Segmentation of Renal Thyroid Follicle Colloid in Common Carp: Insights into Perfluorooctanoic Acid-Induced Morphometric Alterations

Abstract

Perfluorooctanoic acid (PFOA) is a globally prevalent contaminant of concern recognised for its persistence and detrimental effects on both wildlife and humans. While PFOA has been established as a disruptor of thyroid function, limited data exist regarding its impact on thyroid morphology. The kidney of the common carp (Cyprinus carpio) harbours numerous thyroid follicles, rendering it a valuable biomarker organ for investigating PFOA-induced thyroid alterations. Renal tissue slides, stained with the Alcian blue/PAS method, were examined from carp in three experimental groups: unexposed, exposed to 200 ng L^-1, and exposed to 2 mg L^-1 of PFOA over 56 days. Thyroid follicle colloids were segmented, and related morphometric parameters, including perimeter, area, and shape descriptors, were obtained. Statistical analyses revealed significant reductions in thyroid follicle colloid perimeter and area in the 200 ng L^-1 PFOA group compared to the unexposed and 2 mg L^-1 PFOA groups. Additionally, the fish exposed to PFOA exhibited a significantly higher follicle count compared to the unexposed fish. These findings collectively suggest that PFOA induces thyroid folliculogenesis, emphasising its impact on thyroid morphology even at an environmentally relevant concentration (200 ng L^-1).

Keywords: animal model; endocrine disruptors; environmentally relevant concentration; folliculogenesis; goitrogenic effect; image analysis; kidney; one health; shape descriptor.

Figure 1

Paraffin-embedded tissue sections of carp kidneys: unexposed (A), exposed to 200 ng L⁻¹ PFOA (B), and exposed to 2 mg L⁻¹ PFOA (C). Alcian blue–PAS. Scale bar = 25 μm. Thyroid follicles are clearly visible in cross-sections as circular to ellipsoidal structures, varying in number and size according to the exposure group. The PAS positivity of the colloid enhances the segmentation process, delineating the follicular lumen with a yellow line. Particularly evident is the higher number and smaller cross-sectional area of thyroid follicles in the 200 ng L⁻¹ PFOA exposure group (B) compared to the unexposed group (A).

Figure 2

Paraffin-embedded tissue sections of carp kidneys from fish exposed to 200 ng L⁻¹ PFOA. Alcian blue–PAS. Scale bar = 25 μm. Segmentation results before (A) and after (B) fine-tuning of segmentation parameters are depicted. Examples of falsely positively segmented structures, such as plasma in a vessel (arrow) and renal tubule brush border (arrowheads), due to their PAS positivity, are shown in panel (A). These structures were no longer visible after fine-tuning, leaving only correctly classified thyroid follicle colloids outlined by yellow lines in panel (B).

Figure 3

Paraffin-embedded tissue sections of carp kidneys from fish exposed to 2 mg L⁻¹ PFOA. Alcian blue–PAS. Scale bar = 25 μm. Segmentation results before (A) and after (B) fine-tuning of segmentation parameters are depicted. Examples of falsely negatively segmented follicle colloids, attributed to the fusion of contiguous follicles (arrows) and the distortion of segmented forms resulting from the adhesion of contiguous follicles (arrowheads), are illustrated in panel (A). These biases were no longer apparent after fine-tuning, leaving only accurately classified thyroid follicle colloids outlined by yellow lines in panel (B).

Figure 4

Violin and box plot of thyroid follicle colloids’ cross-sectional area (pixel²), perimeter (pixel), circularity, and roundness (dimensionless) according to experimental exposure group (n = number of screened thyroid follicle colloids). Each coloured bullet denotes a single data value for each morphometric parameter/experimental group. Significant differences, where present, are indicated by horizontal whiskers along with the respective p-values.

Figure 5

Violin and box plot of thyroid follicle colloids’ convexity, compactness, and solidity (dimensionless) according to the experimental exposure group (n = number of screened thyroid follicle colloids). Each coloured bullet denotes a single data value for each morphometric parameter/experimental group. Significant differences, where present, are indicated by horizontal whiskers along with the respective p-values.

Figure 6

Violin and box plot of the number of segmented thyroid follicle colloids according to the experimental exposure group (n = number of screened tissue slides). Each coloured bullet denotes a single data value for each experimental group. Significant differences, where present, are indicated by horizontal whiskers along with the respective p-values.