G protein-coupled receptors are important drug targets that engage and activate signaling transducers in multiple cellular compartments. Delineating therapeutic signaling from signaling associated with adverse events is an important step towards rational drug design. The glucagon-like peptide-1 receptor (GLP-1R) is a validated target for the treatment of diabetes and obesity, but drugs that target this receptor are a frequent cause of adverse events. Using recently developed biosensors, we explored the ability of GLP-1R to activate 15 pathways in 4 cellular compartments and demonstrate that modifications aimed at improving the therapeutic potential of GLP-1R agonists greatly influence compound efficacy, potency, and safety in a pathway- and compartment-selective manner. These findings, together with comparative structure analysis, time-lapse microscopy, and phosphoproteomics, reveal unique signaling signatures for GLP-1R agonists at the level of receptor conformation, functional selectivity, and location bias, thus associating signaling neighborhoods with functionally distinct cellular outcomes and clinical consequences. Agonists of the glucagon-like peptide-1 receptor are used to treat diabetes and obesity. Here, Wright et al. investigate the subcellular location of the receptor’s signaling events and uncover associations between signaling profiles and adverse drug reactions. Among these are the mutational and epigenetic landscape of drug targets, polypharmacology and an incomplete knowledge of functional selectivity or biased signaling. In particular, the functional selectivity of existing compounds that target G protein-coupled receptors (GPCRs)—the largest family of drug targets—remains largely unexplored and is often limited to a couple of downstream, signal-amplified readouts2. The dynamic nature of GPCR architecture enables diversity across ligand binding pockets and intracellular binding sites—increasing the number of pathways that can be engaged5. This inherent diversity has implications for rational drug design, which aims to balance efficacy and adverse drug reactions (ADRs).As clinicians, healthcare systems, and the pharmaceutical industry try to move away from generic disease management towards precision medicine, several fundamental issues need to be addressed in order to maximize therapeutic efficacy and minimize adverse events7. Biologics that target this receptor, such as semaglutide and liraglutide have proven clinically successful in regulating blood glucose levels, but they are not without drawbacks that include daily or weekly injections and reports of ADRs ranging from nausea and diarrhea to pancreatitis7. In order to produce an orally available GLP-1R agonist, increased efforts have been directed towards the development of small molecules. Incretin mimetics engage both the extracellular and transmembrane domains (TMD) of GLP-1R, whereas small molecule GLP-1R agonists are more restricted to the traditional GPCR binding pocket in the TMD11. Differences in binding pose have been shown to lead to unique signatures in transducer engagement for which emphasis has been placed on Gs and β-arrestin (βARR). Recent work from our group demonstrated that the endogenous agonist for GLP-1R, GLP-1 (7-36), activates members from all G protein families at the plasma membrane5. However, our knowledge about the signaling signatures of preclinical and clinical GLP-1R agonists and the location of this activity is incomplete.In recent years, the glucagon-like peptide-1 receptor (GLP-1R) has emerged as an important target in the treatment of type II diabetes and obesity21 and there is convincing evidence that subcellular signaling can lead to ADRs25. In light of the increasing attention that GLP-1R has received as a drug target, we sought to better characterize the signaling fingerprint of this receptor after treatment with preclinical and clinical GLP-1R agonists. To this end, we developed an integrative and comprehensive approach to monitor transducer promiscuity with subcellular resolution based on bioluminescence resonance energy transfer (BRET). This strategy allowed us to decode the specific transducer recruitment signatures of GLP-1R at the plasma membrane, early endosomes, the Golgi apparatus, and the endoplasmic reticulum and the resulting signaling fingerprints correlated with ADR risk in the FDA Adverse Event Reporting System (FAERS). At the molecular level, differences in transducer engagement entailed altered kinetics and connectivity of phosphoproteomic signaling networks. These findings open up new possibilities for finetuning drug development and suggest that preclinical pipelines to assess efficacy and safety should be reassessed to ensure that the benefit to the patient outweighs any risk.The ability of GPCRs to signal from different subcellular compartments is now well-establishedPDB: 5VEX) and PF-06372222 (PDB: 6KJV), the peptide agonists GLP-1 (7-36) (PDB: 6X18), exenatide (PDB: 7LLL) and semaglutide (PDB: 7KI0) as well as the small molecule agonist danuglipron (PDB: 6X1A)  and GLP-1 (7-36)], the GLP-1 analogues liraglutide and semaglutide, synthetic GLP-1 mimetics from the reptile Heloderma suspectum (exenatide and lixisenatide) and the small molecule GLP-1R agonist danuglipron.Based on the structural differences observed in the residue contact analysis, we selected a series of homologous peptide agonists  in addition to the small molecule agonist /Gβγ and βARR , GRK/Gβγ and βARR  stimulation5, underlining the importance of investigating polypharmacology. GLP-1 (7-36), liraglutide, and exenatide strongly activated members of the Gi/o family compared to other tested drugs. Semaglutide and lixisenatide strongly engaged the Gs, GRK/Gβγ, and βARR pathways at the plasma membrane, while danuglipron, a small molecule agonist in clinical trials, displayed a unique fingerprint of pathway engagement that appeared to be biased away from βARR towards Gs and Gi/o. We then quantified the similarity and diversity of transducer engagement across drugs using the Jaccard index for efficacy and potency  anchored to the plasma membrane using the CAAX box from KRAS. Using this approach, translocation of the biosensor from the cytosol to the plasma membrane results in energy transfer, which is indicative of pathway activationARR Fig. . Surpris27. Receptor trafficking or subcellular localization of GPCRs has functional implications for drug action. In particular, endosomal GLP-1R has been proposed to contribute to the generation of cAMP following receptor activation28. However, sustained signaling from internalized GLP-1R does not promote further insulin release and has instead been linked to gastrointestinal ADRs30. We sought to track active GLP-1R by confocal microscopy using the fluorescent biosensor mini Gs (mGs) that detects the receptor conformation that binds Gs31. Using this tool, we could monitor the trafficking dynamics and intracellular signaling neighborhoods that accommodate signaling  and trend of substrate enrichment varied greatly . Exploration of these signaling neighborhoods revealed pharmacodynamic differences across pathways and compartments for the 8 tested drugs  Fig. . From thter Fig. . Semagluk-means cluster centers revealed the cluster-specific molecular underpinnings to predict both protective and undesirable signaling signatures. Overall, these findings provide the molecular framework linking the differences in receptor conformation and signaling neighborhoods to clinical responses.We hypothesized that the differences in drug-induced signaling profiles might be related to ADR risk. We thus used the FDA adverse event reporting system (FAERS) to evaluate adverse event reports for all approved GLP-1R agonists included in our study: semaglutide, lixisenatide, liraglutide and exenatide Fig. . Our ana42. Despite our increased understanding of how these proteins operate and their recognized value for disease management, the drugs that target these receptors are not without their drawbacks, which include lack of efficacy or safety issues. Moreover, the absence of scalable and sensitive tools to measure emerging paradigms like pathway selectivity and location bias—aspects of signaling that can contribute to ADRs—has made it difficult to integrate these properties into drug development programs.GPCRs are the most intensively studied of all drug targets and represent the largest target class of approved drugs and agents in clinical trials44. Our data unequivocally show differences in the signaling fingerprints of GLP-1R agonists at the levels of transducer engagement, signaling kinetics and subcellular localization. However, the functional outcome of this pluridimensional signaling remains to be understood—especially in a native, tissue-specific context. Activation of GLP-1R in different tissues leads to insulin release, suppression of glucagon secretion, slowing of gastric emptying and increase in satiety7. It is therefore plausible that variation in the stoichiometry of the drug-specific GLP-1R transducerome across tissues could mediate differential outcomes and our work paves the way for future studies into such tissue-specific signaling profiles. Yet, coupling pathway-selective biosensors with compartment-specific markers, as we have done here, was sufficient to group GLP-1R agonists and correlate these groups with ADRs, suggesting that ADRs may, in part, be explained by pathway and compartment selectivity.The work presented here demonstrates how pluridimensional signaling can be measured by a singular robust, yet modular ebBRET-based biosensor platform, allowing for the characterization of pathway activation in an organelle-specific manner—an important aspect of GPCR biology that may become the new frontier in rational drug design46. Future research should therefore explore the consequences of different dosing regimens and pharmacokinetics of GLP-1R agonists in vivo on both signaling profiles and biological outcomes. Importantly, the firm establishment of causal relationships between transducer signatures and ADR risk requires future evaluations on a larger scale.Moving forward, it will be important to extend the concept of signaling neighborhoods to primary human tissues to evaluate whether the profiles are conserved. Also, secretin receptor family crosstalk or interpatient variability are important parameters to consider in future work to fully comprehend the physiological impact of pathway-selective compartmentalized signaling. Another intriguing aspect of GLP-1R signaling that deserves attention is the potential for kinetic bias of drugs acting on this receptor, as this may modulate efficacy and potency51. Using BRET and phosphoproteomics, we show that the signaling profiles of peptide and small molecule agonists of GLP-1R are rather different, which is consistent with our observation that they engage distinct residue contact networks. Our analyses reveal that GLP-1R agonists can elicit drastically different downstream signaling profiles. For instance, we show that semaglutide and lixisenatide had a lower efficacy for Gi/o than liraglutide and exenatide specifically at the plasma membrane and coincidentally have a lower incidence of pancreatitis. In addition to patterns specific to individual compounds, drug class-specific differences were observed. Specifically, βARR recruitment at the plasma membrane and receptor endocytosis, which have been linked to nausea29, were revealed to be common defining features of all incretin mimetics, but not the small molecule drug danuglipron. However, the impact of danuglipron-induced GLP-1R activation at the Golgi and endoplasmic reticulum, but not at endosomes, coupled with extensive changes to the cellular phosphoproteome remains unknown. This information has enormous implications for drug development and clinical practice as oral small molecule compounds that target Class B GPCRs are increasingly sought after due to improved adherence11.In addition to the plasma membrane, GPCR transducers like heterotrimeric G proteins, GRK and βARR can also be found in subcellular compartments. GPCR activation can dynamically affect the subcellular distribution and activity of these transducers, which may or may not be coupled with the trafficking properties of the receptor52. Using a different approach, our analyses reveal that sequence modifications that prevent degradation or increase protein binding had a greater effect on defining drug fingerprints than their homology to the human endogenous peptide. Specifically, exenatide, which is derived from a peptide found in the venom of the Gila monster that is separated from human biology by around 320 million years of evolution (~50% sequence identity), closely resembles transducer signatures and safety profiles of liraglutide—a modified human incretin. This demonstrates that, for the subset of compounds tested in this study, GPCR signaling neighborhoods serve as a better predictor of clinical outcomes than the structural homology of compounds. Future conscious efforts aimed at shaping these signaling neighborhoods through data-driven drug development may increase our chances of minimizing adverse events.In the present study, we show that location- and pathway-selective signaling profiles correlate with compound safety. These results are consistent with recent findings that ADRs correlated better with drug signaling profiles than with pharmacokinetic propertiesD-PBS, DMEM, Trypsin, PBS, penicillin/streptomycin, fetal bovine serum (FBS) were from Gibco . Polyethylenimine (PEI) was purchased from Alfa Aesar . Glucagon was from Sigma Aldrich . GLP-1 (1-37), GLP-1 (7-36), exenatide, liraglutide, semaglutide, lixisenatide and danuglipron were from either BACHEM  or MedChemExpress LLC . Dyngo-4A was from ApexBio . Coelenterazine 400a was purchased from Nanolight Technologies .Rluc853, p63RhoGEF-RlucII, Rap1Gap-RlucII, PDZRhoGEF-RlucII5, GRK2-D110A-RlucII54, β-arrestin-2-RlucII55, rGFP-CAAX and rGFP-FYVE43 and tdrGFP-Giantin and tdrGFP-PTP1B17 have been described previously. Human GLP-1R was generously given by Domain Therapeutics NA Inc. SNAP-GLP-1R was from Novo Nordisk. GLP-1R-RlucII was generated by amplifying human GLP-1R from Domain Therapeutics NA Inc. with flanking restriction sites for NheI and BamHI followed by subcloning into pcDNA3.1-(GFP10)-RlucII to replace GFP10 with GLP-1R. Human Gαi1, Gαi2, Gαi3, GαoA, GαoB, Gαz, Gαq, Gα11, Gα14, Gα15, Gα12 and Gα13 were purchased from cDNA.org . Rluc8-mGs and mRuby2-mGs were generated by subcloning from NES-Venus-mGs31 into Rluc8-C1 and mRuby2-C1 vectors using EcoRI and XhoI. All plasmid constructs were verified by Sanger sequencing.GLP-1R-2 and 90% humidity. Cells  were transfected in suspension with 1.0 μg of plasmid DNA complexed with linear polyethyleneimine  and seeded (3.5 x 104 cells/well) in white 96-well plates. All cell lines were regularly tested for mycoplasma contamination.HEK293 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, propagated in plastic flasks and grown at 37 °C in 5% CORluc8-mGs, GRK2-D110A-RlucII, or β-arrestin-2-RlucII and rGFP-CAAX, rGFP-FYVE, tdrGFP-Giantin or tdrGFP-PTP1B. To monitor the recruitment of effectors to transducers (GEMTA), HEK293 cells were transfected with GLP-1R, Gα, p63RhoGEF-RlucII, Rap1Gap-RlucII or PDZRhoGEF-RlucII and rGFP-CAAX, rGFP-FYVE, tdrGFP-Giantin or tdrGFP-PTP1B. Receptor trafficking was measured in HEK293 cells that had been transfected with GLP-1R-Rluc and rGFP-CAAX, rGFP-FYVE, tdrGFP-Giantin or tdrGFP-PTP1B. After a 48-hour incubation, cells were washed once with HBSS and maintained in the same buffer. Prior to BRET measurements, cells were stimulated with agonist and incubated with coelenterazine 400a . See Supplementary Data To monitor the recruitment of transducers, HEK293 cells were transfected with GLP-1R, 2: 400/70 nm (donor) and 515/20 nm (acceptor) for detecting the RlucII/Rluc8 (donor) and rGFP (acceptor) light emissions, respectively.Plates were read on a Tecan Spark multimode microplate reader  equipped with the following filters for BRET50 was outside of the concentration range tested or if the span was negative. One-sided extra sum-of-squares F test was employed to test whether top parameter of the non-linear regression was statistically distinguishable from the bottom. The Bonferroni correction was applied to account for multiple comparisons across the eight drugs in each pathway (P < 0.00625). All further data processing was performed in R (version 4.2.1).Best fit values were excluded if ECHEK293 cells were transfected in suspension with GLP-1R, mRuby2-mGs, and rGFP-CAAX, rGFP-FYVE or tdrGFP-Giantin and grown in four compartment 35 mm glass bottom dishes. After 48 h, the cells were washed with HBSS before adding 1 μM semaglutide. Cells were imaged every 15 s for 30 min. Confocal images were acquired using a Zeiss 980 laser scanning microscope. Images were acquired using a 40×, 1.2 NA C-Apochromat objective and the 488 nm and the 594 nm laser lines were used to excite rGFP and mRuby2 fluorophores, respectively. To quantify colocalization between mRuby2-mGs and tdrGFP-Giantin, green particles were selected as regions of interest and the mean intensity of red fluorescence per area was measured throughout the duration of the timelapse in ImageJ2 2.9.0.HEK293 cells seeded on 8 well μ-Slide (ibidi) were transfected with SNAP-GLP-1R and compartment markers fused to rGFP. The following day, cells were labeled with 5 μM SNAP-Surface® Alexa Fluor® 647 (New England Biolabs) for 15 min at 37 °C. Cells were then washed and exposed to 1 μM semaglutide for the following incubation times: 10 min for PM (rGFP-CAAX), 20 min for EE (rGFP-FYVE) and 30 min for GA (tdrGFP-Giantin). Finally, cells were fixed with PFA 2% before adding ibidi Mounting Medium with DAPI. Confocal microscopy was carried out using an SP8 LIGHTNING confocal microscope (Leica Microsystems) with a 63× oil immersion lens, . Images were treated using ImageJ2 2.9.0 software.56 as provided by Open Targets (https://platform.opentargets.org/)57 and which corrects for the prevalence of a given drug and the frequency of an event across drugs. In total, 48 unique ADRs were retrieved and mapped to the Medical Dictionary for Regulatory Activities (MedDRA), which was further aggregated and summed into 9 “System Organ Class” (SOC) ontology-levels.Post-marketing adverse events for all approved and available GLP-1R-targeting agents included in our study were obtained. Significant adverse reactions (ADRs) have been estimated from reports submitted to the FDA Adverse Event Reporting database (FAERS) by healthcare professionals by transformation into a log likelihood ratio (LLR)PDB:6X1811], semaglutide [PDB:7KI058], exenatide [PDB:7LLL59] and non-peptide agonist PF-06882961/danuglipron [PDB:6X1A11]. The two inactive structures in complex with two negative allosteric modulators, respectively PF-06372222 [PDB: 6KJV60] and NNC0640 [PDB: 5VEX61]. Structures in complex with a signaling protein were set as the reference structures for the active state (100%-degree activation) and structures with a highly closed conformation were set as the reference structures for the inactive state (0%-degree activation) based on a maximum measured distance between 2x46 to 6x37. The Cα atom distance pairs for each structure were compared to the reference structures and the mean distance to the active structures and the mean distance to the inactive structures were calculated. These distances to the reference structure sets were then converted into an “activation score” by subtracting the mean distance to the inactive-state structures from the mean distance to the active-state structures. The activation score was converted into a percentage activation based on the minimum and maximum activation scores for all structures in that class (https://docs.gpcrdb.org/structures.html?highlight=activation#structure-descriptors). In our analysis, all four selected active structures displayed 100% normalized degree activation. Conversely, the two reference inactive state structures both displayed 0%-degree activation. See Supplementary Data Six GLP-1R structures were selected from the Protein Data Bank, four in active and two in inactive conformations, respectively. The active structures are in complex with: GLP-1 (7-36) [https://review.gpcrdb.org/structure_comparison/comparative_analysis)62. Briefly, contact analysis was performed considering sidechain-sidechain and sidechain-backbone interactions among inter-segment residues and only sidechain-sidechain interactions among intra-segment contacts (as the GPCRdb webserver default settings). For each receptor residue, non-hydrogen atoms in close proximity of non-hydrogen atoms from neighboring residues are taken into account. The potential contacts are further evaluated based on atom and residue types and their distance. For each of the contact types, the default maximum distances are ionic (4.5 Å), polar (4 Å), aromatic (stacking 5.5 Å and cation-π 6.6 Å), hydrophobic (4.5 Å) and Van der Waals contacts (1.1 times their combined VdW radii).Both the analyses of the receptor contact pairs and the interaction types were performed using the GPCRdb webserver for comparative structure analysis (see Corresponding residue positions in the structures were identified with the structure-based GPCRdb generic residue numbering system that is based on the structure-based generic residue numbering system for class B1 (Wootten numbering). This avoids gaps and mismatching after structural alignments of receptors due to missing residues and/or different helix bulges/constrictions among the structures.Following this numbering system, not all residues can be assigned to generic numbers; in fact, only the TM helices, H8 and structurally conserved loop segments with annotated generic residue numbers were included, excluding the extracellular domain. The included structures varied in their coverage from 263 (5VEX) to 390 (6X1A) resolved residues . We hence restricted the residue contact analysis to only the shared 219 residues among all 6 structures.Supervenn) where each row is a set of PDB-specific contact pairs, and the overlapping parts (groupings) correspond to intersections of sets (i.e. shared residue-pairs among sets). The sizes of sets and their groupings are proportional, but the order of elements is not preserved. A combinatorial optimization algorithm was applied that rearranges the groupings (the columns of the array plotted) by occurrence (groupings that are in more sets go first). The columns on the right represent, for each PDB, respectively: the set sizes (N. contact pairs), the residues included in the analysis , and the unshared residues excluded from the analysis (N. excluded not shared residues). The numbers on the top (Number of overlapping structures) show how many sets fit into this intersection (i.e. how many PDBs show that particular grouping of contact pairs). The numbers below  represent a scale to give indications of the proportion of the number of residue contacts.The obtained shared contact pairs were represented in a Venn-like diagram .All structural representations were produced using UCSF Chimera66. In short, 200 μL of the lysis buffer preheated at 95 °C (4% sodium dodecyl sulfate in 50 mM Tris adjusted to pH 8.5) was added to the cells. The samples were then denatured at 95 °C for 10 min, placed on ice and then sonicated using a probe sonicator  (Branson). Lysate protein concentration was determined using the Pierce bicinchoninic acid assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The lysates were reduced using 5 mM dithiothreitol for 1 h at room temperature and alkylated using 15 mM iodoacetamide for 1 h at room temperature in the dark. Proteins were precipitated and digested on beads using MagReSyn Amine magnetic beads (ReSyn Biosciences) according to the manufacturer’s instructions. Protein digestion was conducted overnight at 37 °C with lysyl endopeptidase   and trypsin . 20% TFA, 2 M glycolic acid in 80% ACN were added to the samples at a 1:1 ratio for phosphopeptide enrichment using MagReSyn TiO2 magnetic beads (ReSyn Biosciences) according to the manufacturer’s instructions. The enriched phosphopeptides were dried overnight in a SpeedVac, resuspended into 5% formic acid (FA), desalted using StageTip (Thermo Fisher Scientific) according to the manufacturer’s instructions and dried in a SpeedVac. The experiment was performed in biological triplicates.HEK293 cells  were transfected in suspension using 3 μg of linear PEI  per 1 μg of plasmid DNA and seeded onto poly-D-lysine-coated 60 mm dishes at a density of 1,750,000 cells per dish. After a 24-h incubation, cells were washed twice with PBS and placed in starvation media (DMEM with penicillin/streptomycin). After a 48-h incubation, cells were washed twice with HBSS and maintained in the same buffer. For timecourse experiments, cells were stimulated for 0, 5, 15, 30 and 60 min with vehicle (1% DMSO), semaglutide or danuglipron (1 μM). For probing the internalization-dependent changes to the phosphoproteome, cells were preincubated with Dyngo-4A (50 μM) for 30 min prior to stimulation for 30 min with vehicle (1% DMSO), semaglutide or danuglipron (1 μM). Cell extracts were prepared as previously describedAll parameters and settings regarding the acquisition of the proteomics data can be found in Supplementary Data 67 applying peak picking of the mass spectra with the vendor-provided algorithm (Thermo Fisher Scientific). The mzML files were analyzed using the FragPipe GUI v18 for both library and DIA search. MSFragger68 was used to search files against the human Swissprot database . Trypsin with up to 2 missed cleavages was set as digestion enzyme, oxidation of methionine, acetylation of the N-terminus and phosphorylation on serine, threonine and tyrosine  were set as variable modifications. Carbamidomethylation of cysteine residues was set as a fixed modification. Peptide length was restricted to 7 to 50 amino acids, and 500 to 5000 Da. The precursor tolerance was set to ±20 ppm, fragment mass tolerance to 20 ppm and MSBooster was enabled. The resulting peptide-spectrum matches were adjusted to a 1% false discovery rate with Percolator69 as part of the Philosopher toolkit (v4.4)70 and converted to a spectra library. DIA files were analyzed by DIA-NN 1.8.172 using the previously generated library and monitor-command mode. Peptide quantification was performed based on the fragment elution profiles from the MS2 level (area under the curve).Acquired raw files were converted to mzML format by MSConvert (version 3.0.21258)k-NN imputation. All statistical comparisons were performed based on homoscedastic two-tailed Student’s t-tests. For the kinase substrate enrichment analysis, the Kinase Library online analytical tool73 was used with predetermined foreground and background sets based on previously determined significance thresholds . All remaining settings were left as default.All further data processing was performed in R (version 4.2.1). Only phosphopeptides with a localization score of at least 0.75 were considered for further analysis. Peptides quantified with different charge states were aggregated and only phosphopeptides with at least 2 quantifications for each time point, treatment and replicate were kept. Intensities were normalized by median centering and log2-scaling. Missing values were imputed by -2 M. All data for potency and efficacy were z-scaled . Combined data sets were then clustered by hierarchical clustering (using the Manhattan distance and ward.D2 clustering) for both drugs and parameters. Drug clusters were determined by k-means clustering. Cluster membership probability was assessed by fuzzy clustering74. The predictiveness of ADRs was based on the dendrogram of sequence homology or signaling neighborhoods compared to adverse event risk from the FDA Adverse Event Reporting database. Specifically, entanglement score was calculated as the L-norm distance between two dendrograms with a score of 1 indicating perfect alignment and a score of 0 indicating an absence of correlation.Data analysis was performed using GraphPad Prism 9.4.0 . Quantitative data are expressed as the mean and error bars represent the standard error of the mean (SEM) unless otherwise indicated. Raw BRET was calculated by dividing the emission intensity at 515/20 nm by the emission intensity at 400/70 nm. netBRET was determined by subtracting the ratio measured from cells expressing only the BRET donor from cells expressing the BRET donor and acceptor. ΔBRET was calculated by subtracting the BRET ratio of vehicle-treated cells from the BRET ratio of agonist-treated cells. Curve fitting was performed by three or four parameter nonlinear regression. Statistical analyses were performed using one-sided extra sum-of-squares F test for comparison of fit parameters with Bonferroni correction. Potency and efficacy were extracted from drug-directed pathway- and compartment-selective activity and represented as a heatmap. For potency, pathway and compartment combinations required at least one measured value and missing values were inputted with 10Further information on research design is available in the Supplementary informationReporting SummaryPeer Review FileDescription of Additional Supplementary filesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Movie 1Source Data


Subnational differences in male fertility within sub-Saharan African countries have not been explored, nor the differences in male fertility according to migration status been sufficiently probed. We study divergences in rural and urban male fertility and investigate the relationship between male fertility and migration across 30 sub-Saharan African countries. We employ 67 Demographic and Health Surveys to estimate completed cohort fertility among men aged 50–64 according to migration status. Overall, we find that urban male fertility has declined faster than rural male fertility, widening the gap between the sectors. Rural-urban migrant men have lower fertility than their rural non-migrant counterparts. Men migrating within the rural sector have similarly high fertility as rural non-migrants, while urban–urban migrant men have even lower fertility than non-migrant urban men. Using country-fixed effects models, we find that among men with at least secondary education, differences in completed cohort fertility by migration status are widest. When we consider the timing of migration in relation to the timing of the birth of the last child, we observe that migrant men are a select group, having around two children less than non-migrant rural men. There is also evidence of adaptation to destination, though to a lesser extent. Furthermore, migration within the rural sector does not seem to be disruptive to fathering. These results indicate that rural-to-urban migration has the potential to delay rural fertility decline, and that urban male fertility is likely to decline further, especially as the proportion of urban-to-urban migration increases. Fertility remains high in sub-Saharan Africa (SSA), with significant differences between urban and rural areas Corker, . AccordiYet, to our knowledge, no research has examined within-country variance in male fertility in SSA. Fertility differences between the rural and urban sectors are expected as each sector progresses through the demographic transition at different paces Dyson, . The levBoth migration and fertility are important events over the life-course. Although previous research has examined the relationship between female migration and fertility  have higher fertility than independent or first-time migrants Ortensi, . MoreoveFertility may have an effect on migration too  remain the main source of information on fertility in SSA countries for both men and women. The DHS collect vast amounts of information from women aged 15–49 on fertility and migration, such as birth histories, place of current residence, place at birth, and time in the new location. From men, such information is mostly collected for the age group 15–64, although in less detail as for women. For instance, men are only asked about the number of children ever born and in some cases the age of the youngest child. Despite this data limitation we are able to estimate a cohort measure of fertility for our main analysis.From the available data in the DHS male records, we compute completed cohort fertility (CCF), which is the arithmetic mean of the values of completed fertility for all individuals in the cohort of 50–64 years old. In computing the CCF, we use the sample weights calculated in the DHS. CCF is a useful measure of fertility since cohort measures are less volatile than period measures, like total fertility rates (TFR),We limit our analysis to cohorts of men who are over age 50 and above for two reasons. First, we do not want to further reduce the sample size by only examining men aged 60 and above, due to the limitations of the upper boundary of the age of men interviewed by the DHS (age 64), and the overall low proportion of men interviewed over age 50. We test for sensitivity of using this lower bound of 50 years old by extending the age group to a lower bound of 45 years and thus increasing the sample size. Second, although biologically men can continue to have children into older ages, the proportion of men who do have children between ages 50 and 60 in SSA has been shown to be small  or within urban areas (urban–urban) are within-sector migrants. We examine ever-migrants in these six categories and exclude from our analysis international migrants. Considering the small sample size within each category of migrants, we test for sensitivity of some of our results by only considering surveys with at least 30 men in each migrant status.We do not limit migrant status by time, including recent migrants and past migrants in the same group. On average, urban-to-rural migrants migrated 15.6 years ago (standard deviation (SD) = 10.5 years), rural-to-urban migrants migrated 18.9 years ago (SD = 11.3), urban-to-urban migrants, 15.4 years ago (SD = 10.9) and rural-to-rural migrants, 16 years ago (SD = 10.8). We acknowledge that this does not consider the stages of adaptation, with the longer the duration of stay in the sector, the closer in behaviour we would expect the migrant to be to the destination fertility. However, considering the relatively small sample of men for whom we can estimate CCF, if we were to consider only recent migrants we would not be able to model male fertility. All the same, we are able to partially account for the timing of migration by considering whether it was before or after the birth of the last child. We also include a measure of duration in current residence in this model. This allows us to unpack whether migrant fertility is different because of selection or adaptation effects.We select our data based on all available DHS which include men of ages 50 and above, and the variables which allow us to identify migration. As a result, we include in our analysis 67 surveys covering 30 SSA countries. Table Using the most recent survey for each country, the lowest CCFs are recorded in Lesotho in 2009 (4.3 children per men) and South Africa in 2016 (3.8), while the highest CCFs are found in Niger in 2006 (10.7), Burkina Faso in 2003 (11.1) and Chad in 1996 (11.2). In countries with multiple DHS surveys, we note a general decline in CCF over time. These differences across countries and over time are in line with female fertility trends  is higher than urban across countries (and years), except for in ten surveys Fig. . These fMale migrants have different CCF levels as compared to non-migrants in both rural and urban areas. In comparison with rural non-migrants Fig. b, d, malWhen we compare migrant CCF to completed fertility of non-migrants at their destination Fig. c, d, we These results are also largely corroborated by examination of TFR. The only contradictory evidence we note is that urban-to-urban migration, based on TFR (a period measure), seems to be higher than that of non-migrants within the urban sector. However, using our cohort measure of fertility, CCF, with the same subsample of surveys, urban-to-urban migrant fertility seems to be lower than non-migrant urban fertility  of these internal migrants, even before they move, are generally different to non-migrant men’s characteristics. Intra-urban migrants have higher educational attainment than urban non-migrants as do rural-to-urban migrants compared to rural non-migrants (see Appendix Table Further research including more precise birth records of men are needed to evaluate the mechanisms behind the relationship between male fertility and internal migration more thoroughly. In particular, precise information about the timing and order of migration events in relation to birth dates would be beneficial. It is possible that these mechanisms may also differ according to country context. Moreover, a limitation to our analysis is that we were not able to account for a full migration history, but only the most recent move: some migrants moved multiple times or were circular migrants. Their fertility may also be different, reflecting “double selection”, diffusion, or “re-adaptation” (Pongi Nyuba, Our analysis focused on movement between rural and urban sectors. Nonetheless, it is possible that fertility differs also between semi-urban or semi-rural sectors, over a gradient of urbanicity. While the DHS includes four categories of previous residence, we were limited to collapsing the categories due to small sample sizes (see Table A final limitation in our analysis derives from using CCF which reflects distant fertility levels, although it does capture smoother trends rather than short-term variation. Our robustness test using TFR for men suggests that the differences in rural and urban male fertility we find are also relevant today.We have contributed to an understanding of within-country male fertility in SSA and taken a first broad look at the relationship between internal migration and fertility among men. Men play a key role in fertility decisions, and even have higher fertility rates than women (Schoumaker,


We are seeing an increasing number of collaborations between professional organisations which have a mutual area of interest for the provision of guidance to healthcare professionals. These collaborations probably have several benefits at multiple levels; they not only reduce cost of production by sharing it but more importantly they bring expertise and knowledge together to explore the most appropriate management options and push the boundaries to the limit. We now have a joint guideline from the European Society of Gynaecological Oncology (ESGO), European Society of Human Reproduction and Embryology and (ESHRE) and European Society for Gynaecological Endoscopy (ESGE) for the fertility-sparing treatment of patients with endometrial cancer simultaneously published in three journals . ESGO haIn 1985 the incidence of endometrial cancer in the UK was 13.5 per 100,000. In 2018 this had doubled to 30 per 100,000 making uterine cancer the most common gynaecological malignancy in the UK . The incThere are some particularly good aspects of this guideline. One of these is the understanding of the need to respect a woman’s perception of the relevant importance of different outcomes . This is often ignored in recommendations for fertility-sparing surgery in cervical cancer where the oncology outcome is seen to trump the desire a woman has to preserve her fertility . This isThere are some aspects that I think could be improved on. One of these is the lack of discussion about obstetric outcomes after fertility-sparing treatment. Conception and fecundity are not the only pregnancy outcomes that need to be discussed as part of an informed consent process.There are two contradictory statements in the guideline. In one recommendation it states “Fertility-sparing treatment is considered for endometrioid patients with endometrial carcinoma with grade 1, stage 1a without myometrial invasion and without risk factors” yet in another it states, “If an early and focal myometrial invasion (1-2mm) is suspected from the resection material a fertility-sparing approach may be discussed on a case-by-case basis”. Are you allowed to offer this approach to a woman with myometrial invasion or not? If a woman declines hysterectomy, surely a doctor can give her another treatment even if it is not the most effective.There is another contradiction in relation to the medical management of endometrial cancer. It states “Medroxyprogesterone acetate (400–600mg/day) or megestrol acetate (160–320mg/day) is the recommended treatment. Treatment with levonorgestrel intrauterine device in combination with oral progestins with or without gonadotropin- releasing hormone analogues can also be considered ”. This contrasts to their other aligned guideline published in this journal  where thDespite these minor issues, this guideline comes towards the top of my league table for recommended practices in gynaecological oncology. This is mainly because of the excellent evidence base on which it was formed, and the dilemmas overcome in interpreting evidence which can be of a low volume, a low quality or often completely absent.The management of endometrial cancer has changed beyond recognition in only the last five years. It has evolved from the domain of the special interest gynaecologist to the sub-specialist team. The old behaviour of an open radical hysterectomy and pelvic lymphadenectomy has moved to laparoscopic surgery and robotics with sentinel lymph node dissection favoured over a full lymphadenectomy. The methods for selecting patients for different types of adjuvant treatment has become more precise. The new molecular profiling classification has helped to further define treatment by the recommendation for testing for p53 abnormalities, POLE staining, mismatch repair (MMR) staining & hypermethylation, and Lynch testing. In light of all these changes, the management of endometrial cancer should be re-analysed to some degree.In my experience, fertility-sparing requests are often initiated by a patient who is not prepared to accept the finality of losing her womb. A woman’s desire to preserve fertility may trump everything else. Irrespective of guidelines a woman often choses her own route. Informed consent is then the responsibility of the doctor and this guideline provides the requisite information to inform that choice. The doctor then has a duty of care to that patient irrespective of her choice. Last week I met Henry . He was six months old. His mother had a high body mass index and presented to me aged 45 with a grade 1 uterine cancer in the background of a multiple fibroid uterus and adenomyosis. She had no partner. In part due to the excellent skill of her fertility specialists and against my advice she conceived Henry who is a happy and loved boy with the world ahead of him. My fertility colleague described him as “a miracle baby”. Anecdote is not really evidence but something to ponder on. His mother has now consented to a hysterectomy.


Wnt signaling in endocrine and metabolic disorders” aims to emphasize the functional role of the Wnt signaling pathway in human endocrinology, focusing on metabolic disease. Endocrine and metabolic disorders encompass a wide range of conditions affecting various organ systems and physiological processes. The Wnt signaling pathway, originally recognized for its role in embryonic development and tissue homeostasis , used for more than 1,300 years in China as a treatment for lumbodynia, may exert its therapeutic effect on steroid-induced ischemic necrosis of the femoral head, is through targeting exosomal microRNAs (miRNAs) to regulate multiple signaling pathways, including Wnt, PI3K-Akt, and MAPK . In another original report investigating miRNAs and Wnt signaling, Tripathi et al. demonstrate that miR-539-3p overexpression in osteoblasts downregulates several components of the Wnt signaling pathway and deteriorates trabecular microarchitecture, leading to decreased bone formation in ovariectomized mice. In the third original article in our Research Topic, a group of investigators led by Xiaolin Tu found that the small molecule C91 (CHIR99021) promotes osteogenic differentiation of bone marrow stromal cells via the activation of Wnt signaling .The first study in this Research Topic clarified that one of the mechanisms by which the “Vilaseca et al. provide an interesting overview of the functional roles of estrogen deficiency in the processes involved in the development of Alzheimer’s disease, including Wnt signaling and glucose transport in the brain, amyloid precursor protein processing to form senile plaques, and Tau phosphorylation forming neurofibrillary tangles.Franco et al. elegantly explain the main differences between the physiological roles of canonical Wnt signaling  and its pathological involvement in the development of several human diseases, including cancer. Correctly interpreting the molecular bases of Wnt signaling and metabolism, ideally in a cell-type and tissue-specific manner (Franco et al.; A very comprehensive review concludes our Research Topic: In conclusion, understanding the intricate interplay between Wnt signaling and endocrine/metabolic disorders holds great promise for the development of targeted therapies and improved patient outcomes.FC: Writing – review & editing. GS: Writing – original draft


Pre-transplant infectious diseases screening is an essential component of the pre-transplant workup. Many centres that offer allogeneic hematopoietic stem cell transplantation (HSCT) routinely screen for human T-lymphotropic virus (HTLV) in all recipients in the pre-transplant setting. The aim of this single centre retrospective chart review was to assess pre-transplant HTLV screening rates in allogeneic HSCT recipients at our institution in Melbourne, Australia and determine the prevalence of HTLV in this cohort.Patients who received an allogeneic HSCT between 1st January 2018 and 31st December 2022 were identified from an institutional database. Patients who received a second HSCT during the study period were excluded from this analysis. Demographic and clinical data were extracted from electronic medical records. Descriptive analysis of the patient cohort was conducted. Data on the number and outcome of HTLV screening tests performed in this cohort between 1st January 2017 and 31st December 2022 were collated in order to determine the rate of pre-transplant HTLV screening and prevalence of HTLV in this cohort.Over the study period 116 patients received an allogeneic HSCT at our institution. Of these, 110 were included in this analysis. 67 (60.9%) were male and 43 (39.1%) were female. The mean age at time of transplant (D0) was 51.0 years. Eight (7.3%) patients received haploidentical transplants, 20 (18.2%) received matched unrelated donor transplants, 44 (40.0%) received sibling transplants and 38 (34.5%) received unrelated donor transplants. 158 pre-transplant HTLV screening tests were performed on included patients over the study period. 75 (68.2%) patients had one pre-transplant HTLV test, 25 (22.7%) had two pre-transplant HTLV tests, seven (6.4%) had three pre-transplant HTLV tests and three (2.7%) had four pre-transplant HTLV tests. All HTLV tests included in this analysis were non-reactive on enzyme immunoassay screening.All patients included in this study had at least one pre-transplant HTLV screening test over the study period. There were no cases of HTLV detected in this cohort. Further research is required on the utility and cost effectiveness of routine pre-transplant HTLV screening in transplant recipients in non-endemic areas.All Authors: No reported disclosures


IIbβ3-initiated outside–in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα−/− mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbβ3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα−/− platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα−/− platelets and the p38 inhibitor blocked the increased integrin-mediated outside–in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα−/− mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside–out and integrin α IIbβ3 to bind soluble fibrinogen and support platelet aggregation. Fibrinogen binding to integrin αIIbβ3 generates a second set of signals referred to as outside–in signaling, which controls the platelet cytoskeletal remodeling and functions such as adhesion and fibrin clot retraction [At the site of vascular injury or ruptured atherosclerotic plaques, exposed extracellular matrix proteins such as collagen and the generated agonist such as thrombin, thromboxane and adenosine diphosphate (ADP) engage with their respective platelet receptors to initiate agonist-induced inside–out signaling . This sitraction ,3. Both Protein kinases have held the center stage in our understanding of the agonist and integrin-mediated signaling pathways. As models of signal transduction have been refined to include protein phosphorylation and dephosphorylation, the contribution of the protein phosphatases to platelet signaling and function has garnered fresh attention . Indeed,IIbβ3 activation, soluble fibrinogen binding, and aggregation in response to low doses of agonist. Concurrently, PP1cα negatively regulates integrin outside–in signaling responses such as platelet adhesion and fibrin clot retraction. Consistent with an opposing role for PP1cα in agonist-induced inside–out and integrin-induced outside signaling, loss of PP1cα did not impact in vivo hemostasis and thrombosis.Protein phosphatase 1 holoenzyme is among the major serine/threonine phosphatases in eukaryotes and contains a catalytic (PP1c) and regulatory subunit (PP1r) . PP1c haIIb) and anti-CD62 (P-selectin) antibodies tagged to fluorescein isothiocyanate (FITC) were obtained from BD Bioscience . Phycoerythrin-tagged antibodies that recognize the active form of murine αIIbβ3 (JON/A) were purchased from Emfret Analytics . Thrombin was brought from Hematologic Technologies Inc. . ADP and collagen were purchased from Helena Laboratories . Thromboxane analog (U44619) and water-soluble p38 SB203580 hydrochloride were ordered from Bio-Techne Corporation . Fibrinogen and Alexa 488-tagged fibrinogen were purchased from Enzyme Research Laboratories Inc.  and Invitrogen, , respectively. Anti-phospho-p38 and p38 antibodies were obtained from Cell Signaling .The reagents used in this study were mostly obtained from Sigma-Aldrich . Anti-CD41(αppp1ca-loxP flox (fl) mice [Pf4 Cre knock-in mice (Jackson Laboratory) [PP1cα knock-out mice. For most studies, 8–16-week-old male and female littermates  and PP1cα fl/fl and Cre negative, referred to as wild type (WT) were employed. The only exception is the use of male mice in intravital microscopy studies on cremaster muscles.Approval from Baylor College of Medicine IACUC  was sought prior to initiating all animal studies. Mice lacking PP1cα conditionally in platelets were generated as we described previously . Brieflyfl) mice  were matoratory)  to generv/v) ratio from the inferior vena cava, as we described [g for 10 min provided platelet-rich plasma (PRP). The second centrifugation of PRP at 754× g for 10 min yielded a platelet pellet that was subsequently washed with D-PBS. Platelets were then gently resuspended in D-PBS containing 0.005 U/mL apyrase. Coulter counter (Beckman–Coulter (Z1), Miami, FL, USA) was used to count platelets, which were adjusted to the final concentration of 2.5 × 108/mL. All studies with washed platelets were initiated after resting for one hour.Mice were anesthetized with isoflurane and blood was collected into the anticoagulant acid-citric acid-dextrose (ACD) in a 1:10 (escribed . Collect7/mL with Tyrode’s buffer containing CaCl2 (1.8 mM) and MgCl2 (0.49 mM). Agonist including thrombin , ADP , collagen  and U46619  was used to stimulate platelets. Depending on the assays to be studied, we employed JON/A tagged PE antibody, Alexa 488-tagged soluble fibrinogen, FITC conjugated CD62P antibody and CD41 tagged PE antibody and analyzed platelets in a flow cytometer . FITC or PE-tagged isotype antibodies served as control. To study agonist-induced release of ATP, luciferin/luciferase reagent was added to agonist-challenged washed platelet and evaluated in a lumi-aggregometer  [8/mL) were stimulated with different concentrations of agonist under stirring conditions (1200 rpm) in an eight-channel Bio/Data PAP-8 aggregometer . Final aggregation after 10 min of agonist stimulation was recorded.For these studies, platelets were further diluted to 2.5 × 10PA, USA) . To studPlatelets were left suspended on BSA (5 mg/mL) substrate for 30 min or allowed to adhere to immobilized fibrinogen (100 ug/mL) for 15 and 30 min. Following washing, platelet lysate was separated on 10–12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to nitrocellulose membrane, and then immunoblotted with antibodies to phospho-Thr180, Tyr182  and p38 MAPK. HRP-conjugated secondary antibodies were used later, and the membrane was developed using chemiluminescence (ECL). Densitometry of p38 signals was performed using Image J (1.53k/Java/8.0_172 964 bits).−/− washed platelets (1 × 107) were incubated for 60 min. Following washing, the acid phosphatase activity of bound platelets was used to quantify platelet adhesion. A standard curve of acid phosphatase 405 absorbance with varying platelet counts was used to obtain the adhered platelet number. Percent platelet adhesion as obtained we have previously reported [8 platelets/mL) containing 3 mM CaCl2 was left untreated or treated with SB203580 (10 μM) and then challenged with 1 U/mL thrombin for 60 min. The volume of liquid that was not incorporated into the clot was measured. To ascertain the volume of the clot, we subtracted the measured volume from the initial 500 μL volume and expressed it as a percentage of the starting volume. For in vitro platelet adhesion and thrombus under flow conditions, whole blood collected in PPACK dihydrochloride from wild type (WT) and PP1cα−/− mice were perfused over collagen type 1 at 1000s−1 using a Bio Flux 1000Z Flexion system . Following 4 min of perfusion, epifluorescence microscopy was used to collect multiple images of platelet adhesion and thrombus formation at different sites by an investigator who was blinded to the genotype. Platelet adhesion and thrombus formation were reported as integrated fluorescence intensity using the Bio flux Montage software (Version 7.8.4.0).For static adhesion assays, ninety-six well plates were covered with fibrinogen (100 μg/mL) and subsequently blocked with 5 mg/mL of Bovine Serum Albumin (BSA). Wells coated with BSA served as controls. Untreated and water-soluble p38 inhibitor (SB203580 10 μM) treated WT and PP1cαreported ,20. For 2 from a 175 W xenon lamp to trigger a photochemical injury that triggers thrombosis. After continuously applying epi-illumination, the time of onset for the initiation of platelet aggregates (thrombus onset) and the time required to occlude the vessel and stop the flow for at least 60 s were monitored. For each mouse, thrombosis was triggered in one or two venules and arterioles, and the average of the results reported. The investigators performing these studies were not aware of the genotype of the mice.To study microvascular thrombosis in the venules and arterioles of the cremaster muscle, we used a light/dye–induced endothelial injury model and evaluated it by intravital microscopy as we previously studied ,22. Brie−/− mice were anesthetized and 1 mm of the tip of the tail was clipped with a sterile blade and the time required to stop the bleeding was recorded, as we described before [WT and PP1cαd before ,20. Aftep-value was <0.05.Results are shown as mean +/− standard deviation. In studies that analyzed two experimental groups, a Students’ paired t-test was considered. For the analysis of three or more experimental groups, one-way ANOVA and Tukey multiple comparisons were employed. Analysis was conducted using GraphPad Prism 9  and studies were considered significant only if the −/− mice and observed reduced platelet aggregation and soluble fibrinogen binding to a lower dose of thrombin (0.02 U/mL). At higher thrombin concentration (1 U/mL), aggregation and soluble fibrinogen binding were similar between wild-type (WT) and PP1cα−/− platelets [−/− platelets, PP1cγ−/− platelets also demonstrated moderately decreased aggregation selectively to low but not high doses of thrombin [We previously generated a conditional platelet PP1cαlatelets . Like PPthrombin . Due to IIbβ3 is a consequence of inside–out signaling triggered by an agonist, we examined the activation status of murine integrin αIIbβ3 using JON/A antibody. Compared to the WT, PP1cα−/− platelets bound significantly less JON/A antibody following thrombin (0.02 U/mL) stimulation (−/− platelets in response to ADP (2.5 μM) (Since activation of integrin αmulation A. Extend(2.5 μM) B and col(2.5 μM) C. −/− platelets. Compared to the WT platelets, PP1cα−/− platelets revealed a moderate but significant decrease in soluble fibrinogen binding to ADP (2.5 μM)  A and col(2.5 μM) B. We hadlatelets . −/− platelets. Similarly, we previously reported reduced aggregation to 0.02 U/mL thrombin in PP1cα−/− platelets [−/− platelets at higher agonist concentrations  A. collaglatelets . Aggregatrations  Taken toIIbβ3 overexpressing 293 cells. Importantly, this effect was specific to PP1cα but not to other isoforms, and to p38 MAPK but not to other MAPKs [180 and Tyr182 of p38 in WT and PP1cα−/− platelets. Phosphorylation of Thr180 and Tyr182 of p38 was induced following adhesion to immobilized fibrinogen in both WT and PP1cα−/− platelets. Importantly, the adhesion of PP1cα−/− platelets led to a robust activation of p38 at 30 min  in PP1cα depleted heterologous αer MAPKs . Therefot 30 min A,B. Thuslatelets A. p38 inbrinogen A. We alslatelets B. Since nhibitor C. Collec−/− platelets exposed to low and high concentrations of thrombin and collagen  and the time to occlude the vessel (occlusion time) in the venules and arterioles of the PP1cα−/− mice were similar to that of WT mice  [−/− platelets and trigger activation of PP1cβ or PP1cγ isoforms to assist platelet activation. Indeed, in our previous study with PP1cγ−/− mice model, we also observed reduced inside–out signaling selectively to a low dose of thrombin [Loss of PP1cα modestly reduced activation of integrin, binding of soluble fibrinogen, and aggregation of platelets especially to a low dose of platelet agonist . These f02 U/mL) . It remathrombin . Thus, uIIbβ3 outside–in signaling functions  [−/− murine platelets indicates that this phenotype is intrinsic to PP1cα and independent of the cell model we previously employed. Importantly, unlike the inhibition of outside–in signaling functions in human platelets treated with inhibitors of Ser/Thr phosphatases, genetic loss of only PP1cα moderately potentiated integrin αIIbβ3 mediated outside–in signaling function.In contrast, loss of PP1cα in murine platelets moderately enhanced integrin αunctions . Interesraction) . Enhance1 protein as one PIP downstream of the G protein-coupled receptor. Thrombin stimulation led to the dissociation of PP1cα from Gβ1, with subsequent association and dephosphorylation of phospholipase C β3 (PLCβ3) at Ser 1105. Since Ser 1105 phosphorylation blocks signaling, dephosphorylation by PP1cα may amplify agonist signaling [IIb [−/− platelets  and subsequent phospho-modulation of specific effectors within the pathway. Indeed, we previously demonstrated the Gβignaling . In the ing [IIb , cytoskeing [IIb  and p38 ing [IIb  represening [IIb  we obserlatelets  and p38 latelets . It is p−/− mice (−/− global mice had no defect in in vivo hemostasis and only moderately reduced time to occlusion in a light-dye injury model [−/− mice. This suggests that platelet PP1cα can crosstalk with injured endothelial cells during the development of thrombosis in vivo and modulate outcome.Despite the critical role of platelet activation in hemostasis and thrombosis, in vivo hemostasis and thrombosis were unaffected in PP1cα−/− mice . Opposinry model . DespiteDespite the subtle phenotype, our studies suggest that PP1cα contributes to platelet signaling in a fashion that is specific to the mode of signaling. Specifically, PP1cα supports agonist induced inside–out signaling and opposes integrin-mediated outside–in signaling. Interestingly, recent studies suggest a role for PP1 signaling in platelet–cancer cross-talk , and alt


Nuclear receptors (NRs) form a family of druggable transcription factors that are regulated by ligand binding to orchestrate multifaceted physiological functions, including reproduction, immunity, metabolism, and growth. NRs represent attractive and valid targets for the management and treatment of a vast array of ailments. Pentacyclic triterpenes (PTs) are ubiquitously distributed natural products in medicinal and aromatic plants, of which ursolic acid (UA) is an extensively studied member, due to its diverse bio-pertinent activities against different cancers, inflammation, aging, obesity, diabetes, dyslipidemia, and liver injury. In fact, PTs share a common lipophilic structure that resembles NRs’ endogenous ligands. Herein, we present a review of the literature on UA’s effect on NRs, showcasing the resulting health benefits and potential therapeutic outcomes. De facto, UA exhibited numerous pharmacodynamic effects on PPAR, LXR, FXR, and PXR, resulting in remarkable anti-inflammatory, anti-hyperlipidemic, and hepatoprotective properties, by lowering lipid accumulation in hepatocytes and mitigating non-alcoholic steatohepatitis (NASH) and its subsequent liver fibrosis. Furthermore, UA reversed valproate and rifampicin-induced hepatic lipid accumulation. Additionally, UA showed great promise for the treatment of autoimmune inflammatory diseases such as multiple sclerosis and autoimmune arthritis by antagonizing RORγ. UA exhibited antiproliferative effects against skin, prostate, and breast cancers, partially via PPARα and RORγ pathways. Herein, for the first time, we explore and provide insights into UA bioactivity with respect to NR modulation.  Encoded by 48 genes, nuclear receptors (NRs) are transcription factors that are categorized into seven subfamilies . They inN-terminal region involves A/B subregions and has a ligand-independent activation function (AF1). The N-terminal is the most divergent among different NRs and is connected to the most conserved C region. The latter represents the DNA binding domain (DBD) that contains two zinc fingers coordinated with cysteines and other basic amino acids. The linker between the C and E region is a flexible, short, and less conservative hinge region, designated as the D region [C-terminal [Sequences of NRs share considerable homology and conserved structures, which are divided into six subregions, as shown in nction AF. The N-tD region ,14. The terminal ,15.Upon ligand binding, conformational changes occur to regulate further transcriptional activity. This happens through recruiting a specific cofactor and binding to a specific DNA-response element (RE) in the corresponding target gene . ImitatiOne of the most explicit examples is the variable pharmacological effect of the widely used estrogen receptor  selective modulator, raloxifene, for the treatment of breast cancer. Raloxifene antagonizes ERα in breast and uterine tissue, although it is an ERα agonist in bone tissue, which makes it useful for increasing bone density. This differential effect of raloxifene is ascribed to the activation of distinct cofactors in different tissues ,5. The cNRs may work as homodimers like steroid receptors, including mineralocorticoid receptors , glucocorticoid receptors , and the receptors for male and female sex hormones, or may function as heterodimers with an obligatory partner, the retinoic acid X receptor α , similar to metabolic NRs ,19. UniqMany FDA-approved drugs are derived from natural sources, especially in the cancer chemoprevention field ,22,23. PRollinia pittieri, is a potent LXRα agonist with EC50 0.18 μM [Unonopsis glaucopetala, is a more potent LXRα agonist with EC50 0.03 μM [To date, many natural products have been reported to possess biological activities due to NR modulation ,34,35,36 0.18 μM . The tet 0.03 μM . This ma 0.03 μM . Celastrus orbicalatus, was identified as a hepatoprotective agent against acetaminophen-induced injury through selective FXR agonism. Owing to its high affinity, hedragonic acid was co-crystalized with FXRα LBD (Protein Data Bank ID: 5WZX) [As anticipated, PTs proved to be prominent NRs ligands, eliciting a multitude of bioactivities due to their high structural similarity to the lipophilic endogenous NRs ligands. A lupane-type PT, betulinic acid, ameliorated non-alcoholic steatohepatitis (NASH) in vivo, via FXR activation . CelastrD: 5WZX) . Its anaD: 5WZX) ,52. The 50 in rodents is quite high: 637 mg/kg for intraperitoneal injection and 8330 mg/kg for oral administration, indicating its high safety margin [Ursolic acid (UA), 3-beta-hydroxyurs-12-en-28-oic acid, is one of the most studied ursane-type PTs due to its safety and diverse bioactivities. UA is orally and topically safe in rodents and humans. UA LDy margin . UA is ay margin ,54,55. UA modulates different pharmacological pathways, leading to multifaceted health benefits and the prevention of chronic diseases ,55,56. IProvoked by our interest in triterpenes chemistry and bioactivity ,49,82,83We retrieved the literature from the Web of Science, PubMed, and Google Scholar databases, using the keywords “ursolic acid” and “nuclear receptors” to perform a comprehensive search. This search was performed without publication year limitations, since there was no previously reported review article on the same topic. The outcome was approximately 100 research articles, review articles, and patents, of which 51 were considered for this review. The remaining articles were not extensively investigated as they focused on other types of receptors or other natural products.PPARs involve three subtypes  that control insulin sensitivity, resistance, and lipid homeostasis, making them valid targets for alleviating metabolic syndrome, hyperlipidemia, and diabetes ,86,87,88The first report on PT modulation of PPARα and their potential pharmaceutical and cosmeceutical role in dermatology was released in 2005 . Concomi50 values of the latter against Ca3/7 or mouse skin papilloma cells MT1/2, as shown by MTT assay. This suggests that the UA cytotoxic effect is partially mediated by PPARα activation [Interestingly, UA’s agonistic effect on PPARα played a pivotal role in its cytotoxic activity against skin cancer through the AMPK pathway. In the mouse squamous carcinoma model, Ca3/7, UA enhanced AMPK phosphorylation at cytotoxic levels, which was reversed by using an AMPK small molecule inhibitor or by AMPK knockdown. As PPARα upregulation has a partial role in skin cancer therapy, the authors investigated this mechanism for UA . Indeed,tivation .Likewise, Jia et al. confirmed UA-induced activation of PPARα in terms of alleviating hypertriglyceridemia. Having said that, they could not confirm that UA directly binds to PPARα LBD using Biacore surface plasmon resonance (SPR) analysis. However, UA treatment remarkably promoted PPARα mRNA concentration in cultured hepatocytes (HepG2), as shown by qPCR. A luciferase reporter gene assay in the same cells revealed that UA is a PPARα activity upregulator. Furthermore, a 20 µM concentration of UA enhanced PPARα binding to its response element in the responsive genes by 46% and promoted PPARα transactivation consequently. In a dose-dependent manner, UA treatment was significantly proved to have significant hypolipidemic effects by reducing intracellular triglycerides (TGL) and cholesterol accumulation in HepG2 cells. This was accompanied by significant upregulation of the fatty acid transport protein 4 (FATP4) gene in both mRNA and protein levels; FATP4 is a major fatty acid transporter in the liver and a known target gene PPARα. The authors emphasized that UA promotes PPARα transactivation by indirect mechanisms, other than simply binding to its LBD .The same research group moved forward with in vivo validation of their previous in vitro results. They found out that UA can regulate lipid and glucose metabolism in high-fat diet (HFD)-fed mice. UA intake reduced lipid accumulation in adipose tissues and the liver, while increasing muscle mass. Biochemical analysis confirmed that plasma levels of TGL and low- density lipoprotein (LDL) levels were reduced in contrast to high-density lipoprotein (HDL) levels. This was accompanied by improved glucose tolerance and insulin sensitivity. In mice tissue, UA treatment resulted in the over-expression of mRNA and protein levels of PPARα, the activation of its responsive genes that regulate fatty acids uptake and β-oxidation, and the suppression of lipogenic genes . AdditioThe anti-hyperlipidemic effect of UA (25 mg/kg) or artesunate (25 mg/kg) alone or in combination (12.5 + 12.5 mg/kg) was assessed in a New Zealand rabbit model on a Western-style diet. UA administration for a couple of months significantly reduced TGL and cholesterol levels in a comparable manner to atorvastatin without a significant effect on LDL level, which was efficiently lowered in the case of UA/artesunate combination. UA alone alleviated hepatocyte steatosis; meanwhile, the combination completely prevented it in the same way as atorvastatin, as displayed by histopathological examination using hematoxylin and eosin (H&E) stains . In liveThe potential role of UA in NASH therapy was further investigated by the Li group using the obese NASH Sprague Dawley rat model. UA administration significantly reversed HFD-induced lipid accumulation, NASH, and liver injury and reduced serum ALT, AST, TGL, FFA, and LDL levels in a dose-dependent manner, as revealed by hepatocyte morphologic, histological, and serum biochemical examination. In the same context, UA promoted mRNA and protein levels of PPARα whose knockdown interrupted UA-induced hepatoprotective effect. UA reduced the expression of hepatic inflammatory cytokines, including different interleukins and the tumor necrosis factor α (TNFα). In this model, UA did not affect the activity of PPARγ, farnesoid X receptor (FXR), or liver X receptor (LXR) . MeanwhiAnother research group studied the PPARα upregulation effect on alleviating peripheral inflammation and inflammatory hyperalgesia in obese animals. Following the injection of carrageenan into obese Sprague Dawley rats, systemic UA administration mitigated thermal hyperalgesia and paw edema, compared with the control group. At the molecular level, UA lowered the expression of inflammatory mediators, including IL-1β, TNF-α, and NF-κB P65 in the spinal cord of the rats, as shown by the Western blot test. Carrageenan injection into rats’ paws significantly reduced spinal cord PPARα levels in the control HFD groups prior to UA administration, which reversed the process and restored PPARα levels. This means that UA could restore PPARα levels in obese rats’ spinal cords and reduce the expression of inflammatory mediators due to peripheral inflammatory stimulation .UA-induced activation of PPARα is not only beneficial in skin diseases and metabolic disorders, but also in right ventricle hypertrophy (RVH) and remodeling . In a SpThe UA effect on PPARγ was described in different aspects of biological activities. PPARγ agonism is well-known to alleviate inflammations in asthmatic animal models . In the Wang et al. explored UA neuroprotection effects through the PPARγ pathway in a model of male Sprague Dawley rats with middle cerebral artery occlusion and reperfusion. UA treatment improved the neurological deficit score, promoted the number of intact healthy neurons, and minimized the infarct size compared to the control animals in a dose-dependent manner. This is accompanied by the upregulation of PPARγ, the downregulation of the inflammatory mediators, matrix metalloproteinase-2/9 (MMP2 and MMP9), the increment of the anti-inflammatory factor tissue inhibitor matrix metalloproteinase (1TIMP1), and the interruption of MAPK signaling pathways in brain tissue, as revealed by Western blot and qRT-PCR. Hence, UA can serve as a neuroprotective therapeutic agent, acting via PPARγ agonism, and optimizing the metalloprotease/anti-metalloprotease balance .Another confirmatory report on UA anti-inflammatory activity in the central nervous system (CNS) via PPARγ activation has been recently published. UA enhanced the phenotypic switch of BV2 cells (murine microglia) from M1 polarization, the pro-inflammatory, to M2 polarization, the anti-inflammatory, through the promotion of PPARγ protein expression. Meanwhile, PPARγ activation resulted in the suppression of MMP2 and MMP9 secretion and the increment of 1TIMP1 secretion, which supports the previous results . NotablyA potential dual role of UA in the treatment of multiple sclerosis (MS) through immunomodulation and neuroregeneration via PPARγ agonism was disclosed by Zhang et al. . In MS mCollectively, UA has a highly promising PPARγ-agonistic character that can be employed for the management of a multitude of diseases, including bronchial asthma, CNS ischemia, and neuro-inflammatory diseases such as MS. The hydrophobic oxysterols are the endogenous ligands of LXRs, which have two subtypes . Both have shared approximately 70% homology with PPARs. They play a paramount role in lipid and glucose homeostasis, atherosclerosis, and NASH development by regulating hepatic de novo lipogenesis ,119,120.Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng, of the family Aquifoliaceae. This bitter-tasting tea contains high amounts of ursolic acid and has been widely used in China for more than 2000 years as a healthy beverage for the management of obesity, cardiovascular disease, hypertension, and hyperlipidemia [Kuding tea or Ku-Ding-Cha leaves are mainly from ipidemia . Fan et ipidemia . In cellLater on, Lin et al. identified UA as an LXRα antagonist, in a similar fashion to its oleanane-type analog OA ,124. In Molecular docking calculations of UA and T090 into the LXRα ligand binding site (Protein Data Bank ID:1UHL)  using thUA activity was assessed in human intestinal cells LS174T, and surprisingly, it upregulated ABCA1 and ABCG1 expression instead of the anticipated downregulation. It is worth noting that ABCG1 gene expression decreased upon co-treatment with UA and T090 in HepaRG cells, confirming the cellular-context paradox. This is further confirmed by the lack of UA effect on cellular contents of TG in LS174T cells. It can be deduced that UA suppressed LXRα activation in hepatocytes but not in intestinal cells, suggesting that UA controls LXRα signaling in a cell- and tissue-specific manner due to differential effects on the recruitment of coregulators .The possible clinical application of UA to mitigate the lipogenic side effects of the ani-epileptic drug valproate was tested. De facto, UA significantly opposed valproate induced LXRα transactivation, lipogenic gene expression, and lipid accumulation in HepaRG cells. As we will mention below, UA may also protect against rifampicin-induced hepatic steatosis . Alongside CAR (NR1I3), PXR (NR1I2) is mainly responsible for xenobiotic detoxification by regulating the expression of the metabolic enzyme cytochrome P450 (CYP 450), including the two main types, CYP3A4 and CYP2B6 ,127. PXRRosmarinus officinalis, activated mouse, rat, and human PXR. In terms of human PXR activation, the EC50 values of UA and carnosol were 10.77 and 2.22 µM, respectively. UA was confirmed to bind within PXR LBD and activate luciferase activity in cells transfected with a plasmid expressing human PXR LBD. In human colon adenocarcinoma cells, LS180, UA promoted the mRNA expression of the major metabolizing enzyme CYP3A4 and a multi-drug resistance protein 1called ATP binding cassette B1 (ABCB1). In the intestine, this effect enhanced the first-pass metabolism and reduced the oral bioavailability of chemicals metabolized by CYP3A4 and transported by ABCB1 [Using a dual-luciferase reporter gene assay in HepaRG cells, UA, alongside carnosol from by ABCB1 .In 2017, two different reports came out, reporting the promising role of UA and OA in attenuating rifampicin/isoniazid-induced cytotoxicity viamodulation of PXR and its sister NR CAR ,130. TheA supporting claim for the outcome for PXR antagonism by UA was introduced by the same research group in 2018; they evaluated the UA effect on PXR transactivation of lipogenic genes, including S14, SCD, FAS, and FAE. It was revealed that UA could effectively oppose the transient activation of promoters S14 and SCD by rifampicin, as shown by reporter assay. In the presence of rifampicin, UA reduced the mRNA and protein expression of S14, SCD, FAS, and FAE genes. Histopathological examination of stained HepaRG cells by phase-contrast microscope showed rifampicin-induced lipid accumulation and steatosis, which was significantly interrupted by UA . Therefo+ cells [As we mentioned above, ROR has three subtypes that possess indispensable roles in immunity, development, and metabolic homeostasis. It is worth noting that the RORγt type is only expressed in immune cells, especially Th17 lymphocytes, where it controls their development and differentiation from CD4+ cells . Th17 ce+ cells ,134. Rec+ cells ,136,137.+ cells ,138. Indeed, methyl corosolate, uvaol, and OA are three triterpenes found in loquat leaves with in vitro inhibitory effects against RORγt, accompanied by an interruption of Th17 differentiation, with a potential application in lupus nephritis . Further50 of 0.68 µM, while its IC50 on Th17 cells was determined to be 0.56 µM. As a result, UA abrogated IL-17 secretion from differentiated Th17 cells of both mouse and human origin. In experimental autoimmune encephalomyelitis mice as an MS model, UA treatment slowed the onset of disease in mice and significantly alleviated the symptoms in comparison with the control group. The CNS of UA-treated mice contained fewer IL-17+ and IFN-γ+ cells, and their spleen showed less IL-17 production. The study paved the way for UA application in autoimmune disorders and Th17-mediated inflammatory diseases [The first report claiming that UA acts as a strong and selective inhibitor of RORγt function came out in 2011 by Xu et al. A preliminary high throughput screening of 2000 compounds identified UA as a human and mouse Th17 development and differentiation inhibitor in a dose-dependent manner. At 2 µM, UA inhibited RORγt-mediated, but not RORαt-mediated, IL-17 and IL-17 expression to almost background level in Th17 cells. UA, in a dose-dependent manner, interrupted the binding of RORγt-LBD, but not RORαt-LBD, to its co-activator peptide. Further experiments led to the conclusion that UA exclusively antagonizes RORγt function with ICdiseases .In addition, UA administration significantly reduced the incidence and severity of collagen-induced autoimmune arthritis in mice models, partially via the inhibition of Th17 differentiation, as shown by flow cytometry. In a dose-dependent manner, mRNA expression of IL-17, IL-21, and RORγt was downregulated in the splenocytes . Owing tA recent interesting report by Zou et al. emphasized the RORγ-dependent anti- proliferative role of UA against triple negative breast cancer (TNBC) cells, HCC70, and prostate cancer (PCa) cell lines C4-2B and 22Rv1 . In the FXRs are involved in lipid and bile acid homeostasis, with a significant role in hepatic inflammation and fibrosis, and are widely distributed in organs such as the liver, kidney, intestinal tract, and adrenal gland ,144,145.A recent report revealed that UA can modulate FXR in rat models with alcoholic liver injury. UA intervention reduced the pathological changes in hepatocytes when examined by hematoxylin–eosin staining. The reduced hepatic steatosis was accompanied by improved cell inflammation and infiltration. In biochemical terms, alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) levels in serum were significantly lessened in comparison to the untreated group. Concomitantly, UA upregulated FXR protein expression and downregulated CYP7A1 and SREBP-1c expression . Selective NR modulation, in the way that introduces health benefits with minimal side effects, is a real challenge due to its conservative structure and unpredictable tissue-specific response. However, finding such a selective modulator can be of huge health benefit in terms of fighting metabolic syndromes that lead to the development of heart and cardiovascular disease. PTs possess a lipophilic structure, making them efficient NR modulators. Indeed, UA demonstrated various pharmacodynamic effects on PPAR, LXR, PXR/CAR, ROR, and FXR. As a result, it exhibited a multitude of health benefits, especially in terms of metabolic disorders, including insulin resistance, diabetes type II, NASH, hyperlipidemia alongside neuroprotection and an anti-asthma effect. Owing to PPARα upregulation, UA can be employed in pharmaceutical and cosmeceutical dermatology and skin cancer. Furthermore, UA showed a comparable anti-hyperlipidemic effect to atorvastatin in vivo. In addition, a significant role of UA in the treatment of autoimmune inflammatory diseases was observed and attributed to RORγt agonism. The latter effect has further conferred anti-proliferative potential to UA in cases of TNBC and PCa. So far, none of the UA semi-synthetic derivatives has been evaluated for NR modulation; therefore, screening of UA derivatives is urgently required, as it may pave the way for finding more potent and selective NR modulators that outperform the parent compound.


Fertility concerns are a pervasive issue but very subtle in patients with cancer. Though various studies have focused on fertility concerns, limited research endeavor has been dedicated to bibliometric analysis. Given this, to visually analyze the hot frontier trends of research related to fertility concerns of patients with cancer using CiteSpace and provide new insights for future research in this field using the bibliometric method. We used CiteSpace software to retrieve the literature related to fertility concerns of patients with cancer in the Web of Science core collection database from the year of establishment to 2022 and conducted visual analysis in terms of authors, countries and regions, research institutions, and keywords. The search resulted in 201 valid articles, and the annual publication volume of literature related to fertility concerns in patients with cancer was generally on the rise; the country with the most publications was the United States, which also had the highest influence; the main research institution was Sloan Kettleson Cancer Research Center; the core research scholar was Jessica R. Gorman; the research hotspots mainly centered on quality of survival, women, survivorship, preservation, breast cancer, adolescence, and infertility. The results of this bibliometric study provide the current status and trends in the fertility concerns of patients with cancer and may help researchers identify the hotspots and frontier trends in this field. In addition to this sobering rise, oncologic disorders are now affecting progressively younger populations, including those unmarried or childless,–6 leading to unique psychological and social distress among these patients, such as reproduction, self/children’s health and raising children issue.,8 This is defined as fertility concerns.Cancer represents a substantial challenge to global public health, with a high incidence rate, mortality, and medical expenditure. Prior research has revealed that many cancer patients are concerned about their future fertility when they are diagnosed, but they do not receive the information they require at that time regarding the risks to fertility and the options for fertility preservation.,12 This lack of attention toward reproductive considerations by healthcare providers has been highlighted in earlier studies. Moreover, tackling fertility concerns involves managing cancer patients’ medical and psychosocial aspects. Research on fertility concerns of different cancer patients exists in various fields. It will be hard to enumerate and summarize them individually. This review will systematically sort out and examine the research results of fertility concerns in cancer patients using the method of bibliometric analysis to more thoroughly assess the hotspots and development trends of the study of fertility concerns in cancer patients.Many cancer patients express concern over fertility, yet clinicians prioritize physical health over addressing these reproductive issues. Despite its widespread application, there remains limited focus on understanding topical hotspots and developments within the realm of fertility concerns in cancer patients. To our knowledge, no previous efforts have been reported to conduct a bibliometric analysis in this field. Motivated by this gap, we aim to examine fertility concerns in cancer patients through a bibliometric analysis, taking into account country, authorship, and keyword dimensions, thereby providing guidelines for scholars in this area to determine the research direction. By doing so, we hope to contribute meaningfully to advancing the current understanding of the subject matter.Bibliometric analysis, a technique involving quantitative assessment, has proven useful in exploring trends and offering direction to researchers in multiple domains.The Web of Science Core Collection database was used as the data source, and the search extended from the establishment of the database to December 31, 2022. Here are the search strategies: 1# TS= ((Fertility concerns) or (Fertility-related concerns) or (reproductive concerns)), 2# TS=((Cancer) or (Tumor) or (Neoplasm) or (Neoplasia) or  or  or ). The retrieval strategies are summarized in Table Article inclusion criteria: published articles; and articles with research topics related to cancer and fertility concerns. Exclusion criteria: repeated articles; and literature from conferences and newspapers, such as letters, and news reports. The literature that was not relevant to the study and duplicate literature were manually removed by reading the title and abstract. 221 papers were finally included and saved as plain text files in download___xx.txt format after retrieval. we analyzed various aspects relating to the authorship, geographical distribution, institutional affiliation, and subject matter associated with the available literature on fertility concerns in cancer patients. These facets were compiled and visually presented in charts to enhance comprehension regarding prominent areas of interest and rising patterns within the field over time. Through this approach, we aimed to facilitate improved understanding and inform future endeavors.Employing innovative Citespace (6.2. R2),The total number of articles published in the literature related to cancer fertility concerns was 201, and the number of articles published from 1999 to 2010 was in the single digits, with stagnant development; the number of articles published from 2011 to 2022 increased, but the overall development was at a low level; the highest number of articles published was 26 in 2020, with an upward trend in the annual number of articles , Sweden , and Canada .The centrality of nodes with circles is greater than 0.1, and the thicker the circle, the greater the centrality, where centrality is an important indicator used to determine the evolutionary process of the discipline and predict the development trend of the discipline, and the higher the centrality indicates the importance of the discipline’s development process.By analyzing the institutions, we can identify whether there is a cooperative relationship among institutions and find the institutions with more issuing volume by comparing the node sizes in the network graph. The node is set to the institution; other selection strategies remain unchanged; the result is run by clicking “GO,” and the cooperation map of institutions is obtained Fig. . A totalHigh-frequency keywords: Keywords are representative words describing the core content of articles, the higher the frequency of occurrence, the higher the research heat, and high-frequency keywords can reflect the hot issues in this research field. Visual analysis of keywords can reflect this field’s research direction, hotspots, and frontiers in various periods Fig. . The topKeywords bursts: The emergent keywords refer to the keywords that are used more frequently in a shorter period and can predict the research trends and research frontiers in the field. Reflecting the changes in fertility worry keywords during 1999 to 2022 ; Loren et al (cited 28 times); Ruddy et al (cited 23 times); Benedict et al (cited 21 times); Oktay et al (cited 20 times); Gorman et al (cited 20 times); Benedict et al (cited 20 times); Benedict et al (cited 16 times); Logan et al (cited 16 times); and Logan et al (cited 16 times). The high citation rate of the article indicates its high impact in the field (Fig. The articles were analyzed for citations, and the authors of the top 10 cited articles Table  were, ineld Fig. .The research on fertility issues in cancer patients mostly takes place in developed nations, with the USA being home to most research efforts. Three American universities lead the pack when it comes to the number of studies they have published: the University of California, San Diego, Oregon State University, and Memorial Sloan Kettering Cancer Center. American researchers are particularly enthusiastic about studying how cancer affects patients’ chances of conceiving children post-treatment, resulting in America having a greater presence than Europe and Asia across all relevant metrics. Nonetheless, Asian nations, particularly China, South Korea, Japan, and India, contribute significantly in terms of volume to the body of research exploring cancer’s impact on human fertility. While the quality of work varies between research groups, collectively, Asia is establishing itself as a major player in investigations into how best to protect patients’ prospects of starting families later. Further examination into the effects of specific therapies continues apace, including radiotherapy to the abdomen/pelvis, pelvic surgery, alkylating agents, and hormone chemotherapy, amongst other targeted treatments, each implicated in possible side effects on reproduction capacity.,28 These works serve to enrich global research on this topic as well as raise awareness regarding fertility preservation options available during the treatment process. A survey by Burgmann et al on 160 young breast cancer patients in Germany showed that 47% of the patients had fertility concerns. American scholars have observed that personal circumstances such as family size and societal ties exert significant influence on fertility concerns. The previous cross-sectional study indicated that educational background, desire for offspring, and preventative steps influence fertility unease. In summary, fertility concerns are influenced by multiple factors.Fertility concerns research following a descriptive trajectory remains prevalent throughout the world, chiefly revolving around women afflicted with cancer, although recent years showcase growing interest in addressing similar challenges encountered by males battling malignancy. Studies probing this subject matter often seek to gauge its impact on reproductive health alongside an exploration of contributing factors. Notably, recent studies expose disconcertion harbored by over 60 percent of mature cancer patients who concurrently confront sexual health dilemmas. According to a randomized controlled trial, using a web-based psychoeducational intervention could improve fertility concerns. In addition, French scholars’ study indicated that fertility preservation methods such as embryo freezing technology, egg freezing technology, and ovarian tissue freezing technology could alleviate the fertility concerns of cancer survivors. In 2006, France included fertility preservation in the law and the American Society of Clinical Oncology has made similar recommendations, but fertility preservation is still controversial in ethics and morality. Overall, there are still relatively few intervention studies on fertility concerns, with small sample size and short intervention time, and its effects need further verification.There are few interventions for fertility concerns at present, and the clinical practice guidelines of the American Society of Clinical Oncology strongly suggest that anxiety can be reduced by providing oncology fertility counseling services to patients. Additionally, research suggests that involving multidisciplinary teams improves cancer survivors’ ability to conceive post-therapy and lessens distress associated with infertility concerns.Cancer patients often encounter complex issues related to fertility. Consequently, managing such problems requires coordinated input across various professional domains within the framework of the modern biopsychosocial medical paradigm. To address this challenge effectively, multidisciplinary teams should work together, comprising experts from oncology, radiation therapy, reproductive medicine, genetics, and psychological counseling, among others. When devising a comprehensive treatment/fertility plan for patients, professionals should first clarify the individual’s childbearing intentions before implementing a strategy crafted jointly by a collaborative group of specialists aimed at mitigating patients’ fertility fears.The results of this sciencemetric review will contribute to the research in this field. However, there are some limitations to this study. First, we only included publications in the Web of Science database. Other databases, such as Scopus or PubMed, should be included in the future. Second, the fertility concerns of different cancer patients need to be analyzed separately in the future.In summary, our study identified hot topics and frontier trends in the research on fertility concerns among cancer patients through CiteSpace, which may guide new directions for further study. More studies on fertility concerns are needed in the future, including prospective cohort studies and high-quality randomized controlled trials.Conceptualization: Jiali Shen.Data curation: Hongzhan Jiang, Huihui Lin.Formal analysis: Jiali Shen.Methodology: Hongzhan Jiang, Huihui Lin.Software: Siyue Fan, Doudou Yu, Liping Yang.Supervision: Lijuan Chen.Writing – original draft: Jiali Shen.