Biological Approaches to Studying Gender Development

Abstract

The origins of sex differences in human behavior have been extensively studied from various theoretical perspectives, and a growing body of evidence has suggested that organization of the brain, and subsequent sex-typed behaviors, are influenced by exposure to sex hormones and the expression of specific genes during early development. Methodological advances in the study of biological bases of sex differences have shed light on mechanisms that influence sex development across the life span, though many questions remain. This chapter provides a general overview of biological approaches to the study of sex differences, with summaries of findings to date and future directions. The emphasis of the chapter is on hormones because that has been the major focus of biological approaches to sex development for decades. The chapter also touches on genetics toward the end given some important emerging work disentangling hormonal effects from genetic effects.

Appendices

Spotlight Feature: The Neurobiology of Gender Dysphoria

• Antonio Guillamon 

1. Department of Psychobiology, Universidad Nacional de Educación a Distancia, Madrid, Spain

   Antonio Guillamon

(The work of AG is supported by grant PGC2018-094919-B-C2 from the Ministerio de Ciencia y Tecnología, Spain)

Genetic, in vivo magnetic resonance imaging (MRI) and postmortem brain studies have been focused on transgender people who fulfill the description of transsexuals in DSM-5 (Guillamon et al., 2016). Research shows there are genetic bases influencing transgender identity. In both transmen (TM) and transwomen (TW), concordance is higher between monozygotic than dizygotic twins (Heylens et al., 2012). Since the pioneering postmortem studies of Swaab on the brain of TW (Zhou et al., 1995), the endocrine bases of transsexuality have been examined from a focus on the sexual differentiation of the brain. This has prompted studies on polymorphisms of the androgen (AR) and estrogen (ERα and ERβ) receptors as well as the aromatase enzyme, all of which contribute to cerebral sexual differentiation. Henningsson et al. (2005) were the first to report that TW differed from cis-men with respect to the mean length of the Cytosine-Adenine repeat in intron 5 of the ERβ gene. Hare et al. (2009) found that a Cytosine-Adenine-Guanine length repeat had a significant association with TW. However, Ujike et al. (2009), in a Japanese population, reported no significant difference in allelic or genotypic distribution of the above-referred genes for either TM or TW. In TW, gender dysphoria (i.e., distress related to an incongruence between experienced gender identity and birth-assigned sex) may have an oligemic component with several genes involved in sex hormone signaling contribution (Foreman et al., 2019). In a recent large study of 2300 cis and trans subjects controlling for early onset of gender dysphoria and sexual orientation, it was shown that TW gender development involves an AR polymorphism accompanied by an ERβ polymorphism (Fernández et al., 2018). An inverse allele interaction between AR and ERβ is characteristic of the TW population: When either of these polymorphisms is short, the other is long. ERα and ERβ polymorphisms are also associated with gender dysphoria in the TM population although no interaction has been observed between these polymorphisms.

A landmark in the study of trans people was the postmortem neurohistological study showing that hormonally treated TW have a feminine (lesser volume and neurons) central part of the bed nucleus of the stria terminalis (BSTc) (Zhou et al., 1995). In rodents, this nucleus is sexually dimorphic (Guillamon et al., 1988), contains AR and ERs (Simerly et al., 1990), and is involved in male sexual behavior (Emery & Sachs, 1976; Claro et al., 1995). The Dutch group, led by Swaab, reported that the volume of the BSTc is larger in cis-men than in cis-women and that TW present a female-sized BSTc and hypothesized that gender identity develops as a result of an interaction between the developing brain and sex hormones (Zhou et al., 1995).

In vivo MRI studies can be classified as structural (sMRI), examining features such as the size, shape, and microstructure of brain regions, or functional (fMRI), examining brain activation in response to stimuli or when at rest. For all, the main methodological problem they may present is the use of non-homogeneous groups of trans and cis people, this being because neither transmen nor transwomen are homogeneous groups with respect to age of gender dysphoria onset and sexual orientation (Blanchard, 1989a, 1989b). It is sometimes quite difficult to achieve a homogenous group design because of the relatively low prevalence of trans people in the population.

Results from sMRI studies in Table 3.1 indicate that, with respect to the main brain parameters, TW and TM show the normative characteristics of their assigned sex at birth. However, when the volume or the thickness of the cortex, the volume of subcortical structures or the white matter microstructure of the main brain bundles are specifically studied, the TW brain is a blend of masculine, feminine, and demasculinized morphological traits just as the TM brain is a mixture of feminine, masculine, and defeminized traits (Rametti et al., 2011a; Guillamon et al., 2016; Kreukels & Guillamon, 2016). It is important to underscore that in TW and TM, structural changes are observed in regions like the insula, parietal lobe, precuneus, and visual cortex (Savic & Arver, 2011; Zubiaurre-Elorza et al., 2013) that are related to body perception. Functional connectivity studies (Table 3.2) suggest that TM and TW each have their own phenotypic circuitry that differs from cis males and females.

Table 3.1 The brain phenotype of transwomen and transmen before cross-sex-hormone treatment

Table 3.2 Brain connectivity profiles in transwomen and transmen

To explain gender dysphoria and gender identity, three complementary hypotheses/theories have emerged from brain studies:

Brain feminization of transwomen. Because the volume of their BSTc is in a feminine range, it was hypothesized that gender identity would develop as a result of an interaction between the developing brain and sex hormones. Hence, TW would experience a process of feminization (Zhou et al., 1995). This hypothesis was the first to focus attention on a differential sexual differentiation of the brain associated with TW. The mechanisms responsible for feelings of gender incongruence in trans people would depend on their prenatal exposure to sex hormones and could lead to atypical sexual differentiation of the brain, with the body and genitals developing in the direction of one sex, while the brain and gender developed in the direction of the other sex (Swaab & Garcia-Falgueras, 2009).

Cortical neurodevelopmental theory. This theory is based on sMRI and resting state functional MRI. This theory considers that (a) there is substantial work showing that the cerebral cortex experiences a life-long thinning process that depends on testosterone and the testosterone receptor (Raznahan et al., 2010), (b) normatively, cis-females show thicker cortex than cis-males, (c) cis-females, TM, and TW present thicker cortex than cis-males, but this occurs in different regions, and (d) these differences give a distinct structural cortical phenotype to each of the four groups. Consequently, it was proposed that cis-females, TM, and TW present a slower cortical thinning process than cis-males. Each of the four groups follows different developmental timings in different regions during cortical development (Zubiaurre-Elorza et al., 2013; Guillamon et al., 2016). Functionally, TM, TW, and cis women have decreased connectivity compared with cis men in superior parietal regions, as part of the salience and executive networks (Uribe et al., 2020).

Body perception disconnection theory. Studies using different methodologies based on functional resting state data have referred to the parietal lobe as a key structure in the own-self body perception processes in TM (Burke et al., 2017; Feusner et al., 2017; Manzouri et al., 2017; Manzouri & Savic, 2018). When comparing intrinsic connectivity in networks involved in self-referential processes and their own body perception and visual processing, TM compared with cis-males and females showed decreased connectivity in several networks (default mode, salience, and visual) related to body perception. This suggests dysconnectivity within networks involved in one’s own body perception in the context of self (Feusner et al., 2017).

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• Heylens, G., De Cuypere, G., Zucker, K. J., Schelfaut, C., Elaut, E., Vanden Bossche, H., De Baere, E., & T’Sjoen, G. (2012). Gender identity disorder in twins: a review of the case report literature. The Journal of Sexual Medicine, 9(3), 751–757.

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Spotlight Feature: Hormonal Treatment Effects on Adolescent Brain Development

• Sarah M. Burke 

1. Brain & Development Research Center, Department of Developmental and Educational Psychology, Leiden University, Leiden, The Netherlands

   Sarah M. Burke

Since their introduction during the 1990s at the gender identity clinic in Amsterdam, puberty suppression by means of Gonadotropin Releasing Hormone analogs (GnRHa), followed by gender-affirming hormone treatment (estradiol and androgen-blocking medication in birth-assigned boys, testosterone in birth-assigned girls), have become standard treatments for adolescents with gender dysphoria (GD; i.e., distress related to incongruence between experienced gender identity and birth-assigned sex; Hembree et al., 2017). GnRHa, which should only be given to youth who have been found eligible after careful diagnostic assessment and who have reached at least Tanner stage 2 (i.e., minimal pubertal development), suppress any further development of the secondary sex characteristics that forms a major source of distress for adolescents with GD. Thereby, GnRHa treatment importantly contributes to improvements in adolescents’—still developing—emotional and social functioning, and more generally their mental health and well-being (Costa et al., 2015). Another advantage of delaying puberty in adolescents with GD is that it provides them with additional time to reflect upon the far-reaching decision of undergoing gender-affirming hormone treatment and, potentially, later surgery as well.

However, a major concern has been that puberty suppression could interfere with significant developmental brain changes, particularly within the prefrontal cortex, that underlie adolescence-specific changes in behavior (e.g., in cognitive control and flexibility, social cognition, working memory, emotion regulation; Juraska & Willing, 2017). More specifically, the rise in pubertal sex hormone levels has been associated with (sex-specific) cortical maturation, and both pubertal stage and timing of pubertal onset have been found to impact brain development significantly (Herting & Sowell, 2017; Juraska & Willing, 2017). Therefore, long-term delay of puberty with GnRHa, and thus prevention of exposure to sex hormones during the early adolescent years, might negatively affect the cognitive and social-emotional development of youth with GD. Indeed, evidence from animal models suggested sex-specific adverse effects of GnRHa on stress-processing, mood and cognition (in females), and locomotion, social behavior (Anacker et al., 2020), and spatial memory (Hough et al., 2017; in males). Furthermore, studies in girls with idiopathic central precocious puberty (reviewed in Hayes, 2017) have suggested deleterious effects of GnRHa treatment on general intelligence and persistent (even after treatment discontinuation) reduction of spatial memory performance. But, although in recent years brain imaging studies have started to accumulate evidence on the shorter term effects of gender-affirming hormone treatments on transgender individuals’ brain functions and structure, knowledge regarding the longer term effects of these treatments on brain and cognition remains very limited, and systematic, prospective studies of puberty suppression in youth with GD are currently lacking. In fact, a recent report, using an expert consensus method, identified the prioritized need for research on the long-term effects of puberty suppression on brain development and on cognitive and social-emotional maturation during the critical transition period of early adolescence (Chen et al., 2020).

One functional magnetic resonance imaging (fMRI) study, including participants with GD ages 14–16 years, aimed to test whether GnRHa treatment might have any adverse effects on adolescents’ executive functions and associated brain activations using an fMRI version of the so-called Tower of London task (Staphorsius et al., 2015). Findings suggested no differences between adolescents receiving GnRHa and treatment-naïve adolescents with GD in task performance. However, because of the relatively small sample sizes of transgender boys (female birth-assigned sex, n = 12) and transgender girls (male birth-assigned sex, n = 8) receiving GnRHa as well as the cross-sectional study design, caution is warranted in drawing any conclusions on the effects of puberty suppressants on cognition and behavior.

In a prospective case study of an 11-year-old transgender girl (male birth-assigned sex), cognitive capacity and changes in diffusion measures of white matter microstructure (indexing myelination, axonal diameter, and white matter integrity, all of which contribute to efficient information transfer) were assessed before initiation of GnRHa treatment and during two follow-up measurements at ages 13 and 14 years, respectively (Schneider et al., 2017). In line with prior studies (Hayes, 2017; Hough et al., 2017; Anacker et al., 2020), performance intelligence quotient and memory deteriorated with treatment, and typical, testosterone-dependent white matter maturation (increase in diffusion parameters) was not observed, suggesting inhibiting effects of GnRHa on neurodevelopment. However, these findings require replication in larger samples and should be compared to the effects of hormone (mainly estradiol) suppression with GnRHa on brain development in birth-assigned females.

In another study, using the same participant samples as in the study by Staphorsius et al. (2015), regional brain volumes of adolescents with and without GD were compared (Hoekzema et al., 2015). It was found that hypothalamus volumes in a sample of 37 transgender girls (male birth-assigned sex) of varying treatment status (n = 11 were treatment-naïve, n = 14 received GnRHa, n = 12 received gender-affirming hormone treatment) tended to be sex-atypical, thus smaller than in cisgender boys. However, given the heterogenous total sample and the small sample sizes of adolescents who had received treatment, it is unclear whether the observed effects on brain structure may be ascribed to the suppression of endogenous hormones, to the addition of estradiol, or being treatment-naïve (i.e., differences could be related to the experience of GD per se).

As of yet, two longitudinal studies, in the same adolescent participant samples, investigated testosterone treatment effects on spatial cognition (Burke et al., 2016) and functional amygdala lateralization (Beking et al., 2020) in youth who experience GD. In the study by Burke et al. (2016), during the first fMRI session, a group of 21 transgender boys (female birth-assigned sex, mean age 16 years) was receiving GnRHa and was compared to groups of treatment-naïve, age-matched cisgender boys (n = 20) and cisgender girls (n = 21). Brain activation patterns of the transgender boys, during an fMRI mental rotation task, were comparable to those of the cisgender boys, but differed significantly from those of the cisgender girls, thus suggesting sex-atypical spatial functioning prior to initiation of gender-affirming hormone treatment. During a follow-up session, after an average 10 months of testosterone treatment, they showed significantly increased activation during mental rotation (in superior parietal, superior frontal cortex) relative to the previous session, similar to the cisgender boys, and significantly different from the cisgender girls, who showed no changes between sessions.

In another task-fMRI study (Beking et al., 2020), using the same participant samples, the effects of testosterone treatment on changes in functional amygdala lateralization were investigated. The two hemispheres of the brain are well-known to differ in structure and function between cisgender males and females, which is thought to develop under the influence of testosterone. In line with the hypothesis, the lateralization index (relative activation differences of individuals’ right and left amygdalae) in transgender boys shifted towards the right amygdala after testosterone treatment. In addition, the cumulative dose of testosterone treatment correlated significantly with amygdala lateralization after treatment although there were no significant differences between the groups. However, because the transgender boys had received GnRHa treatment during the first fMRI sessions, both studies lacked a baseline (treatment-naïve) control condition. Therefore, possible advantageous effects of suppressing estrogens (which are known to have detrimental effects on mental rotation performance) in the transgender boys (female birth-assigned sex) versus the cisgender girls cannot be ruled out. Nevertheless, together with other findings from hormone administration studies (Heany et al., 2016), these data highlight the influence of testosterone on the human brain.

To conclude, the existing literature suggests long-term adverse effects of GnRHa on adolescent cognitive development, but evidence is too limited to allow definitive conclusions. In addition, it remains an open question whether the window of sex hormone-sensitive brain reorganization might be closed by the time adolescents with GD start using gender-affirmative hormones. Future follow-up studies of existing samples of GD youth and new longitudinal studies that consider typical brain developmental trajectories as well as include a pre-pubertal baseline condition are highly recommended.

Spotlight references

• Anacker, C., Sydnor, E., Chen, B. K., LaGamma, C. C., McGowan, J. C., Mastrodonato, A.,…Denny, C. A. (2020). Behavioral and neurobiological effects of GnRH agonist treatment in mice—potential implications for puberty suppression in transgender individuals. Neuropsychopharmacology. doi: 10.1038/s41386-020-00826-1

• Beking, T., Burke, S. M., Geuze, R. H., Staphorsius, A. S., Bakker, J., Groothuis, A. G. G., Kreukels, B. P. C. (2020). Testosterone effects on functional amygdala lateralization: A study in adolescent transgender boys and cisgender boys and girls. Psychoneuroendocrinology, 111, 104461. doi: 10.1016/j.psyneuen.2019.104461

• Burke, S. M., Kreukels, B. P. C., Cohen-Kettenis, P. T., Veltman, D. J., Klink, D. T., & Bakker, J. (2016). Male-typical visuospatial functioning in gynephilic girls with gender dysphoria—organizational and activational effects of testosterone. Journal of Psychiatry & Neuroscience, 41, 395–404.

• Chen, D., Strang, J. F., Kolbuck, V. D., Rosenthal, S. M., Wallen, K., Waber, D. P.,…Garofalo, R. (2020). Consensus parameter: Research methodologies to evaluate neurodevelopmental effects of pubertal suppression in transgender youth. Transgender Health, 5, 246–257.

• Costa, R., Dunsford, M., Skagerberg, E., Holt, V., Carmichael, P., & Colizzi, M. (2015). Psychological support, puberty suppression, and psychosocial functioning in adolescents with gender dysphoria. Journal of Sexual Medicine, 12, 2206–2214.

• Hayes, P. (2017). Commentary: Cognitive, emotional, and psychosocial functioning of girls treated with pharmacological puberty blockage for idiopathic central precocious puberty. Frontiers in Psychology, 8, 44.

• Heany, S. J., Van Honk, J., Stein, D. J., & Brooks, S. J. (2016). A quantitative and qualitative review of the effects of testosterone on the function and structure of the human social-emotional brain. Metabolic Brain Disease, 31, 157–167.

• Hembree, W. C., Cohen-Kettenis, P. T., Gooren, L., Hannema, S. E., Meyer, W. J., Murad, M. H., … T’Sjoen, G. G. (2017). Endocrine treatment of gender-dysphoric/gender-incongruent persons: An Endocrine society* clinical practice guideline. The Journal of Clinical Endocrinology & Metabolism, 102, 3869–3903.

• Herting, M. M., & Sowell, E. R. (2017). Puberty and structural brain development in humans. Frontiers in Neuroendocrinology, 44, 122–137.

• Hoekzema, E., Schagen, S. E. E., Kreukels, B. P. C., Veltman, D. J., Cohen-Kettenis, P. T., Delemarre-van de Waal, H., & Bakker, J. (2015). Regional volumes and spatial volumetric distribution of gray matter in the gender dysphoric brain. Psychoneuroendocrinology, 55, 59–71.

• Hough, D., Bellingham, M., Haraldsen, I. R., McLaughlin, M., Robinson, J. E., Solbakk, A. K., & Evans, N. P. (2017). A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment in sheep. Psychoneuroendocrinology, 77, 1–8.

• Juraska, J. M., & Willing, J. (2017). Pubertal onset as a critical transition for neural development and cognition. Brain Research, 1654, 87–94.

• Schneider, M. A., Spritzer, P. M., Soll, B. M. B., Fontanari, A. M. V., Carneiro, M., Tovar-Moll, F., … Lobato, M. I. R. (2017). Brain maturation, cognition and voice pattern in a gender dysphoria case under pubertal suppression. Frontiers in Human Neuroscience, 11, 528. doi: 10.3389/fnhum.2017.00528

• Staphorsius, A. S., Kreukels, B. P. C., Cohen-Kettenis, P. T., Veltman, D. J., Burke, S. M., Schagen, S. E. E., … Bakker, J. (2015). Puberty suppression and executive functioning: An fMRI-study in adolescents with gender dysphoria. Psychoneuroendocrinology, 56, 190–199.