Quetiapine, an atypical second‐generation antipsychotic drug, is approved for treatment of schizophrenia, bipolar disorder, and depression. Due to the limitations of clinical trials, the association between quetiapine and rare cardiac adverse events (AEs) is still unclear. This study is to evaluate quetiapine‐associated cardiac AEs through data mining of FDA Adverse Event Reporting System (FAERS).Reporting odds ratio (ROR) was used to quantify the signals of quetiapine‐related cardiac AEs from the first quarter (Q1) of 2018–2022 Q1. Serious and nonserious cases were compared, and signals were prioritized using a rating scale.A total of 1004 cases of quetiapine‐associated cardiac AEs were identified, with 31 signals being detected, among which 13 AEs were identified as new and unexpected signals. Besides, nine and 22 cardiac AEs were identified as moderate and weak clinical priority. The median TTO for moderate and weak clinical priority signals were 0 and 4 days, respectively. All of the cardiac AEs had early failure type characteristics, suggesting that most of the patients developed cardiac AEs in a few days after quetiapine treatment, and that the risk of cardiac AEs occurrence would be gradually decreased over time.Our study provided valuable evidence for health‐care professionals to mitigate the risk of quetiapine‐associated cardiac AEs based on an extensive analysis of a real‐world, large‐sample FAERS database, and prioritize cardiac AE signals. A total of 1004 cases of quetiapine‐associated cardiac adverse events (AEs) were detected, with 13 AEs were identified as new and unexpected signals. Besides, nine and 22 cardiac AEs were identified as moderate and weak clinical priority. The antipsychotic activity of quetiapine is achieved by blocking central dopamine Dp = 0.004). Both researches by Wenzel‐Seifert et alEvidence is accumulating from the reports and mechanism studies to support a possible association between quetiapine and cardiotoxicity. A case‐crossover study using a nationwide population‐based sample obtained from Taiwan's National Health Insurance Research Database reported that antipsychotic drug use such as quetiapine was associated with a 1.53‐fold increased risk of ventricular arrhythmia and/or sudden cardiac death.Due to the limitations of clinical trials, such as specific population, relatively small sample size, limited duration of follow‐ups, and strict inclusion and exclusion criteria, the association between quetiapine and rare cardiac AEs is still unclear, suggesting continuous post‐marketing surveillance is urgent.22.1https://fis.fda.gov/extensions/FPD‐QDE‐FAERS/FPD‐QDE‐FAERS.html. To get the latest reports, all reports recorded in FAERS covering the period between the first quarter (Q1) of 2018 and the 2022 Q1, were extracted in our study.A disproportionality analysis, designed as a case/non‐case study, was used to quantify the association between quetiapine and cardiological AEs. It calculated the proportion of occurring target AEs between a target drug (case) and all other drugs (non‐case).2.2The database included seven data files, namely patient demographic information (DEMO), drug/biologic information (DRUG), adverse events (REAC), patient outcomes (OUTC), report sources (RPSR), start/end dates of drug therapy (THER), and indications for drug (INDI).Subsequently, descriptive analyses were carried out to summarize the clinical characteristics of all quetiapine‐associated reports. Detailed information will be calculated if the data are available, such as gender, age, weight, reporting countries, indications, outcomes, and time‐to‐onset, etc. A flowchart including the multistep process of data extraction, processing, and analysis is shown in Figure 2.3Based on the disproportionality analysis using a 2 × 2 table Table , the rep2) or Fisher's exact test, and Mann–Whitney U test was applied for continuous non‐normal distribution data, such as age and weight. Data were analyzed using SPSS , and statistical significance was set at a two‐tailed p < 0.05. To further explore the influence of different stratification regimens on the correlation between quetiapine and cardiac disorders, we performed subgroup analysis by gender , age , weight , and reporters  separately.The outcomes of AE reports were classified as serious or nonserious in FAERS. The serious outcomes included death (DE), life‐threatening (LT), inpatient hospitalization or prolongation of existing hospitalization (HO), disability (DS), congenital anomaly (CA), required intervention to prevent permanent impairment/damage (RI), as well as other serious/important medical event (OT). Some reports might list more than one specific outcome , which recorded in the OUTC file. However, the serious and nonserious reports were also compared to clarify the severity of the detected safety signals and identify risk factors  in patients. Proportions were compared using a Pearson's chi‐squared (χ2.4025) values, proportion of death, characterization as designated medical events (DMEs) or important medical events (IMEs), and evidence evaluation.AEs with significant disproportionality were scored and ranked according to clinical priority in our study. A semiquantitative score was performed to prioritize disproportionality signals by assessing five different features, including number of AE reports, the lower limit of the ROR 95% CI (ROR2.5Time‐to‐onset (TTO) was calculated as the duration between the occurrence date of AEs (EVENT_DT in DEMO file) and start date of quetiapine use (START_DT in THER file).In order to predict the risk of increase or decrease of these quetiapine‐associated cardiological AEs over time, we calculated the median TTO and WSP of signals with moderate or weak clinical priority after quetiapine use. The WSP tests were performed using Minitab statistical software .Moreover, we also considered the effect of concomitant drugs on quetiapine‐related cardiac AEs, and concomitant therapy was defined as quetiapine being the “primary suspect.” and other drugs were listed as “second suspect,” “concomitant,” or “interacting.” Subsequently, the sensitivity analysis was performed after the exclusion of the reports associated with potential suspicious drugs.33.1n = 443, 90.22%). Most reports were submitted by health‐care professionals  and consumers , respectively. The country with the most cardiac AE reports was USA , followed by United Kingdom  and Canada .From January 2018 to March 2022, the FAERS database has recorded 6,437,182 AE reports after exclusion of duplicates, including 1415 quetiapine‐associated cardiac AEs in 1004 patients. The clinical characteristics of quetiapine‐induced AEs are described in Table 3.2n = 205), tachycardia (n = 138), palpitations (n = 57), and cardiorespiratory arrest (n = 57). Figure 025 of 1.48. Further, investigation of cardiac AE signals revealed that 31 cardiac AEs showed statistically significant signal strengths after quetiapine treatment compared with non‐quetiapine‐related cardiac AEs, with values of signals ranging from a ROR025 of 1.19 (supraventricular tachycardia) to 52.96 (Brugada syndrome). Other quetiapine‐related nonpositive cardiac AE signals are shown in Table During the study period, a total of 31 different PTs of quetiapine‐associated cardiac AEs were identified in FAERS database in at least five cases Figure . The mos3.32 = 1.395, p = 0.237), age , and body weight  between severe and non‐severe cases of cardiac AE patients receiving quetiapine. Dizziness , cardiorespiratory arrest , and palpitations  were more likely to be reported as serious AEs, while other 28 AEs were more tended to be reported as nonserious AEs with p > 0.05, such as tachycardia , arrhythmia , and myocarditis (p = 0.161), etc. Of note, all AEs outcomes for cardiorespiratory arrest (n = 57), atrial septal defect (n = 19), torsade de pointes (n = 13), ventricular extrasystoles (n = 11), ventricular tachycardia (n = 9), ventricular septal defect (n = 8), ventricular fibrillation (n = 8), Brugada syndrome (n = 7), ventricular arrhythmia (n = 5), and tachyarrhythmia (n = 5) were severe cases.Data in Table 3.4025 was found to be 1.48, whereas it was 1.20 in males, indicating a significant association of quetiapine with cardiac AEs in both male and females. Moreover, it suggested that women were more likely to experience cardiotoxicity with quetiapine than men. The majority of quetiapine‐associated cardiac AEs cases were found in the 18–65 years age groups. However, it did not show significant association between cardiac disorders and quetiapine treatment when stratified by age and body weight, primarily in subgroups of patients >65 years old and body weight <80 kg and >100 kg.As shown in Figure 3.5n = 57, ROR025 = 4.64) and myocarditis  were graded as moderate clinical priority with the highest priority scores of 7. Besides, 18 AEs were evaluated as strong clinical evidences with “++.”In total, 14 of the 31 AEs (45.16%) with statistically significant disproportionality signals were categorized as IMEs, whereas only two represented DMEs (6.45%), including torsade de pointes and ventricular fibrillation , sertraline hydrochloride , mirtazapine , aripiprazole , and lamotrigine . The sensitivity analysis was performed after exclusion of drugs  that belonged to the second‐generation antipsychotics. Although the number of quetiapine‐related cardiac AE reports were decreased from 1004 to 868  after removing the reports of suspicious drugs, it still had a significant association between quetiapine and cardiac disorders. Moreover, five PTs no longer showed significant signals, including long QT syndrome, supraventricular tachycardia, ventricular arrhythmia, extrasystoles, and bundle branch block right. The detailed changes are shown in Table Among quetiapine‐induced cardiac AEs reports, there were 721 cases (71.81%) in combination with other drugs. The reports of quetiapine combined with one, two, and three drugs were recorded in 157 (15.64%), 124 (12.35%), and 97 (9.66%) cases, respectively. The top 20 concomitant drugs are shown in Table 4This pharmacovigilance study comprehensively and systematically provided the latest findings of quetiapine‐associated cardiac safety profiles by post‐marketing based on the FAERS database. Our results are in line with previous clinical trials and literature reviews highlighting the association between exposure to antipsychotics and cardiac AEs. According to a recent epidemiological study, Weeke et aln = 205, ROR = 1.60), tachycardia , palpitations , and cardiorespiratory arrest , which were consistent with clinical trials.Fortunately, we innovatively used multidimensional analytic approaches, such as subgroup analysis, clinical priority of signals and the serious outcomes, to provide insight into the primary results. In our study, a total of 1004 reports of quetiapine‐associated cardiac AEs were obtained, and 31 significant AEs with at least five reports were detected. The most frequently reported cardiac AEs of quetiapine were dizziness (p < 0.05) in three AEs among the 31 cardiac AEs, when compared serious cases with nonserious cases. Patients gender (p = 0.237), age (p = 0.093), and weight (p = 0.476) may not be associated with increased risk of quetiapine‐related cardiac AEs severity. However, the descriptive analysis  than those aged >65 years  and <18 years , which were consistent with a FAERS study that the median patient age among quetiapine abuse‐related event reports was 44 years (IQR = 31–55).p = 0.093). However, Yunusa et alIn our study, patients aged 18–65 years reported more frequently cardiac AEs (025 = 1. The other two groups (<80 and >100 kg) did not show an association between quetiapine and cardiac AEs because of ROR025 < 1. A Bayesian meta‐analysis of 41 short‐term trials of second‐generation antipsychotics found a significant mean weight gain  during treatment with quetiapine.We conducted subgroup analysis by body weight , and found that only the subgroup of body weight 80–100 kg presented significant signal strength with RORn = 138, score 6), palpitations , sinus tachycardia , arrhythmia , torsade de pointes , and cardiomyopathy , which were consistent with the drug label. Of note, 12 of the 31 AEs were serious cases , our analysis is subject to inevitable and unquantifiable biases. Second, because the total number of patients using quetiapine is not available in FAERS, we cannot calculate AE incidence and establish causality. Third, we focus only on AEs in cardiac disorders, and the deep relationship between quetiapine and other systemic organ classes remain unknown. Notwithstanding the above limitations, we systematically and comprehensively reveal the association between quetiapine and cardiotoxicity through an extensive analysis of a real‐world, large‐sample FAERS database, and prioritize cardiac AE signals, which provides valuable evidence for healthcare professionals to mitigate the risk of quetiapine‐associated cardiac AEs.5Our pharmacovigilance study systematically explored and quantified the potential cardiotoxicity induced by quetiapine, which obtained novel safety information about quetiapine. Among the 31 cardiac AEs, 13 new and unexpected AEs signals are confirmed. Besides, nine and 22 cardiac AEs were identified as moderate and weak clinical priority. The median TTO for moderate and weak clinical priority signals were 0 and 4 days, respectively, and all of the cardiac AEs had early failure type characteristics. The characteristics of cardiotoxicity spectrum found in this study are helpful for clinicians to recognize different cardiotoxicity and guide clinical practice. Some cardiac AEs have not received enough attention, and more cardiac examinations are recommended for high‐risk patients.Qilin Zhang and Yamin Shu contributed to conception and study design, and took responsibility for the collection, integrity, and accuracy of the data. All authors drafted the article, participated in data analyses and interpretation, and revisions of the article, and approved the final version.This study was supported by grants from National Natural Science Foundation of China (No. 82104476).The authors declare that they have no conflicts of interest.Data S1.Click here for additional data file.


Due to the novelty of CrossFit, there are few studies on the effect of nutritional strategies on the acute response to this type of sports activity. This study examined the effect of caffeine (CAF) and sodium bicarbonate  before completing the Cindy protocol . Capsules containing 6 mg/kg body weight (BW) CAF were consumed 50 min before the Cindy workout while 0.3 g/kg BW NaHCO3 was consumed for 3 days, leading to 120, 90, and 60 min before the Cindy workout. Performance, RPE, muscular power (MP), handgrip strength (HGS), and maximum heart rate (MHR) were measured before and shortly after the Cindy.In a double-blind, crossover, randomized, placebo-controlled trial, 20 CrossFit participants underwent five experimental conditions, including control (CON), placebo (PLA), CAF, NaHCO3, and CAF + NaHCO3 (P > 0.05). In contrast, RPE during and at the end of the Cindy was significantly decreased following CAF + NaHCO3 consumption compared to PLA and CON . However, MP (P = 0.82) and HGS (P = 0.52) were not significantly different between conditions. Also, MHR was significantly greater following CAF, NaHCO3, and CAF + NaHCO3 consumption than CON (P = 0.01).The performance of CrossFit participants during the Cindy protocol was not significantly improved following CAF, NaHCO3 supplementation decreased RPE despite significantly increased MHR, but with no significant effect on performance, HGS, or MP. Therefore, CrossFit participants may benefit from the ergogenic effects of CAF and NaHCO3 when consumed separately or together.CAF + NaHCO Therefore, CrossFit combines intense and resistance training .+) as a byproduct of the anaerobic glycolysis pathway because of the limited oxygen availability caused by the increased rate of glycolysis in active muscle cells  = 0.40, P = 0.80, F = 3.62,P < 0.001, P = 0.01) and CAF + NaHCO3  compared to CON (191 ± 6.23 Rpm). Non-significant effect of supplements was also observed on HGS F  = 0.52, P = 0.71, F  = 0.37, P = 0.82, The results did not show a significant effect of supplementation on performance in CrossFit participants during the Cindy protocol , F  = 32.08, P ≤ 0.05. Pairwise comparison revealed that RPE was significantly less in CAF  compared to NaHCO3 (6.41 ± 0.48) and CON (6.75 ± 0.48). A significant difference was also observed in RPE between NaHCO3 , CAF + NaHCO3 (5.89 ± 0.42), and CON (6.75 ± 0.48). Likewise, RPE was significantly less in CAF + NaHCO3  compared to PLA (6.37 ± 0.63) and CON (6.75 ± 0.48) (The effects of different supplements on RPE in CrossFit participants during the Cindy protocol  were sig ± 0.48) .Table 23.3.3 and NaHCO separately compared to the PLA group, but these complications were temporary, were not severe, and did not affect participants’ performance. Hooper total score was similar between conditions before the Cindy protocol.The results showed significant GI side effects with the combination of CAF and NaHCO4.3 supplementation when consumed together or separately on performance, MP, HGS, MHR, and RPE before, during, and after the Cindy protocol. We hypothesized that co-ingestion of CAF and NaHCO3 would have a greater effect on athletic performance than CAF and NaHCO3 alone. However, the findings rejected the primary hypothesis and showed no additional benefits of co-ingesting CAF and NaHCO3 compared to CAF or NaHCO3.This study investigated the ergogenic effects of CAF and NaHCO3 (4.0%) than CAF (2.6%) or NaHCO3 (1.1%) when compared to PLA. This result is in contrast with Pruscino et al. [3 was consumed separately or with CAF [3 supplementation on participants’ performance during CrossFit training. The results were consistent with our findings. The results showed that acute SB supplementation did not improve the performance of professional CrossFit participants during the Cindy protocol [Performance in this study was defined as the repetition of performing all exercises in each round until voluntary exhaustion. Our results indicated that all conditions improved the performance compared to PLA during the Cindy protocol. Although the differences between conditions were not statistically significant, a close review of the results showed a greater improvement in performance in CAF + NaHCOo et al. , Christeo et al. , Carr eto et al. , and Felo et al. . Pruscinwith CAF . In othewith CAF ,30. In awith CAF  concludewith CAF . Toledo with CAF  conducteprotocol . Also, oprotocol , who shoprotocol .3 supplementation, separately or in combination, did not significantly improve HGS of CrossFit participants. There was no difference between the supplementation conditions compared to PLA and CON. Also, comparing the handgrip test scores in all conditions after the Cindy protocol compared to before indicated that CAF and NaHCO3 supplementation separately and in combination did not affect HGS in fatigue period caused by the implementation of the Cindy protocol in each condition  but does not affect heart rate while riding a bicycle with an intensity of 50% VO2 max [Our results showed that NaHCOicipants . Contrar bicycle . Bell et VO2 max .3 and CAF supplementation combined with NaHCO3 caused significant GI side effects compared to the PLA group (3 because no significant GI side effects were observed in the CAF condition. These complications were temporary and did not impair participants’ performance. Acute consumption of NaHCO3 is associated with GI upset [Finally, our findings showed that NaHCOLA group . These iGI upset . In the GI upset . Our resGI upset  and DrilGI upset , the finGI upset . ContradGI upset .Limitations of this study do exist. Before the trial started, participants were well familiarized with the Cindy protocol. Even though the findings were not statistically significant, a practice effect might have impacted them. The small sample size of our study is another limitation. Participants were selected from just one CrossFit gym to ensure uniformity of training regimens throughout the research. More research with a larger sample size may be required to confirm the conclusions of this study. In addition, the primary limitation identified in the present study pertains to the lack of control over extracurricular physical activities that participants engaged in outside the scope of the Koohpaye Box club. Additionally, the variable values of sleep before each intervention session also presented a potential confounding factor. Klier et al. reported a significant positive effect of sleep quality on sports performance in CrossFit participants .5.In general, the results of the present study confirm some previous studies and contradict others on the changing power, strength, MHR, RPE, and exercise performance. The study showed that the supplementation of CAF and SB together could not provide further benefits in improving performance and performance-related factors in CrossFit participants. Furthermore, due to the lack of significant changes in athletic performance and MHR, it can be concluded that CAF and SB supplementations, separately and in combination, have the same effect on performance and MHR in CrossFit participants during the Cindy protocol.


Mustela aistoodonnivalis Wu & Kao, 1991, was originally described as being from Taibai Mountain and Zhashui county, Shaanxi, China. Subsequently, it was considered a subspecies or synonym of Mustela nivalis. In a faunal survey of northwestern Sichuan, eight specimens of M. aistoodonnivalis were collected. A molecular phylogenetic analysis of one mitochondrial and six nuclear genes clustered the specimens as a distinct clade and not with M. nivalis. Morphologically, the lack of the second lower molar differentiated them from M. nivalis, and genetic distances were typical of discrete species. These analyses confirmed that M. aistoodonnivalis is an independent species in the genus Mustela.The lacked‐teeth pygmy weasel,  Mustela aistoodonnivalis is of great importance to the study of ecosystem. In this study, we determined the taxonomic status of M. aistoodonnivalis as an independent species through morphological and molecular biological studies and gave a detailed morphological description of M. aistoodonnivalis.The taxonomic status of  The genus occurs in Europe, North Africa, Asia, North America, and northern parts of South America. These small or middle‐sized weasels occupy diverse habitats ranging from tropical rainforests to tundra and from steppe and desert to riparian biotopes and coastal waters , Baoxing , Li County , Jiuzhai , and Pingwu , Sichuan, from 2001 to 2019. Another 16 specimens of the same family (Mustelidae) were collected from 10 localities in China, including 4 individuals of M. altaica, 6 individuals of M. sibirica, 4 individuals of M. nivalis, and 2 individuals of Neogale vison  and six nuclear gene fragments (apolipoprotein B (APOB)), transporting alpha polypeptide (ATP7A), brain‐derived neurotrophic factor (BDNF), recombination‐activating gene 1 (RAG1), recombination‐activating gene 2 (RAG2), and transmembrane protein 20 (TMEM20)). During the Mammal Tree of Life Project (2011), these genes were found to provide sound phylogenetic information.Employing the methods of Koepfli et al. , Sato etCYTB amplifications consisted of an initial denaturing step at 94°C for 5 min followed by 38 cycles of denaturation at 94°C for 45 s, annealing at 49°C for 45 s, extension at 72°C for 90 s, and then a final extension step at 72°C for 12 min. PCR conditions for the nuclear genes were basically the same as those of the CYTB, with a few modifications. We adjusted the annealing temperature for each nuclear gene: APOB: 49°C; RAG2, ATP7A, and TMEM20: 52°C; RAG1: 54°C; BDNF: 56°C. We checked the PCR products on a 1.0% agarose gel and subsequently purified them using ethanol precipitation. Purified PCR products were directly sequenced using the BigDye Terminator Cycle Kit v 3.1 (Applied Biosystems) and an ABI 310 Analyzer (Applied Biosystems).All the gene fragments were amplified with published primers Table . PCR ampMustela and to evaluate the species status of M. aistoodonnivalis, we downloaded homologous sequences of additional species of Mustela from GenBank for comparison  the concatenated six nuclear genes, and (3) each nuclear gene. Modeltest v3.7  and 10,000,000 generations for single gene datasets and 30,000,000 generations for concatenated gene datasets. All runs were sampled every 10,000 generations. Convergences of runs were accepted when the average standard deviation of split frequencies fell below 0.01. Tree nodes were considered to be strongly supported when Ultrafast bootstrap values (UFBoot) were ≥95 and posterior probabilities (pp) were ≥0.95  interspecific distances for 2.5M. nivalis and M. aistoodonnivalis or skull measurements because measurements of the male individual fell among those of females. Thus, all genders of M. nivalis and M. aistoodonnivalis were used for morphometric analyses. For the other species, we used adult females only for a principal component analysis (PCA). Moreover, since some species distribute in untraversed areas , some have the small population size or narrow distribution area in China , and some have large individual size and uneasy to be captured (M. eversmanii), resulting in the small sample sizes for these species. Nonetheless, 59 complete skulls of intact specimens were collected for morphological measurements, including 4 specimens of M.altaica, 6 specimens of M. aistoodonnivalis, 5 specimens of M. erminea, 6 specimens of M. eversmanii, 7 specimens of M. kathiah, 5 specimens of M. nivalis, 21 specimens of M. sibirica, and 5 specimens of M. strigidorsa. Details of collected localities and housed museums are listed in Table To avoid age‐related morphological differences, we only used adult individuals for morphological analysis. No sexual dimorphism was found in Chinese Following Yang et al. , we meas2.6For the geometric morphometric analyses, dorsal, ventral, and lateral views of the cranium, as well as lateral views of the mandible, were photographed  ‐SL14# were six equal‐diversion points from landmarks 3 (L3#) to L4#, SL15#‐SL16# were three equal‐diversion points on the outer edge of the zygomatic arch from L4# to L5#, SL17# was determined on L5# to L6#, and SL18# was determined on L6# to L7#  in the software Coordgen 8 , 21 RAG1 (1064 bp), 21 RAG2 (456 bp), 21 ATP7A (636 bp), 21 BDNF (536 bp), 21 TMEM20 (596 bp), and 21 APOB (907 bp). In addition, 45 CYTB, 12 RAG1, 10 RAG2, 11 ATP7A, 11 BDNF, 8 TMEM20, and 12 APOB sequences were downloaded from GenBank for phylogenetic analysis. All newly obtained sequences were deposited in GenBank . Mustela kathiah, which belongs to the same subgenus Gale as M. altaica and M. nivalis, formed an isolated clade with strong support (pp = 1.00). Mustela aistoodonnivalis clustered with M. richardsonii, M. haidarum, and M. erminea, but with very low support (pp = 0.75). Importantly, M. aistoodonnivalis did not cluster with M. nivalis as anticipated based on traditional relationships. Mustela altaica and M. nivalis were sister groups that were strongly supported (pp = 1.00). Mustela itatsi, M. sibirica, M. nigripes, and M. eversmanii also constituted a monophyletic lineage that was strongly supported (pp = 0.99), but M. lutreola clustered with M. putorius, however, and was not well supported (pp = 0.67).The s Figure . Both M.CYTB (dataset 1), the topological structure was slightly different, and both the analyses strongly clustered M. aistoodonnivalis away from M. nivalis (pp = 1.00). Mustela lutreola was monophyletic with strong support (pp = 1.00) as was M. sibirica (pp = 1.00). Mustela itatsi, M. nigripes, M. eversmanii, and M. putorius had mixed associations with weak support, and the samples of M. putorius divided into two different branches. Due to the lack of nuclear gene sequences of M. haidarum, its position remained uncertain.The Bayesian reconstruction using the six nuclear genes concatenated (dataset 2) revealed 14 monophyletic clades Figure . In cont3.3M. aistoodonivalis was the same as in the mitochondrial and nuclear gene trees with strong support (pp = 0.95). Mustela richardsonii, M. nigripes, M. nivalis, M. altaica, and M. lutreola also obtained high support in the *BEAST species tree (pp = 1).The topology of the *BEAST species tree differed slightly from those of the mitochondrial and nuclear gene trees Figure . The posMustela ranged from 0.008 to 0.158  distances between the species of 3.4Morphological index measurement data are shown in Table χ2 = 1703.319, p = .000), which meant the data were spherical and the variables were somewhat independent of each other. KMO was 0.913, indicating that there was a strong correlation among the variable data of skull morphology, which was suitable for factor analysis. Two principal components, explaining 92.506% of the morphological variation, were extracted from the analysis. Its factor loading values were all positive and most were greater than 0.95 缺齿伶鼬 Que Chi Ling You (Lacked‐teeth pygmy weasel).Measurements. W: 39–48 g; HBL: 124–155 mm; TL: 58–70 mm; HFL: 20–25 mm; EL: 10–12 mm.Type locality: Taibai Mountains, Shaanxi, China, 2750 m above sea level.Holotype specimen: Herbarium of the Shaanxi Zoological Research Institute, Collection No. 84009. Female.Mustela, with a body shape similar to M. nivalis and distinguished from other species of Mustela by the following characteristics: tail long, about half the head–body length; back and end of tail dark brown; white throat and light yellowish abdomen tinged with rusty red; forelimb fur brown; sagittal suture faint or almost lacking; mandible slender; and second lower molar lacking.Diagnosis: A smaller species in the genus M. aistoodonnivalis is a Mustela species with smaller individuals, weighing 39–48 g, body length 124–155 mm, and tail length 58–70 mm. The hindfoot length is 20–25 mm, and ear length 10–12 mm. The general appearance is similar to M. nivalis; however, the tail length is more than 1/3 the head–body length, and summer dorsal hairs are dark brown from the upper lip edge through the cheeks backward, as well as along the side of the body to the tail and the outside of the limbs. The ears are dark brown, covered with short hairs of the same color. The ventral part of the chin to the throat is white. The neck to the abdomen is yellowish, tinged with rusty red. Individual specimens have less obvious rusty red patches, and the dorsal and ventral boundaries are clear. The dorsal surfaces of the front and hindfeet are slightly lighter in color, with some individuals having white medial forefeet and a few white hairs covering the dorsal surfaces of the carpal palps and the dorsal surfaces of the hind toes. Pelage exhibits no seasonal variation; a specimen collected in beginning of November (SAF181732) had the same pelage color as in specimens collected in summer.Description: The dorsal surface of the skull is relatively flat, the cerebral skull is broad, and the muzzle is narrow and short and significantly recurved. The interorbital width is quite large, and there is a less pronounced postorbital protrusion. The bony sutures between the nasal bone, frontal bone, parietal bone, interparietal bone, and squamous bone are healed and poorly defined. The occipital foramen magnum is broadly rounded and oval, with a small occipital condyle. On the lateral side of the skull, the anterior orbital foramen is obvious (located below the slender anterior margin of the orbit) and the zygomatic arch is slender and curved upward. At the ventral surface of the skull, the incisor foramen is very short, and the hard palate is triangular in shape and consists of the maxilla and premaxilla. The basioccipital bone is almost fused with the anterior pterygoid and basi‐pterygoid, with indistinct sutures and a narrow rectangular shape. The auditory vesicle is large and full, long and oval, with a flat, nearly parallel inner margin.The tooth pattern is 3.1.3.1/3.1.3.1 = 32. The upper incisors are transverse, the third incisor is thicker, flatter, and inclined backward. The canines are longer than the incisors, slightly curved, and twice as tall as the third incisor. The first and second premolars are tricuspid, with the first premolar having an inconspicuous anterior cusp and a small protocusp and being slightly higher than the canine root. The second premolar has a more pronounced protocusp, orthogonally oriented upward, with a more rounded anterior and posterior cusp. The third premolars are “Y”‐shaped, with the inner lobe of the anterior margin larger than the outer lobe. The original cusps are broad and sharp. The upper molars are transverse, “dumbbel” shaped, with the inner lobe rounded and flattened with a small cusp and the outer lobe with two small cusps. The mandibular incisors are arranged in an inverted zigzag pattern, with the second incisor located behind the first and third incisors. The canine teeth are stubby and more curved, shorter than the maxillary canines. The first and second premolars are both unicuspid. The third premolar has three cusps, with the primary cusp being more pronounced and much larger than the first and second premolars. The lower molars consist of three lobes in a “W” shape, which forms a well‐developed cleft tooth with the maxillary third premolars. There is no second lower molar.M. aistoodonnivalis is similar in body shape to M. nivalis. Mustela nivalis is brown to dark brown on the back and tail end, and M. aistoodonnivalis is dark brown . On measurements, M. aistoodonnivalis has longer tail length. The longest tail of the type series of M. nivalis russelliana is 54 mm, about 40% of head–body length, but in M. aistoodonnivalis, the shortest tail length is 58 mm (average: 63 mm), approximately 46% of head–body length. A t‐test using data for M. nivalis russelliana from original description Thomas (p = .004 < .05). Thus, we reject the hypothesis that M. aistoodonnivalis and M. nivalis russelliana are the same taxa.In Sichuan, Kingding (Tatsienlu) is the type locality of y Thomas . We faili Allen, . Accordin Thomas  and Gao n Thomas  obtains M. aistoodonnivalis mainly occurs in the Qinling Mountains of Shaanxi and in the Minshan and Qionglai mountains of Sichuan, including Pingwu, Jiuzhai, Huanglong, Baoxing, and Heishui ; data curation ; formal analysis (lead); investigation ; methodology (lead); software (lead); validation ; visualization ; writing – original draft (lead); writing – review and editing (lead). Yingting Pu: Conceptualization ; data curation ; formal analysis ; methodology ; software ; validation ; writing – original draft ; writing – review and editing . Shunde Chen: Data curation ; formal analysis ; funding acquisition ; resources . Xuming Wang: Formal analysis ; investigation ; methodology ; validation . Robert Murphy: Writing – review and editing (lead). Xin Wang: Investigation ; methodology ; resources . Rui Liao: Data curation ; investigation (lead); project administration . Keyi Tang: Formal analysis ; methodology (supporting); software (supporting); visualization (supporting). Bisong Yue: Formal analysis ; methodology ; resources ; supervision ; validation ; writing – review and editing . Shaoying Liu: Data curation ; formal analysis (lead); investigation (lead); methodology (lead); project administration ; resources (lead); supervision (lead); writing – review and editing (lead).This research was funded by the National Natural Science Foundation of China .The authors declare no conflicts of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


Moreover, bleeding resulted associated to thyroid nodule > 3 cm (p = 0.015), although all bleedings but one occurred in the remote-access site from the axilla to the neck. In experienced hands, robotic transaxillary thyroidectomy is feasible and safe even in patients with large thyroid nodules or thyroiditis.In 2016, the American Thyroid Association published a statement on remote-access thyroid surgery claiming that it should be reserved to patients with thyroid nodule ≤ 3 cm, thyroid lobe < 6 cm and without thyroiditis. We retrospectively enrolled all patients who underwent robotic transaxillary thyroidectomy between February 2012 and March 2022. We compared surgical outcomes between patients who presented a thyroid gland with a nodule ≤ 3 cm, thyroid lobe < 6 cm and without thyroiditis (Group A) and patients without these features (Group B). The rate of overall complications resulted comparable ( Since the introduction of robot-assisted gasless transaxillary thyroidectomy (RATT) in South Korea in 2009 , severalIn this uncertain context, The American Thyroid Association (ATA) Statement on Remote-Access Thyroid Surgery of 2016 acknowledged that the robotic transaxillary approach may be performed by experienced surgeons in selected cases; notwithstanding, RATT was recommended only in patients with well-circumscribed nodule ≤ 3 cm, thyroid lobe < 5 to 6 cm in the largest diameter and absence of thyroiditis [The aim of the present study is to assess surgical outcomes of RATT in a large European population, comparing patients who fulfill the ATA suggestions with those with thyroid features beyond these recommendations.This is a retrospective study performed using a prospectively designed database for data collection and analysis. We enrolled all patients who underwent RATT from February 2012 to March 2022 at the University Hospital of Pisa, Endocrine Surgery Unit. All patients received an extensive explanation of the procedure and provided informed consent. This study had been approved by the internal research board.All procedures were performed with the da Vinci robotic system, both the SI and the XI versions, using a single axillary incision and three robotic arms. Data regarding patient demographics, thyroid volume, diameter of the largest nodule, extent of surgery, operative time, postoperative length of hospital stay, postoperative complications and histology were recorded. The preoperative evaluation of patients consists of neck ultrasound (NUS), thyroid function test and US-guided fine-needle aspiration cytology if required. We routinely provided counseling about the conduct, risks and alternatives of RATT and conventional thyroidectomy.Selection criteria for RATT were: sonographically determined nodule diameter ≤ 75 mm for benign nodules and ≤ 35 mm for malignant nodules and expressed preference of the patients for transaxillary approach. Exclusion criteria included previous neck surgery or radiotherapy, locally advanced cancer, Graves’ disease and shoulder or arms disease. The choice of surgery  was in concordance with the American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer at the time [Operative time was defined from the skin incision to the skin closure. We defined postoperative hypocalcemia as an albumin-corrected calcium level of < 8.0 mg/dl or inability to interrupt calcium therapy [Surgical operations were performed as previously described .Patients were divided into two groups on the basis of the ATA statement on remote-access thyroidectomy : patientThe primary endpoint of the study was the overall rate of postoperative complications; secondary endpoints of the study were operative time and length of hospital stay.t test for continuous variables (SPSS v.28 technology). Significance was fixed at 0.05.Categorical data were described by absolute and relative (%) frequency; continuous data were summarized by mean and standard deviation. Statistical analysis was performed using the Chi-square test for categorical variables and p < 0.001). Features of the two groups are summarized in Table Between February 2012 and March 2022, 650 RATT were performed at University Hospital of Pisa, Endocrine Surgery Unit. Overall, 650 operations were performed on 614 patients, 610 (99.3%) of which were females, whereas 4 (0.7%) were males. 293 (45.1%) out of 650 cases fulfill the ATA recommendations (Group A), whereas 357 (54.9%) were beyond these criteria (Group B). The two groups resulted comparable in terms of age, body mass index (BMI), type of surgery and histology. The estimated thyroid volume resulted significantly higher in group B (p = 0.225) and total thyroidectomy , hospital stay  and overall complications rate . Moreover, the rate of transient  and permanent  hypocalcemia in patients who underwent total thyroidectomy or completion thyroidectomy resulted comparable between Group A and B. Furthermore, the two groups resulted comparable in terms of rate of transient  and permanent  RLN palsy, rate of cervical hematoma , rate of bleeding which requires reoperation , rate of seroma  and rate of unconventional complications . Specifically, unconventional complications included 2 (0.3%) tracheal injuries (1 in Group A and 1 in Group B), 2 (0.3%) brachial plexus palsy (1 in Group A and 1 in Group B), 1 (0.2%) track seeding in Group B and 1 (0.2%) Bernard-Horner syndrome in Group A. In our case series, only 1 (0.2%) conversion to open thyroidectomy occurred at the beginning of the experience; the patient was included in Group B.Table p = 0.018).162 (24.9%) out of 650 cases resulted affected by thyroiditis. As reported in Table 52 (8.0%) out of 650 cases presented a thyroid lobe size ≥ 6 cm. As reported in Table p = 0.015). 2 (1%) out of the 3 bleedings occurred in the surgical track from the axilla to the neck, whereas only 1 (0.5%) occurred in the thyroid bed. Moreover, a longer operative time, either for hemithyroidectomy and total thyroidectomy  was associated to nodule size > 3 cm. No other surgical outcomes differ significantly.205 (31.5%) out of 650 case presented a nodule size > 3 cm. As reported in Table Robotic gasless transaxillary thyroidectomy has emerged as a popular remote-access approach, widely diffused in the Far East. In Asian countries, RATT has been shown to be safe with comparable operative outcomes to open thyroidectomy and resulted associated to excellent cosmesis and reduced postoperative pain , 11.Nonetheless, in U.S. after initial interest for the technique, the enthusiasm was dampened after withdrawal of industrial support . Indeed,p = 0.02) [Hinson et al. published in 2015 an analysis of trends of RATT in U.S. [ = 0.02)  and proh = 0.02) .Furthermore, differences between Eastern and Western patients in terms of body habitus, cultural issues, funding reimbursement and thyroid features raised issues regarding the adoption of this technique in U.S. and Europe. In particular, wide differences emerged regarding the thyroid features of Asian and American patients, mainly related to different approach of the health care systems to thyroid disease and the Korean screening program for thyroid cancer , 18. AccThe American Thyroid Association Statement on remote-access thyroid surgery, published in 2016 by Berber et al. [Although anthropometrics’ and thyroid’s features of European patients are similar to those of Americans, in Europe some high-volume institutions perform RATT routinary reporting case series of hundreds of patients , 21, 22.We started performing RATT in 2012. At the beginning of our experience there were no accepted guidelines for preoperative patient selection for robotic procedures . Considep = 0.399), the rate of transient and permanent hypocalcemia , transient and permanent RLN palsy , cervical hematoma (p = 0.058), bleeding which requires reoperation (p = 0.116), seroma (p = 0.561) and other unconventional complications (p = 0.808) resulted comparable. Moreover, we stratified patients on the basis of each ATA statement parameter and found no statistically significant differences between patients with or without thyroiditis and patients with thyroid lobe < or ≥ 6 cm. Regarding nodule size, no statistically significant differences were documented between patients with nodule ≤ or > 3 cm, except for bleeding which requires reoperation which resulted higher for nodules > 3 cm. Nonetheless, all bleedings but one occurred in the subcutaneous flap from the axilla to the neck, thus without correlation to the thyroid gland.Our experience showed no statistically significant differences in terms of morbidity between cases in accordance to the ATA statement on remote-access thyroidectomy and those not. In particular, the overall complication rate . Overall, after a mean follow-up of 54.2 ± 30.6 months, all patients are free from structural recurrence at ultrasound examination. Moreover, considering only patients who underwent total thyroidectomy and completion thyroidectomy, after excluding 21.9% of patients lost at follow-up, 66.2% of patients underwent radioactive iodine (RAI) therapy. Furthermore, thyroglobulin (Tg) levels resulted < 0.2 ng/ml in 68.2% of patients, between 0.2 and 1 ng/ml in 20.4% of patients and ≥ 1 ng/ml in 11.5% of patients. Besides, the mean Tg level in patients who underwent RAI therapy was 0.3 ± 0.7 ng/ml, whereas it was 0.9 ± 1.6 ng/ml in patients who underwent thyroidectomy alone.In 2018 we already described our results on differentiated thyroid carcinomas proving RATT as oncologically safe . MoreoveOverall, our study demonstrated the feasibility and safety of RATT in patients with large thyroid nodules and thyroiditis, making this approach applicable to a large portion of the Western population, on contrast of Minimally-Invasive Video-Assisted Thyroidectomy which is reserved to a minority of patients , 35. NonThis study harbors limitations. First of all, the retrospective nature of the study may have led to selection bias. Moreover, we did not assess the oncologic outcomes of groups since the study was focused only on surgical outcomes. Third, this series was performed at a single institution, thereby limiting the generalizability of the results.All in all, in high-volume institutions RATT is feasible even in cases beyond ATA recommendations . Nonethe


Bladder cancer (BCa) is the 10th most common type of cancer worldwide, and human papillomavirus (HPV) is the most common sexually transmitted infection. However, the relationship between HPV infection and the risk of BCa is still controversial and inconclusive.This systematic review and meta‐analysis were conducted following the PRISMA 2020 reporting guideline. This study searched four bibliographic databases with no language limitation. The databases included PubMed (Medline), EMBASE, Cochrane Library, and Web of Science. Studies evaluating the interaction between HPV infection and the risk of BCa from inception through May 21, 2022, were identified and used in this study. This study estimated the overall and type‐specific HPV prevalence and 95% confidence intervals (95% CI) using Random Effects models and Fixed Effects models. In addition, this study also calculated the pooled odds ratio and pooled risk ratio with 95% CI to assess the effect of HPV infection on the risk and prognosis of bladder cancer. Two‐sample mendelian randomization (MR) study using genetic variants associated with HPV E7 protein as instrumental variables were also conducted.p = 0.0011; HPV 18 E7 protein: IVW OR per unit increase in protein level = 1.0003 [95% CI: 1.0001–1.0005]; p = 0.0089).This study retrieved 80 articles from the four bibliographic databases. Of the total, 27 were case–control studies, and 53 were cross‐sectional studies. The results showed that the prevalence of HPV was 16% (95% CI: 11%–21%) among the BCa patients, most of which were HPV‐16 (5.99% [95% CI: 3.03%–9.69%]) and HPV‐18 (3.68% [95% CI: 1.72%–6.16%]) subtypes. However, the study found that the prevalence varied by region, detection method, BCa histological type, and sample source. A significantly increased risk of BCa was shown for the positivity of overall HPV , which was also influenced by study region, detection method, histological type, and sample source. In addition, the study found that HPV infection was significantly associated with the progression of BCa . The two‐sample MR analysis found that both HPV 16 and 18 E7 protein exposure increased the risk of BCa (HPV 16 E7 protein: IVW OR per unit increase in protein level = 1.0004 [95% CI: 1.0002–1.0006]; In conclusion, HPV may play a role in bladder carcinogenesis and contribute to a worse prognosis for patients with BCa. Therefore, it is necessary for people, especially men, to get vaccinated for HPV vaccination to prevent bladder cancer. Studies have found that HPV is the most common sexually transmitted infection among humans.The urinary tract is anatomically close to the genital tract. As a result, HPV has a high probability of infecting the urinary epithelium of the bladder.Mendelian randomization (MR) is a method that uses genetic variants as instrumental variables of the exposure to evaluate the causal effects of the exposure on an outcome to exclude the influence of the confounding factors inherent in observational studies.22.1The inclusion and exclusion criteria were as follows: (1) The exposure of interest was HPV infection in bladder tissues; (2) The outcome of interest was bladder cancer; (3) This study only included the latest patient datasets reported in different articles; (4) Immunosuppressed patients such as those accepting organ transplant or with immunodeficiency were excluded; (5) Patients with secondary malignant tumors of the bladder and cervical lesions simultaneously were excluded; (6) Studies using cell or animal models and case reports were excluded; (7) Studies without full text or lack of valuable data were excluded. The search was not limited to any language or publication status.2.2This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta‐analyses (PRISMA) 2020 reporting guideline.2.3This study used a designed data extraction sheet to extract information from the included studies. The data extraction sheet consisted of bibliographic information and background information. Bibliographic information had author's name, year of publication, journal name, and title. Background information included the country where the study was carried out, the inclusion and exclusion criteria for patients, age, percentage of male patients, and the smoking rate of the patients. In addition, background information had sample source, detection method, types of HPV detected, histological types of bladder cancer, the prevalence of HPV  in bladder tissues, matching criteria if the study design was case–control and follow‐up duration, and the outcome of patients. Moreover, if the prevalence of HPV was detected using two or more methods, such as polymerase chain reaction (PCR) and in situ hybridization (ISH), the results of PCR were used for further analyses. The detailed characteristics of the articles included in this study are shown in Table 2.4The evaluation criteria for an observational study of the Agency for Healthcare Research and Quality (AHRQ) were adapted to evaluate the quality of cross‐sectional studies.2.5χ2 (Cochrane's Q) test with a significance level of α = 0.10 and the I2 test.I2 describes the percentage of variation across studies, and an I2 statistic value of ≥50% indicates considerable heterogeneity.p < 0.05 was considered significantly different.This study estimated the prevalence of overall and type‐specific HPV and 95% CI using the size and number of HPV‐positive infections in BCa patients using Random Effects (RE) models or Fixed Effects (FE) models.2.6https://gwas.mrcieu.ac.uk/; version: v6.5.2‐2022‐04‐11), developed at the MRC Integrative Epidemiology Unit at the University of Bristol.The data resources were obtained from MRC IEU OpenGWAS . Five MR methods were used: Weighed median regression, Inverse variance weighting (IVW), Mendelian randomization‐Egger (MR Egger), Simple mode, and Weighed mode. The IVW method with RE was the main statistical model. This study applied the SNP‐specific Wald ratio to evaluate the causality. The pleiotropy test tested the pleiotropic effects, and p ˃ 0.05 was regarded as having no pleiotropic effects. Heterogeneity was tested using the Cochrane Q value. Supporting Information: Tables Supporting Information: Table p values were two‐tailed, and the R software (version 4.2.0) with the Two Sample MR packageAll 3This study retrieved 546 publications from electronic databases and other sources. However, after applying the inclusion and exclusion criteria in Figure 3.1The characteristics of the 80 articles included in this study are shown in Table In these studies, most patients were more than 60 years, and the percentage of male patients was greater than 80%. It was found that only one study focused on female patients with BCa.3.2This study adopted the Agency for Healthcare Research and Quality (AHRQ) evaluation criteria to evaluate the quality of cross‐sectional studies, as shown in Supporting Information: Table 3.3I2 = 96%, RE model), as shown in Figure The 80 studies had 6065 patients with BCa, in which the prevalence of HPV ranged from 0% to 100%, and the overall HPV prevalence was 16% . However, Begg's test did not show a significant publication bias .A contour‐enhanced funnel plot was constructed because the results found that the articles included in this study had significant heterogeneity. However, the result in Supporting Information: Figure p = 0.2153) and the smoking rate (p = 0.8540), as shown in Supporting Information: Table Besides, meta‐regression was also performed to find out the latent confounding variables. The study used the percentage of male patients and the smoking rate to construct the univariate meta‐regression model, as shown in Supporting Information: Figure 3.4I2 = 60%, RE model). A subgroup analysis stratified by continent, detection method, histological subtype, and sample source was also performed, as shown in Figure A total of 25 case–control studies comprising 1621 BCa patients and 685 used were included to investigate the association between HPV infection and the risk of BCa. Two of these studiesz = −0.66, p = 0.5091) and Egger's test . In addition, the cumulative meta‐analysis and sensitivity analysis were performed by stepwise addition and deletion of studies. There were no observable changes in the POR, as shown in Supporting Information: Figures A funnel plot shown in Supporting Information: Figure p = 0.0078) and might serve as a latent confounding factor. On the other hand, the models constructed based on age (p = 0.5547), smoking rate (p = 0.5918) and the publication year (p = 0.3295) had no statistical significance, as shown in Supporting Information: Table In univariate meta‐regression, the study found that the percentage of male patients was significantly associated with the increased risk of BCa   and Egger's test .The funnel plot did not show good symmetry, as shown in Supporting Information: Figure p = 0.0326), while the models constructed based on follow up duration (p = 0.3950), publication year (p = 0.6786), age (p = 0.4156), the percentage of male patients (p = 0.8388), smoking rate (p = 0.0867) and HPV‐16 prevalence (p = 0.5226) all lacked statistical significance, as shown in Figure In univariate meta‐regression, the study found the prevalence of HPV‐18 was negatively associated with the risk of BCa (3.6p = 0.7473), as shown in Supporting Information: Table The data source for this two‐sample MR study was shown in Supporting Information: Table p = 0.0011; HPV 18 E7 protein: IVW OR per unit increase in protein level = 1.0003 [95% CI: 1.0001–1.0005], p = 0.0089).The study also performed the leave‐one‐out analysis to exclude the influence of individual SNPs on the overall causal estimate by the stepwise removal of each SNP and repeating the MR analyses. The leave‐one‐out analysis showed a relatively stable outcome after removing each SNP, as shown in Figure 4An early meta‐analysis published in 2011p = 0.0011; HPV‐18 E7 protein: IVW OR per unit increase in protein level = 1.0003, p = 0.0089), implying that this interaction represents a causal effect of HPV infection.A total of 6065 BCa cases were included in this study, in which the prevalence of HPV ranged from 0% to 100%, yielding a pooled HPV prevalence of 16% (95% CI: 11%–21%), consistent with the previous meta‐analysis.BCa is the 10th most common cancer worldwide with high morbidity and mortality rates. Smoking and exposure to several chemicals are known risk factors for BCa and it is important to identify the latent risk factors to prevent BCa. HPV is a family of sexually transmitted DNA‐based viruses that infect the stratified squamous epithelia, and more than 150 HPV subtypes have been identified,p = 0.8540). Differences in these outcomes are because: First, smoking status was not accurate in some studies because of information bias, and the duration as well as smoking dose were unknown in most studies, which was significantly associated with HPV prevalence. Second, most of the included patients were male, however, HPV prevalence was higher among female smokers. Finally, only a limited number of studies reported on the smoking status of patients, therefore, the result should be interpreted with caution.Smoking is an acknowledged risk factor for bladder cancer and has been highly associated with higher incidences of HPV infection , which was dose‐dependent and more significant for female patients  and high‐risk HPV subtypes.Globally, HPV infections account for 630 000 new cancer cases per year, among which 83% are cervical cancer cases.HPV‐associated oropharyngeal squamous cell carcinoma (OPSCC) was associated with worse overall survival outcomes, relative to non‐HPV‐associated OPSCC, even after adjusting for smoking status.Then, we investigated the causal relationship between HPV E7 protein levels and BCa risk. The meta‐analysis for different bladder cancer datasets showed that genetically elevated HPV E7 levels are causally associated with increased BCa risk for both HPV‐16 and HPV‐18, consistent with the findings from previous meta‐analyses of conventional observational studies after exclusion of latent bias caused by confounding factors. Although statistically significant, the OR values for HPV‐16 and HPV‐18 E7 proteins on BCa were close to 1 and dependent on E7 protein levels, therefore, this result should be carefully interpreted. Besides, clinical assessment of E7 protein levels is difficult and lacks practical significance. Apart from these limitations, findings from two‐sample MR analysis are in accordance with the result of this systematic review and meta‐analysisSchistosoma hematobium infections, which could probably influence the outcomes of this study. Third, we only found a dataset for HPV E7 protein levels but did not find other instrumental variables for HPV infections. Fourth, there was a great heterogeneity in the characteristics of the study cohort. Fifth, detection methods for some studies were not sensitive enough and did not detect the specific HPV subtypes, which may have interfered with the final outcome. Finally, there were various limitations regarding our analysis of HPV and BCa progression. The number of studies included in this section was limited. Recurrence and death were not valid outcomes for prognosis, especially when the causes of death were not confirmed and specified and when the HPV persistence/clearance was unknown during BCa treatment/follow‐up. Several potential confounders during prognosis were unknown and were not controlled for in the analysis. Therefore, functional studies using cell lines and animals as well as clinical studies should be performed to confirm our findings.This study has various limitations. First, due to the lack of original data, we did not investigate the role of HPV infection in different stages of bladder cancer. Second, most SCC cases were associated with 5HPV infection is significantly associated with increased risk and worse prognosis of bladder cancer. Therefore, HPV vaccination is necessary to prevent bladder cancer, especially among men. Future studies should investigate the association between HPV infection and bladder cancer using large‐scale sampling of various populations. Moreover, the mechanisms behind these phenomena should be elucidated.Jian‐Xuan Sun, Jin‐Zhou Xu, Chen‐Qian Liu, Qi‐Dong Xia, and Shao‐Gang Wang contributed to developing the main research question, carrying out the literature search, collecting the included studies’ information, and describing the results. Jian‐Xuan Sun, Jin‐Zhou Xu, and Chen‐Qian Liu performed the meta‐analysis and wrote the first draft of the manuscript. Xing‐Yu Zhong, Na Zeng, Hao‐Dong He, and Si‐Yang Ma contributed to developing the main research question and revised the manuscript. Ye An, Meng‐Yao Xu, Zheng Liu, and Jia Hu revised the manuscript. All authors contributed to the article and approved the submitted version. Jian‐Xuan Sun, Jin‐Zhou Xu, Chen‐Qian Liu, Qi‐Dong Xia, and Shao‐Gang Wang contributed equally to this study.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Circulating tumor cells (CTCs), an indispensable liquid biopsy classifier, can provide extra information for the diagnosis and prognosis of hepatocellular carcinoma (HCC).n = 270) for CTC number, Ki67 index reflecting the proliferative CTC percentage (PCP), and CTC clusters correlated with the characteristics of malignant HCC tumors.We systematically analyzed the peripheral blood of preoperative HCC patients (n = 73). CTC number and PCP were associated with tumor size, microvascular invasion (MVI), presence or absence of multiple tumors, and thrombosis significantly. CTC number and PCP robustly separated patients with and without relapse, with a sensitivity of 88.89%/81.48% and a specificity of 72.73%/94.81% in the training set (n = 104) and corresponding values of 80.00%/86.67% and 78.38%/89.19% in the validation set (n = 52), showing a better performance than that associated with the alpha‐fetoprotein (AFP) level. CTC number, PCP, CTC clusters, and MVI were independent significant risk factors for HCC recurrence . A nomogram model based on these risk factors showed a considerable prediction ability for HCC recurrence (area under the curve = 0.947). PCP  and CTC clusters  were more significantly correlated with worse recurrence‐free survival than CTC number .Driver gene mutations were found with high levels of consistency between CTCs and primary tumors (PCP and CTC clusters may predict HCC recurrence and improve the performance of the serum biomarker AFP. Tumor‐derived proliferative CTC and CTC clusters predict aggressiveness and early recurrence in hepatocellular carcinoma. Proliferative CTC and CTC clusters may predict HCC recurrence and improve the performance of serum biomarker alpha‐fetoprotein. The number and phenotype of CTCs are related to the progression of primary tumor lesions and can help elucidate the nature of the tumor lesions.CTCs originating from different tumor sources have high heterogeneity and are characterized by different surface markers and are different sizes. With the development of new identification techniques, more CTC phenotypes have been revealed. Metastasis‐related mesenchymal‐like CTCs are associated with poor overall survival and have spatial heterogeneity in the circulatory system.Ki67 is regarded as a proliferation index of the primary HCC in evaluations of tumor malignancies and is related to tumor size, high AFP level, and TNM stage. Ki67 is an important prognostic factor for the overall survival and progression‐free survival of patients with HCC.To date, no relevant study has explored the correlation between Ki67‐positive CTCs and poor prognosis of HCC. Herein, a cohort of 270 HCC patients was recruited and CTCs were isolated. Immunofluorescence (IF) and RNAscope were applied to identify and enumerate the CTC number, the Ki67‐positive proliferative CTC percentage (PCP) and CTC clusters. The preoperative CTC number and Ki67‐positive PCP are correlated with the characteristics of malignant HCC tumors, such as tumor diameter, tumor number, satellite nodules, thrombosis, microvascular invasion (MVI), and AJCC and BCLC stages. After performing verification in an independent sample set, we found that a CTC number >15/7.5 mL, a Ki67‐positive PCP > 29.6%, and CTC clusters could serve as HCC prognostic biomarkers. In addition, these factors were correlated with a worse recurrence‐free survival (RFS) rate and were independent significant risk factors for HCC recurrence. In addition, compared with the CTC number, the Ki67‐positive PCP and CTC clusters were better predictive biomarkers for the early recurrence of HCC.22.1From October 2014 to June 2018, a total of 270 HCC patients from Cancer Hospital of the Chinese Academy of Medical Sciences, Beijing You‐An Hospital and Beijing Friendship Hospital were enrolled for CTC detection. The inclusion criteria included a pathologic or radiographic diagnosis of HCC. Tumor stage was determined according to the AJCC and BCLC staging systems. Tumor differentiation was defined according to the Edmondson grading system. Liver function score was measured by Child‐Pugh system. The clinical information of patients with HCC, such as age, sex, imaging data, biochemical results from liver function examinations, and pathological results, was collected. The clinical characteristics of the 270 HCC patients included in this study are summarized in Table 2.2+ white blood cells were depleted by applying them to an LS column  with magnetic separation. The supernatants were transferred into a new tube and centrifuged at 1000g for 10 min, and unlabeled CD45− cells were spotted on glass slides for IF staining.Peripheral blood (7–10 mL) was collected from each participant in Streck tubes (Streck). First, peripheral blood mononuclear cells (PBMCs) were collected by density gradient centrifugation using histopaque‐1077 (Sigma–Aldrich). Second, both negative selection and positive selection strategies were used to enrich and isolate CTCs. For negative selection, the PBMCs were mixed with microbeads (Miltenyi Biotec) conjugated to anti‐human CD45 antibodies. CD452.3After fixation and air drying, the cells were subjected to IF staining with an antibody cocktail against pancytokeratin (CK8/18/19)‐FITC  and CD45‐PE  for 1 h at room temperature. The slide was washed three times with phosphate‐buffered saline. The nuclear dye DAPI (Sigma‐Aldrich) was applied, and stained cells were observed and recorded with a fluorescence microscope (Nikon).2.42O2 for 10 min, and then treated with Protease Plus  at 40°C for 30 min. Next, the C3 probes were diluted in C1 probes at a 1:50 ratio for hybridization at 40°C for 2 h. Then, AMP1, AMP2, and AMP3 were added to the slides and incubated at 40°C for 30 min. After HRP treatment, C1 probes detected with TSA‐Plus FITC and C3 probes detected with TSA‐Plus Cy5 were added to the slides sequentially, with HRP blockers applied after every step. Finally, DAPI (Sigma‐Aldrich) was added to label the nuclei, and the slices were mounted. Proliferative CTCs were defined as DAPI+/Ki67+/CK+ cells, and regular CTCs were defined as DAPI+/Ki67–/CK+ cells.RNAscope ISH assays were performed on formalin‐fixed CTC slides. The RNAscope probes Homo sapiens‐Hs‐panCK‐C3 (404751‐C3) and Homo sapiens 2.5 LS Probe‐Hs‐MKI67‐C3 (591778‐C3) were synthesized by Advanced Cell Diagnostics. The location and expression level of the target RNA were detected by the RNAscope Multiplex Fluorescent Reagent Kit v2  according to the manufacturer's instructions. In brief, the CTC slides were dehydrated in an ethanol gradient, treated with H2.52O were added, and the reaction was promoted for 4 h at 30°C. Phi29 DNA polymerase had strand displacement activity with high replication fidelity. For quality control, the amplification products were used as the template for secondary amplification, in which, GAPDH, TP53, and CTNNB1 primers were designed for the target segment, and the CTC pool with at least one positive result was used for subsequent sequencing.The identified CTCs from the positive enrichment strategy were separated into phosphate buffer solution by a micromanipulator with a 10 μM capillary needle and stored at −80°C for genomic analyses. Single‐cell whole‐genome amplification of CTCs and quality control genomic DNA amplification were carried out using the REPLI‐g Single Cell Kit (Qiagen) according to the manufacturer's protocol. The CTC pool was sized up to 4 μL using phosphate buffer, 3 μL denaturant (DLB: DTT = 11:1) was added, and degeneration was performed for 10 min at 65°C. Then, 3 μL stop solution was added to terminate degeneration. After oscillation and blending, 29 μL of reaction buffer, 2 μL of Phi29 DNA polymerase, and 9 μL of H2.6P < 0.05 was considered significant .Statistical analysis was conducted with R software  and GraphPad Prism . All measurement data are presented as the median±standard deviation (median±SD), and the minimum and maximum values are shown in parentheses. Differences between two groups were determined by the Mann–Whitney rank test. Differences among more than two groups were assessed with analysis of variance followed by a multiple comparisons test. The “pROC” package was applied to establish the receiver operating characteristic (ROC) curve to evaluate the prognostic values of the CTC number and PCP. “Survival” and “survminer” were used to perform univariate and multivariate Cox regression analyses and visualization of the results. Before the log‐rank test for RFS, continuous parameters, including of the CTC number and PCP, were dichotomized by optimal cutoff values determined based on their ROC curves. The nomogram model was constructed based on the final multivariate Cox regression model using the “rms” package. The performance of the nomogram was assessed using a calibration curve with 1000 bootstrap resamples for internal validation to assess their predictive accuracies. Decision curve analysis (DCA) was conducted with the “rmda” package to evaluate the efficacy of the nomogram. Analysis of similarity (ANOSIM) was performed using the “vegan” R package, and mutation state dissimilarity in ANOSIM was calculated based on the Euclidean distance matrix. The “wilcoxsign_test” of the “coin” package was applied to conduct permutation testing with 1000 resamples. All statistical tests were two‐tailed, and 33.1n = 139) or liver transplantation (n = 131) surgery. The clinical information and pathological characteristics of the patients are summarized in Table A total of 270 patients with HCC from two hospitals were enrolled, and they underwent hepatectomy  were found CTC positive Figure , and the3.3P = 0.0220; hazard ratio [HR] 7.31, 95% confidence interval [CI] = 0.99–53.75; Figure TP53 , TERT promoter , ARID1A , CTNNB1 , and AXIN1  between the tumor tissues and the preoperative CTCs was 93.75% (15/16), 71.43% (30/42), 92.31% (12/13), and 62.50% (5/8), respectively, both were considerable , 174 alterations were identified in the primary tumors, 107 (61.49%) of which overlapped with the homologous CTC pool. Among the patients with mutations in the tumor, 85.25% (52/61), 70.49% (43/61), and 47.54% (29/61) of them had at least one, half, and all identical mutations detected in the matched pool of whole‐genome amplification products of CTCs , we found that the RFS of patients with TP53 mutation in CTCs was significantly different from that of patients without TP53 mutation in CTCs , tumor number (P < 0.0001), largest tumor diameter (P = 0.0003), MVI (P < 0.0001), thrombosis (P < 0.0001), AJCC stage , and BCLC stage , indicating that CTC number was associated with HCC tumor burden and could be used to assess HCC progression , multiple tumors (P = 0.0017), larger tumor diameter (P = 0.0002), MVI (P < 0.0001), thrombosis (P < 0.0001), AJCC stage , and BCLC stage , revealing that the CTC number and PCP could be used to assess disease progression in HCC patients . During follow‐up, 42 patients (26.92%) exhibited disease recurrence confirmed by imaging, 39 patients underwent hepatectomy and exhibited a recurrence rate of 28.00% (35/125), and 7 patients underwent liver transplantation and exhibited a recurrence rate of 22.58% (7/31).n = 27) from nonrecurrence patients (n = 77), with areas under the curve (AUCs) of 0.890 and 0.858, respectively, which were much higher than the AUC for AFP of 0.546 . In all, 13 of 14 (92.85%) HCC patients with CTC clusters exhibited recurrence, which was more serious than that in 29 of 142 HCC patients without CTC clusters . Similarly, 35 of the 43 (81.40%) patients with a positive PCP exhibited disease recurrence, compared with 7 of the 113 patients with a negative PCP . In addition, the RFS of patients with positive CTC numbers preoperatively was significantly different from that of patients with negative CTC numbers preoperatively in both the training data set and validation data set  and validation data set  and validation data set  patients with positive CTCs exhibited disease recurrence compared with 7 of the 88 (7.95%) patients with negative CTCs , CTC number , CTC cluster , and PCP  were significant independent risk factors for HCC recurrence, while multiple nodules, thrombosis, and BCLC stage were not  in predicting tumor relapse and its weak associations with patient survival.CTCs are capable of vessel invasion and the formation of metastatic loci; thus, studies have focused on exploring the biological characteristics of CTCs from multiple dimensions.The molecular characteristics of CTCs, which represent a state between primary and metastatic tumors, could assist in accurate classification, exploration of tumor cell subgroups with high malignancy, and targeted drug selection.The CTC number could be used for early diagnosis, prognosis evaluation, and evaluations of response to antitumor drugs in patients with multiple tumors.OCT4, SOX2, Nanog, and SIN3A) are hypermethylated. CTC clusters are associated with poor prognosis but are sensitive to some targeted drugs, such as Na+/K+‐ATPase inhibitors, affording a theoretical basis for precision therapy. It seems that neutrophils are involved in the formation of CTC clusters and promote the CTC cell cycle process to promote metastasis.Stem‐like CTCs have stronger drug resistance and anti‐apoptosis effects and contribute to tumorigenesis and metastasis by self‐seeding, which has been identified in animal models. In addition, stem‐like CTCs have been demonstrated in large cohorts to have more diagnostic value.Our research showed that tumor size (positive defined as >5 cm), AFP level (positive defined as >20 ng/mL), and BCLC staging (positive defined as >A stage), which are available preoperatively, were not effective in the prediction of postoperative relapse . The remaining reference pathological parameters, such as MVI, thrombosis, and satellite nodules, depend on the postoperative surgical tumor specimens. However, the CTC number and PCP as well as liquid biopsy convey related information derived from the primary tumor; therefore, this strategy could more consistently predict relapse cases before surgery than other clinical biomarkers . Although the detection rate of CTC clusters was only 10.74% in patients with HCC, the specificity of this parameter for predicting postoperative recurrence was as high as 98.61% (71/72) in the total cohort. In addition, a nomogram prediction model was constructed based on the independent risk factors for the CTC number, PCP, CTC clusters, and MVI in the multivariate analysis. As evaluated by the DCA, ROC curves, and survival curves, the comprehensive nomogram prediction model was proven to be more accurate in prognosis prediction.These results illustrate that both the CTC number and specific phenotype could work as biomarkers of early recurrence in patients with HCC and help prolong survival and eventually reduce mortality. It has been reported that short‐term CTC culture could provide an opportunity to monitor drug susceptibility and response to adjuvant therapies.In summary, CK and CD45 staining were used to identify regular CTCs and CTC clusters in the peripheral blood of patients with HCC. Furthermore, Ki67 was used as a marker of proliferative CTCs to identify the most active subtype of CTCs. The preoperative CTC number, CTC clusters, and PCP were significantly associated with the RFS rate in HCC patients and regarded as independent significant risk factors for HCC recurrence. The PCP showed a better performance in predicting recurrence than the CTC number, and both were significantly better than the AFP level. Inevitably, there are some shortcomings in our study: (1) only two subtypes of CTCs were analyzed in this study, so the overall heterogeneous CTC situation in HCC patients may not be accurately illustrated, and the spatial heterogeneity of CTCs from different vascular sites was not discussed; (2) the number of cases used for the survival analysis was relatively small in this study; and (3) the molecular characteristics of CTCs, both at the genomic and transcriptomic levels, need to be further explored, which we aim to address in future studies. In conclusion, our study revealed that both the number and specific phenotype of CTCs could be used to support the clinical management of HCC patients and thus provide a foundation for future studies.Lina Zhao: Data curation ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft . Jinge Song: Data curation ; formal analysis ; investigation . Yulin Sun: Data curation ; formal analysis ; funding acquisition ; investigation . Qiang Ju: Data curation ; formal analysis ; investigation ; visualization . Hong Mu: Data curation ; formal analysis ; investigation ; visualization . Xiu Dong: Data curation ; formal analysis ; investigation . Jing Ding: Data curation ; investigation ; resources . Yunhe Liu: Data curation ; investigation ; resources . Xuebing Wang: Data curation ; investigation ; resources . Liying Sun: Data curation ; investigation ; resources . Jianxiong Wu: Data curation ; investigation ; resources . Yuchen Jiao: Data curation ; formal analysis ; funding acquisition ; methodology ; supervision . Shichun Lu: Data curation ; formal analysis ; investigation ; resources ; supervision . Xiaohang Zhao: Conceptualization (lead); funding acquisition (lead); project administration (lead); resources ; supervision (lead); writing – review and editing .This work was partly supported by the National Key R & D Program of China (2017ZX10203205‐003‐001 and 2018YFC1313101 to XZ), the National Natural Science Foundation of China (81872033 to XZ and 82073327 to YS), and the CAMS Innovation Fund for Medical Sciences .The authors declare that they have no conflicts of interest.This study was carried out with the approval of the Institutional Review Board of the Ethics Committee of Cancer Hospital of Chinese Academy of Medical Sciences (ID: NCC3094 and 2021101216113802) and Beijing Friendship Hospital (ID: 1018‐768). Informed consent was obtained from participants in accordance with respective committee regulations and the study was performed in accordance with the guidelines of the Declaration of Helsinki.Table S1–S6Click here for additional data file.Figure S1–S6Click here for additional data file.


Coronavirus disease 2019 (Covid‐19) following venous thromboembolism (VTE) and blood hyperlactatemia are associated with higher mortality. However, reliable biomarkers for this association remain to be elucidated. This study investigated the associations of VTE risk and blood hyperlactatemia with mortality among critically ill Covid‐19 patients admitted to the intensive care unit (ICU).In this single‐centre retrospective study, we included 171 patients aged ≥18 years with confirmed Covid‐19 admitted to the ICU at a tertiary healthcare clinic in the Eastern region of Saudi Arabia between 1 March 2020 and 31 January 2021. Patients were divided into two groups: survivor and non‐survivor. The survivors have been identified as the patients discharged from the ICU alive. The VTE risk was defined using a Padua prediction score (PPS) >4. The blood lactate concentration (BLC) cut‐off value >2 mmol/L was used to determine the blood hyperlactatemia.p = 0.050; HR = 3.87, 95% CI = 1.12–13.45, p = 0.033, respectively). The Area under the Curve for VTE and blood hyperlactatemia were 0.62 and 0.85, respectively.Multi‐factor Cox analysis showed that PPS >4 and BLC >2 mmol/L were more likely to be significantly associated with higher odds of ICU mortality in critically ill Covid‐19 patients (hazard ratio [HR] = 2.80, 95% confidence interval [CI] = 1.00–8.08, VTE risk and blood hyperlactatemia have been associated with a higher mortality risk in critically ill Covid‐19 patients who are hospitalized in the ICU in Saudi Arabia. According to our findings, these people needed more effective VTE prevention strategies based on a personalized assessment of their risk of bleeding. Moreover, persons without diabetes and other groups with a high risk of dying from COVID‐19 may be recognized by measuring glucose as having elevated glucose and lactate jointly. Critically ill Covid‐19 patients treated in the ICU in Saudi Arabia had a greater mortality risk when they had VTE risk and blood hyperlactatemia. These findings indicated that these individuals required more efficient VTE preventive methods based on evaluating their bleeding risk and monitoring together glucose and lactate, including in people without diabetes. Because public awareness about VTE has been reported as poor in Saudi Arabia.22.1This single‐centre retrospective cross‐sectional study examined medical records of confirmed Covid‐19 patients admitted to ICU at tertiary healthcare clinic in the Eastern region of Saudi Arabia between 1 March 2020 and 31 January 2021.2.2We included 171 patients aged ≥18 years with Covid‐19 who had not received the Covid‐19 vaccine. The reason to include patients without vaccination was that recent studies2.3The PPS was created to determine the likelihood of VTE risk in hospitalized patients. A PPS of ≤4 represents a low risk of VTE. This is one of the acknowledged scoring techniques suggested for evaluating the risk of VTE in non‐surgical patients both during and after hospitalization.2.4For this study, the following several covariates were collected from critically ill patients with Covid‐19 at admission to ICU who had not received the Covid‐19 vaccine: demographics , and clinical signs, such as temperature, systolic and diastolic blood pressure, the fraction of inspired oxygen (FiO2), presence of comorbidities, Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE) III scores, weight loss, last body mass index (BMI), heart rate (HR), respiratory rate (RR), an anticoagulant used, and ventilator use.2.5t‐test, a one‐way analysis of variance for continuous variables. In contrast, the Mann–Whitney, Kruskal–Wallis or Chi‐square tests were utilized for definite variables. The clinical features and laboratory test results, including normal ranges, were shown in count and median (IQR).The constant variables were briefed in mean and standard deviation (SD) or median and interquartile range (IQR) based on the data distribution for survivors and non‐survivors. The categorical variables were displayed in count and percentages. Significance between the groups was defined using the independent Student's p < 0.05.Cox regression was used to evaluate the association between VTE risk, blood hyperlactatemia and ICU mortality. The association was estimated in two steps: univariate and multivariate. Multivariate model adjusting for age, gender, creatinine clearance, creatinine, platelet count, ferritin level, and baseline D‐dimer levels were all adjusted for in a multivariate model. The data were reported as hazard ratio (HR) and 95% confidence interval (CI). Survivor was used as a reference. The area under the curve was generated to identify predictors of mortality based on the highest sensitivity and specificity in four groups: (1) non‐survivor with VTE, (2) non‐survivor without VTE, (3) survivor with VTE and (4) survivor without VTE. The survival curve was drawn using the Kaplan–Meier method. The variable group represents the patient's risk category, the ICULOS represents the ICU stay, and the variable Status is the censoring indicator. The log‐rank test established the significance of survival trends in various groups. All the analyses were conducted using SAS version 9.4  for Windows. The statistical significance was determined as 3Of the 171 patients, 83% were survivors and 27% were non‐survivors. Although the average age of the total patients was 65.9 years, non‐survivors (58.9 years old) were 14 years younger than the survivors (73 years old). Most non‐survivors had a higher median score for the SOFAS (3) and FO2 (32). Moreover, non‐survivors had less creatinine clearance (median: 26.2 mL/min) and higher creatinine (mean: 2.4 mg/dL). Non‐survivors had lost platelets (mean: 278.1 cells/mm3), including those with high lactic acid (median: 3 mmol/L) and iron (mean: 85.3 g/dL). In addition, non‐survivors had higher ferritin levels (median: 1079 mg/L). The median length of hospital and ICU LOS for these patients was 18 and 8 days, respectively (Table p < 0.0001). The BLC >2 mmol/L was also significantly associated with greater odds of ICU mortality in this group . The multivariate model adjusted for age, gender, creatinine clearance, creatinine, platelets, ferritin level and baseline D‐dimer revealed that PPS >4 and BLC >2 mmol/L were associated with higher odds of ICU mortality  . At 18 days of ICU LOS, the survival probability estimates were about 0.3 for non‐survivors with PPS >4. For the other groups, the probability of survival after 30 days of ICU LOS was approximately 0.1  for PPS >4 and BLC >2 mmol/L were 0.62 and 0.85, respectively Table . There w4The present study investigated the association between VTE risk, blood hyperlactatemia and ICU mortality risk among critically ill Covid‐19 patients in Saudi Arabia. The results of this study demonstrated that VTE risk and blood hyperlactatemia were significantly associated with greater odds of ICU mortality among non‐survivors than survivors of Covid‐19 who had not received the Covid‐19 vaccine, even after adjusting for age, gender, creatinine clearance, creatinine, platelets, ferritin level and baseline D‐dimer.The AUC of blood hyperlactatemia was close to 0.8, indicating that the predictive abilities of mortality risk were acceptable.The current study found that 72.4% of Covid‐19 non‐survivor patients experienced VTE risk in the ICU, which was high compared to the prevalence reported in the previous studies.In the present study, the finding from the multivariate analysis shows that VTE significantly had 80% more likely associated with mortality risk among non‐survivors than among survivors in Saudi Arabia. This finding is consistent with an earlier meta‐analysis showing that VTE incidence was 31% among critically ill Covid‐19 patients, followed by DVT, PE and arterial thromboembolism, 28%, 19% and 5%, respectively.Consistent with the literature,The primary strength of this study is that it first investigated the association between risk in VTE, blood hyperlactatemia and ICU mortality risk among critically ill Covid‐19 patients in Saudi Arabia using a PPP >4 and BLC >2 mmol/L as biomarkers. Some studies found PPP >4 to be well in populations of Italy5VTE risk and blood hyperlactatemia were linked to a high mortality risk among critically ill patients with Covid‐19 who were hospitalized in the ICU. The single‐centre retrospective methodology and our study's small sample size were both limitations. Our findings, however, imply that more successful VTE prevention methods based on an individual evaluation of the risk of bleeding were required for these individuals. Additionally, a group with a high chance of dying from COVID‐19, including those without diabetes, may be identified by measuring glucose as having increased glucose and lactate together. More research is required to examine all potential factors implicated in mortality. Clinical trials are necessary to ascertain if preventive or therapeutic anticoagulation reduces the mortality risk.Conceptualization: Hassan M. AlQarni and Vishal Vennu. Data curation: Hassan M. AlQarni and Vishal Vennu. Formal analysis: Vishal Vennu. Funding acquisition: Imran Ali Khan. Investigation: Vishal Vennu. Methodology: Hassan M. AlQarni and Amal F. Alshammary. Project administration: Hassan M. AlQarni. Resources: Hassan M. AlQarni. Software: Vishal Vennu. Supervision: Vishal Vennu. Validation: Amal F. Alshammary, Raed Farzan and Imran Ali Khan. Visualization: Amal F. Alshammary, Raed Farzan and Imran Ali Khan. Writing—original draft: Vishal Vennu and Hassan M. AlQarni. Writing—review and editing: Amal F. Alshammary, Raed Farzan and Imran Ali Khan. Approval of final manuscript: Amal F. Alshammary, Hassan M. AlQarni, Raed Farzan, Imran Ali Khan and Vishal Vennu.The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSPD2023R735), King Saud University, Riyadh, Saudi Arabia for funding this project.There are no conflicts of interest.This study was performed in line with the principles of the Declaration of Helsinki. This study was approved by the National Committee of the Bio‐Ethics at John Hopkins Aramco Healthcare (reference #H‐05‐DH‐044) on 7 April 2021. Written consent was obtained from the patients before enrolling in this study. All methods were carried out following the necessary laws and regulations.


The term laminopathies refers to a group of congenital diseases characterized by accelerated degeneration of human tissues. Mutations in LMNA, LMNB, ZMPSTE24, and other genes lead to structural and functional abnormalities associated with lamins. One subtype of laminopathy is the generalized lipodystrophy‐associated progeroid syndrome (GLPS), which occurs in patients with heterozygous mutations of the LMNA gene c.29C>T(p.T10I). This paper reports the first case of GLPS in China and compares the clinical features of other GLPS patients with literature reports. A 16‐year‐old male patient was treated for diabetic ketoacidosis, presenting with premature aging appearance, systemic lipodystrophy, severe fatty liver, and decreased bone density. After peripheral blood DNA extraction and second‐generation sequencing, a heterozygous mutation of exon 1 of the LMNA gene c.29C>T(p.T10I) was detected. This case of GLPS may provide a diagnostic and therapeutic basis for potential patients. This paper reports the first case of generalized lipodystrophy‐associated progeroid syndrome (GLPS) with LMNA c.29C>T mutation in China. LMNA is the most common pathogenic gene causing laminopathies, and the clinical phenotypes caused by different mutation sites vary. Hussain The patient is a 16‐year‐old Han male who was admitted to the hospital in 2022, due to abdominal pain, nausea, and vomiting for 3 days. Diabetic ketoacidosis was diagnosed in the emergency department.He was born at term in a non‐consanguineous family without a history of dystocia. His birth weight was 3.5 kg, but his height and weight development had been lagging behind his peers. The patient's parents and two sisters were in good health, and there were no similar patients in the family.2. He presented with a progeria face , total cholesterol levels of 25.63 mmol/L (0–5.7), low density lipoprotein cholesterol levels of 16.74 mmol/L (0–3.62), and high density lipoprotein cholesterol levels of 0.34 mmol/L (1.16–1.42). The HbA1c was 15.9%. The level of 25‐hydroxy vitamin D3 was 16.9 ng/mL (>20 ng/mL). The abdominal CT . The sequencing results Figure  showed aLaminopathies include clinical phenotypes such as atypical progeria, insulin‐resistant diabetes mellitus, generalized lipodystrophy, severe fatty liver, and diffuse black and white dermatoid papules.The treatment process is shown in Table Laminopathy is a rare monogenic disease caused by mutations in the LMNA, LMNB, ZMPSTE24, and other genes that encode lamininsThis report describes a juvenile case of laminopathy with diabetic ketoacidosis at the onset. Genetic analysis and testing confirmed a sporadic case of LMNA(p.T10I) mutation. Clinical manifestations include progeria facies, generalized lipodystrophy, insulin‐resistant diabetes, severe fatty liver, muscle atrophy, and skin papulosis. Due to the lack of lonafarnib for the treatment of progeria and metreleptin for the treatment of lipodystrophy in Chinaet al.et al.et al. analyzed the clinical data of 10 patients with the p.T10I mutation and shared common clinical characteristics. Therefore, it was recommended that patients with the p.T10I mutation be considered as a subtype of laminopathy called generalized lipodystrophy‐associated progeroid syndrome.Diagnosing laminopathy requires three featuresThe authors declare no conflict of interest.Approval of the research protocol: N/A.Informed consent: All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for publication of this case report and accompanying images.Registry and the registration no. of the study/trial: N/A.Animal studies: N/A.