Reviewer ’s report  
 
Title: The influence of probiotics on genital high -risk human papilloma virus clearance, 
associated lesions and quality of cervical smear: A randomized placebo -controlled trial  
 
Version: 0 Date: 25 Mar 2019  
 
Reviewer: Jo-Ann S. Passmore  
 
Reviewer's report:  
The st udy by Ou et al. describes a randomized, double -blinded, placebo controlled trial to 
evaluate the efficacy of a Lactobacillus rhamnosis GR -1 and L. reuteri RC -14-containing oral 
probiotic in clearance of high -risk HPV infections (evaluated by Hybrid Captur e II) in 121 
women with HR_HPV infections at enrollment, followed up for 15 months. While the oral 
probiotic treatment did not appear to influence rate of HPV clearance in the study group, the 
probiotics did influence cervical disease clearance (LSIL) in t he 21 women who had cervical 
disease at enrollment. Overall, the study is interesting although some important details and 
limitations should be added.  
1. Why was vaginal pH or BV status not reported in the study? Since these are 
acknowledged by the author s to be important variables in HPV risk, it seems appropriate 
that this information would be collected?  If it wasn't, this should be acknowledged as a 
limitation of the study design.  
2. Please justify selection of 50% power for power calculations.  This i s considerably lower 
than a power of 80% that is the minimum that should be used for clinical trials. Thus, 
they were underpowered to show a difference between the groups, and I think their 
negative result may be misleading. Furthermore, the main positive result from this study 
is the improved clearance of LSIL.  The study certainly was not powered for this 
outcome, with only 21 women having LSIL.  
3. Probiotic was given orally.  Justify why U -relax was selected as the product to be used? 
Please justify why oral administration was selected if vaginal colonization was intended?  
Please provide more detail about the U -relax product: dose, co -formulation, QC that was 
done on the product to ensure organisms were live, how long the women were intended to 
take the probiotics for and what proportion of them reported regular dosing/incomplete 
dosing? Was antibiotic treatment (for any condition) noted? As this would influence 
probiotic viabilility?  
4. They don't mention how the placebo was manufactured. Please provide details of this.  
5. They only mention ethics approval but no regulatory approval for the trial. Can this be 
clarified? Who did the randomization? Please provide more detail on 
inclusio n/exclusion/screening criteria.  
6. About a third of the cohort were post -menopausal.  Authors should comment on whether 
these women were on hormone replacement therapy?  Comment on BV and HPV 
prevalence post menopause and whether low estrogen levels would influence probiotic 
efficacy?  
7. Discussion - page 10 line 7 -10.  Please provide evidence that oral probiotics make their 
way through the GI tract after oral administration to colonize the genital tract in women?  
8. Comparison is made in the discussion wit h the Verhoeven study - please provide more 
detail about probiotic product used by Verhoeven, dose, duration, strains etc.  
9. Discussion page 11 line 14 - authors state that probiotics may influence different strains 
of HPV differently.  Please provide mor e justification for why you think this would be 
likely?  
10. Limitations section in the Discussion needs to be expanded to include/acknowledge other 
limitations: probiotics were orally delivered, vaginal colonization was not assessed, BV 
status was not asse ssed by standard methods, vaginal pH?  Study not powered for the 
LSIL finding since numbers were so small? Commenting on whether individuals took 
their probiotics for the full duration of the study or whether this information was even 
collected? Comment on  the fact that the isolates in the probiotic were not vaginal 
isolates? Ie, were they appropriate for a vaginal probiotic study? HPV was measured by 
Hybrid capture that is not type specific, and semi -quantitative.  
11. Please include information about ASCUS /LSIL prevalence in Table 1.  
 
Minor comments:  
Page 8 line 58 - what confounding factors were adjusted for in the multivariate logistic 
regression analysis?  
Page 10 line 20 - Many studies have reported…. They only cite one study.  Please correct this.  
Are the methods appropriate and well describ ed? 
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately  supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended,  please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the public ation of this 
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3. Do you hold or are you currently  applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other fi nancial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give detai ls below.  
 
None.  
 
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