Towards defining parameters for a successful single embryo transfer in frozen cycles

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1 Human Reproduction Vol.21, No.5 pp , 2006 Advance Access publication January 12, doi: /humrep/dei490 Towards defining parameters for a successful single embryo transfer in frozen R.Tang 1,2, J.Catt 1,3 and D.Howlett 1 1 Monash IVF, Clayton, Victoria, Australia and 2 Centre for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan, PR China 3 To whom correspondence should be addressed at: Monash IVF, 1/252 Clayton Rd, Clayton, Victoria 3168, Australia. jcatt@monashivf.edu.au BACKGROUND: Twin pregnancies in IVF should be avoided by transferring embryos one at a time, even for frozen. In this study, we investigated the effect of blastomere lysis and cleavage in singleton frozen embryo transfer (sfet). Outcomes were compared with the transfer of two embryos in frozen transfer (dfet). METHODS: A retrospective analysis was performed on 891 FET, involving 404 sfet and 487 dfet. RESULTS: Overall, in sfet, the pregnancy and implantation rates were 8.9 and 8.7%. When blastomere lysis was more than 25% but no greater than 50%, the pregnancy and implantation rates were 3.2%. If blastomere lysis was greater than 50% there were no pregnancies. If blastomere lysis was less than 25%, but with no cleavage, the pregnancy and implantation rates were 4.1%. The results significantly improved (P = 0.007) in the group with less than 25% lysis, when cleavage occurred. The pregnancy and implantation rates for this group were 17.3 and 16.6%. This was not significantly different from unselected two embryo transfers (22 and 12.7%, P = 0.2 and 0.19, respectively). There were 21 twins with dfet (19.6% of pregnancies) and none in sfet. CONCLUSION: Both blastomere lysis and cleavage affect the outcome in sfet. To avoid the risk of twins, sfet should be considered when the embryo shows less than 25% blastomere lysis and at least one blastomere cleaves. Key words: blastomere cleavage/blastomere lysis/cryopreservation/fet/dfet/sfet Introduction Single embryo transfer (SET) or elective single embryo transfer (eset) has been advocated to decrease the occurrence of twin pregnancies in fresh transfers. eset in a selected group of patients leads to an acceptable pregnancy rate and a reduction in the twin pregnancy rate (Martikainen et al., 2001; Tiitinen et al., 2001; Catt et al., 2003; Kovacs et al., 2003; Salumets et al., 2003a; Tiitinen et al., 2003; de Boer et al., 2004; Hyden-Granskog and Tiitinen, 2004; Thurin et al., 2004a,b; Bergh, 2005; Gerris, 2005; Van Montfoort et al., 2005). In most IVF centres, usually more than one embryo is transferred in frozen embryo transfer (FET), as it is believed that cryopreserved embryos have a lower potential for implantation and therefore were not considered to be good quality embryos, which is a necessary condition for eset. With improvements in embryo culture, eset has been suggested to decrease the number of embryos transferred in FET so as to decrease the risk of multiple pregnancy (Ziebe et al., 1998; El-Toukhy et al., 2003; Anderson et al., 2005). A recent report (Hyden-Granskog et al., 2005) has shown that singleton frozen embryo transfer (sfet) can be successfully used to reduce multiple pregnancies. Blastomere survival after thawing and the resumption of mitosis after overnight culturing of the frozen-thaw embryos have been suggested as two valid parameters for grading and selection of embryos to be transferred in FET (El-Toukhy et al., 2003; Van den Abbeel et al., 1997; Van der Elst et al., 1997; Ziebe et al., 1998; Burns et al., 1999; Guerif et al., 2002; Archer et al., 2003; Pal et al., 2004). There were two objectives of our retrospective study, which enrolled 404 single embryo transfers in 891 FET. The first was to investigate the effect of blastomere lysis and cleavage of single thawed embryos so as to ascertain a selection strategy for sfet. The second was to compare the outcome of sfet with 487 double embryo transfer (dfet) frozen to determine whether guidelines could be proposed for sfet to decrease twin pregnancies after FET. Materials and methods In this retrospective study, we reviewed the outcome of 1378 transferred frozen-thawed embryos in 891 FET performed from January 2004 to December 2004 at Monash IVF. There were 598 couples included. The mean age of the women, at the time of embryo cryopreservation, was 35 ± 4.5 years (range years). A total of 98 of the 598 women were 40 years of age. One hundred and one embryos in 60 FET were derived from oocyte donation. The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved For Permissions, please journals.permissions@oxfordjournals.org

2 R.Tang, J.Catt and D.Howlett Ovarian stimulation, oocyte collection, insemination (standard IVF or ICSI) and embryo culture were performed according to routine protocols. Oocytes were aspirated h after HCG injection and inseminated 4 6 h later. Sixteen to 20 h later, the oocytes were assessed for fertilization. One or two embryos were transferred usually at day 3, occasionally on day 2 or exceptionally on day 1. All surplus embryos satisfying the freeze criteria were cryopreserved on the day of fresh embryo transfer. The freeze criteria were appropriate development (i.e., 2 cells or greater on day 2 or 6 cells or greater on day 3) and less than 40% cytoplasmic fragmentation, provided blastomere criteria were met. Cryopreservation was carried out using 1,2-propanediol (PROH) and sucrose as cryoprotectants following a slow-freezing protocol (Testart et al., 1986). The embryos were frozen singly, in 0.25-ml plastic straws (IMV, L Aigle, France) using a Cryologic machine (Cryologic, Melbourne, VIC, Australia). Upon completion of freeze ( 35 C), the straws were removed from the cryochamber, plunged directly into nitrogen and stored until required. Embryo thawing was performed the day before scheduled embryo transfer. Briefly, one straw at a time was quickly removed from liquid nitrogen, thawed for 40 s in air and then plunged into a 30 water bath for another 30 s. The contents of the straw were recovered, the embryo located and transferred to the sequential thaw solutions. The thawed embryo(s) were transferred to culture medium for 18 h until transfer. Cleavage medium, either Sage (Cooper Surgical, Trumball, MN, USA) or Cook (Cook IVF, Brisbane, QLD, Australia), was used to culture day 2 embryos and Blastocyst media (either Sage or Cook) used to culture day 3 embryos. The procedure was repeated until the requested number of embryos to be thawed/transferred was achieved. The clinician, in conjunction with the patient, decided this number. Sage media were used before 10 June 2004, and Cook culture media was used after that time. Three main types of clinical protocols were used for endometrial preparation: natural, HRT or Clomiphene. Natural were used for women with regular ovulatory menstrual. The time of ovulation was calculated as 1 day after the LH surge. Thawed embryo transfer was scheduled for 3 days after ovulation for day 3 embryos and 2 days after ovulation if day 2 embryos were used. If the progesterone level at the transfer day was >10 nmol/l on a day 2 transfer or >13 nmol/l on day 3 transfer, no luteal support was used. Otherwise, progesterone pessaries (200 mg BD) were used, starting the night of transfer and continuing until the day 16 pregnancy test. HRT included both down-regulation/hrt and no down-regulation/ HRT (direct HRT). The former was prescribed to those with anovulatory menstrual and the latter to those with ovarian failure. If the endometrial thickness was 6 mm or more by an ultrasound scan after approximately days of HRT (Progynova Schering, Sydney, NSW, Australia) administration, progesterone pessaries were commenced and embryo transfer was arranged according to the embryo age, which was 3 days later for day 3 embryos and 2 days later for day 2 embryos. HRT was continued until day 16 pregnancy test. If the pregnancy test was positive, the HRT was continued until approximately 10 weeks gestation. A Clomiphene protocol was used for women with irregular ovulation or anovulatory as an alternative to more complicated HRT. Chlomiphene citrate (Aventis, Sydney, NSW, Australia) was administered orally at 100 mg per day for 5 days, commencing between day 2 and day 6 of the cycle, or induced withdrawal bleed for anovulatory patients. Ultrasound was used between day 10 and 12 to measure follicular diameters. When the dominant follicle was estimated to be 17 mm or more, blood E 2 was rising and >500 pmol/l and baseline LH had not risen and 5000 IU of hcg (Profasi, Serono, Australia) was administered. Alternatively, no hcg was administrated and the LH surge monitored. Ovulation was estimated at 36 h following 1180 Profasi or after the time of LH surge. In a few, Gonal-F with or without GnRHa or with GnRHant were used, and these were classified as other protocols. The ovulation and embryo transfer was timed as the Clomiphene protocol. After thawing, each embryo was evaluated for the number of surviving blastomeres and then cultured overnight for approximately 18 h. Just prior to transfer, a second evaluation was performed. Blastomere lysis for each embryo was calculated as the percentage of blastomeres lysed between freezing and thawing, and the cleavage percentage was the change in blastomere number between thaw and transfer. If 50% or more of the blastomeres survived the thaw, and at least one blastomere cleaved overnight, the embryo was considered optimal for transfer. If less than 50% of blastomeres had survived, then the transfer was cancelled unless the patient insisted on a transfer, even after counselling for poor prognosis. Clinicians, in conjunction with the patients, decided on whether to transfer one (elective sfet) or two (dfet) embryos prior to thawing. If only one thawed embryo survived, or there was only one embryo to thaw, then obligatory sfet was undertaken. Embryo transfer was performed with a K-Soft (Cook) catheter, often aided by abdominal ultrasound scan guiding. was tested by serum HCG 16 days after transfer. Transvaginal ultrasound evaluation was performed 5 weeks after transfer to ensure the presence of an intrauterine pregnancy and to assess the number of sacs. rate was defined as the proportion of pregnancy of the total transfer. The implantation rate was defined as the fraction of transferred embryos resulting in an intrauterine gestational sac with a fetal heart visualized by ultrasound. Statistical analysis of the results was performed using the Student t-test, and chi-square test or Fisher s exact test of probabilities where applicable. A P value greater than 0.05 was considered statistically significant. Results One thousand three hundred and seventy-eight frozen-thawed embryos were transferred in a total of 891 FET from 598 patients. The results are shown in Table I. Only 15 of the transfers were with day 2 embryos and three with pronucleate stage frozen zygotes. The average number of embryos transferred per cycle was 1.5 ± 0.5. There were 143 where pregnancy was initiated with 159 sacs with fetal heartbeat revealed by transvaginal ultrasound scanning. The overall pregnancy rate was 16.1%, with the implantation rate being 11.5% per embryo transferred. Twenty-one women showed two sacs under ultrasound scanning, giving a twin pregnancy rate of 14.7% (21/143). There were no significant differences in the age, endometrial preparation protocols, insemination types (standard IVF or ICSI), oocyte recipients and the embryo age and degree of fragmentation at freezing between pregnant and non-pregnant. However, the number of embryos being transferred in pregnancy was significantly higher than that in non-pregnancy (Table I; P = 0.02). The analysis of singleton embryos transferred in frozen is summarized in Table II. Four groups of blastomere lysis were initially used. Group 1 was defined as no lysis of blastomeres, group 2 as some lysis but less than 25% of blastomeres, group 3 where 25% or more but less than 50% blastomeres lysed and group 4 where 50% or more blastomeres lysed. The pregnancy and implantation rates of group 2 was

3 Single embryo transfer in frozen Table I. General results of all frozen embryo transfer (FET) Table III. The effect of blastomere cleavage on singleton frozen embryo transfer Total (embryos) (sacs) Non-pregnancy PR% (IR%) Blastomere cleavage Cycles Sacs Implantation Cycles 891 (1378) 143 (159) (11.5) Age (years) 35 ± ± ± 4.4 No. of embryos 1.6 ± ± 0.4 a 1.5 ± 0.5 a EPP Natural 567 (855) 94 (108) (12.6) HRT 225 (370) 36 (38) (10.3) CC 82 (124) 13 (13) (10.5) Other 17 (29) 0 (0) 17 0 (0) Insemination type IVF 241 (357) 44 (49) (13.7) ICSI 650 (1021) 99 (110) (10.8) Embryo age Day (1349) 137 (153) (11.3) Day 2/1 18 (29) 6 (6) (20.7) Oocyte recipients Yes 60 (101) 14 (17) (16.8) No 831 (1277) 129 (142) (11.1) EPP, endometrium preparation protocols; IR, implantation rate; PR, pregnancy rate. HRT included down-regulation/hrt and direct HRT and CC indicated Clomiphene or Clomiphene/HCG. Other protocols included GnRHa/ FSH/HCG short protocol or GnRHant/CC/HCG. a P = Table II. The effect of blastomere lysis on singleton frozen embryo transfer (original and combined groups) Blastomere loss Cycles a,b P = c P = d P = Sacs Implantation Group 1 (0%) Group 2 (>0 to <25%) a b Group A c d (combined 1 and 2) Group 3 (25 to <50%) a b Group 4 (50% or more) Group B c d (combined 3 and 4) significantly higher than that of group 3, (P = 0.03) However, there was no significant difference between group 1 and group 2, as well as between groups 3 and 4. Therefore, groups 1 and 2 were merged to become group A (less than 25% lysis), and groups 3 and 4 were merged to become group B (25% or more lysis). Both pregnancy and implantation rates were significantly higher in group A than in group B (P = and P = 0.001, respectively). The development of embryos after overnight culture was then analysed. Initial analysis of the degree of blastomere cleavage indicated that those embryos that had blastomeres lysing overnight and those that had no division were not significantly different. Therefore, two groups were used for further analysis (summarized in Table III). Group 1 were those with blastomere division and group 2 were those embryos with extra lysis or no division after overnight culture. Both the pregnancy rate and implantation rate in group 1 were statistically higher than those in group 2 (P = and P = , respectively, Table III). Group a b (some cleavage) Group a b (no cleavage) a P = b P = Table IV. The effect of both blastomere lysis and cleavage on singleton frozen embryo transfer Blastomere loss/cleavage Cycles a P = b P = c P = d P = e P = f P = Sacs Implantation Group (i) a,c,e b,d,f Group (ii) a b Group (iii) c d Group (iv) e 0 0 f Table V. The outcome of singleton frozen embryo transfer (sfet) and double embryo transfer (dfet) Outcome Total sfet sfet (good quality embryo) a P = b P = c P = d P = Same superscript numbers denote significant difference. dfet FET No. of embryos a 17.2 a 22 a Sacs Implantation b 16.6 b 12.7 b Twin sacs Twin c 0 d c,d For the analysis of combined blastomere lysis and cleavage, four final groups were used. Groups (i) and (ii) had less than 25% blastomere lysis, with group (i) at least one division and group (ii) no cleavage. Groups (iii) and (iv) had 25% or more blastomere lysis, with group (iii) at least one division and group (iv) no division. The results are summarized in Table IV. Both pregnancy and implantation rate in group (i) was significantly higher than those in the other three groups. The comparison of sfet with dfet is summarized in Table V and Figure 1. Single embryo transfers accounted for 45.3% of 1181

4 R.Tang, J.Catt and D.Howlett Figure 1. Graph showing the pregnancy and implantation rates for sfet and dfet and selected good prognosis embryos for sfet. IR, implantation rate; PR, pregnancy rate. the total. The mean age of the women in sfet was 34.7 ± 4.3 years at the time of cryopreservation, showing no significant difference compared with dfet (35.1 ± 4.4). Overall, both the pregnancy rate and the implantation rate were significantly higher in dfet than those in sfet (22.0 and 12.7% versus 8.9 and 8.7%, P = and 0.03, respectively). The twin pregnancy rate in dfet was 19.6% of the established pregnancies compared with no twins with sfet. When a good quality embryo was transferred in sfet (i.e. blastomere lysis less than 25% and cell division), both the pregnancy and implantation rates showed no significant differences for unselected dfet (22 and 12.7% versus 17.2 and 16.6%, P = 0.20 and 0.19). Discussion Overall, success rates for frozen embryo transfer are approximately half to two-thirds of those observed in fresh transfer in most centres (15 25%, Speroff and Fritz, 2005). Results of our study showed the overall pregnancy rate per transfer cycle was 16.1%, and the implantation rate per transferred embryo was 11.5%. This is an overall rate and includes 16% of patients over 40 years of age, at the time of cryopreservation, and with almost half the being sfet. It also includes some embryos that would normally be outside of our acceptable criteria but were transferred because of patient request. The data presented here is skewed towards day 3 embryos rather then day 2, as reported by Salumets et al. (2003b), and was not sufficiently powered to detect a less than 20% difference in the pregnancy and implantation rates with the age of embryo at cryopreservation. Both pregnancy and implantation rates showed no significant differences with patient age, endometrial preparation protocol or original insemination method, similar to that reported by Oehning et al. (2000). There were no significant differences with the origin of the embryos (whether donor oocyte or not). The pregnancy rate following FET has been shown to be related to such embryological parameters as the number of 1182 Outcome of sfet,dfet and sfet for "good quality" embryos PR SET IR DET SET-"good embryo" blastomeres and their morphological appearance, the growth rate of embryos prior to freezing, the extent of embryo damage after thawing and the resumption of post-thaw blastomere division (Van den Abbeel et al., 1997; Ziebe et al., 1998; Burns et al., 1999; Edgar et al., 2001; Guerif et al., 2002; Archer et al., 2003; El-Toukhy et al., 2003; Pal et al., 2004). A reduction in the number of blastomeres as a result of cell lysis is a well-recognized consequence of the cryopreservation of human early cleavage-stage embryos. It is common practice to consider an embryo as surviving and suitable for transfer, after cryopreservation, if at least half of the initial number of blastomeres remain intact (Van den Abbeel et al., 1997; Burns et al., 1999; Edgar et al., 2000a,b; Guerif et al., 2002). The consequences of blastomere loss in thawed embryos include impaired preimplantation development in vitro and reduced cell number in resultant blastocysts (Archer et al., 2003). Previous reports (Van den Abbeel et al., 1997 and El-Toukhy et al., 2003) advocated the transfer of fully intact embryos, if these were available. In the data reported here, when blastomere lysis was 25% or more, a significantly lower pregnancy and implantation rate was found than when blastomere lysis was less than 25%. This suggests that the competency was seriously affected when 25% or more of blastomeres lysed, implying that, for sfet in our centre, blastomere lysis should be less than 25%. The difference between this and the two studies above could be a reflection that the majority of our embryos were day 3 rather than day 2 and perhaps more able to tolerate the loss of one blastomere. In this study, both the pregnancy and implantation rates were significantly higher for those embryos that had resumed cell division compared with no division. This is similar to previous reports. Ziebe et al. (1998) reported a retrospective study in which a total of 1408 thawed day 2 embryos were transferred in 612 with one to three embryos transferred. Both the pregnancy and implantation rates in the cleaved embryo transfer group (in which at least one of the transferred embryos had cleaved) were significantly higher than those obtained in the non-cleaved embryo group (28 versus 11% and 10 versus 4%, respectively). In a more recent study, Guerif et al. (2002) reported the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 and 3%, respectively). These data suggest that only those embryos that have resumed division should be transferred. As the competence of cryopreserved embryo is lower than that of fresh embryos, usually more than one embryo is transferred in FET. Despite this reduced implantation potential, multiple pregnancies are also relatively frequent in frozen embryo transfer (approximately 20% of established pregnancies, Kolibianakis et al., 2003). Similarly, in this study, the twin pregnancy rate was 19.6% of the established pregnancies when two embryos were transferred. In recent publications Tiitinen et al. (2003) and Hyden- Granskog et al. (2005) reported that the percentage of sfets in their clinic increased from 28 to 66% from 1996 to Concommittantly, the pregnancy and implantation rates improved and the multiple delivery rates have decreased from 22 to 8%. The delivery rate was 26% for transfers with two embryos and

5 Single embryo transfer in frozen 19% for single embryo transfers. Their criteria for transfer were more rigid than those reported here, as our programme allows for embryos to be transferred if they have not cleaved. We believe that our criteria need to be reassessed because noncleaving embryos have an implantation rate of only 2.8% compared with 13.4% for embryos that have resumed division and 16.6% if blastomere lysis was less than 25%. El-Toukhy et al. (2003) reported that the clinical pregnancy rate for those patients having a single, intact embryo transferred was comparable to that in those patients who had at least one partially damaged embryo transferred (19 and 14%, respectively). Our study supports the recommendation that frozen embryos should be thawed individually and if the thawed embryo survives with all its blastomeres intact (and preferably divides after thawing) then serious consideration should be given to transferring this embryo alone, saving surplus frozen embryos for future. It has always been our protocol to freeze embryos individually, thereby enabling us to adopt the individual embryo thaw policy. We conclude that both blastomere lysis and cleavage after overnight culture affect the FET outcome in singleton thawed embryos and sfet should be performed when the embryo shows less than 25% blastomere loss and at least one blastomere cleaves overnight. This will eliminate the risk of dizygotic twin pregnancies. If the embryo has not cleaved overnight then another should be thawed and transferred, either with or without the original embryo. References Anderson AR, Wilkinson SS, Price S and Crain JL (2005) Reduction of high order multiples in frozen embryo transfers. Reprod Biomed Online 10, Archer J, Gook DA and Edgar DH (2003) Blastocyst formation and cell numbers in human frozen-thawed embryos following extended culture. Hum Reprod 18, Bergh C (2005) Single embryo transfer: a mini-review. Hum Reprod 20, Burns WN, Gauder TW, Martin MB, Leal YR, Schoen H, Eddy CA and Schenken RS (1999) Survival of cryopreservation and thawing with all blastomeres intact identifies multicell embryos with superior frozen embryo transfer outcome. Fertil Steril 72, Catt J, Wood T, Henman M and Jansen RJ (2003) Single embryo transfer in IVF to prevent multiple pregnancies. Twin Res 6, de Boer K, Catt J, Jansen RJ, Leigh D and McArthur S (2004) Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertil Steril 82, Edgar DH, Bourne H, Jericho H and McBain JC (2000a) The developmental potential of cryopreserved human embryos. Mol Cell Endocrin 169, Edgar DH, Bourne H, Speirs AL and McBain JC (2000b) A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryo. Hum Reprod 15, Edgar DH, Jericho H, Bourne H and McBain JC (2001) The influence of prefreeze growth rate and blastomere number on cryosurvival and subsequent implantation of human embryos. J Assist Reprod Genet 18, El-Toukhy T, Khalaf Y, Al-Darazi K, Andritsos V, Taylor A and Braude P (2003) Effect of blastomere loss on the outcome of frozen embryo replacement. Fertil Steril 79, Gerris JMR (2005) Single embryo transfer and IVF/ICSI outcome: a balanced appraisal. Hum Reprod Update 11, Guerif F, Bidault R, Cadoret V, Couet ML, Lansac J and Royere D (2002) Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal. Hum Reprod 17, Hyden-Granskog C and Tiitinen A (2004) Single embryo transfer in clinical practice. Hum Fertil (Camb) 7, Hyden-Granskog H, Unkila-Kallio L, Halttunen M and Tiitinen A (2005) Single embryo transfer is an option in frozen embryo transfer. Hum Reprod 20, Kolibianakis EM, Zikopoulos K and Devroey P (2003) Implantation potential and clinical impact of cryopreservation a review. Placenta 24 (Suppl. B),S27 S33. Kovacs G, Maclachlan V, Rombauts L, Healy D and Howlett D (2003) Replacement of one selected embryo is just as successful as two embryo transfer, without the risk of twin pregnancy. ANZ J Ob Gyn 43, Martikainen H, Tiitinen A, Tomas C, Tapanainen J, Orava M, Tuomivaara L, Vilska S, Hyden-Granskog C, Hovatta O and the Finnish ET study Group (2001) One versus two embryo transfer after IVF and ICSI: a randomized study. Hum Reprod 16, Oehning S, Mayer J and Muasher S (2000) Impact of different clinical variables on pregnancy outcome following embryo cryopreservation. Mol Cell Endocrinol 169, Pal L, Kovacs P, Witt B, Jindal S, Santoro N and Barad D (2004) Posthaw blastomere survival is predictive the success of frozen-thawed embryo transfer. Fertil Steril 82, Salumets A, HydeÂn-Granskog C, MaÈkinen S, Suikkari A-M, Tiitinen A and Tuuri T (2003a) Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures. Hum Reprod 18, Salumets A, Tuuri T, MaÈkinen S, Vilska S, Husu L, Tainio R and Suikkari AM (2003b) Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer. Hum Reprod 18, Speroff L and Fritz MA (2005) Clinical Gynecology Endocrinology and Infertility, 7th edn. Lippincott, Williams and Wilkins, Philadelphia. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD and Frydman R (1986) High pregnancy rate after early human embryo freezing. Fertil Steril 46, Thurin A, Hausken J, Hillensjo T, Jablonowska B, Pinborg A, Strandell A and Bergh C (2004a) Elective single-embryo transfer versus double-embryo transfer in vitro fertilization. NEJM 351, Thurin A, Hausken J, Hillensjo T, Jablonowska B, Pinborg A, Strandell A and Bergh C (2004b) Elective single embryo transfer in IVF, a randomized study. Hum Reprod 19 (Suppl. 1),i60. Tiitinen A, Halttunen M, Harkki P, Vuoristo P and Hyden-Granskog C (2001) Elective single embryo transfer: the value of cryopreservation. Hum Reprod 16, Tiitinen A, Unkila-Kallio L, Halttunen M and Hyden-Granskog C (2003) Impact of elective single embryo transfer on the twin pregnancy rate. Hum Reprod 18, Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P and Van Steirteghem AC (1997) Viability of partially damaged human embryos after cryopreservation. Hum Reprod 12, Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P and Van Steirteghem AC (1997) Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 12, Van Montfoort APA, Dumoulin JCM, Land JA, Coonen E, Derhaag JG and Evers JLH (2005) Elective single embryo transfer (eset) policy in the first three IVF/ICSI treatment. Hum Reprod 20, Ziebe S, Bech B, Petersen K, Mikkelsen AL, Gabrielsen A and Nyboe Andersen A (1998) Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum Reprod 13, Submitted on August 28, 2005; resubmitted on November 14, 2005; accepted on November 21,