Introduction

A series of dynamic and complex events are triggered following sperm–oocyte interaction that sequentially leads to fertilization and the formation of a zygote. These events include sperm penetration, sperm–oocyte fusion and oocyte activation, male and female pronucleus (PN) development and gradual migration of the pronuclei (PNs) to a central position in the oocyte.

Induced by the sperm penetration and subsequent calcium oscillations, the fertilized oocyte undergoes maternal-to-zygotic transition as a result of major changes in the molecular signals that control the arrest of meiotic development at the metaphase II stage of the second meiotic division (Ajduk et al., 2011). In humans, the sperm centriole has the leading role in organizing the microtubules, which direct the migration of PNs and their rotation within the cytoplasm. In this way, PNs position their axis toward the second polar body and achieve a proper orientation at syngamy by controlling the plane of the first mitotic division. During PN formation, nuclear precursor bodies (NPBs) become evident and start to migrate and merge into nucleoli in a time-dependent manner. The NPBs do not form a functionally active nucleolus at the zygote stage; however, they can be used as an indirect measure of the location and the grade of condensation of DNA within the PNs. Nucleoli are the sites of synthesis of pre-rRNA and its availability is extremely important since newly synthesized rRNA is necessary for the translational processes when the embryonic genome becomes active (Gianaroli et al., 2003). Asynchrony in the timing of any of the events associated with fertilization could compromise embryo development.

Once PNs are aligned onto a polar axis, parental chromosomes then separate in preparation for mitosis. The human zygote's mitotic potential is paternally inherited with the spermatozoon delivering the centrosome (Palermo et al., 1994; Sathananthan et al., 1996).

A zygote's morphological characteristics are accepted to be an inherent indicator of both gamete quality and subsequent embryo implantation potential (Alpha Scientists in Reproductive medicine and ESHRE Special Interest Group of Embryology, 2011). Many studies have underlined the predictive value of zygote morphological assessment through correlations with chromosome constitution and the incidence of zygotic arrest (Gianaroli et al., 2003; Balaban et al., 2004; Edirisinghe et al., 2005; Zamora et al., 2011). Recent strategies in embryo selection include sequential morphology assessment where PN scoring has been shown to play an important role as an indicator of gamete constitution as well as a prognostic tool for embryo competence. Scoring of PNs has also proved to be useful in countries where restrictive legislation mandates selection at the zygote stage for embryo transfer and consecutive elimination or cryopreservation of sibling zygotes (Senn et al., 2006; Zollner et al., 2005).

Although numerous studies have associated positive clinical results with the implementation of PN scoring, other reports have questioned the predictive value of PN scoring systems and see no benefits or improvement in the outcome (Nicoli et al., 2010; Weitzman et al., 2010). The Istanbul consensus workshop comprised a worldwide panel of experts who just recently evaluated the current practice of oocyte to embryo scoring and established common criteria for assessment (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Fertilization check is to be performed at 17 ± 1 h post-insemination which may establish uniformity in the future and eliminate the variability in PN scoring regimens that have confounded comparative analyses. It must be noted, however, that the processes associated with fertilization by conventional insemination lags 1h behind fertilization using ICSI (Nagy et al., 1998). A more complete elucidation of events during the zygote stage, however, can be expected with the application of continuous monitoring through the introduction of time-lapse imaging instead of the traditional isolated observations using light microscopy (Montag et al., 2011).

Normal fertilization is assessed by the presence of two centrally positioned, juxtaposed PNs with clearly defined membranes and two polar bodies. If an abnormal PN number is observed whether it be 1, or 3 or more, PNs, a low viable pregnancy is to be expected thus the transfer of these zygotes is to be avoided (Reichman et al., 2010). Aberrant PN size and position have been correlated with developmental arrest and aneuploidy and are represented by PNs of unequal size (>4 μm), localized far apart or peripherally or with the presence of fragmented or additional micronuclei (Munné and Cohen, 1998; Sadowy et al., 1998; Garello et al., 1999; Nagy et al., 2003; Scott et al., 2007). Panel experts also agreed and strongly advised that the assessment and elimination of dysmorphic zygotes with smooth endoplasmic reticulum (SER) discs (see Chapter One) due to the association with severely adverse clinical outcomes (Otsuki et al., 2004; Ebner et al., 2008).

Correct alignment of PNs onto the polar axis is considered a fundamental feature for the completion of the first cleavage division and normal sequential development (Gardner, 1996, 1999, 2001; Edwards and Beard, 1997, 1999; Payne et al., 1997; Garello et al., 1999; Scott, 2001). Cell cycle-related dynamics of PN events have led many authors to investigate and correlate the presence, pattern and number of NPBs to embryo developmental potential. As shown for mitotic cells, the inequality in number, size or distribution of NPBs is correlated with abnormal development (Pedersen, 1998). Therefore, the panel experts from the consensus workshop established three categories for PN scoring that are based on the morphology of NPBs and PNs, namely: (i) symmetrical, (ii) non-symmetrical and (iii) abnormal (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Category 1 includes zygotes presenting with equal numbers and size of NPBs, either aligned at the junction between PNs or scattered in both PNs. Category 2, non-symmetrical, comprises all other patterns including peripherally localized PNs. Category 3, abnormal, includes single NPB (‘bull's eye’) or total absence of NPBs. The latter were found to be correlated with imprinting errors and delayed onset of functional NPBs and nucleoli formation in animal models (Svarcova et al., 2009).

Reports on early cleavage checks have been demonstrated to be a beneficial tool in selecting embryos with high implantation potential and decreased chromosomal anomalies (Sakkas et al., 1998; Lundin et al., 2001). The consensus opinion on a second, Day 1 observation was to leave the choice to the operator's discretion, but if applied, is to be performed 26 ± 1 and 28 ± 1 h post-insemination for ICSI and IVF embryos, respectively.

Sequential morphology assessment through time-lapse cinematography will certainly shed light on the discrepancies in the literature with respect to PN scoring and an in-depth analysis and correlation with the clinical background behind the gametes forming the zygote will possibly reveal even more reliable prognostic tools to improve clinical outcomes.