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The brain underpinnings remain understudied. Here, we investigated the role of age, puberty and mental health in determining the longitudinal development of sex differences in brain structure. We captured sex differences in limbic and non-limbic structures using machine learning models trained in cross-sectional brain imaging data of 1132 youths, yielding limbic and non-limbic estimates of brain sex. Applied to two independent longitudinal samples (total: 8184 youths), our models revealed pronounced sex differences in brain structure with increasing age. For females, brain sex was sensitive to pubertal development (menarche) over time and, for limbic structures, to mood-related mental health. Our findings highlight the limbic system as a key contributor to the development of sex differences in the brain and the potential of machine learning models for brain sex classification to investigate sex-specific processes relevant to mental health.<\/jats:p>","DOI":"10.1038\/s42003-025-08866-3","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T12:28:51Z","timestamp":1762345731000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Longitudinal development of sex differences in the limbic system is associated with age, puberty and mental health"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1770-1765","authenticated-orcid":false,"given":"Gloria","family":"Matte Bon","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2227-6378","authenticated-orcid":false,"given":"Jonas","family":"Walther","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erika","family":"Comasco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0133-4486","authenticated-orcid":false,"given":"Birgit","family":"Derntl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4003-1018","authenticated-orcid":false,"given":"Tobias","family":"Kaufmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,5]]},"reference":[{"key":"8866_CR1","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1016\/S0140-6736(20)31561-0","volume":"396","author":"F Mauvais-Jarvis","year":"2020","unstructured":"Mauvais-Jarvis, F. et al. 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