Abstract. Introduction. RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 21 August 2009

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 21 August 2009 Article Increasing dehydration of human cleavagestage embryos prior to slow cooling significantly increases cryosurvival Dr David Edgar is a graduate of the University of Glasgow and University College London. He completed a post-doctoral fellowship in Cambridge before being appointed as Lecturer at the University of Dundee where he established the Assisted Conception Laboratories in the 1980s. Since 1994 he has been Scientific Director of Reproductive Services, Melbourne IVF, Australia and is also Senior Lecturer in the Department of Obstetrics and Gynaecology, University of Melbourne. His main scientific interests lie in the areas of preimplantation embryonic development and the application of cryopreservation in reproductive medicine. Dr David Edgar David H Edgar 1,2,3, Jerustin Karani 1, Debra A Gook 1,2 1 Reproductive Services, Melbourne IVF, Royal Women s Hospital, Cnr Grattan Street and Flemington Road, Parkville, Vic. 3052, Australia; 2 Department of Obstetrics and Gynaecology, University of Melbourne, Australia 3 Correspondence: david.edgar@mivf.com.au Abstract Increasing the proportions of embryos and blastomeres which survive cryopreservation would be expected to make a significant contribution to the outcome of assisted reproduction treatment. Despite this, the methodology used for slow cooling of human cleavage-stage embryos has remained largely unchanged for over two decades. Previous studies have demonstrated the value, in terms of cryosurvival, of increasing the extent of intracellular dehydration by increasing the concentration of non-permeating cryoprotectant prior to slow cooling of oocytes and embryos which have been biopsied for preimplantation genetic diagnosis. The present study extends the use of this approach to the slow cooling of non-biopsied day-2 embryos. Dehydration in the presence of 0.2 mol/l sucrose significantly increased the proportions of surviving embryos, surviving blastomeres and fully intact embryos (92.6%, 91.1%, and 80.4%, respectively) relative to those observed after dehydration in 0.1 mol/l sucrose (78.5%, 74.1%, and 54.6%, respectively, all P < 0.001). Post-thaw resumption of mitosis in vitro and implantation were not adversely affected by the increased prefreeze dehydration. This improved method for slow cooling of cleavage-stage embryos should have a major impact on clinical outcome. Keywords: cryoprotectant, cryosurvival, dehydration, human embryo, slow cooling, sucrose Introduction Cryopreservation of human embryos is now a routine component of clinical IVF treatment. It allows potential clinical use of all the embryos generated from a cycle of ovarian stimulation without the need to transfer multiple embryos simultaneously and, as a consequence, can lead to a reduction in the incidence of multiple pregnancy. The value of embryo cryopreservation has become more apparent as a result of an increasing trend towards transferring a single fresh embryo (Tiitinen et al., 2001). Importantly, it has been shown that oocyte collection cycles in which single embryo transfer is performed can give rise to equivalent cumulative pregnancy rates to those achieved following the transfer of two embryos when the outcomes from transfer of cryopreserved embryos are included (Thurin et al., 2004; Lundin and Bergh, 2007). The implantation potential of cryopreserved cleavage-stage embryos is dependent on a number of embryonic factors, including the prefreeze development of the embryo (Edgar et al., 2000, 2007), blastomere survival following freezing and thawing (Van den Abbeel et al., 1997; Burns et al., 1999; Edgar et al., 2000, 2007; Guerif et al., 2002) and post-thaw resumption of mitosis in vitro (Van der Elst et al., 1997; Ziebe et al., 1998; Guerif et al., 2002; Edgar et al., 2007). The impact of cryopreservation on the implantation potential of a population of thawed embryos is, however, predominantly related to blastomere survival since it 521 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 522 has been demonstrated that fully intact thawed embryos have similar implantation rates to equivalent fresh embryos (Edgar et al., 2000). Increasing blastomere survival would, therefore, be expected to result in increased clinical efficiency of embryo cryopreservation. Although the first reports of pregnancy and live birth from human cryopreserved embryos involved the use of dimethylsulphoxide as a permeating cryoprotectant, by far the most common current method employs 1,2-propanediol (PROH) as the permeating cryoprotectant in conjunction with the non-permeating cryoprotectant sucrose (Lassalle et al., 1985). Although PROH is routinely used at supramolar concentrations (predominantly 1.5 mol/l) during dehydration of cleavage-stage embryos prior to slow cooling, sucrose has conventionally been used at the significantly lower concentration of 0.1 mol/l. While over three-quarters of all cleavage-stage embryos can survive with at least 50% of their blastomeres intact after slow cooling under the above conditions, the proportion of fully intact embryos is closer to 50% (Edgar et al., 2000; Balaban et al., 2008). Recently, vitrification has been shown to increase the proportion of surviving and fully intact cleavage-stage embryos compared with traditional slow cooling (Balaban et al., 2008) but this technology involves exposure to extremely high concentrations (c. 5 mol/l) of permeating cryoprotectants and is, as yet, unproven in terms of safety. Follow-up data on babies born from embryos cryopreserved after slow cooling is reassuring (Wada et al., 1994; Sutcliffe et al., 1995; Wennerholm et al., 1998; Aytoz et al., 1999). Similar data will be required on outcomes following vitrification. Similarly, specific considerations relating to the risk of infectious disease transmission during cryostorage must be addressed in vitrification systems (Vajta and Nagy, 2006). Despite the fact that 1.5 mol/l PROH in the presence of 0.1 mol/l sucrose has been applied as a routine protocol for dehydration of human cleavage-stage embryos prior to slow cooling for over two decades, there is no direct evidence to suggest that the level of dehydration achieved is optimal for cryosurvival. It has been suggested from studies on human oocytes (Fabbri et al., 2001) and human embryos which have been biopsied for preimplantation genetic diagnosis (Jericho et al., 2003) that increasing prefreeze dehydration by increasing the sucrose concentration may improve cryosurvival after slow cooling. Since the rate of post-thaw rehydration is usually regulated by reducing the extracellular concentration of PROH in the presence of an elevated concentration of sucrose, it is also advisable to maintain this differential by increasing the extracellular concentration of sucrose in the rehydration phase (Jericho et al., 2003; Bianchi et al., 2007). The aim of this study was to determine whether increasing the extent of prefreeze dehydration by increasing the sucrose concentration from 0.1 mol/l to 0.2 mol/l increases the cryosurvival of day-2 embryos. Materials and methods All embryos included in this study (n = 945) were cryopreserved in the study centre s laboratories between October 2007 and July 2008 and thawed prior to the end of July All embryos were cryopreserved on day 2 post insemination using PROH and sucrose as cryoprotectants (Lassalle et al., 1985). Patients having oocyte collections in the unit are randomly allocated to one of two theatre sites, each with its own adjacent laboratory. Patient allocation was based on logistic considerations unrelated to patient characteristics. Methodology and historical control values for all outcomes, including cryopreservation, are identical in the two laboratories and all the embryology staff regularly rotate between both laboratories. All patients having embryos cryopreserved signed a written consent form approved by the institutional research and ethics committee. Embryos cryopreserved over the study period in one laboratory were dehydrated prior to cryopreservation in the presence of 0.1 mol/l sucrose and in the other laboratory in the presence of 0.2 mol/l sucrose. The mean female age at the time of embryo cryopreservation was 37.1 years and was not significantly different between the 0.1 mol/l sucrose and the 0.2 mol/l sucrose groups. The cryopreservation methods used were as previously described (Edgar et al., 2007). Only embryos consisting of less than 30% fragmentation were considered suitable for cryopreservation. Embryos were dehydrated in a single step in the presence of 1.5 mol/l PROH + sucrose for 10 min at room temperature before being loaded singly into plastic straws and transferred to programmable freezers (Kryo 10 Series; Planer Products, Sunbury-on-Thames, UK). Straws were cooled at a rate of 2 C/min to 7 C, at which point ice nucleation was induced manually. Cooling was then continued at rates of 0.3 C/min to 30 C and 50 C/ min to 150 C before plunging and storing in liquid nitrogen. Embryos were thawed rapidly by exposure to air for 30 s followed by immersion in a water bath at 30 C for 45 s and rehydrated by sequential incubation in 0.5 mol/l, 0.2 mol/l and 0.0 mol/l sucrose (each for 5 min at room temperature) before washing and overnight incubation at 37 C in Quinn s Advantage Cleavage Medium containing 4 mg human serum albumin per ml (SAGE, Trumball, USA). Blastomere survival was assessed after post-thaw rehydration and resumption of mitosis was assessed after overnight culture. Embryos were transferred as previously described (Edgar et al., 2007). Only embryos that survived thawing with at least 50% of their prefreeze blastomeres intact were considered suitable for transfer, based on historical data (Edgar et al., 2000). When possible, and for the majority of procedures (>80%), embryos were transferred in natural menstrual cycles 3 days after detection of an LH surge. In cases of irregular or anovulatory cycles, hormone replacement was achieved with oestradiol valerate and micronized progesterone. Implantation rates were calculated as number of fetal hearts per embryo transferred and per embryo thawed. Statistical analysis used chisquared tests.

3 Table 1. Outcomes after thawing of cryopreserved embryos dehydrated in 0.1 mol/l or 0.2 mol/l sucrose prior to slow cooling. Fully intact (100% blastomere survival) 259 (54.6) a 379 (80.5) a Partially intact (50 99% blastomere survival) 113 (23.8) 57 (12.1) Did not survive (<50% blastomere survival) 102 (21.5) b 35 (7.4) b Values in parentheses are percentages. a,b Values with the same superscript letter are significantly different (P < 0.001). Results Details of the post-thaw survival of embryos cryopreserved after dehydration in 0.1 mol/l or 0.2 mol/l sucrose are shown in Table 1. A significantly (P < 0.001) lower proportion of embryos failed to survive in the 0.2 mol/l sucrose group (7.4%) than in the 0.1 mol/l sucrose group. In other words, by conventional definition, 92.6% of embryos in the 0.2 mol/l sucrose group were suitable for transfer compared with 78.5% in the 0.1 mol/l sucrose group. Overall blastomere survival is shown in Table 2. The mean number of cells per embryo was very close to four in both groups, as would be expected for embryos cryopreserved on day 2 of development. In the 0.2 mol/l sucrose group (n = 471), 70.1% of embryos contained exactly four blastomeres, 14.9% contained more than four blastomeres and 15.0% contained less than four blastomeres. The corresponding proportions in the 0.1 mol/l sucrose group (n = 474) were 72.2%, 15.4% and 12.4%, respectively. The proportion of surviving blastomeres in the 0.2 mol/l sucrose group (91.1%) was significantly (P < 0.001) higher than in the 0.1 mol/l sucrose group (74.1%). Additionally, a significantly (P < 0.001) higher proportion of embryos cryopreserved at exactly the four-cell stage on day 2 were suitable for transfer in the 0.2 mol/l sucrose group (94.8%; 313/330) than in the 0.1 mol/l sucrose group (79.8%; 273/342). The frequency of post-thaw resumption of mitosis in surviving embryos is shown in Table 3. There was no significant difference in the proportion of embryos that underwent subsequent cell division between the 0.2 mol/l sucrose group and the 0.1 mol/l sucrose group. This was also evident in terms of the increase in total blastomere numbers in the embryos from both groups. In the 0.2 mol/l group there was a 50.2% increase in the total number of blastomeres after overnight culture, with the increase being 51.9% in the 0.1 mol/l sucrose group. Transfer of 410 thawed embryos in the 0.2 mol/l sucrose group resulted in the detection of 74 fetal hearts, i.e. an implantation rate of 18.0% per embryo transferred and 15.7% per embryo thawed. In comparison, transfer of 365 thawed embryos in the 0.1 mol/l sucrose group resulted in the detection of 61 fetal hearts, i.e. an implantation rate of 16.7% per embryo transferred and 12.9% per embryo thawed. In order to control for the potential impact of female age in this comparison, the results for women under the age of 36 years in both groups are shown in Table 4. In this patient age group, 80% of all transfers were single embryo transfers. The implantation rates per embryo transferred and per embryo thawed were 24.9% and 22.1%, respectively, in the 0.2 mol/l group and 23.0% and 17.5%, respectively, in the 0.1 mol/l sucrose group. This represents a 26.3% increase in the yield of fetal hearts per embryo thawed in the 0.2 mol/l sucrose group. Discussion The value of embryo cryopreservation in assisted reproduction is self evident and it has played a pivotal role in the evolution of strategies to reduce multiple pregnancy rates (Tiitinen et al., 2001; Thurin et al., 2004; Lundin and Bergh, 2007). However, the loss of blastomeres, and even whole embryos, during freezing and thawing will inevitably result in loss of implantation potential (Van den Abbeel et al., 1997; Burns et al., 1999; Edgar et al., 2000; Guerif et al., 2002). Minimizing blastomere loss from cryopreservation is, therefore, the challenge for those who wish to maximize the efficiency of assisted reproduction. Table 2. Blastomere survival after thawing of cryopreserved embryos dehydrated in 0.1 mol/l or 0.2 mol/l sucrose prior to slow cooling. Mean no. of blastomeres per embryo No. of blastomeres thawed No. of surviving blastomeres (%) 1421 (74.1) a 1704 (91.1) a a P <

4 Table 3. Post-thaw resumption of mitosis in surviving embryos cultured overnight. No. of surviving embryos cultured overnight No. of cultured embryos resuming mitosis (%) 290 (78.0) a 345 (79.1) a a Not significantly different. Table 4. Outcomes from transfer of thawed embryos in women under the age of 36 years. No. of embryos thawed No. of embryos transferred No. of fetal hearts detected Implantation rate per embryo transferred (%) Implantation rate per embryo thawed (%) The current methodology for cryopreservation of early cleavage-stage embryos via slow cooling involves prefreeze dehydration using PROH and sucrose. The concentration of the permeating cryopotectant PROH is in the supramolar range (1.5 mol/l) whereas the non-permeating cryoprotectant is present at the significantly lower molar concentration of 0.1 mol/l, based on early publications (Lassalle et al., 1985; Testart et al., 1986). This methodology has been retained in practice and results in the majority of cleavage-stage embryos surviving with at least half of their prefreeze blastomeres intact (Edgar et al., 2000; Balaban et al., 2008). However, under these circumstances, only around half of all thawed embryos survive with all their blastomeres intact. Combined experience of slow cooling of human oocytes suggests that survival can be significantly enhanced by increasing the extent of dehydration prior to cryopreservation via an increase in the concentration of the non-permeating cryoprotectant, sucrose (Fabbri et al., 2001; Gook and Edgar, 2007). This is not surprising given the variation in membrane water permeability observed between individual oocytes (Hunter et al., 1992). Similarly, it has been previously shown that the well-documented reduction in survival of biopsied cleavage-stage embryos after slow cooling (Joris et al., 1999; Magli et al., 1999) could be reversed by a modified method which included dehydration in an increased concentration of sucrose (Jericho et al., 2003). This method has now been in routine use with biopsied embryos in the study centre s laboratories for over 5 years. Other than a preliminary study using 0.2 mol/l sucrose in the presence of ethylene glycol (Chi et al., 2002), there have been no reports of the use of increased sucrose concentrations for the dehydration of human non-biopsied cleavage-stage embryos. Common to the demonstration of improved cryopreservation outcomes after exposure to an increased concentration of sucrose (0.2 mol/l) prior to freezing is the use of a further elevated concentration of sucrose during the initial postthaw rehydration stages to buffer against excessive rates of water inflow (Jericho et al., 2003; Bianchi et al., 2007). The rehydration protocol used in this study, and previously described for use when thawing cleavage-stage embryos (Edgar et al., 2007), involves the removal of PROH in the presence of decreasing concentrations of sucrose with the initial concentration of 0.5 mol/l being appropriate for embryos dehydrated in a range of sucrose concentrations. This allowed us to compare the effect of increasing the dehydration concentration of sucrose from 0.1 mol/l to 0.2 mol/l in the absence of any confounding variables. The proportion of embryos with at least 50% of their blastomeres surviving, the overall proportion of surviving blastomeres and the proportion of all thawed embryos which were fully intact in the 0.1 mol/l sucrose group are all consistent with historical controls over many years and very similar to published data based on thousands of thawed embryos from both of the study centre s laboratories (Edgar et al., 2000). The consistency of this control data adds emphasis to the results obtained using 0.2 mol/l sucrose for dehydration. The impact of increased prefreeze dehydration, as shown in the results from the present study, manifests as an 18% increase in the proportion of embryos available for transfer (92.6% versus 78.5%), a 47% increase in the proportion of all embryos which survive fully intact (80.4% versus 54.6%) and a 23% increase in the proportion of all frozen blastomeres which survive (91.1% versus 74.1%). Considering the close association between loss of blastomeres and loss of implantation potential (Van den Abbeel et al., 1997; Burns et al., 1999; Edgar et al., 2000; Guerif et al., 2002) and the demonstration that fully intact embryos have similar implantation potential to equivalent non-frozen embryos (Edgar et al., 2000), the most dramatic finding was the increase in the proportion of fully intact embryos. The 17% increase in the proportion of surviving blastomeres suggests that approximately 17% of blastomeres in the 0.1 mol/l sucrose group may have been inadequately dehydrated prior to cryopreservation. These results with 0.2 mol/l sucrose slow cooling match those reported following vitrification of cleavage-stage embryos (Balaban et al., 2008), a technique which involves exposure to very high concentrations of cryoprotectant

5 prior to ultrarapid freezing. The fact that post-thaw resumption of mitosis in the surviving embryos is not compromised in embryos dehydrated in 0.2 mol/l sucrose confirms retention of normal physiology following rehydration. The primary aim of this study was to assess the impact of application of the study centre s method on cryosurvival. However, the observed implantation rate per embryo thawed suggests that this method will have a significant impact on clinical outcome after embryo cryopreservation. Although the clinical results did not reach statistical significance in the course of this study, the unequivocal demonstration of significantly improved cryosurvival has resulted in the recent adoption of the modified method as routine practice in the study centre s laboratories since it was considered unethical to withhold the potential benefits from patients. In summary, a significant improvement has been demonstrated for the survival of cleavage-stage embryos when they are dehydrated in the presence of an increased concentration of sucrose prior to slow cooling. This represents a major advance in the methodology for slow freezing of cleavagestage embryos which will have a significant impact on clinical outcome from embryo cryopreservation and allow the adoption of single embryo transfer with greater confidence. References Aytoz A, Van den Abbeel E, Bonduelle M et al Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection. Human Reproduction 14, Balaban B, Urman B, Ata B et al A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. 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Lancet 351, Ziebe S, Bech B, Petersen K et al Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Human Reproduction 13, Declaration: The authors report no financial or commercial conflicts of interest. Received 15 January 2009; refereed 3 February 2009; accepted 12 June