Endometrial Microbiota Research Articles

Altered Endometrial Microbiota Profile Is Associated With Poor Endometrial Receptivity of Repeated Implantation Failure.

To gain insight into the endometrial pathophysiology of unexplained repeated implantation failure (RIF), we examined the characteristics of genital tract microbiota and explored the correlation between the microbiota and endometrial receptivity. Vaginal secretion (VS) and endometrial biopsy (EB) samples were collected from patients with RIF (RIF group, n=32) and those with infertility who had achieved pregnancy during their initial embryo transfer cycle (control group, n=18). 16S ribosomal RNA sequencing and quantitative PCR were performed to characterize the microbiota of the two groups. Spearman's correlation analysis was performed to determine the relationship between endometrial receptivity markers and endometrial microbiota. Endometrial microbiota exhibited distinct characteristics from vaginal microbiota, with a higher alpha-diversity. Alpha-diversity of the endometrial microbiota was higher in the RIF group than in the control group. Compared with the control group, the RIF group had a significant decrease in endometrial Lactobacillus abundance and an increase in Gardnerella and Acinetobacter abundances. The expression levels of endometrial receptivity markers, including homeobox A11, integrin αvβ3, leukemia inhibitor factor, matrix metalloproteinase-9, and vascular endothelial growth factor, were lower in the RIF group than in the control group. Moreover, the expression levels of these markers were correlated with endometrial Lactobacillus, Gardnerella, and Acinetobacter abundances. RIF is characterized by endometrial microbiota dysbiosis and poor endometrial receptivity. Moreover, abnormal endometrial microbiota is associated with impaired endometrial receptivity, which may be a potential cause of unexplained RIF.

Microbiota and Recurrent Pregnancy Loss (RPL); More than a Simple Connection.

Recurrent Pregnancy Loss (RPL) affects 1-2% of women, and its triggering factors are unclear. Several studies have shown that the vaginal, endometrial, and gut microbiota may play a role in RPL. A decrease in the quantity of Lactobacillus crispatus in local microbiota has been associated with an increase in local (vaginal and endometrial) inflammatory response and immune cell activation that leads to pregnancy loss. The inflammatory response may be triggered by gram-negative bacteria, lipopolysaccharides (LPS), viral infections, mycosis, or atypia (tumor growth). Bacterial structures and metabolites produced by microbiota could be involved in immune cell modulation and may be responsible for immune cell activation and molecular mimicry. Gut microbiota metabolic products may increase the amount of circulating pro-inflammatory lymphocytes, which, in turn, will migrate into vaginal or endometrial tissues. Local pro-inflammatory Th1 and Th17 subpopulations and a decrease in local Treg and tolerogenic NK cells are accountable for the increase in pregnancy loss. Local microbiota may modulate the local inflammatory response, increasing pregnancy success. Analyzing local and gut microbiota may be necessary to characterize some RPL patients. Although oral supplementation of probiotics has not been shown to modify vaginal or endometrial microbiota, the metabolites produced by it may benefit patients. Lactobacillus crispatus transplantation into the vagina may enhance the required immune tolerogenic response to achieve a normal pregnancy. The effect of hormone stimulation and progesterone to maintain early pregnancy on microbiota has not been adequately studied, and more research is needed in this area. Well-designed clinical trials are required to ascertain the benefit of microbiota modulation in RPL.

P-293 Influence of the endometrial microbiota composition on the clinical outcomes of egg donation PGT-A cycles with controlled endometrial receptivity

Abstract Study question Does the endometrial microbiota composition and diversity influence embryo implantation and pregnancy evolution in assisted reproduction treatment (ART) cycles? Summary answer Endometrial microbiota composition influences ART success. Detection of endometrial pathogens is associated with poor outcomes whereas sterile endometria show the highest pregnancy and live-birth rates What is known already The endometrial microbiota has emerged as a possible influencing factor in the results of ART. Recent research suggests its impact on female reproductive health, particularly in patients with chronic endometritis (CE), where the presence of microbial pathogens associated with this condition has been linked to infertility. In turn, the bioregulatory role of species of Lactobacillus in this tissue has been explored, although its essentiality or benefit for female reproductive function continues to be topics of debate. Study design, size, duration This is a case-control retrospective study analysing for the first time the influence of the endometrial microbiota composition on the clinical outcomes of 220 women undergoing egg donation cycles with Preimplantation Genetic Testing for Aneuploidy (PGT-A) and controlled endometrial receptivity between March 2016 and April 2021 in IVF-Life Alicante (Spain). ART results after the transfer of a single euploid blastocyst in the WOI of patients with different endometrial microbiota profiles were analysed and compared. Participants/materials, setting, methods Presence of 18 microbial CE pathogens and 4 Lactobacillus species was determined by high-throughput qPCR (MicrobioMap®, iGLS) in endometrial samples obtained during the WOI of a hormone replacement therapy cycle. In all cases, PGT-A by next generation sequencing (NGS) (EmbryoMap, Vitrolife) and study of endometrial receptivity by gene expression analysis (ER Map, iGLS) were performed. Clinical outcomes (β-hCG+, β+ and live birth, LB) in relation to the endometrial microbiota profile were analysed and compared. Main results and the role of chance In the study, 31.8% of patientś endometria were identified without microbial detection, 40% exclusively with Lactobacillus, 13.2% with pathogens only, and 15% with both. Clinical results revealed significant differences in β+ rates among the groups (p < 0.05). The absence of microorganisms was associated with higher success rates (β + 72.9%), while the presence of pathogens significantly reduced pregnancy success (β + 41.4%). A similar trend was observed in LB rates, although it did not reach statistical significance (44.3% and 24.1%, respectively). The presence of Lactobacillus alongside pathogens improved clinical outcomes (β + 57.6% LB 48.5%). Interestingly, exclusive presence of Lactobacillus showed lower implantation rates than patients without microbial detection (β + 58%). These findings highlight the impact of microbial composition on the clinical outcomes of ART cycles, underlining the association between endometrial microbiota patterns and pregnancy success. Limitations, reasons for caution This study is constrained by its retrospective nature, emphasizing the need for further validation through randomized controlled trials, non-selection studies, or other investigative approaches to confirm the extent of any clinical benefits. Endometrial biopsies were obtained transcervically, while executed with utmost care, risk of contamination cannot be completely excluded. Wider implications of the findings The composition and diversity of the endometrial microbiota is key for ART success. A sterile endometrial environment offers optimal conditions for implantation and pregnancy. The study highlights the potential of Lactobacillus in counteracting the negative impact of pathogenic presence. Trial registration number not applicable

P-307 Evaluation of pregnancy rates post-probiotic treatment in patients with varied Lactobacillus species in pre-transfer endometrial microbiome analysis

Abstract Study question Are there differences in clinical outcomes based on the predominant species of Lactobacillus in the endometrium of patients treated with probiotics? Summary answer No discernible variations in clinical outcomes were noted based on the Lactobacillus species identified in the pre-transfer microbiome analysis after personalized probiotic treatment. What is known already Lactobacillus is the genus with the greatest presence in the female reproductive tract. They are responsible for acidifying the environment and play an important role in the prevention of excessive proliferation of pathogens through different mechanisms (competitive inhibition or production of antimicrobial compounds). Among the species of Lactobacillus, crispatus, iners, jensenii and gasseri are the most frequent in the endometrium. Lactobacillus species share some genus-specific characteristics. However, there are differences in the production of antimicrobial compounds among the species: L. iners has a lower production of hydrogen peroxide and lower anti-inflammatory capacity and therefore has lower protective capacity against pathogens. Study design, size, duration A retrospective study analyzed with 209 endometrial samples from 164 patients with previous implantation failure or miscarriage (average age= 41.47-/+4.66 years). Patients underwent endometrial microbiota testing between May 2021 and December 2022. Following analysis, patients received personalized probiotic treatment based on results, particularly tailored for L. iners overgrowth. One hundred and thirty-five patients underwent an embryo transfer after completing the treatment. Participants/materials, setting, methods DNA from endometrial biopsies was analyzed to detect bacteria, fungi and viruses. To evaluate the potential impact of L. iners presence, clinical outcomes were calculated across patient groups with varying percentages of this Lactobacillus species. These rates were compared to those of L. crispatus, known as the most favourable species. Furthermore, the analysis subdivided the detected percentages of each species into differente ranges to discern potential variations in clinical outcomes based on the percentage levels. Main results and the role of chance The overall implantation rate following probiotic and antibiotic treatment (as needed) was 52.59%, with a clinical pregnancy rate of 46.67%. Notably, no statistically significant differences were observed in between patients with a dominant Lactobacillus microbiome (>90%) and those without prior to the treatments. These findings were anticipated, given the probiotic treatment’s inclusion of Lactobacillus strains, which foster an acidic environment conducive to the establishment of endometrial Lactobacillus species. Interestingly, regardless of the percentage of L. crispatus detected in the endometrial biopsy prior to probiotic treatment, implantation rates remained consistently above 50%, with no statistically significant differences observed. Conversely, patients with a high percentage of L. iners (>75%) exhibited a lower implantation rate of 38%, though this disparity was not statistically significant compared to patients with a high percentage of L. crispatus. No statistically significant differences were observed in the clinical outcomes comparing L. crispatus and L. iners. However, high % of L. crispatus was associated with a lower miscarriage rate compared to high % of L. iners (13%vs22%, respectively). This may be attributed to the treatment administered prior transfer, which aims to rebalance Lactobacillus composition, especially in cases of L. iners dominance. Limitations, reasons for caution All patients included in this study underwent personalized probiotic treatment before embryo transfer, limiting the ability to directly assess the impact of L. iners on pregnancy outcomes. Additionally, increasing the number of patients would strengthen the study’s findings and confirm the observed findings. Wider implications of the findings Microbiota studies can effectively identify various species within the same genus (Lactobacillus spp). Personalized probiotic treatment appeared to balance endometrial microbiota, not only increasing the percentage of Lactobacillus in patients with a non- dominant Lactobacillus environment, but also modify the type of Lactobacillus species that are present in the endometrium. Trial registration number not applicable

P-315 Microbiota in endometriosis: culturomics analysis in receptive-phase endometrium

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

P-395 Vaginal probiotics and vitamin D, as immunomodulatory approaches in improving the implantation and live birth in repeated implantation failure (RIF) women with imbalanced immune system

Abstract Study question Are vaginal probiotics and vitamin D (Vit.D) capable of improving implantation and live birth rate in RIF women with imbalanced immune system? Summary answer Vaginal probiotics and Vit.D are capable of improving the rate of implantation and live birth in RIF women with imbalanced immune system. What is known already RIF, determined as failure in achieving pregnancy after at least three in-vitro fertilization (IVF), is related to various factors including maternal and embryonic factors. Any abnormalities in maternal or embryonic part, such as maternal imbalanced immune profile, pose a risk for implantation. It has been estimated that endometrial microbiota and Vit.D would affect immune profile of endometrium and consequently, the process of implantation. Considering the effects of probiotics in normalizing the endometrium microbiota, it is likely that probiotics besides Vit.D regulate the imbalance of immune system in endometrium of RIF patients, to improve the rate of implantation and live birth. Study design, size, duration 148 women with a history of at least 3 consecutive implantation failures (from Feb 2021- Sep 2023), who referred to infertility centers of Valiasr International Hospital of Tabriz, were enrolled. Blood samples were taken, T helper 1 (Th1)/Th2 ratio (CD4+ interferon-γ (IFNγ)+/CD4+ interleukin-4 (IL-4)+ ratio) was evaluated by flowcytometry. Pregnancy (by the positive β- human chorionic gonadotropin (HCG) test and ultrasound) and live birth rate were also determined. Participants/materials, setting, methods The group of RIF women with elevated Th1/Th2 ratio (n = 65) was divided into 2 subgroups. The study group (n = 41) received probiotics (two capsules vaginally, every day for 6 months) and Vit.D (50,000 international unit (IU) every week for 10 weeks prior to the transfer). The control group (n = 24) received no treatment. At the end of the treatment, the embryo transfer was conducted. Finally, the pregnancy and live birth rate was evaluated. Main results and the role of chance Evaluation of pregnancy rate among the groups indicated that RIF women who received probiotics and Vit.D had a significantly higher rate of pregnancy (20/41, 48%) in comparison with no-treatment group (5/24, 20%). Live birth rate was also significantly higher in probiotics- and Vit.D-receiving RIF women (18/41, 43%) than no treatment group (3/24, 12%). Limitations, reasons for caution Lack of evaluation of endometrium microbiota, Vit.D level and local immune system profile prior to the study are the limitations of the current study. Wider implications of the findings The crucial role of balanced immune profile and presence of normal microbiota in the endometrium is undeniable for a successful implantation and pregnancy. Providing an optimum microbial microenvironment by probiotics and a favorable immunologic balance with Vit.D, as an immunomodulator, improves the implantation and pregnancy outcome of RIF patients. Trial registration number not applicable

P-276 Endometrial microbiome diversity and the local macrophages populations during the mid-secretory phase

Abstract Study question Are there any quantitative associations between endometrial macrophages populations and microorganisms’ diversity? Summary answer The quantities and spatial distribution of endometrial macrophages populations correlated significantly with several microbiome diversity indices. What is known already Numerous studies have linked the relative abundance of Lactobacilli with a receptive endometrium, while the presence of multiple other bacterial species has been implicated in implantation failure. Another determinant of endometrial receptivity is the local immune cell composition and its inflammatory status. Knowing that the microbiome affects the immune system, it is worth studying the potential interplay between bacterial composition and immune cell populations in the endometrium during its receptive state. The present study aimed to investigate associations between the quantity and distribution of endometrial macrophages populations and the local microbiome diversity during the mid-secretory phase of the menstrual cycle. Study design, size, duration This prospective observational study was conducted between April 2021 and January 2023. Endometrial biopsies were collected from 25 female patients of reproductive age on day 7 after luteinising hormone surge. Eligibility criteria included no endometrial pathologies, no history of recent or chronic inflammatory or infectious disease, no recent antibiotic treatment, and no endocrinological, autoimmune or oncological conditions. Participants/materials, setting, methods Half of each biopsy was used for immunohistochemistry with CD11c + (Proteintech) and CD163 + (Quartett) to reveal pro- and anti- inflammatory macrophages, respectively. Their quantities and spatial distribution were analysed by HALO® image analysis software. The remaining endometrial tissue underwent DNA isolation and subsequent 16S rRNA (v4-v5 region) gene sequencing to determine endometrial microbiota composition. Alpha diversity indices - Good’s coverage, Shannon and phylogenic diversity - were evaluated and tested for correlation with the macrophages metrics. Main results and the role of chance The observed median percentage and range of the studied macrophages populations in the endometrial stroma were: 1.16% (2.98) CD11c+ macrophages and 1.96% (4.48) CD163+ macrophages. The evaluated alpha diversity indices were: 0.99±0.01 Good’s coverage, 2.53±1.20 Shannon and 38.56±15.43 phylogenic diversity.The CD11c+ macrophages percentage was not related to the microbial diversity of the studied samples. The percentage of the CD163+ macrophages, however, showed significant correlation with the Good’s coverage alpha diversity index (R = 0.397, p = 0.04). The median distance between CD11c+ and CD163+ cells in the endometrial stroma was 63.7µm (108.36) and it was significantly related to the Shannon alpha diversity index (R=-0.347, p = 0.05) and phylogenic diversity whole tree index (R=-0.367, p = 0.05).In summary, the anti-inflammatory macrophages percentage in the mid-secretory endometrial stroma is positively related to the local microbial diversity. Moreover, the distances between the pro- and anti-inflammatory macrophages populations in the endometrium are negatively associated to Shannon and PD whole tree alpha diversity indices. Limitations, reasons for caution The results of this study should be confirmed in a larger study population. Wider implications of the findings This study’s findings indicate that there is a correlation of the endometrial macrophages quantity and stromal distribution with the endometrial microbiome diversity. The association between these determinants of endometrial receptivity could help in understanding the fine tuning of the endometrium for accepting the embryo. Trial registration number The current research was funded by the National Science Fund, Ministry of Education, Bulgaria, Contract № KP-06-N53/14/16.11.2021.

P-333 Combining deep shotgun metagenomic sequencing and culturomics for in-depth characterization of a potential endometrial microbiome

Abstract Study question Does the endometrium host a microbiome, and if so, are these taxa distinct from the vaginal/cervical microbiome? Summary answer Multifaceted analysis of endometrial tissue reveals no distinct microbial taxa compared to the vaginal/cervical samples, questioning whether detected microbiota are endometrium-related or result from cross-contamination. What is known already Recent studies suggest a role for the endometrial microbiota in reproductive health, yet the existence of an endometrial microbiome and its specific bacterial taxa remain enigmatic. The female reproductive tract, with a bacterial abundance gradient, is predominantly Lactobacillus-dominated in the lower region. Inconsistent findings on Lactobacillus dominance in endometrial samples are often ascribed to cross-contamination and methodological constraints, highlighting the need for precise methodologies in studying the low-biomass endometrial environment. Study design, size, duration Fifteen women, undergoing a diagnostic hysteroscopy and endometrial biopsy, as part of the subfertility work-up, were included in this study. Microbiota profiles of endometrial biopsies were compared to paired vaginal and cervical swabs from nine patients using 16S amplicon sequencing. Furthermore, the microbial composition of six additional endometrium biopsies was characterized using both a culturomics-based approach and deep metagenomic sequencing. Participants/materials, setting, methods Endometrial biopsies (n = 15) were taken with a sterile Pipelle de Cornier and snap-frozen in liquid nitrogen, followed by -80 °C storage and 16S amplicon sequencing (n = 9) targeting the V4-region or deep shotgun metagenomic sequencing (n = 6) (Prebiomics, Italy). Simultaneously, in nine women, paired cervical and vaginal swabs (Copan Diagnostics, Italy) were taken and preserved in eNAT medium. In six participants, an additional endometrial biopsy was collected and stored in Amies medium for immediate culturomics. Main results and the role of chance We observed a high similarity in the microbial composition of endometrial biopsies, cervical, and vaginal samples using 16 amplicon sequencing, but also some taxa were only found in the endometrial samples. Notably, the presence of Gemmobacter, a waterborne genus, was only found in endometrial biopsies and could be a source of contamination from water used during hysteroscopy to flush the uterus. Most endometrial microbiome profiles were Lactobacillus dominated, with exceptions observed in three patients. No significant differences for alpha diversity measures (richness and evenness) of the microbiota composition were observed between the different niches. For the first time we applied both culturomics and deep shotgun metagenomic sequencing for the characterization of the microbial composition in the same endometrial biopsies. Commonly identified genera using both techniques include Lactobacillus, Prevotella, Bifidobacterium, Anaerococcus, and Cuticubacterium. Remarkably, at the species level, the two approaches revealed almost no overlapping species within each biopsy, underscoring the importance of combining multiple methodologies in examining low-biomass niches like the endometrium. Limitations, reasons for caution A limitation of this study is the low-biomass nature of endometrial biopsies which are prone to cross-contamination with the lower female reproductive tract. This challenges downstream analysis, as it becomes difficult to distinguish between a relevant microbiological signal and contaminating noise. Wider implications of the findings This study emphasizes the need to consider a multifaceted microbiome approach when exploring low-biomass environments, like the endometrium. The critical consideration of potential cross-contamination by the lower female reproductive tract and its implications for downstream analysis is paramount in clinical microbiology research and demands meticulous handling. Trial registration number not applicable

Chronic endometritis and the endometrial microbiota: implications for reproductive success in patients with recurrent implantation failure

BackgroundChronic endometritis (CE) is associated with poor reproductive outcomes, yet the role of endometrial microbiota in patients with recurrent implantation failure (RIF) and CE remains unclear. This study aims to characterize endometrial microbiota in RIF patients with CE and assess its implications for reproductive outcomes.MethodsIn this prospective study, we enrolled RIF patients both with and without CE. Endometrial and cervical samples were collected for 16 S rRNA gene sequencing. Microbiota composition was compared between groups using diversity indices, phylum, and genus-level analysis. Canonical correlation analysis (CCA) and Spearman’s correlation coefficients were used to assess relationships between CE, reproductive outcomes, and microbiota. Predictive functional profiling was performed to evaluate metabolic pathways associated with CE.ResultsEndometrial microbiota in CE patients exhibited greater diversity and evenness compared to non-CE patients. Principal coordinates analysis (PCoA) revealed distinct clustering between CE and non-CE groups. Linear discriminant analysis (LDA) identified Proteobacteria, Aminicenantales, and Chloroflexaceae as characteristic of CE, while Lactobacillus, Acinetobacter, Herbaspirillum, Ralstonia, Shewanela, and Micrococcaceae were associated with non-CE. CCA demonstrated associations between CE, adverse reproductive outcomes, and specific bacterial taxa. Microbial metabolic pathways significantly differed between CE and non-CE groups, with enrichment in pathways related to cofactors, vitamins, secondary metabolites, and the immune system in CE patients.ConclusionRIF patients with CE exhibit distinct endometrial microbiota compositions associated with adverse reproductive outcomes. The increased microbial diversity and altered metabolic pathways in CE suggest a potential correlation with reproductive outcomes, although further studies are necessary to elucidate the causal relationship between microbiota alterations and fertility. Modulating the endometrial microbiome may represent a novel therapeutic strategy to improve IVF outcomes in patients with CE.

Endometrial Cancer: A Pilot Study of the Tissue Microbiota.

The endometrium remains a difficult tissue for the analysis of microbiota, mainly due to the low bacterial presence and the sampling procedures. Among its pathologies, endometrial cancer has not yet been completely investigated for its relationship with microbiota composition. In this work, we report on possible correlations between endometrial microbiota dysbiosis and endometrial cancer. Women with endometrial cancer at various stages of tumor progression were enrolled together with women with a benign polymyomatous uterus as the control. Analyses were performed using biopsies collected at two specific endometrial sites during the surgery. This study adopted two approaches: the absolute quantification of the bacterial load, using droplet digital PCR (ddPCR), and the analysis of the bacterial composition, using a deep metabarcoding NGS procedure. ddPCR provided the first-ever assessment of the absolute quantification of bacterial DNA in the endometrium, confirming a generally low microbial abundance. Metabarcoding analysis revealed a different microbiota distribution in the two endometrial sites, regardless of pathology, accompanied by an overall higher prevalence of pathogenic bacterial genera in cancerous tissues. These results pave the way for future studies aimed at identifying potential biomarkers and gaining a deeper understanding of the role of bacteria associated with tumors.

A Standard Pipeline for Analyzing the Endometrial Microbiome.

The endometrial microbiome is a rapidly advancing field of research, particularly in obstetrics and gynecology, as it has been found to be linked with obstetric complications and potential impacts on fertility. The diversity of microorganisms presents in the endometrium, along with their metabolites, can influence reproductive outcomes by modulating the local immune environment of the uterus. However, a major challenge in advancing our understanding of the endometrial microbiota lies in the heterogeneity of available studies, which vary in terms of patient selection, control groups, collection methods and analysis methodologies. In this study, we propose a detailed pipeline for endometrial microbiome analysis, based on the most comprehensive prospective of 64 studies that have investigated the endometrial microbiome up to the present. Additionally, our review suggests that a dominance of Lactobacilli in the endometrium may be associated with improved reproductive prognosis, including higher implantation rates and lower miscarriage rates. By establishing a standardized pipeline, we aim to facilitate future research, enabling better comparison and correlation of bacterial communities with the health status of patients, including fertility-related issues.

Comparative analysis of endometrial, vaginal, and gut microbiota in patients with and without adenomyosis.

Alterations in microbiota composition have been implicated in a variety of human diseases. Patients with adenomyosis present immune dysregulation leading to a persistent chronic inflammatory response. In this context, the hypothesis that alterations in the microbiota may be involved in the pathogenesis of adenomyosis, by affecting the epigenetic, immunologic, and biochemical functions of the host, has recently been postulated. The aim of the present study was to compare the microbiota composition in the vagina, endometrium, and gut of individuals with and without adenomyosis. Cross-sectional study including 38 adenomyosis patients and 46 controls, performed between September 2021 and October 2022 in a university hospital-based research center. The diagnosis of adenomyosis was based on sonographic criteria. Fecal, vaginal, and endometrial samples were collected. Study of the microbiota using 16S rRNA gene sequencing. Patients with adenomyosis exhibited a significant reduction in the gut microbial alpha diversity compared with healthy controls (Chao1 p = 0.012, Fisher p = 0.005, Observed species p = 0.005). Beta-diversity analysis showed significant differences in the compositions of both gut and vaginal microbiota between adenomyosis patients and the control group (Adonis p-value = 0.001; R2 = 0.03 and Adonis p-value = 0.034; R2 = 0.04 respectively). Specific bacterial taxa were found to be either overrepresented (Rhodospirillales, Ruminococcus gauvreauii group, Ruminococcaceae, and Actinomyces) or underrepresented in the gut and endometrial microbiota of adenomyosis patients compared with controls. Distinct microbiota profiles were identified among patients with internal and external adenomyosis phenotypes. The study revealed reduced gut microbiota diversity in adenomyosis patients, accompanied by distinct compositions in gut and vaginal microbiota compared with controls. Overrepresented or underrepresented bacterial taxa were noted in the gut and endometrial microbiota of adenomyosis patients, with variations in microbiota profiles among those with internal and external adenomyosis phenotypes. These findings suggest a potential association between microbiota and adenomyosis, indicating the need for further research to comprehensively understand the implications of these differences.

Endometrial microbiota alteration in female patients with endometrial polyps based on 16S rRNA gene sequencing analysis.

The potential role of the endometrial microbiota in the pathogenesis of endometrial polyps (EPs) warrants further investigation, given the current landscape of limited and inconclusive research findings. We aimed to explore the microecological characteristics of the uterine cavity in patients with EPs and investigate the potential of endometrial microbiota species as novel biomarkers for identifying EPs. Endometrial samples were collected from 225 patients who underwent hysteroscopies, of whom 167 had EPs, whereas 58 had non- hyperproliferative endometrium status. The endometrial microbiota was assessed using 16S rRNA gene sequencing. We characterized the endometrial microbiota and identified microbial biomarkers for predicting EPs. The endometrial microbial diversity and composition were significantly different between the EP and control groups. Predictive functional analyses of the endometrial microbiota demonstrated significant alterations in pathways involved in sphingolipid metabolism, steroid hormone biosynthesis, and apoptosis between the two groups. Moreover, a classification model based on endometrial microbial ASV-based biomarkers along with the presence of abnormal uterine bleeding symptoms achieved powerful classification potential in identifying EPs in both the discovery and validation cohorts. Our study indicates a potential association between altered endometrial microbiota and EPs. Endometrial microbiota-based biomarkers may prove valuable for the diagnosis of EPs. Chinese Clinical Trial Registry (ChiCTR2100052746).

General Overview of Endometrial Microbiota

Here, we aimed to summarize the current knowledge describing the endometrial microbiota. The uterine cavity was considered sterile until the second half of the 20th century. The development of molecular biology techniques such as polymerase chain reaction and sequencing enabled many studies to characterise microbial communities inside the uterine cavity. Although the average number of bacteria in the uterus is lower than in the vagina by 2-4 decimal logarithms, their diversity is still remarkable. The physiological core of endometrial microbiome has not been clearly delineated because of technical limitations including possible contamination during sampling, heterogeneity of sequencing techniques, and variations of experimental design. Bacteria might colonise the uterus from vagina, with semen, by translocation through the blood, or during the respective medical procedures. Generally, major alterations of the endometrial microbiota are triggered by gynecological diseases or assisted reproduction. Hitherto, the impact of endometrial microbiota on human health remains controversial. Understanding the relationships between the endometrial microbiota and human diseases can lead to better diagnosis, treatment and prevention of gynecological diseases. To achieve the success, future studies should employ standardized protocols, from sample collection to bioinformatics analysis.

Unlocking the Uterine Code: Microbiota, Immune Cells, and Therapy for Recurrent Reproductive Failure.

The uterine microbiota has been the subject of increasing study, but its interaction with the local immune system remains unclear. Successful embryo implantation relies on endometrial receptivity, which is pivotal for immunological tolerance to fetal antigens and precise regulation of inflammatory mediators. Emerging data suggest a dynamic interplay between endometrial microflora and the immune system, making dysbiosis a potential determinant of pregnancy outcomes. Imbalances in the regulation of immune cells in the endometrium and decidua have been associated with infertility, miscarriage, and obstetric complications. A thorough comprehension of the immune system in the female reproductive tract shows potential for improving women's health and pregnancy outcomes. The objective of this study was to evaluate the patterns of endometrial microbiota in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) and to explore their implications for endometrial immune cells and chronic endometritis (CE). Immune cells in biopsies from 107 RIF and 93 RPL patients were examined using flow cytometry. The endometrial microbial composition was analyzed using real-time polymerase chain reaction (RT-PCR). The research uncovered disrupted endometrial microbiota in most women with RIF and RPL, which was often associated with significant effects on lymphocytes, T cells, and uNK cells.

Impact of vaginal microecological differences on pregnancy outcomes and endometrial microbiota in frozen embryo transfer cycles.

This prospective study investigates the correlation between vaginal microecology and pregnancy outcomes and explores their impact on endometrial microbiota composition during frozen embryo transfer (FET) cycles. Additionally, the impact of transvaginal Lactobacillus supplementation on reproductive outcomes in patients with previous failed cycles was assessed. A total of 379 patients undergoing FET at a reproductive medicine center were categorized into clinical pregnancy (CP), miscarriage (MISC), and non-pregnant (NP) groups. Vaginal specimens were collected for microecological evaluation prior to embryo transfer. Endometrial microbiota samples were obtained during embryo transfer for 16S rRNA gene sequencing analysis to assess endometrial microbiota composition. Vaginal microecological indicators, including pH, Lactobacillus dominance, and leukocyte esterase activity, were measured. Transvaginal Lactobacillus supplementation was investigated in 60 patients with previous failed cycles. Vaginal microecology significantly correlated with pregnancy outcomes, with normal microecology associated with a higher clinical pregnancy rate. Vaginal pH and leukocyte esterase activity were significantly associated with clinical pregnancy. Furthermore, vaginal microecological differences significantly impacted endometrial microbiota composition. However, no significant differences were observed in endometrial microbiota composition among the CP, MISC, and NP groups. Notably, transvaginal Lactobacillus supplementation increased the clinical pregnancy rate without affecting the miscarriage rate. This study highlights that normal vaginal microecology, characterized by lower pH and leukocyte esterase negativity, is associated with a higher likelihood of clinical pregnancy following FET. Importantly, vaginal microecological differences influence endometrial microbiota composition. Moreover, transvaginal Lactobacillus supplementation appears promising in improving clinical pregnancy rates in patients with previous failed cycles. These findings contribute to a better understanding of the interplay between vaginal and endometrial microbiota and offer potential interventions to enhance reproductive success in assisted reproductive technologies.

The Endometrial Microbiota’s Role in Fertility: Updates and Future Directions

The Endometrial Microbiota’s Role in Fertility: Updates and Future Directions

Preconceptual administration of doxycycline in women with recurrent miscarriage and chronic endometritis: protocol for the Chronic Endometritis and Recurrent Miscarriage (CERM) trial, a multicentre, double-blind, placebo-controlled, adaptive randomised trial with an embedded translational substudy

IntroductionRecurrent miscarriage is a common condition with a substantial associated morbidity. A hypothesised cause of recurrent miscarriage is chronic endometritis (CE). The aetiology of CE remains uncertain. An association between...

Clinical application of the endometrial microbiota test combined with personalized treatment in patients with repeated implantation failure

Objective: The objective of this study was to investigate the clinical value of the endometrial microbiota test (EMT) combined with personalized treatment in patients with repeated implantation failure (RIF). Methods: From March 2019 to August 2022, the clinical data of patients with RIF at the Reproductive Medicine Research Center of the Sixth Affiliated Hospital of Sun Yat-sen University were retrospectively analyzed. A total of 356 patients underwent the EMT using 16S rRNA sequencing and were included in the EMT group. The SPSS statistical software propensity score was set at 1:1 to match the age, anti-Müllerian hormone (AMH), antral follicle count (AFC), years of infertility, and body mass index (BMI) of the women who had not undergone the EMT; subsequently, 354 patients were classified in the non-EMT group. We compared the clinical characteristics of the two groups and analyzed the clinical pregnancy outcomes after personalized treatment. Results: The clinical pregnancy rate (CPR) of the EMT group was significantly higher than that of the non-EMT group (47.81% vs. 13.38%, P <0.05). Among 356 patients who underwent the EMT, 55 patients (15.45%) in group A with normal endometrial microbiota composition did not test positive for the pathogenic bacteria causing chronic endometritis, 34 patients (9.55%) in group B with abnormal endometrial microbiota composition did not test positive for the pathogenic bacteria causing chronic endometritis, 35 patients (9.83%) in group C with normal endometrial microbiota composition tested positive for the pathogenic bacteria causing chronic endometritis, and 232 patients (65.17%) in group D with abnormal endometrial microbiota composition tested positive for the pathogenic bacteria causing chronic endometritis. After administration of probiotics or antibiotic plus probiotics treatment, the pregnancy rates of groups C and D were higher than those of group A (44.00% vs. 20.00% and 55.03% vs. 20.00%, respectively; both P <0.05). Conclusion: The EMT combined with personalized treatment in patients with RIF has solid clinical value as a new treatment strategy.

Cellulomonas endometrii sp. nov.: a novel bacterium isolated from the endometrial microbiota

An isolate of a bacterium recovered from an endometrial biopsy failed to be identified by MALDI-TOF mass spectrometry and was subjected to 16S rRNA sequencing. The obtained sequence was compared by BLASTn against the NCBI database, which revealed that the most closely related species was Cellulomonas hominis and Cellulomonas pakistanensis, with 98.85% and 98.45% identity, respectively. Phenotypic characterisation and genome sequencing were performed. The isolate was facultative anaerobic, gram-positive, motile, non-spore forming, and rod-shaped. Cell wall fatty acid profiling revealed that 12-methyl-tetradecanoic acid was the most abundant fatty acid (36%). The genome size was 4.25 Mbp with a G + C content of 74.8 mol%. Genomic comparison of species closely related to this strain showed that all digital DNA–DNA hybridisation (dDDH) and mean orthologous nucleotide identity (OrthoANI) values were below published species thresholds (70% and 95–96%, respectively). Based on these data, we conclude that this isolate represents a new bacterial species belonging to the family Cellulomonadaceae and the phylum Actinomycetota. We propose the name Cellulomonas endometrii sp. nov. The type strain is Marseille-Q7820T (= CSUR Q7820 = CECT 30716).