P-420 Dysregulated microRNAs in uterine fluid from women with recurrent implantation failure are linked to endometrial receptivity and implantation
Authors: Von Grothusen C , Frisendahl C , Vijayachitra M , Parameswaran Grace L , Peters M , Faridani O , Andres S , Rao Boggavarapu N , Gemzell-Danielsson K 
Published: June 2022

Abstract

Study question
Is the microRNA content different in uterine fluid (UF) from women with recurrent implantation failure (RIF) compared to healthy fertile women?

Summary answer
The miRNA content is altered in the UF of women with RIF compared to healthy fertile women.

What is known already
Previous studies indicate that microRNAs secreted from endometrial cells into the UF are involved in endometrial receptivity and embryo implantation. Moreover, endometrial miRNAs are dysregulated in women with RIF and poor endometrial receptivity has been suggested as a putative cause of the condition.

Study design, size, duration
This is a descriptive experimental case-control study where microRNA abundancy in UF was compared between women with RIF (n = 34) and healthy fertile women (n = 17). Study participants were recruited at two university clinics in Stockholm, Sweden, and Tartu, Estonia. UF samples were collected vaginally in the receptive phase on day LH + 7-9 by flushing the uterine cavity with sterile saline. RIF was defined as three failed in vitro fertilization (IVF) cycles with good-quality embryos.

Participants/materials, setting, methods
To identify miRNAs in UF we performed small RNA sequencing. Differential expression analysis (DESeq2) was used to compare the abundancy of miRNAs in UF between the two groups. Dysregulated miRNAs were externally validated using relevant published datasets and further analyzed using tools such as target gene prediction (miRTarBase) and biological KEGG pathway analysis (g:Profiler). Technical validation was performed on two miRNAs with quantitative real-time PCR (RT-PCR).

Main results and the role of chance
In total, we identified 61 differentially abundant UF microRNAs with a false discovery rate of &lt; 0.05 and fold change (FC) of -2 or 2. Out of these, 34 were up-regulated and 27 were down-regulated. External validation based on published literature showed that several of the dysregulated miRNAs are expressed in endometrial epithelial cells and have been described in extracellular vesicles from the endometrium and in the context of endometrial receptivity and RIF. Using publicly available single-cell transcriptome data, we found that the predicted target genes of our microRNAs are expressed both in the trophectoderm of human blastocysts and in epithelial cells of mid-secretory endometrium. Furthermore, the predicted miRNA target genes were significantly enriched in 25 pathways critical for endometrial receptivity and embryo implantation. Technical validation of hsa-miR-486-5p (FC -20.32; p-value=0.004) and hsa-miR-92b-3p (FC -9.72; p-value=0.004) were successfully in line with the sequencing data.

Limitations, reasons for caution
The sample size of this descriptive study was limited. A larger study cohort should be used to validate the differentially abundant microRNAs. Moreover, further in-vitro and in-vivo studies are needed to establish the role of identified miRNAs and their predicted target genes and enriched pathways in the pathogenesis of RIF.

Wider implications of the findings
RIF represents a true challenge in the IVF clinic. We show that total miRNAs can be comprehensively mapped in UF and constitute a promising source of non-invasive biomarkers for RIF that could be further evaluated for its clinical utility. Our findings also give insight into the molecular mechanisms of RIF.

Trial registration number
Not applicable