PROLONGED EXPOSURE OF HUMAN BLASTOCYSTS TO HYALURONAN-ENRICHED TRANSFER MEDIA HAS NO EFFECT ON PERI-IMPLANTATION STAGE EMBRYO DEVELOPMENT DURING IN VITRO CULTURE

Abstract

Our objective was to determine whether the prolonged exposure of human blastocysts to EmbryoGlue (EG) is beneficial for human peri-implantation stage development in vitro. Additionally, we investigated whether the addition of a cocktail of estradiol (8nM), progesterone (200 ng/mL), pyruvate (1mM), and lactic acid (0.22% v/v) to EG would benefit human embryo development during the peri-implantation stage in vitro. Vitrified human blastocysts donated for research (WIRB study no. 1179872) were warmed and recovered in EG or EG with additives (EGA) for either 10 min or 3 h (EG10m, EGA10m, EG3h, and EGA3h). Embryos from each group were then fixed with 4% paraformaldehyde and stained for DAPI and antibodies against cleaved caspase-3 to examine apoptotic stress. Separate blastocysts were also treated (EG10m, EGA10m, EG3h, and EGA3h) and then introduced to an extended embryo culture (EEC) system (Deglincerti et al., Nature 2016) and cultured in vitro until EEC day 5. Embryo attachment, morphology, and trophectoderm outgrowth areas were assessed on each day during EEC. Finally, we performed surgical ET in mice to assess implantation and fetal developmental potential of in vitro produced CF1 embryonic day 3.5 mouse blastocysts exposed to EG or EGA for 3 h. Implantation and fetal development were assessed at day 17.5 post fertilization. No differences in total (EG10m: 5.71 ± 0.98 n=24; EGA10m: 6.62 ± 0.92 n=21; EG3h: 9.50 ± 2.59 n= 24; EGA3h: 8.14 ± 1.29 n= 21) or % apoptotic cells (EG10m: 7.99% ± 1.52%; EGA10m: 11.13% ± 1.53%; EG3h: 13.22% ± 4.04%; EGA3h: 12.63% ± 1.93%) were noted amongst treatments. There were also no differences in attachment, percent of normal development, or outgrowth areas during EEC. Finally, there were no differences in fetal development following surgical ET in mice (Fetus/Implantation: EG3h 21%, n=51; EGA3h 33% n=50). Prolonged exposure of human blastocysts to EG has no effect on peri-implantation stage embryo development during in vitro culture.