P-388 Isolation and characterization of human uterine fluid lavage-derived extracellular vesicles by three different methods for optimal embryo transfer timing discovery
Authors: Saraee F , Shekari F , Moini A , Sadeghi M , Ghaznavi P , Nazari A , Ghaheri A , Totonchi M , Eftekhari-Yazdi P 
Published: June 2022

Abstract

Study question
What is the suitable method for isolating extracellular vesicles (EVs) from a small volume of Uterine Fluid Lavage (UFL)?

Summary answer
It seems that the qEV column can help in faster, precise, and gentle isolation of EVs from small amounts of UFL.

What is known already
Several studies have discovered that existing EVs in the UFL, which are informative carriers, reflect cytological and molecular conditions of the uterine environment. The relative amounts of the EV components are different in various physiological situations. So, they are the ideal source for precise molecular analysis of the uterine condition to identify the Window of Implantation (WOI) for appropriate embryo transfer (ET) timing. There is limited information describing the most efficient method for EV isolation from a low-volume sample of biological fluids. In particular, EV separation from some biological fluids such as UFL is more complicated because of the viscosity.

Study design, size, duration
The study was conducted in two steps; first, the pre-implantation UFL sampling effect was studied on the implantation and clinical pregnancy rates. UFLs were obtained from 20 IVF Frozen Embryo Transfer (FET) patients as the case group. The control group consisted of 20 FET patients without lavage. In the second step, UFLs (n = 30) were collected and pooled. The EVs were extracted by ultracentrifugation (UC), Sucrose cushion (Suc), and Size-exclusion chromatography (by the qEV column) methods.

Participants/materials, setting, methods
First, UFL was collected two days before frozen blastocyst ET from the case group using an intrauterine insemination catheter attached to a syringe. Then, patients were compared with control at the seventh week. Second, EVs were extracted from pooled UFL samples and characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western Blot (WB). Albumin contamination was detected by SDS-PAGE. Total RNA was extracted, and Real-time PCR was performed for six housekeeping genes.

Main results and the role of chance
A comparison between case and control groups indicated no significant difference for implantation and clinical pregnancy rates. Moreover, we have confirmed the presence of EVs in UFL that were successfully isolated, by the UC, Suc, the qEV methods. The SEM images showed that the collected vesicles were morphologically spherical. The DLS analysis revealed that the intensity peak of isolated particles with the UC and Suc methods were 281 nm and 216 nm, respectively. Extracted EVs using the qEV technique were smaller and homogenized in size, with an intensity peak of 67.7 nm. SDS-PAGE of EVs showed a weaker albumin band in the qEV column (with a 100 kDa filter) method than the UC, Suc, and qEV column (with a 30 kDa filter) methods. Although, their quantification showed no significant differences. WB analysis indicated common EV markers (D81, CD9, CD63, and TSG101) showed better expression using the qEV extraction technique. But CD9 and TSG101 had lower expression levels in EVs isolated using Suc and UC, respectively. Calnexin was not detected in the isolated EVs. The GAPDH, 18SrRNA, ACTIN, GUSB, HMBS, and YWHAZ genes were detected by real-time PCR in all three EV extraction methods. The 18SrRNA gene showed better CT values.

Limitations, reasons for caution
Uterine fluid samples collection from qualified study groups and the patient’s follow-up for ensuring successful implantation and clinical pregnancy are time-consuming. The low volume of uterine fluid lavage sample and consequently the low number of extracellular vesicles inside it was also the limitation of this study.

Wider implications of the findings
Endometrial receptivity assay is difficult to study non-invasively. Herein, endometrial secretome EVs as a minimally invasive source for uterine environment molecular assessment is presented. High-throughput analysis of EV components can offer biomarkers for individual determination of ET timing, providing an additional method to use with transvaginal-ultrasonography for assessing endometrial receptivity.

Trial registration number
not applicable