The realities of emergency care and resuscitation research involving nursing home (NH) residents suggest an overuse of resuscitation attempts in NHs. A complete analysis of all NH resident deaths is needed to provide a complementary perspective of potential underuse. The present research investigated whether residents of different NH homes died at the NH during attempted resuscitation or after transfer to hospital.A full survey of resuscitation attempts and deaths among NH residents, via retrospective analysis of data from the death registry and the German Resuscitation Registry for the years 2018 to 2021.Over the 4-year study period, 14,598 individuals died, of whom 3,288 (22.5%) were residents of 31 different NHs. The mean age of the deceased NH residents was 87 years (±8.6); 2,196 (66.8%) were female, 118 (3.6%) underwent a resuscitation attempt, and 58.5% died at the NH. NH averages were as follows: deaths per NH: 106 ; number of beds: 102 ; deaths per bed per year 0.27 ; resuscitation attempts per 1,000 beds per year: 9.5 ; and ratio of futile resuscitation attempts to deaths: 6.0% (0–12.5%). Considering the entire study region before and during the COVID-19 pandemic, a slight underuse of resuscitation attempts with female NH residents emerged. On a facility level, substantial disparities and opposing trends were found. The incidence of deaths and resuscitation attempts, as well as the place of death and the ratio of futile resuscitation attempts to deaths, varied considerably.Resuscitation attempts are rarely administered to dying NH residents. However, their frequency varies considerably between NHs. Cardiopulmonary resuscitation is rarely attempted in nursing homes (NH) and, when it is attempted, it rarely does any good.In support of this impression, most of the literature on cardiac arrests in NHs refers to patients who received aCPR by an out-of-hospital medical team.Despite possible survival rates in excess of 1%, aCPR for NH residents may be perceived as inappropriate and overused. However, the evidence does not necessarily support this assessment. Equally important to consider is the potential for underuse. Indeed, both overuse and underuse—referring to the delivery of unnecessary services and the failure to deliver needed services, respectively—are global concerns that can simultaneously affect the same healthcare system, organisation or patient.Any enquiry into the prevalence of prehospital aCPR among NH residents at the end of life should consider only residents who died within the NH facility. This is because the transfer of dying NH residents to a hospital shifts the decision-making about aCPR from the NH to the hospital. While some research on the transfer of dying residents from the NH to the hospital and their place of death (PoD) has been published, many open questions remain.[15]In this context, the significance of the PoD lies in its influence on the frequency of prehospital aCPR.Since the deaths of NH residents unrelated to aCPR are neither a primary focus of research nor a common experience for EMS personnel, the present study aimed at contributing to a broader view, expanding the perspective from death during aCPR to consider all deaths of NH residents within various NH settings. Thus, the research considered whether residents of different NH died after being transferred to hospital or in their NH facility and whether prehospital aCPR was attempted. Additionally, it explored potential changes in these patterns during the Covid-19 pandemic.The present research was based on a retrospective analysis of data from the death registry and the German Resuscitation Registry, covering the years 2018 to 2021. The study region was the city of Braunschweig, located in Lower Saxony in Germany. Braunschweig encompasses 192 kmResidents of various NHs were identified in the death registry data through a comparison of the residential addresses of the deceased with the addresses of the various NHs. To ensure an exact identification, missing address details were investigated in the population register. PoD was categorized into five groups, with reference to the death certificate of Lower Saxony. The categories “at home” and “in nursing home” were merged to “NH,” while the categories “transport” and “other places” were combined into “other.” The category “hospital” was retained. Thus, this research classified all PoDs as “NH,” “hospital” or “other.”The EMS of the city of Braunschweig has been prospectively collecting data about aCPR since the year 2011. This data collection process is linked to the German Resuscitation Registry and overseen by the Medical Director of the EMS. Data for each aCPR event includes the location, (e.g. NH).Residential long-term care facilities and their bed capacity were identified using data from the local authorities. To ensure the anonymity of each of the investigated NHs, no specific facility data was reported here. The size of each NH was classified according to bed capacity, using the following categories: “up to 90,” “91 to 120,” and”over 120” beds. NH ownership was classified as “public,” “non-profit,” “private-local” or “private-nationwide,” based on information from the facility’s online presence.In addition to the number of beds, deaths, and aCPR events linked with each NH, various indicators were generated for the NH resident population across the study region and within individual NH facilities. These included: (a) deaths per bed per year, (b) PoD distribution as a percentage of all deaths, and (c) aCPR incidence per 1,000 beds per year. The analysis also calculated the correlation between aCPR and death, as a measure of overuse.The data processing, anonymization and analysis followed established standards for secondary data analysis.The study comprised part of a larger interdisciplinary research project (acronym”NOVELLE”) that aimed at developing and implementing emergency management guidelines for NHs. Ethical approval was granted by Ostfalia University of Applied Sciences on July 2, 2020. Data processing and analysis were performed using the software packages MS Access, MS Excel, and IBM SPSS Statistics 28.SD ± 8.6), and 2,196 (66.8%) were female. Seventy (2.1%) individuals were younger than 65 years, 1,082 (32.9%) were aged between 65 and 85 years, and 2,136 (65.0%) were at least 85 years old.In the study region 14,598 individuals died between 2018 and 2021, including 3,288 (22.5%) residents of long-term care facilities. Of these NH residents, 1,925 (58.5%) NH-residents died at PoD „NH“ and 1347 (41.0%) died at the PoD „hospital“. The mean age of the deceased NH residents was 87 years  were administered aCPR with the involvement of EMS personnel. The origins of their cardiac arrests were classified as cardiac in 43 (36.4%) cases, hypoxic in 20 (16.9%) cases, and “other” or not known in 55 (46.6%) cases. The initial rhythm was shockable in 11 (9.3%) individuals. Collapse was not observed in 70 (59.3%) cases; however, it was observed in 38 (32.2%) cases by nursing staff and in 10 (8.5%) cases by EMS personnel. Chest compression was initiated by nursing staff in 60 (50.8%) cases and by EMS personnel in 58 (49.2%) cases. A return of spontaneous circulation was achieved in 35 (29.7%) cases, while 84 (71.2%) individuals died at their NH and 34 (28.8%) died after transfer to hospital. None of the NH residents who underwent aCPR survived to hospital discharge. SD ± 8.6), and 1,006 (67.7%) were female.SD ± 8.5), and 969 (65.7%) were female.During the pandemic period, 1,475 NH residents died, including 60 (4.1%) in the context of aCPR. Of these, 891 (60.4%) died at the PoD “NH” with 43 (4.8%) dying during aCPR. Additionally, 574 (38.9%) died at the PoD “hospital” including 17 (3.0%) following prehospital aCPR. The mean age was 87 years . During the reference period, 1,006 (67.7%) of the deceased NH residents were female, of whom 601 (59.7%) died in the NH and 404 (40.2%) died in hospital. Thirty (3.0%) of these female NH residents underwent aCPR. The remaining 479 (32.3%) were male, of whom 262 (54.7%) died in the NH and 216 (45.1%) died in hospital. Twenty (4.2%) of these male NH residents underwent aCPR.Throughout the pandemic period 969 (65.7%) of the deceased NH residents were female, of whom 612 (63.2%) died in the NH and 349 (36.0%) died in hospital. Thirty-six (3.7%) of these female NH residents underwent aCPR. The remaining 505 (34.2%) were male, of whom 279 (55.2%) died in the NH and 224 (44.4%) in hospital. Twenty-five (5.0%) of these male NH residents underwent aCPR.Among the female NH residents who died at the NH, the ratio of resuscitation attempts to deaths increased from 5.0% during the reference period to 5.9% during the pandemic period (RD 0.9%). In male residents the risk of receiving aCPR increased from 7.6% to 9.0% (RD 1.4%).SD ± 39; min–max 34–210). Among these, 13 (41.9%) provided up to 90 beds, 8 (25.8%) had 91 to 120 beds, and 10 (32.3%) had more than 120 beds. Nineteen (61.3%) of the facilities were under non-profit ownership, five (16.1%) were owned privately on a nationwide scale, and seven (22.6%) were privately owned on a local scale. None was in public ownership.A total of 31 NHs providing residential long-term care were identified, including one facility under non-profit ownership founded at the turn of the year 2019/2020. The average number of beds per NH was 102 , resulting in 0.27  deaths per bed per year. The average number of aCPR events was 3.8 , with 9.5  aCPR events per 1,000 beds per year. The average ratio of aCPR to deaths was 6.0% (0–12.5%). During the reference period the average number of deaths per bed per year in the different NHs ranged from 0.14 to 0.49, with an average of 0.26 (SD ± 0.07). The percentage of PoD “NH” averaged 59.9% (37.7– 85.7%). Finally, there was an average of 8.1  aCPR attempts per 1,000 beds per year, and an average ratio of aCPR events to deaths of 5.8% (0–15.0%).Throughout the pandemic period, the average number of deaths per bed per year was 0.26 . The percentage of PoD “NH” was 60.3% (44.8–77.8%). A total of 10.9  aCPR events per 1,000 beds per year occurred, and the average ratio of resuscitation attempts to deaths was 7.1% (0–25.0%). The present research surveyed the aCPR events and PoDs of residents of the different NHs in an urban district in Germany. The results provide a picture of aCPR overuse and underuse in these facilities and a broad view on the end of life situation of NH residents. Furthermore, the research compared periods just before and during the Covid-19 pandemic.Data from diverse populations can be used to construct indicators. However, the application of such indicators as a direct measure of quality or for benchmarking purposes is questionable. Quality assessment in long-term care necessitates a systemic understanding of quality.[23]To address the potential underuse of aCPR, the present analysis standardized deaths and aCPR events based on the number of beds in each NH . Additionally, the ratio of aCPR events to deaths provided insight into potential overuse,Regarding the entire cohort of deceased NH residents, the number of deaths varied annually but did not increase during the pandemic period. However, the incidence of aCPR increased from 8.8 during the reference period to 10.9 during the pandemic period. The absolute risk of receiving futile aCPR for residents who died at the NH increased by 1.3%. The demographic data showed little to no variation. Considering the study region as a whole, the results do not indicate that NH residents were significantly discouraged from being transferred to hospital. While the balance between aCPR overuse and underuse did shift slightly towards overuse during the pandemic, it is essential to note that, during the pandemic, excess mortality was not observed in Braunschweig, in contrast to the general population of Lower Saxony and Germany more widely.The majority of the deceased NH residents were female. Compared to their male counterparts, these female residents died at an older age, more frequently at the NH and less frequently in the context of aCPR. During the pandemic, the sex related difference in the balance between aCPR overuse and underuse at the end of life increased: RD for futile aCPR was 0.9% for female residents and 1.4% for male residents. Sex-related differences in the situations, treatments and outcomes of cardiac arrest have previously been reported.However, the underlying reasons for these differences remain unclear. The data sample did not provide insight into individual cases, including whether transportation to the hospital (or aCPR) was not indicated, declined by the patient, or simply omitted by health care providers.Significant variations were observed between different NHs. During the 4-year study period, the incidence of death varied by a factor of more than three, and aCPR incidence ranged from 0 to more than 20 per 1,000 beds per year. Moreover, the absolute risk of receiving aCPR at end of life ranged from 0% to 12.5% across the different facilities. In the comparision of the reference and pandemic period, specific facilities demonstrated significant changes. For instance, facility no. 1 experienced half as many incidents of death and a decrease in the number of residents passing away at the NH. Additionally, in this facility, no aCPR was conducted during the pandemic, while 15 aCPR per 1,000 beds per year were conducted prior to the pandemic. Moreover, the absolute risk of futile aCPR decreased (-9%). In contrast, facility No. 26 showed no substantial changes in the place and incidence of death, but the incidence of aCPR and the absolute risk of futile aCPR increased (RD 25%). There are numerous potential causes of the substantial differences and contrasting trends that were observed across facilities, including the differing medical indications and individual care preferences of NH residents, as well as differences in NH policies, staff competence and availability, access to physicians and other factors.The present study is unique in its analysis of aCPR in relation to deaths in NH residents over a 4-year period, shedding light on the potential overuse and underuse of aCPR. One strength of the research is the complete data collection for the full survey. However, it is important to also acknowledge the limitations of this research, which stem from the retrospective analysis of data. Furthermore, information about the different NHs was limited, and data regarding individual cases were not available. Finally, the main limitation refers to the questionable generalizability of the results from a single urban district in Germany to broader populations.The frequency with which aCPR is administered to dying NH residents is generally low. However, the present study revealed significant variation between NHs with regard to PoD and the overuse and underuse of aCPR. EMS personnel and researchers should be aware that, in NHs, residents receiving aCPR represent only a small minority of the residents who pass away in these settings.The “NOVELLE” project received funding from the Innovation Fund of the German Federal Joint Committee (G-BA), under Grant No. 01NVF18007. The funding body was not involved in the execution of the present study. With the exception of the affiliations and funding mentioned, the authors declare no further conflicts of interest.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In humans, arterial grayscale ultrasound texture features independently predict adverse cardiovascular disease (CVD) events and change with medical interventions. We performed this study to examine how grayscale ultrasound texture features and elastin fibers change in plaque-free segments of the arterial wall in a murine model prone to atherosclerosis.tm1Unc/J mice  were imaged at 6, 16, and 24 weeks of age. Two mice were euthanized at 6 and 16 weeks and the remaining mice at 24 weeks. Texture features were extracted from the ultrasound images of the distal 1.0 mm of the common carotid artery wall, and elastin measures were extracted from histology images. Two-way analysis of variance was used to evaluate associations between week, sex, and grayscale texture features. Texture feature and elastin number comparisons between weeks were conducted using the sex-by-week two-way interaction contrasts. Sex-specific correlations between the number of elastin fibers and grayscale texture features were analyzed by conducting non-parametric Spearman's rank correlation analyses.A total of 10 Apoep = 0.026). Spatial gray level dependence matrices-homogeneity (SGLD-HOM) also correlated with carotid artery plaque score . Elastin fibers in the region of interest decreased from 6 to 24 weeks for both male and female mice, although only significantly in male mice. The mean (SD) number of elastin fibers for male mice was 5.32 (1.50) at 6 weeks and 3.59 (0.38) at 24 weeks (p = 0.023). For female mice, the mean (SD) number of elastin fibers was 3.98 (0.38) at 6 weeks and 3.46 (0.19) at 24 weeks (p = 0.051).Arterial wall homogeneity changed significantly in male mice from 6 to 24 weeks, with a mean (SD) of 0.14 (0.03) units at 6 weeks and 0.18 (0.03) units at 24 weeks  were observed during this time and may differ by sex. AlthoSome investigators have postulated that arteries that are more echolucent (dark gray) are indicative of infiltration of lipids and inflammatory cells based on associations between ultrasound characteristics of carotid plaques and endarterectomy specimens , 3–17. FAlthough these early studies demonstrated relationships between arterial texture, CVD risk factors, and adverse CVD events, their actual relationship to arterial wall microstructure and cellular composition in plaque-free segments have not been well studied. If these texture features are related to arterial wall microstructure and cellular composition due to early injury , texture features have the potential to become a powerful research tool for studying early arterial injury and remodeling associated with arterial disease and treatment of risk factors. Ultrasound is safe, widely available, and inexpensive compared to other imaging modalities; thus, it is an ideal tool for monitoring arterial health across the life span , 8.Subsequently, we and other investigators extracted texture features from plaque-free segments of the common carotid artery wall and demonstrated that grayscale texture features and measures of echolucency are associated with several traditional and inflammatory CVD risk factors  and predict incident adverse CVD events , 19–21. Our objective was to examine how novel uses of ultrasound grayscale texture features are associated with an arterial structure based on the presence and quantity of elastin fibers. We hypothesized that with age-related elastin fragmentation and arterial injury due to atherogenesis, arterial walls will demonstrate fewer shades of gray resulting in a more homogenous phenotype with lower contrast and higher homogeneity, a phenotype that has been associated with an increased risk for adverse CVD events in humans .2.2.1.tm1Unc/J mice  were imaged at 6, 16, and 24 weeks of age. After baseline imaging, all mice were fed a high-fat diet  ad libitum until 24 weeks. Euthanasia was performed via CO2 inhalation for two mice at 6 weeks , two mice at 16 weeks , and six mice at 24 weeks   . Tissue  female) . It was 2.2.In vivo image acquisition methods have been described previously  , 25–30. 2.4.en bloc from the thoracic cavity and placed in the cassette in a similar manner, with the rostral ends of the transected common carotid arteries identified with small amounts of tissue ink. The tissue cassette was then placed in 10% neutral buffered formalin until fixed. The cassette then underwent standard paraffin processing in an automatic tissue processor. The tissues were embedded as flat as possible and as close to the anatomic imaging position as possible within a paraffin block by the submitting pathologist. The branch point segments were sectioned entirely through the regions of interest in continuous 5 μm sections, with each section captured sequentially on serial slides. All slides were stained with hematoxylin and eosin (H&E). Cytoseal™ 60 mounting medium  was used with #1.5 coverslips. A pathologist reviewed all H&E-stained histologic sections, evaluating the branch point segments for plaque presence in the region of interest (ROI) and nearby. The ROI was defined and identified as the approximately 1 mm region on the near  wall of the CCA wall as it extends toward the external carotid artery (identified by tissue ink). Sections were evaluated for the presence of foam cells, cholesterol clefts, spindle cells, eosinophilic material, media expansion, and any other abnormalities. All H&E slides including the ROIs were digitally scanned (see below). The central slide(s) for each mouse which demonstrated the best view of the ROIs were selected for additional combined staining with Masson's trichrome and Verhoeff's Van Gieson. After examining all sampled levels of each carotid segment, 17 slides were selected by the pathologist to include the level best representing each ROI  for H&E destaining followed by re-staining to identify collagen (Masson's trichrome) and elastin (Verhoeff's Van Gieson). Selecting in this way ensured the best possible match to the ultrasound plane ROI . In some cases, a mouse's left and right ROIs were both sampled well at the same level, such that only one rather than two slides per mouse were needed. The pathologist identified the distal 1.0 mm of the near wall of the CCA as it branched into the external carotid artery and digitally segmented the intima–media and adventitia layers of the common carotid artery wall for microscopic imaging. Combination-stained Masson's trichrome and Verhoeff's Van Gieson slides were also examined by the pathologist, who then assisted in annotating elastin fibers for analysis of fiber separation. A bilateral plaque score was calculated for each mouse (0–8) with one point awarded for plaque presence in each carotid arterial segment .The abdomen and thorax were opened through a ventral midline incision, and the ventral chest plate was removed. The thymus and lungs were removed to allow for visualization of the aortic arch and branching vessels. Grossly identifiable plaques were described and measured. After exposing the branch point segments  bilaterally, the branch point segments adhered to a small piece of tissue wrap  in anatomic orientation. Small amounts of differently colored tissue ink  were applied to the exposed internal and external carotid arteries to aid in the orientation of the tissue specimens once the arteries were removed. The internal, external, and common carotid arteries were each transected, and the branch point segments were removed in anatomic orientation and adhered to the tissue wrap. Each branch point segment was sandwiched with another piece of tissue wrap between two biopsy foam pads  and placed in a tissue cassette. The remaining portions of the common carotid arteries, in addition to the proximal aortic arch, were removed 2.5.At the time of gross pathology examination, the pathologist reported plaque presence and location. Four arterial segments on both the right and left sides  were used to determine the plaque score. One point was awarded for plaque presence in each arterial segment. Thus, plaque scores could range from 0 to 8.2.6.H&E slides were scanned at 20× using an Aperio CS2 Digital Pathology Scanner  by the Translational Research Initiatives in Pathology (TRIP) Laboratory at the University of Wisconsin-Madison. A pathologist annotated the ROIs including the adventitia and intima–media layers on the Aperio images. A traditional bright-field light microscope  with Olympus UPlanFL 40×/NA = 0.75 objective lenses (Olympus Corp.) was used to image the corresponding ROIs on the combination-stained Masson's trichrome and Verhoeff's slides to provide higher resolution bright-field images of the elastin fibers. Based on the manual annotations of the elastin fibers performed in collaboration by the imaging scientist and pathologist, a Fiji  plugin “in vivo ultrasound image to the histopathology image.Methods for identifying the ROI were reviewed by a single pathologist, imaging scientist, and ultrasound reader. The methods described above were employed to standardize and best match the ROI from the 2.7.p-values are two-sided, and p < 0.05 was used to define statistical significance. Statistical analysis was conducted using SAS software , and version 9.4. Origin  was used for box and whisker plots.Grayscale texture features  were summarized by means and standard deviation, stratified by sex and week. A two-way analysis of variance (ANOVA) was used to evaluate associations between week, sex, and grayscale texture features. Comparisons of grayscale features between weeks were conducted using the sex-by-week two-way interaction contrasts. The comparisons of the number of elastin fibers between weeks were conducted using the same analytic approach. Residual plots were examined to verify the model assumptions. The results were presented in graphical format using box and whisker plots. The sex-specific correlations between the number of elastin fibers and grayscale texture features and plaque score were analyzed by conducting non-parametric Spearman's rank correlations analyses after splitting the data file by sex and using both the left- and right-side measurements. All the reported 3.3.1.n = 3 male, n = 3 female). All six mice had atherosclerotic plaques in their carotid arteries. The range of plaque scores for male and female mice at 6, 16, and 24 weeks is shown in The gross examination of the two mice euthanized at 6 weeks demonstrated no atherosclerotic plaques in the common carotid, carotid bulb, and internal or external carotid artery segments. At 16 weeks, the gross examination of the same regions demonstrated no atherosclerotic plaque in the female mouse and three atherosclerotic plaques in the carotid arterial system of the male mouse. At 24 weeks, six mice were euthanized (3.2.3.2.1.p = 0.003). However, the change from 104.57 (17.99) units at 6 weeks to 116.79 (25.75) units at 24 weeks was not significant (p = 0.295). In male mice, the GSM did not change significantly over time. The mean GSM values at 6, 16, and 24 weeks were 100.67 (20.62) units, 81.15 (36.62) units (p = 0.240), and 94.80 (42.92) units (p = 0.741), respectively ] GSM significantly decreased from 104.57 (17.99) units to 69.59 (23.76) units at 16 weeks (ectively .3.2.2.p = 0.399), and 4.49 (0.18) units at 24 weeks (p = 0.614). The mean entropy values for males were 4.51 (0.18) units at 6 weeks, 4.42 (0.19) units at 16 weeks (p = 0.261), and 4.40 (0.09) units at 24 weeks (p = 0.217)  grayscale entropy value for the females was 4.45 (0.17) units at 6 weeks, 4.52 (0.13) units at 16 weeks (= 0.217) .3.2.3.p = 0.277 and p = 0.751 for 6–16 weeks and 6–24 weeks, respectively). For male mice, the mean GLDS-CON value was 169.61 (82.36) units at 6 weeks, 155.93 (47.54) units at 16 weeks (p = 0.668), and lower at 100.01 (52.61) units at 24 weeks (p = 0.055) , 167.71 (65.93), and 146.28 (49.78) units with no statistically significant difference (= 0.055) .3.2.4.p = 1.000, for 6–16 weeks and p = 0.649 for 6–24 weeks). The mean SGLD-ASM value for male mice was 0.0015 (0.0004) units at 6 weeks, 0.0017 (0.0005) units at 16 weeks, and 0.0018 (0.0003) units at 24 weeks   .3.2.5.p = 0.355 and p = 0.509 for 6–16 and 6–24 weeks, respectively). For male mice, the mean value for SGLD-HOM was 0.14 (0.03) units at 6 weeks and 0.15 (0.04) units at 16 weeks (p = 0.760 for 6–16 weeks) and significantly increased to 0.18 (0.03) units at 24 weeks (p = 0.026 for 6–24 weeks)  units at 6 weeks, 0.13 (0.03) units at 16 weeks, and 0.14 (0.03) units at 24 weeks (4 weeks) .3.3.p = 0.071 and p = 0.051 for 6–16 and 6–24 weeks, respectively). For male mice, the mean number of elastin fibers was 5.32 (1.50) at 6 weeks, 3.08 (0.23) at 16 weeks, and 3.59 (0.38) at 24 weeks  (p = 0.019)  . Qualita= 0.019) . From 163.4.sr = 0.707, p = 0.033) (p > 0.05). None of the grayscale texture features were significantly correlated with plaque score or the number of elastin fibers  in female mice  . No otheale mice .3.5.sr = −0.390, p = 0.266; females sr = −0.422, p = 0.258) nor were any other elastin parameters   .One male mouse at 6 weeks did not have left carotid artery measurable grayscale texture features, and one female mouse at 24 weeks did not have right-side elastin data.3.6.One reader measured 20 mouse carotid arterial wall images twice, blinded to the first reading. Images were selected randomly at each time point to ensure that images were selected over the course of the study, and both the right and left CCAs for that time point were measured. A total of 10 images from male mice and 10 images from female mice were intentionally selected for the reproducibility study to ensure equal representation of each sex. The average time between readings was 837 (SD 49) days. The same procedure was followed at time 2 as described in the “Materials and methods” section for grayscale texture feature extraction. Images were normalized (as described above), and the pixel density was confirmed to be 190 pixels/mm. The average (SD) GSM reading for time 1 and time 2 measurements were 88.50 (30.40) units and 91.97 (29.25) units, respectively. The intra-observer absolute difference was 5.80 (4.76) units, and the intra-observer within-subject standard deviation was 5.25. The GSM intraclass correlation coefficient (ICC) was 0.97 . The average (SD) entropy reading for time 1 and time 2 measurements was 4.45 (0.18) units and 4.49 (0.17) units, respectively. The intra-observer absolute difference for entropy was 0.07 (0.05) units and the intra-observer within-subject standard deviation was 0.06. The ICC for entropy was 0.87 . The average (SD) GLDM-CON measurement for time 1 and time 2 measurements was 151.01 (68.32) and 149.16 (66.21), respectively. The intra-observer absolute difference for GLDM-CON was 14.07 (9.61), and the within-subject SD was 11.95. The ICC for GLDM-CON was 0.97 . The average (SD) for SGLD-ASM time 1 and time 2 measurements was 0.0015 (0.0004) and 0.0015 (0.0004), respectively. The intra-observer absolute difference for SGLD-ASM was 0.0001 (0.0001), and the within-subject SD was 0.0001. The ICC for SGLD-ASM was 0.92 . The time 1 and time 2 measurements for SGLD-HOM were 0.14 (0.04) and 0.14 (0.04), respectively. The intra-observer absolute difference for SGLD-HOM was 0.01 (0.01), and the within-subject SD was 0.01. The SGLD-HOM ICC was 0.95 .4.As novel grayscale ultrasound image analysis techniques become increasingly used in research studies, it is important to understand what these measures represent at a tissue level and how their changes, which reflect changes in CVD risk, reflect changes at the tissue level. In this study, we used a murine model that is designed to develop atherosclerosis . Our maiMale mice demonstrated a significant decrease in elastin fibers from 6 to 24 weeks. Arterial wall homogeneity for the male mice also was associated positively with plaque score, indicating that arteries that are more homogenous are associated with greater atherosclerotic plaque burden. Female mice showed a significant decrease in GSM from baseline 6 weeks to 16 weeks and an increase, although not significant, in GSM from 6 weeks to 24 weeks yet also developed plaque and had plaque scores higher than those of male mice at 24 weeks. Other pre-clinical mouse studies examining plaque formation in the aortic root and aorta have demonstrated that female mice have larger plaque areas compared to those of male mice that were fed atherogenic diets and sacrificed between 24 and 26 weeks of age –38. Our In both human  and pre-tm1Unc mice, we identified a quantifiable change in elastin fiber number from 6 to 16 and 24 weeks, which is much earlier than reported by others  changed from baseline to 24 weeks in the mice and to see if these changes were associated with grayscale texture features. Others , 44 repo−/− mice , 44. How−/− mice , 44. The−/− mice , 39–41. 5.p-values were not statistically significant. Because we used atherosclerosis-prone Apoetm1Unc/J mice that were fed a high-fat diet to intentionally accelerate atherogenesis, we cannot separate changes in elastin fibers associated with aging from those associated with atherogenesis in this mouse model. We only studied mice that were prone to atherosclerosis since we were studying arterial injury and studying arteries that were healthy over time was not a prudent use of animal resources given the scientific questions to be answered.The number of mice we studied was small; however, our preliminary findings identified several significant associations between grayscale measures, plaques, and elastin, indicating a consistent effect size for several measures. It is likely that even more associations would have been statistically significant with a larger sample size, since several of the correlations had a moderate effect size of >0.30, but the corresponding 6.Grayscale ultrasound texture features that are associated with increased risk for CVD events in humans were used in a murine model, and the grayscale texture feature SGLD-HOM was shown to change in male mice from 6 to 24 weeks. Structural alterations of the arterial wall (change in elastin fiber number) that reflect arterial injury during atherogenesis also were observed during this time frame and may differ by sex.


Therapeutics to reduce intracranial pressure are an unmet need. Preclinical data have demonstrated a novel strategy to lower intracranial pressure using glucagon-like peptide-1 (GLP-1) receptor signalling.a priori at less than 0.1.Here, we translate these findings into patients by conducting a randomized, placebo-controlled, double-blind trial to assess the effect of exenatide, a GLP-1 receptor agonist, on intracranial pressure in idiopathic intracranial hypertension. Telemetric intracranial pressure catheters enabled long-term intracranial pressure monitoring. The trial enrolled adult women with active idiopathic intracranial hypertension  who receive subcutaneous exenatide or placebo. The three primary outcome measures were intracranial pressure at 2.5 h, 24 h and 12 weeks and alpha set 2, intracranial pressure 30.6 ± 5.1 cmCSF). Exenatide significantly and meaningfully lowered intracranial pressure at 2.5 h −5.7 ± 2.9 cmCSF (P = 0.048); 24 h −6.4 ± 2.9 cmCSF (P = 0.030); and 12 weeks −5.6 ± 3.0 cmCSF (P = 0.058). No serious safety signals were noted.Among the 16 women recruited, 15 completed the study (mean age 28 ± 9, body mass index 38.1 ± 6.2 kg/mThese data provide confidence to proceed to a phase 3 trial in idiopathic intracranial hypertension and highlight the potential to utilize GLP-1 receptor agonist in other conditions characterized by raised intracranial pressure. et al. report that the GLP-1 receptor agonist exenatide significantly reduced intracranial pressure in patients with idiopathic intracranial hypertension, both acutely and after 12 weeks of dosing, in a randomized placebo-controlled trial. The data support moving to a phase 3 trial in idiopathic intracranial hypertension.Mitchell  GLP-1 stimulates glucose-dependent insulin secretion and inhibits glucagon release, thereby lowering blood glucose but not causing hypoglycaemia.2 GLP-1 receptor agonists also signal at the hypothalamus to regulate satiety and weight.3 This has led to GLP-1 receptor agonists being licensed to promote weight loss in the setting of obesity.4Glucagon-like peptide-1 (GLP-1) is a gut neuropeptide secreted by the distal small intestine in response to a meal.6 GLP-1 receptors are expressed in the choroid plexus, the predominant CSF-secreting structure in the brain.8 Our preliminary data have shown that GLP-1 receptor agonism reduces CSF secretion and intracranial pressure (ICP) in an in vivo rodent model with elevated ICP.7 The reduction in ICP was of a greater magnitude to that observed with the commonly used drugs in idiopathic intracranial hypertension (IIH).9Relevant to this trial is the role of GLP-1 receptor agonists in regulating fluid secretion. GLP-1 receptor agonists have been shown to reduce sodium reabsorption and promote diuresis through actions in the renal proximal tubule.11 Weight loss is disease modifying and if maintained can induce remission.13 As the incidence of IIH is rising14–16 in line with global obesity trends,17 targeted treatments are an unmet clinical need.IIH is characterized by increased ICP with no identifiable cause. Recent weight gain is the major risk factor for development of the condition and its occurrence is most commonly observed in women of reproductive age with obesity.18 with up to 25% suffering marked visual impairment.19 Chronic disabling headaches occur in the majority and have adverse impact on quality of life.21 Cognitive deficits linked to raised ICP have been documented.22 Currently, there is no licenced therapy for IIH.23 The most commonly used off-label medicine is acetazolamide; other medicines have included topiramate, frusemide, spironolactone and octreotide.23 Owing to side effects and treatment failures new therapies are needed. Patient groups have highlighted the importance of prioritizing novel targeted treatments for IIH.24Visual loss is observed in greater than 90% of those with IIHThe aim of the IIH:Pressure trial was to translate the preclinical data demonstrating efficacy of GLP-1 receptor signalling to reduce ICP into patients with raised ICP by conducting a randomized, placebo-controlled, double-blind trial in IIH. The trial aimed to evaluate both acute effects on ICP as well as effects over a 3-month time horizon.The trial was a prospective, randomized, parallel group, placebo-controlled trial in women with active IIH. Patients with a diagnosis of active IIH were identified and recruited from a single tertiary referral hospital . This study was approved by the West Midlands—Solihull Research Ethics Committee (17/WM/0179) and all subjects provided written informed consent according to Declaration of Helsinki principles. The trial was registered with ISTCRN (12678718).25 All had normal brain imaging, including magnetic resonance venography or CT venography . All eligible patients had optic nerve head swelling in at least one eye and ICP >25 cmCSF. Those with significant comorbidities, prior CSF diversion procedures, those currently using GLP-1 receptor agonists or dipeptidyl-peptidase 4 (DPP-4) inhibitors or taking drugs that were thought to reduce ICP were excluded. Those taking drugs that might influence ICP discontinued these at least a month prior to enrolment. Pregnant patients or those planning pregnancy were excluded, urine human choroid gonadotrophin (HCG) was checked at each study visit. Detailed enrolment criteria are provided in Women aged 18–60 years who met the diagnostic criteria for IIH were recruited.Following enrolment, a 28-day headache diary was completed . A telemetric ICP catheter (Raumedic) was implanted prior to the baseline visit through a 4 mm burr hole into the right frontal lobe.2] were recorded and a urine pregnancy test performed. Visual assessments included logarithm of the minimum angle of resolution (logMAR) visual acuity and intraocular pressure as measured with the iCare IC200 (Main-line); perimetric mean deviation (PMD) using the Humphrey visual field analyser [24–2 Swedish Interactive Threshold Algorithm (SITA) standard test pattern using a size III white stimulus]. Papilloedema was confirmed on a dilated slit lamp examination by a neuro-ophthalmologist. It was quantified by spectral domain optical coherence tomography  using the global peripapillary retinal nerve fibre layer (RNFL). Patient-reported outcome measures were assessed [Headache Impact Test-6 (HIT-6)26 and 36-item short from survey (SF-36) Rand version].27 Assessments were repeated at 12 weeks. Drug compliance was monitored based on the remaining exenatide in the pen injector device.At baseline, medical history, examination (including blood pressure and heart rate) and body mass index , and standard error (SE) and 95% CI where appropriate. Hierarchical linear regression models were used to analyse repeated measures of the primary and secondary outcomes and to estimate differences adjusted for baseline values. In these models, population-level effects  comprised the intercept, time as a factor variable and the two-way interaction of treatment arm and time as a factor variable to model changing treatment effects over time. Group-level effects  comprised patient-level adjustments to the intercept. The threshold for statistical significance was pre-specified at 0.1.Anonymized individual participant data will be made available along with the trial protocol and statistical analysis plan. Proposals should be made to the corresponding author and will be reviewed by the Data Sharing Committee in discussion with the Chief Investigator. A formal Data Sharing Agreement may be required between respective organizations once release of the data is approved and before data can be released.n = 7) or the placebo group (n = 8)  .One participant was withdrawn before randomization due to disease progression requiring urgent CSF shunting . All ranBaseline characteristics confined a cohort of patients with active IIH . Age, BMP = 0.048 and at 24 h was −4.7 (2.1), P = 0.030. The effect was sustained at 12 weeks with an ICP difference of −4.1 (2.2), P = 0.058  [mean (SD) mmHg],  = 0.058 . This waP = 0.069 compared to the placebo arm −1.5 (4.8) days, P = 0.404  days [mean (SD)],  = 0.404 , althoug = 0.404 . There w = 0.404 . Headachhe trial .P = 0.036. There was no significant change in PMD, difference between arms 1.0 (0.8) dB, P = 0.188. OCT RNFL did not change significantly between arms, −40.2 (47.2) µm, P = 0.396  logMar units,  = 0.396 .P = 0.910 ,  = 0.910 .Analysis of quality of life, using the SF-36, showed no significant changes in either the physical or mental component scores .2 and 38.1(4.9) kg/m2 at 12 weeks, whereas in the exenatide arm baseline was 37.6 (7.9) kg/m2 and 37.5 (7.4) kg/m2 at 12 weeks. There was no significant difference in BMI between arms at 12 weeks, P = 0.854 (BMI in the placebo arm baseline was 38.6 (4.7) kg/m = 0.854 .No significant changes were seen in the safety monitoring blood tests .Twelve adverse events were reported during the trial. Eight adverse events occurred in the exenatide arm, seven of which were nausea related to exenatide initiation. Four adverse events occurred in the placebo arm. One unrelated serious adverse event, thyrotoxicosis, was reported in the placebo arm. No patients withdrew due to adverse events . All parNo anti-exenatide antibodies were detected in samples collected at baseline. In total, two of the seven patients on exenatide developed antibodies by Week 12. At Week 2 one patient had antibodies present (> 0.1 µg/ml) and these were also present at 12 weeks (> 0.1 µg/ml). A further patient was noted to have antibodies at 12 weeks (>1.0 µg/ml).The two patients with anti-exenatide antibodies at 12 weeks had higher (3- and 6-fold higher) than predicted exenatide concentrations at 12 weeks and were excluded from the 12 weeks data analysis. At baseline, Week 2 and Week 12, there was a sharp increase in exenatide concentrations in patient serum at 2.5 h following subcutaneous administration of exenatide. This was followed by a sharp decline in peptide concentrations at 6 h. The mean exenatide concentrations at 2.5 hours are higher at baseline than at Week 2 and Week 12 , which is expected as 20 μg of exenatide was administered at baseline, compared to 10 μg administered at Weeks 2 and 12 . The numNo hypoglycaemia was encountered during the trial. There was no significant difference in fasted glucose and fasted insulin between the two trial arms at 12 weeks .P = 0.088, but there was no significant change within arms between baseline and 12 weeks , 12 weeks .P = 0.028] and 120–150 min  (We looked in more detail at the changes in ICP in the first 2.5 h following drug administration. ICP was significantly lower in recordings taken over time points 90–120 min  .36 In the placebo arm the ICP rose overnight as expected by 3.1 (4.1) mmHg, P = 0.13. In the exenatide arm, the overnight rise in ICP was supressed to 0.1 (3.2) mmHg, P = 0.97. There was a significant difference in the ICP measurement between the exenatide and placebo arms at the last reading of the night .ICP was then measured overnight between 24.00 and 07.00 h on the first day of dosing . ICP is We determined that IIH patients had a similar fat mass, lean mass and android–gynoid ratio in those treated with exenatide as compared to those on placebo with no significant differences detected at 12 weeks .This is the first randomized controlled trial comparing the efficacy of the GLP-1 receptor agonist exenatide, with placebo to reduce ICP in patients with active IIH. We have found a significant and clinically meaningful reduction in ICP in the treatment arm as compared to the placebo arm both acutely and after 12 weeks of dosing. The drug was safe and well-tolerated. This trial establishes the successful translation of preclinical data into humans and is the first to demonstrate a new drug treatment for IIH that significantly reduces ICP in IIH.37 Therefore, to evaluate the efficacy of exenatide the primary outcome for this trial was chosen as ICP.38 A significant reduction in ICP was seen acutely at 2.5 h of −4.2 mmHg , which corresponds to peak exenatide serum levels. The reduction in ICP was also noted at 24 h [reduction of 4.7 mmHg ; 39 and hence a drug with rapid onset of action is clinically advantageous for IIH. A drug treatment that can rapidly reduce ICP is also of potential relevance for other conditions characterized by raised ICP, such as following traumatic brain injury and stroke.The hallmark of IIH is raised ICP, which causes headaches and visual loss (through compression of the optic nerve and papilloedema).38 There have been four previous randomized control trials in IIH40–42 and one prospective crossover trial.13 Four of these have used ICP as an outcome measure, with the magnitude of treatment effect between trial arms ranging from 5.9 cmCSF (acetazolamide and diet),41 −6.0 (1.8) (by bariatric surgery)12 and −6.2 cmCSF .13 The efficacy in these studies is akin to what we have observed following treatment with exenatide for 3 months (−5.6 cmCSF).A significant reduction in ICP was also noted after prolonged dosing at 12 weeks ; those in the placebo arm did not have any significant improvement .53 This technology has led to the accurate demonstration of ICP lowering both with single dosing and with repeated dosing. The intracranial telemetric catheters were safe, with only one failing to register by 12 weeks.This is the first trial, to our knowledge, to use telemetric intracranial monitoring to measure ICP in IIH. Patient input into the trial design advocated for telemetric ICP monitoring over multiple lumbar punctures to facilitate data validity and reduce patient discomfort from multiple lumbar punctures. This has permitted detailed characterization of ICP changes and the ability to measure ICP for prolonged periods over several weeks without further invasive procedures.This study has several limitations. As an early phase study, it was powered to the primary end points of ICP reduction with single and repeated dosing. Thus, the study was underpowered to detect differences in patient-centred outcomes and secondary clinical end points such as headache and visual field perimetry. Powering the study for those end points would have required a large increase in number of participants; however, telemetric ICP monitors would not be feasible in large trial cohort. No stratification was used at randomization and groups were unmatched for baseline headache days and visual field perimetry. Additionally, patients with minimal visual field defects were not excluded as in prior studies, thus making the study more clinically representative, but visual effects more difficult to detect due to the ceiling effect of the measurement. With the main side effect of exenatide being nausea, patients could have been unmasked; however, the primary end point was a physiological measure, which should minimize this risk.54The results of this study may have wide reaching implications for many other diseases of raised ICP as the drug does not target the specific pathogenesis of IIH, but CSF secretion. Given the tolerability of exenatide and its direct and early effect on ICP, it may be beneficial in other conditions such as hydrocephalus, traumatic brain injury, raised ICP in stroke and meningitis and space flight–associated neuro-ocular syndrome. In addition, GLP-1 receptor agonists have been shown to demonstrate neuroprotective properties and hence may have additional benefits in conditions such as traumatic brain injury.7 This preclinical work has now been translated to a population of patients with raised ICP due to IIH. The data demonstrated the efficacy of exenatide to significantly lower ICP. A new efficacious therapy is a clear unmet patient need,24 and this is the first trial of a new drug treatment for IIH that significantly reduces ICP. The data presented here support further evaluation of exenatide in a large randomized controlled trial, powered to evaluate clinically relevant outcome measures.We had previously identified a novel pathway to modulate ICP in an animal model.awad003_Supplementary_DataClick here for additional data file.


Ambient temperature, relative humidity, ambient levels of fine particles (PM2.5) and nitrogen dioxide (NO2) assessed during the same period were adjusted for as sensitivity analyses. Furthermore, we explored the potential different effects by urbanicity and park accessibility through stratified analysis. We found that higher greenness exposure at the second trimester of pregnancy and averaged exposure during the entire pregnancy were associated with higher birthweight and birthweight Z-score. Specifically, a 0.1 unit increase in second trimester averaged NDVI value was associated with an increase in birthweight of 10.2 g (95% CI: 1.8–18.5 g) and in birthweight Z-score of 0.024 (0.003–0.045). A 0.1 unit increase in an averaged NDVI during the entire pregnancy was associated with 10.1 g (95% CI: 1.0–19.2 g) increase in birthweight and 0.025 (0.001–0.048) increase in birthweight Z-score. Moreover, the associations were larger in effect size among urban residents than suburban residents and among residents without park accessibility within 500 m compared to those with park accessibility within 500 m. Our findings suggest that increased greenness exposure, particularly during the second trimester, may be beneficial to birth weight in a metropolitan area.Few studies have examined the association between greenness exposure and birth outcomes. This study aims to identify critical exposure time windows during preconception and pregnancy for the association between greenness exposure and birth weight. A cohort of 13 890 pregnant women and newborns in Shanghai, China from 2016–2019 were included in the study. We assessed greenness exposure using Normalized Difference Vegetation Index (NDVI) during the preconception and gestational periods, and evaluated the association with term birthweight, birthweight  CIConfidence intervalOROdds ratioNDVINormalized Difference Vegetation IndexPBPreterm birthSESSocioeconomic status1.et alet alDeveloping countries have experienced rapid urbanization over the past decades. For example, the urbanization rate in China increased drastically from about 18% in 1978 to more than 60% in 2018  and large-for-gestational age (LGA) and test potential pathways through which greenness exerts beneficial effect on birth outcomes health through mediation analysis. Our research can add new scientific evidence to the literature on the association between residential greenness and birth outcomes by identifying critical exposure period during the pregnancy. In particular, by assigning greenness exposure to each period of preconception and three trimesters, our study could improve the understanding of the critical exposure time window that affects birth weight.During pregnancy, living in a green area can potentially benefit health through promoting physical activities, mitigating air pollution and improving mental health  or early pregnancy (N = 15 615) at preconception or prenatal clinics from 28 maternity institutions in 10 districts of Shanghai, between March 2016 and December 2018. The SPCC study has been described in detail elsewhere  study utilized the birth outcome data based on an existing birth cohort of Shanghai Preconception Cohort  which enrolled −2 and <24 kg m−2), overweight (BMI ⩾ 24 kg m−2 and <28 kg m−2), and obese (BMI ⩾ 28 kg m−2) referring to the definition of the Chinese population . Additionally, we used the routine antenatal care electronic medical record data from the Maternal Clinic Antenatal Medical Record System (MCAMRS), which allowed to get access the information of gestational weight and height, last menstrual period, gestational week measured by ultrasound or last menstrual period. Based on the maternal height and weight, we calculated the maternal body mass index (BMI) at early pregnancy and further categorized into underweight (BMI < 18.5), normal weight . Additionally, we estimated the gender and gestational age-specific birth weight percentiles of each newborn using recorded birth weight, gestational age at delivery and reference percentile chart for Chinese representative children  service. All locations were manually checked on the map, and we categorized addresses into two groups based on whether there was a park within a 500 m circular buffer. This distance represents the physical activity function of greenspace. We retrieved our satellite data from Google Earth Engine (https://earthengine.google.com/) using the geemap package  and R .We conducted a buffer distance analysis to summarize the greenness and park features of the residential surrounding environment. To represent participants’ exposure to surrounding greenness, we used the NDVI, a satellite-based index. Specifically, we utilized the smoothed and gap-filled composite Moderate Resolution Imaging Spectroradiometer (MODIS) NDVI data and combined measurements from Terra and Aqua satellite instruments. When retrieving the data, we removed the negative values of NDVI to reduce the effects from water and cloud , and averaged PM2.5 and NO2 exposure over pregnancy periods for each participant as the greenness exposure assessment. The air pollution models provide PM2.5 and NO2 estimations at 3 day temporal resolution and 1 km spatial resolution in Shanghai between October 2012 and December 2019.Additionally, we estimated ambient air pollution levels in this study based on residential addresses using the temporal-spatial model, , Summer , Autumn , and Winter . Maternal social variables , smoking history (yes or no), exposure to environmental smoke (yes or no) and delivery mode . Educational levels include three categories: (1) high school and below, (2) college, and (3) postgraduates and above. Occupations includes seven categories of (1) manager (2) technician (3) entrepreneur (4) worker (5) farmer (6) self-employed, and (7) other. We further classified the occupations into three groups of group A , group B , and group C (other or unemployed). Missing data of education levels, occupations, smoking history and environmental smoke exposure were coded as a separate category for these variables to incorporate all participants in the analysis. We did not consider altitude and slope in this study because Shanghai is an alluvial plain with an average altitude of 4 m.Z-score. We used multivariate logistic regression models for SGA and LGA as categorical variables . We employed multivariate linear regression models to investigate the association between environmental exposures and birth weight and s figure . We perf2.5 and NO2) during the whole pregnancy in the model. To test individual air pollutant’s effect, we only controlled one pollutant at a time in the model, as model PM2.5 and model NO2, respectively. Considering air pollution levels were potentially correlated with NDVI values during the same trimester due to seasonal effects, we further controlled the air pollution level throughout the entire pregnancy as well as the meteorological factors of the same pregnancy period across trimesters. We further stratified the participants by urbanicity (urban vs suburban) and park accessibility (park within 500 m buffer) and test the consistency of our findings of different residential environments. Urban area was defined as the area within outer circle (or ‘Waihuan’) highway.We conducted a number of sensitivity analyses to test the robustness of our findings. Firstly, we tested the greenness association with birth outcomes by quartile of NDVI values through different stages of pregnancy. Secondly, we further controlled contemporaneous meteorological conditions (relative humidity and temperature) and air pollution  and the whole pregnancy among participants who lived in urban areas, we conducted a mediation analysis using the ‘mediation’ package in R  based on greenness exposure (X) and any necessary control variables (C). The outcome model: A model predicting birthweight (Y) based on greenness exposure (X), NO2 levels (M), and any necessary control variables (C). Then we estimated the average causal mediation effects (ACME), average direct effects (ADE), total effects, and the proportion mediated for each exposure period. The ACME represents the indirect effect of greenness on birthweight through NO2 levels, while the ADE represents the direct effect of greenness on birthweight after accounting for NO2 levels. The total effect is the sum of the direct and indirect effects, and the proportion mediated represents the proportion of the total effect that is attributable to NO2 levels. The covariates adjusted for are the same as the main model of the adjusted model (figure To investigate whether NOp < 0.05 level (two-tail). Statistical analyses were conducted with R (www.r-project.org/ version 4.1.3).Results were considered statistically significant at the 3.3.1.−2 when measured during the first antenatal care visit at around 14 weeks of gestational age. The children in our study were born between 2017 and 2019, with more than half (58.2%) born in 2018. The majority of births (30.5%) occurred in the fall. Folic acid supplementation was used by less than a third (29.0%) of the participants during preconception and early pregnancy. The average birth weight for full-term births was 3358.7 g (SD: 401.7 g).Tables During pregnancy, more than half of the participants resided in urban areas (61.7%), consistent with the high population density in those areas . The majority of participants (73.5%) did not reside within 500 m to a park. Participants had moderate levels of greenness exposure during pregnancy .3.2.2.5 and NO2 at residential address level and measured temperature and relative humidity represent the entire city for the preconception, 1st trimester, 2nd trimester, 3rd trimester and whole pregnancy averages, respectively. The mean concentrations of NO2 and PM2.5 at the participants’ residences throughout pregnancy were 37.6 μg m−3 (SD: 3.6) and 42 μg m−3 (SD: 6.3), respectively. These levels were above the WHO guidelines for PM2.5 annual concentration of 5 μg m−3 and NO2 annual concentration of 10 μg m−3. The correlations among air pollution, meteorological conditions and greenness throughout the pregnancy periods are included in figure S3. For instance, temperature is highly positively correlated with NDVI values showing a seasonal trend.In table S1, we presented the predicted levels of PM3.3.Z-scores during certain time periods of pregnancy. Specifically, the adjusted model estimates suggest that higher NDVI during the 2nd trimester and whole pregnancy is associated with higher birthweight, and higher NDVI during the 1st trimester, 2nd trimester, and whole pregnancy is associated with higher Z-scores. However, no significant associations were observed between exposure to greenness and the pregnancy outcomes of LGA and SGA during any period of pregnancy. Quartile greenness analysis also show that birthweight was significantly associated with greenness exposure during the 2nd trimester and whole pregnancy (table S2). We did not observe a nonlinear relationship between exposure to greenness and birthweight according to the GAM model results (table S3).Table Z-score, even after accounting for meteorological conditions (Model Meteorology). However, we did not observe any significant association between greenness exposure and birth outcomes after controlling for air pollution levels (Model Air Pollution-combine). Then we controlled individual air pollutant of PM2.5 or NO2. The results indicate that PM2.5 was not affecting the greenness effects with minimal impact on the estimate and significance of greenness effects on the birth weight and z-score during the second trimester and whole pregnancy (Model PM2.5). However, after controlling NO2, the effect size of the association between greenness exposure during the second trimester and birth weight decreased from 9.8 grams (95% CI: 1.1–18.6) to 8.5 grams (95% CI: −0.3–17.3), and the association became non-significant (Model NO2). Finally, when controlling both air pollution and meteorology, a significant association only observed during the whole pregnancy  (table S4). For sensitivity analysis, we only controlled the air pollution levels averaged during the whole pregnancy, we found a significant association between greenness during the second trimester  and the entire pregnancy  with birthweight (table S5). And, for Z-score, a significant association was observed only during the second trimester  (table S5).Our findings, presented in table Z score during the 2nd trimester, 3rd trimester and whole pregnancy only for the participants who lived in the urban areas (table S6). Specifically, a 0.1 unit increased in NDVI value was associated with an increase in birthweight of 14.2 g (95% CI: 2.9–25.5 g), 15.2 g (95% CI: 3.8–26.5 g) and 16.0 g (95% CI: 3.5–28.5) and in Z-score of 0.037 (0.008–0.066), 0.041 (0.011–0.070) and 0.042 (0.010–0.074), in 2nd trimester, 3rd trimester and the whole pregnancy, respectively. We also find a positive association between LGA and greenness exposure during the third trimester. For the urban participants, we also observed per 10 μg m−3 increase of exposure to NO2 during the 2nd and whole pregnancy trimester associated with a decrease in birthweight of 15.7 g  and 23.7 g  (table S7). We performed NO2 mediation analysis because we did not observe any significant associations for PM2.5. Results from this analysis suggested a potential mediating effect of NO2 in the relationship between greenness and birthweight during the 2nd trimester of pregnancy among urban participants . The mediation analysis for participants living in urban areas showed that 22.7%  of the association between greenness and second trimester birthweight and 16.8%  of the association between greenness and whole pregnancy birthweight were mediated by NO2 (table S9). The remaining direct effect of greenness on birthweight during the second trimester was not statistically significant (p = 0.056), while the direct effect for the whole pregnancy was significant (p = 0.046).In the stratified analysis by urbanicity figure , we founZ-score of children who had no park within 500 m (figure Z-score (95% CI: 0.013–0.062), respectively. In contrast, the smallest effect was seen during the 3rd trimester, with a 11.1 g increase in birth weight (95% CI: 1.7–20.6 g) and a 0.027 increase in Z-score (95% CI: 0.002–0.051). Additionally, we found that higher greenness during the 1st and 2nd trimesters were significantly associated with the lower risk of SGA, respectively.Figure 4.Z-scores and birthweights both during the entire pregnancy and the 2nd trimester. Our study identified a potential critical window of greenness exposure and added new evidence on the association of exposure to residential greenness with birth outcomes using a longitudinal approach throughout pregnancy. The results of this study also suggest a mediation effects of NO2 toward greenness exposure effects during the 2nd trimester among the urban participants.In this study, we investigated the associations between greenness and birth outcomes in urban and suburban Shanghai, a mega metropolitan area in China. It is the first study to investigate the association between greenness environment and birth weight outcomes segmented by pregnancy time period, including preconception and the three trimesters. Our findings indicate that residential greenness exposure within a 500 m radius was associated with birthweight et alet al  during the entire pregnancy was associated with a 10.1 g (95% CI: 1.0–19.2 g) increase in birthweight among full-term birth children. Our results are consistent with the findings of a previous systematic review and a meta-analysis that suggested a potential positive association between residential greenness and higher birth weights, ranging from 7.99 g (95% CI: 4.29–11.70) to 15.35 g (95% CI: 11.41–19.29) per 0.1 NDVI increase Hu et al. Comparialet al  found thl .et alet al2 (0.9%) in 2005 to 26.1 km2 (9.0%) in 2015. By the start of our study in 2016, Shanghai had constructed large public green spaces in the central city thanks to these plans  which typically contains a large area of green space and/or recreational facilities for activities purposes. In addition, some parks in Shanghai require payment for access, which could limit local residents to visit and enjoy these public spaces. Public policies should be designed to maximize the use of green parks, especially by susceptible people such as pregnant women.Our finding supports the urban planning policy of Shanghai that urban areas should develop green infrastructure for recreational purposes. From 2000 to 2016, the Shanghai government committed to increasing urban greenery by constructing green spaces in the central city, in line with Shanghai’s Five-Year Plans for ecological and environmental protection ) . In the stratified analysis of urban participants (n = 8575), a 10 μg m−3 increase in NO2 exposure during the second trimester was associated with a decrease in birth weight of 15.7 g (95% CI: 3.9–27.4 g). Our mediation analysis suggests that NO2 may be a mediator of effects of greenness exposure among urban participants during the second trimester . However, it does indicate that NO2 is a potential mediator  among urban participants during the whole pregnancy, referring to the ACME  and proportion of mediation . In urban areas, traffic is typically the primary source of NO2, which typically reaches background levels at a distance of approximately 380 m from the road  as reported by Zhu et al , Restorative Functions , and Capacity Building  as summarized by (Markevych hu et al , and prel (et al . The imp


BackgroundSame-level recurrent disc herniation remains a challenge in spine surgery. Although most surgeons agree on discectomy as the treatment of choice for primary lumbar disc herniation, the management of recurrent disc herniation remains ambiguous and largely depends on the operating surgeon. Many surgeons recommend repeat discectomy over fusion because it is cheaper and less invasive. In this study, we analyzed 50 patients who underwent a repeat discectomy.Materials and methodsThe patients in the study had previously been managed for lumbar disc herniation and then presented with either recurrent same-level herniation or symptoms attributed to the same level. The patients were then managed with a repeat discectomy without fusion. We analyzed the preoperative and postoperative Oswestry Disability Index (ODI), duration of surgery, blood loss, duration of hospitalization, and complications.ResultsFifty patients were included: 27 females (54%), and 23 males (46%). They were followed up for an average of 2.81 years (range: 1-4). The mean duration of hospitalization was 4.06 ± 1.5 days (range: 2-8). The operative time was 104.60 minutes (range: 50-195), with an intraoperative blood loss of 85.40 mL (range: 50-150 mL). Durotomy occurred as a complication in eight (16%) patients. The recurrence rate was 26%, with 36% progressing to fusion. The change in preoperative ODI and postoperative ODI was 20.94 ± 7.24 (6-37), with a p-value of 0.04. There were no long-term complications recorded.ConclusionRepeat discectomy is a good management option for same-level recurrent disc herniation. The procedure is associated with low intraoperative blood loss and a short operating time, but there is a significant risk of durotomy. The risk of recurrence remains a concern due to the progression of degenerative changes, especially in the presence of Modic-2 changes. These advantages and disadvantages should be discussed with patients. Degenerative disc disease and facet joint problems affecting the lower back are common in the aging population and are major causes of disability. Statistics indicate that approximately 40% of individuals over 40 years old and up to 80% of those over 80 years old suffer from this condition . HoweverA common manifestation of degenerative spine disease is a herniated disc, which is typically treated through discectomy. Several discectomy methods have been developed, including conventional open surgery, microdiscectomy, endoscopic discectomy, and others ,5. HowevThis study is a retrospective case series conducted at a single center, including a total of 50 patients who experienced recurrent disc herniation and underwent repeat discectomy without fusion between the years 2018 and 2022. Recurrent intervertebral disc herniation was defined according to the criteria established by Yao et al. in 2016 [There were a total of 50 patients included in this study. Of these participants, 27 (54%) were female, while 23 (46%) were male. The mean duration of follow-up was 2.81 years (1-4). The mean age was 50.94 years (33-72). The mean ages for males and females were 50.75 and 51.15 years, respectively (Table The type of discectomy the patient underwent prior to this study was recorded. The patients previously underwent microscopic discectomy (36%), open discectomy (50%), and endoscopic discectomy 14%). Forty percent of the patients had one previous discectomy, while 60% had multiple discectomies. The highest number of discectomies was five. Upon admission to our institution, the patients underwent either an open discectomy 58%; n = 29) or a microdiscectomy . The mean intraoperative blood loss was 85.40 ml (50-150 ml)  Figure .Analysis of the preoperative MRI showed Modic-2 changes in 78% (n = 39). Of the 50 patients, 26% (n = 13) had a recurrence following the repeat discectomy Figure .Of note is that 83% of these patients had Modic-2 changes on preoperative MRI. Progression to fusion following repeat discectomy was eventually performed in 36% of patients during the follow-up period. The mean preoperative ODI was 30.52 (16-41), while the postoperative mean ODI was 9.58 (0-26). The change in preoperative ODI and postoperative ODI was 20.94 ± 7.24 (6-37) . The duration of hospitalization was 4.06±1.5 days (2-8 days).Recurrent lumbar disc herniation (RLDH) is a challenge in spinal surgery. There is still no standardized international protocol for the management of these patients. With the development of various equipment and technology in spinal surgery, the options for approaches to managing lumbar intervertebral disc herniations have increased . These iAny repeat operation comes with additional risks compared to first-time surgeries. The presence of adhesions and distortion of anatomy makes identification and dissection of the structures a challenge . In RLDHRepeat surgeries usually require longer operative times due to the difficulties in tissue dissection and the risks of intraoperative complications and hemorrhage . This stThis study has some limitations. The number of patients is small; a larger multicenter study would provide more information. The absence of a comparative group is another weakness of the study. This study analyzes the single-center experience of repeat discectomy. In the future, a multicenter study comparing various management options will be more informative in creating a management protocol.Repeat discectomy is a good management option for same-level recurrent disc herniation. The procedure is associated with low intraoperative blood loss and a short operating time. However, there is a significant risk of durotomy. The risk of recurrence remains a concern due to the progression of degenerative changes, especially in the presence of Modic-2 changes. These advantages and disadvantages should be discussed with the patients.


Cell Prolif. 2022;55:e13196. doi:10.1111/cpr.13196Ma S, Hu H, Wu J, et al. Functional extracellular matrix hydrogel modified with MSC‐derived small extracellular vesicles for chronic wound healing. This note aims to correct two errors of Figure 3B and Figure 8B in the version of this article initially published. The Figure 3B mislabelled the legend name. The immunohistochemical result of Figure 8B was misplaced. The other elements of Figures 3 and 8 remain the same, and the interpretation of the results remains unchanged.The corrected images for Figures 3 and 8 are below.We apologise for the errors.FIGURE 3.FIGURE 8.


To determine the frequency of endocrine disorders in Beta-Thalassemia Major (BTM) patients presenting for Endocrine Evaluation to the Department of Diabetes, Endocrinology and Metabolic Diseases, Hayatabad Medical Complex, Peshawar, Pakistan, a tertiary care hospital.This descriptive study was conducted in the Department of Diabetes, Endocrinology and Metabolic Diseases, Hayatabad Medical Complex, Peshawar from October 2019 to August 2021. All patients with BTM presenting for endocrine evaluation were included in the study. Height and weight were assessed and plotted on the standard charts. For secondary sexual characteristics tanner staging was used. Blood samples for hormonal profile were taken according to standard protocol and sent for endocrine assessment.2, mean age of transfusion started was 6.7±3.99 months, mean duration of transfusion 13.6±4.03 years and mean duration of chelation therapy received 6.1±4.5 years. Regarding endocrine complications, out of 135 patients assessed, one hundred (74.1%) had height less than 5th centile and fifteen (11.1%) had diabetes mellitus. For thyroid and parathyroid function, 58 and 13 were tested respectively, out of which 16 (27.6%) and 6 (46.2%) had thyroid dysfunction and hypoparathyroidism. Out of 91 patients assessed for pubertal delay, 61 (67.03%) had delayed puberty.A Total of 135 patients BTM were enrolled in the study comprising of 70 (51.9%) males and 65 (48.1%) females. Their mean age was 14.8±3.9 years, mean height 138.5±13.01 cm, mean weight 35.9±8.4 kg, mean BMI 18.6±2.8 kg/mHigh percentage of endocrine complications were found in patients with BTM. Severity and multiplicity of endocrine organs involvement was dependent on duration of the disease and lack of compliance with chelation therapy. Beta thalassemia is an autosomal recessive inherited disorder characterized by defect in beta chain synthesis which can either be homozygous or heterozygous. In homozygous state it is referred as Beta thalassemia major (BTM) while in heterozygous state it is known as thalassemia minor. BTM is a lifelong blood transfusion dependent disorder.4Beta thalassemia is prevalent in many countries, but the highest carriers are found in Cyprus (14%), Sardinia (10.3%) and Southeast Asia.7Endocrine complications are the commonest among BTM patients and the attributing factor is iron deposition in endocrine organs due to frequent blood transfusions along with sub-optimal iron chelation. These endocrine complications adversely affect the quality of life and increase the burden of disease on patient both physically and financially.th October 2019. A written informed consent was obtained from all the patients enrolled in the study. Using non-probability consecutive sampling technique, BTM patients both male and female, who were on regular blood transfusion and age range from 10-30 years were enrolled in the study. After taking detailed history and physical examination, all the relevant information was recorded on a pre-designed questionnaire. Patients with acute kidney injury, sepsis, malignancies, critical illnesses, and those using calcium/Vitamin D supplements or under any treatment for endocrinopathies or with history of intake of traditional medications like herbal and hakeemi medications were excluded from the study.This descriptive study of 22 months duration was conducted in the Department of Diabetes, Endocrinology and Metabolic Diseases, Hayatabad Medical Complex, Peshawar from October 2019 to August 2021. The study was approved by the institutional research and ethics committee under Ref. No. 230/HEC/B&PC dated 4th percentile for their age were labelled as short stature. Secondary sexual characteristics were assessed and staged according to the Tanner staging system. Pubertal status was assessed in girls over 13 years and in boys over 14 years by Tanner’s classification. Delayed puberty was indicated by absence of breast development in girls by age 13 years and absence of testicular enlargement in boys (less than 4 ml) as measured by Prader’sorchidometer by the age of 14.Height and weight of the patients were plotted on the Centre for Disease Control and Prevention (CDC) 2000 age/gender specific growth charts and patients whose heights were below the 5For statistical analysis data was stored and analyzed by the statistical program SPSS version 19.0. Mean ± standard deviation was calculated for quantitative data whereas percentages were calculated for categorical data. The relationship of endocrine complications with the duration of transfusion and compliance to chelation therapy was determined by performing chi-square test. P-value of less than 0.05 was considered statistically significant. All the results are presented in tables accordingly.2. Mean age of transfusion started was 6.7 ± 3.99 months, mean duration of transfusion was 13.6 ± 4.03 years and mean duration of chelation therapy received was 6.1 ± 4.5 years. Mean serum ferritin was 5219.94 ± 3076.3 ng/ml, mean FBS was 109.8 ± 56.97 mg/dl and mean TSH was 4.9 ± 4.3 uIU/ml. Mean serum calcium, phosphorus and iPTH were 9.04 ± 1.03 mg/dl, 4.7 ± 1.2 mg/dl and 25.4 ± 20.4 pg/ml respectively. Similarly mean LH, FSH and serum testosterone were 3.5 ± 3.8 mIU/ml, 3.6 ± 3.7 mIU/ml and 10.7 ± 54.4 ng/ml respectively.A total of 135 participants were enrolled for the study, of whom there were 70 (51.9%) males and 65 (48.1%) females. They had a mean age of 14.8 ± 3.9 years, mean height 138.5 ± 13.01 cm, mean weight 35.9 ± 8.4 kg and mean BMI 18.6 ± 2.8 kg/mRegarding endocrine complications, out of 135 patients assessed, one hundred (74.1%) had height less than 5th centile and fifteen (11.1%) had diabetes mellitus. Regarding gonadal function, out of 91 patients, sixty one (67.03%) had delayed puberty. For thyroid and parathyroid function, 58 and 13 were tested respectively, out of which 16 (27.6%) and six (46.2%) had thyroid dysfunction and hypoparathyroidism. Overall, 74.1% (28/135) of patients suffered from at least one endocrine complication and only 3.7% (5/135) of patients knew about the long-term benefits of chelation therapy. Short stature was the most common endocrinopathy while diabetes mellitus was the least common endocrinopathy. The frequency of endocrine complications is presented in The relation of short stature, delayed puberty, diabetes mellitus, thyroid and parathyroid dysfunction with the duration of transfusion was assessed and is presented in th centile on a CDC growth chart. Another study was performed in Iran in 2019 which showed that the prevalence of short stature in BTM patients was 72.6%.rd centile whereas the criteria selected in our study was that of less than 5th centile.Around 74.1% 128/135) of the study participants suffered from at least one endocrine complication. This indicates a high burden of endocrinopathies amongst patients of BTM. Short stature was the most common endocrinopathy in the study participants with a frequency of 74.1 %, followed by delayed puberty which was seen in 67.03% of the study population. These are very similar to the findings of a study conducted in Karachi which showed that 75 % and 70.8% of the participants had short stature and delayed puberty respectively.8/135 of Regarding Thyroid and Para thyroid dysfunction, a study conducted in 2020 revealed hypothyroidism in 26.8% of the participants, while our study showed prevalence of thyroid dysfunction of 26.6% including both hypothyroid (19%) & Subclinical Hypothyroid (8.6%).16Regarding Diabetes mellitus (DM), a study conducted in Iran in 2017 showed prevalence of DM and pre-diabetes as 10% and 58%, respectively.12In the current study, 74.1% patients have at least one endocrine disorder as compared to a study done in Iran in 2019 which demonstrated that 86.3% patients have at least one endocrinopathy.19Although this study exclusively studied patients with BTM for endocrine complications, it has few limitations. Firstly, this study has the weakness of a cross-sectional study where definitive conclusion between the associations and correlations are difficult to obtain. Secondly, this study was conducted at a single center in a tertiary care hospital and the results of this study cannot be generalized unless confirmed by multicenter studies performed at large scale with a larger sample size. Thirdly, certain investigation like growth hormones, IGF1 and estradiol were not done because of financial constraints and were not covered in Sehat Sahulat (insurance card). Finally, only 58 and 13 patients were assessed for thyroid and parathyroid dysfunction respectively because of insurance coverage limitations. Despite these limitations, more than 50% of patients had one or more endocrine complication.This study revealed a high percentage of endocrine complications in patients with BTM. Severity and multiplicity of endocrine organs involvement was dependent on duration of the disease and lack of compliance with chelation therapy. This emphasizes upon the timely and routine screening of these patients for endocrinopathies, and other complications and this standard of care should be adopted by all those centers caring for and treating patients with BTM.


The decrease in the OAB symptom score remained statistically significant: 3  before the surgery vs. 2  at 6 years (p = 0.0150). However, this improvement was significantly less pronounced than at 1 year: 0  (p = 0.002). Conclusions: Bariatric surgery seems to be effective at treating SUI and OAB with a long-lasting effects, still noted at 6 years.Background: Bariatric surgery is known to improve stress urinary incontinence (SUI) and overactive bladder disorders (OAB). However, there is little long-term follow-up in the literature. Objective: To determine the long-term effect of bariatric procedures on SUI and OAB and their impact on quality of life, we applied the ICIQ and USP questionnaires. Setting: The research was conducted at a French university hospital with expertise in bariatric surgery. Methods: We performed an updated follow-up at 6 years of a prospective cohort of 83 women who underwent a bariatric procedure between September 2013 and September 2014. The women completed the USP and ICIQ questionnaires before surgery, 1 year and 6 years after the surgery. Results: Of the 83 patients, 67 responded (80.7%) in full. SUI remained improved at 6 years: the USP score decreased from 3  before surgery to 0  ( BMI changed from 42.5  kg/m2 before the surgery to 29.1  kg/m2 one year after the surgery, then to 30.8  kg/m2 six years after the surgery (p < 0.0001).Weight loss at 6 years remained significant despite weight regain between the first and sixth years: %EWL increased from 73.0  to 61.0  (p = 0.57) or %TWL (p = 0.51)No significant difference in evolution was found between pre-surgery and six years post-surgery according to the type of surgery (LSG or LRYGB) for BMI (p = 0.001) at 1 year after surgery and 0  (p = 0.0253) at 6 years. There was no deterioration of the USP score between one and six years (p > 0.9999). There was no correlation between the evolution of SUI and the evolution of %EWL  or %TWL . The type of surgery (LSG or LRYGB) did not influence the evolution between the results before surgery and at six years (p = 0.72).SUI remained improved at 6 years: the USP score decreased from 1  before surgery to 0  (p = 0.0150). However, this improvement was significantly less pronounced than at one year: 0  (p = 0.002). There was no correlation between the evolution of OAB and the evolution of %EWL  or %TWL . The variation of the score before surgery vs. at six years was not influenced by the type of surgery (p = 0.12).The decrease in the OAB symptom score remained statistically significant: 3  before the surgery vs. 2  at six years (p = 0.8997) and 0  at six years (p = 0.7306).Despite weight loss, dysuria was not improved at one and six years. The score was 0  before the surgery and was still 0  at one year (p = 0.0001)) was not found at six years: 0  (p = 0.3918). There was a significant deterioration of the ICIQ (p = 0.0325). The results before surgery vs. at six years did not differ significantly depending on the type of surgery (p = 0.39).The improvement in QoL related to urinary symptoms at one year  on urinary incontinence in women. This effect seems to diminish in the long term compared to at one year but is still notable. The improvement does not seem to correlate with %EWL or %TWL. Management of SUI and OAB in obese patients should require them to join a bariatric surgery program.


Puccinia coronata f. sp. avenae P. Syd. & Syd (Pca) is a major constraint to oat (Avena sativa L.) production in many parts of the globe. The objectives of this study were to locate Pc96 on the oat consensus map and to develop SNP markers linked to Pc96 for use in marker-assisted selection. SNP loci linked to the crown rust resistance gene Pc96 were identified by linkage analysis and PACE assays were developed for marker-assisted selection in breeding programs. Pc96 is a race-specific crown rust resistance gene originating from cultivated oat that has been deployed in North American oat breeding programs. Pc96 was mapped in a recombinant inbred line population (n = 122) developed from a cross between the oat crown rust differential known to carry Pc96 and the differential line carrying Pc54. A single resistance locus was identified on chromosome 7D between 48.3 and 91.2 cM. The resistance locus and linked SNPs were validated in two additional biparental populations, Ajay × Pc96  and Pc96 × Kasztan . Based on all populations, the most probable location of the oat crown rust resistance gene Pc96 on the oat consensus map was on chromosome 7D approximately at 87.3 cM. In the Ajay × Pc96 population, a second unlinked resistance gene was contributed by the Pc96 differential line, which mapped to chromosome 6C at 75.5 cM. A haplotype of nine linked SNPs predicted the absence of Pc96 in a diverse group of 144 oat germplasm. SNPs that are closely linked to the Pc96 gene may be beneficial as PCR-based molecular markers in marker-assisted selection.Oat crown rust caused by  Avena sativa L.) is a widely grown cereal, used for grain, forage, and straw production ..Pc genesPc58 complex genes, Pc58a and Pc58c are positioned at 10.8 cM [Pc96. Whereas Pc58b is at 110.4 cM [Pc96 is implausible as a Pc58 cluster gene. Using association mapping methods Esvelt Klos et al. [QPc.CORE.02 on linkage group Mrg02. This QTL was identified in the CORE collection of elite oat lines and influenced variation in crown rust disease response in field nurseries in Louisiana, North Dakota, and Manitoba in 2010 and 2011. QPc.CORE.02 was best represented by the SNP GMI_GBS_94371 at 28.1cM, at least 30 cM proximal to Pc96, suggesting that QPc.CORE.02 is unlikely to represent the effect of the Pc96 gene. It seems that chromosome 7D contains multiple Pc genes . These characteristics make chromosome 7D a promising target for further investigation. For example, the map positions of Pc62 and Pc63 are still only roughly defined.Of the  10.8 cM  on the c110.4 cM , 21, 51.s et al.  reportedPc96 differential line, with a perceptible affect appeared to be segregating in the Ajay × Pc96 population. This was originally suggested by the lack of fit to a single segregating gene model  between Pc_MBTG_6C and a single SNP with location corresponding to 110.4 cM on the consensus map. This corresponds to report of a minor QTL, QPc.APR-6C, on chromosome 6C at 625 Mb [Pc differential line to be found to be segregating for multiple crown rust resistance genes. For example, the Pc50 differential line has been found to carry Pc50-2 [Pc50-4 [Pc50-5 [Pc58a, Pc58b and Pc58c) and several genes conferring quantitative types of resistance [Pc gene differential lines currently available were developed by phenotypic selection which would allow additional unobserved genes to remain in the line during development.The mapping crosses with the Pc96 differential were made under the assumption that this line carried a single gene conferring seedling resistance to crown rust. Interestingly, an additional gene, also originating from the ne model . In addit 625 Mb . Seedlint 625 Mb  study. Iy Pc50-2 , Pc50-4  [Pc50-4  and Pc50 [Pc50-5 . The Pc5sistance , 21, 50.Pc96 gene could be of interest to oat breeders for use in marker assisted selection (MAS). The complications inherent to identifying markers suitable to MAS in a new cross are illustrated by our attempts to characterize inheritance of Pc96 in the Pc96 × Kasztan population . Given that the frequency of virulence remains low in natural populations, it may be beneficial to use Pc96 in oat breeding for crown rust resistance. It has been previously suggested that Pc96 is a good candidate gene for combining with other effective Pc genes including Pc42, Pc45, Pc48, Pc50, Pc62, Pc68, and Pc91 [Pc96 gene in the Matt Moore Buckthorn Nursery, we also observed moderate effectiveness of genes Pc58, Pc64, Pc91 and Pc94. In a recent report of three years data , lines carrying those Pc genes had 19–32% rust severity in the field . This information suggests potential for utilization in combination with Pc96.Historical reports of the effectiveness of nd Brown  found thnd Brown  observedre 0.857 . The effre 0.857  and was re 0.857 . Within and Pc91 , 57, 58.Pc96, which confers race-specific crown rust resistance in oat, to a region which corresponds to the oat consensus linkage group Mrg02 (chromosome 7D) approximately at 87.3 cM. This increases the number of crown rust seedling resistance genes placed to the oat genome in the context of modern high-throughput molecular markers. This study also showed that the Pc96 differential line was segregating for another Pc gene on chromosome 6C.In conclusion, we mapped S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.