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Neuroimaging studies have reported differences in brain structure and function between homosexual and heterosexual men. The neural basis for homosexual orientation, however, is still unknown.This study characterized the association of homosexual preference with measures of fractional amplitude of low-frequency fluctuation (fALFF) and functional connectivity (FC) in the resting state.We collected echo planar magnetic resonance imaging data in 26 healthy homosexual men and 26 age-matched heterosexual men in the resting state.Sexual orientation was evaluated using the Kinsey scale. We assessed group differences in fALFF and then, taking the identified group differences as seed regions, we compared groups on measures of FC from those seeds. The behavioral significance of the group differences in fALFF and FC was assessed by examining their associations with the Kinsey scores.Compared with heterosexual participants, homosexual men showed significantly increased fALFF in the right middle frontal gyrus and right anterior cerebellum, and decreased fALFF in the left postcentral gyrus, left lingual gyrus, right pallidum, right postcentral gyrus, left interior parietal gyrus, right superior temporal gyrus, left cuneus, and left inferior frontal gyrus. Additionally, fALFF in the left postcentral gyrus and left cuneus correlated positively with Kinsey scores in the homosexual participants. When the seeds in the left cuneus, left cuneus, and left superior parietal gyrus also had reduced FC in homosexual participants, FC correlated positively with the Kinsey scores.Differences in fALFF and FC suggest male sexual preference may influence the pattern activity in the default mode network. Hu S, Xu D, Peterson BS, Wang Q, Lai J, Hu J, Wei N, Zhang M, and Xu Y. Differing default mode network activities in men with homosexual or heterosexual preferences. J Sex Med 2014;11:2474–2484.