Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

Overview

• This study evaluated an ex vivo hemoperfused model of the equine uterus by analyzing gene and protein expression in the endometrium to confirm its functional stability.
• It also assessed the effectiveness of using the QuPath software for computer-assisted immunohistochemical analysis of equine endometrial tissue.

Background and Purpose

• Traditionally, equine reproductive research has been conducted in vivo, but there is growing interest in in vitro and ex vivo models for more controlled study conditions.
• The ex vivo hemoperfused uterus model allows for perfusion of isolated equine uteri with autologous blood, enabling study of physiological and molecular processes outside the living animal.
• This study aimed to:
• Validate the functional stability of this ex vivo model through molecular markers at both gene (mRNA) and protein levels.
• Determine how perfusion and reproductive cycle stages affect expression of key endometrial markers.
• Evaluate the software QuPath for its suitability in analyzing immunohistochemically stained equine endometrium, comparing computer-assisted results with visual scoring.

Methods

• Sample Collection and Preparation:
• Twelve equine uteri were collected post-slaughter and flushed with preservation solution.
• Uteri were transported on ice to the laboratory and perfused for 6 hours with the horse’s own blood (hemoperfusion).
• Cycle Stage Determination:
• Ovaries were examined for Graafian follicles or corpora lutea.
• Plasma progesterone levels were measured to distinguish estrus (4 horses), diestrus (4 horses), and anestrus (4 horses) stages.
• Sampling Times:
• Samples were taken directly after slaughter, after transport, and at intervals during perfusion (240, 300, 360 minutes).
• Gene Expression Analysis:
• RT-qPCR was used to measure mRNA levels of:
• Progesterone receptor (PGR)
• Estrogen receptor alpha (ESR1)
• Oxytocin receptor (OXTR)
• Marker of cell proliferation (MKI67)
• Marker of apoptosis (CASP3)
• Protein Expression Analysis:
• Immunohistochemical (IHC) staining was performed on endometrial tissue sections for the corresponding proteins.
• Staining was evaluated both visually by a pathologist and using the QuPath software for automated quantification.

Key Findings

• Gene Expression Results:
• PGR mRNA expression was significantly affected by both perfusion time and cycle stage (P < 0.05).
• ESR1 mRNA expression varied depending on the cycle stage but was not influenced by perfusion.
• OXTR mRNA expression remained constant regardless of perfusion or cycle stage.
• MKI67 mRNA, indicative of cell proliferation, decreased after 360 minutes of perfusion compared to pre-perfusion samples.
• CASP3 mRNA, indicative of apoptosis, changed after transport and at the 240-minute perfusion time point.
• Protein Expression and Histology:
• Immunohistochemistry demonstrated that steroid hormone receptors (PGR and ESR1) in stromal and glandular cells were influenced by perfusion.
• Expression of Ki-67 (proliferation marker) and active Caspase 3 (apoptosis marker) proteins was stable and not significantly affected by perfusion.
• OXTR protein was detected throughout all layers of the endometrium and remained stable during perfusion.
• Evaluation of QuPath Software:
• Computer-assisted scoring results were similar to visual evaluation by experts.
• Correlation coefficients between QuPath and visual scores were greater than 0.5 for most cell types and antigens, indicating good agreement.

Conclusions

• The ex vivo hemoperfused equine uterus model maintains molecular and cellular stability of key reproductive markers up to 6 hours of perfusion, supporting its use for functional reproductive studies.
• The cycle stage influences steroid hormone receptor gene expression, but the model remains functionally viable across stages.
• QuPath software is a reliable tool for immunohistochemical evaluation in equine endometrial tissue and can complement or streamline traditional visual assessments.
• This study provides molecular validation for an ex vivo equine uterus model and demonstrates the utility of combining advanced molecular techniques with computer-assisted histopathology analysis.