Endometrial Biopsy Research Articles

Results of Endometrial Biopsy and Its Impact on Survival Data in Patients with High-Risk Uterine Sarcoma.

Background: There are conflicting data regarding the detection rate of high-risk uterine sarcoma (HRUS) by endometrial biopsy. In addition, there are no studies in the literature on its impact on the chosen surgical approach and survival. Methods: This study includes 415 patients with HRUS. Of these, 178 (42.9%) patients had undergone endometrial biopsy. We analyzed the detection rate of endometrial biopsy and its impact on surgical approach and survival data. Results: Correct specific histologic diagnosis was achieved in only 30.0% of LMS and 33.3% of HGESS/UUS. Other uterine sarcoma, unspecified malignant mesenchymal tumor, carcinosarcoma or carcinoma were found in 45% of LMS and 78.2% of HGESS/UUS. As a result of the histologic findings, the rate of inadequate surgery was reduced by up to 19.9%. As tumor morcellation was performed significantly less often with biopsy (32.5% with vs. 55.4% without biopsy, p < 0.001), the locoregional recurrence-free interval remained unaffected between the two groups (p = 0.81). This is obviously an advantage of biopsy, although it does not affect the local recurrence rate in morcellated patients. Conclusions: Indicated endometrial biopsy is an important step in the diagnosis of HRUS, despite its low detection rate. It helps to avoid inappropriate surgical procedures but does not affect OS.

Vaginal microbiome dysbiosis as a novel noninvasive biomarker for detection of chronic endometritis in infertile women.

To identify the vaginal microbial signature in women with chronic endometritis (CE) and investigate the potential of vaginal microbiome characterization as a novel diagnostic tools for CE. A cross-sectional study was conducted to compare the characteristics of the vaginal microbiome in 98 women who underwent endometrial biopsy for routine clinical inspection of infertility (49 women diagnosed with CE and 49 with non-CE). The vaginal microbiome was analyzed using 16S ribosomal RNA gene amplicon sequencing. The study included an analysis of diversity, bacterial abundance, and microbial function. In addition, microbial markers were identified, and a CE classifier was developed. The relative abundances of genera, including Bifidobacterium, Prevotella and Gardnerella, were found to be different between the two groups. Analysis of the Kyoto Encyclopedia of Genes and Genomes pathways reported differential expression in metabolism-related pathways in the two groups. We identified four microbial markers of CE (Enterobacter, Prevotella, Faecalibacterium, and Phascolarctobacterium) and developed a predictive classifier for diagnosing CE, achieving an area under the curve of 83.26%. The results of the current study revealed that, compared with the non-CE controls, patients with CE have a different vaginal microbiota, highlighting the diagnostic significance of the vaginal microbiome as a promising noninvasive biomarker in detecting CE.

Evaluation of the Vascular Endothelial Growth Factor and Smooth Muscle Actin Expression With Microvessel Density and Morphometric Analysis of Endometrial Vessels in Patients With Abnormal Uterine Bleeding.

Abnormal uterine bleeding (AUB) that occurs in a structurally normal uterus with regular menstrual cycles and without other identifiable etiology is often caused by a primary endometrial disorder (AUB-E). Altered vascular morphological changes and expression of markers of angiogenesis have been implicated as an underlying cause in these cases. The study was conducted to investigate the expression of vascular endothelial growth factor (VEGF) and smooth muscle actin-alpha (SMA-α), and to perform microvessel density (MVD), andmorphometric evaluation of endometrial vessels in patients with AUB-E. Endometrial biopsies and hysterectomy specimens of 40 patients clinically diagnosed with AUB-E were included in the study with 40 age-matched controls. Immunohistochemistry (IHC) with VEGFandSMA-αwas performed, and the expression and staining pattern was recorded as the number of positive vessels per 10 high power fields and intensity scores. Morphometric analysis was performed on CD34 stained sections using Leica Application Suite, version 4.4.0 software (Leica Microsystems, Wetzlar, Germany). MVDwas calculated by the vascular hotspot method. A statistically significant increase in VEGF vessel count (p-value<0.001) and a decline in SMA-α expression (p-value=0.23) was seen in cases as compared to the control group.There was a statistically significant increase in microvessel caliber (p-value=0.01) and MVD (p-value <0.001) in cases as compared to controls. These findings support aberrant vascular proliferation and impaired vessel maturation, contributing to the pathology of AUB-E. Alterations in angiogenesis in these patients reveal potential therapeutic targets for AUB.

Revolutionizing chronic endometritis diagnosis: real-time polymerase chain reaction unveils microbial pathogens in Indian women with abnormal bleeding and reproductive challenges

BackgroundThis study aimed to assess the utility of real-time-polymerase chain reaction (PCR) for diagnosing chronic endometritis (CE) by targeting 11 prevalent pathogens and to compare the outcomes with conventional culture-based diagnosis. Study DesignA retrospective analysis was conducted on 500 patients with clinical conditions such as abnormal bleeding, in vitro fertilization failure, recurrent implantation failure, recurrent miscarriage, and recurrent pregnancy loss. The prevalence of 11 key pathogens associated with CE was evaluated in endometrial biopsy samples. ResultsIn our study, PCR identified 318 cases (63.6%) positive for at least one of the 11 investigated pathogens, while culture-based methods detected 115 cases (23%). Predominant pathogens detected by PCR included Enterococcus faecalis (E. faecalis) (19%), Escherichia coli (E. coli) (6.8%), Staphylococcus aureus (S. aureus) (9%), Mycoplasma hominis (5%), Mycoplasma genitalium (6.2%), Streptococcus agalactiae (S. agalactiae) (4.2%), Ureaplasma urealyticum (4%), nontuberculous Mycobacterium (5.2%), Mycobacterium tuberculosis (1.2%), Neisseria gonorrhoeae (0.6%), and Chlamydia trachomatis (2.4%). Standard culture methods identified E. faecalis (10.8%), S. aureus (6.2%), E. coli (3.8%), and S. agalactiae (2.2%). ConclusionThe DICE panel proves itself as a swift, precise, and cost-effective diagnostic tool for detecting both culturable and nonculturable endometrial pathogens in CE. Demonstrating superiority, the Molecular method outshines microbial culture, ensuring accurate and sensitive detection of CE-associated pathogens, harmonizing seamlessly with histology and hysteroscopy findings.

P-325 Comparing slow-freezing and vitrification endometrial biopsy cryopreservation protocols for tissue cryodamage and functional organoid formation

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

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

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

P-399 Beyond chromosome screening: The impact of progesterone and in vitro oxidative stress on failed euploid blastocyst transfers

Abstract Study question To assess the effect of serum progesterone levels on the production of radical oxygen species (ROS) by endometrial immune cells in patients with non-genetic miscarriages. Summary answer Elevated serum pre-transfer progesterone levels favour the development of an endometrial microenvironment that is not conducive to a healthy pregnancy, by increasing ROS production. What is known already Endometrial immune cells, especially uterine Natural Killer (uNK) cells, play an essential role in the proper development of pregnancy, promoting angiogenesis and adequate placentation. The maturation of these cells is indirectly determined by progesterone levels, which, during the secretory phase, enhance the synthesis of key cytokines for uNKs’ functionality. Previous studies have already indicated that increases in progesterone levels during the follicular phase can alter the endometrial microenvironment, affecting its receptivity. However, the molecular effect that imbalances of this hormone may have on the endometrium on the day of embryo transfer and directly in relation to uNK is still unknown. Study design, size, duration We conducted a prospective exploratory study, between September 2022 and January 2024, including 15 patients with previous miscarriages after euploid single embryo transfer (SET), ruling out the genetic cause for such gestational losses. We obtained endometrial biopsies of all patients, after 5 days of progesterone, in a cycle with hormone replacement therapy. The same day of the biopsy, serum progesterone levels were measured in all the patients included. Participants/materials, setting, methods In vitro cultures enriched in uNK cells (CD56+CD16-) were established from the patients’ endometrial biopsies and cell enrichment was confirmed by flow cytometry. These cells were co-cultured with the JEG-3 cell line (placental cells), simulating the microenvironment of early pregnancy. Cellular ROS production was measured by fluorescence detection 40 hours post-culture using a commercial kit. A linear regression model was used to determine statistically significant correlations (p &amp;lt; 0.05) between ROS and progesterone levels. Main results and the role of chance The mean age of the participating patients was 38.9 years and the BMI was 23.2 kg/m2. The mean progesterone level the day of the biopsy was 15.6 ng/ml. The cell cultures obtained from patients were successfully enriched in the desired cell population, with an average percentage of 89.6% uNK cells (CD56+CD16-) of total leukocytes (CD45+) obtained by flow cytometry. After culturing the same number of cells from each patient with the same number of spheroids of the JEG-3 cell line, an average ROS production of 31341.2 arbitrary units (a.u.) was obtained. Taking into account the co-cultures stablished from the 15 patients, a positive correlation between cellular ROS production and progesterone level was observed (r = 0.6728, p = 0.006). Limitations, reasons for caution Although the results shown may provide clues about the molecular dynamics of the endometrium during early pregnancy, they should be taken with caution, as it is not yet known whether this progesterone elevation is the cause or rather the consequence of an unfavourable endometrial microenvironment with high oxidative stress. Wider implications of the findings This study provides new information on the endometrial microenvironment of patients suffering non-genetic miscarriages and, although much remains to be studied, this association between hormone levels and oxidative stress may point the way to future studies investigating a possible explanation for these undesirable reproductive outcomes. Trial registration number Not Applicable

P-322 Development of a CD138-based histopathological scoring system to diagnose chronic endometritis

Abstract Study question Does a standardized histopathological classification system lead to a robust interobserver agreement to diagnose chronic endometritis (CE) and can an accurate computation algorithm be developed? Summary answer The proposed scoring system has a particularly good to excellent interobserver agreement among pathologists. A machine-learning algorithm outperformed pathologists in the clinically most relevant 2-tier-system. What is known already Plasma cell (PC) presence in the stromal endometrium has been proposed as a marker for CE, yet no standardized classification system that considers the amount or distribution of PCs exists. Also, interobserver agreement has been insufficiently reported in the previous publications on the topic. Without a robust histopathological score, the potential clinical impact of CE on ART treatment outcomes cannot be investigated. This issue is strongly reflected in the heterogeneity of the current diagnostic and therapeutic approaches, specifically for patients experiencing repeated implantation failure (RIF) or recurrent pregnancy loss (RPL). Study design, size, duration A 5-tier classification system based on CD138-positive PCs (Roche B-A38/automated Ventana system) was established: class0 (no PCs), classIPC (isolated PCs), class1 (³1 cluster of 5-19 PCs/0.25mm2), class2 (³1 cluster of 20-49 PCs/0.25mm2), class3 (³1 cluster of 50 or more PCs/0.25mm2). A test set of 78 endometrial biopsies (collected over two years) was selected by a certified pathologist who sorted &amp;gt;1000 biopsies to ensure the full spectrum of PC counts/distribution was accounted for. Participants/materials, setting, methods The 3DHistech-platform digitized the slides (pixel size 0.2738x0.2738mm) and a scoring session was set up in the Pathomation software suite. Six pathologists scored independently after training. Overall agreement was expressed by Fleiss’ Kappa values. Pairwise interobserver agreement was calculated using Cohen’s Kappa statistics. A supervised machine-learning algorithm was developed in QuPath(V0.4.2) and considered a seventh observer in the interobserver agreement calculations. Main results and the role of chance The CD138-based semiquantitative scoring system showed particularly good to excellent interobserver agreement among all observers. Overall agreement values were 0.722 for the 5-tier system and 0.858 for the 2-tier system (0+IPC versus 1 + 2+3, which was considered the clinically most relevant classification, i.e. CE absent versus diagnosed). Pairwise interobserver agreement was 0.871-0.951. Intra-class correlations varied from 0.727 to 0.839 in the 5-tier system and 0.893 to 0.965 in the 2-tier system. All findings were statistically significant (p &amp;lt; 0.001). The supervised machine-learning algorithm was able to compete with human pathologist observers and outperformed them in the 2-tier system. The proposed classification system provides a framework for assessing the presence and severity of endometrial PC infiltration and can guide further research. Limitations, reasons for caution Inconsistent staining intensity, non-specific epithelial staining and background interference are common problems that might limit interlaboratory use of the algorithm. Furthermore, the algorithm has not yet been associated to patient/clinical parameters. Accurate analysis of an endometrial biopsy might still be insufficient to provide a formal diagnosis of CE. Wider implications of the findings To unravel the association between PCs in endometrial biopsies (as a proxy for the diagnosis of CE) and the reproductive outcomes following ART, a standardized methodology is of crucial importance for future studies. Higher accuracy, efficieny and automation are advantages when using computer-aided diagnostics for CE. Trial registration number BUN 143202042810

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 (&amp;gt;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 (&amp;gt;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-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.

O-100 Chronic endometritis - research and practice

Abstract Chronic endometritis is an inflammation of the endometrium characterized by the presence of plasma cells. Plasma cells can be identified on H&amp;E and CD138 immunohistochemical staining by their characteristic appearance. There is a large body of evidence supporting the association between chronic endometritis and recurrent pregnancy loss (RPL), with a prevalence of approximately 30% among women with RPL (McQueen 2021). A limitation of the current literature, however, is heterogeneity of diagnostic criteria utilized and there is no consensus on the exact definition or number of plasma cells required. The treatment of chronic endometritis is oral antibiotic therapy and removal of any associated uterine pathology such as retained pregnancy tissue. Studies show that a single course of antibiotics has a cure rate of 60-94% when endometrial biopsies are repeated following treatment (Johnson 2010, McQueen 2014). Following antibiotic treatment, if resolution of chronic endometritis confirmed, women with RPL have improved live birth rates (Kimura 2019; Pirtea 2021).

O-278 The vaginal and endometrial microbiome: do they tell the same story?

Abstract Study question Is the composition of the endometrial microbiome comparable to that of the vagina and can vaginal sampling provide sufficient information to characterize the uterine microbiome? Summary answer The compositions of the endometrial and vaginal microbiome are largely comparable and therefore, in most cases, vaginal sampling is sufficient to characterize the uterine microbiome. What is known already A dysbiotic endometrial microbiome (i.e., a shift from a predominance of beneficial bacteria to less favourable species) has been reported in association with adverse pregnancy outcomes in assisted reproductive treatment (ART). If dysbiosis is detected, antibiotics or probiotics can be administered to restore an optimal microbiome. However, characterization of the endometrial microbiome is challenging, involving invasive sampling, and technical difficulties due to the low microbial mass in the endometrium. In contrast, vaginal sampling is considered minimally invasive and microbial analysis is technically less complex. It is important to clarify whether the microbiomes of the endometrium and the vagina are comparable. Study design, size, duration A prospective multi-centre study including 60 ART patients was conducted from June 2022 to August 2023. During the luteal phase (on average day-19), paired vaginal swabs and endometrial biopsies were collected sequentially and stored in a preservation medium for further analysis. Patients and physicians were also asked to complete a questionnaire to identify any lifestyle or medical factors that may have differentially affected the microbial composition of the vagina and endometrium. Participants/materials, setting, methods A carefully validated qPCR methodology was used to accurately assess the presence and relative abundance of the four major lactobacillus species associated with eubiosis (crispatus, gasseri, iners, and jensenii) and the 15 most common bacterial species implicated in dysbiosis. Additionally, the relative abundance of microbial and human DNA was examined. Of note, endometrial analysis required additional tissue lysis. Furthermore, due to the low microbial DNA content in endometrial samples, preamplification was necessary prior to qPCR. Main results and the role of chance The proportion of microbial versus human DNA was approximately 90 times higher in vaginal samples compared to endometrial samples (p &amp;lt; 0.0001). However, the microbial compositions of both the endometrium and the vagina showed remarkable similarities. Lactobacillus species were predominant at both sites, accounting for an average of 86% and 89% of the bacterial population in the vagina and endometrium, respectively (p = 0.6). Of the dysbiotic species, Gardnerella vaginalis was the most common, accounting for 8% (vagina) and 7% (endometrium) of the total bacterial population (p = 1). There was no difference in the average number of distinct microbial species between the two niches (1.8 vs. 2.1; p = 0.3). Direct comparison of paired samples highlighted the high similarity of microbial compositions between both sites and showed strong correlations of species abundances in individual patients (L.crispatus: r = 0.9, L.iners: r = 0.9, L.gasseri: r = 1, L.jensenii: r = 0.9, G.vaginalis: r = 0.7; p &amp;lt; 0.0001/all species). However, the microbial composition differed in four patients (6.6%), with three having a dysbiotic vaginal profile (≥50% dysbiotic species) but a eubiotic endometrial profile (≥50% lactobacillus species) and one showing the reverse pattern. The medical conditions of two patients, including chronic endometritis and use of antibiotics and probiotics in the preceding cycle, may account for the observed differences. Limitations, reasons for caution The species analysed were carefully selected to ensure a comprehensive analysis. Nevertheless, the potential presence of unevaluated species could impact the results. While the included patients displayed diversity in their reproductive histories, it is important to acknowledge that the observed results may still be influenced by underlying causes of infertility. Wider implications of the findings The close similarity between the microbiomes at the two sites investigated suggests that minimally invasive vaginal sampling may be sufficient to characterise the endometrial microbiome. The ease of vaginal sampling and processing is expected to improve patient recruitment in microbiome research and facilitate easier application of clinical microbiome tests. Trial registration number not applicable

O-265 Mapping decidualization resistance at multi-omic level

Abstract Study question What are the cellular and molecular features of decidualization resistance (DR) at single-cell resolution? Summary answer There is an absence of the stromal-epithelial transition with aberrant crosstalk among cell types in essential pathways (e.g., endoglin, WNT) confirmed at the protein level. What is known already DR refers to the inability of the endometrium to undergo decidualization changes preparing the endometrium for a conceptus, noted in disorders like endometriosis, recurrent pregnancy loss, miscarriage and preeclampsia. The molecular mechanisms causing this disorder remains unknown. It has been demonstrated that there is a transcriptomic signature of DR in the endometrium of former preeclampsia patients, during and years after suffering from the disease. Our objective is to dilucidated what is DR at single-cell resolution. Study design, size, duration This cross-sectional study compares late secretory phase endometrial biopsies from patients that previously have suffered severe Preeclampsia (sPE). The sample size consisted of 11 sPE patients and 12 control participants for single-cell RNA sequencing (scRNA-seq), 8 sPE patients and 8 controls for spatial transcriptomic mapping covering 95 tissue regions, and 10 sPE patients and 10 controls for proteomic analysis. The study spanned 2 years, from sample collection to omics analysis. Participants/materials, setting, methods Late secretory phase endometrial biopsies from sPE patients and controls underwent molecular analyses. Methods included 10x Genomics protocol for single-cell RNA sequencing, MiloR to calculate cell abundance, CellChat to infer cell-to-cell communication and ClusterProfiler for gene ontology. We solved spatial mapping at transcriptomic level using Nanostring. Proteomics data was collected by laser-capture microdissection coupled mass spectrometry. Protein-protein networks, involving PR and ESR1 receptors as master regulators of decidualization, were built using String. Main results and the role of chance Initial morphological analyses of endometrial biopsies collected during the late secretory phase revealed gross anatomical defects in glandular morphology that were histologically confirmed. scRNA-seq revealed sPE-associated alterations in cell composition involving stromal and epithelial subtypes, denoting a higher proportion of cells expressing proliferative markers in the stroma (MMP11, SFRP1) and epithelium (IHH, EMID), and a near absent epithelial-stromal transition that is normally linked to decidualization. Further, abnormal abundance and gene dysregulation were detected in the three decidualized stages identified. Mapping cell-cell communication networks confirmed aberrant crosstalk among cell types involved in essential pathways (e.g., endoglin, WNT, SPP1). Spatial transcriptomic in a replication cohort of samples revealed a landscape of dysregulated genes in stromal, luminal and glandular enriched regions, corroborating sPE-associated DR. Laser capture microdissection/mass spectrometry of samples revealed differential protein expression at a global level and confirmed many of the scRNA-seq results, such as sPE-exclusive proteins related to proliferation and the absence of a stromal to epithelial transition at a pathway level. These data lend insights into potential molecular mechanisms underlying the pathogenesis of DR. Limitations, reasons for caution DR analysed in this work is associated with sPE but we do not know if this decidualization defect would be the same in other DR associated conditions such as endometriosis, recurrent pregnancy loss, or miscarriage. Wider implications of the findings Here we present the most comprehensive snapshot of DR to date, providing a full range of markers potentially responsible for this decidualization disorder linked to sPE. We concluded that stromal and epithelial cell populations are altered revealing a near absence of the stromal-epithelial transition, cellular crosstalk and glandular constitution. Trial registration number not aplicable

P-736 The therapeutic effect of hysteroscopic surgery on chronic endometritis in infertile women with intrauterine disorders

Abstract Study question Can chronic endometritis (CE) be treated by hysteroscopic surgery without antibiotic treatment in women with intrauterine disorders? Summary answer Most CE cases with intrauterine disorders were cured through hysteroscopic surgery without antibiotic therapy, regardless of the type of intrauterine abnormalities. What is known already Persistent inflammation of the local endometrium, CE, interferes with embryo implantation. CE is primarily caused by intrauterine infection; therefore, broad-spectrum antibiotic therapy is the current treatment for CE. Nevertheless, most cases with CE and endometrial polyps can be cured via hysteroscopic polypectomy without antibiotic therapy in our previous study. Study design, size, duration This study was a prospective cohort study approved by the Ethics Committee. To evaluate the therapeutic effect of hysteroscopic surgery on CE, the endometrial specimens for the immunohistochemistry analysis of the plasma cell marker CD138 were obtained to diagnose CE at surgery between November 2018 and June 2021. In the women diagnosed with CE, endometrial biopsy was performed without use of antibiotics in the subsequent menstrual cycle. Participants/materials, setting, methods The study population consisted of 337 consecutive infertile women who underwent hysteroscopic surgery. Eighty-nine consecutive patients without intrauterine disorders were also recruited as control group. CE was diagnosed as the presence of ≥ 5 CD138-positive cells in 10 nonoverlapping random stromal areas of high-power fields (HPF) in this study. Main results and the role of chance The prevalence of CE with ≥5 CD138-positive cells in women with no intrauterine disorder, endometrial polyps, myomas, intrauterine adhesion (IUA), and septate uterus were 15.7%, 85.7%, 69.0%, 78.9%, and 46.2%, respectively. Multivariate analysis revealed that CE was diagnosed significantly more often in the endometrial polyp (p &amp;lt; 0.0001, odds ratio [OR] 27.69, 95% confidence interval [CI] 15.01–51.08) and IUA groups (p &amp;lt; 0.0001, OR 8.85, 95%CI 3.26–24.05). The CE recovery rates by hysteroscopic surgery in the women with endometrial polyps, myomas, IUA, and septate uterus were 89.7%, 100%, 92.8%, and 83.3%, respectively (p = 0.45). Limitations, reasons for caution This study has limitations. First, some patients received a combination of estrogen and progestin or dienogest before surgery. These medicines may influence the number of CD138-positive cells. Second, ≥5 CD138-positive cells per 10 HPF were defined as the diagnosis of CE; however, different diagnostic criteria may generate different results. Wider implications of the findings CE is primarily caused by intrauterine infection due to a wide variety of microorganisms; however, most CE cases with intrauterine abnormalities were cured by hysteroscopic surgery without antibiotic use. Most CE cases associated with intrauterine abnormalities may be noninfectious CE. 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-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 &amp;lt; 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-306 GnRH agonists exert a positive influence on regulatory immune cells in the endometrium of women with adenomyosis

Abstract Study question How does treatment with GnRH-agonist (GnRH-a) prior to frozen embryo transfer (FET) in women with adenomyosis affect immune cell populations in endometrium and peripheral blood? Summary answer GnRH-a induces a decrease in monocytes and an increase in CD4+T-cells in peripheral blood and a decrease classical monocytes and T-regulatory cells in the endometrium What is known already Adenomyosis is associated with lower implantation and higher miscarriage rates. Observational studies have reported improved pregnancy outcomes in women with adenomyosis after assisted reproductive technologies (ART) and pre-treatment with GnRH-a prior to FET. However, the specific mechanisms of GnRHa action beyond central hormonal suppression remain unclear, but may be mediated through local and/or systemic immunomodulation. Study design, size, duration Prospective cohort study including infertile women with adenomyosis and GnRH-a treatment ≥3 months prior to FET. Pre- and post-treatment analysis of blood and endometrium samples were compared. Recruitment is ongoing with n = 30 women included thus far, 16 of whom have completed the study, while the remaining are still undergoing GnRH-a therapy. Participants/materials, setting, methods Adenomyosis was diagnosed sonographically if ≥ 2 criteria published by the MUSA group were met. Peripheral blood and endometrial samples were obtained before and after treatment with GnRH-a. Plasma cells and uterine Natural Killer (uNK) cells in endometrial biopsies were quantified with immunhistochemistry (IHC), and leukocyte populations in all samples were analysed using flow cytometry (FC). Pre- and post-treatment levels of immune cells and percentage of leukocyte populations were compared using Wilcoxon rank-sum tests (p &amp;lt; 0.05 significant) Main results and the role of chance Interim analysis of immune cell populations in peripheral blood and endometrium of the 16 women who already completed the study was performed. At baseline, IHC showed elevated plasma cell concentrations indicating chronic endometritis in 3/30 patients (10%), while uNK cells were elevated (&amp;gt;300/mm2) in 13/30 patients (43.3%). FC analysis of peripheral blood showed a significant decrease in percentage of monocytes (p = 0.01) and increase in percentage of T-cells (p = 0.03), specifically concentration of CD4+T-cells (p = 0.03), after treatment with GnRH-a as compared to pre-treatment levels. In the endometrium, following GnRH-a therapy, we observed a decrease in both the percentage of classical monocytes (p = 0.03) and T-regulatory cells (p = 0.03). All 16 participants who completed the study already underwent FET, resulting in ten pregnancies (62.5%), although four resulted in miscarriage, while the outcome of the remaining pregnancies is still pending. Limitations, reasons for caution These are preliminary results whose generalizability needs to be confirmed in a larger study population. No correction for multiple comparisons was performed and significant findings have to be interpreted with caution. Wider implications of the findings Elevated uNK on IHC cells seen at baseline may be associated with implantation failure, miscarriage. CD4+ and DN T-cells are important mediators of reproductive immune tolerance. In women with adenomyosis, GnRH-a may have a positive impact on these regulatory immune cells in peripheral blood as well as in the endometrium. Trial registration number Not applicable

P-283 Spatial transcriptomics identifies dysregulated pathways in adenomyosis proposing potential re-purposed lesion-specific treatments

Abstract Study question How does the transcriptome of adenomyosis lesions compare to the matched eutopic endometrium, and can they be targeted with lesion-specific treatments? Summary answer Adenomyosis lesions are transcriptionally distinct from matched eutopic endometrium, exhibiting upregulated cell motility pathways, increased cilia, and altered hormonal regulation, providing targets for lesion-specific treatments. What is known already Adenomyosis affects 10% of women of reproductive age and is caused by the presence of ectopic endometrium within the myometrium, causing heavy periods, chronic pain, and subfertility. The only definitive treatment is hysterectomy and there are no current fertility-sparing treatments available. Spatial transcriptomics allows interrogation of the transcriptome with spatial context, overcoming the limitations of bulk and sc-RNA sequencing technology. In the endometrium, spatial transcriptomics has providing insight into how cellular functions differ depending on their location and communication with neighbouring cells, however it has not yet been used to investigate adenomyosis. Study design, size, duration A retrospective observational study of ten women undergoing hysterectomy for heavy, painful periods, with a histological diagnosis of adenomyosis. All women were in the secretory phase of menstrual cycle and had not taken exogenous hormones for the preceding three months. Paired full-thickness endometrial and myometrial biopsies containing adenomyosis lesions were obtained from each woman. The presence of adenomyosis lesions was confirmed by the presence of endometrial glands and stroma &amp;gt;2.5mm from the endo-myometrial junction. Participants/materials, setting, methods Whole transcriptome profiling of the following regions of interest was performed: luminal epithelium, functionalis, basalis, and adenomyosis lesions, and immunofluorescent morphology markers were employed to identify epithelial, stromal, endothelial, and immune cells within each region. A generalised linear-mixed model was designed and unsupervised clustering, differential gene expression comparison, gene set enrichment analysis (GO and Reactome), ligand-receptor signalling, and in silico drug screening was performed. Statistical analyses were adjusted for a false discovery rate of (&amp;lt;0.05). Main results and the role of chance Adenomyosis lesions were more similar to endometrial basalis than functionalis, with 102 significantly DEG’s between adenomyosis lesions and endometrial basalis, and 282 significantly DEG’s between adenomyosis lesions and endometrial functionalis (adjusted p &amp;lt; 0.05). Adenomyosis lesion epithelium, stroma, and immune cells consistently revealed significantly downregulated DEG’s involved with cellular proliferation and apoptosis (FOS, JUNB, DUSP1) compared to the endometrium (adjusted p = 0.002). Pathway analysis revealed that adenomyosis lesion epithelium was positively enriched for terms relating to ciliation compared to the endometrium (adjusted p &amp;lt; 0.0001), and immunostaining confirmed an increased number of ciliated cells in adenomyosis lesions compared with eutopic endometrium in the same samples (n = 10, p &amp;lt; 0.01). Androgen receptor signalling was negatively enriched in the stroma of adenomyosis lesions compared to endometrial functionalis (adjusted P &amp;lt; 0.0001). Ligand-receptor signalling analysis revealed prominent crosstalk between endothelial-epithelial, and immune-epithelial cells in adenomyosis lesions, and a loss of stromal-epithelial cross-talk. TNFSF10 ligand from endothelial and immune cells showed high regulatory potential of gene receptors on epithelial cells known to be involved in cellular response to stress, regulation of growth, and apoptosis (DUSP1, DUSP5, EGR1, FOS, IER3, JUNB, PPP1R15A, SGK1). In-silico drug repurposing analysis revealed key compounds which may target adenomyosis lesions and spare the eutopic endometrium. Limitations, reasons for caution This is an observational study with a small sample size of ten which is a limitation, although is larger than the sample size of most published transcriptional studies of this type. Further validation work is needed to confirm the exploratory transcriptional findings outlined. Wider implications of the findings Fertility sparing, effective, lesion-specific treatments are urgently needed for women with adenomyosis. This study provides region and cell-type specific comparative transcriptional data between adenomyosis lesions and eutopic endometrium, identifying lesion-specific dysregulated pathways, insight into their biological relevance, and potential treatment targets to be trialled in future pre-clinical studies. Trial registration number Not applicable

O-098 The transcriptome of endometrial organoids of fertile and subfertile women exposed to seminal plasma of fertile men

Abstract Study question What is the effect of seminal plasma (SP) from fertile males on the transcriptome of endometrial epithelial organoids of fertile and subfertile women? Summary answer Transcriptomic changes in fertile and subfertile endometrial organoids are observed upon treatment with SP, related to hormone- and immune-regulation, reproduction and cellular organisation. What is known already Clinical studies have demonstrated the potential beneficial effect of intrauterine SP (liquid fraction of semen) insemination during assisted reproductive technology (ART), resulting in higher implantation- and pregnancy rates. However, the molecular mechanism by which SP exerts its function remains unclear. SP components such as TGF-β1 or IL-8 have the potential capacity of conditioning the uterus for pregnancy. SP has shown to induce significant transcriptional changes in 2D endometrial epithelial cells. 3D self-organising human endometrial organoids have been established over the last years, mimicking a more in vivo-like physiology, and therefore may better represent the interaction of the endometrium with SP. Study design, size, duration Endometrial tissue biopsies from fertile (n = 5) and subfertile (n = 5; subfertile defined as women not having reached clinical pregnancy after at least 12 months of regular unprotected intercourse) were used to establish endometrial epithelial organoids. Subsequently, SP from fertile males (n = 5) was collected and pooled. Endometrial organoids were cultured in complex media supplemented with 1 µM 17β-estradiol (E2) and incubated with or without (control) 1% SP for 6 h. Participants/materials, setting, methods RNA sequencing was performed using NextSeq600 (Illumina). Significant DEGs upon SP exposure were identified using DESeq2 (Fold Change (FC) of ≥ 1.5 or ≤ -1.5). Eight selected DEGs were validated by qPCR in one cell line. Principal Component Analysis (PCA) was performed to compare endometrium donor correlation. Gene Set Enrichment Analysis (GSEA; v4.3.2) was then used to identify overrepresented pathways and gene ontology (GO) gene sets (P &amp;lt; 0.05; False Discovery Rate (FDR) &amp;lt; 0.25). Main results and the role of chance High heterogeneity between donors was observed, regardless of their fertility phenotype. PCA analysis indicated clustering based on donor rather than SP treatment or phenotype. Moreover, the number of significantly DEGs ranged from 629 to 2332. Treatment with SP resulted in identification of 105 significant DEGs (p &amp;lt; 0.05) among all donors (fertile and subfertile) from which 80 DEGs were upregulated and 25 DEGs were downregulated. Several of these DEGs were also reported to be affected by SP in other 2D epithelial cultures studies. Interesting DEGs include progesterone-associated endometrial protein (PAEP; log2FC 2.0 ± 1.0), interleukin-6 (IL6; log2FC 4.5 ± 1.5), insulin-like growth factor-binding protein 3 (IGFBP3; log2FC 1.1 ± 0.2) and mucin 6 (MUC6; log2FC 3.7 ± 1.6). GSEA analysis identified gene set enrichment of pathways and biological processes related to hormone- and immune regulation, reproduction, and cellular organisation in all endometrial organoids treated with SP. Preliminary RNAseq validation with qPCR showed comparable expression patterns for 8 genes (e.g. PAEP and IL6). Moreover, expression of these genes was uniform when using a distinct SP batch (n = 6; fertile males). No clear differential response between fertile and subfertile organoids upon SP exposure was observed. Limitations, reasons for caution The apical-inside organisation of the organoids (i.e. SP interaction from the basolateral side) should be taken into account, since in vivo interaction will be apically. Moreover, high heterogeneity between donors was observed between and within the fertility phenotypes. Therefore, conclusions based on fertility phenotypes should be drawn with caution. Wider implications of the findings Understanding the effect of SP on the endometrium will shed light on male-female reproductive tract crosstalk, which is omitted during ART. Future research should focus on identifying key regulatory SP molecules and functional studies should include apical SP interaction. Potentially providing therapeutic options for improving success rates in ART. Trial registration number Not applicable