RBMOnline - Vol 19. No 1. 2009 126-140 Reproductive BioMedicine Online; www.rbmonline.com/Article/3857 on web 8 May 2009

Review Current trends, biological foundations and future prospects of oocyte and embryo cryopreservation Ashok Agarwal is a Professor in the Lerner College of Medicine at Case Western Reserve University and the Director of the Center for Reproductive Medicine, and the Clinical Andrology Laboratory at The Cleveland Clinic, Cleveland, Ohio, USA. He has published over 400 scientific articles, reviews and book chapters in different areas of andrology, male/ female infertility and fertility preservation. His research program is known internationally for its focus on disease-oriented cutting edge research in the field of human reproduction. His team has presented over 700 papers at national and international meetings and more than 150 scientists, clinicians and biologists have received their training in his laboratory.

Dr Ashok Agarwal Alex C Varghese1, Zsolt Peter Nagy2, Ashok Agarwal1,3 1Center for Reproductive Medicine, Glickman Urological and Kidney Institute and Department of Ob-Gyn and Women’s Health Institute, Cleveland Clinic, Cleveland, Ohio, USA; 2Reproductive Biology Associates, Atlanta, Georgia, USA 3Correspondence: e-mail: [email protected]

Abstract

For a long time, the cryopreservation of gametes and embryos remained a major hurdle for the clinicians and scientists in terms of success. However, recent technical advancement in the field of cryobiology has opened up various options for freezing gametes and embryos at different developmental stages. The tendency of the IVF world to switch over to natural- cycle IVF and to elective single-embryo transfer has put cryotechnology in the forefront of research. Still, the intricacies of the cold-induced changes in human gametes and embryos that could affect the intracellular and developmental processes need to be known. The transcriptomics, proteomics and metabolomic platforms hold promise for elucidating these complex processes during cryopreservation processes.

Keywords: cryopreservation, embryo, infertility, IVF, oocytes, ovarian tissue

Introduction

The first pregnancy using cryopreserved mature oocytes and successfully cryopreserve gametes, embryos and ovarian was reported by Chen (1986). Since then, cryopreservation tissues for use in assisted reproductive technologies. This review protocols for gametes, embryos and ovarian tissues have will discuss recent developments in cryopreservation regimens, evolved substantially. Researchers have studied many variables their advantages/disadvantages and biological backgrounds and that can affect the success of the process including temperature avenues for future prospects. (during cryoprotectant addition, freezing and thawing), type of cryoprotectant, additives, vessels for holding the gametes/ Cryopreservation of oocytes embryos, type of machinery, osmotic properties of solutions and several mathematical models for developing an effective protocol. However, except for some very recent (unpublished) There are several advantages of freezing gametes and embryos achievements, the pregnancy rate with frozen–thawed gametes/ for later use in assisted reproduction. For example, the embryos is still below that of fresh cycles. process reduces concentrations of gonadotrophins for ovarian stimulation and protects oocyte competence and reduces the At the same time, worldwide rates of infertility continue overall cost for multiple IVF attempts. In an interesting study to rise. Indeed, infertility now affects every sixth couple in done by the French national register on in-vitro fertilization western countries. Moreover, in some countries, up to 3% of all (FIVNAT, 1994), 465 pregnancies from frozen embryos were registered births result from assisted conception. Thus, there is compared with 8757 pregnancies from fresh embryo transfers. 126 a stronger-then-ever need for freeze–thaw protocols that safely There were fewer premature infants and small-for-gestational

© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK Review - Oocyte and embryo cryopreservation - AC Varghese et al. age (SGA) infant rate was lower in the group of pregnancies oolemma permeability of human oocytes to commonly used originating from frozen embryos. In singleton pregnancies, cryoprotective agents (Van den Abbeel et al., 2007). That study the SGA rate of newborns originating from frozen–thawed also indicated that there may be a considerable difference in embryos was half that of the pregnancies coming from fresh the tolerance level to cryoprotective agents between oocytes of embryo transfers. It is remarkable to note that the main in-vivo or in-vitro origin other than the genetic influence for difference between the two groups of patients was that ovarian the expression of important cryobiological characteristics of stimulation was mild or even absent in the frozen-embryo mammalian oocytes. Several lines of evidence also indicate that replacement cycles (Olivennes et al., 2002). mammalian oocytes express water channels (aquaporins), the expression pattern of which may affect how oocytes at various Oocyte cryopreservation has wider clinical implications than stages of maturity respond to cryopreservation (Ford et al., embryo freezing. Women who have no spouse, who wish to 2000; Edashige et al., 2003). delay their motherhood or stand to lose ovarian function due to surgery or radio/chemotherapy can maintain their fertility via When using the same freezing and thawing procedures, oocyte cryopreservation. It also avoids ethical, religious and mature human oocytes had survival rates that were lower legal issues surrounding embryo cryopreservation. Moreover, than that of embryos (Tucker et al., 1998a). The cytoplasmic oocyte cryopreservation remains the only option in countries membranes of oocytes, which have fewer submembranous actin where embryo freezing is forbidden by local regulations microfilaments, are more fragile to cryopreservation (Gook et such as Italy, where strict laws have been in place since 2004 al., 1993) than cleavage-stage embryos. The volume-to-surface (Benagiano and Gianaroli, 2004). ratio of oocytes is greater, making the dehydration process more difficult and more likely to lead to meiotic spindle disturbances, chromosomal dispersion, failure of normal fertilization and Experimental evidence and failure to develop (Eroglu et al., 1998a). biological factors that determine In one study, PolScope analysis revealed a limited amount oocyte cryosurvival of meiotic spindles in living human oocytes after cooling– rewarming (Wang et al., 2001). It has been suggested that a It has been well documented that oocytes experience both 3h post-thaw incubation could help restore meiotic spindles reversible and irreversible damage during cryopreservation. after slow freeze and thawing cycles, thus facilitating normal This includes hardening of the zona pellucida, premature release fertilization, preferably by intracytoplasmic sperm injection of cortical granules, depolymerization of the microtubules (ICSI) (Chen et al., 2005). Considerable variations in the and misalignment of the chromosomes. Immunofluorescent spindle recovery rate have been documented based on the type studies on fixed cells, and very recently PolScope-based live of cryopreservation protocols that are used. One method, ultra- imaging of the spindle apparatus, indicate that spindles from rapid cooling (vitrification), seems to help maintain the spindle both animal and human oocytes are susceptible to the freeze– apparatus (Gardner et al., 2007). However, well-designed slow- thaw process. The spindle is a dynamic structure composed of freezing protocols that use a higher sucrose concentration have heterodimeric units of α- and β-tubulin. They are assembled into also been shown to maintain spindle integrity (Nottola et al., 13 protofilaments arranged side by side, which are assembled 2006; Coticchio et al., 2007) continuously at one end and removed at the other in a treadmill fashion. The polymerized tubulin, the major component of the In an attempt to improve the survival of denuded or cumulus spindle microtubules, is in equilibrium with the free tubulin pool enclosed metaphase II (MII)-stage oocytes, Fabbri et al. within the ooplasm (Vincent and Johnson, 1992). For an oocyte (2001) increased the sucrose concentration in cryopreservation spindle to be considered normal, the chromosome must be media to 0.3 mol/l and the exposure time to 15 min in the slow located at the metaphase plate and have intact bonds between the freeze–rapid thaw method that would dehydrate the oocytes/ microtubules and kinetochores. Stray chromosomes may result cumulus-enclosed oocytes more adequately. They achieved a from perturbations inflicted upon the spindle polymers from the survival rate of 82%, which was significantly higher than the freeze–thaw process. The major challenge in cryopreservation rates associated with the 0.1 mol/l (34%) and 0.2 mol/l (60%) stems from the limited ability of human oocytes to repair their sucrose concentrations. A higher sucrose concentration during spindle upon return to physiological temperatures, which is in the rehydration step of the thawing procedure may keep osmotic contrast to the mouse oocyte (Mandelbaum et al., 2004). The stress within tolerable limits and could also be a contributor latter is more resistant to freeze damage (Stachecki et al., 2004), to increased survival (Fabbri et al., 2001). It is important which casts doubts on its suitability as a model for optimization to completely remove the cryoprotectant and establish the of human oocyte cryopreservation regimens. original water content and thereby a normal physiological state, and currently practiced thawing procedures and their effects Although the chromosomes reassemble themselves and align on volume excursions in oocytes during rehydration need along the spindle equator at cell rewarming, there is a risk of refinement to avoid any osmotic stress-induced vulnerability chromosomal loss and aneuploidy during the first maturation (Coticchio et al., 2007). division (Pickering et al., 1998). Human oocytes can withstand a wide range (~600–3000 mOsm) of aniosmotic conditions, Coticchio et al. (2006) provided evidence that during but in terms of membrane integrity and enzymatic functions, slow cooling, spindle and chromosome configuration are the tolerance level of meiotic spindles to non-physiological safely preserved provided that a higher sucrose (0.3 mol/l) conditions is considerably narrow (Mullen et al., 2004). A concentration in the freezing solution is used. It is interesting recent investigation reported significant variations in osmotic to observe that cryopreservation with either the 0.1 or 0.3 response and tolerance limits to changes in osmolarities and in mol/l sucrose protocols has given rise to different spontaneous 127

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abortion rates (20.0% and 14.2%, respectively) (Borini et al., In spite of the pitfalls arising from the technique of oocyte 2006b; Bianchi et al., 2007). Studies using PolScope show low cryopreservation, other biological factors such as the quality and high spindle birefringence, an indicator of differences in of the gametes, age of the patient and the in-vivo and in-vitro spindle organization (or tubulin fibre density), during freeze– microenvironment of growing oocytes has to be considered thaw cycles (Bianchi et al., 2005). It has been suggested that the (Mandelbaum et al., 2004; Edgar and Gook, 2007). Among reduced ability for spindle reformation during the post-thaw of the various indicators of developmental potential of embryos human oocytes may be linked to the fact that they contain fewer derived from cryopreserved oocytes, day-3 blastomere number foci of pericentriolar material – the site containing microtubule and an early cleavage at 25-h after insemination can be useful organizing centres (MTOC) – than do murine oocytes (Paynter, markers of selection (Edgar et al., 2000; Bianchi et al, 2005). 2000). Further insights from analysis of microtubule regulatory One major obstacle is the extent to which the clinics can use proteins (like protease activated receptors PAR1 and PAR3) various biological indicators of oocyte competence and embryo might reveal damage inflicted on the spindle apparatus viability on cryopreserved oocytes, based on the strict legal during oocyte freeze–thaw process (Sun and Schatten, 2006). restriction imposed in some European countries. Disturbances Assessment of tubulin organization by confocal laser scanning in the maintenance of Ca2+ signalling due to freezing and microscopy in the same thawed oocyte that has been analysed thawing-induced defects in mitochondrial polarity have also for its birefringence pattern may extend the knowledge on been reported (Jones et al., 2004). Using fluorescent ∆ψm fine cytoarchitecture of microtubular structures and optimal reporter JC-1, it has been documented that cryopreservation fertilization time required after oocyte thawing. can diminish the ability of pericortical mitochondria to retain high polarity and to form J-aggregates. The observation that A low sodium/sodium-depleted, choline-based medium can some oocytes of infertile patients are J-aggregative negative significantly improve oocyte storage outcome in terms of and, therefore, developmentally compromised (Van Blerkom et survival after thawing, fertilization rates and in-vitro and al., 2002) need to be considered during oocyte cryopreservation in-vivo developmental competence (Stachecki et al., 1998, experiments. 2002). The replacement of sodium with choline in the base solution may prevent salt-induced cryopreservation injury Oocyte vitrification: replacing the (solution effect), resulting in improved survival rates and embryonic development in vitro. It is argued that equilibration traditional slow cooling strategies? in commonly used freezing media causes an excess of sodium to be pumped into the ooplasm. Since the functions of Na-K Vitrification is a cryopreservation method during which pumps may be impaired during the freeze–thaw process, Na solutions become solid, forming a glass-like state, which could accumulate intracellularly, which is detrimental to cell avoids crystallization (Rall and Fahy, 1985; Shaw and Jones, viability. Unlike the sodium ion, the choline ion is not thought to 2003). By preventing the formation of ice, vitrification also cross the cell membrane and it may have a stabilizing effect on prevents the progressive concentration of salt and other the membrane by protecting against freezing damage. Another solutes during cooling but at the expense of potential damage protective mechanism of choline-based medium is believed to from a much higher initial concentration of cryoprotectant relate to an increase in viscosity at low temperatures. A total of (Pegg, 2005). By increasing the cooling and warming rates 11 human live births have been reported in the literature using to 20,000–200,000°C/min, however, this cryoprotectant the choline-based freezing method (Quintans et al., 2002; concentration can be diminished to a less harmful level. Azambuja et al., 2005). The vitrification technique is simpler, more convenient Boldt et al. (2003) compared two slow-freezing protocols: and more effective than the slow-cooling method (Vajta sodium-depleted (choline-substituted) freezing medium and Nagy, 2006). In 2001, Kuwayama et al. modified the containing 1.5 mol/l 1,2 propanediol and 0.2 mol/l sucrose vitrification technique by increasing the high rates of cooling and seeding at –6°C versus a sodium-based freezing medium and warming to reduce the concentration of cryoprotectants containing 1.5 mol/l propanediol and 0.1 mol/l sucrose with to 15% dimethyl sulphoxide (DMSO), 15% ethylene glycol a seeding at –7°C. Among the two groups, the choline group (EG) and 0.5 mol/l sucrose in an a minimum volume cooling showed a significantly higher survival rate than sodium-based plate (Kuleshova et al., 1999; Katayama et al., 2003). group (74.4% versus 12.3%) after thawing. In the sodium- This technique minimizes the damage associated with depleted group, the fertilization rate was 59%, and of 11 embryo cryopreservation such as intracellular and extracellular ice transfers, four pregnancies and five live births were reported crystal formation, chilling injuries and osmotic shock (Lucena (Boldt et al., 2003). It is difficult to say whether the better et al., 2006). Over the years, the use of various types of outcome in the choline-group was attributable only to sodium- vessels has been considerably refined to minimize the volume depletion in the freezing medium. The authors have used a higher of solution needed to quickly submerge the carrier in liquid sucrose concentration along with a higher seeding temperature, nitrogen. These vessels include cryoloop (Lane et al., 1999), both of which have been reported to increase oocyte survival open pulled straws (Vajta et al., 1998), cryotop (Kuwayama after thawing (Trad et al., 1999; Fabbri et al., 2001). Despite the and Kato, 2000), cryotip (Kuwayama et al., 2005a), cryoleaf suggestions put forward that choline-based media could prevent (Chian et al., 2005), flexipet denuding pipette (Liebermann et the solution effect, the better survival and pregnancy outcome al., 2002), electron microscope grids (Steponkus et al., 1990), reported with sodium-rich basal media by several groups invite direct cover vitrification (Chen et al., 2006). The cooling and further well-controlled studies comparing sodium-containing warming rates for cryostraw (25 µl) are 2500 and 1300°C/ and sodium-depleted cryopreservation media on human oocytes min, respectively (Rall and Fahy, 1985), whereas these values (preferably based on a good number of oocytes) or in non- for open pulled straws (1 µl), cryotop (0.1 µl) and cryotip (1 128 human primate models on freeze–thaw outcome. µl) are 16,700 and 13,900°C/min (Vajta et al., 1998), 23,000

RBMOnline® Review - Oocyte and embryo cryopreservation - AC Varghese et al. and 42,100°C/min (Kuwayama et al., 2005b) and 12,000 abnormalities in the vitrified oocytes than in the slow-frozen and 24,000°C/min (Kuwayama et al., 2005c), respectively. oocytes. In spite of that, the incidence of aneuploidy was In another study, Liebermann and Tucker (2002) found the similar between the two cryopreservation groups. Using a hemistraw system to be a superior carrier than cryoloop and mouse model, Bos-Mikich et al. (1995) also found no increase obtained a significantly higher (37.7% versus 29.4%) embryo in the incidence of aneuploidy following oocyte vitrification. compaction rate. Compared with vitrification, oocytes cryopreserved using slow freezing produced blastocysts with significantly fewer It is said that for a given cell, the maximum initial concentration cells (including a lower inner cell mass:trophectoderm ratio) that can be used without impairment of viability depends on and reduced viability following embryo transfer (Lane and the temperature, the rate at which the cryoprotectant is added Gardner, 2001). and removed and the total time of exposure (Pegg, 2005). Although in the last few years, the use of slow-freezing The results from the study by Huang et al. (2007) also indicate protocols have increased the survival rate to 90% (Stachecki that most vitrified oocytes regain normal meiotic spindles and et al., 2006), the pregnancy rate per thawed oocyte remains chromosome configuration after 1-h post-thaw incubation. poor (Boldt et al., 2006: 4.2% 15/361; Borini et al., 2006a: In contrast, a significantly higher percentage of oocytes in 2.3% 16/705), appearing to be much lower in comparison the slow-freezing group exhibited abnormal morphology of with vitrification (Kuwayama et al., 2005a: 11.2%, 12/107; spindle organization and chromosome alignment than the Lucena et al., 2006: 8.2%, 13/159; Antinori et al., 2007: vitrified oocytes. 11.8%, 39/330). Antinori et al. (2007) have also reported an implantation rate of 13.2% using cryotop vitrification of A number of studies have speculated that commonly used human oocytes (Figure 1). Very recently, Cobo et al. (2008) cryoprotectants and the type of cryopreservation process described an oocyte donation programme where the results play a role in the deregulation of cell cycle machinery such achieved with vitrified versus fresh oocytes did not differ as calcium oscillation and calcium signalling pathways from those of unvitrified controls, with pregnancy rates (Takahashi et al., 2004; Larman et al., 2006; Larman et over 60% in both groups after blastocyst transfer. Moreover, al., 2007). Larman et al. (2006) demonstrated that two according to a recent investigation, 200 infants conceived cryoprotectants commonly used in vitrification protocols following oocyte vitrification were not associated with an (DMSO and ethylene glycol) cause transient increases in increased risk of adverse obstetric and perinatal outcomes intracellular calcium in mouse MII oocytes. The increase in (Chian et al., 2008). calcium is sufficient to cause zona hardening, presumably through triggering cortical granule exocytosis, which is Huang et al. (2007) compared vitrification with slow freezing a calcium-dependent event (Kline and Kline, 1992). By using mouse oocytes and choline-based media. Survival, removing extracellular calcium, it was demonstrated that fertilization and early embryonic development rates were the source of the calcium increase in response to ethylene higher in the vitrification group. Analysis of spindle integrity glycol was mainly extracellular in contrast to intracellular and chromosome alignment indicated that there were fewer for DMSO. In a continuation of their study, Larman et al.

Figure 1. Human metaphase II oocytes prior to, during and after various steps of vitrification using the Cryotop method. (a) Before vitrification; (b) in equilibration solution; (c) in vitrification solution; (d) in dilution solution; (e) in washing solution; (f) in the culture media 5 min after the final washing step. 129

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(2007) verified that propanediol also causes a significant 1994). The inherent ability to survive such extreme conditions rise in intracellular calcium. Since calcium is a ubiquitous correlates with a spectrum of physiological adaptations that cellular signalling messenger, strict homeostatic regulation include the accumulation of large amounts of intracellular sugars. of the calcium concentration is of utmost importance for Among the sugars, trehalose, sucrose and glucose have been found cellular functions. Moreover, integral cell cycle proteins such to be particularly effective in the protection of macromolecules as M-phase-promoting factor and mitogen-activated protein and subcellular structures from the harmful effects of excessive kinase (MAPK) have been shown to be regulated by calcium dehydration (Crowe et al., 1992). The addition of sugars to (Marangos and Carroll, 2004). Osmotic pressure changes cryoprotectants has multiple benefits: sugars can cause an osmotic to the oocyte could induce oocyte activation (Whittingham, gradient across the cell membrane, which augments dehydration 1980) through Ca2+ release and thereby a high rate of cortical of the cell before freezing of extracellular water, and sugars are granule exocytosis and pronuclear formation. Amiloride, a capable of preserving the structural and functional integrity of pharmacological agent and an inhibitor of Na+/H+ exchange, cell membrane at low water activities (Hotamisligil et al., 1996). has been shown to inhibit cortical granule exocytosis and Adding sugars to the vitrification solution could enhance viscosity, protein phosphorylation caused by osmotic pressure changes whereby incubation of cells in this solution before vitrification will associated with cryopreservation (Inagaki et al., 1996). The help withdraw more water from the cells and reduce their exposure treatment has also been reported to limit the chromosomal to the toxic effects of the cryoprotectants (Orief et al., 2005). dispersion to some extent. Furthermore, it has been reported that a vitrification solution that contains added sugars could significantly improve the survival Although ICSI can circumvent zona hardening due to oocyte rate of vitrified bovine blastocysts (Saito et al., 1994). Toth et al., cryopreservation (Kazem et al., 1995; Porcu et al., 2000), (1994) showed that the maturation rate of frozen human immature most thawed oocytes that have undergone a cortical granule oocytes improved when sugars were added. An appropriate reaction have already undergone an activation process prior osmotic stress in the vitrification solution is an important factor to actual fertilization that leads to deregulation of cell for improving the maturation rate of vitrified–thawed immature cycle control proteins. The deregulation of cortical granule oocytes, which could be regulated by sugars. Fabbri et al. (2001) exocytosis by the freeze–thaw process and possible upstream demonstrated that increasing the sucrose concentration from 0.1 perturbations in Ca2+ signalling pathways and downstream mol/l to 0.3 mol/l in a freezing medium improved the survival rate fetal development anomalies should be addressed in future of human oocytes after a slow-freezing rapid-thawing programme studies based on the report by Ozil and Huneau (2001). These and prevented the formation of intracellular ice. On the other authors reported the consequences of experimentally induced hand, Mullen et al. (2004) verified that an increased sucrose Ca2+ signalling on the morphology of fetuses derived from concentration imposed a greater osmotic stress and increased the parthenogenetically activated rabbit oocytes. The observation likelihood of damage to the spindle. Ultrastructural studies using that the use of calcium-free media during the freeze–thaw different concentrations of sucrose in the freezing media indicate process reduces the abnormal increase in intracellular calcium that a lower concentration of sucrose (0.1 mol/l) causes a more and associated detrimental physiological effects justifies its conspicuous loss of cortical granules than a higher concentration use with common cryoprotectants (Larman et al., 2007). (0.3 mol/l) (Nottola et al., 2006), which might partly explain the It should be noted, however, that excellent results can be better results in cryopreservation of MII oocytes employing a achieved with common, calcium-containing media in human higher sugar concentration (Fabbri et al., 2001). oocyte vitrification, as mentioned earlier. In a study by Eroglu et al. (2002), the use of a combination of Overall, babies born through the use of cryopreserved human small amounts (0.15 mol/l) of intracellular trehalose (a sugar oocytes do not have an increased incidence of abnormal being utilized by some organisms to cope with extreme freezing karyotypes or congenital malformations (Porcu et al., 2000). and desiccation) with its moderate extracellular (0.5 mol/l) However, the case number is still relatively small (around 600 concentration effectively protected human oocytes against to 1000 worldwide) and more data are needed to verify the cryopreservation-associated stresses in the absence of any other results. Even though an upsurge of experimental evidence in cryoprotectant. In a subsequent study, they showed that the the current literature has substantiated the beneficial effect of microinjected trehalose was progressively eliminated during well-designed vitrification protocols for mature oocytes, it is mouse embryonic development (Eroglu et al., 2005). However, possible (although not very likely) that better results may be such invasive procedures need to be further assessed with achieved using classical controlled-rate freezing of oocytes, extensive molecular and developmental biological studies before provided further improvements in each step of the procedure they can be applied to human oocytes. An interesting observation are made, especially in the rehydration step after thawing, a is that bovine cumulus–oocyte complexes (COC) that were less well addressed area of research. matured in vitro in the presence of trehalose had better membrane stability (assessed by the propidium iodide/Hoechst differential staining) after vitrification–thawing procedures than the controls Sugars, antifreeze proteins and (Berlinguer et al., 2007). However, there were no differences in antioxidants: key players for a the fertilization or cleavage rates between the test and control groups. Trehalose has been considered to be beneficial either in prospective outcome? counteracting the osmotic effect or specifically interacting with membrane phospholipids (Crowe and Crowe, 1984). Kim et al. In nature, a variety of organisms such as arctic frogs, tardigrades, (1986) reported an improvement in the viability of mouse morula salamanders, some nematodes, rotifers, insects, brine shrimp, frozen ultra-rapidly in the presence of trehalose and found that bacteria, yeasts, fungi and their spores and various plant seeds trehalose is superior to sucrose during freezing. However, Abe et 130 survive extreme dehydration conditions (Crowe et al., 1992; Potts, al. (2005) obtained similar results with either trehalose or sucrose

RBMOnline® Review - Oocyte and embryo cryopreservation - AC Varghese et al. during bovine germinal vesicle (GV)-stage oocyte vitrification (15–20 mg/ml) maintains myocyte structure and mitochondrial using a stepwise protocol. integrity (Amir et al., 2003). It is interesting to note that type- III AFP from ocean pout (Zoarces americanus, a demersal eel- Another candidate that holds promise for cryopreservation is like species found in the northwest Atlantic from Labrador to antifreeze proteins. Many species of ectothermic animals, plants Delaware), when expressed in transgenic mice ovaries, was able and microbes inhabiting cold environments produce antifreeze to protect the fecundity of whole ovaries after vitrification (Bagis proteins/polypeptides to protect themselves from freezing et al., 2008). damage (Fletcher et al., 2001; Marshall et al., 2004). Since their discovery 35 years ago (DeVries and Wohlschlag, 1969), there Since oocyte plasma membranes contain significant amounts has been a growing interest in the various families of antifreeze of polyunsaturated fatty acids, they are particularly vulnerable proteins (AFP), antifreeze glycoproteins and their mutants. AFP to oxidative attack. It has been reported that lipid peroxidation can lower the freezing temperature of a solution noncolligatively of membrane phospholipids is adversely involved in embryo without affecting the melting temperature (thermal hysteresis) development. Some reports have indicated that cryopreservation and thus can prevent freezing of body fluids (Liu et al., 2007). can damage the antioxidant enzymes protecting against lipid AFP can also inhibit ice recrystallization, of which the large ice peroxidation and that freeze–thaw stress can be modified by crystals grow at the expense of smaller ones, thus preventing cell incubating the embryo in the presence of inhibitors of membrane damage during freeze–thaw cycles. It is generally accepted that lipid peroxidation (Tarin and Trounson, 1993). These results AFP function through adsorption of their flat ice-binding surfaces suggest the possibility that the process of cryopreservation onto particular planes of ice crystals and prevent or inhibit further induces the production of reactive oxygen species (ROS) or ice growth (Fletcher et al., 2001). Cryomicroscopic observations alters the antioxidant enzyme potential of oocytes, leading to indicate that antifreeze glycoproteins cause phenomena that lipid peroxidation of their plasma membranes and resulting are typical of vitrification (absence of visible crystallization, in reduced survival potential by a perturbation of membrane transparency of medium) (Karanova et al., 1995), which is structure and permeability. Loss of cell membrane function via distinctly different to the cryoprotectant DMSO. Microinjection of lipid peroxidation might interfere with transport systems such AFP into teleost embryos has been shown to increase the survival as pH regulatory systems on the cell membrane, and disruption rate significantly (Robles et al., 2006, 2007). AFP have unique of organelle membranes could affect transport systems such properties: they can protect cell membranes from cold-induced as mitochondrial transport systems essential for oxidative damage (Rubinsky et al., 1991) and inhibit ice recrystallization phosphorylation, the major energy-generating pathway of the (Knight and Duman, 1986). It should be noted, however, that the early embryo (Lane et al., 2002). initial enthusiasm regarding the use of AFP has diminished in the new millennium, partially because of the achievements made In addition, it is possible that loss of survival and fertilization using new vitrification techniques, where such supplementation competence following oocyte cryopreservation is also mediated did not result in a considerable improvement. by the mechanism of the cytotoxic action of peroxynitrite and

NO2. Supplementation of 50 IU/ml of superoxide dismutase In the early 1990s, considerable work was published on AFP (SOD) or the concomitant addition of SOD and haemoglobin in cryopreservation. Recently, interest in AFP has given rise to to the freezing and thawing media has been shown to improve new research amongst cryobiologists. Currently, a number of survival and fertilization of mouse oocytes (Dinara et al., 2001). experimental approaches are being pursued to understand how It has been suggested that peroxynitrite, formed by the interaction –• antifreeze proteins interact with ice and thereby decipher the of O2 and NO2, may exert a cytotoxic effect on mouse metaphase underlying molecular mechanism (Sander and Tkachenko, 2004; II oocytes during cryopreservation. In addition, inclusion of Liu et al., 2007; Wierzbicki et al., 2007). Theoretical modelling Lycium barbarum polysaccharide in the vitrification solution and molecular dynamic computer simulations of fish type-I kinetic was shown to reduce the production of ROS, thereby preventing ice inhibitor protein at the ice–water interface have recently been plasma membranes from lipid peroxidation and stabilizing studied by some groups. This has provided new insight into the membrane structure and permeability of porcine oocytes (Huang unique molecular properties that determine the ability of AFP to et al., 2008). inhibit ice growth at the ice–water interface (Jorov et al., 2004; Yang and Sharp, 2005; Wierzbicki et al., 2007). The work by Lane et al. (2002) demonstrates that including 0.1 mmol/l ascorbate when cryopreserving mouse cleavage-stage Adding antifreeze glycoproteins at 1 mg/ml has been shown to and blastocyst-stage embryos is beneficial to subsequent embryo improve the fertility rates of mouse oocytes cryopreserved in 6 development and maintenance of normal cell function. Ascorbate mol/l DMSO (O’Neil et al., 1998). The same authors have also assists embryo development by stimulating development of the reported that antifreeze glycoproteins could also be bound to the inner cell mass following cryopreservation. The beneficial effects surface of growing ice-crystals, inhibiting the addition of any of ascorbate were most evident in mouse embryos that were slow further water molecules. Positive results were also obtained with frozen compared with those that were vitrified, which substantiates porcine embryos, which are highly susceptible to chilling injury the fact that the damage from oxygen radicals is greater after slow because of their high lipid content (Chen et al., 1995; Fei et al., freezing. Ascorbate also reduced hydrogen peroxide generation 1995). Hays et al. (1996) reported that antifreeze glycoproteins significantly among vitrified embryos but failed to do so in the found in the blood of polar fish could inhibit leakage of slow-frozen ones. These results indicate that the slow-freezing intracellular contents across membranes during thermotropic procedure augments the production of hydrogen peroxide phase transitions. AFP also holds promise in the preservation of considerably more than the ultra-rapid vitrification procedure. ultrastructural functions of organs during the freeze–thaw process. Future studies on antioxidative enzymes and NO scavengers may Electron microscopy has shown that subzero cryopreservation of lead to a better understanding of the biochemical processes that mammalian hearts for transplantation using type-I or type-II AFP occur during oocyte/embryo cryopreservation. Estimating the 131

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total antioxidant capacity and measuring ROS concentrations in The freeze–thaw process can rupture the zona pellucida, cause the follicular fluid of retrieved oocytes will help select patients cumulus cells to detach, and interrupt gap junctions between the whose gametes require antioxidant supplementation during cumulus cells and oocyte, further influencing subsequent oocyte cryopreservation and subsequent in-vitro cultures. The implication development and maturation (Wu et al., 2006). Such structural that ROS-mediated damage to oocyte developmental competence changes might be attributed to severe dehydration resulting and embryo viability in infertile patients have raised concerns from osmotic effects of the cryoprotectants. To avoid formation and need further research to identify the pathology behind this of intracellular ice crystal during vitrification, it is essential for condition (Agarwal et al., 2005). the oocytes to dehydrate (Fabbri et al., 2000). On the other hand, dehydration more or less affects cellular structures. Therefore, The expression of various biomarkers of oxidative stress such as damage to microvilli resulting from vitrification could reduce the SOD, Cu-Zn SOD, Mn SOD, glutathione peroxidase, glutamyl viability of oocytes. Vitrification of immature porcine oocytes synthetase and lipid peroxides has been demonstrated in normally lead to a reduction of their mitochondrial matrix density, but cycling human ovaries (Agarwal et al., 2005). A delicate balance the extent to which these changes affect oocyte development is exists between ROS and antioxidant enzymes in ovarian tissue. unclear (Wu et al., 2006). Because hypoxic conditions following retransplantation of ovarian tissues induce ROS generation and the freeze–thaw cycle A porcine GV oocyte vitrification study by Wu et al. (2006) depletes the tissue’s antioxidant capacity, antioxidant treatment employed an open pulled straw vitrification method using 20% EG might arguably be warranted. Studies of bovine ovarian tissue (equilibration) and 40% EG with 0.6 mol/l sucrose (vitrification transplantation showed that ascorbic acid reduced apoptosis solution). However, successful production of blastocysts was (Kim, 2003). reported by Aono et al. (2003) following ultra-rapid vitrification with stepwise equilibration of GV-stage mouse oocytes. It is Cryopreservation of immature interesting to note that, in the above study, the blastocyst hatching rates were 8.8% for single-step equilibration, 15.6% for two- oocytes: imminent challenge for step equilibration and 22.8% for 10-step equilibration. This suggests that the process of gradual equilibrium conversion of the cryobiologists cryoprotectant agent seems to adjust the permeability of the plasma membrane, which may contribute to maintaining the connection Establishing technology that can vitrify oocytes at the prophase-I between the oocyte and cumulus cells and/or a decrease in rapid (GV) stage and other stages of maturation may also be important changes in osmotic pressure. The three-dimensional COC is for clinical and experimental use. Because cumulus cells must be likely to be particularly prone to physical disruption caused by connected to oocytes through gap junctions for immature oocytes ice crystal formation. Even in the absence of ice crystal formation to mature, vitrification protocols may prove beneficial. Developing during vitrification, the vast difference in size between the oocyte techniques that can successfully cryopreserve GV-stage oocytes and its associated cumulus cells means that they are likely to will also aid GV transplantation, which requires a large number react differently to the stresses applied during cryopreservation. of donor GV oocytes. Removing GV oocytes from older women The same group has reported a modified protocol for stepwise and transplanting them into younger enucleated oocytes has been pre-equilibration called SWEID, which ultrarapidly vitrifies proposed as a novel option for reducing aneuploidies (Takeuchi mouse GV oocytes. After cryopreservation, the GV oocytes were et al., 2001). This will also lead to an in-depth elucidation of the fertilized in vitro and cultured. Some of the 2-cell-stage embryos differences between nuclear and cytoplasmic maturation processes. were then transferred, resulting in live birth (Aono et al., 2005).

In GV oocytes, chromatin remains in a decondensed state. In Abe et al. (2005) reported the favourable effect of stepwise pre- addition, microtubule-organizing centres are not formed, and the equilibration prior to vitrification of bovine immature oocytes. microtubular system is not yet organized. The survival rate and They showed that after stepwise exposure to cryoprotectants, developmental potential of these oocytes decrease after freezing. the in-vitro maturation rate and the normality of cytoplasmic Cryopreservation of immature oocytes has been reported with organelles in the vitrified GV oocytes were most similar to those varying degrees of success in several mammalian species such as in the control, resulting in production of viable blastocysts. In the mice (Eroglu et al., 1998b; Moffa et al., 2002), cattle (Suzuki et single-step exposure, most oocytes showed a highly vacuolated al., 1996), buffalo (Wani et al., 2004), pigs (Fujihira et al., 2004) cytoplasm with many ruptured mitochondria (Abe et al., 2005). and humans (Tucker et al., 1998b; Isachenko et al., 2006). Birth Since vitrification is a non-equilibrium cryopreservation method after slow freezing of immature human oocytes (Tucker et al., that needs relatively high concentrations of cryoprotectants, a 1998b) and biochemical pregnancy after vitrification of immature stepwise addition of cryoprotectants may reduce the toxic effect human oocytes (Wu et al., 2001) has been reported. However, at of cryoprotectants and minimize damage due to extreme cell- least one study reported irreversible damage to the cytoskeleton volume expansion (Rall, 1987). of porcine GV and MII-oocytes after vitrification, which could be an important factor affecting developmental competence (Rojas Chilling injury, a major cause of irreversible changes to the et al., 2004). Protocols for the collection of fully grown GV-stage cytoplasmic membranes, occurs at subphysiological temperatures human oocytes have been devised by modifying mature oocyte during the transition of lipids from the liquid crystalline phase to collection techniques (Trounson et al., 1994) and by reducing the gel phase (lipid phase transition). The phospholipid:cholesterol hormonal stimulation. Several lines of evidence indicate that ratio and nature of the fatty acid composition of oocyte cytoplasmic immature oocytes collected from stimulated ovaries are more membranes – which show varying seasonal as well as inter- and resistant to cryopreservation and have lower incidences of intra-species variation – could also account for the complexity chromosomal and spindle abnormalities than immature oocytes of oocyte survival during the freeze–thaw process. Experimental 132 from unstimulated cycles (Baka et al., 1995; Park et al., 1997). manipulation of the lipid-phase transition temperature by

RBMOnline® Review - Oocyte and embryo cryopreservation - AC Varghese et al. polyunsaturated fatty acids has been shown to reduce chilling a lower destabilizing effect of glycerol when the addition was injuries to oocytes and embryos (Hochi et al., 1999; Zeron et combined with low temperature. al., 2002). It is believed that, in addition to being an integral part of the cell membrane, cholesterol is also present in the lipid- The use of DMSO has produced similar effects on oocyte enriched microdomains (lipid rafts), which can influence receptor microfilaments similar to glycerol (Vincent et al., 1990). In functions (Westover et al., 2003). Since the maturation process of addition to the changes in F-actin organization, an irregular frozen–thawed immature oocytes requires several growth factors shrinkage pattern was observed when non-human primate GV and proper interaction between the oocytes and receptors, the oocytes were exposed to hypertonic solutions. Interestingly, MII integrity of the cell membrane, with its functional ligand-receptor oocytes under similar conditions showed a uniform shrinkage system, needs to be preserved during cryopreservation. pattern. The irregular shrinkage of GV oocytes is said to be caused by differences in the microfilament organization in Embryo development status improved when bovine oocytes were oocytes at various maturational stages, particularly in reference to treated with cholesterol-loaded methyl-β-cyclodextrin (CLC) the transzonal projections, which anchor the plasma membrane of prior to vitrification (Horvath and Seidel, 2006). Moreover, when GV and MI oocytes to the zona pellucida and corona cells (Younis oocytes were exposed to cyclodextrin containing 4-nitrobenzo-2- et al., 1996). GV oocytes treated with ethylene glycol tetraacetic oxa-1,3diazol-labelled cholesterol for about 1 h, fluorescence was acid – a calcium chelator that disengages junctional contacts – observed in both the COC and oocytes, indicating that oocytes showed a uniform shrinkage pattern after exposure to hypertonic as well as cumulus cells incorporated the cholesterol from CLC conditions. Ultrastructural study has revealed that the disruption through diffusion. However, the results also showed that CLC may of the actin filament system caused by DMSO was associated with be effective in incorporating the cholesterol into cell membranes changes in the lengths and distribution of oolemma microvilli. only when it is added to chemically defined media. Otherwise, the cholesterol may be sequestered in the presence of additives such The freeze–thaw process could cause irreversible changes in as fetal calf serum. nuclear and cytoplasmic organization in immature oocytes in spite of an apparent normal progression to the MII stage shown Isachenko and Nayudu (1999) have reported a high survival by polar body extrusion (Boiso et al., 2002). Since microtubules rate and meiotic normality together with good preservation play a major role in mediating cytoplasmic maturation during of attached cumulus cells using a simple new vitrification the early stages of meiosis by facilitating mitochondrial and procedure. They showed that 37°C pre- and post-freeze exposure mRNA distribution and cortical positioning of the MI spindle, could significantly improve both survival and normal spindle disruption of the microtubule assembling or the depolymerization/ configuration of mouse GV oocytes after in-vitro maturation. polymerization dynamics during cryopreservation could have a Egg yolk (a natural complex mixture of phospholipids and major negative impact on development. The MI block and erratic antioxidants) was found to produce further beneficial effects on MII spindle usually observed following the post-thaw maturation both the oocyte and cumulus cell integrity, with the best effects stage are suggested to be due to detrimental influences on MAPK being obtained at 37°C with the inclusion of egg yolk both before activities that are initiated after GV breakdown and remain at a and after the freezing. With this protocol, the normal survival high level throughout the transition from MI to MII (Lu et al., on post-thaw was greater than 80% with an intact and attached 2002; Comizzoli et al., 2004). Comizzoli et al. (2004) recently cumulus complex. In addition, there was an 84% maturation rate showed that exposure of cat GV-stage oocytes to 1.5 mol/l and a 45% normal metaphase configuration. However, concerns propanediol at 25°C had little effect on cell cycle regulatory regarding possible disease transmission hampered widespread mechanisms as observed by a normal developmental pattern and use of egg yolk, and a similar effect could also be achieved with chromosome integrity, which was similar to that of the controls. other protein supplements. It is suggested that, based on the However, EG appeared to be more harmful to the cat oocyte than interrelationships of the cytoskeleton and the cellular membrane propanediol and was suggested to have a possible detrimental (Younis et al., 1996), additives that protect the integrity of the effect on M-phase promoting factor (MPF) and MAPK regulators cellular membrane during each step of cryopreservation might (c-mos, cyclin B) or other factors influencing meiotic progression also be expected to produce an improvement in the survival and and cytoskeletal organization (Wu et al., 1999; Kim et al., 2000) normality of the oocytes after exposure to cryoprotectants and during the MI–MII transition (Comizzoli et al., 2004). Dobrinsky cooling (Isachenko and Nayudu, 1999). et al. (2000) reported increased survival of porcine blastocysts following treatment with cytochalasin B before vitrification. It is well documented that the oocyte cytoskeleton is damaged Verlhac et al. (2000) showed that the Mos-MAPK pathway by the cryopreservation process, which could lead to significant controls the activity of the actin microfilament network, perhaps changes in the organization and trafficking of molecules and through myosin IIA in mouse oocytes, and proper positioning and organelles (Vincent and Johnson, 1992). The completion of active movement of organelles are essential for oocyte growth, several dynamic events, including homologous chromosome maturation and fertilization. separation, spindle anchorage, spindle rotation, vesicle and organelle transport and pronuclear apposition (mouse) requires The literature documents contrasting results regarding human interaction between microfilaments and microtubules in oocytes oocyte survival rate after cryopreservation with or without the (Sun and Schatten, 2006). Another study also emphasizes that cumulus oophorus. In one study, the presence of the cumulus the dynamic equilibrium between polymerized and free tubulins mass or the partial or total removal of the cumulus cells did not in mammalian oocytes is extremely sensitive to temperature significantly modify the oocyte survival rate (Mandelbaumet al., change (Zenses et al., 2001). Younis et al., (1996) studied the 1988). Similar results were obtained by Fabri et al. (2001) who deleterious effect of glycerol (not a common cryoprotectant for used a higher sucrose concentration. However, Gook et al. (1993) female gametes) at different equilibration temperatures on F-actin observed reduced survival of oocytes that were cryopreserved organization of rhesus monkey oocytes (GV stage). They found along with the cumulus corona mass. In contrast, Imoedemhe and 133

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Sigue (1992) reported a better survival rate in oocytes that were the oocyte has been shown to affect the developmental capacity frozen intact with cumulus cells (54%) than in denuded oocytes of the derived embryos in fresh IVF cycles. A similar adverse (27%) where they used 0.25 mol/l sucrose in the cryoprotectant effect is reflected in freeze–thaw cycles with the developmental agent. It seems that the cryopreservation regimen dictates the capacity of frozen–thawed embryos related to the morphology of survival rate of cumulus-enclosed oocytes. It is suggested that the the oocyte from which they originate (Balaban et al., 2006). It has cumulus mass may offer some protection against sudden osmotic been suggested that the success rate of embryo cryopreservation changes and stresses that could occur by rapid influx/or efflux of can be improved by selecting embryos for transfer based on their the cryoprotectant agent during freezing and thawing procedures capacity for sustained cleavage during a 24-h culture period in (Fabri et al., 2001). One explanation for the greater osmotic vitro. In this context, freezing embryos at the zygote stage may sensitivity in the GV oocytes is that their hydraulic conductivity is be an interesting option based on recent reports showing high two times lower than that of in-vitro matured MII oocytes (Agca survival and pregnancy rates using zygote-stage vitrification and et al., 2000). Since oocyte–somatic cell interactions are vital for post-thaw culture for 24 h before embryo transfer (Al-Hasani et the cytoplasmic and nuclear maturation of immature oocytes al., 2007): in this study of 849 vitrified pronuclear zygotes, 339 (Albertini et al., 2001), it is important to design freeze–thaw pronuclear zygotes were thawed, resulting in an 89% survival rate protocols for an intact COC that are beneficial in view of recent and a pregnancy rate of 36.9%, both of which are higher than developments in in-vitro maturation systems. Lack of proper those obtained with the slow-freezing method (10.2%). The post- meiotic competence, fertilization and embryo development of thaw embryo cleavage rate in vitro is also a good predictor of frozen–thawed and in-vitro cultured GV-stage oocytes, even in the embryo implantation rate (Van der Elst et al., 1997). Based the presence of fresh cumulus cells, indicate that intercellular on the observation that freeze–thaw cycles may have some connections (gap junctions) mediated transfer of paracrine factors deleterious (hardening) effects on the zona pellucida, partial are mandatory for in-vitro maturation of cryopreserved cumulus- zona dissection or laser-assisted hatching may be beneficial in enclosed immature oocytes (Ruppert-Lingham et al., 2003). improving the implantation rate following transfer of frozen– thawed embryos. Even though partially damaged embryos The study by Ruppert-Lingham et al. (2003) on the effect of following cryopreservation can give rise to term pregnancies, all DMSO exposure and cryopreservation of GV COC indicates that recent studies involving high numbers of frozen-embryo transfer cumulus–oocyte cross-connections remain intact when exposed cycles agree that the developmental potential of partially damaged to the cryoprotectant, but the disruption is initiated due to the frozen–thawed embryos is inferior to that of embryos that remain freeze–thaw procedure. Two other studies have also reported fully intact after thawing. It is postulated that damaged blastomeres a loss of cumulus cells from COC following the freeze–thaw exert a toxic effect on the growth potential of intact ones. In a process using slow-cooling programmes (Cooper et al., 1998; recent randomized prospective study, there was no difference Goud et al., 2000). Low-sodium (choline-based) media were in pregnancy/implantation rates when lysed cell removal was used by Goud et al. (2000) on immature human oocytes, and performed on thawed embryos (Prados et al., 2006). they found no differences in the maturation rates when cumulus- enclosed GV oocytes were compared with the controls. They Another line of investigation has suggested that the type of did, however, report a considerable loss of cumulus cells after ovarian stimulation regimen affects oocyte and embryo quality thawing. Although ICSI has been advocated for the fertilization and the success of frozen-embryo transfer cycles. The type of of cryopreserved and thawed oocytes, classical IVF must be gonadotrophin preparation used for ovarian stimulation may promoted along with the need for a cryopreserved and intact influence embryo quality and its potential to implant in fresh COC (Li et al., 2005; Coticchio et al., 2007). Cellular and cycles. A recent study showed that a higher proportion of embryos genetic perturbations emanating from the stressful process of derived from IVF patients stimulated with highly purified human cryopreservation combined with the effects of microinjection menopausal gonadotrophin survived cryopreservation than cannot be ruled out as having an effect on the molecular structure those stimulated with recombinant FSH and appeared to have a of oocytes and causing long-term post-natal consequences. One higher potential for development and implantation (Ziebe et al., recent study found untoward effects of the ICSI procedure on the 2006). These studies confirm that it is not only the refinements wellbeing of children born by this procedure and the deregulation in cryopreservation protocols that will improve results of frozen- at the epigenetic level (Varghese et al., 2007a). embryo transfer cycles but also derivation of good-quality oocytes and thereby embryos with optimal genetic integrity and Zygote and cleavage-stage physiological status via proper clinical management (such as ideal gonadotrophin treatment). embryos: are slow-cool freezing Although embryos subjected to vitrification are likely to be injured methods giving way to vitrification? by the toxicity of the high concentration of cryoprotectant, many vitrification methods have been refined in recent years and proved Since the first pregnancy after replacement of frozen–thawed effective for the cryopreservation of embryos at various stages of human embryos was reported (Trounson and Mohr, 1983), embryo development in laboratory and domestic species. In 1998, it was cryopreservation has become a routine technique in assisted shown that vitrification using an EG-based vitrification solution conception programmes. The use of frozen embryos together (EFS40) (Kasai et al., 1990) with conventional cryostraws was with refinements in embryo quality evaluation have facilitated effective for human embryos at the 4–8-cell stage (Mukaida et al., the current shift of embryo transfer policy towards single-embryo 1998). The effectiveness of vitrification was confirmed for human transfer with the consequent reduction of the risk of unwanted embryos at the 8–16-cell stage (Saito et al., 2000) and the morula multiple pregnancies. However, overall, cryopreservation leads stage (Yokota et al., 2001) also using EG-based vitrification to a 30–40% reduction in the implantation potential (Edgar et solutions. Human blastocysts are much less permeable to 134 al., 2000; El-Toukhy et al., 2003). Cytoplasmic dysmorphism of cryoprotectant and water and have been observed to shrink more

RBMOnline® Review - Oocyte and embryo cryopreservation - AC Varghese et al. slowly than mouse and bovine blastocysts when placed in the transcriptosomes are said to compensate for cold-related reduction cryoprotectant solution. This suggests that they are more likely in enzyme activities and to synthesize stress-protective molecules. to be injured by intracellular ice. One major reason for this has Interestingly, of all the genes studied for cold response, a higher been attributed to the blastocoelic fluid. Artificial reduction of the number of differentially expressed genes (22/180, 12.2%) were blastocele cavity prior to the vitrification procedure has yielded located in chromosome 7 in mice. This study identified several favourable results. Among a total of 725 vitrified blastocysts, high new candidate marker genes for cryosurvival. In cryopreserved rates of survival (80%) and pregnancy (37%) were reported by mouse embryos and ovarian tissues, cold response gene Mukaida et al. (2003) using cryoloop-based vitrification. expression patterns have indicated that embryo growth is arrested and that DNA damage-inducible genes are present (Liu et al., A recent study demonstrates that the average characteristics 2003; Mamo et al., 2006). The results of these studies may help of pregnancies and labour as well as perinatal health exhibited further improve cryopreservation protocols. by infants (n = 34) born as a result of embryo transfer after vitrification are not different from that of the overall population In addition, recent analysis of the oocyte proteome using time-of- average (Potapov et al., 2006). To vitrify the embryos, this group flight mass spectrometry following cryopreservation revealed that used a solution containing EG, trehalose, Ficoll, hyaluronan slow freezing significantly affects protein expression (Larman et and human serum albumin and 0.25 ml cryostraw as the vessel; al., 2007). When proteomics was used to compare slow freezing the total exposure time was 5 min. The embryos were then and vitrification methods, the results showed that vitrified MII plunged into liquid nitrogen. As mentioned earlier, babies born oocytes closely resembled the non-frozen controls whereas the after vitrification of oocytes did not exhibit increased rates of MII oocytes that underwent slow freezing exhibited markedly developmental abnormalities (Chian et al., 2008) different protein profiles with either up- or down-regulation of 19 positively charged and 21 negatively charged proteins (P < 0.05). Genomic and post-genomic Further analysis of the oocyte’s proteome revealed that it was during the dehydration stage prior to seeding in the slow-freezing approaches in cryotechnology protocol when these alterations occurred (Gardner et al., 2007). The observation that specific proteins were significantly up-regulated Global embryonic gene expression in humans occurs at the 4–8 after slow freezing is said to be associated with the length of time cell stage (Braude et al., 1988). The maternal proteins and mRNA the oocytes are exposed to the toxic cryoprotectant. This exposure that were synthesized during oogenesis are responsible for the may induce stress-related responses and the up-regulation of initial cleavages of mammalian embryos. Alterations in gene stress proteins and/or apoptosis. In contrast, vitrification has a expression patterns detailed by reverse transcription polymerase minimal impact, indicating that it has a fundamental advantage in chain reaction or microarray analysis can be of help to elucidate the cryopreservation of oocytes. the effects of ovarian stimulation, culture conditions and any in- vivo or ex-vivo manipulations such as cryopreservation. Kim et al. (2006) studied the proteome of cryopreserved bovine ovarian tissue. They found alterations in protein profiling and Tachataki et al. (2003) has shown that there is a gradual decrease expression in ovarian grafts after transplantation. Most of the in the expression pattern of the TCS2 gene throughout human significant proteins identified after transplantation were proteins preimplantation development; the level of expression is high related to tissue survival and metabolism such as actin, laminin and during the unfertilized state and falls to a low level when global antioxidant (glutathione S-transferase). Glutathione-S-transferase activation of the embryonic genome occurs. This gradation of the is an enzyme that is usually up-regulated in oxidative stress, and transcriptosome expression pattern was lost in unfertilized aged mRNA for this enzyme has also been shown to be up-regulated in oocytes. There was a considerable drop in TCS2 mRNA when mouse embryos under cold response (Mamo et al., 2006). Among D-2 embryos were rapidly thawed. This drop was not significant the slow-freeze and vitrification groups, changes in the protein when D-3 embryos were subjected to such protocols. The study profile of the vitrified and transplanted ovarian tissue were more revealed that human embryos frozen on day 2 appeared to be consistent in the fresh controls (Kim et al., 2006). more susceptible to temperature change than embryos frozen on day 3, which may be a consequence of the activation of the Low-molecular-weight metabolites signify the end-products embryonic genome. of cell regulatory processes and therefore reveal the response of biological systems to a variety of environmental or genetic The transciptome analysis of cells and tissues by microarray influences. As physiological changes to a cell resulting from techniques and further elucidation of the molecular and gene overexpression or deletion/silencing are amplified through functional status of cells under particular conditions by proteomic the hierarchy of the transcriptome and proteome, these changes and metabolomic approaches hold great promise in assisted are said to be more readily measured through the metabolome reproductive technologies to improve the success rate (Katz- (Singh and Sinclair, 2007). Moreover, the metabolome is Jaffe and Gardner, 2007; Singh and Sinclair, 2007; Varghese downstream of gene function and therefore considered to be et al., 2007b). Temperature reductions are known to negatively a superior measure of cellular activities at the physiological affect the stability of RNA secondary structures, which have been level compared with transcriptomic and proteomic approaches. suggested to lead a rate-limiting step of translation initiation in The metabolic profiling of frozen–thawed embryos has been prokaryotes (Zangrossi et al., 2000). The gene expression profile reported as a novel marker of embryo viability and growth as assessed by microarray techniques showed that vitrification potential irrespective of the embryo grade (Stokes et al., of embryos results in a broad spectrum of consequences: cell 2007). In spite of reassuring data on the post-natal safety of metabolism and regulatory- and stress-related genes seem to be human embryo cryopreservation, animal studies indicate that most effected (Mamo et al., 2006). Several of these enhanced frozen–thawed embryos could affect metabolism in the late biological processes as revealed by overrepresentation of preimplantation development (Emiliani et al., 2000) and have 135

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more subtle long-term, as well as post-natal, effects that are References manifested late in development (Dulioust et al., 1995). It is suggested that cryopreservation-induced stress may alter Abe Y, Hara K, Matsumoto H et al. 2005 Feasibility of a nylon-mesh homeostasis, metabolism, cell integrity and developmental holder for vitrification of bovine germinal vesicle oocytes in potential (Stokes et al., 2007). Cryopreserved embryos with the subsequent production of viable blastocysts. Biology of Reproduction capacity to develop to the blastocyst stage had a lower rate of 72, 1316–1420. Agarwal A, Gupta S, Sharma RK 2005 Role of oxidative stress in female amino acid depletion, appearance and turnover than arresting reproduction. Reproductive Biology and Endocrinology 3, 28. embryos. Metabolic parameters are therefore imperative Agca Y, Liu J, Rutledge JJ et al. 2000 Effect of osmotic stress on the when identifying treatment protocols that least interfere with developmental competence of germinal vesicle and metaphase key cellular functions of the oocytes (Gardner et al., 2007). II stage bovine cumulus oocyte complexes and its relevance to Furthermore, when monitoring metabolism through pyruvate cryopreservation. Molecular Reproduction and Development 55, uptake is considered, mouse oocytes and developing embryos 212–219. following slow freezing were shown to be more metabolically Albertini DF, Combelles CMH, Benecchi E et al. 2001 Cellular basis for impaired than those that were vitrified (Lane and Gardner, paracrine regulation of ovarian follicle development. Reproduction 121, 647–653. 2001; Lane et al., 2002). Analysis of membrane integrity Al-Hasani S, Ozmen B, Koutlaki N et al. 2007 Three years of routine through the retention of the enzyme lactic dehydrogenase vitrification of human zygotes: is it still fair to advocate slow-rate also indicated that slow freezing induces significantly more freezing? Reproductive BioMedicine Online 14, 288–293. plasma membrane damage than vitrification (Lane et al., Amir G, Rubinsky B, Kassif Y et al. 2003 Preservation of myocyte 2002). Non-invasive footprinting of glycolytic activity (Lane structure and mitochondrial integrity in subzero cryopreservation of and Gardner, 1996) and knowledge of fatty acid metabolism mammalian hearts for transplantation using antifreeze proteins – an and uptake of embryos (Haggarty et al., 2006) may also be electron microscopy study. European Journal of Cardiothoracic used one day to help select cryopreserved/vitrified embryos. Surgery 24, 292–296. Antinori M, Licata E, Dani G et al. 2007 Cryotop vitrification of Research so far indicates that vitrification procedures have human oocytes results in high survival rate and healthy deliveries. less of an impact on oocyte/embryo proteome and energy Reproductive BioMedicine Online 14, 71–78. metabolism than slow freezing. Aono N, Abe Y, Hara K et al. 2005 Production of live offspring from mouse germinal vesicle–stage oocytes vitrified by a modified stepwise method, SWEID. Fertility and Sterility 84, 1078–1082. Conclusions Aono N, Naganuma T, Abe Y et al. 2003 Successful production of blastocysts following ultrarapid vitrification with step-wise ‘A tendency to adopt techniques widely when they show promise equilibration of germinal vesicle stage mouse oocytes. Journal of often occurs at the expense of further refinement.’ Gook DA and Reproduction and Development 49, 501–506. Edgar DH (1999). 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In this regard, offspring by orthotopic transplantation of cryopreserved mouse non-human primate models (Songsasen et al., 2002) may offer ovaries. Molecular Reproduction and Development 75, 608–613. a useful experimental system because of the close relationship Baka SG, Toth TL, Veeck LL et al. 1995 Evaluation of the spindle between Homo sapiens and other primates based on an apparatus of in-vitro matured human oocytes following evolutionary and genetic point of view. Moreover, there is much cryopreservation. Human Reproduction 10, 1816–1820. to learn from studying cold adaptation in some organisms – which Balaban B, Yakin K, Isiklar A et al. 2006 Developmental ability of Human is the result of millions of years of evolution – and the organic cryo-thawed embryos derived from dysmorphic oocytes. Reproduction 21 (Suppl. 1), i38. molecules that protect their protoplasm from extreme desiccation. 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Declaration: The authors report no financial or commercial conflicts of interest.

Received 4 August 2008; refereed 28 August 2008; accepted 4 February 2009.

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