1 Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy and 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain
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Abstract |
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Key words: blastocyst transfer/embryo quality evaluation/embryo transfer policy/pronuclear scoring
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Introduction |
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However, other studies have reported comparable pregnancy (Scholtes and Zeilmaker, 1996; Coskun et al., 2000
; Huisman et al., 2000
; Hsieh et al., 2000
) and implantation (Coskun et al., 2000
; Huisman et al., 2000
) rates after day 3 and day 5 embryo transfers. It is also known that, with the current techniques, most embryos do not become blastocysts during extended culture, and it is not clear how many of these embryos would have implanted if replaced at the cleavage stage (Alper et al., 2001
). In view of these conflicting data, the question of the overall benefit of blastocyst culture and transfer needs to be revisited.
The use of different criteria for the selection of embryos for transfer may be at least partly responsible for the discrepancies between different studies comparing day 3 and day 5 embryo transfer outcomes. In the past few years the possibilities of viable embryo selection at the early cleavage stages have been improved substantially by introducing non-invasive scoring criteria applicable as early as the pronuclear stage (Scott and Smith, 1998; Tesarik and Greco, 1999
) and by refining the scoring criteria for cleaving embryos (Gerris et al., 1999
; Van Royen et al., 1999
, 2001
). This prospective randomized study reports pregnancy and implantation rates achieved with the use of combined pronuclear and cleavage-stage evaluation criteria and day 3 embryo transfer as compared with those achieved with day 5 blastocyst transfer. These comparisons are made for fresh embryo transfers only, for cryopreserved embryo transfers only and for cumulative outcomes from fresh and cryopreserved embryo transfers.
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Materials and methods |
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Assisted reproduction techniques
Controlled ovarian hyperstimulation was performed with the use of recombinant human FSH (Puregon; Organon, Oss, The Netherlands) after pituitary suppression with buserelin acetate (Suprefact; Suprefact Hoechst; Marion Roussel, Milan, Italy) started in the late luteal phase of the previous cycle as described (Ubaldi et al., 1999). Ovulation was induced by 10 000 IU HCG (Profasi; Serono, Rome, Italy) when at least three follicles had reached a diameter of >18 mm, and transvaginal follicle aspiration was performed, under ultrasound guidance, 36 h later (Ubaldi et al., 1999
).
Oocytes were freed from the cumulus oophorus by a brief incubation (1530 s) at 37°C in 40 IU/l hyaluronidase solution (Hyase; Vitrolife, Gothenburg, Sweden), followed by mechanical removal of the corona radiata with the use of finely drawn denuding pipettes (SAGE BioPharma, Bedminster, NJ, USA) and subjected to ICSI using previously described techniques and instruments (Rienzi et al., 1998). Fertilization was assessed by three sequential inspections of the sperm-injected oocytes performed between 12 and 16 h after ICSI. Only those oocytes that showed two pronuclei and two polar bodies during at least one of these inspections were considered further for eventual embryo transfer. Abnormally fertilized oocytes (1 or 3 pronuclei) were excluded from further consideration.
Normally fertilized oocytes (zygotes) were cultured in G1.2 medium up to day 3 after ICSI and in G.2.2 medium (both media purchased from Vitrolife) from day 3 to day 5 where applicable. The cultures were carried out at 37°C, and the media were equilibrated with 5% CO2 in air. Two best-scoring embryos, selected with the criteria described below, were transferred to the patient's uterus on either day 3 or day 5 according to the study design. When the pronuclear and cleavage-stage scores were not in agreement for day 3 transfers, the pronuclear score was given priority. For day 5 transfers, blastocyst morphology was given priority to pronuclear score in case of discrepancy. The remaining good quality embryos were cryopreserved if they did not show a developmental blockage (no developmental change throughout the last 24 h of culture). Day 3 and day 5 embryo cryopreservation was performed with freeze-kit 1 and freeze-kit 2 (both purchased from Vitrolife) respectively, according to the manufacturer's instructions.
Zygote and embryo quality evaluation
Zygote quality was evaluated during three observations performed between 12 and 16 h after ICSI with the use of previously described criteria (Tesarik and Greco, 1999). Zygotes were considered morphologically normal when at least 3 nucleolar precursor bodies (NPB) were present in each pronuclei; when the difference in number of NPB between the two pronuclei did not exceed 3; and when the NPB were similarly distributed (random or polarized) in both pronuclei. Zygotes showing abnormal pronuclear patterns were assigned to a single group as reported (Tesarik et al., 2000
).
Cleaving embryos were evaluated on days 2 and 3 after ICSI with the use of a cumulative embryo classification scheme taking into account cleavage speed, blastomere symmetry, extent of fragmentation, and the presence or absence of multinucleated blastomeres (Table I). Those embryos that received the lowest number of points in each cohort were considered to have the highest implantation potential.
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Statistical analysis
Significance of differences in the success rates between the day 3 and day 5 transfer protocols was evaluated by 2-test using StatView II statistical package (Abacus Concepts, Berkeley, California, USA).
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Results |
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The basic characteristics of the patient were similar between the two groups (day 3 and day 5 embryo transfer): mean age ± SD (31.6 ± 3.1 versus 32.2 ± 2.5; not significant), mean ± SD oocytes retrieved (12.7 ± 7.1 versus 13.1 ± 5.2; not significant), fertilization rate (71.2 versus 71.8%; not significant) and cleavage rate (92.8 versus 91.3%; not significant).
Two cleaving embryos and two blastocysts were transferred to each patient of the day 3 and day 5 group respectively. The blastocyst formation rate per fertilized oocyte obtained in the day 5 embryo transfer group was 44.8% (211/470). Cryopreservation was performed for those patients for whom supernumerary good quality embryos or blastocysts were available. This occurred more frequently in the day 3 group (42/48 patients) as compared with the day 5 group (18/50 patients; P < 0.01).
When the two presumably best embryos were being chosen from each patient's embryo cohort for day 3 transfer, the pronuclear score agreed with the day 3 score in 216 out of 258 embryos evaluated (84%) and disagreed in the remaining 42 (16%) embryos.
The comparison of success rates between day 3 and day 5 transfer did not show any difference for either fresh (Table II) or cryopreserved (Table III
) embryos. When cumulative success rates from fresh and cryopreserved embryo transfers were calculated per embryo transfer, there was no difference between the day 3 and day 5 transfers as to the pregnancy rate, clinical pregnancy rate and delivery rate, but the implantation and birth rates were slightly higher (P < 0.05) for the day 5 transfer (Table IV
). On the other hand, cumulative pregnancy, clinical pregnancy and delivery rates were substantially higher for the day 3 transfer as compared with the day 5 transfer when they were calculated per oocyte recovery attempt (P < 0.01) (Table V
).
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Discussion |
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It is also known that the implantation rate after blastocyst transfer is influenced by blastocyst quality (Balaban et al., 2000; Gardner et al., 2000b
). On the other hand, it appears to be useless to wait up to day 6 if blastocyst quality is not ideal on day 5 (Shapiro et al., 2001
). In this study two blastocysts showing the best morphology from the available embryo cohort were always transferred on day 5. No attempt was made to improve blastocyst quality by extending the culture period up to day 6.
The success rates for cryopreserved embryo transfer were also similar for day 3 and day 5 embryos in this study. However, this comparison was flawed by asymmetry of the two groups with only a few cases in which cryopreserved blastocysts were available for transfer. This asymmetry was also responsible for the slightly higher cumulative implantation and birth rates, calculated per embryo transfer, for day 5 embryos as compared with day 3 embryos. These data do not demonstrate any real advantage for day 5 embryos; they simply reflect a `dilution' of higher-chance fresh embryo transfers, highly prevailing in the day 5 group, by the much higher contribution of the lower-chance cryopreserved embryo transfers in the day 3 group.
Yet it was just because of the contribution of the cryopreserved embryo transfers that the cumulative success rates per oocyte recovery attempt turned clearly in favour of the day 3 transfer policy. With equivalent success rates for both the fresh and cryopreserved embryo transfers, the higher number of cryopreserved embryo transfers in the day 3 group led to a significant improvement of per-oocyte-recovery pregnancy, clinical pregnancy and delivery rates as compared with the day 5 group. Because the clinical and laboratory procedures related to embryo formation are more expensive and represent a higher degree of clinical risk and physical discomfort to the patient than the procedures related to embryo storage and transfer, the day 3 transfer policy, in our hands, was clinically more efficient and more cost-effective as compared with day 5 transfers in the selected population of patients analysed.
High implantation rates for day 3 embryo transfer, similar to those observed in this study, have also been reported by other authors using similar embryo selection criteria (Gerris and Van Royen, 2000; Scott et al., 2000
; Gerris et al., 2001
). In the current trend towards single embryo transfer, the extended culture and blastocyst transfer are thus not necessary prerequisites any more.
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Notes |
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References |
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Submitted on January 9, 2002; accepted on March 3, 2002.