AMPK/mTOR Down-regulated Autophagy Enhances Aberrant Endometrial Decidualization In Folate Deficient Pregnant Mice

Adverse Pregnancy Outcome(APO)generally refers to the abnormal results of all perinatal adverse environments,heredity and other factors on the pregnant mother and fetus.Such as miscarriage,stillbirth,fetal growth restriction.Nutrient requirements increase for pregnant women.Vitamins are essential nutrient elements for fetal growth and development.Inadequate or excessive of vitamins in pregnant women will lead to birth defects and other APOs.Folate(vitamin B9)plays an important role in maintaining normal cell functions.Existing studies have shown that folate deficiency in pregnant women lead to neural tube defect of fetal.Supplementing folate for mother can effectively reduce the risk of APOs such as fetal neural tube defects and cleft lip and palate,etc.As we all known,the pregnancy process in mammals involves many stages such as the establishment of endometrial receptivity,embryo implantation,endometrial decidualization,embryonic development and childbirth.Maternal folate level is crucial for the maintenance of pregnancy.For the first time,we pay attention to the effect of folate deficiency on endometrial function during pregnancy.Our early studies showed that uterine receptivity was normal,while decidualization was impaired by folate deficiency;however,the mechanism is still unclear.The aim of this study is to evaluate the adverse effect of folate deficiency on endometrial decidualization during early pregnancy both in animal and cellular experiments.The main research results are listed as follows:1.Construct animal model and cell model of folate deficiency.Serum folate concentration of folate-deficient pregnant mice was significantly lower than that of the control group in vivo.It indicated the successful establishment of a folate-deficient mouse model.And then,the m ESCs were isolated from folate deficient and normal mice respectively and were artificially induced decidualization by hormone in vitro.The culture medium without folate was further used to construct a folate deficiency cell model.2.Endometrial decidualization was impaired by folate deficiency in early pregnant mice and in primary m ESCs.The real-time PCR and western blot results showed that the key factors expressions of decidualization,such as Bmp2,Mmp2,Mmp9,Hoxa10 and PR,were significantly decreased in the folate deficient group,compared to the control group in vivo and in vitro.It suggested that endometrial decidualization was impaired by folate deficiency.3.Autophagy was inhibited by folate deficiency during endometrial decidualization in early pregnant mice and in primary m ESCs.Compared with the control group,the number of endometrial autophagosomes was drastically decreased in folate deficient group in vivo and in vitro.The immunofluorescence,real-time PCR and western blot results showed that the expression levels of autophagic marker LC3 were significantly decreased in folate deficient group in vivo and in vitro.These data indicated that autophagy was significantly down-regulated by folate deficiency.Additionally,we examined proteins expressions of autophagy at different stages using western blot.Our results indicating that folate deficiency not only impaired autophagosome formation and autophagosome-lysosome fusion,but also disturbed lysosomal degradation.4.Aberrant decidualization induced by folate deficiency was reversed by activating autophagy in early pregnant mice and in primary m ESCs.To further address whether folate deficiency-impaired endometrial decidualization was mediated by inhibited autophagy,autophagy was activated by rapamycin and was blocked by 3-MA in vivo and in vitro.The decreased number of embryo implantation sites on D6 by folate deficient,were enhanced by rapamycin and reversed by 3-MA,respectively.Western blot analysis showed that the folate deficient-decreased decidualization related protein expression levels were significantly increased by rapamycin and were further decreased by 3-MA.These indicated that autophagy play a significant role in folate deficiency-impaired endometrial decidualization.5.Endometrial AMPK/mTOR signaling was disrupted in the impaired decidualization by folate deficiency in early pregnant mice and in primary m ESCs.To determine whether AMPK/mTOR signaling pathway is involved in the folate deficiency-mediated regulation of autophagy,the AMPK/mTOR-related protein expression levels were evaluated by western blot in vitro and in vivo.The results showed that AMPK/mTOR was disrupted by folate deficiency.And,rapamycin significantly reversed the inhibitory effect of folate deficiency on the endometrial decidualization-related protein expression levels.Furthermore,the protein interaction between Cathepsin L and Hoxa10 was notably reduced in folate deficiency group.Conclusions:In conclusion,here we explored the role of autophagy in the aberrant endometrial decidualization induced by folate deficiency.It found that AMPK/mTOR down-regulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice,which could result in APOs.This study provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency during early pregnancy.