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

The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis Chia-Yu Hu, Angel Hsin-Yu Pai, Yi-Wen Wang, Pei-Chen Lu, Hsien-Ming Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4975125/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background 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. Methods 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. Results 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/m 2 , 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. 2 a). The distribution histogram shows the relative abundance of the top-10 major phyla of gut microbiomes between two groups (Fig. 2 b). The compositions of microbial species at each hierarchical level are illustrated using heat trees (Figs. 2 c, 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. 3 a), Simpson diversity index (Fig. 3 b), and species richness (Fig. 3 c) between the two groups. Meanwhile, beta diversity, as assessed by unweighted (Fig. 3 d) and weighted UniFrac distance (Fig. 3 e), 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. 3 f), 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. 3 g). Genera such as Megamonas , [ Eubacterium ] coprostanoligenes_group , Allisonella , Ruminiclostridium_5 , [ Eubacterium ] hallii_group , Negativibacillus were significantly more abundant in the ovarian endometriosis group (Fig. 3 h). 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. 4 a, b). The compositions of microbial species at each hierarchical level are illustrated with heat trees (Figs. 4 c, d). No significant differences were seen in alpha diversity, as indicated by the Shannon-Wiener diversity index (Fig. 5 a), Simpson diversity index (Fig. 5 b), and species richness (Fig. 5 c). However, lower beta diversity was found in the vaginal samples of patients with ovarian endometriosis, as shown by the unweighted UniFrac distance (Fig. 5 d, p < 0.001) and weighted UniFrac distance (Fig. 5 e, 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. 5 f). Discussion 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. Conclusions 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. Abbreviations gmGUS Gut microbial β-glucuronidase GnRH Gonadotropin-Releasing Hormone LDA Linear discriminant analysis LEfSe Linear Discriminant Analysis Effect Size OTU Operational Taxonomic Unit Declarations 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 References Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. The human microbiome project. Nature. 2007;449(7164):804-10. Pai AH, Wang YW, Lu PC, Wu HM, Xu JL, Huang HY. Gut Microbiome-Estrobolome Profile in Reproductive-Age Women with Endometriosis. Int J Mol Sci. 2023;24(22). Vitale SG, Ferrari F, Ciebiera M, Zgliczynska M, Rapisarda AMC, Vecchio GM, et al. 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The role of gut and genital microbiota and the estrobolome in endometriosis, infertility and chronic pelvic pain. Hum Reprod Update. 2021;28(1):92-131. Beaud D, Tailliez P, Anba-Mondoloni J. Genetic characterization of the beta-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus. Microbiology (Reading). 2005;151(Pt 7):2323-30. Dabek M, McCrae SI, Stevens VJ, Duncan SH, Louis P. Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria. FEMS Microbiol Ecol. 2008;66(3):487-95. Wei Y, Tan H, Yang R, Yang F, Liu D, Huang B, et al. Gut dysbiosis-derived beta-glucuronidase promotes the development of endometriosis. Fertil Steril. 2023;120(3 Pt 2):682-94. Svensson A, Brunkwall L, Roth B, Orho-Melander M, Ohlsson B. Associations Between Endometriosis and Gut Microbiota. Reprod Sci. 2021;28(8):2367-77. Huang L, Liu B, Liu Z, Feng W, Liu M, Wang Y, et al. Gut Microbiota Exceeds Cervical Microbiota for Early Diagnosis of Endometriosis. Front Cell Infect Microbiol. 2021;11:788836. Chadchan SB, Cheng M, Parnell LA, Yin Y, Schriefer A, Mysorekar IU, et al. Antibiotic therapy with metronidazole reduces endometriosis disease progression in mice: a potential role for gut microbiota. Hum Reprod. 2019;34(6):1106-16. Ayako Muraoka MS, Tomonari Hamaguchi, Shinya Watanabe, Kenta Iijima, Yoshiteru Murofushi, Keiko Shinjo, Satoko Osuka, Yumi Hariyama, Mikako Ito, Kinji Ohno, Tohru Kiyono, Satoru Kyo, Akira Iwase, Fumitaka Kikkawa, Hiroaki Kajiyama, Yutaka Kondo. Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts. Sci Transl Med. 2023;14(15):700. Khan KN, de Ziegler D, Guo SW. Bacterial infection in endometriosis: a silver-lining for the development of new non-hormonal therapy? Hum Reprod. 2024;39(4):623-31. Shan J, Ni Z, Cheng W, Zhou L, Zhai D, Sun S, et al. Gut microbiota imbalance and its correlations with hormone and inflammatory factors in patients with stage 3/4 endometriosis. Arch Gynecol Obstet. 2021;304(5):1363-73. Perez-Prieto I, Vargas E, Salas-Espejo E, Lull K, Canha-Gouveia A, Perez LA, et al. Gut microbiome in endometriosis: a cohort study on 1000 individuals. BMC Med. 2024;22(1):294. Hernandes C, Silveira P, Rodrigues Sereia AF, Christoff AP, Mendes H, Valter de Oliveira LF, et al. Microbiome Profile of Deep Endometriosis Patients: Comparison of Vaginal Fluid, Endometrium and Lesion. Diagnostics (Basel). 2020;10(3). Lu F, Wei J, Zhong Y, Feng Y, Ma B, Xiong Y, et al. Antibiotic Therapy and Vaginal Microbiota Transplantation Reduce Endometriosis Disease Progression in Female Mice via NF-kappaB Signaling Pathway. Front Med (Lausanne). 2022;9:831115. Chao X, Liu Y, Fan Q, Shi H, Wang S, Lang J. The role of the vaginal microbiome in distinguishing female chronic pelvic pain caused by endometriosis/adenomyosis. Ann Transl Med. 2021;9(9):771. Ata B, Yildiz S, Turkgeldi E, Brocal VP, Dinleyici EC, Moya A, et al. The Endobiota Study: Comparison of Vaginal, Cervical and Gut Microbiota Between Women with Stage 3/4 Endometriosis and Healthy Controls. Sci Rep. 2019;9(1):2204. Perrotta AR, Borrelli GM, Martins CO, Kallas EG, Sanabani SS, Griffith LG, et al. The Vaginal Microbiome as a Tool to Predict rASRM Stage of Disease in Endometriosis: a Pilot Study. Reprod Sci. 2020;27(4):1064-73. Cox AJ, West NP, Cripps AW. Obesity, inflammation, and the gut microbiota. Lancet Diabetes Endocrinol. 2015;3(3):207-15. Yang T, Santisteban MM, Rodriguez V, Li E, Ahmari N, Carvajal JM, et al. Gut dysbiosis is linked to hypertension. Hypertension. 2015;65(6):1331-40. Rajilic-Stojanovic M, Biagi E, Heilig HG, Kajander K, Kekkonen RA, Tims S, et al. Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology. 2011;141(5):1792-801. Duan M, Wang Y, Zhang Q, Zou R, Guo M, Zheng H. Characteristics of gut microbiota in people with obesity. PLoS One. 2021;16(8):e0255446. Zhou J, Zhang Q, Zhao Y, Zou Y, Chen M, Zhou S, et al. The relationship of Megamonas species with nonalcoholic fatty liver disease in children and adolescents revealed by metagenomics of gut microbiota. Sci Rep. 2022;12(1):22001. Kunaseth J, Waiyaput W, Chanchaem P, Sawaswong V, Permpech R, Payungporn S, et al. Vaginal microbiome of women with adenomyosis: A case-control study. PLoS One. 2022;17(2):e0263283. Cao T, Wang Y, Huimin S. Causal effects between gut microbiota and endometriosis: a two-sample Mendelian randomisation study. J Obstet Gynaecol. 2024;44(1):2362415. Qin R, Tian G, Liu J, Cao L. The gut microbiota and endometriosis: From pathogenesis to diagnosis and treatment. Front Cell Infect Microbiol. 2022;12:1069557. Jiang Y, Greenwood-Van Meerveld B, Johnson AC, Travagli RA. Role of estrogen and stress on the brain-gut axis. Am J Physiol Gastrointest Liver Physiol. 2019;317(2):G203-G9. Wu Z, Pfeiffer RM, Byrd DA, Wan Y, Ansong D, Clegg-Lamptey J-N, et al. Associations of Circulating Estrogens and Estrogen Metabolites with Fecal and Oral Microbiome in Postmenopausal Women in the Ghana Breast Health Study. Microbiology Spectrum. 2023;11(4):e01572-23. Coburn SB, Stanczyk FZ, Falk RT, McGlynn KA, Brinton LA, Sampson J, et al. Comparability of serum, plasma, and urinary estrogen and estrogen metabolite measurements by sex and menopausal status. Cancer Causes Control. 2019;30(1):75-86. Johnson KV, Burnet PW. Microbiome: Should we diversify from diversity? Gut Microbes. 2016;7(6):455-8. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4975125","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":346666955,"identity":"f67cba4c-971a-48f9-84d6-8e392e4f0f80","order_by":0,"name":"Chia-Yu Hu","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chia-Yu","middleName":"","lastName":"Hu","suffix":""},{"id":346666956,"identity":"cdeb1506-425a-4412-ac94-1875568577b8","order_by":1,"name":"Angel Hsin-Yu Pai","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Angel","middleName":"Hsin-Yu","lastName":"Pai","suffix":""},{"id":346666957,"identity":"5e8e736f-73d7-4420-971f-06626fa17a80","order_by":2,"name":"Yi-Wen Wang","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi-Wen","middleName":"","lastName":"Wang","suffix":""},{"id":346666958,"identity":"2e72e9fe-0bbc-40b1-8402-0abec1128067","order_by":3,"name":"Pei-Chen Lu","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei-Chen","middleName":"","lastName":"Lu","suffix":""},{"id":346666959,"identity":"f4ac05f1-8515-4e48-9e70-9b1440b710ea","order_by":4,"name":"Hsien-Ming Wu","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hsien-Ming","middleName":"","lastName":"Wu","suffix":""},{"id":346666963,"identity":"3f3f3aff-5184-4c42-94ea-463bc8fca153","order_by":5,"name":"Hong-Yuan Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACAxiDH4iZSdMi2QDXQkAnXIvBAWK1mLP3Hn7N22aXZ3z8+NXNBQw2+fIO/Mck8Gmx7DmXZs3bllxsdian7PYMhjTLjQeY2fBqMbiRY2bM28acuO0GT9ptHobDBoYNzGw3iNBSn7h5BglajB/zth1O3CDBfgysRZ6BkJYzZ8wY55w7njjjTA7bbR6DNAMDZmbzH3i1HO8x/vCmrDqxv/34s9s8FTYG8u2Njw3waQECNikeMM1jAI4mg8ME1AMB80eIO9gfgCn5BsJaRsEoGAWjYGQBAA9wSFBXWWWFAAAAAElFTkSuQmCC","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hong-Yuan","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-08-26 04:18:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4975125/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4975125/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66867581,"identity":"fdf888f0-f7cb-4e29-b5ac-90d95bf2b9b8","added_by":"auto","created_at":"2024-10-17 09:21:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48116,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic assay with results given in U/L. Boxplots: box indicate the 1st and 3rd quartiles, dash lines the upper and lower whiskers, cross indicates mean, horizontal bold lines the median, and dots signifies outliers.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/adfe5d08e500705a9064b425.png"},{"id":66866993,"identity":"4becc41e-7692-4cce-bd3d-54fdc624467a","added_by":"auto","created_at":"2024-10-17 09:13:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":371883,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance and composition of gut microbiomes between control and patients with ovarian endometriosis. (a) The top-5 major phyla of bacteria in gut microbiota of Taiwanese women. (b) Relative abundance of the top-10 major phyla of gut microbiota in two groups are shown in distribution histogram. (c-d) Circular heat trees represent the sequence abundance of different hierarchical taxa, displayed from the center outwards. The innermost circle represents the highest taxonomic level, the bacterial domain (Bacteria). Moving outward from the center, the taxonomic levels decrease, and the number of sequences annotated to different taxonomic levels decreases accordingly. Sequence abundance is represented by node size, branch thickness, and color. Species with higher abundance are indicated by larger nodes, thicker branches, and colors closer to brown. The legend in the lower-right corner shows the sequence count along with their corresponding colors and node sizes. (c) Control and (d) Ovarian Endometriosis. CES = control, ES = ovarian endometriosis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/b3f37ba089524dc77653e71c.png"},{"id":66867582,"identity":"8250e51a-229b-4942-8170-c30b2f96b18e","added_by":"auto","created_at":"2024-10-17 09:21:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171705,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of gut microbiota composition between the two groups. (a-c) Alpha diversity analysis on a single sample reflects the richness, evenness, and diversity of microbial communities within that sample. Shown with box plots, there were no significant differences observed using Shannon (a), Simpson (b) and species richness (c) indices. These signified that the alpha diversity of gut microbiota was similar between the two groups. (d-e) Beta diversity studies use OTU representative sequences to construct phylogenetic trees and calculate unweighted UniFrac and weighted UniFrac (when relative abundance of species within sample was considered) distances in order to evaluate differences between samples. Smaller values indicate less differences in species diversity between samples. Results are represented with box plots: (d) unweighted UniFrac and (e) weighted UniFrac. T-test was used to calculate for significant differences. (f) F/B (Firmicutes / Bacteroidetes) ratio of the control and ovarian endometriosis group shown in box plot. (g) LEfSe (linear discriminant analysis (LDA) Effect Size) was performed to define microbial biomarkers for ovarian endometriosis group with statistical differences. The representative microbiotas for each group are shown with the LDA scores (left panel). The phylogenetic trees of dominant microorganisms are revealed in cladogram (right). (h) Histogram of abundance comparison of species at genus level. CES = control, ES = ovarian endometriosis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/f74b49b2fdc8db25ed228230.png"},{"id":66866996,"identity":"b6c81804-5487-4674-bc38-8ffd6b300d32","added_by":"auto","created_at":"2024-10-17 09:13:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304751,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance and composition of vaginal microbiomes between control and patients with ovarian endometriosis. (a) The top-5 major genus of bacteria in vaginal microbiota of Taiwanese women. (b) Relative abundance of the top-10 major genus of vaginal microbiota in two groups are shown in distribution histogram. (c-d) Circular heat trees represent the sequence abundance of different hierarchical taxa, displayed from the center outwards. (c) Control and (d) Ovarian endometriosis. CEV = control, EV = ovarian endometriosis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/d092390aca789d57aac9bb30.png"},{"id":66866995,"identity":"7ecb2706-79da-425d-b960-ab2913739706","added_by":"auto","created_at":"2024-10-17 09:13:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143494,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of vaginal microbiota composition between the two groups. Shown with box plots, no significant differences were observed using Shannon (a), Simpson (b) and species richness (c) indices, which suggested that the alpha diversity of vaginal microbiota was similar in the endometriosis and control group. (d-e) Beta diversity studies use OTU representative sequences to construct phylogenetic trees and calculate unweighted UniFrac (d) and weighted UniFrac (e) distances in order to evaluate differences between samples. Smaller values indicate less differences in species diversity between samples. T-test was used to calculate for significant differences. (f) LEfSe (linear discriminant analysis Effect Size) was performed to define microbial biomarkers for endometriosis group with statistical differences. The representative microbiotas for each group are shown with the LDA scores (left panel) The phylogenetic trees of dominant microorganisms are revealed in cladogram (right). CEV = control, EV = ovarian endometriosis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/835789fc8ca5ea7045679517.png"},{"id":66869919,"identity":"a1146d0a-3bba-4622-936c-1512a037d5b3","added_by":"auto","created_at":"2024-10-17 09:37:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1699331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4975125/v1/bd597a0a-3e63-45bf-9c05-65b8e73479f3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis","fulltext":[{"header":"Background","content":"\u003cp\u003eGenetic material produced by the vast number of organisms making up the human microbiota create the microbiome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Growing evidence suggests the vital role of microbiota in sustaining bodily systems like the skin, gut, and genital tracts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndometriosis 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 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], it commonly inflicts clinical symptoms like dyspareunia, infertility, dysmenorrhea, and severe pelvic discomfort. Retrograde menstruation, also known as the Sampson\u0026rsquo;s theory [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], is the most widely accepted pathogenesis. However, with 90% of women experiencing retrograde menstruation and only 10% developing the condition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], other possible mechanisms relating to genetic, anatomical, endocrine, inflammatory, and environmental factors, are being explored.\u003c/p\u003e \u003cp\u003eEstrobolome describes genes carried by microbiota that are capable of metabolizing estrogens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe female reproductive tract hosts distinct microbiota that constitute about 9% of the body's total microbial population [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, altered gut microbiota profiles are also identified in animal models of endometriosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. 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 \u003cem\u003eErysipelotrichia\u003c/em\u003e class and fecal folds of four estrogens/estrogen metabolites [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Cohort and Sample collection\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis case-control study included 38 women of reproductive age (20\u0026ndash;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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Categorization of deeply infiltrated endometriosis was based on the Enzian score [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProtein Extraction and Enzyme Activity Assay (β-glucuronidase)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProtein extraction was conducted using a published protocol [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. 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\u0026deg;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\u0026ndash;20 \u0026micro;L of fecal lysate, adjusted to 90 \u0026micro;L with assay buffer for each reaction. Following the addition of the provided Substrate Mix (Abcam, Cambridge, UK) for 0\u0026ndash;60 min at 37\u0026deg;C, fluorescence was measured (Ex/Em\u0026thinsp;=\u0026thinsp;330/450 nm) using a multi-well ELISA spectrophotometer (Tecan Infinite M200 Plate Reader, Ramsey, MN, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eLiquid Chromatography and Tandem Mass Spectrometry (estrogens and metabolites)\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor 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.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA Extraction and 16S rRNA Gene Sequencing (fecal \u0026amp; vaginal DNA, rRNA)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMicrobial 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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe raw data were merged using FLASH and filtered through QIIME to obtain clean data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Chimeric sequences within the clean data were identified and removed with UCHIME, yielding the final set of effective tags [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eUsing 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 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To identify potential biomarkers with statistical differences between the groups, metagenomic features were analyzed using Linear Discriminant Analysis Effect Size (LEfSe) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, taxa with a linear discriminant analysis (LDA) score greater than 2 (log 10) were considered significant.\u003c/p\u003e\u003cp\u003eStatistical methods such as t-test, ANOVA, and Wilcox test were performed to analyze the significance of difference between groups. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant for all statistical tests employed.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eOf 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 \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. There was no significant difference in average age (38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 vs. 35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6 years, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.129) and BMI (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 vs. 23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 kg/m\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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\u0026thinsp;\u0026plusmn;\u0026thinsp;63.4 vs 27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 U/mL, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007). There were also significant differences in gravida (0 vs 2.5, p\u0026thinsp;=\u0026thinsp;0.004) and parity (0 vs 2.0,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the two groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBaseline characteristics of control and endometriosis group.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEndometriosis (n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.5 (\u0026plusmn;\u0026thinsp;5.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.3 (\u0026plusmn;\u0026thinsp;6.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.5 (\u0026plusmn;\u0026thinsp;4.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.4 (\u0026plusmn;\u0026thinsp;4.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.450\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGravida\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50 (0.00-3.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00 (0.00-1.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.00 (0.00\u0026ndash;3.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00 (0.00\u0026ndash;0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA-125 (U/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.4 (\u0026plusmn;\u0026thinsp;4.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.4 (\u0026plusmn;\u0026thinsp;63.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erAFS score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.83\u0026thinsp;\u0026plusmn;\u0026thinsp;36.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndometriosis stage (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.3% (2/24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.2% (1/24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0% (0/24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.7% (10/24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.8% (11/24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eSimilar \u0026beta;-Glucuronidase Activity between Endometriosis and Control\u003c/h3\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eGut microbial \u0026beta;-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 \u0026beta;-glucosidase activity (-14.82 U/L vs. 23.15 U/L, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.76) or \u0026beta;-glucuronidase activity (5851.95 U/L vs. 5122.04 U/L, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.70) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003eDifferences in Estrogen Metabolites Were Seen in the Urine and Vagina but Not Fecal Samples from Patients with Ovarian Endometriosis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAnalyses of 14 estrogens and its metabolites demonstrated significantly lower folds of 4-methoxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), 2-methoxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and 2-hydroxyestrone-3-methyl ether (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) in the vaginal samples of patients with ovarian endometriosis when compared to that of the control group. Meanwhile, 16\u0026alpha;-hydroxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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 \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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mF9FrEvt4xjbo/7E9gJllKexyPPVDiCvqr+qQ2kcQX2VPKgr1kc5lc1lG+tXmtpIyMdQY6QyahPldE+Oar/aRoj31DnlYluNMcaYnIU7XrmToLgWPY5aqIDFSgsesJDFeL64QqyP8vlix/VaLCddX5UfoZzqEromLtyxDHHlReQYxLJ5fwXlqhZxriWP6wRpsSz1xfwI/eH6POj6vD2kq73qd6RK1hFkQVocr7r6QXVoPNTfPEBeFqrkmbeBe+bykVwoG89z8jzqyesyxhhj6lj4N163b99OR73W+fnzZ/oA+/Xr1yn++fPnhf46serXdOfPn0+/glwEtD/nzJkz6fjr1690pD+82lKf3717l34YIPTa6smTJ6Ni0S9Tp0Ovzj5+/DjXB+0aj2LsT4RHjx6VJepRf+uoytdYV8mxDYwjIW/vPBwdHZVnfzl79mw6/v79O+lw4fClgMybXh8yprwe1+tGvYI0xhhjqljKx/VytPjm5dy5c6O7d++mxYlFCado0Xz69Kk8+wuL5qJg4a9aULVYw71799IinMOijXOIsxCdsWnQN0vUMa/TynjgvM3DJOei6vsy7jsLjOO3b9/K2OJAH6uQU60x29nZGT1+/PjU92GCMaWcHP1F6p0xxpj1YymOlxytr1+/pt0DFicWpBs3bowuX75clmqGxb1usYs8ffo03SvuSrx69SqlR9jJAJW7c+fOqfqrdja0E0TbBTtaLMjRkaKfLL7sbt26datMPUaLMlQ5iW2IO3vzOE5qZ/xgHFk37eoI7WbGa/khBPXhGGqnSB+9AzImTddOy/3799NYRWcv/xAe2rRfPHz4MB31QT68efMmPTDg2NJm1ff+/fs01nVQB23jujjOxhhjTCXF0/pSoGp9NwT5tzxAnHKEYtFLaZQjTp6+p1HI46q/qlyEb3DiNXmZuusiKkOo+6aHPlTlxWspo/PY/3id0gi0t1jQT6Spjni90mNfc3mLvD6Vy9sT20qAqrbk94n1xLxJ9efjqO/kqsqJmJbXD3l7dW1VPwRl4j2Vl7ePOqTXSlOb9f2ZdNQYY4yBXv/lemOMMcaYdaJ3f0DVGGOMMWZdseNljDHGGNMRdryMMcYYYzrCjpcxxhhjTEfY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI+x4GWOMMcZ0hB0vY4wxxpiOsONljDHGGNMRdryMMcYYYzrCjpcxxhhjTEfY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI+x4GWOMMcZ0hB0vY4wxxpiO2CrCn+NTY4wxxhizLP78+eMdL2OMMcaYrtgqvC/veBljjDHGdIB3vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42VWxrNnz0ZbW1spvH37NsV//PhR5s7PgwcPUt0XL14sU4xZLuixdFohT79582ZKq4N866xZBLKBCthY+PLly6m0WVD987CJdtqOl1kJTPzHjx+PDg8P03+h8Pnz5xSPsFjN44i9ePGiPDOmG27fvp10GnZ2dpJug9L39vZG79+/T2nGLBts4O7ubjpHHx89epTOr1y5Mjo4OEj6qLRVsYl22o6XWQm/fv0qz46JBgJwuO7cuVPGjNkccMy+f/9exoyZDzk2Hz58OPEgiw2e1+mibv/nN9Njx8ushEuXLqXj9vb2eIuZSXzhwoW0G0Y6cGTnC3gFU7c9rnQC1xvTZ+KrHp0zD+LrdyCNc17H6DzqPgupyk96hWk2Fz3Uvnv3Lh2riPY1vvaLuqpA2fj6HD3U9Rx1jt6KvB7Z9Y2E/6vRmFVwcHDAo9I4FMZhnL6zs5PSDg8PU9re3t44vr+/n84pB5QtHLR0Th3kCc6VZ0wXoKPoHXoZIR09FtJVAjotPeUoHY5zhOvJi/rMueYN59RjTI70SLqT64lsKkfZWtnXqHOyyyLaaem9rs3LxnpIj/ODuPI2Ae94mZXBdwaFDqZvDeDly5fpKYj0nFevXqUjO2Jnz55N52/evElPWmyh6wnt/Pnz6bjRT1Omt5w7d648+wv6zzdgTa8X2bFA9yPsIBSLXZo37CBwzreSxuRgUwvHJukIepOD/mGLf/78Of7WNv8cBKSD8ZVlTuFQVdpw9JuAriq+qdjxMisBx0gGQA4YMPGNGTK5gyR+//5dns0Hi2cEp4z5Q9jED5VNO+7fv5+OT58+HT+8RuQQ8cF95OPHj0nnyMfJ39/fr9XxKuSk6TW6bP0mY8fLrIyrV6+OJ6WOly9fTsccGQ3K6Uns7t27yQDwhKWnp6Ojo3TkCc6YVYFOshMbdxfu3bu3UL3UrgKLITA34jc1xkRu3bqVjtjKfEdK3w1ifz99+pTOxbdv35KzJed+Vh1mJ415UbXjtnEUgjSmc/iWgO8AeK+PGhLi9y/65oCgbw34JqCqLCg9lo/f0OTljVk2UbcJh4fH3ysCOqr0+G2Lvq8hoL+xjjgn9F1XXT3GVIGOVNnCXLd0DtHuKqB38RrKxHJ5HkiXieuctmyind7in6LDxhhjjDEn4LOQ/E/7FM6T/x7dHPhVozHGGGMqweniGy/2aAj7U37jZU7jHS9jjDHGVMJ3g/qOELa3tzf6F4mLwI6XMcYYY0xH+FWjMcYYY0xH2PEyxhhjjOkIO17GGGOMMR1hx8sYY4wxpiPseBljjDHGdIQdL2OMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkIO17GGGOMMR1hx8ssjLdv3462trZqQxPx2h8/fpSpxzx79myc9+XLlzLVmOlAd6RHVSHXu0i8NtdB66dpCzomXakKTfrTpIPRfnJu+o0dL7Mwbt++PTo8PCxjoxH//7rCzs5OmVoN19aVefTo0Wh3d7eMGTMbV65cSboo0FXp5yT94tq6MtZP05YLFy4kfdve3k7xg4ODsQ7u7e2ltDqadBD7Oel60x/seJlOeP/+fXlmTP948eJFWhSNWRU48DhXZv2x42WWCq9h8m3xmzdvjrfFya8jvsIxZhnwWiZ/NWP9NF2Cfcz17MGDB2Pd4ryO+Irx6OioTDV9x46XWRoYg8ePH5exYzAwHz58SK959vf3U37Vdw2kkcf2OdvwL1++LHOMWQy87rlz504ZO8b6abrk6tWrKURwptAnXkMSOK/6bovvxdBfXj+igx8/fixzTN+x42WWRtV3C69evUpHXuucPXs2nb958yYdI1+/fk3Hc+fOpaO/oTGLRs5VxPppukTOVeT169fpeObMmRRAaZFv376l4/nz59Px/v376Wj6jx0vs1T83YLpM3yUTDBmVWAfsZNmc7DjZTpFT2Vsk//69Sud3717Nx0jly9fTsfPnz+nY9NP/Y1ZFNZPs2ru3buXjr9//04BlBa5dOlSOn769Ckd/Y3XgPhjzILY39/nt/onwsHBQZn7l52dnXH+3t5eSovXkg/kKW17e3t8NGYW0EXpkwJ6l1Oln/Fa6aD100zL4eHhWGcUpGOR3d3dcT7nUKWD0W5KBwmm32zxTzFQxhhjjDFmyfhVozHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHa8N4e3bt6MHDx6Mtra2KgN5bXn27Nn4uh8/fpSpq4H70w76Nw1tr7t58+bUdZvp+fLlS5L1xYsXx7oVA3ltYbx0HfWuGtrBnJmWNtcxb2ep25zGOniaNtdZB6fHjtcGgBF48uTJ6MWLF6M/f/6Mtre3U+CccHh4WJZsx6NHj0a7u7tlbLnQ9lU6dx8+fBjdvn27jK0OjL4MOUHOYDTwSqsC469yfVgIIrTn6tWro/fv34++f/8+2tnZSenST8I0MF57e3tlbLnQ9lXK8+PHj6Nbt26VscWzznoXsQ7OzrJ1cBKD1NFCocwaUzhVWIw/BwcHZcqfP4XTlcI8FI5Xqpf6l4Xavsx7NIHMCgNcxlYPbUEeyD6yv7+fwiQ0ZlEX+gBtiu1XP+ehWPQ66esq5cm8mHcet2Fd9S5C+6yD09OVDk5iaDrqHa815927d+l45cqVdMzJt8+1zR5fS4r4ZJATny702pLtZ+LUqfpIU7rKQdVTB7tyOv73v/8d5+s1IfWovXqiUd3KB5UnUB7y65RPEG/evBndu3evjK0edizh5cuX6Rjpw67cLEj+ly5dSsccdCTueOrpNuqn8uM4Hx0dpTSxDP2ULrFTEusnX3WqvdLFqnaA0giQX5fnA3P7/v37ZWx5rKPeRbrSwahDsrtRH6LeKV3lINatNs+ig1XtAMornXvl1+X50JUOTmJwOlo6YGZNwZPPn0iIM/QKETx+0iijcz0xkMaTBehanni0M0W5eA66F3E9lfAkqCcMiNcQdG+VIR/0BEm+6qC8rlV7OcZzlQW1P15HULqOwH1072UgedA2ncf7V6FytBloH32MSOZ5fZKnZKIykj9B9UKsZ1moHRH1USGOQWyrzqkDuI42g9pOX1WOvsXzWE46oPokK4jXELiGtChPII84+dRBfeSpzrp2EDROOsbrqvJB9U8L11H3JutdRG2KqL8KtFXEduucOoDraD+oH5JTLJdfo7hkxVHt0r1VRrpB+rQ6CFXnlFG7qZO643VV+aD6F41kwr10zrEJlRuCjnrHa83Rk0lOoThozPhbhpz8KYZ6CiUcXbhwIcULRU1H+PbtWzqePXt2nP/58+d0BO4Vnzr4RkxQr66/c+dOCtznzJkzKa2K169fpzr0lCO+fv2ajlyr60nj+wOehHiC4xuOHNrGt1w80SlfT4zqzzLQvfhGgm/wigmf2lE3ZqAdOGQAyC7uZvJ0ivwKA5LGqK4+5Me4AH3k3hHtxqAj1KP4osl3BSK6dxVV6RpDiPq7DP2E8+fPp2PO06dPUx25rtW1o0r/IlX5tItvkep2spuw3p2kCx3EnsC5c+fSERl8+vQpnYuodw8fPizPRqPfv3+Pr3/8+HHa3QLSp9XBunZoLNnNwq7mdq8qfx4dnMS666gdrzUnn0A5cWLWKfXPnz/Ls+VSPFEkZSY0tbvJKauCuqgTMBxVkH/jxo3xNjoO2/Xr18vc5SAjiKGIBqKp73IQmowGfeE1qbbdMdDTwrUYKeTBOQZwGdQtHCIuAnX62bRwLpK2+oljNS25/uXk+SwqxGfBeneSIekgToB0sMnhmUUHcaKoH9lLRyJ5/jw6OIl111E7XmsORgUlmQRee5NSk8dTgBQ6Kra+jfj169c4/dq1a+nYBl2vJ5VZn3IvX76cjkwmTSjSeNLh+wecTPqQGxXyKIOBZQcQg8JToOpbFtqhk2F59erVeAeSSa2Qy0NP2qTn36Ugf65B/nVP6m1BVjLyGN1loCfvKkMZYXwmgezUzrgQ9kU/69pRpX+Rqnx2yqbpQ8R6d5IudFCy1kMsNnmaB7s4VkBbJrW3irp2YBMZV5wTxk86Iqry59HBSay9jhYXmjWHYda7bs6rQqFIKZ+j4oWij/OBOmJ5joUCpzx9WxDTiqejcVrdeVVZvUuPdf7vf/8bn9MuoXYQIL+P0tQXXRuv4z7KJ70wRum4bOi77qN2c+9JaByiHAR1qh71UWOvPMWVD+q/ZK+4ylbda1HQDt1XbaoKoHYR1B8C/SUornrU7qhLVTpXd15VVm2N8yGec28R20v7qtpBmsrp2njdv//971P5nM8K91U9m6x3EdqkNqh9VQHURoL6Rpikg1FHlBb1oU4H68pCrDOec28R20v7qtpBWiwHMf6f//znVL6Oy2DddXR5kjO9AYWJE9H0A8YkGrM2hkVEQxCJBlvGhPvIICkO0ZDHdgjFCdO0bVrUNtMN1rvTWAf7xbrr6Bb/FBebNYfXFWwN87rCrB62vYtJnj701Hb6JsOrDD4Izj8GNovFelePdbAfbIKO+huvDYEPD+109Qd9xzPtDwXWFQysF7zlY72rxzrYDzZBR+14GdMxfBjLnyWAZf0qyJgc653pO5uio37VaIwxxhjTEd7xMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLzMzNmzdHW1tbKTx48CD9r/L8X1tdwz1pA+3hfBVtMP0BXZReohNK65qol2/fvl1JG8xq6IsOone04eLFi9bBHmHHy8wEk/nDhw8j/qtPwrVr10bb29tl7uzM4jQ9evSoPDs+//Tpk52vDYUF5uXLl6PDw8Okl0+ePEm6Oi/z6uXt27fT0Qvf+tMnHUTvZJetg/3BjpeZGk3cg4ODdAQmdYzPgp4M5+XFixejx48fpx04szmwMLHY7e/vjy5cuJDSrly5ktLmAX0/OjoqY7ODXrIgf/nypUwx64Z10LSi8MiNmQrUZpLqFIZnXG53dzel7e3tpXjxBPZnZ2cnnVMOKBPLK59riHMNVNULxLlGUD7mm/WHMUcPikWuTDnNtHqpPAJ5XKNrlQdt9ZLzGDfrRRsdLB5Qx7oiXcj1hyP6BW11sKpeoE0EYR1cPXa8zFRgUJjYcSLnqAzGJJ6DDBNGgnMZABkQGZt4H+U11ct5NCY2LpsHOkCoYxa9JJ/03JmKevm///1vXNckvaQerjXrCeNNaIJ82bl4jp4QR3c4l5600UHpXVW9lIs6Zx1cPX7VaKZC2+fFRE/HKr59+5aOZ8+eHZf//PlzOkIx6dP2e+Ty5cvl2Ulu3LiRvpUpdHVivTnfv38vz8wmgF5B3SvmWfTy0qVL5dlJ+I5Hevn//t//S2lt9bJp7phhM0kH9Yrv3Llz6Uh5vkmN6Fss0UYHf//+ndKa6o1YB1eLHS8zNcXTWDpWfScw7bcDk5yj8+fPl2fTg2EymwNOOsjBiixaL+VgzYIWZ7N+LFIHJzlH1sHhYsfLTA0faMK9e/fSUeDosGOgJ7Rfv36Nn/z41eO8TFMvC+c8hskMD+nlnTt30lHwo40zZ870Qi/J9wPB+jJJB7Wj+vPnz3TEubp+/Xo6n4dp6rUO9gDeNxozC6hPDMVkL3OaP2JWms7j9zAEyul7B8I09ZJP+fw6szkUT/NjfSDw3ZaYRS9VH3kxf5p6VVd+nVlPmnSQc6Xr+79cf3SODkEbHZxUr+rKrzPds8U/xUAYszbwdMnTXvw7SsasGv0ZFu2KGNM11sF+4FeNZq3g7+jY6TJ9g78aDl7wzKqwDvYH73gZY4wxxnSEd7yMMcYYYzrCjpcxxhhjTEfY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI7aK4F81GmOMMcYsGf6QhP+chDHGGGNMR/hVozHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xG9drwePHgw2traqgw/fvwoS83GzZs3R2/fvi1j9Tx79iy1w8zGxYsXkwzboLH98uVLinOd0urGgHEkv+09+ob619R+9FTlqnQWGVTJR/OHMTDHujhJHlHWCtJHjkqrGy+uH4q9mNQXIZnkRFnl9liyrrpuk4k2Lc7lqFu5flbppIJ0c51Al9Q/bFsk6lXe95gXQ15HL+APqPaZ/f39Pzs7O2XsmL29vT+Hh4dlrJrd3d3ybHa4NyJaRF2byPb2dpIf49WE5MxRHBwcjK9jrPN8pQ15bJAP/QR0XOcR+qk+kk+fI1xXJQdkpzTktuk6TP9lM5BZnU5GHYNoe3Qu3VN9kaHoZBvdA83NXO8oTx2AHHQO1Cc5IueYZ45BJlU6VKc70rkIZUnLdXZdQHfoXz5Xm+YXebG85NY3HRyk4zWJWa6pg0EcgiHtK4xDPnEimhi5AcrJJxQTacgGJy5cUKezuVyqyiCbXEepOy6mbWS8zsS+o0d1jkYuI8m17TgMwV601T1BefQnor4Krtd8zMsSr5P3poL8CLlDUKc76F8uV6B8Vfo6gH6hU7n+1MkIyIt6CZJdnr5KBveNF1u1cVtbW7dsO7LFz7bsnTt3Rh8+fEjplCVdr1207ci2ZNyqjNuUcQvYLJfnz5+PiskyKgxQkn3Ta49z586lo8bn9evXJ8Z0SHz9+vXEK4WzZ8+Ovn//Xsb+cuHChfLs+NXh+/fvy1g96HxhbEZnzpwpU0ZJvr9//y5jm4fkKNtx5cqVdMyJ8kbPrl27ls5jOjr64sWLMnYMtkRl+05b3Wvi48eP4/kIyOfnz59j+UYbje6Z00iH5nk1/fDhw3Rcx1eOcOnSpVHhMI2uXr1apkwPuska8+nTpzJl9QzC8ZITRXj8+HGZegzCLBzI0dOnT1P89u3bo8JLHhVPYCn93bt3o5cvXyZDQZyFi4WahUmg+Czi5JOO47auitw3GBdA9sVTTRrfKtljyBlb+Pz58+jGjRujJ0+epOvQjyaHra/ExXwS6D56HBfMSUxT/yaAjuAEoGNtHq7QM+mcQP5cnzsTb968OVW2zyxCN3DYcqgX2WB3zWSwf8zreR/2f/36VZ6tH48ePUrr+bwP2NM+XCyTQThecqIIeL85LEo4VPlTKDBoeLv3798vU0apbDScKL6egDEc3IOnQrN8cHT11MYYMNa5Ecl3GHDC2F3QmDFeR0dH6XxdkWOKvLwjOxvYAuSIjuG0TyLu2giMtx7a5OxzlA6b451onFM9LCOvuh3GTYf1ho0CHvar9K0tVU7wOsGaPe8D9jQPrctmcK8aMZ7xaY0BwZgywefxiOdRejM7OMBNr8DkZMQx55zXGkPm8uXL490+wNlkF68JFi8eIiaBfJBr3Dn04veXqge0HGR37969MnYS5BsfAF+9epXkjQ3C4Zh2Z7JrZtG9HMrHh1PsJ/UCeqYHZfS16mHZ/IWdUuQ07RgAn2rAJsxtvRGJutsGdJM5ef369TJl9QzO8QIEGb/VAiY5HvEs8AQsBQZeX966dauMmWXCTqReEzOujKFe2bD48boHZxv0LcTdu3fTBBQsfKQNCRlKOUfswLTpAwakzSst5MrrL+ApsY3DtinwGizugFeB7Pi+pA50TjaCXTA5GjgZyLpPrzVyZtW9COWRAVAP/c0Xf9lozV9TT5uHgRzsIfaAHbNNAP1ifmnHuQ2sKTwUEXqlh4Wx6C2FAUu/RqgK+pVD4TSN04pBSWnkKS3WoV/dxGuKQUxpxcCM01S3flFBUN2mPVVjA3m8ajzIV5oCYylivsZ1aNBX9SHKQ32L5wo5Ub+jfEByRbc3HcmIEGUNVWnILhJtAUF6mkM9+Tj0kTa6B9GWxnRQWUKUh9KGOi+XSZQ7IUJe1B3mLfFc92LIIS3X5SES14Rcj+LcjPoa1/AYcnnkOr4KtvinaIQxxhhjjFkyg3zVaIwxxhgzROx4GWOMMcZ0hB0vY4wxxpiOsONljDHGGNMRdryMMcYYYzrCjpcxxhhjTEdsFcF/TsIYY4wxZsnwF7z8d7yMMcYYYzrCrxqNMcYYYzrCjpcxxhhjTEfY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI+x4GWOMMcZ0hB0vY4wxxpiO6IXjdfHixdHW1tapQPqy+fLlS7qXePbs2ejBgwdlrB6u66J9QwcZIdMmbt68OR7zt2/flql/+fHjR62sde2ke/QV9bup/ehjXbmmvDiv0NdNBh2SLAhtQLei3GQrCLmsY/1VOtxH6voSadKvOG8jlFO6AvLZVKQbVXMwztE6vYnyzOvQ9bl9jPrYZj3rK+pDk47CpHKSU5WMyetcP/kDqn1gd3f3z97eXhk7hrQmDg8PT10zLYhgWjFwX67Z3t4uU0wVyAc5NY0ReTs7O+n84OCgcizIz2WtMZikI32GPtFnoI86j+zv76cAko/KkS65SB4cAblINrHcpiIdA85jvApkFmUNuiaXNcQ4so55fWRe3WPe6jzqGugaMUnW64x0JdclQE6T9IRrZD8pH+cxMtf1yDja2SjzqnsPgTY6Ck3lOKf/dWsQ6eR3PV97/arxxYsX5Vk1z58/L89mpxiY8qw9Fy5cmOm6TeP79++jYiKUsXqQJ5w5cyYdIzyhXL9+vYz95caNG6PCEE3Ukb6iJ9crV66k471790ZPnz5N55Hbt2+nAJRFnr9+/Upx0JOuZBg5f/58Op49ezYdN5n379+XZ6PRkydPkm428fnz51Fh0MvY8Q6C6kDWjINkjo7G+P379xdim5bFInTv1q1b4+uvXbuWgrh06VJ5diy3qvm7KaATxTpbxk5y586dpGNNuznYxEePHqVz5IrdEw8fPhzrHDK+fPlyOodoFxk3jdVQaKujk8pdvXo1rdWSYaTzXa5Abx2vfHtU29oIGkNH/suXL0ePHz8eLz7ka2ucMpTlXCGircc3b96UKcdwfZwIqo+Qt8nMDwaccWSs3r17l5ypCAtgNCig7eLXr1+ncUE3hsbXr1/Hegs4R5OcASFHikXxw4cPSR6E4ultbIjv3r2b5gbGBR1HVuYvUfY5zPPcoZdcAfsQ89HRmH/u3LmVGvVJLEL3Yn/pvxw0iHnM6Xz+muNFH4eMwDytW1uiLHHmo94pT7oWnSvlYRd40BgabXW0qRzzFKcTZ6xq/UaeVQ5ZF/TK8UIB5eR8/PixTD32avHoUVI8WEABd3d302KDoCV8nvIphyHA82UhJ84AaMFmoSaP9AgDhRMgKK/6CLSp6enETA8GgicSjWs04FULIGDoefLDoDAuOB9DHJdoVKchGtjDw8P05IxMohGhDLrPEzW7EfGaTQdjXbcYYWvi7k0OdgY7FXfDQLuLQ2ERuod9xFZjM+vm39HRkXWvgih/bBgyrHPWSZec84dM5I4uopNa3wRlsQ3Y1j4/CNTRVkfryn369CnlsW5jJ5GfZMSRh9NV0SvHCydKTk7cUmXiolgoEnlxcRbyctlBEbwaoCxKy+IMKCDnqiMKn4ULZ06wSxCf1ogzmGaxsCPDuMYnv6YFkDGMzgR6g4Ffd5BNfGUGPLUhO4xLbpRxxuSY+YHhGC1Adc4AulhlXwR2BpnCJsm0SveQE7qHzWTu5gxxsV8V2LDfv3+XsZPgPCBnHqRYu7CNgjWLPDYW8ocJxos86POr72XBXGV9R34E9PTnz58pD9u4ygeC3r5qzHc6UCAUCyeqrcHDWFBeijkL8Xsas3gYI+0WME48lWBYWABxGBg/nthY7DjHmDOJNIGGCg593NVFz+LDRg46nzuicR5gZAh6osMJozyyQnYsjF4Ij78NrHu9gN6hf+gZAbmhe7m9QaYslAI5xwcydLPP3zUtQvciVbvS8O3bt5XuKgyNqm9cIzi6detY3RhA/vnGEGiro03l2J2uWr+xkXGeA7uG+Y7hMun1x/UIgkmPQWSBxkPltVTbXSeEq6dTgdEEGVNeO0C+WwAYz/gUQVneF5vFko8nBghDgiNGYMyZGJwzfvp+Sbx69WpwBl5PW3p6Rc/q+iCDoJ0Y5oKIRgf0DQ7wVBeR7m8qGGLtjCP33NAyJtI5AjpX92EuOqfddcaFnQg5tuhm3HnvG4vSPUGZ+KZA8IZglbsKQwG90WuxJiiH/lbJlG/p+FFHFYzDutrHpnKs1XH9xh9gXmqnVgHwE6TjnVDceOUUBi79pLMqQGH80s9ElUYciqfOcVqsQxTG4FQ+1xKUrnoh1lc8JaS0WAf5UAzSOI3rTTVxzCQ7yONx7CT3CONFmUjVWA2NqEdRHuobRP2rKlsnY4jXaM5sIlHOMQjOc9kBOie5oWPxWuqMRJsyBH2cV/finKVcDvVXpW8iUX5V+pTbNuSmtDgGeTmlE+IYRl0kDHXut9FRqCsHUX51ciAvn8/LZot/ihsbY4wxxpgl0+tXjcYYY4wx64QdL2OMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkIO17GGGOMMR3B38v33/EyxhhjjFky/OlU/wFVY4wxxpiO8KtGY4wxxpiOsONljDHGGNMRdryMMcYYYzrCjpcxxhhjTEfY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI3rneP348WO0tbVVGR48eFCWMkPh4sWLo2fPnpWxZjTOX758SfGoCzdv3kxpOdTdtv4+ov419eHt27fjcpwL5KT0qnyIMuR8k0EXkQPHJurKMUaSZZ0tIr0pv0+oL5Pmj/QvR9cTct2SHCbJetNoq4NAmXxsJNcYIpRfJ5mrj7PYR2DdUF4k2s5VyKt3jteFCxfSX3bd3t4eHRwcpHOFSTA4m7649AkU+vDwsIzVo4mzv7+fxvnKlSspHR3geo19vpgx1o8fPy5jwwP5SMc/ffo0djgj9PHz58+pDGXv3LlT5oxGv379SukKu7u7o0uXLpW5x/MhziPm1qaC7nz8+DHJoWpBE3XlNDako5MvX748ZeTRYcaLMi9evChT+0kb3QP6GHVOsKBxLQG9u3HjRplzrHdA3tOnT0/N202lrQ4C+VW289q1a2O5E3Z2dsqcY7kP2R7mtNHRJvuIPJ48eZLy0NGoh+ilZAid62hx415SLBh/CkGWsXbQnUJZy5jpA4Vh+LO3t1fGTsN4VY0bY8+1gniursVkSqGp/r5Cf9BxUTidJ/orcrnEMnkeshBV8tpkoqzQlzrb0rZcrneT9LxPtNU9MUmXkFnMz2131fzeRNrqlsh1Kpch1zN2EeJxbIdKWx3NZVJnH7k+l5Woq3uZDOYbr9wj5emSpy68Wrxe4lAMVkrDO8Zj1lYjZUjjXEHoeh11Lqgnv8YshufPn6enEcYN+eopkN2c79+/p3M4c+ZMeXYMT+J3794tY8Pj69evSa/E2bNnT/RXxF0q5sD79+/L2Mk8dPv8+fNl7PiJDrlKb/PdmU1DstK81q5qTttycO7cuXRE9h8+fBgdHR0lWcdx7SNtdW8amL+A3IoF78R8Je/3799lbHOZRreqiPMd3rx5c2KHe51YtH1kV+z27dtl7DS5bJdNrx2vq1evjheOCItz4RGnbURtrRZOZDoy6W/dupWu5VxbjQj23r176ZxQPEmMB1ZGg+1y8grvd/Tt27eUhuOmbUmuIW4WB1vvgHwZU8aThQyDwvhxXgUTaVrD1TemmezMAV5v1S3qGCr0XuAI4Igh1+KJLm3Bx4eJTQS7wVxHx5oc0TblkKUMObLHydVrIOj81cWULHKhwVZiIyNdL2RDoa0OtmWd5bwI+4iMlaeH+hzWkocPH5axbui148VCjCGTMROXL19OjhUGj7x8gIhzLQquxZkBiN8hPHr0KB31hAbRo/7582c6soCxaDF4TJZ5nwzNSZC9lJ6xwullt4sxxNGV881Ykgcsan3/hmbRoOfoNPKqMth8A6F5IAdLOo6DgPw2fdcBeSBH9IgHsjomlcOAR/1jpwsnV44YTsgmObmTdhPMX9rq4CTyHe5Np84+opfk8WDE+p2DHHlgmsbJWwSDedUYDR0LNMIEFuW6XZGcWY0hg8n9CHa8FkuTQyAjRaCcDBVPL4y7nmSYUEPbieThQbt9gLMZHwyqQO8xIDno9fXr18vY3yfFTVr8p6Gt015VTkY9GmoWQJyvoTCL7tXBDkOUE3JhrkabjP0c+u70ommrg3XkO9zrxiLtI1TJG/vIQ9IqHhoG43gBOx1MaJ44MYAszOyKoIST4NUVu1cyCAgdozHJ0+XJJL426PsrhKFx//798WsKxoQxyicCDhZlZLzljBGYaOhAfLc/BNQX6SNOZZtv1nA0c/m8e/cuGaoIcuH7OdA9vPgdg7zQu0nk5ZAjuzvYHZAtYAFkXOTovn79On3W0Fdm1b0cHna0ONJ3vcpBZnx/BKTVLYabTFsdrCPucK8ji7SPgL+Q6yGOnNYN8nWvTigWr15RPB2lX8FUhcIJSmWKhTadK10UT1op/s8//5y6Bg7KX+cQKCtiWjE447h+BaF6Y5qZTBwjxkzk8ViO8Yc4VkqrgvGKdQ2JqOuxD5yTFs8Vqog6Hol6u+lEGeb6EtPqyuXjQED3BHZB6Xn9faSN7kGchzE9pinEeao5He3sphNlleuI1h5RZzuBManSsaiD6yD3ee1jtH9RtrlOE7qW1xb/FDc2xhhjjDFLZlCvGo0xxhhjhowdL2OMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkI/hNE/zkJY4wxxpglw1/w8t/xMsYYY4zpCL9qNMYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOqIzx2tra2v09u3bMnaSZ8+epfybN2+WKYuB+uruOS8XL14cffnypYzV8+DBg9S/TePHjx9pTBWaQI51ZZEzacgxIp0hLGuMu0B9aKMjyCmfI7mciUdifp63SUyjj8i4SWboG3noplB5hVxf+4jaOkn36srlMpWs4tzM88yxTZsk8zodjDLPdUx6SVj0Wroq1J9Z7aOuJ+Q6GOUV53IXdOJ40bE66PCrV6/SX3N9//59mboYqO/27dtlbDomGc7v37+Prly5UsaqQVlevnxZxjYL5MeYEnZ2dhoNwdevX8dlCYJrnj59Ok6Tg8Xx06dPKf3w8HB0586dVk5w30D3Dw4OUj/oz6Q+3Lt3rzz7y7t378ZyI1y4cKHMOda/7e3t8T1i3qbRVh8pd/369VRuf38/yS/Cdegb+dgAQB9Vt667du1ayusrbXWvqVyd7p07d+5EOvLeZN2LIE9sVhNNOhj1mLVF44FTgV5SN3nopuzlUJnXPjJXJavd3d3RjRs3ypxjfv78Oc7XXO6M4qadUCjPn0KJytgxhTBS6Bt7e3sLaxf1UN8mU0yeNP5VoBOoIaEwGmXqn3Qe1TPWgUyjLvVVj5rIZUJ/igWqjJ0GHSLEMtQh2XEeUZ45TS77COlRD4lL13L5i1ge+q6LbXWvqZz0q0r38nm86fYvBxk2yaRJB2N6HDPS43wnT9cMkbY6KqrsYySXD2Wlv6tgZd944aFrN0jbfU3g7aocXr/AK45bhtHLJy96ySpDutC2OOWol/jjx49T20gHPGfyiOvenMetS+IKkzzzTSTKPHLp0iU0f/xkJ9l9+/btxJPemTNnxk+KyJ2nFXH+/PnybDiwyxdlcvbs2dqnLukZOwkRZILsCiM1unr16gndZ6ewcADGOjn0p99FU6eP6Njv37/L2Mly2AV2biRTjUu+mxPtQh9pq3tN5Zp0L8qDXbHLly+XMdOGJh2UbJH3kydP0jmQXjgdaX2S/s36tqcPLMI+5sT15NatW0l/CwfsxFzuipU5XiysKApb8giA87rtf4TC5KYc4ePHj0nxKI+SooCks3iz3QrKEwwiRoJybDnKgdIrKxYqePToURoMFi3SKffhw4fUBuIvXrw4oRCge5EvQ9T1QPYZJlE0EhEZEowE46dxgFzOgm14Xk8PnXzBruP58+dJL3N0Pa+80bsoY3QWhxSd1LywTh7TpI/YoTdv3pSxv/BAgOGWvcI+5K8ugHJVr4T7RlvdqyvXpHuRo6OjiZ9kmJPU6aBgvWE+5+sMn9awNqKXi/5sZxXMax8j+BtxbVHdWu+po0tW5nixY0Hn5ZUzces8WoQGetLEyeF6lAtj+Pr165RPXdo1UR6gnFyDonI9u1lRYUmjPE5VDmlMhGhMYzuph3g0RBhltXnTkZzbGN+2T2iaZNIHdiL6/k3NrPCAcffu3TJWD/KVoyqZS06aF/EpelOZpI/Md+12E3Bg2ZX99etXkq909OHDhyce7AQLJuU3iah7kWhjTXvqdFCwVuH8Q3QYeFsjx4vrNoG29vHz58+16wt2smtdXZnjxbZgXWfx6KV02pnC+UHZFCZ5uFXE6/VEICXmXtx3FqoMsDkGIzDNWMmBxdBgcAROAzogcHYZN562cSraOm19gdcvGEnBwl61g8JDhR4YeMpFJnVGVbLT0QvfaSbpI7KTjdDON2lNrzpyJP++0lb32paDqj7z8NlmUTQnqdPBHHayBZsNvAWgHI4b12jtHCKLtI88FFRtqkS6nrMrc7xYKBGSvunhSfH+/fvpXM4QAYFpEdZ3BCwoePeCVweguvKnWYTK4hwVUed6UuNecaFvi+qO7UFhhuYILANkq8WKsdH41YEMtXOFXHG0dA3bxPkrHPKYdG0XxD4hHZXOsuNbtUjFuYCh1QNIDrKI37phePU0XDcvNo1p9BH7wGcIMtiSna7h2yVkHKHOIXxv2Fb32pbLdU+wKG66zs1DroM5yDeOBxsA8WFriN++ira6N8k+spkiBy73GwRyrqp7qRSNXDqFYzL+BUEhnDL15C9jCiNWplYTyxIEdRfCHqcXypfSq9IUJ1AfxHLF00VKo41K++eff8bnuib2RyhO0P2oT2mx3+sO/Y/yUBCcS9ZV8o8oL+oH56Rx7ZCJcop9l97koEOxz5IDoWr+VOnpJjKNPkpmVboY66nSPcZAc7/vtNW9unKTdI/rqtI3nTp7h95JXnU6mK+BWo+Exo4wdNsIbXVU5PZR18ag+Rlt4yrW5i3+KW4+WHiK9ZOVMcYYY4bAyl41GmOMMcZsGoN2vPRnHPKf1RpjjDHG9JHBv2o0xhhjjBkKftVojDHGGNMRdryMMcYYYzrCjpcxxhhjTEfwJ179jZcxxhhjzJLhs3p/XG+MMcYY0xF+1WiMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnREpeP148eP0dbW1ujLly9lykkePHiQ8jnOAtdyj1l5+/bt6OLFi2XM9BnG6dmzZ2XsNIwl+hBDrnfoSt1437x5M13TdI8+oz63lRHnEaUT4pyivpiX528y6EydbQPyotxETM/HSzaTkI9RX6nrS05dOc09QiTKqWrekr+p9jvOy6gnTTKrspEKXLduxLmEjkWQTV3fY14MeR29gD+gGjk8POQPqqZwcHBQpv6F9J2dnTI2Pdvb26kO7jMLah/19JXd3d3ybLPRWO/t7ZUpp9nf3y/PjqnSLdLy8ZYeDFnW9ElzjD5WzTf6qT6ST59FlBVloozayHUTQS7IsErWok5fJUPpXrRhMc44xLw+0kb3oK4cMtI5uhfnYdQ1ro95kr2uiXmbBHLJdQjq5CGdi1CWtHyurwvoGP3L52OTzpAXy0tuyLtPnNrxunDhQvqT9lXgORadGr1//75MmZ7v37+XZ7NB+wpFK2P9Y9LT4ybBWBdGuIxVc+nSpfLsGMY3wtPe9evXy9hfbty4kfTgxYsXZcqw0NPalStX0vHevXujp0+fpvMc9ZGyUZ5xHj58+HBUGJkydlKuPEFWyXAT+fz586gwwmXsNOjb48eP05Ny3CHkXPJGRxkH6SrXxPj9+/dHz58/T+d9pK3uNZW7devWOP3atWspiKiXlI9yZG0h7eXLl+maoc7fecF+FU5COs4KsqOOO3fulCnrBzae+Tjrzh5zErtI6NPa3PobLzr+4cOH0dHRUTJK+XaotvUoh4MWt1Gr+P3794lr4naqhMx5vI/yMZ4R0vT6U/dVWYLqi1vjpOn8v//97/hc12vbUig/tkfbxtTF/YmjJBgVXcs981ezsR1RGVRHXh6ifPqkQPMSHS36GA04MNaXL18uY8dojF6/fp3kgTyHxtevX0/o0tmzZysfSqJ80Iemh57oUMTr3r17d0qGmwjym7TQ47DiHGDwkadsR5Qn8y/Wg47G/HPnzp1wNvpGW91rKhf7S/9v375dxk4TyzJfnzx5khwGros2btOQDs0jAx64QHq6bjAf2ey5evVqmTI96B/69unTpzJl9bR2vJiENJ6FUTtiUhgWPgwV6W/evEkOWr6TkcPTE+Wpk7qZuAcHB+nJUU9SCFwTHQNAPtdEoybDcP78+ZRHPbQHD5c41zBoemLlfhhU7R5Q5l//+lcqT7oMCE+t5EG8N08o6jcDSZqeAh89epTazD1Ipxyy4N7EmWgYbfWdgKOGI0E6DhtxZMz9Pn78mOplUmGs4jXrONFyA478qhZKyjEOkgkyHqIzGhekSbBgoR9xIYx8+/atctcCeFjSnNpUmC+5U1+FxgQ9xKblMkX+zL981wz7MyTa6l5TOT0Mopd184+5KucAmK+AbWNuV83vTQI5ID89TM7Kr1+/yrP1g3WVtXreB+yqh4tV0drxwnhjXLQwxi1kOiRH6+7du8koTZrYciqAukGLQ+5UKK58HBchYbL1DbSJNN2fa3CEWJiAiY7xxGDE3QPKs5jnE4D6cMpw3mRk1G9QPVUGhDQUJrb31atXJ5xSjDvGCeWinThukjH3BRxTzrkXAdZxokW5Ni2UlCNP+oDMpEPrCgsWzjh6UGWkc6dVRJluMjwQVsmniary2BbNyyE6+4sE+aCX2C2c0RzN4XwtYN72aRFcJciGNYDXhfPMVXYj1xnW2HkfsOseWldBa8cLp6vN4oZDEN9by1kgtPHqcVIwkpSVMzUtMox1oOw4h/kWL04jr68wGPmrGQyMghw2jsTp2zTeOK9Zp0W7aArTLiJ9B5lHBxUdwBghW5xeOZ4YJ8bv58+fZclhgn7Fh4983lShh4gcDErdzgEPHOj1JoNu8cAkO4QuoVNtjHjuNABpOPsC5yK+xkA3+/xNXVvda1uuSveYpzycr5udWgbIiHk9af5XoW8J9RC6zvDgiYMfdbIN6CLzv1dzsljEKyGr6GgZO/1Lnp2dnfGvKeJ54dCkXxZQTmk5sR7KEiLUQZ1C91Z9lCeu62J9wPWFYSxjx3FBvSqrtkbIj+UhL6fzWI42APeNZaNsgLzYN+KSM+dqN2mqU+cqx7FOtn2DvsaxqIO+xzGM0N8o6ygbIE+yGRKx3W37EPsNUZ855rKOumaOaStrZFk3z6gj6ivjong87yttda9NOWQUbR5QVpDfRt6bRC4vQGZV6YA+5XOfsqQNZS2YFuZfPo9Iy+UQQSbRBkpuUR/7QGUPaKhCnDAMsNKrOkdQxzmvAgHEOuK54DzGId6bxUSCjPVFlEbQ4EVFjfVFZSc9xoXKEiQT2qE0tTfW+88//4zPoxzjdZSHXBY6Vz9jvX1Tojqq5AN5HJocBGSX9znKSDIcGnHeRHmob/FcIRLTFaKh4rxKlzcddCnOR+Qm+dfpbJx/hChnoD7lDUEf2+ge1JWLdjfqWJSDwlDsVRdEeRIi+XxFbsRz3Yshh7Q4TkOlao0Uca2I+hp1MoZcHrmOr4It/ika0St4BcBrxqpt/mXDK07el2/C1q0xxhhjuqX1N15dwrdkq3C6gI+U7XQZY4wxZhn0xvHiAzh9/Bp/ftwVuvemf4hsjDHGmOXRy1eNxhhjjDHrSC9fNRpjjDHGrCN2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCP7jP39cb4wxxhizZPg9o3/VaIwxxhjTEX7VaIwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkIO17GGGOMMR1hx8sYY4wxpiPseBljjDHGdERnjtfW1tbo7du3ZeyYixcvpnSFmzdvljmLgfryey4K2v7ly5cyVs+DBw9Gz549K2ObCeMwSVbIs05OjCH6QRn48ePHCb0hDFXGbdqPDtWVy2VBXOg6yc0cM48+cl0uZ4hjQED2fUdtnTR36so16aXSF23Thw5ykmzi2iS9IuTzVfavKrRZg4ZGtGm5/iCbur7HvBh6qYP8AdVlw20I+/v7ZcqfPwcHB38ODw/L2HF8b2+vjK2e3d3d8mx26A/97lO/uoYxRwaMbx3b29u1ctrZ2Ul5kahHwHVN9fcV+q1208+qPtBX9Zf8XJZ1ukW6dJjrF6HP68A8+ij5E6LtyvUxjllfaaN7UFeO/pEHyCLKhHKCMn2XxSqQjkU9grp5KhlHKEvausqX+Uf/8nnYZMvIi+UlN+lqX+hkx6u4z6joeBk75syZM6MLFy6UsdHo69evo1u3bpWx1TLpCbAtjx49GhWKUMY2k8+fP58a+5zv37+PCmNdxv6icUB/IpcuXSrPjjk6OhpduXKljA0DPa2p3ffu3Rs9ffo0nUdu376dAlAWOf369SvFqePx48fpqS5/+nv16tXo7t276ZzrX758eWqXZhOZRx+Rf2HIy9hfcn3kHhqzPtJW9yaV085MtOPw4sWL8mw0unHjxin5mGO5sDZwnBXkTB137twpU9aPwqlMNi63b21BN5mzhEWt64tgZd945ZP106dPp9IiCF5bh3Ebn8kft2Lj9i15ccBURgYDtPVLOeolzkCzUJEObFVqW1335jwuZMQVZlWSdQNZRSM8LYwDOiG5St5RT4bqTPCgEfXw7NmzacFvA2WBhxec0oODg9HVq1fHuo9MMDTkC5yN379/l7HNZF59rCO3W33Xyba611QOx/LDhw9J5wh7e3tjOeiIHbx27dop+ZhjpItaU2bh4cOH6biuaw5OO7qFfZsV9A8HFR+jL/Ti43oM1fXr18vYachH8CwyhI8fP6bJjkPEAvPkyZOUjncs7195AgPCAkU5njKk7AwGaXqSY5eKgWagSKccBoY2EGeyRGMEuhf5WgT7bnyXjYzurHA9zgJ1INe6p8Nv376Nd3aGxqwLknYgdD1x9I55EPGC95d59bEt3Iedob7TVjeaymHzsLfs8GE3I9hN7CD56+oULALWMh7y44bBLGgXfB1Bt9iBnvdbrbYPtl3QC8fr3bt3o8uXL5ex07C4gnY+mPA/f/4cvX//Pi3Or1+/Tvk8hRFnoisPcIK4BkPA9flrF9IoX/U0TBqDHo1pHEDqIR4XQZwEtXlTefPmzVyvWzAkOLiqgye76EgLxl6OyLrDYoaeVoEM8gcC85d59bEt3GdTXq09f/48PRThPOSLInaTPGxw1WtMcwzrhjYM5nlY1y74uoLdYwNknteFfbKPvXC88m90mMRysrQzhfPDRFbIn7DaEK/XAsaROPea1aOucgg2GRxfvaolyOmdZtK0ffU21F0dHjRYsASOZtP3Hshu0o6NZMFRDyCCMdgUBzVnEfo4DX3Xyba611Quyo55SqjatdFDsamHB4K6Hf1J4PzCJsxtdvX5/CTqZBtwaJn/TW/VumbljleVly9niMCTE0+Q+p4AuCZOfL5FAC00uRJqIZITBzqXF8y9uMe0qO7YHhSji6frvoL8NX4E5MOkmcZZ1hhqzNkVxThFyOvi9dEyUP+ks7wmrHtlKhlIp6IeC8qcP3++jI1G9+/fT7svgG7mstskFqGPbWAs4xj0lba6N6lcvgBW7bqgg0N49bpqqt62TAI7gEPBjtkmgD7yGdA0Gx34Csx3wqLn+1wUhmjpFJ1OP+kk5D995aefhREsY/VQRnXEZlP3TvknBwjFoKT0qjTFCbpnLEdbgDYq7Z9//hmf65rYH6E4QfejPqXl/d4kkFccY+QhWUPVGAByVDplcqrShkTsX+y39AYKh2lcJi8b8zjPkVyRv/nLIvSRoHkuGIM8ra+00T2oKwdVcspltMl2r4pcPhHy4jxGT4nH9SgPOaTl4zREom7lOhTtftTXuC7HkMsjXrMqtvinaMRgYcdqk77zMcYYY8xw6cU3XsYYY4wxm8CgHS/9GQf/+QZjjDHGDIHBv2o0xhhjjBkKftVojDHGGNMRdryMMcYYYzrCjpcxxhhjTEfwv0D7Gy9jjDHGmCXDZ/X+uN4YY4wxpiP8qtEYY4wxpiPseBljjDHGdIQdL2OMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOmIRsfry5cvo62trRQuXrxYplbz7Nmz0YMHD8rYbHCPt2/flrH1A/lEGd28ebNVfxch26758ePHWHcIbUAe6JwgHuvIdTDmxeuGhNrPGNeBjqhcri8xD5lHlI4czUl7RmgCXasbE8k86mOslzCE+aq2NukeqL9VoFtVfSWNayatG5sGspbc41xuWmvjHM/DUO1eE3HtyG0Xsqnre8yLoZf2jz+gWsfOzk559ufP9vb2n93d3TJ2kv39ff4Ia21+G7iWOqirj+zt7f05PDwsY9Mzq4wWIdtVEHWH8xivQv08ODhIcWStcyAeZcB4DB3mlPqIfGJ/Rew3+chIEKcOoJzOIZ+7fZ1XXdJWZ5AXcq4qj1zjGEAuW+J9l3cb3QPNy7zPIFnktgm5KY3rh2a7ukA6lq8pdbKiXD4GlCWt77o2K+gR/cvnYZM+kRfLS27RNvaBRscrwuBGY54TJ9us9HmBqJok04J8ZpHRImS7SqKDUAf9i4tBLmv0IuYxHkM2OrlM6uZXLodYBplFI0Oe5BGvo9y8ujt0kIt0po0skGVu8Im3GaO+z9W2uicoj9yqoK95f+M8hrYy3yQkt9wu1ukO8qsaA8rXjc3QYb5p3kZ9qpMRkJfPW8kuT18lU33jdeHChfKsHm0xa3tP23+Ka6tV26yK59uBpOfb1XGbVuXjNiznuh9lKcO5rldZ8iDWp/Zom5N7awudOBSTJF3DFid1qn6uUVt1rYhbyDnUEbdLVY6wblvIGoMqkNeLFy/K2DG5rr1+/Xp05cqVMpYszaiYUKM7d+6Mx3NIfP369YRMzp49O/r+/XsZ+0uUA3J6//59GRuNPn78ODp37lwZOy778+fP8TmgR9euXWs1d9eZS5cuJZ0pDHmax7PMr8ePHyc5ao7q1W4u2/yVb99oq3uzQN+Zl2fOnClTju3m79+/y5gRsnlxvZiWhw8fpuO6rReCeVs4TKOrV6+WKdPD/CwcstGnT5/KlNXT2vH6/PnzeJDrePnyZTLyTLwPHz6kNCZ08TQ1evLkSYrfunUrGb/bt28nZwdhYBDJ5zogHSNHfeRRB4p1dHSU4gRAYamn8IZTHANy48aNVM+jR4/SIoXAnz59mvIpyyCSF++tBRyj8fz58xSnH9wfdD/SaT9KwDltVp7aSrquoz7Kkk5QOuC0UVYoTjn6w3V9N+BtwdBr/HPkGEwiLm4654jM0JUhki/YTbDQoz+5A4vO18H8QI/Q7XU1zG2RrLEB2B/ZhLYgPxwIdJU5il3B1uRQ7t69e2Wsv0yje7Ow7PrXBR6emNd68J+VX79+lWfrB+s1PoQ2W2ZlUQ8Xi6CV46XFcdJkwhhh2IQcBxZdGbp3796Ny7CLISPFbgaGDeQgUZ948+bNiQWapwWUFrgWY8oik7cTZ1GLPsqN4wQ4kjiHLGi6L09lXK84BjaHunGMKKMdGNIoS/26Fuhr7EM8xylUWeSEUqjd1EvZb9++pfiQkQ7E3aoI4xp1pgrkGsc+gsyQ1bo4qXXIIcfRbGukmSNch55N62isM5P0rQoWNpxeXYtdiQ9OAn3mKd2YNmC/WLv04D8rTQ9g6wDrJev1PG83mt66dM1ExwtlwGhHYxVf0bXpDIsujsW8i6Neo1RBHgswChxBsWkjziNl5NwAzh0LkwLtpJ+c45jF1wmToCzXVBnjNsx6Xd9hV4AnlioYE572pEvIAOc5n1zIddJiGcd1CFy+fHn84AAs7FU7KBE55ILy7CYKdJV6c3jAMSeZVl+meR3Xd12cRffaQt9x9Jnbgnld9+Bljh8EmNezjAFvaGAT5MuDJ283ou62AbvIOnP9+vUyZfVMdLxQBn1XwpM2E4qFVM5KW2OE80Zd+Tcp2o1COEzQOs+fHY/4SondoPv376dz7QDwhI8zxSIe4R759r/q073UN17PcKSutrtOXMtWqN7ZC/rKgOseddvKMlbR4aDsJGej7+DwSj+Qad5vjIX0iIAMmFy5o1alDyIa+CEhQ6n2o6N3795N502gP9ILyr969SqdUw+yrjLA7MIM4fVXVzDP6nZQ65BcpcP5bjYwBufPny9j/WVW3WsLdhmdA2Sdy8mcJl872sBahT1gx2wTQG9Z36fZpGDtYF0h1G0ArIRiwaukWADTLwFiKBpf5p6kGPhxmUIwJ66J5HGgTtI5Eqgrr0/EuovJnNLUTv0qR/mECHUXA1bGjon16Xrq5Zy02F+1859//jl1DShN5XQt9cXyarfuQVC7FI9pdbLoM7Q99kVBcF7VF2TGeEakD5GqcRsiUU5RHupfPFfIifnSmVz+ufw2kTjfoqwhT6srG+VapXfMbY1B32mje5CvA5Fo22TXhGQYbag5PTcj5EU5IjvicQ3IQw5puX4PkTgHc/sV517UV629ecjlkev4Ktjin6IRnYCHPotnvwhWeW9jjDHGGJjqz0nMA1v0i9zOnga21Kd9tWCMMcYYs2iW7nix06QPz6u+P1km+hFA/uMAY4wxxphV0OmrRmOMMcaYTaazV43GGGOMMZuOHS9jjDHGmI6w42WMMcYY0xH8pVF/42WMMcYYs2T4rN4f1xtjjDHGdIRfNRpjjDHGdIQdL2OMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkIO17GGGOMMR0xleP14MGD0dbWVgrPnj0rU6u5ePHi6MuXL2Vsen78+JHuw9EME3SAMeRYx9u3b8c6pRD1Juoc5wL9Uzp1DBX1oWk+Nc07zROFOF90XZP8N5GbN2822ibyokxFTM/HIerjPHavS+r6klNXrkkvlY6szV+inrRBZaNOKS2f18haeUO2iRH1Z1YdbbKPce3p2ka2drxo5LVr19JfXT04OBg9fvy41sCgAIeHh2VsNm7cuFGe9ZPoBJjTIJ+PHz8mfUGpmyYOZRR2dnZGV65cSelMEukc4cWLFykdXfz06VNKQ8/u3LkzmMUuglyYS/SD/lT1YdK8e/fu3Vg+hAsXLqR0yZu0p0+fWl9LkOeHDx/KWDVfv349IVOBHImjc4yDjLjGg7z9/f3RvXv3UrzPtNE9qCuHHDW/c3lg/0knfP/+PZU1J+0WMm1ySimLQ4A+UV42MY4Ha6TqYL4/efIkpXMNNjE6GUNkXh2FOvsIP3/+HKejp51S3HQmigXyTzHAZew029vbfwphlLHpKSYzFi8d+wb9on+mnjhue3t7tbqQj+/u7m55dqxj6ADHCGWi7hGP1w2BXIfoT97PKuK8ow7kQ8jlm8+/vs6lrkFPmmwTspVMo7xy2cWxysv1XRfb6l5TufyaKK8oD2SRy25TiXMX6vQQeVXNV+JxPOrKQZOOD4FF6Ch5msu5LFiTlLcK5vrG6+zZs+VZPdrKw4PHA1dcT+TaHo1PS8SfP3+e4gKvVlvb+bUxLW5/4/nq/L///e/4fNK9Y5qePDhSN3VevXo1PeWRTpx03Zc6gHuoLqUB6ZRVfsyL7aXMkNGTheSoJ7ac+ASCjNndEexwFTqazhl/QZ08rYjz58+XZ8OBXZXYJ+ZS26cuzbszZ84k+RRGJekk8gPkg36SLwrjNPr9+3cZ20yYU9o1rePSpUtJpoUBTzLT03PUU+ZurCfmYbcm3WPVtNW9pnK3b99OO4foHKFYyMZy0BHZMZ+jfDYZ5BXXTGT769evMvYXdKhwWJP+sRZovWL+Mq/FJLnG+T80FqGjdfYRbt26lfLQW2Ssdaor5nK86hZTQWfVuc+fPydFIQ6PHj1KR7blUSbyMIzXr19PZeJiinMihSOPa1FGriVOYKsbwWL0uB/QvsL7Tfn/+te/0hEm3Rujq1edr1+/Tmm0n8GhTgaSSUE6A//y5cvxtvv79++TwTk6OkpxAlA/7aOdlAfurdce1C15EagvKsoQYYyQk8ZmEsgYgy5kWJApk0t1ME6vXr1K50NmkuGsQ/NO10snedUQmbX+dUROwCQkM/QQO8DrxQh6iD6j1xHmLwacuR0fpvpKW91oKof94pUW81Y2VWDvsGdD/QxgWbRxhrD9wDrAvEbfkKHm/SRbii6yfg19/s+ro0qvso/KQ2/xF/KNnmUzk+PFpGIxnASdFdGjpKPy4tm5kBAwWniioCNwLwzd3bt3y5RRWnh5OhUYSQwAIEyeGDCE+dNn3b1RdhScazAWcvTIJ+3hw4eVfda97t+/X6aMRm/evDm1c0P9GHPuT/loqJDNt2/f0jn3InD/uKszROgjxgPnN3cKqog6ksP1kodkJ1kxbm0W1aHTNO8wLvHJz5yEORmd+jZUledpWrZBdgSwE+g6doiHqU1wNlis9JCYO5vYPPKw27nzappBv1hvgHmN/dTOGPrF+iTbh3y1hokh7Lp2TZN9ZD1pWnuWwdSOF8YmX+SYdFIEFodJ4FThONHZc+fOlanTM+nVCQqbt6fp3jiKGAsFkAHhCWKahW0Wp0m7cwrRORsybYwAC9Wkj5LjeLEAIiPGDOMz7aK6ai5fvjx+sgUMa9MPSqrmXY4MMEdkEhd/jLmemDcN5MBDnWwUsmA3JjpOdeSLGpCmXfUc9JB53Gfa6l5TuSg75iKhaieGNwbmGPRCD9iAzOLmgWDu1q1t6JfWB+rLnVrWu7hBMVQWoaM5VXNZNOUtg6kcL00sLXJyangKlzK0WWTpJMIhRMVD4bTlJwWtEyLpUenY7ZLCSfm0OxCfxuruTTwu/OqbnC0mCbTxjFkg2YUR9CXuiFVBW/TNBHCfNgvDEOCXJZP6z45ElRESjE3uXCErFlCNzZCQEyTniB29OoNZN+8ilImv55E3MgX0iF3WTQVZyz4RsDM47JMebJqcXR7e4q68YN6ij312ctvq3qRyccGD+P2SQAcnPVBtCshBjijzlbWlasFn7mptQ59YF3Lbx7V8chHTpa8atyo7MRQWpaMit48R+QudUhiiVhSGmy2gE6F46itzT1J44ifK6Jw6RGH4Tv1KoXgSHZdVHaTF+oiLmL5f/lpE9+PIPZQf71V1b4j1qW+x32p/bGdhxMfnagNU9Zt8pcV8AsT2Km2oxH5IlqIqLR+PKGNCHHeNSdUYDonYxygP6QZE/cvLVulmRPqMjpq/IA/mmogyrbIBEOcuoUofCUORdRvdg7pyUCWrWJ4QbaI5qSsR9CbO4Shb6VqUbdRfiOUVhi77eXW0yT7WrdtdscU/xc07Bw+VbcHck++CVd7bGGOMMZvLXL9qnIdZPnZdFKu8tzHGGGM2l84dL95N84HrpA+Fl8Eq722MMcYYs7JXjcYYY4wxm8bKXjUaY4wxxmwadryMMcYYYzrCjpcxxhhjTEfY8TLGGGOM6YitIvjjemOMMcaYJcPvGf2rRmOMMcaYjvCrRmOMMcaYjrDjZYwxxhjTEXa8jDHGGGM6wo6XMcYYY0xH2PEyxhhjjOkIO17GGGOMMR1hx8sYY4wxpiNmcry+fPkyunnzZhmr5uLFi6ncrPz48WO0tbWVjmaYoAOMIcc6NM4KkZiX69uDBw/GeUPWEfXh2bNnZcpp3r59Oy7HeSTKIZ9v8bqmMdgU2uhjBJ2rsmGSa1U9Gg+OfUe60aR7oP5WgYyq+ipZ1123qSDrNnJBriqX2zfl5TYx1j3P2tsn1J8mHY02MC+ndELdOoGudr2GzOR43bt3rzyrBoU4PDwsY7Nx48aN8qyfDMGwrhLk8/Hjx/RXelHsuolDOcoQdnZ2ThiT7e3tpEf6G7+SOQvBtWvXUvre3t5gxwK5HBwcpH58+vSp0lhiED5//pzKUPbOnTtlzrGhBeVdvXr1hAH5+fNnyiN8//69TN1M2uqjQMc+fPhQxv6CfjIGVTKVcSfvxYsXZWo/aaN7gByizkWQRZ2Mnj59mupmfnIvcyxLZI1ckH20dRF09fr166nc/v5+soMi5nGUHmv8dM2kNXoItNHRuBZQ9vHjx+NyyJd0wu7ubqVPgfzm9VVmomjUVBQTKYVikSxTqimU5U8hiDI2PYUwWG3TsW/QL/pn6onjhr600YUoV86jjhGXusa6Saf+oRH7CoWxrJxTuf7HMpxTjyAuWXBEXjNM8bVkWn0sDPUpG8Z1VWMEUfZ9p63uCcrX6RFyIkTyssQnyXsTQMbIWuT6JUiP+kpc1yFL5XGUrGN5zvMxGRrT6qjIZSyirARpspNRfl0w1Y6XnqbPnTuXjm3QNh+eKdcrLk8dr1RPijH+/PnzFBd4v3j7VdfGNJUh4Pnq/L///e/4fNK9YxrtJs6RuqmTnYVioFI6cdJ1X+oA7qG6lAakU1b5MS+2lzJD5sKFC+koOV65ciUdJ6Gn41+/fp3YUThz5kx59rduePPmzejRo0dlbDh8/fr1xE7A2bNnK3elYl/Riffv35exUSqPnEQse+vWLaxM2nFAnzQOm8o0+oicq3aseJqmHs1R1cW8Zefn6Ogopfd9h6et7s2CZBL1rVhAy7PNBh1B1oIxiPNXsLb8/v27jP21iegZSJejTse5z9rZ9x3XScyjo1HGkVwPkdOq1o6pHK9pG4qDIuPP6xKUgzioHrZEUTTyMHjaRj1//nzKB5wTygB5XIvjwrXECRhFnCMUjvsBxrXwgFP+v/71r3SESfcuPObxtuTr169TGu1Hwamz8MbTIJKOgrx8+XL8GoOFkQmCESZOAOqnfbST8sC9tVVP3ZIXgfooP2QYI+SksZkEsnzy5Ek6v3TpUpKPjE2OnFRkiWyHSDSWk1BfozFCR9HPKlQ3us58yB9kNpE2+ohe8eoih3Su1WuN+OoCvSWuPOi7Tk6je9NAvcjp3bt3ZYqJxAfIOlizeKCsAtnWwRoiOxEf6IfKrDpa9VD17du39PpbMP/v3r1bxlZAYShawfadtkXbbPsVCjIuz3ZeLE+coHNBc4rFNp1zjPFYHxBXHtCmuL3Kebxe1N2b+igfA1TVk2+DUibWRTxud3KtylNO7SRddVM+3psQ6xwyjH2UVxXIIO8v8SiPKp1TmTg+QyDqAeQ6VQVl6GvUrSgfQpwjkUnzdZNo0sc4JpSRPHObp7kL+fzPy/aNaXVPelcF9cS6QOVjMCfXN0BH4lwW0q0YSKsap7xO0Foi3R0is9jHXA8jeV6MV8lw2bTe8eLJmh0ZPGo+tmSnhnPAu+ac0OZJj9cgr169Sh76NK8tc+J2bBWFYp9qT9O9i8HFQowDsIPGOU+3cbdhEnzYPC20N95/mt3FPtNm2xv55v0lLlkUk268GxahDHlD4/Lly2lXU/DKQTsodfAkVxiMMnaM5FMY26Q/da/QlrXDMUTq9JFdLXYLZMsKY5xsHjtlTa862J1nh3sozKJ704AOSi/R12IRLXM2G+YnOy8CfWJnP4e5KvkhO2RIGnJFJ1m7gCO2L5/bt2/fTvcaMtPqKHO0aqcaWLfjnGe3K85zQI6dvmEqBndq2jzR4Z3K48Z7zcvjcVImeprE5YnKaycNYn1AuVgnceXHc8q0ubfSBHGIabqGuvOy9FGo7YK48jmqbuqiHEedU1Z5sc4hQz+a+hJliWwlAxHlkkP5SbrYV+i39DSeNxH1SiDbKMMc9K1N3ZvCJH0U+ZgQlx5yfdU8BvSxTl/7wjS6R16V3gEykBxykENd3iaCTshWxfM6kF1ehjTpbjyPoIdN9mAotNVRZBnlEHUO+Wle1q2pce52xVIcL/LoDIGO6jwKBCHmddB5lVUdpMX6ooBiugyd7sdRBoMQ71V1b4j1aYBos9LU/thOFELnagNU9Zt8pcV8AsT2Km2oxH5IlkJpUY4xQJRFHHOI41Q1jkMh9j/KSLoRzxUikkOcV6JOLzcVyYJQp485ubGP45XrXT63+04b3YMmm1RlG0Fp1rvTRJlF0DXJUHO3To+UH3Uw1kv+OtBGR2O/87J5OiFfS6AufZls8U9x485hW5/tQ7ZFu2aV9zbGGGPM5jLTH1BdBPxqY1WOzyrvbYwxxpjNpXPHiw/d+KCt7kO4ZbLKextjjDHGrOxVozHGGGPMprGyV43GGGOMMZuGHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOoK/l++P640xxhhjlgy/Z/SvGo0xxhhjOsKvGo0xxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI5o5XhtbfF3VrvjwYMHo2fPnpWx9YJ+3bx5s4y17+vbt29PXDcULl68OLF/9AsdI/z48aNM/Qt9J4+6BOe65suXL2Xq8FAf2ugA/Yw6QFzXKyAroL48r0q2m0YbfczlKmJ6XkcsT2Be9x21dRbdgzhvI9a9emS3oi3Lkb2LAfmLmF4lV3SPvCHo4CTUzyYdVX+byknuso+ga1axrk50vLoaPN0Hwb18+TKd9w0GLU6AaeHax48fl7FjXrx4MXr06FEZq4br7ty5U8aGA8p+eHhYxqph3K9fv57+mu/+/v5oe3u7zDmGSUHfyf/+/XtK45qPHz+mtL29vdHVq1dT+tBAPgcHB6kfnz59mqhb9+7dK8+O+fXrV7pWYXd3d3Tp0qWUd+7cuRN5Ozs7owsXLqS8TaWNPsLXr19PyE48ffo0xamDeaxFD7sQy6PH165dS3l9ZV7dw04/efIkXY/exXXCuldNtFvIv8mZyOV35cqVlI49xOaRzjHKHXAk0EvyWVuGTBsdZe4x1yhDWeZlLMc5Mrl//34qc/v27ZSOHIkTWFeiQ9YFjY4XjT5//nwZWx50WkYMJ4SJ3EcwNPPA5GGyTAvXYcyHBgqN0WgCQ3Tr1q10zqTA8dIkkGFickQwKDLkclqH9kQt4yCDysLGwl4HssB4RORkRSSXmIdscG43nTb6iO5hvLWACc7fv3+fzpFxdCbycfj8+fPYwPeRRegec1bXs/BFR9O6V83Dhw/HOoNMLl++nM5zcn3SNQLHNh6FnDLp6ZBpq6PMM801yjIveSAVPJTjkOWbG9EpvXHjRqUtXSaNjtebN2/Gi2ITeN1MTo4YLI7yNAkRlSFQBkPHjsaHDx9SWjR2KBJp0aunvK7HI4Z4L9oRr4v3i2VVZ6xPCz0Qpx7SaJOelhlIrqUe0nQvylBWdZEuyFP60dFRmXoM5eRoAHWqbExfV5Dp79+/y9hx/wULIEZH8oi6kZMbp77Drkrs69mzZ8c7ejnqd25oY5/Rx/iQFPPevXtXa+TNSTDAOPrafUWuEOXJPI+GO9e9Jj3tA4vWvdzRtO5VI7lIpnIqcqL8WAOiU4sDorcfr1+/HushesoayvqCrYzjO0Sm0dEcygLzFEcMmSGT6EdIxsgN+UaZd0JhZCopPOc/xaKYQkOxVI58QmGs/hTeZTovOpzyC+OV0oGyu7u76VzlgHyVB8qQRxkCdQBtycsprnZSF/fhOkGcIHQe6wbdU9cTSKNuoCxpupfKi1i27pw61Gb1U/Ihrvp0D8VzGQ0F2hxln0O+dAKI01f6HXWHMnGsBOWa6u8rtDn2W/2tQuWadID6pGM58T6bziR9jFTJmzFiXhKqYByls31lUbpHmmRRJ1Pr3kmQk2TWRk+q5Kc6GDehMVWdjOeQZT+NjkaijnJOHdhFradR5uRpLKIsu6Byx0seeRsvUK8GC0GdeOrRdmf0WnlPy/dbeJ/6Lqfu6ZD69ERQCC0deXqK29Z4+3j5QFsL4Y2fBuLTBG3Ut1V4uHoCw6umbu2oANuU5NM+feuRy0H3KhThxH0oy+6N6gKeWAoFGNcRt+xpP3kC2ag+yiMD2rDOIAPpBIHxZNeBcUB3pFNs00sPIuzK5tvI6wT6c/fu3TJWD3Mr11Po++5Ln4n2TPDULT2MO+QCfez6tcWymKR7yAebh/2XfY1Y906DrUJm2P02n67kMlSc9Yc1Sm9F2Olix1s6y2u5TZM/O1rxNStzFf3FLhLQ058/f5a5x2sPY8E63vSqfRlUOl44OPrOgUYB5zgt8dVdfJ3WlsLjTJ1VqFosmshf1UVYrGUEcqUjHSXFkYnOEumxPSgu+ZyD+t0GHAUGUNfOwqZNFsZfssfRZDxIa7O1jP7hkA0RnHu+bxPoLt8a5PA6AQOLHsZX8hF0pu47mm/fvrVy3Ew1VfaJNHS1jmltWtcsUvdAr7tyrHv11MkswrrDa7II48T4sUbFjQacrqa1cWi01VHBQ1B8JQusx1w3CfS8ayodL3nlBD3dcc5gy0skTPsRH4uDFAUQ1jSOBoPB7oiuQTFZqHXOtwa0D+cOhzHWzQLNE0ZUTtUnx4ojzhnt4ogc2u46qY5cJjgQGCzlszNBvOppmaeg58+fl7Hjsm2+sVsHcOjprwySnGM90fEwoLEGyjOeWuTi+/shoP5JL+hL1SKFPmm+odfoCOeRpu9oMCq6l5mOKmMuXr16dWpuMpZd/BhpXhape8AcjXNTWPfqYc7mP1jIqds9jeuRNkbQxbg2IvvcaRsSbXUUtEZot09rAf3nOoF8qtZT5Ny5rIqJ1EjheKV3oHUUjknKJ8TzQiHSO1bF9Q61mKDjNM6BvJim87w+4B2t0qgf1EaV4agy5AnKqx0i1qfruW9su1BarF9tgJiuc4j9oLyuiX1Vu2IdSsvlMxSiDJGBiHH1N+YLjSuhTs4K+bgOgdi/2H/pSw66GuUgqtKA+oekL8umjT7WlYl2ghDtikDWVel9ZF7di3OwSsese6eRvAhR5oA8c3lVzes4boSob1FH8/qHSBsdRWYqU1U25muNyGWI3Lpmi3+Km28EeMJttniNMcYYY5ZB45+TWCfYgh3CawBjjDHGrC9rv+PF+1++K9ve3m79d0CMMcYYY5bBRr1qNMYYY4xZJRvzqtEYY4wxZtXY8TLGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI/j/H/xxvTHGGGPMkuH3jP5VozHGGGNMR/hVozHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xG1jtfbt29HW1tbKdy8ebNM7YYHDx6Mnj17VsbWC/oV5dm2r4xH1+MwDz9+/BjrD6EJ+l9XDvkoj3Nx8eLFcfqXL1/K1OGhPkzSgbpyuZyJiziHkZc51ptJskafokwF8y+mR5mu0l7Oitrbxv4gk7p+MS/pv4jzWSHq5SYju9VmPlbp6iQ9U/2EOCZDRX1p0tG4RuTlmvKUvpL5yh9QzTk8PPyzu7tbxtIfWP2zt7dXxpaD7sd9urjfLOzv7/85ODgoY9PDtfRtZ2enTGnHrNetkthWzuvajkyVRz9jOfSQ/Bx0hTyQvgyR7e3tsT7R7zrdairXNE/6OIdWCXJsY1uq8tG3KPdoIzmnXukk96nS2z7RVvcE5ePcFFxH32N/875XXbeJRLuFTJr0sEpXo85Bno/cVf860EZH6bP0TbqocqRTB+RzNOokZXKdXTatViwGNw74oqHTURDcq0kpV0VUhFmhX7MYolxGQwKZaQLk0Keo9PlkY7JM6necUEMhl0nd+DaVI4++R2Mj0DPlmb8guybbgnwlt6hTuX5RTjKXURfcI+p032irewJ51dktbHXe31xuTfLeJKJckEk+Z3Mm6Sp5uSNGWAd5T6ujIupifk2c03EskGGMd0Hrb7zOnz9fnp2G7Ty28bStxzFu10fi1h9l2A69c+fO6MOHDyktbklra59rRNxq1XZtvBftiNfF+8WyqjPWF7ciiVMPabSJexWDM7p69Wq6lnpI070oQ1nVFbcvyVP60dFRmXoM5eKW8LptFQuNVQ7jfvbs2TJ2XO7Xr1/p/MWLF6xm6bzuenHhwoXybBh8/fr1RJ+Qwffv38vYX5rKnTlzJsmnMFJJL6O+3Lp1K+UVRjjpkl/1tOPSpUtJboXRHhWGP81zyPXr9evXoytXrqRz8goDn+yC5Hz79u107CNtdQ/Un3PnzqVjBHv38OHDMvaXKKt3796NLl++XMY2G8lFMpX+zIPWZepEbwmPHz8er29DZRodzdF6whxkfcEuErCFGgMdmd/Xrl07obOdUAzURJo8TbxrqiHgYeKpcq5r8FrlgUYPXeUg90wpQx5loueLV5qXU5w8tYH7cJ0gThA6j3WD7qnrCaTJG6YsabqXyotYtu6cOtRm9VPyIa76dA/FcxkNiXw8IlE2QB8lj0hdOvXGsR0KtFlzAehH1EXRtlxdOuR1bDLoUVt9aZpzVfJE/nVj0Cfa6hSoXC4L5qzkWDc3wXp3EmSGzYt2v45Julqnm5Db1aExjY5GcploHa3SQ9I0FtTfJRN3vPAUnzx5UsZO8+jRo1HRgeRNxqe89+/fp2P0Wj99+jR6+fJlegLnCR3qnsSpT08EhfDSkaen69evp3NgRwSPFvBYC+Gl3TOITxO0kacAwMPVExheNXVrhwnYbSGf9pFfyOiUN6x7FYpw4j6U/f3797guQH6FMozruH//fjoC7SdPIJv4FI0MaMOQWdTTHTr48+fPMvaXN2/epPHddJBvnGsR5FM3z0w9dbtWzGmekiPs/nz8+HF048aNE/N/yNDPu3fvlrGTPH/+fOK8s86dBpmxTmD3m9bVSUxal1k7WIs2CXb55HcI9BR5MzfjWyjQGxXW8adPn5ap3dDoeOGkfP78+cSiSefkqOQdaUPh5afOKuROzSTyV3URnCacQJysfNKTjrLiyMT+kB7bg7Eln3Ogn3rdMAkWPgZQ187CuhkrFqImA40B+vbtWxkbpe1kXvdUkb/uQP+qXnUMAZx7jIFAd5FVTtty0DSXpp1n5pgquWETo1OGfXj16lUqizHHpvT5VU9bneJ1Kg+g2MD8cxA9QBNIJz/vM/O6znHbdNCTWalal6vgU4ShMo3dAx58qh6GBOsKAR8gBz3vmlrHi8mFEyEFoROkyUsk5N7lJNit0o4UqM62MBhMeF2DAmLkdI4y0j6cO7zYWDcLNE8I0XFTfXKsODIwtIsjDkPbXSfVkcuE980YJuWz60c8KoXACcFDF5TlW52hgiOq9/KSbc69e/fGik8+11QtdpSLix1GnvFU2T4vdFXIaEov6EvVItW2HLKr+w4T2XgBnB7maG7MocpmsXMe05u+iV01bXUKWyZbj03FPnHOnFM6gXTyc2cifgdnTsLbm/j2oy3oWNW6HCHO2lFlR4dCWx0FrStaH+JaEJ030PdfEd6asL50SjFxTsG7Z7JiKCZXmXuSwjEZl4nnvI/lGsX1DrVwlMZpnAN5MU3neX0Q26Y26T2uynBUGfIE5fN3ubE+Xc99Y9uF0mL9agPEdJ1D7AfldU3sq9oV61BaLp++o/HIg+AcmYgoB5HXEccxykghH9chEPsY5SF9EXXlotxyvYgyQsfN3/lLiHKM8boyAllWyVNjRog2oa+01T1Bn+v6RXouE+ofgq3qEsk7lzkwX6O8qvQQGcc6CBqTmEdd60AbHY02sKpslRxjvYSq+bxstvinuPlGgCecP5UZY4wxxnRF6z8nMXTYfu3z9r8xxhhj1p+13/Hi/S/flW1vb7f+OyDGGGOMMctgo141GmOMMcasko151WiMMcYYs2rseBljjDHGdIQdL2OMMcaYjuD/tvA3XsYYY4wxS4bP6v1xvTHGGGNMR/hVozHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xFTO15v374dPXjwoIx1w9bW1ujHjx9lzAyFixcvprHj2MSXL19SOYUcxj6vI17D+VBRH549e1am1EM/b968WcaOYT6qjqo5QlpT/rojPcn7HuWCDOuI8s1lz5gpL0e637WtnAa1fZLuSQY5k3QPkMMm6l0dTToTqSuHPik9z8/T+6x7bVFfJunopHLIomqeRzsQ9RS7MWndmoepHa87d+6UZ8sDAUiAy+z8vKyDYi8LZPPx48f0V3oZw6aJ8/Xr11ROISeXM/px9erVcXnOhwhyOTg4SH349OnTRAfy3r175dkxlH/y5Em6/vDwcHTjxo0y5xhkvr29Pb7HhQsXypzNAPnU6QZyQWbIBRlWOQekYe9U7vv372PjzZExIx35RqeM86dPn6Y8qDL4q6at7tH2Kps/SfcA/SPPHNOkM5GmcteuXUvpCjs7Oymda2L6/v5+Kjtk2uropHLEX758Wcb+UmcfccIAnWbtWco6X9ywNbu7u3/29vbScZnoPoJmFhO4jPWDYrD+FINWxkxOHC/GEnlVURiINL51Y0w+10dZ5zqY68sQyPWHfhZGtIydhv4RYpm83+RRD1D/lNN7LUGnct3KZZ3rk9C1Iso3ngNjiczza/poJ6bVvSpdatI9QA7k57LfZOp0JqdJtyKkqVyeV6XPQ6KtjrYphyxymXJdrtMRypMfr1kkrXe88BrbetDattMWHtfiNXKee48qg9cK5OOdPn78eJwGv3//PlGfUL0E1Y0nG8vqnKcCnausrledsT6lqQ7qJZ84T9KFso/LKY+42h3vF/tCOk8xyo95UW51T0RDQE8P2km4cuVKOuZcunQJ7U9PaMUEGstcfP78eXT58uUydgxPNefPny9jo3R+dHRUxoYBu3xx3M+ePZt2VKqQDM+dO5eOgh3FmIbMf/78mc7ZcSkMzliX0DVzDDoVd/+QoWQcoUxhgNO8Vv7t27fT8cOHD2nMBGP569ev0bdv35IeizNnzvRu12ca3aujSffg+fPno0ePHpUxA3U6k1NXLuosvHnzJtlPyPOq9HlItNXRSeVYlx8+fFjG/tJkH4mzm0s+toL5v2haO14MsoxOExICxocFlcbjpOC0FV7miS0/ypJGOW3rvXjxIl1TPC2dECCvWVQfwgaExaJLOgFjgKCZ8CzkgOHDeJJP+znSNg3G3bt3UxtwDLgWKEOaXlPE1wbcg7Lkq49MChxF+kacdqP4r1+/TnEC/cORwrFg657JxaCSh2HWRKFO4qSD2jREaDv9QTZ1C78MBmPDmCFrIX2oIndChkhuLOtoWsSigY6gX5obyBWdG7oxXiTRcW/i/fv3ac4zfzmPYFuqiAtBX2mre03U6R5zHbtqTlOnMzlty1WNI2tM/lnCEGmro3XlZO+q8pvso9Ze5j3rT90aNA+tHK86r7EKOUvxKQ9niYU1KhOdpAzODR4mTkvTwoAQhHY3cGzibghxdkOA++G8sfDjvUZY3FnMACdOuzFcSztoj5wu2nT9+vWUxr2qvG7di36Kd+/epesEg8dgcy8GGmcwDig7etrtoc3cj/JD28mJ4CygxPQ1H4MqkKNAFkP/RmERzLKIaR7JWUOu6BQ6ZqYD2yfHizlpJsMDZd0Ot1kM2Me6h4e4E7bJ1D2wtrGP6G/VWr8oWjler169GjsD2tnRUx1H0gmz7M6wMCvkT5RtqNqqFTgtOEO594+g485bBKcotglvWQ4EDts0xncWpwk5x/svw9vummn6oKcTjAdPIcg7vtaVI4xxF8h5aE4aTnx8mECPWdxzeJjQw4l2SqWDlNfuLyAb6pUMZWDMSdAVPaABr8jiQ5JgccP2IU90GFui1w48TPBaUWCkWewIjJHAmFO2T7TVvSbqdI8HBT28Sk+xaXU73ptEnc7ktCmH7G/dulXGTlO1yzMk2upoXTn0MeohcxL7yfzthX0sFvep4IPJwgCVsWqotlgo0zllVZ60eMtiQp6oS+ccuY+oq48y1CGIF47TqXPKx3Kgaw8O/n7cSFpsH3HuW0yE8f055xpCfm+1C8iP7SaufNpFPYJyagfn1CVinUOF/sQ+1UEZjVkkl3XUo3g+NKL+5bpYRa43US65jNAb6U6et0lIPzQPRUyrygfkFvOQp/Q4jkU+LpxLj+N5n5hG9ySHSNSpeJ5TJ9tNpElnIm3K1V3LWLSxtUOgrY62KYe84jxctX2cesViUNXgKugAk41AWZ1zjc5jJ5VGkMDidXX1SblivUpDwMTJY9IrP96X9Cphx/rUT45qhxQ+1vt///d/43O1AdQOgq6jj0qL9RKoM9artCES+xBlAjENudSVE1UTI8q2aqINgTjWse+ck5ZTZYRVlpDrStStTaRpLsV5mBvkqGtRvrnso63IUbpsSN9oq3tRTjEdmnRPNOVtInU6g85FXWnSLcYkjlmE69ZF3m11tK5chLkb5zms0j5u8U9x442DbUa+w6p6B2yMMcYYswym/gOq6wJOV9M7cmOMMcaYRbNxjhd/0kEffQ79A0RjjDHGDIuNfdVojDHGGNM1G/uq0RhjjDGma+x4GWOMMcZ0hB0vY4wxxpiOsONljDHGGNMR/LzPH9cbY4wxxiwZfs/oXzUaY4wxxnSEXzUaY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEd0drxunnz5mhrayuFt2/flqndwD1//PhRxsxQ+PLly1hnCHU8e/astsyDBw/GeZznSC+pY4iob03tZ76pXD73mBfKI8R5Eq+7ePFimbqZRFkooJ9V1OlttIGEKNNYP+WGgNo7ae6obzm6npDbZ83bTde7nCZbF6nTwTZ6JtlX2cuhob5O0tFJ5ZAFsovomio5Iv9l6m4rx4vOPHnyJP3F1f39/dGdO3eW6ghRtwTY54m7Doq9TL5+/Zp0RqEKJsOnT59S/sHBwYlJgB5cu3ZtfP2LFy/KnL8Ox4ULF1Leo0ePypzhgG7TZ9qPDKocAfr5+fPnsXyYe5F3796N5UNAHuLnz5/j9O/fv5epm0uU087OzujKlStlzkmq9JZxkA0kHB4ejm7cuDHOY1xIIw9Z50a+b7TRPaAfuc4B81Sy2N3dHcsCZLvJe/r0qe1kSZOty6nTQdkCwocPH045GthEypEf7eUQaaujk8oRf/nyZRk7JupvPl+RIaDT6O5S9Le48dRsb2//KTpaxhZPMZH/7O3tlbFCMkUzC6NWxvoB/UcOpprCQU/jNmnsigUwlRVRt8jjeo45lIvXDY1cf+hLVT9z2cUy1CEZ5/OR+aM8c1qO2Jgq6vQ2v55ykjl5Uc65TveNtronpGd15P2PcxhyWW4quV7kchJ1OpjDHI96TP1x3RwybXW0TTlklMs+ypX8XM6UR/7xmkUy8zdeZ86cKc9OI6+bIwGPE6+R89x7VBntbJGPd/r48eNxGvz+/ftEfUL1ElQ3TwGxrM7xanWusrpedcb6lKY6qJd84levXk1PuCqnPOJqd7xf7AvpeNzKj3lRbk1PRH3n0qVLWOK0Q1pMjLEsc3hqO3v2bBk7fnr59etXOueJjToglx+8fv16sHLiiTb2CRlU7UrFHSz06/3792XseA4in8L4JH2UXODWrVsprzDESUbo1SYT5Yic2Emtok5v4/WA7mnHjLzCUKfxkZxv376djn2kre5NA7IC+o9djOsDedjvTafJ1kXa2k44f/58OlKG+o+Ojk6tKUOkrY5OKse6/PDhwzL2F81n5IYtiPMb+bG7XThkaYeReb1opna8mFhsweWGSEgIKAzKQ+NZFOgcC0Tc8qMsaZTTth6LLdewYEQB3rt3b1wfwgYMKIpHOuHjx49J0Lx2QmkBA4BRJB9jyJG2aTDu3r2b2oAR5VqgjBYzYLucNOAelCVffWTy4CjSN+K0GzlhnIkT6B8OAgPN1j2TRNvGGCoZbOokTjqoTUND+oHMGQtkWEedE686cDbQFTkWyA156tVP1Zb7EKibQ1VgDNCvaGR0vfQReQjlMReYS8+fP09xc6w/dY5RW73Nxw4dxTagl9E57ivT6N4kvn37dkpOi6x/nWjasBBtdZDXavrEgjWRtVGfZsAyHIYuaatDdeW0ptblIx/WeNbj6NxKfsxn/JFlvLKd2vHCgDc1RM4SzoNAIVCiqHR6MqLjWlQkqCoQgsCrBxyby5cvp3MgjjIC92PBwZGJCxKgyFqIUFg9uXIt7aA9crpo0/Xr11Ma96ryunUv+in49obrBDLDQeBeTCacwShHngg1+LSZ++kJZuggn3lhDPlmCRgTDIzGDdmvg5yawBjgXDFn4s6WQBbRKYtgnJvm1qbRVhZ1elu1Y4bjL8eLubtJNDmyZj6adDCua9g/NiFUnjVu0+c8a3zTt7+sv9hV1tvcucWeVq31i2IqxwsPkR2iCMYeQ0OYZdeBjivM8qRYtVUrUEacIXbLIihn3HmL4BTFNuEtM3icMzjTGNVZnAHtoiksw9teBXVPHTigPDELlJ2t9irOnTuXjtQlJ2yo4MTHhwn0mEW7CYxBdO5z6mQMTXmbBA83uT1ookpuuaNBna9evUplma+MUZ93G2bRvTqw/9FGIQNsWNxB4GFBD0mbzDS2LpLrILJFB6NMcbrW6eGzrY7WlcPp1CYKgU0Mdraq5iUbNl3T2vHCqYq7DOoAyiMnocm7zNEEjYKY1lixoxS9fnavZFTVXowCA5HvBrBLQhoDJ6gv/oKHOhhAXhFyxDFk8kSjUgf1xl08rmlaNAHZYqSiA9tnA94WjUUVjJcUn6c4xqRqsaOcFjucf17tCha9/IGg72geSZfQ4zZ9QKeqnoKRnb73yEGHhiafZfHmzZtWix3U6W3VTgLzNqbXjUUfmFX3crCLWvTou+zW/fv3k5yBtEl2b1Noa+siuQ4iZzYA5OySTxrfdMb1hvtM84DRN9rqaF055Bo3MFi32VSp2shAVzuXVdGoiRSN5qXniVB0osw9SeFMjcsUzs34vJh843PKCKURDspfeMTr6urjHGK9SqNtxMkrDOI4P96X9BgXsT7OlaZ2IAuI9f7f//3f+FxtALWDoOvoo9JivQTqjPUqbYhEnYkygTwtylxMkgPXK69OF/tO7GOUh/oWzxUiVboqol4NVT7LQPMwgowk/ya9BWRZJc84TlX36BttdA+ivYrpMU0hzlHJscrGbjJVtg6Qk+ZwnQ7G9UQh6lrMr9LdodFWR+vKRZCT5m0sT6iaz8tmi3+Km28cPBnwHdY0u3TGGGOMMfMw85+TGDo4XWzPGmOMMcZ0xcY5XnyXoA/kJ71fN8YYY4xZJBv7qtEYY4wxpms29lWjMcYYY0zX2PEyxhhjjOkIO17GGGOMMR1hx8sYY4wxpiP4eZ8/rjfGGGOMWTL8ntG/ajTGGGOM6Qi/ajTGGGOM6Qg7XsYYY4wxHWHHyxhjjDGmI+x4GWOMMcZ0hB0vY4wxxpiOsONljDHGGNMRdryMMcYYYzrihOP19u3b0dbWVgo3b94sU6t58ODB6NmzZ2VsNi5evJjuaU6DfDUWP378KFONMcYYM2TGjheL++fPn9NfVSV8+PCh1rEi/eXLl2VsNnAsDg8Py1j/oH2rAmf02rVraRz29vZW2hZjjDHGLI6x43XhwoXRixcvytgoLfhHR0dl7CSPHj0a7e7ulrHZ4F7b29tlrF/ghM7rWM7DpUuXRrdv307nly9fHl2/fj2dG2OMMWbYNH7jdf78+fKsHl5J8jpMuzJ6PaY4R+JfvnxJcXbLiOevMimnsspT2ZgWX4dyzutKzimr8/x6vc6M9SkNJ4u47k9cDqHKEahT9QP9UV1KA9IpF/MjSqNMHTjB4s2bN8nRNcYYY8wawP/VWMXOzk55Vs3u7i7/x+Ofg4ODFApnpcz5k84PDw/TufJhf39/XC9pXE/a3t5eOo91kM89BNcprmtVRvcCyul+pFM/xHuTzvUcdT35pMV80L1UXsS20n71WWV1L9LVnnjOfWP/cuJ9m8oZY4wxZjhUOl44IXIQ6sAZwOEAOQmC6+UsqAzgjMgRAhwRxWN9QDyWxamJzg553DOWAdoipyevT46MAmWr6omOF5CnOoG4+ifkeFFnbKf6GJ0yhVhnHfSBslxvjDHGmGFz6lUjr8j4yP7KlStlyslf2OWvCKvg+6RFfCP18+fP8uw05BXOz+jOnTtlyjG0+/v375W/BCycGLypcaAsbeWcPtO/quuqaFsuJ97//fv341edCnolK3jNWDhv6XxSWWOMMcb0mxOOFwv706dPxx/Z800UacSjs9AGnBy+Y+LjcMG3S0+ePEnn1Ht4eJgcJ85z+FXf48ePy9ho9O3bt9H9+/fTub7Pol3cByckQh9u3LgxOnfuXJnytz7dizpwXHAqOVIXjhz3mQQfv/OrTzk+1Elf47dZOeThQHE/wTnp0RmLDi9wD9U9qawxxhhjek6xgCf0yi2Guldhev1FiOfxFRuvxmJckKayBO4b6yAuYrpe7em1ptqmfEIkj0OsT9dTL+ekxfaqHPnxXKgdBF0XXydSp+olUB4Uj2k58Tq10xhjjDHDZ4t/igV+4bAL9O7du5X9Io/dJO3cGWOMMcb0gcY/JzEPOF23bt0qY93Ca8S7d++WMWOMMcaYfrBwx4uP7/XNVdM3T8tAPwLIfxxgjDHGGNMHFu548fE9by9X8YpRPwLwK8b+wA8D+JHGJKrK8cMC/YIzz6O88vQjhyFS178cdnEpl6MHnao84JW/8qt+xLJJRFnpBzpVTCqnsUAHBeOna4aij2rvrLqnB92qOpQXZWRO6kkTTeWUXidbyZ7j0FFfJ+loXTnpLqHK/tXZR/2obWnwjZcxy0A/pOBHDU3UldMPC/SjBf0tM37koHP9YGKI0G/9wIK+1v3YIv7wJULfdQ0yiT/+AMmmrt5NAllIn5BHnc5MKkdenkY5rgPGKv5Ip6/Mq3uxn/n8RBbSRcrlermpIIuoWzrPaSoXxw255nUwDnX1Do22OlpXjmPUUZ2LOvuoNNnUZehvtfUxZkEwEbQoNZGXkxEXTcYkGv2hEI0CRGNbBeXpZyT2mesJoqr8JhOdAeRWJ5umcuRVjVEcB86XYagXySJ0L7+GfMkhLoQQ8zYZ5BXnaC4nUVcOGcZxIx5ly3VtbO0QaKujTeWYh1EeUa5cl+t0hLLkx3FYJEv7uN6YeYjfB7J9POn1cdffE87L169fT2xlnz17Nv3h32mIfea7Rv3H6sDfsisMz3gbvenV2ibAD334o868QuCHP4VBLXNO0lSOvwOIzPNXE3Ecnj9/3vtPHRahe+gaf8sQvSIUC1ySAzIpHIHRmTNnypKj9PcLf//+XcY2F+SFrAVj8OvXrzL2l7pyyBDZiqh36CvXHR0dJd1c6muyDmiro03lPn78eOJveSIv/VH2JvtInL83Sj52dRmvbO14mV7DpGLBw3hXgcHB6A+RaDhnRd8w4CzE7xswwvwn98XDVXIe6v5Q8aaArIun3NHVq1dTPDqpkbpy6Bk6yB9iRqYYZf5Is9C3IoxDm//dY9UsQvdwAtArFqf8m95F1L+ORIe0iapy+sFY1UMUDgg6Kf2EoX/j1VaHmspFBzbSZB8lPxw3HqKW8SBlx8v0Gp5e9JRX9YHlmzdvVva34vqA/ssrjK7+pwcZEMmFMt51ONYVZIWcmhalqnLsOPAQIEfs4cOHp3YfZMQx6jhq6w67e/SZBWoIzuY6ICdBOzXMa3SPnS4cCeknOzqb/KDVRBv7iJM77S7wNNjxMr0Hw1K1q4WxZwEcIvxXWixYgoU97qBMS3wq0xOgDe9fcKBYmABnQa8Tc+rK1b3qyMGI7+zslLF+sgjdiw9ByIXATgy6xyIWZYuD6j/vM0p6Ef9LOmTGfz+X01QO/UIvCZTDwQJ0FudrXWiro03lOLITKLCHlO+DfbTjZQbBq1evTvxBXhZI3sNrEjXtYPQRLURaoOjLPH/0l0WPXS/BOTsSoHts+uL36dOn8uyYutc+VeXy1zx8/xXlLTDmLJR9lvWidC8ueKDXOvyfuuwaAg5alZw2kXv37o1ev36dztEjdlBlvyJtypF2/fr18Q6Xvk2UM8H11DNU2upoUzmOrBtAfpyXK7ePhedszFLQL0MI8dclebyq3P7+yf87tHhqTulQPFGfyCPwK5WhQZ/U/igPzkkT+gWOgohyKAxJmfqXmG9OygP9EsSj/OvKxfFCZwWyVzrXDoF5dQ/q5jcobyjy6IqoKxHkFOdwVbk4ZlX2LtrMfDyGSFsdrSsHKkugXCTO865Z2v/VaIwxxhhjTuJXjcYYY4wxHWHHyxhjjDGmIwbnePGx5iwfUvMLOH0YOwuz3tcYY4wxRgzC8ZLDg+Okv1U0DVzP39aZlnnva4wxxhgT6b3jxc9j+Zks8NPZvRn+Sjl/42hnyr+ts4j7GmOMMcZElu546RUdR/7SLq/8cGr0l3f1d0dAZQjsMpG3vX38X8UoTVAnafqLyRyJ8/dNgLLEqXMS89wXYn9iOm1R+TbtMMYYY8x6s1THC4eFV3TaOTo8PEyv/PiLsvwVC3ah+EOEQFn+cCHplOO/RQDOgXT9sTjq4/+kUn3w/v379EfR9Jd8tUs16b+Tmfe+gJNGGmUBJwsHLNaxyf+tjTHGGGOOWarjJecHhyg6HvqLx/Gv8fIfreLMsDuk3aa6/1uO+uQMgXbN+O9j+Mu1gEMV/9J5HfPeV3/1lmupg7r4rxtwBEmb56+RG2OMMWa96NU3Xjhp7A4pTPsn/HHkeL2HM/Tz58/K/46hinnvi4MVr1/G/2ZujDHGmOHTG8eLV3i8ltTuFTtW2k2aBna8pvk/qua9L04arxTjN1z6NaQxxhhjzAn+LJH8/47SOSH+X1T6/9Bimfh/oSltO/zfSnl9EcoVzlAZO/n/XlX9H1fz3pd7xTTi+r/KFDfGGGOMWcv/q5EdJ7/uM8YYY0zf6NU3XouA14S8PjTGGGOM6Rtr43jpb3Hx5yTiLw+NMcYYY/rCWr5qNMYYY4zpI2v3qtEYY4wxpq/Y8TLGGGOM6Qg7XsYYY4wxHbFVBH/jZYwxxhizZPis3jtexhhjjDEdYcfLGGOMMaYj/OckjDHGGGM6wjtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jjDHGmI6w42WMMcYY0xF2vIwxxhhjOsKOlzHGGGNMR9jxMsYYY4zpCDtexhhjjDEdYcfLGGOMMaYj7HgZY4wxxnSEHS9jBsrNmzdHW1tbKfz48aNMHY2ePXs2Tn/w4EGZekzMy0OsYyggA/rUN5Dnly9fylgzlLt48WIZm4+oE7FO7qH0t2/flqnVUGbRuoAe1sljUX1vkuMiZdyWWe7J+E0anzpmuR/l68alLXkd6I50jf7M06e15Y8xZrAcHh7+YRpvb2+XKcccHBz82d/fL2MnIS8vv7Ozk+ohrw/s7u6WZ/Xs7e2lNnOcRJv66qB+5NyWaWSpcrRPYV5obz6+gD7U6YTgOto0TX8ngTyaxkg6vMh7gsZN9VfJpC1tx2Xa8WM8FjHnFtHHRUEb6NOyxnUd8I6XMQOnWGDSMd/dmob379+PigVydO/evTJldfDE/PLlyzJWz6NHj1KbJzHvjtjjx4/Ls3Ygy7YUNnj09OnT1N9r166NXrx4UebMDnIpFrvKXavbt2+XZ9V8//69PFsM7HRcuHAhtakO8ouFei79rULjpvpnpa0+zqJnT548Kc/mY94+LhJ078yZM6lN6DdHcxI7XsasAR8/fkyLwzxb+iwCdQu2iK8R8sArhRwWIxZUveKMZfK6iBOKJ+aUT1pVf0gjr+q1CvXH+rgvCzCyIQ14LaIycbGP6WqnrqFNWli5RuXiKxalT+NAUB657+7ujj5//lx7LX2lrPpOaIL6nj9/XsaO+fnzZ3l2jOohVL1u0phxjGMlYlskmxz6dvfu3TL2d8zpZ+zrlStX0jFvRxwTrpUcND4ab6A9TeMGk+SXt494ro9RFgTi3CPXs9geIJ245ElfmG9Xr14dyyJ/baf+6j4Q+0B+G7gn5ak7yj3WG2WtwL1A5ZQe26g85QMyU9/n6RN1SI7k1Y1l7JP6SlD7e0fa9zLGDBK28fUah9cWTGnS2Oqf5lUjkM71HBeB2kPQa59ocmIbVBbycpHYdpVT/3nNE2WhcqTpFVBVGyibp3MuOXBOPsS6JC+I6epLWznGPtWhMry2A45N9ce2gfooYnvjOcT+kk4+xDrzNlf1N5cpUB/pcbwF99G9ItSrfkPsO/WA2hbLEVc/lK9+0va8vVDVvrwfUSbxnKPqz9tDHmlKV7tiOyhLXoyrf9SruuM18ZxjHJOI2sEx1lPXltgvlSNAVZ7qgBifp0/k6b7EuU79IE450PhwjGViet/wjpcxawKvkQpjNbpx40aZMhu8JqgjPvXmIT7dA+0pDHRqU3zVRB08AccnW8oWBvPEk3EVb968Gd2/fz+d8wqDawS7fuw60JY7d+6k3YScb9++paPaTBl2gkinnaKwjeNdmMinT5/GuxrsVAD9efXq1XhnZ9LrvBzuM+kVH/1mXNu+xqTOYmEaP/GzmxZf+TAevNZkzKZ9lQpfv35NspMc4devX+kofv/+Pd4tErxOVRoyzjk6OirP/kJfPnz4MN4dQVbIA7SLR5li0U3ndXDf+Co3by9Mah9QB/JDf+teQebtuXz5ctIX5Ea9Va/fGNsoL/osXeKeajv9Z45Kf9tCee4R64nQnygTjYXKxXvleXXM0yfymN/IEXmC7occox2QTNFz7kE9ui962DfseBmzRsiQzfKtFt8aYayqFgUhI1cVpvm2CSYZ7VkonoRPtKkKjHksI6dQC/skWAji9U3yWhQ4lQ8fPkznOKfIjsUovraJr2GA8eRVH/06f/58mXqMXtOQj3M8CziqUQ5tHE7KUJYFkvu3lTn3wjmm3a9fv651eOalTfv0Kosxic56E4wV9QLXTnrAEFX3x1nGCae+6NQ0wdykPPfOH5AEtoMHFvVNtmTRLKpPVU46oM/UoyCnrU/Y8TJmzcBoTvMkDBg+nhRZ1LoAY0gb4zcYciaawIGIOzS0mTgLGYY7Opzxuw9x6dKldI3uyyLAQqp0LYikx7aJ69evp8VJcC1lcYBwdEB1aEdsXqiP+uXgUS9jDMhMC0z+ATtOBDJGDrdu3SpTj2Gc405CE1rg2GUAdIUdHJwf9ZVjLi92MHI9pC2UZVGXMxXJHUTBThRtZoeLNuM8I3vasUgmtQ/Y3cTBn8bhpq3IhzHCMZAsm6CP8Ts96Rp6Ou1DC/oD6AnXV0HfpUvLeCiCRfVJ+se1wJgR0BPsgdKROem9oxCyMWaAFAtn+oaBUBi0MvUY8ghQLH6pDHG+zdA1MRRPmamsiNfMSmxffl/QPWKaUBrX5dBX5XMey8Q8pcd2cE++D1GcIGK5mI5siBcLcYpzVBmlgdLUBu4DXB/LTUu8H4E+tIVrc92A2Ff1D3npnABRVuqXyOuogmskB1B7qq7Jy+ZwDfcEjvH62E7JWn35559/xnncQ/cn5Pera5/KIyOC4roH7Yny+M9//jM+p06uifcVSsvbpTFWnKC+x3K6///+979xGvk58RrNC11LgFhGgXbEcrHvnMe8fD7nfZ62T3Hc4n3Vv5jGNaKqrMZmmrmzTLb4p2iQMcaYHsJuBTtcXbzSXDTsOPDabtJrK3Yo2HGZ9nW1WRzsPsVdU8aEHb82r5DNdPhVozHG9BRek8TXjENDizaLehPb2yc/fDfdgoPMD0ci7969S6/gzeKx42WMMT0EZ4Xvufj+ZZLj0mdwqPhOrO5bGxxLXrwM1blcB+Qg82G9AnhMloNfNRpjjDHGdMJo9P8DyXU9woi8oLoAAAAASUVORK5CYII=\" width=\"606\" height=\"593\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eGut Microbial Landscape and Diversity Analysis of the Study Cohort\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003ePaired-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. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. In this present study, the predominant bacterial phyla in the gut microbiomes of Taiwanese women with endometriosis were \u003cem\u003eBacteroidetes\u003c/em\u003e (49%), \u003cem\u003eFirmicutes\u003c/em\u003e (41%), and \u003cem\u003eProteobacteria\u003c/em\u003e (8%) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). The distribution histogram shows the relative abundance of the top-10 major phyla of gut microbiomes between two groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The compositions of microbial species at each hierarchical level are illustrated using heat trees (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Both groups displayed similar relative bacterial abundances.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eWith its various indicators, alpha diversity, which is a measure of microbial diversity, richness, and evenness within samples, showed no significant differences in Shannon\u0026ndash;Wiener diversity index (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), Simpson diversity index (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb), and species richness (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) between the two groups. Meanwhile, beta diversity, as assessed by unweighted (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed) and weighted UniFrac distance (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee), showed similar microbial composition between the two groups.\u003c/p\u003e\n \u003cp\u003eA balanced gut microbiota that compose mostly of the \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e phyla supports the gut epithelial barrier and overall homeostasis [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Higher Firmicutes/Bacteroidetes ratio (F/B) in the gut suggests dysbiosis [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. As depicted in the box plot (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef), the median F/B ratio of the ovarian endometriosis group was marginally higher than that of the control group (0.98 vs. 0.93, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.88), but it did not reach statistical significance.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDistinct Gut Microbiomes in Healthy Women and Ovarian Endometriosis Patients\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo 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\u0026thinsp;\u0026gt;\u0026thinsp;2 to identify significant characteristic flora at the genus level. The LDA scores and cladogram highlighted Rothia genus within the \u003cem\u003eMicrococcales\u003c/em\u003e lineage as more prevalent in the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg). Genera such as \u003cem\u003eMegamonas\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ecoprostanoligenes_group\u003c/em\u003e, \u003cem\u003eAllisonella\u003c/em\u003e, \u003cem\u003eRuminiclostridium_5\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ehallii_group\u003c/em\u003e, \u003cem\u003eNegativibacillus\u003c/em\u003e were significantly more abundant in the ovarian endometriosis group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eVaginal Microbial Landscape and Diversity Analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eSimilar 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.\u003c/p\u003e\n \u003cp\u003eThe lower female reproductive tract has been shown to be primarily populated by \u003cem\u003eLactobacillus spp.\u003c/em\u003e, which protects it against pathogens by creating an acidic environment and producing bacteriocins and hydrogen peroxide [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Compatibly, \u003cem\u003eLactobacillus\u003c/em\u003e was the predominant genus observed in the vaginal microbiota of both the endometriosis and controls groups. In the ovarian endometriosis group, \u003cem\u003eLactobacillus\u003c/em\u003e accounted for 64.4%, \u003cem\u003ePrevotella\u003c/em\u003e 6.6%, and \u003cem\u003eGardnerella\u003c/em\u003e 5.5%. In contrast, the control group had 48.5% \u003cem\u003eLactobacillus\u003c/em\u003e, 6.8% \u003cem\u003eGardnerella\u003c/em\u003e, and 5.8% \u003cem\u003ePrevotella\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). The compositions of microbial species at each hierarchical level are illustrated with heat trees (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). No significant differences were seen in alpha diversity, as indicated by the Shannon-Wiener diversity index (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea), Simpson diversity index (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), and species richness (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). However, lower beta diversity was found in the vaginal samples of patients with ovarian endometriosis, as shown by the unweighted UniFrac distance (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and weighted UniFrac distance (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, p\u0026thinsp;=\u0026thinsp;0.006). The results signified lower species annotation and OTU abundance in the vaginal samples of patients with endometriosis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMicrobial Flora of the Vaginal Microbiota That Associates with Ovarian Endometriosis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eLinear discriminant analysis with effect size (LEfSe) analyses on the phylogenetic trees of the dominant microorganisms, using a LDA threshold\u0026thinsp;\u0026gt;\u0026thinsp;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 \u003cem\u003eRuminococcaceae\u003c/em\u003e families (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047), \u003cem\u003eEggerthellaceae\u003c/em\u003e families (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009) and \u003cem\u003eBeijerinckiaceae\u003c/em\u003e families (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047) were more dominant in the control group when compared to the ovarian endometriosis group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which is an enzyme commonly found in several bacterial genera, such as \u003cem\u003eBacteroides, Bifidobacterium\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A review article in 2023 concluded that gut microbial β-glucuronidase may become a potential biomarker for early diagnosis of estrogen-dependent diseases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, mounting data in human and animal studies supported an association between dysbiotic gut or genital microbiome and endometriosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Many animal models had also demonstrated a bidirectional relationship between endometriosis and microbial alterations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Yuan \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] 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 \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] 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 \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] demonstrated a promotional pathogenic role of \u003cem\u003eFusobacterium\u003c/em\u003e 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 \u003cem\u003eFusobacterium\u003c/em\u003e 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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur previous study aimed to find associations of the gut microbiota with endometriosis through bacterial analyses, enzymatic assays and targeted metabolites quantification [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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 (\u003cem\u003eUBA1819\u003c/em\u003e, \u003cem\u003eEisenbergiella\u003c/em\u003e, \u003cem\u003eHungatella\u003c/em\u003e, and \u003cem\u003eErysipelatoclostridium\u003c/em\u003e) and exhibited increased levels of four estrogen metabolites (estriol, 16-epiestriol, 16alpha-hydroxyestrone, and 2-methoxyestradiol) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In line with our previous results, several gene-based researches had associated gut microbes with endometriosis. Svensson \u003cem\u003eet al\u003c/em\u003e. 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Besides, abundance of 12 bacteria belonging to the classes \u003cem\u003eBacilli, Bacteroidia, Clostridia, Coriobacteriia\u003c/em\u003e, and \u003cem\u003eGammaproteobacter\u003c/em\u003e 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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In their study, genera such as \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eDorea\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e, were significantly more abundant in the endometriosis group in comparison to the controls, whereas lower levels of \u003cem\u003eLachnospira\u003c/em\u003e and \u003cem\u003eEubacterium eligens\u003c/em\u003e 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 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding 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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), 2-methoxyestrone (p\u0026thinsp;=\u0026thinsp;0.043), and 2-hydroxyestrone-3-methyl ether (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) in the vaginal samples and higher fold of 16α-hydroxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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 \u003cem\u003eMegamonas\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ecoprostanoligenes_group\u003c/em\u003e, \u003cem\u003eAllisonella, Ruminiclostridium_5\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ehallii_group\u003c/em\u003e, and \u003cem\u003eNegativibacillus\u003c/em\u003e. Furthermore, patients with ovarian endometriosis exhibited relatively lower bacterial abundance and diversity in their vaginal samples, whereas the control group displayed higher abundances of \u003cem\u003eRuminococcaceae\u003c/em\u003e and \u003cem\u003eBeijerinckiaceae\u003c/em\u003e families.\u003c/p\u003e \u003cp\u003ePrevious studies analyzing the role of the reproductive tract microbiome had demonstrated that opportunistic pathogens, such as \u003cem\u003eStreptococcaceae\u003c/em\u003e and \u003cem\u003eStaphylococaceae\u003c/em\u003e, were enriched in the cystic fluid of females with ovarian endometrioma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies from Brazil and China noted lower \u003cem\u003eLactobacillus\u003c/em\u003e abundance in endometriosis patients compared to controls [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, Ata \u003cem\u003eet al\u003c/em\u003e. highlighted differences in vaginal microbiota between severe endometriosis patients and healthy individuals, noting the absence of \u003cem\u003eGemella\u003c/em\u003e and \u003cem\u003eAtopobium spp.\u003c/em\u003e in vaginal samples from the endometriosis group at the genus level [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Perrotta \u003cem\u003eet al\u003c/em\u003e. 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 \u003cem\u003eAnaerococcus\u003c/em\u003e (phylum \u003cem\u003eFirmicutes\u003c/em\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eConsistent with prior research that showed an association of gut microbial disequilibrium with increased Firmicutes / Bacteroidetes ratio (F/B ratio), leading to obesity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], hypertension [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and irritable bowel syndrome [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], our current study revealed higher abundances of certain bacterial genera, namely \u003cem\u003eMegamonas\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ecoprostanoligenes_group\u003c/em\u003e, \u003cem\u003eAllisonella\u003c/em\u003e, \u003cem\u003eRuminiclostridium_5\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ehallii_group\u003c/em\u003e, and \u003cem\u003eNegativibacillus\u003c/em\u003e, in the gut microbiota of patients with ovarian endometriosis. Interestingly, all of which belonged to the \u003cem\u003eFirmicutes\u003c/em\u003e phylum. The \u003cem\u003eMegamonas\u003c/em\u003e genus, which is anaerobic, non-motile, and non-spore-forming, has been positively associated with obesity [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and several diseases such as nonalcoholic fatty liver disease [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, in previous vaginal microbiota studies, Kunaseth \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] reported that \u003cem\u003eAlloscardovia\u003c/em\u003e, \u003cem\u003eOscillospirales\u003c/em\u003e, \u003cem\u003eRuminococcaceae\u003c/em\u003e, \u003cem\u003eOscillospiraceae\u003c/em\u003e, \u003cem\u003eEnhydrobacter\u003c/em\u003e, \u003cem\u003eMegamonas\u003c/em\u003e, \u003cem\u003eSelenomonadaceae\u003c/em\u003e, and \u003cem\u003eFaecalibacterium\u003c/em\u003e 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 \u003cem\u003eRuminococcaceae\u003c/em\u003e 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 \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. They suggested that distinct abundances of short-chain fatty acids (SCFA)-producing bacteria including genus \u003cem\u003eRikenellaceae RC9 gut group\u003c/em\u003e, family \u003cem\u003eRuminococcaceae\u003c/em\u003e, genus \u003cem\u003eRuminococcaceae UCG005\u003c/em\u003e, genus \u003cem\u003eEubacterium ruminantium\u003c/em\u003e, and family \u003cem\u003eClostridiales vadinBB60 group\u003c/em\u003e were acting as the protective factors in endometriosis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince published studies suggested that enterohepatic recirculation affected systemic estrogen levels and could ultimately lead to reproductive diseases [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], 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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), 2-methoxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and 2-hydroxyestrone-3-methyl ether (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) in the vaginal samples and higher levels of 16α-hydroxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032) in the urine samples of the ovarian endometriosis group. Similar to previous research [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], 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.\u003c/p\u003e \u003cp\u003eAs 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 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eWhile 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBy 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3707%;\"\u003e\n \u003cp\u003egmGUS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84.6293%;\"\u003e\n \u003cp\u003eGut microbial \u0026beta;-glucuronidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3707%;\"\u003e\n \u003cp\u003eGnRH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84.6293%;\"\u003e\n \u003cp\u003eGonadotropin-Releasing Hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3707%;\"\u003e\n \u003cp\u003eLDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84.6293%;\"\u003e\n \u003cp\u003eLinear discriminant analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3707%;\"\u003e\n \u003cp\u003eLEfSe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84.6293%;\"\u003e\n \u003cp\u003eLinear Discriminant Analysis Effect Size\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3707%;\"\u003e\n \u003cp\u003eOTU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84.6293%;\"\u003e\n \u003cp\u003eOperational Taxonomic Unit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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\u0026mdash;original draft preparation, C.-Y.H \u0026nbsp;and A.H.-Y.P.; writing\u0026mdash;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research works was supported in part under Chang Gung Memorial Hospital Research Grant (CMRPG3G1981, CMRPG3K0782).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed 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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan City 333423, Taiwan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e College of Medicine, Chang Gung University, Taoyuan City, Taiwan\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTurnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. 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Gut Microbes. 2016;7(6):455-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"gut microbiota, vaginal microbiota, ovarian endometriosis, dysbiosis, β-glucuronidase, 16S ribosomal-RNA gene, estrogen metabolites","lastPublishedDoi":"10.21203/rs.3.rs-4975125/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4975125/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA 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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWhile 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 \u003cem\u003eRothia\u003c/em\u003e genus whereas genera such as \u003cem\u003eMegamonas\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ecoprostanoligenes_group\u003c/em\u003e, \u003cem\u003eAllisonella\u003c/em\u003e, \u003cem\u003eRuminiclostridium_5\u003c/em\u003e, [\u003cem\u003eEubacterium\u003c/em\u003e] \u003cem\u003ehallii_group\u003c/em\u003e, and \u003cem\u003eNegativibacillus\u003c/em\u003e were significantly more abundant in the ovarian endometriosis group. Meanwhile, besides statistically lower folds of 4-methoxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), 2-methoxyestrone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and 2-hydroxyestrone-3-methyl ether (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006), vaginal samples of patients with endometriosis also revealed significantly lower bacterial abundance, diversity, richness, and evenness.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWhile 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.\u003c/p\u003e","manuscriptTitle":"The Endobiota-estrobolome Study in Reproductive aged Women with Ovarian Endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-17 09:13:20","doi":"10.21203/rs.3.rs-4975125/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f332e757-9533-4811-9dd7-7c931ea0b016","owner":[],"postedDate":"October 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-07T07:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-17 09:13:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4975125","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4975125","identity":"rs-4975125","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}