Despite its study being long hampered by the lack of suitable animal and cellular models and inter-species differences, a substantial body of literature on the subject has built up in the last three decades and the physiology of β3-AR is unraveling quickly. As will become evident in this work, β3-AR is emerging as an appealing target for novel pharmacological approaches in several clinical areas involving metabolic, cardiovascular, urinary, and ocular disease. In this review, we will discuss the most recent advances regarding β3-AR signaling and function and summarize how these findings translate, or may do so, into current clinical practice highlighting β3-AR’s great potential as a novel therapeutic target in a wide range of human conditions.The beta-3 adrenergic receptor (β New aspects of β3-AR have recently been described, involving it in urine concentrating mechanisms [3-AR agonist mirabegron is the first of this class of compounds that has been approved for the treatment of overactive bladder syndrome and one of the few alternatives to anticholinergic medications [3-AR expression and signaling in districts such as myocardium and adipose tissue, mirabegron is also being tested in repurposing trials for cardiovascular and metabolic conditions  and LDL-C (Low Density Lipoprotein-Cholesterol) accumulation rates and glucose tolerance [3-AR role in some of the main tissues where its expression has been reported. In each subsection, we will review the state of the art in signaling pathways and potential therapeutic approaches with a focus on studies in humans and rodents.Soon after the subdivision of β-adrenoceptors into subtypes βchanisms , and infchanisms ,6 thus oications . Given βolerance ,11. Afte3-AR has been identified in several species such as rat [1- and β2-AR amino-acid sequences, respectively. This is mostly limited to the transmembrane domains and membrane-proximal regions of the intracellular loops, parts of the receptor respectively involved in ligand binding and G-protein interactions [3-AR are 81% identical, with the highest homology in the transmembrane domains (94%), and the lowest in the C-terminal tail and third intracellular loop. Consistently, their C-terminal regions differ in sequence and length, ranging from 6 (human) to 12 (mouse and rat) additional residues. β3-AR gene also contains introns. The number of exons/introns of the human and rodent genes differs and can be summarized as follows: the human β3-AR gene is composed of two exons and a single intron while in mouse, there are three exons and two introns [3-AR gene, whereas two have been characterized in the mouse [The gene encoding βh as rat , mouse [h as rat , bovine,h as rat , and dogh as rat . In humaractions ,17. The  introns . In both introns . Despitehe mouse ,20,21.3-AR gene encodes a single 408–amino acid residue–long peptide chain that, together with β1- and β2-AR, belongs to the G-protein coupled receptor (GPCR) family characterized by seven transmembrane (TM) domains, with three intracellular and three extracellular loops. The N-terminal region of β3-AR is extracellular and glycosylated, whereas the C-terminus is intracellular. Unlike in β1- and β2-AR, it lacks sites for phosphorylation by protein kinase A (PKA) and β-adrenoceptor kinase (βARK) thereby making β3-AR relatively resistant to desensitization [3-AR an interesting therapeutic target that would potentially be suitable for chronic treatments. From a molecular point of view, desensitization of β3-AR might not be mediated by internalization/degradation of the receptor, as for β1-AR [βtization ,22,23. Aor β1-AR , but rator β1-AR  or even or β1-AR . The disor β1-AR .3-AR pharmacological response have been defined in several studies [3-AR has high affinity and potency for selective agonists such as mirabegron, vibegron, solabegron, and ritobegron; partial agonist activity of β1- and β2-AR antagonists, such as CGP12177A, bucindolol, and pindolol; an atypically low affinity for β-AR antagonists such as propranolol and nadolol; and lastly, poor stereoselectivity for reference agonist and antagonist enantiomers in respect to the values reported for traditional β1- and β2-AR. Several studies that clarify β3-AR pharmacology were published in the last 20 years and are summarized in a comprehensive review by Donny Strosberg [The criteria that are used to define a characteristic β studies ,30,31,32trosberg . Structutrosberg , the lintrosberg . A greattrosberg ,37.3-AR agonists fall in two classes depending on the time of their discovery [βiscovery : the fir3-AR agonists in both human and mouse settings, but its pharmacology is controversial. It displays a higher affinity for rodent β3-AR vs. human β3-AR and also higher potency measured as the ability in stimulating adenylyl cyclase [3-ARs are involved in the relaxation of human detrusor muscle and of human corpus cavernosum [3-AR vs. β1- and β2-AR [3-AR: >128- and 10-fold higher than β1- and β2-AR, respectively [3-AR, as its potency to stimulate cAMP formation in transfected CHO cells was found to be higher with mouse vs. human β3-AR (pEC50 8.7 vs. 4.3) [2-AR, the predominant isotype in skeletal muscle [BRL37344 is possibly the most widely used of all of the β cyclase ,48. Nonevernosum ,50. Morevernosum ,51. Finand β2-AR  some othnd β2-AR ,53. CL31ectively . CL31624vs. 4.3) . CL31624vs. 4.3)  althoughl muscle . It was l muscle .3-AR (EC50 ≈ 22 nM), with intrinsic activities comparable to reference agonist isoproterenol. When tested on pre-contracted human bladder strips, mirabegron produced a concentration-dependent relaxation with an EC50 of 0.78 μM, similar to isoproterenol (EC50 = 0.28 μM) [3-AR expression in the central nervous system. Finally, when β3-AR presence was reported in humans and rodents [3-AR, and overall lack of side effects [Second-generation agonists include FDA-approved molecules like mirabegron (YM178), yet discontinued ones such as amibegron SR58611A) and other molecules that are currently being validated in phase II and III clinical trials, such as solabegron (GW427353) and ritobegron (KUC-7483). Mirabegron, solabegron, and KUC-7322 (the active metabolite of ritobegron) have been tested for their abilities to induce human detrusor relaxation. Mirabegron and solabegron were proven to be highly selective in stimulating cAMP accumulation in CHO cells transfected with human β0.28 μM)  while ri0.28 μM) . Solabeg0.28 μM) . It also0.28 μM) . Subsequ0.28 μM) , but the rodents ,61, seve1A and ot effects ,63,64,653-AR gaining more and more importance as a therapeutic target, the screening for new compounds is progressing at a fast pace [3-AR agonists and to identify common structural characteristics in the active molecules [With βast pace ,67. Of nolecules .3-AR was identified later than β1- and β2-AR is its much lower affinity for classical β-AR antagonists. For instance, propranolol and bupranolol showed 140- and 17-fold higher affinity for β1- and β2-AR and >500- and 30-fold higher affinity for β1- and β2-AR, respectively [3-AR antagonists are SR59230A and L748337. SR59230A belongs to the class of aryloxypropanolaminotetralins [3-AR antagonist in rat gut while also displaying interesting properties in rat Brown Adipose Tissue (BAT) blunting β3-AR-mediated cAMP increase [3-AR-mediated responses [3-AR vs. β1- and β2-AR [3-AR receptor [3-AR [3-AR responses, at least when not assessed in combination with other compounds.A key reason that βectively ,31. Two etralins  and was increase . Despiteesponses ,72,73, Snd β2-AR ,74. It areceptor  as well receptor . Lastly,or [3-AR . These f3-AR and also reduced glycerol production in rhesus monkey adipose tissue. It was reported to have a 100-fold higher affinity (Ki = 4 nM) for the human β3-AR vs. β1- and β2-AR, and also vs. the rat β3-AR [3-selective radioligand [3-AR might render L748337 not fully suitable for experimental studies on the receptor [3-AR on the market.Pre-incubation with L748337 blunted cAMP accumulation in CHO cells expressing human βat β3-AR . Accordiioligand . As seenioligand . The lowreceptor . Further3-AR has been detected in several human tissues such as myocardium, retina, myometrium, adipose tissue, gallbladder, brain, urinary bladder, and blood vessels. Its expression has been recorded both at the mRNA and protein levels, and its activation involves a variety of cellular pathways. A recent comprehensive quantitative analysis of the human transcriptome reported β3-AR expression to be far more restricted than previously hypothesized [3-AR expression has been studied so far, their associated signaling pathways and clinical implications in humans will be discussed. Since its discovery in the late 1980s, β3-AR has been localized in kidneys [β kidneys  includin kidneys , urethra kidneys , prostat kidneys , and bla kidneys  where it kidneys  although kidneys .1- and β2-AR agonists but only with β3-AR agonists such as BRL37344A. Consistently, β3-AR antagonists SR58894A and L748337 both induced a rightward shift in the concentration–relaxation curve of isoproterenol-treated detrusor muscle preparations [3-AR transcript was first reported in human detrusor smooth muscle cells [3-AR mRNA to be the most represented (97%) against 1.5% and 1.4% of β1- and β2-AR respectively [The presence of an atypical β-receptor in human detrusor was first suggested in the 1970s  and was arations ,88. The le cells . Determiectively . However3-AR signaling pathway in the bladder, several different and not always consistent hypotheses have been offered. Beta adrenergic signaling often relies on the cAMP/PKA pathway [3-AR agonist FR165101 have been shown to induce a concentration dependent decrease in muscle basal tension and an increase in the cAMP levels. Furthermore, pre-incubation with adenylate cyclase inhibitor SQ22536 markedly suppressed both of these effects [2+ activated K+ channel (BKCa) [3-AR activation strikingly inhibits EFS-evoked contraction and acetylcholine (Ach) release from cholinergic nerves in a concentration-dependent manner in human detrusor bladder strips [3-AR signaling [3-AR stimulation reduces neurotransmitter release. β3-AR modulation of ACh release is also supported by a recent immunohistochemistry study showing β3-AR expression on the detrusor cholinergic fibers in close proximity to sympathetic bundles innervating the human bladder. This arrangement is physiologically logical, as adrenergic fibers can release noradrenalin necessary to activate β3-AR [As for the β pathway ,90,91 an effects . However effects . Along tl (BKCa) . Both Ucr strips . The facr strips  and thatignaling ,94 mightte β3-AR .3-AR presence in the kidney has been reported up to date, with some publications describing functional and transcriptional evidence [3-AR to the vasopressin-sensitive segments of the mouse nephron. The agonist BRL37344 was used on freshly isolated kidney tubules and slices from wt and β3-AR knock out (KO) mice to demonstrate β3-AR coupling to Gs/adenylyl cyclase and its positive effects on key water and solute transporters, such as Aquaporin 2 (AQP2) and Na-K-2Cl (NKCC2). All effects reported were blocked upon pre-incubation with the antagonist L748337 or PKA inhibitor H89, suggesting a potential activation of the cAMP/PKA axis. Ex vivo findings are strengthened by in vivo data, showing that KO mice for β3-AR are mildly polyuric and highlighting a role for β3-AR in renal homeostasis. Interestingly, administration of BRL37344 to a mouse model of X-linked nephrogenic diabetes insipidus (NDI), known to be heavily polyuric, completely reverted the phenotype within 1 h from injection [3-AR as therapeutic target in conditions where water-solute homeostasis and/or vasopressin signaling are impaired  is currently the most promising agent in over 30 years. This drug has been extensively studied, with a good number of Phase II and Phase III trials all over the world, exhibiting a good balance between efficacy and tolerability in all of them [3-AR agonist, vibegron, has recently passed the later stages [βstenosis , chronicstenosis . The β3- of them . It was  of them . Severalr stages  and has r stages .3-AR is abundantly expressed in rodent white (WAT) and brown (BAT) adipose tissue where it mediates lipolysis and thermogenesis [3-AR mRNA levels appeared to be much lower in these tissues. The transcript was mainly identified in infant peri-renal BAT and in various deposits of WAT in the adults [3-AR role and function in humans is represented by the little presence of BAT in adults; however, studies in the last decade are overturning these findings, showing proof for a metabolic role of BAT in healthy adults [3-AR stimulates lipolysis in human isolated white adipocytes [βogenesis ,102. In e adults ,103 while adults ,105. Of y adults ,107. Simipocytes ,109.3-AR, which couples to the α-subunit of Gs-proteins and triggers the activation of the cAMP-PKA axis. Final targets of this cascade include lipid droplet proteins, like hormone-sensitive lipase (HSL) and perilipin, that start the lipolytic process in white adipocytes. However, β3-AR signaling in adipocytes is promiscuous in that HSL activation can also be triggered by β3-AR coupling to Gi and consequent initiation of the ERK1/2 MAP kinase cascade [3-AR activation can also lead to lipolysis by increasing the transcription/expression of the inducible nitric oxide synthase (iNOS) and thus nitric oxide levels in a PKA-dependent fashion [A major trigger of both lipolysis and thermogenesis is the sympathetic nervous system. From a molecular point of view, noradrenaline (NA) released by sympathetic nerve endings binds to β cascade ,111. The fashion .3-AR activation was recently found to be involved in the generation of hyperthermia arising from WAT, instead of the more classically thermogenic BAT, with simultaneous WAT remodeling and increased beiging in mice with impairment in triglyceride storage capabilities [3-AR KO mice also showed impaired cold-induced thermogenesis with a reduction in white adipocyte beiging [3-AR activation induces trans-differentiation of mature white adipocytes but that cold-induced beiging occurs via β1-AR [3-AR is dispensable for thermogenesis, showing that adaptive response to cold is not affected in KO mice [βbilities . β3-AR K beiging . This isia β1-AR . In cont KO mice .3-AR KO mice lack a major phenotype, with only moderate fat accumulation in females vs. males and no altered adipose-related functional response to β-agonists [1-AR in WAT and BAT and they are able to survive cold exposure through β1-AR mediated thermogenesis and UCP1 increase [3-AR KO mice generated on the same background show an increased susceptibility to high fat diet, developing more severe obesity with white adipocyte hypertrophy and inflammation in comparison to wild type mice [3-AR axis is a key player in obesity-driven increases in lipolysis and that administration of MEK inhibitors is efficient in blunting both in vivo lipolysis in diet-induced obese mice and ex vivo lipolysis induced by β3-AR agonist treatment on human and murine WAT explants [Regardless of these effects, βagonists . KO miceincrease ,117. Morype mice . Consistexplants .3-AR agonists with normalizing effects on hyperinsulinemia, increases in resting energy expenditure (REE) and decreases in circulating FFAs, fat/non-fat mass ratio and body weight gain in rats or in obese mice [3-AR expression level is reduced in obese patients [3-AR and different contributions of white and brown adipocytes in rodents and humans [Lipid and carbohydrate metabolism are influenced by βese mice ,120,121.patients . These opatients ,123, butpatients ,125 wered humans .3-AR in modulating adipogenesis in humans than in rodents or due to the little presence of BAT in adults, although this concept has been challenged in the last decade [3-AR as a target for metabolic conditions. A short review by Jonathan Arch. discusses this long-standing issue, proposing some alternatives like combined administration of β3-AR agonists and other compounds aimed at increasing BAT amount in patients [1-AR off-target binding. Finally, even in cases where weight reduction is not achieved, β3-AR stimulation could generally exert positive metabolic effects [3-AR role as a possible candidate for the treatment of obesity and metabolic diseases, highlighting the necessity for the development of more subtype-selective agonists so as to limit side effects to a minimum.No agonist has yet progressed beyond phase II clinical trials. It is unclear whether this is because of the lack of compounds with suitable drug properties , because of a lesser role for βt decade ,107. Howpatients . A recenpatients . Despite effects . This st3-AR agonists to proceed into a clinical stage and more insights into the receptor physiology are ultimately needed for this purpose.Along the same line and based on all these pre-clinical and clinical considerations, new beta-phenylethylamine compounds were recently synthetized and tested in rats with alloxan-induced type II diabetes. All of these compounds markedly decreased levels of total cholesterol, LDL cholesterol, and triglycerides and increased the values of antiatherogenic HDL cholesterol. Moreover, the effects were significantly more intense than the reference substance BRL37344 . In ligh3-AR transcript [3-AR activation [3-AR was shown to exert a marked negative inotropic effect upon stimulation with agonists such as BRL37344, amibegron, and CL316243. However, these compounds were shown later on to exert non-specific effects through β1- and β2-AR [3-AR stimulation on cardiac contractility is abrogated by pre-incubation with either a non-specific NO blocker or a nitric oxide synthase (NOS) inhibitor and treatment with BRL37344 induces an increase in both NO and cGMP, known mediators of the NO pathway. The NOS isoform most likely involved is the endothelial NOS (eNOS), abundantly expressed in human cardiac myocytes [3-AR can also modulate NO signaling via neuronal NOS (nNOS) [l-NMMA (a NOS inhibitor), or ODQ (a soluble guanylate inhibitor) abolished the effect of BRL37344 [+ channel-mediated current (ICa-L) in isolated human atrial myocytes upon β3-AR stimulation with BRL37344 and partial agonist CGP12177. These effects were neither due to Gi nor to NOS activation but were instead mediated by a cAMP/PKA pathway, as pre-incubation with the selective PKA inhibitor H89 abolished them completely [1- and β2-AR stimulation. This is possible because BRL37344 has been reported to induce preferential activation of β2-AR in human atria and its effect is only mildly antagonized by L748337 while it is heavily blunted by β2-AR (and β1-AR) antagonists [3-AR stimulation and ICa-L in both atrial and ventricular chambers [3-AR role in the human myocardium and all the hidden dangers in the interpretation of its pharmacology has been recently published by Arioglu-Inan and colleagues [3-AR partial responses in the myocardium might suggest that the therapeutic use of β3-AR agonists does not pose a risk to cardiac function; on the other, Mo et al. recently reported that mirabegron exerts both a cardiostimulant and a cardiodepressant effect seemingly unrelated to β3-AR activation. In this study, stimulation of human right atrial appendages with mirabegron caused an increase in contractile force that was not only antagonized, but even reversed to negative inotropy upon incubation with β1-AR blocker CGP20712A. However, neither effect was influenced by treatment with β3-AR antagonist L748337. Besides discrediting a role for β3-AR in mediating cardiac inotropy the authors also point out that, given mirabegron structure, a direct interaction with β1-AR is rather unlikely in favor of a more indirect action as a sympathomimetic drug. Indeed, preincubation of the samples with neuronal uptake blockers induces the same negative inotropic effect achieved with CGP20712A [3-AR agonists intended for clinical use.The presence of βanscript ,129 and anscript ,105 in canscript . Signifitivation . In linend β2-AR . Since tmyocytes . The effmyocytes ,135,136.S (nNOS) ,138. IntS (nNOS) . FunctioBRL37344 . Unexpecmpletely . Howeveragonists . Some otchambers . Of notechambers . A thorolleagues . If on tGP20712A . It is, 3-AR signaling pathway in human myocardium can thus be summarized as follows: its stimulation leads to the activation of eNOS or nNOS, probably by means of Gi protein, increasing NO production; NO, in turn, activates a soluble form of guanylate cyclase leading to cGMP production followed by PKG activation that ultimately enhances myocyte relaxation and causes negative inotropy, possibly through the phosphorylation of troponin I, phospholamban, and L-type Ca2+ channels [3-AR downstream signaling the heart might also involve L-type Ca2+ channels stimulation through a cAMP/PKA mechanism leading to positive inotropy at least in human atria [2+ channels, some studies fail to show any effect on inotropy at all [The βchannels . Howeveran atria . Finallyy at all ,1443-AR produces a negative inotropic effect in the human myocardium that is more pronounced in ventricles than in atria and opposite to the one mediated by β1- and β2-AR. Unlike the other two isotypes, β3-AR is expressed at a low level in the normal heart but several reports showed changes in its expression levels at both protein and transcript level in pathophysiological conditions such as diabetes, heart failure, sepsis, and myocardial fibrosis [3-AR is considered to be a promising therapeutic target for cardiac conditions. For instance, both nebivolol and metoprolol, FDA-approved β1-selective blockers, have been shown to exert their cardio-protective effects via β3-AR [3-AR could serve as a ‘safety valve’ at the high levels of sympathetic stimulation typical of cardiac conditions; on the other, its cardio-depressant effect might become maladaptive during later stages and lead to further myocardial dysfunctions. β3-AR antagonists and especially agonists have been proposed as potential strategies for treatment or prevention of heart failure and of ischemic damage [3-AR-PKG signaling cascade inhibits hypertrophy in transgenic mice models with cardiac specific expression of the human receptor and that selective β3-AR stimulation confers protection in the failing heart through a specific cGMP/NO signaling pathway [3-AR in the heart attenuates cardiac hypertrophy via the activation of nNOS. This effect is exerted through paracrine mediators secreted by β3-AR-expressing cardiac myocytes that ultimately attenuate the pro-fibrotic phenotype in cardiac fibroblast [Hence, βfibrosis ,149,150.ia β3-AR ,152. On c damage ,149. It  pathway . NO has  pathway  that can pathway . Recentlbroblast .3-AR agonism on HF progression and development  and the “Assessment of Efficacy of Mirabegron, a New Beta 3-Adrenergic Receptor in the Prevention of Heart Failure” , are evaluating the effects of β1-AR [3-AR mRNA in the rat brain [3-AR agonist”, amibegron, with high efficacy and potency at the rat and human β3-AR, demonstrating its robust anxiolytic-like effects in rats [3-AR in several areas of rat brain and also NE release via activation of central β3-AR probably located in the locus coeruleus, [3-AR-mediated antidepressant effect [Antidepressant effects mediated by a so-called atypical β-receptor in rat brain , but att1-AR , were deat brain  and in dat brain  was repoat brain , Stemmel in rats . Indeed,eruleus, . Other st effect ,160. Amit effect .3-AR is also densely expressed in the murine hippocampus [3-AR in regulating the action potential firing of hippocampal pyramidal neurons via a cAMP-independent pathway involving hyperpolarization-activated cyclic nucleotide gated channels [3-AR agonist CL316243 to day-old chicks, a process most likely due to an increase in glucose uptake via GLUT3 [3-AR KO mice show a decrease in mRNA level of GLUT3 transporter in the amygdala and impairments in long- and short-term memory formation [βpocampus  where itchannels . Gibbs eia GLUT3 . Interesormation .3-AR has also been implicated in the generation of neuropathic pain as the mediator of ATP release from dorsal root ganglia neurons upon NE stimulation [3-AR are involved in the generation of pain and neuroinflammation by eliciting an increase in the release of pro-inflammatory cytokines such as TNFα and Interleukin 1β [Finally, βmulation . Recent eukin 1β . Given teukin 1β .3-AR also exerts vasorelaxant and antiangiogenic effects in rodent retinal blood vessels [3-AR pro-proliferative effect is likely mediated by a signaling cascade involving mitogen-activated protein kinase (MAPK) pathway components rather than by the cAMP/PKA axis, while β3-AR induced cell migration relies instead on a Src-PI3K pathway. Interestingly, functional studies on human choroidal cells showed that β3-AR stimulation evoked a substantially lower proliferative response in these cells compared to retinal endothelial cells and that this is likely mediated by PKG [3-AR as a therapeutic target in retinopathies [3-AR regulation of VEGF production in mouse retinas, showing that β3-AR protein levels are increased in hypoxic retinas and that siRNA silencing of the receptor blunts hypoxia-induced VEGF increase [3-AR stimulation with BRL37344 significantly increased VEGF release in hypoxic retinas vs. normoxic and untreated hypoxic samples. Conversely, its blockade prevented VEGF increase in the same hypoxic conditions, suggesting a potential role for β-blockers in the treatment of retinal diseases. However, given the physiological role of VEGF as a growth factor, it has to be taken into account that changes in its levels could be associated with cell death as reported by Casini et al. 2014 [3-AR blockade, pinpointing NO as a key downstream effector in this pathway. Of note, expression of the inducible isoform of NOS (iNOS) is not only unaffected by β3-AR stimulation but even reduced upon β1-AR activation in human retinal endothelial cells [3-AR effect in human retina could involve different NOS isoforms such as eNOS or nNOS, as in the myocardium [β vessels . Protein vessels . A targed by PKG . Rodent opathies . In retiincrease . β3-AR sal. 2014 . Strikinal cells , suggestocardium ,138 and 3-AR mRNA and protein has been reported across different tumors, including vascular tumors, colon and breast cancer, and human leukaemia cells [3-AR presence is detected at very high levels in human melanoma sections [3-AR in the context of melanoma. They showed that β3-AR blockade increases apoptosis and reduces tumor cell proliferation and vascularization, thereby slowing down melanoma growth in treated mice [3-AR blockade, suggesting that the reduced tumor vascularization is probably due to the increase in apoptosis and that the chance of systemic side-effects is minimal. β3-AR was also shown to enhance the proliferative advantage of tumor cells by promoting a metabolic shift towards aerobic glycolysis, known as the Warburg effect, in melanoma stem cells. Notably this effect is reverted by SR59230A treatment [3-AR as immune-modulator agent in melanoma. In the study, the authors show that β3-AR antagonism with SR59230A and siRNA injections reduces melanoma growth in vivo. This effect is due to an increase in the number of natural killer cells (NK) and CD8 T cells (CD8) as well as their cytotoxicity, and to the abrogation of the immune-suppressive cells, the so-called myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), in the tumor microenvironment [3-AR in immune-tolerance, reinforcing the hypothesis that its targeting could actually represent an effective therapy to overcome melanoma growth. In addition to cancer cells β3-AR is highly expressed in stromal, inflammatory, and vascular cells of the melanoma microenvironment where it favors melanoma aggressiveness through recruitment of stromal cell precursor to the tumor site and induction of inflammatory cytokines secretion and de novo angiogenesis [3-AR role in melanoma has recently been published by Dal Monte et al. [The presence of βia cells ,176,177 sections ,176. Thisections . Of noteted mice . Importareatment . Anotherironment . Thus, te et al. . Main li3-AR in cancer therapy or at least in melanoma treatment.Besides the limitations described above these results may still lead the way to further explorations β3-AR gene that yields a Trp64Arg substitution at the intracellular end of TM1 [3-AR has a prominent role in the human bladder [3-AR polymorphism Trp64Arg has been recently studied in relation to urate and gout with a positive ethnicity-dependent correlation [Each β-AR is known to have some commonly recognized polymorphisms in humans . Early d of TM1 ,185. Homd of TM1 . A corred of TM1 . The pred of TM1  and HDL-d of TM1  accumulad of TM1  and has d of TM1 . A strond of TM1 . β3-AR h bladder . A meta- bladder . However bladder ,193. For bladder , althoug bladder . The disrelation , and in relation ,198,199 3-AR has yet been targeted. More are still under development and need to be explored. For instance, β3-AR mRNA and protein expression were surprisingly reported in the female reproductive system  as one of the highest hits in a recent comprehensive quantitative analysis of the human transcriptome [3-AR in such tissues remains unclear, its presence and function in human near-term myometrium was previously described and specific stimulation of the receptor caused marked relaxation of myometrial contractions via a cAMP/PKA mechanism [3-AR in the management of preterm labor and of its involvement in chronic inflammatory diseases. It is a known fact that inflammation is one of the regulators of preterm labor. Indeed, Hadi et al. recently described how β3-AR stimulation evokes a dose-dependent anti-oxidant effect that ultimately blunts inflammation in myometrial biopsies. The study also reports, for the first time, β3-AR presence and anti-oxidant properties in human macrophages obtained from healthy donors [The therapeutic approaches described herein are only some of the ways βcriptome . Althougechanism ,201. Sucy donors .3-AR can act as upstream regulator of key proteins for water and solute reabsorption. It is fair to note that the β3-AR signaling pathway shares a high degree of similarity with the vasopressin pathway, leading to increases in water and solute uptake that are mirrored by the activation of water and solute transporters via cAMP. In addition, β3-AR selective stimulation rescues AQP2 function in the absence of arginine vasopressin (AVP) action and dramatically reduces the urine volume in a mouse model of X-linked NDI [3-AR agonists in vivo, we recently confirmed that human β3-AR is resistant to desensitization in kidney epithelial cells and that it elicits long-lasting downstream effects on its targets, by comparing its desensitization rate to human AVPR2 upon stimulation with selective agonists [Finally, as previously mentioned, βagonists .3-AR is rapidly being revealed, making this receptor an interesting target for novel therapeutic approaches. Its role in humans is, however, controversial and studies have suffered from the late discovery of β3-AR, the consequent lack of selective detection tools, and inter-species differences. Finally, thanks to advances made over the last two decades, targeting β3-AR has been proposed for the treatment of several conditions, with some drugs undergoing Phase II and III clinical trials and others already on the market. Restricted tissue expression and relative low tendency to desensitization are some of β3-AR features that would allow for a prolonged pharmacological stimulation with low systemic off-target effects. Its low sensitivity to catecholamines also suggests its potential role in pathological conditions , thereby making it even more appealing in such a context. The data reviewed here summarize the evidence that paved the way to the use of β3-AR in pharmacologic treatments and that might lead to speculations on future applications of β3-AR as a therapeutic target in the treatment of multiple conditions.The physiology and function of β


To investigate the effect of asthma rehabilitation at high altitude  compared to low altitude .For this randomized parallel-group trial insufficiently controlled asthmatics (Asthma Control Questionnaire (ACQ) > 0.75) were randomly assigned to 3-week in-hospital rehabilitation comprising education, physical-&breathing-exercises at LA or HA. Co-primary outcomes assessed at 760 m were between group changes in peak expiratory flow (PEF)-variability, and ACQ) from baseline to end-rehabilitation and 3 months thereafter.50 asthmatics were randomized .p = 0.033), ACQ − 1.4, and after 3 months by − 3% and − 0.9. The HA-group improved PEF-variability by − 10%, ACQ − 1.1, and after 3 months by − 9% and − 0.2. The additive effect of HA vs. LA directly after the rehabilitation on PEF-variability was − 6%(− 14 to 2), on ACQ 0.3(− 0.4 to 1.1) and after 3 months − 5%(− 14 to 5) respectively 0.4(− 0.4 to 1.1), all p = NS.The LA-group improved PEF-variability by median(95%CI) -7% contains supplementary material, which is available to authorized users. Asthma is a major global health problem affecting over 300 million people worldwide with increasing prevalence in developing countries . Asthma The current Global Initiative for Asthma (GINA) has outlined that patients with asthma should be managed, according to their level of asthma control, which includes symptoms during the day, symptoms during the night, use of reliever medication, limitation of daily activity, pulmonary function and exacerbations .Many patients with asthma can be adequately controlled with bronchodilators in combination with inhaled corticosteroids. However, some asthma patient suffer from refractory disease despite medical therapy  and evenRehabilitation contains not only physical training and breathing exercises but also education, including smoking cessation , 4 aiminFor decades, asthma patients have been sent to altitude clinics aiming to improve asthma control , 7. At hKyrgyzstan is a lower middle- income country with less access to advanced drug therapies . Thus, aAs in the Swiss Alps, since many years asthma patients have been taken to Tuja Ashu, a high-altitude clinic located at 3100 m asl in the mountain area of the Kyrgyz Republic to improve asthma control. However, randomized controlled trials, which compare the effect of asthma rehabilitation at high compared to low altitude are completely lacking. Whether the reported positive effect of asthma rehabilitation at Alpine resorts is due to hypobaric hypoxic environment at altitude with less allergen exposure or due to the comprehensive rehabilitation program, including patient education and exercise or both is not clear to date.We aimed to study a specific in-patient asthma rehabilitation program and its effectiveness on asthma control and investigated the effects of performing the rehabilitation at the Tuja Ashu high altitude clinic (HA), 3100 m, in comparison to the same program performed at low altitude in Bishkek (LA), 760 m. We hypothesized that a 3-week rehabilitation at high altitude improves PEF-variability and ACQ compared to a low altitude 3-week rehabilitation. Additionally, we hypothesized the effects would be sustained at 3 months follow-up.In this randomized parallel-group trial asthmatics were assigned to in-patient rehabilitation either at LA  or HA , between May and October 2016. The study was approved by the local Kyrgyz Ethics Committee (01–8/151) and the Ethics Committee Zurich (2016–00076).Adults ≥18 years) diagnosed with atopic (positive prick test) or non-atopic asthma according to the GINA-guidelines were eligible if there disease was not well controlled, defined as asthma control questionnaire (ACQ) score > 0.75  and that8 years dThe 3-week rehabilitation program  in the score of the ACQ and the PEF-variability from baseline to the end of the 3-week rehabilitation and to 3-month follow-up (FU). The PEF-variability was computed as [(day’s highest-day’s lowest)/mean of day’s highest+lowest] . PEF meaSecondary outcomes were the percentage of patients with well controlled or partially controlled asthma (ACQ score < 1.5), the between-group differences of the change in the following assessments to end of rehabilitation and FU: forced expiratory volume in 1 s (FEV1), PEF-variability, asthma-related quality of life (AQLQ), generic quality of life assessed by the short-form 36 questionnaire (SF-36), exercise performance assessed by the sit-to-stand (STS), 6 min walking distance (6MWD), airway inflammation assessed by the exhaled nitric oxide (FeNO)  and blooThe HA-group additionally filled out the Environmental Symptom Questionnaire cerebral score (AMS-c) at the second day at altitude to assess safety of altitude exposure symptoms of acute mountain sickness .With an estimated standard deviation of the ACQ-score of 1  and a miA list of the eligible patients was sent to an independent coordinator who randomly allocated patients to rehabilitation at LA or HA by a computer program  with the plugin “rct_minim”) providing minimisation with regard to atopy . Blindinp < 0.05 was considered statistically significant.The analysis was performed on data from all participants undergoing rehabilitation at one of the two locations. Data are summarized as median . The primary and secondary outcomes were evaluated with the Wilcoxon test for paired samples within group and the Mann-Whitney-U-Test for the difference between groups. Difference between the change in PEF-variability and change in ACQ from baseline to 3 weeks and from baseline to 3 months follow-up were calculated and presented as median differences with 95% confidence intervals. A Linear regression analysis was used to estimate the effect of the patients’ characteristics on the change of the PEF-variability. Statistical analyses were conducted with SPSS 22 .The data is reported in adherence with the CONSORT guidelines.Of 159 eligible patients 50 were included in the study: 25 per group with one patient in each group not receiving the intervention , see study flow chart Fig. At baseline, the elevated values of ACQ and PEF-variability confirmed that the asthma was poorly controlled according to selection criteria. Both outcomes significantly improved during the 3-week rehabilitation program at LA although PEF-variability was reduced to below 10% in the HA-group only. After 3 months, the improvements in ACQ were maintained in both groups while PEF-variability remained significantly below baseline and below 10% in the HA-group only. Moreover, the absolute values of PEF-variability at 3 weeks and 3 months were lower in the HA-group compared to the LA-group. There was no significant difference between groups (HA vs. LA) with regard to the changes of the ACQ and the PEF-variability from baseline to end of rehabilitation at 3 weeks and FU at 3 months Table , 3.Tablep < 0.001) after the rehabilitation and to 54% (p = 0.003) at 3 months; corresponding values in the HA-group were 16, 71% (p < 0.001) and 48% (p = 0.008) respectively).The percentage of patients with well controlled or partly controlled asthma (ACQ < 1.5)  increasep = 0.025, HA p = 0.002) and to a similar extent in both groups  g/L, 2 0.666, p < 0.001].Linear regression analysis Table  revealedOf the 25 patients undergoing rehabilitation at HA, symptoms of AMS defined as score ≥ 0.7 were present in 11 patients in Bishkek (760 m) and 6 patients at altitude with only two having a higher score at HA. One patient needed medical treatment for an AMS with severe headache and a very high AMS score of 4.46.During the rehabilitation program the following events occurred, which were not directly linked to the training: In the LA rehabilitation group, one patient had a slight cold, one had temporary fever and one had a sore throat; in the HA one patient was treated with acetazolamide for 1 day for relevant AMS (including headache), one had slight headache, one had intermittent diarrhea and one had an asthma exacerbation defined as a decline in PEF of > 12%. None of the patient had to interrupt the whole rehabilitation program for > 1 day due to symptoms.This is the first randomized controlled trial which investigated the additional effect of undergoing a 3-week rehabilitation program at HA (3100 m) vs. LA (760 m) on asthma control in patients with uncontrolled asthma. We found that a comprehensive 3-week rehabilitation program including education, endurance training, muscle- and breathing exercise significantly improved asthma control both at low and high altitude. The effect of the rehabilitation at HA was not superior compared to the same rehabilitation program at LA in ameliorating asthma control as assessed by the ACQ and PEF-variability. Nevertheless, rehabilitation at HA was well tolerated and led to a better improvement of exercise capacity (STS) and airway inflammation assessed by FeNO. In addition, regression analysis revealed that the overall improvement in PEF-variability from baseline to 3 weeks was better in the HA vs. LA-group when controlling for relevant confounders such as age, gender and the baseline absolute PEF. Thus suggesting that patients with a higher baseline PEF-variability may benefit more from a rehabilitation at altitude.For many years, asthma patients are taken to HA in order to improve their asthma control in a presumptively cleaner environment with less allergen exposure. The effect of training at altitude (1600 m asl) on asthma control was investigated in a prospective, uncontrolled study by Rijssenbeek-Nouwens and coworkers , which sIn the present randomized trial, we could not show an additive effect of providing the rehabilitation at HA vs. LA on our primary outcome. This extends data from a previous non-randomized study suggesting that that allergen avoidance during HA sojourns improved bronchial hyperresponsiveness in adolescents with severe asthma . AdditioIn our study, the vast majority of participants (92%) had atopic asthma, but the improvements found with the rehabilitation at HA were similar as with the same program performed at LA. Nevertheless, we found a significantly greater reduction of FeNO in patients undergoing HA- vs. LA rehabilitation. This may point towards less airway inflammation in the HA-group. The findings are especially remarkable as FeNO measurements were performed at 760 m on the day after return from HA presumably when patients were already re-exposed to some allergens and other noxious stimuli. Our data are in line with previous reports of beneficial effects of HA rehabilitation in adults and children on FeNO and asthma control in both, allergic and non-allergic asthmatics according to nonrandomized studies , 22.Several randomized trials showed a positive impact of physical exercise and training on asthma control , 23–25 a2 max and the total treadmill time [We could show that exercise capacity assessed by the 6MWD and the STS improved significantly with our comprehensive 3-week rehabilitation program and the improvements were maintained up to 3 months follow-up, potentially due to the continuation of daily exercise according to the programs instruction in this highly motivated asthma collective. The improvement in exercise capacity found in both our groups where slightly smaller compared to other studies, assumedly because the rehabilitation lasted 3 weeks compared to 12 weeks . Anotherill time . Whereasill time . Of inteThe significant improvement of asthma specific quality of life reflects an important effect of rehabilitation that was achieved to a similar degree in the HA and LA group. Also all sub scores of the AQLQ improved after the rehabilitation and remained at this level at 3 months follow up in both groups without difference between groups.The program was well tolerated by patients at both locations, expect one patient at altitude who suffered from mild AMS (headache), no adverse events occurred. Intrinsically, it was not possible to blind investigators and patients to the intervention, and thus other factors than altitude and rehabilitation such as being away from home or factors concerning comfort might have played a role in the outcomes. However, this would be the case with any rehabilitation program all over the world. Among the participants of our study, there were no current smokers. Thus, our results may not be transferable to smoking asthmatics. The current study population revealed a relatively low and thus favorable daily PEF-variability and it may well be that the effect of a rehabilitation program including education and training would have been different, potentially even better, in a more compromised asthma collective. Despite these limitations, this study is of particular importance because of its robust design as a randomize trial.In the present randomized trial, we could show that a 3-week comprehensive asthma rehabilitation program including a clear action plan, peak flow diary, patient education, respiratory exercises, force and endurance training is highly effective in sustainably improving asthma control in terms of the Co-primary outcome asthma-related quality of life (ACQ) and PEF-variability, but also exercise performance and this positive effect was irrespective of the altitude is the rehabilitation program was performed.Additional file 1:Asthma rehabilitation at high vs. low altitude: randomized controlled parallel-group trial. (DOCX 32 kb)


Intratumoral steroidogenesis and its potential relevance in castration‐resistant prostate cancer (CRPC) and in cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1)‐inhibitor treated hormone‐naïve and patients with CRPC are not well established. In this study, we tested if substrates for de novo steroidogenesis accumulating during CYP17A1 inhibition may drive cell growth in relevant preclinical models.CYP17A1 were used to block CYP17A1 enzyme activity. The antiandrogen RD162 was used to assess androgen receptor (AR) involvement. Cell growth was measured by 3‐‐2,5‐diphenyltetrazolium bromide assay. AR‐target gene expression was quantified by reverse transcription polymerase chain reaction (RT‐PCR). Nuclear import studies using cells with green fluorescent protein (GFP)‐tagged AR were performed to assess the potential of precursor steroids to directly activate AR.PCa cell lines and their respective CRPC sublines were used to model CRPC in vitro. Precursor steroids pregnenolone (Preg) and progesterone (Prog) served as substrate for de novo steroid synthesis. TAK700 (orteronel), abiraterone, and small interfering RNA (siRNA) against Preg and Prog stimulated cell proliferation and AR target gene expression in VCaP, DuCaP, LNCaP, and their respective CRPC sublines. The antiandrogen RD162, but not CYP17A1 inhibition with TAK700, abiraterone or siRNA, was able to block Preg‐ and Prog‐induced proliferation. In contrast to TAK700, abiraterone also affected dihydrotestosterone‐induced cell growth, indicating direct AR binding. Furthermore, Prog‐induced AR translocation was not affected by treatment with TAK700 or abiraterone, while it was effectively blocked by the AR antagonist enzalutamide, further demonstrating the direct AR activation by Prog.Activation of the AR by clinically relevant levels of Preg and Prog accumulating in abiraterone‐treated patients may act as a driver for CRPC. These data provide a scientific rationale for combining CYP17A1 inhibitors with antiandrogens, particularly in patients with overexpressed or mutated‐AR. ARandrogen receptorCRPCcastration‐resistant prostate cancerCYP17A1cytochrome P450, family 17, subfamily A, polypeptide 1DCCdextran‐coated charcoal‐stripped fetal calf serumDHTdihydrotestosteroneFCSfetal calf serumMTT3‐‐2,5‐diphenyltetrazolium bromidePCprostate cancerPregpregnenoloneProgprogesteroneqPCRquantitative PCR1CYP17A1]), abiraterone acetate (Zytiga), a 17‐α hydroxylase and 17,20‐lyase specific enzyme inhibitor blocking steroidal synthesis from androgen precursorsCastration‐resistant prostate cancer (CRPC) continues to rely on androgen receptor (AR) signaling for its growth, evidenced by the majority of patients with CRPC still responding to novel AR signaling pathway targeted agents. Both the antiandrogen enzalutamide (Xtandi),Clinical data have shown that a subgroup of patients with CRPC progressing on abiraterone still responds to enzalutamide,Preclinical studies have postulated CYP17A1‐dependent intratumoral de novo steroid synthesis as a driver of CRPC and CYP17A1‐inhibitor resistant disease. These studies reported de novo dihydrotestosterone (DHT) synthesis in LNCaP and VCaP cell linesIn the present study, we assessed if CRPC cell growth could be driven by androgen precursors (pregnenolone [Preg] and progesterone [Prog]) at clinically relevant levels found in aging men22.1l‐glutamine (Cambrex BioWhittaker), and antibiotics. PC346C (wild‐type AR) were maintained in prostate growth medium (PGM) based on Dulbecco's modified Eagle's medium (DMEM)‐F12 medium with several PC growth factors and antibiotics as described in Marques et alT877A) cells were maintained in PGM supplemented with 2% dextran‐coated charcoal‐stripped FCS (DCC) instead of FCS, without R1881 and with the addition 1 μM of hydroxyflutamide. LNCaP cells  were maintained in RPMI supplemented with 5% FCS and antibiotics. The adrenal cancer cell line H295R (ATCC) was maintained in DMEM‐F12 supplemented with 5% FCS and antibiotics. The cell lines PC346C, PC346C FLU1, and PC346C FLU2 were generated in our laboratory and are authenticated and described in Marques et al.VCaP  and DuCaP , both carrying wild‐type AR, were grown in Roswell Park Memorial Institute (RPMI) 1640  with 10% fetal calf serum (FCS) and antibiotics. Hep3B stably expressing the green fluorescent protein (GFP)‐AR2.2VCaP and DuCaP cells were cultured in RPMI with 10% DCC to deplete the serum from steroids in the presence of antiandrogens bicalutamide (1 μM) or hydroxyflutamide (1 μM), with n = 10 per condition for >20 months to generate a panel of castration‐resistant clones. When cell growth resembled that of the parental cells growing under standard culture conditions, clones were considered CRPC. For current experiments, clones expressing elevated levels of wild‐type AR and CYP17A1 compared with parental lines were selected for further experiments without further authentication.2.3For cell proliferation assays, 5,000 cells per well were plated in 96‐well dishes in their respective medium with DCC. After overnight attachment, the synthetic androgen R1881, steroids, and compounds were added to reach the indicated concentrations in a final volume of 200 μL. After 9 days, cell proliferation was assessed by 3‐‐2,5‐diphenyltetrazolium bromide (MTT)‐assay as described previously.2.4For cell culture assays, RD162, a nonsteroidal antiandrogen was used . It is closely related to and was selected from the same drug‐screen as MDV3100 . In vitro and in vivo, it has equal potency in AR‐antagonism as enzalutamide and no significant difference in bioavailability in preclinical testing.Steroids were obtained from Steraloids  and dissolved in ethanol. RD162, enzalutamide, TAK700  or abiraterone  were all dissolved in dimethyl sulfoxide (DMSO). Similar amounts of DMSO (0.1%) were added to control cells. Concentrations used were based on levels reported in Belanger et al2.5CYP17A1 inhibitors TAK700  or abiraterone (Johnson & Johnson) for 48 hours. Medium from wells without cells served as blanks. Three replicates were used per condition. After 48 hours of culture, the medium was collected and frozen at −20°C. ∆4‐Androstenedione concentrations were determined using the IMMULITE 2000 automated assay system  with a detection limit of 1.05 nM. The results are shown as means ± SE of three independent experiments. Inhibitory concentration (IC50) values were determined by nonlinear regression using the GraphPad Prism software with Y = 100/(1 + 10X−logIC50).H295R cells at 100.000 cells per well were seeded into 24‐well tissue culture plates and allowed to attach overnight in medium with FCS, after which medium was replaced by the serum‐free medium with or without the 2.6After overnight attachment, cells were transfected with CYP17A1 or nontargeting small interfering RNA  using Lipofectamine RNAiMax  according to the manufacturer's instructions. Twenty‐four hours after transfection, the medium was replaced by DCC medium with indicated steroids. RNA was isolated after 48 hours or proliferation determined at day 6.2.7GAPDH or PBGD and vehicle treated cells.For quantitative polymerase chain reaction (qPCR) studies, RNA was isolated using RNA‐Bee  from cultures treated for 48 hours with indicated compounds/steroids, 24 hours after seeding in DCC medium at 100.000 cells per well. Reverse transcriptase and qPCR runs were performed as described previously2.8Nuclear translocation of the AR has been studied in time as well as in end‐point measurements using fluorescence confocal microscopy on PC346C cells stably expressing enhanced green fluorescent protein (EGFP)‐AR.The ratio of AR nuclear localization was expressed as:2. Time‐lapse imaging was performed using a Zeiss LSM510 confocal microscope , equipped with a 63 × 1.3 NA oil immersion objective. EGFP‐AR was visualized using 488 nm excitation of an Argon‐laser line and detection of emission between 500 and 530 nm. For time‐lapse imaging, images were acquired with 5 minutes interval during 130 minutes at multiple locations of the same sample. After 5 to 10 minutes of imaging, Prog (100 nM), Preg (100 nM), or R1881 (1 nM) was added to the medium to investigate AR nuclear translocation. Average fluorescence intensities in the nucleus and cytoplasm were measured at every time point.For the analysis of AR‐translocation dynamics, cells were seeded on glass coverslips in six‐well plates. After overnight attachment, cells were treated with TAK700 (3 µM) or vehicle for 12 hours and subsequently transferred to a live‐cell chamber and maintained at 37°C and 5% CO2.9t test. AR translocation was analyzed using one‐way analysis of variance (ANOVA) with subsequent Tukey's multiple comparisons on logarithmically transformed values to equalize variances. Analyses were carried out using the GraphPad Prism version 5.03 . P < 0.05 was considered statistically significant.MTT and qPCR results were normalized to control and compared using the two‐sided Student 33.1To determine IC50 values of the CYP17A1 inhibitors under the conditions used in this study, human adrenal H295R cells were incubated in steroid stripped medium with increasing amounts of abiraterone or TAK700. ∆4‐Androstenedione synthesis—which is directly dependent on CYP17A1 activity —was effectively blocked in H295R cells (expressing CYP17A1 1100‐fold vs prostate cells) with IC50 values of 15.5  and 67.7  nM for abiraterone and TAK700, respectively .3.2CYP17A1 and AR gene expression level compared with their parental cell lines (Table S3). Preg and Prog at levels of 1 nM and upstimulated cell growth in all CRPC clones tested . Similar results were obtained for LNCaP , and PC346C CRPC cells that are characterized by an overexpressed wild‐type AR (PC346C FLU1) or by T877A AR mutation . Of note, CYP17A1 messenger RNA (mRNA) could not be detected in LNCaP nor in PC346C (Table S3). Also, we were unable to detect CYP17A1 protein in VCaP despite detectable mRNA levels. . The AR‐driven effects in the presence of TAK700 indicate that growth of these CRPC clones, despite upregulated CYP17A1 mRNA, was not dependent on the increased activity of de novo steroidogenesis.To assess the biological relevance of CYP17A1‐mediated cell growth in CRPC, we selected CRPC cell lines of VCaP  and DuCaP (BIC‐H) based on their elevated Prog and Preg stimulate cell growth of castration‐naïve VCaP and DuCaP cells via AR activation to test if Preg and Prog could also facilitate cell growth of castration‐naïve parental VCaP and DuCaP, characterized by relatively low levels of CYP17A1 Table S3), cells were incubated with 10 and 100 nM Prog and Preg. Indeed, cell growth was significantly stimulated Figure A with co, cells wCYP17A1. Similarly to incubation with TAK700, treatment with CYP17A1 siRNA—resulting in undetectable levels of CYP17A1 mRNA—did not affect Prog stimulated levels of PSA mRNA in parental VCaP . To further substantiate these findings, we evaluated AR translocation in the human prostate cancer cell line PC346C, which naturally expresses ARwt and lacks CYP17A1 expression. Incubation with Prog, but not Preg, induced translocation of AR to the nucleus in PC346C cells stably transfected with GFP‐ARwt. This translocation could not be inhibited by overnight preincubation with 1 μM TAK700 or abiraterone, but only by pretreatment with 1 μM of the AR antagonist enzalutamide  CRPC resistance that is driven by accumulating precursor androgens that may directly mediate AR‐regulated cell growth, particularly in tumors overexpressing AR. Indeed, recent studies on cell‐free DNA in patients with metastatic CRPC have demonstrated that AR copy number gain at the initiation of treatment with second‐line hormonal agents is indicative of primary resistance to these agents.Here, we show that clinically relevant levels of androgen precursors Preg and Prog stimulated cell growth of parental VCaP and DuCaP and of their respective CRPC cell lines that are characterized by overexpressed levels of wild‐type Data on precursor steroid levels in patients treated with abiraterone are scarce. As a surrogate measure of systemic steroid concentrations, urinary Preg and Prog metabolites in patients treated with abiraterone without exogenous glucocorticoids have previously been shown reported to be 2.5 to 44 and 3.8 to 61 times higher compared with baseline.CYP17A1 were able to reverse Preg‐ or Prog‐induced cell growth of CRPC at clinically relevant levels shown in our study underscore reports that intratumoral de novo steroidogenesis is not essential for CRPC growth.With CYP17A1 expression being a magnitude lower in prostate cells compared with adrenal cells  explained by direct AR‐antagonism, albeit at peak concentrations. This is consistent with data that abiraterone can bind to and antagonize AR in LNCaP and VCaP,To date, two retrospective studies reported PSA response rates of 27% and 30% for enzalutamide in abiraterone progressive patients.Although our study may be limited by the use of in vitro models, it is important to note that these different cell lines were selected based on highly clinically relevant characteristics, including the absence of intratumoral CYP17A1 expression in the context of high AR expression. The studies were performed using clinically relevant levels of steroids and drugs as reported from relevant patient cohorts, to recapitulate the negative consequences of enhanced substrate levels of preg and prog in patients with CRPC treated with CYP17A1 inhibitors. The in vitro system allows for defined assessment of the potential of different steroids that will otherwise be obscured in in vivo models. These data provide basic mechanistic evidence to combine steroid synthesis inhibition with antiandrogens to fully extinguish ligand‐dependent AR activation in tumors that have become hypersensitive to minute levels of androgen or alternative steroidal ligands (like accumulating progestagens due to systemic CYP17A1 inhibition) via AR amplification or mutations in the absence of intratumoral CYP17A1 activity. Thus, prospective data on PSA response and possible survival benefit of combining abiraterone with enzalutamide from the start of second‐line hormonal therapy are eagerly awaited.5In summary, our study demonstrates that in castration‐naïve and CRPC cell lines, androgen precursor steroids Preg and Prog are able to directly activate wild‐type and mutated‐AR, independent of CYP17A1‐mediated conversion into testosterone Figure . These fRW receives consultancy and speaker fees from Sanofi, Millennium, Merck, Roche. RS receives speaker fee from Sanofi, Janssen Pharmaceuticals. WW receives grant supports from Sanofi, Millennium, Janssen Pharmaceuticals, Servier.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Rehabilitation is crucial for the realization of the right to health and a proper concern of global health. Yet, reliable information to guide rehabilitation service planning is unavailable in many countries in part due to the lack of appropriate indicators. To ensure universal health coverage and meet the central imperative of “leaving no one behind” countries must be able to assess key aspects of rehabilitation policy and provision and monitor how they have discharged their human rights responsibilities towards those most disadvantaged, including people with disability. This article describes the process of developing an expert guided indicator framework to assess governments’ efforts and progress in strengthening rehabilitation in line with the Convention on the Rights of Persons with Disabilities.A systems methodology - concept mapping - was used to capture, aggregate and confirm the knowledge of diverse stakeholders on measures thought to be useful for monitoring the implementation of the Convention with respect to health related rehabilitation. Fifty-six individuals generated a list of 107 indicators through online brainstorming which were subsequently sorted by 37 experts from the original panel into non overlapping categories. Forty-one participants rated the indicators for importance and feasibility. Multivariate statistical techniques where used to explore patterns and themes in the data and create the indicators’ organizing framework which was verified and interpreted by a select number of participants.r = .58) with experts prioritizing the indicators contained in the clusters of the Governance and Leadership domain. Two of the most important indicators arose from the Service Delivery, Financing and Oversight domain and reflect the need to monitor unmet needs and barriers in access to rehabilitation. In total, 59 indicators achieved above average score for importance and comprised the two–tiered priority set of indicators.A concept map of 11 clusters of indicators emerged from the analysis grouped into three broader themes: Governance and Leadership (3 clusters); Service Delivery, Financing and Oversight (6 clusters); and Human Resources (2 clusters). The indicator framework was comprehensive and well aligned with the Convention. On average, there was a moderately positive correlation between importance and feasibility of the indicators  contains supplementary material, which is available to authorized users. Rehabilitation is an integral component of the therapeutic continuum that every health system must strengthen on the path to universal health coverage (UHC) . Today tAre human rights respected, protected and fulfilled in service delivery?Are the key principles of human rights met in policy formulation and implementation– Is the right to access to rehabilitation realized equally for all, with the active participation of service users, effective accountability mechanisms and without discrimination?What barriers do persons with disabilities experience in accessing appropriate rehabilitation? Are efforts being made to progressively remove those barriers?Is there an enabling environment for the implementation of human rights - Is the social, institutional, legal, organizational and economic environment conducive to the realization of the right to health and rehabilitation?Indicators, broadly speaking, are succinct measures that aim to provide the most comprehensive picture of the health system (or a subcomponent of it) possible with the least amount of unnecessary detail . At the The development and use of such indicators is particularly pertinent to heath systems, because some of the most chronic and entrenched deficiencies in rehabilitation services are a direct consequence of the denial of human rights of persons with disabilities and result from frailties in the way health policies are formulated and implemented and laws are developed and enacted .It is generally agreed that indicators must be embedded in a clear and robust underlying conceptual framework that specifies system domains or target areas for improvement. However, in the literature there is a gap on indicator frameworks that can help obtain a comprehensive picture of the weaknesses in rehabilitation service delivery and identify systemic failures in policy development and CRPD implementation. Approaches to monitoring rehabilitation systems and services vary considerably and there is very little agreement over the domains and elements of a global indicator framework. Without a strong empirical understanding of what such a framework should comprise of– including its key domains, subdomains and measures – progress with the implementation of the CRPD and other widely agreed recommendations on rehabilitation will be difficult.Specifically, much of the existing research has focused on the development of clinical governance measures for various disease groups for the purpose of assessing improvements in quality of HRR –33. AlonThree recent systematic efforts have developed approaches to monitor the status, performance and quality of HRR services respectively: the International Classification of Service Organization in Rehabilitation (ICSO-R) , the RehTo help countries meet their obligations under the CRPD and achieve greater and more equitable improvements in service delivery, there is an urgent need to develop a conceptually robust indicator framework for HRR. To be useful both for human rights accountability and monitoring States’ efforts to strengthen and scale up HRR services within the context of Sustainable Development Goals (SDG), such a framework must faithfully account for a wide range of legal, policy and programmatic factors that influence equitable access to rehabilitation and assistive technologies. To achieve this, the indicators included in the framework should be informed by key provisions of the CRPD pertaining to rehabilitation to drive the collection of data consistently with international human rights standards . FurtherIn light of these considerations, we conducted a study to develop an expert-informed indicator framework for assessing country efforts to strengthen rehabilitation through implementation of the CRPD. Specifically the objectives of this study were: (a) to elicit and synthesize the knowledge of experts on indicators that would be useful for monitoring the implementation of the HRR aspects of the CRPD; (b) to integrate and confirm this knowledge through feedback to develop a shared conceptual model for rehabilitation sector assessment; and (c) to prioritize a set of important indicators to guide future evaluation and research.In meeting the objectives above, we have adopted the following definitions:In an effort to acknowledge the critical connections between rehabilitation services and the health system we use the term “health related rehabilitation” in the same way as in the context of Article 25 of the CRPD , which tBecause rehabilitation, by definition, targets persons whose functions are limited as a result of illness, injury or chronic disease, it is very rare for a HRR intervention or service to benefit only persons with long term impairments and disabilities. Thus an approach to defining “persons with disabilities” for the purpose of monitoring HRR should be broad enough to account for the variety of disease groups that may benefit from HRR. To maintain consistency with the CRPD, “persons with disability” are understood in this research “to include those women, men, girls and boys with long-term physical, mental, intellectual or sensory impairments which, in interaction with various barriers, may hinder their full and effective participation in society on an equal basis with others” . Howevergroup concept mapping, to develop the indicator framework which we call the Rehabilitation Systems Diagnosis and Dialogue (RESYST) framework. Group Concept mapping (GCM) is a mixed method that integrates sound qualitative group procedures  with multivariate statistical analyses to help a group describe their ideas on the topic of interest and represent these ideas visually through a series of related maps  anThere were methodological issues that may have impacted on our results and lessons learned will help adjust the design of similar studies in the future. Despite the implementation of evidence based measures to increase participation in our study  participation rates were lower than the average concept-mapping project , for botThis research had a strong focus on the rehabilitation related dimensions of the right to health of persons with disabilities. We have therefore adopted a practical definition for HRR to facilitate a common understanding of the programmes and services that stakeholders think of as “health related rehabilitation”. This definition, along with evidence generated from a previous legal analysis of States obligations under the CRPD, helped participants identify potential measures that address several factors that impact on access to services and the enjoyment of disabled people’s right to health. This emphasis on the health care aspects of rehabilitation may have biased our selection of potential eligible participants in favour of those who are more active in the health domain. This has resulted in an overrepresentation of rehabilitation professionals and disability researchers in the group of sorters and raters. Additionally, given the general scepticism over contemporary approaches and definitions of rehabilitation by the disability community, and the subsequent lack of trust in rehabilitation research endeavours, it is possible that the emphasis on HRR has deterred some representatives of people with disabilities from participating in the sorting and rating of indicators. However our decision to concentrate on these aspects is justified by the widespread and often insurmountable barriers people with disabilities confront in accessing rehabilitation treatment services. It could be argued that an approach that would place a greater focus on social aspects of rehabilitation, especially CBR, may have created a more enabling environment and provided greater opportunities for persons with disabilities - especially those from low and middle income countries - to express their voice on this important topic and may have allowed to capture a small but significant segment of rehabilitation service systems in low income countries that is disproportionately focused on community inclusive development. Given however the expressed concerns over the ability of CBR as a strategy to bring broader systemic changes in line with the CRPD and remove barriers in access to services , it is dNotably, experience from research shows that the issue of participation of service users or community members in the development of measures and indicators is highly controversial (as is the definition of stakeholders in community based research) as there are no specific guidelines regarding participation rates and the optimal balance between stakeholders groups , 109. ThInterpretation of the conceptual map took place with a convenience sample of experts who participated in the sorting phase coming mainly from high-income countries. The lack of involvement of participants from low- and middle-income countries in the group interpretation may have introduced a culture bias as it is possible that the results would have evolved differently if their insights on the grouping of clusters into thematically related domains have been incorporated into the final model. Discussion of the cluster map and the indicator set with a larger number of participants - especially policy decision makers and service users –is recommended as part of future local adaptation and/or validation processes, as this may enhance their relevance for implementation as well as the sense of ownership of the results of the monitoring activity.Although the framework incorporates a large number of factors previously found to be important in assessing progress in the implementation of the HRR aspects of the CRPD, it is possible that some indicators that are standardly used in rehabilitation service evaluations and quality assessments may have been missed. This was expected as the emphasis of this research was on human rights indicators intended to complement, rather than replace existing performance measures. It is likely that with further research, including testing and validation of the framework considering local context and stakeholder priorities, additional indicators may be included in the framework.The RESYST presents both similarities and differences with the EQUIFRAME, a policy analysis framework that has been previously developed to facilitate the examination of the compatibility of health policies with human right guarantees for vulnerable populations , includiIt will be important to test the validity and practical utility of the indicators in different contexts, assess the methodological constraints in data collection as well as the organizational costs and benefits associated with the use of the proposed indicators. This will require the drafting of indicator specification sheets  and the development of multi-item assessment instruments to facilitate the collection, verification and analysis of information. The use of empirically developed and field tested indicators will provide opportunities to appraise rehabilitation policies and compare service organization across countries and thus move the scientific evidence base on comparative rehabilitation systems research forward. The practical insights offered by this study are both timely and strategically relevant as leading health agencies and professional organizations strive to integrate rehabilitation in health systems through capacity building and assessment initiatives. It is therefore recommended that health agencies, especially the WHO, professional organizations and international research consortia use the RESYST framework as an evidence source in future projects aiming to develop service monitoring and capacity assessment tools as well as to stimulate debate on methodological issues pertaining to the construction of system level indicators for rehabilitation.To our knowledge, this is the first systematic attempt to conceptualize the constituent domains and elements of a system level framework that details what, beyond traditional clinical outcomes or quality indicators, should be monitored to enable health programme planners implement evidence informed strategies to shape a more inclusive and pragmatic response to population rehabilitation needs as well as introduce and implement disability rights compliant policy reforms. As a conceptual device, the RESYST framework enables practitioners, researchers and advocates derive a complex understanding of the issues that must be considered in comprehensive rights based analyses and service audits. As a systems methodology, GCM was used to visualize and simplify the representation of multiple correlated legal, policy and programmatic variables that influence the implementation of the right to access to rehabilitation and helped make the connections between rehabilitation services and health systems more explicit. As a group decision tool, GCM allowed the collective thinking and priorities of a select group of experts and global scientists to surface which can more meaningfully direct efforts and inform future assessment of rehabilitation systems and policies.The results reported here contribute to expanding the relatively limited evidence base of rehabilitation systems research and thus building stakeholders’ capacity for monitoring and evaluation. Future research should build on the experience of this study aiming at empirically testing the framework and the proposed measures and adapting it to local circumstances. Implementation of the RESYST after proper validation may help governments, and those seeking to support them, strengthen policy surveillance to gain a clearer and more comprehensive picture of the main weaknesses in rehabilitation services and align national strategies with obligations and commitments on disability rights and inclusion, thus leading to better and more equitable outcomes for all.Additional file 1:Indicators per cluster. Rights based indicators for rehabilitation contributed by stakeholder-participants with Bridging Index and rating scores for relative importance and feasibility to implement, arranged by cluster in descending order of importance. (DOCX 37 kb)Additional file 2:Stakeholder panel membership. Organizational affiliations of participants in the sorting and rating phase. (DOCX 77 kb)


Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, with a prevalence of up to 12% of women of reproductive age, and is in part characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium. A high percentage of PCOS patients suffer from infertility, a condition that appears to be linked to mistimed and incomplete decidualisation critically affecting events surrounding embryo implantation. The aim of this study was to examine the involvement of MAGEA11, and the genome-wide role of AR in PCOS. We determined that elevated androgen levels on PCOS cells had an impact on the delayed and incomplete decidual transformation of endometrial cells. The AR co-regulator MAGEA11, a known enhancer of AR function, was constitutively overexpressed throughout the menstrual cycle of PCOS patients, co-localised in the nucleus of PCOS stromal tissue and cells and formed a molecular complex with AR. Genome-wide AR analysis in PCOS stromal cells revealed that AR targets included genes involved in cell death and apoptosis, as well as genes commonly dysregulated in endometrial cancer. Enhanced MAGEA11 and AR-mediated transcriptional regulation may impact on a correct endometrial decidualisation response, subsequently affecting endometrial receptivity in these infertile women.MAGEA11 and AR are overexpressed in hyperandrogenic PCOS patients.MAGEA11-AR overexpression in PCOS correlates with delayed decidualisation.AR and MAGEA11 associate in a molecular complex.AR directly regulates a unique set of genes controlling gene differentiation.The online version of this article (10.1007/s00109-019-01809-6) contains supplementary material, which is available to authorized users. Polycystic ovary syndrome (PCOS) is a common gynaecological disorder affecting 4–12% of reproductive age women. Up to 44% of unexplained infertility cases display a PCOS morphology linked with ovulatory dysfunction and systemic hyperandrogenaemia. PCOS patients accumulate androgens due to defects in aromatase activity that prevents the conversion of androgens to oestrogens . In addiPreviously, we have shown that levels of apoptosis are reduced in stromal cells in both ovulatory PCOS and androgen-treated fertile samples . This isThe effects of androgens are mediated through androgen receptors (AR), nuclear hormone receptors that bind to and regulate the expression of specific target genes. AR functions through interactions with co-regulator proteins that result in the repression or activation of target gene transcription through modification of chromatin structure and regulation of RNA polymerase activity . AR co-rHere, we show that the addition of androgens disrupts the differentiation of endometrial stromal cells during the process of decidualisation in vitro. This process is accompanied by increases in the expression of MAGEA11, which is recapitulated in the endometrium of PCOS patients. Following treatment with dihydrotestosterone (DHT), both MAGEA11 and AR accumulate and co-localise in the nucleus both in vitro and in vivo in endometrial tissues obtained from PCOS patients. Genome-wide analysis of AR interactions using chromatin immunoprecipitation revealed that AR interacts with genes involved in the regulation of cell death and apoptosis including EGFR and WT1, supporting the notion that elevated androgens result in delayed decidualisation processes, and implicating AR and MAGEA11 in incorrect endometrial differentiation. This could impact on the critical timing process of embryo implantation, and thus human reproduction.5 cells) were cultured in 6-well plates for 24 h prior to treatments. Confluent ESC monolayers were treated with cAMP , DHT , and DHT + cAMP (10−8 M + 500 μM) for 24 h, 48 h, and 72 h. RNA was extracted after treatments and cell morphology assessed throughout the duration of the experiment.Primary endometrial stromal cells (ESCs) were isolated from endometrial biopsies as previously described , yieldinPrimary cells were washed in PBS and fixed for 15 min in 4% paraformaldehyde buffered in 0.1 M sodium cacodylate (pH 7.2). Standard light microscope images  were captured and shape index was used to calculate changes in cell shape. ImageJ software was used to calculate the circularity of the cells and this was performed as previously described . A perfeHuman prolactin/IGFBP-1 DuoSet ELISA kits  were used to analyse the presence of prolactin/IGFBP-1 in cell culture media. The target medium was collected from confluent monolayers of ESC cells treated for 48 h with cAMP and DHT. The protocols were performed as per manufacturer’s instructions and each sample was measured in triplicates.in vitro studies.Patient recruitment and consent was carried out at the gynaecology clinics at Singleton Hospital, Swansea. Endometrial biopsies from women in a natural menstrual cycle were obtained for immunohistochemistry and in vitro studies. Patients had not received exogenous hormonal therapy at least two months before the procedure. Women with systemic diseases, sexually transmitted infections, or evidence of endometritis, endometrial hyperplasia, or endometrial polyp were excluded. The presence of chronic endometritis was ruled out by immunohistochemical analysis of biopsy samples. The phase of the natural menstrual cycle was confirmed by ultrasound and histological criteria; urinary luteinising hormone (LH) was used to document ovulation. The control group included women with proven fertility and regular menstrual cycles. The study group consisted of infertile women with PCOS; patients in control and study groups were matched with regard to body mass index (BMI) and smoking habits. PCOS was diagnosed based on the Rotterdam criteria of ultrasound and clinical and biochemical features of hyperandrogenism . Anovulan = 33), infertile ovulatory PCOS ] and 37 patients in the proliferative phase. . Endometrial samples were also taken from 21 PCOS women who were anovulatory (anovPCOS). There were no statistically significant differences in the mean age or body mass index between the groups  samples, nuclei-staining, and immunohistochemistry was performed as previously described . PositivGAPDH). Expression was calculated as a ratio between treated and control samples for each gene. Densitometry analyses were performed using Image Lab software .RNA isolation, quantification, and qRT-PCR analysis using gene-specific primer-pairs (available on request) were performed as previously described . Express5 cells) were grown to 80% confluence on 8-well chambered borosilicate coverglass . At 24 h post induction of decidualisation (+DHT), cells were fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100 (Sigma), and blocked with 3% BSA in PBS for 1 h. Primary antibodies, anti-AR , and anti-MAGEA11  diluted in 3% BSA PBS were incubated overnight at 4 °C, followed by 1 h incubation with secondary antibodies, Alexa488  and Alexa TexasRed . Cells were washed with PBS extensively between all steps, counterstained before imaging with NucBlue reagent (Life Technologies) and imaged using an LSM710 confocal fluorescence microscopy system .ESCs (2 × 10Protein blotting was performed as previously described . For co-6 cells) were cultured in 75-cm2 flasks for 24 h prior to DHT treatment (48 h). DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions. The resulting pellet was sent to Active Motif® for sequencing, alongside with an anti-AR antibody (ab9474), which was used to probe for AR-target region enrichment. Raw ChIP-seq data is deposited in the GEO Dataset, with accession number GSE119432.PCOS ESCs  was used to discover relatively enriched motifs, comparing the AR ChIP-seq sequences with control shuffle sequences. Next, the motif comparison tool Tomtom  was used to compare the DREME-enriched motifs against a database of known motifs, providing a list of matches and their correspondent Alongside with the correspondent FASTA files, Active Motif® provided a list of 2798 genes. In most cases, the gene sequence encompassed the associated ChIP-seq peak location, although some upstream or downstream peaks were linked to gene targets through the identification of nearby promoters. Each gene was associated with a peak value, an average of peak values in case the gene contains several peaks, which were filtered to obtain a list of 577 high-intensity peaks . The list of 577 high-intensity peaks was compared with a list of AR binding sites in human prostate cancer tissues  using a Venn diagram .The filtered genomic localisation of 6296 AR-PCOS peaks  was loaded onto Genomic Regions Enrichment of Annotations Tool (GREAT)  in order to analyse the functional significance of ChIP-seq-identified cis-regulatory regions (gene ontology).U test applied in a post hoc to determine statistical significance. For normally distributed data, an ANOVA test followed by a t test was used to determine significant differences between groups. The test statistic and corresponding p value were reported. All data analyses were performed using SPSS version 13.0 .Data distributions were assessed for normality using the Kolmogorov Smirnov test. Non-normally distributed data was analysed with the Mann-Whitney Decidual transformation involves a mesenchymal to epithelial (MET)-like phenotypic transition in hESC in response to cyclic AMP (cAMP) in a normal menstrual cycle . We assep < 0.01; IGFBP-1: p = 0.025) and cAMP + DHT . In accordance with the morphological response observed in PCOS patients  and cAMP + DHT . These results are consistent with previous observations and indicate that the decidual response is either delayed or inhibited in PCOS patients.Prolactin and IGFBP-1 are known indicators of MET in the context of hESC ; accordints Fig. , PCOS cep < 0.01), which decreased after 48 h and 72 h   and second messengers (cAMP) regulate the expression of the AR co-regulator MAGEA11 in human endometrial cell lines [2 h Fig. . For PCO01) Fig. . This dep < 0.01; 48 h: fold change = 0.24, p < 0.01; 72 h: fold change = 0.48, p < 0.01), and when used in combination with cAMP, was further reduced after 48 and 72 h   Fig. . This suTo determine whether the expression of MAGEA11 in vivo was similar to that observed in vitro, biopsies obtained from proliferative endometrium of fertile and PCOS patients were assessed using immunohistochemistry (IHC).p = 0.03) and stromal tissue (p < 0.01) compared with proliferative phase samples in fertile samples .MAGEA11 was present in epithelial and stromal compartments of fertile endometrium, with expression restricted to the cytoplasm Fig. , and onlles Fig. , confirmp < 0.01) and stromal (p < 0.01) compartments showed high levels of expression, which were further increased in the secretory phase . Similarly, MAGEA11 levels were significantly elevated in anovPCOS samples in both the epithelial (p < 0.01) and stromal (p < 0.01) compartments  and anovPCOS (p = 0.0011) compared with fertile sample levels  and anovulatory PCOS (anovPCOS), MAGEA11 levels were significantly higher than in fertile patients Figs. . Prolifents Fig. . MAGEA11ls Figs.  and 3c.AR staining in fertile endometrium was higher during the proliferative phase of the cycle and predominantly expressed within the stromal compartment Figs.  and 3d. Detailed analysis of AR and MAGEA11 distribution in PCOS samples revealed that they accumulate at high levels in both cytoplasmic and nuclear compartments Figs.  and 3, wHaving demonstrated the temporal and spatial co-location of the two proteins, their direct molecular interaction was assessed, as such an interaction would support the notion that MAGEA11 functions as an AR nuclear co-regulator in the endometrial stroma, similar to its function in prostate cancer cells. Fertile and PCOS primary stromal cells were grown to confluence, and total protein extracted. Protein blot analysis revealed a major 50-kDa MAGEA11 isoform, corresponding to the canonical isoform one of the protein, which was expressed in both cell types Fig. . The banAACR) using the MEME software [p = 3.85E− 05)  and Sp1 (data not shown). JUN proteins are components of the AP-1 transcription activator family that have previously been shown to interact with, and enhance AR activity in prostate and prostate cancer cells [AR expression in PCOS stromal cells is likely to drive transcriptional re-programming and, as such, has a direct role in the delay of endometrial cell differentiation Figs.  and 3. Wsoftware  and demo05) Fig. . In addier cells .Table 2TGene ontology analysis revealed that AR targets were predominantly involved in processes related to the positive regulation of cell death, including apoptosis  with AR targets from a prostate-specific data set (1371 genes)  revealedFinally, as PCOS patients have an increased risk of developing endometrial cancer (EC), we considered whether any AR targets were implicated in this disease. Comparison of our data with a list of EC-related genes obtained from PubMed databases revealed a set of known oncogenic targets  and medroxyprogesterone acetate to induce decidualisation, as well as siRNA to knock down KLF9 expression, both KLF13 and BMP2 mRNA levels increase. Conversely, in the same decidualised hESC model, siRNA to knock down KLF13 results in the increase of KLF9 expression and reduction of BMP2. This KLF-BMP appears important as the endometrial stromal cells of bmp2-deleted mice are infertile [AR chromatin immunoprecipitation experiments using PCOS stromal cells stimulated with DHT identified the direct targets of the nuclear hormone receptor. Krüppel-like factor (KLF)-9 and 13 transcription factors (KLF9/KLF13) were both major AR targets as determined by the depth of read. KLF9 is expressed in endometrial stromal cells but controls the response of adjacent luminal epithelial cells to progesterone . In murinfertile . Disruptnfertile . KLF9/13Interestingly, as well as binding to AR response elements, the nuclear receptor is also recruited to JUN response element-containing regulatory regions, presumably by a JUN response element-binding protein such as c-Jun, which interacts directly with AR in prostate cancer cells and enhances the trans-activation function of AR , 26.Gene ontology analysis of AR chromatin immunoprecipitation data revealed that AR interacts with genes involved in the regulation of cell death and apoptosis including EGFR and WT1, the expression of which is reduced in PCOS , suggestTogether, these data support the notion that elevated androgens result in delayed decidualisation and implicate AR and its co-regulator MAGEA11 in incorrect endometrial differentiation, the critical timing process of embryo implantation, and thus human reproduction.ESM 1(DOCX 4642 kb)ESM 2(XLSX 819 kb)ESM 3(XLSX 11 kb)