hsa-miR-375-3p in embryo culture medium exosomes as a preimplantation non-invasive biomarker for predicting live birth after IVF.

RESEARCH QUESTION: Can non-coding RNAs in exosomes from IVF embryo culture media serve as non-invasive markers for evaluating embryo quality?

DESIGN

Spent culture medium (SCM) was prospectively collected from grade 1 eight-cell-stage embryos that resulted in pregnancy success (defined as live birth, n = 34) or failure (n = 33) among patients who underwent IVF and single embryo transfer at a single centre over 5 months. Exosomes were isolated by precipitation, followed by high-throughput small RNA sequencing to profile exosomal non-coding RNAs, including miRNAs, tRFs and piRNAs. The diagnostic potential of validated differentially expressed miRNAs was then evaluated by receiver operating characteristic (ROC) analysis. Specific inhibitors targeting differentially expressed miRNAs were designed and microinjected into mouse embryos to assess their effects on embryonic development.

RESULTS

Single-particle immunofluorescence analysis confirmed the presence of exosome-specific markers (CD63, CD9 and CD81) in isolated exosomes. Small RNA sequencing of exosomes identified 51 miRNAs, 547 piRNAs, and 39 tRFs, among which five, 11 and eight were differentially expressed, respectively. Validated by qRT-PCR, expression of hsa-miR-375-3p and hsa-miR-215-5p in SCM-derived exosomes was higher in the successful pregnancy group than in the pregnancy failure group. The ROC curve analysis indicated that hsa-miR-375-3p (AUC = 0.763, sensitivity 85%, specificity 70%) is a potential marker of embryo quality, laying the foundation for a miRNA-based predictive model to improve IVF outcomes. Inhibition of miR-375-3p significantly reduced the eight-cell mouse embryo formation rate (P = 0.0358).

CONCLUSIONS

hsa-miR-375-3p in exosomes from SCM of embryos is a non-invasive predictive biomarker of live birth after IVF. miR-375-3p is critical for embryonic development during the four- to eight-cell stage transition.