This report includes work arising from the following clinical protocol: NCT01434368. We have recruited approximately half of the proposed sample, and several of the children in the original 12-13-year-old cohort have completed the study through to their 18th birthday. To date we have studied 57 prepubertal children (33 boys and 24 girls), who are rigorously characterized as pre-gonadarchal by Tanner staging and bone age. These children also have plasma levels of estradiol and testosterone at the lower limits of detectability and have a bimodal distribution of plasma adrenal androgen levels (e.g., DHEA-S, androstenedione) indicating both boys and girls are in either early or later stages of adrenarche. Preliminary neuroimaging findings point to sex-differences particularly in the medial prefrontal cortex (mPFC), suggesting the presence of some sex-differences in functional connectivity prior to HPG axis (re)activation. A connectome-wide association study (CWAS) in this prepubertal cohort identified sex-differences in resting state functional connectivity in the mPFC, a key region in the default-mode network (DMN), a brain network implicated in autobiographical/self-referential memory. Using this mPFC region as a seed, post hoc analysis showed several clusters in the DMN and executive control network in which girls showed higher mPFC connectivity compared to boys, whereas no voxels showed higher connectivity in boys. These preliminary results suggest that there may be neural circuits that exhibit sex-differences prior to gonadarche and HPG axis activation. However, further analysis is needed to determine the factors contributing to sex-differences in this cohort. For example, in addition to possible independent sex-chromosomal effects, sex-differences could reflect greater exposures to adrenal androgens in those children who are in the later stages of adrenarche (which we can examine given the dichotomous distribution of adrenal androgen levels in this sample), or earlier sex steroid exposure secondary to the initial post-natal activation of the HPG axis which lasts 1-2 weeks in boys but up to two years in girls. Initial findings in our combined pre- and post-pubertal cohorts have identified several landmarks in reproductive aging that could have important implications for brain development. We have observed that post-menarcheal girls experience the commencement of fluctuations in estradiol secretion (presumably corresponding to the maturation of the normal architecture of the menstrual cycle). Given our findings of the importance of changes in ovarian steroid levels in RERMDs, these observations could signal an important physiologic event during which functional neurocircuitry undergoes reprogramming. We also have observed in both boys and girls that MRI-measured gonadal volumes increase across the pubertal transition. The individual inflection points in these volume curves identify another important reproductive event (i.e., the expansion of the gonad) that is a sentinel event indicating the initial activation of the GnRH pulse generator, gonadotropin secretion, and the beginning of gonadarche. As enrollment continues and our pre-pubertal children mature, the longitudinal analysis of the development of these sex-differences in brain activity (as well as developmental trajectories in brain that are not sexually dimorphic) could identify specific puberty-related reproductive or metabolic events relevant to normal brain development. These data also will serve as an important archival data set for studies of children at high risk for the development of behavioral disorders. In our larger cohort that includes both the pre-pubertal children as well as children who were enrolled mid-puberty (i.e., ages 12-13 years), preliminary results of multi-modal neuroimaging studies suggest that pubertal stage contributes to several measures of adolescent functional brain development and accounts for differences between adolescent and adult brain development that are both brain region- and sex-specific. First, our evaluation of reward circuitry function shows a main effect of pubertal status in functional connectivity during reward anticipation between nucleus accumbens (NAcc) and several brain regions, including prefrontal cortex, hippocampus and inferior parietal lobule, with increased modulation between NAcc and all regions in pre-pubertal children compared to older mid-late pubertal adolescents. However, we have not observed either sex-differences or interactions between pubertal status and sex in the reward system development. Second, our evaluation of the executive control network using a n-back working memory task, we observed no sex or age differences in working memory (WM) performance or reaction time. However, boys activated, but girls deactivated the right hippocampus regardless of pubertal status. Furthermore, prepubertal children showed greater activation than older mid-late pubertal adolescents in the left dorsolateral prefrontal cortex (DLPFC) regardless of sex. No sex-by-pubertal-stage interaction was observed in WM-related regions. In pre-pubertal children analyzed alone, boys showed greater activation than girls in the right inferior parietal lobule, medial prefrontal cortex and DLPFC. The hippocampal sex difference observed regardless of pubertal status was also confirmed in the prepubertal children analyzed alone. In our evaluation of stress responsivity in these children (both prepubertal and mid-late pubertal samples) we have observed a sex difference in the morning wakening cortisol response with girls having a greater salivary cortisol response compared to boys that persists into later puberty. Therefore, our findings suggest (albeit preliminarily) that sex differences in HPA axis appear prior to gonadarche. These findings are analogous to previous adult data from our group in which sex differences in HPA axis responsivity were observed after men and women were made hypogonadal by leuprolide treatment, in response to either exercise or corticotropin releasing hormone (CRH) stimulation compared with women. Finally, given the multidisciplinary nature of this study, we have observed one incidental finding related to pelvic fluid levels in the abdomen of normally developing children that has not previously been reported. Prior to this report pelvic fluid (PFF) in the abdomen was considered an accompaniment of malignancy or inflammation. In these otherwise healthy children, however, MRI of the pelvis (obtained to measure gonadal volume) showed the normal presence of pelvic fluid that was significantly higher in girls. Thus, among healthy pubescent children, the prevalence of PFF is significantly higher in females. The volume of PFF may reach volumes greater than 10 mL during normal puberty, especially in 4th stage, and can be assumed normal in the absence of active disease. These findings will inform the clinical assessments of pelvic fluid abnormalities in normal girls.
