Supplementation With High-dose Cholecalciferol Throughout Pregnancy Induces Fetal Growth Restriction Through Inhibiting Placental Proliferation And Trophoblast Epithelial-Mesenchymal Transition

Backgrounds and ObjectivesVitamin D deficiency has been associated with adverse pregnant outcomes.Several studies investigated the effects of maternal vitamin D3 supplementation on fetal development with inconsistent results.The aim of this study was to investigate the effects of maternal supplementation with different doses of vitamin D3 on fetal development.MethodsPregnant mice were administered with different doses of cholecalciferol(0,2,000,10,000,40,000 IU/kg/day)by gavage throughout pregnancy.Fetal weight and crown-rump length were measured.Placental proliferation and mesenchymal characteristics were detected.Experiment three,HTR-8/SVneo cells were incubated in absence or presence of calcitriol(500 nmol/L)to evaluate the effects of active vitamin D3 on migration and invasion of human trophoblast cells.This study consists of three independent experiments.Experiment one,to investigate the effects of supplementation with cholecalciferol on placental and fetal development,pregnant mice were administered with different doses of cholecalciferol(0,2,000,10,000,40,000 IU/kg/day)by gavage throughout pregnancy.Pregnant mice were sacrificed on GD18.The number of live fetuses,dead fetuses,resorbed fetuses and implantation sites were recorded.And for live fetuses,placentas and fetuses were weighted.Simultaneously,crown-rump length and placental diameter were measured.Placentas were fixed in 4% paraformaldehyde for histopathology.Experiment two,to investigate the effects of high-dose cholecalciferol on placental proliferation and EMT,pregnant mice were treated with cholecalciferol(0,40,000 IU/kg/day)by gavage from GD0 to GD8.Half pregnant mice(N=6)were sacrificed on GD9.All placentas were separated aseptically for real time RT-PCR and Western Blotting.The remaining pregnant mice(N=6)were sacrificed on GD15.Some placentas were fixed in 4%paraformaldehyde for immunohistochemistry.The remaining placentas were separated aseptically for real time RT-PCR.Experiment three,HTR-8/SVneo cells were incubated in absence or presence of calcitriol(500 nmol/L)to evaluate the effects of active vitamin D3 on migration and invasion of human trophoblast cells.ResultsAlthough a low dose of cholecalciferol was safe,fetal weight was reduced in dams treated with high-dose cholecalciferol throughout pregnancy compared to control group(1.16 ± 0.07 g vs 1.39 ± 0.02 g,P=0.002).Crown-rump length was decreased in high-dose cholecalciferol group compared to control group(20.87 ± 2.24 mm vs 23.35± 0.98 mm,P=0.004).Placental weight was reduced in mice administered with high-dose cholecalciferol compared to control group(0.09 ± 0.01 g vs 0.10 ± 0.02 g,P=0.051).And labyrinth thickness was reduced in mice administered with high-dose cholecalciferol(1.04 ± 0.08 mm vs 1.34 ± 0.077 mm,P=0.006).An obvious calcification was observed in placentas of mice administered with high-dose cholecalciferol.Ki67-positive cells,a marker of placental proliferation,were reduced in mice treated with high-dose cholecalciferol compared to controls(8.25% ± 1.95% vs23.07% ± 2.35%,P=0.004).N-cadherin and vimentin,two mesenchymal markers,were decreased in cholecalciferol-treated mouse placentas and calcitriol-treated human trophoblast cells.MMP-2 and MMP-9,two matrix metalloproteinases,were downregulated in cholecalciferol-treated mouse placentas and calcitriol-treated humantrophoblast cells.In addition,trophoblast migration and invasion were suppressed by calcitriol.ConclusionSupplementation with high-dose cholecalciferol induces fetal growth restriction partially through inhibiting placental proliferation and trophoblast epithelial-mesenchymal transition.