AQP3 and AQP9 expression in extracellular microvesicle-enriched samples of plasma and supernatant of placental explants from healthy pregnant women at term

FERREIROS, ALBERTO; DAMIANO, ALICIA E; COROMINAS ANA; CASALE, ROBERTO; SZPILBARG, NATALIA; RUIZ, JULIETA

Bogotá (Virtual)

Simposio; IX Simposio de la Sociedad Latinoamericana de Interacción Materno-Fetal y Placenta; 2022

Sociedad Latinoamericana de Interacción Materno-Fetal y Placenta

Objectives: To detect AQP3 and AQP9 protein expression in extracellular microvesicle-enriched samples of plasma and supernatant of placental explants from healthy pregnant women at term.Methods: EDTA anticoagulated blood samples and placentas were obtained from 6 women with healthy term pregnancies, under Ethics Committee protocol and signed informed consent at Hospital Posadas in Buenos Aires. Explants were prepared and cultured for 24 hours under standard culture conditions. Plasmas and the corresponding supernatants of placental explants from each patient were subjected to a protocol of differential centrifugation, filtering, and ultracentrifugation to obtain samples enriched in extracellular microvesicles. Each sample was analyzed by Dynamic light scattering (DLS) in a Zetasizer Nano-ZSP equipment (Malvern Instruments, UK) equipped with a He-Ne laser (633nm) and a digital correlator, model ZEN5600, to determine the size of the microvesicles. Subsequently, the samples were analyzed by Western blot to determine the expression of AQP3, AQP9, and PLAP using specific antibodies: anti-PLAP HD 11F7 (1:200, Santa Cruz), anti AQP3 (1:1000, Alpha diagnostics), anti- AQP9 (1:1000, Alpha diagnostics) and secondary antibodies (Jackson), using homogenates from each placenta as positive controls.Results: One or two (depending on the case) predominant microvesicle populations, whose size ranged between 40 and 220nm, were obtained in plasmas and their corresponding supernatants of placental explant cultures. Using Western blot, the presence of PLAP (syncytiotrophoblast marker), AQP3 and AQP9 was evidenced in all the samples analyzed.Conclusion: These preliminary results allow us to lay the foundations to be able, in the future, to determine the presence of AQPs in microvesicles of placental origin in the plasma of pregnant women at any stage of pregnancy. In this sense, monitoring the expression levels of these proteins in healthy and pathological pregnancies may also let us learn more about their functions during pregnancy.