Research suggests that diabetic peripheral neuropathy (DPN) is related to plasma fibrinogen (Fib) concentrations, although its correlation with Fib function has not been reported. Here, the k value and angle α, reflecting the plasma Fib function, were used to analyse its correlation with DPN, and their potential as biological indicators for diagnosing DPN was explored.This prospective observational clinical study enrolled 561 type 2 diabetes mellitus (T2DM) patients, who were divided into the diabetes with symptomatic neuropathy (161 cases), diabetes with asymptomatic neuropathy (132 cases) and diabetes with no neuropathy (268 cases) groups. Meanwhile, 160 healthy unrelated subjects were recruited as controls.P < 0.001; odds ratio 1.131 [1.063–1.204], P < 0.001). The k value and angle α levels were closely correlated with neuropathy stage . The optimal cut-off point for k value levels to distinguish patients with diabetic neuropathy from those without was 1.8 min, with a sensitivity of 73.7% and a specificity of 83.2% (AUC = 0.873). The optimal cut-off point for angle α levels was 60°, with a sensitivity of 41.0% and a specificity of 95.6% (AUC = 0.669).Fib levels increased slightly in diabetic subjects with neuropathy compared with those without. The angle α levels increased slightly in subjects with asymptomatic DPN compared with those with no neuropathy and increased greatly in subjects with symptomatic DPN compared with those without. The k value levels slightly decreased in subjects with asymptomatic DPN compared with those with no neuropathy and greatly decreased in subjects with symptomatic DPN compared with those without. The association of the k value and angle α with diabetic neuropathy was independent of the hyperglycaemic state and other potential confounders (odds ratio 0.080 [0.051–0.124], The k value and angle α are closely associated with DPN. The levels of the k value and angle α may be helpful in the early diagnosis of DPN. Diabetes mellitus (DM) is the fifth leading cause of death in the world, with an average global prevalence rate of 8.6% –3. DiabeThe currently accepted and reliable methods for the diagnosis of DPN in the clinic are neuroelectrophysiological examination and small neurofibropathy examination , 6. HoweAt present, DPN is known to be associated with oxidative stress, metabolic abnormalities, and immune and inflammatory responses . At the A total of 561 subjects who met the 1999 World Health Organization (WHO) type 2 diabetes diagnostic criteria and were registered consecutively as outpatients or inpatients with our hospital along with 160 healthy control subjects registered with our hospital physical examination centre between August 2018 and March 2021 were randomly enrolled in the study. All volunteers signed informed consent. The study was approved by the hospital and university scientific and ethics committees. The exclusion criteria were age < 20 years or  > 75 years, severe liver or kidney damage, trauma, surgery, tumour, acute infection, pregnancy or lactation, diabetic ketosis, blood disease, long-term alcohol abuse, other nondiabetic causes  that could cause neurological damage, and other diseases that may be confused with the clinical symptoms of DPN, such as vitamin deficiency, osteoarthritis, peripheral vascular disease, and trauma surgery. The inclusion criteria for healthy control subjects included no history of diabetes, fasting blood glucose < 5.6 mmol/L, and glycated haemoglobin < 5.6%.Testing was performed on each participant by the same experienced physician according to standard procedure. All tests were conducted in a quiet laboratory. First, all patients had a complete history of neurological symptoms taken and were given a physical examination.For somatic and cardioautonomic neuropathy, symptoms were documented, including numbness, paraesthesia, burning, deep aching, unsteadiness in walking, unexplained resting tachycardia and postural fainting .The assessment by a professional medical staff member  included 10 g nylon wire (pressure sense), tuning fork (vibration sense), temperature sense, acupuncture pain, and tendon reflex; positive findings on 2 of the 5 tests indicate abnormal signs of the nervous system , 20.All patients were examined using an electromyography (EMG) instrument . All subjects were tested in a quiet environment. The distal latency (DML), motor conduction velocity (MCV) and motor nerve conduction amplitude (CMAP) of the bilateral median nerve, ulnar nerve, tibial nerve and common peroneal nerves were detected. The sensory conduction velocity (SCV) and sensory nerve action potential amplitude (SNAP) of the bilateral median nerve, ulnar nerve, superficial peroneal nerve and sural nerve were detected. At the same time, the bilateral tibial nerve H reflex and the ulnar nerve F wave latency were detected.2. The right-arm blood pressure of each seated subject was obtained after 10 min of rest using a mercury sphygmomanometer. Retinal conditions were evaluated by ophthalmologists using a combination of clinical examination, stereoscopic retinal photographs, optical coherence tomography and fluorescein angiography.Body weight and upright height were measured on the same scales and wall-mounted stadiometer in light clothing without shoes before breakfast. Individual BMI was then calculated as weight (kg)/height (m)All subjects stopped anticoagulant and antiplatelet drug use 2 weeks prior, and venous blood was collected in the morning from the antecubital vein after the subjects fasted for 10 to 12 h. The k value and angle α were assessed by a thromboelastography analyser . Fib was measured using a blood coagulation meter . Fasting plasma glucose, serum creatinine, blood lipids, and liver and kidney function were measured by an automatic biochemical analyser . HbA1c was measured using high-performance liquid chromatography . Serum vitamin B12 was measured using an automated assay . The urinary albumin concentration was assessed using immunonephelometry . The urinary creatinine concentration was quantified using the alkaline picrate method. The individual urinary albumin-creatinine ratio (UACR) was then calculated as albumin (mg)/creatinine (g). Endogenous creatine clearance (Ccr) was calculated to estimate the glomerular filtration rate according to the Cockcroft equation: Ccr = {[140 – age (years) × body weight (kg)]/[0.818 × serum creatinine ]} for males and × 0.85 for females.Diabetic neuropathy was diagnosed according to the American Diabetes Association recommendation . PolyneuP < 0.05 was considered indicative of statistical significance.We used SPSS version 19 for statistical analysis. The data are expressed as the mean (SD) for normally distributed data. The chi-square test was used to compare the count data. Multiple comparisons among groups were assessed using one-way analysis and comparisons between two groups (LSD method) for variables. A t test was used for comparison between the two groups. k value and angle α were later added to a logistic regression model, controlling for possible confounders. The relation of the k value and angle α levels to the stages of neuropathy was calculated using Spearman’s correlation analysis. Receiver operating characteristic (ROC) analysis was conducted with MedCalc Software version 15.2 to assess the accuracy of serum k values and angle α levels in distinguishing between patients with and without diabetic neuropathy. The optimal cut-off point was identified by calculating the area under the curve (AUC). P = 0.191). The k value was slightly reduced in the subclinical DPN group (group B) (P < 0.001) and further decreased in the confirmed DPN group (group A) (P < 0.001). Compared with the normal control group (group D), the angle α levels were not significantly increased in the diabetic group without DPN (group C) (P = 0.404), but there was a significant increase in the subclinical group (group B) and the confirmed DPN group (group A) (P < 0.001). Moreover, the confirmed DPN group (group A) had significantly higher angle α levels than the subclinical DPN group (group B) (P = 0.002) . Correspondingly, k value levels were negatively correlated with diabetic peripheral neuropathy , and angle α levels were positively correlated with diabetic peripheral neuropathy . Fib levels were also correlated with diabetic peripheral neuropathy  (Table P < 0.001), and 58.4° for the angle α with a sensitivity of 95.6%, a specificity of 41.0%, and the highest AUC equal to 0.669 (P < 0.001)  P = 0.04, but tThe results of this study demonstrated that the k value and angle α are potential biomarkers of DPN. In this study, patients with DPN had lower k values than diabetic patients without peripheral neuropathy, and the levels of angle α in patients with DPN were significantly higher than those in diabetic patients without peripheral neuropathy. More importantly, the k value and angle α were changed in the early stage of diabetic peripheral neuropathy .DPN refers to clinical and/or electrophysiological evidence of peripheral neuropathy in patients with a confirmed diagnosis of diabetes, excluding other diseases. The incidence of DPN can be as high as 60 to 90%, and the incidence rate reported in China is as high as 85%. At present, the pathogenesis of DPN remains unclear. Many studies suggest that DPN is associated with abnormal metabolic pathways, microvascular disease, nerve growth factor, autoimmunity, inflammation and oxidative stress. Among them, metabolic pathway abnormalities and microvascular changes are considered to play an important role in the occurrence and development of DPN , 23. A lDiabetic peripheral neuropathy has a high incidence, and its clinical symptoms are also diverse. Due to the low degree of neurological damage in the early stage of DPN, typical symptoms and signs are not evident, and no symptoms may be present. This is easily overlooked, and once the disease progresses, there may be adverse outcomes such as diabetic foot . TherefoAt present, the most commonly used methods for the diagnosis of DPN are neuromuscular electromyography , nerve biopsy, the Toronto clinical score and the Michigan neuropathy screening table, all of which have limitations and low clinical support. According to different screening methods, the prevalence of DPN was 2.4–74.8% . In contThis study further confirmed that abnormal blood coagulation may lead to the occurrence and development of diabetic peripheral neuropathy. The k value, angle α and Fib serve as indicators of coagulation function and may serve as potential blood biomarkers for the diagnosis and treatment of diabetic peripheral neuropathy. Importantly, the k value and angle α may be helpful for the early diagnosis of diabetic peripheral neuropathy. Clinicians can monitor the k value, angle α and Fib to detect diabetic peripheral neuropathy in time. When the monitoring indicators are abnormal, adding drugs to improve blood viscosity on the basis of diabetes treatment may have a certain role in preventing and delaying the occurrence and progression of diabetic neuropathy, benefitting the patients. However, there are still some limitations to this study. The sample size of this study was not large, which may have had an impact on the research. Further comprehensive research with large sample sizes is needed.


Multiple logistic regression analysis in the fully adjusted model revealed both BMI‐z  and PFF  were independent risk factors for hypertension. In conclusions, in addition to BMI‐z, ectopic pancreatic fat is an important risk factor for hypertension in children with NAFLD.Although nonalcoholic fatty liver disease (NAFLD) is known to be a risk factor for cardiovascular diseases, few studies have reported an association between ectopic fat deposition and metabolic complications, including hypertension, in children with NAFLD. The present study evaluated the risk factors for hypertension in children with NAFLD from the aspect of ectopic fat. This cross‐sectional retrospective study investigated 65 children with NAFLD , who underwent liver biopsy and magnetic resonance imaging‐based fat fraction measurement for ectopic hepatic and pancreatic fats, as well as anthropometry, blood pressure, laboratory tests, and body composition analysis. A logistic regression model was used to identify the risk factors for hypertension. Through a simple logistic regression analysis, age (OR 1.392), BMI‐z (OR 3.971), waist circumference‐to‐height ratio (OR 1.136), fat‐free mass index (OR 1.444), γ‐glutamyl transferase (OR 1.021), quantitative insulin sensitivity check index (OR 0.743), dyslipidemia (OR 5.357), and pancreatic fat fraction (PFF) (OR 1.205) were associated with hypertension. The optimal cut‐off of PFF to divide children with NAFLD into two groups with and without hypertension was 4.39% (area under the curve 0.754,  Correspondingly, obesity‐induced metabolic complications, including nonalcoholic fatty liver disease (NAFLD), cardiovascular diseases (CVD), type 2 diabetes mellitus (DM), and metabolic syndrome (MetS) have also increased.Mounting evidence highlights that NAFLD is an independent risk factor for CVD in adults. However, to date, this association has not been fully substantiated in children with NAFLD.When circulating triglycerides (TG) and free fatty acid levels exceed the metabolic capacity through adipocyte hypertrophy and hyperplasia, ectopic fat accumulation in non‐adipose tissues such as the heart, liver, pancreas, and skeletal muscles in patients with obesity becomes an increasing risk.Along with ectopic hepatic fat in NAFLD, pancreatic fat is also considered as an obesity‐induced ectopic fat depot, which may contribute to cardio‐metabolic disturbances such as NAFLD, HTN, DM, dyslipidemia, and MetS.Therefore, the present study aimed to investigate the risk factors associated with HTN in pediatric patients with NAFLD from the aspect of ectopic fat.22.1This retrospective cross‐sectional observational study included 65 children and adolescents with liver biopsy‐proven NAFLD who underwent both liver biopsy and abdominal MRI performed within 2 days from March 2014 through February 2020. All children underwent anthropometric measurements, blood pressure (BP), body composition, and laboratory evaluation tests. NAFLD was diagnosed as the presence of steatosis in ≥5% of hepatocytes in the absence of evidence of other causes of liver disease. Secondary etiologies of hepatic steatosis, systemic disease, and pancreatic disease were excluded.st edition of the Nelson textbook of Pediatrics, the definition of adolescence includes three groups based on age: early adolescence indicates the approximate age range of 10–13 years, middle adolescence is 14–17 years, and late adolescence is 18–21 years.In our study, six patients were ≥18 years of age. These six patients were enrolled in this study despite their relatively higher age because they had been followed up continuously in the “pediatric gastroenterology, hepatology, and nutrition center” of our hospital since they were diagnosed with NAFLD at an early age. In the 21The study conformed to the ethical guidelines of the Declaration of Helsinki  and the recommendations of the Ethics Committee of Seoul National University Bundang Hospital. This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB No. B‐2103‐670‐105).2.2th percentile and overweight as BMI between the 85th and 95th percentiles, adjusted for age and sex, according to the Korean National Growth Charts 2017.th percentile, adjusted for age and sex, according to the Korean National Growth Charts 2007.Anthropometric parameters, including height, weight, and waist circumference (WC), were measured in all children using standardized methods; we calculated the waist circumference‐to‐height ratio (WHtR), body mass index (BMI), and BMI standard deviation score (z‐score). In the Korean national growth charts, WC z‐score (WC‐z) has not yet been developed; therefore, we used WHtR instead of WC‐z. Obesity was defined as BMI ≥952).Body composition was measured using bioelectrical impedance analysis , and the total body fat mass and fat‐free mass were recorded. The fat mass index (FMI) and fat‐free mass index (FFMI) were calculated as the fat mass (kg) and fat‐free mass (kg), respectively, divided by the square of height , TG, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, γ‐glutamyl transferase (GGT), uric acid, fasting plasma glucose (FPG), insulin, and glycated hemoglobin (HbA1c) levels.The homeostasis model assessment of insulin resistance (HOMA‐IR) was calculated using the following formula: FPG (mg/dl) × fasting insulin (μU/ml)/405,Prediabetes and DM were defined based on the American Diabetes Association guidelines.Dyslipidemia was defined as having at least one of the following: T‐chol ≥200 mg/dl, LDL‐C ≥130 mg/dl, HDL‐C < 40 mg/dl, TG ≥100 mg/dl (age ≤9 years), or ≥130 mg/dl (age ≥10 years).th age‐, sex‐, and height‐specific percentiles, WC ≥90th sex‐specific percentile, and FPG ≥100 mg/dlMetS was diagnosed in children who met at least three of the following five criteria: TG ≥110 mg/dl, HDL‐C ≤40 mg/dl, BP ≥902.5Liver biopsy was performed by an experienced pediatric radiologist with ultrasound guidance, and the findings were interpreted by an expert liver pathologist who was blinded to the patients’ clinical data. All biopsy specimens were evaluated based on the NAFLD Clinical Research Network criteria, and the NAFLD activity score (NAS) was assessed.2.6Abdominal MRI examinations were performed using a 3.0 T MR scanner . The modified DIXON‐Quant sequence was obtained in a single breath hold, which automatically reconstructed a proton density fat fraction (PDFF) map. We obtained the maps of water, fat, fat fraction, R2*, and T2* by post‐processing the acquired images using the software provided by the manufacturer. All data were transferred to the IntelliSpace Portal software . Selection of the region of interest (ROI) and fat fraction measurements were performed by an expert pediatric radiologist who was blinded to the patients’ clinical and histopathological data. Hepatic fat fraction (HFF) measurements were performed by drawing two different ROIs in the right and left hepatic lobes. MRI‐PDFF is known to have an excellent diagnostic value for the assessment of hepatic fat content in patients with NAFLD.2.7t‐test for parametric variables and the Mann‐Whitney U‐test for non‐parametric variables. Categorical variables were compared using chi‐square or Fisher's exact tests. Correlation between the continuous variables was tested using the Pearson's correlation matrix. Receiver operating characteristic (ROC) curves were constructed to examine the appropriate cut‐off for PFF and BMI z‐score (BMI‐z), respectively, to divide children with NAFLD into two groups, with and without HTN. Pairwise comparison of ROC curves was performed to confirm diagnostic superiority. A logistic regression model was used to identify the risk factors for HTN. Through a simple logistic regression analysis, we identified several variables associated with HTN. Multiple logistic regression analysis was performed in the fully adjusted model after selecting noncollinear covariates to predict the risk factors associated with HTN. A two‐sided p value of < .05, was considered statistically significant. All statistical analyses were performed using PASW Statistics software  and MedCalc software .Descriptive characteristics are presented as mean ± SD for normally distributed variables or medians and ranges for non‐normally distributed variables. Categorical measurements are expressed as absolute numbers and percentages. Intergroup differences were evaluated using an independent 33.1th percentile) . Most children were overweight or obese ; 13 (20.0%) children were overweight, 47 (72.3%) were obese, and only five (7.7%) children were within the normal range (BMI < 853.2p = .002).As for ectopic fat on MRI, the median HFF was 24.3%  and the median PFF was 3.8% . HFF was not significantly different according to the status of obesity, HTN, central obesity, dyslipidemia, prediabetes/DM, and MetS status. Moreover, PFF did not differ according to obesity, central obesity, dyslipidemia, prediabetes/DM, and MetS status. However, PFF was significantly different according to HTN status. The median PFF of the HTN group was 6.7% (range 1.0%–26.9%) and that of the non‐HTN group was 3.2% (range 0.4%–15.8%) (r) = 0.125, p = .327 and between PFF and BMI‐z, r = 0.143, p = .264). Furthermore, HFF was not significantly correlated with PFF .Both HFF and PFF did not correlate with BMI‐z , BMI‐z , WHtR , FMI , FFMI , GGT , dyslipidemia state , QUICKI , MetS  and PFF  . Likewise, as FMI and FFMI were relative to each other, a high multicollinearity between FMI and FFMI was shown (correlation matrix for the parameter estimates output 0.82). Because the p = .010) and PFF  were independent risk factors for HTN after adjusting for FFMI, GGT, dyslipidemia status, and QUICKI in children with NAFLD (Table p = .044).Both BMI‐z  at a cut‐off PFF of 4.39%, a sensitivity of 82.4%, a specificity of 73.9%, a positive predictive value of 53.9%, and a negative predictive value of 91.9%)  was 0.754  . Therefore, PFF was not inferior to BMI‐z in the diagnosis of HTN. MRI‐measured PFF performed similarly in predicting HTN compared with BMI‐z. PFF had high values for predicting patients with HTN as much as BMI‐z.When a pairwise comparison of the ROC curves was performed to confirm diagnostic superiority, BMI‐z was not superior to PFF for dividing the patients into two groups: those with and without HTN .As it is presumed that IR may be associated with ectopic fat accumulation in the pancreas, we hypothesized that pancreatic steatosis may also affect the development of CVD through IR and undiscovered mechanisms. According to a meta‐analysis on pancreatic ectopic fat, non‐alcoholic fatty pancreas disease was associated with a significantly increased risk of arterial HTN  and five (7.7%) were within the normal range (BMI < 85th percentile). A systematic meta‐analysis showed the prevalence of NAFLD in children and adolescents with normal weight to be 2.3% , in overweight patients to be 12.5% , and in obese patients to be 36.1%  in studies conducted among the general population.2 from the West and BMI < 25 kg/m2 from the East) was considered, epidemiological data indicated that the prevalence of non‐obese NAFLD in adults was 10%–30% (7%–21% for Western studies and 3%–27% for Eastern studies).Our study included 18 (27.7%) patients with non‐obese NAFLD. Of these, 13 (20.0%) were overweight  or subcutaneous adipose tissue (SAT) content was not measured because of limitations in the software used in the present study. VAT plays an essential role in the development of IR and may serve as a missing link between IR and fatty pancreas.In conclusions, in addition to BMI‐z, ectopic pancreatic fat deposition is an important risk factor for developing HTN in children with NAFLD. Furthermore, PFF was not inferior to BMI‐z in suggesting patients with HTN. This study shows the importance and necessity of interpreting MRI‐measured PFF as well as HFF in conjunction with anthropometric and laboratory findings of obesity in assessing pediatric patients suspected of NAFLD. Measuring PFF as well as HFF on MRI could help predict the development of HTN in children with NAFLD. It would be possible to give early preventive measures to these young people to halt the development of HTN. Further large‐scale prospective studies are warranted to compare patients with NAFLD with age‐ and sex‐matched normal controls or overweight/obese children without NAFLD. Future studies should analyze VAT and SAT together with HFF and PFF to confirm the association between HFF, PFF, and metabolic components. More research is needed in the future to study the association between PFF and atherosclerosis parameters in children with NAFLD to confirm PFF as a risk factor for HTN.The authors thank the Division of Statistics in Medical Research Collaborating Center at Seoul National University Bundang Hospital for assisting in statistical analysis. This study was supported by the 2019 Research grant of the Korean Pediatric Society (Seokcheon Research Award).The authors have no potential conflicts of interest to disclose.Conceptualization: Hye Ran Yang, Eun Hye Lee, Data curation: Eun Hye Lee, Ji Young Kim, Formal analysis: Eun Hye Lee, Hye Ran Yang, Methodology: Hye Ran Yang, Ji Young Kim, Investigation: Eun Hye Lee, Ji Young Kimm, Supervision: Hye Ran Yang, Writing‐original draft: Eun Hye Lee, Writing‐review & editing: Eun Hye Lee, Hye Ran Yang. All authors read and approved the final manuscript.


To date, only one case of SARS‐COV‐2 B.1.1.7 reinfection has been reported.Ct 10 on Abbott m2000). On January 6, he was moved to the intensive care unit (ICU) and intubated. On January 10, the patient was moved to the ICU of Milan hospital for logistical reasons, pronated, and finally extubated on January 20. He was then moved to the COVID ward: the radiological pattern of pneumonia showed marked improvement, and NPSs tested negative for SARS‐CoV‐2 RNA on January 23, January 31, and February 2. On February 3, he was moved to a different hospital for rehabilitation. On February 4, a new NPS‐tested positive for SARS‐CoV‐2 RNA , while anti‐SARS‐CoV‐2 immunoglobulin G (IgG) was 194 AU/ml , and C‐reactive protein rose to 64 mg/L, but without clinical worsening. On February 5, serology was repeated on a new sample with the concordant result (169 AU/ml), but additional NPSs tested negative for SARS‐CoV‐2 RNA on February 5, February 6, and February 11. At this point, we confirm the real‐time polymerase chain reaction (PCR) results testing a new aliquot of the positive NPSs starting from an independent extraction and we sequenced twice the RBD fragment of the spike gene from different aliquots of each NPS sample dated January 4 and February 4, as previously reported.Case 1 was a 56‐year‐old immunocompetent male, a former smoker with obesity and dyslipidemia. He was employed as a truck driver, moving across Switzerland, Austria, and Germany. On December 31, 2020, he presented at Varese hospital's emergency room with moderate dyspnea; he was discharged and treated at home with levofloxacin, corticosteroids, and low molecular weight heparin. Clinical conditions worsened, and he was readmitted to the hospital on January 3, when interstitial pneumonia was diagnosed and treated with continuous positive airway pressure (CPAP) ventilation was initiated. On January 4, he tested positive for SARS‐CoV‐2 RNA on the nasopharyngeal swab (NPS) , with anti‐RBD IgG > 400 AU/ml, and without clinical worsening. On February 10 the patient was moved to the subacute medical unit. At that point, we confirm the real‐time PCR results for the above patient and we decided to sequence twice the RBD from aliquots of NPS samples dated January 19 and February 6: the January 19 S sequence showed absence of mutation if compared with SARS‐CoV‐2 isolate Wuhan‐Hu‐1 (deposited in GenBank as MW599251), while the February 6 strain resulted in B.1.1.7, with aminoacid substitution N501Y and A570D (deposited in GenBank as MW599954).Case 2 was a 58‐year‐old immunocompetent male who tested positive for SARS‐CoV‐2 RNA on NPS on January 7, 2021. He was treated at home with azithromycin, enoxaparin, and prednisone, which required hospital admission on January 18, where interstitial pneumonia was diagnosed. On January 19, he tested positive for SARS‐CoV‐2 RNA on NPS (1302 RLU on Hologic Panther), and he was moved to the COVID ward for oxygen support, progressing to CPAP ventilation on January 21. CPAP was discontinued on January 26, and two follow‐up NPSs on January 31, and February 2 were negative for SARS‐CoV‐2 RNA. New NPSs on February 4 and 6 were positive for SARS‐CoV‐2 RNA (1181 RLU on Hologic Panther, and According to Facebook mobility data,The authors declare that there are no conflict of interests.Federica Novazzi, Andreina Baj, Daniele Focosi, and Fabrizio Maggi contributed to study design, data collection, data interpretation, the literature search, and writing of the paper. Daniela Della Gasperina, Walter Ageno, Paolo Severgnini, and Francesco Dentali contributed to patients' recruitment, data collection, and clinical management. Angelo Genoni, Pietro G. Spezia, Alberto Colombo, Gianluca Cassani, Cristian Zago, and Renee Pasciuta managed the laboratory diagnostic procedures and collected data. Fabrizio Maggi supervised the clinical activity of the project. All authors reviewed and approved the final version of the report.


Four studies involving 2552 White American participants were conducted to investigate bias based on the race-based phenotype of hair texture. Specifically, we probed the existence and magnitude of bias in favor of Eurocentric (straight) over Afrocentric (curly) hair and its specificity in predicting responses to a legal decision involving the phenotype. Study 1 revealed an implicit preference, measured by an Implicit Association Test (IAT), favoring Eurocentric over Afrocentric hair texture among White Americans. This effect was not reducible to a Black/White implicit race attitude nor to mere perceptual preference favoring straight over curly hair. In Study 2, the phenotype (hair) IAT significantly and uniquely predicted expressions of support in response to an actual legal case that involved discrimination on the basis of Afrocentric hair texture. Beyond replicating this result, Studies 3 and 4 (the latter preregistered) provided further, and even more stringent, evidence for incremental predictive validity: in both studies, the phenotype IAT was associated with support for a Black plaintiff above and beyond the effects of two parallel explicit scales and, additionally, a race attitude IAT. Overall, these studies support the idea that race bias may be uniquely detected by examining implicit attitudes elicited by group-based phenotypicality, such as hair texture. Moreover, the present results inform theoretical investigations of the correspondence principle in the context of implicit social cognition: they suggest that tailoring IATs to index specific aspects of an attitude object  can improve prediction of intergroup behavior. Social evaluation draws upon a multitude of cues, many of them seemingly irrelevant, and even opposed, to the task at hand. Notably, aspects of physical appearance reliably predict judgments about ability and character, even when there is no objective basis for such prediction: men with beards are more likely to be seen as angry  versus Afrocentric (curly) hair texture. To our knowledge, no published work to date has measured implicit attitudes toward specific phenotypic traits rather than toward racial categories in general . We believe that incorporating implicit judgments into the study of how hair texture influences social perception and behavior stands to benefit both our understanding of racial phenotypicality bias . However, such studies seem to violate the so-called correspondence principle according to which criterion behaviors and the attitudes used to predict them should match each other in terms of specificity , the relationship between explicit and implicit attitudes toward Afrocentric hair texture (Studies 2–4), and the unique predictive power of explicit and implicit hair attitudes with regard to a relevant intergroup behavior (Studies 2–4).Study 1A was designed to provide an initial test of implicit attitudes toward Eurocentric hair texture versus Afrocentric hair texture and to probe the relationship of these phenotype-based attitudes with more general implicit attitudes toward White and Black racial categories. Study 1B, in turn, addressed a potential confound emerging from Study 1A: specifically, even if the hair attitude IAT were to reveal an implicit preference for Eurocentric hair texture over Afrocentric hair texture, this preference may be due to a more general preference for straight over curly hair or even for straight over curved shapes. To eliminate this alternative explanation, in Study 1B a hair IAT similar to the one featured in Study 1A was administered to participants, but this time using racially ambiguous (light-skinned and clearly non-Black) targets. In this and all remaining studies, we focus on White American participants given well-documented pro-White/anti-Black racial biases in this group . We report all data exclusions and all measures in all studies. Study 4 was preregistered (https://aspredicted.org/tn45b.pdf). In Studies 1–3, the sample size was determined a priori without conducting formal power analyses. In Study All materials, data files, and analysis scripts are available for download from the Open Science Framework (http://implicit.harvard.edu/). 320 participants were recruited to participate in Study 1A, and 319 participants were recruited to participate in Study 1B. In Study 1A, 14 participants who did not complete the Implicit Association Test . Analyses involving African American participants are reported in a section below collapsing across all studies. As is usual for online samples, women (73.50%) were more heavily represented in the sample than men (26.50%), younger individuals were more heavily represented than older individuals , and liberal individuals (47.48%) were more heavily represented than conservative individuals (22.71%). These and other demographic variables are available for reanalysis to interested investigators for this and all remaining studies.Given theoretically expected differences across White American and non-White American participants , a new set of images was developed for this IAT. “Straight hair” and “curly hair” were chosen as category labels with full knowledge that there are several other terms to denote hair texture. We selected these terms to clearly distinguish two hair types without relying on terms such as “nappy”, “kinky”, or “wooly”, which may be unknown to White American participants, seen as derogatory, or ambiguous in meaning.N = 201; Supplementary Study 1 in Supplement 1), the majority of participants categorized each of these faces as Black (median = 88.56%). As such, in line with our intentions, this IAT measured the effects of hair texture specifically in the context of Black female targets. In this and all remaining studies, only female faces were used on the hair attitude IAT because women are the main targets of discrimination on the basis of hair texture  manipulated, thereby controlling for facial identity (see Table r = . 70).These stimuli were used to measure implicit attitudes toward Eurocentric versus Afrocentric hair texture in a standard 7-block Implicit Association Test  a specific evaluation of Eurocentric hair texture versus Afrocentric hair texture, (b) a more general evaluation of straight versus curly hair, or (c) perhaps even a relatively low-level perceptual preference for straight over curved shapes, an alternative version of the hair attitude IAT was created. Specifically, instead of the line drawings of dark-skinned women, this IAT used line drawings of light-skinned women as target stimuli, varying their hair type between straight and curly (see Table r = 0.64).White/Black race attitude IAT To further probe the relationship between implicit evaluations of Eurocentric hair texture versus Afrocentric hair texture and implicit evaluations of White and Black racial categories more generally, a standard White/Black race attitude IAT was also created. The two categories were represented using black-and-white photographs of the internal facial features of six individuals from each group .For ease of comparability, the procedure of each study is summarized in Table Data from these and all remaining studies are openly available for reanalysis from the Open Science Framework (OSF). Additional results for this and all remaining studies are reported in Supplement 1.Mean levels Descriptive statistics for all variables from Study 1A and all remaining studies are provided in Table t(217) = 10.50, p < 0.001, BF10 = 1.10 × 1018, Cohen’s d = 0.71. Moreover, in line with well-established findings of implicit ingroup preference among White Americans  = 15.82, p < 0.001, BF10 = 4.95 × 1034, Cohen’s d = 1.07.Relationship between the hair attitude and race attitude IATs Next, we probed whether implicit hair attitudes and implicit race attitudes are completely independent of each other, related to each other but not redundant, or fully redundant. Scores on the hair and race attitude IATs were found to be significantly correlated, r = 0.28 , t(216) = 4.32, p < 0.001, BF10 = 9.61 × 102, thus providing evidence against the idea of complete independence. Those who showed greater preference for straight hair relative to curly hair also showed preference for White Americans relative to Black Americans.p = 0.15 , a large portion of meaningful variance remained unexplained, p = 0.61 . As such, these analyses reveal a significant association, but no redundancy, between implicit race and hair attitudes.However, given attenuation as a result of imperfect reliability  = 4.64, p < 0.001, BF10 = 1.92 × 103, Cohen’s d = 0.32. This result suggests that at least some of the preference for straight over curly hair obtained in Study 1A may not have been specific to Black targets. However, if the effect were fully reducible to a decontextualized aesthetic preference, responding on the non-Black hair IAT (Study 1B) and on the Black hair IAT (Study 1A) should have been indistinguishable from each other. Contrary to this prediction, implicit preferences on the non-Black hair IAT were significantly less strong than implicit preferences on the Black hair IAT, t(395.44) = − 2.44, p = 0.015, BF10 = 1.86, Cohen’s d = 0.23. As such, the two studies combined suggest that, to a considerable degree, the IAT administered in Study 1A captured implicit preferences specifically in favor of Eurocentric over Afrocentric hair texture.The non-Black hair attitude IAT revealed an implicit preference for straight over curly hair, Study 1A established an implicit preference of considerable magnitude for Eurocentric hair texture over Afrocentric hair texture among White American participants. This preference was moderately related to, but not redundant with, more general implicit evaluations of the underlying racial categories. The results of Study 1B, in turn, suggest that the phenotype-based implicit attitudes observed in Study 1A cannot be fully reduced to an aesthetic preference for straight over curly hair or, even more generally, straight over curved shapes given that the bias was significantly attenuated when the targets were light-skinned racially ambiguous (but clearly non-Black) women rather than dark-skinned Black women. Whether the difference in implicit attitudes between the two studies emerged due to skin tone, facial features, the combination of both, or some other reason, can be more specifically explored in future work. From our perspective, the important finding is that of discriminant validity, i.e., a markedly stronger implicit preference for straight over curly hair texture in the context of Black, rather than non-Black, targets.Interestingly, the preference for straight over curly hair was not eliminated even when racially ambiguous light-skinned non-Black women, rather than Black women, served as targets on the IAT. This result echoes findings obtained using explicit measures in recent work . 43 participants who did not complete the IAT and 12 participants whose response latencies suggested inattentive responding were excluded from further analyses, resulting in a final sample size of 864. The analyses reported below focus on 622 White American participants. Similar to Study 1, these participants tended to be female (68.08%) rather than male (31.92%), younger rather than older , and liberal (53.54%) rather than conservative (19.14%).The participants were 919 American volunteers recruited from Project Implicit (r = 0.73), some new materials and measures were created for use in this study, including the description of a legal case involving an African American plaintiff alleging discrimination on the basis of hair texture, and three explicit scales measuring self trait judgments and societal trait judgments about women with Afrocentric hair texture, as well as participants’ views about the legal case mentioned above. All materials and measures used in this study, including the verbatim text of the legal case, are included in Supplement 2.In addition to the Black hair IAT already used in Study 1A  was created. The case involved Chastity Jones, an African American woman, who was about to start working at a company in Alabama. However, after she had accepted the position, the corporation informed her that she would need to cut her dreadlocks to comply with company policy. When Jones refused to do so, the company rescinded its offer of employment. Jones sued the company for discrimination. The case was presented to participants without describing the court’s decision, which was to rule in favor of the corporation.The description of the case provided excerpts from the briefs on behalf of the employer and the plaintiff. Specifically, the employer argued that although “Title VII prohibits discrimination on the basis of immutable (unchangeable) characteristics, such as race, color, or natural origin”,… “[a] hairstyle, even one more closely associated with a particular ethnic group, is a mutable (changeable) characteristic.” In addition, they referred to their corporate policy prohibiting “excessive hairstyles or unusual colors.” The plaintiff, in turn, argued that dreadlocks were a natural part of the bodies of many people of color. As such, “… discrimination against natural Black hair is equivalent to discrimination against skin color.”Self trait judgments A four-item scale was created to measure participants’ personal trait judgments of women with Afrocentric hair texture in terms of how attractive, caring, competent, and professional they are. For each of these traits, participants were presented with the incomplete sentence “I BELIEVE that dreadlocks and afros make women look…” and selected one of seven response options. For instance, for the attractiveness trait, the response options read “much less attractive”, “moderately less attractive”, “slightly less attractive”, “no different in terms of attractiveness”, “slightly more attractive”, “moderately more attractive”, and “much more attractive.” For the remaining traits, the word “attractive” was replaced with the corresponding trait adjective. Responses were scored from 1 to 7 such that higher scores correspond to higher levels of explicit bias against Afrocentric hair texture. The self trait judgment scale exhibited satisfactory internal consistency (Cronbach’s α = 0.75).Societal trait judgments A four-item scale was created to measure participants’ perceptions of general societal views of women with Afrocentric hair texture. For each of the four traits mentioned above, participants were presented with the incomplete sentence “SOCIETY BELIEVES that dreadlocks and afros make women look…” and selected one of seven response options, as described above. Responses were scored from 1 to 7 such that higher scores correspond to higher levels of perceived societal bias against Afrocentric hair texture. The societal trait judgment scale exhibited satisfactory internal consistency (Cronbach’s α = 0.78).Views about the legal case A five-item scale was created to measure participants’ views regarding the legal case described above. The items measured participants’ agreement with (a) the plaintiff being correct for suing the company, (b) the plaintiff showing disregard for the company, (c) the company showing race bias against the plaintiff, (d) discrimination based on hair being equivalent to racial discrimination, and (e) the company being within its rights to not hire the plaintiff based on her hairstyle. Participants rated their agreement or disagreement using a 7-point Likert scale, with the response options anchored by “strongly disagree” and “strongly agree.” Responses were scored such that higher scores indicated higher levels of agreement with the company, i.e., items (a), (c), and (d) were reverse scored. The legal judgment scale exhibited satisfactory internal consistency (Cronbach’s α = 0.85).Participants completed the Black hair attitude IAT, read the legal case, and completed the legal judgment scale. The legal judgment scale always directly followed the legal case, and the order of the IAT and the legal case and judgments was counterbalanced. Finally, participants completed the self and societal judgment scales. The two scales were interleaved with each other such that items for self and society for the same trait always followed each other in individually randomized order.Given satisfactory internal consistency of each scale, the analyses involving mean levels were performed on composite variables. In line with established practice, the structural equation model (SEM) included each item separately as an indicator of a latent variable. For the IAT, two indicator variables were created by calculating D scores using odd-numbered and even-numbered trials, respectively. The correlation matrix forming the basis of the SEM analysis is reported in Supplementary Table 2 (Supplement 1).t(614) = 16.51, p < 0.001, BF10 = 3.01 × 1047, Cohen’s d = 0.67, replicating the result obtained in Study 1A.The hair attitude IAT revealed significant implicit preference in favor of Eurocentric over Afrocentric hair texture, t(598) = 1.60, p = 0.109, BF01 = 6.06, Cohen’s d = 0.07. That is, participants reported that Afrocentric hair texture did not influence their social judgments of women in a positive or negative direction. By contrast, mean societal trait judgments were significantly higher than the midpoint of the scale, t(598) = 29.97, p < 0.001, BF10 = 2.13 × 10177, Cohen’s d = 1.22, indicating awareness of societal bias against Afrocentric hair texture. The mean level of perceived societal bias significantly exceeded the mean level of bias reported for the self, t(598) = 24.31, p < 0.001, BF10 = 3.70 × 1087, Cohen’s d = 0.99. This finding is reminiscent of the bias blind spot, i.e., the tendency to perceive no bias in the self along with reports of high levels of bias in others  = − 23.63, p < 0.001, BF10 = 7.28 × 1083, Cohen’s d = 0.97, indicating general agreement with the African American plaintiff rather than the employer.Finally, mean legal judgments were significantly below the midpoint of the scale, χ2(84) = 285.93, p < 0.001. However, given inherent conceptual difficulties in interpreting a nonsignificant test as an indication of good model fit . 34 participants who did not complete the IAT and 9 participants whose response latencies suggested inattentive responding were excluded from further analyses, resulting in a final sample size of 710. The analyses reported below focus on 507 White American participants. Similar to Studies 1–2, these participants tended to be female (65.22%) rather than male (34.78%), younger rather than older , and liberal (57.99%) rather than conservative (17.36%).The participants were 753 American volunteers recruited from Project Implicit , with the order of both IATs counterbalanced. To prevent participant fatigue, the number of IAT blocks was reduced from seven to five .r = 0.59) and the White/Black race attitude IAT (r = 0.55) showed adequate split-half reliability, although internal consistency was attenuated relative to Studies 1 and 2, presumably due to the smaller number of trials. The self trait judgment scale (Cronbach’s α = 0.78), the societal trait judgment scale (Cronbach’s α = 0.80), and the legal judgment scale (Cronbach’s α = 0.85) exhibited good-to-excellent internal consistency. The correlation matrix forming the basis of the SEM analysis is reported in Supplementary Table 4 (Supplement 1).The Black hair attitude IAT  = 257.40, p < 0.001, all alternative measures of model fit indicated good fit (see Supplementary Table 3). Therefore, we proceed with model interpretation.5Similar to Study 2, a latent legal judgment variable was predicted by a latent self trait judgment variable, a latent societal trait judgment variable, and a latent implicit hair attitude variable. Moreover, a latent implicit race attitude variable was newly added. Despite a significant difference between the model-implied and empirical correlation matrices, β = 0.43 , z = 6.74, p < 0.001, and uniquely and negatively predicted by societal trait judgments, β = − 0.25 , z = − 4.06, p < 0.001. That is, the higher their level of self-reported preference for Eurocentric over Afrocentric hair texture and the lower their level of acknowledgement of societal bias, the more likely participants were to agree with the corporation rather than with the plaintiff.With regard to self trait judgments, societal trait judgments, and implicit hair attitudes, the results of the SEM model Fig.  replicatβ = 0.12 , z = 2.13, p = 0.033. However, the effect of implicit race attitudes on legal judgments was not statistically significant, β = 0.09 , z = 1.76, p = 0.078, suggesting superior predictive validity of the implicit measure specifically tailored to the behavior to be predicted. At the same time, we note that the effect size associated with the two tests is highly similar and, as such, this result should be interpreted with caution, especially in the absence of a formal power analysis.Remarkably, in spite of no shared method variance, implicit hair attitudes were again found to be significantly and uniquely associated with legal judgments, Study 3 provides an independent replication of the surprising result obtained in Study Given the novelty of the approach of applying implicit measures to the study of racial phenotypicality bias, Studies 1–3 should be seen as exploratory. In line with recent recommendations . 81 participants who did not complete the IAT and 17 participants whose response latencies suggested inattentive responding were excluded from further analyses, resulting in a final sample size of 1509. In line with the preregistered analysis plan, the analyses reported below focus on 978 White American participants. This target sample size was determined based on a resampling-based simulation approach. Specifically, we aimed to have at least 0.80 power to detect the incremental predictive validity effect associated with the hair attitude IAT in the SEM analysis of Study 3 (β = 0.12). Similar to Studies 1–3, participants tended to be female (67.79%) rather than male (30.36%), younger rather than older , and liberal (52.69%) rather than conservative (25.03%).The participants were 1607 American volunteers recruited from Project Implicit  and the race attitude IAT (r = 0.64) showed adequate split-half reliability, with higher values than those observed in Study The hair attitude IAT (t(964) = − 16.61, p < 0.001, BF10 = 1.23 × 1051, Cohen’s d = − 0.53.The results involving mean levels of all variables were in line with the results obtained in Studies 1–3, with one notable exception. Specifically, in Studies 2–3, White participants expressed neutrality on the self trait judgment scale, whereas in Study χ2(109) = 717.29, p < 0.001, all alternative measures of model fit indicated excellent fit (see Supplementary Table 3). Therefore, we proceed with model interpretation.7Similar to Study 3, a latent legal judgment variable was predicted by a latent self trait judgment variable, a latent societal trait judgment variable, a latent implicit hair attitude variable, and a latent implicit race attitude variable. Despite a significant difference between the model-implied and empirical correlation matrices, β = 0.37 , z = 8.94, p < 0.001, and uniquely and negatively predicted by societal trait judgments, β = − 0.24 , z = − 6.37, p < 0.001. That is, the higher the level of self-reported preference for Eurocentric over Afrocentric hair texture and the lower the level of acknowledgement of societal bias, the more likely participants were to agree with the corporation rather than with the plaintiff.The results of the SEM model Fig.  replicatβ = 0.12 , z = 3.69, p < 0.001. Moreover, unlike in Study β = 0.10 , z = 2.81, p = 0.005, thus providing further evidence for the idea that the two IATs measure related but separate constructs. At the same time, these results also suggest that the correspondence principle may not apply to implicit social cognition in a straightforward manner. We return to this issue in the General discussion below.Remarkably, in spite of no shared method variance, implicit hair attitudes were again found to be significantly and uniquely associated with legal judgments, In Study In addition, Study 4 also produced two unexpected secondary findings. First, White American participants expressed a significant preference for Afrocentric hair textures on an explicit scale. This finding is in contrast with the results of Studies 2–3 in which participants remained neutral on a similar measure. We can only speculate as to the reasons for this discrepancy across studies.It is conceivable that participants were more reluctant to express negative evaluations on an absolute  rather than a relative scale . In addition, Studies 1–3 were conducted prior to the national reckoning on race that began in the summer of 2020, whereas data for Study 4 were collected in June 2021. Explicit expressions of positive regard for Afrocentric hair texture may reflect the vast change in at least such verbal statements supportive of Black American attributes. In addition, research has more generally documented decreases in the expression of explicit prejudice on the Project Implicit website over the past decade , β = 0.10 , z = 5.16, p < 0.001. However, the size of the effect did not differ across the two IATs in either model 2, β = − 0.02 , z = − 0.51, p = 0.612, or model 4, β = − 0.02 , z = − 0.51, p = 0.606. Taken together, these results suggest that the hair attitude IAT and the race attitude IAT measure independent constructs. At the same time, under the correspondence principle, it may have been expected that the more specific phenotypicality IAT would be more predictive of responses to a legal case involving that phenotype than a generic race IAT. These data are not in line with this prediction, which makes it questionable whether the correspondence principle can be straightforwardly applied to implicit attitudes, at least under the present conditions.Overall, the results of the four models are straightforward. The two IATs showed incremental predictive validity both above and beyond explicit measures (model 1), n = 520). The results from African American participants were analyzed using Bayesian meta-analyses, with results from White American participants presented for comparison  racial phenotypicality bias, focusing specifically on implicit attitudes toward Afrocentric versus Eurocentric hair texture. Across three high-powered studies, the last of which was formally preregistered, we found that although implicit and more commonly used explicit measures of racial phenotypicality bias were positively correlated with each other, this correlation, similar to the relationship between implicit and explicit race attitudes  and explicit predictors (β = 0.11) was small. In addition, also in line with recommendations by Kurdi et al.  may be more predictive of the legal judgments in the present studies than general attributes (such as “good” vs. “bad”) given that the legal case centers specifically on the question of whether natural Black hair and professionalism are compatible with each other. However, in this context, considerable added complexity may be created by the fact that stereotype IATs using clearly valenced attributes are usually highly correlated, or even redundant, with attitude IATs  that was, to a large extent, under participants’ volitional control. As such, the present project provides evidence in favor of another idea proposed by Kurdi et al. , namely Given that, to our knowledge, the present paper reports the first peer-reviewed studies demonstrating the existence and downstream consequences of implicit phenotypically bias more generally and implicit bias against Afrocentric hair more specifically, the present studies should be understood as providing an existence proof argument. We hope that research in social cognition will continue to investigate implicit racial phenotypicality biases and their relationship with social behavior and further clarify some issues left unaddressed here.One such issue concerns stimulus variation. Specifically, although Studies 2–3 and Study 4 yielded converging results despite differences in stimulus materials, future work may further explore other possible variations in the IAT procedure. For example, in the present studies we used only relatively dark-skinned Black targets; in the future, investigators may want to ask whether the effect extends to light-skinned Black targets. Similarly, in the present studies we used only female targets; future work may want to examine generalization to targets of other genders. Moreover, the mean level and predictive validity estimates provided by IATs relying on real facial images may be different from the estimates provided by the IATs in the present studies, which all relied on line drawings. Finally, although the present data suggest that hair texture bias emerges especially strongly in the context of Black  targets, future work may want to more systematically explore variation by race both in terms of mean levels and in terms of predictive validity.Given that the studies reported above rely exclusively on the Implicit Association Test, it is unclear to what extent implicit attitudes toward Eurocentric versus Afrocentric hair texture are automatically activated without specific instruction to categorize stimuli along this dimension. Future work using alternative implicit measures of attitudes, such as the evaluative priming task (Fazio et al., Finally, recent work in implicit social cognition has demonstrated striking similarities between response time-based measures of implicit attitudes administered to individual participants and algorithmically derived measures of semantic association obtained from large corpora of online text (e.g., Caliskan et al.,


Median patient age was 61 years (range 57–76 years). MGA lesions were estrogen receptor (ER)‐negative, progesterone receptor‐negative, HER2‐negative, and S100‐positive. DNA CN alterations (CNAs) were complex or limited to few gains and losses. CN gain on chromosome 2q was the most common CNA and was validated by FISH in five of eight cases. NGS demonstrated an average of two mutations per case (range 0–5) affecting 10 different genes . CNAs and mutations were similar in MGA and adjacent BC, indicating clonal relatedness. DNA methylation profiling identified aberrant hypermethylation of CpG sites within GATA3, a key transcription factor required for luminal differentiation. Immunohistochemistry showed regular GATA3 protein expression in the normal mammary epithelium and in ER‐positive BC. Conversely, GATA3 was reduced or lost in all MGA cases tested (8/8). In conclusion, MGA is characterized by common CN gain on chromosome 2q and loss of GATA3. Epigenetic inactivation of GATA3 may provide a new clue to the peculiar biology of this rare neoplasia.Microglandular adenosis (MGA) represents a rare neoplasm of the mammary gland, which in a subset of cases may be associated with triple‐negative breast cancer (BC). The biology of MGA is poorly understood. In this study, eight MGA cases ( Microglandular adenosis (MGA) of the mammary gland was first described in 1968 and was initially considered a benign lesion . HistoloImmunohistochemically, MGA shows a peculiar phenotype characterized by the absence of estrogen receptor (ER) and progesterone receptor (PR) expression but positive staining for S100 . MGA alsAfter the initial descriptions, invasive breast cancer (BC) was found to subsequently develop years after the diagnosis of MGA , 7. In aet al have demonstrated variable and partially complex copy number (CN) alterations (CNAs) in MGA [et al described complex CNAs in MGA and aMGA [et al, gains on chromosome 2q were detected in 7 of 10 (70%) cases of MGA with adjacent invasive triple‐negative BC, but not in MGA without BC (0/2 cases) [So far, only a very limited number of molecular studies on MGA has been reported , 11, 12.) in MGA . Recurre) in MGA . In a siet al also studied MGA for somatic mutations in 236 genes known to be recurrently mutated in BC [TP53 was shown to be the sole highly recurrent gene mutation in MGA. TP53 was affected in six of seven (86%) cases of MGA with adjacent invasive triple‐negative BC, but not in MGA without BC (0/2 cases) [et al concluded that MGA belongs to a spectrum of rare, low‐grade, triple‐negative neoplasms of the mammary gland [Using next‐generation sequencing (NGS), Guerini‐Rocco ed in BC . TP53 wa2 cases) . The ove2 cases) . Gueriniry gland . To datery gland .In this study, eight cases of MGA, including four lesions without adjacent invasive BC, were analyzed. NGS was applied for mutational profiling, and molecular inversion probe (MIP) array analysis was used for high‐resolution genome‐wide DNA CN profiling. Unlike previous studies, DNA methylation patterns were also assessed and included in the molecular characterization of MGA.Formalin‐fixed paraffin‐embedded (FFPE) specimens of MGA were retrieved from the archives of the Institute of Pathology at the Hannover Medical School (MHH). Most cases (7/8 cases) were submitted to the MHH from other institutes for histopathological consultation, and final reference diagnosis was made by Prof. Hans Kreipe (HK). This study was conducted in accordance with the guidelines of the local ethics committee .For immunohistochemistry, 1 μm‐thick sections of FFPE tissue blocks were mounted on superfrost slides . Next, slides were deparaffinized and rehydrated conventionally and were subjected to immunohistochemical staining on a Benchmark Ultra  automated stainer. The CC1 mild program was used for antigen retrieval, and the ultraView DAB kit (Ventana) was used for signal detection. Antibodies and scoring methods used for immunohistochemistry are summarized in supplementary material, Table n = 10, 8 μm each) from corresponding FFPE blocks. Another HE stain, prepared after cutting off the unstained sections, confirmed unaltered tissue representation on deeper sections of the blocks. Next, genomic DNA and RNA were extracted with the Maxwell RSC DNA and RNA FFPE kits  using a Maxwell RSC instrument (Promega) according to the manufacturer's recommendations. DNA was quantified using a Qubit 2.0 fluorometer  and the Qubit dsDNA HS assay kit .Genomic DNA was extracted as described previously . In brieWhole‐genome DNA CN profiling was performed using MIP arrays  and approximately 80 ng total DNA, as described previously . OSCHP fMutational analysis was carried out by NGS as described previously . NGS wasin situ hybridization (FISH), 4 μm‐thick sections of FFPE tissue blocks were mounted on superfrost slides (Thermo Fisher Scientific). Next, slides were deparaffinized and rehydrated conventionally and were permeabilized and denatured in a microwave oven for 30 min with sodium citrate buffer (pH 6.0) . Then, slides were cooled to room temperature, rinsed in distilled water, and were incubated in a pepsin solution for 15 min at 37 °C. Subsequently, slides were passed through ascending concentrations of ethanol  and were hybridized with a commercial FISH probe for the ERBB4 gene locus on chromosome 2q34 and the AFF3 gene locus on 2q11 close to the centromeric site of chromosome 2 . ERBB4 signals were quantified in 30 tumor cells each. The average ERBB4 CN per cell was calculated as the sum of the counts of signals divided by 30. The threshold used for the definition of an ERBB4 CN gain was CN ≥3.0, which is a common threshold for CN gain in diagnostic FISH applications [For fluorescence ications .‐positive/HER‐negative BCs, for comparison  and an additional nine specimens, including normal mammary gland tissue, unrelated triple‐negative BCs, and ERn = 14), triple‐negative breast carcinomas (n = 18), and normal mammary glands (n = 13). Clinicopathological characteristics of included carcinomas are summarized in supplementary material, Table n = 45) were treated with sodium bisulfite using the EZ DNA Methylation Kit™  according to the manufacturer's instructions and then eluted in 40 μl of elution buffer. Bisulfite pyrosequencing analysis was performed as previously described [For validation of DNA methylation characteristics, DNA samples already subjected to DNA methylation analysis by Infinium EPIC 850 K array were analyzed by bisulfite pyrosequencing. In addition, we enlarged the group of hormone receptor‐positive breast carcinomas , CN profile could not be obtained due to insufficient DNA amount or quality. Overall, CN profiles showed variable characteristics  served as a reference cohort, providing 42 nonclonal lesion pairs from independent patients for this statistical analysis. LR values, reflecting the odds that two lesions are clonal, ranged from 1.7 × 10−8 to 5.2 × 103 (median 2.9 × 10−6) in lesions from independent patients .The microdissected tissue of adjacent triple‐negative BCs was also subjected to DNA CN profiling. Adequate CN profiles were obtained from a total of four adjacent BCs . In three cases , CN profiles from both lesions (MGA and invasive BC) were obtained. CNAs of MGA and adjacent BC were highly similar, indicating clonal relatedness . Subsequently, CN gain on chromosome 2q was validated by FISH using a commercially available probe for the ERBB4 gene locus (chromosome 2q34). CN gain of the ERBB4 gene locus was validated by FISH in five of eight MGA lesions and in three of four adjacent invasive BCs , both of which were associated with invasive BC. Both TP53 mutations were truncating mutations (p.W146* and p.Q104*) , triple‐negative BCs , and hormone receptor (HR)‐positive BCs  for comparison  was identified in all three MGA specimens under study  could be included in this exploratory analysis because of the high minimal DNA amount required for this assay. For the identification of differentially methylated positions (DMPs), we also included microdissected normal mammary gland tissue , HR‐positive BCs (n = 14), and triple‐negative BCs (n = 18)   Figure . MGA les) Figure . Hence, GATA3 in MGA, we hypothesized that GATA3 protein expression is reduced in MGA lesions. Therefore, GATA3 was assessed by immunohistochemistry. In accordance with the molecular findings with regard to GATA3 gene hypermethylation, immunoreactivity for GATA3 was constantly reduced  or completely missing in all MGA lesions  and adjacent BC  confirmed CN gain on chromosome 2q in five of eight MGA cases. However, CN gain on chromosome 2q34 was not associated with enhanced ERBB4 protein expression, as determined by immunohistochemistry (data not shown).Consistent with the study of Shin d losses . CN gains [et al . FISH anet al and Geyer et al, mutational characteristics were highly variable in MGA [TP53, PTEN, FGFR2, ATM, PIK3CA, and PMS2 [CTNNB1, ARID1A, FBXW7, and MET genes. Interestingly, TP53 mutations were less frequent than previously reported. In the study of Guerini‐Rocco et al, 7 of 12 (58%) MGA cases harbored a TP53 mutation [TP53 mutations were detected in only two of seven (29%) MGA cases. Both mutations were truncating mutations, and none of the MGA lesions showed nuclear p53 accumulation by immunohistochemistry. We conclude that immunohistochemistry for p53 is of limited value for the histological differential diagnosis of MGA.In accordance with the studies of Guerini‐Rocco e in MGA , 12. Genand PMS2 , 12. Newmutation . These mmutation . In the et al, Guerini‐Rocco et al, and Geyer et al, CNAs and mutational characteristics of BCs adjacent to MGA indicated clonal relatedness. On morphological grounds, a relationship between MGA and adjacent BC has already been described because both lesions may exhibit similar cytoplasmic clearing, a similar growth pattern, and a similar triple‐negative immunophenotype [et al. Alternatively, MGA and invasive BC could originate from a common neoplastic stem cell through different paths of clonal evolution. Although we and others found considerable overlap between CNAs and mutational profiles in MGA lesions and adjacent BCs, there were also mutations restricted to either MGA or adjacent BC. Because our NGS panel included only a limited set of genes (n = 161), linear or parallel evolution of MGA and adjacent BC remains an open and still unsolved issue [Consistent with the studies of Shin henotype . These mnes n = 11, linearNeither histological nor molecular characteristics appeared to enable differentiation between MGA with and MGA without associated BC. As a consequence, when encountered in diagnostic core needle biopsies, it is reasonable to categorize MGA as a B3 lesion  . CompletGATA3 gene in MGA. Subsequent immunohistochemical analyses confirmed that GATA3 protein expression is constantly reduced or lost in MGA, as well as in associated BCs. GATA3 is a critical transcription factor, which regulates the development of the skin and the mammary gland [GATA3 is also one of the most frequently mutated genes in BC. GATA3 mutations are typically gain‐of‐function mutations and, in most instances, can be observed in ER‐positive BCs [For the first time, DNA methylation patterns of MGA have been analyzed. Increased DNA methylation suggested an epigenetic inactivation of the ry gland , 23, 24.ry gland , 23, 24.tive BCs , 27. Muttive BCs , 29. It GATA3 gene.In conclusion, we present evidence that MGA represents a mammary gland neoplasm of uncertain malignant potential, which is characterized by CN gain on chromosome 2q and loss of GATA3 protein expression in conjunction with epigenetic inactivation of the MR, MC and HK designed the study and assessed histomorphological characteristics. JLvL, HS, JB and DS performed DNA CN profiling. SB and UL carried out mutational analysis. MR, HC, LR, ML and CW performed FISH analyses. SB, HS, IG and UL carried out DNA methylation profiling. All authors contributed to data collection and final data analysis.Figure S1. Immunophenotypic characteristics of MGAFigure S2. Whole‐genome CNA profiles of MGA casesFigure S3. Clonal relatedness of CN profiles in MGA and adjacent BCFigure S4. Quantitative methylation analysis by bisulfite pyrosequencing of GATA3 in MGAFigure S5. Loss of GATA3 expression in MGAClick here for additional data file.Table S1. Antibodies used for immunohistochemistryTable S2. List of gene targets in Oncomine Comprehensive Assay v3Table S3. Characteristics of specimens for DNA methylation profilingTable S4. Clinicopathological characteristics of carcinomas used for pyrosequencing assayTable S5.GATA3 pyrosequencing assay primersTable S6. CN gains/losses in MGA and adjacent BCTable S7.ERBB4 CN by FISHTable S8. NGS detailsTable S9. List of genes examined in the 850 K array analysisTable S10. CpG sites within GATA3 examined in the 850 K array analysisClick here for additional data file.


The K+ < 3.86 mmol/L group showed significantly decreased N1 , LST , and periodic leg movement during sleep related to microarousals (MA) /arousal (PLMS-A)  and increased REM  compared to the K+ ≥ 3.86 mmol/L group. A subdivided analysis by gender showed that these changes were more statistically significant in men than in women. Significant positive correlations were identified between K+ and N1 , as well as PLMS-A  in subjects. Compared to women, a significantly strong correlation was identified between K+ and REM sleep in men . Multiple linear regression analysis indicated that K+ is significantly associated with N1 in all subjects (p = 0.03) and with REM in men (p = 0.008), even after adjusting for confounders. Decreased K+ may disturb the homeostasis of the sleep architecture, and gender may interfere with their links in the hypertensive population.The aim is to investigate the association between alterations in the serum potassium (K+) concentration and sleep architecture parameters in essential hypertensives. Two hundred ninety-two hypertensives undergoing polysomnography and providing blood samples were recruited. The sleep architecture was composed of sleep stages 1 (N1), 2 (N2), 3 (N3), 4 (N4) and REM. The light sleep stage (LST) was composed of N1 + N2, and the deep sleep stage (DST) was composed of N3 + N4. The potentialrelationships between electrolytes and sleep parameters were determined via univariate and multivariate analyses. The subjects were divided into two groups via the serum K Sleep accounts for approximately one third of the human lifetime, a complex phenomenon that comprises two substrates, non–rapid and rapid eye movement sleep (NREM and REM). NREM is further divided into stages, such as N1, N2, N3 and N4; the latter two stages are also referred to as slow wave sleep [SWS] , insomnia or shift workers. Only patients who were previously diagnosed as hypertensive and were taking anti-hypertensive medications or were clinically assessed with an auscultatory blood pressure ≥ 140/90 mm Hg for at least 3 times  were con+ = 3.86 mmol/L, resulting in the K+ < 3.86 mmol/l group (n = 145) and the K+ ≥ 3.86 mmol/l group (n = 147). Weight, height, smoking habits and alcohol use were assessed on the day of polysomnography (PSG). The body mass index (BMI) was calculated as kilograms per meter squared. The data used were approved by the Ethics Committee of the previously cited hospital . All participants provided written informed consent.Two-hundred ninety-two patients were ultimately selected for the present study and were divided into two groups via a median serum KAll patients underwent PSG  in a dedicated sleep laboratory, as previously described . The subEchocardiography was performed using similar methods as previously described . Cardiac+), potassium (K+), magnesium (Mg2+), calcium (Ca2+), chlorine (Cl-), total of CO2 (TCO2), glucose, and creatinine were measured on a C16000 automated biochemistry analyzer .Fasting blood samples were collected at 8:00 in the morning. To avoid factitious increases in the measured potassium concentrations, we used a syringe and needle. After venipuncture was achieved, blood was withdrawn in a slow and careful manner for at least 10 s and separated within 30 min . The serχ2 test was employed for categorical variables. Correlations between the serum potassium and sleep parameters were calculated with Spearman or Pearson correlations, depending on whether the bivariates were normally distributed. Variables with p value < 0.05 were subsequently entered in a multiple liner regression procedure to determine the independent association of K+ and sleep parameters . A P value < 0.05 was consideredstatistically significant. All statistical analyses were performed with SPSS statistical software, version 17.0 .The subjects’ baseline characteristics, electrolytes and sleep parameters were expressed as the mean ± SD. or median (interquartile range). Continuous variables were compared by two-sample t test or Wilcoxon rank sum test, depending on whether the sample had a normal distribution, and the p = 0.232), age , BMI, Na + , Cl-, Ca2+, Mg2+, total CO2, fasting glucose, 24 h urinary creatinine and uric acid, urinary pH, and systolic and diastolic blood pressure. The two groups were also similar in the usage and combination of anti-hypertensive agents, with the exception that the subjects in the K+ < 3.86 group took more angiotensin receptor blockers . In addition, the subjects in both groups showed a similar LV function status.The demographic and polysomnographic characteristics of the subjects in the two groups are shown in Table + < 3.86 mmol/l group showed significantly lower N1 , LST , PLMS-A  and significantly higher REM  than the K+ ≥ 3.86 mmol/L group. No statistically significant differences were identified in the AHI, TST, sleep efficiency, sleep latency, WASO, N2, N3, N4, and DST between the groups.As shown in Table + < 3.86 mmol/l showed significant decreases in the N1, LST, and PLMS-A and a significant increase in REM sleep compared to the women.As shown in Fig. + and N1        Fig.  and PLMS01) Fig.  in all p04) Fig.  and wome41) Fig. . Moreove28) Fig.  in men, 42) Fig. .Fig. 2a + is associated with N1 in all subjects (p = 0.03) and with REM in men (p = 0.008), even after adjusting for age, BMI, AHI, SBP, DBP, Na+, Cl-, Ca2+, Mg2+ and fasting glucose with/without smoking history as potential confounders.As shown in Table +, the N1% was shortened and K+ had a significantly positive independent association with N1% in EH subjects with lower serum K+. (2) Compared to EH men with higher serum K+, REM sleep was prolonged and the serum K+ had a significantly negative independent association with REM sleep in those with lower serum K+.To our knowledge, this investigation is the first study to propose circulating K + concentrations may disturb the sleep pattern assessed by PSG in essential hypertension. The primary findings are as follows: (1) compared to EH subjects with higher serum K+ < 3.86 mmol/l group than inthose with higher serum potassium. It is established that the extracellular K+ concentration plays a pivotal role in mediating the movement of potassium in and out of skeletal muscle. Therefore, patients with hypokalemia coincide with lower excitability and contractility of the skeletal muscles. Accordingly, we speculate that it is not easy to wake up individuals with lower serum K+ because of a possibly high arousal threshold, and the decrease in the PLMS-A may be the crux of the problem. Furthermore, potassium channels play an important role in regulating the activity of neuronal pathways by influencing the resting membrane potential of neurons. The activity of potassium channels has been found to both regulate neurotransmitter release and mediate the effects of neurotransmitter activation [+ may possibly be due to a decrease in neurotransmitter release and the conduction of neuronal pathways.It was seemingly difficult to wake up individuals with a lower potassium from sleep onset in our population. N1 sleep is a transition from wakefulness or nocturnal arousal to sleep onset. Normally, when arousal occurs, N2, N3, N4 or REM sleep is likely to change to N1 sleep according to the Rechtschaffen and Kales scoring rules . Therefotivation . Therefo+ < 3.86 mmol/l group. In clinical settings, essential hypertensive patients often present with hypokalemia. The prevalence of hypokalemia (K+ < 3.5 mmol/l) in 292 essential hypertensive patients is 13.7%, which is less than Cohn’s report  [+ channel-mutated mice [+ level may influence the activity of potassium channels; therefore, we speculate that the activity or density of potassium channels might be decreased and the K+ conductance may also be reduced with declined K+, which, in turn, may also lead to a reduction in REM sleep, followed by compensatory REM rebounds. Consequently, the total REM sleep time may be pathologically lengthened.The REM sleep time might be lengthened pathologically in the Katients) . Potassiatients) . Pang etted mice . The K+ The clinical implication of our findings is that compared with NREM sleep, REM sleep is associated with greater sympathetic activity and cardiovascular instability in healthy subjects and EH patients , 34. AltGender is the most important interfering factor for the sleep structure, the mechanisms of which remain unclear. Consistent with previous data , our finWhile explaining the current results, the effects of anti-hypertensive agents and renal and heart function on serum kalemia should be considered. It is reported that >20% of hospitalized patients have hypokalemia (<3.5 mmol/L) . However+ and sleep parameters may be due to the lack of healthy controls.There are several limitations in this study. First, the nature of the cross-sectional study does not enable conclusions to be drawn. Second, the sleep architecture is measured during a single night PSG, which may not be representative of the habitual sleep pattern of subjects. Taking full account of this problem, study subjects underwent PSG in a dedicated sleep laboratory with suitable temperature and humidity, and patients with ≥65% of the sleep efficiency and ≥300 min of TST were included in the study. Another potential limitation of our study is that the weak link established between KOur findings provide evidence that hypokalemia may disturb the sleep architecture in hypertensive individuals, particularly in men with EH.


To compare the donor outcomes of living donor kidney transplantation between standard donors (SDs) and marginal donors (MDs) including diabetic patients (MD + DM).2, (d) 24‐h creatinine clearance ≥70 to <80 ml/min/1.73 m2, and (e) hemoglobin A1c > 6.2 or ≤6.5 with oral diabetic medicine. Fifty‐three of 114 donors were MDs. We compared donor kidney functions until 60 months postoperatively.MDs were defined according to Japanese guideline criteria: (a) age >70‐years, (b) blood pressure ≤130/80 mmHg on hypertension medicine, (c) body mass index >25 to ≤32 kg/mp = .02), 49 vs. 40 , 48 vs. 42 , 47 vs. 38 ) and the percentage of residual eGFR (SD vs. MD + DM: 63 vs. 57 (1 (month), p < .01), 63 vs. 57 , 64 vs. 56 , 63 vs. 57 , 63 vs. 52 ). However, when MD with a single risk factor of DM was compared to SD, the difference disappeared. Nine out of 12 (75%) MD + DM had ≥2 risk factors.No kidney function parameters were different between SDs and MDs. When comparing SD and MD + DM, MD + DM had a lower postoperative eGFR (48 vs. 41 (1 (month), Although long‐term observation of donor kidney function is necessary, careful MD + DM selection had the potential to expand the donor pool. This study demonstrates the safety and potential of kidney donations for marginal donors. Careful Mmarginal donors with diabetes mellitus selection has the potential to expand the donor pool. ACRurine albumin‐creatinine ratioBMIbody mass indexBPblood pressureDMdiabetes mellituseGFRestimated glomerular filtration rateESRDend‐stage renal diseaseHbA1chemoglobin A1cHTNhypertensionIQRinterquartile rangeMDmarginal donorMD + DMmarginal donor with diabetes mellitusMMFmycophenolate mofetilPCRurine protein‐creatinine ratiosCrserum creatinineSDstandard donorTac‐ERextended release tacrolimus1Kidney transplantation is the most useful treatment for patients with end‐stage renal disease (ESRD). The prevalence of ESRD in Japan was the second highest in the world in 2016, with 2599 cases per million of the general population.In 2014, the original marginal donor (MD) criteria were established in Japan with reference to the standards for live‐donor kidney transplants put forth by the Amsterdam Forum.A total of 19.6% of kidney donors in Japan had a history of HTN and 4.1% of kidney donors had a history of diabetes in 2018 and those rates are incrising.It is essential that a donated kidney is functionally adequate, and also that the selected MD's kidney function and health condition is not affected after the donation in the long‐term. The definition of marginal criteria differs by reports; however, several reports have indicated the usefulness and safety of kidney donation from MDs.This study investigated the adequacy of the current selection criteria for MDs based on the Japanese guidelines, including those with DM, and assessed the changes in renal function of donors after donation.22.12, (d) 70 ≤ creatinine clearance <80 ml/min/1.73 m2 per 24‐h urine collection, (e) 6.2 < hemoglobin A1c (HbA1c) ≤ 6.5 without oral medication for diabetes or ≤6.5 with oral medication for diabetes.In this study, we retrospectively evaluated 114 patients who underwent donor nephrectomy at our institution between May 2009 and April 2020. One hundred ten cases (96%) of donor nephrectomies were performed laparoscopically. We compared the backgrounds of donors at the time of transplantations including age, gender, marital status and each marginal factor. Based on the guidelines for living‐donor renal transplantations in Japan, we defined donors as marginal when at least one of the following conditions was met: (a) age >70 years, (b) blood pressure (BP) ≤ 130/80 mmHg with taking antihypertensive drugs, (c) 25 < body mass index (BMI) ≤ 32 kg/mOur retrospective study complied with the standards of the Declaration of Helsinki and current ethical guidelines, and was approved by the Okayama University Institutional Review Board (Registration No.: 1908‐026). We obtained written informed consent from all patients for the use of their clinical records.2.2Triple therapy, including extended‐release tacrolimus (Tac‐ER), mycophenolate mofetil (MMF), and prednisolone plus basiliximab, was the induction therapy. Basiliximab was administered on the day of the transplantation and 4 days later. ABO major mismatch, ABO minor mismatch, preformed DSA, and focal segmental glomerulosclerosis cases received a 200 mg/body dose of rituximab 1–2 weeks before the date of renal transplantation in addition to induction therapy. Tac‐ER, MMF, and prednisolone were also used as maintenance therapy.2.3Donors kidney function transitions were compared. To evaluate kidney function, serum creatinine (sCr), estimated glomerular filtration rate (eGFR), and urine protein‐creatinine ratio (PCR) were measured preoperatively and postoperatively at 1, 12, 24, 36, 48, and 60 months. The percentage of residual eGFR from baseline was also calculated. The baseline eGFR was defined as preoperative one. ACR was also assessed preoperatively and at discharge. MD + DM were subsequently compared with SDs in the same way.2.4U test was used for continuous variables. p < .05 were considered significantly different.All statistical analyses were performed using EZR version 1.41 , a graphical user interface for R.3p < .01), but gender and marital status were similar between groups. Median age of MD + DM was 71 (interquartile range [IQR]: 68–74) and that of MD without DM was 62 (IQR: 56–66). HTN and high BMI were the most common risk factor among MD (n = 25) and suboptimal kidney function was the least common risk factor (n = 5).There were 61 SDs and 53 MDs. As shown in Figure p = .16) and 8 versus 9 (p = .55). All donors had a good postoperative course, with no cases of dialysis induction or death.Preoperatively, and up to 60 months postoperatively, donor kidney function did not differ significantly between the SD and MD groups Figure . In thatp = .04; at discharge: 8 vs. 9, p = .99). However, when compared to MD + DM without multiple risk factors for 36 months, the difference disappeared. Nine out of 12 (75%) MD + DM had ≥2 risk factors per the MD criteria (Figure The comparison between SD and MD + DM in donors could be analyzed for 48 months. Postoperative eGFR was lower in MD + DM, and residual rate of eGFR from baseline in MD + DM was significantly higher at any point compared with those in SDs. No significant differences between SDs and MD + DM were found in sCr, PCR Figure  and ACR 4The data from our study suggested that donor kidney function did not differ significantly between the SD and MD groups. However, we should carefully follow MD + DM with multiple risk factors since they had a lower kidney function compared with SD.Previous reports comparing eGFR between SD and MD groups showed that MDs had a lower eGFR preoperatively and postoperatively; however, no significant differences in the rate of residual of eGFR were reported.We also compared the changes in renal function between SD and MD + DM. Our results showed that the postoperative eGFR was significantly lower in MD + DM. However, the differences disappeared comparing with the MD + DM without multiple risk factors. Our results indicated that a kidney transplantation from MD + DM was possible. It will help expand the donor pool. However, one should be careful to use MD + DM with multiple risk factors especially when they were young. The average age of MD + DM was 71 years in our study.Systolic BP > 140/90 mmHg is important cardiovascular risk for both the recipient and donors during and after kidney transplantation.2 is a contraindication for kidney donation according to the Japanese criteria.2 after dieting.Obesity is one of the significant risks affecting long‐term outcomes after kidney donation. BMI > 32 kg/mAll donors have visited the hospital at 1 month, 2 months, and then every year after kidney transplantation to check their health status, including HTN, obesity, decreased kidney function and DM. When the results of blood and urine examination and vital checks indicate the need for intervention, we have actively referred them to medical specialists and provided nutritional counseling. Our strict MD selection and careful postoperative follow‐up has contributed to no donor on dialysis after kidney transplantation.All donors have visited the hospital at 1 month, 2 months, and then every year after kidney transplantation to check their health status, including HTN, obesity, decreased kidney function and DM. When the results of blood sampling and vital checks indicate the need for intervention, we have actively referred them to medical specialists and provided nutritional counseling. These severe MD selections and careful postoperative follow‐up have resulted in no donor being placed on dialysis intervention after kidney transplantation.Comparing with the fact that MDs were 23%–29% in previous Japanese reports,This study has some limitations. First, this was a small, retrospective study performed at a single center and thus has the potential for selection bias. Second, our observation period is only 5 years. Third, Japan has as high as 90% of living‐donor kidney transplantations, whereas in most countries kidneys are acquired from deceased donors.Despite these limitations, this study demonstrates the safety and potential of kidney donations for MDs. The kidney function of MDs was not different when compared with SD during the first 5 years. Long‐term observation is needed in the future. Greater attention needs to be taken in the selection of MD + DM in terms of postoperative kidney function, particularly when MD candidates present with multiple risk factors.The authors declare that there are no conflict of interests.Motoo Araki, Koichiro Wada, and Yasutomo Nasu were involved in study design and data interpretation. Kasumi Yoshinaga, Yuki Maruyama, Yosuke Mitsui, and Takuya Sadahira were involved in data analysis, drafted the manuscript and designed the figures. Takanori Sekito, Shogo Watari, Risa Kubota, Shingo Nishimura, Kohei Edamura, Yasuyuki Kobayashi, Katsuyuki Tanabe, Hidemi Takeuchi, Masashi Kitagawa, Shinji Kitamura, Jun Wada, Masami Watanabe, and Toyohiko Watanabe were were involved in critical revision of the manuscript for important intellectual content. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.