A Sex Difference in the Human Brain and its Relation to Transsexuality

Authors – J.-N. Zhou, M.A. Hofman, L.J. Gooren and D.F. Swaab

Source – © NATURE, 378: 68-70 (1995)

Transsexuals have the strong feeling, often from childhood onwards, of having been born the wrong sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy.. The possible psychogenic or biological etiology of transsexuality has been the subject of debate for many years [1,2]. Here we show that the volume of the central subdivision of the bed nucleus of the stria terminalis (BSTc), a brain area that is essential for sexual behaviour [3,4], is larger in men than in women. A female-sized BSTc was found in male-to-female transsexuals. The size of the BSTc was not influenced by sex hormonesHormones, such as oestrogen and testosterone, affecting sexual and reproductive development or function. in adulthood and was independent of sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual.. Our study is the first to show a femaleA sex, usually assigned at birth, and based on chromosomes (e.g. XX), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. vagina, uterus). brain structure in genetically maleA sex, usually assigned at birth, and based on chromosomes (e.g. XY), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. penis, testicles). transsexuals and supports the hypothesis that gender identity• One’s innermost concept of self as male or female or both or neither – how individuals perceive themselves and what they call themselves. One’s gender identity can be the same or different than the sex assigned at birth. Individuals are conscious of this between the ages 18 months and 3 years. Most people develop a gender identity that matches their biological sex. For some, however, their gender identity is different from their biological or assigned sex. Some of these individuals choose to socially, hormonally and/or surgically change their sex to more fully match their gender identity.
• The gender to which one feels one belongs.
• Internal and psychological sense of oneself as a woman, a man, both, in between, or neither.
develops as a result of an interaction between the developing brain and sex hormonesHormones, such as oestrogen and testosterone, affecting sexual and reproductive development or function. [5,6].

Investigation of genetics, gonads, genitalia or hormone level of transsexuals has not, so far, produced any results that explain their status [1,2]. In experimental animals, however, the same gonadal hormonesChemical substances that control and regulate the activity of certain cells or organs; see also: sex hormones. that prenatally determine the morphology of the genitalia also influence the morphology and function of the brain in experimental animals in a sexually dimorphic fashion [6,7]. This led to the hypothesis that sexual differentiation of the brain in transsexuals might not have followed the line of sexual differentiation of the body as a whole. In the past few years, several anatomical differences in relation to sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. and sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual. have been observed in the human hypothalamus (see [6] for a review), but so far no neuroanatomical investigations have been made in relation to the expression of cross-gender identity (transsexuality).

Figure 1: Schematic frontal section through two subdivisions of the bed nucleus of the stria terminalis (BST) that are hatched. III: third ventricle; AC: anterior commissure; BSTc and BSTv: central and ventral subdivisions of the BST; FX: fornix; IC: internal capsule; LV: lateral ventricle; NBM: nucleus basalis of Meynert; OT: optic tract; PVN: paraventricular nucleus; SDN: sexually dimorphic nucleus; SON: supraoptic nucleus.

We have studied the hypothalamus of six male-to-female transsexuals (T1-T6); this material that was collected over the last eleven years. We searched for a brain structure that was sexually dimorphic, but not influenced by sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual., as male-to-female transsexuals may be “oriented” to either sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. with respect to sexual behaviour. Our earlier observations showed that the paraventricular nucleus (PVN), sexually dimorphic nucleus (SDN) and suprachiasmatic nucleus (SCN) did not meet these criteria ([6] and unpublished data). Although there is no accepted animal model for gender identity• One’s innermost concept of self as male or female or both or neither – how individuals perceive themselves and what they call themselves. One’s gender identity can be the same or different than the sex assigned at birth. Individuals are conscious of this between the ages 18 months and 3 years. Most people develop a gender identity that matches their biological sex. For some, however, their gender identity is different from their biological or assigned sex. Some of these individuals choose to socially, hormonally and/or surgically change their sex to more fully match their gender identity.
• The gender to which one feels one belongs.
• Internal and psychological sense of oneself as a woman, a man, both, in between, or neither.
alterations, the bed nucleus of the stria terminalis (BST) turned out to be an appropriate candidate to study for the following reasons. First, it is known that the BST plays an essential part in rodent sexual behaviour [3,4]. Not only have oestrogen and androgen receptors been found in the BST [8,9], it is also a major aromatization centre in the developing rat brain [10]. The BST in the rat receives projections mainly from the amygdala and provides a strong input in the preoptic-hypothalamic region [11,12]. Reciprocal connections between hypothalamus, BST and amygdala are also well documented in experimental animals [13-15]. In addition, sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. differences in the size and cell number of the BST have been described in rodents which are influenced by gonadal steroids in development [16-18]. Also in humans a particular caudal part of the BST (BNST-dspm) has been reported to be 2.5 times larger in men than in women [19].

The localization of the BST is shown in figure 1. The central part of the BST (BSTc) is characterized by its somatostatin cells and vasoactive intestinal polypeptide (VIP) innervation [20]. We measured the volume of the BSTc on the basis of its VIP innervation (Fig. 2).

Figure 2: Representative sections of the BSTc innervated by vasoactive intestinal polypeptide (VIP). A: heterosexual manA human being who self-identifies as a man, based on elements of importance to the individual, such as gender roles, behaviour, expression, identity, and/or physiology.; B: heterosexual womanA human being who self-identifies as a woman, based on elements of importance to the individual, such as gender roles, behaviour, expression, identity, and/or physiology.; C: homosexual manA human being who self-identifies as a man, based on elements of importance to the individual, such as gender roles, behaviour, expression, identity, and/or physiology.; D: male-to-female transsexualThis is typically used to describe people who identify as transgender who are transitioning toward the gender with which they identify. This may include socially presenting (e.g., clothing, hair, mannerisms, overall gender expression) as the gender with which they identify, or it may include more extensive changes like taking hormones and/or surgical procedures to modify their body.. Bar=0.5 mm. LV: lateral ventricle. Note there are two parts of the BST in A and B: small sized medial subdivision (BSTm), and large oval-sized central subdivision (BSTc).

The BSTc volume in heterosexual men (2.49±0.16 mm3) was 44% larger than in heterosexual women (1.73±0.13 mm3) (P0.15), indicating that the observed small size of the BSTc in transsexuals was not due to the fact that they were, on average, 10 to 13 years older than the hetero- and homosexual men.

The BST plays an essential role in masculineDescribes socially and culturally constructed aspects of gender (e.g. roles, behaviour, expression, identity) typically associated with boys and men. sexual behaviour and in the regulation of gonadotrophin release, as shown by studies in the rat [3,4,21]. There has been no direct evidence that the BST has such a role in human sexual behaviour but our demonstration of a sexually dimorphic pattern in the size of the human BSTc, which is in agreement with the previously described sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. difference in a more caudal part of the BST (BNST-dspm) [19], indicates that this nucleus may also be involved in human sexual or reproductive functions. It has been proposed that neurochemical sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. differences in the rat BST may be due to effects of sex hormonesHormones, such as oestrogen and testosterone, affecting sexual and reproductive development or function. on the brain during development and in adulthood [22,23]. Our data from humans however, indicate that BSTc volume is not affected by varying sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. hormone levels in adulthood. The BSTc volume of a 46-year-old womanA human being who self-identifies as a woman, based on elements of importance to the individual, such as gender roles, behaviour, expression, identity, and/or physiology. who had suffered for at least 1 year from a tumour of the adrenal cortex that produced very high blood levels of androstenedione and testosterone, was within the range of that of other women (Fig. 3: S1). Furthermore, two postmenopausal women (aged over 70 years) showed a completely normal female-sized BSTc (Fig. 3: M1, M2). As all the transsexuals had been treated with oestrogens, the reduced size of the BSTc could possibly have been due to the presence of high levels of oestrogen in the blood. Evidence against this comes from the fact that transsexualThis is typically used to describe people who identify as transgender who are transitioning toward the gender with which they identify. This may include socially presenting (e.g., clothing, hair, mannerisms, overall gender expression) as the gender with which they identify, or it may include more extensive changes like taking hormones and/or surgical procedures to modify their body. T2 and T3 both showed a small, female-like BSTc (Fig. 3), although T2 stopped taking oestrogen about 15 months before death, since her prolactin levels were too high and T3 stopped hormone treatment since a sarcoma was found about three months before death; also a 31-year-old manA human being who self-identifies as a man, based on elements of importance to the individual, such as gender roles, behaviour, expression, identity, and/or physiology. who suffered from a feminizing adrenal tumour which induced high blood levels of oestrogen, nevertheless had a very large BSTc (Fig. 3: S2).

Figure 3: Volume of the BSTc innervated by VIP fibres in presumed heterosexual males (M), homosexual males (HM), presumed heterosexual females (F) and male-to-female transsexuals (TM). The six transsexuals are numbered T1-T6. The patients with abnormal sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. hormone levels are numbered S1-S4. M1 and M2: postmenopausal women. Bars indicate mean±SEM. Open symbols: individuals who died of AIDS.

Methods

Brains of 42 subjects matched for age, postmortem time and duration of formalin fixation were investigated. The autopsy was performed following the required permission. For immunocytochemical staining of VIP, the paraffin sections were hydrated and rinsed in TBS (Tris-buffered-saline: 0.05 M tris, 0.9% NaCl, pH 7.6). The sections were incubated with 200 µl anti-VIP (Viper, 18/9/86) 1:1000 in 0.5% triton in TBS overnight at 4° C. The immunocytochemical and morphometric procedures were performed as described extensively elsewhere [25-27]. In brief, serial 6 m m sections of the BSTc were studied by means of a digitizer (Calcomp 2000) connected to a HP-UX 9.0, using a Zeiss microscope equipped with a 2.5x objective and with 10x (PLAN) oculars. Staining was performed on every 50th section with anti-VIP. The rostral and caudal borders of the BSTc were assessed by staining every 10th section in the area. The volume of the BSTc was determined by integrating all the area measurements of the BSTc sections that were innervated by VIP fibres. In a pilot study, the size of the BSTc was measured on both sides in eight subjects (five females and three males) and no left-right asymmetries were observed: the left BSTc (1.71±0.16 mm3) was comparable in size to that of the right BSTc (1.83±0.30 mm3) (P=0.79). No asymmetry was observed in the BNST-dspm either [19]. The rest of our study was therefore performed on one side of the brain only. Brain weight of the maleA sex, usually assigned at birth, and based on chromosomes (e.g. XY), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. penis, testicles). transsexuals (1385±75 g) was not different from that of the reference males (1453±25 g) (P=0.61) or that of the females (1256±35 g) (P=0.23). The cause of death of the six transsexuals was suicide (T1), cardiovascular disease (T2,T6), sarcoma (T3), AIDS, pneumonia, pericarditis (T4) and hepatitic failure (T5). Sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual. of the subjects of the reference group (12 men and 11 women) was generally not known, but presumably most of them were heterosexual. Sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual. of nine homosexuals• Men and women who form their primary loving relationships with people of the same gender. Many gay people prefer that the terms “gay men and lesbians” or “gay people” be used to describe homosexuals as a group rather than the term “homosexuals”.
• An outdated term that was historically used to describe people who were attracted to other people of the same gender; replaced by the more inclusive and respectful term, ‘gay’ or ‘lesbian’, which are not considered offensive by many.
was registered in the clinical records [28]. Differences among the groups were tested two-tailed using the Mann-Whitney U test. A 5% level of significance was used in all statistical tests.

Our results might also be explained if the female-sized BSTc in the transsexualThis is typically used to describe people who identify as transgender who are transitioning toward the gender with which they identify. This may include socially presenting (e.g., clothing, hair, mannerisms, overall gender expression) as the gender with which they identify, or it may include more extensive changes like taking hormones and/or surgical procedures to modify their body. group was due to the lack of androgens, because they had all been orchidectomized except for T4. We therefore studied two other men who had been orchidectomized because of cancer of the prostate (one and three months before death: S4 and S3, respectively), and found that their BSTc sizes were at the high end of the normal maleA sex, usually assigned at birth, and based on chromosomes (e.g. XY), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. penis, testicles). range. The BSTc size of the single transsexualThis is typically used to describe people who identify as transgender who are transitioning toward the gender with which they identify. This may include socially presenting (e.g., clothing, hair, mannerisms, overall gender expression) as the gender with which they identify, or it may include more extensive changes like taking hormones and/or surgical procedures to modify their body. who had not been orchidectomized (T4) ranged in the middle of the transsexualThis is typically used to describe people who identify as transgender who are transitioning toward the gender with which they identify. This may include socially presenting (e.g., clothing, hair, mannerisms, overall gender expression) as the gender with which they identify, or it may include more extensive changes like taking hormones and/or surgical procedures to modify their body. scores (Fig. 3). Not only were five of the transsexuals orchidectomized, they all used the antiandrogen cyproterone acetate (CPA). A CPA effect on the BSTc does not seem likely, because T6 had not taken CPA for the past 10 years, and T3 took no CPA during the two years before death and still had a female-sized BSTc.

Results

In summary, our observations suggest that the small size of the BSTc in male-to-female transsexuals cannot be explained by differences in adult sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. hormone levels, but is established during development by an organizing action of sex hormonesHormones, such as oestrogen and testosterone, affecting sexual and reproductive development or function., an idea supported by the fact that neonatal gonadectomy of maleA sex, usually assigned at birth, and based on chromosomes (e.g. XY), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. penis, testicles). rats and androgenization of the femaleA sex, usually assigned at birth, and based on chromosomes (e.g. XX), gene expression, hormone levels and function, and reproductive/sexual anatomy (e.g. vagina, uterus). rats indeed induced significant changes in the number of neurons of the BST and suppressed its sexual dimorphism [17,18].

Considered together with information from animals, then our study supports the hypothesis that gender identity• One’s innermost concept of self as male or female or both or neither – how individuals perceive themselves and what they call themselves. One’s gender identity can be the same or different than the sex assigned at birth. Individuals are conscious of this between the ages 18 months and 3 years. Most people develop a gender identity that matches their biological sex. For some, however, their gender identity is different from their biological or assigned sex. Some of these individuals choose to socially, hormonally and/or surgically change their sex to more fully match their gender identity.
• The gender to which one feels one belongs.
• Internal and psychological sense of oneself as a woman, a man, both, in between, or neither.
alterations may develop as a result of an altered interaction between the development of the brain and sex hormonesHormones, such as oestrogen and testosterone, affecting sexual and reproductive development or function. [5,6]. The direct action of genetic factors should also be considered on the basis of animal experiments [24].

We found no relationship between BSTc size and the sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual. of transsexuals, that is, whether they were male-oriented (T1,T6), female-oriented (T3,T2,T5), or both (T4). Furthermore, the size of the BSTc of heterosexual men and homosexual men did not differ, which reinforced the idea that the reduced BSTc size is independent of sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual.. In addition, there was no difference in BSTc size between early-onset (T2,T5,T6) and late-onset transsexuals (T1, T3), indicating that the decreased size is related to the gender identity• One’s innermost concept of self as male or female or both or neither – how individuals perceive themselves and what they call themselves. One’s gender identity can be the same or different than the sex assigned at birth. Individuals are conscious of this between the ages 18 months and 3 years. Most people develop a gender identity that matches their biological sex. For some, however, their gender identity is different from their biological or assigned sex. Some of these individuals choose to socially, hormonally and/or surgically change their sex to more fully match their gender identity.
• The gender to which one feels one belongs.
• Internal and psychological sense of oneself as a woman, a man, both, in between, or neither.
alteration per se rather than to the age at which it becomes apparent. Interestingly, the very small BSTc in transsexuals appears to be a very local brain difference. We failed to observe similar changes in three other hypothalamic nuclei, namely, PVN, SDN or SCN in the same individuals (unpublished data). This might be due to the fact that these nuclei do not all develop at the same time, or to a difference between these nuclei and the BST with respect to the presence of sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. hormone receptors or aromatase. We are now studying the distribution of sexBiological attributes and legal categories used to classify humans as male, female, intersex or other categories, primarily associated with physical and physiological features including chromosomes, genetic expression, hormone levels and function, and reproductive/sexual anatomy. hormone receptors and the aromatase activity in various hypothalamic nuclei in relation to sexual orientationPatterns of emotional, romantic, and/or sexual attractions to groups of people (e.g. men, women, trans people), a person’s sense of identity based on those attractions, related behaviors, and membership in a community of others who share those attractions; for example pansexual, bisexual, LGB, heterosexual. and gender• However gender is far more complicated. It is the complex interrelationship between an individual’s sex (gender biology), one’s internal sense of self as male, female, both or neither (gender identity) as well as one’s outward presentations and behaviours (gender expression) related to that perception, including their gender role. Together, the intersection of these three dimensions produces one’s authentic sense of gender, both in how people experience their own gender as well as how others perceive it.
• Gender is expressed in terms of masculinity and femininity. It is largely culturally determined and is assigned at birth based on the sex of the individual. It affects how people perceive themselves and how they expect others to behave.
• Socially and culturally constructed roles, behaviours, expressions and identities of girls, women, boys, men, and trans people.
.

Acknowledgements

We thank Mr. B. Fisser, Mr. H. Stoffels, Mr. G. van der Meulen, and Ms. T. Eikelboom and Ms. W.T.P. Verweij for their help, and Drs. R.M. Buijs, M.A. Corner, E. Fliers, A. Walter and F.W. van Leeuwen for their comments. Brain material was provided by the Netherlands Brain Bank (coordinator Dr. R. Ravid). This study was supported by NWO.

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