Abstract. Introduction. RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 4 February 2005

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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 4 February 2005 Ethics, legal, social, counselling Belgian legislation and the effect of elective single embryo transfer on IVF outcome Sylvie Gordts (born in 1973) attended the Medical School at the Catholic University of Leuven from 1991 to 1998 and graduated magna cum laude. She undertook her further education as a specialist in obstetrics and gynaecology at the same university. With her special interest in reproductive medicine and genetics she recently joined the Leuven Institute for Fertility and Embryology as a collaborator. Dr Sylvie Gordts S Gordts 1, R Campo 1,4, P Puttemans 1,2,4, I Brosens 1, M Valkenburg 1, J Norre 1, M Renier 1,2, D Coeman 3, S Gordts 1,4,5 1 Leuven Institute for Fertility and Embryology, Tiensevest 168, 3000 Leuven; 2 AZ Waasland, Campus Stadskliniek, Lodewijk De Meesterstraat 5, 9100 Sint Niklaas; 3 AZ Klina, Augusteinslei 100, 2930 Brasschaat; 4 Heilig Hart Ziekenhuis, Naamsestraat 105, 3000 Leuven, Belgium 5 Correspondence: lifeleuven@lifeleuven.be Abstract In order to reduce the number of multiple pregnancies following IVF, the Belgian government agreed to reimburse laboratory expenses for six IVF cycles up to the age of 42 years, in exchange for restriction of the number of embryos replaced. Data on assisted reproduction outcome before and after the introduction of this new legislation were analysed retrospectively in terms of implantation, pregnancy and multiple pregnancy rates. After the introduction of the new law, the percentage of single embryo transfer increased from 14 to 49%. Implantation rates were 25.9 and 23% respectively. There was no difference in the overall pregnancy rate before and after the introduction (36 versus 37%). Twin pregnancies, however, decreased from 19 to 3%. These findings indicate that elective single embryo transfer significantly decreases the twin pregnancy rate without a reduction in the overall pregnancy rate. Keywords: in-vitro fertilization, multiple pregnancy, single embryo transfer 436 Introduction Although IVF offers many couples the possibility to achieve a pregnancy, so far the major iatrogenic complication of assisted reproduction remains the high multiple pregnancy rate, with its own high rate of obstetrical and neonatal complications and developmental consequences (Dhont et al., 1999; Keith and Oleszcuk, 1999; Bergh et al., 2000; Elster, 2000). It is estimated that in the United States, 0.9% of all babies are born as a result of assisted reproduction. The rate of twinning, however, is 22 times higher than in the general population and the rate of triplets and high order pregnancies is 50 times normal (Adamson and Baker, 2004). Data from the Studiecentrum voor Perinatale Epidemiologie (SPE) indicate that in Belgium about 4.6% of children are born as a result of assisted reproduction treatment, i.e. 46% following IVF/intracytoplasmic sperm injection (ICSI), and 54% as a result of ovulation induction or ovarian stimulation with intrauterine insemination. Treatment with IVF/ICSI was responsible for 25% of twins and for 42% of all triplets. Twin pregnancies also have to be considered as a complication of assisted reproduction, in view of the reported 6-fold increase in fetal mortality compared with singleton pregnancies (Luke and Keith, 1992). Because of the high morbidity seen in the surviving children of multiple births, the medical, social and financial consequences are considerable and constitute a major health issue that requires urgent action in order to limit these effects (ESHRE Capri Workshop, 2000). Recent data demonstrate that a reduction in the number of transferred embryos results in a sharp decrease in the multiple pregnancy rate without impairment of the clinical pregnancy rate per transfer (Nijs et al., 1993; Templeton and Morris, 1998b; ESHRE Campus Course Report, 2001).

2 In order to reduce the number of multiple pregnancies, the Belgian government agreed to reimburse the laboratory expenses for six IVF cycles at a rate of 1182 Euro/cycle in exchange for the restriction of the number of embryos replaced. This law became effective 1 July The influence of this legislation upon the outcome of an assisted reproduction programme has been evaluated, before and after its introduction. Materials and methods Belgian legislation After discussion with a group of Belgian opinion leaders in the field of reproductive medicine, the Belgian Minister of Social Affairs proposed that the government should reimburse six IVF cycles in a lifetime, up to a woman s 43rd birthday. This proposition was accepted and became effective on 1 July For all patients younger than 36 years, single embryo transfer (SET) is obligatory at the time of their first IVF attempt. During the second cycle, SET also has to be performed as a first choice, but double embryo transfer (DET) is allowed when only embryos of poor quality are available. From the third cycle on, DET is allowed without restriction (Table 1). For patients between 36 and 39 years, DET can be performed on the first and second occasions. From the third cycle on, replacement of up to three embryos is allowed. For patients older than 39 years, there is no legal limit to the number of transferred embryos. Patients A retrospective analysis of data on IVF outcome in 2002 (n = 454) and 2003 (n = 503) was performed. In all, 397 embryo transfers were performed in 2002 and 408 in For the year 2003, results were analysed for the first and second semester of the year, i.e. before and after the introduction of the new law: group I (n = 216 cycles) for the period 2 January to 30 June 2003 and group II (n = 287 cycles) from 1 July to 31 December Data on assisted reproduction outcome were analysed in terms of implantation, pregnancy and multiple pregnancy rates. A cycle was considered a conception cycle when human chorionic gonadotrophin (HCG) values of >10 miu/ml were obtained on day 12 following embryo transfer and any day thereafter, indicating the beginning of implantation. A clinical pregnancy was defined as a conception cycle with at least one fetal sac with a positive heartbeat. Treatment protocol Patients were treated with a long or short gonadotrophinreleasing hormone agonist (GnRHa) protocol using buserelin (nasal spray, µg/day; Suprefact, Hoechst, Frankfurt, Germany) starting in the mid-luteal phase of the cycle preceding the IVF attempt for a period of at least 2 weeks in the long protocol, or starting on the first day of menstruation following the intake of a contraceptive pill for 2 3 weeks in the short protocol. Hormonal stimulation was performed with urinary gonadotrophins (Menopur; Ferring, Limhamn, Sweden) or recombinant FSH (Puregon; Organon, Oss, The Netherlands). When follicles reached a diameter of 18 mm, 10,000 IU HCG (Pregnyl; Organon) was given and ultrasound guided oocyte aspiration was performed 35 h later. Embryo transfer was performed 2 or 3 days after insemination using a Cook soft catheter (K-soft 5100; Cook, Brisbane, Australia). During 2002 and the first half of the year 2003, two or three embryos were replaced routinely; from 1 July 2003 on, embryo transfer was performed according to the restrictions of the new legislation. In all cycles, luteal phase was supported with mg of micronized natural progesterone (Utrogestan; Besins International, Brussels, Belgium), administered vaginally. Blood samples were taken on day 12 following embryo transfer, for analysis of serum oestradiol, progesterone and HCG concentrations. Morphological selection criteria Morphological selection criteria were based upon the number of blastomeres present, the regularity of blastomeres, the percentage of fragmentation and the absence of multinuclei (Van Royen et al., 1999). Furthermore, pronuclear zygote morphology, evaluating the localization of the nucleoli and pronuclei and their polarity, was incorporated into the routine assessment for the selection of the best embryo (Gianaroli et al., 2003). Statistics Different groups were compared using chi-squared tests and two-tailed t-tests with P < 0.05 as the limit of significance. Since the study relied on the available number of patients, a sample calculation and power analysis was not done. Results Analysis of the different groups showed no statistical difference for the mean age; the causes of infertility were also comparable between groups (Table 2). The cancellation rate, the number of oocytes retrieved and the mean number of embryos available were also similar (Table 3). Patients in group I aged under 35 years showed a conception rate of 42% from 132 cycles and 120 embryo transfers. The corresponding figures for these patients in the 2000 group and group I taken together were 41%, 422 and 401 respectively. The figures were similar for patients in this age group in group II, i.e. 43%, 180 and 167 respectively. The overall conception rate was 36% in 2002 and 38 and 37% in groups I and II respectively, with an ongoing pregnancy rate of 29% in all three groups. Implantation rate and miscarriage rate were not significantly different (26, 21, 23 and 19, 23 and 21% in 2002, group I and group II respectively). Twin 437

3 Table 1. Belgian legislation: strategy of embryo transfer in order to reduce multiple pregnancy rate. SET = single embryo transfer; DET = double embryo transfer; TET = triple embryo transfer. Maternal age (years) Attempt number Strategy 35 1 SET 2 SET (if high quality embryos are available) 2 DET (if only poor quality embryos are available) 3 6 DET a >35 to 39 1 and 2 DET a 3 6 TET a >39 to No limit to number of transferred embryos a Maximum. Table 2. Causes of infertility in the three groups studied. Causes of infertility 2002 Group I Group II Female (%) 23 (104) 24 (46) 25 (67) Male (%) 46 (207) 32 (62) 40 (108) Mixed female/male (%) 13 (57) 19 (37) 15 (39) Unexplained (%) 19 (86) 25 (47) 20 (55) Mean age (years) 33.4 ± ± ± 4.4 Values in parentheses are numbers of patients. Group I: period 1 January to 30 June Group II: period 1 July to 31 December Table 3. Results of IVF cycles in the three groups studied Group I Group II Cycles (n) Cancellation rate (%) 12.5 (57) 11.0 (24) 6.0 (18) Mean no. oocytes 10 ± ± ± 6.2 Mean no. embryos 5.6 ± ± ± 4.4 Mean no. embryos transferred Conception rate (%) 36 (142) 38 (64) 37 (52) Implantation rate (%) 26 (206/796) 21 (70/331) 24 (91/383) Ongoing pregnancy rate (%) 29 (115) 29 (49) 29 (69) Twins (%) 19 (28) 9 (6) 3 (3) Triplets (%) 2 (1.4) 0 0 Values in parentheses are relevant numbers of cycles or embryo transfers. Group I period: 1 January to 30 June Group II period: 1 July to 31 December

4 pregnancies decreased from 19% in 2002 to only 3% in group II (P = 0.007). There were two triplet pregnancies in 2002 and none in 2003 (both groups). The mean number of embryos replaced decreased from 2.0 in 2002 to 1.6 in group II in The percentage of SET increased from 14% in 2002 to 49% in the second half of 2003 (Figure 1). After SET, the pregnancy rates for 2002 in group I and group II were 25, 22 and 37.3% respectively. In 2002 and during the first half of 2003, SET was usually performed if only one embryo was available for transfer, explaining the lower pregnancy rates. The pregnancy rates for a day 2 embryo transfer were 39, 37 and 39%, and for a day 3 embryo transfer 35, 42 and 34% (2002, group I and group II respectively). The distribution of embryo transfers on day 2 versus day 3 was equal in the different groups. In 94 patients in group II, younger than 36 years and having more than one embryo available, an elective SET was performed, resulting in a pregnancy rate of 41%. Depending upon the quality of the replaced embryo, the pregnancy rates varied between 46% for an embryo of grade I quality, 35% for a grade II (intermediate) and 22% for a grade III (poor quality) embryo (Table 4). DET was performed in 288 cycles in 2002 (72.5% of all transfers), resulting in a pregnancy rate of 40%, including 21% twin pregnancies and one triplet pregnancy. In group I, 120 cycles with DET (71%) resulted in a pregnancy rate of 39% with 10% twins; in group II, 102 DET cycles (42%) resulted in a pregnancy rate of 38% with only 5% twins. The mean ages of the patients receiving DET were 33, 32 and 35 years in 2002, group I and group II respectively. In 48 cycles in 2002, three embryos were transferred, resulting in 14 pregnancies (29%) with three twins (21%) and one triplet (7%), versus 19 transfers of three embryos resulting in five pregnancies (26%), including one twin pregnancy (20%) in group II. Discussion The need to prevent multiple pregnancies is widely accepted, although transfer of several embryos is still standard policy in many IVF centres. Competition between different centres and the fear of compromising pregnancy rates by decreasing the numbers of embryos transferred deterred many centres and clinicians from reducing the number of replaced embryos. Their fear was inspired by the reported poor pregnancy rates when only one embryo was available. Following reports that triplets can be avoided by replacing two embryos (Staessen et al., 1995; Templeton and Morris, 1998), the issue of SET has now been addressed by several authors, demonstrating that by replacing only one embryo, twins can be avoided without impairment of the mean pregnancy rate (Martikainen et al., 2001; Gerris et al., 2002; Tiitinen et al., 2003). In selected groups of patients, increasing the number of embryos transferred does not improve the overall pregnancy rate any further, but only results in a higher multiple pregnancy rate (Martin and Welch, 1998). The study by Land and Evers (2004) also showed that a given pregnancy rate in a country does not reflect the mean number of replaced embryos and that those countries with a high mean number of replaced embryos frequently have a low pregnancy rate. They state that only toplevel clinics, where treatment efficacy is guaranteed, are able to decrease the number of embryos transferred without compromising their pregnancy rate. SET can never be mandatory in all patients, but the percentage of elective SET performed by a particular assisted reproduction treatment centre does reflect its quality. In the study reported here, the drop in the twin pregnancy rate to 9.4% in group I of 2003 can be seen as a bias of the traditional transfer policy in view of the upcoming law during the months preceding 1 July Although the percentages of SET in 2002 and the first half of 2003 were comparable (14 and 16% respectively), there was a small shift towards more SET in younger patients than in patients who only had one embryo to transfer. This is, however, not statistically significant. Laboratory experience and embryo selection are of the utmost importance. It is generally accepted that the number and shape Figure 1. Influence of the increase in percentage of single embryo transfers (14% in 2002, 16% first half of 2003, up to 49% in second half of 2003) on mean conception rate and twin pregnancy rate. Table 4. Group II: outcome of single embryo transfers related to embryo quality. Embryo quality Pregnancy rate/embryo transfer (%) Grade I 46 (22/48) Grade II 35 (18/52) Grade III 22 (4/18) Values in parentheses are number of embryo transfers. 439

5 440 of blastomeres, the percentage of fragmentation and the absence of multinuclear blastomeres (Van Royen et al., 2001; Land and Evers, 2004) are important parameters for the selection of those embryos that have the highest potential of implantation. The scoring of pronuclear zygote morphology was reported to correlate well with the proportion of chromosomally normal embryos (Gianaroli et al., 2003). The characteristics of patients and embryos with high implantation potential, however, are still poorly defined and further research on embryo morphology, genetic evaluation and metabolic processes is needed to allow an even more accurate selection of the best quality embryo. Transfer of a single blastocyst has been proposed as a method of choice resulting in higher pregnancy rates compared with day 2 or day 3 transfers (Gardner and Lane, 2003). Conflicting data, however, have been reported by others (Coskun et al., 2000; Rienzi et al., 2002; Kolibianakis et al., 2004; Utsunomiya et al., 2004). Furthermore, blastocyst transfer can be held responsible for the higher rate of monozygotic twinning (Unger et al., 2004; Wright et al., 2004). Compared with normal conception, monozygotic twinning is increased after assisted reproduction, with the occurrence of triplet and even quadruplet pregnancies after transfer of a single embryo (Rísquez et al., 2004). Although several mechanisms have been suggested as a possible cause, for example maternal age, zona pellucida handling and ovarian stimulation, in-vitro culture conditions (Cassuto et al., 2003) and the prolonged cultures have generally been held responsible for the higher rate after blastocyst transfer (Alikani et al., 2003). A prospective health economy study (Gerris et al., 2004) has shown that the total cost, maternal and neonatal, per child up to 3 months of age and born after a first IVF/ICSI treatment, is substantially higher when two embryos are transferred (DET) than when a single embryo transfer (SET) is applied. The ESHRE campus course report (ESHRE Campus Course Report, 2001) considers a twin pregnancy rate of >25% unacceptable and recommends reducing this incidence to 10% or lower. A pregnancy rate of 30% is considered as very acceptable and is still higher than the natural cycle conception rate. With the introduction of this IVF legislation in Belgium these recommendations could be achieved. As such, all centres were obliged to follow a strict policy concerning the number of embryos replaced, in order to eliminate the competition between centres traditionally based upon the number of replaced embryos and forcing them to elaborate on improving the quality of the embryos and refining the laboratory handling as the preferred way to obtain high pregnancy rates. Although the value of this study is limited because it is a retrospective analysis reflecting first experience, with a short duration of follow-up not enabling inclusion of the pregnancy outcome, the data confirm the Scandinavian experience (Martikainen et al., 2001; Tiitinen et al., 2003) and the findings of others (Gerris et al., 2002; De Neubourg and Gerris, 2003). While in 2002 only 14% of all transfers were SET, this percentage increased to 49% in group II in In the present IVF/ICSI programme, no statistically significant difference in pregnancy rate was observed on the one hand, but there was a significant reduction of the twin pregnancy rate from 19 to 3% on the other (Figure 1). Already in the first half of 2003 (group I), the reduction in the mean number of replaced embryos and the slight increase in elective SET resulted in a fall in the twin pregnancy rate and the absence of triplets. No difference in pregnancy rate was noted between day 2 or day 3 transfer. As the percentage of SET increases, more embryos will become available for freezing. Since the follow-up period was too short, an adequate evaluation of the cumulative pregnancy rate in this series could not yet be made, but as mentioned in other papers, an increased cumulative pregnancy rate per cycle can be expected (Tiitinen et al., 2004). As the general public is unaware of the risks of multiple pregnancies and the consequences of pre-term delivery, most infertile patients with a strong desire for a pregnancy and a child underestimate these risks. Good counselling should include realistic information, not merely on the risks of twin gestation but also on the long-term consequences of multiple births. References Adamson D, Baker V 2004 Multiple births from assisted reproductive technologies: a challenge that must be met. Fertility and Sterility 81, , discussion. Alikani M, Cekleniak NA, Walters E, Cohen J 2003 Monozygotic twinning following assisted conception: an analysis of 81 consecutive cases. Human Reproduction 18, Bergh T, Ericson A, Hillensjö T et al Deliveries and children born after in-vitro fertilization in Sweden : a retrospective cohort study. 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6 or a day 5 transfer? Human Reproduction 19, Land JA, Evers JL 2004 What is the most relevant standard of success in assisted reproduction? Defining outcome in assisted reproduction: a Gordian knot of safety, efficacy and quality. Human Reproduction 19, Luke B, Keith LG 1992 The contribution of singletons, twins and triplets to low birth weight, infant mortality and handicap in the United States. Journal of Reproductive Medicine 37, Martikainen H, Tiitinen A, Tomas C et al One versus two embryo transfer after IVF and ICSI: a randomized study. Human Reproduction 16, Martin PM, Welch G 1998 Probabilities for singleton and multiple pregnancies after in vitro fertilization. Fertility and Sterility 70, Rienzi L, Ubaldi F, Iacobelli M et al Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Human Reproduction 17, Rísquez F, Gil M, D Ommar G et al Monochorionic triplets after single embryo transfer. Reproductive BioMedicine Online 9, Nijs M, Geerts L, van Roosendaal E et al Prevention of multiple pregnancies in an in vitro fertilization program. Fertility and Sterility 59, Staessen C, Nagy ZP, Liu J et al One year s experience with elective transfer of two good quality embryos in the human invitro fertilization and intracytoplasmic sperm injection programmes. Human Reproduction 10, Templeton A, Morris JK 1998 Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. New England Journal of Medicine 339, Tiitinen A, Unkila-Kallio L, Halttunen M et al Impact of elective single embryo transfer on the twin pregnancy rate. Human Reproduction 18, Tiitinen A, Hyden-Granskog C, Gissler M 2004 What is the most relevant standard of success in assisted reproduction? The value of cryopreservation on cumulative pregnancy rates per single oocyte retrieval should not be forgotten. Human Reproduction 19, Unger S, Hoopmann M, Bald R et al Monozygotic triplets and monozygotic twins after ICSI and transfer of two blastocysts: case report. Human Reproduction 19, Utsunomiya T, Ito H, Nagaki M, Sato J 2004 A prospective, randomized study: day 3 versus hatching blastocyst stage. Human Reproduction 19, Van Royen E, Mangelschots K, De Neubourg D et al Characterization of a top quality embryo, a step towards single embryo transfer. Human Reproduction 14, Van Royen E, Mangelschots K, De Neubourg D et al Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model. Human Reproduction 16, Wright V, Schieve LA, Vahratian A, Reynolds MA 2004 Monozygotic twinning associated with day 5 embryo transfer in pregnancies conceived after IVF. Human Reproduction 19, Received 22 November 2004; refereed 15 December 2004; accepted 13 January