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. 2022 May 3;130(5):057002. doi: 10.1289/EHP10273

Figure 1.

Figure 1 is a flowchart having three parts, namely, Processes, Indicators and methods, and Aims. There are six steps. Step 1: Process: Model validation; Indicators and methods: Morphologic observation keratin 7, G A T A 3 (Immunofluorescence) human chorionic gonadotropin, and estradiol (Elisa); and Aims: Validating the previous and optimized trophoblast organoid models. Step 2: Process: High-throughput screening of 46 organophosphate flame retardants (10000 nanomolar); Indicators and methods: Antigen K I-67 (Immunofluorescence) and Sytox Green; and Aims: Organoids proliferation and Cell survival. Step 3: Process: Short-term exposure (100, 1000, 10000 nanomolar 2-ethylhexyl-diphenyl phosphate); Indicators and methods: Antigen K I-67, tumor protein 63, transferrin receptor, human leukocyte antigen protein-G (Immunofluorescence), Aerobic respiratory (Mitochondrial stress assay), pyruvate dehydrogenase complex (western blotting), Antigen K I-67 of organoids from 5 donors (1000 nanomolar 2-ethylhexyl-diphenyl phosphate, Immunofluorescence); and Aims: Organoids proliferation, villous cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell, Aerobic respiratory levels, tricarboxylic acid cycle, and Individual differences. Step 4: Process: Long-term exposure (100, 1000, 10000 nanomolar 2-ethylhexyl-diphenyl phosphate); Indicators and methods: Antigen K I-67, tumor protein 63, transferrin receptor, human leukocyte antigen protein-G (Immunofluorescence), human chorionic gonadotropin, and estradiol; and Aims: Organoids proliferation, villous cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell, and Secretion functions of organoids. Step 5: Process: Mechanism (100, 1000, 10000 nanomolar 2-ethylhexyl-diphenyl phosphate without special instructions); Indicators and methods: ribonucleic acid sequence, phosphorylated-insulin growth factor 1 receptor, insulin-like growth factor 1 receptor, phosphorylated-protein kinase B, protein kinase B (western blotting), insulin-like growth factor 1, insulin-like growth factor 2 (Reverse transcription-quantitative polymerase chain reaction), Directly binding effect (1000 and 10000 nanomolar 2-ethylhexyl-diphenyl phosphate, Immunoprecipitation), Inhibition of tyrosine kinases activity in vitro (1, 10, 100, 1000, 10000 nanomolar 2-ethylhexyl-diphenyl phosphate and 0.001, 0.01, 0.1, 1, 10, 100 nanomolar linsitinib), Antigen K I-67, tumor protein 63, transferrin receptor, human leukocyte antigen protein-G (50 nanomolar linsitinib, Immunofluorescence); and Aims: Mechanism study, insulin growth factor 1 receptor- protein kinase B signaling activity, insulin growth factor 1 and insulin growth factor 2 expression, 2-ethylhexyl-diphenyl phosphate directly binds to insulin growth factor 1 receptor and inhibit its function in vitro, Organoids proliferation, villous cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell in positive insulin growth factor 1 receptor inhibitor exposure. Step 6: Animal study (0.4, 2, 10 milligrams per kilogram per day 2-ethylhexyl-diphenyl phosphate without special instructions); Indicators and methods: Caudal-type homeobox 2, Antigen K I-67, (10 milligrams per kilogram per day 2-ethylhexyl-diphenyl phosphate, Immunofluorescence), trophoblast-specific protein alpha, transcription factor activating protein 2 gamma, Heart And Neural Crest Derivatives Expressed 1 (10 milligrams per kilogram per day 2-ethylhexyl-diphenyl phosphate, Immunofluorescence), phosphorylated-insulin growth factor 1 receptor, insulin-like growth factor 1 receptor, pyruvate dehydrogenase complex, cytochrome C (western blotting), phosphorylated-insulin growth factor 1 receptor (10 milligrams per kilogram per day 2-ethylhexyl-diphenyl phosphate, Immunofluorescence), ribonucleic acid sequence, Implanted embryos and survival embryos counting, fetal growth restriction analysis, and oral glucose tolerance test; and Aims: Abnormal placentation in immature placenta, Disorder in mature placenta, 2-ethylhexyl-diphenyl phosphate inhibit insulin growth factor 1 receptor and aerobic respiratory in placenta, and Outcomes.

Summary of methods. Note: Akt, protein kinase B; CD71, transferrin receptor; Cyto C, cytochrome C; E2, estradiol; EHDPP, 2-ethylhexyl-diphenyl phosphate; EVT, extravillous trophoblast cell; FGR, fetal growth restriction; GATA3, GATA-binding protein 3; HAND1, crest derivatives-expressed protein 1; hCG, human chorionic gonadotropin; HLA-G, human leukocyte antigen protein-G; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; KRT7, keratin 7; OGTT, oral glucose tolerance test; OPFRs, organophosphate flame retardants; OSI-906, linsitinib; p-Akt, phosphorylated Akt; PD, pyruvate dehydrogenase complex; p-IGF1R, phosphorylated IGF1R; RNA-seq, RNA sequencing; SCT, syncytiotrophoblast; TCA, tricarboxylic acid cycle; TFAP2C, transcription factor AP-2γ; TP63, tumor protein 63; TPBPA, trophoblast-specific protein α; VCT, villous cytotrophoblast.