PFAS Exposure and Male Reproductive Health: Implications for Sperm Epigenetics

Abstract

Per- and polyfluoroalkyl substances (PFASs) are persistent environmental contaminants found in human tissues and persist in the environment, posing significant risks to reproductive health. This review examines the impact of PFAS exposure on male reproductive health, with a focus on sperm epigenetics. PFASs disrupt endocrine function by altering key reproductive hormones and impairing sperm motility, quality, and viability. Epidemiologic and animal studies highlight inconsistent yet concerning associations between PFAS exposure and semen parameters, as well as altered gene expression and DNA methylation patterns. Moreover, PFAS exposure during critical windows of development has been linked to differential impacts on male versus female pubertal development, cognitive outcomes, and reproductive physiology, emphasizing the complexity of PFAS interactions. This comprehensive analysis highlights the need for continued research into the mechanisms by which PFASs influence reproductive health and development with potential implications for sperm epigenetics. The review emphasizes the importance of understanding the epigenetic mechanisms behind these disruptions, particularly DNA methylation and its role in heritable changes. Investigating the epigenetic modifications driven by PFAS exposure is crucial for elucidating the mechanisms by which these chemicals influence reproductive health. Future research should focus on understanding these epigenetic changes in both immediate fertility outcomes and transgenerational health risks.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous chemicals used for their nonstick, surfactant, and firefighting characteristics and have been measured in the blood of individuals at levels approaching the parts per million range (μg/mL). PFASs are a class of man-made chemicals, encompassing an estimated 4,000 to over 20,000 individual compounds and their breakdown products.^1,2 These PFAS mixtures are present in the environment, human tissues, and serum, and in commercial products like aqueous film-forming foams. The primary human exposure route for PFAS is through drinking water, with dietary ingestion of contaminated foods and via PFAS-laden food packaging also critically important. Moreover, dermal and inhalation exposures have also been observed in humans. Initially, these fluorinated chemicals were manufactured with relatively long carbon backbone chains and with head groups mirroring carboxylate or sulfonic acids.³ Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), both C8 compounds, were the most commonly manufactured up through the early 21st century with ~3,500 metric tons of PFOS being manufactured in 2000 and 1,200 metric tons of PFOA being manufactured in 2004, for example.⁴ Although C8 compounds make up likely the most important legacy exposure across the United States, more than 85,000 tons of PFAS are produced domestically each year with many of these now being short-chain fluorotelomers or fluorotelomer-based side-chain fluorinated polymers (Fig. 1a).⁵ Epidemiological studies focusing on occupational exposures, contaminated drinking water, and national surveys, such as the National Health and Nutrition Examination Survey,⁶ have repeatedly observed significant associations with PFAS exposure and increased risk of multiple diseases related to the immune, cardiovascular, and reproductive systems.^7,8 These observations have been validated with mechanistic studies using in vitro and in vivo systems. This review will focus primarily on reproductive toxicity related to male PFAS exposures and their impact on male reproduction and the molecular mechanisms related to the endocrine-disrupting nature of PFAS and sperm epigenetics.

Fig. 1.

(a) Structures of long- and short-chain PFAS substances. (b) Illustration of a perfluorinated compound’s effects on male fertility, showing the mechanisms involved and proposed toxicity pathways. Green arrows represent stimulatory effects, red arrows with blunt ends indicate inhibitory regulation, and purple arrows suggest epigenetic regulation of endocrine function and semen quality. (Created in https://BioRender.com.)

Multiple PFAS have been associated with endocrine disruption in epidemiologic and animal studies that focused on impacts on reproduction-associated signaling molecules including testosterone, estradiol, and thyroid-related hormones.^9,10 PFAS may disrupt normal sperm homeostasis by disrupting the production of these key hormones or by aberrantly antagonizing or activating their respective transcription factors (Fig. 1b). As with most PFAS, associations with endocrine activity and legacy long-chain PFAS are more readily reported, with a majority related to maternal exposures.¹¹ Recent in silico and in vitro studies also implicate emerging PFAS as possible endocrine-disrupting chemicals. For example, a recent study that incorporated machine learning approaches predicted 159 PFAS to be ERA antagonists and 104 to be antagonists of androgen receptor.¹² Emerging PFAS including (9-(nonafluorobutyl)–2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)–3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl) pentanamide (NNN) were confirmed to be antiandrogenic in vitro by inhibiting AR transactivation via competitive binding and impacts on receptor expression.¹³ In addition to AR, estrogen receptors are also critical for proper testosterone production in the teste.¹⁴ In multiple cell types, long-chain PFAS have been shown to disrupt normal estrogen functions with PFOA, PFOS, perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) all enhancing ERα transcriptional activation.¹⁵ Additionally, there are established PFAS species and chain length differences that have been observed for ER disruption.¹⁶ Furthermore, PFAS may also impact on estradiol (E2) levels themselves. For example, in male rats, PFOA increased serum E2; this finding was also observed in Japanese men who were occupationally exposed.¹¹ Also, other endocrine-related transcription factors including the thyroid receptor are also critically important for proper male reproduction. Both TRa and TRb are expressed in the testis and proper activation is required for the proliferation and differentiation of Sertoli and Leydig cells and for subsequent spermatogenesis and steroidogenesis.¹⁷

Multiple epidemiologic and preclinical studies have observed significant associations with PFAS exposure and thyroid disease or thyroxine levels.¹¹ Finally, it must be highlighted that PFAS are established PPAR activators, and at a minimum, PPARα is expressed in Leydig cells, spermatocytes, and other regions of the testis.¹⁸ Interestingly, PPAR activation can modulate the expression of estrogen and androgen receptors and PPARα within Leydig cells, which can interact with estrogen receptors to regulate steroidogenesis via modulation of lipid metabolism.¹⁹ In general, it is established that individuals with hyperlipidemia and high circulating LDL cholesterol may also have detrimental male reproductive effects, including low seminal fluid and abnormal sperm count and morphology; however, less is known on the impacts of increased lipids at the site of the testis themselves on male fertility.²⁰ Our group, to our knowledge, was the first to show that PFAS can accumulate in testis tissue,²¹ but it is unknown if our observed impacts on sperm are related to in situ PPAR activation or through the other interconnected mechanisms. Mixtures of legacy and emerging fluorinated chemicals are found in the environment, human blood, and reproductive organs such as testis and they may elicit toxicity through direct or indirect mechanisms, which will be discussed throughout this review. This review will examine various species, including humans (Fig. 2), mammalians (Fig. 3a), and aquatic species (Fig. 3b).

Fig. 2.

Overview of PFAS exposure effects on human health, highlighting impacts on pregnancy outcomes, developmental processes, semen quality, levels in biological fluids, endocrine disruption, and DNA methylation changes. (Created with https://BioRender.com.)

Fig. 3.

(a) Overview of PFOS and PFOA exposure effects on mammalian systems, including rodents, bull sperm, and Landrace boars, illustrating impacts on spermatogenesis, hormone regulation, oxidative stress, and gene expression, with a focus on reproductive and endocrine disruptions. (b) Summary of PFOS/PFOA effects on aquatic systems, including zebrafish, guppies, largemouth bass, and turtles, highlighting behavioral changes, gene expression alterations, and hormonal disruptions with potential impacts on reproduction and stress responses. (Created with https://BioRender.com.)

PFAS Exposure on Reproductive and Developmental Health

Exposure to PFAS mixtures during pregnancy has been linked to impaired sperm quality and altered hormone levels in young male offspring.²² Studies show that PFAS can affect reproductive health by disrupting sperm function, altering hormone levels, and damaging testicular cells.^23–26 Epidemiologic research highlights inconsistent findings on the impact of PFAS on semen parameters but suggests that prenatal exposure to PFAS mixtures, particularly during pregnancy, is associated with lower sperm count and altered hormone levels in young men.^27,28 Moreover, paternal PFAS exposure also impacts the F1 generation, affecting gene expression related to cholesterol metabolism, xenobiotic responses, and cell cycle regulation in the liver and fat tissues of the offspring.²¹

Investigating the influence of PFAS on pubertal development is vital for uncovering how environmental contaminants might disrupt critical growth processes and long-term health outcomes. In females, PFAS concentrations correlated with delayed pubic hair and breast development and later menarche.²⁹ However, no significant associations were found between higher maternal PFAS levels and pubertal development in males. Exploring the effects of both maternal and paternal PFAS exposure on children’s cognitive development (up to age 8) is crucial for understanding potential long-term impacts on educational and psychological outcomes. Maternal PFAS levels were associated with higher Full-Scale Intelligence Quotient (FSIQ) scores in children, though overall cognitive function did not show adverse effects.³⁰ The effect varied by sex, with higher prenatal PFOA linked to better FSIQ scores in females but not in males.

PFAS Exposure on IVF and Birth Outcomes

Exploring paternal PFAS exposure is vital due to its direct impact on IVF outcomes and birth metrics. In a study involving 96 couples from Zhejiang University, plasma samples from both partners were analyzed for 10 PFAS, including PFOA, PFOS, PFNA, and PFHxS. Results indicated that higher plasma PFOA concentrations in women were associated with fewer retrieved oocytes, mature oocytes, 2 PN zygotes, and good-quality embryos. In men, higher PFNA and PFDA levels were linked to decreased sperm concentration, while higher PFOSA and PFHpA levels were associated with lower total sperm count and abnormal morphology, respectively. Notably, higher paternal PFOA levels were significantly associated with a reduction in fertilization rates; however, no correlation was observed between PFAS concentrations and pregnancy outcomes such as implantation or live birth.³¹

In a study involving the Environment and Reproductive Health (EARTH) study, subfertile couples were evaluated for fertility at Massachusetts General Hospital from 2005 to 2019. Blood samples from 312 women and 145 men were analyzed for various PFAS, including PFOA, PFOS, PFHxS, and others. Men generally had higher PFAS levels than women, with correlations between their PFAS concentrations varying from weak to moderate.³² Moreover, maternal preconception levels of PFOS and PFHxS were linked to lower birthweight, while paternal levels of these PFAS were associated with higher birthweight, although these associations were imprecise. No significant associations were found between PFAS levels and gestational age or preterm birth. Sensitivity analyses revealed that some associations were sex-specific, with PFAS concentrations affecting birthweight differently for male and female infants. Coadjusting for partner PFAS levels enhanced the associations found.

PFAS exposure is associated with delayed pubertal development in females but not males,²⁹ and maternal PFAS levels are linked to higher IQ scores in females.³⁰ Birthweight outcomes vary by sex and PFAS source, with maternal PFAS generally decreasing birthweight and paternal PFAS showing complex effects.³² Maternal PFAS exposure also altered fetal endocrine hormone levels, while paternal exposure had minimal impact.^27,28

Mammalian Systems

Research on mammalian systems provides crucial insights into how PFAS, specifically PFOS and PFOA, affect reproductive health and development. Shi et al’s review highlights several impacts of PFOS and PFOA on rodents,³³ detailing mechanisms such as apoptosis in spermatogenic and Leydig cells, oxidative stress, and disruptions in hormone synthesis and cellular communication in both males and females. Moreover, PFAS also exhibited comparable reproductive toxicities and may present even greater risks to placental health in rodent models. In pregnant rats, exposure to PFOS (5 mg/kg/day) and PFOA (5 mg/kg/day) negatively affects neonatal survival, slows down postnatal development, and leads to teratogenic outcomes in offspring.^34,35 Luebker et al found that PFOS exposure (3.2 mg/kg/day) decreased gestational length, reduced embryonic sites, and increased the incidence of stillbirths or pup mortality during the first few days of lactation.³⁶ Juvenile rats exposed to environmental levels of PFOA and PFOS (0.1 and 1 mg/kg/day) showed disrupted hypothalamic–pituitary–gonadal (HPG) regulation, leading to early puberty in females and altered reproductive physiology.³⁷ However, PFOS exposure did not affect the estrus cycle but did reduce the number of gravid sites in female rats.³⁶ In adult male rats, PFOS affected various aspects of HPG function, including hormone levels and receptor expression.³⁸ In mice, exposure to PFOA (10 mg/kg/day) for 3 weeks resulted in lower testosterone levels and sperm counts, likely due to reduced expression of hormonal receptors and impaired testicular function.^39,40 PFOS impacted sex hormone levels more significantly, while PFOA affected reproductive organ development, causing testicular damage in male offspring and delayed vaginal development in females.^41,42 PFOA exposure also impaired placental development and function, reducing placental efficiency and causing fetal growth retardation and necrosis.⁴³ Additionally, chronic low-dose PFOS exposure (0.1 mg/kg/day) inhibited estrogen production, affected follicular development and ovulation, and decreased primordial follicle numbers, consistent with human findings.⁴⁴

Aquatic Systems

In a study conducted by Haimbaugh et al in 2022, researchers examined the effects of PFOS and PFOA on early development, F0 embryos were exposed for 5 days to varying concentrations of PFOS (24, 240, 2,400 ng/L), PFOA (7, 70, 700 ng/L), and a combined mixture where each compound was present at half of the individual concentrations (e.g., 12, 120, 1,200 ng/L for PFOS and 3.5, 35, 350 ng/L for PFOA).⁴⁵ On day 5, larvae were rinsed with buffered water to end exposure before assays. No significant abnormalities or mortality were observed, though ultra-low PFOS exposure showed a near-significant increase in abnormalities. PFOA reduced larval swimming distance in both dark and light, except at low light, while PFOS had no effect in the dark but reduced activity at higher light concentrations. Transcriptomic analysis revealed differential gene expression from ultra-low PFOA and PFOS exposure, affecting xenobiotic metabolism, lipid metabolism, and immune function, though pathway analysis was limited by the number of DEGs.⁴⁵ Researchers proposed that epigenetic changes, triggered by F0 exposure, likely drive the altered behavioral and transcriptomic profiles observed in unexposed F1 and F2 generations. These phenotypic changes, which involve disruptions in neuronal pathways, immune function, and steroid metabolism, suggest that PFAS interferes with normal biological processes, potentially impacting both aquatic ecosystems and human health.

Taken together, PFAS exposure significantly disrupts development in various animal models, including rodents and zebrafish, impacting reproductive and developmental processes. These studies underscore the broader implications of PFAS exposure, indicating potential risks to both aquatic ecosystems and human health through its effects on biological functions and epigenetic changes.

PFAS and Semen Parameters

Human

Human studies on PFAS exposure and semen parameters reveal mixed results. Some research suggests positive correlations between serum PFAS levels and sperm concentration,²² while another study reported that elevated PFOA and PFOS levels are associated with reduced sperm motility.⁴⁶ In a study conducted by Pan et al in 2015 to 2016, seminal levels of PFOA, PFOS, and 6:2 Cl⁻PFESA were significantly linked to a lower percentage of progressive sperm and a higher percentage of DNA fragmentation (false discovery rate-adjusted p-values <0.05).⁴⁷ In contrast, the associations between serum PFAS levels and semen parameters were generally weaker, except for DNA stainability, which showed a stronger correlation with serum PFAS levels than with those in semen. Researchers from the Department of Toxicology at Gazi University, Ankara, Turkey, conducted a study on semen samples from three healthy nonsmoking men (aged 27–34 years), collected after 2 to 7 days of abstinence.⁴⁸ Semen analysis followed WHO guidelines (2010).⁴⁹ Sperm cells were treated with noncytotoxic concentrations (100, 300, and 1,000 μM) of PFAS chemicals dissolved in DMSO, with viability remaining above 85%. Lymphocytes also showed over 90% viability in the trypan blue test. Genotoxicity tests using the comet assay revealed that none of the tested chemicals (PFOS, PFOA, PFNA, PFHxA) caused significant DNA damage in sperm cells, with damage levels remaining similar to the control group. Positive controls, however, showed a marked increase in DNA damage. The results indicated no genotoxic effects from the PFAS chemicals despite their differing chain lengths.

A study conducted by Luo et al, between 2013 and 2015, consisted of 740 men of both preconception and pregnancy cohorts.⁵⁰ Researchers found varied associations between different PFAS compounds and semen parameters. PFBS was consistently negatively associated with sperm count and concentration, whereas PFHpS and other isomers showed positive associations. However, 6:2 Cl⁻PFESA was inversely associated with motility rates. Quantitative and machine learning analyses identified PFBS, PFHxS, and PFHpS as key contributors to negative associations with sperm quality, while PFHxS and PFBS were linked to a reduction in sperm count and concentration. Similarly, a study involved participants from the EARTH study, a prospective cohort of subfertile men and women seeking fertility evaluation and treatment at the Massachusetts General Hospital Fertility Center, United States, from 2005 to 2019.⁵¹ Blood samples collected at enrollment were centrifuged to isolate serum, which was then analyzed for n-PFOA, branched PFOA isomers (sb-PFOA), PFNA, PFDA, PFUnDA, and MeFOSAA. In the EARTH study, baseline serum PFAS measurements were obtained from 572 females and 279 males, making up a total of 249 couples. Analysis of PFAS highlighted higher PFAS concentrations in males compared with females. In addition, males with male factor infertility exhibited higher PFAS levels, whereas those with unexplained infertility had lower PFAS levels. Moreover, diet played a significant role, with fish and shellfish consumption positively associated with PFAS concentrations in both genders.

In a study by Di Nisio et al, researchers conducted a cross-sectional study involving 212 males exposed to PFCs from the Veneto region, one of the four globally significant areas of PFC pollution, along with 171 non-exposed control subjects. Comparing exposed and control groups revealed significant differences in testicular volume, penile length, and semen parameters, including lower sperm motility and increased immotile sperm counts in exposed individuals.⁵² Additionally, a cross-sectional study in 2003 investigated semen quality among young Danish men entering the military, who provided both semen and venous blood samples. From the 546 men examined that year, 105 were selected to explore the link between PFAAs and testicular function. This group comprised 53 men with the highest testosterone levels (median: 31.8 nmol/L; range: 30.1–34.8 nmol/L) and 52 men with the lowest testosterone levels (median: 14.0 nmol/L; range: 10.5–15.5 nmol/L). Danish men showed tendencies toward reduced morphologically normal sperm in high PFAA groups, though not statistically significant.⁵³ Additionally, PFHpS was negatively associated with the percentage of progressively motile sperm, but no other significant associations were found between serum PFC levels and semen quality parameters or testicular volume.⁵⁴ In 2015, Buck Louis et al utilized the LIFE study cohort to evaluate the relationship between 7 PFCs and 35 semen quality parameters, aiming to identify potential associations. A total of 501 couples who were trying to conceive were recruited from 16 counties in Michigan and Texas. Researchers observed diminished semen quality and significant associations between PFOSA, MePFOSA-AcOH, and PFOA with various semen quality endpoints.⁵⁵ Specifically, PFOSA was linked to a smaller sperm head area and perimeter, increased numbers of immature and bicephalic sperm, and decreased DNA stainability, while MePFOSA-AcOH was associated with higher rates of sperm neck/midpiece abnormalities and immature sperm, and decreased DNA stainability. Moreover, PFOA was significantly associated with reductions in the percentage of sperm with coiled tails, indicating possible structural abnormalities. This suggests that PFOA, along with PFOSA and MePFOSA-AcOH, may contribute to impaired semen quality through various effects on sperm morphology and maturity.

In 2012, a study enrolled male partners of pregnant women until ~200 men had provided semen samples from each region. Initially, 3,833 pregnant women and their male partners were invited to participate during their first antenatal care visit at one of three locations: local hospitals in 19 cities and settlements across Greenland, a large central hospital in Warsaw, Poland, and three hospitals along with eight antenatal clinics in Kharkiv, Ukraine.⁵⁶ Researchers found that PFOS was significantly associated with an increase in the percentage of sperm positive for the proapoptotic marker, Fas, particularly in Polish men, where the trend was statistically significant (p = 0.03). Serum PFOA was also linked to higher sperm DNA fragmentation in men from Greenland, with a trend toward increased TUNEL-positive sperm cells, but no such associations were observed in men from Poland or Ukraine.⁵⁶ Moreover, associations between PFOS exposure and semen quality identified no significant associations for semen volume or sperm motility across Artic and European populations.⁵⁷

In 1988 to 1989, pregnant women were recruited in Aarhus, Denmark, to provide a blood sample. In 2008, their sons were invited to complete an online questionnaire about their health and lifestyle. Additionally, from 2008 to 2009, their sons also participated in a physical examination, during which they provided a home-collected semen sample, a blood sample for reproductive hormone analysis, and self-measured their testicular volumes. This study involved 169 participants and found that in utero exposure to PFOA and PFOS was associated with reduced sperm concentration and count.²⁸ Although no significant effects were found on progressive spermatozoa percentage, computer-assisted semen analyses suggested lower sperm concentrations in higher exposure groups.

A meta-analysis conducted by Wang et al includes data from seven cross-sectional studies, encompassing 2,190 subjects, conducted across Denmark, the United States, Greenland, Poland, Ukraine, the Faroe Islands, and China between 2009 and 2019. Overall, the meta-analysis included seven studies examining the impact of various PFAS on semen quality, analyzing sperm concentration, motility, semen volume, and morphology. The studies focused on PFOS, PFDA, PFHxS, PFNA, and PFOA. Overall, PFOS, PFDA, and PFHxS showed no significant correlations with semen parameters, though PFNA exposure was negatively associated with sperm progressive motility. Subgroup analyses by geographic location and sample size revealed that PFOA’s effect on sperm concentration and count varied with significant associations in Asian countries and larger sample sizes but not in Western countries or smaller samples. PFOS exposure showed a positive correlation with sperm count only in larger studies and with semen samples specifically. There were no notable differences in semen quality linked to PFNA when stratified by sample size, but a significant negative correlation with sperm morphology was observed in larger studies.⁵⁸

Some studies suggest that PFAS exposure impacts semen quality, including sperm concentration and motility, but findings are inconsistent, with certain PFAS showing both detrimental and neutral effects.³³ In 2024, Alamo et al investigated PFOA using 50 healthy men who visited the Division of Andrology and Endocrinology at the University of Catania for sperm analysis. Sperm samples were incubated with PFOA at concentrations of 0.01, 0.1, and 1 mM. Following incubation, spermatozoa were assessed to determine how PFOA affected sperm motility and biofunctional parameters, including total and progressive motility, in accordance with WHO 2010 guidelines.⁴⁹ Specifically, PFOA significantly inhibited total and progressive sperm motility in a concentration-dependent manner, with the strongest inhibition at 1 mM. Additionally, PFOA exposure increased abnormal chromatin compactness, lipid peroxidation, and mitochondrial superoxide production in sperm, while it had no significant effect on sperm viability, mitochondrial membrane potential, or DNA fragmentation. Research on PFOA specifically found significant inhibition of sperm motility at higher concentrations, with increased oxidative stress but negligible effects on sperm viability.⁵⁹ Moreover, a study included 10 healthy donors with normozoospermia, aged 20 to 35 years, who participated in an infertility survey at the University Andrology Unit in Italy. Each donor provided semen samples across four sessions, ensuring at least 3 days of sexual abstinence between donations to minimize variability. Additionally, cervical mucus samples were collected from 10 women living in highly exposed areas of the Veneto region and 10 age-matched controls from areas with low exposure. Researchers selected three concentrations of PFOA for the study: 0.1, 1, and 10 ng/mL. Incubation with PFOA showed minimal effects on sperm viability, regardless of the concentration or exposure time. However, PFOA significantly reduced sperm progressive motility even at the lowest concentration of 0.1 ng/mL, as the percentage of sperm with forward motion decreased markedly after 1 hour compared with the control condition.⁶⁰ In addition, sperm analysis revealed substantial reductions in hypermotility, curvilinear velocity, straight-line velocity, and average path velocity compared with controls, indicating a clear impact of PFOA on sperm motility.

The reviewed studies highlight a complex and inconsistent relationship between PFAS exposure and sperm parameters. While some evidence points to negative impacts on sperm parameters, other findings are inconclusive or suggest positive correlations. These discrepancies emphasize the need for further research with standardized methods and larger sample sizes to better understand PFAS effects on semen parameters and overall male reproductive health.

PFAS and Semen Parameters: Insights from Model Systems

Mammalian Systems

Limited data are available on the effect of PFAS on semen parameters in animal studies. In a study conducted by Kumaresan et al, bull sperm was exposed to PFOS at concentrations of 10 μM (average population level) and 100 μM (high-exposure level) for 4 hours at 37 °C to assess fertility functions. Initially, 70% of sperm was motile, 72% viable, with 13.3% having high intracellular calcium, 76.3% showing high mitochondrial membrane potential, 71.7% positive for mitochondrial ROS, and 41.6% displaying protein tyrosine phosphorylation (PTP).⁶¹ After exposure to 100 μM PFOS, sperm motility decreased by 87.5%, while a 50% reduction was seen at 10 μM. Sperm viability dropped by 36 and 70% in the 10 and 100 μM groups, respectively. PFOS exposure significantly reduced live sperm with high intracellular calcium and mitochondrial membrane potential in a dose-dependent manner, with decreases of 41% at 10 μM and 64% at 100 μM. Mitochondrial ROS levels were only significantly impacted at 100 μM. Conversely, PFOS exposure increased sperm with PTP, with rises of 64% at 10 μM and 41% at 100 μM.⁶¹

In a mouse study, C57BL/6J mice were divided into four groups: control, PFOS-treated, PFOS with GSK3787, a PPARδ antagonist, and PFOS with GW6471, a PPARα antagonist. After a 7-day acclimation, PFOS was administered in drinking water at 5 mg/kg body weight for 35 days. Mice in the GSK3787 and GW6471 groups also received daily intraperitoneal injections of either compound, while control and PFOS-only groups received a vehicle solution.⁶² In PFOS-treated mice, PPARδ activation played a key role in spermatocyte loss, contributing to male reproductive toxicity. Mice treated with PFOS for 35 days showed significant reductions in testicular and epididymal indices, sperm concentration, motility, and spermatocyte numbers. Histopathological analyses revealed disorganized seminiferous epithelium and increased germ cell loss. Blocking PPARδ with GSK3787 alleviated most of these impairments, while blocking PPARα with GW6471 had minimal effects. Further in vitro studies on spermatocytes exposed to PFOS demonstrated endoplasmic reticulum (ER) stress, apoptosis, and disrupted calcium homeostasis. PFOS also activated the PPAR signaling pathway, which was linked to metabolic stress and ER stress in spermatocytes. Blocking PPARδ or inhibiting ER stress markers improved calcium homeostasis and reduced apoptosis in both in vitro and in vivo models, suggesting PPARδ as a key mediator of PFOS-induced reproductive toxicity.⁶²

Semen samples from 10 fertile Landrace boars were collected and sperms were incubated under capacitation conditions with varying concentrations (500–3,000 μM) of PFOS and PFOA for 4 hours. PFOS and PFOA showed dose-dependent toxic effects on sperm viability, with PFOS having higher toxicity than PFOA. The median lethal concentrations (LC50) for PFOS and PFOA were 460 and 1,894 μM, respectively, and their inhibitory concentrations of capacitation (ICC50) values for inhibiting capacitation were 274 and 1,458 μM. PFOS significantly impaired capacitation and induced acrosome reaction (iAR), while PFOA had a milder effect. PFOS increased intracellular calcium levels, disrupting calcium homeostasis, membrane potential, and cholesterol efflux.⁶³ Additionally, both PFAS compounds altered membrane microdomain distribution and actin cytoskeleton reorganization, critical for sperm capacitation and mobility. PFOS’s toxic effects were generally more rapid and pronounced compared with PFOA, highlighting its potential for causing significant reproductive damage during sperm capacitation.

Aquatic Systems

In 2024, researchers conducted an experiment where guppies were exposed to PFOA, GenX, or a control in a static system for 21 days at ecologically relevant concentrations. The guppies were descendants of wild-caught individuals from the Lower Tacarigua River in Trinidad. After exposure, male guppies were weighed, measured for body size, and photographed to assess their color. Sexual behavior was tested in 25 males per group, and sperm traits were analyzed, including sperm swimming performance, viability, and number. The study found that exposure to GenX and PFOA significantly reduced sperm swimming velocity compared with the control group but had no impact on sperm production or viability.⁶⁴ This indicates that while PFAS exposure can impair sperm motility, it does not necessarily impact overall sperm production or viability in guppies.

Research has shown that PFAS, including PFOS and PFOA, impair sperm quality in various species, with effects worsening at higher doses. In mice, PFOS reduced sperm motility, viability, and mitochondrial function, while in boars, PFOS and PFOA were toxic, with PFOS having a greater impact on sperm capacitation. Guppies exposed to GenX and PFOA experienced reduced sperm swimming velocity, but no changes in production or viability, emphasizing the detrimental effects of PFAS on male reproductive health across species.

PFAS Concentrations in Serum and Semen

Humans

Among the many biological matrices in which PFAS can accumulate, serum and semen represent two critical fluids for assessing both general exposure and reproductive risks. Measuring PFAS concentrations in serum provides an indicator of systemic exposure, while analyzing PFAS levels in semen offers insights into their potential impact on male reproductive health. Understanding the distribution of PFAS in these fluids is essential for exploring the link between environmental exposure and reproductive outcomes, especially in men. Between 2015 and 2016, male partners of couples attending their initial visit to the Reproductive Medical Center at Nanjing Jinling Hospital in Nanjing, China, were recruited for a study. Sixteen target PFAS compounds were measured, including perfluorobutanoate (PFBA), perfluoropentanoate (PFPeA), perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), PFOA, perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnDA), perfluorododecanoate (PFDoDA), perfluorotridecanoate (PFTriDA), perfluorotetradecanoate (PFTeDA), perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), PFOS, and 6:2 and 8:2 Cl⁻PFESAs. PFOA, PFOS, and 6:2 Cl⁻PFESA were detected at the highest concentrations in both semen and serum, with median concentrations of 0.23, 0.10, and 0.06 ng/mL in semen, respectively, and a semen-to-serum ratio of 1.3:3.1. The correlations between PFAS concentrations in semen and serum were strong, PFOA (R = 0.70), PFNA (R = 0.72), PFDA (R = 0.74), PFUuDA (R = 0.79), PFOS (R = 0.80), 6:2 Cl⁻ PFESA (R = 0.83), and 8:2 Cl⁻PFESA (R = 0.75).⁴⁷ Moreover, analysis of PFAS concentrations in matched serum and semen in men showed similar distributions with PFOS being the highest in serum and PFOA in semen (Table 1). The correlation coefficients ranged from 0.646 to 0.826.⁶⁵ Most other PFAS were readily detected in serum, with PFOA, PFOS, and 6:2 Cl⁻PFESA being the most dominant compounds, accounting for ~84% of total PFAS with detection rates reaching 100% for PFOA and 6:2 for Cl⁻PFESA.⁴⁷ In another study conducted by Joensen et al, in 2013, fourteen PFCs were measured, with PFHxS, PFHpS, PFOS, PFOA, PFNA, and PFDA being detected in greater than 99% of samples.⁵⁴ Serum–semen paired samples were analyzed for PFAS profiles, with PFBA, PFPeA, and PFHxA not being detected in any samples.

Table 1.

Concentrations (ng/mL) of PFAS in paired serum and semen samples

Pan et al 2019	Median (IQR)		Correlation coefficient
n = 664	Serum (ng/mL)	Semen (ng/mL)	Pearson’s correlation
PFOA	8.57 (6.80, 11.04)	0.23 (0.15, 0.36)	0.70
PFNA	1.47 (1.01, 2.22)	0.02 (0.01, 0.04)	0.72
PFDA	1.24 (0.71, 2.03)	0.02 (0.01, 0.03)	0.74
PFUuDA	0.75 (0.45, 1.20)	0.02 (0.01, 0.03)	0.79
PFOS	8.38 (5.57, 13.09)	0.10 (0.06, 0.18)	0.80
6:2 Cl_PFESA	6.09 (3.36, 9.90)	0.06 (0.04, 0.12)	0.83
Cui et al 2020	Median (IQR)		Correlation coefficient
n = 651	Serum (ng/mL)	Semen (ng/mL)	Spearman’s rank correlation
PFOA	8.57 (6.83, 11.11)	0.23 (0.15, 0.37)	0.65
PFNA	1.47 (1.03, 2.23)	0.02 (0.01, 0.04)	0.72
PFOS	9.94 (6.65, 15.65)	0.15 (0.09, 0.27)	0.79
6:2 Cl_PFESA	6.10 (3.36, 9.90)	0.07 (0.04, 0.12)	0.83

The exposure profiles of periconception couples to PFAS in Shanghai, China, from 2013 to 2015, were comprehensively examined. Among the targeted PFAS, 21 substances were detected; n-PFOA (20.4 ng/mL) and n-PFOS (12.1 ng/mL) were the dominant PFAS in plasma; and emerging PFAS such as 6:2 Cl⁻PFESA (10.5 ng/mL), 6:2 diPAP (0.41 ng/mL), and branched PFOS or PFOA isomers were also commonly detected. PFAS concentrations were generally correlated within couples, but significant gender differences were observed, with men showing higher plasma levels and different isomer profiles than their partners. Household factors like income, floor type, water source, and proximity to farmlands were linked to couple-based PFAS exposure.⁶⁶ Couples with higher incomes and those using bottled water as their primary drinking source generally had lower PFAS levels, while other environmental factors like floor type and nearby farmland also influenced exposure. A study conducted by Toft et al, involving multiple Arctic and European countries, from Greenland, Ukraine, and Poland, included 588 individuals who provided both semen and blood samples for PFC analysis between 2002 and 2004. These studies showed that PFOS and PFNA were detectable in all serum samples.⁵⁷ Analysis of PFC exposure distribution revealed varied levels, with Greenland exhibiting the highest exposure to PFOS, PFHxS, and PFNA. However, median PFOA levels were marginally higher in Poland, while the lowest levels were found in Ukraine. Serum concentrations of PFAS in men differed significantly among regions, with Greenland exhibiting the highest levels. PFOS and PFOA were the most predominant PFAS across all countries, and their serum levels were highly correlated with semen quality.

Despite regulatory restrictions, PFOS and PFOA remain dominant over other PFAS in humans from most regions, reflecting the consequence of long-term and extensive applications, long half-life, as well as their bioaccumulative nature.^{52,57,67–71} Serum analyses in men revealed correlations between PCB congeners and PFAS, such that strong correlations were observed with PFOS, while moderate correlations were observed with PFOA and other PFAS.^57,72,73 Males exhibited higher levels of PFAS compared with females, with factors such as lower household income, reduced water quality, poor diet, and higher BMI leading to increased PFAS exposure. Moreover, households who consumed higher quantities of fish exhibited higher levels of PFAS.^66,71 In a study conducted by Tsai et al, samples collected from 1992 to 2000, age appears to impact PFAS concentrations as the 18- to 30-year-old age group exhibited higher mean serum PFOS concentrations compared with the 12- to 17-year-old group.⁷¹ The authors proposed that this age-related difference may arise from developmental factors, as the younger group was in puberty—a hormonally driven, critical stage that could increase their vulnerability may make them more vulnerable to endocrine-disrupting chemicals like PFAS, potentially affecting reproductive hormone levels differently than in adults. An investigation into occupational exposure found that initial serum concentrations of PFOS, PFHS, and PFOA had a wide range, which decreased after 5 years without exposure.⁷⁴ Overall, these findings highlight the pervasive presence of PFAS in human populations and the various factors influencing exposure levels, emphasizing the need for continued monitoring and regulatory measures.

Mice and Turtles

A study by Maxwell et al, in 2024, investigating PFAS exposure in mice measured PFAS concentrations in the plasma, liver, and testes, detecting PFOS, PFOA, PFNA, PFHxS, and GenX, with all but GenX found in the testes. In plasma, PFHxS shows the highest levels (894.4 ng/mL), followed by PFNA (458.1 ng/mL), and PFOS (334.4 ng/mL). HFPO-DA levels are lower (16.2 ng/mL), and PFOA is present at 109.9 ng/mL. In testes, PFOS and PFNA are significantly lower (110.1 ng/g for PFOS; 107.7 ng/g for PFNA) compared with plasma, with PFOA also lower (12.96 ng/g). PFHxS is lower in testes as well (36.63 and 32.75 ng/g). In the liver, PFNA and PFOS are highest, with 2,716.5 ng/g for PFNA and 1,399.4 ng/g for PFOS, indicating much higher accumulation compared with plasma and testes. HFPO-DA levels in the liver were 4.77 ng/g, while PFOA levels were 382.8 ng/g, and PFHxS was 157.5 ng/g, all notably higher than in plasma and testes.²¹ Similarly, a study from 2024 analyzed serum PFAS in turtles and observed detectable levels in all turtles from both impacted and control sites, with PFOS being the dominant constituent.⁷⁵ Notably, in turtles, sex-specific differences in PFAS profiles were found, with males generally exhibiting higher ΣPFAS concentrations than females, and no significant association was observed between total blood PFAS and body size. These findings suggest that PFAS exposure is widespread across different species and environments, with notable sex-specific differences in PFAS accumulation. The persistence of PFOS as a dominant compound underscores the need for continued scrutiny of its environmental and biological impacts.

Impact of PFAS Exposure on Protein and Gene Expression: Insights from Model Systems

Mammalian Systems

Proteomic analysis in bull sperm revealed 16 differentially expressed proteins between the control group and the group treated with 10 μM PFOS.⁶¹ Exposure to PFOS at 10 μM resulted in changes to sperm proteins associated with spermatogenesis and chromatin condensation. At a concentration of 100 μM, PFOS impacted proteins related to motility and fertility. Researchers identified 299 phosphopeptides from 116 proteins, with 45 showing differential expression between the control and PFOS-exposed groups. PFOS disrupted the phosphorylation of several key proteins (ACRBP, PRKAR2A, RAB2B, SPAG8, TUBB4B, ZPBP, and C2CD6) that play crucial roles in sperm capacitation, the acrosome reaction, sperm–egg interaction, and fertilization. Additionally, PFOS influenced the phosphorylation of other proteins (AQP7, HSBP9, IL4I1, PRKAR1A, and CCT8L2) involved in sperm stress resistance and cryotolerance. Notably, four proteins (PRM1, ACRBP, TSSK1B, and CFAP45) showed altered regulation at both proteomic and phosphoproteomic levels. Flow cytometric analysis verified that PFOS increased protein phosphorylation in sperm and led to reductions in sperm motility, viability, calcium levels, and membrane potential, while increasing mitochondrial ROS in a dose-dependent manner. The affected proteins are predominantly localized in the cytosol, mitochondria, acrosome, and flagellum.

Aquatic Systems

Guppies were exposed for 21 days to ecologically relevant concentrations of PFOA, GenX, or control in a static system without solvents. Reproductive traits such as coloration, sexual behavior, and sperm characteristics were assessed. Experimental tanks were set up to achieve final concentrations of 1 μg/L of PFOA or GenX. Transcriptomic analysis in guppy testes revealed significant differential expression of genes between control and treated fish.⁶⁴ Both GenX and PFOA exposure influenced immune-related genes, apoptotic pathways, and genes involved in inflammation and cholesterol biosynthesis. Shared differentially expressed genes (DEGs) between GenX and PFOA treatments included those associated with gonad differentiation, embryonic development, immune response, and inflammation. GenX uniquely regulated genes related to immune functions, inflammatory processes, antioxidant activity, and genes possibly involved in fish reproduction and sperm quality. PFOA specifically activated genes associated with complement pathways, lipid metabolism, calcium response, cellular defense against reactive oxygen species, and estrogenic modulation.

Male largemouth bass of reproductive age were collected over 2 days in September 2009 from three impacted lakes and two reference lakes with low PFAS concentrations. The sites were chosen based on PFAS analyses conducted by the Minnesota Pollution Control Agency (MPCA) in 2007 and 2008.⁷⁶ Largemouth bass from reference and impacted lakes exhibited distinct patterns, indicating potential geochemical similarities among lakes within each group. Expression analyses of the liver and testis tissue revealed numerous altered genes from the bass of impacted lakes compared with reference lakes, suggesting that exposure to higher contaminant levels alters gene expression. Notably, affected biological processes differed between reference and impacted lakes, with upregulation of xenobiotic metabolism in impacted lakes’ livers and downregulation of apoptotic signaling pathways in their testes. The study also identified detoxification biomarkers associated with PFOS exposure, indicating altered expression of genes involved in drug metabolism and kidney excretion, further highlighting the impact of environmental contaminants on fish health.

These studies collectively illustrate the widespread effects of PFAS on various species and their biological functions in sperm, testes, and the liver. These findings highlight the broad ecological and physiological impacts of PFAS contamination, emphasizing the need for continued research on their environmental and health effects.

PFAS Impact on Hormones

Humans

PFAS have drawn significant attention for their potential impact on human health, particularly in relation to male reproductive hormones. As persistent environmental contaminants, PFAS may disrupt endocrine function, causing alterations in hormone levels that are essential for maintaining reproductive health. To explore this further, one study examined 902 male partners from the preconception cohort of the Shanghai Birth Cohort (SBC) between 2013 and 2015, offering valuable insights into the connection between PFAS exposure and male hormone regulation.⁷⁷ The study quantified various perfluoroalkyl substances (PFAS) in plasma. This included nine carboxylic acids (BA, PFHpA, PFNA, PFDA, PFuDA, PFDoA, PFTrDA, PFTeDA, and nPFOA), four linear perfluoroalkyl sulfonic acids (PFSAs) (PFBS, PFHxS, PFHpS, and n-PFOS), seven branched PFOS isomers (1m-PFOS, 4,4m2PFOS, 4,5m2-PFOS, 3m-PFOS, 4m-PFOS, 5m-PFOS, and 6m (iso)PFOS), two branched PFOA isomers (5m-PFOA and iso-PFOA), and two chlorinated alternatives to PFOS (6:2Cl-PFESA and 8:2Cl-PFESA, also known as F-53B). Associations with reproductive hormones were assessed using multiple linear regression and adjusted elastic net regression, revealing significant negative associations between E2 and several PFAS, particularly branched PFOS and long-chain PFAS, even after correcting for multiple comparisons.⁷⁷ Additionally, Bayesian kernel machine regression demonstrated PFAS mixtures were inversely associated with E2 and E2/TT levels, primarily driven by PFuDA, while PFOA exposure was negatively associated with male TT levels. Conflicting results for PFOS exposure on E2 and TT levels were found across multiple studies, but a meta-analysis revealed a significant negative association between PFOS exposure and male TT levels. In contrast, PFNA showed no significant associations with reproductive hormones, whereas PFHxS was positively associated with female E2 levels. Furthermore, serum FSH levels were significantly decreased in association with different percentile categories of PFOS in males aged 12 to 17 years, with similar trends for female FSH levels across different percentile categories of PFUA. Notably, no associations were found between PFAS concentrations and estrogen, luteinizing hormone (LH), or free testosterone levels.⁷⁸ Finally, marginal correlations were observed between plasma PFOS and T3, and plasma PFOA with free testosterone and LH.

In a study conducted by Cui et al, 651 male partners from couples who visited the Reproductive Medical Center at Nanjing Jinling Hospital in Nanjing, China, were recruited. Each participant provided both a venous blood sample and a semen sample simultaneously. The unprocessed seminal fluids and serum samples were directly analyzed to measure the levels of 16 target PFASs, including two emerging PFASs (6:2 and 8:2 Cl⁻PFESAs) and 14 legacy PFASs. Linear regression models revealed significant negative trends between all PFAS and total T levels.⁶⁵ Negative correlations were also found with free T and E2 levels, total T/LH ratios, and SHBG levels. In semen, significant negative relationships were identified between PFAS concentrations and total T levels, as well as inverse associations between PFOA concentrations and free T. Significant negative correlations were observed between PFOA and 6:2 Cl⁻PFESA concentrations and E2 levels, and between all studied PFAS and total T/LH ratio.

A meta-analysis of 2,853 studies, narrowed down to 12 cross-sectional studies involving 7,506 subjects, from the United States (2011–2016), Denmark (2009–2023), and China (2016–2023), examined PFAS exposure and male sex hormone levels. The meta-analyses found that, in serum, PFOA exposure was negatively correlated with testosterone levels, while no significant associations were found with other sex hormones.⁷⁹ These findings highlight the potential endocrine-disrupting effects of PFOA on adverse male reproductive health and emphasize the importance of studying these associations, given PFAS’s widespread presence in the environment and its potential to alter hormone levels that play crucial roles in male development and fertility. Serum PFOS concentrations showed no correlation, and PFNA exposure was negatively associated with testosterone levels, while PFHxS exhibited no significant correlation with testosterone or estradiol levels. Regional studies revealed inconsistent associations between PFAS and reproductive hormones, suggesting region-specific effects of PFAS exposure on sperm quality and reproductive health. Trends of higher FSH and LH levels with higher PFOA exposure were observed, with inverse associations between PFOA and total testosterone, PFOS and estradiol, and PFNA and IGF-1 in boys.⁸⁰ Age-specific positive associations were seen between PFOS and PFNA and thyroid-stimulating hormone (TSH) in males aged 12 to <20 years, and PFOA, PFOS, and PFNA were positively related to free thyroxine (FT4) in females aged 20 to <40 years.⁷³

PFAS exposure is generally linked to negative effects on reproductive hormone levels, particularly testosterone and estradiol. Compounds like PFOA and PFOS are associated with lower testosterone in males, though the results for other PFAS, such as PFNA, vary. PFOS has shown inconsistent effects on hormones, while PFHxS is positively associated with female estradiol levels. In countries like the United States and Denmark, PFOS exposure is linked to lower testosterone, but no significant association is found in China, possibly due to differences in PFAS regulations and exposure levels. Overall, PFAS exposure may disrupt endocrine function and reproductive health across populations.

Turtles

In a study published in 2024, researchers explored the impact of PFAS on Eastern short-necked turtles in Queensland, Australia.⁷⁵ Blood samples were taken to measure PFAS levels, hormones, and functional omics markers. Turtles from the affected area showed elevated PFAS levels, with PFOS being the most prevalent. Males had higher PFAS concentrations in serum than females, and hormone levels also varied between the sexes. Significantly lower progesterone levels were observed in turtles without eggs compared with those with eggs, suggesting possible reproductive disruption to PFAS exposure. Elevated testosterone levels were noted in male turtles from impacted sites, potentially indicating delayed mating or prolonged spermatogenesis. Additionally, stress hormone (CORT) levels were higher in males compared with females, with a potential positive relationship between blood PFAS and CORT in males. Overall, these findings suggest that environmental impacts, particularly related to PFAS exposure, may affect both reproductive and stress responses in turtles.

PFAS Impact on Epigenetics

Spermatogenesis, the complex process of sperm development within the seminiferous tubules of the testes, involves distinct stages including mitosis of spermatogonial stem cells, meiosis of primary and secondary spermatocytes, and the transformation of round spermatids into mature spermatozoa through spermiogenesis, which includes chromatin condensation, acrosome formation, and flagellum development. Mature spermatozoa are released into the seminiferous tubule lumen during spermiation and further mature in the epididymis. Specific stages in spermatogenesis are identified as “windows of susceptibility,” whereby disruptions from environmental exposures or endocrine disruptors may impact germ cell quality and reproductive health through epigenetic mechanisms.^81–83 During prenatal development and crucial for germ cell establishment, epigenetic alterations can affect later reproductive disorders. Puberty is another critical window, where hormonal changes drive spermatogenesis and disruptions can impact fertility through epigenetic modifications.⁸⁴ In adulthood, factors such as toxins, radiation, heat stress, medications, and lifestyle choices affect spermatogenesis and sperm quality through epigenetic mechanisms. Aging also plays a significant role, with advanced paternal age linked to increased genetic mutations in sperm, and may negatively affect fertility and offspring health.⁸⁵ Understanding these windows of susceptibility and their interaction with epigenetic factors is vital for grasping the comprehensive impact of environmental, hormonal, and lifestyle influences on male reproductive health across different life stages.

Methylation in Humans

In a study conducted by Petroff et al, in 2023, PFHxS, PFOS, PFOA, and PFNA were highly detectable, while PFDA was detected in 60% of cases in serum. Analysis of first-trimester maternal blood analyzing methylation at 744,926 CpG sites via EPIC array revealed significant site-specific methylation differences for each PFAS with two CpGsites (cg15429214 and cg20360148) that were significant across different PFAS.⁸⁶ Sex-specific differences showed significant associations for all PFAS, with PFHxS, PFOS, PFNA, PFDA, and PFUnDA affecting methylation in various CpG sites. Interestingly, the comparison of 5-mC and 5-hmC revealed that PFAS generally showed more associations with 5-hmC than with 5-mC. In males, AGPAT1, RNF5, and RNF5P1, located on chromosome 6, were highly associated with PFHxS, highlighting a region sensitive to methylation changes potentially linked to PFAS exposure. KEGG pathway analysis indicated neuroendocrine system pathways were commonly affected. Regarding birth outcomes, PFHxS, PFNA, and PFUnDA exhibited associations with decreased gestational age and Fenton z-scores, with some mediation effects observed between PFAS exposure and birth outcomes through DNA methylation changes. Overall, the study identified significant correlations between PFAS and both DNA methylation and birth outcomes with varying effects across different PFAS and methylation and hydroxymethylation of CpGs.

Overall, these limited studies indicate that PFAS exposure may impact DNA methylation and mediate adverse birth outcomes, thus highlighting the potentially complex interplay between PFAS exposure, epigenetics, and health effects.

Mammalian Systems

Our previous work is the first to examine sperm methylation using reduced representation bisulfite sequencing (RRBS) in PFAS-exposed mice. Adult C57BL/6 male mice between 3 and 5 months old were exposed to a mixture of 5 PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX) at a concentration of 20 μg/L for 18 weeks. RRBS analysis revealed 2,861 sperm differentially methylated regions (DMRs).²¹ These DMRs were predominantly hypermethylated and enriched in genic regions and CpG islands. Functional enrichment analysis of these DMRs highlighted terms related to neuron projection development and cell morphogenesis, indicating potential impacts on reproductive health and developmental processes. Moreover, with the introduction of a new methylation array for mice, we assessed its efficacy in examining the impact of PFAS mixture exposure on the sperm methylome and compared results with those from RRBS. From over 285,000 CpGs analyzed, we identified 12,772 CpG clusters and observed 83 DMRs in sperm with PFAS exposure, all showing significant hypermethylation except for one hypomethylated DMR. Enrichment analysis revealed that PFAS-induced sperm DMRs were less common in open sea regions but more prevalent in CpG islands, shores, and shelves. Functional enrichment highlighted terms related to organophosphorus response, phospholipid metabolism, and lipid modification. Concordance analysis between RRBS and the array showed only four overlapping DMRs at stringent thresholds, but increasing sensitivity revealed 12 overlapping DMRs, all showing consistent directional methylation changes with only moderate correlation in methylation values (r = 0.48).

Overall, these findings underscore the complex interactions between PFAS exposure and DNA methylation patterns in both humans and mice, revealing potential implications for reproductive outcomes and neurodevelopmental processes. Given the limited research in this area, further investigation is crucial to fully understand these effects and their broader impact.

Conclusion

The comprehensive review of PFAS exposure across various species reveals significant and multifaceted impacts on male reproductive and developmental health. In humans, PFAS exposure, particularly to compounds like PFOS and PFOA, is associated with altered serum and sperm PFAS concentrations, disrupted hormone levels, and inconsistent effects on semen quality. Animal studies further illustrate the pervasive effects of PFAS with evidence of reduced sperm motility and viability in mice, and reproductive toxicity in guppies and zebrafish. Epigenetic studies underscore the importance of epigenetic changes with PFAS exposure linked to distinct alterations in methylation patterns in both human and mouse sperm. The evidence collectively highlights a concerning relationship between PFAS exposure and adverse reproductive outcomes, including potential epigenetic modifications and developmental impacts, underscoring the urgent need for continued research in exploring how PFAS is altering the epigenome.