Article Outcome of blastocyst transfer according to availability of excess blastocysts suitable for cryopreservation

Transcription

1 RBMOnline - Vol 7. No Reproductive BioMedicine Online; on web 10 October 2003 Article Outcome of blastocyst transfer according to availability of excess blastocysts suitable for cryopreservation Dr Bulent Urman After his residency training in Obstetrics and Gynecology in the University of Hacettepe in Ankara, Dr Urman completed a 3-year fellowship programme in Reproductive Endocrinology and Infertility in Vancouver, Canada. He returned to Hacettepe University in 1991 and participated in the foundation of one of the first IVF clinics in Turkey. He worked as an Associate Professor until 1996 in the same institution. Dr Urman resigned from the university in 1996 and founded the Assisted Reproduction Unit of the American Hospital of Istanbul, one of the biggest IVF centres in the country. His major areas of interest are clinical assisted reproduction, laparoscopic and hysteroscopic surgery. He has published extensively in these fields, having over 70 articles published in renowned international journals. Bulent Urman 1, Basak Balaban, Kayhan Yakin, Aycan Isiklar American Hospital of Istanbul, Assisted Reproduction Unit, Istanbul, Turkey 1 Correspondence: Abdi Ipekci Cad, Milli Reassurans Han II, Kat 5, Macka, Istanbul, Turkey. Tel: ; Fax: ; burman@superonline.com Abstract The purpose of this study was to assess the outcome of blastocyst transfer in relation to the presence or absence of excess blastocysts available for cryopreservation. The study was designed as a retrospective case series in a tertiary care private hospital. The study group consisted of 450 blastocyst stage embryo transfer cycles. In 139 cycles there were excess freezeable blastocysts (group 1), in 78 cycles there were excess but unfreezeable blastocysts (group 2), and in 233 cycles there were no excess blastocysts (group 3). A mean of three blastocysts was replaced in all groups. Treatment cycle characteristics, implantation and pregnancy rates following fresh and cryopreserved blastocyst transfer were assessed in each group. More embryos reached the blastocyst stage in group 1 and more blastocysts were of good quality. In group 1, clinical pregnancy and implantation rates (71 and 41%) were significantly higher compared with groups 2 (56 and 27%) and 3 (43 and 19%). Embryos that were selected for transfer among a cohort of good quality blastocysts yielded the highest implantation and pregnancy rates. Given a clinical pregnancy rate of 71%, an implantation rate per embryo of 41%, and a multiple pregnancy rate of 58%, serious consideration should be given to a single blastocyst transfer in these patients. Keywords: blastocyst, blastocyst cryopreservation, embryo transfer Introduction Blastocyst transfer offers the advantage of prolonged in-vitro observation of cleavage stage embryos. Transfer of embryos at later stages of development has been associated with higher implantation and pregnancy rates in some but not all studies (Scholtes and Zeilmaker, 1996; Gardner et al., 1998; Coskun et al., 2000; Huisman et al., 2000; Karaki et al., 2002; Levron et al., 2002; Rienzi et al., 2002; Utsunomiya et al., 2002). Advocates of blastocyst transfer claim higher pregnancy rates due to better embryo endometrium synchronization and improved ability to select more viable embryos (Gardner et al., 1998; Karaki et al., 2002). It appears that the selection of the embryo endowed with the potential to implant is the crucial factor leading to success of late embryo transfer. Most of the reported studies dealt with highly selected patient populations, thus making it difficult to assess the real value of blastocyst transfer in a general IVF/intracytoplasmic sperm injection (ICSI) population. Randomized studies that dealt with an unselected patient population suffered from inappropriate randomization protocols, use of a single medium for day 3 as well as day 5 transfers, or use of several different sequential media during the study period (Scholtes and Zeilmaker, 1996; Utsunomiya et al., 2002). Younger women having more than a certain number of cleavage stage embryos are the ones usually counselled for blastocyst transfer. These women not infrequently will have several embryos that have progressed to the blastocyst stage and will opt for cryopreservation. Being able to select among a cohort of blastocysts will undoubtedly increase the success of blastocyst transfer, as blastocyst grade appears to make a substantial difference to success rates (Balaban et al., 2000). 587

2 588 The aim of this study was to evaluate the success of blastocyst transfer in couples with and without excess blastocysts suitable for cryopreservation following fresh transfer on day 5. Materials and methods Patients A total of 450 blastocyst transfer cycles undertaken during a period of approximately 2 years ranging from 1998 to 2000 was evaluated. These were a selected group of patients who had five or more grade 1 or 2 cleavage stage embryos on day 3 who opted for embryo transfer on day 5. Blastocyst transfers formed 29.2% of the 1536 IVF/ICSI embryo transfer cycles performed in the institution during the same period of time. Embryo transfer cycles were analysed according to the availability of excess blastocysts. Group 1 included embryo transfer cycles where freezeable excess blastocysts were available. Group 2 included embryo transfer cycles with excess but unfreezeable blastocysts. In group 3, there were no excess blastocysts. Ovarian stimulation Ovarian stimulation was undertaken using a long gonadotrophin-releasing hormone (GnRH) analogue protocol combined with pure or recombinant FSH. Human chorionic gonadotrophin (HCG) was administered when the leading follicle reached 20 mm with two or more follicles >16 mm in mean diameter. Follicular aspiration was performed with transvaginal ultrasound guidance under local anaesthesia and intravenous sedation. ICSI was performed according to standard protocols. In-vitro culture of embryos In-vitro culture of embryos was undertaken as previously described (Balaban et al., 1998, 2001a; Gardner et al., 1998). Sequential media system (G1 and G2 media; Vitro Life, IVF Science Scandinavia, Goteborg, Sweden) designed for further embryonic development was used. Embryos were individually cultured in microdroplets containing G1 media on days 1 and 2. After the assessment of the cell number and morphology, embryos were transferred to G2 medium for further culture up to the blastocyst stage. Laser zona opening was performed on all embryos on day 3. Grading of cleavage stage embryos and blastocysts Cleavage stage embryos were graded as follows: grade 1 embryo: no fragmentation with equal sized homogenous blastomeres, grade 2 embryo: <20% fragmentation with equal sized homogenous blastomeres, grade 3 embryo: 20 50% fragmentation with unequal sized blastomeres, grade 4 embryo: >50% fragmentation with unequal sized blastomeres. Blastocyst grading was according to Dokras et al. (1993). Grade 1 blastocysts were characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophoectoderm layer. Grade 2 blastocyst exhibited a transitional phase where single or multiple vacuoles were seen which over subsequent days developed into the typical blastocyst appearance of the grade 1 blastocysts. Grade 3 blastocysts were defined as blastocysts with several degenerative foci in the inner cell mass, with cells appearing dark and necrotic. Blastocyst cryopreservation and thawing Only grade 1 and 2 blastocysts were deemed appropriate for cryopreservation. Blastocyst cryopreservation protocol was modified from Hartshorne et al. (1991). Briefly, a six-step slow freezing and quick thawing protocol was used utilizing Freeze Kit 2/Thaw Kit 2 medium (Vitrolife Fertility Systems) respectively. Blastocysts were initially incubated in 1 8% glycerol and frozen with a programme starting from ambient temperature to 7 C at a rate of 1 C per minute using the Planer Series III. Manual seeding was achieved at 7 C. Slow cooling was performed at a cooling rate of 0.3 C until 30 C and rapid cooling from 30 C to 150 C. The straws were then plunged immediately into 196 C liquid nitrogen (LN 2 ). The thawing process was as follows. The straws were removed from LN 2 and air thawed for 30 s. The straw was then plunged into 37 C water for s. Following water thawing, the embryos were moved into 8% glycerol solution, of which the concentration was decreased stepwise to 1%. The embryos were rinsed in the incubation medium and cultured in G2 medium until transfer. Embryo viability was verified according to expansion and/or hatching of the blastocysts. Embryo transfer Embryo transfer was performed on day 5. Observation of hatching was not awaited for embryo transfer; however, whenever there was a hatching blastocyst, this was included in the batch of embryos selected for transfer. On average, three (four in women >38 years) blastocysts were transferred using Wallace or Frydman embryo transfer catheters. All embryo transfers were performed under transabdominal ultrasound guidance. Tetracycline 200 mg b.i.d. (Monodoks; DEVA, Istanbul, Turkey) and methylprednisolone 16 mg/day (Prednol 16 mg; Mustafa Nevzat Ilaç Sanayi, Istanbul, Turkey) were administered for 5 days starting from the day of oocyte retrieval. Luteal phase was supplemented with intravaginal natural progesterone at a dose of 600 mg divided in three doses (Utrogestan; Laboratories Besins Iscovesco, Paris, France) starting on the day of oocyte retrieval. Pregnancy was defined as two β-hcg titres assessed 12 and 14 days after embryo transfer showing appropriate doubling. Clinical pregnancy was defined as the presence of gestational sac/s with a viable embryo shown on vaginal ultrasonography performed approximately 24 days after embryo transfer. Statistics Results were analysed using one-way analysis of variance and chi-square tests. A P-value of <0.05 was accepted as significant. Results Mean age of the women, duration of infertility, the incidence of previous failed embryo transfer cycles, and the mean number of retrieved oocytes were similar between groups

3 (P > 0.05) (Table 1). Likewise, 2PN fertilization rate and cleavage rate were also similar (P > 0.05). There were significantly more G1 + G2 cleavage stage embryos in groups 1 and 2 compared with group 3 (P < 0.05). Furthermore, in groups 1 and 2, the incidence of 8-cell embryos on day 3, rate of progression of cleavage stage embryos to the blastocyst stage, and the incidence of grade 1 and 2 and hatching blastocysts were significantly increased compared with group 3 (P < 0.05). A mean of three blastocysts were transferred. Significantly more grade 1 and 2 blastocysts were transferred in group 1 compared with groups 2 and 3, and in group 2 compared with group 3. Clinical pregnancy rates per embryo transfer were 71.2, 56.4, and 43.7%; implantation rates per embryo were 40.7, 27.2, and 18.6% in the three groups respectively (group 1 is significantly different from groups 2 and 3 and group 2 is significantly different from group 3). The rate of twin and higher order multiples was also significantly higher in group 1 (P < 0.05). Of the 139 blastocyst transfer cycles associated with excess freezeable blastocysts, 99 cycles ended in a clinical pregnancy and 90 with a term or preterm delivery (Table 2). Of the patients with frozen blastocysts, 30 opted for replacement following thawing. Of these thaw cycles, four (13.3%) resulted in a clinical pregnancy. Two of these patients aborted and two (6.7%) delivered at term. The clinical pregnancy and implantation rates for day 3 frozen thawed embryo transfer cycles at the same period were 19 and 8% respectively. During the same time period, 1086 day 3 embryo transfer cycles were performed. Of these, 308 cycles were associated with excess and freezeable cleavage stage embryos. In 531 cycles there were excess embryos of poor quality that were not suitable for cryopreservation, and in 247 cycles there were no excess embryos. Clinical pregnancy rate per embryo transfer and implantation rate in these groups were 63.3 and 30% versus 45.2 and 16.7% versus 30.3 and 9.1% respectively. Results of day 3 transfers compared with day 5 transfers are given in Table 3. In all groups, clinical pregnancy rates per embryo transfer were similar for day 3 and day 5 transfers (P > 0.05). Day 5 transfers were, however, associated with higher implantation rates in all groups (P < 0.05). Abortion rates were similar. In the group with no excess embryos, day 5 transfers resulted in higher multiple pregnancy rates despite the transfer of fewer embryos. Discussion Blastocyst transfer is associated with high implantation and clinical pregnancy rates (Gardner et al., 1998; Balaban et al., 2000; Langley et al., 2001; Kolibianakis, 2002; Wilson et al., 2002). It is, however, still unresolved at this time whether higher success is due to late transfer of embryos and its associated purported benefits, or due to better embryo selection. A recent Cochrane Review indicated that blastocyst transfer confers little overall benefit over and above that associated with cleavage stage embryo transfer (Blake et al., 2002). Caution was advised when offering prolonged embryo culture to all patients due to increased cancellation rates of Table 1. Patient and embryo characteristics of patients undergoing blastocyst transfer. Embryo transfer Embryo transfer Embryo transfer cycles with excess cycles with excess cycles with no freezeable blastocysts non-freezeable excess blastocysts blastocysts Number Mean age (years) Duration of infertility (years) Percentage of couples with previous failed IVF/ICSI cycles No. oocytes retrieved 1966 (14.1) 1108 (14.2) 3216 (13.8) (mean) Injected oocytes PN fertilization (%) 1036 (70.0) 584 (70.0) 1648 (69.2) Cleaved embryos (%) 1019 (98.3) 573 (98.1) 1618 (98.1) G1 + G2 embryos 724 (71.0) 361 (63.0) 826 (51.0) on day 3 (%) a Embryos 8 cell 655 (64.2) 299 (52.1) 632 (32.0) on day 3 (%) b Blastocysts (%) a 675 (66.2) 328 (57.2) 744 (45.9) BG1 + BG2 468 (68.3) 180 (54.8) 298 (40) blastocysts (%) a Hatching blastocysts 237 (35.1) 89 (27.1) 141 (18.9) (%) a a Group 1 is significantly different (P < 0.05) from group 3. b Group 1 is significantly different (P < 0.05) from groups 2 and

4 Table 2. Outcome of blastocyst transfer in patients with no excess, excess but unfreezeable and excess and freezeable blastocysts. Embryo Embryo transfer Embryo transfer cycles cycles with transfer cycles with freezeable excess non- with no excess excess blastocysts freezeable blastocysts blastocysts Number Blastocysts 417 (3.0) 235 (3.0) 744 (3.1) transferred (mean) BG1 + BG2 blastocysts 383 (91.8) 180 (76.5) 298 (40) transferred (%) Clinical pregnancy/ 99/139 (71.2) 44/78 (56.4) 102/233 (43.7) embryo transfer (%) a Implantation/ 170/417 (40.7) 64/235 (27.2) 139/744 (18.6) embryo (%) a Clinical abortions (%) 8/99 (8.1) 5/44 (11.3) 18/102 (9.0) Multiple pregnancies 57/99 (58) 19/44 (43.2) 37/102 (36.3) (%) b Multiple pregnancies 44/45/12/0 24/17/2/0 65/37/0/0 (S/Tw/Tr/Quad) a Group 1 is significantly different (P < 0.05) from groups 2 and 3. b Group 1 is significantly different (P < 0.05) from group 3. Table 3. A comparison of day 3 and day 5 embryo transfers according to the availability of excess embryos suitable for cryopreservation. Embryo transfer cycles with Embryo transfer cycles with Embryo transfer cycles excess freezeable embryos excess non-freezeable embryos with no excess embryos Day 3 Day 5 Day 3 Day 5 Day 3 Day 5 Number Embryos 1170 (3.7) 417 (3.0) 2058 (3.8) 235 (3.0) 865 (3.5) 744 (3.1) transferred (mean) Clinical pregnancy/ 195/308 99/ /531 44/78 75/ /233 embryo transfer (%) (63.3) (71.2) (45.2) (56.4) (30.3) (43.7) Implantation/ 351/ / / /235 79/ /744 embryo (%) a (30) (40.7) (16.7) (27.2) (9.1) (18.6) Clinical abortions (%) 20/195 8/99 25/240 5/44 11/75 18/102 (10.2) (8.1) (10.4) (11.3) (14.6) (9.0) Multiple pregnancies 115/195 57/99 95/240 19/44 4/75 37/102 (%) b (59) (58) (39/5) (43.2) (5.3) (36.3) Multiple pregnancies 80/74/41 44/45/12/0 145/85/10 24/17/2/0 71/4/0 65/37/0/0 (S/Tw/Tr/Quad) a In all groups, day 5 transfers are significantly different (P < 0.05) from day 3 transfers. b In embryo transfer cycles with no excess embryos, day 3 transfer is significantly different (P < 0.05) from day embryo transfer and possible decrease in blastocyst cryopreservation rate (Blake et al., 2002). It is becoming clear that the success of blastocyst transfer is heavily dependent on the number and quality of the blastocysts available (Balaban et al., 2000). The results from day 3 transfers undertaken during the same time period indicate that success of cleavage stage embryo transfer is associated with the availability of a good cohort of embryos. Highest pregnancy rates were obtained where there were excess and freezeable cleavage stage embryos. As has been shown with cleavage stage embryos (Grimbizis et al., 1998), selective and elective blastocyst transfers yield very different pregnancy rates. The ability to select among a number of available blastocysts appears to have an added advantage over and above that associated with the natural selection of cleavage stage embryos during prolonged in-vitro culture. The present results indicate that in patients with excess freezeable blastocysts, implantation and clinical pregnancy rates are much higher compared with patients who do not have any excess blastocysts or excess blastocysts suitable for freezing. These results indicate that similar to day 3 cleavage stage transfers, blastocyst transfer is more successful in the presence of excess blastocysts, thus providing a cohort of selectable

5 embryos. Although the study did not intend to compare day 3 with day 5 transfers, day 5 transfers resulted in higher implantation rates than day 3 transfers undertaken during the same time period. Day 3 transfers were also grouped similar to day 5 transfers. Therefore it may be reasonable to assume that day 3 transfers associated with freezeable embryos were similar to those in patients selected for day 5 transfer. However, prolonging the culture period appeared to confer an added benefit over and above that associated with the availability of a large cohort of good quality embryos. Success of blastocyst transfer has resulted in the discussion of single blastocyst transfer in favourable patients (Gardner and Lane, 2003). Assessment of morphological criteria and providing algorithms for embryo scoring will increase success of both day 3 and day 5 embryo transfers, concurrently with a decrease in the number of transferred embryos (Racowsky et al., 2003). Women with good quality blastocysts are candidates for single blastocyst transfer that will yield high pregnancy rates. Better blastocyst cryopreservation techniques will undoubtedly further increase cumulative pregnancy rates in women undergoing blastocyst transfer (Mukaida et al., 2003). It is a matter of debate whether cumulative pregnancy rates are compromised when embryo transfer is undertaken after prolonged in-vitro culture, thus potentially limiting the number of embryos available for cryopreservation (Kolibianakis, 2002). Pantos reported lower rates of blastocyst cryosurvival and implantation (56 and 5.3%) when embryos were frozen at the blastocyst stage, compared with embryos that were frozen at the cleavage stage and transferred at the blastocyst stage following thawing (89 and 24.5%) (Pantos et al., 2001). However, Behr et al. reported a 16% implantation rate following the transfer of thawed blastocysts that were frozen either on day 5 or 6 (Behr et al., 2002). A higher implantation rate was reported by Langley et al. in a retrospective analysis comparing day 3 versus day 5 transfer of frozen thawed embryos (10.1 versus 21.9%) (Langley et al., 2001). Furthermore more embryos were transferred in the day 3 group (3.1 versus 2.3). It remains to be shown in prospective studies whether cumulative pregnancy rates are affected by prolonged in-vitro culture of embryos. Undoubtedly, the cohort of embryos will be smaller after prolonged in-vitro culture that will limit the number of selectable embryos for transfer and embryos suitable for cryopreservation. Better cryopreservation techniques and culture media that yield higher blastocyst survival and higher implantation rates of thawed blastocysts will undoubtedly increase the efficiency of blastocyst transfer (Vanderzwalmen et al., 2002; Reed et al., 2002; Lane et al., 2003; Son et al., 2003). Mukaida and colleagues in a recent study, reported 80% survival and 37% clinical pregnancy rate following transfer of 583 blastocysts that were vitrified using cryoloops (Mukaida et al., 2003). In the present study, low pregnancy and implantation rates associated with frozen thawed blastocyst transfer may be attributed to the technique used during the time period in which the study was conducted. Blastocyst transfer has been proposed as a means to select the most appropriate embryo for transfer, the one that is endowed with the highest capacity to implant (Balaban et al., 1998, 2001b). Progression of the cleavage stage embryo to the blastocyst stage appears to be influenced by maternal age, culture conditions in the embryology laboratory, the composition of the culture media utilized and last, but not least, the genetic constitution of the cleavage stage embryo as analysed by FISH techniques (Sandalinas et al., 2001). Furthermore, pronuclear pattern, grade, and cleavage rate are also indicators of survival during prolonged in-vitro culture (Balaban et al., 2001c). In the present study, patients with freezeable excess blastocysts had significantly more grade 1 and 2 embryos and embryos with >8 blastomeres on day 3. These were translated into a higher rate of blastocyst and hatching blastocyst formation. Therefore, the most favourable group was selected early in the course of in-vitro culture. In the group with excess freezeable blastocysts significantly more grade 1 and grade 2 blastocysts were transferred, leading to higher implantation and pregnancy rates compared with the group in which a similar number of blastocysts were transferred, however, without the presence of freezeable blastocysts. The group with no excess blastocysts fared worst, due to a more limited number of grade 1 and grade 2 blastocysts available for transfer compared with the other groups. In conclusion, the results of the present study show that blastocyst transfer in the presence of excess freezeable blastocysts is associated with very high implantation and pregnancy rates. Consideration should be given to a single blastocyst transfer in this subset of patients. Lower implantation and pregnancy rates are obtained when there are excess but unfreezeable blastocysts and when there are no excess blastocysts at all. References Balaban B, Urman B, Sertac A et al Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection. Human Reproduction 13, Balaban B, Urman B, Sertac A et al Blastocyst quality affects the success of blastocyst stage embryo transfer. Fertility and Sterility 74, Balaban B, Urman B, Isiklar A et al. 2001a Blastocyst transfer following intracytoplasmic injection of ejaculated, epididymal, or testicular spermatozoa. Human Reproduction 16, Balaban B, Urman B, Alatas C et al. 2001b Blastocyst stage transfer of poor quality cleavage stage embryos results in higher implantation rates. Fertility and Sterility 75, Balaban B, Urman B, Isiklar A et al. 2001c The effect of pronuclear morphology on embryo quality and blastocyst transfer outcome. Human Reproduction 16, Behr B, Gebhardt J, Lyon J, Milki A 2002 Factors leading to a successful cryopreserved blastocyst transfer program. Fertility and Sterility 77, Blake D, Proctor M, Johnson N, Olive D 2002 Cleavage stage versus blastocyst transfer in assisted conception. Cochrane Database Syst Rev 2, CD Coskun S, Hollanders J, al Hassan S et al Day 5 versus day 3 embryo transfer: a controlled randomized trial. Human Reproduction 15, Dokras A, Sargent I, Barlow DH 1993 Human blastocyst grading: an indicator of developmental potential. Human Reproduction 8, Gardner DK, Lane M 2003 Towards a single embryo transfer. Reproductive BioMedicine Online 6,

6 Gardner DK, Schoolcraft W, Wagley L et al A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Human Reproduction 13, Grimbizis G, Vandervorst M, Camus M et al Intracytoplasmic sperm injection results in women older than 39 according to age and the number of embryos replaced in selective and nonselective transfers. Human Reproduction 13, Hartshorne G, Elder K, Crow J et al The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Human Reproduction 6, Huisman G, Fauser B, Eijkemans M, Pieters M 2000 Implantation rates after in vitro fertilization of a maximum of two embryos that have undergone three to five days of culture. Fertility and Sterility 73, Karaki R, Samarraie S, Younis N et al Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertility and Sterility 77, Kolibianakis E 2002 Blastocyst culture: fact or fiction. Reproductive BioMedicine Online 5, Lane M, Maybach J, Hooper K et al Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan. Molecular Reproduction and Development 64, Langley M, Marek D, Gardner D et al Extended embryo culture in human assisted reproduction. Human Reproduction 16, Levron D, Farhi J, Nahum H et al Prospective evaluation of blastocyst transfer vs zygote intrafallopian tube transfer in patients with repeated implantation failures. Fertility and Sterility 77, Mukaida T, Nakamura S, Toniyama T et al Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Human Reproduction 18, Pantos K, Stefanidis K, Pappas K et al Cryopreservation of embryos, blastocysts, and pregnancy rate of blastocysts derived from frozen thawed embryos and frozen thawed blastocysts. Journal of Assisted Reproduction and Genetics 18, Racowsky C, Combelles C, Nureddin A et al Day 3 and day 5 morphological predictors of embryo viability. Reproductive BioMedicine Online 6, Reed M, Lane M, Gardner D et al Vitrification of human blastocyst using the cryoloop method: successful clinical application and birth of offspring. Journal of Assisted Reproduction and Genetics 19, Rienzi L, Ubaldi F, Iacobelli M et al Day 3 embryo transfer combined with pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Human Reproduction 17, Sandalinas M, Sadowy S, Alikani M et al Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Human Reproduction 16, Scholtes M, Zeilmaker G 1996 A prospective randomized study of embryo transfer results after 3 or 5 days of embryo culture in invitro fertilization. Fertility and Sterility 74, Son W, Yoon S, Yoon H et al Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction 18, Utsunomiya T, Naitou T, Nagaki M 2002 A prospective trial of blastocyst culture and transfer. Human Reproduction 17, Vanderzwalmen P, Bertin G, Debauche C et al Birth after vitrification at morulae and blastocyst stage: effect of artificial reduction of the blastocoeleic cavity before vitrification. Human Reproduction 17, Wilson M, Hartke K, Kiehl M et al Integration of blastocyst transfer for all patients. Fertility and Sterility 77, Received 13 May 2003; refereed 10 July 2003; accepted 22 August