Vitrification of blastocyst stage embryos

1. Blastocyst
Vitrification
S.SASIKUMAR ,M.SS.,(PhD)
Senior Embryologist
NOVA IVI
FERTILITY,Chennai

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

4. Problems Associated with
Traditional Vitrification Procedures
• High levels of cryoprotectants are toxic to
embryos
• (4-10 M compared to 0.5-1.0M)
• Procedure must be performed at 4o
C
• 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 37o
C

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

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

7. Vitrification Slow cooling
Control of solute penetration Yes No
Control of dehydration rate Yes No
Duration out of the incubator 10min. 3 hrs.
Prolonged temperature shock No Yes
Fracture of ZP No Possible
Capture by growing ice
crystals
No Possible
Equipment and running costs Inexpensive Expensive
Vitrification & Slow-cooling
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; 0.5-0.7 mm in diameter)
Thin film of cryoprotectant
solution by surface tension
Embryos are placed by pipette

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

14. 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

15. 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

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

17. Faster re-expansion after thawing with vitrification method

18. Slow Freezing Vitrification
No of.
blastocysts
72 81
Survival Rate(%) 56.9 (41/72) 84 (68/81)*
Higher survival rates with blastocyst vitrification

19. 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

20. 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

21. 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

22. Successful Vitrification