Cryopreservation: slow freezing or vitrification?

Outline

• Introduction

• Effects of cryopreservation on physiology

• Slow freezing versus vitrification: clinical results

• Open versus closed carrier system

• Cryoprotectant mixtures

• Summary

Introduction

1983Slow freezing of human embryos

1985Vitrification of mouse embryos

1984First birth from slow frozenhuman embryo

1986First birth from slow frozenhuman oocyte

1992Advent of ICSI

1997ICSI implemented withslow frozen human oocytes

1999First birth from vitrifiedhuman oocyte

1998First birth from vitrifiedhuman cleavage-stage embryos

2001First birth from vitrifiedhuman blastocyst

Martino et al., 1996EM grids

Vajta et al., 1998Open-pulled straws

Dinnyes et al., 2000Solid surface

Lane et al., 1999CryoLoop

2004Cryotop

2005Cryoleaf and Cryotip

History of embryo cryopreservation

1972Slow freezing of mouse embryos

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

Slow freezing versus vitrification

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

Slow-freezing

• low levels of cryoprotectants

• slow controlled rates of cooling (0.1-0.3°C/min)

• slow dehydration of cells to minimize ice crystal formation and damage

• freezing machine required (calibration, expenses)

• takes hours

Vitrification

Vitrify/Vitrification from Latin vitrum (glass)

Base medium

Base medium+

Cryoprotectant

Effects of Cryopreservation on physiology

Outcome of cryopreservation

USA 2006 Europe 2005

http://www.cdc.gov http://www.ESHRE.com

Outcome of cryopreservation

Effect of cryopreservation on development

Blastocyst / oocyte (%)

Blastocyst cell number

Survival (%)

Fertilization (%)

Control Vitrification Slow-Freeze0

20

40

60

80

100

*

**

**

**

Lane and Gardner, (2001) Mol. Reprod. & Dev 58, 342-7

*: p<0.05

**: p<0.01

4000 4500 5000

0

100

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300

uA M2 in-v iv o oocy te

0

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uA M2 v itrif ication oocy te

0

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uA M2 slow f reezing oocy te

4000 4500 5000

4000 4500 5000

M2 in-v iv o oocy te

M2 v itrif ication oocy te

M2 slow f reezing oocy te

4000 4500 5000

MII In vivo Oocyte

MII In vivo Oocyte

MII Vitrified Oocyte

MII Vitrified Oocyte

MII Slow Frozen Oocyte

MII Slow Frozen Oocyte

LinePlot

Da

Da

Larman et al., (2007) Human Reproduction 22, 250-259

Oocyte Protein Profiles

Larman et al. (2007) Human Reproduction 22, 250-259

Bef

ore

Aft

er

Vitrification Slow Freezing Vitrification

Maintenance of the spindle after vitrification

Larman et al., (2007) RBM Online 15, 692-700

BEFORE VITRIFICATION AFTER VITRIFICATION

0

0.5

1

1.5

2

2.5

Spin

dle

Ret

ard

ance

(n

m)

Mouse oocytes Human oocytes

Spin

dle

Ret

ard

ance

(n

m)

0

0.5

1

1.5

2

Vitrification Slow Freezing

Larman et al., (2007) RBM Online 15, 692-700

Maintenance of spindle retardance after vitrification

Protein Leakage by 2-cell Mouse Embryos following Cryopreservation

Ascorbate (mM)

0 0.1 0 0.10

10

20

30

40

50

a

b

c c

Slow freezing Vitrification

Different letters; P<0.05

LD

H L

eaka

ge (

pmol

NA

DH

oxi

dize

d/h)

Lane et al. (2002) Human Reproduction 17, 2686-2693

Pyruvate uptake in human day 3 embryos

Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

**: p<0.01

Slow freezing versus vitrification: clinical results

Scientific evaluation

Is vitrification superior?

• Randomized controled trials

• Sample size

• Correct statistical evaluation

• Meta-analysis

• Summary of RCT’s

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. 2008 Jul;90(1):186-93. Epub 2007 Nov 5. Review.

• Pubmed search: 873 titles

• Number included: 4

• Increased survival after vitrification

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. 2009 Jun;21(3):270-4. Review.

• 3 additional studies included

• Survival superior after vitrification

• No difference in pregnancy rates

Oocyte vitrification

Oocyte vitrification

Fadini et al. RBM Onl. 2009, 19 (2); 171-80

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. 2005 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. 2009 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. 2008 May;16(5):608-10.

Open versus closed carrier systems

Open versus closed carrier systems

Youssry et al. RBM Onl. 2008, 16 (2); 311-20

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

Open versus closed carrier systems

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

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.

Rapid-i™

• Good survival rates and development after vitrification of mouse embryos as well as poor quality human embryos

• Clinical testing initiated

Cryoprotectant solutions for vitrification

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?

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?

DMSO-free vitrification

Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

DMSO-free vitrification

Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

DMSO-free vitrification

Patients Embryos

Warming 238 738

Survival 667 (90.3 %)

100 % survival 441 (66.1 %)

Embryo transfers 238 548 (2.3)

Clinical pregnancies 101 (42.4 %)

Implantation 136 (24.8 %)

Updated clinical results American hospital, Istanbul 1/1/2009

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