The endometrium undergoes rapid cycles of menstrual breakdown and repair. In each cycle, oestrogen-dependent proliferation followed by progesterone-dependent differentiation of the endometrium culminates in a sterile inflammatory tissue response, termed the decidual reaction, at the start of the embryo implantation window. Analysis of timed endometrial biopsies is widely used to investigate a spectrum of reproductive disorders, including recurrent implantation failure in IVF and recurrent miscarriage. Deep profiling of whole slide images (WSIs), capturing the spatial and functional organization of key histological structures, such as nuclei, glandular and luminal epithelium, subluminal stroma and spiral arterioles, holds significant promise for automated endometrial assessment. To address the lack of such methodologies, we developed ASTER, a multi-task deep learning model for simultaneous segmentation of multiple histological structures in immunostained endometrial WSIs. ASTER has been developed and validated over a large dataset of 2,652 endometrial whole slide images, including 35,135 annotated objects obtained using a pathologist-in-the-loop methodology. The model demonstrates strong performance across all segmentation tasks. Further, analysis of an independent set of 2,082 unseen WSIs showed that ASTER-derived features correlate with cycle-dependent endometrial gene expression. This represents the first systematic study linking segmented morphological characteristics to molecular and temporal markers of endometrial function, demonstrating its effectiveness in enabling comprehensive and automated profiling. This highlights the potential of ASTER to support personalized management of women experiencing reproductive failure. Segmentation results are available for interactive exploration at https://tiademos.dcs.warwick.ac.uk/bokeh_app?demo=ASTER.

In every menstrual cycle, progesterone acting on estrogen-primed endometrium elicits an inflammatory decidual reaction, rendering it poised for embryo implantation and transformation into the decidua of pregnancy. Here, we show that the sequential functions of the decidual reaction-implantation and decidualization-pivot on the time-sensitive loss of progesterone-resistant DIO2+ stromal cells that form a specialized implantation niche and reciprocal expansion of progesterone-dependent PLA2G2A+ predecidual cells. Simultaneously, uterine natural killer (uNK) cell proliferation results in the accumulation of immunotolerant subsets. Examination of endometrial biopsies from 924 women revealed that the recurrence risk of miscarriage closely aligns with the incidence of a weakened or stalled decidual reaction, more so than poor uNK cell expansion. Analysis of paired biopsies obtained in different cycles and modeling in assembloids intimated that prior miscarriages disrupt intercycle endometrial homeostasis and calibration of the decidual reaction. Our findings show that erosion of the decidual reaction following a miscarriage drives the recurrence risk irrespective of maternal age.

Does the gut microbiota play a role in changing the local immune response to an adverse vaginal microbiota? We show that in those with an adverse vaginal microbiota, a healthy gut can protect you and dampen inflammation at the local cervicovaginal level. There is growing evidence to implicate the role of the vaginal microbiota in reproductive health. We have recently shown that an adverse vaginal microbiota accompanied by a heightened inflammatory response is linked to euploid miscarriage. However, it is unclear what modulates this immune response as some women with suboptimal vaginal microbial composition go on to deliver term pregnancies. This was a prospective observational cohort study based at Queen Charlotte’s and Chelsea Hospital, Early Pregnancy Unit, London between July 2017 and February 2019. We analysed the vaginal and rectal microbiota in 47 miscarriage patients (29 euploid and 18 aneuploid, determined following cytogenetic analysis) and 49 viable pregnancies. Here we investigate the role of the rectal microbiota in relation to miscarriage and the local cervical vaginal immune response. We used 16S rRNA gene based metataxonomics to interrogate the vaginal and rectal microbiota in 29 euploid and 18 aneuploid miscarriages and 49 viable pregnancies. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid. There was no difference in the richness or diversity of the rectal microbiota in the different pregnancy outcomes. In patients with an adverse vaginal microbiota, there was a significantly higher Firmicutes/Bacteroidetes ratio (a marker of gut dysbiosis) in euploid miscarriage compared to aneuploid miscarriage (P=0.02). Finegoldia magna, Prevotella bivia, Dialister invisus and Peptoniphilus gorbachii were enriched in euploid miscarriage whereas relative abundance of Clostridium XIVa and Bacteroidetes was higher in aneuploid miscarriage. In the whole cohort, anti-inflammatory cervical vaginal cytokines were significantly higher in patients with a healthy gut microbiota (low F/B ratio). In those with a healthy vaginal microbiota, rectal dysbiosis was not associated with cervicovaginal cytokines. However, in patients with an adverse vaginal microbiota a low F/B ratio was associated with significantly increased concentrations of anti-inflammatory cervicovaginal cytokines (IL4 and IL10) compared to patients with a high F/B ratio (P=0.01 and 0.04 respectively). This study provides initial insights into the potential role the gut microbiota has to play in miscarriage. The next steps would involve longitudinal preconception profiling of the patient cohort alongside stool cultures and detailed diet and lifestyle questionnaires to further advance our knowledge on the topic. We postulate there may be crosstalk between the vaginal microbiota and gut dysbiosis in euploid miscarriages with an effect of gut dysbiosis on inflammatory responses. This provides a rationale for healthy eating to maintain gut health, and using live biotherapeutics orally and vaginally to improve reproductive health and pregnancy outcomes. No

Understanding the link between endometrial gene expression and corresponding histological phenotypes is crucial for assessing luteal phase timing and reproductive success. While current profiling techniques do not typically utilize tissue whole slide images (WSIs), this study underscores their importance, demonstrating significant potential benefits such as deeper spatio-molecular and temporal profiling, cost reduction, and scalability. We present Endonama, a graph neural network (GNN) that predicts the expression of 13 key genes directly from 1,834 CD56-stained endometrial WSIs, comprising over 5 million image patches, thereby reducing reliance on molecular assays. This represents the largest endometrial histology study to date, as well as the first to predict receptivity directly from WSIs. The model also accurately estimates clinically relevant biomarkers, including the SLC15A2/GPX3 ratio for personalized embryo transfer timing (Spearman's. = 0.731, 95% CI: 0.616-0.814) and the PLA2G2A/DIO2 ratio, an age-independent marker of miscarriage risk, supporting targeted interventions and long-term monitoring of recurrent pregnancy loss. Leveraging its graph-based design, Endonama produces interpretable spatial heatmaps that reveal localized and temporal variations in gene expression, providing insights into tissue heterogeneity and function. Endonama offers a powerful tool for personalized fertility assessment and paves the way for new advances in reproductive medicine. Interactive visualizations of Endonama's predictions are available at https://tiademos.dcs.warwick.ac.uk/bokeh_app?demo=Endonama.

To understand the relationship between endometrial CD138 expression and the reproductive tract microbiota. CD138+ staining of endometrium is associated with general delay in endometrial maturation but not the composition of the microbiota. Chronic endometritis (CE) is a purported cause of recurrent miscarriage (RM). This asymptomatic inflammation of the endometrium has been associated with RM, with several studies demonstrating a higher prevalence than the general population. CE has previous been associated with a reduction in Lactobacilli species and a dysbiotic endometrial microbiota. CD138 is a widely used plasma cell marker utilised to aid the diagnosis of CE. However, CD138, a transmembrane heparan sulfate proteoglycan, is expressed by other cells in human endometrium. Its expression has previously been shown to vary across the menstrual cycle. The implications of this remain under-explored. Translational cohort study embedded within a randomised controlled trial with a subset of 103 samples derived from a trial population of 737 women. Women aged ≥18 to < 42 yrs, with a history of two or more first trimester consecutive miscarriages were recruited from specialist Recurrent Pregnancy Loss (RPL) clinics. A timed endometrial biopsy alongside endometrial microbial sampling was performed following ovulation (10±4 days). Additional samples were obtained from the Tommy’s National Reproductive Health Biobank. Samples underwent CD138 immunostaining, histological and molecular dating analysis, immune profiling, cytokine analysis and bacterial metataxonomic profiling with 16S ribosomal RNA (rRNA) sequencing analysis. Non-immune expression of CD138 was demonstrated across all endometrial cell populations. Stromal expression was very high (>200 CD138-positive stromal cells/10mm2) in 26 out of 27 proliferative endometrial samples. While CD138 immunoreactivity in the stroma declines markedly following ovulation (Mann Whitney U-Test; p < 0.005), gene expression analysis demonstrated persistently elevated SDC1 (CD138) transcript levels in a subset of non-immune stromal cells. Immunohistochemistry revealed three patterns of CD138 immunoreactivity: punctate staining, diffuse staining, and a mixed pattern. When compared to CD138 negative samples, conspicuous diffuse staining in the stromal compartment was associated with significantly earlier histological dating (p < 0.01) and lower molecular dating ratios (p < 0.01). Sequencing of paired vaginal and ectocervical swabs and endometrial Tao brush samples collected from 114 patients demonstrated tightly interconnected microbial ecosystems across the low reproductive tract. There was no association between the pattern or severity of CD138 immunoreactivity in luteal phase endometrium and vaginal, ectocervical or endometrial community state types (p < 0.05). Analysis of supernatants of vaginal and ectocervical swabs and Tao Brush revealed an inverse correlation between the pattern and severity of stromal CD138 immunoreactivity in endometrial stroma and secreted levels of TNF-α, CXCL/BRAK and VEGF (q-value < 0.05). This study relied on self-reported ovulation testing using commercially available urine LH tests. In addition, multiple statistical testing was utilised across differing data types. The consistency of the results seen, however, adds credence to the findings. This study challenges the assumptions underlying the reliability of CD138 IHC to detect plasma cells infiltration. It suggests the possibility of a confounding relationship of delayed endometrial maturation within the literature on CE to date. CD138-based CE testing and treatment should not therefore be performed outside of a research context. Yes

Recurrent implantation failure (RIF) is defined after three or more good quality embryo transfers following in vitro fertilisation without a successful pregnancy outcome. Many factors contribute to RIF however, the endometrial contribution remains unclear. Previous work by our group has identified a micro-RNA (miRNA) miR-185-5p as conserved across placental mammal irrespective of implantation strategies. We tested the hypothesis that miR-185-5p and the pathways it regulates, may be disrupted in the endometria of women with RIF. A human endometrial epithelial cells line (Ishikawa cells) was transfected with mimics or inhibitors for miR-185-5p for 24 (for implantation assay) or 48 hr (for proteomic analysis) along with non-targeting controls. There was a significant different in percentage attachment of BeWoW spheroids to cells transfected with miR-185-5p mimic compared to inhibitor (P<0.05). Transfection of epithelial cells with miR-185-5p altered expression of 1450 (mimic alone) and 509 (inhibitor alone) proteins respective of which, 146 were modified by both. Comparison of predicted targets of miR-185-5p, proteins modified by this study, and key endometrial genes from the literature determined genes and proteins associated with CNP family were further investigated in biopsies from individuals with (n=10) and without RIF (n=9) with CSNK1D expression significantly lower (p<0.05) in individuals with RIF. Collectively these data demonstrate that miR-185-5p modifies pathways that are important for successful implantation in humans. ### Competing Interest Statement The authors have declared no competing interest.

Early human trophoblast development has remained elusive due to the inaccessibility of the early conceptus. Non-human primate models recapitulate many features of human development and allow access to early postimplantation stages. Here, we tracked the pre- to postimplantation transition of the trophoblast lineage in superficially implanting marmoset embryos in vivo. We differentiated marmoset naive pluripotent stem cells into trophoblast stem cells (TSCs), which exhibited trophoblast-specific transcriptome, methylome, differentiation potential, and long-term self-renewal. Notably, human TSC culture conditions failed to support marmoset TSC derivation, instead inducing an extraembryonic mesoderm-like fate in marmoset cells. We show that combined MEK, TGF-β/NODAL, and histone deacetylase inhibition stabilizes a periimplantation trophoblast-like identity in marmoset TSCs. By contrast, these conditions differentiated human TSCs toward extravillous trophoblasts. Our work presents a paradigm to harness the evolutionary divergence in implantation strategies to elucidate human trophoblast development and invasion.

Understanding the process of human embryo implantation is impeded by the inability to study this phenomenon in vivo, thus limiting opportunities to gain knowledge to in vitro modeling. Previous models have relied on monolayer co-cultures, which do not replicate the complexity of endometrial tissue. Here, we detail the establishment of three-dimensional endometrial assembloids, comprising gland-like epithelial organoids in a stromal matrix. Endometrial assembloids mimic endometrial tissue structure more faithfully and can be used to study human embryo-endometrial interactions. Co-cultures of human embryos and endometrial assembloids will enhance our fundamental understanding of these processes as well as allowing us to study the mechanisms of persistent reproductive failure.

Summary Decidualization denotes the differentiation of endometrial stromal cells into specialized decidual cells, essential for embryo implantation and pregnancy. The process requires coordinated activation of both progesterone and cAMP signaling pathways, which converge on downstream transcription factors. PGE2 and relaxin, acting respectively through their Gαs-coupled GPCRs EP2 and RXFP1, are putative candidates responsible for generating cAMP in differentiating stromal cells. Here, we show that PGE2 is less efficacious than relaxin in elevating intracellular cAMP levels in primary stromal cells but more effective at driving the expression of canonical decidual genes. Both PGE2- and relaxin-induced cAMP generation involves receptor internalization, but EP2 is endocytosed into very early endosomes (VEEs). Perturbation of the VEE machinery dysregulates PGE2-dependent cAMP profiles and disrupts key decidual signaling pathways, resulting in a disordered differentiation response. We demonstrate that the spatial location of EP2 is essential for coordinated activation of the downstream signaling cascades that govern decidualization.

The human endometrium is a dynamic entity that plays a pivotal role in mediating the complex interplay between the mother and developing embryo. Endometrial disruption can lead to pregnancy loss, impacting both maternal physical and psychological health. Recent research suggests that the endometrial microbiota may play a role in this, although the exact mechanisms are still being explored, aided by recent technological advancements and our growing understanding of host immune responses. Suboptimal or dysbiotic vaginal microbiota, characterized by increased microbial diversity and reduced Lactobacillus dominance, has been associated with various adverse reproductive events, including miscarriage. However, the mechanisms linking the lower reproductive tract microbiota with pregnancy loss remain unclear. Recent observational studies implicate a potential microbial continuum between the vaginal and endometrial niche in patients with pregnancy loss; however, transcervical sampling of the low biomass endometrium is highly prone to cross-contamination, which is often not controlled for. In this review, we explore emerging evidence supporting the theory that a dysbiotic endometrial microbiota may modulate key inflammatory pathways required for successful embryo implantation and pregnancy development. We also highlight that a greater understanding of the endometrial microbiota, its relationship with the local endometrial microenvironment, and potential interventions remain a focus for future research.

Decidualization denotes the differentiation of endometrial stromal cells into specialized decidual cells, essential for embryo implantation and pregnancy. The process requires coordination of progesterone and cAMP signaling, which converge on downstream transcription factors. PGE2 and relaxin, acting, respectively, through Gαs-coupled GPCRs EP2 and RXFP1, are putative candidates for generating cAMP in differentiating stromal cells. Here, we show that PGE2 is less efficacious than relaxin in elevating intracellular cAMP levels in primary stromal cells but more effective at driving the expression of decidual genes. PGE2-and relaxin-induced cAMP generation involves receptor internalization, but EP2 is endocytosed into very early endosomes (VEEs). Perturbation of VEE machinery through depletion of key trafficking proteins; APPL1 and GIPC, dysregulates PGE2-dependent cAMP profiles and disrupts key decidual signaling pathways, resulting in a disordered differentiation response. We demonstrate that regulation of EP2 via internalization is essential for coordinated activation of the downstream signaling cascades that govern decidualization.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.

Decidualization denotes the process of inflammatory reprogramming of endometrial stromal cells (EnSC) into specialized decidual cells (DC). During this process, EnSC are subjected to endoplasmic reticulum (ER) stress as well as acute cellular senescence. Both processes contribute to the proinflammatory mid-luteal implantation window and their dysregulation has been implicated in reproductive failure. Here, we evaluated the link between ER stress, decidual differentiation and senescence. In-silico analysis identified HSPA5 gene, codifying the ER chaperone BiP, as a potentially critical regulator of cell fate divergence of decidualizing EnSC into anti-inflammatory DC and proinflammatory senescent decidual cells (snDC). Knockdown of HSPA5 in primary EnSC resulted both in decreased expression of DC marker genes and attenuated induction of senescence associated βgalactosidase activity, a marker of snDC. Stalling of the decidual reaction upon HSPA5 knockdown was apparent at 8 days of differentiation and was preceded by the upregulation of ER stress associated proteins IRE1α and PERK. Further, HSPA5 knockdown impaired colony-forming unit activity of primary EnSC, indicative of loss of cellular plasticity. Together, our results point to a key role for HSPA5/BiP in decidual transformation of EnSCs and highlight the importance of constraining ER stress levels during this process.

Endometrial cancer is a multifactorial disease with inflammatory, metabolic and potentially microbial cues involved in disease pathogenesis. Here we sampled different regions of the reproductive tract (vagina, cervix, endometrium, fallopian tubes and ovaries) of 61 patients and showed that the upper genital tract of a subset of women with and without endometrial cancer harbour microbiota quantitatively and compositionally distinguishable from background contaminants. A microbial continuum, defined by detection of common bacterial species along the genital tract, was noted in most women without cancer while the continuum was less cohesive in endometrial cancer patients. Vaginal microbiota were poorly correlated with rectal microbiota in the studied cohorts. Endometrial cancer was associated with reduced cervicovaginal and rectal bacterial load together with depletion of Lactobacillus species relative abundance, including L. crispatus, increased bacterial diversity and enrichment of Porphyromonas, Prevotella, Peptoniphilus and Anaerococcus in the lower genital tract and endometrium. Treatment of benign and malignant endometrial organoids with L. crispatus conditioned media had minimal impact on cytokine and chemokine profiles. Our findings provide evidence that the upper female reproductive tract of some women contains detectable levels of bacteria, the composition of which is associated with endometrial cancer. Whether this is a cause or consequence of cancer pathophysiology remains to be elucidated.

Abstract Study question Is de-differentiation of committed endometrial stromal and epithelial cells in response to decidual senescence involved in regulating stemness of cycling human endometrium? Summary answer Transient decidual senescence promotes endometrial tissue rejuvenation by reprogramming stromal and epithelial cells into progenitor stem-like cells whereas chronic senescence causes stem cell depletion. What is known already In wound healing, acute but not prolonged senescence, a cellular state characterised by permanent cell cycle arrest and production of a complex secretome rich in inflammatory mediators, ECM proteins and proteinases, growth factors and angiogenic modulators, has been shown to promote de-differentiation of committed cells into stem-like progenitors, thereby enhancing tissue regeneration upon immune clearance of senescent cells. Menstrual repair depends on endometrial progenitor cells but whether they represent stromal and epithelial cells that have de-differentiated in response to acute premenstrual decidual senescence is not known. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from mid-luteal endometrial biopsies. The assembloids were then subjected over 36 days to 4 cycles of decidualization, a process associated with acute inflammatory senescence, followed by hormonal withdrawal. To induce chronic senescence, assembloids were decidualized continuously for 14 days. Stemness of assembloids was assessed in undifferentiated and decidualized cultures at the end of each ‘cycle’. Participants/materials, setting, methods 'Instant' assembloids, which closely recapitulate native endometrium, were established in collagen hydrogels from 10 midluteal biopsies and subjected to cyclical or prolonged decidualization. Decidualization was monitored by RT-qPCR analysis, using epithelial and stromal cells isolated at regular timepoints. The level of stemness of stromal and epithelial cells was measured by colony-forming unit (CFU) and organoid formation efficacy (OFE) assays, respectively. Main results and the role of chance Repeated cycles of hormonal stimulation and withdrawal resulted in cyclical decidualization of instant assembloids, as characterised by the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes when compared to parallel undifferentiated assembloids. Cyclicity enabled cells to maintain a healthy state and preserved the structural integrity of the assembloids. Further, cyclical decidualization of assembloids enhanced CFU activity and OFE activity of stromal and epithelial cells, respectively, indicative of active de-differentiation of committed cells. By contrast, chronic senescence, as induced by a prolonged decidualization, resulted in stem cell depletion in both glandular and stromal compartments and progressive loss of structural integrity of assembloids. We observed a reduced expression of the decidual marker gene PRL and an enhanced expression of IGFBP1 representing the increased stress state of cells. Taken together, induction of acute decidual senescence resulted in a robust de-differentiation response and increased abundance of stromal and epithelial progenitor cells, whereas prolonged senescence caused stem cell exhaustion in both cellular compartments. Limitations, reasons for caution Although the cellular responses observed in our ‘instant’ assembloid model were robust, caution is warranted when extrapolating from in vitro observations. Further, although the ‘instant’ assembloid model enables co-culturing of endometrial endothelial and immune cells, current hydrogels greatly limit their migratory capacity. The mechanisms of cellular de-differentiation are incompletely understood. Wider implications of the findings Our findings indicate that the level of premenstrual decidual senescence in the superficial layer regulates stemness in the basal layer, thus ensuring inter-cycle endometrial homeostasis. Conversely, prolonged decidual senescence associated with clinical miscarriages may plausibly increase the risk of further pregnancy loss by depleting stemness of the regenerative basal layer. Trial registration number N/A