Neurodevelopmental Blueprint of Gender: From Embryo to Identity -Bridging Biology and Beyond

Genetic and chromosomal factors

more than just XX or XY. Sexual differentiation is a complex and multifactorial process that involves not only sex chromosomes (allosomes) but also many genes located on autosomes.^[6] It refers to the developmental events that guide the formation of testes or ovaries from an initially “bipotential” or undifferentiated gonadal structure.^[7] This process begins around the 7th week of embryonic life. At first, both sexes share similar genital precursors, but transcription factors act as critical switches that direct gonadal development. Initially, two pairs of ducts (mesonephric and paramesonephric) are present; depending on genetic and hormonal influences, one set proliferates while the other regresses.^[6]

XY pathway and testicular development

In those carrying an XY chromosomal set, expression of the ‘SRY gene’ drives the transformation of primitive gonadal tissues into testes. These testes form Leydig and Sertoli cells.^[6]

Leydig cells produce testosterone, which binds to androgen receptors and stabilizes the mesonephric (Wolffian) duct, driving male reproductive development.^[8] Expression of ‘steroidogenic factor’ marks one of the earliest events in the gonadal cell differentiation.^[9] Sertoli cells, under the influence of SRY, SOX9, and steroidogenic factor 1 (Sf1), release anti-Müllerian hormone (AMH), leading to the regression of the paramesonephric (Müllerian) ducts, thereby preventing female reproductive structures from forming.^[10,11]

XX pathway and ovarian differentiation

Conversely, in XX embryos, the lack of SRY, testosterone, and AMH leads to a distinct development trajectory. The paramesonephric ducts develop into female reproductive structures, while the mesonephric ducts degenerate in the absence of testosterone.^[6] Genes such as Wnt4 (Wingless-related integration site gene) and Rspo1 (R-spondin 1)—key players in the Wnt signaling pathway—become more active, suppressing SRY-box transcription factor 9 (SOX9) and promoting ovarian development.^[12] Mutations in Rspo1 can disrupt this balance, sometimes leading to conditions like XX testicular differences in sexual development (DSD).^[13]

However, even receptor level or hormonal resistance can show variations in sexual development as seen in Androgen Insensitivity Syndrome.

Disorder of sexual differentiation (androgen insensitivity syndrome )

DSDs represent a spectrum of variations in sex development.^[6] Androgen insensitivity syndrome (AIS) is one such condition, caused by mutations or deletions in the gene regulating androgen receptor (AR) activity.^[14,15] In AIS, although the individual has XY chromosomes, tissues cannot respond fully to androgens. This may result in a female phenotype, ambiguous genitalia, or a spectrum of presentations depending on the extent of receptor sensitivity.^[6]

Interestingly, genetic studies also suggest associations between certain gene variations and gender diversity. For example, studies have shown that women with non cis gender identities tend to have longer cytosine–adenine– guanine (CAG) repeats in the gene encoding the androgen receptor compared to cis gender women.^[16] Likewise, a specific mutated variant of the CYP17 gene, which regulates sex hormone production, was observed more commonly in men with non cis gender identity.^[17] While no single “ gene” has been pinpointed as definitive, these observations suggest the role of complex genetic and hormonal interactions in shaping gender identity.

Thus, biological sex differentiation is not determined solely by chromosomes (XX or XY). Instead, it involves an intricate interplay of genes, hormones, and developmental pathways that begin in early fetal life. This complexity underscores the natural diversity in human sex and gender development, moving beyond a simplistic binary model.

Endocrine disruptors

The United States Environmental Protection Agency (EPA) defines an endocrine-disrupting compound (EDC) as “any substance that interferes with the synthesis, secretion, transport, binding, or metabolism of natural hormones that regulate homeostasis, reproduction, growth, development, and behavior”.^[18] These compounds are diverse^[19] and broadly categorized into two groups.^[20]

1. Naturally occurring EDCs – such as phytoestrogens.

2. Synthetic EDCs – including chemicals and secondary products generated through industrial processes, plastics, plasticizers, pesticides, fungicides, and certain pharmaceutical agents.^[20,21]

While much of the evidence regarding the effects of EDCs comes from animal studies, human data remain limited. Compounds such as dichlorodiphenyltrichloroethane, bisphenol A (BPA), and di (2-ethylhexyl) phthalate (DEHP) have been shown to cause epigenetic modifications, including histone changes, DNA methylation, and altered expression of non-coding RNAs.^[22-24] These findings raise the possibility that environmental exposures may influence brain sexual differentiation, particularly when exposure occurs during sensitive developmental stages.^[25] For example, pregnant mice exposed to BPA at doses below current human safety limits produced offspring with long-lasting alterations in sex-related behaviors.^[26,27] Moreover, some of these effects appear to be transgenerational, affecting subsequent generations even without direct exposure, suggesting an epigenetic mode of inheritance.^[28-31] At present, these observations remain hypotheses, and no definitive conclusions can be drawn from the available evidence.

These findings suggest that environmental exposures may add another layer of complexity to the biological foundations of gender identity, complementing genetic and hormonal pathways.

Neuroanatomical correlates

Research on gender identity has explored several domains, including structural neuroanatomy, functional neuroimaging, genetics, and prenatal hormone exposure.^[3] A common limitation across studies is the relatively small sample sizes and the assumption that gender-diverse individuals form a homogeneous group, when in fact they represent multiple subgroups.^[3,32] An example of a few such anatomical sites has been mentioned below-

Bed nucleus of the stria terminalis (BSTc): One of the earliest and most consistently studied regions is the Bed Nucleus of the Stria Terminalis (BSTc). Differences are noted between cisgender males, cisgender females, and transgender women in ‘the bed nucleus of the stria terminalis (BSTc)’. In trans women,both ‘BSTc’ size and neuronal count resemble those of typical females.^[33] ‘Somatostatin-expressing neurons’ in the ‘BSTc’ follow a similar pattern: trans women and typical females have comparable numbers, while typical males have nearly twice the count compared to females. Trans men show counts similar to typical males. These findings appear independent of hormone therapy.^[34]

Interstitial nucleus of the anterior hypothalamus (INAH3 and INAH4): Building upon these early observations, subsequent research examined other hypothalamic nuclei -particularly the Interstitial nuclei of the anterior hypothalamus. These nuclei demonstrate sexual dimorphism. ‘INAH3’ volume and neuron count are greater in cisgender men than in cisgender women. In transgender women, values align more closely with cisgender women, while transgender men resemble cisgender men—even after discontinuation of testosterone therapy for several years, suggesting the effect is not merely hormonal.^[32] Similarly, the INAH4 shape differs by sex: elongated in cisgender and transgender men but spherical in cisgender women.^[32]

Kisspeptin system: Additional hypothalamic systems, such as the kisspeptin network, provide further insight into neuroendocrine integration in gender identity formation. Postmortem studies have shown that kisspeptin-expressing neurons in the infundibular nucleus were comparable between cisgender men and transgender men, suggesting that brain sex characteristics and body sex can develop along different trajectories.^[35]

In functional neuroimaging: All the above-mentioned studies present static observations, whereas functional neuroimaging studies extend this understanding by exploring how these neural circuits operate dynamically in living individuals.Resting-state fMRI studies in adolescents with GD have shown connectivity patterns that diverge from their birth-assigned sex and more closely resemble those typical of their experienced gender. Interestingly, such differences are not observed in prepubertal children, implying that these brain connectivity patterns may emerge during puberty.^[36,37]

Together, structural and functional neuroimaging findings suggest that gender identity is rooted in neurodevelopmental organization rather than purely environmental learning. This neurobiological evidence is complemented by genetic and familial studies that further illuminate inherited influences.

Genetics study and twin evidence

The role of genetics in gender identity has been examined through family and twin studies.^[3] Early reports described parent-child and sibling pairs where more than one family member identified with GD, suggesting a possible genetic influence.^[38] However, larger studies indicate that while familial occurrence exists, it is relatively rare.^[39] Twin studies provide stronger evidence: concordance rates for GD are higher in monozygotic twins compared to dizygotic twins, consistent with a genetic contribution.^[3,40,41]

Cytogenetic research has produced mixed results. A large study in Spain reported a five-fold increase in chromosomal variations among transgender women and men versus the general population, while another study in adolescents found no such increase.^[42-44] Taken together, genetic data support biological predisposition but indicate that genes interact with environmental and hormonal factors in a complex way.

Prenatal hormone exposure: Prenatal androgen exposure has been suggested as one factor influencing later gender-related expression.^[3] One commonly studied proxy for such exposure is the 2D:4D finger length ratio, considered an indirect marker of prenatal androgen levels.^[45] One study examined 96 individuals recorded as male at birth and 51 recorded as female at birth who had ‘GD ‘, and compared them with 90 male-at-birth and 112 female-at-birth controls without the condition.The research also included a comparison of 67 boys and 34 girls with ‘GD’ against 74 boys and 72 girls without the condition, highlighting specific differences between the groups. Among assigned males, both in adult and child groups, analysis of the 2D:4D ratio revealed no meaningful difference between the GD and control groups. However, among assigned females, adults with GD showed a more “masculinized” digit ratio compared to controls.^[46] This has been interpreted as suggestive of differing prenatal hormone exposure in this group. Other investigations have speculated that reduced prenatal testosterone may be involved in the origin of ‘GD’ in assigned females, though similar findings have not been consistently demonstrated in birth-assigned males.^[47] While not definitive, these results support the possibility that variations in prenatal androgen exposure may have an impact on the development of gender identity.^[3] Although biological determinants lay the foundation, the final consolidation of gender identity is also shaped by early life experiences, social affirmation, and cultural context.

Psychosocial perspectives: Psychosocial influences on gender identity development involve a combination of parental responses, cognitive development, associated psychosocial conditions, and underlying psychodynamic factors. Parents often react to early cross-gender behavior with neutrality or mild encouragement, perceiving it as a harmless phase until it becomes persistent or socially concerning.^[48] Young children’s incomplete grasp of gender constancy – mistaking gender for outward expressions like clothing or activities – may contribute to ongoing confusion about gender roles.^[49,50] Emotional or behavioral problems, including anxiety, and family stress. Or autism spectrum traits can intensify gender related behavior by fostering rigid thinking or obsessive interests.^[51,52] From a psychodynamic perspective, unresolved parental conflicts or traumatic experiences can unconsciously influence a child’s gender identity. This signifies that gender identity evolves through a complex interaction of social, cognitive, emotional, and familial processes.^[53]