Evaluation of a commercial multiplex immunoassay for assessing uterine inflammation in mares susceptible to persistent breeding-induced endometritis.

Overview

• This study evaluated a commercial equine-specific multiplex immunoassay to measure cytokine proteins in mare uterine fluid, aiming to determine its effectiveness in assessing uterine inflammation related to persistent breeding-induced endometritis (PBIE).
• The research focused on whether cytokine levels detected by this assay correspond to established clinical and cytologic indicators of inflammation after breeding in susceptible mares.

Background and Purpose

• Persistent breeding-induced endometritis (PBIE) is an inflammatory condition in mares characterized by a prolonged and inappropriate immune response after breeding.
• Cytokines, which are signaling proteins involved in immune responses, play a central role in this inflammation.
• Traditionally, studies of uterine inflammation and cytokine involvement in horses have relied on measuring gene expression rather than directly quantifying cytokine protein levels.
• Multiplex immunoassays, such as those based on the Luminex platform, allow simultaneous measurement of multiple cytokines in small fluid samples.
• However, their application for equine uterine samples, especially in low-volume lavage (LVL) fluids, was not well characterized before this study.
• This research aimed to assess if cytokine concentrations measured by an equine-specific multiplex immunoassay align with known clinical and cytological markers of uterine inflammation in mares prone to PBIE.

Methodology

• Six mares previously identified as susceptible to PBIE were selected.
• Each mare underwent three breeding challenges during estrous cycles, totaling 18 events.
• Intrauterine insemination was performed using freeze-killed spermatozoa to standardize the breeding challenge and minimize variability due to live sperm.
• Post-insemination uterine inflammation was assessed using several methods:
  • Transrectal ultrasonography to evaluate uterine fluid accumulation.
  • Endometrial cytology from cytobrush samples to measure neutrophil presence.
  • Low-volume uterine lavage (LVL) to collect fluid for cell counting and cytokine measurement.
  • Systemic blood samples were also collected to measure cytokine levels in circulation.
• The cytokines quantified using the Luminex multiplex assay included CXCL-8, IL-1β, IL-6, and TNF, which have known roles in inflammation.

Key Findings

• Among the cytokines studied, CXCL-8 showed the strongest association with uterine neutrophil infiltration, an important indicator of inflammation.
• Statistical correlations demonstrated:
  • A strong positive relationship between luminal (uterine) CXCL-8 concentrations and neutrophil counts obtained by LVL (R² = 0.66, highly significant).
  • A moderate positive correlation between luminal CXCL-8 and neutrophils measured by cytobrush cytology (R² = 0.37).
  • A weaker but still statistically significant association between CXCL-8 levels and uterine fluid accumulation (R² = 0.15).
• IL-1β showed only a weak association with inflammatory indicators.
• IL-6 and TNF did not demonstrate any significant association with uterine inflammation in this context.
• No correlation was found between luminal and systemic CXCL-8 levels or between systemic CXCL-8 and uterine inflammation markers, suggesting local cytokine levels are more indicative of uterine status than blood levels.

Conclusions and Implications

• The study validates the utility of an equine-specific commercial multiplex immunoassay on the Luminex platform to accurately quantify CXCL-8 protein in low-volume uterine lavage fluid.
• Cytokine measurement, particularly CXCL-8, closely reflects neutrophil-mediated uterine inflammation post-breeding in mares susceptible to PBIE.
• This method offers a practical and sensitive means to monitor uterine inflammatory status in mares, which could improve diagnosis and management of PBIE.
• The findings imply that local uterine cytokine levels provide more precise information about uterine inflammation than systemic blood cytokine concentrations.
• The lack of correspondence for IL-6 and TNF in this context suggests these cytokines may not be reliable markers for PBIE inflammation under the tested conditions.

Study Limitations and Future Directions

• The sample size was limited to six mares, so larger studies could help confirm these results.
• Use of freeze-killed spermatozoa standardized the immune challenge but may not fully replicate natural breeding effects.
• Future research could explore longitudinal cytokine dynamics over time and include other inflammatory markers to provide a more comprehensive inflammatory profile.
• Potential development of diagnostic panels based on these findings could enhance equine reproductive health management.