The observed sex disparity in bladder cancer (BlCa) argues that androgen receptor (AR) signaling has a role in these malignancies. BlCas express full-length AR (FL-AR), constitutively active AR splice variants, including AR-v19, or both, and their depletion limits BlCa viability. However, the mechanistic basis of AR-dependence is unknown. Here, we depleted FL-AR, AR-v19, or all AR forms (T-AR), and performed RNA-seq studies to uncover that different AR forms govern distinct but partially overlapping transcriptional programs. Overlapping alterations include a decrease in mTOR and an increase of hypoxia regulated transcripts accompanied by a decline in oxygen consumption rate (OCR). Queries of BlCa databases revealed a significant negative correlation between AR expression and multiple hypoxia-associated transcripts arguing that this regulatory mechanism is a feature of high-grade malignancies. Our analysis of a 1600-compound library identified niclosamide as a strong ATPase inhibitor that reduces OCR in BlCa cells, decreased cell viability and induced apoptosis in a dose and time dependent manner. These results suggest that BlCa cells hijack AR signaling to enhance metabolic activity, promoting cell proliferation and survival; hence targeting this AR downstream vulnerability presents an attractive strategy to limit BlCa. BlCa is classified as non-muscle invasive or muscle invasive and may be of high or low grade. Muscle invasive BlCa (MIBC) is most problematic and ultimately leads to fatalities2. BlCa displays a well-documented gender disparity where men are 3–4 times more likely to develop disease than women3. Numerous studies reported that the AR has a significant role underlying this disparity6. Immunohistochemical (IHC) analysis of BlCa tissue from males and females found that AR expression is higher in males7. Animal models confirmed that androgens contribute to bladder carcinogenesis. AR conditional expression in mouse urothelium and supplementation of female rats with testosterone promoted N-butyl-N-(4-hydroxybutyl) nitrosamine-induced tumor incidence while urothelium targeted AR knockout reduced tumor incidence9. Epidemiological studies found that men with BlCa treated with 5α-reductase inhibitors, which block conversion of testosterone to the more potent dihydrotestosterone, exhibited a lower risk of BlCa related death10, further supporting a role for AR in BlCa.BlCa in the US accounted for over 83,000 new cases of cancer and ~ 17,200 deaths in 202111. The AR normally exerts a regulatory role in multiple tissues including muscle, bone, brain, and prostate, but in malignancies this role can be perverted to benefit tumor cell proliferation and survival12. AR mediated androgen signaling is pivotal in prostatic malignancies, hence much of our knowledge on the mechanistic basis of AR signaling is derived from prostate cancer (PCa) studies. In PCa, the strong reliance of cells on AR signaling has been exploited in the development of therapeutics, which target the LBD to limit AR signaling, restricting tumor growth. Initially, these cancers respond to therapy, but eventually castration resistant malignancies emerge driven by multiple mechanisms, including aberrant splicing leading to expression of constitutively active low molecular weight (LMW) AR isoforms13 refractory to LBD targeting therapeutics.AR is a member of the 48-member steroid receptor superfamily, potent signaling molecules governing multiple cellular processes. All members share a basic structure consisting of activation and DNA binding regions linked to a ligand binding domain (LBD), conferring ligand-dependent regulation of transcription14. Importantly, siRNA mediated depletion of total-AR (T-AR) reduces viability and induces apoptosis in multiple BlCa cellular contexts14, confirming a dependence on AR signaling. Hence, limiting AR signaling is an attractive therapeutic strategy. However, the presence of constitutively active splice variants limits the efficacy of AR targeting drugs14. Given the accumulating evidence that the AR has a key role in BlCa tumorigenesis, but would be refractory to current therapies, defining AR downstream targets may identify actionable vulnerabilities.We recently reported that while FL-AR is in some BlCa, most BlCa cells also express LMW splice variants. We identified a novel variant, AR-v19, which localizes to the nucleus, is expressed in multiple BlCa cell lines and tumors and transactivates transcription from an AR-regulated promoter in a dose dependent mannerStudies herein describe the transcriptomes regulated by depletion of T-AR, depletion of FL-AR or AR-v19, and cells overexpressing the AR-v19 splice variant. Key findings show that loss of all AR signaling alters oxidative phosphorylation components resulting in an elevation of HIF1A and its target genes, and a deregulation of pro and anti-apoptotic molecules. mTOR expression is repressed following depletion of all AR isoforms. Accordingly, depletion of AR alters mTOR downstream signaling and pharmacological targeting of mTOR effectively limits cell viability. Perusal of large high-grade BlCa datasets revealed that multiple HIF1A pathway components negatively correlate with AR, arguing that increased AR expression represses hypoxic responses. This aligns with our results that reduced AR signaling decreases OCR. Uncoupling oxidative phosphorylation with the antihelminth, niclosamide, was highly effective in reducing BlCa cell viability. Overall, these studies point to AR augmentation of metabolic processes as a targetable driver in AR-dependent BlCa.15, and AR-v19 specific siRNAs designed by us A14. Niclosamide and sapanisertib were obtained from Selleck Chemicals  and resuspended in DMSO.UM-UC-3, TCCSUP, J82, T24, and RT4 were obtained from ATCC . All cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), mmol/liter glutamine and 100 units/ml penicillin, and 100 ug/ml streptomycin  at 37 °C and 5% CO2. Cells used in studies were under passage 20 from the time they were received from ATCC. siRNA transfections were carried out using Lipofectamine RNAiMAX (Invitrogen) or PolyPlus (at an oligonucleotide concentration of 50 nM unless otherwise specified) maintained in the above media, and harvested 72 h post transfection, unless stated otherwise. The following oligonucleotides from Dharmacon (Thermo Scientific) were used for transfection; siAR-smart pool (L003400-00), control non-targeting oligonucleotide (D-001210–01), LBD targeting (J-003400–06)14. qRT-PCR was performed using the primers listed in Supplementary Fig. 1. Briefly, RNA was extracted using the RNeasy kit  and cDNA was generated using QuantiTect  reverse transcription based on the manufacturer’s protocol. qRT PCR was performed using primers from Invitrogen. Standard curves were generated for each primer set using serial dilutions of cDNA to standardize variations in PCR reactions. In addition, after each run, melting curves were used to verify the melting temperature of the amplicon. All qPCR reactions were performed in triplicates. HPRT levels served as a quantification control. cDNAs were in ddH2O and added to QuantiTech SYBR Green PCR master mix (Qiagen) and 200 nM of each primer. PCR conditions had initial denaturation step at 95 C for 2 min 30 s, 40 cycles at 95 C for 13 s. Data were collected by the Mastercycler ep Realplex .RNA and cDNA were prepared, and qPCR studies were conducted as previously describedCells were placed in a 4 °C RIPA lysis buffer containing a protease inhibitor mixture (Sigma). Thirty to fifty micrograms of protein were separated on 8%, 10%, or 12% SDS-PAGE gels, transferred to 0.45 µm nitrocellulose membranes (Thermo Scientific), and blocked with 5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween 20 (PBS-T) or OneBlock Western-CL blocking buffer . If membranes were probed for more than one protein, then the membranes were cut prior to incubation with specific antibodies. Membranes were incubated with primary antibody overnight at 4C. The following antibodies were used: AR (N20), actin, GAPDH, cyclin A (239), E2F1 (251), and Cyclin D3 (182) , AR A303  or A303 , cleaved PARP, phospho-AKT (4060), phospho-p70S6K (9234), total AKT (4685), total p70S6K (9202), total mTOR (2972), cleaved PARP, and cyclin E (4129) . Tubulin, pAKT, and S70 , The following day, membranes were washed with PBS-T 3 times, incubated with secondary antibody conjugated to HRP, and development was carried out using SuperSignal West Femto chemoluminescence (Thermo Scientific) or were imaged using LI-COR near-infrared western blot detection. Gel loading was assessed either by GAPDH or tubulin. Uncropped images are in Supplementary figures.Cells were plated at 10,000–20,000 cells/well in a 12 well plate and 24 h later treated with niclosamide, sapanisertib (Selleckchem), or DMSO control for 2 or 4 days. Viability was assessed using Cell Counting Kit – 8 (CCK-8) following manufacturer’s recommendations . For experiments assessing the growth of cells transfected with total AR siRNA, cells were plated in a 24 well plate at 10,000–20,000 cells/well and transfected 24 h later with a control oligonucleotide or AR targeting siRNA. Proliferation was assessed 72 h later using CCK-8 assay. All experiments were performed in triplicate. Data is displayed as the mean +/− standard deviation.Cells were plated in 6-well plates at 15,000 cells/well. The following day, cells were transfected with control or AR specific siRNA and were propagated for 10–12 days post transfection. Colonies were fixed and stained with crystal violet (0.5%) in 25% methanol/water, dried, and photographed. Image J was used to quantify the images.17 for mapping/alignment of raw sequence reads (FASTQ format) to the reference human genome assembly (GRCh37/hg19), transcript assembly, and quantitation of gene and transcript expression as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Data were annotated for unique genes/transcripts with Ensembl Release 82 (GRCh37/hg19). Group comparisons were conducted with Cuffdiff, and genes meeting a statistical threshold of an adjusted P-value < 0.05 were assigned as differentially expressed. Log2(fold change) and adjusted P-values were used as input for volcano plots, and normalized FPKM values utilized for hierarchical clustering and heatmap visualization. The ToppGene Suite18 and EnrichR19 platforms defined enrichment for molecular functions, biological processes, cellular components, and pathway alterations.RNA was isolated and submitted to Novogene. RNA purity and concentration were measured using the NanoPhotometer Pearl (Implen). Following ribo-depletion, RNA-Seq libraries were prepared using a protocol to capture both mRNA and lncRNA transcripts and sequenced on an Illumina NovaSeq 6000 System . Data analysis was performed with a TopHat2-Cufflinks-Cuffdiff pipelineAnalyses were performed using an Agilent Seahorse XFe24 analyzer (Agilent Technologies). Cells were plated onto the Seahorse plate  at 90,000 cells/well and cultured in 250 μL RPMI media (10% FBS and 1% Penicillin Streptomycin). Seahorse XF24 Sensor Cartridge was hydrated overnight using Seahorse XF Calibrant Solution (Seahorse XFe24 Flux Pak). Cells were placed in Seahorse XF RPMI assay medium supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Seahorse XF Cell Mito Stress Test Kit  was performed according to the manufacturer’s protocol, using the final well concentrations of 1.5 μM Oligomycin, 2 μM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and 0.5 μM Rotenone/Antimycin A. On completion cells were counted using an Automatic cell counter  for data normalization. Data analysis used the Seahorse Wave (RRID:SCR_014526) Desktop software and was exported using the Seahorse XF Cell Mito Stress Test Report Generator. a two tailed two sample equal variance student’s t-test was used to assess differences between samples. For RT-qPCR experiments, the delta-delta Ct method was used to calculate fold change in gene expression as described before (https://www.sciencedirect.com/science/article/pii/S1046202301912629). A p < 0.05 was considered as statistically significant. Statistical analyses were preformed using GraphPad Prism 8.For all assays,20, publicly available on cBioportal21 show that AR alterations are evident in 10–11% of bladder malignancies. AR gene alterations are present in 3% of malignancies, the most common being deletion or missense mutations , TCGA PanCancer Atlas and MSK TCGA 2020UM-UC-3 which express FL-AR and the AR-v19 splice variant and show a strong response to total AR depletion . Pathways in cancer includes cell cycle components, mTOR and transcription factors. MAPK pathway includes the CREB1 transcription factor and AP1 transcription factor family . Changes in the apoptotic network includes an increase in pro-apoptotic proteins  and a decrease in anti-apoptotic BCL2A1, shifting the homeostasis towards apoptosis. The apoptotic pathway is linked to mitochondrial proteins, which are instrumental in oxidative phosphorylation. Notably, multiple HIF1α pathway components are upregulated, indicative of hypoxia.22. Altered deleted in lymphocytic leukemia 2 (DLEU2) expression has been detected in multiple malignancies23. Nuclear paraspeckle assembly transcript 1 (NEAT1), scaffold RNA and component of paraspeckles, is elevated. Previous studies reported that in BlCa cells reducing NEAT1 expression augments a reduction in cell proliferation and increases apoptosis on cisplatin treatment24, therefore the importance of NEAT in BlCa is unclear.Long non-coding RNAs (lncRNA) are altered on T-AR depletion where 10 are repressed and 14 are elevated (Supplementary Dataset File). Analysis of the lncRNA targets did not identify significantly altered molecular functions, biological processes, cellular components, or pathways, but this cohort includes several lncRNA previously studied in the cancer context. The repressed small nucleolar host gene 14 (SNHG14), has been reported to promote BlCa cell proliferation and silencing this lncRNA restrains cell viability25. On FL-AR , transcripts inversely regulated, upregulated following AR-v19 depletion, downregulated on AR-v19 overexpression  or downregulated on AR-v19 depletion and upregulated on AR-v19 overexpression  and transcripts commonly altered in both datasets (grey) Fig. F. A heatFifty-five transcripts are commonly regulated on depletion of T-AR, FL-AR and AR-v19 Fig. A. The mo26. Immunoblot analysis confirmed that mTOR protein is reduced on AR depletion , caspase 9 (CASP9), syndecn 1 (SDC1), ST3 beta-galactoside alpha-2,3 sialyltransferase 1 (ST3GAL1), catenin Beta 1 (CTNNB1), and (FOXO3) . Statistically significant decreases in disease free survival are evident in individuals where malignancies have elevated expression of CREB1, KDM7A, and forkhead box O-3 (FOXO3), and a decrease in overall survival with higher expression of TNF receptor associated factor 4 (TRAF4) and CASP9, suggesting that these genes may have a role in BlCa tumorigenesis .Interrogation of cBioportal and GEPIA228. The anti-helminth, niclosamide, previously described as a mitochondrial uncoupler in tapeworms and in human cells31 exhibits very potent ATPase inhibitory activity , adrenomedulli (ADM), lactate dehydrogenase A (LDHA), aldolase, fructose-bisphosphate A (ALDOA), and N-myc downstream regulated 1 (NDRG1) Fig. A. The T-ity Fig. C. In addity Fig. D.Figure UM-UC-3 cells treated with niclosamide show a dose and time-dependent decline in cell viability Fig. E. Simila36. An abrupt cessation of all AR signaling results in the downregulation of transcripts associated with proliferative biosynthetic processes and coordinately, an elevation of transcripts encoding proteins that negatively regulate these processes, pro-apoptotic proteins, and markers of hypoxia arguing that AR is instrumental in promoting metabolic activities.The current study defined BlCa AR-driven transcriptional output to identify exploitable downstream vulnerabilities. We found that T-AR and AR isoforms regulate distinct cohorts of genes, but with an overlapping transcript population, similarly to what has been reported for prostatic malignancies38 and proliferation is unaltered in steroid depleted media (data not shown), arguing that either very low androgen levels are sufficient for growth, that the cell synthesize sufficient testosterone, or that the AR, but not androgens are essential for cell growth. However, siRNA-mediated depletion of FL-AR retarded cell viability14, suggesting that this isoform is active. FL-AR activity is further substantiated by the current study where depletion of the FL-AR elevates expression of multiple histone transcripts. The consequences of an abrupt elevation of multiple histones are not well studied, but in yeast abnormal histone accumulation interferes with chromosome segregation and promotes whole genome duplication39, while overexpression of replication-dependent histone genes confers malignancy to dedifferentiated liposarcoma40. Restraining FL-AR in the BlCa context may have similar consequences, but this needs to be further explored. The large transcriptome changes following AR-v19 depletion argue that this specific splice variant has a major role in gene expression. Surprisingly, depletion of AR-v19 altered expression of far more transcripts than depletion of T-AR or FL-AR. The reason for this is unclear, but an abrupt depletion of all AR forms in cells that are addicted to this signaling pathway may precipitate an acute stress response that culminates in a rapid induction of programmed cell death. Depletion of a single isoform, while deleterious, may be compensated by the remaining AR, thus slowing the decline in cell viability. Comparison of transcription alterations on AR-v19 k/d and overexpression reiterated that depletion and overexpression of a transcription factor does not drive gene expression in an inverse manner.Our previous studies found that treatment with enzalutamide has a small effect on UM-UC-3 viability suggesting that FL-AR is active in these cells. Notably, the cells are culture in 10% FBS (0.1 nM testosterone)41. ATXN1 regulates epithelial mesenchymal transition (EMT) in cervical carcinoma cells where its depletion promotes migration and invasion42. ARL4C, a target of Wnt/β-catenin and Ras signaling, positively correlates with EMT on AR depletion in PCa43. H6PD is elevated in prostatic malignancies treated with AR-limiting enzalutamide44, and SRD5A3 is elevated in PCa when AR signaling is restrained44. SRD5A3 converts testosterone to the more potent dihydrotestosterone45 thus SRD5A3 upregulation on AR depletion suggests that a decline of AR signaling triggers a compensatory increase in SRD5A3 to restore signaling. While certain transcripts are similarly regulated in BlCa and PCa, there are notable differences. Myc, highly AR-regulated in PCa12, is not AR regulated in BlCa. KLK2 and 3 are not expressed while TMPRSS2 is expressed at very low levels and not AR regulated, hence some well described AR targets in PCa, are not AR-regulated in BlCa.There is a modest overlap in transcripts commonly regulated on depletion of T-AR, FL-AR and AR-v19. Several elevated transcripts have previously been associated with malignancies. WDTC1 has oncogenic properties in colorectal cancer46. RNA from identically treated cells was used in expression microarray studies leading to the identification of 96 direct AR target genes. A comparison of the identified direct AR target genes with transcripts altered by depletion of T-AR, FL-AR or AR-v19 found that of the 51 of the 96 were regulated by on T-AR, FL-AR or AV-v19 depletion. This included FKBP5 and CD44 . However, most of the identified transcripts were not inversely regulated on AR depletion vs R1881-dependent AR activation, suggesting the two cell lines while similar has distinct features or additional factors have a substantial role in regulating AR-dependent gene expression.A previous study used a different approach where a UM-UC-3 subclone with highly elevated AR expression when compared to UM-UC-3 parent cells, was stimulated with 10 nmol/L R1881 and subjected to CHiP-seq studies to identify AR binding regions47 where it optimizes muscle protein levels by regulating the balance of mTOR and AMPK48. In PCa, elevated or deregulated mTOR signaling is a major cause of castration resistance, hence targeting this pathway is an attractive strategy49. Interaction between AR and mTOR includes direct binding of nuclear mTOR to AR to affect gene transcription50. In PCa cells limiting AR signaling promotes an increase of mTORC1 downstream target p70S6 phosphorylation, but not the mTORC2 target AKT. Moreover, C1 and C2 regulate each other, where depletion of the critical C1 protein raptor enhances C2 signaling, while depletion of the C2 protein rictor elevated C1 signaling51. In BlCa AR depletion also increases phospho p70S6 levels, therefore there are similarities between AR mTOR regulation between PCa and BlCa. However, thus far, clinical trials limiting mTOR activity have shown limited efficacy52, but studies using novel mTOR targeting agents or a combination of agents are ongoing and may identify formulations that can effectively limit BlCa tumorigenesis.mTOR is decreased on depletion of T-AR, FL-AR and AR-v19 and the AR/mTOR correlation expends to high grade malignancies, arguing that this linkage is a common feature of BlCa. The importance of AR in driving mTOR is documented in the context of muscle cells. In castrated rodents, testosterone supplementation improved muscle and bone strength by mTOR activation53, while in PCa cells mitochondrial AR serves to reduce mitochondrial function by regulating OXPHOS enzymatic activity54. In castration resistant PCa, MPC2 is a direct AR target and its inhibition restricted OCR55. Our analysis did not identify MPC2 as an AR regulated transcript, but AR inhibition has a similar effect on OCR, suggesting that oxidative phosphorylation may be regulated by alternative mechanisms in BlCa, but the overall outcome is similar.Increased expression of HIF1A network components on AR depletion was reinforced by analysis of high grade BlCa. Multiple transcripts indicative of hypoxia strongly negatively correlate with AR. An elevation of a hypoxic signature and reduction of OCR on T-AR depletion is indicative of compromised mitochondrial activity and suggested a targetable vulnerability. AR interplay with mitochondria is complex. AR can directly regulate mitochondrial gene expression, where in muscle C2C12 cells AR promotes mitochondrial gene transcription driving mitochondrial biogenesis57. Niclosamide was developed to target parasites and early studies reported its effect on uncoupling oxidative phosphorylation58. Subsequent studies described additional niclosamide targets and it is unclear if there are multiple niclosamide targets or if there is substantial crosstalk between affected pathways. Niclosamide affects mTOR activity and is a potent inhibitor of Wnt/β-catenin and STAT3 signaling60. In PCa, niclosamide inhibits AR splice variants to overcome enzalutamide resistance61, but we did not observe this effect in BlCa cells, suggesting that niclosamide’s effects on AR are cell context specific. However, niclosamide’s suitability is hindered by its poor solubility and brief plasma half-life62. Encouragingly, newer formulations are overcoming these obstacles, repurposing this drug as a cancer therapeutic that may be utilized for the treatment of high grade BlCa57.Several feature of the antihelminth niclosamide make it particularly attractive drug for mimicking the effect of AR depletion. (1) Niclosamide has a robust effect on ATPase and treatment of BlCa cells reduce OCR. (2) Niclosamide exhibits mTOR binding. (3) In preclinical and a limited number of clinical trials niclosamide or novel niclosamide formulations exhibit an acceptable toxicity profileIn conclusion, the BlCa studies herein identified T-AR and isoform dependent transcriptomes and uncovered molecular processes and pathways associated with the regulated transcriptomes. Cross referencing with AR regulated transcribes using the cBioportal database validated transcripts that are positively and negatively associated with AR expression. mTOR positively correlated with AR expression, mTOR signaling is deregulated on AR-depletion and pharmachological targeting of mTOR resulted in a significant decline in cell viability. Conversely transcripts that are indicative of hypoxia are negatively associated with AR expression, arguing for an AR effect on mitochondrial activity. Depletion of AR resulted in a sharp decline in OCR and targeting mitochondrial activity with the potent ATPase inhibitor niclosamide decreased OCR and reduced cell viability in a dose and time dependent manner. Taken together these studies found that in BlCa the AR drives metabolism and revealed that compromising mitochondrial activity is a targetable vulnerability.Supplementary Information.


Elite performance and sporting success are often the result of optimal integration and synergy of all components of sports preparedness , which evolve because of systematic long-term sports preparation. However, the relative importance of these characteristics varies between individual and team sports. While some individual sports require a high standard of bioenergetic and neuromuscular abilities and capacities, team sports performance is closely related to technical and tactical skills, which may compensate for weakness within the fitness level . NonetheSports may be categorized according to the degree of predictability of the environment that they are played in . Team spPerformance in team sports is affected by several factors that affect the organization of training and competitions. These include, for example, COVID-19 cases , PCR tesElite team sports usually face match-congested schedules  during the preseason and the competitive season where the teams can be involved in two or more competitions simultaneously  , 11. ForTeam performance may be considerably affected by player availability . Thus, dFrom the teamś perspective, managers could create squads with larger number of players to deal with (and complete) these ﬁxtures distribution. Moreover, understanding player pacing and its relationship to the introduction of appropriate player turnover can be a valuable tool for coaches and performance staff in developing tactical strategies during match-congested schedules and making better decisions to improve team performance . Some reThe role of the International Olympic Committee (IOC), International Federations and National Governing Bodies in responding this challenging issue (preventing fatigue and maintain performance during match-congested schedules) in sport is a key factor. For instance, the recommendation of the International Olympic Committee consensus, which indicated that soccer matches should be interspersed by at least 96 h , 24, hasAn injury is probably the most important factor that would interfere with the readiness of an athlete to participate in competition. The cause of an injury is multifactorial and depends on intrinsic and extrinsic factors. For instance, in the NBA context, greater load and fatigue, more years of NBA experience and shorter height are associated with a higher injury risk . In addiFrequently, coaches complain in the media about how the increased injury rates are affecting the competition outcome in team sport . For exaContemporary sudden stops throughout the season, such as individual COVID-19 cases and restrictions imposed by the governments to avoid the spread of COVID-19 , may genThe relevance of decision making to practice is something that is likely to resonate with sport coaches. This is evident in, for example, team selection, managing competition performance, devising strategy, managing the delivery of interventions, planning, responding to crises, providing appropriate feedback, and interacting with athletes . HoweverThe emerging collective properties of teams cannot be assigned to any single player . HoweverAdditionally, team sport coaches need to identify fatigued players that underperform in a match as early as possible to substitute or adapt their playing style. However, the specific rules, regulations and schedules of each team sport may also influence the coaches' decisions. For instance, a handball player performing an offensive tactical variation would affect the winning or losing status , but thiIt is also important to consider that elite sport team athletes are frequently required to embark on long-haul transmeridian travel for competition purposes  exposing them to travel fatigue and jet lag. Travel fatigue can accumulate over time , 56, and1)Planning constraints during trainingThe levels of fatigue, the emotional state, congested schedules, or the opponent's behavior are only a few examples of constraints which demand continuous adjustments of training plans . Therefo–Integrating the following perspectives : player,–Identify training methodologies that help player achieve the hypothetical  optimal load, for instance reducing the physical load from both a physiological and neuromuscular aspect possibly positively impacting the cognitive  match preparation .–Identify windows of opportunities to train. Practices, although greater in frequency, should be closely monitored for total volume and should include tactical and strategical concepts, skill-based activities and minimal conditioning sessions.–Seizing the opportunity of the transition period to improve different aspects of performance.–Including a variety of challenging constraints and training variability, which improve psycho-emotional factors , the health status of performers  and redu–Consider the plausible effects of individual training strategies at private facilities, without their team's coaching staff .2)Workload MonitoringA primary goal of workload monitoring should be to assist and inform player/coach/manager decision making on player unavailability for training . In this–Creating, implementing, and establishing a personalized system of fitness, workload and well-being monitoring on a daily basis .–Complementing quantitative methods  with qualitative assessments .–Simplifying the information when reporting, limiting it to a few key and complementary metrics .–Reporting the key metrics in an easy and understandable manner using appropriate data visualization tools.–Establishing possible benchmarks for athletes based on the sport-specific context .3)Recovery StrategiesTeams should dedicate and concentrate their efforts to improve the player's recovery-stress balance . Enhanci–Prescribing the optimal load during training sessions could be the first recovery strategy .–Taking into consideration how physiological stress and physiological and biochemical markers are affected when the same players start in sequential matches and how they differ from those who remain on the bench , 68.–Using individualized protocols based on the players' characteristics such as actual fitness level, injury history and preferences , 69.–Educating players to take responsibility for their recovery.–Providing players with recommendations and tools  for their recovery management , 70, 71.–Consider that some recovery strategies preferred by players may improve their happiness while other recovery strategies may be needed to improve their wellness . Thus, i4)Squad RotationSquad rotation is also a key issue in coping with the high demands of contemporary training and match-play. From a practical perspective, the following suggestions should be considered:–Having a clear criterion to clarify when to propose player rotations (probably before and not during the matches).–Using players from secondary teams for potentially winnable matches.5)Travel-Related FatigueThere is no research-based evidence to manage travel fatigue in athletes, and low-quality evidence exists for effective interventions to recover from jet lag in athlete populations . Therefo–Traveling with private charter flights when possible.–Avoiding making player's travel who are not going to compete.–Minimizing international travel for injured players who are only going to be evaluated by national medical staff.6)Return-to-SportReturn-to-sport decision-making is a complex process and is often characterized by uncertainties such as re-injury risk, time pressure induced by competition schedule and social stress from coaches, families and supporters . An inad–Creating, implementing, and controlling personalized preventive and corrective training programs to protect and improve locomotor health of players .–Improving the prevention process and communication among players, coaches and medical staff, because there is often little agreement between players and coaches regarding return-to-play decisions .7)CommunicationThe quality of communication within head coaches/managers, strength and conditioning coaches, sport scientists, and medical team members  should also be improved to increase player availability. Some practical recommendations are:–Ensuring well-developed communication within an interdisciplinary team of experts with clearly defined rules, roles, and responsibilities.–Establishing well-developed communication with the players who are the main protagonists of success and who should be provided with appropriate professional and scientific support.–Adopting an effective coach leadership, communicating frequently with individuals of all staff disciplines . This maAccording to the above-mentioned concerns, player availability seems to affects performance in elite team sports. Therefore, the following practical suggestions and recommendations should be considered during the systematic long-term sports preparation with the aim to increase player availability:Player availability, defined as keeping players injury-free and ready to participate in competition, is extremely important in the current elite team sport scenarios, because it is related to team performance. In fact, individual player availability may be more important than each player being at their individual level of peak performance. However, this topic needs to be explored more in future research. Moreover, it is imperative to highlight the prevailing need to preserve the health and wellbeing of professional players when facing a high frequency of extremely demanding matches. Thus, efforts of head coaches/managers, strength and conditioning coaches, sport scientists, and medical team members  should really focus on strategies for optimization of player availability while minimizing factors like fatigue.


Migration and marriage are major life events that might interact and be jointly decided. Places with good labor market opportunities may or may not provide good marriage options. In this paper, I quantify gains and losses in marriage prospects for unmarried migrants and natives during the population redistribution driven by internal migration. I also examine how the experiences differ by individual characteristics and regional factors. The analysis measures marriage prospects using the availability ratio (AR) with adaptive assortative matching norms for every unmarried individual from sample data of the 2010 China population census. The AR quantifies the intensity of competition for suitable partners in the local marriage market. I compare (1) migrants’ current AR with an alternative AR if the migrant returned to the hometown and (2) natives’ AR with a hypothetical AR if all migrants returned to their hometown. The first comparison shows that among migrants moving for labor market opportunities, most women have higher ARs (better marriage prospects) in the place of residence than in their hometown, especially those of rural origin. In contrast, migrant men’s ARs mostly decrease after migration except for the best educated. The second comparison reveals small negative externalities of internal migration on ARs for native women but positive impacts for some native men. The results suggest a conflict between labor market opportunities that dominate internal migration decisions and marriage market opportunities in China. This study demonstrates a method to quantify and compare marriage prospects and extends the literature on how migration and marriage interact.The online version contains supplementary material available at 10.1007/s10680-023-09658-3. People migrate for a degree, a job, a significant other, or in general a better future. Sometimes these ends point toward one direction but sometimes not. When they are not, people face trade-offs. One dilemma that is common for young adults may be between labor market opportunities and marriage market options  with adaptive local assortative matching norms regarding age, education, and residency status. The AR essentially quantifies the intensity of competition for suitable partners, and a higher AR means a more favorable situation  were women. Among these migrant women, 68% migrated for work, 10.4% for education or vocational training, and 15.2% for marriage or with family. The respective percentages among migrant men were 83.4%, 9.4%, and 3.2%, showing that more men migrated for economic opportunities and more women migrated for family ties NBS, . The patMarriage is nearly universal in China . The long-form survey collected information about migration history and marital status, in addition to the basic demographic characteristics collected in the short form, including sex, age, education level, and household registration residency status (hukou type). The data contain information about both residency at the census time and the household registration address, so they can be used to study marriage prospects for migrants in the current place of residence and their hometown.The analytical sample consists of 121,179 young adults aged 20–34 who were unmarried at the census time, including the never married (97.6%), divorced, and widowed. Restricting the sample to the never married makes little difference. The sample is representative of the population aged 20–34 in mainland China except in Xinjiang, Tibet, and Qinghai provinces, from which the observations are excluded due to very low population density (< 15 persons per square kilometer). In the analytical sample, 21.9% are migrants who moved across prefectures and had been away from their hukou address for over 6 months. Among them, 95.8% of men and 94.5% of women moved for reasons related to labor market opportunities, including for work, business, education, and vocational training. Others moved with family members, for marriage, or for other reasons. The percentage of migrants moving for labor market related reasons among the population aged 20–34 is much higher than that in the total population of migrants. Table The calculation uses information from the same data about unmarried persons aged 14–41 who could be potential partners for those in the analytical sample. It also uses information about marriages formed after 2005 and lasted till the census time in 2010 as recorded in the census data (referred to as “the marriage sample”) to infer prevailing local assortative matching patterns and calculate weights. Same-sex marriage is not recognized in China, so this analysis focuses exclusively on opposite-sex marriage.This study defines the local marriage market at the prefecture level. A prefecture in China, formally known as a prefectural-level division, is the second level of the administrative structure, ranking below a province and above a county. There are 333 prefectures in mainland China. According to the population census in 2010, the smallest 10% of the prefectures had on average 480 thousand people in each prefecture and the largest 10% of the prefectures had on average 10 million. Within a prefecture, social and economic connections construct a reasonably well-connected social network. Two individuals living in the same prefecture have a realistic chance to meet each other via daily contacts, work connections, family relationships, and friendship circles, which are the common ways of meeting a partner. Online-dating websites use the prefecture-level location in their matching algorithms so that matched users may develop their relationships offline. Nonetheless, defining the geographic scale of the local marriage market in a world with increasing connectivity is conceptually difficult and always requires some degree of subjectivity. The choice is also constrained by the granularity of the data in an empirical study. The other two options, namely province and county, are too large or too small, respectively, to define a local marriage market.The analysis measures local assortative matching patterns at the province level, a level above the prefecture, using marriages formed after 2005 and lasted till the census time in 2010. Allowing assortative matching patterns to vary by region alleviates the endogeneity problem that matching patterns adapt to local marriage market opportunities. The decision to use the province instead of the prefecture as the unit is a compromise between using information about marriages from recent years and proximate geographical areas. Some prefectures do not have enough observations of recent marriages to infer robust matching patterns and variations in matching patterns across prefectures within a province tend to be small. For each of the 30 provinces, I calculate the empirical distribution of the age gap and the education gap between the husband and the wife. The distributions show similar curves with moderate variations across provinces (see Online Resource 1).The analysis measures individual marriage prospects using an extended version of the Availability Ratio (AR) developed by Goldman et al. , which qThe weighted AR has some special features. First, matching is reciprocal, meaning that if a man is defined as a match for a woman, then the woman is also a match for the man. Second, the AR incorporates competition from other groups of the same sex, approximating various assortative matching preferences. For example, assuming that men prefer a woman of the same or younger age, then the AR for a 27-year-old woman has a 27-year-old man as one of the matches in the numerator, and the number of women aged 25 as part of its denominator because these women can be competitors, i.e., matches for the 27-year-old man. Therefore, the AR for a woman of certain traits would not be the reciprocal of the AR for a man of the same traits. Third, the AR uses weights to reflect the degree of preference for a certain match, allowing for the fact that a potential match with less favorable traits adds less to one’s marriage prospects. The weights are derived from the empirical distribution of local matching patterns as described in Sect. The AR is a more sophisticated measure of marriage prospects than population sex ratios .Second, two persons need to reside in the same prefecture and have the same hukou type to become a matched pair. The hukou restriction depicts the urban–rural boundary in the marriage market  accounts for over 3% of the couples, and in total 87.8% have an age gap in this range, with a mean of 1.97. Therefore, I define a match to have an age gap between – 2 and 6 years and use the percentage of marriages with the age gap among marriages with an age gap between – 2 to 6 as the weight (see Online Resource 1). Those outside the range are counted as zero matches.Fourth, a matched pair should have equal or neighboring education levels, assuming educational assortative matching. Education is classified into four levels: illiterate and primary school, junior high school, senior high school, and college and above. According to the marriage sample, over 95% of couples have equal education or a one-level gap in their education. Therefore, I define a pair with an education gap of no more than one as a matched pair. The weight is the scaled percentage of marriages between the person’s education and the potential match’s education among all marriages involving the person’s education (see Online Resource 1).Let For non-matched pairs, i.e., For matched pairs, The matching rule is represented by province-specific weights: let The weighted count of suitable women for The AR for and the AR for The final weight of a specific match is the product of the weights of age and education. It is assumed that the two weights are independent because the marriage sample size in each province is not large enough to infer a joint distribution. The scale of the weight does not affect the interpretation of AR as the weight appears on both the numerator and the denominator of the ratio. The calculation of the AR is performed in the following steps:For each person, I calculate an AR based on their current prefecture of residence and a hypothetical AR as the potential outcome for comparison. The hypothetical scenario differs by migration status.For migrants whose prefecture of residence is different from the prefecture of their household registration address , I assign the person to the hometown prefecture to calculate an alternative AR, keeping the person’s other traits and everyone else’s residence constant. I use weights that reflect the matching patterns in the hometown province for the hypothetical AR. This AR for migrants thus measures the marriage prospects the migrant would have if he/she returned to the hometown. Comparing this hypothetical AR and the migrant’s current AR after migration reveals changes in marriage prospects for migrants due to migration.all migrants to their hometown prefecture and keep everything else constant in the calculation. Differences between the real AR and this hypothetical AR for natives thus reflect the impact of internal migration on their marriage prospects. This hypothetical AR also applies to migrants, but its interpretation is different from the previous one, in which only the migrant of interest moves back to the hometown. This strategy is similar in concept to the hypothetical “migration-adjusted” AR developed in Eckhard and Stauder  migrant women mostly improve their marriage prospects via migration, and those of rural origin gain more, (2) migrant men with a college degree are likely to have better marriage prospects after migration, whereas those without the degree lose, and (3) natives’ experiences are different from their migrant peers of the same sex and traits.The findings reveal a potential conflict between labor market opportunities and marriage prospects in China. For migrants, migrant men substitute for native men in the labor market but might not as bridegrooms due to female status hypergamy Stark, . This isInternal migration also affects the marriage prospects of natives. As Hypothesis In addition to clear contrasts by sex and migration status, there are also differences by education in terms of gains and losses from internal migration. For men, the best-educated men gain the most among migrants. This is because they are in short supply in the marriage market featured by female status hypergamy and therefore could strategically maximize their marriage prospects. On the other side, the least-educated men have limited options and they hardly improve their marriage prospects through moving to other places. For women, the less educated have more favorable ARs and also tend to gain more as migrants and lose less as natives than the better educated.The conditional average ARs reveal some structural problems in the marriage market. First, the fact that on average women mostly have higher ARs than their men counterpart reflects a national shortage of marriageable women. The national shortage originates from decades of skewed sex ratios at birth and finds no solution in internal migration Below is the link to the electronic supplementary material.


Background and Objectives: Patients who survive severe COVID-19 require significant pulmonary rehabilitation. Heart rate (HR) has been used as a safety variable in the evaluation of the results of interventions in patients undergoing pulmonary rehabilitation. The aim of this research was to analyse HR during a pulmonary rehabilitation program in post-severe COVID-19 patients who survived mechanical ventilation (MV). The study includes the initial and final evaluations and aerobic training sessions. Materials and Methods: Twenty patients  were trained for 8 weeks. A 6-minute walk test (6 MWT) was performed and, subsequently, a supervised and individualised training plan was created. Resting heart rate (RHR), heart rate recovery (HRR), heart rate at minute 6 (HR6 min) and the product of HR6 min and systolic blood pressure (HR6 minxSBP) were measured at 6 MWT. In addition, HR was measured at each training session. Results: After 8 weeks of pulmonary rehabilitation, patients decreased their RHR from 81.95 ± 9.36 to 73.60 ± 9.82 beats/min (p < 0.001) and significantly increased their HRR from 12.45 ± 10.22 to 20.55 ± 7.33 beats/min (p = 0.005). HR6 min presented a significant relationship with walking speed and walked distance after the pulmonary rehabilitation period . HR6 minxSBP presented a significant relationship with walking speed and walked distance after training . In the pulmonary rehabilitation sessions, a significant decrease in HR was observed at minutes 1, 6 and 15 (p < 0.05) between sessions 1 and 6 and at minute 1 between sessions 1 and 12. Conclusions: Eight weeks of individualised and supervised pulmonary rehabilitation were effective in improving RHR and HRR in COVID-19 patients surviving MV. HR is an easily accessible indicator that could help to monitor the evaluation and development of a pulmonary rehabilitation program in COVID-19 patients who survived MV. COVID-19 is a disease caused by the SARS-CoV-2 virus, belonging to the coronavirus family . The proDue to severe respiratory symptoms and, in some cases, acute respiratory distress, patients with COVID-19 may require prolonged mechanical ventilation (MV). In addition to MV being an invasive procedure, there are cases where its disconnection can take time. The consequences of prolonged connection to MV generate the need for pulmonary rehabilitation during and after hospitalization . These MSome symptoms of COVID-19 can last beyond the period of acute infection, with exercise intolerance standing out as the most frequent finding ,8,10. ThBaseline fitness assessment through the 6-minute walk test (6 MWT) and aerobic training are part of most pulmonary rehabilitation programs ,11. ThusxSBP) were measured in this test. Ventilatory capacity through spirometry was also performed before and after 8 weeks of pulmonary rehabilitation. In addition, an incremental and continuous test was carried out to design the pulmonary rehabilitation sessions. HR was also measured at each training session.This was a prospective study, where the sample was for convenience and the recruitment of participants to the pulmonary rehabilitation program was in accordance with bronchopulmonary dysplasia. Twenty patients  diagnosed with COVID-19 were included in the present study. This research was approved by the Scientific Ethics Committee of the Central Metropolitan Health Service (Resolution No. 048975). In addition, all patients read and signed an informed consent agreement. The inclusion criteria were as follows: (a) diagnosis of severe/critical COVID-19, (b) requirement of MV with orotracheal intubation, (c) hospital discharge, (d) control with a cardiologist and normal electrocardiogram and (e) being in health control at the Carmen Hospital in Maipú, Santiago, Chile. Patients who did not understand commands were excluded. All participants underwent 8 weeks of individualised and supervised pulmonary rehabilitation. In these 8 weeks, 2 evaluation sessions (PRE and POST), 12 exercise sessions and 2 control sessions (after completion of pulmonary rehabilitation) were included. Before (PRE) and after (POST) pulmonary rehabilitation, the walked distance with the 6 MWT was evaluated, and the RHR, HRR, heart rate at minute 6 (HR6 min) and product of the HR6 min with systolic blood pressure (SBP)  and then performed a spirometry test. After a 10-minute rest, a 6 MWT was performed with the measurement of the parameters indicated above before and after the test. The 6 MWT was used to measure the walked distance by each participant and their walking speed, which allowed the incremental test to be dosed according to the conditions of each participant. The continuous test was used to evaluate the duration of the maximum speed obtained in the incremental test. The three tests  were performed on the same day, with 10 min of rest between each one and always in the same order.Spirometric measurements were standardised according to the standards of the American Thoracic Society , incorpo®, Nonin Medical, Minneapolis, MN, USA). The walked distance was recorded in metres. HR was recorded using the Polar® system . An elastic belt  was attached to the participant’s chest at the level of the lower third of the sternum. In the 6 MWT, HR was recorded at rest, during the 6 MWT and in the recovery period (one minute after completion of the 6 MWT) .Each session ended with flexibility exercises that consisted of muscle stretching for each muscle group worked (2 series × 15 s of maintenance), concentrating on the inhalation and exhalation process . If the This was performed at home with a threshold valve  IMT (Inspiratory Muscle Trainer), twice a day , for each day of the eight-week pulmonary rehabilitation program. The threshold valve was set at 30% of the initial maximum inspiratory pressure (MIP). The established training protocol consisted of three series of 3 min of training with 2 min of rest; breaths should be slow and deep. The evaluation and progression of the training had to be recorded in a daily record guideline, which was evaluated by the physical therapists in charge of each face-to-face session. Training load was readjusted weekly and MIP was reassessed at the end of the program  28]..28].2 box with an exhaust fan, under the care standards implemented by the de Hospital El Carmen in Maipú, Santiago, Chile, during the pandemic, where only the kinesiologist and the patient were present. Rotation was every 60 min and all instruments were cleaned with 70% isopropyl alcohol. The kinesiologist used an N95 mask and face shield and the patient used an N95 mask .Work was carried out in a 24 mt-test or Wilcoxon test for paired samples was used. For the analysis of HR between training sessions, ANOVA was used for repeated measures. For the analysis of dyspnoea and fatigue data, the Friedman test was used. Correlations were established using the Pearson or Spearman coefficient, depending on the normality of the data. A significance level of p < 0.05 was considered.The results are presented as means, standard deviation and 95% confidence intervals. The statistical program used was STATA 16 . The normality of data was determined through the Shapiro–Wilk test. For the statistical analysis of HR in the 6 MWT, the Student Twenty-two patients were trained, but two did not complete the eight-week intervention due to voluntary withdrawal, so the results of 20 patients (11 men and 9 women) were analysed. The baseline anthropometric and lung function characteristics of the patients evaluated are shown in p = 0.0008). HRR increased significantly from 12.45 ± 10.22 (95% CI: 7.66–17.23) to 20.55 ± 7.33 beats/min (95% CI: 17.12–23.98) (p = 0.005). HR6 min and HR6 min× SBP did not show significant differences after the pulmonary rehabilitation program (p > 0.05). Both walking speed and walked distance in the 6 MWT increased significantly from 4.70 ± 1.15 (95% CI: 3.98–5.41) to 5.73 ± 0.99 km/h (95% CI: 5.26–6.19) (p < 0.001) and from 451.5 ± 152.2 (95% CI: 380.2–522.7) to 549.3 ± 83.04 (95% CI: 510.4–588.1) metres (p < 0.001), respectively (RHR significantly decreased from 81.95 ± 9.36 (95% CI: 77.57–86.33) to 73.60 ± 9.82 (95% CI: 69.00–78.20) beats/min (ectively . p = 0.040) and from session 1 to 12 (p < 0.05) (p < 0.05) and from sessions 1 to 12 (p < 0.05)  A,C. Simi < 0.05) A,C. The  < 0.05) B. The su < 0.05) C.p = 0.015 and r = 0.528, p = 0.016, respectively. After the pulmonary rehabilitation program, the relationships of HR6 min with speed and walked distance were r = 0.555, p = 0.011 and r = 0.613, p = 0.011, respectively (p = 0.040)  and distance travelled   . The HR6= 0.008) .The aim of this research was to analyse the usefulness of HR during the evaluation and development of a pulmonary rehabilitation program in severe COVID-19 patients who survived an MV stay. The main findings were the significant decrease in HRH and the significant increase in HRR after 8 weeks of an individualised and supervised pulmonary rehabilitation program in patients who were severe COVID-19 survivors of MV. There was also a direct and significant relationship between HR6 min, walking speed and walked distance in the 6 MWT, before and after the rehabilitation process. In addition, a significant decrease in the HR of velocity and slope was observed between sessions 1, 6 and 12, accompanied by a significant decrease in the perception of dyspnoea and fatigue. In this line, the importance of this study is that it makes it clear that the beneficial results obtained here reinforce the concept of personalised training. p = 0.01) and the control group  [p = 0.381), the RHR decreased significantly after the pulmonary rehabilitation, a fact that is considered one of the benefits of physical exercise on the cardiac system [Gruet et al., set out to determine whether maximal HR during the 6 MWT could be used to predict the gas exchange threshold HR during a maximal cardiopulmonary exercise test in patients with cystic fibrosis. Their results showed that there were no significant differences between HR6 max at 6 MWT and gas exchange threshold HR at maximal cardiopulmonary effort. They also observed a direct and high relationship between the maximum HR in the 6 MWT and the gas exchange threshold HR in both patients with cystic fibrosis  . This coc system . This cop < 0.05) and lower HRR (p < 0.04) in relation to athletes–non-smokers. From these results, they concluded that these changes contribute to the adaptation of cardiovascular function to training requirements [On the other hand, the recovery period also showed a significant relationship with the walked distance in the 6 MWT after the pulmonary rehabilitation program. In this regard, Pepera and Panagiota investigated the effects of habitual smoking on heart rate response and HRR after the step test in athletes. Their results indicate that athletes–smokers had a higher RHR . This showed that the designed training plan improved cardiovascular fitness in COPD patients [×SBP could be due to the series of secondary disorders of COVID-19 in the cardiovascular system [Although there were no significant differences in HR6 minpatients . The difp = 0.14) and during exercise (p = 0.12). However, it was observed that the HR significantly increased during the recovery state after the intervention (p = 0.036). Furthermore, following the pulmonary rehabilitation program, HR variability increased by 68–75% at rest, exercise and during the recovery period [Although the HR in training increased during the 30 min of forced walking between sessions 1 and 6, it showed a significant decrease in the three evaluations , despite there being a significant increase in speed and slope. Senanayake et al., investigated the effects of a 6-week pulmonary rehabilitation program on HR response and metabolic demand in patients with pulmonary fibrosis. After the pulmonary rehabilitation program, there was no significant variability in HR pre-exercise (y period . The resy period . This diy period . Thus, tFinally, the results show that dyspnoea exhibits the same behaviour as HR during the training sessions A, B. On 2max. This research has limitations that need to be discussed. During the critical period of the COVID-19 pandemic, one of the decisions made by the Chilean health authorities was the use of beds in critical units for COVID-19 patients only, so it was not possible to include a control group. This could have generated a potential selection bias. Moreover, the sample of patients studied is low, but it has the strength of being patients who complied with the exercise protocols for 8 weeks and suffered from COVID-19, in addition to being subjected to MV during their illness. The dynamics of health personnel and the redistribution of resources to the closed health system delayed the start of pulmonary rehabilitation, which affected the time of admission to the program. This could have affected the evaluations of lung function and exercise capacity.Future research that could complement the results obtained here would entail observing the impact on quality of life and have more complex measures such as HR variability or VOEight weeks of an individualised and supervised pulmonary rehabilitation program were effective in improving RHR and HRR in COVID-19 patients who survived MV. HR is an easily accessible indicator that could help to monitor the evaluation and development of a pulmonary rehabilitation program in COVID-19 patients surviving MV.


Sex steroid hormones, such as androgens and estrogens, are known to exert organizational action at perinatal periods and activational effects during adulthood on the brain and peripheral tissues. These organizational effects are essential for the establishment of biological axes responsible for regulating behaviors, such as reproduction, stress, and emotional responses. Estradiol (E2), testosterone, and their metabolites exert their biological action through genomic and non-genomic mechanisms, bounding to canonical receptors, such as estrogen receptor (ER)α, ERβ, and androgen receptor (AR) or membrane receptors, such as the G protein-coupled estrogen receptor (GPER), respectively. Expression of ERs and AR was found to be different between males and females both in the brain and peripheral tissues, suggesting a sex-dependent regulation of their expression and function. Therefore, studying the ERs and AR distribution and expression levels is key to understand the central and peripheral role of sex steroids in the establishment of sex-specific behaviors in males and females. We investigated the organizational effects of estrogens and androgens in the pituitary and adrenal glands of adult male and female rats. For this, selective blockade of AR with flutamide or 5α-reductase with finasteride or aromatase with letrozole during the first 5 days of life has been performed in male and female pups and then quantification of ERs and AR expression in both glands has been carried out in adulthood. Data show that inhibition of dihydrotestosterone (DHT) and E2 production during the first five postnatal days mainly decreases the ER expression in male to female values and AR expression in female to male levels in the pituitary gland and increases AR expression in female to male levels in the adrenal gland. In contrast, blocking the action of androgens differentially modulates the ERs in males and females and decreases AR in both males and females in both glands. Altogether, the results suggest that neonatal modifications of the androgen and estrogen pathways can potentially lead to permanent modifications of the neuroendocrine functions of the pituitary and adrenal glands in the adulthood of both sexes. The anterior lobe is part of the parvocellular neuroendocrine system that controls the release of numerous hormones , whereas the posterior lobe is part of the magnocellular neuroendocrine system that regulates the production and secretion of oxytocin and vasopressin Dorton, . AdrenalAll experiments were designed according to the guidelines presented in the “Guidelines for the Use of Animals in Neuroscience Research” by the Society for Neuroscience, the European Union legislation (Council Directives 86/609/EEC and 2010/63/UE), and the Spanish Government Directive (R.D. 1201/2005). Experimental procedures were approved by our Institutional Bioethical Committee . Special care was taken to minimize animal suffering and to reduce the number of animals used to the minimum necessary.ad libitum. For mating, a male was placed in a cage with two females for 1 week. Pregnant females were housed individually in plastic maternity cages with wood shavings as the nesting material. Twelve pregnant females were used in order that at least 2–3 mothers give birth within the same 24 h. On the postnatal day 1 (P1), pups from these litters born within the same 24 h were weighed, sexed, and randomly distributed to the different experimental conditions, assigning five females and five males to each mother.Wistar albino male and female rats from our in-house colony were kept on a 12:12-h light–dark cycle and received food and water From P1 to P5, pups received a daily s.c. injection of flutamide , or finasteride , or letrozole . Control animals were treated s.c. with vehicle (corn oil). The doses were selected based on the previous studies  followed by the manufacturer's instructions to measure ERα, ERβ, G protein-coupled estrogen receptor (GPER), and AR mRNA levels. 18S rRNA was selected as the control housekeeping gene. The first cDNA strand was synthesized using 2 μg of extracted RNA by reverse transcription in a final volume of 15 μl using the RevertAid H Minus first-strand cDNA synthesis kit (MBI Fermentas) following the manufacturer's instructions. After reverse transcription, the cDNA was diluted in a ratio of 1:10, and 5 μl were amplified by real-time PCR in 20 μl using SYBR Green master mix (Applied Biosystems) in an ABI Prism 7500 sequence detector (Applied Biosystems), with conventional Applied Biosystems cycling parameters . All samples were amplified in duplicate. Primer sequences were designed using Primer Express (Applied Biosystems) and, after amplification, the size of the quantitative real-time PCR products was verified by electrophoresis on 2% (w/v) ethidium bromide-stained agarose gel, and the identity of PCR products was ascertained by sequencing. Bands were excised and cDNA was purified using the QIAquick PCR purification Kit . An amount of 100 ng ERα, ERβ, GPER, and AR cDNA samples were sequenced  with the same sense and antisense primers. The obtained sequence was aligned with the expected sequence obtained from the GenBank. In addition, dissociation curve analysis was also performed after each PCR reaction to ensure that a single product and no primer dimers were present. Primers for ERα, ERβ, GPER, AR, and 18S in rats were used as previously described by our group . The levels of significance were denoted as *P < 0.05; **P < 0.01; ***P < 0.001. A level of P < 0.05 was considered statistically significant. Levene's test was used to assess the equality of variances. Data in the figures are represented as mean ± SEM.Statistical significance was assessed by two-way ANOVA to analyze the effect and the interaction between the variable “sex” and the variable “treatment” and by one-way ANOVA to analyze the receptors' relative amounts and treatment differences among other groups, followed by the Bonferroni's F = 69.309; P = 0.000], and subsequent Bonferroni's post-hoc test showed the ERα in comparison with ERβ (P = 0.000), GPER (P = 0.000), and AR (P = 0.000)  = 149.864; P = 0.000] and subsequent Bonferroni's post-hoc test revealed a higher amount of ERα compared to ERβ (P = 0.000), GPER (P = 0.000), and AR (P = 0.000), and a higher amount of GPER in comparison with ERβ (P = 0.037)  and GPER as well as AR are expressed in the pituitary gland of both adult male and female rats. Interestingly, the analysis of the different receptors' expression levels in the pituitary highlights the differences in their relative amount in both males and females. One-way ANOVA of the receptors' relative amounts revealed significant differences in males , ERβ , GPER , and AR . The t-test comparison between control groups showed higher ERα  (t(7) = 9,122, p = 0.000] (t(7) = 5,541, p = 0.001] (t(7) = −3.873, p = 0.006], where females showed higher levels compared to males  = 27.604; P = 0.000], ERβ , GPER , and AR . Subsequent Bonferroni's post-hoc comparison among controls and treated animals showed that flutamide decreased ERα (P = 0.007), ERβ (P = 0.000), and AR (P = 0.000), whereas flutamide increased GPER (P = 0.000) expression, finasteride decreased ERα (P = 0.000), ERβ (P = 0.000), and GPER (P = 0.001) and increased AR (P = 0.039) expression, and letrozole decreased all receptors' levels, namely, ERα (P = 0.000), ERβ (P = 0.000), GPER (P = 0.000), and AR (P = 0.000).Selective inhibition of AR, 5α-reductase, and aromatase modulates ER and AR expression in the pituitary. Two-way ANOVA of receptors' expression showed a significant effect of early-time exposure to flutamide, finasteride, and letrozole in all receptors, namely, ERα , ERβ , GPER , and AR .A significant interaction between sex and treatment was found by two-way ANOVA in the expression of ERα , and subsequent Bonferroni's post-hoc test showed an extremely higher amount of GPER in comparison with ERα (P = 0.000), ERβ (P = 0.000), and AR (P = 0.000)  = 118.210; P = 0.000] where GPER displayed as well an extraordinarily higher expression compared to ERα (P = 0.000), ERβ (P = 0.000), and AR (P = 0.000)  and G protein-coupled ER (GPER) as well as AR are expressed in the adrenal gland of both adult male and female rats. Interestingly, the analysis of the expression levels of different receptors in the adrenal gland underlines differences in their relative amount in both males and females. One-way ANOVA of the receptors' relative amounts revealed significant differences in males , GPER , and AR . The t-test analysis of receptors' expression between male and female control animals revealed significant differences in ERα  (t(5) = 9,628, p = 0.000] (t(5) = 13,953, p = 0.000]  = 95.790; P = 0.000], ERβ , GPER , and AR . Subsequent Bonferroni's post-hoc test showed that flutamide and finasteride decreased ERα levels (P = 0.000), whereas letrozole decreased ERα (P = 0.000) and increased GPER expression (P = 0.000).Selective inhibition of AR, 5α-reductase, and aromatase modulates ER and AR expression in the adrenal glands. Two-way ANOVA of receptors' expression after flutamide, finasteride, and letrozole treatments showed a significant effect in all receptors, namely, ERα , GPER , and AR .A significant interaction between sex and treatment was found by two-way ANOVA in the expression of ERα [P = 0.000) and AR (P = 0.000) expression, as well as finasteride for ERα (P = 0.000), but this latter additionally promotes dimorphism, not showed in controls, increasing GPER expression levels in male animals (P = 0.001) (P = 0.006) and abolished the sexual differences of ERα  . Finallys of ERα .via its metabolites E2 or DHT and interactions with estrogen or ARs , with a second period of divergent expression starting with the pubertal onset, and PRL mRNA levels are significantly higher in females starting with the pubertal onset .DG, HP, and PC designed and supervised the experiments. NL, JF-G, NB, AB, BC, M-AA, HP, and DG performed the experiments. DG and NL prepared the figures for publication and wrote the first draft of the manuscript. All authors read and approved the manuscript.This study was supported by Agencia Estatal de Investigación, Spain, and Fondo Europeo de Desarrollo Regional (FEDER) , Universidad Autónoma de Madrid - Comunidad Autónoma de Madrid, Programa de estímulo a la investigación de jóvenes doctores (Project SI3-PJI-2021-00508), Centro de Investigación Biomédica en Red Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Madrid.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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