Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Single-cell analysis of fertile and infertile endometrial epithelial organoids identified novel genes associated with endometrial receptivity (#193)

Michaela Sacco 1 2 , Sefi Prawer 3 , Wei Zhou 1 2 , Changqing Wang 4 5 , Wan Tinn Teh 2 6 7 , Kate Stern 2 6 7 , Tarana Lucky 6 8 , Catarina Ang 2 6 , Matthew E Ritchie 4 5 , Michael Clark 3 , Eva Dimitriadis 1 2
  1. Gynaecology Research Centre, Royal Women's Hospital, Melbourne, Victoria, Australia
  2. Department of Obstetrics, Gynaecology and Newborn Health, The University of Melbourne, Melbourne, Victoria, Australia
  3. Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Victoria, Australia
  4. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
  5. Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  6. Reproductive Services, Royal Women's Hospital, Melbourne, Victoria, Australia
  7. Melbourne IVF, Melbourne, Victoria, Australia
  8. Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, Victoria, Australia

Epithelial cells of the human endometrium line the uterus and are the point of embryo contact during the window of implantation (WOI). These cells allow embryo adhesion, yet until now we have not been able to define how this occurs. Single-cell RNA sequencing (scRNA-seq) has revealed unique patterns in endometrial epithelial cell subtypes. We aimed to determine gene expression in fertile and infertile endometrial epithelial organoids (EEOs) using scRNA-seq to identify potential cell-type specific markers and causal factors of implantation failure/infertility.

We performed the first scRNA-seq of EEOs from fertile (n=3) and primary unexplained infertile (n=3) endometrium modelling the WOI. Organoids were digested to single cells, processed using 10x Genomics and Nanopore protocols, and analyzed via FLAMES and Seurat pipelines. DESeq2 was used for pseudobulk differential expression analysis, followed by DAVID functional annotation. 10,945 cells were sequenced and filtered for doublets, cells expressing >200 genes and <20% mitochondrial content. Resulting in analysis of 7,923 cells.

scRNA-seq results identified five epithelial cell clusters: unciliated, secretory, ciliated, ciliated proliferative, and proliferative, classified according to confirmed markers. Analysis of differentially expressed genes (DEGs) in all clusters revealed 453 genes dysregulated within the infertile cohort (fold change>1.5, P<0.05). Functional enrichment analysis demonstrated dysregulation of cell adhesion and metabolic pathways. Key DEGs identified included genes involved in endometriosis pathogenesis. Focused analyses of ciliated clusters, cells associated with embryo contact, displayed dysregulated RNA transcription, extrinsic apoptotic signalling, and non-motile cilium assembly pathways.

This study characterises the molecular changes of endometrial epithelial cell subtypes, shedding light on their function in receptivity. In particular, we identified key pathways dysregulated in infertile luminal epithelial cells that previously have not been possible to determine. This is of critical relevance as this is where embryos adhere, initiating implantation, and can be used as personalised biomarkers and treatment targets of implantation failure/infertility.