Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, PO Box 140, 00029 HUS, Finland
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Abstract |
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Key words: cryopreservation/embryo transfer/in-vitro fertilization
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Introduction |
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Cryopreservation of embryos following IVF/intracytoplasmic sperm injection (ICSI) provides further possibilities of pregnancy in addition to those achieved from the fresh cycle. The contribution of embryo cryopreservation to the results of assisted reproductive techniques over a long period of time is not usually reported. The efficacy of IVF, which is more important for the infertile couple, as well as for the health administrators, is the cumulative birth rate after completion of the IVF cycle. The contribution of cryopreservation to pregnancy has been reported to increase the take-home baby rate by 5.2% (Kahn et al., 1993), to 11% (Wang et al., 1994
) or even 19% (Bergh et al., 1995
).
The objective of our follow-up study was to analyse data from our elective single embryo transfer programme and to expand the findings by combining the results from frozenthawed cycles. In an attempt to establish quality criteria for embryos, we examined the characteristics of the embryos that resulted in ongoing implantation both in fresh and frozenthawed transfers.
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Materials and methods |
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The oocytes and embryos were cultured in microdrops under oil (Ovoil; Scandinavian IVF Science, Gothenburg, Sweden) in IVF-500 medium (Scandinavian IVF Science,) or Sydney IVF fertilization medium and Sydney IVF cleavage medium (Cook IVF, Brisbane, Australia). The spermatozoa were prepared using both density gradient centrifugation (PureSperm; NidaCon, Gothenburg, Sweden) and swim-up. Insemination was carried out 45 h after oocyte retrieval with 25 00075 000 progressively motile spermatozoa. If ICSI was performed, denudation of oocytes was carried out with hyaluronidase (HYASE-10X; Scandinavian IVF Science). ICSI was done with PVP (ICSI; Scandinavian IVF Science). On the next day, 17 h after the insemination, oocytes were examined for the appearance of pronuclei. Unfertilized oocytes and oocytes containing only one pronucleus were examined again 45 h later. The embryo grading and cleavage rate was assessed 24 h later at x400 magnification. The embryos were graded according to the number of blastomeres, the degree of fragmentation, regularity of blastomeres and mono-/multinuclearity in the blastomeres. Embryos were suitable for transfer or cryopreservation if they had <50% fragmentation and, if nuclei were visible, only one nucleus per blastomere. The best embryo was selected for fresh transfer. Elective transfer of one embryo was performed only if there was at least one four-cell embryo on day 2 after oocyte retrieval.
Freezing and thawing were carried out using the standard protocol (Testart et al., 1986) with 1,2-propanediol (PROH), modified according to the manual of the media provider (Freeze-kit1 and Thaw-kit1; Scandinavian IVF Science) in vials. Usually two or three embryos were frozen in the same vial for practical reasons and to ensure the possibility for transfer after thawing. Two-cell embryos were thawed and cultured overnight and transferred only if they cleaved (Van der Elst et al., 1997
). Four-cell embryos were thawed and transferred on the same day. One embryo was always transferred if there was a medical reason. Two-embryo transfer was allowed after careful counselling of the couple, if two good quality embryos in the same vial were alive after thawing. The frozenthawed embryos were transferred during a natural cycle, if possible, 3 days after a luteinizing hormone (LH) surge measured by a home test kit (Clearplan; Unipath, Bedford, UK). Micronized vaginal progesterone was administered for 2 weeks after embryo transfer. In cases of anovulatory cycles, hormone replacement with oestradiol valerate (Progynova, Schering) and micronized progesterone was used.
During the years 19981999, we performed 708 IVF or ICSI cycles with embryo transfer. In 487 cases, two embryos were transferred and in 221 cases one embryo was transferred. In 127 cycles, a single embryo was transferred and at least one embryo was cryopreserved, whereas in 94 cycles only one embryo was available.
The main indication for elective single embryo transfer was the couple's wish to avoid twins, and in some cases various medical reasons, which include diabetes mellitus, uterine malformation, a history of cervical incompetence and indication for prenatal diagnosis. The mean age of the women in this group was 31.3 years (range 2040 years), and 57 (44.9%) had primary infertility. Eighty-nine (70.0%) cycles were first attempts at IVF. The implantation rate and pregnancy rate per transfer and cumulative pregnancy rate per oocyte retrieval was calculated. The correlation between clinical implantation rate and embryo quality as revealed by cleavage and fragmentation status was analysed.
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Results |
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Discussion |
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The ultimate goal of assisted reproductive techniques is to get the woman pregnant with a singleton pregnancy (Coetsier and Dhont, 1998; Hazekamp et al., 2000
). The increased efficiency of IVF and ICSI programmes in many centres has produced overall pregnancy rates of 3040% per cycle, with multiple rates of 2535%. The need for a high success rate conflicts with a low complication rate. It is essential to give objective information to the couples concerned. In aiming for an individualized transfer policy, we have to consider the probability of the embryo implanting and the subject conceiving. The obstetric and perinatal risks should also be considered, as well as the long-term social and psychological impact on the whole family. Competition between the centres should not result in multiple embryo transfers and thus multiple pregnancies.
How is an embryo with high implantation potential selected? This is mainly accomplished by looking at two parameters: cleavage speed and fragmentation. The implantation rate increases with better quality embryos in cycles where only one embryo is available (Giorgetti et al., 1995; Ziebe et al., 1997
). Van Royen et al. describe the implantation potential of a well-defined embryo in their retrospective analysis: the top quality embryo has no multinucleated blastomeres, four or five blastomeres on day 2 and <20% fragmentation (Van Royen et al., 1999
). Using these embryos, they achieved an implantation rate of 49% (Van Royen et al., 1999
) and 45% in a prospective randomized study (Gerris et al., 1999
). In our earlier retrospective study (Vilska et al., 1999
), the highest implantation rates were obtained when four- to five-cell embryos with fragmentation <20% were transferred on day 2 (36%) or when six- to eight-cell stage embryos were transferred on day 3 (45%) or embryos with no fragmentation irrespective of cleavage stage were transferred (34%). The implantation rate in the present study was 39.8% with the same criteria as applied in the Belgian study.
The scoring of the embryos could be improved even more (Edwards and Beard, 1999). Besides the commonly used morphological criteria, polarity of the pronuclei (Scott and Smith, 1998
; Garello et al., 1999
), or variations in the thickness of zona pellucida (Palmstierna et al., 1998
) might be applicable. Tesarik et al. have shown implantation rates of 30.2% in embryos judged normal at the pronuclear stage (Tesarik et al., 2000
). Until now, we have not used this criterion for selection of the embryo for transfer. Careful evaluation of all blastomeres in four-cell embryos is also essential. It has been demonstrated that embryos displaying multinuclearity have impaired implantation potential (Pickering et al., 1995
; Pelinck et al., 1998
). In our practice, embryos with multinuclear blastomeres are not used for transfer unless they are the only embryos available.
In our previous study (Vilska et al., 1999) the selection criteria for patients for single embryo transfer were not as strict as in the Belgian study (Gerris et al., 1999
), which produced an ongoing pregnancy rate of 40%; we had a pregnancy rate of 30%. In the present study, the criteria for elective single embryo transfer were even more liberal. We cautiously counsel our patients who come for the first IVF/ICSI treatment cycle and who are under 38 years of age, to accept only one embryo for fresh transfer, in order to avoid multiple pregnancies. In cases where good embryos are not produced, transfer of two embryos is allowed. This means that a change in attitude is necessary both in patients but, more importantly, in doctors (Gerris et al., 1999
). We have not performed any three-embryo transfers during the last 5 years, because we think that triplets should not be allowed in a responsible fertility centre. During 19981999, 31% of our fresh transfers were single embryo transfers. This year our aim is to perform at least 50% of all cycles as single embryo transfers.
Whether the laborious blastocyst culture will result in a high pregnancy rate while lowering the number of twins has to be studied in a prospective setting, in which transfer of the best embryo on day 2 is compared with the transfer of one blastocyst, and cumulative pregnancy rates per oocyte retrieval with frozenthawed embryos are analysed. Until now, there has been no evidence that single blastocyst transfer is a cost-effective method of avoiding twins. Moreover, the cryopreservation of blastocysts is difficult.
The ability of embryo cryopreservation to enhance the cumulative pregnancy potential after oocyte retrieval is somewhat underappreciated (Damario et al., 2000). Good cryopreservation and thawing techniques are necessary in elective single embryo transfers (Horne et al., 1997
; Vilska et al., 1999
). Many variables may influence the outcome of embryo cryopreservation and frozen embryo transfer. Multicell embryonic survival of cryopreservation and thawing with all blastomeres intact might identify embryos with superior implantation potential (Burns et al., 1999
). In our study, we had similar implantation rates irrespective of the fragmentation stage of the embryo or whether it had all blastomeres intact. We transferred two embryos to selected patients, achieving an on-going pregnancy rate of 32.5%, of which 14.8% were twins. This means that we have to consider single embryo transfer in frozenthawed cycles as well. In order to do this, the selection criteria for embryos to be frozen should be optimized, preferably only one top quality embryo, possibly accompanied by a lower grade embryo in the same vial.
More than half of our 125 patients have delivered, and 18 non-pregnant couples still have frozen embryos. It can be speculated that if the implantation rate is the same with these cryopreserved embryos, ultimately 57% of the couples will have a live birth after single oocyte retrieval cycle. However, we can already state that embryo cryopreservation is effective in increasing the pregnancy rate per oocyte recovery and thus lowering the ultimate cost per delivery. This information should be used when counselling patients and formulating health care policy (Van Voorhis et al., 1995). IVF results should be presented as `birth per embryo transferred' and this term should be given the status of `the criterion of assisted reproductive techniques excellence' (Hazekamp et al., 2000
).
We conclude that even better results can be achieved if the embryo culture and freezing techniques can be improved. Single embryo transfer combined with freezing of extra embryos is highly effective and lowers the ultimate costs per delivery.
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Notes |
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References |
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Submitted on November 13, 2000; accepted on February 20, 2001.