P-412 3D live-imaging reconstruction of the human embryo implantation ex vivo

Abstract Study question How do human embryos implant in physiological conditions and develop beyond blastocyst stage? Summary answer Human embryos apply forces during invasion of the matrix, the mural trophectoderm undergoes a quick compaction leading to a fast radial expansion of polar side What is known already Human embryos can be cultured beyond blastocyst stage using supplements of animal origin, such as fetal bovine serum. However, two main issues limit our understanding of the implantation of the human embryo. First, human embryos do not express fluorescent proteins, severely limiting the access to advanced live-imaging tools. Second, the animal serums may not represent the physiological peri-implantation conditions of the human embryo, thus limiting the reproducibility of the in vitro experiments. Here we combine label-free multiphoton imaging with media formulations including clinical-grade protein supplements from human plasma unlocking the observation of the human embryo in 3D and physiological conditions Study design, size, duration We have cultured 150 human embryos from D3 until blastocyst stage and also more than 600 mouse embryos from zygote until blastocyst stage and then transferred them into a 3D implantation platform. All embryos were supplemented with clinical-grade human-derived serum, which is rich in globulins and growth factors, and commercial basal media. The implantation was monitored for 3-to-5 days up until day 11. Traction-force microscopy and molecular imaging was employed to quantify implantation markers. Participants/materials, setting, methods Implantation was measured using our novel version of traction-force microscopy, which reveals the force applied by entire embryos during live-imaging experiments. Multiphoton illumination of autofluorescent molecules allows to reconstruct images of live human embryos on 3D at unprecedented resolution. Key molecular markers for the formation of the epiblast rosette (OCT4), extra-embryonic visceral endoderm (GATA4), extra-embryonic ectoderm and trophoblast (CDX2) and primary yolk sac and pre-amniotic cavity were reconstructed in 3D using high resolution confocal imaging. Main results and the role of chance The implantation of human embryos was visualized in 3D movies at high resolution, during time-resolved experiments and compared to implantation in mouse embryos. Human embryos undergo a remarkable compaction upon attachment of the polar trophectoderm and thereafter they implant by en