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Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.

Study Question: Can a co-culture of three cell types mimic the in vivo layers of the uterine wall?, Summary Answer: Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo., What Is Known Already: We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells., Study Design, Size, Duration: Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min., Participants/materials, Setting, Methods: The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm., Main Results and the Role of Chance: We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization., Large Scale Data: N/A., Limitations, Resons for Caution: The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects., Wider Implications of the Findings: Numerous in vitro models were developed in order to mimic the human endometrium and endometrium-myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases., Study Finding/competing Interest(s): This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Intrauterine growth restriction (IUGR) pathophysiology is driven by abnormal uterine natural killer cell (uNK) activity leading to placental dysfunction. Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can improve experimental IUGR by mechanisms not fully understood. We sought to examine TRASCET's effects in downstream products of uNKs in a model of IUGR: 15 Sprague-Dawley dams were exposed to alternating hypoxia (10.5% O 2 ) from gestational day 15 (E15) until term (E21). Their fetuses ( n = 189) were divided into four groups. One group remained untreated ( n = 52), whereas three groups received volume-matched intraamniotic injections of either saline (sham, n = 44) or a suspension of amniotic fluid-derived MSCs, either in their native state (TRASCET, n = 50) or "primed" to an enhanced antiinflammatory phenotype (TRASCET-Primed, n = 43). Normal fetuses served as controls ( n = 33). At term, various analyses were performed, including ELISA for surrogates of placental inflammation and uNK activity. Statistical comparisons included Bonferroni-adjusted criterion. Overall survival from hypoxia was 74% (140/189). Placental efficiency was lower in untreated and sham but normalized in both TRASCET groups ( P < 0.01-0.47). Interleukin-17, a stimulator of uNKs, was elevated from normal in all groups ( P < 0.01 for all). Interferon-gamma, released from activated uNKs, was elevated in all groups except sham but lower than the untreated in both TRASCET groups ( P ≤ 0.01-0.06). Tumor necrosis factor-alpha, also produced by uNKs, was elevated in untreated and sham ( P < 0.01 for both), but normalized by TRASCET ( P = 0.05) and even lowered from normal in TRASCET-Primed ( P < 0.01). Vascular endothelial growth factor, also released by uNKs, was elevated in untreated and sham but lower than normal in both TRASCET groups ( P < 0.01 for all). We conclude that TRASCET with MSCs modulates the activity of placental uNKs in experimental IUGR, with distinct effects on their downstream products. This mechanistic insight may inform the development of novel strategies for the management of this disease.

During early pregnancy in mice, the establishment of uterine receptivity and endometrial decidualization require the extensive proliferation and differentiation of endometrial epithelial cells or stromal cells. Pin1 has been suggested to act as a molecular 'timer' of the cell cycle and is involved in the regulation of cellular proliferation and differentiation by binding many cell-cycle regulatory proteins. However, its physiological role during early pregnancy is still not fully understood. Here, we employed immunohistochemistry to determine the spatiotemporal pattern of Pin1 expression during early pregnancy. We found that Pin1 was mainly localized in subluminal stromal cells on day 4, in the decidual zone on days 5 to 8 of pregnancy and in artificial decidualization. Using a uterine stromal cell culture system, we found that progesterone, but not estrogen, induced the expression of Pin1 in a progesterone receptor-dependent manner. Inhibition of Pin1 in the uterus leads to impaired embryo implantation and decidualization in mice. Notably, a decrease in Pin1 activation affected the functional execution of several implantation- or decidualization-related factors. These findings provide new evidence for a previously unknown function of Pin1 in mediating embryo implantation and decidualization during successful pregnancy establishment and maintenance., (© 2024 Federation of American Societies for Experimental Biology.)

Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns., Competing Interests: Declaration of competing interest None, (Copyright © 2023. Published by Elsevier Ltd.)

The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The invasive trophoblast cell lineages in rat and human share crucial responsibilities in establishing the uterine-placental interface of the hemochorial placenta. These observations have led to the rat becoming an especially useful animal model for studying hemochorial placentation. However, our understanding of similarities or differences between regulatory mechanisms governing rat and human invasive trophoblast cell populations is limited. In this study, we generated single-nucleus ATAC-seq data from gestation day 15.5 and 19.5 rat uterine-placental interface tissues, and integrated the data with single-cell RNA-seq data generated at the same stages. We determined the chromatin accessibility profiles of invasive trophoblast, natural killer, macrophage, endothelial and smooth muscle cells, and compared invasive trophoblast chromatin accessibility with extravillous trophoblast cell accessibility. In comparing chromatin accessibility profiles between species, we found similarities in patterns of gene regulation and groups of motifs enriched in accessible regions. Finally, we identified a conserved gene regulatory network in invasive trophoblast cells. Our data, findings and analysis will facilitate future studies investigating regulatory mechanisms essential for the invasive trophoblast cell lineage., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR) 1-3 . However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells., (© 2023. The Author(s).)

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively ( n = 182) and negatively ( n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively ( n = 22) and negatively ( n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure., Competing Interests: The authors have declared that no competing interests exist.

Zearalenone (ZEN)-contaminated diets induce detrimental effects on the bovine reproduction. Recently, we reported that active sperm induce pro-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. This study aimed to investigate the impact of presence of ZEN on the sperm-uterine crosstalk in vitro. BEECs monolayers were stimulated by ZEN (10, 100, and 1000 ng/mL) for 0, 3, 6, 12, or 24 h and gene expressions were analyzed by real-time PCR. Moreover, BEECs were pre-exposed to ZEN (10, 100, and 1000 ng/mL) for 24 h then, co-incubated with sperm for 6 h. Conditioned media (CM) from a sperm-BEECs co-culture, after pre-exposure to ZEN, were harvested and exploited to challenge either polymorphonuclear cells (PMNs) or sperm. Both PMNs phagocytic activity toward sperm and sperm motility parameters were then assessed. Results showed that ZEN alone induced pro-inflammatory responses in BEECs through the induction of mRNA expressions of pro-inflammatory cytokines (TNFA and IL1B) and PGES1 at different time points. Pre-exposure of BEECs to ZEN, amplified the sperm-triggered upregulation of pro-inflammatory cytokines (TNFA and IL1B) and chemokine IL8 mRNA abundance in BEECs. Sperm-BEECs conditioned media, primed by ZEN, stimulated the PMNs phagocytosis for sperm whereas suppressed sperm motility parameters. Taken together, these findings indicate that the presence of ZEN augments the pro-inflammatory cascade triggered by sperm in BEECs, provokes PMNs phagocytosis for sperm, and reduces sperm motility parameters. Such immunological reactions may create a hostile environment for sperm competence and survival in the bovine uterus, thus impair fertility., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the adult uterus of mice, rats and humans, the initially closely packed muscle bundles of the inner myometrium (muscular tissue that encircles the endometrium where the conceptus implants) undergo a pregnancy-induced dispersal that is clinically significant and hypothesized to regulate important pregnancy events. However, where, when and how this dispersal occurs, what its functions are, as well as its spatial relationship to the mouse metrial gland/mesometrial lymphoid aggregate of pregnancy (MG/MLAp), are unknown. The MG/MLAp, is a pregnancy-induced uterine structure required for successful rodent pregnancy located mesometrial to (above) the decidua basalis (pregnancy-modified mesometrial endometrium) and defined by its accumulation of maternal lymphocytes known as uterine Natural Killer (uNK) cells. To begin to understand how mouse inner myometrium dispersal (IMD) occurs, we spatiotemporally described it by observing the distribution of its muscle bundles and measuring their volume fraction (VF), as well as the VF of uNKs and stromal cells of inner myometrium. We discovered that (a) IMD (defined as reduction in VF of inner myometrium muscle bundles) is restricted to the mesometrial half of the uterus, is first evident at Embryonic day (E) 5.5 (early postimplantation) but not at E3.5 (preimplantation), further increases between E6.5 and E7.5 and remains unchanged from E7.5 to E10.5, (b) IMD initiation (observed between E3.5 and E5.5) occurs in the absence of uNKs and is associated with VF increases of pre-existing inner myometrium stromal cells and (c) the IMD observed between E6.5 and E7.5 is not associated with VF increases of uNKs or stromal cells. To get functional clues about IMD, we examined whether stromal cells between the dispersed muscle bundles undergo decidualization (important for correct fetomaternal interactions) and provide evidence that they do by E10.5, based on their production of Desmin (decidualization marker). Lastly, we examined whether mouse MG/MLAp only comprises the dispersed inner myometrium or additionally includes the mesometrial triangle (a triangular-like area mesometrial to the inner myometrium at the mesometrium-uterus attachment site), as is the case in rats. Our data supports that the dispersed inner myometrium is the only tissue that makes up the mouse MG/MLAp. In conclusion, we provide novel cellular and spatiotemporal insights about IMD that will contribute to understanding its mechanism and function and allow more informed inter-species comparisons about this process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice., Methods: The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG., Results: The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells., Conclusions: This new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience., (© 2021. The Author(s).)

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Retinoic acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to a lack of genetic data, it was unclear whether RA signaling is truly required for implantation and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant-negative form of RA receptor (RAR) specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/bone morphogenetic protein 2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with a pan-RAR antagonist to show that in vitro decidualization is impaired. RNA interference perturbation of individual RAR transcripts in hESCs revealed that RARα in particular was essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling was crucial for mammalian embryo implantation, and its disruption led to failure of uterine receptivity and decidualization, resulting in severely compromised fertility.

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry., (© 2021 Federation of American Societies for Experimental Biology.)

Decidualization is a crucial step for human reproduction, which is a prerequisite for embryo implantation, placentation and pregnancy maintenance. Despite rapid advances over recent years, the molecular mechanism underlying decidualization remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of a mouse uterus during decidualization. By analyzing the undecidualized inter-implantation site of the uterus as a control, we were able to identify global gene expression changes associated with decidualization in each cell type. Additionally, we identified intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. Our data provide a valuable resource for deciphering the molecular mechanism underlying decidualization.

The gut microbiota is influenced by environmental factors such as food. Maternal diet during pregnancy modifies the gut microbiota composition and function, leading to the production of specific compounds that are transferred to the fetus and enhance the ontogeny and maturation of the immune system. Prebiotics are fermented by gut bacteria, leading to the release of short-chain fatty acids that can specifically interact with the immune system, inducing a switch toward tolerogenic populations and therefore conferring health benefits. In this study, pregnant BALB/cJRj mice were fed either a control diet or a diet enriched in prebiotics (Galacto-oligosaccharides/Inulin). We hypothesized that galacto-oligosaccharides/inulin supplementation during gestation could modify the maternal microbiota, favoring healthy immune imprinting in the fetus. Galacto-oligosaccharides/inulin supplementation during gestation increases the abundance of Bacteroidetes and decreases that of Firmicutes in the gut microbiota, leading to increased production of fecal acetate, which was found for the first time in amniotic fluid. Prebiotic supplementation increased the abundance of regulatory B and T cells in gestational tissues and in the fetus. Interestingly, these regulatory cells remained later in life. In conclusion, prebiotic supplementation during pregnancy leads to the transmission of specific microbial and immune factors from mother to child, allowing the establishment of tolerogenic immune imprinting in the fetus that may be beneficial for infant health outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Brosseau, Selle, Duval, Misme-Aucouturier, Chesneau, Brouard, Cherbuy, Cariou, Bouchaud, Mincham, Strickland, Barbarot and Bodinier.)

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huhn, Zhao, Esposito, Moffett, Colucci and Sharkey.)

The uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies.

Various gynecologic diseases and chemoradiation or surgery for the management of gynecologic malignancies may damage the uterus and ovaries, leading to clinical problems such as infertility or early menopause. Embryo or oocyte cryopreservation-the standard method for fertility preservation-is not a feasible option for patients who require urgent treatment because the procedure requires ovarian stimulation for at least several days. Hormone replacement therapy (HRT) for patients diagnosed with premature menopause is contraindicated for patients with estrogen-dependent tumors or a history of thrombosis. Furthermore, these methods cannot restore the function of the uterus and ovaries. Although autologous transplantation of cryopreserved ovarian tissue is being attempted, it may re-introduce malignant cells after cancer treatment. With the recent development in regenerative medicine, research on engineered biomaterials for the restoration of female reproductive organs is being actively conducted. The use of engineered biomaterials is a promising option in the field of reproductive medicine because it can overcome the limitations of current therapies. Here, we review the ideal properties of biomaterials for reproductive tissue engineering and the recent advancements in engineered biomaterials for the regeneration of female reproductive organs.

Impairment of uterine structure and function causes infertility, pregnancy loss, and perinatal complications in humans. Some types of uterine impairments such as Asherman's syndrome, also known as uterine synechiae, can be treated medically and surgically in a standard clinical setting, but absolute defects of uterine function or structure cannot be cured by conventional approaches. To overcome such hurdles, partial or whole regeneration and reconstruction of the uterus have recently emerged as new therapeutic strategies. Transplantation of the whole uterus into patients with uterine agenesis results in the successful birth of children. However, it remains an experimental treatment with numerous difficulties such as the need for continuous and long-term use of immunosuppressive drugs until a live birth is achieved. Thus, the generation of the uterus by tissue engineering technologies has become an alternative but indispensable therapeutic strategy to treat patients without a functional or well-structured uterus. For the past 20 years, the bioengineering of the uterus has been studied intensively in animal models, providing the basis for clinical applications. A variety of templates and scaffolds made from natural biomaterials, synthetic materials, or decellularized matrices have been characterized to efficiently generate the uterus in a manner similar to the bioengineering of other organs and tissues. The goal of this review is to provide a comprehensive overview and perspectives of uterine bioengineering focusing on the type, preparation, and characteristics of the currently available scaffolds.

This study investigated the changes in the mRNA expression of transforming growth factor beta (TGF-β), plasminogen activators (PAs), and interleukin (IL) caused by sperm, as well as the regulatory mechanism of PA activity through TGF-β, in porcine uterine epithelial cells. The cells were isolated from the uterine horn of pig and co-incubated with Percoll-separated boar sperm (45% or 90%), or TGF-β for 24 h. The mRNA expression of TGF-β isoforms (TGF-β1, 2 and 3) and their receptors (TGF-β R1 and R2), PAs (urokinase-type, uPA; tissue-type, tPA; uPA receptor, uPAR; type 1 PA inhibitor, PAI-1), IL-6 and IL-8 was analyzed using real-time PCR. Supernatant was used to measure PA activity. Co-incubation with sperm from the 90% Percoll layer increased TGF-β1 mRNA, whereas TGF-β2 and TGF-β3 were decreased (P < 0.05). However, both TGF-βRs were not changed by the presence of the spermatozoa. Expression of tPA, PAI-1, IL-6, and IL-8 mRNA was down-regulated by 90% Percoll-separated sperm (P < 0.05), and sperm from 45% Percoll increased uPA expression (P < 0.05). TGF-β decreased tPA and IL-8 mRNA expression, and increased uPAR and PAI-1 mRNA (P < 0.05). The suppressive effect of TGF-β on PA activity was blocked by Smad2/3 and JNK1/2 signaling inhibitors (P < 0.05). In conclusion, sperm separated in 90% in porcine uterus could suppressed inflammation via modulation of TGF-β and down-regulation of PAs and ILs. Therefore, the regulatory mechanism of inflammation by sperm in the porcine uterus could be associated with interactions between numerous cytokines including TGF-β., Competing Interests: Declaration of Competing Interest No potential conflict of interest relevant to this article was reported., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Data on how the immune system reacts to decellularized scaffolds after implantation is scarce and difficult to interpret due to many heterogeneous parameters such as tissue-type match, decellularization method and treatment application. The engraftment of these scaffolds must prove safe and that they remain inert to the recipient's immune system to enable successful translational approaches and potential future clinical evaluation. Herein, we investigated the immune response after the engraftment of three decellularized scaffold types that previously showed potential to repair a uterine injury in the rat. Protocol (P) 1 and P2 were based on Triton-X100 and generated scaffolds containing 820 ng mg -1 and 33 ng mg -1 donor DNA per scaffold weight, respectively. Scaffolds obtained with a sodium deoxycholate-based protocol (P3) contained 160 ng donor DNA per mg tissue. The total number of infiltrating cells, and the population of CD45 + leukocytes, CD4 + T-cells, CD8a + cytotoxic T-cells, CD22 + B-cells, NCR1 + NK-cells, CD68 + and CD163 + macrophages were quantified on days 5, 15 and 30 after a subcutaneous allogenic (Lewis to Sprague Dawley) transplantation. Gene expression for the pro-inflammatory cytokines INF- γ , IL-1 β , IL-2, IL-6 and TNF were also examined. P1 scaffolds triggered an early immune response that may had been negative for tissue regeneration but it was stabilized after 30 d. Conversely, P3 initiated a delayed immune response that appeared negative for scaffold survival. P2 scaffolds were the least immunogenic and remained similar to autologous tissue implants. Hence, an effective decellularization protocol based on a mild detergent was advantageous from an immunological perspective and appears the most promising for future in vivo uterus bioengineering applications., (Creative Commons Attribution license.)

As a crucial step for human reproduction, embryo implantation is a low-efficiency process. Despite rapid advances in recent years, the molecular mechanism underlying embryo implantation remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of embryo implantation sites. By analyzing inter-implantation sites of the uterus as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Additionally, we predicted signaling interactions between uterine luminal epithelial cells and mural trophectoderm of blastocysts, which represent the key mechanism of embryo implantation. We also predicted signaling interactions between uterine epithelial-stromal crosstalk at implantation sites, which are crucial for post-implantation development. Our data provide a valuable resource for deciphering the molecular mechanism underlying embryo implantation.

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher ( p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.

Aquaporins (AQPs) are integral membrane proteins, which play an important role in water homeostasis in the uterus. According to the literature, the expression of aquaporins in reproductive structures depends on the local hormonal milieu. The current study investigated the effect of selected PKA kinase inhibitor H89 and MAPK kinase inhibitor PD98059, on the expression of AQP1 , 2 , 5 , and 7 , and steroid hormones (E 2 ), progesterone (P 4 ), and arachidonic acid (AA) in the porcine endometrium on days 18-20 and 2-4 of the estrous cycle (the follicular phase where estrogen and follicle-stimulating hormone (FSH) are secreted increasingly in preparation for estrus and the luteal phase where the ovarian follicles begin the process of luteinization with the formation of the corpus luteum and progesterone secretion, respectively). The luminal epithelial cells were incubated in vitro in the presence of the aforementioned factors. The expression of mRNA was determined by the quantitative real-time PCR technique. In general, in Experiment 1, steroid hormones significantly increased expression of AQP1 , 2 , and 5 while arachidonic acid increased expression of AQP2 and AQP7 . On the other hand, MAPK kinase inhibitor significantly decreased the expression of AQP1 and 5 . In Experiment 2, E 2 , P 4 , or AA combined with kinase inhibitors differentially affected on AQPs expression. E 2 in combination with PKA inhibitor significantly decreased expression of AQP1 but E 2 or P 4 combined with this inhibitor increased the expression of AQP5 and 7 . On the contrary, E 2 with PD98059 significantly increased AQP5 and AQP7 expression. Progesterone in combination with MAPK kinase inhibitor significantly downregulated the expression of AQP5 and upregulated AQP7 . Arachidonic acid mixed with H89 or PD98059 caused a decrease in the expression of AQP5 and an increase of AQP7 . The obtained results indicate that estradiol, progesterone, and arachidonic acid through PKA and MAPK signaling pathways regulate the expression of AQP1 and AQP5 in the porcine luminal epithelial cells in the periovulatory period.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Natural killer (NK) cell assessment has been attempted since the 1990s and, apart from antibody testing, is probably the commonest immune test available to clinicians. It is clear that simple enumeration of uterine NK cells by immunohistochemistry is inadequate, although better methodology such as flow cytometry may prove to be more beneficial in the future. Blood testing is an appealing noninvasive test that may be a marker for immune dysfunction, rather than a guide to uterine numbers per se. It is currently performed in women with repeated reproductive failure and should be done using tests of activation. Patients value investigation and clinicians should prefer it to blind empirical immune therapy in repeated reproductive failure cases. But, in addition to blood NK testing, a fundamental new NK genetic test (the KIR/HLA-C interaction) is likely to provide an even more effective diagnostic tool. Insights from KIR/HLA-C analysis imply that new immune therapy trials will need to take KIR/HLA-C results into account., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P 4 ) promotes CCA in the uterine epithelium and previous studies indicated that P 4 activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine-specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1-deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1-induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre-implantation P 4 supplementation is sufficient to restore these defects and embryo invasion. In Rb1-deficient uterine epithelial cells, TNFα-primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P 4 through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1-induced CCA is involved in TNFα-primed epithelial necroptosis at the implantation site for successful embryo invasion., (© 2021 The Authors.)

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

To determine the effects of Follicle Stimulating Hormone (FSH) treatment and subsequent withdrawal on uterine proliferation and estrogen receptor (ESR), Brahman crossbred heifers (n = 12) were twice daily injected with FSH (4, 3, and 2 mg/injection) on Days 17-19 of the estrous cycle (FSH 3 days) and (4 and 3 mg/injection) on Days 17-18 (FSH 2 days) and withdrawal with saline on Day 19 and (4 mg/injection) on Day 17 (FSH 1 day) and withdrawal with saline on Days 18-19. Uterine tissue was subjectively collected on Day 20 and microscopically classified to four regions: endometrial stroma (ES), surface endometrial gland (EG), deep endometrial gland (DG), and myometrium (Myo). The cell proliferation marker, Ki-67, was quantified as labeling index (LI) in uterine regions, and tissues were immunostained to detect ESR2 followed by image analysis. The LI of ES, EG, and DG was greater (P = 0.0018, P = 0.0005, and P = 0.0103; respectively) in heifers received FSH for 3 days. The expression of ESR2 protein on ES and EG was greatest (P < 0.0001 and P = 0.0036, respectively) in FSH 3 days-treated group. Thus, FSH administration during proestrus stimulates uterine cell proliferation, and ESR2 expressions are affected by FSH during proestrus and differentially distributed in the uterine regions., (© 2021 Japanese Society of Animal Science.)

We have earlier reported pluripotent, very small embryonic-like stem cells (VSELs) and slightly bigger endometrial stem cells (EnSCs) in adult mouse uterus and their regulation by gonadotropin and steroid hormones. VSELs can differentiate into cells of all three lineages in vitro; however, they neither expand readily in vitro nor compliment a developing embryo. In the present study, a robust protocol is described to enrich uterine stem/progenitor cells along with their characterization and variation across estrus cycle. After enzymatic digestion of adult mouse uterus, single-cell suspension obtained was spun at 1000 rpm (250 g) to pellet majority of cells. Stem cells remain buoyant at this speed and were pelleted by spinning supernatant at 3000 rpm (1000 g). Spherical, darkly stained VSELs (2-6 μm) with high nucleo-cytoplasmic ratio and EnSCs (> 6 μm) expressed OCT-4, NANOG, SSEA-1, SCA-1, and c-KIT. OCT-4-positive cells co-expressed SSEA-1, ERα, ERβ, PR, and FSHR. Transcripts specific for pluripotent state (Oct-4, Oct-4a, Sox-2, Nanog), primordial germ cells (Stella, Fragilis), and receptors for pituitary and steroid hormones (ERα, ERβ, PR, FSHR 1 and 3) were studied by RT-PCR in 3000 rpm pellet. Cell pellet collected at 3000 rpm showed 10-fold enrichment of VSELs (2-6 μm, viable cells with surface phenotype of LIN-CD45-SCA-1+) by flow cytometry and upregulation of pluripotent transcripts by qRT-PCR compared with 1000 rpm pellet. VSELs were maximal during estrus and metestrus phases of estrus cycle. To conclude, VSELs/EnSCs can be enriched from adult uterus using the strategy described here, vary in numbers across estrus cycle, and are vulnerable to endocrine disruption as they express steroid receptors.

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority., Competing Interests: Luc Leybaert holds a patent entitled ‘Vessel protect – use of connexin channel inhibitors to protect grafts’, PCTEP2013076001, US patent 14/650,553; there are no potential conflicts of interest to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ambient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies., (© 2020 Wiley-VCH GmbH.)

Purpose: Infertility is a debilitating situation that millions of women around the world suffer from, but the causal relationship between infertility and endometriosis is still unclear. We hypothesize that the immune cell populations of uterine natural killer cells (uNK) and plasma cells (PC) which define chronic endometritis could differ in patients with or without endometriosis and therefore be the link to endometriosis-associated infertility., Methods: Our retrospective study includes 173 patients that underwent an endometrial scratching in the secretory phase of the menstrual cycle and subsequently immunohistochemical examination for uNK cells and PC. Sixty-seven patients were diagnosed with endometriosis, 106 served as the control cohort., Results: The risk for an elevated number of uNK cells in women with endometriosis is not increased as compared to the control group. Our findings suggest that patients with endometriosis are 1.3 times more likely to have chronic endometritis (CE) as compared to those without and that the treatment with doxycycline might increase pregnancy rates. Endometriosis and an increased number of uNK cells seem to be unrelated., Conclusions: In contrast to the lately published connection between endometriosis, infertility and increased uNK cells, we could not find any evidence that patients with endometriosis are more prone to elevated uterine uNK cells. Counting of PC in endometrial biopsies might be a new approach in the search of biomarkers for the nonsurgical diagnosis of endometriosis since our findings suggest a connection.

Successful implantation is associated with a unique spatial pattern of vascular remodeling, characterized by profound peripheral neovascularization surrounding a periembryo avascular niche. We hypothesized that hyaluronan controls the formation of this distinctive vascular pattern encompassing the embryo. This hypothesis was evaluated by genetic modification of hyaluronan metabolism, specifically targeted to embryonic trophoblast cells. The outcome of altered hyaluronan deposition on uterine vascular remodeling and postimplantation development were analyzed by MRI, detailed histological examinations, and RNA sequencing of uterine NK cells. Our experiments revealed that disruption of hyaluronan synthesis, as well as its increased cleavage at the embryonic niche, impaired implantation by induction of decidual vascular permeability, defective vascular sinus folds formation, breach of the maternal-embryo barrier, elevated MMP-9 expression, and interrupted uterine NK cell recruitment and function. Conversely, enhanced deposition of hyaluronan resulted in the expansion of the maternal-embryo barrier and increased diffusion distance, leading to compromised implantation. The deposition of hyaluronan at the embryonic niche is regulated by progesterone-progesterone receptor signaling. These results demonstrate a pivotal role for hyaluronan in successful pregnancy by fine-tuning the periembryo avascular niche and maternal vascular morphogenesis.

Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation-a fast, scarless healing process in healthy individuals-will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, "Menstruation: Science and Society" with an aim to "identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field." Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration-and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids-to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent "menstrual equity" and "period poverty" movements spreading across high-income countries., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Bioengineered uterine tissue could provide a treatment option for women with uterine factor infertility. In large animal models, reconstruction of the uterus has been demonstrated only with xenogeneic tissue grafts. Here we use biodegradable polymer scaffolds seeded with autologous cells to restore uterine structure and function in rabbits. Rabbits underwent a subtotal uterine excision and were reconstructed with autologous cell-seeded constructs, with nonseeded scaffolds or by suturing. At 6 months postimplantation, only the cell-seeded engineered uteri developed native tissue-like structures, including organized luminal/glandular epithelium, stroma, vascularized mucosa and two-layered myometrium. Only rabbits with cell-seeded constructs had normal pregnancies (four in ten) in the reconstructed segment of the uterus and supported fetal development to term and live birth. With further development, this approach may provide a regenerative medicine solution to uterine factor infertility.

Luminal uterine epithelial cells (UEC) have a surge in vesicular activity during early uterine receptivity. It has been predicted these vesicles exit the UEC via exocytosis resulting in secretion and membrane trafficking. The present study investigated the changes in SNARE proteins VAMP2 (v-SNARE) and syntaxin 3 (t-SNARE) localisation and abundance in UECs during early pregnancy in the rat. We found VAMP2 and syntaxin 3 are significantly higher on day 5.5 compared to day 1 of pregnancy. On day 5.5, VAMP2 is perinuclear and syntaxin 3 is concentrated in the apical cytoplasm compared to a cytoplasmic localisation on day 1. This change in localisation and abundance show VAMP2 and syntaxin 3 are involved in vesicular movement and membrane trafficking in UECs during early pregnancy. This study also investigated the influence of cytoskeletal disruption of microtubules and actin filaments on VAMP2 and syntaxin 3 in UECs grown in vitro, since microtubules and actin influence vesicle trafficking. As expected, this study found disruption to microtubules with colchicine and actin with cytochalasin D impacted VAMP2 and syntaxin 3 localisation. These results suggest VAMP2 and syntaxin 3 are involved in the timely trafficking of vesicular membranes to the apical surface in UECs during early pregnancy, as are of microtubules and actin.

The human uterus is composed of three layers: endometrium, myometrium and perimetrium. It remodels during the monthly menstrual cycle and more significantly during the complex stages of reproduction. In vivo studies of the human uterine wall are yet incomplete due to ethical and technical limitations. The objective of this study was to develop in vitro uterine wall models that mimic the in vivo structure in humans. We co-cultured multiple cellular models of endometrial epithelial cells, endometrial stromal cells and smooth muscle cells on a synthetic membrane mounted in multi-purpose custom-designed wells. Immunofluorescence staining and confocal imaging confirmed that the new model represents the in vivo anatomical architecture of the inner uterine wall. Hormonal treatment with progesterone and β-estradiol demonstrated increased expression of progestogen-associated endometrial protein, which is associated with the in vivo receptive uterus. The new tissue-engineered in vitro models of the uterine wall will enable deeper investigation of molecular and biomechanical aspects of the blastocyst-uterus interaction during the window of implantation.

Structural organization of the microcirculatory bed and mast cells in the uterus of mature female Wistar rats (n=60) were examined every 2 h over 24 h. During night hours, many vessels characterized by intensive reaction to NADPH-diaphorase and endothelial NO synthase were detected in the endometrium, and the density of these vessels and mast cells was quite high. During the day and evening hours, the capillaries with high levels of enzymes involved in NO synthesis and mast cells were less numerous. NO directly regulating the diameter of arteries and the intensity of transcapillary exchange can act as a signal molecule in the temporal dynamics of the vasoceptive action of estrogens and their receptors in the uterus.

Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.

UCHL1 is expressed specifically in the brain and gonads of almost all studied model organisms including Drosophila, zebrafish, amphibians, and mammals, suggesting a high degree of evolutionary conservation in its structure and function. Although UCHL1 has been involved in spermatogenesis in mice, its specific expression in mammal placenta remains elusive. Our previous work has revealed that UCHL1 is highly expressed in oocytes, and has been involved in mouse ovarian follicular development. Here, we further examined UCHL1 expression change in endometria during early natural pregnancy, with different stages of the estrous cycle and pseudopregnancy as control. The UCHL1 gene deletion model showed that UCHL1 protein is associated with endometrial development, and its deletion leads to infertility. Notably, we demonstrate evidence showing the distinct expression pattern of UCHL1: weak expression over the uterine endometria, strong expression in decidualized stromal cells at the implantation site with a peak at pregnancy D6, and a shift with primary decidualization to secondary decidualized zones. Using the delayed implantation, the delayed implantation activation, and the artificial decidualization models, we have demonstrated that strong expression of UCHL1 occurred in response to decidualization and estrogen stimulation. These observations suggest that during the early proliferation and differentiation of mouse uterine decidua, UCHL1 expression is up-regulated, and formed an unique intracellular distribution mode. Therefore, we proposed that UCHL1 is involved in decidualization, and possibly in response to estrogen regulation.

Tolerogenic dendritic cells (tDCs) play a central role in the development of paternal antigen-specific regulatory T cells (Tregs) during pregnancy. We examined whether uterine CD11c + antigen presenting cells (APC) induced paternal antigen-specific tolerance in allogeneic pregnant mice. Female BALB/c mice were mated with male DBA/2 mice, and their surface markers of APCs were studied using flow cytometry. After allogeneic mating, the uterine APCs exhibited significantly decreased expression of major histocompatibility complex (MHC) class II on day 3.5 post-coitus (pc) and day 5.5 pc. To analyze how seminal fluid affects surface markers of APCs, female BALB/c mice were mated with male mice that had undergone seminal vesicle excision (SVX). No reductions of MHC class II expression on APCs were seen in these mice. To analyze APC functions, a mixed lymphoid reaction (MLR) assay to paternal splenocytes was performed. Uterine APCs from allogeneic pregnant mice significantly suppressed the MLR reaction, but APCs from SVX mated mice did not suppress the MLR reaction Uterine APCs induced paternal antigen (Mls1a)-specific Treg development in vitro, but not in mice that mated with allogeneic SVX mice. These findings suggest that seminal fluid priming expands the paternal antigen-specific Treg population by inducing APCs development., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pregnant women with obesity are at increased risk of parturition dysfunction; however, the biological mechanism has remained unknown. We hypothesized that molecules circulating in the serum of pregnant women with obesity may induce the aberrant expression of contraction-associated proteins (CAPs), leading to insufficient uterine contractions. This study aimed to investigate the effects of maternal serum on CAPs expression by human uterine smooth muscle cells (UtSMCs) and elucidate the influence of maternal obesity. Blood samples were collected from singleton pregnant women at 36-41 weeks of gestation before the onset of labor. UtSMCs were incubated in the serum, and the mRNA expressions of PTGFR, OXTR, GJA1, and PTGS2 were examined by RT-PCR. Progranulin (PGRN) is a circulating glycoprotein associated with insulin resistance characterized by the accumulation of visceral fat. The serum PGRN levels of the samples were measured by ELISA. After incubated with PGRN (100-1,000 ng/mL), mRNA expression of PTGFR, OXTR, and GJA1 and protein expression of CX43 were examined by RT-PCR and western blotting, respectively. The mRNA expressions of PTGFR, OXTR, and GJA1 showed significantly negative correlations with gestational weight gain (GWG). Serum PGRN levels showed a significantly positive correlation with GWG. High levels of PGRN suppressed the mRNA expression of GJA1 and the protein expression of CX43. The change in maternal serum induced by GWG suppressed the CAPs expression by UtSMCs. PGRN is one of the factors in the serum responsible for inhibiting the expression of CX43.

Embryo implantation involves a sterile inflammatory reaction that is required for the invasion of the blastocyst into the decidua. Adenosine triphosphate (ATP) released from stressed or injured cells acts as an important signaling molecule to regulate many key physiological events, including sterile inflammation. We found that the amount of ATP in the uterine luminal fluid of mice increased during the peri-implantation period, and this depended on the presence of an embryo. We further showed that the release of ATP from receptive epithelial cells was likely stimulated by lactate released from the blastocyst through connexin hemichannels. The ATP receptor P2y2 was present on uterine epithelial cells during the preimplantation period and increased in the stromal cells during the time at which decidualization began. Pharmacological inhibition of P2y2 compromised decidualization and implantation. ATP-P2y2 signaling stimulated the phosphorylation of Stat3 in uterine luminal epithelial cells and the expression of early growth response 1 ( Egr1 ) and prostaglandin-endoperoxide synthase 2 ( Ptgs2, also known as Cox-2 ), all of which are required for decidualization and/or implantation, in stromal cells. Short exposure to high concentrations of ATP promoted decidualization of primary stromal cells, but longer exposures or lower ATP concentrations did not. The expression of genes encoding ATP-degrading ectonucleotidases increased in the decidua during the peri-implantation period, suggesting that they may limit the duration of the ATP signal. Together, our results indicate that the blastocyst-induced release of ATP from uterine epithelial cells during the peri-implantation period may be important for the initiation of stromal cell decidualization., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During pregnancy, the uterus undergoes intense neovascularization and vascular remodeling to supply oxygen and nutrients to the embryo. During this period, progesterone secreted from the ovary has effects on vascular remodeling in the endometrium and interacts with angiogenic factors. However, the exact mechanism of uterine vascular remodeling during pregnancy is poorly understood. Therefore, the aim of the present study was to investigate the association between angiopoietin-2 (Ang-2), one of the angiopoietins, and intrauterine vessel remodeling during pregnancy, and to determine the effect of progesterone on Ang-2 levels. Changes in Ang-2 expression were observed according to quantitative modification of progesterone using pregnant mice and human uterine microvascular endothelial cells. As a result, Ang-2 was observed mainly in the mesometrial region (MR) of the uterus during the period between implantation and placentation. Furthermore, a substantial amount of Ang-2 also appeared in endothelial cells, particularly of the venous sinus region (VSR). Interestingly, Ang-2 expression was increased by progesterone, whereas estrogen had limited effects. To confirm the association between Ang-2 and progesterone, the function of the progesterone receptor (PR) was inhibited using RU486, a blocker of PR. Ang-2 expression and vascular remodeling of the VSR in the uterus were decreased when the functions of progesterone were inhibited. Overall, the regulation of Ang-2 by progesterone/PR was associated with vascular remodeling in the VSR during pregnancy. The present study proposed a solution to prevent pregnancy failure due to a lack of vascularity in the uterus in advance.