Bovine embryonic disc characterisation in in vitro-produced (IVP) embryos at early developmental stages

Anim Reprod. 2025; 22(3) THEMATIC SECTION: 41ST ANNUAL SCIENTIFIC MEETING OF THE ASSOCIATION OF EMBRYO TECHNOLOGY IN EUROPE (AETE) EMBRYOLOGY AND DEVELOPMENTAL BIOLOGY Bovine embryonic disc characterisation in in vitroproduced (IVP) embryos at early developmental stages Sara Núñez Torvisco1, Trudee Fair1, Pat Lonergan1 1University College Dublin, Ireland E-mail: [email protected] Pregnancy loss in cattle during the first trimester is primarily associated with impairments in blastocyst development and conceptus elongation and attachment. Additionally, evidence suggests that the method used to produce the embryo can alter endometrial responses by modifying both the endometrial transcriptome and embryonic gene expression. However, little is known about the phenotypic alterations that may arise from the origin of the embryo. Therefore, this study aims to phenotypically characterize bovine in vitro produced (IVP) embryos at early developmental stages, focusing on embryonic disc progression during the process of blastocyst expansion and hatching. Bovine blastocysts were produced from abattoir-derived ovaries from multiple collection days using commercial media for in vitro maturation, fertilisation and culture (Stroebech Media, ARTSMedia Denmark ApS). Mean blastocyst rate from starting cumulus-oocyte-complexes varied from 35 to 45%.The expression of SOX2 and SOX17, established biomarkers for inner cell mass (ICM)/ epiblast and hypoblast cells, respectively, was evaluated in IVP blastocysts at days 7 (D7, blastocysts, n=7), 8 (D8, hatched blastocysts, n=4), and 9 (D9, hatched blastocysts, n=6), using wholemount immunofluorescent labelling. Isotype-matched IgG negative controls were included for each biomarker. Z-stacks were acquired using an LSM800 Airyscan confocal microscope and analysed with Fiji (Schindelin, et al., Nature Methods, 9(7), 676–682, 2012). Statistical analyses (one-way ANOVA followed by Tukey’s HSD test) were performed in R. Significance was defined at P<0.05. The number of SOX2+ cells decreased significantly across time. From D7 (52.4%, 33/63) to D8 (28.3%, 56/198), there was a 24.1% reduction, followed by a further 16.8% decrease from D8 to D9 (11.5%, 24/209), resulting in a total reduction of 40.9% from D7 to D9. This reduction coincides with the developmental transition from the blastocyst stage to blastocyst expansion and hatching from the zona pellucida, during which the ICM begins to differentiate into epiblast and hypoblast lineages, with SOX2 becoming specific to epiblast cells. SOX17 expression was detected in D8 blastocysts and appeared to co-localise with SOX2. Additionally, by D9, 66.7% presented SOX17 labelling in cells directly underneath SOX2+ cells and in cells migrating towards the blastocoel, potentially indicating the initiation of hypoblast migration. Continued investigation is ongoing to fully characterize these developmental changes as the embryo progresses through ovoid, tubular and filamentous stages and to determine whether, and how, the origin of the embryo (in vivo derived vs. IVP) contributes to phenotypic variation during early development that may underlie embryonic loss. Acknowledgments: This research was funded by AFRODITA that has received funding from the European Union’s Horizon Europe programme under the MSCA Doctoral Network grant agreement No 101120126. Keywords: bovine, embryonic disc, IVP.