Contents: Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out theirDNAin response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains ofStreptococcus zooepidemicus, Escherichia coliorStaphylococcus capitis, known to cause endometritis in mares were able to induceNETs releasein vitroby equinePMNto different extents. An intriguing dilemma is the dual action ofNETs. WhileNETs play a desirable role fighting micro‐organisms in mare uterus, they may also contribute to endometrial fibrosis. A long‐termin vitroexposure of mare endometrium explants toNETs components (myeloperoxidase, elastase and cathepsin G) up‐regulated fibrosis markersTGFβand Tissue inhibitor of metalloproteinase (TIMP‐1). Also, pro‐fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1‐α,IL‐1β,IL‐6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role ofCTGF,ILandNETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Equine endometrosis is a major cause of infertility in mares and is characterized by degenerative, functional and fibrotic changes in the endometrium with increased collagen (COL) deposition. Transforming growth factor (TGF)-β1 is one of the major pro-fibrotic factors involved in the excessive deposition of extracellular matrix (ECM) components in the equine endometrium. It has been demonstrated that ovarian steroids, specifically 17β-estradiol (E2) and progesterone (P4), not only regulate the cyclicity of the estrous cycle, but also have been implicated as anti- or pro-fibrotic factors. This study aimed to evaluate (i) the effect of E2 and P4 on the expression of ECM-associated genes including COL1A1, COL3A1, matrix metalloproteases (MMPs): MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13, and tissue inhibitors of MMPs (TIMPs): TIMP-1 and TIMP-2 in equine endometrial fibroblasts, and (ii) the effect of ovarian steroids on TGF-β1-induced COL1 expression in equine endometrial explants from the follicular and mid-luteal phases of the estrous cycle. The mRNA expression of ECM-associated genes in endometrial fibroblasts and TGF-β1-induced COL1 expression in endometrial explants was modulated by ovarian steroids, with variations depending on the type of steroid and the duration of treatment. Moreover, P4 decreased TGF-β1-induced COL1 protein abundance in the mid-luteal phase of the estrous cycle after 48 h (p < 0.05). The results of our study indicate that during the estrous cycle, the ovarian steroids E2 or P4 may act directly on endometrial fibroblasts, thereby affecting the expression of genes involved in tissue remodeling, namely MMPs and TIMPs. Furthermore, P4 appears to affect not only the ECM-associated genes in endometrial fibroblasts, but also to attenuate the pro-fibrotic action of TGF-β1 in the mid-luteal stage of the estrous cycle.

Contents: We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular‐FP (n = 8) and mid luteal–MLP (n = 7) phases explants were cultured for 24–48 hr with medium alone (Control), ELA (0.5 μg/ml,1 μg/ml), sivelestat ‐ ELA inhibitor (INH,10 μg/ml), or ELA (0.5 μg/ml,1 μg/ml) + INH (10 μg/ml). COL1 gene transcription was done by qRT‐PCR and PGE₂ and PGF₂α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF₂α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE₂ production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF₂α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE₂ (p < 0.05). PGF₂α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up‐regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF₂α production (p < 0.05), but PGF₂α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE₂ production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti‐fibrotic PGE₂ and inhibiting pro‐fibrotic PGF₂α.

Although proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as Cathepsin G Inhibitor I (INH; β-keto-phosphonic acid). Metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or mid-luteal phase were treated for 24 or 48h with CAT, inhibitor alone, or both treatments. In explants, transcripts (qPCR) of COL1A2, MMP2 and MMP9, as well as the relative abundance of COL1 protein (western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24h treatment, and in mid-luteal phase at 48h (P < 0.05), and on the relative abundance of COL protein in follicular phase and mid-luteal phase explants, at 48h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT induced COL1 formation, and to hamper endometrosis establishment.