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

Article information

Environ Anal Health Toxicol. 2025;40.e2025s03

Publication date (electronic) : 2025 April 4

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

Doyeong Kim ¹, Suin Ji ¹, Kwangsik Park ¹^,

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

^*Correspondence: kspark@dongduk.ac.kr

Recommended by: Prof. Jung-Hwan Kwon

Received 2025 January 21; Accepted 2025 February 25.

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.

Figure 1.

Molecular initiating event (MIE) and Key events (KEs) of Adverse outcome pathway (AOP) for the endocrine disruption.

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).

Figure 2.

Steroidogenic enzymes in the pathway from cholesterol to testosterone and estradiol.

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.

Table 1.

Effects of PFOA/PFOS on the steroidogenic enzyme activity and hormone levels of 17β-estradiol (E2) and testosterone (T).

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.

Table 2.

Binding affinity of PFOA/PFOS to the estrogen receptor.

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.

Table 3.

Effects of PFOA/PFOS on the transcriptional activity of estrogen receptor (ER) and androgen receptor (AR).

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.

Table 4.

Effects of PFOA and PFOS on the cell proliferation.

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].

Table 5.

In vivo effects of PFOA/PFOS on the steroidogenic enzyme activity and hormone levels of 17β-estradiol (E2) and testosterone (T).

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.

Table 6.

Epidemiological studies on hormonal changes of 17β-estradiol (E2) and testosterone (T) caused by PFOA and PFOS.

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.

Table 7.

Epidemiological studies of PFOA/PFOS on human adverse outcomes.

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.

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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

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

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

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

Ref.	Location	Number (gender, age)	Study Period	Results
				description	PFOA		PFOS
				description	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
[65]	Denmark	169 (m, 19-21)	1988–1989	• PFOS did not appear to be associated with any of the outcomes assessed	NA	NA	NA	N
[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	↑	↓	↓	↓
[69]	The U.S.	2292 (m/f, 6-9)	2005-2006	• 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
[70]	The U.S.	200 (f, > 35) (m/f, 12)	2003-2006	• 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)
[71]	China	540 (m/f, 12-30)	2006-2008	• 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)
[72]	Taiwan	225 (m/f, 13-15)	2009-2010	• 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
[8]	China	240 (f, 20-40)	2013-2016	• 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.	↑	-	↑	-
[73]	China	942 (m/f, neonate)	2013-2014	• 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	↓
[9]	Norway	178 (f, 25-35)	2000-2002	• 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
[76]	Japan	189 pairs (f, 30)	2002-2005	• 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.	-	↑	-	↑

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)

Outcomes	Ref.	Location	Number (gender, age)	Study Period	Explanation
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	↓	↓
	[79]	Denmark	105 (m, 19)	2003	• 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
	[65]	Denmark	169 (m, 19-21)	1988–1989	• 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	↓	↓
	[82]	China	664 (m, 29)	2015-2016	• 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.	-	↓
	[67]	Denmark	864 (m, 19-21)	2017-2019	• 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.	↑	↑
	[89]	The U.S.	2732 (f, 20-65)	1999-2010	• 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).	↑	↑
	[91]	Greenland, Poland, Ukraine	1623 (f, 19-49)	2002-2004	• 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
	[92]	The U.S.	501 (f, 18-40)	2005-2009	• 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
	[93]	China	950 (f, 30)	2013-2015	• 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
	[94]	Denmark	267 (f, 0-20)	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.	↓	↓
	[95]	Singapore	382 (f, 18-45)	2015-2017	• 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.	↓	↓
	[96]	Multinational	13 studies, 11,030 subjects	2009-2019	• 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.
			• PFOS: 3,996 (pg, 20-34)		• 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
	[102]	Multinational	21 studies, 28,765 subjects	2009-2020	• 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	↓	↓
	[103]	The U.S.	293 (m/f, neonates)	2004-2005	• 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.	↓	↓
	[104]	China	369 (family, -)	2010-2013	• 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
	[105]	Denmark	1,400	1996-2002	• 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	↓
	[101]	The U.S.	• 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	↑
PCOS	[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.	↑	↑