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

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 24 . 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. Fig. 1 Abnormal high expression of PANX1 in endometrium tissues from patients with RIF. Immunohistochemical test showed that PANX1 was expressed in both mesenchymal and epithelial cells in endometrium. Scale bar = 20 μm ( A ). However, compared with the normal patients, RIF patients showed a significantly increased expression of PANX1 in endometrium ( A - C ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test，** P< 0.01 (n=5) Abnormal high expression of PANX1 in endometrium tissues from patients with RIF. Immunohistochemical test showed that PANX1 was expressed in both mesenchymal and epithelial cells in endometrium. Scale bar = 20 μm ( A ). However, compared with the normal patients, RIF patients showed a significantly increased expression of PANX1 in endometrium ( A - C ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test，** P< 0.01 (n=5) Normally, spontaneous decidualization of the human endometrium results from the increased local cAMP, which due to the high level of progesterone. Thus, we treated HESCs 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 h, 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 h to 72 h, we found that the expression of PANX1 increased firstly and reached the peak at 24 h, but decreased gradually from 24 h to 72 h (Fig.  2 E). Consistent with our findings, the extracellular ATP (eATP) level was also increased firstly and gradually decreased after 24 h (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. 25 . Fig. 2 The expression of PANX1 decreased and eATP changed during the decidualization of HESCs. After 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 ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, * P <0.05, ** P< 0.01, *** P <0.001 (n=3). The expression of PANX1 decreased and eATP changed during the decidualization of HESCs. After 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 ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, * P <0.05, ** P< 0.01, *** P <0.001 (n=3). 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 h, 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 elongated stromal cells 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. Fig. 3 Overexpression of PANX1 impaired HESCs decidualization. The 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 . Scale bar = 2 μm ( E ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, * P <0.05, *** P <0.001 (n=3). Overexpression of PANX1 impaired HESCs decidualization. The 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 . Scale bar = 2 μm ( E ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, * P <0.05, *** P <0.001 (n=3). 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 h, 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). Fig. 4 PANX1 knockdown impaired decidualization of HESCs. After 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. Scale bar = 20 μm ( E ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, *P<0.05, **P<0.01,***P<0.001 (n=3). PANX1 knockdown impaired decidualization of HESCs. After 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. Scale bar = 20 μm ( E ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, *P<0.05, **P<0.01,***P<0.001 (n=3). 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). Comprehensive histological examination, primarily utilizing hematoxylin and eosin (H&E) staining, demonstrated a significantly attenuated endometrial epithelium in the RIF murine model. Notably, this structural alteration was accompanied by dense inflammatory cell infiltration within the stromal compartment. Furthermore, immunohistochemical analyses indicated a marked upregulation and high expression of PANX1 in these pathological tissues (Fig.  5 E and F). These results further demonstrate that the elevation of PANX1 might result in the RIF. Fig. 5 Mice with RIF showed a increased expression of PANX1. Compare 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 ). Histological analysis revealed that the endometrium of RIF mice was thinner, accompanied by massive inflammatory cell infiltration and high expression of PANX1 ( E , F ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 (n=3). Mice with RIF showed a increased expression of PANX1. Compare 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 ). Histological analysis revealed that the endometrium of RIF mice was thinner, accompanied by massive inflammatory cell infiltration and high expression of PANX1 ( E , F ). Data are presented as mean±standard deviation (mean±SD). Differences between two groups were compared using unpaired Student’s t-test, ns means no statistical significance, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 (n=3).

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 ( n  = 25). Patients with tubal obstruction or unexplained infertility who achieved clinical pregnancy after the first embryo transfer were assigned to the Control group ( n  = 30). The clinical data of all the patients from 1 st 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. 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 14/30(46.7%) 13/25(52%) 4BB 10/30(33.3%) 8/25(32%) Clinical statistical characteristics of the subject population. The healthy menstrual cycle comprises four sequential phases: the menstrual phase, proliferative phase, ovulatory phase, and luteal phase. Primary human endometrial stromal cells (HESCs) were obtained from healthy female volunteers ( n  = 3) of reproductive age with natural regular menstrual cycles, and endometrial biopsies were performed during the proliferative phase of menstruation, a stage when endometrial decidualization occurs. 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 μm 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. Dissolve cAMP (Sigma) in sterile nuclease-free water and MPA (MCE) in DMSO (sonicate to aid dissolution) in a biosafety cabinet; store at −80 °C. For use, prepare decidualization-inducing medium containing 0.5 mM 8-Br-cAMP and 1 µM MPA 22 . Seed human endometrial stromal cells (HESCs) into 6-well plates and culture in medium supplemented with charcoal-stripped fetal bovine serum. When the cell confluency reaches 80%, replace the regular medium with the decidualization-inducing medium. In routine experiments, maintain decidualization treatment for 72 h; in dynamic observation experiments, treat the cells for 0 h, 24 h, 36 h, 48 h, 60 h, and 72 h respectively. Fresh human endometrial tissue was fixed with 4% paraformaldehyde for more than 24 h, 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. 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. 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’. The pCDH-CMV-MCS-EF1-copGFP (PCDH) plasmid was used to produce the PANX1 overexpression vector. The 293 T cells were inoculated in 6-well plates with a density of 70%, and the density reached 80%−85% after 12 h 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 h 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 h after transfection. 48 h 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 h after infection. Cells were seeded in 6-well plates at a density of 2*10 6 /well and cultured for 12 h. 2 h 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 h after transfection. The siRNA sequences for PANX1 used the following: siPANX1-01 : CAGGAGATCTCGATTGGTA, siPANX1-02 : CTCGATTGGTACACAGATA, siPANX1-03 : GGTCAAGTCATACAAGTGT. 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. 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 h. The ordinary complete medium in different cell wells was replaced with 100 µL of decidualized medium every 8 h, and the previous wells were replaced with new medium before each change. After 48 h, all wells were changed to normal complete culture medium, and wells without decidualization were used as control group. After 8 h, 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. Mifepristone (Solarbio) was solubilized in dimethyl sulfoxide to achieve a 0.8 mg/ml concentration and subsequently stored at −20 °C for preservation. 12 female ICR mice (6–8 weeks old) were housed under controlled environmental conditions with free access to water and food and 12 h 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. Finally, the uterus was collected to observe the embryo implantation and to detect the related proteins 23 . 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.

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 26 . 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 27 , 28 . 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 29 . 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 30 – 33 . It has been known that suitable concentratione ATP is very important for the initiation and maintenance of decidualization 21 , 25 . In our study, we found a short-term high concentrations of ATP in the beginning of HESCs decidualization and gradually declined after 24 h, interestingly, the expression of PANX1 was coincident with the ATP changes. 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]. 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 25 . 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, on the contrary, it further suppressed the mRNA expression levels of the decidualization markers IGFBP-1 and PRL . We hypothesize that the silencing of PANX1 by siRNA leads to excessive ATP accumulation in HESCs, thereby causing functional impairment of these cells, 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.

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. Fig. 6 Pattern of decidualization damage caused by abnormal high level of PANX1. Overexpression of PANX1 in endometrial stromal cells leads to massive eATP efflux, and induction with 8-Br-cAMP and MPA fails to induce decidualization changes in these cells. which is a possible reason of RIF. Pattern of decidualization damage caused by abnormal high level of PANX1. Overexpression of PANX1 in endometrial stromal cells leads to massive eATP efflux, and induction with 8-Br-cAMP and MPA fails to induce decidualization changes in these cells. which is a possible reason of RIF.

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 endometrium may play a important role in embryo implantation 7 . This process 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 elongated stromal cellsinto secretory cells with large, round, polygonal nuclei 9 . In early pregnancy, this process is traditionally induced by estrogen and progesterone levels, as well as other factors, such as cyclic adenosine monophosphate (cAMP) 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 . 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 . eATP 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.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1