208 Preliminary evaluation in equine endometrial explants with grade IIA and IIB endometrosis of the antifibrotic effect of adipose or endometrial stem cells pre-conditioned with PGE2

Endometrosis in mares, a condition stemming from chronic uterine inflammation and characterized by periglandular fibrosis, has thus far lacked an effective treatment. However, the exploration of a novel approach using mesenchymal stem cells (MSC) as a potential treatment offers a promising avenue. By modulating inflammation at the endometrial level and stimulating cell regeneration, this approach could pave the way for new and effective treatments. In addition, MSCs have antifibrotic properties, further enhancing their potential as a novel treatment. Our work aimed to evaluate the antifibrotic effect of the interaction of the secretome of MSCs on endometrial explants obtained from mares with grade IIA and IIB endometrosis according to the Kenney and Doig classification score. Equine MSCs of adipose (AT-eMSC) or endometrial origin (ET-eMSC) were cultured in high-glucose DMEM supplemented with 10% FBS at 37°C and 5% CO₂ in 12-well plates until reaching 90% confluency. At this point, eMSCs were washed three times with PBS1× and cultured in high-glucose DMEM conditioned with 3 µM PGE₂ 24 h before co-culture with endometrial explants. Endometrial biopsies were taken from mares, and 30 mg (~3-mm explants) were processed and used in the experiments. Biopsies were taken in duplicates: one for histologically assessing endometrosis, and the other for culture. Co-culture was carried out in a Transwell system in which the explants were placed in the upper chamber and the eMSCs in the lower one for 48 h. The control group included explants but no cells in the lower chamber (only culture medium). At the end of this time, RNA/proteins were extracted from the explants. The relative expression of different genes related to fibrosis (αSMA, COL1A2, COL3A1, CTGF, MMP2, MMP9) was measured by RT-qPCR. The expression levels of the smooth muscle α-actin protein were also determined by western blot. An ANOVA (Kruskal-Wallis test followed by Dunn’s multiple comparisons test) was used to determine significant differences between the experimental groups. The relative expression of COL3A1 was reduced in grade IIB explants co-cultured with ET-eMSCs conditioned with PGE₂. We also observed an increase in the relative expression level of MMP9 and MMP2 in the explants with grade IIB when co-cultured with AT-eMSCs and ET-eMSCs conditioned with PGE₂. We did not observe any effect on the level of CTGF expression. Our study demonstrated an antifibrotic impact of ET-eMSCs on the explant model mediated by a decrease in the levels of αSMA and COL3A1 and an increase in the levels of MMP2 and MMP9. There were significant differences in the anti-fibrotic response of the explants depending on the degree of fibrosis; explants with grade IIB responded better to treatments.

Funding for this study was provided by Fondecyt Regular 1210349, ANID, Gobierno de Chile.