EMBRYO FREEZING AND BLASTOCYSTS VITRIFICATION

Mol. Bio.Zafer Nihat Candan

Memorial Hospital, ART & Reproductive Genetic Center, Istanbul, TURKEY

CRYOPRESERVATION OF LIFE

• To enable to interfere biological clock and to stop it for a while.

• Basically, cryopreservation enable to storage the gamet cells, embryos and somatic cells by arresting or slowing metabolic activities until the subsequent thawing procedure.

HISTORY OF EMBRYO CRYOPRESERVATION

• Cryopreservation era arised with the accidentally understanding of the value of cryoprotectants in 1949 by Christopher Polge.

• By the early 1970s, Wilmut and Whittingham developed independent methods for freezing mouse embryos in DMSO.

• By the 1980s, the freezing of human embryos emerged as a common procedure in the treatment of infertile couples.

• First successfull pregnacy with the frozen-thawed cleavaged stage embryo transfer was reported by Trounson and Mohr in 1983.

• Two year later, Cohen published new procedure for human blastocyst stage embryo freezing.

• In 1985, new approach, termed vitrification, in which highly concentrated crypective agents were used, was successfully applied on mouse embryos.

WHY CRYOPRESERVE HUMAN EMBRYOS?

• Reduced multiple pregnancy in any one treatment cycle and provides storage of good quality surplus embryos for later use.

• Preserved embryos in initial attempt obviate the need for repeated surgery and high hormone induction to obtain eggs in subsequent trial.

• Thaw cycle is cost-effective, aproximately 1/5 of that of normal IVF cycle.

• Freezing provides tha chance to detect infectious disease and genetic abnormalities, by offering extended time for proper screening and analysis.

• Cyropreservation of embryos, which would otherwise be discarded, allow parents to have additional child if they wish.

• Overian hyperstimulated syndrome can be prevented by freezing all embryos instead of transfering.

WHY CRYOPRESERVE HUMAN EMBRYOS?

SLOW FREEZING

VITRIFICATION

Two procedures have been used so far for gamet and embryo cryopreservation

SLOW FREEZING

Expensive equipment Higher incidence of

intracellular ice formation Low concentration of

cryopropectant, less toxic Not cost-effective ??

VITRIFICATION

No ice crystal

Absence of mechanical injuriy

Possible osmotic stress

Liquid Phase Solid phase

Amorphous state/ Vitrified state /Glassy state

Vitrification is the solidification of a solution at low temperature without ice crystal formation

CRYOPROTECTANT TOXICITY??

Use of high concentration of cryoprotectant provides:

liquid/solid transition temperature

high viscosity

impossibility for the molecules to re-arrange

High speed of cooling, which results from

low volume of cryoprotectant (~1-2µl)

type of carrier

How to avoid ice crystal formation?

Amorphous

state

SLOW FREEZING VERSUS

VITRIFICATION

-35°C-35°C

-50°C/min

RTRT

-6°C-6°C

LN2LN2 (196(196°C°C))

Slow cooling Programmed equipment (Planner)

Equilibration with the cryoprotectantsEquilibration with the cryoprotectants

Drop in LN2

Time

Seeding-2°C/min

- 0.3 °C/min

1-3hr Time

Vitrification Vitrification Dewar container

Drop in LN2

5-10min

Concentration of cryoprotectantConcentration of cryoprotectantss

PermeablePermeable

Low MWLow MW

Non PermeableNon Permeable

Low MWLow MW

High MWHigh MW

VitrificationVitrification

Ethylene glycol Ethylene glycol

DMSODMSO

ErythritolErythritol

Intercellular

cryoprotectant

SucroseSucrose

TrehaloseTrehalose

Dehydration

Ficoll, Ficoll, PEGPEG

Extracellular

cryoprotectant

10-20 M

0.5-0.75 M

10 mg/ml

Slow coolingSlow cooling

11,,2 propanediol2 propanediol

DMSODMSO

GlycerolGlycerol

SucroseSucrose

TrehaloseTrehalose

0.2-1 M

1- 1.5 M

-6°C

-35°C

CP + CP + salt salt ++proteins proteins

11

1 1 Osmolarity increase outsideOsmolarity increase outside

2 2 Efflux Efflux of H20 of H20

33 Osmolarity increase insideOsmolarity increase inside

Solution

CP + salt

2233

- 0.3°C/min

Equilibrium freezingEquilibrium freezing

Slow freezing EquiliSlow freezing Equilibbrium freezingrium freezing

Rate of coolingRate of cooling

Ice crystal

Seeding

LN2

cristallisationmechanical damage induce by ice crystal high

Intracellular water efflux

VitrificationVitrification

Rate of coolingRate of cooling

LN2

RT

2.000°C -20.000°C/min

High conc. Cryprotectants

High cooling rate

High viscosity

amorphous state (vitrified state)

mechanical damage induce by ice crystal low

Cooling speed of vitrification

• Directly, depending on the type of used carriers;

– , Rapid : 2.500 °C/min

– Ultra-rapid: 20.000 °C/min

Conventional vitrification in straw Rapid

An Optimum Rate of Cooling

This results from the balance of two phenomena;

– At rates of cooling slower than the optimum, cell death is due to the long periods of exposure to hypertonic conditisions

– At the rates of cooling faster than the optimum, cell death is associated with intra cellular ice formation

Special Carriers of Ultra-Vitrification

Open Pulled StrawEMGridsCryoloopHemi-StrawCryotips

Open Pulled Straw

EMGrids

Hemi-Straw

Cryoloop

Advantages of Ultrarapid Vitrification

• Increase the cooling and warming rates.

• Direct contact between a small volume of vitrification solution and LN2.

• Thereby, prprevent intracellular ice formation event intracellular ice formation and reduce the toxicity of high concentration and reduce the toxicity of high concentration of cryoprotectant and chilling damageof cryoprotectant and chilling damage

BLASTOCYST VITRIFICATION

First pregnancy in 1985First pregnancy in 1985Cohen JCohen J

Slow Freezing protocolsSlow Freezing protocolsSeeding –6°C 0.3°C/min -37°C

Glycerol 10%

Regarding the poor results with slow blastocyst freezing, many groups report to switch back to day 1 and day 2-3 freeze/thaw programs

How can we improve the results of blastocyst freezing?

move to vitrification procedure? Pantos et al.,2001

The survival rates of blastocysts cryopreserved by vitrification and slow-freezing were 68.33 and 65.52 per cent (p = 0.89), whereas hatching rates were 51.22 and 44.74 per cent, respectively (p = 0.64).Therefore, vitrification of blastocyst-stage-embryos may be a useful, economic method for freezing the excess blastocysts in some centers where blastocysts are routinely transferred. (Wanvisa et al.,2003)Stehlik et al.,2005

Important parameters of blastocysts vitrification

• Blastocyst quality

• Day of vitrification, 5 to 6

• Artificial shrinkage application

blastocoel could be a source of ice crystal

===> reduction in the survival rates

Art.ShrinkageArt.Shrinkage BBlastlast-E-Exp.xp.BB Vtirification cyclesVtirification cycles 3939No.of thawed B-ExpBNo.of thawed B-ExpB 7171Viability, %Viability, % 53 (70.6%)53 (70.6%)Delivery /vitrif.Delivery /vitrif. 21%21%ImplantationImplantation 18.4%18.4%

VDZ Hum. Reprod 2002

Artificial Shrinkage

No Art.ShrikageNo Art.Shrikage BBlastlast-E-Exp.xp.BB Vtirification cyclesVtirification cycles 3939No.of thawed B-ExpBNo.of thawed B-ExpB 108108Viability, %Viability, % 43.3%43.3%Delivery /vitrif.Delivery /vitrif. 12.812.8%%ImplantationImplantation 4.54.5%%

Needle used shrinkage

Laser pulse shrinkage

Mukaida et al., 2006

Artificial Shrinkage

10%DMSO-10%EG10%DMSO-10%EG20%DMSO-20%EG20%DMSO-20%EGFicoll 400 MW – 0,65M suc.Ficoll 400 MW – 0,65M suc.

2 min-4min 40 sec.

Cleavaged Stage Embryo/Blastocyst Vitrification System in Memorial Hospital IVF Labs In-house made solution

equilibration with the cryoprotectant solution - vitrification

Direct Plunge into LN2

Artificial Laser Shrinkage Hemi-straw system

Thawing is carried out 4h or 24 h before transfer at RT (22-25°C)

Sucrose 0,5MSucrose 0,5M

4-5 min.

PBS - PBS - 20% 20% HSAHSA

Sucrose 0,25MSucrose 0,25M

2 min.

LN2

Culture for 4 h or 24 h in Culture for 4 h or 24 h in culture media culture media before embryo transferbefore embryo transfer

Cleavaged Stage Embryo/Blastocyst Thawing Procedure in Memorial Hospital IVF Labs

Sucrose 0,Sucrose 0,1125M25M

2 min.

1 min.

Artificial Shrinkage

Before laser shot

After laser shot,

3-5min

After laser shot, 30s, 1min

Efflux of blastocoel solution

5hr incubation

Thawed Blastocysts

3-4 Cell Stage3-4 Cell Stage 6-8 Cell Stage6-8 Cell Stage

No.of thawing cyclesNo.of thawing cycles 8181 5252

Patients’age,Patients’age,♀♀ 30.2 30.2 ±± 4.1 4.1 32.1 32.1 ±± 4.3 4.3

Survived / Vitrified rate (%)Survived / Vitrified rate (%) 78.1 %78.1 % 73.4 %73.4 %

No.of transfer / ETNo.of transfer / ET 3.03.0 33

ΒΒ-hCG pregnancy rate-hCG pregnancy rate 55.6 55.6 aa % % 40.840.8bb % %

Clinical pregnancy rateClinical pregnancy rate 40.7 40.7 aa % % 26.526.5bb % %

Implantation rateImplantation rate 18.5 18.5 aa % % 8.78.7bb % %

Comparation of Survival and Pregnancy Rates of 2nd and 3rd Day Cleavaged Stage Embryos Cryopreserved by Slow

Freezing

Memorial Hospital ResultsChi-square test; p < 0.05

Slow Freezing Slow Freezing

(2-3 Day)

Vitrification Vitrification

(3-4 Day)(3-4 Day)

No.of thawing cyclesNo.of thawing cycles 138138 7676

Patients’age,Patients’age,♀♀ 30.7 30.7 ±± 4.2 4.2 31.7 31.7 ±± 5.3 5.3

Survived / Vitrified rate (%)Survived / Vitrified rate (%) 76.6 %76.6 % 85.2 %85.2 %

No.of transfer / ETNo.of transfer / ET 3.03.0 2.82.8

ΒΒ-hCG pregnancy rate-hCG pregnancy rate 50.7 50.7 aa % % 40.140.1bb % %

Clinical pregnancy rateClinical pregnancy rate 34.7 %34.7 % 32.9 %32.9 %

Implantation rateImplantation rate 18.5 %18.5 % 16.2 %16.2 %

Survival and Pregnancy Rates with Cleavaged Stage Embryos Cryopreserved by Either Slow Freezing As

Compared with Vitrification

Memorial Hospital ResultsChi-square test; p < 0.05

VitrifiedVitrified FreshFresh

No.of thawing cyclesNo.of thawing cycles 167167 416416

Patients’age,Patients’age,♀♀ 30.4 30.4 ±± 5.2 5.2 30.1 30.1 ±± 4.5 4.5

Survived / Vitrified rate (%)Survived / Vitrified rate (%) 755 / 851 (88.7)755 / 851 (88.7)

No.of transfer / ETNo.of transfer / ET 2.62.6 2.32.3

ΒΒ-hCG pregnancy rate-hCG pregnancy rate 56.3 %56.3 % 67.5 %67.5 %

Clinical pregnancy rateClinical pregnancy rate 44.9 %44.9 % 64.4 %64.4 %

Implantation rateImplantation rate 23.6 %23.6 % 39.6 %39.6 %

Survival and Pregnancy Rates with Blastocysts Cryopreserved by Vitrification

Memorial Hospital Results

No.of thawing cyclesNo.of thawing cycles 3838

Patients’age,Patients’age,♀♀ 32.4 32.4 ±± 3.6 3.6

Survived / Vitrified rate (%)Survived / Vitrified rate (%) 85.285.2

No.of transfer / ETNo.of transfer / ET 2.6 2.6 ±± 0.7 0.7

ΒΒ-hCG pregnancy rate-hCG pregnancy rate 52.6 %52.6 %

Clinical pregnancy rateClinical pregnancy rate 42.1 %42.1 %

On-Going pregnancyOn-Going pregnancy 26.3 %26.3 %

Implantation rateImplantation rate 17.0 %17.0 %

Survival and Pregnancy Rates with Blastomere Biopsied Cleavaged Stage Embryos Cryopreserved by Vitrification

Memorial Hospital Results

76,6

85,2 88,7

34,7 32,9

44,9

0

10

20

30

40

50

60

70

80

90

Survival Rate Clinical Pregnancy Rate

Slow Freezing(Cleavaged stage)

Vitrification(Cleavaged Stage)

Vitrification(Blastocyst Stage)

a

d

b

a

d c

a,b; p<0.05

c,d; p<0.05

Cleavaged stage Embryos Cryopreservation Vs Blastocysts Vitrification

Evolution of the vitrification techniques

0

5

10

15

20

25

30

35

40

Ongoing pregnancyImplantation rate

Classical virificationClassical virification Ultra-rapid virificationUltra-rapid virification

vitrification

Vitrification+

shrinkage

Ultra-rapidVitrification

Ultra-rapidVitrification

+Zona hatching

Conclusions Conclusions

Primary Benefits of Primary Benefits of Ultra-rapid vitrificationUltra-rapid vitrification

•Utilizes higher concentration of cryoprotectant that allows shorter exposure times to the cryoprotectant

• Rapid vitrification/warming; reduce the cryopreservation procedure up to 10 min.

• Loading embryos in carrier in a small volume of cryoprotectants provides a significant increase in the cooling rate from 20.000-30.000 °C/min

• Minimizes osmatic injuries

• Very simple protocols

• Eliminates the cost of expensive programmable equipment

Conclusions Conclusions

Variables of Variables of vitrificationvitrification• Type and concentration of cryoprotectants, even all cryoprotectants are toxic

• Temperature of vitrification solution at exposure

• Lenght of time embryos are exposed to the final cryoprotectant before plunging into liquid nitrogen

• Variability in the volume of cryoprotectant solution surrounding the embryos

• Technical proficiency of the embryologists

• Direct contact of the LN2 and the CP medium containing the embryos, which may be a source of contamination,

• Sealed (high security straw) use

• Sterile LN2

• Storage invapour LN2

Can be solutions to avoid contamination

Conclusions Conclusions

• Which cryopreservation technic is being used dependening on the strategy of embryo transfer day.

• Slow freezing comparing the vitrification is more effective for 2nd day embryos regarding the survival and pregnancy rates.

• On the other hand blastocysts should be vitrified unless the new slow cooling technique is adapted.

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