Persistent Breeding-induced Endometritis Research Articles

Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Use of equine embryo-derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development and implantation would potentially improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis. Early gestation tissue provides a unique source of multipotent stem cells that exhibit immunomodulatory, proliferation, and differentiation properties. Extracellular vesicles (EVs) are nanometre sized, membrane enclosed structures released by cells that play a crucial role in mediating cellular communication through the shuttling of their cargo of bioactive molecules. This study involved two clinical trials aimed to utilise D32 embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in mares. The aim of Experiment 1 was to determine the efficacy of an intrauterine deposition of EDMSCs and their EVs 24 h prior to being challenged by intrauterine insemination with a dose of pooled dead sperm PBIE-susceptible (Troedsson and Woodward. Reproductive Biology. 2016;16:8-12) mares (n=5) in a controlled cross-over designedexperiment. In Experiment 2 a cohort of client-owned thoroughbred mares (n=37; 40 estrous cycles) from farms that were previously involved in a large-scale epidemiological study in which the average first cycle pregnancy rate in mares was 53.6% (Firth, et al. New Zealand Veterinary Journal. 2012;60:329-334) were utilized. The mares were identified as being susceptible to PBIE with a history of failing to become pregnant on >2 consecutive estrous cycles despite intense breeding management and use of a fertile stallion/s received intrauterine EDMSCs or their EVs 24 h prior to natural mating. A significant reduction in uterine fluid volume was observed at both 12 and 24 h after insemination in Experiment 1 when mares were pre-treated with either EDMSCs or their EVs compared to placebo (control) treatment (p>0.05). In Experiment 2, 9 of 19 (47%) mares treated with EDMSC and 13 of 20 (65%) mares treated with EVs prior to natural mating were pregnant after the first cycle, and 12 of 18 (67%) mares treated with EDMSCs and 15 of 19 (79%) mares treated with EVs had conceived by the end of the breeding season. These preliminary clinical studies are the first report of the use of EDMSCs or their EVs as a potential therapy for the management of PBIE in susceptible mares.

Equine endometrial organoids treated with embryo-derived mesenchymal stem cell-secreted extracellular vesicles as a treatment for endometritis

A healthy uterine environment is essential for normal mare fertility and is critical for ensuring a positive financial return on breeding in the equine industry. Approximately 20% of Thoroughbred mares undergo persistent breeding-induced endometritis (PBIE), creating a uterine environment that is unfavorable for pregnancy. Despite extensive in vivo studies, the exact mechanisms causing endometritis and the most efficacious strategies for preventing or resolving PBIE are unknown. Organoids are physiologically-relevant 3D cell cultures that allow disease processes to be studied at the cellular level and eliminate confounding factors present in vivo. A potential therapeutic for PBIE is extracellular vesicles (EVs) secreted by embryo-derived mesenchymal stem cells (EDMSCs). EVs are lipid-bound nanoparticles containing proteins, mRNAs, and miRNAs that are naturally secreted by cells for intercellular communication. We hypothesized that EVs secreted by EDMSCs have anti-inflammatory properties to prevent PBIE. Our objectives were i) to assess EVs produced by EDMSCs for the presence of anti-inflammatory miRNAs via PCR and ii) to assess transfer of the anti-inflammatory miRNAs from EVs to endometrial organoids stimulated for inflammationwith frozen-killed sperm (FKS) or lipopolysaccharide (LPS). Results demonstrated the presence of reported anti-inflammatory miRNAs miR-10a, miR-21, miR-24, miR-145, and miR-146a in EVs secreted by three different EDMSC cell lines. Then, endometrial organoids (n=3 mares) were co-cultured with EDMSC EVs or PBS (control) for 24 h and subsequently exposed to two inflammatory stimuli (FKS or LPS). After 12h of exposure to inflammatory stimuli, organoids were washed and assessed for the presence of anti-inflammatory miRNAs by PCR (data was normalized using U6 snRNA). MiR-10a and miR-24 trended to be increased in organoids treated with EDMSC EVs compared to PBS controls in groups stimulated with either FKS or LPS. Furthermore, uptake of EVs by the organoids was corroborated by the co-culture of organoids with fluorescently-labelled (PKH26) EVs. EV uptake was confirmed by presence of a fluorescence signal in recipient organoid cells. While studies are ongoing, data suggests that EDMSC EVs contain anti-inflammatory miRNAs that may be delivered to endometrial cells to prevent PBIE in mares. Funding was provided by the Grayson-Jockey Club Research Foundation.

Intrauterine vitamin D3 improves inflammation resolution in mares susceptible to persistent breeding-induced endometritis

Persistent breeding-induced endometritis (PBIE) is one of the leading causes of subfertility in mares, affecting 10-15% of the population. The objective of this study is to identify a novel, antibiotic-free treatment to improve inflammation resolution in mares susceptible to PBIE. Mares susceptible to PBIE tend to be older animals with an impaired immune response, factors that are associated with vitamin D3 (D3) deficiency in humans. As our previous research suggests that vitamin D is actively transported to the site of inflammation where it plays a central role in resolving inflammation, we hypothesized that local administration of vitamin D3 would aid in improved uterine clearance and enhance inflammation resolution in mares susceptible to PBIE. To this end, susceptible (n = 6) and resistant (n = 4) mares were bred with centrifuged, extended frozen-killed semen containing either D3 or vehicle (Con) in a randomized cross-over design, with a rest cycle between. Status was assigned based on endometrial biopsy score (modified Kenney-Doig) and results of a test breeding cycle at the beginning of the season, with susceptible mares having a 2B or 3 biopsy score coupled with 96h+ of fluid retention, while resistant mares had a 1 or 2a biopsy score and cleared fluidby 48 h. Plasma was obtained at the time of insemination (0h), 6h post-breeding (6h) and 7d post-breeding (7d), with endometrial biopsies taken at both 6h and 7d. Susceptible mares showed a nearly 65% decrease (P < 0.001) in the days to uterine clearance when treated with D3 (2.2 ± 0.48) compared to their own Con cycle (6.0 ± 3.1), bringing days to uterine clearance to a level which was not significantly different than the control cycle for resistant mares. Enhanced inflammation resolution was also evident on a histological level, with mucosal inflammation and leukocyte infiltration significantly higher in the control cycle, as compared to the D3 cycle and the pre-breeding biopsy (P <0.05). Lastly, plasma concentrations of IL-1α, IL-6 and IL-10 were altered, with pro-inflammatory IL-1α significantly decreased in D3-treated mares, while anti-inflammatory IL-6 and IL-10 showed a significant increase, with the highest measured levels seen at 7d post-breeding (P<0.05). In conclusion, vitamin D3 shows promising results as a potential treatment for PBIE in mares, showing greatly improved resolution of breeding-induced inflammation. Work is currently underway to determine whether pregnancy rates are influenced by D3 treatment, as well as mechanistic pathways.

Susceptibility to persistent breeding-induced endometritis and positive embryo flushing are associated with differential endometrial gene expression and uterine extracellular vesicles cargo in mares

Persistent breeding-induced endometritis (PBIE) is the leading cause of subfertility in mares; embryo donor mares are prone to become susceptible to PBIE due to excessive uterine manipulation after repeated breeding-flushing cycles, coupled with age-related deterioration of the cervix and uterus. Therefore, the study's objectives were to determine: (i) if a differential endometrial response to the embryo exists (i.e., positive embryo flush) on Day-8 post-ovulation and (ii) if this endometrial response is different in mares susceptible to PBIE. Twenty mares were screened for susceptibility to PBIE using a two-billion-frozen-killed sperm model coupled with Kenney & Doig's scoring; and, based on their responses, classified as susceptible (n=6) or resistant (n=6) to PBIE or intermediate (n=8). Mares were bred with two-billion sperm from one fertile stallion. On Day-8 post-ovulation, mares were submitted to low-volume uterine lavages (i.e., 100 mL), followed by embryo flushing (4 L) and collection of endometrial biopsies. Two embryos were collected from susceptible and three from resistant mares; seven breeding cycles yielding a negative embryo flushing were included as controls. Endometrial luminal (LE) and glandular epithelium (GE) were micro-dissected with ZEISS PALM Microbeam and analyzed by RNA-sequencing. Uterine extracellular vesicles (uEVs) were isolated from uterine fluid, and their RNA content was analyzed. The comparison betweenpositive- and negative embryo-flushing revealed 305 differentially expressed genes (DEGs) for LE (FDR 5%) while 50 DEGs for GE (FDR 20%, P<0.001), indicating that the main response is present in the LE. The most significantly overrepresented functional terms for genes upregulated in LE on Day-8 were related to ‘response to hormones,’ ‘lipid or extracellular stimulus,’ ‘cell cycle,’ and ‘T-cell activation.’ The analysis of uEVs revealed 442 differentially abundant (DA) RNAs (FDR 5%) between mares with positive- and negative-embryo flushing. At the miRNA level, miR-500-5p and miR-1246 were found in higher concentrations in uEVs from mares yielding positive-embryo flushing. Overrepresented functional terms were related to, e.g., ‘translation,’ ‘lipid binding,’ ‘cell cycle, ‘cytokine binding,’ and ‘angiogenesis.’ The comparison between susceptible and resistant mares resulted in 80 DEGs for GE (FDR 20%, P<0.001) and 65 DEGs for LE (FDR 28%, P<0.001). Functional analysis revealed enrichment for: ‘lipid metabolism and biosynthesis,’ ‘response to the hormone,’ ‘RNA splicing,’ and ‘cell morphogenesis.’ Analysis of uEVs revealed 7 DA RNAs (FDR 20%) and one DA miRNA (eca-miR-181a-5p) between susceptible- and resistant-mares, which has been described as associated with fertility. In conclusion, this is the first study showing a transcriptional response to the presence of an embryo on Day-8 in terms of alterations in endometrium and uEVs cargo and revealing differences between mares susceptible and resistant to PBIE.

Mitigation of post breeding endometritis in mares treated with platelet rich plasma

Persistent breeding-induced endometritis (PBIE) is one of the major causes of infertility in mares, characterized by a lingering inflammatory response in the uterus, which leads to accumulation of uterine fluid and polymorphonuclear cells (PMN), harming the reproductive efficiency of the animal. The aim of this study was to determine if the presence and volume of intrauterine fluid and the percent of endometrial neutrophils decreased after intrauterine application of blood platelet-rich plasma (PRP) in mares with post-mating endometritis. Healthy estrous crossbreed mares (n = 9) aged between 6 to 12 years, with optimal body condition and under the same nutritional management, had two cycles randomly assigned to a crossover trial. Each received either intrauterine infusions of lactate ringer solution (LRS) or autologous PRP 4h after artificial insemination (AI) with fresh semen of low sperm quality from the same stallion. All the animals went through all the treatments. A total of 100 mL of citrated whole blood was collected for PRP production and was centrifuged at 2315g for 15 minutes then 579g for 10 minutes. The presence of intrauterine fluid (IUF) was analyzed by ultrasonographic examination and the percentage of endometrial neutrophils was determined through endometrial cytology, both before insemination (in the presence ofa preovulatory follicle ≥35mm and endometrial edema) and 24h after treatment application. Statistical analysis was performed by a Mixed model (SAS, 2023) for repeated measures with treatment and time as between subject factors. The presence of intrauterine fluid had an effect of time (p ≤ 0.049), where fluid size was smaller before AI than 24h after treatment; however, there was no treatment effect (p ≥ 0.264) over intrauterine fluid. The percentage of endometrial neutrophils had an effect of time (p ≤0.001), less inflammatory cells were found before AI than 24 hours after treatment; there was an effect of treatment (p ≤0.001) as well, where PRP decreased the number of endometrial polymorphonuclear cells compared to the control group infused with LRS. The increase in intrauterine fluid with time suggests that semen itself increases blood perfusion that generates intrauterine fluid accumulation. After induction of physiological endometritis, endometrial polymorphonuclear cell numbers were reduced successfully with PRP treatment. PRP is rich in peptides and signaling proteins that suppress the expression of proinflammatory cytokines such as IL-1 and TNF, inhibits polymorphonuclear neutrophil migration, and thus regulates intrauterine inflammation through decreased endometrial COX-2 expression.

140 Intrauterine vitamin D3 improves inflammation resolution in mares susceptible to persistent breeding-induced endometritis

Persistent breeding-induced endometritis (PBIE) is one of the leading causes of subfertility in mares, affecting 10–15% of the population. Mares susceptible to PBIE tend to be older animals with an impaired immune response, factors that are associated with vitamin D3 (D3) deficiency in humans. As our previous research suggests that vitamin D is actively transported to the site of inflammation where it plays a central role in resolving inflammation, we hypothesized that local administration of vitamin D3 would aid in improved uterine clearance and enhance inflammation resolution in mares susceptible to PBIE. To this end, susceptible (n = 6) and resistant (n = 4) mares were bred with frozen-killed semen containing either D3 or vehicle (ethanol; Con) in a randomized crossover design, with a rest cycle between. Status was assigned based on endometrial biopsy score (modified Kenney-Doig) and results of a test breeding cycle at the beginning of the season. Plasma was obtained at the time of insemination (0h), 6h post-breeding (6h) and 7d post-breeding (7d), with endometrial biopsies taken at both 6h and 7d. Susceptible mares showed a nearly 65% decrease (P < 0.001) in the days to uterine clearance when treated with D3 (2.2 ± 0.48) compared with their own Con cycle (6.0 ± 3.1), bringing days touterine clearance to a level which was not significantly different than the control cycle for resistant mares. Resistant mares also showed a significant improvement with D3, going from 3.12 ± 0.4 d (Con) to 1.3 ± 0.21 d (D3; P < 0.01). Enhanced inflammation resolution was also evident on a histological level, with mucosal inflammation and leukocyte infiltration significantly higher in the control cycle, as compared with the D3 cycle and the pre-breeding biopsy (P < 0.05). Lastly, plasma concentrations of IL-1α, IL-6 and IL-10 were altered, with pro-inflammatory IL-1α significantly decreased in D3-treated mares, while anti-inflammatory IL-6 and IL-10 showed a significant increase, with the highest measured levels seen at 7d post-breeding (P < 0.05). No significant differences were seen between cytokine levels in resistant and control mares. In conclusion, vitamin D3 shows promising results as a potential treatment for PBIE in mares, showing greatly improved resolution of breeding-induced inflammation. Work is currently underway to determine whether pregnancy rates are influenced by D3 treatment, as well as mechanistic pathways.

Relationship between Clinical Uterine Findings, Therapy, and Fertility in the Mare

Persistent breeding-induced endometritis (PBIE) is a major cause of subfertility in horses and the susceptibility is increased by several factors. The aim of this study was to determine the effects of clinical uterine findings and PBIE therapies, respectively, on pregnancy rate in mares. The analysis included records from 220 mares (390 cycles) inseminated at an artificial insemination (AI) center in Switzerland. Gynecological examinations were performed repeatedly before and after AI to determine cervical tone, uterine edema, and intrauterine fluid accumulation. Pregnancy rate was lower (p < 0.001) in barren mares compared to mares of other reproductive status. A more flaccid cervix (p = 0.009) was observed at the time of ovulation in pregnant cycles, but there was no difference (p > 0.05) regarding uterine edema. Intrauterine fluid accumulation reduced pregnancy rate (p = 0.002). Oxytocin administration had beneficial effects on pregnancy rate (p = 0.015), especially for barren mares, while uterine lavage did not have any effect (p > 0.05). The results show that cervical tone and intrauterine fluid accumulation, but not its degree, are useful parameters for assessment of fertility in mares. Oxytocin treatment improved pregnancy rates in mares with PBIE while uterine lavage had a limited effect.

Identification of genes associated with susceptibility to persistent breeding-induced endometritis by RNA-sequencing of uterine cytobrush samples

This study aimed to investigate the susceptibility to persistent breeding-induced endometritis (PBIE). Cytobrush samples were collected from 81 broodmares 1–3 days before artificial insemination (AI). Susceptibility to PBIE was evaluated by the presence of ≥ 2 cm of intrauterine fluid 24 h after AI, besides the fertility was determined by a sonographic pregnancy diagnosis 2 weeks after ovulation. RNA expressions were compared between susceptible non-pregnant (SNP) mares (n=9) and resistant pregnant (RP) mares (n=9) as well as between susceptible pregnant (SP) mares (n=9) and susceptible non-pregnant (SNP) mares. 66 differentially expressed genes (DEGs) were identified between SNP and RP mares and 60 DEGs between SP and SNP mares. In SNP compared to RP mares, transcript levels of genes regulating steroid hormone metabolism and neutrophil chemotaxis were lower, while higher for genes participating in uterine inflammation.Transcripts of genes related to extracellular matrix degradation, tissue adhesions, and fibrosis were lower in SP mares than in SNP mares, while higher for genes related to uterine cell proliferation, differentiation, and angiogenesis in SP mares than SNP mares. In conclusion, increased transcript levels of apolipoprotein E (APOE) and roundabout 2 (ROBO2), cluster domain 44 (CD44), integrin beta 3 (ITGB3), and epidermal growth factor (EGF) are possible biomarkers for susceptibility to PBIE. While higher expression of fibroblast growth factor 9 (FGF9), kinase domain receptor (KDR), and C-X-C motif chemokine ligand (CXCL) 16, collagen type V alpha 2 (COL5A2) and fibronectin (FN1) are suggested indicators of fertility in susceptible mares if they receive proper breeding management.

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares.

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (−48 and −24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilβ, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey’s post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property.

Intrauterine infusion of killed semen adversely affects uterine blood flow and endometrial gene expression of inflammatory cytokines in mares susceptible to persistent breeding-induced endometritis

Persistent breeding-induced endometritis (PBIE) is a leading cause of infertility in mares. The objective of the study was to assess genital perfusion and endometrial gene expression of inflammatory cytokines in mares classified as susceptible (n = 5) or resistant (n = 5) to PBIE. Ten mares were examined daily during estrus until 6 d after hCG-induced ovulation for two estrous cycles. Twenty-four hours after application of 1500 IU hCG, 4 mL of killed (by repeated freezing in liquid nitrogen and thawing at 50 °C) deep-frozen semen or sterile saline was instilled into the uterine body and examinations were carried out immediately before and 3, 6, and 12 h after intrauterine infusion. Examinations included blood sampling to determine plasma progesterone (P4) concentrations, and transrectal ultrasonography in B- and color Doppler mode to determine follicular and luteal size and blood flow, the extent of intrauterine fluid, as well as time-averaged maximum velocity (TAMV), blood flow volume (BFV), and blood flow resistance (expressed as pulsatility index, PI) of the uterine arteries. Additionally, endometrial biopsies were obtained at 24 h before, and 2 and 7 d after infusion, and mRNA expressions of IL1B, IL6, IL8, IL10, TNF, CASP3, and COX2 were determined by qRT-PCR. Statistical analyses were performed with mixed models. Intrauterine fluid retention (diameter >20 mm for at least 3 d) was found after infusion of killed semen in five susceptible mares. There was no treatment effect (semen vs saline; P > 0.05) on genital blood flow, plasma P4 concentration, and endometrial gene expression. In comparison to resistant mares, susceptible mares had an increased (P = 0.04) BFV of the uterine arteries at 24 h before intrauterine infusion of killed semen, and an increased (P = 0.03) PI at 2 d after infusion. The TAMV, plasma P4 concentrations, and follicular and luteal size and blood flow did not differ (P > 0.05) between resistant and susceptible mares. Endometrial mRNA expression of IL1B increased (P = 0.05) at 2 d after the infusion of killed semen in the susceptible mares, and the expression of IL10 increased (P = 0.003) at 7 d after the infusion within the resistant mares. Interleukin 6 mRNA was increased (P = 0.05) in susceptible compared to resistant mares at 2 d after infusion. In summary, an intrauterine infusion of killed semen increases uterine blood flow resistance and alters endometrial gene expression of inflammatory cytokines for at least 7 d but does not affect ovarian blood supply and luteal function in mares susceptible to PBIE.

Diagnostic and Treatment Practices of Equine Endometritis-A Questionnaire.

Endometritis is a major cause for impaired fertility in mares. The objectives of this study were to collect information on diagnostic and treatment practices performed by veterinarians and to investigate possible effects of professional experience, caseload, and geographical location on the practitioners' management of endometritis cases. For this purpose, equine practitioners (n = 680) were asked to fill out an online survey (34 questions). The online survey yielded 117 responses by veterinarians practicing in all parts of Germany. Most respondents came from Lower Saxony and managed <20 mares per year. For the diagnosis of chronic infectious endometritis, uterine sampling for microbiological examination was performed manually with a swab by the majority of practitioners whereas only few used the speculum technique. The incidence of antibiotic resistant pathogens was reported to be <5% by almost all respondents. Most practitioners relied on systemic antibiotic treatment with trimethoprim-sulfadiazine. Only occasionally veterinarians used intrauterine antibiotic therapy. Uterine lavages were performed routinely by almost half of the respondents in case of positive uterine cultures, mostly with 0.9% saline solution. Irritant solutions (e.g., iodine, chlorhexidine, kerosene) were used less often. Collection of an endometrial culture after completion of the treatment was common practice. While only a very limited association of the geographical location of practitioner (e.g., on selection of mares for endometrial culture, p < 0.05) was observed, the number of managed mares affected the answers notably (e.g., for use of irritating intrauterine treatment, p < 0.05). The management of persistent breeding induced endometritis (PBIE) was influenced by the number of managed mares (e.g., for use of oxytocin, p < 0.05) and 29.6% of respondents administered antibiotics as part of their PBIE management. In summary, treatment strategies in the field vary considerably and include also non-evidence-based methods, but most German practitioners apply the recommended suitable treatments. Following the guidelines for antibiotic usage, most veterinarians administer antibiotics dependent on endometrial culture results but do not use cytology, low-volume lavage, or biopsy routinely. Antibiotic resistant uterine pathogens are reported to be isolated infrequently and equine practitioners prefer systemic to local antibiotic treatment of endometritis.

Equine endometritis: a review of challenges and new approaches.

Endometritis in the mare begins as a normal physiological inflammatory response to breeding that involves both a mechanical and immunological response pathway activated to rid the uterus of semen and bacteria. With successful resolution of this inflammation, the mare's uterus will provide a hospitable environment for the development of the semi-allogenic conceptus. If the mare fails to resolve this inflammatory response within 48 h of breeding, she will become susceptible to persistent breeding-induced endometritis (PBIE) which will have detrimental effects on her fertility. This condition can then predispose the mare to bacterial or fungal endometritis leading to further degeneration of the endometrium. Optimisation of the mare's fertility requires a fine balance between allowing the natural immune response of the endometrium to its exposure to allogenic semen to run its course, and yet preventing its progression to PBIE or the involvement of infectious agents. This review discusses the challenges presented by PBIE, latent infections, biofilms, fungal infections and the need to utilise diagnostic methods available and implement targeted treatments to optimise fertility in the mare.

Effect of Mycobacterium cell wall fraction on endometrial histomorphometry of mares resistant and susceptible to persistent breeding-induced endometritis

Mycobacterium cell wall fraction (MCWF) is a biological component made up of molecules with immunostimulant properties, which is therapeutically used to modulate persistent breeding-induced endometritis (PBIE). The aim of this study was to analyze the effect of this immunomodulator on the endometrial histological structure during the diestrus of PBIE-resistant and -susceptible mares that either received treatment with MCWF or not. The experiment was conducted with 10 resistant mares (RM) and 9 susceptible mares (SM). In the first estrous cycle of the trial, all mares were inseminated with dead semen as an inflammatory stimulus (Group A); at the next cycle, all mares were inseminated with dead semen and treated with a MCWF commercial immunomodulator (Group B). In both groups, endometrial biopsies were taken on day 7 post-ovulation (diestrus). Endometrial biopsies of untreated-RM (UTRM, n = 6), untreated-SM (UTSM, n = 7) MCWF-treated-RM (TRM, n = 6) and MCWF-treated-SM (TSM, n = 6) were evaluated. They were randomly chosen as representative mares of Group A and B, respectively. The height of lining and glandular epithelia, glandular diameter, glandular density and glandular area were evaluated. The histological structure revealed lymphocytic infiltration and dilated, tortuous glands with some glandular nests, particularly in UTSM. The histomorphometrical results showed no differences (ρ > 0.05) between the analyzed groups. This would indicate that post-service treatment with the MCWF immunomodulator does not modify the endometrial histoarchitecture but, apparently, its action would be mainly based on the stimulation of the cellular and humoral immune responses.

The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.

Persistent Breeding-Induced Endometritis in Mares - a Multifaceted Challenge: From Clinical Aspects to Immunopathogenesis and Pathobiology.

Post-breeding endometritis (i.e., inflammation/infection of the endometrium), is a physiological reaction taking place in the endometrium of mares within 48 h post-breeding, aimed to clear seminal plasma, excess sperm, microorganisms, and debris from the uterine lumen in preparation for the arrival of an embryo. Mares are classified as susceptible or resistant to persistent breeding-induced endometritis (PBIE) based on their ability to clear this inflammation/infection by 48 h post-breeding. Mares susceptible to PBIE, or those with difficulty clearing infection/inflammation, have a deficient immune response and compromised physical mechanisms of defense against infection. Molecular pathways of the innate immune response known to be involved in PBIE are discussed herein. The role of the adaptive uterine immune response on PBIE remains to be elucidated in horses. Advances in the pathobiology of microbes involved in PBIE are also revised here. Traditional and non-traditional therapeutic modalities for endometritis are contrasted and described in the context of clinical and molecular aspects. In recent years, the lack of efficacy of traditional therapeutic modalities, alongside the ever-increasing incidence of antibiotic-resistant microorganisms, has enforced the development of non-traditional therapies. Novel biological products capable of modulating the endometrial inflammatory response are also discussed here as part of the non-traditional therapies for endometritis.

95 N-Acetyl cysteine as a potential treatment for equine persistent breeding-induced endometritis

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Transient uterine inflammation is a normal response to breeding; however, PBIE-susceptible mares do not clear this inflammation in a timely fashion. Uterine inflammation at the time of embryonic descent from the oviducts ultimately results in early embryonic death and is seen clinically as infertility. Several risk factors for PBIE have been identified and include age, parity, anatomical conformation, such as poor perineal conformation and cervical fibrosis, as well as other reproductive tract abnormalities. N-Acetyl cysteine (NAC), a mucolytic used to treat endometritis in mares, has anti-inflammatory properties, affects inflammatory cytokines, and is a mild inhibitor of nitric oxide synthase. Increased nitric oxide, causing smooth muscle relaxation and alterations in inflammatory cytokines, has been documented in PBIE mares. The objective of our study was to determine if NAC treatment would lower nitric oxide and inflammatory cytokine levels, thereby resolving PBIE. A randomised, blinded, crossover design clinical trial was performed utilising PBIE-susceptible mares (n=10). Mares were screened for bacterial endometritis before enrolment in the study and only mares that had negative bacterial cultures were used. No other treatments were given to mares while they were enrolled in the study. Intrauterine infusion of 180mL of 3.3% NAC was performed 12h before insemination, when a follicle &amp;gt;35mm was present. Mares were sampled for endometrial cytology and endometrial fluid to determine inflammatory cytokine (ELISA) and nitric oxide (colourimetric assay) levels at 12 and 72h post-insemination. Endometrial biopsies were taken at the same time points post-insemination to determine gene expression of inflammatory cytokines by qPCR. Clinical signs of endometrial oedema and intrauterine fluid volumes were assessed at 12 and then every 24h post-breeding. Statistical assessment of the data was performed using a repeated-measures ANOVA. Uterine inflammation, as determined by percent number of neutrophils on endometrial cytology (P=0.0006), and interleukin 6 gene expression (P=0.003) were highest at 12h. Uterine oedema was greatest at 12 and 24h (P=0.02) and uterine fluid volumes were highest at 24h (P=0.02). Interestingly, interleukin-6 protein levels were not in accordance with gene expression, and were highest at 72h. In this clinical trial, pre-breeding intrauterine treatment with NAC did not affect nitric oxide levels, cytokine gene expression, or clinical signs of PBIE. However, the pattern of inflammation noted in this study is consistent with that described in PBIE mares. Nevertheless, the assessment of inflammatory cytokines, both at the gene and protein level at different time points post-AI, in combination with clinical signs will add to the understanding of the alterations in inflammatory cytokine levels in mares susceptible to PBIE.

Influence of cycle stage, age and endometrial biopsy score on oxytocin receptor distribution and gene expression in the cervix and uterus of non-pregnant mares

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Insemination Dose and Site With Frozen Semen Affects the Sperm Transport and Inflammatory Response in Mares?

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

The anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares

The deposition of semen into the uterus of the horse induces a transient innate immune response that lasts 24–36 h in the normal mare. There exists a subset of mares that are unable to resolve this inflammation in a timely manner, and are classified as susceptible to the disease of persistent breeding-induced endometritis (PBIE). Lactoferrin is a protein of interest as a potential therapeutic for this persistent inflammation due to its anti-inflammatory and bactericidal properties. The addition of human recombinant lactoferrin (hrLF) to the insemination dose was previously shown to suppress mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α at 6 h after insemination, but no studies have shown the effect of lactoferrin when infused post-breeding. Therefore, the objectives of this study were to (1) assess the safety of intra-uterine infusion of hrLF, (2) evaluate the effect of intrauterine infusion of hrLF post-breeding as a modulator of the immune response to breeding in the susceptible mare, and (3) determine the most effective concentration of hrLF. For the first experiment four normal mares received an intrauterine infusion of 500 μg/mL hrLF resuspended in 10 mL lactated Ringer's solution (LRS) and heart rate, rectal temperature, respiration, and endometrial quality were evaluated. For the second experiment, six mares classified as susceptible to PBIE were bred during estrus with 500 × 106 progressively motile sperm comprised of the ejaculates from two stallions, which were centrifuged over Androcoll-E to remove seminal plasma. Each insemination dose was resuspended in 30 mL LRS. Six hours after breeding, a 1L LRS uterine lavage was performed prior to treatments. Four treatments were administered over four consecutive estrous cycles in randomized order of: 10 mL LRS (vehicle control), 50 μg/mL hrLF resuspended in 10 mL LRS, 250 μg/mL hrLF resuspended in 10 mL LRS, and 500 μg/mL hrLF resuspended in 10 mL LRS. Twenty-four hours after breeding the mares were evaluated via transrectal ultrasonography for fluid retention. A low volume uterine lavage (250 mL LRS) was performed and the effluent was evaluated for polymorphonuclear neutrophils (PMNs). Finally, an endometrial biopsy was obtained for qPCR analysis of selected inflammatory cytokines. Lactoferrin had no significant overall effect on vital signs or endometrial quality. The addition of hrLF (50 μg/mL, 250 μg/mL, 500 μg/mL) did not significantly affect the amount of fluid detected post-breeding, but suppressed the ratio of PMNs to epithelial cells at all three concentrations compared to controls. In addition, all three concentrations of hrLF increased the mRNA expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN), while the 50 μg/mL dose significantly suppressed mRNA expression of the pro-inflammatory cytokine interferon gamma (IFNγ). In conclusion, the infusion of hrLF post-breeding was found to modulate the inflammatory response to breeding in the mare, and appears to be most effective at the 50 μg/mL concentration.