P-300 Exosomes derived from uterine fluid in patients with repeated implantation failure inhibit embryonic adhesion, invasion, and migration to endometrium
Authors: Yin W , Feng C , Yan Y , Jin M , Chen Y , Jin Y 
Published: July 2024

Abstract

Study question
By collecting clinical samples, we focused on the effect and mechanism of exosomes derived from uterine fluid on embryo implantation during the window of implantation.

Summary answer
Exosomes from uterine fluid in repeated implantation failure group inhibited adhesion, invasion, and migration of trophoblast cells via MAPK, VEGF, estrogen, Rap1 signal pathways.

What is known already
Implantation of the embryo requires the endometrium to be in an optimal state of reception for simultaneous development of normal blastocyst. Repeated implantation failure can result from embryonic quality and disturbed crosstalk between embryo and endometrium. Exosomes are extra cellular vesicles, they can be secreted into the uterine cavity, and transfer key information to recipient cells. At present, plenty of studies have shown that exosomes derived from uterine fluid worked on the maternal-fetal interaction during the window of implantation. Whether and how exosome from uterine fluid of RIF patients caused transplantation failure remained to be explored.

Study design, size, duration
During 2023.2-2023.8, 6 women with repeated implantation failure (RIF) and 9 women who had successfully conceived after the first transplant were recruited as RIF and control groups. All the included women were selected strictly according to the inclusion and exclusion criteria, and confounding factors such as age, BMI, and menstrual cycle were excluded, the window of implantation was determined by artificial cycle preparation.

Participants/materials, setting, methods
Exosomes were identified by Transmission electron microscope(TEM), Western blot(WB) and Nanoparticle Tracking Analysis(NTA). Immunofluorescence test scratch test and transwell experiment were used to verify the effects of exosomes on cell migration and invasion, cell implantation model culture and real-time cells analysis by xCELLigence were used to verify the effect of exosomes on cell adhesion. Finally, Micro-RNA sequence was conducted on Illumina HiSeq 6000 instrumen between two groups to find out the key potential signal pathway.

Main results and the role of chance
a. Isolation and identification of exosomes: TEM: Exosomes derived from the uterine fluid from the two groups showed a complete disc-like structure; NTA: The average diameter of exosomes from two groups were similar; WB: CD9, CD63, TSG101, HSP70 were expressed in both groups.
b. Exosomes can be taken up by trophoblast cells: Immunofluorescence assay showed that exosomes derived from uterine fluid could be taken up by trophoblast cells, and the amount of uptake increased with time.
c. Exosomes affect cell migration and invasion: Scratch test and transwell experiment showed that compared with the control group, the migration ability of trophoblast cells in RIF group was reduced, and the number of migrating cells decreased(P＜0.05).
d. Exosomes affect cell adhesion: cell implantation model culture and real-time cells analysis by xCELLigence showed that compared with the control group, the adhesion ability of trophoblast cells decreased in the RIF group(P＜0.05).
e. miRNA profile between RIF and control group: Before and during the implantation window period, there were significant differences in 43 genes between two groups, these genes regulate embryo implantation by mediating the MAPK, VEGF, estrogen, Rap1, mucin type O-glycan biosynthesis pathway.

Limitations, reasons for caution
1.Due to the strict recruitment criteria, the number of women enrolled in the study was limited.
2.Through the analysis of differentially expressed micro-RNAs, relevant downstream pathways have been found. However, there is a lack of corresponding mechanism studies on how gene expression affects maternal-fetal interaction.

Wider implications of the findings
Through in vitro cell experiment and biological information data analysis, this study allows us to better understand the mechanism of action of exosomes in the embryo implantation process, detect and diagnose RIF populations at an early stage, and provide new ideas for promoting the treatment of infertility and implantation failure.

Trial registration number
Not applicable.