Effect And Mechanism Of DEHP On The Decidualization Of Uterus

Objectives:Di-(2-ethylhexyl)phthalate(DEHP)is widely used as a plasticizer in polyvinyl chloride(PVC)plastics.DEHP is a lipophile compound that cannot be stably bound to the plastic matrix,so it is easy to be released into the environment with the process of repeated use,heating and cleaning of plastic products,and enters the body through skin,digestion and respiration,and results in the damage to multiple human and animal systems.In recent years,the toxic effects of DEHP on the female reproductive system have attracted widespread attention.Studies have shown that exposure to DEHP during pregnancy can lead to the damage to reproductive system,including the downregulation of endometrial receptivity,low pregnancy rate,high abortion rate and fetal weight loss.However,whether are these adverse pregnancy outcomes closely related to the impaired endometrium decidualization process caused by DEHP exposure during pregnancy? So far,no relevant studies have been found.Therefore,we intend to establish the pregnant animal model and administer different doses of DEHP by gavage to explore the effect and mechanism of DEHP on endometrial decidualization in pregnant mice.Methods:1.Establishment of animal models and treatment:(1)Normal pregnancy model:female mice of the same strain were mated with male mice in a cage,and the detection of vaginal plug was regarded as the first day of gestational day(GD 1).Pregnant mice were randomly divided into 4 groups(n=10 mice per group): control group,20 mg/kg/d,200 mg/kg/d,500 mg/kg/d DEHP-treated group.Then,pregnant mice were given with vegetable oil and different concentration of DEHP prepared with vegetable oil every day.The pregnant mice were sacrificed on the eighth day of gestation(GD 8),and tissue samples of uterus,ovary,liver and kidney were collected.(2)Pseudopregnancy model: female mice of the same strain were mated with ligated male rats in cages,and the detection of vaginal plug was regarded as pseudopregnant day 1.Then,pseudopregnancy mice were randomly divided into 4 groups(n=10 mice per group): control group,20 mg/kg/d,200 mg/kg/d,500 mg/kg/d DEHP-treated group.Pseudopregnant mice were given vegetable oil and different concentrations of DEHP prepared with vegetable oil in every day.On the fourth day of gestation(GD4),mice were induced artificial decidualization by infusing oil into one side of uterine horn on GD 4.On GD 8,the uterus,ovary,liver and kidney tissue samples were collected.2.The effect of DEHP exposure on the Polyploidization of decidual cells was observed by HE staining in GD 8 mice.3.The effects of DEHP exposure on the decidualization and proliferation of decidua cells were studied by immunohistochemistry in GD 8 mice.4.The effects of DEHP exposure on serum estradiol and progesterone levels were analyzed by enzyme linked immunosorbent assay in GD 8 mice.5.The effects of DEHP exposure on the expression of estrogen and progesterone receptors,angiogenesis,the numbers and maturation of u NK cells of uterine decidua was studied by immunohistochemistry in GD 8 mice.6.The effects of DEHP exposure on the expression level of cytokine secreted by u NK cells in decidual tissue were studied by real-time quantitative PCR in GD 8mice.Results:1.In normal pregnancy model,compared with the control group,DEHP treatment of different doses had no significant effect on the number of uterine implantation sites(P>0.05).However,the ratio of uterus weight to body weight decreased significantly in 200 mg/kg/d and 500 mg/kg/d DEHP-treated groups(P<0.05),and the ratio of implantation site weight to body weight decreased significantly in 200 mg/kg/d and 500 mg/kg/d DEHP-treated groups(P < 0.05 or 0.01).In pseudopregnancy model,compared with the control group,The uterine weight of oil-induced side and the ratio of oil-induced side to non oil-induced side were significantly decreased in 500 mg/kg/d DEHP-treated groups(P<0.05 or 0.01).2.The results of HE staining showed that the number of polyploid decidual cells in the decidual tissue decreased significantly with the increase of DEHP concentration(P<0.01 or 0.001)compared with the control group in normal pregnancy and pseudopregnancy model.3.Immunohistochemical results showed that the number of PCNA positive cells in each DEHP-treated group was significantly lower than that in the control group(P<0.01 or 0.001)in normal pregnancy and pseudopregnancy model.The number of cyclin D3 positive cells in DEHP-treated groups was significantly decreased compared with the control group(P<0.05 or 0.01).4.The results of enzyme linked immunosorbent assay showed that there was no significant difference in serum estradiol level between the control group and DEHP-treated group,but the levels of progesterone were significantly increased in200 mg/kg/d and 500 mg/kg/d DEHP-treated groups(P<0.05 or 0.001).5.The results of immunohistochemistry showed that compared with the control group,the expression level of estrogen and progesterone receptors in decidua of mice treated by 200 mg/kg/d and 500 mg/kg/d DEHP were significantly decreased(P<0.01 or 0.001).6.Immunohistochemical results showed that in normal pregnancy and pseudopregnancy model,compared with the control group,the areas of blood vessels in the decidual tissue of mice in 200 mg/kg/d and 500 mg/kg/d DEHP-treated groups were significantly reduced.7.Immunohistochemical results showed that there was no significant difference in the number of u NK cells of normal pregnancy between the 20 mg/kg/d group and control group.However,the numbers of u NK cells in the 200 mg/kg/d and 500mg/kg/d groups were significantly decreased(P < 0.01 or 0.001).In the pseudopregnancy model,the numbers of u NK cells were significantly decreased in DEHP-treated groups compared with the control group(P < 0.05 or 0.01).In DEHP-treated group,u NK cells became smaller and immature with increasing DEHP concentration in normal pregnancy and pseudopregnancy model.8.The results of real-time quantitative PCR showed that,the m RNA expression levels of Lif and IFNγ in the DEHP-treated uteri were significantly decrease compared with the control group(P<0.001).Conclusion:Gestational exposure to DEHP can significantly inhibit the process of uterine decidualization by inhibiting the proliferation and multinucleation of decidual cells,disregulation of serum progesterone level,downregulation of estrogen and progesterone receptor,reducing the formation of blood vessels and the number of u NK cells and the expression of related cytokines in decidual tissues.