Enhancing Oocyte Quality in Aging Mice: Insights from Mesenchymal Stem Cell Therapy and FOXO3a Signaling Pathway Activation.

Ovarian aging reduced the quality of oocytes, resulting in age-related female infertility. It is reported that mesenchymal stem cells (MSCs) therapy can improve age-related ovarian function decline and the success rate of in vitro maturation (IVM) in assisted reproductive therapy. In order to investigate the effectiveness and mechanisms of MSCs to enhance oocyte quality of cumulus oocyte complexes (COCs) in advanced age, this study focus on the respective functional improvement of oocytes and granulosa cells (GCs) from aging mice and further to explore and verify the possible mechanisms. Here, we studied a popular but significant protein of follicular development, Forkhead box O-3a (FOXO3a), which is a transcription factor that mediates a variety of cellular processes, but the functions of which in regulating oocyte quality in MSCs therapy still remain inconclusive. In this study, the RNA-seq data of metaphase II (MII) oocytes and GCs isolated from COCs confirmed that, GCs of immature follicles show the most potential to be the targeted cells of bone marrow mesenchymal stem cells (BMSCs) by FOXO3a signaling pathway. Furthermore, we demonstrated the effectiveness of BMSCs co-culture with aging COCs to enhance oocyte quality and found its mechanism to function via ameliorating the biological function of GCs by alleviating FOXO3a levels. These results provide significant fundamental research on MSCs therapy on ovarian aging, as well as offering guidance for raising the success rate of assisted reproductive technology such IVM in clinical and non-clinical settings.

MiR-484 promotes nonalcoholic fatty liver disease progression in mice via downregulation of Sorbs2.

OBJECTIVE: MicroRNA 484 (miR-484) plays a pivotal role in the development and progression of different diseases and is typically described as a mitochondrial regulator. Whether miR-484 is involved in lipid metabolism or exerts a role in nonalcoholic fatty liver disease remains unclear. METHODS: miR-484 levels were examined in the livers of male mice fed a high-fat diet and in hepatocytes treated with free fatty acids. Sorbin and SH3 structural domain-containing protein 2 (Sorbs2) were identified as a novel target of miR-484 by sequencing mRNA in the livers of miR-484 knockout mice. Sorbs2 liver-specific knockdown mice were constructed by tail vein injection of adeno-associated virus vector to miR-484 knockout mice. In addition, genetic manipulation of SORBS2 was performed in human hepatocyte lines, mouse primary hepatocytes, and the liver. RESULTS: Serum and hepatic miR-484 levels are upregulated in nonalcoholic fatty liver disease mice. miR-484 knockdown ameliorated hepatocyte steatosis, whereas miR-484 overexpression increased hepatocyte lipid load. miR-484 knockdown-mediated alleviation of hepatic steatosis, liver injury, inflammation, and apoptosis was compromised after high-fat diet-induced knockdown of Sorbs2 in mouse liver and free fatty acid-induced primary mouse hepatocytes. CONCLUSIONS: These results identify Sorbs2-mediated mitochondrial ß-oxidation and apoptosis that promote miR-484 knockdown-mediated remission of hepatic steatosis.

Erratum: Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis: Erratum.

[This corrects the article DOI: 10.7150/ijbs.68028.].

hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells: promising bio-markers of live birth after IVF/ICSI.

BACKGROUND: MicroRNAs (miRNAs) are considered potential biomarkers for various diseases. This study investigated whether hsa-miR-320a-3p and hsa-miR-483-5p levels in human ovarian granulosa cells derived from follicular fluids are associated with embryo developmental competence. METHODS: We collected 195 granulosa cells samples and analyzed the treatment outcomes in patients undergoing in vitro fertilization (n = 147) or intracytoplasmic sperm injection (n = 48) cycles. The hsa-miR-320a-3p and hsa-miR-483-5p levels in granulosa cells were measured using quantitative reverse transcription-polymerase chain reaction. RESULTS: Patients were subdivided into four groups according to the granulosa cells hsa-miR-320a-3p and hsa-miR-483-5p levels quartiles (Q1-Q4). Embryo developmental competence was compared using the chi-square test. Patients in Q3 were less likely to achieve a normal fertilization rate for in vitro fertilization and blastocyst formation than those in Q1 as they expressed high levels of hsa-miR-320a-3p and hsa-miR-483-5p (P < 0.05). Patients in Q3 and Q4 were less likely to achieve a good-quality embryo as they expressed high levels of hsa-miR-483-5p and hsa-miR-320a-3p (P < 0.05). The hsa-miR-320a-3p and hsa-miR-483-5p levels were not associated with clinical pregnancy. However, multiple regression analysis indicated that in Q3 and Q4 intervals had experienced a decreased chance of live birth due to high expression levels of hsa-miR-320a-3p and hsa-miR-483-5p levels. The relative hsa-miR-320a-3p expression levels in granulosa cells were weakly and positively correlated with the patient age (P = 0.0033). Moreover, both the basal follicle stimulating hormone (P = 0.0003) and ovarian stimulation protocols (P = 0.006 and P = 0.004) significantly and positively affected hsa-miR-320a-3p levels. The days of stimulation was negatively correlated with the relative hsa-miR-320a-3p expression level (P = 0.047). CONCLUSIONS: The hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells negatively correlated with the good-quality embryo rate and live birth, indicating that hsa-miR-320a-3p and hsa-miR-483-5p can be used as potential negative indicators to predict good-quality embryos and live births.

Multi-biofunctional graphene oxide-enhanced poly-L-lactic acid composite nanofiber scaffolds for ovarian function recovery of transplanted-tissue.

In this study, we successfully constructed the new graphene oxide/poly-L-lactic acid (GO/PLLA) nanofiber scaffolds with a hydrophilic surface and porous network structure that were highly favorable for cell infiltration. When employed these new nanofiber scaffolds for a wide range of tissue engineering applications, it was expected to promote graft tissue survival and angiogenesis. The new GO/PLLA nanofiber scaffold with an appropriate concentration of 1.0 wt% was applied for the restoration of ovarian function and reserve in mice with primary ovarian insufficiency (POI). After co-transplanting the normal ovarian cortex loaded on these new nanomaterials into the in situ ovarian tissue of POI mice, the fusion of transplanted ovarian cortex with damaged ovarian tissue was improved, as well as the ovarian function and the follicle numbers. Moreover, angiogenesis was observed clearly and proved to exist in the transplanted tissue and nanomaterials, with the most conspicuous effect after co-transplantation with 1.0 wt% GO/PLLA nanofiber scaffold. In addition, nitric oxide (NO) production by phosphorylated endothelial nitric oxide synthase (p-eNOS) in vivo was proven to be involved in the effect of GO and PLLA on the improved survival rate of the transplanted ovarian cortex. This study provides a new method for the fertility preservation of ovarian tissue cryopreservation and transplantation, as well as a new strategy for the transplantation of other organs.

A comparative study of Mesenchymal Stem Cells transplantation approach to antagonize age-associated ovarian hypofunction with consideration of safety and efficiency.

Introduction: The transplantation of mesenchymal stem cells (MSCs) in patients with premature ovarian failure (POF) could lead to clinical improvement. The transplantation to the ovaries among other transplantation methods have been reported in various animal models, however, there is little evidence regarding the optimal method, including the clinical safety and the efficiency for the treatment of age associated ovarian hypofunction. Objectives: To establish the most effective transplantation route of MSCs, explore the resistance to therapy, its safety and role in the natural aging process of the ovaries. Methods: Highly purified MSCs were injected intraperitoneally, directly into the ovaries or tail-intravenously in mice animal model. The ovarian function, quantity and quality of oocytes, cell viability/apoptosis, were evaluated, applying chemiluminescence analysis (CLIA), western blotting, immunofluorescence staining, transmission electron microscope (TEM), TdT mediated dUTP Nick End Labeling (TUNEL) assay and other techniques. The organ tumorigenicity was also evaluated by long-term observation and histopathological examination. The efficiency of MSCs was further verified in non-human primates by the most effective transplantation route. Results: The 32nd week was ultimately determined as the time point of MSCs transplantation. Our results showed that the intra-ovarian injection was the best transplantation method with a more conspicuous effect. With deeper investigations, we found that the transplanted MSCs showed an effective influence on the follicular number, promoted follicle maturation and inhibited cell apoptosis, which was further verified in non-human primates. In addition, the long-term observation and the histopathological examinations ruled out neoplasms or obvious prosoplasia after MSCs transplantation. Conclusion: MSCs transplantation by intra-ovarian injection could within a month exert the most conspicuous anti-age-associated ovarian hypofunction effects, which may improve the quantity and quality of oocytes by changing the mitochondrial structure, regulating mitochondrial function and attenuating cell apoptosis to increase the storage of the follicle pool without a remarkable potential of tumorigenicity.

Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis.

Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines in vitro. We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR.

N6-Methyladenosine Modifications in the Female Reproductive System: Roles in Gonad Development and Diseases.

N6-methyladenosine (m6A) is the most prevalent chemical modification in eukaryotic messenger RNAs. By participating in various RNA-related bioprocesses including RNA decay, splicing, transport and translation, m6A serves as a pivotal regulator of RNA fate and plays an irreplaceable role in cellular activities. The m6A modifications of transcripts are coordinately regulated by methyltransferase "writers" and demethylase "erasers", and produce variable effects via different m6A reading protein "readers". There is emerging evidence that m6A modifications play a critical role in a variety of physiological and pathological processes in the female reproductive system, subsequently affecting female fertility. Here, we introduce recent advances in research on m6A regulators and their functions, then highlight the role of m6A in gonad development and female reproductive diseases, as well as the underlying mechanisms driving these processes.

Advances in the study of CDC42 in the female reproductive system.

CDC42 is a member of the Rho-GTPase family and is involved in a variety of cellular functions including regulation of cell cycle progression, constitution of the actin backbone and membrane transport. In particular, CDC42 plays a key role in the establishment of polarity in female vertebrate oocytes, and essential to this major regulatory role is its local occupation of specific regions of the cell to ensure that the contractile ring is assembled at the right time and place to ensure proper gametogenesis. The multifactor controlled 'inactivation-activation' process of CDC42 also allows it to play an important role in the multilevel signalling network, and the synergistic regulation of multiple genes ensures maximum precision during gametogenesis. The purpose of this paper is to review the role of CDC42 in the control of gametogenesis and to explore its related mechanisms, with the aim of further understanding the great research potential of CDC42 in female vertebrate germ cells and its future clinical translation.

The potential of Zishen Yutai pills to facilitate endometrial recovery and restore fertility after induced abortion in rats.

CONTEXT: Abortions damage the endometrium in women. Currently, therapeutic options for endometrial recovery are limited. Zishen Yutai Pill (ZYP) was found to promote endometrial blood supply as a traditional Chinese medicine. However, whether ZYP promotes endometrial recovery post-abortion has not yet been explored. OBJECTIVE: This study evaluated the role of ZYP in rat endometrial recovery after induced abortion and explored its mechanism of action. MATERIALS AND METHODS: Sprague-Dawley rats were divided into three groups: no-operation group, control group, and ZYP group. The rats in the control and ZYP group were induced abortion, and then treated with normal saline or ZYPs, respectively, for 1-3 oestrous cycles. Morphological changes in the endometrium were examined. Expression levels of the factors related to endometrial recovery were analyzed. The duration of this study was almost seven months. RESULTS: The endometrial thickness (7.3 ± 0.17 mm) and number of glands (5.5 ± 0.20) increased significantly in the ZYP group compared with those in the control group (5.5 ± 0.15 mm and 3.5 ± 0.18; p < 0.05). Fibrosis of the endometrium was ameliorated by ZYP administration (45 ± 6% vs. 58 ± 7%; p < 0.05). ZYPs treatment increased the expression of VEGF, ER, MMP-9, LIF, and HB-EGF, but decreased TGF-ß expression. Moreover, the average number of pups in the ZYP group (9.0 ± 1.5) was greater than that in the control (4 ± 1.3). DISCUSSION AND CONCLUSION: ZYPs accelerate endometrial recovery and restored fertility in rats, suggesting its potential to promote human endometrial repair.

Therapeutic Potential of HUMSCs in Female Reproductive Aging.

With the development of regenerative medicine, stem cells are being considered more frequently for the treatment of reproductive aging. Human umbilical cord mesenchymal stem cells have been reported to improve the reserve function of aging ovaries through their homing and paracrine effects. In this process, paracrine factors secreted by stem cells play an important role in ovarian recovery. Although the transplantation of human umbilical cord mesenchymal stem cells to improve ovarian function has been studied with great success in animal models of reproductive aging, their application in clinical research and therapy is still relatively rare. Therefore, this paper reviews the role of human umbilical cord mesenchymal stem cells in the treatment of reproductive aging and their related mechanisms, and it does so in order to provide a theoretical basis for further research and clinical treatment.