Endometrium Of Mares Research Articles

The path to fertility: Current approaches to mare endometritis and endometrosis.

The path to fertility in the mare requires an understanding of the hormonal influences, the immune response, genetics, and epigenetic mechanisms involved not only in physiological reproductive processes, but also such pathologies as endometritis and endometrosis. Endometritis may lead to endometrosis establishment. In the presence of endometritis, neutrophils arrive at the mare endometrium, and form neutrophil extracellular traps. While NETosis plays pivotal roles, prolonged inflammation can lead to chronic endometritis, endometrosis, and fertility issues. Matrix metalloproteinases and epigenetic changes influence the course of endometrosis. Inhibitors of specific enzymes involved in NETosis and epigenetic inhibitors have shown potential in reducing pro-fibrotic effects. Collagen type III (COL3) has emerged as a putative biomarker, correlating with endometrosis and useful in fertility assessment. Thus, COL3 may offer a non-invasive diagnostic tool, as a complement to histopathological methods. Epigenetic modifications and miRNA expressions offer new avenues for therapeutic strategies, emphasizing the importance of understanding the cellular mechanisms at play in mare endometrial fibrosis.

Expression of oestrogen and progesterone receptor and intermediate filament proteins in the oviduct of mares with endometrosis

Equine endometrosis is one of the major causes of equine infertility, in the course of which periglandular fibrosis signs occur in the endometrium. As an oviduct is a key place of early embryo development, we hypothesized that loss of contact between mucosal cells and perturbation in hormonal action may coexist in the uterus and oviducts. Thus, this study aimed to investigate the expression of the intermediate filaments (vimentin and desmin) as well as the estrogen receptor alpha (ERα) and progesterone receptor A (PRA) in the stroma and epithelia of endometrium and oviduct in healthy mares and these with endometrosis. Out of 56 culled-in slaughterhouse mare endometrium samples were annotated in two groups (i) healthy endometrium (n=26) and endometrium with signs of moderate and severe endometrosis (n=20). The expression of vimentin, desmin, ERα, and PRA was investigated immunohistochemically and compared between the above groups as well as between the phase of the estrous cycle. The expression of both vimentin and desmin was higher in the endometrial stroma of endometrosis group compared to healthy endometrium. The expression of ERα was higher in the healthy endometrial stroma and luminal epithelium during the follicular phase than luteal, however, no differences were noted in endometrium of mares with endometrosis. Moreover, PRA expression was significantly higher in the stoma oviduct during the follicular phase than luteal. It seems possible that mucosal cells through their altered interactions may support the occurrence of moderate and severe endometrosis. In addition, the oviduct appears to be less sensitive to hormonal changes than the uterus. The changed expression of intermediate filaments and steroid hormones may be some of the leading factors which can contribute to the oviduct functional status in endometrosis.

Transcriptomic profiling of mare endometrium at different stages of endometrosis

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-β1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members’ gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-β1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

Immunohistochemical expression of myeloperoxidase in the equine endometrium suffering from endometrosis

Myeloperoxidase (MPO) is a pro-oxidant enzyme with potent bactericidal activity playing an important role in the innate immune response against microbial pathogens. MPO is a marker of neutrophil activation, but it is also expressed by epithelial cells. In the endometrium of mares in physiological conditions, MPO is immunohistochemically expressed in a hormonal-dependent fashion with maximal expression in mid and basal glands during estrus (Parrilla Hernández et al. Animals. 2023;13(3):375). This suggests that MPO, as a part of the uterine mucosal immune system, contributes to the prevention of equine endometrial contamination and inflammation. Thus, an alteration of the MPO expression may have an impact on uterine immune defense. A deviation in the endometrial expression pattern of some other proteins has already been associated with endometrosis (Hoffmann et al. Theriogenology. 2009;71:264-274) and it has been proposed as one of the reasons for the reduced fertility in mares suffering of this condition. Therefore, the aim of this study was to investigate the immunohistochemical expression pattern of MPO in the estrous equine endometrium affected by endometrosis. Endometrial biopsies (n=7) obtained from mares in estrus with histological detection of three or less neutrophils per field(400x) and presenting a moderate endometrial fibrosis were studied. Immunohistochemical analysis using MPO-specific horse antibody was performed. The uterine glandular epithelium within fibrotic foci mostly presented a non-steroidal dependent immunostaining pattern reaction. In comparison to unaffected glands, most glandular epithelial cells surrounded by periglandular fibrosis exhibited no or decreased MPO immunostaining. No differences were observed between single glands or nests. These results are in accordance with previous studies for other endometrial expressed proteins. This decrease of MPO expression in the presence of periglandular fibrosis may be produced by a lack of paracrine communication between stromal and epithelial cells, which is necessary for the regulation of uterine secretory proteins by sex hormones (Pierro et al. Biology of reproduction. 2001;64(3):831-838). Although further investigation with different degrees of endometrosis is necessary, the reduced expression of MPO compared to that expected in estrus we observed in this study suggests that endometrosis may lead to disturbance in the uterine immune environment, which in turn may predispose to an increase of susceptibility to uterine infections and endometritis in mares.

Immunohistochemistry for natural killer cells, endometrial biopsy grade, and fertility in mares with repeated early embryonic death

Early embryonic death (EED) is a potential cause of infertility in mares. Causes include embryo related chromosomal defects or disturbances in the oviductal and uterine environment, but often the cause remains unknown. In humans, most of the endometrial lymphocytes are natural killer (NK) cells (CD56-positive lymphocytes), and their phenotypes, surface receptors, and cytokine production have been associated with early embryonic death and endometrial angiogenesis. (Fuchinoue et al. J Obstet Gynaecol Res. 2016;42:1541-1552). The aims of this study were to 1) describe the relationship between endometrial biopsy grade and future fertility in mares with repeated EED, and 2) to identify and localize equine natural killer cells in the endometrium of mares with repeated EED. The study included 32 formalin-fixed paraffin-embedded endometrial biopsies of mares with a history of two or more EEDs. Immunohistochemistry for the identification of NK-cells was done on endometrial biopsies, using polyclonal NK cell surface receptor antibodies NK2GD and CD56 (Thermo Fisher Scientific, MA, USA) according to manufacturer instructions. Mouse brain and spleen samples functioned as positive controls. Information on biopsies and post-biopsy foaling were collected and analysed with Fisher'sexact test to evaluate dependence between variables (age group, biopsy grade, later abortions, previous parturitions, number of EED, and live foals). A p-value of < 0.05 was considered significant. The biopsies were graded according to Kenney & Doig (In: Morrow, DA, ed. Current Therapy in Theriogenology. 1986; 723–729). Of all mares, 3.1% were grade IIA, 43.8% grade II, 53.1% grade IIB. In total, 43.8%were younger than 13 years and 56.3% 13 years or older, 71.9% had had two EEDs and 28.1% more than two. In total 71.9% of the mares had foaled previously, 68.8% were bred again of which 59.1% foaled a live foal. The age group (< 13 vs. ≥ 13), biopsy grading (grade I-II vs. IIB-III) or later abortions (abortion at over 50 days of pregnancy) were found not to have a significant effect on the later live foal rate. Previous foalings and the number of EEDs (< 3 vs. ≥ 3) seemed to affect the foaling result but failed to have statistical significance in this small study population. Natural killer cells were not identified in the endometrial samples with the selected antibodies, suggesting potential species differences in surface receptors. In conclusion, repeated EED was not related to lower future foaling rates, implying mainly incidental causes of EED in this population.

Endometrial fibrosis in mares: Picrosirius Red and Collagen-2 and 3

In mares, endometrial fibrosis results from abnormal collagen deposition in the stroma and around the endometrial glands. The pathogenesis of endometrial fibrosis remains unknown, but the different types of collagens are a fundamental part of the process. The Picrosirius Red (PSR) staining is widely studied and necessary for collagen fiber and tissue remodeling. The present study aimed to determine the collagen deposition and the gene expression of collagen-2(COL-2) and collagen-3(COL-3) in the endometrium of mares with different degrees of fibrosis. Cyclic mares (n = 34) with a mean age of 14 ± 6.4 years old were used. A uterine biopsy from each mare was obtained at diestrus (D5–D10). Mares were grouped according to the degree of uterine fibrosis described by Kenney (1978): Grade I (n = 12), Grade II (n = 12), and Grade III (n = 10). Collagen in the equine endometrium was morphometrically quantified under PSR staining. The morphometric quantification of the images was performed from each sample after digital microphotographs were taken at 200x magnification, processed, and evaluated using the ImageJ 1.52a software. COL-2 and COL-3 expression was performed using qPCR. Grades were considered independent factors. Collagen-stained area and transcript expression were considered dependent factors. Data were analyzed with ANOVA andt-test. A Pearson correlation was performed between age and collagen deposition. The percentage of tissue collagen area stained with PSR revealed a significant increase between all groups in collagen deposition according to the degree of fibrosis: Grade I (11.7 ± 1.4%), Grade II (17.8 ± 1.3%), and Grade III (24.1 ± 1.9%). Age and the percentage of collagen deposited in the endometrial tissue correlated (r = 0.5) (P = 0.015). Grade II samples presented higher expression of COL-2 transcripts compared to Grade I (P = 0.026) and Grade III (P = 0.003). However, COL-3 did not show differences between groups. This study agrees with other studies that indicate that the worsening of degenerative changes coincides with advanced mare age. Sample evaluations emphasizing degenerative changes rely on an experienced evaluator, while the PSR, followed by computerized analysis, presents quantitative data. In conclusion, the increase in PSR analysis in Grade II and Grade III mares compared to Grade I demonstrates that this technique could play an important role in routine analyses for the detection, quantification, and dynamics of collagen in specific tissues. The study revealed that COL-2 might be involved in the progression and differentiation of healthy endometrium into an endometrium with severe fibrosis.

Metagenomic characterization of the equine endometrial microbiome during anestrus

Recent studies have confirmed that healthy uteri, including the equine uterus, harbour a microbiome. Studies on mares have been performed in estrus and therefore under the influence of high estrogen concentrations. Multiple human studies investigating the vaginal, as well as the gut microbiome have shown that the microbiome is dynamic throughout the estrous cycle and is influenced by differences in estrogen levels. This is in contrast to a study performed on Arabian mares which indicated that the equine vaginal microbiome is stable throughout the estrous cycle.In this study the resident uterine microbiome of healthy anestrous mares was characterized by metagenomics from double-guarded endometrial swabs and compared to the microbiome in the same mares during estrus.Double guarded endometrial swabs from 15 healthy estrous mares were taken and stored at -80°C until extraction, in the following non-breeding season 8 mares from this group were sampled in similar fashion during anestrus. Blank samples were obtained during both sample period. Genomic DNA was extracted using Qiagen DNeasy PowerSoil extraction kits and 16S rRNA gene was amplified using primers 515f-806r. An amplicon sequence variant table was made and chimeras were removed and the genomic16S rRNA from the blank samples was substracted. Sequences were processed and analysed using 16S rRNA SILVA v138.1 database. Contaminating reads were removed from the samples by subtracting the reads from the blank samples using Microdecon and downstream analysis performed using the Phyloseq package. Alpha and beta diversity were calculated using microbiome, amplicon and vegan packages.Results suggest that the equine uterine microbiome in estrus has a low diversity, low richness, and a low abundance at phylum level. The equine uterine microbiome in anestrus has a higher diversity, higher richness, and higher abundance compared to the estrus uterine microbiome. However, both the estrus as well as the anestrus endometrial microbiome is dominated by Proteobacteria, Firmicutes, and Bacteroidota. PCoA analysis displays a strong dissimilarity between estrus and anestrus samples.The difference in the microbiome between estrus and anestrus can possibly be explained due to the variance in immune responsiveness of the endometrium. The estrous mare's endometrium is under the influence of estrogens, allowing for a high immune response and possibly less diverse microbiome.

Molecular mechanisms underlying equine endometrial cup regression: a transcriptomic analysis

The gonadotropin-secreting equine endometrial cups (EC) have a major role in pregnancy maintenance, but an intriguingly short lifespan. First seen in the maternal endometrium at days (d) 38-40 post-ovulation, the EC undergo degeneration via poorly understood mechanisms starting around d 60-70, with complete dehiscence at approximately 120-150 d of gestation. Pregnancy losses during the EC physiological lifespan and EC pathological persistence after abortion or uncomplicated parturition usually lead to irregular estrous cyclicity and subfertility, with only anecdotal and inconsistent therapeutic options currently available. A holistic understanding of the cellular signaling leading to EC regression may provide insight for development of effective therapies for retained or persistent EC. We therefore hypothesized that comparing the transcriptome of EC during development and regression could elucidate key regulators and molecular pathways leading to EC trophoblast cell demise. Samples of EC were dissected from the adjacent endometrium of pregnant pony mares during early development (DEC, 42-47 d post-ovulation, n = 5), and regression (REC, 96 – 120 d, n = 5). Next generation RNA sequencing was performed with Illumina NovaSeq6000 and reads mapped to EquCab3.0 (STAR-2.5.2b). Differentially expressed genes (DEGs) were identified with Cuffdiff-2.2.1 (FDR < 0.05). A total of 2,504 DEG were identified, being 1,648 upregulated and 856 downregulated in REC when compared to DEC. Pathway Analysis of DEGs was also performed (IPA, Qiagen). Interestingly, the main pathways affected in REC included oxidative phosphorylation (P = 1.08e-38);mitochondrial dysfunction (P = 2e-25); estrogen receptor signaling (P = 2.2e-10); Sirtuin signaling pathway (P = 9.64e-16), which has central regulatory roles in cell senescence and apoptosis; and Granzyme A Signaling (3.29e-15), associated with Cytotoxic T Lymphocyte and Natural Killer-mediated apoptosis via caspase-independent pathways. Upstream regulator analysis highlighted the roles of mitochondrial protein ubiquinol-cytochrome c reductase complex-3; rapamycin-insensitive companion of mTOR, an essential molecule in cellular homeostasis; Torin-1, a potent mTOR inhibitor; dead box protein 5, which may act as a co-activator of estrogen receptor signaling; and transducing-like enhancer protein-3, a member of a family of proteins that regulate cell fate through the Notch signaling pathway. Importantly, the main upstream regulators and downstream molecules are also involved in cancer cell biology (P = 1.32e-07 – 1.71e-122), including the downstream effectors of neoplastic and epithelial cell death secreted phosphoprotein 1 (Spp1), and secreted protein acidic and cysteine rich (Sparc). Along with aromatase, Spp1 and Sparc were upregulated in REC in comparison to DEC. In summary, we have highlighted key molecular pathways and regulators of EC regression, which merit further molecular and functional investigations. Since multiple identified molecules have also been targeted in human oncological treatments, further investigation of their effectiveness in hastening EC demise is warranted.

Does the uterine inflammatory response differ between mares and donkey jennies inseminated with frozen donkey semen?

Frozen donkey semen produces poor fertility rates in jennies (0-11%) while showing normal fertility in mares (33-53%). This may be related to interspecific differences in the interaction of the uterus with frozen semen. This study aimed to evaluate and compare the inflammatory process in the endometrium of donkey jennies and mares inseminated with cryopreserved donkey semen. A total of 36 estrous cycles from 12 Pega donkey jennies and 36 estrous cycles from 12 mares (Breton and Mangalarga), aged between 3 and 14 years old were used. Once ovulation was detected, the animals were inseminated deeply in the horn ipsilateral to ovulation with thawed donkey semen containing 1 billion sperm. The following parameters were evaluated in both species at ovulation/before insemination (time 0h) and after insemination (times 6h, 12h, 24h, 48h, 72h, 96h and 120h): uterine vascularization, endometrial cytology, serum progesterone levels and pregnancy rate. Normally distributed data were analyzed using a mixed-model ANOVA and a Wilcoxon test for non-normally distributed data, using R software. Donkey jennies showed a higher percentage of colored pixels in the uterine body region at T6 compared to mares. Also, the percentage of inflammatory cells was higher in the donkey jenniescompared to the equine cytology at T12 (52.75 versus 37.44) and T48 (16.00 versus 3.98). The inflammatory peak was reached at T6 in both species (55.77 versus 57.31) but remained elevated until T12 only in donkeys. Although a complete resolution of the inflammation occurred within 48 hours after AI in both species, the percentage of inflammatory cells remained higher in jennies than in mares. An eosinophilic uterine infiltrate was present in donkeys, while it was not observed in mares. In addition, donkey jennies showed a significant increase in monocytes at T12, compared to the other times. Also, jennies showed a higher mean P4 level (13 ng/ml), compared to the mares (6 ng/ml), both species had progesterone concentrations compatible with the maintenance of pregnancy. The pregnancy rate 14 days after AI was higher in mares than in donkeys, reaching a rate of 54.55% (6/11) versus 0% (0/12), respectively. In conclusion, the inflammatory uterine response in the presence of frozen donkey semen differs between mares and jennies. Jennies have a later inflammatory resolution, marked by the presence of intense polymorphonuclear and eosinophilic infiltrates. These differences may contribute to the lower fertility rates in this species.

Embryo-maternal interaction is associated with the location of the embryo during the mobility phase in mares

Despite advances in the molecular analysis of the endometrium during early equine pregnancy, little is known about embryo-maternal interaction during the embryo mobility phase. It has been demonstrated that the embryo stimulates upregulation of prostaglandin synthase (PTGES) locally in the endometrium. This suggests that endometrial gene expression is influenced by the location of the embryo in the uterus. The aim of this study was to characterize the transcriptome of the endometrium of mares based on the location of the mobile embryo and to compare the gene expression of the endometrium between the uterine horns of pregnant (n = 8) and non-bred (n = 8) mares. On day 12 (D0 = ovulation), a cytobrush was used to collect endometrial samples from the middle of the uterine horn of pregnant mares that did (PE) and did not (PWE) contain the embryo, and from the middle of both uterine horns of non-bred mares (NB). RNA was extracted, followed by library preparation and total RNA-sequencing (PE-150bp). On average, 40 million reads were generated per sample. Reads were trimmed and mapped to the current equine reference genome (Ecab 3.0). Differentially expressed genes (DEGs) were identified using Deseq2 (P value < 0.01) and gene ontology (GO) analysis was performed using Shinny Go. Based on the location of the embryo (PE vs PWE), 56 DEGs were identified, 41 upregulated and 15 downregin PWE relative to PE. The DEGs included two genes (LOC100064092, FADS3) involved with fatty acid metabolism, and seven genes (RIPOR2, SMAD6, ROBO1, TENM1, SELL, ADORA2A, and AGR2) involved in cell-to-cell interaction. Based on GO, the DEGs in the pregnant group were mainly associated with cell adhesion, response to chemokines, inflammatory responses, connective tissue replacement involved in inflammatory responses, and connective tissue replacement. Additionally, there were 669 and 707 DEGs, in PE vs. NB and PWE vs. NB comparisons, respectively. The DEGs were mainly involved in cell metabolism and signaling. Moreover, there were 13 overlapping genes among the PE vs. NB and PE vs. PWE groups. There were two genes (PLIN5, LOC100064092) involved with fatty acid metabolism, one with hematopoiesis (SERPINB10), one with vascularization and development (ATOH8), one gene related to osmotic pressure (MUC5B), and one gene related to endometrial remodeling (TIMP1). In conclusion, these results suggest that the embryo initiates local maternal-embryo dialogue, embryo development and immune system regulation during the early pre-fixation phase of pregnancy. This project was supported by the John P. Hughes Endowment and the Center for Equine Health with funds provided by the State of California satellite wagering fund and contributions by private donors.

139 Cyclic changes in uterine epithelial sodium channel in mares

Epithelial sodium channel (ENaC) has been localized within many organs of various species, including the uterus of mice, rats, and humans. Additionally, ENaC expression within the rodent uterus was differentially influenced by 17β-estradiol (E2) and progesterone (P4), and has been proposed to be the major mechanism regulating uterine fluid absorption and secretion. In the equine, ENaC has been observed in the trachea, hooves, and endometrium; however, the potential relationship between E2, P4, and ENaC in the mare endometrium has yet to be elucidated. The aim of this study was to investigate possible interactions between E2, P4, and ENaC from cycling mares using immunohistochemistry (IHC). Estrus was synchronized for all mares (n = 13) by daily administration of oral altrenogest (ALT) for 15 consecutive days. On the last day of ALT treatment, all mares received 5 mg of dinoprost IM. Following synchronization, all mares were teased daily with a stallion. Transrectal ultrasonography (US) for observation of ovarian activity and day of ovulation (OV; d0) were completed every other day until OV. On d10, US, blood collection via jugular venipuncture, and a uterine biopsy sample were collected. Biopsy samples were fixed to later complete ENaC evaluation by IHC. A uterine biopsysample and blood collection had also been similarly collected and processed for each mare during estrus when she simultaneously had a ≥ 30mm Graafian follicle present and was displaying estrual behaviors. For statistical comparison, a paired t-test was applied. Mares averaged an estrus duration of 4.85 ± 1.86 d. Plasma E2 was 2.27 ± 0.45 pg/mL on d10 and 3.58 ± 0.48 pg/mL on estrus sample day. Plasma P4 concentration was significantly higher (P < 0.01) on d10 when compared with estrus sample day (8.53 ± 1.17 ng/mL vs 0.04 ± 0.02 ng/mL, respectively). The percentage of positively stained ENaC cells was significantly higher (P < 0.01) on d10 when compared with estrus sample day (81.27 ± 3.19% vs 58.84 ± 3.23%). In conclusion, our findings indicate that sex steroids, specifically P4, may regulate ENaC within the mare endometrium, with ENaC being increased during diestrus and decreased during estrus. Collectively, the changes of ENaC concentrations during the estrous cycle might impact the critically sensitive fluid environment within the mare uterus.

5-Aza-2′-Deoxycytidine (5-Aza-dC, Decitabine) Inhibits Collagen Type I and III Expression in TGF-β1-Treated Equine Endometrial Fibroblasts

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts and the use of 5-aza-2'-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-β1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-β1 upregulated DNMT3A transcripts and collagen secretion. In TGF-β1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-β1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.

Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine Endometrium.

Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies.

Safety and Effects of a Commercial Ozone Foam Preparation on Endometrial Environment and Fertility of Mares.

Mares' subfertility represents a complex diagnostic and therapeutic challenge and both clinical and subclinical endometritis are considered major causes of impaired fertility. Thanks to its properties, ozone has a big potential as a treatment for equine endometritis. Therefore, the aim of this study is to describe the safety and the effects on endometrium and reproductive parameters of mares of a commercial ozone foam preparation (Riger Spray®). Twenty-four mares were treated during estrus: ozone group with an intrauterine instillation of ozone foam preparation (OG, n=16) and control group with 20 ml of lactated Ringer's solution (CG, n=8). Samples for endometrial cytology were collected before the ozone treatment (T0), after 24 h (T1), after one week (T2), two weeks (T3), and when the subsequent estrous phase was detected (T4). Furthermore, samples for histological examination and uterine swab for bacteriological examination were collected at T0 and T4. At T1, a statistically significant increase of endometrial inflammation in the OG mares compared to T0 (P<.05) and to CG at same time point (P<.05) was observed, but it was already resolved at T2. No differences in endometrial inflammation in CG, biopsy grade before and after the treatment in the two groups, number of mares pregnant at the end of the season and number of mares pregnant at the first cycle were observed. However, the number of inseminations required for pregnancy tended to be lower (P=.0711) in the OG (1.69±0.06) than in CG mares (2.60±0.89).

The index for assessing and predicting fertility in mares

The evaluation of reproductive qualities of mares is an important element of horse breeding technology. Histological analysis of the endometrium provides information about the mare’s ability to conceive and carry a pregnancy to term. The article presents the results of histological studies of the endometrium in mares of &lt;10 (n=6) and ≥10 (n=13) years old (yо). Two morphometric parameters were used for the analysis: diameter (GDm) and density (GDs) of endometrial glands in diestrus phase of the cycle. There was noted an increase in GDm (20.5 - 62.9 μm, p&lt;0.001) and a decrease in GDs (16.6 - 10.9 units per field of view (ufov), p&lt;0.1) in the endometrium of mares older 10 yo. An endometrial index EI (GDm / GDs, μm/ufov) characterizing the functional state of the endometrium has been proposed. The results of pregnancy rate and the level of life foal birth in mares after histological studies were analyzed. A significant relationship EI with age (p=0.0133) and fertility (p=0.0059) of mares was established. Among mares with EI&lt; 3 µm/ufov only one mare was older than 10 years and all the mares had foaled successfully. The mares with EI&gt;3 µm/fov were older than 10 years and had problems with conception and gestation. In our studies, EI in the range from 3 to 6 µm/ufov could be interpreted as reversible state of the endometrium and after correct treatment it is possible to increase the fertility of mares.

Galectinology of Equine Pregnancy

Galectins are a family of proteins that bind to glycans, acting in a cytokine-like manner throughout the body. In the majority of mammalians, galectins have been found to be involved in pregnancy maintenance, but few studies have evaluated this in the horse. Therefore, the objective of this study was to examine the expression of various galectins in pregnant and nonpregnant mares. Next-generation RNA sequencing was performed on the chorioallantois and endometrium of healthy pregnant mares at 120, 180, 300, and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4), postpartum chorioallantois (n = 3), and diestrus endometrium (n = 3). In the endometrium, galectin-1 and galectin-13 were found in the highest expression in the nonpregnant mare, with decreasing levels of expression noted throughout gestation. In contrast, galectin-8 and galectin-12 were found to be the lowest in the nonpregnant mare and reached the highest expression levels in mid-gestation before declining as parturition neared. In the chorioallantois, galectin-1, galectin-3, and galectin-3BP were found to have heightened expression levels at 45 d of gestation, with lesser expression levels noted throughout gestation. In contrast, galectin-9, galectin-12, and galectin-13 experienced the highest expression levels in the late-term chorioallantois (300 d/330 d), with lesser expression noted in early- to mid-gestation. Of note, galectin-1, galectin-3BP, galectin-9, galectin-12, and galectin-13 all experienced the lowest expression levels in the postpartum placenta, with heightened expression noted during gestation. In conclusion, galectins appear to be involved in equine pregnancy, and this is dependent on both the tissue within the feto-maternal interface and the specific galectin involved.

Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares—A Preliminary Study

Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student t tests. The mean percentage of endometrial CD4+FOXP3+ Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4+FOXP3+CD25+ Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus (p = 0.0947). The mean proportion of CD3-NKp46+ lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4+FOXP3+CD25+ Tregs and CD3-NKp46+ NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.

Tumor necrosis factor signaling during equine placental infection leads to pro-apoptotic and necroptotic outcomes

Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-cervical inoculation of S. equi spp. zooepidemicus in pregnant mares (n = 6 infected / n = 6 control) and tissues and serum were collected to evaluate TNF-related transcripts. IHC was performed to confirm protein localization of TNFR-1 and TNFR-2. In healthy pregnancy, TNFR-1 appears to be the predominant TNF-related receptor. Following induction of disease, TNF concentrations increased in maternal serum, but expression did not alter at the tissue level. While both TNFR-1 and TNFR-2 increased following induction of disease, alterations in downstream pathways indicate that TNFR-1 is the dominant receptor in ascending placentitis, and is primarily activated within the chorioallantois, with minimal signaling occurring within the endometrium. In conclusion, TNF appears to be involved in the pathophysiology of ascending placentitis. An increase in this cytokine during disease progression is believed to activate TNFR-1 within the chorioallantois, leading to various pro-apoptotic and necroptotic outcomes, all of which may signal for fetal demise and impending abortion.

SAFETY OF UTERINE LAVAGE WITH OZONATED SALINE IN MARES

This study aimed to assess the safety of uterine lavage using ozonated saline in mares. Each liter of saline (NaCl 0.9%) was ozonated at 50 µg of ozone per mL for 7 min using a medical ozone generator. Then, uterine lavage was performed during estrus or 4h post-breeding and each liter of saline remained inside the uterus for 10 min before being recovered. In experiment 1, two cycles of six mares were used in a crossover design. Lavage was performed with ozonated (Treated) or non-ozonated (Control) saline 4h after the infusion of dead sperm, and cytology samples and biopsies were collected 6 and 24h later. Experiment 2, endometrial biopsies were collected before lavage (T0) and at 15 (T15), 30 (T30), and 60 (T60) days post-treatment with ozonated (n=11) or non-ozonated (n=3) saline. In experiment 3 (n=10), the pregnancy outcome was evaluated subsequent to lavage with ozonated saline 4h after insemination. In Experiment 1, there were no difference at 6h post-breeding in PMNs count on endometrial cytology between treatment and control groups (P&gt;0.05). However, PMNs tended (P=0.09) to be superior in the experimental treatment compared to the control at 24h post-breeding. In addition, the number of PMNs tended to be lower in the control cycle at 6 h (P=0.06) and 24 h (P=0.08) in endometrial biopsies. In Experiment 2, the endometrial architecture, the numbers of lymphocytes and PMN were not affected by the treatment (P&gt;0.05). In Experiment 3, nine out of 10 mares were confirmed to be pregnant after treatment, and there was not pregnancy loss up to 60 days. In conclusion, uterine lavage with ozonante saline solution may cause a mild endometrial inflammation, however, doesn’t harm the endometrium or impact pregnancy outcomes in healthy mares.

The NF-κB-signalling pathway in mare's endometrium infiltrated with the inflammatory cells.

Endometritis is an important issue decreasing mares' fertility. In the case of endometritis, both inflammatory cells infiltration and proinflammatory molecules production are regulated by various cellular and gene regulatory mechanisms, including the nuclear factor‐κB (NF‐κB)‐dependent pathway. NF‐κB‐signalling pathway has been recently studied in the equine endometrium in the context of endometrosis. Thus, this study aimed to determine gene transcription of NF‐κB subunits (RelA; NF‐κB1; NF‐κB2), proinflammatory molecules (MCP‐1; IL‐6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) in endometritis and compare them with the intensity and type of inflammatory cell infiltration. Endometrial samples, collected post‐mortem from cyclic mares in oestrus or dioestrus, were classified histologically and examined using quantitative PCR. Transcription NF‐κB subunits genes did not differ with either inflammatory intensity or type of inflammatory cell infiltration. Transcription of MCP‐1 and IL‐6 genes increased with the severity of inflammation, with the involvement of HAS 3 and HAS 2 genes, as opposed to HAS 1 genes. These proinflammatory molecules and hyaluronan synthases in the equine inflamed endometrium do not seem to be regulated by the NF‐κB pathway. Hence, separate signalling pathways for the development and progression of equine endometritis and endometrosis may be suggested.