Male Twin Pairs Research Articles

Hypertrophic pyloric stenosis in twins: same genes or same environments?

The aetiology of infantile hypertrophic pyloric stenosis (IHPS) remains obscure. Cases in twins, usually monozygotic, have been sporadically reported as evidence for the genetic origin of the disease. We present a case of IHPS in a pair of dizygotic male twins together with a review of the literature, focusing on the question of whether the twin cases actually support a genetic or an acquired nature of IHPS.

21.12 A throw-and-catch pattern in postural sway doesnot exclude continuous feedback control

Heritability of the variation of the plasma concentrations of total and unbound cortisol, cortisol binding globulin (CBG), and dehydroepiandrosterone sulfate (DHEA-S) was investigated in 20 monozygotic (MZ) and 20 dizygotic (DZ) male twin pairs. Three plasma samples collected between 8 am and 9:30 am were pooled for the assays. Heritability was calculated from the intraclass correlation [2(rMZ - rDZ)]. The mean age, total and unbound cortisol, CBG, and DHEA-S were not significantly different between the MZ and DZ groups of twins. The heritability index for variability of the plasma content of steroids was 45.4% (p < .05) for total cortisol, 50.6% (P < .05) for unbound plasma cortisol, 57.8% (P < .05) for DHEA-S, and 32.4% (P > .05) for CBG. The data were analyzed by factor analysis, and heritability estimates were corrected for factors including age, smoking, drinking, exercise, and degree of obesity. These factors did not account for the variation in hormone values in twin pairs. Factor analysis of the three quantitative measurements, cortisol, percent free cortisol, and DHEA-S, provides no evidence for shared factors. The correlation coefficients between age and CBG and total and unbound plasma cortisol concentrations were insignificant. The correlation coefficient between total plasma cortisol levels and CBG was 0.57, which indicates that CBG accounts for 32% of the variation of plasma cortisol concentrations. The results suggest that genetic factors have a decided influence on the variation of the concentration of cortisol of DHEA-S in normal adult men.

Energy expenditure, body composition and insulin response to glucose in male twins discordant for the Trp64Arg polymorphism of the β3‐adrenergic receptor gene

The tryptophan to arginine change in position 64 (Trp64Arg) polymorphism of the beta3-adrenergic receptor (beta3AR) gene has been associated with an increased prevalence of obesity, insulin resistance and type 2 diabetes. In this, decreased rates of energy expenditure and impaired insulin secretion could play a role. In 10 male twin pairs discordant for the Trp64Arg polymorphism, we examined insulin response to glucose by an oral glucose tolerance test (OGTT), a frequently sampled intravenous glucose tolerance test (FSIGT), body composition by the bioimpedance method, dual-energy X-ray absorptiometry scanning and energy expenditure by indirect and direct calorimetry. Twins heterozygous for the Trp64Arg polymorphism showed significantly lower fat mass independent of the method used, and significantly lower fasting insulin and glucose concentrations compared with their homozygous wild-type co-twins. Correspondingly, insulin resistance and insulin secretion determined by homeostasis model assessment were significantly lower in twins carrying the Trp64Arg polymorphism. However, there were no significant differences in adiponectin levels, insulinogenic index assessed by OGTT, or insulin sensitivity, acute insulin response to glucose, glucose effectiveness or insulin disposition index assessed by minimal modelling of the FSIGT. Furthermore, there were no differences in sleeping, resting or post-prandial energy expenditure. In male twins with a high similarity in genetic and environmental background, the Trp64Arg polymorphism of the beta3AR gene is associated with lower fat mass, fasting insulin levels and an appropriate insulin response to glucose. Thus, heterozygosity for the Trp64Arg variant is unlikely to increase the risk of obesity, insulin resistance or type 2 diabetes.

Cardiac structure and function in monozygotic twin pairs discordant for physical fitness

Cross-sectional studies in athletes and untrained subjects suggest that exercise training induces adaptations in cardiac structure and function. However, the role of genetic variation on the results has largely been ignored in these studies. The purpose of this study was to investigate the effects of long-term volitionally increased physical activity on electrocardiographic and echocardiographic parameters in male monozygotic twin pairs discordant for physical activity and fitness. On the basis of the mailed questionnaires, a telephone interview, and the inclusion criteria, 12 pairs of young adult male monozygotic twins were recruited from a Finnish twin cohort. All subjects completed a maximal oxygen uptake (.VO2 (max)) test and electrocardiography and echocardiography studies. Nine pairs had at least 9% difference in .VO2(max) and were selected for further analysis and for a second echocardiography study. Twins were divided into the more (MAG) and less active group (LAG), according to their VO2(max). On average, MAG had 18% higher VO2(max) compared with LAG. In electrocardiography, MAG had 29% (P = 0.02) higher Cornell voltage and 37% (P = 0.01) higher right-side hypertrophy index. In echocardiography, no significant differences were observed between the groups, and left ventricular mass index was only 7% (P = 0.16) higher in MAG. These results show that the volitionally increased physical activity that has led to an 18% increase in cardiorespiratory fitness induces greater changes in electro- than echocardiographic parameters. Electrocardiographic changes were suggestive of left ventricular hypertrophy, and echocardiography showed a similar but statistically nonsignificant trend.

Diet and exercise during growth have site-specific skeletal effects: a co-twin control study

Exercise and improved nutrition offer safe, low-cost and widely applicable approaches to potentially reduce the burden of fractures. We conducted a cross-sectional study of 30 monozygotic and 26 dizygotic male twin pairs, aged 7-20 years to test the following hypotheses: (1) Associations between bone mass and dimensions and exercise are greater than between bone mass and dimensions and protein or calcium intakes; (2) exercise or nutrient intake are associated with appendicular bone mass before puberty and axial bone mass during and after puberty. Total body and posteroanterior (PA) lumbar spine bone mineral content (BMC) and mid-femoral shaft dimensions were measured using dual energy X-ray absorptometry (DEXA). Relationships between within-pair differences in nutrient intake (determined by weighed-food diaries) or exercise duration (determined by questionnaire) and within-pair differences in BMC and bone dimensions were tested using linear regression analysis. In multivariate analyses, within-pair differences in exercise duration were associated with within-pair differences in total body, leg and spine BMC, and cortical thickness. Every-hour-per-week difference in exercise was associated with a 31-g (1.2%) difference in total body BMC, a 10-g (1.4%) difference in leg BMC, a 0.5-g difference in spine BMC and a 0.1-mm difference in cortical thickness ( p <0.01- p <0.1). A 1-g difference in protein intake was associated with a 0.8-g (0.4%) difference in arm BMC ( p <0.05). These relationships were present in peri-pubertal and post-pubertal pairs but not in pre-pubertal pairs. Exercise during growth appears to have greater skeletal benefits than variations in protein or calcium intakes, with the site-specific effects evident in more mature twins.

Genetic and environmental influences on eating behavior: the Swedish Young Male Twins Study

Eating behavior may be implicated in the increasing prevalence of overweight and obesity, presumably in relation to easy access to energy-dense and highly palatable foods. The aim of the present study was to disentangle genetic and environmental influences on eating behavior in a population-based cohort of male twins. The study included 326 dizygotic and 456 monozygotic male twin pairs aged 23-29 y from Sweden. The revised 21-item version of the Three-Factor Eating Questionnaire (TFEQ-R21) was used to assess eating behavior. This validated instrument consists of 3 dimensions: cognitive restraint, emotional eating, and uncontrolled eating. Structural equation modeling was used to estimate the heritability of eating behavior. Cognitive restraint was the only TFEQ-R21 scale that significantly correlated with BMI (r = 0.39, P < 0.0001). The best-fitted models gave a heritability of 59% (95% CI: 52%, 66%) for cognitive restraint, 60% (95% CI: 52%, 67%) for emotional eating, and 45% (95% CI: 36%, 53%) for uncontrolled eating. These results show the great importance of genetic factors in the eating behavior of a large, unselected population of young adult male twins. Nonshared environmental factors were also important, whereas shared environmental factors did not contribute to eating behavior.

Smell identification ability in twin pairs discordant for Parkinson's disease

Olfactory dysfunction has been proposed to be a sign that may precede the motor features of Parkinson's disease (PD). To determine whether smell identification deficits predict subsequent PD, we studied smell identification ability using the University of Pennsylvania Smell Identification Test (UPSIT) in 62 members of male twin pairs discordant for PD at baseline. Smell identification ability was reduced at baseline in the twins with PD compared to their unaffected brothers (23 vs. 31 of 40; P = 0.001). UPSIT scores were not reduced in the twins without PD when compared to age- and gender-specific normal values. After a mean interval of 7.3 years, 28 unaffected twins were still alive and 19 agreed to a second evaluation. Two had newly developed PD. Neither twin had impaired smell identification at baseline. The average decline in UPSIT percentile scores in these 2 twins was greater than in the 17 twins who did not develop PD (-68% vs. -24%; P = 0.01). In subjects who did not meet Core Assessment Program for Intracerebral Transplantations diagnostic criteria for PD at baseline, the presence of cardinal signs of parkinsonism was not associated with lower baseline UPSIT scores nor with a subsequent decline. Smell identification ability may not be a sensitive indicator of future PD 7 or more years before the development of motor signs, even in a theoretically at-risk population.

The Heritability of Hypothalamus Pituitary Adrenal Axis Responses to Psychosocial Stress Is Context Dependent

Individual differences in the response of the hypothalamus pituitary adrenal axis to stress are known to play an important role in health and disease risk. The origins, or determinants, of these individual differences are not well understood. To date, no study has examined the effects of context on the heritability of psychoendocrine stress responses. In the present study, 58 male twin pairs were exposed to a psychosocial stressor (Trier Social Stress Test) three times at weekly intervals, and their salivary and total cortisol, ACTH, and heart rate responses were assessed. Modest heritabilities were observed for all measures at the first stress exposure (all h2 < 0.33), but heritability estimates increased substantially with repetition of the stressor (T3: all h2 > 0.97). Concurrently, only the first but not the second and third Trier Social Stress Test exposure induced a significant increase in state anxiety (T1: P < 0.01, T2 and T3: n.s.), suggesting low heritabilities in a new, anxiety-evoking context and high heritabilities in a familiar, low-anxiety context. These findings challenge previous reports on a low heritability of markers of stimulated hypothalamus pituitary adrenal axis activity and are the first to document the relevance of context for psychoendocrine heritability estimates.

Genetic and environmental influences on antisocial behavior and alcohol dependence from adolescence to early adulthood

Genetic and environmental influences on symptoms of adult antisocial behavior (AAB) and alcohol dependence at ages 17, 20, and 24 were examined cross-sectionally and longitudinally in 188 monozygotic and 101 dizygotic male twin pairs. A moderate genetic influence on both AAB and alcohol dependence was found at each age, with a substantial proportion of this influence common to the two disorders, suggesting they share susceptibility genes. Biometrical models showed that continuity effects accounted for most of the stable variance in symptoms of both AAB and alcohol dependence, indicating that genetic and environmental effects associated with each of these disorders were similar at each age. Significant cross-lag effects (effects of alcohol dependence contributing to variance in AAB and vice versa) were observed at ages 20 and 24 for both disorders. The largest and theoretically most interesting of these effects indicated that one sixth of the genetic influence on AAB at age 20 was due to genetic effects associated with alcohol dependence at age 17. Thus, alcohol dependence symptoms at age 17 in particular had an effect on antisocial behavior symptoms at age 20, suggesting that alcohol involvement in adolescence may ensnare otherwise desisting youth in persistent antisocial behavior.

Progressive facial hemiatrophy: a complex disorder not only affecting the face. A report in a monozygotic male twin pair

Progressive facial hemiatrophy (PFH) is a ubiquitous disease, characterized by hyperpigmentation of skin followed by unilateral craniofacial atrophy of subcutaneous tissues, including fat, muscle and bone. Hereditary factors have been postulated to be involved in the aetiology of PFH. Yet, the occurrence of PFH in one of two identical male twins reported here makes this possibility unlikely. PFH usually occurs in the first two decades of life, and the clinical presentation resembles linear scleroderma. PFH may be complicate by autoimmune, neurological, ocular and dental disorders. Management of PFH comprises a long term follow-up of somatic disorders, and prevention of psychological problems. Treatment of PFH is symptomatic and consists of plastic surgery after the disease activity has stopped. The occurrence of PFH in one of monozygotic twin pair suggests that genetic factors are not involved in its aetiology. Early diagnosis of PFH and accurate follow-up is essential to disclose the occurrence of complications.

Progressive facial hemiatrophy: a complex disorder not only affecting the face. A report in a monozygotic male twin pair

Progressive facial hemiatrophy (PFH) is a ubiquitous disease, characterized by hyperpigmentation of skin followed by unilateral craniofacial atrophy of subcutaneous tissues, including fat, muscle and bone. Hereditary factors have been postulated to be involved in the aetiology of PFH. Yet, the occurrence of PFH in one of two identical male twins reported here makes this possibility unlikely. PFH usually occurs in the first two decades of life, and the clinical presentation resembles linear scleroderma. PFH may be complicate by autoimmune, neurological, ocular and dental disorders. Management of PFH comprises a long term follow-up of somatic disorders, and prevention of psychological problems. Treatment of PFH is symptomatic and consists of plastic surgery after the disease activity has stopped.The occurrence of PFH in one of monozygotic twin pair suggests that genetic factors are not involved in its aetiology. Early diagnosis of PFH and accurate follow-up is essential to disclose the occurrence of complications.

Illicit drug use and abuse/dependence: modeling of two-stage variables using the CCC approach

Drug use and abuse/dependence are stages of a complex drug habit. Most genetically informative models that are fit to twin data examine drug use and abuse/dependence independent of each other. This poses an interesting question: for a multistage process, how can we partition the factors influencing each stage specifically from the factors that are common to both stages? We used a causal-common-contingent (CCC) model to partition the common and specific influences on drug use and abuse/dependence. Data on use and abuse/dependence of cannabis, cocaine, sedatives, stimulants and any illicit drug was obtained from male and female twin pairs. CCC models were tested individually for each sex and in a sex-equal model. Our results suggest that there is evidence for additive genetic, shared environmental and unique environmental influences that are common to illicit drug use and abuse/dependence. Furthermore, we found substantial evidence for factors that were specific to abuse/dependence. Finally, sexes could be equated for all illicit drugs. The findings of this study emphasize the need for models that can partition the sources of individual differences into common and stage-specific influences.

Habituation of cortisol responses to repeated psychosocial stress—further characterization and impact of genetic factors

Although a rapid response habituation to repeated stress exposure is a key characteristic of the hypothalamus-pituitary-adrenal (HPA) axis, several studies document a substantial inter-individual variability of such HPA response patterns. In order to further investigate the individual differences in the habituation of this important neuroendocrine system to psychosocial stress, 54 male twin pairs were exposed to moderate psychosocial stress on three occasions, each exposure separated by a 1-week interval. Additionally, an ACTH(1-24) stimulation test (1 microg) and a dexamethasone suppression test (0.5mg) were performed. Although on average the expected decrease of mean cortisol and ACTH responses across stress exposures was observed, only 52% of the subjects showed this well-documented general decline and almost 16% of the participants even showed a response sensitization across sessions. Furthermore, a weak habituation was related to low cortisol responses to both the first stress exposure as well as the ACTH challenge. Moreover, genetic analyses did not reveal any evidence for a substantial heritability of the individual cortisol response habituation or an association between this habituation and two common polymorphisms in the glucocorticoid receptor gene.

The ratio of animal protein intake to potassium intake is a predictor of bone resorption in space flight analogues and in ambulatory subjects

Bone loss is a critical concern for space travelers, and a dietary countermeasure would be of great benefit. Dietary protein and potassium-associated bicarbonate precursors may have opposing effects on the acid-base balance in the body and therefore on bone loss. In 2 studies, we examined the ability of dietary protein and potassium to predict markers of bone metabolism. In the first study, 8 pairs of male identical twins were assigned to 1 of 2 groups: bed rest (sedentary, or SED, group) or bed rest with supine treadmill exercise in a lower-body negative pressure chamber (EX group). In a second study, groups of 4 subjects lived in a closed chamber for 60 or 91 d, and dietary data were collected for two or three 5-d sessions. Urinary calcium, N-telopeptide, and pyridinium cross-links were measured before bed rest; on bed rest days 5-6, 12-13, 19-20, and 26-27; and daily during the chamber studies. Data were analyzed by Pearson's correlation (P < 0.05). The ratio of animal protein intake to potassium intake was significantly correlated with N-telopeptide in the SED group during bed rest weeks 3 and 4 (r = 0.77 and 0.80) and during the 91-d chamber study (r = 0.75). The ratio of animal protein intake to potassium intake was positively correlated with pyridinium cross-links before bed rest in the EX group (r = 0.83), in the EX group during bed rest week 1 (r = 0.84), and in the SED group during bed rest week 2 (r = 0.72) but not during either chamber study. In both studies, these relations were not significant with the ratio of vegetable protein intake to potassium intake. The ratio of animal protein intake to potassium intake may affect bone in ambulatory and bed-rest subjects. Changing this ratio may help to prevent bone loss on Earth and during space flight.

A twin study of early cannabis use and subsequent use and abuse/dependence of other illicit drugs

Cannabis use is strongly associated with the use and abuse/dependence of other illicit drugs. Gateway and common liabilities models have been employed to explain this relationship. We sought to examine this association using a combination of the discordant twin design and modeling methods. We assess the relationship between early cannabis use and the subsequent use and abuse/ dependence of other illicit drugs in a population-based sample of male and female twin pairs using four analyses: (i) analysis of the association between early cannabis use and other illicit drug use and abuse/dependence in the entire sample of twins, (ii) assessment of the influence of early cannabis use in twin 1 on twin 2's use or abuse/dependence of other illicit drugs, (iii) use of twin pairs discordant for early cannabis use in a discordant twin design and (iv) a model-fitting procedure. We found: (i) a strong association between early cannabis use and use and abuse/dependence of other illicit drugs in the sample, (ii) twin 1's early cannabis use is significantly associated with the twin 2's other illicit drug use, (iii) the role of correlated genetic factors with some evidence for a causal influence, and (iv) the correlated liabilities model fits the data well. Early cannabis use is strongly associated with other illicit drug use and abuse/dependence. The relationship arises largely due to correlated genetic and environmental influences with persisting evidence for some causal influences.

Effect of environmental and genetic factors on education-associated disparities in weight and weight gain: a study of Finnish adult twins

Disparities in body mass index (BMI) between persons with different educational levels in Western countries are well documented, but the background of these education-associated disparities remains poorly understood. The objective was to examine the influence of environmental and genetic factors on education-associated disparities in self-reported BMI and weight change. Longitudinal postal surveys were performed in 1975, 1981, and 1990. The data were analyzed by using multivariate genetic models for twin data. The data derived from the Finnish Twin Cohort included 2482 monozygotic and 5113 dizygotic same-sex male and female twin pairs born between 1915 and 1957. Education-associated differences in BMI and in weight change were clear in 1975 and 1981, respectively, whereas no differences were seen in weight change between 1981 and 1990 when age and baseline BMI were adjusted for. The trait correlation between baseline BMI and educational attainment (-0.15 in men and women) was mainly due to correlations between additive genetic factors that contributed to BMI and education in men (-0.20; 95% CI: -0.25, -0.14) and women (-0.32; 95% CI: -0.40, -0.25) when adjusted for age. Among women, a weaker positive correlation was found for the unshared environmental effects contributing to the 2 traits (0.06; 95% CI: 0.02, 0.12). The same factors that affected the association between education and BMI in 1975 largely explained the association between education and weight change in 1981. The results suggest the possibility that common genetic factors affect educational attainment and body weight, which contribute to education-associated disparities in BMI in adulthood.

A twin study of genetic and environmental influences on tobacco initiation, regular tobacco use and nicotine dependence

Numerous twin studies have reported significant genetic contributions to the variability of tobacco initiation (TI), while fewer studies have shown similar results for the persistence of smoking behavior, or nicotine dependence (ND). As the development of ND requires regular tobacco use (RTU) which in turn requires TI, a conditional approach is necessary. We used structural equation modeling of multi-step conditional processes to examine the relationship between genetic and environmental risk factors for TI, RTU and ND. The tobacco variables were assessed by personal interview in female, male and opposite-sex twin pairs from the population-based Virginia Twin Registry. The results suggested that the liabilities to TI, RTU and ND were correlated. Over 80 % of the variance in liability to TI and RTU were shared, and a smaller proportion was shared between RTU and ND. The heritabilities were estimated at 75 %, 80 % and 60 % respectively for TI, RTU and ND. The variance specific to liability to RTU was entirely accounted for by additive genetic factors. Only a modest part of the heritability in liability of ND was due to genetic factors specific to ND. Shared environmental factors were not significant. No sex differences were found for the sources of variation or causal paths, but prevalences were significantly greater in males versus females. This study showed significant overlap in the contribution of genetic factors to individual differences in TI, RTU and ND. Furthermore, there was evidence for significant additional genetic factors specific to RTU and ND.

Neuropsychological consequences of regular marijuana use: a twin study

Results of previous research examining long-term residual effects of marijuana use on cognition are conflicting. A major methodological limitation of prior studies is the inability to determine whether differences between users and non-users are due to differences in genetic vulnerability preceding drug use or due to the effects of the drug. Fifty-four monozygotic male twin pairs, discordant for regular marijuana use in which neither twin used any other illicit drug regularly, were recruited from the Vietnam Era Twin Registry. A minimum of 1 year had passed since the marijuana-using twins had last used the drug, and a mean of almost 20 years had passed since the last time marijuana had been used regularly. Twins were administered a comprehensive neuropsychological test battery to assess general intelligence, executive functioning, attention, memory and motor skills. Differences in performance between marijuana-using twins and their non-using co-twins were compared using a multivariate analysis of specific cognitive domains and univariate analyses of individual test scores. Dose response relationships were explored within the marijuana-using group. Marijuana-using twins significantly differed from their non-using co-twins on the general intelligence domain; however, within that domain only the performance of the block design subtest of the Wechsler Adult Intelligence Scale--Revised reached a level of statistical significance. Out of the numerous measures that were administered, only one significant difference was noted between marijuana-using twins and their non-using co-twins on cognitive functioning. The results indicate an absence of marked long-term residual effects of marijuana use on cognitive abilities.

Latent class typology of nicotine withdrawal: genetic contributions and association with failed smoking cessation and psychiatric disorders

Nicotine withdrawal is associated with failed smoking cessation and thus contributes to continuance of the habit and increases risk of smoking-related illnesses. Withdrawal is also associated with psychiatric disorders such as depression and alcoholism. However, relatively little is known about how to characterize the severity of withdrawal, including whether withdrawal subtypes exist in male smokers. If so, do these subtypes represent quantitative or qualitative differences? Smoking and withdrawal data were obtained from 4112 male-male twin pairs of the Vietnam Era Twin Registry during a 1992 administration of the Diagnostic Interview Schedule. Latent Class Analysis (LCA) was used to derive significantly different nicotine withdrawal profiles, and their association with psychiatric disorders was assessed. Genetic and environmental contributions and the correlation between these contributions were evaluated using bivariate biometrical modeling of the withdrawal phenotype and failed smoking cessation. The LCA model which best fit the data was a four-class severity continuum. Psychiatric disorders were significantly associated with more severe classes and the magnitude of the association increased as withdrawal severity increased. Genetics accounted for 31% and 51% of the variance in risk for withdrawal and failed cessation, respectively. The genetic contributions were significantly correlated (r = 0.37). Nicotine withdrawal classes are characterized by quantitative differences. The strong association between psychiatric disorders and withdrawal severity and the significant genetic correlation between withdrawal and cessation highlight the importance of withdrawal severity. Further refinement of the DSM definition of withdrawal to incorporate severity ratings may be warranted.

The Role of Genetics and Environment in Lifting Force and Isometric Trunk Extensor Endurance

Our understanding of what different back performance tests are measuring is limited. The purpose of this study was to investigate the relative contributions of genetics and unique and common environmental factors for 3 tests of back muscle performance in a classic twin analysis. The subjects were a population-based sample of 122 monozygotic and 131 dizygotic male twin pairs aged 35 to 69 years (mean=49.9, SD=7.7). Variance component analysis was applied to estimate genetic and environmental influences on isokinetic and psychophysical lifting and isometric trunk extensor endurance test performance. The Cholesky decomposition genetic factor model was used to estimate genetic and environmental correlations of these variables. Path analysis was applied to study determinants of isokinetic and psychophysical lifting and isometric trunk extensor endurance test performance. Genetic effects accounted for 60%, 33%, and 5% of the total variance of isokinetic and psychophysical lifting forces and isometric trunk extensor endurance, respectively, and unique environmental factors accounted for 35%, 49%, and 61% of the variance. Genetics had a dominant role in isokinetic lifting and unique environmental factors in isometric trunk extensor endurance. The relatively high role of genetics in lifting force suggests the potential to increase and sustain changes in back muscle force in the general population may be particularly challenging.