The Role Of Polarization And Functional Changes Of Macrophages Interfered By Carbon Disulfide In Embryo Implantation Failure

BackgroundCarbon disulfide（CS₂）is the most commonly used raw material in the production of viscose fiber industrial products.Because of the unique excellent performance of viscose fiber,it is widely used in the apparel industry.The apparel industry is a labor-intensive industry dominated by female workers.In recent years,the impact of CS₂ on the female reproductive system has attracted wide attention.Epidemiological survey found that,compared with non-occupational groups,reproductive system of CS₂ occupational exposure females is subject to varying degrees of damage.Our previous epidemiological survey of professional female workers found that compared with the female population in the control group,CS₂ occupational exposure female workers showed a higher early pregnancy loss and a more obvious delay of conception time.Meanwhile,in mice animal model,we found that the most sensitive period of CS₂-induced reproductive toxicity was embryo implantation.However,the toxic mechanism is not clear.The immune microenvironment of maternal-fetal interface can ensure the environment of receptive uterine,thus becoming conducive to the success of pregnancy.The macrophages play an important role in the establishment of the maternal-fetal microenvironment.During the embryo implantation period,the macrophages gather in the maternal-fetal interface,remove the apoptosis cells,secrete cytokines and induce immune tolerance.Meanwhile,macrophages also secrete vascular endothelial growth factor to promote the formation and remodeling of the vascular of maternal-fetal interface and build the placental vessels for the transportation of nutrients for the embryonic development.In addition,the macrophages also take part in the formation of the ovarian corpus luteum to ensure the secretion of the estrogen and progesterone.In the previous study,we found that the exposure of CS₂ could lead to the increased secretion of inflammatory cytokines in the uterine tissue of pregnant mice,and the decreased estrogen and progesterone expression levels in serum of the pregnant mice.These results suggest that the macrophages are associated with the failure of the embryo implantation,which is induced by CS₂ in mice.Above all,the polarization and functional changes of macrophages after the implantation of CS₂ during the implantation period might be one of the important mechanisms of embryo implantation failure.Therefore,in order to reveal the role of polarization and functional changes of macrophages in embryo implantation induced by CS₂ exposure,this study is aimed to use an embryo implantation disorder model of the mice to detect the macrophage polarization and functional changes in uterus and ovaries.The model of embryo implantation failure is designed through exposing to CS₂ at different point and observed at different point sections.ObjectivesMice embryo implantation disorder model was established at different points of CS₂ exposure during the implantation period after conception in mice.The changes of macrophage polarization and the expression levels of IL-6,IL-10,IL-12,TGF-β1 and Vegfa of uterine tissues were determined at different end points.Meanwhile,the changes of macrophage polarization and the expression levels of TNF-a,Cox2,Hifl-a and Vegfa of ovarian tissue were determined.The above experiment was aimed to elucidate the role and mechanism of macrophage polarization and functional changes in mouse embryo implantation disorder induced by CS₂.Methods1.The establishment of animal modelsThe sexually mature Kunming mice aged 8-12 weeks were used after a week’s adaptive feeding.Mating was confirmed by the presence of the female mice were examined at 8:00 the next morning and the positive vaginal plug was defined as gestational day 1（GDI）.Three single intraperitoneal injection exposure groups were designed according to our previous study,the GD3,GD4,and GD5 respectively.The observation endpoints were designed at the continuous time point after exposure（the GD4,GD5,GD6,and GD7,a total of 13 observation endpoints）,and there were 12 mice in each observation endpoint.The dose of CS₂ was 631.4 mg/kg（0.4LD50）for the pregnant mice of exposure group.The CS₂ was dissolved in olive oil for intraperitoneal injection and the control group was injected intraperitoneally with equal volume of olive oil as a solvent control.2.The collection and determination of tissue samplesThe tissues of uterus and ovaries of pregnant mice were collected at each observation endpoint.Flow cytometry was performed to detect the macrophage polarization of endometrial tissue（F4/80:macrophages,CD16/32:M1 macrophages and CD206:M2 macrophages）.The protein and mRNA expression levels of IL-6,IL-10,IL-12,TGF-β1 and Vegfa in uterine tissues were determined by ELISA and Quantitative RT-PCR.The paraffin sections of ovarian tissue were prepared,and the HE staining and pathology analysis were performed,and the pathological evaluation indicators were quantitatively analyzed.Meanwhile,the levels of the M1 and M2 types of macrophages were immunohistochemically stained with iNOS and Arg-1 respectively,and the results were quantitatively analyzed.The expression levels of TNF-a,Cox2,Hifl-a and Vegfa mRNA in the ovarian tissue were determined by ELISA and Quantitative RT-PCR.3.Statistical analysisThe data were recorded with Excel,and analyzed by SPSS 19.0 statistical analysis software with two-tailed tests of significance（P<0.05）.According to original experimental group data divided by control group data,the original results were standardized and tested by normality first,the normal distribution indicators were represented as mean 士 standard deviation.Before comparison,the variance homogeneity test was performed.If the variances show homogeneity,one-way ANOVA was used to analyze and Dunnett’s T test for pairwise comparison.If not,the Welch test was used to analyze and Dunnett’s T3 test for pairwise comparison.Moreover,the GraphPad Prism software was used to draw figures.Results1.The influence onhe structure and function of the ovarian corpus luteum after exposed to CS₂Compared with the control group,being exposed to CS₂ during peri-implantation period,the structure and function of the pregnant mice’s ovarian corpus luteum received different degrees of damage.In the GD4-treated GD5 observation group of,vasodilation and congestion of the pregnant mice’s ovarian corpus luteum were more obviously（P<0.05）,and the structural damage was more obviously（P<0.05）.Meanwhile,the expression of Cox2 and Hifl-a mRNA in the corpus luteum of pregnant mice showed the tendency of initial increase and then decrease.Cox2 mRNA expression was significantly higher in the GD3-treated GD4 observation group and the GD4-treated GD5 observation group（P<0.05）,while the expression was significantly lower in the GD5-treated GD7 observation group（P<0.05）.Hifl-a mRNA expression was significantly higher in the GD3-treated GD4 observation group and the GD4-treated GD5 observation group（P<0.05）.The results indicated that CS₂ exposure could lead to the damage of the corpus luteum in pregnant mice.2.The influence on the macrophage polarization balance of the ovarian corpus luteum after exposed to CS₂Compared with the control group,exposure of CS₂ in the GD3-treated GD5 observation endpoint during the peri-implantation period,both the Ml and M2 types of macrophages significantly increased（Ml:P<0.05 and M2:P<0.01）,and subsequently,the Ml type of macrophages reduced to normal level while the M2 type of macrophages dramatically increased in the GD6 observation endpoint（P<0.01）.Meanwhile,the M2 type of macrophages markedly decreased both in the GD7 observation endpoint（GD4-treated and GD5-treated,P<0.05）.During the window of implantation,the polarization balance of M1/M2 macrophages shifted from the Ml type of macrophages（M1>M2 in the GD3-treated GD4 observation endpoint,P<0.01）to the M2 type of macrophages（M1<M2 in the GD5-treated GD6 observation endpoint,P<0.01）.3.The influence on the macrophage polarization balance of the uterine endometrium after exposed to CS₂Exposure to CS₂ in the GD3,GD4,and GD5 during peri-implantation period,the M2 type of macrophages were significantly lower than that of the control group,especially in the GD7 observation endpoint（P<0.01）;the M1 type of macrophages was significantly higher in the GD4-treated GD6 observation endpoint（P<0.05）.During the initial implantation period,the dominance of the polarization balance of M1/M2 macrophages was shifted from the M1（in the GD3-treated GD4 observation endpoint,P<0.05）,to the M2（in the GD3-treated GD5 observation endpoint,P<0.01）,and then to the M1（in the GD3-treated GD6 and GD7observation endpoints）.4.The influence on the expression of immune factors in the uterine and ovarian tissue after exposed to CS₂After administration of CS₂ in the GD3-treated GD4 observation endpoint during peri-implantation period,the expression of IL-6 mRNA and protein of the uterine tissues increased significantly（P<0.05）;while the expression of IL-12 protein decreased significantly（in the GD5 observation endpoints,P<0.01;in the GD7 observation endpoints,P<0.05）.On the other hand,the expression of protein IL-10 in the GD3-treated GD4 observation endpoint（P<0.05）and in the GD4-treated GD7 observation endpoint（P<0.01）were significantly higher,while the expression of protein decreased in the GD5-treated GD6 observation endpoint（P<0.05）.For TGF-(31,the mRNA expression decreased significantly in GD7 observation endpoints（P<0.01）while the protein expression increased significantly in the GD4 observation endpoint and the GD6 observation endpoints（P<0.05）.In ovarian tissue,TNF-αmRNA expression increased significantly（in the GD3-treated GD4 observation endpoint,P<0.01;in the GD4-treated GD7 observation endpoint,P<0.01,and in the GD5-treated GD6 observation endpoint,P<0.05）.5.The influence on expression of Vegfa in the uterine and ovarian tissue after exposed to CS₂Compared with the control group,being exposed to CS₂ in the GD3 during peri-implantation period,the Vegfa mRNA expression of the uterine tissues were significantly higher in the GD4 observation endpoint while were significantly lower in the GD6 observation endpoint（P<0.05）.Meanwhile,the protein expression of Vegfa mRNA were significantly lower in the GD5 observation endpoint（P<0.05）.In ovarian tissues,the Vegfa mRNA expression was significantly higher in the GD4 observation endpoints and the GD7 observation endpoints（P<0.01）Conclusion1.After being exposed to CS₂ during peri-implantation period,the polarization balance of M1/M2 macrophages showed a transiently inflammation both in the ovarian corpus luteum and the uterine endometrium,suggesting macrophages were intended to regulate the unfavorable state by inducing tolerance.2.Being exposed to CS₂ during peri-implantation period,the structure and function of the ovarian corpus luteum were damaged,and this mechanism may be due to the change of the polarization balance of M1/M2 macrophages.3.Being exposed to CS₂ during peri-implantation period,the protein expression of IL-10 and TGF-β1 increased in the uterine tissue,and we demonstrated that the partly normal implantation and embryo development in the uterine tissue might depend on the tolerance regulation.