Equine granulosa cell tumours among other ovarian conditions: Diagnostic challenges.

BACKGROUND: Granulosa cell tumours (GCT) are the most common ovarian tumours in mares. While the classical presentation may not represent diagnostic challenges, diagnosis is not easy in the early stages. OBJECTIVES: Illustrate the variability in the presentation and serum biomarkers associated with ovarian abnormalities in the mare. STUDY DESIGN: Retrospective case series. METHODS: Nonclassical cases of GCTs and other ovarian conditions were identified and behaviour, GCT endocrine results, palpation and ultrasonographic findings are described and the diagnostic value of each is discussed. RESULTS: Mares in this case series with GCTs had been presenting clinical signs ranging from no behavioural changes to behaviours including aggression, stallion-like and inability to work under saddle. Hormonal profiles of endocrinologically functional GCTs can be erratic and unpredictable. The clinical form and ultrasonographic appearance may also vary with time from an initially enlarged/anovulatory follicular structure that later develops a multicystic 'honeycomb' appearance. Mares with GCTs can also present with persistent anovulatory follicles or apparent luteal tissue that are unresponsive to treatment. If both ovaries are of relatively normal size and symmetry, but hormonal biomarkers are markedly increased (AMH >10 ng/mL, inhibin B and/or testosterone >100 pg/mL; 0.37 nmol/L), it is likely that a functional GCT is present. Still, it can be a challenge to decide which ovary to remove. Post-surgical endocrine testing can be helpful, especially if histopathology is not performed or a GCT is not found. MAIN LIMITATIONS: Cases limited to 14. CONCLUSIONS: Granulosa cell tumours present with a wide variety of clinical signs that do not fit what is commonly described as 'classic'. Only if AMH, testosterone and inhibin B concentrations are markedly increased, and there is an abnormally enlarged ovary, the diagnosis of a GCT is more confident. In the presence of normal size ovaries, normal hormonal biomarkers and abnormal behaviour, it is more likely that the ovaries are not involved.

Model of Chronic Equine Endometritis Involving a Pseudomonas aeruginosa Biofilm.

Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux-engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux-labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria (P < 0.05). A greater incidence (P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences (P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased (P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly (P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus.

In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent "biofilm-like" matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo.

Comparison of equine endometrial cytology samples collected with uterine swab, uterine brush, and low-volume lavage from healthy mares.

BACKGROUND: There have been no studies evaluating and comparing the quality of equine endometrial cytology samples obtained via the 3 most common sampling techniques from healthy mares. OBJECTIVES: The objective was to compare the quality and contents of equine endometrial samples obtained by 3 different sampling techniques: double-guarded uterine swab, double-guarded uterine brush, and low-volume lavage (LVL), all collected from clinically healthy mares. METHODS: Samples were collected from 24 healthy mares in early estrus. In 19 mares, samples were obtained in a sequential manner, first with the swab, then with the brush, followed by LVL. Cytologic evaluation included estimates of quality, cellularity, and presence of inflammatory cells. The clinical pathologist performing the evaluations was blinded to the collection technique. The Friedman test with Dunn's multiple comparisons was used to compare rankings of quality, cellularity, and the presence or absence of inflammatory cells. Observed cytologic differences were described. RESULTS: All techniques provided diagnostic samples, but swabs yielded the lowest quality sample. In our hands, the uterine brush provided the highest quality sample. Low-volume lavage samples contained higher numbers of neutrophils, although, in general, < 1 neutrophil/400× field is expected for all endometrial sampling techniques in healthy mares. CONCLUSIONS: All sampling techniques can be adequate methods for endometrial cytology, but the brush technique consistently provided the best sample. Sample contamination or poor slide quality can adversely affect interpretation. The most accurate criteria for determining what constitutes mild endometritis in mares have yet to be established.

Use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares.

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating-induced endometritis.

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH).

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤ 20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥ 35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17ß, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥ 35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17ß, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.