The Protective Role of Magnoliae Flos in Preventing Ovotoxicity and Managing Ovarian Function: An In Vitro and In Vivo Study.

Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.

Donepezil protects against cyclophosphamide-induced premature ovarian failure in mice: A focus on proinflammatory cytokines and NLRP3/TLR-4/NF-κB interplay.

BACKGROUND AND AIM: Cyclophosphamide (CP) chemotherapy is a significant iatrogenic component of premature ovarian failure (POF). The aim of this work was to evaluate the potential protective effects of donepezil, a centrally acting acetylcholinesterase (AChE) inhibitor, on CP-induced POF in mice. METHODS: 40 female Swiss albino mice were split into 5 equal groups: group 1 (control), group 2 (CP-POF); induced by intraperitoneal injection of CP on 8th day of the experiment, and group (3-5); mice received oral donepezil daily (1, 2, or 4 mg/kg, respectively) 8 days before CP injection. Mice were euthanized after 24 h of CP injection, and blood samples were collected to assay serum anti-Mullerian hormone (AMH) levels. Ovarian tissues were dissected, and the right ovary was processed for further assays of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), nucleotide-binding domain-like receptor family, the Pyrin domain-containing 3 (NLRP3) inflammasome, and Toll-like receptor 4 (TLR-4), while the left one was processed for histopathological and immunohistochemical examination of nuclear factor-Kappa beta (NF-κB) and caspase-3. RESULTS: Donepezil, in a dose-dependent manner particularly (4 mg/kg), has an inhibitory action on NO (40 ± 2.85 vs. 28.20 ± 2.23, P < 0.001), proinflammatory cytokines (P < 0.001), the TLR-4/ NF-κB / NLRP3 inflammasome pathway (P < 0.001), and apoptosis (P < 0.001), with a significant elevation in the AMH levels (4.57 ± 1.08 vs. 8.57 ± 0.97, P < 0.001) versus CP-POF group. CONCLUSION: Donepezil may be a potential protective agent against CP-induced POF in mice, but further research is needed to fully understand its therapeutic function experimentally and clinically.

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

BACKGROUND: Premature 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. METHODS: We 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. RESULTS: Our 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. CONCLUSION: Our study highlights the significant therapeutic effects of HUCMSC transplantation on CTX-induced POF in a Tibetan miniature pig model.

Assessing risks and knowledge gaps on the impact of systemic therapies in early breast cancer on female fertility: A systematic review of the literature.

The therapeutic landscape for early breast cancer (eBC) has expanded by introducing novel anticancer agents into clinical practice. During their reproductive years, women with eBC should be informed of the potential risk of premature ovarian insufficiency (POI) and infertility with the proposed systemic therapy. Although the topic of female fertility is becoming increasingly relevant in patients with cancer, limited information is available on the gonadotoxicity of new agents available for eBC treatment. Analyses from clinical trials and prospective data on ovarian function biomarkers are lacking. The purpose of this systematic review is to report the available preclinical and clinical data on female fertility risk with the use of the new agents that are part of clinical practice use or under development for eBC management. This review highlights the clear need to perform additional research efforts to improve our understanding on the gonoadtoxicity of new anticancer agents.

Bergenin attenuates triptolide-caused premature ovarian failure in mice based on the antioxidant activity.

Tripterygium wilfordii (TW) preparations have been utilized in China for treating rheumatoid arthritis and autoimmune diseases. However, their clinical use is limited due to reproductive toxicity, notably premature ovarian failure (POF). Our study aimed to investigate the effect and mechanism of bergenin in attenuating POF induced by triptolide in mice. POF was induced in female ICR mice via oral triptolide administration (50 µg/kg) for 60 days. Mice received bergenin (25, 50, 100 mg/kg, i.g.) or estradiol valerate (EV) (0.1 mg/kg, i.g.) daily, 1 h before triptolide treatment. In vitro, ovarian granulosa cells (OGCs) were exposed to triptolide (100 nM) and bergenin (1, 3, 10 µM). Antioxidant enzyme activity, protein expression, apoptosis rate, and reactive oxygen species (ROS) levels were assessed. The results showed that triptolide-treated mice exhibited evident atrophy, along with an increase in atretic follicles. Bergenin (50, 100 mg/kg) and EV (0.1 mg/kg), orally administered, exerted significant anti-POF effect. Bergenin and EV also decreased apoptosis in mouse ovaries. In vitro, bergenin (1, 3, 10 µM) attenuated triptolide-induced OGCs apoptosis by reducing levels of apoptosis-related proteins. Additionally, bergenin reduced oxidative stress through downregulation of antioxidant enzymes activity and overall ROS levels. Moreover, the combined use with Sh-Nrf2 resulted in a reduced protection of bergenin against triptolide-induced apoptosis of OGCs. Together, bergenin counteracts triptolide-caused POF in mice by inhibiting Nrf2-mediated oxidative stress and preventing OGC apoptosis. Combining bergenin with TW preparations may effectively reduce the risk of POF.

Transcriptome analysis during 4-vinylcyclohexene diepoxide exposure-induced premature ovarian insufficiency in mice.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman's physical health and fertility. Investigating VCD's pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

Human umbilical cord mesenchymal stem cells alleviate chemotherapy-induced premature ovarian insufficiency mouse model by suppressing ferritinophagy-mediated ferroptosis in granulosa cells.

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis.

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

BACKGROUND: Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. METHODS: hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. RESULTS: hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. CONCLUSION: hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.

Bu-Shen-Ning-Xin decoction inhibits macrophage activation to ameliorate premature ovarian insufficiency-related osteoimmune disorder via FSH/FSHR pathway.

Limited studies are associated with premature ovarian insufficiency (POI)-related osteoimmune disorder currently. Bu-Shen-Ning-Xin decoction (BSNXD) displayed a favorable role in treating postmenopausal osteoporosis. However, its impact on the POI-related osteoimmune disorder remains unclear. The study primarily utilized animal experiments and network pharmacology to investigate the effects and underlying mechanisms of BSNXD on the POI-related osteoimmune disorder. First, a 4-vinylcyclohexene dioxide (VCD)-induced POI murine model was conducted to explore the therapeutical action of BSNXD. Second, we analyzed the active compounds of BSNXD and predicted their potential mechanisms for POI-related osteoimmune disorder via network pharmacology, further confirmed by molecular biology experiments. The results demonstrated that VCD exposure led to elevated follicle-stimulating hormone (FSH) levels, a 50% reduction in the primordial follicles, bone microstructure changes, and macrophage activation, indicating an osteoimmune disorder. BSNXD inhibited macrophage activation and osteoclast differentiation but did not affect serum FSH and estradiol levels in the VCD-induced POI model. Network pharmacology predicted the potential mechanisms of BSNXD against the POI-related osteoimmune disorder involving tumor necrosis factor α and MAPK signaling pathways, highlighting BSNXD regulated inflammation, hormone, and osteoclast differentiation. Further experiments identified BSNXD treatment suppressed macrophage activation via downregulating FSH receptor (FSHR) expression and inhibiting the phosphorylation of ERK and CCAAT enhancer binding proteins ß. In conclusion, BSNXD regulated POI-related osteoimmune disorder by suppressing the FSH/FSHR pathway to reduce macrophage activation and further inhibiting osteoclastogenesis.

Yu Linzhu alleviates primary ovarian insufficiency in a rat model by improving proliferation and energy metabolism of granulosa cells through hif1α/cx43 pathway.

BACKGROUND: Yu Linzhu (YLZ) is a classical Chinese traditional formula, which has been used for more than 600 years to regulate menstruation to help pregnancy. However, the mechanism of modern scientific action of YLZ needs to be further studied. METHODS: Thirty SD female rats were divided into three groups to prepare the blank serum and drug-containing serum, and then using UHPLC-QE-MS to identify the ingredients of YLZ and its drug-containing serum. Twenty-four SD female rats were divided into four groups, except the control group, 4-vinylcyclohexene dicycloxide (VCD) was intraperitoneally injected to establish a primary ovarian insufficiency (POI) model of all groups. Using vaginal smear to show that the estrous cycle of rats was disturbed after modeling, indicates that the POI model was successfully established. The ELISA test was used to measure the follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) levels in the serum of rats. HE stain was used to assess the morphology of ovarian tissue. The localization and relative expression levels of CX43 protein were detected by tissue immunofluorescence. Primary ovarian granulosa cells (GCs) were identified by cellular immunofluorescence. CCK8 was used to screen time and concentration of drug-containing serum and evaluate the proliferation effect of YLZ on VCD-induced GCs. ATP kit and Seahorse XFe24 were used to detect energy production and real-time glycolytic metabolism rate of GCs. mRNA and protein expression levels of HIF1α, CX43, PEK, LDH, HK1 were detected by RT-PCR and WB. RESULTS: UHPLC-QE-MS found 1702 ingredients of YLZ and 80 constituents migrating to blood. YLZ reduced the FSH while increasing the AMH and E2 levels. In ovarian tissues, YLZ improved ovarian morphology, follicle development, and the relative expression of CX43. In vitro studies, we found that YLZ increased the proliferative activity of GCs, ATP levels, glycolytic metabolic rate, HIF1α, CX43, PEK, HK1, LDH mRNA, and protein levels. CONCLUSIONS: The study indicated that YLZ increased the proliferation and glycolytic energy metabolism of GCs to improve follicular development further alleviating ovarian function.

The mechanisms of MicroRNA 21 in premature ovarian insufficiency mice with mesenchymal stem cells transplantation : The involved molecular and immunological mechanisms.

Umbilical cord-derived mesenchymal stem cell (UCMSC) transplantation has been deeply explored for premature ovarian insufficiency (POI) disease. However, the associated mechanism remains to be researched. To explore whether and how the microRNA 21 (miR-21) functions in POI mice with UCMSCs transplantation, the autoimmune-induced POI mice model was built up, transplanted with or without UCMSCs transfect with the LV-hsa-miR-21-5p/LV-hsa-miR-21-5p-inhibition, with the transfection efficiency analyzed by QRT-PCR. Mice hormone secretion and the anti-Zona pellucida antibody (AZPAb) levels were analyzed, the ovarian morphological changes and folliculogenesis were observed, and the ovarian apoptosis cells were detected to evaluate ovarian function. The expression and localization of the PTEN/Akt/FOXO3a signal pathway-related cytokines were analyzed in mice ovaries.Additionally, the spleen levels of CD8 + CD28-T cells were tested and qualified with its significant secretory factor, interleukin 10 (IL-10). We found that with the LV-hsa-miR-21-5p-inhibition-UCMSCs transplantation, the mice ovarian function can be hardly recovered than mice with LV-NC-UCMSCs transplantation, and the PTEN/Akt/FOXO3a signal pathway was activated. The expression levels of the CD8 + CD28-T cells were decreased, with the decreased levels of the IL-10 expression. In contrast, in mice with the LV-hsa-miR-21-5p-UCMSCs transplantation, the injured ovarian function can be reversed, and the PTEN/AKT/FOXO3a signal pathway was detected activated, with the increased levels of the CD8 + CD28-T cells, and the increased serum levels of IL-10. In conclusion, miR-21 improves the ovarian function recovery of POI mice with UCMSCs transplantation, and the mechanisms may be through suppressing the PTEN/AKT/FOXO3a signal pathway and up-regulating the circulating of the CD8 + CD28-T cells.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

Effect of dexpanthenol on cyclophosphamide-induced ovarian toxicity: a histological and molecular study in rats.

RESEARCH QUESTION: Does dexpanthenol work as an effective therapeutic agent against cyclophosphamide (CYC)-induced premature ovarian failure (POF) in rats? DESIGN: A total of 28 female Wistar Albino rats were randomly divided into four groups (n = 7 per group). The POF and POF plus dexpanthenol groups were intraperitoneally administered CYC at an initial dose of 50 mg/kg, followed by 8 mg/kg for 14 days. The dexpanthenol and POF plus dexpanthenol groups were both intraperitoneally administered dexpanthenol at a dose of 500 mg/kg/day for 15 days. RESULTS: In the group administered CYC, the following was observed: a decrease in the ovarian index; a decrease in the numbers of primordial, primary, secondary and antral follicles; an increase in the number of corpus luteum and atretic follicles; a decrease in proliferation cell nuclear antigen immunoreactivity; a significant reduction in anti-Müllerian hormone and oestradiol levels; and an increase in serum FSH levels compared with controls. Dexpanthenol, on the other hand, reversed these effects. Quantitative reverse transcription polymerase chain reaction analyses showed that dexpanthenol increased Bcl-2, Akt1, mTOR, Nrf2 and HO-1 in CYC-induced ovarian tissues, but decreased Bax, Cas3, Hsp27, Hsp70, and Hsp90. Dexpanthenol treatment has a potential for inhibiting the intrinsic apoptotic pathway and oxidative stress levels in ovarian tissues via the downregulation of the mRNA expression of heat shock proteins and the activation of Nrf2/HO-1 pathways. CONCLUSIONS: Our findings demonstrated that dexpanthenol is an effective agent against POF caused by CYC; however, further experimental and clinical data are needed to use it effectively.

Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice.

Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 µM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 µM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.

α-Ketoglutarate Improves Ovarian Reserve Function in Primary Ovarian Insufficiency by Inhibiting NLRP3-Mediated Pyroptosis of Granulosa Cells.

SCOPE: Premature ovarian insufficiency (POI) is a common female infertility problem, with its pathogenesis remains unknown. The NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis has been proposed as a possible mechanism in POI. This study investigates the therapeutic effect of α-ketoglutarate (AKG) on ovarian reserve function in POI rats and further explores the potential molecular mechanisms. METHODS AND RESULTS: POI rats are caused by administration of cyclophosphamide (CTX) to determine whether AKG has a protective effect. AKG treatment increases the ovarian index, maintains both serum hormone levels and follicle number, and improves the ovarian reserve function in POI rats, as evidence by increased the level of lactate and the expression of rate-limiting enzymes of glycolysis in the ovaries, additionally reduced the expression of NLRP3, Gasdermin D (GSDMD), Caspase-1, Interleukin-18 (IL-18), and Interleukin-1 beta (IL-1ß). In vitro, KGN cells are treated with LPS and nigericin to mimic pyroptosis, then treated with AKG and MCC950. AKG inhibits inflammatory and pyroptosis factors such as NLRP3, restores the glycolysis process in vitro, meanwhile inhibition of NLRP3 has the same effect. CONCLUSION: AKG ameliorates CTX-induced POI by inhibiting NLRP3-mediated pyroptosis, which provides a new therapeutic strategy and drug target for clinical POI patients.

Ameliorative effect of salidroside on the cyclophosphamide-induced premature ovarian failure in a rat model.

BACKGROUND: Oxidative stress injury is an important pathological factor of premature ovarian failure (POF). Salidroside, extracted from the Chinese herb-Rhodiola rosea, has advantages in antioxidant characteristics. However, their therapeutic efficacy and mechanisms in POF have not been explored. PURPOSE: This study aims to assess the therapeutic effects of salidroside in chemotherapy-induced ovarian failure rats. METHODS: A POF rat model was established by injection of cyclophosphamide, followed by treatment with salidroside. The therapeutic effect of salidroside was evaluated based on hormone levels, follicle count, and reproductive ability. Oxidative stress injury was assessed by the detection of SOD enzyme activity and MDA levels. Differential gene expression of Keap1, Nrf2, HMOX1, NQO1, AMH, BMP15, and GDF9, were identified by qRT­PCR. The protein expression of Keap1, Nrf2, P53, and Bcl-2 were detected by western blot. RESULTS: Salidroside treatment markedly restored FSH, E2, and AMH hormone secretion levels, reduced follicular atresia, and increased antral follicle numbers in POF rats. In addition, salidroside improves fertility in POF rats, activates the Nrf2 signaling pathway, and reduces the level of oxidative stress. The recovery function of high dose salidroside (50 mg/kg) in a reproductive assay was significantly improved than that of lower dose salidroside (25 mg/kg). Meanwhile, the safety evaluation of salidroside treatment in rats showed that salidroside was safe for POF rats at doses of 25-50 mg/kg. CONCLUSIONS: Salidroside therapy improved premature ovarian failure significantly through antioxidant function and activating Nrf2 signaling.

Metformin improves d-galactose induced premature ovarian insufficiency through PI3K-Akt-FOXO3a pathway.

PURPOSE: Metformin (MET), a first-line treatment for type 2 diabetes mellitus, restores ovarian function in women with polycystic ovary syndrome. MET has been shown to increase the rate of success for in vitro fertilization when utilized in assisted reproductive technologies. This study was designed to examine the impact of MET on ovarian function and fertility in a mouse model of galactose-induced premature ovarian insufficiency (POI). We further investigated the underlying mechanisms. MATERIALS AND METHODS: Female mice were divided into 4 groups: saline, d-galactose, d-galactose â€‹+ â€‹MET, and MET. Body weight, ovarian index, and fertility were assessed. The hormonal profile was done. Advanced glycation end products (AGEPs), receptor for advanced glycation end products (RAGE), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), forkhead box O3a (FOXO3a) expression were measured. Ovarian follicle counting and morphology were analyzed. Immunohistochemistry of cleaved caspase-3 expression was performed. RESULTS: Our findings demonstrated that MET reversed irregularities in the estrus cycle, enhanced the ovarian index, and improved the abnormal levels of hormones and AGEs induced by d-galactose. Furthermore, the expression levels of PI3K, Akt, FOXO3a, and RAGE were upregulated with d-galactose. However, MET attenuated their expression levels. The primordial follicles ratio was improved, whereas atretic follicles and apoptotic-related cleaved caspase-3 expression were decreased in the d-galactose â€‹+ â€‹MET group compared to the d-galactose group. CONCLUSION: This study demonstrates that MET partially rescued ovarian dysfunction and apoptosis induced by d-galactose via a mechanism involving PI3K-Akt-FOXO3a pathway. Our finding proposed that MET may be a promising alternative treatment for POI.

PDCD4 deficiency improved 4-vinylcyclohexene dioxide-induced mouse premature ovarian insufficiency.

RESEARCH QUESTION: What role does programmed cell death 4 (PDCD4) play in premature ovarian insufficiency (POI)? DESIGN: A PDCD4 gene knockout (PDCD4-/-) mouse model was constructed, a POI mouse model was established similar to human POI with 4-vinylcyclohexene dioxide (VCD), a PDCD4-overexpressed adenovirus was designed and the regulatory role in POI in vitro and in vivo was investigated. RESULTS: PDCD4 expression was significantly increased in the ovarian granulosa cells of patients with POI (P ≤ 0.002 protein and mRNA) and mice with VCD-induced POI (P < 0.001 protein expression in both mouse ovaries and granulosa cells). In POI-induced mice model, PDCD4 knockouts significantly increased anti-Müllerian hormone, oestrodiol and numbers of developing follicles, and the PI3K-AKT-Bcl2/Bax signalling pathway is involved in it. CONCLUSION: The expression and regulation of PDCD4 significantly affects the POI pathology in a mouse model. This effect is closely related to the regulation of Bcl2/Bax and the activation of the PI3K-AKT signalling pathway.

Gonadotropin upregulates intraovarian calpains-1 and -2 during ovarian follicular recruitment in the SD rat model.

Calpain role has been shown in the cumulus cell-oocyte complexes and, corpus luteum. We investigated the association of calpains-1 and -2 in ovarian folliculogenesis using the Sprague-Dawley (SD) rat model and steroidogenesis in the human granulosa cells (hGCs). We induced PCOS in 42-day-old SD rats by letrozole oral gavage for 21 days. Premature ovarian failure (POF) was induced in 21-day-old SD rats by 4-vinylcyclohexene diepoxide (VCD). Ovulation and ovarian hyperstimulatory (OHS) syndrome were induced by pregnant mare gonadotropin (PMSG) + human chorionic gonadotropin (hCG) treatments in 21 days SD rats, respectively. Steroidogenesis is stimulated in human granulosa cells (hGCs) by forskolin and the response of 17-beta-estradiol (E2) on calpains expression was checked in hGCs. The protein expression by immunoblotting and activity by biochemical assay of calpains-1 and -2 showed an oscillating pattern in the ovarian cycle. PMSG-induced follicular recruitment showed upregulation of calpains-1 and -2, but with no change during ovarian function cessation (POF). Upregulated calpain-2 expression and calpain activity was found in the hCG +PMSG-induced ovulation. Letrozole-induced PCOS showed downregulation of calpain-1, but upregulation of calpain-2. PMSG+hCG-induced OHS led to the upregulation of calpain-1. Letrozole and metformin separately increased the expression level of calpains-1 and -2 in the hGCs during luteinization. In conclusion, the expression levels of calpains -1 and -2 are increased with ovarian follicular recruitment by PMSG and calpain-1 is decreased in the PCOS condition, and letrozole and metformin upregulate the expression of calpains-1 and -2 during luteinization in the hGCs possibly via E2 action.

Fertility protection: a novel approach using pretreatment with mesenchymal stem cell exosomes to prevent chemotherapy-induced ovarian damage in a mouse model.

BACKGROUND: Primary ovarian insufficiency refers to the loss of ovarian function before the age of 40 years and leads to amenorrhea and infertility. Primary ovarian insufficiency has diverse causes, but a common cause is exposure to gonadotoxic chemotherapy used in cancer treatment. Because of the risk for developing primary ovarian insufficiency, patients who want to preserve their fertility may consider various procedures for fertility preservation. However, current fertility preservation options are highly invasive, carry substantial risks, and have uncertain success rates. Recent studies from our group and others reported that mesenchymal stem cells and mesenchymal stem cell-derived exosomes can restore ovarian function in preclinical models of primary ovarian insufficiency by restoring damaged cells and inhibiting apoptosis. Although the restorative effect of mesenchymal stem cell-derived exosomes has been well reported in previous studies, the potential of mesenchymal stem cell-derived exosomes in preventing ovarian damage has not been fully elucidated. OBJECTIVE: This study hypothesized that the antiapoptotic potential of mesenchymal stem cell-derived exosomes may protect ovarian tissue from chemotherapy-induced damage. STUDY DESIGN: In this study, we delivered mesenchymal stem cell-derived exosomes directly into the ovaries of mice before administration of chemotherapy. A total of 60 mice were divided into 3 groups (20 per group), which were labeled the control, chemotherapy, and fertility protection groups. Only the fertility protection group mice received exosomes, whereas the control and chemotherapy group mice received saline. After exosome injection, the chemotherapy and fertility protection groups of mice were subjected to chemotherapy to induce ovarian damage. After chemotherapy, we evaluated the protective effects of exosome treatment on ovarian function, such as estrous cyclicity, serum hormone levels, and the fertility rate, by comparing these outcomes between the chemotherapy and fertility protection groups. These outcomes were also compared with those of the control group for comparison with outcomes under healthy conditions. RESULTS: After intraovarian injection of exosomes before chemotherapy, the mice were able to maintain their estrous cycle (4- to 5-day cyclicity), serum anti-müllerian hormone level (66.06±26.40 ng/mL, not significantly different from that of the healthy controls), folliculogenesis (32.2±11.3 in the chemotherapy group vs 46.4±14.1 in the fertility protection group; P<.05), expression of the steroidogenic acute regulatory protein gene (a the steroidogenesis marker) (0.44±0.11-fold expression in the chemotherapy group and 0.88±0.31-fold expression in the fertility protection group; P<.05), and fertility (2 of 8 in the chemotherapy group and 5 of 8 in the fertility protection group), thereby showing prevention of chemotherapy-induced damage. We found that exosome treatment before chemotherapy can preserve ovarian function and protect fertility through the overexpression of ATP synthase-binding cassette transporters, such as ABCB1b (10.17±17.75-fold expression in the chemotherapy group and 44.14±33.25-fold expression in the fertility protection group; P<.05) and ABCC10 (3.25±0.59-fold expression in the chemotherapy group and 5.36±1.86-fold expression in the fertility protection group; P<.05). CONCLUSION: In this study, we present a novel fertility protection method using mesenchymal stem cell-derived exosomes. We concluded that mesenchymal stem cell-derived exosomes are a promising and simple treatment option for fertility protection in reproductive-aged patients who are receiving gonadotoxic chemotherapy.