To evaluate the performance of third‐trimester ultrasound for the diagnosis of clinically significant placenta accreta spectrum disorder (PAS) in women with low‐lying placenta or placenta previa.This was a prospective multicenter study of pregnant women aged ≥ 18 years who were diagnosed with low‐lying placenta  or placenta previa  on ultrasound at ≥ 26 + 0 weeks' gestation, between October 2014 and January 2019. Ultrasound suspicion of PAS was raised in the presence of at least one of these signs on grayscale ultrasound: (1) obliteration of the hypoechogenic space between the uterus and the placenta; (2) interruption of the hyperechogenic interface between the uterine serosa and the bladder wall; (3) abnormal placental lacunae. Histopathological examinations were performed according to a predefined protocol, with pathologists blinded to the ultrasound findings. To assess the ability of ultrasound to detect clinically significant PAS, a composite outcome comprising the need for active management at delivery and histopathological confirmation of PAS was considered the reference standard. PAS was considered to be clinically significant if, in addition to histological confirmation, at least one of these procedures was carried out after delivery: use of hemostatic intrauterine balloon, compressive uterine suture, peripartum hysterectomy, uterine/hypogastric artery ligation or uterine artery embolization. The diagnostic performance of each ultrasound sign for clinically significant PAS was evaluated in all women and in the subgroup who had at least one previous Cesarean section and anterior placenta. Post‐test probability was assessed using Fagan nomograms.A total of 568 women underwent transabdominal and transvaginal ultrasound examinations during the study period. Of these, 95 delivered in local hospitals, and placental pathology according to the study protocol was therefore not available. Among the 473 women for whom placental pathology was available, clinically significant PAS was diagnosed in 99 (21%), comprising 36 cases of placenta accreta, 19 of placenta increta and 44 of placenta percreta. The median gestational age at the time of ultrasound assessment was 31.4  weeks. A normal hypoechogenic space between the uterus and the placenta reduced the post‐test probability of clinically significant PAS from 21% to 5% in women with low‐lying placenta or placenta previa in the third trimester of pregnancy and from 62% to 9% in the subgroup with previous Cesarean section and anterior placenta. The absence of placental lacunae reduced the post‐test probability of clinically significant PAS from 21% to 9% in women with low‐lying placenta or placenta previa in the third trimester of pregnancy and from 62% to 36% in the subgroup with previous Cesarean section and anterior placenta. When abnormal placental lacunae were seen on ultrasound, the post‐test probability of clinically significant PAS increased from 21% to 59% in the whole cohort and from 62% to 78% in the subgroup with previous Cesarean section and anterior placenta. An interrupted hyperechogenic interface between the uterine serosa and bladder wall increased the post‐test probability for clinically significant PAS from 21% to 85% in women with low‐lying placenta or placenta previa and from 62% to 88% in the subgroup with previous Cesarean section and anterior placenta. When all three sonographic markers were present, the post‐test probability for clinically significant PAS increased from 21% to 89% in the whole cohort and from 62% to 92% in the subgroup with previous Cesarean section and anterior placenta.Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.Grayscale ultrasound has good diagnostic performance to identify pregnancies at low risk of PAS in a high‐risk population of women with low‐lying placenta or placenta previa. Ultrasound may be safely used to guide management decisions and concentrate resources on patients with higher risk of clinically significant PAS. © 2022 The Authors.  abstract to view the translations.This article's abstract has been translated into Spanish and Chinese. Follow the links from the  What are the novel findings of this work?This is the first multicenter prospective study showing that grayscale ultrasound has good diagnostic performance to identify pregnancies at low risk of placenta accreta spectrum disorders (PAS) in a high‐risk population of patients with low‐lying placenta or placenta previa. The probability of clinically significant PAS decreased from 21% to 11%, 9% and 5% when the uterine serosa–bladder wall interface was normal, when placental lacunae were absent and when the hypoechogenic retroplacental space was non‐interrupted, respectively.What are the clinical implications of this work?In patients with low‐lying placenta or placenta previa in the third trimester, ultrasound may be safely used to guide management decisions and concentrate resources on patients with higher risk of clinically significant PAS.Placenta accreta spectrum disorder (PAS) is a pregnancy complication that occurs when the chorionic villi invade the myometrium. On the basis of the depth of myometrial invasion, it is classified into placenta accreta, placenta increta and placenta percretaattributed to a combination of limited sample size, retrospective design, variability in study inclusion criteria and whether confirmation of diagnosis of PAS is at delivery or by histopathologyClinicalTrials.gov (NCT02442518). The study was approved by the ethics committee of the coordinating center (University of Brescia) and all local ethics committees. Written informed consent was obtained from all study participants. The study was not funded.This was a multicenter prospective observational study  involving 16 Italian hospitals, conducted from October 2014 to January 2019. The study was registered with We included pregnant women with a low‐lying placenta  or placenta previa (covering the os), aged ≥ 18 years, who underwent ultrasound assessment at ≥ 26 + 0 weeks to assess placental location. All patients underwent a further scan at 34–36 weeks' gestation for repeat assessment of placental location. During the antenatal visits, maternal demographic characteristics and medical and obstetric history were recorded, and ultrasound examination was performed to assess the placenta. Both transabdominal and transvaginal examinations were performed in all cases, using only grayscale ultrasound. First, transabdominal imaging was performed to obtain an overview of placental location and start assessing the regions of concern. Then, a transvaginal scan was performed, inserting the transducer carefully into the vagina up to a short distance from the cervix, under continuous observation of the image. A sagittal scan of the whole length of the cervix and lower part of the uterus was obtained in each woman. The internal cervical os and the lower part of the uterine wall were visualized clearly in all cases, moving the transvaginal probe from side to side. Measurements were obtained by tracing the distance between the lower edge of the placental tissue and the margin of the internal cervical os in the absence of uterine contraction. The thickness of the lower placental edge was measured within 1 cm of the meeting point of the basal and chorionic platesUltrasound suspicion of PAS was raised in the presence of at least one of these signs: 1) obliteration of the hypoechogenic space between the uterus and the placenta; (2) interruption of the hyperechogenic interface between the uterine serosa and the bladder wall; (3) abnormal placental lacunae, defined as the presence of numerous lacunae including some that are large and irregular (Finberg Grade 3), often containing turbulent flow visible on grayscale imaging obliteraTo assess the ability of ultrasound to detect clinically significant PAS, a composite outcome comprising the need for active management at delivery and histopathological confirmation of PAS was considered as reference standard. In the presence of any ultrasound sign of PAS, the placenta was labeled as possibly accreta. Clinical suspicion of PAS at the time of delivery was raised whenever the placenta did not separate at all or only partially at delivery and/or the attempt to remove it led to brisk hemorrhageU‐test. Categorical variables were presented as n (%), and comparisons were performed using the chi‐square test or Fisher's exact test, as appropriate. P‐values < 0.05 were considered significant. Sensitivity, specificity, diagnostic accuracy, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, positive predictive value and negative predictive value (NPV) were calculated for each ultrasound sign and their combination, in all women for whom pathology data were available and in the subgroup with at least one previous Cesarean section and anterior placenta. Fagan nomograms were used to estimate how the result of each ultrasound sign changed the probability that a patient had clinically significant PASContinuous variables were presented as median and interquartile range (IQR), and comparisons were performed using the Kruskal–Wallis test or Mann–Whitney After a pilot estimate of the prevalence of PAS in our population as 0.2 (unpubl. data), we estimated that, for a predetermined value of sensitivity of 80% and α = 0.05, with precision of estimate set at 10%, specificity of 95% and an expected dropout rate of 10%, a sample size of 348 would be needed. Statistical analysis was performed using Stata version 13.1 . The results of this study are reported in accordance with the Standards for Reporting Diagnostic Accuracy (STARD) statementA total of 568 women with low‐lying placenta or placenta previa were included in the study. Of these, 95 delivered in local hospitals, and placental pathology according to the study protocol was therefore not available. The frequency of ultrasound signs of PAS and active management in the study population are shown in Table in‐vitro fertilization. There were 215/473 (45.5%) women with anterior placenta, of whom 78/215 (36.3%) had clinically significant PAS. Of the women with anterior placenta, 103 had at least one previous Cesarean section, of whom 64 had clinically significant PAS. Of the 78 women with clinically significant PAS and anterior placenta, 31 had a history of dilatation and curettage and four conceived by in‐vitro fertilization. The median gestational age at the time of ultrasound assessment was 31.4  weeks. All patients underwent an additional scan at 34–36 weeks' gestation to reassess placental location. In nine of the 473 cases, the distance between the lower placental edge and the internal cervical os was ≥ 20 mm; three of these had clinically significant PAS and underwent hysterectomy. Table Characteristics and detailed clinical outcomes of the study cohort, according to their history of Cesarean section, are presented in Table n = 103) is shown in Table The diagnostic performance of the three ultrasound markers  and their combination for the antenatal diagnosis of clinically significant PAS in all women with available placental pathology is shown in Table et al.et al. found that the most effective ultrasound criteria for detection of morbidly adherent placenta were loss or irregularity of the clear space between the placenta and uterus and hypervascularity of the uterine serosa–bladder wall interface, with NPVs of 96.7% and 97%, respectivelyWe have shown that grayscale ultrasound findings in the third trimester have a good NPV for clinically significant PAS in a high‐risk population. Normal hypoechogenic space between the uterus and the placenta reduced the post‐test probability of PAS from 21% to 5% in women with low‐lying placenta or placenta previa in the third trimester Figure  and fromet al. in a recent meta‐analysis of population‐based studieset al.The main strengths of the present study are its prospective design, the use of a standardized approach to collect and evaluate ultrasound markers of PAS, and its multicenter nature, which are likely to increase the external validity of our findings. A limitation of this study is its potentially low generalizability, as we evaluated a high‐risk population in the third trimester of pregnancy, with a high overall prevalence of PAS (21%), particularly among women with anterior placenta and previous Cesarean section (62%). Another limitation is that a standardized pathological examination of the placenta was not available in 16.7% of the initial cohort. These women delivered in their local hospital after assessment in one of the referral centers participating in the study, and had more favorable outcomes than women included in the analysis, with no hysterectomy. This patient selection process may explain the higher prevalence of PAS in our cohort of women with placenta previa or low‐lying placenta compared to the 11.1% reported by Jauniaux Although color Doppler had the best predictive accuracy for PAS in a systematic review and meta‐analysis including 3707 patients at risk for invasive placentationIn conclusion, a low anterior placenta with invasive placentation poses great challenges for peripartum management. Such pregnancies are most likely to experience complications, and therefore prenatal diagnosis is likely to have the greatest impact. This multicenter prospective study showed that grayscale ultrasound has good ability to identify pregnancies at low risk of PAS in this otherwise high‐risk population. Ultrasound may be used safely to guide management decisions, concentrating resources on patients with the higher risk of clinically significant PAS.Figure S1 Fagan nomograms showing post‐test probability of clinically significant placenta accreta spectrum disorder for interrupted hypoechogenic retroplacental space (a), interrupted hyperechogenic uterus–bladder interface (b), presence of abnormal placental lacunae (c), interrupted hypoechogenic retroplacental space in addition to abnormal placental lacunae (d), and presence of all three markers (e) in 103 women with low‐lying placenta or placenta previa who had at least one previous Cesarean section and anterior placenta. In one case, the operator was not able to assess the retroplacental hypoechogenic space.Click here for additional data file.Table S1 Ultrasound findings and management at delivery in the whole study population, according to whether placental pathology was availableTable S2 2 × 2 tables for diagnostic performance of third‐trimester ultrasound markers for antenatal diagnosis of clinically significant placenta accreta spectrum disorder (PAS) in 473 women with low‐lying placenta or placenta previaTable S3 Diagnostic performance of third‐trimester ultrasound markers for antenatal diagnosis of clinically significant placenta accreta spectrum disorder (PAS) in 103 women with low‐lying placenta or placenta previa who had at least one previous Cesarean section and anterior placentaClick here for additional data file.


The case is a 67‐year‐old female with autosomal dominant polycystic kidney disease who was followed up regularly. CT scan showed a mural nodule growing over the past 4 years inside the hypodense region surrounded by hepatic cysts. Surgery was performed and the pathological diagnosis was StageI gallbladder cancer. The prognosis of ADPKD has been prolonged, accordingly the number of patients with malignant tumors is expected to increase. There are few reports on the association between ADPKD and gallbladder cancer, and further accumulation of data will be needed. Approximately 70% of patients develop end‐stage renal failure at a median age of 58 years.2Transcatheter arterial embolization of the left hepatic artery was performed in 2017 for symptoms of abdominal distension associated with multiple hepatic cysts. The patient was followed up regularly in our renal center. A computed tomography (CT) scan of the liver in 2019 showed a mural nodule inside the hypodense region Figure ,B. Hepat3ADPKD is mainly caused by mutations of polycystic kidney disease 1 (PKD1) and polycystic kidney disease 2 (PKD2), which encode for polcystin‐1 (PC‐1) and polycystin‐2 (PC‐2), respectively. PC‐1 and PC‐2 form a complex on the plasma membrane and function as a calcium channel involving in various pathological pathways, including the cAMP and the mTOR pathways.Hepatic cysts are one of the most frequent complications of ADPKD, with a reported incidence of approximately 75% in patients aged over 60 years.The reason behind the difficulty in diagnosing gallbladder cancer was the anatomical deviation of gallbladder due to multiple hepatic cysts. Magnetic resonance cholangiopancreatography (MRCP) and drip infusion cholecystocholangiography CT (DIC‐CT) are generally used to evaluate the travel and anatomy of the bile and gallbladder ducts. However, it was difficult to distinguish the cyst from the gallbladder, even though MRCP was performed before treatment in this case. DIC‐CT can show bile ducts in more detail and may have been useful in recognizing the anatomy of the gallbladder.Gallbladder carcinoma is considered to be a tumor with a poor prognosis due to the lack of symptoms and the tendency to invade the gallbladder bed, where the serosal layer is deficient.We experienced a case of gallbladder cancer associated with ADPKD that was difficult to diagnose because of the anatomical deviation of the gallbladder and the absence of cholecystitis after TACE. ADPKD is now expected to have a long‐term prognosis. Therefore, it is important to perform periodic imaging examinations all while taking into consideration malignant complications.Hisashi Murakami: Conceptualization; writing – original draft; writing – review and editing. Satoshi Okubo: Supervision; writing – review and editing. Masahiro Kobayashi: Writing – review and editing. Miho Akabane: Writing – review and editing. Masaru Matsumura: Writing – review and editing. Junichi Shindoh: Writing – review and editing. Masaji Hashimoto: Writing – review and editing.The authors have no conflicts of interest directly relevant to the content of this article.All methods were performed in accordance with the relevant guidelines and regulations including the Declaration of Helsinki.Formal consent is not required for this type of study.


Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).Maintenance of vigorous exercise habits from young to old age is considered protective against hip fractures, but data on fracture risk in lifelong vigorous exercisers are lacking. This longitudinal cohort study examined the hazard of hip fractures in 1844 male former athletes and 1216 population controls and in relation to exercise volume and intensity in later years. Incident hip fractures after age 50 years were identified from hospital discharge register from 1972 to 2015. Exercise and covariate information was obtained from questionnaires administered in 1985, 1995, 2001, and 2008. Analyses were conducted using extended proportional hazards regression model for time‐dependent exposures and effects. During the mean ± SD follow‐up of 21.6 ± 10.3 years, 62 (3.4%) athletes and 38 (3.1%) controls sustained a hip fracture. Adjusted hazard ratio (HR) indicated no statistically significant difference between athletes and controls . In subgroup analyses, adjusted HRs for athletes with recent high  and low (<15 MET‐h/week) exercise volume were 0.83  and 1.04 , respectively, compared with controls. The adjusted HR was not statistically significant between athletes with low‐intensity exercise (<6 METs) and controls . Athletes engaging in vigorous‐intensity exercise (≥6 METs at least 75 minutes/week) had initially 77% lower hazard rate  than controls. However, the HR was time‐dependent ; by age 75 years the HRs for the athletes with vigorous‐intensity exercise reached the level of the controls, but after 85 years the HRs for these athletes increased approximately 1.3‐fold annually relative to the controls. In conclusion, these data suggest that continuation of vigorous‐intensity exercise is associated with lower HR of hip fracture up to old age. © 2022 The Authors.  Some studies which have assessed exercise habits from younger to older ages suggest that engagement in strenuous exercise during the formative years from adolescence to young adulthood may also have a significant protective effect on the risk of hip fracture in later life. Evidence suggests that youth is the optimal time for intense exercise to improve peak bone mass, as well as balance and muscular strength, which are important synergistic predictors of future falls and fractures. However, most training‐induced physical adaptations are transitory, which suggests that the continuation of exercise throughout adult life may be necessary to sustain the gains made in young ages. Although the research on physical exercise and hip fractures in the general population have included participants with differing exercise patterns, there appear to be no follow‐up studies of older individuals who have engaged in vigorous levels of activity throughout their lives. Lack of fracture data on lifelong exercisers is a notable gap in the literature given that habitual engagement in relatively vigorous exercise is widely recommended as a means of promoting general health and function, while minimizing the risk of skeletal frailty and fractures.Several population studies in older people indicate that a higher level of physical exercise in the later years is associated with lower risk of hip fracture.3, 4 In an earlier examination of the present male Finnish former elite athlete cohort, the hazard of hip fracture was slightly lower in the former athletes than population controls aged 50 years or older. Other studies conducted in Sweden have also shown a trend toward lower hip fracture hazard in retired older male athletes compared to peers in the general population. However, these few available studies in former athletes may have lacked statistical power due to having no or a low number of participants over age 70–75 years; ie, a life phase when hip fracture incidence rates begin to increase exponentially. Moreover, the previous studies with former athletes have not addressed the possible influence on hip fracture risk of variation in exercise volume and intensity after an active sport career.Former competitive athletes represent a unique research cohort for studying the association of prolonged early‐life training exposure with bone traits and fractures later in life.21 In Primary objective was to investigate the association of hip fracture hazard with high‐volume and vigorous‐intensity exercise determined at four time points from midlife to old age.Secondary objective was to examine whether the original findings on fracture risk between the entire group of athletes and their controls remain unchanged over an additional 10 years of observational time when most of the participants had reached an age when hip fractures become more common.The tertiary (supportive and exploratory) objective was to evaluate the relation of the exercise volume and intensity to hip fractures in pooled cohort of athletes and controls.Consequently, this study continued and extended the follow‐up of the male Finnish former athlete cohort to provide additional information on the association of long‐term exercise habits with the risk of hip fractures in older ages. The objectives were as follows:Our hypothesis was that among former athletes, continuation of exercise at both high‐volume and vigorous intensity levels would be related to lower hazard for hip fracture compared with less active controls. We also expected that hazard for hip fracture would be lower in the entire group of athletes than in their controls. Briefly, the original athlete cohort consisted of 2448 former male athletes who had represented Finland at least once in the Olympic Games, World or European Championships, or inter‐country competitions between 1920 and 1965 in endurance , power , team game , other track and field , or shooting events. Shooters were not included in the present study. The athletes were identified through athletic organizations’ yearbooks and registers and 97.7% of eligible participants were traced. All the selected sports disciplines and training regimens included weight‐bearing dynamic activities, with probable load on the proximal femur. Due to the low number of fracture cases in the different sports categories, and hence to maximize statistical power, all the athletes were collapsed into one group for the main analyses.The selection of athletes and their sports events and population controls matched for age and area of residence has been detailed in previous publications.28 Bri Selection of the controls was carried out when 85.3% of the athletes were identified. No controls for athletes traced afterward were obtained that partially explains lower number of the controls than athletes in the present study. Participants were traced primarily through records of local parishes. All study participants were of European ancestry.The control group consisted originally of 1712 healthy Finnish men from the same age cohort and area of residence as the athletes. Controls were selected from among men classified as having A1 health  at the medical examination into military service at age 20 years. This allowed the analyses to be conducted free from possible confounding effect on the relationship between lifelong exercise and fracture incidence caused by including individuals with chronic diseases and impairments at a young age.29 Sel In this updated analysis, in contrast to the previous study by Kettunen and colleagues, we excluded an additional 303 athletes and 251 controls due to fracture or death before age 50 years or prior to the start of the fracture follow‐up in 1972, or due to missing birth and death data, or to relocation abroad during the follow‐up. After these exclusions, the final study population for the first fracture analyses comprised 1844 athletes and 1216 controls. To control for the potential effect of lifetime work history , we collected data on the participants’ longest held occupation mainly from the Central Population Registry of Finland and also from questionnaires. For the occupation‐based analyses, we categorized the participants into six groups: executives, clerical staff, skilled workers, unskilled workers, farmers, and other or unspecified occupation. The study was approved by the Ethics Committee of the University of Jyväskylä and conformed to the principles of the Declaration of Helsinki. Permission for record linkages was received from the Finnish Institute for Health and Welfare and Statistics Finland.The present analyses were limited to participants aged 50 years and older, as only a few fragility fractures occur at younger ages.26 In n = 1985), 1995 (n = 1431), 2001 (n = 1136), and 2008 (n = 575). Overall, 67% of the participants responded at least once over the four waves of surveys. The questionnaires included items on personal characteristics known to affect fracture risk, including anthropometry , living situation , alcohol use (g/month), smoking status , and physical exercise. Leisure‐time physical exercise was assessed by items on the average intensity, average duration, and monthly frequency of exercise activities during the previous year. Average exercise intensity and total exercise volume were expressed as metabolic equivalent units (METs: work metabolic rate/resting metabolic rate) and by a sum score of MET hours of exercise per week (MET‐h/week), respectively. For the subgroup analyses, physical exercise was divided into two exercise volume and two exercise intensity categories. The cut‐point for high exercise volume was defined as at least 15 MET‐h/week, an exercise level that has been found to associate with increased bone mineral density (BMD) at the proximal femur in adult men . The cut‐point for vigorous exercise intensity was defined as MET score of 6 or greater. In addition, the time criterion for vigorous exercise intensity was set to at least 75 minutes per week . Occupational and non‐sportive activities  were not included in the measure.Our new analysis included a subgroup of athletes and controls who participated in later follow‐up questionnaire studies conducted in 1985 . Participants with the high‐energy hip fractures were excluded from the study. Fractures were collected from the register based on both primary and secondary fracture diagnoses. If a participant had sustained multiple hip fractures (two participants), only the first fracture was included in the analyses. The Care Register for Health has been acknowledged as a very reliable source in its accuracy and completeness for the diagnosis of severe injuries such as hip fractures.The follow‐up data for hip fractures were drawn from the Care Register for Health  and the search covered the records of inpatient care from January 1, 1972 to December 31, 2015. The hospital discharge records were linked with information on dates of deaths collected from the National Death Register, Statistics Finland. Hip fractures were identified by the International Classification of Diseases (ICD) codes . Only fragility hip fractures caused by moderate to minimal trauma  were included in this study. The literature reports that over 90% of all hip fractures are caused by falls from standing height.39 FraAs sample characteristics we report means and proportions together with 95% confidence intervals (CIs). As descriptive statistics of fractures data, we report person‐years of exposure, number of fractures, and fracture rates per 100 person‐years. We used incidence rate ratios (IRRs) together with their 95% CIs to examine an unadjusted relationship across pairs of groups with the control group as the reference group. We analyzed group differences in average ages at fracture with variance analysis.IRRs provide a crude estimate for the development of fractures because they do not account for differences in timing of the fractures. Thus, to study the importance of participants’ age at the fracture hospitalization, we used the Cox regression model . As a secondary objective, we studied hip fracture hazard between all former athletes and controls using Cox proportional hazards regression models adjusted for occupation. This analysis included participants with fracture data available for the years 1972 to 2015. and we also modeled the association of the exercise with the ratio of fracture hazards as a time‐dependent effect based on a polynomial function of time.In our primary objective analysis, we used Cox regression models to evaluate the association of exercise variables and covariates on hip fractures, including data available from follow‐up questionnaires in 1985, 1995, 2001, and 2008. Comparisons were made between athletes in two categories of exercise volume (<15 MET‐h/week versus ≥15 MET‐h/week) and exercise intensity (<6 METs versus ≥6 METs at least 75 minutes/week) and controls. We used age as survival time, and the participants were followed starting from the first questionnaire administration (athletes) or from 1985 (controls) until time of death, diagnosis of hip fracture, or end of follow‐up on December 31, 2015. The exercise categories were allowed to vary as time‐dependent exposures,44 andh) for different study groups. For a time‐dependent effect, the hazard ratio of the covariate is separated into two components: (i) an effect that remains constant over time, and (ii) an interaction effect of the covariate with a function of time. The former defines the level of the covariate effect at the point where the follow‐up time is zero, whereas the latter captures the changes occurring in the HR over time. More details are given in the Methodological Supplement. If, based on the scaled Schoenfeld residual test and plot, significant HR modification was observed, we reported the time‐dependent effects. The models were adjusted for height, weight, living situation, occupational class, alcohol use, and smoking status. All the covariates, excluding occupational class, were treated as time‐dependent exposures. Assessment of nonresponse using missing data indicators suggested that missingness was not related to any of the variables under study, and hence missing data in confounders were not imputed. For the study objectives, Nelson‐Aalen cumulative hazard plots were used to illustrate the unadjusted accumulation number of expected fractures in the study groups over time.We report the proportional hazards model results as hazard ratios (HRs), which are pairwise comparisons of hazard rates , epiR (version 1.0‐15) and emmeans (version 1.5.1) in the R programming environment . More details related to the data and methodology can be found in the Methodological Supplement.In the supplementary and exploratory analysis, the same multiple regression model was also used to compare fracture hazard rates of the two exercise volume and two exercise intensity groups created from the pooled population of athletes and controls. All the analyses were limited to those aged 90 years or below to avoid problems due to sparse data at high age. Statistical significance level was set to 0.05. Descriptive analyses were performed using SPSS Statistics software . Fracture‐time analyses were done using a custom script utilizing the packages survival (version 3.1‐12),46 lub the former athletes had a twofold greater exercise volume (MET‐h/week) and higher exercise intensity (MET), duration and frequency than controls . Significant differences in exercise volume and intensity as well as participation rate in the high volume (≥15 MET‐h/week) and vigorous intensity of exercise (≥6 METs at least 75 minutes/week) in favor of the former athletes persisted at all time points . However, mean age at the time of the fracture event was 77.6 years  among the athletes compared to 73.8 years  among the controls. The difference of 3.84 years  was statistically significant (p = 0.019).Figure p = 0.046 and 0.154, respectively).Cumulative hip fracture incidence curves for the time‐dependent measurements of total exercise volume exposure in our primary objective analysis are shown in Fig. p = 0.031), but not between the low‐intensity athletes and controls . A sensitivity analyses where the athletes and controls were treated identically with regard to start of being at risk did not materially change the results of exercise volume and intensity in primary hypothesis study .The association of the two time‐dependent exercise intensity levels with hip fracture incidence in our primary objective analysis is shown in Fig. A for effect size). Moreover, no statistically significant difference was observed in the level effect of the fracture hazard rate in the vigorous compared to low exercise intensity group . However, a significant time‐dependent curvilinear increase was found in the fracture hazard rate in the vigorous compared to low exercise intensity group: using the approximate Wald test , a protective effect in favor of the vigorously active participants was observed until approximately 67 years of age. Between ages 67 and 84 years the hazard rates did not differ significantly; however, from the age of 84 years onward the hazard rate rose in a curvilinear fashion in the vigorous relative to lower exercise intensity group , similar to the result we found in the primary objective analysis  in the vigorous exercise intensity group, although based on a small sample, suggests that high age may modify the association between vigorous exercise and fracture hazard rate.This extended follow‐up of the Finnish male former elite athlete cohort examined, to our knowledge for the first time, whether early‐life athletic participation and continued physical exercise at higher volume and intensity levels in later life are associated with hip fracture risk. In line with the original study of this same cohort,22 the but not all studies, in former high‐performance athletes suggests that years of training and competing in weight‐bearing sports can lead to positive adaptations in femur bone mass, size, and structure that are not completely lost even many decades after cessation of training. However, the answer to the critical question whether former athleticism can ultimately reduce the risk of hip fractures in old age remains unclear. The original fracture follow‐up of the present cohort as well as earlier reports from Sweden have shown either no difference in hip fracture risk in retired male athletes compared to matched population controls or only a nonsignificant trend toward a lower risk in the athletes.. Here the additional 10 years of follow‐up data allowed us to follow the entire cohort into a more advanced age during which the proportion of hip fractures increased by 54% percent. The present result was in line with the previous finding in this cohort, which found the hip fracture hazard rate was slightly, but not significantly, lower in the combined athlete group than controls.Evidence from some studies,52 butImportant new (primary and tertiary) objectives of this study were to examine whether variation in total exercise volume and average exercise intensity in later life is associated with hip fracture hazard. Our hypothesis that fracture hazard would be associated with both higher volume and more intense exercise was only partially supported. The results indicated that among the athletes, a higher exercise volume of at least 15 MET‐h/week was not statistically significantly associated with lower fracture hazard rate compared to controls. In turn, the athletes who engaged in vigorous intensity exercise ≥6 METs at least 75 minutes/week initially had a 77% lower hip fracture hazard rate than controls. However, this hazard changed around age 75 years, when the fracture hazard rate became similar to that of the control group. After age 85 years, the rate of the vigorously active athletes, which until then had remained lower, began to rise. Thus, among the vigorous‐intensity athletes, the cumulative fracture hazard remained notably lower than among controls even up to the ninth decade see Fig. B. Our findings also showed consistency with a prospective 21‐year study of a Finnish male population reporting that participation in vigorous exercise at baseline (age 50 years) was associated with a 62% reduction in the hazard rate of hip fracture. In that investigation exercise volume and fracture hazard rate were not associated, except that among the men in the second quartile of exercise volume, the rate was reduced. A meta‐analysis of 13 prospective population studies concluded that moderate‐to‐vigorous exercise is related to a hip fracture risk reduction of 45% and 38% among men and women. More recently several studies, but not all studies, have confirmed the protective effect of exercise during later adulthood against hip fractures in men. Note, however, that many population studies have classified vigorous exercise according to overall volume  rather than intensity of leisure physical activity. It must also be emphasized that the majority of population studies have not examined time‐varying effects of exercise on fracture hazard and lack data on participation in sportive activities in young ages and may thus not be directly comparable with our results.Very few studies have dealt with the importance of the volume and intensity of exercise as determinants of the fracture risk. However, a recent study found a significant 35% lower hazard rate of hip fractures among vigorously active older men and women (aged 65–90 years) participating in long‐distance cross‐country ski races as compared to age‐matched controls.55 Our In addition, some studies in middle‐aged and older men indicate that the maintenance of a high osteogenic index or loading intensity, rather than total time spent in physical activities, is associated with greater skeletal benefits at the loaded sites. Therefore, it is possible that among former athletes, even a modest amount of participation in vigorous‐intensity exercise (75 minutes/week), may have been effective in counteracting age‐related decline in femoral neck strength, thus reducing the probability of a hip fracture in the case of a fall. Another explanation might be that continued vigorous exercise helps to protect against fractures through other mechanisms such as preserved rapid muscle force capacity and dynamic postural balance that could reduce the frequency of falls and/or facilitate effective protective responses during a fall. Furthermore, the association may also reflect better functional and health status, which could contribute to maintaining intensive activity and thus influence the fracture risk.Our study allows no conclusions to be drawn on the mechanisms by which the maintenance of relatively vigorous exercise intensity might be more protective against later hip fractures than high total exercise volume. However, studies in animals have indicated that bone adaptation to exercise seems to be intensity‐dependent, whereas the duration and number of loading cycles is of less importance.57 In  Thus, it may be that in very old age some former athletes reach the fracture threshold at the same time as they confront increasing physiological limitations due to aging processes , whereas their wish to engage in year‐round leisure exercise exposes them to hazardous situations, such as slippery winter conditions, where falls and fractures can more easily occur.On the other hand, our model of the time‐dependent effects of exposure suggests that the advantage with respect to fracture hazard of vigorous exercise in athletes may decrease after approximately age 85 years and the cumulative hazard rate increase to the level of the controls. It should be remembered that over half of all hip fractures occur in individuals who are not osteoporotic but are classified as having osteopenia or low bone mass.62 ThuA possible confounding factor when comparing former athletes and population‐based controls is that the nonathletic group includes some participants with vigorous leisure activity (≥6 METs). To address this issue, we performed the tertiary objective analysis to evaluate fracture hazard in the two exercise volume and intensity groups in the pooled cohort of athletes and controls. With this reclassification the effect from higher exercise intensity on fracture HR appeared to partly support the finding from our primary hypothesis study: the time effect indicated hazard modification in the vigorous‐intensity versus low‐intensity groups in the early and final follow‐up years. The finding that the results of time effect remained similar as in the primary objective analysis is somewhat surprising and unclear given that the controls most probably had trained at a lower intensity than the athletes in young ages. Therefore, it could be speculated that although training in weight‐bearing sports in youth is important in achieving high starting bone strength and physical abilities, maintenance of vigorous‐intensity activity in later years may also play a significant role in hip fracture protection in non‐athletes.Studying former elite athletes, who in later life were still exercising intensively relative to age‐appropriate maximum capability, provided us with a “natural experimental setting” that would be difficult to emulate with any other approaches to study of lifetime vigorous exercise. Key methodological strengths in this study include the long follow‐up time, a complete national cohort of international‐level former athletes in the main sporting events, an age‐ and area‐matched general population control group, and reliable national hospital register data on fractures and trauma types (low versus high energy). A further important strength of our study was the evaluation of exercise and covariates up to four time points during follow‐up that allowed us to capture potential variation in exercise exposure or confounding factors from mid‐to‐late life; fracture‐preventive association would have remained undetected had we not considered the time‐dependent effect of exercise on fracture hazard rates. We also acknowledge that MET and MET‐h/week values in the absence of information on the exercise type must be considered at best as crude proxy measures of bone loading intensity and volume. Furthermore, the questionnaire studies did not collect data on the age at which competitive sporting activities were commenced and ceased. However, in a recent follow‐up survey of this cohort, the athletes (n = 214) reported having begun regular training at 10.9 ± 3.6 years of age and competing at 13.5 ± 3.4 years of age (unpublished data), which is in line with other studies of former athletes. The mean age when ceasing to compete was 38.9 ± 15.6 years, with some athletes continuing their sports at a competition level as veterans. A further limitation of the study is that our database of fractures did not include information about the circumstances causing the injury, and possible comorbid conditions and medications which could affect the ability to exercise and change the risk of falls and fractures. However, the response rates in different time points were only slightly lower in controls than former athletes. Our analysis of missing data found no major differences in terms of age or socioeconomic variables, suggesting that the missing data was random subset of data. Finally, although our conclusions can be criticized for being limited to the most highly trained and best adapted male athletes, it should also be remembered that training regimens have evolved over the past decades. The former athletes in this study participated in competitions over half a century ago and it is possible that the training level during their active careers was lower than it is among modern professional athletes and perhaps more comparable to that of committed recreational exercisers.The main limitation of this study is the relatively small number of fracture events, which may explain some of the null results and prevented us from carrying out subgroup analyses on athletes subjected to different loading modalities and multiple doses including extreme training to clarify ideal and safe upper limit of exercise in old ages. One limitation is the use of self‐reports of physical exercise, which are susceptible to errors.65 We In conclusion, no difference was observed in fracture hazard rates between the entire male athlete cohort and controls. Among a subgroup of the former athletes, the maintenance of vigorous exercise intensity was associated with reduced initial fracture hazard rate (77%), whereas total exercise volume had no significant association with hazard rate. Furthermore, in the vigorous‐intensity athletes the fracture events were postponed on average by 5.5 years compared with controls. Our analysis suggests, however, a further possible interaction between age and exercise for fracture hazard whereby the protective association on hazard with vigorous‐intensity exercise may be attenuated from approximately age 85 years onward. Together, these findings suggest that vigorous‐intensity exercise throughout life, not sports participation in youth alone, is associated with decreased hazard of hip fracture in later years. The finding that the fracture‐protective association of vigorous exercise diminished in very old age may imply that careful attention should be paid to safe exercise forms and conditions, but not that participation in sportive activities should be avoided.Marko T. Korhonen: Funding acquisition; investigation; methodology; writing – original draft; writing – review and editing. Urho M. Kujala: Investigation; methodology; project administration; writing – original draft; writing – review and editing. Jyrki Kettunen: Investigation; methodology; writing – review and editing. Olga V. Korhonen: Investigation; writing – original draft; writing – review and editing. Jaakko Kaprio: Funding acquisition; investigation; methodology; project administration; writing – original draft; writing – review and editing. Seppo Sarna: Investigation; methodology; project administration; writing – original draft; writing – review and editing. Timo Törmäkangas: Funding acquisition; investigation; methodology; writing – original draft; writing – review and editing.All authors state that they have no conflicts of interest.The data that support the findings of this study are available upon reasonable request from the corresponding author. The registry data are not provided outside the University of Jyväskylä or the University of Helsinki due to privacy and ethical restrictions.https://publons.com/publon/10.1002/jbmr.4624.The peer review history for this article is available at Appendix S1. Supplementary InformationMethodological SupplementClick here for additional data file.Fig. S1. Total exercise volume (A) and average exercise intensity (B) by group, and proportion of athletes and controls reaching cut points of high total exercise volume of ≥15 MET‐hour/week (C) and vigorous average exercise intensity of ≥6 METs at least 75 minutes/week (D) at different time points. Values within bars are means  and mean percentages  with 95% confidence intervals in parentheses. p < 0.001 in all pairwise comparisons between groups (A–D). Total exercise volume .Click here for additional data file.Fig. S2. Nelson‐Aalen cumulative fracture hazard curves of hip fractures for the pooled sample (athletes plus controls) stratified into two groups based on total exercise volume (A) and average exercise intensity (Panel B). Hazard ratios (HRs) and their 95% confidence intervals (CIs) are shown in the inset tables. The models were controlled for occupation, height, weight, living situation, alcohol use and smoking history. Nelson‐Aalen curves of hip fracture risks were adjusted by possible changes in exercise level during follow‐up. Time‐dependent effect observed for exercise intensity (B) is partitioned into two components: initial HR is the hazard ratio at the start of the follow‐up period and time HR refers to risk modification over time. Hip fractures were followed starting from the first questionnaire participation until time of death, diagnosis of hip fracture or end of follow‐up on December 31, 2015. Exercise and other characteristics were obtained from questionnaire studies in 1985, 1995, 2001 and 2008. See Table Click here for additional data file.


To investigate the accuracy of additive manufacturing (AM) by means of internal fit of fixed dental prostheses (FDPs) fabricated with two AM technologies using different resins and printing modes  compared to milling and direct manual methods.Sixty 3‐unit interim FDPs replacing the first mandibular molar were divided into 6 groups (n = 10): manual (Protemp 4), milled (Telio‐CAD), and AM groups were subdivided based on AM technology (direct light processing (Rapidshape P30 [RS]) and stereolithography (FormLabs 2 [FL])) and the polymer type (P‐Pro‐C&B [St] and SHERAprint‐cb [Sh]) . Validated (RS‐Sh and RS‐St) or nonvalidated (FL‐St and FL‐Sh) modes were adopted for AM. The specimens were scanned to 3D align (GOM inspect) according to the triple scan method. The internal space between the FDPs and preparation surfaces in four sites  was measured using equidistant surface points (GOM Inspect). Statistical analysis was done using Kruskal Wallis and Dunn post‐hoc tests. (α = 0.05).p < 0.05). FDPs with St resin (FL‐St and RS‐St) displayed bigger marginal space than milled, FL‐Sh, and RS‐Sh. The nonvalidated printing mode showed better mean space results (p < 0.05) with higher predictability and repeatability (p < 0.001).One AM group (FL‐Sh) and milling exhibited better adaptation compared to manual and RS‐St at molar site (The AM interim FDPs tested provided valid alternatives to the milled ones in regard to their accuracy results. The printing mode, resin, and the AM technology used significantly influenced the manufacturing accuracy of interim FDPs, particularly at the marginal area. The nonvalidated printing mode with lower‐cost 3D printers is a promising solution for clinical applications. Additive manufacturing (AM) is arguably one of the fastest developing technologies with great potential in the restorative/prosthodontic domain. With this expansion, a wider range of three‐dimensional (3D) printers and printable materials are being introduced to the market. This results in the creation of new clinical applications of the 3D printed products, thereby increasing demand and ultimately reducing costs of AM. The fabrication of interim restorations is one of the clinical applications that can benefit the most from the developments in 3D printing technology.An interim restoration should offer certain mechanical stability, while maintaining the biological health of the teeth and their surrounding tissues.Even though 3D printing offers certain advantages over subtractive manufacturing, namely reduced initial cost of the device and waste material, the possibility of fabricating extended dental appliances such as dental models, as well as fabricating a large number of objects in a shorter time period,Vat‐polymerization is one of the most used AM methods for fabrication of dental devices, and can be subcategorized based on the light source employed on the printer: stereolithography (SLA), direct light processing (DLP), and liquid crystal display based (LCD) printers also called daylight polymer printing (DPP).Moreover, for the clinicians who prefer the use of AM as a chairside option instead of higher cost milling devices, there is unquestionably a demand for the use of low‐cost 3D printers that allow a wide range of material alternatives. Thus, investigations comparing so called “nonvalidated” manufacturing of the dental devices to the validated printing mode are needed.Therefore, the purpose of this in vitro study was to evaluate the accuracy of AM by means of internal and marginal adaptation of interim FDPs that are fabricated using two different AM technologies employing validated and nonvalidated printing modes with different resins compared to the interim FDPs manufactured by subtractive CAM method and direct manual method. The null hypotheses were that the interim internal and marginal adaptation would not be influenced by the manufacturing method  and that the internal and marginal adaptation of the AM interim FDPs would not be influenced by the type of 3D printer, the printable resin used, or by the printing mode .Three‐unit posterior interim FDPs were manufactured by means of manual, subtractive manufacturing (milling) and AM replacing the first mandibular molar. Two different vat‐polymerization 3D printers, DLP  and SLA , were used to print specimens out of two different commercially available printable resin materials. The six study groups , materials used, and manufacturing specifications are detailed in Table A mandibular typodont model  was modified by removing the tooth #46 to create an edentulous area. Teeth #45 and #47 then were prepared following the preparation principles for complete‐coverage restorations.A digital impression of the AM master model was taken  and FDPs were CAD  in a full contour shape with 1mm of marginal and axial wall thickness and, 1.5 mm of occlusal thickness. The overall cement space was set to 30 µmThe milled and four AM group specimens were fabricated using the same STL file. The milled FDPs (Mil) were nested and milled with a CAM unit Table . Specime21: the scanning of the preparation (preparation scan); scanning of the specimen, intaglio and outer surface of the FDPs (FDP scan); scanning of the assembly, and the FDP was positioned on the master model (assembly scan) . An anti‐reflective powder coating  was sprayed at a fixed distance of 20 cm on the preparation, intaglio and outer surfaces of the specimens. The accuracy of the scanner was reported as 4 µm by the manufacturer. The scanning was made following a modified triple scan methodInitially, the preparation scan was imported, and the abutment surfaces of the preparation scan were separated applying three surface curves and corresponding sections in the inspection software  to create 3 areas‐‐ marginal, axial and occlusal Fig . The marThe internal and marginal adaptation of the FDPs at three defined areas  for each abutment tooth (premolar and molar) were measured at multiple points throughout the surface using the surface comparison option  Fig . The numSpe) which corresponded to the level of homogeneity of the internal and marginal space throughout the inspection surfaces.The single point measurements were exported to a software program  and were used to calculate the mean space between the marginal, intaglio , and total areas of the abutment teeth. The standard deviations for each specimen per inspection site were also calculated   Figs  and 5. Rp < 0.05).The mean, standard deviation (SD), median and interquartile range (IQR) of mean internal  and marginal spaces and their corresponding mean deviations for study groups were analyzed using descriptive statistics. The mean internal and marginal spaces were tested for normality by Shapiro‐Wilk test. Kruskal‐Wallis test was used for comparisons of internal and marginal mean spaces in between the groups. A Dunn post hoc test was later applied to detect the differences across the groups for each site of abutment teeth (p < 0.05). Mann Whitney U and t‐tests were used to analyze and compare the total 3D adaptation and the mean deviations of the AM interim FDPs fabricated by two 3D printers. The statistical analysis was performed using a statistical software program A general linear model using mean deviations as the dependent variable and tooth type and tooth site as fixed effect factors was run. One‐way analysis of variance (ANOVA) test was further performed to highlight differences between the groups for total surface mean deviations. Tukey B post‐hoc test was done to identify the groups that shows significant differences. , and two abutment teeth (premolar and molar) are shown in Table p < 0.05).Marginal space results of FL‐Sh and RS‐Sh groups were similar to Mil group and exhibited significantly better marginal adaptation than FL‐St, RS‐St, and Man groups for both premolar and molar abutment teeth analysis Table . The resp < 0.05)  , and the interim material influenced the total 3D adaptation of the interim FDPs at the molar abutment. Mil and FL‐Sh specimens were significantly better adapted than Man and RS‐St specimens  exhibited better adaptation results compared to Man and RS‐St groups. The difference between the manufacturing methods at the premolar site was not significant. Therefore, the first null hypothesis was partially rejected. Similar if not smaller marginal mean space results were obtained from the FL‐Sh group compared to Mil specimens, thus AM can be regarded as a promising alternative to milling for interim FDPs.Previous investigations on comparison of the accuracy of milling and AM reported similar if not better marginal and internal adaptation for AM interim restorations,The printer accuracy is determined by the resolution of the x‐, y‐, and z‐axis, which is related to the characteristics of the printer's light source. Contrary to the x‐ and y‐axis resolution, the resolution along the z‐axis is modifiable depending on the material and determines the layer thickness. The z‐axis resolution is reported to influence the degree of conversion which showed better results with lower layer thicknesses and eventually reduced the distortion due to photopolymerization shrinkage.Spe were compared. In other words, the nonvalidated printing mode showed higher mean accuracy results with better predictability and repeatability. The mechanical stability results based on the second part of this investigationPrinting parameters such as layer thickness, shrinkage between layers,Additionally, the influence of the AM technology , on the accuracy of AM dental devices should not be overlooked.The triple scan method is a validated nondestructive alternative to the replica method.The results of this study should be interpreted with care as the in vitro design has limitations to reflect all clinically relevant factors, the limited number of interim dental materials and manufacturing procedures tested, and the specific setting printing parameters. Further in vitro and clinical trials are needed to broaden the analysis of the usability of additively manufactured interim FDPs.Within the limitations of this in vitro study, the AM interim FDPs demonstrated acceptable accuracy results, similar to milled ones. The printing mode, resin, and the printing technology used have influenced the printing accuracy of the interim FDPs, particularly at the marginal area. The use of nonvalidated printing mode with lower‐cost 3D printers, can be an interesting solution for clinical applications.Authors declare no conflict of interest.


However, the group of patients taking RAAS inhibitor drugs appeared to be more critical at ICU admission, and this difference became statistically significant in the subgroup of non‐hypertensive patients. ICU mortality in the subgroup of non‐hypertensive patients treated with RAAS inhibitor drugs also tended to be higher. Overexpression of the angiotensin‐converting enzyme 2 (ACE2) in human cells, induced by RAAS inhibitor drugs, promotes viral entry‐replication of SARS‐CoV‐2 and alters the basal balance of the RAAS, which may explain the findings observed in the present study. These phenomena may be amplified in non‐hypertensive patients treated with RAAS inhibitor therapy.Previous studies suggested that ongoing treatment with renin–angiotensin–aldosterone system (RAAS) inhibitor drugs may alter the course of SARS‐CoV‐2 infection and promote the development of more severe forms of the disease. The authors conducted a comparative, observational study to retrospectively analyze data collected from 394 patients admitted to ICU due to SARS‐CoV‐2 pneumonia. The primary aim of the study was to establish an association between the use of RAAS inhibitor drugs and mortality in the ICU. The secondary aims of the study were to establish an association between the use of RAAS inhibitor drugs and clinical severity at ICU admission, the need for tracheal intubation, total days of mechanical ventilation, and the ICU length of stay. The authors found no statistically significant difference in ICU mortality between patients on RAAS inhibitor drugs at admission and those who were not  in human cells, induced by RAAS inhibitor drugs, promotes viral entry‐replication of SARS‐CoV‐2 and alters the basal balance of the RAAS, which may explain the more severe course of COVID‐19 suggested by the results of this study. These phenomena may be amplified in non‐hypertensive patients treated with RAAS inhibitor therapy. Major efforts are underway to discover effective treatments for the virus and the disease it produces .In a scenario where SARS‐CoV‐2 becomes endemic, it is likely that some patients would still develop severe forms of the disease, such as bilateral interstitial pneumonia and ARDS, and it is important to identify manageable factors that could influence this unfavorable clinical course. This knowledge could also be applied to other coronaviruses now and in the future.Basic research has already demonstrated that the angiotensin‐converting enzyme 2 (ACE2) serves as a specific target for SARS‐CoV‐2 and that the lung appears to be the most vulnerable target organ , the need for tracheal intubation, the total days of mechanical ventilation, and the ICU length of stay (LOS).We conducted a comparative, observational study to retrospectively analyze data collected from the records of patients hospitalized in adult ICUs of the Geneva University Hospitals  and the Sion Hospital  between 01.03.2020 and 31.05.2021 due to SARS‐CoV‐2 pneumonia. We excluded patients who tested positive for SARS‐CoV‐2 but were admitted to the ICU for another medical reason unrelated to SARS‐CoV‐2 pneumonia as well as patients transferred to other units and/or hospitals during their stay and having incomplete data. Patients on RAAS inhibitor therapy at hospital admission were compared with patients not taking RAAS inhibitor drugs. The primary aim of the study was to establish an association between the use of RAAS inhibitor drugs and ICU mortality in patients admitted due to SARS‐CoV‐2 pneumonia. The secondary aims of the study were to establish an association between the use of RAAS inhibitor drugs and clinical severity at ICU admission .For data analysis, the Mann–Whitney U test was used to compare continuous variables between both groups. Chi‐squared or Fisher's exact test was used for categorical variables as appropriate. To investigate the association between RAAS inhibitor drugs and ICU mortality, we performed a multivariable logistic regression adjusting the estimates for SAPS II and the presence of hypertension, heart disease, or diabetes mellitus in the clinical history of the patient. The results of the logistic regression are expressed as odds ratios (OR) and 95% confidence intervals (CI 95%). Two‐tailed 3p < 0.01), had a higher prevalence of diabetes mellitus , hypertension , or cardiomyopathy  than patients not taking a RAAS inhibitor.Of the 394 patients admitted to the adult ICUs of the Geneva University Hospitals and the Sion Hospital during the study period due to SARS‐CoV‐2 pneumonia, 150 patients (38.1%) were on RAAS inhibitor therapy at the time of hospital admission. Patients on RAAS inhibitor therapy were older . This difference becomes statistically significant in the subgroup of non‐hypertensive patients   Table .p 0.06).Moreover, ICU mortality in the subgroup of non‐hypertensive patients treated with RAAS inhibitors tended to be higher  and angiotensin II (Ang II) into the vasodilator fragments angiotensin 1–9 and angiotensin 1–7, respectively, while decreasing the concentration of Ang II to further facilitate vasodilatory and anti‐hypertensive effects  or protein levels and ACE2 activity, particularly for angiotensin receptor blockers , Genève (Switzerland) – BASEC number: 2020–00917. Regional Research Ethics Committee (CCER), Vaud (Switzerland) – BASEC number: 2021–01299.


Villagers reported known risk factors for ASFV transmission – such as free‐ranging management systems and wild boar access – in all four villages. The villagers reported increased pig trader activity from Vietnam before the outbreaks; however, the survey did not determine a single outbreak source. The outbreak caused substantial household financial losses with an average of nine pigs lost to the disease, and Monte Carlo analysis estimated this to be USD 215 per household. ASFV poses a significant threat to food and financial security in smallholder communities such as Thapangtong, where 40.6% of the district's population are affected by poverty. This study shows ASFV management in the region will require increased local government resources, knowledge of informal trader activity and wild boar monitoring alongside education and support to address intra‐village risk factors such as free‐ranging, incorrect waste disposal and swill feeding.African swine fever virus (ASFV) causes a deadly disease of pigs which spread through southeast Asia in 2019. We investigated one of the first outbreaks of ASFV in Lao People's Democratic Republic amongst smallholder villages of Thapangtong District, Savannakhet Province. In this study, two ASFV affected villages were compared to two unaffected villages. Evidence of ASFV‐like clinical signs appeared in pig herds as early as May 2019, with median epidemic days on 1 and 18 June in the two villages, respectively. Using participatory epidemiology mapping techniques, we found statistically significant spatial clustering in both outbreaks ( ASFV is a DNA virus present in all secretions, blood and tissues of affected animals , a layperson trained in basic animal health management who reports to their District Agriculture and Forestry Office (DAFO). The DAFO communicates with their local Provincial Agriculture and Forestry Office (PAFO), which then informs the Department of Livestock and Fisheries (DLF) and the National Animal Health Laboratory (NAHL) in Vientiane. The DLF handled epidemiology and control measures, while the NAHL performed the laboratory‐based diagnosis of ASFV . The NAHL used the TaqMan quantitative real‐time polymerase chain reaction (rt‐PCR) for confirmation of cases  was provided by the acting head of the Savannakhet PAFO Livestock division through semi‐structured interviews conducted in English followed by a written survey.Village Chiefs (VC) and VVW first reported abnormal pig deaths in Densateung and Phouphanang‐Khampia in late May–early June 2019 to the Thapangtong DAFO. The DAFO then reported these deaths to the Savannakhet PAFO on 25 June 2019. Together the PAFO and DAFO investigated the cases on 29 June 2019 .PAFO staff collected whole blood from between 1 and 4 pigs per village, using jugular venepuncture on live animals showing clinical signs. In each of the two affected villages, the PAFO team collected all samples from a single household. The samples were transported by land from the Savannakhet PAFO to the NAHL in Vientiane . Following formal diagnosis from NAHL, PAFO and DAFO staff began control activities on 3 July. They completed stamping out measures in the two affected villages by 6 July 2019, and movement controls in the 5 km surrounding the district continued until early August 2019 .2.2This study was conducted in the Thapangtong district of Savannakhet province, which was the second location in Lao PDR to report an ASFV outbreak and is adjacent to Salavane province, where the first outbreak occurred. For this study, an ‘affected village’ was defined as a village with one or more PCR‐confirmed ASF cases. An ‘affected household’ was defined as a household that owned one or more pigs with clinical signs of ASFV in an ‘affected village’ during the high‐risk period until the end of the DLF investigation of the outbreak. Not all affected households were PCR‐confirmed.The ‘high‐risk period’ was when the ASFV outbreak might have existed in the affected villages including the time before the first report from the Thapangtong DAFO to Savannakhet PAFO. Based on farmer interviews, clinical signs and laboratory findings, this period was estimated to be from 1 May to 2 July 2019. The period prior to viral detection was changed from the Nurmoja et al.  approachOf the district's three villages with confirmed cases (as at mid‐September 2019), two of similar size, Densateung and Phouphanang‐Khampia, were chosen for this study. The NAHL had confirmed the Densateung and Phouphanang‐Khampia outbreaks on 1 July 2019. Due to the high reported pig mortality rate in the affected villages, two unaffected villages, Napaxard and Xaysomboun, were selected as controls. The control villages had healthy pig populations at the time of the survey, were of similar human population size and close to the same major road as the affected villages. The study, although initially designed for traditional risk factor analysis, was changed to one that was descriptive about the impact and spread of the disease at the household level, while describing management practices at the village level. The number of surveys per village was set at 25 for simplicity of study design as a protocol needed to be created for both case and control villages.2.3The survey had two phases: a pilot followed by a final questionnaire. Questions found to be poorly understood or in need of additional information in the pilot were adapted and included in the final questionnaire. An independent company, experienced in medical and agricultural translations, translated the questionnaire into Lao, then NAHL staff experienced in animal health extension programs back‐translated the questionnaire into English for confirmation. The questionnaire included 28 questions on how many animals they owned and their value in Lao Kip (LAK); purchasing/selling behaviour; biosecurity practices; pig management practices and pig health practices. Where literature existed about possible answers (such as housing methods and feeding), the question styles were closed. Where no literature existed, a short structured‐open question was used, such as ’How do you normally dispose of household food scraps?’ Instructions for the interviewers to guide the questioning style added clarity. The questionnaire covered the recent history of disease outbreaks in the village, including the number of animals affected and when they were affected.Subjects were chosen from all the pig‐raising households in the selected villages. In Densateung and Phouphanang‐Khampia, almost all households were ASFV‐affected Table , disease2.4After the individual surveys, villagers worked with the investigators to map their village, marking their households' locations, significant landmarks and known areas of wild boar activity. This map was hand‐drawn on a large sheet of paper, and each household represented in the survey contributed to the development of the maps.2.5glmer function and the binomial logit method  was used as a prior in the 2.7We mapped the outbreak to investigate the spatial component of disease spread in the village. The map data were analyzed with a space–time permutation (STP) scan statistic  to full‐time enclosures (n = 19), some of the latter being communal rather than private. Of note were the farmers who kept their pigs in enclosures near their rice paddies (n = 6) some distance from the village, which removed their pigs from the village ecosystem (Table n = 35 villagers confirmed contact), including with neighbours' pigs and feral pigs or wild boar. In Lao PDR, feral pigs and Eurasian wild boar are called muu paa (forest pig), and both closely resemble the domestic village pigs.Sampled farmers listed pig housing methods with a range of biosecurity levels, from all‐day free ranging (em Table . Reporten = 2) reported feeding pork or kitchen swill to their pigs. All surveyed farmers reported feeding a mixture of rice bran and the water used to prepare sticky rice as the pigs' primary diet. Water sources (other than the rice water) included household water supplies, communal wells and rivers. Of the farmers surveyed, 79 used a communal water source for their pigs and 17 used private water sources. When asked an open‐ended question about how they disposed of their kitchen rubbish, farmers gave various responses, including burying waste. However, the most common method was to burn kitchen waste. Most surveyed households butchered animals inside the house after slaughter, but 14.9% butchered animals outside the house. Many farmers gave the leftover bones to their dogs (50.7%). Another possible transmission source was using the same syringes and needles to treat multiple sick animals during the outbreak as reported by the VVWs. Several farmers attempted antibiotic therapy, and during a semi‐structured interview a VVW explained that they sometimes washed the syringe with soap and water between uses rather than disposing of the syringe.Only two farmers (3.2n = 41), 330 pigs died with clinical signs of ASFV during the high‐risk period. No pigs died in the control villages during the same period.We surveyed 49 ASFV‐affected households and 50 control households. Of these 99 households, eight households surveyed in the ‘affected villages’ did not meet the definition of an ‘affected household’ (outlined in the Section 2). These households were not included in calculations relating to outbreak characteristics, outbreak losses, spatial modelling or epidemic statistics. However, these eight households were included for the purposes of describing management styles and practices. Across the ASFV‐affected households surveyed (3.3n = 41). The majority of pigs died and were either buried or burned rather than culled, and only deaths recorded on or after 3 July were culled and buried by authorities.During the household surveys, an obvious route of disease entry did not become apparent. Direct contact through the purchase of an infected pig seems unlikely as none of the affected farmers in this survey purchased new pigs in the high‐risk period or the 4 weeks prior. However, all (both affected and unaffected) reported Vietnamese pork traders during the risk period. Farmers were asked about their initial diagnosis, and 21% identified the cause of the deaths as a seasonal disease. However, many were unsure of the cause of the sudden increase in pig deaths (51%). The VVWs were also uncertain about what disease was causing the outbreak. An average of nine pigs died or were culled in affected households surveyed (n = 330), the most common early clinical signs were depression (21.5%), fever (15%), inappetence (15%) and shivering/trembling (15%). Late clinical signs included seizures/convulsions (21.1%), shivering (11.1%) and ‘looking cold’ (6.7%). Many farmers noted death or sudden death (28.9%). The median clinical interval from onset of clinical signs to death was less than one day (IQR = 2 days), meaning that farmers observed their pigs becoming sick and dying within 24 h. The mean clinical interval was 4.4 days (SD ± 6.1). In Densateung, the median epidemic day was 1 June 2019, with an interquartile range (IQR) of 35 days. In Phouphanang‐Khampia, the median epidemic day was 18 June 2019 with an IQR of 5.5 days  were kept in enclosures adjacent to rice paddies and were therefore not included in the stamping‐out measures.Of the affected animals . This is likely because smaller herds were significantly associated with the two control villages, Napaxard (p < 0.05) and Xaysomboun (p < 0.001), while the two ASFV‐affected villages had more households with larger herds.The quasi‐complete separation of ASFV outcome by the villages made the data unsuitable for logistic regression  with SEM ± USD26.85  were detected in Densateung village. The first cluster noted ASFV symptoms in the second week of May 2019 and was the earliest cluster affected in the Thapangtong region   Figure . The fir4This study describes the epidemiologic characteristics, including financial losses, associated with ASFV outbreaks in selected villages in Lao PDR. The study highlights knowledge that could be implemented to reduce the impact of ASFV and similar transboundary animal diseases on smallholders in similar resource limiting contexts. By performing this study, we also explored extant challenges and preliminary strategies to reduce the opportunity for inter‐ and intra‐village spread of ASF. These strategies will benefit policymakers and researchers beyond ASFV in the control of other high‐impact and zoonotic diseases.The major potential pathways for introducing ASFV discussed here include traders of live pigs/pig products, iatrogenic spread and wild boar. This study did not identify any single, obvious route of ASFV entry into the villages. However, many plausible hypotheses present themselves, and all should be addressed in future disease prevention activities. The study made obvious that conditions within the villages were ideal for the spread of ASFV. A combination of inter‐ and intra‐village control measures will be required in future to prevent the spread and establishment of ASFV in smallholder communities.A putative source for the ASFV outbreak in southern Lao PDR is the ASFV outbreak in Vietnam that began in early 2019. Both Thapangtong district and the first‐affected Toomlan district are on the same major road to Vietnam. Despite a lack of evidence that any ASFV infected live pigs were purchased from traders in the high‐risk period or the month prior, the reports of Vietnamese traders suggest increased activity from a region known to have had ASFV in that same period. Whilst the traders did not sell the villagers any pigs, the traders would have been able to contaminate the villages with ASFV contaminated pork meat products, pig wastes from trucks or even by dropping off contaminated carcasses. Previous social network analyses in the Northern Province of Xayabouri suggest that semi‐commercial piggeries interact almost exclusively with 1–2 traders  and the number of animals with ASFV‐like clinical signs in the ‘high risk period’ (n = 330). The reported clinical signs, whilst typical of acute and peracute ASFV are also typical of classical swine fever, erysipelas, salmonellosis, and highly pathogenic porcine respiratory and reproductive syndrome, all of which are endemic to Lao PDR. Only five animals were sampled and definitively diagnosed as ASFV cases using PCR, highlighting difficulties in a centralized testing system. In the pilot survey, several farmers reported that pig disease was common during June and July (Lao PDR' wet season). They initially thought the deaths were due to this endemic disease syndrome. The familiar clinical signs may have also delayed reporting and action. None of the villagers included ASFV in their initial diagnoses for the pig deaths despite information materials being made available to local authorities by the OIE in early 2019.ASFV outbreaks require prompt and thorough investigation. The epidemiologic findings suggest that ASFV was well established in the two villages before local authorities were able to act. The disease notification system used by the DLF (outlined in Section 2.1) relies on VVWs to identify and report cases to the DAFO, reporting to the PAFO for investigation. There are no standardized processes across the provinces, and funding for disease outbreak investigation is limited to the private veterinary incomes of the PAFO and DAFO staff. Weaknesses in this ‘ground‐up’ reporting approach emerged in the 2015 Vientiane FMD outbreak where numerous FMD‐affected villages that were presumed to be ‘FMD free’ by DAFO due to no reports from VVWs, yet retrospective serology determined otherwise (Miller et al., The sampling strategy described here allowed us to speak with many household representatives in each village; however, it has its limitations. Households were selected from a list provided by the VC, who may have favoured owners that were more educated, more receptive to government communications, owned more pigs or had positive management traits, leading to selection bias. Herd size skewed right across all villages, with most households owning small herds of three to four pigs and a few exceptional individuals owning larger herds. The ASFV‐affected villages had more households with large herds than the control villages Table . The relDiscussions on the impact of the 2019 ASFV outbreak on global markets have focused on pork prices, demand for alternative sources of protein and demand for intensive livestock feed products such as soya beans (Croz et al., The findings of this study should be utilized in future decisions about the management of ASFV in the region. Trader, VVW and villager behaviour must be managed and control measures put into place for contact between village pigs and wild boar. The resources available to local government authorities must be assessed for them to act promptly in cases of emergency disease outbreaks. When designing control and education strategies, local farming practices, as well as the disease ecology must be considered together in order to develop effective materials to aid in the prevention and management of ASFV outbreaks into the future.The authors declare that there were no conflicts of interest in the development of this work.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. Ethics approval for the surveys was obtained from the University of Sydney Human Ethics committee, approval number [2019/725].


Antirrhinum majus) and many other plant species. Here, we tested for the reproducibility of signals extrapolated from germination results between open and understorey microhabitats in two parapatric snapdragon plant subspecies (A. m. striatum and A. m. pseudomajus) characterized by a similar elevation range by using common garden experiments at different elevations. Signals observed under one microhabitat systematically differed in the other. Most scenarios could be inferred, with signals either shifting, appearing or disappearing between different environments. Our findings imply that caution should be taken when extrapolating the evolutionary significance of these types of experimental signals because they are not stable from one local environmental condition to the next. Forecasting the ability of plants to adapt to environmental changes based on common garden and reciprocal transplant experiments must account for the multivariate nature of the environment.Experimental studies on local adaptation rarely investigate how different environmental variables might modify signals of adaptation or maladaptation. In plant common garden experiments, signals of adaptation or maladaptation to elevation are usually investigated in open habitats under full light. However, most plants inhabit heterogeneous habitats where environmental conditions differ. Understorey microhabitats are common and differ in terms of tree shade, temperature, water availability, microbiota, allelochemicals etc. Germination is a fitness‐related trait of major importance for the adaptation of plants to contrasted climate conditions. It is affected by shade in snapdragon plants ( Antirrhinum majus) common garden experiments differ between microhabitats (open habitat and understorey), thereby suggesting a lack of reproducibility of adaptation signals obtained in simplified experimental environments.Potential signals of adaptation to elevation obtained in snapdragon plants ( Each common garden experiment was conducted in an open microhabitat under full light and was reproduced in another type of microhabitat under the shade of the canopy of understorey trees, where the temperature is usually lower and water availability usually higher. This is a proof of concept study where the focus was made on two germination‐related traits—seed germination and timing of germination—widely recognized for their role in plant adaptation . We conducted common garden experiments at high and low elevation on snapdragon plants (A. m. ssp. striatum and ssp. pseudomajus) that inhabit closely similar ecological niches in the south of France and are characterized by a similar elevation range  inhabiting exclusively heterogeneous habitats occurring between 6 and 1564 m  is a hermaphroditic, self‐incompatible, short‐lived perennial species producing inflorescences and small seeds dispersed by gravity a few metres apart from the plant  were sampled in 2011  were the same, so that the four gardens (open habitat and understorey in both low‐ and high‐elevation sites) were composed by a similar gene pool. In every garden, every seed family was represented by six or seven seeds Table . This ex2.5Seeds from every population originating from each of the low‐ (from sea level to 750 m) and high‐elevation strata (from 750 to 1600 m) were set to germinate in two sites Figure . One sit2.6Picea abies), silver fir spruce , beech (Fagus sylvatica) etc. at high elevation and European chestnut (Castanea sativa), common hazel (Coryllus avellana), European filbert (Cornus sanguinea) etc. at low elevation. On each site, the common gardens in different microhabitats (open habitat and understory) were within 200 m of each other to keep the local environmental conditions that are not affected by microhabitat variation as similar as possible. Along with the shade induced by understorey vegetation, soil moisture, temperature and biotic interactions  were undoubtedly different between the two types of microhabitat. Open and understorey microhabitats were artificially separated in our experimental design, whereas in nature, snapdragon plants germinate and develop in heterogeneous habitats where both these types of conditions are mixed. Differential adaptation to these microhabitats between populations was therefore not expected. We estimated the contrast between microhabitat types by measuring the photosynthetically active radiation (PAR) in each common garden, which was strongly and significantly reduced in the understorey . On each site, seeds were exposed to the two different microhabitats: open habitat under full light and understorey trees providing shade. We used natural vegetation to induce differential environmental conditions based on the presence and absence of understorey trees: common spruce  and the time to germination . We monitored germination in common gardens three times per week during the spring and summer 2015 (from June to October). In accordance with the current knowledge on the species, we considered that seeds not germinating during the first year harboured no potential for germination in future years and did not play a role in a particular ecological strategy of delayed germination across seasons.2.8p values  on germination‐related traits for each subspecies separately. The fixed effects included the site elevation, the elevation of origin of the populations , the microhabitat ‘treatment’ (‘open habitat’ vs. ‘understorey’) and their interactions. The population and the family were included as random effects. Traits were modelled differently depending on their distribution: (a) germination  was modelled as a binomial variable (with a logit link function) and (b) the time to germination was modelled as a Gaussian variable . We presented 95% confidence intervals and used their lack of overlap with zero or lack of overlap between treatments to define what effects were statistically significant in our GLMM approaches, and this is a conservative approach that provides information on the statistical effects  that is not provided by i.e. phenotypic responses of same genotypes between high‐ and low‐elevation gardens) of populations originating from high‐ and low‐elevation habitats is the adaptive signal for measurements monotonically related to fitness. Since our aim was not to assess adaptation but to evaluate the potential modification of this type of adaptive signals by an additional environmental variable, we also tested for interaction effects with the microhabitat. We also represented and tested for the significance of effects analysed in isolation; for example high vs low elevation of origin within each garden by using Wilcoxon tests (non‐parametric data). We checked that the results were consistent across populations originating from a same elevation  of potential adaptive scenarios drawn from common garden experiments comparing multiple populations at different elevations. Opposite signals were found that suggest heterogeneous selection inside wild snapdragon populations where plants are generally exposed to mixed microhabitat conditions. Experimental signals of plant adaptation detected in common garden studies are usually obtained in open habitat under full‐light conditions. Lack of reproducibility between microhabitats is likely to affect conclusions from similar assessments conducted in other species naturally occurring in heterogeneous environments. Our findings imply that adaptive scenarios built upon the experimental simplification of the natural environment must be discussed with a lot of caution. They also imply that forecasting the ability of plants to adapt to environmental changes based on common garden and reciprocal transplant experiments must account for the multivariate nature of the environment.The authors of this article declare that they have no financial conflict of interest with the content of this article.BP designed the research programme. SM, JA, MI, GO, AlG and BP carried out the experiments; AG and PM analysed the data; AG and BP wrote the manuscript.https://publons.com/publon/10.1111/jeb.13973.The peer review history for this article is available at Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.