Cell-free RNA in spent embryo medium for non-invasive assessment of preimplantation embryo quality: a review

Embryo quality assessment of preimplantation was a key step that affects the treatment outcomes of assisted reproductive technology (ART). Conventional morphological grading and time-lapse imaging (TLI) were widely used in clinical embryo selection. However, these methods mainly rely on morphology and developmental kinetics. They cannot fully reflect the intrinsic transcriptional activity, metabolic homeostasis and stress status of embryos. Recently, cell-free RNA (cfRNA) has attracted increasing attention as an important molecular biomarker in liquid biopsy. It was non-invasive, dynamic and functionally informative. During in vitro culture, embryos can release RNA fragments into the spent embryo medium (SEM). These RNA molecules may reflect the developmental status of embryos. This review summarizes the molecular types, biological sources, release mechanisms and stability of cfRNA. It also describes the potential associations between cfRNA in SEM and embryo morphology, developmental potential, chromosomal status, implantation competence and pregnancy outcomes. In addition, we discuss the application value of cfRNA in non-invasive embryo quality assessment. Current evidence suggests that cfRNA may serve as an important complement to morphological grading, TLI and cfDNA detection. It may provide a new strategy for building a multidimensional, non-invasive and precise embryo selection system. However, this field still faces several challenges, including the low sample volume of SEM, RNA degradation, difficult contamination control, inconsistent detection workflows and insufficient clinical validation. Future studies should promote standardized detection procedures, combined analysis of multiple cfRNA types, multi-omics integration and artificial intelligence-based modeling. These efforts may accelerate the clinical application of cfRNA in ART.