This study is part of the European Hypiend project, which aims to explore the effects of co-exposure to endocrine disruptor chemicals (EDCs) on the function and epigenetic programming of the hypothalamic-pituitary-adrenal (HPA) axis in order to define intervention strategies to minimize exposure and consequences on the neuroendocrine system during the perinatal and prepubertal phases. The intervention at the perinatal phase is registered under the Unique Protocol ID: 101137440 - Hypiend - Clinical study to evaluate the effectiveness of a multicomponent behavioural intervention to reduce EDC exposure during the perinatal period in women and their offspring.

The intervention presented here concerns the prepubertal phase. The primary objective is to demonstrate that a multi-component behavioral intervention (MBI) implemented in primary schools in two different European countries over two and a half school years is effective in reducing the presence of EDCs in the urine of prepubertal children using the Health Action Process Approach (HAPA) and the Positive Behaviour Support (PBS) models to improve the intervention and parents' knowledge of these contaminants, as well as to advance scientific knowledge.

1. BACKGROUND AND JUSTIFICATION

   1.1. SCOPE AND ASSESSMENT OF CURRENT KNOWLEDGE DIRECTLY RELATED TO THE SCIENTIFIC QUESTIONS TO BE ANSWERED IN THE CLINICAL STUDY Several epidemiological studies have identified worrying links between high EDC exposure and adverse health effects in children and adolescents. As an illustrative example, in the framework of the Human Biomonitoring for Europe (HBM4EU) project, Lange et al. (2022) analysed the risk assessment of combined exposure to anti-androgen phthalates (Di(2-ethylhexyl) phthalate (DEHP), Di-isobutyl phthalate (DiBP), Di-n-butyl phthalate (DnBP), Butyl benzyl phthalate (BBzP) and Di-isononyl phthalate (DiNP) in children (6-11 years) and adolescents (12-18 years) from different European countries. The authors reported that DnBP and DiBP were identified as the main drivers of the increased hazard index (HI) of a mixture of phthalates in a large cohort of European children and adolescents. Interestingly, however, for 63% of these subjects who had a HI > 1, this risk would not have been detected if the risk for each chemical had been analysed individually, illustrating the importance of conducting risk assessment analyses of mixtures. In addition, the authors found differences in the exposure-related HI between European regions, with children and adolescents in eastern regions being more exposed than those in the south and west, highlighting the importance of including countries with different degrees of exposure in order to obtain more holistic conclusions and to implement adapted measures, policies and recommendations. Different epidemiological studies have suggested that the increase in exposure to EDCs with hormonal activity in recent decades, especially to substances with direct estrogenic activity, could be among the causes attributed to the observed increase in the onset of puberty in girls and boys in developed countries. In this regard, a very recent cross-sectional study of 1539 Spanish children found that higher concentrations of certain non-persistent pesticides were associated with a greater probability of developing earlier puberty in both boys and girls, and that this association depended on the child's body mass index (BMI).

   To the knowledge of the investigators, there are very scarce and inconclusive data on EDCs aimed at reducing exposure to these substances in prepubertal children, including most of them under 100 children, analysing a maximum of two different families of EDCs and lacking a control group.

   HYPIEND aims to evaluate whether a multicenter, multicomponent controlled intervention (implementation of plastic-free lunchroom in elementary school + school-based educational program for children + parent workshops + mobile application for parents designed to limit EDC exposure) conducted in primary schools in prepubertal children is able to reduce urinary concentrations of different EDCs and this may prevent the earlier onset of the pubertal phase associated with high EDC exposure. In addition, different anthropometric measures will also be evaluated. The effects will be compared with a control intervention group, which will only receive placebo workshops with information about the dangers of screens, the benefits of physical activity and the importance of good sleeping habits. The intervention will be implemented over two and a half consecutive school years (from November 2025 (start of the intervention) to December 2027) and will consider appropriate behavior change techniques and models to achieve high adherence to the intervention.

   Schools will be recruited from different environmental zones (rural and urban) to account for different exposures to environmental contaminants. The investigators will assess the concentration of biomarkers of exposure to different families of EDCs in children's urine at three different time points (at baseline and at the end of the first and third school year). A risk assessment of EDC exposure will be performed, including all data obtained in urine at different time points. In addition, multivariate analyses will be conducted to explore the possibility of determining EDC exposure through predictive variables, such as dietary habits, behavioral habits related to EDC exposure, school environment and socioeconomic status. Finally, the study will be conducted in two different countries (Belgium and Spain). Overall, this approach will contribute to obtain a more accurate picture of EDC exposure and the degree of EDC exposure reduction in response to the implemented intervention, taking into account different important factors involved in EDC-related burden.

   1.2. RESULTS OF COMPLETED CLINICAL STUDIES AND NUMBER OF ONGOING CLINICAL STUDIES USING THE SAME INTERVENTION IN THE SAME INDICATION.

   Recently, Sessa et al. (2021) demonstrated that, a school lunchroom intervention (implementation of a plastic-free service) for 5 days per week for 6 months significantly decreased urinary levels of bisphenol A among the groups and also within the group enrolled in the plastic-free school lunchroom. However, larger MBI studies involving schools and also families, with the support of experts in behavior change and educational sciences are needed to promote long-lasting effects, in order to minimize overall exposure to different EDCs in a more conclusive manner.

   1.3. LEVEL OF EVIDENCE RELATED TO THE MECHANISM OF ACTION OF THE INTERVENTION IN THE PLANNED CLINICAL STUDY POPULATION.

   EDC-Mix-Risk and ENDpoiNTs are two outstanding Horizon 2020 projects that combined experimental research in both cells and animals with epidemiological data. Researchers from both projects and others demonstrated very well, using a statistical model for multivariate regression with data obtained in the SELMA pregnancy study, that different EDCs (including phthalates, alkylphenols and perfluoroalkyl substances) analysed in the urine or serum of women at week 10 of pregnancy were associated with language delay in children at 2.5 years of age. Subsequently, the identified EDCs were mixed and tested in human brain organoids, as well as in Xenopus leavis and Danio rerio to elucidate the molecular and functional impact of exposure. Finally, the authors integrated experimental and epidemiological data and conducted a risk assessment approach, finding increased odds of language delay in up to 54% of offspring who had prenatal exposures above experimentally derived levels of concern. Despite these highly relevant findings, a limited number of human studies have shed light on the mechanisms underlying EDC-mediated dysfunctions in the HPA axis, including induction of epigenetic changes in key genes involved in neuroendocrine regulation and precocious puberty, as well as alterations in inflammatory response and gut microbiota (dysbiosis). EDCs may increase the risk of neurodevelopmental disorders in children by interfering with estrogen and thyroid hormone signaling or metabolism in early life. Very recently it was shown that increased urinary levels of a mixture of different environmental chemicals (phenols, toxic metals and non-persistent pesticides) measured in adolescents aged 15-17 years were associated with higher circulating levels of kiss54 protein and lower levels of methylation at different Cytosine-phosphate-Guanine (CpGs) in the kiss1 gene. In addition, adolescents who showed higher serum kiss54 had higher Luteinizing Hormone (LH) levels, and third tertile CpG1, CpG2, and total CpG methylation percentages were associated with lower Follicle-Stimulating Hormone (FSH) and Estradiol (E2). These findings suggest that kiss proteins, which is a precursor-derived neuroactive peptide (pro-hormone, pre-pro-kisspeptin) released by neurons in the hypothalamus, could be a promising biomarker of the effect of environmental chemical mixtures on the reproductive hormone profile. However, despite this evidence, to the knowledge of the investigators, no human intervention studies aimed at reducing EDC exposure have analyzed the molecular impact that may accompany these changes in EDC exposure. Further research is needed to shed more light on these questions, including molecular and omics analyses in humans to elucidate whether human intervention studies aimed at reducing EDC exposure are able to favorably modulate these molecular changes.

   In this project, different molecular and omics approaches will be carried out to shed more light, in the framework of the HPA axis, on the epigenetic drivers most susceptible to alteration and the interaction between gut microbiota and the impact of EDC exposure on systemic inflammation. These analyses will be performed on samples obtained in the study taking into account results obtained in preclinical models exposed to EDC mixtures that attempt to mimic real human exposure, to increase the chances of obtaining robust and reliable results using a targeted approach. Specifically, in prepubertal children, in addition to gut microbiota, the investigators will also assess HPA axis-related hormones, methylation of different CpG sites of key genes involved in the HPA axis and circulating kisspeptin levels using dried blood droplets. Overall, these analyses will allow us to delve deeper into the mechanisms by which EDCs exert their harmful effects, as well as how intervention studies aimed at reducing exposure to these chemicals can modulate these biological pathways and the identified molecular biomarkers of exposure. To the knowledge of the investigators, this approach has not yet been carried out in large human intervention studies in the EDC setting.

   1.4. HYPIEND PROJECT The intervention presented here is part of the European HYPIEND Project. This project is coordinated by the Technological Center of Catalonia (Eurecat), funded by the Horizon Europe framework program (Grant agreement ID: 101137440).

   The HYPIEND project investigates the effects of exposure to multiple EDCs on the function and epigenetic programming of the HPA axis to delineate intervention strategies to minimize exposure and consequences on the neuroendocrine system during the perinatal and pre-pubertal stages.

   The multidisciplinary HYPIEND consortium consists of 14 partners from eight European countries. Among them: Stichting Radboud Universiteit (Netherlands), Institut de Investigació en Ciencies de la Salut Germans Trias i Pujol (Spain), Sciensano (Belgium), Kungliga Tekniska Hoegskolan (Sweden), Katholieke Universiteit Leuven (Belgium), University of Granada (Spain), Centrum Medyczne Ksztalcenia Podyplomowego (Poland), Protoqsar 2000 sl (Spain), Enco srl (Italy), Fundacion para la investigacion del hospital universitario la Fe de la Comunidad Valenciana (Spain), Université de Liège (Belgium), Universite de Geneve (Switzerland), King's College London (UK).

   More information about this project can be found at the following links: https://hypiend.eu/ Here is an overview of the Hypiend methodology. Data from public databases will be combined to develop a knowledge base linking selected EDC exposure to health outcomes related to the HPA axis. Quantitative structure-activity relationship (QSAR) methodologies will be combined with data analysis to identify patterns of co-exposure. EDC mixtures with high prevalence in the target populations will be defined, both qualitatively and quantitatively, according to the coexposure patterns. Candidate EDC mixtures will be tested in cell-based models to define dose-response patterns and dosing for subsequent studies. Zebrafish models recapitulating HP axes will be used to select those EDC mixtures with the greatest impact. Next, selected EDC mixtures will be tested in placenta and BHE models to assess the diffusion of EDC mixtures and characterize the fractions reaching both the foetus and hypothalamus. Effects on hypothalamus and pituitary development and function will be assessed in stem cell-derived organoids and OoC, with particular attention to effects on whole genome DNA methylation and hormone functionality. The results will be used to further select complex mixtures of EDCs according to their HP epigenetic programming and disruptive potential. Subsequently, laboratory animals will be exposed to EDC mixtures from gestation to first generation to assess the programming and cumulative effects of EDC exposure on the HPA axis. Physiological and behavioral outcomes of HP disruption will be monitored. In parallel, effects on DNA methylation of hypothalamus, pituitary and peripheral blood cells, as well as changes in specific circulating ECV will be determined. An AI-based systems biology approach will be used to describe associations between hypothalamus/pituitary function, hematoperipheral blood cell methylation patterns, and multiomics data. These associations will be used to establish specific signatures associated with HP disruption by EDC exposure. Signatures will be explored in samples from the target population (i.e., children in their first 1000 days and prepubertal) to explore their translatability and application as biomarkers. Human studies will not be limited to cross-sectional determinations, but are intended as novel interventions to promote decreased EDC exposure and to assess cause-effect relationship. This will allow the definition of biomarkers based on data obtained from the different work packages and a systems biology approach to unravel conserved mechanisms of action.

   The work has been proposed as a sequence of tasks aimed at defining the impact of realistic EDC co-exposure on the HPA axis of two target life stages, i.e. prepubertal and perinatal. The different levels proposed by organizations such as OECD, including NAMs and computational toxicology have been adopted to minimize animal research. This design combines translational research with intervention studies.

2. HYPOTHESIS OR STUDY QUESTION A MBI in primary schools in two European countries (Spain and Belgium) over 28 months will reduce urinary EDC levels in prepubertal children and increase knowledge about these contaminants.

3. OBJECTIVES

3.1. MAIN OBJECTIVE The overall goal of HYPIEND is to understand the effects of EDC co-exposure on HPA axis function and epigenetic programming in order to delineate interventional strategies to minimize exposure and neuroendocrine consequences during the perinatal and prepubertal stages.

In this framework, the main objective of the clinical study is to demonstrate that a MBI implemented in primary schools in two different European countries during 25 months is effective in reducing the urinary EDC levels of prepubertal children using the HAPA constructs to improve the intervention and the knowledge about these contaminants of their parents, as well as to deepen the scientific knowledge.

3.2. SECONDARY OBJECTIVES

1. To determine whether an intervention designed to reduce EDC exposure can prevent some of the deleterious effects related to altered HP in prepubertal children aged 6 to 8 years (earlier onset of the pubertal phase and alterations in BMI and cognitive function).
2. To better understand the relationship between urinary EDC levels and HP-related hormone levels, as well as DNA methylation patterns and gut microbiota composition, shedding more light on the mechanisms by which EDCs may exert their deleterious effects.
3. To explore, by multivariate analysis and risk assessment analysis, predictors of elevated urine EDC levels.
4. To determine whether an intervention based on the combined HAPA1 and PBS2 models in primary schools is effective in improving HAPA constructs, knowledge and behaviour of parents, children and educational teams towards these pollutants, against a placebo intervention.

Inclusion Criteria:

• For children: aged between 6 and 8 from participating schools, and their parents/legal guardians.

• For parents/legal guardians:

  • Understanding the local languages (either French in Belgium, or Spanish/Catalan in Spain) and being able to complete forms in that language;
  • Be in possession of a smartphone. However, if participants do not have a smartphone and wish to do so, a smartphone will be made available to them for the duration of the study.

Exclusion Criteria:

• For schools :

  • The number of participating children in the school is less than 5.
  • Not being ready to devote time to the project.

• For children :

  • If children do not agree to participate (ICF for children), the parents will be asked to discuss the matter with them before the child is excluded.
  • If the parents or legal guardians do not sign the informed consent.

• For parents/legal guardians :

  • If the parents refuse to participate.
  • If the parents do not wish to use a mobile phone as part of the project, they will not be included in the sample.

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