Evidence of biomechanical and collagen heterogeneity in uterine fibroids

Abstract

Objective: Uterine fibroids (leiomyomas) are common benign tumors of the myometrium but their molecular pathobiology remains elusive. These stiff and often large tumors contain abundant extracellular matrix (ECM), including large amounts of collagen, and can lead to significant morbidities. After observing structural multiformities of uterine fibroids, we aimed to explore this heterogeneity by focusing on collagen and tissue stiffness.

Methods: For 19 fibroids, ranging in size from 3 to 11 centimeters, from eight women we documented gross appearance and evaluated collagen content by Masson trichrome staining. Collagen types were determined in additional samples by serial extraction and gel electrophoresis. Biomechanical stiffness was evaluated by rheometry.

Results: Fibroid slices displayed different gross morphology and some fibroids had characteristics of two or more patterns: classical whorled (n = 8); nodular (n = 9); interweaving trabecular (n = 9); other (n = 1). All examined fibroids contained at least 37% collagen. Tested samples included type I, III, and V collagen of different proportions. Fibroid stiffness was not correlated with the overall collagen content (correlation coefficient 0.22). Neither stiffness nor collagen content was correlated with fibroid size. Stiffness among fibroids ranged from 3028 to 14180 Pa (CV 36.7%; p<0.001, one-way ANOVA). Stiffness within individual fibroids was also not uniform and variability ranged from CV 1.6 to 42.9%.

Conclusions: The observed heterogeneity in structure, collagen content, and stiffness highlights that fibroid regions differ in architectural status. These differences might be associated with variations in local pressure, biomechanical signaling, and altered growth. We conclude the design of all fibroid studies should account for such heterogeneity because samples from different regions have different characteristics. Our understanding of fibroid pathophysiology will greatly increase through the investigation of the complexity of the chemical and biochemical signaling in fibroid development, the correlation of collagen content and mechanical properties in uterine fibroids, and the mechanical forces involved in fibroid development as affected by the various components of the ECM.

Fig 1. Representative photographs of tissue slices showing differences in gross appearance of fibroids.

A: Classical irregular whorled pattern; B, C, D: Patterns of nodules; E, F: Trabecular structures; G: Characteristics of multiple patterns. This example shows a trabecular/nodular pattern; H: Not categorized. This example shows a tightly gyrated pattern. I: Myometrial tissue shown for comparison. Note the seedling fibroid embedded in the tissue (white appearance). Ruler (cm) shown for size.

Fig 2. Representative samples of Masson trichrome-stained fibroid tissues (collagen stained blue-green; muscle cells stained red) examined under digital microscopy (20x).

Samples (approx. 1x1 cm) from 2 different fibroids were chosen representing a high collagen content (A:14–3) and a relatively low collagen content (B:15–2). The circular holes are due to 5 mm punches taken for rheometry before samples were fixed and stained. Collagen was quantified using pixel counts and is denoted underneath each sample.

Fig 3. Stiffness and percent collagen in fibroids.

Columns represent mean tissue stiffness (complex shear modulus [kPa]) in 19 fibroid slices from 8 different subjects. X-axis labels indicate the subject number followed by the fibroid number. Five subjects contributed more than one fibroid to the study. Error bars indicate within-fibroid variability (SD). The pink line represents percent collagen in each fibroid slice as determined by analysis of Masson trichrome staining. The correlation coefficient of stiffness to percent collagen was 0.22.

Fig 4. SDS-PAGE analysis of collagen in a representative fibroid sample.

Lane A: Total collagen extract under non-reducing conditions. Lane B: Total collagen extract under reducing conditions (with TCEP). Lane C: Collagen extract depleted of type V by selective salt precipitation. Lane D: Collagen extract enriched in type V by selective salt precipitation. Sample shown is from 395-E.