Background
Substantial evidence supports models of autism spectrum disorder (ASD) as a condition characterized by altered brain connectivity [
1,
2]. Yet, the striking heterogeneity of ASD has challenged efforts to profile the mechanisms underlying ASD-related dysconnections. One significant source of heterogeneity in ASD is biological sex [
3,
4], as highlighted by the 3–4:1 male preponderance in prevalence [
5,
6]. However, the impact of biological sex on ASD-related dysonnections remains poorly understood. In recent years, resting-state functional magnetic resonance imaging (R-fMRI) has demonstrated its feasibility in capturing typical sex differences in various aspects of the intrinsic connectome [
7‐
9] and has emerged as a robust tool for substantiating the functional dysconnectivity hypothesis of ASD [
1,
2]. This has motivated initial R-fMRI studies to characterize the contribution of biological sex to the neurobiology of ASD [
10,
11]. Preliminary evidence suggests that both sex-dependent and sex-independent dysconnections coexist in ASD [
12].
Here, in considering sex-dependent factors, we focused on investigating the neurophenotypic convergence of ASD-related characteristics and typical sex differences [
13,
14]. Our focus was motivated by (1) genetic evidence emphasizing shared mechanisms between vulnerability to ASD and typical sexual differentiation [
15] and (2) two current theoretical models whose predictions on neurophenotypic convergence can be assessed with neuroimaging. One model is the Extreme Male Brain (EMB) theory, which emerges from observations at the cognitive-behavioral level that individuals with ASD, regardless of sex, show “masculinized” or “hypermasculinized” profiles of empathizing and systemizing [
16,
17]. Neurophenotypic prediction from the EMB model is that ASD brain characteristics will be associated with enhanced brain maleness (i.e.,
shift-towards-maleness) in both males and females with ASD. The other model is the Gender Incoherence (GI; [
18]) theory. This is grounded on anthropometric and physiological findings that adult males and females with ASD are more androgynous than same-sex neurotypical peers [
18]. The neurophenotypic prediction of the GI model is that a
shift-towards-maleness is limited to females with ASD and that males with ASD instead show a
shift-towards-femaleness.
While neuroimaging studies have begun to consider the mechanisms underlying sex differentiation in ASD in the context of these two models [
14], only two studies have focused on intrinsic functional properties of the connectome. Both R-fMRI studies converged on a neural shift-towards-maleness in females with ASD, consistent with a prior volumetric study [
3]. However, these earlier studies diverged in regard to males with ASD. Ypma et al. [
11] reported a shift-towards-maleness within the default network (DN) in individuals with ASD regardless of their biological sex—a pattern consistent with EMB predictions. Results showed that both males and females with ASD exhibited hypo-connectivity within DN compared to typical males, who, in turn, showed lower intrinsic functional connectivity (iFC) than typical females. Alaerts et al. [
10] found a similar pattern of a shift-towards-maleness in females with ASD in the voxel-wise iFC of the posterior superior temporal sulcus and posterior cingulate cortex (PCC). However, they also observed a shift-towards-femaleness in males with ASD—as predicted by GI. The disparities in these R-fMRI studies may reflect their moderate sample sizes. They employed a two-factorial design (i.e., testing sex-by-diagnosis interactions), with sample size generally including
n ~ 40 individuals per cell. This is due to the scarcity of available ASD female datasets even in large repositories such as the Autism Brain Imaging Data Exchange (ABIDE).
Conjunction (spatial overlap) analyses can be a useful alternative and complementary approach in the identification of potential associations between ASD-related atypicalities and typical sex difference maps [
3,
14]. This is particularly true in instances of limited availability of female datasets. Factorial designs can provide information about factors related to typical sex differentiation in regions that
differ between males and females with ASD relative to NT males and females (i.e., by testing for regions showing significant sex-by-diagnosis interactions) and brain regions showing main effects of sex (ignoring diagnostic group) or ASD (ignoring sex). However, it is still possible that regions showing ASD-related atypicalities do not differ between males and females with ASD relative to NT males and females but are still related to typical sex differentiation. These nuances can be captured in the examination of specific spatial overlap scenarios of effects predicted by hypothetical links between ASD atypicalities (separately for ASD males and ASD females, whenever data is available) and typical sex differences.
We applied conjunction analyses to the large ABIDE I male R-fMRI dataset, thus allowing to address concerns on sample size as well as prior inconsistencies in sex-dependent intrinsic functional brain properties in males with ASD [
10,
11]. We focused on five key whole-brain voxel-wise R-fMRI indices in regard to (a) typical sex differences in a neurotypical (NT) sample from the 1000 Functional Connectome Project (FCP) [
19] and (b) ASD-related differences among males from ABIDE I [
1]. We conducted an unbiased whole-brain exploration given reports of multiple networks involved in ASD including and beyond DN. To ensure that findings were not dependent on the specific NT sample employed, we examined replicability using the Brain Genomics Superstruct Project (GSP) [
20] sample.
Discussion
To characterize the neurophenotypic convergence of ASD-related intrinsic brain characteristics and typical sex differences, we examined the spatial overlap between neurotypical sex-related and ASD-related intrinsic brain properties in large independent samples [
1,
19]. Results provided insights into two competing models of such convergence predicting either a shift-towards-maleness (EMB) [
16] or a shift-towards-femaleness (GI) [
18] in males with ASD. Across R-fMRI metrics, analyses revealed evidence consistent with both models, yet involving distinct functional neural networks. A shift-towards-maleness in males with ASD mostly involved DN and FP networks serving higher-order socio-emotional and cognitive control processes. In contrast, a shift-towards-femaleness in males with ASD predominantly centered around the SM network. These patterns remained stable across analytical strategies adjusting for differences in preprocessing pipelines and samples. The results suggest that previously reported R-fMRI abnormalities in males with ASD may partly result from atypical sexual differentiation in the brain, and these mechanisms act in a network-specific manner.
In typical individuals, a mosaic of brain region-specific masculinization and feminization exists across the sexes [
42‐
45]. Our findings of coexisting shift-towards-maleness and shift-toward-femaleness of intrinsic brain properties in males with ASD suggest that biological mechanisms involved in sex mosaicism partly contribute to the neural characteristics of ASD. These may involve hormonal and non-hormonal factors [
44,
46]. For example, estradiol can induce opposite effects in distinct brain regions by either initiating or preventing cell death and synaptogenesis, as well as by enhancing or dampening excitation [
47]. Beyond hormonal factors, brain regional sexual differentiation is also driven by sex differential gene expression [
46,
48]. Recent studies have shown that male differential expression of astrocyte and microglial genes are upregulated in ASD [
13]. How these molecular phenomena affect the macro-scale intrinsic functional brain organization should be the focus of multilevel approaches that generate a unifying model of the relation between typical sex differentiation and ASD.
In regard to the macro-scale networks and functional processes involved, our most consistent finding was the shift-towards-maleness of DN, a network widely implicated in ASD [
11,
23,
49‐
51]. In line with prior work [
10,
11], we found that significant overlaps with typical sex differences encompassed PCC-iFC decreases along the DN midline. By extending our exploration to the whole brain, analyses revealed similar shift-towards-maleness exist for a range of intrinsic properties in the PCC. These included ASD-related decreases in local connectivity (ReHo) [
52], homotopic inter-hemispheric interactions (VMHC) [
30], and fractional amplitude of low frequency fluctuations (fALFF) [
32]. These findings suggest that examinations of DN in ASD should consider sex-dependent biological factors.
Beyond DN ASD-related R-fMRI decreases, a shift-towards-maleness also involved other processes, especially ASD-related increases in FP local connectivity (ReHo). Notably, DN and FP networks subserve higher-order processes that are core to EMB theory’s postulation that individuals with ASD are weaker “mentalizers” and stronger “systemizers” [
16,
17,
53]. Consistent with the DN’s role in social cognition and mentalizing, the shift-towards-maleness of atypical intrinsic properties in ASD mapped onto cognitive components associated with theory of mind and emotion recognition processes. Impairments in these domains are referred to as atypical mentalizing and characterize individuals with ASD [
53]. On the other end, the FP network mapped onto inhibitory control and cognitive flexibility. Impairments in these domains have been attributed to weak central coherence, perseveration, and hyper-systemizing in ASD [
54].
Our systematic examination of the intrinsic functional brain also revealed evidence of a shift-towards-femaleness in the male ASD brain in the SM network comprising motor and auditory cortices. Altered sensory-motor processing has often been observed in ASD [
55,
56], and associated atypical intrinsic brain properties are emerging [
57‐
61]. Our findings suggest that a biological shift-towards-femaleness in the SM network may underlie these atypicalities [
62,
63]. Alternatively, a SM shift-towards-femaleness might result from experience (e.g., being less engaged in motor activities). Longitudinal studies are required to clarify the impact of experiential factors [
64], sex-specific biological factors, and their interactions. Finally, although in NT individuals motor and language processes are hemispherically specialized, NT females have greater bi-hemispheric integration compared to NT males [
65]. Given prior reports of a reduction of typical asymmetries in males with ASD [
58,
66,
67], our findings of a shift-towards-femaleness involving motor and auditory domains in males with ASD suggest that biological sex-related factors are likely involved in atypical inter-hemispheric interactions in this population.
With respect to specific R-fMRI features and atypical ASD-related differences, we note that shift-towards-femaleness or shift-towards-maleness in ASD did not affect any of the metrics differentially. Instead, echoing recent large-scale studies reconciling prior mixed findings of hypo- and hyper-connectivity in ASD [
1,
24], results varied by the functional network involved. Our findings further suggest that sex-related biological factors contribute to the complex presentation of atypical iFC in ASD.
Results should be interpreted considering several limitations. First, a sufficiently large dataset was only available for males with ASD. While focusing on a large ASD male sample allowed us to address prior inconsistencies about a shift-towards-femaleness in males with ASD [
10,
11], future large-scale characterizations of both females and males will provide insights into the role of typical sexual differentiation in ASD for both sexes [
14,
68]. Second, while patterns of shift-towards-maleness and shift-towards-femaleness were similar across analytical strategies, aspects of the shift-towards-femaleness were more variable between the primary strategy and those adjusting for differences in age range. Speculatively, the shift-towards-maleness in ASD may therefore be related to
organizational effects on neurodevelopment, as predicted by the EMB model that posits elevated prenatal steroidogenic processes [
16,
69,
70]. An ASD-related shift-towards-femaleness in males could reflect later events even during and beyond puberty. This would be in line with the GI model which was mostly conceptualized based on postpubertal physiological measures [
18]. As such, a shift-towards-femaleness may be more variable depending on the sample age. A cross-sectional examination of age effects would require a larger and more homogenous age distribution across data acquisition sites than the present one. Nevertheless, as we included age as a nuisance covariate in the statistical models used to generate the
Z-maps overlapped, potential confounds on the present results are limited. Third, it was not possible to address the role of comorbid psychiatric conditions in our findings due to limited availability of this information across the ABIDE I datasets. Many comorbid conditions within ASD, such as attention deficit hyperactivity disorder, show a sex-biased prevalence ratio themselves [
71,
72]. Further, recent studies considering comorbidities in individuals with ASD show brain connectivity patterns that are specific to ASD comorbidities [
27]. This calls for characterization of both ASD core and comorbid symptoms in neuroimaging studies of ASD. Finally, given that FCP and ABIDE I aggregate data retrospectively across multiple sites, unknown confounds due to site differences may exist. We limited this concern by including sites as covariates at the group-level comparisons generating
Z-maps used to assess spatial overlaps.