Development to the Blastocyst Stage

An interest in culturing human embryos to the blastocyst stage has always existed, in particular as there were always concerns as to the logic of transferring early-cleavage-stage embryos to the uterine environment. Limited data existed that indicated an improved pregnancy rate when placing blastocysts into the uterus. Buster et al. (21) recovered in vivo-developed human blastocysts by uterine lavage and transferred the same blastocysts to achieve a high implantation rate (3/5, 60%), well above that currently observed in most IVF cycles. Similar implantation rates have now been published using sequential embryo culture media by a number of clinics (22-24).

Figure 4.2. Outcome of embryo transfer related to the number of early cleaving (EC) two-cell human embryos. Open bars, pregnancy rate; solid bars, implantation rate. *Significantly different (p<.01) from the no early cleavage group. The number of cycles are in parentheses.
Figure 4.3. Ideal features of the cleavage-stage embryo. (From Sakkas [46], with permission of Martin Dunitz Press.)

The time of blastocyst development is evidently important, but forming a blastocyst per se is not the criterion most strongly associated with pregnancy outcome (Figure 4.4). In the mouse model several aspects of blastocyst development and physiology were quantitated and related to subsequent fetal development (25). It was determined that total cell number, inner cell mass (ICM) cell number, and glycolysis had the strongest correlation with blastocyst viability. Blastocyst formation and hatching were poorly correlated with pregnancy outcome.

This theory has been further substantiated by a number of studies that adopted a scoring method for the blastocyst transferred. This is highlighted in the study by Gardner et al. (26), who showed that blastocysts of high quality led to the highest pregnancy and implantation rates. Blastocysts were scored according to the expansion state of the blas-tocoel cavity and the number and cohesiveness of the ICM and trophectodermal cells. Blastocysts with a full blastocoel cavity, a well-populated and tightly formed ICM, and a cohesive trophectoderm with many cells were given a score of 3AA or greater and designated as the top-scoring blastocysts (Figure 4.5). When two such blastocysts were transferred, pregnancy rates were >80%, and implantation rates were 70%. The transfer of one top-scoring blastocyst in the cohort of two still led to pregnancy rates >60% and implantation rates of 50%. The importance of blastocyst quality in relation to pregnancy outcome has also been shown by Balaban et al. (27). The other factor important in the assessment of blastocysts is the time of blastocyst formation. In a previous study when cases where only day 5 and 6 frozen blastocysts were transferred were compared to those frozen on or after day 7 and transferred, the pregnancy rates were 7/18 (38.9%) and 1/ 16 (6.2%), respectively (28). In these cases expanded blastocysts with a definable ICM and trophectoderm were frozen. These results showed that even though blastocysts could

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Glycolytic Activity (pmol/embryo/h)

Figure 4.4. Correlation between parameters for embryo assessment and fetal development after transfer. The plots show the relationship between seven parameters used to determine the effectiveness of different culture media to support mouse blastocyst development, and the resultant viability after transfer (25). Dots represent mean values. (a) blastocyst formation and fetal development (p > .1); (b) blastocyst hatching and fetal development (p > .1); (c) total blastocyst cell number and fetal development (p < .01); (d) inner cell mass cell number and fetal development (p < .05); (e) trophectoderm cell number and fetal development (p > .1); (f) inner cell mass outgrowth and fetal development (p < .01); (g) glycolytic activity and fetal development (p < .07). The relationship between glycolysis and viability has been further analyzed (36). The techniques used to assess embryo development and metabolism can be found in chapters 3, 8, and 9. (From Lane and Gardner [25], with permission from Reproduction.)

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Glycolytic Activity (pmol/embryo/h)

Figure 4.4. Correlation between parameters for embryo assessment and fetal development after transfer. The plots show the relationship between seven parameters used to determine the effectiveness of different culture media to support mouse blastocyst development, and the resultant viability after transfer (25). Dots represent mean values. (a) blastocyst formation and fetal development (p > .1); (b) blastocyst hatching and fetal development (p > .1); (c) total blastocyst cell number and fetal development (p < .01); (d) inner cell mass cell number and fetal development (p < .05); (e) trophectoderm cell number and fetal development (p > .1); (f) inner cell mass outgrowth and fetal development (p < .01); (g) glycolytic activity and fetal development (p < .07). The relationship between glycolysis and viability has been further analyzed (36). The techniques used to assess embryo development and metabolism can be found in chapters 3, 8, and 9. (From Lane and Gardner [25], with permission from Reproduction.)

Figure 4.5. Blastocyst scoring system used to select embryos for transfer. (a) Initially blastocysts are given a numerical score from 1 to 6 based on their degree of expansion and hatching status: (1) early blastocyst; the blastocoel being less than half the volume of the embryo; (2) blastocyst; the blastocoel being greater than or equal to half of the volume of the embryo; (3) full blastocyst; the blastocoel completely fills the embryo; (4) expanded blastocyst; the blastocoel volume is now larger than that of the early embryo and the zona is thinning; (5) hatching blastocyst; the trophec-toderm has started to herniate though the zona; (6) hatched blastocyst; the blastocyst has completely escaped from the zona. The initial phase of the assessment can be performed on a dissection microscope. The second step in scoring the blastocysts should be performed on an inverted microscope. For blastocysts graded as 3-6 (i.e., full blastocysts onward) the development of the inner cell mass (ICM) and trophectoderm can then be assessed. ICM grading: (A) tightly packed, many cells; (B) loosely grouped, several cells; (C) very few cells; (D) degenerate ICM. Trophectoderm grading: (A) many cells forming a cohesive epithelium; (B) few cells forming a loose epithelium; (C) very few, large cells; (D) degenerate cells. (b) Photomicrograph of a human blastocyst (score 4AA morning of day 5). The blastocoel cavity is expanding and the zona is thinning (score of 4). The trophectoderm is composed of numerous cells (score of A). Blastocyst diameter is 140 mm. (c) Photomicrograph of the same human blastocyst in panel b taken in a different focal plane. The ICM is visible and is composed of numerous tightly packed cells (score of A). (Adapted from Gardner and Schoolcraft [45] with permission of Parthenon Publishing.)

be obtained, the crucial factor was when they became blastocysts. When taking this into account, the best blastocysts would be those that develop by day 5. In the bovine model it has also been demonstrated that those embryos that form blastocysts earlier are the most viable (29).

A number of studies have attempted to investigate whether the embryos that are growing the best at the earlier stages of development are also those that reach the blastocyst stage. Although this seems logical, the results are not conclusive. For example, Graham et al. (30) reported that the criteria for embryo selection on day 3 seem to be inadequate for selecting blastocysts. In contrast, Shapiro et al. (31) found a predictive value of 72-h blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocyst development. Similarly, Langley et al. (32) showed that there was a relationship between embryo cell number on day 3 and the potential to form a blastocyst (Figure 4.6).

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