Gut Microbial Composition on Dienogest Therapy in Patients with Endometriosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Veronika et al. demonstrates that future studies of the effects of dienogest on the intestinal and reproductive tract microbiota will likely yield useful information and may offer insights into the treatment of endometriosis. Therefore, this report is suitable for publication in Microbiology Research. However, a number of points need to be clarified/corrected before the manuscript can be considered for publication.

 

1. The Introduction states

"So, this article examines demonstrated that dienogest (DNG) exerts dual hormonal and microbiomial effects...."

It is not clear what "examines demonstrated" means. It is already well known that progestins exert hormonal effects.

Also, it should be mentioned that dienogest is a progestin medication.

Also, this sentence needs to indicate that the present study investigated the effects of dienogest on the gut (not the reproductive tract) microbiome.

Also, the authors should note why the gut microbiome and not the reproductive tract microbiome was investigated.

The passage could be revised to something like

"Since it is well known that progesterone affects the reproductive tract microbiome (suitable reference), the present study concentrated on the effects of dienogest (DNG), a progestin medication, on the gut microbiome. We demonstrated that DNG affects the gut microbiome, exerting novel dual hormonal and microbiomial effects and revealing novel mechanisms by which progestin therapy may achieve therapeutic benefits in endometriosis."

 

2. The Materials and Methods states

"Microbiological examination of faecal samples was performed at two points: baseline before therapy and after 6 months of therapy DNG 2 mg daily orally in continuous regimen, comparison was made in pairs (mean duration of therapy was 6 (6;7) months)."

The phrase "comparison was made in pairs" suggests that faecal samples obtained after 6 months of therapy were compared with samples obtained before therapy for each patient. However, the results for each patient are not presented separately, rather, the results of the microbiological examination of faecal samples for all patients at baseline were compared with the results of the microbiological examination of faecal samples for all patients after therapy.

Also, point 7 of the exclusion criteria states

"7. Medication use for less than 6 months"

This point appears to refer to oral ingestion of DNG.

Therefore, the sentence should be revised to something like

"Microbiological examination of faecal samples was performed at two points: at baseline before therapy and after daily oral ingestion of DNG 2 mg for at least 6 months.

 

Point 9 of the exclusion criteria states

"9. Presence of gastrointestinal symptoms that could interfere with sample collection, such as diarrhea, constipation, rectal bleeding associated with organic pathology (hemorrhoids, anal fissures, etc.) or of unspecified etiology; menstruation."

This suggests that women who menstruated were excluded from the study.

Either "menstruation" should be removed from point 9 or the type of menstruation that resulted in exclusion from the study needs to be defined.

Also "The" should be added before "Presence". -> "9. The presence...."

 

Section 2.1 states

"A tenfold dilution of the substrate in saline solution was per-

formed from 10⁻¹ to 10⁻⁹."

The meaning of 10⁻¹ to 10⁻⁹ is not clear.

 

Section 2.1 states

"Petri dishes with Sabouraud agar were inoculated and placed in a thermostat at 30°C and 37°C."

Does this mean that paired petri dishes with Sabouraud agar were inoculated and placed in two thermostats at 30°C and 37°C?

 

Table 2 lists the Number of microbial species per patient as 12.0 at baseline and 13.24 after 6 months of therapy.

This is not correct. These patients will have many more bacterial species than the very small number listed.

 

The Discussion refers to "Gammaproteobacteria". This should be "Gammaproteobacter.

 

After discussing studies by Svensson et al. and Shan et al. the Discussion states

"An increase in the number of bacteria from the genus Prevotella was also detected in a similar study by Svensson et al., where a significant correlation with intestinal symptoms in patients with endometriosis was found [23]."

This suggest that the authors are referring to another study by Svensson et al.

The sentence should be revised to

""An increase in the number of bacteria from the genus Prevotella was also detected in the study by Svensson et al., where a significant correlation with intestinal symptoms in patients with endometriosis was found [23]."

 

The Discussion states

"These results present interest, because to present day known is role of estrogens and estrogen-containing preparations (combined oral contraceptives, hormone replacement therapy including intravaginal forms etc.), and also their influence on composition of microbiota of intestine and female reproductive tract [1]."

Does this mean that currently, the influence of estrogens and estrogen-containing preparations on the microbiota of the female reproductive tract is not known?

Also, Ref [1] does not address the effect of sex hormones on the microbiota of the female reproductive tract.

Also, the current study investigated the effects of a progestin medication, not estrogen, on the gut microbiota. Estrogen levels were not measured, and therefore the effect of estrogen on the gut microbiota were not specifically investigated.

Possibly, "This study for the first time obtained data about influence of DNG on composition of gut microbiota in patients with endometriosis" can be added to the next paragraph and the point about estrogen can be added to the second to last paragraph of the Discussion.

Also, the abbreviation "GM" is used only twice in the manuscript text. Therefore "GM" can be replaced by gut microbiota in the Discussion.

Therefore, the final paragraphs of the Discussion can be revised to something like

"To date, data on the effects of dienogest on the composition of the intestinal microbiota are limited, and the mechanisms of action of dienogest associated with microbial composition are also unclear. The present study obtained data for the first time about the influence of DNG on the composition of the gut microbiota in patients with endometriosis. Based on the results of the present study, there was a noted tendency of improvement of a series of microbiological parameters in patients with endometriosis on DNG therapy. This, may be associated with direct anti-inflammatory activity of DNG through inhibition of the production of proinflammatory cytokines by endometrioid cells. DNG may also have a direct effect on intestinal barrier permeability. Zhou Z. et al. demonstrated that exogenous introduction of progesterone increased transepithelial electrical resistance in the large intestine due to enhancing the expression of occludin and decreasing tight junction permeability, and this effect was dose-dependent. This indicates that progesterone mediated induction of occludin expression reduced the permeability of the intestinal barrier, thereby preventing systemic microbial translocation and development of chronic inflammation [2].

In addition, DNG may influence endometriosis by reducing the degree and prevalence and activity of endometrioid foci due its antiproliferative and antiangiogenic activity [3]. In addition, as noted above, Shan J, et al. reported a decrease in the alpha diversity of the gut microbiota and an increase in the Bacillota/Bacteroidota ratio in patients with endometriosis, and in our study, there was a noted tendency to increase species richness and a statistically significant reduction in the Bacillota/Bacteroidota ratio (p = 0.0421). The Bacillota/Bacteroidota ratio is a significant indicator for assessing the gut microbial composition. Reduction of the Bacillota/Bacteroidota may cause a reduction in the absolute quantity of facultative-anaerobic opportunistic pathogens Staphylococcus spp. (type Bacillota) and increase the number of bacteroids (type Bacteroidota). DNG therapy also resulted in an increase of the commensal bacteria Lactobacillus spp. and Collinsella aerofaciens. C. aerofaciens is one of the representatives of the normoflora of the large intestine of humans, known for its ability to ferment a series carbohydrates of plant and animal origin, produce H2, ethanol, short-chain fatty acids (SCFA) and lactate [4]. In particular, C. aerofaciens possesses the ability to produce butyrate, a 'useful' SCFA, supporting epithelial barrier integrity [5-6]. Notably, patients with irritable bowel syndrome exhibit a reduction of C. aerofaciens, supporting a role of C. aerofaciens as a predictor of a positive response in patients on therapy probiotics [7].

Another likely mechanism by which DNG affects the gut microbiota is via its effects on estrogen (appropriate reference). In addition, DNG therapy may affect estrogen indirectly by reducing the number and titer of opportunistic pathogens which participate in the metabolism of estrogens. Estrogens and estrogen-containing preparations (combined oral contraceptives, hormone replacement therapy including intravaginal forms etc.) have been shown to influence the composition of microbiota of the intestine [1]. This suggest a novel mechanism for DNG’s efficacy, potentially involving regulation of microbial estrogen metabolism.

The results of the present study indicate, that taking DNG promotes changes in the composition of the gut microbiota with increased bacterial diversity and production of 'useful' SCFAs by bacteria-symbionts. DNG also reduces the number and titer of opportunistic pathogens capable of producing endotoxins and which participate in the metabolism of estrogens."

 

The Conclusions state

"Thus, DNG therapy for 6 months is effective not only in reducing the severity of clinical manifestations in patients with endometriosis, which is common knowledge, but also in improving the composition of the intestinal microbiota. There was a trend towards an increase in species and taxonomic diversity, a decrease in the Bacillota/Bacteroidota index and Staphylococcus spp. colonisation, an increase in the number of symbiont bacteria Lactobacillus spp. and Collinsella aerofaciens."

The conclusions presented in the Abstract should be added to these conclusions.

For example, something like

"DNG therapy for 6 months is effective not only in reducing the severity of clinical manifestations in patients with endometriosis but also in improving the composition of the intestinal microbiota. There was a trend towards an increase in species and taxonomic diversity, a decrease in the Bacillota/Bacteroidota index and Staphylococcus spp. colonization, an increase in the number of symbiont bacteria Lactobacillus spp. and Collinsella aerofaciens. Thus, DNG therapy modulates the gut microbiota reducing opportunistic pathogens and increasing symbionts, favoring a composition associated with anti-inflammatory and barrier-protective effects."

 

Strengths and Limitations state

"The data obtained in the future have more advantages in using in real clinical practice, in contrast to sequencing, which is an expensive and limited method of use."

Does this mean

"The data obtained by these methods have advantages in clinical practice, in contrast to sequencing, which is an expensive and limited method."

 

Strengths and Limitations state

"performed in the only national centre of the Russian Federation."

does this mean

"performed in the National Centre of the Russian Federation."

 

Finally, references need to be cited in order.

The first reference cited is [1], the second reference cited is [2], etc.

Therefore, all of the citations and References need to be re-numbered.

Comments on the Quality of English Language

There are a number of passages that need to be revised. In my comments, I have suggested revisions to these passages.

Author Response

Thank you very much for reviewing our article and providing in-depth analysis! Indeed, when you look at your article with fresh eyes - you can see the flaws) We have attached a file with comments on your comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The problem of women with endometriosis is increasingly being studied.  It is not only ovarian or cervical cancer that is finally being described and studied. As we know, endometriosis leads to pelvic pain, painful menstruation and infertility. In addition, studies of the intestinal microbiome show the impact of dysbiosis on the development of many diseases in the fields of gynecology, neurology, gastroenterology, diabetology, etc. and disorders of the microbiome affect the deregulation of the entire body including the intestinal economy. A study of very interesting use of Dienogest (DNG) in the treatment of endometriosis and its potential impact on the gut microbiome and hormonal balance.  Promising results are provided by changes in the gut microbiome after DNG administration. 
Tables Clear
Charts Clear
Notes:
Change in microbiota to microbiome.
The question is whether 17 patients are sufficient to conclude an actual positive contribution of DNG.

 

Author Response

Comments 1: Change in microbiota to microbiome.

 Response 1: agree. Have been changed

 

Comments 2: The question is whether 17 patients are sufficient to conclude an actual positive contribution of DNG.

Response 2: Fully agree. Although statistically significant results were obtained for some parameters, for full conclusions it is really necessary to increase the sample, which will be carried out in further studies.