O5 - Reproducción

Isolation and characterisation of extracellular vesicles from in vitro-produced porcine blastocysts: impact of insulin-transferrin-selenium and metformin

Luchetti, Carolina Griselda ^1,2 - Caamaño, Diego ³ - Ibañez, Belén ³ - Bilbao, María Guillermina ^1,4 - Lorenzo, María Soledad ^1,2 - Cabezas, Joel ⁵ - Martinez, Ioanna ³ - Mendez-Perez, Lídice ³ - Ovidio Castro, Fidel ³ - Rodriguez-Alvarez, Lleretny ³ - Lombardo, Daniel Marcelo ^1,2

1) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, Buenos Aires C1425FQD, Argentina
2) Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal, Cátedra de Histología y Embriología, Chorroarín 280, Buenos Aires C1427CWO, Argentina
3) Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepción, Av. Vicente Mendez 595, Chillán 3780000, Chile
4) Universidad Nacional de La Pampa, Facultad de Ciencias Veterinarias, La Pampa, Argentina
5) Facultad de Medicina Veterinaria, Universidad San Sebastián, Concepción, Chile
Contacto: dlombard@fvet.uba.ar

Extracellular vesicles (EV) released into embryo culture media offer a promising non-invasive approach for assessing embryo quality, avoiding embryo damage (biopsy, fixation or homogenization). The objective was to evaluate the effects of supplementation with Insulin-Transferrin-Selenium (ITS) and metformin (Me) during porcine embryo culture on the size and concentration of EV obtained from the culture media.
After in vitro fertilization (IVF), porcine embryos were cultured in the following groups: C (without supplement, n = 9), M (Me 10-4 M, n = 2), ITS (ITS 0.1% v/v, n = 5), M+ITS (Me 10-4 M + ITS 0.1% v/v, n = 2). On day 7, expanded blastocysts were selected and individually cultured in 10µl drops of albumin-free medium until day 10. The culture media were stored at −20°C until use. EV were successfully isolated using Amicon® Ultra 0.5 mL centrifugal filters (Merck) and analysed for size and concentration using Nanoparticle Tracking Analysis (NTA). Expression of EV markers (TSG101, Alix, CD9) was confirmed by Western blot. Differences in median EV size and concentration between groups were analysed using the Kruskal-Wallis test, followed by Dunn's post-hoc test.
No significant differences were observed in EV size, with a median size of 275.5 nm and an IQR of 182 nm across all groups. Median EV concentration in Group ITS (112069 particles/mL, IQR 2171236) was higher (P <0.001) than in Group C (24276 particles/mL, IQR 962710). Further studies, including transmission electron microscopy (TEM) and increased sample sizes, are needed to confirm these findings.
This study reports, for the first time, the isolation and characterisation of EV from IVF-produced porcine blastocysts. ITS supplementation was associated with an increase in EV concentration, reflecting enhanced embryonic metabolic activity. Although preliminary, these findings are promising and suggest that EV could serve as potential indicators of embryo quality.

Palabras clave: ANIMAL REPRODUCTION, EMBRYONIC DEVELOPMENT, GLUCOSE METABOLISM