Title: Comparative analysis of serum and saliva samples using Raman spectroscopy: A high-
throughput investigation in patients with polycystic ovary syndrome and periodontitis
Version: 2 Date: 03 Jun 2023
Reviewer's report:
Dear Authors,
Thank you for submitting your manuscript for review. Your study explores the association
between polycystic ovary syndrome (PCOS) and periodontal disease, providing valuable
insights into the potential link between these two conditions.  your work shows promise and has
the potential to contribute to the existing body of knowledge on PCOS and periodontal disease.
We look forward to reviewing the revised version of your manuscript.
I have carefully reviewed the manuscript and have identified some under-focused points that I
believe should be addressed:
1.I suggest alternative titles that could be more informative:
• "Comparative Analysis of Serum and Saliva Samples Using Raman Spectroscopy: A High-
Throughput Investigation in Patients with Polycystic Ovary Syndrome and Periodontitis"
• "Diagnostic Potential of Raman Spectroscopy: Evaluating Serum and Saliva Samples in
Patients with Polycystic Ovary Syndrome and Periodontitis"
2.Abstract main notes:
• Sample Size: The sample size in this study is relatively small, consisting of 66 females
with PCOS and 22 systemically healthy volunteers. A larger sample size would improve the
generalizability of the results and increase statistical power.
• Lack of Longitudinal Design: The study employs a cross-sectional design, which limits the
ability to establish causal relationships between PCOS and periodontal disease. A longitudinal
design would provide stronger evidence of the temporal relationship between these variables.
• Limited Generalizability: The study includes only female participants, which restricts the
generalizability of the findings to a broader population. Including male participants and diverse
demographic groups would enhance the external validity of the study.
• Potential Confounders: The study does not account for potential confounding factors such
as oral hygiene practices, socioeconomic status, and lifestyle factors (e.g., smoking), which
could influence the association between PCOS and periodontal conditions. Controlling for these
variables would strengthen the validity of the results.
• Raman Spectroscopy Analysis: While the use of Raman spectroscopy is innovative, the study
does not provide detailed information about the specific variables measured or the methodology
employed. Further explanation and validation of the Raman spectroscopy analysis would
enhance the reproducibility and reliability of the results
3.Main background notes:
• Limited Background Information: Although the background section provides some
information about the prevalence and impact of PCOS and periodontal diseases, it lacks a
comprehensive overview of existing literature and gaps in knowledge. A more extensive and up-
to-date review of relevant studies would strengthen the rationale for conducting this research.
• Lack of Specific Research Objectives: The study does not clearly state the specific research
objectives or hypotheses that will be addressed. Clearly defined objectives would provide a
focused direction for the study and aid in the interpretation of the findings.
• Unclear Methodological Approach: The study mentions the use of Raman spectroscopy
to analyze serum and saliva samples, but it does not provide sufficient details about the
methodology employed. Lack of information on the specific parameters measured, the
instrumentation used, and the analysis techniques employed raises concerns about the reliability
and validity of the results.
• Insufficient Sample Size Justification: The study does not provide a rationale for the chosen
sample size of the participants. A justification based on power analysis or previous studies would
enhance the robustness of the sample size determination.
• Lack of Control Group: The study only includes participants with PCOS, but does not
include a control group of individuals without PCOS. A control group would allow for a
comparison of periodontal conditions and metabolic profiles between PCOS patients and
individuals without PCOS, strengthening the findings and providing a clearer understanding of
the unique contributions of PCOS.
• Absence of Statistical Analysis Details: The study mentions comparing periodontal status with
hormonal and metabolic profiles, but it does not specify the statistical tests or analyses that will
be used. Providing details about the statistical methods and software used would enhance the
transparency and reproducibility of the study.
• Lack of Discussion on Limitations: The study does not discuss potential limitations associated
with the methodology, sample selection, or data analysis. Addressing limitations and potential
sources of bias would strengthen the interpretation of the findings and provide a more balanced
assessment of the study's results.
• Missing Ethical Considerations: The study does not mention ethical considerations such as
informed consent, approval from an ethics committee, or potential conflicts of interest. Ethical
considerations are important to ensure the protection of participants' rights and to maintain the
integrity of the research.
• Limited Generalizability: The study focuses on a specific population of women with PCOS,
which may limit the generalizability of the findings to other populations or genders. Discussing
the potential implications and generalizability of the findings would enhance the relevance of
the study.
Addressing these weaknesses would improve the clarity, rigor, and validity of the study,
allowing for more robust conclusions to be drawn regarding the association between PCOS,
periodontal diseases, and hormonal/metabolic profiles.
4.Main Methodology notes:
• Lack of Detailed Calibration Information: The methods section mentions that the examiner
performing the periodontal assessment was calibrated, but it does not provide specific details
on how the calibration was conducted. The lack of information on the calibration process raises
concerns about the reliability and consistency of the periodontal measurements.
• Incomplete Description of Data Collection: While the section mentions that interviews,
questionnaires, and physical examinations were conducted to gather demographic and clinical
data, it does not provide sufficient details about the specific questions asked or the procedures
followed during the physical examination. A more detailed description of the data collection
process would enhance the transparency and replicability of the study.
• Insufficient Information on Biochemical Analysis: The section briefly mentions that various
serum biochemical parameters were retrieved and quantitated, but it does not provide specific
details about the biochemical analysis techniques used. Additional information on the specific
assays, equipment, and protocols employed would improve the clarity and reproducibility of
the study.
• Lack of Justification for Raman Spectroscopy Parameters: The section provides some details
on the acquisition of Raman spectra, but it does not explain why specific parameters, such as
laser power, spectral range, and acquisition time, were chosen. Providing a rationale for the
selection of these parameters would help readers understand the experimental design and the
potential impact on the results.
• Inadequate Description of Statistical Analysis: Although the section mentions the use of
various statistical tests, it lacks specific details on the variables included in each analysis
and the rationale for choosing a particular statistical test. Providing more information on the
specific variables analyzed and the justification for the chosen statistical tests would enhance
the transparency and interpretation of the results.
• Lack of Information on Data Preprocessing and Analysis: The section mentions that Raman
spectroscopy datasets were preprocessed and analyzed using software tools, but it does not
provide specific details on the preprocessing steps or the specific analysis techniques employed.
A more detailed description of the data preprocessing and analysis methods would improve the
reproducibility and reliability of the results.
• Incomplete Reporting of Visualization and Analysis Software: The section mentions the use
of Labspec 5 software for preprocessing the Raman spectroscopy datasets, but it does not
provide information on the specific analyses or visualizations performed using this software.
Additionally, while the section mentions the use of the R environment for further analysis
and visualization, it does not specify the specific R packages or functions utilized. Providing
specific details on the software tools and packages used would aid in replicating the analyses
and interpreting the results.
• Lack of Discussion on Potential Limitations: The methods section does not include
a discussion of potential limitations associated with the chosen methodologies or data
collection procedures. Identifying and discussing potential limitations would provide a more
comprehensive understanding of the study's findings and implications.
Addressing these defects would improve the clarity, reproducibility, and reliability of the study's
methods, allowing for a more accurate interpretation of the results.
5.Main results feed back points :
• Small sample size: The study included a relatively small number of participants, with 66
PCOS patients and 22 healthy subjects. A larger sample size would provide more robust and
generalizable results.
• Limited generalizability: The study participants were recruited based on specific inclusion and
exclusion criteria, which might limit the generalizability of the findings to a broader population.
• Imbalanced group sizes: The distribution of participants across the four groups (H-H, H-P, P-
H, P-P) is imbalanced, with some groups having significantly fewer participants than others.
This could potentially affect the statistical power and reliability of the results.
• Lack of information on recruitment methods: The study does not provide details about how
participants were recruited, which could introduce selection bias. It is important to ensure that
the recruitment methods are appropriate and representative of the target population.
• Potential confounding variables: The study does not account for potential confounding
variables that could influence the relationship between periodontal conditions and other factors
such as hormonal, metabolic, and inflammatory profiles. Controlling for these variables would
strengthen the validity of the study findings.
• Cross-sectional design: The study design is cross-sectional, which means that it captures data
at a single point in time. This limits the ability to establish causal relationships between variables
and only allows for associations to be observed.
• Lack of information on statistical tests: The study mentions the use of statistical tests (ANOVA
or Kruskal-Wallis test) for analyzing inter-group differences, but does not provide specific
details about the post-hoc tests, adjustments for multiple comparisons, or the significance levels
used. This information is important for interpreting the statistical findings accurately.
• Missing information on dental and medical history: The study lacks detailed information on
the dental and medical history of the participants, which could be relevant for understanding the
observed periodontal conditions and potential confounders.
• Limited information on the methodology: The study does not provide sufficient details about
the methodology used for data collection, measurements, and assessments. It is important to
have clear and standardized protocols to ensure the reliability and reproducibility of the results.
• Lack of longitudinal follow-up: The study only captures a snapshot of the participants'
conditions at a single time point. Longitudinal follow-up would provide valuable insights into
the progression or changes in periodontal conditions and associated factors over time.
6.Discussion feedback notes:
• Lack of clarity in the research objective: The section does not clearly state the main objective
of the study or research question, which makes it difficult for readers to understand the purpose
of the investigation.
• Inadequate sample size: The text acknowledges that the limited sample size of the study may
have influenced the results and introduced selection bias. Larger sample sizes are recommended
to provide more representative findings.
• Lack of significance in certain comparisons: The section mentions that there was no significant
difference in the proportion of PCOS patients with bleeding on probing (BOB) or regular dental
screenings compared to non-PCOS subjects. However, the significance or implications of this
finding are not discussed.
• Inconsistent findings: The section acknowledges inconsistencies with previous studies
regarding the correlation between LH, FSH, and E2 levels and periodontal status in PCOS
patients. However, the section does not provide a clear explanation or interpretation for these
discrepancies.
• Insufficient discussion on underlying mechanisms: While the section briefly mentions the
involvement of inflammatory mediators and hormones in periodontal diseases and PCOS, it
lacks a comprehensive discussion on the underlying mechanisms connecting these conditions.
The text suggests further investigations using multi-omics approaches to identify these
mechanisms.
• Lack of clarity in Raman spectroscopy findings: The section mentions correlations between
Raman spectroscopy peaks and biomarkers of periodontal diseases, but it does not provide
detailed explanations or interpretations of these correlations. This makes it difficult for readers
to understand the significance of these findings.
• Incomplete conclusion: The section concludes that periodontal status can affect biomolecular
profiles in PCOS patients, but it does not provide specific implications or recommendations for
clinical practice based on these findings.
• Suggestions for future research: The section briefly mentions the need for longitudinal
investigations and larger sample sizes, but it does not elaborate on other potential avenues for
future research or highlight specific research gaps that need to be addressed.
To improve this section, it would be helpful to address these defects by providing clearer
objectives, discussing the limitations in detail, providing more context and explanations for the
findings, and offering specific recommendations for future research and clinical practice.
7.General Comments  regarding the selection and writing of references:
• Inconsistent citation style: The references provided follow different citation styles (e.g.,
Vancouver style, APA style), which indicates a lack of consistency in the citation format. It is
important to use a consistent citation style throughout the document.
• Missing publication details: Some references lack important publication details such as volume
number, issue number, and page numbers. It is important to include complete publication details
to ensure the accuracy and reliability of the references.
• Lack of recent references: The majority of the references are from 2016 or earlier, with only a
few from 2020-2023. It is important to include more recent references to ensure the up-to-date
relevance and accuracy of the information presented.
• Limited diversity of sources: The references primarily consist of research articles from
scientific journals. It would be beneficial to include a broader range of sources such as
systematic reviews, meta-analyses, clinical guidelines, and authoritative textbooks to provide a
comprehensive and well-rounded review of the topic.
• Overemphasis on specific topics: Some references focus on specific aspects of the topic, such
as the relationship between periodontal disease and polycystic ovary syndrome. While these
specific topics are relevant, it is important to provide a balanced representation of the broader
subject matter.
• Missing key references: There may be key references or landmark studies in the field that
are missing from the list. It is important to conduct a thorough literature search and include
influential and widely cited references to provide a comprehensive overview of the topic.
• Lack of critical appraisal: The references are listed without any critical evaluation or
assessment of their quality, relevance, or methodology. It would be beneficial to provide a brief
critique or summary of each reference, highlighting its strengths, limitations, and relevance to
the topic.
• Limited use of primary sources: Some references appear to be review articles or secondary
sources rather than primary research studies. While review articles can provide valuable insights,
it is important to include primary research studies to support the claims and arguments made
in the document.
• Insufficient number of references: Depending on the scope and depth of the document, the
number of references provided may be insufficient to adequately cover the topic. It is important
to ensure that an adequate number of references are included to support the claims and provide
a comprehensive overview of the subject matter.
• Lack of diversity in authorship: The references primarily include authors from a limited
geographic region or institutional affiliation. It would be beneficial to include references from a
diverse range of authors and institutions to provide a more comprehensive and global perspective
on the topic.
Best regards
Dr.Bahaa Aldeen AbdulRahman Hadi