Stress hyperglycemia ratio (SHR), a novel biomarker of stress hyperglycemia, was proved to be a reliable predictor of short‐term adverse outcomes in patients with acute coronary syndromes (ACS). However, its impact on long‐term prognosis remained controversial.A total of 7662 patients with ACS from a large nationwide prospective cohort between January 2015 and May 2019 were included. SHR was calculated by the following formula: SHR = admission glucose (mmol/L)/(1.59 × HbA1c [%]−2.59). The primary end point was a major adverse cardiovascular event (MACE) during follow‐up, a composite of all‐cause death, myocardial infarction, and unplanned revascularization. The second end point was the separate components of the primary end points.During a median follow‐up of 2.1 years, 779 MACE events occurred. After multivariable adjustment, ACS patients with the highest SHR tertile were significantly associated with increased long‐term risks of MACE , all‐cause death  and unplanned revascularization . Although significant associations between the highest SHR tertile and risks of MACE and all‐cause death were assessed in both diabetic and nondiabetic patients, the patterns of risk were different in these two groups.Elevated SHR was independently associated with a higher risk of long‐term outcomes irrespective of diabetic status, suggesting that SHR was a potential biomarker for risk stratification after ACS. HighlightsThe association between stress hyperglycemia ratio (SHR) and long‐term outcomes was still controversial.Based on a large prospective multicenter cohort, we found that high SHR levels were independently associated with increased risk of major adverse cardiovascular events (MACE), all‐cause death, and unplanned revascularization. Associations between high SHR levels and risk of MACE and death remained positive in patients with or without diabetes.SHR was a potential biomarker for risk stratification after acute coronary syndromes. From January 2015 to May 2019, a total of 18 701 adults with coronary artery disease (CAD) were recruited from Fuwai hospital  and eight other medical centers throughout China. Patients were excluded in the present analysis if (1) a diagnosis of chronic coronary syndrome was established on admission; (2) baseline data on glycosylated hemoglobin (HbA1c) or admission glucose level were missing; and (3) follow‐up data were incomplete. Finally, 7662 patients with ACS  were included , which considered various factors related to coronary lesion data.Baseline data on demographic and clinical characteristics, laboratory results, and medications were collected at each clinical center by well‐trained independent clinical research coordinators. After coronary intervention was completed, details related to the procedure were recorded by two experienced interventional physicians. To assess the severity of CAD, SYNergy between percutaneous coronary intervention (PCI) with TaXus and cardiac surgery (SYNTAX) score was calculated using an online calculator , a composite of all‐cause death, MI, and unplanned revascularization. Secondary end point included individual components of the primary end point. The definition of myocardial infarction was based on the Fourth Universal Definition of Myocardial Infarction as a clinical condition characterized by signs or symptoms of newly developed myocardial ischemia with an increase in cardiac enzyme level.2.4p values <.05. Statistical analyses were performed using R version 4.2.0 .Continuous variables were presented as mean ± SD or median and interquartile range (if variables were skew distribution). Categorical variables were presented as frequency and percentage. one‐way analysis of variance, Kruskal‐Wallis H test, or Pearson chi‐square test were used to assess the baseline differences across SHR tertiles as appropriate. Correlations between SHR and other variables were assessed by Spearman's rank correlation coefficient. Cumulative incidences among tertiles were drawn by Kaplan–Meier curves and compared by log‐rank test. Univariable and multivariable Cox regression models were applied to identify the independent associations between SHR and MACE or all‐cause death, whereas the associations with MI and unplanned revascularization were calculated by Fine–Gray hazard models for the competing risk of death. Covariates for adjustment in multivariable model include age, sex, clinical presentation , body mass index, eGFR, hemoglobin, and SYNTAX score. Covariates were selected from Table 33.1r < 0.4) were presented between SHR and white blood cells, high‐sensitivity C‐reactive protein, cardiac troponin I, creatine kinase‐myocardial band, and LVEF   who met the inclusion and exclusion criteria were included in this study. Of these patients, 1773 (23.1%) were female and 3178 (41.5%) had diabetes. The mean age and SHR levels were 61.0 ± 10.6 years and 0.91 ± 0.27, respectively. Baseline characteristics across SHR tertiles are presented in Table 1) Table .3.2During follow‐up time , 779 MACE events (10.2%) and 349 death events (4.6%) occurred, with response rates of 98.7% at first year and 97.1% at second year. Cumulative incidences of clinical outcomes across SHR tertiles are illustrated in Figure 3.3p for interaction = .396; all‐cause death: p for interaction = .499). The third SHR tertile was not significantly associated with the risk of MI and unplanned revascularization in patients with or without diabetes   trials reported that patients receiving insulin infusion had significantly lower rates of death compared with control groups, other studies including DIGAMI 2, HT‐5 (Hyperglycemia: Intensive Insulin Infusion in Infarction), and BIOMArCS‐2  trials failed to find a persistent beneficial effect of intensive glucose‐lowing therapy.Our study also had several limitations. First, due to its observational nature, although numerous covariates were adjusted in the model, potential unmeasured confounders could still influence the results. Second, as we included only patients from China, the generalizability of our findings was limited to other ethnic populations. Third, data on glucose‐lowing therapy at discharge were incomplete in our study. Furthermore, glucose fluctuation and management of hyperglycemia during hospital stay and follow‐up were unavailable, which may be the source of bias. Finally, the definition of diabetes was not precise, for we did not distinguish type 1 and type 2 diabetes, and also did not include new‐onset diabetes due to a lack of data on oral glucose tolerance tests.5In this large nationwide cohort, our results suggest that high SHR was independently associated with long‐term poor prognosis irrespective of diabetic status, indicating that SHR is a reliable biomarker for risk stratification. Further study are warranted to confirm the results.Jinqing Yuan, Yaling Han, and Guyu Zeng contributed to the conception and design of the work. Ying Song, Zheng Zhang, Jingjing Xu, Zhenyu Liu, Xiaofang Tang, XiaoZeng Wang, Yan Chen, Yongzhen Zhang, Pei Zhu, Xiaogang Guo, Lin Jiang, Zhifang Wang, Ru Liu, Qingsheng Wang, Yi Yao, and Yingqing Feng contributed to data collection and analysis. Guyu Zeng drafted the manuscript. Jinqing Yuan and Yaling Han critically revised the manuscript. All authors read and approved the final manuscript.This study was supported by The National Key Research and Development Program of China (grand No. 2016YFC1301300 and 2016YFC1301301), the National Clinical Research Center for Cardiovascular Disease, Fuwai Hospital, Chinese Academy of Medical Sciences (grand No. NCRC2020013) and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grand No. 2020‐I2M‐C&T‐B‐049).The authors declare no conflict of interest.Table S1. Collinearity analysis of covariates.Table S2. Spearman correlation analysis between stress hyperglycemia ratio and other variables.Table S3. The association between admission glucose, HbA1c, and long‐term major adverse cardiovascular events (MACE).Table S4. C‐statistic of stress hyperglycemia ratio.Figure S1. SHR on a continuous scale and adjusted risk of major adverse cardiovascular events and all‐cause death in the entire population.Figure S2. Adjusted risk of myocardial infarction and unplanned revascularization across SHR tertiles in patients with diabetes and without diabetes. CI, confidence interval; HR, hazard ratio; SHR, stress hyperglycemia ratio.Figure S3. Receiver operating characteristic curves of stress hyperglycemia ratio for prediction of major adverse cardiovascular events in patients with diabetes and without diabetes.Click here for additional data file.


Coeliac disease (CD) is a life-long food-related disorder with a global prevalence of approximately 1%. Patients with CD often experience reduced health-related quality of life that could improve with a strict adherence to a gluten-free diet (GFD).To obtain visual analogue scale (VAS), time trade-off (TTO) and willingness-to-pay (WTP) values amongst patients with CD.In 2020–2021, a cross-sectional online survey was conducted amongst 312 adult CD patients in Hungary. Patients completed the Gastrointestinal Symptom Rating Scale (GSRS) and evaluated their current health and three hypothetical health state vignettes defined based on dietary adherence using VAS, conventional 10-year TTO and WTP. Multivariate regressions were used to explore the effect of patients’ demographic and clinical characteristics on utility and WTP values.p < 0.05).Mean VAS values for current health and ‘CD with strict adherence to GFD’, ‘CD with loose adherence to GFD’ and ‘CD without GFD’ hypothetical health states were 79.69 ± 18.52, 85.36 ± 16.18, 62.44 ± 19.91 and 36.69 ± 25.83, respectively. Corresponding mean TTO utilities were: 0.90 ± 0.19, 0.91 ± 0.20, 0.87 ± 0.23 and 0.76 ± 0.29. Mean annual WTP values for returning to full health were: €845 ± 1077, €648 ± 1002, €862 ± 1135 and €1251 ± 1496. Older age at diagnosis, male sex, more severe gastrointestinal symptoms (GSRS) and having comorbidities were associated with lower VAS and TTO or higher WTP values for current own health (This is the first study to report TTO utilities for CD health states. Strict adherence to the GFD may result in substantial health gains in symptomatic patients. Utilities and WTP results can be used to estimate benefits of GFD in cost-utility and cost–benefit analyses. Coeliac disease (CD) is an immune-mediated systemic disorder activated by the ingestion of gluten in genetically susceptible individuals . It is tA large number of studies reported impaired health-related quality of life (HRQoL) in CD patients before diagnosis –16. A reSo far, the EQ-5D has been the most commonly used instrument to derive health utilities in CD patients , 24–27. When generic preference-accompanied measures are not feasible to assess utilities, vignette-based methods may be considered. A vignette is a description of a hypothetical health state that is directly valued by using a preference elicitation method . As vignThis study therefore aims to assess health states by visual analogue scale (VAS), TTO and WTP values in CD patients using a vignette-based study design.An online cross-sectional survey was performed between November 2020 and January 2021 in Hungary. Permission for conducting the study was granted by the Research Ethics Committee of the Corvinus University of Budapest (reference no. KRH/390/2020). A convenience sample of CD patients were recruited through 30 different patient organisations and social media groups. Participation was voluntary and anonymous, no remuneration was provided. The survey was programmed in Qualtrics . To be included in the study, participants were required to be aged 18 years or over and to give their informed consent. All questions in the survey were mandatory with the exception of the income question, therefore respondents could not proceed to the next question without answering the previous one.Both current own health and the hypothetical health state vignettes were valued by VAS and TTO and by contingent evaluation asking WTP. In the last part of the questionnaire, sociodemographic data, including age, sex, employment, place of residence, net household income and education, were collected.The questionnaire consisted of four parts. The first part included questions about CD-related clinical characteristics, including disease duration, comorbidities and adherence to GFD. This section was built on two earlier national surveys involving CD patients in Sweden and the UK , 16. TheThe following three hypothetical health state vignettes were developed for this study: 1) CD without GFD, (2) CD with loose adherence to GFD and (3) CD with strict adherence to GFD. The vignettes were presented from a second-person perspective. The vignettes were primarily developed based on existing literature reviews , 39–41.  CD withoFor the hypothetical health states, a horizontal VAS was used with the endpoints of ‘the worst health you can imagine’ (= 0), and ‘the best health you can imagine’ (= 100). To measure patients’ own current health, we used the EQ VAS in an earlier section of the questionnaire. Note that the EQ-5D-5L including the descriptive system and EQ VAS were both completed by the patients; however, only the EQ VAS data were used for the present study . The EQ The TTO method elicits utility values for imperfect health states by asking patients to make a trade-off between quality and length of life . We opteU = (10–2)/10 = 0.8. There was no worse-than-dead task in this study, therefore utilities ranged from 0 (being dead) to 1 .Suppose, for example, that a patient indicated to give up 2 years, yielding a WTP measures the maximum amount of money an individual would be willing to pay to be free from their own symptoms or those described in the vignettes . In our U test. WTP responses were converted into a yearly value and then to euros, based on the European Central Bank’s closing conversion rate for February 2021 (EUR 1 = HUF 361.01). Nonsensical WTP responses (e.g. ‘I can not tell’) were excluded from the data analysis. Descriptive statistics  were computed for VAS, TTO and WTP values. The differences in VAS, TTO and WTP values across the patients’ own health and the three hypothetical health states were tested by Friedman test. Predictors of VAS, TTO and WTP values were explored by using multivariate regression models . Insignificant variables were removed from the models by backward stepwise elimination. Before the regressions, a logarithmic transformation was applied to normalise the distribution of the WTP responses. Heteroscedasticity was evaluated by Breusch–Pagan test and corrected by using robust standard errors. For all analyses, a p < 0.05 was considered statistically significant. Data were analysed using Stata 14.0  and R 4.2.0 .To ensure homogeneity and high quality of data, respondents that (1) have not been diagnosed with CD by a physician or (2) filled in the questionnaire in less than eight minutes were excluded. Socio-demographic and clinical characteristics of the patients were analysed using descriptive statistics. The difference in GSRS scores between female and male patients was tested by Mann–Whitney p = 0.001).Out of 734 individuals that opened the questionnaire, 455 62.0%) finished it. Of these, 143 respondents were excluded based on the exclusion criteria .Of the 1248 WTP responses given in the four tasks, 22 (1.8%) nonsensical answers (from 13 patients) were excluded. The mean annual WTP values were €845 ± 1077 for current health, €648 ± 1002 for the ‘CD with strict adherence to GFD’ health state, €862 ± 1135 for the ‘CD with loose adherence to GFD’ health state and €1251 ± 1496 for ‘CD without GFD’ health state. Overall, 79.5%, 68.9%, 84.9% and 87.8% were willing to pay to regain full health. The maximum WTP was €16,620 annually . A total of 20 patients (6.4%) refused to pay for any of the health states, including their own health. Using any of the three methods , there was a statistically significant difference across patients’ valuations for the four health states (p < 0.001), and a 1-year older age at diagnosis with a 0.173 decrease in VAS values (p < 0.05). Concomitant depression and rheumatic disease substantially reduced patients’ VAS values (− 10.373 and − 12.197 p < 0.05). These variables together explained 30.9% of the overall variation in VAS values.The multivariate regressions showed that patients with higher income had significantly higher VAS values (p = 0.006) Table . A one-pp < 0.05) (Table p = 0.005). These three variables explained 8.6% of the variance of TTO utilities.Females and those with higher income were, on average, willing to trade less life years, resulting higher TTO utilities (5) Table . Similarp = 0.022). Patients’ WTP increased by 6.8% with + 1 GSRS score (p = 0.002), by 3.9% with a 1-year increase in age at diagnosis (p = 0.047) and by 251.6% in case of concomitant gastroesophageal reflux disease (p = 0.012) (Table p < 0.001).The amount patients were willing to pay decreased by 70.45% in females compared to males (p < 0.05) (Tables p < 0.05). Having rheumatic or inflammatory bowel disease decreased the patients’ VAS valuations and having higher income resulted in higher TTO and WTP values (p < 0.05). Furthermore, every 1-year increase in the patients’ age increased the TTO utility by 0.002 (p = 0.045). These variables explained 49.0%, 8.7% and 10.9% of the overall variation in VAS, TTO and WTP values, respectively.Both the ‘CD without GFD’ and the ‘CD with loose adherence to GFD’ hypothetical health states were associated with significantly lower VAS and TTO and higher WTP valuations compared to the ‘CD with strict adherence to GFD’ health state  Tables , 5. A + This study aimed to provide VAS, TTO and WTP values in adult patients with CD. To our knowledge, this is the first study to report TTO utilities in CD patients. The health state ‘CD with strict adherence to GFD’ had lower utilities than expected, possibly reflecting the psychosocial aspects of the disease, for example, the patients being prevented from eating with their peers. Older age at diagnosis, male sex, more severe gastrointestinal symptoms and having comorbidities were associated with lower VAS and TTO or higher WTP values for current own health. Compared to the TTO and WTP values, a relatively higher proportion of the variance of VAS values was explained by sociodemographic and clinical predictors. A possible explanation for this observation is that our study did not collect data on some likely predictors of TTO, including cultural values, self-esteem, marital status, having children, religious beliefs and attitudes towards life –56. SimiIt is worthwhile to reconcile our findings with those of previous studies with CD patients. The mean GSRS score in our study (28.3) was in the range of other patient populations on GFD from different countries (21.0–30.4) , 59. FemPreference measurement also helps to shed light on the HRQoL burden associated with a disease. In addition, utilities may be used to calculate quality-adjusted life years (QALYs) in cost-utility analyses, whilst WTP values may be used in cost–benefit analyses of GFD and possible new treatments in the future . PrefereCD without a strict adherence to GFD might result in a sizeable QALY loss at a societal level. Preference-accompanied measures, such as the EQ-5D, may not be able to fully capture the health impact of CD , therefoMany countries offer various forms of reimbursement for CD patients, such as tax reduction , cash transfer , food provision (some provinces in Argentina and Spain), prescription for gluten-free food  and subsidy  to reduce the individual financial burden of GFD , 78. HowThis study has a few limitations that should be noted. First, the questionnaire was administered online and relied on self-reported clinical data that were not verified by physicians. Secondly, selection bias might have occurred as the majority of our study population were from the middle- and high-income social groups, with college or university degrees, and females were somewhat overrepresented in the study population. It is also possible that patients voluntarily filling in such a questionnaire may differ in their clinical characteristics, and thus, the sample may not be representative of the whole population of CD patients in Hungary. In the valuation tasks, three different hypothetical health states were valued; however, the clinical manifestation may vary widely and other CD health states with atypical symptoms could have also been selected. Given that the majority of patients respond to GFD, we only included hypothetical health states that improve after following the GFD. However, a substantial minority may develop persistent or recurrent symptoms even after following GFD . FurtherTo conclude, this study provides a better understanding of the burden of CD by reporting VAS, TTO and WTP values for patients’ current health and three hypothetical GFD-related health states. Utilities and WTP results from the present study may be useful in economic evaluations examining and comparing the value of GFD, screening strategies, subsidies and new alternative treatment options in the future.


As they age, people living with HIV (PLWH) must face new challenges, such as accelerated ageing and higher rates of comorbidities. This study described the characteristics of HIV acquisition among treatment‐naïve PLWH aged ≥50 years and <50 years in Beijing from 2010 to 2020, exploring associated risk factors for comorbidities.t‐test, Mann−Whitney U test and chi‐square test. Temporal trend data were analysed via joinpoint regression. A multivariate logistic regression model was conducted to analyse the associated factors with PLWH having one or more comorbidities.In this cross‐sectional study, differences in HIV‐related and non‐HIV‐related characteristics were compared using the The proportion of PLWH aged ≥50 years has significantly increased since 2013, with a corresponding increase in homosexual transmission in this age group over the past decade. The proportion of individuals with CD4 counts <200 cells/μl significantly decreased from 2010 to 2013 among PLWH aged ≥50 years and from 2010 to 2014 among those aged <50 years. Delayed initiation of antiretroviral therapy (ART) improved for both age groups over the course of the decade, especially from 2014 to 2020. Compared to PLWH aged <50 years, those aged ≥50 years had a higher proportion of CD4 counts <200 cells/μl, higher levels of plasma HIV RNA load and a higher prevalence of non‐HIV‐related risk factors. Multivariate analysis revealed that PLWH aged ≥50, male, not single, transmission through heterosexual contact or drug injection, WHO Stage IV, coinfection with hepatitis B virus/hepatitis C virus and CD4 counts <200 cells/μl at the initiation of ART were associated with higher risk of the presence of an HIV comorbidity.Due to the persistent burden of HIV‐related characteristics or symptoms and the increasing prevalence of coexisting comorbidities among treatment‐naïve PLWH aged ≥50 years, physicians should provide the highest‐quality screening, prevention, treatment and management of coexisting comorbidities, adopting a multidisciplinary approach. HoweverAlthough ART has significantly decreased HIV‐associated morbidity and mortality , AIDS‐reOlder PLWH face other challenges as well, such as accelerated ageing and higher comorbidity rates . A previThis study compared HIV‐related characteristics and comorbidities between ART‐naïve PLWH aged ≥50 years and <50 years to explore the associated factors with one or more comorbidities based on 11‐year surveillance data from Beijing.22.1This was a cross‐sectional study, conducted using data collected from a surveillance system that tracks clinical information on PLWH receiving ART and long‐term follow‐up care at the STD/AIDS Prevention and Treatment Institute of the Beijing Centre for Diseases Prevention and Control (CDC) in China. Clinical data were reported by clinicians in four designated HIV treatment hospitals in Beijing: Peking Union Medical College Hospital, Beijing You'an Hospital, Beijing Ditan Hospital and 302 Military Hospital of China. Our analysis included all treatment‐naive PLWH who initiated therapy at one of these hospitals between 1 January 2010 and 31 December 2020 . The datPLWH aged 18–80 years who initiated ART in Beijing, China, were included in this study. Two age groups were identified: PLWH aged 18–49 years and those aged ≥50 years. Confidentiality was protected for individual information processing. The study protocol was approved by the Research Ethics Committee of the Beijing CDC. The data were based on clinical records that had already been de‐identified, so informed consent was unnecessary.2.22, following the Working Group on Obesity in China  3.90%, 9.19%; p = 0.001). The demographic characteristics of the participants, stratified by age, are shown in Table p = 0.077), with a statistically significant increase over 2010–2016  and a fluctuation from 2016 to 2020. In both age groups, more were Beijing residents than were not. A much higher proportion of PLWH were married or cohabitating in the older group. As with younger PLWH, older PLWH were primarily infected through homosexual contact, followed by heterosexual contact, PWID and other (including unknown). Figure p = 0.008). However, the increase was only statistically significant in the older group . Figure p = 0.280), with a significantly decreasing trend observed from 2010 to 2015  and fluctuations observed from 2015 to 2020.In all, 23,622 PLWH who initiated ART between 2010 and 2020 were included in this study, of whom 2261 (9.57%) were aged ≥50 years, and 21,361 (90.43%) were aged <50 years. Most (72.27%) older PLWH were aged 50–59 years, 20.21% were aged 60–69 years and 7.52% were aged 70–80 years. As shown in Figure The HIV‐related and non‐HIV‐related characteristics of the participants by age are shown in Table p = 0.002), with inflection points in 2013 for the older and 2014 for the younger groups. Both groups experienced a sharp decrease in the initial trend , followed by a stable period. Temporal trends in the rate of delayed ART initiation among PLWH by age group are shown in Figure p = 0.463), and the initiation delay improved for both groups over the decade, especially from 2014 to 2020 . Nevertheless, a difference remained between the two groups, and the proportion of delayed ART initiation was 10% lower in the older group. All the aforementioned trends among treatment‐naïve PLWH in Beijing, China from 2010 to 2020, as determined by joinpoint analysis, are presented in Table Temporal trends by age group in the proportions of CD4 counts <200 cells/μl in treatment‐naïve PLWH are shown in Figure As the timeframe of this study includes year of 2020, when the COVID‐19 epidemic existed, which might  influence the diagnosis and treatment of HIV acquisition due to lockdowns and reduced access to clinical care, a sensitivity analysis was conducted that excluded data from 2020, presenting in Table A significantly higher prevalence of comorbidities was seen in the older group Figure . FurtherNo significant differences were seen in ART selection between older and younger PLWH. Between 2010 and 2020, efavirenz, lamivudine and zidovudine EFV+3TC+AZT) were the most commonly used ART regimens, accounting for approximately 50%. This shifted to EFV+3TC+tenofovir (TDF) from 2013 to 2020; the proportions administered fluctuated but were over 50% at all times  per month in 2017 . HIV casIn our study, although the rate of MSM was lower in the older group than the younger group, it remained the most common route of HIV in this group and was much higher than that found in a cross‐sectional study in Taiwan (49.0%)  and a coLate initiation of ART was defined as having CD4 counts <200 cells/μl or having a clinical AIDS diagnosis at ART initiation . The proNevertheless, older PLWH had a significantly higher proportion of CD4 counts <200 cells/μl, and experienced a slower decrease than the younger group. In addition, the proportion of CD4 counts <200 cells/μl in older PLWH is substantially higher than that reported in a report covering 31 European countries, where the corresponding proportion was 27.4% . AlthougThis study found that older PLWH were more than twice as likely to have one or more comorbidities than younger PLWH at the initiation of ART, similar to older PLWH with access to ART in many Western countries . It is aHIV acquisition and the associated immune activation are independent risk factors for many comorbidities . Our mulThe intersection of ageing‐related and HIV‐related social services and support systems should be enhanced. We strongly recommend that clinicians pay greater attention to comorbidities in older PLWH at the initiation of ART to better prevent and manage further comorbidities. Furthermore, it is necessary for health systems to reconsider care strategies and meet the growing needs of older PLWH. For example, improved screening algorithms are needed to detect metabolic disease in older PLWH. At present, there are no validated CVD risk assessment tools specifically designed for PLWH. The most prominent ones developed hitherto for the general population include the US‐based pooled cohort equations, the Framingham risk functions and the Europe‐based systematic coronary risk evaluation . HoweverOur study had several strengths. First, the data were routinely collected through a standard hospital‐based data‐collection platform, ensuring their validity and reliability. In addition, all subjects were ART‐naïve PLWH; thus, we excluded any adverse effects caused by ART regimens to focus on the effects of ageing and HIV status. However, our study also had notable limitations. First, only already available clinical or demographic parameters were analysed, and some variables had a missing rate of more than 10%, causing us to create a separated “missing” group in the regression model. Second, some comorbidities were not systematically examined, including hypertension, osteoporosis and malignancy, as the information examined was mainly from laboratory data, accumulated during clinical care. However, laboratory data are more reliable than measurements. Finally, the healthy lifestyle factors and socio‐economic status were not included. Future studies should pay closer attention to the more detailed characteristics of PLWH.5Our study highlights a significant increase in the proportion of PLWH aged ≥50 years and the important role of MSM in Beijing's HIV epidemic among older PLWH. In addition, widespread testing and earlier diagnosis should be implemented to address the late initiation of ART among older PLWH. Furthermore, the significant proportion of older PLWH in Beijing with multimorbidities indicates that HIV physicians should provide high‐quality care for screening, preventing and treating age‐related diseases, ideally through a multidisciplinary approach. Finally, under universal health coverage, older PLWH have lifelong access to comprehensive services, prioritizing ending HIV transmission through a strong monitoring system, integrated care and specialist access. This approach provides holistic care, ensuring accessibility and sustainability for older PLWH.DW, MZ, PW, JZ, YM, JL and FC disclose no competing interests.The authors’ responsibilities were as follows: DW wrote the manuscript. MZ contributed to conceptualization, writing, review, and editing. PW, JZ and YM contributed to the statistical analysis, discussion and reviewed/edited the manuscript. MZ, JL and FC were involved in the study design and data collection, contributed to the discussion, and reviewed and critically revised the manuscript.This work was supported by the National Key R&D Program of China during the 14th Five‐Year Plan Period [Project No. 2021ZD0114102], the National Natural Science Foundation of China [Project No. 71874100], Beijing Municipal Science & Technology Commission [Project No. D171100006717002], Incubation Fund of Vanke School of Public Health at Tsinghua University [Project No. 2021PY002] and Sanming Project of Medicine in Shenzhen [No. SZSM202111001].Table S1: Missing rates of each variable and management of missing data.Table S2: Trends in the proportion of older, male, homosexual, heterosexual, CD4 counts <200 cells/µL, and delayed ART initiation among treatment‐naïve PLWH in Beijing, China from 2010 to 2020, as determined by Joinpoint analysis.Table S3: Trends in the proportion of older, male, homosexual, heterosexual, CD4 counts <200 cells/µL, and delayed ART initiation among treatment‐naïve PLWH in Beijing, China from 2010 to 2019, as determined by Joinpoint analysis.Figure S1A: Temporal trend in the proportion of treatment‐naïve PLWH aged ≥50 years in Beijing, China, 2010–2019.Figure S1B: Temporal trend in the proportion of male treatment‐naïve PLWH by age group in Beijing, China, 2010–2019.Figure S1C: Temporal trend in the proportion of transmission through homosexual contact by age group in Beijing, China, 2010–2019.Figure S1D: Temporal trend in the proportion of transmission through heterosexual contact by age group in Beijing, China, 2010–2019.Figure S1E: Temporal trend in the proportions of treatment‐naïve PLWH with CD4 counts less than 200 cells/µL by age group in Beijing, China, 2010–2019.Figure S1F: Temporal trend in the proportion of delayed ART initiation among treatment‐naïve PLWH by age group in Beijing, China, 2010–2019.Click here for additional data file.


Thyroid nodules are common diseases of the endocrine system, with a 5% prevalence rate in the general population. This study aimed to identify prevalence, clinical, cytological and ultrasonographic features of incidental thyroid cancer and its associated factors in Vietnam.This cross‐sectional descriptive study consisted of 208 patients with incidental thyroid nodules detected by ultrasound at the Endocrinology Department, Bach Mai Hospital, Hanoi, Vietnam between November 2019 and August 2020. Clinical information, sonography characteristics of thyroid nodules, results of fine‐needle aspiration biopsy (FNAB), postoperative pathology and lymph node metastasis were collected. A multiple logistic regression model was used to estimate factors associated with thyroid cancer.A total of 272 thyroid nodules (from 208 participants) were included in this study. The mean age was 47.2 ± 12.0 (years). The rate of incidental thyroid cancer patients detected was 17.3%. Nodules <1 cm in size were significantly more prevalent for malignant nodules. The size of more than half of thyroid cancer nodules was 0.50–0.99 cm. Postoperative pathology of all nodules with Bethesda V and VI was papillary thyroid cancer which was consistent with cytological results. 33.3% of thyroid cancer patients have lymph node metastasis. The regression model showed that thyroid cancer was more likely to occur at a younger age , taller‐than‐wide nodules  and hypo‐echoic nodules .The study showed that the prevalence of incidental thyroid cancers was 17.3%, of which 100% was papillary carcinoma. People under the age of 45 and the presence of ultrasound characteristics, such as taller‐than‐wide and hypoechoic nodules increased risk for malignancy. People under the age of 45 and the presence of ultrasound characteristics, such as taller‐than‐wide and hypoechoic nodules increased risk for malignancy. In the absence of accurate clinical signs or predictors of malignancy, many of the nodules will require fine‐needle aspiration biopsy (FNABs), which appears not to be cost‐effective and diagnostic challenges, for example indeterminate and non‐diagnostic cytology. Clinical evaluations and sonographic variables should be used in combination to determine whether the clinician should proceed with further confirmatory tests or with periodical follow‐up.The 5‐year survival rates for thyroid cancers are nearly 100% for localized tumours, 97% for tumours with regional metastases and 57.3% for distant staged tumours.22.1The study was designed as a cross‐sectional investigation including 208 subjects with incidental thyroid nodules, who presented to the Endocrinology Department, Bach Mai Hospital, Hanoi, Vietnam between November 2019 and August 2020.2.22.2.1All subjects had incidental thyroid nodules detected by thyroid ultrasound during routine check‐ups. They had no history, palpable or other clinical signs of thyroid disease and agreed to participate in this study.2.2.2Participants were excluded from the study if they had any of the following conditions: (1) a history of thyroid diseases  (2) known primary cancer of other organs, (3) symptomatic thyroid nodules  (4) missing laboratory results .n = Z21‐ α/2 * [p*(1‐p)/d2], with n = the required sample size, Z1‐ α/2 = 1.96  and d = precision (assumed as 0.05). With p = .15 ,The sample size was calculated using a single population proportion formula: The study was approved by the Ethics Committee of the Bach Mai Hospital Review Board, (Reference Number: 837/BVBM‐HDDD). Written informed consent was obtained from all participants. This study complied with the Declaration of Helsinki.2.32.3.1A questionnaire was designed to collect information from participants, including age, gender, past history , family history , familial adenomatous polyposis (FAP), Gardner syndrome, Cowden syndrome, Carney). A resident doctor was trained to collect the data.2.3.2All participants were taken blood to measure FT4 and TSH by Electrochemiluminescence immunoassay (ECLIA) at the Department of Biochemistry, Bach Mai hospital. The normal range of FT4 and TSH is 12–22 pmoL/L and 0.27–4.2 μU/ml, respectively. Thyroid function was classified according to ATA 2016.Thyroid ultrasonography was conducted with a Korean Samsung Medison ultrasound system (Model UGEO G60 ‐ USS‐G60NF40/WR) with a 7,5 MHz probe. The ultrasound technique was standardized to include transverse and longitudinal images of both lobes of the thyroid gland as well as the adjacent structures in the neck. All nodules were measured in 3 planes, and hard copy images were created to document nodule location and orientation. Thyroid nodules were characterized by the number, location, echogenicity, margin and shape of nodules. Thyroid nodules were classified according to the American College of Radiology Thyroid Imaging Reporting and Data Systems (ACR‐TIRADS) 2017.* Ultrasound‐guided fine‐needle aspiration (US‐guided FNA) was performed with a 25‐gauge needle and a 5‐mL disposable syringe. The sterile gel was used as the coupling agent, and the needle was inserted along a path within the scanning plane so that the nodule and the needle were continuously visualized. When the needle reached the target, aspiration biopsy was performed with an in‐and‐out movement while suction was applied. Suction was released before the needle was removed from the nodule. The collected material was expelled onto glass slides, fixed immediately by a trained cytotechnologist and placed into a CytoLyt solution for centrifugation. FNA results were interpreted by pathologists at the Pathology Center, Bach Mai Hospital, and classified by Bethesda 2018.* The thyroid ultrasound and US‐guided FNA protocols were developed, standardized and trained for three experienced and well‐trained endocrinologists at Endocrinology Department, Bach Mai Hospital. After training, these physicians were re‐tested before starting the study to confirm the results were similar. Each patient had an ultrasound and FNA biopsy by one of these three endocrinologists.All patients with Bethesda V and VI were performed thyroidectomy in three hospitals, which were Bach Mai Hospital, Vietnam National Cancer Hospital and National Hospital of Endocrinology. The postoperative pathology results were evaluated using the WHO 2017 classification.2.4t‐tests. Univariate logistic regression was performed on general factors and other potential factors that can be associated with thyroid cancer based on the literature such as the size of a thyroid nodule, and ultrasonographic features. Only variables that had a p‐value <.2 on univariate analysis were selected for multivariate analysis. Multivariate analyses were conducted with logistic regression, the results were presented as an odds ratio (OR) with a 95% confidence interval (CI). All values of p < .05 were considered statistically significant.The analysis was performed with SPSS 20. Descriptive statistics were used: continuous variables were presented as mean ± standard deviation (SD) and categorical variables as frequency and percentage. Proportions were compared by using Chi‐squared tests with continuity correction or Fisher's exact test when appropriate, continuous variables were compared by using Student's 3A total of 272 thyroid nodules (from 208 participants) were included in this study. The mean age was 47.2 ± 12.0 (years). The proportion of patients with thyroid cancer detected was 17.3%. The rate of thyroid cancer in the age group ≤45 (years) was statistically significantly higher than that in the age group >45 (years) (Table p < .05). More than half of thyroid cancer nodules were 0.50–0.99 cm in diameters. Malignant nodules were more likely to be solid , taller‐than‐wide (33.3% vs. 3%), hypo‐echoic , microcalcification  and blurred margins  nodules.The sonographic characteristics of those with cancer versus non‐cancer ones were compared in Table Cytological characteristics of FNA, postoperative pathological and lymph node metastasis are shown in Table Of those having surgery, 33.3% of thyroid cancer patients had lymph node metastasis. According to the AJCC 8th classification of differentiated thyroid cancer, there are 35 patients with stage I, and 1 patient with stage II.On multiple logistic regression analysis Table , thyroid4To our knowledge, the current study is the first work to mention incidental thyroid nodules in a hospital‐based study in Vietnam. The results showed that the rate of thyroid cancer was 17.3% out of 208 patients with thyroid nodules detected incidentally on ultrasound. Thyroid cancer patients were mostly aged ≤45 and most of the nodules with diameter ranged from 0.50 to 0.99 cm. The sonographic features of nodules associated with a high risk of malignancy were those with taller‐than‐wide dimensions and hypo‐echogenicity.The incidence of malignancy in incidental thyroid nodules varies depending on the diagnosis technique being computed tomography (CT)/magnetic resonance imaging (MRI), ultrasound or positron emission tomography (PET), ranging from 5% to 13%,Age is an important prognostic factor of differentiated thyroid cancer as outlined in classification systems such as TNM, AMES and MACIS.Many studies have attempted to show that certain sonographic features are associated with a higher risk of malignancy, especially in nonpalpable thyroid nodules to avoid unnecessary FNA or surgery.Nonpalpable thyroid nodules are usually small in size. According to American Thyroid Association 2015, FNA is recommended for those nodules ≥1 cm and TIRADS 4, 5.In our study, cytological and histopathology results were 100% papillary thyroid carcinoma (PTC), possibly due to the size of nodules and ethnicity. Pamela L. etThere are some strengths and limitations to our study. This has been the first study in Vietnam to discover random thyroid nodules. We found it possible to use ultrasound—a cost‐effective and easily accessible tool—to detect thyroid cancer with great accuracy and relevance in developing countries. However, this was a cross‐sectional study that could not show a causal relationship between risk factors and outcomes. Therefore, it is essential for further studies to explore these issues.5In conclusion, the current study emphasizes that incidental thyroid cancers were not uncommon with a prevalence of 17.3%, in which 100% was papillary carcinoma. People under the age of 45 and the presence of ultrasound characteristics, such as taller‐than‐wide and hypoechoic nodules increased risk for malignancy. Ultrasound imaging could be used to assist in the clinical risk assessment of thyroid cancer.Bay Quang Nguyen: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Hau Thi Tran: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Huong Thi Thu Nguyen: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; validation ; visualization ; writing – original draft ; writing – review and editing . Thanh Xuan Nguyen: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Thu Thi Hoai Nguyen: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Tam Ngoc Nguyen: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Huyen Thi Thanh Vu: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectorsThe author reports no conflicts of interest in this work.


Atelopus zeteki; PGFs) likely extirpated from the wild, ensuring long-term sustainability of captive populations is crucial in order to conserve this critically endangered species. Unfortunately, PGFs display a unique reproductive behavior involving a prolonged period of amplexus leading to challenges in their successful captive propagation. The Maryland Zoo in Baltimore has observed high levels of mortality during the breeding season and suboptimal reproductive success leading to the use of hormone stimulation to aid in reproduction and health management.With Panamanian golden frogs (n = 174) were given GnRHa either in various concentrations (Experiment 1) or combined with metoclopramide (Experiment 2), and oviposition success, latency, and mortality were measured as binary response variables.This project aimed to develop induced ovulation and health management protocols by (1) evaluating different doses of gonadotropin releasing hormone analogue (GnRHa), (2) comparing the efficacy of GnRHa and GnRHa + metoclopramide, (3) determining latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Female PGFs (Overall, the use of exogenous hormones significantly decreased mortality when compared to the control data of natural egg-laying females. GnRHa doses of 0.05 μg/g body weight produced similar ovulation rates compared to higher doses, and the addition of metoclopramide did not increase oviposition success compared to GnRHa alone. Lastly, results indicate the majority of female PGFs will release eggs within 48 h following the initial pulse of hormones with a small percentage ovipositing after a second pulse.Findings from this study will benefit captive management of PGFs by documenting the increased survival of females when given hormone stimulation and defining appropriate GnRHa doses and expected latency to spawning. Atelopus zeteki) is a critically endangered amphibian [Batrachochytrium dendrobatidis (Bd) [pers comm.), and with the PGF population presumed to be exclusively in captivity, maximal reproductive output with optimal genetic sustainability and minimal risk to existing individuals is crucial for species survival. Management efforts to promote captive breeding capitalize on knowledge of natural breeding behavior and reproductive cycles [Atelopus species display a unique reproductive behavior, in that amplexus is initiated reliably by the male and lasts for extended periods of time, sometimes up to 60 days or more [The Panamanian golden frog  –6. In 20dis (Bd) , 8. The e cycles , 10; howe cycles , 11. Sev or more , which i or more . In capt or more –16. Thes or more , and thrThe high level of morbidity and mortality in the captive population of PGFs primarily occurs during the breeding season, which appears to be related to the failure of gravid females to undergo oviposition , 16. BetNatural oviposition results from environmental stimuli triggering the neuroendocrine cascade of the hypothalamus-pituitary-gonad axis (HPG-axis), whereby endogenous gonadotropin releasing hormone (GnRH) causes release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) , 28, 29.Lithobates pipiens) [Lithobates sevosa) [Determining the optimal hormone regimen is a critical step for promoting gametogenesis and spawning, but responses to specific hormones, their combinations, or doses vary widely across species , 34. Forpipiens) , 38 and  sevosa) , as well sevosa) . The add sevosa) , 38. AltAnaxyrus baxteri) [Anaxyrus fowleri) [Pseudophryne guentheri) [Pseudophryne corroboree) [Peltophryne lemur) [(Lithobates chiricahuensis) (Kouba per. communication). Previous studies by our lab on hormone priming effects on spawning rates [Depending on the species and the stage of gametogenesis, a single exogenous hormone stimulation can often successfully induce ovulation. However, there are amphibian species in which priming or pulses of hormones are required to facilitate the spawning process. The beneficial impacts of primed or pulsed hormones on increased spawning rates have been described for Wyoming toad (baxteri) , Fowler’fowleri) , Güntherentheri) , corroboroboree) , dusky groboree) , 40, Puee lemur)  and Chirng rates , 25, 40 ng rates . A seconThe goal of this study was to develop an effective hormone protocol for induction of ovulation and spawning in captive female PGFs that serves to both enhance assisted breeding as well as alleviate the risk of death from egg binding. To address both these issues, the objectives of this study were to: (1) evaluate different doses of GnRHa, (2) compare the efficacy of GnRHa and GnRHa + MET, (3) determine latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Since the MZIB’s breeding program uses hormone stimulation as a method for maintaining both the health and reproduction of female frogs, this study focused primarily on number of spawning females and survival as the end goal and does not attempt to quantify the effects different hormone regimens have on clutch size, fertilizing capacity, or larval development.n = 431) allowed to breed naturally. Naturally breeding females were those that spawned without hormones in the first 2 weeks of the natural breeding period or which never received hormones and were allowed to continue natural breeding past the 2-weeks for up to 60 days. These extended natural breeding instances occurred primarily in the early breeding seasons 2009–2011. In total, 102 females laid eggs without hormone therapy in this 7-year time frame. Within the group of females that were amplexed by a male and naturally spawned, 23% laid eggs within 24 h, 45% within 1–7 days, 22% within 8–14 days and 10% past 2 weeks. For the majority of natural spawning incidences, 80% of those cases occurred during the early breeding years between 2009 and 2011. Only eight females laid eggs naturally within the first 2 weeks of amplexus for the two seasons spanning 2014–2016, when our two experiments were conducted. We observed a decreasing trend in reproductive capacity over time from 2009 to 2016, possibly due to extended time in captivity or hatching of new animals into captive settings with no residual knowledge of natural stimuli. Females that did not naturally spawn within the first 2 weeks of amplexus, during the 2014–2016 reproductive season, were used for hormone-induced ovulation Experiments 1 and 2 described below.The control data set was obtained from historic PGF breeding records collected between 2009 and 2016 on all females , 48.0% (n = 12/25), 59.1% (n = 13/22), and 45.0% (n = 9/20) when given GnRHa concentrations of 0.05, 0.1, 0.2, and 0.4, μg/g BW, respectively  = 2.406, p = .49). Although nearly 20% more females spawned when given 0.05 compared to 0.4 μg/g BW, the effect was not significant  = 1.773, p = .18). Overall, hormone therapy using GnRHa at any concentration between 0.05–0.4 μg/g BW can induce spawning above the rate of untreated controls, and with equal efficacy.The efficacy of GnRHa at four concentrations was tested to determine the optimum dosage needed to achieve efficient spawning in the PGF. Females laid eggs at a response rate of 65.2% n = 5/23, 48.n = 90) and compare to the control (n = 431), where animals were allowed to naturally spawn for 2 weeks. Overall, treatment of female PGFs with GnRHa elicited a significantly higher  = 34.27, p = .00002) spawning response compared to natural spawning controls (Table n = 70/90) or 4 , upon being paired with a male. No effect of gravidity  = 1.029, p = .31) between grades 4 or 5 was found for ovipositing females in response to hormone treatment.There was no significant difference between the various GnRHa treatments on number of spawning females, which allowed us to combine all hormone treatment animals together  = 0.742, p = .86), such that 47.8% (n = 11/23), 40.0% (n = 10/25), 50.0% (n = 11/22), and 40.0% (n = 8/20) of all females laid eggs when given GnRHa dosages of 0.05, 0.1, 0.2, and 0.4 μg/g BW, respectively. The second pulse of GnRHa (at the same concentration) induced a smaller proportion of the remaining females (n = 50) to oviposit, specifically 33.3% (n = 4/12), 13.0% (n = 2/15), 18.2% (n = 2/11), and 8.3% (n = 1/12), and also with no effect of concentration  = 2.893, p = .41). In addition, we examined the response based on the cumulative amount of hormone received from all pulses and also found no difference  = 5.544, p = .24), although a general inverse-trend was observed. For instance, the proportion of females ovipositing decreased  as the cumulative amount of GnRHa , required to induce oviposition, increased , data not shown. Thus, spawning can be induced using the lowest tested GnRHa concentration of 0.05 μg/g BW (1 or 2 pulses); whereas, higher concentrations of GnRHa, either as 1 or 2 pulses, did not increase the overall percent of the population that will lay eggs.The overall spawning rate includes females receiving either one or two pulses of hormone at the given concentration of GnRHa Table . The span = 8/9) dropped eggs after the first pulse, compared to 73.3% (n = 11/15) when given 0.05 μg/g  = 0.551, p = .46) (Table n = 8/49) or laid eggs naturally ,  = 0.599, p = .44). Overall, females have the highest risk of mortality if they do not lay eggs and have been in amplexus for at least 3.5 weeks, regardless of hormone treatment. Interestingly, females that did not lay eggs and were not hormonally treated after 2 weeks of amplexus, had a significantly higher mortality rate  = 26.949, p < .00001) than females that were treated with GnRHa, and did not lay eggs. Furthermore, within the GnRHa treated group, mortality was similar whether females oviposited  or not ,  = 2.163, p = .14). Moreover, the mortality rate was similar for frogs treated with any of our GnRHa concentrations tested, regardless of number of pulses  = 0.982, p = .80). Post- spawning deaths ranged from 9.1–25.0% regardless of GnRHa concentration. Females that did not oviposit at all had the highest proportion of fatalities across all GnRHa treatment groups, 37.5, 23.1, 22.2 and 36.4% for 0.5, 0.1, 0.2 and 0.4 μg/g BW respectively, but there was no significant effect of concentration  = 1.028, p = .79). As a result, the chances of a female frog dying is independent of whether the female was hormonally treated, the concentration administered, or the number of pulses given, while the reward of increased spawning using hormone therapy is significant.Finally, we compared the rate of mortality across GnRHa concentrations and the number of hormone pulses administered to determine if there was an unforeseen risk in using hormone concentrations in this range compared to control. The mortality rate for hormone treated females was no different than the background mortality of non-treated females (χn = 50) or GnRHa + MET (n = 34) there was a higher proportion of spawning than the background control rate of natural breeders  = 15.460, p = .00008). Interestingly, there was no additional effect of adding MET to the GnRHa,  = 0.029, p = .86) compared to spawning numbers for GnRHa alone (Table n = 18/50) of all treated females to release eggs, compared to 26.4% from GnRHa + MET (n = 9/34). The second pulse of GnRHa caused 10.0% (n = 5/50) more females to spawn while a second pulse of GnRHa + MET induced an additional 17.7% (n = 6/34) to spawn. Importantly, over half of all females failed to spawn, even after two pulses of exogenous hormones at these concentrations (mean GnRHa = 0.25 μg/g BW), which is in line with the spawning rate observed in Experiment 1 for a GnRHa dosage between 0.2–0.4 μg/g BW. Overall, adding the dopamine antagonist MET did not result in any more female PGFs to spawn beyond the response rate of GnRHa alone.We tested if there was an effect of adding 150 μg of MET to 4 μg GnRHa on either oviposition or mortality rates of female PGFs compared to GnRHa alone. All females in this experiment were a grade 5 on the gravidity scale. Overall, we found that of the 84 female frogs treated with either GnRHa ( MET n = 4 there wn = 23) did so at a constant rate over time, with half of all remaining females that were going to spawn doing so at each 24 h period. Specifically, 52% of all spawning females oviposited < 24 h, the next 26% from 24 to 48 h, the next 13% between 48 and 72 h and the final 9% of spawning females laid between 72 and 96 h. No females responded after 96 h from the initiation of hormone therapy. Overall, 78% of all GnRHa responsive females laid eggs after the first pulse, and the second pulse induced the final 22% of laying females to oviposit. By contrast, the latency to ovipositon with GnRHa + MET was more evenly distributed over time such that the proportion of females laying in each 24 h period was 33, 27, 13 and 27%, respectively. Only 60% of all laying females responded to the first pulse of the GnRHa + MET compared to 78% with GnRHa alone, which was significantly lower  = 7.574, p = .0059) when considered on a proportional basis  = 0.779, p = .67),  = 4.420, p = .109). Thus, there is no apparent detrimental effect to female frogs’ health with respect to the addition of MET to the GnRHa treatment, nor with respect to the number of pulses that included additional MET. As seen previously  = 17.155, p < .0002) if the females were give GnRHa alone or with MET, compared to those left untreated.Compared to the background mortality rate (18.0%), there was no effect to adding the MET (χ), Table . MoreoveWith the presumptive disappearance of the wild population of Panamanian golden frogs in the past decade, having a sustainable captive population is critical to the long-term survival of the species, especially with the goal to eventually release animals back into the wild with maintained genetic diversity. Thus, the decline in natural reproduction over the last decade and high mortality rate during the breeding season has necessitated the use of exogenous hormones to stimulate spawning in PGFs. Results from this study reveal that GnRHa significantly increased the number of spawning females compared to those that were allowed to breed naturally prior to hormone administration. Moreover, of the GnRHa concentrations tested, all induced spawning with similar response rates, and combining GnRHa with MET did not increase the number of spawning females compared to GnRHa alone. Fortunately, the hormone treatments did not present higher levels of mortality compared to background rates that occur during natural spawning. Although we did not find that hormone treatments were a panacea for inducing all females to spawn or for reducing mortality loss when females laid eggs, it does appear to make a significant difference in non-spawning female survivability. Additional research is needed to increase overall spawning rates, but hormone treatments may significantly decrease mortality during the breeding season of egg-bound females. Altogether, the hypotheses that increasing GnRHa concentrations, supplementing with MET, or administering additional pulses of hormones would induce substantially more females to ovulate over time, were rejected. These findings provide new knowledge on female PGF reproductive physiology and impact best management practices for the captive propagation and conservation of this critically endangered species.Oviposition depends on the degree of oocyte development, which is a function of species-specific biotic and abiotic cues related to the environment, nutritional plane, hibernation stimuli or combinations of these factors. The stages of oogenesis , can be Female PGFs are thought to be ready for breeding when they reach grade 5 based on anecdotal observations, reflecting a prominent egg mass of large oocytes that significantly displace the female’s organs by filling a majority of the abdomen, which becomes distended . SimilarAnaxyrus boreas boreas) [This is the first study to determine if a dose response relationship was present between increasing concentrations of GnRH and spawning rates in PGFs, with the goal of developing an optimized breeding protocol. Findings from this study suggest a dose of 0.05 μg/g BW GnRHa induces oviposition at comparable rates as higher doses in female PGFs. Similar low doses of GnRHa (0.1 μg/g) have successfully induced ovulation in several Bufonidae species including the Puerto Rican crested toad  and bore boreas) . However boreas) , 22, 36.The mechanistic response to GnRHa and initiation of the hormone cascade depends on the number, and binding efficiency, of hormone receptors and their patterns of expression, which may vary across amphibian species. Several investigators studying hormone response in amphibians , 34, 45 n = 5.7, median = 5.6) and we distributed older age females  evenly across treatments with all individuals having similar body weights (16.3 ± 0.5 g). Unlike the boreal toad, the PGF response to exogenous hormones appears to be independent of female age, weight or previous spawning history, similar to reports of hormone response in the dusky gopher frog [Roth et al. 2010) found that age, body size, and condition were all important factors that influenced the success of GnRHa effectiveness in induced spawning by boreal toads . To deteher frog , the sou10 found her frog . HoweverSequential administration of exogenous hormones has typically been discussed in terms of priming for improved fecundity and spawning rates of female amphibians , 26, 46.We observed that a second pulse of GnRHa induced another ~ 10% of the treated females to lay eggs up to 96 h later, resulting in an overall ovulation rate of ~ 54%. While not significant, more females who were administered a lower concentration of GnRHa responded to the second pulse compared to the highest concentration, 17% vs. 5%, respectively, possibly suggesting receptor down regulation and desensitization at the higher concentrations. Anecdotal evidence is widely available within zoo and aquarium captive breeding programs, where multiple pulsed injections are given to female anurans; if they do not spawn on the first resolving dose it is a low probability that they will spawn after the second pulse, and almost no animals spawn after 3 hormone pulses. Although several papers have been published on the benefits of priming hormones  for femaThe purpose of supplementing GnRHa with MET, is to reduce the dopamine inhibition of the GnRH-induced release of LH and FSH to the gonads, effectively increasing the efficacy of GnRH binding. Previous studies on Northern leopard frogs advocated that the addition of MET to GnRHa promoted ovulation in females; however, there was no direct comparison to the GnRHa alone to determine the effect of MET supplementation , 38. A dPelodryadid tree frogs (spp. Litoria). Female PGFs ovulate and spawn moderately well (> 50%) when treated with GnRHa alone, similar to other species within the Bufonidae family including Anaxyrus fowleri, A. baxteri, A. americanus, A. houstonensis, A. boreas boreas, and Peltophryne lemur [pers. comm. Diane Barber) and thus reflect response patterns observed in the Australian ground frogs. Spawning and ovulation rates have been significantly advanced in recent years in several species of the family Bufonidae by selecting only females with advanced mature oocytes through ultrasound visualization [Interestingly, male PGFs produced greater sperm concentrations when given GnRHa + MET compared to GnRHa alone . Howeverne lemur , 27, 49 lization , 43, 44.In addition to developing a standardized hormone protocol for spawning in PGFs, the following study was valuable in that it evaluated whether or not mortality during the breeding season could be reduced following treatment with GnRHa and oviposition. Hormone treatment did not appear to lower mortality rates beyond the established background rates for natural breeding when females successfully spawned; however, hormone treatment did decrease the mortality rate for females that did not oviposit after spending weeks in amplexus with a male compared to females that were not hormone treated. This study yielded a mortality rate between 18 and 30% regardless of GnRHa treatments or combination with MET, which was statistically different from background rates (89%) during natural breeding for non-ovipositing females. Of those females that were hormone treated, those that laid eggs had a mortality rate of 16% while 29% of those that didn’t lay eggs died after a month or more in amplexus, possibly due to egg-binding. These results suggest there was a benefit to stimulating females to lay eggs and that spawning provided some protection from egg binding and death. Sadly, very few studies using reproductive hormones report whether mortalities were observed, so we have little to compare our results within the published literature. Future research is needed to determine if additional hormones (e.g. oxytocin) or combinations of hormones can increase spawning rates above 60%, while decreasing mortality further.PGFs exhibit a unique reproductive ecology with pairs remaining in amplexus for long periods of time , which ipers. comm.). In captivity, female PGFs begin visible oogenesis around August, males will begin amplexus around October and egg laying can extend from November through May [Between 2001 and 2003, a North American breeding population of PGFs was established with the importation of 19 males, 19 females, and 12 juveniles of unknown gender. The Maryland Zoo in Baltimore has been intensively involved in the reproduction of this species, having produced > 2000 offspring that have been distributed around the country. PGFs managed in captivity are split into two closely managed populations by locale and phenotype and are managed by a Species Survival Plan (SSP). According to early field studies, females lay on average ~ 370 eggs in strings on the underside of rocks or underwater cavities in swiftly flowing streams . Breedinough May .n = 174: age ≥ 2 years) from the F1-F3 generations of captive born PGFs. During the breeding season, females were regularly checked for egg development and added to the study groups as they reached a level 4–5 on the gravidity scale as previously described [3) with a water depth of 30 cm and an elevated moss-covered platform (60 × 25 cm2). Terra cotta pots and faux plants provided cover on land and provided egg-laying sites under water. All paired frogs were allowed to breed naturally, and remained in amplexus until either the female oviposited or 2 weeks had passed. Females that do not naturally release eggs within 2 weeks are then administered exogenous hormones while the breeding pair remains in amplexus. There was no predetermined sample size for either experiment as the number of frogs undergoing hormone stimulation was unknown until female frogs developed adequate gravidity, and after the conclusion of the natural breeding attempt. All animals were returned to their standard enclosures after the end of the experiment. The MZIB’s Institutional Animal Care and Use Committee (IACUC) reviewed and approved all experimental procedures.Experiments were conducted at the MZIB during the 2014–15 and 2015–16 breeding seasons with adult female frogs  that did not oviposit during the 2-week period of natural breeding were randomly assigned to one of four GnRHa treatment groups, 0.05 (n = 22), 0.1 (n = 25), 0.2 (n = 23), and 0.4 (n = 20) μg/g BW. The first hormone pulse was given in the morning and a second hormone pulse given 48 h later to any females that had not oviposited. Following each hormone pulse, breeding pairs were allowed to spontaneously spawn and tanks were checked daily for eggs. Breeding pairs that spawned were allowed to remain in amplexus until they naturally separated shortly after egg-laying was completed. Following the second hormone pulse, breeding pairs were allowed to remain in amplexus for up to 7 days. If spawning did not occur, they were manually separated, and individuals were closely monitored over the next 30 days for negative signs of health or mortality.Experiment 1 was conducted to evaluate the optimal GnRHa concentration based on body weight (BW) needed to induce oviposition. Females (n = 84/group) following 2 weeks of natural breeding attempts. Females were administered either 4 μg GnRHa (Treatment 1) or 4 μg GnRHa + 150 μg MET (Treatment 2) via intracoelomic injection while still in amplexus with the male as described above. For both treatments, the same concentration of hormone was applied to each female regardless of body weight in order to evaluate efficacy under standard breeding conditions; weight varied little for the females in our study. Using the average weight of females in the study, MET was administered at approximately 9.2 μg/g BW and GnRHa at 0.25 μg/g BW, similar to published recommendations [Experiment 2 compares the efficacy of GnRHa alone to GnRHa + MET for inducing oviposition in female PGFs. PGF females (avg. weight = 16.3 ± 0.5 g) were randomly assigned to a treatment group , hormone concentration (0–0.4 μg/g BW), the number of hormone pulses needed for a response (1 or 2), and latency period . The latency period references the time since the initial hormone administration to oviposition and consisted of five categories: females that laid eggs in ≤24 h, 25–48 h, 49–72 h, 73–96 h, and 96 h to 7 days after initiation of hormone therapy. Females in the last three latency categories received a second pulse of the designated hormone treatment, thus latency in the present study is a categorical reference rather than a true time response. Only one female laid between 2 and 7 days after the second pulse in any experiment, so this category for latency was not included in the statistical analysis. Lastly, mortality refers to females that died within 30 days of the last hormone treatment received.2) reported as: χ2  = χ2 statistic value, p = p value, with results considered significant at an α <  0.05. Effects were evaluated against the control and between treatments. The control data was obtained from records collected between 2009 and 2016 on breeding females that were not treated with hormones, and females that spawned during the 2-week natural breeding period and/or died prior to hormone therapy. A Yates-correction was used in the analysis to account for the high number of females in the non-hormone treated reference set. In Experiment 1, which tested efficacy of GnRHa at 4 different concentrations, a 2 (observed binary responses) X 4 (treatments) contingency analysis was applied for each parameter: total females ovipositing, hormone pulse, and latency category, for each response variable . Similarly, data in Experiment 2, which examined the effect of GnRHa + MET, was analyzed using a 2 X 2 contingency table for oviposition and a 3 × 2 contingency table for mortality. Statistical analysis was conducted using program R and JMP version 14.3.Response data was analyzed using a two-tailed (N-1) Chi-squared test (χ


Bifidobacterium genus as test case. Strain tracking analysis based on 1664 shotgun metagenomics datasets of healthy infant faecal samples were employed to identify bifidobacterial strains suitable for in silico and in vitro analyses. Subsequently, an ad hoc bioinformatic tool was developed to screen local strain collections for the most suitable species‐representative strain alternative. The here presented approach was validated using in vitro trials followed by metagenomics and metatranscriptomics analyses. Altogether, these results demonstrated the validity of the proposed model for reference strain selection, thus allowing improved in silico and in vitro investigations both in terms of cross‐laboratory reproducibility and relevance of research findings.The genomic era has resulted in the generation of a massive amount of genetic data concerning the genomic diversity of bacterial taxa. As a result, the microbiological community is increasingly looking for ways to define reference bacterial strains to perform experiments that are representative of the entire bacterial species. Despite this, there is currently no established approach allowing a reliable identification of reference strains based on a comprehensive genomic, ecological, and functional context. In the current study, we developed a comprehensive multi‐omics approach that will allow the identification of the optimal reference strains using the  Historically, the term ‘type strain’ is employed to denote bacterial clonal descendants of the first isolated member of a novel species, also indicated as ‘nomenclatural type’ ’ based on the closest genomic relative to the newly assigned reference bifidobacterial strains. The validity of the identified novel representative bifidobacterial model strains in terms of microbe–microbe and microbe–host interactions was investigated by means of metagenomic and metatranscriptomic methods. The latter approaches were applied using in vitro models simulating real‐life settings involving (i) co‐occurrence with complex bacterial communities encompassing other members of the infant gut microbiota and (ii) molecular interactions with human intestinal cells.All publicly available datasets corresponding to infant faecal samples sequenced through a shotgun metagenomics approach were selected and downloaded from the NCBI SRA repository using NCBI SRA Toolkit 2.11.0 faster‐dump . Collected filtered reads were then taxonomically classified through the METAnnotatorX2 pipeline  using the fastq‐mcf script  gellan gum, 0.25% (w/v) xanthan gum, and 0.2% (w/v) sodium citrate, as previously described  was added to the medium  with a working volume of 400 ml inoculated with 40 ml (10% v/v) faecal beads. Fermentation started in a batch mode by aseptically replacing spent medium with fresh medium every 12 h for 3 days, after which the fermentation was switched to a continuous mode by feeding the bioreactor with fresh medium at flow rates of 66 ml hB. adolescentis 713B, B. bifidum PRL2010, B. breve 1895B, B. dentium 181B, B. longum subsp. longum 39B and B. pseudocatenulatum 1896B, as well as the type strains and the identified sub‐optimal strains for each selected bifidobacterial species were in batch cultivated in a complex medium mimicking the infant intestine  (Concept Ruskinn) at 37°C.Specifically, bifidobacterial strains were revitalized from glycerol‐based stock in MRS broth medium supplemented with 0.05% (w/v) Each aliquot of every single obtained batch fermentation, together with the control sample, that is, the 10‐day stabilized infant gut microbial community were subjected to DNA extraction using the QIAmp DNA stool mini kit following the manufacturer's instructions . The extracted DNA was prepared using the Illumina Nextera XT DNA Library Preparation Kit and following the Illumina NexteraXT protocol. Specifically, DNA samples were enzymatically fragmented, barcoded, and purified using magnetic beads. Subsequently, samples were quantified using a fluorometric Qubit quantification system , then loaded on a 2200 Tape Station Instrument  and normalized to 4 nM. Paired‐end sequencing was performed using an Illumina MiSeq sequencer with MiSeq Reagent Kit v3 . Taxonomic reconstruction of shallow shotgun data was performed as described above for shotgun metagenomic data analysis.For total cell counts, each culture replicate was 100,000 times diluted in physiological solution (phosphate‐buffered solution [PBS]). Subsequently, 1 ml of the obtained bacterial cell suspension was stained with 1 μl of SYBR®Green I  , vortex‐mixed and incubated at 37°C in the dark for at least 15 min before measurement. All enumeration experiments were carried out through an Attune NxT flow cytometry  equipped with a blue laser set at 50 mW and tuned at an excitation wavelength of 488 nm. Multiparametric analyses were performed on both scattering signals, that is, forward scatter (FSC) and side scatter (SSC), while SYBR Green I fluorescence was detected on the BL1 530/30 nm optical detector. Cell debris was excluded from acquisition analysis by setting a BL1 threshold. In addition, the gated fluorescence events were evaluated on the forward‐sideways density plot to exclude remaining background events and to obtain an accurate microbial cell count. All data were statistically analysed with the Attune NxT flow cytometry software.−1) and 10 mM of sodium pyruvate, respectively, as previously described  was seeded in DMEM + FBS at a density of ≈105 cells cm−2 into cell culture inserts with membrane filters (pore size 0.4 μm) for Falcon 24‐well‐multitrays , and cultured for 21 days with a medium replacement every 3 days until a tight monolayer was formed (TEER > 600 Ω • cm2).Human colorectal carcinoma‐derived Caco‐2 cells (purchased from ATCC) and HT29‐MTX , that is, a human colon carcinoma‐derived, mucin‐secreting goblet cell line, were cultured in Minimum Essential Medium (MEM) and Dulbecco's Modified Eagle's medium (DMEM) with high glucose (4.5 g L8 cells ml−1) were added to Caco‐2/HT29‐MTX cell monolayers, as previously described , cells were enumerated through Thoma cell counting chamber (Herka), possibly diluted to reach a final concentration of 108 cells ml−1, washed in PBS, resuspended in DMEM without antibiotics, and seeded on Caco2/HT29‐MTX cell monolayers. Furthermore, for each bifidobacterial strain, a control sample, that is, the bifidobacterial strain resuspended in DMEM and maintained under the same incubation conditions of the 24‐well plates without any contact with human cell lines, was obtained.For these experiments, each bifidobacterial strain, that is, Total RNA of each considered condition was isolated using a previously described method  was treated to remove rRNA by using the QIAseq FastSelect—5S/16S/23S following the manufacturer's instructions (Qiagen). The yield of rRNA depletion was checked by using a 2200 TapeStation . Then, a whole transcriptome library was constructed using the TruSeq Stranded mRNA Sample preparation kit (Illumina). Samples were loaded into a NextSeq high‐output v2 kit (150 cycles) (Illumina) as indicated by the technical support guide. The obtained reads were filtered to remove low‐quality reads  as well as any remaining ribosomal loci using the METAnnotator X2 pipeline , consisting of metagenomic data sets of faecal samples from individually selected healthy infants, ranging in age from a few days to 3 years, sequenced through Illumina‐based technology in order to avoid any bias related to different sequencing platforms Figure  Table S Table 1.B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. longum, B. dentium and B. pseudocatenulatum were shown to be the most representative Bifidobacterium species across the IGMC, confirming previously published data  to reconstruct (near complete) genomes. This procedure allowed the reconstruction of 239 bifidobacterial metagenomes assembled genomes (MAGs), including 105, 47, 30, 19, 16, 13, and nine chromosomal sequences belonging to the B. longum, B. bifidum, B. breve, B. pseudocatenulatum, B. dentium, B. adolescentis, and B. catenulatum species, respectively , in total encompassing 1297 bifidobacterial chromosomes of the abovementioned key microbial players of the infant gut microbiota Figure . This daB. longum, B. bifidum, B. breve, B. adolescentis, B. pseudocatenulatum, B. dentium and B. catenulatum species, respectively, for a total of 490 bifidobacterial genomes showing a prevalence >0.1% across the IGMC datasets   B. longum subsp. longum ATCC 15707T, B. longum subsp. infantis ATCC 15697T, B. dentium DSM 20436T, B. catenulatum DSM 16992T, B. breve ATCC 15700T, B. bifidum JCM 1255T and B. adolescentis ATCC 15703T show a much lower A x P value than the RefBif‐IS , as described in the S Figure . AlthougS Figure   in order to identify phylogenomically related alternatives that labs may use as alternative reference models. For this reason, we developed a tool named RefBifSelector, which allows a rapid screening of local biobanks for bifidobacterial strains that are closely related to the optimized reference strains defined by RefBif‐IS (https://probiogenomics.unipr.it/cmu/) . While ANI analysis investigates the nucleotide identity between genome pairs, PPOS is a score retrieved from Blastp analysis that uses the following formula to compare the translated amino acid sequences: [ + (number of similar matches)]/. Therefore, a final score equal to the value of ANI * Average_PPOS was obtained, considering a minimum score threshold equal to 9600 (corresponding to an ANI and Average_PPOS of 98). Strains below this minimum threshold cannot be assessed adequately similar to RefBif‐IS and cannot be considered suitable choices.While RefBif‐) Figure . This toB. bifidum PRL2010, B. longum subsp. longum 39B, B. breve 1895B and B. pseudocatenulatum 1896B displayed the best choice in terms of ANI and Average PPOS value with respect to the RefBif‐IS references, thus representing the LocalRefBif‐IV (in vitro), while B. catenulatum, B. adolescentis, B. dentium and B. longum subsp. infantis were excluded from the analysis since no strains of human origin were available in the bifidobacterial strains repository that we employed   and only eight downregulated (fold change < 0.25) genes, resulting in extensive activation of genes involved in the metabolism of carbon sources (24) and trans‐membrane transport (11), as well as replication and signalling (10) along with two genes putatively involved in extracellular adhesion  equal to 0.0632 and 0.8448, respectively .Click here for additional data file.Table S3: List of genomes used as input for the creation of the SSDbs (through the use of strainGST software).Click here for additional data file.Table S4: Additional information about Bifidobacterium genomes publicly available on online NCBI genomes repository.Click here for additional data file.Table S5: StrainGST results and analysisClick here for additional data file.Table S6: Comparison between local available strains and RefBif‐IS throught RefBifSelectorClick here for additional data file.Table S7: Taxonomical profiling of LocalRefBif‐IV fermentation in simulated intestinal environment obtained through the use of METAnnotatorX2 software.Click here for additional data file.Table S8: RanSeq results of tested strains, grown singularly with and without contact with human cells line.Click here for additional data file.


The optimal control thresholds for systolic blood pressure (SBP) and diastolic blood pressure (DBP) in patients with white matter hyperintensity (WMH) are still unclear.A longitudinal retrospective study of patients with brain magnetic resonance imaging (MRI) scans with intervals of more than 3  years was conducted. Blood pressure records during hospitalization and from outpatient visits between baseline and the last MRI scan were collected. The outcome was the change in total WMH from baseline to the final visit.n = 121), mild WMH group (n = 126), and moderate to severe WMH group (n = 210). Both baseline and longitudinal mean SBP, DBP, and SBP SD were significantly associated with WMH severity (p < 0.05). An average SBP of 130-140 mmHg  was associated with a higher risk of WMH progression. DBP ≥ 90 mmHg  was associated with a higher risk of WMH progression, but was not after adjusted for other covariates. Longitudinal BP variability was not significantly associated with WMH progression.Among the 965 patients with MRI scans, 457 patients with detailed longitudinal blood pressure records were ultimately included and classified into the WMH absent group (Both SBP and DBP had a stronger relationship with the severity of WMH. A target mean SBP of <130 mmHg and mean DBP of <80 mmHg was associated with a lower risk of WMH progression. An observational study revealed that a systolic blood pressure (SBP) higher than 160 mmHg or lower than 140 mmHg was associated with higher risk of WMH progression compared to an SBP between 140 and 160 mmHg in patients over 80 years old , indicatCurrently, whether diastolic blood pressure (DBP) plays an important role in the development and progression of WMH remains controversial . SeveralTo provide more evidence for SBP and DBP control in patients with WMH, this study mainly focused on longitudinal SBP and DBP data from baseline to the final MRI scan. Second, in this study, we aimed to determine the optimal threshold of mean SBP and DBP, and BP variability associated with a lower risk of WMH progression.2.2.1.This was a longitudinal retrospective study involving patients admitted to Dalian Central Municipal Hospital between January 1st, 2008, and January 1st, 2022, with dynamic MRI scans with intervals of more than 3 years and detailed blood pressure records between the baseline and last magnetic resonance imaging (MRI) both in hospitalization and outpatient visits. The inclusion criteria were as follows: (1) age over 18 years old; (2) BP records of more than 5 times every year. The exclusion criteria were as follows: (1) WMH with a defined or suspicious diagnosis of gene disease such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); (2) WMH with a defined or suspicious diagnosis of acute inflammation or immune-mediated small vessel disease; (3) patients with large area cerebral infarction that influenced the analysis of WMH; and (4) patients without detailed clinical data.2.2.Baseline demographic variables, including age, sex, and education, were collected. Clinical variables, including history of hypertension, diabetes mellitus, hyperlipidemia, hyperhomocysteinemia, smoking, alcohol consumption, cardiovascular disease, atrial fibrillation, heart failure, kidney dysfunction, chronic respiratory disease, autoimmune disease, thyroid disease, anxiety, and depression, were all collected. The average interval of MRI from baseline to the last scans was calculated between groups. Etiology of hospitalization were also collected.2.3.BP values were collected from inpatient records , outpatient visit records, community monitoring records, and home monitoring records . BP values were calculated as baseline mean SBP and DBP values (mmHg), mean SBP and DBP values for longitudinal control (mmHg), and two commonly used BP variability indices, including SDs and coefficients of variation (CVs). The intervals of mean SBP were stratified into <130 mmHg, 130–140 mmHg, 140–150 mmHg, and ≥ 150 mmHg; the intervals of mean DBP were stratified into <80 mmHg, 80–90 mmHg, and ≥ 90 mmHg. BP variability were calculated using the SD (mmHg), and CV  . BP SD a2.4.All patients had undergone dynamic MRI scans . The presence and severity of WMH was assessed based on the baseline and last MRI by two neuroimaging specialists according to the semiquantitative Fazekas scale . If the 2.5.Baseline characteristics of the study population, categorized based on the severity of WMH, were compared with standard statistics. Continuous variables are expressed as the mean (SD) or median . Categorical variables are presented as counts (percentages). Fisher’s exact tests or chi-square tests were used for categorical variables. ANOVA or Kruskal–Wallis tests were used for continuous variables. Ordinal logistic analysis was used to evaluate the associations between different BP values and the severity of WMH. Binary logistic regression was used to analyze the association between different BP thresholds and the risk of WMH progression, of which Model 1 was not adjusted and Model 2 was adjusted for age, education level, diabetes mellitus, hyperhomocysteinemia, smoking status, stroke, kidney dysfunction, and etiology of hospitalization.p value <0.05 was considered significant. All statistical analyses were performed using SAS version 9.4 .A two-sided 3.3.1.n = 121), mild WMH group (n = 126), and moderate to severe WMH group (n = 210) (p < 0.001). Other risk factors with significant differences between groups were education level, hypertension, diabetes mellitus, hyperhomocysteinemia, smoking status, history of stroke, kidney dysfunction. The mean intervals of MRI from baseline to the last scan were 6.52 ± 2.97 years in total, 6.83 ± 2.90 years in the WMH absent group, 6.47 ± 2.83 years in the mild WMH group, and 6.38 ± 3.09 years in the moderate to severe WMH group (p = 0.24). Etiology of hospitalization were also different between groups, especially hospitalization due to cerebral vascular disease (p < 0.001). The median number of BP measurement time points was 16  . Demogras was 16 .3.2.p < 0.001, p < 0.001, The mean baseline SBP was 129.25 ± 10.52 mmHg in the WMH absent group, 131.33 ± 11.32 mmHg in the mild WMH group, and 141.03 ± 13.72 mmHg in the moderate to severe WMH group , and patients with a history of stroke , but not in patients with age over than 70 years or without a history of stroke , p = 0.01], male patients , and patients with a history of stroke  but not in patients aged over 70  years, diabetic patients, female patients, or patients without a history of stroke , f stroke . A longif stroke .4.In this retrospective study, both SBP and DBP values were strongly associated with the severity of WMH. A mean SBP value lower than 130 mmHg, a mean DBP value lower than 80 mmHg were associated with a lower risk of WMH progression, but only mean SBP was significant after adjusted for age, education level, diabetes mellitus, hyperhomocysteinemia, smoking status, stroke, kidney dysfunction, and etiology of hospitalization. Although longitudinal SBP variability and DBP variability were associated with the severity of WMH, but were not related to risk of WMH progression.In the SPRINT study , intensiUntil now, the association between DBP and WMH has remained controversial. Two studies using the data from the Northern Manhattan study revealed a significant association between DBP and WMH progression  and the In our study, SBP SD, SBP CV, DBP SD, and DBP CV was not associated with the risk of WMH progression. Although SBP SD >11.1 mmHg and an SBP CV of >12.24% was associated with the risk WMH progression, but this was not significant after adjusting for age, education level, diabetes mellitus, hyperhomocysteinemia, smoking status, stroke, kidney dysfunction, and the etiology for hospitalization. Previous studies had reported an independent association between 24 h BP variability and WMH volumes/severity . There wSubgroup analysis in our study revealed that the risk of WMH progression was significantly reduced when the SBP value was lower than 130 mmHg and the DBP value was lower than 80 mmHg in patients younger than 70 years old and patients with a history of stroke. This was consistent with a previous meta-analysis that indicated a higher risk of WMH progression in patients under 70 years old than in patients over 70 years old when their DBP increased . A cliniPatients with hypertension tend to develop lower myelin content, have more severe brain microstructure impairment , have a This study had several differences and limitations compared with other studies. The first was that we used the Fazekas scale to semi-quantitatively analyze severity and WMH progression rather than WMH volume. As the Fazekas scale is a commonly used tool both in the clinic and in clinical studies, it may be easier and applicable to help clinicians understand the severity of WMH and the velocity of WMH progression. The second was that we did not analyze a lower SBP of <120 mmHg or < 110 mmHg and DBP <60 mmHg as there were very few patients with a mean SBP of lower than 120 mmHg and DBP lower than 70 mmHg in our study. The third limitation of the retrospective study was that the total number of blood pressure records and the time interval between blood pressure measurements, which might differ among different patients as the number of hospitalizations and outpatient visits were different. These blood pressure records may not be adequate to represent the patients’ average daily control of blood pressure.5.In this longitudinal retrospective study, both SBP and DBP had a strong relationship with the severity of WMH. A target mean SBP lower than 130 mmHg and a mean DBP lower than 80 mmHg were associated with a lower risk of WMH progression. An SBP SD value <11.1 mmHg and an SBP CV value <12.24% were associated with lower risk of WMH progression, but DBP variability was not.The original contributions presented in the study are included in the article/All procedures were performed in accordance with relevant named guidelines and regulations and were approved by the ethics committee of the Dalian Central Municipal Hospital. Written informed consent was not required to participate in this study in accordance with the local legislation and institutional requirements.SL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Writing – original draft. MW: Data curation, Formal analysis, Supervision, Validation, Visualization, Writing – original draft. DG: Data curation, Investigation, Validation, Writing – original draft. YL: Data curation, Investigation, Validation, Writing – original draft. XZ: Data curation, Investigation, Validation, Writing – original draft. HL: Data curation, Investigation, Validation, Writing – original draft. CJ: Investigation, Validation, Writing – original draft. XN: Investigation, Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Writing – review & editing. JL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – review & editing.