Evaluation of IGF1 and MKI67 mRNA expression in relation to histopathological features of equine endometrosis

This study demonstrates that in equine endometrosis, increased TGFB1 transcription and reduced MKI67 expression are associated with early fibrotic lesions and epithelial damage, respectively, while IGF1 expression inversely correlates with the severity of histopathological changes and inflammatory infiltration.

The Gist

Imagine a mare's uterus as a highly sophisticated, self-repairing garden. In a healthy garden, the soil is soft, the flower beds (glands) are lush, and the fence (the lining) is intact. When the garden gets damaged, it has a natural ability to heal itself, regrow plants, and fix the fence.

Equine Endometrosis is like a garden that has stopped healing properly and instead is slowly turning into a concrete parking lot. The soft soil gets replaced by hard, fibrous concrete (scarring), the flower beds get crushed or disappear, and the fence gets torn. This "concretization" is the main reason many mares struggle to get pregnant.

This study was like sending a team of molecular detectives into these gardens to see what the "construction workers" (genes) were saying while the damage was happening. They specifically looked at three key workers:

1. The "Growth Manager" (IGF1): This worker usually tells cells to grow, survive, and repair the garden.
2. The "Construction Crew Chief" (MKI67): This worker counts how many cells are actively dividing and building new tissue.
3. The "Scar Maker" (TGFB1): This worker is responsible for laying down the hard, fibrous concrete (scarring).

Here is what the detectives found, translated into everyday terms:

1. The "Growth Manager" is on Strike

The researchers found that as the garden got more damaged (more "concrete" and more inflammation), the Growth Manager (IGF1) was shouting less and less.

• The Analogy: Imagine a construction site where the foreman stops giving orders to fix the potholes. The more broken the site gets, the quieter the foreman becomes.
• The Finding: In mares with severe scarring and heavy inflammation (like a garden overrun by weeds and rocks), the gene for IGF1 was very quiet. This suggests the uterus is losing its ability to say, "Hey, let's repair this!"

2. The "Construction Crew" Stops Working When the Fence Breaks

They looked at the Construction Crew Chief (MKI67), who tracks cell division.

• The Analogy: If the garden fence is torn down (erosion), the construction crew stops showing up to build new plants.
• The Finding: In samples where the inner lining of the uterus was torn or eroded, the MKI67 gene was much lower. It seems that when the physical barrier is broken, the cells stop trying to multiply and repair.

3. The "Scar Maker" is Loud Early On

The Scar Maker (TGFB1) is the one who creates the fibrosis (the bad scarring).

• The Analogy: You might think the scar-maker is only loud when the garden is already a parking lot. But the study found this worker was actually shouting the loudest in the early stages of damage (Category I+), even before the whole garden was ruined.
• The Finding: The gene for TGFB1 was higher in mares with very early, subtle signs of scarring compared to healthy ones. This suggests the "concretization" process starts very early, perhaps even before we can see it with a microscope.

4. The Connection Between Hormones and Construction

They also found a strong link between the Construction Crew (MKI67) and the Hormone Receptors (ESR1).

• The Analogy: The construction crew only shows up when the "Sun" (Estrogen) is shining. If the hormone signals are off, the crew doesn't work.
• The Finding: The genes for cell division and estrogen receptors moved in perfect lockstep. This confirms that the uterus's ability to repair itself is tightly tied to the mare's hormonal cycle.

The Big Picture

Think of the uterus as a house that needs constant maintenance.

• Healthy House: The maintenance crew (IGF1) is active, the builders (MKI67) are working, and the walls are solid.
• Damaged House (Endometrosis): The maintenance crew goes silent (low IGF1), the builders stop working when the walls crack (low MKI67), and the "concrete crew" (TGFB1) starts pouring hardening cement too early.

Why does this matter?
This study tells us that infertility in mares isn't just about "scars." It's about the loss of the ability to repair. The uterus stops trying to fix itself (low growth factors) and starts turning into rigid scar tissue (high fibrosis signals) very early in the disease process.

The Takeaway: To help mares get pregnant, we might need to find a way to wake up the "Growth Manager" (IGF1) and stop the "Scar Maker" (TGFB1) from taking over the construction site too soon.

Technical Summary

1. Problem Statement

Equine endometrosis is a primary cause of subfertility in mares, characterized by progressive fibrotic remodeling of the endometrial stroma and glandular alterations ("fibrotic nests"). While the disease is routinely graded using the modified Kenney and Doig (KD) histopathological system, the molecular mechanisms linking specific histological features to local transcriptional changes remain incompletely defined.

Existing research has heavily focused on profibrotic pathways (e.g., TGF-β1 signaling). However, there is a gap in understanding how markers of tissue maintenance and cellular proliferation—specifically Insulin-like Growth Factor 1 (IGF1) and Ki-67 (MKI67)—vary with disease severity and specific histopathological lesions. The study aims to determine if the progression of endometrosis is accompanied by a reduction in regenerative potential and altered expression of growth factors and proliferation markers.

2. Methodology

Study Design and Sample Collection:

• Source: Post-mortem endometrial tissue samples were collected from 72 mares of various breeds at a commercial abattoir in Poland (August 2023).
• Selection: From the initial 72 samples, a subset of 47 samples was selected for RT-qPCR analysis. Selection criteria aimed to represent the spectrum of endometrosis while excluding samples with predominant active inflammation (endometritis) or technical artifacts.
• Grouping: Samples were categorized into five analytical groups based on histology:
  • KD I: Control (normal, no endometrosis/inflammation).
  • I+: Early/minor endometrosis (focal remodeling not meeting full KD IIA criteria).
  • KD IIA, IIB, III: Progressive stages of endometrosis.

Histopathological Assessment:

• Standard Grading: Samples were graded using the Kenney and Doig (KD) system.
• Feature-Based Assessment: A standardized panel of 15 additional histopathological features was evaluated on H&E-stained sections. These included:
  • Glandular degeneration and periglandular fibrotic foci.
  • Basal lamina disruption.
  • Luminal epithelial erosion and degeneration.
  • Inflammatory infiltration intensity (overall, lymphocytic, neutrophilic, macrophage, eosinophilic).
  • Vascular changes (dilation, perivascular fibrosis).
• Blinding: Two independent observers performed scoring blinded to molecular results.

Molecular Analysis (RT-qPCR):

• Target Transcripts: mRNA expression was quantified for:
  • Primary Targets: IGF1 (tissue maintenance), MKI67 (proliferation).
  • Secondary Targets: TGFB1 (fibrosis), ACTA2 (myofibroblast marker), ESR1 (Estrogen Receptor), PGR (Progesterone Receptor).
• Normalization: Relative expression was calculated using the $2^{-\Delta\Delta Ct}$ method, normalized against the geometric mean of reference genes SDHA and GAPDH.
• Statistical Analysis: Non-parametric tests (Kruskal-Wallis, Mann-Whitney U) were used for group comparisons, and Spearman's rank correlation ($\rho$) was used for associations between transcripts and histological scores.

3. Key Contributions

• Novel Molecular Markers: This is one of the first studies to specifically evaluate IGF1 and MKI67 mRNA expression in relation to the detailed histopathological spectrum of equine endometrosis.
• Refined Phenotyping: The study introduces and validates an "I+" category to capture early, focal endometrosis lesions that are often missed by the standard KD system, revealing distinct molecular signatures in these early stages.
• Feature-Specific Correlations: Instead of only correlating gene expression with broad KD categories, the study links specific molecular changes to precise histological features (e.g., basal lamina disruption, epithelial erosion).

4. Key Results

Gene Expression by KD Category:

• TGFB1: Expression was significantly higher in the I+ (early) group compared to both KD I (control) and KD III (severe) groups ($p = 0.041$). This suggests a peak in profibrotic signaling during the initiation of lesions.
• IGF1, MKI67, ACTA2, ESR1, PGR: No significant differences were found when comparing expression levels across the broad KD categories (I, I+, IIA, IIB, III) alone.

Gene Expression vs. Specific Histological Features:

• MKI67 (Proliferation): Significantly lower in samples with luminal epithelial erosion ($p = 0.049$).
• TGFB1: Significantly higher in samples with glandular basal lamina disruption ($p = 0.020$).
• ACTA2: Significantly higher in samples with luminal epithelial degeneration ($p = 0.044$).

Correlation Analysis:

• IGF1: Showed significant negative correlations with:
  • KD Category severity ($\rho = -0.401$).
  • Glandular degeneration ($\rho = -0.340$).
  • Overall inflammatory infiltration ($\rho = -0.387$).
  • Lymphocytic ($\rho = -0.426$) and Neutrophilic ($\rho = -0.448$) infiltration.
  • Interpretation: As disease severity and inflammation increase, the expression of this tissue maintenance factor decreases.
• MKI67: Showed a very strong positive correlation with ESR1 ($\rho = 0.887$) and moderate positive correlations with ACTA2 and PGR. This confirms the hormonally regulated nature of endometrial proliferation.
• No Correlation: IGF1 did not correlate significantly with MKI67, TGFB1, or ACTA2.

5. Significance and Conclusion

Biological Implications:

• Loss of Regenerative Capacity: The inverse relationship between IGF1 expression and disease severity/inflammation suggests that progressive endometrosis is characterized by a failure in tissue maintenance and repair mechanisms. The decline in IGF1 may contribute to the irreversibility of advanced fibrosis.
• Early Profibrotic Signaling: The elevated TGFB1 in the "I+" group indicates that profibrotic pathways are activated very early in the disease process, potentially before extensive fibrosis is visible under standard grading.
• Epithelial Damage and Proliferation: The reduction in MKI67 associated with epithelial erosion suggests that structural damage to the luminal lining is accompanied by a local suppression of cellular cycling, hindering recovery.

Clinical Relevance:

• The findings support the hypothesis that endometrosis is not just a fibrotic process but also a failure of regenerative homeostasis.
• The "I+" category definition offers a more sensitive tool for early detection and molecular study of the disease onset.
• Therapeutic strategies might need to focus not only on inhibiting fibrosis (TGF-β1) but also on restoring tissue maintenance pathways (IGF-1) and epithelial integrity to improve fertility outcomes.

Limitations:

• Cross-sectional design (no temporal progression data).
• Bulk tissue analysis (cannot distinguish cell-type specific expression).
• Lack of protein-level validation (mRNA does not always equate to functional protein activity).
• Cycle phase assigned via gross morphology rather than endocrine profiling.