The incidence of anal squamous cell carcinoma (SCC) has been steadily growing globally in the past decade. Clinical data on anal SCC from China are rare. We conducted this study to describe the clinical and epidemiological characteristics of anal SCC in China and explore prognostic factors of outcomes among patients with anal SCC.We audited demographic characteristics, relevant symptoms, risk factors, treatment modalities and outcomes for patients diagnosed with anal SCC at 11 medical institutions in China between January 2007 and July 2018.n = 93, 64.6%), noticing a lump , and pain . The proportion of patients at the American Joint Committee on Cancer (AJCC) stages I-IV were 10 (6.9%), 22 (15.3%), 61 (42.4%) and 8 (5.6%), respectively, and AJCC stages in 43 (29.9%) patients were unknown. Thirty-six patients (25.0%) underwent abdominoperineal resection initially. Univariable analysis showed that T stage predicted recurrence-free survival (RFS) , and age group , AJCC stage , and N stage  predicted overall survival (OS).A total of 144 patients  were diagnosed with SCC during this period. Median age at initial diagnosis was 52.0 (interquartile range: 46.0–61.8) years. The most common symptoms were bleeding (T stage was identified as prognostic factor of RFS, and age, AJCC stage, and N stage were identified as prognostic factors of OS. Improving symptom awareness and earlier presentation among patients potentially at risk for anal SCC should be encouraged. Familiarity with the standard treatment among health care providers in China should be further improved. Anal cancer is a malignancy accounting for 1–2% of digestive tract tumours and 2–4% of colorectal and anal tumours . In the Before mid-1980s, the standard treatment for SCC was abdominoperineal resection (APR), however because of several disadvantages, such as permanent stoma, this method as first line therapy was abandoned . In 1974One of the key factors that influence the outcomes of anal SCC is the stage at diagnosis , 20. A sIt is not clear whether the findings from studies carried out in Europe or North America are generalizable to China where literature on anal SCC is scarce. We conducted this study to understand the clinical and epidemiological characteristics of anal SCC and prognostic factors of outcomes in patients with anal SCC in China.We audited anal SCC patients recorded in 11 medical institutions in China between January 2007 and July 2018. , Sun Yat-sen University Cancer Center, The Second Affiliated Hospital of Zhejiang University, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Nanfang Hospital, Southern Medical University, Guangdong General Hospital). Patients with anal SCC were identified using the International Classification of Disease (ICD)-10 codes of anal SCC (C 21.0–21.8) . AdenocaFor all identified patients, medical records were reviewed by author PY Li for demographic information , relevant symptoms , risk factors , information of the tumour , treatment received  and outcomes. AJCC stage was determined according to the American Joint Committee on Cancer, 7th edition .p value less than 0.05 was considered to be statistically significant.Data obtained from the medical records were summarized using descriptive statistical analysis. Frequencies and percentages were used to describe categorical variables and median and interquartile range (IQR) were used to describe continuous variables. Chi-square test was used to compare proportion of categorical variables. For analysis, age was divided into two groups: ≤50 years and >50 years. Tumour size was divided into two groups: ≤20 mm and >20 mm. Recurrence-free survival (RFS) was defined as the interval between diagnosis and recurrence . Overall survival (OS) was defined as the interval between diagnosis and death from any cause or last follow-up , 28, 29.This study was approved by the Ethics Committee of the University of New South Wales (IRB ID: HC180393) and the Ethics Committee of the School of Public Health, Sun Yat-sen University (IRB ID: 2018–026). Because the study was retrospective and the data was de-identified, the informed consent requirement was waived.p = 0.637). When grouped by age at initial diagnosis, 41.7% of patients (n = 60) were less than or equal to 50 years, and 58.3% of patients (n = 84) were older than 50 years.A total of 144 patients were identified with anal SCC diagnosed between January 2007 and July 2018 (Table Only one (0.7%) patient  was recorded as HIV positive. Four (2.8%) patients had a history of other cancers . Two (1.4%) patients were diagnosed with sexually transmitted diseases  3 with syphilis and genital warts) while being diagnosed with anal cancer. One (0.7%) patient had a history of genital warts. Among 113 (78.5%) patients with a record of their sexual behaviors, no one reported a history of receptive anal intercourse or homosexual behavior. Seventeen (11.8%) patients were smokers (including current smokers and ex-smokers).n = 93, 64.6%), noticing a lump , pain , altered bowel habit , perianal itch , and tenesmus . Other symptoms  also have been observed. One patient who was diagnosed by physical examination reported no symptom.Median duration of symptoms until initial diagnosis was 90 days (IQR: 30–180 days). The most common symptoms were bleeding  had a tumour less than or equal to 20.0 mm. There were 13 (9.0%) patients diagnosed at T 1 stage, 40 (27.8%), 28 (19.4%), and 36 (25.0%) at stages T 2–4, respectively. The frequencies and proportion of patients in N0–3 stages were 52 (36.1%), 33 (22.9%), 22 (15.3%) and 8 (5.6%), respectively. The frequencies and proportion of patients in AJCC stages I-IV were 10 (6.9%), 22 (15.3%), 61 (42.4%) and 8 (5.6%), respectively, and AJCC stages in 43 (29.9%) patients were unknown.The median tumour size was 30.0 (IQR: 20.0–40.5) mm. About one in four patients (n = 107) of patients, and radiotherapy to 70.1% (n = 101) of patients. Ninety-seven (67.4%) patients underwent CRT. Thirty-six patients (25.0%) underwent APR initially, of which four, five, eight, and zero patients were in AJCC stage I, II, III, and IV, respectively, and AJCC stages of the remaining patients were unknown. Of the 36 patients who underwent surgery initially, 24 patients had performed only APR, and eight, three, and one patient had performed CRT, chemotherapy, and radiotherapy after APR, respectively. Tumour sites treated with APR were anal canal or anal canal and margin. Fifteen (10.4%) patients were treated with local mass resection. Only two patients underwent local mass resection were diagnosed at stage I, and sites of tumour were anal margin.As for diagnosis and treatment modalities, two patients were misdiagnosed with haemorrhoids and underwent haemorrhoidectomy. Chemotherapy was administered to 74.3% , other regiments, such as fluorouracil plus cisplatin (PF), FOLFOX and Capox were also involved. Among the patients diagnosed at M1 stage, the radiation dose varied from 5400 cGy to 6000 cGy, and the frequency of radiotherapy varied 25 to 30 times. Volumetric intensity modulated arc therapy (VMAT) and intensity modulated radiotherapy (IMRT) were the most often adopted technique. Among these patients, five died of cancer, one survived, and one lost to follow-up.The chemotherapy regimens were not uniform and varied across different medical institutions. Among all the 144 patients, TP, PF and fluorouracil (5-FU) plus mitomycin were the most widely used. FOLFOX, Capox, capecitabine monotherapy, 5-FU monotherapy, and cisplatin monotherapy were also used in some hospitals. Concurrent chemotherapy was applied to more than half of the patients, and the others were treated with induction chemotherapy, adjuvant chemotherapy or both. For radiotherapy, apart from some earlier patients who adopted 3-dimensional conformal radiation therapy (3D-CRT), the rest patients all adopted VMAT or IMRT technique. Radiation dose had a wide range, fluctuating between 3780 cGy and 7000 cGy. The median of radiation dose was 5600 cGy (IQR: 5000–6000 cGy). The frequency of radiotherapy varied from 21 to 35. Every single dose, with maximum at 240 cGy and minimum at 180 cGy, was performed five times per week.p = 0.032; Table p = 0.028), AJCC stage , and N stage  predicted OS (Table Within a median follow-up of 44 months (IQR: 25–67 months), 22 patients died of anal SCC and 25 patients developed a recurrence. Estimated 5-year RFS was 79.4%, and 5-year OS was 82.8%. The univariable analysis of RFS showed that T stage was a significant prognostic factor of RFS  had a history of smoking. The smoking rate among males was much higher than that among females. In 2010, Chinese Center for Disease Control and Prevention carried out the Global Adult Tobacco Survey (GATS) in China and reported that smoking rate was 28.1%  among Chinese adults . In the Among 144 SCC patients, only a small proportion were diagnosed at an early stage (10 6.9%] at AJCC stage I and 13 [9.0%] at T1 stage). This was similar to a previous study of anal cancer (129 [71.7%] patients were adenocarcinoma) in China that indicated only 7 of 126 (5.6%) patients were diagnosed at stage I .9% at AJ. The proIn our study we found that about two-thirds of patients received the standard treatment for anal SCC, which was significantly higher than that reported in a previous study carried out in 2011 by Peng et.al where only 9.5% (2/21) received CRT. Peng et al. reported that the reason so few received CRT was that doctors were not familiar with this method as a standard treatment of anal SCC . HoweverClinical practice guidelines suggest that patients who received the standard CRT could achieve a response rate of 80–90%. The remaining 15% of patients whose regional lesion do not respond to CRT can receive APR as salvage treatment . Among aAnal SCC is strongly associated with HPV infection. Recent study regarding cancer burden attributable to HPV infection used 100% as the population-attributable fraction of HPV in anal SCC, which meant that the authors thought nearly all anal SCC were caused by HPV infection . And recIn 2012, HPV 16/18 were responsible for 87.0% anal cancer globally, and proportion rose to 95.9% for HPV 6/11/16/18/31/33/45/52/58 . The HPVFour factors were identified as prognostic factors of outcomes. In our current analysis, we found that advanced T stage at diagnosis was associated with shorter RFS. Previous conducted in Norway reported that advanced T stage significantly increased the risk of recurrence . Ghosn ePrevious studies also mentioned that HPV infection and its surrogate (i.e. p16) were strongly associated with the outcome of anal SCC –54. CompAnal intercourse, a known risk factor for anal SCC, is practiced in a significant proportion of heterosexual couples (6 to 40%)  and nearThere were several limitations that need to be considered when interpreting the data. Our study was a retrospective audit of clinical records. The rarity of anal SCC and the lack of centralised reporting system for anal SCC restricted our ability to find more records which may lead to compromised representativeness of our sample. There is a lack of essential SCC-relevant variables in the current clinical recording system, including detailed sexual behaviors, HPV testing result, HPV-related biomarkers, history of other HPV-related cancers, and HIV status. The tumor registration system in many medical institutions did not systematically separate anal SCC from adenocarcinoma, leading to underreported SCC patients. Meanwhile, the patients in our study were mainly HIV-negative and female. Because of the restricted population, the results of this study may not be generalized. Our study also had several strengths. Our study was the largest one on anal SCC in mainland China. Another strength was the clinical records of patients were collected from 11 medical institutions in various parts of China, which is more representative compared with records obtained from a single institution.Our data illustrated that T stage of anal SCC was a predictive factor of RFS, and age, AJCC stage, and N stage were identified as prognostic factors of OS. Among anal SCC patients, only a small fraction were diagnosed at an early stage. The proportion of patients receiving CRT increased over the past decade. However, the usage of CRT still needs to be improved. Further research about identifying other predictive factors of outcomes e.g. biomarkers, improving the rate of early diagnosis and improving the usage of CRT should be conducted. More efforts are needed to collect necessary information regarding HPV infection, biomarkers of HPV infection, history of HPV-related cancer, and sexual behavioral from SCC patients.


N = 1,500) irrational response to green technological innovation so as to promote their pro-environmental behaviors. Our experiments identify students' mental construal level as an important psychological factor that, when combined with a proper message framing strategy of introducing new green technologies, can remedy their irrational response to new green technologies. Our findings suggest that highlighting the new technology as playing a preventive/promotional role related to climate change can mitigate risk compensation behavior and eventually promote students' pro-environmental behaviors when they are at a high/low mental construal level.Although green technological innovation is designed to combat climate change, recent research suggests that increased attention to technological innovations might decrease climate change risk perception and reduce pro-environmental behaviors due to the feeling of being assured, which is referred to as risk compensation behavior. Although there has been a growing interest in reducing the risk compensation effect related to climate change, the academic literature in this area is very limited. In this study, we propose a psychological intervention to mitigate a sample of university students' ( Most simulations suggest that the change in the global surface temperature between 1,850 and the end of the twenty-first century is likely to exceed 1.5°C. The World Meteorological Organization (WMO) says that if the current warming trend continues, temperatures could rise 3–5°C by the end of this century. Temperature rises of 2°C have long been regarded as the gateway to dangerous warming. The Intergovernmental Panel on Climate Change report of 2014 states that a 70% reduction in anthropogenic GHG emissions between 2010 and 2050 is needed to limit global warming to 2°C above preindustrial levels  based on construal level theory, which is widely used in social psychology, in conjunction with the message framing method to remedy people's irrational responses to new “green” technologies, or “risk compensation” behavior.This paper presents the results of three experiments conducted on 1,500 university students in Chongqing China. The results show that highlighting a new technology as playing a promotional/preventive role related to climate change can significantly mitigate university students' risk compensation behavior so as to promote their pro-environmental behavior when they are at low/high mental construal level , respectively. We examined participants' perceived climate change risk and pro-environmental behavior using a survey before and after interventions. To the best of our knowledge, this is the first study to apply a combination of construal level theory and message framing method on the mitigation of risk compensation behavior among university students related to climate change. We also contribute to the literature that examines the effectiveness of promotional-framed and preventive-framed messages in various contexts. Previous studies have focused on smoking cessation  or a more temporal distance benefit  is highlighted.Lee and Aaker  and ThomTo mitigate risk compensation behavior, we propose that when individuals are in a low construal mindset, learning about a green technological innovation that is framed as playing a promotional role related to climate change can mitigate their risk compensation behavior, due to the belief of abstract thinkers that the preventive action is not an immediate “solution” to climate change, whereas when individuals are in a high construal mindset, learning of a green technological innovation that is framed as playing a preventive role related to climate change can also reduce their risk compensation behavior, due to the belief of abstract thinkers that the preventive action is only a partial and temporary “solution” to climate change. Overall, the incongruent combination of message framing and construal level results in less persuasive messaging and makes participants believe that the conveyed technology is not an efficient remedy for climate change. As a result, this leads to less risk compensation behavior.Experiment one tests for the predicted interaction between mental construal (abstract vs. concrete) and the type of benefit  that is highlighted when advocating the effect of a new “green” technology on the shift of public climate change risk perception.H1a:Advocating “green” technological innovation undermines climate change risk perception due to the feeling of being assured.H1b:The response to “green” technological innovation  varies with individual construal level .H1c: When individuals form an abstract thought , learning of a green technological innovation that is framed as playing a preventive  role related to climate change can mitigate their reduced climate change risk perception (risk compensation behavior) due to the belief of abstract thinkers that preventive action is only a partial and temporary “solution” to climate change and that the new technology is less likely to mitigate climate change risk.H1d:When individuals form a concrete thought , learning of a green technological innovation that is framed as playing a promotional  role related to climate change can mitigate the reduced climate change risk perception (risk compensation behavior) due to the belief of concrete thinkers that the promotional action is not an immediate “solution” to climate change and the technology is not an efficient way to solve the climate change issues.www.wenjuan.com to follow the procedure as follows:The experiment was a 2  × 3 (non vs. mitigation vs. adaptation) mixed design see . In thisStep 1: In the pretest survey, the participants answered a few basic demographic questions and questions from the survey on climate change risk perception and pro-environmental behavior  a group that was asked to complete a task  that manipulated their mental representations into high-level construals; (2) a group that was asked to complete a task  that manipulated their mental representations into low-level construals.Step 3: Then, the participants randomly received one of three versions of the articles , which is the dependent variable. ANOVA was used to test for the significance of differences among the group means in our sample, and regression analysis showed the direction  and magnitude  of these differences.H1athat learning of a “green” technical innovation can undermine climate risk perception and provides suggestions for a risk compensation effect. For those with high-level construals , preventive messaging advocating for technology by highlighting the mitigating benefit of the technology on climate change results in the lowest feeling of assurance  group, and M = 5.01 in the promotional message group) and thus moderates the reduced risk perception  group and M = −3.78 in the promotional message group). This is because the combination of a high construal mindset and a preventive frame is an incongruent intervention. This combination is not effective in persuading participants to believe that the new technology is able to mitigate the climate change risk.M = −1.02 vs. M = 2.62 in the pure technology message (control) group and M = −4.50 in the preventive message group). Overall, the low construal group presented promotional messages advocating green technologies experience a decreased compensation effect and thus exhibit a less reduced risk perception. These findings provide supportive evidence for hypotheses H1b, H1c, and H1d.Likewise, those with low-level construals  were the least assured by promotional messages highlighting the climate-change adaptation (long term) benefit of the technologies. Again, this combination results in an incongruent intervention that is not persuasive enough to make participants believe that the technology is an efficient remedy for climate change. Consequently, this leads to less reduction in risk perception  = 9.85, p = 0.0001], and F is the F statistic. In contrast, the main effects of construal level and message type were not significant  = 0.20, p = 0.6533; and F = 0.33, p = 0.7171]. To further corroborate the interaction effect, we checked the simple main effect of message type by mental construal level and the simple main effect of construal level for the message type. All p-values [see Panel A (a)–(d)], except those for preventative messaging, indicate significance.Next, we examine the regression results in Taken together, we confirm the interaction effect of mental construal level and the way “green” technology is advocated on the shift of public perceived climate risk. In particular, the lack of fit between a mental representation and a message advocating technology as solutions to climate change helps remedy the decrease in climate risk perception. Meanwhile, the results reported in We demonstrated in experiment one that a mismatch between a mental representation and a message framing could effectively attenuate people's lowered climate change concerns caused by risk compensatory behavior. However, it is unclear if greater climate change risk perception would translate into increased individual action to preserve the environment. Consequently, we ran experiment two to examine whether people's heightened concern about climate change would lead to more pro-environmental action intentions.H2a: Changes in climate risk perception can transfer to pro-environmental behavioral intentions.H2b: When individuals form an abstract thought , learning about a green technological innovation that is framed as playing a preventive  role related to climate change can attenuate the reduced pro-environment behavioral intentions caused by the alleviation of lowered climate change risk perception.H2b: When individuals form a concrete thought , learning about a green technological innovation that is framed as playing a promotional  role related to climate change can attenuate the reduced pro-environment behavioral intentions caused by the alleviation of lowered climate change risk perception.www.wenjuan.com. The experimental procedure was the same as that described in Experiment 2, but two differences were presented. First, for the robustness test, we use an alternative way of manipulating participants' mental construal levels. The participants were presented with 10 tasks and asked to indicate either “why” we need to complete the tasks  or “how” we can complete the tasks  Förster et al. -(d)]. The regression results are presented in Panel B. Once again, we observe that learning of “green” technologies reduces people's pro-environmental behavioral intentions  but highlighting mitigating/adapting benefits for people with high/low level mental construals can significantly correct the reduction by 0.764 and 1.097 points, respectively. Taken together, we demonstrate in experiment 2 that individuals' increased climate change concern results in increased pro-environmental action intentions which is consistent with our hypothesis.One inherent challenge associated with surveys eliciting information about pro-environmental behavior is that they are not externally verifiable. Student participants may overstate or fabricate their actual pro-environmental behavior in the survey , learning of a green technological innovation that is framed as playing a preventive  role related to climate change can mitigate the reduced actual pro-environment behavior due to the belief of abstract thinkers that the preventive action is only a partial and temporary “solution” to climate change.H3b: When individuals form a concrete thought , learning of a green technological innovation that is framed as playing a promotion  role related to climate change can mitigate the reduced actual pro-environment behavior due to the belief of concrete thinkers that the promotional action is not an immediate “solution” to climate change.In this experiment, 300 students were recruited from a university in Chongqing, China. The student participants completed the same manipulation of mental construal level as described in Experiment 1 and were then randomly given one of the three versions of the articles. Then, they were asked to play a word decoding effort game. The word game is based on a word decoding effort task, comparable to Dorner , Erkal eEach completed word lowers the amount of money donated to charity for that round. The initial donation for any given round was set at 15 RMB per participant. The charity is a local tree planting organization, and participants are aware that every two RMB given to the organization results in the planting of one seedling. Additionally, participants are aware that each completed word lowers the charity contribution by 2 RMB, which is deducted from the donation of 15 RMB. However, participants may minimize the negative impact of that word on the charity by adding additional letters in the middle right of the paper. The instructions make it obvious to participants that these extra letters are optional. One additional letter reduces the damage to the charity by one-third, two additional letters reduce the damage to the charity by two-thirds, and all three additional letters reduce the damage to the charity to nothing. As a result, participants must choose a trade-off between their private profits and the amount of harm they are prepared to inflict to the charity payout in each round. The current round's cumulative earnings and damage are displayed in the top center of the paper. At the conclusion of the experiment, students were compensated for the amount earned during the activity. The money saved by the participants was then donated to a tree planting charity. The money-incentivized task has the advantage of reflecting participants' real intention to engage in pro-environmental activities.We administered the word game to the participants via paperwork, and one round of a sample game is shown in p = 0.0001. All the p-values for simple main effects, except for those of the effect of message type on people with high construal levels and the effect of construal level when using preventive messages [see Panels A (a)-(d)] on advocating “green” technologies, were significant. The regression results are presented in Panel B. In addition to the demographic characteristics, we also include the productivity indicator we obtained from the practical test to control for the intrinsic word processing ability of the participants. We observe that, on average, presenting preventive/promotional messages about technologies that have mitigating/adapting benefits to people with high/low construals can significantly moderate the donation deduction by 1.084 and 1.921 yuan, respectively. The results are consistent with the hypothesis. The study's design has several advantages, but it also comes with some drawbacks. To begin, we conducted our research on a group of university students, many of whom were living on their own for the first time. This may have influenced their susceptibility to interventions, as people are more inclined to change their habits following a move. This begs the question of whether our intervention's effects can be generalized to more stable household circumstances as well. Nonetheless, this group may be particularly interesting because they represent future environmentalists. Future research should examine the effectiveness of a similar intervention in diverse settings, such as a factory or a household.Another limitation is that participants were aware that they were being monitored throughout the trial in our study. This, of course, could have an impact on their behavior. However, because individuals in different treatment groups received identical information but not interventions, we could account for potential effects associated with the concept of monitoring. Although we randomly assigned participants into different treatment groups to account for potential monitoring effects, it would be interesting to observe if comparable results occur when individuals are unaware they are being monitored.The role of peer effects in decision making has been largely explored in many contexts, such as green product adoption, saving and borrowing decisions  with a targeted messaging method to alter university students' risk compensation behavior. Specifically, we conducted three experiments on mitigating risk compensation behavior in the setting of climate change risk perception and pro-environmental behavior with 1,500 university students from both an online survey platform and a university in Chongqing, China, in 2019. The sample size in our study is substantially greater than comparable studies. For example, Wang et al.  investigThe results demonstrate that highlighting a new technology as playing a preventive/promotional  role related to climate change can mitigate the risk compensation behavior of individuals with high/low mental construals . We provide evidence that this mitigation is driven by the lack of fit between the participants' abstract/concrete thinking and the immediate/long-term solution to climate change. The incongruent combination makes messages less persuasive and allows participants to believe that the technology is not an efficient remedy for climate change. Consequently, this significantly promotes university students' pro-environmental behavior.This paper also has implications for policymakers. The need for measures to address climate change  and efforts to reduce greenhouse gas emissions have been widely acknowledged by scientists and policymakers IPCC, . HoweverThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by School of Finance, Chongqing Technology and Business University. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This research is supported by The National Social Science Fund of China: Research on Mechanism Innovation and Effect Evaluation of Ecological Poverty Alleviation from the Perspective of Targeted Poverty Alleviation (17BJY131).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Scientific Reportshttps://doi.org/10.1038/srep13677, published online 02 September 2015Retraction of: The Editors have retracted this Article.Partial overlap of panels between Figures 2G and 4G, and Figures 2H and 4HDuplication of parts of panels within Figure 4E and 4FDuplication of parts of panels within Figure 2E and 4E, and Figure 4F and 2FThe Editors therefore no longer have confidence in the integrity of the data presented.After publication, it was brought to the Editors' attention that there were irregularities in a number of figures, specifically:Zhaohui Gong disagrees with the retraction of the Article. Lihua Yang, Jie Yang, Jingqiu Li, Xingkai Shen, Yanping Le, Chengwei Zhou, Shaomin Wang, Shun Zhang, and Dazhi Xu did not respond to correspondence in regard to the retraction of this Article.


Adult-onset citrullinaemia type II (CTLN2) is a rare disease in Chinese patients. As a subtype of citrin deficiency (CD), it is an autosomal recessive disease related to the SLC25A13 mutation on chromosome 7q21.3. In this study, we report a case of CTLN2 presenting with paroxysmal altered consciousness and refractory hyperammonaemia. The diagnosis was finally confirmed by gene analysis. The patient recovered after liver transplantation. It can be learned from this case that CD should be considered in patients with refractory hyperammonaemia and paroxysmal mental disorder without a history of liver disease. Metabolic encephalopathies are a group of disorders defined as brain malfunction secondary to systemic metabolic derangements. Hyperammonaemia is one of the aetiologies. As the liver is the main organ of ammonia metabolism, hyperammonaemia is often considered a common complication of chronic liver disease. In fact, any disease involved in the urea cycle can cause hyperammonaemia. Citrin, a liver-type mitochondrial aspartate–glutamate carrier, plays an important role in supplying aspartate to argininosuccinate synthetase in the cytosol to generate argininosuccinate in the urea cycle. Citrin deficiency (CD) can cause urea circulation disorder, which leads to hyperammonaemia. There are two kinds of CD, neonatal intrahepatic cholestasis (NICCD) and adult-onset citrullinaemia type II CTLN2) . In Chin . In Chi2An 18-year-old man with paroxysmal altered consciousness for half a year was admitted to our hospital. The patient complained of episodic delirium, screaming, convulsion, and vomiting every 5–8 days, each time lasting for a few hours before the symptoms spontaneously alleviated. The patients did not provide any precipitating factors before the onset of these symptoms but confessed a preference for foods such as nuts, peanuts, eggs, and meat, as well as an aversion to rice or sweet desserts. The initial local hospital examinations revealed no abnormalities in the brain or abdominal computed tomography but a significant increase in plasma ammonia levels , and a slight elevation of alanine aminotransferase  and aspartate aminotransferase . Tests for EBV, CMV, HAV, HBV, HCV, HIV, thyroid function, ceruloplasmin, markers of autoimmune liver disease, and α-antitrypsin were negative. Data from another hospital 1 month prior also showed a significant increase in plasma ammonia levels (209 μmol/L), and a slight elevation of ALT (77 U/L) and AST (80.15 U/L) levels. Metabolic encephalopathy was diagnosed and treated. As the symptoms recurred with an increased frequency of paroxysmal altered consciousness, the patient was transferred to our hospital.On admission, no abnormalities were found on physical examination. The levels of plasma ammonia (543.9 μmol/L), ALT (88 IU/L), and AST (149 IU/L) were still high. Despite the use of arginine and ornithine aspartate, the patient slipped into a coma without precipitating factors. Both pupils were equally round at approximately 2.5 mm each with a slow light reflex. Active tendon reflexes and positive Babinski signs were elicited and interpreted as the result of cerebral oedema. Mannitol and furosemidum were administered, and the patient recovered consciousness. Brain magnetic resonance imaging (MRI) showed a region of high signal intensity in the bilateral frontal lobes, which indicated the possibility of metabolic encephalopathy or infarction . Urine oSLC25A13 gene analysis showed that the patient was a compound heterozygote for c.851-854del and IVS16ins3kb. His mother had a heterozygous mutation in c.851-854del. Adult-onset citrullinaemia type II (CTLN2) was diagnosed. After conservative treatment including arginine, ornithine aspartate, sodium pyruvate, glutathione, ursodeoxycholic acid, and a low-carbohydrate, high medium-chain triglycerides (MCT) and lactose-free diet, the patient recovered significantly. One month later, the patient received liver transplantation, after which his blood ammonia level dropped to 61 µmol/L. Approximately 1 month after the surgery, brain MRI indicated cerebral atrophy . FortunaConsent for the publication of this case has been obtained from the patient.3+)/NADH ratio through the malate–citrate shuttle and can reduce oxidative stress [CTLN2 is a rare disease in the Chinese population. As a subtype of CD, it is an autosomal recessive disease related to the SLC25A13 mutation on chromosome 7q21.3. Several genotypes of SLC25A13 have been discovered, such as c.851-854del, IVS16ins3kb, IVS6 + 5G > A, and c.1177 + 1G4A . MCT cane stress .Because of the non-specificity of symptoms, CTLN2 is easily misdiagnosed. Elevation of the plasma ammonia concentration is common in CTLN2. Serving as an aspartate–glutamate transporter in the mitochondria, citrin is essential for the urea cycle and NADH shuttle . CD leadIn our case, the patient was very young with a short disease course and rapid progression. Liver cirrhosis and brain atrophy are usually seen in a long disease course. In addition, it is uncommon to find hepatic cirrhosis in a young man with a short duration of CTLN2. Chen reported a case of CTLN2 with a course of 6 months. Brain MRI revealed bilateral symmetric lesions of the cingulate cortex, which disappeared after liver transplantation .4In conclusion, our case indicated that CTLN2 should be considered in patients who present with paroxysmal altered consciousness and refractory hyperammonaemia. Unusual food preferences may provide some clues for the diagnosis. Some conservative treatments may induce a temporary remission of hyperammonaemia and altered consciousness but failed to achieve long-term improvement, and most of the patients finally died of severe brain damage . Therefo


Background: Acromegaly is a chronic disease caused by an abnormal secretion of growth hormone (GH) by a pituitary adenoma, resulting in an increased circulating concentration of insulin-like growth factor 1 (IGF-1). The main characteristics are a slow progression of signs and symptoms, with multisystemic involvement, leading to acral overgrowth, progressive somatic changes, and a complex range of comorbidities. Most of these comorbidities can be controlled with treatment. The literature reveals that the most evident and early signs are those related to soft tissue thickening and skeletal growth, especially in the head and neck region. Methods: The authors reviewed the available literature on the clinical oro-dental features of acromegaly, selecting articles from PubMed and Google Scholar. The aim of this review was to summarize all the reported clinical oro-dental features of acromegalic patients. Results: The most common facial dimorphisms involved the maxillo-facial district, with hypertrophy of the paranasal sinuses, thickening of the frontal bones, and protruding glabella, which may be associated with joint pain and clicks. Regarding the oro-dental signs, the most frequent are dental diastema (40–43%), mandibular overgrowth (22–24%), mandibular prognathism (20–22%), and macroglossia (54–58%). These signs of acromegaly can be significantly reduced with adequate treatment, which is more effective when initiated early. Conclusions: Increased awareness of acromegaly among dentists and maxillo-facial surgeons, along with the early identification of oro-facial changes, could lead to an earlier diagnosis and treatment, thereby improving patients’ quality of life and prognosis. Acromegaly is a rare chronic disease caused by an excessive secretion of growth hormone (GH), mostly due to an adenoma of the anterior pituitary gland and resulting in an increased circulating concentration of insulin-like growth factor 1 (IGF-1), the main effector of GH activity. It is characterized by a slow progression of signs and symptoms and multisystemic involvement, leading to physical alterations, progressive somatic changes, and a complex range of systemic comorbidities ,7,8,9,10The somatic feature changes are slow and often cause a delay in diagnosis; facial dysmorphism, oro-dental signs, enlargement of extremities, and soft tissue thickening are the most common and earliest manifestations of acromegaly ,14,15,16Focusing on facial changes and oro-dental alterations, these manifestations, in particular, the development of malocclusion due to mandibular prognathism, are associated with reduced self-esteem and decreased quality of life (QoL) ,25, and The authors reviewed the available literature and selected articles using PubMed, Web of Science and Google Scholar. The following keywords were entered separately and in combination: “acromegaly,” “oral manifestations,” “dental manifestations,” “dental manifestation,” “oro-dental acromegaly,” “facial manifestation of acromegaly,” “dentist,” “dentist acromegaly,” and “orthodontics.”All articles published between 1950 and 2021 were considered for inclusion. We included only papers published in English. We excluded case reports and papers for which only the abstract was available. The literature was analyzed specifically for the collection and rationalization of all the reported clinical oro-dental features shown by the acromegalic patients. The aim of this review was to collect all the clinical oro-dental features observed and reported in acromegalic patients.Acromegaly prevalence ranges from 20 to 130 people per million inhabitants ,13,14,15The most frequent signs and symptoms of acromegaly are: menses abnormalities, physical changes , headache, paresthesia, impaired glucose metabolism, erectile dysfunction, arthropathy, hypertension, fatigue, daytime sleepiness, and increased perspiration . SymptomThe suspicion of acromegaly in a subject is based on the clinical manifestations and it can be confirmed with testing. The standard blood tests include IGF-1 levels and GH nadir after an oral glucose tolerance test. Imaging of the pituitary gland and magnetic resonance or computerized tomography (if MR is not applicable) are also used to confirm diagnosis. According to the guidelines, screening for excess of GH and IGF-1 is important in patients with clinical features of acromegaly associated with multimorbidity  who are unresponsive to treatment. Similarly, if comorbidities that cause GH excess have an earlier than usual onset or if these comorbidities are associated with a pituitary mass , patients should also be tested for acromegaly.Treatment of GH pituitary adenoma includes different therapeutic approaches, such as neurosurgery (the first-line treatment), medical treatments , and radiotherapy in select patients. Therapeutic goals in patients with acromegaly are the biochemical control of GH and IGF-1 secretion, the control of systemic comorbidities, the reduction of mortality, and the restoration of QoL.Among acromegaly symptoms, oral manifestations and maxillo-facial features are reported by most patients to have been present up to 10 years before diagnosis, preceded only by increased size of hands and feet. These manifestations may also be present in the later phases of the disease depending on duration of active disease, entity of diagnostic delay, and GH and IGF-1 secretion levels. Mandibular growth, reported in 22–24% of patients, can lead to prognathism in 20–22% of these patients and the development of a class III dental and skeletal pattern , panel AMacroglossia is seen in 54–58% of acromegaly patients ,6,19,22;Acromegaly patients can also develop a dental malocclusion , panel CRegarding dental elements, acromegaly patients may report diastemata , panel ESome studies also describe an increased mineralization of the alveolar bone at the level of the roots of the molar elements, which outlines the features of hypercementosis ,24,28,29The effects of the disease on the alteration of gingival tissues and on the development of periodontal and periradicular pathologies are still being debated, and the link with the disease remains questionable . Some auAt the palatine and mandibular level, a study  identifiClinically, not only dental components are modified by GH and IGF-1 excess but also soft tissue may be involved in facial changes due to acromegaly. In fact, it has been found that acromegalic patients commonly have enlarged submandibular glands (regardless of the activity of the disease) ,32,33, tFinally, hypertrophy of the paranasal sinuses, especially of the frontal ones, may also occur. This aspect, together with laryngeal hypertrophy, can cause a deepening and sound resonance of the patient’s voice ,6,9.These oro-facial alterations in acromegaly patients may be associated with pain in the mouth, especially in the maxillo-facial area  and joinBiochemical treatment for the limitation of GH hypersecretion can only partially improve facial appearance, especially regarding soft tissues, but in most cases, oro-facial changes are not completely reversible and persist despite pharmacological treatment ,25. TablSome studies were also conducted using teleradiography examination to capture the cephalometric characteristics of acromegalic patients. The results showed that these patients generally report an increase in overall facial height and in the length of the cranial bases, an increase in the gonial angle, and an increase in the size of the branch  and of tAccording to the literature, orthopantomography does not play a significant role in identifying the pathognomonic signs of the disease. It has been reported that only the panoramic and intraoral radiographs of these patients can reliably show radiopaque lesions at the level of the roots of the molar elements, defined as “hypercementosis” ,29,43,47Nevertheless, clinicians should pay attention to these aspects when reading the results of radiographic examinations. Dentists and orthodontists  are adviAbout 80% of acromegaly patients present with oro-dento-facial signs, such as dental diastema, mandibular overgrowth, mandibular prognathism, and macroglossia. Dentists, orthodontists, and dental hygienists are in an ideal position to detect these signs early in acromegaly patients, which can lead to earlier diagnosis and improved prognosis. Moreover, with early diagnosis and subsequent appropriate endocrinological treatment earlier in the disease course, these signs can be significantly reduced. This would significantly impact patients’ QoL and self-esteem. It should be noted that even if the signs are not reduced by endocrinological treatment, they can be addressed with specific oral and maxillo-facial treatments.For these reasons, oro-facial alterations, as distinctive and early signs of acromegaly, should be considered a primary target to be identified and evaluated properly. It is imperative to increase awareness among specialists, including dentists, orthodontists, and dental hygienists, with the specific goal of recognition of the pathology and earlier diagnosis of the disease. This would result in earlier initiation of treatment, which has been shown to improve patient outcomes in a myriad ways.


Stem cell therapy is one of the novel and prospective fields. The ability of stem cells to differentiate into different lineages makes them attractive candidates for several therapies. It is essential to understand the cell fate, distribution, and function of transplanted cells in the local microenvironment before their applications. Therefore, it is necessary to develop an accurate and reliable labeling method of stem cells for imaging techniques to track their translocation after transplantation. The graphitic quantum dots (GQDs) are selected among various stem cell labeling and tracking strategies which have high photoluminescence ability, photostability, relatively low cytotoxicity, tunable surface functional groups, and delivering capacity. Since GQDs interact easily with the cell and interfere with cell behavior through surface functional groups, an appropriate surface modification needs to be considered to get close to the ideal labeling nanoprobes. In this study, polyethylene glycol (PEG) is used to improve biocompatibility while simultaneously maintaining the photoluminescent potentials of GQDs. The biochemically inert PEG successfully covered the surface of GQDs. The PEG-GQDs composites show adequate bioimaging capabilities when internalized into neural stem/progenitor cells (NSPCs). Furthermore, the bio-inertness of the PEG-GQDs is confirmed. Herein, we introduce the PEG-GQDs as a valuable tool for stem cell labeling and tracking for biomedical therapies in the field of neural regeneration. Stem cell based therapy is currently a promising and advanced research field that attracts attention as a critical player for the treatments of intractable diseases, such as cancer, neurological disorders, cardiac diseases, diabetes, and liver diseases ,2. ThereQuantum dot (QD) is a promising solution for more capable bioassay and bioimaging technology. QDs emerged as a result of the discovery that the size of the nanomaterials determines the properties of QDs ,9. In te2 or OH functionalized ones. However, all three different surface chemistry influenced the biological pathway, such as phosphorylation [Recently, the cytotoxicity related to the surface functional group and the size which influences the biological effects and the uptake of GQDs were addressed. Weihu Shang et al. showed that GQDs smaller than 10 nm demonstrated direct and easy penetration into stem cells without affecting their viability, proliferation, or differentiation capacity . Furtherrylation . Thereforylation , drug derylation ,26, and rylation . PEG funrylation ,29,30,31rylation ,33,34.For these reasons, the PEG-GQDs were fabricated and investigated as a platform of the theranostic agents for the vulnerable and fragile nerve tissues. The GQDs were fabricated by the graphite intercalation compounds mediated exfoliation method and the PEGylation proceeded on the GQDs’ surface through the solvothermal method. The size, surface charge, surface functional groups, and photoluminescence property of GQDs before and after PEGylation were investigated. Then, the experiments that dealt with the concentration dependant cytotoxicity established the available dose of material when the fetal rat NSPCs were employed. Moreoever, it was confirmed that the PEG-GQDs are well internalized into the cell. Finally, the presence of the PEG-GQDs’ influences on cell differentiation was closely investigated. Through this study, a method to utilize the low-cytotoxic, bio-inert, and bioimaging nanoparticle is proposed.4H4O6·4H2O, Sigma Aldrich, Burlington, MA, USA) precursor was introduced in the graphite by mixing and the exfoliation proceeded at 250 °C for 24 h. The size distribution of the GQDs was controlled by sieving with a dialysis membrane . The particles were dried for several days under vacuum conditions. The surface functional groups of GQD were reacted with polyethylene glycol (PEG-bis(amine), MW: 2000, Sigma Aldrich, Burlington, MA, USA) via an amide bond [The GQDs were fabricated by the graphite intercalation compounds (GICs) method described previously ,36,37. Bide bond . The aqu−1) analyses were conducted.X-ray diffractometer , dynamic light scattering and zeta potential , X-ray photoelectron spectroscopy , transmission electron microscopy , Fourier transform infrared spectroscopy . The amount of 100 µL PEG-GQDs was added to the 96-well plate and fluorescence images were taken by a fluorescence microscope .5 rNSPCs was seeded into a 6 cm low-attachment culture dish and incubated in a 37 °C, 5% CO2, and 100% humidity incubator. Every 2 days, half of the media was replaced with a fresh one. Cells were passaged once a week when the neurospheres were larger than 200 µm in diameter. For consistency, cells between passage 2 and 5 were used in this study.Neural stem/progenitor cells (NSPCs) were prepared using a previously reported method ,40. The The complete growth media contains DMEM: F12 , L-GlutaMAX , 2% B27 minus vitamin A , 1% Penicillin/ Streptomycin , 20 ng/mL epidermal growth factor , and 20 ng/mL basic fibroblast growth factor .The differentiation media contains Neurobasal plus medium , 2% B27 Plus Supplement , 0.5 mM L-GlutaMAX, and 1% Penicillin/Streptomycin.For identification of the differentiation potential of rNSPCs, cells were firstly seeded in the 96-well plate and incubated for 24 h under the growth media. Then, the growth media was changed into differentiation media to induce the differentiation of rNSPCs and cells were cultured for another 7 days. The media was changed every 2–3 days.4 rNSPCs in 100 µL growth media was seeded in a precoated 96-well plate and permitted to grow for 24 h. Subsequently, different concentrations of PEG-GQDs  were added to the cells. Twenty-four hours later, after discarding the supernatant, cells were carefully washed once with warm PBS and treated with CCK-8 reagent  diluted with prewarmed media and incubated for another 2 h. Then the absorbances were measured with a microplate reader at 450 nm. For detecting the viable cells, the LIVE/DEAD™ Viability/Cytotoxicity Kit  was used. The cells were incubated with Calcein AM (2 µM) and Ethidium homodimer-1 (4 µM) and were diluted with prewarmed DMEM: F12 media for 30 min at 37 °C. Before capturing, cells were washed once with prewarmed DMEM: F12 media. Images were captured with a fluorescence microscope  at 40×.For the culture of NSPCs, the plates were coated with 1% Matrigel , a good basement membrane biomaterial. In detail, the original Matrigel was diluted to 1% with ice-cold DMEM/F12 media. The amount of 100 µL of 1% Matrigel solution was added to the 96-well plate and the plate was then placed into an incubator at 37 °C for 1 h. Before seeding cells, the plate was gently washed once with DMEM/F12 media. The amount of 1 × 104 cells in 500 µL growth media) in a 24-well plate and incubated for 24 h. PEG-GQDs were added to the cells at a concentration of 320 µg/mL. After 24 h of incubation, the supernatant was removed and cells were washed twice with PBS to remove residual PEG-GQDs. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA)  for 30 min, following three times of PBS washing. For better observation of cell morphology, Alexa Flour 488 Phalloidin  solution was added to cells and incubated for another 30 min at room temperature. After washing it three times in PBS, the fluorescence images were captured using a Zeiss LSM 700 confocal microscope .To check the visualization ability and intracellular location of PEG-GQDs, confocal laser-scanning microscopy  was used. The rNSPCs were seeding on the 1% Matrigel pre-coated 15 mm cover-glasses , and then resuspended in cell staining buffer . The uptake of PEG-GQDs into rNSPCs was analyzed by fluorescence-activated cell sorting (FACS) analysis , based on the blue fluorescence signals of PEG-GQDs.The rNSPCs were seeded in a 24-well plate at 1.25 × 10For identifying the stemness and differentiation potential of primary culture rNSPCs, immunocytochemical experiments were performed and the data are shown in n = 15). The total cell numbers were quantified by Hoechst staining.For quantitative analysis, Tuj1 and GFAP-positive cells were manually quantified in a visual field at 400× (p < 0.05) between groups.All results are presented as means ± standard deviations (SD). For all assays, one-way ANOVA and Tukey’s comparison tests were performed to determine possible significant differences , sp3 (~284.8 eV), and C–O or C–OH (~286.6 eV); and functional groups amide (N–C=O) (~288.7 eV), carboxyl (COOH), or ester (~290 eV), respectively. The binding energy of GQDs presented a predominant peak near 284 eV. This is close to the sp2 binding energy with an asymmetric shape, which is preferably derived from graphite or graphene samples. After the PEG coating, the portion of sp3 was increased. Moreover, the peak of the PEG and amide group was revealed at around 286.6 and 289 eV. These XPS results confirm the presence of PEG on the GQDs after successful encapsulation.The XPS spectra of carbon 1s were investigated to obtain more information about the surface functional groups. As shown in −1; C=C, C=O stretching, ester, aliphatic COOH, and conjugated C=O ~1600 cm−1; C-H stretching ~3000 cm−1; and O–H stretching ~3500 cm−1, respectively. After surface modification, the distinctively changed locations were highlighted. The C–O stretching epoxide peak at 1024 cm−1 appeared prominently in PEG-GQDs. The ratio between C-H stretching band (~2880 cm−1) and the C–O stretching band (1130 cm−1) was also changed after the PEGylation. On the surface of PEG-GQDs, the ratio of the C–H/C–O was lowered and the carboxyl groups at 1660 cm−1 completely disappeared. These results are evidence of the successful modification of the PEG layer on the GQDs.This result was also crosschecked by the FT-IR spectra analysis and is shown in In summary, it was confirmed that the PEG modification successfully proceeded on the GQDs surface. It is expected that they could increase the utilization of nanoparticles as a bio labeling probe without an accumulation of unwanted locations caused by nonspecific binding on the cell and proteins and simultaneously reduce the biological interaction than the pristine GQDs.The samples were prepared at various concentrations in the DW to investigate the visualization ability of the PEG-GQDs. The images of PEG-GQDs were captured with a fluorescence microscope with three different wavelengths. As shown in In neural regeneration, neural stem/progenitor cells (NSPCs) are one of the major cell types for stem cell based therapies because the NSPCs’ self-renewing and multipotent characteristics can generate the critical phenotype cells of the nervous system such as neurons, astrocytes, and oligodendrocytes ,45,46,47After seeding rNSPC for 24 h, various concentrations of PEG-GQDs  were added and then incubated with cells for an additional 24 h. As shown in Fluorescence-positive cells were sorted by performing FACS analysis to quantify the rate of intracellular uptake ratio of PEG-GQD in rNSPC with respect to incubation time. Percentages of blue fluorescence-positive cells were then calculated based on the FACS analysis. As shown in The differentiation potential of rNSPCs with internalized nanomaterials is an essential characteristic for stem cell based therapy that should not be changed by the containing material for further bio applications. To investigate whether PEG-GQDs impair the differentiation potential of rNSPCs, we induced a differentiation process of rNSPCs. After 7 days of differentiation, neuron-specific class III beta-tubulin (Tuj1) and glial fibrillary acidic protein (GFAP) were examined using immunocytochemistry. As shown in The successful PEGylation did not interfere with the photoluminescence properties of GQDs while passivating the surface from other reactive substances. The assays related to the biological effects on rNSPCs showed that the PEG-GQDs exhibited excellent biocompatibilities, bioimaging property without weakening the rNSPCs’ activity, and differentiation ability under concentrations up to 320 µg/mL. Moreover, the PEGylation of GQDs may extend the in vivo circulation time and improve the penetration efficiency to nerve tissue, leading to enhanced bio-imaging, delivering, and therapeutic effects to the nerve tissue injury sites.It was confirmed that the interaction between PEG-GQDs and rNSPCs may provide some useful information for future explorations of a biocompatible and visible on-target probe for the different therapies for neurological disorders. The uniform-sized, water-soluble, and light-emitting GQDs are successfully combined with the PEG that is used to enhance the colloidal stability with a hydrophilic surface modification. Our approach takes advantage of the photoluminescence property of GQDs without distinct losses and takes advantage of the non-reactive hydrophilic surface of the PEGs. The synthesized material showed no specific cytotoxicity up to a concentration of 320 µg/mL and exhibited an ability to be imaged when the material was internalized inside the cells. The embryonic rNSPCs that internalized the substances were investigated on whether they sustain their differentiation ability. The result showed that the differentiation potentials of rNSPCs were not adversely affected by PEG-GQDs.In conclusion, PEG-GQDs showed proper bio labeling ability and biocompatibility without attenuation of rNSPCs differentiation, suggesting that PEG-GQDs are useful material tools at the visible nano-delivery system for the regeneration therapies against neurological disorders.


There is an increasing recognition of the importance of sex in susceptibility, clinical presentation, and outcomes for heart failure. This review focusses on heart failure with reduced ejection fraction (HFrEF), unravelling differences in biology, clinical and demographic features and evidence for diagnostic and therapeutic strategies. This is intended to inform clinicians and researchers regarding state-of-the-art evidence relevant to women, as well as areas of unmet need.Females are well recognised to be under-represented in clinical trials, but there have been some improvements in recent years. Data from the last 5 years reaffirms that women presenting with HFrEF women are older and have more comorbidities like hypertension, diabetes and obesity compared with men and are less likely to have ischaemic heart disease. Non-ischaemic aetiologies are more likely to be the cause of HFrEF in women, and women are more often symptomatic. Whilst mortality is less than in their male counterparts, HFrEF is associated with a bigger impact on quality of life in females. The implications of this for improved prevention, treatment and outcomes are discussed.This review reveals distinct sex differences in HFrEF pathophysiology, types of presentation, morbidity and mortality. In light of this, in order for future research and clinical medicine to be able to manage HFrEF adequately, there must be more representation of women in clinical trials as well as collaboration for the development of sex-specific management guidelines. Future research might also elucidate the biochemical foundation of the sex discrepancy in HFrEF. Heart failure (HF) continues to increase in prevalence in our community in men and women, estimated to affect 64.34 million people globally . The preThere has been a growing need, and widespread call, for increased representation of women in clinical trials, for studies to be powered for sex disaggregated analyses, and for guidelines to consider and reflect sex differences. Here, we review the most recent studies and literature and summarise the sex differences in demographics, mechanism, clinical presentation, biomarkers, outcomes, clinical pathways, management and representation in the literature for HFrEF.In addition to referring to key early studies in the field, we systematically searched databases on Embase using the search strategy (HF with reduced ejection fraction OR HFrEF) and , limited to the English language, humans and the years 2016 to 2021. After removing duplicates, there were 488 results. After abstracts were screened, there were 42 studies and after reading the full texts, 33 were relevant to this review, with key themes summarized in  –7. They New data from the UK has found that women are more obese at presentation, and this obesity portends worse outcomes in women compared to men . This maThere are key sex differences in the underlying mechanisms contributing to HFrEF, as well as differences in the susceptibility to each and the relative contribution to the burden of HFrEF.Whilst a driving precipitant of HFrEF in males is macrovascular coronary artery disease, myocardial ischaemia and infarction , HFrEF iThere are some HF aetiologies that are unique to women, such as peripartum cardiomyopathy, with risk factors including advanced maternal age, and pre-eclampsia . AdditioIn patients with underlying myocardial dysfunction and cardiomyopathy, inflammation is increasingly recognised as playing a key role in a sex-specific manner. Sex discrepancies have recently been demonstrated in antibody-mediated immune response on cardiac remodelling in HFrEF, affecting progression of HF. We know that IgG can be found in the myocardium in patients with end-stage HFrEF associated with ischaemic heart disease (IHD). One study indicated that, in the early stages of remodelling, IgG1 and IgG3 levels differ between men and women . FurtherTakotsubo cardiomyopathy is also more common in postmenopausal women. Whilst classically understood to be an acute and reversible condition, there is increasing appreciation of longer-term myocardial abnormalities and many patients continue to suffer symptoms which may be attributed to the recovery process , 17.More evidence is emerging on the impact of sex hormones on sex discrepancy in HFrEF. There is increasing research on the protective effects of oestrogen on HF. Additionally, an oestrogen depletes postmenopausal state may contribute to women having higher left ventricular systolic and diastolic stiffness when compared to men as oestrogen is involved in blood pressure and arterial tone regulation . FurtherIn terms of presenting features of HFrEF, most recent data concurred with older data , 21 showEchocardiography analysis highlights that women have higher ejection fractions with smaller left atria, higher longitudinal strain and higher circumferential strain , 22. AsiAs coronary microvascular and macrovascular dysfunction have similar risk factors, unique biological risk factor profiling is a promising method of early diagnosis . CurrentBiomarker identification in HF could identify potential targets for HF prevention in higher-risk individuals . One bioImportantly, data from 12 years’ worth of follow-up, after adjustment of risk factors, showed that a more androgenic hormone profile among post-menopausal women was associated with increased risk of HF . Whilst International guidelines such as those of the European Society of Cardiology  or the ANumerous recent studies have demonstrated that women are less likely to receive evidence-based medical therapy for treatment of HFrEF compared to their male colleagues , 6, 34. Differences in tolerability and efficacy between drug therapies provide evidence that women require different guidelines for medical therapy of HFrEF , 38. ForAdvances in electric and mechanical devices have continued with substantial benefits for patient symptoms, hospitalisation and outcomes, with strong evidence in both sexes  Table 11. The raThere is strong evidence, reflected in the guidelines, for cardiac rehabilitation (including education), and lifestyle modifications including salt reduction, weight loss, and exercise (strong evidence) and a whole grain plant-based diet (less evidence) , in imprp = 0.001), and this survival difference was not observed in men [The literature reports on multiple outcomes in HF, exploring mortality, quality of life, rates of hospitalisation and readmission, susceptibility to arrhythmia, and recovery of LV function. The previous body of evidence found no difference in-hospital mortality between sexes, which also correlated with several recent studies , 44. A fd in men .Previous literature had described that females with HF report lower quality of life, and this was reiterated in new data . DespiteRegarding rates of hospitalisations, most data, including more recent studies, showed that women have a lower risk of hospitalisation . Only onp = 0.04) [Although many studies have explored predictive factors for HF with recovered ejection fraction, few have sought to understand whether there is a gender discrepancy. More recent data has identified a link between female sex and left ventricular ejection fraction recovery –54. One  = 0.04) . Since p = 0.04) .An important consideration in the interpretation of the literature in regard to heart failure outcomes is the often-poor stratification of analyses and reports by acute versus ambulant or chronic heart failure, with less data available regarding sex differences in the acute heart failure setting. Data that is available suggests that, whilst co-morbidities at the time of presentation differ between the sexes, acute in-hospital outcomes are similar. For example, in the ALARM-HF multinational survey of over 4000 patient hospitalizations, men were more likely to have acute coronary syndrome as the precipitating event and underlying coronary artery disease, and women were more likely to have concomitant AF, obesity, anaemia and depression. In hospital, mortality, however, was similar in both sexes (11%) . This coFour studies closely analysed and quantified female representation in trials. Female participation in landmark HF trials ranged from 0 to 40% with an average of 20% . In an aUnder representation of females in clinical trials and lack of sex-disaggregated data means studies are often underpowered to apply results to clinical practice. For more than 25 years, there has been an acknowledgement of the sex discrepancy in HFrEF , and desThe sex differences in HF with reduced ejection fraction are undeniable. The pathophysiology, types of presentation, morbidity and mortality differ between the sexes. However, without more representation of women in clinical trials, the development of sex-specific management guidelines appears improbable.


Heart disease is the leading cause of death in both men and women in developed countries. Heart failure (HF) contributes to significant morbidity and mortality and continues to remain on the rise. While advances in pharmacological therapies have improved its prognosis, there remain a number of unanswered questions regarding the impact of these therapies in women. Current HF guidelines recommend up-titration of neurohormonal blockade, to the same target doses in both men and women but several factors may impair achieving this goal in women: more adverse drug reactions, reduced adherence and even lack of evidence on the optimal drug dose. Systematic under-representation of women in cardiovascular drug trials hinders the identification of sex differences in the efficacy and safety of cardiovascular medications. Women are also under-represented in device therapy trials and are 30% less likely to receive a device in clinical practice. Despite presenting with fewer ventricular arrythmias and having an increased risk of implant complications, women show better response to resynchronization therapy, with lower mortality and HF hospitalizations. Fewer women receive advanced HF therapies. They have a better post-heart transplant survival compared to men, but an increased immunological risk needs to be acknowledged. Technological advances in mechanical circulatory support, with smaller and more hemocompatible devices, will likely increase their implantation in women. This review outlines current evidence regarding sex-related differences in prescription, adherence, adverse events, and prognostic impact of the main management strategies for HF. Men and women have the same risk of developing heart failure (HF) throughout life. However, it is well-known that women develop the disease later in life. In addition, women have a higher prevalence of HF with preserved ejection fraction (HFpEF), the prevalence of which increases with age. This may partly explain the under-representation of women in pharmacologic and device therapy trials designed to treat HF with reduced EF (HFrEF) .Sex based differences in pharmacokinetics and pharmacodynamics of pharmacological agents may explain the variable effects in men and women. However, given the smaller number of women included in clinical trials of HFrEF, where they represent less than one-third of the study population, we do not have accurate information. Unfortunately, the results of large clinical trials are often not analyzed separately by sex and we only have subgroup analyses so they cannot be fully extrapolated to women . The samThere are important sex-dependent differences in pharmacokinetics (PK) and pharmacodynamics (PD) that need to be acknowledged to understand how specific cardiovascular drugs can affect women and men differently. The differences can affect absorption, metabolism, distribution, and elimination.For orally administered drugs, two main factors need to be acknowledged: compared to men, women (1) produce less gastric fluid, which can lead to a decrease in the absorption of weak acids and an increase in the absorption of weak bases and (2) have longer intestinal transit time , 4. The Total body water is greater in men, while women have a higher proportion of adipose tissue. Therefore, distribution volume for hydrophilic or lipophilic drugs varies according to sex.Plasmatic proteins involved in drug transport can be modulated by estrogens, resulting in a sex-dependent distribution , 6.Lower hepatic flow in women, sex-dependent activity of metabolic enzymes, increased proportion of adipose tissue and lower basal metabolic rate can explain differences in drug metabolism , 7, 8.In general, glomerular filtration, tubular secretion, and tubular reabsorption are higher in men , howeverLiver enzyme activity decreases in presence of elevated female hormone levels which may decrease drug elimination. Therefore, metabolism can change throughout the menstrual cycle, during pregnancy, with oral contraceptives intake or after menopause .An increased risk of death in women was reported in the DIG trial. Although it may have been related to higher digoxin levels in women, it could not be proven since digoxin levels were available in less than one third of the study patients .Women have higher plasma levels of beta-blocker (BB) due to decreased renal clearance (Cl) and smaller distribution volume (Vd) . Despitemax) and area under the curve (AUC) were found in women, the differences disappeared when adjusted for weigh (Sex-based differences have not been identified on the antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEI), angiotensin-receptor blockers (ARB) and aliskiren . Althougor weigh .The potential effects of age and sex on the PK of Sacubitril/Valsartan were assessed in a study that enrolled 36 subjects, 50% male and 50% female: No sex-dependent differences were found in PK .max and AUC of torsemide are 30–40% higher in women due to reduced elimination . But safety and efficacy studies suggest the importance of dose adjustment based on body weight. In a DOAC meta-analysis including 66,389 patients (37.8% women), DOACs were associated with a significantly lower risk of major bleeding in women compared to men  and a higher risk of stroke and systemic embolism compared with men   established guidelines for the inclusion of women and minorities in clinical research. They recommend that clinical trials should enroll equal numbers of men and women in order to understand sex differences. Shortly thereafter, Congress approved these recommendations, and they became law. The Food and Drug Administration (FDA) published another regulation requiring detailed information by sex in clinical trials investigating new drugs, and therapies .Clinical trials however, unfortunately, remain underpowered to identify statistically significant treatment effects in both sexes.A recent study assessed the enrollment of women in 36 cardiovascular trials evaluating different drugs approved by FDA from 2005 to 2015. Adequacy between the percentage of women included in the trials and the prevalence of the female sex in the disease studied, was evaluated using the participation to prevalence ratio (PPR). A relationship between 0.8 and 1.2 was considered to reflect a good representation of women population. It should be noted that in the 3 HF trials included in this study women inclusion ranged from 22 to 40%. The overall PPR was 0.5, reflecting an inclusion of women in the trials well below their prevalence of the disease. More recent trials show the same pattern, ranging from 21 to 29% inclusion of women .In heart failure (HF) clinical trials, women represent approximately a quarter of patients with HFrEF and over half of those with HFpEF. However, epidemiologic data demonstrate a much higher proportion of women suffering the disease in the real world , 23 Fig.The differences between real world proportion of women with heart failure and their representation in clinical trials may depend on a variety of factors: more comorbidities, older age in women, fulfilling exclusion criteria more frequently, lower proportion of HFrEF, less investigator counseling or less personal availability, and willingness to enroll.The first clinical trials with ACEI date back to the late 1980s. A sub-analysis of the SOLVD trial  revealedWhen large trials of ARB in HFrEF populations  explored sex-specific treatment effect they found no differences in mortality or HF hospitalization between women and men . ELITE Ip < 0.0001). Conversely, there was no survival difference between ACEI or ARB in men   and 9,475 men , showed that women on ARBs had a better survival than those on ACE inhibitors, with a 31% relative risk reduction in all-cause mortality  .Hormone effects on angiotensin II receptor expression or differences in adverse events may explain the potential superiority of ARB in women.The studies assessing the role of mineralocorticoid receptor antagonist (MRA) in HFrEF (spironolactone in RALES and eplerenone in EPHESUS), showed no sex differences in prognosis , 31. In p = 0.051)  .The PARADIGM-HF trial, showed superiority of sacubitril/valsartan compared to enalapril at reducing mortality and HF hospitalization in patients with HFrEF . In subgp = 0.017)  compared to men 1.03  , primariIn conclusion, PARAGONF_HF subanalysis suggest that sacubitril-valsartan may lead to greater reduction in HF hospitalizations in women with HFpEF.post-hoc pooled analysis confirmed similar and significant benefits of BB  on combined end-point of all-cause mortality and all-cause hospitalizations in both women and men  in sinus rhythm with heart rate >70 bpm, showed a reduction in the composite primary outcome of CV death or hospital admission for worsening HF. Subgroup analyses did not show any sex-differences in efficacy or safety of ivabradine .In the last 6 years, several large cardiovascular outcome trials evaluated the effect of iSGLT2 in patients with type 2 diabetes and established cardiovascular disease or those with high cardiovascular risk, they have consistently shown to reduce the risk of hospitalization for heart failure –49.p interaction ≥ 0.17).A meta-analysis of SGLT2i including patients with type 2 diabetes enrolled in the EMPA-REG OUTCOME, CANVAS Program, DECLARE TIMI-58, and CREDENCE trials, showed  no sex dRecently, a meta-analysis condensing two single large-scale trials  in patients with HFrEF with or without diabetes assessing the effects of SGLT2i on cardiovascular outcomes have been published. SGLT2i reduced hospitalizations for HF and death, with an improvement in renal outcomes, regardless of sex and other conditions such as age, diabetes status, or baseline heart failure medications .The effects of diuretics on mortality and morbidity in chronic heart failure have not been studied in large clinical trials. There are no reported sex-related differences with diuretic therapy. Observational studies have shown a relationship between diuretics dose and mortality risk, which was maintained after adjusting for sex .p = 0.011) and no excess mortality in women at serum concentrations between 0.5 and 0.9 ng/ml, whereas serum concentrations ≥1.2 ng/ml was harmful .In the DIG trial, digoxin was associated with a significantly higher risk of death among women , with no increased risk in men . SubsequOverall, whereas higher digoxin levels tend to increase mortality in women, low concentrations seem to be safe and associated with improved symptoms.The A-HEFT trial enrolled more than 5,000 black women  with moderate to severe heart failure (NYHA class III-IV)  to test Treatment with hydralazine and isosorbide showed a significant reduction in mortality, first heart failure hospitalization, and change in quality of life at 6 months, with no differences between men and women.Women are known to have an increased adverse reaction (AR) to cardiovascular drugs compared to men (1.5–1.7-fold)  and haveWomen experience greater electrolyte imbalance with diuretic use, which in turn increases the arrhythmic risk. For instance, women have an increased risk of drug-induced torsades de pointes (2.3-fold) related to a longer corrected QT interval induced by the effects of estradiol on potassium and calcium channel modulation .A post hoc analyses of the DIG study  showed aWomen present higher plasma levels of beta-blocker due to a lower distribution volume  and a slower clearance. Dosage needs to be adjusted according to these differences to prevent AR.An increased risk of angioedema and cough (2-fold) has been described in women . MoreoveNo significant sex-differences in risk of kidney impairment, hypotension, or hyperkalemia have been described with the use of Losartan .There seems to be a higher withdrawal rate in men due to the appearance of gynecomastia (seen in 5.3% of the men) .Implantable Cardioverter Defibrillators (ICD) have shown to reduce sudden death risk in heart failure patients with reduced ejection fraction, especially of ischemic etiology. Therefore, they have a class 1A indication according to current guidelines for primary prevention in patients with left ventricular ejection fraction (LVEF) <35% despite optimal medical therapy .p < 0.001) were offered ICD implantation. Of note, the same proportion of men and women underwent the implant once it was advised . In another observational study 32.2 per 1,000 men and 8.6 per 1,000 women received ICD therapy. After controlling for demographic variables and comorbidity, men were 2.44 (95% CI 2.30–2.59) times more likely to receive an ICD compared to women.These recommendations are based on classical studies, such as SCD-HeFT , MADIT IThe reduced rate of ICD implantations in women may be related in part to the controversies regarding efficacy and higher risk of complications in women compared to men.p ≤ 0.001) compared to men and no significant benefit on mortality. In a European study . The reasons for the differences in the complication rate are unclear but could be related to delayed presentation or greater severity of illness. Smaller vessel size and a thinner walled right ventricle may explain a higher rate of pneumothorax or perforation. Increased bleeding risk have also been reported in women.ICD related complications have been reported more frequently in women. In the Ontario ICD Database  women weOverall, studies show sex differences in arrhythmic risk and ICD-related complications. Nevertheless, there is a risk of sudden death in women with HF and reduced LVEF that could be prevented by ICD implantation. Careful and individualized assessment is required to identify patients that would benefit the most from this therapy.Cardiac Resynchronization Therapy (CRT) has shown to improve functional capacity and survival amongst patients with LVEF <35%, left bundle block >130–150 ms and NYHA functional class II-IV and therefore has a class 1A indication in current HF guidelines . Severaln = 752, 28% female) and COMPANION  trials demonstrated similar reduction in mortality and time to hospitalization in both sexes after CRT implant. In the MASCOT  women showed better left ventricular remodeling and lower mortality and HF hospitalizations after adjustment for cardiovascular risk factors. Remarkably, women showed wider QRS and smaller left ventricle size at enrollment. In another study that only included patients with non-ischemic cardiomyopathy  CRT response among women was greater  than in men, despite similar baseline QRS duration (p = 0.025) and showed a trend toward lower heart failure hospitalization. In a metanalyses , QRS duration was the only independent predictor of CRT benefit. Further analysis showed the benefit was even more significant at lower height. There was a higher proportion of women amongst the wider QRS and shorter patients.The CARE-HF   mainly related to pneumothorax, infection or bleeding. The main risk factor for complications seemed to be size and body mass index both in women and men.As we discussed for ICD, complication rate seems to be higher in women after CRT implant. In the MADIT-CRT trial, women were twice as likely as men to experience a major procedure-related adverse event  female sex was associated with an increased risk of first neurological event , with no difference in other complications. In a later paper focusing on stroke rates during support with continuous-flow LVAD, female sex was also a predictor of stroke . The same was reported in an analysis of more than 900 HeartMate II outpatients  is the therapy of choice to improve survival in patients with end-stage HF. Mean survival after HT nowadays is 12.5 years for the adult population and 12–21 years for the pediatric population . Rejectip < 0.001)  .Women are younger than men at the time of listing (mean 48 year for women vs. 56 years for men), have less ischemic heart disease and more idiopathic dilated cardiomyopathy, and fewer cardiovascular risk factors such as smoking, diabetes mellitus, hypertension, or tobacco use . On the In the post HT period, women are at a lower risk of coronary allograft vasculopathy and malignancy. These differences could explain longer survival in female HT recipients.Pre-HT sensitization and post-HT rejection risk are higher in women, related predominantly to the presence of circulating preformed HLA antibodies due to sensitization from previous pregnancies . Donor-rIn summary, fewer women receive HT, despite their better long-term survival. Sex specificities need to be considered in the pre-HT evaluation , at the time of transplant (sex and size donor-recipient matching) and in the long-term post-HT follow-up  .Adherence to long-term therapies for chronic diseases in developed countries averages only about 50–75% . Inadequn = 7,599) and they found that 11% were poor adherers . Poor adherers were more likely to be women , have a higher heart rate, and a greater number of concomitant illnesses  between January 2000 and December 2008 who had a follow-up >1 year after HF drug treatment initiation. In this study women were more likely than men to be adherent to their treatment  (We can conclude that non-adherence to disease-modifying drugs is associated with an increased mortality and HF readmissions, but adherence seems to be similar between sexes .Although women represent 50% of the world population and despite similar overall prevalence of heart failure among men and women  women arWomen are generally more symptomatic than men when they present with HFrEF , which cWomen are also less likely to receive lifesaving therapies such as LVAD  and HT. Reproductive health counseling, teratogenic effect of HF medications and pregnancy management for women with HF are some important topics that affect women uniquely and that need to be a focus for future research and discussion, especially for those in need of advanced HF therapies and devices.Despite high prevalence of HF in women, there is lack of data on the use of drugs and HF therapies, with limited enrolment in randomized control trials and limited access to lifesaving strategies. Future trials should focus on greater enrollment of women in heart failure therapeutics and devote resources to understand the pathophysiology of the sex differences and disparities in access to advanced therapies.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.