; CBL). After 48h in outgrowth, attachment was assessed and media was replaced with IVC2. Media was replaced daily until 96h of outgrowth (D10), at which point embryos were fixed and imaged to measure outgrowth area. Embryos were then stained with F-actin, DAPI, and POUF51, and imaged using confocal microscopy to determine outgrowth volume, total cell number, and epiblast cell number, respectively. RESULTS: Blastocyst development was not different between 3PN embryos cultured in CON and RN medium on D5 (30.4% and 24.0%) or D6 (34.7% and 28.0%). All embryos placed into outgrowth were attached by 48h (CON n¼11; RN n¼7; CBL n¼7). There was no difference in the area of outgrowth between treatments, either at 48h (0.05AE0.02mm 2 , CON; 0.09AE0.04mm 2 , RN; 0.06AE0.02mm 2 , CBL) or 96h (0.12AE0.06mm 2 , CON; 0.23AE0.10mm 2 , RN; 0.20AE0.10mm 2 , CBL). Of embryos placed into outgrowth, 73% CON, 60% RN, and 100% CBL had a 3D volume that was assessed using confocal microscopy. Of these embryos, 50% of CON, 67% of RN, and 40% of CBL embryos contained a visible epiblast; these embryos had similar average numbers of epiblast cells (59, CON; 41, RN; 67, CBL). CONCLUSIONS: This data demonstrates that an environment of reduced nutrient concentration successfully supports the development of human zygotes to the blastocyst stage, with equal developmental potential to both those cultured in control medium and those cultured in standard clinical conditions. In addition, 3PN zygotes developed to the blastocyst stage and successfully organized peri-implantation embryonic development equivalent to normally fertilized embryos. This innovative approach to safely investigating novel culture conditions for human embryos could significantly enhance research in the development of more effective embryo culture media for human ART.
