The epigenetic clock is the core tool to quantify the process of biological aging, and the scientific interpretation of its detection results is the key link between the research and development of connection technology and clinical application. With the development of precision medicine, the clock has expanded from a simple age prediction model to a multi-dimensional indicator for assessing health risks, monitoring disease progress, and measuring intervention effects. However, the interpretation of its results is often challenged due to data complexity, individual heterogeneity, and differences in technology platforms, and it is urgent to establish a standardized and easy-to-operate interpretation path.

Therefore, constructing a clear and scalable interpretation method is not only a prerequisite to improve the efficiency of research data transformation, but also an important basis to help clinicians and researchers quickly extract core information and formulate targeted programs, which is of great practical significance to promote clinical transformation in the field of epigenetic aging.

The article outlines four key aspects (core metrics, IEAA/EEAA, confidence intervals, advanced clocks) for interpreting epigenetic clock results and their significance in aging research and clinical use.

The Key Metrics: DNAmAge and Age Acceleration (AA)

In the field of epigenetic clock research, DNA methylation age (DNAmAge) and age acceleration (AA) are the core quantitative indicators to evaluate the process of biological aging. DNAmAge is calculated by the methylation pattern of specific CpG sites, which can objectively reflect the biological age of the body. AA, as the difference between DNAmAge and actual chronological age, can accurately capture the individual difference in aging rate. Together, they constitute a key tool for analyzing aging mechanisms, predicting disease risk, and evaluating intervention effects, and provide a standardized quantitative dimension for aging-related research.

Defining DNAmAge: the Internal Timer of Life

DNAmAge is a biological age estimated based on DNA methylation patterns, and it is an important concept in aging research. DNA methylation is an epigenetic modification that plays a key role in cell development, differentiation, and aging by adding methyl groups to regulate gene expression without changing the DNA sequence. With the increase in age, DNA methylation patterns will change regularly, recording the aging process of individuals.

• Algorithms and models: Scientists analyze a large amount of DNA methylation data of individuals of different ages, establish algorithms and models, and estimate the biological age of individuals according to the level of DNA methylation. For example, the Horvath clock is based on the methylation level of 353 CpG loci, and the prediction error of age is only 3.6 years. The Hannum clock is based on 71 CpG loci, and it performs well in predicting the age of a specific population.
• Important position: DNAmAge is extremely important in the study of aging, which provides an index to quantify the degree of aging and can more accurately evaluate the aging state of individuals. The traditional measurement of aging degree by actual age can not fully reflect the individual's physiological state and health status, and the aging degree of different people varies greatly when they are at the same actual age. And the influence of DNAmAge on aging can better reflect the real aging degree of the body.

Interpretation of AA: Fast-forward and Slow-forward Keys of Aging

AA refers to the difference between DNAmAge and actual age, which is the core index to evaluate the aging speed and health risk of individuals. AA is positive, indicating that DNAmAge is older than the actual age, aging is accelerated, and health risks are high. AA is negative, indicating that DNAmAge is younger than the actual age, aging is slowed down, and the health status may be better.

• AA is very important in health risk assessment. Numerous studies have shown that AA is closely related to the risk of various chronic diseases: for every year of accelerated epigenetic age, all-cause mortality increases by 9%.
• In patients with type 2 diabetes mellitus, AA was positively correlated with HOMA-IR (p<0.001). The higher AA value, the more severe insulin resistance, and the higher risk of diabetic complications.
• It is found that high-level moderate-intensity physical activities (such as brisk walking, jogging, cycling, slow swimming, etc.) can effectively delay aging; However, excessive physical activity has the opposite effect, and lipids play an intermediary role in it. AA, as a key evaluation index, reveals the relationship between exercise intensity and aging, and provides a scientific basis for making a reasonable exercise plan.
• In addition, AA is an important risk predictor in cardiovascular disease research. Long-term follow-up study of a large-scale population shows that the risk of cardiovascular disease is significantly increased in individuals with higher AA. This may be a series of pathophysiological changes, such as impaired vascular endothelial function, enhanced inflammatory response, and abnormal blood lipid metabolism, which may increase the risk of cardiovascular diseases.

Age acceleration versus number of somatic mutations in the TCGA data (Horvath et al., 2013)

Intrinsic vs. Extrinsic Age Acceleration (IEAA vs. EEAA)

Internal and external age acceleration (IEAA and EEAA) are the core quantitative indicators in the field of epigenetic clock, which together reveal the difference between biological aging and actual chronological age.

• IEAA focuses on the internal aging process dominated by genetic background, and its fluctuation is mostly related to endogenous factors such as genome stability and cell metabolism.
• EEAA pays more attention to the impact of environmental exposure, covering exogenous variables such as lifestyle and pollutant exposure.

Clarifying the definition and difference between them is an important theoretical basis for analyzing the aging mechanism and carrying out disease risk assessment.

Intracellular Secrets: IEAA

IEAA is an important concept in aging research, which helps to understand the process of cell aging. It refers to the accelerated aging phenomenon driven by internal factors of cells, which is not interfered with by external factors such as immune cells.

The key factors affecting IEAA include telomere shortening and mitochondrial dysfunction. Telomeres, located at the end of chromosomes, play a key role in cell division. With cell division, telomeres gradually shorten to a certain extent, and cells enter an aging state. Mitochondria are energy factories of cells. With the increase in age, their functions decline, energy production decreases, and a large number of reactive oxygen species (ROS) are produced. ROS will oxidize and damage intracellular biomolecules and accelerate cell aging.

IEAA is unique in many diseases:

• Neurodegenerative diseases, such as Alzheimer's disease, in patients with brain neurons, IEAA is accelerated, telomeres are shortened, and mitochondrial function is impaired, resulting in abnormal energy metabolism, influence on neurotransmitter synthesis and release, and cognitive impairment and memory loss.
• In cardiovascular diseases, the IEAA of vascular endothelial cells in patients with atherosclerosis is accelerated, the function of endothelial cells is damaged, the vasodilation and contraction cannot be maintained, and the vascular wall is thickened and hardened, which increases the risk of cardiovascular diseases.

Ripple of the Immune System: EEAA

EEAA is different from the IEAA, which is mainly influenced by the changes of immune cell composition and function, and has a far-reaching impact on the aging process. Immune cells are an important part of the human immune system and play a key role in maintaining immune balance and resisting pathogen invasion. However, with the increase in age, changes in the composition and function of immune cells lead to changes in EEAA.

• Changes of immune cells: The variety and quantity of immune cells change significantly with the increase of age, such as the decrease of the number of T and B cells and the increase of the proportion of memory T and B cells, which leads to the decline of immune system function. At the same time, the ability of immune cells to activate, proliferate, and secrete cytokines decreases, which affects the normal function of the immune system.
• Inflammation: Inflammation plays a key role in EEAA. Age makes the body appear in a chronic low-grade inflammatory state, that is, inflammatory aging, and the level of inflammatory factors in the body continues to rise, sending out inflammatory signals, leading to cell and tissue damage and accelerating the aging process. Inflammation also affects the function of immune cells, produces more inflammatory factors, and aggravates EEAA.
• Related to diseases: EEAA is closely related to the occurrence and development of many inflammation-related diseases. In patients with rheumatoid arthritis, due to abnormal activation of the immune system, the inflammatory reaction is intensified, EEAA is accelerated, the level of inflammatory factors in the body continues to rise, and immune cells attack joint tissues, resulting in joint swelling, pain, and deformity. In cardiovascular diseases, inflammation damages vascular endothelial cells, promotes the formation of atherosclerosis, and the increase of EEAA aggravates the condition.

Comprehensive analysis of IEAA and EEAA is of great significance to comprehensively evaluate individual health status and the aging process. In practical application, detecting their levels combined with blood biochemical indicators, genetic test results and other health indicators can more accurately assess health risks and aging degree, help to find health problems at an early stage, formulate personalized health management programs, and take intervention measures such as lifestyle adjustment, nutritional intervention and drug treatment to delay aging and prevent and control disease development.

Estimated parameters by quantile with 95% confidence limits for the effect of the MetS severity score on IEAA (Nannini et al., 2019)

Context is King: Understanding the Confidence Interval

The extensive application of the epigenetic clock promotes the development of quantitative evaluation of aging, but it is not perfect. Attention should be paid to the error range and confidence interval when using it, which has an important impact on evaluating individual aging degree, health decision-making, and research conclusions. The key role is discussed below.

Boundary of Test: Error Range of the Epigenetic Clock

In the field of science, no test is absolutely perfect, and neither is the epigenetic clock. Although it provides strong support for evaluating the biological age of individuals, it is limited by the current technical level and has an error range of 3-5 years.

From a technical point of view, the reasons for the error are as follows:

• There are limitations in DNA methylation detection technology. Although the commonly used microarray technology and high-throughput sequencing technology can detect a large number of CpG loci, their accuracy and sensitivity need to be improved. The former is affected by probe hybridization efficiency, and the methylation level of some sites is not accurate. The latter may have a base mismatch when sequencing, which affects the judgment of the methylation level.
• Differences in experimental operation will introduce errors. The way and time of sample collection, processing, and preservation, as well as the stability and accuracy of experimental instruments, may lead to errors. Different laboratories will have different test results due to different experimental conditions and operating procedures, which will increase the uncertainty of epigenetic clock prediction.

Individual genetic differences and the diversity of the living environment are also important factors leading to errors:

• The genetic background is unique, and genetic factors affect the pattern and rate of DNA methylation, which makes the prediction of the epigenetic clock biased. Some genetic variations lead to abnormal gene methylation levels, which affect the calculation of epigenetic age.
• Living environment factors, such as diet, exercise, psychological stress, and environmental pollution, will have an impact on DNA methylation. Individuals who have been exposed to a polluted environment for a long time have changed DNA methylation patterns, and the difference between epigenetic age and actual age has increased.

The power of Trends: Tracking Changes Rather Single Data

In aging research and health management, although the epigenetic clock can estimate the biological age of an individual, the single detection data is disturbed by factors such as detection error, fluctuation of physiological state, and short-term change of living environment, and it is difficult to fully and accurately reflect the health status and aging process of an individual, which has limitations.

Tracking the change trend of the epigenetic clock with time can reflect the change in individual health more accurately. Multiple tests can average the impact of accidental factors on the data, making the real trend of the individual aging process clearer. The accelerated growth of epigenetic age may mean that the body has undergone adverse changes. If the trend is stable or even slows down, it may indicate that the health condition is improving or the health intervention measures are effective.

Studies have shown that the growth rate of epigenetic age of the elderly who maintain a healthy lifestyle is slower than that of the elderly who have an unhealthy lifestyle. The study of cancer patients found that the changing trend of the epigenetic clock can be used as an important index to evaluate the therapeutic effect of cancer and the prognosis of patients.

In practical application, the health management strategy can be adjusted in time according to the changing trend of the epigenetic clock. If the epigenetic age increases rapidly, it is necessary to deeply analyze the reasons and take targeted measures to delay the aging process and maintain health.

Bivariate twin models for epigenetic age metrics (Miao et al., 2024)

Beyond a Single Number: The Phenotypic Age and GrimAge Clocks

With the development of science and technology, scientists in the field of aging research pursue more accurate and comprehensive aging assessment tools to deeply understand the essence of aging and help human health. In this process, a new generation of clocks, such as the phenotypic age clock and GrimAge clock, appeared, which broke through the traditional limitations, revealed a new dimension of aging, and pushed aging research into a new stage.

Transcending Tradition: New Perspective on the Phenotypic Age Clock

The phenotypic age clock is an important innovation in aging research, which provides a new perspective for evaluating aging. It breaks the traditional concept of measuring aging only by time, and comprehensively reflects the actual aging degree of the body by integrating the inflammatory level, glucose metabolism, renal function, and hematological indicators.

Compared with the epigenetic clock based on DNA methylation, the phenotypic age clock has unique advantages. It not only pays attention to the changes of cell molecules, but also comprehensively considers the state of various systems of the body from the overall physiological function. When evaluating aging, the epigenetic clock changes according to the DNA methylation pattern, and the phenotypic age clock is included in the inflammatory index, because it plays a key role in the occurrence and development of chronic diseases and can directly reflect the health status and aging process.

In disease prediction, the phenotypic age clock is excellent:

• The study of cardiovascular disease found that the estimated biological age is closely related to the risk of cardiovascular disease. When the phenotypic age is significantly higher than the actual age, the risk of cardiovascular disease increases greatly, because its comprehensive blood pressure, blood lipid, and blood sugar are important risk factors for cardiovascular disease, which can provide early warning and provide a basis for prevention and early intervention.
• In the study of diabetes, it also has a good predictive performance. The phenotypic age of diabetic patients is higher than that of ordinary people, and it is related to the occurrence of complications. Because of the metabolic disorder caused by diabetes, it can capture these changes and provide valuable information for the health management of patients.

Early Warning of Death Risk: Insight into the GrimAge Clock

The GrimAge clock is an important breakthrough in aging research. Based on DNA methylation patterns and integrating lifestyle factors such as smoking history, a model for predicting the time of individual death, cancer, and coronary heart disease is constructed by a complex algorithm.

• Calculation method and principle: A large number of DNA methylation sites related to aging and diseases are deeply analyzed, and the laws of their changes with age and lifestyle are used to model, with special attention to smoking-related methylation sites, to more accurately assess health risks.
• Advantages: Unlike the traditional epigenetic clock, which focuses on estimating biological age, the GrimAge clock directly focuses on the prediction of death risk and disease susceptibility. The research shows that the death risk increases by about 50% and the cancer risk increases by 20% with high accuracy and sensitivity.
• Application: In cancer screening, individuals with high predictive value can be classified as high-risk groups of cancer, and high-level screening is recommended first to improve the early diagnosis rate. In the aspect of cardiovascular disease prevention, it can help identify individuals at high risk of coronary heart disease and intervene in advance to reduce the risk of onset.

Associations between frailty and different epigenetic clock measures (Verschoor et al., 2021)

Conclusion

With the global population aging, aging research has become the core topic of life science. Epigenetic clock based on DNA methylation provides a new perspective and method for aging research. Its DNAmAge and AA can quantify aging and assess risks, and IEAA and EEAA can deepen the understanding of the aging mechanism. Pay attention to the 3-5 year error in the application, and track the change trend more accurately. A new generation of clocks, such as the phenotypic age clock and GrimAge clock, has made significant progress.

In the future, aging research is expected to make breakthroughs in technical aspects and intervention strategies, and technologies such as cellular-resolution methods and multi-omics will help to analyze the aging mechanism, and it is expected to develop new drugs and other intervention measures. Aging research has a far-reaching impact on individual health and the social economy, and it is expected to realize the transformation from treating diseases to promoting healthy aging.

Key Insights for Epigenetic Clock Result Interpretation

FAQ

1. If AA is positive, does it mean I will definitely develop chronic diseases soon?

No. A positive AA indicates that your biological age is older than your actual age and your aging rate is accelerated, which will increase the risk of chronic diseases. However, it does not mean that you will definitely develop chronic diseases soon.

2. Which is more suitable for me to use, the PhenoAge clock or the GrimAge clock?

It depends on your core needs. If you want to comprehensively evaluate your overall physiological function and predict the risk of chronic diseases, the PhenoAge clock is more suitable. If your focus is on predicting long-term health risks, the GrimAge clock is a better choice.

3. Can IEAA and EEAA be used together to assess aging, and what should be paid attention to when using them?

Yes, combining IEAA and EEAA can more comprehensively assess the aging process. IEAA reflects the internal aging driven by factors, while EEAA reflects the aging impact of external factors such as immune cells and inflammation.

References

1. Horvath S. "DNA methylation age of human tissues and cell types." Genome Biol. 2013 14(10): R115.
2. Nannini DR, Joyce BT, Zheng Y, et al. "Epigenetic age acceleration and metabolic syndrome in the coronary artery risk development in young adults study." Clin Epigenetics. 2019 11(1): 160.
3. Miao K, Liu S, Cao W, et al. "Five years of change in adult twins: longitudinal changes of genetic and environmental influence on epigenetic clocks." BMC Med. 2024 22(1): 289.
4. Verschoor CP, Lin DTS, Kobor MS, et al. "Epigenetic age is associated with baseline and 3-year change in frailty in the Canadian Longitudinal Study on Aging." Clin Epigenetics. 2021 13(1): 163.