From Research Bench to Clinical Practice

16 Pages·2002·0.57 MB·English

JournalofAndrology,Vol.23,No.6,November/December2002
Copyright(cid:113)AmericanSocietyofAndrology
Oxidative Stress and Male Infertility: Review
From Research Bench to Clinical Practice
RAMADAN A. SALEH AND ASHOK AGARWAL amount necessary to maintain normal cell function. It
is not surprising that a battery of different antioxidants
Center for Advanced Research in Human Reproduction,
is available to protect spermatozoa against oxidants
Infertility, and Sexual Function, Urological Institute,
(Sies, 1993).
The Cleveland Clinic Foundation, Cleveland, Ohio.
Seminal oxidative stress (OS) develops as a result of
an imbalance between ROS generating and scavenging
ExtensiveresearchinourcenterattheClevelandClinic activities (Sikka et al, 1995; Sharma and Agarwal,
indicates that the seminal oxidative stress test has di- 1996; Sikka, 2001). Spermatozoa are particularly sus-
agnostic and prognostic capabilities beyond those of ceptible to OS-induced damage because their plasma
conventional tests of sperm quality or functions. An membranes contain large quantities of polyunsaturated
oxidative stress test can accurately discriminate be- fattyacids(PUFAs;AlvarezandStorey,1995)andtheir
tween fertile and infertile men and identify patients
cytoplasm contains low concentrations of scavenging
with a clinical diagnosis of male-factor infertility who
enzymes (Jones et al, 1979; Aitken and Fisher, 1994;
are likely to initiate a pregnancy if they are followed
deLamirandeandGagnon,1995;SharmaandAgarwal,
over a period of time. In addition, the test can help
1996). In addition, the intracellular antioxidant en-
select subgroups of patients with infertility in which
zymes cannot protect the plasma membrane that sur-
oxidative stress is a significant factor, and who may
rounds the acrosome and the tail, forcing spermatozoa
benefit from antioxidant supplementation. Incorpora-
to supplement their limited intrinsic antioxidant de-
tion of such a test into routine andrology laboratory
fenses by depending on the protection afforded by the
practice may be of particular importance to the future
seminal plasma (Iwasaki and Gagnon 1992; Zini et al,
management of male infertility.
1993). Oxidative stress attacks not only the fluidity of
In recent years, the generation of reactive oxygen
the sperm plasma membrane, but also the integrity of
species (ROS) in the male reproductive tract has be-
DNA in the sperm nucleus (Aitken, 1999).
come a real concern because of their potential toxic
effects at high levels on sperm quality and function. Even though OS has been established as a majorfac-
ROS are highly reactive oxidizing agents belonging to torinthepathogenesisofmaleinfertility,thereisalack
the class of free radicals (Aitken, 1994). A free radical of consensus as to the clinical utility of seminal OS
is defined as ‘‘any atom or molecule that possesses one testing in an infertility clinic. One important reason for
or more unpaired electrons’’ (Warren et al, 1987). Re- the inability to use an OS test in clinical practice may
cent reports have indicated that high levels of ROS are be the lack of a standard protocol toassessseminalOS.
detected in semen samples of 25% to 40% of infertile The role of OS in male infertility has been the focus of
men (de Lamirande et al, 1995; Padron et al, 1997). our research center at the Cleveland Clinic Foundation
However, a strong body of evidence suggests thatsmall for the past 10 years. The main objective of our re-
amounts of ROS are necessary for spermatozoa to ac- search inthisareawastotransferthisimportantknowl-
quire fertilizing capabilities (Aitken, 1999). edge from the research bench to clinical practice. We
Spermatozoa, like all cells living in aerobic condi- designed studies with the following aims: 1) to under-
tions, constantly face the oxygen (O ) paradox: O is
2 2 stand the precise mechanism by which OS develops in
requiredtosupportlife,butitsmetabolitessuchasROS
semen, which we thought could help develop strategies
can modify cell functions, endanger cell survival, or
to overcome the problem, 2) to establish assays for ac-
both (de Lamirande and Gagnon, 1995). Hence, ROS
curate and reliable assessment of seminal OS, and 3)
must be continuously inactivated to keep only a small
to identify the clinical significance of testing seminal
OS in the male infertility practice. In this review, we
Correspondenceto: Ashok AgarwalPhDHCLD,Director,Centerfor summarize the efforts of our program to explore the
AdvancedResearchinHumanReproduction,InfertilityandSexualFunc- role of OS in male infertility. We also include detailed
tionUrologicalInstitute,TheClevelandClinicFoundation,9500Euclid
information on the protocolsintroducedrecentlybyour
Avenue,DeskA19.1,Cleveland,OH44195(e-mail:[email protected])
center for the assessment of seminal OS in a clinical
ReceivedforpublicationFebruary28,2002;acceptedforpublication
June10,2002. andrology laboratory.
737
738 Journalof Andrology · November/December2002
(cid:82)(cid:101)(cid:97)(cid:99)(cid:116)(cid:105)(cid:118)(cid:101)(cid:79)(cid:120)(cid:121)(cid:103)(cid:101)(cid:110)(cid:83)(cid:112)(cid:101)(cid:99)(cid:105)(cid:101)(cid:115)(cid:97)(cid:110)(cid:100)(cid:83)(cid:112)(cid:101)(cid:114)(cid:109)(cid:80)(cid:104)(cid:121)(cid:115)(cid:105)(cid:111)(cid:108)(cid:111)(cid:103)(cid:121) bound to oxygen reduction products, they can produce
secondaryandmorereactiveoxidants,particularlythehy-
Until recently, ROS were exclusively considered toxic to
droxyl radical (OH•) (Halliwell, 1990).
human spermatozoa. The idea that limited amounts of
Interception—Free radicals have a tendency toward
ROS can intervene in a physiological manner in the reg-
chainreaction(ie,acompoundcarryinganunpairedelec-
ulation of some sperm functions was first evoked in a
tron will react with another compound to generate an un-
study by Aitken et al (1989). The authors found that low
paired electron, ‘‘radical begets radical’’). Hence, the ba-
levels of ROS can enhance the ability of human sper-
sic problem is to break this chain reaction by the for-
matozoa to bind with zonae pellucida, an effect that was
mation of nonradical end products (Sies, 1993). Alpha-
reversed by adding vitamin E. Other studies have found
tocopherol (vitamin E), a chain-breaking antioxidant,
that incubating spermatozoa with low concentrations of
inhibits LPO in membranes by scavenging peroxyl (RO•)
hydrogen peroxide (H O ) stimulates sperm capacitation,
2 2 and alkoxyl (ROO•) radicals. The ability of (cid:97)-tocopherol
hyperactivation, and the ability of the spermatozoato un-
tomaintainasteadystaterateofperoxylradicalreduction
dergo the acrosome reaction and oocyte fusion (de La-
in the plasma membrane depends on the recycling of (cid:97)-
mirande and Gagnon, 1993; de Lamirande et al, 1993;
tocopherol by external reducing agents such as ascorbate
Griveau et al, 1994; Aitken et al, 1995; Kodoma et al,
or thiols (Wefers and Sies, 1988). In this way, (cid:97)-tocoph-
1996; Aitken, 1997). ROS other than H O ,such asnitric
2 2 erolisabletofunctionagainasafreeradicalchain-break-
oxide and superoxide anion (O •(cid:50)), have also been shown
2 ing antioxidant, even though its concentration is low
to promote sperm capacitation and the acrosome reaction
(Buettner, 1993). For efficient interception, the radical to
(Griveau et al, 1995a; Zini et al, 1996).
be intercepted must have a relatively long half-life (Sies,
1993). The peroxyl radicals are major reaction partners
(cid:77)(cid:101)(cid:99)(cid:104)(cid:97)(cid:110)(cid:105)(cid:115)(cid:109)(cid:111)(cid:102)(cid:65)(cid:110)(cid:116)(cid:105)(cid:111)(cid:120)(cid:105)(cid:100)(cid:97)(cid:110)(cid:116)(cid:80)(cid:114)(cid:111)(cid:116)(cid:101)(cid:99)(cid:116)(cid:105)(cid:111)(cid:110)(cid:105)(cid:110)(cid:83)(cid:101)(cid:109)(cid:101)(cid:110)
because their half-life extends into the range of seconds
It is interestingthatseminalplasmaiswellendowedwith (7seconds).Incontrast,thehydroxylradical,withitshigh
an array of antioxidant defense mechanisms to protect reactivity and extremely short half-life (10(cid:50)9 seconds),
spermatozoa against OS (Sikka, 1996; Smith et al, 1996; cannot be intercepted with reasonable efficiency (Sies et
Armstrong et al, 1998). These mechanisms compensate al, 1992).
forthedeficiencyincytoplasmicenzymesinsperm(Don- Repair—In some situations, the damage caused by ox-
nelly et al, 1999). Seminal plasma contains a number of idants may be repaired. Unfortunately, spermatozoa are
enzymatic antioxidants such as superoxide dismutase unable to repair the damage induced by OS because they
(SOD; Alvarez et al, 1987), the glutathione peroxidase/ lack the cytoplasmic enzyme systems that are requiredto
glutathione reductase (GPX/GRD) system (Chaudiere et accomplish this repair. This is one of the features that
al, 1984), and catalase (Jeulin et al, 1989). In addition, make spermatozoa unique in their susceptibility to oxi-
seminal plasma contains a variety of nonenzymatic anti- dative insult (Alvarez et al, 1987; Aitken et al, 1989).
oxidants such as ascorbate (Fraga et al, 1991), urate
(Thiele et al, 1995), (cid:97)-tocopherol (Aitken and Clarkson, (cid:80)(cid:97)(cid:116)(cid:104)(cid:111)(cid:103)(cid:101)(cid:110)(cid:101)(cid:115)(cid:105)(cid:115)(cid:111)(cid:102)(cid:83)(cid:101)(cid:109)(cid:105)(cid:110)(cid:97)(cid:108)(cid:79)(cid:120)(cid:105)(cid:100)(cid:97)(cid:116)(cid:105)(cid:118)(cid:101)(cid:83)(cid:116)(cid:114)(cid:101)(cid:115)(cid:115)
1988; Moilanen et al, 1993), pyruvate (deLamirandeand Thetermoxidativestressisappliedwhenoxidantsout-
Gagnon, 1992), glutathione (Lenzi et al, 1994), taurine, number antioxidants (Sies, 1993), peroxidation products
and hypotaurine (Alvarez and Storey, 1983). develop (Spitteler, 1993), and when these phenomena
It has been reported that seminal plasma from fertile causepathologicaleffects(Janssenetal,1993).Oxidative
men has a higher total antioxidant capacity (TAC) than stresshasbeenimplicatedinnumerousdiseasestatessuch
seminal plasma from infertile men (Lewis et al, 1995). as cancer, connective tissue disorders, aging, infection,
However, pathological levels of ROS detected in semen inflammation, acquired immunodeficiency syndrome,and
from infertile men are more likely a result of increased male infertility (Clark et al, 1986; Aitken et al, 1992,
ROS production rather than reduced antioxidant capacity 1995).
of the seminal plasma (Zini et al, 1993). Antioxidant de- In the context of human reproduction, a balance nor-
fense mechanisms include three levels of protection: 1) mally exists between ROS generation and scavenging in
prevention, 2) interception, and 3) repair (Sies, 1993). the male reproductive tract. As a result, only a minimal
Prevention—Prevention of ROS formation is the first amountofROSremains,whichisneededtoregulatenor-
lineofdefenseagainstoxidativeinsult.Anexampleisthe mal sperm functions such as sperm capacitation, acro-
binding of metal ions, iron, and copper ions in particular, some reaction, and sperm-oocyte fusion (Gagnon et al,
which prevents them from initiating a chain reaction 1991; de Lamirande and Gagnon, 1994; Griveau and Le
(Sies, 1993). Chelation of transition metals is a major Lannou, 1997). Excessive ROS production that exceeds
means of controlling sperm lipid peroxidation (LPO)and critical levels can overwhelm all antioxidant defense
DNA damage. When transition metals become loosely strategiesofspermatozoaandseminalplasmacausingOS
SalehandAgarwal · OxidativeStressandMaleInfertility 739
(Sikka et al, 1995; Sharma and Agarwal, 1996; de La- zation, both of which are associated with a reduction in
mirande et al, 1997; Sikka, 2001). All cellular compo- membrane fluidity that is necessary for sperm-oocyte fu-
nents including lipids, proteins, nucleic acids, and sugars sion (de Lamirande and Gagnon, 1995). Another hypoth-
are potential targets for OS. The extent of OS-induced esis is that H O can diffuse across the membranes into
2 2
damage depends not only on the nature and amount of the cells and inhibit the activity of some enzymes such
ROS involved but also on the moment and duration of as glucose-6-phosphate-dehydrogenase (GPD). This en-
6
ROS exposure and on extracellular factors such as tem- zyme controls the rateofglucoseflux throughthehexose
perature, oxygen tension, and the composition of the sur- monophosphate shunt, which in turn, controls the intra-
rounding environment including ions, proteins, and ROS cellular availability of nicotinamide adenine dinucleotide
scavengers. phosphate (NADPH). This in turn is used as a source of
Lipid Peroxidation of Sperm Plasma Membrane—Lip- electrons by spermatozoa to fuel the generation of ROS
id peroxidation is broadly defined as ‘‘oxidative deterio- by an enzyme system known as NADPHoxidase(Aitken
ration of PUFA’’ (ie, fatty acids that contain more than et al, 1997). Inhibition of G PDleads to adecreaseinthe
6
two carbon-carbon double bonds; Halliwell, 1984). The availability of NADPH and a concomitant accumulation
LPO cascade occurs in two fundamentalstages:initiation of oxidizedglutathioneandreducedglutathione.Thiscan
and propagation. The hydroxyl radical (OH•) is a pow- reduce the antioxidant defenses of the spermatozoa and
erful initiator of LPO (Aitken and Fisher, 1994). Most increase peroxidation of membrane phospholipids (Gri-
membrane PUFAs have unconjugated double bonds that veau et al, 1995a).
are separated by methylene groups. The presence of a Oxidative Stress-Induced DNA Damage—Two factors
double bond adjacent to a methylene group makes the protect sperm DNA from oxidative insult: the character-
methylene C-H bonds weaker and, therefore, hydrogenis istic tight packaging of the DNA and the antioxidants
more susceptible to abstraction. Once this abstractionhas present in seminal plasma (Twigg et al, 1998a). Studies
occurred, the radical produced is stabilized by the rear- in which the sperm was exposed to artificially produced
rangementofthedoublebonds,whichformsaconjugated ROS resulted in a significant increase in DNAdamagein
diene radical that can then be oxidized. This means that the form of modification of all bases, productionofbase-
lipids, which contain many methylene-interrupted double free sites, deletions, frame shifts, DNA cross-links, and
bonds, are particularly susceptible to peroxidation (Blake chromosomal rearrangements (Duru et al, 2000). Oxida-
tive stress has also been correlated with high frequencies
et al, 1987). Conjugated dienes rapidly react with O to
2
form a lipid peroxyl radical (ROO•), which abstracts hy- of single and double DNA strand breaks (Twigg et al,
1998a; Aitken and Krausz, 2001).
drogen atoms from other lipid molecules to form lipid
Strong evidence suggests that high levels of ROS me-
hydroperoxides(ROOH).Lipidhydroperoxidesarestable
diate the DNA fragmentation commonly observed in
under physiological conditions until they contact transi-
spermatozoa of infertile men (Kodama et al, 1997; Sun
tion metals such as iron or copper salts. These metals or
et al, 1997). This information has important clinical im-
their complexes cause lipid hydroperoxides to generate
plications, particularly in the context of assisted repro-
alkoxyl and peroxyl radicals, which then continue the
ductivetechniques(ART).SpermatozoaselectedforART
chain reaction within the membrane and propagate the
most likely originate from an environment experiencing
damage throughout the cell (Halliwell, 1984). Propaga-
OS,andahighpercentageofthesespermmayhavedam-
tion of LPO depends on the antioxidant strategies em-
agedDNA(Kodamaetal,1997;Lopesetal,1998).There
ployed by spermatozoa. One of the by-products of lipid
is a substantial risk that spermatozoa carrying damaged
peroxide decomposition is malondialdehyde. This by-
DNA are being used clinically in this form of therapy
product has been used in biochemical assays to monitor
(Twigg et al, 1998b). When intrauterine insemination or
the degree of peroxidative damage in spermatozoa (Ait-
in vitro fertilization is used, such damage may not be a
ken et al, 1989; Aitken and Fisher, 1994). The results of
causeofconcernbecausethecollateralperoxidativedam-
such an assay exhibit an excellent correlation with the
age to the sperm plasma membrane ensures that fertiliza-
degree to which sperm function is impaired in terms of
tion cannot occurwithaDNA-damagedsperm.However,
motilityandthecapacityforsperm-oocytefusion(Aitken
whenintracytoplasmicsperminjection(ICSI)isused,this
et al, 1993; Sidhu et al, 1998).
natural selection barrier is bypassed and a spermatozoon
Impairment of Sperm Motility—The increased forma-
with damaged DNA may be directly injected into theoo-
tionofROShasbeencorrelatedwithareductionofsperm
cyte (Twigg et al, 1998b; Aitken, 1999).
motility (Aitken et al, 1989; Iwasaki and Gagnon, 1992;
Lenzi et al, 1993; Agarwal et al, 1994a; Armstrong et al, (cid:83)(cid:111)(cid:117)(cid:114)(cid:99)(cid:101)(cid:115)(cid:111)(cid:102)(cid:69)(cid:120)(cid:99)(cid:101)(cid:115)(cid:115)(cid:105)(cid:118)(cid:101)(cid:82)(cid:101)(cid:97)(cid:99)(cid:116)(cid:105)(cid:118)(cid:101)(cid:79)(cid:120)(cid:121)(cid:103)(cid:101)(cid:110)(cid:83)(cid:112)(cid:101)(cid:99)(cid:105)(cid:101)(cid:115)
1999). The link between ROS and reduced motility may (cid:80)(cid:114)(cid:111)(cid:100)(cid:117)(cid:99)(cid:116)(cid:105)(cid:111)(cid:110)(cid:105)(cid:110)(cid:83)(cid:101)(cid:109)(cid:101)(cid:110)
be due to a cascade of events that result in a decrease in Morphologically abnormal spermatozoa and seminal leu-
axonemal protein phosphorylation and sperm immobili- kocyteshavebeenestablishedasthemainsourcesofhigh
740 Journalof Andrology · November/December2002
ROS production in human ejaculates (Aitken and West, diated by the cytosolic enzyme G PD (Figure 1) (Aitken,
6
1990; Kessopolou et al, 1992). Virtually every human 1999).
ejaculate is contaminated with potential sources of ROS Recent studies by Ollero et al (2001) and Gil-Guzman
(Aitken,1995).Itfollowsthatsomespermcellswillincur et al (2001) have shown that levelsof ROSproductionin
oxidative damage and a concomitant loss of function in semen were negatively correlated with the percentage of
every ejaculate. Thus, the impact of OS on male fertility normal sperm forms as determined by World Health Or-
isaquestionofdegreeratherthanthepresenceorabsence ganization (WHO, 1999) classification and by Kruger’s
of pathology. strictcriteria(Krugeretal,1987).Thecorrelationofsem-
Clear evidence suggests that human spermatozoa pro- inal ROS levels with morphologically abnormal sperm
duce oxidants (Aitken et al, 1992; Hendin et al, 1999; was also evident when morphology slides were scored
Gil-Guzmanetal,2001).SpermatozoamaygenerateROS using a sperm deformity index (SDI) (r (cid:53) .31; P (cid:53) .01;
in two ways: 1) the NADPH oxidase system at the level (unpublished data). The SDI was introduced by Aziz et
of the sperm plasma membrane (Aitken et al, 1992), and al (1996) as a novel expression of sperm morphological
2) the NADH-dependent oxido-reductase (diphorase) at abnormalitiesthatwerefoundtobehighlycorrelatedwith
the level of mitochondria (Gavella and Lipovac, 1992). fertilization in vitro. This new method for sperm mor-
The mitochondrial system is the major source of ROS in phology assessment uses a multiple entry scoring tech-
spermatozoa in infertile men (Plante et al, 1994). niqueinwhichanabnormalspermisclassifiedmorethan
Effect of Sperm Morphology on ROSProduction—Go- once if more than one deformity is observed. SDI is cal-
mez et al (1998) have indicated that levels of ROS pro- culated by dividing the total number of deformities ob-
duction by pure sperm populations were negatively cor- served by the number of sperm randomly selected and
related with the quality of sperm in the original semen. evaluated, irrespective of their morphological normality.
The link between poor semen quality and increased ROS In our study, SDI was significantly higher in a group of
generation lies in the presence of excess residual cyto- infertile men who had high levels of seminal ROS than
plasm (cytoplasmic droplet). When spermatogenesis is in a group of infertile men with low ROS and in a group
impaired, the cytoplasmic extrusion mechanisms are de- of fertile sperm donors (Table 1). The link between sem-
fective, and spermatozoa are released from the germinal inal ROS and high SDI may be causal and related to the
epithelium carrying surplus residual cytoplasm. Under greater capacity of morphologically abnormal spermato-
these circumstances, the spermatozoa that are released zoa to produce ROS.
duringspermiationarebelievedtobeimmatureandfunc- Effect of Sperm Preparation Techniques on ROS Pro-
tionally defective (Huszar et al, 1997). Retention of re- duction—Studies from our center have indicated that hu-
sidual cytoplasm by spermatozoa is positively correlated man spermatozoa significantly increase levels of ROS
with ROS generation via mechanisms that may be me- production in response to repeated cycles of centrifuga-
Figure1.MechanismofincreasedproductionofROSbyabnormalspermatozoa(spermatozoawithcytoplasmicretention).
SalehandAgarwal · OxidativeStressandMaleInfertility 741
Table1. Comparisonofsemenparametersandspermdeformityindex(SDI)infertiledonors,infertilemenwithlowROS,andinfertile
menwithhighROS
InfertileMen InfertileMen
withLowROS withHighROS
FertileDonors ((cid:35)1(cid:51)106cpm; ((cid:46)1(cid:51)106cpm;
Parameters (n(cid:53)8) n(cid:53)14) n(cid:53)25) A B C
SeminalROS((cid:51)106cpm) 0.1(0.1,0.5) 0.3(0.1,0.5) 5(3,60) 0.51 0.03 0.03
Seminalleukocytes((cid:51)106/mL) 0.2(0.1,0.2) 0.1(0.0,0.2) 1.2(0.3,2.4) 0.61 0.005 0.007
Spermconcentration((cid:51)106/mL) 68(42,90) 56(38,72) 20(12,26) 0.08 0.004 0.07
Spermmotility(%) 73(67,76) 59(39,63) 38(33,52) 0.002 (cid:44)0.001 0.07
NormalmorphologybyWHO
(1999)(%) 33(31,40) 23(17,32) 16(12,23) 0.02 (cid:44)0.001 0.07
NormalmorphologybyKruger(%) 12(8,14) 7(5,11) 6(3,8) 0.12 0.008 0.14
Spermdeformityindex 1.6(1.5,1.7) 1.6(1.5,1.9) 2(1.7,2.2) 0.86 0.009 0.003
ROS (cid:53) reactive oxygen species measured in washed sperm suspensions (after simple wash and resuspension inPBS) bychemiluminescence
assay.Valuesaremedianandinterquartilerange(25thand75thpercentiles).A(cid:53)PvalueofdonorsversusinfertilemenwithlowROS;B(cid:53)Pvalue
ofdonorsversusinfertilemenwithhighROS;andC(cid:53)PvalueofinfertilemenwithlowROSversusinfertilemenwithhighROS.Wilcoxonrank-sum
testwasusedforcomparison,andstatisticalsignificancewasconsideredatP(cid:44).05.
tion involved in the conventional spermpreparationtech- from seminiferous tubules to the epididymis may be an
niquesusedforART(Agarwaletal,1994a).Theduration important cause of male infertility. If this is the case, per-
ofcentrifugationwasfoundtobemoreimportantthanthe hapsinterventionsdirectedto1)increaseantioxidantlevels
force of centrifugation for inducing ROS formation by inimmaturegermcellmembranesduringspermatogenesis,
human spermatozoa (Shekarriz et al, 1995b). These data and 2) isolate spermatozoa with intact DNA by in vitro
aresignificantbecauseexposingspermatozoatohighlev- separation techniques should be of particular benefit to
els of ROS may cause DNA fragmentation, which can these patients in whom a defect in the normal regulation
have adverse consequences if they are used forART,par- of spermiogenesis leads to an abnormal increase in the
ticularly ICSI (Lopes et al, 1998). This may be true par- production of ROS-producing immature sperm.
ticularly in light of a recent report by Ziniandcoworkers Effect of Temperature on ROS Production—Prelimi-
(2000) who found that improvement in sperm motility nary data from a recent unpublished study in our center
after Percoll processing was not associated with asimilar indicatethatlevelsofROSproductioninsemenmayvary
improvement in sperm DNA integrity. The authors rec- accordingtothetemperatureatwhichsemenisincubated.
ommended that the current sperm preparation techniques For the purpose of this study, semen samples were col-
be reevaluated with the goal of minimizing sperm DNA lected from a nonselected group of infertility patients (n
damage. In a recent study in our center, the recovery of (cid:53) 12) and from a group of healthy sperm donors (n (cid:53)
sperm with intact nuclear DNA was found to be signifi- 12) after 2 to 3 days of sexual abstinence. We examined
cantly higher after the swim-up technique than after the levels of ROS in washed sperm suspensions immediately
ISolate gradient technique (unpublished data). after liquefaction (basal ROS) and compared the results
Our group has also reported that ROS production by with ROS levels in aliquots of the same samples after 1-
human sperm increases with sperm concentrationandde- hour incubation at 3 different temperatures (4(cid:56)C, 25(cid:56)C,
creaseswithtime(Shekarrizetal,1995b,c).Resultsfrom and 37(cid:56)C). Our results indicate that ROS levels are sig-
our recent studies indicated that ROS production signifi- nificantly lower in aliquots of semen incubated at 37(cid:56)C
cantlyvariesinsubsetsofhumanspermatozoaatdifferent for 1 hour compared to aliquots of the same samples in-
stages of maturation (Ollero et al, 2001; Gil-Guzman et cubatedat4(cid:56)Cand25(cid:56)C(Table2).Spermmotionkinetics
al, 2001). Following ISolate gradient (Irvine Scientific, (curvilinear velocity, straight-line velocity, average path
Santa Ana, Calif) fractionation of ejaculated sperm, ROS velocity, and amplitude of lateral head displacement) as
production was found to be highest in immature sperm determined by a computer-assisted semen analyzer
withabnormalheadmorphologyandcytoplasmicretention (CASA)weresignificantlyhigherinsamplesincubatedat
and lowest in mature spermand immaturegermcells.The 37(cid:56)C than at 4(cid:56)C (P (cid:53) (cid:44) .001, (cid:44) .001, (cid:44) .001, and
relativeproportionofROS-producingimmaturespermwas (cid:44).001, respectively) although not significantly different
directly correlated with nuclear DNA damage values in from samples incubated at 25(cid:56)C (P (cid:53) .69, .51, .95, and
mature sperm and inversely correlated with the recovery .79, respectively). Therefore, it may be speculated that
of motile, mature sperm. These interesting findings led to 37(cid:56)C could be the optimum temperature at which semen
the hypothesis that oxidative damage of mature sperm by can be collected and processed for different diagnostic
ROS-producing immature sperm during their comigration and therapeutic purposes. Handling semen samples at
742 Journalof Andrology · November/December2002
Table2. ComparisonofROSlevelsindonorsandinanonselectedgroupofinfertilitypatientsatdifferenttemperatures(valuesare
medianandinterquartilerange)
ROSAfter ROSAfter ROSAfter
1-Hour 1-Hour 1-Hour
Incubation Incubation Incubation
BasalROS at4(cid:56)C at25(cid:56)C at37(cid:56)C
Variable ((cid:51)106cpm) ((cid:51)106cpm) ((cid:51)106cpm) ((cid:51)106cpm) A B C D E F
Donors 1.0 1.0 0.4 0.1 0.60 0.84 0.002 0.75 0.008 0.004
(n(cid:53)12) (0.2,2.6) (0.1,3.1) (0,7.2) (0,0.9)
Infertilitypatients 2.2 2.2 1.8 1.3 0.60 0.18 0.001 0.40 0.004 0.04
(n(cid:53)12) (0.3,67.2) (0.4,61.2) (0.3,58.5) (0.2,28.4)
ROS (cid:53) reactive oxygen species measured in washed sperm suspensions (after simple wash and resuspension inPBS) bychemiluminescence
assay.A(cid:53)PvalueofbasalROSvs.ROSafter1-hourincubationat4(cid:56)C.B(cid:53)PvalueofbasalROSvs.ROSafter1-hourincubationat25(cid:56)C.C(cid:53)
PvalueofbasalROSvs.ROSafter1-hourincubationat37(cid:56)C.D(cid:53)PvalueofROSafter1-hourincubationat4(cid:56)Cvs.ROSafter1-hourincubation
at25(cid:56)C.E(cid:53)PvalueofROSafter1-hourincubationat4(cid:56)Cvs.ROSafter1-hourincubationat37(cid:56)C.F(cid:53)PvalueofROSafter1-hourincubationat
25(cid:56)Cvs.ROSafter1-hourincubationat37(cid:56)C.Wilcoxonrank-sumtestwasusedforcomparison,andPvalue(cid:44).05wassignificant.
37(cid:56)C may help avoid deleterious effects of ROS on hu- leads to respiratory burst and production of high levels
man spermatozoa. of ROS (Blake et al, 1987). Such an oxidative burst isan
Reactive Oxygen Species Production by Seminal Leu- early and effective defense mechanism in cases of infec-
kocytes—With respect to all nonsperm cells, the majority tion for killing the microbes (Saran et al, 1999).
of the so-called ‘‘round cells’’ consist of immature germ Sperm damage from ROS that is produced by leuko-
cells with less than 5% leukocytes under normal condi- cytes occurs if seminal leukocyte concentrations are ab-
tions (Eggert-Kruse et al, 1992). Peroxidase-positive leu- normally high (ie, leukocytospermia; Shekarriz et al,
kocytes were found to be the major source of high ROS 1995a), if the patient has epididymitis,or ifseminalplas-
production in semen (Tomlinson et al, 1993; Aitkenetal, ma was removed during sperm preparation for assisted
1995; Rajasekaran et al, 1995; Shekarriz et al, 1995a; reproduction (Ochsendorf, 1999). However, Sharma et al
Ochsendorf, 1999). Peroxidase-positive leukocytes in- (2001) observed that seminal leukocytes may cause OS
clude polymorphonuclear (PMN) leukocytes, which rep- even at concentrations below the WHO cutoff (ie, (cid:44)1 (cid:51)
resent 50% to 60% of all seminal leukocytes and mac- 106 peroxidase positive leukocytes/mL semen) value for
rophages, which represent another 20% to 30% (Wolff leukocytospermia. This may be due to the fact that sem-
and Anderson, 1988; Fedder et al, 1993; Thomas, et al, inal plasma contains large amounts of ROS scavengers
1997). Peroxidase-positive leukocytes in semen are con- but confers a variable (10% to100%) protection against
tributed largely by the prostate and the seminal vesicles ROS generated by leukocytes (Kovalski et al, 1992).
(Wolff, 1995). Recent yet unpublished data from our center indicated
Activatedleukocytesarecapableofproducing100-fold that levels of ROS production by purespermsuspensions
higher amounts of ROS than nonactivated leukocytes from infertile men with a laboratory diagnosis of leuko-
(Plante et al, 1994). Leukocytes may be activated in re- cytospermia were significantly higher than those in infer-
sponse to a variety of stimuli including inflammationand tile men without leukocytospermia (Table 3). This obser-
infection (Pasqualotto et al, 2000a). Activated leukocytes vation led to the postulation that seminal leukocytes play
increase NADPH production via the hexose monophos- a role in enhancing sperm capacity for excessive ROS
phate shunt. The myeloperoxidase system of both PMN production either by directsperm-leukocytecontactorby
leukocytes and macrophages is also activated, which soluble products released by the leukocytes. Studies are
Table3. BasalROSlevelsinoriginalcellsuspensions(containingspermandleukocytes)andinpurespermsuspensions(leukocyte-free
spermsuspensionsaftercompleteremovalofleukocytesusinganti-CD coatedparamagneticbeads)in3studygroups
45
Donors Nonleukocytospermic Leukocytospermic
Variable (n(cid:53)13) (n(cid:53)32) (n(cid:53)16) A B C
BasalROS 0.4 (0.1,2.5) 2.7 (0.53,12) 178(32,430) 0.06 0.0001 (cid:44)0.0001
((cid:51)106cpm)
PurespermROS 0.06(0.01,0.2) 0.31(0.09,1.2) 3.3(0.5,7.4) 0.05 0.001 0.002
((cid:51)106cpm)
Valuesaremedian(25th,75thpercentiles).ROS(cid:53)reactiveoxygenspeciesmeasuredbychemiluminescenceassay.A(cid:53)Pvalueofdonorsversus
nonleukocytospermic,B(cid:53)Pvalueofdonorsversusleukocytospermic,andC(cid:53)Pvalueofnonleukocytospermicversusleukocytospermic.Wilcoxon
rank-sumtestwasusedforcomparisonandstatisticalsignificancewasassessedatP(cid:44).05level.
SalehandAgarwal · OxidativeStressandMaleInfertility 743
Figure2.MeasurementofROSinwashedspermsuspensions(containingspermatozoaandseminalleukocytes)bychemiluminescenceassay.
currently underway to investigate this hypothesis. How- spermsuspensionsusingachemiluminescenceassay(Ko-
ever, this new finding may have significant implications bayashi et al, 2001; Figure 2). With this protocol, lique-
for the fertility potential of sperm both in vivo and in fied semen is centrifuged at 300 (cid:51) g for 7 minutes, and
vitro. Excessive production of ROS by sperm in patients the seminal plasma is separated and stored at (cid:50)80(cid:56)C for
with leukocytospermia implies that both the free-radical measurement of TAC. The pellet is washed with phos-
generating sperm themselves and any normal sperm in phate-buffered saline (PBS) and resuspended in the same
the immediate vicinity will be susceptible to oxidative washing media at a concentration of20 (cid:51) 106 sperm/mL.
damage. Furthermore, once the process of LPO is initi- Four-hundred-microliter aliquots of the resulting cellsus-
ated, the self-propagating nature of this process ensuresa pensions (containing sperm and leukocytes) are used to
progressive spread of the damage throughout the sperm assess basal ROS levels. Eight microliters of horseradish
population. peroxidase (HRP) (12.4 units of HRP Type VI, 310 U/
mg; Sigma Chemical Company, St Louis, Mo) are added
(cid:65)(cid:115)(cid:115)(cid:101)(cid:115)(cid:115)(cid:109)(cid:101)(cid:110)(cid:116)(cid:111)(cid:102)(cid:83)(cid:101)(cid:109)(cid:105)(cid:110)(cid:97)(cid:108)(cid:79)(cid:120)(cid:105)(cid:100)(cid:97)(cid:116)(cid:105)(cid:118)(cid:101)(cid:83)(cid:116)(cid:114)(cid:101)(cid:115)(cid:115)(cid:105)(cid:110)(cid:116)(cid:104)(cid:101)
to the cell suspension. The HRP sensitizes the assay so
(cid:65)(cid:110)(cid:100)(cid:114)(cid:111)(cid:108)(cid:111)(cid:103)(cid:121)(cid:76)(cid:97)(cid:98)(cid:111)(cid:114)(cid:97)(cid:116)(cid:111)(cid:114)(cid:121)
that it measures the extracellular H O . Ten microlitersof
2 2
Extensiveresearchinthefieldofmaleinfertilityhasbeen luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione;Sig-
conducted to develop adequate indices of OS that would ma), prepared as 5 mM stock in dimethyl sulfoxide
helpdetermine,withaccuracy,whetherOSisasignificant (DMSO), are added to the mixture and serve as a probe.
contributortomaleinfertility(Sharmaetal,1999).Levels Luminol is an extremely sensitive oxidizable substrate
of OS vary greatly in infertile men (Alvarez et al, 1987). that has the capacity to react with a variety of ROS at
Because OS is an imbalance between levels of ROS pro- neutral pH. The reaction of luminol with ROS results in
duction and antioxidant protection in semen, it is con- production ofa light signal that isthenconvertedtoelec-
ceivable that assessment of OS will rely on the measure- trical signal (photon) by a luminometer. A negative con-
ment of ROS as well as TAC of semen. trolispreparedbyadding10(cid:109)Lof5mMluminolto400
Protocol for Measurement of ROS by Chemilumines- (cid:109)LofPBS.LevelsofROSareassessedbymeasuringthe
cence Assay—Levels ofROScanbemeasuredinwashed luminol-dependant chemiluminescence with the lumi-
744 Journalof Andrology · November/December2002
nometer(modelLKB953;WallacInc,Gaithersburg,MD)
in the integrated mode for 15 minutes. The results are
expressed as (cid:51)106 counted photons per minute (cpm)per
20 (cid:51) 106 sperm. Normal ROS levels in washed sperm
suspensions range from 0.10 to 1.0 (cid:51) 106 cpm per 20 (cid:51)
106 sperm.
ProtocolforMeasurementofTotalAntioxidantCapac-
ity in Semen by Enhanced Chemiluminescence Assay—
Total antioxidant capacity in the seminal plasma can be
measured using an enhanced chemiluminescence assay
(Kolettis et al, 1999). Frozen samples of seminal plasma
arethawedatroomtemperatureandimmediatelyassessed
for TAC. Seminal plasma is diluted 1:20 with deionized
water (dH O) and filtered through a 0.20-(cid:109)m filter (Al-
2
legiance Healthcare Corporation, McGaw Park, Ill). Sig-
nal reagent is prepared by adding 30 (cid:109)L of H O (8.8
2 2
molar/L),10(cid:109)Lofpara-iodophenolstocksolution(41.72 Figure3.LinearrelationshipbetweentheconcentrationsofstandardTro-
(cid:109)M), and 110 (cid:109)L of luminol stock solution (3.1 mM) to lox solutions (25 (cid:109)M, 50 (cid:109)M, and 75 (cid:109)M) and 10% recovery time (in
seconds)oftheinitialchemiluminescence.
10 mL of Tris buffer (0.1 M pH 8.0). Horseradish per-
oxidaseworking solution ispreparedfromtheHRPstock
solutionbymakingadilutionof1:1ofdH O.Lightemis- y (cid:53) (Mx (cid:54) C) (cid:51) d
2
sionoccurswhenthechemiluminescentsubstrateluminol
In this equation, M refers to the slope increase in the
is oxidized by H O in a reaction catalyzedbyHRP.HRP
2 2 value of Trolox equivalent for 1-second increase of the
catalyzes the reaction between a hydrogen acceptor (ox-
recovery time, whereas C accounts for the daily back-
idant) and a hydrogen donor. Under normal circumstanc-
ground variability. The results are multiplied by the di-
es,thisreactionproduceslow-intensitylightemissionthat
lution (d) factor and expressed as molar Trolox equiva-
maydecayrapidly.Thecharacteristicsofthereactioncan
lents (Sharma et al, 1999).
bealteredsubstantiallybytheadditionofpara-iodophenol
Quality Control of Oxidative Stress Indices (ROS and
as an enhancer that gives a more intense, prolonged, and
TAC)—It was of utmost importance to standardize the
stablelightemission.Thecontinuouslightoutputdepends
measures that we used as indices for OS, including mea-
on the constant production of free radical intermediates
surement of ROS in washed sperm suspensions and TAC
derived from para-iodophenol (enhancer), luminol (sub-
in seminal plasma. We have demonstrated that the lumi-
strate), and H O (oxidizer) in the presence of HRP.
2 2 nol-dependent chemiluminescence assay for ROS mea-
Trolox(6-hydroxyl-2,5,7,8-tetramethylchroman-2-carbox-
surement in washed sperm suspensions is both accurate
ylic acid), a water-soluble tocopherol analogue, is prepared
and reliable when the sperm concentration is greaterthan
as a standard solution (25, 50, and 75 (cid:109)M) for TAC cali- 1 (cid:51) 106/mL and when the samples are analyzed within 1
bration. With the luminometer in the kinetic mode, 100(cid:109)L
hour after collection (Kobayashi et al, 2000). Similarly,
of signal reagent and 100 (cid:109)L of HRP working solution are
assessmentofTACinseminalplasmausingtheenhanced
addedto700(cid:109)LofdHOandmixed.Themixtureisequil-
2 chemiluminescence assay was found to be both accurate
ibrated to the desired level of chemiluminescence output
and reliable, with very low intraassay, interassay, and in-
(between 2.8 and 3.2 (cid:51) 107 cpm) for 100 seconds. One terobserver variations (P (cid:36) .8) (unpublished data). The
hundred microliters of the standard Trolox solution is im-
intraassay reliability was 91% [coefficient of variation
mediatelyaddedtothemixture,andthechemiluminescence (CV) (cid:53) 5%], the interobserver reliability was 89% (CV
ismeasured.Suppressionofluminescenceandthetimefrom (cid:53)13%),andinterassayreliabilitywas92%(CV(cid:53)13%).
theadditionofstandardTroloxsolutionto10%recoveryof ROS-TAC Score: An Accurate Method for Assessment
the initial chemiluminescence are recorded. Plotting the of Seminal Oxidative Stress—The fact that neither ROS
threeconcentrationsofTroloxsolutionversus10%recovery alonenorTACalonecanadequatelyquantifyseminalOS
time results in a linear equation (Figure 3). led to the logical conclusion that combining these two
The same steps are repeated after the Trolox solutions variables may be a better index for the diagnosis of over-
arereplacedwith100-(cid:109)Laliquotsofthepreparedseminal all OS affecting spermatozoa. This conclusion led to the
plasma. The assay is conducted in a dark room because introduction of the ROS-TAC score as a new method for
light affects the chemiluminescence. Seminal TAC levels assessment of OS status in infertile men (Sharma et al,
are calculated using the following equation: 1999). The new ROS-TAC score is a statistical formula
SalehandAgarwal · OxidativeStressandMaleInfertility 745
derivedfromlevelsofROSinwashedspermsuspensions
and TAC in seminal plasma using principal component
analysis. The resulting score minimizes the variability in
the individual parameters of OS (ROS alone or TAC
alone). The ROS-TAC score is an accurate measure of
seminal OS and low ROS-TAC scoresindicatehighsem-
inal OS (Sharma et al, 1999). A cutoff value of 30 was
determined as the lower limit of normal range for the
ROS-TAC score, and individuals with scores below this
cutoff value were found to be at particular risk for pro-
longed inability to initiate pregnancies.
Assessment of ROS in Whole (Unwashed) Ejaculates:
A Reliable Alternative for ROS-TAC Score—Our group
has recently introduced an additional test of OS in which
ROS levels are directly measured in the whole (un-
washed) ejaculate following liquefaction (unpublished
data). Semen samples in our study were collected from
34 infertile men and from 9 normal, healthy donors. Do-
nors were selected on the basis of normal genital exam-
ination and normal standard semen parameters according
to WHO (1999) guidelines. All semen specimens were
producedbymasturbationafterthepatientshadabstained
from sex for a period of 48 to 72 hours.Eightmicroliters
of HRP were added to 400 (cid:109)L-aliquots of liquefied se-
men. Levels of ROS were measured by the chemilumi-
nescence assay using luminol (10 (cid:109)L) as the probe with
the luminometer set in the integrated mode for 15 min-
utes. The results were expressed as (cid:51)104 cpm per 20 (cid:51)
106 sperm. In addition, levels of ROS in the washed
spermsuspensionsandTACinseminalplasmaweremea-
sured in aliquots from the same samples in order to cal-
culate the ROS-TAC scores.
Our results indicated that levels of ROS (median [25th
and75thpercentiles])inthewholeejaculateofthenormal
donors ((cid:51)104 cpm/20 million sperm/mL) were signifi-
cantly lower than in infertile men (0.3 [0.2, 0.9] vs. 5.8
[1.0, 81.0] (cid:51) 104 cpm/20 million sperm/mL; P (cid:53) .004).
Figure4.CorrelationofROSlevelsinwhole(unwashed)ejaculatewith
The maximum level of ROS in the whole ejaculate of
(A) levels of ROS in washed sperm suspensions (following cycles of
normal donors was 1.5 (cid:51) 104 cpm/20 million sperm/mL. simplewashandresuspensioninPBS)(r(cid:53).87,P(cid:44).001),and(B)the
Sixty-eight percent (23 of 34) of infertile men had ROS statistical formula, reactive oxygen species—total antioxidant capacity
levels (cid:46)1.5 (cid:51) 104 cpm/20 million sperm/mL and were score(r(cid:53)(cid:50).75,P(cid:44).001)derivedfromlevelsofROSinwashedsperm
suspensionsandantioxidantsinseminalplasma.
classified as OS-positive. The remaining 32% (11of 34)
of infertile men had ROS levels (cid:35)1.5 (cid:51) 104 cpm/20 mil-
lion sperm/mL and were accordingly classified as OS- [51, 55], P (cid:53) 0.4). In addition, levels of ROS in whole
negative. (unwashed) ejaculate were correlated positively with lev-
Leukocytospermia(concentrationsgreaterthan1(cid:51)106 els of ROS in washed sperm suspensions and negatively
leukocytes/mL of semen) was found in 9% (1 of 11) of with ROS-TAC scores (Figure 4).
OS-negativesamplesandin39%(9of23)ofOS-positive ROS levels in whole ejaculates were correlated nega-
samples. It is interesting that ROS-TAC scores were sig- tively with sperm concentration (r (cid:53) (cid:50).52, P (cid:53) .0003),
nificantlyhigher(ie,lowerOS,[49{47,52}]inthegroup motility (r (cid:53) (cid:50).41, P (cid:53) .006) and morphology by the
classified as OS-negative based on ROS levels in whole WHO method (r (cid:53) (cid:50).34, P (cid:53) .02), and positively with
[unwashed] ejaculates than the OS-positive group [36 seminal leukocyte concentrations (r (cid:53) .65, P (cid:44) .0001).
{32,44}],P(cid:53).001).TheOS-negativegroupscoreswere Furthermore, quality control studies of assessment of
not significantly different from the normal controls (53 ROS in whole ejaculates indicated low variability (in-
746 Journalof Andrology · November/December2002
traassay [CV (cid:53) 17, with 99% reliability] and interassay 100 spermatozoa and 100 neutrophils were scored using
[CV (cid:53) 18, with 96% reliability]). Therefore, this testcan 100(cid:51) magnification. Leukocytes were scored as follows:
be used as an alternative to ROS-TAC score for accurate cells filled with formazan ((cid:49)(cid:49)(cid:49)), intermediate density
and reliable assessment of seminal OS in the andrology ((cid:49)(cid:49)), scattered or few formazan granules ((cid:49)), and no
laboratory.The strongpositivecorrelationbetweenlevels detectableformazan((cid:50)).Spermatozoawerescoredasfol-
of ROS in whole ejaculates and seminal leukocyte con- lows: formazan occupying (cid:35)50% of cytoplasm ((cid:49)) and
centrationssuggeststhattreatmentofOS-positivepatients (cid:46)50% of cytoplasm ((cid:49)(cid:49)).
should include plans for effective control of excessive The most important finding from our study was a sig-
leukocyte infiltration, antioxidant supplementation, or nificant increase in the percentage of NBT-positive leu-
both. kocytes in semen from leukocytospermic samples (70%)
Nitroblue Tetrazolium Test for Identification of ROS- as compared with nonleukocytospermic (14.5%) and do-
Producing Cells in Semen—It is important to determine nor samples (7%) (P (cid:53) .02 and 0.03, respectively). In
the source of ROS in a given semen sample because the addition, a strong positive correlation was found between
clinical implications of infiltrating leukocytes are quite levels of ROS, as measured by chemiluminescence, and
different from those of pathological conditions in which the percentage of NBT-positive neutrophils (r (cid:53) .7; P (cid:44)
spermatozoa themselves are the source of ROS (Aitken, .0001) and sperm with cytoplasmic retention (r (cid:53) .72; P
1994). The luminol-dependent chemiluminescence assay (cid:44) .0001). Furthermore, the results of the NBT test were
that was described above helps assess levels of seminal inversely correlated with ROS-TAC scores. These results
OSbymeasuringthetotalamountofROSinsemenusing indicate that the NBT reduction test can help assess the
a luminometer. This assay, however, does not provide in- differential contribution of leukocytes and defectivesper-
formation on the differential contribution of spermatozoa matozoa to the amount of ROS generation in semen. The
and leukocytes to ROS production in semen or on the NBTtestiseasilyperformedandcanbeusedasaroutine
state of activation of individual cells. Nitroblue tetrazo- andrology laboratory procedure to assess seminal OS
lium (NBT) is a yellow, water-soluble dye that can be without the need for expensive equipment such as the
reduced by accepting electrons in the presence of free luminometer.
oxygen radicals to form a blue-black water-insoluble
(cid:67)(cid:108)(cid:105)(cid:110)(cid:105)(cid:99)(cid:97)(cid:108)(cid:83)(cid:105)(cid:103)(cid:110)(cid:105)(cid:174)(cid:99)(cid:97)(cid:110)(cid:99)(cid:101)(cid:111)(cid:102)(cid:65)(cid:115)(cid:115)(cid:101)(cid:115)(cid:115)(cid:109)(cid:101)(cid:110)(cid:116)(cid:111)(cid:102)(cid:83)(cid:101)(cid:109)(cid:105)(cid:110)(cid:97)(cid:108)
compoundknownasformazan(Baehneretal,1967).The
(cid:79)(cid:120)(cid:105)(cid:100)(cid:97)(cid:116)(cid:105)(cid:118)(cid:101)(cid:83)(cid:116)(cid:114)(cid:101)(cid:115)(cid:115)
cytoplasmic NADH, which is produced by oxidation of
glucose through the hexose monophosphate shunt, serves Currently, no single laboratory test can accurately and
asanelectrondonor.Theoxidasesystemsavailableinthe precisely assess a man’s total fertility (Evenson et al,
cytoplasm help transfer electrons from NADH to NBT 1999). The entire purpose of performing a diagnostictest
and reduce NBT into formazan (Baehner et al, 1976). in the evaluation of the male partner of a suspected in-
Thus,theNBTreactionindirectlyreflectstheROSgen- fertile couple is to learn whether or not his fertility is
erating activity in the cytoplasm of cells and can help impaired (Matson, 1997). Therefore, any test must then
determinethecellularoriginofROSinasuspensioncon- have a threshold above and below which it will provide
taining a variety of cells such as semen. Earlier studies discrimination and predictive capabilities with little over-
have shown that NBT reduction and formazandeposition lap between fertile and infertile men. Determining which
in blood neutrophils are related to their phagocytic activ- tests are valid in the evaluation of male infertility is dif-
ity (Park et al, 1968; Segal and Levi, 1973). In the same ficult for a number of reasons. First, the term ‘‘male in-
studies, the state of activation of neutrophils was deter- fertility’’ does not constitute a defined clinical syndrome
mined by scoring the blue-black formazan granules de- but rather a collection of disparate conditions with a va-
posited in the cytoplasm. In a recent prospective study riety of causes and prognoses (Aitken et al, 1995). In
(unpublished data), we assessed ROS generating activity addition, various clinical diagnoses are unable to deter-
in semen from a group of infertile men using the NBT minetheunderlyingcauseofspermdysfunctionandpath-
reduction test and also by the chemiluminescence assay. ophysiology of infertility. Relatively few men will have
Nitroblue tetrazolium (0.1%) was prepared by adding 10 conditions causing absolutesterilitysuchasazoospermia.
mg of NBT powder to 100 mL of PBS (pH 7.2) and Rather,themajorityofinfertilemenhaveabnormalsemen
stirringatroomtemperaturefor1hour.TheNBTsolution quality of varying severity and poorly understood etiol-
was filtered using a 0.2-(cid:109)m filter. Equal volumes ofNBT ogy. Until the causes of male infertility are better under-
solution (0.1%) and unwashed semen were incubated for stood, it is unlikely that any given descriptive test of
30 minutes at 37(cid:56)C. After another 30 minutes of incu- spermqualityorspermfunctionwillpredictwithabsolute
bation at room temperature, the mixture was centrifuged certainty that a man will be fertile or infertile in a given
at 250 (cid:51) g for5 minutes.Smearswerepreparedfromthe time period (Van Voorhis and Sparks, 1999).
pellet, air-dried, and stained with Wright stain. A total of The second reason that testing a man’s fertility is dif-