Unexplained male infertility

15 Pages·2011·1.46 MB·English

2 Reviewarticle
Unexplained male infertility: potential causes and management
Alaa Hamadaa, Sandro C. Estevesb and Ashok Agarwala
aCenterforReproductiveMedicine,ClevelandClinic, Objective
Cleveland,USAandbANDROFERT–Andrologyand
Tohighlighttheconceptofunexplainedmaleinfertilityanddiscussthepotentialcauses
HumanReproductionClinic,Campinas,Brazil
andits propermanagement.
CorrespondencetoAshokAgarwal,PhD,HCLD,
Design
LernerCollegeofMedicine,Director,Centerfor
ReproductiveMedicine,ClevelandClinic,DeskA19.1, Reviewofliterature.
9500EuclidAvenue,Cleveland,Ohio44195,USA
Results
Tel:[phone];
fax:[phone]; Maleinfertility of unknownorigin is a condition inwhichfertility impairmentoccurs
e-mail:[email protected]
spontaneously or duetoan obscureor unknowncause. Itincludes two categories,
unexplained male infertility andidiopathic male infertility.Thedividing linebetween
themis semenanalysis,whichisnormal inthe unexplained categoryandabnormal
Received28February2011
Accepted12March2011 inidiopathic infertility.After rulingout female infertility factors, erectile problems and
coitalfactors, modern andrology may helptoanalyze theunexplained male fertility
HumanAndrology2011,1:2–16
problemonthebasisofcellularandsubcellularmechanisms.Furthermore,thisanalysis
mayleadtotheselectionofpropertreatmentoptionsfittingtheneedsofpatientswith
unexplained infertility.
Conclusion
Despite the advances andinnovation ofsophisticated laboratory tests inthefield of
andrology,further researchisstill needed tosolve thedilemma ofinfertility.
Keywords:
male infertility,normospermia, sperm function, oxidativestress,autoimmune infertility,
assistedreproductive techniques
HumAndrol1:2–16
(cid:2)c 2011 HumanAndrology
2090-6048
ruled out [8]. The reported prevalence of UMI ranges
Introduction
from 6 to 27% [5] and it strongly depends on how
Infertilityisacommonclinicalproblemaffecting13–15%
exhaustiveistheevaluationofthepatient.Menclassified
ofcouplesworldwide[1].Theprevalencevariesthrough-
as having idiopathic male infertility have an unexplained
out developed and underdeveloped countries, being
reduction in semen quality with no history associated
higher in the latter in which limited resources for
with fertility problems and have normal findings on
diagnosis and treatment exist [2]. A male factor is solely
physical examination and endocrine laboratory testing.
responsible for infertility in approximately 20% and
Theirroutinesemenanalysisshowsdecreasednumberof
contributory in another 30–40% of couples; as such, a
spermatozoa (oligozoospermia), decreased motility (asthe-
male factor is implicated in more than 50% of couples
nozoospermia), or an increased proportion of abnormal
attempting to conceive [3]. A reduction in the male
forms (teratozoospermia). These abnormalities usually
fertility potential may be due to congenital or acquired
occur together and are described as the oligoasthenoter-
conditions such as urogenital abnormalities, varicocele,
atozoospermia syndrome [6]. This category comprises
infections of the genital tract, genetic abnormalities,
approximately 31% of infertile men [6]. Table 1 clearly
endocrine disturbances, testicular failure, immunologic
shows the frequency distribution of different causes of
problems,cancer,systemicdiseases, altered lifestyle,and
male infertility [8]. Furthermore, the frequency of male
exposuretogonadotoxicfactors[4].Thecauseoffertility
infertility of unknown origin is different between
impairment cannot be determined in several cases,
countries. In a group of 2383 subfertile male individuals
despite the advances in diagnosis by the introduction of
attending one of the investigators’ (S.C.E.) tertiary
novel sophisticated tests.
center for male reproduction, 12.1% of the individuals
were categorized as having infertility of unknown origin
Infertility of unknown origin includes unexplained male (Table 2).
infertility and idiopathic male infertility; it is a condition
inwhichfertilityimpairmentoccursspontaneouslyordue Inbrief,theinitialassessmentofsubfertilemenincludes
to an obscure or unknown cause. Infertility of unknown history, physical examination, and at least two semen
origin accounts for 37–58% [5–7]. The category ‘un- analyses after 12 months of unprotected intercourse.
explained male infertility’ (UMI) is reserved for infertile However, the initial male work-up may be carried out
men with infertility of unknown origin with normal earlier, particularly in the cases of advanced female age
semen and in which female infertility factors have been (more than 35 years), the presence of known male
2090-6048(cid:2)c 2011HumanAndrology DOI:10.1097/01.XHA.0000397686.82729.09
UnexplainedmaleinfertilityHamadaetal. 3
Table 1 Distribution of final diagnostic categories found in resultsinanticipationofspontaneousconception?Second,
maleinfertilityclinic[8] whatshouldbedonenextafterperformingacomprehensive
Category Frequency(%) work-uporwhenfailingtoidentifythecausesofinfertility?
Normal semen analysis results do not guarantee fecundity.
Immunological —
A significant proportion of patients with normal semen on
Idiopathic 32.6
Varicocele 26.6 routine analysis remain childless over several months
Obstruction 15.3 attempting to conceive [13]. In one study involving 430
Normalfemalefactor(unexplainedmaleinfertility) 10.7
couples,45%ofmenwithaspermconcentrationofgreater
Cryptorchidism 2.7
Ejaculatoryfailure 2.0 than 40 million sperm/ml were unable to impregnate their
Endocrinologic 1.5 wives [14]. It had been demonstrated that the routine
Drug/radiation 1.4
semen analysis was unable to detect sperm functional
Genetic 1.2
Testicularfailure 1.1 deficienciesin40%ofmenpresentingwithsubfertility[15].
Sexualdysfunction 0.7 Table 3 shows the frequency of semen analysis abnormal-
Pyospermia 0.5
ities in 8758 infertile patients attending the fertility clinic.
Cancer 0.4
Systemicdisease 0.3
For men with unexplained infertility and normal semen
Infection 0.2
Torsion 0.1 analyses the following possibilities should be considered:
Ultrastructural 0.1 (i) presence of a female factor, (ii) inappropriate coital
Total 100.0
habits, (iii) erectile dysfunction, (iv) the presence of
antisperm antibodies (ASAs) (autoimmune infertility),
and (v) sperm dysfunction [12]. To exclude the first
Table 2 Distribution of diagnostic categories in a group of
three conditions, proper history taking as well as a
infertilemenattendingamaleinfertilityclinic
thorough gynecological evaluation is needed, whereas
Category N Percentage
modern andrology could be of help in managing the last
Varicocele 629 26.4 two conditions.
Infectious 72 3.0
Hormonal 54 2.3
Ejaculatorydysfunction 28 1.2 Autoimmune infertility
Systemicdiseases 11 0.4 Autoimmune infertility has long been postulated as one
Infertilityofunknown 289 12.1 of the causes of subfertility [16]. It represents approxi-
origin
mately 4.5% of male factor infertility [6]. Why does a
Immunologic 54 2.3
Obstruction 359 15.1 man’sbodytreathisspermasforeigninvaders?Toexplain
Cancer 11 0.5 this,threetheoriesarehypothesized.Thefirststatesthat
Cryptorchidism 342 14.3
sperm are not present at the time of embryological
Genetic 189 7.9
Testicularfailure 345 14.5 development during which the immune system estab-
Total 2383 100.0 lishes tolerance to self antigens [17]. The second claims
that spermatozoa are haploid and have a different
chromosomal make up from the somatic cells [16].
infertility risk factors (such as an undescended testicle),
Meanwhile, the third theory, called ‘immunosuppression
orifamanquestionshisfertilitypotential[3].Inroughly
theory’, postulates that Tsuppressor lymphocytes, which
half of the patients, the initial assessment will identify
inhibit immune responsiveness, are activated by small
the cause of infertility, whereas many other patients will
amounts of spermatozoal antigens continuously leaked
need to go through several complementary tests to find
fromthegenitaltract[18].Assoonasspermatozoa,which
its cause.
are considered immunologically foreign cells, are formed
The goals of meticulous evaluation of subfertile men are during puberty, they must be completely isolated from
(i) identification of the cause of subfertility, which is a the immune system. This isolation occurs within the
prerequisite for the correct indication for appropriate testis,oneoftheimmunologicallyprivilegedsites,bythe
surgical or medical treatment, (ii) identification of blood–testis barrier. In other regions of the male genital
inadequatelifestyleorsexualbehaviortoallowcounseling tract, the epithelial lining, probably supplemented by a
towardanimprovementinthereproductivepotentialand local immunosuppressive barrier, is responsible for this
overallhealthstatus,and(iii)identificationofsignificant isolation [19]. Despite its immune-privileged status, the
medical pathologies that threaten a man’s overall health
or life (endocrinopathies, testicular and prostate cancer,
Table 3 Distribution of abnormalities of semen parameters in
brain andspinalcord tumors),which can beidentified in
8758patients[8]
up to 6% of infertile men [9–12].
Abnormalityinsemenparameters Frequency(%)
Azoospermia 4
Predominanceofasingleabnormalparameter 29
Potential etiologies of unexplained male Motility 18
infertility Volume 2
Morphology 7
Twovitalquestionscomeacrosstheclinicians’mindwhen
Density 2
dealingwithasubfertilemalewhosemedicalevaluationis Defectsintwoormoreparameters 37
unrevealing.First,how predictivearethe semenanalyses Allparametersnormal(unexplainedmaleinfertility) 30
4 HumanAndrology
testis is clearly capable of mounting inflammatory even when all ejaculated spermatozoa are antibody
responses,asprovenby its effectivecellular and humoral coated. Therefore, sperm agglutination, although extre-
defenseagainstinfections.Inpathologicalcircumstances, mely suggestive of sperm autoimmunization, does not
the imbalance between the tolerogenic and the efferent represent an important mechanism of the antibody
limbs of the testicular immune response can lead to the interference with fertility in most cases. Apart from
development of ASAs, and in rare instances, to auto- spermagglutination,thereislittleevidencethatsuggests
immuneepididymoorchitis[16,20–22].Bothhumoraland acause/effectrelationshipbetweenASAandtheabnorm-
cellularimmunityhavebeenimplicatedintheetiologyof alityofsemenparameters[39].AnegativeimpactofASA
immune infertility. on sperm motility/vitality, for instance, should involve a
complement-mediated sperm injury, which is prevented
Humoralimmune infertility by anticomplementary activity in human seminal plasma
Approximately10%ofallinfertilemenmayhaveASA(vs. [41,42]. Nevertheless, adequate amount of complement
2% of fertile men) [23]. Unexplained male factor is present in the cervical fluid, which can be activated
infertility may be related to ASAs. Moghissi et al. [24] through antibody antigen reaction and can exert a toxic
confirmed that the incidence of sperm antibodies was effect on sperm.
significantly higher (42.5%) among patients with un-
The diagnosis of immunological infertility requires two
explained and persistent infertility. The pathogenesis of
conditions to be satisfied [43].
the formation of ASAs is still a matter of debate.
Antisperm immune responses occur probably as a result
(1) Fifty percent or more of the motile spermatozoa
of the disruption of the epithelial or blood–testis barrier
(progressive and nonprogressive) have attached
[25,26],animmunosuppressiondefect[27],orasaresult
of an insult to the genital tract that would provide an beads. It should be noted, however, that particle
excess of spermatozoal antigens that could override the bindingrestrictedtothetailtipisnotassociatedwith
mechanismofimmunosuppression[28].Amajorcauseof impaired fertility and can be present in fertile men;
ASA is vasectomy [29–31]. Other causes include vas (2) Sperm-bound antibodies interfere with sperm func-
obstruction [29], testicular trauma, torsion, malignancy, tion; thisisusually demonstrated byusing functional
infection of the genital tract, semen deposition at
testssuchasthesperm–mucuspenetrationtest,zona
nongenital tract sites (homosexuality), and perhaps,
binding assays, and the AR [43].
varicocele [31–33] and intolerance to heavy metals [34].
It is still unclear whether ASA is produced locally in the
Frequently, antibody-coated sperm may appear as a poor
genital tract or transuded from serum. ASA can be found
postcoital test (PCT). Complement, which is normally
in serum, seminal plasma, and can be sperm bound.
foundinhigheramountsinthecervicalmucusthaninthe
Among these, sperm-bound antibodies are the most
seminal plasma, is needed to immobilize spermatozoa.
clinically relevant. The antibody classes that appear to
The antibody complement reaction may take at least 6h
be clinically relevant include immunoglobulin G (IgG)
tomanifest.PhysiciansperformingaPCTwithin2hafter
and IgA. IgG antibody is derived from local production
intercourse or using in-vitro mucus penetration assays
and from transudation from the bloodstream. IgA, in
may miss the immobilizing antibodies. Consequently,
contrast,isthoughttobepurelylocallyderived[35].The
these patients may appear as having an absence of male
epididymis is postulated to be the production site in
factor infertility. It is therefore advisable to perform a
cases of obstruction because of the combined effect of
PCT after at least 6h after intercourse [44]. ASA can
increased intraluminal pressure and leak of spermatozoa
cause infertility without obvious problems with cervical
antigens. In contrast, a woman can also produce ASAs in
mucus penetration. Such antibodies may interfere with
her cervical fluid. Such antibodies have been reported in
the AR and may inhibit sperm penetration into the zona
7–17% of infertile women, varying with the type of test
pellucida and fusion with the oocyte [45].
performed and the population screened [36,37]. ASA
impair sperm function by induction of apoptosis, or by Currently, the most popular tests to identify sperm-
inducing premature acrosome reaction (AR). ASA may bound ASA are both the direct immunobead test (IBT)
also interfere with fertilization by inhibition of cervical and the direct mixed agglutination reaction [46]. In the
mucus penetration, zona pellucida (ZP) binding or direct IBT, beads coated with covalently bound rabbit
sperm–oocyte fusion. ASA may change some macromole- antihumanimmunoglobulinsagainstIgGorIgAaremixed
cular and subcellular function by altering chaperon directly with washed spermatozoa. The binding of beads
function, protein folding, and disulfide bonds [38]. The with antihuman IgG or IgA to motile spermatozoa
endresultisthatpregnancyratesmaybereducedbyASA indicatesthepresenceofIgGorIgAantibodiesonsperm
[39]. When dealing with patients with unexplained male surface [43]. IBT is more time consuming but it
infertility, one should keep in mind that the presence of identifies the proportion of antibody-bound sperm in a
elevated levels of ASA may occur even in the face of given sample, the antibody class, and the location of
normal semen analysis. Sperm agglutination is the only antibodies on the sperm surface. In contrast, the direct
well-establishedsemenalterationrelatedtothepresence mixed agglutination reaction test is an inexpensive,
of ASA [40]. However, sperm agglutination, which is a quick, and sensitive screening test in which sheep
time-dependentphenomenon,onlyrarelyinvolvesalarge erythrocytes are used instead of immunobeads to detect
proportion of motile spermatozoa soon after liquefaction, and localize antibody-bound sperm [47,48].
UnexplainedmaleinfertilityHamadaetal. 5
Cellular immune infertility men misdiagnosed as having unexplained and idiopathic
Thereisevidence that cell-mediatedimmunity mayplay infertility. Abnormalities causing male infertility include:
a role in immunological infertility. Histopathological
studies demonstrated the presence of inflammatory cells (1) Chromosomal complement changes in number (e.g.
infiltrating the contralateral testis in animal models and aneuploidy) or structure such as translocations or
patients with unilateral testicular torsion because of inversions.
release of germ cell inoculums in response to ischemia (2) Gene mutation and polymorphisms.
and necrosis of the torsed testis [49,50]. Despite this (3) DNA integrity defects.
immunologic event, ASA are rarely seen in such cases.
Other supporting evidence come from immunoreactivity Chromosomal complement: the risk of sperm chromosomal
studies using purified macrophages isolated from indivi- aneuploidy is inversely related to sperm concentration and
dualswithrepairedunilateralandbilateralcryptorchidism total progressive motility [58,59]. The overall frequency of
and exposed to homologous sperm. It had been found chromosomally abnormal sperm in the general population is
that 50% of patients with unilateral and 80% with estimated to be 7%. The mean frequency of disomy for
bilateral surgically repaired cryptorchidism had cell- autosomes and sex chromosomes are 0.13 and 0.37%,
mediated immunoreactivity [51]. Sperm granuloma at respectively, whereas the rate for diploidy is 0.2%. For
the vasectomy site is another evidence of cell-mediated normospermicinfertilemen,correspondentfiguresare0.11,
immunity;itrepresentsadynamicstructure andasite of 0.44, and 0.3–1%, respectively [60,61]. Increased sperm
spermatozoal phagocytosis. Intraluminal macrophages, aneuploidy rates may impact male fertility and pregnancy
also known as spermatophages, absorb degradation viability. Their causes are unknown, but smoking, alcohol,
products rather than the whole sperm. T-type lympho- chemotherapy,and agingmayplayarole.Interchromosomal
cytes, in addition to ASA, may contribute to testicular variationintheratesofdisomieshasbeenobservedwithsex
damage after vasectomy [52]. Spermatozoa exposed to chromosomesandchromosomes21and22;thehigherrateof
cytokines such as tumor necrosis factor and interferon g abnormalities related to such chromosomes may be due to
show impairment in motility and inability to penetrate their lower rate of meiotic recombination, which renders
hamster eggs [53,54]. Despite all this, the full-blown them more prone to nondisjunction [62].
spectrum of cell-mediated immunity is difficult to prove
Abnormal spermatozoa that retain excess cytoplasm show
using laboratory tests and its role in unexplained
a greater extent of aneuploidy and diploidy than those
infertility is still speculative [47]. More sophisticated
withoutexcesscytoplasmfromthesameejaculate,whether
investigation is needed to detect the impact of cellular
selected by density gradient centrifugation [63], swim-up
immunity in men with unexplained infertility.
[64], or binding to hyaluronic acid [65]. In contrast, both
morphologically normal and abnormal spermatozoa can be
Deficientsperm function
disomic or diploid [66], or contain damaged DNA [67]; as
Conventional semen parameters such as sperm count,
such, selecting normal-looking spermatozoa for assisted
motility,vitality,andmorphologyareinadequatetomonitor
reproductive technique (ART) does not guarantee the
sperm function and to be used as markers of fertility
absence of chromosomal abnormalities.
potential [55]. Conversely, sperm function tests may
provide more clinically useful prognostic and/or diagnostic Chromosomal inversions, deletions, balanced or unba-
information. Such tests may be used to distinguish lanced translocations, and Y-chromosome microdeletions
between fertile and infertile men and to aid in showing areoftenassociatedwithabnormalsemenparametersand
the cause of male subfertility and in suggesting therapeu- higher rates of abortion, and in some cases, with a higher
tics. Sperm function tests available in the andrology riskforthebirthofaseverelyhandicappedchild[68].In
armamentarium include assays that investigate sperm Y-chromosome infertility, the AZFc region is prone to
DNA integrity, seminal reactive oxygen species, AR, many smaller subdeletions that are thought to be caused
hyperactivated motility, and ZP binding and penetration. by intrachromosomal recombination [69]. These partial
deletions produce a wide array of phenotypes, ranging
Sperm chromosomal complement andDNA integrity defects from normospermia to azoospermia, because of factors
Germcellsundergomeioticdivisionstoformfourhaploid that include the interaction of the environment and the
spermatids. During meiosis, shuffling of some genes genetic background [70].
occurs between homologous chromosomes giving rise to
Most chromosomal abnormalities may be detected by
genetic diversity. During spermiogenesis, the haploid
using one of the following methods: (i) Sperm karyotyp-
sperm chromatin undergoes significant changes in which
ing for detection of numerical chromosomal abnormal-
most histones are replaced first by transition proteins,
ities; (ii) Fluorescence in situ hybridization analysis,
and then by positively charged protamines [56]. By this
which can be used to assess numerical and structural
remodelingprocess,thespermDNAcondensessotightly
chromosomal changes by using specific probes; and (iii)
that it is resistant to mechanical stresses such as
quantitative polymerase chain reaction, which is a
sonication [57] and even to boiling [57], which destroy
promising technique to detect and quantify damage to
the DNA in somatic cells. The condensation of sperm
nuclear and mitochondrial DNA [71].
DNA protects it during its transit through the male and
female reproductive tracts. Cytogenetic analysis and Specific gene defect (mutations and polymorphisms): the
molecular biology genetic testing may identify subfertile definitionofnucleotidesequenceforthehumangenome
6 HumanAndrology
has facilitated the identification of human fertility- Reactiveoxygen species
related genes and this will open the way to identify It has been shown that 40–88% of nonselected infertile
these genes in the future. Nevertheless, DNA sequence patientshavehighlevelsofseminalROS[85].Moreover,
analysis is rarely performed in the evaluation of male normospermic infertile men have higher ROS levels and
infertility [72]. In animal studies involving mice, up to reduced total antioxidant capacity levels than the
300 null mutations and 50 conditional targeted deletions normospermic fertile counterpart [86]. However, the
have produced models of male infertility. Not only the true prevalence of oxidative stress problem among
DNA sequence has effect on male infertility but there is normospermic men remains to be determined. Mamma-
also a role for epigenetic events and modifiers of gene lian spermatozoa are redox cells that are able to produce
expression. With regard to unexplained male infertility, oxygen radicals and to export them to the extracellular
genescertainlyplayaroleastheycontrolmeiosisevents, medium [87–91].
spermiogenesis, remodeling, motility, capacitation, and
The main source of oxygen radicals in spermatozoa
fertilization. It is now possible to monitor the expression
appears to be the mitochondria, as the result of the
of thousands of genes simultaneously with DNA micro-
monovalent reduction of molecular oxygen during oxida-
array analysis. Garrido et al. [73] used the microarray
tive phosphorylation [88]. ROS refer to a group of
technologyintheanalysisofspermatozoafromfertileand
metabolites formed by reduction of oxygen, including
infertile men with normal semen analysis. They found
that hundreds of gene sequences (targets) were differ- free radicals such as superoxide anion (O2–K), the
hydroxylradical(OHK)aswellaspowerfuloxidantssuch
entially expressed between groups. Preliminary results
ashydrogenperoxide(H O ).Italsoincludesreactantsof
confirmed that there are few genes that are over- 2 2
carbon-centered radicals with molecular oxygen, includ-
expressed, whereas all others are underexpressed in
ingperoxylradicals(ROOK),alkoxylradicals(ROK),and
infertile men.
organic hydroperoxides. ROS may also include other
powerful oxidants such as peroxynitrite or hypochlorous
DNA integrity defects: sperm DNA integrity is increasingly
acid as well as the highly biologically active free radical,
being recognized as an important marker of fertilizing
nitric oxide (KNO). ROS in semen originate from
efficiency, and it is associated with better diagnostic and
immature spermatozoa and seminal leukocytes [92].
prognostic values than standard sperm parameters [74].
These free radicals have considerable reactivity and the
Saleh et al. [75] reported that anincrease of spermatozoa
ability to react with and modify the structure of many
with abnormal chromatin structure or DNA damage
differentkindsofbiomolecules,includingproteins,lipids,
(expressed as DNA fragmentation index) negatively
and nucleic acids. The wide range of targets that can be
correlated with intracytoplasmic sperm injection (ICSI)
attackedbyROSisacriticalfacetoftheirchemistrythat
and in-vitro fertilization (IVF) outcomes.
contributessignificantlytothepathologicalimportanceof
these molecules [92]. ROS in low levels have a
Populations of sperm with DNA damage are more often
physiological role. They are required by sperm to attain
seen in subfertile/infertile men than in fertile ones
their functional maturity and are essential for capacita-
[76–78] Spermatozoa with damaged DNA may lead to
tion, hyperactivation, and AR [93,94]. ROS also exert
paternal transmission of defective genetic material with
their effect on sperm–oocyte interaction. Low levels of
adverse consequences to embryo development [79,80].
lipid peroxidation cause modifications of plasma mem-
Approximately 8% of infertile men have abnormal DNA
branes facilitating sperm adhesion to the oocyte [95].
integrity despite normal semen parameters [81].
However, the physiological ROS levels are still undeter-
mined.
DNA damage is often assessed by the determination of
chromatin compaction or DNA fragmentation. The A natural antioxidant defense system offers protection
former examines the accessibility of dyes (Toluidine against ROS. It consists of radical scavengers, chain-
blue, aniline blue, and chromomycin A3) to nucleopro- breakingantioxidants,andROS-metabolizingenzymesin
teins or chromatin after challenging spermatozoa with thevicinityofthespermatozoaduringtheirsojourninthe
physical insults; as such, it reflects how susceptible the malereproductivetract.Radicalscavengersincludesmall
DNA is, or has been, to noxious agents [82]. Toluidine molecular mass such as vitamin C, uric acid, tryptophan,
and aniline blue stains bind to lightly packed chromatin and taurine [96,97]. Chain-breaking antioxidants include
and to lysine residues of histone that are not fully membrane-associated antioxidants epitomized by a-
replaced by protamines, respectively. Chromomycin A3 tocopherol, a hydrophobic vitamin that is capable of
binding is specific for protamine-deficient areas because terminating the peroxidation chain reaction [98]. Sper-
of its affinity to guanine–cytosine (GC)-rich areas of matozoa also harbor antioxidant enzymes such as super-
DNA [83]. In contrast, DNA fragmentation is measured oxide dismutase and those of the glutathione cycle, but
by detecting single-strand or double-strand DNA breaks. very little catalase. Imbalance between the oxidant load
Transferase-mediated dTUP nick-end labeling, comet andnaturalantioxidantdefensesystemhasapathological
assay,acridineorangetest,andspermchromatinstructure effect on sperm function by causing damage to sperm
assay are methods clinically available to detect DNA DNA in the nucleus and mitochondria as well as by
fragmentation.Althoughtheydifferincostsandmethods, inducing lipid peroxidation in the sperm plasma mem-
most of the mentioned tests are clinically significant and brane [99]. DNA damage includes single-stranded or
correlate with sperm function and fertility [84]. double-stranded DNA breaks, DNA base-pair oxidation,
UnexplainedmaleinfertilityHamadaetal. 7
chromatin cross-linking, chromosome microdeletions, and obvious sperm abnormalities such as asthenozoospermia
evenvarioustypesofgenemutationssuchasdeletions,point and teratozoospermia, many patients have normal semen
mutations, or polymorphisms that may result in decreased analysis and subtle sperm defects that affect sperm–ZP
semen quality [100,101]. 8-Hydroxy-2-deoxyguanosine is interaction. These defects cannot be shown by routine
consideredakeybiomarkerofoxidativeDNAdamage[102]. semen analysis but are apparent with sperm–ZPinterac-
ROSmayinducelipidperoxidationandhencelossofsperm tion tests [113].
motilityandmayeveninitiateachainreactionbyactivating
caspases that ultimately lead to apoptosis [103]. Zona pellucida binding defects: sperm binding to the ZP is
attributed to the presence of complementary binding
The most often used methods for detecting ROS in an
sitesorreceptorsonthesurfaceofthegametes;typically,
andrology setting are divided into two major categories,
these receptors manifest a high degree of species
that is, direct methods such as chemiluminescence and
specificity [114–116]. Human ZP (hZP) is composed of
flow cytometry, and indirect methods such as the
four major glycoproteins (hZP1, hZP2, hZP3, and hZP4)
colorimetric one. Chemiluminescence uses the probes
[117]. The ZP3 of human oocytes is believed to be the
lucigenin or luminol to detect ROS [104,105]. Luminol
primaryZPreceptorforcapacitatedacrosome-intactsperm
(C H N O ) is a versatile chemical that shows chemilu-
8 7 3 2 binding [117,118]. In contrast, the exact nature of human
minescence when mixed with an appropriate oxidizing
sperm receptors for the ZP has not been established.
agent. It can penetrate inside the cell and react with
Althoughanumberofcandidatespermproteinshavebeen
intracellular reactive oxygen species, in addition to
foundtobeabletointeractwitheithersolubilizedorintact
extracellular ones. This probe has to undergo a one
ZP, it is not clear whether or not they are the primary
electron oxidation before it becomes sensitized to the
receptors for sperm binding to the ZP [118–121]. Sperm
presence of ROS, which can be accomplished by the
binding to ZP3 induces a signal transduction cascade
addition of horseradish peroxidase to promote luminol
within the spermatozoon, involving multiple proteins and
oxidation in the extracellular space. In the absence of
other factors, including protein kinases A and C pathways
exogenoushorseradishperoxidase,theassayisdependent
[122],thatleadstotheAR.Acrosome-reactedspermatozoa
onthepresenceofintracellularperoxidasetoactivatethe
are believed to bind to ZP2, which facilitates the
probe [104–107]. The one electron oxidation of luminol
penetration to the zona matrix and progression into the
leadstothecreation ofaradicalspecies(LK).Thelatter
perivitelline space [123]. Defective ZP-bound sperm are
then interacts with ground-state oxygen to produce O2K present in approximately 15 and 25% of subfertile men
thatinducesthe oxygenationofLKtocreate anunstable
with a normal semen analysis and with an abnormal one,
endoperoxide, which ultimately breaks down with the
respectively [124–126]. Such individuals have a reduced
release of light. Lucigenin is a positively charged
chance of achieving successful fertilization when under-
moleculeandhenceitcannotenterthecellandtherefore
goingIVF[126].Mackennaetal.[127]reportedthattwoof
could only measure the extracellular ROS only. Photons
18 men with unexplained infertility showed lack of sperm
produced are converted to an electrical signal and
binding to the zona despite having sperm morphology and
measured using a luminometer [108], with ROS genera-
hyperactivation status similar to fertile individuals. The
tionbeingmeasuredascountedphotonsperminute.The
presence of defective sperm–ZP binding in infertile men
normal range is less than 0.2(cid:3)106cpm per 20 million
with normal semen may be due to defective signal
spermatozoa[109].IntracellularROScanbemeasuredby
transduction pathways upstream of protein kinases A and
flow cytometry using different fluorescent probes such
C. However, most defective sperm–ZP binding infertile
as 20, 70-dichlorofluorescin-diacetate, hydroethidine, that
menwith normalsemenand thosewith severe teratozoos-
react with ROS to emit a red fluorescence [110]. The
permia are likely to have downstream disorders, structural
colorimetric technique is also widely used for indirectly
defects, or absence of sperm receptors for binding the ZP.
quantifying ROS. It is based on the principle of spectro-
photometry and measures lipid peroxide end products,
mainly malondialdehyde, lipid hydroperoxides, and iso- Twotestsofspermbindingtothehumanzonahavebeen
prostanes [111]. described: (i) the hemizona assay and (ii) the sperm–
zona binding ratio test. In the former, a single zona is
bisectedandeachzonahalfisincubatedwithcontroland
Fertilization defects
patient sperm suspensions [128]. In the latter, a
The sperm fertilizing potential is related to its ability of
completezonaisincubatedwithequalnumbersofmotile
undergoing capacitation, which includes the acquisition
spermatozoa from control and test populations, each
of hyperactivated motility, and the acrosomal reaction to
labeled with a different fluorescent dye [129]. In each
penetrate the ZP and its ability to fuse with oolema.
case, the number of spermatozoa from each population
Normospermic infertile men may have defective sperm
bound per whole or halfzonaiscounted andthe number
that are unable to fertilize. This assumption is based on
oftestspermisexpressedasaratioofthatofthecontrol.
the observation of low success rates of IVF and
intrauterine insemination (IUI) in certain cases of Capacitation defects: capacitation is a combination of
unexplained infertility. The major cause of fertilization concomitant processes; mainly, the sperm acquisition of
failure in conventional IVF [112] is due to abnormalities a motility pattern known as hyperactivated motility,
of sperm–ZP binding and penetration. Although most which enables efficient zona drilling and allows sperma-
sperm–ZP binding and penetration defects are due to tozoa to reach the oolema, and preparation for the AR
8 HumanAndrology
[130]. Defects in capacitation may explain subfertility in which is the migration of spermatozoa toward a higher
some normospermic infertile men. concentration of chemoattractant [142].
Assessment of hyperactivation motility in vitro involves
Defects in hyperactivation: hyperactivation is considered the
theuseofcomputerizedmotionanalysisinconjunctionto
firststepof the complexcapacitationprocess. It involves
kinematics module to distinguish different subpopula-
a typical swimming pattern of movement shown by most
tionsofmotilespermatozoa.Hyperactivatedspermatozoa
spermretrievedfromtheoviductalampullaatthetimeof
can be distinguished from nonhyperactivated ones by
fertilization [131]. Hyperactivated sperm show high-
their high curvilinear velocity (VCL), low linearity
amplitudeandasymmetricalflagellarbendingmovement.
(calculated as straight-line velocity/VCL), and large
Hyperactivation is characterized by switching of sperm
amplitude of the lateral head displacement. The clinical
movement from progressive motility to more vigorous
significance of such data is reflected by their correlation
(nonprogressive) flagellar motion. The role of hyperacti-
with IVF outcomes and spontaneous pregnancy rates
vation is to enhance the ability of sperm to detach from
[143]. Munire et al. [144] showed that there is a
the oviduct wall, to move around in its labyrinthine
significant decrease in the percentage of hyperactivated
lumen, to penetrate into the cumulus oophorus, and
sperm,spermmotility,progressivemotility,andVCLfrom
finally,topenetratetheZPoftheoocyte[132].However,
infertile men in comparison with sperm from fertile
littleisknownaboutthemechanismsthatleadthesperm
donors after overnight incubation with capacitating
to hyperactivation. It is speculated that specific signals
conditions,whereaslinearitywasincreasedintheformer.
appearwithintheoviductshortlybeforeovulation.There
Computerized assessment of follicular fluid (FF)-in-
is evidence that various components of the female
ducedhyperactivationhasbeenprovedtobesignificantly
reproductive tract serve as physiological stimuli of
lower in patients with unexplained infertility in compar-
hyperactivation, such as hormones (e.g. progesterone),
ison with normal fertile men [128]. The absence of
ions, and secretions in the oviduct luminal fluid [133].
hyperactivation after the addition of FF was observed in
When the oocyte enters the oviduct, it usually brings
39% of patients with unexplained infertility [127]; it is
alongcumulusoophorusandFFthathavebeenshownto
likely that spermatozoa from such patients have reduced
influence sperm motility. A number of physiological
abilitytopenetratethroughtheoocytevestmentsandZP
factors such as Ca2+, c-AMP, bicarbonate, and metabolic
as a result of this abnormal hyperactivation response to
substrates have been found as essential for the initiation
FF. In fact, Avenarius et al. [145] discovered that male
or the maintenance of hyperactivated motility in vitro
patients with mutated CatSper1 gene are infertile with
[134]. Recent studies have demonstrated that increased
poorhyperactivationresponsedespitetheirnormalsperm
intracellular calcium entry through voltage gated calcium
count, morphology, and even their initial sperm motility.
channels (Cation channel of sperm; CatSper1–4) in the
Furthermore, an animal study on mice concluded that
principal piece of the sperm flagellum is the prime
mutation in each of CatSper (1–4) ion channel protein
mechanism for hyperactivation [135–137]. This entry is
can lead to infertility despite normal semen parameters,
induced by intracellular alkalinization because of extru-
normal testicular histology, size, and weight [146].
sion of H+ through voltage gated proton pumps, which
Interestingly,therearetwoknownCatSper2gene-related
are also located in the principal piece of the flagellum
mutations in humans that cause male infertility, termed
[135]. Increased intracellular pH and intracellular Ca+
CatSper-related nonsyndromic male infertility and deaf-
regulatenotonlythehyperactivationprocessbutalsothe
ness–infertility syndrome [147]. However, both syn-
ARandtheabilityofthespermtofertilizetheegg[135].
dromes are associated with gross semen abnormalities.
Interestingly, molecular studies on CatSper ion channel
Further investigation is needed to show the genetic and
show that it is a novel protein complex composed of six
molecular nature of fertilization in patients with defec-
subunits.Ofthese,fourareasubunits(CatSper1–4)with
tive hyperactivation response and unexplained infertility.
calcium-selective pore and two are transmembrane
Moreover, minor mutations in human CatSper (1–4)
proteinswithlargeextracellulardomains,calledCatSperb
genes are yet to be deciphered in men with unexplained
and CatSperg, of unknown functions [138,139]. For
infertility.
humans, hyperactivation is not well defined as it is for
other species [134], and only a small proportion of the Acrosome reaction: the AR is defined as the process of
sperm population may be hyperactivated at each time. fusion of sperm plasma membrane with the outer
The extent of hyperactivated motility in a population is acrosomal membrane leading to the release of exocytotic
positivelycorrelatedwiththeextentofzonabinding,the proteolytic enzymes (acrosine and hyaluronidase) in
AR, zona-free oocyte penetration, and fertilizing capacity response to sperm–ZP binding. ZP3 is considered the
invitro[140].Migrationofspermatozoatowardtheoocyte natural stimulus for the AR, which leads to the
in the oviduct’s ampulla is probably assisted by thermo- proteolytic dissolution of the ZP. Human sperm initiate
taxis, which is the migration of spermatozoa toward a primary binding to the ZP with intact acrosome [148].
thermal gradient. The ampulla has a higher temperature Glycoprotein (ZP3), present inthe ZP, isinvolved inthe
than the isthmus of the fallopian tube and this may induction of the AR [149]. Artificial stimuli used in vitro
mediatelong-rangemigrationtotheampulla[141].Ithas to challenge the AR are calcium ionophore A23187 and
beenshownthatonlycapacitatedspermatozoarespondto progesterone.TherearetwotypesofdefectiveAR,which
these influences [141]. The spermatozoa may also be haveclinicalsignificance.ThefirstistheARprematurity,
directed to the egg within the cumulus by chemotaxis, which is defined as a high level of spontaneous AR
UnexplainedmaleinfertilityHamadaetal. 9
(>20% of spermatozoa showing spontaneous AR). The Furthermore, it is possible that other prostatic secretions
second is the AR insufficiency, which is defined as poor (e.g. citric acid) might affect zinc levels or even sperm
responsiveness to AR stimulants (when <15% of function. Zinc level determination in the seminal plasma
spermatozoa responded to the ionophore A23187 chal- is unlikely to be clinically useful for the prediction of
lenge) [150]. Both conditions are associated with poor defective ZPIAR; currently, only spermatozoa–ZP inter-
fertilization capacity on conventional IVF treatment. action tests using human oocytes have been shown to be
Under normal conditions, more than 15% AR in response accurate [159].
to ionophore treatment is expected [151]. Various
Defect ofthe fusogenic ability of the acrosome-reacted sperm with
techniques are used for visualizing the human sperm
the oolema: the ability of the equatorial region of the
acrosome,includinglectins,monoclonalantibodies,orthe
acrosome-reactedhumanspermtofusewiththevitelline
triple stain [152–156]. Replicate (minimum two) slides
membrane of the oocyte is tested by using the sperm
must be scored for each determination, with at least 100
penetration assay (SPA) also known as the zona-free
spermatozoa counted per slide. Some patients with
hamster oocyte penetration test. Although this test does
unexplained infertility that have normal sperm–ZP
not assess sperm–ZP, it measures the spermatozoon’s
binding have defective ZP-induced AR (ZPIAR), which
ability to undergo capacitation, AR, fusion and penetra-
will result in reduced sperm–ZP penetration and failure
tion through the oolema, and decondensation within the
of fertilization. Patients with this condition usually have
cytoplasm of an oocyte. The ZP is removed from a
a long duration of unexplained infertility, normal semen
hamster oocyte, which is then incubated with human
analysis, and normal sperm–ZP binding, but show failure
spermatozoa. In the original test, scoring is achieved by
of ZP penetrationby sperm andhave zero or lowratesof
calculating the percentage of ova that are penetrated;
fertilization with standard IVF [157]. The diagnostic
normal sperm are able to penetrate 10–30% of hamster
feature isthat verylow proportionsof sperm undergo AR
ova [68]. Recent refinement of this test is performed by
after binding to the ZP. However, these patients achieve
incubating sperm in more potent capacitating media,
high fertilization and pregnancy rates with ICSI [158].
which allow the majority of ova to be penetrated; scores
Although the frequency of defective ZPIAR was high in
are obtained by calculating the number of sperm that
subfertilemenwithidiopathicoligozoospermia(65%)and
penetrate each ovum [68]. Aitken et al. [170] reported
severe teratozoospermia (62%, strict normal sperm
that 34.1% of patients with unexplained infertility had
morphology r5%), defective ZPIAR was found in only
less than 10% oocyte penetration against 0% in a control
25% of normozoospermic subfertile men [125]. Although
group of fertile men. Various studies have evaluated the
the exact mechanisms of defective AR are unknown,
ability of the SPA to predict success or failure of IVF.
defective ZPIAR is more likely to be related to major
Some investigators have shown no correlation with an
structural defects of the sperm head, such as small or
abnormal test [171], whereas others have claimed 100%
abnormal acrosomes, or associated abnormalities in the
predictability [172]. Taking an average from different
overlying plasma membrane in severe teratozoospermic
studies, a normal SPA may have 70% predictability of
subfertile men. In normozoospermic men, it has been
fertilization in vitro [44]. Nevertheless, semen samples
shown that the seminal zinc concentration was signifi-
that fail to fertilize hamster ova usually are unable to
cantly higher in men with defective ZPIAR. Addition of
fertilize humanova[68].AlthoughtheSPAisconsidered
0.5mmol of zinc to the culture media had no effect on
a research tool, it may be of clinical value for men with
spermatozoa–ZP binding, but significantly reduced
unexplainedinfertilitywithpoorfertilizationrateonIVF.
ZPIAR rates in vitro. Zinc has a role as a decapacitation
factor by binding to the sperm plasma membrane; as
such, high seminal zinc concentration may have an A practical approach for the assessment of
adverse effect on the ZPIAR [155]. Addition of zinc to men with unexplained infertility
the culture medium during capacitation of human PCT, if appropriately timed and performed, can be the
spermatozoa inhibited spontaneous AR as well as the initial test for couples with unexplained male infertility.
ARinducedbythecalciumionophore[159].Itispossible Cervical mucus is normally hostile to sperm, except near
that zinc binding to the sperm plasma membrane affects the time of ovulation. The absence of sperm on a PCT
calcium influx through ion competition during capacita- in the presence of normal semen parameters suggests
tion. It is known that the seminal plasma contains incorrect coital technique or failure to ejaculate into
decapacitation factors as its addition to the culture vagina, whereas the presence of normal sperm numbers
medium inhibits sperm capacitation and hyperactivated but reduced motility or a shaking motion on a PCT is
motility,aswellasspermatozoa–ZPbindingandpenetra- suggestiveof the presenceof ASAs [173].The finding of
tion in vitro [160–163]. It is therefore apparent that a normal PCTraises the possibility of a functional sperm
defectiveZPIARmaybecausedbydifferentmechanisms defect.Assessmentofspermfunctioncanbedividedinto
in men with or without severe sperm morphological two steps. The first step should be to check the
defects.Inmenwithnormalspermmorphology,defective competence of the sperm before the fertilization event
ZPIAR is most likely to be due to subtle biochemical or by measuring the levels of ROS and DNA integrity
molecular defects in ZP receptors, signal transduction defects. The second step should include the assessment
pathways,inefficientcholesterolorzincremovalfromthe ofthefertilizationpotentialofsperm,especiallyforthose
plasma membrane during capacitation, actin polymeriza- patients with a history of failure of conventional
tion, or acrosomal enzyme activation [116,164–169]. IVF. These tests include sperm–ZP binding assay,
10 HumanAndrology
capacitation, hyperactivation motility, inducibility of AR, steroids remains unclear as most studies lack appropriate
and the ability of sperm to fuse with the vitelline placebo controls or have used different regimens and
membrane (zona-free hamster egg penetration test). drugs. Despite these shortcomings, two prospective and
Figure 1 shows the appropriate management plan for randomized placebo-controlled studies were conducted
unexplained male infertility. and showed conflicting results. In the study of Hendry
etal.[178],40mgofprednisolonewasgivenfora6-month
period from cycle days 1–10 of the female partner, and
Treatment strategies
then was tapered rapidly for the next 2 days. The PR of
The treatment of men with unexplained infertility does
treatedanduntreatedgroupswas31and9%,respectively.
not follow the typical rules for standard clinical practice
In another study, the investigators reported similar PR
decision making. Such a convention requires a specific
after administration of methylprednisolone for three
scientificplantoidentifyandcorrectaknowndefectand
cycles, despite a significant decrease in sperm-associated
thecalculationofrisksversusbenefits.Owingtothefacts
IgG(butnotIgA)inthesteroidtreatmentgroup[179].It
thatnorecognizablereasonforinfertilityisidentifiedand
has been shown that steroids may be only effective in
few randomized clinical trials are available, no uniform
removing sperm-bound ASA in the presence of low
protocol could be followed.
antibody titer [180]. Treatment with high-dose steroids
Counseling is an important part of management, particu- for long time is associated with side effects that include
larly with regard to orientation about the physiology of moodchanges,fluidretention,dyspepsia,gastrointestinal
ovulation and the need to time intercourse with the bleeding,asepticnecrosisofthehipjoint,andsignificant
periovualtoryperiod.Adetailedmedicalhistorymayhelp decrease of bone mineral density in up to 60% of the
to disclose any hidden problems such as sexual dysfunc- patients [178,181,182].
tion and inadequate coitus habits.
Alternatively, methods to remove ASA already bound to
Expectant management sperm include sperm washing and IgAproteasetreatment.
Expectantmanagementisadvisedforyoungcoupleswith The effectiveness of these techniques in recovering
a short duration of infertility. Pregnancy may occur antibodies-free spermatozoa is conflicting; most reports
spontaneously without any interventions in cases of show limited success because of the difficulty of eluting
unexplained infertility [174]. Hull et al. [175] found a the sperm cell surface by any washing method [183].
cumulativepregnancyrate(PR)rangingfrom50–80%over Esteves et al. [184] demonstrated that the population of
a3-yearperiodasafunctionoffemaleageand30–80%PR antibody-free spermatozoa was increased by 29% after
asafunctionofinfertilityduration.CumulativePRsof60% discontinuous colloidal gradient centrifugation. However,
maybeachievedwithin2years.However,infertilityperiods the investigators observed that sperm washing was
longerthan3yearsareassociatedwithverylowPRof1–3%, ineffective to remove ASA in approximately 30% of the
particularly if the female partner is aged 35 years or older cases,andadvisethatthepotentialbenefitofthisstrategy
[174].Forcoupleswhosetimetoconceiveislongerthan3 has to be tested individually. Microinjection of the
years, the cumulative PR decreases by 2% for each year of compromised spermatozoa into the oocyte cytoplasm
age after 25.7 years [176]. Owing to the costs of infertility (ICSI) bypasses sperm–oocyte membrane interaction, and
treatments and given high proportion of couples with ICSI has been shown to increase fertilization when
unexplained infertility who spontaneously conceive within compared with conventional IVF in cases of male
a2-yearperiod,itisadvisabletodefertreatmentofcouples immunologic infertility. Nagy et al. [185] analyzed the
in this time period, unless the female partner is aged 35 outcomeofICSIin37menwithaproportionofantisperm
years or older. antibody-bound spermatozoa of 80% or higher. They
concluded that fertilization, cleavage, and PRs after ICSI
Interventional management were not influenced by the percentage of ASA-bound
Interventions,whichincludemedicationand/orsurgeryor spermatozoa, by the dominant type of antibodies present,
assisted conception, are justified in cases of unexplained or by the location of ASA on the spermatozoa. However,
infertility of long duration and/or advanced maternal and embryo quality was lower in the ASA-positive group. In
paternal age. another study, similar results were observed but a higher
rate of first trimester pregnancy loss was observed in the
Immunological infertility ASA-positive group [186]. Clarke et al. [187] and Check
Treatment of immune infertility includes methods to et al. [188] studied 39 patients with a strong positivity on
either decrease ASA production or to remove sperm- IBT (>80%) and 93 patients with various degrees of
bound ASA. ASA titers may be decreased by using autoantibodies, respectively. They found that fertilization
condoms and systemic steroid. Condoms are of theore- and PRs were comparable between different levels of ASA
ticalbenefitbecausetheymayhelptolessenthechances onsperm.Estevesetal.[189]analyzedalargecohortof351
for frequent exposure of sperm to the female reproduc- patientsandconfirmedthatfertilization,cleavage,andPRs
tive tract and hence decrease the sensitization and afterICSIwerenotinfluencedbytheASAlevelsonsperm.
formation of ASA in the cervical mucus [177]. Immuno- These investigators observed neither the negative impact
suppressivetherapyhadbeentriedinearlyyearsbutitis ofASAonembryoqualityandcleavageratenoranincrease
seldom used nowadays, mainly because of the high in pregnancy loss, as reported by other investigators.They
incidence of side effects [178]. Moreover, efficacy of also compared ICSI outcomes between patients with ASA
UnexplainedmaleinfertilityHamadaetal. 11
Figure1
Work-upplanforunexplainedmaleinfertility.ROS,reactiveoxygenspecies;ICSIintracytoplasmicsperminjection;andART,assistedreproductivetechniques.
positivity and a group of patients in which ICSI was addition, therapeutic dosing, duration, and the toxic
indicated for other reasons. Fertilization, embryo develop- levels of ROS are still to be determined.
ment, pregnancy success, and miscarriage rates after ICSI
in men showing varying levels of autoimmunity against
spermatozoawerewithinthesamerangeasourpopulation DNAdamage
of ICSI patients with severely abnormal seminal para- The management of unexplained male subfertility
meters.TheinvestigatorsconcludebysuggestingthatASA because of DNA damage often requires ART. The
may become inactive within the ooplasm after microinjec- probability of fertilization in vivo and by IUI seems to
tion,orthatasegregationprocessmayoccurduringthefirst be low when the proportion of sperm cells with DNA
cleavage divisions, similar to the inactivation and segrega- damage exceeds 30 and 12%, as detected respectively by
tionprocessesthatalsooccurwiththeacrosomeandsperm sperm chromatin structure assay or transferase-mediated
tail after microinjection. dTUP nick-end labeling [192,193]. Sperm DNA damage
is negatively correlated with embryo quality and blas-
tocystformationinIVFcyclesandwithfertilizationrates
Excessive oxidative stress
both in IVFand ICSI cycles [194]. However, successful
Men with unexplained infertility may have higher
pregnancies in IVF/ICSI cycles can be obtained using
oxidative stress than controls [86,190]. Lines of therapy
semen samples with a high proportion of DNA damage.
include lifestyle habit modification, use of antioxidants,
Bungum et al. [195] demonstrated that significantly
and ART. Patients are advised to quit smoking, eat
higher clinical PRs (52.9 vs. 22.2%) and delivery rates
antioxidant-rich food, and avoid pollutant environmental
(47.1 vs. 22.2%) were obtained after ICSI as compared
conditions.Anantioxidanttherapyhasattractedattention
with IVF when semen samples with high levels of sperm
in the recent years. Antioxidants are compounds and
DNA damage were used, as previously suggested.
reactants that dispose, scavenge, and suppress the
formation of ROS, or oppose their actions. Various The activation of embryonic genome expression occurs
antioxidants such as carnitine, vitamin C, vitamin E, at the four-cell to eight-cell stage in human embryos.
coenzyme Q10, selenium, glutathione, N-acetyl cysteine, Therefore, the paternal genome may not be effective
carotenoids, and trace metals are available. A recent untilthisstageanditisspeculatedthatanelevatedlevel
Cochrane review on the use of antioxidants for male of sperm DNA strand breaks seems to be of importance
subfertility suggests that antioxidant supplementation inthelaterstagesofembryonicdevelopment.Aitkenand
may improve the outcomes of live birth and PR for Krausz [196] proposed that sperm DNA damage is
subfertile couples undergoing ART cycles, but further promutagenic and can give rise to mutations after
head-to-head comparisons are necessary to identify the fertilization, as the oocyte attempts to repair DNA
superiority of one antioxidant over another [191]. In damage before the initiation of the first cleavage.