Many rapid nucleic acid testing systems have emerged to halt the development and spread of COVID‐19. However, so far relatively few studies have compared the diagnostic performance between these testing systems and conventional detection systems. Here, we performed a retrospective analysis to evaluate the clinical detection performance between SARS‐CoV‐2 rapid and conventional nucleic acid detection system.Clinical detection results of 63,352 oropharyngeal swabs by both systems were finally enrolled in this analysis. Sensitivity (SE), specificity (SP), and positive and negative predictive value  of both systems were calculated to evaluate their diagnostic accuracy. Concordance between these two systems were assessed by overall, positive, negative percent agreement  and κ value. Sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was further analyzed with respect to the viral load of clinical specimens.p = 0.000). Daan Gene was 99.31% sensitivity for specimens with high viral load (Ct < 35) and 50% for low viral load (Ct ≥ 35).Sensitivity of Daan Gene was slightly lower than that of conventional detection system (0.86 vs. 0.979), but their specificity was equivalent. Daan Gene had ≥98.0% PPV and NPV for SARS‐CoV‐2. Moreover, Daan Gene demonstrated an excellent test agreement with conventional detection system (κ = 0.893, While showing an analytical sensitivity slightly below than that of conventional detection system, rapid nucleic acid detection system may be a diagnostic alternative to rapidly identify SARS‐CoV‐2‐infected individuals with high viral loads and a powerful complement to current detection methods. Many rapid nucleic acid testing systems have emerged to halt the development and spread of COVID‐19. However, so far relatively few studies have compared the diagnostic performance between these testing systems and conventional detection systems. Here, we performed a retrospective analysis to evaluate the clinical detection performance between SARS‐CoV‐2 rapid and conventional nucleic acid detection system. While showing an analytical sensitivity slightly below than that of conventional detection system, rapid nucleic acid testing systems may be a diagnostic alternative to rapidly identify SARS‐CoV‐2 infected individuals with high viral loads and a powerful complement to current detection methods. Rapid spread of SARS‐CoV‐2 and its variants had brought a huge burden to the global economy and medical system.Chinese expert consensus on the rapid nucleic acid testing of SARS‐CoV‐2. The analytical and clinical performance of these newly rapid nucleic acid testing for SARS‐CoV‐2 have been rarely evaluated based on the Chinese population thus far. Even if there are similar studies, they generally have some weaknesses, such as small scale of clinical validation trials and insufficient scientific data support.COVID‐19 caused by SARS‐CoV‐2 is a new infectious disease following SARS and MERS. Globally, as of 5:54 pm CEST, September 6, 2022, there have been 603,164,436 confirmed cases of COVID‐19, including 6,482,338 deaths, reported to WHO (22.1n = 18). After these exclusions, 63,352 cases of Shenzhen Hong Kong Cross Border truck drivers' nucleic acid test results were finally included in the analysis. Nucleic acid test results of both systems  and reported results after CDC review were collected from laboratory information management system records. The study design is displayed in Figure Oropharyngeal swabs specimens, which were obtained for rapid and conventional nucleic acid detection, collected from a total of 63,370 Shenzhen Hong Kong Cross Border truck drivers at six SARS‐CoV‐2 nucleic acid testing laboratories in Shenzhen China from March 1, to 16, 2022. The ages of the drivers enrolled in this study were between 18 and 65 years and gender unlimited. Exclusion criteria were as follows: <48 h after receiving SARS‐CoV‐2 vaccine. Samples that needed to resample after CDC re‐tested . Briefly, the supporting kit is a single tube and single copy packaging specification, and there is no need to prepare the reaction system. The reagent can be transferred to the amplification detection area after instantaneous centrifugation for 15 s, shaking and mixing for 10 s, and then instantaneous centrifugation for 15 s. Amplification procedure: 50°C 2 min → 95°C 2 min →  → .2.3The process of specimen collection and nucleic acid extraction was the same as described in Section In Huanggang port laboratory (HGP) and Channel 1 laboratory of Futian Free Trade Zone (FTFTZ) of the Eighth Affiliated Hospital of Sun Yat‐sen University, conventional nucleic acid detection was carried out on ABI7500 fluorescence quantitative PCR instrument followed the instructions of the SARS‐CoV‐2 nucleic acid detection Kit (Sansure Biotech Inc.). PCR amplification cycle parameters were set as follows: 50°C for 30 min →95°C for 1 min → .2.4the Technical Guidelines for COVID‐19 Laboratory Testing (Ninth Edition) issued by China CDC, the case confirmed as positive in the laboratory was further sequenced to monitor the mutation of virus genome, provide experimental data for the change of COVID‐19 nucleic acid detection reagent, vaccine research and development strategy, and also provide support for the work of epidemiological control and traceability.For the specimens suspected to be positive or positive in the initial test of port laboratory, conventional nucleic acid detection was carried out using both SARS‐CoV‐2 nucleic acid detection kits  to re‐test the specimens by Shenzhen CDC. According to 2.5the Technical Guidelines for COVID‐19 Laboratory Testing (Ninth Edition) issued by China CDCTrue positive cases in this study were confirmed by Shenzhen CDC in reference to the Technical Guidelines for COVID‐19 Laboratory Testing (Ninth Edition) issued by China CDCTrue negative cases in this study were confirmed in reference to 2.6Guideline for evaluation of qualitative test performance (WS/T505‐2017) issued by National Health and Family Planning Commission of the people's Republic of China and the Guidance on the Performance Verification for Molecular Diagnostic Procedures (CNAS‐GL039) issued by China National Accreditation Committee for Conformity Assessment. To evaluate the diagnostic accuracy of these systems, sensitivities (SE), specificities (SP), positive predictive value (PPV), negative predictive value (NPV), and 95% confidence interval (CI) were calculated based on the diagnostic accuracy criteria. CI for proportions were calculated by the Wilson method. Concordance between these two systems was assessed by the overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa value (κ) which was performed using SPSS™  and classified as follows: 0.81–1, almost perfect; 0.61–0.80, substantial; 0.41–0.60, moderate; 0.21–0.40, fair; 0.0–0.20, slight.p value <0.05 was considered to be significant. Ct values of the inconsistent specimens between the two systems were recorded and presented in the form of median ± IQR.Analysis was performed according to the 33.1Clinical detection results of 63,352 oropharyngeal swabs by both systems were finally enrolled in this analysis. As described in Table Taken together, our results indicated that both systems exhibited a perfect agreement with diagnostic accuracy criteria, and SARS‐CoV‐2 conventional nucleic acid detection system shown a more preferable agreement.3.2p = 0.00), with an OPA of 0.999 (95% CI: 0.998–1.0), PPA of 0.824(95% CI: 0.769–0.868), NPA of 0.999 (95% CI: 0.999–1), as reported in Table In general, agreement between these two systems for individual SARS‐CoV‐2 nucleic acid detection were almost perfect  and SARS‐CoV‐2 conventional nucleic acid detection system is displayed in Figure Ct values , ranged from 33.67 to 44.26, with median of 38.14, IQR of 2.95.The distribution of 3.4Ct values and determined the sensitivity of rapid nucleic acid detection system (Daan Gene) under different viral loads , 50% for low viral load (Ct ≥ 35). The sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was 100% for specimens with a high viral load (Ct < 35) in STJP, LTP, WJDP, HGP, and FTFTZ medical laboratories, and 98.89% in SZBP medical laboratory. Moreover, for samples with a low viral load (Ct ≥ 35), the diagnostic sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was 100%, 40%, 72.72%, 52.38%, and 9.09% in STJP, LTP, WJDP, SZBP, and HGP medical laboratories, respectively.The diagnostic ability of detection system is closely related to the virus load of the tested sample. We further divided the viral loads of samples by s Figure . Overall4Etiological examination is an important basis for the diagnosis of SARS‐CoV‐2 infection, including viral nucleic acid and antibody. Virus nucleic acid detection is the earliest and most commonly method applied to confirm the infection of SARS‐CoV‐2, assessing the efficacy of clinical treatment, population screening in the event of an outbreak, and epidemiological investigation.SARS‐CoV‐2 rapid testing reagent of Daan gene and its supporting equipment have been approved by the National Health Commission of China. From the statement provided by the manufacturers, as compared to the conventional nucleic acid detection system, the SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) is equipped with a fluorescent quantitative PCR system with the characteristics of small size, light weight, easy operation, and fast speed (the amplification detection time only takes 30 min). In addition, the supporting reagents add an anti‐pollution system that can degrade the pollutants generated in the PCR process and reduce the false positives caused by the PCR product pollution, including UDG enzyme and dUTP enzyme; as well as a high‐efficiency enzyme system that can improve the specificity and sensitivity of PCR reaction, including hot start Taq antibody enzyme and c‐mmlv enzyme.Overall, results of this study showed that the sensitivity of the SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was slightly lower than that of the conventional detection system, but their specificity for SARS‐CoV‐2 was equivalent.p = 0.000). The diagnostic sensitivity of a nucleic acid test kit is closely related to the viral load of the sample.Ct values , ranged from 33.67 to 44.26, with median of 38.14, IQR of 2.95. Among the six different nucleic acid testing laboratories, the SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) showed the lowest sensitivity and positive coincidence rate in HGP medical laboratory. The inconsistent results of molecular detection in this laboratory were also 14 samples with low concentration of viral RNA, whose Ct values ranging from 35.07 to 39.41, with median value of 37.55, and IQR of 2.71.And the SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) had ≥98.0% PPV and NPV for the detection of SARS‐CoV‐2. Moreover, the SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) demonstrated an excellent test agreement with the widely used conventional nucleic acid detection system , the Ct values of N gene and ORF gene in two consecutive SARS‐CoV‐2 nucleic acid tests were all ≥35. We further divided the viral loads of samples by the Ct values and calculated the sensitivity of these two systems under different viral loads. Results showed that the sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was 99.31% for specimens with high viral load (Ct < 35) and 50% for low viral load (Ct ≥ 35). SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) performed less well at specimens with Ct values ≥35; however, the reduction in sensitivity is relatively unimportant since high Ct values probably indicate a low transmission risk.Ct > 30 is negative, and these infected individuals are non‐infectious.Ct values ≥35 was still substantially higher than that most of the SARS‐CoV‐2 rapid antigen tests. Michael Kleines et al concluded that the SARS‐CoV‐2 Rapid Antigen Test (Roche)'s sensitivity for samples with a Ct value of <25, 25–30, 30–35, and ≥35 was 100%, 95%, 44.8%, and 22.2%, respectively.Ct < 28 and decreased to 38.5%, 26.7%, and 9.1% in the other ranks .Ct ≤ 24.22), but 0%–11.1% sensitivity for low viral load specimens (Ct ≥ 31.43).Additionally, according to one of the criteria for lifting quarantine management and discharging from hospital in 5In summary, although the sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system is not as high as that of SARS‐CoV‐2 conventional nucleic acid detection system, SARS‐CoV‐2 rapid nucleic acid detection system is a rapid and simple detection method that can identify COVID‐19 patients with high transmission risk. For emergency and symptomatic patients, or for large‐scale population screening at customs, railway stations, airports, etc., it is a better choice to use the SARS‐CoV‐2 rapid nucleic acid detection system for early triage and rapid management of the suspected population, and then use the conventional nucleic acid detection system for confirmation. It is believed that with the development of rapid nucleic acid detection system, more stable and high‐quality SARS‐CoV‐2 rapid nucleic acid detection technologies will emerge and become an indispensable force in clinical diagnosis and treatment and epidemic prevention and control.All authors have made substantial contributions to all of the following: Chan Mo and Xiuming Zhang, the conception and design of the study; Kamfai Lo, Ying He, and Bo Peng, acquisition of data; Feifan Guo, Zhou Zheng, and Ruiwei Jiang, analysis and interpretation of data; Chan Mo, Yihua Cai, Yumin Li, Dongyue Guo, and Bing Zhang, drafting the article; Tong Ou and Dan Xiong, revising the article critically for important intellectual content; Chan Mo, Dan Xiong, and Xiuming Zhang, final approval of the version to be submitted.This work was supported by Shenzhen Key Medical Discipline Construction Fund (Grant No. SZXK054), Science and Technology Planning Project of Shenzhen City of China .The authors declare no conflict of interest.


The study aimed to explore the value of CT findings and inflammatory indicators in differentiating benign and malignant gallbladder polypoid lesions before surgery.The study comprised a total of 113 pathologically confirmed gallbladder polypoid lesions with a maximum diameter ≥ 1 cm , all of which were enhanced CT-scanned within 1 month before surgery. The CT findings and inflammatory indicators of the patients were analyzed by univariate and multivariate logistic regression analysis to identify independent predictors of gallbladder polypoid lesions, and then a nomogram distinguishing benign and malignant gallbladder polypoid lesions was developed by combining these characteristics. The receiver operating characteristic (ROC) curve and decision curve were plotted to assess the performance of the nomogram.p < 0.001), plain CT value (p < 0.001), neutrophil–lymphocyte ratio (NLR) (p = 0.041), and monocyte-lymphocyte ratio (MLR) (p = 0.022) were independent predictors of malignant polypoid lesions of the gallbladder. The nomogram model established by incorporating the above factors had good performance in differentiating and predicting benign and malignant gallbladder polypoid lesions (AUC = 0.964), with sensitivity and specificity of 82.4% and 97.8%, respectively. The DCA demonstrated the important clinical utility of our nomogram.Base status of the lesion (CT findings combined with inflammatory indicators can effectively differentiate benign and malignant gallbladder polypoid lesions before surgery, which is valuable for clinical decision-making. Gallbladder polypoid lesions (GPs) are clinically common gallbladder diseases, affecting about 5% of the adult population in Western countries , 2, but CT examinations have been increasingly used to evaluate gallbladder polypoid lesions –10. But,In recent years, inflammation has got a lot of attention because it plays an important role in tumor growth by promoting tumor cell proliferation, invasion, and angiogenesis . NeutropThis retrospective study was approved by the Ethics Committee of Third Affiliated Hospital of Naval Medical University, and the requirement for informed patient consent was eliminated. Between January 2015 and December 2021, the researchers collected pathologically confirmed GPs with complete preoperative enhanced CT data, and 113 patients were eventually enrolled in the study, with 45 malignant GPs and 68 benign GPs. The inclusion criteria were as follows: (1) the largest diameter of the lesion was ≥ 1 cm; (2) the upper abdominal enhancement CT examination was performed in our hospital within 1 month before surgery; (3) complete pathological data were available after cholecystectomy. The following were the criteria for exclusion: (1) the lesion had invaded surrounding tissues, such as the liver and hepatic hilar lymph node; (2) patients underwent some operations and treatment before surgery including radiochemotherapy and percutaneous transhepatic gallbladder drainage; (3) the image was indistinct. The flow chart of patient registration is shown in Fig. The CT scan was performed on a GE Discovery 750HD multi-row spiral machine. The scanning parameters were as follows: tube voltage, 120 kV; reference tube current, 250–280 mAs; matrix, 512 × 512; pitch, 1.0; scan thickness and layer spacing, 5 mm. The patient fasted for at least 4 h before the CT scan, and the examination was performed in the traditional supine position, with an unenhanced scan first and subsequently an enhanced scan. Enhancement scans were performed using ioversol  ratio with a total injection volume of 1.5 ml/kg, injected through the elbow vein at a rate of 2.5–3.5 ml/s. Following the acquisition of unenhanced CT images, arterial phase, portal venous phase, and delayed phase scans were performed for 23–26 s, 50–60 s, and 120–160 s, respectively.2); (d) liver functional parameters  the following data were obtained from the neutrophil count, lymphocyte count, monocyte count, platelets: NLR (neutrophil to lymphocyte ratio), PLR (platelet to lymphocyte ratio), MLR (monocyte to lymphocyte ratio), SIRI (neutrophil × monocyte/lymphocyte); (f) tumor biomarkers  demographic characteristics ; (b) clinical symptoms: (absent or present); (c) BMI  and were independently evaluated by two radiologists imaging features. The two radiologists did not know the clinical indicators or pathological findings. A consensus was reached by joint discussion when there was disagreement between the two physicians. The main observations were the maximum length of the lesion; its location ; the number of the lesion (single or multiple); the base status ; gallblt test was used to compare continuous variables with a normal distribution, and the Mann–Whitney U test was used to analyze variables with an abnormal distribution. Categorical variables were compared using the χ2 test or Fisher’s exact test. NLR, PLR, LMR, MLR, and SIRI were converted to categorical variables based on receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression analyses were used to determine independent predictors of benign and malignant GPs. A two-sided P value < 0.05 was considered statistically significant. Based on the results from the multivariate logistic regression analysis, a nomogram model for differentiating benign and malignant GPs was developed, which underwent internal validation using the bootstrap method. Sensitivity, specificity, and area under the ROC curve of the nomogram were calculated. The accuracy and clinical utility of the nomogram were evaluated using decision curve analysis.All statistical analyses were performed using SPSS  and R software (Version 3.6.1). Student’s n = 45, benign n = 68) were enrolled in this study, of which approximately 73 (64.6%) were women and 40 (35.4%) were men. Age (P < 0.001), Lymphocyte (P = 0.016), NLR (P = 0.006), MLR (P < 0.001), SIRI (P = 0.002), ALB (P < 0.001), and CA19-9 (P = 0.008) were significantly different between the benign and malignant groups (P < 0.05), according to the results of the recorded clinical information. Other clinical indications did not differ significantly between the two groups , base status (P < 0.001), and the CT values of each phase  (P < 0.001) were significantly different between the two groups among the recorded CT characteristics , MLR , base status , and plain CT value  were independently associated with malignant GPs , MLR (P = 0.022), the base status of the lesion (P < 0.001), and plain CT value (P < 0.001) were independent risk factors for GPs, and the nomogram model based on this result could effectively distinguish and predict benign and malignant GPs ≥ 1 cm (AUC = 0.964). In addition, the model demonstrates better accuracy, which can provide important information for medical decision-making.The emphasis of this investigation was on GPs with a maximum diameter of ≥ 1 cm, and GPs with a diameter of ≤ 1 cm are usually managed by observational follow-up in clinical practice due to their low risk of malignancy . BecauseUltrasound is the preferred method for diagnosing gallbladder polypoid lesions; however, it can be difficult to distinguish between benign and malignant GPs. CT is also often used to examine gallbladder disease because contrast enhancement allows the lesion to be distinguished from the surrounding tissue and aids in the diagnosis of gallbladder disease. The base appearance was found to be an independent predictor of benign and malignant GPs in this analysis, which is consistent with prior studies , 12. TheP < 0.05). These differences could be attributable to sample bias or sample size disparities.CT values at all phases were associated with malignant GPs in our study, yet only the plain CT value was found to be an independent predictor. This is consistent with the findings of a recent study , which dOn the other hand, we have found some inflammatory indicators to be of diagnostic value in distinguishing benign and malignant GPs. Previously, researchers have concentrated more on the prognostic relationship between NLR and gallbladder carcinoma, in contrast to the limited studies focusing on NLR and preoperative diagnosis. The role of NLR has been investigated in several retrospective studies and was significantly associated with poor prognosis in GBC patients undergoing radical operation , 17. BesWe considered the constructed nomogram to be a useful and convenient method for preoperative differential and prediction of benign and malignant GPs due to the objective and precisely measurable acquisition of inflammatory indicators and the convenience and availability of CT examinations for preoperative examination. The model has an AUC of 0.964, indicating more diagnostic efficiency than prior investigations (AUCs of 0.856 , 0.875 [Despite the more satisfactory findings, this study has several limitations. First, this was a retrospective study with a small number of cases, which might lead to bias. Second, the study enrolled the lesions which were maximum diameter ≥ 1 cm in size, which limits the applicability of the model results. Finally, because this is single-center research, more multicenter and large sample studies are required for external validation.In conclusion, this study presents a novel model based on CT findings and inflammatory indicators for the effective preoperative differentiation and prediction of benign and malignant GPs, which could guide the preoperative decision-making of malignant GPs ≥ 1 cm in size. In addition, it still needs to find more evidence and be confirmed by further clinical and multicenter studies.


Breast cancer (BC) remains the leading cause of cancer‐related deaths worldwide. High recurrence risk Luminal BC receives adjuvant chemotherapy in addition to standard hormone therapy. Considering the heterogeneity of Luminal B BC, a more accurate classification model is urgently needed.low , ER+PR+Ki67high , and ER+PR−Ki67high . The cox proportional hazards regression model was used to evaluate the correlation between each variable and outcomes. Besides, discriminatory accuracy of the models was compared using the area under the receiver operating characteristic curve and log‐rank χ2 value.In this study, we retrospectively reviewed the data of 1603 patients who were diagnosed with HER2‐negative breast invasive ductal carcinoma. According to the expression level of PR and Ki‐67 index, the Luminal B (HER2‐negative) BCs were divided into three groups: ER+PR−Ki67p < 0.001) and disease‐free survival (DFS) (p < 0.001). Interestingly, patients in the ER+PR−Ki67high subgroup have the worst survival outcome in Luminal B (HER2‐negative) subtype, similar to Triple‐negative patients. Besides, the ER+PR+Ki67high has worse 5‐year DFS compared with Luminal A group. There was a significant relationship between the regrouping subtype and the recurrence score index (RI) (p < 0.001). Moreover, the results showed that patients in ER+PR–Ki67high subtype were more likely to have high RI for distance recurrence (RI‐DR) and local recurrence (RI‐LRR). Our newly defined classification has a better discrimination ability to predict survival outcome and recurrence score of Luminal B (HER2‐negative) BC patients, which may help in clinical decision‐making for individual treatment.The analysis results showed that there was a significant correlation between subtypes using this newly defined classification and overall survival  breast cancer, which may help in clinical decision‐making for individual treatment. However, the role of PR in BC etiology is still uncertain and whether its value can be used as a predictor in BC remains controversial. Furthermore, it is noteworthy that in the Luminal B subtype, both PR expression level and Ki‐67 index have a considerable range. Therefore, we assume that the Luminal B subtype is a more biological and clinically heterogeneous group that requires detailed classification to make individual treatment options.The application of adjuvant chemotherapy in BC patients is generally based on molecular subtypes and other clinicopathological features . More recently, genomic tests such as the Oncotype Dx recurrence, Mammaprint, and EndoPredict have been used to guide the application of adjuvant chemotherapy. However, these tests have been developed mainly based on European and American populations and whether these tests are appropriate for Asian populations needs further validation.The purpose of this study is to investigate whether luminal B cancer can be divided into subgroups by PR values and Ki‐67 index to precisely predict clinical outcomes and support treatment planning. We also analyzed the association between recurrence score (RS) and our newly defined subgroups to investigate whether some patients may avoid the RS test before the starting of possible adjuvant chemotherapy due to high costs and a long time to get results.22.1We conducted a retrospective analysis of 1603 patients diagnosed with ER+HER2− early stage invasive BC who underwent surgery in the Tianjin Medical University Cancer Institute and Hospital from August 2014 to December 2015. All patients received surgery. None of them received neoadjuvant therapy or had a prior history of cancer or bilateral tumors. Informed consent was obtained from all the patients above, and the Ethics Committees approved the research protocol for this study at the Tianjin Medical University Cancer Institute and Hospital.Tumors were classified into four different subtypes according to the 2013 St. Gallen Consensus.2.2p < 0.05 derived from univariate CPHRM were further carried out to perform forward stepwise regression. As a result, there were two variables screened out, including the T stage and N stage. We then built up a multivariate CPHRM adjusted for the T and N stages to evaluate the association between molecular subtypes and outcomes. Additionally, we predicted the 3‐ and 5‐year survival rates using the multivariate CPHRM, and the time‐dependent ROC curve was generated using the nearest neighbor method.Cox proportional hazards regression model (CPHRM) was used to analyze the association between each variable and study outcomes, including overall survival (OS) and disease‐free survival (DFS). These variables with 33.1high disease are less likely to have lymph node metastasis, compared with the other two subgroups (p < 0.001). Moreover, in the ER+PR−Ki67high subgroup, patients are more likely to be histologic Grade III than the other two subgroups (p < 0.001). These results showed that patients with PR‐negative and high Ki‐67 index represent tumor biology more similar to that seen in Triple‐negative patients.A total of 1603 patients with early stage invasive BC were included. Clinicopathological features and treatment modalities are summarized in Table 3.2p < 0.001) and T stage (p < 0.001) were significantly associated with DFS and OS both in univariate and multivariate survival analyses. We then built up a multivariate CPHRM adjusted for the T stage and N stage to evaluate the association between molecular subtype and outcomes (Table p < 0.001). The log‐rank χ2 value of the regrouping model was higher than that of the traditional model in the adjusted survival analysis. As shown in Table high subgroup have the worst survival outcome in Luminal B (HER2‐negative) subtype, similar to Triple‐negative patients. Besides, the ER+PR+Ki67high has worse 5‐year DFS compared with the Luminal A group. There was no significant difference in terms of 5‐year DFS between Luminal A and ER+PR−Ki67low subgroup. Likewise, Kaplan–Meier survival curves compared the regrouping subtype and 5‐year OS in Figure At the last time of follow‐up, 1448 90.3%) patients were alive and disease free, 103 (6.4%) were alive with recurrent cancer, and 52 (3.3%) died of recurrent cancer. As shown in Tables % patientes Table . Kaplan–3.3To further validate the predictive value of the regrouping model, the nearest neighbor method was used to construct a time‐dependent ROC curve. As shown in Figure 3.4p < 0.001). And the results showed that the Luminal B BC patients with ER+PR−Ki67high tumors were more likely to have a high risk both for RI‐DR and RI‐LRR: 18 of 27 (66.67%) patients had a high risk for RI‐DR and 16 of 27 (59.26%) patients had a high risk for RI‐LRR.As shown in Tables 4BC can be divided into different subtypes based on gene expression profiling, and each subtype represents a distinct clinical outcome.high tumors had the worst survival among Luminal B (HER2‐negative) patients.The results showed that patients with PR‐negative have worse survival outcomes compared with PR‐positive in high ki‐67 index Luminal B HER2‐negative) BC  BC treatment. However, a significant proportion of patients may experience endocrine resistance from the beginning or during the treatment process.high group usually had a higher risk of grade III and worse survival outcomes similar to the Triple‐negative group. However, not all ER+PR− groups had poor survival outcomes. As shown in Figure low group had similar survival outcomes to the Luminal A group, a subset recognized for its indolent behavior. This result was consistent with the published data, which showed that some ER+PR− tumors had ER+PR+ gene expression profiles. A proportion of ER+PR− tumors showed a pattern of ER−PR− gene expression. Similar to our findings, a recently published study showed that 16% of IHC ER+HER2− BCs could be reclassified as Basal molecular subtypes by multigene expression profiling.RNA profiling has determined that ER+PR− tumors represent a subgroup of BC with poor survival outcomes and high invasive features, although being clinically ER+ by IHC test.Currently, multigene expression profiles, such as Oncotype DX, MammaPrint, and Prosiga, have become widely used to predict the risk of recurrence and adjuvant chemotherapy benefits.high tumors to omit the gene expression profiling tests before receiving adjuvant chemotherapy. So, to some extent, the regrouping model can replace the expensive genomic assay in Luminal B patients.Moreover, the purpose of the MINDACT study is to select patients from high clinical relapse risk using a 70‐gene signature to avoid unnecessary adjuvant chemotherapy. Nevertheless, this study did not include the expression of PR and Ki‐67, which leads to an incomplete definition of high‐risk patients. Our study used the expression levels of PR and Ki67 to supplement the definition of high‐risk groups. More importantly, discordances in estimates occur between the genomic tests due to their different emphasis on ER‐related features and proliferation features,Our study has several strengths. First, according to the PR status and Ki‐67 index, the Luminal B (HER2‐negative) BC were divided into three subgroups. And we have proved that this new classification could better predict the prognosis of Luminal B patients than the traditional classification. Our research optimizes the traditional molecular subtype and shows that a high Ki‐67 index and PR‐negative status are potential predictive factors for high RS in Luminal B HER2‐negative BC patients. PR‐negative and high Ki‐67 index may represent tumor biology more similar to that seen in Triple‐negative patients. So, Ki‐67 index and PR status may be considered before using gene expression profiles, which would allow some patients with high Ki‐67 index and PR‐negative status tumors to omit the gene expression profiling tests before receiving adjuvant chemotherapy. Therefore, reorganizing Luminal B (HER2‐negative) BC by measuring the expression of PR and Ki67 instead of gene expression has clinical significance for accurately assessing the prognosis of BC and providing a reliable basis for clinical treatment.low BC is quite a few, with no death and only one local recurrence observed in our present study. Therefore, our findings should be validated in a large, multi‐center study.Some limitations should be taken into account when applying our results. First, we used a retrospective design with small sample size. Thus, the results should be considered exploratory and hypothetical. Second, due to a limited follow‐up period, the number of patients with ER+PR−Ki675high tumors differ from the other two groups in survival outcomes and treatment response, who cannot be continued treated as a homogeneous group. Besides, these patients were more likely to have high RS scores to receive adjuvant chemotherapy. Thus, we recommend that Luminal B BC patients with PR‐negative and high Ki67 index may avoid RS test and accept intensive adjuvant chemotherapy.The reclassification of Luminal B (HER2‐negative) BC using the expression of PR and Ki67 is of clinical significance. Patients with ER+PR−Ki67Zheng‐Jun Yang: Investigation, data analysis and interpretation, methodology, visualization, writing—original draft, writing review, and editing. Yu‐Xiao Liu: Investigation, data analysis and interpretation, methodology, visualization, writing—original draft, writing review, and editing. Yue Huang: Investigation, data acquisition, interpretation, editing. Zu‐Jin Chen: Investigation, data acquisition, interpretation, editing. Hao‐Zhi Zhang: Investigation, data acquisition, interpretation, editing. Yue Yu: Investigation, data acquisition, interpretation, editing, and supervision. Xin Wang: Investigation, data acquisition, interpretation, editing, and supervision. Xu‐Chen Cao: Investigation, data acquisition, interpretation, editing, and supervision.This study was supported by the National Natural Science Foundation of China .None.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Limited data are available with reference intervals of serum anti‐Müllerian hormone (AMH) level in Korean women.We retrospectively reviewed serum AMH test results performed with automated electrochemiluminescence immunoassay in Korean women who visited health promotion centers between January 2019 and December 2020. Serum AMH results by age group were compared with previously reported reference intervals.During the 2‐year study period, a total of 1953 AMH test results from Korean women (age 20–49 years) undergoing general health checkups were obtained. Serum AMH level differed significantly by age group. Peak AMH level was observed at age 25–29 years and decreased to undetectable for subjects older than 44 years. The 2.5th, 5th, and 10th percentile values of the present study were comparable with previously assessed lower limits of reference intervals. The upper limit of the reference interval defined as the 97.5th percentile value in women younger than 35 years was higher than that of Western populations. The 90th percentile value of the present study population was similar to the 95th or 97.5th percentile value of reference intervals for Western populations of women younger than 35 years.Understanding patient populations and differences in reference intervals by age group and measurement method can help guide clinical decisions and clinical laboratory analysis. All data were anonymized prior to statistical analysis. Repetitive serum AMH results measured in the same individuals were excluded for statistical analysis. Serum AMH results were investigated by age group as follows: 20–24, 25–29, 30–34, 35–39, 40–44, and 45–49 years.Serum AMH was analyzed using an automated electrochemiluminescence Elecsys AMH immunoassay  on an e411 analyzer (Roche). The analytical measurement range of the assay was 0.01–23.0 ng/ml. Accuracy of the AMH assay was verified by a proficiency testing/quality management program of a College of American Pathologists survey in the United States and by the Korean Association of External Quality Assessment Service.https://www.medcalc.org; 2020). p values were considered significant at the 0.05 level.The non‐parametric test was adopted when appropriate for non‐normally distributed continuous variables to compare serum AMH levels by age group. Serum AMH data were presented as 2.5th, 5th, 10th, median, 90th, 95th, and 97.5th percentile values.https://kosis.kr/eng/) were collected. In addition, another public database for annual numbers of patients diagnosed with and managed for irregular menstrual cycle in Korea was reviewed through Healthcare Bigdata Hub by the Health Insurance Review & Assessment Service (HIRA) using the 10th revision, Clinical Modification of the International Statistical Classification of Diseases and Related Health Problems (ICD‐10‐CM) code N91 for absent, scanty, and rare menstruation and code N92 for excessive, frequent, and irregular menstruation (available at: http://opendata.hira.or.kr/op/opc/olap4thDsInfo.do).Previous studies about serum AMH reference intervals in Korean women were reviewed in parallel with the results of the present study. Because clinical information of study subjects was limited in the present study, data from a public database for the entire Korean population by Statistics Korea .3During the 2‐year study period, a total of 1953 AMH test results from Korean women (age 20–49 years) undergoing general health checkups were obtained. Numbers of study subjects and AMH level by age group are summarized in Table p < 0.0001). In post hoc analysis, the serum AMH levels in women aged 20–24 years and in women aged 25–29 years were not significantly different (p ≥ 0.05). The serum AMH levels in women from other age groups differed significantly from each other after post hoc analysis (p < 0.05).Among age groups, the number of tested subjects was highest in women aged 30–34 years. The number of women aged 20–24 years was <120. Serum AMH level was significantly different by age group (The peak serum AMH level was observed at ages 25–29 years and declined to undetectable for subjects older than 44 years (45–50 years). In young age groups, the serum AMH level was widely distributed. The 2.5th, 5th, 10th, 90th, 95th, and 97.5th percentile values of serum AMH of the present study population were reviewed in parallel with previously described reference intervals and their definitions  since the study would not adversely affect the rights and welfare of the subjects due to its retrospective design and no more than minimal risk to the subjects.


Neutrophil‐to‐lymphocyte ratio (NLR) is a routinely available biomarker that reflects systemic inflammation. The study evaluated the predictive value of NLR for ischemic stroke and atrial fibrillation (AF) in patients with type 2 diabetes mellitus.This was a population‐based cohort study of patients with type 2 diabetes mellitus and complete blood count tests at baseline between 1 January 1st, 2009, and 31 December, 2009, at government‐funded hospitals/clinics in Hong Kong. Follow‐up was until 31 December, 2019, or death.p < .001, HR: 1.41 [1.32–1.51], p < .001 and HR: 1.38 [1.29–1.47], p < .001) and AF  compared to quartile 1. On multivariable analysis, NLR remained a significant predictor of ischemic stroke risk for quartiles 2 and 3  but not quartile 4 . NLR was not predictive of AF after adjusting for confounders .A total of 85,351 patients  were included. Univariable Cox regression found that increased NLR at quartiles 2, 3 and 4 was significantly associated with higher risks of new‐onset ischemic stroke (hazard ratio [HR]: 1.28 [1.20–1.37], NLR is a significant predictor of new‐onset ischaemic stroke after adjusting for significant confounders in Chinese type 2 diabetes patients. NLR is a significant predictor of new onset ischaemic stroke but not AF after adjusting for significant confounders in diabetes melltius. Patients fulfilling any of the following inclusion criteria were recruited: (1) diagnosis of T2DM using the International Classification of Disease, Ninth Edition (ICD‐9) coding system, (2) patients meeting the laboratory criteria for diabetes based on HbA1c or two successive fasting or random glucose tests as per ADA criteria, or (3) prescribed any anti‐diabetic medication between 1 January, 2009, and 31 December, 2009, at any public hospitals or outpatient clinics managed by the Hong Kong public sector, and (4) with baseline neutrophil and lymphocyte results. The data were retrieved from the Clinical Data Analysis and Reporting System (CDARS), an electronic medical database for shared comprehensive patient records across public hospitals and clinics. The system has been used for cohort studies by our team and other teams in the past,2.2Diagnoses were obtained from ICD‐9 codes. The primary outcome was incident AF (427.31) or ischemic stroke (434–435). The code 433 . This is due to the very low proportion of patients coded with 433 had acute stroke.2.3p‐Value were reported for the Cox regression. Univariate predictors with p‐Value <.10 were entered into a multivariate model. The Kaplan–Meier curves were plotted to reflect the outcome of stroke and AF stratified by high and low risk of NLR.To ensure the robustness of our findings, we performed the cause‐specific hazard model and Fine and Grey's subdistribution hazard model with consideration of mortality as a competing risk event. Statistical significance was defined as p‐Value <.05. Statistical analyses were performed using RStudio software (Version: 1.1.456) or Prism (Version: 9.0.0).Statistical descriptive statistics summarized the baseline and clinical characteristics of the type‐2 diabetes patients. Continuous variables were presented as mean  and categorical variables were presented count (%). Univariate Cox regression was used to identify predictors for incident episodes of both AF and ischemic stroke. Hazard ratio (HR), 95% confidence interval (CI) and 33.1The study cohort consisted of 85,351 patients with T2DM and neutrophil and lymphocyte values at baseline, between 1 January, 2009, and 31 December, 2009. This cohort was used for predicting the risks of new‐onset ischemic stroke (stroke cohort). This cohort was 48.8% male with a mean age of 67.6 years old (standard deviation [SD]: 13.2). During the follow‐up, 7458 patients exhibited new‐onset ischemic stroke (8.74%) and 7121 patients presented with new‐onset AF (8.87%). The baseline characteristics of the study cohort are summarized in Table p = .0315)   but significance was lost for quartile 4 . The hazard ratios for the risks of new‐onset ischemic stroke as a function of NLR levels are shown in Figure p < .0001) , mortality (red) and no events (green) are shown  Table . Higher  1.41 1.3–1.51, p  1.41 1.3–1.51, p 3.2p = .0001)  . Yet, the cause‐specific model and the subdistribution hazard model showed that the NLR is statistically significant in predicting the risks of new‐onset AF (Table p < .0001)  Table . Higher AF Table . The Kap8 1.20–1.7, p < .04The main finding of this paper is that higher NLR was a predictor of new‐onset ischemic stroke, with a positive correlation between NLR levels and level of risk.Inflammation contributes to the risk of stroke via a number of mechanisms, including activation of leukocytes, accumulation of pro‐inflammatory cells such as monocytes and macrophages, and platelet aggregation.A pro‐inflammatory environment is known to play essential roles in the pathophysiology of stroke. Our data demonstrated that the NLR is predictive of incident ischaemic stroke in T2DM. Prior studies have similar reported the utility of NLR in predicting new‐onset strokes in other settings.Our data showed the NLR was predictive of new‐onset AF on univariable analysis but not after adjustment for significant confounders. Nevertheless, patients who suffered from AF showed higher NLR at baseline compared to those who did not develop AF. Previous literature has found that the higher level of the NLR is predictive for the increased risks of AF occurrence or recurrence.4.1This study has several strengths. Firstly, this study is powered by a large number of patients who were followed up for a long observational period. Secondly, the territory‐wide healthcare database of Hong Kong included both outpatient clinic data from both family medicine and secondary specialist care, and hospital data. Therefore, diagnoses and attendances are recorded comprehensively in our database. Thirdly, the regression results were confirmed using competing risk analyses, including the cause‐specific and subdistribution hazard models. Lastly, the long follow‐up period allows capturing the temporal variability and long‐term outcomes.However, several limitations should be noted. Firstly, given this is a retrospective study, there is a lack of analysis of clinical outcomes during real‐time follow‐up. Furthermore, this retrospective study can only show the association but not the causation relationship. Secondly, given the lack of coding of the common confounders such as smoking status, diet, and adherence to the prescribed medications, those critical factors were not adjusted in our models. Thirdly, Unfortunately, in this study, only ICD‐9 codes of 434  and 435 . It was difficult to separate out large vessel occlusions from small vessel occlusions based on the ICD codes alone. Therefore, we did not perform subgroup analysis based on the subtypes of stroke by the TOAST classification. Future studies should be conducted to determine whether NLR can predict various stroke subtypes. Finally, this cohort is recruited from a single region such that external validation is needed to take account of the geographical heterogeneity.5Higher NLR is associated with an increased risk of ischemic stroke in a Chinese population of T2DM patients.Carlin Chang: Conceptualization ; data curation ; formal analysis ; investigation ; writing – original draft . Jiandong Zhou: Data curation ; formal analysis ; software ; validation ; visualization ; writing – original draft . Oscar Hou In Chou: Data curation ; formal analysis ; writing – original draft ; writing – review and editing . Justin Chan: Data curation ; formal analysis ; investigation ; writing – original draft . Keith Sai Kit Leung: Data curation ; formal analysis ; resources ; writing – original draft . Teddy Tai Loy Lee: Conceptualization ; data curation ; formal analysis ; methodology ; writing – original draft . Wing Tak Wong: Formal analysis ; funding acquisition ; methodology ; supervision ; writing – review and editing . Abraham Ka Chung Wai: Data curation ; formal analysis ; methodology ; project administration ; resources ; software ; supervision ; writing – review and editing . Tong Liu: Formal analysis ; investigation ; methodology ; project administration ; resources ; supervision ; visualization ; writing – original draft ; writing – review and editing . Qingpeng Zhang: Formal analysis ; investigation ; methodology ; project administration ; resources ; supervision ; writing – review and editing . Sharen Lee: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; supervision ; writing – original draft ; writing – review and editing .None.Appendix S1.Click here for additional data file.


Rising child obesity rate creates a need for tools quantifying changes in children suffering from obesity, for purposes of detection or prevention of comorbidities. A candidate for such a role seems to be microRNAs, which in vivo serve as the suppressing factors in gene expression.This study aimed at reviewing recent discoveries in this field and concluding directions of research or application of studied molecules.Repeated browsing of databases and screening of results, led to final approval of 16 articles. Filtered studies examined differences in microRNA expression between subjects with obesity and children suffering from its comorbidities.Studies concerning endothelial dysfunction identified molecules miR‐320a and miR‐630 as a possible diagnosis and treatment option. Search for the alternative markers in diagnosis of non‐alcoholic fatty liver disease suggested value of molecules: miR‐199a‐5p and miR‐122. miR‐486, miR‐146b, and miR‐15b may serve in grading the development of type 2 diabetes in children, although further research raised doubts. Panel of molecules was indicated as useful in early detection of metabolic syndrome and insulin resistance associated alterations. No valid link between studied microRNAs and atherosclerosis was found.MicroRNAs seem to be promising prognostic markers for diagnosis of endothelial dysfunction, non‐alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome and insulin resistance in children. ABCA1ATP‐binding cassette transporters A1ABCG1ATP‐binding cassette transporters G1AMPadenosine monophosphateASatherosclerosisAUCarea under the receiver–operator curveBMIbody‐mass indexBMI‐zbody‐mass index Z‐scoreC2C12myoblasts cultureCGcontrol groupCGSWHO Child Growth StandardsCK18cytokeratin 18dbmice with diabetesEDendothelial dysfunctionEGexperimental groupELISAenzyme‐linked immunosorbent assayETendothelinEVextracellular vesicleFPGfasting plasma glucoseGCGerman cohortHbA1glycated haemoglobinHbA1c%measurement of percentage of HbA1cHOMA‐IRhomeostatic model assessment insulin resistanceICItalian cohortICAM‐1intercellular adhesion molecule 1IRinsulin resistanceMALAT1metastasis‐associated lung adenocarcinoma transcript 1MetSmetabolic syndromeMIN6murine beta‐cells culturemiRmicroRNAmiRNAmicroRNAmRNAmessenger ribonucleic acidNAFLDnon‐alcoholic fatty liver diseaseNASHnon‐alcoholic steatohepatitisNEFnormal endothelial functionNOnitric oxideNWpatients without obesityOBchildren with obesityobmice with obesityOWchildren who were overweightPATperipheral arterial tonometryPICOSparticipants, interventions, comparisons, outcomes, study designRHIreactive hyperaemic indexRNAribonucleic acidROCreceiver–operator curveSCSlovenian cohortSDstandard deviationT2DMdiabetes mellitusTmaxtime of peak reperfusionUSGultrasonographyVATvisceral adipose tissueVCAM‐1vascular cell adhesion molecule 11Obesity in children is one of the most demanding challenges emerging in the modern world of paediatric medicine. It is described in relation to patient's body‐mass index (BMI). In children a threshold of obesity is BMI above 95th percentile in a group of the same age and sex. Despite various prevention programs, analyses, and research, its occurrence is still high,Research on miRNA is nowadays one of the more popular scopes of interest in numerous branches of medicine. Recently the differences in miRNA profiles between the children with and without obesity have been investigated.2Subject group: ‘child obesity’ OR ‘childhood obesity’ OR ‘obese children’ OR ‘paediatric obesity’ OR ‘overweight children’ OR ‘adolescent obesity’Entry terms were formulated according to the following scheme:Comorbidity: ‘complications’ OR ‘comorbidities’ OR ‘NAFLD’ OR ‘non‐alcoholic fatty liver disease’ OR ‘liver’ OR ‘fatty liver’ OR ‘CVD’ OR ‘cardiovascular diseases’ OR ‘endothelial dysfunction’ OR ‘type 2 diabetes’ OR ‘hypertension’ OR ‘metabolic syndrome’ OR ‘puberty’ OR ‘asthma’ OR ‘obstructive sleep apnea’ OR ‘‐’ANDMarker: ‘miRNA’ OR ‘miR’ OR ‘microRNA’ OR ‘circulatory microRNA’ANDAdequate filters regarding publication dates (up to 5 years prior to search) and text availability were applied. The repeated results of search were ruled out.Retrieved data underwent further screening on a basis of titles, abstracts, and keywords (performed by MH and JH) according to inclusion and exclusion criteria, which led to exclusion of reviews, systematic reviews, author manuscripts, personal communication, letters to editor, conference material, and case reports. Articles concerning the miRNA as the markers of obesity itself in children, as well as the studies of miRNA not obtained from blood or plasma were ruled out – in order to be plausible for the role of markers, miRNAs must be easily accessible (blood or plasma) and correlate with comorbidities of obesity (rather than the obesity itself). Inadequate matching of experimental and control groups in the matter of age or exceeding upper age threshold were also the discriminating factor.p‐values .At the stage of data synthesis the retrieved studies were assessed by two independent researchers (AZ and PM) in terms of methodology quality, as well as relevance of the results. The principal measures of differentiating potential were area under the receiver–operator curve (ROC) (AUC) score, fold change, and 3Multiple browsing of databases, such as PubMed, askMEDLINE, Wiley Online Library, and Elsevier, conducted in the span of January 2020 – September 2021 using search strategy presented in Materials and Methods section, returned a total of 1754 results. That number was reduced to 245 individual articles by excluding 1509 repetitions. The remaining have been found eligible for further title and abstract valuation, of which 34 studies were accepted as fitting for whole text reading according to the inclusion and exclusion criteria, which were as follows:English literature published in the last 5 yearsStudies in human subjectsPaediatric population (<19 yo)Studies examining miRNA profiles of patients with obesityInclusion criteria:Publication type: reviews, systematic reviews, author manuscripts, personal communication, letters to editor, conference material, and case reportStudies based on miRNA from body fluids other than bloodStudies not linking miRNA of patients suffering from obesity to any comorbiditySubjects exceeding at the study time the age criterion of paediatric group (≥19 yo)Control group inadequate in terms of age to the study groupSubjects diagnosed with comorbidity other than the examined oneExclusion criteria:In total 16 studies fully met the inclusion and exclusion criteria.The process of data collection for this review is presented in Figure Main characteristics of selected articles can be found in Table Enrolled studies focused on associations between changes in miRNA profiles of patients with obesity and presence of 6 obesity comorbidities. Their methodology is discussed in the following sections separately for each complication.3.1The highest number of articles was linked to ED – a total of four studies , which examined among others the miRNAs: miR‐126, miR‐320a, miR‐125a‐5p, miR‐342‐3p, miR‐365b‐3p, and miR‐630. ED is a pathological state characterized by impaired maintenance of vascular tone and oxidative stress, resulting from dysregulation of synthesis of endothelial mediators – nitric oxide (NO), endothelin (ET), and others.3.2NAFLD is a disease comprising of two main clinical features – steatosis and inflammation of liver. It is a condition resulting from factors other than alcohol consumption.3.3The goal of study 7 was the comparative analysis of miRNA levels in paediatric groups of children with (OB) or without obesity (CG) and children who were overweight (OW); adult groups of patients with type 2 diabetes/normal glucose tolerance (T2DM/NGT)  and animal model groups of mice with obesity/diabetes (ob/db). First stage was miRNA profiling of nine patients from OB and CG groups each, validated further in full‐size final paediatric groups (study 2) and in groups of adults with T2DM/NGT. MiRNAs which were chosen according to the results of the experiment  were then transfected using lentivirus into human preadipocyte, murine beta‐cell (MIN6), and myoblast (C2C12) cultures in order to examine their influence on respectively: adipocytes cell proliferation, insulin secretion, and glucose intake. Obesity and overweight in children were measured with BMI standard deviations (SDs), according to WHO Child Growth Standards (CGS) – children under 60 months: obesity >3 SDs, overweight >2 SDs; children over 60 months: obesity >2 SDs, overweight >1 SDs. Measure of T2DM was fasting plasma glucose (FPG) >7.0 mmol/L. Information on FPG as well as age, serum triglyceride, and cholesterol levels of subjects were collected from hospitals.p < 0.05). ROC of miRNAs and serum betatrophin were created in order to assess the potential of differentiating between the groups.Study 8 was conducted as a response to the results of study 7. Serum levels of three selected miRNAs , as well as of the betatrophin, were measured and compared between the groups of children with (>95th BMI percentile) or without (5th to 85th BMI percentile) obesity, and children suffering from diabetes . Venous blood samples intended for measurement of percentage of HbA1c (HbA1c%), betatrophin, cholesterol, glucose, and miRNAs relative expression levels were collected after overnight fasting. Correlations of miRNAs expression, betatrophin, and other parameters were tested by Pearson correlations coefficient  potentially useful in detection of T2DM development in children. Results of study 8 are less satisfactory than those of study 7, showing lower AUC score values. However, it should be pointed out, that in study 7 the cumulative effect of simultaneous measurement of two or three of aforementioned molecules was not examined.Another two miRNAs  were examined in study 9 in terms of differences of their levels in children without obesity, with obesity but without T2DM, and in those with obesity and T2DM. Two hundred and ninety eight patients, aged 9–15 years, were divided into above groups based on their BMI and WC (obesity vs. non‐obesity) and glucose levels , or HbA1c% >6.5%. The groups were significantly different in terms of diabetes parameters , whereas children with obesity and without T2DM did not differ significantly in WC and BMI percentiles from children with obesity and T2DM.3.4Similarly to the obesity, we can observe the increasing metabolic syndrome (MetS) prevalence worldwide. This term represents the cluster of metabolic changes, which stem directly from long‐term positive energy balance. These alterations include obesity, followed by hypertension, and atherogenic dyslipidemia.n = 50); with obesity but without MetS (n = 45); with obesity and MetS (n = 52). Cut‐off points of metabolic parameters for the MetS were as follows: FBG ≥5.6 mmol/L or type 2 diabetes; SBP ≥130 mmHg and/or DBP ≥85 mmHg; TG ≥1.7 mmol/L; HDL‐c <1.03 mmol/L.A potential of miR‐24‐3p molecule in differentiating children with obesity (BMI ≥95th centile according to Working Group on Obesity in China) from children without obesity, as well as children with MetS from ones without MetS, was examined in study 11. The population of study consisted of three groups: children without obesity  were examined in terms of panel of 16 circulating miRNAs , hepatic transaminases, anthropometric, and blood metabolic parameters. Similar to study 10, the associations between the miRNA profile and adiponectin, as well as obesity‐related inflammatory markers levels were investigated. The population of the study varied in age  with a disproportion for sex , therefore the results were adjusted for age and sex, which led to differences between the sexes in significance of correlation of some miRNAs with studied parameters.3.5Three studies  concentrated on the correlation between miRNAs and insulin resistance (IR). In study 13, the children and adolescents aged 10–17 years were divided into two groups: children with obesity  and children with obesity and IR , based on the HOMA‐IR score . Their anthropometric parameters and biomarkers values  were measured and compared between groups. Forty miRNAs were measured in serum of patients and compared with values of biomarkers and anthropometric parameters.Study 14 in turn examined the role of miRNA‐27a in the development of IR. For that purpose, group of 45 children with obesity (OB) and group of 45 age‐matched children without obesity (CG) were examined in terms of serum levels of miR‐27a and biochemical parameters: fasting blood glucose, adiponectin, leptin, resistin, subfatin, visfatin, and TNF‐α, IL‐6. The correlations between miRNA‐27a and above biomarkers were calculated. Further research was conducted on animal model, comparing the influence of high‐fat and low‐fat diet, as well as knocking‐out gene for miRNA‐27a on mRNA expressions of IRS‐1 and GLUT4 in skeletal muscle tissue.Analysis of glucose metabolism parameters and their association with miRNA profile in a group of 58 children with overweight or obesity was conducted in study 15. There was no statistical difference in mean age or insulin levels and HOMA‐IR between the sexes, though the latter parameters were insignificantly higher in girls. Two examined miRNAs: miR‐191‐3p and miR‐375, were also further analysed in terms of interactions with their respective pathway targets.3.62). Cholesterol efflux capacity reduction leads to creation of foam cells, thus being crucial for the development of atherosclerosis.Relations between BMI, miRNA levels  and cholesterol efflux capacity of THP‐1 derived macrophages were examined in study 16, which was based on population of adolescents (12–19 years) with  and without obesity  from various biological materials, we begin to recognize an important role of miRNAs in modulation of nearly every process in human body. Observing changes of miRNA levels and profiles in various physiological and pathological conditions creates the possibilities to establish new ways of disease diagnosis, treatment, as well as the prediction of their comorbidities and complications. Comparison of miRNA profiles in groups of adults and children with obesity and/or its comorbidities is an important issue for critical assessment of miRNAs as markers reflecting the development of obesity and its comorbidities. These molecules modulate expression of genes involved in metabolic pathways common for both age groups. Dysregulation of given molecule, associated with a comorbidity in one group, should also be observed in the other, with differences in its extent attributable to entering further stages of disease and its associated alterations. Such comparison would be a valid method of evaluating the findings for both age groups (adults and children), nonetheless it exceeds the scope of this review, which concentrated solely on studies conducted in the paediatric population.Examined miRNAs, their level changes, as well as their significance and AUC score are presented in Table 4.1Condition of endothelium as a whole can be inspected via levels of endothelial products, such as NO and ET‐1, their ratio (NO/ET‐1) or through functional test – RHI measurement, whose score is dependent on endothelial reaction engaging NO.p < 0.01 in t‐test comparison of pre‐ vs. post‐intervention results). Simultaneously the miRNA‐126 levels also increased, with positive correlation to BMI decrease and to the growth of RHI and NO/ET‐1 .Physical activity is one of the factors contributing to healthy endothelium. MiRNA‐126 is widely regarded as a molecule linked with the functionality of this tissue and as such was chosen for the subject of study 1. It examined a possible role of this molecule in the mechanism of ED renewal during 6 weeks of interventional lifestyle program (dietary and exercise routine change) in population of adolescents with obesity. Intervention resulted in an increased quality of endothelial condition, associated with statistically significant improvement of RHI and NO/ET‐1 parameters  in patients who apart from changes in diet changed also their life habits. When comparing levels of these molecules in exercise group before and after 12‐week intervention, the same changes were observed for either of the molecules (p < 0.001). Changes in miR‐320a expression correlated negatively with changes in levels of ED markers: VCAM‐1 , ICAM‐1 , and E‐selectin , whereas changes in MALAT1 expression correlated positively with these markers . MiR‐320a was also significantly negatively correlated with parameters connected with obesity, including BMI , waist circumference , hip circumference , TC , LDL‐C , and HOMA‐IR . This molecule may potentially be considered as the marker of obesity‐related pathological changes in endothelium.Similar to study 1, study 2 was also an interventional study, which compared the effects of 12‐week dietetic intervention or 12‐week dietetic and exercise intervention on anthropometric measurements, levels of blood indices, ED markers, and metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1)/miR‐320a axis in group of obese adolescents. Expression of miR‐320a increased, and expression of MALAT1 decreased significantly more  for three miRNAs whose expressions were found to be significantly different in groups. The procedure was repeated for groups of children with ED and with NEF, but without obesity, in order to rule out the influence of BMI‐z score. MiRNA profile in these groups was similar to the one in groups with obesity.Gene mapping revealed putative targets of these three molecules , involved in transforming growth factor β signalling, cytokine‐cytokine receptor interactions, and activin receptor‐like kinase pathways in cardiac myocytes, included in MED13 and MED14 mediator complexes. This study suggested the possible value of aforementioned three miRNAs as endothelial dysfunction indicators, but unfortunately they seem to be unrelated to obesity's involvement in development of this comorbidity.ED group when compared to subjects of OBNEF group. EVs obtained from patients with ED disrupted integrity of cell membranes in endothelial cell culture and significantly (p < 0.001) reduced expression of mRNA coding endothelial NO synthase. Injection of EVs derived from OBED subjects into murine circulation prolonged Tmax significantly (p < 0.001) in juxtaposition to the effects of injection of both CG‐ and OBNEF‐derived EVs.Study 4 identified significantly underexpressed 4 miRNAs  and one overexpressed (miR‐4665‐3p) in children of OBzonula occludens‐1 distribution were altered. Gene targets specific for miRNA‐630 are engaged in 10 pathways, among them in the ones involved in nuclear factor erythroid 2‐related factor 2 – mediated oxidative stress responses, adenosine monophosphate (AMP) kinase, and tight junction signalling.MiRNA‐630 was chosen for further examination due to its highest fold change value. Endothelial monolayer culture was treated with EVs transfected with miR‐630 mimic or inhibitor, while the mixture of two was used for control. Mimic induced restoration of the culture from ED cells, while in NEF cells culture (influenced by the inhibitor) the resistance and Relevance of results of this study can be limited due to relatively small size of examined group. Nonetheless, administration of miR‐630 presented a direct regenerative effect on endothelium , which suggests its potential properties in terms of endothelial regeneration as part of ED treatment strategy.4.2Diagnosis of NAFLD is based on rating the function and condition of liver with liver‐associated enzymes – ALT, AST, GGT,p < 0.05; >2‐fold change) overexpressed in children with NAFLD when compared to healthy controls. Discriminating potential was examined using ROC, leading to assessment of AUC parameter. Twelve molecules had AUC greater than 0.9, although three molecules: miR‐122‐5p (0.9833), miR‐21‐5p (0.9813), miR‐199a‐5p (0.975), and miR‐23a‐3p (0.9708) had the highest scores. Associations between these markers, BMI, and serum transaminases was also analysed – its results are presented in Table Results of study 5 presented a panel of 16 (out of 20 tested) miRNAs that were significantly , followed by third highest value of AUC parameter. There was however no significant correlation between this marker and BMI, ALT, and AST parameters. But bearing in mind its role in preadipocyte proliferation and differentiation, insulin signalling, and correlation with state of fibrosis in human liver, miR‐199a‐5p still poses as a strong candidate for the potential NAFLD development marker.r = 0.50) and ALT (r = 0.60). Previous studies in adults established its protective role in non‐alcoholic steatohepatitis (NASH),In the contrast, miR‐122 scored the highest AUC and second highest fold‐change (12.48) between the groups and was also positively related with both AST . Although the findings might be connected directly to NAFLD, the influence of obesity itself on the results cannot be ruled out.MiR‐122 was also solely investigated in study 6. in comparison to serum markers of NAFLD – AST, ALT, and CK18. In population of two pre‐pubertal cohorts, German and Italian, miRNA  and serum markers – CK18 , ALT  – had the similar potential of differentiating patients with NAFLD from healthy ones (as measured with AUC).r) with BMI , AST , ALT , and CK18 , thus suggesting its efficacy in NAFLD grading. Levels of miR‐122 were significantly different between patients with different grades of NAFLD in GC (p < 0.05), although no such significance was found in IC.In both groups the miR‐122 matched the quality of CK18, but scored lower when compared to ALT. MiR‐122 was also found to be positively correlating . They were also positively correlated to FPG values . ROCs and AUC values were calculated for each miRNA, as well as for their combination . It stands in opposition to the results of study 6, where these molecules scored significantly higher, although it is worth mentioning, that the population of the 2018 study was significantly more numerous. Also, the cumulative effect of molecules in differentiating was not established, contrary to the aforementioned study.p < 0.001) between the groups of children in study 9, with 0.21 fold change of miR‐29a and 0.37 fold change of miR‐122 between children with obesity and diabetes compared to the ones with obesity but without diabetes. In the whole population of the study the significant (p < 0.01) positive correlations between miR‐29a and the metabolic parameters: HbA1c% (r = 0.570), glucose (0.654), insulin (0.356), C‐peptide (0.254), and HOMA‐IR (0.651) were observed. Analysis of miR‐122 levels showed similarly significant (p < 0.01) correlations between this molecule and the above parameters . In patients with obesity the levels of both studied miRNAs were significantly correlated with each other. This study suggests the possible role of these molecules in the pathogenesis of T2DM in children with obesity. Its results might be of high significance, considering the reasonable size of the examined groups, as well as a good design of the groups, allowing differentiation of the influence of obesity and diabetes on the miRNA profiles.MiRNA profiles differed significantly  and 5 underexpressed  molecules, all of which were positively linked with the concentrations of adipokines. The upregulated molecules correlated positively with values of BMI, diabetic, and lipid profile parameters, whereas downregulated ones correlated negatively with them. A difference in levels of obesity‐related parameters  could be observed between the groups, which reflected the down‐ and upregulation of studied molecules.p < 0.001), whose expression of miR‐24‐3p was highest of all groups. They also correlated with some of the parameters, including FBG , TG , SBP , and DBP , but not with TC, LDL, and HDL . Though this study is well designed and conducted on group of relatively sound size (n = 147), it failed to prove correlation of miR‐24‐3p levels with parameters of dyslipidemia, which are an important component of MetS. Nonetheless, miR‐24‐3p presented the considerable specificity (71.2%) and sensitivity (95.6%) for detecting MetS, with AUC of 0.890. Further trials are needed to evaluate potential of this molecule.In study 11, the analysis of potential of miR‐24‐3p in detection of obesity and MetS, expressed as AUC – respectively 0.951 and 0.890. Levels of this molecule were significantly lower in children without MetS than in those with MetS , IL‐1Ra , and procalcitonin , and negatively with adiponectin . Similar correlations were observed for miR‐34a and TNFα , and procalcitonin . It suggests that these three miRNAs may play a role in obesity‐related inflammatory reaction, which is an important feature of MetS pathogenesis. Moreover miR‐33b, ‐193b, and ‐1290 were shown to be associated with lipid parameters and hepatic transaminases, although after adjusting for age and sex, the associations of miR‐193b and miR‐93 with CK‐18, as well as of miR‐33a and TG decreased. Interestingly, miR‐122 was the only miRNA which correlated with HOMA‐IR . An insignificant trend (p = 0.10) for this molecule was observed in patients with MetS, whereas miR‐93 expression was significantly lower in these patients than in those without MetS. Similar findings were presented for miR‐192 in patients with metabolically unhealthy obesity . This study presented a set of miRNAs, which were shown to be firmly correlated with metabolic pathologies stemming from obesity. Nonetheless, the limitation of the study is inclusion of patients with prediabetes (19.3% patients), T2DM (4.6%), and NAFLD (57.8%). Only a 13.9% of patients suffered from MetS, therefore despite the high association of studied molecules with components of MetS, it is difficult to rule out the influence of the obesity comorbidities other than MetS on the results of the study.Results of study 12 showed that miR‐122, previously described as involved in hepatic steatosis, and miR‐192 correlated with TNFα , fasting insulin (p < 0.05), TG (p < 0.05), and TG/HDL‐C ratio (p < 0.05). Out of 40 examined miRNAs, levels of 12 were significantly different in the patients of OB and OB‐IR groups . Moreover, four of these molecules  correlated with insulin  and TG/HDL‐C  values. Three of these molecules  correlated with values of other parameters of IR, such as HOMA‐IR, homeostasis model assessment‐adiponectin (HOMA‐AD), and adipose insulin resistance (Adipo‐IR) index.Despite the small size of groups in study 13 , they were significantly different in terms of HOMA‐IR  lacked the significant difference in fasting blood glucose levels (mean 4.59 ± 0.58 vs. 5.03 ± 0.49 mM), which of all examined parameters is the one that is the most associated with development of IR. Therefore, it is difficult to evaluate if increased miR‐27a levels in OB groups reflect impaired insulin signalling or rather the grade of obesity.p < 0.001 for either molecule with both HOMA‐IR and insulin), as well as when the patients were divided according to sex and analysed separately. Both miRNAs were found to interact with Hippo signalling pathway, which is responsible for pancreatic β‐cell proliferation and functionality.In population of study 15, levels of examined miRNAs (miR‐119‐3p and miR‐375) correlated with HOMA‐IR and insulin level, both when the group was analysed as a whole  are involved in this process.p < 0.05) correlated with cholesterol efflux capacity , and four which correlated negatively .Study 16 through the multivariate analysis identified two miRNAs which significantly .Although the study identified a panel of miRNAs significantly different levels between the subjects with high and low cholesterol efflux capacity, these results lack the correlation with BMI and obesity in subjects, which is essential in order to treat them as relevant in prediction of risk of developing atherosclerosis in children with obesity.In total, miRNAs have been identified as potential markers identifying, grading the development and even treating five diseases: ED , NAFLD , T2DM , MetS , and IR . The potential clinical implications of these molecules seem to be promising and require further examination.5Results of reviewed studies indicated miR‐320a as a potential marker useful in diagnosis of ED, whereas miR‐630 was shown to influence the condition of endothelium, therefore posing as a strong candidate for future therapeutic strategy for ED.Two miRNAs: miR‐199a‐5p and miR‐122, presented a good potential in diagnosis of NAFLD and grading its development, however further evaluations are recommended.Studies concerning type 2 diabetes mellitus suggested miR‐486, miR‐146b, and miR‐15b as markers of disease progression, although the efficacy of these molecules differed between the studies (AUC: 0.88–0.98 in study 7 vs. 0.54–0.62 in study 8). MiR‐122, associated with NAFLD, and miR‐29a were correlated with levels of glucose metabolism parameters. They may also be involved in development of T2DM.MiRNAs examined in studies 10, 11, and 12 showed significant correlation with parameters of metabolic syndrome , which might be used for early detection and planning adequate prevention of this disease.Studies 13, 14, and 15. concentrated on miRNAs significantly associated with the development of IR, suggesting the role of several miRNAs  in its pathogenesisResearch on a link between miRNAs and cholesterol efflux capacity, which is an important factor in AS development, returned no satisfying results.Review of literature provided us with a good insight into the possibilities of applying miRNAs in diagnosis and prevention of obesity comorbidities.Results of above studies indicate that miRNA might play an important role in diagnosis of obesity comorbidities in paediatric population, especially ED, NAFLD, and T2DM. Possibility of early detection of developing obesity‐related pathologies is crucial for creation of efficient strategy aimed at slowing or even inhibiting their progression.The authors declare no conflicts of interest.Michał Hutny and Jagoda Hofman performed the data collection through database browsing, results filtering, and prepared the initial manuscript. Paweł Matusik and Agnieszka Zachurzok conducted the analysis of the gathered results and revised the manuscript versions. All authors were involved in designing the study, writing the manuscript and had final approval of the submitted version manuscript.


Autistic people have worse mental health (MH) than non-autistic people. This proof-of-concept study explored feasibility of longitudinal research with autistic university students, focusing on their MH and coping styles across an academic year.Twenty-two students took part at all timepoints. They completed four rounds of online MH questionnaires.Over 80% of students were retained. They started the year with high levels of all MH issues, which remained stable across the year. Network Change analysis showed the connections between MH and coping style changed over time.Autistic students are engaged participants who are likely to take part in longitudinal research. While MH levels were stable, it may be that coping styles are a useful target for intervention. In the general UK population, up to one in four adults will experience mental health problems at any one point in time   showed decreases in anxiety and depression from Semester One to the end of the academic year, whereas those using ‘avoidant’ strategies showed an increase in both over the same period  had an autism diagnosis and (2) were a current University of Bristol student (the institution where the research was carried out). Individuals responded to advertisements put out over social media, direct emails from university departments/services, poster advertisements around campus or heard about the study via word-of-mouth. The School of Education’s ethics committee within UOB granted the study ethical approval, and all participants gave informed consent before taking part.Twenty-seven autistic students volunteered and completed questionnaires at Time 1 (October 2019). Twenty-six took part at Time 2 (December 2019), 24 at Time 3 (February 2020), and 22 at Time 4 (May 2020). This represents an 81% participant retention rate, which is highly successful – especially considering the length of time involved, and that the COVID-19 pandemic occurred during data collection, which disrupted many participants lives. There were no apparent differences in age or other factors between those who dropped out during the process and those who remained involved – those who dropped out were mostly female (four of five) and mostly undergraduates . All were of White British ethnic origin. Those who dropped out had similar MH scores to those who completed all rounds in terms of anxiety, depression, social anxiety, ADHD and OCD, and their behavioural scores  were also similar. Statistical tests comparing participants who were retained with those who dropped out were not carried out due to the small numbers of participants in the latter group.n = 22). This constituted a sample of 13 female individuals, 8 male individuals and 1 nonbinary individual, with an age range from 19 to 36 years at T4 , all of whom reported having a formal clinical diagnosis of being on the autism spectrum, said diagnosis being the basis of Disability Support Statements for all participants. The sample was majority White British (18 participants), with two participants identifying as White other, one as Mixed ethnicity, and one as Latino. Sixteen participants were undergraduates , four were Masters/PGCE students, and two were PhD students . Nine participants were in Science, Technology, Engineering and Mathematics departments, and thirteen participants were in Art, Humanities and Social Sciences departments. Fourteen participants disclosed at least one existing mental health diagnosis , with ten participants having two or more mental health diagnoses.All participants who completed all rounds of questionnaires were included  across the academic year. At T1, participants provided demographic information and were asked for changes to this at further time points. Participants were emailed a link to the study page and were paid £20 in Amazon vouchers per round of participation – each of which took 20 to 30 min. The study took place during the emergence and early stages of the COVID-19 pandemic, which undoubtedly would have had a significant impact on our participants. However, because the research had always been designed to be conducted online, our procedures did not have to change in response to the pandemic.SCID Depression screening : The SCID is a widely used clinical screening measure, asking participants to report the frequency of depression-related cognitions and behaviours, rated from 0 to 3 (e.g. ‘I do not feel sad’ to ‘I am so sad and unhappy that I can’t stand it’). The 21-item measure exhibits good validity and reliability : Participants answered five ‘yes/no’ questions covering the core behaviours of anorexia and bulimia nervosa. The scale has been validated with university students , and showed good reliability with autistic women .Participants were asked to rate how often they had recently encountered difficulties related to ADHD. The scale has strong internal and concurrent validity , and is accurate for detecting ADHD-specific symptoms amongst people with clinical depression .The AUDIT centres on how often - over the last year (T1), or since the last set of questionnaires (T2-T4) - participants engaged in varyingly extreme drinking activity. Psychometric properties of the AUDIT are favourable when compared to other self-report measures of disordered alcohol use .Participants answered ten ‘yes/no’ questions about drug-taking habits over the last year (T1), then since the last set of questionnaires. The DAST-10 exhibits good specificity and sensitivity amongst individuals with other mental health diagnoses . Participants made judgements on a four-point Likert scale regarding how often they employ 14 different approaches to coping with hardship. These sub-scales can be grouped under two validated and reliable coping styles – ‘avoidant’  and ‘approach’  .This study was actively carried out with members of the autism community. Autistic consultants, including an autistic university student, were involved in the funding application and research questions, design of the study, checked measures for accessibility and reviewed the manuscript presenting our findings. Following T1 (Oct 2019), we had further discussions with this group and some participants, and then decided to include the Brief-COPE at all other timepoints as the initial battery had been less demanding for participants than we had anticipated.ps > 0.05), bar depression (p = 0.036) and social anxiety (p = 0.048). These data were therefore retained in all further analyses, and the change in depression scores is discussed below. Linear mixed effects models from the package lme4 . T1 levels of each mental health condition were calculated, then compared to each subsequent time point. As the COVID-19 global pandemic took hold between T3 and T4, we explored whether T4 was statistically significantly different to other timepoints and found that it was not on all constructs , and similarly strong correlations between all Avoidant strategies across all timepoints (ps < 0.05). Therefore, to reduce the number of tests conducted on a small sample, we did not run tests for each strategy within each overarching style. There was a main effect of coping style, with Approach being used more than Avoidant  = 18.96, p < 0.001, BF = 775.66 ± 2.14) with the BF providing strong evidence in favour of this result. There was no main effect of time on coping  = 0.51, p = 0.60, BF = 0.14 ± 1.56) and no time by coping style interaction  = 0.51, p = 0.603, BF = 0.18 ± 6.04) with the BFs providing moderate evidence in favour of the null hypothesis. This suggests that coping style could be introduced as a predictor alongside time to examine the impact of these two variables on mental health and alcohol and drug use.Prior to conducting the linear mixed models with coping style as one of the predictors we examined whether there were any significant changes in coping style over time. If coping style varied as function of time including both variables as predictors could introduce multicollinearity in the linear mixed models. There were strong correlations between all Approach strategies across all timepoints (2 (1) = 1.89, p = 0.169, BF = 0.20), or Drug use (X2 (1) = 0.07, p = 0.787, BF = 0.13). The associated BFs provided anecdotal to moderate evidence in favor of the null hypothesis suggesting the present data were 1.2 to 7.9 times more likely under the null hypothesis.That analysis showed that there was a significant effect of coping on Depression  = 18.30, p < 0.001, BF = 721.44), Anxiety  = 7.20, p = 0.010, BF = 8.43), Social Anxiety  = 6.88, p = 0.012, BF = 5.75), and ARFID scores  = 6.90, p = 0.011, BF = 7.48). In all cases lower coping scores, indicating increased reliance on avoidance, was associated with more mental health difficulties. The corresponding BFs indicated that there was moderate to very strong evidence in favor of the alternative hypotheses. There was no significant effect of coping on Anorexia/Bulimia scores  = 2.32, p = 0.135, BF = 0.59), ADHD scores  = 1.01, p = 0.319, BF = 0.83), OCD scores  = 1.70, p = 0.197, BF = 0.54), Alcohol use (XX2(2) = 12.90, p = 0.002, BF = 22.64) with the BF providing moderate evidence in favour of this result and suggesting the data were approximately 22.6 times more likely under the alternative hypothesis . Post-hoc tests revealed that this was driven by a significant decrease in Alcohol Use between T2 and T4, z = 3.37, p = 0.002. There was no main effect of time for Depression scores  = 2.36, p = 0.107, BF = 0.50), Anxiety scores  = 0.66, p = 0.521, BF = 0.28), Social Anxiety scores  = 1.66, p = 0.203, BF = 0.25), Anorexia/Bulimia scores  = 0.22, p = 0.894, BF = 0.15), ARFID scores  = 1.26, p = 0.295, BF = 0.50), ADHD scores  = 1.12, p = 0.337, BF = 1.1), OCD scores  = 0.69, p = 0.508, BF = 0.16), or Drug Use (X2 (2) = 1.32, p = 0.501, BF = 0.02). The BF associated with all but the ADHD scores provided anecdotal to strong evidence in support of the null hypothesis and suggested that the data were 2.0 to 58.0 times more likely under the null hypothesis. Interestingly, the BF associated with the ADHD scores pointed towards anecdotal evidence for the alternative hypothesis. This suggests that overall, drug use and most mental health variables remained stable across the course of the study.There was a main effect of time for Alcohol Use (2 (2) = 1.77, p = 0.413, BF = 0.13), or Drug use (X2 (2) = 1.94, p = 0.379, BF < 0.0001). The associated BFs provided anecdotal to strong evidence in favor of the null hypothesis.There were no significant coping by time interactions for Depression scores  = 1.04, p = 0.360, BF = 0.17), Anxiety scores  = 0.05, p = 0.950, BF = 0.12), Social Anxiety scores  = 0.83, p = 0.442, BF = 0.28), Anorexia/Bulimia scores  = 2.02, p = 0.146, BF = 0.83), ARFID scores  = 0.07, p = 0.928, BF = 0.12), ADHD scores  = 0.32, p = 0.727, BF = 0.21), OCD scores  = 0.76, p = 0.475, BF = 0.30), Alcohol use , suggesting excellent longitudinal participant retention rates are possible for research of this kind with autistic students. We were able to examine whether this global pandemic had an impact on autistic student mental health. Interestingly, it appeared to have relatively little impact overall, with no significant worsening of any specific measure, although it should be noted that this may be because our participants went into the pandemic with poor mental health. The one thing that changed was a reduction in alcohol consumption, possibly due to a decrease in the number of social opportunities where students would often drink together before lockdown. Interestingly, we also found that greater reliance on avoidance-based coping was associated with more depression, generalised anxiety, social anxiety and ARFID symptoms and these associations were not influenced by time.This lack of impact of COVID-19 emphasises the overall message of stability over time in the mental health status of our participants. Autistic students arrive at the university  with poor mental health, in line with other research identifying high levels of depression and anxiety . Services should recognize that reaching out and asking for help can be more difficult for autistic students and find ways to make this process easier for them. Future research around supporting autistic students to develop and maintain positive coping strategies has the potential to significantly improve their experiences of university.It is worth highlighting that our study shows that despite the challenges autistic students face at university, they are keen to contribute to research. With 81% of our participants staying engaged and completing all rounds of data collection, we can make reasonable assumptions, based on Bayesian analysis, of the likely validity of our findings of no change over time in levels of mental health difficulty. Achieving quite this level of participant retention was unexpected, considering the demands and distractions of university, and then of life during COVID. However, it is excellent news for future, larger, longitudinal studies with this population, and suggests that such work is feasible and practicable.There are some limitations to this study. The sample is small, especially for longitudinal work, and cannot necessarily be taken as representative of all autistic students. However, our participants comprise approximately 10% of all autistic students registered at the university , a range of genders, and of study levels, which strengthens the findings. The high level of participant retention means that we have captured the mental health of all our participants across the academic year, making our study as comprehensive as possible. Furthermore, the sample is majority female, with just over half of participants identifying as such. This is potentially unexpected in autism research, considering that more males are diagnosed as autistic than females (Loomes et al., This proof-of-concept project is the first study to track the mental health of a group autistic university students across an academic year, highlighting the stability of mental health issues in this population – those who arrived with mental health issues did not see significant improvements, although they also did not see significant worsening of the challenges they face. This was the case despite the emergence of the COVID-19 pandemic, which is an unexpected finding – although the reduction in social anxiety makes sense considering the restrictions on meeting others and entering social spaces at university such as lectures and seminar rooms. The findings highlighted that, while there was little change in mental health over time for the students involved, coping style was strongly associated with mental health. This suggests that autistic students may acclimatise to university life over the course of the academic year, as they become familiar with their course, peers, and independent living, but that current systems do not do enough to support them in improving their mental health.