High Sucrose and Cholic Acid Diet Triggers PCOS-like Phenotype and Reduces Enterobacteriaceae Colonies in Female Wistar Rats

Round 1

Reviewer 1 Report

 

The manuscript demonstrates the effect of high sucrose and cholic acid (HSCA) diet on presence of PCOS-like conditions and gut microflora changers in rats. The topic is of importance because of controversial results of experimental and clinical data regarding dietary factors and gut dysbiosis as causes of PCOS. Nevertheless, there are some remarks:

We propose to change the term "productive women" (line 37) to more standard (women of reproductive age or premenopausal women).

The experimental diet is described as not only high- sucrose, but also as a higher-fat one when comparing with control diet (percentage of calories from fat 32 vs 17%, lines 87-88). These issues may cause misunderstanding and should be detailed.

Regarding the conclusion that “Enterobacteriaceae in HSCA rats was significantly lower than that in control rats”. To our opinion, the data presented in the fig 1D do not support the effects of diet on Enterobacteriaceae, because dynamic fecal sampling is not mentioned in the Methods section.

 

The conclusion is: minor revision

 

 

Author Response

Point 1:                We propose to change the term "productive women" (line 37) to more standard (women of reproductive age or premenopausal women).

Response 1: Thank you for this suggestion. We agree to the suggestion and change this term into premenopausal women.

 

Point 2: The experimental diet is described as not only high- sucrose, but also as a higher-fat one when comparing with control diet (percentage of calories from fat 32 vs 17%, lines 87-88). These issues may cause misunderstanding and should be detailed.

Response 2: Thank you for this feedback as we mistakenly wrote the diet. This should be 32% of glucose. The control rats were treated with a normal food while the other rats were treated with a modified food with 32% of glucose. We will revise this. I appreciate that the reviewers raised this point. Thanks to the reviewer.

 

Point 3: Regarding the conclusion that “Enterobacteriaceae in HSCA rats was significantly lower than that in control rats”. To our opinion, the data presented in the fig 1D do not support the effects of diet on Enterobacteriaceae, because dynamic fecal sampling is not mentioned in the Methods section.      

Response 3: Thank you for raising this point. Since the beginning, we hypothesized that the number of Enterobacteriaceae increases in the HSCA group. When performing fecal sampling and growing the samples into appropriate bacterial media, however, we found that the bacterial colonoies were lower compared to the control. We do understand what the reviewer asks is to provide more convincing data. Shotgun metagenomics sequencing (1) or RT-PCR (2) can be used. However, we managed this study with a very tight funding. The financial source is mainly contributed from private fund of the first author (from research experiments to publication).

 

References

1. Chu W, Han Q, Xu J, Wang J, Sun Y, Li W, et al. Metagenomic analysis identified microbiome alterations and pathological association between intestinal microbiota and polycystic ovary syndrome. Fertil Steril. 2020;113(6):1286-98.e4. doi: https://doi.org/10.1016/j.fertnstert.2020.01.027.
2. Guo Y, Qi Y, Yang X, Zhao L, Wen S, Liu Y, et al. Association between Polycystic Ovary Syndrome and Gut Microbiota. PloS one. 2016;11(4):e0153196-e. doi: 10.1371/journal.pone.0153196. PubMed PMID: 27093642.

 

Reviewer 2 Report

Revision for language required. Follicle de graaf is not the appropriate use for Graafian follicle.

Author Response

Point 1:                Revision for language required. Follicle de graaf is not the appropriate use for Graafian follicle.

Response 1: In our country, the term of Follicle de Graaf is widely used and we do not realize that the term was not standardized. Thank you for the feedback and we will change this term. Regarding the language, the reviewer may notice that we are not English-native speakers, therefore, we plan to use the publisher service to improve the language quality. Once again, we appreciate these valuable suggestions.

Reviewer 3 Report

Comments:

1. Could the authors please clarify how and why did they choose this animal model for their experiments please?
2. Could the authors please clarify how the number of animals for each experiment were determined please i.e., how was the study powered?

Author Response

Response to Reviewer 3 Comments

 

Point 1:                Could the authors please clarify how and why did they choose this animal model for their experiments please?

Response 1: There are some considerations to determine appropriate animal model for this research. Non-human primates are suitable model for PCOS as these animals have similar ovarian physiology as women have. However, the use of nonhuman primates is strictly regulated due to ethical and sampling number issues (Ko & Lim 2021). The use of rodent models is commonly preferred because of larger sample sizes, lower costs, simplicity, and short-time duration of induction methods, although the physiology of rodent and human is slightly different (Tamadon et al. 2018). Shojaei-Zarghani and Rafraf (2021) systematically reviewed the animal studies carried out to assess the effect of resveratrol on PCOS. The study result showed that all studies used Sprague Dawley or Wistar rats. Therefore, we thought that using Wistar rats in the present study is reasonable.

Point 2: Could the authors please clarify how the number of animals for each experiment were determined please i.e., how was the study powered?

Response 2: We followed the formula for sample size calculation as described by Charan and Kantharia (2013). The formula is as follows:

  n = 2 x ((Zα + Zβ)SD / d)²

Where Zα is 1.96 at type 1 error of 5% and Zβ is 0.84 at 80% power. The calculated n is 15.68 which means that each group requires 16 samples. However, we also consider 20% of dropout, thus the number of each group is 20. To best of our knowledge, it is uncommon to present the details about the calculation of sample size in animal studies, therefore there was no description about the size calculation in the manuscript.

 

 

References:

Charan, J & Kantharia, ND 2013, 'How to calculate sample size in animal studies?', Journal of pharmacology & pharmacotherapeutics, vol. 4, no. 4, pp. 303-306.

Ko, MJ & Lim, CY 2021, 'General considerations for sample size estimation in animal study', Korean J Anesthesiol, vol. 74, no. 1, Feb, pp. 23-29.

Shojaei-Zarghani, S & Rafraf, M 2021, 'Resveratrol and Markers of Polycystic Ovary Syndrome: a Systematic Review of Animal and Clinical Studies', Reproductive Sciences, 2021/07/26

Tamadon, A, Hu, W, Cui, P, Ma, T, Tong, X, Zhang, F, Li, X, Shao, LR & Feng, Y 2018, 'How to choose the suitable animal model of polycystic ovary syndrome?', Traditional Medicine and Modern Medicine, vol. 01, no. 02, 2018/06/01, pp. 95-113.

Please see the attachment.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

The discussion could be complemented with possible explanations for the study's findings. It is not explained why they made the correlation of the tertiary follicles with the corpus luteum. It is not discussed.

Author Response

Point 1:                The discussion could be complemented with possible explanations for the study's findings. It is not explained why they made the correlation of the tertiary follicles with the corpus luteum. It is not discussed.

Response 1: We really appreciate this suggestion as we did not include Figure 4 in the discussion. Please see our revision as follows:

In lines 297 … We speculate that our findings as shown in Figure 3A&D are an early sign of folliculogenesis deterioration, as longer periods of HSCA treatment could result in more prominent PCOS phenotypes. In addition, we found there is a correlation between tertiary follicle and corpus luteum (Figure 4). Deterioration in the follicular phase could be related to abnormality in the luteal phase (Abdulla et al. 2018). However, in the present study, corpus luteum counts between two groups did not show significant differences (Figure 3B). The luteal phase may also be affected during longer exposure to HSCA. More studies are required to confirm our speculation by evaluating the ovarian every 4 weeks with 16 weeks of treatment periods.

 

Reference

Abdulla, SH, Bouchard, TP, Leiva, RA, Boyle, P, Iwaz, J & Ecochard, R 2018, 'Hormonal Predictors of Abnormal Luteal Phases in Normally Cycling Women', Frontiers in public health, vol. 6, pp. 144-144.

Round 2

Reviewer 3 Report

The authors have satisfactorily responded to my comments.