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
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Abstract |
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Key words: assisted reproduction/embryo score/human embryo/IVF/single embryo transfer
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Introduction |
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Although a correlation between zygote and embryo morphology has been shown to exist (Ludwig et al., 2000; Scott et al., 2000
), 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, 1998
). Furthermore, the developmental rate of embryos has been suggested to be indicative of implantation potential (Edwards et al., 1984
; Cummins et al., 1986
; Racowsky et al., 2000
). One group (Racowsky et al. 2000
) 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.
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Materials and methods |
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Study design
The zygote scoring system was developed based on previously published methods (Figure 1A). 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., 1998
; Scott and Smith, 1998
; Tesarik and Greco, 1999
; Scott et al., 2000
; Wittemer et al., 2000
) [Figure 1A(i)
and Table I
]. Second, the morphology and orientation of nucleoli was scored, equal numbers of nucleoli opposed in the zygote centre considered top quality (Scott and Smith, 1998
; Tesarik and Greco, 1999
; Scott et al., 2000
; Wittemer et al., 2000
) [Figure 1A(ii)
and Table I
]. 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, 1998
; Tesarik and Greco, 1999
; Wittemer et al., 2000
; Scott et al., 2000
) [Figure 1A(iii)
and Table I
]. In addition, a system for scoring embryos was developed (Figure 1B
). Again, three parameters were scored. First, blastomere volume and synchrony of cleavage was assessed (Edwards et al., 1980
, 1984
; Cummins et al., 1986
; Puissant et al., 1987
; Visser and Fourie, 1993
; Giorgetti et al., 1995
; Ziebe et al., 1997
; Hu et al., 1998
; Desai et al., 2000
; Hardarson et al., 2001
; Van Royen et al., 2001
) [Figure 1B(iv)
and Table I
]. 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., 1980
, 1984
; Cummins et al., 1986
; Puissant et al., 1987
; Visser and Fourie, 1993
Giorgetti et al., 1995
; Ziebe et al., 1997
; Hu et al., 1998
; Desai et al., 2000
; Hardarson et al., 2001
; Van Royen et al., 2001
) [Figure 1B(v)
and Table I
]. Third, the extent of fragmentation was considered (Alikani et al., 1999
, 2000
) [Figure 1B(vi)
and Table I
]. 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 I
). The maximum score obtainable was 15 for zygotes and 15 for each day of embryo culture (Table I
). 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 1014 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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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.
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Results |
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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., 2000). 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 3
, Table II
). 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 3
, Table II
). 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 3
, Table II
). 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 II); 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 II
). 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 II
).
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., 1984; Cummins et al., 1986
). The rate of development alone was predictive of success after IVF in the present data; pregnancies achieved after day 2 transfer (4041 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 (6061 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., 2000
; Scott et al., 2000
). 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 II
). 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 3
, Table II
). 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 3
, Table II
). 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 3
, Table II
), 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 3
, Table II
). Group 2 achieved 14/31 pregnancies (45.2%) and an implantation rate of 13.4% (3.5 ± 0.5 embryos transferred; Figure 3
, Table II
). 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 3
, Table II
).
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 III). 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 III
)). In cases where four top quality embryos were transferred, a total of six pregnancies was achieved in eight patients (75%) (Table III
). The implantation rate was equally high in these cases, with 28.1% of transferred embryos implanted into the uterus (Table III
). 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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Discussion |
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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., 2000). 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., 1996
).
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Acknowledgements |
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Notes |
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References |
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Submitted on August 3, 2001; resubmitted on February 11, 2002; accepted on May 1, 2002.