231 Blood plasma-derived extracellular vesicle characterization as a predictor of superovulatory response in sheep
M. C. C. Morais A , A. P. P. Schmidt A , L. F. L. Correia A , P. M. S. Rosa B , J. da Silveira B , F. Z. Brandão A , A. S. Alcântara-Neto A and J. M. G. Souza-Fabjan AA
B
Small extracellular vesicles (sEV) are nanoparticles delimited by plasma membrane phospholipid bilayer that play a crucial role in intercellular communication through the transference of molecules between cells. Analysis of sEV from blood provides a noninvasive method to identify biomarkers of reproductive status, offering potential applications in assisted reproduction. This study characterizes the abundance, size, and protein concentration of blood plasma-derived sEV from ewes showing either high or low response to superovulation (SOV). Santa Inês ewes (n = 20) underwent oestrus synchronization followed by a Day 0 protocol SOV program (Taira et al. 2022 Theriogenology 181, 140–146). Artificial inseminations were performed three times at 12-h intervals. Blood plasma samples were collected in the oestrous cycle’s follicular (Day 2) and luteal (Day 9) phases. Corpora lutea (CL) were counted by transrectal ultrasound and eight ewes were allocated into two experimental groups: high-response (HR; ≥13 CL; n = 4) or low-response (LR; ≤9 CL; n = 4). The sEV from blood plasma were isolated by two 15-minute centrifugations (at 300g and 12 000g) followed by two 90-minute ultracentrifugations (both at 100 000g). Protein quantification (Nanodrop®) and nanoparticle tracking analysis (NTA; Nanosight®) were performed. Data were submitted to the normality (Shapiro–Wilk) test, unpaired t-test (HR vs LR within luteal or follicular phase), and paired t-test (luteal vs follicular phase within HR or LR). Protein concentrations were similar (P > 0.05) between HR and LR groups, both in follicular (1.77 ± 0.58 vs 0.87 ± 0.17 mg mL−1, respectively) and luteal phases (0.88 ± 0.32 vs 0.98 ± 0.29 mg mL−1, respectively), with no differences (P > 0.05) within the same group, when comparing both phases as well. The NTA results showed that the concentration of sEV (particles/mL) was similar (P > 0.05) between HR and LR groups, both in follicular (3.3 × 109 ± 8.1 × 108 vs 3.9 × 109 ± 1.5 × 109) and luteal (2.2 × 109 ± 3.6 × 108 vs 2.5 × 109 ± 3.5 × 108) phases, respectively. Regarding size (nm), in the follicular phase, sEV from HR had a higher mode (P = 0.038) compared with the LR group (150 ± 4.2 vs 137 ± 2.4, respectively), indicating a possible relation between the size of sEV and the antral follicle count. Additionally, the HR group exhibited a higher mode size (P = 0.014) in the follicular (150 ± 4.2) than in the luteal (132 ± 6.5) phase, and a tendency to superiority (P = 0.075) in mean size during the follicular (179 ± 4.6) compared with the luteal phase (172 ± 5.6). In conclusion, this study provides initial evidence that blood-derived sEV may be further used as biomarkers for superovulatory response, showing promising potential for selecting females more responsive to traditional embryo transfer programs.
Financial support was provided by CAPES (001), CNPq, FAPERJ, and FAPESP # 15/21829–9.
