Study On Gene Expression Related To Endometrial Receptivity In Patients With Endometriosis

Endometriosis(endometriosis,EMT)is that endometrial tissues(glands and stroma)appear outside the uterus.Endometriosis is a hormone-dependent disease.The pelvic is the most common site of occurrence.Globally,5%～10% of women are affected by endometriosis,of which 20%～30% suffer from infertility.Endometriosis can interfere with normal pregnancy from all aspects of pregnancy such as follicle development,ovulation,development of fertilized ova,transportation,and implantation,leading to infertility.To explore the pathogenesis of infertility caused by endometriosis is of great significance to the fertility function of endometriosis patients.Endometrium receptivity refers to the receptivity of the endometrium to the embryo,which allows the embryo to adhere to the endometrium until the implantation is completed.It is closely related to endometrium cell decidualization and the secretion of estrogen and progesterone.Studies have shown that estrogen and progesterone changes in patients with endometriosis,which may affect endometrial stromal cell decidualization and endometrial receptivity,thus leading to embryo implantation failure.The decrease of endometrial receptivity in patients with endometriosis is one of the main reasons leading to infertility,which is a research hotspot in the field of reproductive medicine.Embryo implantation is a process in which a blastocyst is embedded into the endometrium,also known as implantation.At the time of implantation,the endometrium is in the secretory phase.After implantation,the blood supply becomes richer,the glands secrete more exuberantly,the interstitial cells become very hypertrophic,rich in glycogen and lipid droplets,and the endometrium further thickens.These changes in the endometrium are called decidual reactions and the mesenchymal cells are called decidual cells.The successful implantation of embryos depends largely on the synchronicity of the development of the embryo and the endometrium.Embryonic implantation begins by attaching the trophectoderm TE of the blastocyst embryo to the epithelial cells of the endometrium containing hormone receptors,and then breaking through the cell layer to infiltrate the stromal cell layer which has undergone decidualization.Due to the limitation of medical ethics,the study of endometrial receptivity using human embryos is restricted.Human choriocarcinoma cells from the human choriocarcinoma cells line can synthesize a variety of hormones,including human chorionic gonadotropin,placental milk source,estrogen and progesterone,which can be used to mimic the biological functions of human embryonic trophoblast cells.Therefore,in this study,we used trophoblast cells of human choriocarcinoma cells instead of embryonic trophoblast cells to interact with decidualized endometrium stromal cells of endometriosis patients to simulate embryo implantation in vitro.Objectives In this experiment,human choriocarcinoma cells and the endometrium of decidual stromal cell from patients with endometriosis were co-culture,which is to observe the adhesion situation of human choriocarcinoma cells ball with endometriosis patients endometrial stromal cell,and then to collect endometrial stromal cells after co-culture,through the RNA-Seq method study the expression differences of gene which related to endometrial receptivity in patients with endometriosis,to explore the reasons for the decrease of endometrial receptivity in patients with endometriosis,and to provide theoretical basis for improving endometrial receptivity in patients with endometriosis and improving embryo implantation rate and clinical pregnancy rate in patients with endometriosis.Materials and Methods 1.Study population and group: In this study,3 cases of endometriosis patients who underwent total laparoscopic hysterectomy were selected as the experimental group and 3 cases of normal endometrium in patients undergoing total laparoscopic hysterectomy with uterine leiomyoma were selected as control group.Inclusion criteria for patients with endometriosis in experimental group:(1)The endometrium was proliferative phase edometrium;(2)previous menstrual regularity;(3)35～40 years old;(4)No hormone use history three months before surgery;(5)By conventional pathological diagnosis of endometriosis.Inclusion criteria for patients with uterine leiomyoma in control group:(1)The endometrium was proliferative phase edometrium;(2)35～40 years old;(3)No hormone use history three months before surgery;(4)The uterine leiomyoma were diagnosed by routine pathology and the endometrium was normal;(5)Have a history of fertility.Exclusion criteria:(1)complicated with other endometrial diseases,such as endometritis,endometrial cancer,etc.;(2)Other endocrine-related diseases;(3)with other inflammatory and infectious diseases related to the reproductive system,such as salpingitis,pelvic peritonitis,cysts with tubo-ovarian,etc.;(4)with other malignant diseases;(5)Preoperative symptoms of menopause.2.Methods:(1)Endometrial tissues of the experimental group and control group were collected.(2)Separation,culture and identification of endometrial stromal cells and epithelial cells.(3)The endometrium stromal cells were induced to undergo decidualization,and the human choriocarcinoma cells spheres and decidualized endometrium stromal cells were co-cultured to simulate the process of embryo implantation in vitro,and the adhesion between the human choriocarcinoma cells spheres and endometrium stromal cells was observed.(3)The co-cultured endometrial stromal cells were collected for total RNA extraction.RNA-Seq was performed and transcriptome analysis was perfermed on the sequencing results to identify the genes related to endometrial receptivity.The functions of differentially expressed genes were identified by GO classification analysis.KEGG pathway analysis was used to identify the pathways related to differentially expressed genes.(4)Real-time Fluorescence Quantitative Polymerase Chain Reaction(PCR)was used for quantitative analysis of differential genes.Results 1.Human choriocarcinoma cell spheres and decidual endometrial stromal cell are co-culture.Endometriosis patients with endometrial stromal cells on the adhesion of human choriocarcinoma cell spheres number of 47.00 ± 4.36,normal endometrial stromal cells on the adhesion of human choriocarcinoma cell spheres number 16.67 ± 1.52,endometriosis patients with endometrial stromal cells and normal endometrial stromal cell adhesion statistically difference(P<0.001).2.RNA-Seq analysis of endometrial stromal cells2.1 RNA-Seq detected 1459 differential genes,including 949 up-regulated genes and 510 down-regulated genes.2.2 In the biological process of GO classification,the five GO functions with the most differentially enriched genes were cellular process,single-organism process,metabolic process,biological regulation and response to the stimulus.The five GO functions with the most differentially enriched genes in cell components are cell,cell part,organelle,membrane and membrane part.The five GO functions with the highest concentration of differential genes in molecular functions were binging,catelytic activity,molecular retrogenic activity,signal retrogenic activity and transporter activity.2.3 ECM-receptor interaction,Focal adhesion and cell adhesion molecules are the three pathways with significant differential gene expression in KEGG pathway,which are related to endometrial receptivity.3.Selecting 10 up-regulated genes(Hand2,MUC16,LPAR1,FGFR2,PRL,RBP4,CNR1,IGFBP-2,PALM and RXFP1)for verification found that 8 genes had increased m RNA expression levels;selected 10 down-regulated genes(FOXF1,KLF12,BMP4,GLI1,IGFBP-5,HOXA13,CYTL1,SCIN,LICAM and GSPG4)for verification and found that 10 genes had decreased m RNA expression levels.Conclusions 1.The decrease of adhesion of endometrial stromal cells in patients with endometriosis may be one of the reasons for the decrease of endometrial receptivity.2.RNA-Seq analysis of endometrium stromal cells in patients with endometriosis showed that the differential gene expression in endometrium stromal cells of patients with endometriosis in GO classification was mainly related to the regulation of cell structure,cell metabolic process and cell migration,and these functions may be related to endometrium receptivity in patients with endometriosis.In KEGG pathway,ECM-receptor interaction,Focal adhesion and cell adhesion molecules may be related to endometrial receptivity changes,which may be the related mechanism of regulating endometrial receptivity.