## 3 Obstacles in Oral Microbiome Studies
### Causality Dilemma  
A critical question in oral microbiome research is whether changes in the microbiome are a cause or a consequence of malignant transformation. This causality dilemma complicates the interpretation of findings and the development of targeted interventions. Determining the directionality of the relationship between microbial shifts and disease progression is essential for understanding the underlying mechanisms and for designing effective therapeutic strategies [168; 169]. One of the primary challenges in addressing this causality dilemma is the complex interplay between the host and microbial communities. Changes in the oral microbiome may precede the development of OPMD and OSCC, suggesting a potential causative role. For instance, certain pathogenic bacteria, such as _Fusobacterium nucleatum_ and _Porphyromonas gingivalis_, have been implicated in promoting carcinogenesis through mechanisms like inducing inflammation, immune modulation, and direct invasion of epithelial cells [168; 170]. These bacteria can create a microenvironment conducive to malignant transformation by disrupting normal cellular processes and promoting genomic instability [170]. On the other hand, malignant transformation and the associated changes in the tissue environment can also influence the composition of the oral microbiome. Tumor development can alter local conditions, such as oxygen availability, pH, and nutrient levels, which in turn can select for specific microbial populations. This suggests that observed changes in the microbiome might be a consequence rather than a cause of malignancy [169]. For example, the metabolic by-products of cancer cells, such as lactate, can provide a favorable environment for the growth of certain anaerobic bacteria, potentially leading to dysbiosis [169]. Longitudinal studies are crucial for disentangling this causality dilemma. By tracking microbial changes over time in individuals at risk of developing OPMD or OSCC, researchers can identify early microbial alterations that may serve as predictive markers of disease onset. These studies can help determine whether microbial shifts precede malignant transformation or occur as a result of it [7]. However, conducting such studies poses significant logistical and ethical challenges, particularly in obtaining repeated invasive samples from participants. Experimental models, including animal studies and in vitro systems, can also provide valuable insights into causality. These models allow for the manipulation of specific microbial populations and the observation of their effects on host tissues in a controlled environment. For example, studies using germ-free mice colonized with specific oral bacteria can help elucidate the direct effects of these microbes on tissue inflammation, immune responses, and tumor development [168]. Advanced analytical techniques, such as multi-omics approaches, can further aid in resolving the causality dilemma. Integrating genomic, transcriptomic, proteomic, and metabolomics data can provide a comprehensive view of the interactions between the host and the microbiome. This holistic approach can reveal how microbial changes influence host cellular pathways and vice versa, offering clues about the directionality of these interactions [169; 170]. Understanding the causality of microbiome changes in the context of OPMD and OSCC is essential for developing targeted interventions. If specific microbial shifts are identified as causative factors, therapeutic strategies could focus on modulating the microbiome to prevent or slow down disease progression. Conversely, if microbial changes are primarily consequences of malignancy, interventions might focus on mitigating their effects to improve patient outcomes [168; 169]. Figure 2 provides a concise summary of all the previously mentioned obstacles in oral microbiome studies, along with proposed solutions for each.