Noninvasive Profiling Reveals Stage‐Specific Cell‐Free RNA Dynamics and the Characterization of Immune Status in Preimplantation Embryos

Previous studies indicated that cell-free RNA (cfRNA) in spent embryo medium (SEM) may serve as a noninvasive biomarker for evaluating embryonic development. However, its stage-specific expression patterns and functional dynamics during preimplantation embryos are still unclear. In view of this, this study performed transcriptome sequencing on cfRNAs from follicular fluid (FF) and SEM at different developmental stages. This result showed that the number of identified cfRNAs showed a trend of decreasing from follicular to cleavage stage, then increasing through blastocyst stage. The proportion of low abundance genes of protein coding RNA (mRNA) and long noncoding RNA (lncRNA) in FF and spent cleavage medium (SCM) was relatively high (0 < FPKM ≤ 10). The proportion of low abundance genes of mRNA and lncRNA in spent blastocyst medium (SBM) was lower than that of high abundance genes (FPKM > 10). The cfRNAs of differentially expressed were detected in FF, including ribosomal proteins and genes involved in protein translation. The genes regulating protein kinase activity were significantly enriched in SCM. Cellular signal transduction, energy metabolism, immunity, RNA transcription, and splicing genes were significantly enriched in SBM. Further studies revealed that immune genes (JAK3, STAT6, DOCK8, CD74) in SBM were closely related to embryo quality, and the expression level of immune genes in high-grade SBM (hSBM) was significantly (p < 0.05) higher than that of low-grade SBM. The Wnt/β-catenin signaling pathway was found to be associated with hSBM, potentially contributing to blastocyst development through the release of immune factors and activation of immune-related pathways. However, these findings were preliminary. Therefore, this study results provide new insights into the molecular dynamics of preimplantation embryos and the impact of immune responses on embryo quality.