DNA methylation was the most extensively investigated epigenetic mechanism, representing approximately 70% of the included studies. Findings consistently demonstrate that methylation patterns at CpG sites provide excellent performance for estimating biological age, with high accuracy in samples such as blood, saliva, teeth, and bones. Models employing machine learning, penalized regression, and neural networks exhibited strong predictive capacity. Furthermore, studies show that methylation signatures enable the identification of biological fluids, distinguishing semen, blood, urine, saliva, and other tissues with high specificity. Another relevant finding concerns the ability to differentiate monozygotic twins, as environmental exposures accumulated over the lifespan generate divergent epigenetic profiles, overcoming limitations of traditional STR markers.

MicroRNAs have emerged as highly promising biomarkers due to their postmortem stability, resistance to environmental variations, and tissue-specific expression. The included studies show that microRNAs are useful for identifying bodily fluids and characterizing tissues in degraded samples. In addition, several articles report that microRNA profiles can estimate the postmortem interval (PMI) with greater accuracy than classical biochemical methods, particularly in cardiac and hepatic tissues. Evidence also indicates their usefulness in identifying specific causes of death, including acute myocardial infarction, trauma, and hypoxia.

Investigations into post-translational histone modifications were less numerous, reflecting an early stage of development in this area. Although biologically relevant, marks such as H3K4me3 and H3K27ac demonstrated limited applicability in real forensic contexts due to protein instability, susceptibility to environmental degradation, and the lack of standardized methods for recovering and analyzing these proteins in compromised samples. However, some experimental studies show that certain histone modifications may provide information about physiological state and pre-mortem gene activity, potentially contributing in the future to inferences related to cause of death.

A significant portion of the studies highlighted that environmental factors—such as smoking, air pollution, chronic stress, drug use, diet, and exposure to pesticides—generate detectable epigenetic signatures capable of reflecting recent or chronic exposures. Although promising, these approaches still lack robust population-level validation and rigorous control of confounding variables, which limits their immediate adoption in forensic practice. Nevertheless, the ability to capture biological markers of exposure positions epigenetics as a potential tool for reconstructing pre-crime events or physiological conditions preceding death.

Overall, the results indicate that forensic epigenetics is an expanding field, with greater maturity in research involving DNA methylation and microRNAs, while histone modifications and integrative analyses represent emerging frontiers. The literature highlights the need for standardization, multicenter validation, pre-analytical protocols, and population-based databases so that these techniques can be applied safely and reproducibly in routine criminal investigations.

The predominance of studies involving DNA methylation reinforces its role as the most established epigenetic mechanism in contemporary forensic practice [7][14][16][21][26]. The most mature and widely validated application is biological age estimation, with models based on CpG sites demonstrating errors of less than 5 years across various biological tissues [11]-[15]. However, relevant methodological limitations were identified, particularly the lack of standardization in CpG panels and the heterogeneity of statistical models, which complicate comparisons between studies [8][24][27][33]. The lack of multicenter validation further highlights the need to expand sample sizes and experimental settings before these biomarkers can be fully implemented in forensic routine practice [25][28].

Differentiation of monozygotic twins represents one of the most compelling advantages of epigenetic analysis over conventional STR-based profiling. While traditional DNA markers are identical between monozygotic twins, accumulating evidence shows that environmentally influenced DNA methylation patterns, microRNA profiles, and other epigenomic features diverge progressively across the lifespan [17][18][20][22].

Recent studies demonstrate that these epigenetic signatures may enable the identification of twin-specific molecular differences with increasing accuracy, particularly when high-resolution methylation arrays or next-generation bisulfite sequencing are used [14][21][23][29]. Although routine forensic adoption will require standardized panels, robust validation, and clearly defined discriminatory thresholds, the ability to distinguish monozygotic twins highlights a transformative application of forensic epigenetics—one not achievable with any current genetic profiling method.

MicroRNAs emerged as the second most investigated group and proved to be highly stable biomarkers, even in degraded samples [17][18]. Their robustness is further supported by broader epigenetic studies demonstrating the resilience of molecular markers under environmental and physiological stress conditions [19]-[21]. Their tissue specificity allows the identification of biological fluids—such as blood, semen, and saliva—complementing traditional protein-based methods and reflecting advances in forensic tissue discrimination and epigenomic profiling [22]-[24]. In addition, microRNA-based approaches have shown promising usefulness in estimating the postmortem interval (PMI) [17][25], and in identifying specific causes of death, including hypoxic and cardiovascular events—phenomena mechanistically linked to epigenetic responses to cellular stress and injury [26][27]. Despite their potential, universally validated panels are still lacking, and further studies applied to real forensic scenarios are needed—an issue aligned with ongoing discussions on methodological standardization and precision epigenomics [17][20][25].

Post-translational histone modifications were less frequently investigated due to protein instability and the difficulty of recovering them from degraded samples [26]. Although biologically relevant, these marks depend on highly controlled laboratory conditions, which limits their practical usefulness in criminal contexts [27]. Recent proteomic technologies, however, suggest potential advances that may support their future forensic application [28].

The included studies indicate that environmental exposures—such as smoking, pollution, stress, and pesticides—leave detectable epigenetic signatures [5][29]-[31]. Such markers may assist in reconstructing previous biological events relevant to criminal investigations [7]. However, interindividual variability and behavioral influences limit their immediate application [31][32]. Additionally, most studies originate from clinical rather than forensic contexts, highlighting important gaps for future research [32].

Many environmentally induced epigenetic signatures are inherently limited by their biological dynamics. Several methylation changes, particularly those associated with short-term exposures, are transient and may revert once the stimulus is removed, while others require prolonged or repeated exposure to stabilize sufficiently for forensic interpretation [1][32]. As a result, most validated exposure-associated markers reflect long-term lifestyle factors—such as smoking or chronic pollution exposure—rather than discrete, acute events. This distinction reinforces why environmental epigenetic profiles are more informative for reconstructing cumulative biological histories than for pinpointing a single momentary exposure relevant to a specific crime [5][32]-[36].

The lack of standardization in the collection, storage, and analysis of epigenetic material remains one of the principal barriers to operational implementation [7][8]. Likewise, the scarcity of multicenter studies with broad population diversity limits the establishment of universal recommendations that ensure reproducibility across laboratories [32]. In parallel, the integration of multiple epigenomic platforms—supported by advances in sequencing technologies and artificial intelligence—is emerging as a powerful direction for forensic science [21][23][37]. This multi-omics strategy enables the extraction of complementary layers of biological information from a single, often limited, evidentiary sample. For instance, DNA methylation markers can provide highly accurate age estimates [14][15], while microRNA profiles offer tissue-specific signatures capable of identifying the biological source of trace material [17][26]. When these modalities are combined, they allow investigators to infer both who the sample may have originated from and what type of tissue was deposited, substantially increasing evidentiary value in degraded or low-quantity samples. Such integrated approaches enhance discriminatory power, improve robustness under challenging forensic conditions, and align with emerging analytical pipelines that merge methylation sequencing, small-RNA profiling, and AI-based classification models [23][37]-[39].

Although epigenetic biomarkers increasingly demonstrate scientific value, their admissibility in judicial proceedings faces substantial practical and legal constraints. Courts applying either the Frye standard (“general acceptance”) or Daubert criteria (“testability, peer review, error rates, standards, and known limitations”) require forensic methods to demonstrate analytical validity, reproducibility, and transparent quality-control procedures—requirements that remain only partially fulfilled for DNA methylation panels, microRNA signatures, histone-based assays, and other emerging platforms [7][28][39]. Recent reviews emphasize that, unlike conventional genetic markers, epigenetic assays are subject to additional biochemical, environmental, and computational sources of variability, complicating claims of stability and reliability [1][2][32].

From a practical standpoint, the absence of harmonized pre-analytical protocols, standardized bisulfite-conversion methods, sequencing workflows, and validated machine-learning models remains a major barrier to operationalization [8][10][23]. The Scientific Working Group on DNA Analysis Methods (SWGDAM) underscores the need for rigorous validation, interlaboratory concordance studies, and full transparency in analytical reporting before any technique can be introduced in court [40]. Moreover, because epigenetic states are dynamic and influenced by environmental exposures, lifestyle, medications, aging, and stochastic variation [30][31][41], defense arguments may question whether these biomarkers possess adequate temporal stability for individual attribution—a challenge observed in debates over early STR interpretation, mitochondrial DNA sequencing, and probabilistic genotyping models [42][43]. Additional evidence demonstrating methylation variability under environmental stress, pollution, or sample degradation further reinforces the need for strict control of analytical conditions [44]-[46].

Legally, admissibility also requires clear articulation of error rates, confidence intervals, and methodological limitations—elements increasingly scrutinized in forensic surveys and judicial analyses [47][48]. Yet, most epigenetic applications, including age estimation, tissue identification, and lifestyle inference, still lack population-scale validation and defined statistical thresholds for uncertainty [13][15][49]. Ethical and privacy concerns introduce an additional dimension of legal scrutiny, as methylation or microRNA profiles may reveal sensitive health, behavioral, or environmental exposure information not directly relevant to the forensic question, paralleling debates in forensic phenotyping and polygenic risk inference [20][30][43].

To progress toward courtroom admissibility, forensic epigenetics must therefore meet several criteria: 1) development of standardized, forensic-appropriate protocols endorsed by accrediting bodies [7][8]; 2) robust multicenter validation using diverse population datasets to establish generalizability and reproducibility [7][32][45]; 3) formal determination of error rates, decision thresholds, and confidence intervals for each specific application [48]; 4) implementation of transparent reporting frameworks that clearly communicate assumptions, analytical steps, and limitations [48][49]; and 5) full compliance with legal disclosure standards to support cross-examination and judicial review. By aligning methodological development with these scientific and legal expectations, epigenetic evidence may progressively advance toward meeting admissibility criteria and supporting responsible, proportionate integration into modern forensic practice [7][8].

The literature consistently highlights that forensic epigenetics holds substantial potential to transform criminal investigation, offering analytical advantages that surpass those of traditional genetic markers, including greater sensitivity, tissue specificity, and the ability to infer biological age, tissue origin, and lifestyle exposures [50]-[52]. Consolidation of these applications, however, depends on achieving rigorous methodological standardization, multicenter validation, and the development of robust population-level reference databases capable of supporting reproducible interpretation across laboratories [53][54]. Recent technological advances are expected to accelerate this maturation process: third-generation sequencing and high-resolution methylome profiling are expanding analytical precision [55]-[57], while artificial intelligence and machine-learning models increasingly enable integration of complex epigenomic, transcriptomic, and exposure-related signals into predictive forensic frameworks [58]-[61]. Together, these developments suggest that epigenetics is moving steadily toward operational feasibility, provided that foundational validation and standardization challenges are effectively addressed.

Thus, this review demonstrates that forensic epigenetics is in a transitional stage, evolving from a promising experimental framework into a scientifically grounded approach with growing potential for integration into routine forensic practice. At the same time, as epigenetic analyses increasingly allow inferences about sensitive lifestyle and health-related information, future applications must carefully address the ethical and privacy implications to ensure that these technologies are implemented responsibly, transparently, and proportionately within the justice system.