Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Analysis of epigenetic clocks links yoga, sleep, education, reduced meat intake, coffee, and a SOCS2 gene variant to slower epigenetic aging.

DNA methylation (DNAm) clocks hold promise for measuring biological age, useful for guiding clinical interventions and forensic identification. This study compared the commonly used DNAm clocks, using DNA methylation and SNP data generated from nearly 1000 human blood or buccal swab samples. We evaluated different preprocessing methods for age estimation, investigated the association of epigenetic age acceleration (EAA) with various lifestyle and sociodemographic factors, and undertook a series of novel genome-wide association analyses for different EAA measures to find associated genetic variants. Our results highlighted the Skin&Blood clock with ssNoob normalization as the most accurate predictor of chronological age. We provided novel evidence for an association between the practice of yoga and a reduction in the pace of aging (DunedinPACE). Increased sleep and physical activity were associated with lower mortality risk score (MRS) in our dataset. University degree, vegetable consumption, and coffee intake were associated with reduced levels of epigenetic aging, whereas smoking, higher BMI, meat consumption, and manual occupation correlated well with faster epigenetic aging, with FitAge, GrimAge, and DunedinPACE clocks showing the most robust associations. In addition, we found a novel association signal for SOCS2 rs73218878 (p = 2.87 × 10-8) and accelerated GrimAge. Our study emphasizes the importance of an optimized DNAm analysis workflow for accurate estimation of epigenetic age, which may influence downstream analyses. The results support the influence of genetic background on EAA. The associated SOCS2 is a member of the suppressor of cytokine signaling family known for its role in human longevity. The reported association between various risk factors and EAA has practical implications for the development of health programs to improve quality of life and reduce premature mortality associated with age-related diseases.

Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks.

BACKGROUND: DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. RESULTS: The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. CONCLUSIONS: We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.

Maternal Age and Behavior during Pregnancy Affect the 2D:4D Digit Ratio in Polish Children Aged 6-13 Years.

BACKGROUND: The length of the second and fourth finger calculated as a ratio (2D:4D) is a marker of prenatal exposure to sex hormones. Higher exposure to testosterone is related to a lower 2D:4D digit ratio, and inversely, higher exposure to estrogen is related to a higher 2D:4D. The digit ratio in humans (prenatally determined by sex hormone levels and androgen receptor activity) is associated with multiple biological, cognitive, and behavioral traits, as well as health in later life. The aim of this study was to verify if maternal traits during pregnancy are associated with 2D:4D among their children. METHODS: We investigated 537 boys, 646 girls, and their mothers. The investigation consisted of a questionnaire and a measurement part. In the questionnaire, we included questions about maternal traits during pregnancy such as: illnesses, active and passive smoking, work activity, psychological trauma (death or serious illness of a loved one, divorce, job loss), and age. We performed length measurements of the second and fourth fingers on both hands for both study groups. RESULTS: The GLM analysis showed that children of smoking mothers were characterized by a higher 2D:4D R as compared to their peers whose mothers did not smoke (ß = 0.10, p = 0.0008). In turn, the offspring of women who worked during pregnancy exhibited lower 2D:4D R values than the children of women who did not work (ß = -0.07, p = 0.0233). It should be noted, however, that the effects of those maternal factors were small, as each of them explained less than 1% of 2D:4D R in the population, adjusted for child age and sex. Pearson's linear correlation revealed that maternal age was negatively correlated with 2D:4D R in daughters (r = -0.11, p = 0.0137), but not in sons (r = 0.02, p = 0.6908). The negative correlation of 2D:4D R with maternal age indicates that the daughters of older mothers exhibited lower values of that ratio relative to the same-age daughters of younger mothers. CONCLUSIONS: For both study groups, the right-hand digit ratio was positively correlated with maternal smoking and negatively with maternal work during pregnancy. Maternal age during pregnancy was negatively related to right hand 2D:4D only among girls.

Evaluation of epigenetic age calculators between preeclampsia and normotensive pregnancies in an Australian cohort.

Advanced biological aging, as assessed through DNA methylation markers, is associated with several complex diseases. The associations between maternal DNA methylation age and preeclampsia (PE) have not been fully assessed. The aim of this study was to examine if increased maternal DNA methylation age (DNAmAge) was shown to be accelerated in women with PE when compared to women who had normotensive pregnancies. The case/control cohort available for study consisted of 166 women (89 with normotensive pregnancy, 77 with PE) recruited previously at the Royal Women's Hospital in Melbourne, Australia. DNA methylation profiles were obtained using the Illumina EPIC Infinium array for analysis of genomic DNA isolated from whole blood. These profiles were used to calculate seven estimates of DNAmAge and included (1) Horvath, (2) Hannum, (3) Horvath Skin and Blood, (4) Wu, (5) PhenoAge, (6) telomere length and (7) GrimAge and its surrogate measures. Three measures of DNA methylation age acceleration were calculated for all seven measures using linear regression. Pearson's correlation was performed to investigate associations between chronological age and DNAmAge. Differences between chronological age and DNAmAge and epigenetic age acceleration were investigated using t-tests. No significant difference was observed for chronological age between women with PE (age = 30.53 ± 5.68) and women who had normotensive pregnancies (age = 31.76 ± 4.76). All seven DNAmAge measures were significantly correlated (p < 0.001) with chronological age. After accounting for multiple testing and investigating differences in DNAmAge between normotensive women and women with PE, only Wu DNAmAge was significant (p = 0.001). When examining differences for epigenetic age acceleration between PE and normotensive women Hannum, Wu, and PhenoAge DNAmAge estimates (p < 0.001) were significant for both epigenetic age acceleration and intrinsic acceleration models. We found that accelerated maternal DNAmAge is increased in women with PE in some models of epigenetic aging. This research underlines the importance for further investigation into the potential changes of differential DNA methylation in PE.

Association of prenatal sex steroid exposure estimated by the digit ratio (2D:4D) with birth weight, BMI and muscle strength in 6- to 13-year-old Polish children.

OBJECTIVES: The aim of this paper was to provide evidence for the impact of prenatal sex steroid exposure on prenatal and postnatal body size parameters, and muscle strength in children. METHODS: The following anthropometric data were studied in a group of 1148 children (536 boys and 612 girls) aged 6-13 years: the 2D:4D digit ratio, birth weight and length, and birth head and chest circumference. Postnatal parameters (6-13 years) included body weight and height, BMI, waist and hip circumference, WHR, as well as grip strength in both hands. All parameters that required it were adjusted for sex and gestational or chronological age. A general linear model, Pearson's correlation, t-statistics and Cohen's Δ were used in statistical analysis. RESULTS: Among birth size parameters, only birth weight was significantly negatively correlated with the 2D:4D digit ratio in children. Higher (feminized) digit ratios were significantly correlated with postnatal parameters such as body weight, BMI, and waist and hip circumference (positively), as well as hand grip strength-a proxy for muscular strength (negatively). CONCLUSION: Problems with maintaining adequate body size parameters and muscle strength may be programmed in fetal life and predicted on the basis of the 2D:4D digit ratio. Body weight at birth and in early ontogenesis are additive correlates of the 2D:4D ratio. The present findings suggest that the 2D:4D digit ratio is related to postnatal phenotypes such as birth weight, overweight, and obesity as well as muscle strength in 6-13-year-old children of both sexes.

Understanding the Associations of Prenatal Androgen Exposure on Sleep Physiology, Circadian Proteins, Anthropometric Parameters, Hormonal Factors, Quality of Life, and Sex Among Healthy Young Adults: Protocol for an International, Multicenter Study.

BACKGROUND: The ratio of the second finger length to the fourth finger length (2D:4D ratio) is considered to be negatively correlated with prenatal androgen exposure (PAE) and positively correlated with prenatal estrogen. Coincidentally, various brain regions are sensitive to PAE, and their functions in adults may be influenced by the prenatal actions of sex hormones. OBJECTIVE: This study aims to assess the relationship between PAE (indicated by the 2D:4D ratio) and various physiological (sex hormone levels and sleep-wake parameters), psychological (mental health), and sexual parameters in healthy young adults. METHODS: This study consists of two phases. In phase 1, we will conduct a survey-based study and anthropometric assessments (including 2D:4D ratio and BMI) in healthy young adults. Using validated questionnaires, we will collect self-reported data on sleep quality, sexual function, sleep chronotype, anxiety, and depressive symptoms. In phase 2, a subsample of phase 1 will undergo polysomnography and physiological and genetic assessments. Sleep architecture data will be obtained using portable polysomnography. The levels of testosterone, estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, prolactin, melatonin, and circadian regulatory proteins (circadian locomotor output cycles kaput [CLOCK], timeless [TIM], and period [PER]) and the expression levels of some miRNAs will be measured using blood samples. The rest and activity cycle will be monitored using actigraphy for a 7-day period. RESULTS: In Poland, 720 participants were recruited for phase 1. Among these, 140 completed anthropometric measurements. In addition, 25 participants joined and completed phase 2 data collection. Recruitment from other sites will follow. CONCLUSIONS: Findings from our study may help to better understand the plausible role of PAE in sleep physiology, mental health, and sexual quality of life in young adults. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/29199.

Thyroid diseases and second to fourth digit ratio in Polish adults.

The association between second to fourth finger ratio and thyroid diseases is unexplained. There is a possible interaction between prenatal exposition to sex hormone and thyroid functions in the adulthood. The study included 175 adults investigated in Lódz in the central Poland. It consisted of two main parts: a survey including questions about occurrence of thyroid gland dysfunction and anthropometric measurements (body mass and height and length of the second and fourth finger, waist and hip circumferences). The women who had thyroid disease had higher 2D:4D digit ratio (left hand) (mean = 1.004; SD = 0.036) than healthy ones (mean = 0.989; SD = 0.030) (t = - 2105; p = 0.038; d = 0.707). The association between thyroid diseases occurrence and prenatal steroid hormone exposition is noticed. Only females who had thyroid diseases tend to have higher 2D:4D digit ratio, for left hand.

Cortisol concentration affects fat and muscle mass among Polish children aged 6-13 years.

BACKGROUND: Cortisol is a steroid hormone acting as a stress hormone, which is crucial in regulating homeostasis. Previous studies have linked cortisol concentration to body mass and body composition. METHODS: The investigations were carried out in 2016-2017. A total of 176 children aged 6-13 years in primary schools in central Poland were investigated. Three types of measurements were performed: anthropometric (body weight and height, waist and hip circumferences), body composition (fat mass FM (%), muscle mass - MM (%), body cellular mass - BCM (%), total body water - TBW (%)), and cortisol concentration using saliva of the investigated individuals. Information about standard of living, type of feeding after birth, parental education and maternal trauma during pregnancy was obtained with questionnaires. RESULTS: The results of regression models after removing the environmental factors (parental education, standard of living, type of feeding after birth, and maternal trauma during pregnancy) indicate a statistically significant association between the cortisol concentration and fat mass and muscle mass. The cortisol concentration was negatively associated with FM (%) (Beta=-0.171; p = 0.026), explaining 2.32 % of the fat mass variability and positively associated with MM (%) (Beta = 0.192; p = 0.012) explaining 3.09 % of the muscle mass variability. CONCLUSIONS: Cortisol concentration affects fat and muscle mass among Polish children. TRIAL REGISTRATION: The Ethical Commission at the University of Lodz (nr 19/KBBN-UL/II/2016).

Associations between second to fourth digit ratio, cortisol, vitamin D, and body composition among Polish children.

Associations between body characteristics (body composition: fat mass, muscle mass, cell, and water mass as well as body proportion-BMI), the 2D:4D digit ratio, and the concentrations of cortisol and vitamin (25-OH)D among Polish children have not been studied before. A total of 133 (73 girls and 60 boys) healthy Polish children aged 7-11 years were examined. The investigation was divided into three parts: measuring (the length of the second and fourth fingers in both hands, body composition, and body height and mass), questionnaires (socioeconomic status), and laboratory investigations (25-OH vitamin D and cortisol concentrations in saliva measured with ELISA methods). Boys with digit ratios below 1 had lower vitamin D concentration than those with digit ratios equal to or higher than 1 (Z = - 2.33; p = 0.019). Only boys with the male-typical pattern of 2D:4D digit ratio tended to have a lower 25-OH vitamin D concentration in saliva. Thus, it might indicate an effect of prenatal programming on the concentrations of steroid hormones in later life. Neither vitamin D, 2D:4D digit ratio nor the cortisol level was associated with the body components or proportions. More studies are needed to evaluate the molecular and genetic background of this phenomenon.

Association of saliva 25(OH)D concentration with body composition and proportion among pre-pubertal and pubertal Polish children.

OBJECTIVES: Due to increasing problems with obesity and vitamin D deficiency among children, studies that tackle both problems together are needed. METHODS: Data were collected from 182 randomly selected children aged 6-13 years in primary schools in central Poland. Measures included anthropometric dimensions, body composition, questionnaires completed by participants' parents, and saliva samples. The level of 25(OH)D was assessed from the saliva samples using an enzyme-linked immunosorbent assay kit. The children were divided into two groups: pre-pubertal (girls below 10 years and boys below 11 years) and pubertal individuals (girls above 10 years and boys above 11 years). RESULTS: The 25(OH)D concentrations were higher in late spring (June) among pre-pubertal children than in the autumn (November-December) among pubertal children. The level of 25(OH)D was positively correlated with body cell mass (BCM,%) among all children (pubertal: R = 0.20, P = .044; pre-pubertal: R = 0.23, P = .041) and inversely associated with waist-to-hip ratio (WHR) among pubertal children of both sexes (R = -0.25; P = .031). The stepwise regression analysis revealed that investigation in spring (June) and breastfeeding was associated with increased muscle mass (MM, %) (beta = 0.253, P = .003 and beta = 0.225, P = .005, respectively) and total body water (TBW, %) (beta = 0.276, P = .004 and beta = 0.246, P = .011, respectively) and was associated with decreased body mass index (BMI; beta = -0.222, P = .024 and beta = -0.269, P = .009, respectively) and fat mass (%) (beta = -0.288, P = .003 and beta = -0.266, P = .005, respectively). CONCLUSIONS: Season of salivary sampling and breastfeeding status were more strongly associated with body components, BMI and WHR, than 25(OH)D concentrations.

Familial factors more importantly modify the age of achieving motor developmental milestones than duration of breastfeeding amongst Polish children.

Breastfeeding in the first year of life is an important factor that may modify the child's later development.The aim of this study was to examine the association between the duration of breastfeeding and the time of achieving motor development of Polish children taking into account their familial background. The study material comprised 460 individuals including 252 boys and 208 girls aged 9 to 56 months who were born at term (37-42 weeks) selected in years 1993-1997. The investigated dependent variables were: first attempts of sitting, standing and walking (months). The independent variables were divided into: explanatory variable - the duration of breastfeeding, and other covariates - birth weight, the paternal and maternal age at the time of childbirth, maternal tobacco smoking during and after pregnancy as well as the parental level of education. The results of regression models after removing the prenatal and familial factors did not indicate a significant relationship between the duration of breastfeeding and sitting up (F = 0.03, p = 0.8569), standing up (F = 0.79, p = 0.3741) and walking variability (F = 0.20, p = 0.6568) in studied group of children. The results showed that parental age and education may affect the offspring's gross motor development, though effect sizes are small and directions of influence vary between fathers and mothers. The study underlines the importance of familial factors over the breastfeeding duration impact on time of developmental milestones.

The association between socioeconomic status, duration of breastfeeding, parental age and birth parameters with BMI, body fat and muscle mass among prepubertal children in Poland.

Objectives: We aimed to indicate simple determinants of abnormal body composition in children, such as socio-economic status (SES), duration of breastfeeding, parental age and birth parameters. Methods: The final data set consisted of 469 healthy prepubertal individuals (247 girls and 222 boys). We studied body mass, body height, and parameters of body composition such as muscle mass and fat mass. The birth parameters and gestational age were obtained from the children's medical record books held by the parents which were completed by medical personnel immediately following birth. Information about socio-economic status (SES), duration of breastfeeding and parental age was obtained by questionnaire. The statistical methods included forward multiple regression and generalized linear models (GLZ) or general linear model (GLM). Results: Higher fat mass (FM) (%) was connected with shorter duration of breastfeeding (< 2 months and lower SES (p < 0.05). Lower muscle mass (MM) (%) was linked with lower SES (p < 0.05) and lower birth weight (p < 0.05). Higher body mass index (BMI) was connected with higher birth weight (p < 0.05), shorter duration of breastfeeding (< 2 months) and lower SES (p < 0.05). Moreover interaction effects were observed in the case of the FM (%) (breastfeeding x SES; breastfeeding x parental age) and the BMI (breastfeeding x paternal age). Conclusions: Body composition can be linked with the duration of breastfeeding, SES, parental age, birth weight and birth length.

Association of the 2D:4D digit ratio with body composition among the Polish children aged 6-13 years.

The 2D:4D digit ratio is formed as a result of genetic factors but also prenatal exposure to sex hormones. The higher index value the higher concentration of the prenatal oestrogen. It is commonly known that testosterone is a hormone connected with muscle mass growth and that oestrogen affects adipogenesis. The aim of this study was to find if the digit ratio can be an informative indicator of the fat mass and muscle mass and body proportions in prepubertal children. Material and methods The analysed cohort included 420 children (221 girls and 199 boys) aged 6-13 years. Pearson's and Spearman's tests were conducted to assess whether 2D:4D was significantly correlated with the body composition measurements. Multiple regression models and stepwise forward regression were applied to select the most important independent variables affecting fat mass (%) and muscle mass (%) as well as the BMI and the WHR. Results The study shows that the digit ratio is negatively correlated with muscle mass (MM%) among girls (p < 0.05).There was no similar relationship in the group of boys. The regression models showed a significant role in determining the body composition and body proportions played by maternal factors such as: maternal level of education and weight gain during pregnancy. Conclusions The 2D:4D digit ratio seems to be an informative indicator of the muscle mass development since girls' early childhood. Moreover, maternal environment is also important in forming the offspring's body composition and proportions.

Meeting Report: Growth and Social Environment. Proceedings of the 25th Aschauer Soiree, held at Krobielowice, Poland, November 18th 2017.

Twenty-two scientists met at Krobielowice, Poland, to discuss the impact of the social environment, spatial proximity, migration, poverty, but also psychological factors such as body perception and satisfaction, and social stressors such as elite sports, and teenage pregnancies, on child and adolescent growth. The data analysis included linear mixed effects models with different random effects, Monte Carlo analyses, and network simulations. The work stressed the importance of the peer group, but also included historic material, some considerations about body proportions, and growth in chronic liver, and congenital heart disease.

Supplementation of vitamin D after birth affects body size and BMI in Polish children during the first 3.5 years of life - an analysis based on two cohorts measured in the years 1993-1997 and 2004-2008.

ABSTRACT: Objectives: The aim of this study was to assess whether the time of vitamin D supplementation after birth, season of birth and the type of feeding affected current body weight, body height and BMI among children aged 3-56 months from two cohorts. Additionally, it was analysed whether birth weight and birth length correlated with current BMI, body height and body weight in both cohorts of children. Methods: The study material included 1930 children from the two cohorts, measured in two following periods: 1993-1997 and 2004-2008. Finally 849 healthy individuals aged 3-56 months were included in the analysis. Multiple stepwise regression model was applied to find the group of the most important variables explaining current body weight, body height and BMI. Moreover generalized linear models for two way interactions were used. Results: The season of birth, time of supplementation of vitamin D, but also birth weight and length might affect current body weight. Individuals' height could be associated with birth length and the season of birth. BMI value was probably regulated by birth length and weight. Furthermore, interactions between the time of vitamin D supplementation and season of birth and also between the time of vitamin D supplementation and type of feeding resulting in variation of body weight and BMI in the first years of life were also observed. Conclusions: The study underlines the importance of a supplementation of vitamin D, season of birth and birth weight and length in current weight, height and body proportion in the first 3.5 years of life.