As a key effector of the Hippo pathway, yes‐associated protein (YAP) is a major regulator of cell proliferation and apoptosis. In this study, 23 hYAP isoforms were identified in HEK293 cells, with 14 isoforms being reported for the first time. These isoforms were classified into hYAP‐a and hYAP‐b isoforms based on the variation in exon 1. The two groups of isoforms showed distinctly different subcellular localizations. hYAP‐a isoforms could activate TEAD‐ or P73‐mediated transcription, affect the proliferation rate, and enhance the cellular chemosensitivity of HEK293 cells. Moreover, different activation abilities and pro‐cytotoxic effects were observed among hYAP‐a isoforms. However, hYAP‐b isoforms were not found to exert any significant biological effects. Our findings add to the knowledge of YAP gene structure and protein‐coding capacity and will help in the elucidation of the function and related molecular mechanisms of the Hippo‐YAP signaling pathway. Fourteen novel isoforms of human YAP have been identified, bringing the total number of isoforms to 23. These isoforms were classified into hYAP‐a and hYAP‐b groups according to differences in exon 1. Functional comparison indicated that YAP‐a isoforms activate TEAD‐ or P73‐mediated transcription, affect proliferation rate, and enhance chemosensitivity. However, hYAP‐b isoforms were not found to exert any significant biological effects. ASalternative splicingFBSfetal bovine serumhP73αhuman P73αhYAPhuman YAPKDknockdownMWmolecular weightsOEoverexpressionORFsopen reading framesTADtranscriptional activation domainTSStranslation start siteYAPyes‐associated proteinAlternative pre‐mRNA splicing is the process by which exons are assembled to form multiple mature mRNAs . The majThe Hippo signaling pathway is an evolutionarily conserved network that plays a crucial role in maintaining organ size. The core components of the canonical Hippo pathway in mammals include MST1/2, SAV1, LATS1/2, MOB1A/B, and YAP/TAZ. YAP is the key effector of this pathway. When the Hippo pathway is inactive, YAP translocate to the nucleus, and forms a complex with the transcription factors to regulate the expression of their downstream target genes, thus affecting cell proliferation, apoptosis, and drug resistance , 9, 10.YAP (hYAP) gene contains nine exons and encodes a 65‐kDa protein. hYAP protein comprises a TEAD factor‐binding domain, one or two WW domains, an SH3‐binding domain, a transcriptional activation domain (TAD), and a PDZ‐binding motif  was added into 1.5 mL EP tubes to resuspend the cells. The tubes were incubated at 37 °C for 30 min. Then, 100 μL of staining solution  was added to the EP tubes. The solution in the tubes was mixed well, incubated in the dark for 10 min, and analyzed using flow cytometry . Data were analyzed using the noveexpress software.After seeding HEK293 cells into 6‐well plates for 72 h, cell layers were digested with trypsin and fixed with 70% ethanol at −20 °C overnight. Subsequently, the cells were washed with phosphate‐buffered saline (PBS), and the cell precipitate was collected. Next, 100 μL of solution [containing 90 μL PBS, 10 μL Triton X‐100, 0.1 μL RNase A (0.2%), and 100 μg·mLspss for Windows package release 25.0, Chicago, IL, USA) was used for statistical analysis of variance via Student's t‐test and Dunnett's t‐test. Data were plotted as mean ± SD, and P < 0.05 was considered statistically significant. Each experiment was conducted at least three times.The Statistical Package for Social Sciences . Each member was named according to the length of their coding amino acids, including newly identified five hYAP‐a isoforms  and nine hYAP‐b isoforms . The occurrence rates for other hYAP‐a/b isoforms were lower than 10% , alternative 5′ splice site of exon 5 (which codes for amino acids DFFFLFIS), alternative 3′ splice site of exon 5 (which codes for amino acids VRPQ) and skipped exon of exon 4 or exon 6. Based on the conservation of AS events, we predicted the existence of rms Fig. . In thes10% Fig. . Quantitrms Fig. .To analyze the expression pattern of hYAP isoforms, we performed nested PCR amplification of the cDNA obtained from various human tissues and cell lines, followed by gel electrophoresis. Both hYAP‐a and hYAP‐b were detected in the lung, liver, testis, brain, pancreas, ovary, spleen, prostate, small intestine, and kidney tissues, but with expression profiles of multiple isoforms. For instance, we can detect hYAP‐a504, hYAP‐a488, and hYAP‐b310 in the lung, while hYAP‐a504 and hYAP‐b326 in the liver. In addition, only hYAP‐a isoforms (hYAP‐a504 and hYAP‐a488) were detected in skeleton and thymus, and only hYAP‐b isoforms (hYAP‐b330 and hYAP‐b326) were detected in the heart. Neither hYAP‐a nor hYAP‐b isoforms were detected in colon and leukocyte samples Fig. . hYAP‐a The function of YAP is closely related to its subcellular localization. To assess whether the sequence of isoforms affected their subcellular localization, 10 isoforms with representative splicing modes and high expression levels were chosen for further analysis Fig. . Each GFAs a cofactor, YAP cannot bind DNA directly . PredomiYes‐associated protein is an important regulator of cell proliferation, but the functional differences among different isoforms remain unknown. Our results showed that hYAP‐a508 OE enhanced the proliferation of HEK293 cells; however, hYAP‐a488 OE was detrimental to cell expansion. The proliferation ability of cells containing hYAP‐a506, hYAP‐a504, hYAP‐a466, and all hYAP‐b OE did not differ significantly from that of control cells Fig. A. Cell cChemotherapeutic agents, such as cisplatin and adriamycin, can lead to cell apoptosis by inducing DNA damage. Previous studies have shown that YAP OE enhanced the sensitivity of cells to these agents . To inveBAX and PUMA) [YAP‐induced chemo‐sensitization probably involves several different mechanisms , 30, 31.nd PUMA) , 34, 35.BAX gene promoter [To further explore the effect of YAP isoforms on P73‐mediated transcriptional activation ability, we constructed a P73‐responsive luciferase reporter system carrying a promoter . The lucAS is an important post‐transcriptional regulation mechanism that enables one gene to produce multiple isoforms, thus increasing the complexity of the transcriptome and proteome , 37. As Previous studies have confirmed the existence of nine isoforms of hYAP , 40. In Yes‐associated protein activity is primarily regulated by its subcellular localization. Extensive research has shown that when the Hippo signaling pathway is activated, YAP is phosphorylated at serine 127, the phosphorylated YAP is then captured by 14–3–3 protein and retained in the cytosol. When the Hippo pathway is inhibited, this YAP site is dephosphorylated and separated from 14–3–3 protein, and the YAP protein is then translocated to the nucleus , 39, 41.The luciferase assay showed that all hYAP‐a isoforms activated TEAD‐mediated transcription but to varying degrees. Among them, hYAP‐a488 exhibited the strongest transactivation ability, while hYAP‐a466 exhibited the weakest. Protein structural analysis showed that only hYAP‐a488 had a complete leucine zipper and two WW domains. The other hYAP‐a isoforms contained disrupted leucine zippers, while hYAP‐a466 also lacked the WW2 domain. These results suggested that the leucine zipper and the WW2 domain contributed to the transactivation ability of hYAP. Thus, we speculated that hYAP‐a496, hYAP‐a494, hYAP‐a492, and hYAP‐a490, all of which carried the complete leucine zipper and two WW structural domains, may also exhibit stronger transactivation ability than the hYAP isoforms that lack these structures. In addition, the sequentially decreasing regulatory ability was observed for hYAP‐a508, hYAP‐a504, and hYAP‐a506, suggesting that the transactivation ability of hYAP enhanced after the alternative 3′ splice site of exon 5 (with the addition of amino acids VRPQ), but repressed by alternative 3′ splice site of exon 4 (with the addition of amino acids GK).P21, BAX, and PUMA) [Previous research has shown that YAP plays a crucial role in cellular sensitivity to chemotherapeutic agents. On the one hand, YAP can promote cell growth and proliferation by interacting with TEADs or AP‐1 or by decreasing AKT‐mediated nuclear localization of P27Kip1 , 46, 47.nd PUMA) , 35, 48.nd PUMA) , 35. InthYAP‐b isoforms were widely distributed in normal human tissues and tumor cell lines. However, none of the hYAP‐b isoforms in this study were found to exhibit any significant biological effects. Previous studies have found that WW1 might be the main domain that mediates the interaction of YAP with other proteins . The hYATaken together, we identified 23 hYAP isoforms in HEK293 cells and found that the different molecular structures resulting from variant splicing modes might affect their subcellular localizations, transactivation abilities, and cellular drug resistance‐imparting abilities. These findings will not only lay the foundation for understanding the function and regulatory mechanisms of YAP but also help elucidate the role of the Hippo signaling pathway in several diseases.The authors declare no conflict of interest.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/2211‐5463.13618.The peer review history for this article is available at LLL and JLZ designed and carried out the experiments, analysis, and interpretation of the data and manuscript writing. JLW, JQW, YPT, YXX, YDC, and MY provided technical assistance. YY, RY, XYW, JJW, WW, CZ, and YH analyzed and interpreted the data. RJ, LX, and YR designed the experiments and directed the study. All authors have read and approved the final version of the manuscript.Fig. S1. Effects of hYAP isoforms on cell proliferation and apoptosis. (A) Effects of hYAP isoforms on cell proliferation were analyzed by CCK‐8 assay. (B‐C) The mRNA and protein expression levels of BAX in the indicate cell lines after cisplatin (B) and adriamycin (C) treatment. Data in A, B, and C were represented as mean ± SD; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. All P‐values were calculated using Dunnett's t‐test.Table S1. Primers for plasmid construction.Table S2. Primers for semi‐quantitative PCR analysis.Table S3. Primers for quantitative real‐time PCR analysis.Table S4. Antibody details.Click here for additional data file.


Cavity shaving (CS) is a surgical technique used in the treatment of breast cancer (BC). It may reduce margin positivity in histologic assessment and consequently reduces re‐ excision rates in breast conserving surgery (BCS). The evidence for this assumption is described in the present review.A systematic review of relevant literature in English from January 1999 to April 2019 was conducted. The analysis included studies on CS and its effects on re‐excision rates and margin positivity. We searched PubMed databases for relevant publications. In total, 22 studies were included in the present review.The benefit from CS on re‐excision rates and histologic margin positivity was variable. Out of 22 studies, 17 reported a reduction in both re‐excision rates and histologic margin positivity in margin shaved patients. Four studies could not find a significant reduction of second surgeries and residual tumor rates. One study suggested that CS after BCS was superior to single BCS only in subgroup analysis in IDC tumors.CS is a surgical technique that was shown to reduce re‐excision and margin positivity rates in most of the studies. Furthermore, it can be a useful tool to assess specimen margins and detect multifocality. Breast cancer (BC) is the most frequent cancer in women worldwide. By now, BC is diagnosed at earlier stages due to wider use and higher sensitivity of BC screening. Thus, the majority of patients are suitable for breast conserving surgery (BCS). Provided that adjuvant radiotherapy is applied, patients with BCS have an equivalent survival rate compared to patients with radical mastectomy . AlthougSeveral studies demonstrated that CS could be an easy and effective procedure to decrease positive margin and re‐excision rates. Whether the classically performed excision of selective margins after radiologic or sonographic intraoperative imaging might be sufficient to improve residual tumor rates (R1/R0) remains unclear. The value of additional CS has been queried as multifocality might outweigh margin status in causing BCS failure. Moreover, excision of more tissue could result in poorer cosmetical outcome. Some authors propose that CS is a tool for assessing tumor margins more accurately and detecting multifocality. It is also claimed that CS may reduce re‐excision rates, which implies avoiding general anesthesia, surgical complications  and shortening of patient´s recovery time. Reducing re‐excision rates may also increase compliance and adjuvant therapy could be administer faster.A systematic search of literature was performed using PubMed databases identifying studies published from January 1999 to April 2020. Studies were eligible for inclusion if they evaluated the role of CS in re‐excision rates in BCS and included at least 80 patients or more. The following groups of key words were used: (“cavity shaving” or “margin shaving” or “cavity margin shaving”) and (“lumpectomy” or “breast‐conserving surgery/therapy” or “breast cancer”). The language was limited to English publications.The studies included vary from comparative studies, including randomized-controlled trials (RCTs) or non‐randomized studies (NRS), self‐control studies, which compared CS with standard lumpectomy in patients with BC undergoing BCS, reviews, and meta‐analyses. BC stages ranged from 0 to III. Different criteria for margin assessment such as “no ink on tumor” but also margin widths from 1 to 5 mm were accepted. However, margins assessed by imaging‐guide techniques were excluded. Only studies assessing the application of CS at the initial surgery were included. When multiple groups were analyzed in one study, the comparison between BCS plus CS and BCS alone was selected. Further evaluation of locoregional or distant recurrence rates was not examined in this review. The main questioning of this study were residual tumor rates and excision rates. Cosmetic outcomes and excised tissue volume were reported as non‐priority aspects.n), study periods, and outcomes of interest. To assess the quality of the studies, the Quality and Oxford Center of Evidence based Medicine (OCEBM) level of evidence (LOE) was used.The following information was extracted from the studies Table : author,172 publications were identified after entering the keywords described above. Irrelevant records, which did not deal with the topics “re‐excision” and/or “cavity margin shaving”, were excluded. Studies in which CS was performed as a second surgery were also excluded. Finally, 22 publications were assessed for eligibility. All of them analyzed the effect of margin shaving on re‐excision rates. Of these, there was one systematic review and meta‐analysis and two randomized‐controlled trials. One study was a prospective follow‐up study.Eighteen studies were retrospective: of these, nine were cohort studies and one was a case‐matched study. The 22 eligible studies were published between 1999 and 2019 with sample sizes from 99 to 976 patients. Patients with BC stages I–III with an indication for BCS were included. The distribution of histological types varied. Twelve studies included invasive ductal carcinoma (IDC), DCIS, IDC with extensive intraductal component, and invasive lobular carcinoma (ILC) –16. SeveDCIS –23, onlyThere was heterogeneity among the selected studies in definitions of margin shaving . Three sp < 0.05], which results in a OR reduction of 59% [p < 0.05]. CS did not seem to increase excised tissue volume compared to BCS alone and it did not decrease locoregional or distant recurrence compared to BCS alone. Although high heterogeneity was present among studies, this claim remained significant for self‐control and comparative studies . This systematic review included 24 retrospective, non‐randomized studies and two randomized-controlled trials; one of them was the trial of Chapgar et al. which represented the most convincing and statistically powerful evidence of the benefit from CS vs BCS alone  and multifocality . The weaknesses of this study were the potential patient and provider bias as well as surgeon‐dependent factors.Mukhtar et al. performed a retrospective cross‐sectional analysis with multivariate model of a cohort of 358 prospectively collected women with invasive lobular carcinoma. The rate of margin positivity was 43% and the re‐excision rate was 25%, which seems to be consistent with the other studies. They compared simple BCS with BCS with oncoplastic techniques or CS for final residual tumor . They caIn the study of Marudanayagam et al., 786 patients were retrospectively analyzed in cohorts and divided into subgroups: 394 patients had a BCS with CS and 392 had BCS only .p < 0.01). Of these 49 patients who needed reoperation, 46 underwent re ‐excision and 44 patients h ad clear resection margins. I n 44 of 49 cases, reoperation was avoided because of CS. Of the 394 patients who obtained BCS with CS, in 52 cases, tumor was found in cavity excision; in 22 cases, re‐excision was necessary; in 30 cases, tumor could be resected in sano. This means that CS could be a useful tool for detecting multifocality and DCIS. A major point of criticism was that differences in re‐excision rates and margin positivity between surgeons were not taken into account. Multifocality as an independent predictive factor for residual tumor on margin was not analyzed.Invasive ductal carcinoma and DCIS were included in the study. The rates of re‐excision were 5.58% (22 patients) in BCS with CS and of 12.5% (49 patients) in standard BCS (p < 0.001). Also, re‐excision rates were significantly lower in the BCS with CS group . OR for positive margin status was 6.2  without CS, while OR for re‐excision was 5.46 . Residual tumor was detected in 20% of patients who underwent BCS with CS, though, in 18.3%, R0‐status was achieved with CS. The re‐excision rates were 18.9% vs 1.9% in BCS and BCS with CS (p < 0.001). A significant reduction of R1‐disease and re‐operation rates was found in luminal A and luminal B such as in triple‐negative breast cancer. In contrast, in HER2‐positive tumors, no benefit from CS was achieved, which is in contrast to other studies. This retrospective cohort study included a relatively high sample volume of patients and the groups had a heterogeneous distribution: the group of BCS with CS included 812 patients, the group of simple BCS 164 patients. Apart from the lack of randomization, only pathologic features were considered in tumors instead of genetic profiling, e.g., besides, there were more patients with luminal than with triple-negative or HER2‐positive breast cancer.In the retrospective cohort study by Corsi et al., 976 patients were evaluated , as well as when larger extent of intraductal component was found in samples . CS was well standardized  in this study, meaning that authors tried to define CS consistently. Though, the small number in group samples (69 patients in each group) and the retrospective nature of this study has to be taken into account.A retrospective, case‐matched and single‐institution analysis by Kobbermann et al. described similar reoperation rates of totally 31.9% . In the N = 33 vs. N = 109 vs. N = 34). The largest excised volume was in the group with pMRI (N = 33) and selective shave margins (SM) (N = 34). In this study, only patients with DCIS were included. So et al.   .p = 0.001] by CS. CS was able to reduce re ‐excision rates in multivariate analysis .Janes et al. recorded data from 217 patients undergoing BCS . 111 patFurthermore, Janes et al. were the only investigators finding that fewer tissue volume was excised in the group of CS compared to standard BCS .p < 0.0001). They reported tumor‐free margins in 90.7% in BCS vs 92.7% in BCS with CS (p = 0.69). No statistically significant difference could be seen between both groups regarding the removed volume.Pata et al. conducted a retrospective case‐matched study of 298 women . 179 of N = 29 patients), in 23 cases after initially negative margins in lumpectomy specimen, and in 6 cases after initially negative lumpectomy margins.Hequet et al. also described a reduction in re‐excision rates after CS in a retrospective study which included 99 patients who underwent BCS and systematic CS . CarcinoN = 25 patients) had a negative cavity shaving histology. In these cases, false-positive results were avoided due to CS. Re‐excision was avoided in 25% of the patients who underwent BCS with CS, as they had positive lumpectomy margins but negative CS margins.Thus, they claimed that CS might be useful in the diagnosis of multifocality. Of all patients with initially positive lumpectomy margins, 25.3% (N = 99) retrospectively and could therefore not analyze whether there was an interpersonal surgeon‐dependent effect on positive margin status or not.Significant association with carcinoma detection in cavity shaving histology could be found in patients with invasive lobular carcinomas, wider carcinoma diameter, positive lymph‐node status, and positive margins in wide local excision. No predictive risk factors for positive margins could be found and there was no group of patients that benefit from margin shaving. CS was performed in four directions in this study, and excision in ventral and dorsal direction was not counted, since the muscular fascia and part of the subcutis were excised. Though, surgery was performed by different surgeons and volume or width of the additional margins taken were not specified. This study analyzed only a relatively small number of patients (p = 0.44) [The reduction of re‐excision rates after CS claimed by Pata et al. was also not statistically significant with rates of 14.3% vs 10.6% in BCS with CS ( = 0.44) . Though, = 0.44) .In multivariate analysis, distance of tumor from initial lumpectomy margins, multifocality, receptor status, and tumor size were related to margin positivity in CS as predictive factors. Concluding, CS was shown to have no prognostic improvement regarding the “no ink on tumor” policy. Though, it provides wider clear margins, reinforces complete tumor removal (R0‐status) and helps overcoming false-positive margins with no wider volume removal.Feron et al. examined 100 patients who underwent BCS and CS. Final histological results clearly showed residual tumor in 25% of cases, whereas R0‐status was obtained in 67% of the cases . InitialThe study of Cao et al. analyzed 126 BCS histopathologic margins regarding their respective CS margins . It showN = 38 patients) of cavity shaved margins in their retrospective study. Of them, 33 patients underwent re ‐excision. When initial margins were positive and CS margins negative, in 20 of totally 107 cases (18.7%), re‐excision was avoided due to careful examination of CS margins. In 13 cases (12.2%), re‐excision was done despite initial margin negativity of tumor bed, rendering again a useful tool in diagnosis of multifocality. Of all analyzed factors, tumor size and non‐menopausal status were the only significant independent predictive factor for margin positivity.Tengher‐Barna et al. selected 107 patients with BCS with CS and analyzed the reoperation rates and the close margin rate . Severalp = 0.008), whereas lumpectomy margins predicted R1‐status with 64,8% accuracy. This predictor had a sensitivity of 50.9% and a specificity of 69.5%. This study correlated with the findings of Heiss et al. [Tang et al. analyzed pathology reports of 242 patients with invasive ductal carcinoma, DCIS and invasive lobular and tubular carcinoma and compared positivity of initial lumpectomy margins and its concordance to CS margins . They fos et al. . CS margN = 83) of all tumor specimens residual tumor was found [N = 42) of specimens were free of tumor. For 48% of patients, re‐excision was avoided due to CS. In 50% (N = 63) of the cases, the technique of CS did not change therapy management, since in 33% (N = 41), initial margins were already negative, and in 18% (N = 22), margins remained positive even after CS. According to this study, re‐excision rates tend to be rather high in the analyzed institution, since 66% (N = 83) of specimens had residual tumor after surgery. No control group with single BCS without CS was included for comparison.Jacobson et al. performed a retrospective study with 125 patients who had undergone BCS with CS. In 66%  were analyzed.p < 0.001) and the existence of ILC or DCIS (p = 0.043) were significant predictors for residual tumor on CS. An important bias of this study was that all surgeries were performed by one surgeon. There was also no comparison with additional CS and single BCS performed without additional shaving.A retrospective, single‐institution study conducted by Keskek et al. analyzed 301 patients with 303 breast cancers . Of thesN = 171 patients with a positive CS, 82 (48%) had final R0. of the 761 patients with R0. 82 (10.8%) had a positive CS. There was poor concordance between positivity of resection margins and CS. Summarizing, CS is an important tool detecting residual and multifocal tumor, since margin and cavity positivity are often not concordant. A main point of criticism of this study is the lacking analysis of interpersonal differences in re‐excision rates between surgeons.In the Breast Unit in Willesborough in the UK, a retrospective study by Hewes et al. gathered 957 patients over 21 years . The iniCS has been claimed to reduce secondary surgery caused by positive margins after BCS. The addition of CS differs from classical BCS in extent and width. Several sampling techniques have been described before. In contrast, CS is a relatively consistent method, with complete resection of the surface and sufficient width of the cavity wall. This review suggested that additional CS had a lower PMR than BCS alone.p = 0.01) [p = 0.02). CS was associated with a 59% OR reduction in the tumor‐involved margin and re‐excision rates [p = 0.65) nor in margin positivity (p = 0.07) [According to the randomized-controlled trial of Chapgar et al. after randomization, there was a significant reduction of histologic margin positivity in BCS with CS compared to BCS alone ( = 0.01) . Re‐excion rates . This ason rates , althougon rates . In anot = 0.07) . This coWhether CS is necessary remains unclear in the study conducted by So et al. in which no significance could be reached when comparing CS and selective shaving margins . Chen etThough, Pata claimed CS avoided re‐excision in some cases, reduced false-positive margin status and contributed to a more accurate margin examination, i.e., for multifocality. These findings correlated with the study of Hequet et al. in which multifocality was diagnosed in 6.1% despite initial negative margins . Feron eOf all 18 retrospective studies, five found a significant reduction in margin positivity rates and, therefore, re‐excision rates, whereas in 13 studies, only a tendency for CS to reduce residual tumor or avoid re‐excision was seen. Summarizing, the majority of studies did not find statistical significance for CS to be a valid technique in reducing re‐excision rates.Some studies described effects of CS only in certain subgroups, such as in invasive ductal carcinoma , multifoOut of 22 studies, 17 reported a reduction in both re‐excision rates and histologic margin positivity in margin shaved patients . Four studies could not find a significant reduction of second surgeries and residual tumor rates , 21, 25.p = 0.0003 and p = 0.005 [Several of the 22 identified studies analyzed predictive risk factors for margin positivity or residual tumor which could predict risk for re‐excision. Kobbermann et al. found that CS and extensive intraductal component were significant risk factors:  = 0.005 . Unzeiti = 0.005 . Keskek  = 0.005 , whereas = 0.005 , triple-.This systematic review has several limitations. As retrospective studies or non‐randomized studies were included, recall bias and selection bias were unavoidable. The definition of positive of shaved margins was not consistent, and the standard of positivity varied between studies with respect to the distance from the cut edge. The width of the cavity shave margins and their orientation for histologic examination were in all studies according to the surgeon's decision and there was no standardization of the excised volume of CS. The surgeon‐based changes in re‐excision and margin positivity rates should be also considered after Valero et al. . AnotherWe conclude that CS is a surgical technique that may reduce re‐excision rates. However, there is limited high-quality evidence that supports general use of this method in BCS. Therefore, more prospective randomized-controlled trials are needed.


Chronic rhinosinusitis with nasal polyps (CRSwNP) was potentially due to the epithelial barrier injury. YES‐associated protein (YAP) is a multifunctional transcriptional factor and plays versatile roles in the regulation and maintenance of epithelial barrier in different organs and tissues. The purpose of this study is to elucidate possible effect and mechanism of YAP on the epithelial barrier of CRSwNP.n = 12) and control group (n = 9). The location of YAP, PDZ‐binding transcriptional co‐activator (TAZ), and Smad7 were estimated by immunohistochemistry and immunofluorescence. Meanwhile, the expression of YAP, TAZ, Zona occludens‐1 (ZO‐1), E‐cadherin, and transforming growth factor‐beta1 (TGF‐β1) were detected by Western blot. After primary human nasal epithelial cells were treated with YAP inhibitor, the expression level of YAP, TAZ, ZO‐1, E‐cadherin, TGF‐β1, and Smad7 were measured by Western blot.Patients were divided into CRSwNP group  level may lead to chronic rhinosinusitis with nasal polyps (CRSwNP) epithelial barrier injury via transforming growth factor‐beta1 (TGF‐β1) signaling pathway and the inhibition of YAP can partially reverse epithelial barrier. Many diseases are associated with disruption of the nasal epithelial barrier.Yes‐associated protein (YAP) and PDZ‐binding transcriptional co‐activator (TAZ) are the core effectors of the Hippo pathway. They play a crucial role in organ size regulation and tumorigenesis.Transforming growth factor‐beta1 (TGF‐β1) is a multifunctional cytokine that typically binds to the TGF‐β1/Smad signaling pathway.The purpose of this study was to investigate the role of YAP in the epithelial barrier of nasal polyps, and to preliminarily study the relationship between YAP and the TGF‐β1 signaling pathway in the epithelial barrier of nasal polyps, so as to provide new ideas for the prevention and treatment of nasal polyps.22.1Nasal polyps tissue from 12 CRSwNP patients and inferior turbinate (IT) from nine control group patients were harvested during ESS from the Department of Otorhinolaryngology in the First Affiliated Hospital of Chongqing Medical University, China. Control group subjects had no history of CRS who required treatment for nasal septum deviation or repair of cerebrospinal fluid rhinorrhea. Nasal polyp patients were diagnosed with CRSwNP based on the criteria of the European Position Paper on Rhinosinusitis and Nasal Polyps 2020. Accordingly, the exclusion criteria were as follows: immune deficiencies, primary ciliary dyskinesia, fungal sinusitis, cystic fibrosis, antrochoanal polyps, vasomotor rhinitis, and asthma. All enrolled patients did not take any glucocorticosteroids or antibiotics at least 1 month before surgery. Additionally, each individual had no upper or lower respiratory infection and other systemic diseases. Detailed information is shown in Table 2.2Tissues were fixed in 4% formaldehyde, embedded in paraffin, and cut into 4 μm per section. These sections were antigen retrieval by buffer solution of citrate, incubated with 3% hydrogen peroxide (Boster Biotechnology) to block endogenous peroxidase activity, and blocked with 10% goat serum to saturate nonspecific staining. Then, sections were incubated with primary antibodies of YAP , TAZ , and Smad7 at 4°C overnight. The next day, slides were washed with PBS, followed by incubation with secondary antibodies at room temperature for 1 h. After DAB treatment, the sections were observed by inverted microscopy. Analysis of average optical density was performed by using ImageJ software.2.32. When pHNECs reached approximately 80% confluence, cells were harvested for further research.Nasal polyps (NPs) tissue and IT mucosa were obtained during general anesthesia surgery and immediately transferred to the lab with liquid nitrogen, washed with cold phosphate‐buffered saline containing penicillin and streptomycin to clear secretion, blood and bone. Here, we isolated pHNECs from tissues by collagenase digestion.2.4Nasal polyps and pHNECs were lysed with RIPA lysate  and 1 mM phenylmethylsulfonyl fluoride  to obtain total protein and protein concentration was measured by BCA protein assay kit . After heat denaturation for 15 min at 100°C, 30 μg total protein was electrophoresed on an 8% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membranes . Then, the membranes were blocked with 5% skim milk at room temperature for 1 h. Primary antibodies including TGF‐β1, TGF‐βRII , Smad2/3, phospho‐Smad2/3, Smad7, YAP, TAZ , ZO‐1, E‐cadherin , and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)  were diluted with fresh blocking reagent and incubated at 4°C overnight. In the following day, membranes were incubated with secondary antibodies (1:2000) at room temperature for 1 h. Then membranes were washed with PBS for three times, and blots were revealed with enhanced chemiluminescence western‐blotting reagent . The photos of the blots were captured and gel documentation of the density of each band was determined using ImageJ software. The ratio of each band/GAPDH was considered as the expression level of the target protein.2.5The paraffin sections and pHNECs were treated with primary antibodies: YAP , TAZ , Smad7 , and ZO‐1 at 4°C overnight. The next day, slides were incubated with Alexa Fluor 594‐conjugated goat antirabbit secondary antibody for 2 h and nuclei was counterstained with 4,6‐diamidino‐2‐phenylindole. Eventually, images captured by using a confocal laser microscope.2.6t‐tests were used to measure p value with a p < .05 and considered statistically significant. Gender comparison was done by χ2 test. All values are presented as the mean ± SD of at least three independent experiments.GraphPad Prism 8.0 software and IBM SPSS 22.0 software were used to analyze the data. Two‐tailed Student's 33.1n = 5 for each group). The results  was conducted. Results Figure  indicate4CRSwNP is a common chronic inflammatory disease in otorhinolaryngology department and patients visit the clinic normally with nasal congestion, decreased smell or anosmia, headache and so on. With increasing levels of pollution, the number of people affected by CRSwNP is on the rise. Therefore, it is urgent to elucidate the mechanism of CRSwNP and provide promising insight into clinical treatment. In this study, we evaluated the different expression of YAP, TAZ, TGF‐β1, TGF‐β RII, pSmad2/3, Smad2/3, Smad7, ZO‐1, and E‐cadherin in CRSwNP and healthy patients. Compared with the control group, the levels of YAP, TAZ, and Smad7 were higher in CRSwNP while TGF‐β1, ZO‐1, and E‐cadherin levels were decreased. After treating pHNECs from NPs with a YAP inhibitor, we observed that the level of Smad7 decreased with increasing levels of ZO‐1, E‐cadherin, and TGF‐β1. These results suggested that YAP may be involved in CRSwNP epithelial barrier injury and the inhibition of YAP could repair nasal epithelial barrier.Nasal epithelial barrier is the first line to protect our body from being invaded by toxins and stimuli, which is mainly composed of TJs. As one of the most important TJ proteins, the lack of ZO‐1 can lead to abnormal formation of TJ and damage of epithelial barrier. Recent studies revealed that MMP‐10 aggravated podocyte dysfunction and proteinuria by disrupting glomerular filtration integrity via degrading ZO‐1 in chronic kidney disease.YAP could take part in the process of epithelial barrier injury and repair. Some research demonstrated that YAP is involved in suppressing TNF‐α‐induced injuries of intestinal epithelial barrier.Numerous studies examined that TGF‐β1 interacted with YAP and TAZ were involved in many diseases, such as vocal cord fibrosisTo further determine the relationship between YAP, Smad7, TGF‐β1, and nasal epithelial barrier injury, we treated CRSwNP epithelial cells with VP which was a potent and selective YAP inhibitor. The results showed that the expression of YAP and Smad7 decreased while TGF‐β1, ZO‐1, and E‐cadherin significantly increased with VP treatment in pHNECs. This indicated that high expression of YAP may contribute to nasal epithelial barrier injury by downregulation of the expression of ZO‐1 and E‐cadherin, while the inhibition of YAP can restore epithelial barrier function by promoting ZO‐1 expression in pHNECs. Interestingly, we also observed a decrease in the level of Smad7 and an increase in TGF‐β1 expression when YAP was inhibited. These results suggest that there may be crosstalk between YAP and TGF‐β1 in CRSwNP, which could be involved in the inhibition of TGF‐β1 signaling pathway by binding Smad7. In brief, our findings indicate that the downregulation of ZO‐1 and E‐cadherin regulated by YAP may contribute to nasal epithelial barrier injury, possibly by inhibiting the TGF‐β1 signaling pathway via Smad7 Figure .Of course, there are some limitations in this study. First, our investigation was limited to the expression of ZO‐1 without exploring other TJ proteins which could also play a role in epithelial barrier function. Additionally, we did not differentiate between eosinophilic CRSwNP and noneosinophilic CRSwNP, which could have an impact on the results. Further studies should be conducted to address this issue. Last, while our study has provided preliminary evidence for the involvement of YAP, Smad7, and TGF‐β1 in nasal epithelial barrier injury, more detailed investigations are necessary to fully elucidate the underlying mechanisms.5In summary, this experiment verified the expression of YAP in nasal polyps. It was found that the high expression of YAP led to or participated in the decrease of the expression of TJ protein ZO‐1 and participated in the epithelial barrier injury. After inhibiting the expression of YAP, the low expression of ZO‐1 was reversed. At the same time, this study also found that YAP and TGF‐β signaling pathway interact in CRSwNP, but the specific mechanism need to be further explored.Xiaocong Jiang: Investigation; resources; software; validation; writing—original draft. Longlan Shu: Software; supervision. Yijun Liu: Software; supervision. Yang Shen: Software; supervision. Xia Ke: Software; supervision. Jie Liu: Software; supervision. Yucheng Yang: Funding acquisition; project administration; supervision; writing—review and editing.The authors declare no conflict of interest.


In older patients with cancer, comorbidities compete with cancer for cause of death. The objectives were to evaluate cancer mortality and factors associated, according to metastatic status.Between 2007 and 2014, patients with cancer aged ≥70 referred for pre‐therapeutic geriatric assessment (GA) were included through the ELCAPA prospective cohort study. The underlying cause of death was defined according to the International Classification of Diseases, 10th Revision. The World Health Organisation definition was used to categorise the cause of death as cancer versus another disease  Competing risk models were used.Mean (SD) age of the 1445 included patients was 80.2 (5.8) and 48% were women. Most common tumour sites were colorectal (19%), breast (17%) and urinary (15%); 773 patients (49%) had metastases. After a 34‐month median follow‐up, 706 cancer deaths were observed among 843 deaths. The 6‐month and 3‐year cancer mortality rates (95% CI) were 12% (9–15) and 34% (29–38) for non‐metastatic patients and 43% (39–47) and 79% (75–82) for metastatic patients, respectively. Dependency in activities of daily living and comorbidities were associated with 6‐month and 3‐year cancer mortality in non‐metastatic  and metastatic patients . Impaired Timed‐Get‐Up‐and‐Go test was associated with 6‐month and 3‐year cancer mortality in metastatic patients . Obesity was negatively associated with 3‐year cancer death in non‐metastatic (aSHR = 0.53 [0.29–0.97]) and metastatic patients (aSHR = 0.71 [0.51–1.00]).The majority of older adults with cancer referred for pre‐therapeutic GA die from cancer. Geriatric parameters are independently associated with cancer mortality and should be considered for prognosis assessment, decision‐making and care. The majority of older adults with cancer referred for pre‐therapeutic GA died from cancer and not from other causes. An altered general status, loss of independency and co‐morbidities were independently associated with cancer death at 6 months and at 3 years, regardless of metastatic status; mobility impairment was independently associated with cancer death at 6 months and at 3 years in metastatic patients. The study report complies with the STROBE guidelines for observational studies.The ELCAPA non‐interventional, observational and prospective cohort study includes patients aged ≥70 years with a solid tumour or haematological malignancy, referred by the oncologist, or specialist for multidimensional geriatric assessment (GA) at one of the 19 geriatric oncology clinics in the Paris area (France). The ELCAPA cohort was created using an age inclusion criterion of ≥70 years, as this is the cut‐off recommended by national and international societies to perform a 2.22 = underweight, between 21 kg/m2 and 24.99 kg/m2 = normal weight, between 25 kg/m2 and 29.99 kg/m2 = overweight, and ≥30 kg/m2 = obesity. Comorbidity burden is assessed by the Cumulative Illness Rating Scale for Geriatrics (CIRS‐G) and was considered high as a total CIRS‐G score above the median value in the study population (≥13).Data collected in the ELCAPA cohort include age, sex, Eastern Cooperative Oncology Group (ECOG) performance status (PS), cancer‐related characteristics , and geriatric data (collected from the GA). The GA, performed by a geriatrician at baseline covers six domains , based on validated tests and scores.2.3The main endpoint was cancer mortality in the short term (6 months) and middle term (3 years). The underlying cause of death (UCD) is defined by the World Health Organisation: ‘the disease or injury that initiated the train of morbid events leading directly to a person's death or the circumstances of the accident or violence which produced the fatal injury’. UCD information was obtained from the French Epidemiological Centre for the Medical Causes of Death (CépiDC), a nationwide database. For each death occurring on French territory, a physician completes an international death certificate form; from this certificate, the CépiDC assigns a single UCD to each death according to internationally adopted rules, coded according to the International Classification of Diseases, 10th Revision. This UCD selection and coding process was performed by coding software's and experienced coders. To identify the UCD of each patient, we linked the ELCAPA cohort to the CépiDC.2.4p‐value below 0.20 in the univariate analysis were considered in the multivariate analysis. Collinearity between PS and CIRS‐G, and between TGUG and ADL, was assessed. A manual stepwise analysis was done to identify confounders. Associations between the above factors and cancer deaths were also analysed using a Cox‐hazard proportional model and reported as cause‐specific hazard ratio (CSHR).Baseline demographic, cancer and geriatric characteristics, and cause of death, were described as number and percentage for qualitative variables, and the mean  or median (interquartile range), where appropriate, for quantitative variables, according to metastatic status. A Fine and Gray competing risk model2.4.1In order to test the stability of our results, we performed Fine and Gray competing risk models in patients with a solid tumour and specific treatment for cancer.p < 0.05. The data were analysed using Stata software .All tests were two‐sided and the statistical significance threshold was set at 33.1Between January 2007 and December 2014, of the 1678 patients recruited in the ELCAPA cohort, 1445 patients had complete metastatic status data and were included in the present analysis. Mean (SD) age was 80.2 ± 5.8 years and 695 (48%) were women. The most common primary tumours were colorectal (19%), breast (17%) and urinary tract (15%). Forty‐nine percent of patients with solid tumours had metastases. The main characteristics of the population are summarised in Table 3.2With a median follow‐up of 34 months, 843 deaths occurred, 706 (84%) of which were due to cancer, 503/561 (90%) in patients with metastasis and 203/282 (72%) in patients without.The cancer mortality rates at 6 months and 3 years were 12%  and 34%  for non‐metastatic cancers, and 43%  and 79%  for metastatic cancers, respectively. The cumulative incidence curves of cancer deaths are shown in Figure 3.3p < 0.05 for all) regardless of metastatic status (Table p < 0.05) (Table p = 0.093) at 3 years. These variables were included in the multivariate analyses.At 6 months and at 3 years, palliative care, supportive care alone, PS 3–4, dependency for ADLs, abnormal TGUG time or inability to perform TGUG, impaired cognitive status, abnormal mini‐GDS score, and comorbidities (CIRS‐G ≥ 13) were significantly associated with cancer death  and the second with ADL and CIRS‐G (Model 2). Fine and Gray competing risk models and Cox models were built with PS, ADL and CIRS‐G as time‐dependent covariates.In non‐metastatic patients, an altered general status (PS 3–4), loss of independency (ADL score ≤5) and comorbidities  were independently associated with cancer death at 6 months and 3 years, after adjusting for age, sex, tumour site, history of cancer and curative treatment vs. palliative care , an impaired mobility (TGUG >20 s or inability to perform TGUG), loss of independency (ADL score ≤5), and comorbidities  were independently associated with cancer death at 6 months and 3 years; cognitive impairment was associated with cancer death at 3 years only in Model 2 . In contrast, overweight and obesity were independently associated with a lower risk of cancer death at 3 years. We found similar associations in a cause‐specific Cox proportional hazards model, with the exception of cognitive impairment, which in the Cox model was associated with a higher risk of cancer death at 3 years in metastatic patients, in the two models Table . Signifi3.4n = 871) gave much the same results as the main analyses . At 6 months, the cancer mortality rate was 12% (from 1% for breast cancer to 27% for pancreatic cancer) for non‐metastatic solid tumours, and 45% (from 26% for breast cancer to 90% for lung cancer) for metastatic solid tumours. The 3‐year cancer mortality rate was 34% (from 6% for breast cancer and 75% for pancreatic cancer) in non‐metastatic tumours, and 83% (from 73% for breast cancer to 95% for lung cancer) in metastatic tumours. In multivariate analysis adjusted for age, sex, tumour site/metastatic status, history of cancer and curative treatment vs. palliative care, an altered general status (PS 3–4), loss of independency (ADL score ≤5), and comorbidities  were independently associated with cancer death in the short to middle term, regardless of metastatic status. Impaired mobility (TGUG >20 s or inability to perform TGUG) and an altered general status (PS = 2) were independently associated with cancer death in the short to middle term only in metastatic patients. Cognitive impairment was associated with cancer death (in one model) in the middle term only in metastatic patients. Overweight and obese patients were less likely to die from cancer than normal weight patients also in the middle term in metastatic patients, and obesity was also associated with a lower risk of cancer death in the middle term in non‐metastatic patients.Few studies on causes of death are available and were performed mostly in prostate and breast cancer. Older men with non‐metastatic prostate cancer had a low 3‐year (5% to 20%) or 5‐year cancer mortality rate (8%), which corresponds to our value of 13%.Dependency, impaired mobility, comorbidities and cognitive impairment are known to have a negative effect on the overall mortality of older patients with cancer, but not on cancer mortality.Underweight was not associated with cancer mortality, but more specific nutritional parameters could not be used because data were missing. Obesity was associated with a lower risk of cancer death in our study only in the middle term. Although a causal relationship is subject to debate, the protective effect of overweight and/or obesity in patients with cancer  has already been described.Our sensitivity analyses produced similar results as the main analyses, supporting the robustness of our findings.The present study has a number of limitations. All patients had been referred to a geriatrician for therapeutic decision, which might have introduced a selection bias. Therefore, we may have overestimated the specific‐cancer mortality rate. Second, misclassification of the cause of death is possible because death certificates are not always filled out correctly. Nevertheless, the degree of agreement between the cause of death recorded on the death certificate and that found in an autopsy is relatively high for cancer (81%).A pragmatic approach might involve the inclusion of geriatric factors in oncologic prognostic scores.In conclusion, the majority of older adults with cancer referred for pre‐therapeutic GA die of cancer and not from other causes. An altered general status, loss of independency, and comorbidities were independently associated with cancer death at short and middle term, regardless of metastatic status. Mobility impairment was independently associated with cancer death at short and middle term in metastatic patients. Patients with obesity were less likely to die from cancer than normal‐weight patients at middle term.Déborah Assouan: Formal analysis ; methodology ; software ; visualization ; writing – original draft ; writing – review and editing . Elena Paillaud: Conceptualization ; funding acquisition ; investigation ; project administration ; writing – review and editing . Philippe Caillet: Conceptualization ; funding acquisition ; investigation ; project administration ; resources ; writing – review and editing . Amaury Broussier: Investigation ; resources ; writing – review and editing . Emmanuelle Kempf: Investigation ; resources ; writing – review and editing . Maxime Frelaut: Investigation ; resources ; writing – review and editing . Etienne Brain: Funding acquisition ; investigation ; resources ; writing – review and editing . Emmanuelle Lorisson: Investigation ; resources ; writing – review and editing . Clelia Chambraud: Data curation ; software ; writing – review and editing . Sylvie Bastuji‐Garin: Funding acquisition ; methodology ; writing – original draft ; writing – review and editing . Olivier Hanon: Funding acquisition ; investigation ; resources ; writing – review and editing . Florence Canouï‐Poitrine: Conceptualization ; funding acquisition ; methodology ; project administration ; resources ; supervision ; writing – original draft ; writing – review and editing . Marie Laurent: Conceptualization ; investigation ; project administration ; resources ; writing – original draft ; writing – review and editing . Claudia Martinez‐Tapia: Conceptualization ; formal analysis ; methodology ; software ; visualization ; writing – original draft ; writing – review and editing .This work was supported by the French National Cancer Institute , Canceropôle Ile‐de‐France and Gerontopôle Ile‐de‐France (Gerond'If). None of the funding bodies had any role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review and approval of the manuscript; or the decision to submit the manuscript for publication.The authors declare no conflict of interest.The study was conducted in accordance with the Declaration of Helsinki and approved by the local independent ethics committee .All patients provided oral informed consent prior to inclusion.ClinicalTrials.gov).NCT02884375 (Data S1:Click here for additional data file.


An 82‐year‐old man was treated with ipilimumab and nivolumab for malignant pleural mesothelioma. Although he was previously treated with prednisolone (1 mg/kg/day) for immune‐related adverse event (irAE) hepatitis by a previous doctor, he still had worsening liver function and was transferred to our hospital. Blood tests and imaging findings were negative for autoimmune and infectious hepatitis, and liver biopsy results were consistent with irAE hepatitis. Steroid pulse therapy improved liver function, but tapering to prednisolone (1 mg/kg/day) again worsened his liver function. Concomitant use of mycophenolate mofetil was initiated, but no improvement in liver function was observed, therefore azathioprine, a thiopurine immunosuppressant, was administered in combination with steroids. During the course of treatment, hepatic dysfunction due to azathioprine was suspected, and the concomitant use of mercaptopurine and prednisolone was started. Afterward, the liver function improved, and the prednisolone dose was gradually reduced to 10 mg/day. This is a rare case in which a thiopurine‐based immunosuppressant was effective against irAE hepatitis, therefore thiopurine‐based immunosuppressants may be effective against steroid‐refractory hepatitis. Coarse of treatment time from initiation of ipilimumab + nivolumab and changes in liver function, aspartate aminotransferase, alanine aminotransferase, and total bilirubin. Steroid pulse therapy means 3‐day administration of 1000 mg of methylprednisolone. The efficacy of nivolumab, a fully human antiprogrammed death‐1 antibody, and ipilimumab, a fully human anticytotoxic T‐lymphocyte antigen‐4 antibody, has been reported for the treatment of unresectable malignant pleural mesothelioma. However, hepatitis, an immune‐related adverse event (irAE), occurred in approximately 6.0% of common terminology criteria for adverse events (CTCAE) of any grade and 4.7% of grades 3 and 4.An 82‐year‐old man was treated with ipilimumab and nivolumab for malignant pleural mesothelioma . On the 15th day after treatment initiation, significant liver dysfunction  was observed, and the patient was admitted to a hospital because of CTCAE grade 4 liver dysfunction. Treatment with prednisolone 60 mg/day (1 mg/kg/day) for irAE hepatitis was initiated, but after 1 week liver function did not improve (AST 434 U/L and ALT 727 U/L), and jaundice developed to CTCAE grade 3 . After starting steroid pulse therapy (3‐day administration of 1000 mg of methylprednisolone), the patient was transferred to our hospital for treatment. Blood tests showed elevated transaminase and bilirubin levels (CTCAE grade 3). Autoantibodies suggestive of autoimmune hepatitis and tests for viral hepatitis were negative  did not suppress the deterioration of liver function, but steroid pulse suppressed it. Concomitant use of mycophenolate mofetil was initiated, but the worsening of liver function could not be suppressed. Eventually, the concomitant use of a thiopurine‐based immunosuppressive agent enabled the tapering of prednisolone, suggesting its effectiveness against irAE hepatitis. Azathioprine was also able to suppress the deterioration of liver function, but considering drug‐induced liver dysfunction due to azathioprine, it was switched to mercaptopurine. Mercaptopurine is a prodrug of azathioprine and was used because it was expected to have the same effect. In addition, mercaptopurine has shown efficacy as an immunosuppressive agent in inflammatory bowel disease.Keita Kawakado: Conceptualization, data curation, writing – review and editing. Kazuhisa Nakashima, Tobita Hiroshi, Nagase Mamiko, Yukari Tsubata, and Takeshi Isobe: Writing – review and editing, supervision. Ken Yoshihara, Mika Horie, Megumi Hamaguchi, and Tamio Okimoto: Writing – review and editing.The authors declare no conflicts of interest.Written patient approval was obtained.


To disaggregate the Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART CORS) age category of “ > 42” and compare age-stratified cumulative live birth rates (CLBR) > 42 years old.Retrospective cohort study of autologous linked ART cycles.United States (US) National ART Database.Women > 42 years old without a history of prior ART cycles who underwent ART between 2014–2020 as reported to the SART CORS database.Disaggregate the SART CORS age category of “ > 42” into age-stratified cumulative live birth rates (CLBR).Age-stratified cumulative live birth rates (CLBR) for women ≥ 43 years old.p < 0.05). Among women 46 and older, CLBR were not significantly different. Women ages 43 and 44 did not exhibit a significant increase in CLBR beyond the 5th cycle. Age 45 and 46 reached CLBR plateau by the 3rd cycle. Age ≥ 47 CLBR plateaued after the first cycle. After adjusting for age, race/ethnicity, BMI, nulliparity, etiology of infertility, number of oocytes retrieved, embryos transferred, blastocyst transfer, use of ICSI, PGT, and ART treatment cycle number, there was no association between markers of ovarian reserve (day 3 FSH and random AMH levels) and live birth for women > 42.Between 2014–2020, 24,650 women > 42 years old without history of prior ART underwent 58,132 cycles, resulting in 1,982 live births. Women ages 43, 44, 45, 46, 47, 48, 49, ≥ 50 achieved maximal CLBR of 9.7%, 8.6%, 5.0%, 3.6%, 2.5%, 1.5%, 2.7%, 1.3%, respectively. CLBR for women between 43–45 were significantly higher compared to those 46 and older (While CLBR of autologous cycles from women 42 or younger generally plateau by cycle number 5, age-stratified cycles from women > 42 plateau after fewer cycles to maximize CLBR. Patient and physician expectations for maximum CLBR beyond 42 may be practically based on fewer planned cycles before reaching an age-specific CLBR plateau than may have been previously expected. The United States lacks contemporary, publicly available, autologous oocyte assisted reproductive technology (ART) age-stratified outcome data for patients > 42 years old. After 2015, the CDC aggregated outcome data beyond age 42 or 43, depending on the year [While donor egg IVF, may be encouraged, many do not accept this option . AttitudThere are a few studies analyzing live birth rates (LBR) in women over the age of 42 –10 and eThis was a retrospective cohort study of initial (no prior history of ART) autologous linked ART cycles of women > 42 between 2014–2020 derived from the SART CORS database. Beginning in 2014 SART CORS, linked all transfers regardless of fresh or frozen to their specific index retrieval. This study was deemed exempt from review by the Yale Institutional Review Board because it relied on anonymous and de-identified data. SART CORS data were collected through voluntary submission, verified by SART, and reported to the Centers for Disease Control and Prevention (CDC) in compliance with the FCSRCA of 1992 (Public Law 102–493). In 2004, SART gained access to the SART CORS data system for the purposes of conducting research. By 2019, 81% of US IVF clinics were SART members reporting 90% of all IVF cycles in the US .The data in the SART CORS are validated annually with some clinics receiving on-site visits for chart review based on an algorithm for clinic selection. During each visit, data reported by the clinic are compared with information recorded in patients’ charts. In 2021, records for 1,945 cycles at 33 clinics were randomly selected for full validation and 262 fertility preservation cycles were selected for partial validation. Nine out of ten data fields selected for validation were found to have discrepancy rates of ≤ 5% . The excSubjects included were women > 42 years old who had no prior history of assisted reproductive technology (ART) and underwent autologous ART treatment between 2014 and 2020, as reported in the SART CORS database. A treatment cycle was defined as any intended retrieval cycle, whether cancelled or not, and all embryo transfers derived from that retrieval. Each treatment cycle for included subjects was extracted from the database. Each subject’s treatment cycle was assigned a sequential number to designate consecutive cycles. CLBR was calculated for each sequential cycle. CLBR was defined as the number of live births up to, and including, a specific cycle divided by the total number of patients undergoing the initial cycle start. This aimed to illustrate the likelihood of achieving a live birth from a specific ART treatment during the study period.Patients were categorized into distinct age groups based on their age at the initiation of their first treatment cycle. Each age group was defined by one-year intervals up to 50 years old. Due to the limited number of subjects older than 50, those aged 50 years and above were grouped together. The sequential CLBR was examined across different age groups and within each age group. The log-rank test was employed to compare the overall CLBR patterns across all cycles between the various age groups, and chi square tests were used to compare specific CLBR between different groups. Within each age group, the 95% confidence interval difference of the mean was utilized to identify any significant differences in outcomes between consecutive treatment cycles. By determining the cycle number at which there were no significant increases in CLBR, we identified a suggested maximum number of ART attempts before considering abandoning autologous ART.The baseline patient demographics and clinical characteristics, including markers of ovarian reserve  and infertility diagnoses, were examined and compared among different age groups using ANOVA and chi-square tests. Infertility diagnoses were categorized into seven distinct groups: diminished ovarian reserve (DOR), tubal factor, uterine factor, ovulatory/polycystic ovarian syndrome, endometriosis, unexplained, and male factor. These were not mutually exclusive categories and patients could have more than one of these diagnoses.Cycle specific clinical information including number of oocytes retrieved, embryos formed, embryos transferred, percentage of cycles with blastocyst transfer, percentage of cycles using ICSI, and percentage of cycles with preimplantation genetic testing (PGT) were compared across each cycle and between age groups using ANOVA tests. Additionally, multivariable logistic regression was performed to identify significant predictors of live birth for ART treatment in women of advanced reproductive age.p < 0.05) but represented a decline from 4.8% to 4.3% (p < 0.01) of the total percent of women who underwent ART at any age.Between 2014–2020, 24,650 women > 42 years old without history of prior ART underwent 54,132 treatment cycles, resulting in 1,982 live births. Overall, these 54,132 cycles represented 6.6% of the total number of ART cycles of all ages in women without history of prior ART performed during this same time period. Between 2014 and 2020 the number of women ≥ 43 years old who underwent ART increased from 3065 to 3755 . Figure p < 0.05). Within age groups 43–47, there was a significant decrease in number of oocytes retrieved and embryos formed with each subsequent ART cycle (p < 0.05). For the population studied, subjects who were younger had significantly higher numbers of transferred embryos, percent blastocyst transfers, and were more likely to use ICSI and PGT (p < 0.001). Subjects in age groups 43 and 44 had fewer numbers of transferred embryos and percent of blastocysts transferred with each subsequent treatment cycle (p < 0.01). Subjects that were 45 years old also had a lower percent of blastocyst transfers with each subsequent treatment cycle (p < 0.05). The other age groups did not demonstrate significant differences in number of transferred embryos, or percentage of blastocyst transfers over each sequential treatment cycle.Table p < 0.05). Maximal CLBR was highest for 43-year-old subjects (~ 9.7%) and lowest for those ≥ 50 (1.3%); p < 0.001. Subjects aged 44, 45, 46, 47, 48, and 49 achieved maximal CLBR of 8.6%, 5.0%, 3.6%, 2.5%, 1.5%, and 2.7%, respectively. CLBR for subjects between 43–45 were significantly higher compared to those 46 and older (p < 0.05). Among subjects 46 and older, CLBR were not significantly different (p > 0.05).Figure p < 0.05). However, beyond the 5th cycle, there was minimal noticeable increase in CLBR . For 45-year-old patients, the CLBR reached a plateau by the 2nd cycle, with approximately 3.8% CLBR, and the maximum CLBR of 5% was achieved by the 7th cycle. Although there was a significant increase in CLBR between cycles 2 and 7 (p < 0.05), the plateau was considered to be at cycle 2 since the CLBR between cycles 2–6 did not show significant differences. For patients who were 46 years old, there was no change in CLBR between cycles 1 and 2, with a CLBR of 2.1%. However, by the 3rd cycle, the CLBR increased to 3.2% (p < 0.05), which was higher than that observed in the first cycle. There was no significant increase in CLBR beyond cycle 3, although a maximal CLBR of ~ 3.6% was reached by the 6th cycle (p > 0.05). For patients ≥ 47 years old, CLBR plateaued after their first cycle.Figure Multivariable logistic regression analysis was performed to identify significant predictors of live birth for women of advanced reproductive age undergoing ART treatment cycles Table . As expeThis is the first comprehensive retrospective analysis of cumulative live birth rates stratified by age in women > 42 using the SART CORS. This study is an analysis of the first 7 years (2014–2020) of linked ART cycle data and CLBR from one of the largest standardized and validated data sets available in the United States.th to 5th ART cycle [It has been reported that CLBR for women 42 or younger generally plateau by the 4RT cycle –17. AccoWomen 45 and 46 years old should be informed that CLBR is approximately 4% and 3%, respectively, and that each subsequent retrieval beyond the second (4% and ~ 3% respectively) affects the CLBR by < 0.5% to yield a maximum CLBR for these ages of ~ 5% and 3.6% respectively. CLBR using autologous eggs are minimal for women ≥ 47 years old, ranging from 1–2% and do not change appreciably beyond the initial cycle. The per cycle LBR is 1% or less for women over the age of 46. These observations are consistent with the fact that at age 43, blastocyst aneuploidy rates approach 85% and continue to increase with advancing age , 19.Our findings reveal a clear trend of decreasing number of embryos transferred and blastocyst transfers with increasing age > 42 Fig. . These fThe current analysis of this large dataset helps frame expectations for maximum CLBR beyond age 42. Our data suggests that women > 42 years old reach a maximum CLBR after 1–5 cycle retrieval cycles, depending on their specific age. For those who do not yield transferable embryos, the value of IVF with autologous eggs could be questioned after fewer attempts. Because all transfer attempts are linked to a single retrieval, each retrieval may yield multiple transfer attempts and therefore number of “cycles” to achieve a live birth may be underrepresented in this study. Accordingly, our findings add credence to the need for early counseling regarding alternatives to autologous ART (donor egg or adoption) when it comes to the decision-making process attempts at autologous ART for women ≥ 44 years of age.The CLBRs presented here are based on cycle starts. The relatively high cancellation rates reported in the setting of autologous eggs beyond age 40 predict lower CLBR . The ASRDiminished ovarian reserve is an inevitable part of the human experience. While there have been anthropological rationalizations for the process, they do not console those who are faced, late in life, with the desire to conceive , 24. We Our study is limited by its retrospective nature. Prospective controlled trials assessing reproductive outcomes at the twilight of reproductive potential are rare and limited. Morgia et al. conducted an RCT to evaluate different COH regimens and noted low implantation rates for women > 40, however, women > 43 were not included . Such ceWe show that beyond age 45 live birth outcomes become quite rare. Meager odds may not dissuade some, but it is incumbent upon providers to be able to provide realistic likelihoods for desired goals. Vaguely stating low odds or providing wide ranges cannot suffice as satisfactory when stakes and costs are as high as they are for a single ART cycle. There remain circumstances when cost to the patient is not an issue, as when insurance coverage is guaranteed no matter the prognosis. In such situations, when treatment becomes futile, there is a clear need for objective data as presented here. As noted by ASRM, not offering treatment in the context of futility is an appropriate expression of professional integrity . Stoppin


S. aureus infected wound and rheumatoid arthritis in rats. In conclusion, this dynamically responsive injectable hydrogel with a triple‐network structure provides an effective strategy to treat IDs, holding great promise in clinical application.Modulating the inflammatory microenvironment can inhibit the process of inflammatory diseases (IDs). A tri‐cross‐linked inflammatory microenvironment‐responsive hydrogel with ideal mechanical properties achieves triggerable and sustained drug delivery and regulates the inflammatory microenvironment. Here, this study develops an inflammatory microenvironment‐responsive hydrogel (OD‐PP@SeNPs) composed of phenylboronic acid grafted polylysine (PP), oxidized dextran (OD), and selenium nanoparticles (SeNPs). The introduction of SeNPs as initiators and nano‐fillers into the hydrogel results in extra cross‐linking of the polymer network through hydrogen bonding. Based on Schiff base bonds, Phenylboronate ester bonds, and hydrogen bonds, a reactive oxygen species (ROS)/pH dual responsive hydrogel with a triple‐network is achieved. The hydrogel has injectable, self‐healing, adhesion, outstanding flexibility, suitable swelling capacity, optimal biodegradability, excellent stimuli‐responsive active substance release performance, and prominent biocompatibility. Most importantly, the hydrogel with ROS scavenging and pH‐regulating ability protects cells from oxidative stress and induces macrophages into M2 polarization to reduce inflammatory cytokines through PI3K/AKT/NF‐κB and MAPK pathways, exerting anti‐inflammatory effects and reshaping the inflammatory microenvironment, thereby effectively treating typical IDs, including  S. aureus infected wound and rheumatoid arthritis in rats.The tri‐cross‐linked dynamic response hydrogel with ROS scavenging and pH‐regulating ability protects cells from oxidative stress and induces macrophages into M2 polarization to reduce inflammatory cytokines through PI3K/AKT/NF‐κB and MAPK pathways, exerting anti‐inflammatory effects and reshaping the inflammatory microenvironment, thereby effectively treating typical IDs, including  Thus, controlling the development of inflammation processes as early as possible is a promising strategy for treatment IDs. However, traditional treatment is based on anti‐inflammatory and immunosuppressive drugs, which are palliative and provide short‐term relief with serious side effects. Therefore, the development of reliable, non‐toxic, long‐lasting, and low‐cost treatments have great potential in the pharmacological market.Inflammatory diseases (IDs) are a general term that covers all diseases in which chronic inflammation perform as the primary manifestation of pathogenesis.1, 2 Hydrogels have been widely used as a treatment for IDs due to their remarkable biocompatibility, tunable biodegradability, and controlled mechanical strength. Whereas, traditional hydrogels have single characteristics, low mechanical properties, high porosity (resulting in rapid gel erosion and drug diffusion), and uncontrollable drug release in pathological environments, greatly limiting their further development. Various functional hydrogels have been developed in recent years, such as injectable, self‐healing, and stimulus‐responsive. Stimulus‐responsive hydrogels, also known as “smart hydrogels”, have become a hot research topic due to their potential applications in biomedicine, responding to external microenvironmental stimuli such as glucose, temperature, pH, reactive oxygen species (ROS), and enzymes. Notably, the inflammatory microenvironment is primarily characterized by increased O2 demand and excessive ROS production, which in turn plays a further crucial role in inflammation exacerbation. For the treatment IDs, pH and ROS are appropriate triggers. Therefore, constructing multifunctional hydrogels with multiple dynamic responses from existing biomaterials is a key challenge.Delivering drugs via biomaterials provides an alternative strategy for IDs treatment. Over the past few decades, synthetic hydrogels with a hydrophilic polymer network structure resembling biological soft tissues have made exciting advances in drug delivery.8, 9 The formation and dissociation of Phenylboronate ester bonds, Schiff base bonds, Disulfide bonds, Acylhydrazone bonds, and Diels‐Alder reactions exhibit unprecedented versatility by simultaneously exhibiting pH and ROS sensitivity. As a class of Lewis acid, phenylboronic acid (PBA) can combine with compounds carrying catechol to form a Phenylboronate ester bonds, which is a reversible covalent bond. As a biodegradable biopolymer, polylysine (PLL) has abundant primary amine groups, low cost, and non‐toxic advantages. Therefore, PBA grafted PLL (PP) combines with 1,2‐diol‐containing polymers to form Phenylboronate ester bonds. Dextran is a polysaccharide often studied for hydrogel preparation and has excellent biocompatibility and biodegradability. The dextran structure is rich in adjacent hydroxyl groups, which could be oxidized to generate aldehyde groups (–CHO). The aldehyde groups on oxidized dextran (OD) can form reversible covalent Schiff base bonds with the amino groups on PP structure chain, which have the characteristics of high reactivity and controllable cross‐linking degree. At the same time, the unoxidized 1,2‐vicinal diol on OD can form a Phenylboronate ester bonds with the phenylboronic acid group on PP. A novel double cross‐linked hydrogel (OD‐PP hydrogel) was developed based on Schiff base bonds and Phenylboronate ester bonds, which can respond to low pH and high ROS, reshape the inflammatory microenvironment, and promote the recovery of IDs. It has been reported that hydrogels containing multiple covalent cross‐links show good mechanical properties. Hydrogels with desirable mechanical properties need to be further explored for their functionality and to broaden the scope of their applications.The functional properties of the hydrogels are vitally dependent on the polymeric composition and the interaction within the network.17 The Selenium nanoparticles (SeNPs) have received extensive attention due to their excellent biocompatibility, antibacterial, antiviral, and anticancer activities, and notable antioxidant and anti‐inflammatory properties. SeNPs triggers intracellular ROS overproduction to induce cell and bacterial apoptosis. Nevertheless, SeNPs have significantly emerged with pro‐oxidant and antioxidant potential dependent on subsequent duration, dose, frequency, and oxidation state. Given that SeNPs have non‐negligible concentration‐dependent therapeutic effects, it is a promising strategy to encapsulate SeNPs in a double covalently cross‐linked hydrogel. In an acidic environment with high ROS, the hydrogels based on Schiff base bonds and Phenylboronate ester bonds can be controllably decomposed and release SeNPs on demand, and maintain the SeNPs concentration at the desired level for clinical use. SeNPs endow additional properties to the hydrogel, such as antioxidant, anti‐inflammatory, and antibacterial. At the same time, the SeNPs work dually as initiators and nano‐fillers, and the polymer network interacts through hydrogen bonds to achieve triple cross‐linking. The addition of SeNPs significantly enhances the mechanical properties of the hydrogels. Tang et al. prepared a multifunctional hydrogel (CCOD‐MgO) by adding MgO to catechol‐modified chitosan and oxidized dextran (OD). CCOD‐MgO exhibits excellent self‐healing, hemostatic function, and low swelling rate. Carcia‐Astrain et al. developed a gelatin hydrogel using silver nanoparticles (AgNPs) as a cross‐linking agent and operating carrier. The addition of AgNPs significantly improved the mechanical properties of the hydrogels.The hydrogels based on reversible covalent chemical bonds have been extensively examined for on‐demand therapeutic delivery of therapeutic agents and cells to treat different IDs. Selenium (Se) is an essential micronutrient that plays a vital role in regulating the immune response. However, the toxicological safety of organic/inorganic selenium compounds limits the applications on multiple diseases treatments.31 Sel In this process, infiltrating inflammatory cells (neutrophils and macrophages) produce high levels of ROS. Excessive ROS will activate nuclear factor kappa‐B (NF‐κB) and mitogen‐activated protein kinase (MAPK) pathways, leading to the release of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6). This causes oxidative stress in surrounding cells, leading to healthy cell apoptosis and eventually has a degenerative effect on the inflammatory lesions. Therefore, the removal of excess ROS is particularly critical in the treatment of IDs. It has been reported that Se can alleviate the inflammatory reaction by suppressing the activation of the NF‐κB and MAPK signaling pathways. The phosphatidylinositol 3‐kinase (PI3K) is a signaling enzyme complex that combines with protein kinase B (Akt) to regulate cellular metabolism. PI3K‐AKT modulates NF‐κB transcriptional activity and stimulates the NF‐κB signaling pathway, which may lead to an inflammatory response and the release of proinflammatory cytokines. As an upstream signal of NF‐κB and MAPK, whether PI3K/Akt is involved in regulating the inflammatory microenvironment by SeNPs hydrogel is not clarified.Although some antioxidant properties of SeNPs have been reported, the specific mechanism is not fully elucidated. Further, exploring the anti‐inflammatory mechanism of SeNPs hydrogel will contribute to reshaping the inflammatory microenvironment and fundamentally curing IDs. Inflammatory sites are characterized by inflammatory cell infiltration and high levels of inflammatory cytokines.42 In S. aureus infected rats with skin injuries and rheumatoid arthritis (RA) rats were assessed, respectively. This study not only lays a function for tri‐cross‐linked hydrogel loaded with SeNPs on anti‐inflammatory mechanisms but also for the clinical applications.In light of the above considerations, we present a high‐performance biomedical hydrogel with triple cross‐linking, namely, OD‐PP@SeNPs, which is injectable, self‐healing, strongly adhesive to tissues, and biodegradable. The critical gelation pathway is to establish triple dynamic cross‐linking among PBA grafted PLL (PP), oxidized dextran (OD), and SeNPs. The injectability and self‐healing characteristics of OD‐PP@SeNPs hydrogels are mainly attributed to dynamic Schiff base bonds, Phenylboronate ester bonds, and hydrogen bonds. The introduction of SeNPs as initiators and nano‐fillers into the hydrogel resulted in extra cross‐linking of the polymer network through hydrogen bonding, and the SeNPs improved the mechanical properties of the hydrogel via reconfiguring various bonds. The presence of Schiff base bonds and Phenylboronate ester bonds leads to OD‐PP@SeNPs hydrogel with dual ROS/pH response, allowing for intelligently controlled release of SeNPs. Meanwhile, Schiff base bonds and Phenylboronate ester bonds consume ROS and adjust pH during covalent bond cleavage. SeNPs, Schiff base bonds, and Phenylboronate ester bonds exert synergistic antioxidant and anti‐inflammatory effects. We prepared OD‐PP@SeNPs hydrogel for the first time and sought to exploit the properties of the material itself to reshape the inflammatory microenvironment, relieve inflammation, and attempt to illustrate the mechanism of ROS clearance. Briefly, we first evaluated in detail the physicochemical properties, drug release characteristics, biocompatibility, in vitro antimicrobial activity, and in vivo retention of OD‐PP@SeNPs hydrogels. Second, the effects and mechanisms of hydrogel remodeling of the inflammatory microenvironment were comprehensively explored at the cellular level. Finally, the therapeutic effects of OD‐PP@SeNPs on 22.1Figure  The surface morphology of SeNPs was observed by TEM  peak in the XPS spectrum of OD‐PP@SeNPs hydrogel indicated that SeNPs were successfully added to the hydrogel system  reveals the elastic properties of the hydrogels and the loss modulus (G″) reveals the viscous properties of the hydrogels. In the range of 0.01 to 1 Hz, G′ was higher than G″ for all groups without any cross trace  allow the hydrogel to retain large amounts of water in its network, enabling a favorable moist environment to be maintained at the application site. The OD‐PP@SeNPs hydrogel kept the tissue moist and absorbed exudate to prevent exudate buildup and sepsis, which was efficient for tissue repair. Based on the results, OD‐PP and OD‐PP@SeNPs exhibited excellent swelling properties, which could accelerate the absorption of excess fluid from hyperpermeable wounds. Generally speaking, excessive swelling will eventually break the hydrogel network and could result in elevated compression on neighboring tissues, this is not optimal for hydrogels. The addition of SeNPs reduced the swellability because SeNPs acted as cross‐linking agents, occupied some hydrophilic cross‐linking sites, and reduced the pore size. Thus, OD‐PP@SeNPs hydrogel exhibited proper swelling capacity, which gave the hydrogel the ability to absorb inflammatory tissue fluids and helped to alleviate oxidative stress. The reasonable degradation rate of biomaterials is an essential indicator for biomedical applications. Furthermore, after 14 d of submersion in pH 7.4 PBS, the weight reduction of the hydrogels was less than 21.67 ± 8.51% , the hydrogels would rapidly disintegrate and change into a concentrated solution, leading to the exposure of BSA‐FITC or SeNPs. The release results indicated that the hydrogel network was broken due to hydrolysis of the Schiff base bonds and Phenylboronate ester bonds under acidic and oxidative conditions. The OD‐PP@SeNPs hydrogel could not only rapidly release PP and OD with antibacterial and antioxidant properties but also sustainably release SeNPs, which have anti‐apoptosis activity. The Schiff base bonds and Phenylborate ester bonds in hydrogels also regulate the pH and ROS of the solution when hydrolyzed. Therefore, OD‐PP@SeNPs hydrogel has good application potential for treating inflammatory diseases. The release kinetics model studied the release characteristics of the hydrogel. The diffusional exponent (n) depended on the release mechanism and the shape of the drug delivery device: 0.45 < n < 0.89 indicates a non‐Fickian diffusion and erosion release mechanism where a combination of diffusion and polymer relaxation controlled release. The swelling and relaxation of polymer chains and the invasion of the matrix polymer were involved during the release process .2.6 Hence, aseptic conditions are essential for parenteral hydrogels to avoid contamination. At the same time, an ideal hydrogel should have antibacterial properties, which help reduce the number of bacteria in the tissue, control inflammation, and accelerate recovery. It has been reported that excess Se exhibits potent antibacterial activity. Thus, E. coli (early infection) and S. aureus (late infection) were used to evaluate the in vitro antibacterial performance of OD‐PP@SeNPs hydrogel and to explore the OD‐PP@SeNPs hydrogel potential antibacterial mechanism. The evaluation methods used were colony formation, OD value, bacterial precipitation, disc diffusion, and live & dead bacteria staining.Tissue infection is an excellent challenge in the clinical setting. Due to the long‐term inflammatory response, more exudate is secreted, which easily induces tissue infection. Bacterial metabolism and infection can generate unique microenvironments with pH differences. Bacterial fermentation produces lactic acid, which can acidify its surrounding pH as low as 3.5.62 Hen As expected, the majority of bacteria were inactivated after contact with materials  for 12 h  were 12.27 ± 1.03 and 13.25 ± 1.24 mm, respectively. Both SeNPs and OD‐PP hydrogel had little antibacterial zones. The integrity of the bacterial cell membrane was examined by SYTO9 (LIVE dye)/propidium iodide (PI) double fluorescent staining and visualized by confocal laser scanning microscopy (CLSM), where green means live bacteria and red means dead bacteria. The Model group showed strong green fluorescence. In contrast, both SeNPs and OD‐PP@SeNPs hydrogels showed extensive red fluorescence , Human Umbilical Vein Endothelial Cells (HUVECs), and Mouse Fibroblast Cells (L929).2O2 were screened by cell activity and oxidative indicators (NO and ROS), respectively. And the final concentration of LPS was 1 µg mL−1  staining, respectively.67 Fir The SOD in the LPS group was significantly decreased, demonstrating the enzyme oxidizing system of macrophages was damaged under inflammation  activity, Glutathione peroxidase (GPx) level, and malondialdehyde (MDA) content assay were performed to address the intracellular antioxidative properties of the OD‐PP@SeNPs hydrogel. SOD is a vital antioxidant metalloproteinase in cells, which can resist oxidative damage caused by external stimuli. Its activity reflects the severity of oxidative damage to cells.68 TheFigure m, SeNPs did not exert a cell migration‐promoting effect (data not shown). Endothelial cell vascularization is a prominent process in angiogenesis. The in vitro tube formation assay was implemented to inspect the angiogenic ability of SeNPs and OD‐PP@SeNPs hydrogel. SeNPs can promote tube formation only at concentrations ≥100 µm , M1, and M2 phenotypes. The M1 phenotype secretes inflammatory cytokines, accelerating oxidative processes and inhibiting tissue repair. And the M2 phenotype secretes anti‐inflammatory cytokines, which reduce the release of inflammatory factors and promote tissue repair and antioxidant activity. An imbalance in M1/M2 balance leads to a chronic inflammatory response. The cells not only secret proinflammatory cytokines such as TNF‐α, IL‐6, and IL‐1β but also accelerate the degradation of the cartilage extracellular matrix. TNF‐α is the predominant proinflammatory cytokine that initiates the inflammatory cascade and is also a central proinflammatory cytokine in the pathogenesis of IDs. Macrophages mainly produce IL‐1β and play an important role in bone erosion, synovial hypertrophy, leukocyte infiltration, and cartilage destruction. It exerts proinflammatory effects principally by regulating the expression of various macrophages, immunomodulatory molecules, cell adhesion molecules, and inflammatory mediators. TNF‐α and IL‐1β have synergistic effects, and both interact with each other to mediate joint damage and inflammatory responses. IL‐10 is a crucial anti‐inflammatory cytokine, which can effectively inhibit the effector T cell response. To study the anti‐inflammatory effects of the OD‐PP@SeNPs hydrogel, we investigated protein expression in RAW264.7 and HUVECs. ELISA assays were used to detect the levels of TNF‐α, IL‐1β, IL‐6, and IL‐10. OD‐PP@SeNPs hydrogel significantly decreased TNF‐α, IL‐1β, and IL‐6 levels and increased IL‐10  polarization of the RAW264.7 macrophages, whereas the addition of OD‐PP@SeNPs hydrogel remarkably induced the M2 phenotype polarization (CD206) in the presence of LPS, demonstrating the shift toward M2 macrophage polarization . All procedures followed ethical standards during the experiment. New Indocyanine Green (IR820) was added to the OD‐PP@SeNPs hydrogel, and the hydrogel was detected in the abdominal subcutaneous and joint cavity retention experiments by the in vivo imaging system. Compared with in vitro degradation, the hydrogel showed a faster degradation rate in vivo, which was attributed to the body temperature of 37 °C and the complex in vivo environment. In LPS (‐) and Healthy group, it is manifest that the OD‐PP@SeNPs hydrogel could be retained locally for at least 14 d  failed to heal themselves  and M1 macrophages (CD86) expression. The Model group showed the most intense green fluorescence, which implies a massive infiltration of M1‐type macrophages and a highly inflammatory state of the wound Figure . More M2S. aureus infection. On the other hand, it reshapes the inflammatory microenvironment. OD‐PP@SeNPs hydrogel continuously released SeNPs. The presence of SeNPs, Schiff base bonds, and Phenylboronate ester bonds induced the polarization of M1 macrophages to M2 type, reduced the level of inflammatory cytokines, decreased the level of ROS in the inflammatory site, shortened the inflammatory phase, reshaped the inflammatory microenvironment, and made the wound enter the cell proliferation stage as soon as possible. In particular, SeNPs promote endothelial cell proliferation and angiogenesis, accelerating wound healing and avoiding excessive vascularization without leaving visible scars.Thus, these two factors illustrate the prominent wound healing properties of OD‐PP@SeNPs hydrogel. On the one hand, antibacterial. SeNPs and PP inhibited the growth of bacteria at the wound site, effectively reduced the inflammation caused by bacteria, and accelerated the wound healing of  Therefore, a CIA rat model was established as a pathological model of RA to assess the role of OD‐PP@SeNPs hydrogel in RA. Methotrexate (MTX), the gold standard for RA, was used as a Positive group.Figure  The fully developed arthritis was confirmed in the Model group after observing noticeable swelling, tenderness, deformity, and ankyloses  model is one of the animal models of RA currently used in preclinical studies of drugs. It is highly similar to human RA regarding clinical features, pathological changes, and immune response.85 Thes Figure . As shows Figure . Specifi The progression or recovery of the RA rats following OD‐PP@SeNPs hydrogel administration was confirmed by evaluating the status of bone, cartilage, and synovium from 57 d. Synovial hyperplasia and joint structure of CIA rats were evaluated by H&E staining , increase IL‐10 secretion, eliminate ROS in joints, and reshape the inflammatory microenvironment. Finally, the OD‐PP@SeNPs hydrogel inhibited synovial hyperplasia/inflammation, reduced osteoclast formation, improved cartilage and bone reconstruction, reduced CIA rat foot swelling, improved immune organ index, and achieved the effect of relieving RA.2.10Hemocompatibility is a fundamental feature of biomaterials since they are in direct or indirect contact with blood. The hemolytic test was performed to evaluate the blood compatibility of the hydrogels. The supernatant of all formulations (OD‐PP@SeNPs hydrogel and SeNPs) with blood cells showed a yellowish to reddish (Figure The biocompatibility of the OD‐PP@SeNPs hydrogel was systematically assessed in vivo. H&E staining exhibited that all the preparation groups were safe for the rats because of the ignorable organ damage and normal cell morphology (Figures 3We prepared for the first time a dynamically responsive hydrogel that integrated multiple functions based on PBA grafted PLL, oxidized dextran, and SeNPs. The OD‐PP@SeNPs hydrogel has a triple‐network structure due to the role of SeNPs as ligands besides Schiff base bonds and Phenylboronate ester bonds, thus resulting in a tough composite hydrogel. The OD‐PP@SeNPs hydrogel has ideal mechanical properties, injectable, self‐healing, adhesion, good flexibility, swelling capacity, biodegradability, stimuli‐responsive active substance release performance, and excellent biocompatibility. The OD‐PP@SeNPs hydrogel with ROS scavenging and pH‐regulating ability protects cells from oxidative stress and induces macrophages into M2 polarization to reduce inflammatory cytokines through PI3K/AKT/NF‐κB and MAPK pathways, exerts anti‐inflammatory effects and reshapes the inflammatory microenvironment. All these features make tri‐cross‐linked dynamically responsive hydrogels as promising biomimetic materials for antibacterial, therapeutic IDs, and other biomedical applications. In the future, we will explore the pharmacological mechanism of OD‐PP@SeNPs hydrogel in modulating the inflammatory microenvironment in vivo and broaden their applications.The authors declare no conflict of interest.Z.G., W.H., and S.W. designed this study. S.W. carried out experiments on synthesis, in vitro cell experiments and animal experiments. Y.L., Q.S., B.Z., C.L., L.G., H.W., and L.C. assisted with the animal experiments and Software. The major contribution of M.J., W.H., and J.G. was the teaching method. S.W. analyzed the data and wrote this manuscript. Z.G. revised this manuscript.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.