Human Reproduction Vol.17, No.7 pp. 1846 1851, 2002 A prospective trial of blastocyst culture and transfer Takafumi Utsunomiya 1, Tae Naitou and Miyuki Nagaki St Luke Clinic, 5, Tsumori-Tomioka, Oita City, 870-0947, Japan 1 To whom correspondence should be addressed. E-mail: St-luke@oct-net.ne.jp BACKGROUND: The aim of this study was to evaluate the effectiveness of the blastocyst culture method compared with the conventional day 3 transfer method using a prospective trial. METHODS: A total of 235 patients with 273 cycles were evaluated for a period of almost 1 year. Depending upon the sequence in which the ovum retrieval was performed, patients were prospectively assigned (alternate allocation) to a culture period of 3 or 5 days duration. Embryos were transferred either on day 3 (after culture in human tubal fluid) or on day 5 (after culture in sequential media). RESULTS: The pregnancy rates after embryo transfer on days 3 and 5 were similar at 26.5 and 25.9% respectively. Among the day 5 embryo transfer group, patients were divided into three groups corresponding to three sequential media. The pregnancy rates were 32.0% using Irvine blastocyst medium, 6.9% using G1.2/G2.2 and 32.4% using Cook blastocyst medium. CONCLUSIONS: The results of our study were not as successful as previous studies had been. Additionally, there may have been problems in day 5 embryo transfer, such as choosing the sequential media, quality control, contamination and so on. From the results of this study, it appears that day 5 embryo transfer has no advantages for ordinary patients of assisted reproductive technology. Key words: assisted reproduction/blastocyst/embryo transfer/pregnancy/sequential media Introduction Although the use of assisted reproductive techniques (ART) has rapidly expanded, the pregnancy rate has not been impressive at ~20% in non-selective cases. Some reports have stated that good results were obtained with blastocyst stage culture using special sequential media (Alves da Motta et al., 1998; Gardner et al., 1998, 2000; Cruz et al., 1999; Graham et al., 2000). A very high pregnancy rate of 87% was obtained using sequential media in patients who had a good response to gonadotrophin stimulation and two top scoring blastocysts (Gardner et al., 2000). The blastocyst-stage transfer was useful even for patients with multiple failures of IVF (Cruz et al., 1999). Very few reports on the use of these sequential media (Gardner et al., 1998; Coskun et al., 2000; Huismann et al., 2000) were designed to evaluate and compare results on a prospective basis. This study was designed to evaluate and compare the results of conventional day 3 embryo transfer using a conventional culture medium of human tubal fluid (HTF) and a new technique based on day 5 embryo transfer using sequential media with strictly prospective and alternative allocation schedules. Material and methods Patients This study was performed under a prospective schedule. The study was open to all patients who consented to enter the trial between September 1999 and September 2000. Depending on the sequence in which the ovum retrieval was performed, patients were prospectively assigned to a culture period of 3 or 5 days duration. Patients were prospectively assigned at the morning of ovum retrieval (day 3: 185 patients, day 5: 185 patients). In one cycle in the day 3 group and five cycles in the day 5 group no oocyte was retrieved. Among the three groups of day 5 embryo transfer, patients were also prospectively assigned depending on the sequence (Figure 1). The total number of eligible patients was 325 (day 3 embryo transfer group: 153, day 5 embryo transfer group: 172) and the number of eligible cycles was 364 (day 3: 184, day 5: 180). Among them, 58 cycles (day 3: 20, day 5: 38) were cancelled because all embryos were cryopreserved to prevent ovarian hyperstimulation syndrome (OHSS). Furthermore, 33 cycles (day 3: 12, day 5: 21) were cancelled because the fertilized oocyte could not be obtained. We recognize that the day 5 embryo transfer group had a higher level of excluded cycles. We believe this to be due to natural phenomena and not the result of human bias. Finally, 235 patients with 273 cycles were registered and divided into two groups (Figure 1, Table I). The number of randomized patients in the day 3 embryo transfer group was 121 and the number of cycles 152. In the day 5 embryo transfer group, the number of patients was 114 and the number of cycles 121.The first group (G-I) consisted of 121 patients with 152 cycles using day 3 embryo transfer, which was used as our control group utilizing HTF (Irvine Scientific, Santa Ana, USA). The second group (G-II) consisted of 49 patients with 53 cycles using the day 5 embryo transfer and Irvine blastocyst medium (Irvine Scientific). The third group (G-III) consisted of 29 patients with 30 cycles using day 5 embryo transfer and G1.2/G2.2 medium (IVF Science Scandinavia, Gothenburg, Sweden). Lastly, the fourth group (G-IV) consisted of 36 patients with 38 cycles using day 5 embryo transfer and Cook blastocyst medium (Cook IVF, 1846 European Society of Human Reproduction and Embryology
Controlled trial of blastocyst embryo transfer Table I. Clinical data and outcome of day 3 and day 5 embryo transfer groups G-I Day 3 embryo transfer G-II, -III, -IV Day 5 embryo transfer Eligible patients (n) 153 172 Eligible cycles (n) 184 180 Cancelled cycles 2PN cryopreservation cycles (n) a 20 38 No. of non-fertilized cycles (n) b 12 21 Registered patients (n) 121 114 Cycles (n) 152 121 Mean age (years; range) c 34.0 4.0 (25 44) 34.0 4.2 (25 45) Mean no. previous ART failure cycles (range) c 4.7 3.8 (1 20) 5.5 5.4 (1 25) Mean no. of oocytes (range) c 9.4 4.8 (1 26) 10.3 5.0 (2 25) Mean fertilization rate (range) c 70.9 22.7 (8 100) 73.4 21.0 (6 100) No. of cancellations (%) c 1 (0.7) 5 (4.1) Mean no. of embryo transfer embryos (range) c 2.9 0.8 (0 5) 3.0 1.0 (0 6) Mean endometrial thickness (mm; range) c 10.9 2.7 (5.8 17) 11.0 3.1 (6.1 22) No. of embryo transfer (%) c 151 (99.3) 116 (95.9) Mean blastulation rate (range) 21.6 25.1 (0 100) No. of pregnancies (%) (per all cycle) c 40 (26.3) 30 (24.8) No. of pregnancies (%) (per embryo transfer) c 40 (26.5) 30 (25.9) No. of abortions (%) c 10 (25.0) 10 (33.3) No. of implantations (%) c 52 (11.7) 33 (9.2) a All 2PN stage embryos were cryopreserved to prevent ovarian hyperstimulation syndrome. b Cycles cancelled because the fertilized eggs could not be obtained. c There is no difference between the two groups statistically. Oocyte retrieval and insemination Oocyte retrieval was performed by transvaginal ultrasound-guided aspiration under i.v. anaesthesia with modified neuro-lepto-analgesia (NLA) 34 h after HCG administration. Oocytes were inseminated by conventional IVF or by ICSI 3 4 h after oocyte retrieval. ICSI was performed if the sperm concentration was 20 10 6 /ml with a total progressive motility 50% and/or sperm morphology 10% normal forms according to strict criteria (Kruger et al., 1988; Menkveld et al., 1991). Sydney, Australia). There was no difference between the day 3 and day 5 embryo transfer groups in mean age, number of retrieved oocytes, number of embryos transferred or endometrial thickness. Controlled ovarian stimulation In all patients, nasal GnRH agonist (Suprecur; Hoechst, Frankfurt, Germany) was administered to achieve pituitary down-regulation from the preceding mid-luteal phase in a long stimulation protocol. Controlled ovarian stimulation was carried out with HMG (Pergogreen; Serono, Japan) or urinary FSH (Fertinorm; Serono). When the mean diameter of the follicles within the cohort reached 18 mm, 10 000 IU of HCG (Profasi; Serono) was administered to induce the final stage of oocyte maturation. Embryo culture Embryos were cultured under mineral oil in each medium at 36.7 C in a 5% O 2,5%CO 2 and 90% N 2 environment. HTF with 10% patient s serum was used for the G-I group. In the G-II group, embryos were cultured in HTF with 10% patient s serum for 3 days, then transferred into Irvine blastocyst medium with 10% patient s serum and cultured for a further 2 days. In the G-III group, embryos were cultured in G1.2 medium for 3 days after insemination and then carefully transferred into G2.2 medium and cultured for an additional 2 days. In the G-IV group, embryos were cultured in HTF with 10% patient s serum for 3 days, then transferred into Cook blastocyst medium and cultured for an additional 2 days. Embryo transfer The embryos were evaluated using embryo scoring (Mills et al., 1992) and blastocysts were graded using a blastocyst scoring system (Gardner et al., 1999). Around three good quality embryos were selected for embryo transfer. If no blastulated embryo was obtained on day 5, embryo transfer was cancelled. Luteal support To support the luteal phase, the patients were injected with 1000 IU HCG on the day of embryo transfer, day 4 and day 7. Additionally, 30 mg of didrogesterone (Daiichi Seiyaku, Tokyo, Japan) was 1847
T.Utsunomiya, T.Naitou and M.Nagaki Table II. Patient profiles, results of conventional IVF and ICSI for each group Day 3 embryo transfer group Day 5 embryo transfer group IVF ICSI IVF ICSI Patients (n) 65 56 66 48 Cycles (n) 76 76 68 53 Mean age (years; range) 33.4 3.6 (26 42) a 34.6 4.3 (25 44) 33.0 4.1 (25 44) c 35.2 4.1 (27 45) Mean no. of oocytes (range) 10.2 4.8 (1 26) a 8.6 4.7 (3 22) 10.3 4.4 (2 25) 10.4 5.8 (2 22) Mean fertilization rate (range) 70.3 22.3 (8 100) 71.5 23.3 (10 100) 71.8 21.1 (22 100) 75.5 21.0 (6 100) No. of cancellations (%) 0 (0.0) 1 (1.3) 4 (5.9) 1 (1.9) Mean no. of embryo 3.0 0.7 (1 5) 2.8 0.8 (0 4) 3.0 1.0 (0 6) 3.0 1.0 (0 5) transfer embryos (range) No. of embryo transfers (%) 76 (100) 75 (98.7) 64 (94.1) 52 (98.1) Mean blastulation rate (range) 26.2 28.9 (0 100) b 16.0 18.1 (0 60) Mean endometrial thickness (mm; range) 11.2 2.6 (6 17) 10.6 2.8 (5.8 16) 11.1 2.8 (6.3 17) 11.0 3.6 (6.1 22) No. of pregnancies (%) 25 (32.9) 15 (19.7) 21 (30.9) 9 (17.0) (per all cycle) No. of pregnancies (%) 25 (32.9) 15 (20.0) 21 (32.8) 9 (17.3) (per embryo transfer) No. of abortions (%) 6 (24.0) 4 (26.7) 7 (33.3) 3 (33.3) No. of implantations (%) 34 (14.8) a 18 (8.3) 23 (11.4) 10 (6.3) a P 0.05: difference between conventional IVF and ICSI in day 3 embryo transfer group. b P 0.05: difference between conventional IVF and ICSI in day 5 embryo transfer group. c P 0.01: difference between conventional IVF and ICSI in day 5 embryo transfer group. administered daily for 16 days. Some of the patients were not administered HCG in order to avoid OHSS. Sixteen days after ovum retrieval, the urinary HCG level was checked. A transvaginal ultrasound visualization of the gestational sac was performed to confirm clinical pregnancy 23 days after ovum retrieval. Statistical analysis Data were compared with the use of the χ 2 - and Student s t-tests. Results Pregnancy rate In the day 3 embryo transfer group, 40 pregnancies were obtained in 152 cycles. The pregnancy rate was 26.5% and the implantation rate was 11.7% per embryo transferred. In the day 5 embryo transfer group, 30 pregnancies in 121 cycles were obtained, with a pregnancy rate of 25.9% and an implantation rate of 9.2%. The abortion rate was 25.0% in the day 3 group and 33.3% in the day 5 group. The cancellation rate was 0.7% in the day 3 group and 4.1% in the day 5 group. There was no difference in pregnancy, implantation, abortion or cancellation rates between the day 3 and day 5 embryo transfer groups (Table I). In the day 3 embryo transfer group, there was no difference in pregnancy, abortion or cancellation rates when using either conventional IVF or ICSI. The same held true for the day 5 embryo transfer group. However, in the day 3 group, the implantation rate of the conventional IVF group was higher than that of the ICSI group (Table II). The patients who participated in day 5 embryo transfer were divided into three groups (G-II, -III and -IV). Among them, the pregnancy rate in G-II was 32% using HTF and Irvine blastocyst medium; in G-III, it was 6.9% using G1.2/G2.2; and in G-IV it was 32.4% using HTF and Cook blastocyst 1848 medium. G-III showed statistically significant lower pregnancy and implantation rates than did the other groups (Table III). Discussion To obtain good results with ART, many techniques and theories have been reported; these include assisted hatching, co-culture, gamete intra-fallopian tube transfer and so on. Recently, the new technique of blastocyst stage embryo transfer using sequential media was reported. The basis for this new method is quite practical and persuasive. There has been no theoretical doubt that the transfer of well-developing embryos in advanced cellular stages should result in high implantation and subsequently high pregnancy rates. Human gene expression may first occur between the 4- and 8-cell stages of preimplantation development (Braude et al., 1988). So, within the hatching stage, the longer the embryos are cultured, the easier it is to select the best embryos. It has been suggested (Ménézo et al., 1998) that it is time to switch from co-culture to sequential media for transfer at the blastocyst stage. However, contrary to our expectations, the results of our prospective study were not as successful as previous studies. A high pregnancy rate of 87% has been reported (Gardner et al., 2000) using day 5 embryo transfer with sequential media. However, in that report the pregnancy rate of the control group using day 3 embryo transfer was also quite high at 66%. We believe this to be because the patients were selected using a criteria based on a number of favourable characteristics, and who therefore had a good chance of responding well. They had a healthy uterine endometrium, no physiological disorders and no sperm abnormalities. Their main aim was to avoid multiple gestations, as the patients had good odds of becoming pregnant. Also, a good result was reported (Milki et al., 2000) using P1 medium plus 10%
Controlled trial of blastocyst embryo transfer Table III. Comparison of clinical data and outcomes for each day 5 embryo transfer culture medium HTF/Irvine G1.2/G2.2 HTF/Cook blastocyst medium blastocyst medium Patients (n) 49 29 36 Cycles (n) 53 30 38 Mean age (years; range) 34.6 4.1 (25 45) 33.9 4.5 (26 44) 33.1 4.1 (25 41) Mean no. of oocytes (range) 9.4 4.7 (2 20) 10.4 5.0 (3 25) 11.6 5.4 (2 22) Mean fertilization rate (range) 73.4 21.4 (26 100) 75.8 20.6 (29 100) 71.6 21.2 (6 100) No. of cancellations (%) 3 (5.7) 1 (3.3) 1 (2.6) Mean no. of embryo 3.0 1.2 (0 6) 3.1 1.0 (0 5) 2.9 0.8 (0 4) transfer embryos (range) Mean blastulation rate (range) 27.3 28.8 (0 100) 18.7 22.4 (0 70) 16.1 20.0 (0 62) No. of embryo transfer (%) 50 (94.3) 29 (96.7) 37 (97.4) Mean endometrial 10.9 3.0 (6.2 17) 11.1 3.3 (6.1 20) 11.2 3.3 (6.1 22) thickness (mm; range) No. of pregnancies (%) 16 (30.2) a 2 (6.7) a 12 (31.6) (per all cycle) No. of pregnancies (%) 16 (32.0) a 2 (6.9) a 12 (32.4) (per embryo transfer) No. of abortions (%) 5 (31.3) 0 (0.0) 5 (41.7) No. of implantations (%) 17 (10.8) 3 (3.2) 13 (11.9) a P 0.05: difference between G 1.2 /G 2.2 and Irvine, Cook blastocyst media. HTF human tubal fluid. synthetic serum supplement and day 5 embryo transfer in patients 40 years old and with three or more 8-cell embryos on day 3. The result of the day 5 embryo transfer pregnancy rate was 68% compared with the day 3 embryo transfer rate of 46%. On the other hand, some reports showed no statistical differences between day 3 and day 5 embryo transfer results using prospective studies (Scholtes and Zeilmaker, 1996; Huisman et al., 2000; Coskun et al., 2000). We too had the same result in this study. In particular, the cancellation rate of the day 5 embryo transfer group was a little higher than that of the control day 3 embryo transfer. Furthermore, the implantation rate of the day 5 embryo transfer group was a little lower than that of the day 3 group, although this was not statistically significant. Our blastulation rate of 21.6% is similar to (Coskum et al., 2000; 28%) and lower than other prospective randomized reports (Huisman et al., 2000; 48%). In this study, the mean age of 34 years was higher (30 years in Coskum et al. and 33 years in Huisman et al.) and the average number of previous ART cycles were 4.7 in the day 3 group and 5.5 in the day 5 group (Table I). These facts showed that there were many difficult cases in this study and this is one of the reasons why the blastulation rate was lower than in other studies. Thus, the day 5 embryo transfer may not become an established clinical practice, due to factors (Tsirigotis, 1998) such as: (i) concerns over nutritional support for embryos cultured long term in vitro; (ii) the lack of specific knowledge with regard to the time needed for the intratubal descent towards the uterus of the naturally conceived embryos and the peri-implantation events (i.e. synchronization of the endometrium); (iii) the lack of established criteria for blastocyst morphology and growth velocity; and not least (iv) the professional worries that embryos will not be available for transfer in ~40% of patients after 5 days of laboratory culture (Scholtes and Zeilmaker, 1998; Shoukir et al., 1998). Currently there are many difficulties associated with day 5 embryo transfer. There is the very tough question as to which sequential media would be the best in which to culture the embryos for 5 days. Even though there are many types of sequential media, there is no paper or study to compare each of them except the report on the controlled comparison of commercial media for human IVF between Ménézo B2 medium, Medi-Cult universal and BM1 medium (Ellios Bio-Media, Igny, France) (Staessen et al., 1998). In this study, we have tried to compare the effectiveness of three media for blastocyst stage embryo transfer. Our study demonstrated that two of the media had better results than the other one. Because our sample was not large enough, we could not arrive at a clear conclusion, and more prospective randomized studies are therefore needed. It is uncertain why the pregnancy rate of G-III was lower than that of G-II and G-IV. One explanation is that it may be due to batch to batch variability. This may be another reason why our blastulation rate of 21.6% (Table II) is lower than that of other reports (Coskun et al., 2000; Huisman et al., 2000). The special medium might play an important role in culturing matured embryos, especially due to its concentration of minerals, electrolytes, vitamins, amino acids and so on. Unfortunately, we do not know the concentration of these materials in some culture media. However, we can guess the concentration of these materials from data obtained from experiments with animals (Gardner and Sakkas, 1993; Gardner et al., 1994; Lane and Gardner, 1998). That being said, the fact still remains that the condition of the culture and the materials for human embryos may differ from those required by animals. Concerning the 5 day culture, a lot of complexities may arise which could easily frustrate situations in the laboratory. We usually evaluate embryo quality every day until the day of embryo transfer. As a result, there are many more culture dishes in the incubators for the 5 day culture than in a conventional 3 day culture. Additionally, the performance of 1849
T.Utsunomiya, T.Naitou and M.Nagaki quality control on the culture media every day for 5 days may be quite difficult and complex. Furthermore, the environment in the dishes may be easily contaminated since it provides a nutritious fluid for bacteria and fungi such as Aspergillus. Even in the conventional day 3 embryo transfer procedure, microbial contamination was reported in IVF and embryo transfer (Cottell et al., 1996). To obtain a good pregnancy rate with ART, an accurate evaluation of embryos is useful. Many reports evaluate embryo quality using simpler methods than the day 5 culture of embryos, including the morphology of the first polar body (PB), pronuclear (PN) morphology, the duration of the 2-cell stage and 72 h blastomere cell number, and these studies have reported good pregnancy rates obtained using these parameters. The morphology of the first PB at the time of ICSI seems to be a suitable indicator for the development potential of zygotes (Ebner et al., 1999). Hence, the elective transfer of embryos selected on the basis of first PB morphology resulted in a higher pregnancy rate of 35.7% compared with that of the control group at 23.3%. PN morphology can be used as a selection criterion for embryo transfer with a high implantation rate occurring when two or more embryos are available with juxtaposed pronuclei with nucleoli aligned at the PN interface and with a halo effect to the cytoplasm (Scott and Smith, 1998). Additionally, they reported that if there were 50% of the pre-embryos with these characteristics, the pregnancy rate would be very low. The morphological parameters used to evaluate the number of nucleolar precursor bodies and their distribution in each pronucleus can be used to predict the developmental fate of embryos (Tesarik and Greco, 1999; Scott et al., 2000). The new evaluation criteria in these reports can be used as early as the PN stage, simply and without requiring repeated observation. In Germany, the German embryo protection law does not allow embryo selection, only the selection of PN stage oocytes. Therefore, the clinical usefulness of PN stage scoring was reported (Ludwig et al., 2000). This method might help to offer patients in Germany the transfer of two selected PN oocytes, which would reduce multiple gestation rates. At the early blastomere stage, the cleavage speed may have a predictive value as to the embryo s viability (Sakkas et al., 1998). The early cleavage of embryos up to the 2-cell stage after ICSI might be an indicator of embryo viability. Also, it has been demonstrated that the more developed 72 h embryos are more likely to become blastocysts and expand (Shapiro et al., 2000). Implantation rates are greater for the transfer of more developed embryos. Contrary to these reports on the blastomere stage, others (Rijnders and Jansen, 1998; Graham et al., 2000) demonstrated that the predictive value of embryo morphology on day 3 for subsequent blastocyst formation was limited. Originally, the idea of blastocyst stage embryo transfer was initiated to avoid multiple gestations in patients who usually obtained a number of embryos, due to the fact that they were good responders (Gardner et al., 1998, 2000). On the other hand, the most important and difficult issue in this field is the treatment of difficult cases who cannot conceive using any approach. The fundamental situation may be different between these difficult cases and blastocyst embryo transfer cases. So, the majority of difficult cases may not be rescued by blastocyst embryo transfer. Therefore, we should choose culture methods with the type of medium that is adequate for patients with high failure rates. We conclude that the pregnancy rates of day 5 and day 3 embryo transfer were equal, while the cancellation rate and the abortion rate of day 5 embryo transfer was a little higher than that of day 3 embryo transfer, and that blastocyst stage embryo transfer is not a viable technique to be used routinely in all patients undergoing ART. The indications for blastocyst stage embryo transfer should be studied further. References Alves da Motta, E.L., Alegretti, J.R., Baracat, E.C., Olive, D. and Serafini, P.C. (1998) High implantation and pregnancy rates with transfer of human blastocysts developed in preimplantation stage one and blastocyst media. Fertil. Steril., 70, 659 663. Braude, P., Bolton, V. and Moor, S. (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature, 332, 459 461. Coskun, S., Hollanders, J., Al-Hassan, S., Al-Sufyan, H., Al-Mayman, H. and Jaloudi, K. 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(1999) In vitro culture of human blastocyst. In Jansen, R. and Mortimer, D. (eds) Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. Parthenon Press, Carnforth, UK, pp. 378 388. Gardner, D.K., Lane, M., Spitzer, A. and Batt, P. (1994) Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol. Reprod., 50, 390 400. Gardner, D.K., Schoolcraft, W.B., Wagley, L., Schlenker, T., Stevens, J. and Hesla, J. (1998) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13, 3434 3440. Gardner, D.K., Lane, M., Stevens, J., Schlenker, T. and Schoolcraft, W.B. (2000) Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril., 73, 1155 1158. 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Controlled trial of blastocyst embryo transfer Ludwig, M., Schopper, B., Al-Hasani, S. and Diedrich, K. (2000) Clinical use of a pronuclear stage score following intracytoplasmic sperm injection: impact on pregnancy rates under the conditions of the German embryo protection law. Hum. Reprod., 15, 325 329. Ménézo, Y.J., Hamamah, S., Hazout, A. and Dale, B. (1998) Time to switch from co-culture. Hum. Reprod., 13, 2043 2044. Menkveld, R., Oettle, E.E., Kruger, T.F. Swanson, R.J., Acosta, A.A. and Oehninger, S. (1991) Atlas of Human Sperm Morphology. Williams and Wilkins, Maryland, USA, pp. 15. Milki, A.A., Hinkley, M.D., Fisch, J.D., Dasig, D. and Behr, B. (2000) Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil. Steril., 73, 126 129. Mills, C.L. (1992) Factors affecting embryological parameters and embryo selection for IVF ET. In Brinsden, P.R. and Rainsbury, P.A. (eds) A Textbook of In vitro Fertilization and Assisted Reproduction. Parthenon Press, Carnforth, UK, pp. 187 204. Rijnders, P.M. and Jansen, C.A.M. (1998) The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 35 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum. Reprod., 13, 2869 2873. Sakkas, D., Shoukir, Y., Chardonnens, D., Bianchi, P.G. and Campana, A. (1998) Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum. Reprod., 13, 182 187. Scholtes, M.C.W. and Zeilmaker, G.H. (1996) A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil. Steril., 65, 1245 1248. Scholtes, M.C.W. and Zeilmaker, G.H. (1998) Blastocyst transfer in day 5 embryo transfer depends primarily on the number of oocytes retrieved and not the age. Fertil. Steril., 69, 78 83. Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 1003 1013. Scott, L.A., Alvero, R., Leondires, M. and Miller, B. (2000) The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum. Reprod., 15, 2394 2403. Shapiro, B.S., Harris, D.C. and Richter, K.S. (2000) Predictive value of 72- hour blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocyst development. Fertil. Steril., 73, 582 586. Shoukir, Y., Chardonnens, D., Campana, A., Bischof, P. and Sakkas, D. (1998) The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum. Reprod., 13, 671 681. Staessen, C., Janssenswillen, C., Clerck, E.D. and Steirteghem, A.V. (1998) Controlled comparison of commercial media for human in-vitro fertilization: Ménézo B medium versus Medi-Cult universal and BMI medium. Hum. Reprod., 13, 2548 2554. Tesarik, J. and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 1318 1323. Tsirigotis, M. (1998) Too soon to abandon current practice? Hum. Reprod., 13, 3285 3289. Submitted on January 16, 2001; resubmitted on September 17, 2001; accepted on March 20, 2002 1851