| Background and Objective:Endometrial receptivity is one of the necessary conditions for successful embryo implantation.Improving endometrial receptivity is an key link to improve in vitro fertilization(IVF),single sperm oocyte cytoplasmic injection(ICSI)-embryo transfer(IVF/ICSI-ET)implantation rate and pregnancy rate in assisted reproductive technology(ART)clinical work,and also very important to success rate of artificial insemination and natural pregnancy.Scholars believe that controlled superovulation may cause damage to endometrial receptivity and negative impact of embryo implantation rate.In our previous work,high-throughput quantitative proteomic isotope labeling relative and absolute(iTRAQ)analysis was applicated to detect the protein expression differences of the endometrium in inplantation window phase of the patients who recieved different ovulation-inducing treatments,and aminopeptidase N(ANPEP/APN/CD13)expression was found to be one of the functional proteins with multiple cellular biology functions.Further bioinformatics analysis indicated that ANPEP as a potential target protein,may be involved in the regulation of endometrium receptivity.The role of aminopeptidase N in regulating endometrial receptivity,especially the function in ovulation-embryo transfer in ART,and the mechanism are still blank.In this paper,ANPEP is used as an entry point to study the regulation mechanism of endometrial receptivity.ANPEP is a membrane-bound zinc-dependent peptidase,belonging to the M1 family of metallomatrix proteinases.It is selectively expressed in human umbilical vein endothelial cells and the endothelial cells that form new blood vessels of tumor and solid tumor and other tissues,but not in the endothelial cells of normal tissues.ANPEP was considered to be an important regulator of neovascularization.Angiogenesis signals such as hypoxia and vascular endothelial growth factor(VEGF)can induce the expression of ANPEP in endothelial cells of neovascular,which can also regulate the angiogenesis signals.The importance of changes in the vascular system of the endometrium in the regulation of endometrial receptivity as well as during embryo implantation and implantation has been widely demonstrated,and the initial phenomenon that can be observed is increased endometrial vascular permeability and increased microvessel density near the implantation point.Thus we proposed that ANPEP may regulate endometrial receptivity by affecting local vascularization.In this study,based on the in vitro culture of human endometrial stromal cell and human umbilical vein endothelial cell line,adenoviral vector infection was used to regulate the expression level of ANPEP in endometrial stromal cells,and the effect of ANPEP protein on endometrial receptivity related factors was investigated,as well as the cell proliferation,migration,angiogenic capacity of HUVECs,to preliminarily explore the regulatory mechanism of ANPEP in regulating endometrial receptivity.Then providing a new idea for improving endometrial receptivity so as to enhance embryo implantation and pregnancy rate in assisted reproductive technology.Materials and Methods:1.Immunohistochemistry and Western blot were used to determine the localization and expression of ANPEP protein in the endometrial tissues of mice at different ages(1 week,8 weeks and 8 months)and in the proliferative,pre-implantation and implantation phases of women.2)Human endometrial stromal cells were isolated and cultured,and the purity of the cells was determined by immunofluorescence.The expression of ANPEP in the primary cultured endometrial stromal cells was regulated by adenovirus infection,and the effectiveness of adenovirus overexpression and interference was verified at the gene and protein levels using real-time quantitative reverse transcription polymerase chain reaction(Real time RT-PCR)and Western blot.3)After the expression of ANPEP in primary cultured endometrial stromal cells was regulated by overexpression/interference of adenovirus infection,the proliferation of endometrial stromal cells was observed by CCK-8 assay,and the changes of cell migration and invasion were observed by Transwell assay.4)HUVECs were co-cultured with endometrial stromal cells infected with ANPEP-overexpressing/interfering adenoviruses using Transwell chambers to observe the changes of migration and invasion ability of HUVECs;HUVECs were cultured with supernatant of endometrial stromal cells infected with ANPEP-overexpressing/interfering adenoviruses to observe their proliferation activity and angiogenic capacity.5)The mRNA levels of endometrial receptivity factors:HOXA10,intergrin β3,LIF,and IL-1 were measured by real time RT-PCR after regulating the expression of ANPEP in primary cultured endometrial stromal cells by ANPEPoverexpression/interference adenovirus infection.6)The mRNA levels of invasion-associated protein metalloproteinases MMP-2,MMP-9 were measured by real time RT-PCR after modulating the expression of ANPEP in primary cultured endometrial stromal cells by ANPEPoverexpression/interference of adenovirus infection.7)The mRNA levels of angiogenic pathway factors VEGF,VEGFR-2,PI3K and Akt were measured by real time RT-PCR after regulating the expression of ANPEP in primary cultured endometrial stromal cells by ANPEP-overexpression/interference adenovirus infection.7)Statistical methods:SPSS 17.0 software was used for statistical processing,and the data were expressed as mean ± standard deviation(Means ± SD).P<0.05 was considered statistically significant.Results:1)ANPEP protein was expressed in the endometrium of mice of all ages,which was lowest in the young age group and highest in the sexually mature group,and then the expression of ANPEP decreased with the decline of fertility with age(P<0.05).ANPEP protein was expressed in endometrial stromal cells in all stages of the human reproductive cycle,and the expression in the implantation window group was significantly higher than that in the proliferative phase group and the pre-implantation window group(P<0.05).2)Human endometrial stromal cells can be passaged,cryopreserved and recovered.Immunofluorescence detection of 2-3 generations of cells showed that the cultured cells were positive for vimentin expression and negative for keratin,with good cell purity.Adenovirus infection could successfully regulate the expression of ANPEP protein at the gene and protein levels and subsequent functional studies could be performed.3)The proliferation,migration and invasion of endometrial stromal cells increased with ANPEP overexpression and decreased with ANPEP downregulation(P<0.05).4)With the up-regulation of ANPEP expression in endometrial stromal cells,the migration and invasion ability of HUVECs co-cultured with them increased and conversely decreased(P<0.05);the proliferation activity andangiogenic capacity of HUVECs increased which cultured in the supernatant of ANPEP up-regulated hEMSCs and inversely decreased(P<0.05).5)ANPEP expression in endometrial stromal cells was up-regulated,which promoted the expression of endometrial receptivity factors HOXA10,intergrin β3,and LIF(P<0.05),while the increasing trend of IL-1 was not statistically significant(P>0.05);downregulation of ANPEP expression in hEMSCs decreased the expression of HOXA10,intergrin β3,and LIF(P<0.05),but IL-1 was not significantly changed(P>0.05).6)The expression of MMP-2 and MMP-9 were upregulated with the up-expression of ANPEP in endometrial stromal cells(P<0.05).When ANPEP expression was downregulated in hEMSCs,the decreased MMP-2 and MMP-9 expression were found(P<0.05).7)ANPEP up-expression in endometrial stromal cells increased the mRNA expression of VEGF,VEGFR-2 and PI3K,AKT(P<0.05).Down-regulation of ANPEP expression in hEMSCs caused a decrease in VEGF,VEGFR-2 and PI3K,AKT expression(P<0.05).Conclusion:1.ANPEP is expressed in mouse and human endometrium and highly expressed in the higher reproductive stage,suggesting that ANPEP is involved in regulating endometrial receptivity and may play an important role in embryo implantation and the maintenance of pregnancy.2.Expression of ANPEP in endometrial stromal cells promotes the expression of endometrial receptivity factors such as HOXA10,intergrin β3,and LIF,thereby affecting endometrial receptivity.3.Upregulation of ANPEP in uterine stromal cells promotes their proliferation,migration and invasion,and increases the proliferation,migration and invasion of human umbilical vein endothelial cells.4.The expression of ANPEP in endometrial stromal cells could promote the angiogenic capacity of umbilical vein endothelial cells,thus affecting the local vascularization of the endometrium and participating in the regulation of endometrial receptivity.Upregulation of VEGF and its receptor could be one of the mechanisms by which it works. |
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