The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis

doi:10.21203/rs.3.rs-4975125/v1

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The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis

https://doi.org/10.21203/rs.3.rs-4975125/v1

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Background

The human body harbors greater than 10 trillion symbiotic, microbial cells that contribute to our bodily functions. Emerging evidences suggest that dysbiosis, especially of the gut or urogenital system, may induce various pathological conditions or altered estrobolome and associate with certain estrogen-dependent diseases like endometriosis. The present case-control study analyzed the enzymatic expressions, bacterial compositions, and variations of estrogen metabolites in fecal, vaginal, and urinary samples of patients with or without ovarian endometriosis.

Methods

A total of 38 women of reproductive age, 24 with pathologically-proven ovarian endometriosis and 14 without (control), were analyzed. Recruited patients provided stool, urine, and vaginal samples before undergoing surgeries for ovarian endometriosis or other benign ovarian tumors. Gut enzymatic assays of β-glucuronidase and β-glucosidase were conducted using an ELISA spectrophotometer. Advanced liquid chromatography and mass spectrometry analyzed estrogen and 14 estrogen metabolites in stool, urine, and vaginal samples. Gut and vaginal microbiota were detected with 16S ribosomal-RNA gene sequencing and further classified with Institute of Genome Sciences bioinformatics pipeline. Analyses for species composition, diversity, and abundance were compared between the ovarian endometriosis and control groups. Statistical significance was determined using t-tests and Wilcoxon tests (p < 0.05).

Results

While similar gut β-glucuronidase activities, microbial diversity, and abundance were observed in the fecal samples of both groups, the gut microbiota of the control group showed higher prevalence of Rothia genus whereas genera such as Megamonas, [Eubacterium] coprostanoligenes_group, Allisonella, Ruminiclostridium_5, [Eubacterium] hallii_group, and Negativibacillus were significantly more abundant in the ovarian endometriosis group. Meanwhile, besides statistically lower folds of 4-methoxyestrone (p = 0.046), 2-methoxyestrone (p = 0.043), and 2-hydroxyestrone-3-methyl ether (p = 0.006), vaginal samples of patients with endometriosis also revealed significantly lower bacterial abundance, diversity, richness, and evenness.

Conclusions

While the current findings did not demonstrate obvious dysbiosis in patients with ovarian endometriosis, variations in certain genera and families of bacteria between the two groups could indicate altered estrogen metabolism or disturbed urogenital and gastrointestinal microbiota.

gut microbiota

vaginal microbiota

ovarian endometriosis

dysbiosis

β-glucuronidase

16S ribosomal-RNA gene

estrogen metabolites

Genetic material produced by the vast number of organisms making up the human microbiota create the microbiome [1, 2]. Growing evidence suggests the vital role of microbiota in sustaining bodily systems like the skin, gut, and genital tracts [3]. Dysbiosis, which often results from disrupted microbial composition or reduced diversity, is associated with a spectrum of health problems, including autoimmune disease, cancer, metabolic cancer, inflammatory bowel disease, and others [4].

Endometriosis is a chronic, estrogen-dependent condition caused by endometrial glands and stroma proliferating outside the uterine cavity. Affecting approximately 6 to 10% of reproductive-aged females worldwide [5], it commonly inflicts clinical symptoms like dyspareunia, infertility, dysmenorrhea, and severe pelvic discomfort. Retrograde menstruation, also known as the Sampson’s theory [6], is the most widely accepted pathogenesis. However, with 90% of women experiencing retrograde menstruation and only 10% developing the condition [7], other possible mechanisms relating to genetic, anatomical, endocrine, inflammatory, and environmental factors, are being explored.

Estrobolome describes genes carried by microbiota that are capable of metabolizing estrogens [8]. For instance, microbial β-glucuronidase converts estrogens from their conjugated to active deconjugated forms, which can subsequently enter the bloodstream and interact with estrogen receptors [8]. Emerging studies have highlighted the estrogen-microbiome axis that interconnects the gut microbiome and host microenvironment. Recent findings suggest that disturbances in the estrobolome may activate estrogen-dependent diseases, such as endometriosis and endometrial cancer [9].

The female reproductive tract hosts distinct microbiota that constitute about 9% of the body's total microbial population [10]. Prior studies have shown that patients with endometriosis exhibit higher levels of bacterial colonization in their endometrial tissues and menstrual blood compared to that of the general female population [11–14]. Moreover, altered gut microbiota profiles are also identified in animal models of endometriosis [15]. However, clinical data on the relationship between endometriosis and the endobiota, including the female reproductive and gastrointestinal tracts, are still limited. We have previously observed that in patients with endometriosis, there were significantly higher abundance of Erysipelotrichia class and fecal folds of four estrogens/estrogen metabolites [2]. This current study aims to further investigate the differences in gut and vaginal microbiota of women with or without endometriosis. Since endometriosis is a heterogeneous disease, ranging from superficial peritoneal implants to ovarian cysts and deep infiltrating endometriosis [16], including all three phenotypes with varying severity may underestimate microbial alterations. Therefore, our study focuses on patients with ovarian endometriosis. By examining their enzymatic expressions, bacterial compositions, and variations in estrogen metabolites, the presence or absence of a urogenital-gastrointestinal crosstalk associating with the development of ovarian endometriosis is explored.

Study Cohort and Sample collection

This case-control study included 38 women of reproductive age (20–48 years old) who underwent surgery for benign gynecological diseases at Linkou Chang Gung Memorial Hospital from August 1, 2020, to March 31, 2022. Postmenopausal women, pregnant individuals, or those with past or current malignancies were excluded. Meanwhile, factors such as parity, body mass index (BMI), previous medical (including hormone replacements) or surgical history, and current surgical indications were not subjected to exclusions. The stage of endometriosis for each patient was classified according to the revised guidelines from American Society for Reproductive Medicine: stage I as minimal, stage II as mild, stage III as moderate, and stage IV as severe [17]. Categorization of deeply infiltrated endometriosis was based on the Enzian score [18]. Participants provided fresh fecal and urine samples any time during their hospitalization before surgery. Additionally, vaginal swab samples were collected from patients upon admission. This study complied with the Helsinki Declaration and received approval from the Institutional Review Board of the Human Investigation and Ethical Committee of Chang Gung Medical Foundation.

Protein Extraction and Enzyme Activity Assay (β-glucuronidase)

Protein extraction was conducted using a published protocol [19]. Around 0.5 g of thawed feces was placed into 10 mL conical tube with 5 mL of extraction buffer and vortexed for 1 min for homogenization. Bacterial cell lysis was achieved through sonication in 30 s intervals at maximum power. All of the steps were performed in an ice bath. The supernatant containing extracted proteins was then collected after centrifuging the lysates at 4°C. The activities of β-glucuronidase and β-glucosidase were measured using commercial assay kits according to the manufacturers' instructions. Specifically, a 96-well microplate was prepared with 5–20 µL of fecal lysate, adjusted to 90 µL with assay buffer for each reaction. Following the addition of the provided Substrate Mix (Abcam, Cambridge, UK) for 0–60 min at 37°C, fluorescence was measured (Ex/Em = 330/450 nm) using a multi-well ELISA spectrophotometer (Tecan Infinite M200 Plate Reader, Ramsey, MN, USA).

Liquid Chromatography and Tandem Mass Spectrometry (estrogens and metabolites)

For analyzing urine and fecal estrogens and their metabolites, samples were transferred on dry ice to Biotools Laboratory in New Taipei City, Taiwan. The lab used advanced liquid chromatography and mass spectrometry for compound separation and analysis. This followed US FDA guidelines for detection and quantification limits, measuring 14 target metabolites including estrone, estradiol, and estriol variations, with final concentrations reported in ng/mL.

Genomic DNA Extraction and 16S rRNA Gene Sequencing (fecal & vaginal DNA, rRNA)

Microbial DNA was extracted from fecal and vaginal samples using the Genomic DNA Isolation Kit (FairBiotech Corp., Taoyuan, Taiwan). The hypervariable regions (V3-V4) of the 16S ribosomal RNA (rRNA) sequence were amplified by PCR, with the detailed procedure and sequences of the universal primers available in a previous study [2]. Fourteen samples from the control group and 24 samples from ovarian endometriosis patients exhibited clear amplified DNA fragments and were prepared for library construction. Amplicon sequencing was performed on the Illumina paired-end platform.

The raw data were merged using FLASH and filtered through QIIME to obtain clean data [20, 21]. Chimeric sequences within the clean data were identified and removed with UCHIME, yielding the final set of effective tags [22]. To analyze the species diversity in each sample, all effective tags were clustered into Operational Taxonomic Units (OTUs) based on 97% DNA sequence similarity using the UPARSE algorithm within the USEARCH pipeline v7.0.1090.20 [23].

Statistical Analysis

Using the QIIME v2 pipeline, the alpha and beta diversities were assessed to elucidate species complexity within and between samples. The relative species, evenness and abundance distribution in each sample were analyzed by alpha-diversity methods, including Shannon, Simpson, and good coverage. To compare microbial communities among groups, beta-diversity was calculated to reflect the dissimilarities between groups, including unweighted UniFrac and weighted UniFrac distance [24]. In order to emphasize the difference of the dominant species between endometriosis and control groups in each taxonomic rank, the top 10 species were selected and distribution histograms were drawn based on relative abundance. To visualize microbial genomes and metagenomes and illustrate evolutionary relationships, species richness, and annotations for a comprehensive phylogenetic tree, GraPhlAn (Graphical Phylogenetic Analysis) was used to construct tree graphs of fecal and vaginal species annotations for the endometriosis group [25]. To identify potential biomarkers with statistical differences between the groups, metagenomic features were analyzed using Linear Discriminant Analysis Effect Size (LEfSe) [26]. In this study, taxa with a linear discriminant analysis (LDA) score greater than 2 (log 10) were considered significant.

Statistical methods such as t-test, ANOVA, and Wilcox test were performed to analyze the significance of difference between groups. A p-value < 0.05 was considered significant for all statistical tests employed.

Of the 38 Taiwanese women included, 24 women had pathologically-proven ovarian endometriosis while the other 14 served as controls. The descriptive characteristics of study participants are summarized in Table 1. There was no significant difference in average age (38.5 ± 5.1 vs. 35.3 ± 6.6 years, p = 0.129) and BMI (24.5 ± 4.4 vs. 23.4 ± 4.3 kg/m², p = 0.450) between the control and ovarian endometriosis groups. Comparison of the pre-operative CA-125 level showed, as expected, significantly higher level in the ovarian endometriosis group than that of the control (67.4 ± 63.4 vs 27.4 ± 4.6 U/mL, p = 0.007). There were also significant differences in gravida (0 vs 2.5, p = 0.004) and parity (0 vs 2.0, p < 0.001) between the two groups.

Table 1

Baseline characteristics of control and endometriosis group.
		Control (n = 14)	Endometriosis (n = 24)	P-value
Age (year)		38.5 (± 5.1)	35.3 (± 6.6)	0.129
BMI (kg/m2)		24.5 (± 4.4)	23.4 (± 4.3)	0.450
Gravida		2.50 (0.00-3.25)	0.00 (0.00-1.75)	0.004*
Parity		2.00 (0.00–3.00)	0.00 (0.00–0.00)	< 0.001*
CA-125 (U/mL)		27.4 (± 4.6)	67.4 (± 63.4)	0.007*
rAFS score		NA	50.83 ± 36.27	NA
Endometriosis stage (%)	O		8.3% (2/24)
	I	NA	4.2% (1/24)	NA
	II	NA	0.0% (0/24)	NA
	III	NA	41.7% (10/24)	NA
	IV	NA	45.8% (11/24)	NA

Similar β-Glucuronidase Activity between Endometriosis and Control

Gut microbial β-glucuronidase (gmGUS) is involved in the metabolism of estrogen (27). It has been shown that stable interaction between gmGUS and estrogen maintains physiological balance while disruption can lead to estrogen-related diseases (27). However, enzymatic activity assays conducted on fecal samples from both the control and ovarian endometriosis group did not show significant differences in the average levels of β-glucosidase activity (-14.82 U/L vs. 23.15 U/L, p = 0.76) or β-glucuronidase activity (5851.95 U/L vs. 5122.04 U/L, p = 0.70) (Fig. 1).

Differences in Estrogen Metabolites Were Seen in the Urine and Vagina but Not Fecal Samples from Patients with Ovarian Endometriosis

Analyses of 14 estrogens and its metabolites demonstrated significantly lower folds of 4-methoxyestrone (p = 0.046), 2-methoxyestrone (p = 0.043), and 2-hydroxyestrone-3-methyl ether (p = 0.006) in the vaginal samples of patients with ovarian endometriosis when compared to that of the control group. Meanwhile, 16α-hydroxyestrone (p = 0.032) was significantly higher in urine samples of ovarian endometriosis patients. However, such trend was not replicated in their fecal samples, as shown in Table 2.

Gut Microbial Landscape and Diversity Analysis of the Study Cohort

Paired-end 16S amplicon sequencing generated raw reads from stool of 14 healthy women and 24 ovarian endometriosis patients. Following quality control, effective tags were used for operational taxonomic unit (OTU) clustering and species annotation. Species composition and abundance were analyzed across samples, with a tree graph displaying species annotations for each group presented in Fig. 2. In this present study, the predominant bacterial phyla in the gut microbiomes of Taiwanese women with endometriosis were Bacteroidetes (49%), Firmicutes (41%), and Proteobacteria (8%) (Fig. 2a). The distribution histogram shows the relative abundance of the top-10 major phyla of gut microbiomes between two groups (Fig. 2b). The compositions of microbial species at each hierarchical level are illustrated using heat trees (Figs. 2c, d). Both groups displayed similar relative bacterial abundances.

With its various indicators, alpha diversity, which is a measure of microbial diversity, richness, and evenness within samples, showed no significant differences in Shannon–Wiener diversity index (Fig. 3a), Simpson diversity index (Fig. 3b), and species richness (Fig. 3c) between the two groups. Meanwhile, beta diversity, as assessed by unweighted (Fig. 3d) and weighted UniFrac distance (Fig. 3e), showed similar microbial composition between the two groups.

A balanced gut microbiota that compose mostly of the Bacteroidetes and Firmicutes phyla supports the gut epithelial barrier and overall homeostasis [28]. Higher Firmicutes/Bacteroidetes ratio (F/B) in the gut suggests dysbiosis [29]. As depicted in the box plot (Fig. 3f), the median F/B ratio of the ovarian endometriosis group was marginally higher than that of the control group (0.98 vs. 0.93, p = 0.88), but it did not reach statistical significance.

Distinct Gut Microbiomes in Healthy Women and Ovarian Endometriosis Patients

To better understand the characteristic gut microbiota associated with patients with ovarian endometriosis, LEfSe analysis was conducted on the phylogenetic trees of the dominant microorganisms in the two groups. We used a LDA threshold > 2 to identify significant characteristic flora at the genus level. The LDA scores and cladogram highlighted Rothia genus within the Micrococcales lineage as more prevalent in the control group (Fig. 3g). Genera such as Megamonas, [Eubacterium] coprostanoligenes_group, Allisonella, Ruminiclostridium_5, [Eubacterium] hallii_group, Negativibacillus were significantly more abundant in the ovarian endometriosis group (Fig. 3h).

Vaginal Microbial Landscape and Diversity Analysis

Similar to gut microbiota, different sections of the female reproductive tract exhibit distinct microbial distributions. The female reproductive tract is divided into a higher segment, consisting of the endocervix and uterus, and a lower segment, comprising of the vaginal canal and ectocervix. Vaginal samples were analyzed for this study in order to evaluate the microbiota of the lower female reproductive tract.

The lower female reproductive tract has been shown to be primarily populated by Lactobacillus spp., which protects it against pathogens by creating an acidic environment and producing bacteriocins and hydrogen peroxide [30]. Compatibly, Lactobacillus was the predominant genus observed in the vaginal microbiota of both the endometriosis and controls groups. In the ovarian endometriosis group, Lactobacillus accounted for 64.4%, Prevotella 6.6%, and Gardnerella 5.5%. In contrast, the control group had 48.5% Lactobacillus, 6.8% Gardnerella, and 5.8% Prevotella (Fig. 4a, b). The compositions of microbial species at each hierarchical level are illustrated with heat trees (Figs. 4c, d). No significant differences were seen in alpha diversity, as indicated by the Shannon-Wiener diversity index (Fig. 5a), Simpson diversity index (Fig. 5b), and species richness (Fig. 5c). However, lower beta diversity was found in the vaginal samples of patients with ovarian endometriosis, as shown by the unweighted UniFrac distance (Fig. 5d, p < 0.001) and weighted UniFrac distance (Fig. 5e, p = 0.006). The results signified lower species annotation and OTU abundance in the vaginal samples of patients with endometriosis.

Microbial Flora of the Vaginal Microbiota That Associates with Ovarian Endometriosis

Linear discriminant analysis with effect size (LEfSe) analyses on the phylogenetic trees of the dominant microorganisms, using a LDA threshold > 2, defined significant characteristics of the flora between the two groups. With LDA scores and cladogram, the present study revealed that, at the family level, the Ruminococcaceae families (p = 0.047), Eggerthellaceae families (p = 0.009) and Beijerinckiaceae families (p = 0.047) were more dominant in the control group when compared to the ovarian endometriosis group (Fig. 5f).

The estrobolome-endometriosis axis is complex and poorly understood. The estrobolome can alter circulating estrogen levels through deconjugation of its conjugated substrates with gut microbial β-glucuronidase [31], which is an enzyme commonly found in several bacterial genera, such as Bacteroides, Bifidobacterium, Escherichia coli, and Lactobacillus [32]. A review article in 2023 concluded that gut microbial β-glucuronidase may become a potential biomarker for early diagnosis of estrogen-dependent diseases [27]. In addition, mounting data in human and animal studies supported an association between dysbiotic gut or genital microbiome and endometriosis [15, 27, 31, 33–36]. Many animal models had also demonstrated a bidirectional relationship between endometriosis and microbial alterations [15, 37]. Yuan et al. [15] conducted a prospective, randomized experiment on an animal endometriosis model induced via the intraperitoneal injection of endometrial tissues. Different compositions of gut microbiota (elevated F/B ratio and increased Bifidobacterium) were detected 42 days after the modeling. Furthermore, Chadchan et al. [37] treated surgically-induced endometriosis mice with broad-spectrum antibiotics or metronidazole and found that oral antibiotic treatment was effective in reducing the progression of endometriosis 21 days after the modeling. Muraoka et al. [38] demonstrated a promotional pathogenic role of Fusobacterium in the formation of ovarian endometriosis by examining vaginal swab samples from 79 women with endometriosis and 76 without. They observed a significant difference: 64% of women with endometriosis tested positive for Fusobacterium while only 7% of healthy women showed the same result. However, the potential use of antibiotics as a non-hormonal therapy for endometriosis remains uncertain [39].

Our previous study aimed to find associations of the gut microbiota with endometriosis through bacterial analyses, enzymatic assays and targeted metabolites quantification [2]. While the results did not detect significant changes in microbial richness, diversity, β-glucuronidase activities, or urinary estrogen metabolites, fecal samples from endometriosis patients showed greater enrichment of specific bacteria (UBA1819, Eisenbergiella, Hungatella, and Erysipelatoclostridium) and exhibited increased levels of four estrogen metabolites (estriol, 16-epiestriol, 16alpha-hydroxyestrone, and 2-methoxyestradiol) [2]. In line with our previous results, several gene-based researches had associated gut microbes with endometriosis. Svensson et al. analyzed the gut microbiome profile of 66 women with endometriosis and 198 control and found overall gut microbial diversity was significantly higher in controls compared to patients with endometriosis [35]. Besides, abundance of 12 bacteria belonging to the classes Bacilli, Bacteroidia, Clostridia, Coriobacteriia, and Gammaproteobacter differed significantly between the patients and controls. Another study showed a lower α diversity of gut microbiota and a higher F/B ratio in 12 patients with moderate-to-severe endometriosis when compared to healthy controls [40]. In their study, genera such as Blautia, Bifidobacterium, Dorea, and Streptococcus, were significantly more abundant in the endometriosis group in comparison to the controls, whereas lower levels of Lachnospira and Eubacterium eligens group were found in women with endometriosis. The largest metagenome study to date analyzed 1000 women from the Estonian Microbiome cohort that included 136 women with endometriosis and 864 control. However, neither distinct compositional or functional gut microbial profiles in women with or without endometriosis nor different estrobolome-associated enzyme sequences were identified [41].

Building from our previous work, this study further explored potential associations between endobiota (urogenital-gastrointestinal microbiota) and estrobolome in the development of ovarian endometriosis. While no significant differences were seen in the gut enzymatic activities of patients with or without ovarian endometriosis, estrogen metabolites showed lower folds of 4-methoxyestrone (p = 0.046), 2-methoxyestrone (p = 0.043), and 2-hydroxyestrone-3-methyl ether (p = 0.006) in the vaginal samples and higher fold of 16α-hydroxyestrone (p = 0.032) in the urine samples of patients with ovarian endometriosis. In addition, different endobiota profiles were observed between the two groups. The gut microbiota of patients with ovarian endometriosis showed higher abundances of certain bacterial genera, including Megamonas, [Eubacterium] coprostanoligenes_group, Allisonella, Ruminiclostridium_5, [Eubacterium] hallii_group, and Negativibacillus. Furthermore, patients with ovarian endometriosis exhibited relatively lower bacterial abundance and diversity in their vaginal samples, whereas the control group displayed higher abundances of Ruminococcaceae and Beijerinckiaceae families.

Previous studies analyzing the role of the reproductive tract microbiome had demonstrated that opportunistic pathogens, such as Streptococcaceae and Staphylococaceae, were enriched in the cystic fluid of females with ovarian endometrioma [12]. Studies from Brazil and China noted lower Lactobacillus abundance in endometriosis patients compared to controls [42–44]. Additionally, Ata et al. highlighted differences in vaginal microbiota between severe endometriosis patients and healthy individuals, noting the absence of Gemella and Atopobium spp. in vaginal samples from the endometriosis group at the genus level [45]. Perrotta et al. broadened their scope to include vaginal community state types (CST) for constructing a random forest classification model that used microbiota to predict r-ASRM endometriosis stages, particularly highlighting the predictive value of changes during the follicular and menstrual phases for identifying disease stages with specific microbial taxa like the genus Anaerococcus (phylum Firmicutes) [46]. Although our results did not demonstrate a causal relationship between endobiota and ovarian endometriosis, the possibility of altered estrogen metabolism or an association with the urogenital and gastrointestinal microbiota should not be dismissed, as variations in certain bacterial genera and families were observed.

Consistent with prior research that showed an association of gut microbial disequilibrium with increased Firmicutes / Bacteroidetes ratio (F/B ratio), leading to obesity [47], hypertension [48], and irritable bowel syndrome [49], our current study revealed higher abundances of certain bacterial genera, namely Megamonas, [Eubacterium] coprostanoligenes_group, Allisonella, Ruminiclostridium_5, [Eubacterium] hallii_group, and Negativibacillus, in the gut microbiota of patients with ovarian endometriosis. Interestingly, all of which belonged to the Firmicutes phylum. The Megamonas genus, which is anaerobic, non-motile, and non-spore-forming, has been positively associated with obesity [50] and several diseases such as nonalcoholic fatty liver disease [51]. Additionally, in previous vaginal microbiota studies, Kunaseth et al. [52] reported that Alloscardovia, Oscillospirales, Ruminococcaceae, Oscillospiraceae, Enhydrobacter, Megamonas, Selenomonadaceae, and Faecalibacterium all exhibited increased abundance in the vaginal microbiota of patients with adenomyosis. Nevertheless, the metabolic pathway contributed by these genera remains to be elucidated. The Ruminococcaceae family, which was found in higher levels in the vaginal samples of our control group, was identified as a protective factor for endometriosis by Cao et al. [53]. They suggested that distinct abundances of short-chain fatty acids (SCFA)-producing bacteria including genus Rikenellaceae RC9 gut group, family Ruminococcaceae, genus Ruminococcaceae UCG005, genus Eubacterium ruminantium, and family Clostridiales vadinBB60 group were acting as the protective factors in endometriosis [53].

Since published studies suggested that enterohepatic recirculation affected systemic estrogen levels and could ultimately lead to reproductive diseases [19, 54–56], we analyzed parent estrogens (estradiol and estrone) and 12 estrogen metabolites in fecal, urinary, and vaginal samples. Our data identified notably lower levels of 4-methoxyestrone (p = 0.046), 2-methoxyestrone (p = 0.043), and 2-hydroxyestrone-3-methyl ether (p = 0.006) in the vaginal samples and higher levels of 16α-hydroxyestrone (p = 0.032) in the urine samples of the ovarian endometriosis group. Similar to previous research [56, 57], we did not find significant correlations in estrogens and their metabolites in the stool samples between the two groups. Interestingly, while stool samples of patients with endometriosis presented with a higher fold of 16α-hydroxyestrone in our previous study, the current study found lower fold of this metabolite in the urinary sample.

As the endobiota is subject to variation among individuals due to numerous factors, this study faced several limitations. Numerous confounders could affect the profiling of an individual's gut microbiota. This feat was made harder since we analyzed the compositions of not only gut but also urine and vaginal microbiota. While the Shannon diversity index is effective for evaluating microbiota diversity in areas with numerous genera, such as stool, it may be less effective in locations with fewer genera, such as the vagina and cervix [58]. Therefore, we attempted to reduce variability by selecting a specific ethnic group of women of similar age and excluding those with strict dietary habits, systemic diseases, past or current cancer diagnoses, and recent antibiotic or probiotic use. In this study, the use of hormone medications was not an exclusion factor, as all participants were undergoing surgical procedures for benign gynecological conditions due to unsuccessful medical treatments or severe symptoms. Consequently, eleven patients in the endometriosis group had been treated with GnRH agonists or progestins within three months prior to their surgeries, whereas none in the control group had received hormone therapy.

While our current findings could not demonstrate specific dysbiosis in patients with ovarian endometriosis, altered estrogen metabolism or potential association with the urogenital and gastrointestinal microbiota should not be dismissed as variations in certain genera and families of bacteria were observed. Larger, more rigorously controlled studies are needed to clarify if an endobiota-estrobolome cross-talk exists. Additionally, the question of whether dysbiosis leads to endometriosis or vice versa remains to be answered.

By analyzing endobiota samples from reproductive-aged, Taiwanese women with and without histologically confirmed ovarian endometriosis, our research contributes to the extensive literature on microbiota and endometriosis. Although the current findings did not identify specific dysbiosis in patients with ovarian endometriosis, variations in certain bacterial genera and families suggested possible alterations in estrogen metabolism or associations with the urogenital and gastrointestinal microbiome. While the results were insufficient to confirm a urogenital-gastrointestinal crosstalk or its associations with ovarian endometriosis, further studies could help clarify these theories.

gmGUS	Gut microbial β-glucuronidase
GnRH	Gonadotropin-Releasing Hormone
LDA	Linear discriminant analysis
LEfSe	Linear Discriminant Analysis Effect Size
OTU	Operational Taxonomic Unit

Acknowledgements

We thank Genomic Medicine Core Laboratory and Laboratory Center of Precise Center, Chang Gung Memorial Hospital for the sequencing and analysis of estrobolome and Biotools Laboratory Institute for providing technical support.

Authors’ contributions

All authors have read and agreed to the published version of the manuscript. Conceptualization, H.-Y.H. and A.H.-Y.P.; methodology, H.-Y.H., Y.-W.W., P.-C.L. and C.-Y.H.; software, Y.-W.W., P.-C.L. and C.-Y.H.; validation, H.-Y.H., C.-Y.H., Y.-W.W., and P.-C.L.; formal analysis, H.-Y.H. and C.-Y.H.; investigation, H.-Y.H., A.H.-Y.P., and C.-Y.H; re- sources, H.-Y.H. and H.-M.W.; data curation, C.-Y.H.; writing—original draft preparation, C.-Y.H and A.H.-Y.P.; writing—review and editing, H.-Y.H., A.H.-Y.P., and C.-Y.H; visualization, H.-Y.H. and C.-Y.H.; supervision, H.-Y.H.; project administration, H.-Y.H. and H.-M.W.; funding acquisition, H.-Y.H.

Funding

This research works was supported in part under Chang Gung Memorial Hospital Research Grant (CMRPG3G1981, CMRPG3K0782).

Availability of data and materials

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to containment of patient identifiers.

Ethics approval and consent to participate

Informed consent was obtained from all subjects involved in the study. The study was reviewed and approved by the institutional review board of the Human Investigation and Ethical Committee of Chang Gung Medical Foundation (CGMHIRB No. 202000462A3).

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Author details

¹ Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan City 333423, Taiwan

² College of Medicine, Chang Gung University, Taoyuan City, Taiwan

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No competing interests reported.

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The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Study Cohort and Sample collection

Protein Extraction and Enzyme Activity Assay (β-glucuronidase)

Liquid Chromatography and Tandem Mass Spectrometry (estrogens and metabolites)

Genomic DNA Extraction and 16S rRNA Gene Sequencing (fecal & vaginal DNA, rRNA)

Statistical Analysis

Results

Similar β-Glucuronidase Activity between Endometriosis and Control

Gut Microbial Landscape and Diversity Analysis of the Study Cohort

Distinct Gut Microbiomes in Healthy Women and Ovarian Endometriosis Patients

Vaginal Microbial Landscape and Diversity Analysis

Microbial Flora of the Vaginal Microbiota That Associates with Ovarian Endometriosis

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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