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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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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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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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 19911997.
Our criteria for transfer of frozenthawed 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 couples 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 1012. 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 1012 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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In DET cycles, the mean PR was 34.3% (range 20.745.0%, 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 19982003, 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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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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References |
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Archer J, Gook DA and Edgar DH (2003) Blastocyst formation and cell numbers in human frozenthawed embryos following extended culture. Hum Reprod 18,16691673
Baker A, Check JH, Lurie D, Hourani C and Hoover LM (1996) Pregnancy achieved with pronuclear-stage embryos that were cryopreserved and thawed twice: a case report. J Assist Reprod Genet 13,713715[CrossRef][ISI][Medline]
Bergh T, Ericson A, Hillensjö T, Nygren K-G and Wennerholm, U-B (1999) Deliveries and children born after in-vitro fertilization in Sweden 198295: a retrospective cohort study. Lancet 354,15791585[CrossRef][ISI][Medline]
Blickstein I, Jones C and Keith LG (2003) Zygotic-splitting rates after single-embryo transfers in in vitro fertilization. N Engl J Med 348,23662367
Burns WN, Gaudet TW, Martin MB, Leal YR, Schoen H, Eddy CA and Schenken RS (1999) Survival of cryopreservation and thawing with all blastomeres intact identifies multicell embryos with superior frozen embryo transfer outcome. Fertil Steril 72,527532[CrossRef][ISI][Medline]
De Neuborg D and Gerris J (2003) Single embryo transferstate of the ART. RBM Online 6,615622
De Sutter P, Van der Elst J, Coetsier T and Dhont M (2003) Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5 year appraisal. RBM Online 6,464469[Medline]
Edgar DH, Bourne H, Jericho H and McBain JC (2000a) The developmental potential of cryopreserved human embryos. Mol Cell Endocrinol 169,6972[CrossRef][ISI][Medline]
Edgar DH, Bourne H, Speirs AL and McBain JC (2000b) A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 15,175179
Edgar DH, Jericho H, Bourne H and McBain JC (2001) The influence of prefreeze growth rate and blastomere number of cryosurvival and subsequent implantation of human embryos. J Assist Reprod Genet 18,135138[CrossRef][ISI][Medline]
EIM/ESHRE (2005) Assisted reproductive technology in Europe, 2001. Results generated from European registers by ESHRE. The European IVF-monitoring programme (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod in press
El-Touhky T, Khalaf Y, Al-Darazi K, Andritsos V, Taylor A and Braude P (2003) Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil Steril 79,11061111[CrossRef][ISI][Medline]
Estes SJ, Laky DC, Hoover LM, Smith SE, Shinfeld JS and Somkuti SG (2003) Successful pregnancy resulting from cryopreserved embryos thawed and cultured to blastocysts, refrozen and transferred. A case report. J Reprod Med 48,4648[ISI][Medline]
Farhat M, Zentner B-S, Lossos F, Bdolah Y, Holtzer H and Hurwitz A (2001) Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage. Hum Reprod 16,337339
Gerris J, De Neubourg D, De Sutter P, Van Royen E, Mangelschots K and Vercruyssen M (2003) Cryopreservation as a tool to reduce multiple birth. RBM Online 7,286294[Medline]
Hydén-Granskog C and Tiitinen A (2004) Single embryo transfer in clinical practice. Hum Fertil 7,175182
Menezo Y (2004) Cryopreservation of IVF embryos: which stage? Eur J Obst Gynecol Reprod Biol 113S, S28S32
Salumets A, Tuuri T, Mäkinen S, Vilska S, Husu L, Tainio R and Suikkari A-M (2003) Effect of the developmental stage of embryo at freezing on pregnancy outcome of frozenthawed embryo transfer. Hum Reprod 18,18901895
Schachter M, Raziel A, Friedler S, Strassburger D, Bern O and Ron-El R (2001) Monozygotic twinning after assisted reproductive techniques: a phenomen independent of micromanipulation. Hum Reprod 16,12641269
Thurin A, Hausken J, Hillensjö T, Jablonowska B, Pinborg A, Strandell A and Bergh C (2004) Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med 351,23922402
Tiitinen A, Halttunen M, Härkki P, Vuoristo P and Hydén-Granskog C (2001) Elective single embryo transfer: the value of cryopreservation. Hum Reprod 16,11401144
Tiitinen A, Unkila-Kallio L, Halttunen M and Hydén-Granskog C (2003) Impact of elective single embryo transfer on the twin pregnancy rate. Hum Reprod 18,14491453
Tiitinen A, Hydén-Granskog C and Gissler M (2004) What is the most relevant standard of success in assisted reproduction? Hum Reprod 19,24392441
Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P and Van Steirteghem AC (1997) Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 12,15131521[Abstract]
Wang JX, Yap YY and Matthews CD (2001) Frozenthawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod 16,23162319
Yokota Y, Yokota H, Yokota M, Sato S and Araki Y (2001) Birth of healthy twins from in vitro development of human refrozen embryos. Fertil Steril 76,10631065[CrossRef][ISI][Medline]
Ziebe S, Bech B, Petersen K, Mikkelsen AL, Gabrielsen A and Nyboe Andersen A (1998) Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum Reprod 13,178181[Abstract]
Submitted on March 10, 2005; resubmitted on May 4, 2005; accepted on May 12, 2005.