Chromosomal and functional characterization of the early stages of human embryogenesis

The main objective of modern IVF is to maximize the effectiveness of the times to achieve a pregnancy and at the same time also manage the risks, looking for new predictive parameters of the embryonic developmental competence. The analysis of embryo morphodynamic growth is not associated with its euploidy or implantation competence. However, some static parameters of embryo quality might exist that could be associated with embryo competence beyond its chromosomal constitution. The aims of this project are: i) To study from a morphodynamic, genetic and clinical point of view, embryos that show an abnormal development during preimplantation growth, in particular, the exclusion of cells (ExC) from embryonic mass at the moment of morulation. ii) Trying to understand if the morphodynamic characterization of euploid blastocyst development allows a higher prediction of live-birth (LB) after single-embryo-transfers (SET). For both the aims set in this Ph.D. project, preimplantation development and morphodynamic growth of embryos were observed in a time-lapse culture system (Embryoscope, Vitrolife). For the first aim, our preliminary data show that the exclusion of cells from the body of the blastocyst could be not-intuitively associated to a higher competence resulting from the embryonic capacity to overcome an abnormal cleavage pattern occurred in the very first divisions before the activation of the embryonic genome (4 to 8cell stage in humans). It is exciting the future perspective of collecting the ExC aiming at analyzing them through the karyomapping technology as well as biochemical assays, to better describe both the chromosomal segregation and the cellular physiology. For the second aim, we have divided the study into two phases in collaboration with 3 IVF Centers. In phase1, 511 first euploid SETs from 1069 patients undergoing preimplantation-genetic-testing-for-aneuploidies (PGT-A) cycles at 2 IVF centers were investigated (training set). All embryos were cultured in a specific time-lapse incubator with continuous media. The data from the time of polar-body-extrusion to time starting-blastulation were collected. Trophectoderm (TE) and inner-cell-mass (ICM) static morphology were also assessed. Logistic regressions were conducted to outline a predictive model of LB, whose power was estimated through a ROC-curve. In phase2, this model was tested in an independent dataset of 319 consecutive SETs from 546 PGT-A cycles at 3 IVF centers (validation set). The average LB-rate in the training set was 40% (N=207/511). Only time-of-morulation (tM) and trophectoderm quality were outlined as putative predictors of LB at both centers. The model showed a significant AUC (area under the curve) of 0.65. In the validation set, the euploid blastocysts characterized by tM<80hr and high-quality trophectoderm resulted in an LB-rate of 55.2% (n=37/67), while those with tM≥80hr and a low-quality trophectoderm resulted in an LB-rate of 25.5% (N=13/51;p<0.01). The ROC-curve analysis pictured an AUC of 0.6. A model including tM and trophectoderm quality involves a better prediction of euploid blastocyst reproductive competence. This model was reproducible across different centers under specific culture conditions. These data support the crucial role of morulation for embryo development, a stage that involves massive morphological, cellular and molecular changes requiring more investigations. Moreover, important guidelines for IVF laboratories that do not conduct a time-lapse-based embryo culture may arise from these two studies.