Endometriosis-like lesions induced by phthalates: new phytotherapic applications to complement traditional cares: a PNRR 2023 project

Endometriosis (E) is a high impact, estrogen-dependent, inflammatory, chronic disease with multifactorial aetiology based on genetic, hormonal, and immunological factors. It is characterized by the presence of extrauterine endometrial tissue, that causes pelvic pain and infertility [ 1 , 2 ]. In Italy, E affects about 3.000.000 of women in reproductive age, with a high impact for the National Health System. Clinical symptoms include chronic, cyclical or non-cyclical pelvic pain and infertility; indeed, in about 30–40% of women, E seriously impairs fertility. Women with chronic pain and infertility have as only choice surgical intervention that can negatively impact ovarian reservoir (already compromised by E). The assisted conception– often used to achieve pregnancy by women affected by E - involves iatrogen estrogen (E2) administration that may worsen E symptoms [ 3 , 4 ]. Steroid responsiveness, inflammatory processes and the peritoneal environment are key factors in E pathogenesis; indeed, E2 stimulates the growth of endometriotic implants and, beyond the ovarian E2 production, in endometriotic lesions E2 local synthesis occurs, mediated by steroidogenic enzymes as aromatase. Moreover, E2 stimulates prostaglandins, resulting in a feedforward mechanism of prostaglandin-mediated E2 production. In addition, resistance to progesterone - that acts as an anti-inflammatory agent - is evident in women with E [ 5 ]. At present, concerns are also arisen by the increased incidence of E at younger ages and by the fact that now E is considered a systemic disease (i) affecting metabolism in liver and adipose tissue, (ii) leading to systemic inflammation, (iii) altering gene expression in the brain and (iv) causing pain sensitization and mood disorders [ 1 , 6 ]. The available therapies to treat E and the related pelvic pain depend on age, side-effect profile, lesion extent and locations, as well as on preliminary treatments. Indeed, the surgery to remove the ectopic E lesions represents the first-line treatment, generally followed by long-term pharmaceutical therapy with Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) and oral contraceptives [ 7 ]. These last induce hypoestrogenism and the inhibition of tissue proliferation and inflammation [ 8 ]. Progestins plus gonadotropin-releasing hormone agonists, that cause amenorrhea, are also used to reduce systemic E2 levels [ 9 ], alleviating disease-related symptoms. However, the conventional therapies usually show limited efficacy for most patients due to several side effects, including peri-menopausal stage symptoms, osteoporosis, lipid profile changes, and liver dysfunction. Furthermore, hormonal drugs work by blocking ovulation and do not allow patients to attempt conception, whereas many of them wish to become pregnant [ 10 ]. For these reasons, in the absence of drugs that cure the disease while promoting fertility, women often resort to non-pharmacological alternatives mainly based on natural substances. In fact, such alternatives, together with a healthy lifestyle characterized by - for example - physical exercise, healthy nutrition, osteopathy and relaxation techniques like autogenic training and meditation, allow to properly control and sometimes revert E symptoms [ 7 ]. Several studies report the beneficial effects of edible and medicinal plants as well as of their bioactive compounds (BCs), mainly polyphenols, in several oxidative, inflammatory and painful diseases [ 11 ]. These compounds, which include different classes of phenolic acids, flavonoids, stilbenes, phenylpropanoids, are natural substances extensively investigated for their antioxidant and anti-inflammatory properties. Concerning E, they may represent an important alternative to NSAIDs due to their cyclooxygenase-2 (COX-2) selectivity, the ability of accelerating the healing process as well as for the lack of side effects that E patients currently experience [ 7 ]. Moreover, polyphenols can interact with oestrogen receptors, due to the structural similarity with E2 or with the synthetic oestrogen diethylstilboestrol, and this aspect appears to be important in E treatment [ 7 ]. In this project, according to the preliminary in vitro and in vivo published studies demonstrating the potential role of PEs or their main BCs against E, the following plant species have been selected: Olea europea L., Verbascum thapsus L., Citrus × limon (L.) Osbeck, Citrus × sinensis (L.) Osbeck and Serenoa repens (W. Bartram) Small [ 11 – 19 ],. Recently, it has been demonstrated that an aqueous extract of olive pulp titrated in hydroxytyrosol (HT) effectively decreased cyst diameter, area and volume as well as collagen deposition and fibrosis in endometriotic lesions. HT causes a reduction in CD34 protein and vascular endothelial growth factor (VEGF) expression preventing the process of angiogenesis [ 20 , 21 ] and also controlling hyperproliferation and apoptosis [ 22 ]. The same extract suppresses neuropathic pain and the release of inflammatory mediators [ 23 ] such as interleukin (IL)-2, IL-1β, tumor necrosis factor (TNF)-α, IL-6 and IL-10 in the peritoneal fluid and endometriotic loci, as well as it restores the oxidant–antioxidant balance by increasing the glutathione reduced levels and decreases lipid peroxidation and superoxide dismutase and myeloperoxidase activity [ 11 ]. Furthermore, it is able to reduce degradation of tight junction proteins and mast cell infiltration in the hippocampus, which plays a pivotal role in the affective and cognitive consequence of neuropathic pain, reducing microglial and astrocytes activation [ 11 ]. Another BC derived from Olea europea , already investigated in E, is the oleuropein (O), which selectively inhibits ERβ activity without altering ERα activity [ 14 ]. This aspect is important because O effectively suppresses the growth of E ectopic lesions without any evident reproductive toxicity. Additionally, O improves the pregnancy rate, repairing the decidualization defect of the endometrium, reduces the hyperinflammatory state, and it has critical effects on bone formation and maintenance [ 24 ]; in this respect, it can be used as an effective remedy to treat the symptoms of osteoporosis, an adverse effect associated with the use of aromatase inhibitors in the treatment of E [ 25 ]. Furthermore, it has been demonstrated that the combination of HT and O effectively suppresses the migration and invasion of ER-positive breast cancer cell lines compared to their monotherapy [ 26 ], suggesting the importance of using a combination of these BCs, or even better an extract titrated in these BCs, to treat E. Verbascum sp. such as V. thapsus and V. xanthophoeniceum contain several BCs, including flavonoids and phenylethanoid glycosides, whose anti-inflammatory, anti-oxidative, anti-tumor and immune-stimulatory activities are well-known [ 27 – 29 ]. One of the most investigated BC is the verbascoside (V), a glycosylated phenylpropanoid ester of caffeic acid. Although currently no studies are available on the potential beneficial activity of V on E, it has been recently demonstrated that caffeic acid decreases the reactive oxygen species (ROS) level as well as haeme oxygenase 1 and NAD(P)H dehydrogenase [quinone] 1 (NQO1) enzyme activities alleviating oxidative stress in ectopic endometrial cells [ 15 ]. Furthermore, it increases the nuclear factor (erythroid-derived 2)-like 2 gene expression, a redox-sensitive transcription factor, which plays a pivotal role in protecting endometrial cells against the progression of E [ 30 ]. Considering this, the study of PEs rich in caffeic acid derivatives such as V could represent a promising strategy to counteract E. Hesperidin (H) is a natural flavanone glycoside found in Citrus fruits such as orange and lemon with various well-known biological activities [ 16 ]. It has been demonstrated that H treatment restores the tissue architecture of uterus after E induction thanks to its antioxidant and anti-inflammatory activity, increasing the total antioxidant status and decreasing the caspase-3, mitogen-activated protein kinase (MAPK) and TNF-α expression, critical markers involved in the pathophysiology of E [ 16 ]. Anti-proliferative and apoptotic effects on two human endometriosis VK2/E6E7 and End1/E6E7 cell lines have been demonstrated also for naringenin (N), another Citrus typical flavanone [ 31 ]. N induces apoptosis decreasing cell proliferation, mitochondrial membrane potential (MMP) depolarization and ROS generation. It induces P38 MAPK and c-Jun N-terminal kinases (JNK) MAPK, but inhibits phosphoinositide 3-kinase (PI3K)/ protein kinase B signal transduction leading to death of endometriotic cells [ 31 ]. Furthermore, it is known that N transiently inhibits Nrf2/HO1/NQO1 signalling, triggering Nrf2 protein depletion and consequent down-regulation of Nrf2-regulated cell defence processes, thereby inhibiting the proliferation of endometriotic cells [ 17 ]. In addition, N exhibits anti-estrogenic activities [ 32 ] that could be useful to counteract development and progression of E. In the end, the selection of S. repens , arises from the dietary recommendations for women at risk or diagnosed with E, who are recommended to increase the consumption of polyunsaturated fatty acids (PUFA), mainly oleic acid and n-3 PUFA, with particular attention to the EPA: AA ratio, because it can be relevant in E and in the reduction of painful symptoms. The most studied plant-complex of this species is the lipid-sterolic extract obtained from the fruits, rich in PUFA and phytosterols. Among them, the β-sitosterol shows a pivotal role because it has been already investigated on E both in vitro and in vivo systems [ 19 ]. In vitro experiments show that β-sitosterol can inhibit the proliferation of hEM15A cells and promote their apoptosis [ 19 ]. Furthermore, β-sitosterol inhibits the transforming growth factor beta 1 expression and suppressed cell proliferation and migration, thereby blocking the formation and progression of E lesions in vivo [ 19 , 33 ]. The combined application of si-Smad7 and β-sitosterol counteracts the positive effects of β-sitosterol allowing to hypothesize that Smad7-mediated TGF-β/Smads signalling pathway could be the key molecular target of β-sitosterol [ 19 , 33 ]. Considering this, it is plausible to hypothesize the efficacy of the lipid-sterolic extract of S. repens in counteracting E. The exposure to widespread environmental contaminants has been indicated as a potential co-factor in E development and progression. Endocrine Disruptors (EDs), due to their targets and mechanisms of actions, are the most likely candidates [ 34 ]. EDs are defined as “exogenous substances that causes adverse health effects in an intact organism, and/or its progeny, consequent to changes in endocrine function [ 35 ]. They can be present in nature (e.g. phytoestrogens), but the majority are chemicals released into the environment by human activities. The general population is ubiquitously exposed to EDs in daily life, through food and water, in indoor and outdoor environments, and through their use as plant protect products, food additives and preservatives, industrial and household products, plastics, detergents, flame retardants and as ingredients of personal care products. The routes of exposure for humans may be oral, inhalation or dermal absorption [ 36 ]. Phthalates (PH) are widely used chemicals added in most plastic products during production, packaging, or delivering. PH are proved to be EDs; in fact, despite their short half-lives in tissues, chronic exposure can adversely influence the endocrine system and the physiology of multiple organs, with negative long-term impacts in adults, as well as in both children and adolescents. Several countries have established restrictions and regulations for some types of PH; however, their widespread use still creates concern [ 37 ]. Bis(2-ethylhexyl) phthalate (DEHP) is the most common PH plasticizer used to make plastic soft and flexible, especially in medical devices, furniture materials, cosmetics, and personal care products [ 38 ]. Since it is not covalently bound, DEHP can leach out of the products and can be found in environmental and human matrices, becoming an ubiquitous pollutant with significant human exposure. Several studies provide evidence for the serious consequences of DEHP exposure for animal and human health [ 39 ]. Indeed, DEHP is classified as ED able to: (i) bind to the nuclear androgen receptor and to the peroxisome proliferator-activated receptors [ 40 , 41 ], (ii) regulate transcription of target genes in the nucleus, (iii) activate signalling pathways in the cytoplasm [ 42 ], (iv) interfere with the synthesis, secretion, transport, binding and elimination of endogenous oestrogens and consequently affecting human health [ 43 ]. DEHP causes inflammation, cytokine changes, increased oxidative stress, endometrial proliferation, and its effects induced prenatally or peri-pubertally are more rapid and irreversible [ 38 ]. Many clinical and experimental studies suggest that exposure and bioaccumulation of DEHP affect female reproductive systems [ 44 ]; in particular, several Authors described the possible association between exposure to some PH and E [ 45 ]. Indeed, since the 2000s, the presence of PH in human matrices of women with E has been recorded, although the understanding of the correlation between E pathophysiology and PH is still limited.

The main expected results of the project can be summarized as follows. The in vitro studies will provide data to select the most promising PE or BC and to identify the main signalling pathways involved in the E pathogenesis allowing the respect of the 3Rs rules and the optimization of time and resources. The in vivo studies will provide data on (i) bioavailability and utero-tropism of the PE and BC and (ii) the preventive and protective effects of the candidate PE and BC against the E induction and progression mediated by DEHP as model environmental factor. In this respect, the in vivo data will lead to the identification and characterization of the role of environmental contaminants on E pathogenesis, and– at the same time - of suitable and targeted natural compounds to prevent and mitigate the impact of these contaminants on E pathogenesis. The ex vivo study on human E primary cells will allow to determine: (i) the DEHP and metabolite concentrations in E patients to be correlated with food and lifestyle data obtained by questionnaires, (ii) the role of DEHP and metabolites on the same signalling pathways as analysed in in vitro and in vivo studies. Overall, the whole project will provide data and tools to develop innovative phytotherapic treatments for E symptoms and to promote fertility. Moreover, the data on potential involvement of environmental contaminants on E pathogenesis will represent a starting point for setting preventive measures of risk reduction, aiming to limit the impact of the E costs on National Health System. Indeed, the integration and the interpretation of whole data will allow to obtain a protocol for the management of E and to implement novel preventive and intervention strategies based on the support of phytotherapic supplements. Scientific-based policy of reduction of exposure could contribute to lower the E impact in the population, which is a significant challenge for the near future, and it represents the basis for the elaboration of preventive measures also focused on susceptible populations sub-group as peripubertal girls. This group represents a key target for E prevention to reduce the impact on National Health System of future problems due to impaired fertility linked to E.

The PNRR 2023 project– “A combined approach to evaluate the effects of Mediterranean plant bioactive compounds on Endometriosis-like lesions induced by phthalates: future prospects for their therapeutic applications to improve and complement the traditional cares” (MCNT2-2023-12377662), focuses on the evaluation of the potential preventive and protective role of selected PEs and BCs, well-known for their antioxidant, anti-inflammatory and anti-cancer activities, on DEHP-induced E-like lesions by a stepwise approach, as follows: i) raw material selection, standardized PE preparation, phytochemical characterization, BC titration and in cell-free assays; ii) in vitro screening on two human endometrial cell lines and in vivo toxicokinetic (TK) to select the best effective PEs or BCs in comparison with traditional NSAIDs; iii) in vivo juvenile toxicity study to test the PE or BC protective and/or preventing activity on DEHP induced E-like lesions, iv) enrolment of patients with E to be subjected to scheduled surgical procedures, administration of structured questionnaire on eating habits and lifestyles, urine, blood and tissue sample during surgery, and v) ex vivo and in vitro studies on human E primary cells and human non-cancerous cells to investigate the DEHP and metabolite concentration, and PE and BC mechanisms, respectively. All the studies evaluate several mechanisms and pathways known to be involved both in E and PE and/or BC activities, including anti -inflammatory, -oxidant, -proliferative and apoptotic, immunomodulatory, and hormone-modulated endpoints. The project was evaluated by ethic committees both for in vivo and human studies. The National Ethic Committee of the Italian National Health Institute approved the protocol of the human study, the documents for the enrolled patients and the informed consent (AOO-ISS − 02/07/2024–0028930– CLASS PRE BIO CE 01.00). The animal study protocols are designed according to the 3R principles and the ARRIVE guidelines 2.0 [ 46 ] and approved by the Italian Ministry of Health (Authorization n°647/2024-PR). During the experimental procedures, all the animal studies are performed in accordance with Directive 2010/63/EU, the Italian Legislative Decree n. 26 of 4 March 2014, the OECD Principles of Good Laboratory Practice. The Operative Unit (UO) 3 of the project, University of Messina– will select and purchase the certified plant material ( O. europea , S. repens , V. thapsus , C. limon and C. sinensis ) through an authorized dealer, will prepare food-grade standardized extracts to be characterized from a phytochemical point of view by LC-DAD-ESI-MS, GC-FID and GC-MS analyses. This procedure will allow not only to highlight the phytochemical profile of the PEs under examination but also to choose the appropriate markers for the subsequent pharmacological and toxicological studies. Furthermore, quantitative analyses of the most abundant BCs will be performed by purchasing LC and GC-grade reference standards. Preliminary in vitro cell-free antioxidant and anti-inflammatory assays will be carried out to select the most promising PEs for subsequent experiments. UO2, Istituto Zooprofilattico Lazio e Toscana, will carry out in vitro screening studies on E-like cell models: (1) human endometrial stromal cells (HESC), stimulated with LPS (10, 100 and 1000 ng/ml) to induce the E-like events, and (2) human epithelial E cell line (12Z), to evaluate the mechanisms and the effects of the PEs and BCs. Several endpoints will be studied: proliferation, cytotoxicity, apoptosis, gene expression of sex steroid receptors, NLR family pyrin domain containing 3 and PGE2-COX2 signalling. The data will be compared with data obtained with conventional NSAIDs, as positive controls, to identify the three most promising and effective PEs and BCs. Concentration-response curves will be calculated, and a negative control (DMSO 0.1%) will be used. The most common NSAIDs such as COXs-inhibitors aspirin, ibuprofen as well as celecoxib will be used at the same concentration as the PEs and BCs. UO1 Istituto Superiore di Sanità– MEGE Center, will perform the in vivo TK study to select the most promising PE or BC in terms of bioavailability and utero-tropism and to establish the most appropriate dose to be used in the in vivo study 2. According to the OECD Guideline 417 [ 47 ], the PEs or BCs (at least 3) showing the best efficacy in in vitro screening will be orally administered by gavage to female rats at two dose levels. The groups, time of exposure and numbers of rat in the TK study are shown in Table  1 . Table 1 Plan of in vivo toxicokinetic study Treatment Single dose, 1 oral treatment, sacrifice after 24 h ( N ° rats) Repeated dose, 5 oral treatments (1 a day), sacrifice 24 h after the last treatment (day 6) ( N ° rats) Control Vehicle only 4 4 PE or BC 1 Low dose 4 4 High dose 4 4 PE or BC 2 Low dose 4 4 High dose 4 4 PE or BC 3 Low dose 4 4 High dose 4 4 Plan of in vivo toxicokinetic study Fifty-six young, healthy female rats (6–7 week of age and 200–250 g of weight) will be randomly divided (simple randomisation − 4 rats/group/exposure time) into 3 treatment groups: control (vehicle only); single dose groups (each PEs or BCs administered for 1 day); repeated dose groups (each PEs or BCs administered for 5 days). control (vehicle only); single dose groups (each PEs or BCs administered for 1 day); repeated dose groups (each PEs or BCs administered for 5 days). PEs and BCs will be dissolved in a food grade vehicle. Two dose levels will be used to gather information to set the in vivo juvenile study 2 [ 47 ]. Both dose levels should be high enough to allow PE or BC metabolite identification in serum and tissues. Where possible, the data of the in vitro screening will be also considered for dose selection. The health status of female rats, body weight and food consumption will be recorded daily. For the determination of TK parameters, 1, 2, 4 and 8 h after the first administration, rats will be anaesthetised with a gaseous solution of isofluorane and blood samples collected through saphenous vein; 24 h after the last treatment (day 2 or day 6), blood samples will be collected by intracardiac puncture. Subsequently, animals will be sacrificed by CO 2 inhalation. Uterus, ovary and liver will be excised, weighed and stored for the determination of PEs, BCs and their metabolite concentration. Uterotropism will be evaluated by UO3 for the achievement of the target tissue in physiological state; plasmatic maximal concentration (C max ), time to achieve the C max , area under the curve and half-time will be calculate by pharmacokinetic software. PEs and BCs and their metabolites will be quantified by LC-DAD-ESI-MS-MS and GC-FID/GCMS after tissue homogenization, solid phase extraction (SPE) and after SPE and/or supported liquid extraction (SLE) for biological fluids. UO1 will perform the in vivo juvenile toxicity study. Nineteen pregnant dams will be divided into 2 groups: A = vehicle only (10 dams) and B receiving the PE or BC selected in the TK study (9 dams). Dams will be treated from gestational day (GD) 7 to 20 (foetal maturation) and from post-natal day (PND) 1 to 22 (lactation) by gavage. The dose level, selected from the TK study, will be calculated on the basis of the dams’ body weight (bw) the first treatment day, whereas during lactation it will be adjusted according to the dam bw gain. During gestation and lactation, the dam health status will be checked every day, bw and food consumption 2 times a week. Dams will be allowed to deliver, and litters counted, weighted, sexed and checked for anomalies and dead foetuses. At PND23 (weaning), every pup will be sexed and weighted; dams and F1 male rats will be no longer used and managed by the ISS animal facility. Female F1 rats will be divided into 5 groups and treated for 28 days, from PND 23 to PND 60 (sexual maturity), 5 times a week by gavage. The group divisions and treatment of dams and F1 females are reported in Fig.  1 . Group 1 will consist of 6 F1 female rats; groups 2, 3, 4 and 5 will consist of 12 F1 female rats. The comparison among the groups will allow to evaluate: (i) the time and severity of DEHP-induced E lesions, (ii) in which stage(s) of pre- and/or post-natal life the PE/BC administration may have the major protective/preventive effect. DEHP will be administered in drinking water at dose levels of 1.2 mg/kg bw per day and the dose level has been selected considering as a key effects the increase of endometrial thickness observed in mice treated with drinking water containing 1330 µg/L DEHP for 10 weeks [ 48 ], using the appropriate mouse-rat conversion factor. PE or BC will be dissolved in a food grade vehicle and orally administered by gavage. Animal health status will be checked every day, bw and food consumption 3 times a week. Vaginal patency, starting from PND 35 and estrus cycle from (PND 40 to 50) will be observed. Magnetic Resonance Imaging (MRI) analyses will be performed longitudinally in anesthetized rats before (PND 23) and at PND 45 to localize and quantify the extent of the E lesions. This MRI-based characterization will give indications on the early beneficial effects of PE or BC tested. At PND 45, rats will be anaesthetised and blood samples collected through saphenous vein for miRNA serum analysis. The miRNAs will be selected based on those found altered in patients, as early indicators of E. Twenty-four hours after the last treatment (PND 60), all rats will be anaesthetised and blood samples will be collected by intracardiac puncture for the determination of serum biomarker. Subsequently, animals will be sacrificed by CO 2 inhalation. Uterus, ovary and liver will be excised, weighed and fixed in buffered formalin for histopathological analysis or flash frozen for molecular and gene expression analysis of PGE2/LTB4, caspase 1, aromatase, E2 and progesterone serum biomarkers; liver metabolomics analyses by NMR spectroscopy (14.1T) will be also performed by dedicated software and multivariate analyses. In the uterus, mRNA/qPCR for gene expression analysis of sex steroids, eicosanoids and inflammatory related signalling pathways and proliferation markers (Ki67) will be evaluated. Levels of PE or BC and liver will be analysed by LC-DAD-ESI-MS- MS and GC-FID/GC-MS after tissue homogenization and SPE, as well as after SPE and/or SLE for blood by UO3. Fig. 1 Group division and treatment of in vivo juvenile study Group division and treatment of in vivo juvenile study The clinicians of UO4 - ARNAS Civico Palermo– will enrol patients with E who are to undergo surgical intervention. The number of the patients (at least n  = 60) will be selected to optimize the ex vivo studies taking into account suitable inter-individual variation and to guarantee sound data from the statistical point of view. Enrolled women will be asked to sign the informed consent and to fill in a specific questionnaire to evaluate their lifestyle and eating habits. A unique alphanumeric code will be assigned to each enrolled patient, to her samples and questionnaire to ensure data anonymization. Specimens from E lesions in different locations, e.g., ovary, peritoneum, retro-peritoneum will be collected together with unaltered uterine tissue during the common surgical procedures; an aliquot of blood for plasma miRNA determination and urine for the analysis of DEHP exposure will be also collected. After surgery, all the tissue samples will be appropriately stored in a specific tissue storage solution and immediately delivered to the UO3 that will provide for primary cell isolation and DEHP determination. Collected biopsy from consenting women will be diced, digested, and filtered through 40 μm filter. Two different procedures will be followed: (i) inverting the filter and collecting the backwash, centrifuged, trypsin-dissociation, neutralization and culture, Human Endometrial Epithelial Cells (HEECs) will be recovered; (ii) collecting the flow through, centrifuged, re-suspended and cultured changing the media to isolate Human Endometrial Stromal Cells (HESCs). The supernatant will be collected, concentrated and chromatographically purified using neutral alumina column chromatography. The DEHP and metabolite concentration in the biopsy will be measured by LC-DAD-ESI-MS analysis in urine samples. Tissue will be weighted and homogenized. Analytes will be recovered by liquid phase ultrasound-assisted extraction before injection into the HPLC system. RNA extracted from isolated cells by UO3 will be shipped to UO1 and UO2 to evaluate the expression of the same signalling pathways investigated in the in vitro and in vivo studies. Moreover, such endpoints will be correlated with DEPH and metabolite concentration. For quantitative variables, the measures of central tendency (mean, median and mode) and dispersion (standard deviation and percentiles) will be calculated. The normality of the distribution will be assessed (Kurtosis and Skewness) to define if the inferential statistics can be done with parametric or non-parametric tests. For qualitative variables, the frequency and the confidence intervals, according to the binomial distribution, will be calculated. Inferential statistical analyses: for quantitative variables, according to the results of the descriptive analyses parametric or non-parametric tests will be applied to analyse the correlation between the outcome variables (internal exposure level) and the other predictive variables. The sample size of the in vivo TK study is selected according to guide line OECD 417 [ 47 ]. The sample size of the in vivo juvenile toxicity study is based on five main comparisons between groups (1 vs. 2, 2 vs. 3, 2 vs. 4, 3 vs. 5 and 4 vs. 5). Group 1 serves as the baseline, consisting of F1 rats without pre- or post-natal treatments or DEHP exposure. Groups 2–5 consist of F1 rats with induced damage and a two-factor design: pre-natal treatment (YES/NO) and post-natal treatment (YES/NO). Group 2 is compared to Group 1 to assess whether DEHP induces increased endometrial thickness (damage) according to Kim et al. [ 48 ]. Since administration of NSAIDs in murine models reduces the endometrial damage by approximately 50% [ 49 ], consequently this is considered as relevant effects also for PE or BC (Cohen’s d = 1.5) when compared with the group 2 (DEHP exposure, without pre- and post-natal treatments). The sample size is calculated using G*Power software (latest ver. 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). A one-tailed Student’s t-test, with alpha = 0.01 (Bonferroni correction applied), and power 1-beta = 0.80, gives a minimum sample size of 10 per group (increased to 12 to account for 10% potential loss and consanguinity). For group 1, 6 female rats are sufficient due to the very large effects (Cohen’s d = 3). In summary, considering that: (i) a primiparous pregnant SD rat generates 5 ± 2 females per litter; (ii) the experimental groups (2–5) consist of 12 F1 females, and 6 F1 female rats in group 1; (iii) no more than 2 consanguineous F1 females will be assigned to the same experimental group; (iv) 50% of the mothers may either not be pregnant upon arrival or may not carry the pregnancy to term, the number of pregnant mothers in group A is 10, and the number of pregnant rats in group B is 9. The number of E patients enrolled will be established starting from urinary concentrations of DEHP metabolites detected in 900 Italian women, representative of the Italian female adult population (P50 (P25-P75) 26.6 (17.6–41.2) µg/g of creatinine) [ 50 ] and considering biologically relevant an increase of 15% than the median population. Setting the experiment with one-tail alpha level = 0.05, and power = 0.80, 50 subjects will be needed to assess a 15% significant increase of DEHP and metabolite concentrations compared to the median female adult population. Considering a possible dropout of about 20%, a minimum of 60 E patients will be enrolled.