1. Cryopreservation: slow freezing or vitrification?

2. Outline Introduction Effects of cryopreservation on physiology
Slow freezing versus vitrification: clinical results
Open versus closed carrier system
Cryoprotectant mixtures
Summary

3. Introduction

4. History of embryo cryopreservation
1986
First birth from slow frozen
human oocyte
2005
Cryoleaf and Cryotip
2004
Cryotop
1985
Vitrification of mouse embryos
Dinnyes et al., 2000
Solid surface
1984
First birth from slow frozen
human embryo
Lane et al., 1999
CryoLoop
1983
Slow freezing of human embryos
Vajta et al., 1998
Open-pulled straws
1972
Slow freezing of mouse embryos
Martino et al., 1996
EM grids
1992
Advent of ICSI
1997
ICSI implemented with
slow frozen human oocytes
This gives you an idea of the timeline of human oocyte and embryo cryopreservation. The first births from cryoprserved oocytes and embryos was in the mid 1980’s. There was somewhat of a hiatus for oocyte cryoprservation after several reports in the literature expressing concerns on the effect of cooling on oocyte physiology, particularly the disruption of the meiotic spindle and subsequent chromosome. Furthermore, until the advent of ICSI there was a problem with fertilizing thawed oocytes due to zona hardening.
Although vitrification has been introduced clinically relatively recently the first report of mammalian embryo vitrification was by Rall and Fahy over 20 years ago. Major changes to the vitrification process have been a reduction in the level of cryoprotectant used. This is predominantly through the use of devices with high temperature conductivity and small volumes with direct application to liquid nitrogen. One such device is the cryoloop which was developed my Michelle Lane whilst working with Barry Bavister. Since then it has been used extensively in the Gardner laboratory.
1998
First birth from vitrified
human cleavage-stage embryos
1999
First birth from vitrified
human oocyte
2001
First birth from vitrified
human blastocyst

5. Slow freezing versus vitrification
OOCYTE / EMBRYO
H2O
CRYOPROTECTANT
SLOW COOLING
RAPID COOLING
SLOW FREEZING
VITRIFICATION
ICE CRYSTALS FORM IN THE EXTERNAL MEDIUM
NO ICE CRYSTALS FORM IN THE EXTERNAL MEDIUM

6. Slow freezing versus vitrification
low levels of cryoprotectants
slow controlled rates of cooling ( °C/min)
slow dehydration of cells to minimize ice crystal formation and damage
freezing machine required (calibration, expenses)
takes hours
Vitrification
high levels of cryoprotectants
extremely fast rates of cooling (>20,000°C/min)
no ice crystal formation or damage; straight to a glass-like structure
no freezing machine required
takes seconds

7. Vitrification Vitrify/Vitrification from Latin vitrum (glass) +
Base medium +
Cryoprotectant
In theory pure water can be vitrified if a cooling rate of around 108 oC is a achieved. However, direct application to liquid nitrogen generates a cooling rate of only 104 oC so clearly the level of water needs to be reduced. A good demonstration of this is to take a drop of the base medium and a drop of the base medium containing an appropriate level of cryoprotectant to prevent ice crystal formation (approx 32%) and drop it directly into liquid nitrogen. The white opaque droplet is due to the ice crystals forming. The translucent glass-like drop is due to lack of organised ice crystal formation.

8. Effects of Cryopreservation on physiology

9. Outcome of cryopreservation
USA 2006
Europe 2005

10. Outcome of cryopreservation

11. Effect of cryopreservation on development
Control
Vitrification
Slow-Freeze 20 40 60 80
100 * **
Survival (%)
Fertilization (%)
Blastocyst / oocyte (%)
Blastocyst cell number
*: p<0.05
**: p<0.01
Using mouse oocytes we can see here that for all the parameters measured slow freezing performed significantly worse than vitrification. Survival, fertilization, number of blasts per oocyte and the number of cells in those blasts was significantly lower following slow freezing
Lane and Gardner, (2001) Mol. Reprod. & Dev 58, 342-7

12. Oocyte Protein ProfilesDa
MII In vivo Oocyte
MII Vitrified Oocyte
MII Slow Frozen Oocyte Da
This is the protein profile ( Da) generated by Time of flight Mass Spectrometry. Extracts were made of mouse oocytes following vitrification and slow freezing and compared to that of in vivo oocyte controls. Some proteins were unaltered (middle peak in blue rectangle), but interestingly some proteins were up or down-regulated compared to in vivo controls and vitrified oocytes. When the protein profiles were compared with statistical software, which groups treatments that are similar, it was found that vitrified oocytes were grouped with in vivo controls and that slow frozen oocytes were grouped separately.
Line
Plot
Larman et al., (2007) Human Reproduction 22,
Larman et al. (2007) Human Reproduction 22,

13. Maintenance of the spindle after vitrification
Slow Freezing
Before
This work was carried out in collaboration with Dr Laura Rienzi in Rome. The first two columns show polarized light images of human oocytes generated by the Oosight imaging system from Cambridge Research Instruments. The third column is of a mouse oocyte which has a much more prevalent spindle.
Images were taken of oocytes before vitrification/slow freezing and immediately after removal from the last warming/thawing solution (all vitrification steps were performed at 37oC). What you can see here is that the spindle is maintained with vitrification and does not depolymerise. With the software provided with the Oosight you can then draw a region of interest around the spindle to get a quantitative measurement of it’s retardance which is an indicator of how organised the microtubules are within the spindle.
After
Larman et al., (2007) RBM Online 15,

14. Maintenance of spindle retardance after vitrification
Mouse oocytes
Human oocytes
0.5 1
1.5 2
2.5 2
1.5
Spindle Retardance (nm)
Spindle Retardance (nm) 1
We found that there was no statistical decrease in spindle retardance in either mouse or fresh human oocytes that have been vitrified. The decrease with the human oocytes maybe explained, in part, by how small the human spindle is, which makes it difficult to find and maneuver into the plane that gives the best signal. This is in contrast to slow freezing. The protracted time spent at room temperature means that the spindle depolymerises and requires 2-5hrs to reform once returned to 37oC.
0.5
Vitrification
Slow Freezing
BEFORE VITRIFICATION
AFTER VITRIFICATION
Larman et al., (2007) RBM Online 15,

15. Protein Leakage by 2-cell Mouse Embryos following Cryopreservation
LDH Leakage (pmol NADH oxidized/h)
Slow freezing 50
Vitrification a 40 30
Different letters; P<0.05 b 20
Lactate dehydrogenase (LDH) is the most abundant intracellular protein and its leakage from the embryo (post-warm/thaw) can be used as an indicator of plasma membrane integrity. Significantly more LDH was found to leak from embryos that were slow frozen than those vitrified. Interestingly when ascorbate was included in the cryopreservation medium the level of LDH leakage was significantly reduced following slow freezing. Presumably ascorbate acts as an anti-oxidant and reduces the level of lipid peroxidation (damage). c c 10
0.1
0.1
Ascorbate (mM)
Lane et al. (2002) Human Reproduction 17,

16. Pyruvate uptake in human day 3 embryos
Balaban et al. Hum Reprod. 2008, 23 (9);

17. Slow freezing versus vitrification: clinical results

18. Scientific evaluation
Is vitrification superior?
Randomized controled trials
Sample size
Correct statistical evaluation
Meta-analysis
Summary of RCT’s

19. Meta-analysis
Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis.
Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC.
Fertil Steril Jul;90(1): Epub 2007 Nov 5. Review.
Pubmed search: 873 titles
Number included: 4
Increased survival after vitrification

20. Meta-analysis
Cryopreservation of human embryos by vitrification or slow freezing: which one is better?
Kolibianakis EM, Venetis CA, Tarlatzis BC.
Curr Opin Obstet Gynecol Jun;21(3): Review.
3 additional studies included
Survival superior after vitrification
No difference in pregnancy rates

21. Oocyte vitrification

22. Oocyte vitrification
Fadini et al. RBM Onl. 2009, 19 (2);

23. Health of children born
Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study.Takahashi K, Mukaida T, Goto T, Oka C. Fertil Steril Jul;84(1):88-92.
Neonatal outcome after vitrified day 3 embryo transfers: a preliminary study. Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Fertil Steril Jul;92(1):143-8.
Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Ruvalcaba Castellón LA, García Amador MI, Montoya Sarmiento JE. Reprod Biomed Online May;16(5):

24. Open versus closed carrier systems

25. Open versus closed carrier systems
Youssry et al. RBM Onl. 2008, 16 (2);

26. Open versus closed carrier systems
Open systems mostly used so far
Cross-contamination risk
Some reports with evidence of cross-contamination
Regulatory requirements
FDA EU

27. Open versus closed carrier systems

28. Development of closed carrier system
Rapid-i™
Rapid-i™ loaded with beads and 1st vitrification solution
Solution has frozen
Rapid-i™ loaded with Vitri-3 and beads and inserted into pre-cooled straw.
Solution has vitrified

29. Rapid-i™ Temperature changes
Cooling/warming rates around ice nucleation temperature sufficient to obtain proper vitrification and warming
Yury A. Tarakanov, Björn O. J. Johansson, Hans J. Lehmann and S. Peter Apell, "Numerical Simulations Demonstrate Safe Vitrification and Warming of Embryos Using the Rapid-i™ Device", Proc. European COMSOL conference 2009.

30. Rapid-i™
Good survival rates and development after vitrification of mouse embryos as well as poor quality human embryos
Clinical testing initiated

31. Cryoprotectant solutions for vitrification

32. Cryoprotectant solutions
Mixture of penetrating cryoprotectants used to reduce possible toxic effects
Mixture used in combination with
Non-penetrating cryoprotectants (sucrose – trehalose)
Molecules increasing viscosity (Ficoll, hyaluronan...)
Most commonly used mixture: DMSO-EG
Has it been proven that DMSO-EG is superior?

33. Cryoprotectant solutions
Cryoprotectants most commonly used for slow freezing
Oocytes: PrOH
Zygotes: PrOH
Multicellular embryos: PrOH, DMSO
Blastocysts: Glycerol
Argumentations on potential toxicity of DMSO
Why using DMSO for vitrification?

34. DMSO-free vitrification
Balaban et al. Hum Reprod. 2008, 23 (9);

35. DMSO-free vitrification
Balaban et al. Hum Reprod. 2008, 23 (9);

36. DMSO-free vitrification
Updated clinical results American hospital, Istanbul 1/1/2009
Patients
Embryos
Warming
238
738
Survival
667 (90.3 %)
100 % survival
441 (66.1 %)
Embryo transfers
548 (2.3)
Clinical pregnancies
101 (42.4 %)
Implantation
136 (24.8 %)

37. Summary Cryopreservation affects oocyte/embryo physiology
Vitrification affects less compared to slow freezing
Meta-analysis comparing slow-freezing and vitrification show increased survival rates for vitrification
Results on outcome of children born after vitrification available so far do not indicate negative effects of the vitrification procedure
Further comparison of efficacy of closed carrier systems as well as different cryoprotectant solutions will help to further optimize vitrification procedures