REGENERATIVE MEDICINE STRATEGIES FOR ENDOMETRITIS IN REPRODUCTIVE MARES:IN VITRO AND IN VIVO STUDIES

Endometritis is a fertility-impairing pathology frequent in reproductive mares. Persistent post-breeding induced endometritis (PPBIE) is caused by a uterine inflammatory imbalance, while chronic degenerative endometritis (CDE) involves endometrial fibrotic alterations and has been associated with advanced age. Causing a considerate alteration of uterine environment, endometritis is believed also to impair fetal-maternal communication. Since conventional therapies often fail to achieve a resolutive endometrial regeneration, this project aimed at identifying novel alternative approaches based on the employment of secretome (conditioned medium: CM) and extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) isolated extra-foetal adnexa as sources of regenerative medicine. Platelet rich plasma (PRP) was also examined as further alternative approach. The initial phase of research involved in vitro studies: 1) Wharton’s jelly-derived conditioned medium (WJ-CM) and PRP were investigated in counteracting in vitro LPS-induced inflammation on equine endometrial cells (ECs). Unexpectedly, PRP did not exert an anti-inflammatory effect on stressed ECs, causing cytotoxicity and increasing ROS levels. Contrarily, WJ-CM showed a significant decrease in PGE-2 release after 24 hours of incubation, confirming its anti-inflammatory activity. Both these regenerative products contain soluble factors and EVs. Therefore, the attention of this thesis was directed on EVs, but specifically those obtained from amniotic mesenchymal stromal/stem cells (AMCs), which are isolated from another extra-foetal tissue, namely the amniotic membrane. 2) The literature provide evidence for the anti-inflammatory and pro-regenerative properties of amniotic-derived extracellular vesicles (AMC-EVs), even in reproductive disorders. However, the mechanism of interaction between AMC-EVs and recipient ECs and the modality of EV-uptake remain to be fully elucidated. Therefore, in vitro analyses were performed to characterize the glycoprofile of ECs and AMC-EVs using a panel of FITC-conjugated lectins. Results show that ECs and AMC-EVs express fucosylated and sialylated glycans on their surface, which are actively involved in the internalization of AMC-EVs by ECs. 3) Given the potential future application of AMCs and EVs in regenerative medicine, a comparative proteomic analysis was conducted to assess the specific functional features of AMCs and their EVs. The results show that AMCs and EVs are characterized by a distinct proteomic profile, showing the expression of different sets of proteins involved in regenerative processes and thus diversified therapeutic properties. Proteins promoting anti-inflammatory activity, oxidative stress resistance and angiogenesis exhibited increased expression levels in AMCs, while ECM organization and deposition are predominantly upregulated in EVs. This emphasizes their important complementary roles in the fine regulation of the main processes involved in tissue regeneration, proposing AMCs and EVs as a dynamic and effective “couple” rather than separated actors with similar effects. Consequently, to date, we are still unable to definitively elect cells over EVs (or vice versa) as better therapeutic candidate. 4) Extracellular vesicles are internalized by ECs and exerted a functional effect in a mare treated via intrauterine infusion, which resulted in a successful pregnancy. But which bioactive molecules are transferred from EVs to target cells? The miRNA cargo of AMCs and their EVs was previously characterized by the research group under which the present project was conducted. However, most of the intercellular communication is likely mediated also by the proteomic cargo of these vesicles. In this context, the proteins involved in AMC-EV effects were evaluated in vitro assessing the effect of their incorporation into equine ECs challenged with LPS to induce inflammation. Treatment with AMC-EVs resulted in the upregulation of several proteins implicated in the modulation of the inflammatory response, components of the extracellular matrix, antioxidant enzymes and mediators, as well as various transcription factors and regulatory molecules. In contrast, a subset of pro-inflammatory mediators—including specific cytokines, oxidative stress-related factors, and inhibitors of matrix metalloproteinases—were found to be downregulated. These findings indicate that AMC-EVs may exert anti-inflammatory effects on LPS-stimulated endothelial cells through a multifaceted mechanism involving cytokine modulation, enhancement of antioxidant defenses, regulation of gene transcription, remodeling of the extracellular matrix, and modulation of key signaling pathways associated with inflammation, confirming the regenerative properties of AMC-EVs and their possible employment as cell-free therapy. Alongside the in vitro experiments, also in vivo studies were performed. 5) Previous studies showed that AMC-EVs decrease pro-inflammatory cytokine levels and improve endometrial cell proliferation in vitro. Moreover, also the in vitro studies performed during this PhD reinforce the potential role of AMC-EVs for endometrial regeneration. Therefore, mare reproductive diseases were approached, including two kinds of endometritis. Among the various forms of endometritis that negatively impact fertility, PPBIE represents a major cause of subfertility in mares. In the present project, stallion semen was supplemented with AMC-EVs at the time of insemination in the attempt of preventing or limiting the development of PPBIE after artificial insemination. Before administration in mares, the effect of AMC-EVs on sperm mobility parameters was assessed to exclude sperm toxicity. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with AMC-EVs (n = 8; EV group). The supplementation of AMC-EVs to the inseminating dose resulted in a reduction in polymorphonuclear neutrophil infiltration as well as intrauterine fluid accumulation. Intrauterine levels of IL-6 were significantly decreased, while anti-inflammatory IL-10 was up-regulated, suggesting successful modulation of the post-insemination inflammatory response and possible future use of this procedure in mares susceptible to PPBIE. 6) Considering the efficacy of AMC-EVs in preventing the onset of PPBIE, an additional in vivo study evaluated AMC-EV treatment in mares affected by CDE. The aim of this project was to attempt restoring embryo-maternal communication and improving endometrial histological conditions by intrauterine administrations of AMC-EVs in twelve mares. These animals were divided into Group 1 (n=7), receiving a single treatment cycle (two administrations), and Group 2 (n=5) receiving two treatment cycles (four administrations). Each administration consisted of 2×10¹⁰ AMC-EVs diluted in 50 ml of sterile saline solution. Eleven mares were able to conceive after the treatment with AMC-EVs without significant difference in pregnancy outcomes between one or two treatment cycles. However, no improvement in endometrial histology was observed, suggesting that under the adopted therapeutical protocol AMC-EVs did not induce a complete tissue regeneration, but likely contributed to restoring functional paracrine interaction between embryo and maternal tissues.