Effectiveness of a multicOmpoNent Behavioural intervenTion to Reduce endocrINe disrUptor Exposure During pErinatal Period (CONTINUE)

Understanding and Preventing the Impact of Endocrine Disruptors on the Hypothalamus-pituitary Axis in Sensitive Populations. Hypiend- Perinatal Study.

The general objective of HYPIEND is to understand the effects of chemical substances called "endocrine disruptors" (EDCs) during the perinatal and pre-pubertal stages. EDCs co-exposure may affect the function of hormones and determine endocrine consequences in vulnerable populations.

The primary objective of this clinical study is to demonstrate that a multicomponent intervention implemented in health care centres from three European countries is effective in reducing the levels of EDCs in different body fluids of pregnant women, breastfeeding and formula feeding women as well as in their infants up to 18 months of age, improving at the same time the level of HAPA constructs (psychological determinants of behaviour) and the knowledge about these chemicals at family level.

Study rationale

1. Extent and evaluation of current knowledge directly linked to the scientific question(s) to be answered by the clinical study.

   Human epidemiological data suggest that EDC exposure during the perinatal period can negatively affect infant growth trajectory and neurodevelopment and possibly play an important role in the rapid epidemiological growth of obesity and diabetes. As an example, one study carried out in 460 mother-infant pairs from Korea showed that Mental and/or Psychomotor Developmental Indices of the Bayley Scales of Infant Development were inversely correlated in 6-month-old males with the maternal urinary levels of the phthalate metabolites MEHHP, MEOHP and MBP at the third trimester of pregnancy. Very recently, it was reported that mothers with overweight displayed increased levels of the highly persistent environmental chemicals polychlorinated biphenyls in breast milk 2-weeks postpartum and that there was a negative association between some of these compounds and the head circumference-for-age, weight-for-age, and weight-for-length z-scores of the infant at the age of 6 months.

   However, there are discrepancies among findings, which can be attributed to differences in the temporal window of EDC exposure assessment (i.e., early versus late gestation or lactation), in the infants' age at which the measurements were carried out or to a misclassification of EDC exposure due to a single-point analyses of their exposure. In fact, one of the limitations of many epidemiological studies is the use of single spot urine samples at one time-point to estimate the exposure to EDCs. The limitation relies on the short biological half-lives of many of these chemicals and their quick excretion in urine, properties that cause a demand for multiple time point for a precise estimation of exposure assessment. Although different epidemiological studies have found an association between higher exposure to EDCs and neuroendocrine and neurodevelopmental alterations in toddlers and children, as far as we know, no clinical interventions have been carried out to elucidate whether reducing the exposure to EDCs can contribute to counteract/ameliorate these harmful effects.

   So far, very few human intervention studies focused on reducing the exposure to EDCs during the perinatal period have been conducted, some carrying out educational approaches and others focused on dietary or other changes. Relevantly, most of them had small sample sizes (<100 people) and analysed a maximum of two different types of EDCs and quite a few were carried out without control groups. Moreover, the level of adherence in this type of interventions is low in general, reflecting the need to implement adequately behavioural change techniques to foster long-term lifestyle habit improvements.

2. Outcomes (efficacy, safety) Of completed and number of ongoing clinical studies utilizing the same intervention in the same indication (including review of public registers)

   Recently, the PREVED project was carried out to improve knowledge, to enhance risk perception and to change exposure behaviour regarding EDCs of 268 women during pregnancy and up to 14 months after birth . The authors reported a significant increase in the evolution of risk perception score and overall psychosocial score in the two intervention groups that received 3 workshops during pregnancy, one group in neutral location (leaflet on EDCs and collective workshops in a meeting room), and the other group in contextualized location (leaflet on EDCs and collective workshops in the real-life pedagogical apartment) when compared with a control group (leaflet on EDCs). However, no differences in consumption of canned food and in percentage of women having a decrease in bisphenol A or parabens concentrations in urine were found between the control and the two intervention groups. In another RCT carried out in 51 women in Korea, a 4-week web-based behavioural intervention reduced the urinary concentrations of BPA, different phthalate metabolites, and parabens in mothers with young children . Moreover, an ongoing study aims to determine if a personalized mobile intervention is able to reduce the exposure to EDCs in adults of child-bearing age (ClinicalTrials ID: NCT05780047). Despite some promising results, these examples illustrate the need to carry out larger multicomponent clinical and community-based intervention studies focused on minimize the exposure to EDCs at short, medium, and long time to obtain results that are more conclusive.

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 the experimental research in both cells and animals with epidemiological data. Researchers of both projects and others nicely showed using a statistical model for multivariate regression with data obtained in the SELMA pregnancy study that different EDCs (including phthalates, alkyl phenols, and perfluoroalkyl substances) analysed in the urine and serum of women at week 10 of pregnancy were associated with language delay of their children at 2.5 years of age. Afterwards, 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 both experimental and epidemiological data and carried out a risk assessment approach, finding increased odds of language delay in up to 54% of the offspring who had prenatal exposures above experimentally derived levels of concern: Despite these very relevant findings, a limited number of studies carried out in humans have shed light on the mechanisms underlying the EDC-mediated dysfunctions on HP axis, including the induction of epigenetic changes in key genes involved in foetal development, neuroendocrine regulation and early puberty as well as the alterations of the inflammatory response and the gut microbiota (dysbiosis). EDCs may increase the risk of childhood neurodevelopmental disorders by interfering with early life estrogenic and thyroid hormone signalling or metabolism. In this sense, in humans, it has been shown that some phthalates may reduce thyroxine and triiodothyronine concentration in pregnant women and children and that the exposure to phthalates produced oxidative stress and can also affect the health of the offspring through epigenetic re-programming of the foetus and placenta. In another study, maternal exposure to heavy metals affected progeny neurodevelopment, and changes in DNA methylation of genes controlling neurodevelopment were observed in cord blood, but not the blood collected at mid-childhood. Analysis of exposure-outcome identified differentially methylated CpG on DAB Adaptor Protein 1 (DAB1) gene as a marker of the effect of prenatal polycyclic aromatic hydrocarbon (PAH) exposure on children's mental development. Increased exposure to PAHs during pregnancy was positively correlated with the methylation of insulin-like growth factor IGF1 and 2, key factors in human growth and development that are maternally imprinted. Incorrect methylation of those genes during early development relates to reduced birth weight and increased predisposition to metabolic problems, like obesity, cardiovascular diseases or diabetes. However, despite this evidence, as far as we know, no human intervention studies aimed at reducing the exposure to EDCs have analysed the molecular impact that can accompany these changes in EDC exposure. Further research is needed to shed more light on these issues, including molecular and omics-based analyses in humans to elucidate whether human intervention studies aimed at reducing EDC exposure can favourably modulate these molecular changes.

Different molecular and omics approaches will be carried out to shed more light in the framework of the HP axis on the epigenetic drivers most susceptible to disruption, the genes and the pathways most differently affected, the interplay between intestinal microbiota and the impact of EDC exposure on systemic inflammation. These analyses will be carried out in samples obtained in the study considering our results obtained in preclinical models exposed to EDC mixtures trying to resemble the real human exposure, to increase the chance to obtain robust and reliable results using a targeted approach. Specifically, in women the analyses will be carried out during pregnancy and after delivery at different time points and will be focused on the analyses of HP axis-related hormones and cytokines, intestinal microbiota, methylation of different CpG sites of key genes involved in HP axis as well as the expression of these genes. In their offspring, in addition to these analyses, the circulating levels of kisspeptin will be also analysed at birth (cord blood) and at the age of 18 months (capillary blood/dry blood spots -DBS-). Overall, these analyses will allow us to go into depth on the mechanisms by which EDCs exert their harmful effects as well as how intervention studies aimed at reducing the exposure to these chemicals can modulate these biological pathways and the molecular biomarkers of exposure identified. As far as we know, this approach has not yet been carried out in large human intervention studies in the framework of EDCs.

Inclusion Criteria:

• Pregnant women with a viable pregnancy confirmed by ultrasound until 13 weeks and 0 days of gestation (recruitment between 6-11 weeks). Their partners will be invited to participate but this is not mandatory for inclusion.
• BMI at Visit 1 between 18.5-40 kg/m²
• Intention to breastfeed
• Being able to read the language of their respective countries (Dutch/French -Belgium-, Spanish and/or Catalan -Spain- and Polish -Poland)
• Being 18 years or older
• In possession of a smartphone. If participants do not have a smartphone because of socio-economic reasons, a smartphone will be at their disposal for the whole duration of the study

Exclusion Criteria:

• Unable to sign informed consent (cultural barriers, psychological conditions)
• Abuse of substances (alcohol, drugs)
• Chronic use (at least, for three months before pregnancy) of any medication that might affect the HP axis:
• Antidepressants
• Insuline.
• Levothyroxine, Methimazole, Propylthiouracil.
• Oral corticosteroids (topical and inhalation formulations will be allowed)
• Arginine vasopressin (AVP)
• Mifepristone.
• Anticortisolic drugs: Metyrapone (Metopirone), Ketokonazole, Osilodrostat (Isturisa), Mitotane (Lysodren), aminoglutethimide (Cytadren) and Levoketoconazole (Recorlev).
• Multiple gestation
• Type 1 or 2 diabetes
• Pregnant women will not be consented into research by any HCP with whom they have a dependent relationship (Declaration of Helsinki) (I would remove this point).