechanism of this ROS-induced sperm damage (Alvarez et al, 1987) leading toinfertility (Fig. 1).

Lipid peroxidation of Spermatozoa-Lipid peroxida-

tion (LPO) is the most extensively studied manifestationof oxygen activation in biology. The most common typesof LPO are 1) nonenzymatic membrane LPO and 2) en-zymatic (NADPH- and ADP-dependent) LPO. The en-zymatic reaction involves NADPH-cytochrome P-450

466 Journal of Andrology . November/December 1995

reductase and proceeds via an ADP-Fe3O2 (perferryl)complex. In spermatozoa, production of malondialde-hyde (MDA)-an end product of LPO induced by ferrousion promoters-has been reported by Alvarez et al (1987).This MDA formation can be assayed by the thiobarbituricacid (TBA) reaction, which is a simple and useful diag-nostic tool for the measurement of LPO for in vitro andin vivo systems.

Biological Implications of SpermatozoaLPO and Ox/dative Stress

The spermatozoa, unlike other cells, are unique in struc-ture, function, and susceptibility to damage by LPO. Inorder to understand the biological mechanisms of LPOin infertility, three important questions need to be ad-dressed: 1) What are the mechanisms of LPO of spermin vivo? 2) What are the consequences of damage to spermmembranes, proteins, and nucleic acids? 3) What regulatesthe antioxidant defense mechanisms in seminal plasma?

In general, the most significant effect of LPO in all cellsis the perturbation of membrane (cellular and organellar)structure and function (transport processes, maintenanceof ion and metabolite gradients, receptor-mediated signaltransduction, etc.). Low levels of NADH and glutathione,as a result of the increased activity of glutathione per-oxidase to remove metabolites of LPO, will further affectcellular Ca2 homeostasis. Minor alterations in spermmembranes in selected cases of dyspermia can be reversedby GSH therapy (Lenzie et al, 1994). Studies on how thesecellular changes caused by LPO affect seminal parametersand sperm function, and the reversal of these effects, areopen to further investigations.

Besides membrane effects, LPO can damage DNA andproteins, either through oxidation of DNA bases (pri-marily guanine via lipid peroxyl or alkoxyl radicals) orthrough covalent binding to MDA, resulting in strandbreaks and cross-linking. ROS can also induce oxidationof critical-SB groups in proteins and DNA that will alterspermatozoal structure and function, with an increasedsusceptibility to attack by macrophages. The oxidativedamage to mitochondrial DNA is well known to occur inall aerobic cells that are rich in mitochondria; this mayinclude spermatozoa. In addition, the redox status of hu-man spermatozoa is likely to affect phosphorylation andATP generation, with a profound influence on its fertil-izing potential. Aitken et al (1995) recently showed thatstimulation of endogenous NADPH-dependent ROS gen-eration in human sperm appears to regulate the acrosomereaction via tyrosine phosphorylation. In general, oxidiz-ing conditions increase tyrosine phosphorylation, with en-hanced sperm function, whereas reducing conditions havethe opposite effect. However, this has been debated for along time, and it is still not clear whether sperm have anNADPH-dependent oxygenase system. Nonetheless, how

these mitochondrial DNA or membrane changes regulatespecific sperm functions in association with altered ty-rosine phosphorylation is an area of interest. These studiesmay open a new series of diagnostic tools to assess spermfunction and damage in clinical infertility.

Antioxidants (Potential Scavengers of ROS)

Antioxidants, in general, are compounds and reactionsthat dispose, scavenge, suppress the formation of ROS,or oppose their actions. A variety of biological and chem-ical antioxidants that attack ROS and LPO are presently

under investigation. Among the well-known biologicalantioxidants, superoxide dismutase (SOD) and its twoisozymes and catalase have a significant role. SOD spon-

taneously dismutates O2 to form 02 and H2O2, whereascatalase converts H202 to 02 and H20.

SOD

202 + 2H -p B2O2 +02

calalasc

H202 - H20 + #{189}O2.

Many studies have been reported in the literature on therole of SOD as an antioxidant in reproductive biology.SOD protects spermatozoa against spontaneous 02 tox-icity and LPO (Alvarez et al, 1987). SOD and catalasealso remove O2 generated by NADPH oxidase in neu-trophils and may play an important role in decreasingLPO and protecting spermatozoa during genitourinaryinflammation.

GSH peroxidase, a selenium-containing antioxidant en-zyme with GSH as the electron donor, removes peroxyl(ROOrn) radicals from various peroxides including H2O2.

GSH reductase then regenerates reduced GSH from ox-idized GSH (GSSG), as shown in the following equation:

pero,idsc.(Se)

2GSH + H2O2 GSSG + 2H20reduclase

GSSG + NADPH + H ‘2GSH + NADP.

A selenium-associated polypeptide, presumably GSHperoxidase, has been demonstrated in rat sperm mito-chondria; it plays a significant role in this peroxyl scav-enging mechanism and, ultimately, in maintaining spermmotility (Calvin et al, 1981). It would be interesting toexplore this antioxidant mechanism in human sperma-tozoa. In addition, GSH peroxidase and GSH reductasemay directly act as antioxidant enzymes involved in theinhibition of sperm LPO. GSH has a likely role in spermnucleus decondensation and may alter spindle microtu-bule formation in the ova, thus affecting pregnancy out-come. In this context, the -y-glutamyl transpeptidase(‘yGGT), considered to be present in the midpiece andacrosomal regions of spermatozoa of certain mammalianspecies (e.g., the boar) may further affect oocyte GSH

Sikka et al . Oxidative Stress and Infertility 467

content at the time of sperm penetration. Thus, because

of the great number of mitochondria present in sperma-tozoa, these antioxidant mechanisms are important in themaintenance of sperm motility, the rate of hyperactiva-tion, and the ability to undergo the acrosome reactionduring sperm preparation techniques, especially in theabsence of seminal plasma. Albumin, used in sperm-washing procedures, is likely to serve as an antioxidant

by providing thiol groups required for “chain breaking”antioxidant activity. A high GSH/GSSG ratio will helpspermatozoa to combat oxidative insults. It seems thatthe role of these GSH enzymes and their associated mech-anisms as related to biological antioxidants in infertilityis an important area for further development.

Within the category of chemical antioxidants, both nat-ural and synthetic products have garnered attention bythe cosmetic, nutrition, and pharmaceutical industries.Their usefulness in reproduction and management of in-fertility has not yet been developed. Vitamins E and Chave shown minimal effects on the improvement in post-thaw sperm parameters, although they may protect sper-matozoa against endogenous oxidative DNA and mem-brane damage. In this regard, carotenoids (beta-carotene)and ubiquinols may also play a role in quenching singletoxygen and reducing lipid derived free radicals with det-rimental effects on sperm LPO.

Hence, the application of ROS scavengers (e.g., SOD,catalase) is likely to improve sperm motility and function.Pentoxifylline, a sperm motility stimulator, can also actas a suppressor or scavenger of ROS (Sikka et al, 1993).High levels of alpha-tocopherols in serum achieved upon

oral administration of vitamin E have been shown toimprove the performance of spermatozoa in the zonabinding test (Kessopoulou et al, 1994). The effect of vi-tamin E supplementation in combination with in vitrofertilization (IVF) techniques is a worthy notion. Furthercontrolled clinical studies will determine if many of theseputative antioxidants can improve infertility in subgroupsof patients.

The Concept of Ox/dative Stress Status

The balance of ROS can be termed as the “balance ofcreation and destruction.” Under normal circumstances,there is an appropriate balance between pro-oxidants andantioxidants. A shift in the levels of ROS towards pro-oxidants in semen and vaginal secretions can induce anoxidative stress on spermatozoa. Concomitantly, a de-crease in antioxidant activities (e.g., SOD, catalase, GSHperoxidase and reductase, GSH) in semen will correlatewith idiopathic infertility. It is possible that an increasedrate of ROS production (suggesting high oxidative stress)may inhibit the action of these antioxidant enzymes, oralternatively the inherent decreased expression of theseantioxidant enzymes may cause increased oxidative stress.

This will result in increased LPO, decreased sperm mo-

tility, viability, and function, and, ultimately, infertility(see Fig. 1).

A situation in which there is a shift in this ROS balancetowards pro-oxidants, because of either excess ROS ordiminished antioxidants, can be classified in terms of pos-itive oxidative stress status (OSS). There is at present notrue ROS detection method available that will evaluatethis balance. However, assessment of OSS, or a similarparadigm when monitored more objectively, would be agood indicator of sperm damage caused by oxidative stress.Chronic asymptomatic genitourinary inflammation canbe regarded as a condition with positive OSS that maybe the real cause of idiopathic infertility in such patients.

Measurement of OSS

Assessment of the rate of ROS productionlgeneration us-ing luminol as a probe can be a dynamic measure ofoxidative stress. However, the clinical evaluation of thisROS generation is limited by a very short half-life of these

free radicals. The potential methods that can be developedfor evaluation of OSS may utilize measurement of anoxidized component that remains in the body fluids (e.g.,TBA-reactive substances, GSH/GSSG balance, the levelsof unaltered tocopherol or ascorbate). Although there havebeen concerns about the specificity, interference, and re-liability of measuring TBA-MDA activity as an indicatorof LPO, this test remains one of the most efficacious meth-ods for assessing oxidative damage to sperm (Alvarez etal, 1987). Eventually, this TBA-MDA measurement willneed to be combined with other assays that suggest ROSproduction and antioxidant protection for the overall as-sessment of OSS in infertility. Measurement of IL-8, e.g.,when combined with SOD or other antioxidants in in-fertile patients with leukocytospermia, will indicate a pos-itive OSS in this population (Rajasekaran et al, 1995) andshould be treated accordingly. Thus, it would be impor-tant to assess OSS either in the semen in the male or thevaginal fluids in the female before, during, and after clin-ical trials. This would be indicative of whether an indi-vidual with low OSS is at a reduced risk of infertility. Ifa positive correlation is observed between OSS and theoutcome of the trial, a predictive value could be deter-mined.

Summary

Oxygen toxicity is an inherent challenge to aerobic life,including spermatozoa, the cells responsible for propa-gation of the species. How this toxicity affects the sper-matozoan in its interactions with the ovum is still un-known. An increase in oxidative damage to sperm mem-branes, proteins, and DNA is associated with alterationsin signal transduction mechanisms that affect fertility. Re-cent evidence suggests that spermatozoa and oocytes pos-

468 Journal of Andrology . November/December 1995

sess an inherent but limited capacity to generate ROS toaid in the fertilization process. Though a variety of defensemechanisms encompassing antioxidant enzymes (SOD,catalase, and GSH peroxidase and reductase), vitamins(E, C, and carotenoids), and biomolecules (OSH and ubi-quinol) are available, a balance of the benefits and risksfrom ROS and antioxidants appears to be necessary forthe survival and functioning of spermatozoa. An assaysystem for the evaluation of OSS needs to be developed.Such an assay will assist the clinician in the assessmentof fertility status of both male and female partners. Thedetermination of this OSS value will also theoreticallyidentify the subgroups of responders and nonrespondersto any putative antioxidant therapy. Though the thera-peutic use of antioxidants appears attractive, cliniciansneed to be aware of exaggerated claims of antioxidantbenefits by various commercial supplements for fertilitypurposes until proper multicenter clinical trials have beencompleted.

AcknowledgmentsWe thanic Drs. Krishna Agrawal and Rajesh Naz for their consultation;

Mrs. June Banks Evans and Ms. Liset Human for editorial corrections

and Mrs. Michon Breisacher Shinn for the preparation of this mimreview.

ReferencesAitken RJ, Clarkson JS. Cellular basis of defective sperm function and

its association with the genesis of reactive oxygen species by human

spermatozoa. J Reprod Fertil 1 987;8 1:459-469.

Aitken Ri, Paterson M, Fisher H, Buckingbam DW, van Duin M. Redox

regulation of tyrosine phosphorylation in human spermatozoa andits role in the control of human sperm function. J Cell Sd 1995; 108:2017-2025.

Aitken Ri, West KM, Buckingham DW. Leukocyte infiltration into thehuman ejaculate and its association with semen quality, oxidativestress, and sperm function. JAndrol 1994;15:343-352.

Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipidperoxidation and production of hydrogen peroxide and superoxidein human spermatozoa: superoxide dismutase as major enzyme pro-

tectant against oxygen toxicity. JAndrol 1987;8:338-348.

Calvin HI, Cooper OW, Wallace EW. Evidence that selenium in rat

sperm is associated with a cysteinre-rich structural proteins of the

mitochondrial capsule. Gamete Res 1981 ;4: 139-145.de Lamirande E, Gagnon C. Reactive oxygen species and human sper-

matozoa. I. Effects on the motility of intact spermatozoa and on spermaxonemes; and II. Depletion of adenosine triphosphate plays an im-

portant role in the inhibition of sperm motility. JAndrol 1992;l3:

368-386.

Gagnon C. Sperm cell biology: reactive oxygen species and sperm func-tion. Post-graduate course-American Society of Andrology 17th An-nual Meeting, Bethesda, MD. 1992: 1-12.

Gagnon C, Iwasaki A, de Lamirande E, Kovalski N. Reactive oxygen

species and human spermatozoa. Ann N Y Acad Sci 1991 ;637:436-

444.Iwasaki A, Gagnon C. Formation of reactive oxygen species in sper-

matozoa of infertile patients. Fertil Sieril I 992;57:409-4 16.Kessopoulou E, Barratt CLR, Powers HJ, Sharma KK, Pearson MJ,

Cooke ID. Rational treatment of reactive oxygen species associatedmale infertility with vitamin E: a double blind, randomized, placebo-

controlled trial. Second International meeting of the British Fertility

Society, Glasgow, U.K. 1994:7-8.Kessopoulou E, Tomlinson MJ, Barratt CL, Bolton AE, Cooke ID. Origin

of reactive oxygen species in human semen: spermatozoa or leuco-cytes? J Reprod Fertil 1 992;94:463-470.

Krausz C, Mills C, Rogers S, Tan SL, Aitken Ri. Stimulation of oxidantgeneration by human sperm suspensions using phorbol esters and

formyl peptides: relationships with motility and fertilization in vitro.

Fertil Steril 1994;62:599-605.

Lenzi A, Picardo M, Gandini L, Lombardo F, Terminali 0, Passi 5,

Dondero F. Glutathione treatment of dyspermia: effect on the lipo-

peroxidation process. Hum Reprod 1994;9:2044-2050.

Rajasekaran M, Helistrom WI, Naz RK, Sikka SC. Oxidative stress and

interleukins in seminal plasma during leukocytospermia. Fertil Steril

l995;64: 166-17 1.

Rajasekaran M, Hellstrom WI, Sparks RL, Sikka SC. Sperm damaging

effects of electric current: possible role of free radicals. Reprod Toxicol

1 994;8:427-432.

Sikka SC, Hellstrom WIG, Naz RK. Pentoxifylline: role in management

of male infertility/mechanisms of action. Mol Androl l993;5:220-

231.