Seeking Arrangements: A New Cleavage-Stage Biomarker from 3D Morphokinetics

Abstract Study question What can we learn from 3D reconstructions of cleavage-stage embryos derived from Hoffman modulation contrast (HMC) time-lapses? Summary answer A simple spatial biomarker extracted from 3D embryo reconstructions at the t4 and t8 stages is associated with blastulation, blastocyst quality, pregnancy and live birth. What is known already Several works have demonstrated significant associations between t4 cell arrangement and blastulation potential. However, no studies have investigated the impacts of cell arrangement beyond the t4 stage in a clinical setting owing to difficulties visualising the 3D structure of embryos in a safe, cost-effective manner. In the previous ESHRE meeting, He et al. presented a deep learning system for the 3D reconstruction of cleavage-stage embryos from HMC focal stacks recorded in standard timelapse incubators. In this work, we use the aforementioned system to understand the spatial networks present in cleavage-stage embryos and investigate their associations with clinical outcomes. Study design, size, duration The study was a retrospective analysis of two imaging datasets from two different clinics. The first dataset (DS1) consisted of 162 t4 embryos with information on blastulation and Gardner grade. The second dataset (DS2) consisted of 202 embryos at t4 and t8 with information on blastocyst grade, pregnancy, live birth and PGT-A. All data was captured at 11 focal planes on Embryoscope incubators between 2018 and 2020. Participants/materials, setting, methods The system proposed by He et al. was used to reconstruct the 3D structure of embryos from their focal stacks. Networks of cell contacts were extracted from the resulting embryo 3D models and each embryo’s mean contacts per cell was computed. Statistical analysis of average cell contacts with respect to outcomes was carried out using unpaired t-tests. Moreover, cell contact networks from different embryos were compared to identify embryos with similar cell arrangements. Main results and the role of chance At t4, a higher average number of contacts per cell was associated with greater rates of blastulation in DS1 (2.59 vs 2.36, blastulated vs non-blastulated, p = 0.029) and blastocyst quality in both DS1 (2.59 vs 2.37, good vs poor, p = 0.010) and DS2 (2.51 vs 2.35, good vs poor, p = 0.017) where a ‘good’ embryo is defined as having an embryologist-provided Gardner grade with EXP>2, ICM>C and TE>C. At t8, a higher average number of contacts was associated with increased blastocyst quality (3.36 vs 3.09, good vs poor, p = 0.017), pregnancy (3.32 vs 2.87, pregnant vs not pregnant, p = 0.003) and live birth (3.40 vs 2.90, live birth vs no live birth, p = 0.0003). No associations were found with miscarriage or aneuploidy. Moreover, average contacts at t4 were not correlated with those at t8 (r = 0.15, 95% CI [-0.043, 0.34]). While 4-cell embryos fell neatly into 9 distinct cell arrangements with the 5 most common (tetrahedral, pseudotetrahedral, planar, closed-Y and linear) accounting for 95% of embryos, 8-cell embryos displayed a great degree of variation with 59 distinct cell arrangements, the largest such group representing only 8 embryos. Limitations, reasons for caution The datasets used in this study were small. Moreover, DS2 was subject to survivorship bias as it only contained embryos with PGT-A results which necessarily entailed successful blastulation. Furthermore, the 3D reconstruction system was not applicable to all cleavage-stage embryos, especially those obscured by the well or having undergone compaction. Wider implications of the findings This work provides evidence for the clinical relevance of cleavage-stage cell arrangement in the human preimplantation embryo beyond the 4-cell stage, which may improve selection techniques for D3 transfers. Moreover, our work provides a strong case for further investigation into spatial biomarkers derived from 3D embryo reconstruction and 3D morphokinetics. Trial registration number N/A