Cell Number/Cleavage Rate

The single most important indicator of embryo viability is the occurrence of cellular division. The ideal cleavage was found to be four cells on Day 2 and eight cells on Day 3, with a markedly lower implantation potential for embryos below that and, to a lesser degree above that (Holte et al. 2007) and is in general agreement with previous observations (Van Royen et al. 1999; Magli et al. 2001). A correlation between ‘normal cell number’ and chromosomal constitution has also been reported (Almeida and Bolton 1996; Magli et al. 2007).

Fragmentation

Mitotic division of embryos can lead 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). Presence of fragmentation is common in human embryos and assessment has been used in almost all embryo scoring systems. Severe fragmentation of the embryo is associated with poor prognosis (Ziebe et al. 1997; Van Royen et al. 1999, 2001).

As well as the percentage of fragmentation in the embryo, fragment size and distribution has been shown to correlate with the probability of implantation (Alikani et al. 2000; Ebner et al. 2001; Van Blerkom et al. 2001). However, it is often unclear how to differentiate fragments from cells and then estimate the relative proportion of the embryo that is fragmented. Large fragments, sometimes resembling whole cells, often distributed randomly, are associated with uneven cells and produced a low implantation rate in some studies (Alikani and Cohen 1995). The loss of important cytoplasmic content, such as cell organelles, proteins, or mRNA may leave cells in largely fragmented embryos too small to be biologically competent (Johansson et al. 2003; Hnida et al. 2004).

The size of fragmented cells has been defined as <45 mm in diameter for Day 2 embryos, and <40 mm in diameter for Day 3 embryos (Johansson et al. 2003; Alpha/ESHRE Istanbul Consensus 2011). As mentioned earlier, these findings may influence scoring criteria; embryos previously scored with uneven‐sized cells might now be scored as partially fragmented embryos. However, it is not easy to measure the size of fragments routinely, and use of TLI may reduce subjectivity.

The presence of small, scattered fragments (10–20%) does not appear to impair further development (Van Royen et al. 1999; Hardarson et al. 2001; Racowsky et al. 2003) and these may disappear through lysis or resorption during culture (Hardarson et al. 2001). Thus, a low degree of fragmentation may be normal and may suggest apoptotic elimination of selected cells in an effort to restore or maintain viability when anomalies are present (Ziebe 1997; Alikani et al. 2000). It is also possible that these fragments are generated during successive divisions and are simply a product of imperfect cytokinesis rather than a specific anomaly.

Cell Size/Cleavage Evenness

When undergoing the first mitotic cell divisions, a zygote cleaving synchronously and symmetrically will present two, four, or eight cells of a similar size. Cells of equal size appear to be another indicator of implantation potential (Steer et al. 1992; Hardarson et al. 2001; Hnida et al. 2004; Paternot et al 2013). For all other early cleavage stages of three, five, six, seven, and nine cells, a size difference in the cells would be expected as the cleavage phase has not been completed (Scott 2001; Magli et al. 2012).

Several studies have identified the phenomenon of uneven cleavage leading to unequal cell size, and this is commonly found in IVF embryos. Unevenly sized cells have also been shown to have an increased prevalence of chromosomal abnormalities (Hardarson et al. 2001; Magli et al. 2001: Ziebe et al. 2003), including multinucleation (Hardarson et al. 2001). The latter study suggested that early cleavage may be hindered by the presence of aneuploidy delaying the cell cycle. This impairment may also be due to uneven distribution of proteins, mRNA, mitochondria, and furthermore may possibly disturb the polarized allocation of certain proteins and genes in both oocytes and embryos (Antczak and Van Blerkom 1999).

However, there are difficulties with routine microscopic assessment as cells scored as unevenly cleaved may actually be large anucleate fragments. As mentioned above in the section on fragmentation, embryos with uneven‐sized cells might be scored as partially fragmented embryos and vice‐versa. This will in turn also affect the reliability of the cell count assessment, e.g. an embryo scored as an uneven eight‐cell may actually be a fragmented six‐cell embryo. TLI may be used as a tool to improve reliability of assessment allowing quantitative measurement.

Multinucleation

A cell containing more than a single interphase nucleus, is defined as being multinucleated (MN) and considered abnormal. A negative impact on the implantation potential of MN embryos has been observed (Van Royen et al. 2003; Saldeen and Sundstrom, 2005) and has been correlated with a high degree of chromosomal aberration (Kligman et al. 1996; Hardarson et al. 2001) with MN cells being significantly larger than nonnucleated cells (Hnida et al. 2004).

However, it was shown that among embryos in which both cells were bi‐ or multinuclear at the two‐cell stage, 30% had only mononuclear cells following cleavage (Staessen and Van Steirteghem 1998). Thus, some MN cells still seem to be able to cleave normally (Meriano et al. 2004).

Factors that have been suggested to affect the rate of MN include culture media (Winston et al. 1991), and poor temperature control especially in relation to oocyte retrieval (Pickering et al. 1990). Different mechanisms leading to MN cells have been suggested including the dissociation of karyokinesis from cytokinesis, partial fragmentation of the nucleus, or defective migration of chromosomes at mitotic anaphase (Munne and Cohen 1993; Staessen and Van Steirteghem 1998). Fluorescence in situ hybridization (FISH) demonstrated that all these mechanisms may be involved.