This document discusses sperm cryopreservation, including an overview of current technologies, factors affecting cryopreservation results, sperm cryodamage, and optimizing sperm cryopreservation. It describes techniques for slow freezing, rapid freezing, and vitrification. Factors affecting optimization include biological factors, technical factors, genetic factors, and cryopreservation medium. Cryopreservation may induce DNA damage, though studies have found mixed results. Studies on reproductive outcomes using cryopreserved versus fresh sperm in ICSI have also found mixed results depending on the source and quality of the sperm. Future work is still needed to fully understand cryopreservation's effects.

1. Sperm
Cryopreservatio
Dr. Yasmin Magdi
Abd-Elkreem

2. Aim of this lecture…………….
• Overviewofthecurrenttechnologiesandapproachesutilizedin
spermcryopreservation.
• Considerthefactorsaffecttoresultsofcryopreservation
• SpermCryodamage.
• Howtooptimizesemencryopreservation.
• Spermpreparationpriorcryopreservation
• Is cryopreservationinduceDNA damage?
• whetherusingfreshratherthancryopreservedspermcellshasthe
sameeffectonreproductiveoutcomeinICSI.
• SpecialandFutureissues

3. What is sperm
cryopreservation?
• A proceduretopreservespermcells (commonlycalledspermbanking)
• Cryopreservationis thefreezingofcellsortissuestosubzerotemperatures,typically-196ºC
(boilingpointofliquidnitrogen).
• Thefirst successfulcryopreservationofspermatozoawasinitiatedover50yearsago.
• Forhumansperm,thelongestreportedsuccessfulstorageis 22years.
• All biologicalactivityis stoppedorpauseduntilitis thawed.
• Thefreezingofspermneedscryopreservationagentsthatminimizedamagetothecells during
thefreezingandthawingprocess

4. Why sperm cryopreservation is
performed ?
1. Semencontainingaverylimitednumberofspermatozoaorabnormalsemenparameters.
2.Forcancerpatientstopreservetheirfertility priortogonadotoxicchemotherapyorradiation.
3.Patientsundergoingcertaintypesofpelvic ortesticularsurgeries
4.Patientswhosufferfromdegenerativeillnesses suchasdiabetesormultiplesclerosis; spinal
corddiseaseorinjury.
5. personsinoccupationswhereasignificant risk ofgonadotoxicityprevails.
6. menundergoingsurgicalsterilizationsuchasvasectomy.
7. allowdonorsemensamplestobequarantinedwhileappropriatescreeningis performedto
preventthetransmissionofinfectiouspathogensduringtherapeuticdonorinsemination(TDI).
8. usedincombinationwithART techniques.

5. How sperm cryopreservation is
performed?
• Thefreezingofspermneedscryopreservationagentsthatminimizedamagetothecellsduring
thefreezingandthawingprocess.
Cryoprotective agents (CPAs):
• lowmolecularweightchemicalsthatservetoprotectspermatozoafromfreezingdamageorice
crystallization bydecreasingthefreezingpointofmaterials.
• CPAs canbetoxicif usedathighconcentrations.
1.PermeatingCPAs (penetratetheplasmamembrane)
 suchasdimethylacetaldehyde;dimethylsulfoxide,glycerol,glycol, ethyleneand
methanol
 stabilizecellplasmamembraneproteinsandreduceconcentrationsof electrolytes.
2.NonpermeatingCPAs (unabletopenetratetheplasmamembrane)
 suchasalbumins,dextrans,eggyolkcitrate,hydroxyethyl,polyethyleneglycols,
polyvinylpyrollidoneandsucrose
 minimizeintracellularcrystallizationbyincreasingviscosityofthesample.

6. Benefitsofcryoprotectants:
1.Maintainingspermviability.
2. Improvementintherecoveryofmotilespermbytheuseofzwitterionbuffershasbeen
attributedtotheirabilitytobindfreehydrogenandhydroxyionsinthesurroundingmedium,
aidinginthedehydrationprocess.
3.Improvespermmembranefluidity.
4.Increasespermlongevityandpercentsurvival.
5.Increaseability ofspermtopenetratecervical mucuspost-thaw.
How sperm cryopreservation is
performed?

7. 1 SlowFreezing:
A. Manualfreezing
- decreasingthetemperatureofthesemenwhileaddingacryoprotectantinastepwisemanner
andafterplungingthesamplesintoliquidnitrogen.
- coolingrateofthespecimenfromroomtemperatureto5°Cis 0.5–1°C/min.
- Thesampleis thenfrozenfrom5°Cto−80°Catarateof1–10°C/min.Thespecimenis then
plungedintoliquidnitrogenat−196°C.
B. Slowprogrammablefreezing
- Liquid nitrogen is poured into the tank, and the machine, once programmed, uses the software
data logging t
o obtain cooling from 20°C t
o −80°C a
t rate of 1.5°C/min and then a
t 6°C/min;
atcompletionofthefreezingthestrawsareremovedandstoredintoliquidnitrogenat
−196°C.This takesabout40min.
- Simpletouse
- doesnotrequirecontinuousoperatorintervention
- increasethereproducibilityofthefreezingoperations
Techniques for Cryopreservation

8. 2-RapidFreezing:
• requiresdirectcontactbetweenthestrawsandthenitrogenvaporsfor8–10minand
immersioninliquidnitrogenat−196°C.
• Insidenitrogenvaporsthereis athermalgradient,asafunctionofthedistanceandthevolume
oftheliquidbelow.
• Thesampleis initially mixedindropwisemannerwithequalvolumeofcoldcryoprotectant;
themixtureis loadedintothestrawsandlefttoincubateat4°Cfor10minutes.
• Thestrawsarethenplacedatadistanceof15–20cm(1hrWHo)abovethelevelofliquid
nitrogen(−80°C) for15min;afterthisstage,thestrawsareimmersedinliquidnitrogen.
• placethestrawsinhorizontalpositiontominimizetheheatdifferencebetweenthetwoends.
• Lowreproducibility
• temperaturedropcurvecannotbecontrolled
• andthefreezingtemperaturesmayvaryfrom−70,−80,and−99°C.
Techniques for Cryopreservation

9. Strawholder
Straws
Forceps
Syringe
Vistube
Techniques for Cryopreservation
Tanks

10. Techniques for Cryopreservation

11. 3. Ultra-rapidfreezing(Vitrification)
• Untilonlyrecently,vitrification ofspermatozoawasunsuccessful,possiblyduetohigh
concentrationsofpermeableCPAs (30-50%comparedto5-7%withslowfreezing)andlow
toleranceofspermatozoatopermeableagents.
• EvenbriefexposuretoahighconcentrationofCPAs canleadtotoxicandosmoticshockand
wouldbelethalforspermatozoa.
• OnepossiblestrategytolowertheconcentrationofCPAs couldbetoincreasethespeedof
coolingandwarmingtemperaturesashigherratesofcoolingandwarming,requirelower
concentrationsofCPAs; theseconditionscanhelpeliminateintracellular icecrystallization,
andfacilitatetheformationofaglassy state.
• Anotheroptionis toaddnon-permeableCPAs--suchascarbohydrates--topermeableCPAs to
minimizeosmoticshockbydecreasingosmoticpressureandstabilizing thenuclear
membrane.
Techniques for Cryopreservation

12. Guidelines

13. Semen preparation pre-freeze
and post-thaw
• Thequalityofejaculatedsemenis alsorelatedtotheoutcomeofcryopreservation.
• Forexample,deadspermatozoaorleukocytesinpre-freezingsemendetrimentallyaffectthe
spermsurvival rateandthefertility potentialafterthawingthroughtheROS generation
process
• Spermpreparationbeforecryopreservationshouldbeconsideredinroutinesperm
cryopreservation.
• Optimizingtheconcentrationofprogressivemotilespermcells beforethefreezingprocessis
recommendedtoamelioratethefertilizingcapacityofthefrozenspermatozoa.
• Semenpreparationbeforefreezingresultedinbetterspermqualityandfewerapoptoticsperm.

16. How to optimize semen
cryopreservation ?
Factors affectingtheoptimizationofhumanspermpreservation:
Biologicalfactors Technical factors
Genetic factors Cryopreservationmedium
Sexualabstinence Addition/removalofcryoprotectant
Seminalfluidquality Cooling rate
Seminalquality packing
Storage
Thawing
storagetemperature

17. Cryopreservation and DNA
Damage
• Thereis noagreementintheliteratureneitheronwhethercryopreservationinducesDNA
damagenorontheamountofdamage.
Because:
(1) differentfreezingprocedures
(2) differentteststoevaluatetheDNA integrity
(3) differentsemenpreparationtechniquesbeforecryopreservation(i.e.,swimupor
densitygradientcentrifugation).

18. Cryopreservation and
Reproductive Outcome
TesticularSpermatozoa:
• No statistically significant differences in all parameters examined (fertilization rate, cleavage
rate, embryo quality, implantation rate, clinical pregnancy rate, and ongoing pregnancy rate)
betweenICSI cycleswithfreshorcryopreservedtesticularspermatozoa.
• Only, De Croo and colleagues stated that fertilization, implantation, and live-birth rates per
embryotransferaresignificantlylowerafterICSI.
EpididymalSpermatozoa:
• TournayeandcolleaguesreportedthattheclinicalpregnancyrateinICSI cycleswas
comparablebetweenfreshandfrozen-thawedepididymalspermatozoa.
• Sukcharoenandcolleaguescametothesameconclusion;alsoCayanandcolleagues[64]
supportedthesameopinion.
• InoppositionShibaharaandcolleaguesstatedthattherewasasignificantdifferenceinall
reproductiveparametersexaminedbetweenICSI cycleswithfreshorcryopreserved
epididymalspermatozoa,comparingICSI cyclesperformedwithfreshandthawedepididymal
spermatozoa.

19. Ejaculated Spermatozoa:
• Kucznynskiandcolleaguesdidnotreportofanystatistically significantdifferencesin
fertilizationratebetweenfreshandfrozensemenpatients.
• ongoingpregnanciesaresignificantlyhigherinICSI patientswhenhumanspermsamplesare
cryopreserved.
this suggeststhatproperly performedcryopreservationselectivelyaffectsdefectiverather
thannormalspermatozoa
• Borgesandhisgroupdemonstratedthat
(1) usingsemenwithnormalmotilitythereproductiveoutcomeobtainedusingfreshorfrozen-
thawedspermatozoais thesame;
(2) insemenwithdecreasedmotilitythefertilization ratewithfreshspermwashigherthanthat
withthecryopreservedone,butnodifferencesweredetectedinimplantationandpregnancy.
Thefreezing-thawingprocedurecausesmoredamagein patientswithalterationsin semen
qualitythanthatin patientswithnormalsemen.However,oncetheoocyteis fertilized,
implantationandpregnancyratesaresimilar in patientswithorwithoutspermanomalies.
Cryopreservation and
Reproductive Outcome

20. Future issues
• Cryopreservationofhumansemenis extremelyimportanttothefield ofmaleinfertility.
However,thereis dissensionregardingthebestcryopreservationprotocolforhumansemen.
• Todatethereis noagreementintheliteratureonwhetherornotcryopreservationaffects
spermchromatinintegrityorontheuseofauniqueandfunctionalprotocolforthefreezing-
thawingprocedure.
• Furtherinvestigationsareneededtofully understandtherealinfluenceofcryopreservationon
spermDNA integrityandtheimpactoftheuseofcryopreservedspermatozoaonthe
reproductiveoutcome,technicalmeasuresshouldbeappliedtoprovidemaximumprotection
tothemalegametes:appropriateuseofcryoprotectantsbeforeandspermselection
technologiesaftercryopreservationseemstohavethegreatestimpactonpreventingDNA
fragmentation,thusimprovingspermcryosurvivalrates.

21. References
1 MarleaDi Santo,NicolettaTarozzi,MarcoNadalini,andAndreaBorini.HumanSperm
Cryopreservation:UpdateonTechniques,EffectonDNA Integrity,andImplicationsforART.
AdvancesinUrology.Volume2012(2012),ArticleID 854837,12pages.
2 2014AndrologyandEmbryologyReviewCourseManualof theAmericanBoardof
Bioanalysis (ABB).
3 WorldHealthOrganisation:DepartmentofReproductiveHealthandResearchWHO
laboratorymanualfortheexaminationandprocessingofhumansemen.5thedition.2010.
4 GardnerDK, WeissmanA, HowlesCM,ShohamZ. TextbookofAssisted Reproductive
Techniques.3rded.Vol.1.In:AgarwalA, ErenpreissJ, SharmaR. Spermchromatinassessment.
UnitedKingdom:Informahealthcare,2009:67-84.

22. Thank You!
For contact:
E-mail: Yas.magdi@hotmail.com