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A comprehensive evaluation of mitral stenosis (MS) severity commonly utilizes two-dimensional (2D) echocardiography techniques. However, the complex three-dimensional (3D) structure of the mitral valve (MV) poses challenges to accurate measurements of its orifice area by 2D imaging modalities. We aimed to assess MS severity by comparing measurements of the MV orifice area using conventional echocardiography methods to 3D orifice area (3DOA), a novel echocardiographic technique which minimizes geometric assumptions.Routine 2D and 3D intraoperative transesophageal echocardiographic images from 26 adult cardiac surgery patients with at least moderate rheumatic MS were retrospectively reviewed. Measurements of the MV orifice area obtained by pressure half-time (PHT), proximal isovelocity surface area (PISA), continuity equation, and 3D planimetry were compared to those acquired using 3DOA.MV areas derived by PHT, PISA, continuity equation, 3D planimetry, and 3DOA (mean value ± standard deviation) were 1.12 ± 0.27, 1.03 ± 0.27, 1.16 ± 0.35, 0.97 ± 0.25, and 0.76 ± 0.21 cm2, respectively. Areas obtained from the 3DOA method were significantly smaller than areas derived from PHT (mean difference 0.35 cm2, P < .0001), PISA (mean difference: 0.28 cm2, P = .0002), continuity equation (mean difference: 0.43 cm2, P = .0015), and 3D planimetry (mean difference: 0.19 cm2, P < .0001). MV 3DOAs also identified a significantly greater percentage of patients with severe MS (88%) compared to PHT (31%, P = .006), PISA (42%, P = .01), and continuity equation (39%, P = .017) but not in comparison to 3D planimetry (62%, P = .165).Novel measures of the stenotic MV 3DOA in patients with rheumatic heart disease are significantly smaller than calculated values obtained by conventional methods and may be consistent with a higher incidence of severe MS compared to 2D techniques. Further investigation is warranted to determine the clinical relevance of 3D echocardiographic techniques used to measure MV area.