Proteomic Signatures of the Eutopic Endometrium of Patients with Deep Bowel Endometriosis

Abstract Authors: Lidia H. J. Myung¹, Rosanne M. Kho², Wagner Fontes³, Carlos A. O. Ricart³, Marcelo V. Sousa³, Raphaela M. Oliveira³, Aline M. A. Martins³, Isabelle S. Luz³, Denise D. Guerreiro⁴, Arabela G. Viana⁴, Maria Julia B. Bezerra⁴, Kathiane L. Augusto⁵, Leonardo R. P. S. Bezerra⁵, Andrea Lomagno⁶, Ishak Yosuf⁶, Dario Di Silvestre⁶, Arlindo A. Moura⁴, Mauricio S. Abrão¹

1 Gynecologic Division, Clinical Hospital, School of Medicine, University of São Paulo; Gynecologic Division, Beneficência Portuguesa de São Paulo, São Paulo, Brazil.
2 Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, USA.
3 Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, Brazil.
4 Department of Animal Science, Drug Research and Development Center, Federal University of Ceará, Fortaleza, Brazil.
5 Department of Women, Child and Adolescent Health, Maternidade Escola Assis Chateaubriand, Federal University of Ceará, Fortaleza, Brazil.
6 National Research Council, Proteomics and Metabolomics Unit, Institute for Biomedical Technologies, Milan, Italy.

Abstract Text: Endometriosis is a gynecological condition characterized by the presence of endometrial stroma and/or glands outside the uterine cavity, affecting 7–15% of the female population of reproductive age. Deep bowel endometriosis is associated with severe symptoms and pain, and high surgical morbidity. Transvaginal ultrasound is accurate for deep endometriosis lesions but with limited efficiency for detecting initial stages of the disease. Minimally invasive methods and molecular markers are needed for improved diagnosis and treatment. This study investigates the proteome of eutopic endometrium in patients (aged 18–45-years) with stage II (n = 8) and stage IV (n = 6) deep bowel endometriosis. Most of these patients exhibited infertility, severe dysmenorrhea, deep dyspareunia, acyclic pelvic pain, and bowel cyclic symptoms. The control group (n = 7) of the study consisted of women in the same age range who underwent tubal ligation and had no evidence of endometriosis or other pelvic diseases. Endometrium samples were collected during laparoscopy and processed for protein extraction, followed by trypsin digestion, desalting and label-free mass spectrometry. The resulting data were analyzed for detection of differentially expressed proteins (DEPs), functional protein modules and interactions. A total of 1,448, 1,623, and 1,616 proteins were identified in the endometrium of the control, stage II, and stage IV groups, respectively, with 255 DEPs. These DEPs were primarily related to protein translation, chromatin organization, RNA splicing/processing, cytoskeleton organization, redox homeostasis, metabolism, protein folding and stress response, immune system, vesicle-mediated transport and proteolysis modules. Most functional modules exhibited an increasing trend from control subjects to stage II and IV patients. However, apolipoproteins, peptidase/protease inhibitors and certain immune system components were downregulated in endometriosis patients. The transition from stage II to stage IV of the disease was associated with upregulation of components of the SnRNP complex, which is involved in RNA splicing/processing. Analysis of protein-protein interactions confirmed the relevance of pathways involved in translation, RNA splicing/processing, protein folding and vesicle-mediated transport. Lipid metabolism was enriched in the control group but downregulated in endometriosis patients. Proteins identified as SNCA, APOB and B2M were found as hubs and bottlenecks in control subjects, while KRAS resulted in specific hub/bottleneck for stage II patients. Progesterone receptor and MAOA were identified as stage II-specific bottlenecks, while RELA was among stage II-specific hubs. CASP3, an apoptosis-related protein, was a stage IV-specific hub/bottleneck, and BAX functioned as a hub in both stages II and IV patients. Additionally, SEC13 and SEC31A were identified as common hub/bottleneck proteins in both stages II and IV of the disease. In conclusion, deep bowel endometriosis caused pronounced alterations in the eutopic endometrium proteome. The reduction in immune system components, particularly B2M, indicates impaired immune surveillance, while decreased expression of apolipoproteins and peptidase/protease inhibitors emphasizes the importance of lipid metabolism, tissue remodelling and immune regulation in stages II and IV of the disease. Notably, the upregulation of components of the SnRNP complex, along with the identification of key hubs and bottlenecks such as KRAS, PGR, MAOA, CASP3, and BAX, marked the transition between stages II and/or IV deep bowel endometriosis. These findings emphasize the potential role of altered progesterone sensitivity and dysregulation of apoptosis-related processes. The characterized proteins and functional modules provide valuable information for further studies focused on early diagnosis of the disease and potential therapeutical targets.