FRI375 Validation Of A Novel Mouse Placenta Explant Protocol For Screening Of Drugs Modulating Circadian Rhythms

Abstract

Abstract Disclosure: J.S. Lee: None. H.M. Hoffmann: None. Introduction Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia. Methods We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&E we validated the explants to be >80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells. Results Through H&E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for <6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ∼4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects. Conclusion We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta. Presentation: Friday, June 16, 2023