Abnormal elevated PANX1 expression hampers endometrial decidualization by upregulating extracellular ATP concentration in patients with recurrent implantation failure

Abnormal elevated PANX1 expression hampers endometrial decidualization by upregulating extracellular ATP concentration in patients with recurrent implantation failure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Abnormal elevated PANX1 expression hampers endometrial decidualization by upregulating extracellular ATP concentration in patients with recurrent implantation failure Xia-Lu Liu, Qi Yang, Pei-Pei Liu, Li-Yun Cao, Xin Li, Zhi-Qin Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7512759/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Backgrounds: Recurrent implantation failure (RIF), a important contributor to infertility in assisted reproductive technologies, is believed to be primarily to abnormal endometrial decidualization. However, its related molecular mechanisms are still incompletely clear. It has been reported that PANX1 is an ATP-permeable channel to regulate the extracellular ATP, and ATP level could influence the progress of endometrial decidualization. Therefore, we conduct this study to explore the potential role of PANX1 during the development of RIF. Methods In this study, we first compared the the expression of PANX1 in RIF patients and control patients. Then, the primary human endometrial stromal cells (pHESCs) was used to the explore the regulatory function of PANX1 on endometrial decidualization by Immunofluorescence staining, Western blot, RT-PCR, Plasmid transfection and ATP detection. Finally, animal model was established to further confirm the results which were found in vitro. Results we observed a significant upregulation expression of ATP release channel protein Pannexin1 (PANX1) in endometrial tissue of RIF patients. Moreover, after overexpressing PANX1 in human endometrial stromal cells (HESCs), we found a significantly high concentration of extracellualr ATP (eATP) and the inhibition of HESCs decidualization, which was characterized as the decreasing expression of prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), as well as the aberrant morphologic cytoskeletion of HESCs. Surprisingly, knockdown the expression of PANX1 in HESCs also impaired the cellular decidualization, indicating the important role of normal PANX1 expression during the process of endometrial decidualization. The subsequent animal study further confirmed our findings in vitro. Conclusions The present study clearly showed that abnormal high expression of PANX1 hindered endometrial decidualization through the upregulation of eATP concentrations, ultimately leading to embryo implantation failure. Our findings suggested a novel cause of RIF and identified a potential therapeutic target for RIF. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Medical research Biological sciences/Molecular biology RIF human endometrial stromal cells PANX1 endometrial decidualization eATP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Assisted reproductive technology (ART) is one of the most rapidly evolving technologies in the field of reproductive medicine in past 40 years. The advent of ART has enabled infertile couples to achieve viable clinical pregnancies, however, some patients still fail to conceive even after transfering high quality embryos. These pregnancy failures are thought to be mainly dued to the embryo recurrent implantation failure (RIF) [ 1 , 2 ] , which is specially to the impaired endometrial decidualization [ 3 ] . Summarizing clinical data from these years, RIF is currently defined as failure to achieve clinical pregnancy after transfering with at least four high quality embryos in at least three fresh or frozen cycles in women under 40 years of age [ 2 ] . Recent studies showed that 15% of patients enrolled in IVF cycles have a history of RIF, and that 1% of these patients who start a pregnancy will eventually have a recurrent miscarriage as well [ 4 ] . Remarkably, there is increasing evidence that insufficient and impaired endometrial cellular metaplasia is the main cause of RIF [ 5 , 6 ] . It is worth noting that high-quality "soil" (endometrium) may be more important than quality "seed" (embryo) in embryo implantation [ 7 ] . The process of embryo implantation involves extensive remodeling of endometrial tissue, including the changes of morphology and structure of endometrial matrix to form decidualization state to ensure successful embryonic implantation [ 8 ] . During this process, endometrial stromal cells differentiated from long fibroblasts into secretory cells with large, round, polygonal nuclei [ 9 ] . Unlike most mammals, human endometrial stromal cells can complete metamorphosis without embryonic implantation, manifesting as the secretory phase of the menstrual cycle [ 10 ] . In early pregnancy, this process is traditionally induced by estrogen and progesterone levels, as well as other factors, such as cyclic adenosine monophosphate (cAMP), prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1) [ 10 – 12 ] . The health of endometrial decidualization is very important for embryonic development, and abnormal decidualization has been associated with many pregnancy disorders, such as intrauterine growth restriction, repeated implantation failure, preeclampsia, and infertility [ 13 ] . Recent studies have found that ATP-P2Y2 signaling promotes the phosphorylation of STAT3 in uterine cavity epithelial cells, thereby increasing stromal cell Early growth response 1 (Egr1) and Prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2) [ 14 ] . Both Egr1 and COX-2 are necessary for decidualization and subsequent implantation of embryos [ 15 ] . ATP-releasing channel protein pannexin 1 (PANX1) is an ATP-permeable channel to regulate the extracellular ATP (eATP) [ 16 ] , which plays a key role in a variety of physiological functions, including blood pressure regulation, apoptotic cell clearance, human oocyte development [ 17 ] , and airway inflammation progression [ 18 ] . eATP exerts its cellular effects by activating the purinergic P2 receptors [ 19 ] , and has an important role in the male and female reproductive organs, such as hormone secretion, penile erection, sperm motility and energy acquisition [ 20 ] . In previous studies, researchers have found that ATP regulates decidualization of endometrial stromal cells in a concentration - or time-dependent manner [ 21 ] , which reminds us that whether PANX1 might be involved with the occurrence and development of RIF. Therefore, we conducted this study to deeply elucidate the regulatory relationship between PANX1 and RIF, which opens up new ideas and methods for clinical optimization of RIF treatment and has important theoretical significance and potential application value. Material and methods Patients and ethics approval The study was conducted in accordance with the recommendations of the Clinical Trial Guidelines of the Ethics Committee of Jiangxi Maternal and Child Health Hospital, and informed consent was obtained from all patients. In this study, patients with no history of pregnancy after at least 3 embryo transfers (including a total of 4 high-quality embryos) were recruited in the RIF group. Patients with tubal obstruction or unexplained infertility who achieved clinical pregnancy after the first embryo transfer were assigned to the Control group. The clinical data of all the patients from 1st January 2024 to 31th December 2024 were retrospectively analyzed. All participants signed an informed consent form. Participants ranged in aged from 20 to 38 years of age and had menstrual cycles between 28 ± 7 days. Patients with polycystic ovary syndrome, endometrial polyps, chronic endometritis, hydrocele, tubal inflammation, polycystic ovary syndrome, endometriosis, and adenomyosis were excluded. Isolation and in vitro decidualization of pHESCs Primary human endometrial stromal cells (pHESCs) were obtained from healthy female volunteers of reproductive age with regular menstrual cycles, and endometrial biopsies were performed during the value-added phase of menstruation. Endometrial tissues were cut into as small pieces as possible and digested with 1% type I collagenase in a constant temperature water bath at 37℃ for 60 min. Next, the digest was passed through a 200 um filter to obtain a cell suspension by centrifugation at 1100 r/min for 10 min. The stromal cells obtained were treated with phenol red-free DMEM/F12 (Wisent) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 µg/mL streptomycin, and were subsequently inoculated into culture flasks and incubated at 37℃ degrees and 5% CO2. Induction of decidualization was initiated when cell density reached 80%. Culture medium containing 10% charcoal adsorbed serum (BI), 100 U/ml penicillin and 100 µg/mL streptomycin, plus 0.5 mM 8-Br-cAMP (Sigma-Aldrich), 1 µM methoxyprogesterone acetate (MPA) (Sigma-Aldrich) treatment. Immunohistochemistry Fresh human endometrial tissue was fixed with 4% paraformaldehyde for more than 24 hours, embedded in paraffin, and sliced continuously. The paraffin sections were dewaxed and boiled with citrate antigen repair solution to repair the antigen. The samples were processed according to the immunohistochemical kit (Zhongshanjinqiao), and the sections were treated with anti-PANX1 antibody incubated overnight. After washing with PBS, the sheep and rabbit secondary antibodies in the kit were incubated at room temperature for 30 min. Finally, the samples were stained with diaminobenzidine and hematoxylin. The slices of the control group were stained with non-specific rabbit IgG, and no non-specific staining was observed. Western blot analysis Proteins in tissues or cells were extracted by RIPA lysis buffer (Applygen). Protein concentrations were measured with a BCA kit (Applygen). Proteins were subjected to polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore). After blocking, the membranes were incubated with primary antibody (PANX1, GAPDH) and the values of relative protein levels indicate the quantification of band intensities of the indicated proteins using Image J against the loading control GAPDH. Quantitative Real-Time PCR Total mRNA was extracted from HESCs using Trizol. The purified total RNA (1 µg) was reverse transcribed to cDNA according to the instructions for the Prime Script RT kit (Takara). cDNA samples were prepared for subsequent assays. Real-time quantitative PCR was performed using TB Green Mix (Takara) and SYBR Green (Takara) on a 7500 instrument. All expression values were normalized against GAPDH. The following primers were used for the tested genes: PANX1: 5’-CAGTTCCAGGTGTTACCGA-3’ and 5’-GCGGCAGCTAATGTACTTG-3’, PRL: 5’-CCACTACATCCATAACCTCTCC-3’ and 5’-TCATCIGTTGGGCTTGCTC-3’, IGFBP1: 5’-CACAGGAGACATCAGGAGAAG-3’ and 5’-GATCCTCTTCCCATTCCAAGG-3’, GAPDH: 5’-GAAGGTCGGAGTCAACGGATTT-3’ and 5’-CTGGAAGATGGTGATGGGATTTC-3’. PANX1 overexpression plasmid construction and lentiviral infectiona The pCDH-CMV-MCS-EF1-copGFP (PCDH) plasmid was used to produce the PANX1 overexpression vector. The 293T cells were inoculated in 6-well plates with a density of 70%, and the density reached 80%-85% after 12 hours culture. Packaged plasmids pVSVG and pSPAX2 were co-transfected with lentiviral vectors in a ratio of 1:3:4:293T. The total plasmid content was 2 µg/ well. 2 hours before transfection, 1 ml/well was replaced with Opti-MEM (Gibco). Dilute 5 µL turboFect (Thermo) in 200 µL Opti-MEM. Then, the configured transfection system was mixed with diluted turboeffect. Each well was supplemented with 5 ml DMEM supplemented with 15% FBS and 10 mM Hepes (Solarbio) replacement medium 12 hours after transfection. 48 hours after transfection, the supernatant was filtered through a 0.45 µm filter. The supernatant was concentrated into concentrated lentivirus using lentivirus precipitation solution (TransGen). Lentivirus was then added to the cell supernatant, and the medium was replaced with complete culture medium 24 hours after infection. SiRNA and transfection Cells were seeded in 6-well plates at a density of 2*10 6 / well and cultured for 12 hours. 2 hours before transfection, 1 ml/well of medium was replaced with Opti-MEM. 20 nM siRNA (Ribose) was diluted in 200 µL Opti-MEM. 5 µL TurboFect (Thermo) was diluted in 200 µL Opti-MEM. Similar to lentivirus transfection, diluted siRNA was mixed with diluted turboeffect. Incubate the mixture at room temperature for 25 min before adding to each well. Medium was replaced with complete medium 12 hours after transfection. The siRNA sequences for PANX1 used the following: siPANX1-01: CAGGAGATCTCGATTGGTA, siPANX1-02: CTCGATTGGTACACAGATA, siPANX1-03: GGTCAAGTCATACAAGTGT. Immunofluorescence staining HESCs grown in 24-well cell culture plates after drug treatment were gently washed with PBS. The culture-medium was washed and fixed with 4% paraformaldehyde at room temperature for 10 min, and washed with PBS for 3 times. The fixed cells were permeated with PBS solution containing 0.2% Triton X-100 at room temperature for 5 min, and then washed again with PBS for 3 times. Next, CoraLite® Plus 488 labeled ghost pen cyclic peptide (Proteintech) was stained at room temperature for 15 min. After washing three times with PBS, the slides were sealed with anti-fluorescence quencher containing DAPI and read under a confocal microscope. Detection of ATP levels in culture supernatants Approximately 0.5×105 cells were seeded in each well of a 48-well cell culture plate and placed in a constant temperature cell incubator for 12 hours. The ordinary complete medium in different cell wells was replaced with 100 µL of decidualized medium every 8 hours, and the previous wells were replaced with new medium before each change. After 48 hours, all wells were changed to normal complete culture medium, and wells without decidualization were used as control group. After 8 hours, the supernatants from each well were collected and transferred to 1.5 mL EP tubes on ice. The cell supernatant was incubated at 500 rpm/min for 5 min at 4°C in a centrifuge, and the system was configured according to the eATP assay kit (Sigma). Absorbance was measured by fluorescence in a multifunctional microplate reader (FLU, λex = 535/λem = 587 nm), and the fluorescent product was proportional to the amount of ATP present. Preparation for Compounds Mifepristone (Solarbio) was solubilized in dimethyl sulfoxide to achieve a 0.8 mg/ml concentration and subsequently stored at -20°C for preservation. Establishment of Animal Model 12 female ICR mice (6–8 weeks old) were housed under controlled environmental conditions with free access to water and food and 12 hours alternating light/dark cycles. (i) We ensured our animal experiment met the standards of the Animal Ethics Committee of Nanchang University and were reviewed and approved by the Animal Ethics Committee of Nanchang University (ethics approval number: NCULAE-20220624011), (ii) we confirmed that all experiments were performed in accordance with relevant guidelines and regulations, (iii) besides, our studies involving live animals comply with the ARRIVE guidelines. Initially, these mice were randomly divided into control group (n = 4), DMSO group (n = 4) and RIF group (n = 4). Animals were housed at 5:00 pm according to a male to female ratio of 1:2 and female mice were checked the formation of vaginal plugs at 9:00 am in the next day. The day finding the vaginal plug was D0. Female mice were intraperitoneally injected with mifepristone solution (0.1 ml/mouse) on D4 and sacrificed carbon dioxide anesthesia on D12. First we place the mouse into a clean, empty chamber filled with normal air. Then gradually introduce 100% compressed CO₂ at a flow rate that displaces 10–30% of the chamber's volume per minute. After breathing stops, maintain exposure for at least one minute to ensure death. Finally, the uterus was collected to observe the embryo implantation and to detect the related proteins. Statistical Analysis Statistical analysis was performed using GraphPad Prism 8 software. The student’s t-test was used to analyze data between two groups, and one-way analysis of variance (ANOVA) was used to analyze data between three or more groups. p < 0.05 was considered statistically significant and marked as *, p < 0.01 was considered as significant difference and marked as **, A p value of less than 0.001 was considered highly significant. All data are expressed as the mean ± standard error of at least three independent experiments. Results Patient characteristics As shown in Table 1 , no significant differences were observed between the control and RIF groups in terms of age, BMI, basal FSH, LH and estradiol levels or mean endometrial thickness. Notably, although there were also no statistically differences in the number of embryos transferred per transfer or in the mean score of transferred embryos, the total number of embryos transferred was significantly higher in the RIF group than in the control group. Table 1 Clinical statistical characteristics of the subject population Variable Control(n = 30) RIF(n = 25) p Age(y) 32.87 ± 2.11 32.92 ± 3.27 0.0641 BMI (kg/m2) 21.52 ± 2.06 21.46 ± 2.26 0.6155 Basal FSH (IU/L) 5.04 ± 1.34 5.39 ± 1.87 0.4283 Basal LH (mIU/ml) 4.04 ± 2.20 4.42 ± 2.82 0.4148 Basal Estradiol (pg/ml) 33.72 ± 16.23 35.62 ± 14.44 0.6581 Endometrial thickness (mm) 10.87 ± 2.39 9.69 ± 2.34 0.0762 Median number of embryo transfers (range) 2.2(1,4) ± 1.03 5.76(4,8) ± 0.79 < 0.0001 Average Number of embryos per transferred 1.5(1,2) 1.5(1,2) 0.1509 Score of transferred blastocyst 4BC 4BB 14/30(46.7%) 10/30(33.3%) 13/25(52%) 8/25(32%) The expression of PANX1 was abnormally increased in RIF patients The statistical difference in the number of embryonic transfers between the RIF and control groups supports our further study of the role of PANX1 in the endometrium. In humans, metaplasia of endometrial stromal cells begins autonomously during the secretory phase of the menstrual cycle independent of embryo implantation [ 22 ] . Immunohistochemical results showed that PANX1 was expressed in both mesenchymal and epithelial cells of midsecretory endometrium (Fig. 1 A), and the expression of PANX1 in the RIF group was significantly higher than that in the control group (Fig. 1 B and C). These results imply that the increased expression of PANX1 might be closely related with the occurence of RIF. Decrease in PANX1 expression induced by 8-Br-cAMP and MPA Normally, spontaneous decidualization of the human endometrium results from the increased local cAMP, which due to the high level of progesterone. Thus, we treated pHESCs with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) and human medroxyprogesterone acetate (MPA) to induce decidualization in vitro. After treatment with 8-Br-cAMP and MPA for 72 hours, the expression of PANX1 was obviously decreased (Fig. 2 A and B). Significantly, HESCs showed a remarkably elevated levels of PRL and IGFBP-1 mRNA, indicating cellular-like decidualization was successfully induced (Fig. 2 C and D). Furthermore, when treated HESCs with 8-Br-cAMP or MPA for 0 hours to 72 hours, we found that the expression of PANX1 increased firstly and reached the peak at 24 hours, but decreased gradually from 24 hours to 72 hours (Fig. 2 E). Consistent with our findings, the extracellular ATP (eATP) level was also increased firstly and gradually decreased after 24 hours (Fig. 2 F). These results suggests that short-term, rather than long-term, high concentrations of eATP is a key factor to promote the initiation of HESCs decidualizatio, which was supported by the study from Chang et al [ 23 ] . Overexpression of PANX1 in vitro significantly impaired decidualization of HESCs To further explore the effect of PANX1 on decidualization of HESCs, we used lentivirus to infect HESCs in vitro to construct PANX1 over-expression HESCs (pCDH-PANX1). After treating cells with 8-Br-cAMP and MPA, we collected the cellular supernatants to detect eATP concentration. The results showed that the overall level of eATP in pCDH-PANX1 group was higher than that in control group (Fig. 3 A and B). Next, we detected the mRNA levels of PRL and IGFBP-1 in these two groups, which were treated with 8-Br-cAMP and MPA for 72 hours, respectively. We found that the expression levels of both PRL and IGFBP-1 were significantly lower in pCDH-PANX1 group (Fig. 3 C and D), indicating that overexpression of PANX1 could remarkably impede the decidualization of HESCs. It has been known that undifferentiated HESCs presented as a long fibroblastlike phenotype, decidual HESCs presented polygonal cells, and F-actin filaments were randomly distributed [ 9 ] . Thus, we detected the cellular morphology by immunofluorescence staining, and obversed that compared with the control group, the number of long fibroblasts in pCDH-PANX1 group was increased, and the number of polygonal decidual cells was decreased, signifing the inhibition of HESCs decidualization (Fig. 3 E). These data suggest that increased PANX1 could inhibite the HESCs decidualization possibly by maintaining abnormally high levels of eATP. To further confirm that the abnormally high expression of PANX1 impairs decidualization of HESCs, we next knocked down the PANX1 expression by using siRNA transfcetion (siPANX1) (Fig. 4 A and B). After treating with 8-Br-cAMP and MPA for 72 hours, the mRNA levels of PRL and IGFBP-1 in the drug treated group were significantly upregulated compared with those in the untreated group. Significantly, compared with the control group, both pCDH-PANX1 and siPANX1 groups showed cellular-like decidualization damage. Interestingly, treating HESCs in pCDH-PANX1 group with siPANX1 could not rescue the decidualization damage of HESCs (Fig. 4 C and D). This finding was affirmed by the results of immunofluorescence staining, which is that knocking down the PANX1 expression obviously inhibited the differentiation of fibroblast-like stromal cells into round and polygonal decidual cells (Fig. 4 E). Increased expression of PANX1 accompanied with embryo implantation failure in mice. To validate the results of the previous results, we detected the number of embryo implantation and the expression of PANX1 in the endometrium from RIF mouse model. We found that the number of embryo implantation in the RIF group was significantly decreased. Furthermore, uterine hematoma formation and embryo degeneration were also observed in RIF group (Fig. 5 A and B). As we excepted, the expression of PANX1 was significantly increased in RIF group compared to control group (Fig. 5 C and D). These results further demonstrate that the elevation of PANX1 might result in the RIF. Discussion Assisted reproductive technology has been in clinical practice for more than 40 years, but more than 60% of women still fail to become pregnant after receiving embryo transfer, and about 20% of the patients suffer from unexplained RIF and recurrent abortion [ 24 ] . In human, during the secretory phase of each menstrual cycle, spontaneous endometrial decidualization occurs due to elevated levels of progesterone, and stromal cells differentiate into secretory decidual cells. This process is a key step for uterus to allow embryos to implant [ 25 , 26 ] . Abnormal decidualization can affect the implantation window period and directly leads to embryo implantation failure. According to previous studies, impaired decidualization of endometrial stromal cells is an important cause of RIF [ 27 ] . In order to explore the possible molecular mechanisms of impaired decidualization in RIF patients, we detected human endometrium samples at the stage of mid-endometrial secretion and found a significantly increased PANX1 expression in RIF patients’ endometrium tissues. Furthermore, when the PANX1 was overexpressed in HESCs, a obvious inhibition of HESCs decidualization was observed. These findings indicate that the abnormal high level of PANX1 might be a important reason causing RIF. However, its related regulatory mechanism are still unclear. Previous studies have proven the function of PANX1, which transportes the ATP from inside the cell to outside [ 28 – 31 ] . It has been known that suitable concentratione ATP is very important for the initiation and maintenance of decidualization [ 21 , 23 ] . In our study, we found a short-term high concentrations of ATP in the beginning of HESCs decidualization and gradually declined after 24 hours, interestingly, the expression of PANX1 was coincident with the ATP changes. Similar with us, Chang et al also found that short-term exposure to high concentration ATP could promote decidualization of mouse endometrial stromal cells through ATP-P2Y2 signal transduction, but long-term exposure to high concentration ATP would inhibit the decidualization process [ 23 ] . These results suggest that short-term, rather than long-term, high concentrations of ATP can promote endometrial decidualization. However, overexpressed the PANX1 could significantly impede the HESCs decidualization, which was showed by the reduced expression of decidual markers PRL and IGFBP-1, as well as the disruption of cytoskeletal changes. These data implied that high level of PANX1 might impaired the decidualization of HSECs by influencing the eATP concentration. Surprisingly, there is no rescue effects when we reduced the PANX1 level in the HESCs with PANX1 overexpression, indicating that abnormal increased or decreased the expression of PANX1 both impaired the decidualization of HSECs. To further confirm our findings in vitro, we subsequently established a RIF mouse model to detected the PANX1 expression in vivo. Consistent with our previous results in HESCs, we found a significantly increased PANX1 expression in mice with embryo implantation failure, supporting the key role of PANX1 in the pathogenesis of RIF. Nevertheless, our study still has some limitations, including: (1) we only explore the relationship between PANX1 and eATP, and did not further investigate the upstream molecules of PANX1; (2) the animal model we constructed is not very stable, and our research only focuses on phenotype. Therefore, the following works will be to explore the reasons of increased expression of PANX1 in RIF patients and to establish a stable RIF animal model. Conclusions In conclusion, abnormal high expression of PANX1 can impede endometrial decidualization by upregulating extracellular ATP concentrations, which results in embryo implantation failure (Fig. 6). Our findings suggested a novel cause of RIF and represented a potential therapeutic target for RIF. Declarations Conflict of interest The authors declared no conflicts of interest. Ethics Statement This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Clinical Ethical Committee of Jiangxi Maternal and Child Health Hospital, and informed consents from patients were obtained before the initiation of the study. All the authors consented to participate in this study. Author Contribution Conception and design of the study: Jun Tan. Cell experiments: Qi Yang. Animal experiments: Xia-Lu Liu. Sample collection: Pei-Pei Liu and Li-Yun Cao. Data analysis and interpretation: Xin Li and Zhi-Qin Zhang. Draft of the manuscript: Xia-Lu Liu and Qi Yang. Supervision and critical revision of the manuscript for important intellectual content: Xian-E Sun, Jun Tan and Yao Cheng. All authors have read and confirm that they meet the authorship criteria. Acknowledgement The authors thank all the staff, nurses, and physicians at the Reproductive Medicine Center for their support in generating this manuscript. This work was supported by the National Natural Science Foundation of China (82360318), the Science Project of Science and Technology Department of Jiangxi Province (20232BAB206029), the Outstanding Youth Fund Program of Science and Technology Department of Jiangxi Province (20224ACB216005), the Youth Fund Program of Science and Technology Department of Jiangxi Province (20232BAB216024) and the JXHC Key Laboratory of Fertility Preservation. 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Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 21 Oct, 2025 Reviews received at journal 21 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Editor assigned by journal 02 Oct, 2025 Editor invited by journal 16 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 First submitted to journal 09 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7512759","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":528495237,"identity":"4b6efd69-762f-4c4a-a2bd-e828883f568f","order_by":0,"name":"Xia-Lu Liu","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xia-Lu","middleName":"","lastName":"Liu","suffix":""},{"id":528495238,"identity":"1840b6b7-d6f4-462a-b838-6f87bb47accf","order_by":1,"name":"Qi Yang","email":"","orcid":"","institution":"Changxing County Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Yang","suffix":""},{"id":528495239,"identity":"69bd7c3f-b607-4a9b-86c1-d87d0e5311ba","order_by":2,"name":"Pei-Pei Liu","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei-Pei","middleName":"","lastName":"Liu","suffix":""},{"id":528495240,"identity":"04c3e443-05ad-4b7e-ac7d-d7303144ba2b","order_by":3,"name":"Li-Yun Cao","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li-Yun","middleName":"","lastName":"Cao","suffix":""},{"id":528495241,"identity":"0e57b269-2324-4de4-9993-b1b0d0e4bbb2","order_by":4,"name":"Xin Li","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health 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02:12:57","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98235,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/dbbecfbcd2159e4df3a5634b.html"},{"id":93726762,"identity":"0b86103e-3970-4d3d-8216-0a7bffa53649","added_by":"auto","created_at":"2025-10-17 02:04:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":423753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbnormal high expression of PANX1 in endometrium tissues from patients with RIF. \u003c/strong\u003eImmunohistochemical test showed that PANX1 was expressed in both mesenchymal and epithelial cells in endometrium (A). However, compared with the normal patients, RIF patients showed a significantly increased expression of PANX1 in endometrium (A-C). **\u003cem\u003eP\u0026lt;\u003c/em\u003e0.01 (n=5).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/be03090292be24554af8e0c9.png"},{"id":93726766,"identity":"d832a439-df1b-447e-976e-02bca958c79d","added_by":"auto","created_at":"2025-10-17 02:04:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":236951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of PANX1 decreased and eATP changed during the decidualization of HESCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter treating HESCs with 8-Br-cAMP and MPA for 72 hours, the expression of PANX1 was remarkably decreased (A, B), and the mRNA levels of PRL and IGFBP-1 were significantly increased (C, D). Interestingly, the expression of PANX1 and eATP concentration were increased firstly and reached the peak at 24 hours, but decreased gradually from 24 hours to 72 hours (E, F). \u0026nbsp;ns means no statistical significance, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u0026lt;\u003c/em\u003e0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 (n=3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/0945697f07bec0b9ac1a4aad.png"},{"id":93726763,"identity":"6798e9c9-09eb-4dce-9358-fd322677706a","added_by":"auto","created_at":"2025-10-17 02:04:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":369036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of PANX1 impaired HESCs decidualization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of PANX1 protein was significantly increased after lentivirus treatment (A). Furthermore, overexpressed PANX1 could elevate the eATP concentration (B), reduce the expression of PRL and IGFBP-1 (C, D), and inhibite the differentiation of fibroblast-like stromal cells into round and polygonal decidual cells after treating with 8-Br-cAMP and MPA for 72 hours (E). ns means no statistical significance, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 (n=3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/a252008839a855cb6bc2b731.png"},{"id":93728767,"identity":"21188144-14f7-4742-91dd-56194325c436","added_by":"auto","created_at":"2025-10-17 02:12:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":196635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANX1 knockdown impaired decidualization of HESCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfecting with siRNA, the expression of PANX1 was reduced (A, B), moreover, the mRNA levels of PRL and IGFBP-1 were aslo decreased (C, D). After treating with 8-Br-cAMP and MPA for 72 hours, PANX1 knockdown inhibited the differentiation of fibroblast-like stromal cells into round and polygonal decidual cells, which was showed by phalloidin immunofluorescence staining (E). ns means no statistical significance, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 (n=3).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/a0eb39a41b2fd4de9d645fa9.jpeg"},{"id":93728766,"identity":"ccec3de2-387f-45f6-97c0-072c39f7078c","added_by":"auto","created_at":"2025-10-17 02:12:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":583388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMice with RIF showed a increased expression of PANX1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompare with the control group, the number of embryo implantation was significantly reduced in RIF group (A, B), and the expression of PANX1 was significantly increased in RIF mouse (C, D). ns means no statistical significance, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 (n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/bc2ffad50baf635bda2d198b.png"},{"id":93730472,"identity":"c182e1ef-de8a-4fbf-8c86-604db710e65f","added_by":"auto","created_at":"2025-10-17 02:20:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":582685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePattern of decidualization damage caused by abnormal high level of PANX1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcessive PANX1 expression causes the endometrial decidualization injury through upregulating eATP concentration, which is a possible reason of RIF.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/29b5f011968a233229eaa2b4.jpeg"},{"id":104250716,"identity":"23c36cde-5fa3-4042-a39e-b6e3f4a9a889","added_by":"auto","created_at":"2026-03-09 16:06:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3420364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7512759/v1/ea785e66-b04e-4725-b1c7-b5b3dbf0d989.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Abnormal elevated PANX1 expression hampers endometrial decidualization by upregulating extracellular ATP concentration in patients with recurrent implantation failure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAssisted reproductive technology (ART) is one of the most rapidly evolving technologies in the field of reproductive medicine in past 40 years. The advent of ART has enabled infertile couples to achieve viable clinical pregnancies, however, some patients still fail to conceive even after transfering high quality embryos. These pregnancy failures are thought to be mainly dued to the embryo recurrent implantation failure (RIF)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, which is specially to the impaired endometrial decidualization\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Summarizing clinical data from these years, RIF is currently defined as failure to achieve clinical pregnancy after transfering with at least four high quality embryos in at least three fresh or frozen cycles in women under 40 years of age\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Recent studies showed that 15% of patients enrolled in IVF cycles have a history of RIF, and that 1% of these patients who start a pregnancy will eventually have a recurrent miscarriage as well\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Remarkably, there is increasing evidence that insufficient and impaired endometrial cellular metaplasia is the main cause of RIF\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt is worth noting that high-quality \"soil\" (endometrium) may be more important than quality \"seed\" (embryo) in embryo implantation\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The process of embryo implantation involves extensive remodeling of endometrial tissue, including the changes of morphology and structure of endometrial matrix to form decidualization state to ensure successful embryonic implantation\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. During this process, endometrial stromal cells differentiated from long fibroblasts into secretory cells with large, round, polygonal nuclei\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Unlike most mammals, human endometrial stromal cells can complete metamorphosis without embryonic implantation, manifesting as the secretory phase of the menstrual cycle\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In early pregnancy, this process is traditionally induced by estrogen and progesterone levels, as well as other factors, such as cyclic adenosine monophosphate (cAMP), prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1)\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The health of endometrial decidualization is very important for embryonic development, and abnormal decidualization has been associated with many pregnancy disorders, such as intrauterine growth restriction, repeated implantation failure, preeclampsia, and infertility\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Recent studies have found that ATP-P2Y2 signaling promotes the phosphorylation of STAT3 in uterine cavity epithelial cells, thereby increasing stromal cell Early growth response 1 (Egr1) and Prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2)\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Both Egr1 and COX-2 are necessary for decidualization and subsequent implantation of embryos\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eATP-releasing channel protein pannexin 1 (PANX1) is an ATP-permeable channel to regulate the extracellular ATP (eATP)\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, which plays a key role in a variety of physiological functions, including blood pressure regulation, apoptotic cell clearance, human oocyte development\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and airway inflammation progression\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. eATP exerts its cellular effects by activating the purinergic P2 receptors\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, and has an important role in the male and female reproductive organs, such as hormone secretion, penile erection, sperm motility and energy acquisition\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In previous studies, researchers have found that ATP regulates decidualization of endometrial stromal cells in a concentration - or time-dependent manner\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, which reminds us that whether PANX1 might be involved with the occurrence and development of RIF.\u003c/p\u003e\u003cp\u003eTherefore, we conducted this study to deeply elucidate the regulatory relationship between PANX1 and RIF, which opens up new ideas and methods for clinical optimization of RIF treatment and has important theoretical significance and potential application value.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients and ethics approval\u003c/h2\u003e\u003cp\u003eThe study was conducted in accordance with the recommendations of the Clinical Trial Guidelines of the Ethics Committee of Jiangxi Maternal and Child Health Hospital, and informed consent was obtained from all patients.\u003c/p\u003e\u003cp\u003eIn this study, patients with no history of pregnancy after at least 3 embryo transfers (including a total of 4 high-quality embryos) were recruited in the RIF group. Patients with tubal obstruction or unexplained infertility who achieved clinical pregnancy after the first embryo transfer were assigned to the Control group. The clinical data of all the patients from 1st January 2024 to 31th December 2024 were retrospectively analyzed. All participants signed an informed consent form. Participants ranged in aged from 20 to 38 years of age and had menstrual cycles between 28\u0026thinsp;\u0026plusmn;\u0026thinsp;7 days. Patients with polycystic ovary syndrome, endometrial polyps, chronic endometritis, hydrocele, tubal inflammation, polycystic ovary syndrome, endometriosis, and adenomyosis were excluded.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation and in vitro decidualization of pHESCs\u003c/h3\u003e\n\u003cp\u003ePrimary human endometrial stromal cells (pHESCs) were obtained from healthy female volunteers of reproductive age with regular menstrual cycles, and endometrial biopsies were performed during the value-added phase of menstruation. Endometrial tissues were cut into as small pieces as possible and digested with 1% type I collagenase in a constant temperature water bath at 37℃ for 60 min. Next, the digest was passed through a 200 um filter to obtain a cell suspension by centrifugation at 1100 r/min for 10 min. The stromal cells obtained were treated with phenol red-free DMEM/F12 (Wisent) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 \u0026micro;g/mL streptomycin, and were subsequently inoculated into culture flasks and incubated at 37℃ degrees and 5% CO2. Induction of decidualization was initiated when cell density reached 80%. Culture medium containing 10% charcoal adsorbed serum (BI), 100 U/ml penicillin and 100 \u0026micro;g/mL streptomycin, plus 0.5 mM 8-Br-cAMP (Sigma-Aldrich), 1 \u0026micro;M methoxyprogesterone acetate (MPA) (Sigma-Aldrich) treatment.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFresh human endometrial tissue was fixed with 4% paraformaldehyde for more than 24 hours, embedded in paraffin, and sliced continuously. The paraffin sections were dewaxed and boiled with citrate antigen repair solution to repair the antigen. The samples were processed according to the immunohistochemical kit (Zhongshanjinqiao), and the sections were treated with anti-PANX1 antibody incubated overnight. After washing with PBS, the sheep and rabbit secondary antibodies in the kit were incubated at room temperature for 30 min. Finally, the samples were stained with diaminobenzidine and hematoxylin. The slices of the control group were stained with non-specific rabbit IgG, and no non-specific staining was observed.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eProteins in tissues or cells were extracted by RIPA lysis buffer (Applygen). Protein concentrations were measured with a BCA kit (Applygen). Proteins were subjected to polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore). After blocking, the membranes were incubated with primary antibody (PANX1, GAPDH) and the values of relative protein levels indicate the quantification of band intensities of the indicated proteins using Image J against the loading control GAPDH.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR\u003c/h3\u003e\n\u003cp\u003eTotal mRNA was extracted from HESCs using Trizol. The purified total RNA (1 \u0026micro;g) was reverse transcribed to cDNA according to the instructions for the Prime Script RT kit (Takara). cDNA samples were prepared for subsequent assays. Real-time quantitative PCR was performed using TB Green Mix (Takara) and SYBR Green (Takara) on a 7500 instrument. All expression values were normalized against GAPDH. The following primers were used for the tested genes: PANX1: 5\u0026rsquo;-CAGTTCCAGGTGTTACCGA-3\u0026rsquo; and 5\u0026rsquo;-GCGGCAGCTAATGTACTTG-3\u0026rsquo;, PRL: 5\u0026rsquo;-CCACTACATCCATAACCTCTCC-3\u0026rsquo; and 5\u0026rsquo;-TCATCIGTTGGGCTTGCTC-3\u0026rsquo;, IGFBP1: 5\u0026rsquo;-CACAGGAGACATCAGGAGAAG-3\u0026rsquo; and 5\u0026rsquo;-GATCCTCTTCCCATTCCAAGG-3\u0026rsquo;, GAPDH: 5\u0026rsquo;-GAAGGTCGGAGTCAACGGATTT-3\u0026rsquo; and 5\u0026rsquo;-CTGGAAGATGGTGATGGGATTTC-3\u0026rsquo;.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePANX1 overexpression plasmid construction and lentiviral infectiona\u003c/h2\u003e\u003cp\u003eThe pCDH-CMV-MCS-EF1-copGFP (PCDH) plasmid was used to produce the PANX1 overexpression vector. The 293T cells were inoculated in 6-well plates with a density of 70%, and the density reached 80%-85% after 12 hours culture. Packaged plasmids pVSVG and pSPAX2 were co-transfected with lentiviral vectors in a ratio of 1:3:4:293T. The total plasmid content was 2 \u0026micro;g/ well. 2 hours before transfection, 1 ml/well was replaced with Opti-MEM (Gibco). Dilute 5 \u0026micro;L turboFect (Thermo) in 200 \u0026micro;L Opti-MEM. Then, the configured transfection system was mixed with diluted turboeffect. Each well was supplemented with 5 ml DMEM supplemented with 15% FBS and 10 mM Hepes (Solarbio) replacement medium 12 hours after transfection. 48 hours after transfection, the supernatant was filtered through a 0.45 \u0026micro;m filter. The supernatant was concentrated into concentrated lentivirus using lentivirus precipitation solution (TransGen). Lentivirus was then added to the cell supernatant, and the medium was replaced with complete culture medium 24 hours after infection.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSiRNA and transfection\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates at a density of 2*10\u003csup\u003e6\u003c/sup\u003e/ well and cultured for 12 hours. 2 hours before transfection, 1 ml/well of medium was replaced with Opti-MEM. 20 nM siRNA (Ribose) was diluted in 200 \u0026micro;L Opti-MEM. 5 \u0026micro;L TurboFect (Thermo) was diluted in 200 \u0026micro;L Opti-MEM. Similar to lentivirus transfection, diluted siRNA was mixed with diluted turboeffect. Incubate the mixture at room temperature for 25 min before adding to each well. Medium was replaced with complete medium 12 hours after transfection. The siRNA sequences for PANX1 used the following: siPANX1-01: CAGGAGATCTCGATTGGTA, siPANX1-02: CTCGATTGGTACACAGATA, siPANX1-03: GGTCAAGTCATACAAGTGT.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eHESCs grown in 24-well cell culture plates after drug treatment were gently washed with PBS. The culture-medium was washed and fixed with 4% paraformaldehyde at room temperature for 10 min, and washed with PBS for 3 times. The fixed cells were permeated with PBS solution containing 0.2% Triton X-100 at room temperature for 5 min, and then washed again with PBS for 3 times. Next, CoraLite\u0026reg; Plus 488 labeled ghost pen cyclic peptide (Proteintech) was stained at room temperature for 15 min. After washing three times with PBS, the slides were sealed with anti-fluorescence quencher containing DAPI and read under a confocal microscope.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDetection of ATP levels in culture supernatants\u003c/h2\u003e\u003cp\u003eApproximately 0.5\u0026times;105 cells were seeded in each well of a 48-well cell culture plate and placed in a constant temperature cell incubator for 12 hours. The ordinary complete medium in different cell wells was replaced with 100 \u0026micro;L of decidualized medium every 8 hours, and the previous wells were replaced with new medium before each change. After 48 hours, all wells were changed to normal complete culture medium, and wells without decidualization were used as control group. After 8 hours, the supernatants from each well were collected and transferred to 1.5 mL EP tubes on ice. The cell supernatant was incubated at 500 rpm/min for 5 min at 4\u0026deg;C in a centrifuge, and the system was configured according to the eATP assay kit (Sigma). Absorbance was measured by fluorescence in a multifunctional microplate reader (FLU, λex\u0026thinsp;=\u0026thinsp;535/λem\u0026thinsp;=\u0026thinsp;587 nm), and the fluorescent product was proportional to the amount of ATP present.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePreparation for Compounds\u003c/h2\u003e\u003cp\u003eMifepristone (Solarbio) was solubilized in dimethyl sulfoxide to achieve a 0.8 mg/ml concentration and subsequently stored at -20\u0026deg;C for preservation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEstablishment of Animal Model\u003c/h2\u003e\u003cp\u003e12 female ICR mice (6\u0026ndash;8 weeks old) were housed under controlled environmental conditions with free access to water and food and 12 hours alternating light/dark cycles. (i) We ensured our animal experiment met the standards of the Animal Ethics Committee of Nanchang University and were reviewed and approved by the Animal Ethics Committee of Nanchang University (ethics approval number: NCULAE-20220624011), (ii) we confirmed that all experiments were performed in accordance with relevant guidelines and regulations, (iii) besides, our studies involving live animals comply with the ARRIVE guidelines.\u003c/p\u003e\u003cp\u003eInitially, these mice were randomly divided into control group (n\u0026thinsp;=\u0026thinsp;4), DMSO group (n\u0026thinsp;=\u0026thinsp;4) and RIF group (n\u0026thinsp;=\u0026thinsp;4). Animals were housed at 5:00 pm according to a male to female ratio of 1:2 and female mice were checked the formation of vaginal plugs at 9:00 am in the next day. The day finding the vaginal plug was D0. Female mice were intraperitoneally injected with mifepristone solution (0.1 ml/mouse) on D4 and sacrificed carbon dioxide anesthesia on D12. First we place the mouse into a clean, empty chamber filled with normal air. Then gradually introduce 100% compressed CO₂ at a flow rate that displaces 10\u0026ndash;30% of the chamber's volume per minute. After breathing stops, maintain exposure for at least one minute to ensure death. Finally, the uterus was collected to observe the embryo implantation and to detect the related proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8 software. The student\u0026rsquo;s t-test was used to analyze data between two groups, and one-way analysis of variance (ANOVA) was used to analyze data between three or more groups. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant and marked as *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered as significant difference and marked as **, A p value of less than 0.001 was considered highly significant. All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of at least three independent experiments.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003ePatient characteristics\u003c/h2\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, no significant differences were observed between the control and RIF groups in terms of age, BMI, basal FSH, LH and estradiol levels or mean endometrial thickness. Notably, although there were also no statistically differences in the number of embryos transferred per transfer or in the mean score of transferred embryos, the total number of embryos transferred was significantly higher in the RIF group than in the control group.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical statistical characteristics of the subject population\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl(n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRIF(n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge(y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0641\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal FSH (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal LH (mIU/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal Estradiol (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.72\u0026thinsp;\u0026plusmn;\u0026thinsp;16.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.62\u0026thinsp;\u0026plusmn;\u0026thinsp;14.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6581\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndometrial thickness (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0762\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian number of embryo transfers (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2(1,4)\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.76(4,8)\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage Number of embryos per transferred\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5(1,2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5(1,2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1509\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScore of transferred blastocyst\u003c/p\u003e\n \u003cp\u003e4BC\u003c/p\u003e\n \u003cp\u003e4BB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14/30(46.7%)\u003c/p\u003e\n \u003cp\u003e10/30(33.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13/25(52%)\u003c/p\u003e\n \u003cp\u003e8/25(32%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eThe expression of PANX1 was abnormally increased in RIF patients\u003c/h2\u003e\n \u003cp\u003eThe statistical difference in the number of embryonic transfers between the RIF and control groups supports our further study of the role of PANX1 in the endometrium. In humans, metaplasia of endometrial stromal cells begins autonomously during the secretory phase of the menstrual cycle independent of embryo implantation\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Immunohistochemical results showed that PANX1 was expressed in both mesenchymal and epithelial cells of midsecretory endometrium (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the expression of PANX1 in the RIF group was significantly higher than that in the control group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). These results imply that the increased expression of PANX1 might be closely related with the occurence of RIF.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eDecrease in PANX1 expression induced by 8-Br-cAMP and MPA\u003c/h2\u003e\n \u003cp\u003eNormally, spontaneous decidualization of the human endometrium results from the increased local cAMP, which due to the high level of progesterone. Thus, we treated pHESCs with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) and human medroxyprogesterone acetate (MPA) to induce decidualization in vitro. After treatment with 8-Br-cAMP and MPA for 72 hours, the expression of PANX1 was obviously decreased (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Significantly, HESCs showed a remarkably elevated levels of PRL and IGFBP-1 mRNA, indicating cellular-like decidualization was successfully induced (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Furthermore, when treated HESCs with 8-Br-cAMP or MPA for 0 hours to 72 hours, we found that the expression of PANX1 increased firstly and reached the peak at 24 hours, but decreased gradually from 24 hours to 72 hours (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Consistent with our findings, the extracellular ATP (eATP) level was also increased firstly and gradually decreased after 24 hours (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggests that short-term, rather than long-term, high concentrations of eATP is a key factor to promote the initiation of HESCs decidualizatio, which was supported by the study from Chang et al\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eOverexpression of PANX1 in vitro significantly impaired decidualization of HESCs\u003c/h2\u003e\n \u003cp\u003eTo further explore the effect of PANX1 on decidualization of HESCs, we used lentivirus to infect HESCs in vitro to construct PANX1 over-expression HESCs (pCDH-PANX1). After treating cells with 8-Br-cAMP and MPA, we collected the cellular supernatants to detect eATP concentration. The results showed that the overall level of eATP in pCDH-PANX1 group was higher than that in control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Next, we detected the mRNA levels of PRL and IGFBP-1 in these two groups, which were treated with 8-Br-cAMP and MPA for 72 hours, respectively. We found that the expression levels of both PRL and IGFBP-1 were significantly lower in pCDH-PANX1 group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and D), indicating that overexpression of PANX1 could remarkably impede the decidualization of HESCs. It has been known that undifferentiated HESCs presented as a long fibroblastlike phenotype, decidual HESCs presented polygonal cells, and F-actin filaments were randomly distributed\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Thus, we detected the cellular morphology by immunofluorescence staining, and obversed that compared with the control group, the number of long fibroblasts in pCDH-PANX1 group was increased, and the number of polygonal decidual cells was decreased, signifing the inhibition of HESCs decidualization (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). These data suggest that increased PANX1 could inhibite the HESCs decidualization possibly by maintaining abnormally high levels of eATP.\u003c/p\u003e\n \u003cp\u003eTo further confirm that the abnormally high expression of PANX1 impairs decidualization of HESCs, we next knocked down the PANX1 expression by using siRNA transfcetion (siPANX1) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). After treating with 8-Br-cAMP and MPA for 72 hours, the mRNA levels of PRL and IGFBP-1 in the drug treated group were significantly upregulated compared with those in the untreated group. Significantly, compared with the control group, both pCDH-PANX1 and siPANX1 groups showed cellular-like decidualization damage. Interestingly, treating HESCs in pCDH-PANX1 group with siPANX1 could not rescue the decidualization damage of HESCs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). This finding was affirmed by the results of immunofluorescence staining, which is that knocking down the PANX1 expression obviously inhibited the differentiation of fibroblast-like stromal cells into round and polygonal decidual cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIncreased expression of PANX1 accompanied with embryo implantation failure in mice.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo validate the results of the previous results, we detected the number of embryo implantation and the expression of PANX1 in the endometrium from RIF mouse model. We found that the number of embryo implantation in the RIF group was significantly decreased. Furthermore, uterine hematoma formation and embryo degeneration were also observed in RIF group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). As we excepted, the expression of PANX1 was significantly increased in RIF group compared to control group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). These results further demonstrate that the elevation of PANX1 might result in the RIF.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAssisted reproductive technology has been in clinical practice for more than 40 years, but more than 60% of women still fail to become pregnant after receiving embryo transfer, and about 20% of the patients suffer from unexplained RIF and recurrent abortion\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In human, during the secretory phase of each menstrual cycle, spontaneous endometrial decidualization occurs due to elevated levels of progesterone, and stromal cells differentiate into secretory decidual cells. This process is a key step for uterus to allow embryos to implant\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Abnormal decidualization can affect the implantation window period and directly leads to embryo implantation failure. According to previous studies, impaired decidualization of endometrial stromal cells is an important cause of RIF\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In order to explore the possible molecular mechanisms of impaired decidualization in RIF patients, we detected human endometrium samples at the stage of mid-endometrial secretion and found a significantly increased PANX1 expression in RIF patients\u0026rsquo; endometrium tissues. Furthermore, when the PANX1 was overexpressed in HESCs, a obvious inhibition of HESCs decidualization was observed. These findings indicate that the abnormal high level of PANX1 might be a important reason causing RIF. However, its related regulatory mechanism are still unclear.\u003c/p\u003e\u003cp\u003ePrevious studies have proven the function of PANX1, which transportes the ATP from inside the cell to outside \u003csup\u003e[\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. It has been known that suitable concentratione ATP is very important for the initiation and maintenance of decidualization\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In our study, we found a short-term high concentrations of ATP in the beginning of HESCs decidualization and gradually declined after 24 hours, interestingly, the expression of PANX1 was coincident with the ATP changes. Similar with us, Chang et al also found that short-term exposure to high concentration ATP could promote decidualization of mouse endometrial stromal cells through ATP-P2Y2 signal transduction, but long-term exposure to high concentration ATP would inhibit the decidualization process\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. These results suggest that short-term, rather than long-term, high concentrations of ATP can promote endometrial decidualization. However, overexpressed the PANX1 could significantly impede the HESCs decidualization, which was showed by the reduced expression of decidual markers PRL and IGFBP-1, as well as the disruption of cytoskeletal changes. These data implied that high level of PANX1 might impaired the decidualization of HSECs by influencing the eATP concentration. Surprisingly, there is no rescue effects when we reduced the PANX1 level in the HESCs with PANX1 overexpression, indicating that abnormal increased or decreased the expression of PANX1 both impaired the decidualization of HSECs.\u003c/p\u003e\u003cp\u003eTo further confirm our findings in vitro, we subsequently established a RIF mouse model to detected the PANX1 expression in vivo. Consistent with our previous results in HESCs, we found a significantly increased PANX1 expression in mice with embryo implantation failure, supporting the key role of PANX1 in the pathogenesis of RIF. Nevertheless, our study still has some limitations, including: (1) we only explore the relationship between PANX1 and eATP, and did not further investigate the upstream molecules of PANX1; (2) the animal model we constructed is not very stable, and our research only focuses on phenotype. Therefore, the following works will be to explore the reasons of increased expression of PANX1 in RIF patients and to establish a stable RIF animal model.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, abnormal high expression of PANX1 can impede endometrial decidualization by upregulating extracellular ATP concentrations, which results in embryo implantation failure (Fig.\u0026nbsp;6). Our findings suggested a novel cause of RIF and represented a potential therapeutic target for RIF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declared no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics Statement\u003c/h2\u003e\u003cp\u003e This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Clinical Ethical Committee of Jiangxi Maternal and Child Health Hospital, and informed consents from patients were obtained before the initiation of the study. All the authors consented to participate in this study.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConception and design of the study: Jun Tan. Cell experiments: Qi Yang. Animal experiments: Xia-Lu Liu. Sample collection: Pei-Pei Liu and Li-Yun Cao. Data analysis and interpretation: Xin Li and Zhi-Qin Zhang. Draft of the manuscript: Xia-Lu Liu and Qi Yang. Supervision and critical revision of the manuscript for important intellectual content: Xian-E Sun, Jun Tan and Yao Cheng. All authors have read and confirm that they meet the authorship criteria.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank all the staff, nurses, and physicians at the Reproductive Medicine Center for their support in generating this manuscript. This work was supported by the National Natural Science Foundation of China (82360318), the Science Project of Science and Technology Department of Jiangxi Province (20232BAB206029), the Outstanding Youth Fund Program of Science and Technology Department of Jiangxi Province (20224ACB216005), the Youth Fund Program of Science and Technology Department of Jiangxi Province (20232BAB216024) and the JXHC Key Laboratory of Fertility Preservation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFranasiak, J. M. et al. A review of the pathophysiology of recurrent implantation failure. \u003cem\u003eJ. Fertil. Steril.\u003c/em\u003e \u003cb\u003e116\u003c/b\u003e, 1436\u0026ndash;1448 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBashiri, A., Halper, K. 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Signal\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSang, Y., Li, Y., Xu, L., Li, D. \u0026amp; Du, M. Regulatory mechanisms of endometrial decidualization and pregnancy-related diseases. \u003cem\u003eJ. Acta Biochim. Biophys. Sin (Shanghai)\u003c/em\u003e. \u003cb\u003e52\u003c/b\u003e, 105\u0026ndash;115 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRusiecka, O. M., Tournier, M., Molica, F. \u0026amp; Kwak, B. R. Pannexin1 channels-a potential therapeutic target in inflammation. \u003cem\u003eJ. Front. Cell. Dev. Biol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1020826 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDahl, G. ATP release through pannexon channels. \u003cem\u003eJ Philos. Trans. R Soc. Lond. B Biol. Sci\u003c/em\u003e \u003cb\u003e370\u003c/b\u003e, (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVultaggio-Poma, V., Sarti, A. 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Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. \u003cem\u003eJ. Embo j.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 5071\u0026ndash;5082 (2006).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RIF, human endometrial stromal cells, PANX1, endometrial decidualization, eATP","lastPublishedDoi":"10.21203/rs.3.rs-7512759/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7512759/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackgrounds:\u003c/h2\u003e\u003cp\u003eRecurrent implantation failure (RIF), a important contributor to infertility in assisted reproductive technologies, is believed to be primarily to abnormal endometrial decidualization. However, its related molecular mechanisms are still incompletely clear. It has been reported that PANX1 is an ATP-permeable channel to regulate the extracellular ATP, and ATP level could influence the progress of endometrial decidualization. Therefore, we conduct this study to explore the potential role of PANX1 during the development of RIF.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, we first compared the the expression of PANX1 in RIF patients and control patients. Then, the primary human endometrial stromal cells (pHESCs) was used to the explore the regulatory function of PANX1 on endometrial decidualization by Immunofluorescence staining, Western blot, RT-PCR, Plasmid transfection and ATP detection. Finally, animal model was established to further confirm the results which were found in vitro.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ewe observed a significant upregulation expression of ATP release channel protein Pannexin1 (PANX1) in endometrial tissue of RIF patients. Moreover, after overexpressing PANX1 in human endometrial stromal cells (HESCs), we found a significantly high concentration of extracellualr ATP (eATP) and the inhibition of HESCs decidualization, which was characterized as the decreasing expression of prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), as well as the aberrant morphologic cytoskeletion of HESCs. Surprisingly, knockdown the expression of PANX1 in HESCs also impaired the cellular decidualization, indicating the important role of normal PANX1 expression during the process of endometrial decidualization. The subsequent animal study further confirmed our findings in vitro.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe present study clearly showed that abnormal high expression of PANX1 hindered endometrial decidualization through the upregulation of eATP concentrations, ultimately leading to embryo implantation failure. Our findings suggested a novel cause of RIF and identified a potential therapeutic target for RIF.\u003c/p\u003e","manuscriptTitle":"Abnormal elevated PANX1 expression hampers endometrial decidualization by upregulating extracellular ATP concentration in patients with recurrent implantation failure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 02:04:51","doi":"10.21203/rs.3.rs-7512759/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T04:50:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T13:41:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77513268489055027897068055717537962799","date":"2025-10-21T16:53:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T06:28:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146177386672406782979201367408927319451","date":"2025-10-16T22:09:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178658403002497739269742073531672691457","date":"2025-10-07T08:08:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-03T02:10:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T01:45:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-16T15:16:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T08:56:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-09T08:53:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f90470e7-89d9-4428-82d0-649e43e826f4","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56166500,"name":"Biological sciences/Cell biology"},{"id":56166501,"name":"Health sciences/Diseases"},{"id":56166502,"name":"Health sciences/Medical research"},{"id":56166503,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-09T16:02:36+00:00","versionOfRecord":{"articleIdentity":"rs-7512759","link":"https://doi.org/10.1038/s41598-026-41800-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-03 15:57:33","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-10-17 02:04:51","video":"","vorDoi":"10.1038/s41598-026-41800-z","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41800-z","workflowStages":[]},"version":"v1","identity":"rs-7512759","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7512759","identity":"rs-7512759","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}