Environmentally relevant concentrations of individual per- and polyfluoroalkyl substances (PFAS) and a PFAS mixture impact proliferation, migration, and gene transcription in a human myometrial cell line.

The effect of individual PFAS on cell viability was assessed using the MTT assay. UT-TERT cells were exposed to 0 – 1000 ng/mL of PFOA, GENX, PFOS, or PFBS for 96 h. An increase in cell viability/cell proliferation was observed at all concentrations of the individual PFAS tested after 96 h ( Fig. 1A – D ). Environmentally relevant concentrations [1 ng/mL, as reported in serum in women ( Jain and Ducatman, 2022 ; Heffernan et al., 2018 ; Hong et al., 2022 ; Petro et al., 2014 ; Mccoy et al., 2017 )] of each PFAS were selected and combined to create the PFAS mixture. After 96 h, treatment with the PFAS mixture resulted in an increase in cell viability/cell proliferation in UT-TERT cells ( Fig. 1E ). To assess the number of proliferating cells following PFAS exposure, KI67 immunofluorescence was used. UT-TERT cells treated with PFOA, PFOS, and the PFAS mixture showed an increase in KI67-positive cells compared to vehicle-treated controls, while GENX and PFBS treatments did not result in differences in KI67-positive cell numbers after 96 hours ( Fig. 2A ). Cell cycle regulation in UT-TERT cells following exposure to individual PFAS compounds and the PFAS mixture was evaluated using flow cytometry. Among the PFAS compounds, only PFOS affected the distribution of cells across the cell cycle phases ( Fig. 3A – C ). PFOA reduced the number of cells in the G 0 /G 1 phase and increased those in the G 2 /M phase ( Fig. 3A – C ). The PFAS mixture led to a decrease in cells in the G 0 /G 1 phase only ( Fig. 3A – C ). GENX and PFBS treatments did not impact any cell cycle phase ( Fig. 3A – C ). Experiments were performed to assess whether PFAS exposure affects UT-TERT cell migration. An 8-hour treatment with PFOS and the PFAS mixture stimulated UT-TERT cell migration, resulting in a higher percentage of area covered by migrating cells compared to the vehicle-treated control ( Fig. 4A , B ). Similarly, treatment with PFOA and PFBS for 8 hours led to a slight increase in cell migration, with a modest rise in the percentage of area covered by migrating cells relative to controls ( Fig. 4A , B ). However, GENX treatment did not significantly affect UT-TERT cell migration after 8 hours ( Fig. 4A , B ). To further evaluate the migratory capacity of UT-TERT cells following PFAS exposure, a Transwell migration assay was conducted. After a 5-hour exposure to GENX, PFOS, PFBS, and the PFAS mixture, the number of cells that migrated through the Transwell insert membrane increased compared to vehicle-treated controls ( Fig. 5A , B ). In contrast, the PFOA-treated group showed no significant increase in cell migration relative to controls ( Fig. 5A , B ). Gap junctions provide cell-to-cell connections between adjacent myometrial cells that coordinate uterine contractions ( Garfield et al., 1980 ; Balducci et al., 1993 ); thus, we investigated if PFAS treatment would alter GJIC in UT-TERT cells. The scrape loading assay revealed that PFOA, PFBS, and the PFAS mixture reduced the area of LY dye transfer relative to the vehicle-treated controls after 24 h of PFAS exposure ( Fig. 6A , B ). Treatment of GENX or PFOS for 24 h did not impact the amount of LY dye transfer in UT-TERT cells after 24 h ( Fig. 6A , B ). To gain deeper insights into the effects of PFAS on UT-TERT cells in vitro, we utilized RNA-seq to analyze the myometrial cell transcriptome following treatment with environmentally relevant levels of individual PFAS and PFAS mixture. We identified upregulated and downregulated genes that were chosen based on a log2foldchange value of 1.0 and ( P < 0.05) ( Fig. 7A ). Relative to control-treated cells, UT-TERT cells exposed to legacy PFAS (PFOA and PFOS), 120 genes increased and 107 genes decreased after exposure to PFOA, while exposure to PFOS increased 122 and decreased 127 genes ( Fig. 7B ; Supplemental Figure 1 , 2 ; Supplemental Table 1 – 4 ). UT-TERT cells treated with alternative PFAS GENX and PFBS had 150 and 123 genes upregulated, respectively, while 123 and 126 genes were downregulated after 96 h exposure compared to vehicle control-treated cells ( Fig. 7B ; Supplemental Figure 3 , 4 ; Supplemental Table 5 – 8 ). Additionally, the PFAS mixture upregulated 131 genes and downregulated the expression of 89 genes ( Fig. 7B ; Supplemental Figure 5 ; Supplemental Table 9 , 10 ). To identify any common factors related to the PFAS treatment we identified 7 genes that were most consistent across all the treatment groups ( Table 1 ). Ingenuity pathway analysis (IPA) was performed to understand the underlying molecular and cellular functions associated with the actions of PFAS. There were five common Molecular and Cellular Functions pathways enriched between PFAS treatments, including Cell-to-Cell Signaling and Interaction (PFOA, GENX, PFOS), Small Molecule Biochemistry (GENX, PFOS, MIX), Lipid Metabolism (GENX, PFOS, PFBS), Cell Death and Survival (PFOS, PFBS, MIX), and Molecular Transport (PFBS, MIX) ( Fig. 7C ). In addition to the common pathways, the Cellular Assembly and Organization, Cellular Function and Maintenance, Cellular Development, and Cellular Growth and Proliferation pathways were unique to PFOA, Cell Cycle and Nucleic Acid Metabolism unique to GENX, Cellular Movement unique to PFOS, Protein Synthesis and Cell Morphology unique to PFBS, and Carbohydrate Metabolism and Drug Metabolism were unique to the PFAS mixture ( Fig. 7C ). Gene set enrichment analysis (GSEA) revealed consistent negative enrichment of the KEGG Gap Junction pathway in PFOA, PFOS, PFBS, and the PFAS mixture, further suggesting that PFAS may suppress intercellular communication via downregulation of connexins and related signaling components ( Supplemental Figure 6A – D ). Pathway-level analysis revealed distinct yet overlapping transcriptional responses to individual and PFAS mixture exposures. GENX exposure was associated with significant activation of cytokine signaling and immune response pathways, alongside predicted inhibition of xenobiotic metabolism ( Supplemental Figure 7 ). PFOS exposure enriched pathways associated with inflammatory signaling, neurotransmission, and cellular stress ( Supplemental Figure 7 ). PFBS exposure led to activation of signal transduction, growth-related, and nervous system signaling pathways ( Supplemental Figure 7 ). The PFAS mixture similarly activated pathways related to intracellular signaling, cell growth, and apoptosis ( Supplemental Figure 7 ). Interestingly, PFOA induced more modest changes, with limited directional predictions ( Supplemental Figure 7 ). Together, these results demonstrate that while each PFAS compound exhibits a unique molecular profile, shared perturbations in signaling, immune modulation, and cellular regulation are evident across exposures.

Undecafluoro-2-methyl-3-oxahexanoic acid (GENX/HFPO-DA; cat# 2121–3–13; 97 % purity) and Perfluorobutanesulfonic acid (PFBS; cat# 6164–3–09; 97 % purity) were acquired from Synquest Laboratories (USA). Perfluorooctanoic acid (PFOA; cat# 171468; 95 % purity) was obtained from Sigma-Aldrich (USA). Perfluorooctanesulfonic acid (PFOS; cat# 009151; 95 % purity) was sourced from Matrix Scientific (USA). Dimethyl sulfoxide (DMSO; cat# D128–500) was purchased from purchased from Fisher Scientific (USA). Stock solutions of individual PFAS (PFOA, GENX, and PFBS) were made at 1000 μg/mL in basal SmGM ™ -2 (Smooth Muscle Growth Medium-2; Lonza, USA). DMSO (5 μl) was added to 5 mL of basal SmGM ™ -2 for complete dilution of PFOS at the 1000 μg/mL concentration. Serial dilutions of the stock 1000 μg/mL were made at 100, 10, 1, and 0.1 μg/mL to formulate the 1000, 100, 10, 1, and 0.1 ng/mL working solutions. Working concentrations were freshly prepared in culture media immediately before each experiment, with a final DMSO concentration of 0.1 % maintained across all treatment conditions. The human myometrial cell line, UT-TERT was a generous gift from Dr. John Risinger and was characterized previously ( Carney et al., 2002 ; George et al., 2019 ). Cells were maintained in growth medium containing SmGM ™ -2 supplemented with 5 % fetal bovine serum (FBS), 0.1 % insulin, 0.2 % basic human fibroblast growth factor (hFGF-b), 0.1 % Gentamicin Sulfate-Amphotericin-1000, and 0.1 % human epidermal growth factor (hEGF) (Lonza, USA) and maintained at cultured at 37 °C in a humidified 5 % CO 2 incubator. Cell lines were tested for mycoplasma using a PCR-based detection kit (Applied Biological Materials, cat# G238). Cell viability was measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were plated in a 96-well plate at a density of 1 × 10 3 /well and cultured in full growth media overnight for attachment. Cells were treated with PFOA, GENX, PFOS, or PFBS in assay medium (basal SmGM ™ -2) the following day at various concentrations (0.1 % DMSO- designated vehicle control, 0.1, 1, 10, 100, or 1000 ng/mL) for 96 h to evaluate cell viability and proliferation. Three hours before the end of the 96 h PFAS treatment, 15 mL of MTT (5 mg/mL in PBS) was added to each well, media aspirated, and DMSO (100 mL) was added to fully dissolve the formed formazan crystals. Absorbance at 590 nm and 650 nm was recorded using a microplate reader (Molecular Devices, USA). Following experiments used the 1 ng/mL concentration of the individual PFAS and the PFAS mixture consisted of 1 ng/mL each of PFOA/GENX/PFOS/PFBS. These concentrations were chosen based on documented PFAS serum levels in women ( Jain and Ducatman, 2022 ; Heffernan et al., 2018 ; Hong et al., 2022 ; Petro et al., 2014 ; Mccoy et al., 2017 ). UT-TERT cells (5 ×10 5 /mL) were cultured and treated with vehicle control, individual PFAS, or PFAS mixture in 100 mm plates for 96 h and detached using TrypLE Express (Gibco, Denmark). After detachment, cells were washed with PBS, fixed in 66 % ice-cold ethanol, and stored at 4°C for further processing. Cells were stained with Propidium Iodide in PBS following the manufacturer’s protocol (ab139418, Abcam, USA). To assess cell cycle distribution, flow cytometry was performed utilizing a BD LSRII Flow Cytometer and ModFit LT V4.0.5 software. A minimum of 20,000 cells per sample were analyzed for cell cycle distribution. Cells were plated onto 15 mm round No. 1 glass coverslips in 12-well culture plates. After 96 h of exposure to the vehicle control, individual PFAS, or a PFAS mixture, the cells were rinsed with PBS and fixed in fresh 4 % paraformaldehyde at 4°C for 30 min. Following fixation, cells were washed in PBS (3 × 10 min), permeabilized for 10 minutes in PBS containing 0.1 % Triton-X (PBSTr), and then blocked for 60 min at room temperature in a solution containing 1 % bovine serum albumin (BSA), 1 % DMSO, 5 % goat serum, and PBS. The primary antibody (KI67, Abcam, cat# ab15580, 1:100) was diluted in fresh blocking solution and applied overnight at 4°C. Samples were then washed in PBS (3 × 10 min) and incubated with the secondary antibody (AlexaFluor488, Invitrogen, cat# A-11008, 1:250) for 90 min at room temperature. Following PBS washes (3 × 10 min), coverslips with labeled cells were air-dried, mounted on glass microscope slides with Vectashield Antifade Mounting Medium containing DAPI (Vector Labs, USA), and stored at 4°C until imaging. Cells were imaged with a Zeiss LSM710 confocal microscope (Zeiss, Germany) fitted with an AxioCam MRc5 camera following immunocytochemistry. Imaging was conducted with a 10x objective to quantify KI67 staining, selecting three random fields per coverslip (n = 6 coverslips per treatment). The Cell Counter module in ImageJ ( https://imagej.nih.gov/ij/plugins/cell-counter.html ) was utilized to count immunopositive cells. The percentage of KI67-positive cells was calculated by dividing the number of KI67-immunoreactive cells by the total number of DAPI-stained nuclei per image, then averaging the percentages across replicates. The total number of cells analyzed for each treatment were: Control – 1112; PFOA – 770; GENX – 1159; PFOS – 1145; PFBS – 1224; and MIX – 1401. Slide blinding was applied to prevent counting bias. Cell migration was evaluated using a wound healing assay. Cells were plated in 6-well plates and cultured to confluence in growth medium. A scratch was made in the cell monolayer with a 100 μL pipette tip, followed by two washes with basal SmGMTM-2 to remove detached cells. The cells were then incubated for 8 h at 37°C in a humidified atmosphere with 5 % CO 2 in basal SmGMTM-2 containing either vehicle control, individual PFAS, or a PFAS mixture. Images of the wound area were taken at the start of the treatment (0 h) and after the 8-hour incubation using an inverted microscope equipped with a DP71 digital camera (Olympus America, USA). The wound area was analyzed and measured using cellSens Standard software (Olympus America, USA). A chemotaxis assay was performed to further confirm the impact of PFAS on UT-TERT cell migration. Cells (4 × 10 5 ) in 100 μL of basal SmGMTM-2, containing either vehicle control, individual PFAS, or a PFAS mixture, were seeded into Transwell inserts (8 μm pore size, Corning, USA). The inserts were placed into wells of a 24-well plate containing 600 μL of growth medium. After a 5-hour incubation, cells on the upper surface of the membrane were removed with a cotton swab. The migrated cells on the lower surface of the membrane were fixed and stained with 0.5 % crystal violet in methanol for 30 minutes. Membranes were then imaged, and 12 images per treatment (n = 4 inserts per treatment) were analyzed. The scrape loading/dye transfer assay (SL/DT) assay was performed to assess gap junctional intercellular communication (GJIC) ( Babica et al., 2016 ). Cells were plated in 6-well plates and cultured to 95 % confluence in growth medium. Cells were rinsed twice in basal SmGM ™ -2 and incubated in basal SmGM ™ -2 media containing the vehicle control, individual PFAS, or PFAS mixture for 24 h at 37 °C in a humidified atmosphere with 5 % CO 2. Cells were gently rinsed three times in PBS with Calcium and Magnesium (CaMg-PBS) and prewarmed Lucifer Yellow (LY) CH dilithium salt (1 mg/mL) added to each well. A scratch was made in the cell monolayer using a 100 μL pipette tip to load the cells with the LY dye. The plate was incubated at room temperature in the dark for 5 min to allow the LY dye to travel through the adjacent cell layers to assess gap junction function. Wells were then washed three times with CaMg-PBS to remove the extracellular dye, fixed in 10 % formalin solution, and stored in the dark at 4 °C until imaging. Cells were imaged using a 10x objective on a Zeiss LSM710 confocal microscope (Zeiss, Germany) equipped with an AxioCam MRc5 camera. The fraction of the control (FOC) was calculated by dividing the area of LY fluorescence of the PFAS-treated cells by the area of LY fluorescence in the vehicle control-treated cells and then averaged across replicates (n = 3 wells per treatment, 2 scratches per well). Total RNA was isolated from control, individual PFAS, or PFAS mixture treated cells using the Zymo Direct-zol Microprep Kit (Zymo Research Corporation, USA). RNA concentration and quality were determined via NanoDrop (ND1000, Nanodrop Technologies, USA). The 5200 Fragment Analyzer System (Agilent, USA) evaluated RNA purity and integrity, with a final integrity score of 10. We performed RNA sequencing (RNA-seq) data analysis on 18 samples of control or PFAS treated cells (n = 3 biological replicates per treatment) from a human myometrial cell line (UT-TERT). The analysis pipeline used was nf-core v3.14.0 (Ewels et al., 2020), within Nextflow v23.10.1 ( Di Tommaso et al., 2017 ). Raw FastQ files were trimmed using Cutadapt and trimgalore to remove adapters, terminal unknown bases (Ns) and low-quality 3’ regions (Phred score < 30). Following trimming, FastQ files were aligned to the hg38 human reference genome using STAR ( Dobin et al., 2013 ). The average number and percentage of uniquely mapped reads were Control – 37,978,473 (91 %); PFOA – 38, 697,852 (90.9 %); GENX – 27,780,581 (90.8 %); PFOS – 32,547,981 (90.8 %); PFBS – 36,763,612 (90.8 %); and MIX – 33,988,235 (90.9 %). Differentially expressed genes (DEGs) were identified using DESeq2 ( Love et al., 2014 ) on the raw count data and genes having counts less than 10 were filtered out before proceeding to the DESeq2 analysis. The Benjamini-Hochberg (BH) procedure was used to account for multiple testing with a cut-off of log2 fold changes ≥ 1 or ≤ −1 and p-values ≤ 0.05, indicating significant upregulated and downregulated genes. All experiments were conducted independently and repeated at least three times. Statistical analyses were carried out using GraphPad Prism 10.0. After confirming normality, a one-way analysis of variance (ANOVA) was applied, and treatment means were compared to the vehicle control mean using Dunnett’s correction. Statistical significance was defined as P ≤ 0.05, with trends considered at P ≤ 0.1. Bar graph data are presented as mean ± standard error of the mean (SEM).

PFAS are extremely persistent in the environment and although their use has declined in some areas, PFAS continue to pose a significant environmental and public health challenge. The myometrium, the smooth muscle layer of the uterus, plays an essential role in reproductive health by controlling the contractile actions during both pregnancy/childbirth and during menstruation. The myometrium is sensitive to various environmental chemicals and these exposures may increase the risk of uterine disorders. There is a lack of research on how environmental contaminants like PFAS affect the myometrium, creating a notable gap in our understanding if their potential impact on uterine health. Moreover, most laboratory studies have concentrated on individual PFAS and/or used concentrations that surpass typical human exposure levels. In this study, we used a human myometrial cell line (UT-TERT) to investigate the effects of environmentally relevant concentrations of individual “legacy” (PFOA/PFOS) and “alternative” (GENX/PFBS) PFAS as well as a mixture of PFAS (PFOA/GENX/PFOS/PFBS) on myometrial cell health including cell viability, proliferation, migration, GJIC, and gene expression. In response to low-level, human exposure relevant concentrations (1 – 1000 ng/mL) of PFOA, GENX, PFOS, and PFBS, cell viability as measured by cellular metabolic activity was increased in UT-TERT cells after 96 h. Additionally, mixtures of the PFAS compounds resulted in an increase in cell viability. Notably, effects were observed even at the lowest dose tested, consistent with the non-monotonic dose-responses often reported for endocrine-disrupting chemicals like PFAS ( Iulini et al., 2025 ; Yang et al., 2022 ; Vu et al., 2023 ). None of the concentrations of PFAS tested in our study demonstrated any cytotoxicity to UT-TERT cells. Interestingly, immunostaining for KI67 revealed increased cell proliferation in cells treated with PFOA, PFOS, and the PFAS mixture but not in the GENX or PFBS treated only cells. This could be attributed to the continuous degradation of KI67 protein levels that occurs in the G 0 /G 1 phases of the cell cycle ( Miller et al., 2018 ). In regard to the observed increased proliferation of the mixture and not the individual treatments of GENX and PFBS, these results could be due to a possible synergistic effect of the mixture. Further supporting this notion is that the number of cells in the GENX and PFBS treated groups do not have any changes in the percentages of cells in the G 0 /G 1 phases of the cell cycle, like the vehicle treated controls. While MTT assays are commonly used to infer changes in cell viability, our findings highlight that increased MTT activity may instead reflect altered mitochondrial metabolism rather than proliferation in the GENX and PFBS treated cells. Congruent with the increases in KI67-positive nuclei in PFOA, PFOS, and Mixture treated cells, the percentage of cells in the G 0 /G 1 phase of the cell cycle was decreased. Interestingly, only the PFOS treated cells had indications of accelerated G 0 /G 1 to S phase transition. While no other studies have looked at PFAS-induced cell proliferation in myometrial cells, investigations utilizing an endometrial cancer cell line (HEC-1) has demonstrated increased survival fraction after exposure to 1 μM (~414 ng/mL) PFOA ( Rickard et al., 2023 ), while PFOA exposure between 0.025 – 2 μM (~10 – 828 ng/mL) in Ishikawa cells did not impact cell proliferation ( Rickard et al., 2023 ; Di Nisio et al., 2020 ; Ma et al., 2016 ). Inconsistent results between cells lines can be attested to possible different molecular profiles of various cell lines, potentially including different proliferative/migratory capacities and receptor expression profiles. While the exact mechanism behind PFAS-induced cell proliferation is unclear in our results, several factors can influence myometrial cell proliferation such as hormonal regulation and apoptosis ( Burroughs et al., 2000 ; Shynlova et al., 2006 ; Wu et al., 2000 ) and various growth factors including epidermal growth factor (EGF), insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF) ( Ciarmela et al., 2011 ). Further work is needed to elucidate if there are different regulatory mechanisms in cell proliferation for the various individual PFAS and how these can contribute to a potential synergistic effect when put together as a mixture. The increased cell proliferation observed, along with the lack of effects on cell apoptosis, suggests that PFAS may promote metastatic cell behavior. To explore this, we assessed the migratory and invasive capabilities of PFAS-exposed UT-TERT cells and found that all the individual PFAS tested and the PFAS mixture significantly enhances both cell migration and invasion. These findings are consistent with other studies involving human ovarian cancer cells ( Gogola-Mruk et al., 2021 ), human breast epithelial and cancer cells ( Pierozan et al., 2018 ; Pierozan and Karlsson, 2018 ; Pierozan et al., 2020 ; Zhang et al., 2014 ), and human endometrial cancer cells ( Ma et al., 2016 ). While uterine fibroids have been weakly associated with PFAS exposures ( Mitro et al., 2023 ), other environmental toxicants have been associated with fibroid cell growth ( Iizuka et al., 2022 ; Yu et al., 2019 ; Li et al., 2022 ; Liu et al., 2024 ; Yang et al., 2023 ). Given that wound healing assays reflect coordinated processes such as cell motility, proliferation, and cytoskeletal dynamics, the observed effects suggest that PFAS may disrupt key pathways regulating cellular behavior. These findings add to growing evidence that low-dose PFAS exposures can trigger biologically meaningful responses, even in the absence of obvious morphological changes. When considered alongside potentially unchecked cell proliferation, the enhanced migration and invasion of myometrial cells could potentially lead to hyperplasia of the myometrium or even tumorigenesis over a prolonged exposure period. As the UT-TERT cell line lacks an active estrogen receptor, additional work on primary myometrial cells would be beneficial to determine what impact PFAS exposure would have on estrogen response and production in the myometrium and if this could potentially contribute to fibroid development and growth. Gap junctions in the myometrium play a crucial role in coordinating uterine contractions, particularly during labor ( Garfield et al., 1978 ; Balducci et al., 1993 ). Gap junctions are specialized intercellular channels that allow direct communication between adjacent cells. They enable the transfer of ions, small molecules, and electrical signals, facilitating synchronized cellular activity. In the myometrium, gap junctions are mainly composed of connexin proteins (with connexin-43 being the most common in this tissue), which form channels between the smooth muscle cells ( Chow and Lye, 1994 ; Geimonen et al., 1998 ). In this study, we found that PFOA, PFBS, and the PFAS mixture significantly inhibited gap junction function after 24 h. Inhibition of GJIC by PFAS has been reported in several other cell types including ovarian granulosa cells ( Zhou et al., 2020 ; Domínguez et al., 2016 ), Sertoli cells ( Li et al., 2016 ), liver cells ( Upham et al., 1998 ; Upham et al., 2009 ), and kidney cells ( Hu et al., 2002 ). Gap junctions can also be hormonally regulated ( Hendrix et al., 1995 ; Garfield et al., 1980 ), so determining any additional influence of steroid hormones on primary myometrial cells and GJIC is another avenue of research that needs to be investigated. Finally, gap junction regulation is essential for normal uterine function, and disruptions in gap junction activity can lead to reproductive complications such as preterm labor or dysfunctional labor ( Kidder and Winterhager, 2015 ). Alongside our functional data, the GSEA findings support the hypothesis that PFAS exposure can deregulate uterine cell signaling. Whether the observed effects on GJIC are a direct or indirect mechanism of PFAS toxicity on uterine cell function remains to be determined. To our knowledge, this is the first study to use RNA sequencing to investigate the effects of PFAS exposure on the transcriptome of human myometrial cells (UT-TERT cells). We observed significant upregulation and downregulation of the expression of over 1200 genes. IPA analysis revealed molecular and cellular function pathways influenced by PFAS exposure including those involved cell viability, cell proliferation and migration, and cell communication. Interestingly, there were 7 common genes related to the PFAS treatment that were consistent across all the treatment groups. Of interest, HOP Homeobox ( HOPX ) was consistently upregulated ~5-fold across the PFAS treatment groups. HOPX is a member of the Homeobox gene family that governs a wide variety of pathways and regulates morphogenesis and cell differentiation ( Caspa Gokulan et al., 2022 ). HOPX acts as a tumor suppressor and transcriptional regulator and has been shown to be downregulated in several cancer types, including endometrial cancer ( Caspa Gokulan et al., 2022 ; Yamaguchi et al., 2009 ). In endometrial cancer, the promoter region of HOPX is hypermethylated with concurrent reduction in HOPX mRNA and protein expression ( Yamaguchi et al., 2009 ). Interestingly, overexpression of HOPX in human endometrial cancer cells reduced cell proliferation, while knockdown of HOPX resulted in increased cell proliferation ( Yamaguchi et al., 2009 ). While we see increased cell proliferation and migration in our results, the upregulation of HOPX in our PFAS treated cells could be a potential protective mechanism in an attempt to regulate PFAS-induced cell proliferation. Conversely, Transferrin ( TF ) was consistently downregulated ~5-fold across all the treatment groups. The TF gene encodes a glycoprotein that transports iron via the blood to various tissues in the body ( Talukder, 2021 ). In the uterus, transferrin aids in the transfer if iron between the placenta and fetus during pregnancy ( Cao and Fleming, 2016 ). In other systems, a decrease in TF gene expression can occur in in cases of iron overload ( Pietrangelo et al., 1992 ) or in cases of hypolipidemic peroxisome proliferators ( Hertz et al., 1996 ). This is of interest as PFAS are known to target peroxisome proliferator-activated receptors ( Evans et al., 2022 ; Szilagyi et al., 2020 , Khazaee et al., 2021 ), though the role of TF in the myometrium has not been well studied and additional regulatory inputs, such as oxidative stress or changes in iron homeostasis, may also contribute to regulation of TF expression. Our study is limited by the use of an in vitro model and a single, brief exposure timepoint. Furthermore, we acknowledge that PFAS adsorption to plastic surfaces and protein binding could influence the bioavailable concentration in culture systems. Although PFAS levels in media or cells were not quantified in this initial study, future work should include LC-MS/MS-based measurements to more accurately assess exposure and cellular uptake. Nevertheless, the biological relevance remains significant, as most PFAS exposures occur continuously throughout a lifetime. Small but consistent changes can signal early disruptions in cell regulation, especially in hormonally responsive cells. These findings, when considered with our other functional data, suggest that even low-dose PFAS exposure may trigger meaningful early cellular responses. While our analysis does not pinpoint a specific mechanism of action, it offers unbiased insights into the processes in UT-TERT cells affected by PFAS exposure. In conclusion, this study highlights functional and transcriptomic alterations in UT-TERT cells following exposure to environmentally relevant concentrations of PFAS as well as an environmentally relevant PFAS mixture.

Per- and polyfluoroalkyl substances (PFAS) encompass a large group of synthetic chemicals that have been used in various industries worldwide since the 1940s. PFAS are highly resistant to environmental and metabolic degradation due to their multiple, highly stable C-F bonds, making them environmentally persistent ( Cousins et al., 2020 ). PFAS are typically classified by chain length such as long-chain (8 or more carbons) and short-chain (7 or fewer carbons) and can also be classified as legacy or alternative/emerging compounds based on their production timeline and evaluation status ( Brennan et al., 2021 ). While the use of legacy PFAS, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), has been phased out in the United States and Europe, these compounds are still manufactured and found in products imported from countries lacking similar regulations ( Sunderland et al., 2019 ). Consequently, alternative PFAS, such as undecafluoro-2-methyl-3-oxahexanoic acid (HFPO-DA/GENX) and perfluorobutanesulfonic acid (PFBS), have been introduced as substitutes for legacy PFAS. Although these alternatives are designed to mitigate risks to human health and the environment, many still lack regulation and thorough testing. Common exposure pathways include consuming contaminated food and drinking water, inhaling dust from treated textiles, absorbing chemicals through the skin from personal care products, or occupational exposures in industrial workers, firefighters, and military personnel ( Dickman and Aga, 2022 ). Notably, nearly all individuals tested in the National Health and Nutrition Examination Survey (NHANES) have detectable levels of at least one type of PFAS chemical ( CDC, 2022 ). In humans, the half-life of these same PFAS compounds have been observed to be ~2.1 – 3.8 years for PFOA, ~3.4 – 5.0 years for PFOS, and 28 days for PFBS ( Fenton et al., 2021 ). Currently, there is no available data on the clearance rate of GENX/HFPO-DA in humans. Emerging research suggests that exposure to PFAS may be linked to various reproductive health issues, including uterine diseases such as endometriosis and uterine fibroids. Observational studies in women have shown that higher serum levels of PFAS increase the risk of endometriosis ( Campbell et al., 2016 ; Wang et al., 2017 ; De Haro-Romero et al., 2024 ; Ao et al., 2024 ; Louis et al., 2012 ). Other observational studies have found little or no changes in the risk of endometriosis in women ( Hammarstrand et al., 2021 ; Ngueta et al., 2017 ). In vitro studies with endometrial cell lines have shown increased migration/invasion ( Ma et al., 2016 ), increased proliferation ( Rickard et al., 2023 ), and an altered response to platinum-based chemotherapies ( Rickard et al., 2023 ). Studies on PFAS and uterine fibroids are extremely limited and conflicting. Serum PFAS level was associated with fibroid growth during pregnancy, although not associated with fibroid prevalence or number ( Mitro et al., 2023 ). Conversely, higher serum PFAS levels were associated with decreased fibroid growth ( Wise et al., 2024 ) and another study found no association of fibroids with exposure to PFAS ( Hammarstrand et al., 2021 ). While existing studies have provided valuable insights between PFAS and uterine disorders, the inconsistencies and gaps in the current body of research require more insight to clarify the impacts of PFAS on the uterus. The myometrium is the smooth muscle layer of the uterus and provides the structural integrity of the uterus, helping maintain its shape and position within the pelvic cavity. During pregnancy, it is responsible for maintaining uterine tone and for the highly contractile activity necessary for childbirth ( Fodera et al., 2024 ). In a non-pregnant state, the myometrium undergoes peristaltic contractions to facilitate sperm motility, support embryo implantation, and assist in the expulsion of menstrual blood ( Fodera et al., 2024 ). Disruptions in myometrial function can lead to severe reproductive complications, including preterm labor ( Phung et al., 2022 ; López Bernal, 2007 ), difficulties in childbirth ( Wray and Prendergast, 2019 ), or myometrial disorders such as adenomyosis and uterine fibroids ( Heller, 2015 ). Despite its importance, there is no research on the impact of environmental contaminants such as PFAS on the myometrium, leaving a significant gap in our understanding of how these chemicals may affect reproductive health. This study aims to address this knowledge gap by investigating the effects of PFAS on myometrial cells, specifically focusing on cellular processes critical for myometrial function, such as proliferation, migration, gap junction intercellular communication (GJIC), and gene expression.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi: 10.1016/j.tox.2025.154173 .