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Title: Folie 1


1
Aseptic vitrification of oocytes and embryos
using the vitrisafe as close carrier device
Vanderzwalmen P, Wierleitner B, Ectors F
IVF Centers Prof. Zech, Bregenz, Austria
Centre Hospitalier Inter Régional Cavell
(CHIREC), Braine lAlleud, Belgium GIGA-Research,
University of Liège, Liège, Belgium Iakentro IVF
Centre, Thessaloniki, Greece pierrevdz_at_hotmail.c
om
2
 Crystallization is incompatible with living
systems and should be avoided whenever possible 
Luyet (1937)
3
Vitrification
  • Solidification of a solution without ice
    crystal formation
  • Involves extreme elevation of viscosity before
    and during cooling,
  • resulting ultimately in a liquid
  • same lack of internal motions as a crystalline
    solid, and thus has no capacity for change
    over time,
  • yet lacks the molecular rearrangements of
    crystallization that are
  • responsible for cell injury and damage.

Drop in entropy No Increase of the latent
surfusion T
Drop in entropy Increase of the latent surfusion
T
Molecular organization in a LIQUID solution
Molecular organization as in a CRYSTAL structure
No molecular re-arrangement in a GLASSY state
Seite 2/3
4
Probability to obtain and maintain a glass-like
state depends on
Cooling and warming rates
x Viscosity (concentration of CPs)
Volume of the sample
Yavin and Arav, 2007
5
T
High cooling - warming rates
Liquid H20
TM
-7C
-40C
Tm melting T Tg Glass transition T
Solid crystalline H20
Tg
Solid Glass - Amorphous vitreous H20
-120C
T O X I C I T Y
Concentration - Viscosity
6
Vitrification to cryopreserve oocytes and embryos
Vitrification - with the use of open carrier
devices - (e.g. EM-grids, cryotop, cryoloop,
cryoleaf, Hemi-Straw) has become a more popular
alternative to the slow rate freezing method,
especially for human oocytes and blastocysts
MII oocytes (Kuwayama 2005, Noyes, 2009, Cobo
2010, Ubaldi 2010, Smith 2010, Nagy 2010, Rienzy
2010) Zygotes (Al Hasani 2006)
Blastocysts (Cho 2002, Son 2003, Mukaida 2003,
Vanderzwalmen 2003, Kuwayama 2005, Liebermann
2006, Stachecki 2008)
7
Current vitrification procedure ULTRA RAPID
VITRIFICATION
cooling / warming rates gt20.000C/min
  • Small straws with thin walls
  • Open-pulled straws (OPS)
  • Open-pulled straws quartz micro-capillary
  • Direct contact between a small volume of
    vitrification solution and LN2.
  • Electron microscope copper grid (EM grids)
  • Cryoloop
  • Hemi-straw system
  • Cryotop
  • Cryoleaf

8
Cooling/warming rates of open devices
Cooling rate gt 20.000C/min
Warming rate gt 20.000C/min
cryotop
9
ULTRA RAPID VITRIFICATION
embryos
10
Vitrification to cryopreserve oocytes and embryos
These protocols involved direct exposure of
oocytes and embryos to liquid nitrogen
11
Risks of direct exposure to LN2
Contamination with pathogens Bacteria or
viruses present in the LN2 Risk for
cross-contamination is debatable and still
discussed in the literature (Kyuwa, Exp Anim,
2003) Recent publication showed 45 risk of
contamination for open devices whereas non of the
probes in aseptical straws were contaminated.
(Criado, Fertil Steril, 2011)
12
Risks of direct exposure to LN2
Toxic compounds present in LN2 LN2 is the
liquefied form of the element nitrogen that is
commercially produced by fractional distillation
of liquid air. Nitrogen is non-toxic, odorless,
and colorless. It is relatively inert. At normal
pressure LN2 boils at -195.8C or
-320.4F. During generation and storage traces
of metals and reactive compounds could occur and
have negative impact on biological material.

13
Risks of direct exposure to LN2
Effect of cryo-storage duration on
cryo-survival, fertilization, embryonic
development of mouse oocytes following
vitrification and warming ESHRE 2010 RC Chian
A cryo-storage of mouse oocytes in contact with
LN2 for more than 6 months affects negatively the
fertilization, the embryonic development to the
blastocyst and number of cells Suggest
physico-chemical toxicity of LN2
14
Risks of direct exposure to LN2
Although the risk of contamination in LN2 with
pathogens and/or toxic compounds under the use
of open devices in vitrification remains
debatable The European Parliament directive
(EU Tissues and Cells Directive 2004/23/EC,
updated in 2006 Commision Directive 2006/86/EC)
dictates that cooling and storage of embryos has
to be performed in a way that minimizing the risk
of contamination aseptic conditions.
15
Minimizing the risks of harm by LN2
  • 1. Go back to slow-freezing
  • 2. Continue with the use of open device
  • Cooling
  • In sterile LN2 (Filtration UV)
  • Storage
  • In vapor (Cobo 2010 HR)
  • 3. Use of hermetically closed system

16
Cooling rate in open vs. closed devices
Cooling rates  open   closed 
Isolation of the sample from LN2 creates a
thermo-insulating barrier
gt 20.000C/min lt 2.000C/min
17
The question
Ultra-rapid vitrification  open  device
Successful results
gt 20.000C/min
short exposure to CP
?
Do we have to modify the procedure ?
 CLOSED  device Reduced cooling and / or
warming rates
lt 2.000C/min
18
Probability to obtain and maintain a glass-like
state depends on
Cooling and warming rates
x Viscosity (concentration of CPs)
Volume of the sample
The higher the cooling rate, the lower the
required cryoprotectant concentration is, and
vice versa.
Yavin and Arav, 2007
19
EFFECT OF A REDUCTION IN THE COOLING RATE ON THE
SURVIVAL RATE

20
Principles of vitrification
High cooling and warming rates reduce the
likelihood of lethal ice crystal formation during
the transit through the crystalline phase
21
Effect of short time exposure to 10-10 DMSO-EG
before vitrification in  open  and  closed 
carrier devices
Mice zygote
Mice zygote
  • Cooling rates
  • gt20.000C/min
  • lt 2.000C/min

survival
survival
Time in 10 - 10 DMSO / EG
Time in 10 - 10 DMSO / EG
20 - 20 DMSO / EG 30
22
Effect of short time exposure to 10-10 DMSO-EG
before vitrification in  open  and  closed 
carrier devices
Human blastocyst
t 24h
t o
?
  • Cooling rates
  • gt20.000C/min
  • lt 2.000C/min

survival
t 24h
Time in 10 - 10 DMSO / EG
20 - 20 DMSO / EG 30
Time in 10 - 10 DMSO / EG
23
DEVELOPEMNT OF A VITRIFICATION TECHNIQUE IN A
 CLOSED CARRIER
24
1 DEVELOPMENT OF AN ASEPTIC DEVICE
  • ? easy to handle
  • ? allow complete isolation of embryos from LN2
  • cooling and storage
  • Ultra-fast warming rate
  • It is more difficult to prevent ice formation
    during rewarming than during cooling so that we
    need more CP for the warming step

25
Vitriplug Hemi Straw
Vanderzwalmen 2003
VitriSafe
Vanderzwalmen 2010
26
Aseptic vitrification kit VitriSafe
0.5 ml CBS straw
VitriSafe plug
Extractor
Cooling 1.300C / min Warming gt 20.000C / min
27
Loading the biological material on the vitrisafe
28
VitriSafe
Droplets of VS with embryos/oocytes lt 1 µl)
29
(No Transcript)
30
VitriSafe
31
VitriSafe
Warming procedure
32
extractor
33
  • The speed of warming remains ultra-rapide
  • 20.000C/min

The key of success lies in rewarming that is
rapid enough to bypass too much nucleation if we
do not work in the range of equilibrium
vitrification
500 µl 1 M Sucrose
34
  • DEVELOPMENT OF A PROTOCOL IN
  • REDUCED COOLING CONDITION

Addition of cryoprotectant in one step or
gradually
35
Development of the method
Developing the method in the mouse model test
 in vitro  and  in vivo  derived
embryos zygotes and blastocysts Vitrification
media FertiPro Support  VitriSafe 
36
Blastocyst rate after slow freezing or aseptical
vitrification of in vitro developed mouse embryos
Mouse strain C57BL/6
37
Blastocyst rate after slow freezing or aseptical
vitrification of in vivo developed mouse embryos
Mouse strain FVB
38
In vivo developement of zygotes and blastocysts
after aseptic vitrification
Survival rate and implantation rate afer
vitrification of zygotes mouse strain C57BL/6 and
FVB.
Nr vitrified zygotes SR (n) Nr blastocysts transferred implantation rate (n)
C57BL/6 210 94 (198) 141 29 (41)
FVB 220 93 (203) 163 38 (62)
Ctl FVB / / 430 19 (81)
Survival rate and implantation rate afer
vitrification of blastocysts mouse strain C57BL/6
and FVB.
Nr vitrified blastocysts SR (n) Nr blastocysts transferred implantation rate (n)
C57BL/6 45 82 (37) 35 (5) 29 (10)
FVB 38 79 (30) 27 (3) 41 (11)
Ctl FVB / / 27 (3) 33 (9)
39
Clinical application of ASEPTIC
vitrification Closed carrier devices Human
oocytes and embryos
40
Aseptic vitrification of 2PN Carrier device
Vitrisafe
41
Aseptic vitrification of 2PN Carrier device
Vitrisafe
Patients Oocyte donors ( 27 ) No sperm available (16) Ethical reasons (1) Age 34.6 4.7
n Cycles 44
n Oocytes vitrified / warmed Survival Rate 289 92
n 2PN d1 Fertilization Rate 226 78
n Embryo-Transfers d2 d3 d5 43 30 13
n Embryos transfered d2d3 d5 102 84 (2.8/ET) 18 (1.4/ET)
Prapas, Zech, Vanderzwalmen, Lejeune
42
Aseptic vitrification of oocytes Carrier device
Vitrisafe
Pregnancy Outcome
ß-HCG positive PR 21 / 43 48,8
Ongoing Pregnancies / vitrification cycle PR 16 / 43 37,2
IR IR per embryo transfered IR per vitrified oocyte 17 6
Recently ongoing pregnancies Live Births 11 4 (2 female 2 male)
Prapas, Zech, Vanderzwalmen, Lejeune
43
Aseptic vitrification of oocytes
Effects of two vitrification protocols on the
developmental potential of human mature oocytes
Paffoni A. et al. RBM Online 2011
compare an open vitrification protocol to a
closed vitrification CRYOTOP CRYOTIP
Irvine Kit (DMSO/EG) 7.5/7.5 20/20 closed
vitrification CRYOTIP Total equilibration time 7
min 75 sec open vitrification protocol
CRYO Total equilibration time 12-15 min 60 sec
44
Aseptic vitrification of oocytes
Paffoni A. et al. Reproductive BioMedicine Online
2011
45
Oocytes vitrification for different indications
and carrier systems
Cobo / Rienzi / Nagy / Lucena /Smith Smith Paffoni Paffoni Our Centers Our Centers
open cryotop open cryotop open cryotop closed cryotip open cryotop closed Vitrisafe
Iindication Donor oocytes Vit sociale Consent patients Italian law Italian law Donor oocytes/ no sperm Donor oocytes/ no sperm
SR 90 - 98 81 83 58 94 92
Clinical PR 45 - 60 38 29 8 21 37
46
Aseptic vitrification of 2PN Carrier device
Vitrisafe
47
Aseptic vitrification of 2PN Carrier device
Vitrisafe
Patients OHSS Age 32.1 5.3 Patients OHSS Age 32.1 5.3 Patients OHSS Age 32.1 5.3 Patients OHSS Age 32.1 5.3
n Vitrification/Warming Cycles 93 93 93
n 2PN vitrified/warmed Survival Rate () 895 95 895 95 895 95
n Embryo Transfers (d5) 89 (94) 89 (94) 89 (94)
Embryo quality at least one Top-Blastocyst Non-Top Blastocyst Morula/ compactions
n Transfers/ Embryos transfered (mean) 41 / 74 (1.8) 37 / 59 (1.6) 15 / 31 (2.1)
Ongoing PR () 23 / 74 (56) 12 (32) 4 (27)
Implantation Rate/ transfered Embryo 25 / 74 (34) 13 / 59 (22) 4 / 31 (13)
Zech, Vanderzwalmen 2010
48
Aseptic vitrification of day 3 Carrier device
Vitrisafe
49
Aseptic vitrification of day 3 Carrier device
Vitrisafe
48 Patients, Age 33.1 4.8
n Vitrification/Warming Cycles 48
n Embryos vitrified/warmed Survival Rate 115 91
n Embryo tranfers (d5) 46 96
n Embryos tranfered (mean) 62 Mean 1.3
ß-HCG positive PR 18 / 46 39
Ongoing PR/ vitrification/warming cycle () 15 /46 31
Implantation Rate/ Embryo transfered 15/62 24
Lejeune, Vanderzwalmen
50
Aseptic vitrification of blastocysts Carrier
device Vitrisafe
Development of an aseptic vitrification
technique application to blastocysts originating
from infertile patients, egg donors and after in
vitro maturation
Vanderzwalmen RBMonline 2010
51
Aseptic vitrification of blastocysts Carrier
device Vitrisafe
Aseptic vitrification of blastocysts
Donor Oocyte Patients Age 30.1 (23 34) Donor Oocyte Patients Age 30.1 (23 34)
n Vitrification/warming cycles 546
n Blastocysts warmed 1335
n Vital blastocysts after warming (SR ) 1228 (92)
n Developing blastocyst before transfer ( 4h ou 24h) () 1137 (85)
n Embryo transfers - d5 (SET e SET DET) 504
n Embryo transfered 877 (1.7)
ß HCG positive 301 55
Ongoing Pregnancies/ vitr. /warming cycle () 265 49
Implantation rate/ embryos transfered IR () 277/877 32
Prapas, Vanderzwalmen
52
Outcome of closed blastocyst vitrification in
relation to blastocyst quality evaluation of 759
warming cycles in a single-embryo transfer
policy Van Landuyt Hum Reprod 2011
CBS-VIT High Security Irvine Kit (DMSO/EG)
7.5/7.5 20/20 Total equilibration time
10 min max 90 sec (one step)
53
Vitrification of hatched or hatching blastocysts
54
Vitrification of Hatching and Hatched Blastocyst
Before vitrification
After Thawing
After 24 hours
55
Vitrification of blastocysts after day 3 biopsy
56
Van Landuyt
Survival day 5 day 6 90 71
Irvine media VHS support Biopsie à J3
57
laser
Trophectoderm biopsy
CGH microarray
Seite 2/3
58
30 min after biopsy
In CP 5/5 DMSO/EG
After thawing
3 hrs after warming
20 hrs after warming
20 hrs after warming
59
Conclusions (I)
  • Vitrification is now on the way to replace the
    slow freezing technique.
  • However, vitrification has suffered from several
    drawbacks one of those is the non-aseptic
    cooling/storage conditions.
  • Vitrification has to be adapt to the
    cooling/warming conditions.
  • More intra and extracellular CP to keep the
    solution in a vitrification state is mandatory
    in case of a reduction in the cooling as well in
    the warming rate.
  • Addition of CPs in several steps reduce the
    osmotic stress during the outflow of water and
    entrance of CP.

60
Conclusions (II)
  • Aseptic Vitrification using the Vitrisafe as
    carrier device is proven to be effective
  • For all stage of embryo development
  • Aseptic vitrification of MII oocytes shows
    encouraging results. Even though the technique
    has to be optimized in case of long term storage
  • The Vitrisafe is an efficient device
  • Is easy to handle
  • Permit very high warming up to 20.000C/min
    (eliminate de risk of devitrification)
  • Easy to store in the LN2 tank
  • Cooling rate of lt 2.000C/min

61
Thank you to the audience and to the co-workers
  • CHIREC
  • Delavl
  • Jareno
  • Lejeune
  • Uraga
  • Vanderzwalmen
  • GIGA
  • Ectors
  • Grobet
  • IAKENTRO
  • Prapas
  • Panagiotidis
  • Achillae
  • IVF Zech
  • Bach
  • Baramsai
  • Neyer
  • Scherda
  • Stecher
  • Wirleitner
  • Zints
  • Zech
  • Institute for Reproductive Medicine and
    Endocrinology, Bregenz, Austria
  • GIGA-Research, Transgenic Platform, University of
    Liège, Liège, Belgium
  • Chirec Cavell IVF unit Brussel Belgium

Pierrevdz_at_hotmail.com
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