Benchmarking the Impact of Anatomical Segmentation on In Vivo Magnetic Resonance Spectroscopy

Purpose Estimation of metabolite concentrations in brain magnetic resonance spectroscopy (MRS) requires correction for differences in tissue water content, relaxation properties, and the proportions of gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). Accurate knowledge of the relative proportions of these tissue classes within the volume of interest is therefore essential for reliable quantification. Commonly used brain segmentation tools differ in their algorithms, priors, and implementation, potentially introducing variability in MRS-derived concentration estimates. This study investigates the impact of segmentation software on estimated absolute concentrations. Methods Three segmentation software tools, ANTs, FSL, and SPM, were evaluated. Segmentations were applied to an in vivo test-retest MR dataset to assess (1) differences in estimated tissue fractions, and (2) how these differences propagate into tissue-corrected metabolite concentrations. As an additional validity check and biological benchmark of segmentation performance, age-related associations with GM and total creatine (tCr) were examined. Results Significant differences ( p < 0.0001) were observed in tissue fraction estimates between segmentation tools, leading to differences in metabolite concentration estimates of up to 9% under identical acquisition and modeling conditions. Although the strength of the correlation varied between segmentation methods, no statistically significant differences were found. Conclusion The choice of segmentation methodology contributed substantially to variability in MRS “absolute” metabolite concentration estimates. These results underscore the need for transparent segmentation reporting to ensure reproducibility and cross-study comparability in MRS research. Quantifying the segmentation-driven variability allows researchers to contextualize cross-study differences, helping determine whether observed effects are methodological or biologically meaningful.