210 Horse allogeneic mesenchymal stem cells perform homing and ameliorate endometrial inflammation after induced endometritis of mares
Authors: Navarrete F. , Saravia F. , Cisterna G. , Rojas F. , Rodríguez-Alvarez L. , Rojas D. , Castro F. 
Published: December 2019

Post-mating induced endometritis (PMIE) is an acute inflammatory response of the endometrium to spermatozoa, linked to an incapability of some mares to drain out the fluids associated with inflammation. This is of pivotal importance for reproductive success in mares. Mesenchymal stem cells (MSCs) are potential candidates for anti-inflammatory uterine therapies. Here, we aimed to study inflammatory markers in the endometrium of healthy mares and of those with induced endometritis, before and after intrauterine inoculation of MSCs, and to characterise their homing potential invivo in an induced endometritis horse model. Nine mares during their ovulatory season were selected after gynaecologic examination (absence of free liquid in the uterus, no polymorphonuclear leucocytes (PMNs) at cytology, negative bacteriology, and grade I in Kenney's scale on uterine biopsies). Mares were infused in the uterine body with 2mL of 500×106 spermmL−1 previously killed by repeated frozen-thawing cycles. At 3h, uteri were flushed with 250mL of sterile saline and the inflammatory response was monitored in the lavages and biopsies. Parameters measured included cytology, protein expression of inflammatory markers (supernatant) after lavage centrifugation (800×g, 10min), ELISA, and immunostaining for interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). The mares were divided into three groups (3 mares each). Then, 24h after dead sperm challenge, group 1 received intrauterine infusion of 2×107 adipose MSC in 0.9% sterile saline; group 2, received the same amount of endometrial MSCs in the same vehicle; and group 3 received only saline. The volume of infusion in the uterine body was 20mL for all groups. Cells (passage 4) were previously labelled with 10μM Vybrant CFDA SE Cell Tracer Kit (ThermoFisher Scientific). After 48h, the same lavages, biopsies, and measurements as described above were performed. Additional biopsies were taken at Days 10 and 30 after intrauterine infusions. Biopsies were split in two, one for confocal microscopy and the other for quantitative PCR. Endometritis was induced in all mares, as judged by cytology and expression of protein markers of inflammation. After 48h, reduction in IL-6 and TNFα was detected by immunostaining of biopsies and confirmed by ELISA in the lavages, as well as by PCR. Homing was detected in all mares infused with MSC and it persisted at Days 10 and 30 after infusion. No homing was found in the control mares. As a result of these experiments, we conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells (adipose or endometrial). Both types of cells were nested in the endometrium at low quantities, although the number of cells actually detected at fixed time points was not quantified. Overall, we can propose that, given the number of homed cells detected and the marked decrease in inflammatory markers after inoculation of cells, MSCs exert their anti-inflammatory function preferentially by a paracrine mechanism and not necessarily by nesting and proliferation, although both events occur.
Funding for this study was provided by Fondecyt 1150757.