Center for Reproductive Medicine, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium
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Abstract |
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Key words: embryo selection/single embryo transfer/twin pregnancy prevention
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Introduction |
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With regard to embryo quality, different groups have addressed the issue from different angles, but the endpoint is the same: the selection of embryos with the highest implantation potential. Some groups adhere to blastocyst culture and transfer (Gardner et al., 2000). Others prefer to critically evaluate pronuclear (Scott and Smith, 1998
; Ludwig et al., 2000
) and cleavage stage embryos on day 2 or 3 (Jackson et al., 1998
; Pelinck et al., 1998
) after fertilization.
We have previously shown (Van Royen et al., 1999) that embryos with a high implantation potential can be detected if the following criteria are met: embryos with four or five blastomeres on day 2 and seven or more cells on day 3, with no more than 20% fragmentation and with the absence of multinucleated blastomeres during the whole observation period.
These criteria were tested in a prospective randomized trial (Gerris et al., 1999) and led to an ongoing pregnancy rate of 38.5% after transfer of one such embryo; these embryos were therefore described as top quality embryos. The most salient conclusion of this study was that using the above described criteria, it was possible to introduce SET into daily clinical practice without a significant drop in the ongoing pregnancy rate. This is an important argument for the patient, especially if the couple considers a twin pregnancy to be a success in itself.
Subsequent to this study, we decided to introduce SET into clinical practice in a prospective cohort study. From our own data, we had calculated that after double embryo transfer (DET) 80% of twin pregnancies occurred in the two first IVF/ICSI cycles and that the implantation potential substantially decreased from 38 years of age onwards. Patients prone to multiple pregnancies have been previously described (Staessen et al., 1993) as women <37 years old in their first three IVF/ICSI cycles with a minimum of six embryos of good quality. Coetsier and Dhont defined good prognosis for pregnancy patients as women <36 years old with more than three embryos with good embryo score available for transfer in their first three cycles (Coetsier and Dhont, 1998
). Strandell et al. showed that the age of the patient and the number of embryos transferred are independent factors to predict multiple birth (Strandell et al., 2000
).
Patients were thoroughly counselled about embryo transfer in their first IVF/ICSI cycle with regard to pregnancy rate and chance of a multiple pregnancy.
The data about the patients choice, patients characteristics, embryo quality, pregnancy chances and multiple pregnancy rates are described.
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Materials and methods |
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Ovarian stimulation protocol
Patients were treated with the long GnRH agonist desensitization protocol, starting in the mid-luteal phase with 6x100 µg buserelin (Suprefact; Hoechst, Germany) intranasally for 3 weeks. Gonadotrophin stimulation (Metrodin HP or Gonal-F; Serono, Geneva, Switzerland) was initiated if basal vaginal sonography showed a thin endometrium and no ovarian cysts. Stimulation was initiated with 150 IU of Metrodin HP or Gonal-F s.c. except in patients with known poor response, where 225 IU was used. The criterion for hCG administration was at least three mature follicles with a diameter of 18 mm. A total of 10 000 IU hCG (Profasi; Serono) was given i.m. exactly 37 h before oocyte retrieval.
IVF/ICSI procedure
Motile sperm were isolated from fresh semen in a two-step protocol: gradient centrifugation followed by migrationsedimentation. The micro-epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) procedures are described elsewhere (Silber, 1997; Tournaye, 1997). In all cases of MESA/TESE, frozenthawed aspirates or biopsies were used.
Oocyte retrieval was performed vaginally under ultrasound guidance. Cumulusoocyte complexes were isolated from the follicular aspirates and washed in Medi-Cult medium. Each was placed individually in a 25 µl microdrop of Ménézo B2 medium (C.C.D., Paris, France) under mineral oil (Sigma, St Louis, MA, USA) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. For standard IVF, 35 h after retrieval every oocyte was inseminated with 20 000 motile sperm and incubated overnight. The ICSI standard procedure was performed.
Embryo quality assessment
Approximately 1619 h after insemination/injection, normal fertilization was checked. All oocytes containing two clearly visible pronuclei were placed together in one fresh 10 µl microdrop of Ménézo B2 medium (maximum 10 oocytes/drop) and cultured for another 24 h. The next day (4043 h after insemination/injection) the embryos were separated and each transferred to a 10 µl drop of Medi-Cult M3 medium for a further culture of 24 h. Every embryo was scored for the total number of cells, and the presence of anuclear fragments and multinucleated blastomeres. From the moment day 2 embryo criteria were recorded, embryos were cultured separately.
On day 3 (6467 h after insemination/injection) embryo quality was evaluated again. Selection for embryo replacement was made according to the top quality embryo selection criteria, defined as follows: four or five blastomeres on day 2; seven or more blastomeres on day 3; and <20% fragmentation and total absence of multinucleated blastomeres at any stage of early cleavage. A final selection of embryos for transfer was based on implantation fractions (Van Royen et al., 2001). Embryos with >50% fragmentation were considered unsuitable for transfer. Supernumerary embryos were frozen. The criteria for cryopreservation of embryos were those which at the time of the transfer had six or more cells and <20% fragmentation with an absence of multinucleated blastomeres. Embryos with four cells on day 2 and seven or more cells on day 3 with up to 30% fragmentation were also cryopreserved.
Counselling about embryo transfer
In this study, patients <38 years old in their first IVF/ICSI cycle or after a previous delivery were thoroughly counselled about the embryo transfer as follows. We informed them of the chances of obtaining a pregnancy after transfer of a single embryo of top quality and how this would not decrease the chance of obtaining a pregnancy as compared with the results for the whole IVF/ICSI patient group. Because of our concern to keep the pregnancy rates stable at the time, we advised couples who chose SET of a top quality embryo but who did not produce such an embryo to have two embryos transferred. The final choice of whether to have one or two embryos transferred was left to the couple.
Embryo transfer technique
All transfers were performed on an out-patient basis using a Wallace embryo transfer catheter (Sims Portex Ltd, Hythe, Kent, UK) consisting of an inner and an outer catheter. The outer catheter was introduced first using a guidewire. Care was taken to limit the introduction of the outer catheter to a maximum of 4 cm into the cervix, in order to minimize potential microtrauma of the uterine cavity. The loaded inner catheter was then passed through the outer catheter until a total distance of 6 cm between the external os and the tip of the inner catheter was reached. The embryo(s) were gently expelled into the cavity in a volume of <30 µl Medi-Cult M3 medium. After removal, the catheter was checked under the microscope to ascertain that the embryo(s) were deposited in the uterine cavity. Strict care was taken that all clinicians followed the instructions for embryo transfer in a similar way.
Luteal phase
In all cycles, luteal phase was supported with 3x200 mg of micronized natural progesterone (Utrogestan; Laboratoires Piette International, Belgium) administered vaginally.
An ongoing pregnancy was defined as a conception cycle with at least one fetal sac with a positive heartbeat reaching beyond 12 weeks amenorrhoea. Consequently, for the calculation of the ongoing implantation rate, biochemical conceptions, clinical miscarriages or extrauterine pregnancies were excluded from the calculation.
For statistical analysis, confidence interval analysis and 2-test were used.
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Results |
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A total of 120 patients who chose to have SET received SET with 105 top quality SETs and 15 non-top quality SETs. Of these 15, 13 were compulsory SETs: there was only one embryo suitable for transfer. The other two patients decided to have SET regardless of the embryo quality. Thirty-six patients received two non-top quality embryos. The clinical outcome for the patients whose choice was SET is listed in Table I. There was an ongoing pregnancy rate of 43% after transfer of one top quality embryo and 40% after transfer of one non-top quality embryo. In the patient group receiving two non-top quality embryos, an ongoing pregnancy rate of 33% was obtained with one twin pregnancy (8%).
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In the group of patients choosing SET, 248 embryos in 156 cycles were cryopreserved and in the group choosing DET, 44 embryos in 87 cycles were cryopreserved. This means 1.59 embryos per cycle for SET versus 0.51 embryos per cycle in the DET group (P < 0.0001). The impact on the cumulative pregnancy rate per cycle could not yet be calculated at this point in the study.
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Discussion |
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Vilska et al. introduced the SET strategy in the Finnish population and compared elective SET (medical reasons, patients choice, risk of ovarian hyperstimulation syndrome) with SET because there was only one available embryo (compulsory SET) and reported pregnancy rates of 29.7% per cycle in the first group and 20.2% in the latter group (Vilska et al. 1999). During the same time period, DETs led to a pregnancy rate of 29.4%, but with a twin pregnancy rate of 23.9%. If the results of frozenthawed embryos of the elective SETs were taken into account, a cumulative pregnancy rate of 47.3% was obtained per oocyte retrieval. On the basis of these data, SET was highly recommended, especially in women <35 years old and particularly if high-grade embryos were available for transfer.
Martikainen et al. reported a multicentre randomized study that compared SET versus DET in a first and second IVF/ICSI cycle in a patient group that had at least four good quality embryos available for transfer. The pregnancy rate was 32% in the SET group and 47% in the DET group (not significantly different, P = 0.01), but with 5 versus 39% twin pregnancies (significantly different) (Martikainen et al., 2001).
The present study showed an ongoing pregnancy rate of 42% in the first IVF/ICSI cycle with a twin pregnancy rate of 11% regardless of the choice of the patient. On the other hand, if patients chose SET of a top quality embryo, or two embryos if there was no top quality embryo available, then the pregnancy rate was 43% with only 2% twin pregnancies.
If two non-top embryos were transferredwhether it be in the SET group with no top quality embryo available or in the DET group with no top quality embryos availablethe twin pregnancy rate was 10%.
The conclusion of this study is that SET can easily be introduced into a twin-prone situation, i.e. patients <38 years old in their first IVF/ICSI cycle, yielding a high pregnancy rate and a low risk of twin pregnancy. Nowadays, many IVF centres as well as patients are convinced that twin pregnancies should and can be avoided in the first IVF/ICSI cycle. Nearly as important as avoiding twin pregnancies is the pregnancy rate. It is only because a high pregnancy rate can be achieved with SET that it became acceptable to clinicians who could then convince their patients.
Embryo selection plays a pivotal role in the success of SET. Embryos labelled as top quality show an ongoing implantation rate of 45.3 versus 18.8% for non-top quality embryos. The non-top quality embryos are also considered as having the second best implantation potential. These data correspond to the implantation potential of top quality embryos as calculated by Van Royen et al. (Van Royen et al., 2001). Presently, no data on the implantation potential of single blastocyst transfers are available. Studies of double blastocyst transfer show a pregnancy rate of 39% (Coskun et al., 2000
) with 38% of twin pregnancies. Gardner et al. have described pregnancy rates of up to 87% with a twinning rate of 61% after transfer of two very high quality blastocysts and concluded that the way is set for SET (Gardner et al., 2000
).
A reduction in the number of embryos transferred leaves a higher number of embryos for cryopreservation. If we take into account the pregnancies that will occur from the embryos that were cryopreserved in the SET cycle and add these to the pregnancy rate of SET (fresh cycle), then a cumulative conception rate may be calculated that probably will further increase the pregnancy rate of SET. This has been shown by Tiitinen et al. who obtained a cumulative delivery rate for elective SET and frozenthawed embryo transfer (one or two embryos transferred) of 52.8% with a 7.6% twin rate (Tiitinen et al., 2001).
The challenge now lies in the implementation of SET into the general practice of IVF/ICSI and into the rest of the IVF/ICSI programme. As twin- and multiple-prone patients have been identified and described by ourselves and others (Staessen et al., 1993; Coetsier and Dhont, 1998
), extension of SET to the second, third and maybe fourth cycle is under evaluation. Any further progress in embryo selection will only make it more easy to do so.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 22, 2002; accepted on July 11, 2002.