Amniogenesis in Human Amniotic Sac Embryoids after Exposures to Organophosphate Flame Retardants

Amniogenesis is a key event in biochemical pregnancy, and its failure may result in human embryonic death. However, whether and how environmental chemicals affect amniogenesis remain largely unknown.

The objective of the present study was to screen chemicals that may disrupt amniogenesis in an amniotic sac embryoid model and to investigate the potential mechanism of amniogenesis failure, with a focus on organophosphate flame retardants (OPFRs).

This study developed a high-throughput toxicity screening assay based on transcriptional activity of octamer-binding transcription factor 4 (Oct4). For the two positive OPFR hits with the strongest inhibitory activity, we used time-lapse and phase-contrast imaging to assess their effects on amniogenesis. Associated pathways were explored by RNA-sequencing and western blotting, and potential binding target protein was identified through a competitive binding experiment.

Eight positive hits exhibiting Oct4 expression were identified, with 2-ethylhexyl-diphenyl phosphate (EHDPP) and isodecyl diphenyl phosphate (IDDPP) showing the strongest inhibitory activity. EHDPP and IDDPP were found to disrupt the rosette-like structure of the amniotic sac or inhibit its development. Functional markers of squamous amniotic ectoderm and inner cell mass were also found disrupted in the EHDPP- and IDDPP-exposed embryoids. Mechanistically, embryoids exposed to each chemical exhibited abnormal accumulation of phosphorylated nonmuscle myosin (p-MLC-II) and were able to bind to integrin $$ ($$).

The amniotic sac embryoid models suggested that OPFRs disrupted amniogenesis likely by inhibiting the $$ pathway, thus providing direct in vitro evidence associating OPFRs with biochemical miscarriage. https://doi.org/10.1289/EHP11958.