Preliminary Study On The Effect Of Butylated Hydroxytoluene Exposure On The Establishment Of Endometrial Decidualization In Mice During Early Pregnancy

Objective: Butylated hydroxytoluene(BHT)is a synthetic phenolic antioxidant(SPA),which is widely used as food additive with strong antioxidant function.Studies have found that BHT can cause damage to various of tissues and organs,and BHT also played cancer-promoter role in the presence of other carcinogens.However,the effects of BHT on embryo implantation and endometrial decidualization in the establishment of pregnancy was still unclear.The pregnancy process in mammals involved multiple links such as embryo implantation,endometrial decidualization and placenta formation,which was an extremely meticulous and complex process.After the embryo implanted endometrium,the endometrial stromal cells located around the embryo began to undergo proliferation and differentiation,which was called decidualization.If the process of established decidualization was impaired,the embryo implantation and subsequent pregnancy process would not be completed normally.Therefore,this study aims to research the effects of BHT exposure in early pregnancy on establishment of decidualization of the mouse endometrium.Methods:1.BHT pregnant mouse model: From the day 1(D1)of pregnancy,the BHT group was given respectively the doses of 200mg/kg/d,400mg/kg/d.The control group was given olive oil by intraperitoneal injection.The mice were killed on D5 morning 9:00,D5 afternoon 18:00,D6 morning 9:00 and D6 afternoon 18:00 by cervical dislocation,and the uteri tissue were immediately stored at-80 ℃.Perform the following experiments:(1)Weighing the weight of pregnant mice;(2)Counting the number of embryo implantation and abnormal embryo spacing;weighing the wet weight of the uterus;observing the morphology of endometrial tissue by H&E staining;(3)Western-blot and Immunohistochemistry(IHC)were used to detect the expression of decidualization markers;(4)Enzyme-linked immunosorbent assay(ELISA)was applied to test serum estrogen(E2)and progesterone(P4)levels;(5)IHC and RT-PCR were used to detect the expression of estrogen receptor α(ERα)and progesterone receptor(PR);2.BHT cell model: Isolating mouse stromal cells,10 n M E2 and1μM P4 were used to induce decidualization of primary endometrial stromal cells.On the basis of inducing decidualization,different concentrations of BHT was used to establish BHT cell models.Perform the following experiments:(1)Immunofluorescence(IF)was used to detect F-actin to observe the morphology of decidual stromal cell;(2)RT-PCR was used to detect the expression of ERα and PR in decidual cells;(3)IF was used to detect the expression of proliferation marker Ki-67 and apoptosis marker Cleaved-caspase3;Results:(1)On D5 morning 9:00 and D5 afternoon 18:00,the maternal body weight was affected by BHT.(2)BHT significantly decreased the number of implantation sites and caused aberrant embryos spacing.(3)BHT exposure in early pregnancy leads to impaired endometrial decidualization in mice.(4)From D5 afternoon18:00 to on D6 afternoon 18:00,BHT caused an increase of E2 and P4 levels in serum,and significantly up-regulated the expression of ERα and PR in the decidua of uterus.(5)BHT exposure damaged decidual morphology of stromal cell,significantly down-regulated of expression of Ki-67 and up-regulated of expression of Cleaved-caspase3.Conclusion:BHT exposure leads to a decrease in embryo implantation rate and caused aberrant embryo spacing.In addition,it caused an up-regulation of estrogen and progesterone in the serum of early pregnancy mice,an increase in the expression of estrogen and progesterone receptors in endometrial tissue.And the establishment of decidualization was impaired.The above results indicate that BHT may be a potential reproductive toxicant.The molecular mechanism needs further study.