Background: Dynapenia and non-alcoholic fatty liver disease (NAFLD) are common, especially in the middle and advanced-age diabetic male population. We aimed to examine the clinical features, NAFLD severity, and parameters associated with the presence of dynapenia in type 2 diabetes mellitus (T2DM) cases. Material and Methods: One hundred thirty-five male patients diagnosed with T2DM between 45 and 65 years of age were included. Patients were staged by ultrasonography according to NAFLD status. Results: There were significant differences in muscle strength, upper arm circumference, calf circumference, and up-and-go test scores between the mild-moderate-severe and non-NAFLD groups . The frequency of dynapenia was lower, and arm and calf circumferences were higher in patients without NAFLD. The muscle strength, upper arm circumference, calf circumference, and up-and-go test scores were significantly lower in the dynapenic group compared to the non-dynapenic group . The prevalence of dynapenia increased along with the increase in NAFLD stages (p < 0.001). Conclusions: We detected a significant association between NAFLD and dynapenia in middle-aged men with T2DM. As muscle strength decreases, the amount of fat in the liver increases, and as the fat in the liver increases, muscle strength decreases. Type 2 Diabetes Mellitus (T2DM) is one of the most common noncommunicable diseases in developing countries. Today, more than 400 million people are known to have T2DM, and this number is estimated to reach 642 million by 2040. NAFLD refers to the presence of hepatic steatosis without a condition to cause secondary hepatic fat deposition. Studies conducted in the USA reported NAFLD prevalence between 10% and 46%. In western industrialized countries where central obesity, T2DM, dyslipidemia, and metabolic syndrome are common, the most common liver disease associated with these diseases is NAFLD. Therefore, it is predicted that NAFLD prevalence will increase along with the increase in T2DM incidence .Insulin resistance is one of the most important risk factors for T2DM and NAFLD. However, the mechanisms underlying the development of NAFLD and T2DM are still not fully clarified. Furthermore, hepatic fat accumulation, changes in mitochondrial function and energy metabolism, and inflammatory cytokines produced from various cell types, including immune cells, lipotoxins, and adipocytokines, have been suggested to play an important role in the development of both NAFLD and T2DM .Dynapenia is a decreased muscle strength without a reduced muscle mass due to ageing. Theories have been presented that dynapenia may be caused by the disturbances in the stimulation–contraction process in the skeletal muscle along with the effect of many neuronal factors such as the deterioration in the ability of the nervous system to innervate the skeletal muscle, having fewer motor neuron units with ageing, and a decrease in the excitability of neurons ,6,7.Dynapenia and NAFLD are becoming increasingly common, especially in the middle and elderly diabetic male population, reducing physical performance and quality of life; The treatment process, costs, and complications constitute an important social problem ,6,7.In our study, we aimed to examine the clinical features, NAFLD severity, and parameters that may be associated with dynapenia in middle-aged male T2DM patients and to reveal the associated factors. Such a clinical study has yet to be completed in the literature, adding value to our study.www.ncss.com) (accessed on 23 September 2021) [This study was a cross-sectional, single-center, and prospective study. All procedures performed in studies involving human participants followed the institutional and national research committee’s ethical standards and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Clinical Research Ethics Committee of the University of Health Sciences, Umraniye Training and Research Hospital . The study’s purpose and procedures were explained to all participants, and written informed consent was obtained from them. The study enrolled one hundred thirty-five male patients due to statistical power analysis. We calculated the power analysis as follows: the minimum number of patients required to complete our study, which was calculated using the descriptive statistics in the article by Zhang et al., with a confidence level of 95% (α = 0.05) and a power of 80% was 131 patients . PASS 11. NCSS, LLC. Kaysville, UT, USA. Blood samples were taken on an empty stomach in the morning and analyzed simultaneously in the same laboratory. T2DM was diagnosed according to ADA criteria .Anthropometric measurements such as height and body weight of the patients were measured according to standardized guidelines: Arm circumference below 22 cm was considered as ‘lower’, and at and above 22 cm, levels were considered as ‘normal’. The calf circumference measurements below 31 cm were considered ‘lower’, and at and above 31 cm were considered ‘normal’ .The physical performance of the patients was evaluated by up and go test. The performance score was evaluated as follows; 1 = normal, 2 = very slightly abnormal, 3 = slightly abnormal, 4 = moderately abnormal, and 5 = severely abnormal .Using a hydraulic dynamometer, the right-left hand muscle strength of the patients was measured (12-0240 Baseline Hydraulic Hand Dynamometer 200 LB Standard W/Case). Muscle strength measurements were performed before blood sampling not to affect the test. Among male patients, more than 30 were normal, and less than 30 were accepted as decreased muscle strength in the measurements [2 was accepted as normal, 8.51–10.75 kg/m2 was accepted as moderate muscle mass reduction, and ≤8.50 was accepted as severe muscle mass reduction for men. Dynapenia was defined as a combination of normal SMMI index and decreased muscle strength, according to Manini and Clark [Patient values such as muscle mass, body fat ratio, metabolism/body fat, and lean body mass volumes were measured using a multi-frequency bioimpedance analysis (BIA) device  device, 2015 . The cut-off value for skeletal muscle mass index (SMMI) ≥ 10.76 kg/mnd Clark . The patA radiologist evaluated the presence and staging of NAFLD through transabdominal ultrasonography (USG) . The criteria of the Turkish Gastroenterology Association were used for the diagnosis of the fatty liver through USG. Hepatic echogenicity typically consists of measurements of the echo difference between the liver and kidneys, evaluation of echo penetration into the deep part of the liver, and determination of the clarity of blood vessel structures. The degree of lipoidosis severity was evaluated clinically as used in daily practice by dividing it into four classes. The ultrasonography findings in hepatic steatosis are presented in 2 test and expressed as observation counts (and percentages). Statistical significance was accepted when the two-sided p-value was lower than 0.05.The statistical analysis was conducted through the MedCalc Statistical Software version 12.7.7 . The normality of continuous variables was analyzed using Shapiro–Wilk’s test. Descriptive statistics were expressed by the mean and standard deviation for normally distributed variables and the median for the non-normally distributed variables. Non-parametric statistical methods were utilized to analyze the values with skewed distribution. The Mann–Whitney U test was used to compare two non-normally distributed groups. The Kruskal–Wallis test was used to compare two non-normally distributed variables. As a pairwise post hoc comparison, Bonferroni corrected Mann–Whitney U test was performed. Categorical variables were evaluated by χThe age average of the participants was 55.2 ± 4.6 (45–65) years, the mean muscle strength was 35.5 kg, the mean HbA1c was 9.9%, and the mean diabetes duration was 5.9 years. USG evaluation of the patients revealed that NAFLD was not detected in 34 (25.2%) patients, mild NAFLD was detected in 34 (25.2%) patients, and moderate and severe NAFLD were detected in 48 (35.6 %) and 19 (14.1%) patients, respectively. Forty-two (31.1%) patients met the criteria for dynapenia, and 93 (68.9%) patients were non-dynapenic. The demographic data, anthropometric measurements, and clinical and biochemical parameters of the patients are summarized in p < 0.001 for all). We detected that arm and calf circumferences were higher in patients without NAFLD; physical performance was better, and dynapenia prevalence was lower. There was no difference between the groups regarding other parameters, which were summarized in There were significant differences in muscle strength, upper arm circumference, calf circumference, and up-and-go test scores between the NAFLD groups . There was not any statistically significant difference between the two groups for age, muscle mass, SMMI index, HbA1c, and fasting glucose measurements. The severe NAFLD ratio was 33.3% in the dynapenic group and 5.4% in the non-dynapenic group.The patients were then compared according to their dynapenic status as dynapenic and non-dynapenic. All the comparisons are summarized in In this cross-sectional study of middle-aged men with T2DM, we investigated the possible relationship between dynapenia and NAFLD, and we detected a clear connection between dynapenia and NAFLD in this patient group. We found that the muscle strength decreased along with the increase of the NAFLD stage in these patients, and the NAFLD stage increased as the muscle strength decreased. Based on the literature review, we believed that our study was valuable as it was the first study to be conducted on this subject among diabetic men. As part of our study, we included patients with T2DM who had insulin resistance, which was considered to occur similarly to NAFLD pathogenesis. Moreover, the study excluded women with different normal ranges of dynamometric measurements and younger men with no history of dynapenia. As a result, a homogeneous and specific patient group was formed. The number of patients with T2DM and NAFLD, commonly observed together, increases globally. NAFLD and T2DM occur with similar metabolic processes, and insulin resistance is considered an essential risk factor in developing both diseases . The intSarcopenia is more common in patients with lower BMI; however, it was reported that dynapenia is more common in patients with higher BMI. It was also reported that dynapeniais is more common in patients with T2DM rather than sarcopenia as a muscle disorder form . Our stuNAFLD was not detected in 25.2% of our patients through USG, whereas NAFLD of various grades was found in 74.8% of patients. Furthermore, 31.1% of our patients were dynapenic, and 68.9% were non-dynapenic. When the patients were compared according to NAFLD stages, there was a significant difference between the groups in muscle measurement parameters such as muscle strength, upper arm circumference, calf circumference, and the up-and-go test. We detected that arm and calf circumferences were higher in patients without steatosis, better physical performance, and lower dynapenia prevalence. Dynapenia was detected in 45.83% of patients with moderate NAFLD and 73.68% with severe NAFLD. Moreover, a significant decrease was observed in muscle strength measurements along with the increase in NAFLD stages. In other words, it was observed in our study that the incidence of dynapenia increased along with the rise in the NAFLD stage.Similarly, long-term progressive resistance training and muscle strengthening training has been shown to stimulate muscle hypertrophy and type II muscle fiber growth, thus preventing or limiting dynapenia .In our study, no significant difference was found regarding fasting glucose, HbA1c, and BMI in all stages of NAFLD. Most guidelines include efforts to reduce oxidative stress and improve insulin resistance and weight loss for the prevention and treatment of NAFLD as management of NAFLD. However, recent studies have shown the importance of maintaining and increasing muscle mass to prevent NAFLD .Our study had some limitations. Our work was conducted within a certain period. Thus, it took a short observation period to establish a causal relationship for the significant association between dynapenia and NAFLD. Our study was single-center; therefore, our results may not represent all patients with T2DM. The patients included in our study were adult, middle-aged men; consequently, it is unclear whether our findings also apply to men and women in this age group. Another limitation of the study is that the measurements and comparisons were carried out only once. Our further studies will be to observe changes in NAFLD scores with liver-protective diets and lifestyle recommendations and then to monitor changes in dynapenia scores in the same patients.Despite these limitations, our study shows the relationship between NAFLD and dynapenia in middle-aged adult T2DM patients and is a valuable study that has not been conducted before.In conclusion, the results of our study revealed that maintaining fasting glucose, HbA1c, and BMI of the patients under control will lead to the improvement of NAFLD disease developed in middle-aged male patients with T2DM along with improving muscle strength However, further studies with more extensive series should be performed to support our hypothesis.


Background and Objectives: Among the extra-pulmonary manifestations of COVID-19, neuromuscular signs and symptoms are frequent. We aimed to assess the correlation between neuromuscular abnormalities  and mobility measures  twice, at least 6 weeks after hospital discharge and 6 months later, taking into account cognitive performance, nutrition, muscle strength, and submaximal exercise capacity. Materials and Methods: 43 patients (51.4 ± 9.3 years old) accepted to participate in the study; they had a dyspnea score ≤ 3 , and no history of neurology/neuromuscular/orthopedic disorders, but high frequency of overweight/obesity and weight loss during hospital stay. The two evaluations included physical examination, cognitive assessment, nutritional evaluation, muscle strength (hand-grip and quadriceps dynamometry), electromyography, Barthel Index, Six-Minute- Walk-Test (6MWT), Berg Balance Scale and Timed-Up-and-Go test. Bivariate and repeated measures covariance analyses were performed (significance level of 0.05). Results: Electrophysiological abnormalities were evident in 67% of the patients, which were associated with diminished performance on the 6MWT, the Berg Balance Scale and the Timed-Up-and-Go test. At each evaluation and between evaluations, scores on the Berg Balance Scale were related to the body mass index (BMI) at hospital admission and the 6MWT , while the time to perform the Timed-Up-and-Go test was related to the electrophysiological abnormalities, weight loss during hospital stay, sex, handgrip strength, and the 6MWT . We concluded that, after hospital discharge, patients with moderate to severe COVID-19 may have neuromuscular abnormalities that can be related to BMI/weight loss, and contribute to mobility decrease. In patients with moderate to severe COVID-19 and high BMI/ large weight loss, neuromuscular and intended mobility assessments could be required to provide early rehabilitation. Apart from the 6MWT, handgrip dynamometry and the Timed-Up-and-Go test were useful tools to quickly assess fitness and mobility. COVID-19 is caused by the beta coronavirus SARS-CoV-2 . In adulAfter the acute illness, which usually lasts 4 weeks from the onset of symptoms, COVID-19 survivors may also show residual symptoms and organ impairment that can persist beyond 12 weeks of the onset of disease . In ordeA systematic review and meta-analysis of 350 studies on the neurological manifestations related to COVID-19, including 145,721 patients, showed 41 neurology manifestations; among them, the most common symptoms were fatigue (32%) and myalgia (20%) . In the After the acute care of COVID-19, the high prevalence of neuromuscular symptoms persists. In Australia, after 6 months of critical illness, 21.7% of 112 surviving patients reported loss of strength , and funThe abilities required to independently care for oneself (activities of daily living) include those activities that enable basic survival and well-being (basic activities) and those required to support daily life at home and in the community  . HoweverAlthough mobility is a fundamental factor to undertake daily life activities, we did not find studies on the contribution of neuromuscular abnormalities (by electrophysiology) to functioning after acute COVID-19, or the cofactors that could have influence on the relationship between these two variables. Then, we primarily designed a study to assess the relationship between neuromuscular abnormalities (by electrophysiology) and mobility measures , at least 6 weeks after hospital discharge due to COVID-19, with a follow-up assessment six months afterwards, taking into account the cognitive performance, nutrition, muscle strength, and submaximal exercise capacity of the patients. The secondary aim of the study was to identify among these cofactors, those that may contribute to the neuromuscular abnormalities and the mobility of patients discharged from hospitalization due to moderate to severe COVID-19.In a general hospital reconfigured to address the surge of patients with COVID-19 during the first two waves of contagions (up to April 2021), 43 consecutive patients  who were alive at least 6 weeks after hospital discharge, and fulfilled the selection criteria, accepted to participate in the study. Their general characteristics are described in Between the two study evaluations, three patients declined participation due to change of residence; they were 53 to 69 years old (2 men/1 woman). Therefore, 43 patients were evaluated at the time of inclusion in the study, and 40 patients at follow-up. A sample size of 41 participants was calculated to detect a statistically significant Pearson’s correlation coefficient of 0.5, considering bilateral type I error of 0.01 and type II error of 0.2.According to the institutional recommendations, during hospitalization, the medical treatment of the participants included dexametasone and antibiotics (ceftriaxone (69%), levofloxacin (37%), alone or combined with azitromicine, claritromicine, menopenem, doxiciclinae or cefotaxam); only four patients had severe disease and required neuromuscular blockade related to mechanical ventilation.General cognitive performance, by the Mini-Mental State Examination ;2;Nutrition, by the Nutritional Risk Screening  or the MThe abilities required to independently care for oneself, by the Barthel Index for daily life activities ;Neuromuscular symptoms, by an in-house short questionnaire to recall symptoms during hospital stay , and to report them if present during the previous week , including myalgia, fatigue, muscle spasms/twitches/tremors, and numbness/tingling/burning sensations;Muscle strength, by three measures: (1) the Medical Research Council scale (MRCs)  ; (2) quaElectrophysiological abnormalities of upper and lower limbs, by electromyography records  by a standardized protocol ,30; abnoSubmaximal exercise capacity by the Six Minute Walk Test (6MWT) ;Mobility by two measures: (1) the Berg Balance Scale  and (2) A detailed description of the procedures is provided in After the first evaluation, the patients received rehabilitation according to their individual capacity .After assessing the data distribution (Kolmogorov–Smirnov test), “t” test (either unpaired or paired) was used to assess comparisons between evaluations and between patients with/without electrophysiology abnormalities; the Pearson’s correlation coefficient was used to explore correlations. Accordingly, repeated measures analyses were performed using Multivariate Analysis of Covariance on the scores of the Berg Balance Scale and the time to perform the Timed-Up-and-Go test. The significance level was set at 0.05. At the two evaluations, all the participants had an adequate performance on the Mini-Mental State Examination (score ≥ 24); the score range was 25 to 30 at the first evaluation, and 28 to 30 at the second evaluation.At the two evaluations, none of the participants was at risk of malnutrition. However, according to the medical records, at hospital admission, 84% of the patients had BMI ≥ 25; while, at hospital discharge, 100% of the patients had lost weight . p = 0.0003).At the first evaluation of the study, 14 of the 43 (32%) patients had BMI ≥ 30 (obesity), and 18 (41%) patients had BMI > 25 ≤ 30 (overweight); at the second evaluation, 17 of 40 (42%) patients had BMI ≥ 30, and 15 (37%) patients had BMI > 25 ≤ 30. Then, the proportional gain at the first evaluation  was from 0% to 62% and, at the second evaluation, the gain was from 0 to 70%, with a significant weight increase between evaluations , and myalgia ; while 16% (95% C.I. 5%–27%) of the patients reported muscle spasms/ twitches/tremors, and 25% (95% C.I. 12%–38%) of them reported numbness/ tingling/burning sensations;At the first evaluation, 77% reported fatigue (95% C.I. 63%–89%) and 44% reported myalgia (95% C.I. 29%–59%), while circa one third of patients reported muscle spasms/ twitches/ tremors as well as numbness/ tingling/ burning sensations;At the second evaluation, the frequency of the symptoms decreased. At the two evaluations, men reported symptoms more frequently than women .p < 0.001), with no change on the handgrip strength (p >0.05). The results on the modified MRCs are shown in Sensory–motor polyneuropathy was diagnosed in seven (16%) patients, including two of the patients who abandoned the study. Three patients had a history of type 2 diabetes, and one was a blacksmith. The recordings were similar at the two evaluations;Motor polyneuropathy was diagnosed in two (4%) patients; one of them with type 2 diabetes, and the other patient required intensive care during their hospital stay, with 45 days of mechanical ventilation. The recordings were similar at the two evaluations;Multiple mononeuropathy  occurred in four (9%) patients, including one of the patients who abandoned the study. Among the four patients, one had a history of diabetes, and two reported the occupational performance of repetitive hand movements. The recordings were similar at the two evaluations.○Median nerves at the level of the carpal tunnel in seven (16%) patients . All the patients reported the occupational performance of repetitive hand movements; in addition, one patient had type 2 diabetes, and another patient reported history of traumatic injury of the affected arm. The recordings were similar at the two evaluations;○Peroneal nerves in seven (16%) patients; below the ankle in four patients, and at the level of the fibular head in three patients, including one patient with bilateral compromise. Two of them reported history of traumatic injury of the affected foot. At the second evaluation, the neuropathy resolved only in one patient with no comorbidities: a 33 year old female, who required 55 days of hospital stay , and had a 14.3% loss of her body mass at hospital discharge.Mono-neuropathy affecting:Generalized myopathy was diagnosed in one patient, a 39 year old man, who required 28 days of hospital stay , receiving dexametasone and antibiotics .The main results are shown in At the two evaluations, all the participants reported independence to perform the basic activities of daily life, except for one patient  who reported limitations for grooming.p < 0.0001), the proportional distance from predicted increased , with less oxygen saturation decrease . Moderate to strong linear correlations were observed on the walked distance, with age, muscle strength, body mass index at hospitalization and weight loss during hospitalization ; additionally, moderate to strong correlations were observed between the proportional distance and the MRCs score , and the weight loss during hospitalization .At the beginning of the two evaluations, all the patients reported no dyspnea, but variable increase during the test (range 0 to 4). At the first evaluation, the distance walked by the patients was only 78.9% (95% C.I. 66.8%–91%) of the predicted distance, with oxygen saturation decrease to 89% (95% C.I. 80%–98%), tachypnea , and tachycardia (120 ± 22/min)) . At the p = 0.001). Moderate linear correlations were observed between the total score at the first evaluation and the MRCs , and the BMI at hospital admission .At the first evaluation, all participants reported no difficulty to stand or sit without support. However, they reported limitations on activities requiring leg strength: standing on one foot, standing with one foot in front of the other foot and reaching forward with outstretched arm . At the p = 0.002); however, four patients performed the test in >10 s, and the three patients who had abandoned the study had performed the test in >10 s at the first evaluation. The time to perform the test at the first evaluation showed moderate linear correlation with age  and muscle strength of both the upper and lower limbs .At the first evaluation, in all the patients, the mean time to perform the test was 8.69 ± 1.48 s. However, 12 patients performed the test in >10 s; they were 39 to 74 years old (six men/six women), all had neuromuscular abnormalities, three had sarcopenia and one had severe COVID-19. At the second evaluation, the time to perform the test decreased (8.05 ± 1.31 s) , had greater weight loss during hospital stay , and had greater weight gain after hospital discharge . At the two evaluations, the frequency of symptoms was similar in the two groups (p > 0.05). At the first evaluation, compared with patients with no electrophysiology abnormalities, patients with any abnormality had lower scores on the MRCs  while, on the 6MWT, they walked a shorter distance , and proportional distance ; they also had a lower score on the Berg Balance Scale . At the second evaluation, a difference persisted on the 6MWT, on the walked distance , while it was borderline on the Berg Balance Scale .Analysis according to electrophysiology abnormalities showed that, compared with patients with no electrophysiology abnormalities, those with any abnormality were older  and the walked distance during the 6MWT (p = 0.01) ; and at the second evaluation the whole model R was 0.65 ; with difference between the two evaluations (p = 0.047), which was related to the walked distance during the 6MWT (p = 0.009).The multivariate analysis of covariance on the scores of the Berg Balance Scale showed that it was related to the BMI at hospital admission (p = 0.001); electrophysiology abnormalities (p = 0.02); sex (p = 0.004); the walked distance during the 6MWT ; and the handgrip strength, either of each hand or the normalized value, but particularly the absolute strength of the dominant hand (p = 0.007), all the patients were right handed ; at the second evaluation, the whole model R was 0.62 ; with difference between the two evaluations (p = 0.007), which was related to the handgrip strength of the right (dominant) hand (p = 0.004), and sex (p = 0.005) since men improved more than women.The multivariate analysis of covariance on the time to perform the Timed-Up-and-Go test showed that it was related to: the proportional weight loss at hospital discharge  persisted. We observed that the results on the two mobility tests  were related to both the 6MWT and the muscle strength. However, the recovery of quadriceps strength was related to improvement on the Berg Balance Scale, while the handgrip strength was mainly related to the time to perform the Timed-Up-and-Go test. A previous study has shown that handgrip strength is related to hospitalization due to COVID-19, independent of age, sex, and health-related conditions , while iWalking and postural transitions are among the basic motor tasks that every person must perform several times a day. In this study, we observed that patients discharged from hospital, with no history or evidence of neurological deficits, may have sub-clinical neuropathy and muscle weakness interfering with balance and mobility. These findings suggest that, in order to select adequate rehabilitation strategies, individual needs should be intentionally evaluated, including neuromuscular and balance tests.The main limitation of this study was the small, convenient sample. The sample size was estimated to assess relationships between variables, while reducing the variability that could be introduced by some recognized co-factors . Another limitation was that we mainly evaluated patients with moderate disease, only four had severe disease, and none had mild disease; the characteristics of each subgroup may imply variability on both neuromuscular abnormalities and mobility. Although the selection bias may have driven to overestimation of performance, it allowed us to identify associations between neuromuscular abnormalities, mobility and the influence of several cofactors; particularly the association among high BMI at hospital admission and weight loss during hospital stay with neuromuscular abnormalities and functional performance, which has not been reported previously in patients with COVID-19. Further studies with broader selection criteria may show the full spectrum of neuromuscular abnormalities and its consequences on mobility.In adults, after hospital discharge due to moderate to severe COVID-19, neuromuscular abnormalities may contribute to mobility decrease; while both BMI at admission and weight loss during hospital stay may contribute to neuromuscular abnormalities. These abnormalities may be identified only after intended evaluations, including electromyoneurography and mobility tests. In addition, apart from the 6MWT, handgrip dynamometry and the Timed-Up-and-Go test could be useful tools for the assessment and follow-up of patients with COVID-19.


Our findings show that key changes in functional connectivity in diabetes occur in the precuneus and executive control networks that evolve before patients develop cognitive deficits. In addition, the current study provides useful information about the role of the thalamus, inferior parietal lobe, and precuneus, which might be potential biomarkers for predicting the clinical progression, assessing the cognitive function, and further understanding the neuropathology of T2DM.In epidemiological studies, type 2 diabetes mellitus (T2DM) has been associated with cognitive impairment and dementia, but studies about functional network connectivity in T2DM without cognitive impairment are limited. This study aimed to explore network connectivity alterations that may help enhance our understanding of damage-associated processes in T2DM. MRI data were analyzed from 82 patients with T2DM and 66 normal controls. Clinical, biochemical, and neuropsychological assessments were conducted in parallel with resting-state functional magnetic resonance imaging, and the cognitive status of the patients was assessed using the Montreal Cognitive Assessment-B (MoCA-B) score. Independent component analysis revealed a positive correlation between the salience network and the visual network and a negative connection between the left executive control network and the default mode network in patients with T2DM. The differences in dynamic brain network connectivity were observed in the precuneus, visual, and executive control networks. Internal network connectivity was primarily affected in the thalamus, inferior parietal lobe, and left precuneus. Hemoglobin A1c level, body mass index, MoCA-B score, and grooved pegboard (R) assessments indicated significant differences between the two groups ( Aging, overeating, a sedentary lifestyle, and lack of physical exercise have led to the present high prevalence of prediabetes and diabetes. Currently, 463 million people worldwide live with type1 or type 2 diabetes mellitus (T2DM), especially in low- and middle-income countries, and 1.8 million people die from this disease annually. Moreover, more than 50 million people live with dementia, and this number is expected to increase threefold by the year 2050 . As bothT2DM, which accounts for approximately 60% of all diabetic cases, primarily characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. Accumulation of advanced glycation end products (AGEs) may lead to inflammation, oxidative stress, protein cross-linking, cerebral insulin resistance and metabolic disorders , damage Previous cross-sectional magnetic resonance imaging (MRI) studies have consistently reported associations between T2DM and reduced global brain volume . In addiTo date, although there are numerous previous T2DM studies on differences in functional connectivity (FC) , especian = 2), missing MoCA-B scores (n = 12), developmental disorders (n = 2), or neuropsychiatric diseases (n = 2) , a total of 82 patients with T2DM and 66 healthy individuals were eventually enrolled in the study. This study was approved by the ethics committee of The first affiliated hospital of Guangzhou University of Chinese Medicine and written informed consent was obtained from all subjects. Subjects with T2DM were selected from among hospitalized patients and outpatients at the Endocrinology Department of The First Affiliated Hospital of Guangzhou University of Chinese Medicine, while NCs were recruited as volunteers over the same period. T2DM was diagnosed when fasting blood glucose was >7.0 mmol/L on two separate occasions or when the 2-h blood glucose level was >11.1 mmol/L during a 75-g oral glucose tolerance test , we had excluded subjects with brain tumors  abuse, moderate-severe hypertension [systolic blood pressure (SBP) = 150 mmHg or diastolic blood pressure = 100 mmHg] or mild hypertension took medication for 3 months prior to the start of the study and during the study, severe hypoglycemia or hyperlipemia, specific abnormalities found on conventional MRI scans, or other factors that might affect brain structure and function .The exclusion criteria for both groups were as follows: cognitive impairments, impaired glucose tolerance or impaired fasting glucose  that not2). Arterial blood pressure was measured on the both arms in a sitting position after rested for 5 min, and hypertension blood pressure was identified (blood pressure 140/90 mmHg).Body weight and height were measured without shoes or heavy clothes. BMI was calculated as weight in kilograms divided by the squared height in meters , MoCA-B (patients with a MoCA-B score <26 were diagnosed with mild cognitive impairment) , MMSE, W2; matrix size, 224 × 256 interpolated to 448 × 512, 192 sagittal slices; slice thickness, 1.0 mm; and voxel size, 0.5 × 0.5 × 1 mm3.All images were acquired on a Siemens  3T Prisma scanner using a standard 64-channel head coil. Functional volumes were acquired using a multiple slice T2-weighted echo planar imaging sequence with the following parameters: repetition time, 2,000 ms; echo time, 30 ms; flip angle, 90°; matrix dimensions, 64 × 64; field of view, 100 mm; slice thickness, 3.5 mm; and number of slices, 33. High-resolution three-dimensional T1-weighted images were acquired using the magnetization-prepared rapid gradient-echo sequence with the following parameters: repetition time, 2,530 ms; echo time, 2.98 ms; inversion time, 1,100 ms; flip angle, 7°; field of view, 256 × 224 mm1 and GRETNA (GRETNA v2.0)2 under Matlab R2013b. All functional images were first corrected for slice timing with the middle slice in the acquisition order as the reference and realigned to the first image to correct head movement. Thirteen subjects showed >2-mm translation and >2° rotation in the localizer task; thus, their data were eliminated from the localizer analysis. Individual functional images were co-registered to the mean functional image. The T1 image was coregistered to a mean functional image and then segmented. The functional images were then normalized to the Montreal Neurological Institute (MNI) space using DARTEL after the segmentation. An anisotropic 6-mm full-width half-maximum Gaussian kernel was used for spatial smoothing.Preprocessing and statistical analyses of imaging data were performed with SPM12 3 voxel size . Condition onset and duration were convolved with the canonical hemodynamic response function (HRF). Realignment parameters were included in the design matrix as covariances to regress out the movement-related variance.3 The number of independent components was estimated using the minimum description length approach, resulting in an average of 41 independent components. The preprocessed resting-state data of all subjects were temporally dimension-reduced using PCA and concatenated. All subjects were then concatenated into one group and passed through another dimension reduction step. The grouped data were decomposed into 20 aggregate components  to extract the functional network of the brain using the Infomax algorithm in GIFT .mponents . Then a mponents .p > 0.05), and there were significant differences in hemoglobin A1c levels, BMI, MoCA-B scores, and grooved pegboard (R) (p < 0.05) between the two groups. There were no significant differences in the other neuropsychological tests results between the two groups (p > 0.05)  .p < 0.05), whereas negative connectivity was mainly observed between the RECN with the SN, DMN, PVN, EVN, and LECN with the DMN (p < 0.05) , the right executive control network (RECN), the primary visual network (PVN), the extrastriate visual network (EVN), the salience network (SN), and the default mode network (DMN). Compared to the NC group, in the T2DM group high positive connectivity was mainly observed between the SN with the PVN and the LECN with the EV ( < 0.05) .p < 0.05, false discovery rate (FDR)-corrected] . Subsequently, to reveal the regions specifically related to these three networks, they were selected as targets for whole-brain FC. Compared to the NC group, the T2DM group showed differences in the right middle cingulate, left paracentral lobule, right angular, left inferior parietal, right middle temporal, and right inferior temporal lobes, left superior anterior cingulate cortex, right precuneus in the LECN; right superior temporal, left middle temporal lobes, and left thalamus in the RECN; right angular, left middle occipital, left inferior parietal lobe, and right precuneus in the precuneus network . In state 3, LN, precuneus network, EVN, PVN, SN, DMN, and RECN connectivity was enhanced, whereas LECN connectivity was weakened. In state 4, LN, precuneus network, EVN, and PVN connectivity was enhanced, whereas connectivity in the LECN with EVN and PVN was weakened. Overall, connectivity in the LN, precuneus network, EVN, and PVN increased, whereas LECN, EVN, and PVN connectivity was lower in the T2DM group compared with the NC group.The k-means algorithm was repeated 1,000 times for each k value, and the clustering results were recorded each time. The clustering evaluation results for different cluster numbers are shown in p = 0.0098, T = 2.6187)  , indicatr = 0.259, p = 0.025) and SBP , respectively. The right thalamus negatively correlated with BMI . The right inferior parietal lobe showed negative correlations with BMI  and DSST . As for the correlation between scales, educational level showed a positive correlation with the AVLT (immediate)  and the AVLT (5 min) . In the NC group, the left precuneus showed a positive correlation with fasting blood glucose   (= 0.036) , and a sThis study found that patients with T2DM-NCI start to exhibit altered brain connectivity in the executive control and the precuneus network, with the main differences, compared with NCs, found in the thalamus, inferior parietal lobe, and precuneus. These observations provide a perspective for follow-up studies in patients with T2DM-NCI. In the present study, we focused on functional network connectivity in patients with T2DM-NCI and NCs. ICA revealed increased connectivity between the SN and VN and decreased connectivity between the LECN and DMN in patients compared to the NC group. These results were further validated by a dynamic network connectivity analysis , which iWe found that the main regions undergoing FC alterations were the inferior parietal lobe, right angular gyrus, left thalamus, left precuneus, left superior parietal and left superior occipital lobes, and left posterior cingulate. The key pathological changes in T2DM-NCI involve the hippocampus, temporal lobe, left inferior parietal lobe, cingulate gyrus, and precuneus . CompareOur analysis of correlations between FC and cognitive scores revealed that the thalamus and the right inferior parietal lobe negatively correlated with BMI, this indicates that obesity can cause serious damage to the thalamus and right inferior parietal lobe. In the NC group, fasting blood glucose levels within the normal range and higher blood glucose levels can stimulate the activity in the left precuneus brain region. The opposite of this phenomenon was observed in our diabetic patients. Thus, within normal ranges, higher fasting glucose levels have an activation effect on the left precuneus, but beyond this threshold, blood glucose levels will cause damage to the corresponding brain regions. A correlation analysis between test scales indicated that education helps with vocabulary memory and reaction ability at the beginning, but has no effect on delayed memory, suggesting that educational level does not play a significant role in cognitive assessment.The thalamus is the higher sense center, as the most important sensory conduction replacement station of the brain. All sensory conduction pathways are projected to the cerebral cortex after neuron replacement in the thalamus. In this study, the thalamus showed a higher correlation with the test scale scores and better overall connectivity. Thalamic activation was more evident compared activation in other brain regions, and thus provides a useful metric for evaluating the progression of type 2 diabetes. Therefore, the thalamus and precuneus should be the focus of future studies on cognitive development in T2DM, given their critical role in cognitive impairment in diabetes.The present study has several strengths. First, to the best of our knowledge, this is the first study to examine the compensatory mechanism of the precuneus in conjunction with other networks in patients with T2DM before the onset of cognitive impairment, and it thus highlights the role of the precuneus network as an important neuroimaging marker in patients with T2DM. Second, by combining functional and network connectivity, we identify the regions of the brain where the differences are observed. Third, patients with cerebral infarction, including lacunar cerebral infarction, were excluded from this study to control for other interfering factors. At the same time, all main analyses were performed with age, sex, and educational level as covariates to exclude the potential influence of these factors. Compared to most earlier studies, the two groups of subjects involved in the current study were relatively young and had a narrow age range, which reduced the heterogeneity of the cohort and thereby the detrimental effect of age.However, this study also has some limitations. First, we did not conduct a sample size estimation before the experiment. Although the sample of patients with T2DM in our study is larger than those of most T2DM studies, it is still somewhat insufficient, especially regarding the number of patients with T2DM with available cognitive domain scores. Since small sample sizes can lower the statistical power and reproducibility of a study , we willIn summary, this study provides novel findings suggesting that higher fasting glucose level may activate the left precuneus within normal ranges in healthy individuals, which means that fasting glucose level is one of the key indicators affecting brain activation. Our findings also suggest that the precuneus network may be involved in a compensatory mechanism in T2DM-NCI, and that the thalamus, precuneus, and inferior parietal lobe may serve as sensitive neuroimaging features of cognitive impairment. Our approach provides a background for predicting cognitive deficits in patients with T2DM of different stages, and an intuitive perspective for follow-up studies on T2DM-NCI. In the future, machine learning methods could be promising for exploring the region-dependent changes in individuals with varying cognitive impairment.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Guangzhou University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SK, XM, XT, HH, YF, YC, and WL contributed to the design of the study. KL, LF, JS, and JW took part in the data processing. LZ, SQ, and DH provided theoretical guidance. JW performed the data analysis and drafted the manuscript. All authors revised the manuscript and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This review aims to sum up the key structural and functional characteristics of Q10, taking stock of its use in people affected by fibromyalgia. A thorough survey has been conducted, using Pubmed, Scifinder, and ClinicalTrials.gov as the reference research applications and registry database, respectively. Original articles, reviews, and editorials published within the last 15 years, as well as open clinical investigations in the field, if any, were analyzed to point out the lights and shadows of this kind of supplementation as they emerge from the literature.The coenzyme Q Sleep disorders are also present, characterized by frequent nocturnal awakenings which exacerbate the fatigue condition. In particular, the so-called alpha-delta anomaly is considered to be specific to FM, as once deep sleep is reached, characterized by delta waves at the electroencephalogram, there is a sharp return to superficial sleep, characterized by alpha waves. Cognitive disorders are present in the majority of patients, being related to difficulty in concentration and short-term memory loss .To date, the prevalence of FM is estimated between 5 and 7%, while the incidence level ranges between 7–11 cases per 1000 people per year. It is more frequent in women than in men, with 11 vs. 7 cases per 1000 people, respectively ,7. MoreoCurrently, the diagnosis of FM is carried out according to the American College of Rheumatology (ACR) 2016 criteria, based on the scores of the widespread pain index (WPI) and the symptom severity scale (SSS) . Mainly For some time now, researchers have been calling for specific biomarkers. As thoroughly reviewed by Ablin and co-workers, it should be possible to highlight both a predisposition to FM (from a genetic point of view) and the manifestations of the disease. However, those investigated so far, including serological alterations and instrumental investigations, are for research purposes only .The etiology of FM has not yet been fully understood, and uncertainty still exists concerning the pathophysiological framework. The preeminent hypothesis calls into question a dysregulation in the mechanisms of pain control by the central nervous system (CNS) ,20,21,2210 levels accompanied by the increased expression of pro-inflammatory interleukins IL-1β and IL-18, higher levels of TNF-α [Anyhow, FM is clearly characterized by mitochondrial dysfunction . Sprott of TNF-α , and theof TNF-α ,28.The multifaceted nature of FM requires a multimodal and multidisciplinary approach, mainly aimed at reducing the severity of the symptoms. However, on the basis of the scientific evidence available to date, there are no fully satisfying protocols. Moreover, poor adherence to the therapeutic indications and the presence of comorbidities are two very frequent factors that can modulate the worsening of symptomatology, leading to its chronicity ,30,31,3210 has the preeminent role of supplementing FM patients, as is thoroughly discussed below.Over the last decade, the use of personalized dietary regimes has become increasingly important. The supplementation of minerals, such as selenium, zinc, iron, and magnesium, but also vitamins like B9, B12, A, and D, as well as antioxidants, is pursued to complement the nutritional strategy, avoiding nutritional deficits, taking control of oxidative stress, and supporting the immune system ,35,36,3710 in our body is produced endogenously, but a small amount is also ingested daily through the diet, being mainly present in meat and fish and, in much lower quantities, in some vegetables. Due to its wide prevalence in living organisms, this coenzyme is further referred to as ubiquinone [Coenzyme Q (CoQ) is a widely distributed naturally occurring lipophilic benzoquinone, firstly discovered by Crane and co-workers more than 60 years ago in beef mitochondria . From a iquinone .10 in the mechanisms of ATP production at the mitochondrial level was deciphered and acknowledged. In particular, the intense research activity carried out by Peter Mitchell, who, thanks to these studies, was awarded the Nobel Prize, clarified CoQ10 role as a key cofactor in the oxidative phosphorylation.Since the first pioneering study by Crane and co-workers, the crucial function of CoQ10 acts as a mobile electron carrier in the electron transfer chain at the inner mitochondrial membrane, but it is also located in blood lipoproteins and cell membranes, where it plays a key antioxidant role once in its reduced form. Indeed, as CoQ10H2, it is able to quench free radicals and regenerate α-tocopherol and ascorbate [2 through a two-electron reaction, and NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase, which are one-electron reductases [As thoroughly reviewed by several authors ,41,42, Cscorbate ,44,45. Sductases ,50,51,5210 influences the expression of a significant number of genes. Investigated in HeLa cells by Gorelick and co-workers, it was proved to alter 264 sequences, enriching, in particular, lipid-related genes [10 treatment increases the expression of 694 genes encoding proteins involved in cell signaling, intermediary metabolism, transport, transcription control, disease mutation, phosphorylation, embryonal development, and binding [In addition to redox activity, more recent data revealed that CoQed genes . Using t binding ,55, thus10 is biosynthesized in all tissues and recovered in all membranes. In human tissues, the highest amount of cofactor is found in the heart, liver, kidneys, and muscles, being the districts with either high energy requirements or metabolic activity [10 is mainly localized in Golgi apparatus, lysosomes, and mitochondria [10 helps to replenish the poor physiological amount of the coenzyme. The recommended daily intake of CoQ10 from exogenous sources ranges from 30–100 mg for healthy people but rises up to 60–1200 mg when it is used as an adjunct supplement in some pathological conditions [10 is characterized by an estimated no observed adverse effect level (NOAEL) of 1200 mg/kg/day, based on a 52-week chronic toxicity study in rats, and an estimated acceptable daily intake (ADI) of 12 mg/kg/day, ensuring a daily intake without an appreciable health risk throughout life. Furthermore, several studies have shown that, following supplementation, CoQ10 does not accumulate in the human body after the cessation of treatment and this does not affect its endogenous synthesis [CoQactivity . At the chondria . Its amochondria . In thesnditions . In any ynthesis ,60,61,6210 may be exploited, represented by food matrices and food supplements. While the former is subjected to the variability of the dietetic regimens, the latter assures the constant, regular, and controlled intake of CoQ10 over time.Two different sources of exogenous CoQ10 among the different food matrices varies significantly, as it is mainly found in meat and fish, while cereals, fruit, and vegetables represent a minority source. The first study to report the CoQ10 levels in food matrices was published in Japan in 1986 [The distribution of CoQ in 1986 . As expe in 1986 . However in 1986 ,66.10 level can be found in reindeer, then beef, pork, and chicken. Despite the differences, meat represents undoubtedly the most important source of CoQ10, representing 64% of the total CoQ10 intake for Danes [10 content were observed, being the coenzyme notably expressed in horse mackerel and sardine and less represented in eels and salmon, despite their significant fat content. However, the consumption of fish and shellfish is very different around the world, and its impact on the dietary intake of CoQ10 ranges from 9% in Northern European countries [10 in chicken eggs ranges from 1 to 4 mg/kg, while dairy products are very low in CoQ10 compared to animal tissues. Regarding products of a plant-based origin, a high CoQ10 amount characterizes parsley and spinach. However, the highest CoQ10 content may be found in oils. The composition of the oils strictly depends on the plant from which they originate, and the concentration of CoQ10 is higher in plants belonging to the Brassicaceae and Fabaceae family [10 content in rice bran and coconut oil proved to be below the detection limit. Various types of nuts and seeds are also quite rich in CoQ10, such as peanuts, sesame seeds, pistachios, walnuts, and hazelnuts, while lower amounts are contained in chestnuts and almonds. In most cereals, the CoQ10 content is negligible, with CoQ9 being the dominant isoform (4–23 mg/kg) [10 was detected in Japanese millet and buckwheat, while its content in barley and oats was not detected. Soybeans are relatively high in CoQ10, but much less CoQ10 can be found in their processed versions, such as tofu, soy milk, and yogurt. Most fruits and berries represent a very poor source of CoQ10, except avocado, where the CoQ10 content, 9.5 mg/kg, is probably linked to its high lipid content. Black currant is another exception, with a CoQ10 level of 3.4 mg/kg.Regarding meat, the highest CoQor Danes , 55% foror Danes . As for ountries . The cone family . On the 3 mg/kg) . CoQ10 w10 concentration [10. Actually, the destruction of CoQ10 was observed during the boiling process, while a reduction of 14–32% occurred during the frying process. As for fat content, foods with a higher quantity of lipids generally have higher quantities of CoQ10. Indeed, fresh whole milk contains 1.2 mg/kg of CoQ10, while the amount of CoQ10 in UHT milk (having a reduced fat content) is more than halved (0.5 mg/kg) [10 are reduced in fermented products such as yogurt, sour milk, and kefir, containing about two-thirds of the CoQ10 detectable in milk with the same fat content, while the content is even lower in reduced-fat products. Interestingly, a lower CoQ10 content (0.3 mg/kg) was found in yogurt derived from goat and sheep milk despite the much higher fat content. Finally, yogurt that is acknowledged as being fat-free contains negligible concentrations of CoQ10.A relationship has been highlighted between the technological processing of food, its fat content, and CoQntration . In this5 mg/kg) . Similar10 have been developed for the oral supplementation of the coenzyme. They show different degrees of bioavailability of the target molecule, depending on the used ingredients and the way they are assembled. Among those investigated, the nano-dimensioned forms proved to be the better choice for clinical practice as they allow a significant improvement in the solubilization and bioavailability of CoQ10 when compared to their non-nano-dimensioned counterparts [Over time, several formulations of CoQterparts . Represe10-loaded nanoparticles. Investigated in vivo for their pharmacokinetic profile, the loaded nanoparticles showed a sustained CoQ10 plasma level for at least 72 h after a single oral administration to Sprague Dawley rats. Significantly, when compared to a non-nanoparticulate formulation, the CoQ10-loaded nanoparticles gave an almost four-fold improvement in the oral bioavailability of the coenzyme. To rationalize the observed results, the authors called into question an electrostatic interaction between the positively charged CoQ10 nanoparticles and the negatively charged sialic acid of mucin , facilitating the subsequent internalization of the particles through endocytosis [10 encapsulating efficiency, were also obtained by developing nanoscale liposomes. Different mixtures of cholesterol, soy-derived phosphatidylcholine, and alpha-tocopherol were used to achieve the sphere-shaped vesicles, all proving to be effective in stably trapping the target coenzyme [10. Compared to other formulations, nanocapsules have the added value of protecting the compound while allowing its controlled release from the matrix. Moreover, the authors found that, in the form of nanocapsules, CoQ10 has greater bioavailability than common tablets. Actually, once administered orally in mice, there was an increase of 176.6% in the bioavailability of the coenzyme after 24 h [Swarnakar and co-workers exploited the polylactic-co-glycolic acid (PLGA) polymer to obtain CoQcoenzyme ,74,75. Z10 by improving its solubility and dissolution rate have been pursued by obtaining solid dispersion [10 dispersion allowed for an increase in both solubility and in vitro dissolution of CoQ10 in water [® 200 allowed for an increase in both the solubility and stability of CoQ10 [10 with the cyclic oligosaccharide γ-cyclodextrin. Administered to healthy adult volunteers under fasting conditions as a single dose containing 30 mg CoQ10, the γ-cyclodextrin complex allowed for a significant increase in the plasma levels of CoQ10 when compared to the same amount of coenzyme administered as a mixture with microcrystalline cellulose [Further attempts to ameliorate the bioavailability of CoQspersion . Bhandarin water . Also, tof CoQ10 . Terao aellulose .antioxi10 bioavailability. In particular, Cheuk and coworkers dissolved CoQ10 in rice bran oil and incorporated the resulting solution into an aqueous octenyl succinic anhydride-modified starch. The high-pressure homogenization of the resulting mixture allowed the workers to obtain a stable nanoemulsion, showing a CoQ10 encapsulation efficiency of higher than 98% [10 into the tissues. However, the TPGS-based nanoemulsion turned out to be more effective in increasing CoQ10 bioavailability when compared to its LC-based counterpart. Significantly, it also guaranteed a more marked CoQ10 distribution to the heart tissue. Actually, five minutes after the intravenous administration of the nanoemulsion, the amount of the coenzyme from the TPGS-based system was 2.8-fold higher than that obtained with the LC-based prototype. Moreover, the observed difference in the concentrations remained unchanged over time, although gradually decreasing [A number of different nanoemulsions were also developed, all demonstrating an improvement in CoQthan 98% . Zhou ancreasing .10 supplementation, the plasma levels of the coenzyme should be monitored to ensure the efficacy of the treatment, especially considering both the different bioavailability between the commercial formulations and the inter-individual variability [When using CoQiability .10 is generally quantified by liquid chromatography (HPLC) after its extraction from the plasma. The reduced form, CoQ10H2, is rapidly oxidized to CoQ10 by oxygen. Therefore, its determination in plasma samples is rather difficult. The percentage of CoQ10H2 and CoQ10 in plasma varies within the wide 51–96% range [10H2 measurement as a routine test. The concentration of CoQ10 in plasma is strongly correlated to the circulating lipids and it has been shown that, when measuring the concentration of CoQ10, the lipids must, in turn, be considered and the ratio between CoQ10 and total or LDL cholesterol calculated.CoQ6% range ,85,86,8710 shares the same fate as lipids. Specifically, the mechanism of CoQ10 absorption appears to be similar to that of vitamin E and is enhanced in the presence of a fatty meal. The digestion process facilitates the release of CoQ10 from food matrices, while it has no impact on the coenzyme from the different forms of the supplements. After being absorbed in the small intestine through mixed micelles, made up of pancreatic secretions and bile, CoQ10 is incorporated into chylomicrons and brought along the lymphatics to the liver for a subsequent redistribution via the bloodstream, mainly within VLDL, LDL, and HDL [10 is reduced to ubiquinol during or after absorption in the small intestine, and approximately 95% of the circulating CoQ10 in humans is represented by the reduced form [max ranging from 5.80 to 8.10 h, depending on the characteristics of the used formulation. The observed values testify to slow absorption, typical of hydrophobic and high molecular weight compounds such as CoQ10. A second plasma peak may be also observed due to the enterohepatic recycling and redistribution from the liver to the circulation [10 in the human body mainly affects tissues with high energy expenditure or metabolic activity, such as the myocardium, kidneys, liver, and muscles, also characterized by a high lipid content. About 40–50% of CoQ10 is present in the mitochondrial inner membrane, and small amounts are found in other organelles and also in the cytoplasm. CoQ10 is metabolized in all tissues, and the resulting metabolites are phosphorylated in the cells, transported through the plasma, and then excreted by the urine. However, the urinary metabolites of CoQ10 represent only a small fraction of CoQ10, as the main route of elimination is biliary and fecal.As a lipophilic substance, CoQ and HDL . Studiesced form . Overallculation . The dis10 as a supplement for FM patients resides in the evidence of its reduced levels in these subjects. A growing body of evidence is demonstrating a causal link between CoQ10 supplementation and the relief of feeling fatigued in these people [Besides its antioxidant efficacy, the rationale for the use of CoQe people . The mai10 for 40 days and up to 3 months in different groups of women affected by FM showed some clinical improvements. In fact, as assessed by both the fibromyalgia impact questionnaire (FIQ) and the visual analogical scale of pain (VAS), pain, fatigue, morning tiredness, and tender points were significantly reduced (p < 0.01). Moreover, CoQ10 supplementation significantly ameliorated the headache impact test (HIT-6). At the same time, recovery of inflammation, antioxidant enzymes, mitochondrial biogenesis, and AMPK gene expression levels, associated with phosphorylation of the AMPK activity, were also observed [In details, investigations carried out by Cordero and co-workers proved that the supplementation of 300 mg/day of CoQobserved ,94,95,9610 supplementation, enriched with B2, B6, B9, B12, and E (DDM Quinone®) vitamins in 22 female patients (53 ± 9.1 years old) affected by FM who received 400 mg/day of CoQ10 for 3 months. The randomized, open-label, cross-over study demonstrated that this supplementation improved most pain-related outcomes by 24–37%, including fatigue (by ~22%) and sleep disturbance (by ~33%) [Di Pierro and co-workers proved the efficacy of oral CoQby ~33%) .10 with magnesium and tryptophan and investigated the effects of this kind of supplementation in a sample of female FM patients aged 18–90 years within the pilot prospective study FATMIA. After 3 months of treatment, a certain improvement in fatigue, sleep quality, and functional capacity was achieved, although the observed differences were not statistically significant. Surprisingly, emotional factors, like depression and anxiety, were not affected, despite the concomitant use of coping strategies and the demonstrated efficacy on the mood of the components of the supplement [Gómez-Centeno and co-workers combined CoQpplement .p < 0.05) in the fatigue scores [Miyamae and co-workers evaluated the efficacy of ubiquinol-10 supplementation in young patients affected by juvenile FM. In the study, 75 patients (13.1 ± 2.45 years old) were randomly assigned to either the intervention group, administered with 100 mg/day of supplement or the control group, administered with a placebo. After 3 months of treatment, the Chalder fatigue scale was used to assess the fatigue status of the participants, who showed a significant improvement  randomly received 100 or 300 mg/day of CoQ10 or a placebo during 8 days of physical activity resulting in the induction of fatigue. Subjective fatigue, measured by the visual analog scale (VAS), turned out to be significantly reduced (p < 0.01) in the group treated with 300 mg CoQ10 when compared to the placebo group [In 2008, Mizuno and co-workers carried out a randomized study to evaluate the effects of CoQbo group .10 or a placebo. When compared to the placebo group, the intervention group did not show any significant reduction in fatigue (p = 0.28), as assessed through the questionnaire on the severity of fatigue scale (FSS). Besides, the authors did not find any evidence that CoQ10 supplementation ameliorated metabolic parameters or inflammatory markers [10 or a placebo for 2 months. The corresponding index did not show any beneficial effect (p > 0.05) in reducing their fatigue during exercise [On the contrary, other investigations failed to demonstrate any efficacy against the same endpoint. Actually, Lee and co-workers carried out a 3-month study on 51 obese subjects with a body mass index (BMI) of ≥25 kg/m, who were randomly assigned to receive either 200 mg/day of CoQ markers . Similarexercise .10 supplementation and the relief of feeling fatigued can be seen only in FM conditions, offering the prospect of using this compound for the management of people affected by this pathology.Therefore, on the basis of the experimental evidence acquired so far, a causal link between CoQ10. As for the latter, low levels of the coenzyme have generally emerged as a common feature in FM patients, thus, opening the way to its reintegration via the use of suitable supplements. The number of investigations aiming to demonstrate the effectiveness of this prescription has increased over time, outlining a positive causal relationship between the amount of CoQ10 supplementation and the relief of FM symptoms, the first and foremost being the feeling of fatigue.FM is a complex syndrome for which the pathophysiological framework is still being defined. Over the last decade, the nutritional status of people affected by FM has been the object of thorough research, putting into evidence the key nutritional deficiencies concerning particular minerals, including Se, Zn, and Fe, but also vitamins and compounds endowed with antioxidant properties, like CoQ10 might be reasonably regarded as the gold standard supplement for people affected by FM. However, a closer look at the available data highlights the lack of a sound and clear scientific basis for justifying this trumpeted claim. Actually, most, but not all, of the authors highlighted the reduced levels of CoQ10 in FM patients; therefore, the consensus of the scientific community on this condition is not unanimous. In particular, Thorsteinsdóttir and co-workers observed no significant differences in the coenzyme content in both the blood and muscle of FM patients when compared to healthy subjects [10 supplementation. Moreover, scientific investigations that claim the functional efficacy of this supplementation suffer from clear and obvious limits. In particular, besides the biased readings of the experimental outcomes, the number of patients enrolled in the studies is often limited, even consisting of a single patient [10 in the management of FM observed in pilot studies.According to the outcomes obtained so far, CoQsubjects . Althoug patient ; this is


HIF means hypoxia-inducible factor gene family, and it could regulate various biological processes, including tumor development. In 2021, the FDA approved the new drug Welireg for targeting HIF-2a, and it is mainly used to treat von Hippel-Lindau syndrome, which demonstrated its good prospects in tumor therapy. As the fourth deadliest cancer worldwide, gastric cancer endangers the health of people all across the world. Currently, there are various treatment methods for patients with gastric cancer, but the five-year survival rate of patients with advanced gastric cancer is still not high. Therefore, here we reviewed the regulatory role and target role of HIF in gastric cancer, and provided some references for the treatment of gastric cancer. The hypoxia-inducible factor (HIF) gene family consists of HIF1, HIF2 and HIF3: HIF1, the most important member of the HIF family, is mainly composed of two subunits, namely HIF1α and HIF1β. When the oxygen concentration is normal, HIF1α is degraded and cannot exist stably. In the case of hypoxia, HIF1α enters the nucleus and combines with HIF1β to promote downstream genes transcription ,5,6 Fig. HIF2 isAccording to the data of the World Health Organization, the number of new gastric cancer patients in the world in 2020 is 1.09 million, ranking fifth, and the number of gastric cancer deaths in the world in 2020 is 770,000, ranking fourth. Therefore, it can be said that gastric cancer seriously harms the health of people around the world . CurrentIn 2016, some scholars found that the antagonist PT2399 targeting HIF2α has good antitumor efficacy in VHL-deficient clear cell renal cell carcinoma ,23,24. TFrom the abstract, we can know that HIF could regulate the occurrence and development of gastric cancer by proliferation, metastasis, apoptosis, drug resistance, angiogenesis, stemness and metabolism of gastric cancer cells . TherefoWe know that tumor cell proliferation plays a huge role in tumor development . HIF alsMany other studies have confirmed that HIF regulates the proliferation of gastric cancer cells ,32,33,34Metastasis causes most cancer deaths . ExtensiInhibiting tumor metastasis by targeting certain genes is also a major strategy for anti-tumor therapy. For example, Entrectinib inhibits the metastasis of non-small cell lung cancer by targeting ROS proto-oncogene 1(ROS1) and neurotrophic receptor tyrosine kinase (NTRK), ROS1 is a proto-oncogene highly expressed in various tumor cells, and ROS1 protein is a type I integral membrane protein with tyrosine kinase activity, and has achieved satisfactory clinical efficacy ,54,55,56Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain the stability of the internal environment. It is not a phenomenon of autologous injury under pathological conditions, but a kind of actively striving death process for better adaptation to the living environment. Apoptosis plays a certain role in the occurrence and development of gastric cancer  Table 3Table 3. In summary, currently the role of HIF in gastric cancer cell apoptosis is controversial, and more studies are required to clarify the role of HIF in gastric cancer.Drug resistance limits the efficacy of cancer treatment, and addressing drug resistance may be a key issue in cancer treatment ,63,64,65The role of HIF in gastric cancer drug resistance is relatively certain: HIF can promote gastric cancer drug resistance, and many other studies have also confirmed this ,73,74,75Anti-angiogenesis has always been an important method in designing anti-tumor drugs. For example, bevacizumab can combine with VEGF to inhibit tumor angiogenesis and achieve the effect of inhibiting tumors ,77. ZhenMany other studies have confirmed that HIF may be a key factor in gastric cancer angiogenesis ,84,85,86Cancer stem cells are defined by the American Cancer Society: A cancer stem cell is a small subset of cells present in a tumor that produces heterogeneous tumor cells with the ability to self-renew . Cancer Zhenqin Luo et al. found that HIF1α can promote the progression of gastric cancer by promoting the stemness of gastric cancer cells  Table 6Table 6. At present, there are not many studies on HIF in gastric cancer stem cells, and there is no successful clinical application of drugs targeting cancer stem cells. Therefore, the role of HIF in gastric cancer stem cells needs more research.Metabolism is closely related to tumors, the metabolism of glucose, lipid and protein in tumors and it is different from normal cells ,101,102.Many studies have suggested that HIF1α plays a key role in the metabolism of gastric cancer ,109,110.We know that 5-FU can exert an anti-tumor effect by inhibiting nucleic acid metabolism ,112. HIFSmall-molecule drugs mainly refer to organic compounds with molecular weights less than 1000, and they have been widely used and mature in theory ,116,117.There are many small molecule drugs that inhibit the progression of gastric cancer by targeting HIF ,136,137.However, there is a piece of exciting news that the US FDA has approved Merck’s innovative oncology drug Welireg, the first HIF2α inhibitor, for the treatment of VHL syndrome-related tumors. VHL syndrome is a rare and serious genetic disorder associated with a high risk of developing cancer in multiple organs. Prior to Welireg, no systemic therapies were approved for the treatment of VHL-related tumors. Patients suffering from VHL-related tumors treated with Welireg demonstrated high response rates and durable responses ,144,145.Given the success of Welireg, a drug targeting HIF2α in treating VHL syndrome-related tumors, HIF plays a huge role in gastric cancer. Can we look forward to the future that scientists discover that HIF-targeting drugs for the treatment of gastric cancer will benefit patients in the clinic?HIF affects the progression of gastric cancer by regulating the proliferation, metastasis, apoptosis, drug resistance, angiogenesis, stemness and metabolism of gastric cancer cells. Many small molecule drugs that inhibit the progression of gastric cancer through HIF have been found in basic experiments, while these drugs have not yet been clinically applied. Given the success of Welireg, a drug targeting HIF2α in treating VHL syndrome-related tumors, HIF plays a huge role in gastric cancer. We look forward to the future where scientists discover that HIF-targeting drugs for the treatment of gastric cancer will benefit patients in the clinic.


There was no difference between GAC and GAM in terms of age/gender, HIF-1/HER2 expression, stage/tumor location. Median survival at 120 months was significantly higher among GAC, with a difference (ΔRMST) of 43.7 months  (p < 0.001) and those with positive HIF-1 expression: ΔRMST 26.6 months  (p < 0.001). Positive HIF-1 expression was associated with better GAM survival, with ΔRMST 33.6 months  (p < 0.001). Conclusion: Despite the limitations of this retrospective study, GA patients in the coastal region and those who expressed HIF-1 exhibited a better prognosis, but this factor was associated with better survival only in the mountain region.Since the incidence of gastric adenocarcinoma (GA) is high in populations living at high altitudes, we evaluated the influence of altitude on the expression of HIF-1 and survival of Ecuadorian GA patients. Method: 155 GA cases were studied: 56 from coastal (GAC) and 99 from mountainous regions (GAM), and 74 non-GA controls (25 coast and 49 mountain). The expression of HIF-1/HER2 was analyzed by immunohistochemistry. Analyses were performed using Fisher's exact and Breslow-Day tests for homogeneity and Kaplan-Meier curves and restricted median survival time ΔRMST. Results: HIF-1 was overexpressed in normal/inflamed gastric mucosa, especially in mountainous non-GA patients ( Cancer incidence and mortality are growing rapidly around the world, becoming a major barrier to increasing life expectancy . GastricEcuador has a varied altitude diversity and there is a differential incidence of cancer between populations living in the Andean or mountainous region when compared to coastal populations or living at low altitude. In Quito and Ambato, cities located at 2800 meters above sea level, GA also has a very high incidence: 19.6 in men and 13.9 in women per 100,000 inhabitants. There is also a high mortality rate: 16.3 in men and 11.0 in women per 100,000 inhabitants . In GuaySeveral factors may act as promoters of gastric cancer including dietary, environmental, and demographic factors; however, there is a broad description of the genetic and molecular factors associated with carcinogenesis and response to the treatment. The TOGA study showed that HER2+ patients, who received chemotherapy plus Trastuzumab, had higher response rates and longer overall survival. It is likely that other receptors, such as HER3 and HER4, also have a similar potential response to other monoclonal antibodies .The association between geographic altitude, regulation of gene expression sensitive to changes in oxygen levels, tissue hypoxia factors and cancer has been the subject of multiple studies . The expIn gastric cancer patients, HIF-1 activation after hypoxia strongly correlates with an aggressive tumor phenotype and poor prognosis. HIF-1 activation has also been reported to occur through mechanisms independent of hypoxia, such as PI3K/AKT/mTOR signaling and ROS production. Critical roles of HIF-1 are found in glucose metabolism, carcinogenesis, angiogenesis, invasion, metastasis, cell survival, and chemo resistance of gastric cancer , 11, 12.Considering that: I) altitude may influence the expression of HIF-1 in GA patients, II) GA is very frequent in Ecuador particularly in areas of high altitudes  when compared to low-altitude areas , we evaluated HIF-1 expression and its impact on patient survival in these distinct Ecuadorian areas.We analysed the medical records of 674 patients from two oncology hospitals and a specialty hospital in the Ecuadorian mountainous region and one oncology hospital in the coastal region. Among these medical records, 438 did not contain complete information on the variables considered essential for the study and only 229 patients met the inclusion criteria , as shown in Since the major goal of the study was the evaluation of HIF-1 expression, the control group was primarily included to observe differences regarding this marker. As expected, HER2 expression was not observed in the control group.p = 0.004).Considering the stratification of GA patients according the regions, the intestinal Lauren histologic type was more frequent than the diffuse type in both regions. Regarding treatment, surgery alone or combined with chemotherapy/radiotherapy was more frequently performed in the coastal area (p = 0.0070), and II) 43 GA patients (43.4%) of the mountainous region and in 47 controls (95.9%) (p > 0.001) The comparisons of GA patients with controls, stratified by region, showed that HIF-1 expression was identified in: I) 37 patients (66.1%) of the coastal region and in 8 controls (32.0%)  and HER2 expression, however, patients from the coastal region and with positive expression of HIF-1 had better survival , Table 4The present study is opportune to verify the relationship between altitude hypoxia and GA, taking advantage of the real circumstances of treatment for patients who reside in the coastal region at 100 meters from sea level and those who reside in the mountainous region at approximately 2,800 meters from sea level.All patients were followed-up and treated in hospital services belonging to the same institution, the Society for Combating Cancer (SOLCA) of the Ecuador. Of the 155 GA cases evaluated, 64 (41.3%) were women and 91 (58.7%) men, and this male predominance is similar to that observed in epidemiological studies . In addiNotwithstanding the gender difference in the coastal region, the GA survival rate was not affected by gender in this area. In contrast, in the mountainous population women had a better survival compared to men. Overall, the increased female survival rate for GA patients is in agreement with previous reports .Patients of the present study exhibited an average age of 60 years in the coastal region and of 59 years in the mountain region. The survival rate for patients with gastric cancer is greater in patients over 60 years . Indeed,In the present study, the HER2 expression in GA patients was only 7% both on the coastal and in the mountainous areas, and was not expressed in patients without adenocarcinoma. HER2 expression has been primarily associated with the intestinal subtype , a resulA recent meta-analysis showed that in 7 out of 15 papers the HER2 positivity was associated with a worse prognosis. In the present study, we observed no significant differences between the survival rate and the HER2 expression in both groups of GA patients, even after adjusting the RMST values for age and gender.The major focus of the present study was the investigation of the role of HIF-1 in the pathogenesis and prognosis of GA patients, taking advantage of having access to Ecuadorian oncological services from coastal and mountainous regions, where patients are submitted to differential degrees of chronic hypoxia.To study the differential HIF-1 expression in gastric samples, we evaluated GA samples from high and low altitudes together with their respective non-GA samples, which served as controls. In short, considering only the non-GA samples, HIF-1 was observed in 32% of the coastal samples and in 95.9% of the mountainous samples. Indeed, we observed the expression of HIF-1 in individuals exhibiting normal biopsies, a finding that differs from other studies, reporting no or low HIF-1 in normal tissue , 19. In H. pylori infection in patients living in the high-altitude regions.Although the control group exhibited gastritis as well as normal mucosa, the overexpression of HIF-1 in mountainous regions compared to the coastal regions indicates that the chronic hypoxia induces the expression of HIF-1, which may be an adaptation of the gastric mucosa to high altitudes, “protecting” the gastric mucosa. Therefore, HIF-1 expression appears to be an early and significant event in normal gastric mucosa or in gastritis, without Noteworthy, compared to controls, mountainous GA patients exhibited an under expression of HIF-1, further indicating that the loss of HIF-1 expression may be detrimental for patients living in high altitudes. The finding that GA mountainous patients exhibiting HIF-1 expression presented increased survival rate when compared to those who did not express HIF-1, even after adjusting the RMST values by gender and age, corroborates this idea.If, by one hand, hypoxia is a common feature for solid tumors, contributing locally and systemically to tumor progression, lack of response to radiotherapy and chemotherapy, increasing the probability of tumor recurrence , on the Concluding, this study suggests that HIF-1 has a differential expression pattern in gastric samples according to geographical features, being highly expressed even in non-carcinomatous cells  from individuals living in regions of high altitude, indicating that the gastric HIF-1 expression may be an adaptation of the individual to high altitudes. Considering that: I) the HIF-1 expression in GA was underrepresented in high altitude areas when compared to controls, and II) the HIF-1 expression in GA samples from high altitudes was associated with better survival rate when compared to samples that do not express HIF-1, irrespective of the age and gender of patients, one may hypothesize that the expression HIF-1 is a protective marker for this population. Finally, studies evaluating the HIF-1 expression should consider altitude as a confusion factor for understanding the role of HIF-1 on GA pathogenesis and prognosis.This is an observational, case-control, cross-sectional, retrospective study. Data were obtained from the electronic medical records of patients diagnosed with GA, and all clinical investigations were carried out in accordance with the principles outlined in the Declaration of Helsinki. The study protocols were approved by the following Ethics Research Committees on Human Beings: I) SOLCA of Quito, II) Central University of Ecuador, III) Hospital of the Unit Oncology of Tungurahua, Ecuador, IV) SOLCA, Guayaquil Hospital, and V) University Hospital of the Ribeirao Preto Medical School, Brazil.We studied 229 individuals, 155 GA cases 99 from the mountainous region of Quito and Ambato and 56 from the Guayaquil coastal region, followed-up in four hospitals from 2005 to 2018. Most of the patients underwent gastrectomy and some of them underwent adjuvant treatment; however, some patients did not accept treatment. No patient underwent neoadjuvant chemotherapy or radiotherapy. Patient inclusion criteria encompassed: I) histopathological diagnosis of GA, II) comprehensive management in one of the SOLCA oncology hospitals , and III) the availability of blocks of paraffin processed with buffered formaldehyde for the immunohistochemical studies. Exclusion criteria included: I) patient management or partial management outside SOLCA cancer hospitals, II) patient death in the staging phase or initial treatment, III) patients who were not treated in these cancer centers, and IV) the presence of histologic gastric cancer features other than GA.In the absence of symptoms, physical examination was performed every 3–6 months during survival time. Follow-up consisted of clinical evaluation, complete blood count, liver function tests, lung, abdominal, and pelvic CT. The date of the first relapse and the date of death were recorded, survival was calculated from the time of histopathological diagnosis, until the last follow-up or death from any cause.Helicobacter pylori. Demographic features of the studied individuals are shown in In parallel, we studied 74 patients (49 patients from Quito and Ambato and 25 patients from Guayaquil) without GA, most of them (89%) exhibited gastritis and the remaining exhibited normal mucosa, and none of the controls exhibited GA samples were obtained from endoscopic biopsies or surgical resection, and stained with the hematoxylin and eosin. Tumors were classified according to Lauren, defining the intestinal, diffuse, mixed and indeterminate types. Diffuse carcinomas consisted of loosely cohesive neoplastic cells with little or no glandular formation. Intestinal carcinomas were formed by glands of different degrees of differentiation. Tumors composed of intestinal and diffuse components in equivalent proportions were called mixed. Undifferentiated tumors were classified as indeterminate , 15.The GA samples previously prepared at the SOLCA hospitals were submitted to deparaffinization, diaphanization and hydration. Antigen retrieval was carried out using hot moist, in the Optisteam Plus steam cooker with the high pH buffer , for 45 minutes. Endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS pH 7.2 buffer. Slides were incubated for one hour with primary antibodies . After incubation, three consecutive washes with TBST buffer were performed, followed by incubation in the EnVision Flex HRP-Dako detection system (code K8002) for 30 minutes. Then, three consecutive washes were performed using TBST buffer. The reaction was run with diaminobenzidine (DAB) from the EnVision Kit (K8002) in 5 minutes. Positive controls were those recommended by the manufacturer for each antibody. Negative controls were obtained using the same histological sections used in the positive controls, replacing the primary antibody with diluent.All immunohistochemical evaluations were performed by experienced pathologists who were unaware of the study groups.Criteria for interpreting HER2 immunohistochemical findings in GA included a full membrane, basolateral, or lateral labelling pattern. Samples exhibiting weak/moderate and strong labelling in ≥10% of the cells in at least 5 pooled tumour cells were considered positive (scores 2 and 3). Samples with lack of reactivity or membranous reactivity in <10% of tumour cells (score 0) or weak or barely perceptible membrane reactivity in <10% of tumour cells in surgical specimens and in groups of at least 5 pooled tumour cells (score 1) were considered negative , 17 Tab.Criteria for the immunohistochemical analysis of HIF-1 included the evaluation of nuclear staining, as defined by the intensity of the staining and the percentage of positive nuclei. A colour intensity value of 0 was classified as negative; 1 represented a weak staining; 2 as moderate; and 3 as strong staining. The percentage of nuclear positivity was stratified into: 0- negative; 1- positive in ≤10% of cells; 2- positive in >10% and ≤50% of cells; 3- positive in >50% and ≤75% of cells; and 4- positive in >75% of cells. The two scores were multiplied and expressed as: 0 negative; 1–4 weak expression; 5–8, moderate expression; and 9–12, strong expression. The mean score was used as a cut off point for expression (mean score = 6). Finally, samples were classified into the following groups: negative - no expression, low expression (scores 1–6), and high expression (scores 7–12) –20. Tablt. The RMST was estimated at t = 24, 60 and 120 months after treatment or after diagnosis [Associations between demographic and clinicopathological features and tumour marker expression were performed using Fisher’s exact test. Kaplan-Meier curves were used to analyse overall survival according to classes of variables of interest . After checking for proportionality of hazards, we verified that standard Cox proportional hazards models and the hazard ratio (HR) measures were inappropriate for our data. Alternatively, restricted median survival time (RMST) was used as a summary statistic for survival curves . The RMSiagnosis . We consiagnosis . RMST va