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 characterization, 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 49 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, the 16S rRNA gene V4 region was sequenced. For RNA-based analysis, 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), and 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 INTERESTS: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.

Endometrial polyps (EPs) are benign overgrowths of the endometrium causing abnormal uterine bleeding and infertility. Despite their clinical significance, the molecular mechanisms underlying their development and recurrence remain poorly understood, warranting comprehensive transcriptomic investigation. We hypothesized that transcriptomic differences, particularly at the single-cell level as revealed through cellular trajectory analysis, distinguish EPs from adjacent endometrium. To investigate this, paired EP and adjacent endometrium (adEN) samples were collected from 12 women undergoing hysteroscopic polypectomy (proliferative phase, n = 9; secretory phase, n = 3) and analyzed using bulk and single-cell RNA sequencing (scRNA-seq). Bulk RNA-seq revealed high transcriptional similarity between EPs and adENs, with only a few differentially expressed genes (FDR < 0.05) in proliferative-phase EPs, including upregulation of KMT2B and DLEC1 and downregulation of COL9A1 and RAB3C, potentially reflecting epigenetic regulation and protective mechanisms against tumorigenesis. scRNA-seq identified eight major cell clusters namely stromal, epithelial, endothelial, immune, perivascular, macrophage, B cell, and ciliated populations in both tissues. Pseudotime analysis revealed a mid-transcriptional arrest and enrichment of MECOM/EYA2-positive intermediate epithelial states in EPs, in contrast to the late, mature epithelial stage seen in the adENs. This aberrant epithelial maturation may be associated with impaired perivascular and endothelial differentiation, potentially contributing to defective vascular remodeling and polyp persistence. In conclusion, while EPs exhibit global transcriptomic similarity to adENs, single-cell and pseudotime analyses suggest subtle but significant disruptions in epithelial differentiation and vascular remodeling that might be involved in EPs development. Study limitations include scRNA-seq restricted to the proliferative phase, which may limit generalizability. Nevertheless, future functional studies using primary epithelial organoids derived from EPs may provide a physiologically relevant model to evaluate targeted therapeutic strategies including hormonal interventions with potential applications in infertility management.

Abstract Background Endometriosis is a complex, estrogen-dependent disease with a strong genetic component. Although genome-wide association studies (GWAS) have identified multiple susceptibility loci, most associated variants reside in noncoding regions, limiting biological interpretation and causal gene identification. Moreover, GWAS gene prioritization is limited by incomplete tissue-specific annotation coverage (e.g., GTEx, ENCODE, fine-mapping, Mendelian randomization, and network-based methods). We therefore applied the AlphaGenome artificial intelligence framework to prioritize endometriosis-associated variants based on predicted uterus-specific regulatory effects. Methods We analysed the top 10,000 endometriosis-associated single-nucleotide polymorphisms (SNPs) identified by previously published GWAS by Rahmioglu et al , using AlphaGenome across multiple genomic output types. Uterus-specific predictions with high-confidence effects (|quantile score| ≥ 0.90) were grouped into major regulatory modalities. AlphaGenome-prioritized SNPs within ±500 kb of known GWAS loci were classified into tiers based on the number of supported regulatory modalities, with broader support indicating stronger multilayer regulatory evidence. Effect allele frequency, linkage disequilibrium (LD), and overlap with previously published endometriosis-associated variants were also assessed. Results AlphaGenome generated uterus-specific, 147,033 high-confidence signals across 10,000 endometriosis-associated variants, spanning six regulatory modalities including gene expression, promoter activity, chromatin accessibility, transcription factor binding, histone modification, and RNA splicing. Within the 42 established endometriosis GWAS loci, AlphaGenome identified 42 alternative sub-threshold SNPs with stronger predicted uterus-specific regulatory effects than the published GWAS lead variants. Nineteen AlphaGenome-prioritized SNPs were classified as tier 1, showing support across all six regulatory modalities, compared with five GWAS lead SNPs. Linkage disequilibrium analysis identified eight tier 1 SNPs with weak-to-low LD (r² < 0.5) relative to the corresponding GWAS lead variants, regulating majority of genes involved in estrogen-driven proliferation and inflammatory signalling, highlighting their potential relevance to endometriosis pathogenesis. Additionally, we identified 167 genome-wide significant SNPs outside 42 published GWAS lead SNP loci including six tier 1 SNPs (rs1482061, rs7772579, rs6557140, rs2982571, rs12631337 and rs79626929), encompassing genes nearby ESR1/ 6q25.1, substantiating biological relevance for endometriosis pathogenesis. Conclusions AlphaGenome-based regulatory prioritization refined endometriosis-associated genome-wide association study loci by identifying variants with stronger predicted uterus-specific functional relevance. These findings provide a regulatory framework for prioritizing candidate variants and genes for downstream functional validation in endometriosis.

The use of semaglutide (SE), a glucagon-like peptide-1 receptor agonist (GLP-1RA) with glucose-lowering and weight-loss effects, has risen rapidly, particularly among women of reproductive age. While preclinical studies suggest benefits for ovarian function via the hypothalamic-pituitary-ovarian axis, its impact on the endometrial-embryo interface remains unclear. Here, we show that GLP-1R is dynamically expressed in human endometrium, restricted to epithelial cells and markedly upregulated during the mid-secretory phase of the menstrual cycle. SE activated intracellular cAMP signalling and enhanced epithelial metabolism, driving a shift toward oxidative phosphorylation. Notably, even in the absence of steroid hormone priming, the highest SE concentration upregulates several endometrial receptivity markers, whereas under hormonal priming, the same concentration modestly reduces expression of a key receptivity marker, PAEP/glycodelin. By contrast, in stromal cells lacking detectable GLP-1R, SE disrupts decidualization, induces endoplasmic reticulum stress and suppresses cell-cycle at G2/M phase. Human embryo models, blastoids, expressed GLP-1R and underwent concordant SE-mediated transcriptional remodeling in epiblast and trophectoderm lineages, encompassing changes in metabolism and epigenetic regulation, but without shifts in lineage proportions. Notably, SE restored blastoid attachment under hormone-deprived conditions at all tested concentrations, but did not further enhance attachment when physiological hormonal priming was already present. Together, these findings reveal a compartment-specific mismatch, as SE augments epithelial and embryonic metabolic activity but compromises stromal support for implantation, with potential consequences for implantation due to stromal dysfunction. ### Competing Interest Statement The authors have declared no competing interest. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Endometrial biopsies of healthy fertile women were collected in South Estonian Hospital (Voru, Estonia) under ethical permissions No. 330/M-8 (16.10.2020) and No. 364/M-9 (16.05.2022) issued by the Research Ethics Committee of the University of Tartu. Ethical permits (454/02 and 2011/745/31-3) issued by Stockholm Region Ethical Review Authority allowed the use of embryonic stem cells to investigate human reproduction and blastoid generation. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Estonian Research Council, PSG1082, PRG1076 Novo Nordisk Foundation, NNF24OC0092384 Swedish Research Council, 2024-02530

The human endometrium undergoes dynamic cyclical remodeling across the menstrual cycle to establish receptivity for embryo implantation. While microRNAs are established regulators of endometrial function and embryo–maternal communication, the contribution of tRNA-derived fragments (tRFs) remains largely unexplored. In this study, we performed comprehensive small RNA sequencing of endometrial biopsy (EB) samples and paired uterine fluid–derived extracellular vesicles (UF-EVs) collected from healthy fertile women across consecutive menstrual cycle phases: mid-proliferative (MP), early secretory (ES), mid-secretory (MS), and late secretory (LS). Using the tRAX analytical framework, we characterized the landscape and compartmentalization of tRF populations. tRFs were detected from all 21 cytosolic tRNA isotypes in both EB and UF-EVs, displaying marked phase- and tissue-specific variation. In EB, tRFs were predominantly derived from tRNA-Lys and tRNA-Val, whereas UF-EVs exhibited greater isotype diversity, including substantial contributions from tRNA-Asp and tRNA-Gly. Fragment-type profiling revealed detection of 5′, 3′-, and internal tRF classes across all phases, supporting broad capture of diverse tRF subtypes. Notably, tRFs derived from tRNA-Val showed the most pronounced menstrual cycle–dependent regulation. Multiple 5′-tRF-Val species, including transcript- and isodecoder-specific fragments from tRNA-Val-CAC and tRNA-Val-AAC, were strongly upregulated during the ES and MS phases compared with the MP phase in both EB and UF-EVs. These coordinated increases suggest selective accumulation and extracellular export of tRNA-Val fragments during the secretory phase, potentially reflecting hormonally driven regulation of tRF biogenesis and vesicle-mediated signaling. Together, these findings provide the first comprehensive characterization of menstrual cycle–dependent tRF expression in the human endometrium and UF-EVs. Our results identify tRNA-Val–derived 5′-tRFs as prominent secretory-phase–associated small RNAs and support a potential role for tRFs as novel regulators and extracellular indicators of endometrial receptivity.

Abstract Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid–endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri-and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo–endometrium crosstalk.

STUDY QUESTION Can a large-scale genome-wide association study (GWAS) meta-analysis identify genomic risk loci and likely involved genes for female genital tract (FGT) polyps, provide insights into the biological mechanism underlying their development, and inform of potential overlap with other traits, including endometrial cancer? SUMMARY ANSWER GWAS meta-analysis of FGT polyps highlights potentially shared mechanisms between polyp development and cancerous processes. WHAT IS KNOWN ALREADY Small-scale candidate gene studies have focused on biological processes such as oestrogen stimulation and inflammation to clarify the biology behind FGT polyps. However, the exact mechanism for the development of polyps is still elusive. At the same time, a genome-wide approach, which has become the gold standard in complex disease genetics, has never been used to uncover the genetics of the FGT polyps. STUDY DESIGN, SIZE, DURATION We performed a GWAS meta-analysis including a total of 36 984 women with FGT polyps (International Classification of Diseases (ICD-10) diagnosis code N84) and 420 993 female controls (without N84 code) of European ancestry from the FinnGen study (11 092 cases and 94 394 controls), Estonian Biobank (EstBB, 14 008 cases and 112 799 controls), and the Pan-UKBB study (11 884 cases and 213 800 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS GWAS meta-analysis and functional annotation of GWAS signals were performed to identify genetic risk loci and prioritize genes in associated loci. To explore associations with other traits, we performed a look-up of associated variants across multiple traits and health conditions, genetic correlation analysis, and phenome-wide association study (PheWAS) with ICD-10 diagnosis codes. MAIN RESULTS AND THE ROLE OF CHANCE Our GWAS meta-analysis revealed 16 significant (P < 5 x 10-8) genomic risk loci. Based on exonic variants in GWAS signals, we prioritized EEFSEC, ODF3, PRIM1, PLCE1, LRRC34/MYNN, EXO1, and CHEK2 which are involved in DNA repair, cell proliferation, and cell growth. Several of the identified genomic loci have previously been linked to endometrial cancer and/or uterine fibroids, highlighting the potentially shared mechanisms underlying tissue overgrowth and cancerous processes. Genetic correlation analysis revealed a positive correlation with body mass index and reproductive traits, that can be classified as symptoms or risk factors of endometrial polyps (EPs), whereas a negative correlation was observed between FGT polyps and both menopause (genetic correlation estimate (rg) = -0.29, SE = 0.08, P = 8.8x10-4) and sex hormone-binding globulin (SHBG) (rg = -0.22, SE = 0.04, P = 2.4x10-8). On the phenotypic level, the strongest associations were observed with endometriosis, uterine fibroids, and excessive, frequent, and irregular menstruation. LARGE SCALE DATA The complete GWAS summary statistics will be made available after publication through the GWAS Catalog (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTION In this study, we focused broadly on FGT polyps and did not differentiate between the polyp subtypes. Considering the prevalence of FGT polyp subtypes, we assumed that most women included in the study had EPs. Further research on the expression profile of FGT polyps could complement the GWAS study to substantiate the functional importance of the identified variants. WIDER IMPLICATIONS OF THE FINDINGS The study findings have the potential to significantly enhance our understanding of the genetic mechanisms involved, paving the way for future functional follow-up, which in turn could improve the diagnosis, risk assessment, and targeted treatment options, since surgery is the only line of treatment available for diagnosed polyps. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by European Union through the European Regional Development Fund Project No. 2014-2020.4.01.15-0012 GENTRANSMED. Computations were performed in the High-Performance Computing Center of the University of Tartu. The study was also supported by the Estonian Research Council (grant no. PRG1076 and MOBJD1056) and Horizon 2020 innovation grant (ERIN, grant no. EU952516). All the authors declared no conflict of interest.

Endometrial polyps (EPs) are localized overgrowths of endometrial glands and stroma, common in reproductive-age and postmenopausal women, and can cause abnormal uterine bleeding and infertility. Here, we investigated the cellular heterogeneity and molecular mechanisms of EPs by integrating bulk and single-cell RNA sequencing (scRNA-seq) of EPs and adjacent endometrial tissues (adENs) from 12 women. Bulk RNA-seq revealed high transcriptional similarity, with few differentially expressed genes including upregulated KMT2B and DLEC1 and downregulated COL9A1 and RAB3C . ScRNA-seq identified eight major cell clusters, such as stromal, epithelial, endothelial, immune, perivascular, macrophage, B, and ciliated cells. Pseudotime analysis showed aberrant stromal-to-epithelial transitions in EPs, marked by MECOM and EYA2 intermediate clusters, indicating incomplete epithelial maturation. These altered differentiation trajectories may disrupt perivascular and endothelial cell development, contributing to abnormal vascular remodeling in EPs, despite minimal overall transcriptomic changes compared with adENs. ### Competing Interest Statement The authors have declared no competing interest. Estonian Research Council, https://ror.org/00jjeja18, PRG1076, PSG1082 Horizon Europe, NESTOR -101120075 Swedish Research Council, https://ror.org/03zttf063, 2024-02530 Novo Nordisk Fonden, NNF24OC0092384

In recent years, high-throughput sequencing technologies have revolutionised reproductive microbiome research. Our understanding of the endometrial microbiome is primarily based on DNA-based 16S rRNA gene profiling, but DNA detection does not imply alive microbe presence. 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. Meta-transcriptomic analysis, an RNA-based approach, addresses these shortcomings by capturing functional transcripts that are actively expressed in living microbes. This study aims to characterize the endometrial microbiota through a dual approach, by integrating 16S rRNA gene sequencing and meta-transcriptomic analyses. By simultaneously analysing microbial composition and gene expression within the female reproductive tract samples, we seek to provide a more comprehensive understanding of its microbiota and their functional potential. A total of 49 women, aged 27-42 years, were enrolled in the study. Vaginal swabs, endometrial brushings, and endometrial biopsy samples were collected from each participant during the mid-secretory menstrual cycle phase, 6-9 days post luteinizing hormone surge. Our findings suggest that in low-microbial-biomass environments like 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, it suggests that microbial functions and genome activity are tissue-specific and dependent on the host tissue environment. Moreover, RNA-based analysis provides higher resolution in detecting certain pathogens, even within the endometrium. In conclusion, our study underscores the need to integrate genomic and transcriptomic data in microbiota studies to better elucidate critical and health-related microbiota-host interactions. ### Competing Interest Statement The authors have declared no competing interest.

Current therapeutics of endometriosis focus on hormonal disruption of endometriotic lesions (ectopic endometrium, EcE). Recent findings show higher glycolysis utilization in EcE, suggesting non-hormonal strategy for disease treatment that addresses cellular metabolism. Identifying metabolically altered cell types in EcE is important for targeted metabolic drug therapy without affecting eutopic endometrium (EuE). Here, using single-cell RNA-sequencing, we examine twelve metabolic pathways in paired samples of EuE and EcE from women with confirmed endometriosis. We detect nine major cell types in both EuE and EcE. Metabolic pathways are most differentially regulated in perivascular, stromal, and endothelial cells, with the highest changes in AMPK signaling, HIF-1 signaling, glutathione metabolism, oxidative phosphorylation, and glycolysis. We identify transcriptomic co-activation of glycolytic and oxidative metabolism in perivascular and stromal cells of EcE, indicating a critical role of metabolic reprogramming in maintaining endometriotic lesion growth. Perivascular cells, involved in endometrial stroma repair and angiogenesis, may be potential targets for non-hormonal treatment of endometriosis.

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.

Abstract Study question Are slow-freezing and vitrification suitable methods for cryopreserving endometrial biopsies intended for downstream research applications and organoid formation? Summary answer Both slow-frozen and vitrified endometrial biopsies resulted in successful organoid formation thus can be considered similar to fresh endometrial biopsies in preserving the cellular viability. What is known already Passive slow freezing (PSF) protocols have demonstrated efficacy for cryopreserving endometrial biopsies with subsequently viable isolated epithelial and stromal cells. Additionally, single-cell transcriptomic analyses and successful generation of endometrial organoids suggest a little impact of cryopreservation. By avoiding ice crystal formation, vitrification (VT) could potentially prevent mechanical injury and improve tissue integrity compared to slow freezing. Study design, size, duration Experimental study using endometrial biopsies collected using a Pipelle from eight healthy volunteers. Samples were retrieved at three cycle phases: proliferative (n = 2), mid-secretory (7 days post-LH surge, LH + 7, n = 2) and late-secretory phase (LH + 11, n = 4). Each biopsy was divided into fresh, slow-freezing and vitrification sub-samples for further comparison of outcomes according to microscopic histological analysis, transmission electron microscopy (TEM), gene expression profiling evaluation and generation of endometrial epithelial organoids (from 2 slow-frozen and 2 vitrified samples). Participants/materials, setting, methods PSF was performed using Mr Frosty with samples embedded in 1x DMEM, 30% FBS and 7.5% DMSO at -80ºC. Vitrification was performed using 40% ethylene glycol (EG); 30% Ficoll; 0.5 M Sucrose and 10 mg/ml HSA, and preserved in liquid nitrogen. Tissue morphology was evaluated by traditional histology, TEM and gene expression profiling. The viability of endometrial cells and the ability to form epithelial organoids were evaluated before and after two different freezing protocols. Main results and the role of chance Our analysis showed that both PSF and vitrification techniques are appropriate for cryopreservation and storage of endometrial biopsies aimed at organoid generation, as no significant differences were found between fresh and cryopreserved tissue samples. However, ultrastructural changes were observed by TEM in PSF samples indicating an impact on mitochondria of epithelial and stromal cells with increased swelling. Changes were also observed in epithelial cells in vitrified samples, with decreased nuclear granularity, clarity, and open karyoplasm. On TEM analyses changes in the chromatin status were more evident in vitrified samples compared to fresh and PSF samples. PSF treated samples showed similarly high quality of epithelial and stromal nuclear staining when compared to the fresh samples in haematoxylin and eosin-stained slides, while VT of samples resulted in a significant decrease of both epithelial and stromal nuclear clarity. However, despite these changes observed in the tissue morphology, all three groups displayed similar transcriptional profiles and endometrial organoids were generated from both slow-frozen and vitrified samples, indicating very little or no impact of the freezing method used for post-thaw tissue functionality. Limitations, reasons for caution Although our study demonstrated relatively mild damages in both freezing protocols, we cannot exclude the absence of additional changes not evaluated in this study, such as those concerning the epigenetic constitution of the cells. Therefore, more extended research with larger sample size is needed for further validation of the results. Wider implications of the findings Our findings indicate that both slow-freezing and vitrification methods applied to endometrial biopsies are effective in maintaining tissue morphology and functionality. Further optimization of cryopreservation methods of endometrium could contribute to future experimental and clinical use of cryopreserved endometrial tissue. Trial registration number not applicable

STUDY QUESTION Can a large-scale genome-wide association study (GWAS) meta-analysis identify the genomic risk loci and associated candidate genes for female genital tract (FGT) polyps, provide insights into the mechanism underlying their development, and inform potential overlap with other traits, including endometrial cancer? SUMMARY ANSWER GWAS meta-analysis of FGT polyps highlighted the potentially shared mechanisms between polyp development and cancerous processes. WHAT IS KNOWN ALREADY Small-scale candidate gene studies have focused on biological processes such as estrogen stimulation and inflammation to clarify the biology behind FGT polyps. However, the exact mechanism for the development of polyps is still elusive. At the same time, a genome-wide approach, which has become the gold standard in complex disease genetics, has never been used to uncover the genetics of the FGT polyps. STUDY DESIGN, SIZE, DURATION We performed a genome wide association study (GWAS) meta-analysis including a total of 25,100 women with FGT polyps (International Classification of Disease, ICD-10 diagnosis code N84) and 207,193 female controls (without N84 code) of European ancestry from the FinnGen study (11,092 cases and 94,394 controls) and the Estonian Biobank (EstBB, 14,008 cases and 112,799 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS A meta-analysis and functional annotation of GWAS signals were performed to identify and prioritise genes in associated loci. To determine associations with other phenotypes, we performed a look-up of associated variants across multiple traits and health conditions, a genetic correlation analysis, and a phenome-wide association study (PheWAS) with ICD10 diagnosis codes. MAIN RESULTS AND THE ROLE OF CHANCE Our GWAS meta-analysis revealed ten significant (P < 5 x 10-8) genomic risk loci. Two signals, rs2277339 (P = 7.6 x 10-10) and rs1265005 (P = 1.1 x 10-9) (in linkage disequilibrium (LD) with rs805698 r2 = 0.75), are exonic missense variants in PRIM1 , and COL17A1 genes, respectively. Based on the literature, these genes may play a role in cellular proliferation. Several of the identified genomic loci had previously been linked to endometrial cancer and/or uterine fibroids. Thus, highlighting the potentially shared mechanisms underlying tissue overgrowth and cancerous processes, which may be relevant to the development of polyps. Genetic correlation analysis revealed a negative correlation between sex hormone-binding globulin (SHBG) and the risk of FGT polyps (rg = -0,21, se = 0.04, P = 2.9 x 10-6), and on the phenotypic level (PheWAS), the strongest associations were observed with endometriosis, leiomyoma of the uterus and excessive, frequent and irregular menstruation. LARGE SCALE DATA The complete GWAS summary statistics will be made available after publication through the GWAS Catalogue (). LIMITATIONS, REASONS FOR CAUTION In this study, we focused broadly on polyps of FGT and did not differentiate between the polyp subtypes. The prevalence of FGT polyps led us to assume that most women included in the study had endometrial polyps. Further study on the expression profile of FGT polyps could complement the GWAS study to substantiate the functional importance of the identified variants. WIDER IMPLICATIONS OF THE FINDINGS The study findings have the potential to significantly enhance our understanding of the genetic mechanisms involved, paving the way for future functional follow-up, which in turn could improve the diagnosis, risk assessment, and targeted treatment options, since surgery is the only line of treatment available for diagnosed polyps. TRIAL REGISTRATION NUMBER Not applicable ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement N.P.G. was supported by MATER Marie Sklodowska-Curie which received funding from the European Union Horizon 2020 research and innovation programme under grant agreement No. 813707. T.L. was supported by the Estonian Research Council grant PSG776. This study was funded by European Union through the European Regional Development Fund Project No. 2014-2020.4.01.15-0012 GENTRANSMED. Computations were performed in the High-Performance Computing Center of University of Tartu. The study was also supported by the Estonian Research Council (grant no. PRG1076 and MOBJD1056) and Horizon 2020 innovation grant (ERIN, grant no. EU952516). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All Estonian Biobank (EstBB) participants have signed a broad informed consent form, and analyses were carried out under ethical approvals 1.1-12/624 and 1.1-12/2733 from the Estonian Committee on Bioethics and Human Research (Estonian Ministry of Social Affairs) and data release application 6-7/GI/630 from the EstBB. For the FinnGen study, we used only publicly available GWAS summary statistics without individual-level data and thus, a separate ethics approval was not needed. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes FinnGen cohort level summary statistics can be accessed as described here: . Protocol for accessing the Estonian Biobank data is described here: . GWAS meta-analysis summary statistics will be made available via the GWAS Catalogue (data upload pending).

Patient-derived endometrial biopsies serve as a crucial source for molecular studies, highlighting the necessity for tissue cryopreservation methods that preserve cell viability and tissue morphology with minimal to no impact. The passive slow freezing (PSF) protocol has demonstrated efficacy for cryopreserving endometrial biopsies, allowing for the subsequent isolation of viable epithelial and stromal cells. Vitrification (VT) enables the avoidance of ice crystal formation and could therefore potentially prevent mechanical injury to tissues. In this study, PSF and VT techniques were applied to endometrial biopsies, and the effects of cryopreservation on tissue samples were evaluated using traditional histology. In addition, transmission electron microscopy (TEM), gene expression profiling analyses, the viability of endometrial cells, and the ability to form epithelial organoids were compared between PSF and VT endometrial biopsies in a subset of samples. The histology and TEM studies demonstrated relatively mild cellular and sub-cellular damage in both cryopreservation protocols which did not affect tissue functionality and the formation of the organoids. Additionally, the cryopreservation methodology did not affect the gene expression profile of the 68 endometrial-receptivity associated genes studied. In conclusion, our findings indicate that although current cryopreservation methodologies need further improvements, they still allow us to achieve acceptable cell viability and functionality, showing promising potential for facilitating the utilization of cryopreserved endometrial tissue samples for research purposes.

BACKGROUND:Modern lifestyle has led to an increase in the age at conception. Advanced age is one of the critical risk factors for female-related infertility. It is well known that maternal age positively correlates with the deterioration of oocyte quality and chromosomal abnormalities in oocytes and embryos. The effect of age on endometrial function may be an equally important factor influencing implantation rate, pregnancy rate, and overall female fertility. However, there are only a few published studies on this topic, suggesting that this area has been under-explored. Improving our knowledge of endometrial aging from the biological (cellular, molecular, histological) and clinical perspectives would broaden our understanding of the risks of age-related female infertility. OBJECTIVE AND RATIONALE:The objective of this narrative review is to critically evaluate the existing literature on endometrial aging with a focus on synthesizing the evidence for the impact of endometrial aging on conception and pregnancy success. This would provide insights into existing gaps in the clinical application of research findings and promote the development of treatment options in this field. SEARCH METHODS:The review was prepared using PubMed (Medline) until February 2023 with the keywords such as 'endometrial aging', 'receptivity', 'decidualization', 'hormone', 'senescence', 'cellular', 'molecular', 'methylation', 'biological age', 'epigenetic', 'oocyte recipient', 'oocyte donation', 'embryo transfer', and 'pregnancy rate'. Articles in a language other than English were excluded. OUTCOMES:In the aging endometrium, alterations occur at the molecular, cellular, and histological levels suggesting that aging has a negative effect on endometrial biology and may impair endometrial receptivity. Additionally, advanced age influences cellular senescence, which plays an important role during the initial phase of implantation and is a major obstacle in the development of suitable senolytic agents for endometrial aging. Aging is also accountable for chronic conditions associated with inflammaging, which eventually can lead to increased pro-inflammation and tissue fibrosis. Furthermore, advanced age influences epigenetic regulation in the endometrium, thus altering the relation between its epigenetic and chronological age. The studies in oocyte donation cycles to determine the effect of age on endometrial receptivity with respect to the rates of implantation, clinical pregnancy, miscarriage, and live birth have revealed contradictory inferences indicating the need for future research on the mechanisms and corresponding causal effects of women's age on endometrial receptivity. WIDER IMPLICATIONS:Increasing age can be accountable for female infertility and IVF failures. Based on the complied observations and synthesized conclusions in this review, advanced age has been shown to have a negative impact on endometrial functioning. This information can provide recommendations for future research focusing on molecular mechanisms of age-related cellular senescence, cellular composition, and transcriptomic changes in relation to endometrial aging. Additionally, further prospective research is needed to explore newly emerging therapeutic options, such as the senolytic agents that can target endometrial aging without affecting decidualization. Moreover, clinical trial protocols, focusing on oocyte donation cycles, would be beneficial in understanding the direct clinical implications of endometrial aging on pregnancy outcomes.

IntroductionThe expression of genes in female reproductive organs is influenced by the cyclic changes in hormone levels during the menstrual cycle. While the molecular changes in the endometrium that facilitate embryo implantation have been extensively studied, there is limited knowledge about the impact of the menstrual cycle on cervical cells. Cervical cells can be easily and routinely collected using a cytobrush during gynecological examination, offering a standardized approach for diagnostic testing. In this study we investigated how the transcriptome of cervical cells changes during the menstrual cycle and assessed the utility of these cells to determine endometrial receptivity.MethodsEndocervical cells were collected with cytobrushes from 16 healthy women at different menstrual cycle phases in natural cycles and from four women undergoing hormonal replacement cycles. RNA sequencing was applied to gain insight into the transcriptome of cervical cells.ResultsTranscriptome analysis identified four differentially expressed genes (DEGs) between early- and mid-secretory samples, suggesting that the transcriptome of cervical cells does not change significantly during the opening of the implantation window. The most differences appeared during the transition to the late secretory phase (2136 DEGs) before the onset of menstruation. Cervical cells collected during hormonal replacement cycles showed 1899 DEGs enriched in immune system processes.ConclusionsThe results of our study suggested that cervical cells undergo moderate transcriptomic changes throughout the menstrual cycle; however, these changes do not reflect the gene expression pattern of endometrial tissue and offer little or no potential for endometrial receptivity diagnostics.

Polycystic Ovarian Syndrome (PCOS) is an endocrine disorder commonly affecting the reproductive capacity of women leading to infertility. PCOS-related infertility is majorly due to anovulation; however, it is not the only cause. The defective endometrium causing recurrent miscarriage and implantation failure can also be accountable for infertility in PCOS women. The unusual levels of hormones and their receptors in the PCOS endometrium have a hostile effect during WOI, making the microenvironment unfavorable for embryo implantation. To date, many studies have been performed to determine the role of candidate genes in endometrial receptivity but very limited data is available using whole genome approach. This review aims at summarizing the existing studies on the basic aspects of endometrial receptivity in PCOS. The review focuses on aberrant levels of hormones and their receptors in the endometrium, affecting the receptivity. Additionally, it explores the novel approach reviewing the effect on treatment options administered for ovulation induction in PCOS on their endometrial receptivity. Overall, this review will help us to understand the molecular milieu in PCOS endometrium and its effect on the receptivity potential. However, to have a thorough understanding of the mechanistic approach of hormonal imbalance in PCOS on endometrial receptivity, there is a need to give more weightage to genome-wide studies in the future. The current review will further guide us to formulate future studies using whole genome technologies for the assessment of endometrial receptivity in different cohorts of PCOS women, which may have future diagnostic implementations.

Recurrent implantation failure (RIF) is one of the major obstacles in IVF. Transcriptomic literature has revealed the various biological processes involved in endometrial receptivity (ER) under different physiological circumstances, especially in natural cycle. We intended to determine the function-specific ER profile under controlled ovarian stimulation (COS) cycle. This can help to back trace the genomic impairment in RIF patients during the IVF cycle and to validate the genes involved in enriched pathways. In our study, retrospective gene expression microarray dataset was reanalysed after the follow-up, in classic non pregnant RIF (cases) vs fertile women (controls) under COS (n = 5/group). Reanalysis of microarray revealed significant downregulation of cell adhesion function (P:3.11E-05) with the maximum gene count. For validation purpose, downregulation of eight genes (COMP, HABP2, ITGAD, CDH3, COL22A1, MFAP4, THBS1and CD300A) involved in enriched cell adhesion pathway having fold change > 3 were assessed by real-time PCR in independent cohorts of cases and controls (n = 24, each). Downregulation of six out of eight genes (COMP, HABP2, ITGAD, CDH3, MFAP4 and THBS1) were confirmed by real-time PCR (P < 0.05) with fold change > 2. This indicates the importance of analysed genes in the ER mechanism under COS, thus mimicking the fresh embryo transfer. The further analysis in larger cohorts would substantiate the study findings in RIF patients undergoing IVF cycle.