BACKGROUND:The main functions of testes are sperm production and androgen secretion in testicular befitting microenvironment. Excessive level of the reactive oxygen species (ROS) from metabolism and cellular events can lead to oxidative stress (OS) and ferroptosis, which injure the functions of mitochondria and endoplasmic reticulum (ER) of testicular cells, leading to the impaired spermatogenesis and spermiogenesis, as well as insufficient androgen. Our previous studies have shown that peroxiredoxin 4 (Prdx4) is a vital ER-located protector against endoplasmic reticulum stress (ERS) in ovarian granulosa cells. In this study, we explored whether Prdx4 played a beneficial role in testes, and the potential application value in male reproduction. METHODS:Prdx4 knockout mice and Prdx4-/y Leydig cells were established using CRISPR/Cas9 system, the natural adult (9 weeks) mice, aging (9 months) mice, and wild-type MLTC-1 cells were used as control. To further investigate the protective effect of Prdx4 in the adult mice, the testicular 43°C water-bath was used to induce an OS model. RESULTS:The male Prdx4-/y mice aged over 9 months showed the age-related pathology of seminiferous tubule, the increased OS and ERS, and the decreased fertility. The adult Prdx4-/y mice under heat stress manifested increased levels of OS, including almost 1.5-fold rise in 4-hydroxynonenal (4-HNE) and 8-hydroxy-2'- deoxyguanosine (8-OHdG), higher ferroptosis, and poorer fertility. Prdx4-/y Leydig cells showed a higher level of ferroptosis after hydrogen peroxide treatment. ACSL4 increased by 18.6% while COX2 increased by 13.5%, and the expressions of GPX4 and SLC7A11 decreased by 49.2% and 24.3%, respectively. Increased levels of ferroptosis in MLTC-1 leads to mitochondria-derived OS and apoptosis, which can be partially restored by deferoxamine and overexpressing SLC7A11. CONCLUSION:We concluded that Prdx4 as a protective factor plays a critical role in preserving spermatogenesis by alleviating testicular OS and ferroptosis, and that Prdx4 may be known as a potential target in mitigating the adverse effects of natural aging and OS injury.

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.

This systematic review and meta analysis was conducted to investigate the reproductive outcomes in the patients with adenomyosis after HIFU treatment, aimed to recommend HIFU treatment for those women with adenomyosis who have reproductive demand. Extensive search was performed through PubMed, EMBASE and Web of Science databases. All available studies that evaluated the reproductive outcomes after HIFU for adenomyosis. Systematic review was performed following PRISMA guidelines. A meta-analysis was performed on data from 15 studies involving 3730 patients with adenomyosis. Pooled results showed that the pregnancy rate (PR) in women who desired to conceive was 54.6

The incidence of premature ovarian insufficiency (POI) is gradually increasing, and currently, no clinical treatments are available to restore ovarian function in POI patients, which severely affects the physical and mental health of young women with fertility needs. Putrescine is a type of polyamine found in the human body and different foods, and research has shown that adding putrescine to drinking water can reduce embryo resorption rates in aged mice. Polyamines are also closely related to apoptosis, with ovarian granulosa cell (GC) apoptosis considered an important factor in follicular atresia, which may lead to POI. Therefore, we investigated putrescine's actions in POI. We generated a drug-induced POI mouse model by intraperitoneally injecting cyclophosphamide (Cy) into animals. Putrescine was then added to their drinking water to explore its effects on ovarian function in mice. In vitro, we added 4-hydroxycyclophosphamide and putrescine to a GC line to examine the specific mechanisms underpinning putrescine actions. We discovered that putrescine improved ovarian function and fertility in Cy-induced POI mice and ameliorated GC apoptosis via P53 signaling. These findings provide potential therapeutic strategies for patients with POI.

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:

Peroxiredoxin 4 (Prdx4) serves as an endogenous antioxidant. In this study, we explored the protection effect of Prdx4 against the testicular Leydig cells senescence. In vitro experiments with Prdx4−/y MLTC-1 cells and in vivo studies using D-galactose-induced and naturally aging mice demonstrated that Prdx4 deficiency exacerbates the oxidative stress(OS)-mediated apoptosis(upregulated Bax, p53, and caspase 3), impairs mitochondrial function and decreases androgen synthesis, while increasing OS markers 4-HNE and 8-OHdG. Conversely, the AAV-mediated Prdx4 overexpression restored testosterone level to 90% of young mice, improved sperm count, motility, and morphology, and alleviated testicular histological damage in aging mice. In the H2O2-treated Leydig cells, Prdx4 regulated autophagy by suppressing the AMPK-mTOR pathway and blocked intracellular Ca2+ influx in the Prdx4-/y cells. Notably, mTOR inhibitor rapamycin and Ca2+ channel blocker amlodipine mimicked Prdx4’s protective effects. These findings indicate that Prdx4 protects against the Leydig cell senescence and mitochondrial dysfunction through the Ca2+-AMPK-mTOR-autophagy axis, highlighting Prdx4 as a potential therapeutic target for male late-onset hypogonadism and reproductive aging.

This study investigated differences in reproductive outcomes and vaginal microbiota profiles between two endometrial preparation protocols-letrozole (LE) combined with human menopausal gonadotropin (HMG) and hormone replacement therapy (HRT) with gonadotropin-releasing hormone agonist (GnRH-a) pretreatment-in women with endometriosis undergoing frozen embryo transfer (FET). Following 1∶1 propensity score matching, a total of 770 FET cycles were analyzed. No statistically significant differences were observed in live birth rates or clinical pregnancy rates between the two groups. However, the LE + HMG group showed a nonsignificant trend toward a lower miscarriage rate (13.7% vs. 19.8%, P = 0.070), as well as lower observed rates of cesarean delivery (64.9% vs. 75.4%, P = 0.020) and hypertensive disorders of pregnancy (4.8% vs. 10.1%, P = 0.039). Previous evidence suggests an association between GnRH-a treatment and reproductive tract microbiota composition. Given the clinical and ethical constraints of endometrial sampling during FET, vaginal microbiota was evaluated as a non-invasive indicator of lower reproductive tract microbial composition. In the prospective arm, vaginal samples from 55 women in the LE + HMG group and 50 in the GnRH-a HRT group were analyzed using 16S rRNA sequencing and droplet digital PCR. While no significant differences were observed in Lactobacillus or Gardnerella abundance, the GnRH-a HRT group showed higher relative abundances of several low-abundance taxa, such as Escherichia-Shigella, Limosilactobacillus, and Staphylococcus. In conclusion, although both protocols achieved comparable live birth outcomes, the LE + HMG regimen was associated with lower rates of cesarean delivery and hypertensive disorders of pregnancy, while the two protocols exhibited differences in several low-abundance vaginal bacterial taxa. However, longitudinal studies incorporating baseline and repeated sampling are needed to clarify temporal relationships and causality.

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.

BACKGROUND:The senescence of testicular Leydig cells (LCs) is a key cause of age-related testosterone deficiency, in which oxidative stress (OS) and mitochondrial dysfunction are critical driving mechanisms. We explore whether the bioactive peptide C248 of PRDX4, an intracellular antioxidant, exerts mitochondrial protection to ameliorate LCs' function. METHODS:Based on the antioxidant domains of the PRDX4 protein, small molecular peptides were designed, and bioactive peptide C248 stood out from the crowd. An OS-induced senescence model of LCs was constructed by treating the MLTC-1 cell line with hydrogen peroxide (H2O2). C248 peptide or nicotinamide mononucleotide (NMN), as the positive control, was administered in the culture medium. The cellular function-related indicators, including DPPH free radical scavenging rate, cell viability, testosterone level, hydrogen peroxide (H2O2) content, senescence-associated β-galactosidase (SA-β-gal) activity, 8-hydroxy-2'-deoxyguanosine (8-OHDG) level, and 4-hydroxynonenal (4-HNE) level, were evaluated. The mitochondrial function and structural indicators, such as mitochondrial membrane potential, ATP production, mitochondrial morphology, and mitochondrial DNA (mtDNA) copy number, were subsequently tested. RESULTS:In vitro experiments confirmed that C248 could scavenge DPPH free radicals in a dose-dependent manner, reduce the levels of reactive oxygen species, and increase antioxidant enzyme activity in LCs (p < 0.01). Both C248 and NMN increased testosterone secretion and improved cell viability (p < 0.01). Both C248 and NMN increased mitochondrial morphology and quantity, mitochondrial membrane potential (p < 0.01), ATP production (p < 0.01), and mitochondrial DNA (mtDNA) copy number (p < 0.01). CONCLUSION:This study reveals that the small molecular C248, a bioactive peptide of PRDX4, is a new candidate molecule for intervening in LC senescence and confirms that mitochondrial protection is a key strategy for improving age-related testicular dysfunction.

The risk of multiple placental thrombosis is significantly elevated in preeclampsia (PE), and the vascular endothelium plays a vital role in coagulation processes through the secretion of ITGA2B, vWF, and TF. However, the underlying pathological mechanisms remain unclear. In this study, placental blood vessels were collected from both PE and normal pregnancies. Protein levels of ITGA2B were found to be up-regulated in PE samples compared to controls, indicating enhanced coagulation function in PE placental tissue. Additionally, Higher cholesterol levels of plasma and lower ALKBH1 expression in placental blood vessels of PE were found, overall DNA N6-methyldeoxyadenosine (6 mA) levels were up-regulated. Interestingly, the DNA 6 mA level at the ITGA2B promoter was decreased. Cholesterol overload experiments in human umbilical vein endothelial cells (HUVECs) demonstrated that cholesterol regulates the protein expression of ALKBH1 and ITGA2B in a concentration-dependent manner. Furthermore, ITGA2B protein expression was increased following ALKBH1 knockout, but no changes were observed when cholesterol was incubated with ALKBH1 knockout cells. These findings provide critical insights into the roles of cholesterol levels, ALKBH1, and ITGA2B in coagulation function within placental blood vessels. This information may contribute to further investigations of potential therapeutic targets and early prevention strategies for placental thrombosis in PE.

The risk of multiple placental thrombosis is significantly elevated in preeclampsia, and the vascular endothelium plays a vital role in coagulation processes through the secretion of Integrin alpha 2b, von Willebrand factor, and tissue factor. However, the underlying pathological mechanisms remain unclear. In this study, placental blood vessels were collected from both preeclampsia and normal pregnancies. Protein levels of Integrin alpha 2b were found to be up-regulated in preeclampsia samples compared to controls, indicating enhanced coagulation function in preeclampsia placental tissue. Additionally, higher cholesterol levels of plasma and lower AlkB homolog 1 expression in placental blood vessels of preeclampsia were found, overall DNA N6-methyldeoxyadenosine levels were up-regulated. Interestingly, the DNA N6-methyldeoxyadenosine level at the Integrin alpha 2b promoter was decreased. Cholesterol overload experiments in human umbilical vein endothelial cells demonstrated that cholesterol regulates the protein expression of AlkB homolog 1 and Integrin alpha 2b in a concentration-dependent manner. Furthermore, Integrin alpha 2b protein expression was increased following AlkB homolog 1 knockout, but no changes were observed when cholesterol was incubated with AlkB homolog 1 knockout cells. These findings provide critical insights into the roles of cholesterol levels, AlkB homolog 1, and Integrin alpha 2b in coagulation function within placental blood vessels. This information may contribute to further investigations of potential therapeutic targets and early prevention strategies for placental thrombosis in preeclampsia.

Premature ovarian insufficiency (POI) is a condition identified by the decline in ovarian function before the age of 40. The treatment of POI patients deserves in-depth research. This study aimed to explore the impacts of probiotics on the gut and vaginal microbiota, ovarian function and metabolic indexes in a mouse model of POI. A mouse model of POI was successfully established by intraperitoneal injection of cyclophosphamide. Subsequently, the mice were divided into the control group, the POI group and the POI gavage group. Mice in the POI gavage group were gavaged for 28 consecutive days with a mixture of 12 probiotics. The anti-Müllerian hormone (AMH) and sex hormone levels, the number of follicles, the serum total cholesterol (TC) and triglycerides (TG) levels and the distributions of gut and vaginal microbiota of the mice were assessed and compared. Compared with the healthy control group, the level of AMH and the number of growing follicles significantly decreased in POI mice (P<0.05), whereas the number of atretic follicles increased significantly (P<0.05). Meanwhile, the gut and vaginal microbiotas were disturbed in the POI group. Bacterial genera such as Allobaculum, Prevotella and Bacteroides were enriched in the gut microbiota, while Proteus, Streptococcus and Rothia were enriched in the vaginal microbiota. However, these bacteria contributed no favorable effect on the host. Interestingly, the increases in these taxa were reversed in POI mice treated with probiotics. Additionally, although there were no significant differences in AMH, estradiol (E2), and follicle stimulating hormone (FSH) levels between POI group without probiotics and POI gavage group (P>0.05), the AMH and E2 levels tended to increase while the FSH level tended to decrease in the gavage group. Besides, the number of growing follicles in the gavage group exhibited a slight increase compared with the POI group without probiotics (P>0.05), whereas the number of atretic follicles in the gavage group showed a decrease (P>0.05). Moreover, the levels of TC and TG in POI group without probiotics were elevated compared with the control group, while this trend was reversed in the POI gavage group (TG: P<0.05, TC: P>0.05). Probiotics may mitigate vaginal microbiota disruption, alleviate lipid metabolism disorders and improve indicators related to ovarian function by modulating the dysbiosis of gut microbiota in POI mice. These results provide some new directions and theoretical foundations for future microecological treatment in POI patients in clinical practice.

Simiao Biejia Decoction (SMBJ), a traditional Chinese herbal formula, has been clinically applied to treat diabetes mellitus (DM)-induced male dysfunction due to its kidney-tonifying, heat-clearing, and blood-enriching properties. However, the precise molecular mechanisms and therapeutic targets through which SMBJ confers protection against DM-induced testicular damage remain to be fully elucidated. Goto-Kakizaki (GK) and wild-type rats were randomly assigned to the wild-type, GK model, and SMBJ low-, medium-, and high-dose groups. SMBJ was administered at low, medium, and high doses (2.44, 4.88, and 9.76 g/kg/day) by gavage for 4 weeks. Serum testosterone, blood glucose, and oxidative stress levels were measured using ELISA and colorimetric assays. Histopathological staining was performed to evaluate testicular damage. In vitro experiments were conducted using the R2C cell line subjected to high-glucose conditions, with intervention using SMBJ-containing serum. RT-qPCR, Western blotting, immunofluorescence, MeRIP-qPCR, RNA stability were employed to elucidate the underlying mechanisms. SMBJ alleviates oxidative stress injury, thereby reducing apoptosis and enhancing testosterone secretion in the interstitial tissue and in high glucose-stimulated R2C cell of diabetic rats. METTL3 expression was downregulated in the testes of diabetic rat and in R2C cell exposed to high glucose. METTL3 knockdown mimicked the effects of high glucose, underscoring its protective role in Leydig cells. Si-METTL3 decreased the stability of PI3K/AKT mRNAs, whereas SMBJ upregulated METTL3 expression, modulating the PI3K/AKT pathway, reversing high glucose-induced damage in R2C cell, and highlighting its therapeutic potential in Leydig cell protection. SMBJ may upregulates METTL3 expression alongside PI3K/AKT activation to ameliorate diabetes mellitus-induced Leydig cell dysfunction. The potential regulatory role of m6A modification in diabetes-induced testicular injury was further elucidated.

ABSTRACTObjectiveTo assess the comprehensive health status of school‐age children conceived through assisted reproductive technology (ART) compared to that of those conceived naturally.DesignA prospective cohort study of children conceived through ART.SettingFirst Affiliated Hospital of Nanjing Medical University, China.Population SampleOne hundred school‐age children were enrolled and followed up (51 conceived via ART and 49 naturally conceived (controls)).MethodsA comprehensive health status assessment was performed in children aged 6–12 years, including anthropometric measurements, glucose‐lipid metabolism, echocardiography, intelligence, behaviour, vision and hearing. Multivariate regression models were used to adjust for sociodemographic and perinatal covariates.Main Outcome MeasuresComprehensive health status of children conceived via ART.ResultsChildren conceived by ART had similar height, weight, blood pressure, blood glucose and lipid profiles compared to the controls. Echocardiography showed similar cardiac geometric morphology and left/right ventricular diastolic‐systolic function between the two groups. ART children had lower carotid intima‐media thickness than controls (0.52 ± 0.11 vs. 0.58 ± 0.11). Visual acuity, hearing and intellectual and behavioural assessments were similar. An alternating covering test for the eyes showed a higher incidence of abnormal eye movement in the ART group than in the control group (87.76% vs. 68.89%).ConclusionsThe similar overall health status of ART‐ and naturally conceived children is reassuring for those receiving ART. However, our study shows a possible increase in the incidence of latent exotropia among ART children. Long‐term follow‐up is warranted to assess the overall health status associated with ART throughout the lifespan.

Objective:Simiao Biejia (SMBJ) granules, a traditional Chinese herbal remedy, have been used to treat erectile dysfunction caused by diabetes mellitus (DMED). However, the molecular mechanisms underlying SMBJ's therapeutic effects remain unclear. This study aimed to investigate the effects and mechanisms of SMBJ in a rat model of DMED using network pharmacology, proteomics, and molecular docking. Methods:A rat model of DMED was established, and SMBJ granules were administered (0, 7.1, 14.2, and 28.4 mg/kg/d, respectively) for 4 weeks. Erectile function was evaluated by measuring intracavernous pressure and mean arterial pressure. The active compounds in SMBJ were analyzed by gas chromatography and identified using network pharmacology and bioinformatics. Potential targets in the penile tissue was identified via proteomics and validated by Western blotting. Molecular docking was used to assess the binding affinity between bioactive compounds and primary targets. Results:SMBJ significantly improves erectile function and ameliorates DMED in rats by reducing corpus cavernosum fibrosis, decreasing eNOS and nNOS levels, alleviating oxidative stress in penile tissue, and mitigating damage to smooth muscle cells (SMCs) and vascular endothelial cells (VECs). Network pharmacology and proteomics identified 24 potential SMBJ targets in DMED. The 4 drug molecules identified were involved in the therapeutic effects of SMBJ, among which luteolin was predicted to be the core drug component. Luteolin bound directly with AKT1, a key differentially expressed protein in the penile tissue of DMED rats. Further analysis showed that luteolin in SMBJ activates the PI3K/Akt pathway and regulation of nNOS and NF-kB expression in the penile tissue of DMED rats to improve erectile function. Conclusion:SMBJ improved oxidative stress damage, vascular endothelial repair, and angiogenesis in the penile tissue of DMED rats. Luteolin is one of the core drug components of SMBJ in DMED treatment that regulates PI3K/AKT-related pathways.

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.

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

The trophoblast epithelial-to-mesenchymal transition (EMT) is a procedure related to embryo implantation, spiral artery establishment and fetal-maternal communication, which is a key event for successful pregnancy. Inadequate EMT is one of the pathological mechanisms of recurrent miscarriage (RM). Whole-exome sequencing revealed that the mutation of bromodomain PHD-finger transcription factor (BPTF) was strongly associated with RM. In the present study, the effects of BPTF on EMT and the underlying mechanism were investigated. We found that the expression of BPTF in the villi of RM patients was significantly downregulated. Gene Ontology (GO) analysis revealed that BPTF participated in cell adhesion. The knockdown of BPTF prevented EMT and attenuated trophoblast invasion in vitro. BPTF activated Slug transcription by binding directly to the promoter region of the Slug gene. Interestingly, the protein levels of both Slug and BPTF were decreased in the villous cytotrophoblasts (VCTs) of RM villi. In conclusion, BPTF participates in the regulation of trophoblast EMT by activating Slug expression, suggesting that BPTF defects are an important factor in RM pathogenesis.