Article Relationship between even early cleavage and day 2 embryo score and assessment of their predictive value for pregnancy

Transcription

1 RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 22 January 27 Article Relationship between even early cleavage and day 2 embryo score and assessment of their predictive value for pregnancy Philippe Terriou was born in France in 196. He received degrees in laboratory biology and human genetics from the University of Marseilles and a degree in reproduction and development from the University of Montpellier (France). He specialized in Assisted Reproduction Technology at the Hôpital de la Conception of Marseilles in 1989 and he has been working since 1991 at the Institut de Médecine de la Reproduction, a private centre for reproductive medicine in Marseilles. His specific areas of interest include intracytoplasmic sperm injection, embryo quality assessment and viral risk in assisted reproduction technology. Dr Philippe Terriou P Terriou 1, C Giorgetti, E Hans, J Salzmann, O Charles, L Cignetti, C Avon, R Roulier Institut de Médecine de la Reproduction, Marseille, France 1 Correspondence: pterriou@aol.com Abstract The aim of this study was, firstly, to define the different patterns of early cleavage (EC) observed at 26 h after either IVF or intracytoplasmic sperm injection (ICSI) and, secondly, to assess the predictive value of one of these patterns, even EC (EEC), on pregnancy rate in combination with day 2 embryo score. In the first part of the study, the relationship between three different EC patterns (EEC, uneven EC and EC with fragmentation of the day 2 embryo) and embryo morphology was determined. EEC was shown to be strongly associated with good embryo morphology. In the second part of the study, it was shown that EEC used in combination with embryo score improved selection of embryos for transfer. The presence of EEC significantly (P <.1) enhanced mean implantation rate in all transfer categories involving identically scored embryos, in both compulsory single embryo transfers and elective single embryo transfers. Multivariate analysis demonstrated that EEC and embryo score had strong complementary predictive value for pregnancy. Based on these findings, it was concluded that even though they are associated, EEC and embryo score could be combined to increase pregnancy rate, especially in elective single embryo transfer programmes. Keywords: early cleavage, embryo score, even early cleavage, single embryo transfer Introduction 294 Identification of embryos with the highest implantation potential is a crucial challenge in assisted reproduction centres. This ability is necessary to optimize success rates and avoid multiple pregnancies by minimizing the number of embryos transferred. Since the first analysis of the factors influencing the success of IVF was performed (Edwards et al., 1984), which demonstrated the influence of embryo quality on pregnancy rate, many authors have studied the influence of embryo morphology (Hill et al., 1989; Scott t et al., 1991), cleavage stage at transfer time (Testard, 1986), or both (Cummins et al., 1986; Claman et al., 1987; Puissant et al., 1987; Erenus et al., 1991; Staessens et al., 1992; Steer et al., 1992; Monks et al., 1993; Shulman et al., 1993) on pregnancy rate after IVF embryo transfer. More recently, it has also been demonstrated that embryo quality is related to euploidy (Baltaci et al., 26) and has a role in predicting early pregnancy loss (Hourvitz et al., 26). In a first series of 957 non-elective single embryo transfers, the predictive value of these embryo parameters for successful pregnancy was studied, and a convenient 4-point embryo score was defined (Giorgetti et al., 1995). In a subsequent study including, multiple and single embryo transfers, it was demonstrated that embryo score was a better predictor of pregnancy than female age and number of transferred embryos (Terriou et al., 21). More recently, early cleavage (EC) has been proposed as a new parameter for evaluating embryo quality and viability after IVF 27 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 (Shoukir et al., 1997) and intracytoplasmic sperm injection (ICSI) (Sakkas et al., 1998). In both these studies, EC was used as the primary criterion in selection of embryos for transfer, and embryo morphology was only a second-choice criterion. Previous data have also demonstrated that embryos showing EC more often develop to the blastocyst stage (Fenwick et al., 22; Neuber et al., 23). However, previous studies do not provide a precise definition of early cleavage in terms of morphological pattern or acute timing after IVF/ICSI. Further study is needed to assess the relationship between EC and day- 2 embryo morphology and to evaluate the respective predictive value of these two embryo parameters for pregnancy. The present study was conducted in two parts. The aim of the first part was to define the different patterns of EC observed after IVF/ICSI and to study their influence on subsequent embryo morphology. Since one of these patterns, even early cleavage (EEC), was found to be significantly (P <.1) associated with good embryo morphology, the aim of the second part of this study was to assess the value of EEC as a secondary parameter in combination with embryo score for predicting implantation after transfer. Embryo score was used as the primary parameter for embryo assessment because of its proven efficacy in the previous series of, embryo transfers. Given the current priority of minimizing the number of embryo transfers at assisted reproduction centres, it was decided to conduct this study in both elective single embryo transfer and multiple embryo transfer groups. Materials and methods Ovarian stimulation All women underwent hypopituitary desensitization using either gonadotrophin-releasing hormone (GnRH) agonist (Decapeptyl; Ipsen-Biotech, France) or GnRH antagonist (Cétrotide; Serono, France). Ovarian stimulation was performed using recombinant- FSH (Gonal-F; Serono or Puregon; Organon, France), human menopausal gonadotrophin (Menopur, Ferring, France) or urinary FSH (Fostimon; Genevrier, France). Ovulation was induced using either recombinant (Ovitrelle; Serono, France) or urinary human chorionic gonadotrophin (HCG; Endo; Organon, France). Oocyte retrieval was performed 36 h after HCG injection. IVF or ICSI was performed between 11: a.m. and 2: p.m. in all cases. Fertilization was checked at 18 2 h and again at exactly 26 h after insemination/injection. Early cleavage assessment Fertilized oocytes checked at 26 h were classified into one of three groups: pronuclear stage, syngamy stage (defined as being no longer pronuclear but not yet blastomeres) and early cleavage (EC). The EC group was further divided into three subgroups: even EC, which comprised embryos with two regularly sized and shaped blastomeres with no fragmentation (EEC); uneven EC, which comprised embryos with two blastomeres of unequal size or shape with no fragmentation; and EC with fragmentation, which comprised embryos with blastomeres showing fragmentation. The relationship between the groups/subgroups and day 2 embryo morphology (as assessed by embryo score, see below) was studied. The mean number of EEC embryos transferred was obtained by dividing the number of EEC embryos transferred by the total number of transferred embryos. Embryo quality assessment Embryo quality was assessed 48 h after oocyte retrieval using the 4-point embryo score described elsewhere (Giorgetti et al., 1995). All cleaved embryos were given 1 point and an additional point was added for each of the following features: absence of fragmentation (or fragmentation involving <25% of embryonic surface), absence of irregularities in blastomere size or shape, 4-cell stage (Table 1). Cumulative embryo score was obtained by adding the individual embryo scores of all transferred embryos (Terriou et al., 21). Mean embryo score was obtained by dividing cumulative embryo score by the total number of transferred embryos. Transfer policy Elective single embryo transfer was proposed before the beginning of ovarian stimulation to couples in which the female partner was younger than 37 years, and who were attempting their first IVF or ICSI cycle. It was performed in consenting couples only if two 3- or 4-point scored embryos were available for transfer. In all cases, embryos with the highest scores were transferred. When two embryos had identical scores, the EEC embryo (if available) was selected for transfer and the other was frozen. Women were treated during their luteal phase by local administration of progesterone (Utrogestan; Besins-Iscovesco, France) at a dose of mg/day for 1 month. Pregnancy was defined as ultrasonography findings showing at least one fetal heart beat 5 weeks after transfer. Implantation rate was defined by the number of fetal heart beats per transferred embryo. Table 1. Calculation of the 4-point embryo score (see text). Feature Score Cleavage Cleaved embryos 1 Uncleaved embryos Division stage 4-cell embryos 1 Other division stages Irregular cells (size and/or shape) Absence 1 Presence Fragmentation (>25% of embryonic surface) Absence 1 Presence Total -, 1-, 2-, 3-, or 4-point embryo 295

3 Statistical analysis The Pearson chi-squared test was used to compare percentages by pairs. The chi-squared test was used to assess the presence of a linear trend in the relationship, i.e. to demonstrate that the frequency of occurrence increases between groups. Multivariate analysis was performed using a forward stepwise logistic regression model. Variables significantly associated in univariate analysis (defined as P <.2) were entered. Calibration was assessed using the Hosmer Lemeshow goodness-of-fit test. Results From May 21 to October 25, EC was assessed in 296 embryo transfers, including 1478 ICSI cycles (71%) and 618 IVF cycles (29%). EC was observed only in ICSI cycles at the beginning of this study. Single embryo transfer (SET) was performed in 371 cycles, including 247 (12%) compulsory SET performed because only one embryo was available and 124 (6%) elective SET performed to avoid multiple pregnancy. Two embryos were transferred in 1146 cycles (55%), three embryos in 522 (25%) and four embryos in 57 (3%). The mean number of transferred embryos per cycle was 2.1. The total number of fertilized oocytes in the first part of this study was 1698 after ICSI and 1434 after IVF. At 26 h, ICSIfertilized oocytes were more often cleaved than IVF-fertilized oocytes (36 versus 19%; P <.1; Figure 1). Regardless of insemination technique, embryos showing even EC developed to 3- or 4-point embryos ( good embryos ) more often than embryos showing uneven EC and EC with fragmentation (respectively, 69 versus 41 and 23%, P <.1 in ICSI cycles and 53 versus 35 and 19%, P <.1 in IVF cycles) (Figure 2). Presence of EEC was also associated with good embryo score more often than with pronuclei or syngamy stages (P <.1). Based on the strong association of EEC with better day 2 embryo morphology observed in the first part of this study, EEC embryos were preferentially chosen for transfer in the second part of the study whenever two identically scored embryos were available. In order to assess the extra predictive value added to embryo score by EEC, homogeneous transfers in which all transferred embryos had identical scores were studied (Figure 3). An increase in implantation rate was observed with increasing embryo score (1-point to 4-point; 8 to 27%, P <.1). In addition, a significant increase in implantation rate within each embryo category was observed whenever at least one EEC embryo was transferred (8 versus 14%, 14 versus 17%, 17 versus 22% and 27 versus 29 % for 1-, 2-, 3- and 4-point embryos respectively; P <.1). The influence of EEC on implantation rate was assessed in a series of 371 single embryo transfers, including 247 compulsory SET and 124 elective SET (Figure 4). EEC significantly increased implantation rate in both types of SET (P <.1). Multivariate analysis of the predictive value for pregnancy was performed for the following parameters: female age, type of infertility (female, male, mixed or unexplained), duration of infertility, year at recruitment, number of retrieved oocytes, oocyte maturity rate, fertilization rate, number of transferred embryos, mean embryo score of transferred embryos and mean number of EEC embryos transferred. In order of decreasing odds ratio, parameters displaying the best predictive value were: number of transferred embryos, mean number of EEC embryos transferred, mean embryo score and female age (Table 2) % pronuclei Syngamy Early cleavage ICSI IVF Figure 1. Distribution of two pronuclei, syngamy and early cleavage stages in zygotes and embryos observed 26 h after intracytoplasmic sperm injection (ICSI) and IVF. Early cleavage was more common in embryos obtained by ICSI than IVF (36 versus 19%, P <.1). % Even Uneven EC with Syngamy 2-pronuclei EC EC fragmentation Figure 2. Percentages of 3- or 4-point scored embryos, i.e. good day 2 embryos, at two pronuclei, syngamy and early cleavage (EC) stages observed 26 h after ICSI (shaded bars) and IVF (non-shaded bars). EC was divided into three subgroups: even EC, uneven EC, and EC with fragmentation. Even EC was associated with good embryo quality more often than other stages for embryos obtained by either IVF or ICSI (P <.1).

4 Implantation rate (%) No EEC 1 EEC Score Figure 3. Implantation rates (%) observed in homogeneous embryo transfer groups, including identically scored embryos. Implantation rate was higher in transfers involving at least one even early cleavage (EEC) embryo than in transfers involving no EEC embryo (P <.1) Implantation rate (%) No EEC EEC 12 cset eset Figure 4. Implantation rates after compulsory single embryo transfers (cset) in which only one embryo was available for transfer and in elective single embryo transfers (eset) in which only one embryo was transferred to avoid multiple pregnancies. Implantation rate was higher in the eset group than in the cset group (P <.1). Implantation rate was higher in cases of even early cleavage (EEC) embryos in both the cset and eset groups (P <.1). 48 Table 2. Predictive value of parameters for pregnancy in decreasing order of odds ratio. Parameter Odds ratio (95% confi dence interval) No. of transferred embryos 1.81 ( ) Mean no. of transferred embryos with EEC 1.65 ( ) Mean embryo score of transferred embryos 1.45 ( ) Female age.94 (.93.96) EEC = even early cleavage. Discussion Recent review articles (Borini et al., 25; Rienzi et al., 25) have underlined the importance of a two-step approach to embryo selection for transfer, namely day-1 zygote and day-2 embryo morphology. Day-1 development may be assessed either by observing pronuclear morphology or EC and day 2 morphology by assessing the number and morphology of blastomeres and presence of fragmentation. The purpose of this study was to propose a convenient, standardized, efficient method of embryo selection combining day-2 embryo morphology and EC assessment. Day-2 embryo assessment was performed using an embryo score shown to be a better predictor of pregnancy than female age and number of transferred embryos (Terriou et al., 21). Assessment of EC (Shoukir et al., 1997) was chosen because it is an easier, more readily standardized method than assessment of pronuclear pattern (Tesarik and Greco, 1999; Scott t et al., 2; Wittemer et al., 2). Day-2 embryo morphology was used as the primary criterion because of its well-known relevance and widespread use by most IVF teams throughout the world. In the present study, EEC was assessed as a second-choice criterion in combination with embryo score. In the first smaller studies reported in the literature, EC was used as the primary criterion (Shoukir et al., 1997; Sakkas et al., 1998). In an interesting study, Van Montfoort et al. (24) demonstrated that EC in combination with the modified cumulative embryo score provides additional information. However, that study presented several drawbacks, e.g. relative complexity of the modified cumulative embryo score, non-standardized timing of EC observation (22 26 h after IVF versus h after ICSI), and lack of a detailed description of EC patterns. By precisely timing assessment of EC in the present study, it was possible to define three simple patterns: even EC, uneven EC and EC with fragmentation. This strategy makes EC assessment much more convenient and standardized. Lastly, Emiliani et al. (26) demonstrated that the pregnancy rate observed in patients for whom EC was assessed was not lower than the pregnancy rate observed in patients for whom EC was not assessed. A possible negative effect of the time embryos remain out of ideal conditions can thus be excluded. 297

5 298 Distribution of the pronuclei, syngamy and EC stages was different after IVF and ICSI cycles. This is probably because observation was performed strictly 26 h after fertilization in both IVF and ICSI cycles. Indeed, Nagy et al. (1994) stated that fertilization events take place sooner after ICSI with a lag time of 2 4 h for IVF. These results are consistent with this claim, and suggest that delaying assessment of IVF zygotes by 2 h would probably have led to a more similar distribution of zygote and embryo stages in IVF and ICSI cycles. One of the strengths of the present study is its large size (296 cases) which allowed the study of homogeneous transfers, compulsory single embryo transfers, elective single embryo transfers and multivariate analysis. Since only 618 IVF transfers were included in this study, separating ICSI and IVF cycles would have automatically diminished the value of all data that need many cycles to be relevant. It was thus decided to present several of the figures and tables mixing IVF and ICSI results. The detailed observations in this study indicated that EC might display different morphological patterns that are unequally associated with day-2 embryo morphology in both IVF and ICSI cycles (Figure 2). The EEC pattern was associated with high embryo scores significantly (P <.1) more often than uneven EC and EC with fragmentation. Based on these findings, it was decided to preferentially transfer EEC embryos whenever two identically scored embryos were available. Restricting transfer in this way demonstrated that EEC added significant (P <.1) predictive value to embryo score in all homogeneous transfer categories (Figure 3). Transfer of one EEC embryo significantly increased implantation rate (Figure 4) in both the elective SET group and the compulsory SET group (respectively 48 versus 35% and 16 versus 12%; P <.1). The implications of this finding were particularly great in the elective SET group, since transfer of one EEC embryo was possible in 5% of patients. Pregnancy rate in this group would probably have decreased if a random single embryo had been transferred. These findings confirm the predictive value of EC on the viability of human embryos in elective SET procedures (Salumets et al., 23; Van Montfoort t et al., 24; Giorgetti et al., 27). Multivariate analysis of the predictive value for pregnancy was performed for the following parameters: female age, type of infertility, duration of infertility, year at recruitment, number of retrieved follicles, oocyte maturity rate, fertilization rate, number of transferred embryos, mean embryo score of transferred embryos and mean number of EEC embryos transferred. According to odds ratios in decreasing order, the best predictors were number of transferred embryos, mean number of EEC transferred embryos, mean embryo score of transferred embryos and female age (Table 2). These results are of particular importance in light of the first part of this study demonstrating that EEC and embryo score are associated. Indeed, the multivariate analysis in the second part shows that these two embryo parameters have additive predictive value for pregnancy. In conclusion, the findings of the present study indicate that EEC is a relevant, convenient embryo parameter that significantly enhances prediction of implantation potential when used in combination with embryo score. Since observation of EEC at a precise moment is easy to perform and EEC is a simply and clearly defined pattern of EC, this technique would provide a standardized method of embryo quality assessment that may be widely used in assisted reproduction centres, especially in those performing elective SET. References Baltaci V, Satiroglu H, Kabukcu C et al. 26 Relationship between embryo quality and aneuploidies. Reproductive BioMedicine Online 12, Borini A, Lagalla C, Cattoli M et al. 25 Predictive factors for embryo implantation potential. Reproductive BioMedicine Online, Claman P, Armant DR, Seibel MM et al The impact of embryo quality on implantation and the establishment of viable pregnancies. Journal of in Vitro Fertilization and Embryo Transfer 4, Cummins JM, Breen TM, Harrison KL et al A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. Journal of in Vitro Fertilization and Embryo Transfer 3, Edwards RG, Fishel SB, Cohen J et al Factors influencing the success of in vitro fertilization for alleviating human infertility. Journal of in Vitro Fertilization and Embryo Transfer 1, Emiliani S, Fasano G, Vandamme B et al. 26 Impact of the assessment of early cleavage in a single embryo transfer policy. Reproductive BioMedicine Online 13, Erenus M, Zouves C, Rajamahendran P et al The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertility and Sterility 56, Fenwick J, Platteau P, Murdoch AP, Herbert M 22 Time from insemination to first cleavage predicts development competence of human preimplantation embryos in vitro. Human Reproduction 17, Giorgetti C, Hans E, Terriou P et al. 27 Early cleavage: an additional predictor of high implantation rate following elective single embryo transfer. Reproductive BioMedicine Online 14, Giorgetti C, Terriou P, Auquier P et al Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Human Reproduction, 1 4. Hill GA, Freeman M, Bastias MC et al The influence of oocyte maturity and embryo quality on pregnancy rate in a program for in vitro fertilization embryo transfer. Fertility and Sterility 52, Hourvitz A, Lerner-Geva L, Elizur SE et al. 26 Role of embryo quality in predicting early pregnancy loss following assisted reproductive technology. Reproductive BioMedicine Online 13, Monks NJ, Turner K, Hooper MA et al Development of embryos from natural cycle in-vitro fertilization: impact of medium type and female infertility factors. Human Reproduction 8, Nagy ZP, Liu J, Joris H et al Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Human Reproduction 9, Neuber E, Rinaudo P, Trimarchi JR, Sakkas D 23 Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development. Human Reproduction 18, Puissant F, Van Rysselberge M, Barlow P et al Embryo scoring as a prognostic tool in IVF treatment. Human Reproduction 2, Rienzi L, Ubaldi F, Iacobelli M et al. 25 Significance of morphological attributes of the early embryo. Reproductive BioMedicine Online, Sakkas D, Shoukir Y, Chardonnens D et al Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm

6 injection as an indicator of embryo viability. Human Reproduction 13, Salumets A, Hydén-Granskog C, Mäkinen S et al. 23 Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures. Human Reproduction 18, Scott L, Alvero R, Leondires M, Miller B 2 The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Human Reproduction 15, Scott RT, Hofmann GE, Veeck LL et al Embryo quality and pregnancy rates in patients attempting pregnancy through in vitro fertilization. Fertility and Sterility 55, Shoukir Y, Campana A, Farley F, Sakkas D 1997 Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Human Reproduction 12, Shulman A, Ben-Nun I, Ghetler Y et al Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertility and Sterility 6, Staessens C, Camus M, Bollen N et al The relationship between embryo quality and the occurrence of multiple pregnancies. Fertility and Sterility 57, Steer CV, Mills CL, Tan SL et al The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Human Reproduction 7, Terriou P, Sapin C, Giorgetti C et al. 21 Embryo score is a better predictor of pregnancy than the number of transferred embryos or female age. Fertility and Sterility 75, Tesarik J, Greco E 1999 The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Human Reproduction 14, Testard J 1986 Cleavage stage of human embryos two days after fertilization in vitro and their developmental ability after transfer into the uterus. Human Reproduction 1, Van Montfoort APA, Dumoulin JCM, Kester ADM, Evers JLH 24 Early cleavage is a valuable addition to existing embryo selection parameters: a study using single embryo transfers. Human Reproduction 19, Wittemer C, Bettahar-Lebugle K, Ohl J et al. 2 Zygote evaluation: an efficient tool for embryo selection. Human Reproduction 15, Received 4 September 26; refereed 21 December 26; accepted 5 January