# Short-Half-Life Chemicals : Maternal Exposure and Offspring Health Consequences - The Case of Synthetic Phenols, Parabens, and Phthalates

Delphine Rousseau-Ralliard, Jeanne Bozec, Marion Ouidir, Nicolas Jovanovic, Véronique Gayrard, Namya Mellouk, Marie-Noëlle Dieudonné, Nicole Picard-Hagen, Maria-Jose Flores, Hélène Jammes, et al.

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Delphine Rousseau-Ralliard, Jeanne Bozec, Marion Ouidir, Nicolas Jovanovic, Véronique Gayrard, et al.. Short-Half-Life Chemicals : Maternal Exposure and Offspring Health Consequences - The Case of Synthetic Phenols, Parabens, and Phthalates. Toxics, 2024, 10.3390/toxics12100710. hal-04713923

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Review

# Short-Half-Life Chemicals: Maternal Exposure and Offspring Health Consequences—The Case of Synthetic Phenols, Parabens, and Phthalates

Delphine Rousseau-Ralliard<sup>1,2</sup>, Jeanne Bozec<sup>1,2</sup>, Marion Ouidir<sup>3 ID</sup>, Nicolas Jovanovic<sup>3 ID</sup>, Véronique Gayrard<sup>4</sup>, Namya Mellouk<sup>1,2</sup>, Marie-Noëlle Dieudonné<sup>1,2</sup>, Nicole Picard-Hagen<sup>4</sup>, Maria-José Flores-Sanabria<sup>1,2 ID</sup>, Hélène Jammes<sup>1,2</sup>, Claire Philippat<sup>3</sup> and Anne Couturier-Tarrade<sup>1,2,* ID</sup>

1 Université Paris-Saclay, UVSQ, INRAE, BREED, 78350 Jouy-en-Josas, France

2 Ecole Nationale Vétérinaire d'Alfort, BREED, 94700 Maisons-Alfort, France

3 University Grenoble Alpes, Inserm U1209, CNRS UMR 5309, Team of Environmental Epidemiology Applied to Development and Respiratory Health, Institute for Advanced Biosciences, 38000 Grenoble, France

4 ToxAlim (Research Center in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31062 Toulouse, France

* Correspondence: anne.couturier-tarrade@inrae.fr; Tel.: +33-1-34-65-24-44

**Abstract:** Phenols, parabens, and phthalates (PPPs) are suspected or known endocrine disruptors. They are used in consumer products that pregnant women and their progeny are exposed to daily through the placenta, which could affect offspring health. This review aims to compile data from cohort studies and in vitro and in vivo models to provide a summary regarding placental transfer, fetoplacental development, and the predisposition to adult diseases resulting from maternal exposure to PPPs during the gestational period. In humans, using the concentration of pollutants in maternal urine, and taking the offspring sex into account, positive or negative associations have been observed concerning placental or newborn weight, children's BMI, blood pressure, gonadal function, or age at puberty. In animal models, without taking sex into account, alterations of placental structure and gene expression linked to hormones or DNA methylation were related to phenol exposure. At the postnatal stage, pollutants affect the bodyweight, the carbohydrate metabolism, the cardiovascular system, gonadal development, the age of puberty, sex/thyroid hormones, and gamete quality, but these effects depend on the age and sex. Future challenges will be to explore the effects of pollutants in mixtures using models and to identify the early signatures of in utero exposure capable of predicting the health trajectory of the offspring.

**Keywords:** endocrine disruptors; gestation; placenta; maternal exposure; offspring phenotype; epigenetics; cardiometabolic outcomes; fertility; adulthood

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**Citation:** Rousseau-Ralliard, D.; Bozec, J.; Ouidir, M.; Jovanovic, N.; Gayrard, V.; Mellouk, N.; Dieudonné, M.-N.; Picard-Hagen, N.; Flores-Sanabria, M.-J.; Jammes, H.; et al. Short-Half-Life Chemicals: Maternal Exposure and Offspring Health Consequences—The Case of Synthetic Phenols, Parabens, and Phthalates. *Toxics* **2024**, *12*, 710. https://doi.org/10.3390/toxics12100710

Academic Editor: Maria João Rocha

Received: 7 August 2024

Revised: 12 September 2024

Accepted: 19 September 2024

Published: 29 September 2024

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**Copyright:** © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Synthetic Phenols, Parabens, and Phthalates

The human population is continually exposed to a wide range of chemical compounds present in the environment, including phenols, parabens, and phthalates (PPPs), which can affect human health.

The most studied phenol is bisphenol A (BPA, 2,2-bis [4-hydroxyphenyl]propane), a synthetic chemical used in consumer products such as the lining of canned food and drinks, the packaging of baby formula and baby bottles, and dental implants [1]. BPA can migrate from cans into food, from plastic bottles into water, or from dental fillings into saliva, leading to an oral exposure. In addition, BPA can penetrate the skin through thermal paper in sales receipts and by inhalation via industry. Despite its ban from plastic baby bottles in 2011 in Europe (https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32011R0321&from=EN, accessed on 12 September 2024) and from all food-grade materials in France from 1 January 2015 (https://eur-lex.europa.eu/legal-content/EN/TXT/)
PDF/?uri=CELEX:32018R0213&from=EN, accessed on 12 September 2024), the exposure to BPA is still widespread in the global population [2]. In the body, BPA is quickly metabolized into BPA-glucuronide in the liver and then mainly eliminated by the kidneys. Noteworthy is that the unconjugated BPA is the bioactive form. BPA has been described as mimicking estrogen by binding to its receptors, leading to its classification as a "reproductive toxicant" in 2016 (https://www.foodpackagingforum.org/news/bpa-classified-as-reproductive-toxicant, accessed on 12 September 2024), and then as an endocrine disruptor in 2018 (https://www.foodpackagingforum.org/news/bpa-identified-as-environmental-endocrine-disruptor, accessed on 12 September 2024), by the European Chemicals Agency (ECHA). BPA is known to induce metabolic disorders in human and animal models, reproductive outcomes, the development of cardiovascular and neurodegenerative diseases, cancer, and genotoxic effects [3,4], but its effect during key periods such as preconception or gestation on the phenotype of first- and second-generation offspring using epidemiological data or animal models is still needed.

Since the banning of BPA in many consumer products [1], substitutes have been proposed by manufacturers over the last decade, including bisphenol S (BPS, 4,4'-sulfonyldiphenol) and bisphenol F (BPF, 4,4'-dihydroxydiphenylmethane). BPS and BPF structures are similar to BPA, suggesting that they can act as endocrine disruptors as well. They have already been shown to cause obesity effects/metabolic effects in animal models [4], but further experiments are needed to assess their impact on organ function and also during pregnancy on the phenotype of the offspring, as these compounds have only been used for a short time.

The phenol family also includes triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy) phenol), a synthetic antimicrobial agent [5]. TCS is used in several personal care products, including toothpaste, antibacterial soaps, dishwashing liquids, deodorant soaps, cosmetic and antiseptic products, and deodorants. TCS is also used in kitchen utensils, toys, bedding, clothes, fabrics, and trash bags. The main route of human exposure is dermal exposure [6], but exposure can also occur through the consumption of water and/or food products [7]. TCS is metabolized to glucuronide and sulfate conjugates, and is mainly excreted in the urine in humans [8]. Several investigators have reported that TCS is an EDC that affects the immune system, and cardiovascular and reproductive functions [7,9–12]. Thus, the percentage of TCS in certain products has been regulated by the EU, and it is also banned in shaving products [13]. Since TCS is used in many products, it is important to know the effects of direct exposure to this molecule on the phenotype as well as the potential intergenerational effects.

Parabens are considered as endocrine disruptors [14] and used to prevent the growth of microorganisms in personal care products such as lotions, deodorants, hair care products, shaving products, pharmaceuticals, textiles, and clothes, but also in food additives such as methylparaben (MeP, food additive E218), ethylparaben (EtP, E214), and, to a lesser extent, propylparaben (PrP, E216), heptylparaben (HeP), butylparaben (n-BuP), and isobutylparaben (i-BuP) [14]. The estimated total intake of parabens from foods achieves 307–940 ng/kg bodyweight [15]. The most common parabens investigated and detected in biological fluids are MeP, EtP, n-PrP, and n-BuP, whereas i-BuP, isopropylparaben (i-PrP), and benzylparaben (BzP) are less investigated. Moreover, the urine concentrations of parabens are higher in women compared to men. Therefore, it is essential to test the effects of exposure to all of these molecules on the phenotypes at key stages of development and during pregnancy so to evaluate the effects on the offspring.

Phthalates are the main plasticizers used in the industry to improve the extensibility, elasticity, and workability of polymers. Phthalates include low-molecular-weight di-n-butyl phthalate (DBP), benzyl butylphthalate (BBP), di-(2-ethylhexyl) phthalate (DEHP), di-methyl phthalate (DMP), and diethyl phthalate (DEP), or high-molecular-weight di-isobutyl phthalate (DiNP) and di-n-octyl phthalate (DiDP). More than 25 phthalates are used in many consumer products, such as toys, clothing, inks, rubbers, adhesives, paints, household materials, pesticides, cosmetics, personal care products, pharmaceutical products, etc. [16–18]. They are present in our environment and are able to reach the
food and beverage chain by migration during processing, storing, transportation, and preparation. Even if ingestion is considered the main source of human exposure, dermal or nasal routes are not negligible. Several phthalates have been classified by the European Commission as having endocrine-disrupting or reprotoxic properties (category 1B). In the EU, several phthalates are now banned from use in cosmetics, including BBP, DEHP, DBP, and bis(2-methoxyethyl) phthalate (DMEP) (EC/1223/2009). The use of several compounds is also regulated in material intended to come into contact with food (DEHP, DBP, and BBP, EC/2007/19) as well as in toys and childcare articles (DEHP, DBP, BBP, DiNP, DiDP, and di-n-octyl phthalate (DnOP), French law 2006-1361) [16,18,19]. In Europe, the REACH list limits the level of exposure to DEHP metabolites, including (mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), MEHP, and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), within 0.02 mg/kg bw/d. Noteworthy, the EFSA has set the tolerable daily intakes (in mg/kg of bw) to 0.05 for DEHP, 0.01 for DBP, 0.15 for DiNP and DiDP, and 0.5 for BBP [20,21]. The half-life of phthalate diesters in blood plasma or urine is less than 24 h, and these compounds are mainly excreted in the urine as conjugated monoesters [22], but some of them, such as DEHP, undergo a secondary metabolism before urinary excretion. Consequently, urine is the more relevant matrix for the biomonitoring of human phthalate exposure [18], even if all the metabolites of a parent molecule cannot be assayed in this fluid for technical reasons. Exposure to phthalates is raising a great concern regarding their impact on human health. For several years, accumulating evidence has suggested that phthalates and their metabolites are suspected to act as EDCs through their interaction with different signaling pathways. Phthalate exposure has been correlated to several health outcomes, among them adverse reproductive disorders in women and men, neurological diseases, cancer, metabolic disorders, asthma, allergies, and toxicity in both the liver and kidney [16], but the intergenerational effects should be investigated further with doses mimicking the human exposure.

In this review, we will focus on PPPs, i.e., their metabolism, the toxicokinetic models for adapting human exposure doses to animal models, and the transplacental transfer of PPPs. The effects of PPP exposure throughout critical periods such as preconception, gestation, and lactation on offspring health, including the placental function, neonatal phenotypes, metabolic outcomes, cardiometabolic disorders, and gonadal functions, will be presented in the context of the developmental origins of health and disease (DOHaD) concept [23]. These data will be based on the literature from epidemiological data, associating human maternal urinary concentrations of these molecules and their metabolites with the health effects on the offspring, and with regard to the long-term postnatal cardiometabolic effects, they will be mainly presented through studies on animal models, since human cohorts currently rarely explore the effects beyond childhood.

## 2. Absorption, Distribution, Metabolism, and Excretion Processes of PPPs

PPPs are considered non-persistent chemicals because absorbed PPPs are rapidly metabolized and primarily excreted in the urine. In humans, ingested BPA and its analogs are rapidly and efficiently absorbed, and almost entirely excreted in the urine as a conjugate [24–27].

Following oral administration to human volunteers, TCS is rapidly absorbed, metabolized to conjugates, and eliminated in the urine within 4 days of exposure, with a terminal plasma half-life of 21 h [28]. Toxicokinetic studies performed in rats have shown that benzophenone-3 (BP-3) is rapidly absorbed after oral exposure and mainly excreted in the urine in conjugated forms [29]. BP-3 can also be transformed into three major metabolites, namely, benzophenone-1 (BP-1), benzophenone-8 (BP-8), and 2,3,4-trihydroxybenzophenone (THB), with BP-1 being the most frequently detected in rats [30,31]. Parabens are well absorbed following oral or dermal exposure and extensively metabolized through hydrolysis into para-hydroxybenzoic acid (PHBA), which is excreted in the urine mainly as glucuronide and sulfate conjugates. A fraction of absorbed parabens is also excreted in the urine in the form of glucuronide and sulfate conjugates [32]. Since PHBA is a non-specific metabolite of
parabens, several biomonitoring studies have assessed the human exposure by measuring the urinary concentrations of conjugated parabens.

For phthalates, mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), and mono-benzyl phthalate (MBzP) are the main urinary metabolites of their respective parent short-chain phthalates, including di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), and BBP, respectively. The monoester metabolites of long-chain phthalates, such as DEHP and DiNP, MEHP, and MiNP, respectively, give rise to secondary oxidative metabolites (mono-2-ethyl-5-hydroxy-hexyl phthalate (MEHHP), mono-2-ethyl-5oxohexyl phthalate (MEOHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), and mono [2-(carboxymethyl)hexyl] phthalate (MCMHP)) for DEHP, and several complex isoforms of monoisobonyl phthalate (MiNP) obtained by the formal condensation of one of the carboxy groups of phthalic acid with the hydroxy group of isononanol, which are the main xenobiotic metabolites excreted in human urine either as free or conjugated compounds [33].

Because PPPs are rapidly metabolized and excreted in the urine, several biomonitoring studies have assessed the human exposure by measuring the urinary concentrations of total (unconjugated plus conjugated) phenols and phthalate monoesters. Indeed, urine represents a relevant biological matrix for the biomonitoring of non-persistent chemical substances such as PPPs because their concentrations in urine are higher than in plasma concentrations, and because its collection is non-invasive. Generalized human exposure to PPPs is therefore reflected by the high frequency of detection of phenols and phthalate monoesters in the urine [2,34–38].

### 3. Toxicokinetic Models for Adapting Human Exposure Doses to Animal Models

Toxicokinetics (TKs) provides critical information for integrating hazard toxicity and exposure assessments to determine the potential risk of exposure to PPPs in humans. TKs is needed for determining the dose/concentration range that should be used in animal or in vitro testing to reproduce the internal exposure that can be expected from realistic human external exposure scenarios.

#### 3.1. Estimation of PPP Daily Intake

PPP urine biomonitoring data have been widely used as biomarkers of exposure for epidemiological purposes [36,39]. Since urine biomonitoring data provide an integrative measure of exposure regardless of the source and route, the measurement of urinary concentrations of total PPP metabolites allows for the direct assessment of the daily PPP intake (DI). The calculation of the DI from urinary biomonitoring data relies on the urinary excretion factors (Fue) of the measured PPPs or metabolites related to the parent chemical ingested (Equation (1), [40]), as follows:

$$DI = \frac{UE \times UV_{norm}}{F_{UE}} \times MW \quad (1)$$

where UE is the molar urinary excretion of the measured PPPs or metabolites (µmol/L); UVnorm is the daily excreted urinary volume (L/kg bw/day), estimated at 0.028 L per kg bw per day for a 70-kg bodyweight [41], and which should be adapted to the targeted population (i.e., pregnant women and infants); FUE is the molar ratio between the amount of PPPs or metabolites excreted in the urine and the amount of parent PPPs taken up; MW is the molecular weight of the parent PPPs.

For phenols, most biomonitoring studies rely solely on the determination of total phenol concentrations in the urine measured after enzymatic hydrolysis. While bisphenols and TCS are primarily eliminated in the urine as conjugates [25,28], the fraction of absorbed parabens excreted in the urine as glucuronides and sulfate conjugates ranges from about 6% for n-BuP to 17% for propylparaben (PrP) [42,43]. The selection of the appropriate biomarkers of exposure to long-chain phthalates such as DEHP and DiNP is more complex. Indeed, while approximately 70–85% of an oral dose of short-chain diester phthalates (DnBP, DiBP, BBP, and DEP) is excreted in the urine as hydrolytic monoester phthalates
(MBP, MiBP, MBzP, and MEP) [44,45], only about 2 and 7% of an oral dose of DiNP and DEHP are excreted in the urine as the hydrolytic monoesters [40,46,47]. The secondary (oxidized monoester) phthalate metabolites of DiNP, such as OH-MiNP, oxo-MiNP, and cx-MiNP, and of DEHP, such as MECPP, MEOHP, MEHHP, and MCMHP, are the major metabolites excreted in human urine as free or conjugated compounds. Therefore, urinary concentrations of hydrolytic monoesters of long-chain relative to short-chain phthalates do not reflect the relative exposure to phthalates [48].

Although urine biomonitoring data provide essential information on the amount of compound that enters the body on a daily intake, regardless of the route of exposure, it is the concentration of the toxicologically active form of PPPs in the blood that determines the amount of chemicals that can reach the target tissues and receptors and exert effects.

For phenols, only the native (unconjugated) form is considered toxicologically active because it is capable of interacting with target tissues and receptors. Since phthalate diesters, such as DEHP, are cleaved to their hydrolytic monoesters in the mouth, stomach, intestines, or blood, plasma concentrations of phthalate diesters are expected to be very low [33]. It is the plasma concentrations of monoester phthalate, namely, MEHP, for DEHP that are considered relevant in terms of risk assessment [49].

## 3.2. Interspecies Extrapolation of PPP Dose

The steady-state plasma concentration ($C_{SS}$) of the toxicologically active form of PPPs therefore represents the most relevant physiological variable for translating the results from animal studies to humans. $C_{SS}$ (ng/mL) is related to the dosing rate (mass per unit of time) by a key toxicokinetic parameter, namely, blood clearance ($CL$, volume per unit of time), in addition to the bioavailability ($F$, ranging from zero to one). This toxicokinetic parameter corresponds to the fraction of compound reaching the systemic circulation without change according to Equation (2), as follows:

$$ \text{Dosing rate} = CL \times C_{SS}/F \quad (2) $$

Using Equation (2), the PPP dosage to be administered orally to reproduce the targeted $C_{SS}$ of the toxicologically active form of PPPs in an animal species can therefore be calculated from the species' CL and F values.

A loading dose (LD) of PPPs can be applied to enable serum concentrations to rapidly reach the targeted $C_{SS}$. This dose is determined from the steady-state volume of the distribution ($V_{ss}$) and F using Equation (3), as follows:

$$ LD = C_{ss} \times V_{ss}/F \quad (3) $$

By permitting the serial sampling of maternal and fetal blood after maternal or fetal administration, pregnant ewes provided a unique model for evaluating the disposition of several drugs during the prenatal period [50]. Among animal models, the rabbit represents an appropriate model for TK purposes. Like in humans, urinary elimination predominates in rabbits. This is unlike other species, such as rats and dogs, that have rather biliary excretion [51]. This interspecies difference is mainly observed for substances with a molecular weight between 300 and 600 Da, such as certain phthalate monoesters.

The main TK parameters of PPPs, i.e., CL, $V_{ss}$, and F, can be determined from the plasma concentration-time profiles obtained following the intravenous and oral administrations of a mixture of EDCs. To achieve this, BPS and DEHP were administered by the oral route in a mixture of eight compounds in female rabbits to compute the loading and daily maintenance doses. The aim was to reproduce in rabbits the steady-state serum concentrations ($C_{SS}$) of the two compounds found in pregnant women. Thus, the mean $C_{SS}$ of female rabbits measured after 17 weeks of exposure to a mixture of the eight compounds administered orally at doses deduced from the estimated TK parameters ranged from 0.77 to 1.21 ng/mL of the targeted $C_{SS}$ [52]. Toxicodynamic modeling in rabbits validated the interspecies extrapolation of human exposure rates to environmental contaminants.
4. Evaluation of Transplacental Transfer of PPPs Using Models

Understanding the materno-fetal transfer of PPPs across the placenta is crucial for
assessing the prenatal exposure risks. To respond to this issue, the placental transfer of
PPPs was investigated using the following two main models, which offer both advantages
and disadvantages:

* The most clinically relevant model to evaluate the placental transfer of compounds is the ex vivo human placental perfusion model at term [53]. This enables placental transfer to be assessed by reproducing maternal and fetal circulation in a few hours and taking samples from each compartment. This model offers several advantages, as the placental barrier maintains its structural integrity and the separate perfusion of the maternal and fetal sides reproduces their respective blood flows. However, this physiological approach does not take into account the nonplacental toxicokinetic factors that could contribute to the level of fetal exposure. These factors include the maternal and fetal metabolism, the dynamic structural conditions of the pregnancy, and the physiological changes throughout the pregnancy (the thickness of the trophoblast, uterine flow, and expression of transporters) [54].

* An integrated pregnant sheep model that enables a direct administration and monitoring of xenobiotics over time in both fetal and maternal blood [55]. Because of important physiological similarities between the sheep and human placental functions, this model has contributed to significant advances in prenatal human medicine [56], despite the interspecies differences in the placental structure (the synepitheliochorial versus hemochorial placental structure) and transporters [56,57].

The ex vivo placental perfusion model had already been used to quantify the materno-
fetal placental transfer of fifteen bisphenols [58]. Despite their chemical-structural sim-
ilarities, these bisphenols differed greatly in the efficiency of placental transport. The
placental transfer rates of bisphenol analogs such as 4,4'-Dihydroxybiphenyl, bisphenol
AP (BPAP), bisphenol E (BPE), bisphenol F (BPF), 3-3BPA, bisphenol B (BPB), and BPA
were similar to those of antipyrine, a molecule that passes the placental barrier by passive
diffusion. This suggests that the exchange of these bisphenols across the placenta primarily
involves passive diffusion and is limited only by the rate of the placental blood flow. In
contrast, the placental transfer rates of bisphenol FL (BPFL) and BPS were very limited and
intermediate for bisphenol BP (BPBP), bisphenol Z (BPZ), bisphenol C (BPC), bisphenol
M (BPM), bisphenol P (BPP), and bisphenol AF (BPAF). This suggests a low diffusional
permeability and/or that their passage might involve efflux transport. Moreover, although
the glucuronide form of bisphenols could be detected in vivo in the fetal compartment,
the placental transfer of glucuronide, evaluated for BPA and BPS, was almost non-existent.
This indicates that, in the fetal compartment, these glucuronides come mainly from the feto-
placental metabolism, at least at the end of gestation [59,60]. A classical QSAR model based
on molecular descriptors of BPs was developed to predict their materno-fetal transfer. But
even if the physicochemical, topological, thermodynamic, and electronic parameters were
able to influence the placental passage of this family of emerging BPs, the placental transfer
efficiency of these 15 structurally related BPs could not be predicted solely from their
physicochemical properties determined in silico [61]. This reinforces the indisputability of
the physiological models.

Using the chronically catheterized fetal sheep model, toxicokinetic studies have been
performed to determine the relative contributions of the placental transfer and the fetal and
maternal metabolism pathways that control fetal exposure to BPA and BPS [55,60,62]. At
the end of pregnancy, about 6% of the maternal dose of BPA reaches the fetal circulation, i.e.,
a dose related to the bodyweight equivalent to the maternal dose. This value was relative
to the estimated 3.1% in humans using an ex vivo placental perfusion model [59]. Most of
the BPA entering the fetal circulation was rapidly eliminated, mainly through the direct
transfer of the BPA from the fetus to the mother (74%), and, to a lesser extent, through the
fetal metabolism of BPA to BPA-glucuronide. In the fetus, entrapped BPA-glucuronide is
eliminated by hydrolysis into the active form of BPA [62]. Therefore, the fetus has a much higher and sustained exposure to BPA metabolites.

It was striking that the fraction of the maternal BPS dose transferred from the mother to the fetus (0.40%) was about ten times lower than that of BPA [62]. This result is in agreement with the 10 times higher transfer efficiency of BPA from the mother to the fetus compared to that of BPS, as demonstrated in the ex vivo placental perfusion model [60]. Once in the fetal compartment, the percentage of the fetal dose of BPS entering the fetal blood eliminated by placental transfer was 26% compared to 74% for BPA. About half of the remaining BPS was metabolized to BPS-glucuronide by the fetus. The removal of BPS-glucuronide from the fetal compartment required its retro-conversion to bioactive BPS, like that of most bisphenol-glucuronides, due to the limited placental transfer. This toxicokinetic model predicted that, despite a lower materno-fetal passage of BPS compared to BPA, the higher persistence of BPS in the fetal compartment leads to expected BPS concentrations in fetal plasma of the same order of magnitude as that of BPA [63]. In agreement with these findings, the comparison of the fetal disposition of BPA, BPS, and BPF in the same pregnant ewe showed that the ratio of total feto-maternal concentrations of BPS was lower than those of BPF and BPA after a single subcutaneous administration of BPS or a mixture of BPA, BPS, and BPF [64].

To our knowledge, the placental transfer rate of TCS has not been studied using an ex vivo placental perfusion model or a pregnant sheep model. Concerning parabens, using a dual-recirculation placental perfusion model on term human placentae, Andersen et al. showed that the four parabens, MeP, EtP, PrP, and n-BuP, and their metabolite PHBA, just like BPA, are rapidly transported across the placental barrier. This implies potential fetal exposure [65].

Phthalate diesters are rapidly hydrolyzed to phthalate monoesters after maternal in- gestion, inhalation, or dermal exposure in humans. In utero exposure to phthalates has been demonstrated in laboratory animals by measuring the concentrations of phthalate diesters or their metabolites in amniotic fluid [66], fetal plasma [66], and embryonic tissues [67]. In pregnant rats, the materno-fetal transfer of ¹⁴C-di(2-ethylhexyl)phthalate (DEHP) and ¹⁴C-diethyl phthalate (DEP) has been evaluated. This study showed that both phthalates and/or their metabolites were present in the placenta, amniotic fluid, and fetus [68]. Addi-tionally, the pharmacokinetics of DBP in this animal model indicated that MBP was the major metabolite in the maternal and fetal plasma. This suggests that the active metabolite of DBP may cross the placenta in the late pregnancy. In amniotic fluid, the major metabolite was initially MBP, but by 24 h after administration, it was MBP-glucuronide [66]. Other studies confirmed that MBP was rapidly transferred to the embryonic tissues and that fetal plasma MBP-glucuronide concentrations were higher than those of the mother [67,69]. After the oral administration of DEHP to pregnant rats, unconjugated MEHP was the predominant metabolite measured in the amniotic fluid [70]. The transplacental transfer of MMP and MEHP, corresponding to the metabolites of DMP and DEHP, respectively, was evaluated in a dual-recirculation placental perfusion model of human placenta. This evaluation showed that MMP can cross the placenta by slow transfer, whereas no placental transfer was evidenced for MEHP. Moreover, MEHP, MBP, and MEP were found in fetal perfusate, corresponding to umbilical cord blood, whereas MMP, MEHHP, MBzP, and MiNP were undetectable. MEP, MBP, and MEHP were detected in placental tissue [71,72].

Altogether, these data demonstrated that the PPPs and their metabolites can cross the placenta, reach the fetal bloodstream, and contribute to the fetal exposome. Due to the short half-life of phthalate metabolites and the variation in the sensibility of the assay, the spot blood materno-fetal concentrations in term are difficult to interpret and cannot allow for the establishment of a relationship between the maternal and fetal concentrations, as shown for BPA [73,74]. Moreover, in the case of phthalates, it is not recommended to measure phthalates in the blood, since there are enzymes that can transform the parent into metabolites after blood collection, leading to false estimation.
## 5. Effects of PPPs on Human Placenta from Epidemiological Data and In Vitro Models and on Fetoplacental Growth from Animal Models

Only a few studies have looked at the association between PPPs and the markers of placental development. These are presented in the following paragraphs.

### 5.1. Epidemiological Data (Table 1)

Of the two studies investigating BPA exposure, one reported sex-specific associations (decreased female placental weight and increased placental weight in males, N = 232/220 male/female) [75], while the other, looking only at male births, showed no association with the placental weight nor with the placental-to-birthweight ratio, a marker of placental efficiency (N = 473) [36]. The single study looking at BPS did not report an association. This could be the result of the small sample size (N = 91) and the low frequency of detection of BPS in the urine (27%), thus limiting the power of the study [76]. Both studies on TCS reported negative associations with the placental weight; however, only one study reported it in male births [36], while the other study only reported it in female births [76]. Of the three studies investigating parabens (individual compounds or the molar sum), two studies reported a positive association with the placental weight in males at birth, either with n-BuP alone [76] or with the molar sum of the four parabens (including n-BuP) [36]. A third study evaluating paraben concentrations in placental tissues instead of maternal urine found opposite results (decreased placental weight with increased paraben placental concentrations) [77]. Of the two studies evaluating BP-3, only one study reported a positive association in male births [36], but the other study, perhaps because of its relatively small sample size (N = 91), did not report a positive association [76].

Regarding phthalates, a large cohort study (N = 2725 mother-child pairs from China) reported that, out of seven metabolites (MMP, MEP, MBP, MBzP, MEHP, MEOHP, and MEHHP), five (MMP, MBP, MEOHP, MEHHP, and MEHP) were associated with placental growth measurements, including the placental thickness, breadth, and length [78]. Most of these associations were positive, suggesting that prenatal exposure to phthalates can lead to a thicker and more circular placenta [78]. This cohort study reported stronger associations among male fetuses than in females. Moreover, the same cohort reported associations between all metabolites (N = 6: MMP, MEP, MBP, MEHP, MEOHP, and MEHHP), either individually or studied as the molar sum, and the placental weight [79]. The associations were also generally positive, particularly when exposure occurred late in pregnancy (the second and third trimesters). This cohort study also explored the effects of mixing, and although most phthalates were associated with placental outcomes when taken individually, no mixing effect was detected.

A few other studies with smaller sample sizes (ranging from 132 to 488) have also reported associations between certain phthalate metabolites and placental weight or the placental-to-birthweight ratio (PFR) [36,75,80]; however, the incriminated metabolites often differed from one study to another. Casas et al. reported a sex-specific association for MBzP that was positively associated with placental weight in boys and negatively associated in girls [75]. No association was reported for this metabolite in the other studies. Philippat et al. reported a negative association between cx-MiNP and MCOP and the placental weight or PFR in male fetuses [36], whereas this association was not reported in another study with a smaller sample size (N = 132, no stratification for child sex), which instead reported a decreased placental weight with prenatal exposure to MEP [80]. Interestingly, this study also reported a negative association between the preconception paternal exposure to DEHP and placental weight, and between the preconception maternal exposure to MEP and the birthweight-to-placental-weight ratio, providing preliminary results indicating the effect of the preconception exposure on placental development. Finally, a study evaluating phthalate metabolites in cord blood reported no association with the placental weight and volume [81].

Using the French mother-child cohort SEPAGES, 4 parabens, 2 bisphenols, 13 phthalate metabolites, and 2 non-phthalate plasticizer metabolites of DiNCH were measured in pools of repeated urine samples collected during the second and third trimesters of
pregnancy. The results suggested negative associations between individual phthalate
metabolites and the placental weight (MBzP and ΣDiNP), placental efficiency (MBzP, MBP,
and ΣDiNP), and placental vascular resistance (MBzP, MBP, and ΣDiNP), indicating the
adverse impacts of phthalate exposure on placental health [82]. Furthermore, using the
same cohort, monoisobutyl phthalate (MiBP) and mono-n-butyl phthalate (MBP) were
positively associated with most fetal growth parameters measured in the second trimester.
Then, in the third trimester, MBP was further positively associated with the biparietal
diameter and femur length [83].
<table><caption>**Table 1.** Effects of phenols, parabens, and phthalates on the placenta using epidemiological data.</caption><thead><tr><th>Population</th><th>Placental Outcomes</th><th>Studied Compounds</th><th>Sample Type, Number, and Timing</th><th>Main Results</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">N = 2723<br>women-child pairs<br>Recruitment: May<br>2013–September<br>2014<br>Country: China</td><td rowspan="10">Placental weight<br>(calculated), PFR,<br>chorionic plate area,<br>disk eccentricity</td><td rowspan="10">MMP, MEP, MBP,<br>ΣLMWP, MEHP,<br>MEOHP, MEHHP,<br>ΣDEHP</td><td rowspan="10">One spot urine<br>sample per trimester</td><td>GM of three trimesters/T1/T2/T3<br>Placental weight:<br>MMP ↑*/↓*/↑*/↑*<br>MEP ↓*/↑*/↓*/↓*<br>MBP ↑*/↑*/↑*/↑<br>ΣLMWP ↑*/↑*/↑*/↑*<br>MEHP ↑*/↓*/↑*/↑*<br>MEOHP ↑*/↑*/↑*/↑*<br>MEHHP ↑*/↓*/↑*/↑*<br>ΣDEHP ↑*/↓*/↑*/↑*<br>Boys/girls stratification (on GM of three trimesters)<br>Boys/girls<br>Placental weight:<br>MMP ↑*/↑*<br>MEP ↑*/↓*<br>MBP ↑*/↑*<br>LMWP ↑*/↑*<br>MEHP ↑*/↑*<br>MEOHP ↓*/↑*<br>MEHHP ↑*/↑*<br>DEHP ↑*/↑*</td><td>PFR:<br>MMP ↑*/↓*/↑*/↑<br>MEP ↓/↑/↓/↑<br>MBP ↑*/↑/↑*/↑*<br>ΣLMWP ↑*/↑/↑*/↑*<br>MEHP ↑*/↓*/↓/↑*<br>MEOHP ↑/↑*/↑*/↑*<br>MEHHP ↑*/↓*/↑*/↑*<br>ΣDEHP ↑*/↓*/↑*/↑*<br>PFR:<br>MMP ↑*/↑*<br>MEP ↓*/↑*<br>MBP ↑*/↑*<br>LMWP ↑*/↑*<br>MEHP ↑*/↑*<br>MEOHP ↑*/↑*<br>MEHHP ↑*/↑*<br>DEHP ↑*/↑*</td><td rowspan="10">[79]</td></tr><tr><td>T1/T2/T3<br>Only chemicals with at least one significant results are shown<br>Overall<br>Placental breadth:<br>MBP ↑*/↓/↑*<br>LMWP ↑*/↓/↓<br>Placental thickness:<br>MMP ↓/↑*/↓<br>MBP ↓/↑*/↑*<br>MEOHP ↑/↑*/↑<br>MEHHP ↓/↑*/↑<br>LMWP ↓/↑*/↑<br>HMWP ↓/↑*/↑</td><td>Boys<br>Placental breadth:<br>MBP ↑*/↓/↑*<br>LMWP ↑*/↓/↓<br>Placental thickness:<br>MMP ↓/↑*/↑*<br>MBP ↑*/↑/↑*<br>MEOHP ↓/↑*/↑*<br>MEHHP ↓/↑*/↑*<br>LMWP ↓/↑*/↑*<br>HMWP ↓/↑*/↑*<br>MEHP ↓/↑*/↑*</td><td>Girls<br>Placental breadth:MMP<br>↑*/↑/↑MBP<br>↑*/↑/↓LMWP<br>↑*/↑/↑Placental thickness:MBP<br>↑*/↑/↑*MEOHP<br>↑*/↑/↓</td></tr><tr><td>N = 2725 (1399 boys and 1326 girls)<br>mother-child pairs<br>Recruitment: May<br>2013 and September<br>2014<br>Country: China</td><td>Placental length,<br>thickness, breadth<br>and surface area</td><td>MMP, MEP, MBzP,<br>MEHP, MEHHP,<br>MEOHP</td><td>One maternal spot<br>urine sample per<br>trimester</td><td>T1/T2/T3<br>Only chemicals with at least one significant results are shown<br>Overall<br>Placental breadth:<br>MBP ↑*/↓/↑*<br>LMWP ↑*/↓/↓<br>Placental thickness:<br>MMP ↓/↑*/↓<br>MBP ↓/↑*/↑*<br>MEOHP ↑/↑*/↑<br>MEHHP ↓/↑*/↑<br>LMWP ↓/↑*/↑<br>HMWP ↓/↑*/↑</td><td>Boys<br>Placental breadth:<br>MBP ↑*/↓/↑*<br>LMWP ↑*/↓/↓<br>Placental thickness:<br>MMP ↓/↑*/↑*<br>MBP ↑*/↑/↑*<br>MEOHP ↓/↑*/↑*<br>MEHHP ↓/↑*/↑*<br>LMWP ↓/↑*/↑*<br>HMWP ↓/↑*/↑*<br>MEHP ↓/↑*/↑*</td><td>Girls<br>Placental breadth:MMP<br>↑*/↑/↑MBP<br>↑*/↑/↓LMWP<br>↑*/↑/↑Placental thickness:MBP<br>↑*/↑/↑*MEOHP<br>↑*/↑/↓</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Population</th><th>Placental Outcomes</th><th>Studied Compounds</th><th>Sample Type, Number, and Timing</th><th>Main Results</th><th>References</th></tr></thead><tbody><tr><td>N = 473<br>mother-infant (only boys) pairs<br>Recruitment: April 2003–March 2006<br>Country: France</td><td>Placental weight, PFR</td><td>MCPP, MBP, MiBP, MBzP, MEP, cx-MiNP, MCOP, MEHP, MEHHP, MEOHP, MECPP, 2,4-DCP, 2,5-DCP, n-BuP, BP-3, BPA, EtP, MeP, PrP, TCS</td><td>One maternal spot urine sample collected between 23 and 29 gestational weeks</td><td>Penalized effects:<br>BP3 ↑ Placental weight<br>TCS ↓ Placental weight<br>ΣPB ↑ Placental weight<br>MCOP ↓ PFR<br>cx-MiNP ↓ Placental weight/↓ PFR</td><td>Unpenalized effects:<br>BP3 ↑ Birthweight/↑ Placental weight<br>TCS ↓ Placental weight<br>ΣPB ↑ Placental weight<br>cx-MiNP ↓ Placental weight/↓ PFR<br>MCOP ↓ PFR</td><td>[36]</td></tr><tr><td>132 mother-child pairs Subfertile population<br>Recruitment:<br>2005–2006<br>Country: USA</td><td>Placental weight assessed at birth</td><td>MEP, MBP, MiBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, MCPP, MCPP, MCOP, cx-MiNP</td><td>Paternal and maternal spot urine samples collected:<br>(1) at recruitment (mother and father);<br>(2) at each fertility treatment cycle (two spots for the mother and one for the father);<br>(3) one spot per trimester of the pregnancy (mother)</td><td>[p-value]<br>MEP ↑ Placental weight [0.80]/↓ FPR [0.02]</td><td></td><td>[80]</td></tr><tr><td>N = 207<br>women-child pairs, recruited between October 2011 and September 2012<br>Chongqing (Southwest China)<br>Recruitment:<br>October 2011–September 2012<br>Country: China</td><td>Placental weight at birth</td><td>DMP, DEP, DMEP, DBP, DEEP, DiBP, DPP, BMPP, DBEP, DCHP, DnHP, BBP, DEHP, DnOP, DNP</td><td>Cord blood sample</td><td>No effect on placental weight or volume</td><td></td><td>[81]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Population</th><th>Placental Outcomes</th><th>Studied Compounds</th><th>Sample Type, Number, and Timing</th><th>Main Results</th><th>References</th></tr></thead><tbody><tr><td>N = 142<br>mother-newborn pairs<br>Recruitment:<br>December<br>2014–December 2016<br>Country: Belgium</td><td>Placental weight at birth</td><td>MeP, EtP, PrP, n-BuP</td><td>Placenta sample</td><td>[p-value]<br>In both sexes:<br>EtP ↓ Placental weight<br>[0.11]<br>ΣParabens ↓ Placental weight [0.08]</td><td>In girls:EtP ↓<br>Placental weight<br>[0.02] Σ Parabens ↓<br>Placental weight<br>[0.03]</td></tr><tr><td>N = 657<br>mother-child pairs<br>Recruitment:<br>2004–2006<br>Country: Spain</td><td>Placental weight at birth</td><td>BPA, DEHP (Σ of MEHP, MEHHP, MEOHP, MECPP), MBzP, LMWP (Σ of MEP, MiBP, MBP</td><td>Two spot urine samples collected at 12 ± 1.7 and 32 ± 1.4 weeks of gestation</td><td>In boys:<br>MBZP ↑ Placental weight</td><td>In girls:<br>MBZP ↓ Placental weight</td></tr><tr><td>N = 130<br>mother-child pairs<br>Recruitment:<br>2006–2008<br>Country: USA</td><td>Placental weight, fetal weight, head circumference, abdominal circumference, and femur length</td><td>2,4-DCP, 2,5-DCP, BP-3, n-BuP, EtP, MeP, PrP, TCS, BPS, TRCB</td><td>One spot urine sample</td><td>Inverse associations were observed between average 2,4- and 2,5-DCP concentrations and birth weight z-scores in males<br>Inverse associations between average TCS exposure over pregnancy and estimated fetal weight combined with birth weight in repeated measures models in males</td><td>[76]</td></tr><tr><td>SEPAGES cohort<br>N = 484 pregnant women<br>Recruitment:<br>2014–2017<br>Country: France</td><td>Placental weight, PFR, placental thickness, and placental vascular resistance</td><td>4 parabens, 2 bisphenols, triclosan, benzophenone-3, 13 phthalate metabolites, and 2 non-phthalate plasticizer metabolites</td><td>Repeated urine samples collected during the second and third trimesters of pregnancy</td><td>Several phthalate metabolites were negatively associated with placental outcomes<br>MBzP: ↓ placental weight and PFR (T2 and T3 trimesters)<br>Negative associations with placental weight and PFR for males only (T3 trimester)<br>MBP: ↓ placental vascular resistance (T2 and T3 trimesters)<br>Σ DiNP: ↓ placental vascular resistance (T3 trimester), ↓ placental weight and PFR in males only<br>No associations between phenols and placental outcomes</td><td>[82]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Population</th><th>Placental Outcomes</th><th>Studied Compounds</th><th>Sample Type, Number, and Timing</th><th>Main Results</th><th>References</th></tr></thead><tbody><tr><td>SEPAGES cohort<br/>N = 484 pregnant<br/>women Recruitment:<br/>2014–2017<br/>Country: France</td><td>Fetal biparietal diameter, femur length, head and abdominal circumferences measured by ultrasound, newborn weight, length, and head circumference measured at birth</td><td>13 phthalates, and 1,2-cyclohexane dicarboxylic acid and diisobutyl ester (DiNCH) metabolite</td><td>Repeated urine samples collected during the second and third trimesters of pregnancy</td><td>MiBP: ↑ biparietal diameter, and head and abdominal circumferences at T2 trimester<br/>MBP: ↑ estimate fetal weight, and head and abdominal circumferences in males<br/>Mixture of phthalate/DiNCH metabolites: ↑estimate fetal weight (T3 trimester)</td><td>[83]</td></tr></tbody></table>

BBP: benzyl butyl phthalate; BMPP: bis (4-methyl-2-pentyl) phthalate; n-BuP: n-butylparaben; BPA: bisphenol A; BP-3: benzophenone-3; DBP: di-n-butyl phthalate; DBEP: bis (2-nbutoxyethyl) phthalate; DCHP: dicyclohexyl phthalate; 2,4-DCP: 2,4-dichlorophenol; 2,5-DCP: 5-dichlorophenol; DEEP: bis (2-ethoxyethyl) phthalate; DEHP: di-(2-ethylhexyl) phthalate; DEP: diethyl phthalate; DnHP: di-n-hexyl phthalate; DiBP: diisobutyl phthalate; DiNCH: di-iso-nonyl-cyclohexane-1,2-dicarboxylate; DMP: di-methyl phthalate; DMEP: bis (2-methoxyethyl) phthalate; DnOP: di-n-octyl phthalate; DNP: dinonyl phthalate; DPP: di-amyl phthalate; EtP: ethylparaben; HMW: high-molecular-weight phthalates; LMW: low-molecular-weight phthalates; MBP: mono-n-butyl phthalate; MBzP: monobenzyl phthalate; cx-MiNP: mono-carboxy-iso-nonyl phthalate; MCOP: mono-carboxy-iso-octyl phthalate; MCPP: mono-3-carboxypropyl phthalate; MECPP: mono-2-ethyl-5-carboxypentyl terephthalate; MEHHP: mono-2-ethyl-5-hydroxyhexyl) phthalate; MEHP: mono-2-ethyl-hexyl) phthalate; MEOHP: mono(2-ethyl-5-oxohexyl) phthalate; MEP: mono-ethyl phthalate; MiBP: mono-iso-butyl phthalate; MMP: mono-methyl phthalate; MeP: methylparaben; PFR: placental-to-birthweight ratio; PrP: propylparaben; TCS: triclosan; TRCB: triclocarban. * indicates statistically significant results, ↓ indicates a decrease, ↑ indicates an increase.
## 5.2. Effects of PPPs on Human Placental Methylation

DNA methylation is the best characterized and most stable epigenetic modification, influencing the chromatin structure and the gene expression. This epigenetic mark typically involves the methylation of the fifth carbon position at a cytosine residue within a CpG dinucleotide (CpG), resulting in 5-methylcytosine (5mC). Recent studies have provided concrete evidence of a link between DNA methylation alterations and certain environmental exposures. Given the evidence that PPPs can cross the placenta, studies have explored the associations between PPP maternal exposure and the evolution of DNA methylation profiles in the placenta. The first studies focused on the locus of imprinted genes, *IGF2* and *H19* [85]. This locus displays an allele-specific expression (the expression of *H19* from the maternal allele and of *IGF2* from the paternal allele). The *IGF2* gene and lncRNA *H19* are together important for embryogenesis and fetoplacental development. The allele-specific methylation is involved in the allele-specific control of expression. Using placental samples collected at delivery from 196 healthy newborns, genomic DNA was used to analyze three differentially methylated regions, IGF2DMR0, IGFDMR2, and H19DMR, considered as imprinting the center region (*H19 ICR*). An increase in the level of methylation of the imprinting center region (*H19 ICR*) was associated with the sum of phthalate metabolites and low-molecular-weight metabolites. An increase in high-molecular-weight phthalate metabolites and DHEP exposure was associated with a deviation of the allele-specific expression of *H19* (10%) only in male placenta. This suggests a potentially sexually dimorphic response. Conversely, the study showed that the sum of phthalate metabolites and high-molecular-weight metabolites was associated with a decrease in *IGF2DMR0*, without significant modification of the methylation level of *IGF2DMR*. In this study, the effects of the exposure to phenols were also investigated, but no significant association was found [85]. Quite different results were obtained by Zhao et al. [86] from the placental samples of 101 healthy newborns and 80 neonates with fetal growth retardation. The placental methylation of *IGF2DMR* was significantly inversely associated with MEHHP and MEOHP concentrations. Associations were much more evident in neonates with fetal growth restriction than in healthy neonates. Using the same mother-newborn cohort, measuring the phthalate exposure during the third trimester, the same authors demonstrated that the placental methylation of LINE-1 was negatively correlated with phthalate exposure levels. This outcome was significantly altered in cases of fetal growth restriction compared to healthy births. Because the LINE-1 repetitive element exhibits a wide distribution in the human genome, it has been frequently used as a surrogate marker of global methylation analysis. The modest hypomethylation of the LINE-1 repetitive element observed after exposure to phthalates could contribute to genome instability and increase the risk of chronic disease later in life [87].

A genome-wide DNA methylation analysis using the Illumina Infinium Human Methylation 450K or 850K Bead Chip was also performed. Although these differently methylated CpG sites constitute a small proportion of the total number of CpG sites in the genome (28 million), they represent a wide distribution of sites [88]. Grindler et al. used this tool to analyze genome-wide DNA methylation marks in placental villi in the first trimester from 16 women with high or low phthalate exposures [89]. They reported 2214 differentially methylated cytosines (DMCs) targeting 1460 unique genes. Taking into account the proximity of the DMCs, 282 differentially methylated regions (DMRs) were identified and found to be associated with 245 unique genes. Interestingly, using the same placental samples for the transcriptomic analysis, the authors identified 163 differentially expressed genes (DEGs) among the targeted genes by DMCs: 124 were downregulated and 39 were upregulated between women with a high-level exposure of phthalates compared to those with a low-level exposure. The authors focused on one particular pathway, erbB signaling. EGFR exhibited placental hypermethylation and decreased expression in women, with a high total phthalate exposure, suggesting that this gene might be the specific target of endocrine disruption by phthalates. Alterations in the erbB signaling methylome have previously been observed in adverse obstetric outcomes such as pre-eclampsia and IUGR.
In two recently published studies [90,91] based on a larger cohort of mother-infant pairs (the French EDEN cohort, N = 202), placental DNA methylation at birth was measured using the Illumina Infinium Human Methylation 450K Bead Chip in combination with 12 urinary phthalate metabolites [91] and 9 phenols [90]. In these studies, only placental samples from boys were assessed for DNA methylation. Since biological functions are in general more strongly associated with genomic regions than single CpGs [92], these two studies aimed to identify DMRs associated with phenol and phthalate concentrations. Most of the associations observed were positive (i.e., increased methylation with increased exposure during pregnancy). TCS exposure was positively associated with 37 DMRs compared to less than 6 for the other phenols and phthalate metabolites studied. Out of the 37 DMRs associated with TCS, 6 encompassed imprinted genes, which represented a significant enrichment. Using the SEPAGES cohort (N = 387 mother-child couples), an exploratory analysis on individual CpGs and DMRs, as well as 20 previously identified CpGs, was performed. In the sex-stratified analysis, 114 individual CpGs (68 in males and 46 in females) were differentially methylated, encompassing 74 genes (36 for males and 38 for females). There was a total of 82 significant DMRs (40 for females and 42 for males). For most DMRs, DNA methylation levels increased with a higher exposure, except for some parabens and DiNP metabolites in males and females, BPA in males, and BPS and DiNCH metabolites in females, which were negatively associated with DNA methylation in most DMRs. Some differentially methylated CpGs and DMRs encompassed imprinted genes, whereas other CpGs were linked to adiposity, the lipid and glucose metabolism, and cardiovascular function [93]. Recently, maternal concentrations of monocarboxyisونonyl phthalate (cx-MiNP), mono-3-carboxypropyl phthalate (MCPP), and BPA were associated with altered methylation in the placenta (the maternal or fetal side). Among them, MCPP was associated with differential CpG methylation [94].

Further studies are needed to better control the analysis conditions (the PPP exposure time, placental sampling, pregnancy stages, genotyping, etc.) and to acquire more causal information between the methylation landscape, gene expression, and placental dysfunction induced by EDCs.

### 5.3. Effects of PPPs on Human Placental Function Using In Vitro Models

In humans, trophoblasts differentiate into extravillous cytotrophoblasts that proliferate, invade the uterine wall, and are important for remodeling the endometrial vasculature and syncytiotrophoblasts. The latter cell type resulting from the fusion of cytotrophoblasts is responsible for the endocrine function of the placenta that produces steroid (progesterone) and polypeptide hormones (human chorionic gonadotropin, namely, hCG, and leptin). To study the effects of PPPs on the human placentation, in vitro studies could be performed using (i) primary cell cultures of trophoblasts from first- or third-trimester placentae [95–99], (ii) placental explants [100,101], and (iii) immortalized trophoblast cell lines such as BeWo, JEG-3, and HTR-8/SVneo [102–105]. The BeWo cell line can fuse and express differentiation markers [106–109], whereas JEG-3 cells can produce placenta-specific hormones, but without cell fusion [110,111]. The HTR-8/SVneo cell line is used as a model of extravillous trophoblasts [112–114].

Using human placenta, the exposure of first-trimester placental explants to BPA (0.5–1 nM) stimulated hCG secretion [115,116] and increased cell apoptosis [116]. The same effects were observed when term primary trophoblasts were exposed to BPA (8 nM–8 mM) [117,118]. Unlike the first-trimester explants, Zou et al. did not observe any change in the level of hCG secretion at any BPA concentration tested in term villous explants. Interestingly, they showed that BPA significantly increased ESRRG expression (estrogen-related receptor gamma) in the female placenta following exposure to 1 µM BPA for 24 h. This suggests that exposure to a low dose of BPA could alter gene expression in human placenta in a sex-specific manner [100]. Moreover, in primary trophoblast cells, data have shown that BPA is also able to (i) induce the expression of corticotrophin-releasing hormone (CRH) and the expression of two enzymes specifically involved in hormone
production (aromatase and 11-β-hydroxysteroid dehydrogenase 2, known as 11β-HSD2), and, conversely, (ii) to reduce the expression of leptin obtained in human term primary trophoblasts [117]. Concerning BPS, this phenol has been shown to block epidermal growth factor (EGF)-mediated trophoblast fusion in term primary trophoblasts [118].

In the first-trimester trophoblast progenitor cells and second-trimester primary trophoblasts, a mixture of four phthalates (MBP, 0.2 µM; MBzP, 3 µM; MEHP, 0.7 µM; MEP, 1.5 µM) decreased hCG expression regardless of the fetal sex. However, the same mixture of phthalates decreased the placental peroxisome proliferator-activated receptors-γ (PPARγ) expression in male cells and increased female cells. This latter finding provides evidence for sex-specific responses to phthalates in human trophoblasts [98]. In primary-term trophoblasts, a recent study demonstrated that, at low concentrations (0.1–1 µM), MEHP decreases the lipid content, hCG secretion, and cell fusion. These effects appear to be associated with a lower activity of (PPARγ). This receptor is a key transcription factor involved in the control of trophoblast differentiation and the lipid metabolism. In contrast, a high concentration (10 µM) of MEHP increases the lipid content, cell fusion, and PPARγ activity, but decreases the hCG secretion. These results highlighted the notion of a non-monotonic dose-response, particularly at environmentally relevant levels of MEHP exposure [97]. Moreover, at this stage, high MEHP doses (100–150 µM) increase the expressions of CRH and cyclooxygenase 2 (COX2) via the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. These results suggest that MEHP could prematurely induce the expression of labor-promoting genes and lead to preterm birth [119].

Using the BeWo cell line, exposure to low doses of BPA (3 nM and 1 µM) has been shown to significantly increase cell proliferation [120,121], whereas exposure to a high dose of BPA (1000 µM) decreased the proliferation rate of cells [121,122]. However, in other studies, the exposure of BeWo cells to BPA did not affect the proliferation or metabolic activity [123]. BPA exposure has also been shown to induce cell cycle inhibition, increase DNA damage [122], and reduce the viability of BeWo cells at concentrations ranging from 100 µM to 1 mM [116]. Regarding β-hCG secretion, a significant decrease was observed at a 30 µM BPA exposure, while the secretion dropped dramatically from 60 to 125 µM BPA [116]. BPA is able to promote the syncytialization in BeWo cells at doses of 1 and 50 mM, known in humans to increase the expression of several syncytin proteins [123], as well as to reduce the invasive capacity of the same cell line at doses as low as 100 µM [124] due to the increased expression of E-cadherin. BPA was also able to reduce the activation of the antioxidant response element [121] and increase the levels of two anti-apoptotic proteins, i.e., B cell lymphoma 2 (Bcl-2) and heat shock protein 70 (Hsp70), and decrease the levels of hypoxia-inducible factor 1-α (HIF-1α) under stress conditions, demonstrating that BPA inhibits trophoblast cell death under cellular stress conditions [121]. Moreover, BPA might exert its various effects mainly by regulating cell-extracellular matrix interactions via transforming growth factor-β (TGF-β) signaling [125]. Cytotoxic effects via cell cycle arrest and apoptotic pathways were also observed in BeWo cells exposed to EtP [126].

As JEG-3 cannot fuse, there are few studies on this cell line. It has only been shown that, at doses of 5–20 µg/mL, BPF induces chromatin condensation [102–104]. Concerning MEHP exposure, the incubation of JEG-3 cells (10 mM for 72 h) exhibited a marked change in the lipid profile, especially in triacylglycerols and glycerophospholipids, with a marked accumulation of triacylglycerols. This once again underlines the detrimental effects of MEHP on the lipidome of human placental cells [127].

Using the HTR-8/SVneo cell line, a high dose of TCS exposure suppressed the viability and migration of HTR-8/SVneo cells but increased the H19 imprinted gene expression and insulin-like growth factor 2 (IGF2) protein secretion in a dose-specific manner. This suggests an altered trophoblast function, gene expression, and DNA methylation status [112]. Regarding the exposure to the paraben family, n-BuP has also been shown to inhibit cell proliferation and induce apoptosis, which could disrupt early placental development [128]. Regarding phthalate exposure, high concentrations of MEHP for 24 h significantly inhibited cell proliferation and viability, promoted apoptosis, and inhibited the cell cycle [129].
In addition, DEHP acts on placental trophoblast cells and inhibits its internalization of transthyretin (thyroxine transport protein), downregulates transthyretin expression, and affects the expression of deiodinase 2, deiodinase 3, and thyroid hormone receptor in the HTR-8/SVneo cell line, as well as in JEG-3 cells [130]. These data suggest that DEHP disrupts the placental transport of thyroid hormones, which could be very detrimental to fetal development, especially at early stages.

In conclusion, depending on the phenol concentrations, BPA may or may not affect placental cell proliferation and hormone production. Parabens, on the other hand, induce cytotoxic effects and apoptosis. Depending on their concentration, phthalates affect the lipid content, hCG secretion, and cell fusion by PPARγ. All of these cell models are useful for testing several concentrations of PPPs and deciphering their mechanism of action. However, these approaches do not allow us to take into account the sub-chronic effects, nor the complexity of the different cell types of the placenta. It is, therefore, essential to supplement the in vitro data with data from in vivo models.

### 5.4. Effects of Phenols and Parabens on Fetoplacental and Neonatal Outcomes Using Animal Models (Table 2)

As a very detailed review of phthalate exposures and placental health has been published by Seymore et al. in 2022 [131], an update has been made over the last two years. Exposure to 20 µg/kg BW/day of DiNP on days 1–7 of gestation in mice led to fetal loss towards the end of gestation. This miscarriage was due to the impaired differentiation of stromal cells associated with a defect in the angiogenic network in the decidua, but also to a disorganization of the placental layers. Interestingly, the labyrinthine area involved in nutrient exchanges was reduced [132].

In rats, oral exposure to BPA (0.0025, 0.025, and 0.250 mg/kg/day) 30 days before pregnancy and during the first 20 days of pregnancy reduced the placental weight at low and high BPA concentrations, while the fetal weight was increased only at low concentrations [133]. In contrast, gestational exposure to BPA in ewes reduced the placental efficiency and fetal weight at mid-gestation [134] and at 110 days [135]. Similarly, the exposure of pregnant mice to BPA at 0.050 mg/kg/day by oral gavage from day 1 to 7 of gestation induced fetal growth restriction at 14 days of gestation, which was preceded by the insufficient remodeling of uterine spiral arteries [136]. In addition, a significant decrease in the number of embryos and the weight of the uterus on days 10 and 12 was reported after the subcutaneous administration of BPA (10 mg/kg/day) to pregnant mice from day 1 of gestation to day 7 [137].

The study of the placental structure was analyzed after exposure to BPA. Tait et al. exposed pregnant mice to BPA at 0.5 mg/kg bw (corresponding to the lowest observed adverse effect level, LOAEL) or 50 mg/kg bw (corresponding to a high range of human BPA exposure) from GD1 to GD11. They demonstrated that a high concentration of BPA induces a significant degeneration and necrosis of giant cells, associated with an increase in vacuolation in the junctional zone and a reduction in the spongiotrophoblast layer at GD12 [138]. Degenerative changes were also found in the trophoblastic giant cells and spongiotrophoblast layer in the pregnant mice exposed to BPA (10 mg/kg/day, from day 1 to day 7) by subcutaneous administration [137]. A low concentration of BPA (0.5 mg/kg bw) induced the development and branching of blood vessels, while a high concentration of BPA inhibited them. Maternal vessels were narrower in placentae exposed to a low concentration of BPA, whereas embryonic and maternal vessels were irregularly dilated in the labyrinthine area of placentae exposed to a high concentration [138]. Doses of 10 and 40 mg/kg BPA (mice exposed from GD0.5 to GD5.5) also decreased the proportion of the labyrinthine and spongiotrophoblast layers, which are associated with large vacuoles [139]. An increase in the retention of smooth muscle cells and a decrease in vascular surfaces at the level of the junctional zone have been reported in mice exposed to BPA at a concentration of 0 to 400 µM in drinking water from GD7 to GD17 [140]. Altogether, these data indicate
that BPA has an effect on the placental structure, which explains the disturbance to the fetal weight.

BPA-related gene expression changes have also been studied. Since BPA is thought to bind to estrogen receptors and regulate the expression of estrogen-responsive genes, BPA may alter the expression of other placental nuclear receptors such as the retinoid Z receptor (RORγ), progesterone receptor (PR), ERβ, LXRα, germ cell nuclear factor (GCNF), steroidogenic factor-1 (SF-1), and so on. The oral administration of BPA at a dose of 0.002 mg/kg/day from GD6.5 to GD17.5 induced the sex-specific placental differences in these genes at GD18.5 [141].

Post-translational modifications of histones, DNA methylation, and the expression of imprinted genes have been studied in mouse placenta concerning BPA exposure. Daily exposure of pregnant mice to BPA between GD8.5 and GD12.5 affected the expression of imprinted genes. At GD13.5, *Rtl1* displayed a slight disturbance of allele-specific expression [142]. A longer maternal exposure time to BPA (two weeks before mating and during gestation) significantly disrupted the expression of imprinted genes in the placenta at GD12.5, such as *Snrpnl* and *Kcnq1*, and altered the methylation levels of differentially methylated regions (DMRs), including the *Snrpnl* imprinting control region (ICR) and *Igf2* DMR1 [143]. Moreover, exposure significantly reduced the genome-wide methylation levels in the placenta, but not in the embryo. The expression of several small RNAs was also found to be disturbed [144]. These epigenetic defects were associated with abnormal placental development [143]. The exposure of pregnant mice from GD7 to GD17 to BPA at a concentration of 0 to 400 µM in drinking water disrupted the expression of 10 genes coding the proteins of epigenetic machinery. These genes include enzymes involved in histone methylation and acetylation, protein phosphorylation, and DNA methylation. Among these enzymes, the DNMT1 mRNA/protein was increased in the placenta, while levels of DNMT3a and -3b, hydroxymethyl transferases (TET1, -2, and -3), and 5-hmc were unaffected. Moreover, high levels of 5-mc indicated an elevated level of methylation in the placental tissues of mice exposed to BPA [140].

As previously described, BPA altered the placental structure, leading to impaired placentation. This could result from increased protein levels of matrix metalloproteinase-9 and 2 (MMP-9 and MMP-2) and decreased levels of the tissue inhibitor of metalloproteinases-3 (TIMP-3), as well as integrin-β1 and integrin-α5, in mouse placenta. These effects were observed in particular in the labyrinthine layer in the event of a low exposure to BPA (5, 10, and 40 mg/kg) from GD0.5 to GD5.5 [139]. Ye et al. confirmed that placental abnormalities were associated with altered invasion-related genes, such as increased tissue inhibitors of metalloproteinases, decreased metalloproteinases, and the Wnt family member WJVT2/β-catenin (mice exposed from GD7 to GD17 to BPA added at a concentration of 0–400 µM in drinking water) [140]. BPA affected several placental transporters, such as cation transport channels. For example, in mice placenta at GD17.5, PMCA1 (ATPase, Ca<sup>++</sup> transporting, plasma membrane 1), hephestin (HEPH), CTR1 (solute carrier family 31, member 1 (copper transporter)), and ATP7A (ATPase, Cu<sup>2+</sup> transporting, alpha polypeptide) were disturbed after exposure to BPA (50 mg/kg/day) from GD11.5 to GD16.5. These disturbances were associated with a decrease in serum calcium/copper/iron levels, which could have an impact on fetal development [145]. Exposure to high doses of BPA (400 and 600 mg/kg) from GD17 to GD19 increased CaBP-9k mRNA/protein in the maternal uterus and placenta in late gestation. CaBP-9k is a vitamin D-dependent calcium-binding protein whose gene carries an estrogen response element (ERE) [146,147]. In rats fed a diet containing BPA (0.0025, 0.025, or 0.250 mg/Kg/day) for a month, plus 20 days during pregnancy, the glucose type 1 transporter was upregulated [148]. BPA modulation of cation transport channels, CaBP, and placental nutrient-glucose transfer could explain the changes in the fetal weight.

As exposure to BPA in pregnant mice (2, 20, and 200 mg/kg bodyweight/day from GD13 to GD16, and euthanized at GD17) increased the plasma estrogen, testosterone, and CRH in dams, the regulation of CRH involved in fetal organ development, the glucose
metabolism, and immune response has been investigated in the placenta. The activation of phosphorylated forms of PKC ζ/λ and δ might promote cAMP-responsive element-binding protein (CREB) phosphorylation. This leads to its interaction with a CBP-responsive element (CRE) located in the CRH gene promoter and to an increase in CRH mRNA, which might be the pathway for the signaling of preterm birth [149].

Other mediators differ between mid- and early gestation in terms of protein expression in the BPA-exposed placenta. Exposure to BPA in ewes at the start of gestation increased interleukin 8, the marker of lipid peroxidation, antioxidants, aromatase, 17 alpha-hydroxylase, estrogen receptor 2, IGF-2 receptor and IGF-binding proteins (IGFBPs), and histone deacetylase 1 and 2, and caused a reduction in tumor necrosis factor-alpha and the IGF1 receptor. Whereas, at mid-gestation, BPA exposure reduced angiogenic factor hypoxia-inducible factor 1 alpha, but increased IL1beta, oxidative stress markers, triglyceride, 17-alpha hydroxylase, IGFBP 1, DNA methyltransferase 3 A, and histone deacetylase 1, which could explain the low birth weight [134]. Exposure to BPA from day 40 through day 110 of gestation in ewes (5 mg/kg/d) was responsible for the placental cytotoxicity, including autophagy, apoptosis, endoplasmic reticulum stress, excessive ROS generation, oxidative damage, and mitochondrial dysfunction [135].

BPA is the most studied phenol so far, but other molecules of the same family are also studied, including BPS, TBBPA, and TCS. BPS is an analog of BPA that is reputedly more inert. Daily exposure to BPS from GD30 to 100 in pregnant ewes reduced maternal circulating pregnancy-associated glycoproteins (PAG1 and PSPB) but did not change the placental weight or placental stereology [150]. However, exposure to BPS in mice (0.200 mg/kg bodyweight BPS or BPA 2 weeks before mating and until day 12.5 of gestation) was shown to reduce the ratio of the spongiotrophoblast zone to trophoblast giant cells within the junctional zone, as well as the exposure to BPA. In addition, BPA and BPS altered placental neurotransmitters such as serotonin and dopamine. BPA and BPS reduced placental serotonin (5-HT) concentrations and 5-HT giant cell immunoreactivity, whereas the concentrations of dopamine and 5-hydroxy indole acetic acid, the main metabolite of serotonin, increased, as well as the dopamine immunoreactivity of the giant cells. Due to BPA and BPS exposure, this imbalance associated with a decrease in docosahexaenoic acid and estradiol could affect the placental-brain axis of the mouse fetus. BPS exposure causes placental effects almost identical to those of BPA, which would justify considering BPS as being as dangerous as BPA [151]. Finally, placentae exposed to BPS in pregnant ewes (from day 30-100) showed a low expression of the protein E-cadherin, few binucleate cells, and a high expression of missing-1 protein in glial cells, suggesting that BPS can affect the trophoblast fusogenic signaling pathway and the placental endocrine function [150].

The administration of TCS at doses of 523 and 785 mg/kg/day on GD1 to GD3 has been observed to impair blastocyst implantation in mice [152]. In animal models, TCS was able to bioaccumulate in the placenta, liver, kidney, ovary, adrenal, spleen, and fat, but with high concentrations for the first four tissues [153,154]. Daily oral exposure of pregnant mice with 8 mg/kg of TCS, but not 1 or 4 mg/kg, from GD6 to GD18 resulted in a decreased fetal bodyweight and an increased rate of fetal loss (spontaneous abortions and fetal growth restriction) [155]. Similarly, a short exposure to TCS from GD5.5 to mid-gestation caused a dose-dependent increase in the rate of fetal loss through abortion [156]. Spontaneous abortion has also been reported in pregnant rats exposed to TCS by gavage with 600 mg/kg/d from GD6 to GD20 [154]. A high TCS concentration decreased the placental weight (exposure by gavage at doses of 1, 10, and 100 mg/kg/day from GD5.5 to GD15.5), and the placental structure was characterized by a thrombus, hemorrhage with tissue necrosis, and junctional zone atrophy [156]. Exposure to 8 mg/kg/day from GD6 to GD18 confirmed the reduction in placental weight and labyrinth volume [155]. Taken together, these data demonstrated that TCS is capable of inducing miscarriage and affecting the placental structure. In addition, TCS negatively modulated the activities and expression of placental System A amino acids or glucose transporters in pregnant mice exposed to 8 mg/kg/day. These negatively modulated activities were associated with a decrease in the
sodium-coupled neutral amino acid transporters (SNAT1/SNAT4) and glucose transporter 1 (GLUT-1) mRNA, respectively [155]. These indicate an effect on nutrient exchanges that may contribute to fetal growth restriction.

Since TCS is an endocrine disruptor, the levels of reproductive hormones and thy-
roid hormones, and their enzymes, have also been studied. In pregnant ewes, as well
as in mice, a high concentration of TCS decreased estrogen sulfonation [153]. Indeed,
estrogen sulfotransferase activities, implicated in both estradiol and estrone sulfonation,
were significantly reduced when pregnant mice were exposed to TCS. Although the serum
estrogen concentration was normal, the ratio of sulfo-conjugated E2 and unconjugated E2
was reduced in mice exposed to TCS. Interestingly, the estrogen receptor antagonist, an
estrogen sulfotransferase activity inhibitor, was able to rescue the platelet aggregation and
placental thrombosis, and limit spontaneous abortion [156].

Feng et al. studied the impact of TCS exposure on placental steroid metabolism
enzymes, including UDP-glucuronosyltransferase 1A1 (UGT1A1), estrogen sulfotransferase
1E1 (SULT1E1), and steroid 5α-reductase 2 (SRD5A2). This study indicated a significant
impairment with high concentrations of TCS as well as progesterone and estrogen receptors.
This could explain why placental hormones such as progesterone, estradiol, testosterone,
and prolactin secreted in maternal blood were reduced in groups exposed to high doses
of TCS [154]. T3 and T4 are known to affect fetal growth and development. In the event
of exposure to a high concentration of TCS (8 mg/kg/day orally from GD6 to GD18),
TCS induced hypothyroxinemia in pregnant mice. As thyroid hormones stimulate the
Akt-mTOR-p70S6K and ERK signaling pathways, which can regulate the activation of
placental amino acid transporters, these pathways have also been investigated. TCS
decreased placental Akt, mTOR, and P70S6K phosphorylation, but this was corrected by
L-thyroxinein (T4). In fact, T4 was able to rescue the activity and expression of amino acid
and glucose transporters and decrease fetal bodyweight [155].

In pregnant rats, following the administration to dams of 100, 200, and 400 mg/kg bodyweight/day from GD7 to 21, parabens (EtP and n-BuP) were distributed in rat maternal plasma, pools of amniotic fluids, placenta, whole-body fetuses, and in the fetal liver. Additionally, high levels of EtP were found in all fluids and tissues compared to n-BuP [157].

All of these experiments relating to maternal oral exposure to PPPs and placental effects were always performed using one pollutant, variable doses, and different timings of exposure, which rarely covered the preconceptional and gestational periods.
<table><caption>**Table 2.** Effects of phenols, phthalates, and parabens on fetoplacental development according to the animal model, the chemicals, the dose administrated, the exposure route and duration.</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.002 mg/kg bw/d</td><td>Oral gavage</td><td>GD6.5-17.5</td><td>GD18.5</td><td>Placental function</td><td>Peroxisome proliferator-activated receptor alpha and gamma (PPARα, PPARγ) and Aryl hydrocarbon receptor (AhR) mRNA expression: no effect<br>RAR-related orphan receptor gamma (RORγ) mRNA expression: BPA ↓ (♀)<br>Estrogen receptor beta (Erβ) and LXRα mRNA expression: BPA ↑ (♂)<br>Progesterone receptor (PR) mRNA expression: BPA ↑ (♂)/↓ (♀)<br>Placental expression of 6 non-nuclear receptor protein mRNAs: BPA ↓ (♂)/↑ (♀)</td><td>COUP-TFα, GCNF, SF-1, and PNR mRNA expression: BPA ↓ (♂)/↑ (♀)</td><td>[141]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.05 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-7</td><td>GD5, 8, 10, 12, 14</td><td>Fetoplacental development/placenta and uterine spiral artery structure</td><td>Placental areas, thickness, and diameter: no effect<br>Placental weight: BPA ↓ (GD14)<br>Fetal bodyweight: BPA ↓ (♀GD14)<br>Percentage of intrauterine growth restriction (IUGR): BPA ↑ (40.5% IUGR)<br>Uterine spiral artery wall thickness: BPA ↑</td><td>Implantation number and abortion rates: no effect<br>Implantation sizes: BPA ↓ (GD12)<br>Uterine artery blood velocity values and uterine natural killer cell numbers: no effect<br>Uterine spiral artery smooth muscle actin staining and wall-to-lumen ratio: BPA ↑</td><td>[136]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.5 or 50 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-11</td><td>GD12</td><td>Fetoplacental development/placental structure</td><td>Placental diameter and embryo length: BPA ↑ (50)<br>Right forelimbs length: BPA ↓ (0.5)/↑ (50)<br>Yolk sac length, first somite<br>longitudinal-transversal diameter, and first branchil arch longitudinal-transversal diameter ratios: BPA ↑ (0.5)/↓ (50)<br>Trophoblast giant cell degeneration and necrosis: BPA ↑<br>Spongiotrophoblast layer: BPA ↓ (50)<br>Maternal blood space area: BPA ↑ (50)/↓ (0.5)<br>Displacement of β-catenin from membrane to nucleus in the spongiotrophoblast and the labyrinthine cells: BPA ↑ (0.5)<br>Numbers of differentially expressed genes (DEGs): 582 (0.5)/701 (50)<br>Numbers of significant gene clusters: BPA (0.5 (11)/50: (13))<br>Gene cluster involvement: vascular/blood vessel development, nucleotide binding, embryonic morphogenesis and proteolysis, peptidase activity, progesterone-mediated oocyte maturation, gap junction pathways, chromosome organization, and chordate embryonic development</td><td></td><td>[138]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0, 2, 20, or 200 mg/kg bw/d</td><td>Oral gavage</td><td>GD13-16</td><td>GD17</td><td>Placental function</td><td>Placental CRH mRNA expression: BPA ↑ (200)<br>Placental CYP19 mRNA expression: no effect<br>Placental cAMP-response element-binding protein (CREB) level: BPA ↑<br>Preterm delivery: BPA ↑</td><td>Plasma CRH, estradiol, and testosterone concentrations: BPA ↑ (20, 200)<br>Protein kinase C ζ/λ phosphorylated forms: BPA ↑<br>Protein kinase C delta (PKCδ) phosphorylated forms: BPA ↑ (200)</td><td>[149]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0, 0.4, 4, 40, or 400 µM BPA in drinking water</td><td>Orally (drinking water)</td><td>GD7-17</td><td>GD13, 16 and 17</td><td>Placental structure and function</td><td>Mean fetal and placental weight, litter size, and placental-to-bodyweight ratio: no effect<br>Placental vessel area: BPA ↓<br>Trophoblast invasion-related gene expression: BPA ↑ (TIMP1, TIMP2)/↓ (MMP2, MMP9, WNT2)<br>Number of differentially expressed genes related to placental epigenetic modifications: BPA ↑ (8)/↓ (2)</td><td>Dams systolic blood pressure: BPA ↑ (4, 40, 400)<br>Dams glomerular atrophy: BPA ↑<br>Maternal bodyweight: no effect</td><td>[140]</td></tr><tr><td>Mouse</td><td>BPA</td><td>200 mg/kg bw/d</td><td>Orally (food)</td><td>GD-15 to GD12.5</td><td>GD12.5</td><td>Placental function</td><td>Placental number of differentially expressed miRNAs: BPA ↑ (22)/↓ (21)<br>Prediction of the number of mRNAs affected by the differentially expressed miRNAs: 142<br>Tissue-specific gene enrichment based on predicted mRNAs affected by the differential expressed miRNAs: thymus, cerebellum, olfactory bulb, brain cortex, E14.5 brain, and heart<br>Pathways affected by the differential expressed miRNAs: neural pathways (neurogenesis, neuron differentiation, and development), cell projection organization, cation transmembrane transport, metal ion transport, and inorganic cation transmembrane transport</td><td></td><td>[144]</td></tr><tr><td>Mouse</td><td>BPA</td><td>10 mg/kg bw/d BPA</td><td>Subcutaneous injections</td><td>GD0-GD7</td><td>GD10, GD12, PND0-56</td><td>Placental structure</td><td>Number of embryos and survival rate: BPA ↓<br>Placental size and proportion of placental decidua basalis: BPA ↓ (GD12)<br>Proportion of placental metrial gland: BPA ↑<br>Proportion of placental labyrinth area, intervillous spaces, and alignment of trophoblast giant cells: BPA ↓</td><td>Uterine weight, uterine-to-bodyweight ratio, and glycogen-containing cells: BPA ↓</td><td>[137]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>5, 10, or 40 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD0.5-5.5</td><td>GD14.5</td><td>Placenta structure and function</td><td>Labyrinthine area and decidua ITGβ1 and ITGα5 protein levels: BPA ↑<br>Placental MMP9 and MMP2 protein level: BPA ↑<br>Placental TIMP3 protein level: BPA ↓<br>Phosphorylated Akt and ERK level: BPA ↑ (5, 40)<br>Labyrinthine area proportion and intervillous spaces: BPA ↓<br>Spongiotrophoblast layer in the placenta: BPA ↓ (10, 40)<br>Presence of large vacuoles in the placenta: BPA ↑</td><td>-</td><td>[139]</td></tr><tr><td>Rat</td><td>BPA</td><td>0.0025, 0.025, or 0.25 mg/kg bw/d</td><td>Orally (drinking water)</td><td>GD-30 to GD20-22</td><td>GD20-22</td><td>Fetoplacental development and uterine artery</td><td>Fetal weight: BPA ↑ (2.5)<br>Placental weight and placenta-to-bodyweight ratio: BPA ↓ (2.5, 250)</td><td>Uterine artery diameter and uterine artery relaxation: BPA ↓<br>Uterine artery gene expression: BPA ↓ (PPARγ/ERα)/↑ (ERβ/VEGF/COX-2)</td><td>[133]</td></tr><tr><td>Rat</td><td>BPA</td><td>0.0025, 0.025, or 0.25 mg/kg bw/d</td><td>Orally (drinking water)</td><td>GD-30 to GD20</td><td>GD20</td><td>Fetoplacental development/placental metabolism</td><td>Fetal weight at birth: BPA ↑ (2.5)<br>Placental weight: BPA ↓ (2.5, 250)<br>Placental efficiency: BPA ↑<br>Glucose transporter 1 (GLUT1)<br>placental expression: BPA ↑ (2.5, 250)</td><td>Non-pregnant rats bodyweight: BPA ↑ (25, 250)<br>3-week pregnancy bodyweight: BPA ↓ (2.5)</td><td>[148]</td></tr><tr><td>Rat</td><td>BPA</td><td>200, 400, or 600 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD17-19</td><td>PND5</td><td>Maternal and fetal uteri gene expression</td><td>Postnatal uterine calbindin-D9K (CaBP-9k) and Estrogen receptor alpha (Era) mRNA expression: ↑ (600)<br>Placental passage of BPA<br>Rapid BPA absorption and distribution in maternal uteri</td><td>Maternal uterine CaBP-9k mRNA expression: BPA ↑ (600)<br>Maternal uterine CaBP-9k protein expression: BPA ↑<br>Maternal uterine Era mRNA expression: no effect<br>Induction of CaBP-9k protein in the endometrium of the maternal uterus: BPA ↑</td><td>[147]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Sheep</td><td>BPA</td><td>0.5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30-90</td><td>GD65, 90</td><td>Placental function</td><td>Fetal weight and placental efficiency: BPA ↓ (GD65)<br>Placental expression of inflammatory genes: BPA ↓ (GD65: TNF)/↑ (GD65: IL8/GD90: IL1β)<br>Placental expression of vascularization genes: BPA ↓ (GD90)<br>Placental expression of steroidogenic metabolism and signaling genes: BPA ↑ (GD65: CYP17, CYP19 and ESR2)/↓ (GD90: CYP17)<br>Placental expression of IGF family: BPA ↓ (GD65: IGF1R)/↑ (GD65: IGF2R, IGFBP1, IGFBP3/GD90: IGFBP2, IGFBP3 and IGFBP4)<br>Placental oxidative stress markers and placentome triglycerides and collagen accumulation: BPA ↑ (GD90)<br>Placental antioxidant genes expression: BPA ↑ (GD65)<br>DNA methyltransferase: BPA ↑ (GD65: DNMT3A)<br>Histone deacetylase: BPA ↑ (GD65: HDAC2 and HDAC1)</td><td></td><td>[134]</td></tr><tr><td>Sheep</td><td>BPA</td><td>5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD40-110</td><td>GD110</td><td>Placental function/fetal development</td><td>Placentome total weight, fetal weight, and placental efficiency: BPA ↓<br>Relative and protein expression level of Bcl-2: BPA ↓<br>Apoptosis rate and relative mRNA and protein expression of caspase-3, 8, and 9: BPA ↑<br>Mitochondrial reactive oxygen species (ROS) production: BPA ↑<br>ATP content and mitochondrial complex activities: BPA ↓<br>mRNA and protein relative expression of antioxidant-related genes and protein: BPA ↓<br>mRNA and protein relative expression of endoplasmic reticulum stress: BPA ↑</td><td></td><td>[135]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA, BPS</td><td>200 mg/kg bw/d</td><td>Orally (food)</td><td>GD-15 to GD12.5</td><td>GD12.5</td><td>Placental structure, function, and metabolism</td><td>Number of fetuses and placental oestrone, corticosterone, testosterone and progesterone (P4) concentration: no effect<br>Percentage of male conceptus: BPA ↑<br>Numbers of differentially expressed genes (DEGs): BPS (11)/BPA (3)<br>Pathways enrichment: Wnt signaling pathway, chemokine signaling pathway, and amino acid metabolism<br>Placental dopamine concentration and percentage of dopamine-positive trophoblast giant cells: BPA ↑/BPS ↑<br>Placental serotonin concentration and percentage of serotonin-positive trophoblast giant cells: BPA ↓/BPS ↓<br>Placental estradiol (E2) concentration: BPA ↓<br>Spongiotrophoblast zone to trophoblast giant cell area ratio: BPA ↓/BPS ↓</td><td>Maternal gestational weight, success of pregnancy, and number of implantation sites: no effect<br>D-fructose, sophorose and glycolic acid concentration: BPA ↓<br>Docosahexaenoic acid (DHA) concentration: BPA ↓/BPS ↓<br>Stearic acid and palmitic acid concentration: BPS ↓<br>D-ribose concentration: BPS ↑</td><td>[151]</td></tr><tr><td>Sheep</td><td>BPA, BPS</td><td>0.5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30 to GD100</td><td>GD120</td><td>Placental function</td><td>Endocrine function (pregnancy-associated glycoprotein 1 (PAG1), pregnancy-specific protein B (PSPB), P4): BPS ↓<br>Placental morphology: no effect<br>Syncytialization: BPS ↓ (E-cadherin)/BPS ↑ (GCM1)<br>Expression of genes involved in trophoblast fusion: BPS ↓</td><td></td><td>[150]</td></tr><tr><td>Rat</td><td>BPA, DEHP</td><td>0.005 mg/kg bw/d BPA, 5 or 7.5 mg/kg bw/d DEHP, or both (mix)</td><td>Oral gavage</td><td>GD6-21</td><td>GD6 (abortion and weight), PND1 to 24 weeks</td><td>Fetal development</td><td>Chest circumference: DEHP ↑ (5)/DEHP + BPA ↑ (5)/DEHP ↓ (7.5)/DEHP + BPA ↓ (7.5)<br>Crown-to-rump length: BPA ↑/BPA + DEHP ↑ (5)<br>Heart weight: BPA + DEHP ↑ (σ: 7.5)</td><td>Abortion rate: BPA + DEHP ↑<br>Thymus weight: BPA ↓ (σ')/DEHP ↓ (σ: 7.5)<br>Thymus apoptosis: BPA + DEHP ↑ (7.5)</td><td>[158]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA, OP</td><td>50 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD11.5-16.5</td><td>GD17.5</td><td>Placental function</td><td>Placental calcium transporter channels: OP ↓ (Trpv6 protein level)/OP ↑ (Pmca1 mRNA and protein levels), BPA ↑ (Pmca1 mRNA and protein levels)<br>Placental copper transporter channel expression: OP ↓ (Crt1 mRNA and protein levels/ATP7A mRNA and protein levels)/BPA ↓ (Crt1 mRNA and protein levels/ATP7A mRNA and protein levels)<br>Placental iron transporter channel expression: OP ↓ (Heph mRNA)/BPA ↓ (Heph mRNA and protein levels)<br>Fetal serum cation levels: BPA ↓/OP ↓ (calcium and copper)</td><td></td><td>[145]</td></tr><tr><td>Rat</td><td>BPA, OP, NP</td><td>200, 400, or 600 mg/kg bw/d</td><td>Subcutaneous injections</td><td></td><td>GD20</td><td>Placental function</td><td>Placental CaBP-9k mRNA expression: BPA ↑ (200)<br>Extraembryonic membrane CaBP-9k mRNA expression: OP ↓ (400/600)/NP ↓ (600)<br>Placental CaBP-9k protein expression: OP ↑ (200)/NP ↑ (200)/BPA ↑ (400, 600)<br>Extraembryonic membrane CaBP-9k protein expression: OP ↓ (400, 600)<br>Fetal uterus CaBP-9k mRNA expression: OP ↑ (400, 600)/NP ↑ (400, 600)/BPA ↑ (600)</td><td>Maternal uterus CaBP-9k mRNA expression: OP/NP ↑ (600)<br>Maternal uterus CaBP-9k protein level: BPA ↑/OP ↑ (600)</td><td>[146]</td></tr><tr><td>Mouse</td><td>BPA, TBBPA</td><td>10 mg/kg bw/d<br>BPA, 0.5 mg/kg bw/d TBBPA</td><td>Orally (food)</td><td>GD-15-GD16.5</td><td>E6.5-E10.5, E16.5</td><td>Fetoplacental development/placental function</td><td>Rate of the hemorrhaging conceptus and fetal loss: BPA ↑/TBBPA ↑<br>Trophoblast giant cells Ido1 mRNA expression: BPA ↓</td><td>Resorption rate: BPA ↑/TBBPA ↑<br>Mean Tregs cell number in the maternal spleen: BPA ↓<br>Mean CD4+ T cell number in the maternal spleen: BPA ↓/TBBPA ↓<br>Percentage of total Ido1 DNA methylation at CpG sites: BPA ↑ (σ')</td><td>[159]</td></tr></tbody></table>
<table><caption>**Table 2. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA, TCS</td><td>0, 87, 262, 523, or 7858 mg/kg bw/d TCS in a single dose; 0, 523, or 7858 mg/kg bw/d TCS in repeated doses; 61 or 122 mg/kg BW/d BPA; 262 mg/kg BW/d TCS + 61 mg/kg BW/d BPA; 262 mg/kg BW/d TCS + 122 mg/kg BW/d BPA</td><td>Subcutaneous injections</td><td>GD1-3</td><td>GD6, 17, PND 0, 4, 7, 14, and 21</td><td>Implantation</td><td>Number of implantation sites: TCS ↓ (repeated-dose GD6: 523, 7858)/TCS ↓ (single-dose GD2: 7858/GD3: 523, 7858)/TCS + BPA ↓ (GD6: 262 + 122)<br>Percentage of dams with normally developing implantation sites: TCS + BPA ↓ (GD6)<br>Gestational length: BPA + TCS ↑ (262 + 122)<br>Postnatal survival and pup bodyweight: no effect</td><td>[152]</td><td></td></tr><tr><td>Mouse</td><td>TCS</td><td>1, 4, or 8 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-18</td><td>GD11-19</td><td>Fetoplacental development/placental function</td><td>Number of live fetuses, fetal bodyweight, and placental weight: TCS ↓ (8)<br>Placental size and volume, labyrinthine area volume, and labyrinth cell proliferation activity: TCS ↓ (8)<br>PCNA and CD3 placental expression: TCS ↓ (8)<br>MeAIB placental expression: TCS ↑ (8)<br>MeAIB transporteur activity (SNAT): TCS ↓ (8)<br>Glucose transporter activity: TCS ↓ (8: SNAT1, SNAT4, GLUT1 mRNA)<br>Phosphorylation of Akt-mTOR-p70S6K signaling: TCS ↓ (8)</td><td>Triiodothyronine (T3), thyroxine (T4) and P4 levels: TCS ↓ (8)<br>Thyroid-stimulating hormone (TSH) level: TCS ↑ (8)</td><td>[155]</td></tr><tr><td>Mouse</td><td>TCS</td><td>4 or 8 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-14</td><td>PND1, 30, 60</td><td>Placenta/ metabolism</td><td>Numbers of pups, offspring bodyweight, and body mass index: TCS ↓ (8)<br>Body length: no effect</td><td></td><td>[160]</td></tr><tr><td>Mouse</td><td>TCS</td><td>0, 10, 50, or 100 mg/kg bw/d</td><td>Oral gavage</td><td>GD7.5-17.5</td><td>GD17.5</td><td>Placental function</td><td>Fetal and placental weight: TCS ↓ (50, 100)<br>Placental expression of PPARγ and PPARγ-regulated genes, and ANGPTL4 and MMP9 proteins: TCS ↓<br>Placental expression of inflammatory genes and IL-1β proteins: TCS ↑</td><td>Uterus size: TCS ↓ (50, 100)</td><td>[161]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>TCS</td><td>75, 150, or 300 mg/kg bw/d</td><td>Oral gavage</td><td>GD8-PND21</td><td>PND3, PND90</td><td>Fetal development/uterine structure/estrous cycle</td><td>Mean of litter weight: TCS ↓ (PND3)<br>Number of estrus cycles and frequency of proestrus phase: TCS ↓<br>Frequency of metestrus phase: TCS ↑</td><td>Maternal bodyweight: TCS ↓<br>The papilliferous appearance of the simple columnar luminal epithelium with vacuolization in the uterus: TCS ↑<br>Uterine cell height and uterine thickness: TCS ↑<br>Inflammatory infiltrate amount (leukocytes): TCS ↑ (150, 300)<br>T4, T3, and TSH levels: TCS ↑</td><td>[162]</td></tr><tr><td>Sheep</td><td>TCS</td><td>0.1 mg/kg bw/d</td><td>Direct infusion into the fetal circulation or through administration to the ewe</td><td>2 days between GD120 and GD130</td><td>3 days after 1st injection</td><td>Placental/liver sulfotransferase activity</td><td>TCS concentration: Placental concentration > Fetal liver concentration<br>Estrogen sulfotransferase activity in placenta and fetal liver cytosol: TCS ↓</td><td></td><td>[153]</td></tr><tr><td>Mouse</td><td>DEHP</td><td>0, 50, or 200 mg/kg bw/d</td><td>Oral gavage</td><td>GD0-6, GD7-12, GD13-17</td><td>GD18</td><td>Fetoplacental development</td><td>Fetal weight: DEHP ↓<br>Crown-rump length: DEHP ↓ (GD7-12/GD13-17)<br>Placental weight, blood sinusoid area in the labyrinth, and placental cell proliferation: DEHP ↓ (GD7-12)<br>Placental diameter: DEHP ↓ (σ: GD7-12)</td><td></td><td>[163]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>DEHP</td><td>125, 250, or 500 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-13</td><td>GD9 and GD13</td><td>Placental development and function</td><td>Embryo implantation: DEHP ↓ (500)<br>Fetoplacental weight, placental-to-body-weight ratio, and ectoplacental cone proportion: DEHP ↓<br>Condensed packed cells of ectoplacental cone: DEHP ↓ (250, 500)<br>Spongiotrophoblast area in the placenta: DEHP ↓<br>Labyrinth area in placenta: DEHP ↓ (500)<br>Gene expression involved in placental development: DEHP ↓ (GD9-13: Ascl2, Esx1, and Fosl1/500 GD9: Eomes)/↑ (GD13: Eomes and Hand1)<br>Formation of the branched fetal vessel in the labyrinthine area: DEHP ↓<br>MAPK signaling pathway and phospho-Erk1/2 levels in placenta: DEHP ↑ (GD9/GD13: 500)</td><td>Protein levels involved in apoptosis process: DEHP ↑ (GD13: Bax casp-3 and Bax caps-8)/↓ (GD13: Bcl2)<br>Number of Ki-67-positive cells: DEHP ↓</td><td>[164]</td></tr><tr><td>Rat</td><td>DEHP</td><td>500 or 1000 mg/kg bw/d</td><td>Oral gavage</td><td>GD7-12</td><td>GD20</td><td>Fetal development/placental function</td><td>Number of living fetuses: DEHP ↓<br>Fetus malformation: DEHP ↑<br>Number of differentially expressed genes (DEGs): DEHP ↑ (500 (1951), 1000 (951))/↓ (500 (1836), 1000 (527))<br>DEG pathways: steroid biosynthesis, PPAR signaling pathway, amino acid metabolism, lipid and drug metabolism, circadian entrainment, and neuroactive ligand-receptor interaction</td><td>Pregnant weight changes at GD18: DEHP ↓<br>Protein-protein interaction: 2682 interaction pairs and 476 nodes<br>Protein-protein interaction enrichment: olfactory transduction, cell cycle pathways, and neuroactive ligand-receptor interactions</td><td>[165]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>DEHP</td><td>750 or 1500 mg/kg bw/d</td><td>Oral gavage</td><td>GD0-19</td><td>GD20</td><td>Placental metabolism</td><td>PPARα and PPARγ mRNA expression in the labyrinth and the junctional zone: DEHP↑ PPARα and PPARγ protein expression: DEHP↑ Essential fatty acid (EFA) transporters in the labyrinth: DEHP↑ (FAT/CD36, FATP1, HFABP) EFA transporters in the junctional zone: DEHP↑ (FATP1, HFABP) EFA metabolic enzyme expression: DEHP↑ (CYP4A1)/↓ (COX-2) Total prostaglandins in the placenta: DEHP↓</td><td>Arachidonic acid (AA) in fetal and maternal plasma: DEHP↓ DHA in fetal plasma: DEHP↓ DHA in maternal plasma: DEHP↑ AA and DHA maternal-fetal ratio: DEHP↑ Fetal organ distribution of AA and DHA: DEHP↓</td><td>[166]</td></tr><tr><td>Rat</td><td>DnHP, DCHP</td><td>0, 20, 100, or 500 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-19</td><td>GD20</td><td>Fetoplacental development</td><td>Fetal bodyweight: DnHP↓ (20, 100)/DnHP↑ (σ*: 500)/DCHP↓ (♀: 500)/DCHP↑ (20, 100) Anogenital distance (AGD): DnHP↓ (♀)/DCHP↓ (♀) AGD/bodyweight ratio: DnHP↑ (100)/DnHP↓ (500)/DCHP↓ (20, 100) Placental weight: DCHP↑/DnHP↑ (20, 100) Placental diameter: DnHP↓/DCHP↓ Placental index: DnHP↑/DCHP↑ Trans-umbilical cord length: DnHP↓ (20, 100)/DCHP↑ (500) Cytoplasmic degeneration of trophoblast giant cells: DnHP↑/DCHP↑ Number and volume of trophoblast giant cells: DnHP↑ (100, 500)/DCHP↑ (500) Degeneration of spongiotrophoblast cells: DCHP↑ (100, 500) Hemorrhage in the labyrinth and basal zone: DnHP↑ (100, 500)/DCHP↑ Vessel formation in the labyrinth: DnHP↑ (500)/DCHP↓ Immunostaining of PCNA, PPARγ, ERα, ERβ, and AR: DCHP↓/DnHP↓</td><td>F0 relative kidney and liver weights: DCHP↑ (20)/DnHP↓ (500) Dams relative organs weights: DCHP↑ (500) Number of implantation sites: DCHP↓ (500) Number of live fetuses: DCHP↓/DnHP↓ (500) Absolute and relative lengths of bone ossification centers: DCHP↓/DnHP↓ Leukocyte, lymphocyte, and monocyte levels: DnHP↑/DCHP↑ N-granulocyte levels: DnHP↓/DCHP↓ Red blood cell parameters: DnHP↑ (σ*: 500)/DCHP↑ (σ*: 20)</td><td>[167]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>n-BuP</td><td>0, 10, 100, or 1000 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-19</td><td>GD20</td><td>Fetal development</td><td>Litter size, survival rate, number of resorptions, sex ratio, and fetal bodyweight: no effect</td><td>Dams bodyweight and bodyweight changes: no effect<br>Dams bodyweight gains: n-BuP ↓ (GD18-20: 1000)<br>Absolute and relative dams feed consumption: n-BuP ↓ (GD6-20: 1000)</td><td>[168]</td></tr><tr><td>Mouse</td><td>n-BuP, PrP</td><td>0, 0.05, 0.5, 5,10, 20, 30, and 35 mg/dams n-BuP; 35 or 40 mg/dams PrP</td><td>Subcutaneous injections</td><td>GD1-4</td><td>PND0, 3, 5</td><td>Fetal development</td><td>Skin irritation after injection: n-BuP ↑ (0.5, 5, 10, 20, 30, 35)<br>Litter size, survival rate, litter mass, gestational length, and number of implantation sites: no effect</td><td></td><td>[169]</td></tr><tr><td>Rat</td><td>DiBP, BP</td><td>600 mg/kg bw/d DiBP or 100 mg/kg bw/d n-BuP</td><td>Oral gavage</td><td>GD7-19 or GD7-21</td><td>GD19-21</td><td>Fetal metabolism</td><td>Plasma leptin level: DiBP ↓/n-BuP ↓<br>Plasma insulin level: DiBP ↓<br>Fetal bodyweight: DiBP ↓ (GD19)<br>Liver PPARα mRNA expression: DiBP ↓ (GD19)<br>Liver and testes PPARγ mRNA expression: no effect<br>Fetal anogenital distance: DiBP ↓ (♂)/↑ (♀)</td><td>Dams bodyweight gain: no effect<br>Testicular mRNA expression of genes involved in steroid synthesis: DiBP ↓ (GD19: SF-1)/↑ (GD21: aromatase)<br>Testes PPARα mRNA expression: DiBP ↓ (GD19)<br>Ovaries mRNA expression of genes involved in steroid synthesis: no effect 17α-hydrolase (P450c17) and PPARγ protein level in Leydig cells, and testicular testosterone production and level: DiBP ↓ (GD19/GD21)</td><td>[170]</td></tr><tr><td>Rat</td><td>EtP, n-BuP</td><td>100, 200, or 400 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD7-21</td><td>GD21</td><td>Placental function</td><td>Number of fetuses, fetus weight, and fetus organ weight: no effect</td><td>Maternal weight: no effect</td><td>[157]</td></tr></tbody></table>
<table><caption>**Table 2. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Fetoplacental Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>EtP, PrP</td><td>0, 400, 800, or 1600 mg/kg bw/d EtP, and 0, 625, 1250, or 2500 mg/kg bw/d PrP</td><td>Oral gavage</td><td>GD1 to GD5-7 or to GD8-9</td><td>GD5, GD7-8, GD10</td><td>Implantation/ endocrine function</td><td>Marker expression of decidualization: EtP ↓ (1600)/PrP ↓ (2500)<br>ER and PR expression: EtP ↓ (1600)/PrP ↓ (2500)<br>Rate of F1 litter size &lt; 7: EtP ↑ (1600)/PrP ↑ (2500)</td><td>Implantation rate: EtP ↓/PrP↓<br>Uterus weight: EtP ↓ (800, 1600)/PrP↓ (2500)<br>E2 and P4 serum levels: EtP ↑ (1600)/PrP ↑ (2500)</td><td>[171]</td></tr></tbody></table>

Results in brackets correspond to the sex in which the effect was observable (♂, male; ♀, female), followed by stage at observation separated by a column from the doses administered at which the effect was observed, and, if so, followed by the number of DEGs in brackets. If one of the above-mentioned characteristics is not specified, the effect was observed for both sexes at the different stages of observation and the different doses used. Arrows indicate the direction of the effect: a downward arrow (↓) indicates a decrease and an upward arrow (↑) indicates an increase. ANGPTL4: angiopoietin-like protein 4; Ascl2: achaete-scute family bHLH transcription factor 2; ATP: adenosine triphosphate; ATP7A: ATPase Cu++ transporting alpha-polypeptide; BPA: bisphenol A; BPS: bisphenol S; n-BuP: butylparaben; bw: bodyweight; cAMP: cyclic adenosine monophosphate; CD3: cyclin D3; Crt1: solute carrier family 31: member 1; COUP-TFα: chicken ovalbumin upstream promoter transcription factor alpha; COX-2: cyclooxygenase-2; CRH: corticotropin-releasing hormone; CYP19: placental aromatase; CYP17: 7 alpha hydroxylase; CYP4A1: cytochrome P450 subfamily 4A1; d: day; DCHP: dicyclohexyl phthalate; DEHP: di(2-ethylhexyl) phthalate; DnHP: di-n-hexyl phthalate; DiBP: diisobutyl phthalate; DNMT3A: DNA methyltransferases; Eomes: eomesodermin; EtP: ethylparaben; ERK: extracellular signal-regulated kinase; ESR2: estrogen receptor 2; Esx1: extraembryonic, spermatogenesis, homeobox 1; F1: first generation; FAT/CD36: fatty acid translocase; FATP1: fatty acid transport protein 1; Fosl1: Fos-like antigen 1; GCM1: glial cell missing factor 1; GCNF: germ cell nuclear factor; GD: gestational day; Hand1: heart and neural crest derivatives-expressed 1; HDAC: histone deacetylases; Heph: hephestin; HFABP: heart cytoplasmic fatty acid-binding protein; Ido1: indoleamine2,3 deoxygenase1; IGF1R: insulin-like growth factor 1 receptor; IGF2R: insulin-like growth factor 2 receptor; IGFBP: insulin-like growth factor-binding protein; IL-1β: interleukin 1 beta; IL-8: interleukin 8; LXRα: liver X receptor alpha; MAPK: mitogen-activated protein kinase; MeAIB: methylaminoisobutyric acid; miRNAs: microribonucleic acids; MMP: metalloproteinase; mRNA: messenger ribonucleic acid; NP: 4-nonylphenol; OP: 4-tert octylphenol; PCNA: proliferating cell nuclear antigen; PMCA1: plasma membrane Ca²⁺ ATPase; PND: postnatal day; PNR: photoreceptor-specific nuclear receptor; PrP: propylparaben; SF-1: steroidogenic factor-1; SNAT: sodium-coupled neutral amino acid transporter; TBBPA: tetrabromobisphenol A; TCS: triclosan; TIMP: tissue inhibitor of metalloproteinase; TNF: tumor necrosis factor alpha; Tregs: regulatory T cells; Trpv6: transient receptor potential cation channels in subfamily V member 6; VEGF: vascular endothelial growth factor; WNT: Wnt family member.
# 6. Effects of PPPs on Offspring Health after Birth from Epidemiological Data and Using Animal Models

Guilbert et al. and Radke et al. suggested that PPPs impact child neurodevelopment (including cognition and behavior) [39,54]. In this review, only the effects of PPPs in terms of obesity, metabolic alterations, cardiovascular disorders, and fertility defects will be explored.

## 6.1. Effects of PPPs on Obesity

### 6.1.1. Epidemiological Data (Table 3)

Few studies have examined the effects of prenatal PPP exposure on postnatal adiposity and growth [113,172–175]. Regarding phenols, we identified 16 studies from nine countries (Canada, China, Denmark, France, Greece, Mexico, Republic of Korea, Spain, and the USA) that examined the association between maternal phenol concentrations measured in urine and postnatal weight and adiposity in children. Sample sizes ranged from 218 to 1301 [173,175,176]. The outcomes measured varied across studies, with weight, BMI, waist circumference, and fat being measured from 6 months to 14 years of age [177]. Of the twelve studies evaluating BPA, eight found no statistically significant associations [113,172–174,176–179], one of 402 mother–child pairs in the USA reported a negative association with BMI, fat mass, and obesity risk at 2 to 9 years in girls [180]. Three other studies found positive associations with obesity markers. These three studies were conducted in Canada (N = 719) [181], China (N = 430) [182], and Republic of Korea (N = 788) [183]. Despite using a limited number of urine samples to assess the exposure (one or two), these studies reported positive associations between prenatal BPA and the waist-to-hip ratio at an average of 3.5 years (range 2–6 years), the waist circumference and an increased risk of obesity at 7 years, and the z-score weight from 2 to 6 years in girls. One study, from the SEPAGES cohort (N = 484), reported a positive association between second-trimester BPS and all infant growth parameters at 3 and 36 months of age [175]. No studies found an association between prenatal exposure to 2,4-dichlorophenol and 2,5-dichlorophenol. However, the number of studies on these compounds was relatively small (two to three). Among the small number of studies examining the association between BP-3 and childhood adiposity, a study of preadolescents (mean age = 11 years) in Spain (N = 1015) reported a positive association between prenatal exposure to BP-3 and a higher BMI z-score at 11 years [174].

Among the five studies of TCS, only one conducted in China reported a positive association with weight at 2 years using three spot urine samples (one from each trimester of pregnancy, N = 850 women–child pairs) [184]. This association appears to be stronger in girls. It is noteworthy that this study was one of the largest in sample size and number of urine samples collected during pregnancy.

We identified six studies regarding parabens. Four of them reported positive associations with postnatal weight, BMI, or the fat percentage at different ages, while the significantly associated paraben compounds differed between studies [172,178,185,186]. Three of these studies reported such effects in boys [178,185,186].

Regarding phthalates, 24 studies were identified. These studies assessed the association between prenatal phthalate concentrations and postnatal adiposity and growth. They were conducted in Australia, the USA, China, France, Germany, Greece, Mexico, Republic of Korea, and Spain. Sample sizes ranged from 180 [187] to 1301 [173]. Most of the studies measured phthalate metabolites in maternal urine collected at 1–3 time points during pregnancy. Serum was used in one study in Australia, resulting in lower detection frequencies and a higher risk of external contamination. In addition to these individual studies, a recent meta-analysis found a negative association between prenatal exposure to DEHP and child BMI [188]. A limitation of this meta-analysis was that only studies assessing associations with the molar sum of all the metabolites of DEHP were considered. This led to the exclusion of studies examining each metabolite separately, while several actually reported positive associations [79,189]. For other high-molecular-weight phthalates, isolated negative associations have been reported in boys between ΣDiNP and lean mass at 20 years [190], between ΣHMWP (high-molecular-weight
phthalates) and weight gain during the first 6 months of life, and with BMI from 4 to 7 years [191]. A few studies also reported associations between exposure to MCPP, cx-MiNP, and MCOP and an increased risk of overweight [172,187], higher BMI [192], waist circumference [193], and lean mass [79]. For MBzP, the results were inconsistent, with four studies reporting positive associations [192–195] and two studies reporting negative associations [177,196] with obesity markers. Among the studies of low-molecular-weight phthalates, two studies reported positive associations between ΣLMWP (low-molecular-weight phthalates) and child BMI. However, this association was observed in boys in one study [190] and in girls in the other [79]. Regarding individual metabolites, of the 19 studies evaluating MEP, 6 studies reported positive associations with BMI [79,193,197], and only 1 study reported an inverse association with fat mass [187]. For MBP, a metabolite of DBP, most of the significant associations reported were positive, suggesting increased adiposity [193,198] and BMI [175,192,195]. Two studies have also reported positive associations between MiBP and BMI [79,190,192].

Overall, most studies examining the associations between prenatal phthalate exposure and child growth or BMI reported associations. However, the sign of the associations and the metabolites involved often differed. A meta-analysis, such as the one carried out for DEHP, could help to understand the links between prenatal exposure to phthalates and child growth. Although phthalates may act through common mechanisms, to date, very few studies have explored them as a mixture [79,113,172,173,199].
<table><caption>**Table 3.** Effects of phenols, parabens, and phthalates on adiposity and growth of offspring using epidemiological data.</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>INMA<br>Spain<br>N = 470</td><td>Phenols and Phthalates</td><td>MEP, MBP, MiBP, MBzP, OH-MiNP, MECPP, MEHHP, MEOHP, MEHP, MCMHP, BPA</td><td>Urine<br>N = 2<br>first and third trimesters</td><td>7 years old<br>Body mass index (BMI) z-scores</td><td>Uni-pollutant and multi-pollutant using PCA</td><td>Yes; p-values for interaction &gt;0.2;</td><td>Compared to the first tertile, 7OHMMeOP was inversely associated with the BMI z-score in the third tertile and MECPP was inversely associated with overweight in both the second and the third tertiles of exposure.<br>In the PCA analysis, nonsignificant negative associations were observed with the zBMI for exposure to the phthalate factor (factor 2) in tertile 3 and tertile 2 compared with tertile 1.</td><td>[113]</td></tr><tr><td>CHAMACOS<br>California, USA<br>N = 309</td><td>Phenols, Phthalates, and Parabens</td><td>MEP, DEP, MBP, DBP, MiBP, DiBP, MBzP, BBP, DEHP, MEHP, MEHHP, MEOHP, MECPP, DiNP, MCOP, cx MiNP, DiDP, MCPP, MeP, PrP, TCS, 2,4-DCP, 2,5-DCP, BP-3, BPA</td><td>Urine<br>N = 2<br>2 prenatal visits</td><td>5 years old<br>BMI z-score and overweight/obesity status</td><td>Uni-pollutant and multi-pollutant using BKMR</td><td>-</td><td>Urinary concentrations of MEP, cx-MiNP, and propylparaben were consistently associated with an increased BMI z-score and overweight/obesity status.</td><td>[172]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>Raine Study Australia<br/>N = 410</td><td>Phthalates</td><td>MEP, MiBP, MBP, MBzP, MEHP, MECPP, MCMHP, MCPP, MiNP, MCOP, MiDP, ΣMBP, ΣDEHP, ΣDiNP, ΣLMW, ΣHMW, Σall.phth.metab</td><td>Serum N = 2<br/>18 and<br/>34 gestational weeks (GWs)</td><td>From birth and up to 20 years of age, longitudinal BMI z-scores, and DEXA at 20 years</td><td>Uni-pollutant, and linear mixed models with an interaction term between the phthalate level and age group (0–2, 2–11, and 11–20 years)</td><td>-</td><td>Compared to the lowest tertile: Childhood BMI was positively associated with the middle tertile of MiBP and adolescent BMI is positively associated with the middle tertile of MiBP and ΣLMW. For fat mass, the highest tertile of MECPP had a lower total fat percentage at 20 years of age. Participants whose mothers had detectable MiDP levels had a higher total fat percent at 20 years of age than those with undetectable MiDP levels. For lean mass, there was a positive association with detectable MCOP levels and an inverse association with the middle or highest tertiles of MEHP and the middle tertile of MiNP or ΣDiNP.</td><td>[190]</td></tr><tr><td>EDEN France<br/>N = 520</td><td>Phthalates</td><td>MEP, MBP, MiBP, ΣLMW, MECPP, MEHHP, MEOHP, MEHP, ΣDEHP, MBzP, MCOP, MCPP, cx-MiNP, ΣHMW</td><td>Urine N = 1<br/>between 22 and 29 GWs</td><td>Measures at birth and 5 years, prediction at 1, 3, and 5 years<br/>Birth weight, BMI, and Jenss–Bayley modeling approach of weight</td><td>Uni-pollutant</td><td>Only boys</td><td>No association between phthalate metabolite concentration and postnatal longitudinal weight. Positive association between MEP and weight and BMI at 5 years old, and weight velocity. Positive association between MBzP and weight at 2 years old and weight velocity at early ages.</td><td>[194]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>ELEMENT<br>Mexico<br>N = 223</td><td>Phthalates</td><td>MEP, MBP, MiBP, MCPP, MBzP, MECPP, MEHHP, MEHP, MEOHP, ΣDEHP</td><td>Urine<br>N = 3<br>one sample per trimester</td><td>Around 10 and 13 years old<br>Skinfold thickness,<br>BMI-for-age z-score; waist circumference (WC)</td><td>Uni-pollutant,<br>generalized estimating<br>equation models with repeated-measures<br>outcome</td><td>Yes, all<br>analyses were<br>sex-stratified</td><td>Among females, positive association between MBP T1, and BMI and MiBP T1, with the 3 outcomes. Negative association between MBzP T2 and skinfold thickness.<br>Among males, only MBzP T2 was positively associated with BMI and waist circumference.</td><td>[195]</td></tr><tr><td>MIREC study<br>Canada<br>N = 719<br>mother-child pairs</td><td>Phenols</td><td>BPA</td><td>Urine<br>N = 1<br>first trimester (mean 12.1 GWs [6.3 to 15])</td><td>Average 3.5 years old (range: 1.9–6.2) weight, height, waist/hip circumference, and subscapular/triceps skinfold thickness</td><td>Uni-pollutant,<br>linear regression</td><td>Yes</td><td>BPA was positively associated with the waist-to-hip ratio among all children.<br>Among girls, BPA was positively associated with the waist circumference (while null in boys) and the subscapular skinfold thickness (while almost inversely in boys).</td><td>[181]</td></tr><tr><td>Mount Sinai<br>USA<br>N = 180</td><td>Phthalates</td><td>MEP, MBP, MiBP, MCPP, MBzP, ΣDEHP</td><td>Urine<br>N = 1<br>T3 trimester (25–40 GWs)</td><td>Each follow-up visit scheduled at approximately ages 4–5.5 (mean, 4.9), 6 (mean, 6.2), and 7–9 (mean, 7.8) years old<br>Fat mass</td><td>Linear mixed-models with random intercepts to account for multiple observations per child;<br>Bayesian modeling framework</td><td>Yes</td><td>No significant association between using continuous chemicals. In tertiles, compared with the lowest ΣDEHP tertile, the fat mass was 3.06% (95% CIs: –5.99, –0.09%) lower in the highest tertile.</td><td>[200]</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>The Mount Sinai Children's Environmental Health Study (MSSM), CCCEH, HOME USA N = 180</td><td>Phthalates</td><td>MEP, MBP, MiBP, MCPP, MBzP, ΣDEHP</td><td>Urine N = 1 third trimester (20–40 GWs)</td><td>At follow-up visits scheduled for approximately ages 4–5.5, 6 and 7–9 years old (MSSM), 5 and 7 years old (CCCEH), and 4, 5, and 7–9 years old (HOME) BMI z-score and overweight/obese status</td><td>Bayesian modeling framework</td><td>Yes</td><td>MCPP was associated with increased odds of overweight/obese status overall. MEP was associated with lower BMI z-scores among girls.</td><td>[187]</td></tr><tr><td>Odense Child cohort Denmark N = 312</td><td>Parabens</td><td>MeP, EtP, PrP, b-BuP, BzP</td><td>Urine N = 1 around 28 GWs (median 28.7 GWs)</td><td>7 years old BMI z-score, total fat, android fat, and gynoid fat</td><td>Uni-pollutant, linear regression</td><td>Yes, all analyses stratified by gender</td><td>Only n-BuP was positively associated with the total and android (visceral and subcutaneous) fat percentage among boys.</td><td>[185]</td></tr><tr><td>TIDES USA N = 780</td><td>Phthalates</td><td>MEP, MBP, MiBP, MCPP, MCOP, cx-MiNP, MBzP, ΣDEHP</td><td>Urine N = 2 around 11 and 32 GWs</td><td>Birth, 1, 3, 4, and 6 years old Weight and BMI</td><td>1—Linear mixed-model with repeated growth measures; 2—Group-based trajectory (patterns of weight or BMI change over time)</td><td>Yes, differences by sex for weight models but not BMI models</td><td>MEP, MBzP, MiBP, and MBP were all inversely associated with weight and BMI at birth. MCPP and MBP were positively associated with BMI at 3 years old. MEP, MBzP, MCPP, and MCOP were positively associated with BMI at 4 years old.</td><td>[192]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>Ma'anshan Birth Cohort (MABC) China N = 990</td><td>Phthalates</td><td>MMP, MEP, MBP, ΣLMW, MBzP, MEHP, MEOHP, MEHHP, ΣDEHP, ΣHMW</td><td>Urine N = 3 each trimester</td><td>Birth, 3, 6, and 9 months, and 1, 1.5, 2, 2.5, 3, 4, 5, and 6 years old BMI for age z-scores</td><td>Uni-pollutant (linear/logistic regression and GEE with repeated measurement) and multi-pollutant (quantile g-computation and BKMR)</td><td>Only girls</td><td>Pregnancy means ΣLMW and MBzP were negatively associated with BMI z-score at birth. Pregnancy means ΣDEHP was positively associated with BMI z-score at 3 months. Trajectories of BMI z-score was associated with first-trimester exposure to MEP (negative association) and MEOHP (positive association), and positively associated with third-trimester exposures to MEP, MBP, ΣLMW, and MEHP. Mixtures analyses did not find any significant associations.</td><td>[201]</td></tr><tr><td>INMA Spain N = 1015</td><td>Phenols, Parabens, and Phtalates</td><td>MEP, MBP, MBzP, DEHP, MeP, EtP, PrP, n-BuP, BP-3, BPA</td><td>Urine N= 2 first (mean 13 GWs) and third (mean 33 GWs) trimesters</td><td>11 years old BMI z-scores</td><td>Uni-pollutants (generalized additive mixed models); multi-pollutant (BKMR)</td><td>Yes</td><td>BP-3 was associated with a higher BMI. In girls, the overall mixture trended with a higher BMI.</td><td>[174]</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>HELIX project<br>European cohort<br>including<br>France, Greece,<br>Lithuania,<br>Norway, Spain,<br>and the UK<br>N = 1301</td><td>45 ED<br>compounds</td><td>Among them,<br>7 high-molecular-weight<br>phthalate<br>metabolites<br>(HMWPs),<br>including<br>4 (DEHP)<br>metabolites, 2<br>DiNP<br>metabolites, and<br>1 metabolite of<br>BBP; 3<br>low-molecular-weight<br>phthalate<br>metabolites<br>(LMWPs);<br>4 parabens;<br>3 phenols</td><td>Urine and blood<br>samples during<br>pregnancy<br>N = 1134</td><td>6 to 11 years old</td><td>Associations<br>were assessed<br>using Bayesian<br>weighted<br>quantile sum<br>regressions<br>applied to<br>mixtures for<br>each chemical</td><td>Yes</td><td>ΣLMW and ΣHMW of phthalates: ↓<br>MetS risk score.<br>Association of prenatal MnBP levels<br>with ↓ child MetS risk score.<br>Phenols and parabens: no association<br>with the MetS risk score.</td><td>[202]</td></tr><tr><td>China<br>N = 436</td><td>Parabens</td><td>MeP, EtP, PrP,<br>n-BuP, BzP</td><td>Urine<br>N = 1<br>3 years old</td><td>3 years old<br>Weight and BMI</td><td>Uni-pollutant<br>generalized<br>linear models</td><td>Yes</td><td>Among all and among boys: urinary<br>EtP concentrations were positively<br>associated with weight z-scores.<br>Nothing significant among girls.</td><td>[186]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>Sheyang Mini Birth Cohort Study China N = 430</td><td>Phenols</td><td>BPA</td><td>Urine N = 1 delivery day</td><td>7 years old Weight, WC, skinfold thickness, and risk of general and central obesity</td><td>Uni-pollutant, generalized linear regression models, and multivariable logistic regression models</td><td>Yes, no significant interaction</td><td>Positive associations with the waist circumference. Higher risk of central obesity in the second and third tertiles of BPA compared to the first tertile.</td><td>[182]</td></tr><tr><td>CHAMACOS California, USA N = 345</td><td>Phthalates</td><td>MEP, MBP, MiBP, MCPP, MCOP, cx-MiNP, MBzP, ΣDEHP</td><td>Urine N = 2 around 14 and 26.9 GWs, averaged for analyses</td><td>5, 7, 9, 10.5, and 12 years old Overweight/obesity, BMI z-score, WC z-score, and percent body fat</td><td>Uni-pollutant, GEE with repeated outcomes</td><td>Yes</td><td>MEP, MBzP, and ΣDEHP were associated with a 20–30% increase in the odds of overweight/obesity at each age point in both boys and girls. MBP was associated with a 30–40% increase in odds in boys only. Consistent positive associations of MEP with BMI, waist circumference, and percent body fat at each time point. ΣDEHP associated with increased waist circumference at 5 years old. MBP, MiBP, MBzP, MCOP, and cx-MiNP were associated with increased waist circumference at 7 and 9 years old.</td><td>[193]</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>CHAMACOS<br>California,<br>USA<br>N = 402</td><td>Phenols</td><td>BPA</td><td>Urine<br>N = 2<br>first (mean<br>13.8 GWs) and<br>second (mean<br>26.4 GWs)<br>trimesters</td><td>2, 3.5, 5, 7, and<br>9 years<br>BMI, WC,<br>percent body fat,<br>and obesity</td><td>Uni-pollutant,<br>association with<br>outcomes at<br>9 years:<br>linear/logistic<br>regressions.<br>Association with<br>longitudinal<br>outcomes: GEE</td><td>Yes</td><td>Association was not significant<br>among all, while there was a negative<br>association among girls (significant<br>negative association with BMI, body<br>fat, and risk of overweight/obesity),<br>and null associations among boys.<br>Longitudinal analysis showed a<br>negative association with the BMI<br>z-score among girls.</td><td>[180]</td></tr><tr><td>CHAMACOS<br>California,<br>USA<br>N = 335</td><td>Phthalates</td><td>MBP, MEP, MiBP,<br>MBzP, cx-MiNP,<br>MCOP, MCPP,<br>MEHP, MEHHP,<br>MECPP,<br>MEOHP</td><td>Urine<br>N = 2<br>around 14 and<br>26.9 GWs,<br>averaged for<br>analyses</td><td>11 follow-up<br>visits between 2<br>and 14 years old<br>BMI trajectories</td><td>PCA</td><td>Yes</td><td>MEP-positive association with BMI<br>through 12 years.</td><td>[197]</td></tr><tr><td>HOME<br>USA<br>N = 220</td><td>Phenols</td><td>TCS</td><td>Urine<br>N = 2<br>around 16 and<br>26 GWs</td><td>8 years old<br>Weight, WC, and<br>body fat<br>percentage</td><td>Uni-pollutant,<br>linear regression</td><td>yes, all<br>p-values = 0.37</td><td>No significant association.</td><td>[203]</td></tr><tr><td>PROGRESS<br>Mexico<br>N = 514</td><td>Phthalates</td><td>ΣDEHP, ΣDiBP,<br>ΣDiNP, ΣDBP,<br>MBzP, MECPTP,<br>cx-MiNP, MCPP,<br>MEP</td><td>Urine<br>N = 2 s and third<br>trimesters</td><td>4, 6 and 8 years<br>old<br>Child adiposity<br>was categorized<br>into 3 trajectories</td><td>Uni-pollutant<br>(linear and<br>multinomial<br>logistic<br>regressions) and<br>mixture<br>(quantile<br>G-computation)</td><td>Yes</td><td>In comparison to the "low-stable"<br>group,<br>ΣDEHP was associated with greater<br>odds of being in the "high-high"<br>trajectory, ΣDiNP was associated<br>with greater odds of being in the<br>"low-high" trajectory, and cx-MiNP<br>was associated with lower odds of<br>being in the "low-high" trajectory.<br>No mixture effects.</td><td>[199]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>EDC Cohort Republic of Korea N = 481</td><td>Phthalates</td><td>MEHHP, MEOHP, ΣDEHP, MBP</td><td>Urine N = 1 second trimester (mean 20.3 GWs)</td><td>6 years old BMI z-score, percentage of fat mass, and FMI</td><td>Uni-pollutant, linear regressions</td><td>Yes</td><td>MEHHP and MnBP were inversely associated with the BMI z-score among girls. All chemicals measured were inversely associated with skeletal muscle index among all children and among girls (not among boys).</td><td>[204]</td></tr><tr><td>MOCEH Study Republic of Korea N = 788</td><td>Phenols</td><td>BPA</td><td>Urine N = 1 third trimester</td><td>Birth, and 6, 12, 24, 36, 60, and 72 months Weight z-score and z-score of weight for length</td><td>Uni-pollutant, linear regression for each outcome, and LMM for growth from 6 to 72 months</td><td>Yes</td><td>BPA was positively associated with the weight z-score at birth among all children and among boys, while positively associated among girls at 24, 36, 60, and 72 months. BPA was positively associated with the z-score of weight for length among all children and among girls at birth, 6, 36, 60, and 72 months. In the longitudinal analysis, BPA was positively associated with the z-score of weight for length among all children and among girls.</td><td>[183]</td></tr><tr><td>Human cohort conducted at Wuhan, China N = 814</td><td>Phthalates</td><td>MECPP, MEHHP, MEOHP, MEHP, ΣDEHP</td><td>Urine N = 3 each trimester</td><td>Birth, 6, 12, and 24 months old Weight, ponderal index (PI), and BMI</td><td>Uni-pollutant— mixed linear models</td><td>Yes</td><td>MECPP and MEOHP were positively related to the average weight z-scores in male offspring. DEHP levels at T3 were positively related to 6-month and 12-month BMI z-scores among all children and boys. DEHP levels at T1 were positively associated with BMI z-scores at 24 months among boys.</td><td>[189]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>CCCEH<br>USA<br>N = 424</td><td>Phthalates</td><td>MEHP, MEHHP, MECPP, MEOHP, MCPP, MiBP, MBP, MBzP, MEP</td><td>Urine<br>N = 1<br>third trimester</td><td>5 (only BMI) and 7 years old BMI z-scores, percentage body fat, fat mass index, and WC</td><td>Analyses by DEHP component and non-DEHP component; GEE for BMI analyses, linear regression for other outcomes</td><td>Yes</td><td>Non-DEHP components were negatively associated with all outcomes of interest among boys.</td><td>[205]</td></tr><tr><td>German LIFE<br>Child cohort study,<br>Germany<br>N = 164</td><td>Phthalates</td><td>MBzP, MEHP, MEHHP, MEOHP, MECPP, MCIOP, oxo-MiNP, OH-MiNP, ΣLMW, ΣHMW</td><td>Urine<br>N = 1<br>late pregnancy (24 or 36 GWs)</td><td>Birth, 1 and 2 years old Weight and BMI</td><td>Uni-pollutant, robust linear regression</td><td>Yes</td><td>ΣHMW was negatively associated with birth weight in all children and among girls (not boys).</td><td>[206]</td></tr><tr><td>Shanghai<br>Obesity and Allergy Birth Cohort Study,<br>China<br>N = 218</td><td>Phenols</td><td>BPA</td><td>Urine<br>N = 1<br>late pregnancy</td><td>2 years old Weight, mid-upper-arm circumference (MUAC), and skinfold thickness (triceps, subscapular, and abdominal)</td><td>Uni-pollutant, linear regression</td><td>Yes, all analyses stratified by gender</td><td>No differences were found in any adiposity measures.</td><td>[176]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>EDEN<br>France<br>N = 520</td><td>Phenols and<br>Parabens</td><td>2,4-DCP,<br>2,5-DCP,<br>BPA, BP-3, TCS,<br>MeP, EtP, PrP,<br>n-BuP</td><td>Urine<br>N= 1<br>between 22 and<br>29 GWs</td><td>6 months, 1, 2,<br>and 3 years old<br>Birth<br>measurement<br>and postnatal<br>Jenss growth<br>trajectories</td><td>Uni-pollutant</td><td>Only boys</td><td>Parabens were positively associated<br>with weight at birth; remained for<br>3 years for methylparaben.</td><td>[178]</td></tr><tr><td>HOME,<br>USA<br>N = 219</td><td>Phthalates</td><td>ΣDEHP,<br>MCPP, MBP,<br>MiBP, MEP,<br>MBzP</td><td>Urine<br>N = 2<br>around 16 and<br>26 GWs</td><td>8 years<br>BMI, WC, and<br>percent body fat</td><td>Uni-pollutant,<br>2 samples<br>averaged for<br>prenatal<br>exposure<br>estimation</td><td>Yes</td><td>MBzP was negatively associated<br>with the BMI z-score.<br>MBzP was negatively associated<br>with body fat when adjusted for<br>creatinine.</td><td>[196]</td></tr><tr><td>Rhea Study,<br>Greece<br>N = 230</td><td>Phthalates</td><td>MEP, MBP,<br>MiBP, MBzP,<br>MEHP, MEHHP<br>MEOHP,<br>ΣDEHP</td><td>Urine<br>N = 1<br>first trimester</td><td>4 and 6 years<br>Weight, BMI,<br>WC, and<br>skinfold<br>thickness</td><td>Uni-pollutant,<br>GAMs to explore<br>shape GEE for<br>associations<br>with repeated<br>outcomes</td><td>Yes</td><td>Only MnBP was associated with a<br>change in waist-to-height ratio at<br>ages from 4 to 6 years old.</td><td>[198]</td></tr><tr><td>Rhea Study,<br>Greece<br>N = 235</td><td>Phenols</td><td>BPA</td><td>Urine<br>N = 1<br>first trimester</td><td>Birth (weight),<br>from 6 to 4 years<br>old (BLI, at<br>4 years for other<br>outcomes)<br>Weight, BMI,<br>WC, skinfold<br>thickness</td><td>Uni-pollutant,<br>first GAM to<br>explore the<br>shape of the<br>relationships,<br>then linear<br>regressions;<br>mixed effects<br>linear regression<br>model for BMI<br>trajectories</td><td>Yes</td><td>No significant association, but<br>patterns of BMI trajectories differed<br>between boys and girls (only<br>visually; not tested).</td><td>[179]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>INMA-Sabadell Spain N = 391</td><td>Phthalates</td><td>ΣHMW, ΣLMW</td><td>Urine N = 2 first (around 13 GWs) and third trimesters (around 34 GWs)</td><td>From birth to 6 months old for weight gain, and 1, 4, and 7 years old for BMI Weight and BMI</td><td>Uni-pollutant and one multi-pollutant model simultaneously adjusted for ΣHMW and ΣLMW; GAMs to explore shape GEE for associations with repeated outcomes</td><td>Yes</td><td>ΣHMW was inversely associated with the weight gain z-score (from 0 to 6 months) and BMI z-scores at 4 and 7 years old among boys, while associations tended to be positive among girls. Zero associations among all children.</td><td>[191]</td></tr><tr><td>HELIX Spain N = 1301</td><td>Phenols, Parabens, and Phthalates</td><td>MEP, MiBP, MBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, OH-MiNP, oxo-MiNP, ΣDEHP, EtP, PrP, n-BuP, BP-3, BPA, TCS</td><td>Urine N = 2 first and third trimesters</td><td>One visit between 6 and 11 years old Weight and BMI</td><td>Uni-pollutant and DSA</td><td>Not with phenols and phthalates</td><td>No significant association.</td><td>[173]</td></tr><tr><td>China N = 850</td><td>Phenols</td><td>TCS</td><td>Urine N = 3 one sample per trimester</td><td>6 months, 1 and 2 years old z-scores of weight</td><td>Uni-pollutant trimester-specific expo and average of expo T1-T2-T3</td><td>Yes</td><td>Prenatal exposure (mean expo) to triclosan was associated with elevated 2-year-old weight z-score in all and in girls. Exposure during T1 and T2 was associated with increased weight at 2 years old.</td><td>[184]</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Population</th><th>Class</th><th>Chemicals</th><th>Sampling Type, Number, and Timing</th><th>Timing of Outcome and Outcome of Interest</th><th>Model</th><th>Sex-Specific Analyses</th><th>Main Findings</th><th>Reference</th></tr></thead><tbody><tr><td>ELEMENT<br>Mexico<br>N = 249</td><td>Phenols and Phthalates</td><td>BPA, MBP, MBzP, MCPP, MEP, MiBP, MEHP, MECPP, MEHHP, MEOHP, ΣDEHP, ΣH MW, ΣLMW</td><td>Urine<br>N= 1<br>third trimester</td><td>One single visit between 8 and 14 years old BMI z-score, WC, and the sum of skinfolds</td><td>Uni-pollutant, linear regression</td><td>Yes, all p-values of sex interaction > 0.2</td><td>Only MBzP was negatively associated with the BMI z-score.</td><td>[177]</td></tr><tr><td>SEPAGES cohort<br>France<br>N = 484<br>pregnant women<br>Recruitment:<br>2014–2017</td><td>Phenols, Parabens, and Phthalates</td><td>4 phenols, 4 parabens, 7 phthalates, and 1 non-phthalate plasticizer</td><td>Weekly pooled urine samples collected from the mother during T2 and T3 trimesters</td><td>BMI, height, weight, and head circumference at 3 and 36 months of age</td><td>Associations with individual chemicals using adjusted linear regression and mixtures of chemicals using a Bayesian kernel machine regression model</td><td>Yes</td><td>BP at T2: ↓ BMI and weight at 36 months of age.<br>BP at T3: ↓ height at 36 months of age.<br>BPS at T2: ↑ BMI, weight, and height at 3 and 36 months of age (tendance).<br>BPS at T3: ↑ BMI, weight, and head circumference at 3 months of age (tendance).<br>ΣDEHP at T3: ↑ BMI and weight at 3 months of age.<br>ΣDiNP at T2: stratification for child sex, ↑ of head circumference in males, and ↓ in females at 3 and 36 months of age.</td><td>[175]</td></tr></tbody></table>

BBP: benzyl butyl phthalate; n-BuP: n-butylparaben; BP-3: benzophenone-3; BPA: bisphenol A; BzP: benzylparaben; DBP: di-n-butyl phthalate; DEHP: di-(2-ethylhexyl) phthalate; DEP: diethyl phthalate; DiBP: diisobutyl phthalate; DiDP: diisodecyl phthalate; DiNP: diisononyl phthalate; EtP: ethylparaben; HMW: high-molecular-weight phthalates; LMW: low-molecular-weight phthalates; MBP: mono-n-butyl phthalate; MBzP: monobenzyl phthalate; MCIOP: mono-(4-methyl-7-carboxyheptyl) phthalate; MCMHP: mono [2-(carboxymethyl)hexyl] phthalate; MCOP: mono-carboxy-iso-octyl phthalate; MCPP: mono-(3-carboxypropyl) phthalate; MECPP: mono-(2-ethyl-5-carboxypentyl) phthalate; MECPTP: mono-2-ethyl-5-carboxypentyl terephthalate; MEHHP: mono(2-ethyl-5-hydroxyhexyl) phthalate; MEHP: mono(2-ethyl-hexyl) phthalate; MEOHP: (mono(2-ethyl-5-oxohexyl) phthalate; MEP: mono-ethyl phthalate; MetS: risk of metabolic syndrome; MiBP: mono-iso-butyl phthalate; MiDP: mono-iso-decyl phthalate; MiNP: mono-iso-nonyl phthalate; cx-MINP: mono-carboxy-iso-nonyl phthalate; MMP: monomethyl phthalate; MNP: mono-isononyl phthalate; MeP: methylparaben; OH-MiNP: mono-hydroxy-iso-nonyl phthalate; oxo-MiNP: mono-oxo-iso-nonyl phthalate; PrP: propylparaben. Arrows indicate the direction of the effect: a downward arrow (↓) indicates a decrease and an upward arrow (↑) indicates an increase.
### 6.1.2. Maternal Exposure to PPPs and Metabolic Outcomes in Experimental Models (Table 4)

Studies of exposure to PPPs were initially interested in the risk of allergies in offspring, and also on the risk of mammary and genital carcinogenesis [207]. These will not be addressed in this review. These studies then focused either on the reproductive toxicity for the offspring, discussed later, or on the neurodevelopmental effects and their repercussions on behavior and learning. Both of these subjects were reported in a very recent review (for the bisphenols and phthalates) [208]. Finally, more recently, studies have focused on the metabolic effects of these PPP exposures and the repercussions on bodyweight, which will be the subject developed here.

Maternal oral exposure to bisphenol during the perinatal period, either A, S, or F, resulted in dose-grading adverse effects in female offspring. These adverse effects on the intestinal and systemic immune response depend on the bisphenol nature, i.e., A, S, or F. In mice, stronger impacts on inflammatory markers in feces were observed with BPS at the dose of 0.005 mg/kg bw/d. Exposure to BPA and BPF at low doses induced significant immune response changes in the offspring. These changes led to both intestinal and systemic Th1/Th17 inflammation [209]. Additionally, maternal exposure to BPS resulted in adverse effects on the triacylglycerol (increase in males), hormone levels (increase in T3 males and T4 females), and behavior (decreased food intake) of the offspring. These effects were observed as a function of the dose with a non-monotonic response [210]. Maternal exposure to BPA induced lighter weaning weights in some males. These males experienced rapid catch-up growth immediately after weaning [211]. So, they represent a subpopulation sensitive and vulnerable to very low fetal serum concentrations of BPA in the pg/mL range, particularly in the case of glucose intolerance. Gestational exposure to BPA has been shown to upregulate offspring pancreatic β-cell division and mass in an ERβ-dependent manner in adult male mice [212]. In C57BL/6J, maternal exposure to BPA mimicking human exposure levels (from 0.010 to 10 mg/kg bw/day) led to dose-specific effects on pancreatic islets in both the first (F1) and second generations (F2) in males only. Moreover, an increase in bodyweight was observed only in the F3 males. In addition, the lowest dose reduced the β-cell mass and smaller islets associated with increased insulin secretion, without a change in glucose tolerance. However, changes in the cytokine levels were reported across the generations in males [213]. In a transgenerational context, female offspring from males exposed in utero and during lactation to BPA were shown to exhibit impaired glucose tolerance despite the absence of compromised insulin sensitivity in vivo or reduced ex vivo glucose-stimulated insulin secretion. However, male offspring showed normal glucose tolerance [214]. Prenatal exposure to BPA (5 mg/kg bw/day) disrupted hepatic lipid homeostasis in a sex- and age-dependent manner. These effects, investigated by lipidomic and transcriptomic approaches, were marked around weaning (the accumulation of lipids and inflammation of the liver), but tended to fade with age, especially in females (bodyweight and total lipid content) [215,216].

In sheep, prenatal exposure to BPA (0–0.05–0.5–5 mg/kg bw/day by subcutaneous injections from days GD30 to GD90; term: 147 days) induced peripheral insulin resistance and adipose tissue disruptions in female offspring of 21 months of age [217]. In addition, lipotoxicity (an increase in blood and tissue triglycerides), accompanied by an increase in oxidative stress, was observed in a non-monotonic manner. In parallel, a reduction in antioxidants was shown in both liver and skeletal muscles, as well as altered proinflammatory markers in the liver (an increase in TNF-α and CD68, but IL-6 and IL-1B) and skeletal muscle (an increase in IL-6, IL-1B, CCL2, and CD38) [218]. These effects contributed to the resistance to insulin. These BPA-induced prenatal metabolic dysfunctions were corroborated for the middle dose (0.5 mg/kg/day) by transcriptomic analyses. These data provided mechanistic clues to explain oxidative stress and lipid accumulation, and potential mitochondrial and fibrotic defects in these tissues [219]. In the same animal model, prenatal exposure to BPA (0.05, 0.5, or 5 mg/kg/day) by subcutaneous injections from days GD30 to GD90 induced a trend toward decreased insulin and β-cell counts associated with an increase in glucagon and α-cell counts. The results were most consistent at the lowest BPA dose in fetal pancreata at GD90 and in adult offspring. These data suggest that early-life BPA exposure poses a likely threat to metabolic health [220].
In mice, maternal subcutaneous exposure to BPA (0.010 or 0.100 mg/kg bw/day) from GD9 to GD16 impaired pancreatic function, with a decrease in the mass of β-cell [221] and impaired glucose homeostasis in 6-month-old male offspring, but not in females [222]. The same replicated protocol showed that such maternal exposure of PND30 increased the retinoid concentrations and gene expression of key elements involved in the retinoid system in the liver in male offspring [223].

In mice, maternal exposure to BPA throughout gestation and lactation (1000 nM via drinking water) increased the cytokine levels in the spleens of the PND21 and PND42 offspring. The levels of cytokines were derived from Th17 cells (IL-17 and IL-21) and were essential for the differentiation of Th17 cells (IL-6 and IL-23). These increases were more pronounced in females and started at the lowest dose of BPA (100 nM), promoting inflammation induced in offspring [224]. When the mice were orally exposed to BPA (0.000050, 0.050, or 50 mg/kg diet) throughout gestation and lactation, primary bone marrow-derived mast cells (BMMCs) presented, after activation, increased cysteinyl leukotriene and TNFα production in all of the exposed groups [225]. Additionally, an increase in prostaglandin D2 and IL-13 production was observed only in the most BPA-exposed group. These BMMCs were generated from the bone marrow culture extracted from the femurs of PND21 progeny [225]. This production of proinflammatory mediators is generally associated with asthma, another harmful effect linked to the exposure of BPA. In mice exposed to BPA (0.010 or 10 mg/kg bw/d) throughout gestation and lactation, males exposed to the lowest dose were lighter at birth. These males then underwent rapid catch-up growth until weaning and showed increased bodyweight after PND117. Furthermore, glucose intolerance and insulin resistance resulted in increased body fat in offspring exposed to the higher dose [226]. Another study exposing the dam to BPA (0–30 mg/kg bw/day) for the same periods as above showed dose-dependent increases in body and liver weights in 20-week-old offspring [227]. A dose-dependent decrease in circulating glucagon in male offspring was also observed. However, in this study, females showed a dose-dependent decrease in body, liver, muscle, and fat pad weights. The latest effects in females were accompanied by decreased adipocyte size, serum lipids, serum leptin, and adiponectin, in parallel with increased physical activity [227]. Maternal exposure to BPA through drinking water (10 mg/L) induced insulitis, i.e., a pancreatic disease caused by lymphocyte infiltration, and accelerated the prevalence of diabetes in the 20-week-old female offspring [228]. In mice, perinatal exposure to BPA via the maternal diet (0.000050, 0.050, or 50 mg BPA/kg diet) induced an increase in the energy expenditure of the offspring during life until the age of 10 months. In female offspring, BPA exposure tended to decrease bodyweight and fats, with improved glucose and insulin profiles, at the highest dose, leading to a hyperactive and lean phenotype [229].

Maternal exposure of mice to the antibacterial TCS (8 mg/kg bw/d) from GD6 to GD14 has been shown to alter prenatal and postnatal growth and development, as well as metabolic phenotypes in male and female offspring. Compared with control offspring, TCS offspring (male or female) initially showed reduced bodyweight at birth, but then showed more rapid bodyweight gain during the fifth day of gestation, which increased over time [160]. Indeed, PND30 overweight TCS offspring showed, at PND60, increased visceral fat and adipocyte size, with delayed glucose clearance and insulin resistance. In rats, maternal oral exposure to TCS (1 mg/kg/day from GD14 to PND20 during lactation) increased bodyweight, blood glucose, and cholesterol in 5-month-old offspring, as well as food intake [230]. The increase in the latter is probably due to the increased hypothalamic expression of orexigenic neuropeptides. Moreover, in aged rats with high in utero exposure to TCS, a decreased hepatic glycogen content was observed, while the serum and hepatic triglyceride content increased with the upregulation of genes involved in carbohydrate and lipid metabolism pathways in the liver [231]. This was also observed in mice, along with an increase in serum and liver triglycerides with the increased gene expression of a protein involved in fatty acid synthesis, but the decreased gene expression of a protein involved in fatty acid oxidation [232].

What has been shown for any of these pollutants, depending on the dose studied, the route of exposure, the group of offspring considered (male or female), their age at the
time of analysis, in addition to the animal model and species, must be rethought when it
comes to combining these pollutants, as all of the cards of effects have to be reshuffled. The
study of cocktail effects is likely to yield surprising results, like those investigated in a few
studies [233], with a reduction in live weight for all of the mixtures in females regardless of
the dose, and in males only at mid-dose.

Maternal n-BuP (parabens) exposure in mice was shown to induce a higher food intake and weight gain in female offspring only, probably reducing hypothalamic POMC expression induced by epigenetic modification [234].

According to the literature, early-life exposure to DEHP is potentially associated
with increased adiposity in rodents [235,236]. Perinatal exposure to different phthalates
will cause different metabolic outcomes in mice, with sex-specific responses. In mice,
females exposed in utero to DEHP exhibited increased body fat and decreased lean mass,
whereas exposure to DiNP induced only a decrease in glucose tolerance. In contrast,
prenatal exposure of males to phthalate did not lead to any significant differences in the
measured metabolic outcomes [237]. However, other studies in mice have reported that
prenatal exposure to DEHP at a low dose (0.2 mg/kg/day) led to a metabolic syndrome
in male offspring, including abnormal adipogenesis, energy expenditure, and glucose
metabolism, by the deregulation of hepatic thiamine transport enzymes [238]. In the mouse
liver, perinatal exposures to phthalates were associated with the short- and long-term
activation of PPAR target genes, which was manifested by increased fatty acid production
in early postnatal life and increased fatty acid oxidation in adulthood [239]. A similar
accumulation of hepatic lipids was observed in rats exposed to DEHP during the perinatal
period [240], as described for TCS [231,241]. Additionally, maternal exposure to DEHP in
rats (0.75 mg/kg bw/day from GD6 to PND21) decreased serum insulin and triglyceride
levels in PND70 male offspring. These outcomes were linked by the authors to the elevated
expression of PPARγ (mRNA and protein) in white adipose tissue [242]. At PND21, male
and female offspring pups exposed in utero to DEHP (700 mg/kg bw/day during the
last third of gestation and lactation) and DBP (500 mg/kg bw/day) showed increased
fasting glucose levels, as well as metabolic alterations [243]. Indeed, gestational exposure
to DEHP has been shown to promote β-cell dysfunction and whole-body glucometabolic
abnormalities in F1 offspring by downregulating the expression of critical genes involved
in β-cell development and function [244]. Maternal exposure to a lower dose of DEHP
(10 and 100 mg/kg bw/day) from GD9 to PND21 (lactation period) by oral gavage in
the male offspring induced hyperglycemia, impaired tolerance to glucose and insulin, as
well as hyperinsulinemia at PND80. This phenotype occurs because the levels of insulin
signaling molecules such as insulin receptors, IRS1, Akt, and its phosphorylated forms are
reduced [245]. In addition, maternal exposure to DEHP throughout gestation has been
shown to disrupt thyroid function in offspring pups. DEHP disrupts thyroid function
by damaging thyroid follicles and affecting thyroid transcription factor 1 (TTF-1), paired
box 8 (PAX8), sodium iodide symporter (NIS), and thyroid peroxidase (TPO) both at the
transcriptional level and at the protein level. In pups, this damage leads to a reduction in
total thyroxine (T4) and an increase in thyroid-stimulating hormone (TSH) [246]. In adults,
this damage was observed without an altered macro-index such as bodyweight in males
at 14 weeks. The males were then likely to develop insulin resistance (hyperinsulinemia),
oxidative stress (increased CAT catalase), and hypothyroidism (decreased T4) [247].

Prenatal exposure to low doses of DEHP or other phthalates has resulted in life-long
metabolic consequences in a sex-dependent manner in offspring. Most of the time, the
results are contradictory, with effects only in females or males. This exposure suggests a
potential risk factor for later obesity and metabolic syndrome development in adulthood.
Additionally, most studies on perinatal exposure to phthalates introduce bias to the extent
that they focus only on the effects on male offspring in rodents, and only on female offspring
in sheep.
<table><caption>**Table 4.** Synthesis of the offspring outcomes related to maternal oral exposure to phenols, phthalates, and/or parabens during the perinatal period (all or part of gestation and/or lactation) according to the animal model, the chemicals, the dose administrated, the exposure route and duration.</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.01, 10 mg/kg bw/d</td><td>Oral route (food)</td><td>Paternal exposure (12 weeks from 5 weeks of age)</td><td>0-20 weeks F1</td><td>Metabolism</td><td>Glucose tolerance and body composition: no effect<br>Glucose tolerance at 4 and 7 months of age: BPA ↓ (♀)</td><td>Insulin-dependent glucose disposal in post-pubertal: BPA ↑ (0.01)<br>Glucose tolerance (6 months) and glucose disposal (1 year): BPA ↓<br>Glucose accumulation: BPA ↑</td><td>[214]</td></tr><tr><td>Mouse</td><td>BPA</td><td>500 mg/kg bw/d</td><td>Oral administration</td><td>GD8-GD14</td><td>PND56</td><td>Reproduction/fertility</td><td>Mortality at birth: BPA ↑<br>Serum testosterone, FSH, and LH level: BPA ↓ (PND56)<br>Serum estradiol level: BPA ↑ (PND56)<br>Bax protein expression in Leydig cells, ovaries, and testis: BPA ↑<br>Ovaries and testis Bcl-2 protein expression: BPA ↓</td><td>Number of mature spermatozoids: BPA ↓ (♂)<br>Number of granular cells: BPA ↓ (♀)</td><td>[248]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.01, 0.1 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD9-16</td><td>PND1-21 + 6 months</td><td>Glucose homeostasis</td><td>Bodyweight at birth and PND21: BPA ↑ (0.01)/↓ (0.1)<br>Bodyweight at 3 months of age: BPA ↓ (♀)<br>Insulin sensitivity and glucose tolerance: BPA ↓ (♂: 6 months of age)<br>Serum insulin level: BPA ↑ (♂)<br>Serum glycerol level: BPA ↑ (♂: 0.1)<br>Glucose-stimulated insulin secretion and islets insulin secretion: BPA ↑ (0.01)<br>Global intracellular calcium entry after glucose stimulation: BPA ↑<br>Pancreatic β-cell area: no effect<br>Pancreatic β-cell proliferation: BPA ↓ (♂)</td><td>Litter size: no effect<br>Maternal glucose intolerance and total mean area under the curve of glucose tolerance: BPA ↑ (0.01)<br>Akt phosphorylation in the maternal liver after insulin stimulation: BPA ↓ (0.01)<br>Insulin and TG serum level: BPA↑<br>Plasma glycerol and leptin level: BPA ↑<br>Maternal bodyweight 3-4 months after delivery: BPA ↑<br>Food intake: no effect</td><td>[222]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>5 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-20</td><td>PND5-3 weeks of age</td><td>Metabolism</td><td>Bodyweight: BPA ↓<br>Serum lipid parameters: BPA ↓ (σ)<br>Serum glucose level: BPA ↑ (σ)<br>Liver number of differentially expressed genes (DEGs): 855<br>Liver Cyp51 (sterol 14-α demethylase) expression (DEG common): BPA ↑<br>GSEA-enriched pathways: lipid metabolism (lipid transport/fatty acid metabolism/cholesterol biosynthesis)/energy metabolism (biological oxidation/tricarboxylic acid cycle)<br>Liver peroxisome proliferator-activated receptor (PPAR) signaling and arachidonic acid pathways enrichment: BPA ↓<br>Liver number of differentially methylated CpGs (DMCs) in males: 476<br>Number of transcription factor (TFs) differentially expressed in the liver: 14 (Esr1, Esrra, Hnf1a, Pparg, Tcf21, and Srebf1)<br>Liver differentially expressed transcription factor involvement: estrogen and PPAR signaling</td><td>Number of differentially expressed genes (DEGs): 86 (adipose tissue)/93 (hypothalamus)<br>Cyp51 expression (DEG common): BPA ↑ (hypothalamus)/↓ (adipose tissue)<br>Number of DMCs in males: 5136 (adipose tissue)/104 (hypothalamus)<br>Top DMC-enriched processes: intracellular and extracellular communication and signaling-related pathways</td><td>[249]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.01, 10 mg/kg bw/d</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>PND7, 14, and 21 weeks of age</td><td>Metabolism</td><td>F2 and F3 bodyweight: no effect<br>Obesity phenotype: BPA ↑ (σF2, F3)<br>Body composition: no effect (♀)<br>Glucose tolerance and insulin sensitivity: no effect (σF3)<br>Glucose-stimulated insulin secretion: BPA ↑ (σF3: 0.01)<br>β-cell mass and proliferation: BPA ↓ (σF3: 0.01)</td><td>Proinflammatory cytokine levels: BPA ↓ (IL-1β and IL-12p70)<br>Immunostaining of CD3 (T lymphocyte markers) and F4/80 (macrophage markers) in the pancreas: BPA ↑ (σF3)<br>Transforming growth factor-beta 1 (TGF-β)1 level: BPA ↑ (10)<br>F2 maternal metabolic milieu: no effect</td><td>[213]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.1, 1, or 10 mg/L</td><td>Oral route (drinking water)</td><td>GD0-PND21</td><td>7 to 28 weeks of age</td><td>Metabolism</td><td>Mean insulitis grade and number of diabetic mice: BPA ↑ (10)<br>Offspring's number, fetal bodyweight, and sex ratio: no effect<br>Number of regulatory T cells: BPA ↑ (♀: 10)<br>Number of F4/80-positive tissue-resident macrophages in pancreatic islets: BPA ↓ (♀: 10)</td><td>Activated caspase-3-positive, insulin-positive, and glucagon-positive apoptotic cells in pancreatic islets: BPA ↑<br>Number of apoptotic cells: BPA ↑<br>LPS-induced interleukin secretion from splenocytes: BPA ↑ (10)<br>IL-2 secretion: BPA ↓ (1, 10)</td><td>[228]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.01 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD9-16</td><td>PND0 and 30</td><td>Pancreatic function</td><td>Pancreatic β-cell area, mass, and proliferation: BPA ↑ (♀)</td><td></td><td>[212]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.01, 0.1 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD9-16</td><td>PND30</td><td>Retinoid signaling pathway</td><td>ATRA (all-trans-retinoic-acid) hepatic concentration: BPA ↑ (0.01)<br>Expression of genes involved in ATRA biosynthesis: BPA ↑ (0.01: Adh1, Aox1, and Cyp1a2)<br>Expression of genes involved in storage and metabolism of retinoids and ATRA biotransformation: no effect<br>Expression of genes involved in the disposition of retinoid metabolites: BPA ↑ (0.1: Bcrp)/↓ (Mrp3)<br>Expression of nuclear receptors modulated retinoid-dependent signaling in hepatic cells: BPA ↑ (0.01: Fgf21)/↓ (0.1: Rxr-β)</td><td></td><td>[223]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.01, 0.1 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD9-16</td><td>PND0, 21, 30, and 120</td><td>Pancreatic function</td><td>Bodyweight: BPA ↓ (PND0/PND21: 0.1)/↑ (PND21-30)<br>Bodyweight gain: BPA ↓ (PND0-21)/↑ (PND21-30)<br>Non-fasting plasma insulin, plasma leptin, and C-peptide level: BPA ↑<br>Insulin release: BPA ↑<br>Glucose-stimulated insulin secretion and pancreatic insulin content: BPA ↓ (0.01)<br>Number and expression of DEGs in the islets of Langerhans: BPA ↑ (325)/↓ (330)<br>Pancreatic β-cell mass: BPA ↑ (PND0/PND21/PND30)/↓ (0.1: 4 months)<br>Pancreatic percentage of β-cell area: BPA ↑ (PND30: 10)<br>β-cell proliferation and apoptosis: BPA↑ (PND30)</td><td>DEGs involvement: cell cycle/mitosis/cell division</td><td>[221]</td></tr><tr><td>Mouse</td><td>BPA</td><td>10, 100, or 1000 nM</td><td>Oral route (drinking water)</td><td>GD0-PND21</td><td>PND21, 42</td><td>Spleen immune response</td><td>Litter size, birth weight, survival rate, and sex ratio: no effect<br>Weaning weight: BPA ↑ (PND21: 10)/↑ (PND35: 1000)<br>T helper 17 cell (Th17) cell frequency in spleen: BPA ↑ (♀: 100, 1000/♂: 1000)<br>RORγt expression: BPA ↑ (♀: 100, 1000/♂: 1000)<br>IL-17 and IL-21 production level by Th17 cells: BPA ↑<br>Serum IL-6 and IL-23 levels: BPA ↑<br>Serum TGF-β level: no effect</td><td>Gestational weight: no effect</td><td>[224]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.00005, 0.05, or 50 mg/kg food/d</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>6 months of age</td><td>Inflammatory mediators/asthma</td><td>Bone marrow-derived mass cell (BMMC) cysteinyl leukotriene secretion: BPA ↑<br>BMMC prostaglandin D2 production: ↑ (50)<br>BMMC tumor necrosis factor-alpha (TNFα) secretion: BPA ↑<br>IL-13 level: BPA ↑ (50)<br>IL-4, IL-5, and IL-6 levels and histamine release: no effect<br>BMMC DNA methylation level: BPA ↓ (50)</td><td>-</td><td>[225]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.01, 10 mg/kg bw/d</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>GD9.5-10.5, GD16.5-17.5, PND1, 14, 21, 28, and 98-117</td><td>Metabolism</td><td>Fetal bodyweight at PND1, 14, and 21: BPA ↓ (σ: 0.01)<br>Fetal bodyweight at PND28 and food intake: no effect<br>Bodyweight between PND98 and 117: BPA ↑ (σ: 0.01)<br>Body fat content between PND98 and PND 117: BPA ↑ (σ)<br>Bone mineral density and content: BPA ↓ (σ: 10)<br>Insulin level: BPA ↑ (σ: 0.01)<br>Glucose intolerance and basal rate of insulin release: BPA ↑ (σ: 10)<br>Maximal glucose-stimulated insulin release: BPA ↓ (σ: 0.01)</td><td>F2 total insulin-like growth factor 2 (Igf2) mRNA expression: BPA ↑ (10)<br>Bodyweight and fat: no effect<br>Glucose intolerance: BPA ↑ (σ: 10)<br>Insulin secretion: no effect<br>Body fat content at PND98 to 117: BPA ↑ (σ: 10)<br>Islets glucose-stimulated insulin secretion: BPA ↓ (0.01)</td><td>[226]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.006, 0.06, or 0.6 mg/kg bw/d</td><td>Subcutaneous capsule</td><td>GD9 to at least 3 weeks</td><td>PND2, 21, and 3 to 12 weeks of age</td><td>Glucose metabolism</td><td>Bodyweight at weaning and 5 weeks: BPA ↓ (σ: 0.006)<br>Bodyweight gain between 3 and 5 weeks: BPA ↑ (σ: 0.006)/↓ (σ: 0.06)<br>Bodyweight gain between 5 and 12 weeks: BPA ↑ (σ: 0.6)<br>Bodyweight at 12 weeks and food intake: no effect<br>Glucose intolerance: BPA ↑<br>Blood glucose level: BPA ↓ (σ: 0.6)<br>Percent change relative to baseline in blood glucose level 30 min after glucose injection (GTT): BPA ↑ (σ: 0.06/0.6)<br>Percent change relative to baseline in blood glucose level 60 min after GTT: BPA ↑ (σ)</td><td>[211]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA</td><td>0.003, 0.01, 0.03, 0.1, 0.3, 1, or 3 mg/kg bw/d</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>PND21, and 5 to 23 weeks of age</td><td>Metabolism</td><td>Litter size, sex ratio, and survival rate: no effect<br>Bodyweight: BPA ↑ (σ' from 6 weeks)/↓ (φ8 weeks)<br>Bodyweight gain, body length, femur length and weight, and relative liver weight: no effect<br>Glucose tolerance: no effect<br>Liver weight: BPA ↑ (σ')/↓ (φ)<br>Interscapular, perigonadal, perirenal, and caudal subcutaneous fat pad weights: BPA ↓ (φ)<br>Perirenal white adipose tissue adipocyte size: BPA ↑ (σ')/↓ (φ)<br>Histopathological liver examination: no effect<br>Circulating glucagon level: BPA ↓<br>Insulin level: no effect<br>Adiponectin, leptin, free fatty acid, and TG levels: BPA ↓ (φ)</td><td>Mortality: no effect<br>Dams bodyweight, bodyweight gain, and food consumption: no effect<br>Brown adipose tissue ucp1 expression: BPA ↑ (φ)<br>Muscle weight: BPA ↓ (φ)<br>Histopathological quadriceps femoris muscle and thyroid gland examination: no effect<br>Histopathological pancreatic islets and adrenal examination: no effect</td><td>[227]</td></tr><tr><td>Mouse</td><td>BPA</td><td>0.00005, 0.05, or 50 mg/kg diet</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>3, 6, 9, and 10 months of age</td><td>Metabolism</td><td>Litter size, survival rate and sex ratio: no effect<br>Fetal wean weight: BPA ↓ (0.00005)<br>Food intake: BPA ↑ (φ6 months)<br>Bodyweight and body fat mass: BPA ↑ (φ6 months: 0.05)<br>Mean baseline glucose and insulin level: BPA ↓ (φ: 50)<br>Mean adiponectin level: BPA ↑ (φ: 0.05)</td><td>Oxygen consumption: BPA ↑ (φ3 months: 50/φ6 months: 0.05, 50/φ9 months: 0.00005/σ'9 months: 0.05, 50)<br>Oxygen consumption corrected for lean body mass: BPA ↑<br>Carbon dioxide production level: BPA ↑ (φ9 months: 0.00005/φ6 months: 50/σ'3 months: 0.05, 50)<br>Respiratory exchange ratio: no effect</td><td>[229]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="12">Rat</td><td rowspan="12">BPA</td><td rowspan="12">0.05 or 5 mg/kg bw/d</td><td rowspan="12">Oral gavage</td><td rowspan="12">GD3 to GD18</td><td rowspan="12">PND21 and PND60</td><td rowspan="12">Lipid metabolism</td><td>Total lipid content: BPA ↓ (♀PND21: 5)</td><td rowspan="12">PUFAs: BPA ↑ (PND21)<br>MUFAs (C15:1 and C18:1): BPA ↑ (♂PND21)<br>Saturated very-long-chain FAs (C32:0 and C34:0): BPA↓ (♂PND21: 0.05/♀PND21: 5)<br>Unsaturated very-long-chain FAs (C36:4): BPA ↓ (♀PND21)</td><td rowspan="12">Potential biomarkers for prenatal BPA exposure: ♀(8 genes, 9 FAs, 1 cholesterol ester, 19 TG, 3 diacylglyceryl-trimethylhomoserine, 1 ceramide, and 1 phosphatidylcholine)/♂(30 genes, 1 FA, 1 TG, 1 phosphatidylmethanol, 2 phosphatidylcholine, and 4 phosphatidylethanolamine)</td><td rowspan="12">[215]</td></tr><tr><td>Lipid accumulation and TG level: BPA ↓ (♀PND21)</td></tr><tr><td>PLS-DA analysis: separations of control, low-BPA-, and high-BPA-dose exposed groups</td></tr><tr><td>Changes in several lipid classes: BPA ↑ (FA, acylcarnitine, cholesterol ester, monoacylglycerol, TG, monogalactosyl diacylglycerol, sphingomyelin, cardiolipin, phosphatidylserine, phosphatidylglycerol, and sulfatide)</td></tr><tr><td>Total free fatty acid and acylcarnitine level: BPA ↑ (PND21)</td></tr><tr><td>Level of total monoacylglycerol: BPA↑ (♂PND21/♀PND21: 0.05)</td></tr><tr><td>Level of total cholesterol ester: BPA ↑ (♀PND21: 5)</td></tr><tr><td>Lipogenesis: BPA ↑ (♀PND1)/↓ (♂PND1 to PND21)</td></tr><tr><td>Ceramide and phosphatidylcholine level: BPA ↓ (♀PND21)</td></tr><tr><td>Cardiolipin level: BPA ↓ (♀PND21)/↑ (♂PND21: 0.05)</td></tr><tr><td>Sulfatide level: BPA ↑ (♀PND21 and PND60)</td></tr><tr><td>Total sphingomyelin, phosphatidylglycerol level: BPA ↑ (♂PND21)</td></tr><tr><td>Digalactosyl and sulfoquinovosyl diacylglycerol level: BPA ↑ (♀PND60)</td></tr><tr><td>Phosphatidylethanolamine level: BPA ↓ (♂PND60)</td></tr><tr><td>Gpd1 gene expression involved in glycerophospholipid metabolism: BPA ↓ (♀)</td></tr><tr><td>Gnpat gene expression involved in glycerophospholipid metabolism: BPA ↓ (♂)</td></tr><tr><td>Bodyweight: BPA ↑ (♀PND1)</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>BPA</td><td>0.05 or 5 mg/kg bw/d</td><td>Oral gavage</td><td>GD3-18</td><td>PND1, 13, 21, 30, and 60</td><td>Liver metabolism</td><td>Bodyweight: BPA ↑ (♀PND1-PND60/♂PND13, PND21-PND30/♂PND60: 5)<br>Liver weight: BPA ↑ (♀PND21)/↓ (♂PND21)<br>Relative spleen weight: BPA ↑ (♀PND1)<br>Relative heart weight: BPA ↑ (♀PND1)/↓ (♂PND60)<br>Relative kidney weight: BPA ↓ (♀PND60)<br>Number of DEGs: 1239 (♀PND1: 0.05)/1672 (♂PND1: 5)/1250 (♂PND1: 0.05)/722 (♂PND1: 5)/217 (♀PND21: 0.05)/337 (♀PND21: 5)/326 (♂PND21: 0.05)/443 (♂: PND21: 5)<br>Principal component analysis: a clear distinction between exposed and control PDN1 males, between exposed and control PND1 females, between exposed and control groups, between high-BPA exposed and control PDN21 females, between exposed and control PND21 males, and between PND21 males and females<br>Number of differentially expressed proteins (DEPs): 101 (♀PND1)/188 (♂PND1, PND21: 0.05)/204 (♂PND1: 5)/176 (♀PND21: 0.05)/159 (♀PND21: 5)/137 (♂PND21: 5)<br>Number of enriched pathways common from DEGs and DEPs in both sexes: 30 (♀ > ♂)<br>Top pathways enriched: fatty acid degradation, steroid hormone biosynthesis, and PPAR signaling pathways<br>Enriched diseases: fatty liver, diabetes, obesity, and cardiovascular diseases<br>TC and cholesteryl ester levels: BPA ↑ (♀PND60: 0.05)<br>HDL cholesterol level: BPA ↑ (♀PND60: 0.05/♂PND60: 5)<br>LDL cholesterol level: BPA ↓ (♂PND60: 0.05)</td><td>Anogenital distance (AGD): BPA ↑<br>Anogenital index (AGI): BPA ↑ (♀PND21)</td><td>[216]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>BPA</td><td>0, 0.05, 0.5, or 5 mg/kg bw/d</td><td>Oral gavage</td><td>GD5-19</td><td>PND1, 21 and 56</td><td>Lipid metabolism</td><td>Bodyweight: BPA ↓ (♀PND56: 0.5)<br>Liver-to-bodyweight ratio: BPA ↑ (♀: 0.05/♀: 0.5)<br>Serum TG and TC level: BPA ↑<br>TG liver level: BPA ↑<br>TC liver level: BPA ↑ (PND21)<br>Liver fatty acid oxidation-related gene expression: BPA ↓ (PND21 PPARα/PND21 CPT1α: 5/PND56 PPARα: 0.5, 5)<br>Liver fatty acid oxidation-related protein expression: BPA ↓ (PND21 PPARα CPT1α: 5/PND56 PPARα 0.5, 5/PND56 CPT1α: 5)<br>Liver fatty acid synthesis-related gene expression: BPA ↑ (SREBP-1, ACC1, FAS, SCD-1)<br>Liver fatty acid synthesis-related protein expression: BPA ↑ (SREBP-1, SCD-1)</td><td>Liver mTOR mRNA expression: BPA ↑ (PND21: 0.05/PND56: 5)<br>Liver mTOR protein expression: BPA ↑ (PND21/PND56: 0.5, 5)<br>Liver CRTC2 mRNA expression: BPA ↑ (PND21/PND56: 0.05, 0.5)<br>Liver CRTC2 phosphorylation level: BPA ↑ (0.5, 5)</td><td>[250]</td></tr><tr><td>Sheep</td><td>BPA</td><td>0.05, 0.5, or 5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30-90</td><td>GD68, 6, 14, 15, 19, 21 weeks, and 13 months of age</td><td>Metabolism</td><td>Fasting glucose level: BPA ↑ (6 weeks: 0.05)<br>Cumulative insulin and insulin/glucose ratio responses: BPA ↑ (13 months: 0.5)<br>Acute insulin response: BPA ↑ (0.5)<br>Glucose tolerance: no effect (15 months)<br>Bodyweight, total fat, visceral fat, and subcutaneous fat: no effect<br>Visceral adipose tissue cell area and diameter: BPA ↑ (♀)<br>Subcutaneous adipose tissue marker of macrophage infiltration CD68 expression: BPA ↑</td><td></td><td>[217]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Sheep</td><td>BPA</td><td>0.5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30 to GD100</td><td>21 months F1</td><td>Liver and muscle metabolic function</td><td>Number of differentially expressed (DEGs) genes in the liver: BPA ↓ (138)/↑ (56)<br>Top 10 DEGs in the liver: BPA ↓ (WFDC2, MSLN, MMP7, COLEC12, and SLC44A4)/↑ (CCDC152 and three other genes with yet-to-be-identified roles, including thioredoxin-like protein 1, putative olfactory receptor 3A4, and elongation factor 1-beta-like)<br>Number of enriched gene pathways in the muscle and liver: 157<br>Pathways enriched in the muscle and liver: mitochondrial, extracellular matrix-related, and oxidative phosphorylation pathways<br>Enriched pathways in the liver: the response to oxidative stress, lipid biosynthetic process, endoplasmic reticulum, and Golgi apparatus structure and function<br>Number of differentially expressed lncRNAs in the liver: BPA ↓ (49)/↑ (28)<br>Number of differentially expressed miRNAs in the liver: BPA ↓ (6)/↑ (8)<br>Number of differentially expressed snoRNAs in the liver: BPA ↓ (63)/↑ (64)<br>Number of differentially expressed snRNAs in the liver: BPA ↓ (15)/BPA ↑ (40)<br>Top 10 of miRNAs in liver: BPA ↑ (MIR200B, MIR409, MIR125B, MIR543, MIR25, MIR22, and MIR191)/↓ (MIR26B and MIR154A)<br>Potential biomarkers of prenatal BPA impact in the liver: BPA ↓ (4 lncRNAs)/↑ (5 lncRNAs)</td><td>Number of differentially expressed genes in the muscle: BPA ↑ (80)/↓ (32)<br>Top 10 dysregulated genes in muscle: BPA ↓ (multidrug resistance-associated protein 4-like genes, ATP-binding cassette subfamily C member 4-like gene, two uncharacterized genes, high mobility group protein 20A-like gene, and 40S ribosomal protein S3a pseudogene)/↑ (HBB)<br>Pathways enriched in the muscle: RNA biosynthetic process, immune function, and collagen synthetic gene pathways<br>Number differentially expressed lncRNAs in the muscle: BPA ↑ (6)<br>Number of differentially expressed snoRNAs in the muscle: BPA ↓ (47)/↑ (18)</td><td>[219]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Sheep</td><td>BPA</td><td>0.05, 0.5, or 5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30-90</td><td>21 months F1</td><td>Metabolism</td><td>Liver IL-1β, IL-6, and chemokine (C-C) ligand 2 expression: BPA ↓<br>Liver CD68 (macrophage marker) expression: BPA ↑<br>Liver TNF-α expression: BPA ↑ (0.05)<br>Visceral adipose tissue IL-1β expression: BPA ↑<br>Visceral adipose tissue CCL2 expression: BPA ↑ (5)<br>Liver oxidative stress marker 3-nitrotyrosine level: BPA ↑<br>Visceral adipose tissue 3-nitrotyrosine level: BPA ↑ (5)<br>Liver and visceral adipose tissue lipid peroxidation marker Thiobarbituric acid reactive substances (TBARS) level: BPA ↑ (5)<br>Liver antioxidant (GSR) mRNA expression: BPA ↓ (0.5, 5)<br>Liver antioxidant superoxide dismutases 1 (SOD1) mRNA expression: BPA ↓ (0.05, 0.5)<br>Liver SOD2 mRNA expression: BPA ↑ (0.05)<br>Visceral adipose tissue glutathione reductase (GSR) and Cyp19 mRNA expression: BPA ↑ (0.5, 5)<br>Liver GSR activity: BPA ↑ (5)<br>Plasma TG content and high-molecular-weight adiponectin level: no effect<br>Hepatic TG content: BPA ↑ (0.5, 5)<br>Plasma low-molecular-weight adiponectin level: BPA ↓ (5)<br>Visceral adipose tissue Cyp17 mRNA level: BPA ↑ (5)<br>Visceral adipose tissue AR mRNA expression and CYP17 and estrogen receptor 1 (ESR1) protein level: no effect<br>Visceral adipose tissue AR protein expression: BPA ↓ (5)<br>Visceral adipose tissue estrogen receptor 2 (ESR2) protein level: BPA ↓ (0.5, 5)<br>Visceral adipose tissue Esr1 mRNA expression: BPA ↑</td><td>Skeletal muscle IL-6 expression: BPA ↑<br>Skeletal muscle CD38 expression: BPA ↑ (0.5, 5)<br>Skeletal muscle IL1B expression: BPA ↑ (0.5)<br>Skeletal muscle 3 nitrotyrosine and TBARS levels: BPA ↑ (0.05, 0.5)<br>Skeletal muscle SOD1 mRNA expression: BPA ↓ (5)<br>Skeletal muscle GSR mRNA expression: BPA ↓<br>Skeletal muscle SOD2 mRNA expression: BPA ↑ (0.05)<br>Skeletal muscle GSR activity: BPA ↓ (0.5)<br>Muscular TG content: BPA ↑ (0.5, 5)</td><td>[218]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Sheep</td><td>BPA</td><td>0.5 mg/kg bw/d</td><td>Subcutaneous injections</td><td>GD30 to GD100</td><td>21 months F1</td><td>Cardiovascular function</td><td>Lung, kidney, and adrenal weight: BPA ↓<br>Heart rate and blood pressure: no effect</td><td>Cardiac failure gene expression in the left ventricle: BPA ↑ (ANP, COL1A1)<br>Cardiac failure gene expression in the right ventricle: BPA ↑ (ANP)/↓ (COL1A1)</td><td>[251]</td></tr><tr><td>Sheep</td><td>BPA</td><td>0.05, 0.5, or 5 mg/kg bw/day</td><td>Subcutaneous injections</td><td>GD30-GD90</td><td>GD65, GD90, 21 months F1</td><td>Pancreas</td><td>Fetal weight: no effect (GD65, GD90)<br>Pancreas weight: no effect (GD65, GD90)<br>Pancreas/fetal weight: no effect (GD65, GD90)<br>Pancreatic islet insulin: BPA 0.5 ↓ (GD90)<br>Beta-cell size: no effect<br>Beta-cell count: BPA 0.5 ↓ (GD90)<br>Pancreatic islet glucagon: no effect (GD60, GD90), BPA 0.05 ↑ (adult F1)<br>Alpha-cell size: BPA 0.5 ↑ (GD90), BPA 0.05 ↑ (adult F1)<br>Alpha-cell count: BPA 0.5 ↑ (GD90)<br>Alpha-to-Beta-cell count: BPA 0.5 ↑ (GD90), BPA 0.05 ↑ (adult F1)<br>Islet collagen accumulation: 0.05 ↑ (adult F1)<br>Pancreatic apoptosis: no effect (GD65, GD90, and adult F1)<br>Gene expression of apoptotic and cell proliferation markers: no effect (GD65, GD90, and adult F1)<br>Fibrosis gene expression: BPA ↓ ACT1 (GD65)</td><td></td><td>[220]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>BPS</td><td>0.01, 0.05 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-PND21</td><td>PND21, PND160-180</td><td>Metabolism, endocrine system</td><td>Plasma triacylglycerol and thyroxine (T4) level: BPS ↓ (σPND21: 0.05)<br>Plasma25-hydroxyvitamin D (25(OH)D) level at PND21 and 180: BPS ↑ (♀: 0.05)<br>Food intake: BPS ↓<br>PND180 visceral fat mass: BPS ↓ (σ)<br>PND180 brown adipose tissue lipid droplets: BPS ↓ (650: ♀)<br>Preference for high-fat diet over high-sugar diet: BPS ↑<br>Plasma triacylglycerol level: BPS ↑ (σPND180: 0.05)<br>Plasma total triiodothyronine (T3) level: BPS ↓ (σPND180: 0.05)<br>PND180 plasma free T4 level: BPS ↓ (♀)</td><td>Plasma testosterone level: BPS ↓ (0.05)<br>Plasma progesterone level: BPS ↓ (♀PND180: 0.01)</td><td>[210]</td></tr><tr><td>Mouse</td><td>BPA, BPS, BPF</td><td>0.005, 0.05 mg/kg bw/d</td><td>Oral route</td><td>GD15-PND21</td><td>PND70</td><td>Intestinal and systemic immune response</td><td>Bodyweight: BPF ↓ (0.05: PND70)<br>Litter size: BPA ↓ (0.05)/BPS ↓ (0.005)<br>Fecal immunoglobulin A (IgA) level: BPA ↓ (♀: 0.05)<br>Lipocalin fecal level: BPS ↑<br>Plasmatic immunoglobulin G (IgG) level: BPA ↓ (♀)/BPS ↓ (♀)/BPF ↓ (♀)<br>Total IgA level: no effect<br>T helper 1 (Th1) subpopulation in the lamina propria: BPA ↑ (♀: 0.005)<br>Th1 frequency in the spleen: BPA ↑ (♀: 0.05)/BPF ↑ (♀: 0.05)<br>Intestinal Th17 frequency: BPA ↑ (0.05)/BPF ↑ (0.05)<br>Small intestine lamina propria cell T cell receptor (TCR)-stimulated IL-17 secretion: BPA ↑ (0.05)<br>T cell frequency in siLP and at systemic level: BPA ↓ (0.05)<br>Principal component analysis: a higher dose BPA- and BPF-exposed groups were the most distant ones and were well separated from the control group/The higher dose BPS-exposed group was less distant to the control group/The high-dose BPA exposed group differed from the control group in terms of the IFN-γ and IL-17 levels and Treg frequency/BPF-exposed groups differed to the control in terms of bodyweight and plasmatic IgA level</td><td>Plasma-specific anti-E. coli IgG: BPS ↑ (0.05)<br>Plasma-specific anti-E. coli IgA: no effect</td><td>[209]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>BPA + NP, OP or IsoBP</td><td>5, 50, or 500 mg/kg bw/d</td><td>Oral gavage</td><td>GD1-21</td><td>PND1, and 21 and 41</td><td>Fetal development</td><td>Survival rate: BPA + OP ↓ (5)<br>Sex ratio: BPA + OP ↓ (50)/BPA + IsoBP (5, 500)<br>Bodyweight: BPA + OP ↓ (♀)/BPA + NP ↓ (♀)/BPA + IsoBP ↓ (♀)<br>Pituitary and liver weight: BPA + OP ↑ (♀: 5, 500)/BPA + IsoBP ↑ (♀: 5, 50)<br>Adrenal weight: BPA + OP ↑ (5, 500)/BPA + IsoBP ↑ (♀: 5, 50)<br>Kidney weight: BPA + OP ↑ (♀: 5, 500)/BPA + IsoBP ↑ (♀: 5, 500)/BPA + IsoBP ↓ (♂: 5, 500)</td><td>Dams gestational day times: BPA + IsoBP ↓/BPA+ NP ↓ (50, 500)/BPA + OP (50, 500)<br>Spleen and reproductive organs weight: no effect<br>Thyroid weight: BPA + NP ↓ (♂)/BPA + OP ↓ (♂: 50)/BPA + IsoBP ↓ (♂: 5/50)</td><td>[233]</td></tr><tr><td>Rat</td><td>BPA, DEHP</td><td>5 µg/kg bw/d (BPA) and/or 5µg and 7.5 mg/kg bw/d (DEHP)</td><td>Oral administration</td><td>GD6-GD21</td><td>PND1, 7, 14, and 21, PND21 to PND84, PND112, and PND168</td><td>Fetal development/food intake</td><td>Maternal gestational weight gain: no effect<br>Post-weaning bodyweights, and food and water intakes: no effect<br>Heart weight: BPA + DEHP ↑ (♂: 7.5)<br>Relative thymus weight: BPA ↓ (♂)/DEHP ↓ (♂: 7.5)<br>Cortex apoptotic index: BPA + DEHP ↑ (7.5)<br>Medulla apoptotic index: BPA + DEHP (♀: 7.5)</td><td>Abortion rate: BPA + DEHP ↑<br>Gestational index: DEHP ↓ (7.5)/BPA + DEHP ↓<br>Litter size and PND21 pup weight and sex ratio: no effect<br>Crown-to-rump length: BPA ↑/BPA+ DEHP ↑ (5)<br>Chest circumference: DEHP ↑ (5)/DEHP + BPA ↑ (5)/DEHP ↓ (7.5)/DEHP+ BPA ↓ (7.5)</td><td>[158]</td></tr><tr><td>Mouse</td><td>TCS</td><td>4, 8 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-14</td><td>PND1, 30, and 60</td><td>Placenta/metabolism</td><td>Bodyweight gain: TCS ↑ (PND5, PND30, PND60: 8)<br>Visceral fat-to-bodyweight ratio and adipocyte size: TCS ↑ (PND30: 8)<br>Fasting plasma glucose and serum insulin level: TCS ↑ (PND60)<br>Areas under the curve of glucose and insulin tolerance test: TCS ↑ (PND60)<br>The phenotype of insulin resistance and hyperphagic obesity: TCS ↑<br>Cumulated food intake within 4 days: TCS ↑ (PND30)<br>Food intakes normalized by bodyweight: TCS ↑ (PND30)<br>Oxygen consumption: no effect</td><td>[160]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>TCS</td><td>1 mg/kg food</td><td>Oral route (food)</td><td>GD14-PND20</td><td>PND1 to 8 months</td><td>Food intake/ metabolism</td><td>Bodyweight: TCS ↓ (σ'2 months)/↑ (σ'4 months/♀8 months)<br/>Food intake: TCS ↑ (5 months)<br/>Liver weight and liver-to-bodyweight ratio: TCS ↑ (σ'4 months)<br/>Serum cholesterol level and glucose concentration: TCS ↑ (8 months)</td><td>Npy and Agrp<br/>hypothalami mRNA expression (involved in the appetite regulatory network): TCS ↑ (σ')</td><td>[230]</td></tr><tr><td>Rat</td><td>TCS</td><td>10, 50 mg/kg bw/d</td><td>Oral gavage</td><td>GD0-PND21</td><td>3, 21, and 52 offspring's weeks of age</td><td>Metabolism/ microbiota</td><td>Glucose clearance: TCS ↓ (52 weeks: 50)<br/>Blood glucose level: TCS ↑ (21 weeks: 50/3 and 52 weeks: 10)<br/>Serum HDL cholesterol level: TCS ↑ (52 weeks/21 weeks: 50)<br/>Serum leptin level: TCS ↑ (52 weeks)<br/>TG and LDL cholesterol level: TCS ↑ (21 and 52 weeks)<br/>Hepatic glycogen level: TCS ↓ (52 weeks/21 weeks: 50)<br/>Number of DEGs: 709 (21 weeks: 10), 699 (52 weeks: 10), 473 (21 weeks: 50), and 1103 (52 weeks: 50)<br/>Involvement of DEGs: lipid metabolic process, fatty acid metabolic process, cellular glucuronidation, linoleic acid metabolism, glycolysis/gluconeogenesis, lipid and carbohydrate metabolism, arachidonic acid metabolism, and biosynthesis of unsaturated fatty acids</td><td>Alpha diversity of the microbial community in gut microbiota: TCS ↓ (50)<br/>Alpha diversity among gut microbiome and abundance of Bacteroides in the microbiota: TCS ↑ (50)<br/>Abundance of Verrucomicrobia and level of Akkermansia muciniphila in the microbiota: TCS ↓</td><td>[231]</td></tr><tr><td>Mouse</td><td>DEHP</td><td>10–12, 55–64, and 119–145 mg/kg bw/d</td><td>Oral route (food)</td><td>GD18</td><td>Fatty acid metabolism</td><td></td><td>α-linolenic acid level: DEHP ↑ (10–12)<br/>Palmitoleic acid level: DEHP ↑ (55–64, 119–145)<br/>Oleic acid level: DEHP ↑ (119–145)<br/>Fads mRNA expression (conversion EFA into LC-PUFA): DEHP ↑ (119–145: PPARα null mice)</td><td>Linoleic and oleic acid level: DEHP ↓ (55–64, 119–145)<br/>α-linolenic and eicosapentaenoic acid level: DEHP ↓<br/>Palmitic acid level: DEHP ↓ (10–12, 55–64)</td><td>[252]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>DEHP</td><td>0.2, 2, or 20 mg/kg bw/d</td><td>Oral gavage</td><td>GD-7 to GD21</td><td>12 weeks of age</td><td>Metabolism</td><td>Bodyweight at 12 weeks of age: DEHP ↑ (σ': 0.2) ↑ (σ': 0.2)<br/>White adipocyte hypertrophy: DEHP ↑ (0.2)<br/>Plasma TC, TG, LDL, and HDL cholesterol levels: DEHP ↑ (0.2)<br/>Lipid deposition in liver cells at 12 weeks of age: DEHP ↑ (0.2)<br/>Number of liver DEGs: DEHP ↑ (σ': 0.2: (932))/↓ (σ': 0.2 (794))<br/>DEGs involvement: metabolism-related pathways (fatty acid metabolism pathway)<br/>Hepatic thiamine and D-glucuronic acid level: DEHP ↓ (0.2)<br/>Hepatic glucose-6-phosphate and N-acethylglutamic acid level: DEHP ↑ (0.2)<br/>The area under the curve of the glucose tolerance test: DEHP ↑ (0.2)<br/>Plasmatic glucose level: DEHP ↑ (σ': 0.2)<br/>Hepatic Slc2a2 expression involved in hepatic glucose secretion and hepatocyte blood glucose uptake: DEHP ↑ (0.2)<br/>Hepatic Slc19a2 expression involved in thiamine transport: DEHP ↓ (0.2)<br/>Cumulative food intake: no effect<br/>Bodyweight gain at 12 weeks of age: DEHP ↑ (0.2)</td><td>Expression of thermogenic genes: DEHP ↓ (0.2 (Ucp1, Cidea, Adrb3))<br/>Differentially generated metabolites involvement: ascorbate and aldarate metabolism, phenylalanine, tyrosine and tryptophane biosynthesis, and thiamine metabolism</td><td>[238]</td></tr><tr><td>Mouse</td><td>DEHP</td><td>25 mg/kg chow/d (5 mg/kg bw/d)</td><td>Oral route (food)</td><td>GD-14 to PND21</td><td>5 months of age</td><td>Cardiac DNA methylome</td><td>Mortality, litter size, sex ratio, bodyweight, and relative heart weight ratio: no effect<br/>Heart DMC and differentially methylated region (DMR): DEHP ↑<br/>DEHP pathways enrichment about hypermethylated DMCs: receptor binding (♀)/neurotransmitter transport (♀)/smooth muscle differentiation (♀)/histone demethylation (♀)/insulin signaling (♀)/meiosis (♀)/glucose transport (♀)<br/>DMC and DMR mapped gene differentially methylated in human heart failure: DEHP ↑<br/>Common genes involved in cardiac fibrosis and development or contribute to sex differences in ischemia-reperfusion injury: PRKCE (♀)/SPRY1 (♀)/GJA5 (♀)/ECE1 (♀)/SMAD7 (♀)/DNMT3A (♀)</td><td>[253]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Mouse</td><td>DEHP</td><td>250 mg/kg bw/d</td><td>Oral gavage</td><td>GD0-PND21</td><td>PND21</td><td>Skeletal muscle development</td><td>Bodyweight change: DEHP ↓<br>Ratios of quadriceps, gastrocnemius, and tibialis anterior muscle weights to tibia lengths: DEHP ↓<br>Average tibialis anterior myofiber cross-sectional areas: DEHP ↓<br>Muscle proteolytic marker (MuRF1 and atrogin 1) expression: DEHP ↑<br>Muscle myogenesis marker (MyoD and Myogenin) expression: DEHP ↓</td><td>[254]</td><td></td></tr><tr><td>Mousse</td><td>DEHP</td><td>30 mg/kg bw/d</td><td>Oral administration</td><td>GD-28 to PND28</td><td>8 weeks of age</td><td>Metabolism</td><td>Diastolic arterial, systolic, and mean blood pressure: DEHP ↑<br>Heart rate: no effect<br>Bodyweight: DEHP ↑<br>White and brown adipose tissue-to-bodyweight ratio: DEHP ↑<br>White adipose tissue adipocyte size: DEHP ↑<br>Brown adipose tissue adipocyte number: DEHP ↑<br>Serum cholesterol level and liver cholesterol content: DEHP ↑<br>Serum TG and glucose level: no effect<br>LDLR, SR-B1, CYP7A1, and ABCG5 hepatic protein expression: DEHP ↓<br>LRP-1 hepatic protein expression: no effect</td><td>Aorta phosphorylated eNOS<sup>thr497</sup>: DEHP ↑<br>Aorta phosphorylated eNOS<sup>Ser1179</sup> and eNOS<sup>Ser635</sup>: DEHP ↓<br>Aorta AT1R protein expression: DEHP ↑<br>Aorta AT2R protein expression: no effect</td><td>[236]</td></tr></tbody></table>
<table><caption>Table 4. *Cont.*</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>DEHP</td><td>300, 600 mg/kg bw/d</td><td>Oral gavage</td><td>PND21 to 24 weeks (males)</td><td>25 weeks</td><td>Metabolism</td><td>Accumulation of DEHP in fat and serum: DEHP ↑ (long-term)<br>Bodyweight changes: DEHP ↑ (24 weeks)<br>TC, TG, ASAT, ALAT, and high-density lipoprotein levels: DEHP ↑ (24 weeks))<br>Liver-to-bodyweight ratio: DEHP ↑ (300)<br>Lipid droplets in the form of subcircular vacuoles in the liver: DEHP ↑<br>Degenerative and necrotic tubular epithelial cells along with fat vacuoles in the liver: DEHP ↑ (600)<br>Separated ellipses of the principal component analysis of the serum metabolic profiling between control and exposed groups<br>Cell necrosis, and fatty and vacuole degeneration in the liver cytoplasm: DEHP ↑<br>UDPGT mRNA expression: DEHP ↓<br>D1 mRNA expression: DEHP ↑</td><td></td><td>[255]</td></tr><tr><td>Rat</td><td>DEHP</td><td>0.75 mg/kg bw/d</td><td>Oral gavage</td><td>GD6-PND70</td><td>PND0-70</td><td>Lipid metabolism</td><td>Bodyweight: DEHP ↑ (GD6-PND21)/↓ (GD6-PND70)<br>TG blood level: DEHP ↓ (GD6-PND21)<br>ALAT blood level: DEHP ↑ (GD6-PND70)<br>TG hepatic level: DEHP ↑<br>Small lipid droplets in hepatocytes: DEHP ↑<br>TG-related gene expression: DEHP ↑ (GD6-PND21: AGPAT1, DGAT1/GD6-PND70: ACSL1, PNPLA2, MGAT1, DGAT1, and PPARα)/↓ (GD6-PND21: SREBP-1c and GPAT)<br>TG-related protein expression: DEHP ↑ (GD7-PND21 and GD6-PND70: DGAT1)/↓ (GD-PND70: SREBP-1c)</td><td></td><td>[240]</td></tr><tr><td>Rat</td><td>DEHP</td><td>300 mg/kg bw/d</td><td>Oral gavage</td><td>GD14-PND0</td><td>PND60, 200</td><td>Blood pressure/heart rate</td><td>Systolic blood pressure: DEHP ↓ (nighttime)<br>Diastolic arterial pressure: DEHP ↓ (low-sodium diet)<br>Heart rate: no effect<br>Aldosterone level: DEHP ↓ (normal salt diet)</td><td>Locomotor activity during daytime: DEHP ↓ (PND60)<br>Locomotor activity during night-time: DEHP ↓</td><td>[256]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>DEHP</td><td>20, 50, 100, 300, and 750 mg/kg bw/d</td><td>Oral gavage</td><td>GD14-PND0</td><td>PND60</td><td>Metabolism</td><td>Circulating aldosterone concentration: DEHP ↓ (100, 300, 750)/↑ (♀: 300)<br>Corticosterone and adrenocorticotropin hormone (ACTH) level: no effect<br>Estradiol level: DEHP ↓ (♀: 300)<br>Liver-made angiotensinogen (Agt) and renin (Ren1) mRNA level: no effect<br>Adrenal morphology: no effect<br>Adrenal weight: DEHP ↓ (750)<br>Adrenal AGTR1a mRNA level: DEHP ↓ (300)<br>Adrenal AGTR1b mRNA level: DEHP ↓<br>Adrenal AGTR2 mRNA level: DEHP ↓ (750, 950)<br>Kidney AGTR2 mRNA level: no effect<br>Adrenal and kidney Agtrap mRNA level: DEHP ↓<br>Adrenal AGTR1A and AGTR1B protein level: DEHP ↓ (300, 750)<br>Adrenal aldosterone gene synthase (Cyp11b2) mRNA level: DEHP ↓ (500, 750)<br>TG, TC, HDL, and LDL cholesterol levels: no effect<br>Adrenal cholesterol uptake receptor (LDLR) mRNA level: DEHP ↑<br>Heart LDLR mRNA level: DEHP ↓ (300)/↑ (750)<br>Adrenal HMG-CoA reductase and synthase (Hmgcr and Hmgcs1) and insulin-induced gene 1 (Insig1) mRNA level: DEHP ↑<br>Heart Hmgcr, Hmgcs1, and Insig1 mRNA level: no effect<br>Adrenal lipid-droplet accumulation: DEHP↑</td><td>Circulating testosterone concentration: DEHP ↓ (100, 300, 750)<br>Sodium, potassium, chloride, calcium, and angiotensin II (AT II) level: no effect</td><td>[257]</td></tr></tbody></table>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th>Animal Model</th><th>Chemicals</th><th>Dose Administered</th><th>Exposure Route</th><th>Exposure Duration</th><th>Observation Stage</th><th>Function Studied</th><th>Metabolic Postnatal Outcomes</th><th>Additional Outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>Rat</td><td>DEHP</td><td>0.75 mg/kg bw/d</td><td>Oral administration</td><td>GD6-PND21 or/and PND22-70</td><td>PND70</td><td>Metabolism</td><td>Serum insulin and TG levels: DEHP ↓ (GD6-PND21)<br>Serum leptin and adiponectin levels: no effect<br>White adipose tissue PPARγ mRNA and protein expression levels: DEHP ↑</td><td></td><td>[242]</td></tr><tr><td>Rat</td><td>DEHP</td><td>10 or 100 mg/kg bw/d</td><td>Oral gavage</td><td>GD9-PND21</td><td>PND80</td><td>Glucose metabolism</td><td>Fasting blood glucose level and insulin resistance: DEHP ↑<br>Serum ASAT, ALAT, alkaline phosphatase, urea, and creatinine levels: DEHP ↑<br>Serum insulin concentration: DEHP ↑<br>Hepatic glycogen concentration and glucogen synthase activity: DEHP ↓<br>Molecules involved in insulin signaling in the liver: DEHP ↓ (Insulin receptor beta (IRβ), IRβ<sup>Tyr1162</sup>, insulin receptor substrate 1 (IRS1), IRS1<sup>Fyr632</sup>, β-Arrestin, Akt, Akt<sup>Ser473</sup>/100: c5rc protein, Akt<sup>Thr308</sup>)<br>Targets of insulin signal transduction: DEHP ↑ (GSK3β, FoxO1)/↓ (GSK3β<sup>Ser9</sup>, FoxO1<sup>Ser256</sup>)<br>Liver G-6-Pase and PEPCK (enzymes involved in gluconeogenesis) mRNA expression: DEHP ↑<br>Birth weight: DEHP ↓ (σ<sup>o</sup>)</td><td>Serum testosterone and estradiol concentration: DEHP ↓</td><td>[245]</td></tr></tbody></table>