{"paper_id":"ed31a04f-2160-404b-84cb-88f84d4c2821","body_text":"Microbiome in women with endometriosis and the in vitro effects of Lactobacillus reuteri on human endometrium\nABSTRACT\nIMPORTANCE\nINTRODUCTION\nMATERIALS AND METHODS\nMicrobiome analysis using next-generation sequencing (NGS) in women with endometriosis: study population and sample collection\nDNA extraction\nLibrary construction and sequencing\nOperational taxonomic unit (OTU) analysis\nASV generation and taxonomic assignment\nANCOM-BC analysis\nIsolation and primary culture of EM cells\nIn vitro co-culture of EM cells with L. reuteri: CCK-8 assay\nAddition of estradiol-17-glucuronide to in vitro co-culture of EM cells with L. reuteri\nProtein isolation and western blot\nELISA-based quantification of β-glucuronidase\nMeasurement of estrogen metabolites using LC-MS/MS system: extraction of estrogens and their glucuronide conjugates\nInstrumental analysis utilizing LC-MS/MS system\nStatistical analysis\nRESULTS\nNGS-based profiling of the vaginal, EM, and peritoneal microbiome in EMS\n| Endometriosis group (n = 27) | Control group (n = 14) | P value | |\n|---|---|---|---|\n| Age, years | 32.4 ± 4.4 | 31.7 ± 4.6 | 0.413 |\n| Height, cm | 161.7 ± 4.5 | 160.8 ± 4.2 | 0.479 |\n| Weight, kg | 54.1 ± 7.2 | 53.1 ± 6.2 | 0.321 |\n| Size of endometrioma, cm | 5.1 ± 3.1 | ||\n| Revised ASRM classification, N (%) | |||\n| I, II | 1 (3.70) | ||\n| III | 16 (59.26) | ||\n| IV | 10 (37.04) | ||\n| *Revised ASRM classification total score | 29 (25–108) |\n| Site | Species | Enriched group | log2FC EMS/control | P value |\n|---|---|---|---|---|\n| Vagina | Dialister micraerophilus | EMS | 6.280005911 | 0.0089 |\n| Porphyromonas bennonis | EMS | 6.61608256 | 0.034 | |\n| Lactobacillus iners | Control | −1.268865256 | 0.015 | |\n| Lactobacillus reuteri | Control | −1.609898408 | 0.0085 | |\n| Endometrium | Acidibrevibacterium fodinaquatile | Control | −6.995873477 | 0.047 |\n| Actinomadura rifamycini | Control | −6.830759741 | 0.047 | |\n| Aeromicrobium panaciterrae | Control | −8.246618483 | 0.047 | |\n| Afifella pfennigii | Control | −8.46598842 | 0.047 | |\n| Altererythrobacter rigui | Control | −5.327575631 | 0.047 | |\n| Arenimonas subflava | Control | −7.183327958 | 0.047 | |\n| Campylobacter ureolyticus | EMS | 10.70206056 | 0.041 | |\n| Chryseobacterium greenlandense | Control | −8.134212895 | 0.0033 | |\n| Corynebacterium tuberculostearicum | EMS | 4.78902972 | 0.02 | |\n| Desulfonatronum alkalitolerans | Control | −7.346036343 | 0.047 | |\n| Desulfovibrio piger | Control | −6.574749807 | 0.047 | |\n| Dongia mobilis | Control | −8.554760688 | 0.047 | |\n| Edaphobacter dinghuensis | Control | −4.773795323 | 0.047 | |\n| Howardella ureilytica | EMS | 5.355473853 | 0.026 | |\n| Insolitispirillum peregrinum | Control | −6.620247319 | 0.047 | |\n| Lactobacillus reuteri | Control | −0.540402898 | 0.0061 | |\n| Lysobacter dokdonensis | Control | −6.157156594 | 0.047 | |\n| Lysobacter niabensis | Control | −6.407144959 | 0.047 | |\n| Mycobacterium marinum | Control | −5.969488254 | 0.047 | |\n| Natranaerovirga pectinivora | Control | −5.232825367 | 0.047 | |\n| Nocardioides alpinus | Control | −7.257245755 | 0.047 | |\n| Pelobacter carbinolicus | Control | −5.337971042 | 0.047 | |\n| Povalibacter uvarum | Control | −6.798125238 | 0.047 | |\n| Prevotella timonensis | EMS | 6.799906456 | 0.018 | |\n| Pseudoduganella violaceinigra | Control | −7.489881158 | 0.047 | |\n| Pseudoflavonifractor capillosus | Control | −10.11202371 | 0.047 | |\n| Pseudoxanthomonas wuyuanensis | Control | −7.55361836 | 0.047 | |\n| Rhodanobacter glycinis | Control | −8.086466305 | 0.013 | |\n| Smithella propionica | Control | −8.229049939 | 0.047 | |\n| Sphingobium mellinum | Control | −7.384932352 | 0.047 | |\n| Streptomyces aomiensis | Control | −3.495528313 | 0.047 | |\n| Streptomyces chlorus | Control | −8.161137516 | 0.047 | |\n| Peritoneal fluid | Thauera chlorobenzoica | EMS | 8.052636498 | 0.026 |\nValidation of differentially abundant taxa using LEfSe and ANCOM-BC\nIn vitro effects of L. reuteri on EM cells: determination of optimal co-culture conditions\nModulation of EMS-related gene expression by L. reuteri and E2G in EM cells\nEffects of L. reuteri and E2G on estrogen pathways in EM cells\nReceptor expression\nEstrogen metabolism\n| E2G | Estradiol | Estradiol/E2G | |\n|---|---|---|---|\n| Control | |||\n| L. reuteri | |||\n| L. reuteri + E2G | 0.36 (0.02) | 3.32 (2.21) | 9.11 (5.75) |\n| E2G | 0.35 (0.08) | 2.97 (2.06) | 8.68 (5.63) |\n| P value | 0.009 | 0.656 | 0.837 |\nDISCUSSION\nConclusion\nACKNOWLEDGMENTS\nSUPPLEMENTAL MATERIAL\n- Download\n- 3.81 MB\n- Download\n- 3.76 MB\n- Download\n- 3.16 MB\n- Download\n- 856.97 KB\n- Download\n- 334.42 KB\n- Download\n- 409.93 KB\n- Download\n- 728.45 KB\n- Download\n- 266.01 KB\n- Download\n- 20.81 KB\nDATA AVAILABILITY\nETHICS APPROVAL\nREFERENCES\nInformation & 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