## 3 Obstacles in Oral Microbiome Studies
### Ethical Considerations  
Ethically, it is challenging to include healthy controls in microbiome studies involving OPMD and OSCC, as invasive procedures may not be justified in healthy individuals. The need to balance the scientific benefits of obtaining control samples with the ethical imperative to minimize harm to participants poses a significant dilemma in the design of such studies [161, 162]. Invasive sampling methods, such as biopsies, are essential for obtaining accurate and representative microbiome data from affected tissues. However, performing these procedures on healthy individuals solely for research purposes raises ethical concerns, including the potential for harm, discomfort, and unnecessary medical risks. Ethical guidelines and institutional review boards often require that the potential benefits of research outweigh the risks to participants, making it difficult to justify invasive procedures in healthy volunteers [161, 163]. To address these ethical challenges, researchers may opt for less invasive sampling methods, such as saliva or oral swabs, which can still provide valuable information about the oral microbiome without posing significant discomfort to participants. While these methods may not capture the full complexity of microbial communities associated with specific lesions, they offer a more ethically acceptable alternative for obtaining control samples [162]. Additionally, non-invasive sampling can facilitate the inclusion of a larger and more diverse cohort of healthy controls, enhancing the generalizability of study findings [163]. However, as mentioned earlier, saliva often fails to capture the lesion-specific microbial communities found in OPMD and OSCC tissues. The microbiological spectrum in biopsy samples is more localized and reflects the tumor microenvironment, including microbial-host interactions that may be missed in salivary analyses [164]. A recent umbrella review has highlighted significant differences between the microbiota profiles obtained from saliva and biopsy samples in OSCC patients. While salivary samples provide an overview of bacterial, viral, and fungal species present in the oral cavity, biopsy samples from OSCC lesions reveal specific microbial signatures more closely associated with the tumor microenvironment, such as _Fusobacterium nucleatum_ and _Candida albicans_. The review emphasized the complementary nature of these methods, with salivary sampling offering a non-invasive approach for broader screening and biopsy sampling providing detailed insights critical for diagnostic and prognostic purposes [19]. Incorporating these findings into ethical considerations is essential for future research. For example, combining non-invasive salivary sampling for initial screening with selective biopsy sampling in high-risk or symptomatic cases could balance ethical imperatives with the need for comprehensive microbiome data [19]. Another ethical consideration is informed consent. Participants must be fully informed about the nature of the study,the procedures involved, and the potential risks and benefits. Ensuring that participants understand and voluntarily agree to participate is crucial, particularly when dealing with invasive procedures. Researchers must provide clear and comprehensive information and allow participants to ask questions and withdraw from the study at any time without penalty [161]. Data privacy and confidentiality are also paramount in microbiome research. The unique nature of microbiome data, which can potentially be linked to personal health information, necessitates stringent measures to protect participant anonymity and data security. Researchers must adhere to data protection regulations and implement robust data management practices to ensure that sensitive information is not disclosed or misused [161]. Furthermore, there is an ethical obligation to ensure that the findings of microbiome research are translated into tangible benefits for the study population, particularly those suffering from OPMD and OSCC.