Salivary Progesterone Levels in Female Patients with a History of Idiopathic Scoliosis: A Retrospective Cross-Sectional Study

Round 1

Reviewer 1 Report

Thank you for allowing me to review this study. This is a cross-sectional study studying the relationship between salivary progesterone, BDNF genomic variants and female patients with AIS.

The introduction is appropriate, covering the reasons for why this was conducted. Some background into salivary progesterone, specifically its good concordance with blood progesterone (this is mentioned in the methods I see, but you should clarify how well it corresponds), and the normal levels of salivary progesterone in pre-menarchal vs post-menarchal vs menopausal patients would be helpful to contextualize its utility as it is not a standard lab test.

The objectives are sensible but I am unsure on how this is clinically relevant.

Methods are clear and appropriate. The figure is blurry in the text but can be viewed in the supplementary material. I am unable to comment on the appropriateness of the genomic analysis and which genes were assessed.

Results, I have some suggestions:

1. The salivary progesterone levels are listed, but it would be easier to view in a table. (cycling/scoliosis/number of patients/average progesterone level)
2. Make sure you clarify that BDNF positive means positive for a genetic variant! This is quite confusing in this section.
3. The non-scoliosis vs scoliosis groups have significant age differences! This will likely make a huge difference in comparisons. Also, pregnancy, breastfeeding and taking oral contraceptive pills will potentially make a big difference in progesterone. These are all confounding factors that need to be considered.
4. No baseline characteristics of all patients is given in this study. This is important to ensure that the relevant population is being evaluated, and to make sense of the t-tests later on as well.
5. Figure 2, a 3D graph is a terrible way to display any data as it is very difficult to visually compare the graphs. Just use a 2D graph with the bars side by side. The Y-axis needs units and a scale, and the title is not needed in the figure. Figure caption is enough
6. Figure 3: Needs units and title is not needed in the figure. Figure caption is enough
7. Table 2: 9 decimal places is far too many, and I am not sure what this is comparing. Is this the P-value? I assume so. This table needs a title. The confidence interval for the t-test is not shown. Can we assume that the data is normally distributed, and what is the standard deviation of the data? The t-tests are on progesterone levels in these two groups?

Discussion:

1. True, scoliosis patients have more genomic variants with BDNF from this data. It makes snese that menstrual cycling status would have any effect on the genomic variants, but I don’t know why this was studied. The expression of BDNF may change with progesterone levels but why should the genomic variants? Are there studies showing a change in genomic variants of BDNF expression vs progesterone?
2. Matsuki looked at BDNF levels, not genomic variants. This is not comparable to this study.
3. Excellent discussion of the shortcomings. I rarely see discussions as thorough and thoughtful as this one. However, I’m unsure what you mean by “menstrual cycling status was indifferent”. Do you mean that it was a statistically insignificant difference between groups from Table 2?
4. The limitation of non-scoliosis patients may have scoliosis is a significant one. I’m not sure how this explanation reduces this limitation. The progesterone levels

Overall, the study makes sense, but it is not clear to me what the relevance is to clinical practice. Scoliosis typically stabilizes by the time patients are in their 20s, so measuring progesterone levels in adults would not change the management of the younger scoliosis patients who require bracing and surgery as mentioned in the introduction. This is the most serious flaw of this study. None of the results will change management, nor does this study point in a direction that indicates progesterone  could be helpful in managing scoliosis. I can see BDNF genomic variants perhaps, but it would be much more convincing to do a study directly on adolescent girls with idiopathic scoliosis.

The actual results are clear but they are presented very unclearly. It is difficult to determine what the baseline characteristics are, and whether the statistical analysis was appropriate because not everything is presented.

The discussion is well written, but the science does not seem quite right. BDNF genomic variation (ie the DNA sequence that expresses BDNF) will not change with progesterone levels. Genes don’t change due to hormones, (aside from in epigenetics which was not studied here) so I am not sure why the genetic testing was compared with progesterone. Comparison with Matsuki et al is similarly inappropriate, as they measured BDNF levels. There are more confounding factors than these in progesterone levels.

While the study itself was interesting, the actual patients being assessed are not appropriate and the presentation of results needs significant improvement to clarify if the right tests were done.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The author studied the association between idiopathic scoliosis and salivary progesterone, or BDNF. The author retrospect 482 patients. For the scoliosis group, 51 female biological genders, AIS medical history, salivary hormone test on file, and genomic test on file. For the control group, 113 patients with female biological genders, salivary hormone test on file, and genomic test on file. The author found that the salivary progesterone level in the control group was higher than that in the scoliosis group, and the BDNF positive patients in the scoliosis group were more than that in the control group.  Interesting work, clear data and well-discussed.

To make the work more profound, I would like to give some suggestions:

• Please compare each SNP of BDNF between the control group and scoliosis group, and calculate each P-value, odds ratio. Please refer to PMID: 20228709.
• Please remake Table1.
• Author showed that the average age of the scoliosis group was 26 years, while the average age of the control group was 44 years. Clearly, the average age of the two groups was different. Does Age Affect Progesterone Levels? Please discuss it in the discussion section.
• Please mark the P-value in Figure 2 and Figure 3.
• Author defined that a patient was considered positive if 10 or more of the 19 BDNF SNPs showed variants.  However, the SNP is the nuclear acid showing different frequencies, it does not show variants, Could the author change the definition, or maybe the author can specify what the variants are.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The author presents a retrospective review on 51 patients with adolescent idopathic scoliosis and a control non-scoliosis group of 113 patients with menarchial and non-menarchial subgroup, where salivatory progesterone levels and  brain derived neurotrophic factor BDNF genomic variants were observed between the two groups. The results suggestes that progesterone is significantly lower in female patients with a history of AIS when compared to female patients with a negative history of AIS. Scoliosis patients were also more likely to have a higher number of genomic variants involving brain-derived neurotrophic factor. 

I suggest to include credentials and affiliations of the author (MD, PhD; Institution). Abstract should be expanded and include some substantial results. Introduction did not sufficiently explain to the reader why did the researcher use only progesterone salivarly levels and BDNF - and why not other sex hormones and its derivates, like in the study which was cited:

Kulis A, Goździalska A, Drąg J, Jaśkiewicz J, Knapik-Czajka M, Lipik E, Zarzycki D. Participation of sex hormones in multifactorial pathogenesis of adolescent idiopathic scoliosis. Int Orthop. 2015 Jun;39(6):1227-36. doi: 10.1007/s00264-015-2742-6. Epub 2015 Mar 25. PMID: 25804208.)

In Materials and Methods - what was the indication of performing salivatory progesterone test in a control group? 

The non-scoliosis patient group had 50 women who were cycling while 63 were not. The average age of this group is 44 years - that would mean that non-cycling women were in the menopause, so that the description non-menarchial does not fit. If the scoliosis group has an average age of 24 and control of 44 and the intention is to examine the effects on progresterone on AIS, then I suppose that both groups should be around the same age. This makes the interpretation of the results difficult. Please explain. Results part is in general too short and the main results should be more thoroughly explained. 

 

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for the opportunity to take a second look at this study. I see that the BDNF genomic data has been removed from this study. That is good, as it was the weakest part of the study.

Unfortunately, the flaws in this study remain. First, a study on the concordance between blood and salivary progesterone cited, but many modern studies show that the correlation is not strong. At best, the correlation is unknown, which is why salivary progesterone is not a routinely ordered lab test. Even serum progesterone is not routinely ordered except in disease-states because the levels fluctuate so significantly. It can range by a factor of 10 within a day, and range from 300 pmol/L to 50000 pmol/L across a menstrual cycle. It is difficult for me to reconcile the salivary progesterone results in this study and the broader science done on progesterone levels.

However, even if we assume the salivary progesterone is stable enough, reliable enough and accurate enough to represent a woman's hormonal status, we still have a problem. The progesterone levels are different between the population you are targeting (patients with AIS as mentioned in the introduction), and the women that you collected data from (women of all ages), regardless of scoliosis status. The sample is not representative of the target population.

Don't get me wrong, I find it fascinating that the salivary progesterone levels seem to be lower in the scoliosis group than the non-scoliosis group. would love for scoliosis screening to be as simple as a salivary progesterone test, but data from patients who are 25+ (up to 65 and post-menopausal even!) cannot be applied to push for a screening test for kids aged 10-14 who have AIS.

However, if you are targeting adult scoliosis, as it seems that your points in that direction, then you need to present the case that scoliosis continues to progress in a clinically significant way. Scoliosis progression slows significantly through adulthood and while a large proportion of adults have scoliosis they do not notice it and it does not affect their function. With greater age, the line between idiopathic and degenerative scoliosis become blurred as well.

As it stands, there are serious scientific flaws in this study. While the topic is fascinating, the variables are not well-controlled enough, the test itself has not been researched enough and the sample is not representative of the target population. It is not enough to collect salivary samples, then compare and contrast if they have scoliosis or not, calculate a t-test and then publish it. I recognize that this could be 'cutting edge' research, because this is a truly novel approach to scoliosis management that could hold some promise, but this study in itself has not been executed in a scientifically sound way, so I cannot accept this paper.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The reviewers remarks have been repplied sufficienty; however I suggest to reconsider the manuscript for publication only if the remarks of the reviewer who rejected the manuscript are clearly answered. 

Author Response

Please see attachment. It is identical to the one sent to Reviewer #1, since Reviewer #3 only wanted to see our responses to Reviewer #1's comments.

Author Response File: Author Response.pdf