The Effect Of Ethylparaben And Propylparaben Exposure On Mice Endometrial Decidualization During Early Pregnancy

Purpose:Parabens(PBs)are a class of chemical preservatives that can widely inhibit various microorganisms.Among them,the most commonly used are methylparaben,ethylparaben(EtP)and propylparaben(PrP).It has long been considered that PBs are safe,but recent studies have found that they have weak oestrogen(E2)effects.E2 is very important to the female reproductive process.Studies have shown that PBs exposure can impair the fertility of female mice,but the specific mechanism is still unclear.Endometrial decidualization is an important part of the pregnancy process.Damage to decidualization can lead to miscarriage,infertility and other adverse pregnancy outcomes.However,the effect of PBs exposure on endometrial decidualization is currently unclear.Our previous research found that the endometrial decidualization in mice would damage after exposure to methylparaben.Therefore,this article aims to study the effects of EtP and PrP exposure on endometrial decidualization in mice during the early pregnancy.Method:1.Establishment of toxicological model of normal pregnant mice.From the first day of pregnancy(D1),pregnant mice were daily gavaged at doses of 0,400,800 and 1600 mg/kg EtP,or at doses of 0,625,1250 and2500 mg/kg PrP.On D5-D8,serum and uterus were collected.The following studies were performed.(1)Taking pictures,counting the number of implantation embryos,weighing uterine weight of pregnant mice.(2)Endometrial tissue morphology was observed by H&E staining.(3)Western blot and immunohistochemistry were used to detect the expression of Homeobox A10(HOXA10),Matrix metalloproteinase 9(MMP9)and Bone morphogenetic protein 2(BMP2)on D7 and D8 in endometrial tissue.(4)Serum E2 and progesterone(P4)levels were detected by ELISA.(5)The expression of receptive marker leukemia inhibitory factor(Lif),mucin 1(Muc1)and epithelial cadherin(E-cad)on D5,and the expression of oestrogen receptor(ER)and progesterone receptor(PR)on D7 in endometrial tissue were detected by Real-time PCR.2.Establishment of artificial induced decidualization model.Mice were daily gavaged with 0 and 1600 mg/kg EtP,or with 0 and 2500 mg/kg PrP from the first day of pseudo-pregnancy(PD1)until PD8.On the morning of PD4,25 ul corn oil was injected into one side of the uterus,which was the deciduation-induced side(stimulated),and the other side of the uterus was not injected as the control side(unstimulated).The uterus was gathered on the morning of PD8 and the following studies were performed.(1)Taking pictures and weighing the weight of the uterus;(2)H&E staining was used to observe the morphology of artificially stimulated uterine tissue.(3)Western blot was used to detect the expression of HOXA10 and MMP9 in stimulated uterine tissue.(4)Real-time PCR was used to detect the expression of decidual/trophoblast PRL-related protein(dtprp)in stimulated uterine tissue.3.Observation of pregnancy outcome.Mice were daily gavaged with 0 and1600 mg/kg EtP,or with 0 and 2500 mg/kg PrP from D1 to D9.After mice parturition,the weight and number of newborn rats were recorded.Results:1.The effect of exposure to EtP on endometrial decidualization in early pregnancy mice.(1)Embryo implantation of mice was impaired after exposure to EtP in early pregnancy.On D7 and D8,the number of implantation sites in all control mice was greater than 7.However,after exposure to 1600 mg/kg EtP,about 36% of the mice at D7 had less than 7 implantation sites,and about 33% of the mice at D8 had less than 7 implantation sites.(2)There was no significant change in endometrial receptivity after mice exposed to 1600 mg/kg EtP in early pregnancy.(3)After mice exposed to 1600 mg/kg EtP in early pregnancy,the expression of BMP2,MMP9 and HOXA10 was significantly decreased on D7 and D8.The expressions of dtprp,MMP9 and HOXA10 were significantly down-regulated in stimulated uterine tissue.(4)After mice exposed to 1600 mg/kg EtP in early pregnancy,the serum E2 and P4 levels were increased at D7,but the expression of endometrial ER and PR decreased significantly.(5)After mice exposed to 1600 mg/kg EtP in early pregnancy,the number of offspring was significantly reduced.2.The effect of exposure to PrP on endometrial decidualization in early pregnancy mice.(1)Embryo implantation was impaired after mice exposed to PrP in early pregnancy.On D7 and D8,the number of embryo implantation sites in control mice was greater than 7.However,after exposure to 2500 mg/kg PrP,36% of the mice at D7 had less than 7 implantation sites,and 25% of the mice at D8 had less than 7 implantation sites.(2)There was no significant change in endometrial receptivity after mice exposed to 2500 mg/kg PrP in early pregnancy.(3)After mice exposed to 2500 mg/kg PrP in early pregnancy,the expression of BMP2,MMP9 and HOXA10 in uterus were significantly decreased on D7 and D8.The expressions of dtprp,MMP9 and HOXA10 were significantly down-regulated in stimulated uterus tissues after exposure to 2500 mg/kg PrP.(4)After mice exposed to 2500 mg/kg PrP in early pregnancy,the serum E2 and P4 levels in mice were increased at D7,but the expression of endometrial ER and PR decreased significantly at D7.(5)After mice exposed to 2500 mg/kg PrP in early pregnancy,the number of offspring was significantly reduced.Conclusion:After mice exposed to EtP and PrP in early pregnancy,endometrial decidualization was damaged and embryo implantation was impaired,but the specific mechanism remains to be further studied.