P-315 Microbiota in endometriosis: culturomics analysis in receptive-phase endometrium
Authors: Canha-Gouveia A , Mändar R , Rööp T , Kõljalg S , Smidt I , Sepp E , Sola Leyva A , Tenorio C  M , Pérez-Prieto I , Vargas E , Molina N , Fontes J , Molina M , Salumets A , Altmäe S 
Published: July 2024

Abstract

Study question
Does the endometrium possess alive, culturable bacteria, and are there any microbes associated with endometriosis?

Summary answer
Thirty-five cultured bacterial species were identified in the receptive-phase endometrium, and an endometriosis-specific bacterial profile was detected.

What is known already
Endometriosis is a chronic inflammatory disease and one of the most prevalent female reproductive disorders, but its etiology remains unknown. Recently, significant attention has been directed towards studying the endometrial microbiome and its potential correlation with endometriosis. These analyses typically employ 16S rRNA gene sequencing, which detects bacterial genera but does not distinguish whether the bacteria are alive and hasn’t provided conclusive results regarding endometrial microbiome composition. Our aim is to develop a culture-based method for identifying live microbes in the endometrium and identifying bacterial species associated with endometriosis.

Study design, size, duration
This pilot cross-sectional study recruited twenty women from couples prior to undergoing infertility treatment at the Reproductive Unit of Universitary Hospital Virgen de las Nieves (Granada) between 2020 and 2022. Ten women were diagnosed with active endometriosis (no treatment), while ten women had male factor infertility, serving as the control group in our study.

Participants/materials, setting, methods
Endometrial biopsies were obtained during the mid-secretory phase, 7-9 days after ovulation (assessed with a urinary LH test), using an endometrial curette device. The biopsies were snap-frozen and stored at -80 °C until analysis. Microbiota cultures were conducted for up to 30 days using nine different media under aerobic, microaerobic, and anaerobic conditions. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) was utilized for bacterial isolate identification.

Main results and the role of chance
In total, 19 out of 20 patients (95%) tested positive for one or more microbes, with only 1 patient (5%) showing no microbial growth. We detected thirty-five bacterial species in these mid-secretory endometrial samples. Among them, fourteen bacterial species were common in both endometriosis and control samples, while five species—Bifidobacterium bifidum, Bifidobacterium breve, Cutibacterium avidum, Lactobacillus vaginalis, and Staphylococcus warneri—were exclusively cultured from endometriosis samples. Conversely, healthy samples harbored fourteen exclusive species, including Actinomyces neuii, Corynebacterium amycolatum, Enterococcus faecalis, and Finegoldia magna, among others. Control endometrial samples, altogether, possessed significantly more bacterial species than endometriosis samples (30 vs. 19 microbial species). These findings are consistent with the initial culturomics analyses of endometrial microbiota, highlighting the method’s efficacy in identifying isolates at the species level. Notably, species such as Actinotignum schaalii, Bifidobacterium dentium, Atopobium vaginae, and Rothia dentocariosa were identified in our samples, marking the first description of these species in endometrial culturomics studies. Among all participants, the bacteria with the highest colony forming units were Cutibacterium acnes, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus iners. Our study confirms the presence of the endometrial microbiota and suggests microbial dysregulation in endometriosis.

Limitations, reasons for caution
This is a pilot study with a small sample size, requiring further investigation with a larger cohort. The developed protocol analyzed frozen samples, which may have failed to detect some of the fragile microbial species.

Wider implications of the findings
The identification of live culturable bacteria through culturomics in endometrial biopsies offers a reliable method for detecting endometritis-associated bacteria. Utilizing culturomics for endometrial microbiota testing shows promise in uncovering specific bacteria associated with gynecological disorders like endometriosis, and it enhances our understanding of potential pathological processes.

Trial registration number
not applicable