1 Sher Institute for Reproductive Medicine, Las Vegas, Nevada, USA and 2 Department of Obstetrics and Gynecology, University of Nevada School of Medicine, Reno, Nevada, USA
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Abstract |
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Key words: blastocyst development/graduated embryo scoring/implantation/IVF
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Introduction |
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However, extended embryo culture has several potential drawbacks. Not all laboratories are equipped to perform blastocyst culture. Extended embryo culture requires a specialized sequential media system, which makes the success rates of IVF programmes that depend upon sustaining high blastocyst conversion rates susceptible to variations in culture media quality. Increased incubator space is necessary to accommodate extended culture and is a limiting factor for many programmes. There is also controversy surrounding the ideal stage for cryopreservation of supernumerary embryos, which many feel is best performed at the cleavage stage rather than as blastocysts. Finally, there is the thorny issue of not having an embryo transfer if no embryos survive to the fifth day.
Many programmes that perform blastocyst transfer have criteria, such as 4 or more 8-cell embryos on day 3 (Milki et al., 1999), for recommending extended culture. These criteria serve to identify patients with a good prognosis for pregnancy, regardless of whether their embryos are transferred on day 3 or day 5. Several authors (Scott and Smith, 1998
; Tesarik and Greco, 1999
; Tesarik et al., 2000
) have proposed criteria to identify embryos at the pronuclear stage, which have a good prognosis for implantation and pregnancy. Several early developmental milestones such as pronuclear symmetry, nucleolar alignment and perinuclear haloing that may be important in predicting implantation potential and pregnancy have been described (Scott and Smith, 1998
); these authors reported a 15% implantation rate in 97 patients, which could be improved to 28% in 48 patients whose embryos had a corrected embryo score of 15 or more. Tesarik and Greco reported a 50% (22/44) pregnancy rate if at least one transferred embryo was pattern 0 (normal development), compared with 9% (2/23) if only pattern 15 (abnormal development) embryos were available for transfer (Tesarik and Greco, 1999
).
It is the policy of our laboratory to culture each embryo separately, rather than in groups. This provides us with the opportunity to follow the developmental progression of individual embryos. The ability to identify on day 3 which embryos will develop into blastocysts offers the potential to increase laboratory efficiency while maintaining the high implantation and pregnancy rates associated with blastocyst transfer. By expanding upon and combining the work of previous authors, we have developed the Graduated Embryo Score (GES), which is comprised of two interval evaluations of early developmental milestones, along with a weighted assessment of conventional morphological characteristics on day 3. The purpose of this study was to evaluate the ability of GES to predict blastocyst conversion, pregnancy and implantation from a large cohort of cleavage-stage embryos derived from patients undergoing treatment with IVF.
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Materials and methods |
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After reviewing the data from the first 685 embryos, the system was modified and simplified to increase its predictive value. The present system awards a total possible score of 100 points, based on three evaluations occurring at 1618, 2527 and 6467 h post insemination (Table I). Several of the original parameters were eliminated, as they did not appear to influence the predictive value. In addition, the relative weight of each parameter was redefined following a review of the pertinent literature.
Based on previous work (Scott and Smith, 1998; Tesarik et al., 2000
; Wittemer et al., 2000
), alignment of the nucleoli along the pronuclear axis at 1618 h was given increased significance (Figure 1a
). Symmetrical cleavage and <20% fragmentation at the first cell division (Figure 1b
) were found to be among the most important parameters and were given increased weight. The relative importance of these two parameters is supported by the work of Zaninovic et al., who reported a 42% blastocyst conversion rate among embryos with cleavage at 2426 h after fertilization, compared with only 11% blastocyst formation among embryos still at the pronuclear stage (Zaninovic et al., 2000
). A 42% blastocyst development from embryos with 79 cells on day 3, compared with 14% if <7 cells, has been reported (Alikani et al., 2000
). Based on this work, a higher weight was given to embryos with 79 cells on day 3, and the highest weight given to embryos with 8 cells (Figure 1c
). After incorporating these modifications, the initial data were reanalysed and found to be significantly more predictive of blastocyst formation (Figure 1d
) and pregnancy rate.
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Results |
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When the data were stratified by the highest scoring transferred embryo, the pregnancy rate was 59% (44/74) among patients whose `best' embryo scored 70, compared with 34% (12/35) if the highest scoring transferred embryo was
65 (P < 0.015) (Table V
). In contrast, when the data were stratified based on the mean GES of all transferred embryos per patient, no significant difference in pregnancy rate was noted (data not shown). The overall implantation rate was 28% (83 sacs/294 embryos transferred) (Table II
). Among a sub-population of patients in whom implantation could be evaluated with certainty, those embryos scoring GES 70100 had an implantation rate of 39% (34/87) compared with 24% (22/92) with GES 065 (P < 0.03) (Table VI
).
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Discussion |
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Extended culture of embryos to the blastocyst stage has been used to improve selection for embryo viability. The development of reliable blastocyst culture techniques has allowed the transfer of fewer embryos, reducing the multiple gestation rate while maintaining high on-going pregnancy rates (Gardner et al., 1998; Milki et al., 1999
). However, blastocyst culture requires specialized media, which can significantly increase laboratory costs. In addition, the ability to extend embryo culture is often limited by the availability of adequate incubator space.
It has been postulated that embryonic developmental potential can be identified as early as the pronuclear stage (Scott and Smith, 1998). Tesarik and Greco (1999) reported that the evaluation of nuclear precursor body symmetry could be more important than cleavage speed (Tesarik and Greco, 1999
). It has further been reported that the implantation potential is greater in embryos with normal pronuclear development compared with those with abnormal pronuclear development, even if the rate of cleavage and morphology are the same (Tesarik et al., 2000
). Single embryo transfer based on early evaluation of embryo cleavage and morphology has resulted in a 42% implantation rate (Gerris et al., 1999
).
Our findings in a large cohort of embryos demonstrated a 36% overall blastocyst conversion rate, with 18% deemed of good enough quality for cryopreservation. However, these numbers are probably an underestimate of the true blastocyst development rates, since 22% (262/1245) of the embryos evaluated were transferred (n = 206) or cryopreserved (n = 56) on day 3. Of those embryos transferred on day 3, 64 gestational sacs resulted (24 singleton, 14 twin, and four triplet), in which at least one additional embryo must have developed to the blastocyst stage.
By ranking embryos based on GES, the blastocyst conversion rate could be significantly increased. Among embryos scoring 90100, grade III blastocyst development occurred in 64%. Among those embryos scoring 7085, 31% developed into grade III blastocysts, while only 11% of embryos scoring 3065 formed good quality blastocysts (Table III). Embryos scoring 70100 had 44% grade III blastocyst formation compared with 9% among embryos scoring 065. In contrast, when the data were analysed by cell number and morphology on day 3, only 34% (156/462) of embryos with
7 cells, regular cleavage and <20% fragmentation developed into grade III blastocysts (Table IV
). These findings suggest that high GES scoring embryos on day 3 can predict high quality blastocyst conversion better than cleavage rate and morphology alone. However, our findings also show that some embryos with low scores will still form grade III blastocysts, especially among those scoring 3065, indicating that further refinement of grading criteria may still be necessary.
When the data were stratified based on the highest scoring transferred embryo, those patients with at least one embryo scoring 70 had a 59% pregnancy rate compared with 34% if the best embryo scored <70 (Table V
). In contrast, the average score of all transferred embryos per patient was not found to be significantly predictive of pregnancy (data not shown). These findings suggest that a threshold of 70 can be used to accurately identify embryos with high potential for causing a pregnancy.
The overall implantation rate was 28%. The implantation rate from day 3 embryo transfer was 32% and from day 5 embryo transfer was 22%. This rate could be significantly improved by using GES to select embryos for transfer. Among the sub-population of patients with 100% implantation, as well as those in whom all transferred embryos scored above or below 70, the implantation rate was 39% among those embryos scoring GES 70100 compared with 24% among embryos scoring 065 (Table VI). These results compare favourably with other reported implantation rates (Gerris et al., 1999
; Tesarik et al., 2000
). The relatively low pregnancy and implantation rates seen with day 5 embryo transfer may be explained in part by our tendency to have transferred good quality, high scoring embryos on day 3, leaving many embryos of questionable quality for extended culture. In addition, the difference in pregnancy rates between day 3 and day 5 embryo transfer (68 versus 42%) suggests that high GES scoring embryos may have some advantage from day 3 embryo transfer over day 5 embryo transfer.
It should also be remembered that the implantation rates reported here are again likely to be an underestimate of the true values, since not all transferred embryos could be evaluated. In addition, eight biochemical pregnancies were excluded (an additional 13% of pregnancies), in which at least one embryo must have initially implanted. While it is tempting to correlate GES with implantation, it must be understood that many variables beyond embryo quality affect implantation, and therefore the idea that any one parameter in isolation can determine outcome must be viewed with extreme caution. Finally, we realize that the data analysis presented here is retrospective and we are currently in the process of prospectively evaluating the ability of GES to predict outcome.
The GES relies on a combination of pronuclear morphology, early cleavage and day 3 morphology to identify embryos with a high potential for blastocyst conversion, pregnancy and implantation. By combining and differentially weighting the significant developmental milestones identified by previous authors, we have been able to create a simple scoring system with a high predictive value for blastocyst development and pregnancy potential. The ability to predict, on day 3, which embryos will to develop to blastocysts may decrease laboratory culture expense, improve efficiency, and allow for earlier cryopreservation of supernumerary embryos. Since embryos with a high GES achieved pregnancy and implantation rates comparable with those associated with blastocyst transfer, the transfer of no more than two embryos scoring 90100 is recommended. We are currently developing a nomogram for determining how many embryos to transfer on day 3 based on GES ranking, in order to maximize the chance of a singleton gestation. Transfer of a single embryo based on a high GES might be used to reduce the chance of multiple gestations in patients at high risk, while transferring three or more embryos might be appropriate in patients with only low scoring embryos available for transfer.
Our early experience also indicates that the individual embryo culture method employed in this study had no adverse effect on the rate of blastocyst development, nor did the repeated removal of the embryos from the incubator for evaluation. We also observed that fragmentation, if absent at the first cleavage, did not occur during subsequent development. We intend to further define those characteristics that are critical to blastocyst development, so that embryos with high implantation potential can be identified with more precision. However, based on our present data, we suggest that it is possible to achieve the high pregnancy and implantation rates associated with blastocyst transfer from the transfer of cleavage-stage embryos selected upon the basis of GES.
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Notes |
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* Presented in part at the 56th Annual meeting of the American Society for Reproductive Medicine, San Diego, CA, USA October 21-26, 2000.
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References |
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Submitted on January 30, 2001; accepted on May 31, 2001.