P-202 Improvement of in vitro implantation competence in human trophoblastic spheroids and mouse blastocysts by extracellular vesicles from human follicular fluid
Authors: Yoon H , Kang I , Kim Y.J , Yang K.M , Lee J , Jun J.H 
Published: June 2022

Abstract

Study question
How do extracellular vesicles (EVs) from human follicular fluid (hFF) affect the implantation process?

Summary answer
The EVs from hFF support the migration of mouse blastocysts and improve the mRNA expression of implantation process in human trophoblastic spheroids and mouse blastocysts.

What is known already
The EVs in hFF contains proteins, mRNAs, and microRNAs (miRs) that can mediate intercellular communication. Some miRs were found to be enriched in EVs of hFF. It has been demonstrated that EVs play different and important roles in the reproductive process such as oocyte maturation, embryo development and implantation. However, the effects of EVs in hFF on the trophoblast migration of in vitro implantation competence remain unclear. We found and analyzed the enriched miRs in EVs of hFF and investigated the effects of EVs on the implantation competence using in vitro outgrowth models using human trophoblastic spheroids and mouse blastocysts.

Study design, size, duration
Mouse 2-cell embryos were collected and then further in vitro cultured up to blastocyst stage. We prepared spheroids with trophoblastic cells, JAr mixed JEG-3 cells (JmJ) of 1:1 ratio. For outgrowth assay, the culture dishes with fibronectin-coated were made 2 hrs before transferring trophoblastic spheroids and mouse blastocysts. After 72 hrs of outgrowth assay, the migration areas of trophoblasts were measured, and qRT-PCR and Western blot were analyzed.

Participants/materials, setting, methods
After collecting the hFF samples in IVF clinic, immediately the EVs were isolated using the conventional ultracentrifugation (UC) method, and stored at -80 °C. Amounts of specific miRNAs were analyzed to confirm the specific miRNA of EVs compared to hFF by qRT-PCR. Protein concentrations were determined and adjusted for supplementation volume.

Main results and the role of chance
The EVs from hFF were prepared by UC, and the size range of EVs were 20∼300 nm (average 136.9±5.6 nm; n = 10) in nanoparticle tracking analyzer. And the EVs were examined via transmission electron microscopy and Western blotting to the exosomal markers CD63, CD81, and TSG101. We conformed miRNAs that enriched in EVs from hFF such as miR10b, miR503, and miR654. In outgrowth assay with human trophoblastic spheroids, the migration areas did not show a difference between control and supplementation of EVs groups. However, the expressions of adhesion molecules ( ITGαV, β3 and β5 ) were increased in supplementation of 2.5 μg/mL EVs compared to control group. In mouse blastocysts, supplementation of EVs significantly increased the trophoblast migration areas than those of control group without EVs. Also the expression patterns of Oct4, Lif, PLGF5 were higher in supplementation of EVs compared to control group. Taken together, EVs from hFF could support the migration of human trophoblastic spheroids and mouse blastocysts in vitro.

Limitations, reasons for caution
Characterization of EVs from hFF was not fully evaluated in various proteins, hormones and nucleic acids. The effects of EVs in hFF on implantation process should be evaluated in primary trophoblastic cells and in vivo mouse model of embryo transfer.

Wider implications of the findings
This study demonstrated that the EVs of hFF could improve the implantation and migration of trophoblasts in vitro. These findings suggest that the EVs of hFF could apply to increase the implantation and the pregnancy rate in human IVF-ET program.

Trial registration number
not applicable