Bacterial endometritis-induced changes in the endometrial proteome in mares: Potential uterine biomarker for bacterial endometritis.

This study compared proteins in uterine lavage fluid from healthy mares and mares with bacterial endometritis to uncover disease-related biological changes and diagnostic biomarkers. It found 38 differentially abundant proteins and a 24-protein biomarker panel pointing to metabolic reprogramming, oxidative stress control, and robust innate immune activation (including NETs and antimicrobial peptides).

Why this research matters

• Equine endometritis is a leading cause of subfertility; objective, mechanism-based diagnostics are limited, and treatments rely heavily on antibiotics.
• Mapping proteome changes in the uterine environment can reveal disease mechanisms and yield measurable biomarkers for faster diagnosis and targeted therapies.

Study design and cohort

• Subjects: 28 mares total; 14 healthy (negative cytology and culture; no clinical signs) and 14 with bacterial endometritis (positive cytology and culture; clinical signs).
• Sample type: Uterine lavage fluid (secretome), which reflects locally secreted and shed proteins from endometrial and infiltrating immune cells.
• Case definition: Standard clinical criteria integrating cytology and culture increased confidence that “endometritis” cases represent active bacterial inflammation.

Proteomics and bioinformatics workflow

• Analytical platform: UHPLC–MS/MS for deep, quantitative proteome profiling of lavage fluid proteins.
• Data analysis: Qlucore Omics Explorer for statistics; multiple-testing adjusted significance (q-value).
• Functional interpretation: Gene Ontology enrichment and pathway analysis using PANTHER and KEGG to contextualize differentially abundant proteins.

Key quantitative results

• Differential abundance: 38 proteins differed between endometritis and healthy mares (fold change > 4.25; q = 0.002), indicating robust shifts in the uterine proteome during infection.
• Biomarker discovery: 24-protein candidate panel identified via a biomarker workbench algorithm, designed to distinguish bacterial endometritis from health.

Biological themes uncovered

• Energy metabolism and REDOX regulation: Upregulated proteins in endometritis mapped to pathways for energy generation and oxidative stress control.
• Innate immune activation: Enrichment of defense response to bacteria, consistent with neutrophil- and macrophage-driven inflammation in the endometrium.
• Metabolic reprogramming (Warburg-like shift): Evidence that endometrial innate immune cells adopt activation-associated metabolic changes akin to aerobic glycolysis, a hallmark of rapid antimicrobial effector responses.

Innate immune effectors among the biomarkers

• NET formation components: Histone H2A and H2B isoforms consistent with neutrophil extracellular trap release during bacterial defense.
• Complement pathway: Complement C3a elevation aligns with opsonization, chemotaxis, and amplification of inflammatory signaling.
• Cytoskeleton remodeling: Gelsolin and profilin, actin-binding proteins essential for actin turnover, cell polarization, and migration of recruited leukocytes.
• Antimicrobial arsenal:
  • Cathelicidin family: Equine cathelicidin 2 and two uncharacterized proteins with high homology to cathelicidins, suggesting expanded peptide defenses.
  • Myeloperoxidase (MPO): Neutrophil enzyme generating hypohalous acids, key for bacterial killing.
  • Lysozyme/“lysosome”: Reported as “lysosome” in the abstract, likely lysozyme—an established antimicrobial enzyme that hydrolyzes bacterial peptidoglycan.

Pathway-level interpretation

• GO/PANTHER/KEGG analyses converged on:
  • Defense response to bacterium and innate immune signaling cascades.
  • Metabolic pathways supporting rapid immune effector function (e.g., glycolysis-linked energy flux and antioxidant systems to buffer respiratory burst).
  • Cytoskeletal dynamics enabling leukocyte trafficking within inflamed endometrium.
• These coordinated changes are consistent with an endometrium actively combating bacterial invasion via neutrophil-driven mechanisms.

Clinical and translational implications

• Diagnostics: A multianalyte protein panel in uterine lavage fluid could complement cytology and culture, potentially enabling earlier, more specific detection of bacterial endometritis.
• Prognosis and monitoring: Longitudinal tracking of select markers (e.g., MPO, cathelicidins, C3a) might reflect treatment response or risk of recurrence.
• Therapeutics: Identification of endogenous antimicrobial peptides and immune pathways opens avenues for non-antibiotic interventions (e.g., peptide therapeutics, immune modulation).

Strengths and limitations

• Strengths:
  • Stringent quantitative thresholds (large fold-change, low q-value) yielded a focused set of robust differences.
  • Integration of unbiased proteomics with pathway analysis strengthened biological interpretation.
  • Sampling the uterine secretome targets disease-relevant proteins at the site of pathology.
• Limitations:
  • Sample size (n=28) warrants external validation and assessment across breeds, ages, and reproductive stages.
  • Cross-sectional design precludes causal inference and kinetic understanding of biomarker dynamics.
  • Potential confounders (estrous cycle phase, prior treatments) are not detailed in the abstract and may influence protein profiles.
  • Biomarker performance metrics (e.g., sensitivity/specificity) are not provided; clinical utility remains to be quantified.

Future directions

• Validate the 24-protein panel in larger, independent cohorts with standardized sampling across cycle stages.
• Develop targeted assays (e.g., ELISA or MRM) for a pared-down subset of the most discriminative markers.
• Assess diagnostic accuracy versus gold standards and evaluate incremental value over cytology/culture.
• Explore functional roles of the uncharacterized cathelicidin-like proteins and their therapeutic potential.
• Longitudinal studies to map biomarker kinetics during infection, treatment, and recovery.

Key takeaways

• Bacterial endometritis in mares is associated with profound shifts in the uterine secretome, highlighting innate immune activation, metabolic reprogramming, and oxidative stress control.
• A 24-protein candidate biomarker panel—including NET components, complement C3a, actin regulators, and antimicrobial peptides—offers a promising foundation for improved diagnostics and non-antibiotic therapeutic strategies.