UNLABELLED: The focus of the presented work was to isolate and characterize circulating endometrial cells (CECs) enriched from peripheral blood (PB) of patients with diagnosed endometriosis. The molecular characteristics of CECs could be supportive for an understanding of endometriosis pathogenesis and treatment decisions in the future. MATERIAL AND METHODS: Blood samples (n = 423) were tested for CECs presence. Subsequently, gene expression analysis (GEA) was carried out for CECs. In parallel, CECs presence and characteristics were tested during menstrual cycle (MC) phases in 11 patients. CECs were enriched by size-based separation. RESULTS: CECs were present in 78.4% of the tested blood samples. In line with the revised American Fertility Society (rAFS) classification, CECs presence was confirmed in all the acknowledged endometriosis stages: minimal, mild, moderate, and severe. Surprisingly, CECs negativity rate was also reported for severe disease in 21.1% of cases. The CECs captured during MC phases displayed different cytomorphology, including epithelial, stromal, and stem cell-like characteristics. The highest CECs numbers were detected in the mid-secretory phase of MC, which corresponds to uterine lining decidualization. CECs captured during mid-secretory periods expressed genes KRT18, NANOG, and VIM in higher amounts when compared to the proliferative phase of MC, where genes KRT19 and ESR1 were mostly elevated. GEA of the super-positive CECs samples (1000 CECs/8 mL PB) revealed high expression of genes KRT18, VIM, NANOG, and FLT1. The expression of these genes was also elevated in the endometriosis tissue samples and endometrioma. CONCLUSION: The panel of the identified CEC genes could be tested in a prospective manner to confirm the role of CECs in endometriosis pathogenesis and diagnostics.

3rd Department of Surgery University Hospital FN Motol and 1st Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague 5 Czech Republic

Academic Unit of Obstetrics and Gynecology Ospedale Policlinico San Martino Genoa Italy Department of Neurosciences Rehabilitation Ophthalmology Genetics Maternal and Child Health University of Genoa Genoa Italy Academic Unit of Gynecology and Obstetrics University of Genoa 16 132 Genoa Italy

Cellpeutics Sp z o o Duńska 9 54 424 Wrocław Poland

Department of Gynecology Military University Hospital and 3rd Faculty of Medicine U Vojenske nemocnice 1200 169 02 Prague 6 Czech Republic

Department of Histology and Embryology Wroclaw Medical University L Pasteur 1 503 67 Wroclaw Poland

Department of Laboratory Genetics Laboratory Diagnostics Faculty Hospital Královské Vinohrady Srobarova 50 100 34 Prague 10 Czech Republic

Department of Obstetrics and Gynecology University Hospital Faculty of Medicine Charles University Alej Svobody 80 301 66 Pilsen Czech Republic

Department of Oncology Wroclaw Medical University Wybrzeże Ludwika Pasteura 1 50 367 Wrocław Poland and Wroclaw Comprehensive Cancer Center Plac Ludwika Hirszfelda 12 53 413 Wrocław Poland

Department of Thoracic Surgery Krajská zdravotní a s Hospital 41100 Ústí nad Labem Czech Republic

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Enrichment of circulating trophoblasts from maternal blood using filtration-based Metacell® technology