Gut microbiota in adults with cystic fibrosis: Implications for the severity of the CFTR gene mutation and nutritional status.

BACKGROUND: Microbial dysbiosis has been linked to cystic fibrosis (CF); however, the composition of gut microbiota in adult CF patients in relation to severity of CF transmembrane conductance regulator (CFTR) gene mutation and nutritional status have not yet been explored. Study aimed to assess the gut microbiota composition in adults with CF, and its relationship with the severity of CFTR mutations, and BMI. METHODS: Gut microbiota of 41 adults with CF, and 26 non-CF controls were compared using whole 16S rRNA gene sequencing. Differences in the microbial community between groups of patients classified according to the severity of CFTR mutations, and BMI were assessed. The alpha diversity, beta diversity, and taxa abundance were identified to reflect gut microbiota composition. RESULTS: Results showed a significant decrease in alpha diversity of bacterial communities in CF compared to non-CF group, but no significant difference between the CF groups distinguished by the severity of CFTR mutations. However, more severe mutations were associated with the higher relative abundance of Bacteroides and Streptococcus and the lower relative abundance of Faecalibacterium and Blautia. Undernourished CF patients showed significantly lower alpha diversity compared to non-CF group and CF patients with BMI within the norm. Significant differences in the structure of the gut microbiota between CF and non-CF groups, as well as between BMI groups were also found. CONCLUSIONS: Our research indicates that CF is associated with alterations in gut microbiota in adults. Additionally, in adult CF patients, the composition of the gut microbiota is also related to BMI.

Why Does Your Uterus Become Malignant? The Impact of the Microbiome on Endometrial Carcinogenesis.

The aim of this review was to describe the uterine microbiome composition that has been analyzed so far and describe potential pathways in the carcinogenesis of the endometrium. The microbiome in the uterine environment is involved in apoptosis and proliferation during the menstruation cycle, pregnancy maintenance, and immune system support. However, bacteria in the uterus could stimulate inflammation, which when chronic results in malignancy. An altered gut microbiota initiates an inflammatory response through microorganism-associated molecular patterns, which leads to intensified steroidogenesis in the ovaries and cancers. Moreover, intestinal bacteria secreting the enzyme ß-glucuronidase may increase the level of circulating estrogen and, as a result, be influential in gynecological cancers. Both the uterine and the gut microbiota play a pivotal role in immune modulation, which is why there is a demand for further investigation from both the diagnostic and the therapeutic perspectives.

Catch-Up Growth as a Risk Factor for Rapid Weight Gain, Earlier Menarche and Earlier Pubertal Growth Spurt in Girls Born Small for Gestational Age (SGA)-A Longitudinal Study.

Most children born small for gestational age (SGA) have rapid postnatal growth. Despite its positive aspects, catch-up growth may affect the level of adipose tissue in the pre-pubertal and pubertal periods and therefore affect the age of puberty. The aim of this study was to determine the associations between size at birth, catch-up growth in infancy, BMI in peripubertal period, age at menarche, and the parameters of adolescent growth spurt of body height in girls born SGA. For 297 girls (22.6% SGA; 77.4% appropriate for gestational age (AGA)) complete body weight and height measurements and age at menarche were obtained. Adolescent growth spurt parameters were estimated using the JPA2 model (AUXAL SSI 3.1). Calculations were made in the Statistica 13 program using the Kruskal-Wallis and Kaplan-Meier tests. Girls born SGA with catch-up had the highest BMIs at the age of 8 years (H = 94.22, p < 0.001) and at menarche (H = 58.21, p < 0.001), experienced menarche earliest (H = 21.77, p < 0.001), same as the onset (H = 6.54, p = 0.012) and peak height velocity (H = 11.71, p = 0.003) of their adolescent growth spurt compared to SGA girls without catch-up and AGA girls. In SGA girls, catch-up growth has far-reaching consequences such as increased risk of fat accumulation and a rapid transition to puberty.

Mutations in GHR and IGF1R Genes as a Potential Reason for the Lack of Catch-Up Growth in SGA Children.

The aim of this review was to describe all of the mutations in the growth hormone receptor (GHR) and insulin-like growth factor-1 receptor (IGF1R) genes that have been discovered so far, and their possible impact on final body height, as well as their relationship with catch-up growth in children born small for gestational age (SGA). Mutations in the GHR gene were found to cause a body height below -2 SD, from the mean for sex and age, whereas the mutations in the IGF1R gene were associated with low body height and intrauterine growth restriction (IUGR), and with being born SGA. After birth, when the child's growth is not restricted by the intrauterine environment, the infant may develop its developmental potential and experience catch-up growth, which makes it possible to catch up with peers born appropriate for gestational age (AGA). Despite this, catch-up growth does not apply to all, but only to about 85% of SGA children, and its mechanism is unknown. It is possible that SGA children who did not experience catch-up growth are carriers of mutations in the GHR and/or IGF1R genes.

The Mediating Role of the Gut Microbiota in the Physical Growth of Children.

Gut microbiota succession overlaps with intensive growth in infancy and early childhood. The multitude of functions performed by intestinal microbes, including participation in metabolic, hormonal, and immune pathways, makes the gut bacterial community an important player in cross-talk between intestinal processes and growth. Long-term disturbances in the colonization pattern may affect the growth trajectory, resulting in stunting or wasting. In this review, we summarize the evidence on the mediating role of gut microbiota in the mechanisms controlling the growth of children.

The determinants of survival among adults with cystic fibrosis-a cohort study.

BACKGROUND: Cystic fibrosis (CF) is one of the most common autosomal recessive diseases. Factors contributing to disease exacerbations and survival rate of CF patients are type of mutation in the CFTR gene, poor nutritional status, lung failure, and infection development by Pseudomonas aeruginosa. The study aimed to evaluate the relationship between the severity of mutation, nutritional status, lung function, and Pseudomonas aeruginosa prevalence and survival rate in adult patients with cystic fibrosis. METHODS: A study of 124 (68 ♀ and 56 ♂) adults with CF aged 18-51 years were evaluated for (a) type of mutation in the CFTR gene, (b) nutritional status (BMI), (c) lung function (FEV1%), and (d) Pseudomonas aeruginosa prevalence. For statistical calculations, Kaplan-Meier analysis of survival, chi-squared test for multiple samples, and logistic regression were used. RESULTS: The type of mutation (χ2 = 12.73, df = 3, p = 0.005), FEV1% (χ2 = 15.20, df = 2, p = 0.0005), Pseudomonas aeruginosa prevalence (χ2 = 11.48, df = 3, p = 0.009), and BMI (χ2 = 31.08, df = 4, p < 0.000) significantly differentiated the probability of survival of patients with CF. The shortest life expectancy was observed in patients with a severe type of mutation on both alleles, FEV1% < 40, subjects in whom Pseudomonas culture was extensively drug-resistant or pandrug-resistant, and patients whose BMI was lower than 18.5 kg/m2. The period from 30 to 40 years of age was the most critical in CF adults' lifespan. The risk of adults with CF death doubled with Pseudomonas aeruginosa prevalence (OR = 2.06, 95% CI 1.29; 2.28) and eightfold when the bacteria acquired antibiotic resistance (OR = 8.11, 95% CI 1.67; 38.15). CONCLUSIONS: All factors included in the study were significantly related to the survival rate of patients with cystic fibrosis.

Pseudomonas aeruginosa, the type of mutation, lung function, and nutritional status in adults with cystic fibrosis.

OBJECTIVE: The aim of this study was to describe the relationship between the prevalence of Pseudomonas aeruginosa (PA) and lung function, as well as the nutritional status and type of gene mutation in adult patients with cystic fibrosis (CF). METHODS: This cross-sectional study evaluated 103 Polish adults with CF the following: • The occurrence of PA and the level of bacterial susceptibility to antibiotics; • Type of mutation in the CFTR gene; • Nutritional status assessed by body mass index (BMI), and • Lung function measured by forced expiratory volume in 1 s (FEV1%). RESULTS: The absence or presence of PA and the level of bacterial resistance were significantly related to the type of gene mutation (P < 0.001). In patients with a severe mutation, PA more often was extensively drug resistant or pandrug resistant compared with Pseudomonas culture-negative patients or patients with mild or unclassified mutations on both alleles. Associations were found between the presence of PA and lower values of BMI (P < 0.001), and FEV1% (P < 0.001). The risk for PA occurrence and the development of bacterial resistance increased twice in the case of severe mutation (odds ratio [OR], 2.51; 95% confidence interval [CI], 1.62-3.89), four times when BMI decreased <18.5 (OR, 4.15; 95% CI, 1.43-10.08). and six times when FEV1% fell <40 (OR, 6.75; 95% CI, 3.11-14.64). CONCLUSIONS: The presence of PA is associated with lower FEV1% and BMI values. Deterioration of lung function, undernutrition, and severe type of gene mutation are linked to a higher probability of PA acquisition and resistance to antibiotic treatment.

Time intervals between the age at menarche and the onset of sexual activity in Polish women born between 1959 and 2001.

OBJECTIVES: The aim of this study was to assess time intervals between the age at menarche (AM) and age at sexual initiation (ASI) in Polish women born before and after the political transformation period in Poland. METHODS: Participants were 518 women born between 1959 and 2001 in Poland. AM and ASI were based on self-report. Three birth cohorts were distinguished based on the year of birth: I (1959-1979), II (1980-1989), III (1990-2001). Three categories of AM were distinguished: early maturing (<12.0 years), average (12.0-13.0 years), and late maturing (≥14.0 years). Temporal intervals between AM and ASI were calculated. RESULTS: Both AM and ASI decreased in subsequent birth cohorts, with the greatest changes observed in the women born after 1989. No significant differences in the time interval between AM and ASI were observed between birth cohorts. However, the time interval between AM and ASI differed by AM category (H = 74.16; P < .001). The longest time period between AM and ASI occurred in early maturing girls (7.13 years) and the shortest in late maturing girls (3.25 years). CONCLUSIONS: Despite the transformation and decline in both AM and ASI, the time interval between these two characteristics remained stable. Significant differences in the time interval were found among women stratified by AM. The time interval between AM and ASI was longer in girls who matured early in comparison to those maturing late or at the average time.

BMI at menarche and timing of growth spurt and puberty in Polish girls - longitudinal study.

Objectives: The purpose of the study was to evaluate the relationship between BMI at menarche and selected growth spurt parameters, such as age at take-off of the pubertal growth spurt (ATO), age at peak height velocity (APHV), growth spurt duration (APHV-ATO), and the pubertal growth rate. Methods: The study group consisted of 243 girls aged 10-16 years. Several measurements of body height and weight have been taken for each girl during the girls' development (longitudinal study). Nutritional status has been assessed using the IOTF (Cole et al. 2000; Cole et al. 2007). Girls were asked about their age at menarche. To investigate the growth rate, the age at TO and PHV for each girl was calculated using AUXAL.3 software and a structural model JPA2. Statistical analyses were made in Statistica 12.0 using Kruskal-Wallis test Kruskal-Wallis test and multiple regression analysis. Results: The mean age at menarche for the entire group was 12.59 (Me = 12.52, Min = 9.9, Max = 16.2). There was a significant statistical relationship between the level of BMI and age at menarche. The age at menarche decreased significantly with increasing levels of nutritional status (H = 33.2, p < 0.001). Also, TO and PHV occurred earlier in girls with higher values of BMI indicator (TO: H = 12.25, p < 0.05; PHV: H = 12.25, p < 0.05). Conclusions: The level of nutritional status at the age of menarche is significantly related to growth and the rate of biological maturation in girls. Higher BMI at menarche is associated with an earlier occurrence of menarche, TO and PHV.

Trends over time in age at sexual debut among Polish women and underlying socio-economic determinants.

ABSTRACT: The aim of the study was to evaluate (1) changes over time in age at sexual initiation among Polish women and (2) underlying socio-economic determinants. 273 women born between 1975 and 1995 in Wielkopolska region (Poland) filled in a questionnaire of sexual behavior. The questionnaire included questions about the age of sexual initiation and women's socio-economic status such as place of residence, level of education, type of school, marital status. Database and statistical analyzes were performed using Statistica 10.0. Age of sexual initiation differed significantly among the cohorts of women stratified by birth cohort (H = 61.4, p < 0.001). In cohort I (born between 1970-1975), mean age of initiation was 18.97 years, in cohort II (1976-1980) 18.67 years, in cohort III (1981-1985) 18.53 years; cohort IV (1986-1990) 18.27 years, and cohort V (1991-1995) 16.41 years. Two socio-economic variables significantly affected the age of the sexual activity onset: the type of school and level of education. The mean age of sexual debut among Polish women has significantly decreased over the last 40 years, with the largest, statistically significant difference observed between the most recent cohort of women (born between 1991-1995) and the other cohorts (born between 1970-1990). The most significant socio-economic determinants of age at first sex were the type of school and level of education.

Perinatal Risk Factors and ADHD in Children and Adolescents: A Hierarchical Structure of Disorder Predictors.

OBJECTIVE: The aim of the study was to hierarchically assess the predictive power of low and high birth weight, pre-term and post-term birth, and low Apgar score as the risk factors for ADHD. METHOD: The data of 132 boys diagnosed with ADHD and 146 boys from control group, aged 6 to 18 years, have been analyzed. The boys were categorized according to term of birth, birth weight, and Apgar score. CART method (Classification and Regression Trees) was used for assessment of the relationship between perinatal factors and the risk of ADHD. RESULTS: Low Apgar score (21.97% vs. 13.01%) and post-term birth (12.12% vs. 0.68%) were more frequent in the sample than in the control group. CART method additionally indicated low birth weight as associated with the risk of ADHD. Among analyzed risk factors, Apgar score had the highest predictive value. CONCLUSION: The decreased Apgar score is the most important perinatal risk factor of ADHD. Research results also indicated a high significance of post-term birth in predicting the disorder.

Digit ratio (2D:4D) and month of birth: A link to the solstitial-melatonin-testosterone effect.

BACKGROUND: Digit ratio (2D:4D) is a sexually dimorphic trait and its determination in utero is influenced by testosterone. The solstitial-melatonin-testosterone hypothesis posits that melatonin inhibits the production of foetal testosterone and melatonin levels are at their lowest in months when light levels are high. AIMS: We test the relationship between 2D:4D, month-of-birth and light levels. STUDY DESIGN: We recruited participants whose year of birth was spread across the 20th Century. SUBJECTS: 323 Polish men and women. OUTCOME MEASURES: Finger lengths, month-of-birth, mean daylight hours per month in and around Poznan, Poland. RESULTS: Our sample was born between 1907 and 1997. In comparison to late-Spring births, late-Autumn births had low right-left 2D:4D (high prenatal testosterone). Regarding light levels, there were significant relationships between low right 2D:4D and right-left 2D:4D (high prenatal testosterone) and long days at the end of the 1st trimester. These relationships were strongest for participants born in the first half of the 20th Century. CONCLUSIONS: Participants born in the late-Autumn and who experienced long days in the 2nd and 3rd prenatal months had low 2D:4D. The effects were strongest for early 20th Century births where photoperiods would be less disrupted by artificial light.

Attention-Deficit/Hyperactivity Disorder is Related to Decreased Weight in the Preschool Period and to Increased Rate of Overweight in School-Age Boys.

OBJECTIVE: Previous studies have associated attention-deficit/hyperactivity disorder (ADHD) with growth deviations and obesity. However, available data regarding the growth of children with ADHD in their early childhood are insufficient. Therefore, we aimed to examine whether there are differences in body size between preschool boys with and without ADHD. METHODS: The study used cross-sectional and retrospective longitudinal data concerning 112 boys with ADHD and a community-based sample of 308 boys without ADHD. The groups were homogeneous in terms of socioeconomic status, place of residence, term of birth, and birth weight. The average age of diagnosis was 8.3 years, and none of boys had been treated with stimulants before they were 7 years of age. Comparisons were made at the ages of 2, 4, and 6 years, for World Health Organization (WHO)-norm-standardized height, weight, body mass index (BMI), prevalence of underweight, overweight, and obesity. Separate analysis were made for the cross-sectional measurements of current body size. RESULTS: Boys with ADHD at the age of 2 had significantly lower z scores for weight (t=-1.98, p=0.04) and BMI (t=-2.09, p=0.04), and at the age of 4 for weight (t=-2.05, p=0.04) than the boys from the control group. A significantly lower percentage of overweight/obesity was observed in boys with ADHD at the age of 2 in comparison with the control group. At the age of 6, boys with ADHD were underweight more often. Cross-sectional analysis of current body size showed that boys with ADHD had lower z scores for height (t=-3.08, p=0.002) and higher z scores (t=3.13, p=0.002) for BMI. Overweight was more frequent in this group. CONCLUSIONS: Preschool boys with ADHD (age of 2-6 years) have a tendency toward lower body weight than their peers. But in subsequent phases of development, they are shorter and more frequently overweight than boys without ADHD, when place of residence, socioeconomic status, term of birth, birth weight, comorbid conditions, and treatment are controlled.

Growth and nutritional status of children from dysfunctional families with alcohol addicted parents in Poland.

The study was aimed at assessment of impact of parents' alcohol addiction on growth and prevalence of underweight and overweight in their children. Two groups of subjects were compared: 80 children of alcohol addicted parents (ChAAP) aged from 7 to 14 years and reference group (RG) of 1000 children selected in terms of age and place of residence. Differences in z scores for height and Body Mass Index (BMI), prevalence of underweight and overweight were assessed. Families of ChAAP were characterized by: lower parents' education, higher unemployment rate, a greater number of children than in RG. The differences between ChAAP and RG in z scores for height (z scores: -0.54 vs. 0.45, t = -7.01, p < 0.001) and BMI (z scores: -0.61 vs. 0.29, t = -6.28, p < 0.001) remained significant when impact of the parents' employment (for height: F = 8.88, p = 0.003; for BMI: F = 21.90, p < 0.001) and the number of children (for height: F = 30.89, p < 0.001; for BMI: F = 21.89, p < 0.001) were controlled. Children raised in families with alcohol addicted parents were shorter and had lower BMI than children of the reference group. Underweight was more frequent in that group, and overweight and obesity were more rare. The observed differences seem to result from other factors than bad living conditions, e.g.: chronic post-natal stress, or adverse events during fetal development.

ADHD and overweight in boys: cross-sectional study with birth weight as a controlled factor.

Population studies indicate a strong relationship between birth weight (BW) and body size in later life. However, BW as a variable was never accounted for in studies on the relationship between attention-deficit/hyperactivity disorder (ADHD) and overweight. This study aims to assess the relationship between ADHD and overweight with control of birth weight and other confounding factors. Prevalence of overweight was compared in clinical sample of 219 boys with ADHD and 396 boys without ADHD, aged 6-18 years. The following factors were controlled: BW, parents income and education level, place of residence, ADHD type, selected comorbid disorders and stimulant treatment. Overweight and obesity were diagnosed according to the criteria proposed by the International Obesity Task Force. Logistic regression analysis was used to estimate the association between ADHD and the prevalence of overweight and obesity. Boys with ADHD differed significantly from the control group in distribution of low BW (8.2 vs. 3.0 %, χ (2) = 8.23, p = 0.02). Low BW was associated with a lower prevalence of overweight than normal and high BW (0 vs. 12.14 %, χ (2) = 4.12, p = 0.04). Overweight was observed significantly more often in boys with ADHD (17.3 vs. 8.3 %, χ (2) = 11.23, p < 0.001) even after adjustment for BW and other variables (OR = 2.44, 95 % CI 1.38-4.29, p = 0.002) and after controlling for ADHD type, stimulant treatment and selected comorbid disorders. Independently to applied analysis, obesity was not associated with ADHD. Lower birth weight is over twice more often observed in boys with ADHD than in control group. Although this phenomenon may reduce the rate of overweight in the studied group, ADHD remains strongly associated with increased prevalence of overweight.

Influence of socioeconomic factors on age at menarche of Polish girls.

The aim of the study is to determine the influence of socioeconomic factors on age at menarche in Polish girls. Questionnaire data of 2016 girls were collected during the cross-sectional research. Within the socioeconomic variables parents' education, urbanization, number of children in the family and date of menarche were considered. To examine the effects of the analyzed socioeconomic factors on age at menarche, the analysis of variance and the Kaplan-Meier method were used. To estimate the mutual relations between the analyzed variables, the method of classification and regression trees (CART) was applied. The socioeconomic factors significantly affect age at menarche. The latest crossed threshold of puberty is observed in girls whose parents inhabit rural areas. Family size also affects age at menarche: girls from large families are the latest who have crossed the pubertal threshold. The method of classification and regression trees indicates that the most important predictive factor is the number of children in the family. The obtained results confirmed the complex effect of the analyzed variables. A factor that conditions occurrence of menarche most of all is the number of children in the family and then the urbanization degree of mother's place of residence. Further research is clearly required--especially research including analyses of mutual relations between variables and their complex effect.

[Assessment of the cognitive, functioning and emotional impairments in patients with Alzheimer's disease in relation to lifestyle behaviours in the stage of life prior to the diagnosis of disease]. / Ocena stopnia zaburzen procesów poznawczych, sprawnosci funkcjonowania oraz stanu emocjonalnego wsród pacjentów z choroba Alzheimera w odniesieniu do ich stylu zycia w okresie poprzedzajacym rozpoznanie choroby.

AIMS: The purpose of the study is to estimate whether the cognitive, functioning and emotional impairments in patients with Alzheimer's disease are related to lifestyle behaviours in the stage of life prior to the diagnosis of the disease. METHODS: Altogether, 65 patients with Alzheimer's disease, 55 women and 10 men, participants of the day center, run by the Wielkopolska Association of Alzheimer's Disease and residents of the Senior Nursing Home in Koprzywnica, were examined. Cognitive, functioning and emotional impairments of patients as well as lifestyle behaviours in the stage of life prior to the diagnosis of AD were estimated using AD-specific questionnaire, which were completed by caregivers. Qantitative and qualitative analyses were run using appropriate statistical procedures available in the Statistica 7.1 programme [StatSoft. Inc.2005 Statistica for Windows]. RESULTS: The findings revealed that patients with AD aged 70 years and older were likely to be more frequently impaired with daily life functioning than their younger counterparts. Patients with a higher attainment of education were more frequently impaired with cognitive than emotional problems and depression. Of all the variables in question, calendar age, educational attainment and intellectual activity in the stage of life prior to the diagnosis of disease were most significant in explaining the variation in the current impairments. Persons who were younger and intellectually active prior to the disease, were likely to be suffering less from the burden of AD than the older and less intellectually active counterparts. CONCLUSIONS: The association between educational attainment and intellectual activity in the stage of life prior to the occurrence of AD, and the burden of AD, found in the study, indicates that a lifelong intellectual activity may help to reduce disabilities and improve the patients' quality of life.

The second to fourth digit ratio and asymmetry.

BACKGROUND: Deviations of physical characteristics from bilateral symmetry, in otherwise symmetric individuals, are supposed to result from environmental perturbations during development. One cause of such perturbations may be sex steroids such as testosterone and oestrogen. AIM: The study examined the relationship between second to fourth digit ratio (2D:4D), a putative negative correlate with prenatal testosterone and a positive correlate with prenatal oestrogen, and asymmetry. METHODS: Eleven traits (including the second and fourth finger lengths) were measured in a sample of 680 English children aged 2-18 years, and second to fifth finger lengths in samples of 120 Austrian and English undergraduate students aged from 17 to 30 years and 213 Polish adults aged from 26 to 90 years. RESULTS: Significant U-shaped curvilinear associations between 2D:4D and all 11 traits were found in English children with the strongest associations between 2D:4D and composite asymmetry of second plus fourth digit, and second to fifth digits. Further investigation of the relation between 2D:4D and digit asymmetries in the sample of Austrian and English undergraduates and the Polish adults confirmed significant U-shaped relationships between 2D:4D and finger asymmetries. CONCLUSION: Our data show that both low 2D:4D (a marker of high prenatal testosterone) and high 2D:4D (a marker of high prenatal oestrogen) are associated with elevated levels of asymmetry and this relationship applies particularly to finger asymmetry.

[Cigarette smoking and the age of natural menopause in women in Poland]. / Palenie papierosów a wiek menopauzy naturalnej u kobiet w Polsce.

The empirical basis for this work has been the material obtained from cross-sectional studies carried out in the years 1998 to 2002, among women living in Poland. The material included data on 3793 women aged from 35 to 69 years. The women who participated in the research varied with regards to the age, education and socio-economic status. Menopausal status and median age at menopause was estimated with the use of retrospective and status-quo methods. It found out that the median age at natural menopause, estimated by probit analysis, was 50.33 years. The main aim of this work is to define whether and to what extent does smoking of cigarettes influence the age of natural menopause. Smoking of cigarettes increases the risk of earlier menopause (value of test F=138.8, p<0.0001). Non-smoking women experience menopause on average two years later than women who are habitual smokers. Also the number of cigarettes smoked every day differentiated the age of menopause, accelerating its occurrence in women who smoked more than five cigarettes per day (value of test F=50.81, p<0.0001).