Hum. Reprod. Advance Access originally published online on June 24, 2005
Human Reproduction 2005 20(10):2935-2938; doi:10.1093/humrep/dei133
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Embryology |
Single embryo transfer is an option in frozen embryo transfer
1 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, PO Box 140, 00029 HUS, Finland
2 To whom correspondence should be addressed. E-mail: christel.hyden-granskog{at}hus.fi
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Abstract |
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BACKGROUND: A good strategy to decrease multiple pregnancy rate in assisted reproduction technology (ART) is the use of single embryo transfer (SET). METHODS: This retrospective study analysed 1647 frozen embryo transfers carried out during 1998–2003 in Helsinki University Central Hospital; of these, 872 were double embryo transfers (DETs) and 775 SETs. The SET group included 140 (18.1%) elective SETs (eSETs). RESULTS: The yearly rate of SETs in frozen cycles increased from 28 to 66%. Overall, the clinical pregnancy rate per frozen embryo transfer was 30.7% and the delivery rate 22.6%. The delivery rate was significantly higher in DET cryocycles than in SET cryocycles (25.7 versus 19.2%, respectively; P < 0.01). In DET cryocycles, the multiple delivery rate was 21.9%, 10 times higher than that observed in cryocycles with SET (2.0%) (P < 0.0001). When eSET was applied, no difference in delivery rate was observed when compared with cryocycles with DET (28.6 and 25.7%, respectively). CONCLUSIONS: SET can be used in frozen cycles to reduce multiple delivery rates.
Key words: cryopreservation of embryos (human)/frozen embryo transfer/multiple pregnancy rate/re-cryopreservation/single embryo transfer
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Introduction |
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The high incidence of multiple pregnancies is the main adverse treatment outcome in assisted reproduction technology (ART) (Bergh et al., 1999
). A good strategy to avoid multiple pregnancies is elective single embryo transfer (eSET) with cryopreservation of spare embryos (Thurin et al., 2004; Tiitinen et al., 2004). The use of eSET in oocyte retrieval cycles has reduced the multiple pregnancy rate (PR) to <15% in various IVF clinics (De Neubourg and Gerris, 2003; De Sutter et al., 2003). Our eSET programme in oocyte retrieval cycles started in 1997 in selected cases, and thereafter eSET became the primary option in 2000. This has reduced the multiple delivery rate (DR) from 25 to 
5% without lowering the overall DR (Tiitinen et al., 2003).
In a randomized study, it was shown that in women under 36 years old, transferring one fresh embryo, followed (if there was no live birth) by the transfer of one frozen embryo, dramatically reduced the multiple DR while achieving a rate of live births that is not substantially lower than that which is achievable with one double embryo transfer (DET) (Thurin et al., 2004). In our programme, the contribution of embryo cryopreservation in eSET cycles has resulted in a cumulative delivery rate of >50% after one fresh and several frozen transfers (Tiitinen et al., 2001; Hydén-Granskog and Tiitinen, 2004). It is obvious that a significant impact of eSET is an increase in the number of embryos available for cryopreservation.
There are no randomized trials comparing SET and DET in frozen cycles. In the study by Thurin et al. (2004), the DR was 16.4% after SET in the frozen cycles. A follow-up study of eSET cycles in our programme showed a DR of 10.9% after SET in the frozen cycle and 32.5% after DET, with 14.8% of deliveries being twins (Tiitinen et al., 2001). However, the problem is that in most ART programmes the PR is lower in frozen cycles than in fresh cycles. In 2001, the DR per frozen embryo transfer varied between 3.6 and 26.5% in different European countries and the multiple DR was between 10.5 and 27.0% (EIM/ESHRE, 2005). SET in frozen cycles could further decrease the multiple PR in ART. Based on our good results with eSET in fresh transfers, we have also increased the rate of SETs in frozen cycles in order to reduce multiple pregnancies while keeping an acceptable pregnancy rate. We have analysed our frozen embryo transfer cycles in order to establish the effect of SET in our cryopreservation programme.
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Materials and methods |
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During 1998–2003, we performed 1940 thawing cycles in total; in 1647 (84.9%) of these, a transfer was performed.
The criteria for cryopreservation of embryos were: at least two blastomeres (on day 2), at least four blastomeres (on day 3), fragmentation <50% and no multinucleated blastomeres (MNBs) observed. Day 2 and day 3 embryos were cryopreserved with 1,2-propanediol (PROH, Vitrolife, Sweden or Cook, Australia). Blastocysts were cryopreserved with glycerol (Cook, Australia). Cryopreservation and thawing were performed according to the recommendations of the manufacturer, the only exception being that ampoules were used. A minority of the thawed embryos had been cryopreserved with dimethylsulphoxide (DMSO), since DMSO was used for cryopreservation during the period 1991–1997.
Our criteria for transfer of frozen–thawed embryos were that at least 50% of original blastomeres were intact (except 2-cell embryos) and fragmentation was <50%. Furthermore, we required that no MNBs were seen at any stage and that cleaving occurred if the embryo was cultured overnight. All embryos with less than four blastomeres were cultured overnight before transfer.
SET was performed in 775 cycles (47%) and DET in 872 cycles (53%). In 1998, most SETs were performed because only one embryo had survived. In 2003, most of the SETs were performed due to the couple’s own wish to avoid a twin pregnancy or based on our recommendation. The SETs consist of two groups; eSET and compulsory SET (cSET). eSET was performed when two or three embryos had been cryopreserved in the same ampoule and more than one embryo fulfilled the transfer criteria after thawing. In all cases, an embryo with a blastomere survival rate of at least 75% was selected for transfer. If possible, the remaining embryos were re-cryopreserved either as early cleavage stage embryos or after prolonged culture until the blastocyst stage. Criteria for re-cryopreservation were 100% blastomere survival after thawing, no MNBs observed and fragmentation <30%. cSET was performed if only one cryo-thawed embryo fulfilled criteria for transfer or there was only one embryo cryopreserved in the ampoule.
Embryo transfer was performed either in a spontaneous cycle (61%) or in a hormonally substituted cycle (39%). In spontaneous cycles, the size of the leading follicle and endometrial thickness were assessed with transvaginal ultrasound measurement on cycle days 10–12. The LH surge was determined at home with commercial urinary LH kits, and embryo transfer was usually performed 3 days after the beginning of the LH surge. All patients used micronized vaginal progesterone 200 mg/day for 2 weeks after the transfer. In hormonally substituted cycles, estrogen (4 mg/day) was started on cycle day 1 in the first cycle and on day 3 in consecutive cycles. Endometrial thickness was assessed with ultrasound after 10–12 days and vaginal progesterone 600 mg/day was started within 1 week depending on endometrial thickness (minimum thickness required 7 mm), and embryo transfer was mostly done on the third day of progesterone use. The serum hCG level was measured 12 days after the transfer. If the pregnancy test was positive, both medications were continued until pregnancy week 10 in substituted cycles.
Statistical methods
For comparisons, the 
2 test was used. A P-value of <0.05 was considered statistically significant.
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Results |
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Overall, the PR per embryo transfer (PR/ET) was 30.7% and the DR 22.6%. The yearly rate of multiple pregnancies was between 18.3 and 7.6% (Figure 1). The clinical PR did not differ between spontaneous and substituted cycles. In 1998, SET was done in 28% of frozen cycles and in 2003 in 66% (Figure 1).
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In all cycles with SET, the mean clinical PR was 26.7% (Table I) (range per year 19.2–33.0%, Figure 1). Monozygotic twins were seen in five of the pregnancies (2.4%). The mean DR was 19.2% and the multiple DR 2.0%. In eSET cycles, the clinical PR was 40.7% (57 out of 140) and the DR was 28.6% (40 out of 140) (Figure 2). The clinical PR was 43.6% (51 out of 117) when a fully intact day 2 or day 3 embryo was transferred and 26.1% (six out of 23) when a partially intact (blastomere survival rate at least 75%) was transferred. No monozygotic twins were seen in these eSET cycles. In cSET cycles, the PR was 23.6% (150 out of 635) and the DR was 17.2% (109 out of 635). Since all monozygotic twins were in this group, the multiple PR was 3.3% and the multiple DR was 2.8%.
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In DET cycles, the mean PR was 34.3% (range 20.7–45.0%, Figure 1) and the multiple PR was 20.1% (Table I). The DR was significantly higher in DET cycles than in SET cycles (25.7 versus 19.2%, P < 0.005). However, when compared with the DR of eSET (28.6%), no difference was observed (Figure 2). The multiple DR was 21.9% (47 twin and two triplet deliveries) and thus the multiple DR was significantly higher in DET cycles than in SET cycles (P < 0.001) (Table I).
During 1998–2003, re-cryopreserved embryos were thawed in 12 cycles. A total of 11 re-cryopreserved early cleavage stage embryos were thawed in eight cycles and four transfers were performed. In two of these cycles, a mixed transfer was done with one re-cryopreserved embryo and one embryo cryopreserved once; both cycles resulted in a clinical pregnancy (spontaneous abortion). In four cycles, embryos re-cryopreserved at the blastocyst stage were thawed. These thawings resulted in three transfers and two deliveries.
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Discussion |
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During the last years, eSET combined with embryo cryopreservation has become an important tool for reducing multiple pregnancies in fresh ART cycles (De Neubourg and Gerris, 2003
; Gerris et al., 2003; Tiitinen et al., 2003, 2004). In our ART programme, embryo cryopreservation is performed in 66% of oocyte retrieval cycles, and about half of our embryo transfers are frozen embryo transfers. More emphasis should be given to the cryopreservation and thawing methodology to improve the results. Further, the aim should be the use of SET in frozen cycles without reducing the pregnancy rate.
In our programme, the delivery rate was significantly lower after SET, but significantly more multiple deliveries were seen after DET. After eSET, the delivery rates were similar compared with DET. It has to be noticed that in SET cycles, 2.4% of the pregnancies and 2.0% of the deliveries were monozygotic twins. This is above the rate of 0.4% reported in the general population, but in the range reported for ART pregnancies (Schachter et al., 2001; Blickstein et al., 2003). It has been recommended that the risk of iatrogenic zygotic splitting and its potential adverse outcome should be explained to the couple, and it is generally accepted that all forms of assisted conception increase the rate of zygotic splitting (Blickstein et al., 2003).
We observed the highest multiple PR in our cryopreservation programme in 2000. In 2002, the multiple PR was reduced to 7.6%, due to the increased use of SET. However, although the amount of SETs in the following year was 66% of all transfers, the multiple PR increased to 13.6% (13 twin pregnancies and one triplet). This increase was partly due to monozygotic twinning after SET (three cases), but partly due to the high multiple pregnancy rate after DET (26.2%, 11 out of 42). Clearly more research is needed in order to identify both the embryos with the highest implantation potential and the women who are at high risk of achieving a multiple pregnancy in frozen cycles.The characteristics of the women that have been associated with a more favourable implantation rate are the success of the previous fresh embryo transfer cycle, age <40 years and non-tubal factor aetiology of infertility (Wang et al., 2001).
Perhaps eSET in frozen embryo transfer cycles is a better strategy than DET, when more than one embryo fulfilling the criteria exists for transfer. No significant difference was observed in PR and DR between eSET and DET cycles. It has been reported that cleavage in post-thaw culture has a positive effect on pregnancy and implantation rates (Van der Elst et al., 1997; Ziebe et al., 1998), so one strategy to decide which embryo to select for transfer might be prolonged culture of the embryos after thawing.
If SET is the aim in the frozen cycles, this should be taken into account already when the cryopreservation is done, e.g.. cryopreserving the embryos one by one. In this way, all surviving embryos can be used for transfer. If several embryos are frozen together and several survive, but only one is transferred, the alternatives for the remaining embryos are destruction or re-cryopreservation. Several deliveries after re-cryopreservation have been reported. In most cases, the first cryopreservation has been performed on day 1, day 2 or day 3 and the second after culturing to the morula (Yokota et al., 2001) or blastocyst stage (Farhat et al., 2001; Estes et al., 2003). At least in one case, zygotes were cryopreserved, thawed and re-cryopreserved again as zygotes (Baker et al., 1996). One possible strategy could be to culture the other surviving embryos to the blastocyst stage and re-cryopreserve them. There are several problems, though, with this strategy, because the safety and efficacy of re-cryopreservation of human embryos are not known (Yokota et al., 2001). It has also been shown that partially intact embryos have an impaired development to the blastocyst stage in vitro and that the resulting blastocysts have a reduced number of cells compared with fully intact embryos (Archer et al., 2003). Before re-cryopreservation is performed, possible risks need to be discussed with the couple (Yokota et al., 2001). In our programme, the only re-cryopreserved embryos that resulted in deliveries were those re-cryopreserved as blastocysts.
Many factors, such as the day of cryopreservation after oocyte retrieval (Salumets et al., 2003), the cryopreservation method used, the cleavage speed and stage (Edgar et al., 2001; Anderson et al., 2003; Menezo, 2004), fragmentation and blastomere survival after thawing (Burns et al., 1999; Edgar et al., 2000a,b, El-Toukhy et al., 2003) may influence the outcome of frozen embryo transfer. To improve the results of SET in frozen cycles, it is especially important to study further these embryo factors affecting the implantation rate after SET. This can only be studied by analysing the embryos transferred in SET cycles.
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Submitted on March 10, 2005; resubmitted on May 4, 2005; accepted on May 12, 2005.
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