By modelling real-world PFAS mixtures found in first-trimester placentas, researchers reveal how everyday chemical exposure can subtly rewire placental hormone release, invasion, and gene pathways critical for early pregnancy.
Study: Real-life per- and polyfluoroalkyl substances mixture impairs placental function: insights from a trophoblast spheroid model. Image Credit: Francesco Scatena / Shutterstock
A recent study in the journal Environmental Research examined placental per- and polyfluoroalkyl substances (PFAS) concentrations in early pregnancy and designed a placenta-relevant PFAS mixture to assess its impact on trophoblast function using a three-dimensional (3D) trophoblast spheroid model.
Human PFAS Exposure and Reproductive Vulnerability
PFAS are chemicals that are ubiquitously present in the environment due to their widespread application in various industries and consumer products. Humans are highly exposed to these chemicals through ingestion, inhalation, and skin contact. PFAS persist and bioaccumulate in different organs, including the lung, kidney, liver, and placenta, partly owing to their long elimination half-lives of 1.5–8.5 years.
Epidemiological research has associated human exposure to PFAS with developmental risk effects and reproductive disorders. Multiple studies have described PFAS as endocrine-disrupting chemicals (EDCs). Pregnant women are especially vulnerable to EDCs because pregnancy-related hormonal, cardiometabolic, and immune changes increase susceptibility. The accumulation of PFAS in the placenta, a temporary organ unique to gestation, allows these chemicals to be transferred to the fetus along with essential nutrients, gases, and metabolites, raising concern for both immediate and long-term health effects.
Although multiple PFAS have been detected in the placenta, data remain limited on their concentration in the first trimester. This is important because organogenesis, cell differentiation, and placenta development occur during this pregnancy phase.
Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are two common PFAS that reduce cell proliferation, viability, and migration in two-dimensional (2D) trophoblast cell lines. These chemicals have also been associated with the upregulation of inflammatory pathways in prior studies. Instead of investigating how single PFAS exposure influences fetal development, and reflecting real-world exposure, it is important to elucidate how a mixture of PFAS accumulation affects placental and trophoblast function.
Quantifying PFAS in First-Trimester Placental Tissue
A total of 31 first-trimester placenta samples were collected from elective terminations of pregnancies. All samples assessed were between 7 and 11 weeks of gestation for PFAS level determination. Although all placenta samples tested positive for PFAS, the quantified levels varied. Out of 56 targeted PFAS, 16 were detected and quantified above the limit of quantitation (LOQ) in more than one sample. The samples were obtained from a single geographic region in Germany, which may limit the generalisability of the measured PFAS concentrations.
Interestingly, even though PFOS use was largely reduced in the industry since 2000, relatively high concentrations (0.13–0.46 ng/g) were detected in placenta samples obtained from 2023. In addition, relatively high levels of perfluorononanoic acid (PFNA) were observed, unexpectedly representing the dominant contributor to the PFAS mixture, with concentrations ranging from LOQ to 9.45 ng/g. PFNA was followed by PFOA and perfluorobutanoic acid (PFBA) concentrations.
Designing a Placenta-Relevant PFAS Mixture
The PFAS concentrations measured in the placenta (ng/g) were converted to cellular exposure concentrations (nM). To generate experimental treatments, a placenta-based PFAS mixture reflecting measured ratios was prepared and then diluted across a wide concentration range. Cells and spheroids were exposed to PFAS concentrations ranging from 0.01 μM to 300 μM. Lower concentrations (0.01–1 μM) were selected to reflect environmentally relevant exposure, while higher concentrations were used to explore potential threshold or non-linear effects.
PFNA, PFOS, perfluorobutanoic acid (PFBA), PFOA, perfluorohexanesulfonic acid (PFHxS), and perfluorodecanoic acid (PFDA) were used to prepare the mixture in the following placenta-based ratio of 80:7:5:4:2:2.
Cytotoxicity of PFAS Mixtures in 2D Trophoblast Cells
To determine whether the PFAS mixture affects trophoblast cell viability, JEG-3 and HTR-8/SVneo cell lines were treated with varying PFAS concentrations (0.01–300 μM), and viability was measured using an MTT assay. Cell viability was significantly reduced in both cell lines at 150–300 μM, demonstrating a clear concentration-dependent effect.
Viability Responses in 3D Trophoblast Spheroid Models
Given that 2D-cultured cells respond differently to chemicals, the effects of the PFAS mixture were evaluated in 3D trophoblast spheroids. After 48 hours of PFAS exposure, JEG-3 spheroids showed increased necrotic cell fluorescence only at 300 μM, while live cell fluorescence remained unchanged. HTR-8/SVneo spheroids displayed no differences in live or necrotic cell fluorescence across concentrations. Caspase 3/7 activity increased only in HTR-8/SVneo spheroids at 300 μM. These findings indicate that trophoblast spheroids exhibit distinct, more resilient viability responses to PFAS compared to 2D cultures.
Disruption of Trophoblast Invasion by PFAS Mixtures
Trophoblast spheroids exposed to PFAS mixtures remained viable, but their invasive function was altered. JEG-3 spheroids showed complete loss of invasion at the highest PFAS concentration and morphological signs of damage, while lower concentrations increased invasion at later time points. In contrast, PFAS mixtures inhibited HTR-8/SVneo spheroid invasion at low and medium concentrations, though high concentrations did not produce statistically significant inhibition. These results suggest PFAS mixtures differentially affect trophoblast spheroid invasiveness depending on cell type and concentration.
PFAS-Induced Reductions in β-hCG and Gene Expression
β-hCG, produced by trophoblast cells, is essential for maintaining early pregnancy. JEG-3 spheroids, representing placental endocrine function, were used to assess PFAS effects on β-hCG secretion. After 48 hours of PFAS exposure, β-hCG secretion decreased at lower concentrations, with a significant reduction observed at 0.01 μM. CGB7 gene expression, which encodes the β-hCG subunit, was also found to be downregulated at 10 μM and 100 μM, indicating that PFAS mixtures impair early endocrine signaling pathways relevant to pregnancy maintenance.
To assess PFAS-induced transcriptomic changes in trophoblast spheroids, mRNA levels of genes related to apoptosis, invasion, migration, and proliferation were measured. PFAS exposure downregulated multiple apoptosis-related genes in JEG-3 spheroids, while increasing CASP3 and decreasing LGALS3 in HTR-8/SVneo spheroids. Genes associated with proliferation, invasion, and migration also exhibited cell-type-specific responses, including downregulation of EGFR, NOTCH3, and PGF in JEG-3 spheroids, and decreased MIF but increased IGF2 in HTR-8/SVneo spheroids. These findings reveal that PFAS mixtures alter gene expression in trophoblast spheroids, consistent with disruptions to pathways supporting placental development and function.
Implications of PFAS Mixtures for Placental Health
A mixture of PFAS was identified in first-trimester placental tissue and, when modelled in vitro, was associated with alterations in trophoblast invasion, endocrine activity, and gene expression, without substantially affecting overall spheroid viability at environmentally relevant concentrations. However, to fully understand the implications of PFAS mixture exposure on gestation and fetal development, further longitudinal in vivo research and mechanistic investigations are needed. Overall, the current study findings underscore the need for stricter risk assessment of chemical mixtures that incorporate pregnancy-relevant endpoints.
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