Application of carnosine injection in improving fertility of elderly female mammals
Carnosine injections address the decline in fertility in older women by improving the ovarian microenvironment and oocyte quality, significantly increasing blastocyst formation rate and follicle count, repairing mitochondrial structure, and enhancing fertility. It is safe and has broad application potential.
Patent Information
- Application Number
- CN202511168816.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-11-14
AI Technical Summary
The problem of declining fertility in older women, especially the low success rate, high cost, and great psychological stress of assisted reproductive technologies, means that current technologies cannot be widely used by the general population, and research on the specific functions of carnosine in ovarian tissue is still lacking.
Carnosine injection improves the ovarian microenvironment, enhances oocyte quality, and optimizes hormone levels. Carnosine injection contains carnosine and saline at a concentration of 10–50 mg/mL. It is administered via intraperitoneal or subcutaneous injection at a dose of 0.05–0.2 mg/g body weight every 3–7 days. Specifically, the dosing frequency is 0.125 mg/g body weight every 5 days.
It significantly improves the early embryonic development potential of oocytes in older women, improves the blastocyst formation rate, increases the number of follicles at each stage in the ovary, repairs the mitochondrial morphology of oocytes, and enhances the fertility of older individuals. It has high safety, strong applicability, and good application prospects.
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Figure CN120939192A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine and specifically discloses the application of carnosine injection in improving the fertility of older female mammals. Background Technology
[0002] With the accelerated pace of modern life, delayed childbearing age, environmental pollution, and changes in lifestyle, reproductive health issues are becoming increasingly serious. According to the World Health Organization (WHO), approximately 15% of couples of childbearing age worldwide face infertility, while in my country, the proportion of infertility among this population has been rising steadily in recent years, even approaching 20%. This not only affects individual health and quality of life but also places multiple pressures on family and social stability. For women, declining ovarian function, decreased oocyte quality, and reduced endometrial receptivity are all significant causes of decreased fertility, especially among older women. Although assisted reproductive technologies (ART) such as in vitro fertilization (IVF) and in vitro oocyte maturation (IVM) are constantly developing, problems such as limited success rates, technical complexity, high costs, and significant psychological stress remain prevalent, preventing widespread accessibility to the general population. In recent years, research on "improving the reproductive microenvironment in vivo to increase natural pregnancy rates" has gradually gained attention. Especially before assisted reproduction, improving the ovarian microenvironment, enhancing oocyte quality, and optimizing hormone levels through nutritional regulation or functional active substances is considered a safer, more convenient, and widely applicable approach.
[0003] Carnosine, an endogenous dipeptide composed of L-histidine and β-alanine, is widely distributed in various tissues of the human body and exhibits good safety and biocompatibility. Its unique structure endows it with a variety of biological activities, including antioxidant, anti-aging, anti-inflammatory, anti-glycation, and maintenance of intracellular pH homeostasis. Studies have shown that carnosine can significantly reduce the level of reactive oxygen species (ROS) in the body and maintain glutathione levels, thereby improving the cellular microenvironment and protecting mitochondrial function and chromosome stability in oocytes.
[0004] It is worth emphasizing that although carnosine has been extensively studied and applied in various animal models, its specific functions in ovarian tissue, especially in improving the quality and fertility of older oocytes, are still lacking. Summary of the Invention
[0005] To address the aforementioned issues, this invention discloses the application of carnosine injection in improving fertility in older female mammals. Carnosine improves the ovarian microenvironment, enhances oocyte quality, and optimizes hormone levels, thereby increasing fertility.
[0006] This invention includes the following technical solutions:
[0007] The use of carnosine in the preparation of a medicament for improving fertility in older female mammals, wherein the medicament is an injectable preparation.
[0008] Furthermore, in the above application, the injectable contains carnosine and physiological saline, wherein the carnosine concentration is 10–50 mg / mL.
[0009] Furthermore, in the above application, the carnosine concentration is 25 mg / mL.
[0010] Furthermore, in the above applications, the dosage form of the drug is an intraperitoneal injection or a subcutaneous injection, the dosage is 0.05–0.2 mg / g body weight, and the frequency of administration is once every 3–7 days.
[0011] Furthermore, in the above application, the dosage is 0.125 mg / g body weight, and the dosing frequency is once every 5 days.
[0012] Furthermore, in the above application, the improvement of fertility is to enhance the early embryonic development potential of oocytes, including increasing the blastocyst formation rate of fertilized eggs.
[0013] Furthermore, in the above application, the improvement of fertility refers to increasing the number of follicles at various stages in the ovary, including the number of cavitary follicles.
[0014] Furthermore, in the above application, the improvement of fertility is achieved by repairing the mitochondrial morphology of oocytes, including reducing mitochondrial swelling and clarifying cristae structure.
[0015] The present invention also discloses a carnosine injection formulation for improving fertility in older female mammals, comprising a therapeutically effective amount of carnosine and a saline carrier.
[0016] Furthermore, the above-mentioned injectable formulation has a carnosine concentration of 25 mg / mL; the dosage form is intraperitoneal injection or subcutaneous injection; and the single-dose dose is 0.125 mg / g body weight.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] This invention is the first to systematically verify the significant role of carnosine (injection) in promoting the development of oocytes in older women and improving fertility, and it has originality and application value.
[0019] This invention proposes to improve ovarian function, enhance oocyte quality, and increase fertility in older individuals through in vivo carnosine supplementation, which has sound scientific basis and promising application prospects. A series of animal experiments have confirmed that carnosine supplementation can significantly improve the overall reproductive capacity of older mice, including increasing the blastocyst formation rate of fertilized eggs, improving blastocyst structure, increasing the number of follicles at each stage in the ovary, and ultimately increasing litter size, indicating that carnosine has a systemic supportive effect on the reproductive system of older adults.
[0020] In summary, this invention has multiple advantages, including a clear mechanism, simple operation, high safety, and strong applicability. It can provide a novel, scientific, and effective solution for fertility intervention in older women and has good prospects for promotion and industrialization potential. Attached Figure Description
[0021] Figure 1 A schematic diagram of the experimental procedure for supplementing carnosine in mice according to the present invention;
[0022] The figure details the animal experimental design process of this study. Aged mice were randomly divided into two groups: a control group (upper dashed branch) received intraperitoneal injections of PBS (solvent control) every five days, and a carnosine treatment group (lower solid branch) received carnosine (Car) injections every five days. As shown on the bottom timeline (Time(d)), the treatment cycle lasted for 20 days. After treatment, PMSG and HCG were injected into both groups on days 20 and 22, respectively, to induce superovulation. Mature MII oocytes were then collected for subsequent experimental analysis.
[0023] Figure 2 This diagram illustrates the early developmental stages of mouse embryos and the effect of carnosine on the developmental potential of older oocytes: The left figure shows the continuous developmental stages of mouse embryos from the 2-cell stage to the hatched blastocyst stage, while the right figure is a stacked bar chart of quantitative statistics. The vertical axis represents the percentage of embryos at different developmental stages (% of cells); different colors in the figure represent the developmental stage of the embryo or whether degeneration has occurred.
[0024] Figure 3 The left side shows representative immunofluorescence staining images, illustrating the cell lineage differentiation of blastocysts from the aged control group (Aged) and the carnosine-treated group (Aged+Car); OCT4 (red) marks the inner cell mass (ICM), GATA3 (green) marks the trophectoderm (TE), and DAPI (blue) marks all cell nuclei; the right side is a quantitative statistical violin plot of the total cell number of blastocysts, with the vertical axis representing the total cell number of blastocysts.
[0025] Figure 4 The left side shows representative H&E stained sections of ovarian tissue from two groups of mice, with red asterisks (*) marking typical follicular structures. The right side shows a quantitative statistical chart of the number of follicles at different developmental stages, with the vertical axis representing the number of follicles in each ovary and the horizontal axis representing different developmental stages of follicles (primordial follicles, primary follicles, secondary follicles, and antral follicles).
[0026] Figure 5 The left side shows representative photos of the offspring (F1 generation) produced after mating between two groups of older female mice and younger male mice; the right side shows a quantitative statistical chart of the number of pups per litter, with the vertical axis representing the number of pups per litter (F1 pups per litter).
[0027] Figure 6 Representative transmission electron microscopy (TEM) images of oocytes from the Aged and Aged+Car groups are presented. Mitochondria in oocytes from the Aged group showed typical signs of aging, such as swelling, vacuolization, and blurred or absent internal cristae. In contrast, after carnosine treatment (Aged+Car group), the mitochondria regained their normal morphology, appearing as elongated ovals or round shapes with a dense matrix and clear and intact internal cristae.
[0028] Figure 7 Representative H&E stained sections of the heart, liver, and periovarian adipose tissue of mice in the Aged control group and the Aged+Car treatment group are shown. Histopathological observation showed no significant differences or pathological damage in the morphology and structure of cardiomyocytes, hepatocytes, and adipocytes in the two groups of mice.
[0029] Figure 8 The left figure shows the litter size of offspring mice from the Aged and Aged+Car female mice after they reached adulthood (and were mated with normal male mice). The results showed no significant difference between the two groups (ns). The right figure shows the weight gain curves of the offspring mice from day 7 to day 56 after birth, with the vertical axis representing weight (g) and the horizontal axis representing age (d). The results showed no significant difference between the growth and development curves of the offspring mice in the two groups (ns). Detailed Implementation
[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] The experimental scheme of this invention is as follows.
[0032] (1) Supplementing carnosine in the body
[0033] like Figure 1 As shown, 10-month-old mice were selected as aging mouse models. At 9 months of age, the mice were randomly divided into two groups. The mice were injected with carnosine every 5 days at a dose of 0.125 mg / g body weight. On the 5th day after the injection, superovulation was performed, and the oocytes obtained from superovulation were used for in vitro fertilization. The control group mice were injected with the same volume and frequency of PBS solution.
[0034] Carnosine was purchased from MCE, catalog number HY-W013494. The preparation method for carnosine injection is as follows: dissolve 25 mg of carnosine in 1 ml of physiological saline to obtain an injection solution with a concentration of 25 mg / mL.
[0035] (2) Perform IVF experiments on superovulated oocytes.
[0036] Epididymal tail sperm are co-incubated with oocytes after capacitation (4×10 5 / mL, 5 hours), observing the double pronuclei as a marker of fertilization. Fertilized eggs were cultured in G1 medium (37℃, 5% CO2) until the blastocyst stage.
[0037] (3) Follicle count and HE staining of heart and other tissues
[0038] Ovarian and cardiac tissues were collected from each group of mice for histological analysis. The tissues were fixed overnight at 4°C with 4% paraformaldehyde (pH 7.5), dehydrated, and embedded in paraffin. Ovarian sections (3 mm thick) were prepared after paraffin embedding and stained with hematoxylin and eosin (H&E). Follicles were counted from both ovaries of three mice in each group.
[0039] (4) Fertility testing
[0040] Two groups of mice supplemented with carnosine were housed with male mice, and the number of offspring in the first litter was counted.
[0041] (5) Immunostaining of oocytes
[0042] Mouse oocytes were fixed with 4% paraformaldehyde (PFA) for 30 minutes, then blocked in blocking buffer (5% BSA and 0.5% Triton X-100 in PBS) at room temperature for 1 hour. After blocking, the oocytes were incubated with primary antibody overnight at 4°C. After washing three times, the oocytes were labeled with secondary antibody at room temperature for 1 hour. Chromosomes were assessed by staining with Hoechst 33342 for 10 minutes. The oocytes were mounted on slides and examined under a laser scanning confocal microscope (LSM 780, Carl Zeiss, Germany).
[0043] (6) Transmission EM
[0044] Fifty GV-stage oocytes were collected from each group, washed twice with PBSPVA, and then pipetted into a granulosa cell suspension. The cells were centrifuged at 1000 rpm for 5 min, and the precipitate was collected and added to a pre-prepared fixative solution (2.0% EM grade glutaraldehyde and 2.5% formaldehyde). The cells were fixed at room temperature for 30 min, and then at 4°C overnight. Samples were processed at Shandong Weiya Biotechnology Co., Ltd.
[0045] The experimental results are shown in Examples 1-6.
[0046] Example 1
[0047] Supplementing with carnosine can significantly improve the early embryonic developmental potential of oocytes in older mice.
[0048] Advanced maternal age women commonly experience decreased oocyte quality and a lower proportion of high-quality embryos suitable for transfer during assisted reproductive technology (ART), severely limiting pregnancy success rates and becoming a long-standing challenge in the field. To verify the effect of carnosine on oocyte developmental capacity, this study used an in vitro fertilization (IVF) experimental system to monitor early embryonic development in fertilized oocytes from older mice. The results showed that supplementing with carnosine during the oocyte maturation stage significantly increased the blastocyst formation rate, indicating that carnosine enhances the early embryonic developmental potential of oocytes from older women. Figure 2 ).
[0049] Example 2
[0050] Supplementing with carnosine can improve the quality of blastocysts in older mice.
[0051] To further verify the effect of carnosine on the developmental quality of older mouse embryos, this study systematically evaluated the blastocyst status using immunofluorescence staining. In the experiment, DAPI staining was used to label chromosomes, OCT4 antibody was used to label the inner cell mass (ICM), and GATA-3 antibody was used to label trophoblast cells (TE) to comprehensively reflect the blastocyst structure and cell differentiation. The results showed that under carnosine supplementation, the proportion of inner cell mass and trophoblast cells in the blastocyst significantly increased, suggesting that carnosine can effectively improve the structural integrity and potential developmental capacity of the blastocyst, thereby improving the overall blastocyst quality. Figure 3 ).
[0052] Example 3
[0053] Supplementing with carnosine can increase the number of follicles at all stages in the ovaries of older mice.
[0054] The morphology and structure of the ovary are crucial for the normal development of follicles. To assess the effect of carnosine on ovarian development in aged mice, this study used hematoxylin-eosin (H&E) staining to observe the morphology of ovarian tissue sections. Follicles at different developmental stages in the ovaries of aged mice showed severe deterioration. The results showed a significant reduction in the number of follicles at each developmental stage and severe structural degeneration in the ovaries of aged mice. In contrast, follicle development was significantly improved in the carnosine-supplemented group. Quantitative statistical results indicated that carnosine significantly increased the number of follicles at various stages in the ovaries of aged mice, especially antral follicles. In conclusion, carnosine supplementation can effectively improve ovarian function and promote oocyte development in aged mice, providing a new strategy for enhancing female fertility. Figure 4 ).
[0055] Example 4
[0056] Supplementing with carnosine can improve the fertility of older mice.
[0057] As women age, the number and quality of their oocytes gradually decline, leading to lower conception rates and an increased risk of spontaneous abortion, a trend that becomes more pronounced after age 38. To simulate the reproductive state of older humans, this study established an advanced reproductive model using 10-month-old mice and assessed their fertility. The results showed that under carnosine supplementation, the number of offspring in older female mice significantly increased, indicating a significant improvement in their reproductive capacity. These results further validate the potential role of carnosine in promoting oocyte function, improving the ovarian microenvironment, and enhancing overall fertility. Figure 5 ).
[0058] Example 5
[0059] Supplementing with carnosine can improve the morphology and structure of mitochondria in oocytes of older mice.
[0060] To explore the potential role of carnosine at the organelle level, this study performed transmission electron microscopy analysis on the mitochondrial morphology of oocytes from aged mice. The results showed that mitochondria in aged oocytes exhibited typical aging characteristics such as swelling, breakage, and blurred cristae, indicating impaired function. In contrast, the mitochondrial morphology in the carnosine-supplemented group was significantly improved, exhibiting a healthy state with uniform size, even distribution, intact membrane structure, and clear cristae. These results suggest that carnosine can exert a protective effect on mitochondria in oocytes, alleviating age-related subcellular structural damage and thus providing support for oocyte development and energy metabolism. Figure 6 ).
[0061] Example 6
[0062] Supplementing with carnosine has no morphological toxicity to various tissues.
[0063] To systematically evaluate the biosafety of carnosine supplementation, this study performed HE staining histological observation on major tissues and organs, including the heart, liver, and ovarian fat pad, of aged mice. The results showed that under continuous carnosine supplementation, the structures of all tissues remained intact, cell morphology was normal, and no pathological changes such as inflammatory cell infiltration, cell necrosis, fatty degeneration, or fibrosis were observed. The tissue arrangement was regular, and there was no significant difference in morphology compared to the control group. These results indicate that carnosine improves fertility in aged mice without producing visible toxic reactions in major organs, demonstrating good tissue compatibility and biosafety. Figure 7 ).
[0064] Example 7
[0065] Supplementing with carnosine does not affect the growth and development of offspring.
[0066] To further evaluate whether carnosine, while improving fertility in older mice, has any potential impact on offspring development, this study systematically followed up on the first litter (F1 generation) of mice born after carnosine supplementation in older mice. The results showed no significant difference in litter size between the experimental and control groups in the F1 generation, indicating that carnosine supplementation did not adversely affect the reproductive capacity of offspring. Furthermore, we monitored the weight changes of the F1 generation for eight consecutive weeks after birth. The weight gain curves showed that the weight change trends of offspring in the carnosine group and the control group were consistent at each time point, with no statistically significant differences, suggesting that carnosine supplementation does not affect the growth and development of offspring. In conclusion, in vivo carnosine supplementation effectively improves the fertility of older female mice without significantly interfering with the physical development and reproductive capacity of offspring, demonstrating good biosafety and promising prospects for intergenerational application. Figure 8 ).
[0067] Summarize:
[0068] Based on the systematic verification of the above embodiments, carnosine injection demonstrated multidimensional reproductive improvement effects in a 10-month-old advanced mouse model. Experiments confirmed that it not only significantly increased the blastocyst formation rate of fertilized eggs and optimized blastocyst structural integrity (increased ratio of inner cell mass to trophoblast cells), but also promoted the development of follicles at all stages within the ovary, especially significantly increasing the number of cavitary follicles. Fertility testing further showed a significantly increased litter size in the intervention group, confirming enhanced overall reproductive capacity. Mechanistic studies indicated that carnosine ensures cellular energy metabolism by repairing the mitochondrial morphology of oocytes (eliminating swelling and clarifying cristae structure). In safety assessments, continuous administration did not induce pathological damage to tissues such as the heart and liver, and the offspring's growth and development indicators were not different from the control group. In summary, this invention fully verified the effectiveness and biosafety of carnosine injection in improving fertility in older mice through animal models. Its multi-target mechanism of action and convenient administration method provide a solid scientific basis for clinical translation.
[0069] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims
1. The use of carnosine in the preparation of a drug for improving fertility in older female mammals, characterized in that, The drug is an injectable form.
2. The application according to claim 1, characterized in that, The injectable solution contains carnosine and saline, wherein the carnosine concentration is 10–50 mg / mL.
3. The application according to claim 2, characterized in that, The carnosine concentration was 25 mg / mL.
4. The application according to any one of claims 1 to 3, characterized in that, The drug is administered in the form of an intraperitoneal injection or a subcutaneous injection, with a dosage of 0.05–0.2 mg / g body weight and a dosing frequency of once every 3–7 days.
5. The application according to claim 4, characterized in that, The dosage is 0.125 mg / g body weight, and the frequency of administration is once every 5 days.
6. The application according to claim 1, characterized in that, The improvement in fertility refers to enhancing the early embryonic development potential of oocytes, including increasing the blastocyst formation rate of fertilized eggs.
7. The application according to claim 1, characterized in that, The improvement in fertility refers to increasing the number of follicles at all stages in the ovary, including the number of cavitary follicles.
8. The application according to claim 1, characterized in that, The improvement in fertility involves repairing the mitochondrial morphology of oocytes, including reducing mitochondrial swelling and clarifying cristae structure.
9. A carnosine injection formulation for improving fertility in older female mammals, characterized in that, It contains therapeutically effective amounts of carnosine and saline carrier.
10. The carnosine injection formulation according to claim 9, characterized in that, The carnosine concentration is 25 mg / mL; the dosage form is intraperitoneal injection or subcutaneous injection; the single dose is 0.125 mg / g body weight.