Use of pyrophosphate in the preparation of a medicament for delaying physiological aging of the ovary

Abstract

The application discloses application of pyrophosphate in preparation of a medicine for delaying physiological aging of an ovary. The application takes old female mice as a physiological ovary aging model, and confirms that the pyrophosphate can improve the ovary aging related phenotypes from multiple biological aspects: increasing the cumulative number of offspring of the old female animals, increasing the number of ovarian follicles at all levels, restoring the endocrine homeostasis of serum FSH and E2, improving the distribution of MII oocyte cortex granules, enhancing the antioxidant stress resistance, maintaining the mitochondrial membrane potential, and improving the oocyte quality. The pyrophosphate is a natural endogenous metabolic small molecule in mammals, and has high safety. The application provides a brand-new medicine strategy for delaying physiological aging of the ovary, and has good clinical application and industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the fields of pathophysiology and biomedicine, specifically relating to the use of endogenous small molecule pyrophosphate in the preparation of drugs for delaying physiological aging of mammalian ovaries, and further relating to ovarian protective drug compositions containing pyrophosphate and their applications. Background Technology

[0002] With the continued decline in global fertility rates and the increasing trend of older women having children, the assisted reproductive technology market is experiencing rapid expansion. However, female reproductive capacity is significantly age-dependent, primarily manifested in the following ways: as women age, ovarian function gradually declines, follicle reserve and development capacity decrease, oocyte quality declines, and ultimately, fertility decreases.

[0003] Ovarian aging is an important biological basis for female reproductive aging. Its occurrence is not determined by a single factor, but is related to multiple factors such as changes in follicular reserve, oocyte nucleoplasmic maturation status, mitochondrial function, redox homeostasis, and reproductive endocrine status. Among these factors, the decline in oocyte quality is an important link in age-related fertility reduction in women, affecting the oocyte's ability to further complete fertilization and early embryonic development.

[0004] Therefore, how to improve the decline in ovarian function caused by natural aging at the in vivo level and further improve the quality of oocytes is an important technical issue in the fields of reproductive medicine and reproductive aging research.

[0005] Current intervention studies on ovarian aging and age-related fertility decline mainly focus on oxidative stress, mitochondrial function, inflammatory response, energy metabolism, and hormone regulation.

[0006] One type of research focuses on improving the state of ovaries and oocytes by supplementing with exogenous compounds with antioxidant effects. For example, studies have reported that nicotinamide mononucleotide (NMN) supplementation can improve the mitochondrial and energy metabolism status of ovaries in aged mice (Liang et al., 2024); melatonin can improve oxidative stress and quality decline in aged oocytes (Ma et al., 2025); and resveratrol has also been used to improve the in vitro maturation status of oocytes in aged mice and humans (Liu et al., 2018).

[0007] Another type of research focuses on studying ovarian aging from the perspectives of the overall metabolic network, mitochondrial metabolism, specific metabolic pathways, and the relationship between metabolism and epigenetic regulation. For example, studies related to mevalonate metabolism suggest that metabolic intermediates can participate in changes in the quality of aging oocytes by influencing intracellular protein modification and signal regulation (Liu et al., 2025).

[0008] Current interventions for age-related fertility decline primarily rely on antioxidants, hormone regulators, or other pharmacologically active molecules, with their effects mainly focused on reducing oxidative stress, alleviating inflammation, or improving single metabolic pathways. However, physiological ovarian aging is a complex process involving multi-level synergistic imbalances in follicle development, endocrine homeostasis, mitochondrial function, and oocyte quality. Existing technologies generally lack comprehensive intervention programs that can simultaneously improve natural fertility, ovarian follicle development, reproductive hormone status, and mitochondrial function and redox homeostasis of oocytes during the MII phase in whole animals. Therefore, how to restore ovarian function and improve oocyte quality through an endogenous metabolite with a sound physiological basis, thereby salvaging the fertility of aging women, remains a key technical problem that urgently needs to be solved in this field.

[0009] Pyrophosphate (PPi) is a class of small inorganic molecules containing a pyrophosphate group, which participate in various phosphate group transfer and metabolic reactions in organisms. Pyrophosphate is typically expressed as P₂O₇. 4- It exists in various forms and can form corresponding salts with different cations.

[0010] Current research on PPi mainly focuses on mineral metabolism and abnormal tissue mineralization. PPi is considered an important endogenous mineralization inhibitor in vivo, and its generation, degradation, and local concentration changes are closely related to the calcification process of blood vessels and soft tissues (Hamczyk and Villa-Bellosta, 2018).

[0011] Previous studies have shown that exogenous PPi supplementation can reduce the degree of abnormal mineralization in specific pathological calcification models. For example, in the ABCC6-deficient mouse model, PPi supplementation can inhibit both chronic and acute soft tissue calcification (Pomozi et al., 2017). Current research and applications of PPi mainly focus on: mineral metabolism regulation, abnormal vascular and soft tissue calcification, specific hereditary calcification diseases, and pathological processes related to the balance between PPi production and degradation. There are currently no reports on the effects of PPi on ovarian function.

[0012] References [1] Liang, J.; Huang, F.; Hao, X.; Zhang, P.; Chen, R. Nicotinamidemononucleotide supplementation rescues mitochondrial and energy metabolismfunctions and ameliorates inflammatory states in the ovaries of aging mice.MedComm 2024, 5, e727. [2] Ma, R.; Xue, M.; Ge, F.; Jureaitetibaike, K.; Zhao, S.; Qian, Z.;He, Z.; Zhang, H.; Tang, T.; Cao, C.; Li, C.; Zheng, L.; Xue, T.; Dong, J.;Jing, J.; Zhong, J.; Ma, J.; Yang, Y.; Huang, Y.; Ge, X.; Yao, B.; Chen, L.Melatonin protects aged oocytes from depalmitoylation-mediated qualityreduction by promoting PPT1 degradation and antioxidant. Redox Biol. 2025,80, 103510. [3] Liu, M.-J.; Sun, A.-G.; Zhao, S.-G.; Liu, H.; Ma, S.-Y.; Li, M.;Huai, Y.-X.; Zhao, H.; Liu, H.-B. Resveratrol improves in vitro maturation ofoocytes in aged mice and humans. Fertil. Steril. 2018, 109, 900-907. [4] Liu, C.; Zhang, H.; Mao, J.; Zhang, S.; Tian, ​​X.; Zhu, Y.; Wang, C.; Fang, J.; Pan, H.; Kang, N.; Zhang, Y.; Zhou, J.; Zhen, X.; Yan, G.; Li, C.; Hu, Y.; Ye, C.; metabolitesboost aged oocyte quality through prenylation of small GTPases. Nat. Aging2025, 5, 2022-2038. [5] Hamczyk, MR; Villa-Bellosta, R. Pyrophosphate metabolism andcalcification. Aging 2018, 10, 3652-3653 [6] Pomozì, V.; Brampton, C.; van de Wetering, K.; Zoll, J.; Calio, B.; Pham, K.; Owens, JB; Marh, J.; Moisyadi, S.; Váradi, A.; Martin, L.; and Acute Calcification in ABCC6-DeficientMice. Am. J. Pathol. 2017, 187, 1258-1272. Summary of the Invention

[0013] To address the problems in existing technologies, this invention aims to provide a novel strategy for rescuing female fertility and delaying physiological ovarian aging through metabolic regulation using the endogenous metabolic intermediate pyrophosphate. Specifically, this invention intends to solve the following technical problems: First, it provides a new use for PPI to improve age-related decline in ovarian function.

[0014] Second, it provides applications for PPi to improve the follicle development status of naturally aging female animals.

[0015] Third, it provides applications for PPi to improve age-related reproductive endocrine abnormalities.

[0016] Fourth, it provides applications for PPi to improve the quality of oocytes in the MII stage of aging.

[0017] Fifth, it provides applications for PPi to improve oxidative stress and mitochondrial function in MII-stage oocytes.

[0018] Sixth, provide applications for PPi to enhance the fertility of naturally aging female animals.

[0019] Therefore, this invention provides, in one aspect, the application of pyrophosphate (as the sole active ingredient) in the preparation of drugs for delaying (physiological) aging of (mammalian) ovaries.

[0020] Preferably, the pyrophosphate is selected from one or more of sodium pyrophosphate, disodium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, magnesium pyrophosphate, dimagnesium dihydrogen pyrophosphate, or their hydrates.

[0021] Most preferably, the pyrophosphate is sodium pyrophosphate decahydrate (Na4P2O7•10H2O).

[0022] Preferably, the drug is used to improve the fertility of older female mammals.

[0023] Preferably, the drug is used to improve the ovarian histology and follicular reserve in aged female mammals.

[0024] Preferably, the drug is used to restore reproductive endocrine homeostasis in aging female mammals.

[0025] More preferably, the drug achieves at least one of the following pharmacological effects: increasing the cumulative number of offspring in older females; increasing the number of follicles at each stage of the ovary; reducing follicle-stimulating hormone (FSH) and increasing estradiol (E2) to restore endocrine homeostasis; improving the distribution of cortical granules in MII oocytes, resisting oxidative stress, and increasing mitochondrial membrane potential.

[0026] Preferably, the drug is used to improve the quality of MII stage oocytes in aged female mammals.

[0027] More preferably, improving the quality of MII oocytes includes at least one of the following: (1) improving the distribution of cortical granules; (2) reducing oxidative stress levels; and (3) increasing mitochondrial membrane potential.

[0028] Preferably, the route of administration of the drug is selected from intraperitoneal injection, intravenous injection, subcutaneous injection, and oral administration.

[0029] Preferably, the molar dose of the drug is 12 × 10⁻⁶. -3 μmol / kg~16×10 -3 μmol / kg, once daily for 14 consecutive days; optimal concentration 16×10 -3 μmol / kg.

[0030] Preferably, the dosage form of the drug is selected from injections, lyophilized powders, sustained-release formulations, liposome formulations, or nanocarrier formulations.

[0031] Preferably, the drug further includes other ovarian protective active ingredients selected from at least one of nicotinamide mononucleotide (NMN), melatonin, and resveratrol.

[0032] On the other hand, the present invention provides a pharmaceutical composition for delaying physiological ovarian aging, comprising: an effective amount of pyrophosphate and a pharmaceutically acceptable carrier or excipient.

[0033] Preferably, the pyrophosphate is selected from one or more of sodium pyrophosphate, disodium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, magnesium pyrophosphate, dimagnesium dihydrogen pyrophosphate, or their hydrates, preferably sodium pyrophosphate decahydrate (Na4P2O7•10H2O).

[0034] Preferably, the pharmaceutical composition further includes other ovarian protective active ingredients selected from at least one of nicotinamide mononucleotide (NMN), melatonin, and resveratrol.

[0035] On the other hand, the present invention provides a method for delaying physiological aging of the ovaries in mammals, comprising administering an effective amount of pyrophosphate to a subject in need.

[0036] Preferably, the pyrophosphate is selected from one or more of sodium pyrophosphate, disodium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, magnesium pyrophosphate, dimagnesium dihydrogen pyrophosphate, or their hydrates, preferably sodium pyrophosphate decahydrate (Na4P2O7•10H2O).

[0037] Preferably, the dosage of the pyrophosphate is 12 × 10⁻⁶. -3 μmol / kg to 16×10 -3 μmol / kg body weight.

[0038] Preferably, the pyrophosphate is administered via intraperitoneal injection for 14 consecutive days, once daily.

[0039] This invention provides a novel application of pyrophosphate in the preparation of drugs to delay physiological ovarian aging. Compared with existing technologies, this invention utilizes small-molecule pyrophosphate, which is naturally present in the body's metabolic processes, as an intervention substance. Using naturally aging female mice as the research subject, a systematic evaluation was conducted on their ovarian function, reproductive endocrine levels, oocyte quality, and fertility. Experimental results show that pyrophosphate can synergistically improve ovarian aging-related phenotypes at multiple biological levels, demonstrating good application value and translational prospects in delaying physiological ovarian aging.

[0040] Compared with existing reported anti-ovarian aging drugs and intervention strategies, the pyrophosphate used in this invention has the following irreplaceable technical advantages: First, regarding safety and source advantages, pyrophosphate is a naturally occurring metabolic intermediate in mammals, not an exogenous synthetic compound. Supplementing with pyrophosphate essentially "restores physiological levels" rather than "introducing exogenous substances," thus having extremely low immunogenicity and systemic toxicity. Its metabolic pathway is well-defined, and the final product is inorganic phosphate, which can be safely excreted through the kidneys, posing no risk of harmful metabolite accumulation.

[0041] Second, it offers the advantage of multi-target synergistic regulation. Most currently reported anti-ovarian aging candidate drugs mainly exert their effects through a single antioxidant or anti-inflammatory mechanism, which is insufficient to address the multi-factor synergistic driving characteristics of ovarian aging. As an endogenous metabolite, pyrophosphate can exert its effects synergistically through multiple mechanisms, such as regulating cellular metabolic homeostasis, improving mitochondrial oxidative phosphorylation function, maintaining redox balance, and protecting cytoplasmic structural integrity, making it more suitable for intervention in the complex biological process of ovarian aging.

[0042] Third, it boasts advantages in formulation development and industrialization. Pyrophosphate preparation technology is mature, raw material sources are abundant, and its chemical properties are stable (not easily oxidized, degraded, or structurally altered), facilitating drug formulation development. It can be prepared into injections, lyophilized powders, sustained-release formulations, liposome formulations, nanocarrier formulations, and other pharmaceutically acceptable dosage forms according to actual clinical needs, demonstrating excellent prospects for industrialization and clinical translation feasibility. Attached Figure Description

[0043] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein: Figure 1 Chemical formula for pyrophosphate (Na₄P₂O₇•10H₂O); Figure 2 Intraperitoneal injection of pyrophosphate; Figure 3 Cumulative number of offspring after injection of pyrophosphate; Figure 4 Counting of follicles at various stages in mouse ovaries; Figure 5 The levels of FSH and E2 in mouse serum; Figure 6 Image of cortical granule distribution and fluorescence intensity in MII stage oocytes; Figure 7 The level of reactive oxygen species (ROS) in MII stage oocytes after H2O2 treatment; and Figure 8 Representative fluorescence images of mitochondrial membrane potential (ΔΨm) in MII stage oocytes. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise indicated, the terms used herein have their general technical meanings as understood by those skilled in the art.

[0046] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0047] In this invention, the terms "comprising" or "including" are open-ended expressions that refer to a specific ingredient or step described, without excluding other ingredients or steps that do not have a substantial effect.

[0048] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0049] In this invention, the term "ovarian aging" refers to the decline of ovarian function in mammals that occurs with natural aging or under the presence of exogenous chemical toxins, surgical damage, or gene knockout pathology. The main characteristics include: reduced primordial follicle reserve and decreased number of follicles at each stage of development; compensatory increase in serum FSH and decrease in E2 secretion; cytoplasmic maturation defects in MII oocytes, increased oxidative stress, and decreased mitochondrial membrane potential; and a gradual decline in individual fertility.

[0050] In this invention, the term "pyrophosphate" refers to salt containing pyrophosphate anions (P₂O₇).4- Pharmaceutically acceptable salts of ) include hydrated and anhydrous salts, such as tetrasodium pyrophosphate decahydrate, tetrasodium pyrophosphate anhydrous, disodium dihydrogen pyrophosphate, potassium pyrophosphate, and magnesium pyrophosphate.

[0051] In this invention, the term "MII stage oocyte" refers to a mature oocyte in metaphase II of meiosis, which is a mature oocyte capable of fertilization obtained after ovulation in mammals.

[0052] In this invention, the term "cortical granules (CG)" refers to specialized membranous organelles of oocytes, which are uniformly distributed in the cortical region of oocytes under normal physiological conditions; in senescent oocytes, they exhibit abnormal aggregation and diffuse distribution; the normal distribution of cortical granules is the structural basis for cytoplasmic maturation, post-fertilization cortical response, and prevention of polyspermy.

[0053] In this invention, the term "mitochondrial membrane potential ΔΨm" refers to the transmembrane potential difference between the inside and outside of mitochondria, which is a core indicator of mitochondrial functional integrity. Senescent oocytes are often accompanied by mitochondrial membrane potential depolarization (decreased membrane potential), insufficient ATP synthesis, and excessive ROS production. JC-1 probe detection: high membrane potential produces red fluorescent aggregates, while depolarized mitochondria produce green monomers; the red / green fluorescence intensity ratio serves as a quantitative indicator of membrane potential, with a higher ratio indicating better mitochondrial function.

[0054] In this invention, the term "cumulative number of offspring" refers to the total number of surviving offspring after the female animal is caged together. It is the gold standard indicator for evaluating the overall reproductive capacity of the female, reflecting the true reproductive outcome in vivo, and is different from indicators based solely on in vitro oocytes.

[0055] In this invention, the term "therapeutic effective dose" refers to a dose that achieves at least one improvement in ovarian aging after administration; in a mouse model, this is 12 × 10⁻⁶ moles of pyrophosphate. -3 μmol / kg~16×10 -3 μmol / kg body weight.

[0056] In this invention, the term "pharmaceutical-acceptable carrier / excipient" refers to conventional solvents, excipients, lyophilization protectants, and osmotic pressure regulators in the pharmaceutical formulation field that have no significant toxicity to organisms, including physiological saline, mannitol, trehalose, water for injection, phospholipids, etc. In this invention, the term "mammal" includes humans, monkeys, cats, dogs, cattle, sheep, horses, donkeys, pigs, rats, rabbits, deer, etc.

[0057] In one specific embodiment, the use of pyrophosphate in the preparation of drugs for delaying physiological ovarian aging is disclosed. This invention is the first to apply pyrophosphate (PPi), an endogenous small molecule metabolic intermediate, to delay physiological ovarian aging and improve fertility in older female mammals.

[0058] In one specific implementation, a specific administration regimen for pyrophosphate is disclosed. Intraperitoneal injection of sodium pyrophosphate decahydrate (Na4P2O7•10H2O) is preferred, with a dosage of 16 × 10⁻⁶. -3 The dosage was μmol / kg body weight, and the administration period was 14 consecutive days of intraperitoneal injection.

[0059] In one specific implementation, the application of pyrophosphate in improving the fertility of aged female mammals is disclosed. This invention verifies through in vivo animal model experiments that pyrophosphate can significantly increase the cumulative litter size of aged female mice after being housed with male mice, thus mitigating age-related natural fertility decline.

[0060] One specific implementation plan discloses the application of pyrophosphate in improving ovarian histological structure and follicular reserve. Pyrophosphate can increase the number of follicles at all stages (primordial follicles, primary follicles, secondary follicles, and cavitary follicles) in aging ovaries and improve the integrity of ovarian tissue structure.

[0061] In one specific implementation plan, the application of pyrophosphate in restoring reproductive endocrine homeostasis is disclosed. Pyrophosphate can reduce the compensatory increase in FSH levels in the serum of aged individuals, while increasing E2 levels, thus promoting the recovery of endocrine function of the hypothalamus-pituitary-ovarian axis.

[0062] In one specific implementation, the application of pyrophosphate in improving the multidimensional quality of MII-stage oocytes is disclosed. This application includes, but is not limited to, improving oocyte cortical granule distribution (cytoplasmic maturity), reducing H2O2-induced oxidative stress levels (antioxidant capacity), and increasing mitochondrial membrane potential (mitochondrial functional state).

[0063] In one specific embodiment, pyrophosphate is disclosed as an active ingredient in a pharmaceutical composition for preventing and / or delaying physiological ovarian aging. The present invention also provides a pharmaceutical composition for preventing and / or delaying physiological ovarian aging, the pharmaceutical composition comprising pyrophosphate and a pharmaceutically acceptable carrier or excipient.

[0064] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All chemicals used in the following reactions are commercially available products unless otherwise specified. Experimental statistics were performed using t-tests. *P< 0.05;** P <0.01; ***P <0.001; **** P <0.0001.

[0065] This invention employs a progressive technical approach of "in vivo intervention—histological evaluation—endocrine detection—cell function verification," using aged female mice as a physiological ovarian aging model to systematically evaluate the multidimensional improvement effect of pyrophosphate supplementation on ovarian function.

[0066] Laboratory Animals and Ethical Statement The experimental mice used in this experiment were specific pathogen-free (SPF) mice with a genetic background of C57BL / 6J. They were housed in the Experimental Animal Center on the 9th floor of the Stem Cell Building at the Institute of Zoology, Chinese Academy of Sciences. The environment was maintained with a 12-hour light / 12-hour dark cycle, a temperature controlled at 22±2℃, and humidity maintained at 50-60%. The animals had free access to food and water. The housing and handling of the experimental mice complied with the relevant standards and policies established by the Experimental Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. All experimental procedures followed the 3R principle of animal experiments (replace, reduce, optimize).

[0067] Example 1: Establishment of an animal model of ovarian aging and drug administration regimen This experiment used 9-month-old aged female C57BL / 6J mice as a physiological ovarian aging model. During the experiment, the aged female mice received daily intraperitoneal injections (ip) of pyrophosphate (sodium pyrophosphate decahydrate, Na4P2O7•10H2O, structural formula see [link to relevant documentation]). Figure 1 (Purity ≥99.5%, purchased from Sigma-Aldrich). The treatment components were administered at two dose levels: medium and high, with doses of 12 × 10⁻⁶. -3 μmol / kg and 16×10 -3 The dosage was calculated based on body weight, and the mice were injected continuously for 14 days at a concentration of μmol / kg. Control group mice received an equal volume of sterile saline daily at the same time as the treatment group. Specific injection protocols are as follows: Figure 2 As shown.

[0068] Example 2 Fertility Assessment - Pyrophosphate Intervention Significantly Improved Overall Reproductive Function in Aged Female Rats To evaluate the effect of pyrophosphate intervention on improving the reproductive capacity of aged female mice, aged female mice (treatment group and control group) after treatment were mated with young, healthy male mice (2-month-old ICR strain, Beijing Vital River Laboratory Animal Technology Co., Ltd.) with confirmed fertility at a female-to-male ratio of 2:1. The mating began on day 14 after the start of drug administration (i.e., the day drug administration ended).

[0069] During the 6-month observation period of continuous co-cultivation, female mice were checked daily for the birth of pups, and the number of pups and their survival rate were recorded in detail. The growth and development of the pups were also observed. The primary evaluation indicator was the cumulative litter size, which is the total number of surviving pups produced by each female mouse at each statistical time point (1 to 6 months) after co-cultivation, used to comprehensively reflect the individual's long-term reproductive capacity. Results were plotted on [date missing]. Figure 3 .

[0070] During the natural aging process, as ovarian function gradually declines, older animals exhibit multiple reproductive decline phenotypes, including decreased ovulation capacity, reduced number of mature oocytes, decreased embryonic developmental potential, and significantly reduced fertility. Experimental data from this invention show that after pyrophosphate intervention (especially at 16×10⁻⁶ ppm), [the following results were observed]. -3 In the high-dose group (μmol / kg), the cumulative litter size of older female mice paired with younger male mice was significantly higher than that of older female mice in the control group, indicating that pyrophosphate intervention significantly improved the overall reproductive function of older female mice. The significance of this effect lies in the fact that cumulative litter size is a comprehensive endpoint indicator of female reproductive function, and its improvement directly confirms that the effect of pyrophosphate intervention is not only manifested in changes in single-cell or histological indicators, but also in improved natural reproductive outcomes at the whole animal level.

[0071] Example 3: Ovarian Histological Analysis and Follicle Counting - Pyrophosphate Intervention Improves the Histological Microenvironment and Follicle Development in Aging Ovaries On day 56 after drug administration, mice were euthanized by cervical dislocation, and both ovaries were quickly dissected and fixed overnight (12-16 hours) in 4% neutral paraformaldehyde (PFA). Subsequently, the ovarian tissue was dehydrated with a gradient of ethanol (70%, 80%, 95%, 100%), cleared with xylene, and then embedded in paraffin. Serial sections of the ovarian tissue were prepared using a paraffin microtome, with a section thickness of 8 µm to fully encompass the entire ovarian structure from the cortex to the medulla.

[0072] Hematoxylin and eosin (H&E) staining was performed on the slides according to standard staining procedures. After staining, using a panoramic histiocytometer, follicles were divided into four developmental stages based on their developmental stage: primordial follicle (PmF), primary follicle (PF), secondary follicle (SF), and antral follicle (AF). Morphological observation and classification counting were performed on all follicles with clearly visible nuclei in every fifth slide to avoid duplicate counting. Figure 4 ).

[0073] Histological analysis of ovarian HE staining showed that the number of follicles at all stages in the ovary increased after high-dose pyrophosphate treatment—the number of primordial follicles, primary follicles, secondary follicles and cavitary follicles in the ovaries of mice in the pyrophosphate treatment group was higher than that in the control group.

[0074] The results have the following important biological implications: First, the comprehensive increase in the number of follicles at all stages suggests that pyrophosphate not only acts on a specific stage of follicle development, but benefits the entire developmental chain from the activation of primordial follicles to the maturation of cavitary follicles. Second, the increase in the number of follicles reflects that pyrophosphate improves the follicular microenvironment within the ovary, creating favorable conditions for the selection and maturation of dominant follicles. Compared to most existing drugs or health products that only intervene in a single aspect of oxidative stress, this invention can simultaneously improve multidimensional pathological changes at the ovarian tissue level, demonstrating a more comprehensive intervention effect on the systemic biological process of delaying ovarian physiological aging.

[0075] Example 4: Serum Reproductive Hormone Level Detection - Pyrophosphate Intervention to Restore Reproductive Endocrine Homeostasis in Aged Female Rats Whole blood samples were collected from mice at the time of sacrifice using the retro-orbital venous plexus method. The procedure was as follows: approximately 0.8-1.0 mL of blood was collected from the medial canthal venous plexus using a capillary tube and placed in a 1.5 mL centrifuge tube without anticoagulant. After allowing the whole blood to stand at room temperature for 60 min to promote coagulation, it was centrifuged at 3,000 rpm for 15 min at 4°C. The supernatant serum (approximately 200 μl) was transferred to a new sterile 1.5 mL centrifuge tube and quickly stored at -80°C for later analysis.

[0076] Commercially available enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Jianglai Biotechnology Co., Ltd.: Mouse follicle-stimulating hormone (FSH) ELISA kit, JL10239-96T; Mouse estradiol (E2) ELISA kit, JL11790-96T) were used to detect serum follicle-stimulating hormone (FSH) and estradiol (E2) levels, strictly following the manufacturer's standard operating instructions. All serum samples were tested in duplicate to minimize operational errors. If the coefficient of variation (CV) between duplicates exceeded 15%, the test was repeated. Hormone concentration results were plotted on [Diagram showing hormone concentrations]. Figure 5 .

[0077] Reproductive endocrine disorders are one of the core characteristics of ovarian aging. With increasing age, the ability of ovarian granulosa cells to secrete E2 declines, weakening their negative feedback inhibition on the anterior pituitary gland, leading to a compensatory increase in FSH levels—the classic "FSH increase - E2 decrease" aging endocrine pattern. Experimental data from this invention show that after pyrophosphate intervention, serum FSH levels in aged female mice showed a decreasing trend, while E2 levels showed an increasing trend, both tending to return to the normal range of youthful states. This provides an endocrinological basis for pyrophosphate's ability to improve reproductive function and further supports its development value as an ovarian function protectant.

[0078] Example 5: Collection of MII stage oocytes To assess the direct effect of pyrophosphate on oocyte quality, mice were subjected to superovulation treatment on day 56 after administration.

[0079] Specific methods: Pregnant mare serum gonadotropin (PMSG) (Ningbo Sansheng, PCL0120) 10 IU / animal was injected intraperitoneally. 46-48 hours later, human chorionic gonadotropin (hCG) (Ningbo Sansheng, PCL0121) 10 IU / animal was injected intraperitoneally. 12-14 hours after hCG injection, cumulus-oocyte complexes (COCs) were collected from the ampulla of the fallopian tube. COCs were transferred using an oral pipette to M2 medium (Sigma-Aldrich, M7167) containing 1 mg / mL hyaluronidase for 1 minute of digestion, followed by gentle pipetting to ensure complete removal of cumulus cells. The resulting MII naked oocytes were then transferred to M2 operating drops and washed twice before use in subsequent experiments.

[0080] Example 6: Detection of cortical granule (CG) distribution in MII stage oocytes Cortical granules are membranous organelles rich in glycoproteins found in the cytoplasm of oocytes. Their normal distribution pattern (uniformly distributed in the cortical region) is an important structural basis for oocyte cytoplasmic maturation and the cortical response after fertilization (blocking polyspermy). Senescent oocytes often exhibit abnormal distribution of cortical granules (distributed in an aggregated or diffuse manner).

[0081] Specific methods: MII stage oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 min, then treated with 0.1% Triton X-100 permeabilization membrane for 15 min to increase antibody / lectin permeability. Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, 9048-46-8) for 30 min. Subsequently, cortical granules were specifically labeled with LCA-FITC (Lens culinaris agglutinin conjugated with fluorescein isothiocyanate) (Thermo Fisher Scientific, L32475) and incubated overnight (12-16 hours) at 4°C in the dark. After staining, the cells were washed three times for 5 min each time to remove unbound free fluorescent probes. Oocytes were mounted on slides with an anti-fluorescence quenching agent (containing DAPI dye) (Beyotime, P0131) and imaged using a high-resolution in vivo imaging confocal laser scanning microscope (Zeiss LSM880 FastAriyscan). The acquired fluorescence images were processed using ZEN software (version 2.3, Carl Zeiss AG) and quantitatively analyzed using ImageJ software (National Institutes of Health, USA), with average fluorescence intensity used as a quantitative indicator of cortical granule content. The results are shown below. Figure 6 .

[0082] Example 7: Detection of oxidative stress (ROS) levels in MII stage oocytes To assess the antioxidant capacity of oocytes under exogenous oxidative stress, MII stage oocytes were first placed in M2 medium containing 100 µM hydrogen peroxide (H2O2) and incubated at 37°C in a 5% CO2 incubator for 30 min to induce exogenous oxidative damage. After incubation, the oocytes were washed three times with pre-warmed M2 medium for 5 min each time. Subsequently, the oocytes were placed in M2 medium containing 10 µM DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) (Beyotime, Reactive Oxygen Spectrometry Kit, S0033S) and incubated at 37°C in the dark for 30 min. After incubation, the oocytes were washed three times with pre-warmed M2 medium (5 min each time) to thoroughly remove any residual extracellular probes. Subsequently, green fluorescence signals (excitation wavelength 488 nm) and bright-field images of oocytes were captured using ultra-high resolution in vivo imaging confocal microscopy, with all imaging parameters maintained consistently across groups. Fluorescence signal intensities were processed using ZEN software and analyzed using ImageJ software to reflect the relative ROS levels of oocytes in each group. Results are shown in... Figure 7 , where BF (bright field) represents the bright field image.

[0083] Example 8: Detection of mitochondrial membrane potential (ΔΨm) in MII stage oocytes To assess the functional integrity and energy metabolism status of oocyte mitochondria, this study used the JC-1 cationic fluorescent probe to detect the mitochondrial membrane potential (ΔΨm) of MII oocytes. The working principle of the JC-1 probe is based on potential-dependent aggregation: in normal mitochondria (high membrane potential), JC-1 forms red fluorescent aggregates (J-aggregates, excitation wavelength 594 nm); in the depolarized (low membrane potential) state of mitochondria, JC-1 exists in the cytoplasm as green fluorescent monomers (excitation wavelength 488 nm). Therefore, the red / green fluorescence intensity ratio can directly reflect the mitochondrial membrane potential level.

[0084] Specific procedures: MII stage oocytes were placed in equilibrated M2 medium containing 10 µg / mL JC-1 (Beyotime, C2005) and incubated at 37°C in a 5% CO2 incubator for 30 min in the dark. After incubation, the oocytes were washed three times with M2 medium for 5 min each time to remove free probes. Subsequently, red fluorescence (excitation wavelength 594 nm) and green fluorescence (excitation wavelength 488 nm) signals of the oocytes were captured using ultra-high resolution in vivo imaging confocal microscopy, with all imaging parameters kept consistent across groups. Fluorescence signal intensity was processed using ZEN software, and the red / green fluorescence intensity ratio of each oocyte was calculated using ImageJ software, which served as the quantitative indicator of ΔΨm. The results are shown below. Figure 8 .

[0085] Examples 5 to 8 demonstrate how pyrophosphate intervention improves multidimensional quality indicators of MII-stage oocytes.

[0086] Decreased oocyte quality is a key factor contributing to reduced fertility in older women, directly determining fertilization success rate, embryonic developmental potential, and ultimately, live birth outcome. The multidimensional experimental evidence obtained in this invention at the MII stage oocyte level strongly supports the core conclusion that pyrophosphate improves oocyte quality. First, regarding the distribution of cortical granules, LCA-FITC staining results showed that the cortical granules of oocytes in the MII stage of aged mice in the control group exhibited a significant aggregated and abnormal distribution, while the cortical granule distribution pattern of oocytes in the pyrophosphate-treated group was significantly improved, tending towards the uniform cortical distribution pattern of young oocytes. Simultaneously, quantitative fluorescence analysis showed that the fluorescence intensity in the treated group was significantly higher than that in the control group. This improvement in cortical granule distribution directly reflects the increased cytoplasmic maturity of oocytes, which is the structural basis for post-fertilization cortical response and the inhibition of polyspermia.

[0087] Secondly, regarding oxidative stress, DCFH-DA probe detection showed that under exogenous oxidative stress stimulation with 100 µM H2O2, the ROS fluorescence signal intensity of oocytes in the pyrophosphate-treated group was significantly lower than that in aged oocytes of the control group. This indicates that pyrophosphate treatment enhances the endogenous antioxidant defense capacity of oocytes, making them more tolerant to exogenous oxidative damage. These results suggest that the effect of pyrophosphate is achieved by maintaining redox homeostasis.

[0088] Third, regarding mitochondrial function, JC-1 probe fluorescence ratio analysis showed that the red / green fluorescence intensity ratio of oocytes in the pyrophosphate treatment group was significantly higher than that in the control group, indicating that mitochondrial membrane potential was restored and mitochondrial function tended to normalize. Given that mitochondria are the main site of energy metabolism and oxidative stress in oocytes, maintaining mitochondrial membrane potential is crucial for ATP supply, calcium homeostasis regulation, and cell survival. These results collectively suggest that pyrophosphate can improve oocyte developmental capacity by maintaining normal energy metabolism and mitigating oxidative damage.

[0089] In summary, this invention presents a novel application of pyrophosphate in the preparation of drugs to delay ovarian physiological aging. This technical approach has received systematic positive experimental evidence across multiple dimensions, including whole animal, tissue and organ, body fluid and endocrine, and cellular and molecular levels. This invention not only provides a novel technical approach for the intervention of natural metabolites in age-related decline in female reproductive function, but also breaks through the traditional limitations of pyrophosphate's application in mineral metabolism and calcification regulation, extending it to the entirely new biomedical field of female reproductive aging intervention. It possesses significant innovation, substantial feasibility, and promising prospects for industrial application.

[0090] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0091] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, and should also be regarded as the content disclosed by the present invention.

Claims

1. Application of pyrophosphate in the preparation of drugs to delay ovarian aging.

2. The application according to claim 1, wherein the pyrophosphate is selected from one or more of sodium pyrophosphate, disodium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, magnesium pyrophosphate, dimagnesium dihydrogen pyrophosphate, or their hydrates.

3. The application according to claim 1 or 2, wherein the pyrophosphate is sodium pyrophosphate decahydrate (Na4P2O7•10H2O).

4. The application according to any one of claims 1-3, wherein: 1) The drug is used to improve the fertility of aging female mammals; 2) The drug is used to improve the ovarian histological structure and follicular reserve in aged female mammals; 3) The drug is used to restore reproductive endocrine homeostasis in aging female mammals; and / or 4) The drug is used to improve the quality of MII oocytes in aged female mammals.

5. The application according to claim 4, wherein improving the quality of MII stage oocytes comprises at least one of the following: (1) Improves the distribution of cortical granules; (2) Reduce oxidative stress levels; and (3) Increase mitochondrial membrane potential.

6. The application according to any one of claims 1-5, wherein the route of administration of the drug is selected from intraperitoneal injection, intravenous injection, subcutaneous injection and oral administration.

7. The application according to any one of claims 1-6, wherein the dosage form of the drug is selected from injections, lyophilized powders, sustained-release formulations, liposome formulations, or nanocarrier formulations.

8. The application according to any one of claims 1-7, wherein the medicament further comprises other ovarian protective active ingredients, preferably the other ovarian protective active ingredients are selected from at least one of nicotinamide mononucleotide (NMN), melatonin and resveratrol.

9. A pharmaceutical composition for delaying physiological ovarian aging, comprising: an effective amount of pyrophosphate and a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition according to claim 9, wherein the pyrophosphate is selected from one or more of sodium pyrophosphate, disodium dihydrogen pyrophosphate, potassium pyrophosphate, dipotassium dihydrogen pyrophosphate, potassium dipotassium dihydrogen pyrophosphate, magnesium pyrophosphate, dimagnesium dihydrogen pyrophosphate, or their hydrates, preferably sodium pyrophosphate decahydrate (Na4P2O7•10H2O).

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