PANX1 Affects Endometrial Decidualization In Recurrent Implantation Failure Patients By Regulating EATP Concentration

Objective:Normal decidualization of Human endometrial stromal cells(HESCs)is one of the decisive factors for embryo implantation,pregnancy establishment and maintenance.Extracellular ATP(eATP)has been confirmed to be involved in endometrial decidualization process.Decidualization damage of HESCs is an important cause of Recurrent implantation failure(RIF),but its mechanism is still unclear.To explore the function and role of PANX1 in Recurrent implantation failure(RIF)patients.Human Pannexin1(PANX1)is an ATP release channel,which can regulate a variety of cell physiological functions by controlling eATP concentration.However,whether PANX1 affects decidualization of HESCs by regulating eATP has not been reported.In this study,by detecting the expression level of PANX1 in HESCs of RIF patients,and by altering PANX1 expression,we investigated whether Panx1 affects human endometrial decidualization by regulating eATP concentration.This study illustrates the function and molecular mechanism of PANX1 in RIF,and provides new ideas and theoretical basis for improving the success rate of human Assisted Reproductive Technology(ART)embryo transfer.Methods:In this study,Human endometrial stromal cells(HESCs)extracted from human endometrial tissue were treated with cyclic adenylate(c AMP)analogs 8-bromo-cyclic adenylate(8-Br-c AMP)and medroxyprogesterone acetate(MPA)to induce deciduating HESCs.Therefore,the effects of decidualization on PANX1 and eATP were examined at the cell level.Further,HESCs were treated with PANX1 lentivirus overexpression plasmid and PANX1 gene-specific knockout small interfering RNA(si RNA)to study the effect of PANX1 on decidualization.1.This study included 55 women aged 22 to 39 with regular menstrual cycles who received treatment at the Assisted Reproduction Center of Jiangxi Maternal and Child Health Hospital from 2019 to 2022.Endometriosis,adenomyopathy,polycystic ovarian syndrome(PCOS),endometrial hyperplasia or endometrial polyps,hydrosalpinx and other infertility disease were excluded.According to the occurrence of recurrent implantation failure,the patients were divided into the recurrent implantation failure group(25 cases)and the control group(30 cases).The basic information collected included age,BMI,Follicle-Stimulating Hormone(FSH),luteinizing hormone(LH),Estradiol(E2),endometrial thickness and the number of embryos per transfer were not significantly different.However,the total number of embryo transfers in RIF patients was significantly higher than that in the control group.2.Mid-secretory endometrial tissues were collected from the experimental group and the control group,and tissue protein was extracted and the expression of PANX1 protein was detected.At the same time,the tissues were fixed and embedded,and the location and expression of PANX1 were observed by immunohistochemistry.3.Endometrial tissues(proliferative phase)were collected from patients undergoing hysteroscopic surgery 3-7 days after the end of menstruation,and primary HESCs were isolated and cultured.Then we use HESCs treated by 0.5 m M 8-Brc AMP and 1 μM MPA for 0 h,24 h,36 h,48 h,60 h and 72 h,respectively.The expression levels of decidualization markers Prolactin(PRL)and human Insulin-like growth factor-binding protein-1(IGFBP-1)and theATP concentration in the cell supernatant.4.The PANX1 overexpression lentiviral plasmid was constructed,and HESCs were infected with the packaging virus for 24 hours to construct the PANX1 overexpression HESCs group.The vector and PANX1 overexpression groups treated with 8-Br-c AMP and MPA for 0 h,24 h,36 h,48 h,60 h and 72 h,respectively.The extracellular ATP concentration and PANX1 protein expression were detected;The m RNA levels of decidualization markers PRL and IGFBP-1 were detected after 72 h treatment with the drug.At the same time,the cytoskeleton of HESCs was stained with Cora Lit@ ghost pen cyclic peptide to observe the morphological changes of HESCs cells after drug treatment.5.Panx1-si RNA was used to knock down PANX1 in empty vector and overexpressing HESCs,and the m RNA levels of decidual markers PRL and IGFBP-1were detected after treatment with 8-Br-c AMP and MPA for 72 hours.At the same time,the cytoskeleton of HESCs was stained with Cora Lit@ ghost pen cyclic peptide to observe the morphological changes of HESCs cells after drug treatment.Result:1.Statistical analysis showed that there were no significant differences in basic information including age,basal follicle stimulating hormone(FSH),basal luteinizing hormone(LH),basal Estrogen(E2),endometrial thickness and number of embryos per transfer between the 25 patients with recurrent implantation failure and the control group.However,the total number of embryo transfers was significantly higher in RIF patients than in controls.2.Immunohistochemistry showed that PANX1 was expressed in the midsecretory phase of both RIF and control endometrium,and it was distributed in both epithelial and stromal cells of endometrial tissue.Compared with the control group,the expression of PANX1 in stromal cells of RIF group was significantly increased.Western Blot also showed an abnormal increase in PANX1 expression in RIF group compared with control group.3.Primary After co-treatment of primary HESCs with 8-Br-c AMP and MPA for72 h,the expression of PANX1 decreased significantly and the m RNA levels of decidualization markers PRL and IGFBP-1 increased significantly;In the process of decidualization induced by 8-Br-c AMP and MPA,PANX1 reached its peak at 24 H after induction,then gradually decreased.With the change of PANX1 expression,eATP level increased from 0h-24 h,and gradually decreased after 24 h.4.After HESCs were infected with lentivirus overexpressing PANX1,PANX1 protein was significantly up-regulated.After treatment with 8-Br-c AMP and PANX1 for 72 h,compared with the vector group,the PANX1 overexpression group showed a sustained thin and high concentration of eATP,and the expression of decidualization markers PRL and IGFBP-1 was significantly decreased.F-actin filaments were distributed randomly after decidualization,and PANX1 overexpressed HESCs retained long fibroblasts after decidualization.5.After HESCs were treated with Panx1-sirna for 24 h,WB results showed that the protein levels of PANX1 in the vector group and the PANX1 overexpression group were significantly decreased,and the level of PANX1 in the vector group was lower than that in the PANX1 overexpression group.After co-stimulation of HESCs with 8-Br-c AMP and MPA for 72 h,compared with HESCs in the vector group,PRL and IGFBP-1 were significantly decreased after decidualization in PANX1 overexpression group and PANX1 knockdown group.Phalloidin immunofluorescence staining showed that PANX1 knockdown HESCs still showed the morphology of long fibroblasts after drug treatment.In conclusion:Our findings suggest that PANX1 is involved in decidualization of human endometrial stromal cells by affecting the process of endometrial decidualization by regulating eATP concentrations.Its abnormal expression in human endometrial tissue can significantly hinder the normal decidualization process of the endometrium,resulting in the failure of embryo implantation,which is one of the important causes of repeated implantation failure.Our findings provide a new potential therapeutic target for recurrent implant failure.