Further Cleavages

As cleavage continues, the first blastomeres of the two-cell embryo divides meridionally followed by approximately equatorial cleavage of the other cell (75). This lesson from mouse embryos may explain the typical crosswise appearance of the 4-cell human conceptus on day two of development. Although a regular tetrahedral configuration of blastomeres with six intercellular contacts is the most common outcome of second cleavage, both the distribution and the relationship between blastomeres may vary, including specimens that are essentially planar. This arrangement involves a reduced number of cell-cell contacts which could impair compaction and delay blastulation of the embryo (28,76).

Apart from the number of blastomeres, routine assessment of embryo quality from day two onward also includes the degree of fragmentation. There is a considerable lack of objective and standardized methods for assessing embryonic fragmentation. In fact, a cell size of 45 mm on day two has been suggested which allowed distinguishing between anuclear fragments and blastomeres. Below this cut-off value, only 3% of the cells contained DNA compared to 67% with a diameter above this cut-off. Similar results were published for day three embryos with the exception that, due to ongoing cleavage, a threshold of 40 mm was indicative in terms of differentiation (77).

It is generally accepted that minor fragmentation does not impair viability of the embryo (78,79) and may disappear during in vitro culture, either by lysis or resorption (80,81). Larger amounts of fragments, however, significantly reduce the chance to achieve pregnancy (82) and, even more importantly, perinatal outcome of babies derived from heavily fragmented embryos (greater than equal to 50% fragmentation) was found to be worse compared with that after transfer of more or less fragment-free embryos (83).

As fragments are structures of blastomeric origin, the actual amount of cytoplasmic fragmentation during cleavage stage can be estimated by the difference between the previous zygote volume and the overall blastomere volume (84). In cases of moderate fragmentation, it appears that different spatial patterns of fragmentation are of more severe developmental consequences than fragmentation per se (81,82). In detail, smaller, more localized fragments did not impair viability, whereas larger and more scattered fragments had a disastrous effect on implantation (82). Theoretically, the detrimental effect of such patterns may be explained by the fact that anuclear fragments lying in close proximity to an assumed cleavage axis may impair further cleavage and/or reduce the number of cell-to-cell contacts required for regular compaction and blastocyst formation. Viability of bad quality embryos may be improved in certain instances if spatial relationship of blas-tomeres is restored by cosmetical removal of the acellular remnants (82).

The higher the degree of blastomeric decay, the higher the risk of chromosomal imbalances, such as mosaicism (63). In these cases, selective fragmentation could function as a means to completely exclude affected blastomeres from further cell aggregation (85). This process is most likely related to programmed cell death (85,86), e.g., the ratio of the two apoptosis-related gene families bcl-2 and bax (85). Others (87) question a direct relationship between apoptotic phenomena and fragmentation and much rather speculate that apoptosis may be triggered if the degree of mitochon-drial and proteinic loss due to fragmentation reaches a certain level.

It is an undisputed fact that two morphological phenomena, namely multinucleation and inequality of cleavage, reduce viability of the cleaving embryos to a minimum. Frequently, both anomalies coincide (80), which may be explained by the larger cell size of multinucleated blastomeres (84). In general, it can be expected that about one-third of all day two and three embryos show at least one multinucleated blastomere (88). The overall incidence, however, will generally be underestimated since nuclei are only visible at interphase. Most previous studies report a disastrous implantation rate of less than 6% after exclusive transfer of bi-or multinu-cleated embryos (88,89). This reduced outcome seems to be a reflection of the chromosomal constitution of the embryos since the vast majority of them (approximately 75%) were chromosomally abnormal (80,90). In detail, cytokinesis may fail during any mitotic division (91) with the worst outcome to be expected if problems arise during the first cleavage ending up with both cells being multinucleated (92).

Even if the embryo is composed of a stage-appropriate number of equally divided mononuclear cells, this does not mean that the texture of the blastomeres will correspond to the assumed normal condition, which is a translucent cytoplasm with moderate granulation. As a result of culture conditions (e.g., media composition), cytoplasm of a day 3 embryo may change to a more mottled appearance showing numerous small (1.5 mm) pits on the surface (93,94). Pitting is physically different from excessive granulation and mostly affects embryos after embryonic genome activation (93). In humans, this switch from maternal genome control is considered to take place around the 8-cell stage (95). Definitely, it is an important hallmark of preimplantation development prior to which it is an assessment of oocyte quality rather than embryo viability.

However, this temporal coincidence of cytoplasmic pitting is not a positive predictor of outcome (96); but much rather it seems to be completely unrelated to implantation and pregnancy. Recent findings, however, suggest a certain influence on viability since some 30% of implantations vanished after exclusive transfer of pitted embryos compared to only 16% of early pregnancy loss in a nonaffected control group (97).

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