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

Background: 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.

Objectives: 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).

Methods: 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.

Results: 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 $\beta 1$ ( $\text{ITG}\beta 1$).

Conclusion: The amniotic sac embryoid models suggested that OPFRs disrupted amniogenesis likely by inhibiting the $\text{ITG}\beta 1$ pathway, thus providing direct in vitro evidence associating OPFRs with biochemical miscarriage. https://doi.org/10.1289/EHP11958.