1. Blastocyst Vitrification
BASAK BALABAN BSc.
American Hospital of Istanbul
Assisted Reproduction Unit
AMERICAN
HOSPITAL

2. Vitrification
Process that produces a glasslike solidification of living cells that completely avoids ice crystal formation during cooling. It completely avoids ice crystal formation in cryopreserved cells during warming to recover the cells for biological applications

3. Vitrification Techniques
Traditional Vitrification
(1998- early 2000s)
Ultrarapid vitrification
( today)

4. Problems Associated with Traditional Vitrification Procedures
High levels of cryoprotectants are toxic to embryos
(4-10 M compared to M)
Procedure must be performed at 4oC
Technically demanding
Advantages of Ultra-Rapid Vitrification
Increases in cooling rates alleviates toxicity of high levels of cryoprotectants
Can be performed at room temperature or 37oC

5. From Kasai et al. RBM Online 2004
Vitrification solutions
DMSO+Acetamide+ propylene glycol
Ethylene glycol+ Ficoll+Sucrose
Ethylene glycol+ DMSO
Ethylene glycol+ glycerol
Slow Freezing solutions
DMSO /1-2 PROH + Sucrose
Glycerol+
Sucrose
Base medium +
Cryoprotectant
From Kasai et al. RBM Online 2004

6. Differences of slow freezing and vitrification
high levels of cryoprotectants
very fast cooling rates
(~20,000oC/min)
fast cooling rates result in solidification of solution into glass-like structure (no crystallization)
takes seconds
Slow-freezing
low levels of cryoprotectants
slow controlled rates of cooling (0.3oC/min)
slow dehydration to minimize ice-crystal formation
takes hours

7. Vitrification & Slow-cooling
Control of solute penetration
Yes No
Control of dehydration rate
Duration out of the incubator
10min.
3 hrs.
Prolonged temperature shock
Fracture of ZP
Possible
Capture by growing ice crystals
Equipment and running costs
Inexpensive
Expensive
Kuleshova et al. F&S 2002

8. Variables in Vitrification
Cooling &warming rates:Ideal vitrification protocol must pass rapidly through the critical temperature zone of 15 to – 5ºC to decrease chilling injuries. High warming rates by directly plunging cells into the warming solution is suggested (-196 to 37ºC)

9. Variables in Vitrification
Concentration of the cryoprotectant:
To achieve high cooling rates requires the use of high concentrations of the cryoprotectant solution which depresses ice crystal formation, so a critical concentration is required but in some cryoprotectants, this minimal concentration (Cv) can lead to either osmotic or chemical toxicity

10. Variables in Vitrification
Sample size and carrier systems
Sample size should be minimized to reduce the duration of vapour coat and to increase the cooling rate, minimizing the volume of the vitrification solution as much as possible is necessary to facilitate vitrification by higher cooling rates
To minimize the volume of the vitrification solution special carriers are used for vitrification process
** Open pulled straws
** Flexipet- denuding pipette
** Microdrops
** Electron-microscopic copper grids
** Hemistraw system
** small nylon coils or nylon mash
** Cryotop,cryotip
** Cryoloop

11. Carriers for vitrification
Cryotop
Cryotip
Cryotip
Kuwayama et al.,RBM Online 2005

12. Cryoloop Hampton Research, Laguna Niguel, CA, USA Nylon loop
(20µm wide; mm in diameter)
Thin film of cryoprotectant solution by surface tension
Embryos are placed by pipette
Hampton Research, Laguna Niguel, CA, USA

13. Advantages of Cryoloop Vitrification
Lack of thermoinsulating layer maximizes heat transfer (>20,000oC/min)
Easy manipulations
Constant visualization of embryo
Cryoloop stored within cryovial
Procedure is performed at 37oC

14. Concerns with Regards to Sterility of Liquid N2 storage
Tedder et al., 1995
Hepatitis B transmission
Bielanski et al., 2000
Viral contamination
Bielanski et al., 2003
Microbial contamination, no viral cross contamination
Kyuma et al., 2003
No microbial or viral cross contamination

15. Necessity of blastocyst vitrification ?
Increasing application of BT especially for some selected cases results with supernumerary blastocysts for freezing to increase cumulative pregnancy rates per oocyte retrieval
A reliable procedure for the cryopreservation of blastocysts is needed, because after fresh ET, only small number of supernumerary blastocysts are likely to be available for cryopreservation
Based on the published cochrane data (2008), vitrification appears to result in significantly higher survival and pregnancy rates

16. Blastocyst vitrification
First pregnancy after human blastocyst vitrification was achieved by Yokota et al., HR 2000
EG- based vitrification solutions are widely used as it has a low toxicity with rapid diffusion into the cell through ZP and cellular membrane
1st. Vit.sol. EG+DMSO
2nd. EG+DMSO+Ficoll+ Sucrose,
Warming: Decreasing concentrations of Sucrose sol. are preferred
Concentration of cryoprotectants are decreased to 7.5% from 25% over the years of experience

17. > blasts. vitrified
Youssry et al.,RBM Online 2008

18. Blastocyst vitrification
Is it the most effective and successful method to cryopreserve embryos at blastocyst stage???

19. Superior results with Vitrification
**Retrospective, carrier: Cryotop
Stehlik et al.,RBM Online 2005

20. Faster re-expansion after thawing with vitrification method
Stehlik et al.,RBM Online 2005

21. Vitrification versus slow freezing method for blastocyst cryopreservation
Liebermann et al., F&S 2006
**Retrospective, carrier: cryotop

22. Vitrification versus slow freezing method for blastocyst cryopreservation
Liebermann et al., F&S 2006

23. Slow freezing & vitrification method for human blastocysts
Kuwayama et al.,
RBM Online 2005

24. Higher survival rates with blastocyst vitrification
Slow Freezing
Vitrification
No of. blastocysts 72 81
Survival Rate(%)
56.9 (41/72)
84 (68/81)*
*p<0.001
Vitrification: 13 patients,
CPR: 53.8%, IR: 23.3%
Huang et al., HR 2005

25. Cryopreservation of human embryos by vitrification or
slow freezing: A systematic review and meta-analysis
Pubmed search: 873, only 4 included!!,
Primary outcome: Postthaw survival rate,
Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR
Loutradi et al., F&S 2008

26. Heterogenity of the studies included
Loutradi et al., F&S 2008

27. Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBR
were NOT feasible

28. Artifical shrinkage by microneedle Artifical shrinkage by laser
Large blatocoele of more developed blastocysts may disturb
the efficacy of vitrification due to
inappropriate Dehydration and permeation of cryoprotectant,
which may cause ice crystal formation in the rapid cooling and
warming steps of vitrification. Ice crystal formation can be a
voided by reducing fluid content of the blastocoele of more
developed blastocysts
Mukaida et al., HR 2006
1st.clin.appl- Vanderzwalmen et al.,
HR 2002,3,4

29. Artifical shrinkage of the blastocoel cavity prior vitrification
Mukaida et al.,HR 2006

30. Better cryosurvival rates with vitrification of blastocysts after artifical zona opening
Zech et al., RBM Online 2005
Galan et al., ESHRE 2003 reported 73% cryosurvival with blastocyst vitrification

31. Similar OPR after ET of biopsied vitrified blastocysts
Escriba et al., F&S 2008
Retrospective study,
Carrier: 0.25ml. French straws

32. Vitrification of blastocysts after PGD yields similar cumulative OPR
Escriba et al., F&S 2008

33. Takahashi et al.,F&S 2005
Liebermann et al., F&S 2006 also reported no adverse effect

34. RESULTS
Vitrification as a cryopreservation method has many primary advantages and benefits based on the published data
Vitrification protocols are now starting to enter the mainstream of human ART
The reports of successfully completed pregnancies following vitrification are encouraging for further research
More studies on vitrification and thawing procedures are needed to develop more efficient and optimal vitrification methods

35. Concerns regarding Vitrification
LN2 still remains to be a potential source of contamination since the technique is based on direct contact between the vitrification solution containing cryoprotectant agents and LN2. So from a clinical point of view:
Is there a need to sterilize LN2? How is it possible to maintain its sterility
Cross contamination with viruses?? ( No publication since 1985, about 450 publications)
Closed systems should be used in clinical human IVF in the future to avoid this concern.(Like CBS HS vitrification straws, Cryotip……) New clinical trials with safer closed systems should be applied
Low toxicity vitrification solutions must be designed in the future
Genetical structure of the vitrified cell?? Chromosal abnormalities, gene expressions More studies are needed to prove the safety of the technique

37. Vitrification of human blastocysts with the Cryoloop
223 cycles, 725 blastocysts!!
Mukaida et al.,HR 2003

38. Successful Vitrification
Liebermann et al., Biol. Reprod 2002

39. Vitrification results with higher cryosurvival rates
for biopsied human embryos
Poor cryosurvival rates (approx. 30%) and clinical outcome reported after conventional slow freezing
of biopsied cleavage stage embryos ( Joris et al., HR 1999, Magli
et al., HR 1999)
Zheng et al.,HR 2005