High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate

Giuseppe De Placido1, Martin Wilding1,2,3, Ida Strina1, Erminia Alviggi1, Carlo Alviggi1, Antonio Mollo1, Maria Teresa Varicchio1, Achille Tolino1, Cristina Schiattarella1 and Brian Dale2

1 Area Funzionale di Medicina della Riproduzione, Università degli Studi `Federico II', Naples and 2 Centre for Reproductive Biology, Clinica Villa del Sole, Naples, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: A scoring system has been developed to determine preimplantation embryo quality, and used to select embryos for transfer into the uterus of patients. METHODS AND RESULTS: The system was used to study early embryo development and to test whether these scores alone can accurately predict IVF outcome. Following zygote and embryo scores through early development, the data showed that a top quality zygote does not necessarily indicate that the resulting embryo will be top quality after in-vitro culture. The embryo quality score can change dramatically when embryos are cultured to day 2 or 3 post-fertilization. Pregnancy rates and implantation rates were compared with the cumulative and separated zygote and embryo scores. Analysis of the predictability of scoring systems suggested that morphological scores alone are relatively unpredictive of IVF outcome. When weighted for in-vitro growth rate, scores were highly predictive, more so than the rate of development alone. CONCLUSIONS: These data suggested that a combination of in-vitro growth rate and morphological analysis both of zygotes and embryos was highly indicative of outcome after IVF. The results can be adopted to the single embryo transfer approach to IVF.

Key words: assisted reproduction/embryo score/human embryo/IVF/single embryo transfer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The current trend in human IVF protocols is to reduce the number of embryos transferred into the uterus in order to eliminate multiple pregnancies while maintaining singleton pregnancy rates at acceptable levels (Coetsier and Dhont, 1998Go; Pennings, 2000Go). Ways of achieving this lie in increasing the reliability of embryo analysis and culture systems. Techniques such as blastocyst transfer have been of assistance (Gardner et al., 1998Go; Gardner and Schoolcraft, 1999Go), although blastocyst culture still requires relatively specialized culture systems and is affected by the loss of a percentage of embryos cultured in vitro (Langley et al., 2001Go). The culture of embryos to day 2 or 3 after fertilization, in contrast to blastocyst culture, is relatively simple. A reliable method of analysis of these embryos could then be highly beneficial in IVF protocols. The selection of human embryos for replacement into the uterus during cycles of IVF depends currently on the examination of embryo morphology on day 2 or 3 post-fertilization (Edwards et al., 1980Go, 1984Go; Cummins et al., 1986Go; Puissant et al., 1987Go; Visser and Fourie, 1993Go; Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Hu et al., 1998Go). However, extended culture of this material has been shown to be unnecessary in many cases (Scott and Smith, 1998Go; Dale et al., 1999Go; Tesarik and Greco, 1999Go; Tesarik et al., 2000Go; Wittemer et al., 2000Go). Recent reports have suggested that an examination of the morphology of human zygotes can predict the outcome of IVF (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Scott et al., 2000Go; Tesarik et al., 2000Go). For example, a pregnancy rate of 49.5% was achieved after the transfer of top quality zygotes, as opposed to 28% after the transfer of medium quality material (Scott et al., 2000Go). Furthermore, a specific pattern of nucleolar alignment has been suggested to indicate high quality, as demonstrated by a pregnancy rate of 50% for zygotes with aligned nucleoli versus a pregnancy rate of only 9% in cases where this was absent (Tesarik and Greco, 1999Go). These authors therefore suggested that pronuclear alignment, morphology of nucleoli and presence of the `cytoplasmic flare' was highly indicative of implantation potential after IVF or ICSI. Although the transfer of human zygotes is successful (Ahuja et al., 1985Go; Quinn et al., 1990Go; Smith et al., 1993Go; Scott and Smith, 1998Go), the technique is limited in that embryo quality cannot be examined and therefore considered in the selection of material for transfer. Despite this negative factor, the transfer of human zygotes in general gives pregnancy rates comparable to the transfer of human preimplantation embryos (Scott and Smith, 1998Go; Dale et al., 2002Go). These data suggest that the morphological analysis of zygotes can be used as a tool to predict further embryo development and the potential for implantation.

Although a correlation between zygote and embryo morphology has been shown to exist (Ludwig et al., 2000Go; Scott et al., 2000Go), the correlation is not perfect, suggesting that some degree of predictability is lost when using either zygote or embryo morphology alone (also see Rjinders and Jansen, 1998Go). Furthermore, the developmental rate of embryos has been suggested to be indicative of implantation potential (Edwards et al., 1984Go; Cummins et al., 1986Go; Racowsky et al., 2000Go). One group (Racowsky et al. 2000Go) for example showed that a pregnancy rate of 33% where no 8-cell embryos were observed on day 3 rose to 51% in cases where patients had three or more 8-cell embryos on this day. In the present study, the utility of individual scoring systems, or a combination of the systems, was examined for predicting IVF outcome.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
Patients attending the IVF clinic for IVF protocols were prepared using standard controlled ovarian stimulation protocols including down-regulation of the pituitary gland with a GnRH agonist (Decapeptyl; Ipsen, Italy) followed by ovarian stimulation with exogenous FSH (Gonal-F; Serono, Italy). A single member of the medical staff co-ordinated all stimulation protocols, thereby ensuring standardization of the stimulation protocols. Oocyte retrieval was performed 36 h after the administration of 10 000 UI hCG when two or three follicles of 18–20 mm diameter were observed by ultrasound examination, and blood 17ß-estradiol levels reached 150–200 pg/ml per follicle over 18 mm diameter. As the Italian State finances the IVF centre in Naples, patients are not required to pay for IVF treatment, and oocytes are routinely treated by ICSI to guarantee optimal fertilization rates. A single team of biologists co-ordinated all procedures, thereby ensuring that both culture protocols and embryo assessment were standardized. Oocytes were processed for ICSI using commercial IVF medium (Medicult, Copenhagen, Denmark) pre-equilibrated to 37°C and 5% CO2. Zygote quality was scored 16–20 h after ICSI. Embryo quality was assessed at 40–41 h after insemination on day 2, and at 64–65 h after insemination on day 3. Embryos were transferred on the second or third day after oocyte retrieval. Embryos were selected for transfer by the following criteria in order of importance: number of blastomeres and embryo morphological score on day of transfer and then zygote score. The establishment of a pregnancy was considered as a positive ß-hCG test >60 IU/l at 14 days after embryo transfer. The implantation rate was calculated by the observation of fetal heart-beats after ultrasound analysis, 8 weeks after the establishment of pregnancy.

Study design
The zygote scoring system was developed based on previously published methods (Figure 1AGo). Briefly, three parameters were considered. First, the position of the pronuclei in relation to the cytoplasm was analysed, considering two opposed pronuclei of equal size in the centre of the cytoplasm to indicate optimal morphology (Sadowy et al., 1998Go; Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Scott et al., 2000Go; Wittemer et al., 2000Go) [Figure 1A(i)Go and Table IGo]. Second, the morphology and orientation of nucleoli was scored, equal numbers of nucleoli opposed in the zygote centre considered top quality (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Scott et al., 2000Go; Wittemer et al., 2000Go) [Figure 1A(ii)Go and Table IGo]. Third, the presence of a `cytoplasmic flare' (a dense area of cytoplasm aggregated around the pronuclei) was considered indicative of high implantation potential and therefore awarded a score of 5 points (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Wittemer et al., 2000Go; Scott et al., 2000Go) [Figure 1A(iii)Go and Table IGo]. In addition, a system for scoring embryos was developed (Figure 1BGo). Again, three parameters were scored. First, blastomere volume and synchrony of cleavage was assessed (Edwards et al., 1980Go, 1984Go; Cummins et al., 1986Go; Puissant et al., 1987Go; Visser and Fourie, 1993Go; Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Hu et al., 1998Go; Desai et al., 2000Go; Hardarson et al., 2001Go; Van Royen et al., 2001Go) [Figure 1B(iv)Go and Table IGo]. Second, the observation of a single nucleus within individual blastomeres was considered top quality, and the level of multinucleation indicators of progressively poorer quality (Edwards et al., 1980Go, 1984Go; Cummins et al., 1986Go; Puissant et al., 1987Go; Visser and Fourie, 1993Go Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Hu et al., 1998Go; Desai et al., 2000Go; Hardarson et al., 2001Go; Van Royen et al., 2001Go) [Figure 1B(v)Go and Table IGo]. Third, the extent of fragmentation was considered (Alikani et al., 1999Go, 2000Go) [Figure 1B(vi)Go and Table IGo]. Basically, embryos with blastomeres of equivalent size, a single nucleus per blastomere and no fragmentation were considered top quality and scored higher than embryos containing one or more defects (Table IGo). The maximum score obtainable was 15 for zygotes and 15 for each day of embryo culture (Table IGo). Cohorts of embryos selected for transfer were categorized into three groups dependent on the mean quality of the cohort. Basically, a group of top quality material (group 1) was permitted a single defect, i.e. a mean score of >=14 points. The medium-scoring group (group 2) included material with two or more defects considered minor in the scoring system, i.e. a mean score of 10–14 points. Group 3 or low-scoring embryos contained defects considered severe (resulting in a mean score of <10 points). Where non-morphological factors were considered (i.e. growth rate), embryos were separated according to day of transfer (i.e. day 2 or 3) and the cohort selected for transfer again divided into three groups. Defining the standard growth rate as two blastomeres on day 2 and five blastomeres on day 3, group 1 embryos were considered advanced for the day of culture (i.e. mean of over four blastomeres on day 2, or seven blastomeres on day 3). Group 2 embryos were considered normally developing (mean of two to three blastomeres on day 2 or four to seven on day 3), and group 3 embryos slow developing (mean of less than two blastomeres on day 2, or less than four on day 3). Weighted scores were calculated as growth rate multiplied by morphology and the obtained value weighted by zygote score. The scoring system was weighted by the equation:



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Figure 1. Design of zygote and embryo scoring system. (A) Zygote scoring. The score is equally distributed into three factors: (i) position of pronuclei; (ii) position and type of nucleoli; (iii) cytoplasmic morphology. (B) Embryo morphology. Again, the score of 15 is equally distributed between three factors: (iv) blastomere volume and synchrony of cleavage; (v) level of multinucleation; (vi) level of fragmentation.

 

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Table I. Cut-off valuesa used in assigning groupings
 
The rationale behind this system was to balance `embryo quality (i.e. morphology with growth rate) and `zygote quality' (i.e. morphology). This scoring system produces a score of (15x4)x15 = 900 for a top quality embryo on day 2 and (15x8)x15 = 1800 points for a top quality day 3 embryo (see Table IGo). The rationale was to facilitate the separation of embryos with similar morphologies and ease the selection procedure for transfer.

The study was a retrospective analysis of the morphological assessment of 154 patients treated in the Polyclinic of Naples between January and December 2000. Patients were unselected for age, body mass index and treatment protocol. Patients diagnosed for endometriosis or polycystic ovarian syndrome were excluded from the study due to the possible influence of these factors on oogenesis or endometrial receptivity. As stated above, all patients were treated by ICSI without reference to sperm quality in order to ensure the comparability of the assessments.

Statistical analysis
All data were plotted as mean ± SD unless stated. All plots and statistical analyses were calculated using the Sigma Plot and Sigma Stat software packages (SPSS; Erkrath, Germany). Regression lines were calculated by the method of least squares, and the significance of the regression lines was tested with the Pearson product-moment test. The z-test with Yates' correction was used to test the significance of proportions where necessary.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Relationship between zygote and embryo score
The scoring system outlined in the present study was used to examine the relationship between zygote and embryo morphology, and to test whether the scores could be used accurately to predict the outcome of cycles of IVF. The formation of human zygotes in vitro and the further development of this material into embryos was followed through to day 2 (40–41 h) or 3 (64–65 h) after oocyte retrieval. The daily scores of individual embryos was highly variable (Figure 2Go). It was noted that scores obtained for zygotes did not always correlate with embryo development (Figure 2Go). In fact, only 18% (211/1171) of top quality zygotes obtaining a score >14 formed low-quality embryos with a score <10 on the day of transfer. In contrast, zygotes achieving a score <10 could also become a high-quality embryo. In addition, 15% (176/1171) of all zygotes with a score <10 (suggesting severe defects; see Figure 1AGo) formed embryos with a score >14. Furthermore, scores for embryos often varied significantly between days 2 and 3 when extended culture was required (Figure 2AGo). A test was conducted (by plotting a regression line) to identify any correlation between morphological score on day 1 and the score from the same embryos on day 2 or 3. The data obtained confirmed the previously described correlation between zygote and embryo scores (Ludwig et al., 2000Go), whether this score was recorded on day 2 or 3 after fertilization (Figure 2BGo). The correlation was not perfect however, suggesting that the zygote score can be used merely to estimate embryo score.




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Figure 2. Embryo quality followed through culture during cycles of IVF-embryo transfer. Individual embryos were cultured until day 3 during cycles of assisted reproduction. The morphological analysis was performed at regular intervals. (A) The data show the variability of score between day 1 and day 3 in a 70-embryo sample out of a total of 1171 embryos cultured in the 154 patients of the present study. (B) Relationship between zygote and embryo score. (i) Zygote versus day 2 embryo score; the data are an analysis of a total of 436 embryos cultured to day 2; the regression line shows a significant correlation coefficient r = 0.2 (P < 0.0001). (ii) Zygote versus day 3 embryo score; the data are an analysis of a total of 735 embryos cultured to day 3; the regression line shows a marginally significant correlation coefficient r = 0.1 (P = 0.05).

 
Does transfer of a `top quality' zygote or embryo increase chances of success after IVF?
The data obtained from 154 cycles of IVF where a total of 551 embryos was replaced on day 2 (40–41 h) or day 3 (64–65 h) after scoring zygote or embryo quality on each day of culture were analysed retrospectively. In previous IVF cycles (where zygote morphology was not considered), a pregnancy rate of 37.4% and implantation rate of 16.5% were achieved (among 182 patients treated between January 1998 and January 1999, with mean maternal age 33.2 ± 8.4 years and mean of 3.5 ± 1.3 embryos transferred). These values were used as baseline to permit accurate evaluation of the scoring system presented in the present data. When zygote scores were considered alone, minimal correlation was observed between quality and predictability. Embryos derived from group 1 zygotes achieved 25/59 pregnancies (42.4%), with an implantation rate of 13.2% (mean 3.4 ± 1.1 embryos transferred; Figure 3Go, Table IIGo). However, embryos derived from group 2 zygotes resulted in 40/89 pregnancies (44.9%) with an implantation rate similar (15.7%) to that in group 1 zygotes (3.5 ± 1.6 embryos transferred; Figure 3Go, Table IIGo). Group 3 zygotes produced poor results, with only one patient achieving pregnancy (pregnancy rate 16.7%, implantation rate 7.1%, 3.4 ± 0.5 embryos transferred; Figure 3Go, Table IIGo).



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Figure 3. Predictability of scoring systems. The IVF outcome (pregnancy rate and implantation rate) of both individual and combined scoring systems is divided by the control IVF outcome from the present laboratory. Therefore, a ratio >1 indicates increased predictability. Solid bars indicate pregnancy rates; shaded bars indicate implantation rates. The three bars for each grouping indicate groups 1–3 (left to right).

 

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Table II. Relationship between embryo scoring systems and outcome of assisted reproduction
 
A further test was conducted to determine whether embryo morphology alone was predictive of IVF outcome; this was achieve by assessing the embryo morphology score obtained on the day of transfer against pregnancy and implantation rates. In the present analysis, a poor correlation between cohort score and predictability was again observed. When a cohort of group 1 embryos was transferred, 19/48 patients achieved pregnancy (39.6%), with an implantation rate of 14.9% (3.6 ± 0.7 embryos transferred; Figure 3Go, Table IIGo). The transfer of group 2 embryos resulted in 46/88 pregnancies (52.3%) and an implantation rate of 15.4% (3.3 ± 0.8 embryos transferred; Figure 3Go, Table IIGo). Group 3 embryos again produced poor results, with only one pregnancy was achieved among 18 patients (pregnancy rate 5.5%, implantation rate 4%, 3.2 ± 0.5 embryos transferred; Figure 3Go, Table IIGo).

The above data suggest that the selection of human zygotes or embryos for transfer on the basis of morphology alone is relatively unpredictive during cycles of IVF. However, these data do not rule out the possibility that such selection of material might be more predictive of success than by using a single scoring system alone (Scott et al., 2000Go). This hypothesis was re-tested retrospectively by analysing the cumulative zygote and embryo score to determine whether the transfer of morphologically `perfect' cohorts of human embryos might be more indicative of success after IVF. Again, only minimal correlation was observed. The transfer of cohorts of embryos with a group 1 cumulative score resulted in 15/38 (39.5%) pregnancies and an implantation rate of 13.0% (3.5 ± 1.0 embryos transferred; Figure 3Go, Table IIGo). Embryos with a group 2 cumulative score resulted in 50/103 pregnancies (48.5%) with an implantation rate of 15.5% (3.4 ± 0.4 embryos transferred; Figure 3Go, Table IIGo). Group 3 cohorts of embryos again produced 1/13 pregnancies (7.7%) with an implantation rate of 5.3% (3.6 ± 1.0 embryos transferred; Figure 3Go, Table IIGo). Taken together, these data suggest that morphological analysis, when considered alone, is relatively inaccurate in the prediction of success rates.

In the present study, embryos were never transferred on day 1, and therefore both zygote and embryo quality were always determined. Where possible, the best quality embryos were selected according to the previously mentioned criteria (see Materials and methods). Whether zygote quality was predictive of embryo implantation independent of embryo quality, was tested by examining defined cohorts of transferred embryos. Cases were examined in which embryos derived from a group 3 cohort of zygotes (>10 points) were selected for transfer (Table IIGo); the embryo morphology score in these cases was 12.1 ± 1.2 (28 embryos in six transfers). As previously stated, only one patient achieved pregnancy, suggesting that poor zygote quality is indicative of a poor prognosis, even when embryo quality is reasonable (compared with group 2 embryos; Table IIGo). As an alternative, cases were examined in which a group 3 cohort of embryos was the only material available for transfer, even after the observation of top quality zygotes. In these cases, one patient in 18 achieved pregnancy (5.5%), and the zygote score for these patients was 13.2 ± 0.8 (76 embryos). Here, the reasonable zygote score did not indicate a good prognosis (compare with group 2 zygotes; Table IIGo).

Is growth rate together with morphology predictive of success?
The above results suggest that little predictive information on pregnancy and implantation rates can be gained from the simple morphological analysis of human zygotes or embryos. Indeed, the rate of development of embryos cultured in vitro is often used as a predictor of success after IVF (Edwards et al., 1984Go; Cummins et al., 1986Go). The rate of development alone was predictive of success after IVF in the present data; pregnancies achieved after day 2 transfer (40–41 h after insemination) were characterized by 3.44 ± 1.26 (117 embryos in 32 patients) blastomeres as opposed to 2.96 ± 1.20 cells (183 embryos in 55 patients) in patients not achieving pregnancy (two-tailed t-test, P = 0.001). The number of blastomeres in couples achieving pregnancy after embryo transfer on day 3 (60–61 h after insemination) was also significantly greater than those not achieving pregnancy (6.04 ± 1.57 blastomeres in 128 embryos from 34 pregnant patients versus 5.06 ± 2.07 blastomeres in 123 embryos from 33 non-pregnant patients; two-tailed t-test, P < 0.001). In the present study, embryos were selected for transfer based on both blastomere number and morphological analysis. A combination of analyses increases the degree of predictability of IVF outcome (Racowsky et al., 2000Go; Scott et al., 2000Go). A test was conducted to determine whether a combination of growth rate with embryo and zygote morphology was more indicative of outcome than morphological analysis alone by calculating the mean weighted score for the cohort of embryos transferred after IVF and comparing these scores with the IVF outcome. A strong correlation was noted between the scores and pregnancy and implantation rates (Table IIGo). Results were subdivided into day 2 and day 3 scores due to the difference in score obtained on these days. Day 2 scores were characterized by a weak correlation between pregnancy and implantation rates. Group 1 embryo cohorts achieved 17/34 pregnancies (50%), with an implantation rate of 11.9% (mean of 3.4 ± 0.8 embryos transferred; Figure 3Go, Table IIGo). Group 2 material was characterized by 7/21 pregnancies (33.3%), and with an implantation rate not significantly different (13.3%) from that of group 1 (3.1 ± 0.4 embryos transferred; Figure 3Go, Table IIGo). On day 2, group 3 embryo cohorts achieved 6/24 pregnancies (25.0%), with an implantation rate of 10.4% (3.5 ± 0.5 embryos transferred; Figure 3Go, Table IIGo), which was not significantly lower than results obtained with group 2 embryo cohorts. In contrast, day 3 scores were marked by a strong correlation between pregnancy and implantation rates. Group 1 cohorts on day 3 were characterized by 20/28 pregnancies (71.4%) and an implantation rate of 24.5% (3.5 ± 0.8 embryos transferred; Figure 3Go, Table IIGo). Group 2 achieved 14/31 pregnancies (45.2%) and an implantation rate of 13.4% (3.5 ± 0.5 embryos transferred; Figure 3Go, Table IIGo). Group 3 cohorts achieved only two pregnancies in 16 cases (pregnancy rate 12.5% and implantation rate 4.2%; 3.7 ± 0.9 embryos transferred; Figure 3Go, Table IIGo).

These data suggest that the combination of embryo morphology, number of blastomeres and zygote score gives a high predictability of pregnancy. However, pregnancies may occur throughout the spectrum of embryo cohorts due to the presence of a single grade 1 embryo even in cohorts with a low mean score. Whether the presence of top quality embryos in transfers influenced pregnancy and implantation rates was tested by examining cases where different numbers of top quality embryos were present. Patients were selected in which four embryos were transferred to enable comparison of the results. In total, 107 patients (total of 47 pregnancies) were included in the analysis (Table IIIGo). Where no top quality embryos were present, eight pregnancies were obtained in a total of 24 patients (33.3%), but the implantation rate was low (9.4%; Table IIIGo)). In cases where four top quality embryos were transferred, a total of six pregnancies was achieved in eight patients (75%) (Table IIIGo). The implantation rate was equally high in these cases, with 28.1% of transferred embryos implanted into the uterus (Table IIIGo). These data demonstrate that embryos scored as top quality using the weighted scoring system presented herein are characterized by a significantly higher potential for implantation than lower-scoring embryos.


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Table III. Impact of high-scoring embryos in the transferred cohort
 
Whether maternal age had any effect on the weighted embryo score was tested by examining the pregnancy and implantation rates together with embryo score in patients categorized by maternal age. Maternal age is known to have a defined negative effect on pregnancy rate in IVF cycles, and this observation was confirmed in the present study. Patients of maternal age <30 years achieved a pregnancy rate of >60%, whether embryos were transferred on day 2 or 3 after fertilization (Table IVGo). In cases when maternal age was >40 years, the pregnancy rate did not exceed 20% (Table IVGo). Embryo score was diminished in relation to maternal age (Table IVGo), though no significant differences were seen between patients aged <30 years and >40 years (Table IVGo). These data suggest that maternal age heavily influences pregnancy and implantation rates during IVF cycles, but this was not due to any effect on embryo score.


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Table IV. Effect of maternal age on weighted embryo score and pregnancy rates
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, an investigation was made as to whether the morphological analysis of human zygotes and preimplantation embryos, either independently or together, might be used to determine IVF outcome. A scoring system for human zygotes and preimplantation embryos was developed, based on previously described factors (Edwards et al., 1980Go, 1984Go; Cummins et al., 1986Go; Puissant et al., 1987Go; Visser and Fourie, 1993Go, Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Hu et al., 1998Go; Scott and Smith, 1998Go; Tesarik and Greco, 1999Go), and the data obtained were examined retrospectively to determine which scoring system was the more accurate. Data obtained from the in-vitro culture of human embryos, including embryos selected for transfer, showed that the quality of human embryos (as determined by the present scoring system) often changed significantly from day to day. In this sense, top quality zygotes could become very low quality embryos after in-vitro culture, while low quality zygotes often became top quality embryos. Although a significant correlation was identified between the two scores, the correlation coefficient when zygotes were compared with either day 2 or day 3 embryos was low, suggesting that the two factors were relatively independent. These data strongly suggest that the static analysis of zygote quality is not highly predictive of the further development of this material. As IVF outcome was equally poor when good quality embryos derived from poor quality zygotes and poor quality embryos derived from good quality zygotes were transferred, a combination of scores is required to maintain reliability when selecting embryos for transfer. In the present study, embryos for transfer were selected on the basis of blastomere number followed by embryo morphology, and finally by day 1 morphological score. In our hands, this system was highly predictive of IVF outcome. When compared with the IVF outcome obtained without using the score, a 1.9-fold increase in predictability was achieved. In fact, a pregnancy rate of 75% was obtained, together with a corresponding implantation rate of 28.1%, when a cohort consisting of 100% top quality embryos was transferred into the uterus. An implantation rate of 28.1% is very reasonable considering that an implantation rate of 40% is expected after blastocyst transfer (Gardner et al., 1998Go; Gardner and Schoolcraft, 1999Go). This implantation rate also compares very well with the relatively low (13–15%) implantation rates obtained globally in the present centre (see Figure 3Go). The data support the hypothesis that zygote and embryo growth are independent variables and both must be considered to enable the best chance of success in IVF protocols.

The data then suggest that neither zygote nor embryo morphology alone, nor developmental rate, are individually as predictive of IVF outcome than a combination of systems. The present system could be used to form a suitable strategy for cycles of IVF, reducing the number of embryos selected for transfer (see Scott et al., 2000Go). This would enable the maintenance of high pregnancy rates with a consequent reduction in the incidence of multiple pregnancies. However, the influence of maternal age on this factor should be taken into consideration, as both the present data and previous reports document a reduction in pregnancy and implantation rates when good quality embryos are transferred into patients of advanced maternal age (van Kooij et al., 1996Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
These studies were funded by grants from Ipsen, Italy to G.De Placido and from Serono Pharma, Italy to B.Dale and G.De Placido. The authors also thank Fondazione Nuovi Orizzonti, Naples, Italy for support. Vincenzo Monfrecola provided invaluable technical support.


    Notes
 
3 To whom correspondence should be addressed at: Area Funzionale di Medicina della Riproduzione, Università degli Studi `Federico II', Via S. Pansini, 5, 80131 Naples, Italy. E-mail: martinwilding{at}katamail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Alikani, M., Calderon, G., Tomkin, G., Garrisi, J., Kokot, M. and Cohen, J. (2000) Cleavage anomalies in early human embryos and survival after prolonged culture in vitro. Hum. Reprod., 15, 2634–2643.[Abstract/Free Full Text]

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Dale, B., Fiorentino, A., De Stefano, R., Di Matteo, L., De Simone, M.L., Wilding, M. and Zullo, F. (1999) Activated oocyte transfer (AOT) as a stress-free approach to the treatment of infertility. Hum. Reprod., 14, 1771–1772.[Abstract/Free Full Text]

Dale, B., Fiorentino A., De Simone, M.L., Di Matteo, L., Scotto di Frega, A., Wilding, M., Fehr, P., Bassan, M., Lo Giudice, C., Maselli, A. et al. (2002) Zygote versus embryo transfer: a prospective randomised multicentre trial. J. Assist. Reprod. Genet. (in press).

Desai, N., Goldstein, J., Rowland, D. and Goldfarb, J. (2000) Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study. Hum. Reprod., 15, 2190–2196.[Abstract/Free Full Text]

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Submitted on August 3, 2001; resubmitted on February 11, 2002; accepted on May 1, 2002.