Euterpe edulis) fruit is a strong candidate for exportation due to its high content of anthocyanins. However, its rapid perishability impairs its potential for further economic exploration, highlighting the relevance of producing ready-to-drink juices by applying innovative processing, such as high hydrostatic pressure (HHP). The effect of HHP  on anthocyanins content and antioxidant activity (AA) by FRAP and TEAC assays, and the most effective HHP condition on overall sensory acceptance and stability of jussara juice, were investigated. While mild pressurization (200 MPa for 5 min) retained anthocyanins and AA, 82% of anthocyanins content and 46% of TEAC values were lost at the most extreme pressurization condition (500 MPa for 10 min). The addition of 12.5% sucrose was the ideal for jussara juice consumer acceptance. No significant difference was observed for overall sensory acceptance scores of unprocessed (6.7) and HHP-processed juices (6.8), both juices being well-accepted. However, pressurization was ineffective in promoting the retention of anthocyanins and AA in jussara juice stored at refrigeration temperature for 60 days, probably due to enzymatic browning.Jussara ( Euterpe edulis), native to the Atlantic rainforest and similar to the açaí palm (Euterpe oleracea), but not as commercialized and consumed, produces a round fruit that has a single seed covered by a thin, shiny, purple- to black-colored layer, which yields a pulp often used to prepare varied beverages and ice creams. Previous work from our research group [O-glucoside and cyanidin-3-O-rutinoside.In the last few years, the Brazilian fruit juice industry has undergone an expansion, especially due to the expansion of international markets for tropical fruit juices exhibiting high nutritional value as well as exotic flavors. The jussara palm , and mixing. To produce ready-to-drink juices, an innovative processing method is high hydrostatic pressure (HHP), a conservation method that enables chilled storage of processed juices. HHP involves subjecting foods to high isostatic pressure (from 100 to 1000 MPa), causing a number of changes in the morphology, cell membrane, or biochemical reactions of microorganisms. All these effects are related to the inactivation of microorganisms, and compared to classical thermal conservation methods such as pasteurization, HHP process tend to preserve the nutritional, functional, and sensorial qualities of perishable foods ,7. HowevThe effects of HHP processing on phenolic compounds are complex. Many factors, such as phenolic compounds’ individual pressure sensitivity, their interaction with other matrix constituents, HHP parameters, and packing materials could influence the contents of phenolic compounds in pressurized food products . Some woSince the effects of HHP processing on food products depend on their matrix, this is the first study aiming at investigating the effect of high hydrostatic pressure processing on anthocyanins content, antioxidant activity, and consumer acceptance of jussara juice. In addition, anthocyanins and antioxidant activity retention were determined during refrigerated storage for 60 days. ® processing company, Resende, Brazil. Fruits were selected, washed, and sanitized in 100 ppm sodium hypochlorite solution for 15 min. Then, jussara fruits were submerged in water at approximately 40 °C for 30 min and subsequently depulped in a vertical depulper with additional water (1 L of water per 2.5 kg of fruit). Jussara juice presented approximately 3 °Brix and a pH of 4.8. The juice was packed in aseptic polyethylene bags and stored at −20 °C.Commercial jussara juice (Euterpe edulis) was produced by Juçaí®, São Paulo, Brazil). These bags were submitted to HHP processing in a pilot equipment . The high pressure vessel  was filled with distilled water and a concentrated carboxylate-based synthetic fluid  as a pressure-transmitting medium. Both this fluid and the juice samples were at room temperature (27 °C). Pressure  and time of processing  were defined according to a 22 full factorial design with a central point, in duplicate, totaling 10 experimental runs (Fifty milliliters of frozen jussara juice (−20 °C) was thawed, transferred to a small five-layer nylon bag with an oxygen barrier, and vacuum-sealed  and filtration through a 0.45 µm PTFE filter unit . Samples were diluted  with 1% aqueous formic acid. The liquid chromatography system  included a quaternary pump LC-20AT, automatic injector SIL-20AHT, diode array detector (DAD) SPD-M20A, system controller CBM-20A, and degasser DGU-20A5. Chromatographic separation of cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R) was achieved using a reversed-phase column C18 , according to Inada et al. [® Kyoto, Japan, version 1.25, 2009).Jussara juice was analyzed directly after centrifugation  of jussara pulp, using the supernatants. Each sample was analyzed in triplicate. The FRAP assay was performed according to Benzie and Strain [2+ equivalents per 100 mL of juice. The TEAC assay was performed according to Re et al. [The AA of jussara juice was assessed by FRAP and TEAC assays directly after centrifugation  aged between 18 and 69 years, on average 30 years, habitually consuming fruit juices at least twice a week, recruited among staff, students, and visitors at the Federal University of Rio de Janeiro, Brazil. Only consumers who reported frequent consumption of fruit juices (at least twice a week) were selected to participate in this test. Unprocessed jussara juice was sweetened with six different sucrose concentrations: 2.5%, 5.0%, 7.5%, 10.0%, 12.5%, and 15.0%. Each consumer evaluated sweetness using the just-about-right (JAR) scale, with intensity ranging in centimeters (cm) from 0  to 9 , with “ideal sweetness” specified as 4.5 cm. Jussara juice samples were offered to consumers at refrigeration temperature in 50 mL plastic cups coded with three-digit numbers. The samples were presented in a monadic sequential random order and each consumer evaluated all six jussara juice samples of different sucrose concentrations. The results were analyzed by linear regression analysis of the score given by the consumers for each different sucrose concentration.After sucrose addition at 12.5%, previously determined as the ideal sucrose concentration, half of the juice volume was pressurized at 200 MPa for 5 min. Unprocessed and HHP-processed jussara juice were stored at 4 °C for two days prior to sensory analysis. An overall impression test was carried out with a group of 80 untrained consumers (47 women and 33 men) aged between 17 and 63 years, on average 29 years, recruited among staff, students, and visitors at the Federal University of Rio de Janeiro, Brazil. Only consumers who reported frequent consumption of fruit juice (at least twice a week) and who enjoyed açaí juice were selected to participate in this test. Each consumer evaluated the overall impression using an unstructured (9 cm) scale, with intensity ranging from 0 (“dislike extremely”) to 9 (“like extremely”), with “indifferent” specified as 4.5 cm. Jussara juice samples were offered to consumers at refrigeration temperature in 50 mL plastic cups coded with three-digit numbers. Unprocessed and HHP-processed jussara juices were presented in a monadic sequential random order.The anthocyanins content and AA (FRAP and TEAC) of unprocessed and HHP-processed (200 MPa for 5 min) juices (both unsweetened) were evaluated during refrigerated storage in the dark, at baseline and after 15, 30, 45, and 60 days of storage. Analyses were performed as previously described.p < 0.05.The experimental design matrix was generated and analyzed using Statistica software, version 7.0 . Analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparison post hoc test was used for comparing the anthocyanins content and antioxidant activity of unprocessed and HHP-processed juices. As the overall impression scores did not present a normal distribution, comparison of unprocessed and HHP-processed juices was performed using a Wilcoxon matched-pairs signed-ranked test. Repeated measures two-way ANOVA followed by Sidak’s multiple comparisons test were used to investigate anthocyanins and AA retention during storage. Statistical analyses were performed using GraphPad Prism software for Windows, version 5.04 . Differences were considered significant when The major anthocyanins reported for jussara ,3, C3G ap < 0.05) losses of 49%, 66%, 40%, and 82% in total anthocyanin content were observed upon pressurization at 200 MPa/10 min, 350 MPa/7.5 min, 500 MPa/5 min, and 500 MPa/10 min, respectively.In fact, the only HHP processing condition that did not affect anthocyanins content was 200 MPa for 5 min . SignifiTerefe et al.  observedAnother relevant factor that affects anthocyanins retention in HHP-processed fruits is the processing conditions. When the same fruit is subjected to different pressures and times of processing, the effect on the anthocyanins content may vary, sometimes increasing, other times decreasing, or even remaining unaltered. Liu et al.  reportedIncreases in anthocyanins content are usually associated with a higher extractability caused by pressurization , which dr = 0.69, p = 0.0135, n = 12), whereas no correlation was observed for FRAP values, suggesting that TEAC assay was able to measure the AA of jussara juice related to the presence of anthocyanins. In summary, pressurization of jussara juice at 200 MPa for 5 min preserved both anthocyanins content and AA.Similarly, several studies reported reduction in TEAC values after HHP processing of strawberry purées, and orange and blueberry juices ,19. As eThe ideal level of sucrose addition to jussara juice was determined in a sensory test with 99 consumers. The proposed linear model showed an excellent fit, explaining more than 96% of the variance A. The idNo significant difference was observed between the overall impression scores for unprocessed (6.7 ± 1.8) and HHP-processed (200 MPa/5 min) (6.8 ± 2.1) jussara juices B, indicap < 0.0001), indicating that HHP processing was ineffective (p = 0.88) in preventing the degradation of anthocyanins. After 60 days of storage, no anthocyanins were found in either unprocessed or HHP-processed juices. Sinela et al. [Anthocyanins content in jussara juices decreased drastically  in the first 15 days during refrigerated storage . Nevertheless, FRAP and TEAC showed different trends during storage: the TEAC loss rate was constant during the whole storage period, whereas the FRAP loss rate decreased after 30 days of storage. This difference may be related to the significant association between TEAC values and anthocyanins content, as previously described. In that sense, the stability of FRAP values after 30 days of storage may be correlated to the presence of other phenolic compounds, possibly those formed as degradation products of anthocyanins .The FRAP and TEAC values of jussara juices showed a gradual decrease during refrigerated storage, with 52% and 38% retention, respectively, by the end of 60 days (p = 0.78), a negative effect was observed for TEAC values (p = 0.021). Even though this effect is of little relevance, it may be explained by a possible increase in polyphenol oxidase activity caused by HHP processing, as reported by Jesus et al. [While HHP processing did not affect FRAP loss during storage  of jussara juice. Sucrose addition at 12.5% was ideal for consumers’ acceptance and HHP processing did not impact the overall acceptance score. However, HHP processing was unable to preserve anthocyanins content and antioxidant activity during refrigerated storage for 60 days. Therefore, this study highlights the relevance of the production of ready-to-drink juices with jussara pulp, applying innovative processes such as HHP in order to maintain its functional composition and acceptance.


Chronic ankle instability, developing from ankle sprain, is one of the most common sports injuries. Besides it being an ankle issue, chronic ankle instability can also cause additional injuries. Investigating the epidemiology of chronic ankle instability is an essential step to develop an adequate injury prevention strategy. However, the epidemiology of chronic ankle instability remains unknown. Therefore, the purpose of this study was to investigate the epidemiology of chronic ankle instability through valid and reliable self-reported tools in active populations.An electronic search was performed on PubMed and Web of Science in July 2020. The inclusion criteria for articles were peer-reviewed, published between 2006 and 2020, using one of the valid and reliable tools to evaluate ankle instability, determining chronic ankle instability based on the criteria of the International Ankle Consortium, and including the outcome of epidemiology of chronic ankle instability. The risk of bias of the included studies was evaluated with an adapted tool for the sports injury review method.After removing duplicated studies, 593 articles were screened for eligibility. Twenty full-texts were screened and finally nine studies were included, assessing 3804 participants in total. The participants were between 15 and 32 years old and represented soldiers, students, athletes and active individuals with a history of ankle sprain. The prevalence of chronic ankle instability was 25%, ranging between 7 and 53%. The prevalence of chronic ankle instability within participants with a history of ankle sprains was 46%, ranging between 9 and 76%. Five included studies identified chronic ankle instability based on the standard criteria, and four studies applied adapted exclusion criteria to conduct the study. Five out of nine included studies showed a low risk of bias.The prevalence of chronic ankle instability shows a wide range. This could be due to the different exclusion criteria, age, sports discipline, or other factors among the included studies. For future studies, standardized criteria to investigate the epidemiology of chronic ankle instability are required. The epidemiology of CAI should be prospective. Factors affecting the prevalence of chronic ankle instability should be investigated and clearly described. Ankle sprain is one of the most common sports injuries in physically active individuals and causes a high financial burden on the healthcare system –3. The iAnkle sprain also predisposes athletes to recurrent ankle sprains and leads to residual symptoms , 7. In sCAI is not only an ankle issue but also systematically affects other joints, causing further physical issues . In the To identify the prevalence of CAI in sporting populations, Attenborough and colleagues conducted a systematic review which defined CAI based on a CAI model published in 2011  and repoThe systematic search was performed in the online search engines PubMed and Web of Science in July 2020 using the keywords and MeSH terms (“ankle instab*” OR CAIT OR IDFAI OR AII) AND . Articles published between 2006 and 2020 were screened, since the three tools  evaluating perceived ankle instability recommended by the International Ankle Consortium were published in 2006, 2006 and 2012 respectively.Studies that met the following criteria were included: (1) peer reviewed studies, (2) using one of the valid and reliable tools  to evaluate chronic ankle instability, (3) determining chronic ankle instability based on the criteria of the International Ankle Consortium, (4) and the outcome represented the epidemiology of CAI. If the studies were not written in English, a review article or the full-text unavailable, they were excluded.The study selection process was performed by two independent reviewers. After removing the duplicated articles, titles and abstracts of the articles were screened based on the pre-determined criteria. The remaining full-texts were reviewed for eligibility and either included or excluded for the current review. A third reviewer was consulted when the two authors could not reach agreements. Authors, published year, studied population, sample size, demographics, the criteria of determining CAI, inclusion criteria, exclusion criteria, and the epidemiological data of CAI were extracted.Two independent reviewers assessed the bias of included studies using an adapted risk assessment tool , 17. TheThe conducted systematic searches resulted in a total of 744 studies Fig. . After rTable N = 1238) [The summarized prevalence of CAI was 25% (ranging from 7 to 53%). The data from one study was not integrated into the overall result because the study applied the prevalence of 65 included participants who sought medical care to estimate the prevalence for the whole population ( = 1238) . Forty-sThe age of the participants among the different study populations ranged from 15 to 32 years. The number of participants aged younger than 18 years was 1399 , 22, 25,The study population consists of military personnel , athleteThe definitions of CAI in the included studies were homogeneous, but the exclusion criteria diverse. Four articles applied the inclusion and exclusion criteria from the International Ankle Consortium to define the presence of CAI (2.3–40%) , 22, 26.The results of the critical appraisal are displayed in Table The current review included nine studies and the results showed that the prevalence of CAI was 25%, ranging from 7 to 53% and the prevalence of CAI within participants with a history of ankle sprains was 46%, ranging from 9 to 76%. Five out of nine included studies had a low risk of bias.The prevalence of CAI from eight included studies ranged from 7 to 53%. The results from one study were not integrated because of the high risk of bias . The widMost of the included studies investigated CAI using a questionnaire and excluded participants with the conditions confounding the presence of ankle instability Table . WithoutIn addition, different characteristics of participants, for example age and population, also vary the prevalence of CAI. Regarding to age, the samples from the included studies come with a wide range of ages (15–32 years). A previous study showed that a younger age is one of the risk factors for recurrent ankle sprain, which in turn is one of the risk factors of CAI . Tanen eFurthermore, different sports disciplines show a varying prevalence of ankle sprain and CAI. Doherty et al. found that indoor/court sports showed the highest prevalence and incidence of ankle sprain  among water/ice sports , filed-based sports  and outdoor pursuits sports  –7.2, 12.. In the Additionally, the investigated populations and the sample size of each sport discipline varied among the included studies. For example, the sample size in the various included sports ranged between five and 163 participants. There were only 10 to 20 participants overall in the categories of swimming, golf, gymnastics, field hockey and rowing/crew, and there were 96 to 108 participants in netball, dance, basketball and soccer  who were recruited into mandatory military service [Some articles investigating the epidemiology of CAI were excluded CAI due to the mismatched definition from the current review. Two systematic reviews defined CAI as self-reported perceived ankle instability, mechanical instability, repetitive ankle sprain and persisting symptoms lasting over 6 months after an acute ankle sprain and surveyed the epidemiology of CAI in sporting populations and children , 29. In  service . Again, Regarding other factors affecting the prevalence of CAI, accessibility of rehabilitation can affect the development of CAI. Exercise therapy showed moderate evidence to treat/prevent recurrent ankle sprain . For insN = 829,791) [p < 0.05) [N = 829,791) [Additionally, there are some other influencing factors that have been discussed in previous studies, for example, body size, gender and competition level. Unfortunately, the evidence is not conclusive. For body size, one cross-sectional study found that participants’ body mass index and height are associated with the presence of mechanical and functional ankle instability in a general population  . However829,791) . The dif < 0.05) . In addi < 0.05) . In cont829,791) .Competition levels may play an essential role on the prevalence of CAI, but the direction is controversial in the current evidence. Tanen et al. found that the prevalence of CAI was higher in the athletes in a lower competitive level (high school athletes) than that in a higher competitive level (collegiate athletes) . AlthougThe criteria to define CAI were applied in each study. Although all studies define CAI based on the standard criteria of the International Ankle Consortium, the inclusion and exclusion criteria were distinct among the included studies. This causes a misestimating of the prevalence of CAI. As mentioned in the previous paragraph, the standard from the International Ankle Consortium is for controlled research, which excludes the participants with other issues confounding identification of CAI. The participants with other conditions (history of a fracture or surgeries or acute injury in previous 3 months) and CAI cannot be clarified. This will definitely affect the results of the CAI prevalence. Therefore, to establish the standard criteria is a prerequisite for conducting epidemiological studies.In regards to the study design, to investigate the epidemiology of chronic injuries, Bahr suggested applying a prospective study design with continuous or serial measurements . HoweverParticipants’ characteristics were missing in four included studies , 24, 25.There were some limitations in the current review. First of all, only nine studies were included. The prevalence might not be representative because of the small sample size. In addition, the included studies were heterogeneous. The surveyed population, countries, competitive level and sports were varying. Three studies presented the prevalence of CAI in different sports , 25, 26.The prevalence of chronic ankle instability in the active population was 25%, ranging between 7 and 53% in different populations. The prevalence of chronic ankle instability within the participants with a history of an ankle sprain was 46%, ranging from 9 to 76%. The wide range of the prevalence was mainly caused by exclusion criteria, age, sports discipline, and other factors. In order to obtain comprehensive epidemiological information about CAI, first of all, prospective studies should be conducted to the symptoms of CAI with valid and reliable tools at regular intervals . To repo


The major signaling pathways in colon cancer are WNT, RAS, and TGF-β. Components of these pathways are mutated in the majority of colon cancers, resulting in aberrantly high or low activity of the pathway. The functional consequences of the mutations reflect the behavior of these signaling pathways in intestinal stem cells. To better understand the roles of each pathway, we cover the basic function as well as points of intersection between the different pathways, to describe how they function individually, as well as together, to regulate cell proliferation.1 into S-phase.Colon cancer tumorigenesis occurs incrementally. The process involves the acquisition of mutations which typically follow an established pattern: activation of WNT signaling, activation of RAS signaling, and inhibition of TGF-β signaling. This arrangement recapitulates, to some degree, the stem cell niche of the intestinal epithelium, which maintains WNT and EGF activity while suppressing TGF-β. The resemblance between the intestinal stem cell environment and colon cancer suggests that the concerted activity of these pathways generates and maintains a potent growth-inducing stimulus. However, each pathway has a myriad of downstream targets, making it difficult to identify which aspects of these pathways are drivers. To address this, we utilize the cell cycle, the ultimate regulator of cell proliferation, as a foundation for cross-pathway integration. We attempt to generate an overview of colon cancer signaling patterns by integrating the major colon cancer signaling pathways in the context of cell replication, specifically, the entrance from G Cancer is a disease in which cells of the body reject the commands of their tissue. This disobedience is a result of the disruption of the signaling pathways, which are no longer able to balance the activities of the cell nor effectively communicate with the extracellular environment. Signaling pathways are disrupted through a myriad of causes, though the most common is the mutation of a gene which produces a critical regulatory protein in the signaling cascade. The mutation severs the connection between the stimulus and its downstream response, because one of the proteins required to relay the appropriate signal is unable to do so. Thereby, the cell loses control over a specific aspect of its behavior and the output of the pathway becomes relatively constant, as opposed to dynamic. This influences the resting state of the cell as well as any dynamic cellular responses which require input of this (now defunct) pathway. As the mutant cell multiplies, more mutations can occur, providing the basis for selection, which results in disease progression as the cancer cell further deviates from normal cell function. Eventually, the cancer cell accumulates a sufficient number of driver mutations, approximately 5–7 in colon cancer, in various pathways, which render it ignorant of its surrounding environment. This forms the basis for metastasis, as the cancer cell is able to traverse the surrounding tissues, withstand secreted growth inhibitory signals, as well as grow in a remote site of the body, independent of the signaling pathway requirements of its tissue of origin.In the healthy intestinal epithelium, the WNT, EGF, Notch, and BMP pathways maintain homeostasis . The intIn colon cancer, the activities of the previously mentioned pathways are altered by mutation and closely mirror the signaling pathway arrangement found within the intestinal stem cell niche, i.e., high WNT, high EGF, and low BMP. However, Notch signaling does not appear to be heavily targeted by mutation in colon cancer . Given tThe WNT signaling pathway is an ancient mechanism necessary for guiding development and maintaining tissue homeostasis throughout multicellular life . In the WNT signaling begins with the production of a small ~40 kDa), cysteine-rich protein, referred to as a WNT ,21. Huma kDa, cysOnce at the target cell membrane, the WNT ligand binds to a Frizzled (FZD) receptor as well as a Low-Density Lipoprotein Receptor-Related Protein (LRP) to form a FZD–LRP coreceptor, thus initiating canonical WNT signaling ,49,50,51TCF7L2, is necessary for intestinal development in mouse embryos as well as intestinal maintenance in adult mice [MYC, CCND1, and AXIN2 are generally considered to be key transcriptional targets of an active WNT signal and serve as WNT reporter genes [Upon successful WNT–FZD–LRP binding, the cytoplasmic domains of FZD and LRP become associated, which results in the recruitment of DVL and AXIN and the phosphorylation of LRP by CKIγ and GSK3, in an incompletely understood mechanism ,65,66,67ult mice ,84. Whiler genes ,90,91.In the WNT OFF state, the FZD and LRP receptors remain unbound by WNT, and therefore, remain unassociated in the cytoplasm. FZD receptors are internalized by ZNRF3 and RNF43, blocking accessibility to WNT ,61. The APC, resulting in a truncated APC protein which can not efficiently facilitate the degradation of β-catenin, resulting in constitutively active WNT signaling [APC appears to follow Knudsen’s two-hit theory, as both alleles have been found to be mutated in the majority of adenomas from patients with familial adenomatous polyposis (FAP) [APC and CTNNB1 account for the majority of WNT mutations in colon cancer [APC and CTNNB1 mutations are mutually exclusive, indicating that they serve equivalent roles in WNT signaling activation [MYC deletion rescues the consequences of APC loss, despite high levels of β-catenin [The majority (93%) of colon cancers harbor mutations in the WNT signaling pathway, which result in altered WNT signaling activity . The majignaling ,103,104 is (FAP) ,108,109.n cancer . These mtivation . Unfortu-catenin .K-RAS, are frequently mutated in cancer [RAS [APC mutation during tumorigenesis, though this order is not invariant [RAS signaling is crucial for mediating cellular growth, differentiation, and survival . As a ren cancer . Approxicer [RAS ,113. In RAS signaling relies on the actions of the Epidermal Growth Factor Receptors (EGFR), which are Receptor Tyrosine Kinases (RTKs), for signal transduction through the plasma membrane. The EGFR family consists of four members: ERBB1 (EGFR), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) ,116,117.FOS, a member of the AP-1 transcription factor [Oligomerization of the EGFR family of receptors results in transphosphorylation of the dimerized, cytoplasmic receptor domains ,131,132.n factor ,156. Addn factor . RAS sigIn the absence of EGF or an EGF-like ligand, EGFR does not dimerize, since the dimerization interface remains sequestered until EGF binding, which induces a conformational change in the receptor ,158. EGFK-RAS mutations, which occur almost exclusively in codons 12, 13, or 61, are found in approximately 50% of tumors [K-RAS mutant cells and significantly reduces the reliance of the cell upon EGF and EGFR for an active RAS growth signal. Mutations in K-RAS are mutually exclusive with mutations in N-RAS or B-RAF, implying similar regulatory functions [FOS [JUN is also mediated by RAS signaling; however, it relies upon the JNK cascade [In colon cancer, f tumors . These mf tumors ,168  and can be divided into the TGFβ–Activin–Nodal and BMP subfamilies . These lInk4b and p21Cip1, the cyclin-dependent kinase inhibitors which mediate the growth-repressive actions of TGF-β [The intracellular workhorses of the TGF-β signaling pathway are the SMAD proteins. There are three functional classes of SMADs: receptor-regulated (R-SMAD), co-mediator (Co-SMAD), and inhibitory (I-SMAD) . The R-Sof TGF-β ,198,199.TGF-β signaling is regulated at several levels. At the ligand level, Noggin and Chordin, are both capable of binding to the BMP ligands, which prevents their association with the BMP receptors ,201,202 TGFBRII, SMAD2, or SMAD4 inactivate the protein and abrogate the ability of TGF-β to regulate proliferation [SMAD4 (10%) are more common than mutations in SMAD2 (6%); however, in hypermutated tumors, mutations in TGFBRII were observed in 51% of tumors [SMAD4 mutations may occur within the MH2 domain, which inhibits the ability of SMAD4 to bind the R-SMAD and transfer to the nucleus, or may occur within the MH1 domain, which results in autoinhibition of the SMAD, which also inhibits SMAD complex formation [TGF-β signaling negatively regulates cell proliferation in the colon and is often mutated in colon cancer ,206. Theferation ,208,209 f tumors . The SMAormation ,211. Ultormation .1 [The cell cycle is initiated by progression of the cells past the restriction point in G1 ,214. Onc1 ,214. The1 ,216. TheAPC, K-RAS, or SMAD4 mutation, results in an imbalance of the signaling pathways and confers a proliferative advantage upon the mutation-harboring cell.The WNT, RAS, and TGF-β signaling pathways are frequently mutated in sporadic colon cancer ,13. WNT MYC and CCND1, which both facilitate progression into S-phase [MYC appears to be the major target of WNT signaling, as MYC deletion rescues APC loss, despite high levels of β-catenin [CDKN1A (p21) and CDKN2B (p15) [MYC, as MYC expression is repressed by SMAD3, which is necessary for TGF-β-induced G1 arrest [CDKN1A and CDKN2B, the genes which are inhibited by MYC [MYC level, with TGF-β dominance resulting in p21 and p15 production and cell cycle arrest, while WNT dominance results in CDKN1A and CDKN2B repression. The APC mutation appears to give WNT a slight advantage. The increased levels of MYC, induced by activating WNT mutations, are complemented by activated RAS signaling via K-RAS [FOS, a member of the AP-1 transcription factor [CCND1 and repressing TP53 [CCND1, by the concerted efforts of β-catenin/TCF4 and AP-1. It has been noted that WNT alone does not appear sufficient to fully express CCND1 [CCND1 expression via AP-1 and MYC phosphorylation, helps to weaken TGF-β signaling by SMAD phosphorylation via ERK, and represses TP53 via AP-1. The high levels of cyclin D, from WNT and RAS, bind to CDK4, a MYC target gene, allowing for the formation of cyclin D and CDK4 complexes [E2F gene promoters, facilitating the activation of the E2F transcriptional program [1 to S, such as CCNE1 [MYC expression, as well as express CDKN1A and CDKN2B, may be the last checks on proliferation, which upon mutation of SMAD4 or one of the TGF-β receptors, results in the complete release of MYC and unregulated proliferation of cancerous cells  ,228. The1 arrest ,230. Furd by MYC ,198. WNTia K-RAS . Phosphon factor ,156. Then factor . Once foing TP53 ,234,235.ss CCND1 . Additioss CCND1 ,154. Furss CCND1 ,238. Oncomplexes . CyclinDomplexes ,241. MYC program . E2F taras CCNE1 . Cyclin as CCNE1 ,244. Theus cells .C. difficile secretes toxins which bind the FZD receptors, inhibiting their ability to bind a WNT ligand [The microorganisms which reside in the colon are often overlooked in regard to their influence on colorectal cancer tumorigenesis. These organisms can generate a profound impact on the gut by the alteration of chemical species, interaction with intestinal cells, or by the release of toxins ,246,247.T ligand ,249. ThiT ligand . EvidentCCND1 through βcat/TCF4 and AP-1, whereas other efforts are complementary, such as MYC expression by WNT and MYC phosphorylation by RAS. TGF-β antagonizes the influences of WNT and RAS on the cell cycle by down-regulating MYC and up-regulating the cell cycle regulators CDKN1A and CDKN2B. The ability of TGF-β to regulate the cell cycle relies on the SMAD transcription factors, which are phosphorylated by the activated RAS pathway to inhibit their nuclear translocation. As colon cancer develops, mutations in the WNT pathway lead to an increase of MYC, which results in small adenoma formation. The coupling of oncogenic RAS to deregulated WNT leads to the formation of a large adenoma, as βcat/TCF4 and MYC are complemented by AP-1 of RAS. Loss of TGF-β, the developmentally designated gatekeeper, allows for the unrestrictive action of MYC and RAS. Taken together, the WNT, RAS, and TGF-β mutations function cooperatively to form a malignant cell.In colon cancer, the mutations of the WNT, RAS, and TGF-β signaling pathways recapitulate the signaling arrangement present in the intestinal stem cells. This stochastic recapitulation of a similar functional program indicates that the coordinated activities of these pathways results in a potent growth-inducing stimulus. These efforts are, at times, overlapping, such as the expression of


Epilepsy is a neurological disease affecting more than 50 million individuals worldwide. Notwithstanding the availability of a broad array of antiseizure drugs (ASDs), 30% of patients suffer from pharmacoresistant epilepsy. This highlights the urgent need for novel therapeutic options, preferably with an emphasis on new targets, since “me too” drugs have been shown to be of no avail. One of the appealing novel targets for ASDs is the ghrelin receptor (ghrelin-R). In epilepsy patients, alterations in the plasma levels of its endogenous ligand, ghrelin, have been described, and various ghrelin-R ligands are anticonvulsant in preclinical seizure and epilepsy models. Up until now, the exact mechanism-of-action of ghrelin-R-mediated anticonvulsant effects has remained poorly understood and is further complicated by multiple downstream signaling pathways and the heteromerization properties of the receptor. This review compiles current knowledge, and discusses the potential mechanisms-of-action of the anticonvulsant effects mediated by the ghrelin-R. Epilepsy is a neurological disease characterized by spontaneous and recurrent seizures . With apOne of these potential options is the orexigenic peptide, ghrelin. Ghrelin exerts both peripheral as well as central effects, and is primarily secreted by X/A-like cells in the stomach , but alsIn the central nervous system (CNS), ghrelin and its receptor are best known for their critical role in food intake, mediated by neuropeptide Y and agouti-related peptide ,18,19 -long peptide, with the active form containing a unique acylation on serine 3 . GhrelinIn human plasma, circulating esterases deacylate ghrelin, and 90% of total ghrelin consists of DAG, while only 10% consists of acylated ghrelin . GhrelinRecently, GOAT was shown to be expressed on the cell surface of mature bone marrow adipocytes, and to be necessary for DAG to promote adipogenesis in mice . In lineGhrelin establishes its numerous effects by interacting with its G-protein-coupled receptor (GPCR), of which two isoforms exist: the full-length (366 amino acids (AA) long) 7-transmembrane GPCR GHSR1a , and a shorter (289 AA long) 3′-truncated variant, GHSR1b ,45. ThisThe ghrelin-R is present in the brain and the periphery. The peripheral sites of ghrelin-R expression include the pancreas, spleen, bone tissue, cardiac tissue, the thyroid gland and immune cells, the adrenal glands, adipose tissue, and the vagal afferents ,48.Centrally, the ghrelin-R is widely expressed in a variety of brain areas and shows high expression levels in several nuclei of the hypothalamus, among which are the arcuate nucleus and the anterior hypothalamic nucleus. The receptor is further expressed in the olfactory bulb, the neocortex, in a variety of nuclei in the midbrain, in the pons, and in the medulla oblongata. These include the globus pallidus, the area postrema, the nucleus tractus solitarius, the substantia nigra, and the ventral tegmental area. In the hippocampus, the ghrelin-R was shown to be modestly expressed in the Cornu Ammonis (CA)1, compared to the higher expression levels in CA2, CA3, and DG of the mouse brain ,49,50,51The expression of the ghrelin-R is highly dynamic, and depends on the developmental stage , the disq/11, Gαi/o, and Gα12/13 signaling, followed by β-arrestin recruitment -substance P, were anticonvulsant in the intrahippocampal pilocarpine infusion rat model. Inverse agonists are typified by their ability to block the intracellular signaling of a receptor, including basal constitutive signaling, which resembles an absence of the receptor. In line with this notion, ghrelin-R knock-out (KO) mice were shown to be protected from seizures ,124, whing model .Ghrelin-R expression is dynamic and may be influenced by the presence of a disease state, or may depend on exposure to ghrelin ,55,134, q signaling and β-arrestin recruitment. The ghrelin-R antagonist, EP-80317, restored the increased hippocampal phosphorylation levels of the other canonical downstream target ERK in the 6-Hz mouse model [Hippocampal Akt signaling was decreased in a pilocarpine rat model, which could be restored by ghrelin administration ,30. Akt se model .50 values compared to ghrelin with regard to the ghrelin-R signaling pathways, but is unable to internalize the ghrelin receptor, and was not able to exert anticonvulsant effects [q and Gα12 selective biased ligand of the ghrelin-R, YIL781, increased seizure severity in a kindling model [Up until now, the exact signaling pathways responsible for anticonvulsant effects downstream of ghrelin-R have remained elusive, but a few possibilities exist, on the basis of previous findings. Not only ghrelin-R agonists, but also ghrelin-R inverse agonists exerted anticonvulsant effects, and ghrelin-R KO mice were protected from seizures ,124. The effects . Because effects . We showng model . Given tHowever, we cannot completely exclude that G-protein-dependent signaling may be required for ghrelin-R-mediated anticonvulsant effects, as Akt and ERK activation have been described ,32,135. Ghrelin increased the number of surviving neurons in the CA1 and CA3 hippocampal regions in the pilocarpine rat model . PilocarThis latter finding was confirmed in the i.p. KA mouse model, in which ghrelin decreased cleaved caspase-3 immunoreactivity in pyramidal CA1 and CA3 neurons, and restored neuronal loss in CA1 and CA3 . TerminaA two-week-long administration of the ghrelin-R agonist, macimorelin, in the IHKA mouse model, exerted anticonvulsant effects on spontaneous recurrent seizures, but did not increase neuronal survival in the CA1, CA3, and DG of the hippocampus. This could be due to the omission of pretreatment, as the onset of the treatment commenced 24 h after SE induction, or due to the additional two-week wash-out in this study . A lowerThe possibility should be considered that ghrelin exerts neuroprotective effects via the activation of a variety of signaling pathways mediated through the employment of G-proteins, whereas the rapid and subsequent internalization via β-arrestin signaling may be responsible for anticonvulsant effects. Unraveling which ghrelin-R downstream signaling pathway is responsible for a particular effect may be obtained by doing further experiments in genetic models, or by using biased ligands that selectively activate a subset of pathways while leaving others untouched.Inflammation is a major hallmark of epileptogenesis, seizures, and chronic epilepsy. This ranges from infiltration of the inflammatory cells and the release of proinflammatory mediators to widespread gliosis ,136,137.Ghrelin significantly reduced the elevated plasma calcitonin gene-related peptide (CGRP), substance P, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in the PTZ rat model ,118. AddSeizures induce oxidative stress, which, in turn, exacerbates seizures (reviewed by ). GhreliWhile several molecular mechanisms-of-action have been described, it remains unknown how these contribute to the anticonvulsant effects of ghrelin, and how they lead to an overall decrease in the brain excitability. The heteromerization of the ghrelin-R with other receptors can lead to the preferential recruitment of other noncanonical signaling pathways . Ghrelin2+ increases through the canonical Gαq protein [2+ in ghrelin-R-expressing neurons. Elevated levels of intracellular Ca2+ are associated with epileptiform activity and epileptogenesis [2+ may be an interesting putative mechanism for seizure suppression in the absence of ghrelin-R signaling. Various studies have shown the differential effects of ghrelin on neuronal excitability and synaptic transmission, which all support that ghrelin acts in a brain-region-specific manner [Ghrelin-R activation results in intracellular Ca protein ,60. One ogenesis ,145. Thec manner ,147,148.q signaling in excitatory neurons via β-arrestin, while increasing Gαq signaling in inhibitory interneurons. Indeed, while GPCRs may be associated with several signaling pathways, these signaling pathways are not always all operative in the same cell. Thus far, the knowledge concerning the cell-specific expression of signaling pathways downstream of the ghrelin-R is lacking and requires further studies.The ghrelin-R is expressed in both excitatory neurons, as well as in inhibitory interneurons in the dorsal CA1. It was recently shown that a selective increased expression of the ghrelin-R in excitatory neurons was detrimental for learning and memory in mice, while an increased expression of the ghrelin-R in interneurons had a beneficial effect . It remaGhrelin is increasingly recognized as a potential important player in seizures and epilepsy. Most studies show lower ghrelin levels in patients suffering from epilepsy, or lower ghrelin levels after a seizure. The exact implications of plasma ghrelin level alterations in epilepsy have remained, up until now, unknown, and should be further investigated in light of its treatments as well, including the KD. It is increasingly evident that there may be important differences between AG and DAG. This may advocate for future investigations of both isoforms of ghrelin in epilepsy, or for studying further whether the contributions of GOAT expression and the local reacylation of DAG are relevant for seizure control.With only a few exceptions, ghrelin and synthetic agonists of the ghrelin-R are anticonvulsant in seizure, epilepsy, and SE models. The notion that both agonists and inverse agonists were anticonvulsant stirred up the discussion concerning the signaling pathways responsible for ghrelin-R-mediated anticonvulsant effects. The hypothesis that β-arrestin recruitment is involved should be more thoroughly investigated to confirm the relevance of this pathway. Overall, the complexity of ghrelin-R signaling, and the extensive list of other factors possibly influencing it, highlight the need for further investigations into the mechanism behind ghrelin-induced anticonvulsant effects.


Atrial fibrillation (AF) is a growing health problem worldwide. In recent years, there has been a rising interest in the relationship between sleep disorders and AF. Several studies have reported higher prevalence and incidence rates of AF in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). However, some believe that OSAHS is not a risk factor for AF; but AF, by itself, is regarded as one of the possible triggers for OSAHS. In this study, the related literature investigating the association between OSAHS and AF was reviewed, and then the possible mechanisms of this interplay were discussed. To conclude, recommendations for further research in this field were presented to researchers and some points were highlighted for physicians. Atrial fibrillation (AF) is considered as a growing health problem worldwide, affecting approximately 1-4% of the general population -5. As thThe association between OSAHS and AF has been investigated so far in more than ten studies. Based on the present review, most of the published articles have used cohort designs -28, follAccording to the latest conducted meta-analysis of cohort studies, OSAHS might increase AF risks , and higher severity of central sleep disorders could be related to a higher risk of AF in the general population. Accordingly, this meta-analysis was conducted on a total number of eight studies, involving 603532 individuals with non-sleep disorders and 14799 patients with OSAHS, which seems enough to conclude. However, they failed to evaluate the influence of variables such as the history of cardiovascular disease and body mass index on the risk of AF with a meta-regression analysis, because these variables had always been unavailable in the included studies .Another Catheter ablation has been recognized as an effective treatment method for AF; however, there is much evidence highlighting the role of OSAHS on the risk of post-ablation AF recurrence -38. A prDifferent pathophysiologic mechanisms have been taken into consideration for the association between OSAHS and AF, indicating the interplay among multiple factors, rather than a singular one . ChangesBesides, an increase in oxidative stress signaling, as well as inflammatory mediators and neurohumoral activation influenced by the quality of sleep are further deemed as the other possible mediators in the association between OSAHS and AF . As wellBased on the results of human studies; repeated OSAHS over several weeks, can lead to atrial fibrosis, atrial connexin 43 downregulation or lateralization, and even AF-promoting electric changes ,53. RemaThe amount of evidence about the relationship between the treatment of sleep disorders and AF incidence is limited to observational studies, and no randomized controlled study has been performed so far.Thus, it is not easy to address these questions: “Does the treatment of sleep disorders have an impact on AF?” and “Does it really decrease the incidence of AF?”. According to the published studies on the effect of the treatment of sleep disorders on AF recurrence after catheter ablation, it seems that continuous positive airway pressure (CPAP) therapies might also be effective for decreasing the burden of AF in patients with sleep disorders .The CPAP as a practical therapy for OSAHS has been insufficiently used, and there have always been uncertainties about the role of CPAP, as a treatment for OSAHS, on the rate of post-ablation AF recurrence .However, beneficial effects of OSAHS treatment with CPAP on AF outcome following ablation have been recently approved in several clinical studies ,61.In some investigations, it was suggested that CPAP therapy could result in 70-80% AF free survival in comparison to 36-47% in non-CPAP users following AF ablation ,62.Moreover, another study showed that AF recurrence was lower in OSAHS-treated patients compared to the non-treated group  .Deng et al. have also shown in their meta-analysis that CPAP treatment for AF patients with OSAHS might have significantly mitigated AF recurrent risks .Although their pooled sample size was limited to only 1217 participants, they showed that after 16.33±10.34 months of follow-up, 33.5% of the patients had faced recurrent AF, and the recurrence rate was significantly different between the CPAP and non-CPAP groups .They also demonstrated that CPAP therapy had decreased the left atrial diameter (LAD) and it had led to an increase in the left ventricular ejection fraction (LVEF) .Besides, it was argued that more clinical randomized controlled trials were required to further support and confirm their findings, mainly due to the small sample size of included studies (3 out of 10 articles).More information is needed concerning accuracy as well as impact, and above all cost-effectiveness of the implementation of OSAHS testing and its treatment strategies in daily AF care.Likewise, more randomized controlled studies are required to confirm the success rate of OSAHS treatment on different parameters in AF burden and AF ablation outcomes.Moreover, the effects of alternative therapeutic options for sleep disorders such as weight reduction or position modification devices in comparison to CPAP on AF burden and symptoms seem to be of great significance.Furthermore, more studies are required to indicate which AHI thresholds should be considered as indications for OSAHS treatment in AF patients. Finally, there is still an important question to be answered; i.e., how ethnicity, regional differences, gender, as well as age may affect the association between AF and sleep disorders.It seems evident that the prevalence and incidence of AF are higher in individuals with OSAHS, especially in middle-aged adults and those with other risk factors. However, the efficacy of any therapeutic interventions among patients with OSAHS in terms of decreasing the risk of AF is not clear yet. Therefore, advising patients receiving OSAHS therapeutic interventions only for reducing the risk of AF needs additional evidence. Moreover, while OSAHS patients are being encountered with greater risks of AF recurrence after catheter ablation than those without OSAHS, the treatment of OSAHS using CPAP is recommended.G.H. and N.D. report research grants from Abbott, Biotronik, Boston Scientific and Medtronic to the institution without personal financial benefits. AA. and A.S.SH have no conflict of interest.


Rationale: Corticosteroid resistance (CR) seriously affects the therapeutic effects of steroids on many chronic inflammatory disorders, including airway allergy. The mechanism of CR development is unclear. Recent research indicates that livin, an apoptosis inhibitor, is associated with the regulation in cell activities. This study investigates the role of livin in the inducing and sustaining CR in the airway mucosa.Methods: Nasal epithelial cells (NECs) were isolated from surgically removed nasal mucosal tissues of patients with allergic rhinitis (AR) and nasal polyps with or without CR. Differentially expressed genes in NECs were analyzed by the RNA sequencing. A CR mouse model was developed to test the role of livin in CR development.Results: The results showed that NECs of AR patients with CR expressed high levels of livin, that was positively correlated with the thymic stromal lymphopoietin (TSLP) expression and the high Ras activation status in NECs. Livin and Ras activation mutually potentiating each other in the inducing and sustaining the TSLP expression in NECs. TSLP induced eosinophils and neutrophils to express glucocorticoid receptor-β (GRβ). Eosinophils and neutrophils with high CRβ expression were resistant to corticosteroids. Depletion of livin or inhibition of TSLP markedly attenuated CR and airway allergy.Conclusions: Livin facilitates CR development in the airways by promoting TSLP expression in epithelial cells and the GRβ expression in eosinophils and neutrophils. Depletion of livin or inhibiting TSLP attenuates CR development and inhibits airway allergy, this has the translational potential to be used in the treatment of airway allergy. There are many diseases, e.g., chronic pulmonary diseases, chronic kidney diseases, inflammatory bowel diseases, rheumatoid arthritis, rely on the steroid therapy that has strong effects on inhibiting inflammation non-specifically The intracellular glucocorticoid receptor-α (GRα) and GRβ mediate the effects of steroids on regulating cell activities + cytotoxic lymphocytes CR is common in chronic airway diseases, including severe asthma, chronic obstructive pulmonary disease, nasal allergy (AR) and chronic rhinosinusitis Patients with chronic rhinosinusitis, nasal allergy and polyps were recruited into this study. The patients were divided into two groups, corticosteroid resistant (CR) group and corticosteroid sensitive (CS) group based on reported criteria Following published data All patients were not used corticosteroid agents, immune suppressive agents and anti-allergy agents at least one month prior to the recruitment. To determine whether the patients were at the CR status, following established procedures Following published procedures Following our established protocols t-test  or the Mann Whitney test . ANOVA followed by the Bonferroni test or Dunnett's test was performed for multiple comparisons. The correlation between two data sets was determined by the Pearson correlation test  or the Spearman correlation test .Each group consisted of 6 mice. Each experiment was repeated at least 3 times with each sample analyzed in triplicate (average of the three readouts was used as one datum). The data are presented as mean ± SEM. The difference between two groups was determined by the Student Some experimental procedures are presented in the We collected surgically removed nasal mucosa from AR-polyp patients with CR or without CR (CS). Nasal epithelial cells (NEC) were isolated from the samples by enzymatic digestion and flow cytometry cell sorting (FCS) and analyzed by the RNAseq. The results revealed 6 differentially expressed genes (DEG), including Livin, KRAS , TSLP, CCL11, IL-8 and IL-13R, in nasal epithelial cells. The DEG levels were significantly higher in AR-CR samples than that in NC samples and AR-CS samples. No statistical difference was detected between the AR-CS group and the NC group  and neutrophils are inflammatory effector cells that play an important role in CR + cells isolated from the CR mouse airway tissues showed high expression of Bcl2  To date, although it has been recognized that high TSLP expression is one of the important cytokines to initiate skewed Th2 responses and plays a critical role in the pathogenesis of allergic diseases The limitation of this study is that the CR was only could be reproduced in an animal model. Prospect studies may focus on modulating the expression of livin in airway epithelial cells to suppress the CR status.In summary, livin is associated with the CR development through promoting TSLP production by epithelial cells. In cooperating with Ras activation, livin is also involved in sustaining the high TSLP expression in epithelial cells that contributes to CR development and CR sustaining. Inhibition of livin attenuates experimental airway allergy and blocks the development of CR.Supplementary methods, figures and tables.Click here for additional data file.


Gynecologic cancers standard treatment often requires the removal of some reproductive organs, making fertility preservation a complex challenge. Despite heightened oncofertility awareness, knowledge about fertility attitudes and decisions of young patients with gynecologic cancer is scarce. The aim of this systematic review was to highlight what is currently known about knowledge, attitudes, and decisions about fertility, fertility preservation, and parenthood among these patients.Peer-reviewed journals published in English were searched in PubMed, Web of Science and EMBASE from January 1, 2000 to July 1, 2020. Childbearing, fertility, fertility preservation, pregnancy, and parenthood attitudes/decisions after gynecologic cancer from women’s perspective were evaluated.A total of 13 studies comprised the review. Most of the women valued fertility preservation procedures that could be regarded as a means to restore fertility. A unique feature identified was that fertility preservation was seen also as a way to restore gender identity perceived to be lost or threatened during diagnosis and treatment. Fertility counseling was suboptimal, with wide variability among studies reviewed. Comparisons between gynecologic cancers and other cancer types about fertility counseling rates were inconclusive. The potential negative impact of impaired fertility on patients’ mental health and quality of life was also documented.Fertility and parenthood were important matters in patients’ lives, with the majority of patients expressing positive attitudes toward future childbearing. Results confirm that the inclusion of patients with gynecologic cancer in research studies focusing on this topic still remains low. Additionally, the provision of fertility counseling and referral by health professionals is still suboptimal. Standard treatment of gynecologic cancer often requires the removal of reproductive organs, making fertility preservation a major concern. This review summarizes the date on the provision of fertility counseling for women with gynecologic cancers, focusing on their knowledge, attitudes, and decisions related to fertility preservation and parenthood. This review recommends that proactive multidisciplinary oncofertility programs that provide optimal fertility counseling should be included routinely in gynecologic cancer care. These programs should be grounded in a robust collaboration among different specialities, with clear pathways for timely referral to fertility specialists. Supplementing these efforts with educational resources, such as Decision Aids, may increase quality of life and patient satisfaction with fertility counseling provided. Timely identification of patients at risk for mental distress and provision of psychosocial interventions are essential to reduce the likelihood of long-term distress. The continuity of oncofertility care should be available to patients at diagnosis and through survivorship.1 However, they share the same threat of potential loss of fertility. Standard treatments involve surgical removal and/or ablative therapies of reproductive tract organs, in addition to adjuvant therapy in the form of pelvic radiation and chemotherapy. These treatments have potential for finite damage to reproductive capacity.2 Although gynecologic cancers are more frequent in women over 50 years of age; a subset of younger women are diagnosed during reproductive years. As such, infertility and subfertility can impact this population.Gynecologic cancers are a heterogeneous group of malignancies in women, each with different pathological features, clinical presentations and treatment modalities.4 Fertility preservation provides an opportunity for women to achieve future biological children. Since standard gynecologic cancers’ treatment often requires removal of some reproductive organs, fertility preservation may be a complex challenge for these patients. Personalized counseling by a multidisciplinary team should be advised for each patient,5-7 including an evaluation of benefits, risks, and safety.2 Established fertility preservation options may be considered in addition to fertility-sparing strategies.8-10 Family building, including egg donation, embryo donation, use of a gestational carrier, and adoption may constitute appropriate alternatives for women who desire to pursue parenthood.11Fertility is a key component of cancer management and quality of life (QOL) in reproductive-aged women. The need for fertility counseling prior to cancer treatments is strongly recommended by clinical guidelines.Literature identifying fertility attitudes and decisions of young patients with gynecologic cancer is scarce. While oncofertility reviews have been addressed in the literature for women with breast cancer or cancer in general, there are no reviews assessing young women with gynecologic cancer who have greater risk for fertility loss. It is paramount to understand fertility issues of gynecologic patients and to devise more comprehensive counseling strategies and psychological/educational interventions tailored to their context. The aim of this systematic review was to capture and summarize all relevant data on the incidence of fertility counseling provision for women with gynecologic cancer and their knowledge, attitudes, and decisions related to fertility, fertility preservation, and parenthood from their point of view.12 Peer-reviewed journals published in English were searched in PubMed from January 1, 2000 to July 1, 2020. The search was conducted using combinations of these phrases or keywords  guidelines.keywords . An idenSearch results were compiled in Covidence  and duplicates removed. Titles and abstracts were screened independently by 4 authors  to identify eligible manuscripts. Differences were resolved through discussion. The same 4 authors independently reviewed full texts for inclusion in the final sample for extraction. Data extracted from each eligible study were recorded in a standardized extraction table and synthesized using narrative description. To answer the research question, results from original authors’ manuscripts were applied in response to the research questions 15-17 Each study was rated according to MMAT guidelines.17 Since this review intended to capture all published data that met the inclusion criteria, no publications were excluded based on quality ratings. Each study’s quality assessment was performed and discussed by 4 authors  , a widely used instrument that allows for quality appraisal of quantitative, qualitative, and mixed methods studies.., M.S.) .In total, 1106 articles were retrieved; after 69 duplicates were removed, 1037 articles remained . Of thosn = 10). Of those, 9 were cross-sectional18-26 and one was a population cohort.27 The studies were of reasonable quality, ranging from 60% to 80% on the MMAT tool. Five studies used heterogeneous samples of different cancer diagnoses. In addition, 5 studies used samples of patients with gynecologic cancer exclusively. Three studies included only cervical cancer,26 of those, 2 used the same sample;19 one study used only patients with ovarian cancer;22 and one study used a mixed sample of different gynecologic cancer diagnoses.23 In total, 910 patients with gynecologic cancers participated in the studies. Of those, 428 had cervical cancer, 217 had ovarian cancer, 147 had endometrial/uterine cancer, and 118 had another gynecologic cancer diagnosis. In the study by Chin et al,27 63 patients of different gynecologic diagnoses  participated; however, the authors did not provide numbers of patients by diagnosis. Across all studies, the mean age at diagnosis ranged from 23 to 41 years. Three studies failed to report this information specific to each cancer site.27 Length of time since diagnosis varied among studies, ranging from 3.76 years to 11.6 years. Campos et al22 solely stated that 69% of participants were diagnosed within 2 years or less and 31% were diagnosed more than 2 years. In addition, 2 studies did not provide this information by cancer site27 and 2 studies failed to report information on this variable.25The majority of studies were quantitative  compared, over 2 years at different time points, patients who underwent radical trachelectomy with patients who had radical hysterectomy. Participants were, on average, 34.54 years at diagnosis. Data were collected by self-reported questionnaires, containing qualitative items exploring fertility and other issues. This study had low quality with a score of 40% on the MMAT.Only one study used mixed methods.27 Fertility counseling satisfaction was also evaluated by 2 studies. Chan et al,23 assessed 470 women, of those 206 (46%) recalled pre-treatment fertility counseling from their oncologist or surgeon; of those, 47% reported satisfactory fertility counseling. Conversely, Shah et al26 reported that among the 18/39 patients (46%) who received counseling prior to RT, 16/18 (89%) believed it was adequate and helped to make informed decisions. Furthermore, counseling regarding pregnancy risks and complications before RT was reported among 26/38 patients (68%), of whom 88% stated counseling was appropriate and 85% reported it helped to make informed decisions.26 The majority of fertility discussions were conducted before cancer treatment,27 predominantly received in person with a minority of women receiving written information21 and initiated by the oncologist,27 the patient or the partner.27 However, with the exception of Shah et al,26 data on these communication issues were not provided by patients but by the entire sample with different diagnoses. Chan et al23 also reported patients received counseling from surgeons, and other sources  and from more than one provider.23 A similar pattern was observed in patients with cervical cancer who received counseling mainly from their oncologists but also from other sources  with some patients receiving counseling from more than one provider.26 Among all counseled patients in the Chin et al 27 study (n = 660), only 12.9% were referred to a fertility specialist. Low rates of referral to fertility specialists were also observed in 3 studies conducted exclusively with gynecologic cancers.30 In this context, Carter et al30 examined patients who spoke to a fertility specialist at 2 points in time. Results showed 15% (n = 5/33) had a consultation in the first year and, this increased by year 2 (n = 6/33). Campos et al22 found 20% (3/16) were counseled by fertility specialists’ presurgery. A slightly higher rate was captured by Carter et al,20 with a subset of 33% (17/51) patients reporting they had spoken to a fertility specialist, and Shah et al,26 who showed that among the 18 patients who received counseling (in a sample of 39 patients), 39% were counseled by a reproductive endocrinologist.Among 6 studies reporting fertility counseling provision, a wide range of rates were observed, from 29.4% to 91%.27 Accordingly, Ameri et al24 compared women who received pelvic radiotherapy, reporting 80% of patients with gynecologic cancer recalled fertility discussions while 25% of women with other diagnoses recalled those discussions. However, this finding was not consistent in another study,20 where gynecologic cancer survivors expressed significantly more fertility-related informational needs than other survivors, such as Bone Marrow/Stem Cell Transplant (BMT/SCT).Across different cancer types, women with gynecologic cancers were more likely to report receiving fertility counseling.29 while concerns or fears about pregnancy and having children after cancer28 were also reported. Carter et al20 found that fertility issues influenced cancer treatment decisions for one-quarter of survivors (N = 51), while the majority stated inadequate time to complete their childbearing plans. For example, Campos et al22 reported 100% of participants had intention to become pregnant in the future. Carter et al20 reported that overall, parenthood was perceived as the highest importance in life by most survivors; however, almost half also worried about the diagnosis and treatment effect on their offspring’s’ health. This worry was also voiced by cervical cancer survivors who underwent RT, with reports of women preferring childless life after surgery.Women expressed positive attitudes toward fertility and parenthood,26 reported 10% of participants (N-4) who underwent radical trachelectomy expressed that if they had to make the decision again, they would choose radical hysterectomy instead of radical trachelectomy namely due to physical symptoms (n = 3) and psychological distress given multiple failed pregnancies (n = 1). Cancer recurrence concerns also equated to avoidance of sexual activity and attempting conception for some.28Complications associated with fertility procedures may determine negative views toward fertility preservation. Shah et al30 cryopreservation of ovarian tissue,29 and family building.20 The qualitative study by Komatsu et al,28 captured the “meaning” women attributed to their reproductive organs, which were perceived as the core of gender identity, parenthood, sexuality, and female identity. The use of radical trachelectomy provided an opportunity to conceive in the future and a means to restore threatened gender identity since the potential loss of their uterus brought feelings of being incomplete. Participants reported high satisfaction with decision making, even if reproductive outcomes were not achieved. This perception was emphasized by some reporting feeling pressured by health providers to pursue childbearing because they underwent a fertility preservation surgery. The authors highlighted the cultural context where the study took place in which social norms and views of family are often influenced by feminine identify. Positive views toward radical trachelectomy were described by Shah et al26; 90% of participants reported they still would choose radical trachelectomy over radical hysterectomy again. Responses to open-ended questions showed gratitude for the possibility to undergo radical trachelectomy and the procedure had a positive impact on their lives. Carter et al30 also identified motives leading to the choice of this fertility preservation surgery instead of radical hysterectomy. The desire to preserve fertility was a decision factor for the majority of women (42/43). In the radical trachelectomy group, women expressed not having had enough time to complete childbearing compared to radical hysterectomy group. Physician recommendations and, to a lesser extent, research were other reasons to pursue radical trachelectomy. About half of radical hysterectomy group  cited fertility as a factor in their treatment choice. In qualitative responses, the radical hysterectomy group referred to physician recommendation, and recurrence, to guide their decisions. Concerns about future conception were examined prospectively in the radical trachelectomy group, showing that at presurgery, 39 patients (91%) had concerns. The numbers of patients with concerns decreased to 24/33 (73%) by Year 2. Women’s perceptions of future successful conception showed presurgery ratings of 61.8%, and at 6 months 53.7%, followed by 55.4% at year 1, and at year 2 was 59.8%.30 Positive views toward fertility-sparing surgery were also expressed by patients with ovarian cancer with the majority of participants (88%) expressing the surgery was important to them.22Women expressed perspectives on fertility preservation decisions, such as radical trachelectomy for early-stage cervical cancer,29 Quotations from these women highlighted a lack of knowledge about the procedure and a need for improved patient education and follow-up care from health providers regarding the process.A desire for future fertility and positive feelings about ovarian tissue cryopreservation, were also expressed in a qualitative study among women diagnosed with gynecologic cancer who underwent this fertility preservation procedure.20 Most of gynecologic cancer survivors were familiar with surrogacy parenting options, such as surrogacy, and had heard of oocyte retrieval and oocyte donation. Oocyte retrieval was considered by 31% (16/51) of gynecologic cancer survivors, while 61% (31/51) considered oocyte donation and 53% (27/51) perceived surrogacy as a viable option. Although concerns about adoption as a survivor were raised by 35% (18/51), the majority  expressed it would be acceptable to explore alternatives such as adoption or fostering a child. Adoption was perceived as the most acceptable alternative, followed by surrogacy, egg donation, and fostering.20Perceptions of lack of availability of fertility preservation options were expressed by gynecologic cancer survivors  compared to BMT/SCT survivors .30 Data analysis of patients’ questionnaires showed that reproductive concerns were associated with poorer QOL, more cancer-specific distress, less social support, lower spiritual well-being, greater gynecologic pain, and poorer sexual functioning in 51 cervical cancer survivors.18 In addition, questionnaires’ data also revealed that reproductive concerns were related to sadness about inability to bear children (31%), inability to talk openly about fertility (30%), frustration related to childbearing inability (25%), and mourning the loss of ability to have children (25%). Compared to controls, cervical cancer survivors expressed significantly more reproductive concerns.19 However, when compared with survivors of lymphoma or Gestational Trophoblastic Disease, differences were not significant.19 Reproductive concerns’ increase was influenced by feelings of anger and grief related to loss of reproductive ability.19 Similarly, Carter et al30 found an increase in reproductive concerns among gynecologic cancer survivors, BMC/STC survivors, and infertile women without cancer, compared with normative data; however, there were no significant differences among groups. Reproductive concerns were one of the predictors of individual differences in QOL in patients with cervical cancer18.Some authors focused on the impact of fertility issues on psychosocial outcomes.23 Fertility-sparing surgery also yielded reduced decisional regret.23 Sobota et al25 reported on the relationship between regret and the impact of cancer treatments on fertility and the increased risk of fertility-related distress. Findings suggested that treatment-related regret mediated the relationship between desire to have children and fertility-related distress. This mediation analyses were further explored regarding socio-cultural influences on regret showing that country of origin acted as a moderator of this relation. Therefore, for British participants, a greater desire to have children contributed to increased treatment outcome regret and indirectly to more fertility-related distress. For Polish women, the desire to have children was related to distress, but did not have effect on regret. Authors speculated the medical system differences between the 2 countries and women’s awareness of fertility preservation opportunities may have been reflected in those results.Fertility counseling and patients’ satisfaction were associated with reduced fertility decisions’ regret.25 and it was associated with increased regret.23 The inability to fulfill this desire was associated with more reproductive concerns and greater need for coping efforts.19 Sobota et al25 studied the relationship between desire for children and fertility-related distress, reporting the psychological value of children, perceived consequences of cancer, emotional burden, and treatment regret mediated this relationship. Wenzel et al19 showed that self-reported infertile women were significantly more likely to express poorer mental health, more cancer-specific distress, reduced overall, physical and psychological well-being that those who did not report fertility problems.A desire for children at diagnosis predicted fertility distress post-treatment25 The later predictor reflected the finding that Polish participants were more likely to experience higher fertility distress. Authors attributed this result to possible cultural differences between Poland and UK regarding family values often attached to religious beliefs.Illness perceptions, namely the emotional representations  rather than the cognitive representations, negative affect, recruitment site (patients’ recruited through clinics rather than online), and country of origin (Poland vs. UK) were also predictors of fertility distress.20 No differences on mood, reproductive concerns, and mental health QOL among this group of cancer survivors and infertile non-cancer women were found. According to the authors, the emotional experience of infertility is similar among women, regardless of the root of infertility, rather than the premise that cancer survivors experience a double-trauma response of having both cancer and infertility compared to women with non-cancer-related infertility.20Women’s perceived knowledge of family building options was associated with depression and distress levels, in a way that women with a perceived need for more information showed significantly more depression and avoidance than women who expressed no informational needs among gynecologic and BMT/STC survivors.4 advocating the vital need to routinely include fertility counseling in cancer management and the growing body of research addressing fertility for reproductive-aged patients, this systematic review confirms the inclusion of patients with gynecologic cancer in research focusing on this topic remains unacceptably low.Despite guidelines31-34 One primary aspect that emerged from this review is the wide variability of fertility counseling rates, which, may be a reflection of studies’ different methodological approaches and shared decision making oncofertility practices among individual clinicians or hospitals. This substantial variability was also described by other systematic reviews reporting on fertility counseling for young patients living with cancer with different diagnosis.33 Comparisons of fertility counseling rates among gynecologic cancers and other diagnosis yielded conflicting results. Two studies in this review documented that patients with gynecologic cancer were more likely to receive fertility counseling27 due possibility to the cancer being located in the reproductive organs and to the greater awareness of gynecologists or gynecologic oncologists about fertility counseling due to their training.34 However, this view was disputed by other studies,21 which corroborated findings suggesting that patients with gynecologic cancers were less likely to have fertility discussions and therefore opportunities for preservation.36 Across all studies, there is the finding that a significant proportion of patients with gynecologic cancer did not receive fertility counseling or were not referred to fertility specialists. The lack or insufficient fertility counseling occurred even among women who underwent fertility preservation procedures, with some reporting that they underwent these procedures with little knowledge of the implications, risks, and possible reproductive outcomes. Many women in the studies expressed desire for more fertility support29. Fertility preservation among patients with gynecologic cancer poses unique challenges.11 It appears preservation methods are offered to selected patients based, for example, on the type and stage of malignancy.5 A multidisciplinary collaboration between gynecologic oncologists and fertility specialists, prior to initiation of cancer treatments, is imperative to determine the suitability of fertility-sparing treatments, assessing reproductive potential, setting realist expectations regarding future pregnancy, and optimizing opportunities for counseling.37The provision of fertility counseling and fertility specialists’ referral by healthcare professionals is still suboptimal. Nonetheless, collectively, the study findings corroborate the universal view that fertility is an important matter in young patients’ lives, and a crucial issue during diagnosis and survivorship.38 This review supports the optimization of the collaborative model of care to guide oncofertility management for patients with gynecologic cancer as well as a means to improve low referral rates to fertility specialists.30 Such models may improve women’s perceived low fertility counseling satisfaction.23 In this review, patients of reproductive age prioritize information provision.39 Educational tools, such as written information has been reported by patients as a valuable resource.40 Unfortunately, the topic of educational information provision was scarcely explored, with only one study reporting on low availability of written information.21 Educational instruments, such as Decision Aids (DAs) are useful to facilitate the decision-making process, especially when tailored to patients’ needs.41 To our knowledge, there are no validated DAs specifically for gynecologic cancers. However, there is a validated DA for all cancer types, in German42 and a Dutch cancer-specific DAs tailored to cancer type, including cervical, endometrial, and ovarian cancers, which is under development.43From a psychological point of view, a collaborative care model between oncology and fertility specialists has been linked to improved patient knowledge and understanding, better decision making, and reduced long-term regret and dissatisfaction with fertility preservation.44 Further, as some patients may require hysterectomy, carrying a future pregnancy would not be an option and would require the use of a gestational carrier, a practice which is financially unaffordable for some and illegal in several countries.46Understandably, there are ethical issues associated with fertility preservation among gynecologic cancers especially those with metastatic disease. While clinical guidelines suggest fertility counseling be offered to all women, regardless of disease type or stage, many clinicians are hesitant to discuss preservation with patients who have a poor chance of survival.30 with the desire to preserve fertility being one of the reasons guiding treatment decisions, such as radical trachelectomy instead of radical hysterectomy in patients with cervical cancer.28 However, pursuing childbearing also trigged patients’ worries namely related to future offspring health and cancer recurrence fears.28 These concerns have also been shown to lead to avoidance of sexual intimacy,47 fear of pregnancy or preference for more radical surgical procedures. A unique feature to gynecologic cancers identified in this review was that retaining the ability of childbearing was a means to restore a woman’s sense of gender identity perceived to be lost or threatened during diagnosis and treatments.28 In this regard, reproductive organs were seen as the core to parenthood, gender identity, and sexuality. To our knowledge, there are no reports of this type of gender identity perception in relation to reproductive organs in other cancer populations. These perceptions were often fuelled by cultural and family beliefs and norms that may shape women’s attitudes and future decisions regarding the choice of fertility preservation procedures.28 This awareness by health professionals of the impact of cancer on women’s own perceptions and attitudes is crucial when providing fertility counseling to patients with gynecologic cancer. The ability to address women’s psychosocial needs and support them in meaningful ways, including addressing their partners or support people is key to patient-physician quality communication.48 For example, findings from this review showed that some women felt pressured by their health professionals to attempt a pregnancy, because they had chosen fertility-sparing surgery.28 patients with gynecologic cancer held positive attitudes toward alternative family building, with adoption being the most acceptable option, in spite of known reports of discrimination in domestic and international adoption related to a history of cancer.50 Since fertility preservation in patients with gynecologic cancer may be challenging, family building may constitute a viable option to pursuing parenthood. More research is needed regarding women’s views and attitudes on this topic taking into consideration the psychosocial implications51 and the remarkable asymmetries in cultural norms, legislation, and accessibility that exist worldwide regarding family building options.52Fertility and parenthood were generally viewed positively by the majority of patients,23 Conversely, other factors may be inevitable, such as the desire for children at diagnosis with the potential for infertility due to cancer treatments. For those, psycho/logical support, should be provided to support patients within the immediate impact of diagnosis and complex fertility decision-making process and at survivorship, bearing in mind that fertility attitudes and decisions and psychosocial adjustment may evolve over time.53Some factors identified in this review related to poor mental health are modifiable, such as providing timely and comprehensive fertility counseling, which may have, among other psychological outcomes, a positive impact on future regret regarding fertility decisions.25 was the cross-cultural differences regarding fertility attitudes, perceptions, and fertility preservation options available after the diagnosis across different countries, which may have varied implications for patients’ mental health and decisional process. Attention to culture and mores is important when devising effective interventions, international guidelines, and counseling models. Larger and rigorous studies are needed to inform comparisons among groups and assess the role of cultural beliefs in oncofertility models. In practice, with the current evidence, health professionals need to be aware of the impact of cultural beliefs and norms regarding fertility when dealing with emigrants, minorities, or ethnical groups, to provide specific support and information they require.54A noteworthy aspect raised by Sobota et al30 the studies evaluated were retrospective in nature, targeted a variety of outcomes, measured by diverse tools or questions lacking uniformity and were rarely multicentre studies. Despite these limitations, this review provides the first collection of data on this topic for patients with gynecologic cancer. The results have potential to translate to clinical practice and strengthen future research.There are some limitations in this review. First, only peer-reviewed articles in English were included. It is possible that unpublished studies or non-peer-reviewed studies in other languages existed. Second, some studies with mixed diagnoses that also included gynecologic cancer had to be excluded because results were not displayed by cancer diagnosis. It is possible that some contributions from patients with gynecologic cancer were discarded. Third, the majority of the studies included in the review were retrospective, so participants’ responses may be affected by recall bias. Finally, the small number of studies included and the premise that studies would not be excluded from the review based on their quality, we need to acknowledge the methodological limitations of some studies, which makes it difficult to draw robust conclusions and, limits generalizability of findings to a wider patients with gynecologic cancer. Studies’ quality was mostly moderate, ranging from 60% to 80%, especially quantitative studies. Small sample sizes, with a considerable number of studies using heterogeneous diagnosis samples with few patients with gynecologic cancer, biased sampling designs and use of measurement tools that lack validation leave room for methodological improvements of future research. In addition, with the exception of Carter et al,To improve clinical practice, this systematic review recommends that a multidisciplinary approach to oncofertility care, integrating psychological support, should be provided to aid patients at the time of diagnosis and throughout survivorship, grounded in a collaboration among different specialities, with clear pathways for timely referral to fertility specialists. Furthermore, the provision of training to the medical team with the necessary communication skills, confidence and knowledge to implement high standard oncofertility care is needed. Supplementing these efforts with the development of educational resources, such as DAs, may increase QOL and patient satisfaction. Additionally, timely identification of patients at risk for mental distress and providing psychosocial interventions is essential to reduce the likelihood of long-term distress.This systematic review also demonstrated that patients with gynecologic cancer have been much neglected in research regarding the impact of the diagnosis and treatments on their fertility from their own perspectives. Therefore, greater efforts should be made to develop large robust multicenter studies that overcome flaws identified in the existing research, and also provide new knowledge regarding gynecologic patients’ attitudes, perceptions, and decision-making on fertility, fertility preservation options, and alternative family building, taking into consideration cultural, religious, and moral beliefs. Moreover, prospective longitudinal studies are essential to capture possible fluctuations in attitudes and the impact of fertility variables on patients’ mental health over time. Future research should also explore barriers that preclude optimal fertility decision-making to promote a high standard of oncofertility care.To the best of our knowledge, this is the first systematic review to assess fertility and parenthood attitudes, perspectives, and decisions of patients with gynecologic cancer. It captures the unique challenges faced by this population regarding treatment impact on reproductive organs and complex fertility decisions, perceptions, and experiences. In reality, a narrow set of patients have the possibility for fertility preservation, since many have advanced disease (often seen in ovarian cancer) and for whom fertility preservation may not be possible. For early stages of disease, the path of pursuing fertility preservation may be also complex. However, it is paramount to consider that for most of the young patients with gynecologic cancer, fertility and parenthood are of high importance, regardless of their clinical circumstances. Therefore, these patients, including the patients for whom fertility preservation is not indicated or requested, should be clinically managed within multidisciplinary oncofertility programs that provide timely, comprehensive, and optimal fertility counseling and support. The continuity of oncofertility care should be available to patients from diagnosis through survivorship.oyab051_suppl_Supplementary_TablesClick here for additional data file.