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Endometrial disorders, such as infertility and endometriosis, significantly impact reproductive health, thus necessitating better models to study endometrial function. Current in vitro models fail to replicate the complexity of the human endometrium throughout the entire menstrual cycle. This study aimed to assess the physiological response of human endometrial organoids (hEOs) to in vitro hormonal treatments designed to mimic the hormonal fluctuations of the menstrual cycle. Endometrial biopsies from three healthy women were used to develop hEOs, which were treated over 28 days with three hormonal stimulation strategies: (1) estrogen only (E) to mimic the proliferative phase, (2) the addition of progesterone (EP) to simulate the secretory phase, and (3) the further addition of cAMP (EPC) to enhance the secretory functions of hEOs. Gene and protein expression were analyzed using qPCR, IHC, and ELISA. The hEOs exhibited proliferation, gland formation, and appropriate expression of markers such as E-cadherin and Ki67. The hormonal treatments induced significant changes in PR , HSD17B1 , PAEP , SPP1 , and other genes relevant to endometrial function, closely mirroring in vivo physiological responses. The prominent changes were observed in EPC-treated hEOs (week 4) with significantly high expression of uterine milk components such as glycodelin (PAEP) and osteopontin (SPP1), reflecting mid- to late-secretory phase physiology. This model successfully recapitulates human menstrual cycle dynamics and offers a promising platform for studying endometrial disorders and advancing personalized treatments in gynecology.

In dairy cows, the processes involved in the resolution of uterine inflammation during the postpartum are closely related to improved fertility during the subsequent lactation period. Little is known, however, about the role and distribution of endometrial immune cell populations during the pre-implantation period. This study was aimed to analyze the endometrial distribution of several mononuclear immune cells (T cells, γδ T cells, B cells and macrophages) in healthy dairy cows during the postpartum, beyond the transition period, looking for its possible association with the parturition-conception interval (PCI) and delayed conception. The quantification of immune cells was evaluated by immunohistochemistry (IHC), and the expression of hormone receptors in immune cells was evaluated by double IHC. Dairy cows were grouped according to their PCI: PCI shorter than or equal to 90 DIM (PCI ≤90 ), PCI between 90 and 120 DIM (PCI 90-120 ), and PCI greater than 150 DIM (PCI ≥150 ). The distribution of endometrial mononuclear immune cells was analyzed by a Generalized Linear Model, and the association of the distribution of mononuclear immune cells with delayed conception was evaluated with a Kaplan-Meier test. The cows from the PCI 90-120 group showed the highest number of endometrial macrophages, and a lower number of B cells than the PCI ≤90 group. Results also showed an association between the lower number of B cells in the endometrium during the pre-implantation period and earlier conception. Also, the present findings indicates that ESR and PR are expressed in the endometrial MØ, T cells, γδ T cells and B cells., Competing Interests: Declaration of Competing Interest I confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. The manuscript has been read and approved by all authors. There are no other persons who satisfied the criteria for authorship but are not listed. I further confirm that the order of authors listed in the manuscript has been approved by all of them. I am the sole contact for the Editorial process (including Editorial Manager and direct communications with the office) as Corresponding Author. I am responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Study Question: Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues?, Summary Answer: We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue., What Is Known Already: As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist., Study Design, Size, Duration: We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed., Participants/materials, Setting, Methods: Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation., Main Results and the Role of Chance: When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes., Limitations, Reasons for Caution: Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models., Wider Implications of the Findings: Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis., Study Funding/competing Interest(s): C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Successful embryo implantation relies on synchronized dialog between the embryo and endometrium, and the role of extracellular vesicles (EVs) in facilitating this cross-talk has been recently established. In our previous study, milk fat globule-EGF factor 8 protein (MFGE8) was identified as increasing in receptive endometrial epithelial cells (EECs) in response to trophoblastic EVs. However, the dynamics of MFGE8 protein in this context are not completely understood. Therefore, we examined its expression and secretion in EECs exposed to estrogen, progesterone, and trophoblastic EVs to gain deeper insights into its potential as an indicator of EV-mediated embryo-maternal dialogue. Our findings revealed that MFGE8 secretion is sensitive to estrogen and progesterone, and that trophoblastic EVs stimulate their release in both receptive and non-receptive EECs. Furthermore, trophoblast EV function was dose and time-dependent. Notably, the secretion of MFGE8 increased within a short timeframe of 30 min after addition of EVs, suggesting the possibility of rapid processes such as binding, fusion or internalization of trophoblastic EVs within EECs. Interestingly, MFGE8 released from EECs was associated with EVs, suggesting increased EV secretion from EECs in response to embryonic signals. In conclusion, increased MFGE8 secretion in this embryo implantation model can serve as an indicator of EV-mediated embryo-maternal dialogue., (© 2024. The Author(s).)

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 pro-inflammatory 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 β-galactosidase 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., (© 2024. The Author(s).)

The human endometrium is a highly regenerative tissue capable of undergoing scarless repair during the menstruation and postpartum phases. This process is mediated by endometrial adult stem/progenitor cells. During the healing of endometrial injuries, swift reepithelization results in the rapid covering of the wound surface and facilitates subsequent endometrial restoration. The involvement of endogenous endometrial epithelial stem cells, stromal cells, and bone marrow-derived cells in the regeneration of the endometrial epithelium has been a subject of prolonged debate. Increasing evidence suggests that the regeneration of the endometrial epithelium mainly relies on epithelial stem cells rather than stromal cells and bone marrow-derived cells. Currently, no consensus has been established on the identity of epithelial stem cells in the epithelial compartment. Several markers, including stage-specific embryonic antigen-1 (SSEA-1), sex-determining region Y-box 9 (SOX9), neural-cadherin (N-cadherin), leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5), CD44, axis inhibition protein 2 (Axin2), and aldehyde dehydrogenase 1A1 (ALDH1A1), have been suggested as potential candidate markers for endometrial epithelial stem cells. The identification of endometrial epithelial stem cells contributes to our understanding of endometrial regeneration and offers new therapeutic insights into diseases characterized by regenerative defects in the endometrium, such as intrauterine adhesion. This review explores different perspectives on the origins of human and mouse endometrial epithelial cells. It summarizes the potential markers, locations, and hierarchies of epithelial stem cells in both human and mouse endometrium. It also discusses epithelial cell-based treatments for intrauterine adhesion, hoping to inspire further research and clinical application of endometrial epithelial stem cells., (© 2024. The Author(s).)

Background: During early pregnancy, glucose is essential for the uterine epithelium and the developing embryo. In cows, progesterone increases the secretion of glucose into the uterine lumen. The uterine epithelium can convert glucose to fructose, but other fates of glucose in the uterine epithelium have been sparsely investigated. Therefore, our objective was to investigate how progesterone influences glucose metabolism in immortalized bovine uterine epithelial (BUTE) cells., Methods: BUTE cells were grown to 80% confluence and treated with vehicle (DMSO) or 10 µM progesterone for 24 h. Cells were collected and analyzed. Immunohistochemistry was performed on endometrial samples collected from the bovine endometrium on days 1 and 11 of the reproductive cycle., Results: Progesterone treatment increased glucose consumption of BUTE cells. RNAseq identified 3,072 genes regulated by progesterone. KEGG analysis indicated that progesterone altered genes associated with metabolic pathways and glutathione metabolism. Manually examining genes unique to specific glucose metabolic pathways identified an increase in the rate-limiting enzyme in the pentose phosphate pathway-glucose-6-phosphate dehydrogenase. Functionally, a major product of the pentose phosphate pathway is NADPH, and progesterone treatment increased NADPH levels in BUTE cells. In cows, immunohistochemistry confirmed that glucose-6-phosphate dehydrogenase levels were higher in the uterine epithelium in the luteal phase when progesterone concentrations are high., Conclusions: Progesterone increased glucose-6-phosphate dehydrogenase expression and metabolism via the pentose phosphate pathway in the bovine uterine epithelium. This metabolism could provide substrates for cell proliferation, molecules to be secreted into the uterine lumen, or maintain reduction/oxidation balance in the uterine epithelium., (© 2024. The Author(s).)

The endometrium undergoes substantial remodeling in each menstrual cycle to become receptive to an implanting embryo. Abnormal endometrial receptivity is one of the major causes of embryo implantation failure and infertility. MicroRNA-124-3p is elevated in both the serum and endometrial tissue of women with chronic endometritis, a condition associated with infertility. MicroRNA-124-3p also has a role in cell adhesion, a key function during receptivity to allow blastocysts to adhere and implant. In this study, we aimed to determine the function of microRNA-124-3p on endometrial epithelial adhesive capacity during receptivity and effect on embryo implantation. Using a unique inducible, uterine epithelial-specific microRNA overexpression mouse model, we demonstrated that elevated uterine epithelial microRNA-124-3p impaired endometrial receptivity by altering genes associated with cell adhesion and polarity. This resulted in embryo implantation failure. Similarly in a second mouse model, increasing microRNA-124-3p expression only in mouse uterine surface (luminal) epithelium impaired receptivity and led to implantation failure. In humans, we demonstrated that microRNA-124-3p was abnormally increased in the endometrial epithelium of women with unexplained infertility during the receptive window. MicroRNA-124-3p overexpression in primary human endometrial epithelial cells (HEECs) impaired primary human embryo trophectoderm attachment in a 3-dimensional culture model of endometrium. Reduction of microRNA-124-3p in HEECs from infertile women normalized HEEC adhesive capacity. Overexpression of microRNA-124-3p or knockdown of its direct target IQGAP1 reduced fertile HEEC adhesion and its ability to lose polarity. Collectively, our data highlight that microRNA-124-3p and its protein targets contribute to endometrial receptivity by altering cell polarity and adhesion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Upon hormonal stimulation, uterine endometrial stromal cells undergo a dramatic morpho-functional metamorphosis that allows them to secrete large amounts of matrix proteins, cytokines, and growth factors. This step, known as decidualization, is crucial for embryo implantation. We previously demonstrated how the secretory pathway is remodelled during this process. Here we show that hormonal stimulation rapidly induces the expression of many mitochondrial genes, encoded in both the mitochondrial and the nuclear genomes. Altogether, the mitochondrial network quadruples its size and establishes more contacts with the ER. This new organization results in the increased respiratory capacity of decidualized cells. These findings reveal how achieving an efficient secretory phenotype requires a radical metabolic rewiring., (© 2024 Dalla Torre et al.)

Background: The human endometrium, lining the inner uterus, regenerates over 400 times uniquely during a woman's reproductive life. Endometrial stem cells (eSCs) enrich the tissue, resulting in a dense vascular network, significant angiogenic potential, and effective regeneration power. Being of natural angiogenic properties and proven effective in the treatment of vascular disorders, eSCs can be considered safe, reliable, and superior to other post-natal stem cells. Cluster of Differentiation 146 (CD146) has emerged as a pivotal marker associated with pericytes and endothelial cells for promoting angiogenesis. Endometrial cells with high CD146 expression could proliferate and differentiate into multiple lineages. This study will explore the role of CD146 in eSCs, focusing on the potential to boost the angiogenic and regenerative functions of the cells. The novelty of this study lies in the investigation of CD146 on eSC function, which may open new possibilities for eSC-based therapy in regenerative medicine and vascular disorders., Methods: The study involved obtaining endometrial biopsies from active reproducing women to isolate and cultivate eSCs. eSCs were assessed for growth factor secretion pattern, characterized for their mesenchymal properties. Finally, eSCs were tested for their angiogenic potential by angiogenic gene expression profile and in-ovo chick embryo model. As aimed, to check the role of CD146 in eSC angiogenesis, CD146 + cells were magnetically sorted and cultured. The sorted cells underwent various analyses, including flowcytometry to identify mesenchymal markers and human growth factor panel to analyze growth factor secretion profiles The study evaluated the angiogenic potential of CD146 + cells using functional assays, including ring formation, endothelial differentiation, and wound scratch assays, to evaluate cell migration and healing capabilities. Molecular insights were obtained through chemokine and cytokine investigations In-ovo Chick model assay was conducted to check the angiogenic potential and evaluated through macroscopic as well as through immunohistochemistry., Result: Endometrial stem cells (eSCs) were successfully isolated using a combination of mechanical and enzymatic digestion, followed by culturing in complete DMEM media. The secretion profile of eSCs revealed significant production of various angiogenic growth factors, including Granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Vascular endothelial growth factor (VEGF), Fibroblast growth factors (FGF), and Platelet derived growth factor AA (PDGF-AA). The angiogenic gene profile indicated upregulation of several angiogenic genes in eSCs. The mesenchymal nature of eSCs was demonstrated through surface marker analysis (Cluster of differentiation 73, Cluster of differentiation 90, Cluster of differentiation 105) and trilineage differentiation. The in-ovo chick model confirmed the angiogenic potential of eSCs. CD146 + cells, isolated via magnetic sorting, exhibited enhanced angiogenic potential. These cells secreted significant levels of angiogenic growth factors such as VEGF. In Matrigel assays, CD146 + cells formed endothelial ring structures more rapidly and persistently than unsorted eSCs. Semi-quantitative PCR confirmed their endothelial differentiation. CD146 + cells express various angiogenic chemokines such as CXCL5, CXCL8, CCL3, and CCL20 and cytokines such as GM-CSF, Interleukin-1β (IL-1β), Interleukin-6 (IL-6), PDGF AA/BB, Epidermal growth factor (EGF), Endothelin 1, Angiopoietin. In-ovo chick model assay showed that CD146 + cells had superior angiogenesis, with more nodes, junctions, and segments compared to eSCs and controls. Immunohistochemistry confirmed increased expression of endothelial markers (Cluster of differentiation 31, VEGF, Vascular associated protein (VAP), Von Willebrand factor (vWF) in CD146 + cells., Conclusion: The study highlights the angiogenic potential of endometrial stem cells, particularly the CD146 + cell population. These cells promote angiogenesis, secreting growth factors and forming stable blood vessel structures. CD146 + cells have higher expression levels of VEGF and TGF-α, key factors in angiogenesis. This suggests CD146 + eSCs may be promising for therapeutic applications in vascular diseases requiring angiogenesis. Further research is needed., (© 2024. The Author(s).)

In cattle, the endometrium during diestrus and early pregnancy displays cellular responses that are consequences of prior, transient stimuli. Goal was to establish a model to study cellular memory in the endometrium. The hypothesis is that stimuli given to endometrium in vivo are retained as a cellular memory that remains after bovine uterine epithelial cells (BUECs) are isolated, cultured, and further stimulated in vitro. Objectives were to measure BUEC proliferation/migration and responsiveness to recombinant bovine Interferon-tau (rbIFNT) in vitro: among cows that showed estrus (experiment 1 [Exp1]), cows that became or not pregnant to artificial insemination (Exp2), cows that received or not supplemental progesterone (P4; Exp3) and cows that received or not a COX-1/2 inhibitor (Exp4). Only cows that displayed estrus were included in studies. For all experiments endometrial cytology was collected 4 days after estrus, BUECs were cultured, propagated, and submitted to rbIFNT treatment and an in vitro scratch assay. In Exp1, different cows spontaneously grouped according to proliferative/migratory capacity and responsiveness to rbIFNT of their respective BUECs. In Exp2, BUECs from pregnant cows showed greater rbIFNT responsiveness and cellular proliferation. In Exp3, BUECs from cows supplemented with P4 presented inhibited proliferation and increased expression of RSAD2. In Exp4, Flunixin Meglumine modified rbIFNT responsiveness of BUECs in an IFN-signaling pathway-specific manner. In conclusion, physiological and pharmacological stimuli received by the endometrium in vivo were retained as cellular memory in BUECs, persisted in culture, and changed BUEC proliferation/migration and responsiveness to rbIFNT, which are characteristics associated with fertility in cattle., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Endometrium receptivity is a multifactor-regulated process involving progesterone receptor-regulated signaling, cytokines and chemokines, and additional growth regulatory factors. In the female reproductive system, macrophages have distinct roles in the regulation of receptivity, embryo implantation, immune tolerance, and angiogenesis or oxidative stress. In the present study, we investigated the effects of PMA-activated THP-1 macrophages on the receptivity-related genes, cytokines and chemokines, growth regulators, and oxidative stress-related molecules of HEC-1A endometrium cells. We established a non-contact co-culture in which the culture medium of the PMA-activated macrophages exhibiting the pro-inflammatory phenotype was used for the treatment of the endometrial cells. In the endometrium cells, the expression of the growth-related factors activin and bone morphogenetic protein 2, the growth hormone EGF, and the activation of the downstream signaling molecules pERK1/2 and pAkt were analyzed by ELISA and Western blot. The secretions of cytokines and chemokines, which are involved in the establishment of endometrial receptivity, and the expression of matrix metalloproteinases implicated in invasion were also determined. Based on the results, the PMA-activated THP-1 macrophages exhibiting a pro-inflammatory phenotype may play a role in the regulation of HEC-1A endometrium cells. They alter the secretion of cytokines and chemokines, as well as the protein level of MMPs of HEC-1A cells. Moreover, activated THP-1 macrophages may elevate oxidative stress protection of HEC-1A endometrium cells. All these suggest that pro-inflammatory macrophages have a special role in the regulation of receptivity-related and implantation-related factors of HEC-1A cells.

Although memory functions of immune cells characterized by increased resistance to subsequent infections after initial pathogen exposure are well-established, it remains unclear whether non-immune cells, especially tissue-resident stem cells, exhibit similar memory mechanisms. The present study revealed that detrimental effects of initial viral antigen exposure (human papillomavirus [HPV]) on diverse stem cell functions were significantly exacerbated upon subsequent secondary exposure both in vitro and in vivo. Importantly, endometrial stem cells exhibited robust memory functions following consecutive HPV antigen exposures, whereas fully differentiated cells such as fibroblasts and vesicular cells did not show corresponding changes in response to the same antigen exposures. Deficiency of angiopoietin-like 4 (ANGPTL4) achieved through small hairpin RNA knockdown in vitro and knockout (KO) mice in vivo highlighted the critical role of ANGPTL4 in governing memory functions associated with various stem cell processes. This regulation occurred through histone H3 methylation alterations and PI3K/Akt signaling pathways in response to successive HPV antigen exposures. Furthermore, memory functions associated with various stem cell functions that were evident in wild-type mice following consecutive exposures to HPV antigen were not observed in ANGPTL4 KO mice. In summary, our findings strongly support the presence of memory mechanism in non-immune cells, particularly tissue-resident stem cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The complex and dynamic cellular composition of the human endometrium remains poorly understood. Previous endometrial single-cell atlases profiled few donors and lacked consensus in defining cell types. We introduce the Human Endometrial Cell Atlas (HECA), a high-resolution single-cell reference atlas (313,527 cells) combining published and new endometrial single-cell transcriptomics datasets of 63 women with and without endometriosis. HECA assigns consensus and identifies previously unreported cell types, mapped in situ using spatial transcriptomics and validated using a new independent single-nuclei dataset (312,246 nuclei, 63 donors). In the functionalis, we identify intricate stromal-epithelial cell coordination via transforming growth factor beta (TGFβ) signaling. In the basalis, we define signaling between fibroblasts and an epithelial population expressing progenitor markers. Integration of HECA with large-scale endometriosis genome-wide association study data pinpoints decidualized stromal cells and macrophages as most likely dysregulated in endometriosis. The HECA is a valuable resource for studying endometrial physiology and disorders, and for guiding microphysiological in vitro systems development., (© 2024. The Author(s).)

Thin endometrium (TE) is closely associated with infertility in reproductive medicine. Estrogen therapy gains unsatisfactory outcomes. In this study, an artificial mucus based on dopamine (L-DOPA)-modified hyaluronic acid combining phytoestrogen cajaninstilbene acid and rat urinary exosomes (CUEHD) is constructed for TE treatment using a rat TE model. In the rat TE model, the dominant elastic behavior and adhesive properties of CUEHD guarantee adequate retention, rendering superior synergistic treatment efficacy and favorable biosafety characteristics. CUEHD treatment significantly increases endometrial thickness and promotes receptivity and fertility. Mechanistically, estrogen homeostasis, inflammation inhibition, and endometrial regeneration are achieved through the crosstalk between ER-NLRP3-IL1β and Wnt-β catenin-TGFβ-smad signaling pathways. Moreover, the therapeutic potential of exosomes from human urine and adipose tissue-derived stem cells (ADSCs) and rat ADSCs are also demonstrated, indicating extensive use of the artificial mucus system. Thus, this study illustrates a platform combining phytoestrogen and exosomes with promising implications for TE treatment., (© 2024 Wiley‐VCH GmbH.)

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24 h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24 h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite significant advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) still occurs in some patients. Poor endometrial receptivity and abnormal human endometrial stromal cell (HESC) proliferation and decidualization have been identified as the major causes. Ubiquitin-specific protease 22 (USP22) has been reported to participate in the decidualization of endometrial stromal cells in mice. However, the role of USP22 in HESC function and RIF development remains unknown. In this study, clinical endometrial tissue samples were gathered to investigate the involvement of USP22 in RIF, and HESCs were utilized to examine the molecular mechanisms of USP22 and Forkhead box M1 (FoxM1). The findings indicated a high expression of USP22 in the secretory phase of the endometrium. Knockdown of USP22 led to a notable reduction in the proliferation and decidualization of HESCs, along with a decrease in FoxM1 expression, while overexpression of USP22 yielded opposite results. Furthermore, USP22 was found to deubiquitinate FoxM1 in HESCs. Moreover, both USP22 and FoxM1 were downregulated in the endometria of patients with RIF. In conclusion, these results suggest that USP22 may have a significant impact on HESCs proliferation and decidualization through its interaction with FoxM1, potentially contributing to the underlying mechanisms of RIF pathogenesis., Competing Interests: Declaration of competing interest The authors declare they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: It has been recognized that the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol has a detrimental effect on clinical outcomes compared to the GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer (IVF-ET) cycles. However, the related mechanisms were unclear., Methods: A total of 18,561 patients, who underwent fresh IVF-ET cycles in the Center for Assisted Reproduction of Jiangxi Maternal and Child Health Hospital from January 2014 to September 2021, were retrospectively analyzed. The propensity score matching (PSM) technique was used to control for confounding factors between the GnRH-ant and GnRH-a groups. Human endometrial stromal cells (hESCs) were collected for primary culture and treated with relevant receptor antagonists and activators. RT-PCR, Western Blot, immunofluorescence staining, cell migration and adhesion assays, and animal experiments were employed to elucidate the molecular mechanism by which GnRH antagonist affects the migration and adhesion ability of hESCs., Results: There was no statistical difference between the two groups in terms of baseline characteristics after matching basal status by propensity score matching. The result showed that the endometrial thickness (10.4 ± 2.35 vs. 11.03 ± 2.61 mm, p  < .001) on trigger day was significantly lower in the GnRH-ant group. Compared with the GnRH-a protocol, the implantation rate (39.71% vs. 50.36%, p  < .001), biochemical pregnancy rate (64.26% vs. 72.7%, p  < .001), clinical pregnancy rate (56.39% vs. 65.24%, p  < .001), live birth rate (45.25% vs. 56.1%, p  < .001) in the GnRH-ant group were significantly decreased. Contrarily, the rate of early miscarriage in the GnRH-ant group (13.95% vs. 9.04%, p  < .001) was higher than in the GnRH-a group. Furthermore, after treating with GnRH-ant, hESCs showed a reduced expression of HOXA10 and MMP-9 proteins, and a weakened migration ability. Subsequently, by establishing the co-culture system of hESCs and JAR trophoblast spheroids, we found that GnRH-ant inhibited the adhesion and invasion ability of trophoblast cells. Moreover, we also found a decreased expression and phosphorylation of c-kit receptor in decidualized hESCs after treating with GnRH-ant. Similar results as observed above were also confirmed when inhibiting the activation of c-kit receptor by imatinib., Conclusions: GnRH-ant could reduce the motility of hESCs by inhibiting the expression and activation of the C-kit receptor, which impaired the process of embryo implantation.

Objective: Impaired endometrial receptivity is the main cause of embryo implantation failure. Little information is available on the role of circRNAs in endometrial receptivity. Here, the effect of circABCC1 on endometrial receptivity and its mechanism were investigated., Methods: GEO database was screened for key biomarkers for recurrent implantation failure (RIF). Endometrial epithelial cells (EECs) were cultured and transfected with circABCC1- and/or FAM155B-related vectors, followed by CCK-8 detection of cell proliferation, western blotting detection of receptivity-related factors LIF and DKK1, and ELISA detection of LIF secretion. An in vitro adhesion model was established to detect trophoblast adhesion to EECs. RIP was used to detect the binding of METTL3 to circABCC1 and FAM155B mRNA, and MeRIP-qPCR was used to detect m 6 A modification of FAM155B mRNA., Results: CircABCC1 and FAM155B were highly expressed in patients with RIF. CircABCC1 or FAM155B overexpression reduced EEC proliferation, LIF and DKK1 expression, LIF secretion, and trophoblast adhesion; circABCC1 or FAM155B knockdown led to the opposite results. CircABCC1 and METTL3 positively regulated FAM155B expression. METTL3 bound circABCC1 and FAM155B mRNA. METTL3 overexpression increased m 6 A modification of FAM155B mRNA. FAM155B overexpression partially eliminated circABCC1 knockdown-induced promotion of EEC proliferation, LIF and DKK1 expression, LIF secretion, and trophoblast adhesion., Conclusion: CircABCC1 binds to METTL3 to regulate FAM155B mRNA modification and promote FAM155B expression, thereby inhibiting EEC proliferation and reducing endometrial receptivity.

Intrauterine adhesion (IUA) resulting from irreversible fibrotic repair of endometrium is the main cause of secondary infertility in women, and current therapeutic approaches to IUA are limited. Increasing evidence has suggested the important role of competitive endogenous RNA (ceRNA) in IUA pathologies. This study aimed to investigate the long noncoding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1)-associated ceRNA in IUA development. We harvested endometrial tissues from patients with or without IUA and extracted endometrial stromal cells (ESCs) from normal endometrial tissues. Transforming growth factor β1 (TGF-β1) was used to induce fibrosis in ESCs. The expression of transforming growth factor β receptor 1 (TGFβR1), α-smooth muscle actin, phosphorylated suppressor of mother against decapentaplegic (p-Smad)2/3, collagen type I alpha 1, MALAT1, and microRNA (miR)-22-3p in endometrial tissues and ESCs was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blotting. Pearson's correlation analysis was conducted to assess the correlation between miR-22-3p expression or TGFβR1 and MALAT1 expression in endometrial tissues. The expression of TGFβR1 in ESCs was also evaluated by immunofluorescence staining. The location of MALAT1 was examined by fluorescence in situ hybridization. Luciferase reporter assays were performed to verify the binding relationship between MALAT1 or TGFβR1 and miR-22-3p. Cell viability was assessed via cell counting kit-8 assays. Our findings revealed that lncRNA MALAT1 and TGFβR1 were upregulated while miR-22-3p was downregulated in IUA endometrial tissues or TGF-β1-stimulated ESCs, and lncRNA MALAT1 expression was negatively correlated with miR-22-3p expression while being positively correlated with TGFβR1 expression in IUA endometrial tissues. Additionally, lncRNA MALAT1 was mainly located in the cytoplasm of ESCs and directly targeted miR-22-3p to regulate TGFβR1 expression. Moreover, knockdown of lncRNA MALAT1 exerted anti-fibrotic effects on ESCs by targeting miR-22-3p, and miR-22-3p overexpression inhibited the fibrosis of ESCs by binding to TGFβR1 3'untranslated region. Collectively, lncRNA MALAT1 promotes endometrial fibrosis by sponging miR-22-3p to regulate TGFβR1 and Smad2/3, and inhibition of MALAT1 may represent a promising therapeutic option for suppressing endometrial fibrosis., Competing Interests: Compliance with Ethical Standards Conflict of Interest The authors declare no competing interests. Ethical Approval The study was approved by the Ethics Committee of Wuhan Third Hospital (Guanggu Campus). Consent to Participate All participants had signed the written informed consent., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mare endometrosis is a major reproductive problem associated with low fertility and is characterized by persistent inflammation, TGFβ-1 signaling, and consequently, extracellular matrix deposition, which compromises endometrial glands. Mesenchymal stem cell-based products (MSCs), such as extracellular vesicles (EVs), have gained attention due to the regulatory effects exerted by their miRNA cargo. Here, we evaluated the impact of preconditioning equine adipose mesenchymal stem cells with TGFβ-1 for short or long periods on the anti-fibrotic properties of secreted extracellular vesicles. MSCs were isolated from six healthy horses and exposed to TGFβ-1 for 4, 24, and 0 h. The expression of anti-fibrotic and pro-fibrotic miRNAs and mRNAs in treated cells and miRNAs in the cargo of secreted extracellular vesicles was measured. The resulting EVs were added for 48 h to endometrial stromal cells previously induced to a fibrotic status. The expression of anti-fibrotic and pro-fibrotic genes and miRNAs was evaluated in said cells using qPCR and next-generation sequencing. Preconditioning MSCs with TGFβ-1 for 4 h enriched the anti-fibrotic miRNAs (mir29c, mir145, and mir200) in cells and EVs. Conversely, preconditioning the cells for 24 h leads to a pro-fibrotic phenotype overexpressing mir192 and mir433. This finding might have implications for developing an EV-based protocol to treat endometrial fibrosis in mares.

Background: An elevated endogenous cortisol level due to the peripartum stress is one of the risk factors of postpartum bovine uterine infections. Selenium is a trace element that elicits anti-inflammation and antioxidation properties. This study aimed to reveal the modulatory effect of selenium on the inflammatory response of primary bovine endometrial stromal cells in the presence of high-level cortisol. The cells were subjected to lipopolysaccharide to establish cellular inflammation. The mRNA expression of toll-like receptor 4 (TLR4), proinflammatory factors, and selenoproteins was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blot and immunofluorescence., Results: The pretreatment with sodium selenite (2 and 4 µΜ) resulted in a down-regulation of TLR4 and genes encoding proinflammatory factors, including interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase. Selenium inhibited the activation of NF-κB and the phosphorylation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase. The suppression of those genes and pathways by selenium was more significant in the presence of high cortisol level (30 ng/mL). Meanwhile the gene expression of glutathione peroxidase 1 and 4 was promoted by selenium, and was even higher in the presence of cortisol and selenium., Conclusions: The anti-inflammatory action of selenium is probably mediated through NF-κB and MAPK, and is augmented by cortisol in primary bovine endometrial stromal cells., (© 2024. The Author(s).)

Decidualization denotes the morphological and biological differentiating process of human endometrial stromal cells (HESCs). Fatty acid pathways are critical for endometrial decidualization. However, the participation of fatty acids as an energy source and their role in endometrial decidualization have received little attention. To identify fatty acids and clarify their role in decidualization, we comprehensively evaluated free fatty acid profiles using liquid chromatography/Fourier transform mass spectrometry (LC/FT-MS). LC/FT-MS analysis detected 26 kinds of fatty acids in the culture medium of decidualized or un-decidualized HESCs. Only the production of octanoic acid, which is an essential energy source for embryonic development, was increased upon decidualization. The expressions of genes related to octanoic acid metabolism including ACADL, ACADM, and ACADS; genes encoding proteins catalyzing the first step of mitochondrial fatty acid beta-oxidation; and ACSL5 and ACSM5; genes encoding fatty acid synthesis proteins were significantly altered upon decidualization. These results suggest that decidualization promotes lipid metabolism, implying that decidualized HESCs require energy metabolism of the mitochondria in embryo implantation.

The endometrium during the sexual cycle undergoes detachment, tissue remodeling, and differentiation during the menstrual cycle. Localized and transient destruction and regeneration of endometrial tissue are also essential for pregnancy. It is possible to attribute many causes of failure in infertility treatment to the implantation stage. To improve the success rate of plateau fertility treatment, it is important to understand the regeneration mechanism of the endometrium, a unique regenerative tissue in the human body. In association with cell proliferation, tissue remodeling requires the relocation of proliferative cells, and the steady-state epithelial cells need to be motile for the relocation. Transient add-on motile activity in epithelial cells is mediated by epithelial to mesenchymal transition (EMT) and reversible mesenchymal to epithelial transition (MET). The destruction and regeneration of endometrial tissue over a period of days to weeks requires a system with a rapid and characteristic mechanism similar to that of wound healing. Here, I review the relationship between the well-known phenomenon of EMT in wound healing and endometrial tissue remodeling during the sexual cycle and pregnancy establishment, which are automatically triggered by menstruation and embryonal invasion.

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.)

Dynamic crosstalk between the embryo and mother is crucial during implantation. Here, we comprehensively profile the single-cell transcriptome of pig peri-implantation embryos and corresponding maternal endometrium, identifying 4 different lineages in embryos and 13 cell types in the endometrium. Cell-specific gene expression characterizes 4 distinct trophectoderm subpopulations, showing development from undifferentiated trophectoderm to polar and mural trophectoderm. Dynamic expression of genes in different types of endometrial cells illustrates their molecular response to embryos during implantation. Then, we developed a novel tool, ExtraCellTalk, generating an overall dynamic map of maternal-foetal crosstalk using uterine luminal proteins as bridges. Through cross-species comparisons, we identified a conserved RBP4/STRA6 pathway in which embryonic-derived RBP4 could target the STRA6 receptor on stromal cells to regulate the interaction with other endometrial cells. These results provide insight into the maternal-foetal crosstalk during embryo implantation and represent a valuable resource for further studies to improve embryo implantation., (© 2024. Science China Press.)

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites., Competing Interests: Declaration of competing interest The authors declare that they have no financial or competing interests concerning this study., (Copyright © 2024. Published by Elsevier B.V.)

Research Question: What impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells?, Design: Cellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications., Results: Native and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC., Conclusions: These findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.

The human endometrial decidualization is a transformative event in the pregnant uterus that involves the differentiation of stromal cells into decidual cells. While crucial to the establishment of a successful pregnancy, the metabolic characteristics of decidual cells in vivo remain largely unexplored. Here, we integrated the single-cell RNA sequencing (scRNA-seq) datasets on the endometrium of the menstrual cycle and the maternal-fetal interface in the first trimester to comprehensively decrypt the metabolic characteristics of stromal fibroblast cells. Our results revealed that the differentiation of stromal cells into decidual cells is accompanied by increased amino acid and sphingolipid metabolism. Furthermore, metabolic heterogeneity exists in decidual cells with differentiation maturity disparities. Decidual cells with high metabolism exhibit higher cellular activity and show a strong propensity for signaling. In addition, significant metabolic reprogramming in amino acids and lipids also occurs during the transition from non-pregnancy to pregnancy in the uteri of pigs, cattle, and mice. Our analysis provides comprehensive insights into the dynamic landscape of stromal fibroblast cell metabolism, contributing to our understanding of the metabolism at the molecular dynamics underlying the decidualization process in the human endometrium., (© 2024. The Author(s).)

The process of endometrial repair after injury involves the synergistic action of various cells including immune cells and stem cells. In this study, after combing Fibrinogen(Fg) with poly(L-lacticacid)-co-poly(ε-caprolactone)(P(LLA-CL)) by electrospinning, we placed Fg/P(LLA-CL) into the uterine cavity of endometrium-injured rats, and bioinformatic analysis revealed that Fg/P(LLA-CL) may affect inflammatory response and stem cell biological behavior. Therefore, we verified that Fg/P(LLA-CL) could inhibit the lipopolysaccharide (LPS)-stimulated macrophages from switching to the pro-inflammatory M1 phenotype in vitro. Moreover, in the rat model of endometrial injury, Fg/P(LLA-CL) effectively promoted the polarization of macrophages towards the anti-inflammatory M2 phenotype and enhanced the presence of mesenchymal stem cells at the injury site. Overall, Fg/P(LLA-CL) exhibits significant influence on macrophage polarization and stem cell behavior in endometrial injury, justifying further exploration for potential therapeutic applications in endometrial and other tissue injuries., (© 2024. The Author(s).)

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-β1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems., (© 2024. The Author(s).)

Background: We previously identified Il17RB, a member of the IL17 superfamily, as a candidate marker gene for endometrial aging. While IL17RB has been linked to inflammation and malignancies in several organ systems, its function in the endometrium has not been investigated and is thus poorly understood. In the present study, we performed a functional analysis of this receptor with the aim of determining the effects of its age-associated overexpression on the uterine environment., Methods: We analyzed IL17RB-related signaling pathways and downstream gene expression in an immortalized human endometrial glandular epithelial cell line ("hEM") forced to express the receptor via lentiviral transduction ("IL17RB-hEM"). We also prepared endometrial organoids from human endometrial tissue sourced from hysterectomy patients ("patient-derived EOs") and exposed them to cytokines that are upregulated by IL17RB expression to investigate changes in organoid-forming capacity and senescence markers. We analyzed RNA-seq data (GEO accession number GSE132886) from our previous study to identify the signaling pathways associated with altered IL17RB expression. We also analyzed the effects of the JNK pathway on organoid-forming capacity., Results: Stimulation with interleukin 17B enhanced the NF-κB pathway in IL17RB-hEM, resulting in significantly elevated expression of the genes encoding the senescence associated secretory phenotype (SASP) factors IL6, IL8, and IL1β. Of these cytokines, IL1β inhibited endometrial organoid growth. Bioinformatics analysis showed that the JNK signaling pathway was associated with age-related variation in IL17RB expression. When IL17RB-positive cells were cultured in the presence of IL17B, their organoid-forming capacity was slightly but non-significantly lower than in unexposed IL17RB-positive cells, but when IL17B was paired with a JNK inhibitor (SP600125), it was restored to control levels. Further, IL1β exposure significantly reduced organoid-forming capacity and increased p21 expression in endometrial organoids relative to non-exposure (control), but when IL1β was paired with SP600125, both indicators were restored to levels comparable to the control condition., Conclusions: We have revealed an association between IL17RB, whose expression increases in the endometrial glandular epithelium with advancing age, and cellular senescence. Using human endometrial organoids as in vitro model, we found that IL1β inhibits cell proliferation and leads to endometrial senescence via the JNK pathway., (© 2024. The Author(s).)

Zearalenone (ZEN) is a mycotoxin known for its estrogen-like effects, which can disrupt the normal physiological function of endometrial cells and potentially lead to abortion in female animals. However, the precise mechanism by which ZEN regulates endometrial function remains unclear. In this study, we found that the binding receptor estrogen receptors for ZEN is extensively expressed across various segments of the uterus and within endometrial cells, and a certain concentration of ZEN treatment reduced the proliferation capacity of goat endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Meanwhile, cell cycle analysis revealed that ZEN treatment leaded to cell cycle arrest in goat EECs and ESCs. To explore the underlying mechanism, we investigated the mitochondrial quality control systems and observed that ZEN triggered excessive mitochondrial fission and disturbed the balance of mitochondrial fusion-fission dynamics, impaired mitochondrial biogenesis, increased mitochondrial unfolded protein response and mitophagy in goat EECs and ESCs. Additionally, ZEN treatment reduced the activities of mitochondrial respiratory chain complexes, heightened the production of hydrogen peroxide and reactive oxygen species, and caused cellular oxidative stress and mitochondrial dysfunction. These results suggest that ZEN has adverse effects on goat endometrium cells by disrupting the mitochondrial quality control system and affecting cell cycle and proliferation. Understanding the underlying molecular pathways involved in ZEN-induced mitochondrial dysfunction and its consequences on cell function will provide critical insights into the reproductive toxicity of ZEN and contribute to safeguarding the health and wellbeing of animals and humans exposed to this mycotoxin., (© 2024 Federation of American Societies for Experimental Biology.)

Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs' morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPβ), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy., (© 2024. The Author(s).)

Endometrial stem/progenitor cells are a type of stem cells with the ability to self-renew and differentiate into multiple cell types. They exist in the endometrium and form niches with their neighbor cells and extracellular matrix. The interaction between endometrial stem/progenitor cells and niches plays an important role in maintaining, repairing, and regenerating the endometrial structure and function. This review will discuss the characteristics and functions of endometrial stem/progenitor cells and their niches, the mechanisms of their interaction, and their roles in endometrial regeneration and diseases. Finally, the prospects for their applications will also be explored., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The pivotal role of human endometrial stromal cells (hESCs) in the development of endometriosis lies in their ability to adopt a pro-invasive and proinflammatory profile upon migration to areas outside the uterus. However, the molecular mechanisms involved in these events remain unclear. In this study, we investigated how angiotensin II (Ang II) affects the plasminogen-plasmin system in hESCs, and the mechanisms underlying cell proliferation, migration, matrix degradation, and inflammation. Precursors, receptors, and peptidases involved in angiotensin metabolism increased significantly in Ang II-treated hESCs. The expression and activity of tissue (tPA)- and urokinase (uPA)- type plasminogen activators and the receptor for uPA (uPAR) were induced in the presence of Ang II. The up-regulation of tPA-uPA/uPAR pathway significantly contributes to heightened plasmin production both on the surface of hESCs and in their conditioned media. As a result, the plasmin generation induced by Ang II enhances the degradation of fibrin and matrix proteins, while also boosting hESC viability, proliferation, and migration through the up-regulation of growth factor expression. Notably, Ang II-induced hESC migration was dependent on the generation of active plasmin on cell surface. Ang II regulates oxidative and inflammatory signalling in hESCs primarily via NADPH oxidase and through the up-regulation of proinflammatory cytokines and adhesion molecules. Interestingly, Ang II receptor (AT1R) blockage, decreased plasmin generation, tPA-uPA/uPAR expression and hESC migration. Our results suggest that Ang II/AT1R axis regulates hESC proliferation and migration through tPA-uPA/uPAR pathway activation and plasmin generation. We propose the Ang II/AT1R axis as a potential target for endometriosis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the preimplantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers connexin-43, insulin like growth factor binding protein 1 (IGFBP1), and prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on extracellular matrix (ECM) remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy. NEW & NOTEWORTHY Endocrine disruptors, such as para-nonylphenol, affect the decidualization of human endometrial stromal cells with an impact on decidualization itself, lysosome biogenesis and exocytosis, and extracellular matrix remodeling. All these alterations may negatively impact embryo implantation with the success of reproduction and the establishment of pregnancy.

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (α v and β 3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑ v β 3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile., (© 2024. The Author(s).)

Background: Intrauterine adhesion (IUA) as a prevalent gynecological disease is developed from infection or trauma. However, therapeutic strategies to repair damaged endometrium are relatively limited. Emerging studies have shed light on the crucial role of endometrial stromal cells (EnSCs) in the process of uterine endometrial regeneration. EnSCs isolated from the uterine endometrium have similar characteristics to mesenchymal stem cells (MSCs). However, it is still unknown whether EnSCs could be used as donor cells to treat IUA. The aim of this study was to evaluate the potential efficacy of EnSCs in treating rat IUA., Methods: Human EnSCs were isolated from the endometrial tissue of healthy female donors and subjected to extensive expansion and culture in vitro. Immunofluorescence, flow cytometry, cell proliferation assay, trilineage differentiation experiment, and decidualization assay were used to characterize the biological properties of EnSCs. We evaluated the immunoregulatory potential of EnSCs by analyzing their secreted cytokines and conducting bulk RNA sequencing after IFN-γ treatment. After EnSCs were transplanted into the uterine muscle layer in IUA rats, their therapeutic effects and underlying mechanisms were analyzed using histological analysis, Q-PCR, fertility and pregnancy outcome assay, and transcriptome analysis., Results: We successfully isolated EnSCs from the endometrium of human donors and largely expanded in vitro. EnSCs exhibited characteristics of mesenchymal stem cells and retained responsiveness to sex hormones. Following IFN-γ stimulation, EnSCs upregulated the anti-inflammatory cytokines and activated immunosuppressive molecules. Xenogeneic transplantation of EnSCs successfully repaired injured endometrium and significantly restored the pregnancy rate in IUA rats. Mechanistically, the therapeutic effects of EnSCs on IUA endometrium functioned through anti-inflammation, anti-fibrosis and the secretion of regeneration factor., Conclusions: Due to their large expansion ability, immunoregulatory properties, and great potential in treating IUA, EnSCs, as a valuable source of donor cells, could offer a potential treatment avenue for injury-induced IUA., (© 2024. The Author(s).)

Adenomyosis (ADS) is a common benign gynecological disease. Abnormal proliferation at the endometrial-myometrial interface (EMI) plays a crucial role in the occurrence and progression of ADS. miR-141-3p is associated with cell proliferation and apoptosis. However, the specific mechanism of miR-141-3p in the etiology of ADS is still unknown. In this study, we explored the effects of miR-141-3p on the proliferation and apoptosis of ADS EMI smooth muscle cells (SMCs). We collected EMI tissues for the primary culture of SMCs from 25 patients diagnosed with ADS and 20 without ADS. Real-time quantitative polymerase chain reaction and western blot were used to measure the mRNA and protein expression levels of miR-141-3p, JAK2, STAT3, phospho-JAK2, and phospho-STAT3 in ADS EMI SMCs. The cell counting kit 8 assay and flow cytometry analysis were used to evaluate the proliferation and apoptosis of EMI SMCs. The miR-141-3p mimic/inhibitor was used to increase or decrease the expression level of miR-141-3p. We added WP1066 to block the phosphorylation of JAK2/STAT3 pathway components. The miR-141-3p levels were decreased, while JAK2 and STAT3 levels were increased in ADS EMI SMCs. miR-141-3p overexpression significantly inhibited the proliferation and enhanced the apoptosis of EMI SMCs, whereas a decrease in miR-141-3p expression level was connected to the opposite results. Meanwhile, inactivated JAK2/STAT3 pathway decreased proliferation and enhanced apoptosis of EMI SMCs after WP1066 treatment. Furthermore, rescue experiments confirmed that the JAK2/STAT3 pathway was the downstream pathway of miR-141-3p and reduced the effect of miR-141-3p on the proliferation and apoptosis of EMI SMCs. These results demonstrate that miR-141-3p regulates the proliferation and apoptosis of ADS EMI SMCs by modulating the JAK2/STAT3 pathway., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The aim of this article was to investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells (EnSCs) through the miR-99b-5p/PCSK9 axis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos was assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model), and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction, western blotting, the CCK8 assay, immunohistochemistry, and dual-luciferase experiments, the potential mechanism by which BMSC-exos-derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSCs and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax, and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, whereas Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy-related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I, and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.

Thin endometrium (TE), which mainly occurs as a result of severe damage to the endometrial basalis, is one of the prominent etiologies of menstrual abnormalities, infertility, and recurrent miscarriage in women. Previous studies have demonstrated that mesenchymal stem cells (MSCs) are considered ideal cells with multipotency for regenerative medicine and exhibit therapeutic effects on TE through their cellular secretions. However, there is limited research on strategies to enhance MSC secretion to improve their therapeutic efficacy. Herein, we isolated menstrual blood-derived mesenchymal stem cells (MenSCs) from menstruation and transformed them into decidualized stromal cells (DSCs), which are specialized cells with enhanced secretory functions. To assess the therapeutic potential of DSCs compared to MenSCs, we conducted a series of experiments in cells and animals. The results demonstrated that DSCs exhibited changes in morphology compared to MenSCs, with a decrease in cell proliferation but a significant improvement in secretion function. Furthermore, DSCs facilitated the restoration of endometrial thickness and increased the number of glands and blood vessel formation. Most importantly, the pregnancy rates in rats were effectively restored, bringing them closer to normal levels. These findings greatly contribute to our understanding of stem cell therapy for TE and strongly suggest that DSCs could hold significant promise as a potential treatment option for TE., (© 2024. The Author(s).)

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA., Methods: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-β1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection., Results: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC., Conclusions: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation., (© 2024. The Author(s).)

Objective: Various stem cell-loaded scaffolds have demonstrated promising endometrial regeneration and fertility restoration. This study aimed to evaluate the efficacy of stem cell-loaded scaffolds in treating uterine injury in animal models., Methods: The PubMed, Embase, Scopus, and Web of Science databases were systematically searched. Data were extracted and analyzed using Review Manager version 5.4. Improvements in endometrial thickness, endometrial glands, fibrotic area, and number of gestational sacs/implanted embryos were compared after transplantation in the stem cell-loaded scaffolds and scaffold-only group. The standardized mean difference (SMD) and confidence interval (CI) were calculated using forest plots., Results: Thirteen studies qualified for meta-analysis. Overall, compared to the scaffold groups, stem cell-loaded scaffolds significantly increased endometrial thickness (SMD = 1.99, 95% CI: 1.54 to 2.44, P < 0.00001; I² = 16%) and the number of endometrial glands (SMD = 1.93, 95% CI: 1.45 to 2.41, P < 0.00001; I² = 0). Moreover, stem cell-loaded scaffolds present a prominent effect on improving fibrosis area (SMD = -2.50, 95% CI: -3.07 to -1.93, P < 0.00001; I² = 36%) and fertility (SMD = 3.34, 95% CI: 1.58 to 5.09, P = 0.0002; I² = 83%). Significant heterogeneity among studies was observed, and further subgroup and sensitivity analyses identified the source of heterogeneity. Moreover, stem cell-loaded scaffolds exhibited lower inflammation levels and higher angiogenesis, and cell proliferation after transplantation., Conclusion: The evidence indicates that stem cell-loaded scaffolds were more effective in promoting endometrial repair and restoring fertility than the scaffold-only groups. The limitations of the small sample sizes should be considered when interpreting the results. Thus, larger animal studies and clinical trials are needed for further investigation., Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO, identifier CRD42024493132., 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 © 2024 Huang, Zheng, Shi and Xu.)

Objective: This study aims to systematically evaluate the protective role of quercetin (QCT), a naturally occurring flavonoid, against oxidative damage in human endometrial stromal cells (HESCs) induced by hydrogen peroxide (H 2 O 2 ). Oxidative stress, such as that induced by H 2 O 2 , is known to contribute significantly to cellular damage and has been implicated in various reproductive health issues. The study is focused on investigating how QCT interacts with specific molecular pathways to mitigate this damage. Special attention was given to the p38 MAPK/NOX4 signaling pathway, which is crucial to the regulation of oxidative stress responses in cellular systems. By elucidating these mechanisms, the study seeks to confirm the potential of QCT not only as a protective agent against oxidative stress but also as a therapeutic agent that could be integrated in treatments of conditions characterized by heightened oxidative stress in endometrial cells., Methods: I n vitro cultures of HESCs were treated with QCT at different concentrations (0, 10, 20, and 40 μmol/L) for 24 h to verify the non-toxic effects of QCT on normal endometrial cells. Subsequently, 250 μmol/L H 2 O 2 was used to incubate the cells for 12 h to establish an H 2 O 2 -induced HESCs injury model. HESCs were pretreated with QCT for 24 h, which was followed by stimulation with H 2 O 2 . Then, CCK-8 assay was performed to examine the cell viability and to screen for the effective intervention concentration. HESCs were divided into 3 groups, the control group, the H 2 O 2 model group, and the H 2 O 2 +QCT group. Intracellular levels of reactive oxygen species (ROS) were precisely quantified using the DCFH-DA fluorescence assay, a method known for its accuracy in detecting and quantifying oxidative changes within the cell. The mitochondrial membrane potential was determined by JC-1 staining. Annexin Ⅴ/PI double staining and flow cytometry were performed to determine the effect of QCT on H 2 O 2 -induced apoptosis of HESCs. Furthermore, to delve deeper into the cellular mechanisms underlying the observed effects, Western blot analysis was conducted to measure the expression levels of the critical proteins involved in oxidative stress response, including NADPH oxidase 4 (NOX4), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p38 MAPK (p-p38 MAPK). This analysis helps increase understanding of the specific intracellular signaling pathways affected by QCT treatment, giving special attention to its potential for modulation of the p38 MAPK/NOX4 pathway, which plays a significant role in cellular defense mechanisms against oxidative stress., Results: In this study, we started off by assessing the toxicity of QCT on normal endometrial cells. Our findings revealed that QCT at various concentrations (0, 10, 20, and 40 μmol/L) did not exhibit any cytotoxic effects, which laid the foundation for further investigation into its protective roles. In the H 2 O 2 -induced HESCs injury model, a significant reduction in cell viability was observed, which was linked to the generation of ROS and the resultant oxidative damage. However, pretreatment with QCT (10 μmol/L and 20 μmol/L) significantly enhanced cell viability after 24 h ( P <0.05), with the 20 μmol/L concentration showing the most substantial effect. This suggests that QCT can effectively reverse the cellular damage caused by H 2 O 2 . Furthermore, the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H 2 O 2 model group compared to those in the control group ( P <0.01). However, co-treatment with QCT significantly reversed this trend ( P <0.05), indicating QCT's potential protective role in mitigating cell apoptosis. ROS assays showed that, compared to that in the control group, the average fluorescence intensity of ROS in the H 2 O 2 model group significantly increased ( P <0.01). QCT treatment significantly reduced the ROS fluorescence intensity in the H 2 O 2 +QCT group compared to the that in the H 2 O 2 model group, suggesting an effective alleviation of oxidative damage ( P <0.05). JC-1 staining for mitochondrial membrane potential changes revealed that compared to that in the control, the proportion of cells with decreased mitochondrial membrane potential significantly increased in the H 2 O 2 model group ( P <0.01). However, this proportion was significantly reduced in the QCT-treated group compared to that of the H 2 O 2 model group ( P <0.05). Finally, Western blot analysis indicated that the expression levels of NOX4 and p-p38 MAPK proteins were elevated in the H 2 O 2 model group compared to those of the control group ( P <0.05). Following QCT treatment, these protein levels significantly decreased compared to those of the H 2 O 2 model group ( P <0.05). These results suggest that QCT may exert its protective effects against oxidative stress by modulating the p38 MAPK/NOX4 signaling pathway., Conclusion: QCT has demonstrated significant protective effects against H 2 O 2 -induced oxidative damage in HESCs. This protection is primarily achieved through the effective reduction of ROS accumulation and the inhibition of critical signaling pathways involved in the oxidative stress response, notably the p38 MAPK/NOX4 pathway. The results of this study reveal that QCT's ability to modulate these pathways plays a key role in alleviating cellular damage associated with oxidative stress conditions. This indicates not only its potential as a protective agent against cellular oxidative stress, but also highlights its potential for therapeutic applications in treating conditions characterized by increased oxidative stress in the endometrium, thereby offering the prospect of enhancing reproductive health. Future studies should explore the long-term effects of QCT and its clinical efficacy in vivo , thereby providing a clear path toward its integration into therapeutic protocols., Competing Interests: 利益冲突　人子宫内膜基质细胞系为厦门大学医学与生命科学学院王海滨教授馈赠。除此之外，所有作者均声明不存在利益冲突。, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Office of Journal of Sichuan University (Medical Sciences).)