Infertility Centre, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
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Abstract |
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Key words: culture/embryo quality/embryo transfer/implantation/pregnancy
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Introduction |
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Several studies comparing embryo transfer on day 2 versus day 3 after oocyte retrieval have been performed but the conclusions are conflicting. One study (Van Os et al., 1989) randomized day 2 and day 3 embryo transfers based on the day of the week on which oocyte retrieval took place and found no difference in pregnancy rates. Another study (Dawson et al., 1995
) concluded from a retrospective study that pregnancy rates were similar between day 2 and day 3 transfers but that the implantation rate in the day 3 group was higher. In a retrospective study (Carillo et al., 1998
) the pregnancy and implantation rates were found to be increased after transfer on day 3. From a prospective randomized study (Ertzeid et al., 1999
), it was concluded that extending the embryo culture period from 2 to 3 days had no effect on implantation and live birth rates. As the topic of preferential transfer on day 2 or 3 remains controversial, it was thought that it would be useful to perform a prospective, randomized study to compare implantation and pregnancy rates between day 2 and day 3 transfers.
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Materials and methods |
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Randomization and minimization for age and the procedure of fertilization (IVF or ICSI) were performed by a computer program. ICSI was performed in ~84% of the cases where IVF was considered as unsuitable (previously failed IVF) or in cases of oligoasthenoteratozoospermia. The setting is a university-based referral centre for male infertility.
Embryo culture and protein supplement
Earle's balanced salt solution was prepared weekly using reagents from Sigma (Bornem, Belgium): Earle's balanced salt solution (E6132), sodium pyruvate (P4562; 1 mmol/l), sodium bicarbonate (S5761; 25 mmol/l), penicillin (P4687; 0.021 mmol/l) and water (W1503). The osmolality of the medium was adjusted to 285 mOsm/kg H2O.
For embryo culture this medium was supplemented with human serum albumin (HSA; Belgian Red Cross) at a concentration of 0.4% (w/v). The serum was guaranteed to be prepared from blood that was non-reactive for HBsAg (hepatitis B surface antigen) or human immunodeficiency virus (HIV).
IVF/embryo transfer procedure
Freshly ejaculated semen was washed in Earle's medium supplemented with HSA 0.4% (w/v) by centrifugation at 1600 rpm for 5 min after a 30 min liquefaction period. The pellet was further processed by the side migration technique for ICSI as described previously (Dozortsev et al., 1996). For IVF, the sperm sample was put on a two-layer Percoll gradient (45/90%) and the pellet of the lower fraction was used for insemination of oocytes. For routine IVF, oocytes were cultured in medium droplets under oil (M8410; Sigma) together with ~100 000 sperm cells/ml at 37°C in a 5% CO2 atmosphere. For ICSI, the cells of the cumulus and corona radiata were removed by incubation of the cumulusoocyte complexes for <1 min in HEPES-buffered Earle's medium containing 80 IU/ml hyaluronidase (H3757; Sigma) followed by gentle aspiration of the cumulusoocyte complexes in and out of a hand-drawn glass pipette with an inner diameter of 150 µm. Denuded oocytes were rinsed several times in culture medium. Until the moment of injection, the oocytes were kept in 25 µl drops of Earle's medium supplemented with HSA in a Petri dish under mineral oil (Sigma, M8410) and stored in an incubator containing 5% CO2 in air at 37°C. Microinjection was carried out on the heated stage of an inverted microscope (Axiovert 135; Zeiss, Zaventem, Belgium). The details of the microinjection procedure have been described previously (Dozortsev et al., 1996
). The presence of two pronuclei and two polar bodies was assessed 1618 h after IVF or ICSI. Patients with a yield of at least seven normally fertilized oocytes were randomized for transfer on either day 2 or day 3 after oocyte retrieval. Stratification was based on type of infertility treatment (IVF or ICSI) and on age of the patient.
In the day 2 transfer group, embryonic development was assessed under the inverted microscope 4244 h after IVF or ICSI. In the day 3 transfer group, embryos were first evaluated 4244 h after IVF or ICSI and then for a second time 24 h after the first evaluation. Embryos were classified based on morphological criteria as described previously (Laverge et al., 1997). Briefly, embryos without anucleated fragments and with equally-sized blastomeres were graded as type I. Embryos with some anucleated fragments (<10%) and/or with unequally-sized blastomeres were graded as type III. Embryos with unequally-sized blastomeres with either
20%, up to 50% or >50% anucleated fragments were classified as type II, IIIII, and III respectively. Embryos of grade I and III were classified as excellent quality embryos, embryos of grade II as good quality embryos and embryos of grade IIIII and III as moderate to poor quality embryos. Embryos with at least one blastomere with more than one nucleus were classified as multinucleated embryos and were considered as not suitable for transfer or cryopreservation. As a rule, two embryos were transferred in all patients aged <38 years if two embryos of excellent or good quality were available. Transfer of a maximum of three embryos was allowed in patients with two previous unsuccessful IVF cycles, patients aged >38 years or when no embryos of good quality were available. Supernumerary embryos up to type II were cryopreserved with a dimethyl sulphoxide (DMSO) slow-freezing protocol.
Pregnancy was defined as positive if the ßHCG measured in venous blood was >20 mIU/ml. Clinical pregnancy was defined as a positive pregnancy test followed by the presence of a fetal sac on transvaginal ultrasound 4 weeks after transfer. Biochemical pregnancy was defined as a positive pregnancy test not followed by the presence of a fetal sac.
Statistical analysis
Statistical analysis was carried out using a 2-test or an unpaired t-test where appropriate. P < 0.05 was considered to be statistically significant. The anticipated difference in pregnancy rate per cycle between day 2 and day 3 transfers was 10%. It was calculated that at least 370 cycles were needed in each group to prove a difference of 10% with a power of 80%.
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Results |
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Embryo quality
A summary of the results on embryo quality is shown in Table I. Overall significantly less embryos were of excellent or good quality after culture for 3 rather than 2 days (10.3 and 40.1% versus 12.9 and 44.2% respectively). The shift to poorer embryo quality on day 3 was found both after ICSI (Table II
) and IVF (Table III
).
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Outcome of pregnancies
The incidence of biochemical and ectopic pregnancies and of miscarriages did not differ significantly for pregnancies established following day 2 or day 3 embryo transfer (Table IV).
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Discussion |
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A number of studies addressing the same issue have already been published and have come to the same or opposite conclusions. In a prospective, randomized study for patients undergoing IVF (Van Os et al., 1989), no significant difference in pregnancy rates between day 2 (n = 434 cycles) and day 3 transfer (n = 324 cycles) was found. These authors also reported that the mean embryo quality (morphological criteria) was slightly better in the day 2 group, although no figures about embryo quality were mentioned in the study. The randomization procedure was based on the day of the week oocyte retrieval took place. Two further retrospective studies arrived at opposite conclusions but may have suffered from selectively allowing better prognosis patients to proceed to day 3, whilst worse prognosis patients had their transfer on day 2. In a retrospective comparison of 661 embryo transfers on day 3 with 567 embryo transfers on day 2 (Dawson et al., 1995
), the patients were compared for age, response to ovarian stimulation, number of oocytes retrieved and embryos transferred. Although the mean number of embryos transferred was significantly higher in patients having embryo transfer on day 2, the pregnancy rate was similar in both groups. The implantation rate was significantly higher following transfer on day 3 than after transfer on day 2. In a retrospective analysis (Carillo et al., 1998
), patients with a relatively large number of retrieved oocytes were selected for embryo transfer on day 3 (n = 176). These patients were then matched on the basis of age, diagnosis and number of oocytes collected with day 2 patients treated in the same year. Pregnancy and embryo implantation rates were significantly higher in patients with day 3 transfers compared with day 2 (51 and 24% versus 29 and 13% respectively). It should be mentioned that glucose- and phosphate-free media were used, which is different from the culture conditions in all other studies. In an earlier study (Edwards et al., 1984
), the incidence of miscarriages after day 3 embryo transfer was reported to be higher but this finding was not confirmed either by our own results or by other authors (Van Os et al., 1989
).
It can be argued that a delay of 1 day is too short to better differentiate the quality of embryos. In recent years, therefore, a more extended delay of embryo transfer, up to the blastocyst stage, has been tried by several investigators. The transfer of cavitating morula stage embryos was found to give an implantation rate of 41% per embryo (Huisman et al., 1994). The disadvantage of culture until day 4 is that only a small fraction of the embryos shows a cavitating morula (18.4% in the study of Huisman et al., 1994). In an early study (Scholtes and Zeilmaker, 1996
), the implantation rate of embryos after 3 and 5 days of IVF culture were compared. They observed that the overall embryo transfer results were comparable. In a more recent study (Scholtes and Zeilmaker, 1998
) these authors analysed the effects of patient age and treatment cycle number on the occurrence of blastocyst transfer and subsequent implantation. They reported a decreasing implantation rate after cycle 2 and observed that biological ovarian age is a determining factor on the frequency of blastocyst transfer or pregnancy rate.
In the above-mentioned studies, a single culture medium formulation was used to support embryo development from the 1-cell stage to the blastocyst stage. More recently, sequential media have been introduced for culture of later stages of embryo development. Gardner et al. developed sequential serum-free media and showed that >50% of embryos became blastocysts. Transfer of these blastocysts resulted in an implantation rate of ~50% (Gardner and Lane, 1998). An improved IVF culture system where the use of sequential `stage-specific' culture media allegedly increased the implantation rate from 11.1% without this system to 30.6% has been described (Mortimer et al., 1998
). The clinical pregnancy rate increased from 19.6 to 45.9%.
The pregnancy and implantation rates for transfer of blastocysts versus transfer of day 2 or day 3 embryos is in the similar range. A pregnancy rate of 38% and an implantation rate of 23% has been reported (Jones et al., 1998). In the present study, the pregnancy rates were 47.9% in the day 2 group and 46.8% in the day 3 group and the implantation rate was 23.8% in both groups. The patient selection in both studies was similar. In the study by Jones et al. only patients aged <40 years with more than five fertilized oocytes were included while in the current study only patients with at least seven fertilized oocytes were included and randomized for age.
In conclusion, this prospective, randomized study demonstrates that (at least in cases where a sufficient number of fertilized oocytes are available), it does not make a difference whether transfer is performed on day 2 or day 3, since similar pregnancy and implantation rates can be achieved. Moreover, these findings indicate that embryo transfers can be safely scheduled at the convenience of the patient and the centre.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 12, 2000; accepted on November 13, 2000.