Organ-specific biological clocks: Ageotyping for personalized anti-aging medicine.

Aging is a complex multidimensional, progressive remodeling process affecting multiple organ systems. While many studies have focused on studying aging across multiple organs, assessment of the contribution of individual organs to overall aging processes is a cutting-edge issue. An organ's biological age might influence the aging of other organs, revealing a multiorgan aging network. Recent data demonstrated a similar yet asynchronous inter-organs and inter-individuals progression of aging, thereby providing a foundation to track sources of declining health in old age. The integration of multiple omics with common clinical parameters through artificial intelligence has allowed the building of organ-specific aging clocks, which can predict the development of specific age-related diseases at high resolution. The peculiar individual aging-trajectory, referred to as ageotype, might provide a novel tool for a personalized anti-aging, preventive medicine. Here, we review data relative to biological aging clocks and omics-based data, suggesting different organ-specific aging rates. Additional research on longitudinal data, including young subjects and analyzing sex-related differences, should be encouraged to apply ageotyping analysis for preventive purposes in clinical practice.

Where are we in the implementation of tissue-specific epigenetic clocks?

Introduction: DNA methylation clocks presents advantageous characteristics with respect to the ambitious goal of identifying very early markers of disease, based on the concept that accelerated ageing is a reliable predictor in this sense. Methods: Such tools, being epigenomic based, are expected to be conditioned by sex and tissue specificities, and this work is about quantifying this dependency as well as that from the regression model and the size of the training set. Results: Our quantitative results indicate that elastic-net penalization is the best performing strategy, and better so when-unsurprisingly-the data set is bigger; sex does not appear to condition clocks performances and tissue specific clocks appear to perform better than generic blood clocks. Finally, when considering all trained clocks, we identified a subset of genes that, to the best of our knowledge, have not been presented yet and might deserve further investigation: CPT1A, MMP15, SHROOM3, SLIT3, and SYNGR. Conclusion: These factual starting points can be useful for the future medical translation of clocks and in particular in the debate between multi-tissue clocks, generally trained on a large majority of blood samples, and tissue-specific clocks.

eXplainable Artificial Intelligence (XAI) in aging clock models.

XAI is a rapidly progressing field of machine learning, aiming to unravel the predictions of complex models. XAI is especially required in sensitive applications, e.g. in health care, when diagnosis, recommendations and treatment choices might rely on the decisions made by artificial intelligence systems. AI approaches have become widely used in aging research as well, in particular, in developing biological clock models and identifying biomarkers of aging and age-related diseases. However, the potential of XAI here awaits to be fully appreciated. We discuss the application of XAI for developing the "aging clocks" and present a comprehensive analysis of the literature categorized by the focus on particular physiological systems.

Associations of circulating GDF15 with combined cognitive frailty and depression in older adults of the MARK-AGE study.

Growth differentiation factor-15 (GDF15) might be involved in the development of cognitive frailty and depression. Therefore, we evaluated cross-sectional associations of plasma GDF15 with combined cognitive-frailty-and-depression in older (i.e. ≥ 55 years) and younger adults of the MARK-AGE study. In the present work, samples and data of MARK-AGE ("European study to establish bioMARKers of human AGEing") participants (N = 2736) were analyzed. Cognitive frailty was determined by the global cognitive functioning score (GCF) and depression by the Self-Rating Depression Scale (SDS score). Adults were classified into three groups: (I) neither-cognitive-frailty-nor-depression, (II) either-cognitive-frailty-or-depression or (III) both-cognitive-frailty-and-depression. Cross-sectional associations were determined by unadjusted and by age, BMI, sex, comorbidities and hsCRP-adjusted linear and logistic regression analyses. Cognitive frailty, depression, age and GDF15 were significantly related within the whole study sample. High GDF15 levels were significantly associated with both-cognitive-frailty-and-depression (adjusted ß = 0.177 [0.044 - 0.310], p = 0.009), and with low GCF scores and high SDS scores. High GDF15 concentrations and quartiles were significantly associated with higher odds to have both-cognitive-frailty-and-depression (adjusted odds ratio = 2.353 [1.267 - 4.372], p = 0.007; and adjusted odds ratio = 1.414 [1.025 - 1.951], p = 0.035, respectively) independent of age, BMI, sex, comorbidities and hsCRP. These associations remained significant when evaluating older adults. We conclude that plasma GDF15 concentrations are significantly associated with combined cognitive-frailty-and-depression status and, with cognitive frailty and depressive symptoms separately in old as well as young community-dwelling adults.

Epigenetics of the far northern Yakutian population.

BACKGROUND: Yakuts are one of the indigenous populations of the subarctic and arctic territories of Siberia characterized by a continental subarctic climate with severe winters, with the regular January average temperature in the regional capital city of Yakutsk dipping below - 40 °C. The epigenetic mechanisms of adaptation to such ecologies and environments and, in particular, epigenetic age acceleration in the local population have not been studied before. RESULTS: This work reports the first epigenetic study of the Yakutian population using whole-blood DNA methylation data, supplemented with the comparison to the residents of Central Russia. Gene set enrichment analysis revealed, among others, geographic region-specific differentially methylated regions associated with adaptation to climatic conditions (water consumption, digestive system regulation), aging processes (actin filament activity, cell fate), and both of them (channel activity, regulation of steroid and corticosteroid hormone secretion). Further, it is demonstrated that the epigenetic age acceleration of the Yakutian representatives is significantly higher than that of Central Russia counterparts. For both geographic regions, we showed that epigenetically males age faster than females, whereas no significant sex differences were found between the regions. CONCLUSIONS: We performed the first study of the epigenetic data of the Yakutia cohort, paying special attention to region-specific features, aging processes, age acceleration, and sex specificity.

Epigenetic aging differences between Wichí and Criollos from Argentina: Insights from genomic history and ecology.

Background and objectives: Epigenetic estimators based on DNA methylation levels have emerged as promising biomarkers of human aging. These estimators exhibit natural variations across human groups, but data about indigenous populations remain underrepresented in research. This study aims to investigate differences in epigenetic estimators between two distinct human populations, both residing in the Gran Chaco region of Argentina, the Native-American Wichí, and admixed Criollos who are descendants of intermarriages between Native Americans and the first European colonizers, using a population genetic approach. Methodology: We analyzed 24 Wichí (mean age: 39.2 ± 12.9 yo) and 24 Criollos (mean age: 41.1 ± 14.0 yo) for DNA methylation levels using the Infinium MethylationEPIC (Illumina) to calculate 16 epigenetic estimators. Additionally, we examined genome-wide genetic variation using the HumanOmniExpress BeadChip (Illumina) to gain insights into the genetic history of these populations. Results: Our results indicate that Native-American Wichí are epigenetically older compared to Criollos according to five epigenetic estimators. Analyses within the Criollos population reveal that global ancestry does not influence the differences observed, while local (chromosomal) ancestry shows positive associations between specific SNPs located in genomic regions over-represented by Native-American ancestry and measures of epigenetic age acceleration (AgeAccelHannum). Furthermore, we demonstrate that differences in population ecologies also contribute to observed epigenetic differences. Conclusions and implications: Overall, our study suggests that while the genomic history may partially account for the observed epigenetic differences, non-genetic factors, such as lifestyle and ecological factors, play a substantial role in the variability of epigenetic estimators, thereby contributing to variations in human epigenetic aging.

Epigenomic signature of accelerated ageing in progeroid Cockayne syndrome.

Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.

Hormesis defines the limits of lifespan.

This commentary provides a novel synthesis of how biological systems adapt to a broad spectrum of environmental and age-related stresses that are underlying causes of numerous degenerative diseases and debilitating effects of aging. It proposes that the most fundamental, evolutionary-based integrative strategy to sustain and protect health is based on the concept of hormesis. This concept integrates anti-oxidant, anti-inflammatory and cellular repair responses at all levels of biological organization (i.e., cell, organ and organism) within the framework of biphasic dose responses that describe the quantitative limits of biological plasticity in all cells and organisms from bacteria and plants to humans. A major feature of the hormetic concept is that low levels of biological, chemical, physical and psychological stress upregulate adaptive responses that not only precondition, repair and restore normal functions to damaged tissues/organs but modestly overcompensate, reducing ongoing background damage, thereby enhancing health beyond that in control groups, lacking the low level "beneficial" stress. Higher doses of such stress often become counterproductive and eventually harmful. Hormesis is active throughout the life-cycle and can be diminished by aging processes affecting the onset and severity of debilitating conditions/diseases, especially in elderly subjects. The most significant feature of the hormetic dose response is that the limits of biological plasticity for adaptive processes are less than twice that of control group responses, with most, at maximum, being 30-60 % greater than control group values. Yet, these modest increases can make the difference between health or disease and living or dying. The quantitative features of these adaptive hormetic dose responses are also independent of mechanism. These features of the hormetic dose response determine the capacity to which systems can adapt/be protected, the extent to which biological performance (e.g., memory, resistance to injury/disease, wound healing, hair growth or lifespan) can be enhanced/extended and the extent to which synergistic interactions may occur. Hormesis defines the quantitative rules within which adaptive processes operate and is central to evolution and biology and should become transformational for experimental concepts and study design strategies, public health practices and a vast range of therapeutic strategies and interventions.

Small immunological clocks identified by deep learning and gradient boosting.

Background: The aging process affects all systems of the human body, and the observed increase in inflammatory components affecting the immune system in old age can lead to the development of age-associated diseases and systemic inflammation. Results: We propose a small clock model SImAge based on a limited number of immunological biomarkers. To regress the chronological age from cytokine data, we first use a baseline Elastic Net model, gradient-boosted decision trees models, and several deep neural network architectures. For the full dataset of 46 immunological parameters, DANet, SAINT, FT-Transformer and TabNet models showed the best results for the test dataset. Dimensionality reduction of these models with SHAP values revealed the 10 most age-associated immunological parameters, taken to construct the SImAge small immunological clock. The best result of the SImAge model shown by the FT-Transformer deep neural network model has mean absolute error of 6.94 years and Pearson ρ = 0.939 on the independent test dataset. Explainable artificial intelligence methods allow for explaining the model solution for each individual participant. Conclusions: We developed an approach to construct a model of immunological age based on just 10 immunological parameters, coined SImAge, for which the FT-Transformer deep neural network model had proved to be the best choice. The model shows competitive results compared to the published studies on immunological profiles, and takes a smaller number of features as an input. Neural network architectures outperformed gradient-boosted decision trees, and can be recommended in the further analysis of immunological profiles.

Uncovering the Relationship between Selenium Status, Age, Health, and Dietary Habits: Insights from a Large Population Study including Nonagenarian Offspring from the MARK-AGE Project.

An inadequate selenium (Se) status can accelerate the aging process, increasing the vulnerability to age-related diseases. The study aimed to investigate plasma Se and Se species in a large population, including 2200 older adults from the general population (RASIG), 514 nonagenarian offspring (GO), and 293 GO Spouses (SGO). Plasma Se levels in women exhibit an inverted U-shaped pattern, increasing with age until the post-menopausal period and then declining. Conversely, men exhibit a linear decline in plasma Se levels with age. Subjects from Finland had the highest plasma Se values, while those from Poland had the lowest ones. Plasma Se was influenced by fish and vitamin consumption, but there were no significant differences between RASIG, GO, and SGO. Plasma Se was positively associated with albumin, HDL, total cholesterol, fibrinogen, and triglycerides and negatively associated with homocysteine. Fractionation analysis showed that Se distribution among plasma selenoproteins is affected by age, glucometabolic and inflammatory factors, and being GO or SGO. These findings show that sex-specific, nutritional, and inflammatory factors play a crucial role in the regulation of Se plasma levels throughout the aging process and that the shared environment of GO and SGO plays a role in their distinctive Se fractionation.

Dose imbalance of DYRK1A kinase causes systemic progeroid status in Down syndrome by increasing the un-repaired DNA damage and reducing LaminB1 levels.

BACKGROUND: People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. METHODS: Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. FINDINGS: Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. INTERPRETATION: Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. FUNDING: Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".

Expanding evolutionary theories of ageing to better account for symbioses and interactions throughout the Web of Life.

How, when, and why organisms age are fascinating issues that can only be fully addressed by adopting an evolutionary perspective. Consistently, the main evolutionary theories of ageing, namely the Mutation Accumulation theory, the Antagonistic Pleiotropy theory, and the Disposable Soma theory, have formulated stimulating hypotheses that structure current debates on both the proximal and ultimate causes of organismal ageing. However, all these theories leave a common area of biology relatively under-explored. The Mutation Accumulation theory and the Antagonistic Pleiotropy theory were developed under the traditional framework of population genetics, and therefore are logically centred on the ageing of individuals within a population. The Disposable Soma theory, based on principles of optimising physiology, mainly explains ageing within a species. Consequently, current leading evolutionary theories of ageing do not explicitly model the countless interspecific and ecological interactions, such as symbioses and host-microbiomes associations, increasingly recognized to shape organismal evolution across the Web of Life. Moreover, the development of network modelling supporting a deeper understanding on the molecular interactions associated with ageing within and between organisms is also bringing forward new questions regarding how and why molecular pathways associated with ageing evolved. Here, we take an evolutionary perspective to examine the effects of organismal interactions on ageing across different levels of biological organisation, and consider the impact of surrounding and nested systems on organismal ageing. We also apply this perspective to suggest open issues with potential to expand the standard evolutionary theories of ageing.

Aging and chronic inflammation: highlights from a multidisciplinary workshop.

Aging is a gradual, continuous series of natural changes in biological, physiological, immunological, environmental, psychological, behavioral, and social processes. Aging entails changes in the immune system characterized by a decrease in thymic output of naïve lymphocytes, an accumulated chronic antigenic stress notably caused by chronic infections such as cytomegalovirus (CMV), and immune cell senescence with acquisition of an inflammatory senescence-associated secretory phenotype (SASP). For this reason, and due to the SASP originating from other tissues, aging is commonly accompanied by low-grade chronic inflammation, termed "inflammaging". After decades of accumulating evidence regarding age-related processes and chronic inflammation, the domain now appears mature enough to allow an integrative reinterpretation of old data. Here, we provide an overview of the topics discussed in a recent workshop "Aging and Chronic Inflammation" to which many of the major players in the field contributed. We highlight advances in systematic measurement and interpretation of biological markers of aging, as well as their implications for human health and longevity and the interventions that can be envisaged to maintain or improve immune function in older people.

Dementia, infections and vaccines: 30 years of controversy.

This paper reports the proceedings of a virtual meeting convened by the European Interdisciplinary Council on Ageing (EICA), to discuss the involvement of infectious disorders in the pathogenesis of dementia and neurological disorders leading to dementia. We recap how our view of the infectious etiology of dementia has changed over the last 30 years in light of emerging evidence, and we present evidence in support of the implication of infection in dementia, notably Alzheimer's disease (AD). The bacteria and viruses thought to be responsible for neuroinflammation and neurological damage are reviewed. We then review the genetic basis for neuroinflammation and dementia, highlighting the genes that are currently the focus of investigation as potential targets for therapy. Next, we describe the antimicrobial hypothesis of dementia, notably the intriguing possibility that amyloid beta may itself possess antimicrobial properties. We further describe the clinical relevance of the gut-brain axis in dementia, the mechanisms by which infection can move from the intestine to the brain, and recent findings regarding dysbiosis patterns in patients with AD. We review the involvement of specific pathogens in neurological disorders, i.e. SARS-CoV-2, human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV1), and influenza. Finally, we look at the role of vaccination to prevent dementia. In conclusion, there is a large body of evidence supporting the involvement of various infectious pathogens in the pathogenesis of dementia, but large-scale studies with long-term follow-up are needed to elucidate the role that infection may play, especially before subclinical or clinical disease is present.

Inflammaging: mechanisms and role in the cardiac and vasculature.

Aging triggers a wide range of cellular and molecular aberrations in the body, giving rise to inflammation and associated diseases. In particular, aging is associated with persistent low-grade inflammation even in absence of inflammatory stimuli, a phenomenon commonly referred to as 'inflammaging'. Accumulating evidence has revealed that inflammaging in vascular and cardiac tissues is associated with the emergence of pathological states such as atherosclerosis and hypertension. In this review we survey molecular and pathological mechanisms of inflammaging in vascular and cardiac aging to identify potential targets, natural therapeutic compounds, and other strategies to suppress inflammaging in the heart and vasculature, as well as in associated diseases such as atherosclerosis and hypertension.

Epigenetic clocks suggest accelerated aging in patients with isolated REM Sleep Behavior Disorder.

Isolated REM Sleep Behavior Disorder (iRBD) is the strongest prodromal marker for α-synucleinopathies. Overt α-synucleinopathies and aging share several mechanisms, but this relationship has been poorly investigated in prodromal phases. Using DNA methylation-based epigenetic clocks, we measured biological aging in videopolysomnography confirmed iRBD patients, videopolysomnography-negative and population-based controls. We found that iRBDs tended to be epigenetically older than controls, suggesting that accelerated aging characterizes prodromal neurodegeneration.

Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Centenarian clocks: epigenetic clocks for validating claims of exceptional longevity.

Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible evidence. Here, we present three DNA methylation-based age estimators (epigenetic clocks) for verifying age claims of centenarians. The three centenarian clocks were developed based on n = 7039 blood and saliva samples from individuals older than 40, including n = 184 samples from centenarians, 122 samples from semi-supercentenarians (aged 105 +), and 25 samples from supercentenarians (aged 110 +). The oldest individual was 115 years old. Our most accurate centenarian clock resulted from applying a neural network model to a training set composed of individuals older than 40. An epigenome-wide association study of age in different age groups revealed that age effects in young individuals (age < 40) are correlated (r = 0.55) with age effects in old individuals (age > 90). We present a chromatin state analysis of age effects in centenarians. The centenarian clocks are expected to be useful for validating claims surrounding exceptional old age.

Long-term human spaceflight and inflammaging: Does it promote aging?

Spaceflight and its associated stressors, such as microgravity, radiation exposure, confinement, circadian derailment and disruptive workloads represent an unprecedented type of exposome that is entirely novel from an evolutionary stand point. Within this perspective, we aimed to review the effects of prolonged spaceflight on immune-neuroendocrine systems, brain and brain-gut axis, cardiovascular system and musculoskeletal apparatus, highlighting in particular the similarities with an accelerated aging process. In particular, spaceflight-induced muscle atrophy/sarcopenia and bone loss, vascular and metabolic changes, hyper and hypo reaction of innate and adaptive immune system appear to be modifications shared with the aging process. Most of these modifications are mediated by molecular events that include oxidative and mitochondrial stress, autophagy, DNA damage repair and telomere length alteration, among others, which directly or indirectly converge on the activation of an inflammatory response. According to the inflammaging theory of aging, such an inflammatory response could be a driver of an acceleration of the normal, physiological rate of aging and it is likely that all the systemic modifications in turn lead to an increase of inflammaging in a sort of vicious cycle. The most updated countermeasures to fight these modifications will be also discussed in the light of their possible application not only for astronauts' benefit, but also for older adults on the ground.

Circulating miR-122-5p, miR-92a-3p, and miR-18a-5p as Potential Biomarkers in Human Liver Transplantation Follow-Up.

The requirement of blood-circulating sensitive biomarkers for monitoring liver transplant (LT) is currently a necessary step aiming at the reduction of standard invasive protocols, such as liver biopsy. In this respect, the main objective of this study is to assess circulating microRNA (c-miR) changes in recipients' blood before and after LT and to correlate their blood levels with gold standard biomarkers and with outcomes such as rejection or complications after graft. An miR profile was initially performed; then, the most deregulated miRs were validated by RT-qPCR in 14 recipients pre- and post-LT and compared to a control group of 24 nontransplanted healthy subjects. MiR-122-5p, miR-92a-3p, miR-18a-5p, and miR-30c-5p, identified in the validation phase, were also analyzed considering an additional 19 serum samples collected from LT recipients and focusing on different follow-up (FU) times. The results showed significant, FU-related changes in c-miRs. In particular, miR-122-5p, miR-92a-3p, and miR-18a-5p revealed the same trend after transplantation and an increase in their level was found in patients with complications, independently from FU times. Conversely, the variations in the standard haemato-biochemical parameters for liver function assessment were not significant in the same FU period, confirming the importance of c-miRs as potential noninvasive biomarkers for monitoring patients' outcomes.