Current results with slow freezing and vitrification of the human oocyte

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1 Reproductive BioMedicine Online (2011) 23, SYMPOSIUM: OOCYTE CRYOPRESERVATION REVIEW Current results with slow freezing and vitrification of the human oocyte Jeffrey Boldt Assisted Fertility Services, Community Health Network, 8040 Clearvista Parkway, Suite 510, Indianapolis IN 46256, USA address: Dr Boldt is Scientific Director of Assisted Fertility Services at Community Health Network in Indianapolis, USA. He is also a clinical associate professor of Medical and Molecular Genetics at Indiana University School of Medicine and Scientific Director for The World Egg Bank, the world s first commercial frozen donor egg bank. He has been involved in assisted reproductive technology since 1983 and has authored more than 30 journal articles and book chapters on mammalian spermatozoa egg interaction, clinical embryology and andrology and oocyte cryopreservation. Abstract The past decade has witnessed renewed interest in human oocyte cryopreservation (OCP). This article reviews the two general methods used for OCP, slow freezing and vitrification, compares the outcomes associated with each technique and discusses the factors that might influence success with OCP (such as oocyte selection or day of transfer). Based on available data, OCP offers a reliable, reproducible method for preservation of the female gamete and will find increasing application in assisted reproductive technology. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cryopreservation, oocyte, slow freezing, vitrification Introduction Oocyte cryopreservation (OCP) has enjoyed a renaissance in the past decade. The advantages of OCP include: (i) avoiding embryo cryopreservation in cases of objections; (ii) fertility preservation for women at risk of medical- or age-associated loss of fertility; and (iii) simplification of donor oocyte cycles. Two basic approaches have been used for OCP: slow or controlled-rate freezing and vitrification. There have been a number of recent reviews on both slow freezing and vitrification for OCP (Borini and Coticchio, 2009; Borini et al., 2007; Chen and Yang, 2009; Cobo et al., 2009; Dessolle et al., 2009; Gook and Edgar, 2007; Porcu et al., 2008; Tao et al., 2009; Vajta et al., 2009). This article provides an overview of OCP using slow freezing or vitrification and highlights variables that may influence the outcomes derived using these techniques. Slow or controlled rate freezing The renaissance of OCP began in the early to mid 1990s when data showed reasonable thaw survival, fertilization and blastocyst development could be achieved with slow freezing (Gook et al., 1995). OCP accelerated with successful outcomes from Italy, where federal laws mandated the use of OCP versus embryo cryopreservation (Porcu et al., 2000) /$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 Oocyte freezing methods 315 Early papers on slow freezing used methods similar to those developed for embryo cryopreservation, with combinations of propanediol (PrOH) or dimethylsulphoxide (DMSO) and sucrose used. Oocytes were incubated for an appropriate time in cryoprotectant to cause cellular dehydration, loaded into an appropriate container (freezing vials or straws), cooled to a subzero temperature of about 7 C and then seeded to induce ice nucleation. Seeding is an essential part of slow freezing: by inducing extracellular ice formation, the remaining unfrozen solution becomes increasingly hyperosmotic, thereby increasing oocyte dehydration. The vial or straw is then cooled to a temperature of about 30 or 40 C, at which point the sample is plunged into liquid nitrogen for long-term storage. Slow-freezing protocols and optimization While reasonable survival rates were obtained (Gook et al., 1995; Porcu et al., 2000), soon alterations to the standard slow-freezing protocols were made to obtain higher post-thaw survival. These modifications were aimed at increasing oocyte dehydration to decrease the possibility of intracellular ice formation. Human oocytes are particularly susceptible to ice damage during freeze thaw, due to their large surface area/volume ratio and plasma membrane permeability properties (Paynter, 2005). Alterations in slow-freezing strategies that have influenced outcomes are discussed below. Sucrose concentration Increasing the sucrose concentration in the freezing and thawing solution from 0.1 mol/l to mol/l has shown an increased survival rate and implantation post thaw by increasing oocyte dehydration before freezing (Bianchi et al., 2007; Chen et al., 2004; Fabbri et al., 2001; Parmegiani et al., 2009). Temperature of equilibration Incubating oocytes in cryoprotectant at 37 C will increase permeability of the permeating cryoprotectants (Yang et al., 1998). Increasing pre-equilibration time Increasing the time that oocytes are held in equilibration media (e.g. from 10 to 20 min) may increase oocyte dehydration. Alteration of electrolyte composition Studies using mouse oocytes showed that substitution of sodium with choline in the freezing medium improved survival rates (Stachecki et al., 1998). The theory includes avoidance of a solute effect, in which excessive sodium accumulates in the cytoplasm, or direct effects of choline on stabilization of the plasma membrane. Use of sodium-depleted, choline-substituted freezing media have resulted in successful human OCP (Boldt et al., 2003, 2006; Quintans et al., 2002). Survival rates The published data on slow freezing indicates that survival rates can vary between 50 60% and 80 90% (Albani et al., 2008; Bianchi et al., 2007; Boldt et al., 2006; Borini and Coticchio, 2009; Grifo and Noyes, 2010; Konc et al., 2008). Likewise pregnancy rates with slow freezing seem to vary widely between clinics. Factors that could influence both thaw survival and pregnancy rates with slow freezing independent of freezing per se are comparable to vitrification and are discussed later in this article. Nevertheless, the results worldwide with slow freezing indicate reproducible survival and pregnancy rates that can be readily adapted for use by laboratories performing slow freezing of embryos (Borini et al., 2007; DeSantis et al., 2007). Summary The evolution of slow-freezing OCP involves strategies that account for the challenges set forth by the oocyte: its size and permeability. Hence, methods that provide more substantial dehydration have improved outcomes and adaptation of knowledge from studies of permeability properties should provide even better results from slow freezing (Paynter, 2005; Van den Abbeel et al., 2007). Vitrification Vitrification is a technique well suited to OCP precisely because of the difficulties the oocyte presents to traditional slow freezing; i.e. its surface area/volume ratio and permeability properties. Vitrification is a process by which an aqueous solution is converted to a solid, glass-like amorphous substance by rapid changes in temperature of the solution. Biophysical processes The biophysical processes of vitrification have been reviewed (Mullen and Fahy, 2011) and for OCP generally involve the following steps. Pre-incubation in relatively low concentrations of cryoprotectant Like with slow freezing, during the vitrification process, oocytes are placed for min in relatively low amounts of cryoprotectant (1 2 mol/l or about 15% v/v) to load the cytoplasm with cryoprotectant and to cause water to exit the cell through osmotic effects. This step helps to ensure that intracellular ice is not formed during the subsequent vitrification step. Exposure to vitrification solution After equilibration, the oocyte is exposed to relatively high concentrations of permeating cryoprotectant (of the order of 5 6 mol/l or 30% v/v) along with extracellular cryoprotectants such as sucrose (about mol/l). The purpose of the high cryoprotectant concentrations is to increase the viscosity of the cryoprotectant solution and to suppress ice nucleation such that intracellular ice formation is avoided. With the relatively high concentrations of cryoprotectant present during vitrification, as the sample is cooled the energy of the molecules in the solution is slowed to the point where an amorphous solid or glass is formed in the absence of ice formation. The balancing act in vitrification is that the high concentrations of cryoprotec-

3 316 J Boldt tant can be toxic to the cell, thus one has to use cryoprotectant concentrations that will allow vitrification to occur, but not at so high a concentration that the cell will die from toxic effects. The concentrations of cryoprotectant used, and the exposure time to cryoprotectant, are directly linked to the next variable in the vitrification process. Rapid temperature decrease With the commonly used vitrification solutions used to date for oocyte vitrification, cooling rates must be quite rapid to induce the vitrified state without ice formation. Vitrification, as currently practised, involves rapid cooling through the melting point of the solution (where ice forms) to the glass transition temperature (the temperature at which glass or amorphous solid forms). If the cooling rate is too slow, ice may form before vitrification takes place. Cooling rates are directly dependent upon the concentration and type of cryoprotectant in the solution. The need for rapid cooling has also led to the development and use of devices that allow for vitrification in very small sample volumes (see below). Rapid warming rate While much attention is paid to the first half of the vitrification process (i.e. the composition of cryoprotectant solutions, the time of exposure to cryoprotectants, choice of device for vitrification, cooling rates), it must be emphasized that the proper warming technique is essential for successful outcomes. The warming rate must be rapid enough to ensure that there is no ice formation or devitrification during the warming process. From a practical perspective, rapid warming rates are achieved through two mechanisms: (i) very small sample volumes (generally less than one microliter) are used to maximize heat transfer during both cooling and warming; and (ii) the device is plunged immediately from liquid nitrogen into warm (generally 37 C) media containing a high concentration of sucrose (generally about 1 mol/l). The rapid warming thus obtained is essential to preventing devitrification and subsequent ice nucleation during warming. One can do an adequate job during the cooling phase of vitrification, but if one is not careful to avoid premature warming (e.g. by allowing the vitrification device to remain in room air briefly before plunging) then ice will form and the oocyte will be damaged. Once warmed, intracellular cryoprotectant is then removed by stepping the oocytes through decreasing amounts of sucrose. Clinical utility The earliest attempts at human oocyte vitrification provided reproducibly high (>80%) survival rates (Kuleshova et al., 1999; Kuwayama et al., 2005; Yoon et al., 2000). A meta-analysis comparing vitrification to slow freezing showed higher survival and pregnancy rates with vitrification (Oktay et al., 2006). Vitrification became a popular method for human OCP and numerous studies now attest to its clinical utility (reviews by Cobo et al., 2009; Nagy et al., 2009a; Noyes et al., 2010; Vajta et al., 2009). The literature on human oocyte vitrification suggest several common themes. Survival rates Vitrification provides higher survival rates than slow freezing, with warming survival rates from 80% to over 90% routinely obtained. Cryoprotectant concentration Various concentrations (generally 30 40% or 5 6 mol/l) of penetrating cryoprotectants such as ethylene glycol (EG), DMSO or PrOH alone or in combination, together with sucrose as an extracellular osmolyte, have been reported to be effective for human OCP (Cao et al., 2009, Nagy et al., 2009b). Warming generally involves use of high (typically 1 mol/l) sucrose, with the oocyte stepped out through decreasing sucrose concentrations. While there are theoretical considerations for the use of specific cryoprotectants (Nagy et al., 2009a), the specific choice of cryoprotectant may not be especially critical as long as there is sufficient concentration of and exposure to cryoprotectant. Vitrification devices Different vitrification devices have been used successfully for OCP including CryoLeafs (Cao and Chian, 2009), CryoTops (Cobo et al., 2008a; Kuwayama et al., 2005; Nagy et al., 2009b), CryoLoops (Lieberman et al., 2003), Cryotips (Grifo and Noyes, 2010), electron microscopy grids (Yoon et al., 2000) and high-security vitrification straws (Camus et al., 2006; Boldt, data not shown). The common theme is that very small volumes of solution containing the oocyte(s) can be placed on or in the device, maximizing heat transfer during vitrification and warming. Vitrification devices can be subdivided into open systems (i.e. CryoLeafs, CryoTops, EM grids, CryoLoops) or closed systems (CryoTips, high-security vitrification straws). The open systems are called such because liquid nitrogen comes into direct contact with the vitrification solution and the oocytes during both cooling and long-term storage. This raises the question of whether there is a risk of contamination of the sample during the vitrification process, either from the liquid nitrogen itself or from cross-contamination by other specimens while in storage (Nagy et al., 2009a). While under experimental conditions, cross-contamination of cryopreserved reproductive cells has been reported (Bielanski et al., 2000), a recent review of this topic indicates that the actual contamination issue with open systems is not a practical concern when working under standard conditions used for assisted reproduction treatment and OCP (Pomeroy et al., 2010). Strategies to reduce any potential risk of contamination with open vitrification systems could include use of filtered or irradiated liquid nitrogen during the vitrification and/or storage process (Nagy et al., 2009a,b; Parmegiani et al., 2010) or vitrification and storage using nitrogen vapour versus liquid nitrogen (Cobo et al., 2010b; Eum et al., 2009; Larman et al., 2006). However, such approaches must balance the unproven risk of contamination under normal working conditions versus the risk of factors such as temperature fluctuations that could cause damage (Pomeroy et al., 2010). Placing the vitrification carrier in an outer straw or holder that could be sealed prior to long-term storage would be the most effective means to guard against any potential for disease transmission and would be accomplished by use of closed systems such as CryoTips or high-security vitrification straws. Such systems can be somewhat more cumber-

4 Oocyte freezing methods 317 some, requiring the additional step of sealing the vitrification carrier before plunging into liquid nitrogen. This raises the possibility of damage from improper heat sealing and potential toxicity to the oocyte because of the longer time it would be exposed to vitrification solution during sealing. In addition, cooling rates could potentially be affected by having an outer carrier surrounding the actual sample, which, if cryoprotectant concentration is held constant, could affect cooling rate and vitrification efficiency. Some of the most heated discussions one can have at a meeting indeed centre on which vitrification device to use! As with most technical aspects of assisted reproduction treatment, the choice of a vitrification device should be made independently by each user and will involve his or her comfort level with a particular system. It should be noted, however, that the above discussion centres on the bulk of current literature available on human oocyte vitrification, in which small sample volumes with specifically designed devices have been used. Such devices may not be absolutely necessary. Oocyte vitrification using conventional freezing straws has been reported (Isachenko et al., 2006). The choice of vitrification device and method must take into account the specifics of the cryoprotectants used, cooling rate and other variables (Mullen et al., 2008). With appropriately designed vitrification conditions (cryoprotectant choice and concentration, exposure times and cooling/warming rates), there is no reason to think that conventional freezing devices such as straws and/or vials could not be used routinely for oocyte vitrification. Time of vitrification Oocytes are placed into vitrification solution for a relatively brief time period (30 60 s) to avoid presumed cryoprotectant toxicity. Note again that, in general, the concentration of cryoprotectants is about 30 40% (v/v) or about 5 6 mol/l, with 0.5 mol/l extracellular sucrose. Whether there is a maximum time for exposure of human oocytes to such vitrification solutions without incurring toxic damage is an open question. One must balance using enough time to equilibrate in vitrification solution to ensure success against potential toxicity. In the author s experience, one can go to at least 60 s exposure without adversely affecting survival and subsequent embryonic development, whereas too limited an exposure time (less than 30 s) may cause higher damage rates post warming. Technical demands Vitrification is a more technically demanding process and requires more experience to master than slow freezing. The technical demands of slow freezing are generally restricted to the seeding process. For vitrification, one must accurately and quickly pipette small volumes of viscous vitrification media containing the oocyte(s) onto the carrier. Summary Fertilization, embryo development and pregnancy rates with vitrification can approach those obtained with fresh oocytes (Almodin et al., 2010; Grifo and Noyes, 2010; Rienzi et al., 2010), particularly in studies using donor oocytes where presumably oocyte quality is higher (Cobo et al., 2008c, 2010a; Nagy et al., 2009b). However, as with any assisted reproductive method success rates can vary dependant on a number of factors (see below). Studies comparing slow freezing and vitrification Oktay et al. (2006) published a meta-analysis of OCP studies published from January 1997 to June The conclusion reached from this study was that vitrification seemed to be a more efficient technique for OCP. There are some pitfalls with respect to such a meta-analysis such as differences in study design and other confounders and, as such, prospective, randomized trials comparing vitrification and slow freezing would be the best way to determine the relative efficiency of each technique. Surprisingly, there is a paucity of studies in the literature directly comparing the two methods. Fadini et al. (2009) compared slow freezing to vitrification in consecutive cycles, with 286 slow freeze versus 59 vitrification warming cycles analysed. Vitrification provided higher survival (78.9% versus 57.9%), pregnancy (18.7% versus 7.6%) and implantation rates (9.3% versus 4.3%) compared with slow freezing. Cao et al. (2009) compared vitrification and slow freezing using 1.5 mol/l PrOH/0.3 mol/l sucrose for slow freezing and 15% EG/15% PrOH/0.5 mol/l sucrose for vitrification on CryoLeafs. These studies were performed when a patient had >15 mature oocytes and appeared to randomize oocytes to either vitrification or slow freezing. After thawing 123 slowly frozen oocytes and warming 292 vitrified oocytes, vitrification provided significantly higher survival rates (91.8% versus 61.0%) and a higher percentage of high-quality embryos (42.3% versus 24.0%) and blastocysts (33.1% versus 12.0%). These data suggest vitrification improves embryonic development rates versus slow freezing. The study centre has observed that embryos derived from slow-frozen oocytes have lower day-3 cell numbers than those derived from either sibling fresh or vitrified oocytes Schumacher and Boldt (data not shown) and Magli et al. (2010) have reported slower developmental rates for slow-frozen oocytes versus sibling oocytes after thawing and subsequent fertilization. In contrast to the above studies, Grifo and Noyes (2010) used slow freezing for 159 oocytes versus vitrification for 163 oocytes, obtained from patients undergoing non-donor IVF. This study involved 23 patients of which 21 had their oocytes divided and preserved by either slow freezing (using PrOH/sucrose) or vitrification (using EG/DMSO/0.5 mol/l sucrose). When thawing/warming was performed, they obtained identical survival rates (88% for slow versus 95% for vitrification), with better blastocyst development with slow-frozen oocytes (52% blastocyst development with slow-frozen versus 35% for vitrified; P = 0.02). The majority of embryo transfers in this study involved embryos from both types of OCP, so it was difficult to determine if either method was superior in terms of pregnancy outcomes. In the study centre, overall survival rates are 80.5% (136/169) with vitrification versus 55.4% (316/570) with sodium-depleted slow freezing and pregnancy rates are

5 318 J Boldt higher with vitrification (36.8% per patient warmed with vitrification versus 28.6% per patient thawed with slow freezing). Number of cancelled cycles, either from no survival post thaw or lack of fertilization or embryo development, was higher with slow freezing: 21% of cycles were cancelled with slow freeze versus 11% with vitrification (Boldt, data not shown). The data did not involve randomization of oocytes within a single cohort; however, they are derived from consecutive series in which only one or the other method was used. Summary Thus, to summarize comparisons between slow freezing and vitrification, data are somewhat lacking. There are only a few studies that attempt to directly compare each technique, and while several show a benefit of vitrification over slow freezing, in one of these studies survival rates were identical between vitrification and slow freezing, with enhanced fertilization and blastocyst development rates when using slow freezing (Grifo and Noyes, 2010). Nevertheless, the trend for OCP is that vitrification has become more the method of choice. Why is this? One can point to: (i) results of the meta-analysis done by Oktay et al. (2006) suggesting greater efficiency for vitrification; (ii) comparative studies of vitrification that in general show higher survival and pregnancy rates than slow freezing (Cao et al., 2009; Fadini et al., 2009); and (iii) studies generally showing higher survival rates with vitrification (Cao and Chian, 2009; Cobo et al., 2008a; Kuwayama et al., 2005; Nagy et al., 2009b) than slow freezing (Albani et al., 2008; Boldt et al., 2006; Konc et al., 2008). True validation of the superiority of one method over the other would necessitate prospective, randomized trials in which oocytes from a single cohort were randomized into each technique, with subsequent thawing/warming and embryo transfer involving only embryos from one protocol versus the other to obtain specific pregnancy and implantation rates for each group. For practical reasons, that is almost impossible to achieve as trials on human assisted reproduction treatment. But are there aspects of OCP, other than simply survival and/or pregnancy rates that might lead one to adopt a specific approach? These are discussed in the following section. Cytoplasmic effects of OCP: slow freezing versus vitrification There are data that suggest vitrification offers an advantage over slow-freezing techniques when considering cytoplasmic effects on the oocyte during freezing and subsequent warming. There has been much discussion and concern over the effects of OCP on the oocyte s metaphase-ii meiotic spindle apparatus. Early studies warned of meiotic spindle damage during oocyte freezing (Pickering et al., 1990) but did not take into account the dynamics of microtubule disassembly and reassembly. The meiotic spindle will disassemble during cooling, but upon thawing or warming will reassemble within several hours (Bromfield et al., 2009; Chen and Yang, 2009; Ciotti et al., 2009; Cobo et al., 2008b; Coticchio et al., 2006; Gao et al., 2009; Rienzi et al., 2004). In studies comparing spindle dynamics in slow-frozen versus vitrified oocytes, data suggest that spindle recovery is accelerated in vitrified versus slow-frozen oocytes (Chen and Yang, 2009; Ciotti et al., 2009) and a higher percentage of spindles are maintained after cryopreservation and subsequent rewarming in vitrified versus slow-frozen oocytes (Cao et al., 2009). It should be noted that different techniques, such as Polscope versus fluorescent staining of tubulin with fluorescent/confocal microscopy, may yield somewhat differing data on spindle formation. Polscope is applicable for viewing the living cell but may not yield comparable detailed images of oocytes fixed and examined by fluorescent/confocal techniques (Coticchio et al., 2010). Also, spindle formation may be divorced from normality of chromosomal distribution on the spindle, with some data suggesting abnormal chromosomal rearrangement after OCP (Cao et al., 2009; Coticchio et al., 2009). Ultrastructural analyses of slow-frozen oocytes suggests that there is loss of cortical granules as well as an increase in cytoplasmic vacuolization after freezing and thawing (Nottola et al., 2007) and with vitrification vacuolization may be less pronounced, although atypical mitochondria and/or smooth endoplasmic reticulum were noted (Nottola et al., 2009). There are also data suggesting vitrification is preferable based on metabolomic and/or proteomic considerations (Gardner et al., 2007). Hence the bulk of the current data on cytoplasmic effects of OCP favour vitrification as being less damaging; however, clearly more data is needed to more fully understand the cellular sequelae of either OCP method. Other factors influencing OCP One of the most frustrating things about developing a new technique for the embryology laboratory is comparing one s own data to others; new techniques can be implemented, only to have them not perform to the same level as published data. OCP is particularly susceptible to this. In addition to the technical challenges offered by the uniqueness of the oocyte and from preservation methods such as vitrification, there are other factors that do not gain either sufficient attention or require more active investigation to optimize outcomes with OCP. Oocyte selection While one of the earliest reports of successful slow freezing involved immature oocytes (Tucker et al., 1998), the norm today is to cryopreserve mature, metaphase-ii oocytes. But which oocytes should be cryopreserved? This brings up the issue of what defines a good-quality versus a poorquality oocyte. An indication of how oocyte quality might influence OCP outcomes can be shown by studies in which use of cryopreserved donor oocytes (i.e. presumably good-quality oocytes) can yield high survival and pregnancy rates similar to those obtained with fresh oocytes (Cobo et al., 2008c, 2010a; Nagy et al., 2009b). Similar to results from frozen-embryo cycles, it stands to reason that if one preserves oocytes regardless of quality, then one might reasonably expect that outcomes would be adversely affected. Some laboratories select for oocyte quality pre-freeze by rejecting oocytes with abnormalities such as excessive gran-

6 Oocyte freezing methods 319 ularity, vacuoles, thickened zonae or fragmented polar bodies. The study centre only rejects eggs with clearly granular foci or excessive vacuoles. The part that oocyte selection would play in improving results merits further research. Stimulation-related factors Stimulation factors go hand in hand with oocyte quality. While it may stand to reason that poor responders would yield oocytes that would not perform as well with OCP, data is lacking. The method of stimulation (e.g. the use of gonadotrophin-releasing hormone agonist versus antagonist) has not been rigorously evaluated. Parenthetically, the study centre has noticed that oocyte survival rates post thaw/warming tend to be lower when oocytes are cryopreserved in cycles where the patient has been hyperstimulated and had more than 20 oocytes retrieved (data not shown). This may be due to an inherent effect on oocyte quality. Timing of cryopreservation relative to retrieval The timing of when oocytes are cryopreserved relative to retrieval can have effects on overall outcomes. Parmegiani et al. (2009) showed when oocytes were slow frozen within 2 h of retrieval, embryo quality, pregnancy and implantation rates were significantly improved over cycles in which oocytes were preserved after 2 h. Thus, in a busy laboratory that may not have the staffing needed to process oocytes quickly, such a source of variation could easily influence outcomes. Number of oocytes thawed/warmed One of the difficulties in comparing OCP data is that many studies arise from programmes where restrictions are placed on the number of oocytes thawed/warmed. For example, many studies are from groups in Italy where, by law, the number of oocytes that can be thawed/warmed and/or inseminated are restricted. In the study centre s programme, patients that request egg cryopreservation do so because they are uncomfortable or have ethical concerns with cryopreserving embryos, so only the number of oocytes that would yield a number of embryos felt to be appropriate for transfer in an individual patient are thawed/warmed, such that excess embryos are not created that would put the couple in an untenable ethical dilemma. Comparing pregnancy outcomes from such programmes to those that do not restrict the number of eggs thawed to a degree compares apples and oranges. While one can compare survival rates, comparison of pregnancy rates is not appropriate in that pregnancy rates would be expected to be higher when starting out with more embryos to select from, just as is seen with fresh IVF. Embryo transfer Many of the studies on OCP have performed embryo transfers on days 2 3. The study centre has adopted this approach because the number of eggs thawed/warmed are limited and, by day 3, the number of embryos for transfer have been generated. Thus, it is presumed there is no selection advantage to pursuing day-5 transfers. Others have seen good pregnancy rates by culturing embryos derived from cryopreserved oocytes to day 5 (Grifo and Noyes, 2010), but in general there are more fertilized eggs generated in these laboratories than in the study centre. Given the strong interest in elective single embryo transfer, there may be merit in pursuing day-5 transfers as the routine and this approach could yield more information as to factors that could influence outcomes from OCP. The number of embryos transferred, as with standard IVF, is also a variable to be considered, with wide variance in the numbers transferred per patient depending on the study (Grifo and Noyes, 2010; Yoon et al., 2007). General factors The downstream variables that can affect pregnancy rate with OCP are the same as those with fresh assisted reproduction treatment: patient selection, technical expertise with intracytoplasmic sperm injection, laboratory environment, proficiency in embryo transfer and the like. Thus, one laboratory may achieve a 50% pregnancy rate with OCP and others a 25% rate, despite both laboratories being extremely proficient in the method and having similar oocyte survival rates. Conclusion The future of OCP is bright. Development of techniques such as vitrification has yielded high survival rates after warming, with data supporting the concept of higher survival and pregnancy rates after vitrification versus slow freezing (Cao et al., 2009; Fadini et al., 2009) as well as pregnancy rates that rival those obtained with either frozen-embryo transfer or fresh IVF (Cobo et al., 2008c; Nagy et al., 2009a). Studies on cytoplasmic effects of OCP seem to favour vitrification as having less damaging effects on the meiotic spindle and cytoplasm. But prospectively randomized trials comparing the two methods are sparse and properly designed experimental trials are needed to definitively show an advantage of a specific method. While vitrification appears to be the hot technique now, does this mean that no one will do slow freezing anymore? No. The choice of a specific freezing method will depend on laboratory and programme specific factors. For example, imagine a clinic with two embryologists that have four egg retrieval cycles on a given day. In two of the cycles, OCP is to be performed and between the two patients there are 50 oocytes to be preserved. Given such a scenario, and the workload associated with vitrification of that many oocytes together with the other cases and associated workload (clearing of eggs for intracytoplasmic sperm injection, micromanipulation, etc.), slow freezing might be a more appropriate alternative, in that the bulk of the time spent for the freezing process is spent within the confines of a slow-freezing unit, rather than at the bench performing vitrification. In such a scenario, slow freezing might have advantages with respect to subsequent embryo development (Parmegiani et al., 2009). Slow freez-

7 320 J Boldt ing has contributed greatly to reproductive medicine (Borini and Coticchio, 2009; Borini et al., 2007; DeSantis et al., 2007) and can provide excellent outcomes (Grifo and Noyes, 2010). Cryopreserved donor egg banks are now a reality (Akin et al., 2007; Cobo et al., 2010a; Nagy et al., 2009b), and more patients are electing to choose OCP for fertility preservation (Porcu et al., 2008). One can envision a scenario where OCP is used to limit the number of embryos routinely cryopreserved, such that the issue of couples having excess embryos cryopreserved after they have completed their family can be decreased. Outcome data suggest there is no increase in birth abnormalities using cryopreserved oocytes, though perhaps the potential for an increase in preterm labour (Chian et al., 2008; Noyes et al., 2009; Wennerholm et al., 2009), and use of molecular genetic analysis techniques could lead to improved outcomes (Keskintepe et al., 2009). As OCP becomes more readily available and justifiably loses its experimental tag (Noyes et al., 2010), it will inevitably find more widespread application in reproductive medicine. References Akin, J.W., Bell, K.A., Thomas, D., Boldt, J., Initial experience with a donor egg bank. Fertil. Steril. 88 (497), e1 e4. Albani, E., Barbieri, J., Novara, P.V., Smeraldi, A., Scaravelli, G., Levi Setti, P.E., Oocyte cryopreservation. Placenta 29 (Suppl. 8), Almodin, C.G., Minguetti-Camara, V.C., Paixao, C.L., Pereira, P.C., Embryo development and gestation using fresh and vitrified oocytes. Hum. Reprod. 25, Bianchi, V., Coticchio, G., Distratis, V., Di Gusto, N., Flamigni, C., Borini, A., Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod. Biomed. Online 14, Bielanski, A., Nadin-Davis, S., Sapp, T., Lutze-Wallace, C., Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 40, Boldt, J., Cline, D., McLaughlin, D., Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum. Reprod. 18, Boldt, J., Tidswell, N., Sayers, A., Kilani, R., Cline, D., Human oocyte cryopreservation: 5 year experience with a sodium-depleted slow freezing method. Reprod. Biomed. Online 13, Borini, A., Coticchio, G., The efficiency and safety of human oocyte cryopreservation by slow cooling. Semin. Reprod. Med. 27, Borini, A., Bianchi, V., Bonu, M.A., Sciajno, R., Sereni, E., Cattoli, M., Mazzone, S., Trevisi, M.R., Iadarola, I., Distratis, V., Nalone, M., Coticchio, G., Evidence-based outcome of oocyte slow cooling. Reprod. Biomed. Online 15, Bromfield, J.J., Coticchio, G., Hutt, K., Meiotic spindle dynamics in human oocytes following slow cooling cryopreservation. Hum. Reprod. 24, Camus, A., Clairaz, P., Ersham, A., Van Kappel Al, Savic, G., Staub, C., The comparison of the process of five different vitrification devices. Gnecol. Obstet. Fertil. 34, Cao, Y.X., Chian, R.C., Fertility preservation with immature and in vitro matured oocytes. Semin. Reprod. Med. 27, Cao, Y.X., Xing, Q., Li, L., Cong, L., Zhang, Z.G., Wei, Z.L., Zhou, P., Comparison of survival and embryonic development in human oocytes cryopreserved by slow freezing and vitrification. Fertil. Steril. 92, Chen, S.U., Yang, Y.S., Slow freezing or vitrification of oocytes: their effects on survival and meiotic spindles, and the time schedule for clinical practice. Taiwan. J. Obstet. Gynecol. 48, Chen, Z.J., Li, M., Li, Y., Zhao, L.X., Tang, R., Sheng, Y., Gao, X., Chang, C.H., Feng, H.L., Effect of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different stages of maturity. Hum. Reprod. 19, Chian, R.C., Huang, J.Y., Tan, S.L., Lucena, E., Saa, A., Rojas, A., Ruvalcaba Castellon, L.A., Garcia, A.M.I., Montoya Sarmiento, J.E., Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod. Biomed. Online 16, Ciotti, P.M., Porcu, E., Notarangelo, L., Magrini, O., Bazzocchi, A., Venturoli, S., Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing. Fertil. Steril. 91, Cobo, A., Bellver, J., Domingo, J., Perez, S., Crespo, J., Pellicer, A., Remohi, J., 2008a. New option in assisted reproductive technology: the CryoTop method of oocyte vitrification. Reprod. Biomed. Online 17, Cobo, A., Perez, S., De los Santos, M.J., Zulategui, J., Domingo, J., Remohi, J., 2008b. Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes. Reprod. Biomed. Online 17, Cobo, A., Kuwayama, M., Perez, S., Ruiz, A., Pellicer, A., Remohi, J., 2008c. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the CryoTop method. Fertil. Steril. 89, Cobo, A., Vajta, G., Remohi, J., Vitrification of human mature oocytes in clinical practice. RBM Online 19 (Suppl. 4), Cobo, A., Meseguer, M., Remohi, J., Pellicer, A., 2010a. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum. Reprod. 25, Cobo, A., Romero, J.L., Perez, S., de los Santos, M.J., Meseguer, M., Remohi, J., 2010b. Storage of human oocytes in the vapor phase of nitrogen. Fertil. Steril. 94, Coticchio, G., DeSantis, L., Rossi, G., Borini, A., Albertini, D., Scaravelli, G., Alecci, C., Bianchi, V., Nottola, S., Cecconi, S., Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow cooling cryopreservation. Hum. Reprod. 21, Coticchio, G., Bromfeld, J.J., Sciajno, R., Gambardella, A., Scaravelli, G., Borini, A., Albertini, D.F., Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes. Reprod. Biomed. Online 19, Coticchio, G., Sciajno, R., Hutt, K., Bromfield, J., Borini, A., Albertini, D.F., Comparative analysis of the metaphase II spindle of human oocytes through polarized light and confocal microscopy. Fertil. Steril. 93, DeSantis, L., Cino, I., Coticchio, G., Fusi, F.M., Papaleo, E., Rabbelotti, E., Brigante, C., Borini, A., Ferrari, A., Objective evaluation of the viability of cryopreserved oocytes. Reprod. Biomed. Online 15, Dessolle, L., LaRouziere, V., Ravel, C., Berthaut, I., Antoine, J.M., Mandelbaum, J., Slow freezing and vitrification of human mature and immature oocytes. Gynecol. Obstet. Fertil. 37, Eum, J.H., Park, J.K., Lee, W.S., Cha, K.R., Yoon, T.K., Lee, D.R., Long term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling. Fertil. Steril. 91, Fabbri, R., Porcu, E., Marsella, T., Rocchetta, G., Venturoli, S., Flamigini, C., Human oocyte cryopreservation: new

8 Oocyte freezing methods 321 perspectives regarding oocyte survival. Hum. Reprod. 16, Fadini, R., Brambillasca, F., Renzini, M.M., Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Reprod. Biomed. Online 19, Gao, S., Li, Y., Gao, X., Hu, J., Yang, H., Chen, Z.J., Spindle and chromosome changes of human MII oocytes during incubation after slow freezing/fast thawing procedures. Reprod. Sci. 16, Gardner, D.K., Sheehan, C.B., Rienzi, L., Katz-Jaffe, M., Larman, M.G., Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 67, Gook, D.A., Edgar, D.H., Human oocyte cryopreservation. Hum. Reprod. Update 13, Gook, D.A., Schiewe, M., Osborn, S.M., Asch, R.H., Jansen, R.P., Johnston, W.I., Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum. Reprod. 10, Grifo, J.A., Noyes, N., Delivery rate using cryopreserved oocytes is comparable to conventional in vitro fertilization using fresh oocytes: potential fertility preservation for female cancer patients. Fertil. Steril. 93, Isachenko, V., Montag, M., Isachenko, E., Dessole, S., Nawroth, F., van der Ven, H., Aseptic vitrification of human germinal vesicle oocytes using dimethylsulfoxide as a cryoprotectant. Fertil. Steril. 85, Keskintepe, L., Agca, Y., Sher, G., Keskintepe, M., Massarani, G., High survival rate of metaphase II oocytes after first polar body biopsy and vitrification: determining the effect of previtrification conditions. Fertil. Steril. 92, Konc, J., Kanyo, K., Varga, E., Kriston, R., Cseh, S., Births resulting from oocyte cryopreservation using a slow freezing protocol with propanediol and sucrose. Syst. Biol. Reprod. Med. 54, Kuleshova, L., Gianaroli, L., Magli, C., Ferraretti, A., Trounson, A., Birth following vitrification of a small number of human oocytes: case report. Hum. Reprod. 14, Kuwayama, M., Vajta, G., Kato, O., Leibo, S.P., Highly efficient vitrification method for cryopreservation of human oocytes. Reprod. Biomed. Online 11, Larman, M.G., Sheehan, C.B., Gardner, D.K., Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact. Reprod. Biomed. Online 12, Lieberman, J., Tucker, M.J., Sills, E.S., Cryoloop vitrification in assisted reproduction: analysis of survival rates in >1000 human oocytes after ultra-rapid cooling with polymer augmented cryprotectants. Clin. Exp. Obstet. Gynecol. 30, Magli, M.C., Lappi, M., Ferraretti, A.P., Capoti, A., Ruberti, A., Gianroli, L., Impact of oocyte cryopreservation on embryo development. Fertil. Steril. 93, Mullen, S.F., Fahy, G.M., Fundamental aspects of vitrification as a method of reproductive cell, tissue, and organ cryopreservation. In: Donnez, J., Kim, S.S. (Eds.), Principles and Practice of Fertility Preservation. Cambridge University Press, New York, pp Mullen, S.F., Li, M., Li, Y., Chen, Z.J., Critser, J.K., Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the application toward vitrification. Fertil. Steril. 89, Nagy, Z.P., Chang, C.C., Shapiro, D.B., Bernal, D.P., Kort, H.I., Vajta, G., 2009a. The efficacy and safety of human oocyte vitrification. Semin. Reprod. Med. 27, Nagy, Z.P., Chang, C.C., Shapiro, D.B., Bernal, D.P., Elsner, C.W., Mitchell-Leef, D., Toledo, A.A., Kort, H.I., 2009b. Clinical evaluation of the efficiency of an oocyte donation program using egg cryobanking. Fertil. Steril. 92, Nottola, S.A., Macchiarelli, G., Coticchio, G., Bianchi, S., Cecconi, S., De Santis, L., Scaravelli, G., Flamigni, C., Borini, A., Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Hum. Reprod. 22, Nottola, S.A., Coticchio, G., Sciajno, R., Gambardella, A., Maione, M., Scaravelli, G., Bianchi, S., Macchiarelli, G., Borini, A., Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop. Reprod. Biomed. Online 19 (Suppl. 3), Noyes, N., Porcu, E., Borini, A., Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod. Biomed. Online 18, Noyes, N., Boldt, J., Nagy, Z.P., Oocyte cryopreservation: is it time to remove its experimental label? J. Assist. Reprod. Genet. 27, Oktay, K., Cil, A.P., Bang, H., Efficiency of oocyte cryopreservation: a meta-analysis. Fertil. Steril. 86, Parmegiani, L., Bertocci, F., Garello, C., Salvarani, M.C., Tambuscio, G., Fabbri, R., Efficiency of human oocyte slow freezing: results from five assisted reproduction centers. Reprod. Biomed. Online 18, Parmegiani, L., Accorsi, A., Cognini, G.E., Cognini, G.E., Bernardi, S., Trollo, E., Filicore, M., Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil. Steril. 94, Paynter, S.J., A rational approach to oocyte cryopreservation. Reprod. Biomed. Online 10, Pickering, S.J., Braude, R.P., Johnston, M.H., Cant, A., Currie, J., Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril. 54, Pomeroy, K.O., Harris, S., Conaghan, J., Papadakis, M., Centola, G., Basuray, R., Battaglia, D., Storage of cryopreserved reproductive tissues: evidence that cross-contamination of infectious agents is a negligible risk. Fertil. Steril. 94, Porcu, E., Fabbri, R., Damiano, G., Giunchi, S., Fratto, R., Ciotti, P.M., Venturoli, S., Flamigni, C., Clinical experience and applications of oocyte cryopreservation. Mol. Cell. Endocrinol. 169, Porcu, E., Buzzouchi, A., Noturangelo, L., Paradisi, R., Landolfo, C., Venturoli, S., Human oocyte cryopreservation in infertility and oncology. Curr. Opin. Endocr. Diab. Obes. 15, Quintans, C.J., Donaldson, M.J., Betrtolino, M.V., Pasqualini, R.S., Birth of two babies using oocytes that were cryopreserved in a choline based freezing medium. Hum. Reprod. 17, Rienzi, L., Martinez, F., Ubaldi, F., Minasi, M.G., Iacobelli, M., Tesarik, J., Greco, E., Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum. Reprod. 19, Rienzi, L., Romano, S., Albricci, L., Maggiuli, R., Capalbo, A., Baroni, E., Colamaria, S., Sapienza, S., Ubaldi, F., Embryo development of fresh versus vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum. Reprod. 25, Stachecki, J.J., Cohen, J., Willadsen, S.M., Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, Tao, T., Zhang, W., DelValle, A., Human oocyte cryopreservation. Curr. Opin. Obstet. Gynecol. 21, Tucker, M.J., Wright, G., Morton, P.C., Massey, J.B., Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil. Steril. 70,

9 322 J Boldt Vajta, G., Nagy, Z.P., Cobo, A., Conceicao, J., Yovich, J., Vitrification in assisted reproduction: myths, mistakes, disbeliefs, and confusion. Reprod. Biomed. Online 19 (Suppl. 3), 1 7. Van den Abbeel, E., Schneider, U., Liu, J., Agca, Y., Critser, J.K., Van Steirteghem, A., Osmotic responses and tolerance limits to changes in external osmolalities and oolemma characteristics of human in vitro matured MII oocytes. Hum. Reprod. 22, Wennerholm, U.-B., Soderstrom-Anttila, C., Bergh, K., Aittomaki, K., Hazecamp, J., Nygren, K.-G., Selbing, A., Loft, A., Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data. Hum. Reprod. 24, Yang, D.S., Winslow, K.L., Blohm, P.I., Improved survival rate after cryopreservation of human fresh and aged unfertilized oocytes using a specially designed oocyte cryopreservation regime. Fertil. Steril. 70 (Suppl. 1), S86 (abstract). Yoon, T.K., Chang, H.M., Lim, J.M., Han, S.Y., Ko, J.J., Cha, K.Y., Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil. Steril. 74, Yoon, T.K., Lee, D.R., Cha, S.K., Chung, H.M., Lee, W.S., Cha, K.Y., Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil. Steril. 88, Declaration: The authors report no financial or commercial conflicts of interest. Received 5 August 2010; refereed 7 October 2010; accepted 23 November 2010.