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
Conclusion
The amniotic sac embryoid models suggested that OPFRs disrupted amniogenesis likely by inhibiting the ITGβ1<math><mrow><mtext>ITG</mtext><mi>β</mi><mn>1</mn></mrow></math> pathway, thus providing direct in vitro evidence associating OPFRs with biochemical miscarriage. https://doi.org/10.1289/EHP11958.
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 <math><mrow><mi>β</mi><mn>1</mn></mrow></math> (ITGβ1<math><mrow><mtext>ITG</mtext><mi>β</mi><mn>1</mn></mrow></math>).