The last decade of genetic medicine has seen a paradigm shift from the investigation of genetic constitution to a focus on active gene expression. The human genome is now understood to be incredibly plastic, as gene expression is not one-dimensional enough to fit into the confines of classical Mendelian genetics. The inception of transcriptomics, the analysis of actively transcribed mRNA molecules in the cell, has allowed for a tangible understanding of genetic dysregulation in the context of human disease. More specifically, the cutting-edge ability to analyze gene expression in individual cells has allowed us to disentangle the expression profiles found in complex tissues. This technology has recently been utilized to decode the cellular heterogeneity of endometriosis, a uterine dysregulation of tissue that is endemic in 10% of individuals with female-assigned reproductive systems. Endometriosis is characterized by endometrial-like tissue (resembling the inner lining of the uterus) proliferating outside of the uterine cavity 1’2. In addition to causing chronic pain, infertility, and inconsistencies in menstrual cycles, endometriosis has been observed to significantly raise an individual’s risk of epithelial ovarian cancer1’3. Fonseca et al at Cedars-Sinai used single-cell RNA sequencing methods to construct a cellular atlas of endometriosis in an effort to characterize the molecular hallmarks that set aberrant endometrial tissues apart1.
Endometriosis is generally characterized into three subtypes: ovarian endometriosis/endometrioma (fig. 1), superficial peritoneal endometriosis (superficial deposits on the lining of the abdominal wall) and deep infiltrating
endometriosis (characterized by lesions that infiltrate 5mm or more under the peritoneal surface)1’4. Numerous and diverse tissue samples were taken from the sample population allowing for a comprehensive analysis of the disease. All these tissues were analyzed using a high-throughput single-cell RNA sequencing method called droplet based scRNA seq: a method that earns its name by utilizing microfluidics to encapsulate individual cells in nanoliter droplet emulsions5’6’7.
With over 9.2 billion reads sequenced, 373,851/432,751 fully profiled individual cells were taken through analysis after stringent quality control1. The sequence data was segregated into different groups by analyzing the expression levels of cell-specific markers, leading to 114 distinctly different cell clusters1. The transcript data was compared across tissue types to deeply investigate the differences in tissue composition between the samples. Analysis confirmed that eutopic endometrium tissues were enriched for epithelial cells and endothelial cells in relation to control tissues. There was a 7-fold depletion in epithelial cells in endometrioma tissues, accompanied by an enrichment in immune cells such as B and plasma cells. Ectopic endometrial tissues were also particularly rich in mast cells and killer T cells. These results affirm our understanding of the aggressive immune response that can accompany the disease, as well as the primary cell types present in dysregulated tissues.
Further stringency in the grouping of the transcript sequence data was accomplished by conducting a principal component analysis (PCA) of the genetic profiles of each tissue type. Cluster 1 was primarily composed of endometriosis, with cluster 2 containing the majority of the endometrioma and eutopic endometrium samples. Unaffected ovarian tissues consistently clustered in group 3. These results reiterate the distinctly different tissue landscapes across different forms of endometriosis. This is a crucial observation, as it presents the possibility that different forms of endometriosis demand the development of different treatments and thus should not necessarily be categorized as the same disease between patients. Furthermore, it was observed that ectopic endometrial tissue and eutopic ovarian tissue from separate ovaries within the same patient grouped to their respective PCA clusters.
The molecular consequence of somatic mutation within endometrial tissues and how such mutations facilitate aberrant cancer expression is relatively poorly understood. It was previously known that somatic mutations within the cancer driver genes ARID1A and KRAS are associated of endometriosis, but the in vivo transcriptional result was especially unclear1. The in vivo transcriptional behaviors of these cancer genes were investigated and better characterized in the study by using immunohistochemical and PCR methods. Heterogenous ARID1A expression was observed, indicating a heterozygous loss-of-function mutation within endometrioma and peritoneal lesion samples. KRAS mutations at codon 12 were also observed within these tissues (fig. 2).
Endometrial tissues strongly differed in expression from unaffected tissues, suggesting aberrant expression and hormone regulation with the disease tissues. Differential gene expression analysis of the tissues examined showed that these tissues go through extensive expression remodeling in conjunction with different stages of the menstrual cycle. There were inverse relationships in gene expression with healthy and disease tissues in concordance with the luteal and follicular phases of the menstrual cycle, suggesting a greater relationship between hormone regulation and disease phenotype.
Endometriosis has remained poorly characterized despite its prevalence in the human population. This study addressed the complexity of endometrial disease with a robust analysis of mRNA transcripts in the context of multiple tissue types, hormonal regulation, and somatic mutation. This was a landmark study in characterizing how the molecular profiles of endometrial type epithelium and stroma differ in expression by locale, affirming the growing body of literature that endometriomas and peritoneal lesions exhibit two distinct disease entities. Specific genes were identified to be upregulated within ectopic endometrial tissues, providing a promising outlook for non-invasive screening assuming these biomarkers can be detected in a blood test. Dysregulation of innate immunity was also observed in the ectopic endometrial tissues studied. Continued large-scale cellular analyses of endometrial tissues is crucial, as the distinction between endometrioma and peritoneal endometriosis that was detected in this study needs to be explored due to different treatment and diagnostic demands.
1. Fonseca, M. A. S. et al. Single-cell transcriptomic analysis of endometriosis. Nat. Genet. (2023) doi:10.1038/s41588-022-01254-1.
2. Amro, B. et al. New Understanding of Diagnosis, Treatment and Prevention of Endometriosis. Int. J. Environ. Res. Public. Health 19, 6725 (2022).
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5. Salomon, R. et al. Droplet-based single cell RNAseq tools: a practical guide. Lab. Chip 19, 1706–1727 (2019).
6. Zhang, X. et al. Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems. Mol. Cell 73, 130-142.e5 (2019).
7. De Rop, F. V. et al. Hydrop enables droplet-based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads. eLife 11, e73971 (2022).
8. Endometriosis: Causes, Symptoms, Diagnosis & Treatment. Cleveland Clinic https://my.clevelandclinic.org/health/diseases/10857-endometriosis.