The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5

Kuo-Chung Lan1, Fu-Jen Huang1,3, Yi-Chi Lin1, Fu-Tsai Kung1,3, Chin-Hsiung Hsieh1, Hsuan-Wei Huang1, Ping-Heng Tan2 and Shiuh Young Chang1,3,4

1 Department of Obstetrics and Gynecology, 2 Department of Anesthesia, Chang Gung Memorial Hospital, Kaohsiung and 3 Medical School, Chang Gung University, Taoyuan, Taiwan

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, 123 Ta-Pei Road, Niaosung Hsiang, Kaohsiung, Taiwan. e-mail: blue{at}adm.cgmh.org.tw


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of this study was to evaluate the ability of using the Z-score alone, or, in combination with the day 3 embryo morphology score, to predict embryo viability at day 5 from a large cohort of embryos derived from patients undergoing treatment with IVF/ICSI. METHODS AND RESULTS: In this retrospective study, a total of 1894 zygotes from 346 treatment cycles (295 couples) was analysed between January 2001 and May 2002. The Z-scoring system was useful in predicting day 5 embryo survival. The mean ± SD day 5 embryo survival rates were 78.2 ± 1.7, 49.0 ± 2.5, 21.4 ± 3.2 and 11.8 ± 5.6% for Z-1, Z-2, Z-3 and Z-4 zygotes groups respectively. Embryos derived from Z-1 scores and grade I day 3 embryo scores showed the best day 5 embryo survival and a very high implantation potential. CONCLUSIONS: These data suggest that a combined evaluation of the Z-score and day 3 embryo morphology is highly predictive of embryo outcome after IVF/ICSI. The Z-score could be of great help in the selection of embryos for cultures extended to later stages. The Z-score alone, or preferably in combination with day 3 embryo morphology, is useful in the determination of the most suitable embryos and the number of embryos for transfer, thus achieving the optimal chance of conception while reducing the risk of high order multiple pregnancy.

Key words: embryo morphology/embryo survival/embryo transfer/implantation/zygote scoring


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the major criticisms of IVF treatment is that it leads to a recognized increase in multiple pregnancies (Hernandez, 2001Go; Schieve et al., 2002Go). Recently, there has been a heightened awareness of the need to control the risk of high order multiple gestations. In several countries these dangers have been reduced by legal restrictions on the number of transferable embryos (Montag et al., 2001Go; Zollner et al., 2002Go). To reduce the number of embryos transferred, a more efficient and rigorous embryo selection process before transfer is needed, as it has been well demonstrated that the number of embryos transferred is closely related to success (Van Royen et al., 1999Go; Fisch et al., 2001Go; Sakkas, 2001Go; Hunault et al., 2002Go; Zollner et al., 2002Go).

The dilemma of a reduction in the multiple pregnancy rate on the one hand, and the economic and emotional impact of a low pregnancy rate on the other, can be overcome if it is possible to select embryos with a very high implantation potential. The benefit of blastocyst transfer has contributed to facilitating selection of good quality embryos and has been reported to generate high implantation rates, while lowering the multiple gestation rates following IVF (Gardner et al., 1998Go; Garcia-Velasco and Simon, 2001Go). However, the effort needed for blastocyst culture, compounded by concerns about the occasional suboptimal performance of sequential media, has led many clinics to maintain their day 2 or day 3 embryo transfer practice.

When embryos are cultured in vitro, ~50% will be arrested during the first week. The reasons for this high rate of embryonic loss during early development are unclear, but could include chromosomal abnormalities, suboptimal culture conditions or inadequate oocyte maturation (Hardy et al., 2001Go). There is also the thorny issue of not having an embryo transfer if no embryos survive to day 5.

Many methods have been suggested for use in evaluating embryo viability in an IVF programme. A limiting factor is that these measurements need to be non-invasive and not time-consuming. Several authors have proposed different criteria to identify embryos with a good prognosis for implantation and pregnancy at the pronuclear stage (Payne et al., 1997Go; Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Wittemer et al., 2000Go; Hunault et al., 2002Go; Zollner et al., 2002Go). It has been demonstrated that the use of a simple, single-observation modified zygote scoring system is positively related to blastocyst development and implantation (Scott et al., 2000Go). The zygotes are described as Z-1, Z-2, Z-3 or Z-4 in the decreasing superiority of zygote quality depending on the size, number and distribution of nucleoli within the nuclei.

Other studies have assessed the characteristics of ‘top quality’ embryos by retrospectively examining embryos that had very high implantation potential (Van Royen et al., 1999Go). These embryos had four or five blastomeres on day 2 and at least seven blastomeres on day 3 after fertilization, no multinucleated blastomeres and <20% of fragments on days 2 and 3 after fertilization.

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 a degree of unpredictability for day 5 embryo viability remains when using either the zygote or morphology alone (Milki et al., 2002Go). In addition, the transfers were sometimes mixed, with both early- and late-cleaving embryos being transferred together. This makes it difficult to draw any conclusions, since it is impossible to ascertain which of the embryos actually was implanted.

If embryos with high implantation potential can be recognized by a combined evaluation of pronuclear and cleav age stage embryo morphology, the application of these criteria in deciding the number of embryos and which embryos should be transferred will not only improve pregnancy rates but will also reduce high order multiple pregnancies by avoiding the transfer of too many superior embryos at the same time.

The purpose of this study was to evaluate the ability of using the Z-score alone, or in combination with the day 3 embryo morphology score, to predict embryo survival on day 5 from a large cohort of embryos derived from patients undergoing treatment with IVF.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
We analysed 346 consecutive cycles in 295 infertile couples that had undergone IVF/ICSI in our institution between January 1, 2001 and May 31, 2002. Couples were included only if at least one oocyte could be collected that showed signs of fertilization after 16–18 h. All cases of unsuccessful oocyte retrieval or polyspermic zygotes were excluded. All the couples completed the standard infertility work-up, which included hysterosalpingography and hormone assays in females and semen analysis in males. Patients were unselected for age, sperm parameters or infertility criteria.

Controlled ovarian stimulation
The protocol for controlled ovarian hyperstimulation followed the standard down-regulation regimen we published previously (Chang et al., 1993Go; Lan et al., 2002Go). All patients received luprolide acetate (LA) (Lupron; Abbott, USA), 1 mg (age <35 years) or 0.5 mg (age >=35 years) s.c. daily, beginning on day 21 of the previous cycle and lasting until cycle day 3, when pituitary down-regulation was evaluated by a determination of serum estradiol (E2) concentration and a transvaginal sonography (TVS) of the ovaries. If the serum E2 level was <35 pg/ml and no follicles >10 mm in diameter were noted on TVS, LA was decreased to half a dose and continued until and including the day of hCG administration. Serum E2 was assayed using a commercially available competitive immunoassay with the Immulite Analyzer (DPC Coat-a Count; Diagnostic Products Corp., USA).

If the pituitary was not suppressed, LA was continued at the same dose and the serum E2 level was rechecked every 3 days until suppression was achieved. Patients received one of the following fixed starting doses (age <35 years: 225 IU/day; age >=35 years: 300 IU/day) of rFSH (Gonal-F, 75 IU/ampoule; Serono, Switzerland) after pituitary suppression with LA. Gonadotrophin was administered daily for 5 days, after which the dose was individualized according to ovarian follicular growth. Monitoring occurred every 2–3 days starting on day 5 of stimulation with TVS and serum E2. hCG (Pregnyl; Organon, The Netherlands) 10 000 IU was administered i.m. when at least two follicles were >=17 mm in diameter with adequate serum (E2) levels. Progesterone was measured only on the day of hCG administration. Oocytes were retrieved 34–36 h after hCG administration.

Oocyte preparation
For IVF, the retrieved oocyte–corona–cumulus complexes were immediately classified according to their maturity. The oocyte– corona–cumulus complexes were cultured in M2 culture medium (Medicult, Denmark) for 3–8 h before insemination. For ICSI, the oocyte–corona–cumulus complexes were denuded and assessed shortly after retrieval. The complexes were placed in a medium with 80 IU/ml of hyaluronidase for 5 s. The cumulus and corona of the cells were removed mechanically by a set of pipettes with consecutive inner diameters of 220, 200, 180 and 160 µm. According to nuclear maturation grading, the oocytes were classified into categories, metaphase II or non-metaphase II. The latter category included oocytes at the metaphase I and prophase I stages. The denuded oocytes were cultured in an M2 culture medium for 3–8 h, and then were examined for the presence of the first polar body. After confirmation of the first polar body, ICSI was performed. The oocytes that did not develop to metaphase II after 8 h of incubation were discarded.

ICSI
The intracytoplasmic microinjection of a sperm into an oocyte was based on the protocol described by Palermo et al. (1992Go), except that polyvinylpyrrolidone was eliminated from the protocol (Tsai et al., 2000Go). The entire procedure was carried out on the heated stage of an inverted microscope.

Assessment of fertilization, embryo culture, zygote and embryo grading
After the ICSI procedure, oocytes were cultured as in the standard IVF procedure and assessed for the presence of pronuclei after 16–18 h of incubation. A total of 1894 zygotes from 346 cycles were investigated. The zygotes were scored according to the Z-score scoring system (Scott et al., 2000Go). The system took account of nuclear size and alignment, and nucleoli number and distribution. Briefly, Z-1 zygotes had equal numbers of nucleoli aligned at the pronuclear junction. The absolute number was not counted but was between three and seven. Z-2 zygotes had equal numbers and sizes of nucleoli (between three and seven) which were equally scattered in the two nuclei. Z-3 zygotes had equal numbers of nucleoli of equal sizes in the same nuclei but with one nucleus having alignment at the pronuclear junction and the other with scattered nucleoli. Zygotes with unequal numbers (a difference of more than one nucleolus) and/or sizes of nucleoli were also considered as Z-3. Z-4 zygotes were those with pronuclei that were not aligned, were of grossly different sizes or were not located in the central part of the zygote. G1.2TM medium (Scandinavian IVF Science) was used for the culture of embryos on days 1–3, and G2.2TM medium (Scandinavian IVF Science) was used for the culture of embryos from day 3 to day 5 or day 6. Veeck’s morphological grading system (Veeck, 1986Go) was modified and adopted for day 3 embryo scoring, as follows: grade I = 8 cells, blastomeres of equal size and no cytoplasmic fragments; grade II = 8 cells blastomeres of equal size and <20% cytoplasmic fragments; grade III = 8 cells with uneven blastomere sizes and no cytoplasmic fragments; and grade IV = 4 or 8 cells with >20% fragmentation. Pre-embryos with few blastomeres of any size and with major or complete fragmentation were scored as grade V. After 2 days of culture in G2.2 medium, blastocyst formation was evaluated. The scoring assessment for the blastocyst was based on the expansion state of the blastocyst and on the consistency of the inner cell mass and trophectoderm cells (Gardner et al., 1999Go). Laser zona-assisted hatching was performed early in the morning of day 3 for all cleavage stage embryos whether transferred or not. The assessment of embryo survival was based on embryo morphology and cleavage speed. Embryos that had the same number of blastomeres at two sequential times of observation, together with those zygotes that remained blocked at the pronuclear stage, were considered as developmentally arrested. A single team of embryologists coordinated all procedures, thereby ensuring that both the culture protocols and the embryo assessment were standardized.

Establishment and follow-up of pregnancy
Embryos were replaced transcervically into the uterus on day 3, 4 or 5 after oocyte retrieval, with individual consideration. In our programme, we have routinely offered blastocyst transfer (BT) to patients with three or more 8-cell embryos on day 3. Patients with fewer than three 8-cell embryos on day 3 received day 3 embryo transfer. Day 4 embryo transfers were done infrequently. Luteal phase supplementation of micronized progesterone (Utrogestan; Piette International Laboratories, Belgium) 800 mg intravaginally daily, was begun on the day of oocyte retrieval, and 5000 IU hCG was administered on day 6 after oocyte recovery in all patients. Pregnancy was confirmed by detecting hCG in the urine 2 weeks after transfer. Clinical pregnancy was determined by identifying a gestational sac at 7 weeks gestation by means of transvaginal ultrasonography. If conception had occurred, micronized progesterone supplementation was provided for an additional 4 weeks.

Statistical analysis
The risk of embryo arrest was assessed by analysing the time to a first event. The starting point for the duration of embryo survival was the date of the presence of pronuclei after 16–18 h of incubation, and the closing point was after 4 days of extended culture. In addition, the endpoints were the duration of overall survival and event-free survival (for the latter, the event was the embryo arrest). In the analysis of embryo arrest, data from patients who had received a day 3 or day 4 transfer were censored after the time of transfer (Rosner, 2000Go). The cumulative probability from the time of zygote scoring was calculated using the product-limit method of Kaplan and Meier (1958Go). For the purpose of this analysis, the effects of clinical and laboratory factors were examined. The statistical significance of each variable was first evaluated by means of the log-rank test (univariate analysis). A multiple regression analysis based on the Cox proportional hazards model with stepwise forward procedure (multivariate analysis) was used to identify independent prognostic factors and to test for the interactive effects of the covariates (Cox, 1972Go). Continuous data are summarized as means ± SD.

The Sigmastat statistical package (Jandel Corporation, USA) was used for data analysis. The top quality embryo transfer study included the unpaired Student’s t-test or Mann–Whitney rank sum test for comparison of means, and Fisher’s exact t-test for proportions. All P-values are two-sided, and P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General characteristics
For the 346 cycles included in this study, general characteristics concerning causes of infertility, maternal age, assisted reproduction technique (IVF or ICSI) and outcome were noted and presented in Table I. The characteristics of 336 embryo transfer cycles, including 136 day 3 transfer cycles, 10 day 4 transfer cycles and 190 day 5 transfer cycles, are shown in Table II. Ten cycles were deleted because there were no embryos for day 3 transfer and/or were cancelled for medical reasons. Statistical comparison between transfer cycles was not performed because of selection biases.


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Table I. Demographic characteristics of 346 cycles that underwent IVF/ICSI
 

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Table II. Laboratory and clinical data for the transfer cycles
 
Relationship between zygote score and embryo survival on day 5
The Z-score scoring system was tested to determine whether or not it could be used accurately to predict embryo survival on day 5. During the study period, 1894 fertilized embryos were produced from 346 cycles undergoing treatment with IVF/ICSI. Univariate analyses using the Kaplan–Meier method showed that day 5 embryo survival was significantly correlated with the number of oocytes retrieved (P = 0.047), cohort oocyte normal fertilization rates (P = 0.006), and the Z-score scoring system (P < 0.0001) (Table III). A Cox regression test for multivariate analysis was conducted to clarify the factors affecting the 1894 zygotes placed in extended culture. It showed that the Z-score scoring system (odds ratio = 26.90) had a significant effect on embryo survival on day 5 (Table IV). The distribution of the zygote score of the 1894 zygotes is shown in Figure 1. The survival rate of the zygotes placed in extended culture up to day 5 was 56.9 ± 1.3% in total (Figure 2A). The embryo survival rate on day 5 of the Z-1, Z-2, Z-3, and Z-4 zygote groups was 78.2 ± 1.7, 49.0 ± 2.5, 21.4 ± 3.2 and 11.8 ± 5.6% respectively (Figure 2B). Aside from the lack of significant difference (P = 0.3135) between the Z-3 groups and the Z-4 groups, there was a significant difference (P < 0.0001) in the day 5 embryo survival rates in two-by-two comparisons in the Z-score groups.


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Table III. Univariate analysis of variables in relation to embryo survival at day 5
 

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Table IV. Multivariate analysis of variables in relation to embryo survival
 


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Figure 1. Distribution of Z-scores for 1894 oocytes.

 


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Figure 2. (A) Zygotes: overall arrest-free survival. (B) Day 5 arrest-free survival according to Z-score.

 
Top quality embryo transfer study
The data obtained from 336 transfer cycles of IVF/ICSI, in which a total of 929 embryos was transferred on day 3, day 4, or day 5 after scoring zygote or embryo quality on each day of culture, were analysed retrospectively. A pregnancy rate of 44.9% and implantation rate of 26.3% were achieved between January 2001 and May 2002 (Table I). Figure 3 demonstrates the relationship between zygote scoring and day 3 morphology grading. It was noted that Z-scores obtained from zygotes were in correlation with day 3 embryo morphology scores (correlation coefficient r = 0.469; P < 0.001). In this series, a total of 832 arrest-free day 5 blastocysts were scored (810 from day 5 transfer cycles and 22 from spare embryos from day 3 or day 4 transfer cycles). The 832 embryos included 567 (68.1%) derived from Z-1 zygotes, 231 (27.8%) from Z-2 zygotes, 30 (3.6%) from Z-3 zygotes and four (0.5%) from Z-4 zygotes. The number of top scoring blastocysts (full blastocysts onward; the development of inner cell mass with many tightly packed, many cells; the trophoectoderm with many cells forming a cohesive epithelium) (Gardner et al., 1998Go) was 539. These blastocysts derived from 443 Z-1, 90 Z-2, six Z-3 and zero Z-4 zygotes.



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Figure 3. Relationship between zygote scoring and day 3 morphology.

 
In order to recognize a subpopulation of embryos with high implantation potential, we strictly defined embryos derived from Z-1 (zygotes had equal numbers of nucleoli aligned at the pronuclear junction; the absolute number was not counted but was between three and seven) and grade I (8 cells, blastomeres of equal size and no cytoplasmic fragments) day 3 embryo morphologies as ‘top quality’ embryos. In 190 day 5 embryo transfer cycles with a total of 1409 zygotes, we found that the 499 top quality embryos had a 91.98% day 5 embryo survival rate. However, embryo viability is best defined as the ability of the embryo to implant successfully and give rise to a normal healthy baby, since embryo development in culture is not always equal to the development of a viable embryo. Therefore the implantation rate and the pregnancy rate in relation to the number of top quality embryo transfer cycles were analysed (Table V). It was observed that a significant trend toward a higher implantation rate, pregnancy rate and multiple pregnancy rate was achieved, based on the numbers of top quality embryos that were available and transferred, regardless of the total embryos transfer numbers and transfer day variability. Of interest is that there were two pregnancies in group V, and they were both high order pregnancies. When comparing group I cycles with only top quality embryo transfers, and group II cycles with only non-top quality embryo transfers, we found the pregnancy rate and implantation rate were remarkably different. Specifically, in one embryo transfer cycle, non-top quality embryo transfers without any pregnancies were achieved (Table VI). In addition, there were no statistically significant differences in pregnancy and implantation rates between the same top quality embryo numbers available, no matter whether or not they were associated with the numbers of non-top quality embryos that were transferred (Table V and Table VI).


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Table V. Analysis of implantation rates and pregnancy rates in relation to numbers of top quality embryos
 

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Table VI. Analysis of implantation rates and pregnancy rates between group I, in which only top quality embryos were transferred, and group II, in which only non-top quality embryos were transferred
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our findings suggest that when experienced embryologists choose embryos derived from the Z-score alone, or a combination with day 3 morphological parameters, they are using a powerful tool for the evaluation of embryo quality and embryo transfer selection strategy.

The transfer of embryos at the blastocyst stage has several advantages, one of which is the better assessment of embryo survival. We employed Cox regression using a forward stepwise selection of the variables as a strategy to identify the clinical and laboratory predictors of embryo survival up to day 5 after IVF/ICSI. The most important predictor identified was the Z-score. This finding is in accordance with a previous study (Scott et al., 2000Go). Hardy et al. also suggested that research on improving IVF success rates should move from its current concentration on optimizing culture media to focusing more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages (Hardy et al., 2001Go). They proposed that the factors predisposing an embryo to develop normally or arrest are largely determined at the 1-cell stage. Environmental effects modulate these, but only to a limited extent, so that there will always be a group of embryos that will arrest and a group that survive, even under significantly substandard conditions.

Figure 2A illustrates the importance of the Z-score in predicting embryo survival and the positive impact of an extended culture. All the zygotes placed in extended culture had a day 5 embryo survival rate of 56.9 ± 1.3%. These predictions are consistent with empirical data found in the literature. Reported rates of blastocyst formation in vitro vary considerably, ranging from 35 (i.e. a 65% arrest rate) in a simple salt solution, to between 60 and 70% (i.e. a 30–40% arrest rate). Hardy et al. (2001Go) have envisaged that 25% of embryos have a low death potential and will develop normally under most circumstances, those with an intermediate death fate (25%) will develop normally under favourable environmental circumstances but arrest under poor conditions, and the remaining 50% will always arrest.

From our data, 78.2 ± 1.7% of the Z-1 zygotes were arrest-free on day 5, suggesting that these zygotes had a low death potential and would develop normally under most circumstances.

The key issue regarding whether blastocyst transfer provides better pregnancy rates than the transfer of day 2–3 embryos remains unsolved. Gardner et al. (1998Go) demonstrated that blastocysts showed a significantly higher implantation rate than cleavage stage embryos transferred on day 3, which is mostly due to a better embryo selection and the improved embryo–uterine synchrony. Not all investigators agree that blastocyst transfer improves implantation rates; however, similar implantation and pregnancy rates were found with blastocysts and cleavage stage embryos in a controlled trial (Coskun et al., 2000Go) and in retrospective studies (Toledo et al., 2000Go). Although many factors influence the result of an IVF cycle (e.g. stimulation response, endometrial receptivity, oocyte maturity, culture conditions, paternal contribution), embryo morphology is regarded as one of the most important. Embryo morphology is easy to evaluate, and has thus been the major parameter for the selection of embryos to transfer. Much effort has been made in trying to find an optimal scoring system for embryo morphology. To address these issues, different embryonic criteria have been used: pronuclear morphology, cleaved embryo morphology (Hsu et al., 1999Go) and kinetic parameters (Shoukir et al., 1997Go).

Selecting embryos with high implantation potential is one of the most important challenges in the field of assisted reproductive technology. Previous studies have suggested a relationship between pronuclear morphology and implantation ability. However, few studies have focused on the non-invasive and time-saving Z-score for zygote evaluation in relation to embryonic development. Our data are in concordance with the concept of human pronuclear embryos being positively related to blastocyst development (Scott et al., 2000Go), and we suggest that Z-3 plus Z-4 may be denoted as a revised Z-3 in terms of the same embryonic developmental viability.

Embryo quality has traditionally been evaluated based on the cleavage rate and blastomere morphology (Puissant et al., 1987Go). However, due to the high rate of genetic wastage, these characteristics alone lack sufficient sensitivity and specificity to predict high pregnancy rates, and have often prompted clinicians to transfer more embryos, which has led to a dramatic increase in multiple pregnancies. When our data were stratified based on the number of transferred top quality embryos, those patients with at least one top quality embryo had a pregnancy rate of 36.7–64.8% and an implantation rate of 20.2–47.1% (Table V). There is a trend toward an increase in the pregnancy and implantation rate based on the increase of top quality embryos being transferred. The number of top quality embryos transferred determined the implantation and pregnancy rates when compared with the same number of top quality embryo transfer cycles that were mixed with non-top quality embryos (Table V and Table VI). In other words, when mixed with top quality embryos, non-top quality embryos provided no further positive impact on transfer outcome. These findings suggest that top quality embryos can be used accurately to identify embryos with high potential for establishing a pregnancy. It appears that the Z-scoring system, when associated with a morphological and kinetic evaluation of embryos on day 3 after oocyte retrieval, allows the selection of embryos with high implantation potential. It is important to note that the incidence of multiple pregnancies was very high when more than one top quality embryo transfer was replaced in a single transfer cycle, irrespective of the number of non-top quality embryos that accompanied them in mixed transfer cycles. The wisdom of limiting the transfer to top quality embryos is easy to understand.

When the blastocyst transfer policy is offered to patients with a good prognosis, such as those of our study population, it is reassuring to see that all cycles progressed to the blastocyst stage embryo transfer, and that no patient suffered complete embryo arrest. Racowsky et al. also showed no cleavage stage arrest in 83 cycles when three or more 8-cell embryos were present (Racowsky et al., 2000Go). However, extended culture and blastocyst transfer are not necessary prerequisites for the current trend towards a single embryo transfer (Rienzi et al., 2002Go). In fact, the morphology of human pronuclear zygotes has been shown to be positively related to blastocyst development (Scott et al., 2000Go), whereas day 3 morphology alone is a poor predictor for blastocyst development in extended cultures (Graham et al., 2000Go). Rijnders and Jansen (1998Go) concluded that the predictive value of day 3 embryo morphology in relation to subsequent blastocyst formation is limited because only 51% of the embryos that have been preselected for transfer on day 3 could reach the blastocyst stage on day 5. We suggest that combining the Z-score and day 3 morphology score may overcome these drawbacks.

Admittedly, we used day 5 embryo survival in culture as our criteria to judge the accuracy of top quality embryos, and it could be argued that embryos probably behave differently in utero or in vitro. However, since implantation potential was tested, we believe that the Z-score comparison is valid.

In conclusion, the Z-score for pronuclear morphology is a valuable additional criterion for selecting embryos for extended culture. The Z-score alone or in combination with day 3 embryo morphology is useful in determining the most suitable number of embryos for transfer, and achieving the optimal chance of conception while reducing the risk of high order multiple pregnancy. Our findings are of value no matter whether day 3 or day 5 transfer is carried out.


    Acknowledgement
 
The authors would like to thank Hsueh-Wen Chang, PhD for statistical analysis and consultation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 5, 2002; accepted on February 18, 2003.