1 Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University,11100 Euclid Avenue, Cleveland, OH 44106, USA
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Abstract |
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Key words: day 3 transfer/embryo score/IVF/morphology/pregnancy prediction
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Introduction |
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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., 1986; Puissant et al., 1987
; Staessen et al., 1992
; Steer et al., 1992
; Roseboom et al., 1995
; Ziebe et al., 1997
). 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, 1995
; Saith and Sargent, 1995
). One such scoring system, the embryo quality or EQS score (Cummins et al., 1986
), 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., 1995
) and the cumulative embryos score (CES) (Steer et al., 1992
; Visser and Fourie, 1993
) 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., 1999) 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., 1996
). 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.
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Materials and methods |
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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 oocytecumulus 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 (75100 µl) under oil (Squibb) at 37°C in a humidified atmosphere with 5% CO2. Pronuclear check was performed 1719 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 3638 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 46 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, ~6567 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., 1999). 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 1
. Figure 2
illustrates an embryo showing early signs of compaction. A decrease can be seen in cell definition between individual blastomeres.
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Results |
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Figure 7 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 6c
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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Discussion |
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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., 1987; Lewin et al., 1994
; Giorgetti et al., 1995
; Ziebe et al., 1997
), 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., 1994
, 1995
; Almeida and Bolton, 1996
, 1998
). The importance of timely cell division is further exemplified in another study (Huisman et al., 1994
), 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., 1996) or to include all transferred embryos as either a cumulative score (Cummins et al., 1986
) or an average score (Roseboom et al., 1995
). 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 6c
). The pregnancy rate for transfers with a score of 66.9 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 7
). 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., 1995). Yet it is clear that fragmenting embryos transferred back to the uterus do in fact implant, albeit at lower rates (Giorgetti et al., 1995
). Recent observations (Alikani et al., 1999
) 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, 1999
). 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 4). 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.
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Notes |
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References |
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Submitted on January 26, 2000; accepted on June 15, 2000.