Early steroid-mediated epigenetic influence can also be observed via genomic or non-genomic influences on neurodevelopmental processes/mechanisms thought to be atypical in autism, such as neurogenesis, apoptosis, neurotransmission and synapse formation/function and the immune system,26,27 and may also be important for understanding early pathophysiological mechanisms for autism.2 For example, steroid receptors upregulate Wnt signaling through their action on β-catenin,28 a molecule that has a crucial role in cell-to-cell adhesion. Atypical cell-to-cell adhesion could potentially underlie minicolumnopathy29 and other abnormalities at the synapse in autism.30 Interestingly, a prominent autism-candidate gene involved in synapse formation, NLGN4X, shows sex-biased differential exon usage that is particularly pronounced during fetal development.31Sex steroids also influence early developmental changes in GABAergic signaling.32Given known abnormalities in GABAergic signaling in autism, elevations in early fetal sex steroids may modulate GABAergic control of the excitatory/inhibitory balance in the developing autistic brain.33,34 Steroid hormones also interact with the immune system and a striking example of this is how early sex steroids influence sexual dimorphism in the brain and behavior through their effects on microglia activation.26 Many studies have observed a wide array of atypicalities in the immune system in autism.35 Converging with the current results, we previously found atypical chemokine and cytokine profiles in both amniotic fluid and neonatal bloodspots of individuals with a diagnosis of autism in the HBC.36, 37, 38 These arguments point to the need for further work investigating interactions between fetal programming effects of steroid hormones and other early pathophysiological mechanisms in autism.
The current results may also be relevant to the literature on prenatal stress and autism. We found that cortisol, a biomarker typically associated with stress, is elevated early in the fetal development of autism. Studies of prenatal stress in autism have been mixed and vary substantially in methodological detail.39,40 While the current results may suggest a link between prenatal stress and autism via heightened fetal cortisol, it is unclear if the association here is due to heightened prenatal stress or is driven by a more primary fetal sex steroid influence that has a side effect of boosting fetal cortisol levels. Furthermore, although cortisol is a prominent stress hormone at later points in development, it may have different functions at earlier stages of brain development. As mentioned earlier, unlike in adulthood, cortisol and sex steroids such as testosterone are positively related to each other in fetal development15,16 and this may suggest a more varied role for cortisol in early brain development. The current work is consistent with prior suggestions of interactions between the fetal hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes, and their known fetal programming effects on later atypical neurodevelopmental phenotypes.41
From a clinical standpoint, the current results say nothing about the potential for such data as a prospective prenatal test of autism risk. The current findings are based on average-level group differences that are useful for hypothesis testing but are not directly applicable for prediction of individual diagnoses. Further work examining the predictive utility of these and other fetal markers is necessary to evaluate their implications for clinical screening. Finally, the present results should not be used as empirical justification for treatments that target sex steroids. The androgen-blocker Lupron has already been inappropriately offered42 as a treatment for autism. Aside from the ethical and safety issues this type of treatment raises, there is little conceptual rationale to justify its use in autism since an elevation in steroidogenic activity in early (fetal) development has no direct implications for the use of androgen-blocking drugs later in life. ‘Organizational’ effects of sex steroids on early fetal brain development tend to create permanent, irreversible changes in how the brain is organized at the cellular level7,27 and such treatments much later in life may be incapable of reversing such effects.
This study has certain limitations. First, data from central registries do not allow for collection of detailed information about clinical symptoms to validate diagnoses against gold-standard instruments. However, other studies have shown that within the Danish Psychiatric Central Register the validity ratio for childhood autism cases is 94% for 1990–1999.43 Second, hormones were assayed from a historic collection of amniocentesis samples stored over several years at −20 °C. Analyte concentration levels are known to change over time, especially when the time period stretches over 10 years.19 However, for the cohort analyzed in this study (born between 1993 and 1999), storage-time-dependent changes are unlikely to be a major issue. For instance, groups did not systematically differ in storage time and inclusion of this and other key variables as covariates did not change any inferences. Furthermore, concentration levels of testosterone were in line with levels typically observed across the literature and robust sex differences in testosterone were apparent as was the known prenatal surge peaking around gestational weeks 14–16 (see Supplementary Figure 7). In addition, the use of PCA-enabled unsupervised separation of various sources of variation in the data and highlighted the latent steroidogenic component. Even if variation due to storage-time-dependent changes affected the data from the first principal component of our analyses, there is no reason to suspect a bias toward inflating the concentration levels in one group relative to the other. Aside from PCA, a multivariate analysis of variance on the raw concentration levels also revealed elevations in the autism group. This suggests that while some storage-time-dependent changes are unavoidable in historic collections, it did not prevent our ability to test the main hypothesis. Finally, the prevalence estimate of autism within this amniocentesis cohort (0.8%) is consistent with prevalence estimates of autism in the Danish population during the same time period.44 Thus, although these results are derived from a selected sample, they may be representative of what would be expected in the general population. Nevertheless, future work on a sample more representative of the general population is necessary, as are tests of autism against other neurodevelopmental conditions that are asymmetrically affected across the sexes.
The current results provide initial support for a fetal steroid theory in explaining some aspects of early epigenetic risk for autism and/or other sex-biased neurodevelopmental conditions occurring during periods of fetal brain development. There are several promising directions for further testing this theory. Other steroid pathways (e.g., the Δ5 pathway, other glucocorticoids hormones), and particularly, estradiol, must be investigated in future work. Estradiol has long been known in non-human species to have potent masculinizing effects on the brain and behavioral development.45 In a prior study, we found genetic association evidence for autism in the CYP19A1 gene, which codes for the protein aromatase that converts testosterone to estradiol. Gene expression levels of an autism-associated gene, retinoic acid-related orphan receptor-α (RORA), are lowered by testosterone and elevated by estradiol, and transcriptionally regulates several autism-associated genes including CYP19A1, thus affected aromatase levels.46,47 Although we were unable to test estradiol in the current study owing to lack of an automated liquid chromatography tandem mass spectrometry assay, we intend to investigate this in future work.
It is also necessary for future work to test if such elevations are specific to autism or are shared by other neurodevelopmental conditions with skewed sex ratios, and to test whether similar elevations exist in females with autism. Given the comorbidity of autism with other sex-biased conditions such as attention deficit-hyperactivity disorder, anxiety disorder and conduct disorder and associations between these conditions and prenatal stress,14,48 it may be that elevations in fetal steroid activity are not specific to autism. However, even if there is no specificity for elevations in autism, this would not necessarily detract from the significance of the results, as the elevations in autism may be more specific to the phenotype when observed in combination with other risk factors for autism that steroid hormones abnormalities may interact with in early development. Finally, given the evidence for positive correlations between all Δ4 steroids and non-Δ4 steroid hormones such as cortisol in early fetal development, future work should investigate interactions between hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes in early brain development.
In conclusion, we report the first direct evidence that steroidogenic activity is elevated in fetal development of those who later receive diagnoses on the autism spectrum. These results raise new questions for understanding a wide array of other observations about the early development of autism, through their interactions with early fetal steroidogenic abnormalities and provide initial support for the importance of fetal steroid hormones as important epigenetic fetal programming mechanisms for autism.
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