AUA Update SeriesLesson 1 Volume 26 2007

Clinical Relevance of Oxidative Stress in Patients With Male FactorInfertility: Evidence-Based Analysis

Learning Objective: At the conclusion of this continuing medical education activity, theparticipant will have a thorough understanding of how oxidative stress affects maleinfertility as well as how treatment of varicocele decreases oxidative stress and improvesfertility.

Ashok Agarwal, Ph.D., HCLDDisclosures: Nothing to disclose

Sushil A. Prabakaran, M.D.Disclosures: Nothing to disclose

Reproductive Research CenterGlickman Urological Institute

Cleveland ClinicCleveland, Ohio

and

Suresh C. Sikka, Ph.D, HCLDDisclosures: Nothing to disclose

Department of UrologyTulane University Health Sciences Center

New Orleans, Louisiana

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KEY WORDS: infertility, male; oxidative stress; DNA fragmenta-tion; apoptosis; antioxidants

INTRODUCTION

Infertility is defined as the inability to conceive after 1 yearof unprotected intercourse by couples of reproductive age. Ac-cording to a national survey of family growth conducted in 1995by the Centers for Disease Control and the National Institutesof Health, male factor infertility is the causative factor in approx-imately 40% of the 2.1 million infertile young married couplesin the United States. Half of these men experience irreversibleinfertility and cannot father children. There are a number ofcommon causes of infertility in men including hormonal andgonadal disorders, medical conditions such as cystic fibrosisand sickle cell anemia, renal diseases and urological conditionssuch as cryptorchidism, varicoceles, testicular cancer, and repro-ductive tract trauma and/or obstruction.. These disorders cantemporarily or permanently cause impaired spermatogenesis,diminish sperm migration in the male and female reproductivetracts due to defective motility, result in poor morphology andaffect sperm function (acrosome reaction, sperm oocyte fusion),all of which can prevent conception . Many of these conditionsincrease the production of reactive oxygen/nitrogen speciescausing oxidative stress, which leads to spermatozoal dysfunc-tion and infertility. This review is designed to assist with theunderstanding of oxidative stress and its role as related to malefactor infertility in clinical practice.

WHAT IS OXIDATIVE STRESS?Oxidative stress is a cellular condition associated with an im-

balance between the production of free radicals, mainly ROS,and their scavenging capacity by antioxidants. When the produc-tion of ROS exceeds the available antioxidant defense, signifi-cant oxidative damage occurs to many cellular organelles bydamaging lipids, proteins, DNA and carbohydrates, thus ulti-mately leading to cell death. Sperm is particularly susceptibleto oxidative damage due to its unique structural composition.In the process of maturation spermatozoa extrude cytoplasm.Since cytoplasm is the major source of antioxidants, lack ofcytoplasm causes a deficiency in antioxidant defense. Ironically,when this process is hindered, residual cytoplasm forms a cyto-plasmic droplet in the sperm mid region. The residual cytoplasmcontains high concentration of some cytoplasmic enzymes(G6PDH, SOD), which are also a source of ROS.1 In addition,the sperm plasma membrane is rich in polyunsaturated fattyacids that readily undergo lipid peroxidation by ROS, resultingin a loss of membrane integrity.

Immature spermatozoa and seminal leukocytes are the majorsources of ROS in semen. In abnormal spermatozoa ROS maybe generated at the level of the plasma membrane (nicotinamideadenine dinucleotide phosphate oxidase system) or mitochondria

ABBREVIATIONS: ART (assisted reproductive technology), ICSI (intracytoplasmic sperm injection), NADPH (nicotinamide adeninedinucleotide phosphate), 8-OHdG (8-hydroxy-2-deoxyguanosine), OS (oxidative stress), PCR (polymerase chain reaction), ROS (reactiveoxygen/nitrogen species), SCSA (sperm chromatin structure assay), SOD (superoxide dismutase), TAC (total antioxidant capacity)

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(NADH dependent oxido-reductase). Seminal leukocytes pro-duce H2O2 through the NADPH oxidase system. H2O2 is themost toxic form of ROS for spermatozoa since it is membranepermeable and readily affects the cellular organelles, whereassuperoxide and the hydroxyl radical are membrane impermeableand take time to exert their effect.2 Nitric oxide reacts withthe superoxide anion to yield the highly reactive metabolitesperoxynitrite and peroxynitrous acid, both of which are strongoxidants. Numerous studies now consider oxidative stress to bea real entity that is likely to have a significant impact on normalsperm function, thus affecting reproduction and fertility (fig. 1).

ROLE OF ROS IN MALE REPRODUCTIVE SYSTEMPhysiological aspects. ROS are required by spermatozoa to

attain functional maturity. Low levels of ROS are essential fornormal fertilization, capacitation, hyperactivation, motility andacrosome reaction.3,4 Spermatozoa acquire fertilizing abilityafter capacitation occurs in a favorable environment.5,6 Capacita-tion is a process that prepares the spermatozoa for interactionwith oocyte leading to fertilization. During this process theconcentrations of intracellular calcium, ROS and tyrosine kinaseincrease, resulting in an increased formation of cyclic adenosinemonophosphate. A high concentration of cyclic adenosine mo-nophophate facilitates the spermatozoa to acquire high motilityknown as hyperactivation.7,8

Only capacitated sperm exhibit hyperactivated motility andundergo a physiological acrosome reaction thereby acquiringthe ability to fertilize.9 Several studies have reported the roleof ROS in promoting sperm capacitation in human and animalmodels. Sperm capacitation is involved in a number of biochemi-cal events including an increase in redox mediated protein tyro-sine phosphorylation. ROS also exert their effect on sperm oo-cyte interaction. Low levels of lipid peroxidation causemodifications of plasma membranes facilitating sperm adhesionto the oocyte.10 However, the physiological level of ROS insemen and/or duration of exposure for normal sperm functionleading to fertilization is not yet established. Knowing the physi-ological levels of ROS seems important especially in under-standing the etiology of idiopathic male factor infertility.

Pathological aspects. Evidence suggests that high levels ofROS mediate damage to many cellular elements in the testisincluding DNA of mature spermatozoa. A common by-productof DNA oxidation, 8-hydroxy-2-deoxyguanosine, has been con-sidered a key biomarker of this oxidative DNA damage.11 Oxida-tive stress causes abnormal denaturation into single-strandedDNA and double-stranded DNA breaks, DNA base-pair oxida-tion, chromatin cross-linking, chromosome microdeletionsetc.12-16 ROS cause various types of gene mutation such asdeletion, point mutation or polymorphism, resulting in decreasedsemen quality.17,18

When the level of DNA damage is less, spermatozoa canundergo self-repair. Oocyte also is capable of repairing damaged

Fig. 1. Etiology and management of oxidative stress. Many factors, including primary pathological condition of male reproduc-tive system, systemic disorders and environmental factors, increase oxidative stress status, which causes spermatozoa dysfunc-tion leading to infertility.

DNA of spermatozoa. However, if the damage is extensive,apoptosis and embryo fragmentation can occur. Decreased fertil-ization rate, and poor embryo cleavage and quality have beenreported in infertility cases where sperm samples contain a highfrequency of damaged DNA.19 DNA damage in the Y chromo-some can cause gene deletion in the Y chromosome of theoffspring leading to infertility.15

The damage to sperm DNA is critical in the context of assistedreproductive technology, which is being increasingly used totreat infertile couples.20,21 The main disadvantage of ART isthat it bypasses the natural selection barriers that are presentthroughout the female reproductive tract before sperm enter theoocyte. With ART, sperm with abnormal genetic material canreach the oocyte with minimal (in vitro fertilization) or no(intracytoplasmic sperm injection) effort. The potential to intro-duce sperm with damaged DNA by ART has triggered a greatdeal of scientific interest and debate among clinicians and basicscientists.22

Recently some studies have reported a significant disruptionof mitochondrial DNA in the testes. Mitochondria are the power-house of human cells including spermatozoa. Any disruption to

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the mitochondrion or its genetic materials causes decreasedsperm motility.23 Some of the proposed mechanisms for oxida-tive damage to mitochondria are loss of DeltaPsim (mitochon-drial electrochemical gradient-dependent membrane potential),low levels of 32 to 30 kDa mature forms of steroidogenic acuteregulatory protein and cessation of cholesterol transport. Thesemechanisms likely contribute to a host of pathophysiologicaleventsevident in testiculardisorders suchas infection, cryptorchi-dism and varicocele.24 The 4977 bp deletion of mitochondrialDNA in spermatozoa was detected by polymerase chain reac-tion,25,26 although how it affects fertilizing capacity is not known.

ROS can also damage mitochondrial DNA to a higher degreethan nuclear DNA.27 Such damage is believed to be the causeof several human diseases including infertility.25,28,29 Higheramounts of 8-OHdG were detected in mitochondrial DNA thanin nuclear DNA.27,28 Closer proximity of DNA to ROS generat-ing electron transport chain in mitochondria, inefficient DNArepair mechanisms, and lack of histones leading to less compactchromatin organization and thereby exposed to free radicalswere suggested as the reasons for greater susceptibility of mito-chondrial DNA.30,31

Apoptosis. Apoptosis (programmed cell death) is a non-inflam-matory response to tissue damage characterized by a series ofmorphological and biochemical changes. It maintains the nurs-ing capacity of Sertoli cells by controlling the over productionof male gametes20 and eliminating abnormal spermatozoa. ROSmay initiate a chain reaction by activating caspases that ulti-mately lead to apoptosis.32 Apoptosis is induced by binding ofFas ligand or agonistic anti-Fas antibody to Fas. Some of theFas labeled cells escape apoptosis, known as abortive apoptosis,failing to clear all of the spermatozoa destined for elimination.This leads to a large population of abnormal spermatozoa in thesemen. Several apoptotic markers such as Bcl2, p53 and annexinV were found in human ejaculate.20 Especially, ejaculates ofmen with abnormal semen parameters (oligozoospermia,azoospermia) bear a large number of spermatozoa that are Faspositive as well as have damaged DNA.32 This relationshipamong apoptosis, sperm DNA damage and infertility is an inter-esting area for further scientific exploration (fig. 2).

ROLE OF SYSTEMIC DISEASES AND OXIDATIVESTRESS IN INFERTILITY

Several systemic diseases, such as cancer, cardiovascular prob-lems, infection and diabetes mellitus, are known to increasethe production of ROS.15,33,34 Commonly used drugs, such asquinines and nitroso-aromatic compounds also generate freeradicals.33 Even the process of hepatic detoxification of thedrugs by cytochrome P450 activity increases ROS production.35

In diabetes mellitus increased production of ROS as a result of

Fig. 2. ROS induced apoptosis. ROS (apoptotic stimulus) trigger mitochondria to release cytochrome C initiating caspase cas-cade. Interaction between Fas and Fas ligand is also necessary in apoptotic mechanism. DNA fragmentation occurs as result ofactivation of effector caspases (caspases 3 and 6) eventually causing apoptosis.

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hyperglycemia affects blood vessels and nerve endings that cancause erectile dysfunction.34 Environmental stressors such asradiation and pollution also increase levels of oxidative stress.By identifying high oxidative stress as a cause of infertility insuch conditions, appropriate therapeutic interventions directedagainst oxidative stress may improve infertility.

INFECTION, OS AND INFERTILITYBacteria, such as Shigella,36 Entameoba histolytica,37 Rickett-

siia rickettsii,38 Salmonella typhi39 etc, have been shown toincrease ROS production. Extracellular ROS are produced byleukocytes, mainly neutrophils.40,41 Low levels of leukocytesare normally present in prostatic and seminal vesicle secretions.During inflammation or infection, leukocyte levels increaseand they are also activated via cytokines, such as tumornecrosis factor-�, interleukins etc, increasing ROS produc-tion42 and causing severe damage to spermatozoa.43,44 Theinvading pathogens or the defense mechanism against themmediated by leukocytes can lead to sperm damage.45 Theenzymes of leukocytes (NADPH oxidase, etc) are responsible forROS generation including hydrogen peroxide, singlet oxygen,perichloric acid, and hydroxyl and superoxide anions. TheseROS are produced to create a defense against the pathogens.46

Similarly, a past infection by the sexually transmitted Neisseriagonorrhea has been demonstrated to cause leukocytospermia(defined as >1 million leukocytes in each ml of semen) that inturn increase ROS production.47 When present in high levels

activated leukocytes produce ROS that leak out of the cell mem-brane causing damage to surrounding cellular elements includ-ing spermatozoa. The mechanism responsible for diseases suchas chronic renal scarring and pyelonephritis48 can lead to infertil-ity. According to a study by Depuydt et al, leukocytospermiaand infection of the male accessory gland reduced the fertilizingpotential by affecting the sperm parameters in vitro and in vivo.49

Several studies have shown that patients with leukocytospermiahave abnormal DNA integrity.21,50 Henkel et al demonstratedthat many patients who had <1 million seminal leukocytes perml could sometimes have abnormal sperm motility and poorDNA integrity.51

VARICOCELE, OS AND INFERTILITYClinical or subclinical varicocele52 causes male infertility in

about 15% of infertile couples.53 These patients have increasedROS in the serum, testes and semen samples.54 ROS in patientswith varicocele are formed due to the excessive presence ofxanthine oxidase, a source of superoxide anion from the substratexanthine, and nitric oxide in dilated spermatic veins. IncreasedNO has been demonstrated in the spermatic veins of patientswith varicocele,55,56 which could be responsible for the sper-matozoal dysfunction.57

Serum proteins are prone to oxidative damage as evidencedby the accumulation of protein carbonyls in blood drawn fromthe spermatic veins of patients with varicocele. Furthermore,increased oxidative products of proteins is correlated with de-creased antioxidant capacity in seminal plasma.58,59 Varicocelec-tomy increases the concentrations of antioxidants, such as super-oxide dismutase, catalase, glutathione peroxidase and vitaminC, in seminal plasma as well as improves sperm quality.60 Thelevels of malondialdehyde, which is a by-product of lipid peroxi-dation, are increased in testicular tissue of infertile patients withvaricocele. It is possible that ROS may have a role in varicoceleassociated testicular dysfunction.54 It is also interesting to notethat the generation of ROS is proportional to the severity ofvaricocele.61 Patients with varicocele have increased 8-OHdGindicating oxidative DNA damage.62,63 Thus, increased OS islikely to be one of the important etiological factors in infertilepatients with varicocele.

CRYPTORCHIDISM, OS AND INFERTILITYCryptorchidism has been associated with increased apoptotic

degeneration of the testes resulting in increased ROS produc-tion.64 Xanthine oxidase is believed to be instrumental in ROSformation in cryptorchid testes. Allopurinol, a xanthine oxidaseinhibitor, decreases apoptosis and promotes spermatogenesis intestes.65 Cryptorchidism also induced heat stress causing DNAfragmentation.66 Evidence from animal models suggests thatdegenerative testicular response to heat is determined, at leastin part, by a genetic component.67 However, it is not knownwhether the heat induced ROS formation is responsible forinfertility in these patients. It is now evident that an increase inNO and a decrease in total antioxidant capacity occur withcryptorchidism and contribute to accelerated germ cellapoptosis. Administration of a nitric oxide synthase inhibitor, N-omega-nitro-L-arginine methyl ester, attenuated apoptosis and

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improved spermatogenesis in experimental (murine) models ofcryptorchidism.68

ROLE OF LIFESTYLE BEHAVIOR AND ENVIRONMENTIN INFERTILITY

Cigarette smoke contains harmful substances such as alkaloids,nitrosamines, nicotine, cotinine and hydroxycotinine, and manyof these substances are considered to generate these free radi-cals.33,69 Several studies have reported that cigarette smoking isassociated with reduced sperm quality (count and abnormalmorphology).70,71 Smoking also affects sperm DNA as evi-denced by the increased level of 8-oxo-deoxyguanosine in sper-matozoa.72 In addition, infertile smokers showed higher levelsof seminal OS than infertile non-smokers.73 Thus, the decreasedsperm quality in smokers could be due to oxidative stress. SinceOS has an adverse effect on fertility potential, it is recommendedthat physicians advise infertile smokers to quit smoking. TheOS effect in smokers could also be due to leukocytes in thesemen, as the number of leukocytes was significantly higher insmokers.

It has been reported that environmental pollution can inducethe production of the ROS ( O2

T — and OHT).74 In a study ofworkers exposed to traffic pollution the time of exposure topollutants was negatively correlated with sperm quality, reduc-ing fertility in young and middle-aged men.75 Tollgate workersshowed poorer sperm quality than age matched men living in thesame area who were not exposed to the same level of automobilepollution. Furthermore, sperm function tests revealed that theseworkers had sperm with lower motility. In the subset of workersabnormal sperm parameters, e.g., motility, viability, membranefunction, nuclear DNA integrity, linearity and amplitude ofsperm lateral head movement, were inversely correlated withmethemoglobin levels, a marker for nitrogen oxide, whereassperm count and viability were inversely correlated with lead(Pb) levels.75

LABORATORY INVESTIGATIONS OF INFERTILITYThe male infertility evaluation starts with a detailed history

on general and gonadal growth and development, medications,infection, previous trauma or surgery, exposure to radiation,steroids, harmful chemicals and sexual history. A thorough his-tory often provides clues to the nature of the infertility problem.Followed by history, a complete physical examination of themale reproductive system is crucial for infertility evaluation.Abnormal genitalia and loss of pubic or facial hair may indicateandrogen deficiency. A proper physical examination helps toidentify varicocele, hernia or epididymal thickening, some of theconditions that affect sperm maturation and transport. Decreasedvolume density of the semeniferous tubule is reflected by de-creased testicular size.

Laboratory evaluation of the infertile male starts with routinesemen analysis, which measures semen volume, pH, sperm con-centration, motility, morphology and leukocytes. Within 1month a second semen sample is collected and analyzed to adjustfor the frequent variability in semen quality. In the presence ofan abnormality in the semen sample serum hormones, especially

testosterone and follicle-stimulating hormone, are measured. Inthe event of a normal semen analysis no further testing is recom-mended, including test for antisperm antibodies, semen culture,sperm-cervical mucus interaction, sperm function tests (com-puter assisted semen analysis, zona-free hamster oocyte penetra-tion test, human zona pellucida binding test etc.), biochemicalanalysis of fructose, reactive oxygen species, levels of antioxi-dants and assessment of sperm DNA damage.

Assessment of oxidative stress. To accurately quantify oxida-tive stress, levels of ROS and antioxidants should be measuredin fresh samples. Direct methods such as pulse radiolysis andelectron-spin resonance spectroscopy have been useful for othersystems of the body. However, the relatively low volume of theseminal plasma, short life span of ROS and need to evaluate infresh samples have led to non-usage of direct methods for themale reproductive system. Several indirect techniques have beenused to measure levels of various ROS and antioxidants.

One of the most widespread methods of measuring ROSis the chemiluminescence assay, which uses sensitive probessuch as luminol and lucigenin for quantification of redoxactivities of the spermatozoa.76 Although the sensitivity ofthese probes is high, they are susceptible to interference. Leuko-cyte contamination is a major confounder. Also, the time ofanalysis after collection (<1 hour) and the high sperm count (>1 × 106/ml) requirement are some of the drawbacks to thistechnique.

Another method of measuring intracellular ROS inside thecell is by flow cytometery. Different probes such as 2’, 7’-dichlorofluorescin-diacetate, hydroethidine are used.77 Spermcells are exposed to the probes that react with ROS to emit ared fluorescence.77 The assay is highly sensitive and requires arelatively low number of cells but is more expensive and requiresexpertise to handle sophisticated equipment.78 The colorimetrictechnique is also widely used for indirectly quantifying ROS.It is based on the principle of spectrophotometry, and measureslipid peroxide end products such as malondialdehyde, lipid hy-droperoxides, isoprostanes etc.

Antioxidant measurement. The presence of low antioxidant inthe seminal plasma is another important reason for increasedOS leading to male infertility. Hence, it is important to measurethe total antioxidant capacity of the semen. Different methodssuch as oxygen radical absorption capacity,79 ferric reducingability of plasma80 and phycoerythrin fluorescence based assayare available for measuring TAC. However, the most widelyused method for measuring TAC in semen is enhanced chemilu-minescence. This method requires expensive instrumentation,and is cumbersome and time-consuming. Another emergingmethod for measurement of TAC is the colorimetric assay.First described by Miller et al in 1993, this method gained itspopularity as the simple, rapid and inexpensive alternative toenhanced chemiluminescence method.81

To accommodate for the variations in ROS and TAC values,our group proposed a concept of combined ROS-TAC score.82

ROS-TAC score was computed using principal component anal-ysis. We calculated ROS-TAC scores from proven fertile menwith low levels of ROS. The composite ROS-TAC scores from

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these men were representative of the fertile group and any score<30 was considered infertile.

Identification of DNA damage and its relevance to infertilityevaluation. Multiple techniques are used to measure the DNAdefects in human spermatozoa.18 Some of the currently availabletests evaluate the integrity of sperm DNA, including terminaldeoxynucleotidyl transferase-mediated deoxyuridine triphos-phate nick end labeling; the sperm chromatin structure assay(SCSA) using various dyes such as acridine orange, aniline blue,toluidine blue and chromomycin A3; comet assay; in situ nicktranslation; and DNA breakage detection-fluorescent in situ hy-bridization assay. However, all of the major DNA fragmentationtests are similar in their recognition of a threshold that placesa man at infertility risk. Quantitative PCR analysis is being usedto identify and quantify the amount of DNA damage. With thistechnique a particular segment of DNA is amplified and thefrequency of repetition of the damaged segment is identified.27

Although sensitive, this technique has limitations associatedwith selection of appropriate PCR segment and other amplifica-tion issues.

It is difficult to determine whether sperm DNA fragmentationcan become a useful test as part of routine semen analysis inan infertility clinic. An association among sperm DNA fragmen-tation (expressed as SCSA DFI), poor embryo quality83 andfailed pregnancy in ICSI83,84 has been reported. Miscarriagerates were the highest especially for ICSI procedures inpatients whose SCSA-DFI was >30%.84,85 The odds of achiev-ing pregnancy with intrauterine insemination is 16 times higherin subjects with SCSA DFI <27% compared to patients withSCSA DFI >27%.86 Thus, SCSA DFI status has the potentialto become a useful tool in infertility evaluation. However, pa-tients must be counseled that a high DFI does not preclude anormal full-term pregnancy and that they should not rush intothe idea of using donor sperm if SCSA DFI is >30%.

MANAGEMENT OF MALE INFERTILITY: ROLE OFANTIOXIDANTS

Lifestyle modification. As reported earlier, infertile couplesdesiring children should be advised to abstain from smoking asit is known to increase OS in seminal plasma. Also it is importantto avoid excessive exposure to environmental pollution in work-ing and living conditions to prevent OS related sperm damage.

Antioxidants. Antioxidants are the main defense against OSinduced by free radicals. These can be classified as preventativeand scavenger antioxidants. Preventative antioxidants, such asmetal chelators and metal binding proteins, block the formationof new ROS, whereas scavenger antioxidants, such as vitamins Eand C, beta-carotene and other antioxidant dietary supplements,glutathione and enzymes, remove ROS already generated bycellular oxidation. Dietary products such as lycopene (tomatoproducts), vitamin C (citrus fruits and green vegetables) andvitamin E (vegetable oils, nuts and seeds) are some of theexcellent source of antioxidants.

Transition metal ions, mainly iron, generate highly reactivehydroxyl radicals by Fenton’s reaction.45,87 These radicals stim-ulate lipid peroxidation by decomposing the peroxides into per-oxyl and alkoxyl radicals, and triggering a chain reaction of

lipid peroxidation. Human semen contains metal chelatorssuch as transferrin, lactoferrin and ceruloplasmin that re-duce lipid peroxidation of the sperm plasma membrane,protecting its integrity.88

Metal chelators, such as DL-penicillamine, 2,3–dimer-captopropan-1 sulfonate and meso-2,3-dimercapto-succinicacid, showed enhancement of sperm quality.89 Zinc replacementof cadmium, which is a class B element and toxic to spermato-genesis, improves success of assisted reproduction technology.Also, protein metallothionein binds to cadmium and eliminatesit from the body, which improves spermatogenesis, maturationand capacitation of spermatozoa.90 Scavenger antioxidants re-move ROS and prevent the propagation of lipid peroxidationchain reaction. Dietary antioxidants are an important componentof this group of antioxidants. Fruits and vegetables as well asdaily dietary supplements constitute the potential sources ofvarious antioxidants.

According to The National Academy of Sciences, 60 mg ofvitamin C a day is recommended for an adult male. Vitamin Edaily requirement varies from 50 to 800 mg depending on theintake of fruits, vegetables, tea or wine. Selenium and carot-enoids work synergistically with vitamin E with recommendeddietary allowances of 70 and 1000 mcg a day, respectively.91

Vitamin supplements, mainly vitamins E and C which are chainbreaking antioxidants, can reduce oxidative stress in the seminalplasma of infertile men.68 Vitamin C in oral doses of 200 to1000 mg a day increases sperm counts in infertile males.92 Arandomized crossover study revealed that oral administration of600 mg a day of vitamin E improved sperm function as assessedby the zona binding test although other sperm parameters werenot affected.93 Quercetin and xanthohumol are some of theimportant dietary flavonoids, and they function synergisticallywith vitamin C.94 Both compounds conserve the alpha-tocoph-erol content of low density lipids and prevent or delay it fromundergoing lipid peroxidation.95

Coenzyme Q-10 found in the sperm midpiece96 recycles vita-min E and prevents its pro-oxidant activity.21 It has been shownto prevent ROS formation with improvement in fertilizationrates.97,96 Glutathione is the most abundant antioxidant foundin the body, and it has an important role in protecting lipids,proteins and nucleic acids against oxidative damage. It has beendemonstrated that 600 mg glutathione therapy intramuscularlysignificantly increased sperm motility, particularly forward pro-gression.98 Carotenoids such as beta-carotene and lycopene alsoform an important component of the antioxidant defense.99 Beta-carotenes protect the plasma membrane against lipid peroxida-tion. Lycopene has been shown to be twice as potent as beta-carotene in inhibiting lipid peroxidation in blood plasma.100

Selenium at a dose of 225 mcg a day orally for 3 monthssignificantly decreased malondialdehyde concentrations in sem-inal plasma and improved sperm motility.101 Carnitines are di-etary antioxidants that decrease mitochondrial ROS productionand improve sperm motility.4,102 Oral intake of L-carnitines for6 months improved motility as well as sperm concentration.98

Apart from the traditional antioxidant therapy other modalitiesof treatment have been successfully tried. When increased

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apoptosis is suspected, specific apoptotic inhibitors have pre-vented cell death and improved survival of the germ cells. Sphin-gosine-1-phosphate, an apoptotic inhibitor, at doses of 1 and 10mmol/l suppressed apoptosis in germ cells of testis.103 Similarly,N-acetyl cysteine when given in concentrations of 125, 100, 50and 25 mmol/l suppressed germ cell death in a dose dependentmanner.104

Assisted reproductive technology. The first ‘‘test-tube’’ baby,Louise Brown, was born in 1978. Since then assisted reproduc-tion technologies have helped many infertile couples to havechildren. In all of these years there has been a phenomenalimprovement in every sphere of ART. In vitro fertilization (eggamete intra-fallopian transfer, zygote intra-fallopian transfer)and ICSI form the major components of ART.

ROS is produced in the ART setting in a number of ways.Oocytes and embryos, cumulus cells and spermatozoa are themajor sources of ROS. ROS can be decreased using spermpreparation techniques to enhance and maintain sperm qualityafter ejaculation.105 Several sperm preparation techniques, suchas migration-sedimentation, density centrifugation gradient andglass-wool filtration, significantly reduce the level of ROS byremoving leukocytes, which are its major source.89,96

Assisted reproductive techniques may show significant im-provement in in vitro supplementation of antioxidants and metalchelators to achieve a better success.22 Excellent results wereobtained with the use of many natural and synthetic compounds,such as rebamipide, pentoxyfylline, vitamins E and C, SOD,catalase etc, at different stages of ART procedures. With theabundance of literature supporting the use of antioxidants duringART procedures, their judicial use is recommended.

Identification of appropriate candidates and procedure is acrucial part since multiple factors influence results, and thiswould prevent the emotional and financial burden associatedwith ART. Adding testicular sperm extraction and percutaneousepididymal sperm aspiration to the armor of ART, and improve-ment in cryopreservation techniques help patients with condi-tions such as azoospermia and cancer.

CONCLUSIONIn the last 15 years we have witnessed a phenomenal growth

in our knowledge of male reproduction, sperm function, anddevelopment of diagnostic tools and treatment modalities formale infertility. Presently there are several different techniquesavailable to predict the fertility status of a man. The WHO isfrequently updating their training manuals with more focus onquality control and standardization of semen analysis proce-dures. In addition, with increased understanding of oxidativestress, several new modalities of treatment are now being triedto improve fertility. Many new antioxidants are now availablethat can decrease oxidative stress and improve sperm quality.However, due to lack of scientific evidence of their effective-ness, these are not yet approved by FDA or other Governingbodies and such an approach raises many concerns.

Although current evidence supports the use of systemic antiox-idants for the management of select cases of male infertility aswell as in vitro supplements during various sperm preparation

techniques, a definitive conclusion cannot be drawn from theavailable studies, mainly because OS is not the only cause ofmale infertility. Therefore, antioxidant therapy should be triedonly in cases with increased oxidative stress status or establishedDNA damage. However, evaluation of OS status is still not aroutine andrology laboratory procedure in clinical practicemainly due to various technical issues.

Our current focus is to validate and simplify the evaluation ofOS status so that it can be performed in a routine andrologyclinic and/or laboratory without the need for sophisticated instru-mentation. Also, dosage and duration of the antioxidant treat-ment are still widely debated and should be tailored to theneed of each individual. In the future large, placebo controlledmulticenter trials will be required to gain a better insight intothis important issue. ART procedures should be recommendedfor carefully chosen infertile couples. This selection process willensure better success especially in cases of idiopathic infertility,prevent the social and psychological trauma associated withthese techniques, and decrease the exorbitant associated costs.

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Study Questions Volume 26 Lesson 1

1. What are the major sources of ROS in semen during in-fection?

a. Leukocytesb. Normal spermatozoac. Morphologically abnormal spermatozoad. RBCe. Epithelial cells

2. What is the most common method of measuring ROS atpresent?

a. Chemiluminescenceb. Flow cytometryc. Electron spin resonanced. Spectrophotometrye. HPLC

3. Which of the following is a metal chelator that has proven todecrease the lipid peroxidation of sperm plasma membrane?

a. D-penicillamineb. Transferrinc. Beta-carotened. Glutathionee. Iron

Fill in answers on answer sheets provided or take this test online at http://www.auanet.org/eforms/cme/

4. Spermatic veins of patients with varicocele show increasedlevels of which compound?

a. H2O2

b. Superoxidec. NOd. Hydroxyl radicale. Catalase

5. What is the suggested DNA fragmentation index rate abovewhich the probability of spontaneous miscarriage is thehighest?

a. 15%b. 35%c. 30%d. 50%e. 90%