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WHAT AN ANDROLOGIST/UROLOGIST SHOULD KNOWABOUT FREE RADICALS AND WHY

ASHOK AGARWAL, SUSHIL PRABAKARAN, AND SHYAM ALLAMANENI

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ree radicals are a group of highly reactivechemical molecules that have one or more un-

aired electrons and can oxidatively modify bi-molecules that they encounter.1 Reactive oxygenpecies (ROS) are free radicals that are derivedrom the metabolism of oxygen. They are presentn all aerobic organisms. ROS play an importantole in many physiologic functions such as phago-ytosis, which is mediated by neutrophils and mac-ophages. They also act as a cell messenger, takingart in many signaling processes, mainly by oxida-ively modifying the compounds.2 However, ROSre highly reactive, and when their levels are high,hey can react with any nearby molecules and causeefects in the functioning of these molecules. Ox-dative stress is a condition that occurs as a result ofxcessive generation of ROS and diminished capac-ty of free radical scavenging by antioxidants.3 Manyiseases, including cancer, are thought to be theonsequence of reactions between free radicals andNA, resulting in germ line mutations that are

ransmitted to offspring.4 Some pathologic pro-esses such as aging, ischemic heart disease, sickleell anemia, and rheumatoid arthritis are also at-ributed to free radical-induced oxidation of vari-us biologic components.5ROS include the various radicals such as super-

xide, hydroxyl, peroxyl, and hydrogen peroxide.n addition, another class of free radicals that areitrogen derived is called reactive nitrogen species.hey are considered a subclass of ROS.6 Nitrousxide, peroxynitrite, nitroxyl anion, and peroxyni-rous acid are some of the most common reactiveitrogen species.

rom the Center for Advanced Research in Human Reproduction,nfertility, and Sexual Function, Glickman Urological Institute,nd Department of Obstetrics and Gynecology, Cleveland Clinicoundation, Cleveland, OhioReprint requests: Ashok Agarwal, Ph.D., Case Western Reserve

niversity, Center for Advanced Research in Human Reproduc-ion, Infertility, and Sexual Function, Glickman Urological Insti-ute, and Department of Obstetrics and Gynecology, Clevelandlinic Foundation, 9500 Euclid Avenue, Desk A19.1, Cleveland,H 44195. E-mail: Agarwaa@ccf.orgSubmitted: February 23, 2005, accepted (with revisions): July

t, 2005

© 2006 ELSEVIER INC. UALL RIGHTS RESERVED

Even though free radicals were first described inpermatozoa by MacLeod in 1943,4 their role inale infertility was not studied extensively until

he last decade. Research in the last two decadesas confirmed the physiologic and pathologic sig-ificance of free radicals in human reproduc-ion.7–9 Studies have shown that 30% to 80% ofnselected infertile patients have high levels ofeminal ROS. It is important for andrologists tonderstand free radicals because of their effect inecreasing male fertility potential in the generalopulation and increasing the risk of testicularancer by altering the sperm DNA.10,11

Although almost all cellular and metabolic pro-esses generate free radicals, an andrologist shoulde more concerned with the sources of ROS in theale reproductive system. Leukocytes, particu-

arly neutrophils and macrophages, have been as-ociated with excessive ROS production, and theyltimately cause sperm dysfunction.12 Another im-ortant source of ROS is immature and morpho-ogically abnormal spermatozoa.13 The productionf ROS is also increased by lifestyle factors such asmoking and pollutions. Smoking increases ROSroduction, causing sperm DNA damage, and sup-resses antioxidants in both semen and serum.14 Atudy from our group has demonstrated that a sig-ificant negative correlation exists between ROSroduction and semen quality.15 Furthermore, ashe concentration of immature spermatozoa in theuman ejaculate increases, the concentration ofature spermatozoa with damaged DNA also in-

reases.15 In assisted reproductive techniques, me-hanical injury to spermatozoa (eg, repeat centrif-gation during sperm preparation) and externalonditions such as high oxygen tension can in-rease ROS production.16

PHYSIOLOGIC ROLE OF FREE RADICALS

Free radicals play a vital role in the physiologicunctioning of the spermatozoa. Research has shownhat spermatozoa require a small amount of ROSor various reactions to achieve fertilizing capaci-

y.17 Hydrogen peroxide stimulates sperm capaci-

ROLOGY 67: 2–8, 2006 • 0090-4295/06/$32.00doi:10.1016/j.urology.2005.07.012

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ation by hyperactivation and acrosome reaction.18

ccording to Zini et al.,19 superoxide and nitricxide also take part in these processes. Free radi-als are also involved in the fusion of spermatozoaith the oocyte.18 Nitric oxide plays a role in the

perm’s ability to fuse with oocyte, but it has noction in zona pellucida binding.20 Low concentra-ions of hydrogen peroxide cause tyrosine phos-horylation, which in turn results in the binding ofhe spermatozoal membrane proteins with ZP-3roteins on the zona pellucida and ultimately,permatozoa-ooctye fusion.21

However, many questions regarding the physio-ogic action of ROS remain unanswered. One ques-ion is whether the inability of spermatozoa to pro-uce free radicals results in a decreased fertilizingapacity of spermatozoa. Another is whether in-reased levels of antioxidants cause scavenging ofhysiologic levels of free radicals and thus spermysfunction.

SPERM DYSFUNCTION AND FREERADICALS IN SEMEN

Excessive levels of free radicals alter normalhysiologic functioning of spermatozoa. ROS ap-ears to have a spectrum of variable effects on sper-atozoa depending on the extent of oxidative

tress.22 For example, a study by Aitken et al.23

eported that a low concentration of hydrogen per-xide did not have any effect on sperm motility butid suppress sperm-egg fusion. This may also ex-lain why patients with normal semen parametersan still experience infertility. In such patients, theOS levels are not high enough to impair basicemen analysis parameters but can cause defects inther processes that are required for fertilization,uch as sperm-oocyte interaction.Excessive ROS levels are related to an increase in

ipid peroxidation of the sperm plasma membrane.24

ipid peroxidation results in loss of membrane flu-dity, which is essential for sperm motility andperm-oocyte fusion. The acrosome reaction is de-ective in spermatozoa with a high concentration ofOS. Griveau and Le Lannou25 reported that thecrosome reaction in human spermatozoa appearedusceptible to ROS and that hydrogen peroxide inac-ivates several enzymes, including glutathione perox-dase, superoxide dismutase, and glucose-6-phos-hate. Several studies have noted that levels of ROSorrelate with motility. Motility is impaired eitherecause of adenosine triphosphate depletion in ax-ns or lipid peroxidation of the sperm plasmaembrane.26 In inflammation and infection, ROS

eneration by leukocytes becomes uncontrollednd leads to excessive ROS levels in tissues. Evenhen the initially stimulated leukocytes become

urnt out, the process of ROS generation continues t

ROLOGY 67 (1), 2006

y the stimulation of surrounding leukocytes andpermatozoa by mediation of cytokines (interleu-in-6 and interleukin-8).

FREE RADICALS ANDPREGNANCY OUTCOME

Clinicians and patients alike want to know whetherevels of ROS in semen can directly predict fertility.ery few studies have analyzed such a direct relation-hip between ROS levels and pregnancy outcome. Inprospective study, ROS generation was shown to beegatively associated with fertility in vivo.27 By usinghe ROS-total antioxidant capacity (TAC) score,nother study found that seminal oxidative stressevels were lower in male patients whose partnerchieved pregnancy than in those patients whoseartner did not (mean ROS-TAC score 47.7 � 13.2ersus 35.8 � 15.0; P �0.01).28

ROS may also play a role in the outcome of preg-ancy as evidenced by the ability of antioxidants to

ncrease the pregnancy rate in infertile patients.ome studies have demonstrated an improvementn the pregnancy rate after antioxidant supplemen-ation. Oral administration of vitamin E (300 mg)wice a day in a randomized, double-blind, placebo-ontrolled trial showed significant improvement inregnancy rates (21%; 11 of 52) in infertile pa-ients, but not in the placebo group.29 Combina-ion therapy of N-acetyl-cysteine or vitamin A and

and essential fatty acids significantly reducedOS and improved pregnancy rates.30 Several stud-

es have demonstrated the ability of carnitines (L-arnitine 1 g and acetyl-carnitine 0.5 g twice a day)o reduce ROS production significantly and increasehe chances of pregnancy.31

FREE RADICALS AND ASSISTEDREPRODUCTIVE TECHNIQUES

Various methods of assisted reproductive tech-iques (ARTs) have recently been developed. Theyere originally designed for treatment of female

actor infertility, but are now used extensively forhe treatment of male factor infertility. Intrauterinensemination, in vitro fertilization (IVF), and in-racytoplasmic sperm injection (ICSI) are some ofhe important ARTs. The levels of ROS in maturepermatozoa correlate significantly with the fertil-zing potential of spermatozoa.32 High ROS levelsre associated with a reduced pregnancy rate afterVF or ICSI and arrested embryo growth.32 A re-ent meta-analysis by our group found that ROSevels correlated significantly with the IVF fertili-ation rate (estimated overall correlation �0.374,5% CI �0.520 to �0.205).33 Measuring ROS levelsn semen specimens before IVF may be useful in pre-icting IVF outcome and in counseling selected pa-

ients with male factor or idiopathic infertility.

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One of the main disadvantages of ARTs is thebsence of a natural selection process. It is possiblehat sperm damaged by free radicals may occasion-lly be used for fertilization, resulting in early em-ryo death when the paternal gene is switched on.CSI is the most promising ART, but it is associatedith the greatest number of miscarriages. This maye because embryologists are unable to choose spermithout DNA damage selectively and as a result,amaged sperm get injected into an oocyte.17

Sperm separation techniques such as density gradi-nt centrifugation, migration-sedimentation, glassool filtration, and conventional swim-up methodlay a critical role in separating out spermatozoaith high motility. The first three methods (den-

ity gradient centrifugation, migration-sedimenta-ion, and glass wool filtration) are more effective inignificantly reducing levels of free radicals thanhe conventional swim-up technique.34 However,epeated centrifugation causes mechanical injuryo spermatozoa and increases ROS production.16

esearchers are currently studying the use of anti-xidants and other substances to prevent ROS gen-ration during sperm preparation processes.

FREE RADICALS IN SPERM DNA DAMAGEAND APOPTOSIS

Free radicals can cause changes in genetic infor-ation by oxidatively modifying the constituents

f DNA. A study by Shen et al. has demonstratedhat smoking increases the DNA damage mediatedy oxidative stress.35 The extent of oxidative dam-ge to DNA can be estimated by measuring 8-hy-roxy-2-deoxyguanosine.10 Normally, when freeadicals damage the sperm plasma membrane andNA, the damaged spermatozoa lose their ability

o fertilize an oocyte. However, this may not be thease with ARTs in which fertilization occurs de-pite dysfunctional plasma membrane; thus, thehance that DNA-damaged sperm will fertilize anocyte is increased. The percentage of sperm withNA damage correlated negatively with the fertil-

zation rate in a study by Sun et al.36 in 298 semenamples from couples undergoing IVF. In a similartudy on patients undergoing ICSI, Lopes et al.37

bserved that the DNA damage measured by theerminal deoxynucleotidyl transferase-mediatedUTP-biotin nick end labeling assay correlatedegatively with the fertilization rate, which was as

ow as 20%. The damaged DNA in spermatozoaay also affect embryo development.38 Oocytes

an repair DNA damage to some extent, but whenhe damage is severe, embryo death, miscarriage,nd congenital malformations occur.Apoptosis is a physiologic phenomenon in the

ody that helps to discard abnormal spermatozoa.

OS generated from abnormal spermatozoa may t

timulate the process of apoptosis, resulting in theeath of spermatozoa. ROS may initiate a chain ofeactions by activating caspases that ultimatelyead to apoptosis.39 When ROS levels are raisedathologically, the process of apoptosis may alsoe initiated in mature spermatozoa. The process ofpoptosis may be accelerated by ROS-inducedNA damage, which ultimately leads to a decline

n the sperm count. Oxidative stress due to exces-ive generation of ROS is presumed to cause sper-atozoa DNA damage and has correlated positivelyith apoptosis.40 According to a retrospective studyy Host et al.,41 apoptosis correlated negativelyith the fertilization rate.

FREE RADICALS AND CLINICALDIAGNOSES OF INFERTILITY

Ongoing research has expanded the list of clini-al diagnoses in patients with infertility in whichOS are implicated as executors of the pathologicndings. Up to 95% of patients with spinal cord

njury are known to have elevated seminal levels ofOS.42 The cause may be a result of prolongedtasis of semen in the reproductive tract and in-ammation. The role of ROS in varicocele has alsoeen extensively investigated. Elevated ROS levelsnd decreased TAC levels have been noted in pa-ients with varicocele.43 Excessive release of nitricxide within dilated spermatic veins has been ob-erved in adults with varicocele, which suggestshat high levels of oxidative stress are present.44

ormation of peroxynitrite from the reaction ofitric oxide with superoxide could be a causative

actor for impaired sperm function in patients witharicocele.45 One study found reduced ROS levelsnd increased antioxidant activity in seminal plasmafter varicocelectomy in infertile men with varico-ele.46 A positive correlation was noted betweenOS levels and the varicocele grade.22

When infection of the male reproductive tractccurs, one can obviously expect levels of ROS toe elevated owing to infiltration by white bloodells. In patients with prostate-vesiculo-epididy-itis, the ROS levels were raised compared with

atients without prostate-vesiculo-epididymitis.47

n the former group of patients, the pregnancy ratemproved after treatment with nonsteroidal anti-nflammatory drugs and antioxidants.48 Idiopathicnfertility in which routine semen parameters areormal and no other cause of infertility is presentay be a result of ROS-mediated damage.49 Pa-

ients with such a form of infertility with elevatedOS are expected to show a good response to an-

ioxidant treatment.

UROLOGY 67 (1), 2006

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INFERTILITY DUE TO ELEVATED FREERADICALS IN SYSTEMIC DISEASES

Excessive generation of ROS may be an impor-ant cause of many diseases such as diabetes melli-us, cardiovascular diseases, systemic infection, andancer.4,5,50 These diseases are known to affect fer-ility in different ways. In diabetes mellitus, in-reased production of ROS is a result of resultantyperglycemia, which in turn affects the bloodessels and nerve endings and can cause erectileysfunction.50 Many drugs, such as quinines and ni-roso-aromatic compounds, generate free radicals.5

oreover, detoxification of even commonly usedrugs such as aspirin and acetaminophen can in-rease ROS production by increasing cytochrome450 activity.51 Environmental conditions such asadiation and pollution also increase levels of oxi-ative stress.52 An andrologist should be aware ofhe above systemic conditions and external factorshat can cause a generalized increase in oxidativetress status in the human body and perhaps also inhe reproductive tract. By identifying high oxida-ive stress as a cause of infertility in such condi-ions, an andrologist can initiate appropriate thera-eutic interventions directed against oxidative stress.

ANTIOXIDANTS

The human body has a number of mechanisms toinimize free radical-induced damage and to re-

air damage that has already occurred. However,permatozoa are unique in human cells in that theyre unable to repair oxidative damage because ofhe lack of cytoplasmic enzymes. Three differentntioxidant protection systems provide the neces-ary protection against the damaging effect of freeadicals: dietary antioxidants that are obtained asart of our regular diet; endogenous antioxidantshat are the part of body’s own defense mechanism;nd the metal-binding proteins that combine withhe metals and prevent their catalytic effect.53

Endogenous antioxidants are the group of anti-xidants consisting of low-molecular-weight com-ounds, such as bilirubin, thiols, uric acid, and coen-yme Q10, and larger molecular enzymes, such asuperoxide dismutase, catalase, and glutathione per-xidase. According to a study conducted by Michielst al.,54 all three of these enzymes are essential fordequate antioxidant defense and all of them act in aynergistic manner. In a study of 38 asthenospermicatients, in vivo administration of coenzyme Q10ncreased the motility of the spermatozoa.55

Diet should consist of an adequate amount ofhain-breaking antioxidants such as vitamin C oritamin E, in addition to beta-carotenes, carote-oids, and flavonoids. In a study conducted byessopoulou et al.,56 oral administration of vita-

in E improved sperm function. Similar studies t

ROLOGY 67 (1), 2006

ave been conducted on other dietary antioxi-ants.Metal-binding proteins, such as albumin, cerulo-

lasmin, metallothionein, transferrin, ferritin, andyoglobulin, form a valuable additive to the anti-

xidant defense system by inactivating the transi-ion metal ions, which would otherwise catalyzehe production of free radicals.1 A study conductedy Twigg et al.57 on healthy normozoospermic do-ors demonstrated the protective effect of albuminither by binding to lipid peroxides or binding toerrous ion promoters. However, it should be notedhat in some circumstances antioxidants can act asro-oxidants. Thus, additional research is requiredn terms of the dose and combination of antioxi-ants to be used clinically.

EVALUATION OF PATIENTS FOROXIDATIVE STRESS

Numerous methods are available to determineOS levels in semen. Of these, chemiluminescence

s the most widely used method. It can discrimi-ate between the production of superoxide andydrogen peroxide by spermatozoa depending onhe probes used.58 Flow cytometry uses the princi-les of light scattering, light excitation, and emis-ion of fluorochrome molecules to generate spe-ific multiparameter data measures. It measureshe fluorescent intensity of different dyes such as,7 dichlorofluorescin diacetate (DCFH-DA), hy-roethidine after oxidation by ROS and thus quan-ifies the amount of ROS present in the semen.Many indirect methods are available for the de-

ection of ROS-induced damage to lipid mem-ranes. The thiobarbituric acid assay is commonlysed. Thiobarbituric acid-reactive substances suchs malondialdehyde are measured by spectropho-ometry analysis.59 The TAC of the semen is deter-ined by either the enhanced chemiluminescence

ssay or the calorimetric assay. To measure theverall oxidative stress, the ROS-TAC score can besed, which was developed by principal compo-ent analysis. A low ROS-TAC score indicates theresence of high oxidative stress and thus spermysfunction.28

Currently, no standard and clinically useful meth-ds are available for estimating oxidative stress.any innovative methods have been used in other

elds of medicine such as electron spin resonancend aromatic trapping for the determination of freeadicals. These methods can be used, along withther currently accepted methods, to evaluate andtandardize the accurate measurement of free rad-cals in andrology. Recently, we attempted to de-ne the basal levels of ROS in normal donors in neatwhole unprocessed) semen specimens and in ma-

ure and immature spermatozoa.60 These basal val-

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es can be used to identify the pathologic levels ofOS in infertile men and may guide therapeutic

nterventions. We have attempted to provide achematic representation of the various factors in-uencing oxidative stress and its managementFig. 1).

OXIDATIVE STRESS: CAUSE OR EFFECT

Do excessive free radicals cause male infertilityr are they a consequence of a pathologic processn the male reproductive tract (ie, are they byprod-cts without any significance)? This is a majoruestion that has not been clearly answered andas major therapeutic implications. If the secondituation is true, no scientific rationale exists forhe use of antioxidants. However, the current evi-ence indicates that the first situation is correct. Theody’s defense mechanism produces leukocytesgainst infections such as prostatitis and urinary tractnfection. Leukocytes are the major producers of freeadicals.61 They are required for the effective de-ense against the invading pathogens. Varicoceleas also been documented to increase the free rad-

cal generation in seminal plasma. Lifestyle behav-ors such as smoking, alcohol abuse, and exposureo radiation and pollution increase ROS produc-ion. Hence, production of free radicals can be con-idered as secondary to the primary disease pro-ess. However, they themselves cause the damageo the spermatozoa resulting in the infertility. Itffects the sperm plasma membrane integrity andamages DNA. Thus, they are the cause of the clin-cal effects of the primary pathologic processes;herefore, methods to decrease their actions have

IGURE 1. Origin of oxidative stress in the etiopatho-enesis of male infertility and its management.

herapeutic implications. 1

CONCLUSIONS

It is crucial for andrologists to understand freeadicals—their sources, mechanism of generation,nd the damage they can cause to the male repro-uctive system. In addition, it is also essential to beware of the various diseases that increase ROSeneration in the blood, plasma, and seminal flu-ds. A multifaceted approach is required for thereatment of male infertility induced by free radi-als. Methods can be used to decrease ROS produc-ion (eg, the addition of antioxidants during spermreparation techniques). With the abundance ofany synthetic and natural antioxidants, it is critical

o use them judiciously. The critical mechanism ofathologic free radical generation and crucial antiox-dant protective systems in the male reproductive sys-em are beginning to be elucidated for therapeuticargeting. Additional research is needed in this direc-ion before they can be used clinically.

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45. Mitropoulos D, Deliconstantinos G, Zervas A, et al: Ni-ric oxide synthase and xanthine oxidase activities in the sper-atic vein of patients with varicocele: a potential role for nitric

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