Autophagy Regulates Abnormal Placentation Induced By Folate Deficiency

Folic acid is a B vitamin which can promote one-carbon unit for the synthesis of DNA and RNA,amino acid metabolism and oxidation.Folate deficiency is a major risk factor for birth defects.The risk rate of neural tube defects,orofacial clefts and congenital heart defects can be reduced by supplying folate before pregnancy and the key time of organ formation.Almost all studies on the impact of folate deficiency on the embryo itself,whereas few people have studied the impairment of the maternal reproductive system and its mechanism.Decidualization is a vital process in the formation of the placenta.Several studies have shown that folate deficiency may result in decreased cell viability and an invasive ability of the HTR-8/SVneo extravillous trophoblast cell line,in addition to inhibiting DNA methylation in mice placentas.Those results suggest that placentation can be damaged by folate deficiency,but the detail mechanism has not been known.Studies have shown that autophagy can be induced by folate deficiency in brain cells,which indicates autophagy involves metabolism of folate.Autophagy is an inducible metabolic reaction for maintaining cellar homeostasis by degrading macromolecules and organelles that are damaged in response to nutrition deprivation and other stimuli.Recent evidences have shown that placental development and gestational diseases,such as preeclampsia and spontaneous abortion,are related to autophagy.Given that autophagy is strongly related to placental development and can be influenced by folate deficiency,this study additionally focused on the effects of folate deficiency on placenta to ascertain whether autophagy is involved in the development of placentas under conditions of folate deficiency.1.The Placental morphology was damaged by folate deficiency in mice.To investigate the effects of folate deficiency on pregnant mice,the embryo resorption rates,anatomy of the uterus and placenta on pregnancy day(D),D10,D12,D14 were recorded.The result showed that embryo resorption rates were significantly increased in the folate-deficient diet groups(F10,F12,F14)compared to the normal diet groups(N10,N12,N14)on D12 and D14;The size of the placentas and embryos in the F10,F12 and F14 groups were distinctly smaller than those in the N10,N12 and N14 groups,respectively.The H&E staining indicated trophoblast giant cells were not observed in the F10 and F12 groups.The cellular contours of the labyrinth were less distinct in the F10,F12 and F14 groups compared to those in the N10,N12 and N14 groups,respectively.In addition,the spongy layer and labyrinth zone were significantly thinner,and the spongy zone was separated to some degree from the decidua basalis in the F12 group.These results indicated that the placental morphological structure was impaired by folate deficiency in mice.2.The endocrine function of the placenta could be influenced by folate deficiency in mice.To explore whether the endocrine function of the placenta was affected by folate deficiency,hormones secreted by the placenta were measured using ELISA.Serum Plgf,Pprh and CG levels were significantly lower in the F10 and F12 groups than those in the N10 and N12 groups,respectively,while Plgf and CG levels were significantly higher in the F14 group compared to the N14 group.P4 was evidently higher in the F10 and F12 groups than those in the N10 and N12 groups,respectively.These findings suggest that the endocrine function of the placenta could be influenced by folate deficiency in mice.3.The development of placenta can be impaired by folate deficiency in mice,human placenta explants and HTR8/SVneo cells.The IHC and Western blot showed that markers of endothelium vasculature,Plgf and CD34 exhibited evidently lower expression in the F10 and F12 groups than in the N10 and N12 groups,respectively.On D14,the expression of Plgf and CD34 was significantly increased in the F14 group.The RT-PCR showed Gcm1,Hand1 and Mash2 were prominently reduced under folate deficiency in mice.To confirm whether human placental explant was also damaged by folate deficiency.The human placental explants were cultured in folate-deficient medium.According to the result of IHC and Western blot,Ki67,GCM1,PCNA and PLGF exhibited obviously decreased expression in human placental explants cultured in folate-deficient medium,Caspase-3 and Cleaved caspase-3 were significantly increased after the human placenta cultured in folate-deficient medium.To further investigate the effect of folate deficiency on HTR8/SVneo cells,the cells were cultured in folate-deficient medium.Transwell and flow cytometry analyses showed that the invasion ability and apoptosis of cells were significantly suppressed by folate deficiency.The results indicated that placental development can be suppressed by folate deficiency.4.The aberrant autophagy in mice and enhanced autophagy in human placenta explants and HTR8/SVneo cells were caused by folate deficiency.To explore whether placental autophagy could be influenced by folate deficiency in mice,the number of autophagosomes determined using TEM was significantly increased in the F10 group,while autophagosomes were markedly reduced in the F12 and F14 groups.the expression of LC3,Atg5 and P62 were dramatically increased in F10,while they prominently declined in the F12 and F14 groups compared to that in the N12 and N14 groups,respectively measured by IF,Western blot and RT-PCR.The autophagosomes in HTR8/SVneo cells were distinctly accumulated in the F group.Dual-fluorescence mRFP-eGFP-LC3 analysis showed autophagy in HTR8/SVneo cells was increased in the F group.And the expression of LC3,ATG5 and P62 in in HTR8/SVneo cells and human placenta explants was obviously stronger in the F group than in the N group by Western blot.These results suggest that placental autophagy may be affected by folate deficiency.5.The impaired placentation caused by folate deficiency was regulated by autophagy.To verify that autophagy could participate in the regulation of folate deficiency-induced damage in placentas,3-MA was used to reduce the autophagy of placentas in mice,human placenta explants and HTR8/SVneo cells.The resorption rate,colour of the uterus and placental size was rescued partially in in the F10 3MA group.And the expression of Gcm1,Plgf,CD34,Hand1 and Mash2 were recovered in the F10 3MA group.the expression of Caspase-3,Cleaved caspase-3 were significantly decreased and PCNA,GCM1,PLGF and Ki67 were distinctly increased in human placenta explants in F-3MA group.The invasion ability and apoptosis of HTR8/SVneo cells were significantly increased in the F-3MA group compared to that in F group.In conclusion,our study further explored the effect of folic acid deficiency on placental development on the basis of the previous study.The animal model,human placenta explant and HTR8/SVneo cell were used in our study.According to the results,placental hyperemia and embolism,abnormal placental morphological structure,endocrine dysfunction and impaired placentation were induced by folate deficiency during pregnancy.In addition,autophagy was involved in the abnormal placental development.Our study provides a new experimental evidence for fully understanding the mechanism of fetal birth defects caused by folate deficiency.