Allantoin/UA ratios in serum and CSF were positively associated with the SCOPA-AUT score  and RBD presence . In conclusion, serum allantoin and allantoin/UA ratio are elevated in patients with de novo PD. Allantoin/UA ratio in serum and CSF is associated with autonomic dysfunction and RBD presence, indicating that higher systemic oxidative stress occurs in PD patients with more diffuse neurodegenerative changes.Oxidative stress supposedly plays a role in the pathogenesis of Parkinson’s disease (PD). Uric acid (UA), a powerful antioxidant, is lowered in PD while allantoin, the oxidation product of UA and known biomarker of oxidative stress, was not systematically studied in PD. We aim to compare serum and cerebrospinal fluid (CSF) levels of UA, allantoin, and allantoin/UA ratio in de novo PD patients and controls, and evaluate their associations with clinical severity and the degree of substantia nigra degeneration in PD. We measured serum and CSF levels of UA, allantoin, and allantoin/UA ratio in 86 PD patients  years; CSF levels were assessed in 51 patients) and in 40 controls  years). PD patients were examined using Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), Montreal Cognitive Assessment (MoCA), Scales for Outcomes in Parkinson Disease-Autonomic (SCOPA-AUT), the University of Pennsylvania Smell Identification Test (UPSIT), one-night video-polysomnography, and dopamine transporter single-photon emission computed tomography (DAT-SPECT). Serum allantoin and allantoin/UA ratio were significantly increased in the PD group compared to controls ( Clinical manifestations of PD are heterogeneous, and several disease phenotypes were defined based on the spectrum of motor and non-motor symptoms. Unbiased clustering analyses showed that patients with dysautonomia and rapid eye movement (REM) sleep behavior disorder (RBD), referred to as the “diffuse malignant” subtype, have a higher risk of cognitive impairment and faster disease progression as compared to patients with only motor involvement, known as the “mild motor-predominant” subtype3.Parkinson’s disease (PD) is the most common form of neurodegenerative synucleinopathy. The main pathological features of PD are the loss of dopaminergic neurons in substantia nigra pars compacta leading to the depletion of dopamine and the formation of alpha-synuclein positive intracytoplasmatic inclusions referred to as Lewy bodies in surviving neurons4. Oxidative stress may be involved in the pathogenesis of PD through several mechanisms such as oxidation of dopamine, calcium influx through L-type calcium channels and its associated excitotoxicity, mitochondrial dysfunction, protein misfolding and aggregation, or ferroptosis5. Oxidative stress arises when the production of reactive oxygen species (ROS) exceeds clearance by endogenous antioxidants6. Uric acid (UA), the end product of purine metabolism in humans, is one of the powerful antioxidants in the extracellular compartment with putative neuroprotective effects. UA is a scavenger of singlet oxygen, peroxy radicals, and hydroxyl radicals7. PD is associated with lower serum UA levels, particularly in the later stages of the disease8. A series of epidemiological studies suggested that higher levels of UA are associated with a decreased risk of disease development and a slower rate of clinical progression in PD10. In line with these studies, a recent dose-response meta-analysis reported that higher serum UA levels reduced the risk for PD11. Consequently, the effects of UA were studied in preclinical disease models. UA within astrocytes prevented dopaminergic cell death and atrophy induced by oxidative and mitochondrial toxins in cellular PD models12. UA showed attenuated toxic effects on dopaminergic cells in a mouse intrastriatal 6-hydroxydopamine model of PD13. Because of the evolutionary loss of the functional uricase enzyme, humans are unable to further catabolize UA to the more soluble compound allantoin. However, UA can be nonenzymatically oxidized into allantoin by ROS under conditions of increased oxidative stress14. Consequently, allantoin has emerged as a reliable biomarker for assessing the oxidative status both in vitro and in vivo15. Whereas serum UA level is influenced by many factors such as diet, renal function, drugs, and variants in the genes coding urate transporters16, allantoin and/or allantoin/UA ratio may be more accurate biomarkers compared to UA alone. In contrast to UA, allantoin has been only marginally studied in PD. One study revealed no differences in 24-hour urine excretion of UA, allantoin, and allantoin/UA ratio between PD and healthy subjects17. In our previous study, we observed increased serum allantoin and allantoin/UA ratio in patients with isolated RBD (iRBD), which is in most cases the prodromal stage of neurodegenerative synucleinopathies, including PD18. This study aimed to compare serum and cerebrospinal fluid (CSF) levels of UA, allantoin, and allantoin/UA ratio in PD and healthy controls, and to evaluate associations of these biochemical parameters with the severity of motor and non-motor symptoms, including the presence of RBD and the degree of substantia nigra degeneration in PD.There is increasing evidence that implicates oxidative stress as a key driver of the complex mechanism underlying neurodegeneration in PDp = 0.50).Of 111 patients consecutively included in the BIO-PD study between 11/2015 and 11/2021, 103 had serum samples available in the biobank; 17 samples were excluded due to the use of medication affecting UA levels. Thus, serum samples from 86 patients were analyzed while CSF was available in 51 of them. Serum and CSF samples from 50 controls included between 11/2015 and 06/2021 fulfilling the inclusion criteria were available in our biobank, while 10 samples were excluded due to a history of using medication affecting UA levels, yielding 40 control samples for comparison. Mean freezer storage-times were comparable for PD and control samples (3.3 (SD 1.7) vs 3.1 (SD 1.3) years; p < 0.001; higher in men) and BMI  on serum UA concentrations while no between-group difference was found. Serum allantoin concentrations and serum allantoin/UA ratio were significantly increased in the PD group compared to controls ; there was a significant effect of storage-time on serum allantoin concentration  and a significant effect of sex on serum allantoin/UA ratio  and allantoin  and significant effect of age on CSF allantoin/UA ratio (p = 0.01); associations with age were positive for all parameters. No between-group differences in CSF parameters were found.CSF UA and allantoin concentrations were significantly correlated with their serum concentrations  and RBD presence , whereby the associations with SCOPA-AUT were mostly driven by the gastrointestinal and urinary subscores . CSF UA levels were positively associated with olfactory function  and CSF  compared to patients without RBD.Adjusted for age and sex, allantoin/ UA ratios in serum and CSF were positively associated with SCOPA-AUT score (p < 0.001) and serum allantoin/UA ratio (p < 0.001) compared to the excluded control group; no between-group differences were observed for the CSF parameters 26 and may be associated with the protective antioxidant activities under systemic oxidative stress. In line with our results, previous research on oxidative stress markers in PD showed significantly higher levels of blood 8-hydroxyguanosine, nitrite, and MDA and lower levels of catalase, glutathione, and total-cholesterol, indicating the exhaustion of the antioxidative defense system in PD27.Elevated levels of allantoin, supposedly related to increased systemic oxidative stress, were previously reported in patients with various disorders, including bacterial meningitis, Behçet’s disease, recurrent aphthous stomatitis or chronic renal failure28, we did not find a significant difference in serum and CSF UA levels between PD patients and controls. This may be due to the inclusion of newly diagnosed, untreated PD patients - a clinical population that did not show consistent alterations in UA levels in previous studies29. Indeed, a meta-analysis confirmed that lower UA levels may be detectable only in later disease stages8. Due to its neuroprotective effects, UA was proposed as a potential therapeutic target in PD13. However, a clinical trial with urate-elevating compound inosine in early PD has not shown any difference in the rate of clinical disease progression over placebo30. Moreover, a genome-wide association study of genetic variants underlying urate levels found no causal associations between UA and PD risk, age at onset, or disease progression severity31. Hence, UA may not be directly involved in the pathogenesis of PD but rather its decreased levels could represent an epiphenomenon32.In contrast to results from two meta-analyses that revealed lower UA levels in PD patients17. In contrast to the latter finding, our previous study revealed increased serum allantoin and allantoin/UA ratio in patients with iRBD, implying increased systemic oxidative stress in prodromal synucleinopathy18. In the current study, we have also found increased serum allantoin and allantoin/UA ratio in PD, but the effect was smaller, yielding 45% increase in serum allantoin in PD compared to its 86% increase in iRBD males. Although allantoin levels were slightly higher in PD patients with RBD compared to those without RBD, we speculate that a larger increase of allantoin/UA ratio in iRBD compared to PD implies that high serum allantoin in synucleinopathies is most pronounced in the prodromal phase and gradually declines during later disease stages when low UA levels become apparent.In PD patients only one study examining allantoin was conducted until now and no difference between PD and healthy subjects in 24-hour urine excretion of UA, allantoin, and allantoin/UA ratio was found33. Hence, the increased allantoin/UA ratio may be the systemic reaction to the spread of α-synuclein in the peripheral nervous system34. The association with autonomic symptoms were mostly found in the gastrointestinal and urinary domains, which may be related to their early occurrence in synucleinopathies. Interestingly, the association between high allantoin levels and autonomic dysfunction mirrors previous findings related to low UA and non-motor symptoms such as fatigue, attention deficit, anxiety, and cardiovascular disturbances in PD35, which we, however, could not replicate. We can speculate that higher oxidative stress levels herald a more severe course in the early stage of disease and it may be worth to further study allantoin/UA ratio as a prognostic biomarker in PD.We have observed moderate associations of UA, allantoin or allantoin/UA ratios in serum and CSF with non-motor symptoms, i.e. autonomic and olfactory function and RBD presence. These results are only exploratory and need to be treated with caution since statistical correction for multiple comparisons was not applied. Autonomic dysfunction as well as RBD are associated with a diffuse spread of synuclein pathology in the nervous system, i.e. the diffuse malignant disease subtypeIn our current work, higher UA and allantoin levels in serum and CSF were observed in PD patients taking medication affecting UA levels (mostly thiazide diuretics). In contrast to UA and allantoin levels, allantoin/UA ratio seems to be relatively independent on the use of medication affecting UA in PD and controls. This fact together with its stronger associations with the severity of non-motor symptoms indicates that allantoin/UA ratio may be a more universal and useful marker in synucleinopathies compared to UA or allantoin alone.36. Yet, under pathological conditions, additional formation of UA from the catabolism of nucleotides, nucleosides, and purine precursors within CNS may occur, as was suggested in patients with bacterial meningitis37. Theoretically, allantoin in the CSF could increase at later PD stages with higher oxidative stress in the CNS compartment, which may be supported by the positive association between CSF allantoin and disease duration in our current study.We did not find significant between-group differences in allantoin or allantoin/UA ratio in CSF. The fact that between group difference in allantoin and allantoin/UA levels in serum was not paralleled in CSF could reflect a higher extent of oxidative stress on a systemic level possibly reflecting alpha-synuclein spread in the peripheral nervous system in early-stage PD. The antioxidative defense in the CNS may also involve different substances than UA. Despite not being increased, the CSF allantoin/UA ratio was significantly associated with SCOPA-AUT score and RBD status in PD patients. It is, however, not clear whether CSF levels of UA and allantoin are truly related to dysautonomia and RBD, or rather these associations reflect just high cross-correlation between serum and CSF levels of UA and allantoin. This finding is in line with previous reports showing that CSF UA is predominantly determined by serum UA, and is modified by the blood-brain barrier integrityTo our knowledge, this is the first study investigating allantoin and allantoin/UA ratio in serum and CSF in PD patients. The limitation of this study is the relatively small size of the control group. Additionally, only a clinical control group could be used for comparison due to difficulties in obtaining CSF from healthy volunteers. This clinical control group partially consisted of patients with altered CNS functioning, such as sleep disorders, anxiety, or chronic pain, conditions where little is known about oxidative stress involvement. However, the inclusion of heterogeneous control samples from patients with various disorders minimizes the likelihood of significant bias.In conclusion, we have shown that serum allantoin and allantoin/UA ratio are elevated in patients with de novo untreated PD. Moreover, we found a positive association of allantoin/UA ratio in serum and CSF with autonomic dysfunction and RBD, indicating that allantoin and allantoin/UA ratio may be biomarkers of a more severe PD subtype with accentuated non-motor symptoms.38. All PD patients were diagnosed based on the Movement Disorder Society clinical diagnostic criteria for PD39 and comprehensively examined before the initiation of dopaminergic treatment.This study is part of a longitudinal project “biomarkers in PD (BIO-PD)” aimed at collecting a large representative sample of de novo PD patients40. To minimize potential bias associated with a homogeneous control group, the selected controls included patients with various neurological disorders including functional movement disorder (n = 14), narcolepsy (n = 3), idiopathic hypersomnia (n = 3), headache (n = 1), nerve root compression (n = 1), anxiety (n = 1), spinal stenosis (n = 1), and patients undergoing surgery in spinal anesthesia for nonmalignant urologic disorders including prostatic hyperplasia (n = 9), hydrocele (n = 6), bladder tumor (n = 6), urethral stricture (n = 3), and phimosis (n = 2).Paired serum/CSF samples from age- and sex-comparable spinal anesthesia subjects and symptomatic controls with noninflammatory, non-neurodegenerative conditions (defined as CSF cell count ≤15 elements/unit of volume and CSF protein concentration ≤1 g/l) were selected from our CSF biobank operated according to Consensus Guidelines for CSF and Blood Biobanking for CNS Biomarker StudiesTo be eligible for this study, all participants had to be free from medical conditions  and medications  that potentially affect UA levels. In controls, a history of neurodegenerative disorders manifesting with a movement disorder or dementia was also an exclusion criterion. Another requirement was the availability of >200 μl serum and (in the case of controls) CSF aliquots stored in the biobank.This study was approved by the ethics committee of the General University Hospital in Prague. Participants with PD were informed about the study and provided written informed consent; participants from the control group provided samples for biomarker research as per universal biobank informed consent.41, Montreal Cognitive Assessment (MoCA)21, Scales for Outcomes in Parkinson Disease-Autonomic (SCOPA-AUT) questionnaire42, olfactory testing using the University of Pennsylvania Smell Identification Test (UPSIT)43, one-night video-polysomnography, and dopamine transporter single-photon emission computed tomography (DAT-SPECT). DAT-SPECT was performed using the [123I]-2-b-carbomethoxy-3b-(4-iodophenyl)-N-(3-fluoropropyl) nortropane  tracer according to European Association of Nuclear Medicine (EANM) procedure guidelines44. The acquisition parameters were as follows: rotational radius 13–15 cm, image matrix 128 × 128, angular sampling with 120 projections at 3° interval and 40 s per view, zoom 1.3, energy window 159 ± 10% keV. Reconstruction of the raw SPECT projection data was performed using the ordered subset expectation maximization (OSEM) algorithm with 8 iterations and 10 subsets including Chang attenuation correction (μ = 0.11 cm−1) and 3D Butterworth post-filtering with FWHM = 8 mm45. Automated semi-quantitative analysis was performed using the BasGan V2 software and specific binding ratios (SBR) in both putamina were calculated according to the formula [(putamen binding–background binding)/background binding]; the lower value from both hemispheres was used for further analyses46. RBD was diagnosed based on clinical history and video-polysomnography according to the International Classification of Sleep Disorders, third edition (ICSD-3)47.The protocol in PD patients included a structured interview, Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) part IIIIn PD patients, venous blood and optionally CSF samples were drawn at the baseline visit in the morning after an overnight fast. In controls, sampling was performed in the morning after an overnight fast either at the neurology or urology department. Lumbar punctures were performed using 22 G atraumatic needles in PD patients and neurologic controls and by 25 G needles in patients undergoing spinal anesthesia.Sera were separated within 60 minutes after collection and then aliquoted. CSF was immediately centrifuged at 3000 rpm under 4 °C, and the supernatant was aliquoted. All samples were frozen at −80 °C until further biochemical analysis.The concentration of UA in serum and CSF was determined using the Beckman Coulter AU system with Beckman Coulter Uric Acid kits  in a standard biochemical laboratory at the Institute of Rheumatology in Prague.13C2, 15N4 as an internal standard) was added to 20 µL of the sample. The mixture was vortexed and centrifuged at 16500×g for 6 min. 50 µL of supernatant was transferred into an LC vial. For the UHPLC-MS/MS analysis, a UHPLC system Agilent 1290 with a Triple Quad 6460 mass spectrometer  was used. The hydrophilic interaction liquid chromatography (HILIC) conditions consisted of the Acquity BEH Amide column; isocratic elution by a mobile phase composed of acetonitrile and 0.1% formic acid 90/10 (v/v). The flow rate of the mobile phase was maintained at 0.3 mL/min, and the injection volume was 2 µL. The temperature of the column was kept at 30 °C and samples were thermostated at 10 °C. The MS/MS spectrometer was operated in a positive mode. The applied conditions of the electrospray ion source were as follows: gas temperature: 300 °C, gas flow: 7 L/min, sheath gas temperature: 300 °C, sheath gas flow: 8 L/min, nebulizer pressure: 310 kPa and capillary voltage: 6000 V. The MS/MS measurement was performed in multiple reaction-monitoring mode (MRM). Two MRM transitions were monitored for allantoin: Quantifier transition was 159 > 116 (collision energy 2 V and fragmentor voltage 50 V) and a qualifier transition was 159 > 99 (collision energy 8 V and fragmentor voltage 50 V). The transition 165 > 120 (collision energy 2 V and fragmentor voltage 50 V) was monitored for allantoin-13C2, 15N4.This method, which has been validated in terms of linearity, lower limit of quantification (LLOQ), upper limit of quantification (ULOQ), accuracy, precision, selectivity, recovery, carry-over effect, matrix effects, robustness, and stability of both quality control (QC) samples and clinical patient samples, was demonstrated to be suitable for its intended purpose. The UHPLC-MS/MS method with one-step sample preparation provided high sensitivity and high sample throughput23.Allantoin concentration in serum and CSF was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with an isotopically labeled internal standard. Before the analysis, protein precipitation was used. Samples were processed as follows: 60 µL of 100% acetonitrile  was used for statistical analysis; graphs were plotted using Graphpad Prism version 9.0 .Comparisons of UA, allantoin, and allantoin/UA ratio in the serum and CSF between PD and controls were performed using a univariate general linear model with group and sex as fixed factors, and with age, body mass index (BMI), and freezer storage-time as covariates. Difference in sample storage-times may influence the concentration of biomolecules and was thus suggested to be included as a covariate in longitudinal cohort studiesFurther information on research design is available in the Supplementary Table 1, Table 2, Table 3, Table 4reporting summary


This population‐based study examined the association between baseline uric acid (UA) and prostate cancer (PCa)‐related mortality amongst PCa patients receiving androgen deprivation therapy (ADT).Adults with PCa who received ADT in Hong Kong between December 1999 and March 2021 were identified. Patients with missing baseline UA were excluded. Patients were followed up until September 2021. The outcome was PCa‐related mortality.p < 0.001), and an inverse association in those with below‐mean baseline UA levels . The former remained significant on competing risk regression, but not the latter.Altogether, 4126 patients (median follow‐up 3.1[interquartile range 1.4–6.0] years) were included. A J‐shaped association was observed between baseline UA level and PCa‐related mortality risk, with a direct association in those with mean(0.401 mmol/L) or above‐mean baseline UA levels (hazard ratio (HR) per standard deviation‐increase 1.35 , A J‐shaped relationship between baseline UA level and PCa‐related mortality risk was identified. This study was mainly limited by potential unmeasured and residual confounders. Further validation studies are warranted. Amongst patients with prostate cancer undergoing androgen deprivation therapy, a J‐shape relationship exists between baseline uric acid level and the risk of prostate cancer‐related mortality, with both high and low uric acid levels being associated with similarly elevated risks when compared against normal uric acid levels. Patient consent was waived as deidentified data were used. All underlying data are available upon reasonable request to the corresponding authors.2.1International Classification of Diseases, Ninth revision (ICD‐9) codes  during 1/12/1993–31/3/2021 were included. Patients with missing baseline UA level were excluded. The endpoint was PCa‐related mortality. Patients were followed up from the date of ADT initiation until death or 31/9/2021, whichever earlier. PCa diagnosis was ascertained using ICD‐9 codes Table , and PCa2.3The following baseline characteristics were collected for all patients: age, the type of ADT, serum uric acid level, comorbid conditions , prior radiotherapy, prior radical prostatectomy, medication prescriptions . Furthermore, ever‐prescription of androgen receptor signaling inhibitors and/or chemotherapy was recorded as a surrogate for metastatic disease2.4Continuous variables were expressed as median and interquartile range. Due to the nature of the database, and as patients with missing baseline UA levels were excluded, there were no missing values in this study. Multivariable Cox regression adjusting for covariates (as abovementioned) was used to evaluate the relationship between baseline UA level and the endpoint, with hazard ratio (HR) and 95% confidence intervals (CIs) as summary statistics. A fractional polynomial curve was used to explore such relationship across the observed range of baseline uric acid levels. As a J‐shaped relationship was observed Figure , the patFor each of these groups, four a priori subgroup analyses were performed. First, to assess the effects across patients of different ages, patients were grouped into those aged 75 years old or above and those younger than 75 years old. Second, as gout is intimately related to UA levels and may be a confounder, patients were grouped by any prior diagnosis of gout. Similarly, UA‐lowering medications affect UA levels and thus may be a confounder, and a subgroup analysis was done accordingly, with grouping by the baseline use of UA‐lowering medication(s). Finally, to explore the respective effects of UA levels in metastatic and non‐metastatic PCa, a subgroup analysis was performed for metastatic vs non‐metastatic PCa, with ever‐prescription of androgen receptor signaling inhibitor(s) or chemotherapy used as a surrogate and thus grouping criterion for metastatic disease.As the high rate of mortality may bias hazards estimated by conventional survival analyses, an a priori sensitivity analysis was performed using competing risk analysis with the Fine‐Gray subdistribution model. Non‐PCa‐related mortality was the competing event. Sub‐hazard ratios with 95% CIs were used as summary statistics.To facilitate clinical translation of our findings, a post hoc analysis was performed where uric acid levels were referenced against the male normal range of 0.24–0.51 mmol/L and categorized into low‐ (<0.24 mmol/L), normal‐ (0.24–0.51 mmol/L), and high‐ (>0.51 mmol/L) uric acid categories. All patients were then analyzed with uric acid as these categories, without the above separation by the mean uric acid level, using multivariable Cox regression.p‐values were two‐sided, with p < 0.05 considered statistically significant. All analyses were performed on Stata .All 3Altogether, 13,537 patients with PCa receiving ADT were identified. After excluding 9411 patients without baseline UA level, 4126 patients were analyzed , higher UA levels were associated with higher risk of PCa‐related mortality (hazard ratio (HR) per SD‐increase 1.35 , p < 0.001). Subgroup analyses (Table p < 0.001).A J‐shaped relationship was observed between UA levels and the risk of PCa‐related mortality Figure . In thoses Table  found diN = 2274), lower UA levels were associated with higher risk of PCa‐related mortality . Subgroup analyses (Table p = 0.071).In those with below‐mean UA levels  and low‐UA  groups had higher cumulative incidence of PCa‐related mortality . Both the high‐UA  and low‐UA groups had significantly higher cumulative incidence of PCa‐related mortality on competing risk regression ; the high‐ and low‐UA groups had similar risks on competing risk regression .Compared to the normal‐UA group, both high‐UA ; data curation ; methodology ; writing – original draft ; writing – review and editing . Jeffrey Shi Kai Chan: Conceptualization ; formal analysis (lead); methodology (lead); visualization (lead); writing – original draft ; writing – review and editing . Chi Ho Leung: Methodology (supporting); writing – review and editing (supporting). Jeremy Man Ho Hui: Investigation ; writing – review and editing (supporting). Edward Christopher Dee: Writing – review and editing (supporting). Kenrick Ng: Writing – review and editing (supporting). Kang Liu: Data curation ; resources ; writing – review and editing (supporting). Tong Liu: Funding acquisition (supporting); supervision ; writing – review and editing (supporting). Gary Tse: Funding acquisition (lead); methodology (supporting); resources ; supervision ; writing – review and editing (supporting). Chi‐Fai Ng: Conceptualization ; funding acquisition (supporting); supervision ; writing – review and editing .This work was partly supported by the Research Matching Grant (reference number 8601454), the Tianjin Key Medical Discipline  Construction Project (Project number: TJYXZDXK‐029A), and a grant from Hong Kong Metropolitan University (Project Reference No. RIF/2022/2.2). The funders played no role in any part of this study.ECD is funded in part through the Cancer Center Support Grant from the National Cancer Institute (P30 CA008748). All other authors have no conflict of interest to report.This prospective cohort study was approved by The Joint Chinese University of Hong Kong‐New Territories East Cluster Clinical Research Ethics Committee and conducted in accordance with the Declaration of Helsinki. Patient consent was waived as deidentified data were used.Table S1.Click here for additional data file.


Osteoarthritis (OA), a chronic joint cartilage disease, is characterized by the imbalanced homeostasis between anabolism and catabolism. Oxidative stress contributes to inflammatory responses, extracellular matrix (ECM) degradation, and chondrocyte apoptosis and promotes the pathogenesis of OA. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central regulator of intracellular redox homeostasis. Activation of the NRF2/ARE signaling may effectively suppress oxidative stress, attenuate ECM degradation, and inhibit chondrocyte apoptosis. Increasing evidence suggests that the NRF2/ARE signaling has become a potential target for the therapeutic management of OA. Natural compounds, such as polyphenols and terpenoids, have been explored to protect against OA cartilage degeneration by activating the NRF2/ARE pathway. Specifically, flavonoids may function as NRF2 activators and exhibit chondroprotective activity. In conclusion, natural compounds provide rich resources to explore the therapeutic management of OA by activating NRF2/ARE signaling. Osteoarthritis (OA), a degenerative and irreversible joint disease, has been associated with chronic inflammation, trauma, and other diseases. No effective strategies are available to cure OA. Clinically, non-steroidal anti-inflammatory drugs (NSAIDs) have been used to alleviate OA symptoms, such as joint pain, stiffness, and movement limitation, which may greatly decrease the quality of life and increase the burden on society and patients . This aw2O2), hydroxyl radical, superoxide anion, nitric oxide (NO), and hypochlorite ion, are free radicals, which have unpaired electrons. ROS can be produced by mitochondrion, non-mitochondrial membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), and xanthine oxidase (XO)  . Overproase (XO) . Thus, sNuclear factor erythroid 2-related factor 2 (NRF2), a transcriptional factor, exhibits a central role in maintaining the homeostasis of the intracellular redox system. NRF2 activation has been reported to counteract ROS generation and oxidative stress by binding to antioxidant response elements (AREs) in the promoters of the downstream antioxidant genes, such as heme oxygenase-1 (HO-1) . PotentiOA elucidates a subset of the pathological alterations that are influencing the joints. The pathogenesis of OA includes three overlapping stages . Firstlyβ-catenin has been found in OA patients, and genetic activation of β-catenin stimulates the expression of catabolic enzymes, such as MMPs and ADAMTSs , GSH, GPx, and SOD, and suppressing oxidative stress in chondrocytes  Figure . In OA cActivation of the NRF2 signaling also contributes to the inhibition of ECM degradation and chondrocyte apoptosis. It has been reported that NRF2 signaling may increase the expression of collagen II and SOX9 and decrease the expression of MMP-13 and ADAMTS5, protecting against IL-1β-induced ECM degradation in OA chondrocytes . ManganeHowever, one study reports that inhibition of the NRF2/ARE signaling can ameliorate LPS-stimulated NLRP3 inflammasome in SW982 cells. Increased expression of NRF2/HO-1 and NLRP3 are also observed in rat OA models . Another2O2-induced oxidative stress and chondrocytes apoptosis by activating the Keap1/NRF2 signaling pathway , and lycopene are the antioxidants isolated from garlic, broccoli, and tomato, respectively, and they may decrease oxidative stress and inflammatory responses. It has been reported that allicin, SFN, and lycopene can effectively inhibit H pathway . Most na pathway . ParticuFlavonoids have various pharmacological activities, including anti-inflammation, anti-oxidation, anti-cancer, anti-apoptosis, and bone system protection . Natural1) acts as a selective estrogen receptor modulator (SERM) due to its structural similarity to estrogen. It has been reported that genistein (10 μM) decreases the expression of COX-2, NOS2, and NO in IL-1β-treated chondrocytes by activating the NRF2 signaling (2), extracted from Garlic  with the availability of 693 mg/kg , Isovitexin , Cannabis , and palm ), Eriodictyol ), Licochalcone A ), and Epigallocatechin-3-O-gallate  exhibit similar effects against inflammatory responses by activating the NRF2 signaling in OA chondrocytes , separated from the root of Pueraria lobata var. Lobata (Willd.) Ohwi (Leguminosae), at the doses of 100 mg/kg/day and 200 mg/kg/day for 14 days by gavage ameliorates the pathological changes of OA cartilage by activating AMPK and NRF2 signaling pathways in MIA-induced rat OA models  of the Moraceae family) decreases IL-1β-induced enhancement of NO and PGE2 production and activation of the NF-κB signaling in human chondrocytes. NRF2 siRNA transfection may abolish the suppressive activity of Morin against inflammatory responses , Hypericum attenuatum Fisch. (Guttiferae), and Hypericum perforatum L. (Guttiferae)) and Cardamonin  consistently protect mouse chondrocytes against IL-1β-induced COX-2 and iNOS expression by suppressing PI3K/AKT/NF-κB and MAPK signaling pathways via activating the NRF2 signaling . Luteolin (12) and 7,8-dihydroxyflavone  inhibit H2O2-induced inflammatory responses in mouse chondrocytes by inhibiting the MAPK pathway and activating the NRF2 signaling (14), isolated from Scutellaria baicalensis Georgi (Lamiaceae), at the doses of 10, 25, and 50 μM has been reported the anti-inflammatory effects, which are not associated with the inhibition of NF-κB and MAPK signaling pathways in IL-1β-treated human chondrocytes , firstly extracted from the root of Veratrum grandiflorum O. Loes (Liliaceae), has been demonstrated to be an activator of Sirt1, which may mediate Wnt/β-catenin and Foxo1 signaling pathways in OA chondrocytes (16), a natural polyphenol from fruits and nuts, has anti-inflammatory and anti-oxidative activities. EA (10 and 25 μM) has been demonstrated to inhibit IL-1β-induced oxidative stress by activating the NRF2/HO-1-NQO-1 signaling pathway in C28/I2 chondrocytes (17) at the doses of 2, 5 and 10 μM exhibits protective activity against 4-hydroxynonenal-induced chondrocyte cell death by activating the NRF2 signaling pathway. In addition, 6-gingerol suppresses inflammatory responses and oxidative stress. However, IL-1β-induced activation of MAPK and NF-κB is not mediated by 6-gingerol , Pterostilbene , Curcumin , piceatannol , and Caffeic acid phenethyl ester , also exhibit inhibitory activities against inflammatory responses by activating the NRF2 signaling pathway in IL-1β-treated chondrocytes (Resveratrol (drocytes . Resveradrocytes . TLR4/NFdrocytes . Ellagicdrocytes . 6-Gingegingerol . Other pdrocytes .2), Moracin (3), and Puerarin (8) can protect against IL-1β-induced upregulation of MMP-13 and ADAMTS5 expression and downregulation of collagen II and aggrecan expression. Knockdown of NRF2 ameliorates the inhibitory effects of myricetin, moracin, and Puerarin on IL-1β-induced ECM degradation, respectively (10) and EGCG (7) inhibit IL-1β-induced ECM degradation by up-regulating collagen II, aggrecan, and SOX9 expression and down-regulating MMP-3, MMP-13, and ADAMTS5 expression via the activation of NRF2 signaling in mouse chondrocytes ) and Wogonin  may alleviate ECM degradation by activating the NRF2 signaling in IL-1β-treated human chondrocytes, as shown by decreased generation of ROS, downregulated expression of MMP-3, MMP-9, MMP-13, and ADAMTS-4, and increased expression of GAG (20) promotes the expression of Col2α, aggrecan, and SOX9, suppresses ECM degradation, and increases chondrocyte viability by inhibiting the NF-κB/HIF-2α signaling  suppresses the expression of MMP-1, MMP-13, and ADAMTS4/5 and enhances the expression of collagen II by inhibiting NF-κB and Wnt/β-catenin signaling pathways in rat chondrocytes (4) at the doses of 25 and 50 μg/mL inhibits MMP-3, MMP-13, and ADAMTS5 expression and increases collagen II and aggrecan expression by activating the NFR2 signaling in IL-1β-treated mouse chondrocytes (19) at the doses of 10 and 20 μM also inhibits IL-1β-induced ECM degradation and chondrocyte senescence by suppressing the PI3K/AKT/NF-κB signaling pathway and reducing senop-associated secretory phenotype (SASP) expression (12) at the doses of 10 and 20 μM may suppress MMP-13 and ADAMTS5 expression and stimulate collagen II and aggrecan expression in H2O2-treated mouse chondrocytes (13) also suppresses the expression of MMP-1, MMP-3, and MMP-13 by activating the NFR2 signaling in H2O2-treated mouse chondrocytes (24) may ameliorate TNFα-induced upregulation of MMP-3/-9 expression and downregulation of collagen II and aggrecan expression in rat chondrocytes (25) at the doses of 10 and 20 μM exhibits protective effects against iron overload-induced cartilage damage by activating the NRF2/HO-1 signaling pathway in IL-1β-treated human chondrocytes  can decrease ROS production, inhibit Bax, cytochrome c, cleaved caspase-9, and cleaved caspase-3 expression, and increase Bcl-xl expression in IL-1β-treated mouse chondrocytes by activating the NRF2 signaling (17) upregulates the expression of ubiquitin-specific peptidase 49 (USP49), promotes the deubiquitination of Axin, and increases the degradation of β-catenin, resulting in the inhibition of Wnt/β-catenin signaling and the attenuation of apoptosis in IL-1β-treated rat chondrocytes (18)  may promote chondrocyte survival by improving mitochondrial homeostasis via increasing the expression of Parkin and NRF2 in H2O2-treated chondrocytes (26) at the doses of 20 and 40 μM can suppress IL-1β-induced apoptosis by mediating the NRF2/Bax/Bcl-2 signaling pathway in rat chondrocytes (12) can suppress H2O2-induced ROS production and cell apoptosis in mouse chondrocytes by activating the NRF2 signaling, as detected by decreased caspase-3 and ssDNA expression  may suppress ROS generation, maintain mitochondrial membrane potential, decrease Bax and cleaved caspase-3 expression, and increase Bcl-2 expression in TNFα-treated rat chondrocytes  inhibits iron overload-induced chondrocyte apoptosis  Bunge, Leguminosae) can regulate iron homeostasis, suppress oxidative stress, and inhibit iron overload-induced mitochondrial dysfunction by up-regulating the NRF2/system xc-/GPX4 signaling pathway in mouse chondrocytes  at the dose of 40 μM exhibits protective activity against OA development by suppressing oxidative stress, inhibiting apoptosis, and stimulating autophagy. The possible mechanism might be associated with upregulation of the NRF2/ARE signaling and downregulation of the NF-κB pathway in C28/I2 chondrocytes (8) also protects chondrocytes by promoting Beclin-1-dependent autophagy and maintaining homeostasis (20) maintains mitochondrial homeostasis, induces mitophagy, and protects chondrocytes by mediating the AMPK/PINK1/Parkin signaling pathway (20) also promotes autophagy and reduces apoptosis by inhibiting the PI3K/AKT/mTOR pathway in TNFα-treated chondrocytes (Engeletin (drocytes . Iron ovpoptosis . Similardrocytes  (Table 2drocytes . Puerarieostasis . Curcumi pathway . Curcumidrocytes .6) may protect mouse chondrocytes against LPS-induced pyroptosis by down-regulating the NRF2 signaling, as shown by decreased expression of caspase-1, IL-1β, IL-18, and NLRP3 inflammasome (11) suppresses IL-1β-induced chondrocyte apoptosis by inhibiting the activity of NLRP3 inflammasome via activating the NRF2 signaling (29), a polyphenolic compound, has anti-oxidative activity. It has been demonstrated that CGA (250 μM) can inhibit the expression of pro-apoptotic markers, such as cleaved caspase-3 and PARP, and stimulate the expression of Bcl-xL by attenuating the NF-κB pathway and enhancing the NRF2/ARE signaling, leading to the amelioration of apoptosis in human C28/I2 cells (30), extracted from Taraxacum mongolicum Hand (Composite), can suppress the expression of pro-inflammatory cytokines, such as IL-6, IL-12, TNFα, COX-2, PGE2, iNOS, NO, and ECM catabolic factors, such as MMP-13 and ADAMTS-5, and increase the production of collagen II and aggrecan by activating the NRF2/HO-1 signaling and inhibiting the PI3K/AKT and NF-κB pathways in C28/I2 cells (31), an ethanolic ingredient from Pinaceae, Leguminosae, and Argyrolobium family, has shown insulin-like effects. It has been reported that pinitol (5 and 10 μM) can ameliorate TNFα-induced chondrocyte senescence and cell cycle arrest by rescuing the NRF2 signaling pathway in C28/I2 cells  may promote NRF2 nuclear translocation and activate the NRF2 signaling in IL-1β-treated human chondrocytes. Ly294002, a PI3K/AKT inhibitor, can block the stimulatory effects of myricetin on the activation of NRF2 signaling (23) and EGCG 7) may downregulate Keap1 expression, upregulate NRF2 expression, and promote the dissociation of Keap1 from NRF2, exhibiting anti-oxidative and chondroprotective activity (12) can interrupt the interaction between Keap1 and NRF2, increase the stability of NRF2, and promote NRF2 nuclear translocation by activating AMPKα1 expression in H2O2-treated mouse chondrocytes (14) can directly interact with the Kelch domain of Keap1 protein and interrupt the association of Keap1 with NRF2, leading to the stability of NRF2 (4) has been reported to interact with NRF2 and promote its nuclear translocation (26) can directly bind to the cavity of NRF2 by forming hydrogen bonds, salt bridge, alkyl, and van der Waals, promoting the stability and nuclear translocation of NRF2 in IL-1β-treated rat chondrocytes n. The structure of terpenes is constructed by the linkage of isoprene units (C5H8)n. Various terpene compounds can be generated by head-to-tail condensation of C5 building blocks. Regarding the number of linked isoprene units, terpenoids can be divided into hemi-, mono-, sesqui-, di-, sester-, tri-, tetra-, and polyterpenes (32) is a diterpene from Andrographis paniculata Burm. f. (Acanthaceae) and has been reported to increase the expression of the NRF2/ARE signaling and decrease the NF-κB, MAPK, and JAK/STAT pathways , an acyclic monoterpene alcohol from comfrey  and cruciferous family, has been demonstrated to suppress IL-1β-induced expression of COX-2/PGE2, iNOS/NO, TNFα, IL-6, MMP-13, and ADAMTS5 and increase the production of collagen II and aggrecan in mouse chondrocytes by activating the NRF2/HO-1 pathway and suppressing the NF-κB pathway , a sesquiterpenoid isolated from Ginkgo biloba L. (Ginkgoaceae), has shown various pharmacological activities, such as anti-inflammatory, anti-oxidative, and anti-apoptosis. In a papain intra-articular rabbit OA model, bilobalide (40 mg/kg and 80 mg/kg by gavage administration) has been shown to decrease the expression of MMP-3 and MMP-13 and improve the biomechanical properties by activating the NRF2/HO-1 signaling pathway (35) is also a sesquiterpene from Pogostemon cablin (Blanco) Benth. (Lamiaceae). It has been reported that PA may ameliorate ECM degradation in D-galactose-induced senescent chondrocytes by stimulating the NRF2/HO-1 signaling pathway in mouse chondrocytes , a natural antioxidant, has been clinically used for treating inflammatory and cancer diseases. In H2O2-treated rat chondrocytes, AP may suppress oxidative stress by activating the NRF2/ARE signaling pathway. Specifically, AP can inhibit H2O2-induced expression of MMP-13 and IL-6 and improve chondrocyte apoptosis (36), derived from Ginkgo biloba L. (Ginkgoaceae), has been demonstrated to inhibit H2O2-induced apoptosis, oxidative stress, and ECM degradation by activating the NRF2/HO-1 pathway and suppressing the NF-κB pathway in rat chondrocytes  is a triterpenoid isolated from some edible citrus fruits and has demonstrated inflammation-modulatory properties. NOM may alleviate the development of OA by decreasing the expression of pro-inflammatory cytokines, such as COX-2/PGE2, iNOS/NO, TNFα, and IL-6, and suppressing ECM degradation by mediating the NRF2 and NF-κB signaling pathways in mouse chondrocytes (38), isolated from lemons and other citrus fruits, has been demonstrated to inhibit IL-1β-induced generation of pro-inflammatory cytokines, such as COX-2/PGE2, iNOS/NO, TNFα, and IL-6, and the expression of MMP-13 and ADAMTS5 in mouse chondrocytes by mediating the NRF2/HO-1/NF-κB signaling pathway , isolated from birch bark, is often used for anti-inflammation treatment. Botulin can decrease IL-1β-induced expression of pro-inflammatory cytokines, such as IL-6, TNFα, COX-2, PGE2, iNOS, and NO, and ECM catabolic factors, such as MMP-13 and ADAMTS-5, by activating the AKT/NRF2 pathway and inhibiting the NF-κB pathway in mouse chondrocytes (40), a triterpenoid saponin isolated from Centella Asiatica L. (Apiaceae), may ameliorate TBHP-induced chondrocyte apoptosis by stimulating the NRF2/HO-1 signaling pathway. Additionally, ASI enhances the production of aggrecan and collagen II and suppresses the expression of MMP-13 and ADAMTS5, improving the degradation of ECM and the progression of OA (41), a pentacyclic triterpenoid saponin, has been reported anti-inflammatory and anti-oxidative activities by inhibiting the JAK2/STAT3/MAPK signaling pathway and activating the Keap1-NRF2/HO-1/ROS/Bax/Bcl-2 axis, leading to the suppression of ECM degradation and upregulation of collagen II and aggrecan expression in IL-1β-treated C28/I2 cells (42), one of the effective metabolites from Glycyrrhiza glabra L. (Leguminosae), is widely used for treating inflammatory diseases. 18β-GA may suppress IL-1β-induced expression of pro-inflammatory cytokines, such as IL-6, TNFα, COX-2, PGE2, iNOS, and NO, and ECM catabolic factors, such as MMP-13 and ADAMTS-5, by activating the activity of the NRF2/HO-1 pathway in mouse chondrocytes (43), an effective triterpenoid from Rhizome of Dipsacus asper Wall (Caprifoliaceae), can suppress inflammatory actions by stimulating the NRF2/HO-1 pathway and inhibiting the NF-κB pathway in mouse chondrocytes, protecting against OA development  is often found in vegetables and fruits and has been demonstrated anti-inflammatory activity against OA development by activating the NRF2/HO-1 pathway and reversing the NF-κB/STAT3 pathway, leading to the decreased degradation of ECM in mouse chondrocytes (45), also named marine carotenoid, exhibits remarkable anti-oxidative activity by activating the NRF2/ARE signaling. AST has been demonstrated to suppress IL-1β-induced inflammatory responses, cell apoptosis, and ECM degradation by inhibiting the MAPK/NF-κB signaling and stimulating the NRF2 pathway in mouse chondrocytes (Tetraterpenoids Lycopene (drocytes . Lycopendrocytes . Astaxandrocytes  Table 144 is oft46), an alkaloid from Corydalis bungeana Turcz. (Papaveraceae Juss.), has been reported to inhibit inflammatory responses and suppress the NF-κB signaling. COR may suppress IL-1β-induced expression of pro-inflammatory cytokines and ECM degradation by binding and activating NRF2 activity in mouse chondrocytes (47), isolated from Sinomenium acutum Thunb. (Menispermaceae), has demonstrated anti-inflammatory effects. It has been reported that SIN reduces the expression of COX-2, PGE2, iNOS, NO, IL-6, and TNFα, inhibits the production of MMPs and ADAMTS5, and suppresses the degradation of collagen II and aggrecan in mouse chondrocytes by activating the NRF2/HO-1 signaling pathway (48) is a bioactive metabolite from Fritillaria thunbergii Miq. (Liliaceae), which is widely used to treat various diseases. PM has been shown to decrease the production of COX-2, PGE2, iNOS, NO, IL-6, and TNFα in mouse chondrocytes. In addition, PM can decrease the expression of MMP-13 and ADAMTS5 and increase the production of collagen II and aggrecan by stimulating the NRF2/HO-1 signaling and inhibiting the AKT/NF-κB pathway , a food flavoring, is a potential NRF2 activator. Coniferaldehyde may ameliorate H2O2-induced IL-1, IL-6, MMP-1, and MMP-13 expression by activating the NRF2/HO-1 signaling pathway in mouse chondrocytes (50) is isolated from the fungus Agaricus campestris. Ergosterol has shown chondroprotective activity by activating the NRF2/HO-1 signaling pathway in mouse chondrocytes (51), an aromatic natural metabolite isolated from red ginseng, has shown various biological effects, including anti-inflammation and anti-oxidative stress. It has been reported that maltol exhibits chondroprotective activity in vivo and in vitro. Specifically, maltol suppresses the production of pro-inflammatory cytokines and the expression of MMP-13 and ADAMTS5 and increases the expression of collagen II and aggrecan by modulating the NRF2 and NF-κB signaling pathways in mouse chondrocytes (52), an azaphilonoid pigment isolated from Monascus purpureus-fermented rice, has demonstrated protectiveness in the development of OA. Specifically, monascin can reduce inflammatory responses and suppress ECM degradation by activating the NRF2 signaling and inhibiting the NF-κB pathway in mouse chondrocytes (53) can be found in Plumbaginaceae, Droseraceae, and Ebenceae family. It has been demonstrated that plumbagin exhibits anti-oxidative stress in H2O2-treated human chondrocytes by activating the NRF2 signaling and inhibiting the NF-κB pathway , an isothiocyanate widely found in the Brassicaceae family and most abundant in broccoli and 3-day-old broccoli sprout extracts  is a semisynthetic triterpenoid derived from oleanolic acid and has been considered one of the most potent activators of the NRF2/ARE signaling pathway. RTA408 has been demonstrated to inhibit radiation-induced oxidative stress by disrupting the interaction between Keap1 and NRF2 , a methyl ester of fumaric acid, is currently the only agent in clinical practice as a NRF2 activator. Notably, DMF increases the activity of mitochondrial tricarboxylic acid (TCA) cycle and the production of ATP. Additionally, DMF and its metabolite monomethyl fumarate have good bioavailability (Dimethyl fumarate (DMF, Tecfidera, lability  and exhilability . Mechanilability . DMF haslability . ERK1, blability . It has lability .Clinicaltrials.gov. The drug development of NRF2 activators has been advanced. Upregulation of the NRF2/ARE signaling pathway may provide beneficial effects in adverse environments. It should be noted that over-activation of the NRF2/ARE signaling pathway also has some potential negative effects. Various challenges, such as target specificity, pharmacodynamic responses, short-term and long-term safety considerations, and the variation of NRF2 activity, may adversely affect the expression of NRF2 (β cells. It can be postulated that over-activation of the NRF2/ARE signaling pathway may be associated with insulin resistance and dysregulated lipid accumulation. In addition, some oxidative modifications of proteins are important for the processes of ubiquitination and protein degradation (Many clinical trials for NRF2 activators, such as plant extracts, dietary supplements, and pure compounds, have been performed and can be found on  of NRF2 . For exa of NRF2 . In addi of NRF2 . ROS at radation . Insulinradation . Thus, tradation .In this review article, we comprehensively discuss the chondroprotective effects of natural compounds by activating the NRF2/ARE signaling pathway . These e


Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus. Human T-cell tropic virus type 1 (HTLV-1) was the first human retrovirus identified ,3,4 as aWe first confirmed the efficiency of HTLV-1 and HIV-1 infection through cell-free and cell-to-cell contacts using HTLV-1 and HIV-1 reporter vectors. As previously described , the celSince retroviral Gag generally interacts with its Env ,19,20,21It has been reported that cytoplasmic domain deletion or mutation affects the incorporation of HTLV-1 Env in the virions since the cytoplasmic domain has two motifs, YXXΦ and PDZ-binding, that regulate Env expression and activity via interactions with cellular proteins . TherefoTo further determine the domain responsible for the efficient incorporation of HTLV-1 Env, we constructed chimeras between HTLV-1 and HIV-1 Gag A since tWe focused on the p6 domain of HIV-1 Gag since the p6 domain in the C-terminal portion of Gag is unique to HIV-1. To evaluate the role of the HIV-1 p6 domain in the incorporation of HTLV-1 Env, we constructed PTAP or YPXL-minus p6 mutants, as well as the entire p6 deletion mutant, A. HoweveGiven that C-terminal HIV-1 p6 Gag mediates the incorporation of HTLV-1 Env into VLPs, it is possible that HTLV-1 Gag fused to the HIV-1 p6 domain facilitates the incorporation of HTLV-1 Env into VLPs. Therefore, we constructed an expression vector for HTLV-1 Gag connected to the HIV-1 p6 domain at the C-terminus (HTLV-1-Gag-p6). Since the PTAP motif located in the C-terminal portion of the MA of HTLV-1 Gag may affect the function of HIV-1 p6, we also constructed an HTLV-1-Gag-p6 with a deleted HTLV-1 PTAP motif (HTLV-1-ΔPTAP-Gag-p6). However, these mutants did not incorporate HTLV-1 Env into the VLPs .It has been reported that the p6 domain recruits the endosomal sorting complexes required for transport (ESCRT) machinery for efficient virus release . For exaHTLV-1 is mainly transmitted through cell-to-cell contact with HTLV-1-infected cells present in body fluids, such as breastmilk, semen, or blood, due to the low infectivity of cell-free HTLV-1 ,35. ThisSeveral models have been suggested for the incorporation of retroviral Env into virions, such as passive incorporation or incorporation via direct or indirect Gag–Env interactions ,42. In oThe fact that HTLV-1 Env was found to be incorporated into HIV-1 Gag VLPs suggests that MA plays a role in the incorporation of retroviral Env via direct or indirect interactions. However, the deletion mutants of MA or chimeras between HTLV-1 and HIV-1 Gag confirmed the absence of the MA domain of HIV-1 Gag for the efficient incorporation of HTLV-1 Env into HIV-1 Gag VLPs. Therefore, it is unlikely that the interaction between MA and HTLV-1 Env enhances the incorporation of HTLV-1 Env into VLPs.We focused our analysis on the p6 domain due to its uniqueness to HIV-1. To determine the role of the p6 domain in the incorporation of HTLV-1 Env into HIV-1 Gag VLPs, it was essential to delete the p6 domain of HIV-1 Gag. However, p6 in HIV-1 Gag possesses two L-domains, the PTAP and YPXL motifs, which are involved in viral release by interacting with TSG101 and ALIX, respectively . NeverthNevertheless, Gag overexpression and the introduction of VA-RNA I in transfected cells enabled us to analyze the effect of p6 deletion mutants on the incorporation of HTLV-1 into HIV-1 Gag VLPs. We found that the deletion of p6 Gag or the PTAP minus mutation, but not the YPXL mutation, abrogated the incorporation of HTLV-1 Env into HIV-1 Gag VLPs. These findings suggest that the PTAP motif in the HIV-1 p6 domain is involved in the efficient incorporation of HTLV-1 Env into HIV-1 Gag VLPs.Because the PTAP motif has been reported to recruit TSG101, a component of ESCRT-I complexes, for efficient viral budding from the plasma membrane, we investigated whether TSG101 is involved in the incorporation of HTLV-1 Env into HIV-1 Gag VLPs. The knockdown of TSG101, but not ALIX, in VLP-producing cells reduced HTLV-1 Env incorporation into HIV-1 Gag VLPs. Furthermore, the cell-free virus produced in TSG101, but not in ALIX knockdown cells, had lower infectivity, while infection by cell-to-cell contact was retained. These results indicate that the interaction of the PTAP motif in the p6 region with TSG101 determines the incorporation of HTLV-1 Env into HIV-1 Gag VLPs. Remarkably, HTLV-1 Gag also has a PTAP motif, which is located in the C-terminal portion of the MA domain, but plays a minor role in viral budding, in contrast to the PPXY motif, which plays an essential role ,46,47,48HTLV-1 Gag fused to HIV-1 p6 did not facilitate the incorporation of HTLV-1 Env , indicatHowever, cell-free HTLV-1 has been reported to efficiently infect myeloid and plasmacytoid dendritic cells (DCs) via heparan sulfate proteoglycans and neuropilin-1 . Thus, iHuman embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) . A glioma cell line expressing human CD4 (NP-2/CD4) and its derivative, NP-2/CD4/CCR5/CXCR4 ,52,53, wThe HTLV-1 Env expression vector pcDNA-1E-RRE was constructed as previously described . HIV-1 aExpression vectors for HTLV-1-Gag-FLAG and -Venus were constructed from pCRVI/HIV-1/Gag-FLAG or pCRVI/HIV-1/Gag-Venus by replacing the HIV-1 gag sequences with HTLV-1 gag sequences amplified by PCR using the pMT-2 HTLV-1 plasmid  as the tThe lentiviral vector particles were produced via the transient transfection of lentiviral vectors expressing shRNAs targeting TSG101 and ALIX with packaging and VSVG-expressing plasmids, as previously described . LentiviFor cell-free or cell-to-cell infection assays for HTLV-1 and HIV-1, an intron Gaussia reporter system (inGLuc) was used, as previously described . BrieflyThe p24 antigen concentration of pseudotyped HIV-1 was determined using p24Ag ELISA  according to the manufacturer’s instructions. The concentration of p24 antigen in the pseudotyped HTLV-1 was determined by ELISA using two monoclonal antibodies against p24 Ag of HTLV-1.For the cell-to-cell fusion assay, 293T cells in a six-well plate were transfected with 3.5 µg of pCRVI/HTLV-1/Gag-FLAG or pCRVI/HIV-1/Gag-FLAG, 1.5 µg of pcDNA-1E-RRE or HIV-1 Env expression plasmid (pCXN-NLenv ), and 0.To produce VLPs, 293T cells were transfected with either 3.5 µg of pCRVI/HTLV-1/Gag-FLAG, or pCRVI/HIV-1/Gag-FLAG, 1.5 µg of pcDNA-1E-RRE, and 0.5 µg of pAdVantage (Promega). The culture supernatant of the transfected cells was recovered, filtered, and used for VLP analysis. To analyze the HIV-1 Gag p6 mutants, the pCRVI/HIV-1/Gag-HA plasmid was used for transfection instead of pCRVI/HIV-1/Gag-FLAG.The 293T cells were transfected with the HTLV-1 Env expression plasmid (pcDNA-1E-RRE) and either pCRVI/HTLV-1/Gag-Venus or pCRVI/HIV-1/Gag-Venus, as described above. The cells were recovered 24 h post-transfection using a cell dissociation solution (Sigma-Aldrich), stained with the anti-gp46 rat mAb LAT-27 [Transfected 293T cells were solubilized in 1% Brij O10 (Sigma-Aldrich) lysis buffer  containing a protease inhibitor cocktail . For the analysis of VLPs by Western blotting, the supernatant of transfected 293T cells was centrifuged, and the resulting pellets were solubilized in 2× sample loading buffer. Cell lysates or solubilized VLPs were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes . The membranes were then incubated with anti-gp46 rat mAb (LAT-27), anti-gp120 polyclonal sheep antibody , anti-FLAG mouse mAb , anti-HA mouse mAb (Wako), anti-cyclophilin A (CypA) rabbit pAb , or anti-β-actin mouse mAb (Sigma), followed by staining with horseradish peroxidase-conjugated anti-mouse , anti-rat (Jackson ImmunoResearch), or anti-rabbit (Jackson ImmunoResearch) IgGs. Signals were detected using Chemi-Lumi One . The images were then acquired using ChemiDoc Touch  and analyzed using Image Lab Software version 6.1.010.9.0 (Bio-Rad).In this study, we investigated the reasons for the poor infectivity of cell-free HTLV-1. Our results indicated that HTLV-1 Env was efficiently incorporated into VLPs produced by HIV-1 but not HTLV-1 Gag, highlighting the role of HTLV-1 Gag in the lower infectivity of cell-free HTLV-1. We further verified that the interaction of the PTAP motif located in the p6 domain of HIV-1 Gag with TSG101 was responsible for the incorporation of HTLV-1 Env into HIV-1 Gag VLPs and enhanced the cell-free infectivity of pseudotyped HIV-1 with HTLV-1 Env. Overall, these findings indicate that the poor infectivity of HTLV-1 can be partly attributed to the inefficient incorporation of HTLV-1 Env into cell-free HTLV-1.


This review article explores the possibility of developing an integrated approach to the management of the different needs of endometrial cancer (EC) patients seeking to become pregnant. Life preservation of the woman, health preservation of the baby, a precocious and—as much as possible—minimally invasive characterization of the health and fertility parameters of the patient, together with the concerns regarding the obstetric, neonatal, and adult health risks of the children conceived via assisted reproductive techniques (ART) are all essential aspects of the problem to be taken into consideration, yet the possibility to harmonize such needs through a concerted and integrated approach is still very challenging. This review aims to illustrate the main features of EC and how it affects the normal physiology of pre-menopausal women. We also focus on the prospect of a miR-based, molecular evaluation of patient health status, including both EC early diagnosis and staging and, similarly, the receptivity of the woman, discussing the possible evaluation of both aspects using a single specific panel of circulating miRs in the patient, thus allowing a relatively fast, non-invasive testing with a significantly reduced margin of error. Finally, the ethical and legal/regulatory aspects of such innovative techniques require not only a risk-benefit analysis; respect for patient autonomy and equitable health care access allocation are fundamental issues as well.  Assisted reproductive technology (ART) refers to several techniques allowing women to achieve pregnancy in a non- (fully) natural way and include in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), gamete intrafallopian transfer (GIFT), and preimplantation genetic diagnosis (PGD) . ART is Endometrial cancer (EC) affects the lining of the uterus and is usually diagnosed in postmenopausal women, but about 5% of cases occur in women under 40, and approximately 20% of cases are diagnosed before menopause . The mosEvaluation of female fertility is an important step for women who want to undergo ART or preserve their fertility after being diagnosed with EC. A pelvic examination should be performed to assess the uterus and adnexa for masses or other abnormalities. The gold standard method for diagnosing EC is hysteroscopy and endometrial biopsy, which have high sensitivity and specificity. Other imaging techniques, such as ultrasound, magnetic resonance imaging (MRI), or computed tomography (CT), can be used to evaluate the disease extent and plan the treatment .In this work, we will examine all the above aspects. We will discuss the etiopathogenesis of EC both from a clinical and a molecular point of view, with a special focus on epigenetic causes and the use of circulating miRs as diagnostic biomarkers. We will report the state-of-the-art in fertility evaluation by means of circulating miR, comparing the two sets of data (circulating miRs in EC and for fertility assessment) and highlight how these two sets have only minimum overlap, providing the possibility to establish both the health status of the patient and her possibility to undergo ART at the same time and with a minimally invasive test (a blood sample). Finally, we will provide an overview of the main ethical and legal aspects of procreation in EC  patients. We set out to elucidate in a succinct and yet comprehensive fashion the underlying factors, determinants, and dynamics at the root of EC, its clinical implications in terms of therapeutics and diagnostics, the prospects for fertility preservation in younger patients, and the prospect for a miR-based, molecular evaluation of patient health status, including both EC early diagnosis and staging and, similarly, the patient’s receptivity. Finally, the complex ethical and legal/regulatory aspects have been weighed. For each of the above-listed subjects, 125 sources have been drawn upon, by using the search strings “endometrial cancer”, “fertility preservation”, “RNA-based molecular classification/evaluation”, “miRNA cancer diagnosis” through scientific databases PubMed/MedLine, PubMed Central, Scopus, ResearchGate, Web of Science. Only articles accounting for EC, infertility/fertility preservation, RNA-based diagnostic and therapeutic approaches, and ethics peculiarities of such innovative techniques have been considered for the fundamental purpose of this review article.EC has long been known as one of the most widespread gynecologic cancers worldwide; it is also the most common cancer affecting the female genital tract in developed countries. This malignancy is localized to the uterus in most patients  .While uterine corpus cancer is currently the most common gynecologic malignancy, endometrial carcinomas constitute the majority of such diagnoses; sarcoma accounts for less than 10% of uterine corpus cancers. As many as 83% of uterine corpus cancers are endometrioid carcinomas. In addition, 4% to 6% of endometrial carcinomas consist of serous and papillary serous carcinomas, while clear cell carcinomas account for 1% to 2%. For a thorough analysis and better management and prevention of such conditions, it is worth drawing a distinction among type 1 endometrioid, type 2 serous endometrial carcinomas, and other highly aggressive non-endometrioid carcinoma histotypes .Abnormal uterine bleeding or postmenopausal bleeding constitutes typical EC presentations. A diagnostic evaluation should be made available to any patient having EC risk factors, which should include the assessment of clinical history, imaging, and endometrial sampling. Standard EC therapeutic pathways may entail hysterectomy, bilateral salpingo-oophorectomy, and surgical staging. Hysterectomy is instrumental in accurately assessing EC prognostic factors, such as stage, grade, myometrial invasion, lymphovascular space invasion, and lymph node status ,13,14.ECs have been found to begin as preinvasive intraepithelial lesions transitioning into full-blown invasive cancers affecting endometrial stroma. A progressive penetration into the myometrium occurs through the lymphatic capillaries, thus spreading cancerous cells into regional lymph nodes. Then, the metastasizing process unfolds via vascular channels. The uterine cervix and stroma are likely affected by tumor progression through lymphatic channels, even if surface spread has been observed to take place from ECs manifesting in the lower uterine segment (LUS). LUS involvement in endometrial carcinoma has often been reported to result in lower survival rates and higher recurrence rates .High levels of free estrogens leading to endometrial hyperplasia have been linked to estrogen-secreting, ovarian tumors, and polycystic ovaries (PCO); both conditions can adversely affect regular ovulation and menstruation. While anovulation obviously results in infertility, nulliparity is linked to a higher EC risk, even after adjusting for infertility . Potential precursors of type I EC  have been found to be atypical endometrial hyperplasia or endometrial intraepithelial neoplasia (EIN). Such dynamics often manifest at an early stage, with rather favorable outcomes. Serous, clear cell, mixed cell, and undifferentiated histologies are all elements reportedly associated with type II ECs, which are estrogen-independent and manifest at an already advanced stage with unfavorable prognosis . The valSuch clinical dynamics entail medical as well as ethical and social concerns: fertility-sparing treatment (FST) can in fact forgo more radical care procedures by prioritizing the patient’s reproductive capabilities. ARTs are often required to that end, which means that EC treatment in patients of reproductive age is uniquely challenging, due to the need to strike a balance between “competing interests” of cancer care and the determination of patients to maintain their reproductive potential. Early menarche and late menopause, with higher levels of lifetime exposure to endogenous estrogens, have been found to lead to higher EC risks.EC type and fundamental traits ought to be thoroughly assessed in order to choose the therapeutic pathway, which best suits each patient, particularly when weighing a conservative management opportunity.Fertility-sparing procedures need to be weighed and counseled when making treatment decisions. That is even truer in light of the potentially harmful psychological dynamics that may be triggered by the loss of fertility following aggressive therapeutic approaches ,20.EC risk of extrauterine spread is an essential aspect to evaluate when assessing patient eligibility for fertility-sparing procedures. For patients who have an interest in preserving their fertility and plan to conceive as soon as possible after remission, fertility-sparing should be always considered, in the absence of contraindications and when there is favorable histopathological cancer makeup .The fertility-sparing decision-making process needs to be weighed against various EC risk factors, such as obesity and polycystic ovary syndrome; such factors are linked to infertility as well; hence, any ART consideration may well be influenced by them. EC stage Ia grade 1 (G1) and EEC are the malignancies for which fertility-sparing treatment is most often chosen. EC type II, on the other hand, often makes patients ineligible for conservative treatments, due to its high level of invasiveness and poor differentiation. Young women with G1, no myometrium and/or adnexal invasion, and without lymphvascular space involvement, are therefore deemed the best candidates for fertility preservation approaches ,23).When outlining any such pathway, it is worth taking into account updated guidelines by scientific societies such as ESGO-ESHRE-ESGE , which have issued specific, evidence-based guidance for fertility-sparing treatment of EC patients, by focusing on the fundamental traits of fertility-sparing treatments. Particularly relevant is the recommendation that EC patients undergoing fertility-sparing procedures are counseled and cared for by a multidisciplinary team relying on oncologists and fertility specialists .Progestin therapies , megestrol acetate (MA), and progesterone-releasing intrauterine device (IUD) are the most widespread and validated EC hormonal treatment (HT) options and constitute the bedrock of the conservative fertility-sparing toolbox. MPA at 250–600 mg daily and MA at 160–480 mg daily are the most widely used regimens and rely on similar potency levels .Recent data have pointed to the novel levonorgestrel intrauterine device (LNG-IUD) as a solid fertility preservation option as well . This dePTEN) gene, which acts on the PI3K/Akt/mTOR signaling pathway, essential for the regulation of the cell cycle and is involved in cell survival, proliferation, and growth [POLE gene encodes the catalytic subunit of DNA polymerase epsilon whose function is implicated in nuclear DNA replication and repair. POLE mutations usually occur in the soma and because of their function, have an extremely high mutation rate but also by excellent prognosis with no recurrence regardless of the FIGO grade. The MSI group also shows a high mutation rate and is mostly caused by MLH1 promoter methylation. MLH1 is a DNA mismatch repair protein, and its gene mutations are frequently associated with microsatellite instability (both facts explaining the high mutation rate in EC) as also observed in hereditary nonpolyposis colorectal cancer . MLH1 mutations cause an EC risk of 25–60% [POLE, absence of MSI, and a low incidence of somatic copy-number variations. This genetic scenario is frequently found in low-grade EEC and the prognosis is intermediate. Finally, the fourth class is characterized by a low mutational rate but a high frequency of copy number variations and a 90% rate of TP53 mutations. This group mainly includes SEC and its patients, who unfortunately have a poor prognosis [PTEN (>77%), PIK3CA (53%), PIK3R1 (37%), CTNNB1 (36%), ARID1A (35%), K-RAS (24%), CTCF (20%), RPL22 (12%), TP53 (11%), FGFR2 (11%), and ARID5B (11%). The most frequently mutated genes in serous carcinomas are TP53 (80–90%), PIK3CA (41.9%), PPP2R1A (36.6%), FBXW7 (30.2%), CHD4 (16.3%), CSMD3 (11.6%), and COLA11 (11.6%), along with loss of heterozygosity (LOH) on many chromosomes. MSI is frequent in type I carcinomas (25–40%) but relatively rare in type II carcinomas (<5%). Interestingly, PTEN mutations are characteristic of type I EC, while TP53 mutations are characteristic of type II EC, affecting just 11% of EEC. Instead, PIK3CA mutations are a common characteristic of both types, being mutated in 40–50% of all EC. This finding suggests that, apart from some shared pathways, types I and II have a very distinct molecular pathogenesis. Similarly, the above-mentioned four subtypes of EC are molecularly different as well, with candidate driver or pathogenic genes accounting for 190 genes in the POLE subgroup, 21 in the MSI subgroup, 16 in the low copy number subgroup, and 8 in the high copy number subgroup [PTEN is the most frequently mutated gene in all except in the copy number high group, where TP53 mutations dominate. Additional genetic causes of EC formation and growth include single nucleotide variants in specific genes [MLH1) [As described before, pathological and demographic parameters allow the identification of two different types of EC: type I EC, also called EEC , usually well differentiated, with average recurrence rates of 20%; and type II EC, which include mainly CCEC  and SEC . It is essential to differentiate type I from type II carcinomas and other highly aggressive non-endometrioid carcinoma histotypes to understand, manage and possibly prevent these diseases . The mold growth ,30. Seved growth  since thd growth . Additiod growth . Back ind growth . Those df 25–60%  and resurognosis . Recentlrognosis . To daterognosis . Accordisubgroup . In thesic genes , shortenic genes , and epinomas 25–% but rel, PIK3CA .9%, PPP2s [MLH1) .In recent years, increasing interest has been devoted to these last molecules and their cross interaction, because of their great potential as EC biomarkers. Focused ncRNA panels are under investigation for creating fast and accurate diagnostic tools; with the aim of being as fast and accurate as possible, but at the same time without using invasive approaches, circulating short ncRNA attracted the attention of researchers.Micro-RNAs (miRNAs or miRs) are a class of single-stranded, non-coding RNAs (ncRNA) characterized by their short length: 20–25 nucleotides on average. They are part of a group of molecules collectively called ‘short non-coding RNAs’ (sncRNA). Their action is modulating gene expression by targeting messenger RNAs (mRNA) through sequence homology. Notably, the match between miR and its target is not always perfect, thus allowing the former to interact with multiple mRNAs; this makes the target discovery challenging and the function identification puzzling . The binp < 0.05). In 2022, Cai and coworkers analyzed both the TCGA and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to reconstruct a lncRNA-mediated ceRNA network for uterine corpus endometrial carcinoma [The literature reports several ceRNA axes in EC  and a loarcinoma . This woarcinoma . In FiguAccording to recent advances in histopathological and molecular characterization, EC can be classified into at least four different classes ,79. In aThe identification of deregulated miRs in EC patients is ongoing, and the discovery of new ceRNA axes is relatively frequent. A meta-analysis published in 2019 by Delangle and co-workers  identifiEismann and coworkers in 2017 showed that the circulating miRs excreted in vitro by EC cell lines are also dependent on exogenous environmental stress, such as hypoxia and acidosis . They shTo date, only a minor proportion of studies analyzed the expression levels of miRs in healthy women to evaluate their potential receptivity.In 2017, Altmae and collaborators performed a meta-analysis using as ‘bait’ a meta-signature of endometrial receptivity involving 57 mRNA genes as putative receptivity markers . SearchiIt is noteworthy to underline that, among all these fertility-related miR, only miR-21, miR-27a, and miR-135b are present in RNA-based measurements are potentially applicable through a wide array of medical areas, including diagnosis, prognosis, and therapy selection. Currently, among the most promising clinical applications, it is worth mentioning not only cancer research  but alsoFertility preservation should only be offered to patients with EC stage Ia grade 1 (G1), who present without myometrial invasion or where cancer has invaded less than 50% of the myometrium, with no evidence of pathological lymph nodes or synchronous/metachronous ovarian tumor . The mosAlthough gynecological cancers are most frequently diagnosed in post-menopausal patients, substantial numbers of younger women of reproductive age are also affected. The importance of thorough counseling of these patients cannot be overstated, including a comprehensive discussion of long-term consequences on fertility of the various treatment options and aiming at fertility preservation as much as possible, if the patient prioritizes this aspect. EC diagnosis at reproductive age points to the risk of a hereditary condition; hence, counseling must address the genetic cancer risk evaluation in order to investigate any known genetic predisposition. Such broad-ranging principles notwithstanding, fertility-sparing therapeutic pathways need to be individually tailored, considering the high level of heterogeneity of gynecological cancers.The chief surgical option for EC is hysterectomy, whereas fertility-sparing management in patients with EC or complex atypical hyperplasia is not deemed the standard approach. Eligibility requirements are age under 40, complex atypical hyperplasia, or grade 1 EC limited to the endometrium. Therefore, an accurate pretreatment staging is non-negotiable .Gallos et al., in a meta-analysis involving 408 women with early-stage EC who underwent fertility-sparing treatments, found a 28% live birth rate, with patients undergoing ART achieving a 39.4% live birth rate, as opposed to 14.9% in the spontaneous conception group .The preservation of fertility and procreative capabilities are ever-more relevant elements in terms of life quality after EC. Patients with no male partner or unwilling to freeze and store embryos can still preserve their oocytes. Such techniques, particularly those relying on freezing by vitrification, lower the risk of incurring multiple pregnancies or ovarian hyperstimulation syndrome (OHSS). Compared to transfer from fresh embryos, no significant differences have been reported in terms of pregnancy rate per cycle and the clinical pregnancy rate per cycle .The decision to store frozen oocytes, as opposed to embryos, can avoid or at least alleviate ethical and legal challenges often arising from cryopreserved embryos and their controversial status. Major improvements in overall outcomes of oocyte cryopreservation have been achieved through more effective cryopreservation techniques such as vitrification rather than the slow-freeze protocol, mostly thanks to the reduction of cellular damage arising from ice crystal formation . MoreoveThe intersection of gonadotoxic therapy and reproduction raises ethical issues for both cancer and fertility specialists, including issues of experimental vs established therapies, the ability of minors to give consent, the welfare of expected children, and posthumous reproduction ,170,171.The newly found possibility to focus both on the health status of the patient, including the definition of EC staging and her fertility, with a fast and minimally invasive approach, as described above, further contributes to enabling such patients to achieve motherhood, through effective management of their treatments.As infertility after cancer has become a recognized survivorship issue, oncologists should be prepared to discuss the negative impact of cancer therapy on reproductive potential with their female patients in the same way as any other risks of cancer treatment are discussed. Patients interested in fertility preservation should be promptly referred to a reproductive medicine expert to offer timely and appropriate counseling and improve the success of fertility preservation. Such aspects are to be viewed as requirements for the medicolegal tenability of any intervention. Reproductive endocrinologists should collaborate with oncologists and molecular biologists, updating them regarding available technologies and facilitating consultations with patients newly diagnosed with cancer. To further these alliances, education about fertility preservation, as well as ethical and legal aspects tailored to country-specific laws, should be incorporated into training programs for oncology and reproductive endocrinology. Just as importantly, the breakthrough constituted by RNA-based diagnostics and therapeutics needs to rely on as broad a consensus as possible, by all stakeholders involved  in order to reconcile the amazing opportunities created by such techniques with the core values that must guide scientific research and medical practice at all times.