One versus two embryo transfer after IVF and ICSI: a randomized study

Hannu Martikainen1,5, Aila Tiitinen4, Candido Tomás1, Juha Tapanainen1, Mauri Orava1, Leena Tuomivaara2, Sirpa Vilska3, Christel Hydén-Granskog4, Outi Hovatta3 and the Finnish ET Study Group,*

1 Department of Obstetrics and Gynecology, Oulu University Central Hospital, Oulu, 2 The Family Federation of Finland, Oulu, 3 The Family Federation of Finland, Helsinki and 4 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The main reason for adverse treatment outcome in assisted reproduction is the high rate of multiple pregnancies. The only strategy to avoid dizygotic twins is to transfer one embryo at a time. METHODS: A total of 144 women, who had had at least four good quality embryos available after IVF/intracytoplasmic sperm injection (ICSI) and who had no more than one previous failed treatment cycle, were randomized to have either one or two embryos transferred. The treatment outcomes including those after frozen embryo transfer were compared between these groups. RESULTS: The clinical pregnancy rate per transfer was 32.4% in the one embryo transfer group and 47.1% in the two embryo transfer group, the difference being not significant. Eleven twin deliveries (n = 39) occurred in the two embryo transfer group and there was one pair of monozygotic twins in the one embryo transfer group. The cumulative pregnancy rate per patient after transfer of fresh and frozen embryos was 47.3% in the one embryo transfer group and 58.6% in the two embryo transfer group. CONCLUSIONS: Our results indicate that among women who have good quality embryos in their first IVF/ICSI, good treatment results can be achieved. They support the idea of changing embryo transfer policy towards one embryo transfer without any remarkable decrease in the success rate, while dizygotic twins can be avoided.

Key words: assisted reproductive technology/IVF/ICSI/multiple pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The high frequency of multiple births is the main factor which leads to adverse outcome in IVF/intracytoplasmic sperm injection (ICSI) treatment (Templeton and Morris, 1998Go; Bergh et al., 1999Go). Transfer of two embryos does not diminish the chance of a birth when compared with three embryo transfer, if more than four embryos are available for transfer (Templeton and Morris, 1998Go). However, in many countries more than two embryos are still generally transferred. For example, in the USA the average number of embryos transferred is four and the multiple pregnancy rate is ~40% in IVF/ICSI treatment (SART Registry, 1999Go). Despite the common policy of transferring only two embryos in Finland, the number of twin pregnancies is still 25%. There is now increasing pressure to reduce even the risk of twins. Selective fetal reduction is not accepted in twin pregnancies in Finland. The only strategy to avoid dizygotic twins is to transfer one embryo at a time.

We have previously studied the efficacy of elective one embryo transfer (Vilska et al., 1999Go). When at least two embryos were available, a high pregnancy rate (29.8) was achieved. When only one embryo was available, the pregnancy rate was lower, 20% per transfer.

It has been estimated that one embryo transfer is more cost-effective than two embryo transfer when all the costs associated with multiple gestations are taken into account (Wolner-Hanssen and Rydhstroem, 1998Go). A prospective randomized multicentre study was carried out to compare the effectiveness of one and two embryo transfer in a good prognosis group of patients. The main outcome measure was the cumulative pregnancy rate after fresh and frozen embryo transfers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
An inclusion criterion in all four centres involved was at least four good quality embryos. A good quality embryo was defined as having even-sized blastomeres and <20% fragmentation on day 2. In Oulu (n = 101), the age of the woman was not taken into account, and the first two cycles were regarded as eligible. In the two units in Helsinki (n = 43), only women younger than 36 years who were undergoing their first treatment cycle were included.

A total of 1301 couples fulfilled the inclusion criteria, and 144 agreed to participate in the randomized study. In all, 187 chose elective one embryo transfer and 970 two embryo transfer (Figure 1Go).



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Figure 1. The outcome of IVF/ICSI cycles during the study period.

 
The study protocol was approved by the Ethics Committees of the University of Oulu, the Family Federation of Finland and the Department of Obstetrics and Gynecology, Helsinki University Central Hospital. The volunteers were carefully informed of the investigational nature of the study. They were randomized to the one or two embryo transfer groups using a computer-generated random number table balanced in sets of 10. Randomization was done just before embryo transfer by the laboratory personnel. The characteristics of the study subjects and the treatment cycles are shown in Table IGo.


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Table I. Characteristics of the study subjects and the treatment cycles
 
Ovarian stimulation was performed after pituitary down-regulation with buserelin (Suprecur®; Hoechst AG, Farnkfurt, Germany) or nafarelin (Synarela®; Syntex Nordica AB, Södertälje, Sweden) for at least 10 days followed by daily injections of highly purified FSH (Fertinorm HP; Laboratoires Serono S.A., Aubonne, Switzerland) or recombinant FSH (rFSH, Gonal-F®, Laboratoires Serono; or Puregon®, Organon, Oss, The Netherlands). Oocyte retrieval was performed 34–36 h after the injection of 5000–10 000 IU of human chorionic gonadotrophin (Profasi®, Ares-Serono; or Pregnyl®, Organon).

Oocytes/embryos were cultured in MediCult medium (Medi-Cult A/S, Copenhagen, Denmark), IVF-500 medium (Scandinavian IVF Science, Gothenburg, Sweden) or Sydney IVF Medium (Cook IVF, Queensland, Australia). One or two embryos were transferred into the uterine cavity 46–50 h after oocyte retrieval. Supernumerary good quality embryos were frozen using a slow freezing protocol with 1,2-propanediol as the cryoprotectant.

Natural progesterone (Lugesterone, Leiras, Finland) was given transvaginally for luteal support for 14 days. Clinical pregnancies were confirmed by transvaginal ultrasonography.

The frozen embryo transfers were carried out in natural or stimulated cycles. In natural cycles, the LH surge was determined using a home kit (ClearPlan; Unipath Ltd, Bedford, England), and embryo transfer was performed 3–4 days later. In stimulated cycles, down-regulation using a gonadotrophin-releasing hormone agonist (Suprecur, Synarela or Zoladex 3.6 mg) was started on day 21–23 of the previous cycle. On day 3 of the cycle, oestradiol valerate (4 mg/day) was started, and vaginal progesterone 600 mg daily was started 3 days before the embryo transfer. The replacement of frozen embryos was not subjected to any protocol policy related to the present study.

Statistical analysis
Variables in the study groups were compared using by {chi}2-tests and two-tailed t-tests.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The two study groups were similar in relation to age, aetiology of infertility and response to ovulation therapy (Table IGo). In the one embryo transfer group, ICSI was carried out in 20 cases (28.6%) and in the two embryo transfer group in 18 cycles (26.5%). The study cycle was the first treatment cycle in 107 cases (75%) (55 in the one embryo transfer group and 52 in the two embryo transfer group) and the second one in 37 cases (19 in the one embryo transfer group and 18 in the two embryo transfer group).

The implantation rate of the fresh embryos transferred was quite similar (33.8 versus 30.7%) in the one and the two embryo transfer groups. The pregnancy rate was slightly but not significantly higher (47 versus 32%) when two embryos were transferred (Table IIGo). There was no significant difference in the incidence of miscarriages or extrauterine pregnancies between the two groups. In the two embryo transfer group 11 (38%) of the deliveries were twins. The number of preterm deliveries (gestation age <37 weeks) was six (21%) in the two embryo transfer and one (5%) in the one embryo transfer group. The number of low birthweight infants (<2500 g) was 10 (26%) in the two embryo and two (9%) in the one embryo transfer group.


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Table II. The outcome of the fresh embryo transfers in the study groups
 
The outcome of the frozen embryo transfers is presented in Table IIIGo. These transfers, in which the study protocol was not followed, resulted in 13 (15%/transfer) (in one embryo group) and nine (16%/transfer) (in two embryo transfer group) clinical pregnancies. The implantation rate of the frozen embryos was 11% in the one embryo group and 9% in the two embryo transfer group. The cumulative pregnancy rates after frozen embryo transfers, 47.3% in one embryo transfer group and 58.6% in two embryo transfer group, were not statistically different at the time of analysis. The cumulative live birth rate per patient was 39% in the one embryo transfer group and 51% in the two embryo transfer group, the difference being not significant.


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Table III. The outcome of frozen embryo transfers in the study groups
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of our randomized, prospective study indicate that an acceptable pregnancy rate (cumulative pregnancy rate 47.3%) was achieved by transferring only one embryo in a selected population. The success rate was not significantly higher when two embryos were transferred in the study population of this size. To show a difference of 10% in the pregnancy rate to be significant with a power of 80% and an alpha of 5%, 360 patients would have been needed. The two study populations were similar as regards age, aetiology of infertility and the assisted reproduction method used indicating that the randomization was successful. The inclusion criteria between the three participating centres were slightly variable regarding the age limit and the rank of the treatment cycle. However, we think that this had little impact on the results obtained because the rank of the treatment cycle and the age distribution of the patients was similar in both study groups. All the women included in this study showed a good response to ovarian stimulation, a high number of oocytes and at least four good quality embryos available for selection.

The pregnancy rate achieved in the one embryo transfer group was much higher than that reported previously by ourselves (Vilska et al., 1999Go) and other authors (Walters, 1996Go) for one embryo transfer in cycles where no embryo selection was possible. During the study period in the participating centres a pregnancy rate of 14% was achieved among women who had only one embryo available for transfer. Among those who fulfilled the inclusion criteria but chose one embryo transfer, the pregnancy rate was 37%, and among those women who chose a two embryo transfer, the pregnancy rate of 40% was associated with a 32% twin rate. These figures match well with the results obtained in the study population.

Recently the results of another prospective randomized study were published, in which one and two embryo transfers were compared in a relatively small and highly selected population (Gerris et al., 1999Go). As in the present study, a high ongoing pregnancy rate (38.5%) was achieved after one embryo transfers. The investigators achieved a very high pregnancy rate (74%) when transferring two embryos accompanied by a high twin rate (30%). The higher pregnancy rate than in our study may be explained by more strict inclusion criteria. They transferred only top quality embryos characterized by four or five blastomeres at day 2 and at least seven blastomeres on day 3, the absence of multinucleated blastomeres and <20% cellular fragments. The twin rate was also high (35.8%) in the non-eligible population, which means that these criteria proved too strict for clinical practice. The cumulative pregnancy rate after frozen embryo transfers was not analysed in the study reported.

In this study, most of the women were younger than 36 years old age. In a retrospective analysis of our overall patient population, a remarkably lower twin rate was observed in women older than 35 years when two good quality embryos were transferred (6 versus 30%, unpublished data). Hence, in our opinion, in order to lower multiple pregnancy rate, one embryo transfer is most effective in the women younger than 36 years old.

According to the literature, the implantation potential of embryos varies greatly. In our study the implantation rate was quite similar in both groups and showed that the implantation process of each embryo was not affected by the presence of another embryo in the uterus. However, even higher implantation rate can be achieved by careful selection of embryos for transfer (Van Royen et al., 1999Go). We have previously shown that the pregnancy/implantation rates were highest if the embryo had reached the 4–5-cell stage on day two or 6–8-cell stage on day 3 after fertilization and, if the embryos had <20% fragmentation (Vilska et al., 1999Go). Besides the commonly used morphological criteria, polarity of embryos at pronuclear stage (Garello et al., 1999Go) or variations in thickness of zona pellucida (Palmstierna et al., 1998Go) might be applicable in clinical practice. It remains to be shown if culturing to blastocyst stage can improve the implantation rate.

All good quality extra embryos were frozen. In the one embryo group, the number of embryos frozen was slightly higher, as expected, than in the two embryo group. Until now, when most but not all these embryos have been transferred, cumulative pregnancy rate of 47.3% has been achieved, which is identical to that obtained in our previous study (Vilska et al., 1999Go). Because two frozen embryos were transferred to those who desired it, one set of dizygotic twins was also achieved. The overall implantation rate of the frozen embryos was much lower (~10%) than that of the fresh embryos. However, in the future, single embryo transfer might also be warranted after embryo freezing.

On the basis of the success rates in this study and in our overall patient population we have estimated that, to achieve the same clinical pregnancy rates in both groups, ~20% (if two embryos are replaced in the subsequent cycle) more treatment cycles must be performed in the one embryo transfer group. These figures are quite close to those estimated previously by other investigators (Coetsier and Dhont, 1998Go).

When the treatment methods are evaluated, the costs associated with maternity and neonatal health care should be taken into account. Our results support the assumption that one embryo transfer would be more cost-effective than two embryo transfer (Wolner-Hanssen and Rydhstroem, 1998Go). Based on the available data, we suggest that one embryo should be transferred in the first treatment cycle to women younger than 36 years who have good quality embryos; 4-cell stage on day 2 or 8-cell stage on day 3 after fertilization, equal-sized blastomeres and <20% fragmentation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank all the staff of the participating IVF clinics for making this study possible.


    Notes
 
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Oulu, Kajaanintie 52, 90220 Oulu, Finland. E-mail: hmartika{at}cc.oulu.fi Back

* The Finnish ET Study Group: Department of Obstetrics and Gynecology, University of Oulu, Oulu: Laure Morin-Papunen, Riitta Koivunen, Ilkka Järvelä, Jouni Lakkakorpi; The Family Federation of Finland, Oulu: Sinikka Nuojua-Huttunen, Kaisa Juntunen; The Family Federation of Finland, Helsinki: Tarja Bützow, Tuija Foudila, Tiina Hakala-Ala-Pietilä, Seija Kaukoranta, Jarna Moilanen, Sirpa Mäkinen, Rita Siegberg, Viveca Söderström-Anttila, Maija Tulppala, Timo Tuuri; Department of Obstetrics and Gynecology, University of Helsinki: Mervi Halttunen, Päivi Härkki-Siren. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bergh, T., Ericson, A., Hillensjö, T. et al. (1999) Deliveries and children born after in-vitro fertilisation in Sweden 1982–95: a retrospective cohort study. Lancet, 354, 1579–1585.[ISI][Medline]

Coetsier, T. and Dhont, M. (1998) Embryo transfer and multiple gestation. Avoiding multiple pregnancies in in-vitro fertilization: who's afraid of single embryo transfer? Hum. Reprod., 13, 2663–2664.[Free Full Text]

Garello, C., Baker, H., Rai, J. et al. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum. Reprod., 14, 2588–2595.[Abstract/Free Full Text]

Gerris, J., DeNeuborg, D., Mangelschots, K. et al. (1999) Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum. Reprod., 14, 2581–2587.[Abstract/Free Full Text]

Palmstierna, M., Murkes, D., Csemiczky, G. et al. (1998) Zona pellucida thickness variation and occurrence of visible mononucleated blastomeres in preembryos are associated with a high pregnancy rate in IVF treatment. J. Assist. Reprod. Genet., 15, 70–75.[ISI][Medline]

SART Registry (1999) Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil. Steril., 71, 798–807.[ISI][Medline]

Templeton, A. and Morris, J. (1998) Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N. Engl. J. Med., 339, 573–577.[Abstract/Free Full Text]

Van Royen, E., Mangelschots, K., De Neuborg, D. et al. (1999) Characterization of a top quality embryo, a step towards single-embryo transfer. Hum. Reprod., 14, 2345–2349.[Abstract/Free Full Text]

Vilska, S., Tiitinen, A., Hydén-Granskog, C. et al. (1999) Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum. Reprod., 14, 2392–2395.[Abstract/Free Full Text]

Walters, D. (1996) The statistical implication of the `number of replacements' in embryo transfer. Hum. Reprod., 11, 10–12.[ISI][Medline]

Wolner-Hanssen, P. and Rydhstroem, H. (1998) Cost-effectiveness analysis of in-vitro fertilization: estimated costs per successful pregnancy after transfer of one or two embryos. Hum. Reprod., 13, 88–94.[Abstract]

Submitted on February 7, 2001; accepted on May 27, 2001.