Sperm DNA fragmentation
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Sperm DNA Fragmentation (Oxidative stress, DNA damage and apoptosis, Test, Techniques, Relation to other semen parameters, Relationship to leucocytes, Relation to ICSI outcomes, Clinical applications, significance and limitations)
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Sperm DNA fragmentation
- 1. Sperm DNA Fragmentation Dr. Yasmin Magdi Abd- Elkreem
- 2. overview • Conventionalsemenanalysistestremainsthemostwidelyused predictorformaleinfertilitydiagnosis. • basedonthedescriptivemicroscopicevaluationofejaculatesperm, includingconcentration,motilityandmorphology. • notdiscriminateeffectivelybetweenthespermoffertileandinfertile men. • DNAintegrityassessmenthasbeensuggestedasanindependent additionalmarkeroffertilitytogivemostconclusiveinformationonthe spermDNA.
- 3. overview • Spermatogenesis:Complex,diverse,74days • Spermspronefordisruptionbypotentialtargets. • Mostsignificant-freeradicals. • Specieswithunpairedelectrons,highlyreactive. • AlthoughspermDNAfragmentationdoesnotconstituteamutationinitsown,it stillisapromutagenicchangeofthemalegenome.
- 4. Etiology of sperm DNA damage • Theterm‘chromatin’referstothemacromolecular complexthatcontainsthe intranuclearDNA. • Thestructureofchromatinisstabilizedbyinteractionsbetweenitsmain compounds,DNAandDNA-bindingproteins. • Spermcellchromatinischaracterizedbyhighlycondensedstructure. • CondensationofspermDNAisnecessaryinordertoaccommodatethepaternal genomeintotherelativelysmallspermcellhead,torepressgeneexpressionduring spermiogenesisandtoprotectthepaternalgeneticmessage makingitinaccessible tonucleases ormutagens.
- 5. Main etiologies of sperm DNA damage TesticularcancerAging EnvironmentaltoxinsObesity HormonaldisturbancesSmoking TesticularhyperthermiaVaricocele Drugs,chemotherapyandradiationInflammation CryopreservationinARTprogramsFebrileillness Spinalcordinjury
- 6. Etiology of sperm DNA damage • TheetiologyofspermDNAdamageismostprobablymulti-factorialbut compromisedchromatinremodeling,oxidativestressandabortiveapoptosisare commonlydescribedtheories. • SpermDNAdamagemayarisefromcombinationsofallthreemechanisms. 1. Abnormalchromatinremodeling 2. Oxidativestress 3. Abortiveapoptosis
- 7. Abnormal chromatin remodeling • Deficienciesinrecombinationandchromatinpackagingduringspermiogenesis. • occurduetodeficientprotaminationorabnormalitiesinprotaminecontent. 1.Duetoabnormalfunctionoftranscriptionalortranslationalregulators (ageneralabnormalityinspermatogenesis). 2.induceapoptosiswhichcouldexplainthelinkbetweenunderprotaminationand poorspermqualityininfertilemen(checkpointregulatorsforspermatogenesis). 3.makesspermatozoamorevulnerabletoattackbynucleases,freeradicalsthatcan causeoxidativestressor(environmental)mutagens. 4.mayalsobecompromisedbyabnormaltopoisomeraseIIactivity (responsiblefor breakandrepairDNAduringprotamination)
- 9. Oxidative stress • ROSproductionisaphysiologicalphenomenonthatisnormallybalancedbyanti- oxidants. • Spermcellshavenodefensemechanism, apartfromthecharacteristictight packagingoftheDNAandanti-oxidantspresentinseminalplasma. • DNAfragmentationcanbecausedbyoxidativestress,duetofreeradicals[reactive oxygenspecies(ROS)].
- 11. Abortive apoptosis • Normalspermatogenesis isadynamicprocessinwhichcellproductionandcell deatharewellbalanced. • Cellstobeeliminatedwillenteracascadeofreactionsleadingtoapoptoticcell death. • CuttingofspermDNAbyendonucleases isoneoftheearliesteventsinthis cascade,thatiscompletedbyphagocytosisandeliminationofappropriately earmarked spermcellsbySertolicells. • Ifthissystemdoesnotoperateefficientlyandtheprocessisnotcompleted, damagedcellsmayescapeapoptosis.
- 12. DNA damage and infertility • spermDNAfragmentation(SDF)testscan differentiatefertilefrominfertile males. • Theassessment ofDNAintegrityinspermcouldbeanindependentmarkerof fertility. • higherlevelsofchromatindamageinmenwithseverespermdefects. • ThenegativeimpactofhighlevelsofspermDNAdamagenegativelyeffecton naturalpregnancy.
- 13. DNA fragmentation and semen parameters • ThereisastrongassociationbetweenthepresenceofnuclearDNAdamage inthe maturespermatozoaofmenandpoorsemenparameters. • spermmotility,concentration,andmorphologyhadsignificantpositive correlationswithnon-apoptoticandDNAnon-fragmented sperm. • Theapplicationofthesefindingsinclinicalpracticecanultimatelyincrease implantationandpregnancyratesinpatientswhereICSIisthetreatmentof choice.
- 15. DNA damage and ART • ThestructureofspermatozoaDNAisveryunique,highlyspecializedinorderto controltime-appropriatematurationofthezygote. • DamagetospermDNAmayoccurasaresultof uniormulti-factors.Thisdamage therebymayhavenegativeeffectsonARTprocedures,andcouldleadtofailureof fertilization. • SpermDNAdamagesignificantlycontributestothegrowingnumberofinfertility cases,andshouldbeapartofamodernandrologylab.
- 16. Assays to quantify sperm DNA damage • Differentmethodsmaybeusedtoevaluatethestatusofthespermchromatinforthepresenceof abnormalitiesorsimplyimmaturity • stainingtechniques: acidicanilineblue(AAB)andtoluidineblue(TB)stains, • fluorescentstainingtechniques:spermchromatindispersion(SCD)test,chromomycinA3 (CMA3),DNAbreakagedetection–fluorescentinsituhybridizationassay(DBD–FISH),insitu nicktranslation(NT),andflowcytometricbasedspermchromatinstructureassay(SCSA). • Someassaysemploymorethanonemethodfortheanalysisoftheirresults.: acridine orange(AO) andterminaldeoxynucleotidyltransferase-mediatedfluorescein-deoxyuridinetriphosphate- nickendlabeling(TUNEL)assays. • Methodslessfrequentlyused:high-performanceliquidchromatography(HPLC).
- 17. 1. Acidic aniline blue (AAB) stain Principle: • TheAABstaindiscriminatesbetweenlysine-richhistonesandarginine/cysteine-rich protamines. • Thistechniqueprovidesaspecificpositivereactionforlysineandrevealsdifferencesinthebasic nuclearproteincompositionofejaculatedhumanspermatozoa. • Histone-richnucleiofimmaturespermatozoaarerichinlysineandwillconsequently • takeupthebluestain. • Ontheotherhand,protaminerichnucleiofmaturespermatozoaarerichinarginine and cysteineandcontainrelativelylowlevelsoflysine,whichmeanstheywillnotbestainedby aniline blue.
- 18. Technique: I. Slidesarepreparedbysmearing5μlofeitherraworwashedsemensample.Theslidesareair- driedandfixedfor30minutesin3%glutaraldehydeinphosphate-bufferedsaline(PBS). II. Thesmearisdriedandstainedfor5minutesin5%aqueousaniline bluesolution(pH3.5). III. Spermheadscontaining immaturenuclearchromatinstainblue,andthosewithmature nucleidonottakeupthestain. IV. Thepercentageofspermatozoastainedwithaniline blueisdeterminedbycounting200 spermatozoaperslideunderbrightfieldmicroscopy. 1. Acidic aniline blue (AAB) stain
- 19. 1. Acidic aniline blue (AAB) stain Clinicalsignificance: • aclearassociationbetweenabnormalspermchromatinendmaleinfertility. • Thecorrelationbetweenthepercentageofaniline blue-stainedspermatozoaandothersperm parametersremainscontroversial. • GoodpredictorforIVF. • Itcannotdeterminethefertilizationpotentialandthecleavageandpregnancyratesfollowing ICSI. Advantagesandlimitations: • simpleandinexpensive • ordinarymicroscope
- 20. 2. Sperm chromatin dispersion test Principle: • TheSCDtestisbasedontheprinciplethatwhenspermaretreatedwithanacidsolutionpriorto lysisbuffer,theDNAdispersionhalosthatareobservedinspermnucleiwithnonfragmented DNAaftertheremovalofnuclearproteinsareeitherminimallypresentornotproducedatallin spermnucleiwithfragmentedDNA. Technique: • Thespermatozoaareimmersedinameltedagarosematrixat37°C.Onaslide,adropof8ulof mixedspermagaroseinitiallytreatedwithanacidsolution(solution1)for7mintodenaturethe DNAwithDNAbreaks,anddirectlytreatedwithlysingsolution(solution2)for20minto deproteinizethenucleoids.
- 21. • Afterremovalofnuclearproteins,fixationwasdoneusingethanolandthenslidewasstained usingsolution3for6minutesandsolution4for7minutes. • Non-fragmentedspermDNAappearswithacoreandwithaperipheralhaloofdispersionof DNAloops.FragmentedspermDNAappearswithverysmallornohaloofDNAdispersion. 2. Sperm chromatin dispersion test
- 22. Advantagesandlimitations: • Itdoesnotrequirethedeterminationofcolororfluorescenceintensity. • Simple,fast,andreproducible,anditsresultsarecomparabletothoseoftheSCSA. • littleisknownaboutitslimitationsanditsclinicalsignificance. 2. Sperm chromatin dispersion test
- 23. Conclusion • Spermchromatinassessmentisanindependentmeasureofspermqualitythatprovidesbetter diagnosticandprognosticcapabilitiesthanstandardspermparametersformalefertility potential(idiopathicinfertility). • Therearemultipleassaysthatmaybeusedforevaluationofthespermchromatinstatus. • Mostoftheseassayshavemanyadvantagesaswellaslimitations.Thechoiceofwhichassayto beperformeddependsonmanyfactors. • Theestablishmentofacutoffpointbetweennormallevelsintheaveragefertilepopulationand theminimallevelsofspermDNAintegrityrequiredforachievingpregnancystillemainstobe investigated.ExceptfortheSCSA.
- 24. References • GardnerDK,WeissmanA,HowlesCM,ShohamZ.TextbookofAssistedReproductive Techniques.3rded.Vol.1.In:AgarwalA,ErenpreissJ,SharmaR. Spermchromatinassessment. UnitedKingdom:Informahealthcare,2009:67-84. • AspinderSinghandAshokAgarwalTheRoleofSpermChromatinIntegrityandDNADamageon MaleInfertilityTheOpenReproductiveScienceJournal,2011,3,65-71. • A.AgarwalandTamerM.Said.RoleofspermchromatinabnormalitiesandDNAdamageinmale infertility.HumanReproductionUpdate,Vol.9,No.4pp.331±345,2003
- 25. Thank You !For contact: E-mail: Yas.magdi@hotmail.com
Editor's Notes
- Condensation of sperm DNA takes place during the final stage of spermatogenesis, along with a remodelling of the nucleus and the loss of most of the sperm cells cytoplasm. Condensation is achieved by the sequential displacement of histones by transition proteins and then by protamines. Protamines are smaller than histones and have extremely strong DNA binding capacity. Further condensation and stabilization of sperm chromatin is obtained by the formation of disulfide cross-links between cysteines abundantly present in protamines. This final stage of chromatin organization takes place in the epididymis
- Lower amount of protamines and abnormal protamine content will negatively affect sperm nuclear compaction. During spermatogenesis a complex and dynamic process of proliferation and differentiation occurs as spermatogonia are transformed into spermatozoa. This process involves a series of meioses and mitoses, changes in cytoplasmic architecture, replacement of histones with protamines (protamination) leading to a highly packed chromatin. During protamination nicks are created to provide relief of torsional stress and aid chromatin rearrangements. These nicks disappear completely at the time when chromatin packaging is Completed. McPherson and Longo (1992) hypothesized that chromatin packaging requires an endogenous nuclease, topoisomerase II, to create and ligatenicks in order to facilitate protamination. In accordance with this hypothesis, it has recently been shown that topoisomerase II plays a major role in linking DNA replication to chromosome condensation and that it interplays with a large protein complex i.e. condensation, which has key functions in mitotic chromosome assembly and organization. In addition, it has also been shown that topoisomerase II is present in the human seminiferous tubules which are the location of spermatogenesis.
- Sperm DNA Organization. Spermatozoa DNA is organized into three main domains: the majority of the sperm DNA is coiled into DNAse-insensitive toroids that are stacked side to side to maximize compaction. This toroid structure is held stable due to the presence of protamines which neutralize the repulsion between the phosodiester backbones. A smaller amount of DNA is associated with histones present in the spermatozoa, with the remaining DNA attached to the nuclear matrix at Matrix Attachment Regions.
- Sperm cells are especially vulnerable for this type of damage, since they have no defence mechanism, apart from the characteristic tight packaging of the DNA and anti-oxidants present in seminal plasma.
- The second hypothesis on the origins of DNA fragmentation in ejaculated spermatozoa describes oxidative stress as the underlying factor for sperm DNA damage. Whilst Barrosso and co-workers (2000) correlated sperm DNA fragmentation with the endogenous generation of ROS, other studies showed correlations with the endogenous generation as well as exogenous stimulus of ROS. In addition to sperm DNA fragmentation, oxidative stress has been implicated in impaired sperm functional competence, including poor fertilization rates in IVF. Reactive oxygen species (ROS) cause peroxidative damage to the plasma membrane, furthermore, ROS are also known to attack DNA, inducing strand breaks and other oxidative based damage in human spermatozoa. DNA damage of mammalian spermatozoa, induced by free radicals, has also been associated with antioxidant depletion in the seminal plasma, presence of transition metals in the sperm culture medium, leukocyte contamination, redox cycling xenobiotics and testicular heating
- Thus, it is important to select morphologically normal sperm by an operator (ICSI) to significantly reduce the risk of injecting sperm with fragmented DNA into the oocyte.