Reproduction in domestic animals = Zuchthygiene2026; 61 Suppl 3; e70279; doi: 10.1111/rda.70279

Layer-Specific Quantitative Assessment of Collagen Distribution in Equine Uteri Using Masson’s Trichrome and Digital Image Analysis: A Comparative Study.

Cerveira-Pinto, M
·
Santos, C
·
Gomes, N
·
Pinho, M
·
Szóstek-Mioduchowska, A
·
Amaral, A

Abstract: Equine endometrial fibrosis is a chronic and progressive equine uterine condition characterized by fibrosis that affects mares' fertility, with a wide range of fertility outcomes being reported for these mares. Therefore, assessing layer-specific collagen (COL) distribution is essential for a comprehensive understanding of endometrial remodelling and its impact on equine reproductive potential. Full-thickness uterine samples, including stratum functionalis (SF), stratum basalis (SB) and myometrium, were collected post-mortem from healthy control mares (Category I; n = 10) and those with moderate endometrial fibrosis (Category IIB; n = 10). Samples were analysed using Masson's trichrome and digital imaging. Significant differences between categories were identified exclusively within the superficial SF, where category IIB displayed higher COL area fraction (%Area) and Normalized Integrated Density than healthy controls. When evaluating variations across layers, both groups exhibited a higher %Area and Normalized Integrated Density in the SB compared to the SF, followed by a significant decrease in Normalized Integrated Density towards the myometrium. Furthermore, the Mean Grey Value progressively decreased into the deeper compartments, indicating higher staining intensity, only in the group with moderate fibrosis. In conclusion, layer-dependent COL deposition underscores the value of multiparametric, quantitative approaches for an objective characterization of equine endometrial fibrosis.

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Publication Date: 2026-10-01 PubMed ID: 42816917DOI: 10.1111/rda.70279Google Scholar: Lookup

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Journal Article
Comparative Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This study quantitatively assessed collagen distribution in the uterine tissue layers of horses, comparing healthy mares to those with moderate endometrial fibrosis using histological staining and digital image analysis.
It identified specific differences in collagen accumulation within uterine layers that may impact fertility and highlighted the importance of detailed, layer-specific evaluation for understanding uterine fibrosis.

Background and Importance

Equine endometrial fibrosis involves excessive collagen accumulation in the uterus, which is a chronic condition that negatively affects reproductive function in mares.
Fibrosis can lead to variable fertility outcomes, and current diagnostic methods do not fully explain how fibrosis in different uterine layers may impact fertility.
Understanding collagen distribution in specific uterine layers (stratum functionalis, stratum basalis, and myometrium) is essential for a nuanced understanding of endometrial remodeling and its effects on reproductive potential.

Methodology

Collection: Full-thickness uterine samples were taken post-mortem from:
- Healthy control mares (Category I; n = 10)
- Mares with moderate endometrial fibrosis (Category IIB; n = 10)
Histology: Samples were stained using Masson’s Trichrome, a method that colors collagen to allow visualization and assessment of fibrotic tissue.
Quantitative Analysis: Digital image analysis was used to objectively assess and quantify collagen distribution in the different uterine layers by measuring:
- Collagen area fraction (%Area) – proportion of collagen within the tissue area
- Normalized Integrated Density – an indicator of the total intensity of collagen staining
- Mean Grey Value – reflects staining intensity, with lower values indicating stronger staining (more collagen)

Key Findings

Significant collagen differences between healthy and fibrotic categories were only observed in the superficial stratum functionalis (SF) layer:
- Category IIB (fibrotic) mares showed higher collagen area fraction and normalized integrated density than controls.
In both healthy and fibrotic mares, collagen distribution across layers showed:
- Higher collagen area and density in the stratum basalis (SB) compared to the superficial stratum functionalis (SF)
- A decrease in normalized integrated collagen density moving deeper into the myometrium (muscle layer)
Only the fibrotic group showed a progressive decrease in Mean Grey Value (indicating increasing collagen staining intensity) deeper into the uterine layers, implying more intense collagen deposition as depth increased.

Interpretation and Conclusion

The study identified a layer-dependent pattern of collagen deposition in equine uteri, with distinct changes occurring primarily in the superficial functionalis layer in fibrotic mares.
The increased collagen in specific layers likely plays a role in impaired fertility associated with endometrial fibrosis.
The findings highlight the importance of a multiparametric and quantitative approach (combining staining with digital image metrics) for an accurate, objective assessment of uterine fibrosis in horses.
This layer-specific evaluation can improve diagnosis, understanding, and potentially management of endometrial fibrosis to better predict and influence fertility outcomes in mares.

Cite This Article

APA

Cerveira-Pinto M, Santos C, Gomes N, Pinho M, Szóstek-Mioduchowska A, Amaral A. (2026). Layer-Specific Quantitative Assessment of Collagen Distribution in Equine Uteri Using Masson’s Trichrome and Digital Image Analysis: A Comparative Study. Reprod Domest Anim, 61 Suppl 3, e70279. https://doi.org/10.1111/rda.70279

Publication

Reproduction in domestic animals = Zuchthygiene

ISSN: 1439-0531

NlmUniqueID: 9015668

Country: Germany

Language: English

Volume: 61 Suppl 3

Pages: e70279

Researcher Affiliations

Cerveira-Pinto, Marta

CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal.
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Lisbon, Portugal.

Santos, Carina

Unidade Local de Saúde de São José-Serviço de Anatomia Patológica, Lisbon, Portugal.

Gomes, Nicole

CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal.
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Lisbon, Portugal.

Pinho, Mário

CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal.
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Lisbon, Portugal.

Szóstek-Mioduchowska, Anna

InLife Institute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, Poland.

Amaral, Ana

CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal.
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Lisbon, Portugal.
Comprehensive Health Research Centre (CHRC), Évora, Portugal.

MeSH Terms

Animals
Female
Horses
Collagen / metabolism
Collagen / analysis
Uterus / pathology
Image Processing, Computer-Assisted
Fibrosis / veterinary
Fibrosis / pathology
Eosine Yellowish-(YS)
Horse Diseases / pathology
Myometrium / pathology
Methyl Green
Endometrium / pathology
Uterine Diseases / veterinary
Uterine Diseases / pathology
Azo Compounds

Grant Funding

UID/00276/2025 / Fundação para a Ciência e a Tecnologia
LA/P/0059/2020 / Fundação para a Ciência e a Tecnologia
2024.13105.PEX / Fundação para a Ciência e a Tecnologia
2023.04679.BD / Fundação para a Ciência e a Tecnologia

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