Key message
Time-lapse and genomic technologies have been used to detect for the first time alternative modalities of embryo
In mainstream IVF treatment, embryos are selected for transfer principally on the basis of their developmental rate and morphological features. Historically, assessment of these embryonic characteristics has been performed statically at isolated time points by conventional transmitted light microscopy. More recently, the challenge of embryo selection has been tackled by extending observation and analysis to several and often novel morphokinetic parameters that can be monitored by the newly introduced time-lapse technology (TLT). Notwithstanding this progress in microscopy technology, the vast majority of old and new developmental parameters remain temporally restricted to day 2, day 3 and, in case of culture to the blastocyst stage, day 5/6. Very rarely, embryos are observed and selected on day 4, at a time when development in vitro has usually progressed to the morula stage. This is due to the difficulty of statically assessing the morphology of the morula, a stage described as an ‘indistinguishable mass of cells’ (ESHRE Atlas of Human Embryology). Therefore, with few exceptions (Iwata et al., 2014; Zakharova et al., 2014; see also below), this phase has received very little attention in clinical embryology as a source of information to predict the ability of the embryo to implant and establish a viable pregnancy.
Indeed, the morula stage emerges from this picture as the ‘Cinderella of IVF’. This is rather paradoxical, considering that pivotal events occur during this stage of development (Coticchio et al., 2019). Metabolism shifts from low to high levels of activity (Leese, 2012); embryo geometry becomes more complex, with the positioning of inner and outer cells (Johnson and Ziomek, 1981); cell shape changes from a spherical to a flattened epithelium-like type, required for embryo compaction (White et al., 2016); and cell fate mechanisms are enhanced, giving rise to two cell lineages (Jedrusik, 2015). These cellular and molecular transitions may represent opportunities for mechanisms of ‘quality control’ of preimplantation development, as suggested by evidence consistent with the existence of self-correction events aimed at reducing the load of aneuploid cells in mosaic embryos (Barbash-Hazan et al., 2009).
A noticeable phenomenon occurring during the morula stage is embryo compaction, which represents a response to the structural imperative to accommodate inner and outer cells within the overall spherical organization of the embryo (White et al., 2017). Interestingly, compaction may involve only part of the embryo, with some blastomeres failing to flatten. Partially compacted morulae (PCM) have been given little attention, despite possible implications for prognosis and treatment (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011; Feil et al., 2008; Fabozzi et al., 2015; Ivec et al., 2011; Tao et al., 2002), and remain obscure from a molecular, cellular and developmental standpoint.
More recently, the advent of TLT, combined with preimplantation genetic testing for aneuploidies (PGT-A), has inspired new approaches for a comparative assessment of full and partial compaction. In a recent study in which chromosome analysis was conducted on biopsied trophectoderm and, at the same time, on cells failing to compact that were obtained from the same embryo, partial compaction was proposed as a possible self-correction mechanism aimed at excluding aneuploid cells from mosaic embryos, thereby reducing or eliminating the aneuploid load (Lagalla et al., 2017). However, much still remains to be investigated. The aim of this study was to investigate the prevalence, underlying morphokinetic mechanisms and possible consequences of partial compaction using TLT.
This retrospective study included 1271 embryos obtained from PGT-A cycles carried out between May 2013 and July 2017. Of these embryos, 982 reached morula stage. Ethical approval for the study was obtained from the local Institutional Review Board (11 March 2013, reference number R 05 PA 19). Patients provided written informed consent to embryo biopsy, chromosome analysis and the anonymous use of clinical data for statistical evaluation and research purposes. Indications for PGT-A were severe
This investigation included the retrospective morphokinetic analysis of 1271 embryos obtained in 350 PGT-A cycles from 233 couples. The mean female age (± SD) of the study group was 40.0 ± 4.4 years (range 26–48 years). From these embryos, 982 morulae were obtained and carefully analysed on the basis of the compaction pattern and morphokinetic behaviour. Of these morulae, 322 developed into blastocysts that were biopsied and assessed chromosomally. Analysis was performed on two data subsets:
Generally speaking, the morula stage has attracted little interest in assisted reproduction, having been considered a transitory, poorly informative stage of embryo development. In addition, its chromosomal status has received little attention due to the difficulty of performing blastomere biopsy after compaction and before formation of the trophectoderm (Zakharova et al., 2014). This is in contrast to the crucial changes that happen during this stage of development (Bissiere et al., 2018;
In final analysis, by adopting a complementary approach involving TLT and genomic technologies, the present study was able to detect for the first time alternative modalities by which incomplete compaction can occur in the preimplantation embryo. Differences between excluded- and extruded-PCM appear to be more profound than mere alternative mechanisms of compaction, overarching the morphokinetic trajectory of preimplantation development and affecting the ability to form a viable blastocyst. The
The authors thank Ms Francesca Pennetta for her technical support. The study was self-funded by the authors’ institution.
Cristina Lagalla developed her career as clinical embryologist at Sant’Orsola Hospital, Bologna and subsequently at SISMER IVF, Bologna. She’s currently senior embryologist at 9.baby Family and Fertility Center, Bologna. Her major interests are PGT and embryo morphokinetics. She published 25 papers and has been lecturer at several university courses. Key message Time-lapse and genomic technologies have been used to detect for the first time alternative modalities of embryo
Altogether, these findings suggest that iDAScore does recognize regular and irregular division, optimal timing of blastomere division, the synchronization of the cell cycle and embryonic morphological characteristics during the cleavage stage. Recently, the compaction at the morula stage has received attention in clinical embryology owing to its pivotal role in blastocyst organization and cell fate determination during morphogenesis (Lagalla et al., 2017; Coticchio et al., 2019; Lagalla et al., 2020). A recent study showed that blastomere exclusion and extrusion, i.e. cell disposal occurring before or during morula compaction, respectively, affected blastocyst yield and quality and pregnancy and live birth rates (Coticchio et al., 2021a).
Cristina Lagalla developed her career as clinical embryologist at Sant’Orsola Hospital, Bologna and subsequently at SISMER IVF, Bologna. She’s currently senior embryologist at 9.baby Family and Fertility Center, Bologna. Her major interests are PGT and embryo morphokinetics. She published 25 papers and has been lecturer at several university courses. Key message Time-lapse and genomic technologies have been used to detect for the first time alternative modalities of embryo compaction. This provides novel information on a developmental stage largely neglected in human assisted reproduction, extending the range of applicability of time-lapse microscopy for studying developmental processes and assessing embryo quality.