Hum. Reprod. Advance Access originally published online on May 11, 2007
Human Reproduction 2007 22(7):1973-1981; doi:10.1093/humrep/dem100
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Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: A prospective study based on 4042 embryos
1 Service de Médecine et Biologie de la Reproduction, CHU Bretonneau, 2 Boulevard Tonnelle, 37000 Tours, France 2 INSERM CIC 202, Université François Rabelais de Tours, CHRU de Tours, France
3 Correspondence address. Tel: +33-247-474-746; Fax: +33-247-478-484; E-mail: royere{at}med.univ-tours.fr
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Abstract |
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BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish.
METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters.
RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability.
CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.
Key words: blastocyst/early cleavage/implantation/embryo morphology/zygote scoring
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Introduction |
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As a result of improved fertilization and early embryo culture techniques, patients may produce better quality embryos, with equivalent cell numbers and morphological scores. Elective single-embryo transfer is increasingly undertaken inorder to prevent adverse outcomes related to multiple pregnancies. Thus, selecting embryos with high implantation potential is one of the most important challenges in the field of assisted reproduction. Moreover, it is important to identify viable and non-viable embryos in order to cryopreserve only the former and to discard the latter. Such identification needs to be accurate in order to avoid massive and useless cryopreservation and to improve the chances of achieving a pregnancy for IVF couples.
Various methods have been proposed to evaluate embryo viability in IVF programmes. A limiting factor in such evaluations is that they should be neither invasive nor time consuming. Selection of the most viable cleavage stage embryo is usually based on embryo morphology on day 2 or day 3, with the rate of development in culture as a guide [for a review see Ebner et al. (2003a) and Scott (2003)]. Embryo morphology is determined by the number, size and shape of blastomeres, the proportion of fragments and the presence of multi-nucleated blastomeres. It has been demonstrated that after 2 days of culture, the 4-cell stage is the optimal cleavage stage (Giorgetti et al., 1995
; Ziebe et al., 1997). Embryos at this cleavage stage with little or no fragmentation and without multi-nucleated blastomeres are associated with a higher implantation rate compared to embryos at other cleavage stages with fragmentation or multi-nucleated blastomeres. However, there is evidence that embryo selection on day 2–3 based on morphological criteria may be unreliable, resulting in the transfer of embryos that are abnormal or that arrest at later developmental stages (Rijnders and Jansen, 1998; Graham et al., 2000; Milki et al., 2002). New parameters such as zygote morphology and early cleavage have recently been proposed to identify embryos with a good prognosis for implantation.
A zygote scoring system on day 1 has been reported to be reliable in terms of viability and implantation. Many morphological transformations can easily be assessed during the development of human pronuclear zygotes by simple non-invasive microscopic observation. Based on empirical observations correlated with pregnancy and previously published observations of zygote morphology (Van Blerkom, 1990), a zygote scoring system was first proposed by Scott and Smith (1998). A strong correlation between this score and the implantation rate was reported. This initial zygote scoring system was later revised (Z1–Z4 score) to allow faster scoring and to make it easy to use by a large number of technicians (Scott et al., 2000). At the same time, a further group proposed another classification based on static observation of human zygotes (Tesarik and Greco, 1999). Six patterns of pronuclear morphology (pattern 0–5) related to zygotes that had equal-sized pronuclei in apposition were described. The normal range of pronuclear variability was defined by analysis of zygotes giving rise to embryos transferred in 100% implantation cycles (pattern 0). Since this date, many different pronuclear scoring systems derived from these classifications have been proposed to select high-quality embryos (Zollner et al., 2002; Neuber et al., 2003; Kattera and Chen, 2004; Senn et al., 2006). Early-cleavage embryos on day 2 were reported to achieve higher implantation rates compared to non-early-cleavage embryos (Lundin et al., 2001; Salumets et al., 2003; Van Montfoort et al., 2004).
Extended embryo culture until the blastocyst stage (day 5/6) was successfully proposed as a means to select the most developmentally competent embryos (Gardner et al., 1998). Delaying transfer until the blastocyst stage should increase the potential for self-selection of viable embryos since only a proportion of embryos reach this stage, as the embryonic genome is activated between the four- and eight-cell stage (Braude et al., 1988). When embryos are cultured in vitro, about 50% will cease development during the first week. The reasons for this high rate of embryo loss during early development are not fully understood. They might include chromosomal abnormalities, suboptimal culture conditions or inadequate oocyte maturation.
Although the best way to evaluate embryo viability is its ability to implant, assessment of embryo development in vitro might make it possible to remain independent of the uterine receptivity that clearly influences implantation success. In addition, the various parameters suggested to date to evaluate embryo viability have mostly been investigated on a group together rather than on individually cultured embryos . Thus, the predictive power of individual sequential assessment of pronuclear morphology, early cleavage and embryo morphology on blastocyst development has hardly been studied (Lan et al., 2003; Neuber et al., 2003; Rienzi, 2005). Moreover, the respective impact of each factor on blastocyst development has never been clearly identified. Using individual culture of >4000 embryos, our aim was therefore first to assess the impact of pronuclear morphology, early cleavage and embryo morphology on the ability to develop to the blastocyst stage at day 5/6, then to evaluate their impact on blastocyst morphology and implantation ability whenever possible.
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Materials and Methods |
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A prospective study was initiated at the IVF unit, Bretonneau University Hospital, Tours, France, between January 2002 and December 2005. Individual sequential assessment from day 1 (zygote stage) to day 5/6 (blastocyst stage) was performed for 4042 cultured embryos from 758 couples. The embryos studied were either from the overall cohort (2270 embryos) or from supernumerary embryos (not transferred or frozen at day 2: 1772 embryos).
The key point in this sequential embryo assessment was the development of a blastocyst after 5 or 6 days of extended embryo culture. In addition to the usual morphological characteristics recorded on day 2 (number of cells per embryo and fragmentation rate), two other morphological features were also studied, i.e. pronuclear morphology at day 1 and first cleavage to the 2-cell stage at day 2. All optical assessments were performed using an inverted microscope with Hoffman modulation contrast (x200 and x 400 magnification). Zygotes and embryos were assessed only once for each time-specific observation.
The ovarian stimulation protocol, IVF and ICSI procedure used and the embryo culture with sequential media have already been reported (Guerif et al., 2004). Fertilization (day 0) was performed in IVF-50TM or G-FertTM (Vitrolife, Gothenburg, Sweden). The following morning (day 1), the oocytes were individually placed in microdrops (25 µl) in IVF-50TM or G1-2TM (Vitrolife) under mineral oil. From day 3 to day 5/6, embryo culture was performed in microdrops in CCM30TM or G2-2TM (Vitrolife) under mineral oil. All cultures were performed in incubators at 37°C with 6% CO2, 5% O2 and 89% N2.
Pronuclear morphology assessment at day 1
Oocytes were checked 18–20 h after insemination/ICSI (day 1). The morphological parameters studied included the number and size of pronuclei and the number of nucleolar precursor bodies (NPB) and their distribution (polarized versus non-polarized) in each pronucleus (Tesarik and Greco, 1999). The criteria initially described by Tesarik were simplified in this study by incorporating the originally described patterns 1–5 (abnormal) into a single group of non-pattern 0 zygotes as opposed to pattern 0 (normal). Briefly, zygotes with the following characteristics were considered as pattern 0: (i) the pronuclei were similar in size and appositional; (ii) the difference in the number of NPB between both pronuclei did not exceed three; (iii) the distribution of NPB was the same in both pronuclei; (iv) there were at least three NPB in each pronucleus.
Early cleavage assessment at day 2
The first mitotic cleavage was assessed 25–27 h post-insemination/ICSI (day 2). Embryos that possessed two cells were classified as "early-cleavage embryos" and those that had not yet cleaved were classified as "non-early-cleavage embryos".
Morphological embryo assessment at day 2
On day 2, individually cultured embryos were evaluated 44–46 h post-insemination/ICSI on the basis of number of blastomeres, fragmentation rate and presence of multi-nucleated blastomeres. Blastomeres were classified into three groups (<cells, 4 cells, >4 cells). The degree of fragmentation was expressed as a percentage of the total oocyte volume occupied by anucleate cytoplasmic fragments. The rate of fragmentation was scored (<20% of the volume of embryo, between 20 and 50% of the volume of the embryo, >50% of the volume of the embryo). Embryos with one or more multi-nucleated blastomeres were excluded from extended embryo culture. Embryos with pattern 0, early cleavage, 4 regular blastomeres, <20% of fragmentation and no multi-nucleated blastomeres were classified as "top quality".
Assessment of blastocyt devlopment at day 5/6
Both the outcome of extended embryo culture and day of blastocyst development (day 5/6) were recorded for each individually cultured embryo. The blastocyst scoring assessment was based on the expansion of the blastocoelic cavity and the number and cohesiveness of the inner cell mass and trophectodermal cells (Gardner and Schoolcraft, 1999). A good morphology blastocyst was defined as having a well-expanded blastocoele cavity (B3-B6) at day 5, a well-defined inner cell mass (A or B) and a single layer of trophectoderm cells surrounding the cavity (A or B) (Gardner et al., 2000).
Blastocyst transfer
In the studied period, 392 IVF/ICSI cycles were programmed for a blastocyst transfer and a transfer was performed in 342 cycles (87%). Women were 33.9 ± 4.0 years of age (range 25–43 years) and the mean number of transferred blastocysts was 1.5 ± 0.5 (range 1–2). The ovarian stimulation protocol was previously described (Guerif et al., 2004). One or two blastocysts were transferred as a function of indication [(single-embryo transfer (n = 119 transfers) or repeated implantation failures (n = 223 transfers)], either at day 5 or at day 6. Two groups of patients were considered as a function of the quality of transferred blastocysts. In one group, at least one good quality blastocyst at day 5 was transferred (n = 204 transfers) whereas in another group included all other transfers at day 5/6 (n = 138 transfers).
Clinical pregnancy was defined as the presence of a gestational sac with fetal heartbeat on ultrasound examination at 7 weeks of pregnancy and the implantation rate was defined as the number of gestational sacs per number of transferred embryos.
Statistical analysis
Statistical comparisons were made using the chi-square test. A stepwise logistic regression model was used to detect independent predictors of blastocyst development. Areas under the receiver operating characteristics (ROC) curve (AUC) were estimated to assess the ability of different combinations of parameters to discriminate between blastocyst development and absence of blastocyst development: the greater the AUC, the better the prediction of the model. Hosmer and Lemeshow (2000) proposed thresholds to help to interpret the AUC:
- 0.9–1: perfect separation;
- 0.8–0.9: excellent discrimination;
- 0.7–0.8: acceptable discrimination;
- 0.6–0.7: low discrimination;
- 0.5–0.6: no discrimination.
Analyses were performed using SAS (SAS Institute Inc., Cary, NC, version 9.1).
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Results |
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Overall characteristics
A total of 4042 embryos from 963 IVF/ICSI cycles were cultured individually from day 1 to day 6. These IVF/ICSI cycles included 392 cycles related to cultured embryos from the whole cohort (whole cohort: 2270 embryos) and 571 cycles related to supernumerary cultured embryos (supernumerary: 1772 embryos).
Rate of blastocyst development
On day 1, the frequency of pattern 0 zygotes was 18%. On day 2, the frequency of early cleavage was 32%, the frequency of <4-cell, 4-cell and >4-cell embryos was 40, 41 and 19%, respectively, and the frequency of embryos with <20% fragmentation, 20–50% fragmentation and >50% fragmentation was 47, 39 and 13%, respectively. The frequency of "top embryos" defined with pattern 0, early cleavage, 4 regular blastomeres, <20% fragmentation and no multi-nucleated blastomeres was 4%.
Univariate associations between each parameter and blastocyst rate are given in Table 1. All parameters studied were individually related to the development of a blastocyst on day 5/6. Each parameter remained strongly related to the success of blastocyst development in multivariate analysis, with greater success in developing to the blastocyst stage for embryos with pattern 0, early cleavage, 4-cells or <20% fragmentation (Table 2). Embryos with pattern 0 zygotes on day 1, combined with early cleavage, a 4-cell stage without any multi-nucleation and <20% fragmentation on day 2 developed to the blastocyst stage on day 5/6 in 72% of cases. Bearing in mind the possible bias resulting from inclusion of supernumerary embryos in this series, we checked the results, using both univariate and multivariate analysis while splitting them between supernumerary and whole cohort embryos. Despite their extended culture peroids, the blastocyst rates for supernumerary embryos were lower than for whole cohort embryos (36.2 versus 49.4%, respectively, P < 0.0001). Differences based on early cleavage or cell number at day 2 were consistently observed in both groups, while pronuclear morphology and embryo fragmentation were less consistently related to blastocyst development using multivariate analysis {supernumerary embryos: non-pattern 0 versus pattern 0 zygote odds ratio (OR) 0.60 [0.46–0.79], P = 0.0003; non-early-cleavage versus early-cleavage OR 0.45 [0.35–0.56], P < 0.0001 (number of cells: 2–3 cells versus 4 cells OR 0.52 [0.41–0.67], P < 0.0001; 5–8 cells versus 4 cells OR 0.57 [0.43–0.76], P = 0.0001); whole cohort embryos: non-pattern 0 versus pattern 0 zygote OR 0.85 [0.67–1.07], P = 0.1594; non-early-cleavage versus early-cleavage OR 0.43 [0.36–0.53], P < 0.0001 (number of cells: 2–3 cells versus 4 cells OR 0.48 [0.39-0.60], P < 0.0001; 5–8 cells versus 4 cells OR 0.42 [0.32–0.54], P < 0.0001)}.
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The AUCROC was used to summarize the predictive power of the combination of day 1 and day 2 parameters (Fig. 1). When parameters were analysed separately, the highest AUCROC (0.634) was obtained with the number of cells on day 2. The best-fitted combination of two parameters involved both number of cells and early cleavage on day 2 (AUCROC = 0.666). Including the rate of fragmentation increased the AUCROC (0.683) for the best fitted combination of three parameters. Finally, a AUCROC of 0.688 was reached when the four parameters were combined altogether. Analyses were also carried on stratified IVF/ICSI techniques with no difference in terms of results (data not shown).
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Blastocyst morphology
Among the 1762 blastocysts obtained, 576 (33%) reached this stage as early as day 5 with a good morphology [(B3–B6) with grade A or B for inner cell mass as well as for trophectoderm]. As in the previous univariate analysis, significant relationships were observed between all parameters studied and blastocyst morphology (Table 3). Altogether, the rate of blastocysts with good morphology at day 5 reached 64% for embryos with pattern 0 zygotes on day 1, combined with early cleavage, a 4-cell stage without any multi-nucleation and <20% fragmentation on day 2. Only early-cleavage embryos and number of cells remained associated with good morphology blastocyst in a multivariate analysis (Table 4). As already mentioned in earlier results, using both univariate and multivariate analysis, we split the results between supernumerary and whole cohort embryos. We observed a clear relationship between early cleavage, number of cells at day 2 and the percentage of good morphology blastocyst at day 5 in both situations using multivariate analysis {supernumerary embryos: non-early-cleavage versus early-cleavage OR 0.48 [0.32–0.72], P = 0.0003; number of cells: 2–3 cells versus 4 cells OR 0.25 [0.15–0.41], P < 0.0001; 5–8 cells versus 4 cells OR 0.59 [0.37–0.94], P = 0.0269; whole cohorts: non-early-cleavage versus early-cleavage OR 0.38 [0.29–0.49], P < 0.0001; (number of cells: 2–3 cells versus 4 cells OR 0.41 [0.28–0.60], P < 0.0001; 5–8 cells versus 4 cells OR 0.62 [0.42–0.91] P = 0.0154)}.
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Implantation as a function of blastocyst morphology and day 1–2 parameters
The IVF/ICSI cycles included 392 cycles related to cultured embryos from the whole cohort. Overall, a transfer of one or two blastocysts was performed in 342 cycles (87%). The clinical outcome as a function of the morphology of transferred blastocysts is shown Table 5. Clinical pregnancy, live birth and implantation rates were significanly higher following transfer of at least one good morphology blastocyst at day 5 compared to transfer of whichever blastocyst(s) at day 5 or 6.
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We checked all parameters of early development with regard to implantation ability of good morphology blastocysts, as compared to all other blastocysts (Table 6). The striking observation was that inside each group neither early cleavage rate, nor cell number at day 2, nor pronuclear morphology, nor fragmentation rate were related to the presence or absence of delivery.
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Discussion |
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The main aim of our study based on 4042 individually cultured embryos was to assess the most predictive combination, using pronuclear morphology, early cleavage, number of cells and fragmentation rate, for in vitro blastocyst development. While all parameters (once adjusted to each other) were significantly related to blastocyst development, early cleavage and number of cells on day 2 appeared more reliable to predict both blastocyst development and morphology. However, the predictive power of the four combined parameters remained weak overall with regard to in vitro developmental potential. Additionally, while implantation rate was clearly influenced by blastocyst morphology in cases of blastocyst transfers, implantation ability of good morphology blastocysts was not related to parameters of early development.
In our study, >4000 embryos were individually cultured in microdrops and evaluated from day 1 to day 6, with the key point being the development of a blastocyst on day 5 or 6. Most studies evaluating embryo viability using pronuclear stage (Tesarik and Greco 1999; Scott et al., 2000), early-cleaving 2-cell embryos (Shoukir et al., 1997; Bos-Mikich et al., 2001; Sakkas et al., 2001) and culture until the blastocyst stage (Gardner et al., 2000) have involved grouped embryos, rather than individually cultured embryos. We chose to culture each embryo separately, rather than in groups, to retain the opportunity to follow the developmental progression of each. One possible concern with sequential embryo assessment, from zygote to the blastocyst stage, is the potential damage done to the embryo by environmental conditions (changes in temperature, additional light exposure and pH shifts) related to performing multiple observations. Limiting the viewing time allowed us to assess all the parameters studied using procedures that were neither invasive nor time consuming.
Studies on pronuclear morphology on day 1 have reported a wide range of observation times (12–20 h), mostly between 16 and 18 h. We chose 18—20 h post-insemination/ICSI to take into account the time difference between ICSI and IVF (Montag and van der Ven, 2001). We used a modified criteria initially described by Tesarik by combining the originally described patterns 1–5 (abnormal) into a single group of non-pattern 0 zygotes as opposed to pattern 0 (normal) (Tesarik and Greco, 1999). Arrest of development beyond day 2 has been most frequently associated with non-pattern 0 zygotes (Tesarik and Greco, 1999; Wittemer et al., 2000). In contrast, studies on pronuclear morphology and blastocyst development have been less controversial. In our study, the highest incidence of blastocyst formation was obtained with pattern 0 zygotes, in agreement with Balaban et al. (2001). Moreover, embryos derived from the Z-1 score, which is included in pattern 0 in Tesarik's classification, resulted in a higher percentage of day 5/6 blastocysts: 76% on day 6 (Scott et al., 2000) and 78% on day 5 (Lan et al., 2003). Such results were confirmed using other detailed descriptions of zygote morphology and scoring systems (Zollner et al., 2002; Neuber et al., 2003). Presence of a cytoplasmic halo at day 1 has been proposed as an additional marker of embryo viability but results remain much debated. It was observed that the presence of a cytoplasmic halo had a significant influence on blastocyst quality irrespective of its aspect without modifying blastocyst development rate (Ebner et al., 2003b), whereas an extreme halo was reported to negatively affect blastocyst development (Zollner et al., 2002).
We also assessed cleavage to the 2-cell stage on day 2 using extended embryo culture as a reliable parameter for embryo viability. Evaluation is performed in most IVF centres 25–27 h post insemination/ICSI. In our study, a higher blastocyst rate was observed in early-cleavage embryos than in uncleaved embryos, as already reported [Day 5: 39 versus 21%, respectively (Neuber et al., 2003), 52 versus 22%, respectively (Windt et al., 2004); Day 6: 66 versus 40%, respectively (Van Montfoort et al., 2004); Day 7: 32 versus 17%, respectively (Fenwick et al., 2002)]. A positive correlation between early onset of cleavage and blastocyst formation had already been reported in mouse (McLaren and Browman, 1973) and bovine embryos (Grisart et al., 1994). Moreover, in these studies the blastocysts with early cleavage were found to have more cells than their later cleaving counterparts. Such results are better explained by the differences in timing of the first cleavage rather than differences in the rates of progression of subsequent cell cycles (McLaren and Browman, 1973). The reasons for better viability of early-cleavage embryos remain largely unexplained, but it may be speculated that such embryos derive from oocytes with adequately synchronized cytoplasmic and nuclear maturation. Early-cleavage embryos should be less likely to be exposed to a critical minimal level of maternal mRNA prior to activation of the embryonic genome (Bachvarova and de Leon, 1980).
With regard to the embryo morphology on day 2, the blastocyst rate was higher for 4-cell embryos compared to faster (5–8 cell) or slower (2–3 cell) embryos. Indeed, the presence of >4 cells at day 2 is not predictive of a greater likelihood of blastocyst formation (Dokras et al., 1993; Shapiro et al., 2000; Langley et al., 2001). Two hypotheses have been suggested to explain this: (i) some blastomeres in these embryos might be rather large anucleate fragments (Ziebe et al., 1997), thus explaining the overestimation of their numbers and (ii) accelerated division might be an indicator of developmental instabilities that would affect the embryo's ability to develop to a blastocyst. Fast-developing embryos have been reported to exhibit higher levels of aneuploidy compared to synchronous embryos (Magli et al., 1998). Blastomere size is also a relevant characteristic that is generally underestimated. When comparing day 2 transfers between 4-cell embryos, it was found that unevenly cleaved embryos had significantly lower implantation rate compared with evenly cleaved embryos (Hardarson et al., 2001). Integration of these criteria remains complex as it is related to the number of cells. For example, transferring a 4-cell embryo with equal-sized blastomeres will be more advisable than a 4-cell embryo with uneven blastomeres, whereas transferring a 3-cell embryo with uneven blastomeres (one bigger and two smaller) will be preferable to a 3-cell embryo with equal-sized blastomeres.
As previously reported, we observed that the rate of blastocyst formation decreased significantly with fragmentation on day 2 (Alikani et al., 2000; Hardy et al., 2003). Several hypotheses have been suggested to explain the detrimental effects of fragments on embryo development. First, fragments might physically impede cell-cell interactions, interfering with compaction, cavitation and blastocyst formation (Van Blerkom et al., 2001). In addition, ultrastructural observations of degeneration in blastomeres adjacent to fragments (Sathananthan et al., 1990) suggest that fragments might release toxic substances and therefore damage nearby cells (Alikani et al., 1999). Alternatively, fragments might also reduce the volume of cytoplasm and deplete the embryos of essential organelles or polarized domains (Antczak and Van Blerkom, 1999). Higher aneuploidy rates were reported for >35% fragmented embryos by Munne et al. (1995) whereas it was also speculated that in the case of moderate fragmentation, various temporal or spatial patterns of fragmentation have quite different effects on embryo development than the occurence of fragments per se (Alikani and Cohen, 1995; Antczak and Van Blerkom, 1999; Van Blerkom et al., 2001).
The main aim of our study was to determine the specific weighting of each parameter to predict embryo development potential, using a stepwise logistic regression model. The possibility of combining or integrating findings from observations at different periods of time to improve the assessment of embryo viability has been explored in many studies. Using pronuclear morphology combined with day 3 morphology, Lan et al. (2003) observed 92% survival at day 5 in 499 top-quality embryos (Z-1 zygotes and grade I embryos). Another study, based on 1550 individually cultured embryos, explored various scenarios to predict blastocyst development from sequential embryo assessment (Neuber et al., 2003). With pronuclear symmetry, early cleavage and subsequent good quality 
4- and >7-cell embryos on days 2 and 3, respectively, they could predict 54% probability for an embryo to reach the blastocyst stage by day 5. A multi-step scoring system has also been proposed to improve the accuracy of such assessment. A graduated embryo score based on a combination of pronuclear morphology, early cleavage and day 3 morphology to identify embryos with a high potential for blastocyst development was proposed following a study using 1245 embryos (Fisch et al., 2001). The highest embryo score had 64% blastocyst formation compared with 11% for the lowest embryo score. Also using a multiple-step scoring system, Rienzi (2005) reported 77% blastocyst formation on day 5 for embryos with normal pronuclear stage and early cleavage on day 1, 4- to 5-cells with equal blastomere size, <10% fragmentation and no multi-nucleation on day 2, and >6 cells with equal blastomere size, <10% fragmentation and no multi-nucleation on day 3. However, the proposed values that defined these scores were attributed quite arbitrarily, since they were only based on observed frequencies. In agreement with previous reports, our results from 4042 cultured embryos showed that the 0 pattern zygote on day 1, combined with early cleavage, a 4-cell stage without any multi-nucleation and <20% fragmentation on day 2 developed to the blastocyst stage in 72% of cases, mainly as early as day 5 (82%). However, as reported earlier by Fisch et al. (2001), the major problem remains the degree of predictability of parameters for non-top-quality embryos, rather than the outcome of the best embryos that combine all the best suited parameters, since unsuitable embryos according to day 1 and day 2 criteria might develop to the blastocyst stage. Moreover, using stepwise logistic regression analysis gave us the opportunity to determine the respective weighting of each parameter. This methodology indicated (i) that each of the four parameters studied influenced the blastocyst rate at day 5/6, with a greater influence of early cleavage and embryo morphology at day 2 on this outcome, and (ii) that only early cleavage and embryo morphology at day 2 had a significant relationship with regard to blastocyst development. Altogether, these findings suggested a limited contribution of pronuclear morphology and fragmentation rate compared to early cleavage and embryo cell number. Moreover, even the combination of all four parameters led to an AUCROC of 0.688. Such a weak AUCROC highlights the limitations of the early morphological parameters available to predict embryo viability. Similarly, when we look at the extended cultures that were achieved during the same study period for the purpose of single-blastocyst transfer, it is important to note that selection of the "right" embryo at day 2 would have been correct in 17% of cases, possible (among various embryos of similar characteristics) in 26%, but incorrect in 52% or impossible (no blastocyst development) in 5% of cases, in agreement with previous reports which observed that day 3 morphology was a poor predictor of blastocyst development in extended culture (Rijnders and Jansen, 1998; Graham et al., 2000; Milki et al., 2002).
A recent prospective randomized, controlled trial found a higher rate of pregnancy among women undergoing transfer of a single blastocyst-stage embryo than in a group undergoing transfer of a single cleavage-stage embryo (Papanikolaou et al., 2006). Bearing in mind that embryo viability is best defined as the ability of the embryo to implant successfully and result in a normal healthy baby, we checked the issues of blastocyst transfers following extended culture of whole cohorts. As expected, transfers of good morphology blastocyst(s) were associated with higher live birth and implantation rates compared to all other blastocyst transfers. As to interpret the value of the reported implantation rate, it is important to note that such a result issued from mostly repeated implantation failures (n = 223; IR = 19%) and partly from single-blastocyst transfer (n = 119; IR = 27%). Interestingly, once looking at implantation ability of at least one good morphology blastocyst, the studied parameters of early embryo development revealed to be unhelpful in predicting implantation.
Our results demonstrated that sequential morphological assessment of pronuclear morphology, early cleavage and embryo morphology at day 2 (number of cells and fragmentation rate) may all be considered as parameters related to development potential. However early cleavage and cell number contribute most to this evaluation. Nonetheless, the predictive power of such morphological evaluation remains quite low, thus arguing for either additional evaluation of already assessed criteria (first polar body morphology, cytoplasmic halo, blastomere size and position of fragments) or definition of new criteria. Bearing in mind our results on absence of predictive value of early embryo development parameters on implantation ability of blastocysts, we suggest that extended culture might be considered as an alternative.
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Submitted on December 11, 2006; resubmitted on March 14, 2007; accepted on March 29, 2007.
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