A novel embryo culture system with reduced nutrients supports development of human 3PN embryos and extended embryo outgrowth

Abstract

In this study, we utilize discarded human zygotes with three pronuclei to investigate a novel system for human embryo culture. Our objective was to determine the efficacy of a medium with significantly reduced nutrient concentrations for human embryo culture to the blastocyst stage, and to investigate quality of the resulting embryos in a peri-implantation extended culture system. Research Study. Human zygotes with 3 pronuclei (PN) were cryopreserved with consent, thawed, and placed into control (CON; n=23) or reduced nutrient (RN; n=25) sequential culture medium, both supplemented with HSA. Embryos were cultured individually in an EmbryoScope and were assessed for blastocyst development on D5 and D6. On D6, blastocysts were placed into fibronectin coated dishes for outgrowth culture in IVC1 (Cell Guidance Systems) medium, along with thawed D6 human blastocyst controls donated to research after culture under standard clinical conditions (Sage CM/BM with SPS; 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. 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 n=11; RN n=7; CBL n=7). There was no difference in the area of outgrowth between treatments, either at 48h (0.05±0.02mm2, CON; 0.09±0.04mm2, RN; 0.06±0.02mm2, CBL) or 96h (0.12±0.06mm2, CON; 0.23±0.10mm2, RN; 0.20±0.10mm2, 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). 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.