MON-168 Validation Of A Novel Mouse Placenta Explant Protocol For Studying Circadian Rhythms In Early, Mid, And Late Pregnancy

Abstract Disclosure: A.M. Yaw: None. K. Jursch: None. J.S. Lee: None. H.M. Hoffmann: None. Preeclampsia, a hypertensive disorder of pregnancy, remains a leading cause of maternal and fetal mortality. Placental dysfunction is a central factor in the etiology of preeclampsia and is heavily associated with the deregulation of clock genes, a family of genes coding for transcriptional regulators which generate 24h circadian rhythms. Placental cells possess these internal molecular clocks, yet their precise role and function remain largely unexplored. To study layer-specific circadian rhythms in the placental decidua, junctional, and labyrinth, we developed and validated sectioning and explant culture protocols for mouse gestational days (GD) 11, 14, and 18 using circadian Per2::Luciferase reporter mice, which express bioluminescence corresponding to the Per2 clock gene. Dissection techniques were established based on size and fragility of the placenta. Whole placenta explant sections were successful at GD11. Due to the larger placenta size, GD18 explants were sub-dissected into 2x2 millimeter sections to isolate the target placental layer. Both dissection techniques were assessed at GD14, and significantly stronger circadian rhythms were observed without sub-dissection. Tissue level circadian rhythms were consistent among all layers across GD11, 14, and 18. Interestingly, there was a fetal-sex difference in junctional zone explants, where circadian period is longer at all gestational timepoints in placentas from female fetuses compared to males. To confirm the enrichment of placental layers in the whole explant method, we used hematoxylin and eosin staining to validate the amount of each layer present. At GD11 and GD14, the explants were enriched with 85-87% of maternal-derived decidua tissue, identified by the presence of neutrophils. Junctional zone explants contained 75-80% junctional zone tissue, identified by spongiotrophoblasts and glycogen trophoblasts, and the fetal labyrinth represented 90-95% of the labyrinth-enriched explants, identified by the presence of red blood cells. To evaluate the potential for this technique to be used as a method for drug screening, we applied epidermal growth factor (EGF). EGF plays a critical role in placental cell processes including proliferation and differentiation. Importantly, the dysregulation of EGF and its receptor (EGFR) have been linked to endocrine disrupting chemicals and preeclampsia. Preliminary results indicated no significant effect of EGF on the circadian period. This work enhances the understanding of placental circadian rhythms and the potential effects of drugs such as EGF on circadian rhythms. Presentation: Monday, July 14, 2025