Ovarian Failure In Mice Research Articles

Collagen (peptide) extracted from sturgeon swim bladder: Physicochemical characterization and protective effects on cyclophosphamide-induced premature ovarian failure in mice

Premature ovarian failure (POF) is a common female reproductive disorder, with oxidative damage playing a significant role in its development. Studies had demonstrated the beneficial antioxidant, anti-aging, and other biological properties of swim bladder collagen (peptide). In this study, acid extraction, water extraction, and enzymatic hydrolysis methods were used to extract collagen (peptide) from sturgeon (Acipenser sinensis) swim bladder, all of which exhibited significant antioxidant activity. Moreover, the effects observed in POF mice were enhanced, including an increasing in the number of growing ovarian follicles, regulation of serum hormone levels, and alterations in signaling pathways, as evidenced by the up-regulation of Phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) and B cell lymphoma-2/ Bcl-associated x protein (Bcl-2/Bax) pathways, alongside the down-regulation of the mitogen-activated protein kinases (MAPK) signaling pathway. Sturgeon swim bladder collagen (peptide) could protect against cyclophosphamide-induced POF in mice, which could be very beneficial in the future advancement of health products.

Deer Blood Hydrolysate Protects against D-Galactose-Induced Premature Ovarian Failure in Mice by Inhibiting Oxidative Stress and Apoptosis.

Premature ovarian failure (POF) is a common disease among women, which can cause many complications and seriously threaten women's physical and mental health. Currently, hormone replacement therapy is the primary treatment for premature ovarian failure. However, the side effects are serious and will increase the chance of breast cancer and endometrial cancer. Deer blood hydrolysate (DBH) is the product of enzymatic hydrolysis of deer blood, has antioxidant, anti-ageing, and anti-fatigue effects, and has the potential to improve premature ovarian failure. In our experiment, a mouse model of premature ovarian failure was established through intraperitoneal injection of 400 mg/kg/d of D-gal for 42 days. At the same time, different doses of DBH were gavaged to observe its ameliorative effect on premature ovarian failure. The experimental findings indicated that DBH could restore the irregular oestrus cycle of POF mice, improve the abnormal amounts in serum hormones follicle-stimulating hormone (FSH), luteinising hormone (LH), progesterone (P) and estradiol (E2), increase the number of primordial follicles and decrease the number of atretic follicles. In addition, DBH also raised the level of superoxide dismutase (SOD) and reduced the level of malondialdehyde (MDA) and reduced the apoptosis of ovarian granulosa cells in mice. The WB assay results showed that gavage of DBH restored the decrease in the indication of nuclear factor erythroid 2-related factor 2 (Nrf2), Heme Oxygenase-1 (Ho-1), and B-cell lymphoma-2 (Bcl-2) proteins and reduced the elevated expression of Kelch-like ECH-associated protein 1 (Keap1), Bcl-2 associated X protein (Bax), and Cysteinyl aspartate specific proteinase-3 (Caspase-3) proteins that were induced by D-gal. To sum up, the present research indicated that DBH can ameliorate D-gal-induced oxidative stress and apoptosis by regulating the Nrf2/HO-1 signalling pathway and the Bcl-2/Bax/caspase-3 apoptosis pathway, which can be used for further development as a nutraceutical product to improve premature ovarian failure.

Clusterin-carrying extracellular vesicles derived from human umbilical cord mesenchymal stem cells restore the ovarian function of premature ovarian failure mice through activating the PI3K/AKT pathway

BackgroundEmerging evidence has highlighted the therapeutic potential of human umbilical cord mesenchymal stem cells (UC-MSCs) in chemotherapy-induced premature ovarian failure (POF). This study was designed to investigate the appropriate timing and molecular mechanism of UC-MSCs treatment for chemotherapy-induced POF.MethodsOvarian structure and function of mice were assessed every 3 days after injections with cyclophosphamide (CTX) and busulfan (BUS). UC-MSCs and UC-MSCs-derived extracellular vesicles (EVs) were infused into mice via the tail vein, respectively. Ovarian function was analyzed by follicle counts, the serum levels of hormones and ovarian morphology. The apoptosis and proliferation of ovarian granulosa cells were analyzed in vitro and in vivo. Label-free quantitative proteomics was used to detect the differentially expressed proteins in UC-MSC-derived EVs.ResultsAfter CTX/BUS injection, we observed that the ovarian function of POF mice was significantly deteriorated on day 9 after CTX/BUS infusion. TUNEL assay indicated that the number of apoptotic cells in the ovaries of POF mice was significantly higher than that in normal mice on day 3 after CTX/BUS injection. Transplantation of UC-MSCs on day 6 after CTX/BUS injection significantly improved ovarian function, enhanced proliferation and inhibited apoptosis of ovarian granulosa cells, whereas the therapeutic effect of UC-MSCs transplantation decreased on day 9, or day 12 after CTX/BUS injection. Moreover, EVs derived from UC-MSCs exerted similar therapeutic effects on POF. UC-MSCs-derived EVs could activate the PI3K/AKT signaling pathway and reduce ovarian granulosa cell apoptosis. Quantitative proteomics analysis revealed that clusterin (CLU) was highly expressed in the EVs of UC-MSCs. The supplementation of CLU proteins prevented ovarian granulosa cells from chemotherapy-induced apoptosis. Further mechanistic analysis showed that CLU-knockdown blocked the PI3K/AKT signaling and reversed the protective effects of UC-MSCs-derived EVs.ConclusionsAdministration of UC-MSCs and UC-MSCs-derived EVs on day 6 of CTX/BUS injection could effectively improve the ovarian function of POF mice. UC-MSCs-derived EVs carrying CLU promoted proliferation and inhibited apoptosis of ovarian granulosa cells through activating the PI3K/AKT pathway. This study identifies a previously unrecognized molecular mechanism of UC-MSCs-mediated protective effects on POF, which pave the way for the use of cell-free therapeutic approach for POF.Graphical

A Review of the Types of Chemotherapy Drugs Used to Induce Premature Ovarian Failure in Mice

Introduction: The most common complication of chemotherapy is infertility due to premature ovarian failure (POF). POF is defined as the loss of normal ovarian function before age 40, characterized by increased gonadotropin levels, decreased estradiol levels, and diminished ovarian reserve, often leading to infertility. Despite the high impact of POF on general health and quality of life, the pathophysiology of this disease is unclear. For this purpose, animal models provide us with the opportunity to hypothetically investigate the pathogenesis of the disease comprehensively. The most common method of creating an animal model of premature ovarian failure is the use of chemotherapy drugs. In this study, the types of chemotherapy drugs and the relevant molecular pathways that play a role in creating the premature ovarian failure model in mice will be investigated Conclusion: According to the studies, cyclophosphamide drug is introduced as the most common gonadotoxic drug in order to induce POF model in mice.

Role of DNA damage response in cyclophosphamide-induced premature ovarian failure in mice.

To investigate the DNA damage response (DDR) in a cyclophosphamide (CTX)-induced mouse model of premature ovarian failure (POF). The POF model was established by injecting mice with CTX. The body, ovarian weights, the estrus cycle, and pathological changes of the ovaries were recorded. The serum levels of 17 β-estradiol (E2) and follicle-stimulating hormone (FSH) were measured. The expression of Ki67, β-galactosidase (β-gal), p21, p53, γH2AX, and pATM in ovarian tissues was detected by immunohistochemistry. The expression of β-gal, γH2AX, and pATM was analyzed by immunofluorescence staining of primary cultured granulosa cells (GCs). The body and ovarian weights decreased, the estrus cycles were erratic, and the FSH level increased, whereas the E2 level decreased in POF mice compared to controls. The pathological consequences of POF revealed an increase in atretic follicles, corpus luteum, and primordial follicles and a decrease in the number of primary, secondary, and tertiary follicles. Ki67 expression was reduced, β-gal, p21, p53, γH2AX, and pATM expression were elevated in the ovaries of POF mice. The expression of β-gal, γH2AX, and pATM increased in GCs with the concentration in a time-dependent manner. In total, CTX induced POF in mice, which was mediated by the DDR pathway of ATM-P53-P21.

Therapeutic effects of curculigoside on cyclophosphamide-induced premature ovarian failure in mice

Objective The main purpose of this study was to elucidate the anti-apoptotic effects of curculigoside (CUR) on ovarian granulosa cells (GCs) in a mouse model of cyclophosphamide (CTX)-induced premature ovarian failure (POF). Method Intraperitoneal injection of CTX (100 mg/kg body weight) induced POF in mice. Thirty-six female mice were divided into six groups: blank group; POF model group; low-dose CUR group; medium-dose CUR group; high-dose CUR group; and estradiol benzoate group. Mice were orally administered for 28 consecutive days. Twenty-four hours after the completion of treatment, mice were weighed and euthanized, and blood was collected from the eyeball under anesthesia. The ovaries were surgically separated and weighed, and the ovarian index was calculated. Hematoxylin–eosin (HE) staining was used to observe follicular development and corpus luteum morphology in the ovaries. Serum levels of follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH) and estradiol (E2) were measured. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) content and malondialdehyde (MDA) levels in ovarian tissue were determined. The GC apoptosis level was measured. Western blotting was used to detect protein expression levels of Beclin-1, LC3, P62, AKT, p-AKT, mTOR and p-mTOR in the ovaries. Results The results showed that CUR can improve body weight and ovarian index; promote follicular development and reduce follicular atresia; improve FSH, AMH and E2 levels; downregulate MDA levels and restore antioxidant enzyme activity; inhibit the autophagy level; activate the AKT/mTOR signaling pathway; and alleviate GC apoptosis. Conclusion CUR improves POF by activating the AKT/mTOR signaling pathway, inhibiting autophagy and alleviating GC apoptosis.

MicroRNA-3061 downregulates the expression of PAX7/Wnt/Ca2+ signalling axis genes to induce premature ovarian failure in mice.

The in-depth mechanisms of microRNA regulation of premature ovarian failure (POF) remain unclear. Crispr-cas9 technology was used to construct transgenic mice. The qPCR and Western blot was used to detect the expression level of genes. H&E staining were used to detect ovarian pathological phenotypes. We found that the expression levels of microRNA-3061 were significantly higher in ovarian granulosa cells (OGCs) of POF mouse models than in controls. The miR-3061+/-/AMH-Cre+/- transgenic mice manifested symptoms of POF. RNA-Seq and luciferase reporter assay confirmed that the PAX7 was one of the target genes negatively regulated by microRNA-3061 (miR-3061-5p). Moreover, PAX7 mediated the expression of non-canonical Wnt/Ca2+ signalling pathway by binding to the motifs of promoters to stimulate the transcriptional activation of Wnt5a and CamK2a. In contrast, specific knock-in of microRNA-3061 in OGCs significantly downregulated the expression levels of PAX7 and inhibited the expression of downstream Wnt/Ca2+ signalling pathway. We also discerned a correlation between the expression levels of mRNAs of the Wnt/Ca2+ signalling pathway and the levels of E2 and FSH in POF patients by examining gene expression in the follicular fluid-derived exosomes of women. We confirmed that overexpression of microRNA-3061 induced proliferative inhibition of OGCs and ultimately induced POF in mice by suppressing the transcription factor PAX7 and downregulating expression levels of its downstream Wnt/Ca2+ signalling pathway genes.

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

Background and aimCyclophosphamide (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. Methods40 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. ResultsDonepezil, 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. ConclusionDonepezil 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.

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.

The effect of high intensity interval training on muscle contractile function 8 weeks following chemically-induced ovarian failure

The negative effects of ovariectomy on muscle contractile function have been well-characterized, however, the effects of gradual ovarian failure (i.e., perimenopausal transition into late-stage menopause) on muscle function over the lifespan have received less attention. Furthermore, whether exercise training can mitigate changes in muscle contractile function associated with menopause is unclear. The objective of this study, using a chemically-induced ovarian failure mouse model (4-vinylcyclohexene diepoxide; VCD), was to investigate time-course changes in muscle contractility of sedentary controls and the potential of high intensity interval training to mitigate any deleterious effects of ovarian failure. Starting at 11wks old, mice were injected with 160mg/kg/day of VCD for 15 days. Twenty-eight (VCD-trained: n=10, VCD-sedentary: n=10, control: n=8) CD1 female mice were used in this study, with the VCD-trained group beginning training at the onset of ovarian failure for a total of 8 weeks. Contractile properties of the plantar flexors were assessed using an in-vivo set-up 8 weeks following the onset of ovarian failure. As well, a fatigue task (repeated maximal isometric contractions until torque decreased by 60%) was performed. Recovery was measured immediately after, and up to 10min following task termination. Upon completion of mechanical testing, mice were sacrificed and intact muscle fibres were isolated from the flexor digitorum brevis, and myoplasmic free Ca2+ (tetanic [Ca2+]i) concentrations were measured across stimulation frequencies of 10-200 Hz and throughout 50 tetanic contractions (70Hz) to replicate our fatigue task. There was no difference in pre-fatigue values across groups for peak twitch torque, peak 100Hz torque, RTD, 10:100Hz torque, tetanic [Ca2+]i during low (10Hz) and high Hz (100Hz) stimulation, and all were reduced similarly immediately following the fatigue task. Repetitions to task failure was similar across groups and tetanic [Ca2+]i during repetitive contractions (n=50) was reduced similarly across groups. As well, all groups recovered similarly across these measures. Torque and RTD did not recover fully by 10min for either measure, while 10Hz, 100Hz and 10:100Hz tetanic [Ca2+]i was recovered immediately following the fatigue task. Given 10Hz torque was relatively maintained following the fatigue task, and there was a ~40% decline in 100Hz torque, the 10:100Hz ratio increased throughout recovery — this, combined with the [Ca2+] data indicate that calcium sensitivity and release are unlikely contributors to impaired force production following the fatigue task across groups. The present study aimed to assess the impact of training on muscle contractility using a mouse model of gradual ovarian failure. Unlike other models of ovarian failure, there does not seem to be any impairment in muscular performance at the joint level in our VCD-mice, and they responded similarly across groups in response to repetitive fatiguing contractions and exercise training. Supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Time course changes in in vivo muscle mechanical function and Ca2+ regulation of force following experimentally induced gradual ovarian failure in mice.

The abrupt cessation of ovarian hormone release is associated with declines in muscle contractile function, yet the impact of gradual ovarian failure on muscle contractility across peri-, early- and late-stage menopause remains unclear. In this study, a 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model was used to examine time course changes in muscle mechanical function. Plantar flexors of female mice (VCD: n=10; CON: n=8) were assessed at 40 (early perimenopause), 80 (late perimenopause), 120 (menopause onset) and 176 (late menopause) days post-initial VCD injection. A torque-frequency relationship was established across a range of frequencies (10-200Hz). Isotonic dynamic contractions were elicited against relative loads (10-80% maximal isometric torque) to determine the torque-velocity-power relationship. Mice then performed a fatigue task using intermittent 100Hz isometric contractions until torque dropped by 60%. Recovery of twitch, 10Hz and 100Hz torque were tracked for 10min post-task failure. Additionally, intact muscle fibres from the flexor digitorum brevis underwent a fatigue task (50 repetitions at 70Hz), and 10 and 100Hz tetanic [Ca2+] were monitored for 10min afterward. VCD mice exhibited 16% lower twitch torque than controls across all time points. Apart from twitch torque, 10Hz torque and 10Hz tetanic [Ca2+], where VCD showed greater values relative to pre-fatigue during recovery, no significant differences were observed between control and VCD mice during recovery. These results indicate that gradual ovarian failure has minimal detriments to in vivo muscle mechanical function, with minor alterations observed primarily for low-frequency stimulation during recovery from fatigue.

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.

Aged and induced-premature ovarian failure mouse models affect diestrus profile and ovarian features

Sex hormones exert a wide influence on several systems of the human body, especially in women, who undergo intense changes in the trans and postmenopausal periods. Different experimental models are used to mimic these conditions; however, the impact on hormonal profile may be different. This study aimed to analyze and compare vaginal cytology of different post-estropausal mice models, along with their microscopical ovarian features. Forty-six C57BL/6J female mice with the ages of 4, 6 and 18 months at the beginning of the experiment, weighing about 25–28 grams, constituted five groups: NC–(negative control) animals with no treatment, OVX-SHAM—sham ovariectomized, OVX–ovariectomized, VCD–medicated with 160 mg/kg/day of 4-vinylcyclohexene diepoxide via IP for 20 consecutive days, and Aged–senescent mice under physiological estropause. Euthanasia was performed at different periods for the removal of the ovaries, and after diestrus was confirmed by vaginal cytology for 10 consecutive days. For daily vaginal cytology, morphological and histomorphometric microscopic analyzes were performed. Aged mice presented significant increased neutrophils when compared to VCD group, as well as increased cornified epithelial cells when compared to OVX mice, and also increased nucleated epithelial cells when compared to VCD and OVX. NC and OVX-SHAM ovaries presented innumerous follicles at different stages of development, while VCD showed marked follicular atresia, depleted of primordial or developing follicles and a predominance of interstitial cells. The ovaries of aged mice were predominantly constituted by corpus luteum degenerated into corpus albicans, with rare antral follicles. All analyzed models led to different permanent diestrus profiles caused by each model, as indicated by ovarian features. This should be carefully considered when choosing a post-estropausal experimental model, in order to better correlate this challenging phase of female’s life with physiological/pathological conditions.

Chitosan microspheres loaded with WJMSCs facilitate the restoration of ovarian function in CTX‐induced premature ovarian failure mice

AbstractPremature ovarian failure (POF) is a complex disease affecting an increasing number of young women. Traditional treatments are limited in low efficacy and side effects. Wharton's jelly mesenchymal stem cells (WJMSCs) therapy holds promise in the treatment of POF. However, the clinical application of WJMSCs is hindered by challenges such as low cell viability, significant loss, and poor survival rates. In this study, we prepared three‐dimensional chitosan microspheres (CSM). CSM loading substantial improved the proliferative ability and cell viability of WJMSCs. Furthermore, transplantation of CSM loaded with WJMSCs into ovaries of POF mice increased serum levels of Estradiol (E2) and Anti‐Müllerian hormone (AMH), alongside decreased levels of follicle‐stimulating hormone (FSH). CSM+WJMSCs transplantation enhanced the number of growth follicles while reduced atresia follicles, and effective alleviated the ovarian fibrosis and apoptosis within the ovarian cortex. CSM also prolonged the residence time of WJMSCs in the ovarian capsule. Moreover, superovulation experiments illustrated that CSM+WJMSCs transplantation enhanced the total number of oocytes and reduced oocyte abnormality rates. Overall, CSM+WJMSCs transplantation promotes follicle development and regeneration, restoring ovarian function by inhibiting ovarian cell apoptosis and reducing levels of fibrosis. This study presents a novel therapeutic strategy that combines biomaterial and mesenchymal stem cells for the clinical treatment of premature ovarian failure.

Microfluidic Encapsulation of Exosomes Derived from Lipopolysaccharide-Treated Mesenchymal Stem Cells in Hyaluronic Acid Methacryloyl to Restore Ovarian Function in Mice.

Premature ovarian failure (POF) features an upward incidence nowadays, and the human umbilical cord mesenchymal stem cells (hUC-MSCs)-derived exosomes (MSC-Exos) have shown applied values in the recovery of ovarian function. Here, a novel exosome-encapsulated microcarrier prepared by microfluidic technology for ovarian repair after chemotherapy damage is presented. The exosomes derived from lipopolysaccharide (LPS)-preconditioned hUC-MSCs are encapsulated with hyaluronic acid methacryloyl (HAMA) via microfluidic electrospray, which is named HAMA/MSC-Exos. Attributing to the biocompatibility and semipermeable property of HAMA, the encapsulated exosomes show great viability and controllable release behavior from HAMA. It is demonstrated that in situ transplantation of HAMA/MSC-Exos can rescue ovarian functions of cyclophosphamide-induced ovarian failure in mice by increasing ovarian volume, improving the number of antral follicles and restoring fertility. It is believed that the transplantation of HAMA/MSC-Exos will provide a new concept for the treatment of POF in clinical practice.

Conservative Hypomethylation of Mesenchymal Stem Cells and Their Secretome Restored the Follicular Development in Cisplatin-Induced Premature Ovarian Failure Mice

Premature ovarian failure (POF) is one of the main causes of infertility in women under the age of 40 years. Recently, epigenetic reprogramming, particularly DNA hypomethylation, has emerged as a promising strategy to enhance the therapeutic potential of mesenchymal stem cells (MSCs). Thus, it is crucial to elucidate how far global hypomethylation of MSCs genome can maintain their pluripotency and viability and improve their therapeutic effect in chemotherapy-induced POF mice. Herein, the genomic DNA of bone marrow-derived MSCs (BM-MSCs) was hypomethylated by the DNA methyltransferase inhibitor (5-Aza-dC), and the degree of global hypomethylation was assessed by methylation-sensitive HepII/MspI restriction analysis. Next, mildly hypomethylated cells and their secretome were independently transplanted (or infused) in POF mice, established via cisplatin-mediated gonadotoxicity. We found that conservative global hypomethylation of BM-MSCs genome with low doses of 5-Aza-dC (≤0.5 μM) has maintained cell viability and MSCs-specific clusters of differentiation (CD). Engraftment of mildly hypomethylated cells in POF mice, or infusion of their secretome, improved the concentrations of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH). Furthermore, mice restored their normal body weight, ovarian size, and ovarian follicle count. This was associated with improved follicular development, where the populations of healthy primordial, primary, secondary, and tertiary follicles were significantly ameliorated, relative to mice transplanted with normally methylated cells. This observational study suggests that transplantation of mildly hypomethylated BM-MSCs cells and their secretome can restore the structural and functional integrity of the damaged ovaries in POF mice. Also, it presents conservative hypomethylation of BM-MSCs and their secretome as a promising alternative to MSCs transplantation.Graphical Transplantation of partially hypomethylated BM-MSCs improved the follicular count and integrity in the POF mouse model. Gonadotoxic drug (cisplatin) was used to establish the POF mouse model. In parallel, BM-MSCs were isolated, authenticated, and then incubated with the DNMTs inhibitor (5-Aza-dC). Partially hypomethylated cells and their secretome were independently transplanted into the POF mice, and both the follicular count, ovarian histology, and the serum levels of the fertility-related hormones (E2, AMH, and FSH) were assessed 1 week after transplantation or infusion. Hypomethylated BM-MSCs and their secretome increased the follicular count, increased the number of healthy follicles, and restricted apoptosis of the granulose cells. Also, the hormonal profile was improved compared to their corresponding level in mice transplanted with normally methylated cells.

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.

P-668 Effects of the active site domain of visfatin on the improvement of ovarian function in 4-vinylcyclohexene diepoxide-induced ovarian failure mice

Abstract Study question Can only the active site of domain of visfatin improve ovarian function in mice with 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure? Summary answer The active site domain of visfatin improves ovarian function and fertility potential in VCD-induced mice by stimulating the ovarian angiogenesis and early follicular development. What is known already Ovarian aging is a representative unmet need in infertility treatment and many studies have attempted to improve oocytes quality by activating ovarian angiogenesis. Visfatin has been reported to have angiogenic activity. Our previous study reported that visfatin improved ovarian function and fertility potential in aged female mice. However, visfatin has many limitations in developing as a drug due to large molecular weight. Visfatin is composed of two structural domains, and the active site is located in the second structural domain (amino acid residues 181 to 390), which were defined as the active site domain (hereafter A-domain peptide) in this study. Study design, size, duration This is a controlled experimental study using a total of mice with 60 VCD-induced ovarian failure. The duration of this study was from January to November 2022. Participants/materials, setting, methods C57BL/6 female mice were randomly divided into six groups: Five groups received 160mg/kg VCD and one group received an equal volume of vehicle. Four VCD groups were intraperitoneally injected three times at intervals of 2 days with 500 and 1000 ng/ml of full-size visfatin and A-domain peptide, respectively. The remaining one group served as a negative control. After final treatment, follicular development, gene expression, AMH level, and fertility potential were examined. Main results and the role of chance In hematoxylin and eosin (H&amp;E) staining, numbers of functional follicles including primordial, primary, secondary, and antral follicles were significantly decreased in the VCD-vehicle group compared to the control. However, treatment of full-size visfatin and A-domain peptide increased the number of primordial and primary follicle. On immnohistochemistry (IHC), expression of BMP15, C-KIT, VEGF, and visfatin in ovarian tissues were significantly reduced in the VCD-vehicle group, but immunoreactivity of these factors strongly stained in the treatment of full-size visfatin and A-domain peptide. This effect was more remarkable in 500 ng/ml of A-domain peptide compared to other treatments. The serum AMH level was significantly lower in the VCD-vehicle group compared to the control group, but it was increased after the treatment with A-domain peptide and full-size visfatin. Limitations, reasons for caution This study does not elucidate a clear mechanism how A-domain peptide increases the ovarian function and fertility of VCD-induced ovarian failure mice although it stimulated the expression of genes associated with early follicular development and angiogenesis. Also, generalizability to humans may be limited because the study was conducted in mice. Wider implications of the findings This study suggests that administration of A-domain peptide could be applied as a new treatment strategy for ovarian aging, presumably by activating ovarian angiogenesis and dormant primordial follicles. Trial registration number Not applicable

Identification of potential quality markers of Zishen Yutai pill based on spectrum-effect relationship analysis.

Introduction: The current quality evaluation of traditional Chinese medicine (TCM) is difficult to attribute to clinical efficacy due to the complexity of TCM. Zishen Yutai pill (ZYP), a well-known traditional Chinese patent medicine, has been widely used to prevent recurrent miscarriage and treat threatened abortion. However, the chemical components of ZYP are unknown, and there is no convincing quality control method applied on ZYP. Although ZYP has been found to promote endometrial receptivity and treat impending abortion, the substantial basis of the therapeutic effects is unclear. The aim of this study was to clarify the quality markers correlated with the potential medicinal activities and provide a theoretical foundation for scientific quality control and product quality improvement of ZYP. Methods: The chemical constituents of ZYP were comprehensively analyzed by offline two-dimensional liquid chromatography-mass spectrometry (2DLC-LTQ-Orbitrap-MS). The efficacy of the 27 ZYP orthogonal groups was investigated using the HTR-8/SVneo oxidative damage model and migration model in vitro, as well as the endometrial receptivity disorder mouse model and premature ovarian failure mouse model in vivo. Based on the efficacy and mass spectral results, spectrum-effect relationship analysis was used to identify the chemical components with corresponding pharmacological activities. Results: A total of 589 chemical components were found in ZYP, of which 139 were not identified in the literature. The potential quality markers for ZYP were successfully identified through orthogonal design and spectrum-effect relationship analysis. By combining mass spectrum data and pharmacological results of 27 orthogonal groups, 39 substances were identified as potential quality markers. Conclusion: The approaches used in this study will provide a feasible strategy for the discovery of quality markers with bioactivity and further investigation into the quality evaluation of TCM.

Red Ginseng Improves D-galactose-Induced Premature Ovarian Failure in Mice Based on Network Pharmacology.

In this study, we evaluated the ameliorative effect and molecular mechanism of red ginseng (Panax ginseng C.A. Meyer) extract (RGE) on D-galactose (D-gal)-induced premature ovarian failure (POF) using network pharmacology analysis. Ginsenosides are important active ingredients in ginseng, which also contains some sugar and amino acid derivatives. We aimed to determine the key proteins through which RGE regulates POF. In this work, we retrieved and screened for active ingredients in ginseng and the corresponding POF disease targets in multiple databases. A PPI network of genes was constructed in the STRING database and core targets were screened using topological analysis. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted in R software. Finally, molecular docking was conducted to validate the results. Female ICR mice were used to establish a POF mouse model for in vivo experiments. Serum levels of relevant estrogens were determined using ELISA and expression levels of relevant proteins in ovarian tissues were detected using immunofluorescence and western blot analysis. Network pharmacology analysis predicted that PI3K, Akt, Bax, Bcl-2, p16, and other proteins were highly correlated with POF and RGE. The results clearly showed that RGE could increase estradiol (E2) and lower follicle-stimulating hormone (FSH) levels in D-gal-fed mice. RGE restored the expression levels of related proteins by reducing Nrf2-mediated oxidative stress, PI3K/Akt-mediated apoptosis, and senescence signaling pathways. Overall, RGE has the potential to prevent and treat POF and is likely to be a promising natural protector of the ovaries.