Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose–response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 10⁶ EVs with 10 × 10⁶ spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Urine-derived mesenchymal stromal/stem cells (USCs) could be a valuable source of cells in regenerative medicine because urine can be easily collected non-invasively. In this paper, USCs were isolated from both healthy dogs and dogs affected by chronic kidney disease (CKD), and the efficacy of collection methods (spontaneous micturition, bladder catheterization, and cystocentesis) were compared. Isolated cells were cultured in the presence of platelet-rich plasma and studied for their proliferative capacity (growth curve, doubling time, and colony forming unit), differentiation properties, expression of mesenchymal markers, and Klotho protein. Morphologically, all cells were elongated and fibroblast-like. USCs isolated from samples collected by spontaneous micturition and bladder catheterization failed to proliferate, whilst USCs obtained by cystocentesis showed a doubling time of 2.04 days in healthy dogs and 1.7 days in dogs with CKD (p < 0.05). Cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lines, showed positive expression to mesenchymal/stem markers, negative expression to hematopoietic markers, and major histocompatibility complex (MHCII) antigen. Klotho protein expression was confirmed. This study confirmed that USCs from healthy and CKD dogs can act as stem cells, with those from sick dogs having greater proliferative ability with the potential for use as autologous therapies.

Extracellular vesicles (EVs) are important mediators of cell–cell communication thanks to their ability to transfer their bioactive cargo, thus regulating a variety of physiological contexts. EVs derived from amniotic mesenchymal/stromal cells (eAMC-EVs) are internalized by equine endometrial cells (eECs) with positive effects on regenerative medicine treatments. As the cellular uptake of EVs is influenced by the glycan profile of both EVs and target cells, this study is focused on the role of surface glycans in the uptake of eAMC-EVs by recipient eECs. Equine ECs were obtained by enzymatic digestion of uteri from healthy mares. Equine AMC-EVs were isolated from amniotic cell cultures according to a standardized protocol. The glycan pattern was studied using a panel of lectins in combination with fucosidase and neuraminidase treatment. Both eECs and eAMC-EVs expressed N-linked high mannose glycans, as well as fucosylated and sialylated glycans. All these glycans were involved in the uptake of eAMC-EVs by eECs. The internalization of eAMC-EVs was strongly reduced after cleavage of α1,2-linked fucose and α2,3/α2,6-linked sialic acids. These results demonstrate that surface glycans are involved in the internalization of eAMC-EVs by eECs and that fucosylated and sialylated glycans are highly relevant in the transfer of bioactive molecules with effects on regenerative medicine treatments.

Background: Equine chronic degenerative endometritis (CDE) is a progressive process characterized by endometrial fibrosis that could be responsible for alterations of uterine environment and foetal–maternal communication. Objectives: The aim of this study was to try to restore this communication by intrauterine administrations of amniotic cell–derived extracellular vesicles (AMC‐EVs) in a case series. Study Design: Twelve mares were selected on the basis of their reproductive history of early embryonic loss or abortion and clinical suspicion of CDE subsequently verified with histopathological examination of endometrial biopsies. Methods: Gynaecological and ultrasound examinations and histopathological examination of endometrial biopsies were performed. Mares were divided into two groups: Seven mares in Group 1 received a single treatment cycle (corresponding to two intrauterine AMC‐EV administrations), whereas five mares of Group 2 received two treatment cycles (corresponding to four intrauterine AMC‐EV administrations). Each administration was of 20 billion AMC‐EVs diluted in 50 mL of sterile saline solution. Results: Eleven mares were able to establish pregnancy after the treatment with AMC‐EVs without significant difference in pregnancy outcomes between one or two treatment cycles (six out of seven mares of Group 1 and all mares of Group 2 were pregnant), suggesting that one cycle may be sufficient. The histological condition of their endometrium did not show any improvement in Kenney–Doig classification, meaning AMC‐EVs did not exert regenerative activity but probably contributed to re‐establishing a functional paracrine interaction between embryo and maternal tissues. Main Limitations: This study has the limitation of the small number of animals enrolled and the lack of a control group. However, considering the large number of past artificial insemination attempts for each animal enrolled in this study, each mare could be considered self‐control. Conclusions: It would seem possible that AMC‐EVs supported and enhanced foetal–maternal communication that was compromised by CDE.

Equine asthma (EA) is a respiratory syndrome associated with the increase of different leukocyte populations in the bronchoalveolar lavage fluid (BALF). Its pathogenetic mechanisms remain unclear. This study aimed to evaluate the associations between the mRNA expression of different cytokines in the BALF, different EA subtypes and lung function. Fifteen horses underwent physical examination, airway endoscopy, BALF cytology and lung function testing (8/15). One horse did not have evidence of EA and was used as healthy reference, while the others were classified as affected by neutrophilic or mixed granulocytic EA. Cells isolated from the residual BALF were used for IL-1β, IL-2, IFN-γ, IL-4, IL-17A genes expression by quantitative RT-PCR., Cytokine expression was compared between groups, and their correlations with BALF leukocyte and lung function were evaluated. IL-1β expression was positively correlated with BALF neutrophils count (p=0.038, r=0.56) and with increased expiratory resistance (p=0.047, r=0.76). IFN-γ was correlated with BALF mast cells (p=0.029, r=0.58). IL-4 was higher in horses with mixed granulocytic EA than neutrophilic (p=0.008), positively correlated with BALF mast cells (p=0.028, r=0.59) and inversely with whole-breath (p=0.046, r=-0.76) and expiratory reactance (p=0.003, r=-0.93). Finally, IL-17A was inversely correlated with expiratory reactance (p=0.009, r=-0.92). These results support that multiple immune responses are involved in EA pathogenesis; innate, Th2, and Th17 responses. Innate immunity appeared associated with neutrophilic inflammation, and Th2 response with increased mast cells. The role of Th1 response in EA remains questionable.

During the preimplantation period, the nutrition of the embryo is dependent on luminal secretions of the uterus, which can be modified by the health status of the animal. The aim of this study was to mimic the paracrine communication between healthy or LPS-stressed epithelial endometrial cells (EECs) and embryos using aa transwell plate. The rate of in vitro embryo production, size, and concentration of extracellular vesicles (EVs), and level of secretion of Galectin-9 (Gal-9) and leukaemia inhibitory factor (LIF) were detected. Embryos were produced with an established protocol of oocyte in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). On day 55 of IVC, one hour before the transfer of morulae in the basolateral compartment of the transwell, EECs were treated with 10 ng/mL of LPS, and IVC was continued until the eleventh day. Extracellular vesicles (EVs) were obtained from IVC medium by ultracentrifugation. Levels of Gal-9 and LIF were evaluated by ELISA. On day 7, the results did not show statistically different blastocyst rates between EECs+Embryo and EECs+LPS+Embryo (34.94 ± 1.95% and 33.06 ± 3.08%, respectively). On day 11, the rate of hatched blastocysts was 23.03 ± 3.18% in EECs+Embryo, while in EECs+LPS+Embryo, no hatching was observed. Nanosight revealed higher values in EV size and concentration in EECs+LPS+Embryo medium compared to EECs+Embryo (p < 0.05). In LPS-treated samples, there was a significant decrease in Gal-9 levels and a significant increase in LIF secretions compared with non-non-LPS-treated samples (p < 0.05). These results highlight how bidirectional secretions between EECs and embryos, crucial for embryo development, can be affected by endometritis.