Meruert Sarsenova ^1,2,3 Nageswara Rao Boggavarapu ¹ Keiu Kask ^2,3 Vijayachitra Modhukur ^2,3 Külli Samuel ³ Helle Karro ^2,4 Kristina Gemzell-Danielsson ¹ Lalit Kumar Parameswaran Grace ¹ Andres Salumets ^2,3,5,6* Maire Peters ^2,3 Darja Lavogina ^3,7,8

• ¹ Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Solna, Sweden
• ² Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, Tartu, Tartu County, Estonia
• ³ Competence Centre on Health Technologies, Tartu, Tartu County, Estonia
• ⁴ Tartu University Hospital Women's Clinic, Tartu, Estonia
• ⁵ Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
• ⁶ Institute of Genomics, University of Tartu, Tartu, Estonia
• ⁷ Institute of Chemistry, University of Tartu, Tartu, Estonia
• ⁸ Department of Hematology and Oncology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia

Background: Endometriosis is characterized by the ectopic growth of endometrial-like cells, causing chronic pelvic pain, adhesions and impaired fertility in women of reproductive age.Usually, these lesions grow in the peritoneal cavity in a hypoxic environment. Hypoxia is known to affect gene expression and protein kinase (PK) activity. We aimed to explore the changes in the transcriptome and PK activity characteristic of eutopic and ectopic endometrium in endometriosis under hypoxia.Methods: Eutopic (EuESCs) and ectopic (EcESCs) endometrial stromal cells were exposed to hypoxia (1% O2) or normoxia (20% O2) for 48 hours. We assessed PK activity and examined transcriptome using mRNA-seq in cells cultured under hypoxic or normoxic conditions.Enzyme-linked immunosorbent assay, quantitative reverse transcription-PCR and immunohistochemistry were performed for the downstream analysis of Transforming Growth Factor Beta Induced (TGFBI) expression.The kinase assay revealed a minor decrease in cAMP-dependent PK (PKAc) and Akt activity and a trend towards an increase in Rho-dependent PK (ROCK) activity in response to exposure to hypoxic conditions in EcESCs. A wider examination of the hypoxia-mediated changes in transcriptomes of cultured cells revealed that the genes related to aerobic glycolysis and cellular metabolism were upregulated in EuESCs exposed to hypoxia. In contrast, EcESCs had a single differentially expressed gene (TGFBI) upregulated under hypoxic conditions. This gene was also found to be overexpressed in EuESCs exposed to hypoxia vs normoxia, and in EcESCs vs EuESCs in normoxia. The level of secreted TGFBI in the spent culture media was accordingly high in the EcESC cultures and in the EuESC culture exposed to hypoxia. In the eutopic endometrial tissue biopsies, TGFBI mRNA and protein expression depended on the menstrual cycle phase, with higher levels observed in the proliferative phase. TGFBI staining showed the protein localized to the stroma and around the blood vessels. In the secretory phase, TGFBI protein expression was stronger in ectopic endometrium compared to paired eutopic endometrium.Conclusions: Within this study, we showed hypoxia-mediated transcriptome changes characteristic of EuESCs and EcESCs and identified TGFBI as a potential therapeutic target for endometriosis due to its role in fibrosis and angiogenesis.