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[1]WorldHealthOrganisation:DepartmentofReproductiveHealthandResearchWHOlaboratory
manualfortheexaminationandprocessingofhumansemen. 5thedition.2010.
[2]2014AndrologyandEmbryologyReviewCourseManualof theAmericanBoardofBioanalysis
(ABB).](https://image.slidesharecdn.com/spermpreparationtechniques-160423112711/75/Sperm-preparation-techniques-27-2048.jpg)
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Sperm preparation techniques
This document discusses various sperm preparation techniques used prior to assisted reproductive technologies. It begins by explaining the reasons for processing sperm samples, such as removing components that could prevent pregnancy and selecting morphologically normal, motile sperm free of debris. Several migration-based techniques are described, including swim-up, density gradient centrifugation, and magnetic activated cell sorting. Glass wool filtration and zeta potential techniques are also covered. The document discusses preparation of epididymal and testicular sperm samples. It concludes by outlining methods for preparing sperm from retrograde ejaculation samples.
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Sperm preparation techniques
- 1. Sperm Preparation Techniques Dr. YasminMagdi Abd-Elkreem
- 2. Introduction • 2–4%ofbirthsindevelopedcountriesinvolvetheuseof(ART). • WithART,semensamplesmustfirstbeprocessedbeforetheycanbeusedforinsemination. Reasons: •Components(e.g.prostaglandins,zinc): areobstaclestotheachievementofpregnancywhennaturalbarriersarebypassedinART, suchasintrauterineinsemination(IUI)orin-vitrofertilization(IVF). • Toyieldafinalpreparationcontainingahighpercentageof: 1. Morphologicallynormal 2. Motilecells, 3. Freefromdebris, non-germcells,deadspermatozoa. • Dilutingsemenwithculturemediaandcentrifugingisstillusedforpreparing normozoospermicspecimensforIUI.
- 3. Introduction • TheWorldHealthOrganization(WHO)recommendsseparatingspermcellsfromtheseminal plasmawithinonehourafterejaculationtolimitdamagefromleukocytesandothercellspresent inthesemen. • IncreasedlevelsofROSresultinDNAdamageinspermatozoa,decreasedspermmotility, increasednumbersofapoptoticspermatozoa,anddecreasedspermplasmamembraneintegrity. •Variousspermseparationorisolationmethodsexisttoselectspermcells. 2.Sedimentationmethods1.Swim-upmethods 4.polyvinylpyrrolidone(PVP)dropletswim-out3.two-layerdiscontinuousgradientcentrifugation 6.Electrophoresisandfluorescencecellsortingmethods.5.pentoxifyllinewash 7.test-yolkbuffer
- 4. Introduction • Steriletechniquesshouldbeusedthroughoutspecimenprocessing. • Whenexaminingthespecimen,itisimportanttopayparticularattentiontoextraneousround cells,debris,andbacteriathatmaybepresent. •Thechoiceofspermpreparationtechniqueisdictatedbythenatureofthesemensample. • Eachlaboratoryshoulddeterminethecentrifugalforceandcentrifugationtimenecessaryto formamanageablespermpellet. • Theefficiencyofaspermselectiontechniqueisusuallyexpressedas: 1.Theabsolutespermnumber, 2.Thetotalnumberofmotilespermatozoa, 3.Therecoveryofmorphologically normalmotilespermatozoa.
- 5. Simple Wash Method •Culturemediumisaddedtotheejaculate. • Centrifugedtoremovetheseminalplasma. • Minimizethedamagecausedbyformationofreactiveoxygenspecies(ROS)bynon-viable spermatozoaandleukocytes. • Characterizedbythepresenceoflargenumbersofnon-viablespermatozoaintheprepared samplecaninhibitcapacitation—aphysiologicalprocessthatconfersspermatozoawiththe abilitytofertilizeanoocyte. • producesthehighestyieldsofspermatozoa,oftenusedforIUIorverypoorparameters(cantuse othermethods). • Whenspermnumbersareextremelylow,itmaybenecessarytomodifythecentrifugalforceor thetime,inordertoincreasethechancesofrecoveringthemaximumnumberofspermatozoa.
- 7. Migration-Based Techniques 1. Swim-Up •Spermatozoamaybeselectedbytheirabilitytoswimoutofseminalplasmaandintoculture medium. • spermsampleswithaverageorgoodmotility. • Liquefiedsemeniscarefullylayeredatthebottomoftheroundbottomtubecontaining the spermwashmedium. • Themediumusedinthistechniqueprovidesthespermwithanourishingenvironmentand attractsthespermcells. • Thetubeisplacedatanangleof45°andincubatedfor60minutes. • Active,motilespermmoveoutofthesampleintotheclearmediumwhichisthenaspirated. • Itcanbeperformedusingacellpellet(calleddirectswimup),inOligozoospermia.
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- 10. Migration-Based Techniques 2. Migration-Sedimentation •Usuallyusedforsampleswithlowmotility. • Spermcellsmigratefromaring-shapedwellintoaculturemedium aboveandthensettlethroughthecentralholeofthering. • SpecialtubescalledTea-Jondettubes. Advantages: 1.agentlemethod 2.theamountofROSproducedisnotverysignificant. Disadvantages: 1.thespecialtubesthatareneededarerelativelyexpensive.
- 11. Migration-Based Techniques 3. Swim-Down •Thistechniquereliesonthenaturalmovementofspermatozoa. • Adiscontinuousbovineserumalbuminmediumisprepared. • Thismediumbecomesprogressivelylessconcentratedmovingfromtoptobottom. • Thesemensampleisplacedontothetopofthemedium,andthetubeisincubatedat 37°Cforonehour. • Duringmigration,themostmotilespermmovedownwardintothegradient.
- 12. Migration-Based Techniques 4. DensityGradient Centrifugation • separatesspermcellsbasedontheirdensity. • Morphologicallynormalandabnormalspermatozoahavedifferentdensities.Amature morphologicallynormalspermatozoonhasadensityofatleast1.10g/mLwhereasanimmature andmorphologicallyabnormalspermatozoonhasadensitybetween1.06and1.09g/mL • Thus,attheendofcentrifugation,eachspermatozoonislocatedatthegradientlevelthat matchesitsdensity. • Thehighlymotile,morphologicallynormal,viablespermatozoaformapelletatthebottomof thetube. • Densitygradientscaneitherbecontinuousordiscontinuous.
- 14. Migration-Based Techniques 4. DensityGradient Centrifugation Advantages: 1.Theleukocytes,celldebrisandmorphologicallyabnormalspermwithpoormotility,arediscarded. 2.producesahigherrecoveryofmotilespermatozoa(>20%) 3.Fast,Easy. 4.easiertostandardizethantheswim-uptechnique,andthusresultsaremoreconsistent. Disadvantages: 1.Productionofgoodinterphasesbetweenlayerscantakesometime. 2.Thereisariskofcontaminationwithendotoxins. 3.Somescientistsfoundthatspermatozoarecoveredafterdensitygradientcentrifugationpossess lowerDNAintegritythanspermatozoarecoveredafterswim-up.
- 15. Magnetic Activated CellSorting • separatesapoptoticspermatozoafromnon-apoptoticspermatozoa. • Duringapoptosis(programmedcelldeath),phosphatidylserineresiduesaretranslocatedfrom theinnermembraneofthespermatozoatotheoutside. • AnnexinVhasastrongaffinityforphosphatidylserinebutcannotpassthroughtheintact spermmembrane. • Colloidalsuperparamagneticbeads(~50nmindiameter)areconjugatedtohighlyspecific antibodiestoannexin VandusedtoseparatedeadandapoptoticspermatozoabyMACS. • AnnexinVbindingtospermatozoaindicatescompromisedspermmembraneintegrity.
- 17. Magnetic Activated CellSorting Advantages: 1.actsatthemolecularlevelasopposedtoroutinespermpreparationtechniquesthatrelyonsperm densityandmotility. 2.theonlytechniquewhichseparatesapoptoticspermatozoafromnon-apoptoticspermatozoa. 4.rapid,convenientandnon-invasive. 5.BeaddetachmentafterMACSisnotnecessary. 6.providesoptimalpurityandrecoverywithreliableandconsistentresults. 7.canusedtooptimizethecryopreservation-thawingoutcomeandenhancecryosurvivalrates. Disdvantages: 1.needstobeusedinconjunction withothertechniquessuchasdensitygradientcentrifugationto removetheothersubstances(leukocytes,andseminalplasma).
- 18. Glass Wool Filtration •Glasswoolfiltrationseparatesmotilespermcellsfromothercontentsofsemenbyfiltration throughdenselypackedglasswoolfibers. • Thefiltrationseparatesoutimmotilespermcells,Leukocytes(87.5%)anddebris. • Afterfiltration,thesemeniscentrifugedtoremoveseminalplasmafromviablespermcells.
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- 20. zeta potential • Maturespermpossessanelectricchargeof–16to–20mVcalledzetapotential(electrokinetic potential). •Washedsperm(0.1mL)ispipettedintothetubeanddilutedwith5mLofserum-freeHEPES– HTFmedium. • Thepositivecharged(+2upto+4kVat1inch)onthetubeismaintainedbyplacingthetube insidealatexgloveuptothecapandbygraspingthecap,thetubeisrotatedtwoorthreeturns andrapidlypulledout. • Theelectrostaticchargeisverifiedusingelectrostaticvoltmeters. • Tomaximizetheisolationofmotilesperm,itisrecommendedthatthespermarepreprocessed ondensitygradient.
- 21. zeta potential (Selectingsperm prior ICSI) Advantages: 1. simpletoperform, 2.inexpensive. 3.permitsrapidrecoveryofspermwithimprovedspermparameters,particularlystrictnormal morphology,DNAnormalintegrity,andaniline bluematurity.;theseparametersareassociatedwith improvedfertilizationandpregnancyafterintracytoplasmicsperminjection(ICSI). 4.Spermprogressivemotilityandhyperactivation(predictiveofsuccessfulpregnanciesafterART procedures)isimprovedinthismethod, Disadvantages: Thismethodisnotbeusefulfortesticularorepididymalspermaspiratesastheylacksufficientnet electricalchargeonthespermmembranesurface.
- 22. Preparation of Epididymaland Testicular Spermatozoa 1. Epididymal Testicular Spermatozoa (PESA) • Thetypicalindicationforepididymalaspirationisobstructiveazoospermiaratherthan testiculardysfunction.Consequently,relativelylargenumbersofspermatozoacanbeharvested fortherapeuticpurposes. • Epididymalaspiratescanoftenbeobtainedwithminimalcontaminationfromredbloodcells andnon-germcells, makingtheisolationandselectionofmotileepididymalspermatozoa relativelystraightforward. • Iflargenumbersofepididymalspermatozoaareobtained,density-gradientcentrifugationisan effectivemethodofpreparingthemforsubsequentuse. • Ifspermnumbersarelow,asimplewashcanbeperformed.
- 23. Preparation of Epididymaland Testicular Spermatozoa 2. Testicular Spermatozoa (TESE) • Testicularspermatozoacanberetrievedbyopenbiopsy(withorwithoutmicrodissection)orby percutaneousneedlebiopsy. • Spermoutenzymaticallyormechanically fromtubules. • Testicularspecimensareinvariablycontaminated withnon-germcellsandlargenumbersofred bloodcells,soadditionalstepsareneededtoisolateacleanpreparationofspermatozoa. • Inordertofreetheseminiferoustubule-boundelongatedspermatids(“testicularspermatozoa”), enzymaticormechanicalmethodsareneeded. • TesticularspermatozoaarepreparedforICSI,sincespermnumbersarelowandtheirmotilityis poor.
- 24. Preparation of Epididymaland Testicular Spermatozoa • Simplewashmethodisusedtoincreasenumberofspermatozoa. • Erythromycinlysissolution(ELB)isusedtoremoveRBCs. • Pentoxifyllineisoccasionallyusedtoincreasethemotilityofepididymalandtesticular spermatozoabeforeICSI. • Coversamplewithmineraloilinaninjectiondish,themotilespermatozoafoundattheinterface betweentheculturemediumandoil. • Fineneedleaspiration(FNA)isthesame.
- 25. Preparation of RetrogradeEjaculation Samples • Insomemen,semenpassesintothebladderatejaculation,resultinginaspermia,ornoapparent ejaculate. • Confirmationofthissituationisobtainedbyexamining asampleofpost-ejaculatoryurinefor thepresenceofspermatozoa. • Ifpharmacologicaltreatmentisnotpossibleornotsuccessful,spermatozoamayberetrieved fromtheurine. • Alkalinizationoftheurinebyingestionofsodiumbicarbonate,forexample,willincreasethe chancethatanyspermatozoapassingintotheurinewillretaintheirmotilitycharacteristics. • TESEorPESAmaybebeneficial.
- 26. • DescribeavarietyofspermpreparationmethodsareavailabletoprocessspermforuseinART. • Determinethebestspermpreparationmethod. •Discusstheefficacyandthesafetyofthespermpreparationtechniques. • Reasonsforprocessing. At the end of lecture you are able to
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- 28. Thank You ! Email:Yas.magdi@hotmail.com
Editor's Notes
- #8 • Allow specimen to liquefy completely for 15–30 minutes in a 37°C incubator before processing. • Measure volume using a sterile 2 mL pipet. • Transfer specimen from a plastic cup to a sterile 15 mL— conical centrifuge tube. If specimen is >3 mL, split the specimen into two aliqouts. • Gently mix the specimen with Quinn’s Sperm Wash Media (HTF) in a ratio of 1:4 using a sterile pasteur pipet. • Centrifuge the tubes at 1600 rpm for 10 minutes. • Examine for sperm count and motility. • Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 3 mL of fresh HTF. Transfer the resuspended sample into two 15 mL sterile round bottom tubes using a sterile serological pipette (1.5 mL in each). • Centrifuge the tubes at 500 rpm for 5 minutes. • Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. • After the incubation period, aspirate the entire supernatant from the round bottom tube. Use a pasteur pipet, with the tip placed just about the pellet surface. • Pool supernatant from the two round bottom tubes into a single 15 mL conical centrifuge tube. Centrifuge the tube at 1600 rpm for 7 minutes. • Aspirate the supernatant from the top of the meniscus using a pasteur pipet. • Resuspend the pellet in a volume of 0.5 mL HTF using a 1 mL sterile pipet. Record the final volume.
- #10 • Allow specimen to liquefy completely for 15–30 minutes in a 37°C incubator before processing. • Measure volume using a sterile 2 mL pipet. • Transfer specimen from a plastic cup to a sterile 15 mL— conical centrifuge tube. If specimen is >3 mL, split the specimen into two aliqouts. • Gently mix the specimen with Quinn’s Sperm Wash Media (HTF) in a ratio of 1:4 using a sterile pasteur pipet. • Centrifuge the tubes at 1600 rpm for 10 minutes. • Examine for sperm count and motility. • Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 3 mL of fresh HTF. Transfer the resuspended sample into two 15 mL sterile round bottom tubes using a sterile serological pipette (1.5 mL in each). • Centrifuge the tubes at 500 rpm for 5 minutes. • Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. • After the incubation period, aspirate the entire supernatant from the round bottom tube. Use a pasteur pipet, with the tip placed just about the pellet surface. • Pool supernatant from the two round bottom tubes into a single 15 mL conical centrifuge tube. Centrifuge the tube at 1600 rpm for 7 minutes. • Aspirate the supernatant from the top of the meniscus using a pasteur pipet. • Resuspend the pellet in a volume of 0.5 mL HTF using a 1 mL sterile pipet. Record the final volume.
- #11 • Allow specimen to liquefy completely for 15–30 minutes in a 37°C incubator before processing. • Measure volume using a sterile 2 mL pipet. • Transfer specimen from a plastic cup to a sterile 15 mL— conical centrifuge tube. If specimen is >3 mL, split the specimen into two aliqouts. • Gently mix the specimen with Quinn’s Sperm Wash Media (HTF) in a ratio of 1:4 using a sterile pasteur pipet. • Centrifuge the tubes at 1600 rpm for 10 minutes. • Examine for sperm count and motility. • Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 3 mL of fresh HTF. Transfer the resuspended sample into two 15 mL sterile round bottom tubes using a sterile serological pipette (1.5 mL in each). • Centrifuge the tubes at 500 rpm for 5 minutes. • Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. • After the incubation period, aspirate the entire supernatant from the round bottom tube. Use a pasteur pipet, with the tip placed just about the pellet surface. • Pool supernatant from the two round bottom tubes into a single 15 mL conical centrifuge tube. Centrifuge the tube at 1600 rpm for 7 minutes. • Aspirate the supernatant from the top of the meniscus using a pasteur pipet. • Resuspend the pellet in a volume of 0.5 mL HTF using a 1 mL sterile pipet. Record the final volume.
- #12 • Allow specimen to liquefy completely for 15–30 minutes in a 37°C incubator before processing. • Measure volume using a sterile 2 mL pipet. • Transfer specimen from a plastic cup to a sterile 15 mL— conical centrifuge tube. If specimen is >3 mL, split the specimen into two aliqouts. • Gently mix the specimen with Quinn’s Sperm Wash Media (HTF) in a ratio of 1:4 using a sterile pasteur pipet. • Centrifuge the tubes at 1600 rpm for 10 minutes. • Examine for sperm count and motility. • Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 3 mL of fresh HTF. Transfer the resuspended sample into two 15 mL sterile round bottom tubes using a sterile serological pipette (1.5 mL in each). • Centrifuge the tubes at 500 rpm for 5 minutes. • Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. • After the incubation period, aspirate the entire supernatant from the round bottom tube. Use a pasteur pipet, with the tip placed just about the pellet surface. • Pool supernatant from the two round bottom tubes into a single 15 mL conical centrifuge tube. Centrifuge the tube at 1600 rpm for 7 minutes. • Aspirate the supernatant from the top of the meniscus using a pasteur pipet. • Resuspend the pellet in a volume of 0.5 mL HTF using a 1 mL sterile pipet. Record the final volume.
- #13 • Allow specimen to liquefy completely for 15–30 minutes in a 37°C incubator before processing. • Measure volume using a sterile 2 mL pipet. • Transfer specimen from a plastic cup to a sterile 15 mL— conical centrifuge tube. If specimen is >3 mL, split the specimen into two aliqouts. • Gently mix the specimen with Quinn’s Sperm Wash Media (HTF) in a ratio of 1:4 using a sterile pasteur pipet. • Centrifuge the tubes at 1600 rpm for 10 minutes. • Examine for sperm count and motility. • Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 3 mL of fresh HTF. Transfer the resuspended sample into two 15 mL sterile round bottom tubes using a sterile serological pipette (1.5 mL in each). • Centrifuge the tubes at 500 rpm for 5 minutes. • Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. • After the incubation period, aspirate the entire supernatant from the round bottom tube. Use a pasteur pipet, with the tip placed just about the pellet surface. • Pool supernatant from the two round bottom tubes into a single 15 mL conical centrifuge tube. Centrifuge the tube at 1600 rpm for 7 minutes. • Aspirate the supernatant from the top of the meniscus using a pasteur pipet. • Resuspend the pellet in a volume of 0.5 mL HTF using a 1 mL sterile pipet. Record the final volume.
- #16 A 100 μL sperm sample is mixed with 100 μL of MACS microbeads and incubated at room temperature for 15 minutes. Themixture is loaded on top of the separation column which is placed in the magnetic field [0.5 Tesla (T) between the poles of the magnet and 1.5 T within the iron globes of the column]; 1 Tesla = 10,000 gauss . The column is rinsed with buffer. All the unlabeled (annexin V-negative) non-apoptotic spermatozoa pass through the column . The annexin V-positive (apoptotic) fraction is retained in the column. The column is removed from the magnetic field, and annexin V-positive fraction is eluted using the annexin V-binding buffer
- #17 to C: Magnetic activated cell sorting: (A) The Octatet magnetic collection device can be used for loading up to a maximum of 8 samples. The tubes are placed between each open slot surrounded by the magnetic field, (B) The apoptotic and the nonapoptotic cells are labeled with the annexin V antibody beads (magnetic). These attach to the outer surface of the sperm that are apoptotic. Annexin V beads (magnetic) do not bind to the sperm that are non apoptotic and have intact membranes, (C) Apoptotic sperm with annexin V beads (magnetic) are retained in the column while the nonapoptotic sperm are eluted out and collected in a tube below the collection device
- #21 To allow adherence of the charged sperm to the wall of the centrifuge tube, each tube is kept at room temperature (22°C) for 1 minute. The tube is held by the cap to avoid grounding the tube. After 1 minute, the tube(s) are centrifuged at 300 g for 5 minutes and each tube is simply inverted to remove the nonadhering sperm and other cell types and excess liquid is blotted off at the mouth of each tube. To detach the charged adhering sperm, serum supplemented HEPES–HTF medium (0.2 mL) is pipetted into each tube allowing the medium to trickle down the side of the tube. The collected medium at the bottom of each tube is repipetted and used to rinse the wall of the same tube several times to increase the number of recovered sperm. • The zeta method can be carried out immediately as sperm cells loose the charge with the onset of capacitation. • To maximize the charge, a new centrifuge tube must be used. • The use of culture medium with a higher percentage of serum or discharging the tube may improve recovery of detached sperm in low sperm concentration situations.
- #24 Incubate the testicular tissue with collagenase (e.g. 0.8 mg of Clostridium histolyticum, type 1A per ml of medium) for 1.5–2 hours at 37 °C, vortexing every 30 minutes. 2. Centrifuge at 100g for 10 minutes and examine the pellet