56 Effects of fetal bovine serum-derived extracellular vesicles on expression of lipid metabolism-related miRNA in oocytes during in vitro maturation

Fetal bovine serum (FBS) is the most commonly used source of macromolecules in culture media during in vitro production (IVP) and is considered one of the main contributors to the increased lipid accumulation in IVP embryos. FBS contains small extracellular vesicles (sEVs) that carry bioactive molecules, including miRNAs. The present study aimed to assess the effects of sEV during IVM on oocyte maturation rates, developmental competence, and lipid metabolism-related miRNA expression levels in oocytes. sEVs were isolated from 1 mL of FBS by size exclusion chromatography (qEV1, Izon) and ultracentrifugation (100 000g for 70 min at 4°C). Bovine cumulus–oocyte complexes (COCs) were collected by aspiration of 3- to 6-mm follicles from abattoir ovaries and subjected to IVM in three groups: (1) cFBS (n = 204; complete FBS, 10%), (2) dFBS (n = 200, depleted of its own sEVs, 10%), and (3) dFBS + sEVs (n = 199, sEVs, 10%) for 22 h, at 38.5°C and 5% CO₂ in air. At the end of IVM, cumulus cells were removed from COCs, and denuded oocytes evaluated for (1) maturation rates by first polar body extrusion, (2) developmental competence (Day 7 blastocyst rates) after standard IVF and IVC, and (3) for expression levels of lipid metabolism related miRNAs by RT-qPCR. Data were analyzed by ANOVA, followed by Tukey’s test, and significance was set at the 5% level. First, concentration and size of sEVs were determined by nanoparticle tracking analysis (NanoSight NS300, Malvern; 1.33 × 10¹⁰ ± 1.03 × 10⁹ particles mL⁻¹, mode size 149.94 ± 13.34 nm and mean 187.8 ± 3.6 nm). The presence of sEV marker proteins was analyzed by flow nanocytometry (CytoFLEX) and was positive for CD81-Calcein (281.66 events/µL) and Syntenin (23.86 events/µL) compared with the negative control (PBS + antibody, 1.78 and 7.20 events/µL, respectively). Transmission electron microscopy was used to analyze sEV morphology and revealed typical cup-shaped structures, consistent with sEVs. The three analyses validated the isolation method. Maturation rates of ~70% (n = 135–169/group, five replicates; P > 0.05) and blastocyst rates of ~47.0% (n = 104–107/group, five replicates; P > 0.05) were not affected by treatments (P > 0.05). From the 41 miRNAs selected as predicted to modulate pathways associated with lipid metabolism, 11 were identified in matured oocytes and only bta-miR-27a-3p had expression affected by the treatments, in which the addition of sEVs increased in treated oocytes (P < 0.05). This miRNA was previously identified in sEVs, suggesting its possible transfer from sEVs to oocytes during IVM. In conclusion, depletion of sEVs from FBS does not affect oocyte maturation or developmental competence or their reintroduction into depleted FBS. However, one miRNA increased in oocytes matured with sEVs and was probably transferred to the oocyte during IVM; it may have been able to regulate oocyte lipid metabolism. These are partial results of an ongoing study and other lipid metabolism parameters are under analysis on oocytes treated with sEVs during IVM.

Financial support was provided by FAPESP grant 2021/06760-3 and PAF postdoctoral fellowship 2024/05583-9.