1 Reproductive Biology Unit, Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, and 2 Melbourne IVF, 320 Victoria Parade, East Melbourne, Victoria 3002, Australia
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Abstract |
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Key words: blastomere loss/cryopreservation/human embryos/implantation rate
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Introduction |
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In order to assess the impact of cryopreservation on human early cleavage stage embryos it is necessary to compare the relative outcome from fresh and frozenthawed embryos. Such comparisons are often complicated by, or misinterpreted due to, difficulties in controlling for differences in the populations of embryos being assessed (Ludwig et al., 1998; Speirs, 1998
; Testart, 1998
).
In the present study we have compared the implantation rates (IR) of fresh early cleavage stage embryos with defined characteristics, in terms of growth rate and embryo quality (degree of fragmentation), to the IR of equivalent cryopreserved embryos in which no blastomere loss had occurred following thawing. Drawing on data from >5000 fresh and >5000 thawed embryos, we have circumvented problems associated with concomitant transfer of embryos with different characteristics by deriving our IR from those subsets of procedures in which only one embryo or only embryos with equivalent characteristics were transferred (`pure' data). In this way we were able to assess whether cryopreservation has an impact on early embryos which is independent of blastomere loss. We then used the same approach to assess the impact of defined levels of blastomere loss on implantation potential. The prevalence of fully intact, different categories of partially intact, and lysed embryos was then combined with the IR of each group (derived from `pure' data) to estimate the expected number of implantations from the entire population of thawed embryos and the value compared to the actual outcome. Having established a model for estimating the effect of cryopreservation on early cleavage stage embryos, we then applied it to our total population of embryos to estimate the impact in terms of `lost' implantations.
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Materials and methods |
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Cryopreservation
Embryos were frozen using 1,2-propanediol and sucrose as the cryoprotectants (Lassalle et al., 1985). Freezing and thawing solutions consisted of cryprotectants in phosphate-buffered saline (PBS) supplemented with HSA (10 mg/ml in freezing solutions and 4 mg/ml in thawing solutions). Embryos were equilibrated in 1.5 mol/l propanediol for 10 min at room temperature before being transferred to 1.5 mol/l propanediol/0.1 mol/l sucrose and loaded individually into plastic straws. Cooling was carried out in programmable freezers (Kryo 10 Series; Planer Products, Sunbury-on Thames, UK) at a rate of 2°C/min to 8°C, at which point seeding was induced manually. Cooling was then continued at rates of 0.3°C/min to 30°C and 50°C/min to 150°C before plunging and storage in liquid nitrogen.
Thawing and transfer
Embryos were thawed rapidly by removing straws from storage, exposure to air for 30 s and immersion in a water bath at 30°C for 45 s. Propanediol was removed in three steps in the presence of 0.2 mol/l sucrose at room temperature. Rehydration was completed by incubation in sucrose-free PBS and embryos were transferred to culture medium at 37°C before being assessed for numbers of remaining blastomeres. Thawed embryos were transferred in cycles as previously described (Bourne et al., 1995).
Analysis of results
Implantation rates (IR) were calculated as the number of fetal heart beats detected by ultrasonic examination per 100 embryos transferred. Proportions of embryos which implanted were compared using Fisher's Exact Test.
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Results |
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The results of this analysis are shown in Table I. In order to be included in any category shown in this analysis, it was necessary for all embryos transferred to fulfil equivalent criteria. Therefore, the restricted subsets of embryos with defined cell numbers on day 2 did not include many embryos from transfer procedures containing embryos of mixed characteristics which could be included in the analysis of all embryos. To account for potential differences due to growth rate, data were separated into the most common developmental stages represented in fresh and thawed embryos, i.e. 4-cell and 2-cell. In addition, any impact of embryo quality (assessed by the extent of pre-freeze fragmentation) was corrected for by restricting the analysis to either only grade A embryos or embryos which were either grades A or B.
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Discussion |
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Our results complement and extend a previous analysis (Mandelbaum et al., 1998) in which an implantation rate of 8.4% per transferred thawed embryo was reported together with a post-thaw survival rate of 73% from >14 000 thawed embryos in four centres over a 10 year period. They further estimated that embryo cryopreservation produced 8% additional births in women having transfers of fresh and frozen embryos. The present study has also attempted to address three questions. Firstly, what is the impact of cryopreservation on human early cleavage stage embryos if we exclude the most obvious effect, i.e. blastomere loss? Secondly, what is the prevalence of blastomere loss and the relationship between defined levels of blastomere loss and implantation potential? Thirdly, how do the answers to the first two questions interact to give us an indication of the overall impact of cryopreservation on a population of embryos? The crucial aspect of our approach was the estimation of the implantation potential of embryos with clearly defined characteristics by restriction of the analysis to embryo transfers which only included one type of embryo. Sufficient homogeneous data, which are necessary to permit unequivocal correlation of embryos with outcome, were available due to the large populations of embryos (>5000) in both the thawed and the control (fresh) group.
Three main points can be made from the data shown in Table I. Firstly, there is no significant difference in the IR of fresh and intact thawed embryos in any of the categories shown, suggesting that an embryo with defined characteristics which survives cryopreservation without loss of blastomeres has the same developmental potential as a similar fresh embryo. This would agree with previous studies (Mandelbaum et al., 1987; and also three of Selick (Selick et al., 1995
), who randomly allocated donated oocytes to frozen and fresh embryo transfers in an attempt to overcome potential embryo quality bias associated with the results reported by Levran et al. (1990). Secondly, we observed a highly significant (P < 0.001) increase in the implantation rates of faster growing (4-cell) embryos relative to those of equivalent slower growing (2-cell) embryos in all categories examined. The association between growth rate and implantation potential in fresh embryos, which also persisted in frozen/thawed embryos in our study (Table I
), confirms the previous findings of Ziebe et al. (1997), who concluded that cleavage stage was more important than fragmentation in predicting the developmental potential of fresh embryos. Thirdly, the extent of fragmentation within the limits investigated (030%) does not appear to have a significant effect on implantation potential since there was no significant difference in the IRs obtained in any category when data were derived from grade A embryos only. The results of Staessen et al., based on relatively small numbers of embryos, suggest that >20% fragmentation is associated with a reduction in implantation (Staessen et al., 1992
). Similarly, the results of Ziebe et al. show a reduction in implantation rate with increasing levels of fragmentation, although the data in this study combine outcomes from all embryos with 1050% fragmentation (Ziebe et al., 1997
). The large data set in our study together with the above reports suggest that fragmentation may influence outcome only when it exceeds a critical threshold, which is difficult to define accurately given the small numbers in some studies, the ranges of fragmentation used in the studies, and the subjective nature of the quantitative assessment.
Although the exact significance of the role, if any, of oestrogen in implantation (Edgar, 1995) and the hormonal control of endometrial receptivity (de Ziegler, 1995
) are still unclear, it is impossible to exclude completely the possibility that endometrial receptivity is altered in cycles in which thawed embryos are transferred relative to those in which fresh embryos are replaced (de Ziegler and Frydman, 1990
). Such differences in receptivity may obscure differences in intrinsic embryo viability but it would seem to be unlikely that the balance of the factors would result in the close similarity seen between the IRs of equivalent fresh and fully intact thawed embryos.
The prevalence of fully intact thawed embryos in our study (55.5%; Table II) and the proportion of thawed embryos which survived with at least 50% of their blastomeres intact (78.3%; Table II
) are at least consistent with the reported results from a large multicentre series (Mandelbaum et al., 1998
) and other studies (Hartshorne et al., 1990
; Horne et al., 1997
; Kowalik et al., 1998
). However, in contrast to the conclusions of Hartshorne et al. (1990) which were based on smaller numbers of embryos, we found a highly significant association between blastomere loss and reduction in implantation potential (Table III
) even in embryos with
50% of their blastomeres intact.
The conclusions drawn from `pure' data, that intact thawed embryos are equivalent to their fresh counterparts and that blastomere loss at each defined level is associated with a defined reduction in implantation potential, are strengthened by the similarity in the actual number of implantations derived from the total population of thawed embryos and the number predicted by applying the values from pure data to the total population (Table IV).
The overall impact of cryopreservation on our population of embryos is apparent from the difference in the number of post-thaw implantations and the number which would have been expected had it been possible to transfer all embryos fresh. The estimated loss is of the order of 30% of the potential implantations. It is, of course, impractical to consider transferring all embryos fresh in order to avoid these losses, although the results suggest that the impact of potential cryodamage should be considered in conjunction with the risk of multiple implantations when deciding how many embryos to transfer fresh and how many to freeze. Further assessment of post-thaw viability based on resumption of cleavage (Van der Elst et al., 1997; Ziebe et al., 1998
) can also help in deciding how to use thawed embryos. Our own unpublished observations, however, agree with the findings of the latter group that pregnancies and births can result following the transfer of embryos which do not resume cleavage, albeit at a lower frequency than following the transfer of cleaving embryos.
The results further emphasize the need to develop and apply methods such as blastocyst culture and transfer (Gardner et al., 1998) and preimplantation genetic diagnosis of aneuploidy (Gianaroli et al., 1997
) which permit the selection of embryos with enhanced developmental potential and reduce the chance of subjecting them to the potentially deleterious effects of cryopreservation. Although Hardy et al. (1990) have reported the lack of an adverse effect on human preimplantation development in vitro following biopsy at the 8-cell stage, our results from thawed embryos with
50% of their blastomeres intact suggest that loss of a minority of cells at early cleavage stages may have an impact on subsequent implantation potential. It is, therefore, important to stress that comparisons of alternative strategies for embryo utilization must include a quantitative assessment of how each aspect of the approach, e.g. embryo biopsy, extended culture or cryopreservation at early or later (blastocyst) stages, will be likely to affect the final outcome from a population of fertilized oocytes.
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Notes |
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References |
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Submitted on June 28, 1999; accepted on October 5, 1999.