Hum. Reprod. Advance Access originally published online on April 7, 2007
Human Reproduction 2007 22(6):1669-1674; doi:10.1093/humrep/dem059
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eSET irrespective of the availability of a good-quality embryo in the first cycle only is not effective in reducing overall twin pregnancy rates
1 Research Institute Growth & Development (GROW), Department of Obstetrics and Gynaecology, Academic Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands 2 Research Institute Growth & Development (GROW), Department of Clinical Epidemiology and Medical Technology Assessment, Academic Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands 3 Research Institute Growth & Development (GROW), Department of Clinical Genetics, Academic Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
5 Correspondence address. Research Institute Growth & Development (GROW), Department of Obstetrics and Gynaecology, Academic Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Tel: +31 43 3874760; Fax: +31 43 3874765; E-mail: avmn{at}sgyn.azm.nl
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Abstract |
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INTRODUCTION: In several clinics, elective single-embryo transfer (eSET) is applied in a selected group of patients based on age and the availability of a good-quality embryo. Whether or not eSET can be applied irrespective of the presence of a good-quality embryo in the first cycle, to further reduce the twin pregnancy rate, remains to be elucidated.
METHODS: In patients <38 years two transfer strategies were compared, which differed in the first cycle only: group A (n = 141) received eSET irrespective of the availability of a good-quality embryo, and group B (n = 174) received eSET when a good-quality embryo was available while otherwise they received double embryo transfer (DET; referred to as eSET/DET transfer policy). In any subsequent cycle, in both groups the eSET/DET transfer policy was applied.
RESULTS: After completion of their IVF treatment (including a maximum of three fresh cycles and the transfer of frozen–thawed embryos), comparable cumulative live birth rates (62.4% in group A and 62.6% in group B) and twin pregnancy rates (10.1 versus 13.4%) were found. However, patients in group A required significantly more fresh (2.0 versus 1.8) and frozen (0.8 versus 0.5) cycles.
CONCLUSIONS: The transfer of one embryo in the first cycle, irrespective of the availablity of a good-quality embryo, in all patients <38 years, is not an effective transfer policy for reducing the overall twin pregnancy rate.
Key words: assisted reproductive technologies/multiple pregnancy/single-embryo transfer
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Introduction |
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In several randomized studies, elective single-embryo transfer (eSET, i.e. the transfer of one embryo in case one could also transfer two or more embryos) has proven its merits for reducing the twin pregnancy rate, while obtaining acceptable overall pregnancy rates in selected patients (see Bergh, 2005
, for review on eSET). Based on these studies, in several clinics, a transfer policy was introduced in which relatively young patients with at least one or two good-quality embryos received eSET and the remaining patients received double-embryo transfer (DET) (referred to as eSET/DET transfer policy). This policy was applied in most studies in the first and second IVF cycles. Recently, it was shown that this policy also led to acceptable results in the third IVF cycle (van Montfoort et al., 2005).
To further increase the implementation of eSET, a new strategy is applied in Belgium. Since July 2003, the legislation prescribes the reimbursement of six cycles in a life time per patient under the condition that in the first cycle of patients <36 years, only one embryo is transferred, irrespective of the availability of a good-quality embryo. In the subsequent cycles and in the cycles in older patients, transfer of a maximum of two or three embryos is allowed (Ombelet et al., 2005). Studies comparing the IVF results before and after the implementation of the new legislation in Belgium showed no difference in the overall ongoing pregnancy rate per cycle, and a reduction in the overall twin pregnancy rate, although studying two different strategies in two subsequent time periods has its limitations (Debrock et al., 2005; Gordts et al., 2005). Besides, in these studies only outcome per cycle in two time periods was compared instead of cumulative outcome per patient.
Our group published a study in which the transfer strategy was comparable to the transfer strategy in the first cycle in Belgium, although in our study no age limit was set (van Montfoort et al., 2006). In this randomized controlled trial (RCT), eSET was applied in the first cycle of an unselected patient population, i.e. irrespective of the availability of a good-quality embryo and age. This strategy, however, led to a significant reduction in the ongoing pregnancy rate as compared to DET or an eSET/DET policy in the first cycle (van Montfoort et al., 2006). Whether or not this decrease in pregnancy rate and the reduction in twin pregnancy rate have their influence on the total IVF outcome per patient after several cycles of an eSET/DET policy remains to be elucidated.
The aim of the present study was, therefore, to compare two treatment strategies, performed in the same time period, which are similar for the second and third cycles (i.e. eSET/DET policy) but differ in the first cycle; one group receiving eSET irrespective of the availability of a good-quality embryo and another group receiving an eSET/DET transfer policy. The two groups, consisting of patients <38 years, were compared with respect to the total number of cycles, the live birth rate and the twin pregnancy rate after a maximum of three cycles.
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Methods |
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Patients
In the IVF clinic of the academic hospital, Maastricht, eSET is routinely performed in patients <38 years when at least one good-quality embryo is present (see below). If one of the criteria is not fulfilled, DET is performed. This transfer policy is referred to as the eSET/DET transfer policy and is applied in all treatment cycles of a patient. From January 2002 until December 2004, an RCT was performed in which consenting patients were randomly allocated to either eSET or DET in their first IVF or ICSI cycle, irrespective of the availability of a good-quality embryo and female age. The exclusion criteria were preimplantation genetic diagnosis, request of SET for medical reasons, language barrier (patients could not be well informed) and less than two 2PN embryos available. Patients fulfilling these criteria but declining to participate received our eSET/DET transfer policy (van Montfoort et al., 2006
). This study was approved by the Institutional Ethical Board of the academic hospital of Maastricht. The present study compared the results of two transfer strategies which only differed in the first cycle: group A receiving eSET irrespective of the availability of a good-quality embryo (i.e. patients randomized for eSET in our RCT) and group B receiving the eSET/DET transfer policy (i.e. patients declining to participate in our RCT). As the age limit for the eSET/DET policy in group B was 38 years, also in group A only patients <38 years were included in the analysis. In the second and third cycles of all patients (a maximum of three cycles are reimbursed in the Netherlands), the eSET/DET transfer policy was applied. In case supernumerary embryos were cryopreserved and no pregnancy was established, the cryopreserved embryos were transferred before a new ovum pick-up (OPU) was performed. The transfer of these cryopreserved embryos was performed in all cycles of both groups according to the eSET/DET policy.
Ovarian stimulation, embryo culture and quality assessment
Ovarian stimulation, IVF, ICSI and embryo culture procedures have been described in detail earlier (van Montfoort et al., 2006). For each embryo originating from a normally fertilized oocyte, an embryo score was calculated on the basis of morphological grade (1–4, with grade 4 being the best grade), number of blastomeres and presence or absence of multinucleated blastomeres (MNBs) (van Montfoort et al., 2005). This score ranged on day 2 from 1 to 16.5 with a higher score correlating to a better embryo quality. Embryos that had reached the 4- or 5-cell stage on day 2, or the 8-cell stage on day 3, in combination with having the best morphological grade (regular, even-sized blastomeres with <20% fragmentation), and an absence of MNBs were classified as good-quality embryos (score 
13.25 on day 2 or 23.25 on day 3) (van Montfoort et al., 2005). Embryos were transferred on day 2 after OPU or in a minority of cases, for reasons of convenience, on day 3. In all cases, including those in the RCT study, embryos with the highest embryo score were transferred.
Cryopreservation of supernumerary embryos was performed on the morning of the third day after OPU depending on their embryo score. The vast majority of cryopreserved embryos had reached the 7- to 8-cell stage in combination with morphological grade 3 or 4.
Statistical analysis
Each patient was followed until a live birth after the transfer of fresh or frozen/thawed embryos was registered, until the patient had completed an IVF or ICSI treatment with a maximum of three OPU (and any frozen–thawed embryo transfers) without success or until the patient dropped out for medical or personal reasons. In this study, a cycle is defined as a stimulation cycle resulting in an OPU. The cycles not resulting in an OPU are left out of consideration. A miscarriage was defined as a proven loss of the pregnancy and/or no fetal heart beat on ultrasound examination at or before 12 weeks gestation in a patient with an initially positive hCG test at 2 weeks after embryo transfer (detection limit 50 IU/l in urine). An independent sample t-test was used to compare continuous variables, and the 
2-test was used for binary variables. A P-value of <0.05 was considered significant.
To analyse whether or not differences in dropout rate between the two groups might interfere with the outcome (i.e. cumulative live birth rates), a life-table analysis was performed (Cooke et al., 1981). As patients dropping out for medical reasons (e.g. poor response on ovarian stimulation) are assumed to have a lower live birth rate irrespective of the treatment strategy, these patients were excluded (Cooke et al., 1981; Daya, 2005).
Patients dropping out for personal reasons (e.g. relocation to other part of country or the burden of treatment) or because of a spontaneous pregnancy can influence cumulative outcome, as these patients are likely to have similar live birth rates as compared to patients not lost to follow-up (Daya, 2005). As the live birth rate of the patients lost to follow-up is unknown, life-table analysis was performed with two different assumptions; the live birth rate in patients dropping out to be 0% and to be similar to that in patients remaining in the study.
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Results |
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Group A consisted of 146 patients and group B of 175 patients. Those who had not completed their treatment at the time of analysis (23 months after the first OPU of the last patient included) were excluded from the analysis (five and one patients, respectively). Therefore, the analysis was performed on 141 patients in group A and 174 patients in group B.
Both groups were comparable regarding patient and first cycle characteristics (Table 1). Table 2 presents the clinical outcomes of the first cycle. In group A, all patients had eSET, and in group B eSET was performed in 100 patients (57.5%). The transfer of two embryos in 42.5% of the patients in group B led to a significantly higher live birth and twin pregnancy rate as compared with group A (33.9 versus 21.3% and 13.3 versus 0%, respectively). After the transfer of cryopreserved embryos in patients not achieving a pregnancy in the fresh cycle, live birth and twin pregnancy rates were comparable (32.6 and 8.5% in group A and 39.7 and 14.3% in group B, Table 2). In Tables 3 and 4, the results of the first fresh cycles of groups A and B are classified according to the transfer of good-or moderate-quality embryos. Although in group A in both cases one embryo was transferred, it did not result in a significant difference in clinical outcome (Table 3). In group B one good-quality embryo or two moderate-quality embryos were transferred (Table 4). With the exception of the twin pregnancy rate, no significant difference was found between eSET and DET in group B. Also no significant difference was found in clinical outcome after eSET with a good-quality embryo between groups A (Table 3) and B (Table 4).
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The outcomes of cycles 2 and 3 did not differ significantly between the two groups. Despite the significant difference in miscarriage rate after a maximum of three fresh cycles, the cumulative live birth and twin pregnancy rate per patient were similar for both strategies (62.4 and 10.1% in group A and 62.6 and 13.4% in group B, respectively, Table 5). Only the mean number of fresh and frozen cycles, patients performed before achieving a pregnancy or before having completed the maximum of three cycles without success, was significantly different (2.0 and 1.8 fresh cycles per patient and 0.8 and 0.5 frozen cycles per patient in group A and B, respectively, Table 5).
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Most cryopreserved embryos in patients not achieving a live birth were thawed during the study period, except for one embryo in group A and two in group B, all from patients who decided to refrain from transfer. In 29 patients achieving a live birth in the study period, embryos are still cryopreserved. Additionally, in two patients from both groups, cryopreserved embryos are still stored as these patients became pregnant spontaneously.
The dropout rate after the first cycle was significantly different between the two groups. In group A, 3.2% (2.1% for medical and 1.1% for personal reasons) of the non-pregnant patients dropped out compared to 15.4% in group B (2.9% for medical and 9.6% for personal reasons and 2.9% because of achieving a spontaneous pregnancy). In group B there was no relation between the number of embryos transferred in the first cycle and the dropout rate, as 15.5% dropped out after eSET and 15.2% after DET. Between the second and third cycles these numbers were 17.5% (4.8% for medical and 9.5% for personal reasons and 3.2% because of spontaneous pregnancies) for group A and 13.6% (1.7% for medical and 11.9% for personal reasons) for group B. To analyse whether this difference in dropout rate can influence cumulative outcome data, a life-table analysis was performed. Assuming a live birth rate in patients dropping out of 0% resulted in a cumulative live birth rate of 64.7 in group A and 64.1% in group B. When assuming a similar live birth rate in patients dropping out and those remaining in the study, these data were 66.5 and 68.6%, respectively (both differences not significant).
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Discussion |
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From the results of this study, it became apparent that treatment strategies consisting of eSET (group A) or eSET/DET (group B) in the first cycle and eSET/DET in the second and third cycles in patients <38 years led to similar live birth rates. To obtain this result, more cycles per patient were required in group A, while the overall twin pregnancy rate was not significantly reduced in group A. Our results put the policy with eSET in the first cycle, irrespective of the availability of a good-quality embryo, at a relative disadvantage, which is remarkable considering that, for example, in Belgium the legislation has been changed towards an eSET ‘irrespective of the availability of a good-quality embryo’ policy in patients <36 years (Gordts et al., 2005
). Although the overall cumulative live birth rate was similar in our study, the live birth rate in the first cycle was significantly different. This difference might be explained by the fact that approximately half of all human preimplantation embryos may be considered chromosomally abnormal (Coonen et al., 1994; Baart et al., 2006). These aneuploidies do not affect the lower-quality embryos only, but also morphologically normal embryos (Baart et al., 2006; Munne et al., 2006). In the case of eSET, approximately half of the embryos will be chromosomally abnormal and therefore not result in pregnancy, even when this embryo is of high morphological quality. This can also be seen in Table 3, where the transfer of one good- or one moderate-quality embryo was shown to lead to statistically similar live birth rates. After the transfer of two embryos in DET, there is a higher chance that at least one chromosomally normal embryo will be replaced, increasing the chance for pregnancy and live birth. It may be argued, however, that in eSET there are also two embryos available for transfer (one is transferred as a fresh embryo and the other after freezing and thawing), with a similar chance for at least one chromosomally normal embryo to be transferred as compared to the transfer of two fresh embryos in DET. In the eSET policy, however, some of the cryopreserved embryos will not survive the thawing process and will be lost before transfer. Therefore, although groups A and B started with the same number of embryos (6.2 and 6.0, respectively), the net number of embryos available for transfer and therefore the net number of euploid embryos, will be lower in an eSET policy (group A) as compared to an eSET/DET policy (group B). This difference, although not significant, did result in one extra cycle per five patients after eSET in our study. In addition, the overall twin pregnancy rate could not be reduced with this strategy. The transfer of cryopreserved embryos and the second and third fresh cycles were all performed according to an eSET/DET policy. The resulting twins annihilated the reduction in twin pregnancies after the first fresh cycle. As 44% of the twins in group A and 20% of the twins in group B originated from the transfer of two frozen/thawed embryos, more attention should be paid to single-embryo transfer with these embryos. Furthermore, more studies are needed to define the morphological criteria of cryopreserved embryos suitable for eSET.
Although the miscarriage rate between the two groups, defined as number of pregnancy losses 
12 weeks of gestation per positive hCG test, did not differ significantly per cycle, there was a trend towards a higher miscarriage rate in group A as compared with group B [respectively 32.6 (15/46) versus 20.0% (15/75) for the first cycle, 24.2 (8/33) versus 11.1% (3/27) for the second cycle and 45.5 (5/11) and 18.2% (2/11) for the third cycle]. Especially in the second and third cycles, the numbers were very small, but they nevertheless resulted in a significantly different cumulative miscarriage rate. The difference in the first cycle miscarriage rate, although not significant, was also reported in RCTs on eSET versus DET [41 versus 9% (Gerris et al., 1999) and 30 versus 20% per clinical pregnancy (Lukassen et al., 2005]. However, other RCTs reported lower miscarriage rates for eSET compared to DET [15 versus 16% (Thurin et al., 2004) and 4 versus 9% (Martikainen et al., 2001)]. The reason for the difference in miscarriage rate in our study between groups A and B remains elusive. As for group A the miscarriage rate is similar for good-and moderate-quality embryos, embryo quality cannot be regarded as the cause for the difference in miscarriage rate. It can, however, partly be explained by the fact that only positive hCG tests resulting in a complete loss of the implantation were counted as a miscarriage. The loss of one conceptus in case of DET (vanishing twin) before or after the detection of a fetal heart beat is not included and the chance of losing both embryos in case of DET is smaller as compared to the loss of one embryo in eSET, putting the latter at a disadvantage.
The dropout rate in the first cycle of group B was significantly higher as compared to A (15.4 versus 3.0%). The most prominent cause for dropout in group B was personal reasons, while almost no couple dropped out in group A for this reason. As in group B the dropout rate was similar after eSET and DET, the number of embryos transferred cannot explain this difference. A more likely explanation is that the first cycle of group A was in a study setting where patients not obtaining a pregnancy in the first three cycles were offered a fourth cycle free of charge, while in group B it was part of standard care. The dropout rate in group B (15.4%) is comparable to the one reported for standard care IVF (26.2%) (Land et al., 1997). Patients agreeing to participate in the RCT study are possibly more motivated to continue treatment and less likely to drop out. Life-table analysis, using both a minimal and a maximal assumed live birth rate in the dropout group, led to comparable cumulative live birth rates in groups A and B. From this, it can be concluded that the difference in dropout rate did not influence the comparison of the two transfer strategies.
From this study, we conclude that in order to reduce twin pregnancy rates, performing eSET irrespective of the availability of a good-quality embryo, in all patients <38 years, in the first cycle only is not effective.
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Footnotes |
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4 Present address: Department of Obstetrics and Gynaecology, University Medical Center Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands
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References |
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Baart EB, Martini E, van den Berg I, et al. (2006) Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF. Hum Reprod 21:223–33.
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Submitted on November 10, 2006; resubmitted on January 9, 2007; accepted on February 13, 2007.
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