This study reports the first evidence that elevated levels of prenatal amniotic oestradiol, oestriol and oestrone are each associated with autism, with oestradiol levels being the most significant predictor of autism likelihood in univariate logistic regression models. These findings complement earlier observations that elevated steroidogenic activity is associated with autism in the same samples derived from the Danish Historic Birth cohort [15]. We also calculated standardised ORs, in order to directly compare the effect sizes of all amniotic steroids measured to date. We found that oestradiol had the strongest positive effect size on autism likelihood, followed by oestrone, oestriol and progesterone (Fig. 3). This finding appears to contradict an earlier report by Windham et al. [32] that showed that lower levels of oestriol in second trimester were modestly associated with a later diagnosis of autism in the offspring. However, our samples correspond to a slightly earlier time point in pregnancy compared to Windham et al. (mean gestational week = 14.9 vs. 17.2 respectively) (see Table 1) [32], which could potentially better capture the steroid surge during the PMW [14]. Furthermore, our samples are of different origin, as Windham et al. assayed maternal serum, rather than foetal amniotic fluid. Steroid hormone levels in maternal serum do not differ relative to the baby’s sex and do not correlate to amniotic levels during the PMW [42]. Therefore, amniotic oestrogens are arguably more relevant to the current research question than are maternal serum oestrogens.

A discrepancy in oestrogen levels between the mother and child could potentially be attributed to the placenta, which acts as an endocrine regulator of the maternal–foetal interface and the main source of oestrogen production for the foetus via the aromatisation of androgens [43]. Several lines of evidence suggest a contributory role for the placenta in the aetiology of autism. First, there is increased placental inflammation in autism [44]. Second, there is atypical placental morphology [45] and increased placental size [46] in cases of autism and at high familial risk respectively. Third, complications related to the placenta (pre-eclampsia [47], hypertensive disorders [48]) are also more frequent in pregnancies that lead to autism. As with autism, placental dysfunction also disproportionately affects males more than females [49].

Given the high pairwise correlations between many of the steroid hormones (Fig. 2, Supplementary Table 3), as well as a lack of difference in aromatisation between cases and controls, our data suggest that an increase in foetal oestrogens is secondary to increased activity along the entirety of the steroidogenic axis in pregnancies that later result in autism [15]. Interestingly, oestradiol was not significantly correlated to testosterone (Pearson’s β = 0.007, p = 0.9103) despite their proximity in steroidogenesis. This discrepancy may be because oestrogens are also de novo produced by the placenta, in addition to being aromatised from foetal and maternal androgens [43, 50]. Thus, a multi-systems approach is needed in order to clarify the causes of elevated foetal oestrogens in autism.

In the brain, oestrogen-mediated signalling on GABAergic neurons in the hypothalamus is required in order to suppress the steroidogenic axis [51]. Inefficient suppression of this axis in autism could be due to inefficient aromatisation of androgens in the hypothalamus, resistance to oestrogen signalling and/or dysfunction of the GABAergic system. Prenatally, foetal genetics (e.g. due to mutations in aromatase [52] or aromatase activators [53]), pregnancy complications (e.g. placental size [46]), as well as maternal risk factors (e.g. PCOS [18]) could all affect various points in this pathophysiologic pathway. These speculations would require further testing. Specifically for aromatisation, ratios based on circulating hormone levels may not be sufficient to capture tissue-specific activity, since aromatase is differentially regulated by separate promoters in the placenta, the adrenals and the brain [54].

High levels of prenatal oestrogens could dysregulate many aspects of prenatal endocrinology and affect prenatal brain development in areas that are not restricted to sexual differentiation. Several lines of evidence support a wider role of oestradiol as a ‘neurosteroid’ with many regulatory properties [55]. For example, disruption of oestrogen signalling in the developing cerebellum of mice reduces Purkinje cell growth in both males and females, but only reduces social behaviour in male mice, suggesting that the cerebellum may react to oestrogenic disruption in a sexually dimorphic way [56]. In early development, oestradiol decreases GABAergic signalling [57] and mediates its postnatal shift from excitation to inhibition [28, 58]. Oestrogens both increase the number of spines on embryonic primary cortical neurons [55] and induce the recruitment of proteins necessary for excitatory synapse formation, such as neuroligin-1, NMDA subunit GluN1, and post-synaptic density protein 95 (PSD-95) to the spines [59]. Higher levels of prenatal oestrogens might therefore increase the number of excitatory synapses in the cortex, increasing the likelihood for autism, as suggested by the excitatory/inhibitory (E–I) theory of autism [60]. The perceptual phenotype in autism is characterised by reduced GABAergic inhibition, as shown using paradigms such as binocular rivalry [61] and attention to detail [62]. Oestrogen signalling could thus be a significant modulator of neuronal inhibition, particularly during early brain development and the ‘critical period’ of cortical plasticity, which is heavily reliant on the GABAergic system [63].

Although oestradiol (aromatised from testosterone) is the main prenatal masculinising agent in most mammals [24], its role in human sexual differentiation remains unclear. Men with aromatase deficiency have typical development of their urogenital tract [64], but may have cognitive disabilities, lack a growth spurt, and have atypical secondary sexual characteristics such as feminised body proportions in adulthood [65]. Oestrogens may therefore both feminise and masculinise humans, depending on the target tissue and developmental milieu. In autism, cognitive styles and sexually dimorphic neuroanatomy show some masculinised phenotypes [7, 9, 10], but functional connectivity and physical growth show a mixed pattern of masculine and feminine shifts [8, 66]. Prenatally though, and particularly during the masculinisation window, the process of sexual differentiation is understood to be directionally masculine over an anatomically and physiologically female default. The observed high levels of foetal oestrogens could thus be contributing to developmental cognitive differences [10], according to the “extreme male brain” theory of autism.

There was no statistically significant univariate, logistic association between autism and testosterone or androstenedione, which act via the androgen receptor. Mechanisms through which androgenic signalling could increase autism likelihood, which may have been missed in this analysis, include additional androgens or other agonists of the androgen receptor (e.g. neurosteroids like dehydroepiandrosterone [67]), interaction effects between androgens and oestrogens (e.g. coactivation of the androgen receptor by oestradiol [68]), as well as non-linear associations of androgens to autism likelihood. Consequently, androgenic activity may still be an important feature in the development of autism, as suggested by related clinical comorbidities [18, 69] and shown in associations of foetal testosterone to autistic traits in a separate cohort [70].

We could not test whether prenatal oestrogens were associated with autism likelihood in females as there were too few diagnosed women in the HBC in this time window. We plan to test this by expanding the time window. Thus, at present, our findings only generalise to males. Furthermore, comparison of the concentrations of androgens and cortisol to oestrogens is potentially confounded by the fact that the latter were analysed at a later time point and underwent an additional freeze–thaw cycle. We have attempted to minimise any potential sources of confounding by using the same assay methodology with the initial analysis (LC-MS/MS), as well as reassessing for any differences in total storage time in this subset of the original cohort (Table 1).

Another limitation of this study is its reliance on clinical diagnoses from the Danish Central Psychiatric Register, which we could not be independently validated. However, a previous validation study of childhood autism diagnoses in the Danish Central Psychiatric Register found that 94% of diagnoses between 1990 and 1999 in the register were valid using a standardised coding scheme [71]. Similarly, we cannot be certain about the source of amniotic steroids, as they could be of foetal, maternal or placental origin. Foetal plasma and amniotic fluid are in osmotic equilibrium until the foetal skin is keratinised (typically by 25 weeks of gestation) [72]. Therefore, steroid concentrations in amniotic fluid accurately reflect those in foetal circulation.

In conclusion, we have demonstrated that prenatal oestradiol, oestriol and oestrone are elevated in in boys who went on to develop autism. This extends our previous finding of elevated prenatal steroidogenesis in the same cohort and provides further evidence for the prenatal steroid theory of autism [15]. High levels of prenatal oestradiol contribute to a greater degree to autism likelihood than other prenatal sex steroids, including testosterone. We conclude that prenatal oestrogenic excess is a characteristic of autism and may interact with genetic predisposition to affect neurodevelopment.