Vitrification Manual

1
Manual for Vitrification of Eggs

and Embryos

using the McGill Cryoleaf
TM

Manual for vitrification of eggs and embryos
2
MANUAL FOR VITRIFICATION OF EGGS AND EMBRYOS

Ri-Cheng Chian, MSc., Ph.D.
Scientific Director
McGill Reproductive Center
McGill University health Center (MUHC)
Assistant Professor
Division of Reproductive Biology
Department of Obstetrics and Gynecology
McGill University
Montreal, Quebec
Canada H3A 1A1

Jack Y.J. Huang, M.D.
Department of Obstetrics and Gynecology
McGill University
Montreal, Quebec
Canada H3A 1A1
3
TABLE OF CONTENTS
Page
PREFACE--------------------------------------------------------------------------------------------------
ACKNOWLEDGMENT---------------------------------------------------------------------------------
VITRIFICATION KIT-----------------------------------------------------------------------------------
1. Equilibration medium (EM)-----------------------------------------------------------------
2. Vitrification medium (VM)-- ---------------------------------------------------------------
THAWING KIT-------------------------------------------------------------------------------------------
1. Thawing medium (TM)----------------------------------------------------------------------
2. Dilution medium 1 (DM-1)----------------------------------------------------------------
3. Dilution medium 2 (DM-2)----------------------------------------------------------------
4. Washing medium 1 (WM-1)--------------------------------------------------------------
5. Washing medium 2 (WM-2)--------------------------------------------------------------

INTRODUCTION----------------------------------------------------------------------------------------
Progress in cryobiology------------------------------------------------------------------------
Embryo freezing---------------------------------------------------------------------------------
Egg freezing--------------------------------------------------------------------------------------
What is vitrification?---------------------------------------------------------------------------

VITRIFICATION PROCEDURE--------------------------------------------------------------------
Preparation of media------------------------------------------------------------------------
Preparation of vitrification container with liquid nitrogen (LN
2
)--------------------

4
Handling tools---------------------------------------------------------------------------------
Equilibration----------------------------------------------------------------------------------
Vitrification-----------------------------------------------------------------------------------
Storage-----------------------------------------------------------------------------------------

THAWING PROCEDURE-------------------------------------------------------------------------
Preparation of media---------------------------------------------------------------------
Thawing---------------------------------------------------------------------------------------
Diluting----------------------------------------------------------------------------------------
Washing---------------------------------------------------------------------------------------
Insemination---------------------------------------------------------------------------------

SURVIVAL AND FERTILIZATION RATES------------------------------------------------

EMBRYONIC DEVELOPMENT IN VITRO-------------------------------------------------

PREGNANCY OUTCOME-----------------------------------------------------------------------

CONCLUSIONS-------------------------------------------------------------------------------------

5
PREFACE

Since Whittingham et al (1972) and Wilmut (1972) first reported successful freezing-thawing of
mouse embryos, cryopreservation of embryos is being applied to animal industry and human
reproductive medicine. In human the first pregnancy and live birth from frozen-thawed embryos
were reported by Trounson and Mohr (1983) and Zeilmaker et al. (1984) respectively. To date,
embryos have been successfully cryopreserved at the pronuclear, multi-cellular, and blastocyst
stages of development. In fact, embryo freezing has become a routine procedure in human
assisted reproductive technology (ART). Based on 5,032 thawed cycles and 14,222 embryos, the
survival rate of day 2 or 3 embryos after freezing-thawing was 73%, the clinical pregnancy rate
per transfer was 16.0% (754/4,590) and the implantation rate per transferred embryo reached
8.4% (864/10,333) (Mandelbaum et al., 1998). It has been reported that there is no difference in
the birth characteristics and perinatal outcomes when comparing babies conceived from
cryopreserved and fresh embryos (Wada et al., 1994). Furthermore, studies focusing on
anomalies and development in children of age 1 to 9 years indicated no difference between
children conceived from cryopreserved embryos and normally conceived children (Stutcliffe et
al., 1995; Olivennes et al., 1996). However, there is no a single protocol that demonstrated to be
universally effective for freezing embryos at different developmental stages or different species.
From the point of efficiency, embryo freezing methodology still needs to be improved.

Although there were case reports and series of live youngs being produced from
cryopreserved eggs in mouse (Whittingham, 1977; Schroeder et al., 1990; Liu et al., 2001) and
human (Chen, 1986; van Uem et al., 1987; Tucker et al., 1998a,b; Kuleshova et al., 1999; Yoon
et al., 2000; 2003; Porcu et al., 2000; Yang et al., 2002; Quintans et al., 2002; Katayama et al.,
2003; Fosas et al., 2003; Boldt et al., 2003; Borini et al., 2004;), the efficiency of egg
cryopreservation in most species remains poor due to the extremely low survival rates after
freezing-thawing (Shaw et al., 2000). In one series, based on 112 IVF cycles using 1,769
cryopreserved mature eggs, the survival rate of the human eggs was 54.1% post-thawing, (Porcu
et al., 1999). Overall, the percentage of live births per thawed egg ranged from 1 to 10%
(Kuleshova and Lopata, 2002). To date, the number of children produced from cryopreserved
6
eggs is limited to less than 100 in the entire scientific literatures. Therefore, an effective
methodology to increase the survival rate of eggs following cryopreservation is required.

We have worked on the project of egg and embryo vitrification for a few years, and
produced relatively promising results. We would like to share our experiences with you, and this
is the purpose of this manual. If something is incorrect in the contents, we would like to have
your feedback.

Finally, we would like to thank Dr. Hans Ingolf Nielsen, R&D, Mr. Henrik Brandt,
Product Manager of International Marketing, and Mr. Lars Ronn, CEO of MediCult Inc. for
inviting us to prepare this manual for vitrification of eggs and embryos. We sincerely hope that
the information it contains will be of value to embryologists in the field of assisted human
reproduction.
7
ACKNOWLEDGMENTS

The contents of this handbook represent the culmination of many years of experience with
vitrification of eggs and embryos in different IVF centers. It would have been impossible for us
to prepare this manual without the support and assistance of our colleagues at the McGill
Reproductive Center, Royal Victoria Hospital, and the Department of Obstetrics and Gynecology
of McGill University. We are grateful to Professor Seang Lin Tan who discovered us and gave
us a chance to perform clinical trials in human egg vitrification at the McGill Reproductive
Center. Our special gratitude goes to our clinical colleagues, Dr. William M. Buckett, for his
valuable collaboration. We are indebted to senior embryologist, Dr. Weon-Young Son for
sharing with us for his dedication and hard work in the egg vitrification projects of at the McGill
Reproductive Center. We also thank all other clinical colleagues, embryologists, nursing staff,
ultrasound technicians and secretaries of our ART team for their cooperation and assistance.
8
VITRIFICATION-KIT

The Vitrification-kit contains two media and five McGill Cryoleafs
TM
. The first is Equilibration
medium (EM); the second is Vitrification medium (VM); and the McGill Cryoleaf
TM
is the tool
to load eggs or embryos for vitrification.

Figure 1. Package of Vitrification kit produced by MediCult.

9

1. Equilibration medium (EM)
This medium is used for equilibration of eggs or embryos before transferring into VM.
Eggs denuded of cumulus cells or embryos can be transferred to this solution directly. This
solution is made of the base medium buffered with HEPES; therefore, the solution pH is not
markedly changed at room temperature and atmosphere. This solution is ready for use following
pre-warming for at least 30 minutes at room temperature.

2. Vitrification medium (VM)
This medium is used for vitrifying eggs or embryos. This medium is also made of the
base medium buffered with HEPES; therefore, the solution pH is not markedly changed at room
temperature and atmosphere. This medium is ready for use following pre-warming for at least 30
minutes at room temperature. Following exposure of eggs or embryos into VM briefly, the eggs
or embryos are ready to load onto McGill Cryoleaf
TM
for vitrification.

10
THAWING KIT

There are five solutions contained in Thawing-kit. The first is Thawing medium (TM); the
second is Diluent medium-1 (DM-1); the third is Diluent medium-2 (DM-2); the forth is
Washing medium-1 (WM-1); and the fifth is Washing medium-2 (WM-2).

Figure 2. Package of Thawing kit produced by MediCult.

11

1. Thawing medium (TM)
This medium is used for thawing of eggs or embryos. This medium is ready for use
following pre-warming for at least 30 minutes at 37C in room atmosphere. The thin leaf of
McGill Cryoleaf
TM
where the eggs or embryos are loaded is directly immersed into TM for
thawing.

2. Diluent medium 1 (DM-1)
This medium is used for diluting the cryoprotectants from the eggs or embryos. This
medium is ready for use following pre-warming for at least 30 minutes at room temperature.

3. Diluent medium 2 (DM-2)
This medium is used for further diluting the cryoprotectants from eggs or embryos. This
medium is also ready for use following pre-warming for at least 30 minutes at room temperature.

4. Washing medium 1 (WM)
This medium is used for washing eggs or embryos following dilution of the
cryoprotectants. This medium is ready for use following pre-warming for at least 30 minutes at
room temperature. This medium is made of base medium buffered with HEPES; therefore, the
medium pH is not markedly changed at room temperature and atmosphere.

5. Washing medium 2 (WM)
This solution is the same as WM-1, but it needs to be pre-warmed for at least 30 minutes
at 37C and room atmosphere.

12
INTRODUCTION

Progress in Cryobiology
Since Whittinghan et al. (1972) and Wilmut (1972) firstly reported successful frozen-
thawed mouse embryo, cryotechnology is being applied to animal industry and reproductive
medicine. In humans, the first pregnancy and livebirth from frozen-thawed embryos were
reported by Trounson & Mohr (1983) and Zeilmaker et al. (1984) respectively. Currently,
embryo cryopreservation has become a commonplace technology in assisted reproductive
technology (ART) of domestic animals and human.

Embryo freezing
The technical requirements for embryo freezing are well documented. However, there is
no single protocol that is universally effective because there are stage- and species- specific
differences among the embryos. Most cryopreservation programs for embryo involve slow-
freeze and rapid-thaw protocols but vary in the concentration of the cryoprotectants. Higher
concentration of cryoprotectants allows for a more rapid freezing and lower cryoprotectant
concentrations require a slower freezing speed. It appears that the permeability of
cryoprotectants changes during the embryonic development. Therefore, selection of
cryoprotectants or freezing program is crucial in order to match the membrane permeability of
the developing embryos. Most embryo freezing procedures involve a cooling rate of 0.3 to
0.5C/min from the seeding temperature (usually 5 to -9C) down to a lower temperature,
usually between 30 and -150C. The embryos are then stored in liquid nitrogen (LN
2
). Embryo
thawing is a rapid procedure that involves plunging the straw containing embryos in 37C water
bath or thawed at the room temperature (>360C/min) (Hunter, 1995).

Embryos have been successfully cryopreserved at the pronuclear, multicellular, and
blastocyst stages of development. Improved survival and implantation rates can be achieved
when embryos are frozen at the pronuclear stage when compared to the 2-cell cleavage stage
(Quinn, 1990; Demoulin et al., 1991; Damario et al., 1999). Recent reports also showed that
embryo cryopreservation at the pronuclear stage optimize the chance for a live born infant
13
following single oocyte retrieval (Damario et al., 2000; Chian et al., 2001). Furthermore, the
survival rate of embryos produced from in vitro matured oocytes is higher when frozen at the
pronuclear stage when compared to the cleavage stage (Chian et al., 2001). The development of
sequential growth media has resulted in a large percentage of human embryos developing to the
blastocyst stage. Although embryos can be frozen at different stages of development, the longer
the duration of culture, the fewer embryos will be available for freezing. This is because not all
embryos are suitable for cryopreservation. It is well recognized that the quality of the embryo
will significantly affect survival rates following freezing-thawing (Kondo et al., 1996; Byrd,
2002).

Based on 5,032 thawed cycles and 14,222 embryos, the survival rate of day 2 or 3
embryo after frozen-thawing was 73%, the clinical pregnancy rate per transfer was 16%
(754/4590) and the implantation rate per transferred embryo reached 8.4% (864/10,333)
(Mandelbaum et al., 1998). Furthermore, Wada et al. (1994) reported no difference in the birth
characteristics and perinatal outcomes of babies conceived from fresh and cryopreserved
embryos. Moreover, studies focusing on anomalies and development in children aged 1-9 years-
old indicated no difference when comparing children conceived from cryopreserved and fresh
embryos (Stutcliffe et al., 1995; Olivennes et al., 1996).

The slow freezing method proved to be effective for embryos of various mammalian
species, including human. However, from the point of efficiency, the method of slow freezing
itself is still need to be improved.

Egg freezing
Since live youngs was first produced from cryopreserved eggs in mouse using slow
freezing method (Whittingham, 1977), there has been very limited success in other species,
including human (Chen, 1986; van Uem et al., 1987; Porcu et al., 1997; Tucker et al., 1998a,
1998b; Young et al., 1998; Yang et al., 1998; Porcu et al., 1999; Yang et al., 2002). Important
problems limiting oocyte cryopreservation include low survival and fertilization rates after
classical insemination, a high incidence of polyploidy and poor developmental ability of
embryos after frozen-thawing. Several factors are responsible for the relatively low success rate
14
of egg cryopreservation. These include damage of the meiotic spindles, particularly the spindles
cannot hold the chromosomes correctly at the metaphase plate prior to second polar body
extrusion (Magistrini and Szollosi, 1980; Gook et al., 1993; Eroglu et al., 1998; Trounson and
Bongso, 1999); damage to the zona pellucida such as cracks and premature hardening (Johnson
et al., 1988; Johnson, 1989); and damage to the cortical granules causing a premature cortical
reaction (Schalkoff et al., 1989; Vincent et al., 1990; Gook et al., 1993).

The spindles are reassembled once the temperature returns to normal physiologic
condition, However, cooling increases the incidence of aneuploidies because chromosomes may
not realign correctly. Although reversible and irreversible changes in spindle was noted at
temperatures as low as 7C below normal body temperature (Pickering and Johnson, 1987;
Friedler et al., 1988; Parks and Ruffing, 1992; Sathananhan et al., 1992; Eroglu et al., 1998b), at
least 60% of surviving eggs was found to have normal spindles and chromosome configurations.
The above findings suggest no evidence of an increase in the frequency of freezing-associated
aneuploidy as assessed by fluorescence in situ hybridization or cytogenetic analysis (Gook et al.,
1993; 1994; Van Blerkom and Davis, 1994).

Studies have shown that cryopreservation induces changes in the zona pellucida and
premature release of the cortical granules, which lead to zona hardening and inhibit fertilization
in mouse model (Johnson et al., 1988; Vincent et al., 1990). However, this phenomenon does not
occur in human eggs after freezing thawing (Gook et al., 1993). Although changes in the zona
pellucida may be caused by premature release of the cortical granules or some other mechanism,
intracytoplasmic sperm injection (ICSI) can be used to overcome the problem of fertilization
associated with the zona hardening following cryopreservation.

The use of immature germinal vesicle (GV) stage eggs avoids the above problems
because the chromosomes are surrounded by a nuclear membrane (Miyake et al., 1993; Cooper
et al., 1998; Isachenko and Nayudu, 1999). However, difficulties are associated with in vitro
maturation of immature eggs after frozen-thawing. Several attempts have been performed with
immature human eggs. Although the survival rates seem improved, poor in vitro maturation and
fertilization are major problems associated with immature egg freezing (Mandelbaum et al.,
15
1988a; 1988b; Toth et al., 1994a; 1994b; Son et al., 1996). Therefore, in all species,
cryopreservation of mature eggs is still more efficient than immature eggs (Son et al., 1996;
Shaw et al., 2000).

Although the developmental potential of mature mouse eggs cryopreserved by slow
freezing and vitrification protocols is comparable to controls (Carroll et al., 1993; ONeil et al.,
1997; Stachecki et al., 1998), these protocols are usually difficult to adapt to other species
because of differences in the size of eggs and the sensitivity to cooling and cryoprotectants. A
recent study reported that the egg survival rate is improved by increasing the sucrose
concentration (from 0.1 to 0.3 M) in the freezing solution using slow freezing procedure (Fabbri
et al., 2001).

Porcu et al. (1999) reported their results based on 112 IVF cycles with cryopreserved
mature eggs using the slow freezing procedure. A total of 1769 mature eggs were frozen and
1502 eggs were thawed. The survival rate was 54.1%; the fertilization rate was 57.7% following
ICSI; the cleavage rate was 91.2%, and 16 pregnancies were achieved following embryo transfer.
Another study using slow freezing procedure reported 70.9% (112/158) of eggs survived post-
thawing. Out of 24 cycles 11 patients became pregnant using donor eggs (Yang et al., 2002).
However, the survival and pregnancy rates remain somewhat variable; the percentage of live
births per thawed egg ranges from 1 to 10% using slow freezing protocol (Tucker et al., 1998a;
Mandelbaum et al., 1998; Kuleshova and Lopata, 2002; Wininger and Kort, 2002). Taken
together, only a few live births have been reported from cryopreserved human eggs using slow
freezing procedure (Chen, 1986; van Uem et al., 1987; Porcu et al., 1997; Tucker et al., 1998a,
1998b; Young et al., 1998; Yang et al., 1998; Porcu et al., 1999; Yang et al., 2002). Better results
are expected with cryopreservation of mature eggs using improved slow freezing procedure.

What is vitrification?
Vitrification is a promising novel technique and may be more effective than slow
freezing procedure for egg cryopreservation (Kuleshova and Lopata, 2002). Vitrification is the
solidification of a solution without crystallization.
16
The development of protocols optimizing the survival rate after exposure to physical and
chemical stresses of cryopreservation remains a major challenge. To successfully cryopreserve
eggs or embryos, they must be preserved at a temperature below the glass transition temperature
of the cytoplasm and the suspending solution. Below -130C is the glass transition temperature of
water. Aside from the cooling procedure, the thawing procedure could also affect oocyte or
embryo survival. Embryos from some species are sensitive to cooling to below 20C (Leibo et
al., 1996). This phenomenom been labelled chilling injury (Hayashi et al., 1989; Kashiwazaki
et al., 1991; Nagashima et al., 1995; Kasai, 2002). However, it appears this chilling injury is not
always fatal to human eggs or embryos. Temperatures between 30C and 0C may compromise
membrane integrity, cell metabolism and cytoskeleton.

Temperature below 0C will introduce the risk of intracellular ice formation (Ruffing et
al., 1993). Even a small amount of ice is likely to re-crystallize becoming larger ice crystals and
destroying the cellular structure. Mammalian eggs or embryos are relatively large cells
occupying a large quantity of water. To prevent the formation of intracellular ice, eggs or
embryos need to be dehydrated so that vitrification occurs below the glass transition temperature.

Generally, in slow freezing procedure, dehydration is achieved by placing the eggs or
embryos in a solution containing 1.0 M to 1.5 M penetrating cryoprotectants so that the eggs or
embryos are concentrated gradually during cooling. Cryoprotectants are small neutral solutes,
such as glycerol, dimethylsulphoxide (DMSO), propylene glycol (1,2-propandiol), and ethylene
glycol (EG). For human embryo cryopreservation by slow freezing method, there is a tendency
to use 1,2-propandiol (PROH) as cryoprotectant to freeze zygotes and early-cleaved embryos
(Lassalle et al., 1985), and glycerol for blastocysts (Cohen et al., 1985; Fehilly et al., 1985).
Although early embryo freezing methods used DMSO as a cryoprotectant, DMSO is less
commonly used now for multicellular embryo freezing because of concerns relating to DMSO
causing spindle polymerization with increased potential for polyploidy (Glenister et al., 1987).

Recently, EG is being used in slow freezing and vitrification methods for
cryopreservation of mammalian embryos, such as rabbit (Kasai et al., 1992), mouse (Ali and
Shelton, 1993; Zhu et al., 1996; Shaw et al., 1995; Emiliani et al., 2000), rat (Jiang et al., 1999),
17
sheep (Cocero et al., 1996), cattle (Donnay et al., 1998; Sommerfeld and Niemann, 1999) and
human (Mukaida et al., 1998; 2001; Choi et al., 2000; Yokota et al., 2000; Chi et al., 2002; Cho
et al., 2002; Vanderzwalmen et al., 2002; Son et al., 2003) due to its low molecular weight, high
permeation ability (Oda et al., 1992; Gilmore et al., 1995; Zhu et al., 1996; Newton et al., 1998)
and low toxicity (Kasai et al., 1992; Sommerfeld and Niemann, 1999; Emiliani et al., 2000).

Therefore, to select a suitable cryoprotectant, the toxicity of cryoprotectants must be
considered for successful freezing of eggs and embryos. In general, rapidly permeating
cryoprotectants are favorable because rapid permeation shortens the exposure time, reduces the
toxic injury and minimizes osmotic swelling during the removal of the cryoprotectants.
Furthermore, the permeation of cryoprotectants is largely influenced by temperature and that
high temperature accelerates the permeation of cryoprotectants.

Rapid freezing can also induce vitrification occurrence (the solidification of a solution
without crystallization) in the eggs and embryos (Rall and Fahy, 1985). In general speaking,
vitrification means rapid freezing procedure. Vitrification involves extremely high cooling and
warming rates to prevent intracellular ice formation. Although directly plunging a plastic
insemination straw (0.25 ml) in LN
2
and warming it by immersion in water (the cooling and
warming rates can be as high as >2,500C/min between 25 and -175C), intracellular ice
crystals can still form, leading to damages of the eggs or embryos (Martino et al., 1996; Vajta et
al., 1998). By modifying the plastic straw, other devices such as electronic microscopy grid,
open-pulled straw, cryoloop, cryotop and cryotip have been used. The cooling and warming
rates can increase to 22,500C/min, and thus successfully avoiding intracellular ice formation
(Martino et al., 1996; Vajta et al., 1997; Lane et al., 1999; Park et al., 1999).

However, most vitrification protocols require very high concentration of cryoprotectants
in order to rapidly dehydrate the eggs or embryos (Rall et al., 1987) and prevent intracellular ice
formation (Martino et al., 1996; Mukaida et al., 2001). Therefore, the toxicity of the
cryoprotectants must be considered. Nevertheless, in human, relatively high survival rates can be
obtained from the eggs and embryos after vitrification (Mukaida et al., 1998; 2001; Hong et al.,
18
1999; Kuleshova et al., 1999; 2000; Yokota et al., 2000; Chung et al., 2000; Chi et al., 2002; Cho
et al., 2002; Vanderzwalmen et al., 2002; Son et al., 2003).

It is also very important during thawing to remove the cryoprotectants from cells or
embryos following either slow freezing or vitrification. If the cells or embryos are directly
exposed to isotonic solution, there is a risk of osmotic swelling, because the inward water
diffusion is more rapid than the outward cryoprotectants diffusion from the eggs or embryos
(Jackowski et al., 1980). The most common strategy for preventing this injury is to thaw the eggs
or embryos in a hypertonic solution containing non-permeating agent to counteract the flow of
excess water (Kasai et al., 1980; Leibo, 1983). Sucrose is usually used to dilute vitrified eggs or
embryos after thawing, acting as an osmotic counterforce to restrict water permeation into the
eggs or embryos, and thus preventing swelling injury of the eggs or embryos (Nowshari et al.,
1994; Fabbri et al., 2001). Particularly, sucrose does not enter the cell membrane. Furthermore,
its existence in the solution increases the osmotic pressure of extracellular solute to draw water
out of the cells (Friedler et al., 1988; Kasai, 1996). However, the protective action of sucrose
may be more complex; the mechanism of the action of sucrose on the egg and embryo freezing
needs to be further investigated.

To date, pregnancies and live births have been reported after cryopreservation of mature
human eggs or embryos using vitrification procedures (Kuleshova et al., 1999; Yoon et al., 2000;
Mukaida et al., 2001; Yokota et al., 2001; Choi et al., 2000; Son et al., 2002; Huang et al., 2005).
Kulesshova et al. (1999) reported a live birth following vitrification of 17 mature eggs using high
concentration of EG. Eleven eggs survived (65%) after vitrification and five pronuclear zygotes
(46%) were obtained after ICSI. Yoon et al. (2000) cryopreserved 90 mature eggs from 7
patients by vitrification. Fifty seven eggs survived (63.3%) post-thawing. The fertilization rate
was 75.4% (43/57) following ICSI. Two healthy live births and 1 ongoing pregnancy were
obtained following transfer of 32 embryos, resulting in an implantation rate of 9.4% (3/32).
These results clearly demonstrate that mature oocyte vitrification can be applied to infertility
treatment. Our preliminary results from vitrification of animal and human eggs or embryos
indicate that the survival rates of eggs and embryos are significantly improved by vitrification
procedure.
19
Therefore, although both slow freezing and vitrification procedures have resulted in the
successful cryopreservation of human embryos and eggs, vitrification is a promising novel
technique and is likely to become safer and more cost effective than slow freezing procedure
(Kuleshova and Lopata, 2002).

20
VITRIFICATION PROCEDURE

All of the cumulus-oocyte complexes (COCs) are stripped prior to the vitrification procedure
using a finely drawn glass pipette following one minute of exposure to a commercially available
hyaluronidase solution. The mature eggs are then subjected to vitrification. The embryos at
different stages can be used directly for vitrification.

Preparation of media
EM and VM should be prepared at least one hour before use and be kept at room
temperature. Briefly, two Petri dishes (Falcon, 35 X 10 mm) can be prepared for each patient
containing 1.5 ml of EM and VM respectively (Figure 3).

Figure 3. Preparing EM and VM dishes for each patient.

21
Preparation of vitrification container with liquid nitrogen (LN
2
)
It is recommended to use medical or pharmaceutical grade LN
2
that is sterilized during its
production (Figure 4). It is also recommended that a sterilized container be used for LN
2
.
Alternatively, Pyrex beaker can be used for this purpose (Figure 5). Pyrex container can be
sterilized by putting it into a heating oven (>180C) for overnight. It is recommended to use
separate sterilized container for vitrification of each patients eggs or embryos.

Figure 4. Vacuum Barrier's unique LN2 filter filters and eliminates contaminants, and is the first
of its kind for LN
2
production (a). Completely vacuumed insulate filters the LN
2
through 0.2
micron filter, sterilizes in place, and is bubble type integrity tested (Vacuum Barrier Corporation
Woburn, MA 01801, USA; Tel: 781-933-3570). It also is important to use sterilized tank to store
the sterilized LN
2
(b).

a b
22

Figure 5. Sterilization of Pyrex LN
2
container. Pyrex beaker (a) is wrapped with aluminum
paper (b), and then put into heat oven (c) for overnight at 180C (d).

a b
c d
23
After heating sterilization, the Pyrex container can be used for containing the LN
2
. To
prevent burns caused by direct contact with LN
2
, the Pyrex container is held by a box with
handles (Figure 6).

Figure 6. Preparation of LN
2
with Pyrex container for vitrification. (a) Following sterilization,
the Pyrex container is inserted into the specially designed box with a hole. (b) This box has two
handles and a cap; (c) Pour LN
2
into the Pyrex container, and let container cools down for a few
minutes. If necessary, the container can be topped up again before vitrification; (d) Bring the
container beside to Stereo-microscope; it is ready to vitrification.

a b
c d
24
It is not easy to pour LN
2
into the Pyrex container. Therefore, it is recommended to use
Brymill Filter as the withdrawal device from sterilized LN
2
dewar (Model 503-F) (Figure 7).

Figure 7. Brymill filter for withdrawal device on LN
2
dewar (Model 503-F).

25
Handling tools
Vitrification procedure requires two forceps, one is big (length >25 cm) and another is
small (length >8 cm) (Figure 8).

Figure 8. It is necessary to have two handling forceps for the vitrification and thawing
procedures.

26
McGill Cryoleaf
TM
is a device that holds the eggs or embryos during vitrification
procedure and for storage (Figure 9). Cryoleaf
TM
is provided with Vitrification Kit.

Figure 9. Actual view of McGill Cryoleaf
TM
. (a) The core part of McGill Cryoleaf
TM
, and the
arrow indicates the loading portion that is made of thin polyethylene stick. The green part can
slide down to protect the loading stick; (b) Protection straw for storage; (a+b) The completed
parts of Cryoleaf
TM
.

1
2
a
b
a + b
27

Equilibration
The mature eggs or embryos can be directly transferred into EM (room temperature) from
culture dish (37C) and be kept for a maximum of 5 minutes at room temperature. The eggs or
embryos will shrink initially and then recover to their former shapes within 3 to 5 minutes.
Following equilibration, the eggs or embryos are transferred to VM (room temperature) for 1
minute.

Vitrification
At this point, the eggs or embryos will shrink again. The eggs or embryos are loaded
onto the tip of the McGill Cryoleaf
TM
and then directly plunge into liquid nitrogen (LN
2
) for
cryopreservation. It is important to avoid exposing the eggs or embryos to VM at room
temperature for more than 1 minutes before plunging the McGill Cryoleaf
TM
into LN
2
. The
illustration of vitrification procedure is shown in Figure 10 and the actual performance is
indicated in Figure 11.

Figure 10. Vitrification procedure for eggs and embryos. The details are described in the text.

28

Figure 11. Vitrification procedure. (a) Preparation of LN
2
with Pyrex container for vitrification;
(b) Loading eggs or embryos onto McGill Cryoleaf
TM
; (c) Immersing the McGill Cryoleaf
TM
directly into LN
2
; (d) Capping the McGill Cryoleaf
TM
under LN
2
for storage.

a
b
c d
29
Storage
After vitrification, it is important to store the eggs or embryos safely with the McGill
Cryoleaf
TM
. The key point is to cap the McGill Cryoleaf
TM
under LN
2
in order to prevent frozen
eggs or embryos from warming after vitrification (Figure 12).

Figure 12. Capping McGill Cryoleaf
TM
under LN
2
(a) and preparing a cane with goblet (b) for
keeping Cryoleaf
TM
(c). McGill Cryoleaf
TM
labeled with patients name and store in LN
2
storage
tank (d).

a b
c
d
30
THAWING PROCEDURE

Preparation of media
TM should be warmed at 37C for at least for 30 minutes before thawing. DM-1, DM-2
and WM-1 and WM-2 should be prepared at least 30 minutes before use and be kept at room
temperature. For preparation of TM, DM-1, DM-2, WM-1 and WM-2, you can use one Petri dish
(Falcon, 35 X 10 mm) containing 1.5 ml of each medium (Figure 13).

Figure 13. Preparation of thawing solutions. (a) TM, DM-1, DM-2, WM-1 and WM-2 each
solution can be prepared with Petri dishes (35 X 10 mm) respectively.

31
Thawing
The eggs or embryos are thawed in 1.5 ml TM in Petri dish (35 X 10 mm). Simply, the
tip of the McGill Cryoleaf
TM
is immersed into TM for 1 minute; the eggs or embryos will fall
into the TM (Figure 14).

Figure 14. Eggs or embryos are thawed with TM. The tip of McGill Cryoleaf
TM
can be directly
immersed into 1.5 ml TM contained in Petri dish (35 X 10 mm). The eggs or embryos will slide
away from the McGill Cryoleaf
TM
within 1 minute and then eggs or embryos can be transferred
to DM-1.

32
Diluting
After thawing with TM, the eggs or embryos are transferred to DM-1 for 3 minutes at
room temperature. At this point, the eggs or embryos return partially to their original shapes.
Following transfer of eggs or embryos to DM-2 for another 3 minutes, the eggs or embryos
further recover their shapes but do not fully return to their former shapes at this stage.

Washing
After diluting with DM-2, the eggs or embryos are transferred to WM-1 for 3 minutes at
room temperature. Within 3 minutes, the eggs or embryos will fully return to their former shapes
(before vitrification). The eggs or embryos are washed in WM-2 for another 3 minutes and then
transferred to the fertilization medium (37C) in 5% CO
2
or tri-gas (5% CO
2
, 5% O
2
, 90% N
2
)
incubator for insemination. The illustration of thawing procedure is shown in Figure 15.

Figure 15. Thawing procedure for eggs and embryos. TM should be kept at 37C and DM-1,
DM-2, WM-1 and WM-2 are kept at room temperature.
33
SURVIVAL AND FERTILIZATION RATES

More than 500 eggs have been thawed after vitrification. The survival rate is >90% based on
assessment of oocyte integrity and morphology after 1 hour of culture (Figure 16). ICSI is
recommended for insemination of frozen-thawed eggs because this method offers a greater
chance of successful fertilization than does IVF. Based on more than 500 eggs inseminated by
ICSI, fertilization rate reached approximately 70%.

Figure 16. Mature (metaphase-II) human egg after freezing-thawing and before insemination.

34
EMBRYONIC DEVELOPMENT IN VITRO

Pre-clinical trial indicated that approximately 30-35% fertilized eggs can develop to blastocyst
stage following culture in vitro (Figure 17, based on more than 100 fertilized eggs). In addition,
approximately >95% of the vitrified embryos (from zygote to blastocyst stage) survived after
freezing-thawing, and these embryos further developed following culture for 24 hours after
thawing.

Figure 17. Blastocysts produced from vitrified human eggs. (a) A blastocyst formed on day 5
following culture in vitro from the vitrified eggs; (b) This blastocyst expanded after further
culture for one day (on day 6).

a b
35
PREGNANCY OUTCOME

Viability and pregnancy outcome of vitrified
oocytes following thawing and ICSI
at McGill Reproductive Center

___________________________________________________________________________

Patients (cycles) 25 (25)
Age 31.8 3.6
No. of eggs thawed 283 (11.3 5.9)
No. of eggs survived (%) 253 (89.4)
No. of eggs fertilized (%) 179 (72.7)
No. of eggs cleaved (%) 155 (80.0)
No. of embryos transferred 92 (3.7 1.1)
No. of clinical pregnancies (%) 11 (44.0)
No. of implantation (%) 15 (20.7)
___________________________________________________________________________
Updated data based on Chian et al. (2005) ASRM Annual Meeting.

36
The details of pregnancies and live births
11
10
9
8
7
6
5
4
3
2
1
Patients
Ongoing Twins 4
Ongoing Twins 4
Ongoing Twins 5
Ongoing Twins 4
Ongoing Twins 3
2 Twins 3
1 Singleton 4
0 Ectopic 4
3 Triplets 4
1 Singleton 5
0 Miscarriage 1
Live births No. of fetus No. of embryos
37

CONCLUSIONS

Human eggs and embryos can be vitrified successfully. Vitrification method can be efficiently
applied for cryopreservation of human eggs and embryos.

38
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Manual for Vitrification of Eggs

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