dehydrogenasesss17.com

AsianJAndrol2005;7(2):121–126.
121.
Novelassociationbetweenspermdeformityindexandoxi-dativestress-inducedDNAdamageininfertilemalepatientsTamerM.
Said1,NabilAziz2,RakeshK.
Sharma1,IwanLewis-Jones2,AnthonyJ.
ThomasJr1,AshokAgarwal11CenterforAdvancedResearchinHumanReproduction,InfertilityandSexualFunction,GlickmanUrologicalInstituteandDepartmentofObstetrics-Gynecology,TheClevelandClinicFoundation,Cleveland,Ohio44195,USA2ReproductionMedicineUnit,LiverpoolWomen'sHospital,LiverpoolL87SSS,UKAbstractAim:Toinvestigatetheimpactofabnormalspermmorphologyusingthespermdeformityindex(SDI)onreactiveoxygenspecies(ROS)productionanditscorrelationwithspermDNAdamage.
Methods:Semensampleswerecollectedfrommenundergoinginfertilityscreening(n=7)andhealthydonors(n=6).
Maturespermatozoawereisolatedandincubatedwith5mmol/Lβ-nicotinamideadeninedinucleotidephosphate(NADPH)forupto24htoinduceROS.
SpermmorphologywasevaluatedusingstrictTygerberg'scriteriaandtheSDI.
ROSlevelsandDNAdamagewereassessedusingchemiluminescenceandterminaldeoxynucleotidyltransferase-mediatedfluorescein-dUTPnickendlabeling(TUNEL)assays,respectively.
Results:SDIvalues(median[interquartiles])werehigherinpatientsthandonors(2[1.
8,2.
1]vs.
1.
53[1.
52,1.
58],P=0.
008).
AliquotstreatedwithNADPHshowedhigherROSlevels(1.
22[0.
30,1.
87]vs.
0.
39[0.
10,0.
57],P=0.
03)andhigherincidenceofDNAdamagethanthosenottreated(10[4.
69,24.
85]vs.
3.
85[2.
58,5.
10],P=0.
008).
HigherDNAdamagewasalsoseenfollowing24hofincubationinpatientscomparedtodonors.
SDIcorrelatedwiththepercentageincreaseinspermDNAdamagefollowingincubationfor24hinsamplestreatedwithNADPH(r=0.
7,P=0.
008)andcontrols(r=0.
58,P=0.
04).
Conclusion:SDImaybeausefultoolinidentifyingpotentialinfertilemaleswithabnormalprevalenceofoxidativestress(OS)-inducedDNAdamage.
NADPHplaysaroleinROS-mediatedspermDNAdamage,whichappearstobemoreevidentininfertilepatientswithsemensamplescontainingahighincidenceofmorphologicallyabnormalspermatozoa.
(AsianJAndrol2005Jun;7:121–126)Keywords:β-nicotinamideadeninedinucleotidephosphate;oxidativestress;spermdeformityindex;spermDNAdamage.
OriginalArticle.
DOI:10.
1111/j.
1745-7262.
2005.
00022.
x2005,AsianJournalofAndrology,ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciences.
Allrightsreserved.
1IntroductionSemenanalysisincludingspermmorphologyremainsthemainpillarformaleinfertilitywork-up.
However,differentmethodologiesforspermmorphologyassess-menthaveremainedcontroversialbecauseofthelackofauniversallyacceptablemethod.
Onedrawbackofat-temptstoclassifyspermintomorphologicalsubgroupsasproposedbyWHOisthateachindividualspermisclassifiedonlyoncebutmayhaveseveraldeformities.
Tygerberg'sstrictcriteriahasbeenproposedtocorre-latewithIVFoutcomeresults[1].
However,itmaynotserveasthebestdiscriminatorbetweennormalandfunc-Correspondenceto:Prof.
AshokAgarwal,CenterforAdvancedResearchinHumanReproduction,InfertilityandSexualFunction,GlickmanUrologicalInstitute,TheClevelandClinicFoundation,9500EuclidAvenue,DeskA19.
1,Cleveland,OH44195,USA.
Tel:+1-216-444-9485,Fax:+1-216-445-6049E-mail:agarwaa@ccf.
orgReceived2004-07-15Accepted2004-12-10.
122.
CorrelationofSDIwithspermDNAdamagetionallyimpairedsamplesduetothelackofacut-offpointfornormalvalues.
InareportbyMenkveldetal.
[2],theaveragepercentageofnormalformsinthefertilepopulationwas6.
5%,whileinsubfertileitwas3.
0%.
Ontheotherhand,successfuloocytefertilizationandpregnancieshavebeenreportedincoupleswith0%nor-malspermmorphology[3].
Thespermdeformityindex(SDI)isanovelexpres-sionofspermmorphologicalassessmentbythestrictTygerberg'scriteriafornormalspermmorphologythatwasreportedtocorrelatewithfertilizationrates[4].
SDIisausefulpredictorintheidentificationoffertileandinfertilesemen,andismorereliablethanthemultipleanomaliesindex,whichinvolvestheassessmentofonlyabnormalsperm[5].
Thefertilizingpotentialofthese-mensamplemaybecompromisedatspermdeformityindex>1.
6despitethepresenceofnormalforms[4].
Indefectivespermiogenesis,thereisfailureoftheremodelingofspermmembranecomponents,whichre-sultsinmorphologicallyabnormalspermatozoathatex-hibitcytoplasmicresidues.
Theenzymeglucose-6-phos-phatedehydrogenase(G6PD)isexcessivelypresentinspermresidualcytoplasmandgeneratesβ-nicotinamideadeninedinucleotidephosphate(NADPH).
Inturn,NADPHisusedasasourceofelectronsbyspermatozoatofuelthegenerationofreactiveoxygenspecies(ROS)production[6,7].
Asignificantpositivecorrelationwasobservedbe-tweenspermROSproductionandtheproportionofspermwithabnormalmorphologycharacterizedbyhighSDIscores[8].
HighlevelsofROSleadtooxidativestress(OS),whichisoneoftheleadingcausesofspermDNAdamage[9].
DespitetheprotectivetightpackagingofthespermDNA,deoxyribonucleicacidbasesandphosphodiesterbackbonesaresusceptibletoperoxidation[10].
Moreover,spermatozoaareparticularlysuscep-tibletoOSduetotheirlimitedantioxidantdefensesandthepresenceoflargequantitiesofpolyunsaturatedfattyacidsintheirplasmamembranes[11].
TheprevalenceofspermatozoawithfragmentedDNAisconsideredamongthemostcommoncausesformaleinfertilitythatmaypassundetected[12].
Thecorrela-tionbetweenspermmorphologyandDNAintegrityre-mainscontroversial.
TheobjectiveofourstudywastoinvestigatetheimpactofabnormalspermmorphologyusingSDIonNADPH-mediatedROSproductionanditscorrelationwithspermDNAdamage.
2Materialsandmethods2.
1SubjectselectionThepresentstudywasapprovedbytheInstitutionalReviewBoardoftheClevelandClinicFoundation.
Se-mensampleswerecollectedfrommenundergoingin-fertilityscreening(n=7)andhealthydonors(n=6).
Sampleswithaspermconcentration1*106WBCs/mLwereexcludedtoavoidROSgenerationfrompotentiallynon-spermatozoalcells.
2.
3AssessmentofspermmorphologyFormorphologicalevaluations,seminalsmearswerestainedwithGiemsastain(Diff-Quik,BaxterScientificProducts,McGawPark,USA).
Slideswerecoded(AndrologyLaboratories,ClevelandClinicFoundation)andevaluatedbytheinvestigator(N.
Aziz,LiverpoolWomen'sHospital,Liverpool,UK).
Atotalof100sper-matozoawerescoredperslideusingbrightfieldillumi-nationandanoilimmersionobjectivewithatotalmagni-ficationof*2000.
Atleasttenhigh-powerfieldsse-lectedatrandomfromdifferentareasoftheslidewereexamined.
Acalibratedmicrometerontheeyepieceofthelightmicroscopewasusedtomeasurespermdimensions.
Allslideswereassessedusingamorphologicalclas-sificationbasedonapplyingthestrictTygerberg'scrite-riafornormalspermmorphology[13].
Amultipleentryscoringtechniquewasadoptedinwhichanabnormalspermwasclassifiedmorethanonceifmorethanonedeformitywasobserved.
TheSDIwascalculatedbydividingthetotalnumberofdeformitiesobservedbythenumberofspermrandomlyselectedandevaluated,irre-spectiveoftheirmorphologicalnormality.
Therefore,theratioofthenumberofdeformedspermtothenum-AsianJAndrol2005;7(2):121–126.
123.
berofdeformitiesineachspermshouldnotaffectthefinalresultsoftheSDI.
2.
4SamplepreparationandinductionofROSbyexog-enousNADPHInordertoseparatepredominantlymaturespermatozoa,theliquefiedsemenwasloadedontoa47%and90%discontinuousISolategradient(IrvineScientific,SantaAna,USA)andcentrifugedat500*gfor20min.
Theresulting90%pellet(maturespermatozoa)wasaspirated,re-suspendedinBiggers,Whitten-Whittinghammedia(BWW,IrvineScientific,SantaAna,USA)andtheassessmentofthespermparametersincludingmor-phologywasrepeated.
Thematurespermsuspensionwasfurthersubdividedintotwoaliquotsandeachali-quotwasincubatedwith5mmol/LNADPH(Sigma,StLouis,USA)for0and24hrespectivelyat37°Cand5%CO2.
EachaliquothaditscorrespondingcontrolwithoutNADPH.
2.
5MeasurementofROSROSlevelsinallfractionsweremeasuredin400Laliquotscontaining>2millionsperm/mLusing4Lof25mmol/Llucigenin(bis-N-methylacridniumnitrate,Sigma,StLouis,USA)atfinalconcentrationof0.
25mmol/L.
Negativecontrolswerepreparedbyaddingequalvolumeoflucigeninto400LofPBS.
ROSlevelsweredeterminedbychemiluminescenceassayusingaluminometer(model:LKB953,BertholdTechnologies,Bad-Wilbad,Germany)for15min,andexpressedas*106countedphotonspermin(cpm)per20millionsperm.
2.
6EvaluationofDNAfragmentationSpermDNAstrandbreakswereevaluatedusingaflowcytometricterminaldeoxynucleotidyltransferase-mediatedfluorescein-dUTPnickendlabeling(TUNEL)assaykit(Apo-Direct,BDBiosciences,Mississauga,USA)asestablishedearlier[14].
Dataacquisitionwasperformedwithin3honaflowcytometerequippedwith488nmargonlaserasalightsource(BectonDickinsonFACScan,SanJose,USA).
Aminimumof10000sper-matozoawereexaminedforeachassayataflowrateof1.
6,while6samplesinthepatientgroup(n=7)hadSDI>1.
6.
TheincreaseinROSlevelsfollowingincubationwascalculatedasthedifferencebetween24-and0-hvalues.
ThemedianincreaseinROSlevelswassignificantlyhigherinaliquotsexposedtoNADPHcomparedtotheunexposedaliquots(1.
22[0.
3,1.
87]vs.
0.
39[0.
1,0.
57],P=0.
03).
However,ROSlevelswerecompa-rablebetweenpatientanddonorgroupsbeforeandaftera24-hincubation,regardlessofNADPHexposure.
Similarly,theincreaseinDNAdamagelevelsfollow-ingincubationwascalculatedasthedifferencebetween24hand0hvalues.
AliquotstreatedwithNADPH(frompatientsanddonors)showedsignificantlyhigherinci-denceofincreasedDNAdamagethanthosenottreated(10[4.
69,24.
85]vs.
3.
85[2.
58,5.
1],P=0.
008).
TheincreaseinDNAdamageseenafter24hfollowingincu-bationwassignificantlyhigherinpatientscomparedwithdonorsinaliquotsexposedtoNADPH(16.
56[11.
29,40]vs.
4.
4[3.
92,5.
25],P=0.
007)andincontrolsaliquots.
124.
CorrelationofSDIwithspermDNAdamagenotexposedtoNADPH(5.
1[3.
87,7.
74]vs.
1.
79[2.
87,3.
36],P=0.
03)(Figure1).
SampleswithanSDIscore>1.
6hadhigherincreaseinDNAdamagedspermcomparedtothosewithanSDIscore1.
6despitethepresenceofequivocalspermconcentrationandmotility.
ExposureofspermatozoatoexogenousNADPHhasbeenshowntoresultinadose-dependentincreaseinROS.
However,highconcentrationsofNADPHarere-quiredtoincreaseitsintracellularconcentrationforsig-nificantROSinductionsincethesubstrateismembraneimpermeable[15].
Basedonresultsofourpilotstudy,wehaveselectedtouseexogenousNADPHinaconcen-trationof5mmol/LasamodelforincreasedROSpro-ductionbyspermatozoa.
Usingthismodel,wewereabletodetectanincreaseinROSlevelswithasimulta-neousincreaseinspermDNAfragmentationfollowingexogenousadditionofNADPH.
Patientsundergoinginfertilityscreeninghadasig-nificantlyhigherincreaseinspermDNAdamagecom-paredtohealthydonors.
SignificantlyhigherSDIscoresandspermwithcytoplasmicresidueswerealsonotedinthesepatients.
Therefore,wehypothesizethatmorpho-logicallyimpairedspermatozoathatretaincytoplasmicTable1.
Summaryofspermcharacteristicsinmaturespermatozoaisolatedbydoubledensitygradientcentrifugation.
SDI:spermdeformityindex.
Resultsareexpressedasmedianandinterquartilevalues(25thand75thpercentiles);bP1.
6,ourpreliminaryfindingssuggestthatsampleswithhighSDIscoresmaybemorelikelytopresentwithprevalentDNAfragmentedsperm.
However,ourstudyhaslimitationsduetosmallsamplesizeandourfindingsrequirefurthervalidation.
Inconclusion,ourpreliminaryresultssuggestthatSDImaybeausefultooltodetecttheprevalenceofspermDNAdamageandtoidentifypotentialinfertilemen.
Infertilepatientswithsemensamplescontaininghighproportionofspermmorphologicalabnormalitiesspe-cificallycytoplasmicdropletsmaybemoresusceptibletodevelopROS-mediatedspermDNAdamage.
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