Background: Commercially-available media have revolutionised preimplantation embryo culture and assisted reproductive technologies across animal species, including humans. However, culturing mammalian embryos beyond the blastocyst stage remains challenging, primarily due to difficulty replicating placental functions. Recent advances have successfully cultured mouse embryos through postimplantation stages, offering powerful tools for characterising developmental processes and potentially producing animals without a uterus - which has applications for conservation and healthcare. Current post-implantation culture media rely on basal cell culture media supplemented with rodent, bovine and human sera.

We hypothesised that uterine fluid (UF) continues to support post-implantation embryo development. Therefore, UF factors could improve culture media formulations for these stages.

Methods: We performed proteomics analysis of mouse uterine fluid at different gestational timepoints to identify candidates for culture media. Bulk and single-nuclei transcriptomics of pregnant mouse uterus identified the source of candidate components. Selected factors were added to ex vivo mouse culture media in temperature- and gas-controlled roller devices, and effects on embryo and yolk sac development were observed.

Results: Although growth factors were rarely detected in UF (likely due to low abundance), their presence was inferred through presence of downstream targets. Uterine transcriptome analysis confirmed that glandular epithelium was the source of these factors. Addition of a cocktail of UF growth factors to media altered yolk sac development in cultured mid-gestation embryos.

Conclusion: These analyses enhance our understanding of how uterine fluid could impact embryo development across gestation, even post-implantation, and will inform the components required for improved embryo culture systems.