Differences between stenotic bicuspid and tricuspid aortic valves: a histological comparison
Authors: Wu H W , El Ouraoui M , Hjortnaes J , Palmen M , Klautz R J M , Jukema J W , Ajmone Marsan N 
Published: October 2024

Abstract

Background
Aortic valve stenosis is a narrowing of the aortic valve opening due to leaflet calcification and thickening. Thickening of the leaflets involves the original leaflet but also the presence of a superimposed tissue (SIT), defined as an extra layer of tissue on top of the original leaflet which has been shown to form mainly in response to mechanical stress. Patients with bicuspid aortic valve (BAV) are known to be at higher risk of developing aortic stenosis and at an early age as compared to patients with tricuspid aortic valves (TAV). However to date, no systematic histo-morphometric comparison has been made between BAV and TAV in order to unravel potential difference in the underlying mechanisms of valve degeneration.

Purpose
Aim of this study was to compare the extent and distribution of calcifications and SIT between stenotic BAV and TAV.

Methods
BAV (n=14) and TAV (n=12) samples were collected from patients undergoing aortic valve replacement due to aortic stenosis. Of each patient, at least one leaflet was used for analyses, for a total of 27 BAV leaflets and 17 TAV leaflets. Samples were fixed in 4% paraformaldehyde solution, and sectioned in the midline of the leaflet. In case of a leaflet with raphe, the leaflet was sectioned in the midline between the raphe and the commissure side of the leaflet. Sections were stained for elastin fibers using Weighert’s Resorcin Fuchsin. Thickness of the leaflets was calculated by dividing the cross sectional area by the length of the leaflet. Calcifications were visualized using Alizarin Red staining and quantified as a percentage of the total cross sectional area of one section. SIT was defined as an extra layer of tissue outside the elastin lamina (Figure).

Results
BAV samples showed in average thicker leaflets as compared to TAV (1.92 vs 1.31 mm, p &lt;0.001). Although the thickness of the original leaflet was the same between the two groups, SIT formation was significantly thicker in the BAV as compared to TAV (0.67 vs 0.30 mm, p &lt;0.001). This difference was mostly due to a significant thicker SIT in the free edge of BAV samples as compared to TAV (0.78 vs 0.39 mm, p=0.003), while the SIT at the level of the leaflet body was comparable between the two groups. The percentage of leaflet calcification was similar between BAV and TAV (23% vs 20% respectively, p=0.669). Interestingly, while calcification in TAV samples was observed only on the aortic side, in BAV samples calcification was also observed on the ventricular side (n=5; 36% Figure).

Conclusions
BAV showed thicker leaflets as compared to TAV due to a larger contribution of SIT formation particularly at the free edge. Extent of calcification was similar between BAV and TAV but calcification at the ventricular side of the leaflets was found only in BAV samples. These observations suggest significant differences in the mechanisms underlying BAV and TAV pathology possibly in relation to distinct mechanical stress.