Oxaliplatin is an effective chemotherapeutic agent for the treatment of malignant tumors. However, severe oxaliplatin-induced peripheral neurotoxicity (OIPN) has been well documented. Traditional Chinese medicine injections (TCMIs) have shown significant efficacy in preventing OIPN. However, it is difficult for clinicians to determine the differences in the efficacy of various TCMIs in preventing OIPN. The aim of this study was to compare the efficacy of various TCMIs in preventing OIPN through a network meta-analysis (NMA) to further inform clinical decision-making.  The Chinese Journal Full Text Database, Chinese Biomedical Literature Database, Wanfang Data Knowledge Service Platform, Chinese Science and Technology Journal Full Text Database, the Cochrane Library, Web of Science, PubMed, and Embase databases were searched for randomized controlled trials (RCTs) of TCMIs for OIPN prevention. The retrieval time was from the establishment of the database to April 12, 2021. NMA was performed using Stata 14.0 software after 2 evaluators independently screened the literature, extracted information, and evaluated the risk of bias of the included studies. Astragalus injection (AI), elemene emulsion injection (EEI), Shenfu injection (SFI), Shenqi Fuzheng injection (SIFZI), Kanglaite injection (KLEI), Huachansu injection (HCSI), and lentinan injection (LI). NMA results showed that AI was superior to AD and SIFZI was superior to ADI in reducing the incidence of grade I neurotoxicity. SIFZI was superior to EEI and ADI, and BJOEI was superior to chemotherapy alone in reducing the incidence of grade II neurotoxicity. SMI was superior to LI and CKSI in reducing the incidence of grade III neurotoxicity. SIFZI was superior to LI, BJOEI, XAPI, EEI, SMI, chemotherapy alone, HCSI, KLEI, and ADI in reducing the total incidence of grade I–IV neurotoxicity. SFI was superior to ADI. Based on the SUCRA values, AI was the most likely intervention to reduce the incidence of grade I neurotoxicity, SIFZI was the most likely intervention to reduce the total incidence of grade II and I–IV neurotoxicity, and SMI was the most likely intervention to reduce the incidence of grade III and IV neurotoxicity.  A total of 45 eligible RCTs involving 3598 cancer patients and 13 TCMIs were included. The 13 TCMIs included Xiaoaiping injection (XAPI), compound kushen injection (CKSI), Aidi injection (ADI), Brucea javanica oil emulsion injection (BJOEI), Shenmai injection (SMI), Kangai injection (KAI),  TCMIs can prevent OIPN to some extent, among which SIFZI, SMI, and AI may be the most promising TCMIs. However, given the limitations of current studies, more well-designed, high-quality clinical trials will be needed in the future to validate the benefits of TCMIs. Howeverute OIPN . It is cute OIPN . Howeverute OIPN , 8. TherAt present, there is no specific method for the prevention and treatment of this kind of peripheral neurotoxicity, and symptomatic treatment of Western medicine is mainly used, such as nerve nutrition, nerve growth factor supplementation, and antioxidant treatment with reduced glutathione –11. In fAstragalus injection and Shenmai injection have shown good clinical effects in preventing the occurrence of OIPN [OIPN belongs to the category of “paralysis” and “impotence” in Chinese medicine. Many studies have shown that Chinese medicine injections (TCMIs) such as  of OIPN –16. Howe of OIPN , 18. ThiNMA was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .Randomized controlled trials(RCTs) were included.Patients with a diagnosis of malignancy confirmed by histopathology and/or cytology or imaging. Treatment with oxaliplatin or oxaliplatin-containing chemotherapy regimens was specified in the chemotherapy regimen.The control group was given chemotherapy alone or chemotherapy with placebo. The experimental group used TCMIs in addition to chemotherapy, and the type, dose, and frequency of TCMIs were not limited.The incidence of OIPN includes the incidence of grade I neurotoxicity, grade II neurotoxicity, grade III neurotoxicity, grade IV neurotoxicity, and the total incidence of grade I–IV neurotoxicity.Republished literatureLiterature with incomplete dataBoth groups used other Chinese medical treatments such as traditional Chinese medicine decoction, Chinese patent medicine, or acupunctureNonrandomized controlled trialThe Chinese Journal Full Text Database, Chinese Biomedical Literature Database, Wanfang Data Knowledge Service Platform, Chinese Science and Technology Journal Full Text Database, the Cochrane Library, Web of Science, PubMed, and Embase databases were searched for randomized controlled trials (RCTs) of TCMIs for OIPN prevention. The retrieval time was from the establishment of the database to April 12, 2021. Search terms included oxaliplatin, neurotoxicity, names of included TCMIs, RCTs, and their synonyms. The search strategy was developed according to the criteria of the Cochrane systematic review handbook. Taking PubMed database as an example, detailed search strategies are shown in supplementary materials .Two researchers independently screened the literature, extracted information, and cross-checked it according to inclusion and exclusion criteria. In case of any disagreement, it was resolved through discussion or referred to a third party for negotiation. The data extraction included (1) basic information of the included studies, including study title, first author, journal of publication, and time; (2) baseline characteristics of the study population, including sample size of each group, age of patients, population origin, and tumor type; (3) specific details of the interventions, including the TCMIs used and the type of chemotherapeutic agents; (4) key elements of bias risk assessment; and (5) outcome indicators and outcome measures of interest, including the measurement tools for OIPN and the incidence of OIPN.The risk of bias for RCTs was evaluated by 2 investigators according to the Cochrane systematic review handbook . EvaluatI2 was used to quantitatively determine the magnitude of heterogeneity. If I2 < 50% and P > 0.1, meta-analysis was performed using a fixed-effects model. If I2 ≥ 50% and P < 0.1, meta-analysis was performed using a random-effects model. Since this study was an indirect comparison of various TCMIs combined with chemotherapy based on chemotherapy, no consistency test was required. Network group commands were used for data preprocessing in NMA. Network evidence plots and “corrected-comparison” funnel plots were drawn for each outcome indicator, and pairwise comparisons of different interventions were performed. Efficacy was ranked according to the surface under the cumulative ranking curve (SUCRA). Stata 14.0 was used for direct comparison meta-analysis, NMA, and graph drawing.Count data were analyzed with relative risk (RR) and 95% confidence interval (95% CI) as efficacy statistics. A total of 4692 literature were retrieved through electronic databases, and 1038 duplicates were removed. 3568 literature were excluded by reading the titles and abstracts. The remaining 246 literature were read through the full text, and finally 45 literature , 21–62 wAstragalus injection (3 items) [The 45 RCTs , 51–62 w3 items) , 16, 30,3 items) , 56, 57,3 items) , 40, 50,3 items) , 55, 59,3 items) , Huachan3 items) , 49, Kan3 items) , 29, 31,3 items) , lentina3 items) , Shenfu 3 items) , 47, 62,3 items) , 60, 61,3 items) , 52. All14 studies were considered low risk for randomization, 4 studies were assessed as high risk because they had incorrect methods of random sequence generation, and the randomization of the remaining 27 studies was unclear. The method of allocation concealment was unclear for all studies. Due to the specificity of TCMIs, it is difficult to do blinding. The blinding method for all studies was unclear. For incomplete outcome data, one study showed high risk of bias. Details of the risk of bias assessment are shown in P < 0.05) compared with chemotherapy; SFI and AI could reduce incidence of grade II neurotoxicity (P < 0.05); SFI and SMI could reduce incidence of grade III neurotoxicity (P < 0.05); ADI, SFI, SMI, SIFZI, CKSI, HCSI, AI, KLEI, LI, and XAPI could reduce total incidence of grade I ∼ IV neurotoxicity (P < 0.05). Results of direct comparative meta-analyses are shown in A meta-analysis of direct comparisons of TCMIs combined with chemotherapy compared to chemotherapy alone was conducted. The results showed that AI can reduce incidence of grade I neurotoxicity ), and SIFZI versus ADI ), and there were no significant differences in other interventions .Incidence of grade II neurotoxicity was reported in 19 studies involving 11 TCMIs and 1462 patients. The results of the NMA showed statistically significant differences for SIFZI versus EEI ), SIFZI versus ADI ), and BJOEI versus chemotherapy alone ), and the difference between the remaining interventions was not statistically significant .Incidence of grade III neurotoxicity was reported in 15 studies involving 8 TCMIs and 1227 patients. The results of the NMA showed statistically significant differences in SMI versus LI ), and SMI versus CKSI ), and the difference between the remaining interventions was not statistically significant .Incidence of grade IV neurotoxicity was reported in 4 studies involving 3 TCMIs and 355 patients. The results of the NMA showed no statistically significant differences in the comparison of the interventions .Total incidence of grade I ∼ IV neurotoxicity was reported in 45 studies involving 13 TCMIs and 3598 patients. The results of the NMA showed that the differences were statistically significant for SIFZI versus LI ), SIFZI versus BJOEI ), SIFZI versus XAPI ), SIFZI versus EEI ), SIFZI versus SMI ), SIFZI versus chemotherapy ), SIFZI versus HCSI ), SIFZI versus KLEI ), SIFZI versus ADI ), and SFI versus ADI ), and the differences between the remaining interventions were not statistically significant .The SUCRA cumulative probability ranking showed that AI was most likely to be the best intervention to reduce the incidence of grade I neurotoxicity. Ranking results of incidence of grade I neurotoxicity were AI (SUCRA = 84.1%) > SIFZI (SUCRA = 78.4%) > SMI (SUCRA = 64.2%) > LI (SUCRA = 58.5%) > chemotherapy (SUCRA = 54.5%)>SFI (SUCRA = 52.5%)>EEI (SUCRA = 49.7%)>XAPI (SUCRA = 44.8)>BJOEI (SUCRA = 34.6%)>CKSI (SUCRA = 33.7%)>ADI (SUCRA = 26.4%)>KAI (SUCRA = 18.6%) . SIFZI wIf no less than 10 studies were included, comparison-corrected funnel plots were drawn to identify the possibility of small-sample effects in the intervention network. The resulting funnel plot was slightly asymmetric, considering the possibility of a small-sample effect or publication bias between studies .We performed sensitivity analyses for outcome indicators that included at least 3 or more literature. Sensitivity analysis showed that SMI plus chemotherapy versus chemotherapy reversed the results of the meta-analysis in terms of incidence of grade I neurotoxicity and incidence of grade II neurotoxicity. The results of the meta-analysis were reversed for AI plus chemotherapy versus chemotherapy in terms of incidence of grade II neurotoxicity. KAI plus chemotherapy versus chemotherapy and XAPI plus chemotherapy versus chemotherapy were reversed for incidence of grade I ∼ IV neurotoxicity. The results of the meta-analysis were reversed for neurotoxicity. No reversal was found for the remaining outcome indicators. The results are presented in the Supplementary Material Figures –S17.OIPN is the primary dose-limiting toxicity of oxaliplatin and is characterized by specific somatosensory features, including cold and mechanical abnormal pain . The patThe NMA evaluated the efficacy of 13 TCMIs for the prevention of OIPN in 3598 cancer patients. 13 TCMIs include XAPI, CKSI, ADI, BJOEI, SMI, KAI, AI, EEI, SFI, SIFZI, KLEI, HCSI, and LI. The NMA results showed that AI was better than ADI and SIFZI was better than ADI in preventing the incidence of grade I neurotoxicity, and the probability ranking showed that AI > SIFZI > SMI > LI > chemotherapy > SFI > EEI > XAPI > BJOEI > CKSI > ADI > KAI. SIFZI was superior to EEI and ADI, and BJOEI was superior to chemotherapy alone in preventing the incidence of grade II neurotoxicity. The probability ranking results showed that SIFZI > CKSI > SMI > LI > SFI > XAPI > KAI > BJOEI > EEI > ADI > AI > chemotherapy. SMI was superior to LI and CKSI in preventing the incidence of grade III neurotoxicity. Probability ranking results show that SMI > SFI > XAPI > LI > ADI > AI > SIFZI > chemotherapy > CKSI. There was no statistically significant difference between the interventions in preventing the incidence of grade IV neurotoxicity. The probability ranking results showed that SMI > AI > ADI > chemotherapy. SIFZI was superior to LI, BJOEI, XAPI, EEI, SMI, chemotherapy alone, HCSI, KLEI, and ADI in preventing grade I ∼ IV neurotoxicity; SFI was superior to ADI. The probability ranking results showed that SIFZI > SFI > AI > LI > KAI > CKSI > BJOEI > XAPI > EEI > SMI > chemotherapy > HCSI > KLEI > ADI. SIFZI, SMI, and AI had the largest SUCRA values and were most likely to be the best treatment options. Considering the moderate quality of the included studies and the limited number of included studies, the probability ranking results are for clinicians' reference only.Astragalus may be a potential nerve growth-promoting factor that helps promote the growth of peripheral nerve axons [Astragalus, attenuates OIPN by modulating neuroinflammation and oxidative stress and downregulating the expression of TNF-α, IL-6, and IL-1β [In vitro and in vivo studies suggest that extracts of ve axons . Astragand IL-1β , 76. A snd IL-1β  showed tnd IL-1β . Therefond IL-1β . From thnd IL-1β , 78. Ophnd IL-1β . The metIn this study, NMA was used for the first time to compare the clinical efficacy of different TCMIs in the prevention of OIPN, with a large number of included studies and a large sample size, showing high statistical efficacy. However, there were also certain limitations: (i) the included studies were all in Chinese, which may have language bias; (ii) the quality of the included studies was average, and most of them did not mention allocation concealment and blinding, which may affect the reliability of the results; (iii) there was some heterogeneity in some results, which may be related to the clinical characteristics of the included studies such as different tumor types and chemotherapy regimens. (iv) The included RCTs were compared on the basis of chemotherapy combined with TCMIs and chemotherapy alone, and there was a lack of direct comparison between TCMI. This may have weakened the strength of the evidence supporting the results. Therefore, future high-quality randomized controlled trials are needed to assess the clinical efficacy of TCMIs for the prevention of OIPN.In summary, the application of TCMIs on top of oxaliplatin-containing chemotherapy regimens can prevent the occurrence of OIPN to some extent. Among them, AI focused on reducing grade I neurotoxic reactions, SIFZI focused on reducing grade II and I ∼ IV neurotoxic reactions, and SMI focused on reducing grade III and IV neurotoxic reactions. However, based on the limitations of this study, the efficacy ranking does not fully indicate the clinical efficacy, and the results of this ranking should be viewed with caution.


T1 colorectal cancers have a low lymph node metastasis rate and good prognosis. Thus, endoscopic resection is an attractive choice. This study aimed to describe the value of poorly differentiated cluster grade in identifying endoscopically curable T1 colorectal cancers.We included 183 T1 colorectal cancer patients who underwent curative resection. Univariate and multivariate logistic regressions were used to identify lymph node metastasis predictors. The Akaike information criterion was used to determine whether poorly differentiated cluster grade was the best predictor. Backward regression was used to screen the variables. Survival analyses were conducted to determine the prognostic predictive power of poorly differentiated cluster grade. Correlations among predictors and concordance between our pathologists were also investigated.G 3 = 0.001; 95% confidence interval [95% CI]G 3 =  < 0.001, 0.139) in T1 colorectal cancer patients; moreover, it had the best predictive value (AIC = 61.626) among all indicators. It was also screened for inclusion in the predictive model. Accordingly, a high poorly differentiated cluster grade independently indicated shorter overall survival  and disease-free survival .Poorly differentiated cluster grade was an independent predictor for lymph node metastasis (adjusted odds ratio [OR]Poorly differentiated cluster grade is a vital reference to manage T1 colorectal cancer. It could serve as an indicator to screen endoscopically curable T1 colorectal cancers. Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide , 2, and We aimed to clarify the value of the PDC grade in determining T1 CRCs requiring surgery. When the risk of LNM is considered minimal in patients with T1 CRC, endoscopic resection is an effective alternative to radical resection. We investigated the influence of PDC grade on LNM and compared its predictive power with that of other indicators. Moreover, we developed and tested a proper model based on the PDC grade and illustrated its unique role in clinical settings. Oncological outcomes among T1 CRC patients were examined to validate the prognostic value of the PDC grade. We also identified and described some correlations among selected parameters that are worthy of mention. Additionally, finding the interobserver agreement for our PDC grading was one of our objectives.This research study was conducted retrospectively from data obtained for clinical purposes and was reviewed and approved by the Ethics Committee of the Affiliate Hospital of Qingdao University (Reference Number QYFYWZLL26957). The procedures used in this study adhere to the tenets of the Declaration of Helsinki. The need for written informed consent was waived by the Ethics Committee of the Affiliate Hospital of Qingdao University due to retrospective nature of the study.The data from patients who underwent curative operations for primary T1 CRC at the Affiliated Hospital of Qingdao University from 2002 to 2020 were retrospectively reviewed and analyzed. All resections were primary surgical resections. Our mean number of lymph nodes (LNs) per patient was 15, when the American Joint Committee on Cancer Staging (AJCC) and National Quality Forum guidelines suggested that at least 12 LNs should be dissected for CRC patients . MoreovePotential variables included the PDC grade, depth of submucosal invasion (> 1 mm vs. ≤ 1 mm), stratification of submucosal invasion (SM 1 vs. SM 2), lesion size (> 20 mm vs. ≤ 20 mm), tumor volume (the mean volume was the cutoff), tumor budding (TB) grade, World Health Organization tumor grade , perineural invasion (PNI), lymphvascular invasion (LVI), mucinous composition, tumor location (right colon vs. left colon vs. rectum), number of lesions , operation method (laparotomy vs. laparoscopy), and postoperative adjuvant chemotherapy . Patient age, sex, body mass index (BMI), hypertension (HP), diabetes mellitus (DM), and coronary heart disease (CHD) are also shown.All tumor tissues were independently reassessed by at least two experienced pathologists who were blinded to the clinical features and original histological reports. In cases of disagreement, a third pathologist (or more) joined the assessment. Majority decisions were considered the final consensus. Moreover, our interobserver concordance of PDC grading was also tested. Pathological parameters were determined by gross specimen analysis rather than endoscopic biopsy to prevent biases caused by insufficient samples. The number of PDCs in a single field with the highest activity was determined and subsequently graded. TB was defined as 1–4 malignant cells at the tumor invasive front, which were evaluated in one field using a 20 × objective lens and counted according to the International Consortium of TB Recommendations (ITBCC) , 33. TB We used a composite endpoint. The primary outcome was the implication of the parameters  on LNM status. The secondary endpoint was the prognostic value of the identified indexes, including overall survival (OS) and disease-free survival (DFS). OS was defined as the date of surgery to the date of death or the follow-up deadline . DFS was defined as the date of surgery to the date of recurrence/distant-metastasis or the follow-up deadline .Postoperative outcomes were investigated through routine scheduled outpatient visits at 3-month intervals during the first 2 years, at 6-month intervals for 3–5 years, and at 12-month intervals thereafter. The following examinations were performed: colonoscopy, abdominal and pelvic computed tomography, and measurement of serum tumor markers levels. In addition, the investigators conducted telephone interviews to estimate the general condition of each patient .2 test or Fisher’s exact test. Moreover, our interobserver concordance of the PDC grading system was shown by the Kappa consistency test to validate the effectiveness of PDC as a predictor. All statistical analyses were conducted with SPSS software . A P value < 0.05 (two-sided) was considered statistically significant.Univariate and multivariate logistic regressions were used to analyze the primary endpoint. Variables that were significant in univariate analyses were introduced into the multivariate analysis. The LNM fit was compared by the Akaike information criterion (AIC) value . Backward regression was used to explore the models. Survival curves were plotted by Kaplan–Meier survival curves (K-M curves), and the differences were evaluated with a log-rank test. Univariate analyses selected covariates to introduce into the multivariate Cox regression models to identify independent risk factors for a poor prognosis. Correlations among variables were evaluated by the χA cohort of 183 patients was included and analyzed in this study. The LNM rate was 10.9%. The percentages of patients with different PDC grades were G1, 71.6% (131/183); G2, 19.7% (36/183); and G3, 8.7% (16/183). Among the patients with LNM, the percentages of patients with different PDC grades were G1, 5.0% (1/20); G2, 30.0% (6/20); and G3, 65.0% (13/20). Among the patients without LNM, the percentages of patients with different PDC grades were G1, 79.8% (130/163); G2, 18.4% (30/163); and G3, 1.8% (3/163).P < 0.001), deeper submucosal invasion (P < 0.001), higher TB grade (P < 0.001), PNI (P < 0.001), LVI (P = 0.002), and postoperative adjuvant chemotherapy (P < 0.001) (Table P < 0.001), deeper submucosal stratification (P = 0.008), higher TB grade (P < 0.001), PNI (P = 0.003), LVI (P = 0.001), greater mucinous composition (P = 0.015), and postoperative adjuvant chemotherapy (P < 0.001)   (Table PDC grade can independently influence LNM in T1 CRC patients. Univariate logistic regressions found that PDC grade was an influencing factor for LNM (P < 0.001), stratification of submucosal invasion , TB grade , PNI , and LVI  all influenced LN status (Table P = 0.034), PNI , and LVI  independently influenced LNM. See Table Regarding the other LNM predictors, univariate logistic regressions showed that the depth of submucosal invasion  , and it was not introduced into multivariate Cox regression models. The results of multivariate analyses are shown in Table Regarding the depth of submucosal invasion, according to the univariate analyses, 12 patients in the > 1 mm group and 8 patients in the ≤ 1 mm group had died at the 5-year follow-up, and the mean OS times  was explored, and backward regression was properly used. The results of this work also indicate that multidisciplinary team (MDT) discussion should be recommended.Our results revealed a correlation between a higher PDC grade and deeper submucosal invasion. Combined with the findings reported in previous studies, ECRCs with deeper submucosal invasion tend to be incompletely resected under endoscopy and even require adjuvant therapy after rection . Our assBd 2 = 0.088, crude ORBd 3 = 0.013); however, an association was not found in the multivariate analysis. Ueno et al. [The depth of submucosal invasion is the basis of T1 CRC treatments in the Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines , 39. Nevo et al.  previouso et al. . As a coo et al. , 42, higo et al. , 43, 44.o et al. . The LN The PDC grade performed well in terms of predictive power. PDCs are cell clusters/nests that are larger and recognizable without specific staining , which sObviously, to obtain firm conclusion that PDC grade is a good reference for selecting management strategies, investigating its predictive value relative to LNM is not enough. Some research has found that local LNM is a precursor for distant metastases  and thatThe procedure used to explore predictive indicators in our work is well established and provides a reference for developing new indexes. We applied the AIC value to compare fit and prove its clinical utility, expanding the use of the fit comparative method of indicators and models. Backward regression was also properly and innovatively used. Moreover, our concordance for the indicator grading was also found by the kappa consistency test. We await the exploration and validation of image recognition tools based on deep learning to further avoid biases caused by histological evaluation.This work has unique clinical value. However, we acknowledge that, as a single-center retrospective study, the limitations of the study type cannot be ignored. Moreover, the small sample size and the low absolute number of patients with oncological outcomes  could have caused bias. Nevertheless, we faithfully recorded the patients’ information in detail. Moreover, all slides were reevaluated according to the definitions in the available consensus to improve the quality of the pathological records. In addition, we used archived slides, and tissue processing was not standardized. However, this defect exactly reflects real-time practice and increases the generalizability of our results to a certain extent. Furthermore, our participants were all from a registered population in Qingdao and the surrounding area; therefore, extrapolating our results requires caution. The interstitium of participants might promote standardized sample collection and minimize possible residual confounding caused by differences in subjects’ genetic backgrounds. Moreover, this study included only surgical resection cases when the endoscopic resection alone cohort was essentially part of our true target. However, potential biases such as insufficient material and blurred margins caused by initial endoscopic resection and the possibilities of unclarified depth/stratification of submucosal invasion and PNI/LVI caused by unradical endoscopic resection followed by secondary surgical resection were avoided to some extent. Further investigations based on endoscopic resection cases alone are still needed.To further confirm our conclusions, multicenter, prospective, and large sample studies are needed. Moreover, the predictive value of PDC grade in biopsy samples also requires further investigation. This might represent a clinical window that allows clinicians to stratify patients by their LNM risk and prognosis at initial biopsy. Then, the subsequent management of patients may be improved. Furthermore, more complete stratification models based on methods such as machine learning are also expected in the future.PDC grade is the best predictive factor for LNM and the oncological outcomes of T1 CRC patients, representing a novel, simple and reliable surgical indicator.


Hirudo nipponia is the only blood-sucking leech included in Chinese Pharmacopoeia having distinct features of anticoagulation, exorcizing blood stasis, and promoting menstruation. Despite such significant characteristics, very little is known about its molecular genetics and related physiological mechanisms. In this study, the transcriptomes of H. nipponia at three developmental stages , revealed a total of 1,348 differentially expressed genes (DEGs), 223 differentially expressed lncRNAs, and 88 novel mRNAs. A significant diverse gene expression patterns were observed at different developmental stages which were analyzed by differential gene expression trends, and the overall gene expression trends consist of three overall down-regulated trends, and two overall up-regulated trends. Furthermore, the GO and KEGG enrichment functional annotation analysis revealed that these DEGs were mainly associated with protein hydrolysis, signal transduction, energy metabolism, and lipid metabolism while growth, development, metabolism, and reproduction-related DEGs were also found. Additionally, real-time quantitative PCR results confirmed deep sequencing results based on the relative expression levels of nine randomly selected genes. This is the first transcriptome-based comprehensive study of H. irudo nipponia at different developmental stages which provided considerable deep understanding related to gene expression patterns and their relevant developmental pathways, neurodevelopmental and reproductive characteristics of the leech. Hirudo nipponia  is widely distributed in many regions of the globe , young (Y), and adult (A) to provide a scientific basis for later research on the breeding, growth, and development of H. nipponia and related functional genes as well as the lncRNAs which are important for chromosomal modification, transcriptional activation, and interference.Many studies have focused on the role of effective components in leech and their cultivation . Recent proteins . But verH. nipponia used in the experiment was collected from Qinzhou Zhanhong traditional Chinese medicine company. The samples were divided into a larval stage (within 10 days of hatching and have not sucked the blood), young insect stage (about 3 months after hatching and sucked blood three times) and adult stage  ( mature) . The leeH. nipponia were observed through paraffin embedding, sectioning, hematoxylin, and eosin staining. The muscles’ ultra-morphology was observed by microscope (EVOS FL auto), and the images were collected via micrograph system (EVOS auto).The histological features of H. nipponia at different developmental stages  using TRIzol reagent . The RNA was purified using the RNeasy Mini Kit . The cDNA library was sent to Beijing Anuo Uda Gene Technology and sequenced using the Illumina HiSeq 4,000 platform , which generated 150 bp paired-end reads. Each sample quality was checked using Fastp (v0.20.1)  . After tv0.20.1)  was usedv0.20.1)  was emplv0.20.1)  was thenv0.20.1)  was usedThe FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) method was used to calculate the expression of all genes in each sample. The differentially expressed genes (DEGs) between different groups were analyzed using DESeq2 software. Genes with FDR  < 0.05 and |log2FC|>1 was set as a criterion to find the DEGs between two stages and Ggplot2 software was used to locate the DEGs.The Tcseq  software was used to analyze the DEGs at three-time points, and after clustering the patterns of DEGs were visualized at different developmental stages. The FDR value <0.05 and |log2FC|>1 was considered as the level of significance to determine the DEGs between the two stages.The functional annotation analysis of DEGs was done by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and genomes) using a cluster profiler . The value of FDR <0.05 was considered to be significantly enriched.Potential lncRNAs were screened by the following highly stringent criteria: 1) transcript length was not less than 200 nt; 2) transcripts were labeled as “i”, “u”, “324” and “x”; 3) the coding potential calculator (CPC2) score was “non-coding”; 4) transcripts containing an open reading frame (ORF) greater than 50 AA were removed; 5) transcripts were identified by alignment with the Swiss lncRNAs act as a cis-regulatory factor and can regulate the expression of adjacent genes. Furthermore, the co-expression networks of candidate lncRNAs and their upstream or downstream 10 kb mRNAs were constructed. Whereas, the ligation and enrichment were performed according to the position-frequency matrix.H. nipponia as suggested by UBQ)   was sele by UBQ) , with prpression .H. nipponia at different developmental stages such as larvae (L), young (Y), and adults (A) are shown in H. nipponia larvae (0.1362 ± 0.0075 g), young insects (1.184 ± 0.031 g), and adults (1.524 ± 0.045 g), were found significantly different in three developmental stages (p < 0.0001) as presented in The formaldehyde-fixed H. nipponia at different growth stages  as shown in We constructed a total of 24 cDNA libraries of p-value = 0.04 (p < 0.05) and then the nine samples were further divided into three sections, showing a tendency to cluster within the same group and a significant separation between the different groups , two overall up-regulated trends (cluster 4 and 6), and three highest trends of DEG expression in HNY   . The chaH. nipponia at different periods as shown in To further unravel the functions of differentially expressed genes, GO analysis was performed and grouped the functional annotation into three categories: molecular function, cellular component, and biological process . The DEGH. nipponia for screening DelncRNAs by using FDR <0.05 and |log2FC|>1 as a criterion and in total, 223 DelncRNAs were obtained , IQ motif containing G (IQCG), β-actin (ACTB), 5-hydroxytryptamine (5-HT), and HOX gene family, etc. were highly expressed in the adult stage (TTLL5), Filamin-A (FLNA), Protocadherin Gamma Subfamily A10 (PCDHGA10), Solute Carrier Family 5 Member 8 (SLC5A8) and NK2 Homeobox 8 (NKX2-8), etc. were expressed in all growth period but were highly expressed in the larval stage (ADAMTS), therostasin (THST), and decorsin (DECO) etc. were highly expressed , ACTB, and UPF3B Regulator of Nonsense Mediated MRNA Decay (REN3B), etc. for RT-qPCR validation. The results show that the expression trends of the genes verified by RT-PCR are in line with the RNA-seq trends  of  leeches . Further leeches .H. nipponia. Our study data provide new insights into the regulatory mechanisms of lncRNAs in the growth and development of H. nipponia.lncRNAs are long-stranded non-coding RNAs that are greater than 200 nt in length and do not have a coding role in living organisms. Although lncRNAs do not encode proteins but they have a crucial role in life activities. It has been demonstrated that lncRNAs are involved in X chromosome inactivation, chromosomal remodeling, transcriptional activation and interference as well as growth and development, metabolism and differentiation, and tumor production. Compared to other species, the study on leech lncRNAs is not well documented. In this study, during three developmental periods  a total of 223 differentially expressed lncRNA molecules were identified. Further analysis of these lncRNA molecules indicated that these lncRNA molecules are likely to play an important role in the growth and development of Ef1a, and GAPDH are used as internal reference genes for RT-qPCR testing, however we selected UBQ as internal reference gene. This is because β-actin or GAPDH are differentially expressed genes according to the results of our RNA-seq sequencing datat therefore we cannot use β-actin or GAPDH as internal reference genes for qRT-PCR analysis. Since there is lack of literature on the selection of internal reference genes in H. nipponia, we looked to other species and found that UBQ could be used as a regular internal reference gene , plays a key function in the muscular tension contraction process (TPM) is the actin-binding protein in muscle filaments is also involved in many vital activities in the body, such as cytoplasmic division and signal transduction. It can bind helically to actin filaments and regulate interactions with myosin (MYH) and the myosin light chain. The MYH family is one of the key members in the regulation of muscle development and affects muscle production and repair after injury, mainly through the regulation of cell proliferation and differentiation  sperm showed that actin was involved in sperm motility and random severance of actin filaments inhibited the flagella motility  throughout its life cycle . Second,activity . In conted blood . Adult lailments  Our findH. nipponia was systematically analyzed in three different developmental stages. Trait and histological observations revealed that adult leeches had significantly higher body length and body weight and a significantly higher number of myenteric tissues than larval and young leeches. Transcriptome analysis revealed that the number of DEGs was highest in L vs. A; DEGs enrichment analysis was mainly related to metabolic pathways; genes related to muscle development, genes related to gonadal development or oviposition regulation and genes related to neural development were significantly period-specific, indicating that these changes were associated with leech growth and development. In addition, we found that hirudin and anticoagulation-related genes were highly expressed in the young stage.In this study, the transcriptome of


The subseafloor is a vast habitat that supports microorganisms that have a global scale impact on geochemical cycles. Many of the endemic microbial communities inhabiting the subseafloor consist of small populations under growth-limited conditions. For small populations, stochastic evolutionary events can have large impacts on intraspecific population dynamics and allele frequencies. These conditions are fundamentally different from those experienced by most microorganisms in surface environments, and it is unknown how small population sizes and growth-limiting conditions influence evolution and population structure in the subsurface. Using a 2-year, high-resolution environmental time series, we examine the dynamics of microbial populations from cold, oxic crustal fluids collected from the subseafloor site North Pond, located near the mid-Atlantic ridge. Our results reveal rapid shifts in overall abundance, allele frequency, and strain abundance across the time points observed, with evidence for homologous recombination between coexisting lineages. We show that the subseafloor aquifer is a dynamic habitat that hosts microbial metapopulations that disperse frequently through the crustal fluids, enabling gene flow and recombination between microbial populations. The dynamism and stochasticity of microbial population dynamics in North Pond suggest that these forces are important drivers in the evolution of microbial populations in the vast subseafloor habitat. Population size can have important consequences for how microbial populations evolve over time, and factors such as genetic drift can have an exaggerated impact on small microbial populations compared to habitats in which microbial populations are larger. Most previous studies of adaptation in natural microbial communities have focused on habitats with high biomass and thus larger population sizes, such as the surface ocean, deep-sea hydrothermal vents, and freshwater lakes , and incubation experiments suggest the North Pond microbial community is growth limited and that increasing temperature and enrichment with carbon sources result in rapid growth , it was possible to replace 22 2018 NORP MAGs with newly reconstructed MAGs with higher completion and/or lower redundancy statistics and to produce 137 completely new MAGs, previously unrecovered from the metagenomic data sets (see Data set S1 at https://doi.org/10.6084/m9.figshare.17698631). Additional results and discussion of these MAG results can be found in the supplemental material  to the original 2018 North Pond (NORP) MAGs  . In total, six MAGs met these criteria. In addition, we identified another four MAGs with high coverage (>30 RPKM) in only one sample. In all four instances, these MAGs had low coverage (<3 RPKM) in the other time points, with three MAGs having extremely low coverage at other time points (<0.15 RPKM)  (15 RPKM) ; Fig. 1.3 to 104 cell mL−1) (−1), nucleotide diversity, and evidence of active replication were used as metrics to determine if any of the populations were candidates for clonal expansion (see Data set S3 and 4 at https://doi.org/10.6084/m9.figshare.17698631). We calculated trough-to-peak ratios (T:P), which can be used as an estimate of genome replication from MAGs  and higher than single species-dominated photosynthetic mats in a connected watershed (ranging between 0.01 and 8.7 SNVs kbp−1)  , 7, 22. ll mL−1) . Few prell mL−1) ; Fig. 1.rom MAGs . It shourom MAGs , T:P ratrom MAGs . Based orom MAGs , a T:P rorphisms  in a fres kbp−1) , 24. BasFor the six MAGs with high abundance across multiple time points, it was possible to compare patterns in allele frequency and reconstruct strain abundance patterns, revealing the coexistence of multiple subpopulations/strains. The MAG populations represented by NORP169 and NORP246 are estimated to contain approximately three strains each .10.1128/mbio.00354-22.2FIG S1FIG S1, PDF file, 2.3 MB.Strain relative abundance as determined using DESMAN for NORP139 (A), NORP147 (B), NORP169 (C), and NORP163 (D). Major allele frequency for SNVs detected in time points of interest for NORP139 (E), NORP147 (F), NORP169 (G), and NORP163 (H). The major allele frequencies have been hierarchically clustered and scaled from 0 to 1. Both 0.0 and 1.0 represent a fixed allele; 0.5 represents a split allele. Download Copyright © 2022 Anderson et al.2022Anderson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The other four MAG populations  differed in that during the time points of interest, at least one strain enters the population that was not present at other time points. For example, the NORP139 population consisted of six strains  similar to those of human pathogens (https://doi.org/10.6084/m9.figshare.17698631). Collectively, this evidence suggests the NORP83, NORP57, NORP147, and NORP169 populations did not undergo high rates of recombination relative to other North Pond populations. For NORP147, the limited number of coexisting strains would reduce the opportunity for recombination, but for NORP83 and NORP169, which have multiple coexisting strains, this may imply that intrinsic biological characteristics prevented high rates of recombination within these populations. Diminished rates of recombination could increase the potential for sympatric speciation and genome-wide sweeps.If population differentiation occurs elsewhere and throughout the aquifer, the degree of recombination between coexisting strains has important implications for speciation and gene transfer. Low rates of recombination could lead to sympatric speciation or more frequent genome-wide sweeps, whereas high rates of recombination between strains could hold lineages together and also lead to gene-specific sweeps and facilitate gene transfer. We used the decay rate of linkage disequilibrium and the four-gamete test as a proxy for detecting recombination within the populations represented by each MAG. Linkage disequilibrium is a measurement that uses the distance between any two allelic variants as a proxy for recombination; variants in close proximity are less likely to become unlinked via recombination. As the recombination rate increases, the number of paired variants decreases as distance between variants increases . Similarly, the four-gamete test is a measure of recombination that searches for pairs of segregating sites . All of the populations had four alleles  at some of the linked biallelic SNV sites. Low H4 frequencies indicate low recombination rates. The mean H4 frequencies for each population ranged from 0.44% to 14.84%. Several of the MAG populations  had H4 frequencies  below the lowest values estimated for single species photosynthetic mats . Linked SNVs are denoted by their predicted mutation type (nonsynonymous [N] and synonymous [S]). Download FIG S2, PDF file, 0.1 MB.Linkage disequilibrium of Copyright © 2022 Anderson et al.2022Anderson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Conversely, the other six MAG populations had distinct patterns of linkage decay and higher frequencies of the H4 allele, suggesting likely recombination; however, the degree of linkage disequilibrium varied between them ; Fig. S232–https://doi.org/10.6084/m9.figshare.17698631). In order to identify genes with abnormal coverage relative to each other, we used a minimum contig length of 2.5 kb to decrease the likelihood that any contig contained a single open read framing (ORF). As such, genes with variable coverage tend to be embedded on contigs between genes that were near the median genomic value.Genes that are unique to individual strains may provide a selective advantage. Genes limited to specific strains were identified by calculating changes in gene copy number based on observed changes in coverage over time  had no KEGG annotation. We observed shifts in gene abundance likely associated with the gradual loss of strain Str4 and the introduction of strain Str2 . Two setThe NORP163 population had 45 putative genes that changed in gene copy number over time . While 2n = 7; n = 18; Shifts in gene abundance for the population represented by NORP246 were highly dynamic over time and accompanied by a change in strain abundance, most notably the increase of strain Str2 over time . Forty-sThere were three general trends in the types of genes that had gene copy variation: (i) genes lacking KEGG annotations, for which function remains unknown; (ii) genes annotated as and associated with extrachromosomal replication  and horizontal gene transfer; and (iii) genes annotated as transporters. While variation in gene copy numbers over time for genes with functional annotations were identified, we cannot state with certainty that these genes provide a selective advantage for a particular variant, but some functions could provide specialization at the intraspecific level. For example, transporters play an important role in substrate availability and acquisition. The correlation between changes in gene frequency and the introduction/removal of a strain from the North Pond populations reinforces the role of dispersal as the likely mechanism for introducing genomic variation between strains of the same population. These populations may compete for and/or maintain partially overlapping ecological niches, such that changes in gene content may reflect the generation of “ecotypes” among the cooccurring strains. In general, the introduction of new strains did not correspond to an increase in total population abundance, perhaps reinforcing the idea that the intraspecific populations shared an ecological niche rather than directly competing for it . NORP83,https://doi.org/10.6084/m9.figshare.17698631).While differences in gene content may result in the evolution of overlapping ecotypes or eventually to sympatric speciation, the higher rates of recombination in some of the North Pond populations should produce evidence of gene-specific sweeps. Genomic regions containing gene-specific sweeps are characterized by low SNV density. Likely due to the increased cutoff values used to determine the location of SNVs, compared to Crits-Christoph et al. , we did dN/dS < mean genomedN/dS) and relaxed selection   that could represent gene-sweep events . Both NO/dS > 1) . While tST by identifying genomic regions of 5+ genes that had variable allele frequencies across populations (see Data set S10 at https://doi.org/10.6084/m9.figshare.17698631). Generally, this metric followed the same pattern observed using nucleotide diversity. NORP163 and NORP169 had several regions with elevated FST when considering a deviation greater than one standard deviation from the mean FST value. Despite its low estimated recombination rate, NORP83 had the second highest number of regions with increased FST (n = 8). For all three genomes, none of the elevated FST regions shared gene content with the putative gene-sweep regions identified with nucleotide diversity. However, increasing the required deviation to greater than two standard deviations from the mean reduced the number of differentiated regions to a single region in the NORP163 population. Due to the constraints placed on calculating the FST metric, which requires genes in two time points to have corresponding biallelic SNVs, many regions were likely excluded from this FST analysis. Additionally, the FST calculations required at least five genes in a variable region, although results from the nucleotide diversity metric demonstrated that the longest stretch in all genomes of decreased diversity was five sequential genes . The low number of SNVs in these regions may arise from the fact that these are often highly conserved core genes, which is consistent with previous studies , Ribosome (BR03011), Ribosome biogenesis (BR03009), Transcription factors (BR03000), and Chaperones and folding catalysts (BR03110) , the battery-powered GEOMicrobeSled (in situ. A complete set (n = 8) of in situ samples were collected from U1382A and provided the necessary samples and resolution for downstream analysis (as described below). Previously described in Tully et al. .To build upon the set of MAGs generated in Tully et al. , the qua10.1128/mbio.00354-22.4TABLE S1Table S1, PDF file, 0.03 MB.Settings used for each of the 7 passes with Binsanity-lc. Download Copyright © 2022 Anderson et al.2022Anderson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00354-22.5TABLE S2Table S2, PDF file, 0.03 MB.Assembly statistics comparing original Tully et al.  result wCopyright © 2022 Anderson et al.2022Anderson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://github.com/Ecogenomics/BamM). Recruitment profiles were merged with anvi-merge. During the anvi-profile step, the flag ‐‐profile-SCVs was set to include identification of single nucleotide variants (SNVs) and single codon variants (SCVs). Each individual MAG anvi’o database had the collection value ‘DEFAULT’ and bin value ‘EVERYTHING’ added using anvi-script-add-default-collection. Using those collection and bin values, anvi-gen-variability-profile  to extract SNV, single amino acid variants (SAAVs), and SCVs, from the profiles of each MAG in each North Pond sample , and MAGs detected in these samples were selected for downstream analysis based on two criteria from the time series: (i) sufficient coverage (≥5 RPKM) in ≥3 time points or (ii) high coverage (≥30 RPKM) in at least one time point. Only time points that had ≥5 RPKM were considered for SNV calculations. This criterion was set to ensure that most base pairs in each MAG had at least 20× read coverage, which was the minimum coverage value to determine SNVs in all downstream analyses (see below).As has been reported previously , 54, 55,Shewanella and shared 98.2% ANI, as determined with FastANI (Two of the MAGs (NORP143 and NORP246) were taxonomically assigned to the genus  FastANI . For con FastANI . The feahttps://github.com/edgraham/GhostKoalaParser) and anvi-import-functions .Protein sequences as determined within anvi’o were submitted to the GhostKOALA KEGG annotation and mapping service  (submittGTDB-Tk  v1.3.0 (−1)  of interest, and if the departure from consensus was ≥10%. Putative SNVs at each time point were converted to single nucleotide variants per kilobase pair (SNVs kbp−1) , a measuhttps://zenodo.org/record/3767070#.Ys1u8XbMI6Y) with the default clustermap settings (average distance linkage method and Euclidean distance metric).We used anvi’o to obtain metrics of microbial population dynamics based on MAGs. anvi’o is a widely used platform for microbial genomics and provides a baseline to determine polymorphisms in the underlying metagenomic reads. The criteria detailed here provide a conservative method to compare features such a SNV frequency, major allele frequency, and gene coverage. For data processed with anvi’o, major allele frequency was determined by counting the frequency of nucleotides in the aligned reads, identifying the consensus allele, and dividing the number of occurrences of that allele by the total coverage for that site . For each MAG, mean major allele frequency was determined by averaging all major allele frequencies for each SNV across the entire MAG. For comparing time points, if a SNV was detected in one time point, but not another (and coverage ≥20×), the positions for which no SNV was recorded were assumed to be fixed at the consensus allele. Hierarchical clustering was performed and plotted using seaborn  and a GFF formatted gene prediction file generated by Prodigal , the distribution of read recruitment identity, per gene and genome average nucleotide diversity (π), which provides a probability for two reads having the same nucleotide at a particular position, and estimate of replication rate . Additionally, the four-gamete test, another signal of recombination, was performed for all biallelic (AB and ab haplotypes) linked SNV locations .In contrast to mcorr, the inStrain profile links polymorphisms over the length of the metagenomic read. Thus, complex populations with extensive recombination, which may have been missed through the use of the mcorr correlation matrix, could be resolved but limited by the length of the read. Along with the accompanying genome statistics, inStrain provides highly precise measures of the metapopulation. inStrain was used to profile the underlying diversity on a genome-wide basis for each MAG and on a gene-by-gene basis using Prodigal predicted ORFs from each time point . Parameter settings, including read percent identity (95%), coverage (20×), and minimum allele frequency (10%), were selected to ensure that SNV detection by inStrain and anvi’o were conducted with the same level of stringency. inStrain includes the ability to filter reads that mapped to multiple locations during recruitment and these reads were removed, if present. Output from inStrain was assessed for the presence of linkage disequilibrium  consistent with Crits-Christoph et al. .For each MAG, gene coverage values were exported from the anvi’o databases using anvi-export-gene-coverage-and-detection. Gene frequency in a sample was calculated by dividing each gene coverage value by the median coverage value of all genes. Genes of interest were identified based on changes in gene frequency such that the maximum frequency value minus the minimum frequency value in the time points of interest was ≥1. Genes identified in this manner lacking a KEGG annotation were compared against GenBank nr  using BLhttps://fhcrc.github.io/seqmagick/). Haplotypes that translated to isoforms with internal stop codons were excluded from further analysis. Haplotypes and corresponding isoforms for each putative gene from all time points of interest were combined. CD-HIT . The output was filtered to include only dN > 0, 0.01 < dS < 1, and dN/dS < 5. dN/dS and dS for all pairwise haplotype comparisons were averaged. The distribution of the number of isoforms predicted for each gene was used to inform the selection of target strains in the DESMAN analysis.The software package Gretel  was used. CD-HIT  was used. CD-HIT  , and the filtered BAM files used in this analysis (https://doi.org/10.6084/m9.figshare.17701254). As detailed in Data set S1, all new/updated MAGs have been submitted to NCBI under the BioProject accession no. PRJNA391950. Scripts created to process data, perform analyses, and create plots have been made accessible through GitHub (https://github.com/bjtully/northpond_evolution).Data for this project can be found deposited at DDBJ/ENA/GenBank under the BioProject accession no. r et al.  constitu


Hysteroscopy is a minimally invasive gynecologic technique that is widely practiced in outpatient procedures. The choice of anesthesia is a key factor for the surgical outcome and postoperative recovery. This study was conducted to assess the effects of different anesthetic modalities based on dexmedetomidine in outpatient hysteroscopic surgery anesthesia.  We did a systematic review and network meta-analysis of outpatient hysteroscopic surgery anesthesia. We searched Pubmed, Embase, and Cochrane-Library from database inception to December 31, 2021. Duplicate literature was excluded and screened separately for initial screening at three tiers: article title, abstract, and full text before deciding whether to include in this study against the above criteria. Results after analysis of categorical variables were expressed as ORR Ratio (95% CI) and continuous variables were expressed as Mean Difference (95% CI). Data collation and analyses were performed using the gemtc package in the R language.  Four trials were finally included with data for 301 participants, three anesthetic drugs, and five anesthetic modalities. A fixed-effects model was used for the different anesthesia modalities without significant heterogeneity  in the analysis of adverse events (AEs), the incidence of respiratory depression, operative time, and time in the post-anesthesia care unit (PACU). Remimazolam tosylate was associated with a lower incidence of AEs versus dexmedetomidine, and significant differences between dexmedetomidine and propofol were absent. Propofol and various doses of remimazolam tosylate resulted in a lower incidence of respiratory depression versus dexmedetomidine, with an absence of differences between propofol and dexmedetomidine. The operative time for different anesthetic modalities was, in descending order, dexmedetomidine < remimazolam tosylate (0.60 mg/kg/h <0.48 mg/kg/h) < propofol < remimazolam tosylate (1.00 mg/kg/h), despite the absence of intergroup differences. Propofol was associated with a longer time in PACU versus dexmedetomidine and remimazolam tosylate (1.00 mg/kg/h); those of dexmedetomidine and remimazolam tosylate (1.00 mg/kg/h) were similar. The time in PACU for different anesthetic modalities, in descending order, was dexmedetomidine < remimazolam tosylate (1.00 mg/kg/h) < propofol. Propofol was associated with a longer time in PACU versus dexmedetomidine and remimazolam tosylate.  In outpatient hysteroscopic surgery anesthesia, dexmedetomidine was associated with a higher incidence of AEs and respiratory depression and a shorter operative time and time in PACU versus remimazolam tosylate and propofol. Remimazolam tosylate showed safety benefits with a similar duration of PACU stay versus dexmedetomidine. Therefore, the choice of anesthetic drugs in outpatient surgery requires consideration of the patient's conditions and preferences. Hysteroscopy provides a clear view of the changes in the uterine cavity to aid physicians for accurate diagnosis . The useα2-adrenoceptor agonist that provides sedation and analgesia with a lesser dose and mild respiratory depression and is now extensively used in clinical practice ) or (uteroscope [Title/Abstract]) or (metroscope[Title/Abstract]) and ((Dexmedetomidine [Title/Abstract]) or (DEX [Title/Abstract])), without language filters for searching. References of the included literature were searched and retrospectively added to potentially missing studies whenever possible.The inclusion criteria are as follows: (1) study type is Randomized Comparison clinical Trial (RCT) or Clinical Trial; (2) study participants are patients who underwent outpatient hysteroscopic procedures with at least two of dexmedetomidine, propofol, or remimazolam tosylate used for anesthesia; (3) outcome measures included hemodynamic status, perioperative indicators ; and (4) the study design was scientific and standardized, with clear grouping and interventions and complete documentation such as follow-up data.The exclusion criteria are as follows: (1) nonclinical studies, case reports, or secondary data analysis; (2) inability to extract relevant outcome indicators; and (3) inclusion of fewer than 15 participants in a single group.Retrieval of data was carried out by two investigators, and literature management was conducted using Endnote. Duplicate literature was excluded and screened separately for initial screening at three tiers: article title, abstract, and full text before deciding whether to include in this study against the above criteria. Quality assessment of the included literature using the Cochrane risk-of-bias tool, and the decision to include was made by a third investigator independently for discrepancies between the two investigators.The following data were extracted by two investigators independently from the included studies: first author's name, year of publication, subject type, number of subjects, treatment, study design, hemodynamic status, perioperative indicators , anesthetic drug dosage, and postoperative adverse events. Postoperative adverse events, operative time, and time in PACU were considered meta-analysis main effect measures.P > 0.1 for both subgroups indicated no heterogeneity in the included studies, and 50% > I2> 0 and P < 0.1 indicated mild heterogeneity. A fixed-effects model was used for all analyses. Results after analysis of categorical variables were expressed as ORR Ratio (95% CI) and of continuous variables were expressed as Mean Difference (95% CI).Data collation and analyses were performed using the gemtc package in the R language. Postoperative adverse events data included sample size and case, and operative time and time in PACU data included mean value, std.dev, and sampleSize. The I2 test was used to evaluate the heterogeneity of the included studies. I2 = 0 and Of 400 original papers retrieved by an electronic search, 312 papers were excluded after literature abstracts reading and exclusion of case reports, abstracts, and reviews, and 58 papers were coarsely included. Following the reading of the full text, studies with duplicate reports and unspecified data were ruled out, and the final 4 pieces of literature were recruited. The four trials included 301 participants, 3 anesthetic drugs , and 5 treatment regimens. There were one 3-arm study of propofol versus remimazolam tosylate, 2 studies of propofol versus dexmedetomidine, and 1 study of remimazolam tosylate versus propofol. The 3-arm study  was converted into 2 two-arm studies (propofol vs. remimazolam tosylate 0.48 mg/kg/h. Propofol vs remimazolam tosylate0.60 mg/kg/h). The basic information of the included literature is shown in Analysis of the incidence of postoperative adverse events was found in 3 studies, with an interstudy heterogeneity of I2 = 16% and no significant heterogeneity found between groups. A fixed-effects model was employed for analysis. Remimazolam tosylate was associated with a lower incidence of AEs versus dexmedetomidine, and significant differences between dexmedetomidine and propofol were absent. The incidence of AEs was, in descending order, remimazolam tosylate (0.48 mg/kg/h < 1.00 mg/kg/h < 0.60 mg/kg/h) < propofol < dexmedetomidine, without significant differences between groups .2 = 17% and no significant heterogeneity found between groups. A fixed-effects model was employed for analysis. Propofol and various doses of remimazolam tosylate resulted in a lower incidence of respiratory depression versus dexmedetomidine, with an absence of differences between propofol and dexmedetomidine. The incidence of respiratory depression for different anesthetic modalities was, in descending order, remimazolam tosylate (1.00 mg/kg/h < 0.60 mg/kg/h <0.48 mg/kg/h) < propofol < dexmedetomidine, without significant differences between groups  < propofol < remimazolam tosylate (1.00 mg/kg/h), despite the absence of intergroup differences ; those of dexmedetomidine and remimazolam tosylate (1.00 mg/kg/h) were similar. The time in PACU for different anesthetic modalities, in descending order, was dexmedetomidine < remimazolam tosylate (1.00 mg/kg/h) < propofol. (Three studies analyzed the time in PACU, with an interstudy heterogeneity of Iropofol. .The results of this study showed that dexmedetomidine was associated with a higher incidence of adverse events and respiratory depression and a shorter operative time and time in PACU versus propofol and remimazolam tosylate. Hysteroscopy is an endoscopic examination that allows direct visualization of intrauterine lesions, provides a rapid and accurate diagnosis of most intrauterine diseases, and has become the diagnostic gold standard for assessing intrauterine lesions . HysteroIn the present study, propofol showed a promising safety profile but a prolonged stay of patients in PACU. Propofol is a short-acting intravenous anesthetic for the induction and maintenance of general anesthesia and is frequently used with epidural or spinal anesthesia, analgesics, inotropes, and inhalational anesthetics . PropofoHere, remimazolam tosylate presented a manageable safety and comparable length of stay in PACU of patients. Remimazolam tosylate is a new anesthetic sedative with rapid onset and breakdown, no significant respiratory or cardiovascular effects, and it is also metabolized by tissue esterases with inactive metabolites that can be antagonized by flumazenil, showing better anesthetic enrichments over midazolam and propofol . ResearcIn outpatient hysteroscopic surgery anesthesia, dexmedetomidine, was associated with a higher incidence of AEs and respiratory depression and a shorter operative time and time in PACU versus remimazolam tosylate and propofol. Remimazolam tosylate showed safety benefits with a similar duration of PACU stay versus dexmedetomidine. Therefore, the choice of anesthetic drugs in outpatient surgery requires consideration of the patient's conditions and preferences. Further research with more literatures should be performed in future study.


French National Cancer Institute-funded prospective sarcoma database NETSARC to report the management and oncologic outcomes in adolescents and young adults (AYAs) patients with sarcoma at the national level.The initial management of patients with sarcoma is a critical issue. We used the nationwide NETSARC database gathers regularly monitored and updated data from patients with sarcoma. NETSARC was queried for patients (15–30 years) with sarcoma diagnosed from 2010 to 2017 for whom tumor resection had been performed. We reported management, locoregional recurrence-free survival (LRFS), progression-free survival (PFS), and overall survival (OS) in AYA treated in French reference sarcoma centers (RSC) and outside RSC (non-RSC) and conducted multivariable survival analyses adjusted for classical prognostic factors.p < 0.001). 3y-OS rates were 81.1% (95%CI 78.3–83.6) in AYA in RSC and 82.7% (95%CI 79.4–85.5) in AYA in non-RSC, respectively. Whereas no significant differences in OS was observed in AYAs treated in RSC and in non-RSC, LRFS and PFS were improved in AYAs treated in RSC compared to AYAs treated in non-RSC (Hazard Ratios (HR): 0.58 and 0.83, respectively).Among 3,227 patients aged 15–30 years with sarcoma diagnosed between 2010 and 2017, the study included 2,227 patients with surgery data available, among whom 1,290 AYAs had been operated in RSC, and 937 AYAs in non-RSC. Significant differences in compliance to guidelines were observed including pre-treatment biopsy , pre-treatment imaging  and R0 margins  (This study highlights the importance for AYA patients with sarcoma to be managed in national sarcoma reference centers involving multidisciplinary medical teams with paediatric and adult oncologists. Sarcomas are a group of rare, highly heterogenous connective tissue cancers with more than 70 histotypes identified and a wide array of clinical presentations . EstimatFrench National Cancer Institute INCa funded the national clinical sarcoma reference network NETSARC gathering 26 reference sarcoma centers to generate a nationwide institutional sarcoma data collection and improve the outcome of sarcoma patients. NETSARC highlighted that a specialized multidisciplinary tumor board (MDTB) presentation before treatment was significantly associated with a better relapse-free survival [Clinical practice guidelines for sarcoma patients recommend dedicated management involving multidisciplinary teams –7. In 20survival , and repsurvival .According to the French healthcare system, any patient < 18 years should be managed in expert centers in order to facilitate improved survival, opportunities for clinical trial inclusion, and ensure access to high-quality pluridisciplinary care –13. RealThis retrospective study queried the nationwide French database NETSARC for patients 15–30 years with sarcoma diagnosed between 2010 and 2017, and compared patient management and outcome in RSC and in non-RSC.In 2009, the French National Cancer Institute (INCa) funded the clinical network NETSARC including 26 French RSC and linked to a dedicated network for expert pathology diagnosis in sarcoma (RRePS) involving 19 pathology reference centers in charge of a second pathological review for each suspected case, in order to improve the outcome of sarcoma patients. In practice, any file from a patient with suspicion of sarcoma should be reviewed by the MDTB. Patient files are presented at any time, before any diagnostic procedure, before initial biopsy, before primary surgery, after primary surgery, at relapse, and/or for eligibility for clinical trial. The current NETSARC + database (netsarc.org) has gathered all sarcoma cases presented to the MDTB from Jan 1st, 2010 and includes data on diagnosis, therapeutic management, and clinical outcomes in terms of relapse and survival . From 20French Sarcoma Group-Groupe d’Etude des Tumeurs Osseuses (GSF-GETO) validated this research project on Jan 18, 2020. Patients received information sheets (https://expertisesarcome.org/espace-patients/adultes/), and non-opposition procedure applied.This study quiered the NETSARC + database for patients with sarcoma diagnosed from 2010 to 2017, and a limited set of anonymized data of patients aged 15–30 years with sarcoma and who have been operated, was used to describe patient and tumor characteristics, quality of surgery, relapse, and survival and to compare patient management in RSC centers and non-RSC centers . The FreNational Federation of Cancer Centres  specified 4 categories for histological grade: grade 1, 2, 3, and ungraded tumors. Sarcomas without grade resulted from histology grading failure or lack of critical elements to complete diagnosis, as determined by experts. The quality of surgical resection used the definition of the Union Internationale Contre le Cancer (UICC) [monobloc resection and clear margins specified in pathological report; R1 margins referred to (possible) microscopic residual disease, with visible tumor cells on resection margins (positive microscopic margins) – in the present study R1 margins indicated margins not confirmed as R0 or R2. R2 resulted from fragmented resections, or operative/pathological reports suggesting or notifying macroscopic residual tumor and/or fragmented resection. Categorization for treatment centers were performed according to the affiliation of the surgeon in charge of the first surgery. A surgeon referenced in the NETSARC network led to consider the patient as treated in French RSC (https://netsarc.sarcomabcb.org). Conversely, a surgeon not referenced in the NETSARC network led to consider the patient as being treated in French non-RSC. Because of surgeon affiliation requirement for categorisation, patients with desmoid tumors who rarely required surgery, and patients with no surgery, or with no information on surgery were excluded.Data collection included patient demographics, disease status  and tumor characteristics. The wider tumor diameter defined tumor size. Soft-tissue sarcoma also included viscera localisation. The r (UICC) , and marData from patient diagnosed from 2010 and not later than 2017 were selected to ensure at least 3-years of follow-up. The data cut-off for data analysis was June 26, 2020.Categorical data were expressed with frequencies and percentages, and continuous data with median and interquartile range. Comparisons were performed with chi-square for categorical variables and Mann-Whitney test (MW) for continuous data.The diagnostic date was the date of pathological diagnosis (biopsy or first surgery). Locoregional recurrence free survival (LRFS) was computed from the diagnosis date to the date of first locoregional progression, or censored at last follow-up. Competing events to LRFS were estimated using a competing risk approach. Progression free survival (PFS) was defined as the time from the date of diagnosis to the date of first local or metastatic progression or death, whichever occurred first, or censored at last follow-up. Overall survival (OS) was defined as the time from the date of diagnosis to the date of death due to any cause, or censored at last follow-up. PFS and OS were estimated using the Kaplan-Meier method. Survival distributions between the 2 groups were compared using a log–rank test and the multivariable analysis used a Cox proportional hazard model. The cumulative incidence function and nonparametric Gray’s test were used to estimate and compare cumulative incidence function between groups and a Fine-Gray model was used for the multivariable analysis , 19. Unin = 338) [n = 384) or patients with no information on surgery (n = 278). In the 2227 patients operated, 1290 patients were treated in RSC, and 937 AYAs were managed in non-reference centers , presented with worse prognosis, had a majority of grade 3  and larger tumor size  compared with AYAs in non-RSC  and lower limb tumors .AYAs with sarcoma treated in RSC were younger p < 0.00, presentp < 0.001) , than in AYAs managed in non-RSC 14%) (4% (p < 0p < 0.001) (Table p < 0.001). Reoperations of AYAs with incomplete surgery after initial resection in non-RSC frequently occurred in the patients with initial R1 margins (56%), and in the patients with initial R2 margins (64%). AYAs initially treated in non-RSC were mostly reoperated in RSC   months.The 3y-LRFS rates for AYA in RSC and AYA in non-RSC were 83.2% (95%CI 80.4–85.5), and 71.3% (95%CI 68.0-74.8), respectively. The cumulative incidence for locoregional progression is shown in Fig. p < 0.001). After adjustment, the multivariable analysis still showed better LRFS patients in RSC: HR 0.58 (0.46–0.73) compared to patients in non-RSC. LRFS was reduced for ungraded tumors . LRFS was increased for lower limb tumors   compared to AYAs in non-RSC , and 50.4% (95%CI 46.4–54.3), respectively , grade 3 tumors , ungraded tumors . PFS was improved in patients with lower limb tumors , and in patients with smaller tumor size (HR 0.79 95%CI 0.67–0.93)  , grade 3 tumors   and 82.7% (95%CI 79.4–85.5), respectively . Indeed, epidemiology based on advanced biological and histopathological characterisation of AYA neoplasms [The present work used the French nationwide prospective database NETSARC to assess the survival of young patients 15–30 years with sarcoma diagnosed between 2010 and 2017. To the best of our knowledge, this study is the 1st to report the survival of young (15–30 years) patients treated for sarcoma at a national level. The present study reported 3y-OS rates in AYA patients with sarcoma of almost 80% which is consistent with the 2y-OS of 80% reported by Raze et al. in 2016 . The stueoplasms , 22, andeoplasms , 23. In eoplasms –26.n = 5) with first surgery in RSC were reoperated for incomplete resection in a non-expert center in this series. Nevertheless, such situations remain marginal and limited, and voluntary patient transition to non-RSC treatment centers for personal reasons cannot be excluded.The initial management of patients with sarcoma is a highly sensitive issue. Reference centers showed better compliance to international guidelines and notably performed pathological review to confirm the absence of microscopically residual disease (R0) , 27, 28.French National Cancer institute INCa to create in 2011 the French academic society for AYA Adolescent and Young Adult Oncology and Hematology Group (GO-AJA) gathering pediatric and adult oncologists and hematologists at the national level [The quality of surgery is known as a major prognostic factor for relapse-free survival and overall survival in bone and soft tissue sarcomas –34. Howeal level . Howeveral level .PFS and LRFS after adjustment for negative prognostic criteria were better in AYA patients treated in RSC compared to AYA managed in non-RSC (HR 0.83 and HR 0.58), respectively. Differences in PFS, and especially in LRFS, may result from a lower compliance with clinical practice guidelines at initial surgery, putting patients at risk for more frequent local and distant relapses. In addition, patients with higher grade tumors had worse PFS (HR 1.88) and LRFS (HR 1.82) compared to patients with grade 1–2 tumors which may reflect the increased aggressiveness of the disease both locally and at distant sites.The absence of differences in OS may result from the current relatively short-term follow-up; a median follow-up of 39 months may be still insufficient to detect a significant difference in OS considering the 2y-OS of 80% and 5y-OS of 60% in AYA patients with sarcoma, aged 15 to 24 years in France [The AYA population in this study showed more STS (60%) than bone sarcomas 40%), which contrasts with the rates previously reported by Raze and colleagues in 2018 in the 15–24 years old patients  0%, which.The NETSARC database does not collect all medical treatments chemotherapy and targeted therapy administered before and after surgery, and do not allow to explore the impact of medical treatments administered in RSC and non-RSC so far. However, from 2016, additional data collection showed that neoadjuvant treatment administration was mostly observed in AYA in RSC (56.9%) and rarely in AYA patients in non-RSC (14%), which may have contributed to better local control and to increase 3y-LRFS in AYA in RSC. Detailed information regarding neoadjuvant treatment  are not specified and impact on 3y-PFS in AYA in RSC are not accessible.Comparisons in sarcoma management in AYAs are a critical issue not only regarding age ranges but also sarcoma histotype representations. If embryonal tumors are obviously more common in children than in AYAs, representation of sarcoma histotypes in AYA is closer to pediatric than to adult sarcomas. Whereas pediatric sarcoma treatment is exclusively performed in reference centers in France, and showed a quality of management recognized as a robust reference, all the AYAs up to 30 years may also derive benefit from a similar high-quality management. Subgroup analyses according to histotype would be highly informative, but have not been achieved so far, still limited by the high heterogeneity of sarcomas.In France, AYA patients with sarcoma managed in an expert center showed better compliance to guidelines at diagnosis and improved LRFS and PFS. With the current median follow-up of 39 months, no significant difference in 3y-OS was observed in our series. The present study outlines the importance of earlier access to reference sarcoma centers with multidisciplinary MDTBs, involving pediatric and adult oncologists, for any patient aged 15–30 years with suspicion of sarcoma.