1. EMBRYO FREEZING AND BLASTOCYSTS VITRIFICATION Khalid Mohammed Karam Obstetrics, Gyne. & ART specialist (PhD)

2. CRYOPRESERVATION OF LIFE
To enable and interfere the biological clock and 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.

3. 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.

4. 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.

5. WHY CRYOPRESERVE HUMAN EMBRYOS?
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.
Ovarian hyperstimulated syndrome can be prevented by freezing all embryos instead of transfering.

6. Two procedures have been used so far for gamete and embryo cryopreservation
SLOW FREEZING
VITRIFICATION

7. SLOW FREEZING Expensive equipment
Higher incidence of intracellular ice formation
Low concentration of cryopropectant, less toxic
cost-effective

8. VITRIFICATION
Vitrification is the solidification of a solution at low temperature without ice crystal formation
Liquid Phase
Solid phase
Amorphous state/ Vitrified state /Glassy state
No ice crystal
Processus par lequel un liquide se solidifie sans formation de cristaux de glace
Possible osmotic stress
Absence of mechanical injury
CRYOPROTECTANT TOXICITY??

9. How to avoid ice crystal formation?
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
Amorphous
state
On essaye d’éviter la formation de cristaux par:
Concentration élevée en CP
diminution du point de fusion
augementation de la viscosité
Vitesse de Refroidissement très rapide qui est influencée par la taille de l’échantillon à congelé et par le type de support des ovocytes ou des embryons.

10. SLOW FREEZING VERSUS VITRIFICATION

11. Slow cooling Programmed equipment (Planner)
-50°C/min RT
-6°C
LN2 (196°C)
Slow cooling Programmed equipment (Planner)
Equilibration with the cryoprotectants
Drop in LN2
Time
Seeding
-2°C/min
- 0.3 °C/min
1-3hr
Time
Vitrification Dewar container
Drop in LN2
5-10min
One of the main difference concerns the concentartion of CP that is very high in the vitrificcation step as compared to the slow freezing procedure.

12. Concentration of cryoprotectants
Slow cooling
1,2 propanediol
DMSO
Glycerol
Sucrose
Trehalose
0.2-1 M M
Vitrification
Ethylene glycol
DMSO
Erythritol
Intercellular
cryoprotectant
Sucrose
Trehalose
Dehydration
Ficoll, PEG
Extracellular
10-20 M M
10 mg/ml
Permeable
Low MW
Non Permeable
High MW

13. Rate of cooling Slow freezing Equilibrium freezing - 0.3°C/min
Seeding
Slow freezing Equilibrium freezing
-6°C
- 0.3°C/min
Intracellular water efflux
-35°C
Ice crystal
The first strategy to prevent ice formation was to adopt a long slow-cooling stage.
The traditional slow-rate freezing can be regarded as an equilibrum freezing. In fact we for each apparition of a new extracellular ice crystal we observed an increase of the extracellular osmolarity followed by an efflux of the water and a reduction of the volume of the cell with as consequence an increasing in the intracellular osmolarity.
LN2
Equilibrium freezing
cristallisation
mechanical damage induce by ice crystal
high

14. Rate of cooling Vitrification High conc. Cryprotectants
2.000°C °C/min RT
High conc. Cryprotectants
High cooling rate
High viscosity
The vitrification technique is a radical change from the conventional equilibrum cooling methods
Vitrification is a process which, by combining the use of concentrated solution with rapid cooling, avoids the formation of ice.The sample is solidified by an extreme elevation in viscosity and reach low T° in a glassy state, which has the molecular structure of a viscous liquid and is not crystalline.
Under such rapid cooling, water molecules don’t have time to arrange themselves into a crystalline lattice structure.
The advantage of vitrification techniques, is that damaging intracellular ice is not formed and effects of extarcellular ice formation, ice crystal growth and thawing are avoided.
LN2
amorphous state (vitrified state)
mechanical damage induce by ice crystal low

15. Cooling speed of vitrification
Directly, depending on the type of used carriers;
, Rapid : °C/min
Ultra-rapid: °C/min
Conventional vitrification in straw Rapid

16. 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

17. Special Carriers of Ultra-Vitrification
Cryoloop
Open Pulled Straw
EMGrids
Cryoloop
Hemi-Straw
Cryotips
Hemi-Straw
EMGrids
Open Pulled Straw

18. Advantages of Ultrarapid Vitrification
Increase the cooling and warming rates.
Direct contact between a small volume of vitrification solution and LN2.
Thereby, prevent intracellular ice formation and reduce the toxicity of high concentration of cryoprotectant and chilling damage

19. BLASTOCYST VITRIFICATION

20. First pregnancy in 1985 Cohen J
Slow Freezing protocols
Seeding –6°C 0.3°C/min °C
Glycerol 10%
Since the first human pregnancy resulting from a frozen blastocyst in 1985 (Cohen), a standardized slow freezing technique has become an integral part in most ART laboratories.

21. 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

22. Artificial Shrinkage Art.Shrinkage Blast-Exp.B Vtirification cycles 39
No.of thawed B-ExpB 71
Viability, % 53 (70.6%)
Delivery /vitrif. 21%
Implantation 18.4%
No Art.Shrikage Blast-Exp.B
Vtirification cycles 39
No.of thawed B-ExpB 108
Viability, % 43.3%
Delivery /vitrif %
Implantation 4.5%
VDZ Hum. Reprod 2002

23. Cleavaged Stage Embryo/Blastocyst Vitrification System in Memorial Hospital IVF Labs
In-house made solution
equilibration with the cryoprotectant solution - vitrification
20%DMSO-20%EG
Ficoll 400 MW – 0,65M suc.
10%DMSO-10%EG
2 min-4min
40 sec.
Hemi-straw system
Direct Plunge into LN2
Artificial Laser Shrinkage

24. Thawing is carried out 4h or 24 h before transfer at RT (22-25°C)
Cleavaged Stage Embryo/Blastocyst Thawing Procedure in Memorial Hospital IVF Labs
Thawing is carried out 4h or 24 h before transfer at RT (22-25°C)
LN2
Sucrose 0,25M
2 min.
Sucrose 0,125M
2 min.
4-5 min.
Sucrose 0,5M
PBS - 20% HSA
1 min.
Culture for 4 h or 24 h in culture media before embryo transfer

25. Artificial Shrinkage

26. Before laser shot After laser shot, 30s, 1min After laser shot, 3-5min
Efflux of blastocoel solution
After laser shot,
3-5min

27. Thawed Blastocysts
5hr incubation

28. Primary Benefits of Ultra-rapid vitrification
Conclusions
Primary Benefits of Ultra-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 °C/min
Minimizes osmatic injuries
Very simple protocols
Eliminates the cost of expensive programmable equipment

29. Variables of vitrification
Conclusions
Variables of vitrification
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

30. 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.