Open access peer-reviewed chapter
Abstract
Gender incongruence (GI) is defined as an individual’s discontent with their assigned gender at birth and their identification with a gender other than that associated with their sex based on physical sex characteristics. The origin of GI appears to be multifactorial. From the extensive research that has been conducted over the past few years, four main factors have been identified as key mechanisms: genes, hormones, epigenetics, and the environment. One of the current hypotheses suggests that GI could be related to a different sexual differentiation of the brain as a result of changes in the DNA sequence of the estrogen receptors ERs and androgen receptor AR genes. These changes in the DNA sequence would imply a variability in the sensitivity of the hormone receptors, causing a genetic vulnerability.
Keywords
- transgender
- cisgender
- healthcare
- gender incongruence
- gender dysphoria
1. Introduction
Chromosomal sex (established at the time of fertilization), gonadal sex (a direct result of the genetic complement), and brain sex (the result of genetic and hormonal actions) tend to be coincident, giving rise to the deep conviction of being male or female. But discrepancies between gender and the sex assigned at birth are also possible. Thus, gender identity could be defined as one’s personal conception of oneself as male, female, a blend of both, or neither [1, 2] that could be coincident or not, with the sex assigned at birth. According to this con- or discordance, we can differentiate into “cisgender” or “transgender” people, respectively [2]. Gender incongruence (GI) in the ICD-11 [3] is characterized by a pronounced and persistent discrepancy between the individual’s experience of gender and their sex assigned at birth.
The brain, like the gonads, is a sexually dimorphic organ, in such a way that genes located on the sex chromosomes will determine the sex of the brain [4], either indirectly by acting on the gonads, which, in turn, will produce different gonadal secretions according to the sex, or through the direct action of the sex chromosome complement XX or XY in brain cells [5].
Once the differentiation of the sexual organs is complete, and gonadal sex is established (ovaries versus testicles), the sexual differentiation of the brain will take place toward the second half of pregnancy. Then, the testicles begin to secrete testosterone, while the ovaries remain quiescent. Testosterone, which is also essential to complete the differentiation of the male sexual organs, will penetrate the brain and act through the androgen receptors (AR), or after its aromatization [6, 7, 8, 9] activating the estrogen receptors ERα and ERβ, respectively involved in masculinization and defeminization of the nervous system [10]. Exposure to testosterone, through activation of the AR during this critical period, is a prerequisite for masculinization of the brain, ensuring that gonadal sex coincides with cerebral sex. The organization of the brain by the action of hormones during this embryonic period, and the subsequent activating effects on sexual behavior in adult life, form the basis of the brain-behavior organizational and activation hypothesis [11].
Since the sexual differentiation of genitalia and the sexual differentiation of the brain occur at different times in intrauterine development, they may take different directions, and in that case, the degree of masculinization of the genitalia may not reflect the degree of masculinization of the brain, giving rise to individuals with XY karyotype and male genitalia, but with feminized brains, or individuals with XX karyotype and female genitalia, but with masculinized brains [12].
2. Mechanisms implicated in brain dimorphism
Transient perinatal exposure to testosterone or its metabolite, estradiol, causes many of the best-studied sex differences in rodent brains, and recent evidence suggests that epigenetic mechanisms underlie many of these hormonal effects [13, 14, 15, 16, 17]. For example, sex differences in the preoptic area of the hypothalamus are altered by injecting a methyltransferase inhibitor directly into the brain of newborn rats or mice during the critical period for sexual differentiation [18, 19]. Similarly, a neonatal disruption of histone acetylation inhibits the development of sex differences in copulatory behavior in male rats [20] and modifies the size of the bed nucleus of the stria terminalis (BNST) in mice, a region of the brain linked to male sexual behavior [21]. These findings suggest that sexual differentiation of the brain requires orchestrated changes in DNA methylation and histone acetylation [13].
In another approach, based on genome-wide studies, both histone methylation and DNA methylation patterns differ by sex in the mouse preoptic area [22, 23]. Testosterone treatment of newborn female mice partially masculinizes the DNA methylation pattern present in adulthood [23], and sex differences in methylation of specific genes are also reversed by neonatal steroid treatment in rats [24]. Therefore, steroid hormones alter the expression or activity of enzymes that place epigenetic marks [19, 25, 26], which may be the mechanism by which hormones affect the epigenome [13].
Another study in rodents infers the role of chromosomal and hormonal sex in brain epigenetics. In rats, mothers lick their newborns more frequently if they are male than if they are female [27], and the amount of maternal attention that a rat pup receives affects the degree of methylation of the estrogen receptor alpha ESR1 gene in the brain [28, 29]. Edelmann and Auger [30] randomly assigned some female newborns to receive the extra attention normally given to males by simulating anogenital licking with a brush. This, in fact, masculinized the methylation pattern of their DNA and modified the expression of the ESR1 gene in the cerebral amygdala of the treated females [30]. In this case, the differential mother’s care is based on the odor of the newborn’s urine [31], which in turn is due to differences in circulating testosterone, and thus to genetic and hormonal sex.
On the other hand, some sex differences in the brain are independent of gonadal hormones and are instead due to the complement of sex chromosomes itself [32, 33]. In this way, sex chromosomes alone influence the expression of epigenetic enzymes and cause sex differences in the epigenome of rodents and flies [32, 34]. Thus, based on animal studies, the two main determinants of biological sex (sex chromosomes and gonadal steroids) contribute to sex differences in the brain epigenome [13].
But in humans, information on sex differences in the brain epigenome is very limited. During some stages of human fetal development, male and female brains differ in both DNA methylation and hydroxymethylation [35]. Because these differences are seen before birth, and presumably before social influences, they are differences due to sex and hormones. There are also differences in epigenetic marks in the prefrontal cortex of adult men and women [36, 37, 38]. However, adults have had many experiences of gender, so it is not clear whether these differences are due to sex or gender.
3. Sexual dimorphism of the brain and gender incongruence
The hypothesis of sexual differentiation of the brain has also been studied by verifying to what extent the brain of people with GI agrees with their natal sex or with their gender [39]. Although the role of brain dimorphism in the development of GI remains unclear, it appears that it is the result of a combination of the effects of hormones in the brain, the expression of certain genes, and epigenetic factors. Due to the complexity of this combination, it is especially difficult to determine the degree of the implication that these elements have separately.
Some in vivo studies suggest that, in general, the brain morphology and cognitive performance of the transgender population show a remarkable congruence with that of their natal sex. However, most literature indicates that the structure and functioning of the brain of the transgender population are more consistent with their gender than with their natal sex and that the trend toward cerebral feminization in transgender women, and toward masculinization in the case of transgender men is an innate quality, independent of hormonal treatment [40].
Hahn et al. [41], in a study on brain connectivity networks in transgender people, found a decreased intra-hemispheric connectivity ratio for transgender women in the subcortical and limbic regions compared to the cisgender population. In the group of transgender men, they found a decrease in intra-hemispheric connectivity between the right subcortical/limbic areas, and in the right frontal and temporal lobes compared to the cisgender population and transgender women. Differences between transgender groups and the direction of brain connectivity suggest that GI is characterized by specific but distinct brain signatures for both transgender groups [41].
Research in the field of gender-affirming hormone treatment (GAHT) has provided much insight into the origin and development of brain sex differences, through the manipulation of gonadal steroids [40]. Sex hormones influence the morphology and functional organization of the brain not only during prenatal and peripubertal development, but studies of gender-affirming therapies have shown that sex hormones can affect the brain even when it is fully developed and that they do so in a non-uniform way. So that some structures tend to be modified in favor of the chosen gender, while others do not, or are located in an intermediate position. It has been found that, in transgender women undergoing GAHT, the volume of the amygdala, corpus callosum, and nucleus putamen do not differ from that of cisgender men, corresponding to their natal sex, while the right insular cortex and right temporal–parietal junction are larger than in the two cisgender groups. However, in transgender men, the third ventricle and the nucleus accumbens are different to in cisgender women, coinciding with the chosen gender and not with their natal sex. Cisgender men, like transgender women undergoing GAHT, have higher overall gray matter volume than cisgender women in the posterior superior frontal cortex, whereas both transgender women and transgender men have lower gray matter volume in the insula than cisgender women [40]. In another study, changes in testosterone levels in transgender men were found to be inversely correlated with gray matter volume in Broca’s and Wernicke’s areas after four weeks of GAHT [42]. Despite the differences in the results, in general, these studies indicate that transgender women present demasculinized patterns in terms of cortical thickness of the white matter microstructure, that transgender men present defeminized patterns, and that both, women and men transgender, have a characteristic and complex sexual differentiation in a mosaic form [39, 43].
4. Major studies on the genetic basis of gender incongruence
Regarding its etiology, GI is considered multifactorial and complex. Thus, there is not a single gene or a single factor that could explain GI. It might be associated with neurodevelopmental processes of the brain, as well as hormonal, genetic, and epigenetic factors, among other possible influences. The main studies on the genetic basis of GI have been focused on the genes involved in the sexual differentiation of the brain: the androgen receptor AR, the estrogen receptor α ESR1, the estrogen receptor β ESR2, the aromatase gene CYP19A1 and the CYP17A1 17-alpha-hydroxylase.
The scientific evidence accumulated in recent years points to a complex etiology with hormonal, genetic, epigenetic disruptors, and immunological mechanisms that cause a specific neuropsychological profile [17]. One of the current hypotheses suggests that GI could be related to a different sexual differentiation of the brain, not concordant with natal sex or sex assigned at birth, as a result of changes in the DNA sequence of the estrogen receptor α- β genes (ESR1 and ESR2) and the AR androgen receptor gene, as well as the CYP19A1 and the CYP17A1 genes [44]. These changes in the DNA sequence would imply a variability in the sensitivity of hormone receptors, causing a genetic vulnerability related to the production of hormone receptors that are more or less sensitive to their ligands (estrogens and androgens).
The ERα-β and AR receptors are proteins that bind their ligands (estrogens and androgens, respectively). These receptors are present in most of our cells (including neurons in the hippocampus and hypothalamus) and their presence allows cells to respond to steroid hormones. Generally, these receptors float in the cell cytoplasm in an inactive form, but when they bind to their ligand, they take on an active form (dimerization) that allows them to enter the cell nucleus and bind to specific DNA regions located near the promoter regions of numerous target genes, modulating the transcription of thousands of genes related to sexual development in a domino effect. Therefore, ER and AR are proteins that can act as hormone receptors and, at the same time, as transcriptional regulatory molecules [45]. All these ideas led to the suggestion of the possible involvement of these receptors in the genetic basis of GI.
The first study on the genetic basis of GI was conducted by Henningsson et al. [46], who analyzed for the first time three repeat polymorphisms located in the ERβ and AR receptor genes and the aromatase enzyme gene (CYP19A1) in a population of 29 transgender women. Specifically, they found longer polymorphisms (with a higher number of repeats) in the trans population. In addition, the logistic regression analysis indicated the existence of interactions between the three analyzed polymorphisms that increase the possibility of gender incongruity. In this way, the results obtained by Henningsson et al. [46] suggest that the risk of presenting GI is also influenced by the other polymorphisms (of the aromatase gene and the ERβ), but the contribution of these other genes is much greater in the presence of short AR alleles. In addition, they found that male carriers of less active AR receptors (long alleles) were more likely to show GI.
Later, Hare et al. [47] replicated the study of Henningsson et al. in a larger population of transgender women, also finding longer AR polymorphisms in the transgender population. However, when Ujike et al. [48] analyzed the same polymorphisms (and others) in a Japanese transgender population, they found no statistically significant data. But we must point out that Ujike et al. [48] incorporated small modifications into the analysis (they used the mean instead of the median to obtain the short and long alleles), which makes it impossible to compare their results with the other investigations.
These and other polymorphisms were also analyzed in a Spanish population. Thus, 974 transgender individuals were analyzed versus a cisgender population of 1,327 individuals [49, 50, 51]. The results confirmed the involvement of both ER α-β receptors in the genetic basis of GI. In addition, crossed associations were also found between the analyzed polymorphisms that were overrepresented in the transgender population [44].
In 2008, Bentz et al. [52] increased the list of the analyzed genes in the GI population with a study of the CYP17A1 gene in a transgender population from Northern Europe (Austria) consisting of 102 transgender women and 49 transgender men, who were compared to 1,671 cisgender individuals (756 men and 915 women) [52]. The results supported the association between the CYP17-rs743572 polymorphism and GI since the mutated allele (A2) was overrepresented in the transgender population compared to the cisgender population. Furthermore, the authors found a sex-dependent allelic distribution in the cis population.
In 2016, our group expanded the study of the CYP17-rs743572 polymorphism in a Spanish population with GI (317 transgender women, 223 transgender men, 264 cisgender women, and 358 cisgender men) [53]. Contrary to Bentz et al., in the Spanish population, the allelic and genotypic frequencies did not show statistically significant differences between the cis and transgender populations. Furthermore, the allelic and genotypic frequencies did not differ significantly between both cisgender groups, contrary to what Bentz et al. [52] had previously suggested. Our results, therefore, contradicted the involvement of the CYP17-rs743572 polymorphism in the genetic basis of GI, based not only on the analyzed population but also on data derived from the 1000 Genomes database and those obtained in a bibliographic review carried out specifically for this study.
The existing discrepancies between our work and the study by Bentz et al. (2008) are due to problems in the selection of the female cisgender sample in the Austrian study since the cis group comprised of women who had made medical consultations for perimenopausal disorders. In this sense, the statistical significance obtained by the Bentz group could be related to diseases dependent on the functioning of estrogens, but not with GI. In conclusion, we can state that, according to our data, the CYP17-rs743572 polymorphism is not associated with GI. Our research group did not confirm the involvement of this CYP17-MspA1 (rs743572) polymorphism or CYP19A1 (rs60271534) in the genetic basis of GI when analyzing a Caucasian sample of Spanish origin. Our results were later confirmed by other groups [54, 55].
Subsequently, the interaction analysis between polymorphisms through a logistic regression study showed the existence of an inverse allelic interaction between ERα and AR in a transgender women population. An association between ERα and ERβ was also found in the group of transgender men. These data confirmed the key role of ERβ in brain gender development in humans [44].
On the other hand, Ramírez et al. [56, 57] analyzed the involvement in GI of the activating molecules of the ERs and AR confirming their involvement in the sexual differentiation of the brain and the fundamental role that estrogens play in it. The authors analyzed 247 polymorphisms distributed in the coactivators NCOA-1, NCOA-2, NCOA-3, NCOA-4, NCOA-5, p300, and CREBBP in a population of 94 Spanish transgender individuals versus 94 Spanish cisgender individuals, with the same geographic origin, race, and biological sex. When they compared the distribution of the allele and genotype frequencies, they found significant differences in 11 polymorphisms that correspond to 4.45% of the total analyzed: three polymorphisms located in NCOA-1, five in NCOA-2, two in p300, and one in CREBBP.
These data are in concordance with a recent work that showed that the nuclear receptor coactivators, NCOA-1, NCOA-2, and p300, are essential for efficient ER transcriptional activity in the brain [58]. Moreover, Auger et al. [59] investigated the consequence of reducing NCOA-1 protein during sexual differentiation of the brain and reported that reducing this protein interferes with the defeminizing actions of estrogens in neonatal rat brains. Auger’s data indicated that NCOA-1 expression is critically involved in the hormone-dependent development of normal male reproductive behavior and brain morphology.
On the other hand, epigenetics offers a very interesting complement to genetic studies because it provides a relationship between genes and the environment. Epigenetic modifications determine which genes are expressed at each moment, in response to specific environmental stimuli. But so far, investigation of the implication of epigenetics in GI has been very limited. Two studies in adult transgender populations have shown that certain environmental factors, such as GAHT, modify the methylation profile of the promoter regions of the ESR1, ESR2, and AR genes [60, 61]. Furthermore, one analysis of global DNA methylation showed that there are differences in the methylome of the transgender population even before GAHT treatment [62]. The main finding of that work was that cis and trans populations have different global CpG methylation profiles, even before GAHT. When comparing trans woman versus cis men, 22 CpGs with significant methylation were located in islands. However, with respect to trans men, significant changes of methylation were found in only 2 CpGs. Furthermore, one of this CpGs, related to the MPPED2 gene, was shared by both transgender populations.
The most significant CpGs in trans women were related to genes WDR45B, SLC6A20, NHLH1, PLEKHA5, UBALD1, SLC37A1, ARL6IP1, GRASP, NCOA6, ABT1, and C17orf79. Among the most statistically significant CpGs, at least four of these genes were involved in brain development and neurogenesis (WDR45B, SLC6A20, NHLH1, and PLEKHA5), and three were related to transcriptional functions (NHLH1, NCOA6, and ABT1). Furthermore, the gene C17orf79 is related to chromatin organization and its activation stimulates the transcription of the AR. Finally, another two genes were related to glutamate synapses (ARL6IP1 and GRASP).
With respect to the MPPED2 gene, it is expressed in most human tissues, and the brain, in men and women, and is expressed predominantly in fetal brains. Furthermore, MPPED2 expression is modulated during development, attributing to this gene an important role in the processes of neuronal differentiation at the embryonic stage [63]. Cg23944405 related to the MPPED2 gene is hypermethylated in both trans populations before GAHT. Thus, the investigation of Ramírez et al. [62] tells us that epigenetics also plays an important role in the etiology of GI.
5. Mitochondrial genes and sexual dimorphism
Mitochondria are intracellular organelles that are fundamentally involved in energy generation processes. Mitochondria have their own genetic material made up of DNA, with certain peculiarities. Among them, it stands out that DNA is double-stranded, circular and occurs in multiple copies (>1000) in the same cell. More than 93% of mitochondrial DNA is coding (compared to only 1.5% of nuclear DNA) and its 37 genes are intron-free [64]. Another of the most relevant aspects of mitochondria is that they are only inherited from the mother since they are found in the cytoplasm of the egg fertilized by the sperm (which only provides the nucleus for the newly formed organism). Therefore, the mitochondrial genetic material of any individual is inherited exclusively through the mother.
Mitochondria perform various interconnected functions, producing ATP and biosynthetic intermediates that contribute to cellular stress responses such as autophagy and apoptosis. They produce ATP through oxidative phosphorylation (OXPHOS) and play a key role in global energy modulation. An increased need for ATP is satisfied by increasing mitochondrial mass and inducing OXPHOS. The regulation of mitochondrial biogenesis is closely coordinated with pathways that induce vascularization and improve oxygen delivery to tissues [65].
Mitochondrial functioning is also sexually dimorphic. Association studies in humans have revealed sex-specific quantitative trait loci (QTLs) that regulate the mitochondrial content of blood tissue [66].
Other work has also suggested that sex hormones play a role in regulating mitochondrial dynamics, metabolism, and cross-talk with other organelles. Specifically, the female sex hormone estrogen has both a direct and indirect role in regulating mitochondrial biogenesis through PGC-1α, a mitochondrial gene coactivator. On the other hand, testosterone is cardioprotective in men and can regulate mitochondrial biogenesis through PGC-1α and PGC-1α.
Both the estrogen receptor ER and the androgen receptor AR are associated with mammalian mitochondria. Estrogens are best known for being antiapoptotic in muscle and neural tissues in the event of stress. Data demonstrate that mammalian sex steroids are potent regulators of stress response at tissue and individual cell levels [67].
To our knowledge, no studies have been published about the role of mitochondria in the genetic basis of GI. Nevertheless, given the association between ER, AR, and mammalian mitochondria, our team deemed it interesting to analyze 242 mitochondrial single nucleotide polymorphisms (SNPs) in a transgender versus a cisgender population, and the results of the whole study are presented here.
The study was conducted in a population of 94 transgender individuals and 94 cisgender individuals, with similar characteristics of race, biological sex, and geographic origin. The inclusion criteria were: presenting gender incongruence according to ICD-11 and having no disorder of sexual development. The exclusion criteria for all participants were DSD (differences in sex development), neurological and hormonal disorder, major medical condition, and history of alcohol and/or drug abuse.
The cisgender population was selected from a country census (Pizarra, Málaga, Spain) matched by geographic origin, race, and sex. Written informed consent was obtained from the transgender group after a full explanation of the procedures. The study was approved by the UNED Ethics Committee.
The analyses were conducted by chromosomic sex, in two independent populations: individuals assigned as females at birth and individuals assigned as males at birth, considering significant a P-value lower than .05. The allele and genotype frequencies were analyzed by the x2 test. The strength of the associations with GI was measured by binary logistic regression, estimating the odds ratio (OR) for each genotype.
Statistically significant differences were found in 26 out of 242 mitochondrial polymorphisms (P ≤ 0.05), but only the Affx-34461653 polymorphism passed Bonferroni correction. This polymorphism is related to the MT-ND4 and MT-ND5 genes that are linked to the effects of oxidative phosphorylation [68]. MT-ND5 interacts with glutamine synthetase (GS) that predominates in the brain, kidney, and liver [69].
In the brain, glutamine synthetase participates in the metabolic regulation of glutamate, in the recycling of neurotransmitters, and the termination of their signals [70, 71]. Glutamine synthetase is an ATP-dependent enzyme found in most species that synthesizes glutamine from glutamate and ammonia. In the brain, glutamine synthetase is primarily located in astrocytes where it works to maintain the glutamate-glutamine cycle as well as nitrogen metabolism. More recent studies indicate that glutamine synthetase may also be present in other CNS cells, including neurons, microglia, and oligodendrocytes [69].
It is estimated that 95% of excitatory neurotransmission in the brain occurs in dendritic spines, and AMPA/kainate glutamate and NMDA receptors are found in a high proportion on the surface of these structures. Furthermore, in the adult mammalian brain, the expression of male sexual behavior correlates with high concentrations of extracellular excitatory glutamate in the preoptic area POA [72]. Blocking the NMDA receptor and, consequently, glutamatergic transmission in this brain region (POA) reduces male sexual behavior in mice, including the number of mounts and intromissions, and does not allow improvement of these measurements with experience [73], while increasing synaptic glutamate has the opposite effect, improving male sexual performance.
Therefore, given the theoretical importance of glutamatergic neurotransmission in adult male sexual behavior, the mitochondrial genes MT-ND4 and MT-ND5 could be involved in the genetic basis of GI. Thus, it has been shown that estradiol induces glutamate release in the hypothalamus to promote defeminization [9]. The ventromedial nucleus (VMN) located in the mediobasal hypothalamus (MBH) is a key brain region for the control of female sexual behavior in mice [74, 75]. Ventromedial nucleus (VMN) neuron dendrites in males branch more frequently and therefore generally have more synapses than females [76, 77, 78].
Estradiol induces both masculinization and defeminization in mice but through different cellular mechanisms. In the MBH, estradiol-induced defeminization begins with rapid (~1 hour) activation of ER-mediated PI3 kinase and enhanced release of presynaptic glutamate. The increase in synaptic glutamate leads to increased activation of postsynaptic NMDA receptors followed by dendritic branching and the construction and stabilization of dendritic spines [79]. These results demonstrate that estradiol-mediated brain sexual differentiation is not an autonomous cellular process in which only ER-containing neurons change morphology in response to steroid exposure. Instead, a neurotransmitter serves as a signaling factor that causes a morphological change in an entire network of cells, suggesting that all neural inputs to sexually differentiated brain regions, such as POA and VMN, are considered and interpreted according to the sex of the individual, regardless of whether the incoming signals are relevant to sex or other functions of the POA. Both the POA and the VMN are implicated in many other functions, including maternal behavior, temperature regulation, and feeding, to name a few [9].
6. Conclusions
In conclusion, our results have shown that mitochondria could play a role in the genetic basis of GI. Furthermore, our data continue to support the hypothesis that GI is a complex multifactorial trait, involving intricate interactions between sex steroids, sex steroid receptors, coactivators, and epigenetics. In addition, mitochondria now come into play as one more factor that could intervene in this complex process through the action of glutamate.
Furthermore, we can conclude that people who question their gender need protection against discrimination, high-quality services, and clear information. In addition to discovering the biological basis of GI, it is necessary to train health professionals to deal with GI. In conclusion, we can say that more studies are needed to give an adequate explanation of the factors associated with GI.
Acknowledgments
This work was supported by grants: Xunta de Galicia ED431 B 019/02 (EP), Ministerio de ciencia, innovación y universidades: PGC2018-094919-B-C21 (AG), and PGC2018-094919-B-C22 (RF, EP). None of these funding sources played any role in the writing of the manuscript or the decision to submit it for publication. We are grateful to everyone who contributed to the study, and to the trans and cis individuals who participated in particular.
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