Premature Ovarian Failure Model Research Articles

Beneficial effects of human umbilical cord mesenchymal stem cell (HUCMSC) transplantation on cyclophosphamide (CTX)-induced premature ovarian failure (POF) in Tibetan miniature pigs

BackgroundPremature ovarian failure (POF), also known as primary ovarian insufficiency, is a common endocrine disease in young women. The emergence of regenerative medicine using stem cells may improve ovarian function and structure, and represents a promising prospect for POF treatment. In his study, we explored the therapeutic effects of human umbilical cord mesenchymal stem cell (HUCMSC) transplantation in a Tibetan miniature pig model of cyclophosphamide (CTX)-induced POF. MethodsWe cultured and identified HUCMSCs, labeled them with DiR iodide red dye, and implanted them into a CTX-induced model of POF in Tibetan miniature pigs. The daily weight changes were recorded, and the levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured on days 0, 7, and 14. At the end of the 21-day observation period, in vivo imaging of the bilateral ovaries was performed, and the ovarian index was measured. Ovarian tissue morphology and follicles were examined by hematoxylin-eosin staining. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to assess cell apoptosis, and immunohistochemistry was used to determine the levels of p-AKT, p-ERK1/2, BAX, and BCL2 expression. ResultsOur analysis indicated successful delivery of HUCMSCs to the ovaries of the POF pig model. Significant increases were observed in body weight, E2 levels, ovarian index, and number of normal follicles (all p < 0.05). Moreover, FSH levels reduced and ovarian tissue morphology improved following HUCMSCs transplantation (all p < 0.05). Importantly, upregulated p-AKT, p-ERK1/2, and BCL2 expression were observed, whereas the expression of BAX was suppressed (all p < 0.05), suggesting the inhibition of ovarian cell apoptosis. ConclusionOur study highlights the significant therapeutic effects of HUCMSC transplantation on CTX-induced POF in a Tibetan miniature pig model.

Inhibition of p38 MAPK/NF-κB p65 signaling pathway activity by rare ginsenosides ameliorates cyclophosphamide-induced premature ovarian failure and KGN cell injury

Ethnopharmacological relevancePanax ginseng C. A. Mey., one of the most used herbs in the world, shows effective treatment in reproductive injury. Recent studies have proven that the processed product, red ginseng, which is more active than ginseng itself. Therefore, it is speculated that its main functional component, rare ginsenosides (heat-transformed saponin, HTS), may be effective in treating premature ovarian failure (POF), but its efficacy has not yet been experimentally confirmed. Aim of the studyTo evaluate whether HTS could attenuate cyclophosphamide-induced inflammation and oxidative damage in POF model rats and the human granulosa-like KGN cell line and protect granulosa cell proliferation. Material and methodsHTS were isolated from ginsenosides and high performance liquid chromatography (HPLC) analysis was used to analyze the HTS components. Cyclophosphamide (CP) was used to establish a POF rat model and KGN cell injury model. Reactive oxygen species (ROS) and antioxidant enzyme production was determined using specific assays, while inflammatory cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). The proliferative function of granulosa cells was assessed using high-content screening and immunohistochemistry to determine the Ki67 protein level. Protein expression in ovarian tissues and KGN cells was analyzed by Western blotting, quantitative real-time PCR (qRT-PCR) was used to determine the transcriptional changes in ovarian tissues and KGN cells. ResultsIn CP-treated POF model rats, HTS significantly decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels, increased glutathione oxidase (GSH) levels, and upregulated Ki67 expression in ovarian granulosa cells. In addition, HTS significantly increased cell survival and Ki67 expression levels in CP-treated cells, and superoxide dismutase (SOD) levels were significantly increased. HTS significantly downregulated IL-6, TNF-α, and interleukin-1β (IL-1β) mRNA expression and significantly inhibited nuclear factor kappa-B p65 (NF-κB p65) and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in POF model rats and KGN cells. Moreover, NF-κB p65 and p38 MAPK levels were significantly increased in ovarian granulosa cells. p65 and p38 protein and gene expression was significantly downregulated. ConclusionHTS ameliorated CP-induced POF and human granulosa cell injury, possibly by inhibiting inflammation and oxidative damage mediated by the p38 MAPK/NF-κB p65 signaling pathway.

Protective effect of Luffa cylindrica fermentation liquid on cyclophosphamide-induced premature ovarian failure in female mice by attenuating oxidative stress, inflammation and apoptosis

Premature ovarian failure (POF) is a leading cause of women’s infertility without effective treatment. The purpose of this study was to investigate the protective effects of Luffa cylindrica fermentation liquid (LF) on cyclophosphamide (CTX) -induced POF in mice and to preliminarily investigate the underlying mechanisms. Thirty-two Balb/c mice were divided into four groups randomly. One group served as the control, while the other three received CTX injections to establish POF models. A 14-day gavage of either 5 or 10 μL/g LF was administered to two LF pretreatment groups. To analyze the effects of LF, the ovarian index, follicle number, the levels of serum sex hormones, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), inflammatory factors, and apoptosis of the ovarian cells were measured. The effects of LF pretreatment on the expression of TLR4/NF-κB and apoptosis pathways were also evaluated. We found that LF pretreatment increased the ovarian index and the number of primordial and antral follicles while decreasing those of atretic follicles. LF pretreatment also increased the serum levels of estradiol (E2) and anti-Müllerian hormone (AMH), while decreasing those of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Furthermore, LF pretreatment increased the levels of SOD and GSH in the ovaries, while decreasing those of MDA, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). LF administration reduced the amount of TUNEL+ ovarian cells and the levels of TLR4 and NF-κB P65 protein expression. In conclusion, LF has antioxidant, anti-inflammatory as well as anti-apoptotic effects against CTX-induced POF, and the inhibition of TLR4/NF-κB and apoptosis pathways may be involved in its mechanisms.Graphical

Zishen Yutai pills restore fertility in premature ovarian failure through regulating arachidonic acid metabolism and the ATK pathway

Ethnopharmacological relevanceThe Zishen Yutai pills (ZYP), a Chinese medicinal formulation derived from the Qing Dynasty prescription “Shou Tai pills", have been documented to exhibit beneficial effects in clinical observations treating premature ovarian failure (POF). However, the anti-POF effects and its comprehensive systemic mechanism have not yet been clarified. Aim of the reviewTherapeutic effects and systemic mechanism of ZYP in POF were evaluated. Materials and methodsAfter pulverization, sieving, and stirring, ZYP was administered intragastrically to cisplatin-induced POF mice at a dose of 1.95 mg/kg/d for 14 days. The anti-POF effects of ZYP were investigated by assessing the number of ovarian follicles at different developmental stages, as well as measuring serum estradiol (E2) levels and ovarian-expressed anti-Müllerian hormone (AMH). Reproductive performance and offspring health were evaluated to predict fertility restoration. Furthermore, a combination of proteomic and metabolomic profiling was employed to elucidate the underlying molecular mechanism of ZYP in treating POF. Western blot (WB) analyses and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to explore the mechanisms through which ZYP exerted its anti-POF effects. ResultsWe have demonstrated that oral administration of ZYP reversed the reduction in follicles at different developmental stages and stimulated the expressions of serum E2 and ovarian-expressed AMH in a cisplatin-induced POF model. Additionally, ZYP ameliorated follicle apoptosis in ovaries affected by cisplatin-induced POF. Furthermore, treatment with ZYP restored the quantity and quality of oocytes, as well as enhanced fertility. Our results revealed 62 differentially expressed proteins (DEPs) through proteomic analyses and identified 26 differentially expressed metabolites (DEMs) through metabolomic analyses. Both DEPs and DEMs were highly enriched in the arachidonic acid (AA) metabolism pathway. ZYP treatment effectively upregulated the protein and mRNA expression of critical targets in AA metabolism and the AKT pathway, including CYP17α1, HSD3β1, LHR, STAR, and AKT, in cisplatin-induced POF mice. ConclusionsThese results indicated that ZYP exerted protective effects against POF and restored fertility from cisplatin-induced apoptosis. ZYP could be a satisfying alternative treating POF.

Metformin protects ovarian granulosa cells in chemotherapy-induced premature ovarian failure mice through AMPK/PPAR-γ/SIRT1 pathway

Premature ovarian failure (POF) caused by chemotherapy is a growing concern for female reproductive health. The use of metformin (MET), which has anti-oxidative and anti-inflammatory effects, in the treatment of POF damaged by chemotherapy drugs remains unclear. In this study, we investigated the impact of MET on POF caused by cyclophosphamide (CTX) combined with busulfan (BUS) and M1 macrophages using POF model mice and primary granule cells (GCs). Our findings demonstrate that intragastric administration of MET ameliorates ovarian damage and alleviates hormonal disruption in chemotherapy-induced POF mice. This effect is achieved through the reduction of inflammatory and oxidative stress-related harm. Additionally, MET significantly relieves abnormal inflammatory response, ROS accumulation, and senescence in primary GCs co-cultured with M1 macrophages. We also observed that this protective role of MET is closely associated with the AMPK/PPAR-γ/SIRT1 pathway in cell models. In conclusion, our results suggest that MET can protect against chemotherapy-induced ovarian injury by inducing the expression of the AMPK pathway while reducing oxidative damage and inflammation.

CXCL10-based gene cluster model serves as a potential diagnostic biomarker for premature ovarian failure.

Premature ovarian failure (POF) is a disease with high clinical heterogeneity. Subsequently, its diagnosis is challenging. CXCL10 which is a small signaling protein involved in immune response and inflammation may have diagnostic potential in detection of premature ovarian insufficiency. Therefore, this study aimed to investigate CXCL10 based diagnostic biomarkers for POF. Transcriptome data for POF was obtained from the Gene Expression Omnibus (GEO) database (GSE39501). Principal component analysis (PCA) assessed CXCL10 expression in patients with POF. The receiver operating characteristic (ROC) curve, analyzed using PlotROC, demonstrated the diagnostic potential of CXCL10 and CXCL10-based models for POF. Differentially expressed genes (DEGs) in the control group of POF were identified using DEbylimma. PlotVenn was used to determine the overlap between the POF-control group and the high-/low-expression CXCL10 groups. QuadrantPlot was employed to detect CXCL10-dysregulated genes in POF. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were conducted on DEGs using RunMulti Group cluster Profiler. A POF model was induced with cisplatin (DDP) using KGN cells. RT-qPCR and Western blot were used to measure the expression of CXCL10, apoptosis-related proteins, and peroxisome proliferator-activated receptor (PPAR) signaling pathway-related proteins in this model, following siRNA-mediated silencing of CXCL10. Flow cytometry was employed to assess the apoptosis of KGN cells after CXCL10 downregulation. The expression of CXCL10 is dysregulated in POF, and it shows promising diagnostic potential for POF, as evidenced by an area under the curve value of 1. In POF, we found 3,362 up-regulated and 3,969 down-regulated DEGs compared to healthy controls, while the high- and low-expression groups of POF (comprising samples above and below the median CXCL10 expression) exhibited 1,304 up-regulated and 1,315 down-regulated DEGs. Among these, 786 DEGs consistently displayed dysregulation in POF due to CXCL10 influence. Enrichment analysis indicated that the PPAR signaling pathway was activated by CXCL10 in POF. The CXCL10-based model (including CXCL10, Itga2, and Raf1) holds potential as a diagnostic biomarker for POF. Additionally, in the DDP-induced KGN cell model, interfering with CXCL10 expression promoted the secretion of estradiol, and reduced apoptosis. Furthermore, CXCL10 silencing led to decreased expression levels of PPARβ and long-chain acyl-CoA synthetase 1 compared to the Si-NC group. These results suggest that CXCL10 influences the progression of POF through the PPAR signaling pathway. The CXCL10-based model, demonstrating perfect diagnostic accuracy for POF and comprising CXCL10, Itga2, and Raf1, holds potential as a valuable diagnostic biomarker. Thus, the expression levels of these genes may collectively provide valuable diagnostic information for POF.

Protective and/or therapeutic effects of berberine in a model of premature ovarian failure induced by cyclophosphamide in rats.

We conducted a biochemical and histological evaluation of whether berberine has a protective and/or therapeutic effect in a cyclophosphamide-induced premature ovarian failure (POF) model. We divided 28 Wistar albino female rats into 4 groups [control group, POF group, cyclophosphamide (CP)+berberine (Bb) group, and POF+Bb group]. The POF model was established by intraperitoneal administration of 50 mg/kg CP on day 1 followed by 8 mg/kg/day CP dissolved in saline for the following 14 days. The CP+Bb group received Bb concurrently for two weeks with CP. The POF+Bb group received berberine for two weeks following the completion of CP administration. Left ovaries were used for histopathological evaluation and right ovaries were used for biochemical analysis [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 levels in tissue]. Ovarian damage scoring was significantly higher in the POF group than in the other groups (p<0.005). In the POF group, primordial and primary follicle counts were the lowest, while secondary and corpus luteum counts were the highest (p<0.005). There was no significant difference between the other groups. The POF group had significantly elevated levels of TNF-α, IL-1, and IL-6 in the biochemistry results (p<0.005). We demonstrated that berberine could be effective in the protection and treatment of POF by reducing proinflammatory cytokines. We believe that our study can make a considerable contribution to the literature in terms of POF protection and/or treatment.

Effect of preconditioning of human umbilical cord mesenchymal stem cells with hydrogen peroxide on the therapeutic potential of MSCs in the cyclophosphamide -induced premature ovarian failure mice model.

Exposure of stem cells to sublethal levels of hydrogen peroxide (H2O2) can prevent oxidative stress-induced apoptosis. In the present study, the effects of H2O2 preconditioning on the therapeutic potential of human umbilical vein cord mesenchymal stem cells (hUCV-MSCs) were evaluated in a murine model of premature ovarian failure. Mature mice were divided into 4 groups, and 10 mice were incorporated into each group. The control (Ctrl) group received phosphate buffered saline (PBS) intraperitoneal (IP), and the CTX group was injected IP with cyclophosphamide (CTX). The CTX+MSC group after receiving CTX was injected with a single dose of hUCV-MSCs labeled with CM-DiI intravenously (IV), whereas the CTX+preMSCs group after CTX injection received preconditioned MSCs with H2O2 IV. Seven days later, the mice were euthanized, and their ovaries were removed for histological studies such as H&E staining and the TUNEL assay. Furthermore, the numbers of CM-DiI-labeled hUCV-MSCs in the different regions of the ovary were calculated. FSH and estradiol values in the serum were measured. Our studies showed that CTX caused degenerative changes and follicular loss in the ovary. The number of follicles in the CTX+MSCs and CTX+PreMSCs groups was significantly higher compared to the CTX group. In addition, in the CTX+PreMSCs group, the numbers of different types of follicles were higher than in the CTX-MSC group. Immunohistochemical studies in the CTX+MSCs and CTX+PreMSCs groups showed little evidence of TUNEL positivity compared with the CTX group. Moreover, the apoptotic index decreased in the CTX+PreMSCs group compared to the CTX+MSCs group. Moreover, CM-DiI-labeled MSCs in the ovary in the CTX+pre-MSCs group were higher than in the CTX+MSCs group. Our experiment offers preconditioning as an effective strategy in stem cell therapy to potentiate MSCs' therapeutic efficacy in ovarian function failure.

The effects of human amnion membrane-derived mesenchymal stem cells conditioned medium on ionizing radiation-induced premature ovarian failure and endoplasmic reticulum stress-related apoptosis mechanism

ObjectivesPremature ovarian failure (POF) is defined as the cessation of menstrual periods for at least 4–6 months before the age of 40 years, accompanied by FSH values measuring over 40 IU/L for a month. Radiation therapy, one of the cancer treatment methods, is known to accelerate ovarian aging by reducing and eliminating the number of primordial follicles in the ovarian follicle pool. Ionizing radiation has been reported to cause POF. The objective of this study is to investigate the impact of mesenchymal stem cell conditioned medium (hAMSCs-CM), which is isolated from the amniotic membrane of human placenta, on premature ovarian failure (POF) caused by whole-body irradiation. The study will focus on the ER stress and apoptosis mechanisms in the process. Study DisaynA POF model was created by exposing rats to 7 Gy of whole-body irradiation. Serum-free hAMSCs-CM were then administered via the tail vein. Follicle count was performed on the ovaries, and immunohistochemistry was used to determine the expressions of GRP78, CHOP, IRE-1, caspase-12, caspase-9, caspase-3. TUNEL was also carried out, and levels of serum FSH, LH, E2, AMH, and oxidative stress marker 8-OHdG were measured. Results and ConclusionThe application of hAMSCs-CM has been found to have a positive impact on follicles affected by radiation. After treatment, the number of primordial, primary, secondary, and graafian follicles, which had previously decreased due to radiation, showed an increase. Furthermore, the number of atretic follicles, which had been increasing due to radiation, showed a decrease. ER is one of the targets affected by ionizing radiation. After ionizing radiation, the expressions of ER stress-related markers and apoptosis markers increased in the ovary. After hAMSCs-CM administration, the expressions of these markers and number of TUNEL-positive cells decreased. Following irradiation, anti-mullerian hormone (AMH) and estradiol (E2) levels decreased, while follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels increased. After administration of hAMSCs-CM, AMH and E2 levels increased, while FSH and LH levels decreased.Amnion membrane-derived mesenchymal stem cell conditioned medium can play a therapeutic role in ionizing radiation-induced premature ovarian failure by reducing endoplasmic reticulum stress and apoptosis.

Immunity and reproduction protective effects of Chitosan Oligosaccharides in Cyclophosphamide/Busulfan-induced premature ovarian failure model mice.

Premature ovarian failure (POF) is a major cause of infertility among women of reproductive age. Unfortunately, there is no effective treatment available currently. Researchers have shown that immune disorders play a significant role in the development of POF. Moreover, growing evidence suggest that Chitosan Oligosaccharides (COS), which act as critical immunomodulators, may have a key role in preventing and treating a range of immune related reproductive diseases. KM mice (6-8 weeks) received a single intraperitoneal injection of cyclophosphamide (CY, 120mg/kg) and busulfan (BUS, 30mg/kg) to establish POF model. After completing the COS pre-treatment or post-treatment procedures, peritoneal resident macrophages (PRMs) were collected for neutral erythrophagocytosis assay to detect phagocytic activity. The thymus, spleen and ovary tissues were collected and weighed to calculate the organ indexes. Hematoxylin-eosin (HE) staining was performed to observe the histopathologic structure of those organs. The serum levels of estrogen (E2) and progesterone (P) were measured via the enzyme-linked immunosorbent assay (ELISA). The expression levels of immune factors including interleukin 2 (IL-2), interleukin 4 (IL-4), and tumor necrosis factor α (TNF-α), as well as germ cell markers Mouse Vasa Homologue (MVH) and Fragilis in ovarian tissue, were analyzed by Western blotting and qRT-PCR. In addition, ovarian cell senescence via p53/p21/p16 signaling was also detected. The phagocytic function of PRMs and the structural integrity of thymus and spleen were preserved by COS treatment. The levels of certain immune factors in the ovaries of CY/BUS- induced POF mice were found to be altered, manifested as IL-2 and TNF-α experiencing a significant decline, and IL-4 presenting a notable increase. Both pre-treatment and post-treatment with COS were shown to be protective effects against the damage to ovarian structure caused by CY/BUS. Senescence-associated β-galactosidase (SA-β-Gal) staining results showed that COS prevents CY/BUS-induced ovarian cell senescence. Additionally, COS regulated estrogen and progesterone levels, enhanced follicular development, and blocked ovarian cellular p53/p21/p16 signaling which participating in cell senescence. COS is a potent preventative and therapeutic medicine for premature ovarian failure by enhancing both the ovarian local and systemic immune response as well as inhibiting germ cell senescence.

Resveratrol alleviates doxorubicin-induced damage in mice ovary

While oocytes and embryos cryopreservation can favor some patients with cancer-induced infertility to achieve pregnancy, the development of effective therapeutic strategies to preserve ovarian function during chemotherapy would be a significant advantage. The aim of the present study is to analyze whether Resveratrol treatment (Res) can preserve ovarian function from doxorubicin (Doxo)-induced gonadotoxicity using a mice model of premature ovarian failure. Res (7 and 15 mg/kg) increased the percentage of primary and antral follicles whilst decreasing the percentage of atretic follicles compared to Doxo alone. Res preserved the number of primordial follicles compared with those in the Doxo group but they did not change from those in the control group. Res treatment increased the number of AMH positive follicles compared to Doxo alone. Res increased proliferation index in follicular cells and reduced the DNA damage and apoptosis in preantral and early antral follicles compared to Doxo alone. Additionally, Doxo administration caused a severe endothelial damage and affected microvasculature stability in the ovary. However, Res was able to increase the recruitment of pericytes and smooth muscle cells in the Doxo-treated group. We also found that Res increased the expression of VEGF compared to Doxo alone. By H&E staining, Doxo-treated mice demonstrated endometrial alterations compared to controls, affecting both epithelial and stromal compartments. Nonetheless, Res restored the architecture of uterine tissue. Moreover, we also showed that Res administration is able to maintain antioxidant defenses through the increase of SOD expression in the Doxo-induced POF model.In conclusion, Res administration prior to and during Doxo treatment might serve as a noninvasive and low-cost protocol to preserve ovarian function in female cancer survivors.

Mechanism of Qiwei Guibao Granules in treatment of premature ovarian failure based on proteomics

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

Regenerative potential of different extracellular vesicle subpopulations derived from clonal mesenchymal stem cells in a mouse model of chemotherapy-induced premature ovarian failure

AimsSome studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. Main methodsGranulosa cells were treated with cyclophosphamide (Cy) in the absence or presence of cMSCs, or cMSCs-derived EV subpopulations (EV20K and EV110K, isolated by high-speed centrifugation and differential ultracentrifugation, respectively). In addition, POF mice were treated with cMSCs, EV20K and/or EV110K. Key findingscMSC and both EV types protected granulosa cells from Cy-induced damage. Calcein-EVs were detected in the ovaries. Moreover, cMSC and both EV subpopulations significantly increased body weight, ovary weight, and the number of follicles, restored FSH, E2, and AMH levels, increased the granulosa cell numbers and restored the fertility of POF mice. cMSC, EV20K, and EV110K alleviated inflammatory-related genes expression (Tnf-α and IL8), and improved angiogenesis via upregulation expression of Vegf and Igf1 at the mRNA level and VEGF and αSMA at the protein level. They also inhibited apoptosis through the PI3K/AKT signaling pathway. SignificanceThe administration of cMSCs and two cMSC-EVs subpopulations improved ovarian function and restored fertility in a POF model. EV20K is more cost-effective and feasible in terms of isolation, particularly in good manufacturing practice (GMP) facilities for treatment of POF patients in comparison with conventional EVs (EV110K).

An evaluation of the effects on the ovaries of hyperbaric oxygen therapy in a rat model of premature ovarian failure created with cyclophosphamide.

To evaluate hyperbaric oxygen therapy (HBO) based on ovarian histology, total antioxidant status (TAS), total oxidant status (TOS), and anti-müllerian hormone (AMH), in the ovarian insufiency (POI) model created with cyclophosphamide (CYP). The rats were separated into 3 groups of the control group (n=6), the CYP group (n=6), and the CYP+HBO group (n=6). The rats in the CYP group and the CYP+HBO group were injected intraperitoneally with 200 mg/kg CYP on day 1, followed by 8 mg/kg/day for 14 days to create POI. From the 15th day onwards, the rats in the CYP+HBO group were placed in a hyperbaric cabin and exposed to 100% oxygen at 2.4 atm pressure for one h, and were then returned to their cages at the end of the hour. A statistically significant decrease was determined in the primordial and primary follicle counts in the CYP group compared with the control group (p<0.05). In the CYP+HBO group, a statistically significant increase was determined in the primordial and primary follicle counts (p<0.05). The serum AMH levels were seen to be significantly decreased in the CYP group compared with both the control group and the CYP+HBO groups. The HBO was seen to decrease TOS and increase TAS. HBO could be an alternative treatment to minimize the effect of ovarian follicle loss caused by CYP, which is used for treating tumors that commonly occur in young females of reproductive age.

Cysteine-rich 61(CYR61) alleviates cyclophosphamide-induced proliferation inhibition in ovarian granulosa cells via suppressing NLRP3/caspase1-mediated pyroptosis.

We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study. Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-β-gal (senescence-associated β-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs. CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.

Pseudotargeted metabolomics analysis of pine pollen intervention in the liver of premature ovarian failure rats

Premature ovarian failure (POF) is a prevalent gynecological disease. In traditional Chinese medicine, it is believed that POF is directly related to abnormal function of the liver and kidneys. As such, regulation of the liver metabolism through the use of medicinal and edible substances is important for the treatment of POF. Pine pollen, a traditional Chinese medicinal and edible pollen variety, contains various active substances, such as sex hormones and phytohormones, which have been used to inhibit inflammation, regulate the immune system, and protect reproductive tissues. Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS), this study examined the influence of pine pollen on the liver metabolome of cyclophosphamide-induced POF model Sprague Dawley (SD) rats. The variations in the metabolites present in the liver tissue of control SD rats, model SD rats, and SD rats treated with various doses of pine pollen or estrogen were analyzed using principal component analysis (PCA) in combination with orthogonal partial least squares discriminant analysis (OPLS-DA) and other multivariate statistical methods to reveal the mechanism of pine pollen intervention in the livers of POF SD rats. An animal model experiment was conducted using six groups of ten-week-old rats. Cyclophosphamide was administered intraperitoneally to the model group and four intervention groups at a dosage of 60 mg/kg for 1 d followed by a dosage of 10 mg/kg for 14 d. Within the following four weeks, each of the four intervention groups received the intragastric administration of 0.1, 0.5, or 1.5 g/kg bodyweight (BW) of pine pollen, or 0.075 g/kg BW of conjugated estrogens (positive control). Equal quantities of normal saline were administered to the control and cyclophosphamide-treated model groups. Subsequently, the rat livers were subject to pseudotargeted metabolomics, and a total of 687 liver metabolites were discovered using both positive and negative ions. The metabolites differing in content were screened using the t-test (p<0.05) and the fold change (FC>2 or <0.5) in univariate analysis, and the variable importance in projection (VIP>1) in multivariate analysis. It was found that in comparison with the control group, the contents of 32 metabolites significantly increased, while those of 28 metabolites significantly decreased in the model group. The majority of these metabolites were involved α-linolenic acid metabolism, vitamin B6 metabolism, and purine metabolism, along with the lysine degradation and glycolysis/gluconeogenesis metabolic pathways. Compared with the cyclophosphamide-induced model group, the estrogen group exhibited increased levels of 47 metabolites and decreased levels of 29 metabolites, wherein 34 metabolites were restored to the levels found in the control group. These metabolites mainly involved the vitamin B6, lysine, glycolysis/gluconeogenesis, arginine and proline, and cysteine and methionine metabolic pathways. In the low/medium/high-dose pine pollen groups, the contents of 34/32/34 metabolites increased, the contents of 30/37/24 metabolites decreased, and the contents of 47/38/34 metabolites were restored to the levels found in the control group, respectively. These metabolites were mainly involved in vitamin B6 metabolism, purine metabolism, and the glycolysis/gluconeogenesis metabolic pathway. These results therefore indicate that the restoring effect of pine pollen is equivalent or superior to that of conjugated estrogen. Additionally, based on the known metabolic pathways, it appears that when estrogen interferes with the liver metabolism, the key metabolic pathways that become affected are the arginine and proline metabolism and cysteine and methionine metabolism pathways. In contrast, pine pollen intervention affected existing metabolic pathways that were known to be disordered by cyclophosphamide. The use of pine pollen may therefore restore the levels of many metabolites. It should be noted that 23 overlaps exist between the estrogen-restored metabolites and the pine pollen-restored metabolites, including a variety of acylcarnitines, such as ACar 10∶0. As a result, pine pollen extract may be able to normalize the liver metabolic abnormalities induced by POF. This study therefore establishes a theoretical reference for the development of functional applications for pine pollen and for the treatment of POF.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells

Ethnopharmacological relevantErxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. Aim of the researchThe study aims to ascertain both in vivo and in vitro rewards of EXD. Materials and methodsEXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. ResultsUsing UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. ConclusionEXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

SİSPLATİN İLE İNDUKLENEN PREMATÜR OVER YETMEZLİĞİ SIÇAN MODELİNDE DİOSMİN UYGULAMASININ ETKİLERİ

m&#x0D; We aimed to examine the potential beneficial effects of diosmin administration on cisplatin - induced premature ovarian failure (POF) in a rat model&#x0D; &#x0D; Material and Methods&#x0D; Twenty-eight rats were divided into four groups. Group A rats (n:7) were determined as the sham group. The remaining rats received an intraperitoneal injection of 1.5 mg/kg/day cisplatin for 10 days to create a POF model. Then, they were randomly divided into 3 subgroups.&#x0D; Group B was determined as POF group. Group C rats were given 100mg/kg/day diosmin for 10 days simultaneously while creating POF model. Group D rats were given 100mg/kg/day diosmin for 10 days after POF model was created. Twentieth day blood samples were taken and left ovaries were resected for examination.&#x0D; &#x0D; Results&#x0D; CIS-induced rats showed reduced levels of SOD, AMH and E2 compared to sham group rats (p0.05) between the sham group, group C and D. No significant (p&gt;0.05) difference in FSH value was observed between group C, D and sham groups.&#x0D; There was no significant (p&gt;0.05) difference in the number of secondary and antral follicles between group C and D compared to the sham group. Primordial follicle count was significantly higher in group C than group B (p

Beneficial Effects of Traditional Chinese Medicine in the Treatment of Premature Ovarian Failure.

Premature ovarian failure (POF) is characterized by hormonal disorders, amenorrhea, and premature loss of fertility potential in women of reproductive age. Several studies have been conducted on the effectiveness of traditional Chinese medicine (TCM) in treating POF. TCM relied primarily on apoptosis, immunity, and aging to treat POF based on the studies of domestic and foreign literature. Zuogui pills inhibited mitochondrial-dependent apoptosis in the treatment of POF. Huyang Yangkun formula regulated the downstream of the Bcl-2 family to resist apoptosis through the aquaporin-1 protein. Modified Bazhen decoction regulated apoptosis in POF by regulating X-linked inhibitors of apoptosis protein. Bushen Tianjing recipe was effective in treating POF by promoting angiogenesis and preventing apoptosis. As for immunity, Bushen Jianpi prescription and Er-Xian decoction cured autoimmunity POF models and increased follicular development-related protein expression. Bushen Huoxue Tang improved ovarian function and reduced ovarian inflammation by regulating the Nrf2/Keap1 signaling pathway and T lymphocytes. Taohong Siwu decoction promoted the proliferation and differentiation of granulosa cells of POF mice by regulating the TGF-β1/Smads signaling pathway. In addition, ginsenoside Rg1 and Jiajian Guisheng formula treated POF by regulating cell aging-related mechanisms. Si Wu Tang treated POF by activating the angiogenesis-related proteins. The goal of this review is to serve as a reference for in-depth research into the treatment of POF with TCM and provide inspiration for new diagnostic methods and treatment options.

Classical music restored fertility status in rat model of premature ovarian failure

Background:The restorative effect of classical music was assessed on the cyclophosphamide-induced animal model of premature ovarian failure (POF).Methods:Mozart’s piano classical music (K.448) was used for up to 4 and 8 weeks. Rats were exposed to music 6 h every day using a stereo system with a volume of 65–70 dB. Sera and ovarian tissue samples were collected for the evaluation of FSH, LH, and E2 and histopathological examination. At the same time points, samples were taken from the hypothalamus and hippocampus to monitor the expression of Ntrk2, Crh, and Pomc using real-time PCR. Mating trial was performed to evaluate the fertility status of POF rats.Results:Histopathological examination revealed a significant increase (p < 0.05) in the numbers of morphologically normal follicles at all the developmental stages in POF rats after music therapy compared to the POF group (p < 0.05). Music therapy decreased FSH and LH levels to near-to-normal levels conidied with elevation of E2 (p < 0.05). Ntrk2, Crh, and Pomc expressions were down-regulated in POF rats. Music therapy increasaed the expression of Ntrk2 in the hypothalamus of POF rats (p < 0.05). In contrast, Crh and Pomc failed to reach the detection limit before intervention and four weeks after the intervention however, these genes were expressed eight weeks after music therapy. Fertility status was increased (p < 0.05) in terms of litter size in POF rats after being exposed to music compared to the non-treated POF control group (p < 0.05).Conclusion:Results showed that music can exert therapeutic effects on POF rats via the alteration of sex-related hormones.