Human uterine fluid lavage-derived extracellular vesicle isolation: a comparative study for minimally invasive endometrial receptivity assessment

Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles?First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized.Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene.The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing.