| BackgroundCarbon disulfide(CS2)is a soluble chemical solventand raw material that is widely used in industry,the number of workers exposed to CS2 is huge.Therefore,occupational hazards caused by CS2 have always been the focus of attention.In recent years,more and more attention has been paid to the harm of CS2 to the reproductive health of the female occupational group.A large number of epidemiological studies have shown that spontaneous abortion,early pregnancy loss and birth defects in female occupational exposure to CS2 were significantly higher than those in the control group.In previous studies,pregnant mice exposed to CS2 showed significantly lower numbers of embryo,when compared with control group.Embryo implantation disorder is one of the most important toxic manifestations of reproductive injury in women induced by CS2,but we do not know the toxicological mechanism.Embryo implantation is a complex physiological process,is the establishment of pregnancy marks and the primary link.Decidualization and angiogenesis play a crucial role in successful embryo implantation.Dendritic cells(DCs),antigen presenting cells(APCs),are closely related to embryo implantation.On the one hand,as a typical immune cell,hapten foetus regulates the immune tolerance of maternal and fetal interface,on the other hand,regulates uterine decidualization and angiogenesis.In recent years,there is abundant evidence that the regulatory function of DCs in the non-immune direction has surpassed its function as an immune cell during early pregnancy in mice.In particular,recent studies have focused on the involvement of DCs in the regulation decidualization and angiogenesis process.A study found that removal of DCs in the uterus of pregnant mice during implantation,the decidualization process was seriously damaged and the embryo implantation failed.It was found that the implantation failure was related to the damaged decidualization process.Plaks et al found that the density of vascular smooth muscle cell markers in mice decreased after DCs was removed,indicating that angiogenesis was damaged.At the same time,dynamic magnetic resonance imaging was used to analyze the function of decidual vessels in mice,it was found that the volume of blood in decidual vessels decreased and the leakage of capillaries increased after the removal of DCs in vivo.These results suggest that the removal of DCs not only reduces vascular permeability,but also impairs angiogenesis and vascular maturation.In addition,DCs in the uterus can produce vascular endothelial growth factor(VEGF)and its receptor VEGFR.ObjectivesIn this study,a mouse model of embryo implantation failure was established by exposure to CS2 at the window of implantation and the changes of the number of DCs in the uterus were observed.The relationship between DCs and embryo implantation was investigated.To explore whether the mice exposed to CS2 caused implantation disorders by changing the number of DCs.Therefore,we studied the number of DCs and the expression levels of IL-11,VEGF and FLt-1 in order to illustrate that the body by changing the number of DCs,further interfere with the process of decidualization and angiogenesis,resulting in embryo implantation disorder after mice exposed to CS2,.Methods1.Grouping and exposure designThe weight of female and male mice was about 30-35 g and 35-40 g respectively.The mice were fed with free diet,natural light,and adaptive feeding for one week before the experiment.According to the female and male 2:1 cage,the next day examination Yin suppository,Yin suppository positive for the first day of pregnancy recorded as GD 1.Two exposure time points of GD4 and GD5 were designed to set up 4 observation endpoints(5th,6th,7th,9th days after pregnancy)and 3 observation endpoints(6th,7th,9th days after pregnancy).The exposed mice were injected intraperitoneally with CS2 at a dose of 631.4 mg/kg(0.4 LD50)at 9 am.The volume of CS2 was 0.1 ml/10 g.while the control group was injected with the same volume of olive oil.2.Sample collection and experimental methodsThe samples were collected at each endpoints,and the uterine tissue were collected immediately and frozen at-80 ℃.It was used for real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Fresh endometrium was collected for flow cytometry.The supernatant was collected and stored in-80 ℃ refrigerator.Serum samples were used for ELISA detection.3.Statistical analysisAll experimental data were analyzed using statistical software(SPSS 19.0).For the two groups experimental data,t-test of two independent samples is used if the data are in normal distribution and the variance is homogeneous.If the data does not fit the normal distribution,the t’test of two independent samples is used.For multiple groups of experimental data,if the variance is homogeneous,one-way ANOVA is used for F test,while Dunnett-t test is selected for comparison between the experimental group and control group.If the variance is uneven,using non-parametric statistical methods.Data are expressed as mean ± standard deviation.P<0.05 indicated the difference was statistically significant.Results1.Effect of CS2 on embryos and maternalEffects on embryos:compared with the control group,the results of embryo development showed that the number of embryos implanted in the GD4 and GD5 exposure groups decreased by 20.6%and 22.4%,the difference was statistically significant(P<0.05),which indicated that the embryo implantation disorder was caused by exposure to CS2 during the implantation window period.The results of embryo development showed that there was no significant difference between the exposed group and the control group in terms of the weight of the uterus,the fossa of the embryo,the average weight of the uterine fossa and the average fossa of the embryo(P>0.05).Effects on maternal weight:there was no significant difference in daily body weight and weight gain between exposed group and control group(P>0.05).Compared with the control group,the body weight of GD4 and GD5 exposed groups began to decrease at the second day after exposure toCS2.The results of study on maternal organs showed that there was no significant difference in heart,lung,kidney,ovary and uterus between the exposed group and the control group(P>0.05).The results showed that CS2 exposure had no effect on maternal.2.Effect of CS2 on the number of uterine dendritic cellsIn the GD4 exposure group,the number of DCs at the endpoint of GD5 was significantly different from that of the control group(P<0.05),and the number of DCs was decreased by 21%.The number of DCs at the endpoints of GD6 and GD7 were decreased compared with the control group,with no statistically significant difference(P>0.05),indicating that CS2 had a significant effect on the endpoint of GD5 in GD4 exposure group.In the GD5 exposure group,there was no significant difference in the number of DCs in the GD6 and GD7 endpoints compared with the control group(P>0.05),indicating that there was no significant effect on the number of DCs in the in the GD5 exposure group.3.Effect of CS2 on the expression of IL-11 in uterineIn the GD4 exposure group,the expression levels of IL-11 mRNA at endpoints of GD5.GD6 and GD7 were significantly different from the control group(P<0.05),and the expression level were decreased by 89%,88%and 60%.The expression levels of IL-11 protein in GD6 endpoint was significantly different from the control group(P<0.05),and the expression level was decreased by 49%.The expression level of IL-11 protein in GD5 endpoint was significantly different from the control group(P<0.01),and the expression level was increased by 83%.In the GD5 exposure group,the expression of IL-11 mRNA at endpoints of GD6 and GD7 were significantly different from the control group(P<0.01),and the expression level were decreased by 78%and 90%.The expression of IL-11 protein at endpoint of GD6 was significantly different from the control group(P<0.01),and the expression level was decreased by 72%.4.Effect of CS2 on the expression of VEGF and Flt-1 in uterineEffects on uterine VEGF expression:In the GD4 exposure group,the expression of VEGF mRNA in GD6 and GD7 endpoints were significantly different from the control group(P<0.05),and the expression level were decreased by 79%and 71%.The expression of VEGF protein in GD6 endpoint was significantly different from the control group(P<0.05),and the expression level was decreased by 30%.In the GD5exposure group,the expression of VEGF mRNA in GD7 endpoint was significantly different from the control group(P<0.01),and the expression level was decreased by 62%.The expression of VEGF protein in GD7 endpoint was significantly different from the control group(P<0.05),and the expression level was decreased by 36%.Effects on uterine Flt-1 expression:In the GD4 exposure group,the expression of Flt-1mRNA at endpoints of GD5,GD6 and GD7 were significantly different from the control group(P<0.05),and the expression level were decreased by 58%,54%and 60%.The expression of Flt-1 protein at endpoints of GD6 and GD7 were significantly different from the control group(P<0.05),and the expression level were decreased by36%and 46%.In the GD5exposure group,the expression of Flt-1 mRNA in GD7 endpoint was significantly different from the control group(P<0.01),and the expression level was decreased by 60%.The expression of Flt-1 protein in GD6 and GD7 endpoints were significantly different from the control group(P<0.05),and the expression level were decreased by 40%and 44%.5.Effect of CS2 on the expression of VEGF in peripheral bloodIn the GD4 exposure group,the expression level of VEGF protein in GD7 endpoint was significantly different from the control group(P<0.01),and the expression level was increased by 73%.In the GD5exposure group,the expression level of VEGF protein in GD7 endpoint were significantly different from the control group(P<0.05),and the expression level was increased by 57%.Conclusion1.The damage of uterine decidualization and angiogenesis may be one of the main causes of mouse embryo implantation disorder.2.Endometrial dendritic cells may be involved in the process of CS2 induced embryo implantation disorder.After exposure to CS2,the embryo implantation failed by reducing the number of DCs in uterus,and then affecting the regulatory function of DCs,that is,regulating decidualization and angiogenesis.3.After exposure to CS2,there was a transient regulatory response to damage to decidualization and angiogenesis.4.VEGF in peripheral blood can be used as a potential biomarker for embryo implantation disorders. |
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