Application of beta-alanine in preparation of medicine for treating ovarian endometriosis

Abstract

The invention discloses a novel application of a compound beta-alanine (beta-alanine) in preparation of a medicine for improving oocyte quality and treating ovarian endometriosis (OEM) related infertility, and belongs to the technical field of biological medicines. An OEM animal model proves that beta-alanine can remarkably improve reproductive function disorder of model animals, specifically, follicular dysplasia caused by endometriosis is effectively relieved, the fertility is improved, and the litter size is increased; in the aspect of oocyte quality, the compound can promote normal discharge of a first polar body in an oocyte in-vitro maturation process, reduce cytoplasm fragmentation, recover spindle body form and microtubule arrangement, enhance mitochondrial membrane potential stability and maintain the integrity of an ultrastructure. The results show that beta-alanine can reverse oocyte quality reduction caused by ovarian endometriosis in multiple dimensions, has a definite treatment potential, and provides an important candidate molecule for developing a novel assisted reproduction intervention strategy for targeting oocyte health.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of β-alanine in the preparation of medicaments for treating ovarian endometriosis. Background Technology

[0002] Ovarian endometrioma (OEM) is the most common and reproductively harmful subtype of endometriosis, with a prevalence of up to 44%. It directly affects ovarian tissue, severely damaging follicular reserve and the microenvironment for oocyte development. According to the 2022 European Society for Human Reproduction and Embryology (ESHRE) guidelines, typical OEM lesions can be clinically diagnosed via transvaginal ultrasound or MRI without surgical intervention. Recent studies have shown that OEM lesions create a pathological microenvironment of chronic inflammation, high estrogen levels, and abnormal angiogenesis in the ovary. This microenvironment can penetrate the follicular barrier, interfering with oocyte maturation, leading to follicular arrest, decreased ovulation, and insufficient luteal support. Although assisted reproductive technologies (such as IVF-ET) can mitigate these adverse reproductive outcomes to some extent, oocytes obtained from EMS patients often have defects, resulting in significantly reduced fertilization rates and embryonic developmental potential. Currently, there is a lack of targeted intervention strategies in clinical practice that can effectively improve the follicular microenvironment, repair oocyte organelle function, and enhance its developmental potential. Therefore, developing safe and effective drugs to reverse OEM-related oocyte quality disorders has become a key issue that urgently needs to be addressed in the field of reproductive medicine.

[0003] While existing treatments have made some progress in alleviating symptoms associated with ovarian endometriosis (OEM), their clinical application remains significantly limited. The core issue is the lack of effective interventions that can target and improve the ovarian microenvironment, repair oocyte organelle function, and enhance developmental potential. Specifically: 1) Single treatment target and significant side effects: Commonly used drugs (such as dinogest and GnRH agonists) can inhibit lesion progression but cannot reverse the already damaged oocyte quality; the long-term reproductive safety of aromatase inhibitors and other regimens in women of reproductive age is unclear, making them difficult to use for fertility protection; 2) Insufficient understanding of the pathogenesis: Current therapies fail to deeply intervene in the core pathological aspects of OEM damage to oocyte quality. Although studies have confirmed that lesion microenvironment dysregulation can induce abnormal post-translational modifications of key proteins, thereby impairing mitochondrial function, spindle stability, and developmental potential of oocytes, the specific target proteins mediating this process, as well as the types and regulatory mechanisms of their post-translational modifications, remain poorly understood. 3) Bottlenecks exist in translational application: Existing therapies mostly focus on lesion control and neglect oocyte intrinsic repair; candidate molecules generally lack ovarian / oocyte targeting, have low bioavailability, or lack safety and efficacy verification in the context of OEM, making it difficult to achieve clinical translation.

[0004] In summary, there is an urgent need in this field to develop a novel treatment strategy that combines ovarian targeting, oocyte organelle protection, and good safety profile to safely and effectively improve fertility outcomes in patients with ovarian endometriosis. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a novel use of β-alanine in the preparation of drugs for treating ovarian ovarian malformation (OEM). The scheme disclosed in this invention overcomes the major limitations of existing treatments, such as poor targeting, difficulty in accurately repairing the intrinsic quality of oocytes and enhancing their developmental potential. It provides OEM patients with a novel fertility-preserving treatment strategy that combines ovarian microenvironment regulation, oocyte organelle protection, and good safety.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses the use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving oocyte quality decline caused by ovarian endometriosis (OEM).

[0008] The present invention also discloses the use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving OEM-related infertility.

[0009] Furthermore, the OEM-related infertility is one or more of the following: follicular development disorder, abnormal oocyte maturation, or reduced fertility.

[0010] The present invention also discloses a pharmaceutical composition for improving oocyte quality damage and reproductive dysfunction caused by OEM, characterized in that the pharmaceutical composition comprises β-alanine and a pharmaceutically acceptable carrier.

[0011] Furthermore, the dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form.

[0012] Furthermore, the dosage of the pharmaceutical composition is a pharmaceutically acceptable dosage.

[0013] The present invention also discloses the use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving oocyte organelle function.

[0014] Furthermore, the improvement of organelle function includes: stabilizing mitochondrial membrane potential, repairing spindle-microtubule structure, and / or reducing cytoplasmic fragmentation.

[0015] The present invention also discloses the use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting oocyte first polar body expulsion rate, germinal follicle rupture rate and embryonic developmental potential.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] This invention is the first to discover and confirm that β-alanine, a known compound with good safety profile, can effectively improve oocyte quality and related reproductive function in OEM model mice, providing a novel application direction for its use in the preparation of drugs to treat oocyte quality-related diseases such as OEM.

[0018] Experiments have confirmed that β-alanine treatment can significantly restore the number of ovulations, improve oocyte maturation rate and fertilization capacity, and enhance mitochondrial function and embryonic developmental potential. Combined with existing research on how lactate accumulation in the microenvironment of OEM lesions can drive abnormal lactation of key proteins, thereby impairing mitochondrial function and spindle stability, this suggests that β-alanine may exert its effect on improving oocyte quality through a key mechanism of regulating local lactate metabolism and the resulting abnormal post-translational modifications. This provides important clues for elucidating its pharmacodynamic mechanism.

[0019] In summary, the β-alanine application scheme provided by this invention has achieved simultaneous improvement of core reproductive dysfunctions (abnormal ovulation, poor oocyte quality, and low fertility) caused by OEM in animal models, providing experimental evidence for the development of a novel fertility protection therapy that can precisely target ovarian microenvironment disorders and actively protect oocyte organelle functions and developmental potential. Attached Figure Description

[0020] Figure 1 This study aimed to quantitatively analyze the lactate content, weight, and gonadal index in the ovaries of zebrafish from the control and model groups.

[0021] Figure 2 Representative H&E staining of zebrafish ovaries in the control and model groups (n=5, scale bar, 50µm).

[0022] Figure 3 Gross morphology of the ovaries of mice in the normal control group (top right) and the model group (bottom right). The red dashed lines represent ectopic uterine cysts on the ovaries (scale bar, 1 cm).

[0023] Figure 4 Representative H&E staining of endometriotic ovaries in the model group, with red asterisks indicating ectopic cysts (scale bar, 1 mm).

[0024] Figure 5 Quantitative analysis of body weight, ovarian weight, and gonadal index of zebrafish in the control group, model group, and treatment group.

[0025] Figure 6Representative H&E staining of zebrafish ovaries (n=5, scale bar, 50µm) was performed in the control group, model group, and treatment group, and the follicle status at each stage was recorded, including developing follicles (stage I, gray arrow; stage II, blue arrow), transitional follicles (stage III, purple arrow), and mature follicles (stage IV, green arrow; stage V, red arrow).

[0026] Figure 7 Representative TEM images of zebrafish oocytes from the control group, model group, and treatment group. Red stars indicate abnormal mitochondria (n=5, scale bar, 500nm).

[0027] Figure 8 The zebrafish oocyte count, fertilization rate, and 72-hour hatching rate (n=10) were compared between the control group, the model group, and the treatment group. ** P =0.0016, *P =0.0141, ***P =0.0001, ***P =0.0003, ***P =0.0001, *P =0.0121).

[0028] Figure 9 The in vitro maturation of zebrafish oocytes in the control group, model group, and treatment group is shown. Red stars indicate oocytes that did not undergo germinal vesicle rupture (GVBD) (n=5, scale bar, 1mm).

[0029] Figure 10 Statistical analysis of ovarian follicles in mice of control group, model group, and treatment group (n=5) **P =0.0020, ****P <0.0001, **P =0.0016, ****P <0.0001, ***P =0.0007, ***P =0.0002, ***P =0.0005).

[0030] Figure 11 Representative images (left) of oocytes from the control group, model group, and treatment group mice, along with quantitative analysis of oocyte number, first polar body protrusion rate, and fragmentation rate (n=6). **P =0.0021, *P =0.0273, ****P <0.0001, **P =0.0029, *P =0.0100, scale bar, 100µm).

[0031] Figure 12 Spindle fibers in mouse oocytes of the control group, model group, and treatment group were stained with α-tubulin and quantitatively analyzed accordingly (n=5, scale bar, 10µm).

[0032] Figure 13 Mouse oocytes from the control group, model group, and treatment group were visualized using TMRE staining to show the mitochondrial membrane potential, and corresponding quantitative analysis was performed (n=5, scale bar, 10µm).

[0033] Figure 14 TEM images of mitochondria in ovarian oocytes of mice in the control group, model group, and treatment group (n=5, scale bar, 500nm).

[0034] Figure 15 Number of offspring mice in the control group, model group, and treatment group (n=8, scale bar, 1cm). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0036] Unless otherwise specified, all reagents and materials used in this invention are commercially available. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0038] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0039] Example 1: Establishing a simulation model of the high lactic acid microenvironment in zebrafish.

[0040] 1. Model building: A high-lactic acid microenvironment simulation model for OEM was built using zebrafish.

[0041] Wild-type (WT, AB strain) zebrafish were purchased from the China Zebrafish Resource Center. The zebrafish were kept in a recirculating aquaculture system at a constant temperature of 28.5±0.5℃, with a photoperiod of 14 hours light / 10 hours dark (light hours: 07:00-21:00). Approximately 10% of the water in the system was replaced daily. The zebrafish were fed freshly hatched brine shrimp twice daily.

[0042] Note: Many model organisms can be used to establish in vivo high lactate models through lactate injection, such as mice and rats. Zebrafish, due to their rapid development and large number of eggs, are a classic model for studying reproductive damage. [1-4] Therefore, by constructing a zebrafish high-lactate model, we can directly verify whether elevated lactate levels are sufficient to lead to a decrease in oocyte quality by precisely introducing high lactate as a single variable.

[0043] To simulate the high lactate microenvironment of the ovary, adult zebrafish (>3 months old, body length >3 cm) were administered sodium lactate working solution I (100 µg / g body weight / day, approximately 0.3 g per fish, single dose 30 µg) via microinjection once daily at a fixed time. The control group received the same dose of carrier solution. All treatments were conducted continuously for 14 days under the same controlled environmental conditions.

[0044] 2. Drug processing: Sodium lactate stock solution (liquid, concentration 798 mg / mL) should be stored at 4℃. Before use, accurately pipette 1 µL of sodium lactate stock solution and add it to 78.8 µL of sterile ddH2O to prepare a sodium lactate working solution I with a total volume of 79.8 µL and a concentration of 10 µg / µL. After preparation, the working solution should be aliquoted and stored at 4℃.

[0045] 3. Sample collection and testing.

[0046] For lactate content analysis, zebrafish were anesthetized, and intact ovarian tissue was rapidly separated, immediately washed in pre-cooled PBS, and then quantitatively measured according to the lactate detection kit instructions. For ovarian weight and gonadal index analysis, zebrafish were anesthetized, and their body length and weight were recorded. Intact ovarian tissue was then excised, and its weight was recorded. The gonadal index (GSI) was calculated as (ovarian weight / body weight) × 100%. Further ovarian morphological analysis was performed by rapidly collecting ovarian tissue from each group and immediately rinsing with pre-cooled PBS to remove residual blood. The tissue samples were then fixed with 4% paraformaldehyde at 4°C for 48 hours, followed by graded ethanol dehydration, xylene clearing, and paraffin embedding. The embedded tissue sections were 4µm thick, dewaxed, hydrated, and then stained with hematoxylin and eosin (H&E). After staining, the sections were sequentially dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. All staining steps were strictly followed according to the reagent manufacturer's instructions. The overall structure of the ovary and the morphological stages of follicle development were evaluated using an optical microscope.

[0047] Experimental results: Sodium lactate treatment significantly increased the relative lactate content in ovarian tissue, confirming the successful establishment of a high-lactate microenvironment model; and compared with the control group, sodium lactate treatment did not significantly change the ovarian weight or gonadal index of zebrafish (see attached). Figure 1Ovarian histological analysis showed that sodium lactate treatment significantly increased the proportion of developing follicles in wild-type zebrafish, while decreasing the proportion of mature follicles (see appendix). Figure 2 ).

[0048] Example 2: Construction of C57BL / 6 mouse OEM model.

[0049] 1. Model construction: An OEM model was constructed using 7-week-old C57BL / 6 mice.

[0050] Seven-week-old female C57BL / 6 mice were selected and housed under specific pathogen-free conditions (12h light / 12h dark cycle, temperature 22±1℃, humidity 60%), with free access to food and water.

[0051] Seven-week-old female donor mice were administered estrogen solution (200 µg / kg / day, 100 µL / time) via intraperitoneal injection once daily at a fixed time for seven consecutive days. Euthanasia was performed 24 hours after the last administration. Uterine tissue was aseptically removed and placed in pre-cooled PBS buffer. After carefully removing surrounding fibrous and fatty tissue, the uterine cavity was longitudinally dissected along the long axis of the uterus, and the endometrial tissue was cut into approximately 1 mm pieces using ophthalmic scissors. 3 The tissue fragments were then transferred to centrifuge tubes containing collagenase digestion solution and digested in a 37°C water bath with shaking for 30 min. After centrifugation at 800×g for 5 min, the precipitate was collected, the supernatant was discarded, and the cell clumps were resuspended in 100µL PBS. The cells were then centrifuged and washed again under the same conditions. The resulting uterine tissue precipitate was immediately used for transplantation.

[0052] Recipient mice, also 7 weeks old, underwent uterine tissue precipitation transplantation after 1 week of environmental acclimatization: Anesthesia was administered via inhalation of isoflurane (induction concentration 2-3%, maintenance concentration 1.5-2%). 5-7 mm skin and muscle incisions were made on both sides of the back to expose the ovaries. 20 µL of PBS was injected into the ovarian bursa space using a 29G insulin syringe to expand and clean the bursa cavity. The bursa wall was then gently opened with a suture needle, and the ovary was freed from the bursa cavity using microscissors and forceps. Half of the endometrial tissue precipitation prepared from one donor mouse was evenly spread on the surface of both ovaries, and the ovary, along with the attached tissue, was returned to the abdominal cavity (one portion of donor precipitation could be transplanted into two recipient mice). Finally, the muscle and skin incisions were sutured layer by layer with 6-0 silk sutures.

[0053] Mice in the sham-operated group also received the same anesthesia, a dorsal incision, and PBS injection into the ovarian bursa, but no tissue was implanted.

[0054] All mice were placed in a standard feeding environment (12h / 12h light / dark cycle) with free access to food and water after surgery and were observed for one week for recovery. Subsequent experimental analysis was performed on day 28 post-surgery.

[0055] 2. Sample collection and testing.

[0056] After modeling was completed according to the established protocol, mice were euthanized, and ovarian tissue was rapidly collected from each group. The tissue was immediately rinsed with pre-cooled PBS to remove residual blood. The ovaries were then grossly observed and photographed. Tissue samples were fixed with 4% paraformaldehyde at 4°C for 48 hours, followed by graded ethanol dehydration, xylene clearing, and paraffin embedding. Embedded tissue sections were 5µm thick, dewaxed, hydrated, and then stained with hematoxylin and eosin (H&E). After staining, sections were sequentially dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. All staining procedures were strictly followed according to the reagent manufacturer's instructions. The overall ovarian structure and ectopic endometrial lesions were morphologically evaluated using an optical microscope. Approximately 40 to 60 serial sections were examined from each mouse to ensure comprehensive coverage.

[0057] Experimental results: Compared with the control group ovaries, the ovaries of the model group mice showed obvious cystic lesions on the surface, which were grayish-white and translucent, and significantly larger in size (see attached). Figure 3 Ovarian tissue morphological analysis showed that the ovarian structure of OEM group mice was significantly disordered, with the cortical area infiltrated by dilated endometrial-like glands and stromal tissue, and the lesions were closely connected to the ovarian parenchyma (see appendix). Figure 4 ).

[0058] Example 3: Effects of β-alanine on oocyte quality and reproductive function in zebrafish under high lactic acid conditions.

[0059] 1. Model building: The same model as in Example 1 is used to build an OEM high lactic acid microenvironment simulation zebrafish model.

[0060] 2. Drug treatment: Preparation of β-alanine stock solution and working solution: Accurately weigh 1g of β-alanine raw material and dissolve it in 40mL of sterile ddH2O. Vortex until completely dissolved to obtain a clear stock solution with a concentration of 280.6mM. This concentration strictly follows the solubility parameter of the compound in ddH2O (471g / L) specified in the product instructions to ensure complete dissolution without exceeding the solubility limit. After preparation, dispense the stock solution and store it at -20℃ to avoid repeated freeze-thaw cycles. Before use, take 10µL of the stock solution and add 15µL of sterile ddH2O to make up to 25µL to prepare a β-alanine working solution with a concentration of 10µg / µL (160µg / kg / d, approximately 0.3g per fish, single dose 50µg).

[0061] Preparation of sodium lactate and β-alanine mixed solution: Accurately pipette 1µL of sodium lactate stock solution (798mg / mL), 52.8µL of β-alanine stock solution (280.6mM) and 79.2µL of sterile ddH2O, mix them and make up to 133µL to obtain a mixed working solution containing 6µg / µL sodium lactate and 10µg / µL β-alanine.

[0062] Adult wild-type zebrafish (AB strain, >3 months old, body length >3cm, average weight approximately 0.3g) were used in the experiment, and the rearing conditions were the same as in Example 1. The drug was administered once daily via microinjection at a fixed time, with a total injection volume of 5µL per fish. The control group received the same dose of carrier solution. All treatments were conducted continuously for 14 days under the same controlled environmental conditions.

[0063] 3. Sample collection and testing.

[0064] For the analysis of ovarian weight and gonadal index, the method described in Example 1 was followed; For the morphological analysis of ovarian tissue, the method described in Example 1 was followed. For electron microscopy analysis of the ovarian ultrastructure, zebrafish ovarian tissue was rapidly dissected after euthanasia and immediately immersed in pre-cooled 2.5% glutaraldehyde fixative for 24 h at 4°C. After initial fixation, the samples were washed three times with 0.1 M phosphate buffer (PB, pH=7.4) at 4°C for 10 min each time. Subsequently, the tissue was post-fixed with 1% osmium tetroxide (OsO4) at 4°C for 2 h. After fixation, the samples were dehydrated with a gradient of ethanol (30%, 50%, 70%, 90%, 100%), followed by epoxy resin infiltration and embedding. 60 nm ultrathin sections were prepared using an ultramicrotome (Leica EM UC7) and placed on a copper mesh support. The sections were stained with uranium acetate for 20 min, followed by lead citrate staining for 5 min. Transmission electron microscopy (TEM) observation was performed under a JEM-1400 Flash microscope with an accelerating voltage of 80 kV. Ultrastructural parameters such as mitochondrial morphology and cortical granule distribution were quantitatively analyzed using ImageJ software in random fields of view of multiple independent samples.

[0065] For in vitro oocyte maturation experiments, sexually mature female zebrafish were euthanized, and their ovaries were quickly removed and preserved on ice in 60% Leibovitz's L-15 medium. Under a stereomicroscope, oocytes with a diameter of 550-650 µm, intact theca membranes, and in full growth phase were manually dissected.

[0066] Sexually mature female zebrafish (4-6 months post-fertilization) were paired overnight in a 1:1 ratio with wild-type (WT) males of confirmed reproductive capacity in a partitioned spawning tank. Spawning was initiated the following morning at 07:00 by removing the partitions. Fertilized eggs were collected 2 hours post-spawning, and the total number of eggs laid by each female was recorded. Degenerate and unfertilized eggs were removed based on morphological criteria (e.g., coagulation, abnormal cleavage patterns). Fertilized eggs were incubated at 28.5°C, and fertilization rate (24 hpf), hatching rate (48 hpf), and juvenile survival rate (72 hpf) were systematically evaluated. After the experiment, the females were euthanized after being anesthetized with MS-222, dried, and their standard body length and weight were measured.

[0067] As previously mentioned, 30 oocytes were randomly selected from each group and seeded into 24-well plates (1 mL of culture medium per well). Oocytes were induced to mature in a medium containing 5 nM 17α,20β-dihydroxyprogesterone (DHP dissolved in 0.1% ethanol, v / v). The culture plates were incubated at 28.5℃ for 5 h, and observed under a microscope every 30 min to record germinal follicle rupture (GVBD). The GVBD rate was calculated using the following formula: GVBD (%) = (Number of oocytes with GVBD / Total number of oocytes) × 100%.

[0068] Experimental results: β-alanine treatment had no significant effect on zebrafish body weight, ovarian mass, or gonadal index (see appendix). Figure 5 Further histological analysis showed that β-alanine could partially alleviate abnormal follicle development caused by a high lactic acid environment (see appendix). Figure 6 ) and decreased oocyte quality (see appendix) Figure 7 The results showed that the proportion of developing follicles tended to be normal and the number of mature follicles increased; in terms of fertility assessment, β-alanine treatment significantly improved the natural mating pregnancy rate and average litter size in lactic acid-exposed zebrafish (see appendix). Figure 8 In in vitro functional experiments, the germinal vesicle rupture (GVBD) rate of its oocytes was also significantly higher than that of the model group (see attached figure). Figure 9 ).

[0069] Example 4: Verifying the intervention effect of β-alanine on reproductive phenotype and fertility in OEM mice 1. Model construction: The C57BL / 6 mouse OEM model was constructed in the same way as in Example 2.

[0070] 2. Drug treatment: Preparation of drinking water: Accurately weigh 6g of β-alanine, dissolve it in sterile drinking water, and bring the volume to 500mL to prepare a 1.2% (w / v) solution (i.e., 12g / L). This solution should be freshly prepared once a week and transferred to the animal's drinking water bottle.

[0071] The entire intervention period lasted 28 days, after which relevant indicators were tested and analyzed.

[0072] 3. Sample collection and testing.

[0073] For morphological analysis of mouse ovarian tissue, follicles at different developmental stages—including primordial follicles, primary follicles, secondary follicles, antral follicles, and tertiary follicles—were classified and quantified to provide a comprehensive assessment of ovarian development and function.

[0074] For electron microscopy analysis of the ultrastructure of the ovary, the operation method is the same as in Example 3.

[0075] To induce superovulation, female mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) 48 hours later. Fourteen hours after hCG injection, the fallopian tubes were dissected to collect cumulus-oocyte complexes (COCs). COCs were incubated in M16 medium containing 0.5 mg / mL hyaluronidase. After removing the cumulus cells, the naked oocytes were counted and collected for subsequent experiments.

[0076] To assess mitochondrial membrane potential status, MII stage oocytes obtained through superovulation were stained with tetramethylrhodamine ethyl ester (TMRE). The specific procedure was as follows: Oocytes after removing cumulus cells were transferred to M16 medium containing 200 nM TMRE and incubated at 37°C in a 5% CO2 incubator for 30 min in the dark. After staining, the oocytes were gently washed three times with preheated M16 medium to remove free dye. The oocytes were then placed in a glass-bottomed imaging dish, covered with a small amount of M16, and mitochondrial morphology was observed using a confocal microscope. Mitochondrial membrane potential levels were quantitatively analyzed using ImageJ software.

[0077] To visualize microtubules, metaphase II (MII) oocytes were treated sequentially as follows: fixed with 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100 PBS for 40 min (room temperature), and blocked in 3% BSA PBS for 30 min. Immunostaining was then performed using anti-α-tubulin primary antibody (1:50 dilution, incubated overnight at 4°C), followed by thorough washing (0.1% PBST) and nucleus counterstaining with DAPI. The treated oocytes were placed in glass-bottomed imaging dishes, covered with PBS, for confocal microscopy observation of spindle morphology.

[0078] To assess the fertility of mice, treated female mice were housed together with healthy male mice at a female:male ratio of 2:1. Mating was confirmed successful the following morning by examining vaginal plugs. On the day of delivery, the number of newborn pups in each litter was recorded to compare pregnancy outcomes and differences in litter size among the groups.

[0079] Experimental results: β-alanine treatment significantly alleviated follicular development disorders caused by endometriosis (see appendix). Figure 10 Superovulation results showed that β-alanine treatment increased the number of ovulations, improved the first-polar ovulation rate, and reduced the proportion of oocyte fragmentation (see appendix). Figure 11 ); staining and morphological observation of oocytes revealed that alanine improved the spindle structure of oocytes (see appendix). Figure 12 ), mitochondrial membrane potential and mitochondrial structural integrity (attached) Figure 13 Fertility experiments further confirmed that alanine treatment significantly increased the number of offspring in endometriosis mice (see appendix). Figure 14 ).

[0080] In summary, this invention, for the first time, expands the application of the known compound β-alanine to the field of reproductive medicine, specifically providing its novel use in the preparation of drugs for improving oocyte quality and treating OEM-related reproductive dysfunction. Through rigorous animal model experiments, this invention demonstrates that β-alanine not only effectively alleviates abnormal follicular development caused by endometriosis, significantly improving fertility and increasing litter size; more importantly, this compound can repair the intrinsic quality of oocytes in multiple dimensions—including promoting normal expulsion of the first polar body, reducing cytoplasmic fragmentation, restoring spindle morphology and microtubule arrangement, and enhancing mitochondrial membrane potential stability and maintaining its ultrastructural integrity. This systematic experimental evidence collectively indicates that β-alanine provides an important candidate molecule with clear clinical translational potential for developing novel assisted reproductive intervention strategies targeting oocyte quality, demonstrating broad therapeutic prospects and application value.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0082] References 1.Bohaud C, Cruz J, Terraza C, Barthelaix A, Laplace-Builhe B,Jorgensen C, et al. Lactate metabolic coordinates macrophage response and regeneration in zebrafish. Theranostics 2022, 12(8): 3995-4009. 2.Tang C, Zhang Y, Zhang F, Sun Y, Zhu Y, Xue F, et al. Evaluatingthe Effects of Neburon Exposure onOvarian Folliculogenesis Using Zebrafishand Mouse Granulosa Cell Line. Environ Health Perspect 2025. 3.Zhou J, Liu T, Guo H, Cui H, Li P, Feng D, et al. Lactatepotentiates angiogenesis and neurogenesis in experimental intracerebralhemorrhage. Exp Mol Med 2018, 50(7): 1-12. 4.Zong Z, Xie F, Wang S, Wu X, Zhang Z, Yang B, et al. Alanyl-tRNAsynthetase,AARS1, is a lactate sensor and lactyltransferase that lactylatesp53 and contributes to tumorigenesis. Cell 2024, 187(10): 2375-2392 e2333.

Claims

1. The use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving oocyte quality decline caused by ovarian endometriosis (OEM).

2. The use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving OEM-related infertility.

3. The application according to claim 2, characterized in that, The OEM-related infertility is one or more of the following: follicle development disorder, abnormal oocyte maturation, or reduced fertility.

4. A pharmaceutical composition for improving oocyte quality damage and reproductive dysfunction caused by OEM, characterized in that, The pharmaceutical composition contains β-alanine and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is pharmaceutically acceptable.

6. The pharmaceutical composition according to claim 4, characterized in that, The dosage of the pharmaceutical composition is a pharmaceutically acceptable dosage.

7. Use of a β-alanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving oocyte organelle function.

8. The application according to claim 7, characterized in that, The improvements in organelle function include: stabilizing mitochondrial membrane potential, repairing spindle-microtubule structure, and / or reducing cytoplasmic fragmentation.

9. The use of β-alanine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for promoting oocyte first polar body expulsion rate, germinal follicle rupture rate and embryonic developmental potential.

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