vitrification manual

8/12/2019 Vitrification Manual

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1

Manual for Vitrification of Eggsand Embryos

using the McGill Cryoleaf TM


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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


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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)--------------------


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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 -------------------------------------------------------------------------------------


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PREFACE

Since Whittingham et al (1972) and Wilmut (1972) first reported successful freezing-thawing ofmouse 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 eggcryopreservation 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


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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. forinviting 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.


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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 forsharing 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.


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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.


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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.


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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.


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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. Thismedium 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.


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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


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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


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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 attemperatures 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.,


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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 (Fabbriet 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.


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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),


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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.,


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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 orembryos 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.


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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).


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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 patientcontaining 1.5 ml of EM and VM respectively ( Figure 3 ).

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


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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 firstof its kind for LN 2 production ( a ). Completely vacuumed insulate filters the LN 2 through 0.2micron filter, sterilizes in place, and is bubble type integrity tested (Vacuum Barrier CorporationWoburn, MA 01801, USA; Tel: 781-933-3570). It also is important to use sterilized tank to storethe sterilized LN 2 (b ).

a b


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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


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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 twohandles 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 thecontainer beside to Stereo-microscope; it is ready to vitrification.

a b

c d


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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).


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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.


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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 thearrow indicates the loading portion that is made of thin polyethylene stick. The green part canslide 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


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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 loadedonto 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.


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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


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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) forkeeping Cryoleaf TM (c) . McGill Cryoleaf TM labeled with patients name and store in LN 2 storagetank ( d ).

a b

c d


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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 eachsolution can be prepared with Petri dishes (35 X 10 mm) respectively.


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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 directlyimmersed into 1.5 ml TM contained in Petri dish (35 X 10 mm). The eggs or embryos will slideaway from the McGill Cryoleaf TM within 1 minute and then eggs or embryos can be transferredto DM-1.


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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.


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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.


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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 5following culture in vitro from the vitrified eggs; ( b ) This blastocyst expanded after furtherculture for one day (on day 6).

a b


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PREGNANCY OUTCOME

Viability and pregnancy outcome of vitrifiedoocytes following thawing and ICSI

at McGill Reproductive Center ___________________________________________________________________________

Patients (cycles) 25 (25)Age 31.8 3.6No. 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.


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The details of pregnancies and live births

11

10

9

87

6

5

4

3

2

1

Patients

On oinTwins4

On oinTwins4

On oinTwins5

On oinTwins4On oinTwins3

2Twins3

1Sin leton4

0Ecto ic4

3Tri lets4

1Sin leton5

0Miscarria e1

Live birthsNo. of fetusNo. of embr os


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CONCLUSIONS

Human eggs and embryos can be vitrified successfully. Vitrification method can be efficiently

applied for cryopreservation of human eggs and embryos.


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