Ubiquitous Flame-Retardant Toxicants Impair Spermatogenesis in a Human Stem Cell Model

Abstract

SUMMARY 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.

Per- and polyfluoroalkyl substances impact human spermatogenesis in a stem-cell-derived model

“…PFOS, PFOA, and PFNA treatment groups were analyzed in comparison to a 0.25% DMSO-only treated negative control for cell viability/apoptosis. This assay has been utilized by our lab in previous studies to assess spermatogenic cell viability status (Easley et al 2015; Steves 2018). As a positive control, cells were treated with a 200 μM hydrogen peroxide (H 2 O 2 ) for a period of six hours to confirm that our system responds to known toxicants (Supplemental Fig.…”

Detrimental effects of flame retardant, PBB153, exposure on sperm and future generations

“…Cell culture. NIH-approved WA01 (H1, WiCell) male, human embryonic stem cells were maintained as described 32,34 . hESCs were pasaged using ReLeSR (STEMCELL Technologies) and plated onto Matrigel (Corning Life Sciences) coated plates as described 32,34 .…”

“…NIH-approved WA01 (H1, WiCell) male, human embryonic stem cells were maintained as described 32,34 . hESCs were pasaged using ReLeSR (STEMCELL Technologies) and plated onto Matrigel (Corning Life Sciences) coated plates as described 32,34 . Differentiation into spermatogenic lineages were conducted as previously described [32][33][34][35] with mouse STO cells for feeder cells or directly onto Matrigel.…”

“…hESCs were pasaged using ReLeSR (STEMCELL Technologies) and plated onto Matrigel (Corning Life Sciences) coated plates as described 32,34 . Differentiation into spermatogenic lineages were conducted as previously described [32][33][34][35] with mouse STO cells for feeder cells or directly onto Matrigel. Medium was gassed every other day with a blood gas mixture (5% carbon dioxide, 5% oxygen, and 90% nitrogen).…”

“…We were then able to simulate male exposure by differentiating WA01 human embryonic stem cells in the presence of 2,2′,4,4′,5,5′-hexabromobiphenyl (PBB153), into spermatogonial stem cells, primary and secondary spermatocytes and, finally, into spermatids. We previously demonstrated that this model mimics major components of human spermatogenesis 32 and have utilized this model to examine impacts of other environmental exposures [33][34][35] . Additionally, our in vitro spermatogenesis model has been independently replicated, highlighting its reproducibility 36 .…”