STUDY QUESTION: Does the molecular composition of uterine fluid extracellular vesicles (UF-EVs) reflect endometrial tissue changes across the menstrual cycle? SUMMARY ANSWER: Concordance between endometrial tissue and UF-EVs exists on miRNA and mRNA levels along the menstrual cycle phases and UF-EV surface proteomic signatures suggest EVs originate from several major endometrial cell populations. WHAT IS KNOWN ALREADY: The clinical value of endometrial receptivity testing is restricted by invasiveness and the use of only one omics level of input. There is promising evidence that UF-EVs can reflect changes in mid-secretory endometrium, highlighting the potential to establish endometrial receptivity testing right before embryo transfer. However, the dynamic changes of UF-EVs molecular cargo have not been directly compared to endometrial tissue on multiple omics levels. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included fertile women from four menstrual cycle phases: proliferative and early-, mid-, and late-secretory phases. In total, 26 paired samples of UF and endometrial tissue were collected. mRNA and miRNA were sequenced, and differential analysis was performed on consecutive phases. UF-EVs were profiled for various protein surface markers associated with different cell types. EVs from epithelial endometrial organoid-conditioned culture media were used as a reference of pure epithelial endometrial EVs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Paired UF and endometrial tissue samples were collected from 26 fertile, reproductive-age women. EV isolation from UF was validated using electron microscopy and western blotting, and particle numbers were measured by nanoparticle tracking analysis. The transcriptome and miRNome of UF-EVs and endometrial tissue were sequenced, and differential expression analysis was conducted on consecutive phases of the menstrual cycle. Bead-based EV flow cytometry targeting 37 surface protein markers was used to characterize EVs from UF and endometrial organoids. MAIN RESULTS AND THE ROLE OF CHANCE: Surface proteome analysis revealed that UF-EVs from the mid-secretory phase had significantly increased expression of natural killer cell marker CD56 (P < 0.005), pan-leukocyte marker CD45 (P < 0.005), pan-T-cell marker CD3 (P < 0.005), and coagulation-related protein CD142 (P < 0.005) compared to those from the proliferative phase, whereas markers associated with endometrial epithelial cells (CD29, CD133, and CD326) did not significantly change across the menstrual cycle. Transcriptomic analysis highlighted differential expression of histone and metallothionein genes that correlated between paired UF-EVs and endometrial tissues in each tested menstrual cycle phase. Principal component analysis of miRNomes of paired UF-EVs and endometrial tissue samples resulted in similar clustering patterns, where mid- and late-secretory samples clustered closely, and proliferative and early-secretory phase samples clustered separately. Half of the differentially expressed miRNAs in each phase in UF-EVs were also differentially expressed in the endometrium. Importantly, nine mid-secretory phase UF-EV DE miRNAs were identified, five of which were common between UF-EVs and endometrial biopsies, including hsa-miR-30d-5p and hsa-miR-200b-3p, both of which were previously implicated in implantation. Notably, three of the nine miRNAs, hsa-miR-200b-3p, hsa-miR-141-3p, and hsa-miR-200a-3p, were predicted to regulate mRNAs in the endometrial tissue and the pre-implantation embryo trophectoderm. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: The clinical dating of the menstrual cycle phase is based on the first day of menstruation and the time of the LH peak, which does not exclude the possibility that the expected endometrial phase was not reached. The wider limitation of our study is the lack of standardized procedures for collecting UF samples in gynaecological practice, which could challenge the replication of our findings. WIDER IMPLICATIONS OF THE FINDINGS: Evidence that UF-EVs reflect endometrial phases of menstrual cycle supports the use of UF-EVs in endometrial receptivity testing. Additionally, further studies of UF-EVs in endometrial pathologies could be beneficial for diagnostics, considering that more invasive tissue biopsies only reflect the biopsy site and not the full endometrium. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the European Regional Development Fund Enterprise Estonia’s Applied Research Program under the grant agreement number 2014-2020.4.02.21-0398 (EVREM), the Estonian Research Council (grant nos. PRG1076 and PSG1082), the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the Swedish Research Council (grant no. 2024-02530), the Novo Nordisk Fonden (grant no. NNF24OC0092384), and the National Recovery and Resilience Plan of the Republic of Bulgaria, project number BG-RRP-2.004-0001-C01. A.S.L. received funding from the Becas Fundación Ramón Areces para Estudios Postdoctorales. All the authors declare no conflict of interest.

Increasing infertility rates represent a growing medical challenge in modern societies resulting from a complex interplay of sociocultural trends, lifestyle factors, exposure to environmental toxins, and underlying health problems. Women's fertility is particularly vulnerable to these shifts. The obesogenic lifestyle not only accelerates weight gain, but also disrupts ovulation driving the rise in infertility. Among several medications used for treating obesity and type 2 diabetes, glucagon‐like peptide‐1 receptor agonists (GLP‐1RAs) show promising improvement in female fertility most likely by stimulating ovulation. However, the effects of GLP‐1RAs on the endometrium remain unclear. Further studies are needed to investigate the impact of GLP‐1RAs on endometrial receptivity and embryo implantation and early development. The aim of this study is to address the knowledge gap regarding the effects of GLP‐1RAs on human reproduction, with special focus on the endometrium. Understanding these mechanisms may help to develop new strategies for improving fertility treatment, reduce implantation failure and address potential safety concerns regarding teratogenicity and adverse developmental outcomes for children born to women conceiving during or soon after GLP‐1RA treatment.

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.

The human endometrium is a highly dynamic tissue. Increasing evidence has shown that microRNAs (miRs) play essential roles in human endometrium development. Our previous assay, based on small RNA-sequencing (sRNA-seq) indicated the complexity and dynamics of numerous sequence variants of miRs (isomiRs) that can act together to control genes of functional relevance to the receptive endometrium (RE). Here, we used a greater average depth of sRNA-seq to detect poorly expressed small RNAs. The sequencing data confirmed the up-regulation of miR-449c and uncovered other members of the miR-449 family up-regulated in RE—among them miR-449a, as well as several isoforms of both miR-449a and miR-449c, while the third family member, miR-449b, was not identified. Stem-looped RT-qPCR analysis of miR expression at four-time points of the endometrial cycle verified the increased expression of the miR-449a/c family members in RE, among which the 5′ isoform of miR-449c–miR-449c.1 was the most strongly up-regulated. Moreover, we found in a case study that the expression of miR-449c.1 and its precursor correlated with the histological assessment of the endometrial phase and patient age. We believe this study will promote the clinical investigation and application of the miR-449 family in the diagnosis and prognosis of human reproductive diseases.

The endometrium, the inner mucosal lining of the uterus, undergoes complex molecular and cellular changes across the menstrual cycle in preparation for embryo implantation. Transcriptome-wide analyses have mainly been utilized to study endometrial receptivity, the prerequisite for successful implantation, with most studies, so far, comparing the endometrial transcriptomes between (i) secretory and proliferative endometrium or (ii) mid-secretory and early secretory endometrium. In the current study, we provide a complete transcriptome description of the endometrium across the entire menstrual cycle and, for the first time, comprehensively characterize the proliferative phase of the endometrium. Our temporal transcriptome analysis includes five time points including the mid-proliferative, late proliferative (peri-ovulatory phase), early secretory, mid-secretory, and late secretory phases. Thus, we unveil exhaustively the transitions between the consecutive proliferative and secretory phases, highlighting their unique gene expression profiles and possible distinct biological functions. The transcriptome analysis reveals many differentially expressed genes (DEGs) across the menstrual cycle, most of which are phase-specific. As an example of coordinated gene activity, the expression profile of histone-encoding genes within the HIST cluster on chromosome 6 shows an increase in cluster activity during the late proliferative and a decline during the mid-secretory phase. Moreover, numerous DEGs are shared among all phases. In conclusion, in the current study, we delineate the endometrial proliferative phase-centered view of transcriptome dynamics across the menstrual cycle. Our data analysis highlights significant transcriptomic and functional changes occurring during the late proliferative phase—an essential transition point from the proliferative phase to the secretory phase. Future studies should explore how the biology of the late proliferative phase endometrium impacts the achievement of mid-secretory endometrial receptivity or contributes to molecular aberrations leading to embryo implantation failure.