The present VBM study investigated GMV and WMV patterns in two TW groups that were well-characterized regarding their sexual orientation and non-conformity to their sex assigned at birth from an early age. The TNTW and TTW subjects were recruited before beginning CHT and at least one year after beginning CHT, respectively, which allowed us to differentiate brain volume findings related to TW or CHT.

The lower total brain volume in CW observed here is consistent with a previously reported profile of sexual differences in the brain10. The finding that the three other groups presented total brain volumes larger than the CW group indicates that these brain volume differences are congruent with the sex assigned at birth in both TW groups27. Additionally, no differences in total brain volume were found between the TTW group and the TNTW and CM groups in the present cross-sectional study, although overall effects of CHT on reductions in total intracranial volume (TIV) and total GMV have been observed in other longitudinal MRI studies of TW undergoing CHT30,31.

Regarding regional GMV, we found lower GMV bilaterally in the insula in both TW groups, in contrasts to the results reported by Savic and Arver (2011), who found higher GMV in the right insula of the TW group relative to controls20. Likewise, Zubiarre-Elorza et al. (2013) verified greater cortical thickness in the right insula in TNTW than in CM controls23. Another MRI study conducted by Zubiarre-Elorza et al. (2014) reported decreased right insular volumes in TW undergoing CHT31. Our finding in the insula is unlikely directly due to CHT because the lower GMV of the insula was observed for both TW groups compared with the CW group. Furthermore, brain volume and cortical thickness are widely considered as complementary brain measurements32,33.

Manzouri et al.34 recently conducted a morphological and functional MRI study including treatment-naïve transgender men - female sex assigned at birth - and cisgender controls. The GMV differences found in the transgender men group followed patterns related to the sex assigned at birth. However, the authors also demonstrated weaker functional connections from the pregenual anterior cingulate to the insular cortex and to the temporo-parietal junction in the transgender men than in controls. These findings also suggest that transgender men differ from cisgender people with respect to the own-body image neural network, which may be a neurobiological substrate related to transgender men34. Similarly, Savic and Arver (2011) and Simon et al. (2013) suggested that regional GMV differences detected in transgender people emphasize the brain regions related to the body perception network20,21.

It is worth mentioning that an important diagnostic criterion for gender dysphoria is the distress that accompanies the incongruity between the body and gender identity, as the secondary sexual characteristics do not belong to the gender with which one identifies35. Decreased insular volumes have been reported in depressed subjects with melancholic symptoms36, and these changes in insular and prefrontal cortical volumes may be specifically associated with the manifestation of psychotic symptoms in major depressive disorder37. The insula is related to all subjective sensations and is the possible foundation of interoception38, including body awareness39. Furthermore, the insula connects the distinct functional systems involved in processing emotions, sensory-motor skills and cognition40. Two studies have evaluated functional connectivity in transgender people and found (i) a pattern of neural connectivity that inferred suffering due to incongruity between sex assigned at birth and gender identity41, and (ii) that the connectivity between the right insula and the somatosensory cortex was negatively related to ratings on a well-being scale in regard to gender dysphoria42. Another study examined brain structural connectivity and observed unique differences in regional network efficiency in the insular area in trans people43. These observations, together with our findings of reduced GMVs in the insula in both hemispheres in two independent TW groups, suggest that such regional GMV differences could be characteristics associated with TW. Furthermore, these alterations in the insula could be related to the neural network of body perception and reflect the distress that accompanies gender dysphoria. The hypothesis that insular volume variations in TW individuals reflect the distress of these individuals should be considered in future studies including large TW samples by evaluating significant correlations between GMV and symptom severity ratings. In addition, it is well known that the symptoms of gender dysphoria improve with CHT35. As a consequence of symptom relief, the TTW group could present a greater insular volume than the TNTW group. However, this possibility was not verified in the current study, which suggests that GMV differences in the insular cortex in both TW groups may be related to a more general “transgender trait”.

One important difference between our VBM study and the study performed by Savic and Arver (2011) is that the latter investigated non-androphilic TW (sexually attracted to females, both males and females, or neither gender)20 according to the Blanchard classification, which is based on the sex assigned at birth of TW44, while our study investigated androphilic TW (sexually attracted to males). Therefore, one might speculate that the differences observed in the insular cortex could be linked to the sexual orientation of TW. It should be noted, however, that sexual orientation and gender identity are distinct phenomena. For this reason, overlapping VBM findings associated with these two conditions are highly speculative. Another VBM study assessing hetero- and homosexual CM did not identify any regional GMV differences between groups. Homosexual CW presented lower GMV in the perirhinal cortex than heterosexual CW45. Considering these speculative findings, further MRI studies comparing androphilic TW with non-androphilic TW are required to reveal further information on this topic.

We also detected lower regional GMV in a large portion of the posterior-superior frontal cortex in the CW controls than in both TW groups and the CM controls. Because our statistical approach was primarily designed to identify regional GMV variations that could be related to gender dysphoria, direct post hoc two-group comparisons between the cisgender controls in our study should not be considered as comprehensive indicators of a predominant profile between the two control groups regarding regional brain volume differences. In addition, the lower GMV detected in the posterior-superior frontal cortex in the CW group is consistent with previously described regional patterns of sexual differences of the human brain10,46. Significantly higher GMV in the posterior-superior frontal cortex was also detected only in the TTW and CM groups relative to the CW group. Conversely, the difference in regional GMV in this frontal region in the TNTW group relative to the CW group was not significant after correcting for multiple comparisons. These results of the present study may indicate that this brain region in TNTW tends to present female characteristics, although this conclusion should be interpreted with caution.

Based on the abovementioned interpretation, the finding of higher GMV in the posterior-superior frontal cortex in the TTW group might seem contradictory, given the overall feminizing effects of CHT. Nevertheless, previous studies have shown increased GMV in several cortical regions in postmenopausal women under oestrogen replacement therapy47, including higher GM density in paracentral and precentral areas48 and in the superior frontal gyrus46. Such findings are believed to reflect the influence of hormonal treatment on brain neuroplasticity.

Overall, our results suggest that the impact of CHT on brain volume may vary across regional and global levels, as we found no difference in total brain volume between the two TW groups, despite the fact that the TTW had undergone prolonged use of sex steroids. A similar study conducted by Hoekzema et al. (2014) revealed that the neuroanatomical characteristics of treatment-naïve transgender adolescents and transgender adolescents undergoing CHT corresponded to those of individuals of the same sex assigned at birth.

Such results are in contrast to the findings of previous studies of TW using repeated MRI measurements before and during CHT. Hulshoff Pol et al. (2006) reported reduced global brain volumes in TW after four months of CHT towards the volumes expected for CW30. Similar to our TW sample, Zubiaurre-Elorza et al. (2014) investigated TW after at least six months of hormonal treatment and reported reduced cortical thicknesses and volumes of subcortical structures in the right hemisphere (specifically in the pallidum and the thalamus) and an enlarged ventricular system31. A recent study showed increased ventricle and reduced right hippocampal volumes in TW after four months of CHT49. These differences between our results and those of previous investigations may be because the present study employed a cross-sectional design, while the previous studies were longitudinal investigations of the same individuals and, thus, may have been more sensitive to detecting CHT-related global volume changes over time in TW.

Our findings, as well as the results of all previous brain imaging studies that have evaluated the influence of CHT, suggest that the effects of CHT on brain volume may differ at global and regional levels and may vary across brain structures. Moreover, the different results obtained for the two TW groups in our study suggest that the association of brain volumes with gender dysphoria and CHT may be complex, and furthermore, the direction of associations of brain volume with these two dimensions may vary.

Conflicting VBM-based findings among MRI studies of TW regarding subcortical GM structures have been reported to date. In the first VBM study to evaluate TW, Luders et al. (2009) identified a greater GMV in the right putamen19. Through SVC-based analyses, Savic and Arver (2011) also reported that TW subjects exhibit a greater GMV in the right putamen than control subjects; conversely, ROI-based measurements indicated reduced volumes of the putamen in TW relative to controls20. In the present study, the SVC-based analysis did not reveal significant group differences in voxels located specifically in the basal ganglia (although relatively larger clusters of lower GMV in the insula extending towards the right putamen were observed in both TW groups, as shown in Fig. 1). Finally, the paucity of between-group differences in the WM analyses in our study suggests that brain volume differences related to the effects of both TW and CHT predominate in the GM compartment of the brain.

Some methodological limitations of our study must be considered. First, because this study employed a cross-sectional design, it was not possible to determine which changes were pre-existing and which resulted from CHT. Second, our sample size can be considered small (n = 80) and may not confer confidence in the results obtained, although the sample size was similar to those in previous MRI studies in this field20,23,43. Third, variability existed in the duration of CHT among individuals in the TTW group. Finally, the stage of the menstrual cycle of the CW was not considered at the time of MRI scanning, which may be a relevant factor considering that brain structures are influenced by hormonal physiological variations50.

In conclusion, significant regional brain volume differences in this VBM study were detected in TW compared with cisgender controls. In TTW, the observed brain volume changes suggested a possible influence of CHT on brain neuroplasticity46,47. Finally, we present a novel finding of GMV alterations in the insula in the two independent TW groups, which may be a characteristic of TW. These alterations in the insula could be related to the neural network of body perception and may reflect the distress that accompanies gender dysphoria.