Per- and polyfluoroalkyl substances (PFASs) represent a highly ubiquitous group of synthetic chemicals used in products ranging from water and oil repellents and lubricants to firefighting foam. These substances can enter and accumulate in multiple tissue matrices in up to 100% of people assessed. Though animal models strongly identify these compounds as male reproductive toxicants, with exposed rodents experiencing declines in sperm count, alterations in hormones, and DNA damage in spermatids, among other adverse outcomes, human studies report conflicting conclusions as to the reproductive toxicity of these chemicals. Using an innovative, human stem-cell-based model of spermatogenesis, we assessed the effects of the PFASs perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and a mixture of PFOS, PFOA, and PFNA for their impacts on human spermatogenesis in vitro under conditions relevant to the general and occupationally exposed populations. Here, we show that PFOS, PFOA, PFNA, and a mixture of PFOS, PFOA, and PFNA do not decrease in vitro germ cell viability, consistent with reports from human studies. These compounds do not affect mitochondrial membrane potential or increase reactive oxygen species generation, and they do not decrease cell viability of spermatogonia, primary spermatocytes, secondary spermatocytes, or spermatids in vitro under the conditions examined. However, exposure to PFOS, PFOA, and PFNA reduces expression of markers for spermatogonia and primary spermatocytes. While not having direct effects on germ cell viability, these effects suggest the potential for long-term impacts on male fertility through the exhaustion of the spermatogonial stem cell pool and abnormalities in primary spermatocytes. Abbreviations: CDC: Centers for Disease Control; DMSO: dimethyl sulfoxide; GHR: growth hormone receptor; hESCs: human embryonic stem cells; PFASs: per- and polyfluoroalkyl substances; PFCs: perfluorinated compounds; PFNA: perfluorononanoic acid; PFOS: perfluorooctanesulfonic acid; PFOA: perfluorooctanoic acid; PLZF: promyelocytic leukemia zinc finger; ROS: reactive oxygen species; HILI: RNA-mediated gene silencing 2; SSC: spermatogonial stem cell

In 1973, the Velsicol Chemical Company, which manufactured FireMaster, a brominated flame retardant, and NutriMaster, a nutritional supplement, mistakenly shipped hundreds of pounds of FireMaster to grain mills around Michigan where it was incorporated into animal feed and then into the food chain across the state. An estimated 6.5 million Michigan residents consumed polybrominated biphenyl (PBB)-laced animal products leading to one of the largest agricultural accidents in U.S. history. To date, there have been no studies investigating the effects of PBB on epigenetic regulation in sperm, which could explain some of the endocrine-related health effects observed among children of PBB-exposed parents. Fusing epidemiological approaches with a novel in vitro model of human spermatogenesis, we demonstrate that exposure to PBB153, the primary component of FireMaster, alters the epigenome in human spermatogenic cells. Using our novel stem cell-based spermatogenesis model, we show that PBB153 exposure decreases DNA methylation at regulatory elements controlling imprinted genes. Furthermore, PBB153 affects DNA methylation by reducing de novo DNA methyltransferase activity at increasing PBB153 concentrations as well as reducing maintenance DNA methyltransferase activity at the lowest tested PBB153 concentration. Additionally, PBB153 exposure alters the expression of genes critical to proper human development. Taken together, these results suggest that PBB153 exposure alters the epigenome by disrupting methyltransferase activity leading to defects in imprint establishment causing altered gene expression, which could contribute to health concerns in the children of men exposed to PBB153. While this chemical is toxic to those directly exposed, the results from this study indicate that the epigenetic repercussions may be detrimental to future generations. Above all, this model may be expanded to model a multitude of environmental exposures to elucidate the effect of various chemicals on germline epigenetics and how paternal exposure may impact the health of future generations.

Sperm counts have rapidly declined in Western males over the past four decades. This rapid decline remains largely unexplained, but exposure to environmental toxicants provides one potential explanation for this decline. Flame retardants are highly prevalent and persistent in the environment, but many have not been assessed for their effects on human spermatogenesis. Using a human stem cell-based model of spermatogenesis, we evaluated two major flame retardants, hexabromocyclododecane (HBCDD) and tetrabromobisphenol A (TBBPA), under acute conditions simulating occupational-level exposures. Here we show that HBCDD and TBBPA are human male reproductive toxicants in vitro. Although these toxicants do not specifically affect the survival of haploid spermatids, they affect spermatogonia and primary spermatocytes through mitochondrial membrane potential perturbation and reactive oxygen species generation, ultimately causing apoptosis. Taken together, these results show that HBCDD and TBBPA affect human spermatogenesis in vitro and potentially implicate this highly prevalent class of toxicants in the decline of Western males' sperm counts.