Hum. Reprod. Advance Access originally published online on June 10, 2008
Human Reproduction 2008 23(9):1976-1982; doi:10.1093/humrep/den222
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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
1 Assisted Reproduction Unit, American Hospital of Istanbul, Guzelbahce Sokak 20, Nisantasi, Istanbul 34365, Turkey 2 Colorado Center for Reproductive Medicine, Lone Tree, CO, USA
4 Correspondence address. Tel: +90-212-3112000; Fax: +90-212-3112339; E-mail: burman{at}superonline.com
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Abstract |
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BACKGROUND: The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification.
METHODS: A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures.
RESULTS: Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively.
CONCLUSIONS: Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010 [ClinicalTrials.gov] .
Key words: cryopreservation/vitrification/propanediol/pyruvate uptake/blastocyst development
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Introduction |
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Embryo cryopreservation plays a significant role in assisted human reproduction. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins and improving cumulative pregnancy rates (Veeck, 2003
; Anderson et al., 2005). Two basic techniques have been employed for the cryopreservation of cells: controlled slow-rate freezing and vitrification. Although slow freezing remains the most commonly used method of cryopreservation in human IVF laboratories around the world, recent studies have reported increasingly successful clinical results with vitrification (Mukaida et al., 2003a, b; Kuwayama et al., 2005a, b; Rama Raju et al., 2005; Desai et al., 2007).
Vitrification is an ultra-rapid method of cryopreservation whereby the embryo is transitioned from 37°C to –196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min). High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass-like state (Vajta and Kuwayama, 2006). To facilitate rapid heat transfer, minimal volumes are used in vitrification, facilitated through the use of minute tools as carriers. The carrier systems that have been developed for the vitrification procedure include the electron microscope grid (Park et al., 2000; Son et al., 2002), pulled and hemi-straws (El-Danasouri and Selman, 2001; Vanderzwalmen et al., 2002, 2003), flexipipet (Liebermann and Tucker, 2002), cryotop and cryotip (Kuwayama, 2007) and the cryoloop (Lane et al., 1999; Mukaida et al., 2001, 2003a, b; Reed et al., 2002; Rama Raju et al., 2005). The use of each technique has recently been reviewed (Chen and Yang, 2007).
Several advantages of vitrification have been discussed in the literature (Kuleshova and Lopata, 2002; Liebermann et al., 2002; Vajta and Nagy, 2006). The main benefits include the lack of ice crystal formation, made possible through increased speed of temperature conduction, reducing associated chilling injuries. A practical advantage is that the speed of the process minimizes the period the embryo is outside of the incubator. Vitrification requires minimal set up time, being performed as needed during the course of the day. Furthermore, there is no need for expensive programmable freezing equipment. One potential disadvantage of vitrification is that it is considered technically more challenging than slow freezing and requires more hands-on time per cryopreserved embryo.
Cryopreservation of human embryos by vitrification or slow freezing was evaluated in a recent review by Loutradi et al. (2007). From the 90 potential studies, only 4 were deemed suitable for analysis (Huang et al., 2005; Kuwayama et al., 2005a; Rama Raju et al., 2005; Zheng et al., 2005). Two of these studies reported a direct comparison of Day 3 cryopreservation with slow freezing and vitrification (Rama Raju et al., 2005; Zheng et al., 2005). Zheng et al. used abnormal, patient donated, research embryos, which underwent subsequent biopsy, and demonstrated significant improvement of embryo survival following vitrification compared with slow freezing. However, there was no statistical difference between the groups for embryo development to the blastocyst stage. In the larger clinical study, Rama Raju et al. cryopreserved approximately 120 embryos using slow freezing or vitrification. Significantly better survival and implantation rates were achieved following vitrification. Thus, vitrification appears to offer improved survival rates over slow freezing, but further studies are required to determine the most efficient cryopreservation method.
In animal models, it has been shown that slow freezing induces significant cellular trauma, including altered metabolism and a reduction in viability (Gardner et al., 1996; Lane and Gardner, 2001; Lane et al., 2002; Sheehan et al., 2006). Embryo metabolism is linked to subsequent viability of the conceptus (Gardner and Leese, 1987; Lane and Gardner, 1996; Brison et al., 2004). Therefore, analyzing effects on physiology, including metabolism, are extremely important in evaluating cryopreservation techniques (Coticchio et al., 2005; Gardner et al., 2007). The aim of this study was therefore to assess the efficacy of slow freezing and vitrification on the survival, metabolism and subsequent development of human cleavage-stage embryos. Furthermore, we report additional clinical data regarding the outcome of transfer of Day 3 human embryos vitrified using the cryoloop [with ethylene glycol (EG) and 1,2-propanediol (PROH) as cryoprotectants] to a cohort of women.
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Materials and Methods |
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The study was performed in the Assisted Reproduction Unit of the American Hospital of Istanbul and Colorado Center for Reproductive Medicine, Denver between October 2006 and October 2007. A total of 466 Day 3 cleavage-stage embryos, donated with consent, from 120 women who did not wish to cryopreserve their surplus embryos were randomly allocated to cryopreservation and subsequent thawing/warming by either slow freezing or vitrification. The clinical outcome of vitrified-warmed embryo transfer was studied in a separate series of 73 treatment cycles.
Laboratory study
Women were stimulated with either the long gonadotrophin-releasing hormone (GnRH) agonist or a GnRH antagonist protocol combined with recombinant follicle-stimulating hormone. Criteria for human chorionic gonadotrophin (HCG) injection, oocyte collection, intracytoplasmic sperm injection (ICSI), laboratory procedures have been described previously (Balaban and Urman, 2005; Urman et al., 2007). Embryos were obtained from women who underwent their first assisted reproduction treatment cycle. Only fresh ejaculated sperm was used for ICSI; embryos derived from surgically retrieved sperm were excluded.
The individual cohort of embryos from each subject was randomized to slow freezing or vitrification arms according to a previously prepared computer-generated randomization list. When each cohort of embryos was recruited, intervention allocation was conveyed to the laboratory team by the study coordinator nurse, who was blinded to the identity of the woman and characteristics of the embryos recruited. Only good-quality embryos having five or more equal-sized and evenly shaped blastomeres, with <20% fragmentation were cryopreserved. Cryopreserved embryos were subsequently cultured up to the blastocyst stage after thawing/warming. Cryosurvival and blastocyst development were compared between the groups. It was not possible to blind the laboratory team to intervention allocation, but outcome assessors were blinded to allocation information. Cryosurvival was assessed according to Rienzi et al. (2002) by an embryologist blinded to the intervention allocation. Embryos were considered to have survived if >50% of the blastomeres were intact or if they had at least three viable cells present at thawing, and showing at least one blastomere divided by 18 h of post-thaw culture. Metabolic analysis of embryos cryopreserved with either technique was subsequently performed. Blastocysts were graded according to Gardner et al. (2000). Following a maximum of 6 days of in vitro culture post-insemination, all embryos were discarded. None of these embryos were transferred.
Cryopreservation techniques
Slow freezing was performed as described previously (Balaban et al., 2007). PROH was used as the cryoprotectant (Freeze Kit-1, Vitrolife, Kungsbacka, Sweden). All procedures were performed at room temperature. A maximum of two embryos were loaded in each straw (CBS High Security Straws, Cryo-Bios, France). Embryos were thawed rapidly at room temperature (Thaw Kit-I, Vitrolife).
The technique previously described by Larman et al. (2007a) was used for vitrification and warming of cleavage-stage human embryos. All vitrification and warming procedures were performed at 37°C. The embryos were held in 1 ml of the base Holding solution (G-MOPS; Lane and Gardner, 2004; supplemented with 12 mg/ml human serum albumin; HSA) for 5–15 min. One to two embryos were placed into the Equilibration solution for 2 min. The Equilibration solution has the same composition as the Holding solution except that it contains 8% (v/v) EG. Once the 2 min had elapsed, the embryos were placed into the Vitrification solution for 30 s. The Vitrification solution has the same composition as the Holding solution except that it contains 16% (v/v) EG, 16% (v/v) PROH, 10 mg/ml Ficoll and 0.65 M sucrose (RapidVitTM Cleave, Vitrolife). The embryos were loaded onto the cryoloop (Hampton Research, Aliso Viejo, CA, USA), transferring as little medium as possible, typically around 50 nl. The cryoloop was then loaded into the cryovial held on a cryocane, which was submerged in liquid nitrogen.
For warming, the cryoloop was removed from the cryovial and dipped into Warming Solution 1. Embryos fell off the cryoloop and were moved through 1 ml volumes of a serial sucrose dilution in G-MOPS supplemented with 12 mg/ml HSA (RapidWarmTM Cleave, Vitrolife): Warming Solution 1 (0.65 M sucrose) for 30 s; Warming Solution 2 (0.325 M sucrose) for 1 min; Warming Solution 3 (0.125 M sucrose) for 2 min and Warming Solution 4 (0 M sucrose) for 5 min. For subsequent embryo development, embryos were then moved into G2.3 (Vitrolife) for 24 (embryo transfer) or 48 h (blastocyst assessment).
Metabolic analysis of embryos
Metabolic analysis was performed on a total of 82 embryos obtained from 21 women who had been consecutively included in the study. Embryos from 8 of these women had been allocated to slow freezing, whereas embryos from 13 women had been allocated to vitrification according to the previously constructed block randomization list.
Quantification of pyruvate uptake by individual human embryos was performed by ultramicro-fluorescence (Leese and Barton, 1984; Gardner and Leese, 1990; Gardner, 2007). Upon thawing/warming, individual embryos were washed through three successive drops of medium G2. Individual embryos were then placed in 1 µl of medium G2 under oil for 3 h. After 3 h, embryos were removed from their drops and cultured individually to the blastocyst stage in 10 µl of medium G2. The remaining medium G2 from the 1 µl drop was then collected in 1 µl capillary tubes and snap-frozen in liquid nitrogen. Samples were labeled and shipped on dry ice from Istanbul to Denver for pyruvate analysis. Researchers performing metabolic analysis were blinded to the technique of cryopreservation.
Clinical data
A total of 73 women underwent vitrified-warmed embryo transfer until the time this manuscript was written. Patient and cycle characteristics were similar to the patients included in the laboratory phase of the study. The mean age of the women was 33.2 years, each undergoing their first ICSI cycle. Controlled ovarian stimulation, oocyte recovery, in vitro culture and embryo transfer were performed as described previously (Balaban and Urman, 2005). ICSI with ejaculated spermatozoa was the mode of fertilization. GIII series sequential culture system (Vitrolife, Sweden) was used for embryo culture. Embryos were cultured in groups in 50 µl of culture medium G1.3 under oil (6% CO2, 5% O2, 89% N2) until vitrification on Day 3. Upon thawing/warming, embryos were placed into G2.3 for 24 h for Day 4 embryo transfer.
Pregnancy was confirmed by measuring serum beta-hCG levels 12 days after embryo transfer. Clinical pregnancy was defined as the presence of a fetus with a heart beat at 6 weeks of gestation; multiple pregnancy was defined as a gestation with more than one fetus; ongoing pregnancy was defined as pregnancy proceeding beyond the 12th gestational week.
Statistical analysis
Power analysis and sample size calculation
Blastocyst development rate was the primary endpoint for the study. Overall blastocyst development rate per cryosurvived embryo is 
50% in our clinic following slow freezing (Balaban et al., 2007). In each group, 182 embryos that survived cryopreservation and thawing would be required to evaluate whether progression to the blastocyst stage after vitrification was increased by 15% in absolute value, with an alpha error rate of 0.05 and beta error rate of 0.2. Assuming 5 blastocysts developed per 10 cryosurvived embryos, a 15% increase in blastocyst development rate corresponds to an increase by one or two in the number of developing blastocysts. Considering the cryosurvival rate of 90% in our laboratory, approximately 200 embryos would have to be cryopreserved and thawed in each group (Balaban et al., 2007). Considering the available resources, it was decided to recruit women for 6 months, which would enable an estimated recruitment of 400 to 500 embryos available for the laboratory study. During the recruitment period, 186 women meeting the inclusion criteria were contacted and 120 of them consented for donating their embryos for research. Finally 466 embryos were included in the laboratory study.
Data analysis
Categorical variables were compared with the 
2 test. Continuous variables were compared with Mann–Whitney test for independent samples. A P-value < 0.05 was considered as being significant. All analyses were performed with a commercial software (Statistical Package for Social Sciences, 13.0, SPSS Inc., Chicago, IL, USA).
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Results |
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A total of 466 embryos from 120 women were analyzed. The age of the women donating embryos and cycle characteristics of the index fresh cycles from which the supernumerary embryos were recruited are given in Table I.
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Embryo development
The overall cryosurvival rate was significantly higher with vitrification than with slow freezing [222/234 (94.8%) versus 206/232 (88.7%), respectively; P = 0.02]. Furthermore, the proportion of embryos with 100% intact blastomeres was significantly higher after vitrification compared with slow freezing [173/222 (77.9%) and 106/206 (51.4%), respectively; P < 0.01]. Progression rate to the blastocyst stage was also significantly higher after vitrification than after slow freezing [134/222 (60.3%) and 102/206 (49.5%), respectively; P = 0.02]. There were non-significant trends for better blastocyst quality [blastocysts
3AA: 70/134 (52.2%) and 43/102 (42.1%), respectively; P = 0.12] and for higher hatching rates [42/134 (31.3%) and 22/102 (21.5%), respectively; P = 0.09] in the vitrification group compared with slow freezing. The results are summarized in Table II.
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Metabolic analysis
Human embryo metabolism following cryopreservation by slow freezing or vitrification is shown in Fig. 1. Pyruvate uptake by the embryos was significantly reduced following slow freezing compared with that in embryos that underwent vitrification (7.50 ± 0.52 pmol/embryo/h; n = 33 and 12.05 ± 0.97 pmol/embryo/h; n = 49, respectively; P < 0.01).
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Clinical outcome
A total of 73 women subsequently underwent vitrified-warmed embryo transfers. A mean number of 3.3 embryos were warmed (n = 241). The cryosurvival rate was 92.1% (Table III). All blastomeres were intact in 72.1% of the embryos after the warming procedure. The mean number of embryos transferred was 2.3 (n = 168). A clinical pregnancy rate and ongoing pregnancy rate of 49.3 and 45.2% was achieved, respectively. The implantation rate was 29.7% (n = 50), resulting with a multiple pregnancy rate of 36.1% (n = 13: 1 triplet, 12 twins). At the time of writing, 8 of the ongoing 33 pregnancies have had successful deliveries of healthy children (2 twins, 6 singletons).
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Discussion |
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The literature on vitrification of human embryos has mainly focused on the blastocyst stage (Lane et al., 1999
; Mukaida et al., 2001, 2003a, b; Reed et al., 2002; Vanderzwalmen et al., 2002, 2003; Hiraoka et al., 2004; Liebermann and Tucker, 2004, 2006; Huang et al., 2005; Kuwayama et al., 2005a, b; Stehlik et al., 2005; Takahashi et al., 2005). There have been fewer publications on the clinical application of human cleavage-stage embryo vitrification (Mukaida et al., 1998; Saito et al., 2000; El-Danasouri and Selman, 2001; Kuwayama et al., 2005a, b; Rama Raju et al., 2005; Desai et al., 2007; Kuwayama, 2007). To date, only Desai et al. (2007) have reported clinically acceptable implantation and pregnancy rates. This study confirms that in terms of embryo metabolism and progression to the blastocyst stage, vitrification of Day 3 human embryos with the cryoloop is superior to slow freezing. The cryosurvival rate with vitrification (95%) is similar to that reported by Rama Raju et al., and higher than that published by Desai et al. (Rama Raju et al., 2005; Desai et al., 2007).
As noted in the systematic review by Loutradi et al. (2007), direct comparison of results between studies is difficult since different base media cryoprotectants and cryotools have been used. The results presented here provide the most extensive analysis and comparison between slow freezing and vitrification of Day 3 human embryos to date. There were 466 embryos from 120 patients used in the initial laboratory study to give an indicator of survival, including how many embryos survived with all blastomeres intact, and viability through to subsequent embryo development. As a way of analyzing the cellular effect of the cryopreservation method, pyruvate uptake was measured in 82 individual embryos post-thawing/warming. The early embryo predominantly uses pyruvate for metabolism, so measuring the amount of pyruvate consumed by the embryo gives an indication of embryonic health. Pyruvate uptake by embryos after vitrification was significantly higher than that after slow freezing. This may, in part, explain the reduced developmental ability of embryos that have been slow-frozen, a phenomenon that was also previously observed in the mouse (Lane and Gardner, 2001; Lane et al., 2002; Sheehan et al., 2006). Given that all of the parameters mentioned earlier were significantly better following vitrification, 241 embryos from 73 patients were vitrified and warmed in the clinical trial, giving a clinical pregnancy rate of 49.3%.
Previously published protocols using the cryoloop with the mouse and human have predominantly used EG and dimethyl sulfoxide (DMSO) as the cryoprotectants (Lane et al., 1999, 2002; Lane and Gardner, 2001; Mukaida et al., 2001; Reed et al., 2002; Larman et al., 2006a, b; Sheehan et al., 2006; Desai et al., 2007). However, concerns with regard to the safety of DMSO have been raised. One of the major potential problems with DMSO is that it is a very potent solvent. Embryo toxic compounds present within the system may be easily introduced into the embryo. Furthermore, DMSO has some untoward effects on intracellular physiology; the cryoprotectants EG, PROH and DMSO have all been shown to be associated with an increase in intracellular calcium in mouse oocytes (Larman et al., 2006a, 2007b). Significantly, however, EG and PROH induce an influx of calcium from the extracellular medium. In contrast, DMSO induces an increase in intracellular calcium through a release of intracellular calcium, most likely through disruption of intracellular organelles (Larman et al., 2006a). Therefore, although the effects of EG and PROH can be alleviated through the use of a calcium-free medium, DMSO induces intracellular aberrations independent of external calcium, making its presence in cryopreservation solutions of greater concern. DMSO has also been shown to cause cellular differentiation and affect DNA methylation in other cell types (Morley and Whitfield, 1993; Iwatani et al., 2006). With these effects in mind, it would appear prudent to find a replacement cryoprotectant for DMSO.
PROH has been used extensively with slow freezing and is effective for vitrification of bovine and human oocytes/embryos (Chian et al., 2004). It is also an efficient substitute for DMSO in bovine and mouse 8-cell and blastocyst vitrification (Gardner et al., unpublished results). It is plausible that the use of PROH, instead of DMSO, explains the increase in survival and blastocyst development, as well as the clinical pregnancy and implantation rates (despite the lower mean number of embryos transferred in this study), over those reported in the study by Desai et al. (2007).
A potential concern regarding the cryoloop is that it is an open system employing direct contact with liquid nitrogen. The possibility of viral contamination of liquid nitrogen has been suggested following the spiking of liquid nitrogen storage vessels with high viral titers (Bielanski et al., 2000). However, to the authors’ knowledge there are no published reports of actual cross-contamination of cryopreserved embryos. Indeed, the publication by Kyuwa et al. (2003) indicates that cross-contamination is unlikely. Kyuwa et al. demonstrated that mouse embryos stored in cryovials, in the same liquid nitrogen vessel as vials containing mouse hepatitis virus or pasteurella pneumotropica for a year, did not become contaminated. Microbial contamination of liquid nitrogen has been reported, but again there is no evidence that an embryo has been contaminated by direct contact with the liquid nitrogen (Fountain et al., 1997; Bielanski et al., 2003; Morris, 2005). With these studies in mind, the concerns of contamination during liquid nitrogen storage are somewhat questionable, even more so when taking into account the fact that slow-frozen embryos have been stored in cryovials for a number of years, which allow liquid nitrogen leakage during long-term storage. However, it is envisaged that closed systems that do not require direct contact of the sample with liquid nitrogen will become the standard. To this end, it has been established that minimal volume techniques, such as employed in this study, can succeed using super-cooled air in cryovials (Larman et al., 2006b). Subsequently, a new closed system using super-cooled air to induce vitrification of the sample is being evaluated clinically.
At the time of writing, 8 of the 33 ongoing pregnancies had resulted in live births of 10 healthy babies (2 twins and 6 singletons). Vitrification is a relatively new clinical technique, so perinatal documentation is important. So far the single largest study by Takahashi et al. (2005) compared the perinatal outcome of 413 cryoloop vitrified-warmed blastocyst transfers with that of 602 fresh blastocyst transfers. No significant differences were reported in the mean gestational age, birthweight, preterm birth rate or congenital birth defect rate. Although this is encouraging, follow-up studies are necessary to ensure the safety of the technique.
We have shown for the first time in human embryos that embryo metabolism is significantly depressed following cryopreservation by slow freezing. Embryo survival and subsequent development was significantly elevated following vitrification compared with slow freezing. Embryos that were transferred following the vitrification technique described herein yield among the highest implantation and pregnancy rates published to date. Given the 29.7% implantation rate of the vitrified embryo reported here, it would seem prudent to warm individual embryos and perform single-embryo transfers in order to avoid the high multiple pregnancy rate observed in this study when two, and in some cases three, embryos were transferred. In conclusion, these data support the hypothesis that vitrification is associated with less cellular trauma than slow freezing and should be considered as the primary method of human embryo cryopreservation.
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Author contribution |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAuthor contributionFundingReferences |
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B. Balaban, Design and institution of the study protocol, collection of the data, drafting the article, approval of the final version.
B. Urman, Design of the study protocol, review and final preparation of the article, approval of the final version.
B. Ata, Design of the study protocol, analysis of the data, drafting, review, final preparation of the article, approval of the final version.
A. Isiklar, Institution of the study protocol and collection of the data.
M.G. Larman, Design of the study protocol, development of the vitrification protocol, drafting and review of the article, approval of the final version.
B. Hamilton, Performing metabolic analyses, collection of the data.
D.K. Gardner, Design of the study protocol, development of vitrification protocol, performing metabolic analyses, drafting and reviewing the article, approval of the final version.
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Funding |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAuthor contributionFundingReferences |
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Vitrolife AB, Gothenburg, Sweden and The American Hospital of Istanbul.
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Footnotes |
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Preliminary results of this trial have been presented at the 63rd ASRM meeting, Washington, USA, October 2007 and at the Nordic IVF Laboratory Society Meeting, Helsinki, Finland, November 2007. 
3 Present address: Department of Zoology, University of Melbourne, Victoria, Australia
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Submitted on February 25, 2008; resubmitted on May 9, 2008; accepted on May 13, 2008.
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