BACKGROUND:Age-related decline in oocyte quality is a significant factor in reduced fertility. This study aimed to evaluate the role of human amniotic mesenchymal stem cell-derived extracellular vesicles (hAMSC-EVs) on oocytes in aged mice and to elucidate the underlying molecular mechanisms. METHODS:An appropriate concentration of hAMSC-EVs was added to the in vitro maturation culture medium of mouse GV-stage aged oocytes. To assess nuclear maturation, the rate of first polar body extrusion and spindle morphology of MII-stage oocytes were evaluated. Following in vitro fertilization (IVF), fertilization rates and blastocyst formation rates were measured to assess cytoplasmic maturation. Intracellular oxidative stress levels were determined by measuring reactive oxygen species (ROS) and glutathione (GSH) levels, while mitochondrial function was evaluated by assessing mitochondrial membrane potential and ATP production. RESULTS:Compared with the untreated aged oocytes, treatment with hAMSC-EVs significantly improved the maturation of aged oocytes and promoted oocyte fertilization and early embryonic development after IVF. Mechanistically, hAMSC-EV was taken up by the cumulus-oocyte complex and increased the expression of SOD2, GPX, and HO-1 in oocytes by regulating the Keap1/Nrf2 pathway, which was significantly associated with attenuated ROS and increased GSH levels and improved mitochondrial function in aged oocytes. CONCLUSIONS:These findings offer a novel theoretical and experimental foundation for the clinical management of age-related diminished ovarian reserve.

Calciphylaxis (calcific uremic arteriolopathy, CUA) is a rare, fatal disorder primarily affecting chronic kidney disease patients, characterized by microvascular calcification, thrombosis, and skin necrosis. In a discovery cohort (3 CUA, 10 uremic), plasma proteomics identified Thrombospondin-1 (THBS1) as the top upregulated hub in CUA, significantly reduced after human amnion-derived mesenchymal stem cell (hAMSC) therapy, alongside latent TGF-β binding protein 1, both linked to coagulation and wound healing. In vitro proteomics indicated that THBS1/TGF-β1 blockade impaired CUA serum-induced endothelial adhesion and coagulation. ELISA in combined discovery and validation cohorts (8 CUA, 20 uremic) confirmed this reduction post-treatment (6 patients), independent of systemic inflammation. Multiplex immunofluorescence revealed THBS1 and CD47 co-localized with CD31 and integrin β3 in injured microvessels. A human microvascular chip showed that THBS1 inhibition or hAMSC-conditioned medium alleviates injury. These findings implicate THBS1 as a key factor and potential biomarker in calciphylaxis, suggesting hAMSC therapy as a promising mechanism-based approach. Video Abstract:

Targeting senescent pancreatic β-cells represents a promising therapeutic avenue for age-related diabetes; however, current anti-senescence strategies often compromise β-cell mass. In this study, human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSC-sEVs) were identified as a novel intervention that can be used to effectively counteract cellular senescence and preserve β-cell integrity. We aimed to systemically delineate the molecular mechanisms underlying hAMSC-sEV-mediated reversal of β-cell senescence in age-related diabetes. In oxidative stress-induced and naturally aged β-cell models, hAMSC-sEVs mitigated senescence-associated phenotypes, restored mitochondrial homeostasis, and enhanced insulin secretion capacity. In aged diabetic mice, administering these vesicles significantly ameliorated hyperglycemia, improved glucose tolerance, and reversed β-cell functional decline by reducing senescent β-cell populations, reinstating β-cell identity markers, and suppressing senescence-associated secretory phenotype (SASP) component production. Mechanistic investigations revealed that the miR-21-5p-enriched hAMSC-sEVs directly target the interleukin (IL)-6 receptor α subunit (IL-6RA), thereby inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation at tyrosine 705 and its subsequent nuclear translocation. This epigenetic modulation alleviated STAT3-mediated transcriptional repression of the mitochondrial calcium uniporter (MCU), rectifying age-related mitochondrial calcium mishandling and insulin secretion defects. Genetic ablation of MCU clearly established the central role of the miR-21-5p/IL-6RA/STAT3/MCU axis in this regulatory cascade. Our findings reveal hAMSC-sEVs as a novel senotherapeutic strategy for age-related diabetes, elucidating the pivotal role of miR-21-5p-driven epigenetic-mitochondrial calcium homeostasis in reversing β-cell dysfunction, establishing a framework for targeting cellular senescence in metabolic disorders.

Premature ovarian insufficiency (POI), characterized by diminished ovarian function in women under 40, has immune factors as one of the significant etiological factors. Metformin, a frontline medication for type 2 diabetes and it may also modulate immune responses, suggesting potential immunomodulatory and anti-inflammatory properties. This study investigates the therapeutic potential of metformin in autoimmune POI. We established an autoimmune POI mouse model by immunizing with the peptide zona pellucida 3 (pZP3) and administered metformin via drinking water to assess its impact on ovarian and immune functions. Flow cytometry revealed an increase in the number of Treg cells in the POI group (pZP3), while the metformin-treated group showed a decrease close to the control group (CON) levels. Under transmission electron micros-copy, the pZP3 exhibited collapse and thinning of the zona pellucida, vacuolization of granulosa cells, and mitochondrial damage, with improvements observed in the metformin-treated group. In paraffin sections of mouse ovaries stained with Sirius Red, the pZP3 displayed a greater number of strong orange signals indicative of fibrosis, and in immunofluorescence, the macrophages in the pZP3 showed a clear polarization ab-normality in M1/M2, which was significantly reduced in the metformin-treated group. Our findings indicate that metformin significantly enhances ovarian function in pZP3-induced autoimmune POI mice and mitigates inflammation and fibrosis within the ovarian microenvironment. Transcriptome sequencing of mouse ovaries revealed 15,250 co-expressed transcription factors (TFs) across all groups. Comparative analysis identified 1098 differentially expressed genes (DEGs) between the CON and pZP3, with 403 upregulated and 695 downregulated genes. Notably, upregulated genes like S100a9 were associated with the IL-17 signaling pathway, while downregulated genes such as Cyp17a1 were linked to steroid metabolism pathways. These results indicate that metformin's therapeutic effects in autoimmune POI are mediated through pathways in-volving hormone biosynthesis, steroid hormone synthesis, and cytokine metabolism. Consequently, metformin's action on the ovarian microenvironment and hormone metabolism could improve both ovarian and immune state in pZP3-induced autoimmune POI mice, presenting a promising therapeutic strategy for POI sufferers.

Objectives Female fertility declines with age, primarily due to a decrease in both oocyte quantity and quality. While putrescine supplementation has been shown to improve oocyte quality in aged mice, the underlying mechanisms remain unclear. In particular, whether putrescine modulates mitochondrial-associated membranes (MAM) and mitigates mitochondrial calcium overload via the IP3R-GRP75-VDAC1 complex has yet to be elucidated. Materials and methods In this study, we investigated the effects of putrescine on oocyte quality using three groups: eight-week-old mice (Young), 40-week-old mice (Old), and 40-week-old mice with 0.5 mM putrescine supplementation during in vitro maturation (Put). Key parameters assessed included oocyte mass, MAM number, mitochondrial calcium levels, mitochondrial function, and apoptosis. Results Aged mice exhibited significantly lower anti-Müllerian hormone (AMH) levels and a reduced oocyte count, accompanied by a decline in oocyte quality. Putrescine supplementation significantly improved first polar body extrusion and blastocyst formation rates in aged oocytes. Additionally, it reduced MAM formation and weakened IP3R-GRP75-VDAC1 interactions, alleviating mitochondrial calcium overload. Consequently, mitochondrial function was enhanced, ATP production increased, and apoptosis reduced. Conclusion Putrescine ameliorates the quality of aged oocytes by modulating Ca 2+ transfer at MAM. These findings provide novel insights into the role of putrescine in improving oocyte quality and suggest its potential as an in vitro maturation (IVM) supplement to enhance reproductive outcomes in older women by modulating MAM.

BACKGROUND:Calciphylaxis, also termed calcific uremic arteriolopathy (CUA) in patients with end-stage kidney disease (ESKD), is a rare and fatal condition characterized by cutaneous ischemic necrosis. METHODS:Three patients with calciphylaxis and metastatic pulmonary calcification (MPC) were treated with human amnion-derived mesenchymal stem cells (hAMSCs). Effects were evaluated using the Visual Analogue Scale (VAS), modified Bates-Jensen Wound Assessment Tool for CUA (BWAT-CUA), wound quality of life questionnaire (Wound-QoL), and histological analysis. MPC was assessed by high-resolution CT (HRCT) and 99ᵐTc-methylene diphosphonate (99ᵐTc-MDP) bone scans.99ᵐTc-labeled macroaggregated albumin (99ᵐTc-MAA) pulmonary perfusion imaging was conducted for the first time in patients with MPC. RESULTS:Three patients exhibited wound healing and improvement in skin symptoms. Two months before CUA, asymptomatic MPC was detected in Patient 1, who was treated with hAMSCs for 15 months. The condition progressed to chest pain and dyspnea. HRCT and 99ᵐTc-MDP bone scans showed worsening calcification, particularly in the upper and mid-thoracic lobes.99ᵐTc-MAA pulmonary perfusion imaging revealed impaired or absent blood perfusion in the areas of metastatic calcification. Patient 1 died from respiratory failure. Patients 2 and 3 had asymptomatic MPC at calciphylaxis diagnosis. After 2 months of treatment, Patient 2, showed no significant imaging improvement and passed away 6 months after discontinuing hAMSC treatment. Patient 3 has shown no significant progression of pulmonary lesions and continues hAMSC therapy. CONCLUSION:We reported personalized early, noninvasive diagnosis and regenerative treatments for calciphylaxis patients with MPC. Although the current hAMSC treatment regimen is effective for skin lesions, its impact on MPC requires further investigation.

Calciphylaxis is a rare, progressive disorder characterized by subcutaneous adipose and dermal microvascular calcifications, microthrombi, and endothelial damage. It mainly affects patients with chronic kidney disease (CKD), which is also known as calcific uremic arteriolopathy. Skin biopsy is the gold standard for diagnosis, but it is an invasive procedure. Calciphylaxis frequently results in ischemic and nonhealing ulcerations with a high mortality rate. A multidisciplinary targeted approach is the primary treatment method. Vascular calcification, which is a common complication in patients with CKD, cannot completely explain the rapid progression of calciphylaxis. This article reviews the advances in the epidemiological characteristics, risk factors, and diagnosis, including non-uremic calciphylaxis and visceral calciphylaxis, pathogenesis, associated animal models, and treatment of calciphylaxis. The scarcity of animal models that mimic the clinical presentation of calciphylaxis hampers the understanding of its pathogenesis. The acute effects on progressive vascular injury, including the induction of severe ischemia and inflammatory responses, have been emphasized. Actively listening to the voices of patients and their families and building a multidimensional research system with artificial intelligence technologies based on the specific molecular makeup of calciphylaxis patients will help tailor regenerative treatment strategies. Mesenchymal stem cells (MSCs) may represent a novel therapy for calciphylaxis because of their regenerative effects, inhibition of vascular calcification, anti-infection and immunomodulation properties, and improvement of hypercoagulability. Safe, effective, accessible, and economical MSC strategies guided by biomarkers deserve consideration for the treatment of this devastating disease.

Pluripotent stem cells (PSCs) have been derived from various species, but most culture systems stabilize only a single PSC type. By contrast, epiblast cells in vivo exist along a continuum and interact dynamically with both embryonic and extraembryonic cells, interactions missing in standard PSC cultures. This absence limits the self-organizing potential of PSCs and leads to disorganized tissue formation in teratomas. To address this, we developed a unified culture system that supports the stable differentiation of epiblast-like cells into multiple key human gastrulating cell types, collectively called human gastrulating stem cells (hGaSCs). hGaSCs, composed of endoderm-like, mesoderm-like, ectoderm-like, amnion ectoderm-like, and primordial germ cell-like cells, maintain a stable balance during long-term culture. In 3D culture, hGaSCs self-assemble into gastruloid-like structures (hGaSC-gastruloids) that model aspects of a Carnegie Stage 7 human embryo, including gastrulation and germ layer specification. Using hGaSC-gastruloids, we modeled the effects of valproic acid (VPA) on human gastrulation and uncovered molecular pathways underlying VPA-induced malformations. When transplanted into the seminiferous tubules, hGaSCs formed embryo-like structures, progressing through fetal tissue and organ development, unlike the disorganized growth seen in teratomas. In conclusion, hGaSCs provide a versatile platform to study human gastrulation, early organogenesis, developmental defects, and drug teratogenicity, with promising applications in tissue and organ generation from cultured stem cells.

Background: Calciphylaxis, which mostly affects individuals with end stage kidney disease (ESKD), is also known as calcific uremic arteriolopathy (CUA). It is a rare and fatal disease that manifests with calcification and thrombosis of microvessels, ischemia, and necrosis in skin tissues(ORPHA:280062). Histopathological features of extracutaneous tissues of CUA patients undergoing human amnion derived mesenchymal stem cell (hAMSC) treatment remain unknown. Methods: A female CUA patient, treated with hAMSCs for 20 months, passed away due to stroke. Histopathological features of her extracutaneous tissues were compared with those of ESKD patients (n = 7). Raman spectroscopy was applied to identify the composition of calcifications. The distribution of hAMSCs, derived from the amnion of a male fetus, in tissues of the CUA patient was determined by detecting the Y chromosome using reverse-transcription polymerase chain reaction. Results: Microvessel lesions were more prevalent in the extracutaneous tissues of the CUA patient than in those of ESKD patients, although the regenerated skin showed normal histological characteristics. The CUA patient exhibited calcifications of microvessel media, including the microvessels in the lungs, kidneys, spleen, pancreas, and uterus. Her mitral valve and kidney displayed severe calcification, identified as calcium phosphate with some calcium carbonate. hAMSCs were not detected in the tissues of the CUA patient. Conclusion: Under the treatment strategy with hAMSCs, based on the effects of skin regeneration, microvascular lesions in the extracutaneous tissues of the CUA patient were more severe than those in ESKD patients. CUA should be considered a systemic disease when identifying treatment targets. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols ### Funding Statement The National Natural Science Foundation of China (81270408, 81570666, 81730041, and 81671447), the International Society of Nephrology (ISN) Clinical Research Program (18-01-0247), Construction Program of Jiangsu Provincial Clinical Research Center Support System (BL2014084), Jiangsu Province Key Medical Personnel Project (ZDRCA2016002), CKD Anemia Research Foundation from China International Medical Foundation (Z-2017-24-2037), Outstanding Young and Middle-Aged Talents Support Program of The First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital), the National Key Research and Development Program of China (2017YFC1001303), the Program of Jiangsu Province Clinical Medical Center (YXZXB2016001, BL2012009), the State Key Laboratory of Reproductive Medicine Program (SKLRM-GC201803), and the Program of Jiangsu Commission of Health (H201605), Jiangsu Province Hospital (the First Affiliated Hospital with Nanjing Medical University) Clinical Capacity Enhancement Project (JSPH-MA-2023-7), Jiangsu Provincial Medical Key Discipline(Laboratory) Cultivation Unit(JSDW202206). All authors declared no competing interests. The study was supported by the ISN Mentorship Program and the authors thank Professor Marcello Tonelli (University of Calgary, Canada) for his helpful comments on the draft of the manuscript. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of the First Affiliated Hospital with Nanjing Medical University in China gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript

Background: Low-level mosaicism is a common trait of early human development. Although mosaic embryos may lead to healthy live births, the direct effects of mosaicism are unknown. While embryo self-correction was demonstrated in mouse models, humans can only study the effects of chromosomal aberrations and blastocyst mosaicism on the early peri-implantation period by extending in vitro embryo culture up to 12 days post-fertilization. Methods: The established culture protocols were followed to generate embryonic stem cells. The 24 high-quality mosaicism/aneuploid mosaicism blastocysts were detected by preimplantation genetic testing for aneuploidy (PGT-A). The isolated inner cell masses (ICMs) were seeded onto feeder-free dishes, and after 10-12 days of culture, there were six blastocyst ICMgenerated stem cell clones. The cells shed during the stem cell growth were harvested, and next-generation sequencing was performed on stem cells and the shed cells. To test pluripotency, a small number of stem cells were isolated and subcultured. Results: The PGT-A status was confirmed from the results of next-generation sequencing of stem cell exfoliated cells and stem cells. The mosaics in five blastocysts were fully repaired while the mosaic in one blastocyst was partially repaired. Simultaneously, the cells were verified to have pluripotency and the ability to differentiate into three germ layers by immunofluorescence, flow cytometry, and in vitro differentiation analyses. Conclusion: Human mosaicism/aneuploid mosaicism blastocysts have the potential for self-correction by eliminating mosaic cells.

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is an orphan disease without proven therapies, we rescued it with human amnion-derived mesenchymal stem cells (hAMSCs). In a discovery cohort of 10 uremic patients and 3 CUA patients, plasma proteomic analysis showed core differentially expressed proteins (DEPs) Thrombospondin 1 (THBS1) and Latent transforming growth factor (TGF)-β binding protein 1 (LTBP1) decreased significantly after 3 days of hAMSC treatment. Single-cell transcriptome sequencing of peripheral blood mononuclear cells (PBMCs) indicated megakaryocytes were the source of THBS1 in CUA patient. Same as the discovery cohort, plasma THBS1 and TGF-β1 levels were increased in seven CUA patients compared to the uremic group (n=20), as measured by enzyme-linked immunosorbent assay (ELISA) in the validation cohort. They can be inhibited after hAMSC treatment and increased as the frequency of therapy decreased. THBS1 and its receptor, CD47, were increased in the CUA skin. THBS1 and TGF-β1 are biomarker candidates for calciphylaxis.

Background Studies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem.Methods In the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay.Results Our study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary.Conclusions The presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn't proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

Calciphylaxis is a rare and highly fatal disease that manifests with calcification and thrombosis of microvessels, ischemia and necrosis in the skin and subcutaneous tissues. Whether it is a systemic process is controversial. We have proposed that human amnionderived mesenchymal stem cells(hAMSCs)are a promising therapy for uremic calciphylaxis, also know as calcific uremic arteriolopathy (CUA). However, the histopathological features and chemical composition of extracutaneous tissues remian unclear.

Diabetic foot ulcers are a serious complication of diabetes and require advanced therapies to promote wound healing. Human amnion-derived mesenchymal stem cells(hAMSCs) have gained attention due to their regenerative properties and potential therapeutic applications. This study aims to evaluate the effectiveness of hAMSCs in the treatment of diabetic foot ulcers in a female uremic patient.

Abstract Background: To study whether ILs/TNFs in the follicular fluid (FF) of women with EMs are responsible for impaired follicular development or (and) ovulation or not, and then to explore the underlying mechanisms. Methods: follicular fluid (containing cumulus granulosa cells) was collected from women with EM and male factor infertility at our Clinical Reproductive Medicine Center, and peritoneal fluid was collected from the above patients with EMs. The expression of ovulation-related genes in cumulus cells was analysed by RT-PCR. Mouse cumulus cells expansion degree was assessed after cultured in follicle fluid from infertile women. Follicle fluid was detected by ELISA. Oocytectmized complex cell model was established, and cultured in vitro medium with addition of 100 IU/ml FSH. TUNEL staining was used to determine the apoptosis of cumulus cells. Then, we explored expression of P-SMAD2/3，key enzyme for retinoic acid metabolism, and methylation of SP1 binding sites in Lhcgr promoter region. Meanwhile, the P-AKT and P-catenin were assessed by Western blot. All experiments were performed independently at least three times, and data are presented as mean ± SEM. Statistical analyses were performed using Graphpad Prism 5 software p<0.05 (* and different letters) were defined as significant differences. Results: In cumulus cells, expression of genes related to ovulation decreased significantly than that in controls (P < 0.05), especially starting from LHCGR . The concentrations of IL-8 and TNF-α in follicle fluid were significantly higher in infertile women with endometriosis than in controls (P < 0.05). The function of follicle fluid and pelvic fluid of endometriosis women have changed. Addition of 500 pg/mL IL-8/TNF-α to medium did not cause significant apoptosis of cumulus cells, but inhibited P-AKT and P-β-catenin. On the other hand, expression of P-SMAD2/3 and retinoic acid production were reduced, while hypermethylation of the Sp1 binding sequence on Lhcgr promoter was identified, and Lhcgr expression was significantly reduced compared to control (P<0.05). Conclusion: Elevated IL-8/TNF-α in follicular fluid of women with endometriosis indirectly maintains Lhcgr promoter hypermethylation through activation of P-SMAD2/3, while inhibiting AKT and β-Catenin phosphorylation, which together reduce LHCGR mRNA expression.