Title: Comparative analysis of serum and saliva samples using Raman spectroscopy: A high-
throughput investigation in patients with polycystic ovary syndrome and periodontitis
Version: 6 Date: 26 Jul 2023
Reviewer's report:
Dear Authors,
I would like to extend our thanks for conducting this comparative analysis of serum and saliva
samples using Raman spectroscopy in patients with polycystic ovary syndrome (PCOS) and
periodontitis. Your study provides valuable insights into the potential application of Raman
spectroscopy as a high-throughput investigation tool for detecting periodontal status in PCOS
patients. I appreciate your efforts in shedding light on the relationship between these two
conditions and the implications for integrated healthcare. Based on the thorough analysis of your
revised work, I would like to offer the following recommendations to enhance the clarity and
impact of your efforts.
1.Abstract feedback :
1. Lack of Context: The abstract does not provide a clear context for the study. It would
be beneficial to briefly mention the prevalence or significance of PCOS and periodontitis to
highlight the relevance of the research question.
2. Sample Size and Representativeness: The abstract does not mention the size of the study
cohorts (PCOS patients and healthy volunteers), and whether they are representative of the
overall population. This information would be useful to assess the generalizability of the study
findings.
3. Methods Overview: While the abstract outlines the use of Raman spectroscopy to analyze
serum and saliva samples, it could benefit from a brief overview of the key steps involved in the
analysis to provide more clarity on the study design.
4. Missing Statistical Significance: The abstract mentions correlations between certain indicators
and periodontitis, but it does not specify the statistical significance of these correlations. Adding
the relevant P-values would strengthen the abstract's scientific rigor.
5. Implications and Recommendations: The abstract would benefit from a sentence or two
summarizing the implications of the study findings and potential recommendations for clinical
practice or future research.
6. Keywords: It is common to include a few specific keywords that highlight the study's key
focus areas. The abstract could include more relevant keywords related to Raman spectroscopy,
periodontal health, and PCOS to increase discoverability.
2.Background feedback comments:
1. Sample Selection: The study includes a relatively small sample size of 66 PCOS patients and
22 healthy volunteers. A larger sample could improve the generalizability and statistical power
of the findings.
2. Lack of Control for Confounders: The study does not account for potential confounding
factors, such as smoking habits, oral hygiene practices, or socioeconomic status, which might
influence periodontal health and PCOS.
3. Limited Generalizability: The study primarily focuses on Chinese women, limiting the
generalizability of the findings to other populations with different genetic and environmental
backgrounds.
4. Cross-Sectional Design: While the cross-sectional design allows for simultaneous assessment
of multiple variables, it cannot establish causality or examine changes over time. A longitudinal
study would provide more robust evidence of the relationships between PCOS, periodontal
health, and hormonal/metabolic profiles.
5. Raman Spectroscopy Interpretation: The study mentions Raman spectroscopy as a useful
tool but does not provide extensive details on its interpretation. Additional information or
collaboration with experts in the field could enhance the analysis.
3.Methods feedback comments:
1. Sample Size: While the study estimates the required sample size, the actual number of PCOS
patients (n=107) is relatively small compared to the healthy control group (n=22). A more
balanced sample size would enhance the reliability of the findings.
2. Generalizability: The study primarily focuses on Chinese patients, limiting the
generalizability of the results to other populations with different ethnic backgrounds and
lifestyles.
3. Cross-Sectional Design: As a cross-sectional study, the research cannot establish causality
or examine changes over time. Longitudinal studies would provide stronger evidence of the
temporal relationships between variables.
4. Potential Confounders: Although the study collects various clinical and demographic data,
certain potential confounding factors, such as dietary habits and socioeconomic status, are not
explicitly addressed.
5. Raman Spectroscopy Interpretation: The study mentions preprocessing steps for Raman
spectra but does not elaborate on the specific analysis of the spectra and the identification of
potential biomarkers, making it difficult to assess the reliability of the results.
6. Data Sharing: While the study mentions that the relevant script code for R analysis is available
at a specific link, it would be beneficial to include this information within the manuscript for
readers' ease.
- Figure1 feedback comments:
1. The image is not labeled clearly. The labels are small and difficult to read, and they do not
provide enough information about the different steps in the analysis. For example, the label for
the step "1000 centrifuged, 10 min" is not clear what it means.
2. The image is low-resolution, making it difficult to see the details. For example, it is difficult
to see the different types of cells in the blood sample.
3. The image is not well-organized. The different steps in the analysis are not clearly delineated,
and it is difficult to follow the flow of the process. For example, it is not clear how the different
steps relate to each other.
3.Results feedback comments:
1 Sample Size and Representativeness: The study includes 66 PCOS patients and 22 healthy
subjects, but the abstract does not clarify how these numbers were determined or whether they
represent a larger population. Providing more details about the sample size calculation and
recruitment process would strengthen the study's validity.
2. Statistical Significance: In the tables and text, it is essential to specify the level of statistical
significance for each comparison. Adding the actual P-values in the tables would make it easier
for readers to assess the significance of the findings.
3. Raman Spectroscopy Results: The Raman spectroscopy results are presented in figures, but
the interpretation of the spectra and their significance in relation to the study's objectives are not
discussed in detail. Elaborating on the specific molecular vibrations associated with PCOS and
periodontitis would enhance the understanding of the Raman spectroscopy findings.
4. Correlation Analysis: The correlation analysis is briefly mentioned, but the specific
correlations between variables (hormones, cytokines, Raman intensities, etc.) and their
implications are not thoroughly discussed. Providing more insights into the correlations
observed and their potential biological relevance would strengthen this section.
5. Limitations: While the results are reported comprehensively, it would be beneficial to include
a brief subsection on study limitations. Discussing potential biases, confounding factors, and
areas of uncertainty would add transparency and credibility to the study.
6. Data Interpretation: The results section presents a wealth of data, but more interpretation and
discussion of the clinical implications of the findings would be helpful. How do the observed
correlations and differences in Raman spectra relate to the overall association between PCOS
and periodontitis?
4.Statistical Analysis feedback comments:
1. Multiple Testing: The study performed numerous statistical tests to analyze various
parameters and outcome measures. However, there is no mention of multiple testing corrections
to control for the increased risk of false positives. Applying methods like Bonferroni correction
or false discovery rate control would be crucial to mitigate the impact of multiple comparisons.
2. Sample Size and Power: While the study included a reasonable sample size, it's essential
to consider statistical power when interpreting the results. A formal power analysis could help
determine if the sample size is sufficient to detect meaningful differences between groups.
3. Age Matching: As mentioned earlier, the age of the PCOS patients and healthy subjects differs
slightly. Since age can influence the results, it's important to address this potential confounding
factor through appropriate matching or statistical adjustment.
4. Correlation Analysis: The correlation analysis performed between clinical parameters and
Raman spectroscopy results is valuable. However, the study should account for multiple
comparisons when interpreting these correlations.
5. Validation of Classification Model: The PLS-DA classification model shows promising
results in discriminating between groups based on Raman spectroscopy results. To strengthen
the validity of this model, it's crucial to perform external validation on an independent dataset.
6. Ethical Considerations: The statistical analysis should also address any ethical considerations,
such as how missing data were handled, and whether any sensitivity analyses were performed.
7. Longitudinal Analysis: Given the cross-sectional design of the study, causal relationships
cannot be established. Future studies could consider longitudinal analysis or intervention studies
to explore causal associations.
5.Discussion feedback comments:
1. Addressing Inconsistencies: The discussion points out that the logistic regression analysis
results are inconsistent with previous studies. It would be helpful to discuss possible reasons for
these discrepancies and potential implications for the interpretation of the current findings.
2. Limitations: The limitations of the study are briefly mentioned, but they could be elaborated
further. For example, the study's cross-sectional design limits its ability to establish causal
relationships between PCOS and periodontitis. Discussing potential confounding factors and the
need for longitudinal investigations in the future would strengthen the discussion.
3. Sample Size: While the authors acknowledge the limited sample size as a potential limitation,
they could further emphasize the importance of larger sample sizes to provide more robust and
generalizable results.
4. Validation of Findings: The discussion mentions the potential of using Raman spectroscopy
for assessing periodontal conditions. However, further validation of these findings on
independent datasets or through longitudinal studies would add credibility to the proposed
approach.
5. Multiomics Approaches: The discussion suggests the use of multiomics approaches to identify
underlying molecular mechanisms. Providing specific examples of how these approaches could
be applied and what additional insights they may offer would enhance the discussion.
6. Clinical Implications: While the discussion mentions the clinical implications of the study, it
could be more explicit in discussing how the early detection of periodontitis in PCOS patients
can lead to improved overall healthcare and management of related conditions.
7. Ethical Considerations: It would be beneficial to briefly discuss any ethical considerations
related to the study, such as informed consent, data privacy, and handling of sensitive
information.
8. Generalization of Findings: The study focuses on a specific group of young women with
PCOS, and the authors acknowledge that the results may not fully represent the general
population. Discussing the potential implications and limitations of the findings in terms of
generalization would be valuable.
6.Conclusions feedback Comments:
1. Generalization: The conclusions should clearly state that the study's findings are based on
a specific group of young women with PCOS and periodontitis and may not fully represent
the general population. Addressing this limitation will provide a more accurate context for the
conclusions.
2. Validation: While the potential of Raman spectroscopy in detecting periodontal status is
discussed, the conclusions should also emphasize the need for further validation and replication
of the findings on larger and diverse cohorts to establish the method's reliability.
3. Ethical Considerations: The conclusions should briefly address any ethical considerations
related to the study, such as informed consent, data privacy, and handling of sensitive
information. This will reinforce the study's ethical integrity.
4. Future Directions: Adding a section on future research directions or potential follow-up
studies would provide insights into how the current findings can be expanded upon and how
they can contribute to the advancement of knowledge in this area.
5. Limitations: The conclusions should reiterate the limitations of the study, including the
cross-sectional design, small sample size, and potential for selection bias. Acknowledging these
limitations will ensure that the readers understand the study's scope and potential constraints.
7.Abbreviations feedback comments:
The list of abbreviations provided is helpful in understanding the various terms and variables
used in the study. It allows readers to quickly reference and comprehend key terms without
repetition. However, there are a few points that could be addressed to enhance clarity:
1. Abbreviation Definitions: While most of the abbreviations are self-explanatory, some less
common abbreviations could benefit from a brief definition or expansion in parentheses when
they are first mentioned in the text. This would aid readers who may not be familiar with
specialized medical or scientific terminology.
2. Abbreviations in Figures and Tables: It is important to ensure that all abbreviations used
in figures and tables are consistent with those listed in the Abbreviations section. Sometimes,
authors may inadvertently use different abbreviations or variations in the main text and figures,
leading to confusion for readers.
3. Order of Abbreviations: In the Abbreviations section, it might be helpful to organize the
abbreviations in alphabetical order for ease of reference. This will make it more convenient for
readers to find specific abbreviations.
4. Inclusion of Units: While some abbreviations represent measurements (e.g., BMI, WHR,
SBP), it could be beneficial to include the corresponding units (e.g., kg/m2, mmHg) alongside
the abbreviations to avoid any ambiguity.
5. Consistency in Presentation: Ensure consistent use of abbreviations throughout the text. For
example, if "PCOS" is used as an abbreviation for "polycystic ovary syndrome," it should be
consistently used in this way throughout the entire manuscript.