Dataset Information

Concentrated exosomes from menstrual blood-derived stromal cells improves ovarian activity in a rat model of premature ovarian insufficiency.

ABSTRACT:

Background

Premature ovarian insufficiency (POI) is one of the major causes of infertility. We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) effectively improved ovarian function in a murine model of POI. Recent studies indicated that mesenchymal stem cell-derived exosomes were important components in tissue repair. In this study, we investigated the therapeutic effects of MenSCs-derived exosomes (MenSCs-Exos) in a rat model of POI and its mechanism in restoring ovulation.

Methods

Ovaries of 4.5-day-old Sprague Dawley rats (SD rats) were cultured in vitro to evaluate the effects of MenSCs-Exos exposure on early follicle development. Furthermore, POI in rats was induced by intraperitoneal administration of 4-vinylcyclohexene diepoxide (VCD). Forty-eight POI rats were randomly assigned to four groups, each receiving a different treatment: PBS, MenSCs, MenSCs-Exos, and Exo-free culture supernatant of MenSCs. Estrous cyclicity, ovarian morphology, follicle dynamics, serum hormones, pregnancy outcomes, and molecular changes were investigated.

Results

Exposure to MenSCs-Exos promoted the proliferation of granulosa cells in primordial and primary follicles in vitro and increased the expression of early follicle markers Deleted In Azoospermia Like (DAZL) and Forkhead Box L2 (FOXL2) while inhibiting follicle apoptosis. In vivo, MenSCs-Exos transplantation effectively promoted follicle development in the rat model of POI and restored the estrous cyclicity and serum sex hormone levels, followed by improving the live birth outcome. In addition, transplantation of MenSCs-Exos regulated the composition of the ovarian extracellular matrix and accelerated the recruitment of dormant follicles in the ovarian cortex and increased proliferation of granulosa cells in these follicles.

Conclusion

MenSCs-Exos markedly promoted follicle development in vitro and in vivo and restored fertility in POI rats, suggesting a restorative effect on ovarian functions. The therapeutic effect of MenSCs-Exos transplantation was sustainable, consistent with that of MenSCs transplantation. Our results suggested that MenSCs-Exos transplantation may be a promising cell-free bioresource in the treatment of POI.

SUBMITTER: Zhang S

PROVIDER: S-EPMC7953711 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Concentrated exosomes from menstrual blood-derived stromal cells improves ovarian activity in a rat model of premature ovarian insufficiency.

Zhang Siwen S Huang Boxian B Su Peng P Chang Qiyuan Q Li Pingping P Song Aixin A Zhao Xinyang X Yuan Zhengwei Z Tan Jichun J

Stem cell research & therapy 20210312 1

<h4>Background</h4>Premature ovarian insufficiency (POI) is one of the major causes of infertility. We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) effectively improved ovarian function in a murine model of POI. Recent studies indicated that mesenchymal stem cell-derived exosomes were important components in tissue repair. In this study, we investigated the therapeutic effects of MenSCs-derived exosomes (MenSCs-Exos) in a rat model of POI and its ...[more]

PMID: 33712079

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Project description:BackgroundPremature ovarian insufficiency (POI) is a refractory disease that seriously affects the reproductive health of women and is increasing in incidence and prevalence globally. There is enormous demand to improve fertility in women with POI, while there is still lack of effective therapeutic methods in clinic. Cell-free fat extract (CEFFE) has been reported to contain thousands of active proteins which possess the ability to promote tissue repair in other diseases. In our study, we aimed to observe the efficacy and biosecurity of CEFFE on the repair of ovarian function and fertility of mice with POI and further explore the underlying mechanism.MethodsIn vivo, POI mice model, established by cyclophosphamide (CTX, 120 mg/kg) and busulfan (BUS, 12 mg/kg), was treated with CEFFE via the tail vein every two days for 2 weeks. Then, the weight of ovaries, estrous cycle and follicle count by H&E staining were measured. The content of AMH, E2 and FSH in serum was measured by Enzyme-linked immunosorbent assay. Fertility was evaluated by the number of oocytes retrieved, the development of embryos in vitro and the litter size. Biosecurity of parent mice and their pups were examined by body mass and visceral index. The proliferation and apoptosis of cells in ovaries were examined by immunohistochemistry and transmission electron microscopy. Furthermore, the mRNA-Seq of mouse ovarian granulosa cells was performed to explore underlying mechanism of CEFFE. In vitro, KGN cell line and human primary ovarian granulosa cells (hGCs) were treated with 250 μM CTX for 48 h with/without CEFFE. The proliferative ability of cells was detected by cell counting kit-8 assay (CCK-8) and EDU test; the apoptosis of cells was detected by TUNEL and flow cytometry.ResultsCEFFE recovered the content of AMH, E2 and FSH in serum, increased the number of follicles and the retrieved oocytes of POI mice (P < 0.05). CEFFE contributed to the development of embryos and improved the litter size of POI mice (P < 0.05). There was no side effect of CEFFE on parent mice and their pups. CEFFE contributed to the proliferation and inhibited the apoptosis of mouse granulosa cells in ovary, as well as in human ovarian granulosa cells (including KGN cell line and hGCs) (P < 0.05).ConclusionsThe treatment of CEFFE inhibited the apoptosis of granulosa cells and contributed to the recovery of ovarian function, as well as the fertility of mice with POI.

Project description:BackgroundIntrauterine adhesion (IUA) is a major cause of female secondary infertility. We previously demonstrated that menstrual blood-derived stromal cell (MenSC) transplantation helped severe IUA patients have pregnancy and endometrium regeneration. We also initiated platelet-rich plasma (PRP) acted as a beneficial supplement in MenSC culturing and a potential endometrial receptivity regulator. Here, we investigated the therapeutic effect of combined transplantation of MenSCs with PRP in rat IUA models and the mechanisms of MenSCs in endometrium regeneration.MethodsRat IUA models were established by intrauterine mechanical injured. Nine days later, all rats were randomly assigned to four groups received different treatment: placebo, MenSC transplantation, PRP transplantation, and MenSCs + PRP transplantation. The traces of MenSCs were tracked with GFP label. Endometrial morphology and pathology, tissue proliferation, inflammation, pregnancy outcomes, and mechanism of MenSCs in the regeneration of endometrium were investigated.ResultsNotably, at days 9 and 18 post-treatment, MenSC transplantation significantly improved endometrial proliferation, angiogenesis, and morphology recovery and decreased collagen fibrosis and inflammation in the uterus. MenSCs had lesion chemotaxis, colonized around the endometrial glands. Gene expression of human-derived secretory protein IGF-1, SDF-1, and TSP-1 was detected in the uterus received MenSCs at day 18. The three treatments can all improve fertility in IUA rats. Moreover, gene expressions of cell proliferation, developmental processes, and other biological processes were induced in MenSC transplantation group. Hippo signaling pathway was the most significantly changed pathway, and the downstream factors CTGF, Wnt5a, and Gdf5 were significantly regulated in treatment groups. PRP enhanced these parameters through a synergistic effect.ConclusionsIn summary, MenSCs could effectively improve uterine proliferation, markedly accelerate endometrial damage repairment and promote fertility restoration in IUA rats, suggesting a paracrine restorative effect and Hippo signaling pathway stimulation. Our results indicate MenSCs, a valuable source of cells for transplantation in the treatment intrauterine adhesion. Combined with PRP, this cell therapy was more effective.

Project description:Ferroptosis is widely present in fibrosis-related diseases. The basic pathology of premature ovarian insufficiency (POI) involves ovarian tissue fibrosis, and there are currently fewer relevant studies addressing the association between ferroptosis and POI. This study aimed to demonstrate that ferroptosis induced by cisplatin (CDDP) caused ovarian tissue fibrosis, leading to POI. Vitamin E (VE), a ferroptosis inhibitor, could repair damaged ovarian function. CDDP was used to establish a rat model of POI, and VE was administered to reverse the reproductive toxicity of CDDP. Ovarian function was assessed by histological section staining, follicle counts, sex hormone levels, as well as fertility assays. The extent of ferroptosis was assessed by transmission electron microscopy (TEM), malondialdehyde (MDA), Perls staining. CCK-8, Ethynyl-2-Deoxyuridine (EdU), and scratch assays were used to determine the effect of CDDP and VE on ovarian granulosa cell (GC) viability. Western blot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were performed to evaluate ferroptosis-related molecular changes. Our results showed that CDDP caused follicle development disorders and ovarian tissue fibrosis, the levels of sex hormones suggested impaired ovarian function, and VE could reverse the reproductive toxicity of CDDP. The results of TEM, MDA and Perls staining suggested that the typical mitochondrial signature of ferroptosis was altered in ovarian GCs from the CDDP group, with significantly higher levels of lipid peroxidation and significant iron deposition in ovarian tissue, whereas VE mitigated the extent of ferroptosis. Molecular experiments then confirmed that the ferroptosis-related molecules acetyl CoA synthetase long chain family member 4 (ACSl4), 15-lipoxygenase-1 (ALOX15), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were differentially expressed in each group. In summary, our study preliminarily demonstrated that CDDP may promote GCs to undergo ferroptosis, cause follicle development disorders, ovarian tissue fibrosis, and induce POI by regulating the expression of ACSl4, ALOX15, SLC7A11, and GPX4, while VE improved impaired ovarian function.

Project description:BackgroundPremature ovarian insufficiency (POI) patients are predisposed to metabolic disturbances, including in lipid metabolism and glucose metabolism, and metabolic disorders appear to be a prerequisite of the typical long-term complications of POI, such as cardiovascular diseases or osteoporosis. However, the metabolic changes underlying the development of POI and its subsequent complications are incompletely understood, and there are few studies characterizing the disturbed metabolome in POI patients. The aim of this study was to characterize the plasma metabolome in POI by using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) metabolomics and to evaluate whether these disturbances identified in the plasma metabolome relate to ovarian reserve and have diagnostic value in POI.MethodsThis observational study recruited 30 POI patients and 30 age- and body mass index (BMI)-matched controls in the Center for Reproductive Medicine, Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University, from January 2018 to October 2020. Fasting venous blood was collected at 9:00 am on days 2-4 of the menstrual cycle and centrifuged for analysis. An untargeted quantitative metabolomic analysis was performed using UHPLC-MS/MS.ResultsOur study identified 48 upregulated and 21 downregulated positive metabolites, and 13 upregulated and 48 downregulated negative metabolites in the plasma of POI patients. The differentially regulated metabolites were involved in pathways such as caffeine metabolism and ubiquinone and other terpenoid-quinone biosynthesis. Six metabolites with an AUC value > 0.8, including arachidonoyl amide, 3-hydroxy-3-methylbutanoic acid, dihexyl nonanedioate, 18-HETE, cystine, and PG (16:0/18:1), were correlated with ovarian reserve and thus have the potential to be diagnostic biomarkers of POI.ConclusionThis UHPLC-MS/MS untargeted metabolomics study revealed differentially expressed metabolites in the plasma of patients with POI. The differential metabolites may not only be involved in the aetiology of POI but also contribute to its major complications. These findings offer a panoramic view of the plasma metabolite changes caused by POI, which may provide useful diagnostic and therapeutic clues for POI disease.

Project description:BackgroundAlthough many reports show that various kinds of stem cells have the ability to recover the function of premature ovarian insufficiency (POI), few studies are associated with the mechanism of stem cell treatment of POI. We designed this experimental study to investigate whether human adipose stem cell-derived exosomes (hADSC-Exos) retain the ability to restore ovarian function and how hADSC-Exos work in this process.MethodsA POI mouse model was established and human ovarian granule cells (hGCs) collected from individuals with POI were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing POI. The hematoxylin and eosin assay method was employed to assess the number of follicles. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of sex hormones. The proliferation rate and marker expression levels of hGCs were measured by flow cytometry (fluorescence-activated cell sorting). Real-time PCR and western blot assays were used to determine the mRNA and protein expression levels of SMAD2, SMAD3, and SMAD5. Western blot assays were used to test the protein expression levels of apoptosis genes (Fas, FasL, caspase-3, and caspase-8).ResultsAfter the hADSC-Exos were transplanted into the POI mice model, they exerted better therapeutic activity on mouse ovarian function, improving follicle numbers during four stages. ELISA results showed that hADSC-Exos elevated the hormone levels to the normal levels. In addition, after hADSC-Exos were cocultured with POI hGCs, our results showed that hADSC-Exos significantly promoted the proliferation rate and inhibited the apoptosis rate. Furthermore, hADSC-Exos also increased the marker expression of hGCs to the normal level. Besides, mRNA and protein assays demonstrated that hADSC-Exos downregulated the expression of SMAD2, SMAD3, and SMAD5 in vivo and in vitro. Western blot assay demonstrated that hADSC-Exos inhibited expression of the apoptosis genes in POI hGCs, and SMAD knockdown increased the protein expression of apoptosis genes.ConclusionsThese findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which exosomes derived from hADSCs improved ovarian function of POI disease via regulation of the SMAD signaling pathway.

Project description:BACKGROUND:Human amniotic epithelial cell (hAEC) transplantation holds great promise in treating premature ovarian insufficiency (POI). However, some deficient biological characteristics of hAECs restrict their application. METHODS:Vitamin C (VC) was added to the culture media of hAECs for 2?weeks. Then, the proliferative ability, migration ability, pluripotency, and self-renewal of VC-treated hAECs (VC-hAECs) were determined. Next, hAECs and VC-hAECs were transplanted into the ovaries of cyclophosphamide (CTX)-induced POI model mice. The ovarian function of POI mice was evaluated after transplantation by counting follicle numbers and measuring the blood levels of AMH, E2, and FSH. The rescue effects of VC-hAECs and hAECs were unveiled by coculturing with CTX-damaged human ovarian granulosa cells (hGCs) and analyzing relative marker expression. Additionally, ovarian marker expression and transplant survival were detected in POI mice after transplantation to verify the beneficial effect of VC-hAECs. The cytokine profiles of VC-hAECs and hAECs were revealed by performing a cytokine array and an ELISA to show their paracrine function. RESULTS:Our results indicated that VC promoted the proliferation, migration, pluripotency, and self-renewal of hAECs in vitro. The most effective concentration of VC was 50??g/ml. After transplantation into the POI mouse model, VC-hAECs reversed ovarian function more powerfully than hAECs. Human granulosa cell marker expression in CTX-damaged hGCs was increased after coculture with VC-hAECs compared with hAECs. In the ovaries of the POI mice, ovarian marker expression was greater after VC-hAEC transplantation than after hAEC transplantation. VC-hAECs showed higher transplant survival than hAECs. Furthermore, VC-hAECs secreted more growth factors than hAECs. CONCLUSION:Treatment with VC promoted the proliferation, migration, self-renewal, and paracrine functions of hAECs. Additionally, VC elevated the therapeutic potential of hAECs in treating POI.

Project description:Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. Premature ovarian insufficiency has few known genetic causes but in familial cases a genetic link is often suspected. A large consanguineous family with three female affected with POI was investigated. All samples including 3 affected and 5 unaffecd underwent whole genome SNP genotyping using Affymetric Axiom_GW_Hu_SNP array. Linkage analysis was carried out using HomozygosityMapper and Allegro softwares.Linkage analysis mapped the disease phenotype to long arm of chromosome 20. Sequence data analysis of potential candidate genes failed to detect any pathogenic variant. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from peripheral blood samples. DNA of eight individuals including three affected subjects was used for homozygosity mapping. Genotyping was performed using the Affymetrix Axiom_GW_Hu_SNP array. Briefly, 250 ng genomic DNA was digested with Digestion Master Mix containing 2 µl NE buffer 2 (10X), 0.5 µl BSA (100X; 10 mg/ml) and 1 µl Nsp1. Digested DNA sample was ligated to Nsp1 adaptor using T4 DNA ligase and amplified by 2 µl of TITANIUM Taq DNA polymerase (50X) and 100 µM PCR primer. PCR products were purified on a Clean-Up plate (Clontech Lab, Madison, USA) and eluted by RB buffer. Purified PCR products were fragmented using Fragmentation Reagent (0.05U/µl DNase 1) for 35 minutes at 37°C followed by labeling of fragmented samples with Labeling Master Mix (30 mM GeneChip DNA Labeling Reagent, 30 U/µl Terminal Deoxynucleotidyl Transferase) for 4 hours at 37°C. Labeled samples were hybridized to Axiom_GW_Hu_SNP array by mixing the sample with Hybridization Master Mix, denatured on thermoblock and loaded on to Array. Array was then placed in a hybridization oven (GeneChip Hybridization Oven 640, USA) for 16-18 hours. After hybridization, array was washed and stained on an automated Fluidic Station 450 followed by scanning on GeneChip Scanner 3000 7G using GeneChip Operating Software (GCOS).

Dataset Information

Concentrated exosomes from menstrual blood-derived stromal cells improves ovarian activity in a rat model of premature ovarian insufficiency.

Background

Methods

Results

Conclusion

Publications

Concentrated exosomes from menstrual blood-derived stromal cells improves ovarian activity in a rat model of premature ovarian insufficiency.

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