Effects of testosterone treatment on hypothalamic neuroplasticity in female-to-male transgender individuals

As control center of the neuroendocrine system, the hypothalamus is the main locus of action of sex hormones in triggering sexual motivation, cognition, and behavior. During perinatal brain development, sex hormones determine the adult hypothalamic neural circuitry into a male pattern or a female pattern (Arnold 2009). Moreover, there is growing appreciation that sex hormones also shape hypothalamic and other subcortical brain structures in adolescence (McCarthy et al. 2009; Campbell and Herbison 2014; Ahmed et al. 2008). In adulthood, these “organizational” effects are then replaced by “activational”, plastic and reversible actions of sex hormones. These include not only feedback actions on gonadotropin releasing hormone (GnRH) neuron firing and GnRH release (Mizuno and Terasawa 2005; Kenealy et al. 2013), but also influence ventromedial nucleus neuron firing involved in the control of glucose and lipid metabolism (Xu et al. 2011), as well as preoptic neuron firing involved in the regulation of body temperature (Silva and Boulant 1986). Estrogen reversibly modulates dendritic spine density, length, and terminal branch number in the ventromedial nucleus of the rat (Madeira et al. 2001). Structural neuroimaging in humans further indicates state-dependent changes in hypothalamus volume across the menstrual cycle (Tu et al. 2013).

Hypothalamic microstructure and function can be investigated using diffusion-weighted imaging (DWI) in humans. In one study, authors observed increased diffusion values in obese compared to non-obese participants in hypothalamus and other subcortical regions (Alkan et al. 2008). Another study compared hypothalamic diffusion parameters in fasted versus fed individuals. Mice as well as humans showed decreased diffusion in the fasted state, which may suggest a cellular swelling response associated with hypothalamic activation (Lizarbe et al. 2013). Indeed, recent DWI investigations indicate a potential link between water diffusion changes and neural activation via accompanying cell swelling (Le Bihan and Iima 2015; Le Bihan et al. 2006). Furthermore, diffusion changes have been associated with astrocyte swelling and used to study short-term gray matter adaptations after learning in the hippocampus (Sagi et al. 2012). However, to our knowledge, only one study so far has probed DWI to quantify sex hormone-induced neuroplastic adaptations in the hypothalamus in humans. Baroncini et al. (2010) investigated the effects of an artificial menstrual cycle on hypothalamic microstructure and observed decreased water diffusion upon inhibition of HPG-axis. Therefore, we aimed to further investigate a potential influence of sex hormones on hypothalamic microstructure using DWI.

The investigation of female-to-male transgender individuals (FtMs) seeking for sex hormone replacement therapy provided us the unique opportunity to accomplish this endeavor in a causal way. Hormone replacement therapy in FtMs consists mainly of high dosages of testosterone to adjust the physical appearance to the desired gender. Masculinization including the induction of body and facial hair growth and lowering of voice pitch is evident within 4 months and continues to develop beyond 1 year (Levy et al. 2003). Therefore, we explored the possibility that hypothalamic water diffusion would significantly change within 4 months of hormone intake.

Here, we show MD reductions in the hypothalamus from female towards male proportions in FtMs undergoing hormone replacement therapy with testosterone. Regression analysis further indicates that increases in free androgen index and in bioavailable testosterone plasma levels are associated with the observed MD reduction in the hypothalamus. The adult hypothalamus has a volume of approximately 0.7 cm³ per hemisphere and its subparts can be reliably measured using MRI, even with machines at the lower field strength such as 1.5 T (Lemaire et al. 2013). Hypothalamic nuclei are well described and anatomical landmarks indicate MD reductions in our study located in the lateral hypothalamic area (Lemaire et al. 2013; Baroncini et al. 2012).

Using diffusion-weighted MRI, the previous studies found activity-related diffusion changes in hypothalamic substructures that are likely generated by astrocytic volume changes (Lizarbe et al. 2013). As hypothalamic activity is strongly modulated by gonadal steroids (Campbell and Herbison 2014), we speculate that the reductions in MD in the lateral hypothalamus in our study were caused by testosterone treatment. Thus, MD reductions after testosterone treatment may indicate increased activity and associated cellular swelling in the lateral hypothalamus (Lizarbe et al. 2013; Le Bihan and Iima 2015). Indeed, early animal research shows significant increases in the firing rate of lateral hypothalamic neurons in response to injections of testosterone (Orsini 1982). Lesion and stimulation studies have associated the lateral hypothalamus with motivation and reward (Stuber and Wise 2016). This is supported by studies elucidating reward-related connections to the dopaminergic ventral tegmental area (Shizgal et al. 1980). Moreover, orexin-producing neurons are known to regulate arousal, as well as feeding and other reward-related behaviors and are restricted to the lateral hypothalamus (Rosin et al. 2003). In particular, effects of testosterone on lateral hypothalamus neurons have been linked to male sexual motivation (Hendricks and Scheetz 1973). This coincides well with the observation that sexual desires are fundamentally increased after initiation of testosterone treatment in FtMs (Wierckx et al. 2011) and that frequency of sexual intercourse is positively correlated with testosterone levels (Costantino et al. 2013). Hence, presumed increases in lateral hypothalamic activity and associated MD reductions may be linked to increased sexual arousal in FtMs. Unfortunately, we were not able to directly test this as sexual arousal was not systematically assessed in our study.

Precaution may also be put forward when it comes to the consistency of anatomical locations of results. Whereas MD reduction was most prominent in the right lateral hypothalamus and only bilaterally significant after 4 months of treatment, testosterone plasma-level increases predicted MD reductions predominantly in the left lateral hypothalamus. In any case, given the imprecision in spatial specificity introduced by motion in small structures such as the hypothalamus, our results might suggest a more general testosterone effect on lateral hypothalamus rather than an anatomically specific and asymmetric influence. Moreover, the action of testosterone on hypothalamic tissue is complex and implies several cellular events. This includes early responses within seconds that can be explained by non-genomic effects as well as late responses, which are compatible with the general model of steroid-hormone action (McEwen et al. 2015). Finally, it is generally assumed that testosterone exerts its influence on male sexual behavior through transformation into estradiol, although see (Antonio-Cabrera and Paredes 2014). Whether this “aromatization hypothesis” also applies for MD changes observed in our study remains to be investigated.

Of note, an earlier study by Hulshoff-Pol et al. (2006) investigated the effects of sex hormone replacement therapy on hypothalamic volume in 6 FtMs and 8 MtFs. They observed that 4 months of androgen treatment in FtMs increased hypothalamus volume and decreased lateral and third ventricle volumes, whereas anti-androgen and estrogen therapy in MtFs had the opposite effect. Although interesting and promising, we refrained from performing an additional volumetric analysis of our data given the significant challenges and shortcomings of MRI investigations on hypothalamus volume in vivo (Schindler et al. 2012). First, ventricular dilation and volume changes of surrounding structures may affect the reliability of hypothalamus volume measurements (Hulshoff-Pol et al. 2006). Second, manual delineation criteria for the hypothalamus region of interest (ROI) differ between studies and are influenced by the delineator. Third, there is strong inter-individual intensity variation of hypothalamus boundaries which even compromise advantages of high-resolution imaging such as at 7T (Schindler et al. 2017). Finally, visible landmarks in MRI data may not coincide with histology which questions the validity of volumetric analyses.

Several limitations of this study should be noted. First, it is important to note that the interaction between group and time did not withstand correction for multiple comparisons. Thus, we cannot conclude that groups were significantly different in their changes of MD over time (Nieuwenhuis et al. 2011). We base our interpretation on the fact that only the group receiving intervention showed MD changes, while the others did not, and because testosterone level changes, i.e., the “size of intervention” correlated with changes in MD. Second, besides testosterone, several other variables—related or not related to hormone replacement therapy—may in principle explain our finding. However, based on our quasi-experimental approach, we believe that it is justified to argue that testosterone treatment or at least some variable associated with the intervention is the most parsimonious explanation for observed effects in the FtM group. This is not to say that there are various possibilities of mediators or moderators (e.g., other hormones) between testosterone treatment and MD changes. In that respect, it is noteworthy that estradiol plasma-level variability was positively correlated with MD changes at an uncorrected p value level, although estradiol plasma levels did not change significantly over the course of treatment. Third, we did not test for behavioral changes associated with our finding. This includes not only sexual arousal as stated earlier but may also apply to changes in mood, mindsets, or habits. Finally, hypothalamic diffusion parameters may vary between fasted and fed states (Lizarbe et al. 2013). Yet, satiety was not investigated in our study which potentially increased variability in our dependent variable.

Together, our data indicate that testosterone treatment reduces mean diffusivity in the adult lateral hypothalamus and that treatment induced increases in testosterone levels are associated with the magnitude of MD reductions. These findings imply microstructural changes and may indicate related changes in neural activity by testosterone in the human hypothalamus of female-to-male transgender persons towards male levels.