Introduction
A precise embryo quality evaluation is of paramount importance to sustain a successful in vitro fertilization (IVF) program. In most IVF clinics around the world, this quality assessment relies mainly on the morphological evaluation of cleavage stage embryos. Embryologists should be able to correlate the features observed at the optical microscope with the implantation potential of each particular embryo (Alikani et al., 2000; Ebner et al., 2003; Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). To achieve this goal, many scoring systems based on the morphological features of the dividing embryo have been developed (Giorgetti et al., 1995; Veeck, 1999; Fisch et al., 2001; de Placido et al., 2002; Baczkowski et al., 2004; reviewed by Rienzi et al., 2005; Torelló et al., 2005; Holte et al., 2007). These embryo classification systems are based on the evaluation of the number of blastomeres, the degree of fragmentation, the symmetry of the blastomeres, the presence of multinucleation and the compaction status. It is very important that the features related to implantation potential are assessed accurately and similarly. The purpose of this chapter is to illustrate morphological aspects useful for the evaluation of the implantation potential of the embryos.
Cleavage stage embryos range from the 2-cell stage to the compacted morula composed of 8–16 cells. The number of blastomeres is used as the main characteristic with the highest predictive value (Van Royen et al., 1999; Alikani et al., 2000; Fisch et al., 2001). Good quality embryos must exhibit appropriate kinetics and synchrony of division. In normal-developing embryos, cell division occurs every 18–20 h. Embryos dividing either too slow or too fast may have metabolic and/or chromosomal defects (Edwards et al., 1980; Giorgetti et al., 1995; Ziebe et al., 1997; Van Royen et al., 1999; Leese, 2002; Munné, 2006; Magli et al., 2007). Recent time-lapse studies indicate that not only the timing of cleavage, but also the time between each cell division is of importance. If all blastomeres divide in exact synchrony, only 2-, 4- or 8-cell embryos would be observed. However, it is frequent to observe 3-, 5-, 6-, 7- or 9-cell embryos, which is an indication of asynchronous development (Scott et al., 2007; Lemmen et al., 2008; Wong et al., 2010; Meseguer et al., 2011; reviewed by Kirkegaard et al., 2012). Time of scoring with respect to the insemination event has to be precisely established for a correct evaluation of the kinetics of cell division (Scott et al., 2007). However, it is also important to keep in mind that the environment in the specific laboratory, such as culture media and temperature, influences the kinetics of development.
Very frequently, the mitosis of embryos leads to externalization of parts of the cell cytoplasm, resulting in the presence of anuclear fragments surrounded by a plasma membrane (Antczak and van Blerkom, 1999). The size and distribution of fragments inside the space surrounded by the zona pellucida (ZP) are variable (Alikani et al., 1999). The amount of fragments is widely used to predict the implantation potential of the embryos and fragmentation has been related to aneuploidy (Ebner et al., 2001; Ziebe et al., 2003; Munné, 2006). If fragmentation does not reach 10% of the total embryo volume it is agreed that it does not have an impact on the embryo's developmental potential (Van Royen et al., 2001; Holte et al., 2007).
Mitosis in blastomeres should produce two equally sized daughter cells. When the division is asymmetric, one of the blastomeres of the next generation will inherit less than half the amount of cytoplasm from the parent blastomere, leading to a defective lineage in the embryo. For example, 4- and 8-cell embryos with equal cell sizes have been shown to have lower multinucleation and aneuploidy rates and increased implantation rates (Hardarson et al., 2001; Van Royen et al., 2001; Hnida et al., 2004; Scott et al., 2007). After two cleavages, the zygote becomes a 4-cell embryo. The four cells of the embryo are normally arranged in a tetrahedron in the spherical space provided by the ZP. However, in some cases, the blastomeres are located close to a single, spatial plane produced by an incorrect orientation of the division axes. This can be associated with altered embryo polarity (Edwards and Hansis, 2005).
Each embryo blastomere should have a single nucleus. Multinucleation has been described to be associated with genetic disorders of the embryo (Kligman et al., 1996; Hardarson et al., 2001). It impairs cleavage rates and the implantation potential of human embryos (Pelinck et al., 1998; Van Royen et al., 2003; Moriwaki et al., 2004) and has been associated with an increased abortion rate (Meriano et al., 2004). Multinucleation can be evaluated on Days 1, 2 and 3 of development. On Day 3, however, this characteristic is more difficult to evaluate due to the more complex structure of a Day 3 embryo (Van Royen et al., 2003). Different morphological anomalies are often associated with each other, and uneven cleavage has been shown to be related to multinucleation (Hardarson et al., 2001) and fragmentation (Hnida et al., 2004).
A clear homogeneous cytoplasm is acknowledged as normal for cleavage stage embryos. The presence of anomalies such as an abundance of vacuoles and aggregation of organelles resulting in clear and granular cytoplasmic regions has to be considered in any embryo quality assessment (Veeck, 1999; Desai et al., 2000; Ebner et al., 2003).
After the embryo reaches the 8-cell stage, the blastomeres begin to show an increase in cell–cell adherence due to the spread of intercellular tight junctions. This is the start of compaction. The process of compaction advances during the next division until the boundaries between the cells are barely detectable (Veeck, 1999). If some of the blastomeres are excluded from this compaction process, the embryo may have a reduced potential for becoming a normal blastocyst (Tao et al., 2002). In a proposed grading system compaction can be classified using the following criteria: the proportion of blastomeres undergoing compaction and the morphology of the compacted embryo (Tao et al., 2002). The validity of this grading system remains to be confirmed.
This cleavage stage chapter seeks to illustrate the morphological aspects discussed in the Istanbul consensus workshop on embryo assessment (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). The aim is to introduce a more accurate and widespread comprehension of the nomenclature applied to the characterization of cleavage stage embryos.
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