Systematic Methods to Resolve Lineage-Specific Stress States in Early Mammalian Embryos and That May Enable Miscarriage Prediction

Early mammalian embryos are highly sensitive to environmental, metabolic, hormonal, and genomic stress, yet embryo assessment during <b>I</b>n <b>V</b>itro <b>F</b>ertilization (<b>IVF)</b> relies largely on morphology and ploidy for embryo assessment, but these tests incompletely predict miscarriage. We present a transcriptomics based framework to classify and quantify lineage-specific stress in early embryos by benchmarking human preimplantation embryos against dose-, time-, and quality-dependent stress programs defined in <b>E</b>mbryonic and placental <b>T</b>rophoblast <b>S</b>tem <b>C</b>ells (<b>ESCs, TSCs</b>) from the implanting blastocyst. Human embryos and stressed ESCs and TSCs are screened using transcriptomic markers from eleven biologically distinct stress <b>G</b>ene <b>O</b>ntology (<b>GO</b>) groups that define functional stress states and enable quantification of pathway presence and upregulation, pathway activity, and downstream outcomes. This framework determines whether the <b>I</b>ntegrated <b>S</b>tress <b>R</b>esponse (<b>ISR</b>), once initiated, resolves to enable the <b>D</b>evelopmentally <b>A</b>ssociated <b>S</b>tress <b>R</b>esponse (<b>DASR</b>). <b>H</b>igh-<b>t</b>hroughput <b>s</b>creening (<b>HTS</b>) titrates stress to define increasingly risky yet biologically equivalent doses for levels of diminished stem cell growth across mechanistically diverse stressors. Then bulk RNA seq derives lineage specific transcriptomic markers putatively respond to common levels of diminished growth and that distinguish weak vs. strong stress and resolved vs. unresolved ISR. These stem cell transcriptomic signatures are applied to bulk RNA seq data from IVF embryos graded for morphology or adhesion, enabling quantitative inference of stress burden, lineage vulnerability, and developmental trajectory.