O2135: Extracellular Vesicles from the Amniotic Fluid as Potential Biomarkers of Neuroinflammation in Human Myelomeningocele

Background: Spina bifida (SB) is a devastating congenital disease, characterized by failure of the neural tube to close, resulting in exposure of the fetal spinal cord. Despite promising advances in antenatal and postnatal interventions, SB is still burdened by poor outcomes, whose causes remain unknown. Recent data have indicated that the inflammation of the spinal cord may play a major role, but our knowledge is limited to experimental studies in rodents and lambs, as it is impossible to obtain neural tissue from human fetuses without injury. Extracellular vesicles (EVs) have recently been proposed as the paradigm for liquid biopsies of tissues and organs that are inaccessible, like the central nervous system. EVs are nanoparticles secreted by all cells and carry cargo (lipids, proteins, RNA species) that reflects the status of the cell of origin. The aim of our study was to profile EVs derived from the amniotic fluid (AF-EVs) fetuses with SB and their cargo.

Methods: Following ethical approval (IRB #2017-2414CFC), amniotic fluid was collected from fetuses with SB aperta (n= 6) at the time of surgical repair (24-25 gestational weeks) and from age- and sex-matched fetuses with twin-twin transfusion syndrome (TTTS, n= 6) that served as controls. EVs were isolated using differential ultracentrifugation and characterized by size (nanoparticle tracking analysis), morphology (transmission electron microscopy), and expression of canonical protein markers (CD63 and TSG101, Western blot). EV cargo was analyzed for expression of inflammatory mediators (Western blot) and small RNA cargo (NovaSeq(S2). Bioinformatics analysis was conducted using featureCounts/R and gene set enrichment analysis (GSEA) adapted for miRNAs was performed using miEAA 2.1.

Results: Compared to TTTS, AF-EVs from SB fetuses had higher density (SB-EVs= 1.07x1011±3x107, TTTS-EVs= 5.24x1010±4x107; p= 0.02) and smaller size (Fig.A, p=0.04). EVs from both populations had expression of EV protein markers and typical double lipid membrane morphology (Fig.B). SB-EVs had evidence of inflammasome activation with upregulation of NLRP3 and pro-caspase-1 (Fig.C). Small RNA-sequencing experiments identified miRNAs (miR-30d, miR-151a, miR-203a), lncRNAs (NEAT1, MALAT1, GAS5), and miscRNAs (RNY1, RNY4P7, RNY4P27) in SB-EVs that are involved in neuroinflammation and inflammasome activation (Fig.D). GSEA analysis identified several miRNAs in SB-EVs related to neuroinflammation via activation of the inflammasome pathway (Table 1).

Conclusion: This study reveals that AF-EVs from SB fetuses contain inflammasome mediators. Further studies are underway to define the exact cellular origin of EVs carrying the inflammatory profile. Nonetheless, the ability to isolate EVs from the amniotic fluid makes them promising biomarker candidates to facilitate early screening, grade disease severity, guide appropriate counseling, and select patients for prenatal surgery.