RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 23 November 2006

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1 RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 23 November 2006 The value of early cleavage (EC) assessment is still being debated. The aim of this prospective study was to examine the predictive value of EC assessment performed exactly 26 h after insemination by IVF or intracytoplasmic sperm injection (ICSI) in a programme of elective single embryo transfer (SET) performed at day 2. If day 2 scoring demonstrated several embryos with high implantation potential, an EC embryo was transferred preferentially. EC was assessed only during normal laboratory hours so that there were two groups: EC assessed, and EC not assessed, the latter being the control. A total of 277 elective SET were performed in women under 37 years undergoing their first IVF or ICSI cycle (mean age 30.5 years, range 21 37). The overall clinical and ongoing pregnancy rates were 40.1% (111/277) and 32.9% (91/277) respectively. Significantly higher overall clinical and ongoing pregnancy rates were obtained after transfer of an EC embryo than a non- EC embryo: 49.4 versus 33.3% (P < 0.05) and 42.4 versus 25.9% (P < 0.02) respectively. However there was no significant difference between the EC assessed and control groups: 40.4 versus 39.3% and 33.2 versus 32.1 respectively. These findings confirm the value of EC assessment for selection of embryos with high implantation potential. Keywords: embryo score, ICSI, IVF, pregnancy rate, single embryo transfer, twin pregnancy Multiple gestation pregnancy is the downside of successful assisted reproduction (Tuppin et al., 1993; Steegers-Theunissen et al., 1998; Blondel and Kaminski, 2002; Land and Evers, 2003; Ombelet et al., 2005; Pinborg, 2005). Transfer of fewer embryos in recent years has resulted in a sharp decrease in the incidence of triplet pregnancies without detriment to the overall outcome of IVF or intracytoplasmic sperm injection (ICSI) (Staessen et al., 1993; Templeton and Morris, 1998; Devreker et al., 1999; Ludwig et al., 2000). However in assisted reproduction centres in Europe, where transfer of two embryos has become standard practice, the risk of twin pregnancy has been estimated at 24% (ESHRE, 2005). Impact of twin pregnancy in terms of maternal, fetal and neonatal complications, as well as socioeconomic implications, has been widely documented in the literature (Dhont, et al., 1999; Blondel et al., 2002; De Sutter et al., 2002; Garceau et al., 2002; Thurin et al., 2006). Elective single embryo transfer (SET) has been recommended as a means of avoiding twin pregnancy (ESHRE, 2000) and has been proposed by some groups (Gerris et al., 1999; Vilska et al., 1999) for several years. Other studies have confirmed the benefits of elective SET (Martikainen et al., 2001; Gardner et al., 2004; Thurin et al., 2004) and have shown that this strategy does not decrease the pregnancy rate, especially when pregnancies obtained after transfer of cryopreserved embryos are taken into account (Tiitinen et al., 2001; Henman et al., 2006). Based on these findings, the authors centre began proposing elective SET for selected women in 2003 and a 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 resulting cumulative delivery rate of 39.5% has been reported (Giorgetti et al., 2006). Successful elective SET depends on selecting the best quality embryo for transfer. The most widely used selection criteria in assisted reproduction centres are day 2 or day 3 embryo morphology and developmental stage (Cummins et al., 1986; Claman et al., 1987; Puissant et al., 1987; Erenus et al., 1991; Staessen et al., 1992; Giorgetti et al., 1995; Ziebe et al., 1997; Van Royen et al., 1999). Recent authors (Scott and Smith, 1998; Tesarik and Greco, 1999; Montag et al., 2001; Zollner et al., 2002; Gianaroli et al., 2003) have used zygote-scoring systems based on assessment of multiple parameters including morphological attributes, location of pronucleus (PN), position or number of nucleolar precursor bodies and presence or absence of cytoplasmic halo. However no conclusion can be drawn from trials using zygote scoring, since two or more embryos have always been transferred at the same time, making it impossible to know which embryo actually implanted. Another parameter that has been shown to have good predictive value for pregnancy is early cleavage (EC) at h after IVF (Edwards et al., 1984; Shoukir et al., 1997) or ICSI (Sakkas et al., 1998; Bos-Mikich et al., 2001; Lundin et al., 2001; Fenwick et al., 2002). Salumets et al. (2003) have proposed the use of EC assessment to select embryos for elective SET. In a retrospective study including both elective and non-elective SET, Van Montfoort et al. (2004) stated that selection for transfer should be based not only on cell number and morphology on the day of transfer but also on EC status. In comparison with zygote scoring, EC assessment is a simple, rapid, reproducible laboratory procedure after both IVF and ICSI. At the authors centre, EC assessment has been used as an adjunct to the day 2 embryo score since The purpose of this study was to evaluate the predictive value of the combination of these two parameters (early cleavage assessment performed at exactly 26 h and day 2 embryo score) in selecting embryos for elective SET. This study included patients who underwent oocyte retrieval at the centre between January 2003 and March The centre has been proposing elective SET to women less than 37 years of age undergoing their first IVF or ICSI cycle since Pituitary desensitization was performed using gonadotrophinreleasing hormone (GnRH) agonist (decapeptyl 3 mg, Ipsen Biotech, France) in 65% of cases and GnRH antagonist (Cetrotide, 3 mg, Serono, France) in 35%. The criteria for injection of agonist and antagonist as well as for triggering of ovulation have been described elsewhere (Roulier et al., 2003). Oocyte retrieval was performed h after human chorionic gonadotrophin (HCG) injection. IVF and ICSI were performed 3 4 h after oocyte retrieval between 11:00 and 14:00 hours. Fertilization was checked 18 to 20 h after insemination or injection. Early cleavage status was assessed at exactly 26 h, between 13:00 and 16:00 hours from Monday to Friday. The time of assessment (26 h after IVF or ICSI) was chosen based on perusal of the literature, which revealed that the most frequent time reported was between 25 and 27 h (Table 1). Cleavage was not assessed during weekends. Depending on whether or not assessment was performed, embryos were divided into two groups, assessed and not assessed. In the assessed group, embryos were classified as either EC or non-ec, according to whether or not they were regularly sized and shaped without any fragmentation. The not assessed embryos were used as the control group. In the control group, the only parameter used to select embryos for transfer was the day 2 embryo score. Table 1. Timing of early cleavage assessment reported in the literature. Publication No. of Procedure Time of EC SET cycles assessment (h) Shoukir et al., IVF 25 No Sakkas et al., ICSI 27 No Bos-Mikich et al., IVF and ICSI No Lundin et al., IVF and ICSI No Sakkas et al., IVF and ICSI No Fenwick et al., IVF No Salumets et al., IVF and ICSI Yes Van Montfoort et al., IVF Yes ICSI Yes Emiliani et al., IVF and ICSI 25 Yes This series 193 IVF and ICSI 26 Yes EC = early cleavage; ICSI = intracytoplasmic sperm injection; SET = single embryo transfer.

3 Day 2 embryo scoring was performed using the centre s scoring system as described previously (Giorgetti et al., 1995). Embryos with a score of 4 (displaying four evenly sized and shaped blastomeres with less than 25% cellular fragments) were considered to have high implantation potential. Multinucleation (Van Royen et al., 2003) was not checked. Embryo transfer was carried out on day 2, 48 to 54 h after oocyte retrieval. Embryos with the highest score were chosen for transfer. SET was performed only if at least one embryo with a score of 4 was obtained for transfer and if at least one additional embryo (score 3 or 4) was available for cryopreservation. In the assessed group, when two embryos with score 4 were obtained, the EC embryo (if available) was transferred preferentially. In the control group, embryos with score 4 were transferred, but it is likely that a proportion of these also exhibited early cleavage. Clinical pregnancy was defined as a βhcg greater than 1000 IU/l or detection of an ovular sac 6 to 7 weeks after transfer. Pregnancies lasting more than 12 weeks after transfer were classified as ongoing. Early pregnancy loss was defined as miscarriage during the first trimester of pregnancy. These parameters were compared between the EC and non-ec transfer groups. Comparison was also made between patients in the assessed and the control groups. The Chi-squared test (qualitative variables) and Student s t-test (quantitative variables) were used to determine differences between groups. A value of P < 0.05 was considered as significant. A total of 277 elective SET were carried out during the study period. Patient characteristics are listed in Table 2. The mean age of women undergoing elective SET with assessed embryos was 30.5 years (range, 21 37). A total of 2715 metaphase II (MII) oocytes were recovered, including PN zygotes (65.9%). The overall clinical and ongoing pregnancy rates were 40.1% (111/277) and 32.9% (91/277) respectively. Cleavage assessment was performed at 26 h in 69.7% of cases (assessed, 193/277) and cleavage assessment was not performed in 30.3% of cases (control, 84/277). There was no difference between the assessed and control groups or between the EC and non-ec groups with regard to age, duration of infertility, aetiology of infertility, ovulation stimulation protocol (long GnRH-agonist versus GnRH-antagonist) and proportion of IVF and ICSI procedures. An EC embryo was transferred in 85 cases (44.0%) and a non-ec embryo in 108 cases (56.0%). The number of oocytes collected (P P < 0.02), MII oocytes ( P < 0.01) and zygotes (P P < 0.05) were significantly higher in patients in whom an EC embryo was observed (n = 85) than was not observed (n = 108) (Table 3). However no difference in these Table 2. Patient characteristics according to type of embryo transferred. Characteristic Assessed group Non-assessed EC Non-EC Total group (control) embryo embryo No. of cycles Female age (years) Mean ± SD 30.6 ± ± ± ± 3.2 Range Duration of infertility (years) Mean ± SD 3.9 ± ± ± ± 2.4 Range Aetiology of infertility Tubal factor (%) 19 (22.4) 24 (22.2) 43 (22.3) 25 (29.8) Male factor (%) 49 (57.6) 62 (57.4) 111 (57.5) 42 (50.0) Endometriosis (%) 8 (9.4) 8 (7.4) 16 (8.3) 5 (5.9) Other (%) 9 (10.6) 14 (13.0) 23 (11.9) 12 (14.3) Protocol GnRH agonist (%) 61 (71.8) 70 (64.8) 131 (67.9) 50 (59.5) GnRH antagonist (%) 24 (28.2) 38 (35.2) 62 (32.1) 34 (40.5) Fertilization procedure IVF cycles (%) 32 (37.6) 43 (39.8) 75 (38.9) 38 (45.2) ICSI cycles (%) 53 (62.4) 65 (60.2) 118 (61.1) 46 (54.8) SD = standard deviation; GnRH = gonadotrophin-releasing hormone; ICSI =.intracytoplasmic sperm injection.

4 Table 3. Comparison of biological and clinical parameters according to the type of embryo transferred. Parameter Assessed group Non-assessed EC embryos Non-EC Total group (control) embryos No. of cycles Oocytes collected Mean ± SD 13.9 ± 7.8 a 12.6 ± 7.0 a 13.1 ± ± 8.7 Range MII oocytes Mean ± SD 10.7 ± 5.8 b 8.9 ± 4.8 b 9.7 ± ± 6.0 Range PN embryos Mean ± SD 6.9 ± 4.0 c 5.8 ± 3.5 c 6.3± ± 4.8 Range Clinical pregnancies 42 (49.4) c 36 (33.3) c 78 (40.4) 33 (39.3) Early pregnancy loss 6 (14.3) 8 (22.2) 14 (17.9) 6 (18.2) Ongoing pregnancies 36 (42.4) d 28 (25.9) d 64 (33.2) 27 (32.1) Values in brackets are percentages; SD = standard deviation; 2PN: two pronucleate. a,b,c,d Values with the same superscript letter are significantly different: P < 0.02, P < 0.01, P < P < 0.02, respectively. parameters was noted between the assessed and control groups. Of all embryos obtained in the assessed group, 28.3% of the ICSI embryos and 15.2% of the IVF embryos exhibited early cleavage. Transfer of a single EC embryo resulted in a significantly higher clinical pregnancy rate and ongoing pregnancy rate than transfer of a single non-ec embryo: 49.4 versus 33.3% (χ 2 = 5.107, P < 0.05) and 42.4 versus 25.9% (χ 2 = 5.792, P < 0.02). A higher early pregnancy loss rate was observed in the non-ec group than in the EC group but this difference was not significant: 22.2% versus 14.3% (χ 2 = 0.289). Clinical pregnancy and ongoing pregnancy rates were not significantly different between the assessed and control groups: 40.4 versus 39.3% and 33.2 versus 32.1% respectively. The clinical pregnancy rate obtained in the present elective SET series was consistent with those reported in the first studies published by teams in Belgium (Gerris et al., 1999) and Finland (Vilska et al., 1999; Martikainen et al., 2001) and confirms the results of the centre s previous study (Giorgetti et al., 2006). The most widely used parameters for selecting embryos for transfer are day 2 or day 3 embryo morphology and developmental stage (Cummins et al., 1986; Claman et al., 1987; Puissant et al., 1987; Erenus et al., 1991; Staessen et al., 1992; Giorgetti et al., 1995; Ziebe et al., 1997; Van Royen et al., 1999). Some patients might benefit from extended culture to day 5 (Jones et al., 1998; Gardner et al., 2000). Two recent reviews emphasized the importance of basing embryo selection on morphological parameters evaluated at different stages of the assisted reproduction cycle (Borini, 2005; Rienzi, 2005). Most data about pronucleus formation and early cleavage pertain to oocytes obtained for ICSI (Payne et al., 1997; Nagy et al., 1998). Studies describing zygote evaluation based on morphological aspects, determination of pronuclearposition or number of nucleolar precursor bodies and presence or absence of cytoplasmic halo have demonstrated the predictive value of zygote scoring for implantation potential. However zygote scoring has two drawbacks: complexity, since it requires examination of several parameters (pronucleus, nucleolar precursor bodies and cytoplasmic halo), and lack of standardization, since several classifications have been described. Scott and Smith (1998) described a five-grade score in a preliminary study and later in a revised system (Scott et al., 2000). During the same period, Tesarik and Greco (1999) proposed a different classification involving six patterns of pronuclear morphology, distinguished according to the number and distribution of precursor bodies. In a subsequent study, the same group (Tesarik et al., 2000) reported that embryos with high implantation potential could be identified by simple, non-invasive examination of pronuclear morphology but only embryos considered to have good morphology were transferred. Despite differences in pronucleus scoring systems, more recent data (Montag et al., 2001; Zollner et al., 2002; Gianaroli et al., 2003) have found that the same number and alignment of precursor bodies in the 2PN embryos are associated with a higher implantation potential compared with embryos deriving from less favourable patterns. However since two or more embryos were always transferred, it is impossible to know which embryo implanted. The first observations relating the time of cleavage to embryo quality were published by Edwards (Edwards et al., 1984). Assessment of early cleavage within 26 h after insemination or injection has been shown to be a good predictor of pregnancy

5 after both IVF (Shoukir et al., 1997) and ICSI (Sakkas et al., 1998). In comparison with zygote scoring, evaluation of cleavage status is a simple, rapid, reproducible laboratory procedure that can be used after both IVF and ICSI. These advantages have been confirmed in several studies (Bos-Mikich et al., 2001; Lundin et al., 2001; Fenwick et al., 2002; Hans et al., 2003). Salumets et al. (2003) proposed use of EC assessment to select embryos for elective SET. In a retrospective study including elective and non-elective SET, Van Montfoort et al. (2004) stated that embryo selection should be based not only on cell number and morphology on the day of transfer but also on cleavage status. In accordance with this recommendation it was decided to use cleavage assessment at 26 h as an adjunct to the usual embryo scoring at 48 h. At the same time, a policy was established of offering elective SET to carefully selected patients. In the centre s elective SET programme, an EC embryo was transferred in 44% of cases and a non-ec embryo in 56% of cases. This proportion is in agreement with a previous study reporting 40.5% and 59.5% respectively (Salumets et al., 2003). The 26-h interval between insemination or injection and cleavage assessment was defined based on perusal of the literature showing the most frequent time was between 25 and 27 h. The fact that cleavage assessment was made at the same time after IVF or ICSI probably accounts for the difference between the early cleavage rates observed after use of the two insemination techniques, 15.2 versus 28.3% respectively. In a recent study in which cleavage assessment was performed h after ICSI and h after IVF (Van Montfoort et al., 2004), no difference was found (39 and 38% respectively). This staggered approach with an additional 2-h delay for assessment after IVF may allow detection of a greater number of EC embryos. These findings confirm that early cleavage was a good predictor of implantation for embryos having the same embryo score. The clinical pregnancy rate for the 85 elective SET carried out with EC embryos was 49.5%. This finding is in agreement with two previous series using EC embryos (Salumets et al., 2003; Van Montfoort et al., 2004), showing a clinical pregnancy rate of 50.0% for 72 transfers and 46.4% for 97 transfers. In this series of elective SET using embryos with high day 2 scores, the clinical pregnancy rate was not significantly different between the assessed and control groups. A study involving logistic regression (Van Montfoort et al., 2004) showed that the ratio between the probability for implantation of EC and non- EC embryos is inversely related to embryo score. In a recent randomized study, a significant and positive correlation between embryo score and early cleavage was observed (Emiliani et al., 2006). Embryo transfer was carried out on day 2, was performed only if an embryo with a score of 4 (4-cell stage with regular blastomere in size and shape, fragmentation involving <25% of embryonic surface) was available. Strict application of day 2 embryo scoring criteria probably accounts for the low predictive value of early cleavage. The non-assessed group served as a control group. Early cleavage was not assessed because the 26 h deadline for assessment occurred outside laboratory hours. This selection criterion was strictly applied since the percentage of embryos in which embryo stage was not evaluated (84/277, 30.4%), corresponds to the expected percentage of Saturdays, Sundays and holidays (31%). The general characteristics of patients (age, duration of infertility, aetiology of infertility, ovulation stimulation protocol, proportion of IVF and ICSI procedures) whose embryos were included in the control group were not different from those whose embryos were included in the assessed group. Furthermore, at the centre, stimulation of ovulation is not programmed, so that timing of follicular retrieval is spread evenly over the 7 days of the week. Based on this similarity, it seems reasonable to assume that embryos in the assessed and control groups presented the same implantation potential. Ability to reliably predict implantation potential has major implications for assisted reproduction. A simple parameter such as early cleavage could be used to optimize the results of elective SET but this would not decrease the number of patients included in twin pregnancy prevention programmes. A more useful application for early cleavage that will require further study would be to predict implantation of embryos with suboptimal day 2 scores. This application could increase the number of patients benefiting from elective SET while maintaining an acceptable pregnancy rate. Another point requiring further study involves the predictive value of early cleavage for early pregnancy loss. In this regard, Lundin et al. (2001) reported a significantly lower early pregnancy loss of EC embryos than for non-ec embryos: 12.1 versus 20.2% (P = 0.036). A similar difference was noted in this study but it was not significant. In conclusion, it has now been established that transfer of a single embryo presenting high implantation potential can achieve satisfactory pregnancy rates in selected patients without a reduction in outcome. This series confirms previous studies showing that early cleavage is a strong indicator of embryo implantation potential. However, in an elective SET programme using only embryos with optimal day 2 embryo scores, early cleavage had no beneficial effect on pregnancy rate. This result is in agreement with a recent randomized study (Emiliani et al., 2006) that showed similar delivery rates for groups with or without assessment of early cleavage. It would be interesting to attempt to assess the predictive value of early cleavage for selecting embryos with suboptimal scores. If successful, this approach could extend the indication for elective SET cycles without impairing pregnancy rates. Blondel B, Kaminski M 2002 Trends in the occurrence, double embryo transfer determinants, and consequences of multiples births. Seminars in Perinatology 26, Blondel B, Kogan MD, Alexander GR et al The impact of the increasing number of multiple births on the rates of preterm birth and low birth weight: an international study. 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