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Environ Anal Health Toxicol > Volume 40:2025 > Article
Kim, Ji, and Park: Systematic review and Integrated Approaches to Testing and Assessment (IATA) of PFOA and PFOS for endocrine disrupting effects based on the AOP framework

Systemic Review

Environ Anal Health Toxicol 2025; 40: e2025s03.

Published online: April 4, 2025

DOI: https://doi.org/10.5620/eaht.2025s03

Systematic review and Integrated Approaches to Testing and Assessment (IATA) of PFOA and PFOS for endocrine disrupting effects based on the AOP framework

Doyeong Kim1, Suin Ji1, Kwangsik Park1, *

1College of Pharmacy, Dongduk Women's University, Seoul, Republic of Korea

*Correspondence: kspark@dongduk.ac.kr

Recommended by: Prof. Jung-Hwan Kwon

Received January 21, 2025       Accepted February 25, 2025

©2025, The Korean Society of Environmental Health and Toxicology & Korea Society for Environmental Analysis

This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been extensively used as surfactants, surface protectors, food packaging materials, and fire-retardants. Due to serious adverse effects on human and environment, they are now considered as legacy compounds. In this study, the effects of PFOA and PFOS on endocrine disruption were assessed using the Integrated Approaches to Testing and Assessment (IATA) methodology based on the adverse outcome pathway (AOP). PFOA/PFOS in vitro data for enzyme activities for steroidogenesis, hormone levels of 17β-estradiol and testosterone, receptor binding capacity, receptor transcriptional activation, cell proliferation and differentiation were collated and assessed to elucidate the association between the data with the human adverse outcomes. Multiple studies indicate that PFOA/PFOS alter enzyme activities, hormone levels, receptor transcription, and cell proliferation. Moreover, associations were found between in vitro data and human outcomes including semen quality, menarche, menopause, menstrual cycle, infertility, miscarriage, cancer, and birth weight. This study effectively links toxic mechanisms to human adverse outcomes of PFOA and PFOS. However, in vitro data based on the molecular initial events (MIEs) and key events (KEs) in AOP frame works are not enough and often inconsistent for integrated assessment, which suggest that more data for endocrine disruption are required for clear and complete IATA of PFOA and PFOS.

Keywords: Perfluorooctanoic Acid, Perfluorooctane Sulfonate, Integrated Approaches to Testing and Assessment, Endocrine Disruption

Introduction

PFAS (polyfluorinated alkyl substances) are fluorinated substances of anthropogenic origin containing at least one fully fluorinated methyl or methylene carbon atom, although exceptions exist. The general structure of PFAS resembles that of fatty acids, with each compound featuring a hydrophobic per- or polyfluorinated carbon tail of varying lengths and a functional end group[1]. Owing to their unique properties, including resistance to water, oil, stains, and fire, they have been and are extensively used in industrial and consumer fields as surfactants, surface protectors for paper and textiles, polishes, food packaging materials, and fire-retardants. The widespread exposure and adverse effects of these chemicals are well documented. Regulatory authorities in many countries have subsequently begun to limit the production and consumption of these compounds, and have initiated programs to reduce product contents and facility emissions. The Stockholm Convention added PFOS to the list of persistent organic pollutants (POPs) to reduce and eventually eliminate its production and uses [2–4]
Exposures to PFOA and PFOS, which are eight-carbon structure with different functional group and well-known legacy compounds, have been reported to cause liver toxicity, reproductive disorders, neurotoxicity, and immunotoxicity. They are also strongly associated with disturbances in hormone homeostasis. Epidemiological studies have identified positive associations between PFOA/PFOS exposures and disrupted serum levels of estrogen, androgen, and thyroid hormones. Additionally, endocrine disruption could lead to infertility, menstrual cycle disorders, and an increased risk of breast cancer [5–7]. In studies involving 120 Chinese women, elevated plasma levels of these compounds were positively associated with the risk of primary ovarian insufficiency (POI), with odd ratios of 3.80 for PFOA and 2.81 for PFOS. Exposure to PFOA and PFOS linked to increased levels of prolactin and decreased thyroxin [8]. Another study involving 178 healthy nulliparous women found that PFOS concentrations were inversely associated with 17β-estradiol, indicating potential effects on women’s reproductive health [9]. Results from the National Health and Nutrition Examination Survey (NHANES 2015-2016) showed significant associations between PFOA/PFOS exposures and increased serum total testosterone concentrations in males, and positive correlations with free testosterone levels in women aged 20-49, while PFOS was negatively associated with total testosterone levels in girls aged 12-19 [10] Endocrine disruption data for PFOS/PFOA show variability, especially in sex hormone levels influenced by gender, age, and compound type. Moreover, the results did not explain the toxicological mechanisms for these epidemiological findings, based on the concept of adverse outcome pathway (AOP) for endocrine disruption [11,12].
In this study, we utilized Integrated Approaches to Testing and Assessment (IATA) for PFOA and PFOS within the framework of AOP for endocrine disruption. IATA is defined as a structured approach that integrates and weighs multiple sources of information to conclude on the toxicity of chemicals and to support regulatory decision-making. It may include existing information from the scientific literature or other resources, along with newly generated data resulting from new or traditional toxicity testing methods to fill data gaps. Although in vivo studies using animals may provide clearer evidence of toxicity in the AOP, new alternative methods have been introduced as tools to avoid animal testing. These include the use of in vitro, in chemico, and in silico assays [13,14]. AOPs for endocrine disruption have recently been proposed by numerous institutions, including the Organization for Economic Cooperation and Development (OECD) and the Environmental Protection Agency (EPA) of the United States of America (USA). These AOPs specify molecular initiating events (MIE) and key events (KE), including enzyme activities for steroidogenesis, sex hormone levels, receptor binding, hormone receptor transcription, and cell proliferation and/or differentiation [15,16]. For the integrated assessment of PFOA and PFOS for endocrine disruption in this study, all data, including both in vitro and in vivo, were collected and evaluated within the IATA framework (Fig.1). Furthermore, the relationship between in vitro/in vivo data and human epidemiological data was assessed to identify connections between the toxic mechanisms and adverse outcomes of PFOA and PFOS.

Methods

Based on the IATA and AOP frameworks for endocrine disruption, components of the MIEs and KEs were identified, and a comprehensive literature search was conducted using PubMed and other resources for relevant English-language papers published up to August 2024 [15,16]. Keywords for chemicals included PFAS, PFOA, and PFOS, while those for endocrine disruption covered steroidogenesis, estrogen, androgen, hormone, receptor binding, transcription, proliferation, differentiation, epidemiology, human, among others. Relevant papers were screened using selective keyword combination. In terms of tests for steroidogenesis, the levels of 17β-estradiol (E2) and testosterone (T), as well as activities of enzymes involved in hormone synthesis, were reviewed. The key enzymes include steroidogenic acute regulatory protein (Star), Cytochrome (cyp) 11a, Cyp17, Cyp19, Cyp21, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD, and 5α-reductase [17]. Additionally, levels of associated proteins and gene expressions were also addressed.
Three researchers independently reviewed and selected studies after discussion. Over 300 papers were scrutinized, and approximately 120 papers were included in this review. Data from in vitro, in vivo, and epidemiologic studies were systematically reviewed and analyzed following the methodologies of previously published reports [18-20]

Results and Discussion

Enzyme activity for steroidogenesis and hormone levels

Steroid hormones, including estrogen and androgens, are derived from cholesterol and exert significant effects on metabolism, reproduction, and various homeostatic processes in the body. For steroidogenesis, cholesterol is transported into mitochondria by StAR (Steroidogenic acute regulatory protein), and the side chain is cleaved by the cytochrome p450 enzyme (Cyp11a) to form pregnenolone. Pregnenolone is a biologically active endogenous steroid and serves as a precursor or metabolic intermediate in the biosynthesis of most steroid hormones, such as progestogens, androgens, estrogens, glucocorticoids, and mineralocorticoids. Multiple enzymes contribute to hormone synthesis (Fig. 2) [17,21]. This study reviewed the effects of PFOA and PFOS on key enzymes in steroidogenesis and hormone levels (E2 and T).
In microsomal fractions from rat testes and intact purified cultured Leydig cells, PFOA inhibited enzymes 3β-HSD and 17β-HSD. The IC50 values for microsomal 3β HSD were 53.2±25.9 μM and for 17β-HSD were 17.7±6.8 μM. Furthermore, it impeded Leydig cell enzymes of 3β-HSD with an IC50 value of 146.1±0.9 μM and 17β-HSD with an IC50 value of 194.8±1.0 μM [22]. Inhibition of these enzymes was notably stronger in human testes microsomes treated with PFOA and PFOS, with IC50 values of 1.35±0.05 μM and 6.02±1.02 μM, respectively [23].
MLTC-1 (mouse Leydig tumor cells) was isolated from the testis of a male patient with a Leydig cell tumor. When MLTC-1 cells were exposed to 0, 50, 100, or 200 μM PFOA for 48 h, biphasic effects on T levels were observed: an increase at 100 μM and a decrease at 200 μM PFOA, while no change occurred at 50 μM. The compound reduced the gene expression of StAR, 3β-HSD, and Cyp17, but did not affect Cyp11 and 17β-HSD [24]. Another study using the same MLTC-1 cell line showed increased T levels even at lower PFOA concentrations of 1 and 10 μM. Increased protein levels of 17β-HSD, which converts androstenedione to T, accounted for the elevated T levels in PFOA-treated MLTC-1 cells [25].
Many steroidogenesis tests have been conducted on cultured H295R cells isolated from the adrenal gland of a female adrenal cancer patient. This cell line can produce adrenal androgens. When H295R cells were exposed to PFOS at concentrations ranging from 6 nM to 600 μM, a dose-responsive increase in E2 was observed without significant changes in steroidogenic enzymes, including Cyp19 (aromatase), suggesting that the altered estradiol steroidogenesis occurred through mechanisms other than modifications of Cyp19 [26]. Other studies using H295R cells indicated that both PFOA and PFOS increased E2 levels but reduced T levels. The mRNA expressions of enzymes StAR, 3β-HSD, and Cyp19 were increased, while those of Cyp11a1 and Cyp17 were decreased. Discrepancies were noted in other enzyme activities between PFOS and PFOA, where PFOA increased Cyp21 and 17β-HSD, but PFOS decreased them [27,28]. Furthermore, two additional studies showed that either PFOS or PFOA increased E2 levels in H295R cells, with an accompanying increase in mRNA expression of Cyp19 [29,30]. However, no effects on E2 levels in H295R cells treated with PFOA or PFOS were also reported, with no changes in the gene expression of those enzymes [31–33]. Biphasic changes in E2 levels depending on the concentration were shown in a study [34].
When the effects on aromatase (Cyp19) activity were assessed using the human choriocarcinoma JEC-3 cells, the enzyme activity remained unchanged by PFOA or PFOS (10-8 M ~ 10-4 M), indicating no apparent effects on E2 levels[35]. Conversely, Cyp19 activity appeared to be inhibited in JEC-3 cells by PFOS and PFOA, although a cytotoxic effect could not be entirely ruled out[36].
Several reports using various test systems have been published, demonstrating the effects of PFOA and PFOS on steroidogenesis. In an assay with primary human placental cytotrophoblasts, PFOS reduced Cyp19 levels and E2secretion[37]. Variations were evident in E2 levels, T, and Cyp19 activity/mRNA expression across different studies [38–41]. All in vitro data related to steroidogenesis are presented in Table 1, highlighting inconsistencies and discrepancies among the findings.

Receptor binding and signal transduction

Hormones interact with their respective receptors located either on the cell membrane surface or inside the cell, triggering signals that exert hormonal effects on target cells. The estrogen receptor (ER) belongs to the nuclear receptor protein superfamily and exhibits transcriptional activity modulated by the binding of ligand agonists or antagonists. This activity influences cell growth, differentiation, and proliferation in estrogen-dependent breast cancer [43]. Only a few studies have explored the binding assay for perfluorinated alkyl substances and ER, as illustrated in Table 2. When the estrogenic activity of PFOA and PFOS was assessed using the Yeast two-hybrid assay with modified yeast cells expressing hER isoforms (hERα and hERβ) and β-galactosidase, no estrogenic effects were detected, indicating a lack of interaction or binding between the chemicals and the ER domain[44]. Utilizing surface plasma resonance (SPR) techniques that differentiate between hERα agonists and antagonists by monitoring protein conformation changes induced by ligand binding, interactions of PFOS and PFOS were detected and identified as agonists with Kd values of 2.19 μM and 107 μM, respectively [45]. A fluorescence polarization (FP) competitive binding assay was employed to measure the binding affinity of PFOA; the chemical was found to competitively inhibit the binding of a reference compound to hERα and hERβ domains. The IC50 values were determined to be 469.5±4.6 μM (hERα) and 384.4±3.5 μM (hERβ) [46]. All binding assays in these studies were indirect, with no direct binding assays reported following the OECD TG 493 guideline [47]. According to the guideline, competition between test chemicals and [3H]-labelled E2 for estrogen receptor binding can be assayed, suggesting direct interaction of test chemicals with the estrogen receptor. Currently, there appear to be no published studies on androgen receptor (AR) binding.

Receptor transcriptional activation

ER and AR regulate gene expression in response to respective hormones and other chemicals acting as agonists or antagonists. Hormone signaling in these receptors may occur via ligand-dependent or ligand-independent pathways. Target genes are regulated through genomic pathways, either by direct receptor complex-DNA interaction or via a tethering mechanism involving other transcription factors. In the classical genomic pathway, ligands bind to the receptor, inducing conformational changes that allow for dimerization and DNA binding, ultimately leading to transcriptional activation (agonists) or inhibition (antagonists). Many PFASs directly impact ER/AR binding, disrupt signaling, and induce transcriptional activity, resulting in endocrine disruption [48]. Studies on the effects of PFOA/PFOS on ER/AR transcriptional activity have shown that outcomes can vary based on cell type, reporter gene constructs, test concentrations, exposure durations, and other measurement conditions, as indicated in Table 3.
When human embryonic kidney (HEK-293T) cells were cultured and treated respectively with PFOA and PFOS for 24 h at concentrations ranging from 1 to 1000 nM, the hER reporter gene (β-galactosidase) was significantly increased to 2~3 times the level of the vehicle control, showing efficacy comparable to E2 [49]. In MVLN cells, which displayed stable expression of firefly luciferase in MCF-7 cells for assaying ER-responsive genes, both PFOA and PFOS enhanced ER transcriptional activity. Without E2 co-treatment, the EC50 values for transcriptional activity were 6.5 x 10-5 M for PFOA and 2.9 x 10-5 M for PFOS, indicating relatively weak estrogenic effects compared to natural estrogen ligands. With E2 co-treatment, these compounds amplified the effects of E2 on ER transcription by 145 to 210% at the highest tested non-cytotoxic concentrations[35]. Using the same cell lines, luciferase transcriptional activity increased in a dose-dependent manner following exposure to PFOA (0~ 800 μM) alone for 12 and 24 h, suggesting agonistic activity of ERs. However, this activity decreased with E2 co-treatment [46]. Similar findings were observed in MVLN cells with varying concentrations of PFOS, yet co-treatment with E2 reduced ER transcriptional activity[50]. Contrasting these increased transactivation effects, a study reported decreased ER transcription in 22Rv1/MMTV cells. In the presence of 10 pM of E2, both PFOA and PFOS exhibited weak antagonistic ER transactivation by reducing luciferase activity compared to 10 pM E2 [30].
However, in other cells such as CV-1 and T47D, ER transcription reporter gene activity was unaffected by PFOA or PFOS treatment (10-9~ 10-7 M), although the combination of these compounds with E2 resulted in additive effects. PFOA alone had no impact on reporter gene expression in CV-1 cells transfected with ER reporter genes when compared with vehicle control, but it increased gene expression 12.8-fold with 1 x 10-9 M E2[42]. PFOS exhibited results similar to those observed with PFOA-treated CV-1 cells [27]. In T47D human breast cancer cells stably transfected with a luciferase reporter gene, PFOA (10-9~10-7 M) and PFOS (10-10 ~10-7 M) demonstrated similar trends as previously described. When the cells were treated only with the compounds, no effects on ER transcriptional activity were observed, but additive effects emerged in the presence of 1nM E2 [51]. Comparable responses were also observed in the T47D-KBluc assay across different concentrations [52]. In the case of HEK293T cells, transiently transfected with either ERα or ERβ and co-transfected with the GAL4-dependent luciferase, the outcomes might vary from those previously reported[49]. In contrast to the increase in ER transcription, there were no effects on ERα and ERβ transcription in cells treated with 100 μM PFOA or PFOS alone. However, additive effects were observed with the co-exposure to 0.4 nM E2, and the compounds significantly increased transcription beyond the levels seen with 0.4 nM E2 alone [33].
Among the results of AR transcription, three instances reported that PFOA or PFOS did not change the transcriptional activity. In the AR reporter gene expression assay using MDA-kb2 cells, none of the test groups exhibited androgenic or anti-androgenic activities at the assay concentrations (10-9~ 10-7M) [27,30,42]. It was also noted that no agonistic or antagonistic activity was observed in the AR transactivation assay using 22Rv1/MMTV cells at concentrations ranging from 10 pM to 10 μM. However, in CHO-K1 cells, the activity decreased, displaying antagonistic effects on AR with or without dihydrotestosterone (DHT). With a positive control treatment of 25 pM DHT, PFOA and PFOS reduced the AR transcription activated by DHT, with IC50 values of 1.1x10-5 M and 4.7x10-6 M, respectively [35]. When the transcriptional activity of ligands was assayed, the results varied with or without testosterone. In HeLa cells transfected with human AR (pSV-AR0) and mouse mammary tumor virus (MMTV)-luciferase, AR reporter gene expression increased with 1 μM PFOA or PFOS without testosterone (10 nM), but these compounds decreased the AR transcription activated by testosterone [53].
All the transcription data for ER and AR did not always demonstrate consistent agonistic or antagonistic activity, but various inconsistencies were observed depending on the test systems, as indicated in Table 4.

Cell proliferation and differentiation

Hormone-related cancers, including breast, endometrium, ovary, prostate, testis, and thyroid cancers, share a unique mechanism where hormones drive cell proliferation and contribute to the accumulation of random genetic errors [58]. In this study, the cell proliferation-promoting capacity of PFOA and PFOS was reviewed. As demonstrated in Table 5, some data suggested that PFOA/PFOS may induce cell proliferation, while other data did not support this finding. There were instances where proliferative activity was apparent only in co-treatment with E2. When MCF-7 cells were treated with 0.01 ~ 100 μM PFOA and PFOS, respectively, for 6 days, cell proliferation was not observed[54]. Similar results were obtained with the same cells and treatment conditions [33].
PFOA and PFOS (10-12 to 10-5 M) without co-treatment of E2, did not demonstrate any proliferation effects on T47D hormone-dependent human breast cancer cells; however, proliferation increased in the presence of 1 nM E2 [51]. In another study of T47D cell proliferation, PFOA (0~ 300 μM) did not stimulate cell growth[55]. Proliferation stimulation was also observed in a few studies. When MCF-10A cells were treated with PFOS (0~ 1000 μM) for 3 days, cell proliferation was elevated approximately 1.5 folds compared to the control group; however, cytotoxic effects were observed at higher concentrations (250 ~ 1000 μM)[56]. It was reported that even at picomolar (10-12 M) concentrations, PFOA increased the proliferation of prostate (DU145) and breast cancer cell lines (MCF-7) [57], and the compound also enhanced the proliferation of trophoblast cells treated for 3 days through a reactive oxygen species-dependent ERK signaling pathway [28]. Currently, the data on PFOA/PFOS-induced proliferation are positive to the association but not conclusive.

Steroidogenesis of in vivo tests

In vivo data on steroidogenesis, potentially altered by PFOS or PFOA treatment, were insufficient compared to the in vitro data. When adult male rats were orally administered PFOS (0.5 mg/kg ~ 6.0 mg/kg), a decrease in serum testosterone concentrations was observed across all doses of the chemical. The study provided no information on steroidogenic enzymes [59]. In an investigation of testicular testosterone, PFOS (5, 20 mg/kg) administered orally from gestational day (GD) 11 to 19 reduced testosterone production in rat fetal Leydig cells at GD 20 and also decreased mRNA expression of StAR, Cyp11, Cyp17, and 3β-HSD [60]. In mice treated with PFOS for 5 weeks at doses of 0.5 and 10 mg/kg (p.o.), serum testosterone levels were significantly reduced, yet the level of E2 remained unchanged[61]. Similar results of decreased testosterone levels were noted in mice treated with PFOA at 1, 5, 10, or 20 mg/kg for 10 days. In this study, Cyp19 was the only enzyme that exhibited increased gene expression, while the expression of other steroidogenic enzymes showed no changes and E2 level remained unchanged [39]. Gender specificity in testosterone levels was also examined. Following maternal exposure to PFOS (0.1, 1.0, and 5.0 mg/kg) during GD 1~17, serum levels of testosterone were decreased while estradiol were elevated in F1 male pups at four and eight weeks of age, but no changes were detected in female pups[62]. However, discrepancies regarding changes in testosterone levels were noted in other studies. Testosterone levels were increased by PFOA [25]. In another study where PFOS was administered in drinking water (10, 50 μg/mL) to pregnant rats from GD 4 ~20, plasma testosterone levels increased and E2 levels decreased. Gene expression of most steroidogenic enzymes was upregulated except for Cyp19. The decrease in mRNA expression of Cyp19 appeared to drive the changes in estradiol and testosterone levels [63]. As demonstrated in in vitro data, results from animal studies also lacked consistency, yet the observed increase in testosterone levels due to PFOS appeared more realistic as indicated in Table 5.

Estrogen and testosterone level in Human studies

Compared to animal studies on the effects of hormone levels induced by PFOA/PFOS, more human data on the association between PFOA/PFOS exposure and hormone levels have been reported. Many participants from various countries including Denmark, the U.S., China, Japan, Italy, Norway, and others have participated in these epidemiological studies, with details such as location, gender, age, study period, and results summarized in Table 6. Generally, individuals involved in an epidemiological study within a single country may exhibit more similarities in terms of food consumption, consumption patterns, social behavior, ethnic status, and other factors. Accordingly, cohort studies or epidemiological surveys were reviewed based on the respective countries where the studies were conducted. In this review, we encountered difficulties in finding consistent results regarding the association between the exposure to perfluorinated compounds and changes in steroid hormone levels.
Four epidemiological studies involving Danish individuals were analyzed in this review. A cross-sectional study of 247 healthy Danish men, with an average age of 19 years, was conducted during 2008-2009. The study results indicated that PFOS levels were negatively associated with testosterone levels, whereas PFOA levels showed no association with this hormone. Neither PFOS nor PFOA exposure showed any association with estradiol levels. Despite the decrease in testosterone levels due to PFOS exposure, semen quality remained unaffected in the study [64]. Blood hormone levels of male offspring (cohort number 169, aged 19 to 21 years) from the pregnant cohort in Denmark and their semen quality, measured in terms of sperm concentration, total sperm count, motility, and morphology, were analyzed. PFOA/PFOS levels in maternal blood samples were also measured from pregnancy week 30. Trends of lower sperm concentration and sperm count with higher FSH and LH levels were observed in the group with higher in utero exposure to PFOA. However, PFOS did not appear to be associated with any of the assessed outcomes. Although PFOA increased levels of FSH and LH, no association was detected between levels of PFOA/PFOS and those of testosterone or estradiol [65]. Another cohort study with Danish participants revealed a contrasting association of high PFOS levels with increased testosterone levels. Although testosterone levels were increased by PFOS exposure, no correlation was found with enhanced endocrine outcomes. In this study, 270 cryptorchidism cases, 75 hypospadias cases, and 300 control cases were analyzed for the association of testosterone levels with PFOS concentration in amniotic fluid [66]. In a further study involving 864 young Danish men, sons in the high tertile of PFOS exposure had lower sperm concentrations, total sperm counts, and percentages of progressive sperm cells, along with higher levels of free testosterone compared with those in the low tertile [67]. From these studies conducted in Denmark, it appears that high levels of PFOA or PFOS may affect sperm quality and reproductive hormone levels, particularly testosterone, although the results were not consistently dependent on age, gender, hormones, and the compounds.
Similar to the studies involving Danish participants, research conducted in the U.S. and China also demonstrated inconsistencies. The relationship between serum PFOA/PFOS and testosterone levels in males and females aged 12 to 80, from the 2011-2012 cycle of the National Health and Nutrition Examination Survey (NHANES) in the U.S., was investigated. No significant relationships were observed in any of the survey models, including age and gender, although these compounds were associated with increased thyroid hormones (Thyroid stimulation hormone, total triiodothyronine, and thyroxine) [68]. However, another study based on the 2015-2016 NHANES cycle reported that PFOS increased serum testosterone concentrations in males. In females, both PFOA and PFOS were positively correlated with the average level of free testosterone in women aged 20-49 years. Conversely, PFOS exposure was negatively associated in girls aged 12-19 years, suggesting age specificity. PFOA was negatively associated with estradiol levels in both boys and girls aged 12 -19 years, while PFOS demonstrated negative associations in girls and positive associations in boys of the same age group. This study indicated that PFAS exposure disrupts sex hormones in a gender-, age-, and compound-specific manner [10]. Another study showed that exposure to PFOA/PFOS was significantly associated with decreased testosterone levels in boys and girls aged 6-9 in the U.S. In terms of estradiol, levels increased with PFOA exposure but decreased with PFOS exposure, highlighting the importance of age-, gender-, and compound-specific differences [69]. A study on prenatal and childhood exposure to PFOA/PFOS (200 mother-child pairs) revealed consistent associations between increased serum levels of these compounds and lower estradiol concentrations at age 12 in girls, as well as delayed pubic hair growth, breast maturation, and age at menarche. Nonetheless, no consistent pattern was observed between PFAS exposure and reproductive hormone levels at other ages [70]. The ethnic diversity of participants in U.S. epidemiological studies, including Mexican American, Asian, African-American, and other multi-racial groups, complicates the interpretation of data.
The results of studies on the association between PFOA/PFOS concentration and hormonal changes in China were similar to those conducted in other countries, indicating a lack of consistent data on age, gender, and compound-specificity. In a study of 540 subjects aged 12-30 years, serum PFOS levels were negatively associated with testosterone levels in young Taiwanese females aged 12-17 years, but not with estradiol [71]. Another report indicated that PFOS was negatively associated with testosterone, while estradiol was positively associated with these compounds in 225 Taiwanese adolescents aged 13-15 years from 2009 to 2010 [72]. PFOA and PFOS were positively associated with primary ovarian insufficiency (POI) in a study of 120 healthy Chinese women and 120 Chinese women with POI, conducted from 2013 to 2016. In the POI group, PFOS was negatively associated with estradiol, but PFOA and PFOS showed no association with serum levels of estradiol and testosterone in the healthy group [8]. Other studies demonstrated a predominantly positive association between PFOA/PFOS and reproductive hormone levels. PFOS was associated with higher serum estradiol levels in neonates in China. PFOA increased estradiol levels and PFOS increased testosterone levels in cord blood in a birth cohort of pregnant women in China. PFOA was positively associated with total testosterone in Chinese women of childbearing age [73–75].
In a study conducted in Norway, 178 women (nulliparous and parous) aged 25-35 were included to assess the association between PFOA/PFOS and reproductive hormones. Among the nulliparous, but not the parous women, PFOS concentrations were associated with reduced E2. However, no association between PFOA and ovarian steroid concentration was observed [9]. In prospective birth cohorts from Japan, 189 mother-infant pairs were recruited between 2002 and 2005. Maternal blood PFOS/PFOA levels after the second trimester and fetal reproductive hormone levels in cord blood were measured to analyze the association between these compounds and hormone levels. As a result, maternal PFOS was significantly associated with increased E2 in male infants [76]. In a UK study involving a population with polycystic ovarian syndrome (PCOS) undergoing infertility treatment, serum PFOS was positively associated with higher incidence of PCOS and irregular menstrual cycles. PCOS cases exhibited higher androgen levels with free androgen index (FAI) and androstenedione compared to controls, although testosterone and estradiol levels were similar. PFOS showed no correlation with E2 and testosterone levels in PCOS cases. However, PFOA might increase testosterone levels in the control group of the study [77]. In a cross-sectional study of 212 perflurocompound-exposed males and 171 non-exposed controls in Italy, increased levels of PFOA/PFOS in plasma and seminal fluid were positively correlated with circulating testosterone and associated with reduced semen quality, testicular volume, penile length, and anogenital distance [53].
When evaluating epidemiological studies on the association of PFOA/PFOS with steroid hormone levels, many results suggested that the correlations or associations between the compounds in serum or semen and hormone levels of estradiol and testosterone were inconsistent, varying based on the specific compound (PFOA or PFOS), age, gender, presence or absence of disease states, among other factors. These findings underscore the need for further research and more extensive data collection.

Adverse outcomes in human by PFOA and PFOS

With evidence of disruptive effects on steroidogenesis, numerous epidemiological studies have been reported. Although major manufacturers ceased production of PFOS in 2002, exposure to PFAS remains a concern as PFAS have biological half-life up to 8.5 years in humans. The NHANES detected PFAS in over 96 % of human samples from participants selected from the 2014~2016 in the survey of the Health of Wisconsin, U.S.A [78]. Additionally, studies in various countries have linked PFAS exposure to symptoms associated with endocrine disruption. Reviews of publications on epidemiological studies have identified the most common adverse outcomes of endocrine disruption caused by PFOS and/or PFOA as affecting sperm quality, menstruation, infertility, miscarriage, fecundity, birth weight, and ovarian/breast cancer. The results of epidemiological outcome studies have shown relatively consistent compared to those of in vitro or in vivo data. These are displayed in Table 7.

Sperm Quality

Several studies have examined the association between exposure to PFOA/PFOS and semen quality. These studies assessed parameters such as semen volume, sperm concentration and count, sperm motility (total and progressive), morphology, and DNA damage. Of eight publications reviewed, six reported that exposure to PFOA and/or PFOS reduced semen quality [65,67,79–82]. However, other studies reported no association between semen quality and exposure to these compounds, both PFOA and PFOS [64,83].

Menarche

Regarding menarche, results were inconsistent. In a study examining the association between chemical exposure and offspring's menarche among pregnant women, involving 218 female offspring aged an average of 11.5 years, exposure to PFOA and PFOS did not appear to alter the age at menarche [84]. Another study showed that serum concentrations of perfluorodecanoate (PFDA) and perfluoroundecanoate (PFUnDA) were associated with early menarche in 921 adolescents aged 15-19 years, whereas PFOA/PFOS were not [85]. However, additional studies indicated an association between menarche and PFOA/PFOS exposure, with chemicals linked to delayed menarche based on serum concentrations in a study of 200 mother-child pairs [70], and a delay of 190 days between the highest and lowest quartile was observed in 2931 girls aged 8-18 years [86]. Cross-sectional epidemiologic studies have shown that PFAS exposure is associated with changes in menarche, irregular menstrual cycles, longer cycle lengths, and earlier menopause [87,88]. Serum PFOA concentration was associated with a lower age at menarche, although the relationship is non-linear and non-monotonic [110].

Menopause and menstrual cycle

Earlier menopause was reported to have association with PFAS exposure [87,88]. In NHANES, the association between PFOA/PFOS levels and age at natural menopause among women aged 20-65 years was investigated. Results indicated that women with higher chemical levels experienced earlier menopause than those with lower levels[89]. Further studies also linked higher PFOA levels with menopause and kidney function[90]. The menstrual cycle appeared to be associated with exposure to perfluorinated compounds, though clear evidence is lacking and inconsistencies remain. Most cross-sectional epidemiologic studies found no association between PFOS exposure and menstrual cycles. Higher PFOA levels were associated with longer menstrual cycles [91–93], but other studies reported no association[94].

Fecundability and infertility

Epidemiologic studies were conducted to examine whether exposure to PFOA and PFOS decreases fecundity in humans. In Denmark, a study measured plasma levels of PFOA and PFOS at weeks 4-14 of pregnancy among 1240 women from the Danish National Birth Cohort recruited from 1996 to 2002 and monitored time to pregnancy (TTP). The results indicated that longer TTP was associated with higher maternal levels of PFOA and PFOS. Compared with women in the lowest exposure quartile, the adjusted odds of infertility increased by 70 ~ 134% and 60 ~ 154 % among women in the higher three quartiles of PFOS and PFOS, respectively [97]. Similarly, reduced fecundity and increased infertility were observed in other studies conducted in Singapore and other countries [95,96]. However, no association between the exposure to the compound and fertility was reported[98,99], although perfluorododecanoic acid (12 carbon) was linked to polycystic ovarian syndrome-related infertility[100].

Miscarriage and Birth weight

Neither PFOA nor PFOS showed any association with miscarriage or preterm birth [101,102]. Birth weight appeared to be associated with the exposure level of PFOA and PFOS. In a cross-sectional epidemiologic study, cord serum samples (n=293) were analyzed for PFOA and PFOS, and the association between the levels and birth weight was evaluated. After adjusting for potential confounders, both PFOA and PFOS were negatively associated with birth weight, but they were not associated with newborn length[103]. Maternal serum levels of PFAS were associated, but paternal levels were not[104]. There were reports that only PFOA or only PFOS was associated with low birth weight whereas the other FPAS were not. The association was very small, limited in precision, and based on self-reported health outcomes [101,105].

Cancer

The association between PFOA/PFOS and cancers has been reported in only a few studies. Estimated cumulative serum PFOS concentrations were positively correlated with kidney and testicular cancer in a study involving 2,507 validated cancers (21 different cancer types) [106]. Similarly, the association of PFOA with kidney, testicular, prostate, ovarian cancers, and non-Hodgkin lymphoma has been documented in another study, which indicated that higher PFOA serum levels might be associated with these cancers. In the study, a total of 25,107 cancer patients participated [107]. In a study conducted in China, 373 breast cancer patients and 657 controls were analyzed for their association with PFOA. The results showed that PFOA was significantly associated with an increased risk of breast cancer [108]
Studies on the association between the levels of PFOA and/or PFOS and conditions such as polycystic ovarian syndrome and premature ovarian insufficiency have been reported, although only a limited number of studies exist [8,77].

Conclusions and future perspective

Test guidelines using animals and high dosages may not always be applicable to human exposure scenarios, and new approach methodologies (NAMs) including in silico, in chemico, and in vitro assays, sometimes have limitations. They are generally used either to complement animal testing or as one component in a compilation of data that needs to be integrated in a relevant, reliable, and unbiased manner. Due to these limitations, the concept of ITS or IATA was introduced to minimize animal testing and amalgamate diverse data in a more mathematically efficient and biologically informed manner [13].
In this study, IATA for PFOA/PFOS were conducted within the frame of AOP for endocrine disruption, particularly focusing on steroidogenesis. The U.S. EPA's endocrine disruptor screening program encompassed various assays including both a screening battery and definitive tests. Tier 1 comprises 11 assays to assess activity, while Tier 2 tests evaluate dose-response relationships and adverse effects. These tests are applied to AOP with MIE, KEs, and Adverse Outcomes. IATA for PFOA and PFOS aimed to provide conclusive remarks on endocrine disruption by comparing animal and human data regarding endocrine disruption outcomes. The analysis was based on enzyme activity for steroid hormone synthesis, levels of steroid hormones, receptor binding and transcription, and cell proliferation/differentiation as depicted in Fig. 1. Despite in vitro data limitations, epidemiological studies consistently associate PFOA/PFOS exposure with adverse outcomes. These outcomes include semen quality, menarche, menopause, menstrual cycle, infertility, miscarriage, cancer, and birth weight. There are many evidences that PFOA/PFOS changes the enzyme activities related with steroidogenesis, E2 and T levels, ER/AR transcriptional activity, and cell proliferation to show estrogenic activity. Many evidences to link the in vitro/in vivo data and human outcomes are observed in the frame of IATA. However, in vitro data regarding the MIE, and KEs are sparse and often inconsistent while it seems evident that PFOA/PFOS exert human adverse outcomes in epidemiological data.
With the integration of multiple toxicity data including in vitro, in vivo and human, the assessment still does not fully satisfies the decision-making criteria of an IATA. To enhance the robustness of IATA for PFOA and PFOS, more consistent in vitro data are needed to improve IATA, especially for MIE and KE in AOP. In addition, more systematic studies integrating quantitative weigh-of-evidence (WoE) approaches would also strengthen the regulatory applicability of these findings. Future research should also explore novel in vitro and computational methods that refine IATA, ensuring a more comprehensive and predictive assessment of endocrine risks.

Notes

Acknowledgement
This work was supported by the Korea Environment Industry & Technology Institute (KEITI) through the Technology Development Project for Safety Management of Household Chemical Products, funded by the Korea Ministry of Environment (MOE) (RS-2023-00215856).
Conflict of interest
The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this paper.
CRediT author statement
DK: investigation, formal analysis. SJ: investigation, formal analysis. KP; conceptualization, validation, writing, supervision.

References

1. Organisation for Economic Co-operation and Development (OECD). Reconciling terminology of the universe of per- and polyfluoroalkyl substances. [cited Jan 21, 2025]. Available from: https://www.oecd.org/en/publications/reconciling-terminology-of-the-universe-of-per-and-polyfluoroalkyl-substances_e458e796-en.html.

2. Lu J, Lu H, Liang D, Feng S, Li Y, Li J. A review of the occurrence, monitoring, and removal technologies for the remediation of per- and polyfluoroalkyl substances (PFAS) from landfill leachate. Chemosphere 2023;332: 138824 https://doi.org/10.1016/j.chemosphere.2023.138824.
crossref pmid
3. Klingelhöfer D, Braun M, Groneberg DA, Brüggmann D. The “forever” per- and polyfluoroalkyl substances (PFAS): A critical accounting of global research on a major threat under changing regulations. Chemosphere 2024;354: 141694 https://doi.org/10.1016/j.chemosphere.2024.141694.
crossref pmid
4. Ruffle B, Archer C, Vosnakis K, Butler JD, Davis CW, Goldsworthy B, et al. US and international per- and polyfluoroalkyl substances surface water quality criteria: A review of the status, challenges, and implications for use in chemical management and risk assessment. Integr Environ Assess Manag 2024;20(1):36-58 https://doi.org/10.1002/ieam.4776.
crossref pmid
5. Radke EG, Wright M, Christensen K, Lin CJ, Goldstone AE, Lemeris C, et al. Epidemiology evidence for health effects of 150 per- and polyfluoroalkyl substances: A systematic evidence map. Environ Health Perspect 2022;130(9):96003 https://doi.org/10.1289/EHP11185.
crossref pmid pmc
6. Shi W, Zhang Z, Li M, Dong H, Li J. Reproductive toxicity of PFOA, PFOS and their substitutes: A review based on epidemiological and toxicological evidence. Environmental Research 2024;250: 118485 https://doi.org/10.1016/j.envres.2024.118485.
crossref pmid
7. Shirke AV, Radke EG, Lin C, Blain R, Vetter N, Lemeris C, et al. Expanded systematic evidence map for hundreds of per- and polyfluoroalkyl substances (PFAS) and comprehensive PFAS human health dashboard. Environ Health Perspect 2024;132(2):26001 https://doi.org/10.1289/EHP13423.
crossref pmid pmc
8. Zhang S, Tan R, Pan R, Xiong J, Tian Y, Wu J, et al. Association of perfluoroalkyl and polyfluoroalkyl substances with premature ovarian insufficiency in Chinese women. J Clin Endocrinol Metab 2018;103(7):2543-2551 https://doi.org/10.1210/jc.2017-02783.
crossref pmid
9. Barrett ES, Chen C, Thurston SW, Haug LS, Sabaredzovic A, Fjeldheim FN, et al. Perfluoroalkyl substances and ovarian hormone concentrations in naturally cycling women. Fertil Steril 2015;103(5):1261-70.e3 https://doi.org/10.1016/j.fertnstert.2015.02.001.
crossref pmid pmc
10. Xie X, Weng X, Liu S, Chen J, Guo X, et al. Perfluoroalkyl and polyfluoroalkyl substance exposure and association with sex hormone concentrations: Results from the NHANES 2015–2016. Environ Sci Eur 2021;33(1):69 https://doi.org/10.1186/s12302-021-00508-9.
crossref pmid pmc
11. Pípal M, Wiklund L, Caccia S, Beronius A. Assessment of endocrine disruptive properties of PFOS: EFSA/ECHA guidance case study utilising AOP networks and alternative methods. EFSA Journal 2022;20(S1):e200418. https://doi.org/10.2903/j.efsa.2022.e200418.
crossref pmid pmc
12. Rickard BP, Rizvi I, Fenton SE. Per- and poly-fluoroalkyl substances (PFAS) and female reproductive outcomes: PFAS elimination, endocrine-mediated effects, and disease. Toxicology 2022;465: 153031 https://doi.org/10.1016/j.tox.2021.153031.
crossref pmid pmc
13. Rovida C, Alépée N, Api AM, Basketter DA, Bois FY, Caloni F, et al. Integrated testing strategies (ITS) for safety assessment. ALTEX 2015;32(1):25-40 https://doi.org/10.14573/altex.1411011.
crossref pmid
14. Casati S. Integrated approaches to testing and assessment. Basic Clin Pharmacol Toxicol 2018;123(Suppl 5):51-55 https://doi.org/10.1111/bcpt.13018.
crossref pmid
15. Browne P, Noyes PD, Casey WM, Dix DJ. Application of adverse outcome pathways to U.S. EPA’s endocrine disruptor screening program. Environ Health Perspect 2017;125(9):096001 https://doi.org/10.1289/EHP1304.
crossref pmid pmc
16. Sakuratani Y, Horie M, Leinala E. Integrated approaches to testing and assessment: OECD activities on the development and use of adverse outcome pathways and case studies. Basic Clin Pharmacol Toxicol 2018;123(Suppl 5):20-28 https://doi.org/10.1111/bcpt.12955.
crossref pmid
17. Chakraborty S, Pramanik J, Mahata B. Revisiting steroidogenesis and its role in immune regulation with the advanced tools and technologies. Genes Immun 2021;22(3):125-140 https://doi.org/10.1038/s41435-021-00139-3.
crossref pmid pmc
18. Bennett KA, Sutherland C, Savage AL. A systematic review and meta-analysis of environmental contaminant exposure impacts on weight loss and glucose regulation during calorie-restricted diets in preclinical studies: Persistent organic pollutants may impede glycemic control. Biochem Pharmacol 2024;225: 116300 https://doi.org/10.1016/j.bcp.2024.116300.
crossref pmid
19. Liu B, Lu X, Jiang A, Lv Y, Zhang H, Xu B. Influence of maternal endocrine disrupting chemicals exposure on adverse pregnancy outcomes: A systematic review and meta-analysis. Ecotoxicol Environ Saf 2024;270: 115851 https://doi.org/10.1016/j.ecoenv.2023.115851.
crossref pmid
20. Lü L, Liu Y, Yang Y, He J, Luo L, Chen S, et al. Bisphenol a exposure interferes with reproductive hormones and decreases sperm counts: A systematic review and meta-analysis of epidemiological studies. Toxics 2024;12(4):294 https://doi.org/10.3390/toxics12040294.
crossref pmid pmc
21. Beevors LI, Sundar S, Foster PA. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays Biochem 2024;68(4):491-507 https://doi.org/10.1042/EBC20240028.
crossref pmid pmc
22. Zhao B, Chu Y, Hardy DO, Li X, Ge RS. Inhibition of 3beta- and 17beta-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid. J Steroid Biochem Mol Biol 2010;118(1-2):13-17 https://doi.org/10.1016/j.jsbmb.2009.09.010.
crossref pmid
23. Zhao B, Hu GX, Chu Y, Jin X, Gong S, Akingbemi BT, et al. Inhibition of human and rat 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances. Chem Biol Interact 2010;188(1):38-43 https://doi.org/10.1016/j.cbi.2010.07.001.
crossref pmid
24. Tian M, Huang Q, Wang H, Martin FL, Liu L, Zhang J, et al. Biphasic effects of perfluorooctanoic acid on steroidogenesis in mouse Leydig tumour cells. Reprod Toxicol 2019;83: 54-62 https://doi.org/10.1016/j.reprotox.2018.11.006.
crossref pmid
25. Huang Q, Luo L, Han X, Li F, Zhang X, Tian M. Low-dose perfluorooctanoic acid stimulates steroid hormone synthesis in Leydig cells: Integrated proteomics and metabolomics evidence. J Hazard Mater 2022;424(Pt D):127656 https://doi.org/10.1016/j.jhazmat.2021.127656.
crossref pmid
26. Kraugerud M, Zimmer KE, Ropstad E, Verhaegen S. Perfluorinated compounds differentially affect steroidogenesis and viability in the human adrenocortical carcinoma (H295R) in vitro cell assay. Toxicol Lett 2011;205(1):62-68 https://doi.org/10.1016/j.toxlet.2011.05.230.
crossref pmid
27. Du G, Hu J, Huang H, Qin Y, Han X, Wu D, et al. Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine-related genes in vitro and in vivo. Environ Toxicol Chem 2013;32(2):353-360 https://doi.org/10.1002/etc.2034.
crossref pmid
28. Du Y, Cai Z, Zhou G, Liang W, Man Q, Wang W. Perfluorooctanoic acid exposure increases both proliferation and apoptosis of human placental trophoblast cells mediated by ER stress-induced ROS or UPR pathways. Ecotoxicol Environ Saf 2022;236: 113508 https://doi.org/10.1016/j.ecoenv.2022.113508.
crossref pmid
29. van den Dungen MW, Rijk JCW, Kampman E, Steegenga WT, Murk AJ. Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells. Toxicol In Vitro 2015;29(4):769-778 https://doi.org/10.1016/j.tiv.2015.03.002.
crossref pmid
30. Kang JS, Choi JS, Park JW. Transcriptional changes in steroidogenesis by perfluoroalkyl acids (PFOA and PFOS) regulate the synthesis of sex hormones in H295R cells. Chemosphere 2016;155: 436-443 https://doi.org/10.1016/j.chemosphere.2016.04.070.
crossref pmid
31. Wang C, Ruan T, Liu J, He B, Zhou Q, Jiang G. Perfluorooctyl iodide stimulates steroidogenesis in H295R cells via a cyclic adenosine monophosphate signaling pathway. Chem Res Toxicol 2015;28(5):848-854 https://doi.org/10.1021/tx5004563.
crossref pmid
32. Rosenmai AK, Taxvig C, Svingen T, Trier X, van Vugt-Lussenburg BMA, Pedersen M, et al. Fluorinated alkyl substances and technical mixtures used in food paper-packaging exhibit endocrine-related activity in vitro. Andrology 2016;4(4):662-672 https://doi.org/10.1111/andr.12190.
crossref pmid
33. Behr AC, Lichtenstein D, Braeuning A, Lampen A, Buhrke T. Perfluoroalkylated substances (PFAS) affect neither estrogen and androgen receptor activity nor steroidogenesis in human cells in vitro. Toxicol Lett 2018;291: 51-60 https://doi.org/10.1016/j.toxlet.2018.03.029.
crossref pmid
34. Rosenmai AK, Nielsen FK, Pedersen M, Hadrup N, Trier X, Christensen JH, et al. Fluorochemicals used in food packaging inhibit male sex hormone synthesis. Toxicol Appl Pharmacol 2013;266(1):132-142 https://doi.org/10.1016/j.taap.2012.10.022.
crossref pmid
35. Kjeldsen LS, Bonefeld-Jørgensen EC. Perfluorinated compounds affect the function of sex hormone receptors. Environ Sci Pollut Res Int 2013;20(11):8031-8044 https://doi.org/10.1007/s11356-013-1753-3.
crossref pmid
36. Gorrochategui E, Pérez-Albaladejo E, Casas J, Lacorte S, Porte C. Perfluorinated chemicals: differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells. Toxicol Appl Pharmacol 2014;277(2):124-130 https://doi.org/10.1016/j.taap.2014.03.012.
crossref pmid
37. Zhang N, Wang WS, Li WJ, Liu C, Wang Y, Sun K. Reduction of progesterone, estradiol and hCG secretion by perfluorooctane sulfonate via induction of apoptosis in human placental syncytiotrophoblasts. Placenta 2015;36(5):575-580 https://doi.org/10.1016/j.placenta.2015.02.008.
crossref pmid
38. Chaparro-Ortega A, Betancourt M, Rosas P, Vázquez-Cuevas FG, Chavira R, Bonilla E, et al. Endocrine disruptor effect of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on porcine ovarian cell steroidogenesis. Toxicol In Vitro 2018;46: 86-93 https://doi.org/10.1016/j.tiv.2017.09.030.
crossref pmid
39. Yang M, Lee Y, Gao L, Chiu K, Meling DD, Flaws JA, et al. Perfluorooctanoic acid disrupts ovarian steroidogenesis and folliculogenesis in adult mice. Toxicol Sci 2022;186(2):260-268 https://doi.org/10.1093/toxsci/kfac005.
crossref pmid pmc
40. Basini G, Bussolati S, Torcianti V, Grasselli F. Perfluorooctanoic acid (PFOA) affects steroidogenesis and antioxidant defence in granulosa cells from swine ovary. Environ Toxicol Pharmacol 2023;101: 104169 https://doi.org/10.1016/j.etap.2023.104169.
crossref pmid
41. Wen C, Chen H, Tang Y, Lin H, Xu C, Ying Y, et al. Per- and polyfluoroalkyl substances inhibit human and rat 17β-hydroxysteroid dehydrogenase 1: Quantitative structure-activity relationship and molecular docking analysis. Ecotoxicol Environ Saf 2024;273: 116173 https://doi.org/10.1016/j.ecoenv.2024.116173.
crossref pmid
42. Du G, Huang H, Hu J, Qin Y, Wu D, Song L, et al. Endocrine-related effects of perfluorooctanoic acid (PFOA) in zebrafish, H295R steroidogenesis and receptor reporter gene assays. Chemosphere 2013;91(8):1099-1106 https://doi.org/10.1016/j.chemosphere.2013.01.012.
crossref pmid
43. Ohno K, Fukushima T, Santa T, Waizumi N, Tokuyama H, Maeda M, et al. Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization. Anal Chem 2002;74(17):4391-4396 https://doi.org/10.1021/ac020088u.
crossref pmid
44. Ishibashi H, Ishida H, Matsuoka M, Tominaga N, Arizono K. Estrogenic effects of fluorotelomer alcohols for human estrogen receptor isoforms alpha and beta in vitro. Biol Pharm Bull 2007;30(7):1358-1359 https://doi.org/10.1248/bpb.30.1358.
crossref pmid
45. Gao Y, Li X, Guo LH. Assessment of estrogenic activity of perfluoroalkyl acids based on ligand-induced conformation state of human estrogen receptor. Environ Sci Technol 2013;47(1):634-641 https://doi.org/10.1021/es304030x.
crossref pmid
46. Xin Y, Ren XM, Wan B, Guo LH. Comparative in vitro and in vivo evaluation of the estrogenic effect of hexafluoropropylene oxide homologues. Environ Sci Technol 2019;53(14):8371-8380 https://doi.org/10.1021/acs.est.9b01579.
crossref pmid
47. Organisation for Economic Co-operation and Development (OECD). Test No. 493: Performance-based test guideline for human recombinant estrogen receptor (hrER) in vitro assays to detect chemicals with ER binding affinity. [cited Jan 21, 2025]. Available from: https://www.oecd.org/en/publications/test-no-493-performance-based-test-guideline-for-human-recombinant-estrogen-receptor-hrer-in-vitro-assays-to-detect-chemicals-with-er-binding-affinity_9789264242623-en.html.

48. Shanle EK, Xu W. Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action. Chem Res Toxicol 2011;24(1):6-19 https://doi.org/10.1021/tx100231n.
crossref pmid pmc
49. Benninghoff AD, Bisson WH, Koch DC, Ehresman DJ, Kolluri SK, Williams DE. Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with human and rainbow trout estrogen receptors in vitro. Toxicol Sci 2011;120(1):42-58 https://doi.org/10.1093/toxsci/kfq379.
crossref pmid pmc
50. Li J, Cao H, Feng H, Xue Q, Zhang A, Fu J. Evaluation of the estrogenic/antiestrogenic activities of perfluoroalkyl substances and their interactions with the human estrogen receptor by combining in vitro assays and in silico modeling. Environ Sci Technol 2020;54(22):14514-14524 https://doi.org/10.1021/acs.est.0c03468.
crossref pmid
51. Sonthithai P, Suriyo T, Thiantanawat A, Watcharasit P, Ruchirawat M, Satayavivad J. Perfluorinated chemicals, PFOS and PFOA, enhance the estrogenic effects of 17β-estradiol in T47D human breast cancer cells. J Appl Toxicol 2016;36(6):790-801 https://doi.org/10.1002/jat.3210.
crossref pmid
52. Evans N, Conley JM, Cardon M, Hartig P, Medlock-Kakaley E, Earl Gray Jr. L. In vitro activity of a panel of per- and polyfluoroalkyl substances (PFAS), fatty acids, and pharmaceuticals in peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, and estrogen receptor assays. Toxicol Appl Pharmacol 2022;449: 116136 https://doi.org/10.1016/j.taap.2022.116136.
crossref pmid pmc
53. Di Nisio A, Sabovic I, Valente U, Tescari S, Rocca MS, Guidolin D, et al. Endocrine disruption of androgenic activity by perfluoroalkyl substances: Clinical and experimental evidence. J Clin Endocrinol Metab 2019;104(4):1259-1271 https://doi.org/10.1210/jc.2018-01855.
crossref pmid
54. Maras M, Vanparys C, Muylle F, Robbens J, Berger U, Barber JL, et al. Estrogen-like properties of fluorotelomer alcohols as revealed by mcf-7 breast cancer cell proliferation. Environ Health Perspect 2006;114(1):100-105 https://doi.org/10.1289/ehp.8149.
crossref pmid pmc
55. Houck KA, Patlewicz G, Richard AM, Williams AJ, Shobair MA, Smeltz M, et al. Bioactivity profiling of per- and polyfluoroalkyl substances (PFAS) identifies potential toxicity pathways related to molecular structure. Toxicology 2021;457: 152789 https://doi.org/10.1016/j.tox.2021.152789.
crossref pmid
56. Pierozan P, Karlsson O. PFOS induces proliferation, cell-cycle progression, and malignant phenotype in human breast epithelial cells. Archives of Toxicology 2018;92: 705-716 https://doi.org/10.1007/s00204-017-2077-8.
crossref pmid pmc
57. Charazac A, Hinault C, Dolfi B, Hautier S, Le Butor CD, Bost F, et al. Low doses of PFOA promote prostate and breast cancer cells growth through different pathways. Int J Mol Sci 2022;23(14):7900 https://doi.org/10.3390/ijms23147900.
crossref pmid pmc
58. Ahn C, Jeung EB. Endocrine-disrupting chemicals and disease endpoints. Int J Mol Sci 2023;24(6):5342 https://doi.org/10.3390/ijms24065342.
crossref pmid pmc
59. López-Doval S, Salgado R, Pereiro N, Moyano R, Lafuente A. Perfluorooctane sulfonate effects on the reproductive axis in adult male rats. Environ Res 2014;134: 158-168 https://doi.org/10.1016/j.envres.2014.07.006.
crossref pmid
60. Zhao B, Li L, Liu J, Li H, Zhang C, Han P, et al. Exposure to perfluorooctane sulfonate in utero reduces testosterone production in rat fetal Leydig cells. PLoS One 2014;9(1):e78888. https://doi.org/10.1371/journal.pone.0078888.
crossref pmid pmc
61. Qu JH, Lu CC, Xu C, Chen G, Qiu LL, Jiang JK, et al. Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice. Environ Toxicol Pharmacol 2016;45: 150-157 https://doi.org/10.1016/j.etap.2016.05.025.
crossref pmid
62. Zhong SQ, Chen ZX, Kong ML, Xie YQ, Zhou Y, Qin XD, et al. Testosterone-mediated endocrine function and TH1/TH2 cytokine balance after prenatal exposure to perfluorooctane sulfonate: By sex status. Int J Mol Sci 2016;17(9):1509 https://doi.org/10.3390/ijms17091509.
crossref pmid pmc
63. Dangudubiyyam SV, Mishra JS, Kumar S. Perfluorooctane sulfonic acid modulates expression of placental steroidogenesis-associated genes and hormone levels in pregnant rats. Reprod Toxicol 2023;118: 108390 https://doi.org/10.1016/j.reprotox.2023.108390.
crossref pmid pmc
64. Joensen UN, Veyrand B, Antignac JP, Jensen MB, Petersen JH, Marchand P, et al. PFOS (perfluorooctanesulfonate) in serum is negatively associated with testosterone levels, but not with semen quality, in healthy men. Hum Reprod 2013;28(3):599-608 https://doi.org/10.1093/humrep/des425.
crossref pmid
65. Vested A, Ramlau-Hansen CH, Olsen SF, Bonde JP, Kristensen SL, Halldorsson TI, et al. Associations of in utero exposure to perfluorinated alkyl acids with human semen quality and reproductive hormones in adult men. Environ Health Perspect 2013;121(4):453-458 https://doi.org/10.1289/ehp.1205118.
crossref pmid pmc
66. Toft G, Jönsson BAG, Bonde JP, Nørgaard-Pedersen B, Hougaard DM, Cohen A, et al. Perfluorooctane sulfonate concentrations in amniotic fluid, biomarkers of fetal Leydig cell function, and cryptorchidism and hypospadias in Danish boys (1980–1996). Environ Health Perspect 2016;124(1):151-156 https://doi.org/10.1289/ehp.1409288.
crossref pmid pmc
67. Hærvig KK, Petersen KU, Hougaard KS, Lindh C, Ramlau-Hansen CH, Toft G, et al. Maternal exposure to per- and polyfluoroalkyl substances (PFAS) and male reproductive function in young adulthood: Combined exposure to seven PFAS. Environ Health Perspect 2022;130(10):107001 https://doi.org/10.1289/EHP10285.
crossref pmid pmc
68. Lewis RC, Johns LE, Meeker JD. Serum biomarkers of exposure to perfluoroalkyl substances in relation to serum testosterone and measures of thyroid function among adults and adolescents from NHANES 2011–2012. Int J Environ Res Public Health 2015;12(6):6098-6114 https://doi.org/10.3390/ijerph120606098.
crossref pmid pmc
69. López-Espinoza MJ, Mondal D, Armstrong BG, Eskenazi B, Fletcher T. Perfluoroalkyl substances, sex hormones, and insulin-like growth factor-1 at 6-9 years of age: A cross-sectional analysis within the C8 health project. Environ Health Perspect 2016;124(8):1269-1275 https://doi.org/10.1289/ehp.1509869.
crossref pmid pmc
70. Liu Y, Calafat AM, Chen A, Lanphear BP, Jones NHY, Cecil KM, et al. Associations of prenatal and postnatal exposure to perfluoroalkyl substances with pubertal development and reproductive hormones in females and males: The HOME study. Sci Total Environ 2023;890: 164353 https://doi.org/10.1016/j.scitotenv.2023.164353.
crossref pmid pmc
71. Tsai MS, Lin CY, Lin CC, Chen MH, Hsu SHJ, Chien KL, et al. Association between perfluoroalkyl substances and reproductive hormones in adolescents and young adults. Int J Hyg Environ Health 2015;218(5):437-443 https://doi.org/10.1016/j.ijheh.2015.03.008.
crossref pmid
72. Zhou Y, Hu LW, Qian ZM, Chang JJ, King C, Paul G, et al. Association of perfluoroalkyl substances exposure with reproductive hormone levels in adolescents: By sex status. Environ Int 2016;94: 189-195 https://doi.org/10.1016/j.envint.2016.05.018.
crossref pmid
73. Liu H, Pan Y, Jin S, Sun X, Jiang Y, Wang Y, et al. Associations between six common per- and polyfluoroalkyl substances and estrogens in neonates of China. J Hazard Mater 2021;407: 124378 https://doi.org/10.1016/j.jhazmat.2020.124378.
crossref pmid
74. Yao Q, Shi R, Wang C, Han W, Gao Y, Zhang Y, et al. Cord blood Per- and polyfluoroalkyl substances, placental steroidogenic enzyme, and cord blood reproductive hormone. Environ Int 2019;129: 573-582 https://doi.org/10.1016/j.envint.2019.03.047.
crossref pmid
75. Lin C, Nian M, Xie M, Zeng L, Qiu W, Zhang J, et al. Associations of per- and polyfluoroalkyl substances and alternatives with reproductive hormones in women of childbearing age. Int J Hyg Environ Health 2023;250: 114158 https://doi.org/10.1016/j.ijheh.2023.114158.
crossref pmid
76. Itoh S, Araki A, Mitsui T, Miyashita C, Goudarzi H, Sasaki S, et al. Association of perfluoroalkyl substances exposure in utero with reproductive hormone levels in cord blood in the Hokkaido Study on Environment and Children's Health. Environ Int 2016;94: 51-59 https://doi.org/10.1016/j.envint.2016.05.011.
crossref pmid
77. Heffernan AL, Cunningham TK, Drage DS, Aylward LL, Thompson K, Vijayasarathy S, et al. Perfluorinated alkyl acids in the serum and follicular fluid of UK women with and without polycystic ovarian syndrome undergoing fertility treatment and associations with hormonal and metabolic parameters. Int J Hyg Environ Health 2018;221(7):1068-1075 https://doi.org/10.1016/j.ijheh.2018.07.009.
crossref pmid
78. Schultz AA, Stanton N, Shelton B, Pomazal R, Lange MA, Irving R, et al. Biomonitoring of perfluoroalkyl and polyfluoroalkyl substances (PFAS) from the Survey of the Health of Wisconsin (SHOW) 2014-2016 and comparison with the National Health and Nutrition Examination Survey (NHANES). J Expo Sci Environ Epidemiol 2023;33(5):766-777 https://doi.org/10.1038/s41370-023-00593-3.
crossref pmid pmc
79. Joensen UN, Bossi R, Leffers H, Jensen AA, Skakkebaek NE, Jørgensen N. Do perfluoroalkyl compounds impair human semen quality? Environ Health Perspect 2009;117(6):923-927 https://doi.org/10.1289/ehp.0800517.
crossref pmid pmc
80. Louis GMB, Chen Z, Schisterman EF, Kim S, Sweeney AM, Sundaram R, et al. Perfluorochemicals and human semen quality: the LIFE study. Environ Health Perspect 2015;123(1):57-63 https://doi.org/10.1289/ehp.1307621.
crossref pmid pmc
81. Song X, Tang S, Zhu H, Chen Z, Zang Z, Zhang Y, et al. Biomonitoring PFAAs in blood and semen samples: Investigation of a potential link between PFAAs exposure and semen mobility in China. Environ Int 2018;113: 50-54 https://doi.org/10.1016/j.envint.2018.01.010.
crossref pmid
82. Pan Y, Cui Q, Wang J, Sheng N, Jing J, Yao B, et al. Profiles of emerging and legacy per-/polyfluoroalkyl substances in matched serum and semen samples: New implications for human semen quality. Environ Health Perspect 2019;127(12):127005 https://doi.org/10.1289/EHP4431.
crossref pmid pmc
83. Toft G, Jönsson BAG, Lindh CH, Giwercman A, Spano M, Heederik D, et al. Exposure to perfluorinated compounds and human semen quality in Arctic and European populations. Hum Reprod 2012;27(8):2532-2540 https://doi.org/10.1093/humrep/des185.
crossref pmid
84. Christensen KY, Maisonet M, Rubin C, Holmes A, Calafat AM, Kato K, et al. Exposure to polyfluoroalkyl chemicals during pregnancy is not associated with offspring age at menarche in a contemporary British cohort. Environ Int 2011;37(1):129-135 https://doi.org/10.1016/j.envint.2010.08.007.
crossref pmid pmc
85. Averina M, Huber S, Almås B, Brox J, Jacobsen BK, Furberg AS, et al. Early menarche and other endocrine disrupting effects of per- and polyfluoroalkyl substances (PFAS) in adolescents from Northern Norway. The Fit Futures study. Environ Res 2024;242: 117703 https://doi.org/10.1016/j.envres.2023.117703.
crossref pmid
86. Lopez-Espinosa MJ, Fletcher T, Armstrong B, Genser B, Dhatariya K, Mondal D, et al. Association of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) with age of puberty among children living near a chemical plant. Environ Sci Technol 2011;45(19):8160-8166 https://doi.org/10.1021/es1038694.
crossref pmid
87. Ding N, Harlow SD, Randolph JF, Calafat AM, Mukherjee B, Batterman S, et al. Associations of perfluoroalkyl substances with incident natural menopause: he Study of Women's Health Across the Nation. J Clin Endocrinol Metab 2020;105(9):e3169-e3182 https://doi.org/10.1210/clinem/dgaa303.
crossref pmid pmc
88. Knox SS, Jackson T, Javins B, Frisbee SJ, Shankar A, Ducatman AM. Implications of early menopause in women exposed to perfluorocarbons. J Clin Endocrinol Metab 2011;96(6):1747-1753 https://doi.org/10.1210/jc.2010-2401.
crossref pmid pmc
89. Taylor KW, Hoffman K, Thayer KA, Daniels JL. Polyfluoroalkyl chemicals and menopause among women 20-65 years of age (NHANES). Environ Health Perspect 2014;122(2):145-150 https://doi.org/10.1289/ehp.1306707.
crossref pmid pmc
90. Dhingra R, Winquist A, Darrow LA, Klein M, Steenland K. A study of reverse causation: Examining the associations of perfluorooctanoic acid serum levels with two outcomes. Environ Health Perspect 2017;125(3):416-421 https://doi.org/10.1289/EHP273.
crossref pmid pmc
91. Lyngsø J, Ramlau-Hansen CH, Høyer BB, Støvring H, Bonde JP, Jönsson BAG, et al. Menstrual cycle characteristics in fertile women from Greenland, Poland and Ukraine exposed to perfluorinated chemicals: A cross-sectional study. Hum Reprod 2014;29(2):359-367 https://doi.org/10.1093/humrep/det390.
crossref pmid
92. Lum KJ, Sundaram R, Barr DB, Louis TA, Louis GMB. Perfluoroalkyl chemicals, menstrual cycle length, and fecundity: Findings from a prospective pregnancy study. Epidemiology 2017;28(1):90-98 https://doi.org/10.1097/EDE.0000000000000552.
crossref pmid pmc
93. Zhou W, Zhang L, Tong C, Fang F, Zhao S, Tian Y, et al. Plasma perfluoroalkyl and polyfluoroalkyl substances concentration and menstrual cycle characteristics in preconception women. Environ Health Perspect 2017;125(6):067012 https://doi.org/10.1289/EHP1203.
crossref pmid pmc
94. Kristensen SL, Ramlau-Hansen CH, Ernst E, Olsen SF, Bonde JP, Vested A, et al. Long-term effects of prenatal exposure to perfluoroalkyl substances on female reproduction. Hum Reprod 2013;28(12):3337-3348 https://doi.org/10.1093/humrep/det382.
crossref pmid
95. Cohen NJ, Yao M, Midya V, India-Aldana S, Mouzica T, Andra SS, et al. Exposure to perfluoroalkyl substances and women's fertility outcomes in a Singaporean population-based preconception cohort. Sci Total Environ 2023;873: 162267 https://doi.org/10.1016/j.scitotenv.2023.162267.
crossref pmid pmc
96. Wang W, Hong X, Zhao F, Wu J, Wang B. The effects of perfluoroalkyl and polyfluoroalkyl substances on female fertility: A systematic review and meta-analysis. Environ Res 2023;216(Pt 3):114718 https://doi.org/10.1016/j.envres.2022.114718.
crossref pmid
97. Fei C, McLaughlin JK, Lipworth L, Olsen J. Maternal levels of perfluorinated chemicals and subfecundity. Hum Reprod 2009;24(5):1200-1205 https://doi.org/10.1093/humrep/den490.
crossref pmid
98. McCoy JA, Bangma JT, Reiner JL, Bowden JA, Schnorr J, Slowey M, et al. Associations between perfluorinated alkyl acids in blood and ovarian follicular fluid and ovarian function in women undergoing assisted reproductive treatment. Sci Total Environ 2017;605-606: 9-17 https://doi.org/10.1016/j.scitotenv.2017.06.137.
crossref pmid pmc
99. Kim YR, White N, Bräunig J, Vijayasarathy S, Mueller JF, Knox CL, et al. Per- and poly-fluoroalkyl substances (PFASs) in follicular fluid from women experiencing infertility in Australia. Environ Res 2020;190: 109963 https://doi.org/10.1016/j.envres.2020.109963.
crossref pmid
100. Wang W, Zhou W, Wu S, Liang F, Li Y, Zhang J, et al. Perfluoroalkyl substances exposure and risk of polycystic ovarian syndrome related infertility in Chinese women. Environ Pollut 2019;247: 824-831 https://doi.org/10.1016/j.envpol.2019.01.039.
crossref pmid
101. Stein CR, Savitz DA, Dougan M. Serum levels of perfluorooctanoic acid and perfluorooctane sulfonate and pregnancy outcome. American Journal of Epidemiology 2009;170(7):837-846 https://doi.org/10.1093/aje/kwp212.
crossref pmid
102. Deji Z, Liu P, Wang X, Zhang X, Luo Y, Huang Z. Association between maternal exposure to perfluoroalkyl and polyfluoroalkyl substances and risks of adverse pregnancy outcomes: A systematic review and meta-analysis. Sci Total Environ 2021;783: 146984 https://doi.org/10.1016/j.scitotenv.2021.146984.
crossref pmid
103. Apelberg BJ, Witter FR, Herbstman JB, Calafat AM, Halden RU, Needham LL, et al. Cord serum concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth. Environ Health Perspect 2007;115(11):1670-1676 https://doi.org/10.1289/ehp.10334.
crossref pmid pmc
104. Yao Q, Gao Y, Zhang Y, Qin K, Liew Z, Tian Y. Associations of paternal and maternal per- and polyfluoroalkyl substances exposure with cord serum reproductive hormones, placental steroidogenic enzyme and birth weight. Chemosphere 2021;285: 131521 https://doi.org/10.1016/j.chemosphere.2021.131521.
crossref pmid
105. Fei C, McLaughlin JK, Tarone RE, Olsen J. Perfluorinated chemicals and fetal growth: a study within the Danish National Birth Cohort. Environ Health Perspect 2007;115(11):1677-1682 https://doi.org/10.1289/ehp.10506.
crossref pmid pmc
106. Barry V, Winquist A, Steenland K. Perfluorooctanoic acid (PFOA) exposures and incident cancers among adults living near a chemical plant. Environ Health Perspect 2013;121(11-12):1313-1318 https://doi.org/10.1289/ehp.1306615.
crossref pmid pmc
107. Vieira VM, Hoffman K, Shin HM, Weinberg JM, Webster TF, Fletcher T. Perfluorooctanoic acid exposure and cancer outcomes in a contaminated community: a geographic analysis. Environ Health Perspect 2013;121(3):318-323 https://doi.org/10.1289/ehp.1205829.
crossref pmid pmc
108. Li X, Song F, Liu X, Shan A, Huang Y, Yang Z, et al. Perfluoroalkyl substances (PFASs) as risk factors for breast cancer: a case-control study in Chinese population. Environ Health 2022;21(1):83 https://doi.org/10.1186/s12940-022-00895-3.
crossref pmid pmc
109. Zhan W, Qiu W, Ao Y, Zhou W, Sun Y, Zhao H, et al. Environmental exposure to emerging alternatives of per- and polyfluoroalkyl substances and polycystic ovarian syndrome in women diagnosed with infertility: A mixture analysis. Environ Health Perspect 2023;131(5):57001 https://doi.org/10.1289/EHP11814.
crossref pmid
110. Guo M, Yu Y, Liu H, Zhu C. Associations between exposure to a mixture of perfluoroalkyl and polyfluoroalkyl substances and age at menarche in adolescent girls utilizing three statistical models. Chemosphere 2023;335: 139054 https://doi.org/10.1016/j.chemosphere.2023.139054.
crossref pmid

Figure 1.
Molecular initiating event (MIE) and Key events (KEs) of Adverse outcome pathway (AOP) for the endocrine disruption.
eaht-40-Special_Issue-e2025s03f1.jpg
Figure 2.
Steroidogenic enzymes in the pathway from cholesterol to testosterone and estradiol.
eaht-40-Special_Issue-e2025s03f2.jpg
Table 1.
Effects of PFOA/PFOS on the steroidogenic enzyme activity and hormone levels of 17β-estradiol (E2) and testosterone (T).
Ref. Test System Conc. Exp. time Results
 Chemical
Enzymes
 Hormone
Star Cyp11a Cyp17 Cyp19 Cyp21 3βHSD 17βHSD E2 T
[22] microsome, Leydig cell 10-7 - 10-3 M 45, 120 min PFOA
Leydig cell 10 - 100 μM 24 h
[23] microsome 10-7 - 10-3 M 30, 90 min - - - - - ↓* ↓* - - PFOA/PFOS
[24] MLTC-1 50 - 200 μM 48 h ↓ N ↓ - - ↓ N - ↑a) PFOA
[25] MLTC-1 1 - 10 μM 48 h ↑ ↑ N - - ↑ ↓ - ↑ PFOA
[26] H295R 6 nM - 600 μM 48 h - ↓ - ↓ - - - ↑ ↑ PFOS
[42] H295R 3 × 10−9 - 3 × 10−7 M 48 h ↑ ↓ ↓ ↑ ↑ ↑ ↑ ↑ ↓ PFOA
[27] H295R 3 × 10−9 - 3 × 10−7 M 48 h ↑ ↓ ↓ ↑ ↓ ↑ ↓ ↑ ↓ PFOS
[29] H295R 30 - 200 μM 48 h N N N ↑ N N ↑ ↑ ↑ PFOS
[30] H295R 10 - 100 μM 48 h N N ↑ ↑ N ↑ N ↑ ↓ PFOA/PFOS
[31] H295R 100 μM 48 h N N N N N N N N N PFOA
[32] H295R 0 - 50 μM 48 h - - - - - - - N - PFOA
[33] H295R 50 - 100 μM 48 h N N N N N N - N - PFOA/PFOS
[34] H295R 1.6 - 50 μM 48 h N N N N N - - ↑b) N PFOA
[35] JEG-3 10−8 - 10−4 M 18 h - - - N* - - - - - PFOA/PFOS
[36] JEG-3 3nM - 500 μM 24 h - - - ↓* - - - - - PFOA/PFOS
[37] placental cytotrophoblasts 0.0001 - 1 μM 24 h - - - ↓ - - - ↓ - PFOS
[38] ovarian Granulosa cells 0.012 - 240 μM c) 24 h - - - - - - - ↓ - PFOA/PFOS
[39] Antral follicles 1 - 100 μg/ml 96 h ↓ ↓ ↓ ↓ - ↓ ↓ ↓ ↓ PFOA
[40] Swine granulosa cells 2 - 200 ng/mL 48 h - - - - - - - ↑ - PFOA
[41] microsome 0 - 200 μM - - - - - - - ↓* - - PFOA/PFOS

* ; data represent enzyme activity, while the others represent mRNA expression or protein levels by Western blotting, N; no effect, ↑; increased, ↓; decreased,

a)b) ; biphasic result was shown,

c) ; PFOA: 0.012 - 24μM, PFOS: 0.12 - 240μM.

Table 2.
Binding affinity of PFOA/PFOS to the estrogen receptor.
Ref. Test System Conc. Exp. time Results Chemical
[44] Yeast cells 1 - 1000 μM 4 h hER α, β: No binding activity PFOA/PFOS
[45] SPR 0 - 250 μM 90 min KD hERα: 107μM for PFOA, 2.19μM for PFOS PFOA/PFOS
[46] hERα, β protetin 1 - 1000 μM 5 min hERα; IC50 = 469.5 ± 4.6 μM, hERβ; IC50 = 384.4 ± 3.4 μM PFOA
Table 3.
Effects of PFOA/PFOS on the transcriptional activity of estrogen receptor (ER) and androgen receptor (AR).
Ref. Test System Conc. Exp. time Results Chemical
[44] HEK-293T 1 - 1000 nM 24 h hERα transcription↑ PFOA/PFOS
[35] MVLN 10−9 - 10−4 M 24 h hER transcription↑ with/without E2, EC50 = 6.5x10-5 M, 2.9x10-5 M PFOA/PFOS
CHO-K1 AR transcription↓ with/without DHT, IC50 = 1.1x10-5 M, 4.7x10-6 M
[46] MVLN 0 - 800 μM 12, 24 h hER transcription↑ at 12 and 24 h without E2 PFOA
hER transcription↓ after 24h with E2
[50] MVLN 0.0001 - 5 mM 48 h hER transcription↑ only by PFOS not by PFOA PFOA/PFOS
hER transcription↓ with E2
[30] MVLN 10 pM - 10 μM 48 h hER transcription↓ PFOA/PFOS
22Rv1/MMTV AR transcription; No effect
[42] CV-1 3x10−9 - 3x10−7 M 24 h rERα transcription; No effect alone but additive effect with E2 PFOA
MDA-kb2 AR transcription: No effect with or without DHT
[27] CV-1 3x10−9 - 3x10−7 M 24 h rERα transcription; No effect alone but additive effect with E2 PFOS
MDA-kb2 AR transcription; No effect with/without DHT)
[51] T47D 10−12 - 10−4 M 24 h ER transcription; No effect alone but additive effect with 1nM E2 PFOA/PFOS
1nM pS2↑ with E2, No effect on PR, pS2 level without E2
[52] T47D 10 - 1000 μM 24 h hER transcription; No effect PFOA/PFOS
[33] HEK-293T 50 - 100 μM 24 h ERα, β transcription; No effect without E2 PFOA/PFOS
ERα transcription; additive effect of PFOS with 0.4 nM E2
ERβ transcription; additive effect of PFOA and PFOS with 0.4 nM E2
MCF-7 No effect on the expression of TFF1, GREB1, PGR, ESR1, ESR2, CTSD
[53] HeLa 1 μM 24 h AR transcription↑ without T (agonistic) PFOA/PFOS
AR transcription↓ with T (antagonistic)

↑; increased, ↓; decreased.

Table 4.
Effects of PFOA and PFOS on the cell proliferation.
Ref. Test System Conc. Exp. time Results Chemical
[54] MCF-7 0.01 - 100 μM 6 Days No effect PFOA/PFOS
[33] MCF-7 50 - 100 μM 6 Days No effect, with/without E2 PFOA/PFOS
[51] T47D 10-9 - 10-7 M 48 h No effect without E2, but proliferation ↑ with 1nM E2 PFOA/PFOS
[55] T47D 0 - 300 μM 0 - 72 h No effect PFOA
[56] MCF-10A 0 -1 mM 24, 48, 72 h proliferation ↑ PFOS
[57] MCF-7/DU145 10-12 - 10-6 M 40 – 80 h proliferation ↑ PFOA
[28] HTR-8/SVneo 100 - 1000 μM 72 h proliferation ↑ PFOA

↑; increased

Table 5.
In vivo effects of PFOA/PFOS on the steroidogenic enzyme activity and hormone levels of 17β-estradiol (E2) and testosterone (T).
Ref. Test System Dose (mg/kg/day) Days Star cyp11 cyp17 cyp19 cyp21 3βHSD 17βHSD E2 T Chemical
[59] SD male rats 0.5 - 6, p.o. 28 days - - - - - - - - ↓ PFOS
[60] SD male fetal rats a) 5 - 20, p.o. GD11 - 19 ↓ ↓ ↓* - - ↓* - - ↓ PFOS
[61] C57 male mice 0.5 - 10, p.o. 5 weeks - - - - - - - N ↓ PFOS
[39] CD-1 female mice 1 - 20, p.o. 10 days N N N ↑ - N N N ↓ PFOA
[62] C57BL/6 mice 0.1 - 5, p.o. GD 1-17 - - - - - - - ↑ ↓ PFOS
[25] SD male rats 0.015 - 1.5, p.o. 60 days - - - - - - - - ↑ PFOA
[63] Pregnant SD rats 10 – 50 μg/ml,p.o. GD 4-20 ↑ ↑ N ↓ - ↑ ↑ ↓ ↑ PFOS

* ; mRNA levels, protein levels, and enzyme activity were decreased,

a) ; PFOS was administered to SD pregnant rats, and the enzyme and hormone levels were measured in their male fetuses. p.o.; oral treatment, N; no effect

Table 6.
Epidemiological studies on hormonal changes of 17β-estradiol (E2) and testosterone (T) caused by PFOA and PFOS.
Ref. Location Number (gender, age) Study Period Results
description PFOA
 PFOS
E2 T E2 T
[64] Denmark 247 (m, 19) 2008-2009 • PFOS levels were negatively associated with T, calculated free FT, FAI and ratios of T/LH, FAI/LH and FT/LH. NA NA NA ↓
[65] Denmark 169 (m, 19-21) 1988–1989 • Trends of higher FSH and LH levels were found with higher in utero exposure to PFOA. NA NA NA N
• PFOS did not appear to be associated with any of the outcomes assessed
[66] Denmark 345(c)/300(p) (f, 32/32) 1980-1996 • The highest tertile of PFOS exposure (> 1.4 ng/mL) in amniotic fluid was associated with a 40% lower INSL3 level and an 18% higher testosterone level compared with the lowest tertile (< 0.8 ng/mL). - - - ↑
[67] Denmark 864 (m, 18.9-21.2) 2017-2019 • higher levels of free testosterone in higher exposed group to PFOS NA NA NA ↑
[68] The U.S. 1682 (m/f, 12-80) 2011-2012 • No statistically significant relationships between PFOA and PFOS exposure and serum T levels. - NA - NA
[10] The U.S. 1868 (m/f, ≥ 12) 2015-2016 • PFOS was significantly associated with increased serum T concentrations in males. ↓(m,f) ↑(f) ↓(f) ↑(m)
• PFOA, and PFOS exposures were positively correlated with FT levels in 20-49 years old women while PFOS exposure was negatively associated with total T levels in 12-19 years old girls. ↑(m) ↓(f)
• E2 was negatively associated with PFOS, and PFOA in 12-19 years old girls, PFOA in 12-19 years old boys while PFOS exposure was positively associated with E2 in these boys.
• PFOS exposure was positively associated with SHBG levels in men older than 20 and in all females.
[69] The U.S. 2292 (m/f, 6-9) 2005-2006 • In boys, PFOA/PFOS concentrations were significantly associated with decreased testosterone levels ↑ ↓ ↓ ↓
• E2 was increased in PFOA but decreased in PFOS exposed group
[70] The U.S. 200 (f, > 35) (m/f, 12) 2003-2006 • In females, no patterns were found linking PFAS levels during pregnancy or early childhood (ages 3 and 8) to reproductive hormone levels, except for lower E2 levels associated with PFAS at age 12. NA NA NA NA
• No pattern was observed for associations of PFAS concentrations with reproductive hormones in males. ↓(f) ↓(f)
[71] China 540 (m/f, 12-30) 2006-2008 • There was a significantly negative association between the different percentile categories of PFOS and the serum T level among the female 12–17-year-old group. NA NA NA ↓(f)
• FSH levels were significantly decreased in association with the different percentile categories of PFOS in the male 12–17-year-old group and the different per-centile categories of PFUA in the female 12–17-year-old group.
[72] Taiwan 225 (m/f, 13-15) 2009-2010 • Among males, PFOS was negatively associated with T level. ↑(m) NA NA ↓(m)
• Male E2 levels were positively associated with PFOA.
[8] China 240 (f, 20-40) 2013-2016 • In patients with POI, levels of PFOS exposure were positively associated with FSH and negatively associated with E2. NA NA ↓ NA
• The concentrations of PFOS and PFOA in the control group did not show an association with E2 and T. ↓(p)
[73] China 942 (m/f, neonate) 2013-2014 • PFOA and PFOS are significantly associated with higher serum levels of E1, E2 in the adjusted model. ↑ - ↑ -
• PFOA alone is significantly associated with higher serum E3 levels.
[74] China 351 (f, 28.35) 2010-2013 • The positive associations between PFOA and E2 levels, and PFOS and T levels. ↑ NA NA ↑
[75] China 433 (f, 29.6) 2014-2016 • PFOA was positively associated with total T. NA ↑ NA NA
• The linear trend was found for PFOA quartiles with total T.
• PFOS was not associated with reproductive hormones including FSH, LH, E2, P4, total T.
[9] Norway 178 (f, 25-35) 2000-2002 • Among nulliparous, but not parous women, PFOS concentrations were inversely associated with E2 and P4. NA NA NA ↓
• No associations between PFOA and ovarian steroid concentrations were found.
[76] Japan 189 pairs (f, 30) 2002-2005 • In the fully adjusted linear regression analyses of the male infants, maternal PFOS levels were significantly associated with increased E2 NA NA ↑ NA
• PFOA levels were positively associated with inhibin B levels.
[77] The U.K. 59 (f, 20-45) 2015 • While PCOS cases showed no association between PFAS and reproductive hormones (T, SHBG, FAI, A4, E2), among controls, PFOA was positively correlated with T and PFOS was negatively correlated with FAI. NA ↑ NA NA
[53] Italy 212(c), 171(p) (m, 19/19) 2017-2018 • Increased levels of PFOA/PFOS in plasma and seminal fluid were positively correlated with circulation testosterone. - ↑ - ↑

Acronyms: A4; androstenedione, E1; estrone, E2 or E; Estradiol, E3; estriol, T; testosterone, TT; total testosterone, FAI; free androgen index, FT; free testosterone, INSL3; insulin-like 3, FAI; free androgen index), P4; progesterone, PCOS; polycystic ovary syndrome, POI; primary ovarian insufficiency, SHBG; sex hormone binding globulin, c; case (normal), p; patient, f; female, m; male, NA; not associated.

Table 7.
Epidemiological studies of PFOA/PFOS on human adverse outcomes.
Outcomes Ref. Location Number (gender, age) Study Period Explanation
PFOA PFOS
Sperm quality [79] Denmark 105 (m, 19) 2003 • Men with high combined levels of PFOS and PFOA had less normal spermatozoa in contrast to men with low PFOS-PFOA ↓ ↓
• Nonsignificant trends with regard to lower sperm concentration, lower total sperm counts and altered pituitary-gonadal hormones among men with high PFOS-PFOA levels.
[65] Denmark 169 (m, 19-21) 1988–1989 • Trends of lower sperm concentration, sperm count were found with higher in utero exposure to PFOA. ↓ NA
• PFOS did not appear to be associated with any of the outcomes assessed, before or after adjustment.
[80] The U.S. 462 (m, 32) 2005-2009 • PFOA and PFOS were associated with 17 semen quality endpoints including sperm viability, sperm counts, morphology etc ↓ ↓
[81] China 103 (m, -) 2012-2013 • Negative correlations were significantly observed between sperm motility and PFOS, PFOA in semen. ↓ ↓
[82] China 664 (m, 29) 2015-2016 • Seminal PFOA and PFOS levels were significantly associated with a lower percentage of progressive sperm and higher percentage of DNA fragmentation ↓ ↓
• Associations between serum PFAS levels and DNA stainability were statistically significant.
[67] Denmark 864 (m, 19-21) 2017-2019 • Total sperm count decreased by 5% and proportion of non-progressive and immotile sperm increased by 4% for each 10ng/mL increase in PFOS. - ↓
• No clear association between exposure to maternal PFAS and testicular volume was observed.
[83] Greenland, Poland, Ukraine 588 (m, 26/31/30/respectively) 2002-2004 • Across countries, sperm concentration, total sperm count, and semen volume were not consistently associated with PFOS and PFOA levels. ND ND
• The proportion of morphologically normal cells was 35% lower for the third tertile of PFOS exposure as compared with the first.
• At the third PFOA exposure tertile, the percentage of motile spermatozoa was 19% higher than in the first.
[64] Denmark 247 (m, 19) 2008-2009 • Both PFOA and PFOS were not significantly associated with semen quality. NA NA
Delayed Menarche [84] The U.K. 448 (f, 8-13) 1991-1992 • No association was found between maternal PFAS exposure and daughters' age of menarche. NA NA
[85] Norway 435 (f, 15-19) 2010-2011 • Increased serum concentrations of PFDA and PFUnDA were significantly positively associated with early menarche while other PFAS were not associated with age at menarche. NA NA
[70] The U.S. 200 (f, > 35) (m/f, 12) 2003-2006 • In females, higher adolescent PFAS concentrations were linked to delayed pubic hair growth, breast maturation, and later age at menarche, with no similar pattern for prenatal or other postnatal concentrations. Delay Delay
• Specifically, PFHxS was associated with later pubic hair and breast growth as well as later age at menarche. Similar associations were observed for PFOA, PFOS and PFNA, although some of these associations did not reach statistical significance.
• No pattern was observed for associations of PFAS concentrations with pubic hair growth in males.
[86] The U.S. 2931 (f, 8-18) 2005-2006 • For girls, higher concentrations of PFOA or PFOS were associated with reduced odds of post-menarche (130 and 138 days of delay, respectively). Delay Delay
Early Menopause risk [87] The U.S. 1120 (f, 45-56) 1999-2017 • Compared with the lowest tertile, women at the highest tertile had hazard ratio for natural menopause of 1.26 for n-PFOS, 1.27 for branched-PFOS, and 1.31 for n-PFOA. ↑ ↑
[88] The U.S. 25,957 (f, 18-65) 2005-2006 • The odds of having experienced menopause were significantly higher in the highest quintile relative to the lowest quintile of PFOA, PFOS in the peri-menopausal and menopausal age groups. ↑ ↑
[89] The U.S. 2732 (f, 20-65) 1999-2010 • Natural menopause showed positive association with PFOA. ↑ ↑
• The odds of hysterectomy were positively correlated with PFOA and PFOS.
[90] The U.S. 3334 (f, ≥ 40) 2008-2011 • A significant increasing trend of reported menopause with increasing measured PFOA though this trend disappeared when using modeled serum PFOA or modeled cumulative PFOA exposure. ↑ -
Risk of Menstrual cycle change [91] Greenland, Poland, Ukraine 1623 (f, 19-49) 2002-2004 • PFOA was positively associated with longer menstrual cycles(≥32 days). ↑ ↑
• A tendency observed toward more irregular cycles with higher exposure to PFOS.
[92] The U.S. 501 (f, 18-40) 2005-2009 • PFOA alone showed negative association with menstrual cycle length. ↑ NA
• Comparing women in the third versus first tertile of PFOA concentration, diminished probability of pregnancy was observed when adjusting for intercourse and cycle length.
[93] China 950 (f, 30) 2013-2015 • Longer and irregular periods were associated with PFOA exposure. Menorrhagia was negatively correlated with both PFOA and PFOS. ↑ NA
• Hypomenorrhea showed no associations with any of PFAS.
[94] Denmark 267 (f, 0-20) 1988-1989 • No statistically significant relationships were found between PFOA or PFOS exposure and cycle length and number of follicles in the subgroup of daughters. NA NA
2008-2009
Fecundability [95] Singapore 382 (f, 18-45) 2015-2017 • A 5-10 % reduction in fecundability per quartile increase of exposure to individual PFAS was found. ↓ ↓
• The decreased odds of clinical pregnancy and live birth per quartile increases of individual PFAS was observed.
[96] Multinational 13 studies, 11,030 subjects 2009-2019 • Meta-analysis showed that PFOA exposure was negatively associated with the female fecundability odds ratio and positively associated with the odds ratio for infertility. ↓ ↓
• PFOS exposure was negatively associated with the fecundability odds ratio.
Infertility [97] Denmark 1,240 (pg, 31) 1996-2002 • Longer Time to pregnancy was associated with higher maternal levels of PFOA and PFOS. ↑ ↑
• The odds of infertility increased in the higher three quartiles of PFOS and PFOA.
• Fecundity odds ratios were virtually identical for women in the three highest exposure groups of PFOS (FOR = 0.70, 0.67 and 0.74, respectively) compared with the lowest quartile. A linear-like trend was observed for PFOA (FOR = 0.72, 0.73 and 0.60 for three highest quartiles versus lowest quartile).
[98] The U.S. 36 (f, 34) 2013-2014 • A relationship was not detected between PFAS levels and fertilization rates or pregnancy outcomes. NA NA
[99] Australia 97 (f, 35) 2006-2009, 2010-2011 • There were few statistically significant associations between PFOA, PFOS concentrations in follicular fluid and infertility factors. NA NA
[100] China 367 (f, 20-40) 2014 • No significant relationship was observed between PCOS-related infertility and PFOA and PFOS. NA NA
Miscarriage [101] The U.S. • PFOA: 1,505 (pg, 20-34) 2000-2006 • Neither PFOA nor PFOS showed any association with miscarriage or preterm birth. NA NA
• PFOS: 3,996 (pg, 20-34) • Preeclampsia was weakly associated with PFOA and PFOS exposures above the median.
• Birth defects were weakly associated with PFOA exposures above the 90th percentile.
[102] Multinational 21 studies, 28,765 subjects 2009-2020 • Maternal exposure to PFOS may have a positive association with preterm birth. NA NA
• No such significant associations were found between PFOA and miscarriage, stillbirth and preterm birth.
Birth weight [103] The U.S. 293 (m/f, neonates) 2004-2005 • Negatively associated with birth weight, ponderal index, and head circumference ↓ ↓
• No associations with newborn length or gestational age.
[104] China 369 (family, -) 2010-2013 • The negative association was found between maternal PFAS and mean of birth weight though the estimated 95% CI included the null. ↓ ↓
• Paternal exposure to PFASs wasn’t associated with fetal growth outcomes.
[105] Denmark 1,400 1996-2002 • Only PFOA levels were inversely associated with birth weight. ↓ NA
• Neither maternal PFOS nor PFOA levels were consistently associated with the risk for preterm birth or low birth weight.
[101] The U.S. • PFOS: 3,996 (pg, 20-34) • PFOA was not associated with an increase in low birth weight, but PFOS showed an increased risk above the median. NA ↓
• PFOA: 1,505 (pg, 20-34)
Cancer [106] The U.S. 17,360 (f, m 53) 2008-2011 • PFOA concentration was positively associated with thyroid, kidney and testicular cancer. ↑ -
[107] The U.S. 25,107 (f, m 67) 1996-2005 • Higher PFOA serum levels may be associated with testicular, kidney, prostate, and ovarian cancers and non-Hodgkin lymphoma ↑ -
[108] China 373(c) , 657(p) (f, 50.6, 53) 2012-2016 • PFOA was positively associated with breast cancer, particularly with the occurrence of estrogen receptor (ER)-, progesterone receptor (PR)-, and human epidermal growth factor receptor 2 (HER2)-positive breast cancer. ↑ -
PCOS [77] The U.K. 59 (f, 20-45) 2015 • Serum PFOS was higher in PCOS cases than controls and in women with irregular vs. regular menstrual cycles NA ↑
[109] China 366(c), 577(p) (f, 28, 28) 2014-2016 • Branched isomers of PFOS were significantly associated with increased odds of PCOS. NA ↑
POI [8] China 240 (f, 20-40) 2013-2016 • Levels of PFOA, PFOS were positively associated with the risks of POI. ↑ ↑

Acronym: HR; hazard ratio, PCOS; polycystic ovarian syndrome, POI; primary ovarian insufficiency, c; case (normal), p; patient, f; female, m; male, pg; pregnant, NA; not associated, ND; not determined
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