Transcriptomic profiling of exRNA from spent human embryonic cleavage culture medium reveals potential in embryo quality assessment: a retrospective study

In brief: Noninvasive embryo quality assessment beyond morphology remains an urgent need. By identifying exRNA in spent embryonic culture medium as a potential noninvasive biomarker, this study provides a foundation for noninvasive strategies based on exRNA in spent culture medium, ranging from embryo quality assessment to applications in reproductive medicine, developmental biology. Abstract: The extracellular RNA (exRNA) transcriptome of the preimplantation embryo culture medium constitutes a comprehensive profile of embryonic RNAs, offering a noninvasive resource for elucidating developmental status. To delineate its relationship with the maternal-to-zygotic transition (MZT) and evaluate its predictive potential for developmental outcomes, we performed systematic exRNA transcriptome profiling of individually cultured cleavage-stage embryos. This study included 34 spent embryo culture medium (SECM) samples for day 3 in-vitro preimplantation cleavage embryos from 30 patients undergoing in-vitro fertilization (IVF)/intracytoplasmic sperm injection. Twenty-four morphologically high-grade samples (grade I, n = 12; grade II, n = 12) and 10 low-grade samples (grade III, n = 6; abnormal pronuclei, n = 2; arrested embryos, n = 2) were included. Embryo transfer after the SECM collection was traced for subsequent clinical pregnancy outcome. ExRNA transcriptome from cleavage SECM showed 81.78% concordance with embryonic gene expression profiles. A total of 1,058 differentially expressed exRNA markers were identified across embryo morphological grading groups, revealing MZT-related molecular dynamics prior to blastocyst formation. A 300-gene signature significantly correlated with embryonic developmental potential was established. The accuracy and robustness of the models were validated using clinical samples. The LogitBoost model built on exRNA profile yielded an area under the curve value above 0.95, indicating encouraging performance for embryo assessment. This study establishes SECM exRNA profiling as a noninvasive method for capturing key molecular events during early embryo development, particularly MZT activation. The identified exRNA biomarkers and machine learning models provide a promising framework for the objective assessment of embryo developmental potential, warranting further validation to overcome the limitations of conventional morphology-based selection in IVF.