Endocrine Abstracts

Pregnancies affected by GDM commonly result in large-for-gestational-age (LGA) infants, which have an increased risk of developing cardiometabolic complications. The mechanisms responsible are unclear but are associated with altered placental physiology. We have previously reported that miRNAs, including pancreatic specific miR-375, are altered in maternal serum extracellular vesicles (EVs) prior to the onset of LGA, and in term placenta. We assessed maternal pancreatic-derived EV/miRNA internalisation into human placenta and whether these could contribute to LGA by influencing placental development/function. QPCR confirmed that miR-375 was not produced in the human placenta by assessing levels of both mature miR-375 and primary transcript (pri-miR-375). Mature miR-375 was present in the placenta, but pri-miR-375 was not detected, suggesting miR-375 is likely transported to the placenta, potentially via EVs from maternal circulation. We next isolated EVs from pancreatic islets. Human pancreatic islet equivalents (20,000 IEQs/patient) were obtained through the Integrated Islet Distribution Program (IIDP) from female donors of reproductive age. Islets were cultured 3 days under normoglycaemic (5.5mM), or mild hyperglycaemic (7mM glucose) conditions. Islet purity/viability were confirmed using dithizone/trypan blue. EVs in conditioned media were isolated by size-exclusion-chromatography and characterised by nanoparticle tracking analysis (NTA; mean diameter, 83.5-163.2nm; 1.28x10⁹-1.82x10¹⁰ particles/ml), Western blotting (EV-proteins) and electron microscopy (cup-shape morphology). QPCR confirmed miR-375 was present in islet-EVs and levels were altered in response to hyperglycaemia. Fluorescent microscopy confirmed placental explant uptake of maleimide-488 labelled islet-EVs. Functional impact of miR-375 on the placenta was assessed following miR-375 overexpression (30-fold, P<0.05; n=6) using specific miRNA-mimics. TMT-mass-spectrometry demonstrated that miR-375 altered the placental proteome. Functional enrichment and Ingenuity pathway analyses revealed differentially expressed proteins were significantly enriched in pathways associated with placental growth, glucose metabolism and vascularisation. Our data provides insight into the potential mechanisms contributing to LGA in GDM pregnancies.