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Phthalates and uterine disorders

  • Shuhong Yang , Shuhao Yang and Aiyue Luo EMAIL logo
Published/Copyright: March 8, 2024
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Reviews on Environmental Health
From the journal Reviews on Environmental Health Volume 40 Issue 1

Abstract

Humans are ubiquitously exposed to environmental endocrine disrupting chemicals such as phthalates. Phthalates can migrate out of products and enter the human body through ingestion, inhalation, or dermal application, can have potential estrogenic/antiestrogenic and/or androgenic/antiandrogenic activity, and are involved in many diseases. As a female reproductive organ that is regulated by hormones such as estrogen, progesterone and androgen, the uterus can develop several disorders such as leiomyoma, endometriosis and abnormal bleeding. In this review, we summarize the hormone-like activities of phthalates, in vitro studies of endometrial cells exposed to phthalates, epigenetic modifications in the uterus induced by phthalate exposure, and associations between phthalate exposure and uterine disorders such as leiomyoma and endometriosis. Moreover, we also discuss the current research gaps in understanding the relationship between phthalate exposure and uterine disorders.

Keywords: phthalates; endometrial cells; uterine disorders; leiomyoma; endometriosis

Introduction

Humans are commonly exposed to environmental endocrine-disrupting chemicals (EDCs), which can have hormonal-like effects on the entire body, and concerns about their e potential toxicity have been raised. Phthalates, a type of EDC, are used extensively as plasticizers or additives in medical devices, food packaging, toys, and personal care products such as cosmetics [1]. Phthalates can leak out of products, enter the human body through ingestion, inhalation, or dermal application and be rapidly metabolized and excreted into urine. Almost every person has detectable concentrations of phthalate metabolites in their urine [2], which reflects ubiquitous exposure that can impact various systems and lead to diseases [3].

Given the negative effects of phthalates on human health, researchers have established the tolerable daily intake values, defined as the daily intake amount of a chemical that has been assessed to be safe for humans on a long-term basis, and the reference dose, defined as the maximum acceptable oral dose of a toxic substance [2], 4]. A female reproductive organ regulated by the Hypothalamic-Pituitary-Ovarianaxis, the uterus is essential for embryo implantation, pregnancy maintenance, and delivery of the uterus. Various cell types (e.g., stromal cells, luminal epithelial cells) of the uterus endometrium and myometrium express the estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR), whose levels vary during the menstrual cycle [5]. The function of the uterus is regulated primarily by hormones, including estrogen, progesterone and androgen.

Phthalates can exhibit estrogenic/antiestrogenic and/or androgenic/antiandrogenic activities, and it is important to investigate their effects on uterine function and associated disorders such as endometriosis (EMs) and fibroids. This review summarizes the following aspects: (1) hormone-like activities of phthalates, (2) in vitro studies of endometrial cells, (3) phthalate and epigenetic modifications, (4) associations between phthalates and leiomyoma, and (5) associations between phthalates and EMs. We focus on the common uterine-associated diseases and their potential links to phthalates.

Materials and methods

For this study, a scoping review of the literature was performed to identify published studies that describe hormone-like activities of phthalates, their effects on in vitro cultured endometrial cells, and the relationship between phthalate exposure and uterine diseases (e.g., leiomyoma and EMs). Full-length articles in English were collected from MEDLINE, PubMed, Google Scholar, Scopus, Web of Science, and ScienceDirect up to November 2023. We searched these databases using the following key terms: (1) “phthalate and estrogenic/antiestrogenic/androgenic/antiandrogenic activity”; (2) “phthalate and endometrial cells”, “phthalate and stromal cells”, “phthalate and endometrial mesenchymal stem/stromal cell”, and “phthalate and endometrial endothelial cells”; (3) “phthalates and uterine DNA methylation” and “phthalates and uterine and miRNA”; (4) “phthalates and uterine leiomyoma” and “phthalates and uterine fibroids”; (5) “phthalates and endometriosis” and “phthalates and adenomyosis”; (6) “phthalates and uterine histology changes”.

The studies selected met the following criteria: (1) studies that examined the effects of exposure to one or more phthalate ester or metabolites on the activity (agonistic or antagonist) of ERs and ARs; (2) experimental studies that reported in vitro, in vivo, or both in vitro and in vivo findings to identify the effects of phthalates on endometrial cells; (3) studies that described the effects of phthalates on uterine epigenetics; (4) human studies that explored the association between phthalate exposure and uterine disorders such as leiomyoma and EMs; and (5) studies that described the relationship between phthalates and uterine histology changes. Studies that met the following criteria were excluded: (1) no methods described; (2) no version available in the English language; (3) no full-text publication available; or (4) content redundancy. Phthalates and their metabolites measured in studies are listed in Table 1.

Table 1:

Phthalates and metabolites measured in studies.

Phthalate abbreviation Name
1. DEHP Bis-2-ethyl hexyl phthalate
 1.1 MEHP Mono-ethyl hexyl phthalate
  1.1.1 MECPP Mono (2-ethyl-5-carboxyphentyl) phthalate, secondary metabolite of DEHP.
  1.1.2 MCMHP Mono-[(2-carboxymethyl) hexyl] phthalate, secondary metabolite of DEHP.
  1.1.3 MEHHP (5OHMEHP) Mono-(2-ethyl-5-hydroxyhexyl) phthalate, secondary metabolite of DEHP.
  1.1.4 MEOHP (5oxo-MEHP) Mono-(2-ethyl-5-oxo-hexyl) phthalate, secondary metabolite of DEHP.
  1.1.5 MECPP Mono (2-ethyl-5-carboxypentyl) phthalate, secondary metabolite of DEHP.
2. DOP (DnOP) Di-n-octyl phthalate
 2.1 MOP (MnOP) Primary metabolite of DnOP, monooctyl phthalate
 2.2 MCPP One oxidative metabolite of DnOP, mono-(3-carboxypropyl) phthalate
3. BBP (BBzP) Benzyl butyl phthalate
 3.1 MBzP Monobenzyl phthalate
4. DCHP Di-cyclohexyl phthalate
 4.1 MCHP Metabolite of DCHP, monocyclohexyl phthalate
5. DMP
 5.1 MMP Metabolite of DMP, 2-(Methoxycarbonyl)benzoic acid
6. DBP, DnBP Dibutyl phthalates (both di-n-butyl and di-isobutyl phthalates, referred to as DBP)
 6.1 MBP, MnBP Primary metabolite of DBP, monobutyl phthalate (mono-n-butyl phthalate)
 6.2 MHBP Metabolite of DBP, mono-hydroxybutyl phthalate
 6.3 MiBP Primary metabolite of DBP, mono-isobutyl phthalate (MiBP)
 6.4 MHiBP Metabolite of DBP, mono-hydroxyisobutyl phthalate
7. DEP Diethyl phthalate
 7.1 MEP Primary metabolite of DEP, mono-ethyl phthalate
8. BzBP (BBzP) Benzylbutyl phthalate
 8.1 MBzP Primary metabolite of BzBP, mono-benzyl phthalate
9. DiNP Di-isononyl phthalate
 9.1 MINP, MNP Primary metabolite of DiNP, monoisonoyl phthalate
  9.1.1 MCNP (cxMINP) Mono carboxyisononyl phthalate
  9.1.2 OHMINP Mono-hydroxyisononyl phthalate
  9.1.3 MCOP Mono carboxyisooctyl phthalate
10. MCPPa Mono-(3-carboxypropyl) phthalate
11. DIBP Diisobutyl phthalate
 11.1 MiBP Monoisobutyl phthalate
 11.2 MHiBP Mono-hydroxyisobutyl phthalate
12. DHP Dihexyl phthalate
13. DiDP, Diiso-decyl phthalate
 13.1 MCNP Monocarboxynonyl phthalate
14. DCHP Dicyclohexyl phthalate
15. DAP Diallyl phthalate
16. DPeP Dipentyl phthalate
17. DiHP Diisohexyl phthalate
18. DIHepP Diisoheptyl phthalate
19. DBzP Dibenzyl phthalate

  1. aMCPP, non-specific metabolite of several phthalates; MnBP, metabolite of mono-n-butyl phthalate; DnBP, secondary metabolite of di-n-butyl phthalate; MnOP, metabolite of mono-n-octyl phthalate; DnOP, secondary metabolite of di-n-octyl phthalate.

The estrogenic/antiestrogenic and androgenic/antiandrogenic activities of phthalates

Phthalates have been shown to act as antagonists and/or agonists via one or more hormonal receptors, including the ERα, ERβ, and AR [6], 7], and these actions can disrupt the endocrine systems [8]. Several in vitro studies have used ER-binding assays, proliferation assays with MCF-7 cells, ER-dependent transcription assays, and ER-mediated reporter gene assays to investigate the estrogenic effects of certain phthalates [9], [10], [11], [12], [13], [14]. Another study used primary-cultured hepatocytes from adult male Xenopus laevis and an enzyme-linked immunosorbent assay (ELISA) to detect vitellogenin (VTG) [15]. In vivo models, such as immature rat uterotrophic and transgenic mice models with an ER-mediated luciferase (Luc) reporter gene system have been used to evaluate the estrogenic characteristic of phthalates [16], 17]. To evaluate AR agonist and antagonist activities, in vitro methods such as the AR-binding assay [18], AR-EcoScreen assay [19], and Luc reporter gene assay [9], 20], 21], and in vivo methods such as the Hershberger assay [22] have been used.

Numerous commercial substances and new chemicals entering the environment often lack data on their potential ER or AR bioactivities, and evaluation of their potential bioactivities and toxicity is urgently needed. However, applying the current validated methods of the U.S. Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) is expensive and would take decades to complete all the testing. As an alternative, high-throughput in vitro screening (HTS) assays and computational toxicology methods have been developed to determine ER bioactivities and to identify potential AR-active chemicals rapidly and cost effectively [23], [24], [25]. Additionally, in silico methods and computational systems have been integrated to explore whether EDCs can bind to nuclear hormone receptors and to identify androgen-active chemicals [26], 27].

A recent study used a suite of in vitro assays and in silico models to identify which method is best for detecting endocrine-disrupting (ED) potential and reported that the effects on the ER and AR were similar. Researchers have suggested assessing the potential of ED effects of chemicals (including phthalates) by combining data from in vitro and in silico assessments, and t information from other sources [28].

studies have shown that phthalates can bind to the ER, PR and AR, and activate their associated pathways, through which they can exert either estrogenic/anti-estrogenic or androgenic/anti-androgenic activities. However, conflicting results between studies, and discrepancies between in vitro and in vivo studies have been observed, as shown in Table 2. For instance, whereas almost all studies have reported that butyl benzyl phthalate (BBP) exhibits estrogenic activity in in vitro assays, some studies have confirmed this action in vivo, but others have reported inconsistent results [8], [10], [11], [12], [13], [14, [29], [30], [31], [32], [33]. These discrepancies may reflect differences in the source of the test sample, solvent used (e.g., ethanol vs. DMSO), variation in the dose or cell lines used, or sensitivity of the assays.

Table 2:

In vitro and in vivo examination of the activities of phthalates.

Phthalates Estrogenic Anti-estrogenic Androgenic Anti-androgenic
In vitro In vivo In vitro In vitro In vitro In vivo
DEHP − [9], 11], 12], 30], 31], 34], + [8] − [12], 31], + [17] − [30], + [8], 14] + [9] − [35], + [9], 22] + [35]
MEHP − [30] − [30] − [35], + [22]
DCHP + [8], 30], 31] − [31] − [30] + [8]
MCHP + [30]
BBP + [8], [10], [11], [12], [13], [14, [29], [30], [31], [32, 34], 36] − [12], 31], 37], + [33] + [8], 30], 36] + [8], 20], 22]
DBP + [8], 9], [11], [12], [13], [14, 31], − [30] − [12], 31], [37], [38], [39] − [30] + [9] + [8], 9], 22]
MBP − [9] + [9] + [9]
DEP + [31], − [30] − [31] − [30]
DHP + [8], 12], − [30] − [12] − [30], + [8] + [8]
DIBP + [8], 11] + [8]
DINP ± [11], − [12] − [12] + [22]
DnOP − [12] − [12] + [22]
DiDP − [12] − [12] + [22]
MBZP − [30] + [30]
DAP + [8]
DPeP + [8] + [8] + [8]
DiHP + [8] + [8] + [8]
DiHepP + [8] + [8] + [8]
DBzP + [36] + [36]

  1. “+” means has potential activity; “−” means not show activity.

Moreover, it is important to note that one phthalate may exhibit several potential activities (e.g., estrogenic/anti-estrogenic or anti-androgenic) in a complex pattern.

Phthalates and epigenetic modifications

Phthalate exposure may exert its effects through epigenetic modifications, which are heritable changes in gene expression that do not involve a DNA sequence change. Epigenetic mechanisms include histone modifications such as methylation, acetylation, ubiquitination, phosphorylation, and SUMOylating, as well as DNA methylation and the expression of noncoding RNAs such as microRNAs (miRNAs). These mechanisms can influence gene expression in most cell types, and growing evidence suggests that epigenetic changes play a crucial role in the impact of environmental chemicals on human health and disease [40].

DNA methylation

As EDCs, phthalates, may exert their effects via epigenetic mechanisms such as DNA methylation, which is a frequently studied epigenetic mechanism that has been linked to both exposure and disease status. Previous studies have explored the association between phthalate exposure and DNA methylation in various organs, including the ovary, placenta, and testis [41], [42], [43], [44], [45], [46], [47], [48], as well as across different life stages, such as during prenatal development, childhood, and adulthood, and transgenerational effects, in different species including humans, mice, and fish [49], [50], [51], [52], [53], [54], [55], [56], [57], [58].

One study focused on the uterus of CD-1 mice treated with dibutyl phthalate (DBP) and reported that DBP disrupted the activity of DNA methyltransferase (DNMT) enzymes in a dose- and time-dependent manner [47]. DNMT activity in the uterus differed significantly between the DBP-treated and vehicle groups at 20 and 30 days. At 20 days of DBP exposure, only the group treated with DBP at 100 mg/kg/day showed an increase in DNMT activity. However, at 30 days, DNMT activity was reduced in all DBP-treated groups compared with the vehicle-treated group. DNMT catalyzes the transfer of a methyl group to DNA, and these changes in DNMT activity may lead to alterations in DNA methylation levels. Thus, it is reasonable to suggest that phthalate exposure may produce epigenetic changes in the uterus, which may increase the risk of developing reproductive diseases. However, direct detection of DNA methylation was not performed in this study, and further investigations are necessary to elucidate the relationship between uterine DNA methylation and phthalate exposure.

Another in vitro study investigated the relationship between phthalate exposure and DNA hypomethylation at the promoter region of the ERɑ gene [59]. MCF-7 human breast cancer cells were treated with BBP or DBP at a concentration of 10−5 M, which led to the demethylation of ERɑ promoter-associated CpG islands. F using a yeast-based ER transcription assay, these authors found that BBP treatment induced the expression of the hERɑ gene. Aberrant DNA methylation of the promoter region of the ERɑ gene might further influence the ER mRNA expression and contribute to the pathogenesis of uterine disorders.

Although several studies have explored abnormalities of DNA methylation in uterine disorders such as leiomyoma and EMs [60], [61], [62], [63], [64], few studies have investigated whether phthalate exposure increases or decreases epigenetic modifications in the uterus. Therefore, additional research is needed to elucidate the superimposed effects of phthalates on the uterus and associated diseases.

Phthalates and miRNA

Phthalates have been linked to epigenetic modifications involving miRNAs. Although the role of epigenetics-miRNA regulatory circuits in organizing the whole – gene expression profile have been investigated [65], limited studies have examined the associations between environmental phthalate exposure and miRNA expression in the uterus and associated diseases. In a recent study, Zota et al. [66] suggested that exposure to certain phthalates may be linked to miRNAs in fibroids and that the associations between exposure and miRNAs may vary according to ethnicity. The researchers quantified the expression level of 754 miRNAs in fibroid tumor samples and measured the concentration of phthalate metabolites in spot urine collected from 45 premenopausal women who underwent fibroid treatment at an academic hospital. Monohydroxy butyl phthalate (MHBP) and mono (2-ethyl-5-hydroxyhexyl) phthalate were positively associated with the expression levels of miR-10a-5p and miR-577, respectively, and some phthalate-miRNA associations varied according to ethnicity. However, in the endometrium, no significant associations were found between phthalates and miRNAs.

Considering these findings, future research should aim to evaluate prospectively the impact of environmental phthalate exposure on alterations in miRNA expression and the development of uterine disorders.

In vitro studies of endometrial cells

Uterine tissues are composed of a variety of cell types, including epithelial, stromal, endothelial and other cells, which express steroid receptors such as the ER, AR and PR. These receptors render uterine tissue susceptible to EDCs such as phthalates. In addition to differentiated cells, the endometrium also contains a distinct population of stem cells that exhibit notable regenerative capabilities during the menstrual cycle. Therefore, investigating the effects of phthalates on these cells and elucidating the potential underlying mechanisms may yield valuable evidence for characterizing these compounds in vitro.

In vitro studies of endometrial mesenchymal stem/stromal cells

Endometrial mesenchymal stem/stromal cells (EN-MSCs) are multipotent stem cells that can be isolated and cultured in vitro. They have the potential to differentiate into various cell lineages such as adipocytes, osteocytes, chondrocytes, and myocytes. Chen et al. [67] isolated and treated EN-MSCs with or without BBP and found that BBP treatment decreased EN-MSCs differentiation. Further evidence suggested that BBP decreased myogenic differentiation of EN-MSC via miR-137-mediated regulation of PITX2. These findings suggest that exposure to phthalates may inhibit the differentiation of EN-MSCs, which may affect endometrial regeneration. However, the precise mechanism requires further exploration.

In vitro study of endometrial endothelial cells

The endometrium is regulated by steroid hormones and undergoes regeneration during each menstrual cycle. A vital component of the endometrium, vascular trees play a crucial role in the control of bleeding and fertility. Endothelial cells lining the luminal surface of all blood vessels modulate their vascular morphology and function through angiogenesis, vascular remodeling, and functional changes. Human endometrial endothelial cells (HEECs) are unique in their expression of the ERβ and PR. Dysfunction of these cells is associated with endometrial disorders including EMs and abnormal uterine bleeding [68], 69].

As previously mentioned, some phthalates exhibit potential estrogenic activity. Because HEECs express the relevant acceptors, it is imperative to investigate whether these compounds affect cell function. Bredhult et al. [70] reported that exposure to DBP decreased the proliferation of in vitro-cultured HEECs; however, the underlying mechanism remains to be elucidated. In a recent study, Lijuan An [71] observed that exposure to mono (2-ethylhexyl) phthalate (MEHP) inhibited the proliferation of human endometrial microvascular endothelial cells (HEMECs) and induced apoptosis and pyroptosis via NLRP3 inflammasome. Given the widespread use of phthalates and the diversity of these compounds, further investigations are needed to explore the effects of other phthalates on HEECs.

In vitro studies of endometrial stromal cells

Endometrial stromal cells (ESCs) are an important cell type in the female reproductive system and their response to phthalates has been studied in vitro [72]. In 2010, Kim et al. cultured ESC under serum-free conditions and exposed them to hydrogen peroxide. They observed for the first time that di-(2-ethylhexyl) phthalate (DEHP) exposure led to increased ESC viability. Additional research confirmed Kim’s these results and demonstrated that DEHP exposure increased the production of reactive oxygen species (ROS) and decreased the expression of glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HO) in human ESCs [73]. In that study, DEHP exposure induced the expression of the ERα, which phosphorylated extracellular signal-regulated kinase (p-ERK)/p-p38 MAPK and NF-κB pathways and mediated the transcription of downstream genes. Another recent study found that DEHP treatment increased ESC proliferation and migration, inflammatory responses, and induction of the epithelial-mesenchymal transition (EMT) and stemness in human endometrial and endometriotic cells through targeting the TGF-β/Smad signaling pathway [74]. In contrast to the data reported by Kim et al. [72], other studies [75], [76], [77] have reported no significant effects on ESC viability following DEHP exposure. These discrepancies may reflect differences in treatment conditions or data analysis methods. For instance, the addition of hydrogen peroxide in the study by Kim et al. may have influenced cell viability.

Other research has investigated the impact of phthalates on the viability of ESCs, and some have shown that treatment with DEHP in in vitro-cultured endometrial cells significantly increased cellular invasiveness [77], activities of matrix metalloproteinase-2 (MMP-2) and 9 [78], 79], phosphorylation of extracellular signal-regulated kinase (Erk), and expression of p21-activated kinase 4 (Pak4) [80]. Further, increased expression of inflammation factors (IL-1β, IL-8, ICAM-1, and COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and aldose-keto reductase (AKR) activity (AKR1C1-3 and AKR1B10) was also observed [75], 81], 82]. All of the aforementioned genes may contribute to various gynecological disorders, such as EMs and endometrial cancer.

In addition to the aforementioned studies, one study [76] of cows has shown that DEHP and MEHP treatment of cultured endometrial cells stimulated the production of prostaglandin F2α (PGF2α) and inhibited the secretion of PGE2α, thereby increasing the PGF2α:PGE2α ratio. Such alterations may result in increased myometrial contractions and accelerate luteal regression, which cold lead to dysmenorrhea and abnormal uterine bleeding. Recent studies have found that DEHP can induce endometrial decidualization defects, which may affect embryo implantation and development, and lead to female reproductive disorders [83], 84].

Overall, the data suggests that exposure to phthalates such as DEHP or MEHP may alter the viability of endometrial cells and change their gene expression patterns (as shown in Figure 1). Such exposure may increase the expression of ERα, increase ROS production, and activate the MAPK/ERK/PAK4 and NF-κB pathways, which may further regulate the expression of genes associated with uterine disorders such as ER, PR, E-cadherin, AKR1C1-3, AKR1B10, MMP2/9.

Figure 1: Potential mechanism(s) of the role of phthalates in uterine disorders. In the “genomic pathway”, many actions of phthalates are mediated by the classical nuclear hormone receptors (NRs), especially estrogen receptors (ERs) and progesterone receptors (PR). After binding to NRs, phthalates can affect the transcription of target genes in the nucleus by binding to the corresponding response element of target genes. The “non-genomic pathway” of phthalates may occur through membrane steroid receptors (ERs, PRs), which may lead to rapid downstream intracellular signaling (MAPK/ERK) or through tryptophan-kynurenine – aryl hydrocarbon receptor (AHR) pathway and regulate the gene transcription. The “epigenetic modification” of phthalates may occur through DNA methylation and then regulate target gene expression.
Figure 1:

Potential mechanism(s) of the role of phthalates in uterine disorders. In the “genomic pathway”, many actions of phthalates are mediated by the classical nuclear hormone receptors (NRs), especially estrogen receptors (ERs) and progesterone receptors (PR). After binding to NRs, phthalates can affect the transcription of target genes in the nucleus by binding to the corresponding response element of target genes. The “non-genomic pathway” of phthalates may occur through membrane steroid receptors (ERs, PRs), which may lead to rapid downstream intracellular signaling (MAPK/ERK) or through tryptophan-kynurenine – aryl hydrocarbon receptor (AHR) pathway and regulate the gene transcription. The “epigenetic modification” of phthalates may occur through DNA methylation and then regulate target gene expression.

Phthalate and uterine leiomyoma (fibroid)

Uterine leiomyoma is the most frequent benign gynecologic tumor, and 70–80 % of women may be affected by this condition over their lifetime. The presence of uterine leiomyoma can affect a women’s quality of life and may require surgery such as myomectomy or hysterectomy. Many risk factors for the development of this tumor including phthalates exposure have been identified. In a comprehensive review of the literature on the effects of phthalates on uterine leiomyoma. We identified 14 epidemiological studies [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98] and one meta-analysis [99], of these studies, only one measured serum phthalate metabolites while the other 13 studies (shown in Table 3) used urine samples. However, the results of these studies were inconsistent.

Table 3:

Evidence profile table of studies of the associations between phthalate exposure and leiomyoma.

Study, year Study design Time period Age range Study population No. of case/control Samples Categories of phthalates and metabolites Summary of findings
Luisi et al. 2006 [89] Case-control Not mentioned Case: 48–50 General population 15/20 Serum DEHP MEHP Serum DEHP and MEHP concentration were lower in women with uterine fibromatosis
Control: not mentioned
Huang et al. 2010 [86] Case-control 2005–2007 27–45

(41.1 ± 6.8)		 Laparotomy population 36/29 Urine MMP, MEP, MnBP, MBzP, 5oxo-MEHP, 5OH-MEHP, MEHP MMP, MEHP and ΣMEHP were significantly higher in the LEI patients than in controls.
Weuve et al. 2010 [92] Cross-sectional 1999–2004 20–54 General population 151/1,020 Urine MBP, MEP, MEHP, MBzP, MEHHP, MEOHP Positive association for MBP and inverse association for MEHP in relation to leiomyoma.
Huang et al. 2014 [85] Case-control 2005–2007 41.1 ± 6.8 Laparotomy population 36/69 Urine MMP, MEP, MnBP, MBzP, 5oxo-MEHP, 5OH-MEHP, MEHP Compared with controls, patients with leiomyoma had significantly higher levels of total urinary ΣMEHP, MnBP, and MEP.
Pollack et al. 2015 [90] Case-control 2007–2009 Case: 37.8 ± 4.9 Laparotomy population 99/374 Urine MBzP, MnBP, MiBP, MCHP, MEP, MEHP, MEHHP, MEOHP, MECPP, MiNP, MCNP, MMP, MCPP, MOP MMP levels were significantly lower in women with than without fibroids.
Control: 31.7 ± 6.9
Kim et al. 2016 [88] Case-control March 2013 and July 2015 Case: 42.6 ± 1.02 Laparotomy population 30/27 Urine MBzP, MiBP, MCHP, MEP, MEHP, MEHHP, MEOHP, MECPP, 2cx-MMHP 5cx-MEPP, MMP, MCPP, MnOP, MnPP, MiDP, MiNP The log transformed creatinine-adjusted levels of MEHP, MEHHP, 2cx-MMHP and ΣDEHP were significantly higher in the leiomyoma group than in the controls.
Con: 34.78 ± 1.90;
Sun et al. 2016 [91] Case-control 2010.10–2012.05 Reproductive age General population 61/61 Urine MMP, MEP, MiBP, MnBP, MEHP, MEHHP, MEOHP, MECPP, MCMHP Cases had significantly higher levels of creatinine-adjusted MiBP, MnBP, MEHP, MEOHP, MEHHP, MECPP, total ΣDEHPmet, and total ΣDBPmet than controls.
Kim et al. 2017 [87] Case-control March 2012 and February 2013 Case: 35.3 ± 0.8 Surgery population 53/33 Urine MEHHP, MEOHP, MnBP, MBzP, MECPP The urinary concentration of MECPP was higher in women with leiomyoma than in controls.
Con: 32.6 ± 1.41;
Zota et al. 2019 [98] Pilot, cross-sectional study During 2014–2017 26–54 Women with symptomatic fibroid tumors and subsequently undergoing surgical management 57 Urine MEP, MnBP, MHBP, MiBP, MHiBP, MBzP, MCPP, MiNP, MCOP, MCNP, MEHP, MEHHP, MEOHP, and MECPP and ΣDEHP Higher levels of MHiBP, MCOP, MCNP, MEHHP, MEOHP, and MECPP and ΣDEHP phthalates were positively associated with uterine volume.
Lee et al. 2020 [93] Case-control During 2015–2016 Case: 38.2 ± 5.3 General population 32/79 Urine OH-MINP, cxMINP, MMP, MEP, MiPP, MiBP, MBP, MPeP, MBzP, MCHP, MHxP, MEOHP, MEHHP, MECPP, MCMHP, MEHP, ΣDEHP metabolites, MCPP The concentrations of MEOHP, ΣDEHP, and OH-MINP in urine were significantly higher in the cases than in the control group.
Con: 37.8 ± 5.11
Lee et al. 2020 [94] Case-control During 2015–2016 25–45 General population 96/337 Urine MMP, MEP, MiBP, MnBP, MBzP, MECPP, MCMHP, MEHHP, MEOHP, ΣDEHP metabolites Urinary concentration of MMP, MECPP, ΣDEHP metabolites were associated with increased odds of leiomyoma.
Fruh et al. 2021 [95] An ongoing prospective cohort study 2010–2012 A prospective cohort of premenopausal Black women aged 23–35 years A population of reproductive-aged Black women 754 Black women Urine MBP, MHBP, MiBP, MHiBP, MBzP, MEHHP, MEHP, MEOHP, MECPP, MEP, MCOP, MNP, MCNP, MCPP. The urinary concentrations of phthalate biomarkers were not significantly associated with higher risk of uterine leiomyoma, either individually or jointly.
Pacyga et al. 2022 [96] A retrospective study Women from NHANES 2005–2016 and MWHS 2006–2015 Ages: 45–54 years Pre- and peri-menopausal women 754 Urine MEHP, MEHHP, MECPP, MEOHP, MCPP, MBzP, MEP, MBP, MiBP ΣDEHP The risk of prior fibroid diagnosis was increased by 13 % for each two-fold increase in ΣDEHP.
Zhang et al. 2023 [97] Case-control 2018–2020 Case: 30.7 ± 4.3 Females who went through ART treatment 226/83 Urine MEP, MiBP, MBP, MBzP, MEHP, MEOHP, MEHHP, MECPP Increased urinary MBzP concentration was associated with an increased risk of uterine fibroids.
Con: 33.6 ± 5.0

  1. Phthalates metabolites with a negative association with fibroids are shown in gray, those with a positive association are shown in bold letters, and those with no significant difference are shown in black normal font letter. MWHS, the Midlife Women’s Health Study; NHANES, the National Health and Nutrition Examination Survey.

In one study by Luisi et al. [89], DEHP and MEHP concentrations were lower in women with uterine fibromatosis. Another study that tested urine samples also found an inverse association between MEHP and leiomyoma [92]. In a separate study, the level of one common phthalate metabolites (MMP) in urine was significantly lower in women with than in those without fibroids (1.78 vs. 2.40 mg/g, respectively) [90]. However, other studies have found that patients with leiomyoma had significantly higher levels of at least one urine phthalates metabolites (as shown in bold in Table 3) compared with control [[85], [86], [87], [88, 91], [93], [94], [95, 97]. A Pilot, cross-sectional study found a positive association between phthalate exposure and uterine volume [98]. Despite conflicting findings, the 2017 meta-analysis by Fu et al. found a significant positive association between DEHP metabolites and uterine leiomyoma [99]. In a study by Pacyga et al. [96], women had a 13 % greater risk of prior fibroid diagnosis for a twofold increase in ΣDEHP (MEHP, MEHHP, MEOHP, and MECPP), which suggested a positive relationship between the presence of fibroids and DEHP. A prospective ultrasound study investigated the association between phthalate metabolites and uterine leiomyoma incidence, and found that MEHP concentration was positively associated with the incidence of uterine leiomyoma, whereas other metabolites had an inverse relationship. However, these associations were weak to moderate and did not reach statistical significance [95]. All of these results suggest that DEHP and its metabolites are positively associated with the development of fibroids.

DEHP is an EDC that is associated with several adverse health outcomes, including adverse reproductive outcomes in both men and women, insulin resistance, type II diabetes, overweight/obesity, asthma, and allergy [100]. In vitro studies [87], 101] have suggested that DEHP may play a role in the pathogenesis of uterine leiomyoma by exerting an antiapoptotic effect, increasing proliferative activity, and increasing collagen production in myometrial and leiomyoma cells. A more recent in vitro study revealed that the principal DEHP metabolite, MEHHP, promotes leiomyoma cell survival by activating the tryptophan-kynurenine-aryl hydrocarbon receptor pathway [102]. A further in vivo study found that NOD/SCID mice implanted with fibroid tissues and fed DEHP had larger volumes of fibroid tissues than control mice that were not exposed to DEHP [103]. Therefore, it is reasonable to propose that this phthalate may have a negative role in uterine leiomyoma. In another study, exposure to certain phthalate biomarkers, such as mono-hydroxyisobutyl phthalate (MHiBP), monocarboxyoctyl phthalate (MCOP), monocarboxynonyl phthalate (MCNP), MEHP, MEHHP, MEOHP, and MECPP, was positively associated with the volume of the uterus; this finding provides further evidence that phthalate exposure may be associated with fibroid outcomes [98].

Phthalate and endometriosis/adenomyosis

EMs is defined as the presence of endometrial-type mucosa outside the uterine cavity. When endometrial tissues invade the myometrium with smooth muscle hyperplasia, it is defined as adenomyosis (AD). EMs is one of the most common benign gynecological diseases in premenopausal women with morbidity 30–50 % in women with infertility and 10–15 % of reproductive-aged women. Patients diagnosed with EMs commonly present with clinical symptoms such as pelvic pain, adnexal masses, or infertility. These patients may also have an increased risk of epithelial ovarian cancer [104], 105]. Endometriosis-associated ovarian cancers, which include endometrioid and clear cell ovarian carcinoma, are generally acknowledged as the most lethal gynecological malignancy [104]. Although the potential pathogenesis of EMs and AD remains unclear, environmental factors such as phthalates are likely to play important roles.

Using a biomimetic hydrogel-based three-dimensional culture model, Kim et al. [74] suggested that DEHP could increase the proliferation and migration of human endometrial and endometriotic epithelial cells, induce the EMT, and promote stem cell properties via the TGF-β/Smad signaling pathway,. This research provides a new potential therapeutic target for DEHP-induced endometriosis.

According to in vitro studies, phthalates exposure may increase the proliferation and viability of endometrial cells, which may lead to the development of EMs via retrograde menstruation. In an in vivo study using the NOD/SCID mouse model, Kim et al. found that the size of endometrial implants was significantly larger in mice fed DEHP compared with those fed with vehicle alone. Although this model cannot fully replicate the human immune system, the findings support the idea that DEHP may stimulate the formation of EMs. A study by Sharma et al. reported that chronic exposure to low-dose BBP promote survival of endometriotic tissue through CD44-expressing on plasmacytoid dendritic cells [106]. In this study, BBP exposure did not promote the growth of endometriotic lesions, although the lesion survival rate in BBP-treated mice was significantly higher than in the control mice.

We conducted a search of all relevant literature for human until Nov 31, 2023 and identified 17 original studies that investigated the relationship between phthalate exposure and EMs (16 studies) [81], 85], 86], 92], 97], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117] or AD (2 studies) [86], 94] which are summarized in Table S1. However, the conclusions of these studies are inconsistent. As shown in Table 4, some studies reported results that are completely opposite, such as those involving DEHP and its metabolites. For example, Cobellis et al. [107], Reddy et al. [109], Kim et al. [111], Nazir et al. [114], and Yi et al. [117] found that women with EMS had significantly higher plasma DEHP concentration than the controls, which suggested a positively association between DEHP concentration and EMs. Upson et al. [113] reported a decreasing trend in the EMs group despite the lack of significant difference in concentration between the Ems and the control group. These differences may reflect differences in samples used (urine and plasma) and study populations. Other studies have also found that the concentrations of MEHP and DHEP are associated with EMs and with advanced-stage EMs [107], 108].

Table 4:

Associations between phthalates and EMs in 16 studies. Level of evidence of phthalate exposure related to EMs was highlighted according to an adapted version of the Oxford Center for Evidence-Based Medicine (CEBM) and Marx et al. [121], [122], [123].

Contradictory results were found more often for DEHP and its primary or secondary metabolites. Phthalate diesters are unstable in human blood and have a short half-life (e.g., 28 min for DEHP). Serum enzymes are reported to hydrolyze DEHP to MEHP during storage [118], 119], and blood sampling can cause phthalate exposure. Given these factors, blood phthalate diesters may be less appropriate biomarkers for evaluating phthalate exposure. Although the results for the metabolites of DEHP in urine were also inconsistent, urinary phthalate metabolites are more stable and relatively free from laboratory contamination.

The evidence supporting a relationship between MBP (MnBP) and EMS is also inconsistent. Five studies [86], 92], 112], 116], 117] have suggested a positive relationship between MBP and EMs, but six other studies [81], 85], 97], 110], 113], 115] have shown no significant difference between the EMs and control groups. Other phthalates such as BBzP, MMP, MiBP, MEP, MBzP also have contradictory effects as shown in Table 4. The discrepant findings reflect differences in exposure assessment and/or case definition. Among these studies, two studies from the same research group have made different conclusions, which may have been influenced by the large standard deviations and individual variability. However, the trend for MBP is the same, with higher concentrations found in the EMs group than in the control groups [85], 92]. No associations have been found between EMs and other phthalates, including MOP, MCHP, and MINP, which suggests that these phthalates may have weak estrogenic activity.

In a meta-analysis, Cai et al. [120] investigated five types of phthalate metabolites (MEHHP, MEP, MBzP, MEHP, and MEOHP) and found that only one metabolite (MEHHP) was potentially associated with EMs. However, this meta-analysis included only eight studies, the quality of the studies was moderate, the available evidence was limited, and there may have been some bias.

No association was found between the exposure to phthalates and AD [86], 94]. However, given that there are only two studies, and their samples were small, further investigation is needed.

In conclusion, given that EMs is an estrogenic-associated disease and phthalates have potential estrogenic activity, there appears to be a strong association between phthalate exposure and this disease. However, further studies are needed to clarify this relationship. Future research should include larger sample sizes and more phthalate metabolites. Additionally, the sample detection time and methods should be taken into consideration to improve the accuracy and comparability of the results.

Phthalates and uterine histology changes

Phthalates exhibit estrogenic activity and can regulate endometrial cells in vitro. In recent years, the effects of phthalate exposure on the uterus have been explored in vivo.

Exposure to phthalates, such as DEHP and DEP, may affect uterus weight, although findings are not always consistent. Some studies have found that DEHP exposure increased uterus weight, but others have reported no significant change, and one study found a decrease in weight in immature rats exposed to DEHP during postnatal day 1–21 [124], [125], [126], [127], [128]. These discrepancies may reflect differences in exposure conditions, such as the time, dose, duration, and age of exposure. DEP, another common phthalate, has also been shown to increase the height of uterine epithelial cells as well as uterine weight in adult female rats. Moreover, DEP exposure up-regulates the expression of several steroidogenic genes and the pS2 gene and activates the MAPK pathway by increasing the p-ERK/ERK ratio [129].

Somasundaram et al. [126] demonstrated that exposing adult rats to DEHP for 30 days at doses of 0–100 mg/kg body weight/day increased the protein levels of ERs, PRs and PPARγ in the uterus. However, no significant changes were observed in the wet uterine weight. Histological studies revealed a decreased diameter and layers of the uterus and disrupted glandular epithelium. These findings are inconsistent with those of Richardson et al. [124], who found that DEHP exposure for 30 days at different doses (20 ug/kg/day to 200 mg/kg/day) increased the number of uterine glands, increased the proliferation of uterine stromal cells at the highest dose, and increased the numbers of weakened, dilated blood vessels in the endometrium. Kim et al. [125] found that chronic low-dose exposure to DEHP had an estrogen-like effect in the mouse uterus. In contrast to the data of Somasundaram et al. [126] but consistent with those of Richardson et al. [124], Kim et al. found that DEHP exposure increased the ratio of uterine weight to body weight (133 μg/L), endometrial and myometrial thickness (133 and 1,330 μg/L), and numbers of luminal epithelial cells (50 μg/L). In addition, the mRNA expression levels of known 17β-estradiol (E2) downstream genes, Esr1, Esr2, Prg, Lox, Egr1 and Muc1 were also changed. Further experiments found that DEHP changed the proliferation status of uterine endometrial cells and modified the translocation of ESR1, ESR2 and PGR [130]. These results suggest that DEHP affects the nuclear activation of steroid hormonal receptors, which may induce histological changes in a dose-dependent manner and have adverse effects on normal uterus function.

DEHP exposure not only has effects on the mother’s generation (F0), but also has long-term reproductive toxic effects on the offspring, F1 and F2, such as expression of the PR, ERα. The expression of estrogen and progesterone regulatory genes such as HoxA11, VEGF A, IHH, LIFR, EP4, PTCH, NR2F2, BMP2, and WNT4 in the uterus were reported to be decreased in DEHP-exposed offspring rats [131]. In another study, female offspring of female mice exposed to DEHP from prenatal to peripubertal times developed endometrial atrophy and fibrosis [132].

Li et al. [133] found that prenatal exposure to a mixture of phthalates in mice (including 5 % BBP, 8 % DIBP, 15 % DINP, 15 % DBP, 21 % DEHP, and 35 % DEP) led to transgenerational and multigenerational effects on uterine morphology and function. The exposure to the mixture (20 μg/kg/day to 500 mg/kg/day) from gestational day 10.5 to parturition resulted in a higher incidence of large, dilated glands, multilayered luminal epithelium, and fibrotic response in all generations. The luminal epithelial cell proliferation was also increased in F2 generation in the 200 mg/kg/day group. A recent study also found that chronic exposure to a phthalate mixture increased the thickness of the myometrial layer and the incidence of collagen deposition in uteri, which may be associated with the upregulation of TGF-β signaling [134]. Although some studies have mentioned the effects of phthalate exposure on uterus weight and cell proliferation, few have explored the underlying mechanisms. Moreover, some studies have shown that prenatal exposure to phthalates such as DiNP has no influence on uterus weight [135], 136]. Further research is needed to examine the effects of phthalate exposure on endometrial hyperplasia and oncogenesis, as well as on the cellular function of the uterus.

Conclusions

In conclusion, studies conducted both in vitro, in vivo and on human populations suggest a strong association between exposure to phthalates and uterus disorders, such as leiomyoma and EMs. Figure 1 summarizes the potential mechanisms underlying this association. However, whether phthalates play a role in uterine carcinomas require further investigation. Abnormal uterine bleeding which is associated with hormone fluctuation and the endometrial environment, including inflammatory factors, may be affected by phthalate exposure. The understanding of the effects of phthalates on the endometrial environment and its secretory functions is currently limited. Research into the epigenetic changes in the uterus caused by phthalate exposure may reveal new insights into the effects of EDCs. Therefore, exploring the effects of phthalate exposure and its underlying mechanisms is crucial for discovering new interventions to prevent and treat uterine disorders in women.

Corresponding author: Aiyue Luo, Department of Obstetrics and Gynecology, National Clinical Research Center for Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 10487 Jiefang Ave, Wuhan 430030, Hubei, People’s Republic of China, Phone: +86 02783663351, Fax: +86 02783663351, E-mail:

  1. Research ethics: Not applicable.

  2. Informed consent: Not applicable.

  3. Author contributions: The first draft of the manuscript was written by Shuhong Yang. Shuhao Yang and Aiyue Luo revised the review. All authors read and approved the final manuscript.

  4. Competing interests: The authors declare no competing interests.

  5. Research funding: Not applicable.

  6. Data availability: Not applicable.

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Supplementary Material

The article contains supplementary material (https://doi.org/10.1515/reveh-2023-0159).

Received: 2023-11-08
Accepted: 2024-01-26
Published Online: 2024-03-08
Published in Print: 2025-03-26

© 2024 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. Mercury and cadmium-induced inflammatory cytokines activation and its effect on the risk of preeclampsia: a review
  4. Prevalence of chronic obstructive pulmonary disease in Indian nonsmokers: a systematic review & meta-analysis
  5. Beyond the outdoors: indoor air quality guidelines and standards – challenges, inequalities, and the path forward
  6. Cadmium exposure and thyroid hormone disruption: a systematic review and meta-analysis
  7. New generation sequencing: molecular approaches for the detection and monitoring of bioaerosols in an indoor environment: a systematic review
  8. Concentration of Tetrabromobisphenol-A in fish: systematic review and meta-analysis and probabilistic health risk assessment
  9. The association between indoor air pollution from solid fuels and cognitive impairment: a systematic review and meta-analysis
  10. Phthalates and uterine disorders
  11. Effectiveness of educational interventions for the prevention of lead poisoning in children: a systematic review
  12. Association between exposure to per- and polyfluoroalkyl substances and levels of lipid profile based on human studies
  13. Summary of seven Swedish case reports on the microwave syndrome associated with 5G radiofrequency radiation
  14. Expanding the focus of the One Health concept: links between the Earth-system processes of the planetary boundaries framework and antibiotic resistance
  15. Exploring the link between ambient PM2.5 concentrations and respiratory diseases in the elderly: a study in the Muang district of Khon Kaen, Thailand
  16. Standards for levels of lead in soil and dust around the world
  17. Tributyltin induces apoptosis in mammalian cells in vivo: a scoping review
  18. The influence of geology on the quality of groundwater for domestic use: a Kenyan review
  19. Biological concentrations of DDT metabolites and breast cancer risk: an updated systematic review and meta-analysis
  20. Letter to the Editor
  21. Ancient medicine and famous iranian physicians
https://doi.org/10.1515/reveh-2023-0159
Keywords for this article
phthalates; endometrial cells; uterine disorders; leiomyoma; endometriosis

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. Mercury and cadmium-induced inflammatory cytokines activation and its effect on the risk of preeclampsia: a review
  4. Prevalence of chronic obstructive pulmonary disease in Indian nonsmokers: a systematic review & meta-analysis
  5. Beyond the outdoors: indoor air quality guidelines and standards – challenges, inequalities, and the path forward
  6. Cadmium exposure and thyroid hormone disruption: a systematic review and meta-analysis
  7. New generation sequencing: molecular approaches for the detection and monitoring of bioaerosols in an indoor environment: a systematic review
  8. Concentration of Tetrabromobisphenol-A in fish: systematic review and meta-analysis and probabilistic health risk assessment
  9. The association between indoor air pollution from solid fuels and cognitive impairment: a systematic review and meta-analysis
  10. Phthalates and uterine disorders
  11. Effectiveness of educational interventions for the prevention of lead poisoning in children: a systematic review
  12. Association between exposure to per- and polyfluoroalkyl substances and levels of lipid profile based on human studies
  13. Summary of seven Swedish case reports on the microwave syndrome associated with 5G radiofrequency radiation
  14. Expanding the focus of the One Health concept: links between the Earth-system processes of the planetary boundaries framework and antibiotic resistance
  15. Exploring the link between ambient PM2.5 concentrations and respiratory diseases in the elderly: a study in the Muang district of Khon Kaen, Thailand
  16. Standards for levels of lead in soil and dust around the world
  17. Tributyltin induces apoptosis in mammalian cells in vivo: a scoping review
  18. The influence of geology on the quality of groundwater for domestic use: a Kenyan review
  19. Biological concentrations of DDT metabolites and breast cancer risk: an updated systematic review and meta-analysis
  20. Letter to the Editor
  21. Ancient medicine and famous iranian physicians
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