Neurobiological characteristics associated with gender identity: Findings from neuroimaging studies in the Amsterdam cohort of children and adolescents experiencing gender incongruence.

This review has been based on my invited lecture at the annual meeting of the Society for Behavioral Neuroendocrinology in 2023. Gender incongruence is defined as a marked and persistent incongruence between an individual's experienced gender and the sex assigned at birth. A prominent hypothesis on the etiology of gender incongruence proposes that it is related to an altered or less pronounced sexual differentiation of the brain. This hypothesis has primarily been based on postmortem studies of the hypothalamus in transgender individuals. To further address this hypothesis, a series of structural and functional neuroimaging studies were conducted in the Amsterdam cohort of children and adolescents experiencing gender incongruence. Additional research objectives were to determine whether any sex and gender differences are established before or after puberty, as well as whether gender affirming hormone treatment would affect brain development and function. We found some evidence in favor of the sexual differentiation hypothesis at the functional level, but this was less evident at the structural level. We also observed some specific transgender neural signatures, suggesting that they might present a unique brain phenotype rather than being shifted towards either end of the male-female spectrum. Our results further suggest that the years between childhood and mid-adolescence represent an important period in which puberty-related factors influence several neural characteristics, such as white matter development and functional connectivity patterns, in both a sex and gender identity specific way. These latter observations thus lead to the important question about the possible negative consequences of delaying puberty on neurodevelopment. To further address this question, larger-scale, longitudinal studies are required to increase our understanding of the possible neurodevelopmental impacts of delaying puberty in transgender youth.

Alterations in the inferior fronto-occipital fasciculus - a specific neural correlate of gender incongruence?

BACKGROUND: Increasing numbers of adolescents seek help for gender-identity questions. Consequently, requests for medical treatments, such as puberty suppression, are growing. However, studies investigating the neurobiological substrate of gender incongruence (when birth-assigned sex and gender identity do not align) are scarce, and knowledge about the effects of puberty suppression on the developing brain of transgender youth is limited. METHODS: Here we cross-sectionally investigated sex and gender differences in regional fractional anisotropy (FA) as measured by diffusion MR imaging, and the impact of puberty on alterations in the white-matter organization of 35 treatment-naive prepubertal children and 41 adolescents with gender incongruence, receiving puberty suppression. The transgender groups were compared with 79 age-matched, treatment-naive cisgender (when sex and gender align) peers. RESULTS: We found that transgender adolescents had lower FA in the bilateral inferior fronto-occipital fasciculus (IFOF), forceps major and corpus callosum than cisgender peers. In addition, average FA values of the right IFOF correlated negatively with adolescents' cumulative dosage of puberty suppressants received. Of note, prepubertal children also showed significant FA group differences in, again, the right IFOF and left cortico-spinal tract, but with the reverse pattern (transgender > cisgender) than was seen in adolescents. CONCLUSIONS: Importantly, our results of lower FA (indexing less longitudinal organization, fiber coherence, and myelination) in the IFOF of gender-incongruent adolescents replicate prior findings in transgender adults, suggesting a salient neural correlate of gender incongruence. Findings highlight the complexity with which (pubertal) sex hormones impact white-matter development and add important insight into the neurobiological substrate associated with gender incongruence.

Postnatal Effects of Sex Hormones on Click-Evoked Otoacoustic Emissions: A Study of Adolescents with Gender Dysphoria.

Click-evoked otoacoustic emissions (CEOAEs) are echo-like sounds, generated by the inner ear in response to click-stimuli. A sex difference in emission strength is observed in neonates and adults, with weaker CEOAE amplitudes in males. These differences are assumed to originate from testosterone influences during prenatal male sexual differentiation and to remain stable throughout life. However, recent studies suggested activational, postnatal effects of sex hormones on CEOAEs. Adolescents diagnosed with gender dysphoria (GD) may receive gonadotropin-releasing hormone analogs (GnRHa) in order to suppress endogenous sex hormones and, therefore, pubertal maturation, followed by cross-sex hormone (CSH) treatment. Using a cross-sectional design, we examined whether hormonal interventions in adolescents diagnosed with GD (62 trans boys, assigned female at birth, self-identifying as male; 43 trans girls, assigned male at birth, self-identifying as female), affected their CEOAEs compared to age- and sex-matched controls (44 boys, 37 girls). Sex-typical differences in CEOAE amplitude were observed among cisgender controls and treatment-naïve trans boys but not in other groups with GD. Treatment-naïve trans girls tended to have more female-typical CEOAEs, suggesting hypomasculinized early sexual differentiation, in support of a prominent hypothesis on the etiology of GD. In line with the predicted suppressive effects of androgens, trans boys receiving CSH treatment, i.e., testosterone plus GnRHa, showed significantly weaker right-ear CEOAEs compared with control girls. A similar trend was seen in trans boys treated with GnRHa only. Unexpectedly, trans girls showed CEOAE masculinization with addition of estradiol. Our findings show that CEOAEs may not be used as an unequivocal measure of prenatal androgen exposure as they can be modulated postnatally by sex hormones, in the form of hormonal treatment.

Potential contribution of progesterone receptors to the development of sexual behavior in male and female mice.

We previously showed that estradiol can have both defeminizing and feminizing effects on the developing mouse brain. Pre- and early postnatal estradiol defeminized the ability to show lordosis in adulthood, whereas prepubertal estradiol feminized this ability. Furthermore, we found that estradiol upregulates progesterone receptors (PR) during development, inducing both a male-and female-typical pattern of PR expression in the mouse hypothalamus. In the present study, we took advantage of a newly developed PR antagonist (ZK 137316) to determine whether PR contributes to either male- or female-typical sexual differentiation. Thus groups of male and female C57Bl/6j mice were treated with ZK 137316 or OIL as control: males were treated neonatally (P0-P10), during the critical period for male sexual differentiation, and females were treated prepubertally (P15-P25), during the critical period for female sexual differentiation. In adulthood, mice were tested for sexual behavior. In males, some minor effects of neonatal ZK treatment on sexual behavior were observed: latencies to the first mount, intromission and ejaculation were decreased in neonatally ZK treated males; however, this effect disappeared by the second mating test. By contrast, female mice treated with ZK during the prepubertal period showed significantly less lordosis than OIL-treated females. Mate preferences were not affected in either males or females treated with ZK during development. Taken together, these results suggest a role for PR and thus perhaps progesterone in the development of lordosis behavior in female mice. By contrast, no obvious role for PR can be discerned in the development of male sexual behavior.

Neural Activation During Mental Rotation in Complete Androgen Insensitivity Syndrome: The Influence of Sex Hormones and Sex Chromosomes.

Sex hormones, androgens in particular, are hypothesized to play a key role in the sexual differentiation of the human brain. However, possible direct effects of the sex chromosomes, that is, XX or XY, have not been well studied in humans. Individuals with complete androgen insensitivity syndrome (CAIS), who have a 46,XY karyotype but a female phenotype due to a complete androgen resistance, enable us to study the separate effects of gonadal hormones versus sex chromosomes on neural sex differences. Therefore, in the present study, we compared 46,XY men (n = 30) and 46,XX women (n = 29) to 46,XY individuals with CAIS (n = 21) on a mental rotation task using functional magnetic resonance imaging. Previously reported sex differences in neural activation during mental rotation were replicated in the control groups, with control men showing more activation in the inferior parietal lobe than control women. Individuals with CAIS showed a female-like neural activation pattern in the parietal lobe, indicating feminization of the brain in CAIS. Furthermore, this first neuroimaging study in individuals with CAIS provides evidence that sex differences in regional brain function during mental rotation are most likely not directly driven by genetic sex, but rather reflect gonadal hormone exposure.

Estradiol-induced neurogenesis in the female accessory olfactory bulb is required for the learning of the male odor.

Odors processed by the main and accessory olfactory bulbs (MOB, AOB) are important for sexual behavior. Interestingly, both structures continue to receive new neurons during adulthood. A role for olfactory neurogenesis in sexual behavior in female mice has recently been shown and gonadal hormones such as estradiol can modulate adult neurogenesis. Therefore, we wanted to determine the role of estradiol in learning the odors of sexual partners and in the adult neurogenesis of female aromatase knockout mice (ArKO), unable to produce estradiol. Female wild-type (WT) and ArKO mice were exposed to male odors during 7 days, and olfactory preferences, cell proliferation, cell survival and functional involvement of newborn neurons were analyzed, using BrdU injections, in combination with a marker of cell activation (Zif268) and neuronal fate (doublecortin, NeuN). Behavioral tasks indicated that both WT and ArKO females were able to discriminate between the odors of two different males, but ArKO mice failed to learn the familiar male odor. Proliferation of newborn cells was reduced in ArKO mice only in the dentate gyrus of the hippocampus. Olfactory exposure decreased cell survival in the AOB in WT females, suggesting a role for estradiol in a structure involved in sexual behavior. Finally, newborn neurons do not seem to be functionally involved in the AOB of ArKO mice compared with WT, when females were exposed to the odor of a familiar male, suggesting that estradiol-induced neurogenesis in the AOB is required for the learning of the male odor in female mice. Aromatase knockout mice (ArKO) presented deficits in olfactory preferences without affecting their olfactory discrimination abilities, and showed no functional involvement of newborn neurons in the accessory olfactory bulb (AOB) in response to the odor of a familiar male. These results suggest that estradiol-induced neurogenesis in the female AOB is required for the learning of the male odor.

Male-typical visuospatial functioning in gynephilic girls with gender dysphoria - organizational and activational effects of testosterone.

BACKGROUND: Sex differences in performance and regional brain activity during mental rotation have been reported repeatedly and reflect organizational and activational effects of sex hormones. We investigated whether adolescent girls with gender dysphoria (GD), before and after 10 months of testosterone treatment, showed male-typical brain activity during a mental rotation task (MRT). METHODS: Girls with GD underwent fMRI while performing the MRT twice: when receiving medication to suppress their endogenous sex hormones before onset of testosterone treatment, and 10 months later during testosterone treatment. Two age-matched control groups participated twice as well. RESULTS: We included 21 girls with GD, 20 male controls and 21 female controls in our study. In the absence of any group differences in performance, control girls showed significantly increased activation in frontal brain areas compared with control boys (pFWE = 0.012). Girls with GD before testosterone treatment differed significantly in frontal brain activation from the control girls (pFWE = 0.034), suggesting a masculinization of brain structures associated with visuospatial cognitive functions. After 10 months of testosterone treatment, girls with GD, similar to the control boys, showed increases in brain activation in areas implicated in mental rotation. LIMITATIONS: Since all girls with GD identified as gynephilic, their resemblance in spatial cognition with the control boys, who were also gynephilic, may have been related to their shared sexual orientation rather than their shared gender identity. We did not account for menstrual cycle phase or contraceptive use in our analyses. CONCLUSION: Our findings suggest atypical sexual differentiation of the brain in natal girls with GD and provide new evidence for organizational and activational effects of testosterone on visuospatial cognitive functioning.

Roles of sex and gonadal steroids in mammalian pheromonal communication.

A brain circuit (the accessory olfactory system) that originates in the vomeronasal organ (VNO) and includes the accessory olfactory bulb (AOB) plus additional forebrain regions mediates many of the effects of pheromones, typically comprised of a variety of non-volatile and volatile compounds, on aspects of social behavior. A second, parallel circuit (the main olfactory system) that originates in the main olfactory epithelium (MOE) and includes the main olfactory bulb (MOB) has also been shown to detect volatile pheromones from conspecifics. Studies are reviewed that point to specific roles of several different steroids and their water-soluble metabolites as putative pheromones. Other studies are reviewed that establish an adult, 'activational' role of circulating sex hormones along with sex differences in the detection and/or processing of non-steroidal pheromones by these two olfactory circuits. Persisting questions about the role of sex steroids in pheromonal processing are posed for future investigation.

Click-evoked otoacoustic emissions in children and adolescents with gender identity disorder.

Click-evoked otoacoustic emissions (CEOAEs) are echo-like sounds that are produced by the inner ear in response to click-stimuli. CEOAEs generally have a higher amplitude in women compared to men and neonates already show a similar sex difference in CEOAEs. Weaker responses in males are proposed to originate from elevated levels of testosterone during perinatal sexual differentiation. Therefore, CEOAEs may be used as a retrospective indicator of someone's perinatal androgen environment. Individuals diagnosed with Gender Identity Disorder (GID), according to DSM-IV-TR, are characterized by a strong identification with the other gender and discomfort about their natal sex. Although the etiology of GID is far from established, it is hypothesized that atypical levels of sex steroids during a critical period of sexual differentiation of the brain might play a role. In the present study, we compared CEOAEs in treatment-naïve children and adolescents with early-onset GID (24 natal boys, 23 natal girls) and control subjects (65 boys, 62 girls). We replicated the sex difference in CEOAE response amplitude in the control group. This sex difference, however, was not present in the GID groups. Boys with GID showed stronger, more female-typical CEOAEs whereas girls with GID did not differ in emission strength compared to control girls. Based on the assumption that CEOAE amplitude can be seen as an index of relative androgen exposure, our results provide some evidence for the idea that boys with GID may have been exposed to lower amounts of androgen during early development in comparison to control boys.

Stress during pubertal development affects female sociosexual behavior in mice.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice.

Although the participation of sex hormones and sex hormone-responsive neurons in aggressive behavior has been extensively studied, the role of other systems within the hypothalamus-pituitary-gonadal (HPG) axis remains elusive. Here we assessed how the gonadotropin-releasing hormone (GnRH) and kisspeptin systems are impacted by escalated aggression in male mice. We used a combination of social isolation and aggression training (IST) to exacerbate mice's aggressive behavior. Next, low-aggressive (group-housed, GH) and highly aggressive (IST) mice were compared regarding neuronal activity in the target populations and hormonal levels, using immunohistochemistry and ELISA, respectively. Finally, we used pharmacological and viral approaches to manipulate neuropeptide signaling and expression, subsequently evaluating its effects on behavior. IST mice exhibited enhanced aggressive behavior compared to GH controls, which was accompanied by elevated neuronal activity in GnRH neurons and arcuate nucleus kisspeptin neurons. Remarkably, IST mice presented an increased number of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV). In addition, IST mice exhibited elevated levels of luteinizing hormone (LH) in serum. Accordingly, activation and blockade of GnRH receptors (GnRHR) exacerbated and reduced aggression, respectively. Surprisingly, kisspeptin had intricate effects on aggression, i.e., viral ablation of AVPV-kisspeptin neurons impaired the training-induced rise in aggressive behavior whereas kisspeptin itself strongly reduced aggression in IST mice. Our results indicate that IST enhances aggressive behavior in male mice by exacerbating HPG-axis activity. Particularly, increased GnRH neuron activity and GnRHR signaling were found to underlie aggression whereas the relationship with kisspeptin remains puzzling.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

The development of female sexual behavior requires prepubertal estradiol.

The classic view of brain and behavioral sexual differentiation holds that the neural mechanisms controlling sexual behavior in female rodents develop in the absence of ovarian sex hormone actions. However, in a previous study, female aromatase knock-out (ArKO) mice, which cannot convert testosterone to estradiol, showed deficient male-oriented partner preference and lordosis behaviors in response to adult ovarian hormones, raising the possibility that estradiol may contribute to the development of these female sexual behaviors. In the present experiments, administering estradiol prepubertally [between postnatal day 15 (P15) and P25] significantly enhanced the ability of ArKO female mice to display lordosis behavior in response to ovarian hormones administered later in adulthood, whereas treatment with estradiol over an earlier postnatal period (P5-P15) had no such effect. Treatment of ArKO females with estradiol between P15 and P25 also rescued their later preference to approach distal cues from an intact male over an estrous female. ArKO females also displayed significantly less female-directed (male-typical) mounting behavior than wild-type control females when treated with testosterone in adulthood. Prepubertal estradiol treatment failed to reverse this deficit in ArKO females, whereas earlier postnatal estradiol augmented later mounting in both genotypes. Our results provide new evidence for an organizing role of prepubertal estradiol in the development of neural mechanisms that control female-typical sexual behavior.

Postnatal and adult exposure to estradiol differentially influences adult neurogenesis in the main and accessory olfactory bulb of female mice.

Neurons incorporated into the adult main olfactory bulb (MOB) and accessory olfactory bulb (AOB) derive from the subventricular zone (SVZ). Despite some recent studies on the role of olfactory neurogenesis in sociosexual behaviors mediated by hormones, data on the implication of estrogens are still lacking. Taking advantage of female aromatase-knockout (ArKO) mice, which are unable to produce estradiol across their life span, we investigated the role of estradiol exposure during early postnatal and adult periods on adult neurogenesis in the MOB and AOB. We found that proliferation of progenitor cells in the adult female SVZ was not influenced by estradiol. However, whereas adult exposure to estradiol influences the turnover of MOB newborn neurons, the survival of those in the AOB depends on exposure to estradiol during the early postnatal period. Finally, based on their expression of Zif268, we showed that newborn neurons in the MOB responded to sociosexual odors, albeit to a lesser extent in ArKO females, suggesting a contribution of estradiol during the early postnatal period to this response. Together, these results suggest that the survival and functional integration of newborn neurons in the adult female MOB and AOB are differentially influenced by estrogens from the early postnatal period to adulthood.

The role of steroid hormones in the sexual differentiation of the human brain.

Widespread sex differences in human brain structure and function have been reported. Research on animal models has demonstrated that sex differences in brain and behavior are induced by steroid hormones during specific, hormone sensitive, developmental periods. It was shown that typical male neural and behavioral characteristics develop under the influence of testosterone, mostly acting during perinatal development. By contrast, typical female neural and behavioral characteristics may actually develop under the influence of estradiol during a specific prepubertal period. This review provides an overview of our current knowledge on the role of steroid hormones in the sexual differentiation of the human brain. Both clinical and neuroimaging data obtained in patients with altered androgen levels/actions (i.e., congenital adrenal hyperplasia or complete androgen insensitivity syndrome [CAIS]), point to an important role of (prenatal) androgens in inducing typical male neural and psychosexual characteristics in humans. In contrast to rodents, there appears to be no obvious role for estrogens in masculinizing the human brain. Furthermore, data from CAIS also suggest a contribution of sex chromosome genes to the development of the human brain. The final part of this review is dedicated to a brief discussion of gender incongruence, also known as gender dysphoria, which has been associated with an altered or less pronounced sexual differentiation of the brain.

Neuroendocrine regulation of female aggression.

Classically the neurobiology of aggression has been studied exclusively in males. Thus, females have been considered mildly aggressive except during lactation. Interestingly, recent studies in rodents and humans have revealed that non-lactating females can show exacerbated and pathological aggression similarly to males. This review provides an overview of recent findings on the neuroendocrine mechanisms regulating aggressive behavior in females. In particular, the focus will be on novel rodent models of exaggerated aggression established in non-lactating females. Among the neuromodulatory systems influencing female aggression, special attention has been given to sex-steroids and sex-steroid-sensitive neuronal populations (i.e., the core nuclei of the neural pathway of aggression) as well as to the neuropeptides oxytocin and vasopressin which are major players in the regulation of social behaviors.

Potential contribution of prenatal estrogens to the sexual differentiation of mate preferences in mice.

The neural mechanisms controlling sexual behavior are sexually differentiated by perinatal actions of gonadal hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estrogens, that exposure to prenatal estrogens completely defeminized their potential to show lordosis behavior in adulthood. Therefore, we determined here whether mate preferences were also affected in female AFP-KO mice. We observed a robust preference for an estrous female over an intact male in female AFP-KO mice, which were ovariectomized in adulthood and subsequently treated with estradiol and progesterone, whereas similarly treated WT females preferred the intact male over the estrous female. Gonadally intact WT males preferred the estrous female over the male, but only when visual cues were blocked by placing stimulus animals behind opaque partitions. Furthermore, when given the choice between an intact male and a castrated male, WT females preferred the intact male, whereas AFP-KO females showed no preference. Finally when given the choice between an estrous female and an ovariectomized female, WT males preferred the estrous female whereas AFP-KO females preferred the ovariectomized female or showed no preference depending on whether they could see the stimulus animals or not. Taken together, when AFP-KO females are tested under estrous conditions, they do not show any male-directed preferences, indicating a reduced sexual motivation to seek out the male in these females. However, they do not completely resemble males in their mate preferences suggesting that the male-typical pattern of mate preferences is not solely organized by prenatal estrogens.

Kisspeptin and neurokinin B expression in the human hypothalamus: Relation to reproduction and gender identity.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are at the core of reproductive functioning. GnRH released into the median eminence regulates the secretion of the gonadotropins from the anterior pituitary, which in turn activates gametogenesis and steroid synthesis by the gonads. The GnRH system displays functional sex differences: GnRH is secreted in pulses at a constant frequency in men, whereas in women, pulse frequency varies over the menstrual cycle. In both sexes, GnRH release is regulated by sex steroid hormones, acting at the level of the hypothalamus and the anterior pituitary in a classic feedback loop. Because GnRH neurons do not express sex steroid receptors, hormone effects on GnRH release are presumed to be mediated indirectly through other steroid-sensitive neuronal systems, which then converge onto GnRH cell bodies and/or terminals. Human genetic studies demonstrated that kisspeptin (KP) as well as neurokinin B (NKB) signaling are both potent regulators of GNRH secretion. In humans, postmortem studies using immunohistochemistry have shown that women have higher KP and NKB expression in the infundibular nucleus than men. Sex differences in KP expression are present throughout life, which is from the infant/prepubertal into the elderly period, whereas sex differences in NKB expression do not emerge until adulthood. KP and NKB are often coexpressed together with dynorphin by the same population of neurons, also known as KDNy neurons in other species. Indeed, significant coexpression between KP and NKB but not with Dynorphin has been observed thereby challenging the KDNy concept in humans. Female-typical expression of both KP and NKB were observed in the infundibular nucleus of trans women (male sex assigned at birth and female gender identity). Taken together, sex differences in KP and NKB expression most likely reflect organizational actions of sex steroid hormones on the developing brain but they also remain sensitive to circulating sex steroids in adulthood. The female-dominant sex difference in infundibular KP and NKB expression suggests that this brain region is most likely involved in both the negative and positive feedback actions of estrogens on GnRH secretion. Finally, the sex-reversal observed in KP and NKB expression in trans women might reflect, at least partially, an atypical sexual differentiation of the brain.