Current knowledge suggests that the uterus harbours its own microbiota, where the microbes could influence the uterine functions in health and disease; however, the core uterine microbial composition and the host-microbial relationships remain to be fully elucidated. Different studies are indicating, based on next-generation sequencing techniques, that microbial dysbiosis could be associated with several gynaecological disorders, such as endometriosis, chronic endometritis, dysfunctional menstrual bleeding, endometrial cancer, and infertility. Treatments using antibiotics and probiotics and/or prebiotics for endometrial microbial dysbiosis are being applied. Nevertheless there is no unified protocol for assessing the endometrial dysbiosis and no optimal treatment protocol for the established dysbiosis. With this review we outline the microbes (mostly bacteria) identified in the endometrial microbiome studies, the current treatments offered for bacterial dysbiosis in the clinical setting, and the future possibilities such as pro- and prebiotics and microbial transplants for modifying uterine microbial composition.

Introduction: Several metabolite classes have been identified in human endometrium, including lipids, nucleotides, amino acids, organic acids, and sugars. The first studies suggest the importance of metabolites in endometrial functions, as imbalance in uterine metabolites has been associated with low implantation rate and endometriosis. Nevertheless, most of studies have put emphasis on specific metabolite classes, and we lack the knowledge of the whole metabolome composition in human uterus. Further, a healthy dietary pattern has been shown to potentially protect against different endometrial dysfunctions and is a potential modulator of metabolomic composition and, consequently, the intrauterine microenvironment. The Mediterranean Diet (MD), characterized by a high intake of fruits, vegetables, cereals, nuts, legumes, fish, and olive oil, and a low consumption of meat, dairy products, and processed foods, has been associated with a wide range of benefits for health. Indeed, the MD pattern has displayed a beneficial role in endometriosis management and fertility; however, the relationship between the MD and the endometrial metabolome is still unknown. In our study, we set out to analyze receptive-phase endometrial metabolome profiles among women with infertility and their associations with MD.Methods: The study included women with male factor infertility (n=8), unexplained infertility (n=10), recurrent implantation failure (n=14), and endometriosis (n=13). The endometrial metabolome was analyzed with ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC–MS/MS). The MD adherence of the participants was assessed using the 14-point MEDAS questionnaire of adherence to the MD. Results: We provide the whole metabolome profile of the endometrium, where 925 different metabolites were identified. Among these metabolites, lipids comprised the largest part, where polyunsaturated fatty acids (PUFAs) prevailed. Women with endometriosis and recurrent implantation failure were found to have lower levels of PUFAs compared to women with male factor and unexplained infertility (i.e., no clear endometrial alterations), identifying a metabolome profile associated with infertility diagnoses where altered endometrial functions are suspected. Moreover, MD adherence seemed to be associated with the endometrial metabolomic profile in a manner dependent on the health status of the uterus. Conclusion: The study findings provide insight into the molecular background of female infertility and lead to identification of potential molecular biomarkers and possibilities for modulating the endometrial microenvironment and, thereby, endometrial functions involved in embryo implantation and infertility.

Microorganisms are important due to their widespread presence and multifaceted roles across various domains of life, ecology, and industries. In humans, they underlie the proper functioning of multiple systems crucial to well-being, including immunological and metabolic functions. Emerging research addressing the presence and roles of microorganisms within human reproduction is increasingly relevant. Studies implementing new methodologies (e.g., to investigate vaginal, uterine, and semen microenvironments) can now provide relevant insights into fertility, reproductive health, or pregnancy outcomes. In that sense, cutting-edge sequencing techniques, as well as others such as meta-metabolomics, culturomics, and meta-proteomics, are becoming more popular and accessible worldwide, allowing the characterization of microbiomes at unprecedented resolution. However, they frequently involve rather complex laboratory protocols and bioinformatics analyses, for which researchers may lack the required expertise. A suitable pipeline would successfully enable both taxonomic classification and functional profiling of the microbiome, providing easy-to-understand biological interpretations. However, the selection of an appropriate methodology would be crucial, as it directly impacts the reproducibility, accuracy, and quality of the results and observations. This review focuses on the different current microbiome-related techniques in the context of human reproduction, encompassing niches like vagina, endometrium, and seminal fluid. The most standard and reliable methods are 16S rRNA gene sequencing, metagenomics, and meta-transcriptomics, together with complementary approaches including meta-proteomics, meta-metabolomics, and culturomics. Finally, we also offer case examples and general recommendations about the most appropriate methods and workflows and discuss strengths and shortcomings for each technique. ^* Both authors contributed equally to this work. Article published online: 06 February 2024 © 2024. Thieme. All rights reserved. Thieme Medical Publishers, Inc. 333 Seventh Avenue, 18th Floor, New York, NY 10001, USA

STUDY QUESTION: Does the analysis of endometrial microbes provide the same information when using DNA or RNA sequencing-based techniques? SUMMARY ANSWER: DNA vs. RNA-based microbial analysis techniques demonstrated significant microbial compositional differences and lack of transcriptionally active Lactobacilli in the endometrium. WHAT IS KNOWN ALREADY: Our understanding of the endometrial microbiome is primarily based on DNA-based 16S rRNA gene profiling, but DNA detection does not imply the presence of living microbes. While this method is cost-effective and widely used, it has notable limitations, including the underestimation of microbial diversity, abundance, and functionality, as well as limited species-level resolution. While the microbiome reflects DNA-based characterisation, the microbiota more precisely captures metabolically active communities. In this context, meta-transcriptomic analysis, an RNA-based approach, addresses these shortcomings by capturing functional transcripts that are actively expressed in living microbes. STUDY DESIGN, SIZE, DURATION: This cross-sectional study consisted of forty-nine reproductive-aged women (27–42 years old) who were receiving ART. By simultaneously analysing the microbial composition and gene expression within female reproductive tract samples, we sought to provide a more comprehensive understanding of the microbiota and functional potential of these samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Vaginal swabs, endometrial brushing, and endometrial biopsy samples were collected from 49 participants during the mid-secretory phase of their menstrual cycle, 6–9 days after the luteinizing hormone surge for parallel 16S rRNA gene sequencing and meta-transcriptome analyses. For DNA-based analysis, 16S rRNA gene V3-V4 regions were sequenced. For RNA-based total RNA was extracted followed by ribosomal RNA depletion. Strand-specific total RNA sequencing libraries were prepared and sequenced. Taxonomy was assigned by using Kraken2 (v2.2.1), Bracken (v2.7). MAIN RESULTS AND THE ROLE OF CHANCE: Our findings suggest that in low-microbial-biomass environments such as the endometrium, the correlation between 16S rRNA gene sequencing and meta-transcriptomics is relatively weak. This highlights the limitations of microbial analysis of low-microbial-biomass samples. Alternatively, microbial functions and genome activity may be tissue-specific and dependent on the host tissue environment. Moreover, RNA-based analysis provides higher resolution in detecting certain pathogens, even within the endometrium. LARGE SCALE DATA: The data presented in the study are deposited in the NCBI SRA Database, accession number PRJNA1247240. LIMITATIONS, REASONS FOR CAUTION: High levels of host RNA and the low abundance of microbial reads in the endometrium complicate microbial identification. Our findings indicate that RNA-seq enables precise profiling of the vaginal microbiome and, in cases of dysbiosis, reveals higher pathogen activity than DNA-based approaches. However, the limited sample size restricts the generalization of these conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Contrary to the general belief of the dominance of Lactobacillus in the human endometrium, our study suggests that the endometrial microenvironment may be harbouring DNA fragments and/or cells of lactobacilli originating from the lower reproductive tract. Our study results indicate a need to re-consider/re-analyse the endometrial microbiome in health and disease. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the projects Endo-Map PID2021-127280OB-I00, ROSY CNS2022-135999 and ENDORE SAF2017-87526-R funded by MICIU/AEI/10.13039/501100011033 and by FEDER, EU. This work was also supported by the Estonian Research Council grants (PSG1082 and PRG1076), Swedish Research Council grant no. 2024-02530 and Novo Nordisk Foundation grant no. NNF24OC0092384. Additionally, A.S.L and I.P.P acknowledge Becas Fundación Ramón Areces para Estudios Postdoctorales—Convocatorias XXXV and XXXVI, para Ampliación de Estudios en el Extranjero en Ciencias de la Vida y de la Materia. A.S. is supported by Horizon Europe (NESTOR, grant no. 101120075) and the Ministry of Education and Research Centres of Excellence grant TK214 name of CoE. All the authors declare no conflict of interest.