P–292 Transcriptome signature of receptive endometrium is not affected by the presence of mild adenomyosis
Authors: Prasnikar E , Kunej T , Gorenjak M , Uroš P , Kovačič B , Knez J 
Published: August 2021

Abstract

Study question
Does the presence of mild adenomyosis, a common acquired uterine anomaly, affect the endometrial gene expression levels during window of receptivity?

Summary answer
Mild adenomyosis has no significant influence on gene expression signature in the window of implantation (WOI).

What is known already
The improvements in imaging techniques have led to frequent detection of adenomyosis in women undergoing investigations for infertility. Although the data are conflicting, some clinical studies have shown that the presence of adenomyosis may interfere with embryo implantation and lead to poor pregnancy outcomes. The knowledge of molecular background that would lead to the phenomenon of altered endometrial receptivity in women with adenomyosis is limited and mainly demonstrated by selected candidate genes. Next-generation sequencing platforms enable genome-wide transcriptomic profiling of desired tissue samples and present a powerful tool to identify differentially expressed genes (DEGs) between women with adenomyosis and controls.

Study design, size, duration
We designed a prospective case-control study comparing women with sonographic evidence of mild adenomyosis (n = 10) and women with normal uteri seeking assisted reproduction due to male factor infertility as the control group (n = 10). All eligible women underwent infertility treatment at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between years 2018 and 2020. For the present study, they were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests.

Participants/materials, setting, methods
Each endometrial biopsy was obtained in the presumed window of implantation (WOI) on days LH + 7 to LH + 9 after LH surge (LH + 0). Isolated total RNA was applied for mRNA + lncRNA sequencing (RNA-seq) by Illumina Novaseq 6000. An aliquot of RNA samples was used to verify the WOI by the endometrial receptivity test “beREADY” (CCHT, Estonia). Gene Ontology and Reactome pathway enrichment analyses were conducted in ClueGO bioinformatics tool to study biological role behind obtained DEGs.

Main results and the role of chance
The R program language and Bioconductor packages were used to align generated RNA-seq reads on the human reference genome assembly (hg19) and to calculate gene expression differences between study groups using normalized counts per million (CPM)&gt;10 in at least 10 samples. A total 233 DEGs (p &lt; 0.05) was identified of which 126 genes were up- and 107 were down-regulated in adenomyosis compared to the control group. However, there was no significantly DEG according to the adjusted p-value. According to the beREADY test, all 20 samples were in receptive phase, however two samples were early-receptive and five were late-receptive. In a sensitivity analysis, all border receptive samples were removed and RNA-seq data sets were re-analysed only by 8 adenomyosis cases and 5 controls. A total of 382 DEGs (p &lt; 0.05) were detected in adenomyosis group (216 up- and 166 down-regulated genes), again with no statistical difference between both groups after adjustment. Functional enrichment analyses of 233 and 382 DEGs identified pathways (adjusted p-value&lt; 0.05) associated with positive regulation of exosomal secretion and expression of IFN-induced genes, respectively. The comparison of 233 and 382 DEGs revealed 28 common genes that may present stronger candidate of adenomyosis-related markers associated with endometrial receptivity.

Limitations, reasons for caution
Only mild adenomyosis was considered in this study, which is most commonly detected in women. The results could differ in women in severe cases of adenomyosis. Multicellular whole-tissue endometrial samples that were used for RNA isolation could mask gene expression differences of specific cell types between study groups.
Wider implications of the findings: According to our results of transcriptome analysis, the presence of mild adenomyosis has no significant influence on the gene expression signature during endometrial receptivity in natural menstrual cycle. Women being investigated for infertility can be reassured that this common acquired anomaly does not significantly influence the chances of successful conception.

Trial registration number
0120–259/2018/16