Embryo cryopreservation: proposal for a new indicator of efficiency

Transcription

1 Embryo cryopreservation: proposal for a new indicator of efficiency Marie Prades, Pharm.D., a Jean-Louis Golmard, Ph.D., b,c Benoit Schubert, M.D., a and Catherine Poirot, Ph.D. a,c a Reproductive Biology Unit and b Statistical Unit, University Hospital, H^opital Pitie-Salp^etriere; and c University Pierre et Marie Curie, Paris, France Objective: To determine whether pregnancy rate (PR) per thawed embryo, accounting for embryo losses, is more relevant than PR per ET, generally used in the literature for expressing embryo cryopreservation results. Design: Systematic review. Setting: University teaching hospital. Patient(s): None. Intervention(s): Analysis of reports from January 1983 to February 2009 involving thawing of human cryopreserved embryos. Of the 1,275 articles that were initially identified, 102 met the inclusion criteria. Main Outcome Measure(s): Comparisons between PR per ET and PR per thawed embryo, contribution of embryo loss to PR. Result(s): Overall PR, expressed per ET and per thawed embryo, was compared according to the different freezing speeds and cryoprotectants used. Statistical analysis revealed significant differences for four comparisons with one approach to expressing results, not identified with the other. All other statistical analyses gave similar results whichever way the results were expressed. The rate of thawed transferred embryos, a measure accounting for embryo losses, is a good prognostic factor for PR. Conclusion(s): Statistical analysis accounting for embryo losses can give results different from those published in the literature. Consequently, the clinical PR per thawed embryo may be a more informative measure for accurate analysis of practices. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Embryo freezing, thawed transferred embryo rate, cryosurvival evaluation, human, pregnancy rate per thawed embryo Embryo cryopreservation undoubtedly has the potential to improve current assisted reproductive technologies. This technique results in clinical pregnancy rates (PR) per frozen ET of between 12.5% (1) and 69% (2). There are several reasons for these differences: embryo cryopreservation varies according to the freeze thawing protocol used and the stage at which the embryo is frozen. Reported values also depend strongly on how embryo survival is evaluated and how the results are expressed. Most groups accept the transfer of cleavage-stage embryos in which at least 50% of blastomeres are intact after thawing (3 5), whereas other teams transfer embryos containing at least a single intact blastomere (6, 7). Some teams thawed up to 12 frozen embryos per patient, from which only the most developed embryos with the best morphologic quality after 24 hours of postthaw culture were selected for transfer (8). Various transfer strategies according to the method of embryo survival evaluation are summarized in Figure 1: group A, at least one intact blastomere; group B, at least 50% of blastomeres intact; and group C, transfer of the most developed embryos after 24 hours of postthaw culture. With single ETs, the number of transfers can be four times that for other approaches with the same number of thawed embryos (four in Fig. 1). It is also easy to imagine that if only one Received July 16, 2009; revised May 20, 2010; accepted May 22, 2010; published online July 5, M.P. has nothing to disclose. J.-L.G. has nothing to disclose. B.S. has nothing to disclose. C.P. has nothing to disclose. Reprint requests: Marie Prades, Pharm.D., unite fonctionnelle de Biologie de la Reproduction et d Assistance Medicale a la Procreation, Groupe Hospitalier Pitie-Salp^etriere, 83 boulevard de l H^opital, Paris cedex 13, France (FAX: ; marieprades@ yahoo.fr). pregnancy occurs in each group, the PR per ET for group C, with the most drastic pretransfer evaluation of survival, would be higher than that in groups A and B, whereas the absolute number of embryos thawed and the number of successful pregnancies would be equivalent. Moreover, the absolute number of pregnancies may be greater in groups A and B, because they involve more than one transfer, whereas this absolute number could not increase in group C. Thus clinical PR expressed per ET, the method of evaluation generally used, does not seem to take thawed but nontransferred embryos into account. The number of thawed embryos is an objective parameter free from the various transfer strategies. So, expressing results as clinical PR per thawed embryo could be more relevant because rates are normalized by this common denominator. We therefore performed a study based on an extensive review of the literature, with the aim of creating a process for standardizing the expression of embryo cryopreservation results. Results were thus determined as clinical PR per thawed embryo and were then compared with those expressed as clinical PR per ET. The contribution of embryo loss, estimated with the number of thawed but nontransferred embryos, to PR was then analyzed to finally evaluate the performance of this parameter. MATERIALS AND METHODS Data Collection We searched the Medline and Embase databases for relevant articles published between January 1983, the year during which the first pregnancy resulting from frozen thawed embryos was reported, and February We used embryo, cryopreservation, and IVF as key words and limited /$36.00 Fertility and Sterility â Vol. 95, No. 2, February doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 FIGURE 1 Examples illustrating the number of frozen thawed ETs according to various survival strategies, in cases of single ETs. the search to human studies with pregnancy as the outcome measure. Reviews were also examined for published articles. Exclusion Criteria The articles that used in-vitro-matured oocytes were excluded from the analysis. Case reports and articles in which embryo cryopreservation was used for preserving fertility were also excluded from the study. Articles were not included if implantation rates, rather than clinical PR, were the main outcomes reported. Data Extraction and Determination of Validity An article was selected if the published data contained the following variables: number of embryos thawed, number of ETs, and number of clinical pregnancies. If these figures were not reported, other data (such as mean number of embryos thawed per cycle, and mean number of embryos transferred per transfer) were used to obtain the appropriate values. Thus, in some cases, data were calculated using the best possible approximation. Selecting Reports for Comparative Analysis We found 1,275 articles that were potentially informative (Fig. 2). A further evaluation, based on article titles, identified 238 publications, of which 216 were full articles, 9 were abstracts, and 13 were extracted from reviews. Only articles written in English were included. Reports not specifying the number of ETs, number of thawed embryos, or number of clinical pregnancies were excluded; the 102 remaining articles were used for our comparative analysis. For trials comparing various freezing methods or embryo stages, values for the reference group and for the test groups were considered separately; thus, data entered into the study in some cases included two or three sets of information (referred to hereafter as studies) from a single report. Additionally, different embryo stages or freezing methods studied in a single article were similarly individualized, such that 137 studies were extracted from the 102 reports (Supplementary Table 1, available online). Comparison Groups The reports were then grouped according to three main items: embryo stage (two pronuclei [2PN], early cleaved, blastocyst), type of cryoprotector agent (CPA) used (1,2-propanediol [PROH], dimethylsulphoxide [DMSO], ethylene glycol [EG], glycerol), and freezing speed. Freezing speed was classified as follows: slow freezing, rapid freezing (method used before the 2000s with a cooling rate of approximately 2,500 C per minute, involving brief exposure of embryos to a high concentration of cryoprotectants, the use of classic closed 0.25-mL plastic straws, and a rapid immersion in liquid nitrogen), or vitrification. If data concerning these three variables were incomplete, the report was not used to analyze the variable for which data was lacking but was used for other analysis. Impact of Embryo Losses on PR Embryo loss, represented by the number of thawed but nontransferred embryos, has two main origins: the survival evaluation and the cryoprocedures applied. The rate of thawed transferred embryos was evaluated to assess the contribution of embryo loss to PR. This was calculated by dividing the number of embryos transferred by the total number of embryos thawed. Statistical Analysis Overall PRs were expressed as percentages. Asymptotic or exact 95% confidence intervals were calculated according to the size of the samples. Most comparisons concerned differences in PR between groups. These differences were tested using two-sample Wilcoxon tests when two groups were compared and the Kruskal-Wallis test for more than two groups, followed by Wilcoxon tests for pairwise comparisons. No correction for multiple tests was performed. Relationships between two quantitative variables were tested using Spearman s rank correlation coefficient. Two multiple linear regressions were performed to assess the contribution of embryo loss to PR. Tests were two sided, and a P value of <5% was considered significant. All computations were performed using the SAS version 8 statistical package (SAS Institute, Cary, NC). 578 Prades et al. Thawed embryo: new indicator of success Vol. 95, No. 2, February 2011

3 FIGURE 2 Flow chart describing the selection of studies for inclusion in the systematic review. Data in italics are reported in the text. RESULTS Data Description Embryo stage was the variable providing the largest differences in PR for the studies included in our comparative analysis; therefore, data were grouped according to this variable. Of the 102 reports, 16 investigated embryo freezing at the 2PN stage, 49 at the early cleaved embryo stage, and 22 at the blastocyst stage alone. Embryos were frozen at the 2PN stage and early cleaved stages in nine studies, at early cleaved and blastocyst stages in three studies, at 2PN and blastocyst stages in one study, and at all three stages in two studies. TABLE 1 Comparisons of PR values according to the two ways of expressing results for each freezing speed and embryo stage studied. Mean PR per ET [95% CI] (No. of pregnancies/no. of ETs) Mean PR per thawed embryo [95% CI] (No. of pregnancies/no. of thawed embryos) Stage R SF V R SF V 2PN (%) [ ] [ ] [ ] [ ] (4/54) (971/3,764) (4/273) (971/15,905) EC (%) 13.4 a 21.8 b 29.7 a,b 3.0 c c [ ] [ ] [ ] [ ] [ ] [ ] (63/472) (2,463/11,274) (195/657) (63/2,125) (2,463/39,749) (195/1,475) Blastocyst (%) [ ] [ ] [ ] [ ] [ ] [ ] (2/12) (568/1,784) (3,192/6,382) (2/30) (568/5,512) (3,192/11,037) Note: Values are compared across row. Superscript letters indicate a significantly statistical difference between pairs. Values with the same superscript are significantly different. The absence of superscript means that values are not statistically significant. CI ¼ confidence interval [lower upper bound]; R ¼ rapid freezing; SF ¼ slow freezing; V ¼ vitrification; EC ¼ early cleaved embryo. a P¼ b P¼ c P¼ Fertility and Sterility â 579

4 TABLE 2 Comparisons of PR values for according to the two ways of expressing the results for each cryoprotectant and embryo stage studied. Mean PR per thawed embryo [95% CI] (No. of pregnancies/no. of thawed embryos) Mean PR per embryo transfer [95% CI] (No. of pregnancies/no. of ETs) Stage PROH Gly EG DMSO DMSODEG PROH Gly EG DMSO DMSODEG 2 PN (%) [ ] [ ] [ ] [ ] (1,187/4,714) (4/54) (1,187/19,349) (4/273) EC (%) 19.8 a,b a,c 11.4 b,c,d 29.3 d 5.9 e,f g 2.5 f,g,h 15.0 e,h [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (3,115/15,720) (27/39) (56/142) (217/1,903) (170/581) (3,115/52,570) (27/202) (56/1,832) (217/8,858) (170/1,133) Blastocyst (%) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (25/81) (711/2,456) (65/163) (3,018/5,982) (25/197) (711/7,204) (65/549) (3,018/9,920) Note: Values are compared across row. Superscript letters indicate a significantly statistical difference between pairs. Values with the same superscript are significantly different. The absence of superscript means that values are not statistically significant. Gly ¼ glycerol. Other abbreviations as in Table 1. a P¼.0315; b P¼.0046; c P¼.0073; d P¼.0351; e P¼.0398; f P¼.0021; g P¼.0336; h P¼ The reports were then classified according to freezing speed and CPA. 2PN Stage Among the 29 studies (28 reports) freezing 2PN stage embryos, 19 used a slow-freezing protocol, and one used rapid cooling. The freezing speed was not specified in the other studies. Among the 29 studies freezing at the 2PN stage, 27 used a solution containing PROH for cryopreservation, 1 used DMSO, and in 1 the CPA was not clearly specified. Early Cleaved Stage A slow-freezing protocol was used in 45 of the 74 studies (63 reports) freezing early cleaved stage embryos, 4 studies used vitrification, and 4 rapid freezing. The freezing speeds were not specified in the other reports. Among the 74 studies, 45 used a solution containing PROH for cryopreservation, and 16 used DMSO. Ethylene glycol was used in three reports, and glycerol and DMSO plus EG were the main CPA in one and two reports, respectively. The other reports did not clearly describe which CPA was used. Blastocyst Stage Of the 34 studies (28 reports) freezing embryos at the blastocyst stage, 11 used a slow-freezing protocol, 17 studies used vitrification as the main method, and 1 study used rapid cooling. The other reports did not specify the freezing speed. Two of these 34 studies used a solution containing PROH for cryopreservation, 10 used DMSO plus EG, 4 used EG, and 14 used glycerol. The CPA was unidentifiable in the other four reports. Comparison of PR for Each Studied Group According to the Two Ways of Expressing Results: PR per ET and PR per Thawed Embryo From extracted data and for each of the variables, the overall PRs were calculated per ET and per thawed embryo (Tables 1 and 2). The two types of measure were compared. Significant differences appeared when comparing freezing variables. Here, only comparisons for which one measure, but not the other, revealed a significant difference were considered informative for further analysis. On the basis of this criterion, four differences were observed for the freezing of early cleaved embryos. First, PR per ET was significantly different between freezing speeds (P¼.0298), whereas PR expressed per thawed embryo was not (P¼.0647). The second difference was observed with a PR of 21.8% after slow freezing, which differed significantly from that of 29.7% obtained after vitrification when expressed per ET (P¼.0335), whereas no significant difference was detected if PR was expressed per thawed embryo (P¼.0571). Third, the PR per ET was 19.8% with PROH, and this was significantly different from the PR per ET of 39.4% obtained with EG (P¼.0315), whereas there was no significant difference between the corresponding PR expressed per thawed embryo (P¼.2774). Fourth, by contrast, the PR per thawed embryo was 5.9% with PROH, which was significantly different from that of 15.0% obtained with DMSO plus EG (P¼.0398), whereas no significant difference was detected if PR was expressed per ET (P¼.0731). All other statistical analysis gave similar results for the two measures. Impact of the Embryo Losses on PR Two linear regression models were performed using two covariates: thawed transferred embryo rate and number of embryos per transfer. 580 Prades et al. Thawed embryo: new indicator of success Vol. 95, No. 2, February 2011

5 The first linear regression model used the clinical PR per thawed embryo as a dependent variable. This rate was linked to each covariate (P¼.0001 for each one), and the final model prediction formula was as follows: PR per thawed embryo ¼ 1.5 þ 0.2 (thawed transferred embryo rate) 2.4 (number of embryos per transfer). The second linear regression model used the clinical PR per frozen ET as a dependent variable. It indicated that this rate was linked to the number of embryos per transfer (P¼.0261) and to the rate of thawed transferred embryos (P¼.0115). The final model prediction formula was this: PR per ET ¼ 6.5 þ 0.2 (thawed transferred embryo rate) þ 3.4 (number of embryos per transfer). DISCUSSION This extensive review of the literature concerning embryo cryopreservation revealed that results are expressed in several different ways: as clinical PR (7, 9, 10), implantation rates (11, 12), or delivery rates (13 15) per ET. Published data seem to show a large diversity in the efficiency of the various practices used. The aim of our analysis was to determine the most relevant way to express results after embryo cryopreservation, allowing standardization and thus comparative evaluation of cryoprocedures. We investigated PR per thawed embryo as a way of expressing results and compared this with PR per ET, as currently used. We believe that this allows a more general view of cryopreservation and accounts for embryo losses, losses that are mainly due to the various transfer strategies or to the procedures used. We found that outcomes were expressed as the clinical PR per thawed embryo in only one of the reports we studied (16). Statistical comparisons of PR between groups gave similar results for 93% (52 of 56) of the tests with the two measures. This may be explained by the use of nonparametric rank correlation coefficients. If we consider the two denominators involved in these two ways of expressing the results, the number of thawed embryos was strongly linked, at 94%, to the number of transfers (P<.0001). Nevertheless, the differences identified suggest that expressing PR per thawed embryo may adjust for some of the variations introduced when results are expressed as PR per ET. For example, the PR for early cleaved embryos obtained after slow freezing vs. that after vitrification was significantly different if expressed per ET. This difference was not detected if PR was expressed per thawed embryo, suggesting that for an equivalent number of thawed embryos, the number of pregnancies resulting from vitrification did not differ from that from slow freezing. Indeed, a PR per ETof 30.5% was reported by a group vitrifying early cleaved embryos (17), and a PR per ETof 17.4% was reported by another group using a slow-freezing program to freeze early cleaved embryos (18). These results are significantly different. If these findings are expressed as PR per thawed embryo they are not significantly different, and furthermore, the values are reversed: the PR was 5.1% for the first team and 11.1% for the second. The success of thawed ET depends on several factors, particularly clinical and biological variables including the embryo implantation potential and the number of transferred embryos per transfer. So, to understand the contribution of embryo losses to results, we used multivariate regression analysis. These linear regressions accounted for the rate of transferred embryos, which reflects the number of embryos that were thawed but not used. These regressions also accounted for the number of embryos per transfer, to remove this major bias from the results. The results of this multivariate regression analysis showed that, when adjusting for the number of embryos per transfer, the chances of achieving pregnancy are better when embryo losses are minimal. Our hypothesis implicates transfer policies: the number of pregnancies may be decreased by applying the most drastic survival evaluations. For example, a PR per ET of 30.2% was reported by a group freezing early cleaved embryos and transferring only embryos with at least 50% of blastomeres intact after thawing (19). A PR per ET of 21.2% was reported by another group transferring embryos even containing at least a single intact blastomere (20). If these findings are expressed as PR per thawed embryo, the PR was 8.9% for the first team and 19.4% for the second. In conclusion, this study, based on an extensive review of the literature, suggests that PR per ET is not an objective measure for evaluating embryo thawing because it does not take into account embryo losses that result, mostly, from the survival evaluations applied or the cryotechnologies used. The rate of thawed transferred embryos is an important positive prognostic factor for the success of pregnancy; it would be better to express results as PR per thawed embryo. Use of this measure would allow a more accurate evaluation and comparison of professional practices. It would also allow a better standardization of published data and consequently could lead to improvements in our freezing techniques. Acknowledgments: The authors thank Alex Edelman for thoroughly revising the manuscript. REFERENCES 1. De Jong D, Eijkemans MJC, Beckers NGM, Pruijsten RV, Fauser BCJM, Macklon NS. The added value of embryo cryopreservation to cumulative ongoing pregnancy rates per IVF treatment: is cryopreservation worth the effort? J Assist Reprod Genet 2002;19: Anderson A, Weikert M, Crain J. Determining the most optimal stage for embryo cryopreservation. Reprod Biomed Online 2004; 8: Salumets A, Tuuri T, M akinen S, Vilska S, Husu L, Tainio R, et al. Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer. Hum Reprod 2003;18: Konc J, Kanyo K, Cseh S. Clinical experiences of ICSI-ET thawing cycles with embryos cryopreserved at different developmental stages. J Assist Reprod Genet 2005;22: Mandelbaum J, Bela ısch-allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3): Hartshorne GM, Wick K, Elder K, Dyson H. Effect of cell number at freezing upon survival and viability of cleaving embryos generated from stimulated IVF cycles. Hum Reprod 1990;5: Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavagestage embryos: post-vitrification development, pregnancy outcomes and live births. Reprod Biomed Online 2006;14: Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997;12: Chi HJ, Koo JJ, Kim MY, Joo JY, Chang SS, Chung KS. Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod 2002;17: Gabrielsen A, Fedder J, Agerholm I. Parameters predicting the implantation rate of thawed IVF/ICSI embryos: a retrospective study. Reprod Biomed Online 2006;12: Macas E, Imthurn B, Borsos M, Rosselli M, Maurer- Major E, Keller PJ. Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection. Fertil Steril 1998;69: Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15: Van den Abbeel E, Camus M, Verheyen G, Van Waesberghe L, Devroey P, Van Steirteghem A. Slow Fertility and Sterility â 581

6 controlled-rate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies. Hum Reprod 2005;20: Tummon IS, Contag SA, Thornhill AR, Session DR, Dumesic DA, Damario MA. Cumulative first live birth after elective cryopreservation of all embryos due to ovarian hyperresponsiveness. Fertil Steril 2004;81: Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce JC. MERIT (Menotrophin vs Recombinant FSH in vitro Fertilisation Trial) Group. Influence of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF. Hum Reprod 2007;22: Van den Abbeel E, Van der Elst J, Van Waesberghe L, Camus M, Devroey P, Khan I, et al. Hyperstimulation: the need for cryopreservation of embryos. Hum Reprod 1988; 3(Suppl 2): El-Danasouri I, Selman H. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001;76: Le Lannou D, Griveau JF, Laurent MC, Gueho A, Veron E, Morcel K. Contribution of embryo cryopreservation to elective single embryo transfer in IVF-ICSI. Reprod Biomed Online 2006;13: Tiitinen A, Halttunen M, Harkki P, Vuoristo P, Hyden-Granskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001;16: Prades M, Golmard JL, Vauthier D, Lefebvre G, Poirot C. Can cumulative pregnancy rates be increased by freezing and thawing single embryos? Fertil Steril 2009;91: Prades et al. Thawed embryo: new indicator of success Vol. 95, No. 2, February 2011

7 SUPPLEMENTARY TABLE 1 References of other reports used for the analysis but not cited in article text. Author(s) Date Journal abbrevation Volume:page range Al-Hasani S, et al Hum Reprod 14: Amarin ZO 2004 Eur J Obstet Gynecol Reprod Biol 117: Balaban B, et al Fertil Steril 87:691 6 Behr B, et al Fertil Steril 77:697 9 Cho HJ, et al Hum Reprod 17: Choi DH, et al Fertil Steril 74:838 9 Cohen J, et al J In Vitro Fert Embryo Transf 3:46 52 Cohen J, et al J In Vitro Fert Embryo Transf 2:59 64 Damario MA, et al Fertil Steril 71:830 5 Damario MA, et al Fertil Steril 72: Damario MA, et al Fertil Steril 73: El-Toukhy T, et al Hum Reprod 18: Emiliani S, et al Assist Reprod Genet 16:405 9 Erasmus EL, et al S Afr Med J 76:613 4 Fehilly CB, et al Fertil Steril 44: Feichtinger W, et al Hum Reprod 6:735 6 Ferraretti AP, et al Hum Reprod 14: Freemann L, et al J In Vitro Fert Embryo Transf 3:53 61 Fugger EF, et al Fertil Steril 50:273 8 Fugger EF 1989 Fertil Steril 52: Fugger EF, et al Hum Reprod 6:131 5 Guerif F, et al Hum Reprod 17: Hammitt DG, et al J Assist Reprod Genet 21:271 8 Hiraoka K, et al Hum Reprod 19: Hoover L, et al Fertil Steril 67:621 4 Horne G, et al Hum Reprod 12:542 7 Hsieh YY, et al Fertil Steril 72:253 6 Huang CC, et al Hum Reprod 20:122 8 Jaroudi KA, et al Fertil Steril 55:835 7 Jericho H, et al Hum Reprod 18: Karlstr om PO, et al Hum Reprod 12: Kattera S, et al Fertil Steril 84: Kuwayama M, et al Reprod Biomed Online 11: Lahav-Baratz S, et al J Assist Reprod Genet 20:444 8 Lee SY, et al J Assist Reprod Genet 23:87 91 Lee JR, et al Fertil Steril 88: Liebermann J, et al Fertil Steril 86:20 6 Lightman A, et al Fertil Steril 67:711 6 Lornage J, et al Hum Reprod 5:60 5 Magli MC, et al Hum Reprod 14:770 3 Magli MC, et al Hum Reprod 21: Mandelbaum J, et al Hum Reprod 3:117 9 Marrs RP, et al Am J Obstet Gynecol 190: Mauri AL, et al Hum Reprod 14: Miller KF, et al Obstet Gynecol 85: Mohr LA, et al J In Vitro Fert Embryo Transfer 3:53 3 Mukaida T, et al Hum Reprod 18: Mukaida T, et al Reprod Biomed Online 6:221 5 Nagy ZP, et al Fertil Steril 84: Obasaju MF, et al Fertil Steril 61: Oehninger S, et al Fertil Steril 57:620 5 Oehninger S, et al Mol Cell Endocrinol 27:73 7 Oyesanya OA, et al Ann Acad Med Singapore 21:471 5 Pantos K, et al J Assist Reprod Genet 18: Pattinson HA, et al Fertil Steril 62: Rama Raju GA, et al Reprod Biomed Online 11:434 7 Rekha P, et al Hum Reprod 13:696 8 Salumets A, et al Hum Reprod 21: Senn A, et al Fertil Steril 74: Fertility and Sterility â 582.e1

8 SUPPLEMENTARY TABLE 1 Continued. Author(s) Date Journal abbrevation Volume:page range Siebzehrubl E, et al J In Vitro Fert Embryo Transfer 3:68 8 Sifer C, et al Hum Reprod 21: Simon A, et al Hum Reprod 13: Son WY, et al Hum Reprod 18:137 9 Stehlik E, et al Reprod Biomed Online 11:53 7 Takahashi K, et al Fertil Steril 84:88 92 Testart J, et al Lancet 8506:569 Testart J, et al Fertil Steril 48: Toner JP, et al Hum Reprod 6:284 9 Toner JP, et al Fertil Steril 56:505 8 Toner JP, et al Fertil Steril 55: Trounson A, et al Fertil Steril 49:822 6 Troup SA, et al Eur J Obstet Gynecol Reprod Biol 38:133 9 Ubaldi F, et al Eur J Obstet Gynecol Reprod Biol 115 Suppl 1:S106 9 Urman B, et al Fertil Steril 87:310 5 Van den Abbeel E, et al Hum Reprod 3 Suppl 2:53 7 Van den Abbeel E, et al Hum Reprod 12: Vanderzwalmen P, et al Hum Reprod 17: Vanderzwalmen P, et al Hum Reprod 18: Van Steirteghem AC, et al Fertil Steril 62: Van Uem JF, et al Lancet 28:752 3 Veeck LL, et al Fertil Steril 59: Veeck LL, et al Fertil Steril 82: Virant-Klun I, et al Fertil Steril 79: Wang XJ, et al Hum Reprod 9:103 9 Wang JX, et al Hum Reprod 16: Warnes GM, et al Hum Reprod 12: Yu Ng EH, et al Hum Reprod 15:250 5 Zeilmaker GH, et al Fertil Steril 42:293 6 Zikopoulos K, et al Fertil Steril 81: e2 Prades et al. Thawed embryo: new indicator of success Vol. 95, No. 2, February 2011