The human T-lymphotropic virus (HTLV) is part of the group of retroviruses that share similar routes of transmission to the human immunodeficiency virus (HIV). Coinfection of these viruses can affect the clinical course of both infections, and reports have shown a quicker progression to AIDS and the development of HIV-related opportunistic infections. The current study investigated the demographic characteristics, prevalence, and the subtypes of HTLV among people living with HIV/AIDS (PLWHA) in the State of Pará, Northern Brazil. Blood samples were obtained from patients who were attending a reference unit that provides medical assistance to HIV-infected individuals in the State of Pará, Brazil, during the period of May 2016 to June 2017. Plasma samples were screened by ELISA tests to detect antibodies anti-HTLV-1/2. DNA and viral types were identified by real-time polymerase chain reaction (qPCR). All samples with viral DNA were submitted to nested PCR and nucleotide sequencing. The overall coinfection rate was 1.4% (5/368), and all samples were from subtype HTLV-1a. No cases of HTLV-2 infection were detected. The prevalence of HTLV-1 was higher in females (80%), individuals between 31 and 50 years of age, heterosexual, unmarried, with low monthly income, with secondary educational level or higher, sporadic condom usage, limited number of sexual partners, and no history of sexually transmitted infections. All samples from HTLV-1-infected patients were identified as strains belonging to the subtype 1a (Cosmopolitan), subgroup A . This study identified that the prevalence of HIV/HTLV coinfection has dropped from 8 to 1.3% in the current investigation. There was a shift of HTLV subtype from a predominance of HTLV-2 infection in the past to an actual exclusively HTLV-1a. There was no significant association between economic, sociodemographic, and behavioral characteristics in HIV/HTLV coinfection. The human T-lymphotropic virus (HTLV) is part of a family of human retroviruses with four types, where the HTLV-1 and HTLV-2 are the most well-studied subtypes of HTLV. The HTLV-1 is associated with some severe diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL), whereas the HTLV-2 was not yet clearly associated with any human disorder . CoincidThe HTLV-1 is the most disseminated globally and is considered endemic in southern Japan, in some regions of Iran and Melanesia, sub-Saharan Africa, the Caribbean, and South America . Brazil Molecular studies based on the long terminal repeat (LTR) region have classified HTLV-1 in seven genetic subtypes (1a–1g) and HTLV-2 in four subtypes (2a–2d) . The HTLIn Brazil, in 2018, 43,941 new cases of HIV infection were diagnosed, being 5,084 cases (11.6%) in the north region of the country, where Pará was the state with the highest number of cases. Related to Brazil, the proportion of new HIV infection cases has remained stable over the last years  . Some stConversely, only two studies have investigated the prevalence of HTLV coinfecting PLWHA in the State of Pará, showing an overall prevalence of HTLV infection of 7.4% in 1998  and 3.5%The present study is descriptive, cross-sectional, and observational. The Human Research Ethics Committee of the Health Sciences Institute, Federal University of Pará, approved the study under number 2.601.161. The participants were informed about the study objectives and agreed to participate in the research; they signed a consent form and then answered an epidemiological questionnaire via a confidential structured interview face-to-face. The questionnaire contained questions regarding age, sex, years of education, marital status, and condom use.Written informed consent was obtained from all five coinfected HIV/HTLV individuals for the publication of any potentially identifiable images or data included in this article.A total of 368 PLWHA who underwent clinical and laboratorial care at the Specialized Reference Unit on Special Infectious and Parasitic Diseases  under the Executive Secretariat of Public Health of the State of Pará (Secretaria Executiva de Saùde Pùblica do Estado do Pará-SESPA) participated in the study from May 2016 to June 2017. The UREDIPE is a reference unit where the clinic–laboratorial attendance of the PLWHA from many of the 144 counties of the Pará state is performed. The subjects participating in the present study were from 63 cities of the Pará state.The inclusion criteria were: ≥18 years old, confirmed HIV infection, agreement to participate in the study, and signing of the patient informed consent form. The exclusion criteria were patients with cognitive impairment who were unable to answer the questionnaire in an appropriate way.g for 15 min, transferred to cryotubes, and frozen at −20°C until serological and molecular analyses.Peripheral blood (10 ml) was collected from each participant in a tube containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The samples were transported to the Virology Laboratory of the Biological Sciences Institute, Federal University of Pará. Plasma and formed elements were separated by centrifugation at 8,944 Plasma samples were screened for anti-HTLV-1/HTLV-2 antibodies using a qualitative enzyme-linked immunosorbent assay  in the Virology Section of Evandro Chagas Institute/Health Surveillance Secretariat, Ministry of Health of Brazil. Reactive or inconclusive results were confirmed using Real-time PCR , which also differentiated between HTLV-1 and HTLV-2 infection.TM Genomic DNA Kit  according to the manufacturer’s protocol to amplify 5′ LTR and pol regions. The qPCR reactions were prepared using the TaqMan® Universal master mix according to protocol: 15 μl of Master Mix, 10.5 μl of water, 1.5 μl of Assay-by-Design (primer and probe set), and 3 μl of DNA in a final volume of 30 μl. The cycling protocol used was one cycle of 50°C for 2 min and 95°C for 10 min and 50 cycles of 95°C for 15 s and 60°C for 1 min. An endogenous control  and the non-homologous regions of the pol gene (186 bp) of HTLV-1 and HTLV-2 were used. The primers used were HTLV-1F 5′-GAACGCTCTAATGGCATTCTTAAAACC-3′), HTLV-1R (5′-GTGGTTGATTGTCCATAGGGCTAT-3′), HTLV-2F (5′-CAACCCCACCAGCTCAGG-3′), HTLV-2R (5′-GGGAAGGTTAGGACAGTCTAGTAGATA-3′), Albumin F (5′-GCTCAACTCCCTATTGCTATCACA-3′), and Albumin R (5′-GGGCATGACAGGTTTTGCAATATTA-3′) as previously described , and 5 units of Taq DNA polymerase. In each amplification reaction, after initial denaturation at 94°C for 5 min, 35 cycles were performed, with 40 s at 94°C, followed by 30 s at 57°C and 1 min at 72°C, followed by a final extension for 10 min at 72°C. The primers used for the first step of reaction were LTR-I.01 (5′-TGACAATGACCATGAGCCCCAA-3′) and LTR-I.02 (5′-CGCGGAATAGGGCTAGCGCT-3′), and for the second step, the primers used were LTR-I.03 (5′-GGCTTAGAGCCTCCCAGTGA-3′) and LTR-I.04 (5′-GCCTAGGGAATAAAGGGGCG-3′). The nested PCR products were visualized after electrophoresis (100 V/45 min) on 2% agarose gel in 1 × TAE buffer  that contained 5 μl of ethidium bromide (10 mg/ml) using a transilluminator with an ultraviolet light source.The samples that were confirmed as positive for HTLV-1 were subjected to nested PCR reactions for amplification of the 5′LTR were sequenced three times in both directions using ABI Prism BigDyeTM Terminator Ready Reaction Cycle Sequencing kit, version 3.1 . After precipitation of the reaction product, the samples were denatured and sequenced in an automated ABI 3130 sequencer (Applied Biosystems) following the manufacturer’s protocol.Both strands of the 1. To verify the clustering of HTLV sequences, maximum-likelihood (ML) phylogenetic trees were reconstructed with the PhyML 3.1 (2. The sequences obtained in this study were deposited in GenBank (MT941570–MT941574).All sequences were edited and aligned using AliView software . The subhyML 3.1  under thhyML 3.1  integrat3. Furthermore, all study data collected were entered into an Excel database and converted to BioEstat. To compare the epidemiological characteristics of the PLWHA in relation to the HIV/HTLV coinfected subjects, the Fisher’s exact test and the G test were used. The Fisher’s exact test and G test were used to compare the epidemiological characteristics of PLWHA in relation to those coinfected with HIV/HTLV. Odds ratio (OR) and associated 95% confidence interval (CI) were used as measures of the strength of independent association between HTLV/HIV infection and individual variables and the variation of this strength. The numbers of HTLV-positive and -negative cases detected in this study were also compared with the results found in two studies conducted with PLWHA in the Brazilian State of Pará in 1998 . However, the value is very close and can be considered safe and representative of the population, as predicted in the calculation.This study accessed information and biological samples of 368 PLWHA treated at the main specialized service for HIV/AIDS in the Brazilian State of Pará. Although the study accessed many PLWHA attended in UREDIPE from May 2016 to June 2017, it was not enough to achieve the estimated minimum sample size (The mean age was 42.3 years (ranging from 18 to 69 years of age). The majority of PLWHA were male, aged between 31 and 50 years, heterosexual, unmarried, had an educational level of secondary school or higher, and were living in cities of the interior of the state. Routine condom use during sexual intercourse was reported by 58.7% and up to one sexual partner over last year for 85.9% of the participants. Sixty percent had no history of other sexually transmitted infection (STI). In 368 PLWHA, five (1.4%) had anti-HTLV-1/anti-HTLV-2 antibodies using ELISA. Amplification of the albumin gene fragment was detected in the five PLWHA samples, indicating success in DNA isolation. HTLV-1 DNA was detected in all samples positive for anti-HTLV-1/HTLV-2 antibodies, which were all genotyped as belonging to the subtype 1a (Cosmopolitan), subgroup A  . Table 2A = -13.02; χ2 = 11.26; p < 0.05). Similarly, a decreasing trend in cases of HTLV-2 infection has also been identified .From the comparison of the numbers of positive and negative cases of HTLV infections of this study with findings from previous studies, a decreasing trend of cases of HTLV infections in PLWHA was detected  is considered hyperendemic for HTLV-2, especially HTLV-2c, which has been described in Amerindian tribes from the 1990s to date . FurtherA remarkable characteristic found here is that the HTLV carriers showed no history of multiple sexual partners and illicit drugs use, which is a different profile from the previous findings in other Brazilian studies . HoweverThe change in the epidemiological scenario observed in the present study, with the predominance of HTLV-1 instead of HTLV-2, may reflect a shift in the coinfection profile by HTLV in PLWHA in the State of Pará. The impact of this change has yet to be assessed, considering that all individuals participating in the present study were asymptomatic, similar to what was previously described in the city of Belém, Pará, where HTLV-2 predominated in this population .Our group is conducting clinical and laboratory monitoring of these individuals in order to better describe the impact of HIV-1/HTLV-1 coinfection in the clinical course of these patients in the future, bearing in mind that this coinfection could cause a greater risk of developing HAM, different from what has already been observed in HTLV-2 coinfection .This study has limitations that should be considered. First, this epidemiological investigation selected a sample of PLWHA attending a reference unit for HIV/AIDS control and may not represent a full picture by excluding those who abandoned treatment. It is important to note that in Brazil, the public health system offers free universal treatment for individuals infected by the HIV. Another limitation is that qPCR tests were only carried out in individuals who tested positive to HTLV-1/2 screening using ELISA, so recent infections with a small concentration of anti-HTLV-1/2 antibodies may not have been detected, thus resulting in false-negative test result . The smaThis study identified that the prevalence of HIV/HTLV coinfection is consistently dropping over the last 22 years in the State of Pará when the first epidemiological survey took place. A shift of HTLV subtypes was also observed—from a predominance of HTLV-2 infection in the past to an exclusively HTLV-1a (Cosmopolitan subtype) in the current investigation. In addition, the presence of HTLV-2 was not detected in PLWHA, especially HTLV-2c, which is hyperendemic in several populations in Northern Brazil. There was no significant association between economic, sociodemographic, and behavioral characteristics in HIV/HTLV coinfection. These results suggest that the prevalence of HTLV infection in the PLWHA population in the State of Pará, North Region of Brazil, is lower than those previously related for the same region and that a shift regarding the HTLV subtype circling in PLWHA may also be occurring, with the predominance of HTLV-1a (Cosmopolitan subtype).All datasets presented in this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Human Research Ethics Committee of the Health Sciences Institute, Federal University of Pará. The patients/participants provided their written informed consent to participate in this study.SA and LM contributed to the conceptualization. MS, CM, RF, and VA contributed to data curation. SA, AR, MB, RL, FF, and VA contributed to the investigation and methodology. SL, AO-F, and LM contributed to the formal analysis. SA, AO-F, and LM contributed to writing the original draft. SA, RL, AO-F, and LM contributed to writing, reviewing, and editing. LM contributed to the project administration. All authors contributed to the development of the research, read, and approved the final manuscript.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.


This cross-sectional study evaluates factors associated with fertility preservation counseling before initiating chemotherapy among patients of reproductive age with recently diagnosed cancer. What factors were associated with discussions about fertility preservation before initiating chemotherapy and referrals to reproductive specialists among patients with recently diagnosed cancer?In this cross-sectional study of 6976 patients of reproductive age, 43.5% had a discussion with their clinician about the risk of infertility associated with chemotherapy; women were more likely to be counseled. Discussions of fertility preservation occurred at higher rates in academic centers compared with private practice settings, and mandated state laws for fertility preservation were associated with improved frequency of discussing fertility risks.The findings suggest that the American Society of Clinical Oncology quality measures should be better disseminated for patients of reproductive age regarding discussion of infertility risk and fertility preservation and appropriate referral to fertility specialists before the onset of chemotherapy. The opportunity to discuss fertility preservation is essential for patients of reproductive age with newly diagnosed cancer before the initiation of treatment.To identify factors associated with fertility preservation counseling among patients of reproductive age before initiating chemotherapy.This cross-sectional study used data obtained from the American Society of Clinical Oncology (ASCO) Quality Oncology Practice Initiative, an oncologist-led quality assessment program that surveys approximately 400 oncology practices biannually, from January, 2015, to June, 2019.The primary outcome was whether reproductive risks were discussed before initiation of chemotherapy. Multivariate logistic regression was performed to identify factors associated with fertility preservation counseling, controlling for age, sex, race/ethnicity, cancer type, year of study, region, clinic type (academic vs private), annual clinic volume, and rates of insurance coverage.P < .001). Factors associated with reduced likelihood of fertility risk discussion included male sex , increasing age , private practice setting , and lack of multidisciplinary team planning . Factors associated with increased likelihood of fertility risk discussion included having breast cancer  and lymphatic or hematopoietic cancers , participating in each subsequent study year , receiving care in an academic clinic , and being a practice offering clinical trial enrollment . States with legislatively mandated coverage of fertility preservation had significantly higher rates of fertility risk discussion compared with states without legislation .Among the 6976 patients of reproductive age  age, 42.5 [7.1] years), with reproductive age ranging from 18 to 40 years for 3405 women and from 18 to 50 years for 3571 men, clinics reported that 3036 of 6976 patients (44%) received counseling regarding the risk of infertility associated with chemotherapy. Women were more likely to be informed (1912 of 3405 [56%]) compared with men (1126 of 3571 [32%]) (The findings suggest that clinicians are more likely to counsel younger patients and female patients about reproductive risks before initiation of chemotherapy. State laws mandating fertility preservation coverage may be associated with improved frequency of fertility counseling before chemotherapy. Further awareness and implementation of ASCO guidelines appear to be needed to improve rates of fertility risk discussion and referrals to fertility specialists before chemotherapy. Whereas chemotherapy and radiotherapy are effective in eliminating cancer cells, adverse effects on the reproductive system persist. In males, this toxic environment can directly damage the germinal epithelium, causing azoospermia or oligozoospermia, or can compromise the neural pathway responsible for erection and ejaculation.7 Although the initial guidelines were published in 2006, ASCO has since released 3 iterations; the most recent guidelines were published in 2018.7 Current guidelines recommend that clinicians address the possibility of infertility as early as possible before treatment starts and refer to an appropriate specialist for patients who express an interest in fertility preservation. Whether a patient is a candidate for or chooses to pursue fertility preservation, having this discussion may be associated with reduced distress and improved quality of life. A study6 evaluating the ASCO Quality Oncology Practice Initiative (QOPI), a nationwide database reporting 150 quality measures, found that only 6% of US patients had a fertility preservation discussion before undergoing chemotherapy between 2006 and 2010. In recent years, there has been increased effort to improve the rates of fertility counseling before chemotherapy, including increased education for oncologists regarding the risks of infertility associated with chemotherapy, additional updates of ASCO guidelines, and an increased number of states passing legislative initiatives requiring insurance carriers to cover fertility preservation for those requiring chemotherapy.8 Given these efforts to improve fertility counseling, we used recent data from the ASCO QOPI to assess current rates of fertility counseling in reproductive-aged patients and evaluated factors associated with discussion of fertility risks and fertility preservation before initiation of chemotherapy.The American Society for Reproductive Medicine and the American Society of Clinical Oncology (ASCO) recommend that health care clinicians caring for adult and pediatric patients with cancer discuss fertility preservation before the initiation of anticancer therapy.STROBE) reporting guideline for a cross-sectional analysis.9This cross-sectional study used data from the ASCO QOPI, which surveys approximately 400 clinical practices yearly, covering 150 evidence-based quality measures. Reporting by clinics is voluntary and not all measures are reported annually. An ethics waiver was obtained from the University of Miami institutional research board, Miami, Florida, because anonymized data were procured from the ASCO QOPI data set. Informed consent to be included in the data set was obtained by participating clinical practices. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  infertility risks associated with chemotherapy were discussed, (2) fertility preservation options were discussed, or (3) referral was made to a reproductive specialist. Patients of reproductive age were defined as women between the ages of 18 and 40 years and men between the ages of 18 and 50 years. Our analysis included patients who were defined as being of reproductive age, received a diagnosis of a cancer that required chemotherapy, and eventually underwent chemotherapy. Data from clinical practices within the US from January, 2015, to June, 2019, were included in our analysis. Patients with a history of infertility or purposeful sterilization were excluded from the analysis.10 To assess whether fertility counseling had occurred, surveyors assessed documentation on whether patients had received information about adverse effects associated with chemotherapy, including infertility. Examples of documentation detailing a risk of infertility included documentation within the electronic medical record, consent forms, treatment plans, and/or drug information sheets on given drugs highlighting serious reactions that require contacting the practice and discussing the risk of treatment, including prevention and management. Discussion of infertility risk or referral to a fertility specialist was determined if such discussion was documented or a referral was made.11The ASCO QOPI follows strict protocols to ensure quality assurance of their data that are received from certified institutions. Two rounds of data acquisition were conducted at the respective center with at least 2 reviewers to ensure data were acquired correctly. A clinician knowledgeable in the QOPI measures was also assigned from each specialty to assist in the data acquisition process. Surveyors looked for documentation that the patient had received information about adverse effects, including infertility risks.Patient-level data available included sex, age, race/ethnicity, type of cancer, year of inclusion, whether the patient underwent discussion regarding infertility risk associated with chemotherapy, and whether the patient was counseled regarding fertility preservation options or referral to a reproductive specialist. In addition to patient-level variables, data were available for each clinical practice. Practice-level data included (1) the number of clinical sites per practice; (2) the region of the practice; (3) the number of new patients seen per year; (4) academic status of the practice; (5) the number of oncology attending physicians, nurse practitioners, and physician assistants per practice; (6) the percentage of patients with commercial insurance, Medicare, Medicaid, or no insurance; (7) the percentage of patients for whom English was not their primary language; (8) whether the practice offered multiple oncologic specialties; (9) whether the practice participated in quality improvement planning; (10) whether the practice participated in multidisciplinary team planning; and (11) whether the practice offered clinical trial enrollment for cancer therapies.The primary objective of this study was to assess whether any patient or practice-level characteristic was associated with increased prevalence of fertility counseling. Given the complex association between these variables, a multivariate logistic regression was performed controlling for the above variables.12Two secondary analyses were subsequently performed. The first analysis assessed the association of counseling with subsequent referral to a fertility specialist. The second analysis assessed the association of state legislative changes requiring insurance coverage of fertility preservation with changes in counseling and referral to fertility specialists. Legislative fertility preservation policies for each state were found on Resolve.org.P < .05.The Kolmogorov-Smirnov test was used to assess normality of numerical variables. Univariate comparative statistics were performed using the Mann-Whitney test and Fisher exact test when appropriate. Multivariate logistic regression was performed using R, version 3.4.1 . Statistical significance was 2-sided and set at P < .001) (P < .001) (P < .001) compared with those from other regions  (P < .001)  identified as being of reproductive age . Overall < .001) . The fre < .001) , B. Ther < .001) , C. Pati < .001) , D. Duri < .001) , E.In multivariate logistic regression, the following factors were associated with reduced likelihood that the fertility risks associated with chemotherapy were discussed : male seThe following factors were associated with increased likelihood that the fertility risks associated with chemotherapy were discussed : breast Of 940 patients younger than 30 years , 636 (67.7%) were counseled about the risk of infertility associated with chemotherapy and 523 (55.6%) were subsequently told about fertility preservation options or referred to a reproductive specialist. Of the 304 patients in this group who were not counseled about the risk of impaired fertility associated with chemotherapy, only 32 (10.5%) discussed fertility preservation or were referred to a reproductive specialist.In 2018, 4 states began legislatively mandated coverage of fertility preservation: Rhode Island, Connecticut, Delaware, and Maryland. In 2019, Illinois joined in legislatively mandated coverage of fertility preservation. In states with legislatively mandated coverage of fertility preservation, patients who were counseled about the risk of infertility were more often counseled regarding fertility preservation or were referred to a fertility specialist after passage of mandated coverage . For states without legislatively mandated coverage of fertility preservation, there was a slight improvement in the rate of discussion of fertility preservation or referral to a fertility preservation clinic after passage of mandated coverage in other states .7 We hypothesized that key factors associated with variability in fertility rate discussions were sex, age, type of center in which the patient received care , and percentage of insurance coverage within a clinic. All of these factors had significant associations with variability in these discussions.Chemotherapy may be associated with patients’ future fertility; thus, counseling patients about these risks before initiation of chemotherapy is necessary. The current study was the largest evaluation to date on whether infertility risk was discussed and preservation options were presented to reproductive age adults antecedent to cancer therapy. Of the 6976 patients in this cross-sectional study using a national quality database, we found that 43.5% of reproductive age patients had a discussion regarding the risk of infertility associated with cancer treatment, despite ASCO recommendations.13 This finding suggests that there exists a need both in the US and elsewhere to identify strategies to increase fertility education and indicated referrals to fertility specialists for adult patients with newly diagnosed cancers.Our findings were consistent with a recent survey of 167 female adolescents and young adults from Japan that demonstrated that only 48% of patients with nonbreast and nonhematologic malignancies had received information about the association of cancer therapy with reproductive organ health.14 included 22 Canadian physicians who were interviewed regarding adherence to ASCO guidelines about fertility preservation conversations with patients. Multiple physicians in this study14 expressed that they did not believe that they were qualified or knew enough about fertility preservation to have conversations about this issue. This study14 found that often physicians attributed the responsibility of fertility counseling to other clinicians, focused on the part of the body being treated, and did not see fertility preservation as their responsibility. In these multispecialty clinics, physicians possibly deferred these conversations, assuming that colleagues would address risks, potentially allowing such conversations to be missed.As expected, counseling and referral were more common in the academic rather than the private clinic setting. Academic institutions might facilitate a more rapid collaboration between oncologists and reproductive specialists. Stemming from this notion of more rapid collaboration, several other variables proved to accompany higher rates of fertility counseling. Specifically, associated factors included whether patients were cared for in multispecialty clinics, were cared for through multidisciplinary team planning, or were seen in a clinic that also offered clinical trials. Surprisingly, multispecialty clinics had reduced rates of fertility risk counseling compared with single specialty clinics. One theory for this decreased rate of counseling in multispecialty clinics could be an unclear delineation of who is responsible for discussing the risk of infertility associated with chemotherapy. A recent qualitative study by Covelli et alOur analysis also found that clinics in which clinical trials were offered had higher rates of discussions regarding fertility risks associated with chemotherapy, even when controlling for private vs academic status. These clinics offering clinical trials may be more familiar with more rigorous oversight, informed consent, and internal institutional guidelines, thus facilitating increased rates of counseling.14 In an interview with oncologists, Covelli et al14 found that clinicians felt an absence of confidence in their ability to engage in fertility preservation discussions, often feeling uncomfortable when patients had questions.14 Ongoing educational alignment between oncologists and fertility specialists may be of value as a strategy to help practices meet ASCO recommendations. Furthermore, additional research should investigate whether unique barriers exist in private practice oncology settings, followed by undertaking quality improvement initiatives.Although oncologists likely understand the association of chemotherapy and radiotherapy with fertility, many may be unaware of contemporary fertility preservation options.We demonstrated that infertility risks were discussed infrequently with patients of reproductive age before chemotherapy despite ASCO guidelines. Protecting options for future parenthood at the time of diagnosis with cancer and appropriate referral to a reproductive specialist should be part of the routine practice of oncologists. Quality improvement initiatives to improve rates of fertility risk discussion and referrals for fertility preservation should be undertaken by clinicians in facilities who are currently deficient on these metrics, and the routine use of ASCO QOPI quality data could track improvements made after implementation of these initiatives. Quality initiatives may include prompts on electronic medical records for patients meeting criteria for fertility discussion and/or referral.Moreover, we demonstrated that clinicians were more likely to counsel younger patients and female patients. Thus, efforts should be undertaken to reduce these biases to ensure equitable care of patients. States with mandated coverage for oncofertility preservation had improved rates of fertility discussion and referral. Therefore, we encourage advocacy efforts to ensure passage of similar laws in other states to improve patient access to reproductive health.11 In addition, data included in this study were recent because study years ranged from 2015 to early 2019. Thus, these data are clinically relevant and representative of the present clinical situation.This study has strengths. This study represents, to our knowledge, the largest sample of reproductive-aged patients from a national database. The ASCO QOPI database provides numerous patient and clinic-level variables, allowing multiple factors to be assessed in our multivariable analysis. Furthermore, the ASCO QOPI uses strict protocols to ensure quality of reported data. Clinical sites undergo an extensive application and trial period with multiple rounds of reviewers assessing submitted data.This study has limitations. Data pertaining to reproductive history of patients  were unknown, and this may be an important factor associated with whether infertility risks or fertility preservation options were discussed. Although the ASCO QOPI uses multiple layers of review to ensure quality of the data, infertility risk and preservation counseling are self-reported from oncology practices and are subject to error. Discussion of infertility risks may have occurred without medical record documentation, or documentation may have occurred without discussion. Our data only represent whether counseling occurred, but do not fully capture the scope or extent of these conversations. Furthermore, data about patients younger than 18 years are not collected in the ASCO QOPI database, limiting analysis of a group of patients for whom fertility counseling is critical. Data are also not collected for those undergoing radiotherapy or surgery instead of chemotherapy, a subset of oncologic patients for whom fertility discussion is also pertinent. In addition, because chemotherapeutic regimens and fertility referrals were chosen by the physician, it was unclear whether the infertility risks associated with a selected chemotherapeutic regimen were associated with whether fertility referral was provided and with clinic accessibility to a fertility center.The findings suggest that clinicians are more likely to counsel younger patients and female patients about reproductive risks before initiation of chemotherapy. State laws mandating fertility preservation coverage may be associated with improved frequency of fertility counseling before chemotherapy. Further awareness and implementation of ASCO guidelines appear to be needed to improve rates of fertility risk discussion and referrals to infertility specialists before chemotherapy.


N = 238, 157 men). We designated the levels of serum UA measured at the time of admission as the independent variable and the degree of neurological impairment at admission as the dependent variable. The National Institutes of Health Stroke Scale (NIHSS) was used to assess the extent of neurological dysfunction: NIHSS ≤ 5, minor stroke; NIHSS > 5, moderate to severe stroke. There was a statistically significant difference in UA levels between patients with mild cerebral infarctions (NIHSS ≤ 5) and those with moderate or severe cerebral infarctions (NIHSS > 5) (P < 0.0001). After adjusting for confounding factors, the serum UA level was found to be nonlinearly related to NIHSS, and the inflection point was 372 μmol/L. The extent of the influence and confidence interval was 0.99  on the left side of the inflection point and 1.00  on the right side. There was a nonlinear relationship between the UA level measured on admission and the degree of neurological impairment in patients with acute primary cerebral infarction. When UA was <372 μmol/L, it was negatively correlated with the degree of neurological impairment in patients with acute cerebral infarction, but when UA was ≥372 μmol/L, the protective effect of UA disappeared.Numerous studies have shown that uric acid (UA) is associated with cerebrovascular disease, but whether UA is a protective factor or worsens the risk of developing cerebrovascular disease remains controversial. This study investigated the relationship between UA levels detected at admission and the severity of acute primary cerebral infarction. This cross-sectional study enrolled patients with acute primary cerebral infarction ( With its highest incidences in Asia—especially in China—and Eastern Europe, cerebral infarction remains a global concern. Moreover, according to the European Stroke Burden Report, no changes in the current incidence of cerebral infarctions will increase the total number of stroke events by more than 30% between 2015 and 2035 . PatientThis cross-sectional study explored the relationship between UA and the severity of acute primary cerebral infarction. The level of serum UA at the time of admission was selected as the independent variable and the degree of neurological impairment at admission as the dependent variable.We collected baseline data from patients who were hospitalized in the Department of Neurology, the First People's Hospital of Changde City, from November 1, 2018, to August 31, 2019. The criteria for inclusion in this study were as follows: (1) incidence time < 48 hours and (2) an acute cerebral infarction that was confirmed by MRI during hospitalization. Exclusion criteria were as follows: (1) a history of cerebral infarction, malignant tumor, gouty arthritis, kidney disease, systemic infection, or an autoimmune disease; (2) the recent intake of drugs related to UA metabolism; and (3) missing data. All data were obtained from the participating hospital's electronic medical record system.The National Institutes of Health Stroke Scale (NIHSS) was used to assess the extent of neurological dysfunction. The NIHSS value was assessed and recorded by a neurologist at the time of admission. An NIHSS score of ≤5 was considered to indicate a minor stroke; NIHSS scores of >5, moderate to severe stroke –12.The specific process of UA measurement was as follows: (1) Venous blood was collected on the first day of the patient's admission and sent to the laboratory through our hospital's transportation system. (2) UA activity was measured using an oxidase method UA kit, the performance of which was approved by the national testing agency.P value of the trend. The purpose of the test was to verify the results of treating UA as a continuous variable and to determine the possibility of nonlinearity. All the analyses were performed with the statistical software package R  and EmpowerStats . P values of <0.05 (both sides) were considered statistically significant.We represent continuous variables in two ways. The continuous distribution of normal variables is expressed as the mean ± standard deviation, while skewed distributions are expressed as medians and interquartile ranges (Q1-Q3). Classification variables are expressed in terms of frequency or percentage. We used a one-way analysis of variance , the Kruskal-Wallis H (skewed distribution) test, and the chi-square test  to examine differences between different UA groups. Our method of data analysis can be divided into two steps. In the first step, we explored the linear relationship between UA and NIHSS by using three linear regression models: Model 1, without covariate adjustments; Model 2, adjusted for social demographic data only, including age and sex; and Model 3, adjusted for all the covariates shown in Based on our strict screening criteria, a total of 238 patients were selected for final data analysis. A total of 51 were excluded, including 10 patients with a history of kidney disease, two patients with a history of cancer, and 39 patients with an onset time of more than 48 hours (see P < 0.05), but no difference in age, systolic blood pressure, diastolic blood pressure, total cholesterol, LDL cholesterol, HDL cholesterol, urea nitrogen, history of hypertension, history of coronary heart disease, history of diabetes, and drinking .The baseline data characteristics of the patients are shown in The results of the univariate analysis are listed in μmol/L. The finding can be explained as follows: the use of the unadjusted model (crude model) indicated an 8% reduction in the risk of moderate to severe cerebral infarction  for every 10 μmol/L increase in UA. In the minimal adjustment model (Model I), the results were interpreted as an 8% reduction in the risk of moderate to severe cerebral infarction  for every 10 μmol/L increase in UA. Finally, after we completely adjusted the variables (Model II), the results could be explained by a 9% increase in the risk of moderate to severe cerebral infarction for each UA increase of 10 μmol/L . For the sensitivity analysis, we also treated UA as a quarantine (quartile) and observed the same trend. As UA concentration increased, the risk of moderate to severe cerebral infarction decreased.The results obtained from the adjusted and unadjusted models are presented in μmol/L, and the influence and confidence intervals of the left and right sides of the inflection point were determined to be 0.99  and 1.00   , respectμmol/L, UA was negatively correlated with the degree of neurological damage in patients with acute cerebral infarction. Yang et al. [This study suggested that when UA was <372 g et al.  found UAg et al. , 15. Chig et al.  posited g et al. , NIHSS sg et al.  found thg et al.  found thg et al.  found thThe neuroprotective effects of UA can be explained in the following ways: (1) UA has an antioxidative effect, preventing the cerebrovascular system from being damaged by oxidative stress , 22. (2)μmol/L, to minimize neurological damage. In particular, UA supplementation may have greater clinical significance in patients with acute episodes who cannot be treated with thrombolytic therapy. This will be our next research direction.Many previous studies have revealed a relationship between UA and the severity of cerebral infarction, and our study provides supporting evidence for the protective effect of UA on nerve function in patients with acute cerebral infarction. In this study, the nonlinear relationship between UA and the severity of cerebral infarction was determined, curve fitting was carried out, and the inflection point was calculated. The results were intuitive. It was found that a correlation remains, despite the adjustment of many confounding factors. A meta-analysis of 12739 patients with acute cerebral infarction suggested that, for patients with acute cerebral infarction, high levels of UA at onset have neuroprotective effects and are markers of a better prognosis, especially for patients undergoing thrombolytic therapy, regardless of the type of thrombolytic therapy . This isThis study also has several limitations: (1) It was small, mainly comprising patients with acute primary cerebral infarction, and all participants were Chinese. (2) As a cross-sectional study, it could not provide evidence of causality. Large prospective cohort studies are needed to investigate this. (3) Previous studies have shown that UA may be related to the size, type, and location of cerebral infarction , 27. Stuμmol/L, it was negatively correlated with the degree of neurological impairment in patients with acute cerebral infarction, but when UA was ≥372 μmol/L, the protective effect of UA disappeared.There was a nonlinear relationship between the UA level measured on admission and the degree of neurological impairment in patients with acute primary cerebral infarction. When UA was <372


Oncolytic viruses provide a biologically multi-faceted treatment option for patients who cannot be cured with currently available treatment options. We constructed an oncolytic adenovirus, TILT-123, to support T-cell therapies and immune checkpoint inhibitors in solid tumors. Adenoviruses are immunogenic by nature, are easy to produce in large quantities, and can carry relatively large transgenes. They are the most commonly used gene therapy vectors and are well tolerated in patients. TILT-123 expresses two potent cytokines, tumor necrosis factor alpha and interleukin-2, to stimulate especially the T-cell compartment in the tumor microenvironment. Before entering clinical studies, the safety and biodistribution of TILT-123 was studied in Syrian hamsters and in mice. The results show that TILT-123 is safe in animals as monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. The virus treatment induces acute changes in circulating immune cell compartments, but the levels return to normal by the middle of the treatment period. The virus is rapidly cleared from healthy tissues, and it does not cause damage to vital organs. The results support the initiation of a phase 1 dose-escalation trial, where melanoma patients receiving a tumor-infiltrating lymphocyte therapy are treated with TILT-123 (NCT04217473). Oncolytic adenovirus therapy was approved for the treatment of advanced head and neck cancer in China in combination with chemotherapy in 2005 . Since tOncolytic adenovirus treatments have generally been well tolerated in humans. The typical adverse events are grades 1 and 2, including flu-like symptoms, headache, nausea, fatigue and dizziness, anemia, leuko- and thrombocytopenia, elevated liver transaminases, and diarrhea. Before using a new therapy on patients, it is important to evaluate the safety in preclinical models. Syrian hamsters serve as a good preclinical model for adenovirus product safety because it is semi-permissive for adenovirus replication, unlike mice, which are nonpermissive . ImmunocHere, we investigate the preclinical safety and biodistribution of an oncolytic adenovirus TILT-123 (Ad5/3-E2F-d24-hTNFa-IRES-hIL2). TILT-123 is a chimeric adenovirus based on type 5 with a fiber knob from type 3, which is used to increase the tumor cell transfection through desmoglein-2 ,9. The EA biodistribution and toxicity study was conducted in Syrian hamsters following Good Laboratory Practices (GLP) at Cellvax S.A.S. . Syrian hamsters, aged 6–7 weeks males and females, were purchased from Janvier Labs  and acclimatized for at least three days before the treatments. The animals received six TILT-123 injections in total . The first injection was either intravenous  or intraperitoneal (i.p.). The other injections were given i.p. The doses were diluted with 0.9% sodium chloride , which was also administered to the mock control animals A,B.Hamster wellbeing was followed daily, and weight development was recorded once a week. In addition, food and water consumption was recorded weekly. On predetermined days, five male and female animals per group were sacrificed to collect blood, secretions , and organs  for biodistribution analyses, histopathology, blood cell analysis, and serum chemistry analyses.5 virus particles (VP), 1 × 107 VP, or 1 × 109 VP (corresponding to approximately 4 × 106–4 × 1010 VP/kg) twice a week, six times in total (9 VP and 100 µg of murine anti-PD-1 (InVivoMAb anti-mouse PD-1 (CD279), BioXcell, Lebanon, NH, USA). The control groups received saline (PBS) or anti-PD-1 alone. Anti-PD-1 administration started after two virus treatments and was given i.p. Five animals per group were sacrificed on day 21 to collect samples  for virus biodistribution, toxicity, and cytokine analyses. The rest 6–7 animals/group were left for survival experiment, where tumor growth and animal weight development were followed.Nude/NMRI female mice  were acclimatized for seven days before experimentation. Human fibrosarcoma tumors (HT-1080) were engrafted subcutaneously and allowed to grow until 0.5 mm in diameter. The animals received 1 × 10in total C. The fi® PCR method for TILT-123 detection and quantification in hamster tissues and biofluids was developed and validated at Accelero Bioanalycs GmbH . The primers for detecting the virus genomes in hamster tissues were designed to amplify a sequence between IRES and IL-2 in TILT-123, a region which does not appear in any naturally occurring naïve specimens: forward primer CGAACCACGGGGACGTGGT; reverse primer GCAAGACTTAGTGCAATGCAAGACAGG; probe FAM-GATAATATGGCCACAACCATGTACAGG-MGB. Before qPCR, an external PCR was first used to amplify the target sequence . The hamster GAPDH housekeeping gene was analyzed as a control [7 VP/sample.TILT-123 distribution in animals was evaluated by qPCR. A real-time TaqMan control . The limMouse tissues were analyzed by detecting the 24-bp deletion region from adenovirus E1A. Beta-acting gene expression served as a reaction control .The collected hamster organs, except for epididymitis and testis, were fixed in 4% formalin for at least 48 h. The epididymitis and testes were fixed in Davidson’s fluid for 48 h, then washed, and stored in 70% ethanol at room temperature before further processing. Mouse tissues were fixed with 10% formalin for 48 h and stored in 70% ethanol. The tissues were embedded in paraffin wax, cut to 4-μm sections, and stained with hemalum, or hematoxylin and eosin. Veterinary pathologists analyzed and scored the slides.Blood was collected by heart puncture from anesthetized or euthanized animals. Blood cells and hemoglobin parameters were analyzed from hamster blood treated with the K2EDTA anticoagulant. Coagulation parameters and blood chemistry were analyzed from hamster plasma separated from the whole blood in citrate tubes or in lithium heparin, respectively. Mouse blood was collected into dry tubes and the serum was separated by centrifugation after blood clotting.Ad5/3-neutralizing antibodies were analyzed from hamster serums 3 days after i.v. injection and two weeks after the treatment period (day 77) as described before . BrieflyThe expression of TILT-123 transgenes human TNFa and human IL-2 were analyzed from hamster and mouse serums with human ELISA kits . The serum was extracted from whole blood in dry tubes. In addition, the transgenes were analyzed from homogenized mouse organs and tumors. The cytokine levels in tissues were normalized against the total protein content in the sample, measured with a Bradford assay.Selective replication of TILT-123 was studied in A549 lung adenocarcinoma cells , human primary hepatocytes , human MRC-5 fibroblasts (ATCC), and human vascular endothelial HUVEC cells (Millipore). The cells were plated on 24-well plates and infected with TILT-123, wild type Adenovirus 5 , Ad5/3 replicative control virus (a similar selection device as TILT-123 but no cytokine payload), or Ad5/3-Luc1 non-replicative control virus ,19. InfeThe virus was released from the cells by repeating freeze–thaw cycles four times in total. DNA was extracted from the samples with the phenol/chloroform/isoamyl alcohol (25:24:1) method and the virus copy numbers determined by detecting the adenovirus E4 copy number in samples by qPCR . Infectip-value smaller than 0.05 was considered significant.Statistical analyses were performed with GraphPad Pism 8.2 using the Mann–Whitney or ANOVA test. Tumor growth curves, animal weight development, and water and food consumption were analyzed using linear mixed model analysis in IBM SPSS Statistics version 24.0.0.2. A 10 VP/kg to 1 × 1012 VP/kg. Repeated TILT-123 injections did not cause premature deaths or visual signs of organ damage in hamsters. All hamsters had positive weight development over time, but the treatment period caused temporary weight loss  corresponded to the ones planned to be used in a clinical setting, ranging from 1 × 10ght loss . The treThe highest treatment dose increased the number of leucocytes in hamster blood only after the first virus injection A. This wIn the middle of the treatment period, the only statistically significant difference in the blood cell populations was a slightly reduced reticulocyte level in animals treated with the highest dose E. LymphoWhen the first injection was given i.v., the changes in blood cells were similar as when the injection was i.p. . The leuBlood clotting was delayed in some animals in both the treatment and the control groups at the last sampling time point (day 185), when the animals received six i.p. injections . IncreasEven though the erythrocyte level was slightly elevated with the highest dose at the last sampling point after i.p. injections, it did not affect the red distribution width . HoweverThe serum chemistry analyses provided data for TILT-123 toxicity evaluation together with histopathological analyses. A veterinary pathologist did not observe any treatment-related abnormalities in healthy hamster tissues. Also, no major differences were observed in the serum chemistry values between the vehicle control and treatment groups.Compared to the control group, i.p. treatment with any dose acutely elevated aspartate aminotransferase and total protein levels in blood  and with anti-PD1 alone. Of note, all cured tumors were among the smallest 25% at the beginning of treatment, indicating that initial size might have influenced the likelihood of a curative response. The control animals gained more weight than the treated animals during the experimental period, but the differences were not statistically significant  E. RegardAs some virus was observed in healthy tissues after treatments, selective replication was studied further in vitro. Human primary hepatocytes, vascular endothelial cells, and fibroblasts were infected with TILT-123 or the control viruses, and the presence of physical and infectious virus particles were studied 72 h post-infection. Neither physical nor infectious TILT-123 particles were detected in the normal cells A,B. HoweNew treatment modalities are urgently needed, especially for metastatic and recurrent cancer. Immunotherapies provide promising results in part of this patient population, but complete responders are still rare . ResponsHere, we evaluated the preclinical safety of TILT-123 as a monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. Treatment doses, schemes, and routes of administration mimic the clinical setting. Monotherapy studies were conducted in Syrian hamsters, which allow human adenovirus replication, including chimeric 5/3 type adenoviruses ,10. The Because the hamster study included long-term follow up, it was not feasible to have the animals bear tumors. Thus, all injections were given i.p. To compare different systemic routes of administration, some of the hamsters received the first injection i.v. Because the aim of the mouse study was to collect acute safety data, tumors could be engrafted and treated i.t. after an initial i.v. treatment, as planned in the clinical setting.10–1 × 1012 VP/kg). The main difference between i.p. and i.v. injection routes was biodistribution: i.v. administration spread the virus more widely in the body. Virus genomes were mainly found from the spleen and gonads after i.p. administration, whereas when given i.v., the gonads were unaffected but most of the virus was located in the liver. The observed i.p. biodistribution profile is compatible with direct transduction of i.p. organs, while i.v. appears to give access to the liver.The results show that TILT-123 is safe in animals when administered in clinically relevant (and above) doses  transgenic mice might allow greater transduction and can cause more effects on healthy tissues. In the used model, the highest copy number levels in normal tissues were observed in the spleen and liver, indicating a similar biodistribution as in hamsters. The results are also in accordance with the observations of the biodistribution of other oncolytic adenoviruses ,29,30,31To summarize, similar safety and biodistribution profiles were seen in immunocompetent hamsters and in immunocompromised mice. Good safety results were not compromised when TILT-123 was combined with anti-PD-1. Overall, the data reported here indicate that the virus does not replicate in normal tissues and that it is rapidly cleared from nontumor organs. These data support clinical translation of TILT-123.The study shows that repeated administration of TILT-123 is safe in animals as monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. Acute changes in blood values indicate that the virus induces immunological reactions in immunocompetent animals but that the values return to normal levels after the acute phase. The virus is also rapidly cleared from healthy tissues, where it cannot replicate. Importantly, the treatment did not cause any tissue-level changes in animals.


Quality of life improvement stands as one of the main goals of the medical sciences. Increasing cancer survival rates associated with better early detection and extended therapeutic options led to the specific modeling of patients’ choices, comprising aspects of reproductive life that correlated with the evolution of modern society, and requires better assessment. Of these, fertility preservation and ovarian function conservation for pre-menopause female oncologic patients pose a contemporary challenge due to procreation age advance in evolved societies and to the growing expectations regarding cancer treatment. Progress made in cell and tissue-freezing technologies brought hope and shed new light on the onco-fertility field. Additionally, crossing roads with general fertility and senescence studies proved highly beneficial due to the enlarged scope and better synergies and funding. We here strive to bring attention to this domain of care and to sensitize all medical specialties towards a more cohesive approach and to better communication among caregivers and patients. The female perspective on fertility after cancer treatment is, nowadays, an important issue. A 2012 study that analyses the information received from cancer survivors has found that there is a sex bias. Thus, men received more information regarding the influence of treatment on fertility 80% vs. 48%, and more men also received information about options to preserve fertility—68%, compared to 14% for women  and the A 2012 review of fertility demographics in USA showed an increase in the number of women giving birth after 30 years of age, with a peak for white women at 35 years . The infChemo-therapeutic agents, known for deleterious effects, include alkylating agents that are considered high risk, such as Cyclophosphamide, Mechlorethamine, Chlorambucil, Busulfan, and Melphalan, whose active metabolites form DNA crosslinks leading to its function and synthesis arrest . They prThe 2018 American Society of Clinical Oncology (ASCO) recommendations paint a clear image of current onco-fertility focusing and emerging possibilities. While patients will first be taken aback by a cancer diagnosis, it is recommended that, early in the therapeutic process, discussions be initiated with the fertile patients concerning the risk of diminished fertility induced by specific treatment and about the options available for fertility preservation. Oocytes and embryo freezing, due to technological progress, are considered the standard of care and are widely available. Considering the conflicting evidence concerning GnRH agonist use to protect ovarian reserve during oncologic treatment , ASCO reIn this context of social pressure, and ultimately, financial stimulus, assisted reproduction technologies had a steep curve of evolution. In this process, IVF has greater visibility and striving for successful pregnancies produced more embryos. A limited number of the better-quality embryos are being selected for intrauterine embryo-transfer, while the remaining good quality embryos were frozen for cryopreservation. The resulting technological prowess led to increasing rates of embryo survival after freeze-thaw, and in turn, to a greater number of pregnancies, similar rates of pregnancies being obtained for fresh embryos and frozen ones. This highlighted the idea that all embryo-transfers should be realized with frozen embryos in a subsequent cycle; in favor of this judgment is the standing lower risk of ovarian hyperstimulation syndrome and a higher endometrial receptivity due to different gene expressions in stimulated versus unstimulated endometrium ,22. It sThere are patients for whom embryo cryo-preservation was not an option, and because oocyte cryo-preservation techniques had slower progress, ovarian tissue-slow-freeze represented the only fertility preservation method. Lately, oocyte cryo-preservation methods achieved good results, and the Practice Committee of the American Society for Reproductive Medicine (ASRM) reclassified oocyte cryopreservation technology as “nonexperimental” in 2013 . While mOvarian tissue cryo-preservation may be an alternative to oocyte preservation, because for some patients’ ovarian tissue cryo-preservation, it may eliminate the need for the oncologic treatment delay needed for stimulation cycles for oocyte retrieval. There is hope that ovarian tissue transplant reinstates not only fertility, but also endocrine function. The slow freeze was compared with vitrification methods for the ovarian tissue  and someDuring the antenatal period, human ovaries lose to follicular atresia 80% of its germinal cells to roughly 500,000–1,000,000 at birth  and reacAs an alternative to ovarian tissue transplants, probing the hypotheses that limited success with ovarian tissue strips is due to the limited and late graft vascularization, there were animal and human trials with whole ovary transplants with limited success . Some liOwing to the dispute that entangles the use of GnRH agonists , alternaThe advent of new technologies brought new fields of research, and while ovarian strip auto-transplant harbors hope for both fertility preservation after oncologic therapy and menopause delay, studies searching for artificial support or even fully artificial ovaries are in full stride. A team is proposing a bioprosthetic ovary that was assembled using 3D printed microporous scaffolds in order to restore ovarian function . AdditioSocietal pressures pushed forward the long and successful experience with ART due to the increasing age of childbearing, which, in turn, exposed women intending to procreate to a higher risk of malign conditions. This is coupled with the better chance and longer disease-free survival by novel chemotherapy schemes that produce a growing population of women trying to conceive at increasing ages, often after oncologic treatment. Oncofertility studies crosslink with female aging studies and general fertility. There are promising technologies from oogonial stem cells activation, artificial scaffold bioprosthetic ovaries, and proactive ovarian tissue extraction for future use that could push female fertility farther away into what is now senescence.


Hyperuricemia is a common metabolic disease that is caused by high serum uric acid levels. It is considered to be closely associated with the development of many chronic diseases, such as obesity, hypertension, hyperlipemia, diabetes, and cardiovascular disorders. While pharmaceutical drugs have been shown to exhibit serious side effects, and bioactive compounds from plant-based functional foods have been demonstrated to be active in the treatment of hyperuricemia with only minimal side effects. Indeed, previous reports have revealed the significant impact of bioactive compounds from plant-based functional foods on hyperuricemia. This review focuses on plant-based functional foods that exhibit a hypouricemic function and discusses the different bioactive compounds and their pharmacological effects. More specifically, the bioactive compounds of plant-based functional foods are divided into six categories, namely flavonoids, phenolic acids, alkaloids, saponins, polysaccharides, and others. In addition, the mechanism by which these bioactive compounds exhibit a hypouricemic effect is summarized into three classes, namely the inhibition of uric acid production, improved renal uric acid elimination, and improved intestinal uric acid secretion. Overall, this current and comprehensive review examines the use of bioactive compounds from plant-based functional foods as natural remedies for the management of hyperuricemia. Hyperuricemia (HUA) is a common metabolic disease caused by an imbalance between endogenous production and excretion of urate . RecentlAs mentioned above, HUA can be caused by either an increase in UA production or a decrease in UA metabolism in the body, with reduced urate excretion being the most common mechanism, accounting for about 90% of cases ,15. XantPlant-based functional foods are derived from natural or unprocessed plant foods, or plant foods modified via biotechnological means . They arFlavonoids are polyphenols with a basic 2-phenyl-chromone structure , and thePueraria lobata (Willd.) Ohwi and Wang et al. randomly divided 120 HUA patients into a control group, a myricetin group, and a puerarin group. After injection with 5 mL/d puerarin injection and 5 mL/d myricetin, the changes in the UA levels in patients with HUA were observed. The obtained results showed that the serum uric acid (SUA) levels of the myricetin and puerarin groups decreased significantly (p < 0.05), indicating that myricetin and puerarin present obvious therapeutic effects on HUA [At present, only a few clinical studies have shown that flavonoids from plant-based functional foods can effectively lower UA levels. For example, following the isolation of puerarin from s on HUA .Sophora japonica L.), exhibits a variety of biological activities. In their study into the effect of quercetin on HUA rats, whereby a rat model of HUA was induced via the administration of potassium oxonate. Xie et al. found that after three weeks of administration, quercetin (10 mg/kg/d) significantly reduced the levels of SUA and inhibited the activities of XOD and ADA in both serum and the liver (p < 0.05) [Quercetin, one of the major flavonols mainly found in onions and sophora japonica ( < 0.05) . In addi < 0.05) . Further < 0.05) . QuercetIpomoea batatas L. possesses a high content of anthocyanins (ACNs) [p < 0.01), in addition to effective decreases in the serum and liver XOD activities in mice (p < 0.05). Furthermore, treatment with high doses of ACNs significantly down-regulated the mRNA expression levels of the URAT1 and GLUT9 (p < 0.001), while up-regulating the mRNA expression levels of OAT1, OAT3, and ATP-binding cassette subfamily G member 2 (ABCG2) (p < 0.05) in the kidney. Moreover, ACNs treatment lowered blood urea nitrogen (BUN) and serum creatinine (Scr) levels, while up-regulating the mRNA expression levels of organic cation transporters (OCT1 and OCT2) and organic carnitine transporter (OCTN1 and OCTN2) compared with the model group (p < 0.01). It has also been suggested that ACNs exhibit hepatoprotective activities and nephroprotective effects, thereby suggesting overall that ACNs are potential treatments for HUA [s (ACNs) . In one  for HUA .The hypouricemic effect of flavonoids has been studied in particular detail compared to other natural products. Although the majority of previous studies have focused on the inhibition of XOD, the number of studies on UA transporters are gradually increasing. Besides, studies on monomeric compounds remain scarce, and in general, there is a lack of high-quality clinical research. For example, it has been shown that stevia residue extract can reduce SUA levels in HUA mice, and this was attributed to the presence of flavonoids , therebyPhenolic acids, which are secondary metabolites, are non-flavonoid phenolic compounds. They represent a substantial part of the human diet . In receCichorium intybus L., Zhu et al. established a quail HUA model to elucidate the active ingredients and mechanism of C. intybus L. in combating HUA. After 21 days of administration, chicory acid (150 mg/kg/d) significantly reduced the quail SUA (p < 0.05). Moreover, the quail serum ADA and XOD levels were also significantly reduced (p < 0.05), which may be related to inhibition of the XOD and ADA activities. Overall, chicory acid significantly reduced quail SUA levels by inhibiting the XOD and ADA activities [In the context of chicory acid, which was isolated from tivities ,73.Coix lachryma-jobi L.) [p < 0.05). Moreover, the BUN and Scr levels were significantly reduced compared with the model control group, and caffeic acid was found to reduce BUN to the normal range. Besides, an in vivo study showed that caffeic acid regulated the transcription levels in a dose-dependent manner by up-regulating the expression of UA secretory transporters OAT1 and ABCG2 mRNA, and down-regulating UA reabsorption transporters URAT1 and GLUT9 mRNA. Furthermore, an in vitro study showed that caffeic acid can inhibit XOD by competitively binding to xanthine, with an IC50 value of 53.45 µM being recorded [Phenolic antioxidants, including phenolic acids, have been isolated from Adlay (jobi L.) . Upon threcorded . TherefoHowever, to date, despite extensive research into phenolic acids, few studies exist regarding the regulation of UA transporters. In addition, there is a lack of anti-HUA clinical data, and few reports have been published on the role of phenolic acids in regulating transport proteins. These issues must, therefore, be solved to enhance the applicability of phenolic acids for the treatment of HUA. The hypouricemic effects and mechanisms of action of the various bioactive phenolic acids found in plant-based functional foods are listed in Alkaloids are a class of nitrogen-containing organic compounds that exist in many organisms . Due to Nelumbinis folium (lotus leaf) for the lowering of the UA levels. UHPLC-Q-TOF-MS and 3D docking analysis were employed to show that roemerine was a potentially active component. More specifically, roemerine bound with XOD through hydrophobic interactions, inhibited the activity of XOD, and reduced the production of UA [p < 0.05), and the excretion of UA increased significantly (p < 0.05) for the high-dose nuciferine group (40 mg/kg). It has since been reported that the mechanism of lowering UA is related to down-regulation of the expression of URAT1, GLUT9, and up-regulation of the expression of OAT1 and ABCG2 in HUA mice [Recently, Sang et al. evaluated the effective components present in a total alkaloid extract from on of UA ,79. FurtHUA mice .Evodia rutaecarpa (Juss.) Benth, and has been shown to exhibit an obvious effect on lowering SUA levels. Tao et al. established animal models of HUA in rats and chickens to observe the effect of evodiamine on lowering SUA after 7 and 14 days of administration. Compared with the model group, the low and high dosage groups (9 and 18 mg/kg) reduced SUA values in rats, while the low dose group showed significantly reduced SUA values in HUA chicken [Evodiamine is the main active component of  chicken . Similar chicken .Although alkaloids have been shown to prevent and control HUA through a variety of mechanisms, exhibiting a significant effect on lowering UA levels, due to a lack of clinical data, larger numbers of studies must be conducted in the context of clinical trials and toxic doses. The hypouricemic effects of alkaloid bioactive components in plant-based functional foods and their mechanisms of action are summarized in Saponins are mainly distributed in terrestrial plants, and small amounts are also found in marine life. Based on their different aglycones, saponins can be divided into steroidal saponins and triterpenoid saponins. In recent years, studies have shown that saponins can reduce UA production by inhibiting the activity of XOD and ADA, and that they can also increase UA excretion by regulating the expression of UA transporters .Dioscorea opposita L. [p < 0.05). In addition, dioscin significantly increased the 24 h cumulative urinary excretion of creatinine (p < 0.05), the protein level of renal mOAT1 in HUA mice treated by dioscin increased significantly at high dosages (1276.86 mg/kg/d), and the protein level of renal mURAT1 decreased. Thus, the mechanism by which dioscin lowers UA levels involves transporter regulation [As an example, the anti-HUA mechanism dioscin, which is mainly distributed in osita L. , was invgulation .Gynostemma pentaphyllum (Thunb.) Makino (GPS). Compared with the model group, the low (15 mg/kg) and high dosage groups (60 mg/kg) exhibited dramatically reduced SUA levels . Moreover, after being treated with a high dosage of GPS, the UA level was close to normal (p < 0.01), and the levels of XOD and ADA in the serum and liver decreased (p < 0.05). Treatment with GPS also increased the kidney index, downregulated URAT1 and GLUT9 expression, and upregulated OAT1 expression in the kidney. GPS may, therefore, be an effective treatment for HUA through the inhibition of XOD and ADA, and an increase in UA excretion by regulation of the URAT1, GLUT9, and OAT1 transporters [HUA rats were also treated with saponins from sporters ,90.Overall, saponin extracts act by inhibiting UA generation, promoting UA excretion, and protecting the kidneys. At present, research into the anti-HUA activities of saponin extracts are in their initial stages, and the identification of additional pharmaceutically active monomer components is necessary. Moreover, investigations into the pharmacological mechanisms of any monomeric species are desirable, as is the development of safe and effective new drugs demonstrating anti-HUA properties.Significant attention has been paid to the extraction and bioactivity of biomacromolecules, such as polysaccharides. Polysaccharides obtained from natural sources tend to exhibit a low toxicity in addition to various bioactivities, such as anti-bacterial, anti-inflammatory activities ,92. In rLonicera japonica (Thunb) is recognized as a medicine food homology species. The L. japonica polysaccharides have been studied for their hypouricemic effect in potassium oxonate-induced HUA mice. Interestingly, with an increase in the polysaccharide dose, the level of UA was significantly lowered, demonstrating that the oral administration of polysaccharides could treat HUA in a dose-dependent manner. In particular, in the high-dose group, the level of SUA was reduced, and compared with the model group, SUA levels in the middle and low dose groups decreased by 47.93% and 43.41%, respectively (p < 0.01). The low, middle, and high dose groups  showed the capability to inhibit the activity of XOD , thereby indicating that L. japonica (Thunb) polysaccharides could significantly attenuate HUA in rats [p < 0.05). The protein expression results showed that the expression of URAT1 was significantly down-regulated compared with the model group, while the expression of OAT1 was significantly increased. These results revealed that PPC increased the reabsorption of UA by down-regulating the expression of URAT1, while up-regulating the expression of OAT1 to reduce the re-secretion of UA [To further study the effect of pachman  on HUA, Wang et al. fed rats with potassium oxonate and ethambutol to establish an animal model for HUA, and then treated with PPC. The high-dose PPC group (2.0 g/kg/d) exhibited an increase in the fractional excretion of UA, while the level of SUA and any pathological changes in renal tubules were reduced compared with the model group  have also been found to exhibit UA-lowering effects. For example, gardenoside and acteoside have been reported to present significant hypouricemic effects ,99. MoreAs mentioned above, the bioactive components of plant-based functional foods prevent UA disorders by inhibiting the enzyme responsible for UA production, by enhancing the excretion of UA, and by preventing its reabsorption. These mechanisms of action are summarized as follows.XOD and ADA are key enzymes that catalyze the production of UA. XOD catalyzes the oxidation of hypoxanthine to xanthine and xanthine to UA . ADA plaL. japonica Thunb. and Dendranthema morifolium (Ramat.) Tzvel., and exhibits its anti-HUA effect by inhibiting the excess production of UA [p < 0.01) [Luteolin is mainly present in on of UA . Hao et  < 0.01) . In anot < 0.01) , therebyp < 0.01, p < 0.05), whereby the UA level was close to normal for the high dosage group (p < 0.01). Moreover, the serum and liver levels of XOD and ADA also decreased (p < 0.05). These results confirmed that GPS significantly reduced the production of UA through inhibition of the XOD and ADA activities [Lipid emulsion-induced HUA rats have been used to study the effects of GPS on lowering UA levels. In this study, the low (15 mg/kg) and high dosage groups (60 mg/kg) presented dramatically reduced SUA levels  resulting in lower UA levels than for the model group (p < 0.05), and a significantly decreased XOD activity for the high dose group (p < 0.05) [Galangin extracted from  < 0.05) . Recentl < 0.05) .SLC22A12 (URAT1) and SLC22A11 (OAT4), and release into the blood through the anion transporter SLC2A9 (GLUT9) from the renal tubular epithelial basolateral membrane [SLC22A6 (OAT1) and SLC22A8 (OAT3)), in addition to GLUT9b on the apical membrane of proximal tubules, play an important role in the secretion of UA [Physiologically, UA production and excretion are in a dynamic balance. However, when UA excretion is reduced and excess UA is produced, thereby leading to HUA . As a remembrane ,24,25. GNumerous studies have demonstrated that the bioactive components of plant-based functional foods increase UA elimination by up-regulating  and down-regulating  UA transporters in the kidneys . They alp < 0.01), while TFDG (20 mg/kg) and TF-3-G (50 mg/kg) also clearly reduced the SUA levels (p < 0.05). These results indicated that the hypouricemic effect of TF was superior to those of TFDG and TF-3-G at the same dosage. With the exceptions of TFDG (20 mg/kg) and TF-3-G (20 mg/kg), BUN was also reduced in other treatment dose groups (p < 0.01), while TF(20 mg/kg), TFDG (20 and 50 mg/kg), and TF-3-G  also decreased Scr (p < 0.001). These components could, therefore, treat renal damage in HUA mice by decreasing BUN and Scr levels. In addition, they down-regulated the expression of the genes and proteins of GLUT9 and URAT1, while up-regulating the gene and protein expression of OCTN1, OCT1, OCT2, OAT1, and OAT2 [Theaflavins are important functional ingredients in black tea. Among them, studies have shown that the three theaflavin monomer, namely theaflavin (TF), theaflavin-3-gallate (TF-3-G), and theaflavin-3-3′-gallate (TFDG) exhibit significant hypouricemic effects on HUA mice. Compared with the model group, the TF , TFDG (50 and 100 mg/kg), and TF-3-G (100 mg/kg) groups notably decreased SUA levels , while GTP dosages of 300 and 600 mg/kg markedly reduced the XOD activities in the serum and liver of HUA mice (p < 0.05). Furthermore, these dosages reduced the expression of URAT1 (p < 0.05), as well as increasing the expression of OAT1 and OAT3 in the kidneys (p < 0.01). Overall, the results suggested that GTP reduced UA levels by inhibiting UA production and increasing its excretion [The effects of licochalcone A from the  of OAT4 . Besidesxcretion .Sophora japonica L.), a potassium oxonate-induced HUA model was established in mice. HUA mice were randomly divided into six groups. Compared to that in control mice, treatment with rutin  caused significant reduction SUA, Scr, and BUN, serum and kidney uromodulin levels, while elevating UA excretion in HUA mice. Further, rutin was administered orally 1 h, significantly downregulated mRNA and protein levels of mice GLUT9 and URAT1, and upregulated mRNA and protein levels of OAT1 and OCTs in the kidney of HUA mice. In conclusion, rutin exerted its hypouricemic and renal function improvement by the regulation of renal organic ion transporters [Furthermore, to examine the lowering UA effects of rutin from Sophora japonica . Compared with the model group, ABCG2 was up-regulated on the jejunum and the ileum. Further research showed that chicory can significantly increase ABCG2 mRNA expression to reduce UA levels in the jejunum and ileum.In addition, Morimoto et al. found that the expression of ABCG2 in an HUA rat group was up-regulated in the intestinal villi and crypt. They confirmed that ABCG2 is involved in the intestinal excretion of UA in humans and rats as an extrarenal excretion pathway, thereby providing some clarification regarding UA metabolism along the intestine by focusing on a novel UA exporter, ABCG2 . FurtherHUA can lead to life-threatening disorders that are rapidly increasing in frequency worldwide, and so the consumption of functional foods could be considered an alternative to medication to prevent or treat HUA. In this context, plant-based functional foods are of particular interest since they contain thousands of naturally beneficial phytochemicals. Numerous in vitro and in vivo experiments have therefore been conducted to elucidate the mechanism by which these plant-based foods lower UA levels, whereby active ingredients such as flavonoids, phenolic acids, and alkaloids reduce the production of UA or promote its excretion. Indeed, it was confirmed that plant-based functional foods are very helpful for the management of UA disorders; however, these studies have their limitations. For example, although animal models can fully reflect the pharmacological actions and metabolic processes of active ingredients, rapid and simple screening is challenging due to the long cycle, and current animal models are limited due to differences in the UA metabolism in humans and animals. In addition, the majority of in vitro experiments carried out to date mainly screen for XOD inhibition, and there is a lack of comprehensive animal model base studies. Furthermore, the anti-HUA effects of many plants have been examined without the determination of the bioactive compounds responsible for their activities, and there is a lack of substantial clinical data and dose-toxicity data to support the applicability of bioactive ingredients in humans. Moreover, there is a lack of relevant research data on intestinal UA elimination by bioactive compounds, and studies focusing on the mechanisms by which such active ingredients act are scarce and vague. Novel approaches are therefore required to identify bioactive ingredients from plant-based functional foods, evaluate their efficacy in human and animal models, and develop a sustainable and natural means of treating or preventing HUA. In this context, molecular docking technology has been used to elucidate the mechanism and structural characteristics of polyphenols inhibiting XOD, which is of great significance for the development and synthesis of XOD inhibitors for the treatment of HUA. Since the function of the UA transporter is essential for the maintenance of normal UA levels, further studies into the function of the UA transporter will provide a new strategy for the treatment of HUA and related diseases. In addition, the intestinal tract should be investigated in further detail as a new route to UA secretion, providing a potential new target for the development of natural drugs against HUA. In combination with clinical trials, a comprehensive study into the bioactive ingredients present in plant-based functional foods is necessary, and the diets of patients at high risk from suffering high UA levels should be altered. In conclusion, a large number of studies have confirmed that many biologically active compounds from plant-based functional foods possess anti-HUA activities, which therefore provides a theoretical basis for the synthesis of novel anti-HUA drugs, and suggests the potential of plant-based functional foods for the future prevention and management of HUA.


Glioblastoma is the most common form of primary brain tumor. Glioblastoma stem cells play an important role in tumor formation by activation of several signaling pathways. Wnt signaling pathway is one such important pathway which helps cellular differentiation to promote tumor formation in the brain. Glioblastoma remains to be a highly destructive type of tumor despite availability of treatment strategies like surgery, chemotherapy, and radiation. Advances in the field of cancer biology have revolutionized therapy by allowing targeting of tumor-specific molecular deregulation. In this review, we discuss about the significance of glioblastoma stem cells in cancer progression through Wnt signaling pathway and highlight the clinical targets being potentially considered for therapy in glioblastoma. Glioblastoma also known as glioblastoma multiforme (GBM) is one of the most lethal and commonly identified forms of brain tumor . Around Wnt signaling pathway is mediated by Wnt family of 19 secreted glycoproteins (bearing22 or 24 cysteine residues) which are essential for cell proliferation, embryonic development, cell polarity, and tissue homeostasis  and non-The current review focuses on the importance of Wnt signaling pathway in glioblastoma stem cell progression and explores targeting of Wnt signaling molecules as a potential therapy for glioblastoma.Glioblastoma stem cells (GSCs) can differentiate into different cell types . GSCs coChemotherapy and radiotherapy are commonly used adjuvant therapies in GBM patients who have undergone surgery. Despite treatment advances, GBM is associated with high recurrence and reduced time for patient survival . GSCs arThe GSC-associated resistance to therapy in GBM is addressed through a triaged approach: one which physically allows drug targets to effectively reach the cells through change in tumor microenvironment –33, secoWnt signaling is known as an important regulator of intercellular interaction, cell fate decision, and migration. Developmental defects are attributed to mutations in the critical components of Wnt signaling whereas aberrant Wnt signaling is associated with cancers. Canonical Wnt components bind to cell surface receptors namely frizzled receptor (FZD) and activate low-density lipoprotein receptor-related protein 5, 6 (LRP5/6) complex. Activation of these receptors leads to destabilization of complex consisting of adenomatous polyposis coli (APC), glycogen synthase kinase-3 (GSK3), and Axin . This st2+-mediated pathway where binding of wnt protein to FZD receptors activates DVL and phospholipase C (PLC) followed by activation of inositol 1,4,5-triphosphate (IP3). IP3 interacts with Ca2+ channels in the endoplasmic reticulum and initiates the release of Ca2+ ions followed by activation of protein kinase C and cdc42, CAMKII, TAK1, NLK, and NFAT [Wnt non-canonical pathway consists of two different pathways depending on the mediators. Disheveled (DVL)-c-jun N terminal kinase (JNK) pathway/planar cell polarity pathway involved in cellular polarity. Binding of wnt to FZD via DVL, JNK kinase activates the pathway responsible for changes in cytoskeleton . The secand NFAT –53.Aberrant Wnt signaling is found in various types of cancers. Activation of Wnt pathway is these tumors may be associated with mutations in sentinel components of the pathway viz, APC, β-catenin, AXIN, WTX, and TCF4. Wnt signaling mutations are most extensively characterized in colorectal cancers. It has been observed that close to 85% of colorectal tumors has a loss-of-function mutation in APC whereas 50% of colorectal tumors which lack an APC mutation have activating mutation in β-catenin. Mutations in Wnt signaling components of colon cancer and medulloblastoma were found to be localized in β-catenin, APC, and AXIN1. Mutations of Wnt components are well characterized in various other cancers including hepatocellular carcinoma. Contrastingly, aberration of main components of Wnt pathway is not of common occurrence in GBM, ovarian, head and neck, breast, and gastric cancers , 55. HowA recent study pointed to the role of proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), a co-regulator of several nuclear receptor proteins, activating β-Catenin, thereby activating the Wnt signaling pathway. Overexpression of PELP1 was observed in 100 % of the GBM samples .miR-138-2-3p and miR-770-5p were differentially expressed. These two have not been studied for their role in Wnt signaling aberration in GBMs, but their ability to modulate β-catenin has been ascertained in other cancers such as hepatocellular carcinoma and laryngeal cancers [In addition to the mutations in the main components constituting the Wnt pathway, few studies have also identified the role of epigenetic modifications that regulate Wnt pathway. One such study compared the Gene Expression Omnibus microRNA profiling of GBM versus the normal brain and found that  cancers .Long noncoding RNAs (lncRNA) namely HOX transcript antisense RNA (HOTAIR), nuclear enriched abundant transcript 1 (NEAT1), and maternally expressed gene 3 were negatively associated; differentiation antagonizing non-protein coding RNA (DANCR) was found to be positively affecting GBM progression through Wnt activation .As discussed above, GBMs are driven by a subset of stem cells that self-renew and attribute tumor heterogeneity in addition to therapeutic resistance. However, the role of Wnt signaling in GBM stem cells remains poorly elucidated. For instance, A study showed that PLAGL2 contributed to stemness in GBM through activation of components of canonical pathway namely, WNT6, FZD9, and FZD2 . OverexpmiR-22-3p and miR-22-5p [miR-34a negatively correlates with patient survival in GBM. In vitro studies in GSCs have established the role of miR-34a expression in targeted degradation of β-catenin. Although the role of miR-34a in GSCs has already been established for notch signaling, its role as a modulator of the Wnt pathway has not been reported earlier [Epigenetic modulation of Wnt pathway in GSCs has also been reported. lncRNA MIR22HG was found to be overexpressed in vitro in glioblastoma stem cell lines and has been functionally shown to be responsible for activation of Wnt signaling, by producing iR-22-5p . Contrar earlier .While cursory studies exploring the activation of Wnt pathways in GBM and GSCs are reported, an in-depth study for identification of Wnt activators/deregulators either directly or through other pathways in GSCs remains elusive. The data remains further unexplored on patient-derived GSCs since most of these studies are in vitro models of GSC lines. Nevertheless, identification of these genetic or epigenetic modifications that result in aberrant Wnt expression in GSCs may serve as therapeutic or even as prognostic targets.Wnt signaling pathway plays an important role in many human cancers. It has been reported that there is continuous activation of β-catenin in tumors though no mutations in the major components of the Wnt pathway or alterations in the signaling pathways. These findings confirm that there are others factors capable of activation of β-catenin and its downstream pathways . EpidermS100β-v-erbB/p53−/− glioma stem-like cells were found to have increased survival and decreased cell renewal when treated with agents brefeldin A and EHT-1864 [Conventional multimodal treatment strategies employed for GBM have failed to prevent the recurrence and improve patient survival. While these strategies may have worked with other cancers, their failure in part, in GBM, is attributed to GSCs. The lack of therapeutic approach to prevent GSC renewal and promote tumorigenesis influences disease outcomes. Contrarily, targeting GSCs is challenging for its resemblance to normal neuronal stem/progenitor cells. We have reviewed studies which explore targeting GSCs in GBM and their implication on treatment outcome. An in vivo study by Almiron et al. targeted the hypoxic pathway in GSCs. Mice allografted with EHT-1864 .KDM2B, JmjC domain histone H3K36me2/me1 demethylase, the chromatin regulator, is highly expressed in GBM and plays a role in GSC survival by modulating β-catenin stability. In vitro studies on patient-derived GBM cells demonstrate that genetic or pharmacological inhibition (GSKJ4) of KDM2B results in a decrease of GSC pool along with increased sensitivity to chemotherapy . SeveralThe existing literature establishes that Wnt plays a significant role in maintaining stemness of normal stem cells thereby allowing repair and regeneration. However, a deregulated Wnt pathway may result in onset of cancer stem cells which eventually results in enlarged tumor mass and proliferation. Despite evidence to the de-regulation of Wnt pathway in GBM-associated GSC, the large number of ligands and receptors for either canonical or non-canonical Wnt pathways or cross talking pathways makes systematic classification difficult. However, from currently reviewed studies, it is evident that direct mutations in components of Wnt signaling pathway  are not significantly reported. Alternatively, genetic or epigenetic modulation of regulatory pathways influencing Wnt signaling in GSCs is more routinely reported. With new evidence, initial impressions may be changing and thus the significance of Wnt signaling deregulation in generation of GSC in GBM cannot be disregarded.GBM is the most lethal form of gliomas involving GSCs. GSCs undergo differentiation, cell proliferation, and self-renewal and maintain stemness by following many signaling pathways. Of these pathways, Wnt signaling pathway is important for differentiation of GSCs. Aberrant Wnt signaling in GSCs renders them resistant to conventional chemo- and radiotherapy thereby making it necessary to assess the efficacy of alternate treatment strategies. While these strategies are experimental in nature, their medical application is limited. The hindrance is the heterogeneity associated with the plasticity of GSC surface markers making recognition and thus targeting of therapy difficult. Additionally, addressing the challenge of similarity between normal neuronal stem cells and GSCs makes treatment-associated toxicity a plausibility. It is suggested that a combinatorial approach which allows cellular targeting of Wnt signaling such as CAR T-mediated drug delivery may be considered to address the challenge.