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 Manual for vitrification of eggs and embryos 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 )--------------------
Manual for vitrification of eggs and embryos 4 Handling tools--------------------------------------------------------------------------------- Equilibration---------------------------------------------------------------------------------- Vitrification----------------------------------------------------------------------------------- Storage-----------------------------------------------------------------------------------------
THAWING PROCEDURE------------------------------------------------------------------------- Preparation of media--------------------------------------------------------------------- Thawing--------------------------------------------------------------------------------------- Diluting---------------------------------------------------------------------------------------- Washing--------------------------------------------------------------------------------------- Insemination---------------------------------------------------------------------------------
SURVIVAL AND FERTILIZATION RATES------------------------------------------------
EMBRYONIC DEVELOPMENT IN VITRO-------------------------------------------------
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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. Manual for vitrification of eggs and embryos 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. Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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., Manual for vitrification of eggs and embryos 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. Manual for vitrification of eggs and embryos 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), Manual for vitrification of eggs and embryos 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., Manual for vitrification of eggs and embryos 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. Manual for vitrification of eggs and embryos 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).
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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).
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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. Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 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.
Manual for vitrification of eggs and embryos 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 Manual for vitrification of eggs and embryos 37
CONCLUSIONS
Human eggs and embryos can be vitrified successfully. Vitrification method can be efficiently applied for cryopreservation of human eggs and embryos.
Manual for vitrification of eggs and embryos 38 REFERENCES Ali J, Shelton JN. Vitrification of preimplantation stages of mouse embryos. J Reprod Fertil 1993; 98: 459-465.
Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod 2003; 18: 1250-1255.
Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004; 82: 601-605.
Byrd W. Cryopreservation, thawing, and transfer of human embryos. Sem in Reprod Med 2002; 20: 37-43.
Carroll J, Wood MJ, Whittingham DG. Normal fertilization and development of frozen thawed mouse oocytes: protective action of certain macromolecules. Biol Reprod 1993; 48: 606- 612.
Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; ii, 884-886.
Chi HJ, Koo JJ, Kim MY, Joo JY, Chang SS, Chung KS. Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod 2002; 17: 2146-2151.
Chian RC. Gulekli B. Buckett WM. Tan SL. Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum Reprod 2001; 16: 1700-1702.
Cho HJ, Son WY, Yoon SH, Lee SW, Lim JH. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod 2002; 17: 2419-2422.
Choi DH., Chung HM, Lim JM, Ko JJ, Yoon TK, Cha KY. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil Steril 2000; 74, 838834.
Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, Han SY, Choi DH, Cha KY. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various mturational stages. Fertil Steril 2000; 73: 545-551.
Cocero MJ, Sebastian AL, Barragan ML, Picazo RA.Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiol 1996; 33:502507.
Cohen J, Simmons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the storage of expanded human blastocysts. J In Vitro Fert Embryo Trans 1985; 2: 59-64.
Manual for vitrification of eggs and embryos 39 Cooper A, Paynter SJ, Fuller BJ, Shaw RW. Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries. Hum Reprod 1998; 13: 971-978.
Damario MA. Hammitt DG. Galanits TM. Stevens SA. Session DR. Dumesic DA. Anonymous oocyte donation performed exclusively with embryos cryopreserved at the pronuclear stage. Fertil Steril 1999; 71: 830-835.
Damario MA. Hammitt DG. Session DR. Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril 2000; 73: 767-773.
Demoulin A. Jouan C. Gerday C. Dubois M. Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum Reprod 1991; 6: 799-804.
Donnay I, Auquer P, Kaidi S, Carolan C, Lonergan P, Mermillod P, Massip A. Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity. Anim Reprod Sci 1998; 52: 93104.
Emiliani S, Bergh MVD, Vannin AS, Biramane J, Englert Y. Comparison of ethylene glycol, 1,2-propanediol and glycol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum Reprod 2000; 15: 905-910.
Eroglu A, Toner M, Leykin L, Toth TL. Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes. J Assist Reprod Genet 1998a; 15: 447-454.
Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil Steril 1998b; 69: 944-957. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411-416.
Fehilly CB, Cohen J, Simmons RF, Fishel SB, Edwards RG. Cryopreservation of cleaved embryos and expanding blastocysts in the human: a comparative study. Fertil Steril 1985; 44: 638-644.
Fosas N, Marina F, Torres PJ, Jove I, Martin P, Perez N, Arnedo N, Marina S. The births of five Spanish babies from cryopreserved donated oocytes. Hum Reprod 2003; 18: 1417-1421.
Friedler S, Giudice L, Lamb E. Cryopreservation of embryos and ova. Fertil Steril 1988; 49: 743-754.
Manual for vitrification of eggs and embryos 40 Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Crister JK. Effect of cryoprotectants solutes on water permeability of human spermatozoa. Biol Reprod 1995; 53: 985-995.
Glenister PH, Wood MJ, Kirby C, Whittingham DG. Incidence of chromosome anomalies in first cleavage mouse embryos obtained from frozen thawed oocytes fertilized in vitro. Gamete Res 1987; 16: 205-216.
Gook D, Osborn S, Johnson W. Cryopreservation of mouse and human oocytes using 1,2 propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8: 1101- 1109.
Gook DA, Osborn SM, Bourne H, Johnston WI. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 1994; 9: 684-691.
Hayashi S, Kobayashi K, Mizuno J, Saitoh K, Hirano S. Birth of piglets from frozen embryos. Vet Rec 1989; 125: 43-44.
Hong SW, Chung HM, Lim JM, Ko JJ, Yoon TK, Yee B, Cha KY. Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril 1999; 72: 142146.
Huang CC, Lee TH, ChenSU, Chen HH, Cheng TC, Liu CH, Yang YS, Lee MS. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005; 20: 122-128.
Isachenko EF, Nayudu PL. Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromosomal normality and cumulus integrity. Hum. Reprod 1999; 14: 400-408.
Jackowski S, Leibo SP, Mazur P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J Exp Zool 1980; 212: 329-341.
Jiang JY. Umezu M. Sato E. Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions. Cryobiol 1999; 38: 160-164.
Johnson MH, Pickering SJ, George MA. The influence of cooling on the properties of the zona pellucida in mouse oocytes. Hum Reprod 1988; 3: 383-387.
Johnson MH. The effect on fertilization of exposure of mouse oocytes to dimethyl sulfoxide: an optimal protocol. J In Vitro Fertil Embry Transfer 1989; 6: 168-175.
Kasai M, Niwa K, Iritani A. Survival of mouse embryos frozen and thawed rapidly. J Reprod Fertil 1980; 59: 51-56. Manual for vitrification of eggs and embryos 41
Kasai M, Nishimori M, Zhu SE, Sakurai T, Machida T. Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures. Biol Reprod 1992; 47: 1134-1139.
Kasai M, Zhu SE, Pedro PB, Nakamura K, Sakurai T, Edashige K. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiol 1996; 33: 459-464.
Kasai M. Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultrarapid vitrification. Reprod Med Biol 2002; 1: 1-9.
Kashiwazaki N, Ohtani S, Miyamoto K, Ogawa S. Production of normal piglets from hatched blastocysts frozen at -196C. Vet Rec 1991; 128: 256-257.
Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223-224.
Kondo I. Suganuma N. Ando T. Asada Y. Furuhashi M. Tomoda Y. Clinical factors for successful cryopreserved-thawed embryo transfer. J Assist Reprod Genet 1996; 13: 201- 206.
Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999; 14: 3077-3079.
Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449-754. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 1985; 44: 645-651.
Leibo SP. A one-step in situ dilution method for frozen-thawed bovine embryos. Cryo-Lett 1983; 4: 387-400.
Leibo SP, Martino A, Kobayashi S, Pollard JW. Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 1996; 42: 4553.
Liu J, Van der Elst J, Van den Broecke R, Dhont M. Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vitro transplantation and in vitro maturation. Biol Reprod 2001; 64: 171-178.
Magistrini M, Szollosi D. Effects of cold and of isopropyl N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980; 22: 699-707.
Mandelbaum J, Belasch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, Alnot MO, Salat-Baroux J. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998a; 13 (Suppl. 3): 161-177. Manual for vitrification of eggs and embryos 42
Mandelbaum J, Junca AM Plachot M. Cryopreservation of human embryos and oocytes. Hum Reprod 1988b; 3: 117-119.
Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059-1069.
Miyake T, Kasai M, Zhu SE, Sakurai T, Machida T. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol based solution by a simple method. Theriogenology 1993; 40: 121-134.
Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998; 13, 28742879.
Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618-620.
Nagashima H, Kashiwazaki N, Ashman RJ, Grupen CG, Nottle MB. Cryopreservation of porcine embryos. Nature 1995; 374: 416.
Newton H, Fisher J, Arnold JRP, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 1998; 13: 376-380.
Nowshari MA, Nayudu PL, Hodges JK. Effect of cryo- protectant concentration, equilibration time and thawing procedure on survival and development of rapid-frozenthawed mature mouse oocytes. Theriogenology 1994; 42: 11931204.
Oda K, Gibbons WE, Leibo SP. Osmotic shock of fertilized mouse ova. J Reprod Fertil 1992; 95: 737-747.
Olivennes F, Schneider Z, Remy V, Blanchet V, Kerbrat V, Fanchin R, Hazout A, Glissant M, Fernandez H, Dehan M, Frydman R. Perinatal outcome and follow-up of 82 children aged 1-9 years old conceived from cryopreserved embryos. Hum Reprod 1996; 11: 1565- 1568.
ONeil L, Paynter SJ, Fuller BJ, Shaw RW. Vitrification of mouse oocytes: improved results following addition of polyethylene glycol to a dimethyl sulfoxide solution. Cryobiology 1997; 34: 295-301.
Park SP, Kim EY, Kim DI, Park NH, Won YS, Yoon SH, Chung KS, Lim JH. Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids. Hum Reprod 1999; 14: 28382843.
Manual for vitrification of eggs and embryos 43 Parks JE, Ruffing NA. Factors affecting low temperature survival of mammalian oocytes. Theriogenology 1992; 37: 53-79.
Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1987; 2: 207-216.
Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724-724.
Porcu E, Fabbri R, Ciotti PM, Marsella T, Balicchia B, Damiano G, Caracciolo D, Giunchi S, De Cesare R, Flamigni C. Cycles of human oocyte crypreservation and intracytoplasmic sperm injection: results of 112 cycles. Fertil Steril 1999; 72 (3) Suppl 1: S2.
Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S, Flamigni C. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169:33-37.
Quinn P. Marrs RP. Stone BA. To publish or not to publish? Fertil Steril 1990; 95: 952.
Quintans C, Donaldson MJ, Bertolino MV, Pasqualini RS. Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 2002; 17: 3149- 3152.
Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196C by vitrification. Nature 1985; 313: 573-575.
Rall WF, Wood MJ, Kirby C, Whittingham DG. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987; 80: 499-504.
Ruffing NA, Steponkus PL, Pitt RE, Parks JE. Osmometric behaviour, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stage. Cryobiology 1993; 30: 562-580.
Sathananhan, A.H.,Kirby, C. and Trounson, A. Philipatos D, Shaw J. The effects of cooling mouse oocytes. J Assist Reprod Genet 1992; 9: 139-148.
Schalkoff ME, Oskowitz SP, Powers, RD. Ultrastructural observations of human and mouse oocytes treated with cryopreservatives. Biol Reprod 1989; 40: 379-393.
Shaw JM. Ward C. Trounson AO. Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow-cooled mouse pronuclear and 4-cell embryos. Hum Reprod 1995; 10: 396-402.
Manual for vitrification of eggs and embryos 44 Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 1990; 89: 43- 50.
Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology 2000; 53: 59-72.
Sommerfeld V, Niemann H. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology 1999; 38: 95-105.
Son WY, Park SE, Lee KA, Lee WS, Ko JJ, Yoon TK, Cha KY. Effects of 1,2-propanediol and freezing on the in vitro developmental capacity of human immature oocytes. Fertil Steril 1996; 66: 996-999.
Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: Case report. Hum Reprod 2002; 17: 2963-2966.
Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137-139.
Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized oocytes: the effect of replacing sodium with Choline in the freezing medium. Cryobiology 1998; 37: 346-355.
Sutcliffe AG, D'Souza SW, Cadman J, Richards B, McKinlay IA, Lieberman B. Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum Reprod 1995; 10: 3332-3337.
Toth TL, Baka SG, Veeck LL, Jones HW Jr, Muasher S, Lanzendorf SE. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994a; 61: 891-894.
Toth TL, Lanzendorf SE, Sandow BA, Veeck LL, Hassen WA, Hansen K, Hodgen GD. Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil Steril 1994b; 61: 1077-1082.
Trounson AO, Bongso A. Fertilization and development in humans. Curr Topics Devel Biol 1996; 32: 59-101.
Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983; 305:707-709.
Manual for vitrification of eggs and embryos 45 Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998b; 70: 578-579.
Vajta G, Booth PJ, Holm P, Greve T, Callesen H. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo-Lett 1997; 18: 191-195.
Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53-58.
Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microscopy Res Technol 1994; 27: 165-193.
Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K and Schoysman R. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002;17, 744751.
Wada I, Macnamee MC, Wick K, Bradfield JM, Brinsden PR. Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum Reprod 1994; 9: 543- 546.
Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to -196 and -269C. Science 1972; 178:411-414.
Whittingham DG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -196C. J Reprod Fertil 1977; 49: 89-94.
Wilmut I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci 1972; 11:1071-1079.
Wininger JD. Kort HI. Cryopreservation of immature and mature human oocytes. Sem in Reprod Med 2002; 20: 45-49.
Van Uem JF, Siebzehnrbl ER, Schuh B, Kock R, Trotnov S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987; i: 752-753.
Vincent C, Pickering SJ, Johnson MH. The zona hardening effect of dimethyl sulfixide requires the presence of an oocyte and is associated with reduction in the number of cortical granules present. J Reprod Fertil 1990; 89: 253-259.
Manual for vitrification of eggs and embryos 46 Yang DS. Improved survival rate after cryopreservation of human fresh and aged unfertilized oocytes using a specially developed oocyte cryopreservation regime. Proc 54th Ann Mtg ASRM, San Francisco, Oct 4-9, Abstract 0-232.
Yang D, Wilslow KL, Blohm PL, Brown SE, Nguyen K, Brubaker C. Oocyte donation using cryopreserved donor oocytes. Fertil Steril 2002; 78 (Suppl.): S14.
Yokota Y, Sato S, Yokota M, Ishikawa Y, Makita M, Asada T, Araki Y. Successful pregnancy following blastocyst vitrification: case report. Hum Reprod 2000, 15, 18021803.
Yokota Y, Yokota H, Yokota M, Sato S, Araki Y. Birth of healthy twins from in vitro development of human refrozen embryos. Fertil Steril 2001; 76:10631065
Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ, Cha KY. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000; 74: 180-181.
Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HJ, Cha KY. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 19: 1323-1336.
Young E, Kenny A, Puigdomenech E, Van Thillo G, Tiveron M, Piazza A. Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report. Fertil Steril 1998; 70: 360-361. Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984; 42: 293-296.
Zhu SE, Kasai M., Otoge H, Sakurai T, Machida T. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J Reprod Fertil (1993); 98, 139145.
Zhu SE, Sakurai T, Edasige K, Machida T, Kasai M. Cryopreservation of zona-hatched mouse blastocysts. J Reprod Fertil 1996; 107: 37-42.