Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study

Nina N. Desai 1,2, Jessica Goldstein1, Douglas Y. Rowland1 and James M. Goldfarb1

1 Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University,11100 Euclid Avenue, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.

Key words: day 3 transfer/embryo score/IVF/morphology/pregnancy prediction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Successful outcomes after IVF procedures depend on many factors, one of the most critical being embryo quality (Saith et al., 1995; reviewed by Saith and Sargent, 1995Go; Roseboom and Vermeiden, 1995Go). Implementation of a good embryo scoring system has many potential benefits, such as: (i) allowing proper selection of embryos for transfer, (ii) standardizing embryo grading between embryologists, (iii) reducing high order multiple births by adjustment of transfer number according to embryo quality, (iv) permitting comparison of embryo quality between patient cycles, and (v) evaluation of different culture regimens.

The three main morphological features considered in embryo grading systems to date have been cell number, blastomere size and shape and degree of fragmentation (Cummins et al., 1986Go; Puissant et al., 1987Go; Staessen et al., 1992Go; Steer et al., 1992Go; Roseboom et al., 1995Go; Ziebe et al., 1997Go). These factors have been combined in a variety of different ways to yield embryo scoring systems to predict pregnancy (see reviews by Roseboom and Vermeiden, 1995Go; Saith and Sargent, 1995Go). One such scoring system, the embryo quality or EQS score (Cummins et al., 1986Go), was based on an assessment of blastomere symmetry, cytoplasmic appearance and amount of fragmentation. These same authors also proposed a formula for describing an embryo's development rate (EDR), based on the ratio of the time at which an embryo was observed to reach a particular stage and the expected time interval. Both the EQS and EDR were found to be inter-related and gave insight into pregnancy potential of individual embryos. Other scoring systems such as the average morphology score (AMS) (Roseboom et al., 1995Go) and the cumulative embryos score (CES) (Steer et al., 1992Go; Visser and Fourie, 1993Go) incorporate number of embryos transferred into the final score. All of these scoring systems were designed for evaluation of embryos on day 2 of culture. A scoring system specific for day 3 embryos has not been extensively looked at.

Culture and transfer of human embryos on day 3 allows for additional assessment of embryonic development and improvement in selection criteria. A recent study on day 3 embryos (Alikani et al., 1999Go) suggests that the pattern of fragmentation on day 3 may in fact give a better measure of implantation potential than the degree of fragmentation, which has always been a predominant feature in day 2 embryo scoring systems. In yet another study, assessing morphological attributes of day 3 embryos giving rise to pregnancies, investigators observed two features which they suggested might be early indicators of embryos likely to undergo embryonic compaction (Wiemer et al., 1996Go). The first was an increase in cytoplasmic granularity and the appearance of tiny pits in the cytoplasm of blastomeres. The second was an increase in cell:cell adherence and loss of definition between individual blastomeres. Another feature that they scored was blastomere expansion. In high quality embryos, individual blastomeres were expanded and touched the zona, leaving very little perivitelline space.

Day 3 marks an important transition point for the human embryo and it is likely that careful attention to additional morphological features may aid in embryo evaluation. The objective of this study was to devise a simple system for embryo grading which incorporates features unique to day 3 embryos and to derive a scoring system for day 3 transfers that is predictive of pregnancy outcome.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design
This was a retrospective study looking at day 3 embryo morphology in patients undergoing IVF treatment at University MacDonald Women's hospital between December 1997 and mid-July 1998.

IVF procedure
Ovarian stimulation for all patients consisted of down-regulation with leuprolide acetate (Lupron: TAP Pharmaceuticals, Chicago, IL, USA) followed by stimulation with human menopausal gonadotrophins (Pergonal) or recombinant follicle stimulating hormone (Gonal F; Serono Laboratories; Randolph, MA, USA). Human chorionic gonadotrophin (HCG; Profasi; Serono, Italy) was administered when two or more follicles reached 18 mm mean diameter. Follicles were aspirated 36 h later using transvaginal ultrasound and oocyte–cumulus complexes were isolated.

Human tubal fluid medium (HTF; Genex International, Madison, CT, USA) supplemented with 6% plasmanate was used for oocyte insemination/sperm preparation, and maternal serum (15%) was used for embryo culture. Conventional IVF or micro-assisted fertilization (intracytoplasmic sperm injection) was carried out using motile spermatozoa prepared by density gradient centrifugation. Oocyte culture was performed in microdroplets (75–100 µl) under oil (Squibb) at 37°C in a humidified atmosphere with 5% CO2. Pronuclear check was performed 17–19 h post-insemination. Normally fertilized embryos were moved to fresh growth medium microdrops containing serum supplemented HTF and monitored daily. During day 2 observations, embryos exhibiting multinucleation were identified. These embryos were not considered for transfer. Embryo transfer was performed on day 3. The number of embryos transferred to the patient was based on the number and quality of embryos available and the age of the patient. In general, three embryos were transferred to patients under the age of 36 years. In cases of increased percentage fragmentation and/or poor cleavage, an additional embryo might be transferred, usually at the patient's request. In the 36–38 years age group, three to four embryos were transferred and at 39 years and above, four or more embryos were transferred. Clinical pregnancy was confirmed by the presence of a fetal heart beat on ultrasound 4–6 weeks after the embryo transfer.

Embryo evaluation
A total of 316 transferred embryos from 93 consecutive patients was retrospectively evaluated using video recordings made on the day of transfer. No patient selection was involved. All embryo observations were performed between 08:00 and 10:00 h in the morning, ~65–67 h post-insemination. Embryo selection for transfer was made at this time and selected embryos were recorded on videotape in several focal planes to allow visualization of all blastomeres. Embryos were hatched using acid Tyrode's solution and returned to the incubator until transfer (usually between 10:30 and 12:30 h).

The parameters to be evaluated were decided on prior to retrospective examination of videotapes of transferred embryos. Parameters were selected based on the laboratory's historical experience with embryo selection for transfer, observations on spare embryos cultured to blastocysts and a survey of current literature. A consensus was then reached on representative examples of the morphological parameters selected for evaluation. Embryo grading was performed by a fifth observer, not associated with our IVF programme and unfamiliar with pregnancy outcomes. Each transferred embryo was evaluated for the following: (i) cell number, (ii) presence of equal sized cells, (iii) good blastomere expansion, i.e. blastomeres touching the zona with minimal perivitelline space, (iv) cellular cytoplasm clear of vacuoles, (v) presence of cytoplasmic pitting, (vi) signs of compaction and (vii) the pattern of fragmentation (FP). The FP was scored as follows, using the criteria previously outlined (Alikani et al., 1999Go). Embryos exhibiting minimal fragments, usually in association with a single blastomere were designated as FP I. Pattern II embryos have some small fragments, localized in the perivitelline space. In pattern III fragmentation, many small fragments can be seen throughout the cleavage cavity and perivitelline space. Pattern IV embryos also exhibit many fragments, usually in association with uneven-sized blastomeres and the fragments are often large, almost the size of a single blastomere. In pattern V, embryo fragmentation is so extensive that blastomeres cannot be distinguished. In our laboratory, we hatch all embryos prior to transfer but do not perform fragment removal. An embryo exhibiting cytoplasmic pitting is depicted in Figure 1Go. Figure 2Go illustrates an embryo showing early signs of compaction. A decrease can be seen in cell definition between individual blastomeres.



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Figure 1. Day 3 embryo exhibiting cytoplasmic `pitting'. Majority of blastomeres have increased cytoplasmic granularity and a `pockmarked' appearance typical of embryos scored positively for `pitting'. Original magnification x400.

 


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Figure 2. Day 3 embryo showing an increase in cell:cell adherence. Definition between individual blastomeres is reduced and the embryo would be scored positive for early signs of compaction. Original magnification x400.

 
Statistical analysis
Differences among variables, in pregnant versus non-pregnant groupings, were analysed using the {chi}2 test for categorical variables (with the Yates' continuity correction) or the Student's t-test for continuous variables, as appropriate. The three different scoring systems were constructed empirically. They were assessed by t-tests of scores in pregnant and non-pregnant groupings and by their ability to predict pregnancy, as measured by sensitivity, specificity and accuracy. We also considered as a benchmark the result of a discriminant analysis of the data set, which theoretically gives the best possible performance of any additive prediction formula for that data set (Lachenbruch, 1975Go). Statistical significance was taken as P < 0.05 and no correction for the multiple testing of data was made, as the statistical tests of parameters were considered to be preliminary model building tasks.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The majority of patients received either three or four embryos (71/93). The clinical pregnancy rate was 49% with three embryos transferred and 40% with four embryos. To understand these results further, we looked at age and number of embryos transferred (Table IGo). We found that with increasing age, more embryos were being transferred but there was a decrease in both pregnancy and implantation rates. However, we did not find a significant difference in age distribution or number of embryos being transferred when we compared the pregnant and non-pregnant patient population. Nor did we find patient diagnosis in the pregnant and non-pregnant groups to be very different. Of the different indications for infertility treatment, tubal factor (31%) and male factor (41%) accounted for the majority of cases in this patient set. Embryo quality and pregnancy rates in cycles with standard oocyte insemination versus ICSI (n = 38) were not significantly different.


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Table I. Relationship between number of embryos transferred, age and clinical outcome
 
The FP of transferred embryos is shown in Figure 3Go. In pregnancy associated cycles, 48% of transferred embryos showed pattern I fragmentation and 26% pattern II. In non-pregnant cycles, we noted a decrease in percentage of FP I embryos and a shift towards more embryos of FP II but differences did not reach statistical significance. In seven women, only embryos with FP III or IV were available for transfer and no pregnancies were obtained in this patient set. Information pertaining to FP in the entire cohort of embryos was not available, since video recordings had only been made of embryos selected for transfer.



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Figure 3. FP (FP) in transferred embryos: pregnancy-associated embryos; embryos in non-pregnant cycles.

 
The relationship between pregnancy outcome and transfer of embryos showing signs of early compaction and/or increased cytoplasmic granularity (`pitting') is depicted in Figure 4Go. Pregnancy rate is plotted against the number of embryos in each transfer exhibiting that morphological attribute. With no compacting embryo being transferred, the pregnancy rate was 40%. The pregnancy rate was observed to increase with the transfer of compacting embryos; however, no statistical difference was found in the rate of pregnancy for those patients receiving no compacting embryos versus those receiving at least one compacting embryo. Compaction was observed in 18.7% of the embryos selected for transfer. No statistical difference was observed in clinical pregnancy rates with the transfer of one or more pitted embryos, yet the pregnancy rates did vary for different numbers of pitted embryos transferred. Interestingly, when we looked exclusively at transfers with either three or four embryos (n = 71), we found the mean cell number to be lower in transfers where no embryonic pitting was observed (6.1 versus 6.8, P = 0.04).



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Figure 4. Association between morphological features and pregnancy rate: number of embryos in transfer exhibiting early signs of compaction or increased cytoplasmic granularity (pitting).

 
Based on these initial data, three different scoring systems were derived to evaluate day 3 embryos. In scoring technique no. 1, we looked only at the average cell number in transferred embryos and its correlation to pregnancy. In the second scoring system (Figure 5aGo), cell number was combined with grading of FP. If an embryo had a FP of >II, we subtracted 2 points from the blastomere number, essentially dropping the embryo down one cleavage level. In the third scoring system (Figure 5bGo), we incorporated not only cell number and FP as in scoring system 2, but also the five morphological features described earlier. In grading embryo morphology, 0.4 points were given for the presence of each positive feature. So if an embryo was positive for all five features a maximum of 2 points would be added, essentially bringing the embryo up by one cleavage level. As should become obvious, a perfect 8-cell with all five positive features would be scored as 10. All transferred embryos were assessed by each of the three methods and the relationship between overall score, pregnancy and implantation rates was analysed.



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Figure 5. (a) Scoring technique no. 2. Each embryo was evaluated for blastomere number and fragmentation pattern (FP). If the FP was greater than II, 2 points were subtracted from the blastomere number to give the embryo score. (b) Scoring technique no. 3. Blastomere number and FP were combined as in scoring technique no. 2 to obtain a basal score. Each embryo was then further evaluated for the presence of the following five morphological features: good blastomere expansion, equal cell size, absence of vacuoles, cytoplasmic pitting and signs of compaction. For each positive feature the embryo was given an extra 0.4 points. Thus if the embryo was positive for all five features a maximum of two points was added to give a final embryo score. This embryo score is referred to as the day 3 embryo quality (D3EQ) score.

 
All three scoring techniques showed a significant difference in mean scores between pregnant and non-pregnant patients (P = 0.02, 0.02 and 0.03 respectively). In Figure 6aGo–c, we looked more closely at the relationship between embryo score of all transferred embryos and pregnancy outcome with each of the grading techniques. Scoring technique no. 1, based on cell number alone is shown in Figure 6aGo. The average cell number in transfer cycles is plotted against pregnancy and implantation rates. Cell number appeared to affect both pregnancy and implantation rates. In transfers where the average cell number of all embryos transferred was eight or higher, the implantation rate per embryo transferred exceeded 35% and the clinical pregnancy rate was >60%. In contrast, if the average cell number in embryos transferred fell below six, the implantation rate per embryo fell to 12% and pregnancy rate was <30%. Figure 6bGo illustrates the same set of transfers, with embryos graded for both cell number and FP (scoring technique no. 2). Fragmentation appeared to counter the positive impact of cell number.



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Figure 6. Relationship between embryo score, pregnancy and implantation rates. Embryos in each transfer were individually scored using each of the described scoring techniques: (i) cell number alone (a), (ii) cell number and fragmentation pattern (FP) (b) and (iii) cell number, FP and morphological (morph) attributes (c). The average embryo score in transfers is plotted against corresponding pregnancy (clin preg) and implantation rates. Pregnant and non-pregnant patients had significantly different embryo scores with all three grading systems (P < 0.05). n = number of transfers.

 
The relationship between day 3 embryo quality score (D3EQ) and pregnancy outcome, using scoring technique no. 3 is depicted in Figure 6cGo. Cell number, fragmentation and morphological parameters were assessed and the individual score for each transferred embryo was calculated as described earlier. The clinical pregnancy rate was seen to decline when the average score of embryos transferred dropped to below 6. In the 5–5.9 range, pregnancy rate was 25% and the implantation rate was 13%. The average embryo quality scores for patients under 36, 36–38 and those 39 years and older were 7.02, 7.03 and 7.54 respectively.

Figure 7Go depicts the relationship between the average D3EQ score of only the two highest graded embryos in a transfer group, rather than all transferred embryos as in Figure 6cGo and pregnancy outcome. We felt this might better reflect pregnancy potential in any given cycle, since the overall score would not be diluted by the presence of a third, fourth or fifth poorer quality embryo. An apparent relationship was observed between the D3EQ score and pregnancy and implantation rates. Using the D3EQ score of the best two embryos in a transfer cohort, the scoring system had a sensitivity of 83% and specificity of 40%, in classifying transfer cycles into pregnant and non-pregnant, with a cut-off point at 7.0 (P < 0.05). The mean score in pregnant patients was 8.25.



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Figure 7. Relationship between the day 3 embryo quality (D3EQ) score of the two highest graded embryos in a transfer and pregnancy outcome. n = number of transfers.

 
A discriminant analysis was also performed on the data set to provide a benchmark for the predictive accuracy of the D3EQ score. The resultant discriminant function was similar in form to the D3EQ additive scoring formula and was able to predict pregnancy with 67% accuracy, thus similar in predictive ability to the D3EQ score that was able to predict pregnancy with 58% accuracy.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is a preliminary report describing techniques for scoring day 3 embryos. To date no scoring system specific for embryos cultured to the 6- to 8-cell stage has been reported on. Incorporation of features unique to day 3 embryos with other traditional scoring parameters may prove useful in classification and selection of embryos for transfer. In our laboratory, we do very little freezing at the pronuclear or early cleavage stage, instead giving preference to blastocyst stage cryopreservation. As a consequence of this laboratory policy, we often have a large cohort of embryos from which we must select two to four for transfer. Often embryos have similar blastomere number and level of fragmentation. More critical observation of embryos for features that may be early indicators of cytoplasmic activity and compaction has aided our laboratory tremendously in ranking and selecting embryos for transfer.

In deriving a day 3 embryo scoring system, the challenge that we faced was taking the many individual observations that we make on an embryo and combining these to yield a single numerical score, that reflects pregnancy potential. To do this, it was necessary first to clarify the relationship between embryo cleavage, morphological attributes and pregnancy outcome. Our findings suggested that cell number was the strongest predictor of embryo vitality and pregnancy outcome. Taken individually, morphological parameters such as fragmentation, pitting, and compaction were not sufficient to predict pregnancy. The D3EQ scoring technique that we devised took these observations into account. Cell number was given the greatest emphasis and represented the basal score. Morphological parameters were used to refine the grading further and served to raise or lower this basal score by a maximum of one cleavage stage or two points. In a laboratory setting, this simplified approach to embryo grading has practical advantages, in that it is quite easily performed and scores are easily interpreted.

Our conclusions as regards the impact of overall cell number on pregnancy are in accord with those of several other investigators (Claman et al., 1987Go; Lewin et al., 1994Go; Giorgetti et al., 1995Go; Ziebe et al., 1997Go), suggesting that slower cleaving embryos influence pregnancy rates in a negative manner. In each of these studies, the transfer of 4-cell embryos as compared to those at the 2-cell stage resulted in a two-fold increase in pregnancy rates. It follows then that with each additional day in culture, embryonic cleavage rate offers an increasing ability to discriminate between normally developing embryos and slower cleaving embryos. Chromosomal studies on embryos that are developmentally lagging or show cleavage arrest suggest an increased incidence of chromosomal anomalies (Munné et al., 1994Go, 1995Go; Almeida and Bolton, 1996Go, 1998Go). The importance of timely cell division is further exemplified in another study (Huisman et al., 1994Go), comparing embryo transfer results after 2, 3 or 4 days of embryo culture in vitro. While 82% of the embryos cultured for 4 days were developmentally lagging, they found that the implantation rate of the small percentage of `normally' developing embryos transferred to patients at the cavitating morula stage was exceptionally high (41%). In the present study, we also obtained high implantation rates (35% or more) in transfers where the mean blastomere number in transferred embryos was at least 8 cells, as would be expected for appropriately dividing day 3 embryos.

A second issue was whether to make an assessment on the basis of the best embryo transferred, as described (Wiemer et al., 1996Go) or to include all transferred embryos as either a cumulative score (Cummins et al., 1986Go) or an average score (Roseboom et al., 1995Go). One of the problems frequently associated with taking every transferred embryo and calculating a single average score is that in mixed transfers involving embryos of good and bad quality, the overall score can be lowered, and not necessarily reflect the potential for pregnancy. We can see this type of biphasic pattern in the current data set (Figure 6cGo). The pregnancy rate for transfers with a score of 6–6.9 was 58%. Between 7.1 and 9.0, the pregnancy rate ranged from 41 to 45% and with scores >9, a pregnancy rate of 67% was achieved. With increasing D3EQ score, the per embryo implantation rate also increased from 3 to 36%. The disadvantage of scoring just the best embryo is that each additional embryo transferred has pregnancy potential for which we are not giving due credit. In the present study, we found that scoring just the two best embryos was sufficient to predict pregnancy outcome (Figure 7Go). Since the majority of patients had either three or four embryos transferred, this approach essentially meant that we were making our prediction based on the quality of one-half to two-thirds of the cohort of embryos being transferred. Additional study of embryo score and multiple pregnancy will be necessary to determine if the average D3EQ score of all transferred embryos versus that of just the best two embryos offers any advantage in predicting risk of multiple pregnancy and optimal number of embryos for transfer.

Amongst morphological features, fragmentation has been consistently included in traditional scoring systems to aid in defining embryo quality (reviewed by Roseboom et al., 1995Go). Yet it is clear that fragmenting embryos transferred back to the uterus do in fact implant, albeit at lower rates (Giorgetti et al., 1995Go). Recent observations (Alikani et al., 1999Go) suggest that the absolute amount of fragmentation may bear less importance than the pattern of fragmentation. The loss of regulatory proteins during blastomere fragmentation may be one mechanism by which the developmental competence of an embryo is affected (Antczak and Van Blerkom, 1999Go). These authors noted that different spatial patterns of fragmentation had different consequences on the blastomere and the developmental potential of the embryo itself. In-vitro study of FP and developmental events, such as ability to blastulate, may aid in further defining the importance of this parameter in day 3 embryo quality assessment.

Embryonic pitting and increased cell:cell adherence as early markers of cytoplasmic activity and potential for embryonic activation also need to be looked at in depth. We noted these features to be associated with embryos most likely to proceed on to the morula/blastocyst stage when studying embryos from our younger patients being transferred on day 5 (unpublished data). In the present study, the impact of these variables was less pronounced (Figure 4Go). One explanation might be that the interaction between variables and their influence on outcome may be different in different patient subpopulations. One such subpopulation might be the 39 years and older patients whose embryos are at greatest risk for aneuploidy. Morphologically, these transferred embryos might exhibit any number of positive characteristics, but clinical outcome would be dependent on the concomitant presence of a normal chromosome complement. Independent analysis of individual variables using discriminant analysis techniques also suggested that patient subpopulations should be examined separately to understand fully the selected variables and their interactions. Because the discriminant formula was constructed using the same data that were used to measure its ability to predict pregnancy, it is not perceived as a proposed scoring system, but rather for reference.

We recognize that the retrospective nature of this study presents certain inherent difficulties. The strongest limitation with this study is that the scoring system was derived on the basis of embryos pre-selected for transfer. As such, morphological attributes deemed undesirable by the embryologists would have been under-represented in the data set analysed. To test the scoring system effectively, both transferred and untransferred embryos need to be examined. We have in fact initiated a prospective study along these lines. Scoring and observation on spare untransferred embryos will also allow further confirmation of the role of individual morphological parameters.

In conclusion, this study for the first time details a grading system specific for evaluating day 3 embryos. The D3EQ score is prognostic of pregnancy outcomes. This model needs to be further evaluated both in our own laboratory and in other in-vitro laboratories. A question of paramount interest would be whether the scoring system can predict in-vitro blastulation. Another potential application of this type of scoring system would be in comparing different culture regimens and their effects on the pattern of in-vitro embryo development.


    Notes
 
2 To whom correspondence should be addressed at: Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106, USA. E-mail: desai_nina{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 26, 2000; accepted on June 15, 2000.