After insemination, mares could develop post-reproductive endometritis triggered by seminal material, debris, or bacteria. Equine endometrium responds to natural mating or artificial insemination with a physiological inflammation that typically resolves within 48 h[1], [2]. However, if the inflammation persists, susceptible mares may develop a condition known as persistent breeding-induced endometritis (PBIE), which may lead to endometrial infection or progress into endometrial fibrosis, with a consequent reduction in pregnancy rates [3]. Indeed, PBIE can negatively impact fertility, as the uterus is not adequately cleaned and prepared for the arrival of the embryo, interfering with pregnancy establishment and significantly affecting maternal recognition of pregnancy [3], [4], [5]. PBIE is a widespread and challenging disorder that is difficult to address, resulting in economic losses for the industry [4].

Over the years, various approaches have been explored as potential solutions to this challenge, utilizing both traditional and non-traditional therapies [4], [6], [7]. Regenerative medicine, including stem cells or their derivatives and platelet-derived products, is emerging as a promising alternative [7].

Mesenchymal stromal/stem cells (MSCs) are self-renewing, multipotent cells derived from various tissues, including bone marrow, adipose tissue, and extra-fetal adnexa [8], [9]. The origin of cells may impact their therapeutic potential [10], [11]. In general, MSCs are regarded as guardians against excessive inflammation by releasing bioactive molecules (e.g., cytokines) that can suppress and modulate the inflammatory response [12]. Moreover, the antimicrobial effect of MSCs has also been reported through indirect (stimulating the immune system) and direct mechanisms, producing antimicrobial substances [13]. The use of MSCs has been recently replaced by their conditioned medium (CM), which is the medium where the stem cells are cultured and contains the complete secretome, including microvesicles and exosomes released by MSCs. This cell-free approach avoids issues related to cell use, such as recipient rejection or regulatory concerns [14].

Another essential product for regenerative medicine is platelet-rich plasma (PRP), produced by a specific centrifugation protocol of whole blood to obtain a concentration of platelet 3-5 times higher than the individual's physiological level. It contains supraphysiologic amounts of growth factors, cytokines, and other proteins, which can help to shift the inflammatory response from an excessive state to a more controlled response [15], [16]. Moreover, peptides contained in platelet granules have antimicrobial properties, including activity against bacteria isolated from mare uteri [17], [18], [19].

The application of regenerative therapies for equine PBIE with these significant regenerative products is limited. The application of MSCs or their secretome in vivo is supported by only two studies: Navarrete et al. [20] and Lange-Consiglio et al. [21]. In the first paper, equine endometrial and adipose MSCs significantly reduced inflammation despite a limited engraftment detectable after one month of infusion [20]. In the second paper, the combined use of extracellular vesicles (EVs) derived from amniotic MSCs and semen during artificial insemination in mares reduced polymorphonuclear neutrophil infiltration, intrauterine fluid accumulation, cytokine concentrations of interleukin (IL)-6 and increase anti-inflammatory IL-10, suggesting successful modulation of the post-insemination inflammatory response [21].

The use of PRP in vivo was tested for the first time in bovine species, specifically on repeat breeders, increasing pregnancy rates [23]. In equines, the in vivo studies exhibit high heterogeneity and present numerous limitations due to interfering factors and a lack of control over natural variables [7], [23]. This can lead to inconsistent outcomes and hinder the identification of specific mechanisms of action.

To the author's knowledge, in vitro studies are underutilized for testing the effects of regenerative products for equine endometritis. Indeed, to date, there are no papers about the in vitro effect of PRP on equine endometrial cells. PRP was tested on bovine endometrial cells [24] and human endometrial cells [25]. The in vitro effects of CM, or EVs derived from MSCs, have primarily been studied by Corradetti et al. [26] and Perrini et al. [27] on equine endometrial cells. All these in vitro studies showed a pronounced effect of these regenerative products (PRP, CM, and EVs) on endometrial cells, resulting in increased cell proliferation rates, enhanced expression of regenerative enzymes, cell migration, and upregulation of genes critical to reproduction.

An in vitro evaluation of regenerative treatments could provide crucial insights into their mechanisms of action and help optimize the design and application of these therapies in vivo. Therefore, the aim of this study was to evaluate in vitro the effect of Wharton’s jelly (WJ) MSC-derived CM and PRP on equine endometrial cells, with or without lipopolysaccharide (LPS)- induced inflammation.