Finding Early Biomarkers to Prevent Unfavorable Long-Term Health Outcomes after Premature Adrenarche: A Multicenter Prospective Cohort Study Protocol

Abstract

Introduction

Adrenarche is a prepubertal developmental phase in humans characterized by increasing levels of adrenal androgens in circulation. While its regulation and biological significance remain poorly understood, the earlier onset of adrenarche – referred to as premature adrenarche (PA) – raises concerns about potential long-term health risks, including metabolic syndrome and polycystic ovary syndrome. Our study aimed to elucidate the regulatory mechanisms underlying adrenarche and PA, specifically investigating whether PA represents a benign variation of normal development, or a disorder associated with increased risks of unfavorable metabolic and reproductive outcomes in adulthood.

Methods

This study employs a longitudinal design to track a well-characterized cohort of children with PA alongside age-matched healthy controls, following them from adrenarche through puberty into early adulthood. Conducted across two independent research centers in Kuopio, Finland, and Bern, Switzerland, the study involves detailed phenotypic assessments, including comprehensive medical histories and body composition analyses. Biological samples undergo multi-omics profiling – encompassing transcriptomics and metabolomics – using advanced techniques such as liquid and gas chromatography tandem-mass spectrometry and RNA sequencing. This integrated approach aims to identify biomarkers predictive of adverse health outcomes in PA, with candidate biomarkers and regulatory factors further validated through in vitro adrenal cell model studies.

Conclusion

This study provides the first comprehensive, longitudinal comparison of PA and healthy controls across critical developmental milestones. By elucidating the molecular factors that regulate the maturation of the zona reticularis, it seeks to resolve the longstanding mystery of adrenarche. Furthermore, by differentiating benign developmental variations from PA cases linked with long-term health risks, the findings could refine current diagnostic criteria and enable early identification of children at risk. Ultimately, this research paves the way for more accurate diagnoses, targeted interventions, and improved long-term health outcomes.

Introduction

Adrenarche is a largely unexplained, gonadal-independent event of human sexual development that progresses slowly in childhood when the innermost layer of the adrenal cortex, the zona reticularis (zR), becomes measurably functional in producing sex steroids (Fig. 1) [1–3]. As a result of zR maturation in mid-childhood, increasing amounts of adrenal androgens are released into circulation and transported to peripheral tissues, where they cause the typical clinical signs of adrenarche – pubic and axillary hair, adult-type body odor, oily hair and skin, and mild microcomedonal or inflammatory acne skin lesions. For a long time, rising levels of dehydroepiandrosterone (DHEA) and its sulfated product were thought to be the most indicative of adrenarche. However, recent studies using expanded mass spectrometric steroid profiling methods have shown that novel androgens produced by alternative pathways (e.g., 11-ketotestosterone from 11-oxy androgen pathway) might be even more characteristic (Fig. 1) [4].

Fig. 1.

Scheme showing the development of the human adrenal cortex from infancy to adolescence, together with corresponding rise of circulating zR-produced adrenal androgens that are characteristic for adrenarche. Schematic illustration for circulating DHEAS and 11-KT concentrations during infancy, childhood, and adolescence is estimates from previously published data. Figure is reproduced from a mini-review originally published in the Journal of Clinical Endocrinology and Metabolism, with permission from the publisher [5]. DHEAS, dehydroepiandrosterone sulfate; 11-KT, 11-ketotestosterone.

The adrenarche remains still largely unexplained. Being unique to humans and a few higher primates [6], its biological role within these species is unknown. Furthermore, details of regulation and trigger(s) for adrenarche remain unsolved [1–3, 7]. So far, the possible trigger was assumed to be either in the adrenal cortex or within the HPA axis, enhancing zR maturation and androgen production [3, 8, 9]. ACTH/PKA signaling seems to play at least a permissive role [10–14], while for intra-adrenal mechanisms the “critical size hypothesis” or inhibition of HSD3B2 by cortisol has been proposed [15, 16]. Alternative hypotheses tested the possibility of triggers in the peripheral circulation or action of androgens likely mediated by the adipose tissue including insulin, IGF-1, leptin, interleukin 6, fatty acids, and prolactin [3, 7–9, 17]. Moreover, we also have an incomplete understanding of the normal developmental biology of the fetal and adult adrenals. The human fetal adrenal cortex consists of 3 layers (called “zones”), of which the inner fetal zone mainly produces DHEA/DHEA sulfate and thus resembles the postnatal zR [2]. Soon after birth, the fetal zone regresses, and the cortex is organized into two zones: the zona glomerulosa producing mineralocorticoids and the zona fasciculata producing glucocorticoids. After the first years of life, the innermost zR develops from focal islets but becomes functionally active later when a continuous layer is formed during adrenarche [18, 19]. How the fetal-postnatal transition and the modeling of the cortex into distinct layers are controlled still needs to be discovered [8].

As physiological adrenarche is an endocrinological mystery, many questions remain concerning the triggers and long-term health consequences of its premature occurrence, a condition called premature adrenarche (PA). PA is currently defined as the appearance of clinical signs of adrenarche with rising adrenal androgens before the age of 8 years in girls and 9 years in boys [1–3, 7, 9, 20]. Diagnosis of PA is made by exclusion of true centrally mediated precocious puberty, androgen-secreting tumors, exogenous androgen exposure, and congenital adrenal hyperplasia (CAH) [3, 20]. Even though some rare monogenetic disorders of steroidogenesis may cause early adrenarche (e.g., deficiencies of PAPSS2, H6PDH, HSD11B1, or non-classic CAH due to CYP21A2 or CYP11B1 deficiencies; summarized in [5]), the underlying factors leading to most of the cases of PA are unknown. Also, the impact of PA on later health is still under debate. Milder forms of PA, manifesting closer to ages 8–9, are discussed as potential extreme variations of normal development [5]. In contrast, more severe forms of PA – such as those occurring earlier and especially with pubic hair – are more often associated with an underlying disorder that may lead to adverse health outcomes [5]. The disrupted pathway leading to PA may originate before birth, beginning with fetal growth restriction, followed by rapid postnatal catch-up growth and weight gain, as well as mildly tall stature and overweight/obesity in early childhood [3, 7, 9, 17, 20].

PA may then lead to abnormal sex development with serious disease consequences in some cases. Thus, PA is thought to be associated with precocious puberty and fertility problems in adult life due to ongoing androgen excess along with metabolic disturbances characterized, e.g., by polycystic ovary syndrome (PCOS) and metabolic syndrome [21–27]. However, with the current clinical approach, it is not possible to identify children with PA at risk for future adverse health outcomes.

As discussed in our recent mini-review [5], the gaps in current knowledge regarding adrenarche and PA highlight the need for a new, prospective study that specifically addresses the most pressing questions in the field. So far, a comprehensive prospective and longitudinal study about sex developmental events from the onset of adrenarche to adulthood is missing. Only a few studies have investigated PA individuals longitudinally [21, 22, 24, 28–33], and none of them compared the characteristics of PA versus controls at time points related to biological development, rather than at fixed ages. We have therefore planned and initiated a study, described in more detail in this study protocol paper, that aims at characterizing the natural course and consequences of PA in children from the onset of adrenarche to the start of puberty into young adulthood. The study collects clinical data and biomaterials of PA individuals and matched controls at time points defined by developmental events; it will compare a wide range of data, including clinical, metabolic, genetic, and transcriptomics data to identify early biomarkers of later disease as well as molecular targets for establishing novel preventive measures. In addition, identified candidate markers will be investigated for their mechanistic impact on developmental biology using molecular and cellular methods.

Methods

Study Design and Setting

This study is a multicenter, prospective cohort study which will be conducted independently, but in parallel, in two different centers: Kuopio University Hospital (Kuopio, Finland; PI Jani Liimatta) and University Children’s Hospital in Bern (Bern, Switzerland; PI Christa Flück).

Participants

Inclusion and Exclusion Criteria

The inclusion and exclusion criteria are presented in Table 1. In general, we will include all healthy children who exhibit either premature or normal timing of adrenarche based on clinical signs such as adult-type body odor, oily skin and hair, acne, and/or axillary and pubic hair. We will also include boys: however, as PA is more common in girls than in boys [34], our cohort will primarily consist of girls. PA cases with known underlying conditions (e.g., non-classic CAH) that affect growth and sexual development will be excluded, as this study focuses on idiopathic PA.

Table 1.

Inclusion and exclusion criteria of the study

​	Inclusion criteria	Exclusion criteria
PA group	Children with any clinical sign of androgen action before the age of 8 years in girls and 9 years in boys	All: any diagnosed disease/syndrome or ongoing medication that might influence growth and sexual development (i.e., a child must be otherwise healthy)
Control group	Age-matched children without any clinical sign of androgen action	PA group: differential diagnoses of PA, i.e., precocious puberty, CAH, androgen-producing tumors, and external exposure of androgens

Sample Size and Power Calculations

Sample sizes have been estimated based on the power calculations on repeated measures ANOVA for steroids and especially the metabolite androstenediol. We selected androstenediol as an example of an adrenal androgen, because longitudinal reference data for girls with normal adrenarche exist [35]. We set the alpha level conservatively at 0.001 without additional corrections to account for multiple comparisons (47 steroid metabolites) and assumed a variance for androstenediol of 1,000 nmol/L/24 h based on data from a previous study [36].

Girls with PA had higher urine androstenediol than controls by a factor of 2.3 (46 nmol/24 h vs. 20 nmol/24 h). With planned measurements and an estimated correlation between measurements of 0.8, minimum sample size of 40 (20 PA children and 20 controls) would be enough to provide a statistical power over 90% to distinguish minimum twofold differences in androstenediol levels between children with PA and controls. For most of the androgen metabolites, we expect similar or higher statistical power, as differences have been similar or more pronounced in the previous studies [5, 36, 37]. Therefore, we plan to recruit in total 60 children in PA group and 120 controls of the same age from our centers (50% from Bern, 50% from Kuopio). As consultation for PA girls is part of usual care and based on our previous experiences from the previous studies [21, 22, 27], we assume a participation rate of 90% or higher and we expect to include 50–55 PA children in our study at baseline. We further assume a dropout rate of 40%, leading to approximately 30 PA participants after long-term follow-up. For control children, we expect that the dropout rate during follow-up might be even higher, and therefore we will use an unbalanced design with a case:control ratio of 1:2.

Recruitment

Participants for the PA group will be recruited from those contacting our health care services and associated networks, seeking consultation because of signs of PA. Age-matched controls will be recruited from the general population through advertisement (flyer, mouth-to-mouth propaganda, social media, and newspaper advertising). For potential participants, information about the study will be given in written, by a short video clip and in person. Final recruitment and written informed consents will then be obtained from those who express their interest after a consideration period.

Timeline and Procedure

The study was launched in 2023 in Bern and in 2024 in Kuopio. The recruitment and baseline phase are planned to last 2–3 years, and the study ends when all participants have reached young adult age of 17–18 years; thus, each participant is followed over a period of about 10–12 years. A scheme of the planned visits and protocol is shown in Table 2.

Table 2.

Scheme of study protocol and visits

​	Baseline (adrenarche in PA group)		Follow-up 1 (at same age in prepuberty)		Follow-up 2 (adrenarche in control group)		Follow-up 3 (pubertal onset: Tanner M2/G2)		Follow-up 4 (menarche in girls/late puberty in boys)		Follow-up 5 (adulthood)
	PA	controls	PA	controls	PA	controls	PA	controls	PA	controls	all girls	all boys
	<8/9 years (girls/boys)	age- and sex-matched	8/9 years (girls/boys)	8/9 years (girls/boys)	​	? years	? years	? years	? years	? years	18 years	18 years
Personal and parental history	X	X	​	​	​	​	​	​	​	​	​	​
Anthropometrics	X	X	X	X	​	X	X	X	X	X	X	X
Physical examination	X	X	X	X	​	X	X	X	X	X	X	X
Body composition	X	X	X	X	​	X	X	X	X	X	X	X
Bone age1	X	​	(X)	​	​	​	(X)	​	(X)	​	​	​
Ultrasound	X	X	X	X	​	X	X	X	X	X	X	X
Plasma/serum	X	X	X	X	​	X	X	X	X	X	X	X
Saliva	X	X	X	X	​	X	X	X	X	X	X	X
Urine	X	X	X	X	​	X	X	X	X	X	X	X
RNA from blood	X	X	X	X	​	X	X	X	X	X	X	X
DNA extraction, incl. parents	X	X	​	​	​	​	​	​	​	​	​	​
Scalp hair	X	X	X	X	​	X	X	X	X	X	X	X
Semen	​	​	​	​	​	​	​	​	​	​	​	X

PA, premature adrenarche.

¹Bone age will be assessed at baseline for PA children who present with pubarche and repeated later only if clinically indicated.

Work Packages

A summary of the work packages (WPs) of the study is depicted in Figure 2.

Fig. 2.

WPs of the study.

WP1: Comprehensive Phenotyping

To assess whether there are differences in history, dietary habits, physical exam, growth, and pubertal development from adrenarche to puberty and into adulthood between PA participants and age-matched controls, we will perform comprehensive phenotyping (Table 2). Personal history will include information on pregnancy and birth (e.g., gestational age, birth weight, and length). Family history will comprise questions on ethnic background and parental health, e.g., height, weight, blood pressure, cardiovascular and lipid disorders, pubertal development, fertility issues (e.g., PCOS), as well as the history of tumors or chronic disorders. In the physical exam of the study subjects, extra attention will be given to growth parameters (height, weight, waist circumference) as well as signs of androgen action and pubertal development, which will be assessed according to Tanner stages. In addition, ultrasound of the gonads and the internal sex organs will be performed. Body composition using InBody method analysis will inform on fat and muscle mass, and grip strength on physical fitness. We will not use dual-energy X-ray absorptiometry method due to ethical reasons because of X-rays. Furthermore, bone age (only for PP children in PA group for ethical reasons) will be assessed for the evaluation of chronological versus biological age and prediction of final height. For comprehensive phenotyping, data on history and physical exam will be recorded according to current standards of Human Phenotype Ontology (https://hpo.jax.org/app/tools/phenomizer) wherever possible. At the end of the study, semen analysis will be performed for boys as an indicator for fertility (quantitative and qualitative characterization).

WP2: Metabolic Profiling

Several studies suggest that PA bears a significant risk for later chronic diseases including metabolic syndrome and PCOS [21–27]. Detection of early markers of adverse outcome will allow to take preventive measures. We therefore plan to perform a broad metabolic profiling of PA and control subjects longitudinally to find early markers of later disease. This profiling will include specific biochemical markers of metabolism and adrenal and sexual development. In addition, we will take a broader unbiased approach and perform metabolome profiling. Metabolic measures that are planned to be investigated are as follows:

• 1.Comprehensive steroid profiling using golden standard gas and liquid chromatography tandem-mass spectrometry methods with several matrixes including plasma/serum, urine, saliva, and hair. These methods have been previously validated and available in our centers [37–43].
• 2.Markers associated with sexual development, including gonadotrophins, anti-Müllerian hormone, and sex-hormone-binding globulin, are measured using commercially available kits.
• 3.Metabolic parameters, including fasting glucose, insulin, lipid profile, and inflammation markers (adipokines, hsCRP), are measured using commercially available kits.
• 4.Unbiased circulating metabolomics approach with nontargeted liquid chromatography tandem-mass spectrometry/mass spectrometry-based method. This method is available at the University of Eastern Finland, Kuopio, Finland [44].

WP3: Transcriptome Profiling and Bioinformatic Network Analysis

The molecular basis of adrenarche remains unresolved, and Genome-Wide Association Studies (GWAS) are lacking. Candidate gene studies have reported associations between PA and polymorphisms in the androgen receptor [45–47], the ACTH (MC2R) receptor [48], genes involved in IGF and insulin signaling [49, 50], as well as in various genes implicated in disorders of steroid biosynthesis, metabolism, and action. Given the multifactorial etiology of PA, characterized by polygenic and environmental contributions as well as a spatiotemporal component, we propose using a transcriptomics approach (in addition to the metabolomics approach described above) to elucidate the underlying regulatory gene expression network and find biomarkers. Additionally, DNA will be extracted and stored from both children and their parents for potential future use in genetic and epigenetic studies, such as methylation analyses.

We will search for differences in the blood-born circulating transcriptome pointing toward underlying genetic and epigenetic events, mechanisms, and targets characterizing normal adrenarche and PA. Our special interest will be in both coding mRNAs and regulatory non-coding RNAs, among them especially in micro(mi)RNAs. MiRNAs are best studied and most frequently assessed for their potential role as biomarkers or therapeutic targets [51]. They contribute to the regulation of a variety of physiological processes such as embryonic development, cell differentiation and proliferation, and metabolism and inflammation. They also play an important role in the pathogenesis of disorders including cancer, cardiovascular and metabolic diseases. More recently, their role has also been described in endocrine disorders. For adrenals, miRNA studies may be found for adrenal tumors [52], aldosterone signaling [53] as well as glucocorticoid action [54, 55]. By contrast, few studies deal with adrenal steroidogenesis [56]. For instance, miRNA-24 has been shown to modulate CYP11B1 and CYP11B2 expression along with alterations in aldosterone and corticosterone secretion [57]. Knockdown of miRNAs using Dicer 1 siRNA in NCI-H295R adrenocortical cells increased mRNA levels of steroidogenic enzymes CYP11A1, CYP21A1, and CYP17A1 and enhanced the secretion of associated corticosteroids [58]. However, no data are currently available on miRNAs and the regulation of adrenal androgens.

Total RNAs and small RNAs will be extracted from blood samples following library preparations and sequencing with high-throughput next-generation sequencing methods. Data will be aligned to human reference sequences and mapped and annotated against reference databases (e.g., NCBI Reference Sequence Database and miRbase). Data will be filtered if needed and normalized before introducing bioinformatic tools to identify candidate RNAs that are differentially expressed in PA. We will also perform target predictions (e.g., between miRNA and human UTR sites) and functional enrichment analysis including network, biological pathway, and gene ontology term enrichment analyses.

Multi-Omics Analyses

Once we have collected the data, we also plan to perform a combined analysis with all clinical, metabolomics, and transcriptomics data of PA and controls as input to find characteristic patterns and the best (diagnostic) discriminator(s) of normal timing of adrenarche and PA and possibly understand the underlying regulatory mechanism(s). For such complex analysis, we will also use machine learning and seek support and collaboration with specialists in bioinformatics (e.g., the Center for Artificial Intelligence in Medicine of the Faculty of Medicine at the University of Bern).

WP4: Validation of the Data

To allow for the data of our cohorts in Kuopio and Bern to be compared with each other, we have planned to use the same study protocol (Table 2) to answer open questions regarding adrenarche. We will run our respective studies independently in both centers, but choosing the same study design will allow us to validate our data vice versa.

WP5: Validation of Identified Biomarkers and Networks through Mechanistic in vitro Studies

While statistical and bioinformatic methods can inform on significance and interrelationship of metabolomics and transcriptomics data, only mechanistic studies can confirm a causative role of specific markers for adrenarche development and mechanisms involved in PA. In best case scenario, we might be able to identify key regulators/triggers of adrenarche and PA through these studies. To perform mechanistic studies for candidate genes/proteins/regulatory molecules/pathways/networks identified through the work described above, we will use established adrenal cell models [57], e.g., “starvation NCI-H295R cell model,” and classic molecular endocrinology experimental approaches. This includes, for instance, specific gene overexpression/knockdown or signaling enhancer/inhibitor experiments with effect readout by RT-qPCR, Western blot, or steroid profiling as used in previous projects [59–62]. For instance, validation of miRNAs targeting adrenal steroidogenesis will be investigated in NCI-H295R cell model using synthetic miRNA mimics and inhibitors and steroid profile as readout. To assess the effects of miRNAs on specific genes (e.g., HSD3B2, CYB5, and SULT2A1), the downstream effects of miRNA expression (both overexpression and inhibition) will be assessed at mRNA (RT-qPCR) and protein level (Western blot).

Data Collection and Management

The data arising from this study will be pseudonymized and stored locally in each center in compliance with current regulations. We will use a REDCap electronic data capture tool hosted by the university of our centers [63, 64]. REDCap is a secure, web-based software platform designed to support data capture for research studies, providing (1) an intuitive interface for validated data capture; (2) audit trails for tracking data manipulation and export procedures; (3) automated export procedures for seamless data downloads to common statistical packages, and (4) procedures for data integration and interoperability with external sources. Statistical analyses will be conducted with software dedicated to analyzing medical data. Data availability will be stated in each publication, and further inquiries with reasonable requests can be directed to the corresponding authors or PIs.

Statistical Analyses and Approaches

We will assess clinical and phenotype outcomes and obtain data from metabolomics measurements for both groups. Clinical/phenotype outcomes will be both binary (e.g., heart problem, yes/no) and continuous (e.g., height in cm), and steroid measurements will be continuous (concentrations, e.g. in nmol/L). We will describe the binary outcomes as absolute numbers and proportions and the continuous outcomes as means/medians and standard deviations/interquartile ranges. For analysis with repeated measurements over time, we will use mixed effects models. These models allow us to fit different measurements per individual. We will use logistic models to estimate binary outcomes and linear models to estimate mean changes in continuous outcomes. If appropriate, we will transform continuous outcome variables to achieve approximate normal distribution of the residuals. The distribution of variables will be assessed visually using histograms, and if the nature of the distribution is not clear, the Shapiro-Wilk test will be used. As random variables, we will include the individual patient to account for variability between patients and the period between assessments. We will then assess the association of change in outcome variables with study group (PA and controls) and baseline clinical factors. These factors will be included in the mixed effects models as fixed effects. For interpretation of the results, we will use an alpha level of 0.001 to account for multiple tests.

For steroid and metabolite profiling, datasets will be first normalized and log-transformed and then analyzed using appropriate models in MetaboAnalyst software (https://www.metaboanalyst.ca) to detect characteristic profile in PA group. We will use fold chance analysis with predefined threshold and a false discovery rate of 0.05 to account for an error raising from multiple testing.

These data can then be further analyzed for group comparison and time series as described above. In addition, unsupervised data overview, cluster analysis, and annotation enrichment analysis will be performed. Statistical analysis will be supported by professional statisticians when needed.

Dissemination

All results arising from this study will be reported in international congresses and peer-reviewed publications. When publishing our findings, we will follow STROBE guidelines [65] and favor the use of an open-access policy. Determination for authorship for each publication will be determined on a case-by-case basis by the PIs while using the authorship guidelines of the journals. Any professional writers or AI-based text generators will not be utilized in manuscript preparation.

Ethics

The study involves examination of the intimate areas of the subjects. This is, however, an important part of research that we cannot avoid. Children and their parents will always be informed why and how the tests are being carried out, and we will carry them all out as sensitively as possible. All researchers conducting study visits are pediatricians, mostly pediatric endocrinologists, for whom these examinations in children and adolescents are routine clinical practice.

We will perform bone age assessments with X-ray taken from one hand. This will be, however, only done for cases with premature pubarche, not for other children in the PA group or for any controls, for ethical reasons. Bone age measurement exposes the child to a very small amount of radiation. For example, this dose is less than a few hours of natural annual background radiation or less than 1 h of the average annual radiation dose received by Finnish citizens [66]. Of note, patients with premature pubarche are generally recommended to be evaluated by pediatricians, and bone age measurements are routine assessments for them in clinical care.

Because the research questions of this study cannot be solved without appropriate biomaterials, we will collect blood samples at each study visit. The blood volume needed to be taken will be, however, in range recommended in World Health Organization’s review of safety limits of blood sample volumes in children in clinical research [67].

Discussion

Adrenarche is a normal event of human development before puberty [3, 20]. It marks the functional activation of the innermost zone of the adrenal cortex to produce androgens [8]. However, its role and regulation remain unclear, alongside with the clinical implications for its premature occurrence. Our study aimed to provide insight into the development and regulation of adrenarche; it is designed to find novel candidate markers and targets for diagnostic, therapeutic, and preventive care in PA children.

Here, we provide a detailed protocol for our study, recognizing the importance of protocol publication for promoting transparency, reproducibility, and methodological integrity. By publicly sharing study protocols in advance, researchers can facilitate critical evaluation of study design, promote consistency across studies, and reduce the risk of analytical pitfalls. This practice is especially valuable in multi-omics contexts, where standardized methodologies are still evolving, and reproducibility remains a challenge. Moreover, protocol publication supports the broader scientific community by providing a framework for replication and adaptation in future research endeavors.

Our study will give rise to a small, but well-defined cohort of children with PA and controls that we plan to follow for over 10 years, from adrenarche to puberty until early adulthood. In contrast to previous studies, this will be the first prospective cohort study to compare data of PA and controls assessed at the time points of the developmental events rather than at fixed ages. This should solve the open question, whether adrenarche in PA children is just time-shifted, or whether it also shows differences in other clinical and biochemical properties. In addition, it will elucidate whether PA influences timing and other characteristics of pubertal maturation. The long-term follow-up data from our study may contribute to a revised definition, allowing for a more accurate distinction between a disorder at the time of manifestation and a normal variant. A more specific classification will help de-medicalize PA in many children and identify only those at risk for future diseases, allowing early interventions and close-up monitoring during development. By doing so, it will enable early interventions and close monitoring throughout development.

Compared to previous works, our study will be unique in its use of a wider range of newer, unbiased methods for characterizing human biomaterials. While targeted and untargeted metabolomic profiling has been performed on samples from PA children [68], our study will enable broad nontargeted metabolomic profiling on follow-up biomaterials. No prior work has conducted transcriptomic analyses of the blood-born circulating RNAs in PA children. Thus, our study may uncover a regulatory role of ncRNAs, such as miRNAs, in zR formation. It may also identify potential marker(s) for PA children at risk of future adverse health outcomes, which can subsequently be validated in larger and more diverse cohorts. The planned multi-omics approach, combined with machine learning-based methods, remains unparalleled in the field of adrenarche research.

A limitation of this study is the relatively small size of the planned cohort, which becomes more pronounced if the dropout rate exceeds expectations. This may hinder the detection of subtle differences between groups. However, as participants will be assessed across at least 5–6 time points, we believe our design remains robust in identifying significant novel findings. Furthermore, since the study is conducted independently and in parallel at two different centers, we can compare and validate the generated data, thereby enhancing the significance of the findings. Another possible limitation is the homogeneous Caucasian European composition of our planned study cohort, which may reduce external validity and increase the risk for selection bias. Given that ethnic background significantly influences the presentation of PA, broader multicenter studies – including non-white ethnicities – will be essential in follow-up to ensure the generalizability of the results. On the other hand, when investigating novel disease mechanisms or markers, a genetically similar cohort might offer the advantage of minimizing external variables (“noise”) beyond adrenarche, allowing for a clearer distinction from PA.

Overall, the impact of the proposed study is on medical practice directly through the identification of early markers of PA with long-term health risks. It addressed the need for a better definition of adrenarche and PA. Depending on the outcome, the study might underscore that development at young age affects health in adult life, and that investigations in children might reveal opportunities for early interventions or even prevention of adult disease. We hypothesize that our work gives rise to new molecular candidates that orchestrate the complex network behind zR formation, opening the door for further in vivo and in vitro studies of adrenal biology. Finally, we might even reveal the long-searched trigger of adrenarche and thereby shed light on a so far obscure developmental event of human sexual development.

Acknowledgments

The authors thank research nurses and project managers Tea Enjala (Kuopio, Finland) and Anette van Dorland (Bern, Switzerland) for their valuable work toward this project.

Statement of Ethics

This study protocol was reviewed and approved by the Ethical Committee of the Canton Bern, Switzerland (BASEC 2022-00653), and by the Medical Research Ethics Committee of Wellbeing Services County of Northern Savo in Kuopio, Finland (835/2023). The study will be carried out, and written informed consents from participants and their parents/legal guardians will be collected, in accordance with the principles stated in the Declaration of Helsinki. Comprehensive information of the study will be given also for participating prepubertal and early pubertal children at a level appropriate to the child’s age. Because this study is planned to continue until adulthood, a child him/herself will also give informed written consent when turning 15–16 years old. In Switzerland, the process is similar, but the age threshold is set at 16.

Conflict of Interest Statement

Sandoz provided support for the creation of professional recruitment materials (e.g., leaflets). Christa E. Fluck was a member of the journal’s editorial board at the time of submission. Otherwise, the authors declare no conflicts of interest. Currently, no study investigators have any conflicts of interest to report, and financial and competing interests will be disclosed in each publication.

Funding Sources

The study described in this manuscript is currently funded by the Foundation for Pediatric Research (Helsinki, Finland), the State Research Funding of Finland, and the Swiss National Science Foundation (320030-207893). Creation of recruitment material has been supported by Sandoz, Switzerland. Other funding sources will be continuously searched. The funding sources have not been involved, nor will they be involved, in: (1) the design and conduct of the study, (2) the collection, management, analysis, and interpretation of the data, (3) review or approval of the manuscripts, or (4) the decision to submit manuscripts for publication.

Author Contributions

J.L., J.J., and C.E.F. conceptualized the study. P.A. and O.N. are PhD students working on the project and wrote the first draft of the manuscript. All authors critically revised the manuscript and approved the final version.

Funding Statement

The study described in this manuscript is currently funded by the Foundation for Pediatric Research (Helsinki, Finland), the State Research Funding of Finland, and the Swiss National Science Foundation (320030-207893). Creation of recruitment material has been supported by Sandoz, Switzerland. Other funding sources will be continuously searched. The funding sources have not been involved, nor will they be involved, in: (1) the design and conduct of the study, (2) the collection, management, analysis, and interpretation of the data, (3) review or approval of the manuscripts, or (4) the decision to submit manuscripts for publication.

Data Availability Statement

No data have been used for this study protocol manuscript. In the upcoming study, we aim to adhere to the FAIR data principles (findability, accessibility, interoperability, and reusability). Data will be made openly available upon publication, except in cases where intellectual property rights restrictions from participating institutions prevent it. Data availability will be stated in each publication and further inquiries with reasonable requests can be directed to the corresponding authors or PIs.