O-032 Primary human endothelial and stromal cells from the uterine endometrium co-cultured in vitro in a 3D-system as a model to study the physiopathology of endometriosis
Authors: Lorenzon A , Guedes C B , Borbely A U , Cardoso E C , Corrêa-Silva S , Bonetti T C , Serafini P C , Motta E L , Freitas V M , Myung L H J , Abraão M S , Bevilaqua E 
Published: June 2022

Abstract

Study question
Is an uterine endothelial-stromal cell 3D-system able to respond to inflammatory/immune factors presented in patient’s endometriotic serum and can this be reversible by hormonal treatment?

Summary answer
The endothelial-stromal cells system is responsive to the serum from women with endometriosis and its cytokine profile may be reverse with hormonal treatment.

What is known already
Endometriosis’s declined fertility is mainly attributed to poor oocyte quality, inhibition of ovulation, an anatomical commitment of tubes and uterus, and loss of endometrial receptivity during the implantation window. Changes in the inflammatory/immune profile in pelvic and peripheral blood also suggest a possible interference of several cytokines playing a role in women's reduced fertility. Three-dimensional (3D) cell cultures open up new study possibilities, by guaranteeing interactions between spatially organized tissues mimicking the natural microenvironment and can significantly contribute to obtaining essential data for understanding endometrial physiology and its associated diseases.

Study design, size, duration
This is a prospective cohort study with oocyte donation women from a private IVF center and patients under endometriosis treatment in an University-affiliated gynecology service. Endometrium biopsy (n = 9) and non-endometriotic serum blood (n = 15) were collected from oocyte donors in the same day of oocyte picked (antagonist protocol), before ovary puncture and serum blood samples were collected from patients diagnostic with endometriosis (n = 15). Samples were collected between Jan/2016 and May/2017 after signing the informed consent form.

Participants/materials, setting, methods
Endometriotic serum samples were obtained from patients with (n = 10) or without (n = 5) estrogen/progestin therapy. Tissue biopsies were digested and submitted to magnetic microbeads. Endothelial and stromal cells layers were added one-by-one to a mixture of extracellular matrix components. The 3D-system received endometriotic or control serum for additional 48h. Supernatants were excluded and the cells homogenized for cytokine evaluation through cytometric bead array. The ANOVA-Tukey’s test were used for statistical analysis, p &lt; 0.05 were considered significant.

Main results and the role of chance
Oocyte donors were 24,2 ± 3,41 years old and had a body mass index (BMI) of 22,7 ± 1,38. Patients with endometriosis were 34,0 ± 3,93 years old (p = 0,21) and had a BMI of 25,4 ± 4,13 (p = 0.02). The morphology assessed by ultrastructural analysis of the 3D-system showed the presence of cells embedded in an abundant extracellular matrix, with no directional organization and with morphology compatible with fibrocytes and fibroblasts. The cells showed characteristics of viable cells with loose chromatin nuclei, evident nucleoli, and well-defined organelles. Eventually, mitosis were observed. Specific presence or absence of stromal and endothelial cells markers (cytokeratin, vimentin, IGFBP1, von Willebrand factor) were confirmed by immunofluorescence and flow cytometry. Cell viability were assessed by Fixable Cell Viability 510 dye staining. Changes in the expression profile of the cytokines TNF-alpha (p &lt; 0.0001), IL-10 (p &lt; 0.001) and IL-2 (p &lt; 0.001) were detected in the endothelial-stromal cells treated with the serum of endometriotic patients in comparison with hormonally-treated and non-endometriotic groups. These results suggest amelioration of the immune response by endometrial cells when submitted to a serum environment under hormonal treatment. Serum of endometriotic patients (hormonally treated and non-treated) also increased the levels of IL-6 and IL-8 in the co-cultured cells.

Limitations, reasons for caution
Endometrial biopsies were collected in the initial secretory phase of young oocyte donor women, which may not reflect to the eutopic endometrial response in patients with endometriosis. The reduced size of patients cohort may also increase the risks of bias in cytokine analysis.

Wider implications of the findings
The possibility of endometrial cells, even in the absence of leukocytes, to become a protagonist in the expression of cytokines and the ability of uterine cells to respond and contribute to a systemic inflammatory/immunomodulating environment with cytokine production may reflect on the pathophysiology of endometriosis and uterine reproductive functions.

Trial registration number
Not Applicable