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Comparison of the in vitro and in vivo profiles of tolterodine with those of subtype-selective muscarinic receptor antagonists.

Tolterodine [(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine ] is a new potent and competitive muscarinic receptor antagonist developed for the treatment of urinary urge incontinence and other symptoms of overactive bladder. In vivo, tolterodine exhibits functional selectivity for the urinary bladder over salivary glands, a profile that cannot be explained in terms of selectivity for a single muscarinic receptor subtype. The aim of this study was to compare the in vitro and in vivo antimuscarinic profiles of tolterodine with those of muscarinic receptor antagonists with distinct receptor subtype-selectivity profiles: darifenacin [(S)-2-[1-[2-(2,3-dihydrobenzofuran-5-yl)ethyl]-3-pyrrolidinyl]-2,2-d iphenylacetamide; selective for muscarinic M3 receptors]; UH-AH 37 (6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo-[b ,e][1,4]diazepine-11-one; low affinity for muscarinic M2 receptors); and AQ-RA 741 (11-([4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-py rido[2,3-b][1,4]benzodiazepine-6-one; high affinity for muscarinic M2 receptors). The in vitro profiles of these compounds were in agreement with previous reports; darifenacin and UH-AH 37 demonstrated selectivity for muscarinic M3/m3 over M2/m2 receptors, while the converse was observed for AQ-RA 741. In vivo, AQ-RA 741 was more potent (1.4-2.7-fold) in inhibiting urinary bladder contraction than salivation in the anaesthetised cat (i.e., a profile similar to that of tolterodine [2.5-3.3-fold]), while darifenacin and UH-AH 37 showed the reverse selectivity profile (0.6-0.8 and 0.4-0.5-fold, respectively). The results confirm that it is possible to separate the antimuscarinic effects on urinary bladder and salivary glands in vivo. The data on UH-AH 37 and darifenacin support the view that a selectivity for muscarinic M3/m3 over M2/m2 receptors may result in a more pronounced effect on salivation than on bladder contraction. The data on AQ-RA 741 may indicate that muscarinic M2/m2 receptors may have a role in bladder contraction.

Animals↗

Therapeutic efficacy of extended release oxybutynin chloride, and immediate release and long acting tolterodine tartrate in children with diurnal urinary incontinence.

PURPOSE: We compare the tolerability and efficacy of extended release oxybutynin chloride, and immediate release and long acting tolterodine tartrate in children with nonneurogenic diurnal urinary incontinence and symptoms of overactive bladder. MATERIALS AND METHODS: Children with a history of diurnal urinary incontinence were arbitrarily assigned to extended release oxybutynin, immediate release tolterodine or long acting tolterodine. The dose was titrated until effective (onset of complete diurnal urinary continence), maximal recommended dosage was achieved or bothersome anticholinergic side effects developed. An independent observer recorded the dose used, anticholinergic side effects and efficacy of therapy (incidence of urinary frequency, urgency, posturing and urinary incontinence). RESULTS: The study included 86 girls and 46 boys. There were no statistically significant differences among the 3 treatment groups regarding the presence of peripheral or central nervous system anticholinergic side effects. Extended release oxybutynin and long acting tolterodine were significantly more effective at reducing daytime urinary incontinence than immediate release tolterodine (p <0.01 and 0 <0.05, respectively). Extended release oxybutynin was significantly more effective then long acting tolterodine for complete resolution of diurnal incontinence (p <0.05). CONCLUSIONS: Extended release oxybutynin and long acting tolterodine are more effective than immediate release tolterodine in decreasing diurnal urinary incontinence. Extended release oxybutynin chloride is more effective than either immediate or long acting tolterodine for control of daytime urinary incontinence and urinary frequency.

Adolescent↗

Predictive value of urethral mobility before suburethral tape procedure for urinary stress incontinence in women.

PURPOSE: We determined whether preoperative urethral mobility predicts the outcome of the suburethral tape procedure outcome in women with urinary stress incontinence. MATERIALS AND METHODS: This retrospective study included 78 women who underwent preoperative cystourethrography while standing. Proximal urethral support was assessed by lateral cystourethrograms at rest and during straining. The 2 images were anatomically superimposed and the angle formed by the 2 proximal urethra axes determined urethral mobility. Surgical outcome was assessed by stress and pad tests. RESULTS: Median followup was 9 months (range 1 to 37) and the objective success rate was 85% (66 of 78 cases). Median rotation of the proximal urethra was 67 degrees in cases without previous surgery for incontinence, 33 degrees in those with 1 and 28 degrees in those with 2 or more procedures (p <0.0001). The success rate was 97% (29 of 30 cases) when urethral mobility exceeded 60 degrees versus 86% (18 of 21) for mobility between 30 and 60 degrees, and 70% (19 of 27) when it was less than 30 degrees (p = 0.023). The success rate was 96% (26 of 27 cases) without previous surgery for incontinence versus 84% (31 of 37) when 1 unsuccessful procedure had been performed and 64% (9 of 14) with 2 or more surgical failures (p = 0.026). Patient age at surgery, menopausal status, mixed incontinence, body mass index, parity, overactive bladder and low maximal urethral closure pressure had no significant prognostic value. CONCLUSIONS: The suburethral sling procedure takes advantage of urethral mobility to avoid leakage. The more the proximal part of the urethra moves while under stress, the better the continence achieved. Risk factors for failure are poor proximal urethral mobility and previous surgery for incontinence.

Adult↗

Increasing costs of urinary incontinence among female Medicare beneficiaries.

PURPOSE: We measured the financial burden of urinary incontinence in the United States from 1992 to 1998 among women 65 years old or older. MATERIALS AND METHODS: We analyzed Medicare claims for 1992, 1995 and 1998 and estimated spending on the treatment of urinary incontinence. Total costs were stratified by type of service (inpatient, outpatient and emergency department). RESULTS: Costs of urinary incontinence among older women nearly doubled between 1992 and 1998 in nominal dollars, from $128 million to $234 million, primarily due to increases in physician office visits and ambulatory surgery. The cost of inpatient services increased only slightly during the period. The increase in total spending was due almost exclusively to the increase in the number of women treated for incontinence. After adjusting for inflation, per capita treatment costs decreased about 15% during the study. CONCLUSIONS: This shift from inpatient to outpatient care likely reflects the general shift of surgical procedures to the outpatient setting, as well as the advent of new minimally invasive incontinence procedures. In addition, increased awareness of incontinence and the marketing of new drugs for its treatment, specifically anticholinergic medication for overactive bladder symptoms, may have increased the number of office visits. While claims based Medicare expenditures are substantial, they do not include the costs of pads or medications and, therefore, underestimate the true financial burden of incontinence on the aging community.

Aged↗

Treatment of urge incontinence in Veterans Affairs medical centers.

Urinary incontinence has far-reaching medical, psychological, social, and economic effects. The objectives of this descriptive study were to examine utilization patterns and discontinuation rates of various pharmacologic agents used to treat symptoms of overactive bladder, primarily urge incontinence (UI), and to estimate the prevalence of urinary incontinence in the study population. Patient-level data regarding specific drugs used to treat UI and the use of diapers or pads over a 9-month period from October 1995 to May 1996 were retrospectively extracted from the medication databases of 9 Department of Veterans Affairs medical centers. A total of 2233 male patients were included in the analyses. Most patients were receiving oxybutynin chloride (39.8%), dicyclomine hydrochloride (16.0%), or imipramine hydrochloride (13.9%), and the remaining 30.3% were using flavoxate hydrochloride, propantheline bromide, hyoscyamine sulfate, and adult diapers or pads. Overall, 72.1% of patients had been prescribed daily dosages within the recommended dosing ranges for these medications. The majority (91.3%) of patients had not switched to another UI medication during the study period. Based on a chronic disease index, 47.6% of patients had 2 or fewer chronic diseases. Using pooled prevalence estimates, the estimated percentage of patients who had ever experienced UI in this population ranged from 7.4% to 20.8%; however, a considerably smaller percentage were taking medications for the treatment of UI. The results of this study suggest that oxybutynin, dicyclomine, and imipramine are the agents most commonly used to treat urinary incontinence within Veterans Affairs medical centers. The majority of patients who received a prescription for one of these drugs did not routinely refill the medication over the course of the study. There are many reasons for patients not to refill a prescription (eg, ineffectiveness, side effects, complications, obtaining the drug from another source), but the present study did not address the causes.

Adult↗

A review of condition-specific instruments to assess the impact of urinary incontinence on health-related quality of life.

To date, severity of symptoms of urinary tract conditions, such as overactive bladder (OAB) and stress incontinence, have been the main indicators used to understand the burden on the patient. However, there is also an impact on patients' health-related quality of life (HRQoL) that should also be assessed. A literature search was conducted and all HRQoL instruments published in peer-reviewed journals were evaluated. Ten instruments were identified: general use (five); urinary urge (OAB) incontinence (four); and stress incontinence (one). Several were identified as valid instruments for assessing HRQoL in urinary incontinence patients.

Health Status Indicators↗

Results of the tension-free vaginal tape technique in the elderly.

OBJECTIVES: To assess the results of the Tension-Free Vaginal Tape (TVT) technique for the treatment of stress urinary incontinence (SUI) in the elderly. PATIENTS AND METHODS: Between March 1998 and February 2001, 76 consecutive women more than 70 years old (median 76) and presenting with SUI were operated with the TVT technique. 28.9% (22/76) of the patients had previous surgery for SUI. 31% (24/76) of the patients had an overactive bladder and 4 patients had detrusor instability controlled by anticholinergic therapy. All patients had preoperative multichanel urodynamic evaluation. RESULTS: No serious complication was noted intra- or postoperatively. At a mean follow up of 24.6 months (range 16-49 months), 67% of the patients were cured (51/76). Among the failures, 10 patients (13.7%) had persistent SUI, 14 patients (18.4%) had urge incontinence and 2 patients were lost to follow up. De novo urgency without incontinence was noticed in 21% of the patients but preoperative urgency symptoms were cured in 46% of the patients. Overall 82% of the patients were satisfied with the result of the surgery, 14% considered the result as incomplete and 4% considered they were worsened by surgery. CONCLUSION: The TVT procedure is safe and efficient to treat SUI in the elderly population even if the rate of de novo urgency appeared to be significant.

Aged↗

Optical micturition interval monitor for experimental animals.

INTRODUCTION: In the past several years, overactive bladder and urinary incontinence have become recognized as an unmet therapeutic need. Some new pharmacologic treatments have recently been described, and new approaches are in development by a number of companies. For preclinical studies, accurate assessment of micturition patterns over a long period of time is important for successful new drug development. Current methodologies rely upon collecting urine in cups positioned upon force displacement transducers and either collecting very large digitized data files, or producing long polygraph tracings. METHODS: The methodology described in this paper utilized an optical device consisting of an infrared photodiode and a matched phototransistor as an electronic drop counter. The device can monitor the appearance of urine flow as it exits the bottom of a metabolic cage. RESULTS: Because the device only collects data when there is an event, the resulting data files are significantly smaller, and there is no need to assure that the urine collection cups do not fill-up and overflow. Data were collected using both methodologies and the results compared. In every experiment, the data derived from the optical device were in very close agreement with the actual micturition pattern recorded by the cup-force transducer method. DISCUSSION: This optical method represents a simple and reliable technique for monitoring micturition patterns in experimental animals.

Animals↗

Pharmacokinetics and metabolism of transdermal oxybutynin: in vitro and in vivo performance of a novel delivery system.

PURPOSE: The purpose of this work was to characterize in vitro/in vivo delivery and pharmacokinetics of oxybutynin (OXY) and its active metabolite. N-desethyloxybutynin (DEO), by a novel matrix transdermal system (TDS). METHODS: Two in vivo, randomized, three-way crossover trials examined single/multiple OXY TDS doses. Abdomen, buttock, and hip application sites were compared and dose proportionality was evaluated. Model independent pharmacokinetics, elimination rate constants, and metabolite/drug ratios were derived from both plasma OXY and DEO concentrations. RESULTS: Single/multiple applications of the OXY TDS to the abdomen yielded mean Cmax OXY concentrations of 3.4 +/- 1.1/6.6 +/- 2.4 ng/mL and median tmax of 36/10 h, with steady state achieved during the second application. Plasma OXY and DEO concentrations decreased gradually after Cmax until system removal. Buttock and hip applications resulted in bioequivalent OXY absorption. AUC ratios of DEO/OXY were 1.5 +/- 0.4 (single dose) and 1.3 +/- 0.3 (multiple dose). Mean in vitro OXY skin absorption (186 microg/h) was comparable to the estimated in vivo delivery (163 microg/h) over 96 h. CONCLUSIONS: Sustained delivery over 4 days and multiple sites allow a convenient, well-tolerated, twice-weekly OXY TDS dosing. A low incidence of anticholinergic side effects is expected during clinical use because of the avoidance of presystemic metabolism and low DEO plasma concentrations. The consistent delivery, absorption, and pharmacokinetics should result in an effective treatment of patients with overactive bladder.

Administration, Cutaneous↗

Primer: questionnaires versus urodynamics in the evaluation of lower urinary tract dysfunction-one, both or none?

What is the role of urodynamic assessments and have we made too much use of them? Is there a correlation between symptoms and underlying pathophysiology? These questions are addressed in this article. There are disparities in the assessment of lower urinary tract dysfunction between the accuracy of symptomatic assessment and the underlying pathophysiology. This is particularly evident with voiding symptoms, in contrast to storage symptoms, and has fuelled the debates that have resulted in the abandonment of the term 'prostatism' in favor of 'lower urinary tract symptoms', whereas the term 'overactive bladder syndrome' has been embraced as a storage symptom complex. Clearly, voiding disorders principally affect men, whilst storage disorders are more common in women. Much has been written and spoken about regarding the use of symptomatic assessment versus urodynamics in the assessment of lower urinary tract symptoms in men, whilst there has been a clearer consensus on the potential usefulness of urodynamics in the assessment of storage disorders, and so this article focuses principally on the latter topic.

Diagnostic Techniques, Urological↗

Mechanisms of Disease: specialized interstitial cells of the urinary tract--an assessment of current knowledge.

Scientists interested in the smooth muscles of the urinary tract, and their control, have recently been studying cells in the interstitium of tissues that express the c-kit antigen (Kit(+) cells). These cells have morphologic features that are reminiscent of the well-described pacemaker cells in the gut, the interstitial cells of Cajal (ICC). The spontaneous contractile behavior of muscles in the urinary tract varies widely, and it is clear that urinary tract Kit(+) interstitial cells cannot be playing an identical role to that played by the ICC in the gut. Nevertheless, there is increasing evidence that they do play a role in modulating the contractile behavior of adjacent smooth muscle, and might also be involved in mediating neural control. This review outlines the properties of ICC in the gut, and gives an account of the discovery of cells in the interstitium of the main components of the urinary tract. The physiologic properties of such cells and the functional implications of their presence are discussed, with particular reference to the bladder. In this organ, Kit(+) cells are found under the lamina propria, where they might interact with the urothelium and with sensory nerves, and also between and within the smooth-muscle bundles. Confocal microscopy and calcium imaging are being used to assess the physiology of ICC and their interactions with smooth muscles. Differences in the numbers of ICC are seen in smooth muscle specimens obtained from patients with various pathologies; in particular, bladder overactivity is associated with increased numbers of these cells.

Humans↗

Animal models in urological disease and sexual dysfunction.

There are several conditions associated with dysfunction of the lower urinary tract or which result in a reduction in the ability to engage in satisfactory sexual function and result in significant bother to sufferers, partners and/or carers. This review describes some of the animal models that may be used to discover safe and effective medicines with which to treat them. While alpha adrenoceptor antagonists and 5-alpha-reductase inhibitors deliver improvement in symptom relief in benign prostatic hyperplasia sufferers, the availability of efficacious and well-tolerated medicines to treat incontinence is less well served. Stress urinary incontinence (SUI) has no approved medical therapy in the United States and overactive bladder (OAB) therapy is limited to treatment with muscarinic antagonists (anti-muscarinics). SUI and OAB are characterised by high prevalence, a growing ageing population and a strong desire from sufferers and physicians for more effective treatment options. High patient numbers with low presentation rates characterizes sexual dysfunction in men and women. The introduction of Viagra in 1998 for treating male erectile dysfunction and the success of the phosphodiesterase type 5 inhibitor class (PDE5 inhibitor) have indicated the willingness of sufferers to seek treatment when an effective alternative to injections and devices is available. The main value of preclinical models in discovering new medicines is to predict clinical outcomes. This translation can be established relatively easily in areas of medicine where there are a large number of drugs with different underlying pharmacological mechanisms in clinical usage. However, apart from, for example, the use of PDE5 inhibitors to treat male erectile dysfunction and the use of anti-muscarinics to treat OAB, this clinical information is limited. Therefore, current confidence in existing preclinical models is based on our understanding of the biochemical, physiological, pathophysiological and psychological mechanisms underlying the conditions in humans and how they are reflected in preclinical models. Confidence in both the models used and the pharmacological data generated is reinforced if different models of related aspects of the same disorder generate confirmatory data. However, these models will only be fully validated in retrospect once the pharmacological agents they have helped identify are tested in humans.

Animals↗

Late-stage clinical development in lower urogenital targets: sexual dysfunction.

In recent years, late-stage clinical drug development that primarily focuses on urogenital targets has centered around four areas of medical need (both unmet need and aiming to improve on existing therapies). These include male sexual dysfunction (MSD), female sexual dysfunction (FSD), prostatic pathology (neoplastic, pre-neoplastic, and non-neoplastic), and improvement in lower urinary tract symptoms. Despite the regulatory approval of compounds to treat erectile dysfunction (ED), benign prostatic hyperplasia, a number of treatments for overactive bladder, and stress urinary incontinence, there remains a deficiency in addressing a number of conditions that arise out of pathophysiological dysfunction resulting in lower urogenital tract sexual conditions. In terms of late-stage clinical development, significant progress has most recently been made in MSD development, especially in understanding further a common and complex sexual dysfunction--that of premature ejaculation. The search also continues for compounds that improve ED in terms of better efficacy and superior safety profile compared to the currently marketed phosphodiesterase-5-inhibitors. Whilst there are no approved medications to treat the subtypes of FSD, there has been significant progress in attempting to better understand how to appropriately assess treatment benefit in clinical trial settings for this difficult to diagnose and treat condition. This review will focus on late-stage human clinical development pertaining to MSD and FSD.

Alprostadil↗

Sexual function in women with urinary incontinence.

To date limited data exists that addresses the impact of urinary incontinence on sexual function. In the present study, we assessed sexual functions in patients with urinary incontinence and compared with healthy continent subjects by means of Female Sexual Function Index (FSFI) questionnaire. A total of 21 premenopausal incontinent women (three stress incontinence, nine overactive bladder and nine mixed incontinence) were enrolled in the study, and 18 healthy continent subjects served as controls. All subjects were asked to complete FSFI questionnaire and each FSFI domain scores including desire, arousal, lubrication, orgasm, satisfaction and pain were calculated. The mean scores in each domain were compared between the groups. Mean age of subjects with urinary incontinence and controls were 39.5+/-6.6 and 32.6+/-9.1 y, respectively. All domain scores were significantly lower in incontinent women except for pain. Among the incontinence types, no significant difference was determined in all domains of FSFI. Urinary incontinence significantly reduces sexual functions in premenopausal sexually active women.

Adult↗

Sexual functioning in patients with lower urinary tract dysfunction improves after percutaneous tibial nerve stimulation.

In this study, we evaluated the effect of lower urinary tract dysfunction and its neuromodulative therapy on sexual functioning. We studied 121 patients with an overactive bladder (OAB) (N = 83), chronic pelvic pain (N = 23) and nonobstructive retention (N = 15), which were treated with neuromodulation (i.e. percutaneous tibial nerve stimulation, PTNS). To obtain information on their sexual function, a self-administered standardized questionnaire was filled out before therapy as well as after 12 weeks of treatment. Before therapy, different aspects of sexual life were considered not normal in 25.3-45.6% of the cases. This improved significantly after treatment. Patients most likely to benefit were women, patients with an OAB and subjective responders. The aspects of sexual life which mostly improved were overall satisfaction, libido and the frequency of sexual activities. Sexual dysfunction is observed in a lot of patients with lower urinary tract disorders and may improve on successful therapy for the latter.

Adult↗

The usefulness of a minimal urodynamic evaluation and pelvic floor biofeedback in children with chronic voiding dysfunction.

OBJECTIVE: To report our experience of assessing children with chronic voiding dysfunction (>6 months' duration) using a minimal urodynamic evaluation, and the management of detrusor-sphincter dyscoordination (DSdc) using pelvic floor biofeedback. PATIENTS AND METHODS: From 1994 to 1997, 120 children (mean age 7.5 years) with three predominant and associated symptoms were referred to one urologist; they had nocturnal enuresis (28 children), urge incontinence (42) or urinary tract infection (50). All patients were assessed by urinary culture, renal ultrasonography and a minimal urodynamic evaluation, i.e. urinary flowmetry with sphincter electromyography (EMG) using perineal surface electrodes. If they had urinary tract infection and/or renal dilatation, they underwent voiding cysto-urethrography. In children with DSdc, urinary training with frequent voiding was instituted initially, with subsequent pelvic floor biofeedback exercises if the improvement was deemed unsatisfactory. RESULTS: DSdc was diagnosed in 33 children (28%), none of whom had isolated nocturnal enuresis. Pelvic floor biofeedback was undertaken by 15 children (12 girls and three boys); it was well accepted because it was administered as a computer game. In all affected patients the DSdc resolved on EMG and there was a significant clinical improvement. Vesico-ureteric reflux was detected in 24 patients, associated with DSdc in 10. The reflux resolved spontaneously on antibiotic prophylaxis in six children and after urinary re-education in four. CONCLUSION: A minimal urodynamic evaluation seems to be useful in the diagnosis of DSdc which caused urinary tract infection and/or bladder overactivity. The results with pelvic floor biofeedback were excellent in these children.

Biofeedback, Psychology↗

Cadaveric fascia lata pubovaginal slings: early results on safety, efficacy and patient satisfaction.

OBJECTIVE: To prospectively evaluate and quantify the efficacy of cadaveric fascia lata (CFL) as an allograft material in pubovaginal sling placement to treat stress urinary incontinence (SUI). PATIENTS AND METHODS: Thirty-one women with SUI (25 type II and six type III; mean age 63 years, range 40-75) had a CFL pubovaginal sling placed transvaginally. The operative time, blood loss, surgical complications and mean hospital stay were all documented. Before and at 4 months and 1 year after surgery each patient completed a 3-day voiding diary and validated voiding questionnaires (functional inquiry into voiding habits, Urogenital Distress Inventory and Incontinence Impact Questionnaire, including visual analogue scales). RESULTS: The mean (range) operative time was 71 (50-120) min, blood loss 78.7 (20-250) mL and hospital stay 1.2 (1-2) days; there were no surgical complications. Over the mean follow-up of 13.5 months, complete resolution of SUI was reported by 29 (93%) patients. Overactive bladder symptoms were present in 23 (74%) patients before surgery, 21 (68%) at 4 months and two (6%) at 1 year; 80% of patients with low (< 15 cmH2O) voiding pressures before surgery required self-catheterization afterward, as did 36% at 4 months, but only one (3%) at 1 year. Twenty-four (77%) patients needed to adopt specific postures to facilitate voiding. After surgery there was a significant reduction in daytime frequency, leakage episodes and pad use (P < 0.05). The severity of leak and storage symptoms was also significantly less (P < 0.002), whilst the severity of obstructive symptoms remained unchanged. Mean subjective levels of improvement were 69% at 4 months and 85% at 1 year, with corresponding objective satisfaction levels of 61% and 69%, respectively. At 1 year, approximately 80% of the patients said they would undergo the procedure again and/or recommend it to a friend. CONCLUSION: Placing a pubovaginal sling of CFL allograft is a highly effective, safe surgical approach for resolving SUI, with a short operative time and rapid recovery. Storage symptoms are significantly improved, and subjective improvement and satisfaction rates are high.

Adult↗

The role of neuromodulation in the management of urinary urge incontinence.

OBJECTIVE: To examine the benefit-risk profile of neuromodulation in treating refractory urinary urge incontinence and other voiding disorders. PATIENTS AND METHODS: The outcome measures from all patients in pivotal clinical trials who had undergone sacral nerve stimulation were analysed retrospectively. RESULTS: Neuromodulation was effective in several clinical studies; the response is durable and the benefit-risk profile good. CONCLUSION: Sacral nerve stimulation is becoming the standard of care for refractory overactive bladder and retention problems. The potential benefit of neuromodulation should be included in female urology and gynaecology training programmes.

Adolescent↗