mother, father, brother, two sisters Mubashir

Dr Hussain Munawar

Soomro

Keywords: Phthalate, prenatal exposure, endocrine disruptor, urinary biomarkers, mother-child cohort, pregnancy induced hypertension

Thèse réalisée dans l'unité INSERM UMR-S 1136:

Equipe EPAR "Epidémiologie des maladies Allergiques et Respiratoires".

research and I thank her for the understanding throughout my PhD study. Her advice and encouragement gave me the confidence to lay a foundation to work independently, without her continuous guidance, it would not have been possible to complete this work.

Isabella, has been highly supportive and allowed me the freedom to pursue various projects without any impediment. I am extremely grateful for her trust on me and the support.

I am very grateful to Professor Carl-Gustaf Bornehag for his consistent support and professional guidance. His personal interest in Endocrine Disruptor's work has been a great source of inspiration for me. I would also like to extend my appreciation to my committee members:

Professor Aziz Sheikh, Professor Dimosthenis Sarigiannis, Professor Anita Burgun, Professor Emeritus Jean-Francois Bernaudin, Dr. Nhan Pham-Thi and Dr. Fleur Cohen for their attendance in my thesis defense, their encouragement, and insightful comments on my thesis manuscript. My deepest appreciations to the administrative staff; Lydie Martorana and Magali Moulé at doctoral school ED 393.

I would like to thank my colleagues Nour BaÏz, Amir Gamil, Fouad Amrani, Shreosi Sanyal, Cara Nicole Maesano, Fatima Benaissa, Laura Judge, Anne-Gwenhael, Ramen Munir Baloch for their assistance and discussions on different research projects. My special thanks to Diogo Griziotti for his help in my everyday life.

I also thank my friends (although too many to list here) especially Dr. Rahim Baig, Dr.

Sohail Ahmed, Dr. Saima, Dr. Mansoor Magsi, Ghulam Abbas Rahar, for their support and the friendship that I needed the most during the course of my research work.

My special thanks to my lovely family. Words cannot express how grateful I am to my mother and father for their love and prayers as well as for nurturing my son. I drew a lot of inspiration from my father Dr. Manzoor Hussain Soomro, himself a Professor and renowned Pakistani scientist for the research leading to improved health of rural communities, which I intend to strive for in future as well. I also wish to thank my two sisters, brother, his wife and their lovely children for their love and moral support as well as for taking good care of my son.

Finally, I am deeply appreciative of my beloved son Mubashir for his patience specially while my being away. Indeed he deserves great appreciation and love for his understanding and patience.

Munawar Hussain Soomro

RÉSUMÉ LONG (Français)

Introduction

Tous les êtres humains sont exposés aux produits chimiques dans la vie de tous les jours, provenant des aliments, de l'environnement intérieur, des cosmétiques et d'autres produits qui nous entourent, que ce soit à la maison et au travail. De plus, l'utilisation de compléments alimentaires, de médicaments et de médicaments phytothérapeutiques peut avoir un effet chimique sur le corps, ce qui peut avoir des effets souhaités ou non. Parmi les milliers de produits chimiques auxquels nous sommes potentiellement exposés au quotidien, il a été démontré que certains agissaient en tant que perturbateurs endocriniens (PE) chez les animaux de laboratoire. Les PE sont des composés synthétiques capables de se lier aux récepteurs cellulaires et d'imiter ou de bloquer le fonctionnement des hormones naturelles, pouvant ainsi entraîner des effets néfastes sur la santé. Cependant, il existe également des composés naturels dotés de telles propriétés. Chez l'homme, il a été démontré que les PE étaient responsables de la cryptorchidie (testicules non descendus dans le scrotum) et de l'hypospadias (malformation des organes génitaux) chez les garçons, de la puberté précoce chez les filles, du cancer du sein et de la prostate, du syndrome métabolique, de l'obésité, du diabète, des maladies cardiovasculaires, d'une moindre qualité du sperme, de l'augmentation de l'incidence du cancer des testicules et de niveaux réduits de l'hormone sexuelle masculine chez l'homme. Certaines des périodes les plus sensibles de la vie humaine sont la vie foetale et l'enfance, car le corps humain se développe de manière significative au cours de ces périodes, ce qui nécessite un équilibre des systèmes endocriniens impliqués pendant les différents stades du développement.

Ces dernières années, des hypothèses ont été émises selon lesquelles les PE pourraient également contribuer au développement de maladies allergiques et respiratoires. En outre, il est probable que les PE pourraient déjà agir pendant la grossesse en provoquant un diabète gestationnel (DG) et une hypertension induite par la grossesse, pouvant également provoquer une hypertension et un diabète plus tard dans la vie.

Ce travail de thèse a permis de mettre en évidence que l'exposition maternelle aux phtalates et aux métaux lourds (plomb, cadmium et manganèse) pendant la grossesse pourrait être associée au diabète gestationnel, à l'hypertension induite par la grossesse et à l'eczéma/dermatite atopique chez l'enfant. Cet objectif sera atteint grâce à la mise en place d'une étude robuste, réduisant ainsi le risque d'une mauvaise classification des maladies et de l'exposition.

Matériel et méthodes

Nous avons utilisé les données de la cohorte Française de naissance mèreenfant, EDEN (Étude des déterminants pré et post-natals du développement de la santé de l'enfant) pour examiner les hypothèses de ce travail de thèse. La cohorte de naissance mère-enfant EDEN a été créée en 2003 et mise en place dans deux hôpitaux universitaires des villes de Nancy et de Poitiers, en France. La participation à cette cohorte a été proposée à toutes les femmes visitant les cliniques prénatales des hôpitaux universitaires avant la 24ème semaine d'aménorrhée. Les critères d'exclusion étaient les grossesses multiples, le diabète connu avant la grossesse, l'analphabétisme ou le projet de quitter la région au cours des trois prochaines années.

Les mères ont été recrutées entre 2003 et 2006. Les mères ont été examinées à trois reprises pour des examens cliniques, une fois entre la 24ème et la 28ème semaines d'aménorrhée, une fois à l'accouchement et une fois 5-6 ans après l'accouchement.

Les enfants ont été suivis pendant cinq ans grâce à des visites dans les centres de recherche et des questionnaires envoyés aux parents.

Les femmes ont été invitées à un examen clinique et ont fourni les urines de leur première miction matinale recueillie à la maison avant leur visite à l'hôpital entre la 24ème et la 28ème semaine de gestation (deuxième trimestre de la grossesse). En cas d'oubli, l'échantillon d'urine a été collecté à l'hôpital. Les analyses des échantillons ont été effectuées au laboratoire national du centre de santé environnementale, aux centres de contrôle et de prévention des maladies (CCM) à Atlanta, Géorgie (États-Unis), pour onze métabolites provenant de huit phtalates: le phtalate de diéthyle (DEP), le phtalate de di-n-butyle (DBP), le phtalate de di-isobutyle (DiBP), le phtalate de di (2-éthylhexyle) (DEHP), le phtalate de benzylbutyle (BBzP), le phtalate de diisononyle (DiNP), le phtalate de di-n-octyle (DOP), le phtalate de di-isodécyle (DiDP).

L'eczéma a été évalué par l'administration d'une version enrichie du questionnaire standardisé et validé de l'étude internationale sur l'asthme et les allergies de l'enfance (ISAAC) complété par les parents lorsque leur enfant était âgé de 1, 2, 3, 4 et 5 ans, sur rendez-vous. Chaque année de suivi, l'eczéma était défini comme un eczéma diagnostiqué par un médecin au cours des 12 derniers mois.

L'eczéma « ever » a été défini comme ayant déjà fait l'objet d'un diagnostic d'eczéma à l'âge de 1, 2, 3, 4 ou 5 ans. En outre, deux phénotypes d'eczéma ont été distingués : l'eczéma d'apparition précoce dans le cas où un eczéma a été rapporté dans un questionnaire quelconque au cours des deux premières années et l'eczéma d'apparition tardive s'il n'a été signalé qu'entre 24 et 60 mois. À l'âge de 5 ans, les IgE sériques totales chez 293 garçons ont été évaluées. Les garçons ont été classés comme atopiques s'ils présentaient des taux d'IgE ≥ 60 UI / mL. Trois métaux, le plomb (Pb), le cadmium (Cd) et le manganèse (Mn) ont été mesurés à partir d'échantillons sanguins de grossesse pendant le deuxième trimestre.

Les femmes ont été invitées à un examen clinique et à fournir un échantillon de sang veineux. Entre la 24ème et la 28ème semaine, les concentrations de glucose dans le sang maternel ont été mesurées une heure après une provocation de glucose à 50 g.

Les femmes ayant une glycémie à 1 heure supérieure à 130 mg/dL à Nancy et à 140 mg/dL à Poitiers devaient subir un test de tolérance au glucose oral de 3 h à 100 g. Le diagnostic de diabète gestationnel a été réalisé en utilisant le test de tolérance au glucose oral selon les critères de Carpenter et Coustan.

Lorsque plus de 2 glycémies étaient supérieures aux valeurs limites suivantes: à jeun = 95 mg / dL, à 1 h = 180 mg / dL, à 2 h = 155 mg / dL et à 3 h = 140 mg / dL. Un diagnostic de tolérance altéré au glucose (IGT) a été posé si l'une des valeurs limites du test de tolérance au glucose par voie orale était atteinte ou dépassée. Les variables DG (diabète gestationnel) et IGT ont été considérées comme variables catégoriques dans l'analyse. Aux fins de l'analyse, une variable DG ou IGT a également été considérée (combinaison de DG et IGT).

La tension artérielle maternelle a été mesurée lors de visites mensuelles de routine, en position de décubitus dorsal, à l'aide d'un tensiomètre à mercure standard.

Une série de mesures a été prise par la sage-femme assignée à l'étude, entre la 24ème et 28ème semaine en utilisant un dispositif différent (semi-automatisé), avec une moyenne calculée sur deux mesures pour déterminer les valeurs à l'issue de cette visite. Les mesures prises au cours de chaque trimestre de gestation (T1, T2 et T3) ont été moyennées pour déterminer les valeurs de pression artérielle systolique (PAS)

Résultats

Notre premier article a montré que la prévalence de l'eczéma « ever » était respectivement de 9,7%, 15,7%, 21,0%, 26,6% et 30,4% à 1, 2, 3, 4 et 5 ans.

L'incidence a diminué de 9,7% à l'âge de 1 an à 5,2% à l'âge de 5 ans. Nous avons mis en oeuvre une régression logistique multiple et avons observé que les métabolites du phtalate de di-isobutyle (DiBP) et du phtalate de di-isononyle (DiNP) étaient positivement associés à l'eczéma d'apparition précoce (de 0 à 24 mois) (15,7%) et tardive (de 24 à 60 mois) (14,7%). L'application du modèle de Cox a montré une association significative entre l'occurrence d'eczéma au cours des cinq premières

Mots-clés:

Phthalate, exposition prénatale, la cohorte mère-enfant, marqueurs urinaires, eczéma, IgE totales, atopique, garçons, métaux-lourds, diabète sucré gestationnel, intolérance au glucose, perturbateur endocrinien, la grossesse hypertension induite par la grossesse.

ABSTRACT (ENGLISH)

All humans are exposed to chemicals in everyday life, from food, indoor environment, cosmetics and other products that surround us at home and at work. Also the use of food supplements, medicines and herbal medicines may cause a chemical effect of the body, which can have both desired and unintended effects. Among the thousands of chemicals, one might be exposed to in daily life, some have been shown to act as endocrine disrupting chemicals (EDCs) in laboratory animals. EDCs are manmade compounds that can bind to cell receptors and mimic or block the functions of natural hormones potentially resulting in adverse health effects. In humans, EDCs have been found to contribute to cause cryptorchidism (undescended testicles to scrotum) and hypospadias (malformation of the genitals) in baby boys, earlier onset of puberty in girls, breast and prostate cancer, metabolic syndrome, obesity, diabetes and cardiovascular diseases and low semen quality, increased incidence of testicular cancer and reduced levels of the male sex hormone in men. Some of the most sensitive periods in the human life are fetal life and childhood, as humans and human bodies during these periods are undergoing a significant development, which requires a balance of the endocrine systems involved in the various stages of development.

In recent years, hypotheses have been put forward that EDCs may contribute also to the development of allergic and respiratory diseases. In addition, it is likely that EDCs could act already during pregnancy by causing gestational diabetes mellitus and pregnancy induced hypertension, which may also trigger to develop hypertension and diabetes mellitus later in life.

This dissertation provides evidence that maternal exposure to phthalates and heavy metals (Lead, Cadmium and Manganese) during pregnancy may be associated with gestational diabetes mellitus, pregnancy induced hypertension and later eczema/atopic dermatitis in children. This will be reached through a robust study design thus diminishing exposure and diseases misclassification.

We used ongoing French EDEN-mother child cohort data for our three studies.

Our first paper showed that the metabolites of di-isobutyl phthalate (DiBP) and diisononyl phthalate (DiNP) were positively associated with early-onset (0-24 months of age) eczema (15.7%) and late-onset (24-60 months of age) eczema (14.7%). Applying the Cox's model showed a significant association of occurrence of eczema in the first 5 years of age with DiBP and DiNP metabolites. Among IgE sensitized boys, metabolites of di-n-butyl phthalate (DBP) and DiBP were significantly associated with ever eczema (Hazard Ratio (HR) = 1.67; 95% Confidence Interval 1.10-2.54; P = 0.01 and HR = 1.87; 95% CI 1.01-3.48; P = 0.04, respectively).

While in paper two, we observed that Lead was significantly associated with gestational diabetes mellitus (Odds-Ratio: 1.83, 95% Confidence Interval: 1.01-3.29, P-value = 0.04) and this persisted after adjusting for confounders (1.65, 0.82-3.34, Pvalue = 0.08). A borderline significance existed also between Cadmium and gestational diabetes mellitus. The association was statistically significant between Cadmium and the combination of gestational diabetes mellitus and impaired glucose tolerance in the adjusted model (1.61, 1.05 -2.48, P-value = 0.03).

Third paper shows that, low molecular weight phthalates metabolites;

Monoethyl phthalate (MEP) and Mono-n-butyl phthalate (MBP) were positively associated with pregnancy induced hypertension in crude , p-value = 0.01) models respectively. We observed a borderline association with one of the high molecular weight phthalate metabolite; Mono (3-carboxypropyl) phthalate (MCPP) in crude (1.46, 0.96-2.24, p-value = 0.08) as well as in adjusted model (1.56, 0.93-2.64, p-value = 0.09).

In conclusion, our results add new suggestive evidence on the relationship between EDCs (phthalates and heavy metals we studied) and maternal and children health, that prenatal exposure to certain phthalates and heavy toxic metals may play a role in the development of gestational diabetes mellitus and pregnancy induced hypertension during pregnancy and later eczema in early childhood. Future research focusing on larger populations and addressing multiple exposures assessed prenatally and postnatally are required to provide more evidence on possible contributions of emerging pollutants to study the role of phthalates and heavy metals.

Keywords: Phthalate, prenatal exposure, mother-child cohort, urinary markers, eczema, total IgE, atopic, boys, heavy metals, mother-child cohort, gestational diabetes mellitus, impaired glucose tolerance, endocrine disruptor, pregnancy induced hypertension.

ARTICLES IN THESIS

Articles published or submitted

INTRODUCTION

In our daily life, we widely use chemical products to facilitate ourselves. These chemical products can be found in the food wrappings, children's toys, cleaning products, medical devices, pharmaceuticals, polyvinyl chloride (PVC) flooring, building materials because of their properties of transparency, durability, and flexibility.

However, the concern about its effects on health are not very clear. These chemicals included in these commonly used products include substances known as endocrine disruptors or endocrine disrupting chemicals (EDCs) 1 , which have the properties to interact with the natural hormone system in humans and as well as in animals and thus impact on their health.

Endocrine

Disrupting Chemicals

Human exposure to phthalates

Phthalates are ubiquitous environmental toxicants, arising from contact with a wide range of consumer products. Normally, phthalates in their pure form are colorless, odorless and oily liquids with high lipophilic properties and low solubility in water.

People can be exposed to phthalates through inhalation, dermal absorption (including air-to-skin transport), contact with contaminated surfaces, and use of personal care products and ingestion (including dietary ingestion and incidental ingestion) or intravenous injection [START_REF] Colacino | Dietary intake is associated with phthalate body burden in a nationally representative sample[END_REF][START_REF] Mitani | Simple and rapid analysis of endocrine disruptors in liquid medicines and intravenous injection solutions by automated in-tube solid-phase microextraction/high performance liquid chromatography[END_REF][START_REF] Mitani | Analysis of phthalate contamination in infusion solutions by automated on-line in-tube solid-phase microextraction coupled with highperformance liquid chromatography[END_REF][START_REF] Schettler | Human exposure to phthalates via consumer products[END_REF][START_REF] Swan | Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans[END_REF].

For many phthalates main route of exposure is through food ingestion [START_REF] Colacino | Dietary intake is associated with phthalate body burden in a nationally representative sample[END_REF], including fatty foods such as dairy products, fish and oil [START_REF] Cheng | Risk assessments of human exposure to bioaccessible phthalate esters through market fish consumption[END_REF][START_REF] Kappenstein | Toxicologically relevant phthalates in food[END_REF][START_REF] Sharman | Levels of di-(2-ethylhexyl)phthalate and total phthalate esters in milk, cream, butter and cheese[END_REF], in drinking water [START_REF] Santana | Preliminary toxicological assessment of phthalate esters from drinking water consumed in portugal[END_REF][START_REF] Wang | Health risk assessment of phthalate esters (paes) in drinking water sources of china[END_REF], and also in wild marine organisms [START_REF] Hu | Phthalate monoesters as markers of phthalate contamination in wild marine organisms[END_REF]. As matrices and solvents, it has been reported that considerable amount of phthalates (up to 5%) are added to a wide range of personal care products, such as cosmetics, lotions, deodorants, perfumes, hair products and nail polish (Al-Saleh and Elkhatib 2016; [START_REF] Huang | Characterization of phthalates exposure and risk for cosmetics and perfume sales clerks[END_REF][START_REF] Just | Urinary and air phthalate concentrations and self-reported use of personal care products among minority pregnant women in new york city[END_REF][START_REF] Koniecki | Phthalates in cosmetic and personal care products: Concentrations and possible dermal exposure[END_REF][START_REF] Silva | Urinary levels of seven phthalate metabolites in the u.S. Population from the national health and nutrition examination survey (nhanes) 1999-2000[END_REF].

Interestingly, DEHP is present in common medical devices e.g. tubing, blood and intravenous bags, catheters, dialysis equipment, and in the manufacturing of disposable and surgical gloves (National Toxicology 2011).

Phthalates are frequently used in children toys because of their flexibility and resistant kinking properties [START_REF] Andaluri | Plastic toys as a source of exposure to bisphenol-a and phthalates at childcare facilities[END_REF][START_REF] Ashworth | Analysis and assessment of exposure to selected phthalates found in children's toys in christchurch, new zealand[END_REF]), however phthalates are can also found in sex toys as they are largely untested and the policies are unregulated [START_REF] Biesanz | Dildos, artificial vaginas, and phthalates: How toxic sex toys illustrate a broader problem for consumer protection, 25 law & ineq[END_REF].

Another source of exposure to phthalates are through the French wines and spirits marketed in Europe or exported has been reported, in which DBP, DEHP and BBP phthalates were detected [START_REF] Chatonnet | Contamination of wines and spirits by phthalates: Types of contaminants present, contamination sources and means of prevention[END_REF][START_REF] Fan | Rapid determination of phthalate esters in alcoholic beverages by conventional ionic liquid dispersive liquid-liquid microextraction coupled with high performance liquid chromatography[END_REF].

Based on large production volumes, widespread use, and environmental contamination, bio-monitoring data suggest that 75-100% of the population is exposed to phthalates on a daily basis [START_REF] Hogberg | Phthalate diesters and their metabolites in human breast milk, blood or serum, and urine as biomarkers of exposure in vulnerable populations[END_REF][START_REF] Silva | Urinary levels of seven phthalate metabolites in the u.S. Population from the national health and nutrition examination survey (nhanes) 1999-2000[END_REF]). Once consumed by humans or animals, phthalates are rapidly metabolized in the gut, liver, and blood by esterases and lipases. Initially, the phthalate diester break down into their respective hydrolytic monoester where only one alkyl-chain remains on the phthalic acid backbone, and interestingly, it is often the monoester metabolites that induce toxicity [START_REF] Braun | Phthalate exposure and children's health[END_REF][START_REF] Koch | Human body burdens of chemicals used in plastic manufacture[END_REF]. Depending on the size of the remaining monoester metabolite, the alkyl-chain can undergo further oxidative metabolism and ultimately glucuronidation depending on the species. These hydrolytic monoester and oxidative monoester metabolites, in addition to the parent phthalates, are used as biomarkers to estimate daily human exposure levels [START_REF] Koch | Human body burdens of chemicals used in plastic manufacture[END_REF].

Health effects of phthalates

Human body is working on a well-functioning endocrine system to regulate hormones release. The endocrine system is made up of glands that play a central role in all vertebrates by producing and secreting hormones, chemical substances produced in the body that regulate the activity of cells or organs.

These hormones regulate the body's growth, metabolism (the physical and chemical processes of the body), and sexual development and function. In vertebrates, the centre for neural control for all endocrine systems is managed by hypothalamus [START_REF] Diamanti-Kandarakis | Endocrine-disrupting chemicals: An endocrine society scientific statement[END_REF][START_REF] Gore | Edc-2: The endocrine society's second scientific statement on endocrine-disrupting chemicals[END_REF]. EDCs may interfere with the body's endocrine system (Figure 2) in the production, secretion, transportation, metabolism, receptor binding, mediation of effects, and excretion of natural hormones which regulate developmental processes and support endocrine homeostasis in the organism and produce adverse developmental, reproductive, neurological, and immune effects in both humans and animals [START_REF] Kavlock | Research needs for the risk assessment of health and environmental effects of endocrine disruptors: A report of the u.S. Epa-sponsored workshop[END_REF][START_REF] Kuo | Immunomodulatory effects of environmental endocrine disrupting chemicals[END_REF][START_REF] Meeker | Exposure to environmental endocrine disruptors and child development[END_REF]).

For many years, when assessing the possible effects of EDs, according to toxicologists, it was said that "the dose makes the poison", implying that higher doses cause greater harm and assuming that effects not seen at high doses are not expected at low doses. In contrast to this dogma in toxicology, multiple studies have revealed that neither threshold nor linear no-threshold models are applicable to the responses seen in connection with EDCs [START_REF] Rhomberg | Low-dose effects and nonmonotonic doseresponses of endocrine disrupting chemicals: Has the case been made?[END_REF][START_REF] Vandenberg | Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses[END_REF]). Dose-response curves associated with EDCs often appear as Ushaped (nonmonotonic) as compared with the linear dose-response curves usually displayed in toxicology [START_REF] Rhomberg | Low-dose effects and nonmonotonic doseresponses of endocrine disrupting chemicals: Has the case been made?[END_REF][START_REF] Vandenberg | Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses[END_REF]. Humans are exposed to many different EDCs simultaneously, each of these chemicals may be being "safe", but mixtures of these EDCs may have both additive and synergistic effects. Therefore, a mixture may be result in more severe endocrine disruption than one chemical at a time [START_REF] Fowler | Impact of endocrine-disrupting compounds (edcs) on female reproductive health[END_REF].

Phthalates effects during pregnancy and early life

During first trimester of pregnancy, the embryo becomes a fetus and vital organs for example kidney, brain and liver are developed and start functioning. During second trimester, fetus start developing eyelids, eyebrows, eye lashes, bones becoming denser and nervous system, reproductive organs and genitalia are fully developed and start functioning. During the third trimester of the pregnancy, the lungs begin to develop and does not start working until several weeks later. If the fetus is exposed to harmful environmental chemicals during this critical developmental period, it may result in adverse health effects that may manifest later in life (Figure 3). Prenatal and early life are critical in the development of the immune system; exposure to EDCs as a toxic pollutants during this period can result in an increased risk of adverse health outcomes later in life. Exposure to these EDCs during the pregnancy and early childhood is potentially sensitive periods for the development of adverse effects on the health of mother causing gestational diabetes mellitus (GDM) [START_REF] Liu | Cadmium body burden and gestational diabetes mellitus: A prospective study[END_REF][START_REF] Oguri | Association between maternal blood cadmium and lead concentrations and gestational diabetes mellitus in the japan environment and children's study[END_REF][START_REF] Romano | A casecohort study of cadmium body burden and gestational diabetes mellitus in american women[END_REF][START_REF] Shapiro | Exposure to phthalates, bisphenol a and metals in pregnancy and the association with impaired glucose tolerance and gestational diabetes mellitus: The mirec study[END_REF], pregnancy induced hypertension (PIH) [START_REF] Cantonwine | Urinary concentrations of bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia[END_REF][START_REF] Werner | The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The home study[END_REF][START_REF] Yazbeck | Maternal blood lead levels and the risk of pregnancy-induced hypertension: The eden cohort study[END_REF]), and reproductive system [START_REF] Aung | Associations between maternal plasma measurements of inflammatory markers and urinary levels of phenols and parabens during pregnancy: A repeated measures study[END_REF][START_REF] Chen | Association between organochlorine pesticide levels in breast milk and their effects on female reproduction in a taiwanese population[END_REF] as well for the childhood neurodevelopmental disorders and obesity [START_REF] Braun | Early-life exposure to edcs: Role in childhood obesity and neurodevelopment[END_REF][START_REF] Ejaredar | Phthalate exposure and childrens neurodevelopment: A systematic review[END_REF][START_REF] Kim | Childhood obesity and endocrine disrupting chemicals[END_REF][START_REF] Whyatt | Maternal prenatal urinary phthalate metabolite concentrations and child mental, psychomotor, and behavioral development at 3 years of age[END_REF], and asthma and allergies (A Just et al. 2012;[START_REF] Stelmach | The effect of prenatal exposure to phthalates on food allergy and early eczema in innercity children[END_REF][START_REF] Whyatt | Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: The columbia center for children's environmental health cohort[END_REF]. Hence, the exposure to chemicals with endocrine disrupting properties is a growing concern for the pregnant mother, fetus, infants, children and adults.

In recent years, growing evidences have suggested that during the different stages of pregnancy, the exposure to phthalates can alter the concentrations of naturally-produced hormones, which help to regulate and control different cells and organs in the body. The exposure during this window periods can effect adversely for the health of the mother as well as play a critical role in early fetal development.

The exposure during pregnancy may cause impaired glucose tolerance or gestational diabetes mellitus [START_REF] Bellavia | Urinary phthalate metabolite concentrations and maternal weight during early pregnancy[END_REF][START_REF] Fisher | Serum phthalate and triclosan levels have opposing associations with risk factors for gestational diabetes mellitus[END_REF][START_REF] Meeker | Pregnancy urinary phthalate metabolite concentrations and gestational diabetes risk factors[END_REF]Zhang et al. 2017), pregnancy induced hypertensive disorders (clinical diagnoses of gestational hypertension, preeclampsia, eclampsia, or HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count) [START_REF] Cantonwine | Urinary concentrations of bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia[END_REF][START_REF] Rahmani | Prenatal exposure to phthalic acid induces increased blood pressure, oxidative stress, and markers of endothelial dysfunction in rat offspring[END_REF][START_REF] Werner | The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The home study[END_REF]; Zlatnik 2016), miscarriage or pregnancy loss [START_REF] Gao | Urinary concentrations of phthalate metabolites in early pregnancy associated with clinical pregnancy loss in chinese women[END_REF][START_REF] Messerlian | Urinary concentrations of phthalate metabolites and pregnancy loss among women conceiving with medically assisted reproduction[END_REF][START_REF] Peng | A study on phthalate metabolites, bisphenol a and nonylphenol in the urine of chinese women with unexplained recurrent spontaneous abortion[END_REF][START_REF] Yi | A pilot study on association between phthalate exposure and missed miscarriage[END_REF][START_REF] Han | In-situ degradation of soil-sorbed 17beta-estradiol using carboxymethyl cellulose stabilized manganese oxide nanoparticles: Column studies[END_REF], poly-cystic ovarian syndrome [START_REF] Rutkowska | Polycystic ovary syndrome and environmental toxins[END_REF], alteration in maternal serum thyroid hormones and sex hormones levels [START_REF] Huang | Early phthalates exposure in pregnant women is associated with alteration of thyroid hormones[END_REF][START_REF] Johns | Urinary phthalate metabolites in relation to maternal serum thyroid and sex hormone levels during pregnancy: A longitudinal analysis[END_REF][START_REF] Kuo | Relationship of urinary phthalate metabolites with serum thyroid hormones in pregnant women and their newborns: A prospective birth cohort in taiwan[END_REF].

In infants, toddlers, children, and adolescents, the routes and sources of phthalate exposure are related to developmental milestones and can be determined by hand-to-mouth activity, mobility, personal care or hygiene practices, diet, and health status throughout the development periods [START_REF] Braun | Phthalate exposure and children's health[END_REF][START_REF] Wormuth | What are the sources of exposure to eight frequently used phthalic acid esters in europeans?[END_REF].

Animals as well as epidemiological studies in infants and children have found various health effects of specific phthalates, including asthma and allergies [START_REF] Bamai | Exposure to house dust phthalates in relation to asthma and allergies in both children and adults[END_REF][START_REF] Berger | Associations between prenatal maternal urinary concentrations of personal care product chemical biomarkers and childhood respiratory and allergic outcomes in the chamacos study[END_REF]Bornehag et al. 2004b [START_REF] Braun | Phthalate exposure and children's health[END_REF][START_REF] Ejaredar | Phthalate exposure and childrens neurodevelopment: A systematic review[END_REF][START_REF] Huang | Fetal and childhood exposure to phthalate diesters and cognitive function in children up to 12 years of age: Taiwanese maternal and infant cohort study[END_REF][START_REF] Whyatt | Maternal prenatal urinary phthalate metabolite concentrations and child mental, psychomotor, and behavioral development at 3 years of age[END_REF].

The prevalence of eczema in childhood, including its allergic-related phenotype, varies globally from 3% to 37% and is steadily increasing globally [START_REF] Asher | Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: Isaac phases one and three repeat multicountry cross-sectional surveys[END_REF][START_REF] Deckers | Investigating international time trends in the incidence and prevalence of atopic eczema 1990-2010: A systematic review of epidemiological studies[END_REF][START_REF] Eichenfield | Guidelines of care for the management of atopic dermatitis: Section 1. Diagnosis and assessment of atopic dermatitis[END_REF][START_REF] Nutten | Atopic dermatitis: Global epidemiology and risk factors[END_REF]. The causes of respiratory diseases, asthma and eczema and of its increased prevalence are still unclear but are likely to be multifactorial in nature and depend on both genetic and environmental factors [START_REF] Aguilar | Computational analysis of multimorbidity between asthma, eczema and rhinitis[END_REF][START_REF] Nutten | Atopic dermatitis: Global epidemiology and risk factors[END_REF][START_REF] Pyun | Natural history and risk factors of atopic dermatitis in children[END_REF]. During in utero life, the skin of the fetus may be exposed to various products absorbed by the mothers, including phthalates given that some phthalates have been detected in amniotic fluid [START_REF] Jensen | Amniotic fluid phthalate levels and male fetal gonad function[END_REF]. Later in life, children's dermal exposure can occur through the use of emollients, personal care products, and dermal contact with plastic products, soil, and dust, which can add to the total intake of certain phthalates through other routes such as inhalation and ingestion [START_REF] Overgaard | Children with atopic dermatitis and frequent emollient use have increased urinary levels of low-molecular-weight phthalate metabolites and parabens[END_REF][START_REF] Wormuth | What are the sources of exposure to eight frequently used phthalic acid esters in europeans?[END_REF]. Phthalates might influence the epidermal barrier development processes, which can take several years to occur, and this influence could be stronger in allergic individuals due to the fragility of their mucosae.

Replacement of phthalate and their alternatives

Over the past few decades, based on research on phthalate exposure and its effects on human and animal health, researchers and policy makers pushed companies to work on phthalate alternatives. This need for alternatives to phthalates has led manufacturers to create alternatives that share properties of phthalate plasticizers without EDCs properties. Manufactures started replacement of DEHP with DINP and DIDP after ban on the usage of DEHP phthalate [START_REF] Attina | Association of exposure to di-2-ethylhexylphthalate replacements with increased insulin resistance in adolescents from nhanes 2009-2012[END_REF].

Further, di-iso-nonyl-cyclohexane-di-carboxylate (DiNCH) is recently introduced as an alternative phthalate plasticizer. Studies have shown that DiNCH exposure to human have limited potential health effects [START_REF] Machtinger | Urinary concentrations of biomarkers of phthalates and phthalate alternatives and ivf outcomes[END_REF][START_REF] Vliet | A review of alternatives to di (2-ethylhexyl) phthalate-containing medical devices in the neonatal intensive care unit[END_REF].

However, more studies are required on large scale to see the adverse effects of these alternative phthalate on human health.

How to reduce phthalates exposure

Plastics commonly used to make food storage containers can inadvertently leach hormone disrupting chemicals into food and drinks. Exposure to phthalates can be reduced by considering following few steps:

-Fresh is best: Phthalates can migrate from the lining of cans and plastic packing into food and drinks. While it is not practical to avoid food packaging altogether, it is better to opt for fresh or frozen instead of canned food as much as possible.

-Store it safe: Storing food and drinks in glass or stainless steel utensils instead of plastic material.

-Do not microwave in plastic: Warmer temperatures increase the rate of chemical leaching into food and drinks. So use heat resistant glass or ceramic containers when microwaved or heat food on the stove.

Human exposure to heavy metals

Metals are one of the major category of globally distributed pollutants and are natural elements. Exposure to heavy metals remains a public health concern for longtime because of their persistence in the environment and their ability to induce toxicity even at low level. In recent decades, their contamination has been dramatically increasing particularly in the developing countries because of their continuous discharge in sewage and untreated industrial effluents [START_REF] Cheng | Heavy metal pollution in china: Origin, pattern and control[END_REF][START_REF] Jan | Heavy metals and human health: Mechanistic insight into toxicity and counter defense system of antioxidants[END_REF].

Naturally occurring metals like lead (Pb), cadmium (Cd) and manganese (Mn) are also classified as EDCs [START_REF] Dyer | Heavy metals as endocrine-disrupting chemicals a[END_REF][START_REF] Georgescu | Heavy metals acting as endocrine disrupters[END_REF][START_REF] Iavicoli | The effects of metals as endocrine disruptors[END_REF][START_REF] Rahman | Infant and mother related outcomes from exposure to metals with endocrine disrupting properties during pregnancy[END_REF].

Exposure to these heavy metals can occur through a variety of routes. Metals may be inhaled as dust or fume in tiny particulate matter, such as the lead oxide particles produced by the combustion of leaded gasoline (Hu 2002). Some metals can be vaporized for example, mercury vapor in the manufacture of fluorescent lamps and inhaled. Metals may also be ingested involuntarily through food and drink. The amount which actually absorbed from the digestive tract can vary widely. It also depends on the chemical form of the metal and the age and nutritional status of the individual (Hu 2002). Metals can escape control mechanisms such as homeostasis, transport, compartmentalization, and binding to specified cell constituents, thus they can have toxic and even lethal effects. Heavy metals can cause malfunctioning of the cellular processes via displacement of essential metals from their respective sites [START_REF] Flora | Heavy metal induced oxidative stress & its possible reversal by chelation therapy[END_REF][START_REF] Jan | Heavy metals and human health: Mechanistic insight into toxicity and counter defense system of antioxidants[END_REF]. Once a metal is absorbed, it distributes in tissues and organs.

Excretion typically occurs primarily through the kidneys and digestive tract, however theses metals tend to persist in some storage sites, like the liver, bones, and kidneys, for years or decades [START_REF] Jarup | Hazards of heavy metal contamination[END_REF].

Cadmium

Cd is a non-essential, toxic heavy metal distributed widely in the environment [START_REF] Barregard | Cadmium exposure in relation to insulin production, insulin sensitivity and type 2 diabetes: A cross-sectional and prospective study in women[END_REF]. The principal source of Cd exposure for the general nonsmoking population is food [START_REF] Swaddiwudhipong | Progress in cadmium-related health effects in persons with high environmental exposure in northwestern thailand: A five-year follow-up[END_REF]. The main source of food contamination with Cd are water wastes, industrial aerosols, fertilizers which are phosphate based and pesticides [START_REF] Georgescu | Heavy metals acting as endocrine disrupters[END_REF]

Manganese

Mn is an essential micronutrient required for the activity of metabolism and antioxidant system (Livingstone 2017). However, the requirements are low and excessive intake or exposure to it can cause neurotoxicity [START_REF] Hutchens | Deficiency in the manganese efflux transporter slc30a10 induces severe hypothyroidism in mice[END_REF][START_REF] Pfalzer | Relationships between essential manganese biology and manganese toxicity in neurological disease[END_REF]. The sources of environmental manganese are sewage sludge, wastewater discharges, mining and mineral processing, emissions from alloy, steel, and iron production, and combustion of fossil fuels [START_REF] Han | In-situ degradation of soil-sorbed 17beta-estradiol using carboxymethyl cellulose stabilized manganese oxide nanoparticles: Column studies[END_REF][START_REF] Iavicoli | The effects of metals as endocrine disruptors[END_REF][START_REF] Van Wendel De Joode | Manganese concentrations in drinking water from villages near banana plantations with aerial mancozeb spraying in costa rica: Results from the infants' environmental health study (isa)[END_REF].

Heavy metals effects on pregnancy, fetus, infancy and childhood

Cadmium

Over the past few decades, epidemiological studies have shown the critical role of prenatal exposure to Cd in the development of human individuals and its impact on public health. High levels of Cd during pregnancy has contributed to the risk of altering organs and tissues in the female reproductive system which leads to reproductive disorders as well as led a significant increase in premature delivery [START_REF] Delemarre | Inducing puberty[END_REF][START_REF] Nishijo | Effects of maternal exposure to cadmium on pregnancy outcome and breast milk[END_REF][START_REF] Parodi | Alteration of mammary gland development and gene expression by in utero exposure to cadmium[END_REF].

In rodents, Cd mimic estrogenic effects, which may leeds to proliferation of uterine and mammary tissues [START_REF] Vaiserman | Early-life exposure to endocrine disrupting chemicals and laterlife health outcomes: An epigenetic bridge?[END_REF]

Manganese

Mn crosses the placenta, which may be adversely affect fetus and infants particularly during high concentrations of Mn, because of homeostatic mechanisms in young children, who absorb and retain a larger fraction of ingested Mn than adults [START_REF] Aschner | Nutritional aspects of manganese homeostasis[END_REF][START_REF] Yoon | Evaluating placental transfer and tissue concentrations of manganese in the pregnant rat and fetuses after inhalation exposures with a pbpk model[END_REF][START_REF] Yoon | Physiologically based pharmacokinetic modeling of fetal and neonatal manganese exposure in humans: Describing manganese homeostasis during development[END_REF]. Based on animal studies, exposure to Mn may affect the normal endocrine system functions, particularly the secretion and production of sexual hormones [START_REF] Hiney | Manganese protects against the effects of alcohol on hypothalamic puberty-related hormones[END_REF][START_REF] Lee | Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats[END_REF][START_REF] Pine | Manganese acts centrally to stimulate luteinizing hormone secretion: A potential influence on female pubertal development[END_REF]. Further in rats an increase in serum luteinizing hormones, follicular stimulating hormones and testosterone, sperm production and efficiency of spermatogenesis has been observed [START_REF] Lee | Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats[END_REF]).

Rationale and hypotheses of thesis

Humans are exposed to various chemicals in everyday life, from food, indoor environment, cosmetics and other products that surround us at work and at home. Because the development of the immune and organs largely takes place in utero and during early childhood. Exposure to environmental factors during these time windows may be particularly critical for the occurrence of diseases in both mother and children.

More specifically, in recent years, hypotheses have been put forward that EDCs may also contribute to the development of allergic and respiratory diseases. In addition, it is likely that EDCs could act already during pregnancy by causing gestational diabetes mellitus (GDM) and pregnancy induced hypertension, which may also trigger to develop hypertension and diabetes mellitus later in life.

However, as a whole, a limited number of studies have been conducted on the impact of EDCs on the health of the childbearing mother and the child in the early years of life. The mother's health is of importance because it can influence child's health later in life, so we conducted three studies and tested our hypotheses by using the French EDEN mother-child prospective cohort study.

The overall aim of this thesis is to improve the knowledge basis for risk assessment and address how exposure to phthalates and heavy metals during pregnancy contributes to the critical issues of maternal and child health. Based on the evidences that EDCs like phthalates and heavy metals may disrupt immunological, and hormonal normal functions to development of the epidermal barrier defects processes.

It improves on previous studies of phthalates and heavy metals which we studied adds substantially to the literature by investigating a poorly understood mechanism that has not been previously tested in humans.

Objectives of the thesis

The objectives of this thesis work are as follows:

General:

To determine the role of EDCs in the childbearing mothers and the children in their first five years of life using birth cohort data.

Specific:

1. To test the hypothesis that exposure to phthalates during in utero increase the risk of eczema in young children through the production of immune markers at various stages of life (Paper I).

2. To test the hypothesis that exposure to heavy metals during pregnancy and its association with gestational diabetes mellitus and impaired glucose tolerance (Paper II).

3. To test the hypothesis that exposure to phthalates during pregnancy and its association with pregnancy induced hypertension (Paper III).

To this extent, we used longitudinal population based data of the pregnant women and children living in two cities of France.

MATERIALS AND METHODS

Study population

The population data set used in this thesis is from the French EDEN (Etude des Déterminants pré et post natals du développement de la santé de l'Enfant) motherchild prospective birth cohort (https://eden.vjf.inserm.fr) to examine the hypotheses of the thesis. Participation in this cohort was proposed to all women visiting the prenatal clinics at the university hospitals before their 24th week of amenorrhea. Exclusion criteria were multiple pregnancies, known diabetes before pregnancy, French illiteracy or planning to move out of the region within the next 3 years. Among the 3758 women invited to participate, 2002 (53%) were enrolled in the cohort (1034 women from Nancy and 968 from Poitiers). Mothers were recruitment during the period of 2003 to 2006. On average, women were included at 15 weeks of amenorrhea (range: 8-26). Mean age was 29 years (range: 18-44). Whereas, 30% of the women were pregnant for the first time.

Introduction of the study protocols

Children were followed for up to 5 years, by visits to research centres and questionnaires mailed to parents. Clinical examinations from pregnancy to 5-6 years of age were performed by specifically trained midwives.

Assessments schedule

Mothers were examined three times for clinical examinations, one between 24 and 28 weeks of amenorrhea, one at delivery and one 5-6 years after delivery (Figure 5). The children were clinically examined for four times: at birth, at 1 year and 3 years and during the 6th year (at 5 years and 8 months, on average), with specific cognitive assessments by psychologists at 3 and 5-6 years. Concomitantly and in between clinical visits, mothers and fathers answered questionnaires, self-administered or administered by midwives, about their offspring, themselves and their household.

Questionnaires were filled up twice during the first year (at 4 and 8 months), at 1 year and every year thereafter until 5-6 years. At 4, 8 and 12 months, the parents provided a 3-day food record for their infants. Biological samples were collected from the mother during pregnancy and at birth, and from the child (or cord) at birth and at 5-6 years.

Blood samples were also collected from fathers at the child's birth. The mothers' recruitment and follow-up procedures, and methodological assumptions have been published in earlier studies.

Figure 5. Assessments and data collection -from mothers included during pregnancy to children 8 years after birth.

Ethical Approval

The study was approved by the relevant ethical committees (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Le Kremlin Bicêtre University Hospital, and Commission Nationale de l'Informatique et des Libertés), and all participating women gave informed written consent for their own participation and both parents for newborn child after delivery.

Data collection

The population in our studies is a subgroup of the French EDEN mother-child prospective birth cohort. The mothers' recruitment and follow-up procedures, and methodological assumptions have been published in earlier studies [START_REF] Heude | Cohort profile: The eden mother-child cohort on the prenatal and early postnatal determinants of child health and development[END_REF][START_REF] Philippat | Exposure to phthalates and phenols during pregnancy and offspring size at birth[END_REF].

Women were given an appointment with a study midwife between the 24 th and 28 th gestational weeks, during which an interview on life style factors was conducted and biological samples were collected. The information on the mothers and their newborns, including parity, mode of delivery, birth weight, gestational age, season of birth, behavioral and environmental data were collected by the use of questionnaires, interviews and from obstetric and pediatric records. Children were followed from birth to the age of 5 years. However, in our first paper only includes all male offspring for whom maternal urine samples had been analyzed for metabolites of phthalates as a part of previous studies [START_REF] Botton | Phthalate pregnancy exposure and male offspring growth from the intra-uterine period to five years of age[END_REF][START_REF] Chevrier | Maternal urinary phthalates and phenols and male genital anomalies[END_REF][START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF], which explains why we had only boys in the study. Restricting to one sex could be an advantage in the context where sex-specific effects of exposures are expected [START_REF] Harley | Prenatal and postnatal bisphenol a exposure and body mass index in childhood in the chamacos cohort[END_REF][START_REF] Wolff | Prenatal phenol and phthalate exposures and birth outcomes[END_REF]); a study restricted to one sex likely has a higher statistical power than a study including both.

EDCs investigated in this thesis

In this thesis we studied the effects of eleven metabolites of eight phthalates:

Diethyl phthalate (DEP), Di-n-butyl phthalate (DBP), Di-isobutyl phthalate (DiBP), Di(2ethylhexyl) phthalate (DEHP), Benzylbutyl phthalate (BBzP), Di-isononyl phthalate (DiNP), Di-n-octyl phthalate (DOP), and Di-isodecyl phthalate (DiDP) and three heavy metals: Lead (Pb), Cadmium (Cd) and Manganese (Mn) are presented (Table 2). Women were invited for a clinical examination and to provide a first morning urine at home before the hospital study visit between the 24 th and 28 th gestational weeks (second trimester of the pregnancy). If forgotten, the urine sample was collected at the hospital. A polypropylene container (FP40VPS) manufactured by Centre Quantification of the phthalate metabolites concentrations was performed using online solid phase extraction high performance liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry [START_REF] Silva | Quantification of 22 phthalate metabolites in human urine[END_REF]). Creatinine concentrations were also quantified following identical analytical procedure as described previously [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF]. The concentrations of metabolites below the limit of detection (LOD) were replaced by instrumental reading values or by the compound specific lowest instrumental reading value divided by the square root of two (LOD/√ -2) when the instrumental value found missing [START_REF] Hornung | Estimation of average concentration in the presence of non-detectable values[END_REF]. The vast majority of the samples/metabolites (>97%) were above LOD. Two-step standardization was performed to reduce variability in biomarkers urinary concentrations owed to sampling conditions as described previously [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF]. There was no significant difference in the concentrations of phthalates in the urines collected at homes compared to those of the urines collected in the hospital.

Phthalates

Heavy metals

Three metals Pb, Cd and Mn were measured from second trimester blood samples of pregnancy. Women were invited for a clinical examination and to provide a venous blood sample. EDTA tubes were used to collect the blood for assay. The blood tubes were prepared within an hour of sample collection. After aliquoting, all biological material was frozen at -80°C. After centrifugation at 3000 rpm for 5 min, maternal blood was aliquoted and frozen at -80°C for later analysis. The blood Pb, Cd and Mn concentrations were measured by electrothermal atomic-absorption spectrometry (model 4100 ZL; Perkin-Elmer, Courtaboeuf, France) with Zeeman background correction as previously described [START_REF] Bonithon-Kopp | Effects of pregnancy on the inter-individual variations in blood levels of lead, cadmium and mercury[END_REF][START_REF] Huel | Assessment of exposure to lead of the general population in the french community through biological monitoring[END_REF][START_REF] Mergler | Surveillance of early neurotoxic dysfunction[END_REF]. The limit of detection was 100%.

Eczema phenotypes

Eczema Further, the prevalence and the incidence of eczema were computed.

Immunoglobulin E (IgE) and atopic status

At the age of 5 years, total serum IgE in 293 boys was assessed by using the CAP assay according to the manufacturers' instructions (Pharmacia CAP System™, Pharmacia and Upjohn Diagnostics AB, Uppsala, Sweden). Boys were classified as atopic if they had IgE levels of ≥ 60 IU/mL [START_REF] Baiz | Early oral exposure to house dust mite allergen through breast milk: A potential risk factor for allergic sensitization and respiratory allergies in children[END_REF]).

Gestational diabetes mellitus and Impaired Glucose Tolerance

At 24-28 weeks, maternal blood glucose concentrations were measured 1 hour after a 50-g glucose challenge. Women with 1-h glucose concentrations over 130 mg/dL in Nancy and 140 mg/dL in Poitiers were scheduled for a 3-h 100-g oral-glucose tolerance test. The GDM was diagnosed by using the oral-glucose-tolerance test according to the Carpenter and Coustan criteria [START_REF] Steer | Maternal blood pressure in pregnancy, birth weight, and perinatal mortality in first births: Prospective study[END_REF]).

When there were >2 blood glucose concentrations greater than the following cut points: fasting = 95 mg/dL, at 1 h = 180 mg/dL, at 2 h = 155 mg/dL, and at 3 h = 140 mg/dL. While a diagnosis of IGT was assigned if one of the oral-glucose-tolerance test cut-off values was met or exceeded.

Maternal Blood Pressure Measurements

Maternal blood pressure was measured during routine monthly visits, with the subject in supine position, using a standard mercury sphygmomanometer. One set of measures was taken by the study midwife between 24 and 28 weeks using a different (semi-automated) device, with two measurements averaged to determine the values for that visit.

Measurements taken within each period of gestational trimester (T1, T2, and T3) were averaged to determine the values for systolic blood pressure (SBP) and diastolic blood pressure (DBP) assigned to each period.

Women were classified as having pregnancy induced hypertension based on SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg measured during at least two visits after the 22nd week of gestation, such that the elevated measures could have occurred during or across any of the three periods, at any point after 22 weeks. Consequently, no diagnosis of pregnancy induced hypertension was made before 24 weeks. All cases of pregnancy induced hypertension were diagnosed by the physicians.

Other Variables

Information on potential confounders was collected which includes; birth weight, gestational age (two-class variable: < 37 gestational week, ≥ 37 gestational week), season of birth, parity, number of siblings (0, 1,2 ≥ 3), exclusive breast-feeding for 4 or more months (yes/no), maternal age at delivery (< 25 years, 25-29 years, 30 -34 years, ≥ 35 years), pre-pregnancy maternal body mass index (BMI, < 18.5, 18.5-24.9, 25.0-30.0, > 30 kg/m 2 ), maternal and paternal history of allergies, maternal and paternal educational level (< 2 years after high school, High school +2 years, ≥ High school +3 years), household income, city of residence (Nancy / Poitiers), mode of delivery (vaginal, caesarean section), smoking during pregnancy (yes/no), maternal alcohol use during pregnancy (yes/no), maternal and paternal physician-diagnosis of asthma, rhinitis, eczema, and food allergies (yes/no), maternal physician-diagnosis of GDM (yes/no) and pregnancy induced hypertension (yes/no).

Statistical analysis

Urinary concentrations of phthalate metabolites and metals were log natural (ln) transformed because of non-normality of the distribution. For the purpose of analysis, MECPP, MEHHP, MEOHP and MEHP were grouped as sum of ΣDEHP (DEHP being their parent compound). We calculated the molar sum of these DEHP metabolites (nanomoles per milliliter) by dividing each metabolite concentration by its molecular weight and then summing the individual concentrations.

The prevalence of eczema, GDM and pregnancy induced hypertension was computed. Additionally incidence was measured for eczema in boys. We applied multiple logistic regression analysis to examine the association between each phthalate metabolite and ΣDEHP and eczema/pregnancy induced hypertension outcomes adjusted for potential confounders. The potential confounders were determined a priori from literature review. Further in our first paper we used Directed Acyclic Graph (http://www.dagitty.net/dags.html) to show the hypothesis relations between biomarkers concentration, confounders and eczema outcomes. The confounders included in our final model were parental asthma/rhinitis/eczema, maternal smoking, maternal age, maternal BMI, maternal education level, gestational age, and number of siblings. Models were additionally adjusted for recruitment centre.

We also applied the Cox proportional hazard discrete time survival model to estimate the association between in utero exposure to phthalates and eczema occurrence in the first 5 years of life after adjustment for the confounding factors.

Results were further stratified in order to see whether phthalate biomarkers were associated with atopic (presence of allergic sensitization according to IgE) or nonatopic (absence of allergic sensitization to IgE) eczema. We also took IgE as an additional variable to see its association with phthalate biomarkers by applying multiple logistic regression analysis. As our statistical analysis involved multiple comparisons so that it became increasingly likely that at least one comparison would have been statistically different due to random sampling error alone, the multiple testing hypothesis was considered through Bonferroni test.

We also applied multiple logistic regression analysis to examine the association between each ln metal concentration and GDM or IGT outcomes adjusted for potential confounders. The confounders included in our final model were maternal smoking, maternal age, maternal BMI, maternal education level, pregnancy induced hypertension, number of siblings and recruitment centre. In addition, quartiles of the concentrations of each metal were estimated to describe mothers' exposure to metals according to 4 levels, namely none, light, moderate and high. Other categorical variables were expressed as the number and percentages (%) and continuous variables were expressed as the mean ± standard deviation (SD). The study population was also compared according to the recruitment centre by applying a chi-squared test (for categorical variables) or a Mann-Whitney U test (for continuous variables).

Paper 2:

Exposure to heavy metals during pregnancy related to gestational diabetes mellitus in diabetes-free mothers.

In our second paper, we show the results on association between maternal exposure to heavy metals during pregnancy and gestational diabetes mellitus. We published our paper in the journal Science of Total Environment. We present the details of this paper in the thesis.

Introduction

Studies have suggested that exposure to endocrine disruptors like phthalates widely used in our daily life (wrapping foods, personal care products, cosmetics, toys, medical devices, polyvinyl chloride flooring, and building materials) may be associated with elevated blood pressure and increased risk of cardiovascular diseases [START_REF] Cantonwine | Urinary concentrations of bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia[END_REF]Trasande et al. 2013). Phthalates are classified into two main groups. Low molecular weight (LMW) phthalates are frequently added to cosmetic products, shampoos, lotions, and other personal care products to the preserve scent. Whereas high molecular weight (HMW) phthalates are used as plasticizers of polyvinyl chloride (PVC) because of their flexibility and resistant kinking properties.

Phthalates being ubiquitous, present in the environment. Exposure to phthalates can occur through inhalation, ingestion (including dietary ingestion and incidental ingestion), dermal absorption (including air-to-skin transport), contact with contaminated surface and use of personal care products [START_REF] Soomro | Prenatal exposure to phthalates and the development of eczema phenotypes in male children: Results from the eden mother-child cohort study[END_REF].

Phthalates are metabolized into monoester metabolites after exposure and conjugated before being excreted in the urine; hence urinary phthalate metabolites can be used as a biomarkers of exposure [START_REF] Swan | Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans[END_REF]. Studies have revealed that phthalates can also cross the feto-placental barrier and enter the amniotic fluid and fetal circulation [START_REF] Ferguson | Associations between maternal biomarkers of phthalate exposure and inflammation using repeated measurements across pregnancy[END_REF][START_REF] Li | Exposure levels of environmental endocrine disruptors in mother-newborn pairs in china and their placental transfer characteristics[END_REF][START_REF] Swan | Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans[END_REF]. Therefore, prenatal and early life are critical in the development of immune system; exposure to toxic pollutants during this period can result in an increased risk of adverse health outcomes during pregnancy as well later in life. However, the exact mechanism through which phthalate exposure may influence cardiovascular health effects is unknown. It is hypothesized that phthalates being endocrine disruptors, affect the metabolism and actions of androgens, thyroid hormones and cortisol [START_REF] Boas | Thyroid effects of endocrine disrupting chemicals[END_REF][START_REF] Ye | Environmental pollutants and hydroxysteroid dehydrogenases[END_REF]. Another mechanism which may cause a pro-inflammatory response, vascular dysfunction and increased oxidative stress [START_REF] Mathieu-Denoncourt | Plasticizer endocrine disruption: Highlighting developmental and reproductive effects in mammals and non-mammalian aquatic species[END_REF][START_REF] Rahmani | Prenatal exposure to phthalic acid induces increased blood pressure, oxidative stress, and markers of endothelial dysfunction in rat offspring[END_REF].

A cross-sectional study in US children and adolescents aged 6-19 years suggested increased urinary phthalate concentrations of HMW phthalate metabolites were associated with higher blood pressure (Trasande et al. 2013). Whearas, another crosssectional study from Sweden in older adults showed increased serum concentrations of monobenzyl phthalate (MBzP) were associated with several cardiovascular risk factors [START_REF] Wiberg | Serum levels of monobenzylphthalate (mbzp) is related to carotid atherosclerosis in the elderly[END_REF]).

To our knowledge, this is the first prospective population based study. However, there is only one prospective birth cohort study from Cincinnati, USA investigated the relationship between phthalate and pregnancy induced hypertensive diseases (EF Werner et al. 2015).

We hypothesized that increased maternal urinary phthalate concentrations would be associated with increased maternal blood pressure and pregnancy induced hypertension. The aim of this study was to evaluate the association between maternal exposure to phthalates during pregnancy and pregnancy induced hypertension.

Methods

Study population and data collection

The population in this study is a subgroup of the French EDEN (Etude des Déterminants pré et post natals du développement de la santé de l'Enfant) motherchild prospective birth cohort (https://eden.vjf.inserm.fr). The mothers' recruitment and follow-up procedures, and methodological assumptions have been published in earlier studies. Briefly, women were given an appointment with a study midwife between the 24 th and 28 th gestational weeks, during which an interview on life style factors was conducted and biological samples were collected. The information on the mothers and their newborns, including parity, mode of delivery, birth weight, gestational age, season of birth, behavioral and environmental data were collected by the use of questionnaires, interviews and from obstetric and pediatric records. The current study includes all maternal urine samples had been analyzed for metabolites of phthalates as a part of previous studies [START_REF] Chevrier | Maternal urinary phthalates and phenols and male genital anomalies[END_REF][START_REF] Soomro | Prenatal exposure to phthalates and the development of eczema phenotypes in male children: Results from the eden mother-child cohort study[END_REF].

Maternal blood pressure was measured during routine monthly visits, with the subject in supine position, using a standard mercury sphygmomanometer. One set of measures was taken by the study midwife between 24 and 28 weeks using a different (semi-automated) device, with two measurements averaged to determine the values for that visit.

Measurements taken within each period of gestational trimester (T1, T2, and T3) were averaged to determine the values for systolic blood pressure (SBP) and diastolic blood pressure (DBP) assigned to each period.

Women were classified as having pregnancy induced hypertension based on SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg measured during at least two visits after the 22nd week of gestation, such that the elevated measures could have occurred during or across any of the three periods, at any point after 22 weeks. Consequently, no diagnosis of pregnancy induced hypertension was made before 24 weeks. All cases of pregnancy induced hypertension were diagnosed by the physicians.

Urine collection and analysis

Women were invited for a clinical examination and to provide a first morning urine at home before the hospital study visit between the 24 th and 28 th gestational weeks (second trimester of the pregnancy). If forgotten, the urine sample was collected at the Quantification of the phthalate metabolites concentrations was performed using online solid phase extraction high performance liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry Creatinine concentrations were also quantified following identical analytical procedure as described previously. [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF] The concentrations of metabolites below the limit of detection (LOD) were replaced by instrumental reading values or by the compound specific lowest instrumental reading value divided by the square root of two (LOD/√ -2) when the instrumental value found missing The vast majority of the samples/metabolites (>97%)

were above LOD. Two-step standardization was performed to reduce variability in biomarkers urinary concentrations owed to sampling conditions as described previously [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF]. There was no significant difference in the concentrations of phthalates in the urines collected at homes compared to those of the urines collected in the hospital.

Other Variables

Information on potential confounders including: gestational age (two-class variable: < 37 gestational week, ≥ 37 gestational week), parity, number of siblings (0, 1,2 ≥ 3),

Statistical analysis

Urinary concentrations of phthalate metabolites were natural log (ln) transformed because of non-normality of the distribution. For the purpose of present analysis, MECPP, MEHHP, MEOHP and MEHP were grouped as sum of ΣDEHP (DEHP being their parent compound). We calculated the molar sum of these DEHP metabolites (nanomoles per milliliter) by dividing each metabolite concentration by its molecular weight and then summing the individual concentrations.

Ethical Approval

The study was approved by the relevant ethical committees (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Le Kremlin Bicêtre University Hospital, and Commission Nationale de l'Informatique et des Libertés), and all participating women gave informed written consent for their own participation and both parents for newborn child after delivery.

Results

Characteristics of study population

Of 2002 women recruited into the EDEN mother child cohort, 604 women with available data on both pregnancy induced hypertension diagnosis and urine analysis for phthalates were included in this study (Figure 1). The majority (98.5%) of these women were educated, 43.5% had less than two years of education after high school while 54.8% had more than that and between 25-34 years of age (29.86 ± 4.8) (Table 1). The average BMI was 23.1 ± 4.5, while 26.4% were overweight or obese (BMI ≥25 kg/m 2 ).

While 13.7% were active smoker and 25% declared some alcohol intake during pregnancy. Four percent had premature newborns, 15.7% had caesarean section at delivery.

The prevalence of pregnancy induced hypertension was found 4.80%. The participants included in this study did not differ in major demographic characteristics except for maternal age, smoking and gestational age (Table S1). However, results did not change when adjusted for them in the final models.

Prenatal exposure to phthalates

Phthalate metabolites were detected in the urine of more than 97% of the mothers.

MEP had the highest geometric mean (110.7 µg/L), while MCNP had the lowest (1.5 µg/L). The limit of detection (LOD) ranged from 0.2 to 0.6 µg/L. A summary of detection frequency, as well as geometric means and select percentiles of urinary concentrations of phthalate metabolites are outlined in Table 2.

Spearman correlation coefficients between ln-transformed phthalate metabolites urinary concentrations are shown in Table S2. The majority of biomarkers were positively correlated, except for the correlation between MEP and MiBP, MECPP, MEHHP, MCOP, MCPP or MCNP. All the other biomarkers were significantly correlated with each other (with correlation coefficients ranging from r = 0.08, p < 0.05 to r = 0.97, p < 0.05). We also observed that DEHP metabolites were highly correlated with each other (r ≥ 0.77, p < 0.05).

products. While MBP is metabolite of Di-n-butyl phthalate (DBP), which is used in diverse range of products such as PVC, paints, printing inks, adhesives, aerosols and cosmetics products: hair spray, a perfume solvent and fixative, skin emollient and a plasticizer in nail polish and finger nail.

Comparison of findings with other published work

To our knowledge, this is the first study to longitudinally assess the phthalate concentrations in relation to pregnancy induced hypertension. However, a prospective birth cohort study from Cincinnati, USA investigated the relationship between phthalate and pregnancy induced hypertensive diseases which includes gestational hypertension, preeclampsia, eclampsia and HELLP syndrome (EF Werner et al. 2015).

This study suggested that maternal urinary MBzP concentrations may be associated with increased diastolic blood pressure and risk of pregnancy induced hypertensive diseases. However with did not observed significant association with pregnancy induced hypertension and MBzP phthalate metabolite in our study except a partial significance in the 4 th quartile (OR: 2.75, CI: 0.95-7.91, p-value = 0.06). While another study from Boston, USA, investigated the relationship between Bisphenol A and phthalate metabolites and the risk of preeclampsia [START_REF] Cantonwine | Urinary concentrations of bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia[END_REF]. Found a significant association between preeclampsia and MEP and DEHP. We found a significant association between pregnancy induced hypertension and MEP (OR: 1.47, CI: 1.01-2.14, p-value = 0.04) or MBP (OR: 1.66, CI: 1.11-2.47, p-value = 0.01) and a borderline significance in MCPP (OR: 1.56, CI: 0.93-2.64, p-value = 0.09).

Other studies on phthalate exposure and blood pressure in children have also shown significant associations (Trasande et al. 2013;Trasande and Attina 2015;[START_REF] Valvi | Prenatal phthalate exposure and childhood growth and blood pressure: Evidence from the spanish inma-sabadell birth cohort study[END_REF]. However, studies from USA (Trasande et al. 2013;Trasande and Attina 2015) were cross-sectional analysis of a subsample, found increased urinary concentrations of MEP and DEHP were associated with increased blood pressure in children age 6-19 years. Whereas, study from Spain [START_REF] Valvi | Prenatal phthalate exposure and childhood growth and blood pressure: Evidence from the spanish inma-sabadell birth cohort study[END_REF] was a population based birth cohort observed that sum of HMW phthalates as well as sum of LMW phthalates were significantly related with lower systolic blood pressure in girls but not in boys at the age of 4 and 7 years. A prospective study on elderly adults from Uppsala, Sweden shown inverse associations between MEP and systolic and diastolic blood pressure [START_REF] Olsen | Associations between circulating levels of bisphenol a and phthalate metabolites and coronary risk in the elderly[END_REF]. Additionally, a cross-sectional study of older adults found increased serum concentrations of MBzP were related to several cardiovascular risk factors [START_REF] Wiberg | Serum levels of monobenzylphthalate (mbzp) is related to carotid atherosclerosis in the elderly[END_REF].

Potential mechanisms

The exact mechanism is still unclear, however it is likely to be multifactorial in nature and depend on diet, living style, genetic and environmental factors. Since the nutritional imbalance or environmental chemical exposures during pregnancy and development can increase the risk of disease and their effects may be share some common pathways [START_REF] Barouki | Developmental origins of non-communicable disease: Implications for research and public health[END_REF]. Indeed, hormones, cytokines and nutritional components can both directly activate receptors that stimulate gene expression and also activate or inhibit the enzymes and pathways that are responsible for DNA methylation, chromatin re-modelling and non-coding RNAs which ultimately control gene expression. Thus, epigenetic regulatory pathways are likely sites for effects of both nutrient and environmental toxicant effects during development and potentially also across the lifespan [START_REF] Barouki | Developmental origins of non-communicable disease: Implications for research and public health[END_REF].

Epidemiological evidence indicates that exposure to phthalates during in utero is associated with increased blood pressure in childhood (Trasande et al. 2013). Studies have revealed that the risk of pregnancy induced hypertension may increases through oxidative stress, up-regulation of inflammatory markers (tumor necrosis factor alpha and interleukin 6), and inhibition of peroxisome proliferator-activated receptor gamma [START_REF] Rahmani | Prenatal exposure to phthalic acid induces increased blood pressure, oxidative stress, and markers of endothelial dysfunction in rat offspring[END_REF][START_REF] Sutherland | Preterm birth and hypertension risk: The oxidative stress paradigm[END_REF][START_REF] Usuda | Peroxisome proliferator-activated receptors for hypertension[END_REF]. The possible mechanism is may be the phthalates mediated deregulation of blood pressure is reduced endothelium-derived nitric oxide production, which is mainly produced by endothelial nitric oxide synthase, one of the key vessel relation factor in regulation of vascular tone [START_REF] Lee | Maternal exposure to di-(2-ethylhexyl) phthalate exposure deregulates blood pressure, adiposity, cholesterol metabolism and social interaction in mouse offspring[END_REF]). In addition to the transcriptional regulation, endothelial nitric oxide synthase activity can be modulated by kinase-dependent phosphorylation at various sites.

Strengths and limitations

This study has several strengths and limitations. The study design of our study is a nested cross-sectional in a population based prospective cohort, which has nutritional, environmental, biological and social factors data starting during the prenatal life are a strength of our study, which allowed us to control for potential confounders and to assess exposure before the health outcomes. A nested cross-sectional cohort design offers advantages over the traditional cross-sectional designs. The diagnosis of pregnancy induced hypertension was conducted by physicians, which ensures the quality and we further adjusted for potential confounders to see the associations. The values of the exposure biomarkers were standardized for urine sampling conditions to reduce subject variability [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: A 2-step standardization method based on regression residuals[END_REF]). However, we were limited to one urine sample per mother during the second trimester of pregnancy, thus we were unable to assess the variability in concentrations of each metabolite across the pregnancy. But it is also not clearly known at what point(s) in gestation the mothers are having higher risk of exposures. Consistency in urinary concentrations during pregnancy has been observed for some environmental exposure biomarkers and other studies have suggested that phthalate biomarkers are relatively stable for a period of weeks to months [START_REF] Hauser | Temporal variability of urinary phthalate metabolite levels in men of reproductive age[END_REF][START_REF] Teitelbaum | Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the united states[END_REF]. Other studies have also used second trimester maternal urinary concentration for biomarkers [START_REF] Gascon | Prenatal exposure to bisphenol a and phthalates and childhood respiratory tract infections and allergy[END_REF][START_REF] Whyatt | Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: The columbia center for children's environmental health cohort[END_REF]. However, increasing the number of urine samples collected would have provided us a more accurate estimate of the average exposure during the gestation period. Finally, we also adjusted for many potential confounders; however residual confounding (if any) cannot be discarded.

Creatinine correction is most often used for urinary biomarkers of phthalates, phenols, and pesticides. There are limitations to the usage of creatinine to normalize for urine dilution; different investigators have used specific gravity as an alternative of creatinine to adjust phthalate urinary biomarkers for urine dilution (AC Just et al. 2012;[START_REF] Whyatt | Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: The columbia center for children's environmental health cohort[END_REF]), but we did not have specific gravity measurements. Nevertheless, specific gravity is extremely correlated with creatinine [START_REF] Barr | Urinary creatinine concentrations in the u.S. Population: Implications for urinary biologic monitoring measurements[END_REF]) and consequently it is not doubtless that we overcorrected for urine dilution. Regarding exposure assessment, it might be important to explore other techniques for assessment of the exposure, particularly those that may offer a more exhaustive and coordinated picture of the individual environment, for instance by assessing particular items use in conjunction with indoor air levels and additionally biomarkers of exposure. Another limitation of our study is that this cohort was conducted in two cities which differ in many respect and which may not represent general French population. However, in the models we adjusted for centre to take into account regional specificities.

The exposures we studied are relatively prevalent, and some biomarker urinary concentrations approach those with significant effects in experimental models. In a healthy cohort such as ours, effects of hormonally active environmental exposures may be small, yet a continuous exposure can affect adversely.

Conclusion

We observed that prenatal exposure to some phthalates which include MEP and MBP, were associated with pregnancy induced hypertension. Our data provide new suggestive evidence that prenatal exposure to certain phthalates may play a role in the development of pregnancy induced hypertension.

DISCUSSIONS

In this discussion, we first summarize the main results reported in our papers and compare them with previous research studies. Then we discuss the strengths and limitations of this PhD work. Finally, we present our conclusions based on this work.

Exposure to phthalates and the development of eczema in children

In our first paper, we reported an association between maternal prenatal urinary concentration of metabolites of DiBP and DiNP and eczema development in offspring preschool boys followed-up between birth and 5 years of age. We observed that the metabolites of DiBP and DiNP were significantly associated with both phenotypes of eczema i.e., early onset and late onset. Our study also shows a significant association between prenatal exposure to various phthalates and allergic eczema in boys at the age of 5 years based on IgE levels. The metabolites of DBP, DiBP and DEHP were significantly associated with ever eczema in IgE sensitized boys and borderline significance was associated with MBzP, the major BBzP metabolite.

The table 3 shows epidemiological studies on relationships between exposure to phthalates and development of eczema in children. found a relationship between atopic dermatitis and MEP in urine, but only in the fourth quartile, and no association with MiBP, a DiBP metabolite, while other metabolites of DiNP were not investigated [START_REF] Callesen | Phthalate metabolites in urine and asthma, allergic rhinoconjunctivitis and atopic dermatitis in preschool children[END_REF].

A Japanese study (Ait Bamai et al. 2016) found the daily phthalate intakes estimated from urinary metabolites were higher in children than their parents. For infants and toddlers, one of the most important phthalate intake sources is floor dust.

Young children ingest up to 10 times more house dust than adults, and are therefore more vulnerable to exposure to phthalates because of their hand-to-mouth behavior on the flooring. House dust is also well known to be a main exposure source to allergens and other EDCs for infants. Similar to indoor air concentrations, levels of phthalates in settled dust have been associated with PVC building materials (Ait Bamai et al. 2016;[START_REF] Bornehag | Phthalates in indoor dust and their association with building characteristics[END_REF]. Similarly, a Taiwanese study [START_REF] Hsu | Predicted risk of childhood allergy, asthma, and reported symptoms using measured phthalate exposure in dust and urine[END_REF] among 101 children between 3 and 9 years of age, BBzP levels in house dust were associated with eczema and asthma examined the home environment along with biomonitoring data. They also noted that exposure to indoor dust-borne BBzP, DBP, and the metabolites MBP and MEHP in urine from children were associated with increased risk of having eczema, asthma and related symptoms.

Health effects of gestational exposure to selected heavy metals

In our second paper, we observed significant associations between maternal concentrations of heavy metals during the pregnancy with GDM or IGT and their combination indicating that the environment the mother encounters during the pregnancy can endanger her health status. More precisely, we found that, after taking into account for potential confounders, Cd was related to both GDM alone and the combination of GDM and IGT and Pb was partially related to GDM.

Cd is a toxic heavy metal distributed widely in the environment [START_REF] Barregard | Cadmium exposure in relation to insulin production, insulin sensitivity and type 2 diabetes: A cross-sectional and prospective study in women[END_REF]. The principal source of Cd exposure for the general non-smoking population is food [START_REF] Swaddiwudhipong | Progress in cadmium-related health effects in persons with high environmental exposure in northwestern thailand: A five-year follow-up[END_REF]. Occupational exposure limits to Cd have been lowered 50-100 fold in the last few decades as the toxic effects of Cd were better understood [START_REF] Wallace | Toxicology and hazard of agrochemicals[END_REF]. Three recent studies from China [START_REF] Liu | Cadmium body burden and gestational diabetes mellitus: A prospective study[END_REF][START_REF] Peng | A nested casecontrol study indicating heavy metal residues in meconium associate with maternal gestational diabetes mellitus risk[END_REF][START_REF] Xing | Relation between cadmium exposure and gestational diabetes mellitus[END_REF] showed significant association between urine Cd or meconium Cd and GDM. A USA case-cohort study [START_REF] Romano | A casecohort study of cadmium body burden and gestational diabetes mellitus in american women[END_REF] showed that women in highest tertile of urinary Cd had two times the risk of GDM as compared with those in the lowest tertile. However, the Canadian birth cohort study did not observed significant results to confirm this finding with Cd measured in blood [START_REF] Shapiro | Exposure to phthalates, bisphenol a and metals in pregnancy and the association with impaired glucose tolerance and gestational diabetes mellitus: The mirec study[END_REF].

Table 4 shows epidemiological studies on relationships between exposure to Cd and gestational diabetes mellitus. [START_REF] Afridi | Comparative metal distribution in scalp hair of pakistani and irish referents and diabetes mellitus patients[END_REF][START_REF] Barregard | Cadmium exposure in relation to insulin production, insulin sensitivity and type 2 diabetes: A cross-sectional and prospective study in women[END_REF][START_REF] Barregard | Cadmium, type 2 diabetes, and kidney damage in a cohort of middle-aged women[END_REF][START_REF] Chen | Plasma metallothionein antibody, urinary cadmium, and renal dysfunction in a chinese type 2 diabetic population[END_REF][START_REF] Hansen | Trace elements in early phase type 2 diabetes mellitus-a population-based study. The hunt study in norway[END_REF][START_REF] Liu | Association of urinary metals levels with type 2 diabetes risk in coke oven workers[END_REF][START_REF] Schwartz | Urinary cadmium, impaired fasting glucose, and diabetes in the nhanes iii[END_REF]. Most of them were either cross-sectional or retrospective [START_REF] Afridi | Comparative metal distribution in scalp hair of pakistani and irish referents and diabetes mellitus patients[END_REF] or case-control [START_REF] Hansen | Trace elements in early phase type 2 diabetes mellitus-a population-based study. The hunt study in norway[END_REF][START_REF] Simić | Trace element status in patients with type 2 diabetes in norway: The hunt3 survey[END_REF] in design, except one study which included limited prospective follow-up after a cross-sectional assessment [START_REF] Barregard | Cadmium exposure in relation to insulin production, insulin sensitivity and type 2 diabetes: A cross-sectional and prospective study in women[END_REF]. Despite a limited number of prospective studies, the research has consistently supported the potential association between increasing blood Cd level and type 2 diabetes mellitus. Studies from different populations of the world have also suggested that women and men with type 2 diabetes mellitus have statistically significant higher levels of Cd in urine [START_REF] Afridi | Evaluation of status of toxic metals in biological samples of diabetes mellitus patients[END_REF][START_REF] Barregard | Cadmium, type 2 diabetes, and kidney damage in a cohort of middle-aged women[END_REF][START_REF] Kolachi | Status of toxic metals in biological samples of diabetic mothers and their neonates[END_REF][START_REF] Schwartz | Urinary cadmium, impaired fasting glucose, and diabetes in the nhanes iii[END_REF], blood [START_REF] Afridi | Evaluation of status of toxic metals in biological samples of diabetes mellitus patients[END_REF][START_REF] Barregard | Cadmium, type 2 diabetes, and kidney damage in a cohort of middle-aged women[END_REF][START_REF] Hansen | Trace elements in early phase type 2 diabetes mellitus-a population-based study. The hunt study in norway[END_REF][START_REF] Kolachi | Status of toxic metals in biological samples of diabetic mothers and their neonates[END_REF] or hair [START_REF] Afridi | Comparative metal distribution in scalp hair of pakistani and irish referents and diabetes mellitus patients[END_REF][START_REF] Kolachi | Status of toxic metals in biological samples of diabetic mothers and their neonates[END_REF].

Pb is another toxic heavy metal and a ubiquitous environmental contaminant [START_REF] Cheng | Atmospheric emission characteristics and control policies of five precedent-controlled toxic heavy metals from anthropogenic sources in china[END_REF][START_REF] Dyer | Heavy metals as endocrine-disrupting chemicals a[END_REF][START_REF] Marx | Global-scale patterns in anthropogenic pb contamination reconstructed from natural archives[END_REF][START_REF] Romero-Freire | Effect of soil properties on the toxicity of pb: Assessment of the appropriateness of guideline values[END_REF].

Occupational exposure can occur through painting, battery, pigments, welding, smeltering, electroplating, and e-waste recycling workshops are exposed to relatively high concentrations of Pb [START_REF] Julander | Formal recycling of e-waste leads to increased exposure to toxic metals: An occupational exposure study from sweden[END_REF][START_REF] Kazi | Occupational and environmental lead exposure to adolescent workers in battery recycling workshops[END_REF][START_REF] Zhang | Elevated lead levels and adverse effects on natural killer cells in children from an electronic waste recycling area[END_REF]).

However, general population is usually exposed to low levels of Pb concentrations via polluted drinking water, cigarette smoking, food and air [START_REF] Augustsson | Metal uptake by homegrown vegetables -the relative importance in human health risk assessments at contaminated sites[END_REF][START_REF] Mohmand | Human exposure to toxic metals via contaminated dust: Bio-accumulation trends and their potential risk estimation[END_REF][START_REF] Pinto | Metals transfer from tobacco to cigarette smoke: Evidences in smokers' lung tissue[END_REF]). Higher Pb exposure may occur through manmade sources, such as in paint around the home, leaded pipes and fixtures that deliver water and soil contaminated with Pb from leaded gasoline.

Recently, other epidemiological studies addressed specifically the association between Pb and GDM [START_REF] Oguri | Association between maternal blood cadmium and lead concentrations and gestational diabetes mellitus in the japan environment and children's study[END_REF][START_REF] Peng | A nested casecontrol study indicating heavy metal residues in meconium associate with maternal gestational diabetes mellitus risk[END_REF][START_REF] Shapiro | Exposure to phthalates, bisphenol a and metals in pregnancy and the association with impaired glucose tolerance and gestational diabetes mellitus: The mirec study[END_REF].

Table 4 shows epidemiological studies on relationships between exposure to Pb and gestational diabetes mellitus. A retrospective case-control study within a Chinese population found a negatively associated significance in the second and third quartile of Pb [START_REF] Peng | A nested casecontrol study indicating heavy metal residues in meconium associate with maternal gestational diabetes mellitus risk[END_REF]. On the other hand, in Canadian study, the observed association was not significant [START_REF] Shapiro | Exposure to phthalates, bisphenol a and metals in pregnancy and the association with impaired glucose tolerance and gestational diabetes mellitus: The mirec study[END_REF].

Controversial findings for type 2 diabetes mellitus in life had been shown by previous research as some investigations revealed that blood Pb [START_REF] Haq | Lead toxicity in relation to diabetes mellitus and chronic renal failure[END_REF], urinary Pb [START_REF] Afridi | Evaluation of status of toxic metals in biological samples of diabetes mellitus patients[END_REF] or hair Pb [START_REF] Afridi | Comparative metal distribution in scalp hair of pakistani and irish referents and diabetes mellitus patients[END_REF] were higher in diabetes as compared to non-diabetes but others reported that blood Pb was not associated with diabetes [START_REF] Hansen | Trace elements in early phase type 2 diabetes mellitus-a population-based study. The hunt study in norway[END_REF][START_REF] Moon | Association of lead, mercury and cadmium with diabetes in the korean population: The korea national health and nutrition examination survey (knhanes) 2009-2010[END_REF], or even inversely associated with it [START_REF] Simić | Trace element status in patients with type 2 diabetes in norway: The hunt3 survey[END_REF]). However, study designs differed among the investigations (most were case control studies) and various biological matrix were used.

Mn is a naturally occurring element and an essential nutrient that plays a role in bone mineralization, protein and energy metabolism, metabolic regulation and antioxidant system (Livingstone 2017). However, the requirements are low and excessive intake or exposure to it can cause neurotoxicity [START_REF] Hutchens | Deficiency in the manganese efflux transporter slc30a10 induces severe hypothyroidism in mice[END_REF][START_REF] Pfalzer | Relationships between essential manganese biology and manganese toxicity in neurological disease[END_REF]. The association between Mn and GDM was not significant in our study which is consistent with other studies conducted in the general populations [START_REF] Hansen | Trace elements in early phase type 2 diabetes mellitus-a population-based study. The hunt study in norway[END_REF][START_REF] Liu | Association of urinary metals levels with type 2 diabetes risk in coke oven workers[END_REF][START_REF] Simić | Trace element status in patients with type 2 diabetes in norway: The hunt3 survey[END_REF]). However, one Korean study revealed that blood manganese levels were negatively associated with increased risk of diabetes [START_REF] Koh | Association of blood manganese level with diabetes and renal dysfunction: A cross-sectional study of the korean general population[END_REF].

Exposure to phthalates and pregnancy induced hypertension

In our third study, we report an association between maternal prenatal urinary concentration of low molecular weight phthalate metabolites including MEP and MBP and pregnancy induced hypertension. We found a significant association between pregnancy induced hypertension and MEP (OR: 1.47, CI: 1.01-2.14, p-value = 0.04)

or MBP (OR: 1.66, CI: 1.11-2.47, p-value = 0.01) and a borderline significance in MCPP (OR: 1.56, CI: 0.93-2.64, p-value = 0.09).

A very limited number of studies have been conducted previously. A prospective birth cohort study from Cincinnati, USA investigated the relationship between phthalate and pregnancy induced hypertensive diseases which includes gestational hypertension, preeclampsia, eclampsia and HELLP syndrome [START_REF] Werner | The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The home study[END_REF]. This study findings shows that maternal urinary MBzP concentrations may be associated with increased diastolic blood pressure and risk of pregnancy induced hypertensive diseases. While in another study from Boston, USA, the relationship between Bisphenol A and phthalate metabolites and the risk of preeclampsia [START_REF] Cantonwine | Urinary concentrations of bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia[END_REF], and reported a significant association between preeclampsia and MEP and DEHP was observed.

Few other studies on phthalate exposure and blood pressure in children have also shown significant associations (Trasande and Attina TM 2015;Trasande L et al. 2013;[START_REF] Valvi | Prenatal phthalate exposure and childhood growth and blood pressure: Evidence from the spanish inma-sabadell birth cohort study[END_REF]. However, studies from USA (Trasande and Attina TM 2015;Trasande L et al. 2013) were cross-sectional analysis of a subsample, found increased urinary concentrations of MEP and DEHP were associated with increased blood pressure in children age 6-19 years. Whereas, study from Spain [START_REF] Valvi | Prenatal phthalate exposure and childhood growth and blood pressure: Evidence from the spanish inma-sabadell birth cohort study[END_REF] was a population based birth cohort observed that sum of HMW phthalates as well as sum of LMW phthalates were significantly related with lower systolic blood pressure in girls but not in boys at the age of 4 and 7 years. A prospective study on elderly adults from Uppsala, Sweden shown inverse associations between MEP and systolic and diastolic blood pressure [START_REF] Olsen | Associations between circulating levels of bisphenol a and phthalate metabolites and coronary risk in the elderly[END_REF]. While, in another Swedish cross-sectional study in which the older adults were found with increased serum concentrations of MBzP were related to several cardiovascular risk factors [START_REF] Wiberg | Serum levels of monobenzylphthalate (mbzp) is related to carotid atherosclerosis in the elderly[END_REF].

8. The nature of our second and third study facilitated to exclude the women with diagnosed pre-gestational diabetes mellitus, renal diseases and hypertension.

And indeed, we could determine that we were dealing with isolated GDM, as no mother had a diagnosis of diabetes at 2 years of age of the child.

Limitations

1. This cohort was conducted in only two cities of France, which may not represent general French population. However, in the models we adjusted for centre to take into account the regional specificities.

2. The study designs of our second and third study were nested cross-sectional within a cohort. However a nested cross-sectional cohort design offers advantages over traditional cross-sectional designs. Further, we can determine that we are dealing with isolated GDM or pregnancy induced hypertensive mothers, as no mother had a diagnosis of diabetes or hypertension at two years of age of the child.

3. The measurement of phthalates and heavy metals was conducted only once.

We were limited to one urine sample per mother during the second trimester of pregnancy, thus we were unable to assess the variability in concentrations of each phthalate metabolite and heavy metal across the pregnancy. It would have been more interesting to study the association between the health related variables and exposure to phthalates and metals during different points in time.

However it is not clearly known that at which point(s) in gestation the mother or infants are at higher risk of exposure.

4. Humans are exposed to a large number of complex mixtures of toxic substances in daily life, so it is hard to see the effect of exposure to a unique phthalate or metal.

5. The diagnosis of GDM is no longer based on two different tests but on only one test, i.e. 75 g-glucose tolerance with only one value above the limit of fasting, 1 h or 2 h. Whether we underestimated or overestimated the relationship cannot be seen in our second study as we had no access to detailed data on glycemia, so the results should be interpreted cautiously.

6. In our first study, we considered only male offspring because the initial purpose was to see the effects of phthalates on malformations of male genitalia [START_REF] Philippat | Exposure to phthalates and phenols during pregnancy and offspring size at birth[END_REF]. Although considering only males limits the generalization of our results, it is not a source of bias. However, from a statistical point of view, focusing on one sex is another way to increase the study accuracy when the sample size is limited.

7. Creatinine was used to normalize urine dilution, other studies have used specific gravity as an alternative to phthalates to adjust phthalate urinary biomarkers.

However, studies have also shown that the specific gravity is highly correlated with creatinine.

8. The questionnaire data were self-reported and thus could have been subject to misclassification; in addition, recall bias was a possibility that might in part account for the high prevalence of eczema in our study population.

Conclusion

This work is based on data from a French Mother-Child prospective cohort study.

We wrote three papers, which are the basis of this PhD work. It has contributed to the critical issues in the area of maternal and child health, which are not studied much previously. These findings are important for public health because humans are continuously exposed to these EDCs (phthalates and heavy metals) chemicals in their everyday life.

In summary, in our first paper, we observed strong associations between phthalates measures during the second trimester of pregnancy and childhood eczema, which was quite strong. The prevalence of early onset and late onset eczema was 15.7% and 14.7%, respectively. We observed strong statistically significant associations between both early and late onset eczema and metabolites of two phthalates in particular; DiBP and DiNP. Among IgE sensitized boys, metabolities of DBP and DiBP were also significantly associated with ever eczema.

In our second paper, the metals namely Pb, Cd, and Mn were measured in blood during second trimester of the pregnancy. We observed the prevalence of GDM and IGT were 7.1% and 10.1% respectively. The Cd was statistically significant to GDM or IGT in the model adjusted for confounders, while Pb to GDM was found at borderline significance.

In our third paper, the prevalence of pregnancy induced hypertension was found 4.80%. We found strong associations between phthalate measures during the second trimester of pregnancy and pregnancy induced hypertension. We observed that low molecular weight phthalate metabolites: MEP and MBP were significantly related to pregnancy induced hypertension.

Our results add new and important information to the growing number of studies that emphasize and providing suggestive evidence on the relationship between EDCs (phthalates and heavy metals we studied) and maternal and children health, that prenatal exposure to certain phthalates and heavy toxic metals may play a role in the development of GDM and pregnancy induced hypertension during pregnancy and later eczema in early childhood. Our results raise concerns on the impact of phthalates and heavy metals that persist long term in the environment on maternal and child health, which opens actions for prevention. Our data also indicates that policy can predict exposure. Minimizing exposure to these EDCs can reduce the disease burden.

Future research focusing on larger populations and addressing multiple exposures assessed at multiple points during pre-natally and post-natally are required to provide more evidence on possible contributions of emerging pollutants to study the role of phthalates and heavy metals.

FUTURE RESEARCH

1. Although direct causal links between exposures to EDCs and disease states in humans are difficult to draw, results from basic research and epidemiological studies make it clear that more screening for exposures and targeting at risk groups is a high priority.

2. The studies in this thesis, we utilized environmental and biological samples to characterize exposure to phthalates and heavy metals and assessed the associations between prenatal exposure and risk of GDM, pregnancy induced hypertension during pregnancy and eczema in children during early childhood.

Future research will bridge across these areas of exposure science, statistical methods and health outcomes to better understand the role of EDCs.

3. In addition to enhancing research in these areas is an important and effective approach is prevention of disease. Chemical policies at local, state, and national levels, as well as globally, need to be formulated, financed, and implemented to ensure the best public health. By communicating these priorities to basic and clinical researchers, physicians, community advocates, and the public at large, we are hopeful that early identification and intervention will be facilitated.

4. A further dimension to consider in future research is multiple exposures of hormonally active agents such as these. In terms of prevention, exposure to these chemicals can be avoided if the contents of products are already known or listed on the products; unfortunately, they are often not listed on the labels because of being non-active ingredients.

5. In long term the exposome approach and Environment-Wide Associations Study (EWAS) is applied for better understanding. This strategy is currently going-on based on large population surveys globally under Pregnancy And Childhood Epigenetics (PACE) project, for which EPAR team is one of the main participants (Figure 6).

  et de pression artérielle diastolique (PAD) assignées à chaque période. Les femmes ont été classées comme ayant une hypertension induite par la grossesse basée sur une PAS ≥ 140 mmHg et / ou une PAD ≥ 90 mmHg mesurée au moins deux fois après la 22ème semaine de gestation, de sorte que les mesures élevées auraient pu être observées au cours de l'une des trois périodes, à tout moment après 22 semaines. En conséquence, aucun diagnostic d'hypertension induite par la grossesse n'a été posé avant 24 semaines. Tous les cas d'hypertension induite par la grossesse ont été diagnostiqués par les médecins. Toutes les analyses statistiques ont été réalisées à l'aide du logiciel statistique Stata 14 (Stata Corp LP, 4905 Lakeway Drive, College Station, Texas 77845, États-Unis) et la valeur p <0,05 était considérée comme statistiquement significative.

  années et les métabolites DiBP et DiNP. Chez les garçons sensibilisés (taux IgE supérieur au seuil), les métabolites du phtalate de di-n-butyle (DBP) et du DiBP étaient significativement associés à un eczéma (risque relatif (RR) = 1,67; intervalle de confiance à 95% (IC) 1,10-2,54; P = 0,01 et HR = 1,87; IC à 95% 1,01-3,48; P = 0,04, respectivement). Dans notre deuxième article, nous avons montré que la prévalence du DG était de 7,06%, alors que celle de l'IGT était de 10,1%. Nous avons observé une association statistiquement significative entre l'exposition au Cd et la combinaison du DG et de l'IGT dans le modèle brut ainsi que dans le modèle ajusté (Odds-ratio (OR): 1,50, CI: 1,50-1,06 et OR: 1,61, CI: 1,05-2,48, respectivement). Des associations à la limite de la signification ont été observées dans le modèle brut et dans le modèle ajusté entre l'exposition au Cd et le DG (OR: 1,64, CI: 0,99-2,76 et 1,81, CI: 0,93-3,50 respectivement). De plus, une association significative a été observée entre l'exposition au Pb et GDM dans le modèle non ajusté (OR: 1,83, IC à 95%: 1,01-3,29), une tendance persistante après ajustement sur les facteurs de confusion (OR: 1,65, IC: 0,82 -3,34). Tout en prenant en compte les quartiles de la distribution des métaux, une augmentation significative de l'Odds-ratio pour le DG et l'IGT combinés a été observée pour une exposition au Cd dans le 4ème quartile après ajustement sur les facteurs de confusion (OR: 2,33, IC: 1,10-4,92). Aucune association statistiquement significative n'a été observée ailleurs après ajustement sur les facteurs de confusion. Dans le troisième article, la prévalence de l'hypertension gravidique était de 4,80%. Nous avons observé que les métabolites de phtalates de bas poids moléculaire comme le phtalate de monoéthyle (MEP) et le phtalate de mono-n-butyle (MBP) étaient positivement associés à une hypertension induite par la grossesse dans les modèles bruts (OR : 1,43, IC : 1,04-1,96, p-value = 0,02 et OR : 1,48, IC :1,10-2,01, p-value = 0,01) et dans les modèles ajustés (OR : 1,47, IC : 1,01-2,14, p-value = 0,04 et OR :1,66, IC : 1,11-2,47, p-value = 0,01), respectivement. Nous avons observé une association à la limite de la significativité pour l'un des métabolites du phtalate de haut poids moléculaire : le phtalate de mono (3-carboxypropyle) (MCPP) dans le modèle brut (OR : 1,46, IC : 0,96-2,24, p-value = 0,08) ainsi qu'en modèle ajusté (OR : 1,56, IC : 0,93-2,64, p-value= 0,09). En prenant en compte les quartiles, nous n'avons pas observé d'association entre le MEP et l'hypertension gravidique. Cependant, une augmentation significative de l'Odds-ratio pour le métabolite de la MBP a été observée pour une exposition dans son 4ème quartile, dans le modèle brut (OR :3,46, IC : 1,10-10,87, p-value = 0,03) ainsi que dans le modèle ajusté sur les facteurs de confusion (OR : 5,91; IC : 0,93-37,79, p-value = 0,06).ConclusionEn conclusion, nos résultats apportent de nouvelles preuves potentielles sur la relation entre les perturbateurs endocriniens (phtalates et métaux lourds étudiés) et la santé de la mère et de l'enfant, selon lesquelles l'exposition prénatale à certains phtalates et métaux toxiques lourds pourrait jouer un rôle dans le développement du diabète gestationnel et de l'hypertension induite par la grossesse pendant la grossesse et, ultérieurement, un eczéma pendant la petite enfance. De futures recherches centrées sur des populations plus importantes et analysant de multiples expositions évaluées avant et après la naissance seraient nécessaires pour fournir davantage de preuves sur les contributions possibles des polluants émergents afin d'étudier le rôle des phtalates et des métaux lourds.



  Soomro MH, Heude B, Bornehag CG, Annesi-Maesano I. The association between maternal urinary phthalate concentrations and pregnancy induced hypertension: Results from the EDEN mother-child Cohort study. (Submitted to Env Interntional)  Soomro MH, Baiz N, Huel G, Yazbeck C, Botton J, Heude B, Bornehag CG, Annesi-Maesano I. Exposure to heavy metals during pregnancy related to gestational diabetes mellitus in diabetes-free mothers. Sci Total Environ. 2019; 656: 870-876.  Soomro MH, Baiz N, Philippat C, Vernet C, Siroux V, Maesano CN, Sanyal S Slama R, Bornehag CG, Annesi-Maesano I. Prenatal exposure to phthalates and the development of eczema phenotypes in male children: Results from the EDEN mother-child Cohort study. Environ Health Persp. 2018; 126(2): 027002. SCIENTIFIC COMMUNICATIONS  2018: Soomro MH, Bornehag CG, Heude B, Annesi-Maesano I. The association between maternal urinary phthalate concentrations and pregnancy induced hypertension. Poster presentation at Annual conference of the Doctoral School (ED393), Saint Malo-2018.  2017: Soomro MH, Baiz N, Huel G, Yazbeck C, Botton J, Heude B, Bornehag CG, Annesi-Maesano I. Exposure to metals during pregnancy and the association with gestational diabetes mellitus. Poster presentation at 10 Th DOHaD world Congress. Rotterdam, The Netherlands.  2016: Soomro MH. Prenatal exposure to phthalates and the development of eczema phenotypes in male children. Oral presentation at HEALS Annual Meeting. Leiden, The Netherlands.  2016: Soomro MH, Bornehag CG, Baiz N, Philippat C, Vernet C, Siroux V, Slama R, Annesi-Maesano I. Prenatal exposure to phthalates and eczema in male children. Poster presentation at 28th ISEE Conference, Rome, Italy.

Figure 2 .

 2 Figure 2. Model of the endocrine systems targeted by endocrine disrupting chemicals. The figure demonstrates that all hormone-sensitive physiological systems are vulnerable to EDCs, including brain and hypothalamic neuroendocrine systems; pituitary; thyroid; cardiovascular system; mammary gland; adipose tissue; pancreas; ovary and uterus in females; and testes and prostate in males.

Figure 3 .

 3 Figure 3. Impact of Endocrine Disrupting Chemicals on human health.

Further

  the use of food supplements and medicines which can have both desired and unintended effects on human body. Studies in humans and laboratory animals have shown that the phthalates and heavy metals can cause a variety of reproductive and developmental problems in offspring, in part by disrupting the normal hormonal functioning systems. The epidemiologic evidence are not strong, however some studies have shown associations with neurodevelopment and reproductive as well as for asthma and allergic conditions. Exposure to phthalates and heavy metals is a global public health concern because several of them have been identified as reproductive and developmental toxicants and EDCs. Even low doses of these chemicals in the environment pose a significant risk to health. Further, the mother's health problems during pregnancy may have repercussions on child's health in early life and later.

  EDEN mother-child prospective birth cohort was set up in 2003 in two university hospitals of the cities, Nancy and Poitiers, in France (see Figure 4), by the local university hospital teams in collaboration with research teams from the National Institute of Health and Medical Research (INSERM).

Figure 4 .

 4 Figure 4. Map shows the study centers Nancy and Poitiers.

  Phthalates or phthalate esters, are esters of phthalic acid are one of the EDCs, which are mainly used as plasticizers. Phthalates are ubiquitous man-made chemical substances, widely used for a variety of purposes in our daily life i.e. medical devices, pharmaceuticals, PVCs, food wrappings, cosmetic products, children's toys and cleaning products. They are added to plastics in order to increase their flexibility, transparency, durability, and longevity[START_REF] Bornehag | Phthalates in indoor dust and their association with building characteristics[END_REF][START_REF] Meeker | Exposure to environmental endocrine disruptors and child development[END_REF] al. 2009; Meeker 2012; Shelby 2006). Phthalates are classified into two major categories based on their number of carbon atoms in the main carbon chains, where different phthalates with different carbon atom numbers have different physical, chemical and even toxicological properties:  Low Molecular Weight (LMW) phthalates: LMW phthalates includes; DEP, DBP, BBzP,and DiBP and their metabolites, which contain three to six carbon atoms in the backbone.  High Molecular Weight (HMW) phthalates: HMW phthalates includes; DiNP, DOP, DiDP, DEHP and their metabolites. HMW phthalates have more than six carbon atoms in their backbone.

  Européen de Biotechnologie SA (CEB) in Angers, France was used. The urine samples were aliquoted and then frozen at -80° C. The analyses of the samples were conducted at the National Center for Environment Health Laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia (USA) for eleven metabolites of eight phthalates: Diethyl phthalate (DEP), Di-n-butyl phthalate (DBP), Di-isobutyl phthalate (DiBP), Di(2-ethylhexyl) phthalate (DEHP), Benzylbutyl phthalate (BBzP), Di-isononyl phthalate (DiNP), Di-n-octyl phthalate (DOP), and Di-isodecyl phthalate (DiDP). The targeted phthalate metabolites were monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monocarboxy-isooctyl phthalate (MCOP), mono(3-carboxypropyl) phthalate (MCPP) and monocarboxy-isononyl phthalate (MCNP).

  hospital. A polypropylene container (FP40VPS manufactured by Centre Européen de Biotechnologie SA (CEB) in Angers, France was used. The urine samples were aliquoted and then frozen at -80° C. The analyses of the samples were conducted at the National Center for Environment Health Laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia (USA) for eleven metabolites of eight phthalates: Diethyl phthalate (DEP), Di-n-butyl phthalate (DBP), Di-isobutyl phthalate (DiBP), Di(2-ethylhexyl) phthalate (DEHP), Benzylbutyl phthalate (BBzP), Di-isononyl phthalate (DiNP), Di-n-octyl phthalate (DOP), and Di-isodecyl phthalate (DiDP).The targeted phthalate metabolites were monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl- 5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monocarboxy-isooctyl phthalate (MCOP), mono(3-carboxypropyl) phthalate (MCPP) and monocarboxy-isononyl phthalate (MCNP).

  maternal age at delivery (< 25 years, 25-29 years, 30 -34 years, ≥ 35 years), prepregnancy maternal body mass index (BMI, < 18.5, 18.5-24.9, 25.0-30.0, > 30 kg/m 2 ), maternal educational level (< 2 years after high school, High school +2 years, ≥ High school +3 years), household income, city of residence (Nancy / Poitiers), mode of delivery (vaginal, caesarean section), smoking during pregnancy (yes/no), maternal alcohol use during pregnancy (yes/no), maternal physician-diagnosis of pregnancy induced hypertension.

Figure 6 .

 6 Figure 6. EXPOSOME approach and the Environment-Wide Associations Study (EWAS) in relation to eczema in children, GDM, pregnancy induced hypertension (From David M. Balshaw, making the case for advancing the EWAS.

Endocrine Disrupting Chemicals and their health effects in humans

	1.1 EDCs are man-made chemicals, found in various materials such as pesticides,
	metals, additives or contaminants in food, and personal care products (Figure 1) used
	as industrial solvents/lubricants and their by-products (polychlorinated biphenyls,
	polybrominated biphenyls, dioxins), plastics (bisphenol A), plasticizers (phthalates),
	heavy metals [(Lead (Pb), Cadmium (Cd), Manganese (Mn), Arsenic (As), Mercury
	(Hg), zinc (Zn)], pesticides [methoxychlor, chlorphyrifos, dichlorodiphenyl-
	trichloroethane (DDT)], fungicides (vinclozolin) and pharmaceutical agents
	(diethylstilbestrol) (Iavicoli et al. 2009; Meeker 2012; United Nations Environment
	Programme and World Health Organization 2013; Unuvar and Buyukgebiz 2012).
		Industrial
	Personal care	Chemicals
	products
		Metals
	1 According to United Nations Environment Programme and the World Health
	Organization, EDCs are defined as:
	Pesticides, "An endocrine disruptor is an exogenous substance or mixture that alters Herbicides, Synthetic
	Fungicides function(s) of the endocrine system and consequently causes adverse health and naturally occurring
	Pharmaceutic effects in an intact organism, or its progeny, or (sub) populations" United Nations hormones
	Environment Programme, World Health Organization. 2013. State of the science al drugs
	of endocrine disrupting chemicals 2012 / edited by åke bergman, jerrold j.
	Heindel, susan jobling, karen a. Kidd and r. Thomas zoeller. Isbn: 978-92-807-
	3274-0 (unep) and 978 92 4 150503 1 (who).

Figure 1. Endocrine disruptors are chemicals or chemical mixtures that interfere with normal hormone actions. They may be of any type of chemical substance.

  Neurodevelopment problems including; lower IQ in children with learning and behavioral problems[START_REF] Fatmi | Lead exposure assessment among pregnant women, newborns, and children: Case study from karachi, pakistan[END_REF][START_REF] Gulson | Mobilization of lead from human bone tissue during pregnancy and lactation--a summary of long-term research[END_REF][START_REF] Hu | Invited commentary: Lead, bones, women, and pregnancy--the poison within?[END_REF][START_REF] Mendola | Environmental factors associated with a spectrum of neurodevelopmental deficits[END_REF]. While other studies have shown that the increased levels of Pb can increase the likelihood of pre-eclampsia or pregnancy induced hypertension related diseases[START_REF] Jameil | Maternal serum lead levels and risk of preeclampsia in pregnant women: A cohort study in a maternity hospital, riyadh, saudi arabia[END_REF][START_REF] Kennedy | Lead exposure, gestational hypertension and pre-eclampsia: A systematic review of cause and effect[END_REF][START_REF] Poropat | Blood lead and preeclampsia: A meta-analysis and review of implications[END_REF][START_REF] Zahran | Maternal exposure to neighborhood soil pb and eclampsia risk in new orleans, louisiana (USA): Evidence from a natural experiment in flooding[END_REF]. A recent study byWu et.al. showed significant associations between prenatal low levels of Pb exposure with newborn DNA methylation, in female infants[START_REF] Wu | Exposure to low levels of lead in utero and umbilical cord blood DNA methylation in project viva: An epigenome-wide association study[END_REF]).

. Cd may also inhibit the transcription of low density lipoprotein receptor mRNA to reduce the supply of cholesterol substrate available for placental progesterone production

[START_REF] Ikeh-Tawari | Cadmium level in pregnancy, influence on neonatal birth weight and possible amelioration by some essential trace elements[END_REF]

. Whereas, higher concentration of Cd in the body may alter the secretory patterns of pituitary hormones, including follicle stimulating hormone and luteinizing hormone

[START_REF] Iavicoli | The effects of metals as endocrine disruptors[END_REF]

. Other adverse effects due to the exposure of Cd are GDM, PIH, low birth weight, impaired fetal growth, spontaneous abortion, still birth, neonatal death, preterm birth. Low birth weight can also leads to increased risk of developing slower cognitive development of the child

[START_REF] Boyce | Academic outcomes of very low birth weight infants: The influence of mother-child relationships[END_REF][START_REF] Rahman | Infant and mother related outcomes from exposure to metals with endocrine disrupting properties during pregnancy[END_REF]

.

Lead

Pb exposure during pregnancy can harm a pregnant woman and fetus. Pb absorbed in the body can be stored in bones for years, which can cause GDM, miscarriage, stillbirth,

Table 1 .

 1 Overview of the study aims, subjects and methods.

	Study	Aim	Study design	Study	Analysis
				population
	I	To test the hypothesis that exposure	Prospective	604	Logistic
		to phthalates during in utero increase	Cohort		regression,
		the risk of eczema in young children			survival
		through the production of immune			analysis
		markers at various stages of life.
	II	To test the hypothesis that exposure	Nested Cross-	623	Logistic
		to heavy metals during pregnancy	sectional Cohort		regression
		and its association with gestational
		diabetes mellitus and impaired
		glucose tolerance.
	III	To test the hypothesis that exposure	Nested Cross-	604	Logistic
		to phthalates during pregnancy and	sectional Cohort		regression
		its association with pregnancy
		induced hypertension.

Table 2 .

 2 EDCs investigated in the thesis.

	Parent compound	Abbrev. Phthalate metabolites	Abbrev.
	Diethyl phthalate	(DEP)	Monoethyl phthalate	MEP
	Di-n-butyl phthalate	(DBP)	Mono-n-butyl phthalate	MBP
	Di-isobutyl phthalate	(DiBP)	Mono-isobutyl phthalate	MiBP
	Di(2-ethylhexyl) phthalate	(DEHP)	Mono(2-ethyl-5-carboxypentyl) phthalate MECPP
	Di(2-ethylhexyl) phthalate	(DEHP)	Mono(2-ethyl-5-hydroxyhexyl) phthalate MEHHP
	Di(2-ethylhexyl) phthalate	(DEHP)	Mono(2-ethyl-5-oxohexyl) phthalate	MEOHP
	Di(2-ethylhexyl) phthalate	(DEHP)	Mono(2-ethylhexyl) phthalate	MEHP
	Benzylbutyl phthalate	(BBzP)	Monobenzyl phthalate	MBzP
	Di-isononyl phthalate	(DiNP)	Monocarboxy-isooctyl phthalate	MCOP
	Di-n-octyl phthalate	(DOP)	Mono(3-carboxypropyl) phthalate	MCPP
	Di-isodecyl phthalate	(DiDP)	Monocarboxy-isononyl phthalate	MCNP
	Lead	Pb
	Cadmium	Cd
	Manganese	Mn

  was assessed by administration of an enriched version of the International Study of Asthma and Allergies in Childhood (ISAAC) standardized and validated questionnaire (ISAAC Steering Committee 1998;[START_REF] Williams | Worldwide variations in the prevalence of symptoms of atopic eczema in the international study of asthma and allergies in childhood[END_REF] to the parents at 1, 2, 3, 4 and 5 years of age by giving appointment. At each year of followup, eczema was defined as eczema diagnosed by a doctor in the last 12 months. Ever eczema was then defined as having ever reported a diagnosis of eczema at 1, 2, 3, 4 or 5 years of age. Additionally, two eczema phenotypes were distinguished: earlyonset eczema if eczema was reported on any questionnaire in the first 2 years of age and late-onset eczema if it was reported only between age 24 months and 60 months.

Table S1 .

 S1 Characteristics of the study population included and excluded in the analysis.

		Sample of analysis	Samples Excluded
	Characteristics	n=623 Mean ± SD	for analysis n=1149 Mean ± SD	P value
		No. %	No. %
	Maternal age (years)	29.02 ± 4.9	29.71 ± 4.8
	<25	121 19.5	160 13.9
	25 -29 30 -34	209 33.5 206 33.1	414 36.1 383 33.3	0.01
	≥35	87 13.9	192 16.7
	Parity
	0	276 44.3	528 45.9
	1	231 37.1	413 35.9	0.80
	≥2	116 18.6	208 18.2
	BMI (kg/m 2 )	23.01 ± 4.2	23.10 ± 4.4
	<18.5	50 8.1	102 8.9
	18.5 -24.9 25 -30	405 65.0 110 17.6	741 64.5 207 18.0	0.89
	>30	58 9.3	99 8.6
	Maternal education
	<2 years after high school	307 49.3	493 42.9
	High school +2 years ≥ High school +3 years	141 22.6 167 26.9	248 21.6 388 33.8	0.01
	Missing	8 1.2	20 1.7
	Active smoking (cig/day)
	0	483 77.5	967 84.2
	1 to 5 >5	94 15.2 36 5.7	127 11.1 47 4.1	0.004
	Missing	10 1.6	8 0.6
	Passive smoking
	Yes	274 44.0	500 43.5
	No	274 44.0	512 44.6	0.97
	Missing	75 12.0	137 11.9
	Alcohol during pregnancy
	Yes	166 26.6	311 27.1
	No	450 72.2	832 72.4	0.36
	Missing	7 1.2	6 0.5
	Gestational age	39.10 ± 1.7	39.20 ± 1.7
	<37 gestational week ≥37 gestational week	37 5.9 586 94.1	62 5.4 1087 94.6	0.63
	Employment
	Yes	451 72.4	877 76.3
	No	168 26.9	263 22.9	0.15
	Missing	4 0.7	9 0.8
	Caesarean section
	Yes No	80 12.8 543 87.2	193 16.8 956 83.2	0.02
	Gender Newborn
	Males Females	327 52.5 296 47.5	603 52.5 546 47.5	0.99
	BMI; Body mass index

Table S2 .

 S2 Associations (Odd Ratio and 95% Confidence Interval) between and blood metals (µg/L) by quartiles and gestational diabetes mellitus or impaired glucose tolerance.

	Abbreviations:
	BMI		Body Mass Index
	GDM (44) vs. Normal glucose (579) 3.3 Paper 3: The association between maternal urinary phthalate concentrations and IGT (63) vs. Normal glucose (560) BBzP Benzylbutyl phthalate	GDM + IGT (107) vs. Normal glucose (516)
	Exposure pregnancy induced hypertension: Results from the EDEN Mother-Child Cohort Study. Unadjusted OR (95% CI) Adjusted OR (95% CI) Unadjusted OR (95% CI) Adjusted OR (95% CI) CI Confidence Interval	Unadjusted OR (95% CI)	Adjusted OR (95% CI)
			Q1 (1-12)	Reference	Reference	Reference	Reference	Reference	Reference
	DEHP		di(2-ethylhexyl) phthalate
	Pb DBP	Q2 (12-17) In our third paper, we show the results on relationships between maternal 0.87 (0.33-2.28) 0.70 (0.19-2.48) 1.13 (0.63-2.04) 1.08 (0.55-2.12) Di-n-butyl phthalate	0.98 (0.56-1.70)	1.28 (0.65-2.53)
			Q3 (17-22)	0.87 (0.30-2.48)	1.16 (0.34-3.96)	1.19 (0.63-2.25)	0.74 (0.36-1.52)	0.82 (0.44-1.51)	1.07 (0.52-2.18)
	Q4 (22-80) exposure to phthalates during pregnancy and pregnancy induced hypertension. We 2.30 (0.97-5.44) 2.25 (0.75-6.77) 1.15 (0.61-2.14) 0.76 (0.38-1.50) † DiBP di-isobutyl phthalate	0.92 (0.52-1.64)	0.97 (0.47-2.01)
	Q1 (0.1-0.5) have submitted our paper in the journal of Environmental International. We present the Reference Reference Reference Reference DiDP di-isodecyl phthalate	Reference	Reference
	Q2 (0.5-0.8) details of this paper in the thesis. 0.55 (0.20-1.55) DiNP di-isononyl phthalate	0.62 (0.18-2.09)	1.86 (1.00-3.48)*	1.47 (0.76-2.84)	1.38 (0.79-2.41)	1.93 (0.92-4.06)	†
	Cd HMW		Q3 (0.8-1.2) high molecular weight 0.88 (0.34-2.26)	1.44 (0.38-3.47)	1.57 (0.81-3.03)	1.39 (0.70-2.73)	1.12 (0.62-2.00)	1.92 (0.90-4.10)	†
	LOD		Q4 (1.2-9.6) limit of detection 1.88 (0.86-4.09)	2.01 (0.73-5.53)	2.17 (1.19-4.03)*	1.49 (0.76-2.92)	1.40 (0.81-2.42)	2.33 (1.10-4.92)*
	LMW		Q1 (1-8) low molecular weight Reference	Reference	Reference	Reference	Reference	Reference
	Mn MEP		Q2 (8-10) Monoethyl phthalate 0.75 (0.22-2.55)	0.65 (0.15-2.81)	1.73 (0.81-3.71)	1.53 (0.66-3.55)	1.43 (0.67-3.06)	1.68 (0.73-3.85)
	MBP		Q3 (10-12) Mono-n-butyl phthalate 1.07 (0.39-2.93)	0.82 (0.25-2.65)	1.13 (0.56-2.25)	1.00 (0.47-2.13)	1.13 (0.57-2.24)	1.15 (0.54-2.44)
	MiBP		Q4 (12-53) Mono-isobutyl phthalate 1.69 (0.02-4.91)	1.08 (0.29-4.01)	1.42 (0.66-3.07)	1.16 (0.49-2.18)	1.04 (0.46-2.33)	1.16 (0.47-2.87)
	Pb = Lead; Cd = Cadmium; Mn= Manganese; GDM = gestational diabetes mellitus; IGT = impaired glucose tolerance
	Models were adjusted for maternal smoking, maternal age, maternal BMI, maternal education level, pregnancy induced hypertension, number of siblings and recruitment centre. MECPP Mono(2-ethyl-5-carboxypentyl) phthalate
	* P<0.05; † P<0.10 MEHHP Mono (2-ethyl-5-hydroxyhexyl) phthalate
	MEOHP	Mono(2-ethyl-5-oxohexyl) phthalate
	MEHP		Mono(2-ethylhexyl) phthalate
	MBzP		Monobenzyl phthalate
	MCOP		Monocarboxy-isooctyl phthalate
	MCPP		Mono (3-carboxypropyl) phthalate
	MCNP		Monocarboxy-isononyl phthalate
	OR		Odds Ratio
	SD		Standard Deviation

  The prevalence of pregnancy induced hypertension was computed. We applied multiple logistic regression analysis to examine the association between each phthalate metabolite and ΣDEHP and pregnancy induced hypertension outcomes adjusted for potential confounders. The potential confounders were determined a priori from literature review. The confounders included in our final model were maternal smoking, maternal age, maternal BMI, maternal education level, gestational age, and number of siblings. Models were additionally adjusted for recruitment centre. All the statistical analysis was conducted using Stata statistical software 14 (Stata Corp LP, 4905 Lakeway Drive, College Station, Texas 77845, USA) where p-value < 0.05 was considered statistically significant.

Table 1 .

 1 Characteristics of the study population and by centre.

		All n=604
	Characteristics	Mean ± SD	Poitiers n= 347 Nancy n= 257 P value
		No. %	No. %	No. %
	Maternal age (years)	29.86±4.8
	<25	79 13.0	48 13.8	12.0
	25 -29 30 -34	218 36.1 205 34.0	123 35.5 120 34.6	37.0 33.1	0.8
	≥35	102 16.9	56 16.1	17.9
	Parity
	0	269 44.5	174 50.1	37.0
	1	222 36.8	117 33.7	42.8	0.005
	≥2	113 18.7	56 16.2	20.2
	BMI (kg/m 2 )	23.1±4.5
	<18.5	57 9.4	27 7.9	11.7
	18.5 -24.9	379 62.9	217 62.5	63.0
	25 -30	110 18.3	68 19.6	16.3	0.4
	>30	49 8.1	30 8.6	7.4
	Missing	9 1.5	5 1.4	1.6
	Maternal education
	<2 years after high school	263 43.5	172 49.6	35.4
	High school +2 years ≥ High school +3 years	132 21.9 199 32.9	72 20.7 98 28.3	23.3 39.4	0.004
	Missing	10 1.7	5 1.4	1.9
	Active smoking (cig/day)
	0	519 86.0	295 85.0	87.2
	1 to 5 >5	60 9.9 23 3.8	39 11.2 12 3.5	8.1 4.3	0.6
	Missing	2 0.3	1 0.3	0.4
	Passive smoking
	Yes	192 31.8	127 36.6	25.3
	No	405 67.1	216 62.3	73.5	0.01
	Missing	7 1.1	4 1.1	1.2
	Alcohol during pregnancy
	Yes	151 25.0	72 20.8	30.7
	No	450 74.5	274 78.9	68.5	0.01
	Missing	3 0.5	1 0.3	0.8
	Gestational age	39.4±1.4
	<37 gestational week ≥37 gestational week	24 4.0 580 96.0	13 3.8 334 96.2	4.3 95.7	0.7
	Employment
	Yes	458 75.9	256 73.8	78.6
	No	142 23.5	89 25.6	20.6	0.3
	Missing	4 0.6	2 0.6	0.8
	Caesarean section
	Yes No	95 15.7 509 84.3	47 13.6 300 86.4	18.7 81.3	0.08
	Season of urine sample
	collection
	Winter	189 31.3	101 29.1	34.2	0.12

Table 2 .

 2 Distribution of phthalate metabolites concentration (µg/L) in maternal prenatal urine (n = 604).

	Parent compound	Abbrev.	Phthalate metabolites	Abbrev.	LOD (µg/L)	%>LOD	Geometric mean (GM)	Minimum			Percentile			Maximum
									5th	25th	50th	75th	95th
	Diethyl phthalate	(DEP)	Monoethyl phthalate (µg/L)	MEP	0.6	100	110.7	7.9	21	51	96	195	714	4093.4
	Di-n-butyl phthalate	(DBP)	Mono-n-butyl phthalate (µg/L)	MBP	0.2	100	51.6	2.8	12	28	43	73	438	2418.8
	Di-isobutyl phthalate	(DiBP)	Mono-isobutyl phthalate (µg/L)	MiBP	0.2	100	42.7	1.5	12	25	39	69	166	689.9
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-carboxypentyl) phthalate (µg/L)	MECPP	0.2	100	40.5	2.6	12	25	38	62	160	2640.5
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-hydroxyhexyl) phthalate (µg/L)	MEHHP	0.2	100	26.9	1.7	6.8	16	27	44	103	2103.8
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-oxohexyl) phthalate (µg/L)	MEOHP	0.2	99.7	22.1	0.6	5.6	14	23	36	84	1441.4
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethylhexyl) phthalate (µg/L)	MEHP	0.5	98	7.2	0.1	1.6	4.4	7.7	15	35	292.3
	Benzylbutyl phthalate	(BBzP)	Monobenzyl phthalate (µg/L)	MBzP	0.3	100	19.7	1.6	4.5	11	18	33	104	970.4
	Di-isononyl phthalate	(DiNP) Monocarboxy-isooctyl phthalate (µg/L) MCOP	0.2	99	4.1	0.1	1.2	2.4	3.9	6.5	19	375.3
	Di-n-octyl phthalate	(DOP)	Mono(3-carboxypropyl) phthalate (µg/L)	MCPP	0.2	100	2.1	0.1	0.7	1.3	1.9	3.4	9.1	78.7
	Di-isodecyl phthalate	(DiDP)	Monocarboxy-isononyl phthalate (µg/L)	MCNP	0.2	97.5	1.5	0.1	0.5	0.8	1.2	2.2	9.5	686.8

Table 3 .

 3 Association (Odd Ratio and 95% CI) between maternal urinary concentrations of phthalate metabolites and pregnancy induced hypertension.

	Phthalate	Unadjusted		Adjusted a
	metabolite	OR 95% CI P-Value	OR	95% CI	P-Value
	MEP	1.43 1.04-1.96* 0.02	1.47	1.01-2.14*	0.04
	MBP	1.48 1.10-2.01* 0.01	1.66	1.11-2.47*	0.01
	MiBP	1.27 0.81-1.99 0.28	1.22	0.69-2.16	0.49
	MECPP	1.26 0.82-1.95 0.29	1.22	0.68-2.21	0.49
	MEHHP	1.21 0.79-1.83 0.37	1.16	0.67-1.99	0.59
	MEOHP	1.19 0.78-1.83 0.41	1.12	0.64-1.95	0.69
	MEHP	0.94 0.66-1.33 0.72	0.84	0.54-1.34	0.48
	MBzP	1.31 0.91-1.89 0.15	1.08	0.66-1.78	0.75
	MCOP	0.81 0.51-1.29 0.38	0.99	0.58-1.69	0.98
	MCPP	1.46 0.96-2.24 † 0.08	1.56	0.93-2.64 †	0.09
	MCNP	1.05 0.72-1.52 0.81	1.24	0.80-1.92	0.33
	ΣDEHP	1.03 0.93-1.15 0.56	1.01	0.88-1.17	0.85
	a OR adjusted for maternal smoking, maternal age, maternal BMI, maternal education level,
	gestational age, number of siblings, and recruitment centre
	MEP = Monoethyl phthalate; MBP = Mono-n-butyl phthalate; MiBP = Mono-isobutyl
	phthalate; MECPP = Mono (2-ethyl-5-carboxypentyl) phthalate; MEHHP = Mono (2-ethyl-5-
	hydroxyhexyl) phthalate; MEOHP = Mono (2-ethyl-5-oxohexyl) phthalate; MEHP = Mono (2-
	ethylhexyl) phthalate; MBzP = Monobenzyl phthalate; MCOP = Monocarboxy-isooctyl
	phthalate; MCPP = Mono (3-carboxypropyl) phthalate; MCNP = Monocarboxy-isononyl
	phthalate; DEHP = di(2-ethylhexyl) phthalate.
	*P<0.05	† P<0.10

Table 4 .

 4 Association (Odd Ratio and 95% CI) between maternal urinary concentrations of phthalate metabolites and pregnancy induced hypertension by quartiles.

					Quartiles
	Phthalate metabolites	Q1	Q2	Q3	Q4
			(Reference)	OR (95% CI) P-value	OR (95% CI) P-value	OR (95% CI) P-value
		Crude	1	1.63 (0.52-5.11) 0.39	0.98 (0.28-3.52) 0.99	2.29 (0.78-6.77) 0.13
	MEP	Adjusted	1	0.99 (0.19-5.12) 0.98	0.63 (0.09-4.24) 0.63	1.24 (0.26-5.92) 0.78
		Crude	1	1.25 (0.33-4.78) 0.73	1.78 (0.51-6.23) 0.36	3.46 (1.10-10.87) 0.03*
	MBP	Adjusted	1	1.78 (0.25-12.77) 0.56	2.76 (0.37-20.40) 0.31	5.91 (0.93-37.79) 0.06 †
		Crude	1	2.75 (0.72-10.61) 0.14	3.49 (0.94-12.97) 0.06 †	2.75 (0.72-10.61) 0.14
	MiBP	Adjusted	1	0.46 (0.07-3.08) 0.42	2.43 (0.49-11.98) 0.27	1.92 (0.35-10.46) 0.44
		Crude	1	1.41 (0.44-4.57) 0.55	2.07 (0.69-6.21) 0.19	1.41 (0.44-4.57) 0.55
	MECPP	Adjusted	1	7.90 (0.83-75.43) 0.07 †	6.09 (0.62-59.70) 0.12	6.47 (0.66-63.11) 0.11
		Crude	1	1.85 (0.60-5.65) 0.28	1.85 (0.60-5.65) 0.28	1.20 (0.36-4.04) 0.75
	MEHHP	Adjusted	1	2.95 (0.51-17.06) 0.22	2.46 (0.40-15.14) 0.33	2.29 (0.33-15.91) 0.39
		Crude	1	4.25 (1.17-15.41) 0.02*	2.75 (0.71-10.61) 0.14	2.04 (0.50-8.31) 0.31
	MEOHP	Adjusted	1	16.08 (1.63-158.12) 0.01*	3.33 (0.26-42.47) 0.35	5.72 (0.52-62.95) 0.15
		Crude	1	0.58 (0.21-1.65) 0.30	0.79 (0.30-2.05) 0.62	0.48 (0.16-1.44) 0.19
	MEHP	Adjusted	1	0.40 (0.07-2.22) 0.29	0.84 (0.19-3.62) 0.81	0.52 (0.09-3.08) 0.47
		Crude	1	1.42 (0.44-4.57) 0.55	0.79 (0.21-3.01) 0.73	2.75 (0.95-7.91) 0.06 †
	MBzP	Adjusted	1	0.81 (0.15-4.21) 0.80	0.50 (0.08-3.11) 0.46	1.79 (0.39-8.25) 0.45
		Crude	1	0.99 (0.36-2.74) 0.99	0.86 (0.31-2.45) 0.79	0.74 (0.25-2.18) 0.58
	MCOP	Adjusted	1	3.99 (0.70-22.68) 0.11	3.41 (0.49-23.49) 0.21	2.42 (0.36-16.17) 0.35
		Crude	1	1.41 (0.44-4.57) 0.55	1.41 (0.44-4.57) 0.55	2.07 (0.69-6.21) 0.19
	MCPP	Adjusted	1	1.89 (0.32-10.71) 0.48	0.95 (0.13-6.53) 0.95	2.49 (0.54-11.52) 0.24
		Crude	1	0.99 (0.31-3.17) 0.98	1.17 (0.38-3.58) 0.77	1.71 (0.61-4.84) 0.31
	MCNP	Adjusted	1	1.11 (0.19-6.51) 0.90	1.30 (0.24-6.92) 0.75	1.71 (0.32-9.11) 0.52
		Crude	1	1.63 (0.52-5.11) 0.39	2.07 (0.69-6.21) 0.19	1.21 (0.36-4.04) 0.75
	ΣDEHP	Adjusted	1	5.45 (0.85-34.84) 0.07 †	3.39 (0.52-22.15) 0.20	3.18 (0.45-22.41) 0.24
	OR adjusted for maternal smoking, maternal age, maternal BMI, maternal education level, gestational age,
	number of siblings, and recruitment centre
	MEP = Monoethyl phthalate; MBP = Mono-n-butyl phthalate; MiBP = Mono-isobutyl phthalate; MECPP = Mono
	(2-ethyl-5-carboxypentyl) phthalate; MEHHP = Mono (2-ethyl-5-hydroxyhexyl) phthalate; MEOHP = Mono (2-
	ethyl-5-oxohexyl) phthalate; MEHP = Mono (2-ethylhexyl) phthalate; MBzP = Monobenzyl phthalate; MCOP =
	Monocarboxy-isooctyl phthalate; MCPP = Mono (3-carboxypropyl) phthalate; MCNP = Monocarboxy-isononyl
	phthalate; DEHP = di(2-ethylhexyl) phthalate.
	*P<0.05		† P<0.10

Table S2 .

 S2 Spearman correlation between ln-transformed standardized ‡ urinary concentrations of phthalate metabolites.

		MBP	MiBP	MECPP	MEHHP	MEOHP	MEHP	MBzP	MCOP	MCPP	MCNP
	MEP	0.13*	0.03	0.04	0.06	0.08*	0.08*	0.10*	0.07	0.02	0.01
	MBP		0.37*	0.26*	0.31*	0.32*	0.24*	0.40*	0.13*	0.62*	0.13*
	MiBP			0.26*	0.29*	0.30*	0.27*	0.38*	0.20*	0.24*	0.11*
	MECPP				0.90*	0.92*	0.77*	0.31*	0.36*	0.31*	0.27*
	MEHHP					0.97*	0.83*	0.36*	0.33*	0.35*	0.25*
	MEOHP						0.84*	0.38*	0.32*	0.36*	0.24*
	MEHP							0.31*	0.25*	0.27*	0.19*
	MBzP								0.19*	0.28*	0.17*
	MCOP									0.28*	0.42*
	MCPP										0.33*
	MEP = Monoethyl phthalate; MBP = Mono-n-butyl phthalate; MiBP = Mono-isobutyl phthalate; MECPP = Mono(2-ethyl-5-carboxypentyl) phthalate; MEHHP
	= Mono(2-ethyl-5-hydroxyhexyl) phthalate; MEOHP = Mono(2-ethyl-5-oxohexyl) phthalate; MEHP = Mono(2-ethylhexyl) phthalate; MBzP = Monobenzyl
	phthalate; MCOP = Monocarboxy-isooctyl phthalate; MCPP = Mono(3-carboxypropyl) phthalate; MCNP = Monocarboxy-isononyl phthalate
	‡ Standardized for urine sampling conditions (creatinine level, gestational age, day and hour of sampling, storage duration at room temperature and year of
	analysis) as described in detail by Mortamais et al (2012)
	* P<0.05

Table 3 .

 3 Epidemiological studies on relationships between exposure to phthalates and development of eczema in children.

	Reference	Study design	Study population	Exposure	Results
	(Shi et al. 2018)	Cross sectional	434 children, age 5-10	MMP, MEP, MiBP,	significant associations for
			years	MnBP,	MECPP,	MEHHP, MEOHP and MBzP
				MEHHP,	MEOHP,
				MEHP, MBzP
	(Berger et al.	Birth cohort	392 children, age 7	MEP, MnBP, MiBP	Non-significant associations
	2018)		years
	(Gascon et al.	Birth cohort	437 children at 6	MBzP, MEP, MiBP,	Non-significant associations
	2015)		months, 421 at 14	MnBP and DEHP
			months, 385 at 4 years,	including	its
			361 at 7 years and	metabolites; MEHP,
			atopy at age 4years in	MEHHP,	MEOHP,
			175 children	MECPP.
	(Smit et al. 2015) Birth cohort	1024 children, age 5-9	DEHP and DiNP	AOR (95% CI) DiNP Current
			years	metabolites		eczema, 0.71 (0.52-0.96) ;
						DiNP ever eczema, 0.90
						(0.74-1.09)
	(Stelmach et al.	Birth cohort	147 children, age 2	DEP, DiBP, DnBP,	Non-significant association
	2015)		years	BBzP, DEHP, DiNP,	for eczema, but significant
				DOP		association for food allergy

Table 4 .

 4 Epidemiological studies on relationships between exposure to Cd, Pb, Mn and gestational diabetes mellitus.

	Reference	Study	Study population	Exposure		Results
		design
	(Oguri et al.	Cross-	16,955 pregnant	Cd, Pb	No		significant
	2019)	sectional	women		association
	(Liu et al.	Birth cohort 2026 pregnant	Cd	The risk ratios (RRs) of
	2018)		women		GDM for the middle
					tertile: 1.04 (95% CI:
					0.74, 1.44), and 1.36
					(95%: CI: 0.98, 1.90) for
					the top tertile.
	(Xing et al.	Birth cohort 6837 pregnant	Cd	The relative risk (RR)
	2018)		women		1.16; (95% CI: 1.03,
					1.33) increase in risk of
					GDM
	(Shapiro et	Birth Cohort 1274	Pb, Cd, As,	No		significant
	al. 2015)			Hg	association with Cd or
					Pb
	(Peng S et	Case-	1359	Pb, Cd, As,	Cd, AOR 16.87 (4.19-
	al. 2015)	control		Hg, Cr	67.86) in 3 rd quartile and
					AOR 11.95 (2.97-48.04)
					in 4 th quartile
					Pb,	a	negative
					association in 2 nd and
					3 rd quartile 0.37 (0.16-
					0.86) and 0.16 (0.06-
					0.44), respectively

ACKNOWLEDGEMENTS

First of all, I submit and bow down to Almighty God for affording me the courage and ability to endeavor for PhD studies and making it a reality! Then I thank all the people who have participated in and contributed to this work. I gratefully acknowledge the Campus France and Higher Education Commission, Pakistan for funding this study and giving me an opportunity to follow this academic programme.

I wish to express my deepest gratitude to my supervisor Dr. Isabella Annesi-Maesano for her great patience, motivation and immense knowledge in guiding my

Acknowledgments

We acknowledge the participating families, midwife research assistants for data collection.

The

EDEN Mother-Child Cohort Study Group I. Annesi-Maesano, J.Y. Bernard, J. Botton, M.A. Charles, P. Dargent-Molina, B. de Lauzon-Guillain, P. Ducimetière, M. de Agostini, B. Foliguet, A. Forhan, X. Fritel, A. Germa, V. Goua, R. Hankard, B. Heude, M. Kaminski, B. Larroque, N. Lelong, J. Lepeule, G. Magnin, L. Marchand, C. Nabet, F Pierre, R. Slama, M.J. Saurel-Cubizolles, M. Schweitzer, O. Thiebaugeorges. We thank the midwife research assistants (L. Douhaud. S. Bedel. B. Lortholary. S. Gabriel. M. Rogeon. and M. Malinbaum) for data collection and P. Lavoine for checking, coding, and entering data.

Fundings:

We acknowledge all funding sources for the EDEN study: Foundation for Medical Research (FRM), National Agency for Research (ANR), National Institute for Research in Public Health (IRESP: TGIR cohorte santé 2008 program), French Ministry of Health (DGS), French Ministry of Research, Inserm Bone and Joint Diseases National Research (PRO-A) and Human Nutrition National Research Programs, Paris-Sud University, Nestlé, French National Institute for Population Health Surveillance (InVS), French National Institute for Health Education (INPES), the European Union FP7 programmes (FP7/2007-2013, HELIX, ESCAPE, ENRIECO, MEDall projects), Diabetes National Research Program (through a collaboration with the French Association of Diabetic Patients (AFD)), French Agency for Environmental Health Safety (now ANSES), Mutuelle Générale de l'Education Nationale (MGEN), French National Agency for Food Security, the EU FP7-ENV funded Health and Environmentwide Associations based on Large population Surveys (HEALS) project (N: 603946) and the French-speaking association for the study of diabetes and metabolism

All the statistical analysis was conducted using Stata statistical software 14 (Stata Corp LP, 4905 Lakeway Drive, College Station, Texas 77845, USA) where pvalue < 0.05 was considered statistically significant. In this part, we show the results on relationships between prenatal exposure to phthalates and development of eczema phenotypes in male children. We published our paper in Environmental Health Perspectives. We present the details of this paper in the thesis.

RESULTS

3

Appendix A. Supplementary data

Exposure to heavy metals during pregnancy related to gestational diabetes mellitus in diabetes-free mothers.

Munawar Hussain Soomro, 1 Nour Baiz, 1 Guy Huel, 1 Chadi Yazbeck, 2,3 Jérémie Botton, 4,5 Barbara Heude, 4,6 Carl-Gustaf Bornehag, 7,8 and Isabella Annesi-Maesano, 1 ; on behalf of The EDEN mother-child cohort study group

Affiliations:

1 Sorbonne Université and

Table of Contents

Table S1. Characteristics of the study population included and excluded in the analysis. (ALFEDIAM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest: none

Abstract

Introduction: Studies have suggested that exposure to endocrine disruptors like phthalates widely used in our daily life (wrapping foods, cosmetics, toys, medical devices, polyvinyl chloride flooring, and building materials) may be associated with elevated blood pressure and increased risk of cardiovascular diseases. Phthalates may cause a pro-inflammatory response and increased oxidative stress and which may be a cause of pregnancy induced hypertension.

Objectives:

We evaluated the association between maternal exposure to phthalates during pregnancy and pregnancy induced hypertension.

Methods: 2,002 pregnant women were initially included in the French EDEN motherchild prospective birth cohort study. Eleven phthalates metabolites were quantified in spot maternal urine samples collected between 23rd and 28th week of gestation. The Association of each urinary phthalate metabolite and pregnancy induced hypertension was assessed with adjusted multiple logistic regression analysis.

Results:

The study population consisted of 604 women with available data on both pregnancy induced hypertension diagnosis and urine analysis for phthalates. While taking into account the quartiles, we do not observed association between MEP and pregnancy induced hypertension. However, a significant increased odd ratio for MBP metabolite were observed in the 4 th quartile in crude (OR: 3.46, CI: 1.10-10.87, P-value = 0.03) as well as in adjusted for confounders (OR: 5.91 CI: 0.93-37.79, Pvalue = 0.06) as shown in Table 4. A borderline association for ΣDEHP (OR: 5.45, CI: 0.85-34.84, P-value = 0.07) was observed in the second quartile.

Discussion

In this study, we report an association between maternal prenatal urinary concentration

Supplementary Material:

The association between maternal urinary phthalate concentrations and pregnancy induced hypertension: Results from the EDEN mother-child cohort study.

Munawar Hussain Soomro, MBBS, MSc, MPhil, 1 Barbara Heude, PhD, 2,3 Carl-Gustaf Bornehag, PhD, 4,5 and Isabella Annesi-Maesano, MD, PhD, DSc, 1 ; the EDEN mother-child cohort study group

Table of Contents

Table S1. Characteristics of the study population included and excluded in the analysis.

Table S2. Spearman correlation between ln-transformed standardized urinary concentrations of phthalate metabolites.