Microbiome in women with endometriosis and the in vitro effects of Lactobacillus reuteri on human endometrium

Jae Hoon Lee, Gee Soo Jung, Kyungmin Kim, Hyemin Park, Yunjeong Park, Inha Lee, Min Jung Lee, Ji-Ho Lee, Young Sik Choi, SiHyun Cho
Microbiology spectrum · 2026 · vol. 14(6) , pp. e0368925 · doi:10.1128/spectrum.03689-25 · PMID:42089668 · PMC13228028
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⚙ AI-generated summary by claude@2026-06, 2026-06-13 ⓘ

This study characterized the microbiome in women with and without endometriosis and found that Lactobacillus reuteri altered apoptosis and inflammation markers in endometrial cells in vitro under estrogenic conditions.

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⚙ AI-generated deep summary by claude@2026-06, 2026-06-12 · read from full text ⓘ

This study profiled the vaginal, endometrium (EM), and peritoneal microbiome in women with endometriosis (n=27) versus controls (n=14) using next-generation sequencing with differential abundance analyses, and it also performed in vitro experiments using primary endometriosis-derived endometrial stromal/EM cells co-cultured with Lactobacillus reuteri. Differential taxa were reported in multiple sites, including enriched species in the endometriosis group and validation by LEfSe and ANCOM-BC, and the in vitro work assessed L. reuteri effects on endometriosis-related gene expression and estrogen metabolism, including conditions with estradiol-17-glucuronide (E2G) and estrogen metabolite measurements by LC-MS/MS. The paper’s key limitation, as implied by its design, is that microbiome associations are cross-sectional and the mechanistic findings are confined to cell co-culture rather than in vivo physiology. This paper is centrally about endometriosis — it links altered multi-site microbiome profiles with in vitro effects of Lactobacillus reuteri on endometriosis-derived human endometrium cells and estrogen-related pathways.

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Abstract

UNLABELLED: Endometriosis (EMS) is a chronic inflammatory disorder affecting ~10% of reproductive-age women, with increasing evidence implicating the microbiome in its pathogenesis through immunomodulation and estrogen metabolism. This study investigated microbiome composition in the vagina, endometrium, and peritoneal fluid (PF) of women with and without EMS and further assessed the effects of Lactobacillus reuteri (L. reuteri) on endometrial (EM) cells in vitro. Samples from 41 patients were analyzed using 16S rRNA gene sequencing, targeting the V3-V4 regions. Western blotting, ELISA, and LC-MS/MS were employed to evaluate protein expression and estrogen metabolism during EM-L. reuteri co-culture with or without estradiol-17-glucuronide (E2G). Microbiome analysis revealed no significant differences in alpha or beta diversity between EMS and controls across all compartments. However, LEfSe analysis identified several taxa with differential abundance, with L. reuteri consistently altered in both vagina and EM. Across the menstrual cycle, EM and vaginal microbiomes were stable, whereas PF microbiota showed phase-dependent variation involving 60 genera and 76 species. In vitro, L. reuteri alone did not alter endometriosis-related proteins, but in the presence of E2G, it reduced BAX/Bcl-2 ratios and increased p-NF-κB, suggesting anti-apoptotic and pro-inflammatory shifts. Progesterone receptor α/β expression decreased, while estrogen receptor levels remained unchanged. L. reuteri increased β-glucuronidase activity but did not enhance E2G-to-estradiol conversion. These findings highlight L. reuteri as a potentially important species in EMS, with in vitro evidence suggesting survival-promoting effects under estrogenic conditions. Further research should explore multi-species interactions and hormonal contexts to clarify microbial contributions to EMS pathogenesis. IMPORTANCE: Although Lactobacillus reuteri appeared more abundant in the vagina and endometrium of controls, suggesting a protective role, in vitro findings paradoxically indicated anti-apoptotic and pro-inflammatory effects under estrogenic conditions, underscoring the need for further investigation of multi-species microbial interactions and hormonal contexts in endometriosis pathogenesis.
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Abstract

IMPORTANCE

Introduction

MATERIALS AND METHODS Microbiome analysis using next-generation sequencing (NGS) in women with endometriosis: study population and sample collection DNA extraction Library construction and sequencing Operational taxonomic unit (OTU) analysis ASV generation and taxonomic assignment ANCOM-BC analysis Isolation and primary culture of EM cells In vitro co-culture of EM cells with L. reuteri: CCK-8 assay Addition of estradiol-17-glucuronide to in vitro co-culture of EM cells with L. reuteri Protein isolation and western blot ELISA-based quantification of β-glucuronidase Measurement of estrogen metabolites using LC-MS/MS system: extraction of estrogens and their glucuronide conjugates Instrumental analysis utilizing LC-MS/MS system Statistical analysis

Results

NGS-based profiling of the vaginal, EM, and peritoneal microbiome in EMS | Endometriosis group (n = 27) | Control group (n = 14) | P value | | |---|---|---|---| | Age, years | 32.4 ± 4.4 | 31.7 ± 4.6 | 0.413 | | Height, cm | 161.7 ± 4.5 | 160.8 ± 4.2 | 0.479 | | Weight, kg | 54.1 ± 7.2 | 53.1 ± 6.2 | 0.321 | | Size of endometrioma, cm | 5.1 ± 3.1 | || | Revised ASRM classification, N (%) | ||| | I, II | 1 (3.70) | || | III | 16 (59.26) | || | IV | 10 (37.04) | || | *Revised ASRM classification total score | 29 (25–108) | | Site | Species | Enriched group | log2FC EMS/control | P value | |---|---|---|---|---| | Vagina | Dialister micraerophilus | EMS | 6.280005911 | 0.0089 | | Porphyromonas bennonis | EMS | 6.61608256 | 0.034 | | | Lactobacillus iners | Control | −1.268865256 | 0.015 | | | Lactobacillus reuteri | Control | −1.609898408 | 0.0085 | | | Endometrium | Acidibrevibacterium fodinaquatile | Control | −6.995873477 | 0.047 | | Actinomadura rifamycini | Control | −6.830759741 | 0.047 | | | Aeromicrobium panaciterrae | Control | −8.246618483 | 0.047 | | | Afifella pfennigii | Control | −8.46598842 | 0.047 | | | Altererythrobacter rigui | Control | −5.327575631 | 0.047 | | | Arenimonas subflava | Control | −7.183327958 | 0.047 | | | Campylobacter ureolyticus | EMS | 10.70206056 | 0.041 | | | Chryseobacterium greenlandense | Control | −8.134212895 | 0.0033 | | | Corynebacterium tuberculostearicum | EMS | 4.78902972 | 0.02 | | | Desulfonatronum alkalitolerans | Control | −7.346036343 | 0.047 | | | Desulfovibrio piger | Control | −6.574749807 | 0.047 | | | Dongia mobilis | Control | −8.554760688 | 0.047 | | | Edaphobacter dinghuensis | Control | −4.773795323 | 0.047 | | | Howardella ureilytica | EMS | 5.355473853 | 0.026 | | | Insolitispirillum peregrinum | Control | −6.620247319 | 0.047 | | | Lactobacillus reuteri | Control | −0.540402898 | 0.0061 | | | Lysobacter dokdonensis | Control | −6.157156594 | 0.047 | | | Lysobacter niabensis | Control | −6.407144959 | 0.047 | | | Mycobacterium marinum | Control | −5.969488254 | 0.047 | | | Natranaerovirga pectinivora | Control | −5.232825367 | 0.047 | | | Nocardioides alpinus | Control | −7.257245755 | 0.047 | | | Pelobacter carbinolicus | Control | −5.337971042 | 0.047 | | | Povalibacter uvarum | Control | −6.798125238 | 0.047 | | | Prevotella timonensis | EMS | 6.799906456 | 0.018 | | | Pseudoduganella violaceinigra | Control | −7.489881158 | 0.047 | | | Pseudoflavonifractor capillosus | Control | −10.11202371 | 0.047 | | | Pseudoxanthomonas wuyuanensis | Control | −7.55361836 | 0.047 | | | Rhodanobacter glycinis | Control | −8.086466305 | 0.013 | | | Smithella propionica | Control | −8.229049939 | 0.047 | | | Sphingobium mellinum | Control | −7.384932352 | 0.047 | | | Streptomyces aomiensis | Control | −3.495528313 | 0.047 | | | Streptomyces chlorus | Control | −8.161137516 | 0.047 | | | Peritoneal fluid | Thauera chlorobenzoica | EMS | 8.052636498 | 0.026 | Validation of differentially abundant taxa using LEfSe and ANCOM-BC In vitro effects of L. reuteri on EM cells: determination of optimal co-culture conditions Modulation of EMS-related gene expression by L. reuteri and E2G in EM cells Effects of L. reuteri and E2G on estrogen pathways in EM cells Receptor expression Estrogen metabolism | E2G | Estradiol | Estradiol/E2G | | |---|---|---|---| | Control | ||| | L. reuteri | ||| | L. reuteri + E2G | 0.36 (0.02) | 3.32 (2.21) | 9.11 (5.75) | | E2G | 0.35 (0.08) | 2.97 (2.06) | 8.68 (5.63) | | P value | 0.009 | 0.656 | 0.837 |

Discussion

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Outcome instruments

rASRM

Condition tags

endometriosis

MeSH descriptors

Endometriosis Endometriosis Endometriosis Endometriosis Endometriosis Endometrium Endometrium Endometrium Endometrium Endometrium Limosilactobacillus reuteri Limosilactobacillus reuteri Limosilactobacillus reuteri Limosilactobacillus reuteri Limosilactobacillus reuteri Microbiota Microbiota Microbiota Microbiota Adult

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