UNIVERSITE LIBRE DE BRUXELLES Faculté de Médecine
CONTRIBUTION OF VITRIFICATION TO HUMAN ASSISTED REPRODUCTION
Giovanna Fasano
Promoteur: Pr. Yvon Englert
Jury:
Président: Pierre-Alain Gevenois Secrétaire:Yvon Englert
Membres: Vanessa Depaepe, Catherine Ledent, Serge Rozenberg Experts: Paul Devroey, Dominique Royère, Etienne Van den Abbeel
Année académique 2012-2013
Dedicata ai miei genitori, esempi di vita che porto nel mio cuore ogni giorno.
Grazie per avermi dato delle ali cosí grandi e forti per arrivare fin qui.
Dedicata a mio padre, a lui tanto simile nella sostanza.
Dedicata a mia madre, che esplode di gioia ogni volta che torno a casa.
Dedicata a mio fratello, che ha vissuto e nascosto la nostalgia della mia assenza affinché potessi realizzare i miei obiettivi.
Vi amo.
This thesis is dedicated to my parents, my role models whom I carry with me in my heart each and every day. Thank you for having given me such large and strong wings which enabled me to be where I am today.
I dedicate this work to my father, whom I am profoundly similar to.
I dedicate it to my mother, who explodes with joy every time I return home.
I also dedicate it to my brother, who missed me but hid it so that I could realise my goals.
I love you.
ACKNOWLEDGEMENTS
As in keeping with tradition, I would like to thank all the people who helped me over the years and shared this wonderful adventure with me.
Prof. Yvon Englert.
Thank you for giving me this incredible opportunity, for believing in my abilities, for always encouraging and supporting me to pursue my ideas, for always finding the time to discuss matters with me and to re-read my work.
Dr. Isabelle Demeestere.
Thank you for welcoming me into your team and opening the doors to the ovarian tissue cryopreservation programme. Thank you for your valuable advice, for correcting my articles and at the same time for your humbleness and for never having said that you did not have the time.
Christine Anuset.
Thank you for your sense of humour and energy in my moments of weakness and fatigue. Thank you for making the administrative red tape so much less difficult for me over the years.
Agnes Echterbille and her Fertility Clinic nursing team.
Thank you for your wisdom and for always finding the right words to encourage me. Thanks to all of you for having spent time discussing my research projects with the patients.
The ART Laboratory team. Thank you for having contributed to my research and clinical projects with your daily work.
The Human Reproduction Research Laboratory team. Thank you for your help and your great warmth.
The Fertility Clinic team and in particular Dr. Anne Delbaere, Dr. Fabienne Devreker and all the medical staff, the secretaries and the psychologists. Thank you for having contributed to this thesis with your many and various skills.
Dr. Philippe Revelard. Thank you for helping me with data extraction which enabed me to properly interpret the results.
And also to those who hindered me along the way and who did not believe in it right to the end. Thank you. Without you, I could never have done it.
Working in this field over the years, every new pregnancy and every new born baby still gives me a strong emotion. My last thought therefore goes to my work that only distracted and superficial people could consider a simple profession.
TABLE OF CONTENTS
INTRODUCTION……….1
LITTERATURE REVIEW 1.1 BASIC KNOWLEDGE ON CRYOBIOLOGY AND CRYOPRESERVATION 1.1.1 Physics of cryopreservation………2
1.1.2 Cryopreservation media………..…6
1.1.3 Cryopreservation techniques……….…………..…9
1.1.3.1 Slow freezing……….9
1.1.3.2 Vitrification……….11
1.1.4 Thawing and warming procedures………...14
1.1.5 Cryopreservation devices………...15
1.1.6 Pratical advantages of vitrification………17
1.2 CRYOPRESERVATION OF GAMETES, EMBRYOS AND GONADAL TISSUES 1.2.1 Spermatozoa………..19
1.2.2 Oocytes………..21
1.2.2.1 Biological aspects of Oocyte Cryopreservation………..21
1.2.2.2 History of Oocyte Cryopreservation……….23
1.2.2.3 Human Oocytes Cryopreservation by Slow freezing and Vitrification………24
1.2.2.4 Assessment of Oocyte quality after warming procedure……….…..26
1.2.3 Embryos………..27
1.2.3.1 Biological aspects of Embryos Cryopreservation………....27
1.2.3.2 History of Embryos Cryopreservation………...29
1.2.3.3 Human Embryo Cryopreservation at different stages of development by Slow freezing and Vitrification……..………30
1.2.3.4 Assessment of Embryo quality after warming procedure……….35
1.2.4 Gonadal Tissues………36
1.2.4.1 Outlines on Cryopreservation of Human Testicular Tissue...36
1.2.4.2 Outlines on Cryopreservation of Human Ovarian Tissue……….37
1.3 Oocyte In Vitro Maturation………..39
1.3.1 Biological aspects of Oocyte In Vitro Maturation……….39
1.3.2 Human Oocyte In Vitro Maturation………..………..41
RESEARCH WORK 2.1 CRYOPRESERVATION OF HUMAN FAILED MATURATION OOCYTES SHOWS THAT VITRIFICATION GIVES SUPERIOR OUTCOMES TO SLOW COOLING (Cryobiology, 2010;61(3):243-247)..…47
2.2 IN-VITRO MATURATION OF HUMAN OOCYTES: BEFORE OR AFTER VITRIFICATION? (J Assist Reprod Genet, 2012;29(6):507-512)...51
2.3 VITRIFICATION OF IN VITRO MATURED OOCYTES COLLECTED FROM ANTRAL FOLLICLES AT THE TIME OF OVARIAN TISSUE CRYOPRESERVATION (Reprod Biol Endocrinol, 2011;9:150-155)……….53
2.4 A RANDOMIZED CONTROLLED TRIAL COMPARING TWO VITRIFICATION METHODS VERSUS SLOW FREEZING FOR CRYOPRESERVATION OF HUMAN CLEAVAGE STAGE EMBRYOS (under revision)………..…56
2.5 ADDITIONAL CLINICAL RESULTS (unpublished data) ………60
DISCUSSION AND CONCLUSIONS 3.1 CONTRIBUTION OF VITRIFICATION TO HUMAN ASSISTED REPRODUCTION 3.1.1 Biological considerations………..67
3.1.2 Therapeutical considerations and future clinical perspectives…………..71 3.1.3 Ethical and legal considerations………75 ANNEXES………81 REFERENCES………87
RESUME
La cryopréservation, dans le domaine de la reproduction médicalement assistée, constitue depuis de nombreuses années une branche suscitant beaucoup d’intérêts et d’espoirs. En effet, de nombreuses équipes de recherche se sont attelées à mettre au point et à améliorer des protocoles permettant de conserver les gamètes, les embryons et les tissus reproducteurs.
Malgré le fait que la cryopréservation soit une technique très attractive, elle peut avoir des effets délétères sur les cellules. Les protocoles expérimentaux visent donc à minimiser ces effets afin d’augmenter la survie et la compétence cellulaire après décongélation.
Les deux méthodes les plus utilisées, la congélation lente et la vitrification, présentent chacune des avantages et des inconvénients. En effet, la première ne permet pas d’éliminer la cristallisation intracellulaire. Quant à la seconde, elle empêche la formation de cristaux de glace mais pourrait provoquer une toxicité due à la forte concentration des cryoprotecteurs.
Cette thèse de doctorat propose plusieurs objectifs :
Présenter les bases de la cryobiologie, de la physique de la cryopréservation cellulaire et l’état actuel des connaissances concernant les techniques de cryopréservation utilisées en médecine de la reproduction assistée humaine.
Présenter les résultats obtenus grâce à plusieurs projets de recherche et cliniques concernant la cryopréservation des ovocytes et des embryons humains au sein de la Clinique de Fertilité de l’hôpital Erasme.
Dans un premier temps, l’efficacité des deux techniques, congélation lente versus vitrification, a été évaluée sur des ovocytes immatures (non utilisables pour les patientes) récupérés lors des ponctions ovocytaires. Ce projet de recherche nous a permis de familiariser avec la technique de vitrification et a suggéré un avantage de la vitrification en terme de survie ovocytaire (Fasano et al., 2010).
Le taux de maturation ovocytaire in vitro a été évaluée ensuite avant et après vitrification. Ce projet de recherche a montré un efficatcité plus élevée lorsque les ovocytes immatures sont d’abord mis en maturation in vitro et puis vitrifiés (Fasano et al., 2012). Cette stratégie a donc été intégrée dans la clinique dans le cadre de notre programme de préservation de la fertilité.
Les ovocytes immatures présents dans le tissu ovarien prélevé dans le cadre d’une cryopréservation avant traitement gonadotoxique ont été récoltés grâce à la ponction des follicules antraux et la filtration du milieu de dissection du tissu, maturés in vitro et vitrifiés. Cette étude clinique a permis de démontrer la faisabilité de la stratégie combinée pour préserver la fertilité des patientes sans limites d’âge ou de phase du cycle (Fasano et al., 2011).
Cette technique offre une alternative thérapeutique pour nos patientes à risque de greffe du tissu et une chance supplémentaire de restaurer la fertilité chez la moitié des patientes. La maturation in vitro et la vitrification des ovocytes a également été appliquée pour la préservation de la fertilité des patientes hors critères pour la cryopréservation du tissu ovarien.
Dans un deuxième temps, l’efficacité de la vitrification a été évalué pour cryoconserver les embryons. Les embryons surnuméraires de bonne qualité obtenus en clinique ont été cryoconservés avec des différentes méthodes pour évaluer l’efficacité de la congélation lente vs vitrification sur le taux de survie et le développement embryonnaire. Cette étude clinique a permis de démontrer la supériorité de la vitrification pour cryopréservation des
embryons à tous les stades de développement, et d’évaluer l’efficacité de différentes techniques de vitrification (en cours de révision). L’application de la vitrification pour cryopréservation des embryons a permis d’augmenter le taux de grossesse cumulatif sur un seul cycle frais en offrant à toutes nos patientes des techniques de cryopréservation plus performantes lors de leurs traitements cliniques.
Enfin, analyser les différentes applications en reproduction médicalement assistée de ces techniques et illustrer des cas cliniques au sein de la Clinique de Fertilité de l’hôpital Erasme. Les aspects thérapeutiques et les perspectives cliniques futures, ainsi que les implications éthiques seront discutées.
INTRODUCTION
This work aimed to evaluate the efficiency of various oocyte and embryo cryopreservation protocols to assess the possibility of using a new promising cryopreservation technique in Assisted Reproduction Technologies (ART): the vitrification.
At the beginning of these doctorate studies, the discarded material obtained from informed patients undergoing in vitro fertilization (IVF) cycles was used for experimental research. After acquisition of the required skills and validation of the procedures using this material, vitrification and oocyte in vitro maturation (IVM) procedures were extended to clinical trials and practice, offering additional efficient technologies to treat patients in our institution. In collaboration with the fertility preservation team, these studies also brought to new developments in the fertility preservation program.
The thesis is structured as follows:
The first part reviews the basic knowledge on cryobiology and cryopreservation, the most important historical events related to the different cryopreservation techniques and the recent results obtained in ART thanks to the new cryopreservation methods.
The second part contains the original research work. This experimental chapter is divided into five sections according to the published or submitted papers and the unpublished data of additional results obtained in our clinical work.
The third part contains the discussion including the biological, therapeutical and ethical considerations of using vitrification in ART, the conclusions and the future perspectives.
The annexes ensure a full understanding of the written text.
LITTERATURE REVIEW
1.1 BASIC NOTES ON CRYOBIOLOGY AND CRYOPRESERVATION
Cryobiology is the science studying the effect of low temperatures on living organisms. Cryopreservation uses this knowledge to develop effective techniques keeping cells and tissues structurally and functionnally intact against- time (Pegg, 1987). Freezing itself is lethal for living systems. However, technologies using the cold to produce stable conditions preserving life have been developed thanks to the understanding of the mechanisms involved in the freezing process (Armitage, 1987; Pegg, 2002). Cryopreservation leads to a stop of the biological time by interferring with the cell physiology. When a cell is frozen and stored at low temperatures, usually at -196 °C in liquid nitrogen (LN2), all metabolic processes are blocked and cell vitality became virtually independent of time (Fuller and Paynter, 2004; Fowler and Toner, 2005; Pegg, 2007).
The possibility to cryopreserve both structures and functions of cells or tissues plays a key role in many areas of biology and medicine, including reproductive biology and ART.
1.1.1 Physics of cryopreservation
The cryopreservation process, first described by Mazur and Luyet around 1960, is linked to the mouvement of molecules through the plasma membrane (PM) according to their specific permeability, to the change of cell volume and to the formation of ice crystals in and outside the cells (Luyet and Rapatz, 1958;
Mazur, 1963; Luyet, 1969; Mazur, 1970; Mazur, 1984).
During cryopreservation, cells face temperatures below zero and, therefore, non- physiological conditions. First, water in its liquid state is a major element for the cells structure and function, but become usually lethal when solidification occurs. Actually, no metabolic reactions occur in aqueous systems at -196°C
because water is maintained at crystalline or glassy physical state: the time is stopped (McGee and Martin, 1962). The only reactions that can occur in frozen aqueous systems at very low temperature are photophysical events such as the formation of free radicals or the breacks in macromolecules induced by ionizing radiations or cosmic rays (Mazur, 1984). Therefore, the challenge for cells during cooling is not the storage at very low temperature, but rather the survival to the cooling steps. During cooling process, chemical and physical phenomena may affect the vitality and functionality of the cells. The main danger for cells during cryopreservation occurs during an intermediate critical temperature zone both during cooling and warming (Dobrinsky, 1996).
Cooling induces:
Reduction of the enzyme activity: temperature variations affect the enzymatic activity and the speed of the reactions they catalyze (Heber, 1968).
Reduction of the active transport: as a consequence of the reduced enzymatic activity, temperature variations affect the active transport (Willis et al., 1978).
Alterations of the cell membrane conformation: temperature variations affect the fluidity, and therefore the molecular structure of membranes (Canvin and Buhr, 1989).
From cooling to freezing, others types of damage can be observed:
Mechanical damages caused by the ice crystals: when water reaches the freezing point, the ice crystals formed can damage cell membranes (Gao and Critser, 2000).
Thermal and osmotic shocks: the thermal shock is related to the mechanical tension inducing irreversible membrane damages and drastic deformation of cellular components, while the osmotic shock is related to the increase of the electrolytes concentration due to the ice formation (Schwartz and Diller, 1983).
Two concepts have to be differentiated regarding cryopreservation:
- Supercooling: it refers to the capacity of a water-based solution to cool below its freezing point without changing state, and, therefore, without going from liquid state to solid state (ice). Latent heat is liberated only when crystallization occurs.
- Freezing point depression: it refers to the capacity of a solute dissolved in a water solution to lower the freezing point of the corresponding pure liquid.
Under spontaneous freezing conditions, solutions would crystalize at determined temperature by a process known as “homogeneous nucleation” (Debenedetti and Stanley, 2003). During supercooling process, the crystalization can be induced by touching the water-based solution with a freezed metal object (Rogerson and Cardoso, 2004). This process, called “heterogeneous nucleation”, also referred as “seeding”, is used in controlled cryopreservation protocols of cells and tissues. The formation of an ice nucleus is induced in the extracellular medium, at a distance from the cell in order to avoid damaging. This ice formation induces a rise in the osmotic gradient, leading to the movement of water out of the cytoplasm causing the cell dehydration. Various experiments have shown that the temperature of the manually-controlled formation of ice represents a key factor in the cell survival during freezing/thawing. Samples enucleated at a temperatures below -9 °C were more damaged than those for which enucleation took place at temperatures between -5 °C and -7 °C in order to avoid excessive supercooling effects (Mazur, 1984).
To be efficient, a cryopreservation protocol must be adapted to the sample. It is therefore extremely important to study the physiology of the different cells and tissues and their reaction to the low temperatures (Armitage, 1987). The following fundamental biological and physical factors must be considered:
Intracellular water content
Membrane permeability
Cell surface area/volume ratio.
The important factors available for preventing the formation of intracellular ice and for achieving osmotic and thermal balance during cryopreservation are:
Cooling and warming speeds: to reduce the quantity of resting water within the cells according to their size and type.
Cryopreservation solutions: to protect the cell during cooling and reducing the damages caused by the formation of ice crystals.
Dilution steps: to allow the optimal restoration of the intracellular micro- environment of the cell.
During cryopreservation, the cells are usually exposed to an increasing concentration of cryoprotectant solutions in order to facilitate the water come out of the cell and to prevent intracellular ice formation. To ensure the optimal cell dehydration, appropriate cooling speed is essentiel. Very low cooling speed would result in an excessive cell dehydration and in an high intracellular concentration of the solutes: cell death may occur in this hypertonic conditions.
On the other hand, when cooling speed is too high, suitable dehydration does not occur: the formation of intracellular ice crystals may induce lethal damages.
Therefore, an optimal cryopreservation process has to adapt the speed to be as high as possible to avoid the formation of intracellular ice and as low as possible to allow adequate cell dehydration (Mazur, 1963; Mazur, 1970).
During warming, the cryopreserved cells pass through the same steps as previously described in a reverse order. To limit the damages that may occur during the change from solid state to liquid, numerous experiments have shown that rapid warming speeds are generally better than slower ones (Meryman, 1974).
1.1.2 Cryopreservation media
In order to optimise cell survival, cryoprotectants (CPs) are added to the cryopreservation medium. CPs can be defined as substances able to protect cells, minimising damages during cooling and warming. These substances have a high solubility in water, a high viscosity and a low toxicity when used at the adequate concentration and temperature. Their permeability is closely related to specific chemical nature and protocol used for cryopreservation (Ashwood-Smith, 1987).
Cryoprotectants protective effect occurs through various mechanisms:
By modifying the intra- and extracellular environment, taking the place of water and decreasing the formation of ice crystals (Echlin et al., 1977).
By lowering the freezing point of the solution, allowing a better cell dehydration (Rall et al., 1978).
By acting directly on the cell membrane, interacting with polar head groups of phospholipids (Cabrita et al., 2001).
CPs can be divided up into three main groups (Fig. 1):
1) Permeable CPs with low molecular weight (<400 Da): methanol, ethylene glycol (EG), 1,2-propandiol (PrOH), dimethyl sulfoxide (DMSO), 2,3-butandiol, glycerol and other alcohols.
2) Non-permeable CPs with low molecular weight (>1.000 Da): galactose, glucose, sucrose, trehalose and other sugars.
3) Non-permeable CPs with high molecular weight (>50.000 Da):
polyvinylpyrrolidone (PVP), polyvinyl alcohol, hydroxyethyl starch, sodium hyaluronate and other polymers.
DMSO ethylene glycol
propanediol glycerol
sucrose PVP Fig. 1 Cryoprotectants commonly used: permeable CPs (DMSO, EG, PrOH, glycerol) and non-permeable CPs (sucrose, PVP).
Each group of CPs has different functions during the cooling and warming processes, although some specific cryoprotectant actions have not yet been entirely clarified.
The permeable CPs replace the water inside the cells during dehydratation process minimising the cell shrinking and the intracellular ice crystals formation.
The non-permeable CPs with low molecular weight induce cell dehydration before cooling by increasing the osmolarity of extracellular solution reducing the ice crystal formation.
These two groups must be combined to ensure the efficiency of the cryopreservation process.
The non-permeable CPs with high molecular weight reduce crystallization and modify the form and size of ice crystals in order to make them harmless.
In addition to these specific actions, CPs also have other functions in protecting the cells during cooling, such as the capacity to stabilise the intracellular structures, proteins and membranes. In general, it can be stated that alcohols protect the protein structures, while some non-permeable disaccharides stabilise the membrane structure. Different studies have been carried out to establish the exact function of each CP, suggesting that the most effective cryoprotective solution may be an appropriate combination of CPs (Ishimori et al., 1993; Van den Abbeel et al., 1994; Vincente and Garcia-Ximenez, 1994; Shaw et al., 1995;
Trad et al., 1999; Bafrani et al., 2003; Lieberman et al., 2003a).
The potential toxicity of CPs is related to their specific physical proprieties and varies according to the temperature and the exposure time. The use of CPs at low temperature and for a short exposure time minimizes the risk of cell damage due to their toxicity.
The first permeable CPs used to cryopreserve oocyte or embryo were EG, PrOH, DMSO and glycerol (Tab. 1). Most of the cryopreservation media combined one or two permeable CPs with one non-permeable CP as sucrose, trehalose or galactose (Miyamoto and Ishibashi, 1977; Schneider, 1986; Rayos et al., 1992;
Voelkel and Hu, 1992; Sommerfeld and Niemann, 1999).
The cryopreservation media are prepared at a stable pH of between 7.2 and 7.4 usually in Phosphate Buffered Saline (PBS), but other tissue culture media or simple physiological solution are also used. The media also include proteins such as serum and albumin, which stabilise the cell membranes.
Tab. 1 Different properties of the permeable CPs commonly used.
1.1.3 Cryopreservation techniques
Two main methods are used for cryopreservation procedures: equilibrium freezing (slow) and non-equilibrium freezing (vitrification). While the concentration of CPs and the cooling steps differ extremely between the two techniques, storage, warming and rehydration processes are similar (Karlsson, 2002).
1.1.3.1 Slow freezing
Slow freezing process is based on a controlled decrease of the temperature up to -196 °C, usually using a programmable freezing machine. In this procedure, the relatively low concentration of the CPs used limits the toxic and osmotic damages, but the formation of ice crystals occurs. Thanks to the seeding the growth of ice is controlled. Extra-cellular ice formation leads to a considerable increase in the concentration of ions, macro-molecules and other components, including the CPs, in the remaining fluid. The low cooling speed of this procedure allows the equilibration of the extra- and the intracellular fluids. This phenomenon gives to the method its name: “equilibrium freezing”.
Conventional slow freezing procedures involve different steps (Fig. 2):
exposure of the cells at room temperature to a low concentrations of CPs and then cooling at a speed of -2°C/min until reaching a temperature of -7 °C;
induction of crystallisation (seeding);
slow controlled freezing at a speed of -0.3°C/min until reaching a temperature of -35°C;
controlled freezing at a speed of -45°C/min until reaching a temperature of - 80°C;
immersion in LN2 and storage at -196 °C.
Fig. 2 Slow freezing: cooling/freezing steps.
The slow freezing procedures can be compared to some physiological protection mechanisms developed by different cold-tolerant animals. Among terrestrially hibernating vertebrates, freeze tolerance has been documented for amphibian and reptile species: frogs, one salamander, and hatchlings of the painted turtle (Storey, 1990). The key factor allowing these animals to survive at low temperatures is the production of a large quantity of CPs with low molecular weight, such as glucose and glycerol, and a variety of other alcohols and sugars.
These compounds prevent the formation of ice crystals during freezing by lowering the freezing point. In these animals, when intracellular fluids begin to reach a balance, specific ice enucleating proteins inducing extra-organ sequestration of ice crystals are produced thanks to specific freeze-induced gene responses at a determined temperature below zero. Additionally, these cold- tolerant animals produce high molecular weight compounds called “anti-freeze proteins” which inhibit recrystallization and stabilize the cell membrane (Carpenter and Hansen, 1992).
1.1.3.2 Vitrification
The term “vitrification” comes from the Latin word “vitrum”. It refers to a physical process whereby a solution goes from a liquid state to a vitreous state at low temperatures, by-passing the formation of ice crystals. Classical terms used in slow freezing techniques as “freezing” and “thawing” cannot be applied for vitrification. During vitrification, the change in state of the solution is obtained without going through the solid phase, but rather directly from liquid to vitreous state (Fig. 3). These phenomena are then called “cooling” and “warming”.
Fig. 3 Different aspects of vitrified (A) and slow freezed solutions (B) (Amorin et al., 2011).
The use of high concentrations of CPs, associated with an ultra-rapid lowering of the temperature, lead to an important dehydration of the cells with a partial
intracellular diffusion of cryoprotectants. These phenomena prevent ice crystals formation. Higher is the cooling speed, lower is the concentration of CPs required. This characteristic gives to the method its name: “non-equilibrium freezing” (Fig. 4).
Fig. 4 Comparison of the oocyte aspect during slow freezing and vitrification methods (Clinique de Fertilité - Hôpital Erasme).
In theory, the extreme increase in the cooling speed up to approximately 107
°C/sec would allow vitrification even in pure water (Fahy et al., 1984). With a low concentration of any CPs (1.5 M), vitrification can also be obtained in classical devices but with a cooling speed of around 15.000-20.000 °C/min.
Limits of technology and high costs of sophisticated equipments reduce the feasibility of these cooling speeds. In order to vitrify a solution at a cooling speed of around 2.000 °C/min, obtained by direct immersion in LN2, very high concentrations of CPs (5-7 M) are required. With vitrification, the formation of ice crystals is avoided thanks to the rapid transition through the critical temperature zone. Vitrification procedure protects the cell against damages due to the cold, but not against osmotic and toxic damages due to the high concentrations of CPs. Therefore, efficient vitrification require high cooling speeds in order to reduce the concentration of CPs. As suggested by the
following equation, the simplest method to increase the cooling speed is to reduce the volume of solution.
Cooling and warming rates x Viscosity _________________________________
Volume
Many studies also attempted to find more permeable and less toxic cryoprotectants: as result, EG became a standard component used in most vitrification protocols. As most of CPs are toxic at the concentration needed to obtain vitrification, many authors proposed combined CPs media. The use of two CPs allows to reduce the concentration of each component, decreasing the specific toxicity of each CP. Permeable CPs have also been tested in various combinations (Ali and Shelton, 1993; Kasai and Mukaida, 2004).
Mono- and disaccharides such as sucrose, trehalose, glucose and galactose are the most commonly used non-permeable CPs (Kasai, 1997; Wright et al., 2004).
Sucrose has the advantage to have a low toxic effect at low temperatures (Rall, 1987; Kasai et al., 1992).
To reduce toxicity of the vitrification solutions, different polymers as PVP, polyethylene glycol, Ficoll, dextran and polyvinyl alcohol, have also been experimented to replace permeable CPs without success (Shaw et al., 1997;
Asada et al., 2002).
To limit the exposure to high CPs concentration, many authors suggested a two- step protocol (Papis et al., 2000; Kuwayama et al., 2005a; Kuwayama et al., 2005b).
Conventional vitrification procedure involves three steps:
initial phase during which the cells are exposed to solutions containing low concentrations of CPs (20-50% of the final solution) for a short period (~ 2-10 min depending on the incubation temperature);
short exposure to the final vitrification solution containing high concentration of CPs (~ 1 min);
immersion in LN2 and storage at -196 °C (cooling speed ranged from ~ 2.500 to >20.000 °C/min highly depending on device used).
The respect of time intervals for each step in the vitrification procedure is essential to ensure optimal results: too long or too short exposition times can have a major impact on survival rates (Vajta et al., 2009).
1.1.4 Thawing and warming procedures
The procedure is carried out by incubate the sample into solutions, with decreasing concentrations of sucrose, in order to balance the osmotic stress and prevent swelling damages.
If cells are slowly warmed, small ice crystals fuse to become larger crystals, more stable at a given temperature. Therefore, warming process is usually performed at high speed (Mazur, 1984; Mazur, 1990; Karlsson, 2001).
Recrystallization is the phenomenon especially observed at thawing of cryopreserved cells using slow freezing method. Moreover, if cells are slowly warmed, the toxicity of the solution can be high because the cells are in contact with CPs. Toxicity is the phenomenon observed especially at warming of vitrified cells.
Conventional thawing steps after slow freezing include:
controlled thawing at a high speed of around 250 °C/min usually in air for 30-40 sec and then in water at 25/30 °C;
removal of the permeable CPs by sequential steps in hypertonic solutions before placing in culture.
Conventional warming steps after vitrification include:
warming at very high speed usually in first warming solution at 37 °C (warming speed ranged from ~ 1.300 to >40.000 °C/min highly depending on devices used);
removal of the permeable CPs by sequential steps in hypertonic solutions before placing in culture.
1.1.5 Cryopreservation devices
All the cryopreservation devices are resistant to the low temperatures and pressures of liquid nitrogen. The cryopreservation storage devices have however to be adapted to each specific sample according to the procedure.
Slow freezing: The straws usually used for slow freezing of oocytes and embryos (0.3 mL straws) are filled by aspiration and cells are often placed between two air bubbles (Fig. 5). In the loading procedure, as the liquid sample comes in contact the cotton filter, the filling is blocked. Directly after loading, the straws are sealed and placed in the programmable freezing machine and stored in LN2at the end of the program.
Fig. 5 Devices used for slow freezing (0.3ml straws) (Cryobiosystem ®).
Vitrification: Most of vitrification methods for oocytes and embryos initially used the traditional devices (0.3 ml straws). The loading volume of these devices however limits the cooling/warming speeds (~2.500 °C/min and ~1.300 °C/min respectively). To increase the cooling speed, the first device adapted to
vitrification was the electronic microscope grid allowing direct contact of the sample with LN2 (Steponkus et al., 1990). Later, the Open Pulled Straws (OPS), was based on a simple, easy-to-develop idea: by reducing the diameter of a normal insemination straw, the volume of solution containing the sample could also be reduced (Vaja et al., 1997). The principles of the OPS have been used to create many new devices exploiting the principle of minimum volumes of vitrification solution (loading volume ranged from ~ 1-2 to <0.01 µl depending on device used) to obtain very high cooling speed (>20.000 °C/min) (Lane et al., 1999; Kuwayama and Kato, 2000; Chian et al., 2005; Kuwayama et al., 2005a) (Fig. 6).
The common characteristic of most of these adapted devices is the direct contact of the vitrification solution containing the samples and the LN2 and, therefore, the risk of microbiological contamination. In a study of Bielanski et al., 21.3%
of vitrified embryos in direct contact with LN2 previously contaminated by bovine virus, were infected. On the contrary, all embryos vitrified in sealed straws were negative to tests for all viruses (Bielanski et al., 2000). The danger of transmitting diseases mediated by LN2 is thus real, even if it is not only restricted to the contact with LN2. At present, no clinical cases of the transmission of diseases mediated by LN2 following the transfer of frozen embryos have been reported in human ART. Several approaches have been described to minimise these risks including the use of filtered sterile LN2. In the case of open systems such as the OPS, aseptic condition can be obtained (OPS have been placed into another 0.5 ml straw) prior to immersion in LN2. Although the cooling speed is lower, the risk of cross-contamination is reduced when closed-systems are used compared to open-systems (Kuleshova and Shaw, 2000; Isachenko et al., 2005a; Isachenko et al., 2005b). Recently, an aseptic vitrification device, the High Security Vitrification kit (VHS kit), have been developed. The VHS kit is considered as safe and efficient device: a microdroplet (<1 µl) of cryoprotectant containing oocytes or embryos is placed
in a gutter before inserting it in a ministraw. The device, which is made of ionomeric resin, is heat-sealed using a special welder which ensures a leak-proof seal before immersion in LN2 (Camus et al., 2006) (Fig. 6). Recent study certified the clinical efficiency of this closed system in clinical work (Stoop et al., 2012).
a b c d e
f g
Fig. 6 Different devices commonly used for vitrification. Open systems: a) electronic microscope grid; b) open pulled straw; c) cryoloop; d) cryotop; e) cryoleaf; and Closed systems: f) cryotip; and g) high security vitrification kit.
(www.reproduction-online.org)
1.1.6 Pratical advantages of vitrification
Both cryopreservation methods have theoretical advantages and limitations.
However, recent studies reported practical advantages of vitrification technique compared to conventional slow freezing (Pegg, 2007; Borini et al., 2008; Leibo, 2008; Vajta et al., 2009).
Vitrification is a very simple procedure and can be acquired after a relatively short training. However, operators have to work rapidly because of the very short time lapse accepted especially during the last step of the procedure (incubation of cells in the vitrification solution and loading before immersion in LN2).
Vitrification is certainly a faster procedure compared to slow freezing.
Actually, the vitrification of a single device takes just a few minutes but considering that each device must be cooled individually, although this time increases according to the total number of cells that have to be stored. In contrast, conventional slow freezing program takes almost 3 hours but all the devices can be simultaneously freezed. However, the timing for vitrification can be optimized as parallel work is possible allowing the handling of more samples at the same time. Moreover, considering that programmable freezing machine is not needed, organization of the routine work is more flexible with vitrification method compared to slow freezing.
Vitrification allows the operator to observe and control the cells during the procedure. Viability of cells can be confirmed when they contract in the first and the second vitrification solution. Moreover, during the warming procedure the presence of contracted cells can be a useful marker for cell survival.
Vitrification requires a stereomicroscope and a simple container for LN2 (0.5 l), while the slow freezing requires an expensive programmable freezing machine and large amount of LN2 (~15-20 l). However, this advantage is moderated because of the high cost of the solutions and of the devices used for vitrification.
1.2 CRYOPRESERVATION OF GAMETES, EMBRYOS AND GONADAL TISSUES
More than 65 years have passed since the publication of the pioneer biologist Basil J. Luyet (1897-1974) “Life and Death at Low Temperature”. Luyet observed that many organisms may be irrevocably damaged by physical and chemical changes during cryopreservation process, but in some cases, he was able to restore normal function after warming. Luyet had already suggested that ultra-rapid cooling and warming could prevent freezing injury (Luyet, 1949).
At present, it could be expected that a clear and functional understanding of freezing injury enables efficient cryopreservation of almost everything.
Unfortunately, even if research to understand and develop new approaches continues to push the actual limits of success, the ultimate goals of applied cryobiology have still to be achieved.
1.2.1 Spermatozoa
Spermatozoa are small cells, quite resistant to cryopreservation as they contain less water compared to other cells. They are suspended in the seminal plasma, a high viscosity solution reducing direct contact between spermatozoa and ice during cryopreservation (Morris, 2002).
The spermatozoa is therefore the first reproductive cell that has been successfully cryopreserved. More than two centuries ago, Spallanzani first reported maintenance of human spermatoza motility after exposure to low temperatures (Spallanzani, 1776). In the middle of last century, slow freezing procedure and cryoprotectants, such as glycerol, were introduced for sperm cryopreservation in zootechnics (Polge et al., 1949). Few years later, normal embryo development was obtained after insemination using frozen/thawed semen previously frozen on dry ice (Sherman and Bunge, 1953). Finally, Perloff
et al. obtained the first pregnancy from human semen frozen in LN2 vapours using glycerol as a cryoprotectant (Perloff et al., 1964) (annex 1). These advances authorized the development of sperm banks all over the western world in the 70ties. Human spermatozoa storage became an incontrovertible tool for the clinical management of infertility and for autologous and donor sperm banking. At present, cryopreservation of semen is a procedure that keeps intact the reproductive potential of the spermatozoa for an indefinite storage time.
Although major improvements have been performed in sperm cryopreservation these last decades, almost 30% of cells still die during cryopreservation procedure. There are many unresolved technical issues and the optimal conditions for human sperm cryopreservation need to be further investigated.
Until recently, vitrification of human spermatozoa was unsuccessful, possibly due to low tolerance of spermatozoa to permeable agents. Thanks to the large amounts of proteins and sugars contained in the intracellular matrix of human spermatozoa, they have been however successfully cryopreserved using only non-permeable CPs (Koshimoto et al., 2000). Successful vitrification procedures of human spermatozoa were firstly reported by Nawroth and Isaschenko (Nawroth et al., 2002; Isachenko et al., 2003). Many studies confirmed that vitrification can effectively protect spermatozoa from cryo-injuries avoiding at the same time toxic effects of permeable CPs (Isachenko et al., 2004a;
Isachenko et al., 2004b; Isachenko et al., 2008). At present, new protocols for sperm vitrification use the combination of extremely high rates of cooling/warming and vitrification media containing proteins and polysaccharides (Morris et al., 2012). Vitrified spermatozoa were successfully used for intracytoplasmic sperm injection (ICSI) with clinical pregnancy resulting in healthy deliveries (Isachenko et al., 2012). A first live birth was also reported following intrauterine insemination (IUI) of vitrified semen (Sanchez et al., 2012).
1.2.2 Oocytes
1.2.2.1 Biological aspects of Oocyte Cryopreservation
The issues of oocyte cryopreservation are related to the specific characteristics of the oocytes. The mature oocyte is the larger cell in mammals with a diameter ranging from 70 to 120 µm according to the species. It is surrounded by a zona pellucida (ZP) and by many layers of granulosa cells, which together form the cumulus-oocyte complex (OCC) (Bjersing, 1982). During OCC development, immature oocyte within the follicle is maintained at the prophase of first meiotic division (germinal vesicle stage, GV) and is characterised by a large diploid nucleus, a dense layer of actin filaments arranged below the oolemma and organelles as mitochondria, endoplasmic reticulum, Golgi apparatus, dotted throughout the ooplasm. The Golgi apparatus of the oocyte gives rise to cortical granules, which are distributed at random in the ooplasm. During the first meiotic division (reductional division), the nuclear membrane disintegrates into vesicles and homologous chromosomes, arranged on the spindle (metaphase I, MI), separate producing a haploid cell. However, the oocyte at the end of the first division contains sister chromatids which are finally split at the end of the second meiotic division (equational division). The ovulated mature oocyte is maintained at the methaphase of second meiotic division (methaphase II, MII) until fertilization and is characterised by the presence of the polar body (PB) and by a spindle oriented perpendicularly to the cortex at the periphery. The spindle is formed by microtubles joined at two poles and the chromosomes are aligned along the metaphasic plate (Moor and Warnes, 1979; Thibault et al., 1987). At MII stage, the cortical granules are in the peripheral area of the ooplasm. They are ready to undergo exocytosis at the time of fertilization to avoid polyspermic penetration of spermatozoa (Cran, 1989). A “good quality” mature oocyte is identified by a perfectly spherical shape, a regular ZP, an intact PB, and a
homogeneous cytoplasm without inclusions. The oocyte is embedded in a well- expanded cumulus mass and surrounded by a radiant array of corona cell layers (Hyttel et al., 1986). It is still controversial whether distinct oocyte morphological characteristics can predict its developmental competence (Coticchio et al., 2004).
Fig. 1 Human mature oocyte morphology (Rosati et al., 1993).
Considerable morphological and functional damages can be induced by the low temperatures and the exposure to CPs during oocytes cryopreservation (Son et al., 1996; Zeron et al., 1999; Paynter, 2005). In human oocytes, changes have been observed in the glycoprotein structure of the ZP due to the premature release of cortical granules, which can be responsible for a decreased fertilisation rate (Mandelbaum, 1991; Ghetler et al., 2006). The use of the ICSI had however overcome this ZP hardening. Other damages include a decrease of the plasma membrane permeability, an extended disorganisation of the ooplasm, changes in the microfilaments and microtubules of the spindle and the presence of vacuoles in the peripheral area (Sathananthan et al., 1987; Chen et al., 2003;
Diez et al., 2005; Valojerdi and Salehnia, 2005; Bromfield et al., 2009).
Moreover, an irreversible loss of mitochondrial polarity potentially affect the
oocytes developmental competence, due to the alteration of adenosine- triphosphate (ATP) levels and cytoplasm ability to regulate intracellular calcium (Jones et al., 2004).
1.2.2.2 History of Oocyte Cryopreservation
The first experiments to cryopreserve oocytes was performed in the middle of last century. In 1957, it was shown that mouse oocytes were able to survive cooling at -5°C in a medium containing glycerol (Lin et al., 1957). The first mouse borned after in vitro fertilization of cryopreserved oocytes was however reported 20 years later (Parkening et al., 1976). The first live birth in humans after in vitro fertilization of cryopreserved oocytes, reported in 1986, used the slow freezing method (Chen, 1986). From the late 1980s, attempts to obtain better survival rates were performed by freezing immature oocytes at the germinal vesicle stage (Mandelbaum et al., 1988; Candy et al., 1994; Toth et al., 1994), but some studies showed that at warming development competence was compromised (Schroeder et al., 1990; Van der Elst et al., 1993). Tucker et al.
reported pregnancy after warming of cryopreserved human immature oocytes and subsequent in vitro maturation (Tucker et al., 1998). The clinical use of vitrification for the cryopreservation of human oocytes only began in the late 1990s. The first human live birth following oocyte vitrification was reported in 1999 (Kuleshova et al., 1999) (annex 1).
1.2.2.3 Human Oocytes Cryopreservation by Slow freezing and Vitrification
In 1995, Gook et al. have tested a slow freezing protocol based on a solution containing 1.5 mol/l of PrOH and 0.1 mol/l of sucrose on human oocytes. They showed that cleaved embryos can be obtained after thawing using this procedure (Gook et al., 1995). Using the same protocol, Porcu et al. obtained a live birth (Porcu et al., 1997), while other studies reported a poor survival rate (Kazem et al., 1995; Borini et al., 2004). The efficiency of this cryopreservation protocol was improved by Fabbri et al. using high concentrations of sucrose in the freezing solution (survival rate of 58% with sucrose at 0.2 mol/l and 83% with sucrose at 0.3 mol/l) (Fabbri et al., 2001). Other authors also obtained high survival, fertilisation and embryonic cleavage rates, but reported unsatisfactory results in terms of pregnancy using the concentration of 0.3 mol/l of sucrose (Chamayou et al., 2006; Levi Setti et al., 2006). In order to obtain better clinical results, another protocol was then proposed to reduce the osmotic stress including two separate concentrations of sucrose, one of 0.2 mol/l for freezing, and one of 0.3 mol/l for thawing. The results obtained showed 75.9%, 76.2%
and 93.8% of survival, fertilisation and cleavage rates respectively out of a total of 403 oocytes. The pregnancy rate per thawing cycle was 18.9%, resulting in 13.5% of implantation rate (Bianchi et al., 2007). Different authors confirmed the benefits of this modified protocol in terms of survival, pregnancy and implantation rates, especially when oocyte are cryopreserved within 2 hours of harvesting (Parmegiani et al., 2008).
An alternative protocol for oocyte slow freezing was reported by Stachecki et al.
who replaced sodium with the less toxic choline chloride for freezing mouse oocytes (Stachecki et al., 1998). Sodium salts are the major components of all media including those used for cryopreservation of mammalian embryos. High intracellular sodium concentrations achieved during freezing process may affect normal cell function. The choline do not cross the cell membrane, therefore it
does not contribute to the intracellular cation load. In addition, choline may also have a possible membrane-protective effect. Quintans et al. reported 63% of oocyte survival rate, 59% of fertilization rate and a 25% of implantation rate using this protocol. Six clinical pregnancies were obtained resulting in the birth of two babies (Quintans et al., 2002). However, both studies referred to a very small number of cases. In 2008, Cobo et al. evaluated the impact of different cryopreservation protocols on the survival and on the repolymerization of MII spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved using four different protocols included Na+ depleted-choline replaced media.
Survival rates and spindle configurations observed did not confirmed that choline has a particular membrane-protective or cell structure-protective effect during human oocyte cryopreservation (Cobo et al., 2008a).
The use of vitrification for the cryopreservation of human oocytes rapidly reported a higher oocytes survival and developmental rates compared to slow freezing (Kuleshova and Lopata, 2002). Yoon et al. observed 21.4% and 6.4%
of pregnancy and implantation rates respectively and all pregnancies resulted in the delivery of healthy babies (Yoon et al., 2003). Kuwayama et al., using a new vitrification method, obtained extremely high oocyte survival, fertilization, and embryo development rates (91%, 90% and 62%, respectively) with 43% of pregnancy rate (Kuwayama et al., 2005a).
In the first randomized study comparing fresh and vitrified oocytes in egg donation program, Cobo et al. reported no difference in fertilization, cleavage and blastocyst development rates between vitrified and fresh oocytes. Embryo morphologies on day 3 and on day 5-6 were similar for vitrification and fresh oocyte groups. Clinical pregnancy and implantation rates, evaluated after 23 embryo transfers, were 47.8% and 40.8% respectively (Cobo et al., 2008b). In another study, published by Rienzi et al., oocytes of IVF patients were randomised in order to be injected with sperm or immediately vitrified (Rienzi et
al., 2010). Results showed that embryo development was not affected by the vitrification procedure and embryo quality was comparable with those obtained from fresh oocytes. The author obtained 30% of clinical pregnancy and 17% of implantation rates, that are comparable with fresh cycles in the infertile population.
In 2011, Cobo and Diaz published a systematic review and meta-analysis of randomized controlled trials (Cobo and Diaz, 2011). Five eligible studies were finally included involving 4.282 vitrified oocytes, 3.524 fresh oocytes, and 361 slow-frozen oocytes. The oocyte survival and the fertilization rates were higher in vitrified compared to slow-frozen oocytes. Vitrification also resulted in a higher embryo cleavage and top-quality embryo rates compared to slow freezing.
The fertilization, embryo cleavage, top-quality embryo and ongoing pregnancy rates were similar in the vitrification and fresh oocytes groups.
In 2009, Noyes et al. reported the birth of around 900 babies without any significant risk of malformations as consequence of the cryopreservation procedure using both techniques (Noyes et al., 2009). A meta-analysis of literature (from 1984 to 2009) regarding the children outcome after oocyte cryopreservation also revealed reassuring results for both slow freezing and oocyte vitrification (Wennerholm et al., 2009). However, controlled long-term follow-up prospective studies on babies born from cryopreserved oocytes is requiered.
1.2.2.4 Assessment of Oocyte quality after warming procedure
The first criterion used to establish viability of oocytes at warming is the morphological assessment allowing the observation of visible degeneration or major cytoplasm abnormalities. However, oocyte viability is not necessarily synonymous of developmental competence (Coticchio et al., 2004).
The spindle apparatus, observed through a polarised microscope, can be used as non-invasive assessment of oocyte quality (Wang et al., 2001). An intact polymerisation after warming may be used as an indicator of the potential developmental competence of the cryopreserved oocyte. Recently, the direct correlation between the fertilization rate and the intact spindle has been confirmed (Cao and Chian., 2009). Besides the visualization of the spindle, polarised microscope also enables the visualization of zona pellucida birefringence (Nottola et al., 2008).
In research studies, other non-invasive and invasive methods to assess oocyte quality have been developped, but they are not easily applicable in clinical practice. These non-invasive assessements include the evaluation before cryopreservation and after warming of the baseline respiration rate correlated with the mitochondrial oxidative capacity and the ability of an oocyte to produce ATP (Vincent et al., 1990). Another non-invasive assessement is the evaluation of the apoptotic gene expression and the incidence of chromosomal abnormalities in cryopreserved cumulus cells for cryopreserved cumulus-oocyte complex (Kumamoto et al., 2005). On the other hand, invasive assessments of oocyte quality, focusing attention on morphological and functional alterations caused by cryopreservation procedures, used a variety of biochemical, molecular and microscopic methods (Larman et al., 2007; Nottola et al., 2008; Succu et al., 2008; Coticchio et al., 2009).
1.2.3 Embryos
1.2.3.1 Biological aspects of Embryos Cryopreservation
Fertilization induces a series of events that have to be considered to successfully cryopreserve embryos. At the pronuclear stage (PN), the two pronuclei are
visible in the cell (zygote). Following the formation of the pronuclei, the permeability of the oocyte membrane to CPs increases and the chromatin organization is modified resulting in a decrease of the embryo sensibility to cooling process (Jackowski et al., 1980; Stricker, 2006 ; Familiari et al., 2008).
Another important factor is the composition of the embryo cell membranes as fluid membranes are generally less damage than rigid membranes during cooling. The increase of the cholesterol content in the membrane appears to improve cryotolerance (Zeron et al., 2001; Seidel, 2006). From the pronuclear stage, rapid cell division started. Embryo cleavage differs from other forms of cell division as it increases the number of cells without increasing the total mass.
Fig. 2 Human Embryos at different developmental stages (www.infertility.net).
The development of the zygote into cleaved embryo thus reduces the surface area/volume ratio of each cell, decreasing the osmotic stress occurring during cryopreservation. At 6-8 cell stage (day 3), the embryonic genome is activated (Braude et al., 1988). At day 4, the blastomeres form a compact mass called morula and the cleavage stage ends with the formation of the blastocele cavity.
After approximately 120 hours (day 5) of development, the healthy human
embryo reaching the blastocyst stage is composed by 50-150 cells: about 20- 30% constitutes the inner cell mass (ICM) and the remainder constitutes the trophectoderm (TE) (Fig. 2). At the blastocyst stage, the embryo is ready for implantation. The future placenta (TE) and embryonic pole (ICM) are already clearly visible (Tesarik, 1988).
Embryos may be cryopreserved at all development stages. The embryonic stage selected for cryopreservation depends on a series of ethical, clinical and practical factors. Cryopreservation of embryos at the pronuclear stage is mainly used in ethical or legal restrictions. Cryopreservation of embryos at cleavage stage versus blastocyst stage has clinical advantages but also limitations. The blastocyst culture allows better selection of embryos leading to a higher implantation rate (Blake et al., 2007). However, the culture of embryo until blastocyst may fail and the culture of embryos until the day 5 requires time and additional work (Alper et al., 2001). As the cleavage stage embryos showed good implantation potential for fresh transfer and high survival rate after cryopreservation based on established morphological criteria, cryopreservation of embryo at cleavage stage was used in most of the clinical programs (Nikolettos and Al-Hasani, 2000).
All factors affecting the survival during cryopreseservation procedures previously described can be apply in embryo, with some specificity regarding the stage of development.
1.2.3.2 History of Embryos Cryopreservation
In 1972 Whittingham et al. obtained the first live birth from mouse embryos cryopreserved in LN2 using the slow freezing method (Whittingham et al., 1972). The first pregnancy from cryopreserved bovine embryos was reported in 1973 (Wilmut and Rowson, 1973). In the field of human assisted reproduction,
the first non-term pregnancy was obtained from cryopreserved embryos in 1983 (Trounson and Mohr, 1983) and the first deliveries in 1984 (Zeilmaker et al., 1984). Starting from the late 1980s, the cryopreservation of human embryos became a routine procedure in ART (Zeilmaker, 1995).
Embryos have been successfully cryopreserved at zygote (Cohen et al., 1988), cleavage (Lassalle et al., 1985) and blastocyst (Fehilly et al., 1985) stages, using various cryopreservation protocols with either DMSO (Mohr and Trounson, 1985), PrOH (Lassalle et al., 1985) or glycerol (Cohen et al., 1985) as cryoprotectant agents. In 2001, successful pregnancies and deliveries after vitrification of human embryos have been reported (El-Danasouri and Selman, 2001) (annex 1).
1.2.3.3 Human Embryo Cryopreservation at different stages of development by slow freezing and vitrification
a) Pronuclear stage embryos
Even if the sizes of zygote and oocyte are similar, the advantages of cryopreservation at pronuclear stage include the lack of the delicate meiotic spindle, the less organized nuclear material and the intact nuclear membrane.
The timing of cryopreservation for PN stage embryos is crucial, as the process must be initiated before reaching syngamy. The PN stage embryo is able to better resist the cooling and warming conditions and the osmotic fluctuations also thanks to the significant membrane permeability changes that occur post- fertilization. On the other hand, cryopreservation of zygotes limits the embryo selection for fresh transfer. Therefore, this option is indicated often when the implantation is compromised during the fresh cycle as in the case of ovarian hyperstimulation syndrome (OHSS) (Nikolettos and Al-Hasani, 2000).
Marrs et al. reported the outcome of 1408 pronuclear embryos slow-frozen in PrOH/sucrose: 78% survived leading to a 26% pregnancy rate per transfer
(Marrs et al., 2004). Conventional cryopreservation of PN stage embryos has been well established in countries such as Germany where cryopreservation of later stage was not allowed by law or by ethic (Al-Hasani et al., 1996). Usually, PN stage embryos are cryopreserved using slow freezing protocols in medium containing PrOH (Testart et al., 1986 ; Cohen et al., 1988).
Recently, vitrification has been successfully used for cryopreservation of PN stage embryos. High survival rates (~ 90%) and clinical pregnancy rate close to 30% with an implantation rate of 17% were reported (Jelinkova et al., 2002;
Liebermann et al., 2002; Al-Hasani et al., 2007).
b) Cleavage stage embryos
The advantage of cryopreservation at cleavage stage include the high cell surface area/volume ratio and the presence of many cells potentially allowing the embryo recovery even if some cells have been destroyed during cryopreservation procedures. Nevertheless, the percent of blastomere loss at warming is an important factor for clinical outcomes. Actually, different authors showed a correlation between the implantation potential and the blastomere loss after warming (Van den Abbeel et al., 1997; Edgar et al., 2004). Obviously, it is important to select embryos before cryopreservation. The degree of fragmentation and the irregular size of blastomeres have a negative effect on cryopreservation results and therefore on implantation (Hartshorne and Edwards, 1991; Edgar et al., 2000). At the present, cryopreservation of embryos at the cleavage stage is used in most of IVF programs in order to enhance overall outcome from ART.
Different slow freezing protocols have been proposed. DMSO and PrOH have been mostly used as permeable CPs (Camus et al., 1989; Mandelbum et al., 199). In a large study analysing results from 132 centres in 18 different
countries, the survival rate of 18.322 thawed embryos reached 56.6% with a clinical pregnancy rate of 11.5% (Van Steirteghem and Van den Abbeel, 1988).
Over the years, implantation rate of slow-frozen embryos increased using original protocols as the removal of degenerated embryo blastomeres after thawing (Nagy et al., 2005; Zheng et al., 2008).
Successful human cleavage embryo vitrification has been recently reported (El-Danasouri and Selman, 2001). Liebermann and Tucker showed post- warming survival rates ranged from 84 to 90% using the cryoloop or the hemi- straw and 33.8% of the survived embryos became compacted (Liebermann and Tucker, 2002).
In 2008, Balaban et al. provided a large prospective randomized study on 466 embryos from 120 patients, comparing slow freezing and vitrification of day 3 human embryos. The overall survival rate was significantly higher after vitrification than after slow freezing (94.8% vs 88.7%). Developmental rate to the blastocyst stage was also significantly higher after vitrification than after slow freezing (60.3% vs 49.5%). Excellent clinical pregnancy rate (30%) following the warming and transfer of vitrified cleavage stage embryos was also reported. In this study, the vitrification protocol, using EG and PrOH as cryoprotectants, had less impact on the embryo metabolism when compared to slow freezing (Balaban et al., 2008).
In a meta-analysis (from 1984 to 2006), identyfing four eligible studies, including 3 randomized controlled trials, Loutradi et al. compared vitrification and slow freezing protocols for 8.824 human embryos. The survival rate of cleavage stage embryos was significantly higher after vitrification as compared with slow freezing (Loutradi et al., 2008).
These results were confirmed by Kolibianikis et al., but the authors didn’t report significant difference in clinical pregnancy rates per transfer between the two cryopreservation methods (Kolibianikis et al., 2009).
In contrast, the recent analysis of AbdeHafez et al. in 2010 showed a significantly higher clinical pregnancy, ongoing pregnancy and implantation rates after vitrification compared to slow freezing. The CPRs for vitrified embryos at cleavage stage in recent studies ranged from 16 to 44% (AbdeHafez et al., 2010).
c) Blastocyst stage embryos
The advantages of cryopreservation at blastocyst stage is the extremely favourable cell surface area/volume ratio and the very high number of cells. In the case of expanded blastocysts, damage could be linked to an increase in the metabolic activity of the blastomeres and an increase in the blastocoele expansion. Any of these factors could induce inadequate dehydratation, ice crystal formation, and cryodamage (Kader et al., 2009). Recently, the progress in blastocyst culture techniques and the high pregnancy rate obtained led to an increase of cryopreservation at blastocyst stage in IVF clinical programs (Sills and Palermo, 2010).
For embryos at the blastocyst stage, most of the slow freezing protocols involve the use of solutions with subsequent steps at increasing cryoprotectant concentrations of glycerol. At the beginning, this technique yielded poor results with survival rates around 50-60% (Hartshorne et al., 1991; Menezo et al., 1992). Thanks to the improved cryopreservation media and protocols, successful slow freezing and thawing of blastocysts with high survival rates (~ 70-90%) has been reported (Behr et al., 2002; Veeck et al., 2004; Desai and Goldfarb, 2005; Van den Abbeel et al., 2005).
Many authors reported pregnancies using the vitrification technique for embryo cryopreservation at blastocyst stage (Liebermann et al., 2003b;
Vanderzwalmen et al., 2003). In 2003, Mukaida et al. obtained very high survival rates using vitrification: out of 725 vitrified blastocysts, 80.4% were intact at warming and the pregnancy rate reached to 37% (Mukaida et al., 2003).
Some experiments including a reduction of the blastocyst cavity using a laser obtained 90% of survival rates and 48% of pregnancy rates (Son et al. 2003).
In 2005, Stehlik et al. published a retrospective study of 86 thawed blastocyst transfer cycles, comparing slow freezing to vitrification for cryopreservation of day 5 and day 6 human blastocysts. Survival rate of slow-frozen blastocysts was 83.1%, resulting in a pregnancy rate of 16.7%, while survival rate of vitrified blastocysts was 100%, resulting in a pregnancy rate of 50% (Stehlik et al., 2005). In 2009, Liebermann published results obtained on 8449 vitrified blastocysts from 2453 patients. The survival rate was 96.3%, the implantation and a clinical pregnancy rates were 29.4% and 42.8% respectively (Liebermann, 2009). In a recent study, Zhu et al. obtained in 136 warming cycles of vitrified blastocyst an implantation rate and a clinical pregnancy rate of 37.0% and 55.1% (Zhu et al., 2011). In 2011, Van Landuyt et al. reported successful cryopreservation of blastocysts from the early cavitating up to expanded blastocyst stages using a closed high security devices (Van Landuyt et al., 2011).
The available data on live births from warmed embryos have not shown any significant difference in the risk of malformations. Regarding the follow-up, several studies on births obtained using cryopreserved embryos confirmed the safety of the procedure whatever the cryopreservation procedure. Congenital abnormalities rate was similar with natural births (Wennerholm, 2000;
Takahashi et al., 2005; Wennerholm, 2009; Wikland et al., 2010).
