Traditional Chinese medicine composition and application thereof in preparation of medicine for improving and / or treating male infertility

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition and application thereof in preparation of medicines for improving and / or treating male infertility. The traditional Chinese medicine composition is prepared from fructus broussonetiae, flatstem milkvetch seeds, roasted herba epimedii, prepared rehmannia roots, raspberries, salted semen allii tuberosi, wine-processed rhizoma polygonati, mulberries, fructus lycii, semen cuscutae, wine-processed fructus schisandrae chinensis, semen plantaginis, fructus rosae laevigatae and honey- Multiple medicinal materials are supplemented on the basis of a traditional prescription, a comprehensive conditioning scheme considering multiple viscera and multiple disease causes and a multi-dimensional dialectical treatment strategy are formed, the treatment purpose of treating both symptoms and root causes is facilitated, multi-cause pathological changes of oligoasthenospermia of male infertility can be more effectively coped with, and the overall treatment effect is improved; and through cooperative application with an assisted reproduction technology, the effect is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition and its application in preparing a medicine for improving and / or treating male infertility. Background Art

[0002] Oligospermia and asthenozoospermia are the most common causes of male infertility. The pathological mechanisms of oligospermia and asthenozoospermia are complex, with existing research primarily focusing on oxidative stress, immune responses, apoptosis, signal transduction, and gene and protein expression. Current treatments include medications (such as antibiotics, hormones, and antioxidants), surgical interventions (for problems like vas deferens obstruction and severe varicocele), and assisted reproductive technologies (ART), including in vitro fertilization with embryo transfer (IVF) and intracytoplasmic sperm injection (ICSI). While medications can improve semen quality, their effectiveness in treating the various etiologies of oligospermia and asthenozoospermia remains limited. Clinical observations have shown that oligospermia and asthenozoospermia often involve multiple internal organs, with kidney deficiency being the most common syndrome. Traditional Chinese Medicine (TCM), with its unique holistic approach and syndrome differentiation and treatment, offers a viable approach to treating oligospermia and asthenozoospermia.

[0003] Wuzi Yanzong Wan, a classic traditional Chinese medicine formula for the treatment of oligoasthenospermia, first appeared in the Tang Dynasty book Xuanjielu and is hailed as the "first prescription of ancient and modern times." This formula has been widely used clinically for the treatment of male infertility, demonstrating excellent efficacy and safety, with no reported side effects to date. Its fundamental therapeutic principle is to tonify the kidneys, focusing on warming and nourishing kidney yang and kidney essence. However, the pathogenesis of oligoasthenospermia is not simply limited to kidney deficiency but involves a complex manifestation of dysfunction across multiple organs, including imbalances in qi, blood, yin, and yang. Consequently, Wuzi Yanzong Wan's therapeutic efficacy remains limited, failing to comprehensively regulate overall function and achieving its desired overall therapeutic effect. Furthermore, while some modern pharmacological studies have tentatively suggested that Wuzi Yanzong Wan may improve spermatogenesis and semen quality by regulating the hypothalamic-pituitary-gonadal (HPG) axis, its specific molecular mechanisms of action in oligoasthenospermia remain unclear.

[0004] In recent years, with the development and application of ART technology, traditional Chinese medicine has gradually penetrated into all aspects of ART. However, the application time of traditional Chinese medicine in in vitro fertilization and embryo transfer (IVF-ET) is short. In particular, the ambiguity of the mechanism of action of Wuzi Yanzong Pills has also restricted its synergistic application in assisted reproductive technology to a certain extent. At present, it still lacks high-quality clinical research support.

[0005] Based on this, this application explores the clinical efficacy of a new Chinese medicine composition in treating oligospermia and asthenospermia patients based on the existing Wuzi Yanzong Pills, and explores its mechanism of action. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention proposes a Chinese medicine composition and its use in the preparation of a drug for improving and / or treating male infertility, thereby solving the problems existing in the prior art.

[0007] In order to achieve the above-mentioned object of the invention, one of the technical solutions adopted by the present invention is: Provided is a traditional Chinese medicine composition, which is prepared from the following raw materials in parts by weight: 10-20 parts of Cuscuta australis fruits, 10-20 parts of Astragalus complanatus seeds, 10-20 parts of roasted Epimedium, 10-20 parts of Rehmannia root, 10-20 parts of Rubus idaeus, 5-15 parts of Allium tuberosum seeds, 10-30 parts of Polygonatum sibiricum wine, 10-20 parts of mulberries, 10-30 parts of Lycium barbarum fruits, 10-30 parts of Cuscuta australis seeds, 10-20 parts of processed Schisandra chinensis, 10-20 parts of Plantago seeds, 10-20 parts of Rosa laevigata fruits, and 5-10 parts of roasted Licorice root.

[0008] Furthermore, the Chinese medicine composition is made from the following raw materials in parts by weight: 15 parts of Cuscuta australis fruits, 15 parts of Astragalus complanatus fruits, 12 parts of roasted Epimedium, 15 parts of Rehmannia root, 15 parts of Rubus idaeus, 9 parts of salted leek seeds, 18 parts of Polygonatum odoratum, 15 parts of mulberries, 30 parts of Lycium barbarum fruits, 30 parts of Cuscuta australis seeds, 12 parts of Schisandra chinensis wine, 12 parts of Plantago seeds, 15 parts of Rosa laevigata fruit, and 6 parts of roasted Licorice root.

[0009] The second technical solution adopted by the present invention is: Provided is the use of the traditional Chinese medicine composition in preparing a drug for improving and / or treating male infertility.

[0010] Furthermore, the male infertility is oligospermia and asthenospermia; the dosage form of the Chinese medicine composition is decoction, powder, capsule, tablet, granule or pill, preferably decoction.

[0011] Furthermore, the Chinese medicine composition is used in the preparation of a drug for treating male oligoasthenospermia by directly targeting Wee1, PLK1, CCNA1 and CCNA2 to regulate the cell cycle pathway in the testis.

[0012] Furthermore, the Chinese medicine composition is used in the preparation of medicines for use in conjunction with assisted reproductive technology.

[0013] Furthermore, the Chinese medicine composition is used in the preparation of medicines for male infertility patients who are treated with assisted reproductive technology.

[0014] Furthermore, the assisted reproductive technology is intracytoplasmic sperm injection or in vitro fertilization combined with embryo transfer technology, and the Chinese medicine composition improves the sperm concentration and motility of patients with oligospermia and asthenospermia, thereby increasing the high-quality embryo rate of patients treated with intracytoplasmic sperm injection (ICSI).

[0015] The above solution of the present invention can bring the following beneficial effects: 1. The Chinese medicine composition of the present invention is a modified Wuzi Yanzong Pill. It is based on the traditional prescription and supplemented with various medicinal materials such as Rehmannia root, Astragalus seed, and Polygonatum odoratum. It not only retains the basic efficacy of the original prescription of tonifying the kidney and replenishing essence, consolidating essence and reducing urination, but also further increases the effects of soothing the liver and relieving depression, replenishing qi and nourishing blood, and strengthening the spleen and blood, forming a comprehensive conditioning program that takes into account multiple internal organs and multiple causes of disease. Its multi-dimensional syndrome differentiation and treatment strategy can holistically regulate the patient's internal organs, tonifying kidney essence, and simultaneously taking into account the spleen and stomach transportation and transformation, liver qi and blood replenishment, achieving the purpose of treating both the symptoms and the root causes, more effectively addressing the multi-factor pathological changes of oligoasthenospermia, and improving the overall treatment effect.

[0016] 2. The modified Wuzi Yanzong Pills of the present invention, starting from a holistic perspective, take into account the dual effects of tonifying the kidney and soothing the liver, nourishing yin and replenishing qi through the combination of drugs such as salt leek seeds, cooked rehmannia root, and polygonatum. Among them, salt leek seeds have the effects of warming and tonifying kidney yang, warming the waist and knees, and promoting qi and dispersing stagnation; cooked rehmannia root is good at nourishing yin and blood, and replenishing kidney essence; polygonatum has the characteristics of tonifying the spleen and kidney, nourishing yin and moistening the lungs, and together they play the effects of tonifying the kidney and replenishing essence, soothing the liver and regulating qi, and nourishing the liver and kidney. This liver-kidney coordination mode of combination makes the prescription more in line with the TCM theory of treating the liver and kidney together, and can effectively alleviate the negative effects of liver depression and qi stagnation or liver blood deficiency on spermatogenesis, achieve liver-kidney tonification and qi and blood coordination, and embody the advantages of TCM's "holistic concept" and "dialectical treatment" in treating male infertility. It has demonstrated a wider adaptability and obvious efficacy advantages in clinical practice. In addition, the modified Wuzi Yanzong Pills, with its treatment approach based on regulating spleen and stomach function, has broken through the shortcomings of simple kidney-tonifying prescriptions and can better meet the needs of patients with different physical conditions in clinical practice.

[0017] 3. The modified Wuzi Yanzong Pills of the present invention adhere to the principle of mutual assistance and mutual generation of yin and yang in terms of drug compatibility. Through the mutual cooperation of different Chinese medicines, it achieves the effect of harmonizing yin and yang, and the balance of water and fire. Among them, Rehmannia root, mulberry, and Polygonatum odoratum are mainly used to nourish yin, which can effectively nourish kidney essence and moisten kidney yin; the drugs such as salt leek seeds and Cuscuta seeds in the prescription can warm and nourish kidney yang, warm and nourish kidney qi, thereby achieving the coordination between nourishing yin and tonifying yang, embodying the treatment method of "seeking yang in yin and seeking yin in yang", and achieving the therapeutic purpose of complementary yin and yang, and the balance of water and fire. The Chinese medicines such as salt leek seeds and Rehmannia root in the prescription theoretically belong to "water", Cuscuta seeds and Rubus idaeus belong to "wood", and Polygonatum odoratum takes into account "earth". The combination of medicinal materials achieves mutual generation and harmonization of internal organs, not only nourishing kidney essence, but also regulating the functions of internal organs and meridians as a whole, and promoting the smooth generation and operation of essence and qi.

[0018] 4. The treatment of oligoasthenospermia requires gradual nourishment rather than short-term excessive nourishment. Compared with traditional Wuzi Yanzong Pills, the modified Wuzi Yanzong Pills of the present invention emphasize gradual and gentle nourishment. The Chinese medicinal herbs such as Rosa rugosa fruit pulp and Cuscuta australis have mild and warming properties, which can not only slowly nourish the kidney essence, but also avoid the drawbacks of qi stagnation or phlegm and dampness stagnation caused by excessive or simple nourishment, thereby achieving the therapeutic effect of "tonifying without stagnation" and "slow and steady is best". Through this mild and nourishing treatment method, the modified Wuzi Yanzong Pills can ensure stable and lasting treatment effects, and achieve long-term improvement in semen quality and reproductive function in patients with oligoasthenospermia.

[0019] In summary, the modified Wuzi Yanzong Pills of the present invention not only focus on short-term improvements in semen quality or quantity, but also prioritize the overall coordination of organ functions throughout the body, ensuring the integrity and effectiveness of the therapeutic effect. This treatment approach is more consistent with modern medicine's understanding of the multifactorial and multi-pathological mechanisms underlying oligoasthenospermia, further demonstrating its scientificity, feasibility, and clinical practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are pictures of the testicles of five groups of mice in the animal experiment of the present invention; Figure 2 Figure 2 is the weight graph of the five groups of mice; Figure 3 The graph shows the testis weights of five groups of mice; **** p <0.0001; Figure 4 HE staining images of testicles of five groups of mice; Figure 5 Microscope images of five groups of mouse sperm; Figure 6 Comparison of sperm concentration and sperm motility among five groups of mice;** p<0.01 , *** p<0.001 ,**** p<0.0001 ; Figure 7 Figure 2 is the expression of FSH and LH in the blood of five groups of mice; **p<0.01, ***p<0.001, ****p<0.0001 ; Figure 8 The results of blood-testis barrier marker mRNA expression in five groups of mice are shown; **p<0.01, ***p<0.001, ****p< 0.0001 ; Figure 9 The mRNA expression diagram of inflammatory markers in five groups of mice; ** p <0.01, *** p <0.001, **** p <0.0001; Figure 10 The expression graphs of germ cell markers mRNA in five groups of mice are shown in Figure 2. p <0.05, **p <0.01, *** p <0.001,**** p <0.0001; Figure 11 The effect of the modified Wuzi Yanzong Pill of the present invention on the cell cycle pathway of oligoasthenospermia mice; p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; Figure 12 This is a flow chart of the clinical study of the present invention.

[0021] in, Figure 1 A in the middle shows the testicles of mice in the control group and oligospermia group, and B shows the testicles of mice in the L-carnitine group, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group. Figure 4 A, B, C, D, and E are the HE staining results of the testicles of mice in the control group, oligospermia group, L-carnitine group, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group, respectively; Figure 5 A, B, C, D, and E in the middle are microscope images of sperm in the testicles of mice in the control group, oligospermia group, L-carnitine group, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group, respectively; Figure 6 Figure A shows the sperm concentration statistics of the five groups of mice, and Figure B shows the total sperm motility statistics of the five groups of mice; Figure 7 Figure A shows the serum follicle-stimulating hormone (FSH) levels of the five groups of mice, and Figure B shows the serum luteinizing hormone (LH) levels of the five groups of mice; Figure 8 A, B, and C are the mRNA expression levels of tight junction protein genes CLDN3, CLDN5, and Cldn11 in the testicular tissues of the five groups of mice, respectively; Figure 10 A shows the mRNA expression level of the key gene SOX9 in the testicular tissues of the five groups of mice, and B shows the mRNA expression level of Wnt4; Figure 11 A, B, C, and D are the mRNA expression levels of genes Wee1, CCNA1, Plk1, and CCNA2 in the testicular tissues of five groups of mice, respectively. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the overall concept of the present invention, the following is a detailed description in conjunction with the accompanying drawings. The embodiments described below are only preferred embodiments of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the embodiments based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0023] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0024] Example 1 This embodiment is a decoction of the Chinese medicine composition of the present invention, and the Chinese medicine composition is named Jiawei Wuzi Yanzong Pills.

[0025] The prescription is: Cuscuta australis 15g, Astragalus complanatus 15g, roasted Epimedium 12g, Rehmannia root 15g, Rubus idaeus 15g, Salted Allium leek seed 9g, Wine-soaked Polygonatum 18g, Mulberry 15g, Lycium barbarum 30g, Cuscuta australis 30g, Wine-soaked Schisandra 12g, Plantago seed 12g, Rosa laevigata fruit 15g, and roasted Licorice root 6g.

[0026] The preparation method is as follows: Decoction Instructions: Soak all the herbs in water in a clay pot for 1-2 hours. For the first decoction, add water until the water level is 2-3 cm above the herbs. For the second decoction, add water until the water level is 1-2 cm above the herbs. Boil all the herbs together over high heat, then reduce heat and simmer for 40-60 minutes to create a decoction.

[0027] Dosage and administration: decoct in water 400mL, once a day, divided into morning and evening.

[0028] Example 2 This embodiment is a decoction of the Chinese medicine composition of the present invention.

[0029] The prescription is: Custard Fruit 10g, Astragalus Seed 10g, Roasted Epimedium 12g, Rehmannia Glutinosa 10g, Rubus 10g, Salted Allium Seed 9g, Wine-soaked Polygonatum 18g, Mulberry 15g, Lycium Barbarum 30g, Cuscuta Seed 30g, Wine-soaked Schisandra 12g, Plantago Seed 12g, Rosa Laevigata Fruit 15g, and Roasted Licorice 6g.

[0030] For the preparation method, usage and dosage, please refer to the preparation and usage in Example 1.

[0031] Example 3 This embodiment is a decoction of the Chinese medicine composition of the present invention.

[0032] The prescription is: Custard Fruit 20g, Astragalus Seed 20g, Roasted Epimedium 20g, Rehmannia Glutinosa 20g, Rubus 20g, Salted Allium Seed 9g, Wine-soaked Polygonatum 20g, Mulberry 15g, Lycium Barbarum 30g, Cuscuta Seed 30g, Wine-soaked Schisandra 20g, Plantago Seed 20g, Rosa Laevigata Fruit 15g, and Roasted Licorice 6g.

[0033] For the preparation method, usage and dosage, please refer to the preparation and usage in Example 1.

[0034] The following animal experiments and clinical observations are used to experimentally study and verify the effects of the Chinese medicine composition of the present invention and samples prepared from reference drugs in treating oligoasthenospermia and oligoasthenospermia caused by male infertility, and their synergistic application with assisted reproductive technology.

[0035] 1. Animal Experimentation 1. Experimental Materials 1.1 Experimental animals Forty C57 male mice, weighing 20 ± 2 g, aged 10 weeks, SPF grade, were obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd., China (License No.: SCXK(Lu)20220099).

[0036] 1.2 Drugs and Reagents 2. Reagent preparation Busulfan test solution: Dissolve 1 mL of 20.8 mg / mL busulfan in DMSO, then add to 4 mL of PEG300 and mix thoroughly. Add 500 μL of Tween-80 to the above system and mix thoroughly. Then, add 4.5 mL of normal saline to make the volume 10 mL to obtain a clear solution with a concentration of 2.08 mg / mL.

[0037] L-carnitine oral solution (1g: 10mL x 6 vials, Approval No. H19990372, Manufacturer: Shenyang First Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group). This solution was adjusted to a concentration of 0.27g / kg / day based on the body surface area coefficient ratio between humans and mice. It was administered orally by gavage to the L-carnitine group, stored in a refrigerator, and thoroughly mixed before use.

[0038] Wuzi Yanzong Pills (produced by Tong Ren Tang Pharmaceutical Factory of Beijing Tong Ren Tang Co., Ltd., specification 60g / bottle, approval number Z11020188. This drug is ground into powder before use and prepared into a solution of 2g per kilogram of body weight per day for gavage based on the body surface area coefficient ratio of humans and mice).

[0039] For the modified Wuzi Yanzong Pill suspension, the modified Wuzi Yanzong Pill decoction prepared in Example 1 was prepared into a lyophilized powder. The lyophilized powder was dissolved in pure water at a concentration of 2 g / mL and the solution was heated at 37°C for complete dissolution. The suspension was stored in a refrigerator at 4°C and shaken evenly before use.

[0040] 3. Animal grouping and drug administration This study used 40 10-week-old C57 male mice, randomly divided into five groups of eight: a normal control group, an oligospermia group, a L-carnitine group, a Wuzi Yanzong Wan group, and a modified Wuzi Yanzong Wan group. For details, see the medication schedule in Table 1.

[0041] Establishment of Oligospermia Models: 32 mice in the oligospermia, asthenospermia, L-carnitine, Wuzi Yanzong Wan, and Jiawei Wuzi Yanzong Wan treatment groups received an intraperitoneal injection of 20 mg / kg busulfan to induce oligospermia models. Fourteen days after model establishment, corresponding drug treatments were initiated.

[0042] The normal control group received intraperitoneal injections of 10% DMSO (the same dose as busulfan) for 14 days. Starting the day after modeling was successfully established, this group of mice received oral administration of purified water (1 mL / kg) daily for 28 days.

[0043] Oligospermia group: After the model was successfully established, starting from the second day, this group of mice were orally administered 1 mL / kg of purified water every day for 28 consecutive days.

[0044] L-carnitine group: Starting from the second day after the model was successfully established, this group of mice began to receive 0.27 g / kg / d of L-carnitine oral solution by gavage for 28 consecutive days.

[0045] Wuzi Yanzong Wan group and Jiawei Wuzi Yanzong Wan treatment group: Starting from the second day after the model was successfully established, these two groups of mice received Wuzi Yanzong Wan and Jiawei Wuzi Yanzong Wan suspensions, respectively, with a dose of 2g / kg, once a day for 28 days.

[0046]

[0047] 4. Experimental process and results SPSS 20.0 statistical analysis software was used to perform statistical analysis of the data. One-way ANOVA was used to compare the data of each group. The rank sum test was used when the variance was uneven. When the P value was less than 0.05, the difference was considered statistically significant.

[0048] 4.1 Effects of Jiawei Wuzi Yanzong Pills on testis, body weight, and histopathology in oligospermic mice After 28 days of intervention with Jiawei Wuzi Yanzong Pills, the mice were anesthetized. After disinfecting the abdomen with alcohol cotton balls, the abdominal cavity was quickly opened and the testicles on both sides were removed. Forceps and surgical scissors were used to gently remove the fat tissue around the testicles, separate the epididymis, and use flat paper to gently absorb the body fluids and blood on the outside of the testicles and epididymis during the separation process. The tissue was placed on a balance and weighed, and the weight of each organ was recorded. After weighing, the testicles were washed with PBS solution, and after absorbing the surface liquid with flat paper, they were quickly placed in testicular tissue fixative and liquid nitrogen for preservation.

[0049] like Figure 1 , showing images of the testicles of five groups of mice. A shows the control group and the oligospermia group, and B shows the L-carnitine group, the Jiawei Wuzi Yanzong Wan group, and the Wuzi Yanzong Wan group. It can be seen that compared to the control group, the testicles of the other four groups of mice were significantly smaller.

[0050] Figure 2 Table 2 shows the changes in body weight of the five groups of mice on day 1, day 15, day 30, and day 44 after enrollment. As the experiment progressed, the weight of the mice in the control group gradually increased. However, on day 30, the weight of the mice in the oligospermia group, the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group decreased compared with the control group. The difference was statistically significant (P < 0.05), and the decrease was most significant on day 44, just before the end of the experiment ( P ﹤0.01), the body weight of mice in the Wuzi Yanzong Pills group, the Jiawei Wuzi Yanzong Pills group and the L-carnitine group did not increase significantly after intervention ( P ﹥0.05).

[0051]

[0052] *Comparison between oligospermia group and control group; #Comparison between L-carnitine, Jiawei Wuzi Yanzong Wan group and Wuzi Yanzong Wan group and oligospermia group; ΔComparison between Wuzi Yanzong Wan group and Jiawei Wuzi Yanzong Wan group. * P ﹤0.05,** P ﹤0.01,*** P <0.001;# P ﹤0.05,## P ﹤0.01,### P ﹤0.001;Δ P ﹤0.05, ΔΔ P ﹤0.01, ΔΔΔ P ﹤0.001.

[0053] Figure 3Table 3 and Table 4 show the statistical data for testicular weights of the five groups of mice. The results showed that the testicular weights of the mice in the oligospermia and asthenospermia group were significantly lower than those in the control group (P < 0.001). However, the testicular weights of the mice in the L-carnitine, Jiawei Wuzi Yanzong Wan, and Wuzi Yanzong Wan groups showed an increasing trend, with the Jiawei Wuzi Yanzong Wan group showing the most significant recovery (Table 4). Compared to the testicular weights of the mice in the oligospermia and asthenospermia group, the increase was statistically significant (P < 0.05). The Wuzi Yanzong Wan group showed the least increase in testicular weight, followed by the L-carnitine group. Specific values ​​are shown in Table 4, "Statistical Table of Testicular Weights of the Five Groups of Mice."

[0054]

[0055] *Comparison between oligospermia group and control group; #Comparison between L-carnitine, Jiawei Wuzi Yanzong Wan group and Wuzi Yanzong Wan group and oligospermia group; ΔComparison between Wuzi Yanzong Wan group and Jiawei Wuzi Yanzong Wan group. * P ﹤0.05,** P ﹤0.01,*** P <0.001;# P ﹤0.05,## P ﹤0.01,### P ﹤0.001;Δ P ﹤0.05, ΔΔ P ﹤0.01, ΔΔΔ P ﹤0.001.

[0056]

[0057] The above results indicate that there is no visible difference between the Jiawei Wuzi Yanzong Wan group and the Wuzi Yanzong Wan group in terms of tissue morphology.

[0058] 4.2 HE staining of mouse testicular tissue sections Mouse testicular tissue was fixed in fixative for 24 hours, then removed from the fixative, placed in a fume hood, and rinsed thoroughly with running water. The tissue was then dehydrated using a gradient of 75%, 85%, and 95% alcohol, followed by two treatments with anhydrous ethanol and two treatments with xylene for dehydration and clearing. The tissue was infiltrated with paraffin three times before embedding. After cooling, the paraffin-embedded tissue was sliced ​​using a microtome, mounted on glass slides, and baked in an oven overnight. After deparaffinization, the tissue was stained with hematoxylin and eosin (HE), dehydrated in a series of steps, and mounted with neutral gum. After mounting, the sections were observed under a 200x and 400x microscope for image acquisition.

[0059] Figure 4The results of HE staining of the testes from five groups of mice are shown. In the control group, the seminiferous tubules showed intact lumina, uniform basement membrane thickness, normal Sertoli cell morphology, and evenly distributed Leydig cells. Spermatogenic cells of varying morphologies were observed, closely arranged from the base to the lumen, with clear layers. Spermatogenesis was normal, with numerous mature spermatocytes visible in the middle of the tubule lumen.

[0060] The lumen structure of the seminiferous tubules of the mice in the oligospermia and asthenospermia group was incomplete, the distance between the seminiferous tubules was large, the lumen morphology was shrunken, deformed and disordered, the interstitial cells connecting the seminiferous tubules disappeared in large numbers, the interstitial cells in some areas were broken, the spermatogenic epithelium was destroyed, the basement membrane became thinner, and many germ cells in the lumen showed apoptosis to form a large number of cavities. Spermatogenic cells and supporting cells at all levels atrophied and fell off in large numbers, leaving only a small number of loosely and irregularly arranged supporting cells and spermatogonia. Inflammatory cell infiltration was present, and there were no mature sperm cells in the lumen.

[0061] The testicular tissue structure of mice in the L-carnitine group and the Jiawei Wuzi Yanzong Pill group was restored, the tubular morphology was improved to a certain extent, the connections between the seminiferous tubules were restored and arranged tightly, the interstitial cells increased, the interstitial cells became thicker, the number of supporting cells increased, and some tubular lumen structures were gradually restored. Cavities formed by apoptosis of germ cells could still be seen inside, mature spermatocytes were present in the tubular lumen, and there was slight inflammatory cell infiltration between the interstitial cells. The degree of damage was less. The asthenospermia group was significantly improved, and the testicular morphology was improved.

[0062] The degree of testicular tissue damage in the Wuzi Yanzong Wan group was between the oligospermia and asthenospermia group, the L-carnitine group, and the Wuzi Yanzong Wan group. The lumen of the seminiferous tubules was enlarged, the connections between the seminiferous tubules were reduced, and the number of interstitial cells, supporting cells, and spermatogonia was small and increased in the asthenospermia group, but less than that in the L-carnitine group and the Jiawei Wuzi Yanzong Wan group. There was a small amount of inflammatory cell infiltration between the interstitial cells, and mature spermatocytes were scarce.

[0063] 4.3 Effects of Jiawei Wuzi Yanzong Pills on Sperm Count and Motility in Oligospermia-Asthenospermia Mice Figure 5 Microscopic images of sperm from the testicles of five groups of mice were displayed, further confirming the significant changes in sperm morphology and quantity under different treatment conditions. In the control group, it was seen that the sperm had regular morphology and a large number, indicating that the spermatogenesis process was normal. The heads of the sperm in the control group showed a typical hook-shaped structure, and the tails were slender, straight, and uniform and smooth in shape, which is a typical phenotype of mature functional sperm. In contrast, in the oligoasthenospermia group, the number of sperm was significantly reduced, and the morphological abnormalities were particularly prominent. The heads of the sperm in the oligoasthenospermia group were mostly pointed, split or shrunken, and the tails were often bent, folded or even broken, and the neck connection area was deformed or shortened, indicating that the sperm differentiation process may have been disturbed, further proving the severity of its spermatogenesis disorder.

[0064] In contrast, some recovery in sperm count was observed in both the L-carnitine and Jiawei Wuzi Yanzong Wan treatment groups, although irregular sperm morphology still existed. Specifically, the morphology of the sperm head and tail improved in the L-carnitine treatment group, but some characteristics of the oligoasthenospermia phenotype were still retained, while the improvement in sperm morphology was more significant in the Jiawei Wuzi Yanzong Wan treatment group. The sperm head structure in this group was more normalized, the tail morphology was more straight, and the neck connection area was more stable. The Wuzi Yanzong Wan group also showed some improvement, but the degree of recovery in sperm count and morphology was slightly lower than that in the L-carnitine and Jiawei Wuzi Yanzong Wan groups. The above results indicate that the Jiawei Wuzi Yanzong Wan group has a significantly higher effect on improving mouse testicular sperm than the Wuzi Yanzong Wan group, and the significant improvement in sperm count and morphology suggests that it has a certain effect in promoting sperm production and improving sperm morphology.

[0065] Figure 6 Table 5 and Table 5 compare the sperm concentration and motility of the five groups of mice, revealing the effects of different treatment conditions on sperm quality. Sperm count: The sperm concentration of mice in the oligoasthenospermia group was significantly lower than that in the control group (P < 0.0001), indicating a significant decrease in sperm count and successful model establishment. In contrast, the sperm concentrations of mice treated with Jiawei Wuzi Yanzong Wan, L-carnitine, and Wuzi Yanzong Wan were significantly higher than those in the oligoasthenospermia group (P < 0.001). The sperm concentration in the Jiawei Wuzi Yanzong Wan group was slightly higher than that in the L-carnitine group. Both groups were superior to the Wuzi Yanzong Wan group, and the sperm concentration in the Jiawei Wuzi Yanzong Wan group was significantly higher than that in the Wuzi Yanzong Wan group, with a statistically significant difference (P < 0.001). This result demonstrates that Jiawei Wuzi Yanzong Wan, L-carnitine, and Wuzi Yanzong Wan can restore sperm count, with the Jiawei Wuzi Yanzong Wan group showing a more pronounced effect.

[0066] Sperm motility: The sperm motility values ​​of all mice in the oligoasthenospermia group were 0, significantly lower than those in the control group (P < 0.0001), indicating that the modeling process successfully reduced sperm motility. Overall, there was no significant improvement in sperm motility in the L-carnitine, Jiawei Wuzi Yanzong Wan, and Wuzi Yanzong Wan groups. The sperm motility of most mice remained 0, with no significant difference compared to the oligoasthenospermia group. However, some mice in the L-carnitine and Jiawei Wuzi Yanzong Wan groups showed some sperm motility, suggesting that Jiawei Wuzi Yanzong Wan may have a certain effect on improving sperm motility. In contrast, the sperm motility of all mice in the Wuzi Yanzong Wan group remained 0, indicating that its effect on improving sperm motility is not yet significant.

[0067] Overall, the sperm count and motility in the oligoasthenospermia group were significantly lower than those in the control group, confirming the successful establishment of the oligoasthenospermia model. Regarding sperm concentration, the Jiawei Wuzi Yanzong Wan group showed the greatest improvement, followed by the L-carnitine group. Although the Wuzi Yanzong Wan group showed some improvement, the results were relatively low. Regarding sperm motility, both Jiawei Wuzi Yanzong Wan and L-carnitine treatments showed some potential for improvement, but this did not yet reach statistical significance. Further extension of the intervention time or increase in dosage may be necessary to clarify their effects.

[0068]

[0069] *Comparison between oligospermia group and control group; #Comparison between L-carnitine, Jiawei Wuzi Yanzong Wan group and Wuzi Yanzong Wan group and oligospermia group; ΔComparison between Wuzi Yanzong Wan group and Jiawei Wuzi Yanzong Wan group. * P ﹤0.05,** P ﹤0.01,*** P <0.001;# P ﹤0.05,## P ﹤0.01,### P ﹤0.001;Δ P ﹤0.05, ΔΔ P ﹤0.01, ΔΔΔ P ﹤0.001.

[0070] 4.4 Effects of Jiawei Wuzi Yanzong Pills on the Endocrine System of Oligospermia-Asthenospermia Mice Figure 7 Table 6 shows the results of ELISA detection of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in the serum of the five groups of mice.

[0071] FSH levels in the oligoasthenospermia group were significantly higher than those in the control group (P < 0.001), suggesting that dysfunction of the hypothalamic-pituitary-testicular axis may be responsible for the abnormally elevated FSH secretion in this oligoasthenospermia model. FSH levels decreased in the Wuzi Yanzong Pill, Jiawei Wuzi Yanzong Pill, and L-carnitine groups. The Jiawei Wuzi Yanzong Pill and L-carnitine groups showed significantly lower FSH levels than in the oligoasthenospermia group (P < 0.01). Furthermore, FSH levels in the Jiawei Wuzi Yanzong Pill and L-carnitine groups approached those in the control group, with no significant differences compared to the control group (P > 0.05). In contrast, although FSH levels in the Wuzi Yanzong Pill group showed a downward trend, the difference was not statistically significant compared to the oligoasthenospermia group (P > 0.05). These results indicate that Jiawei Wuzi Yanzong Wan is more effective than Wuzi Yanzong Wan in reducing FSH levels, suggesting that it may restore normal FSH secretion by more effectively regulating the function of the hypothalamic-pituitary-testicular axis.

[0072] The LH level of the oligospermia group mice was significantly higher than that of the control group ( P ﹤0.01), indicating that the damage to testicular function not only affects spermatogenesis, but also interferes with the normal secretion of LH. Compared with the oligospermia group, the LH levels of the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group were significantly lower, and the differences were statistically significant ( P ﹤0.05). Among them, the reduction in the Jiawei Wuzi Yanzong Pills group and the L-carnitine group was more significant ( P ﹤0.001), and the effect was better than that of the Wuzi Yanzong Pill group. This further indicates that Jiawei Wuzi Yanzong Pill can more effectively improve the endocrine disorder of oligoasthenospermia mice.

[0073] Wuzi Yanzong Pills, Jiawei Wuzi Yanzong Pills, and L-carnitine all modulated FSH and LH secretion levels in oligoasthenospermia mice, with Jiawei Wuzi Yanzong Pills demonstrating significantly superior efficacy. Jiawei Wuzi Yanzong Pills may significantly alleviate endocrine disorders and restore normal FSH and LH secretion by more effectively regulating the hypothalamic-pituitary-testicular axis, thereby providing an effective endocrine environment for improved spermatogenesis.

[0074]

[0075] *Comparison between oligospermia group and control group; #Comparison between L-carnitine, Jiawei Wuzi Yanzong Wan group and Wuzi Yanzong Wan group and oligospermia group; ΔComparison between Wuzi Yanzong Wan group and Jiawei Wuzi Yanzong Wan group. * P ﹤0.05,** P ﹤0.01,*** P <0.001;# P ﹤0.05,## P ﹤0.01,### P ﹤0.001;Δ P ﹤0.05, ΔΔ P ﹤0.01, ΔΔΔ P ﹤0.001.

[0076] 4.5 Effects of Jiawei Wuzi Yanzong Wan on the expression of blood-testis barrier marker mRNA, mouse inflammatory factor marker mRNA, and germ cell marker mRNA in oligospermic mice and its influence on cell cycle pathways 4.5.1qPCR detection: ① Total RNA was extracted from tissue samples using the TRIzol extraction kit according to the manufacturer's instructions.

[0077] ② RNA concentration measurement: After centrifugation, mix and measure the total RNA. (Wash three times, then blanch and measure the concentration.) Divide the measured concentration by 1000. If the concentration is too low, divide it by 500 / the lowest concentration of the sample measured. Make up to 7 μl with ddH2O.

[0078] ③ Removal of genomic DNA Prepare the reaction mix on ice using the following ingredients. To ensure accurate reaction mix preparation, prepare an additional 2x Master Mix based on the desired number of reactions and distribute it among the individual reaction tubes. Finally, add the RNA sample to each tube. See Tables 7, 8, and 9 for details.

[0079]

[0080] ④ 42℃ 2min (or room temperature 5min) ⑤ Perform the reverse transcription process on ice to ensure the stability of the reaction mixture. To improve the accuracy of the preparation, prepare a Master Mix for two additional reactions based on the number of reactions required and distribute it evenly among the reaction tubes. After mixing, start the reverse transcription process immediately.

[0081]

[0082]

[0083] 4.5.2 Effects on mRNA Expression of Blood-Testis Barrier Markers Figure 8 The results showed that the mRNA expression levels of tight junction protein genes Cldn3, Cldn5, and Cldn11 related to the blood-testis barrier (BTB) in the testicular tissues of five groups of mice were detected by real-time fluorescence quantitative PCR (qPCR). Using the normal control group mice as the reference standard, the results showed that the expression level of the Cldn3 gene was significantly reduced in the oligospermia group mice ( P﹤0.05). Cldn3 (Claudin-3), as one of the key components of the tight junction of the blood-testis barrier, participates in maintaining the structural stability and integrity of the testicular basement membrane, and plays an important role in maintaining the homeostasis of the testicular microenvironment and in the process of spermatogenesis. Therefore, the significant downregulation of Cldn3 expression indicates that the structural integrity of the testicular basement membrane of oligoasthenospermic mice is impaired, thereby affecting the normal function of the blood-testis barrier. At the same time, it was found that Cldn5 and Cldn11, as important members of the tight junction protein family, also play a key role in maintaining the function of the blood-testis barrier. Cldn5 is mainly expressed in the endothelial cells of the blood vessels in the testis and participates in regulating the vascular permeability and microcirculatory homeostasis of the testicular tissue. Cldn11 is widely distributed in the supporting cells (Sertoli cells) and their junctions with spermatogonia, participating in the formation of the special microenvironment required for spermatogenesis and maintaining the functional barrier between the supporting cells and germ cells. In this study, the mRNA expression levels of Cldn5 and Cldn11 in the oligoasthenospermic mice were significantly increased ( P ﹤0.05), this abnormal expression may be a compensatory response of the body to the barrier damage caused by the decreased expression of Cldn3, in an attempt to maintain the stability of the blood-testis barrier function.

[0084] In the testicular tissue of oligospermia mice treated with Jiawei Wuzi Yanzong Pills and L-carnitine, the expression level of Cldn3 gene was significantly increased compared with that in the oligospermia model group ( P This result suggests that Jiawei Wuzi Yanzong Pills can improve the structure of the testicular basement membrane and restore the blood-testis barrier function.

[0085] The above results preliminarily revealed the expression changes of Cldn3, Cldn5 and Cldn11 in the oligoasthenozoospermia mouse model and their effects on the structure and function of the blood-testis barrier, and further clarified that Jiawei Wuzi Yanzong Pills may improve the testicular tissue microenvironment and partially restore the blood-testis barrier function by regulating the expression of tight junction proteins, providing new experimental basis and research ideas for the treatment of male oligoasthenozoospermia with traditional Chinese medicine.

[0086] 4.5.3 Effects on mRNA Expression of Inflammatory Factor Markers in Mice Figure 9 The results showed that the expression levels of CD45 mRNA in the testicular tissue of five groups of mice were detected by real-time fluorescence quantitative PCR (qPCR). Compared with the normal control group, the expression of CD45 gene in the testicular tissue of the oligospermia model mice was significantly increased ( PCD45 is a transmembrane glycoprotein widely present on the surface of immune cells. It is often used to reflect the infiltration and activity of immune cells. Increased expression of CD45 is often closely associated with immune system activation and enhanced inflammatory responses. In this study, the significant upregulation of CD45 expression suggests that the testicular tissue of oligoasthenospermic mice may have significant immune cell infiltration and localized inflammatory responses, suggesting that the stability of the testicular microenvironment is disrupted.

[0087] Damage to the blood-testis barrier can lead to immune cell infiltration into the testicular interstitium and seminiferous tubules, triggering a local inflammatory response, further exacerbating testicular tissue damage and disrupting spermatogenesis and the normal physiological function of Sertoli cells. It is speculated that upregulated CD45 expression may be closely related to damage to the blood-testis barrier, suggesting that immune-mediated inflammatory responses play a significant role in the pathogenesis of oligoasthenospermia. Long-term immune cell infiltration may lead to tissue fibrosis and cell apoptosis, ultimately severely impacting testicular development and spermatogenesis.

[0088] After intervention with Jiawei Wuzi Yanzong Wan, the expression level of CD45 was significantly reduced compared with the asthenospermia model group ( P <0.05). In comparison, L-carnitine and L-carnitine treatment also significantly reduced CD45 expression, but the magnitude of the decrease was less than that observed in the Jiawei Wuzi Yanzong Wan group. This result suggests that Jiawei Wuzi Yanzong Wan has promising therapeutic potential for improving the testicular immune microenvironment and inhibiting inflammatory responses, and is superior to Wuzi Yanzong Wan.

[0089] The above studies further confirmed the important role of immune inflammatory response in the pathogenesis of oligoasthenospermia mouse model through changes in CD45 expression, and Jiawei Wuzi Yanzong Pills may improve the testicular microenvironment by reducing immune cell infiltration and thus promote sperm production, which provides an experimental basis for the treatment of oligoasthenospermia with traditional Chinese medicine.

[0090] 4.5.4 Effects on germ cell marker mRNA expression Figure 10 The results showed that the mRNA expression levels of SOX9 and Wnt4, key genes related to spermatogenesis, were detected in the testicular tissues of five groups of mice by real-time fluorescence quantitative PCR (qPCR). The results showed that the expression of SOX9 and Wnt4 genes in the testicular tissues of oligospermia group mice was significantly increased ( P ﹤0.05).

[0091] SOX9, an important transcription factor, plays a crucial role in testicular development and maintenance of function, particularly in regulating the differentiation and function of Sertoli cells. SOX9 promotes Sertoli cell differentiation, maintains their morphology and function, and plays a crucial regulatory role in the maintenance and further differentiation of spermatogonia. In this study, upregulation of SOX9 expression may be a compensatory response to Sertoli cell dysfunction, attempting to maintain Sertoli cell number and function by activating SOX9 expression, thereby protecting and repairing the microenvironment of spermatogenesis.

[0092] Wnt4 is a key member of the Wnt signaling pathway and is widely involved in biological processes related to spermatogenesis, such as seminiferous tubule development, spermatogonia differentiation, and spermatocyte maturation. Abnormally elevated Wnt4 gene expression may disrupt normal Wnt signaling pathway activity, leading to abnormal spermatogonia differentiation and spermatocyte maturation during spermatogenesis, resulting in a significant decrease in sperm quality and quantity.

[0093] After intervention with Jiawei Wuzi Yanzong Wan, the mRNA expression levels of SOX9 and Wnt4 were significantly lower than those in the asthenospermia group ( P The results showed that the effect of Jiawei Wuzi Yanzong Wan on spermatogenesis was significantly better than that of Wuzi Yanzong Wan, indicating that Jiawei Wuzi Yanzong Wan may improve spermatogenesis and restore the stability of the testicular microenvironment in oligoasthenospermia mice by regulating the expression levels of SOX9 and Wnt4. The results also showed that Jiawei Wuzi Yanzong Wan had a better effect than Wuzi Yanzong Wan, demonstrating its potential advantages in improving the testicular microenvironment and promoting spermatogenesis.

[0094] In summary, the important roles of SOX9 and Wnt4 in the pathogenesis of oligoasthenospermia mouse model were further confirmed, and new experimental basis was provided for the effect of Jiawei Wuzi Yanzong Pills on improving spermatogenesis in male oligoasthenospermia.

[0095] 4.5.5 Impact on Cell Cycle Pathways Figure 11 The results showed that the mRNA expression levels of Wee1, Plk1, CCNA1 and CCNA2, genes related to the cell cycle pathway, in the testicular tissues of five groups of mice were detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the expression levels of the above genes in the testicular tissues of the oligospermia model group were significantly decreased compared with those in the normal control group ( P ﹤0.05).

[0096] Cell cycle regulation is crucial for spermatogenesis. Wee1 and Plk1 play key roles in regulating cell cycle progression, spermatogonia proliferation, and spermatocyte differentiation. Wee1 is a cell cycle checkpoint kinase that negatively regulates the transition from the G2 to M phase of the cell cycle, ensuring genomic stability and normal cell division. Plk1 (Polo-like kinase 1) plays important roles in multiple phases of the cell cycle and is particularly essential for regulating meiosis and spermatogenesis. CCNA1 and CCNA2, members of the cyclin A family, are involved in regulating the transition from G1 to S phase and from G2 to M phase, and are crucial for germ cell cycle regulation and normal spermatogenesis.

[0097] In this study, the expression of Wee1, Plk1, CCNA1 and CCNA2 in the oligospermia and asthenospermia mouse model group was significantly reduced, indicating abnormal cell cycle regulation, which may lead to significant inhibition of the proliferation and differentiation of spermatogonia and spermatocytes, thereby further interfering with the normal spermatogenesis.

[0098] After intervention with Jiawei Wuzi Yanzong Wan, the expression levels of the above cell cycle-related genes were significantly increased compared with the asthenospermia group ( P ﹤0.05), and the expression level was higher than that in the Wuzi Yanzong Wan group, suggesting that Jiawei Wuzi Yanzong Wan may improve the spermatogenesis function of oligospermia mice by regulating the expression of genes related to the cell cycle pathway and promoting the normal division and differentiation of spermatogonia and spermatocytes.

[0099] The above research results revealed for the first time the mechanism by which Jiawei Wuzi Yanzong Pills may improve spermatogenesis through the cell cycle pathway, providing a theoretical basis and experimental support for further in-depth research on the treatment of male oligoasthenospermia with traditional Chinese medicine.

[0100] 2. Clinical Research Trials 1. Retrospective cohort study 1 Materials and Methods Patient data for this study were obtained by searching the ART Management System (ARMS) of the Reproductive Department of a Shandong hospital. The data included patients who underwent ICSI between January 2020 and December 2024. Inclusion criteria for the observation group were: ① The male met the diagnostic criteria for oligoasthenozoospermia; ② had previously received treatment with the Jiawei Wuzi Yanzong Pill. Inclusion criteria for the control group were: ① The male met the diagnostic criteria for oligoasthenozoospermia; ② had received only conventional Western medication. A total of 578 patients were enrolled, including 367 in the observation group and 211 in the control group.

[0101] 2 Statistical methods This study used R language (version 4.1.3, R Foundation for Statistical Computing, Vienna, Austria) for statistical analysis. The test methods included Wilcox test, etc. The significance level was P <0.05 indicated that the difference was statistically significant.

[0102] 3 Results 3.1 Comparison of basic data of patients in the observation group and the control group There was no statistically significant difference between the observation group and the control group in basic characteristics such as age, body mass index, abstinence time, sperm concentration and forward motility of sperm ( P >0.05), the two groups were comparable, and the specific data are shown in Table 10. There was no significant difference in the basic data of the female patients in the two groups, such as age, years of infertility, and body mass index ( P ﹥0.05), and are also comparable. Detailed information is shown in Table 11.

[0103]

[0104]

[0105] 3.2 Comparison of laboratory results between the observation group and the control group The results showed that the observation group was superior to the control group in multiple embryo development indicators. The number of D3-I embryos, D3-II embryos, normal fertilized eggs, cleavage number, 2PN cleavage number, D1-2PN number, total number of effective embryos and total number of high-quality embryos in the observation group were significantly higher than those in the control group, and the differences were statistically significant ( P ﹤0.001). In addition, the total number of effective blastocysts formed and the total number of effective blastocysts in the observation group were not statistically significant compared with the control group ( P ﹥0.05), but it showed an upward trend. Detailed information is shown in Table 12.

[0106]

[0107] 4. Summary The results of this study found that the observation group had significantly better embryonic development indicators than the control group, including the number of D3-I embryos, the number of D3-II embryos, the number of normal fertilized eggs, the number of cleavage eggs, the number of 2PN cleavage eggs, the number of D1-2PN embryos, the total number of effective embryos, and the total number of high-quality embryos. P<0.001), significantly improving sperm fertilization ability, embryo quality, and early developmental potential. These studies demonstrate the positive effects of Jiawei Wuzi Yanzong Wan in improving embryonic development in patients with oligoasthenospermia and demonstrate its clinical value in treating male infertility with assisted reproductive technology. This provides important theoretical basis and experimental support for the application of Traditional Chinese Medicine in clinical assisted reproductive technology.

[0108] (2) Prospective randomized controlled studies 1 Clinical Data 1.1 Case Source This study has been approved by the Medical Ethics Committee of a Shandong hospital and is registered with the China Clinical Trial Registry. A total of 90 patients with infertility due to oligospermia and asthenospermia who visited the reproductive center of a Shandong hospital between January 2023 and December 2024 were enrolled. This prospective, randomized, parallel-controlled trial design was used. The 90 patients undergoing infertility treatment were randomly divided into two groups, an observation group and a control group, each consisting of 45 patients, using R language version 3.5.1. The control group received L-carnitine, while the observation group received the modified Wuzi Yanzong Pills (decoction) of the present invention in addition to the control group's treatment.

[0109] L-Carnitine Oral Solution (1g: 10mL x 6 vials, Approval No. H19990372, Manufacturer: Shenyang First Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group). Adjust dosage based on weight: For those weighing 60-100 kg, take 2-3g daily; for those weighing less than 60 kg, take 1g daily, with a maximum daily dose of no more than 3g. Take twice a day, morning and evening, or three times a day, after meals.

[0110] 1.2 Sample size estimation Previous studies have found that after treatment, the concentration of active sperm in the observation group was 10×10 6 / mL, and under the alternative hypothesis it is 9×10 6 / mL (the concentration of motile sperm in the control group was 9×10 6 Sample size estimation was performed using the TwoSample T-Test in PASS 2021 software. The significance level (α) was set at 0.05, the power (1-β) was set at 80%, and the intergroup ratio was set at 1:1. Assuming a 15% dropout rate, the final enrollment target was 90 participants, 45 in each group.

[0111] 2 Research Methods 2.1 Test flow chart see Figure 12 .

[0112] 2.2 Study population Inclusion criteria: (1) Men aged 22-35 years; (2) The diagnostic criteria for oligospermia and asthenospermia were based on the Manual of Laboratory Examination of Human Semen and Sperm-Cervical Mucus Interactions (Fifth Edition)

[54] , with the average of three semen tests as the basis for judgment: sperm density ≤ 15 × 106 / mL or progressively motile sperm (PR, grade a + grade b) less than 32%; (3) No other serious complications, such as heart disease, liver disease, kidney disease, etc.

[0113] Exclusion criteria: (1) Men under 22 years of age or over 35 years of age; (2) Those who have received any medication for oligoasthenospermia in the past 6 months; (3) Those with other diseases or conditions that may affect the study results, such as diabetes, hypertension, heart disease, liver disease, kidney disease, etc.; (4) Those with a history of severe drug allergies; (5) Those with azoospermia; (6) Those with bad habits such as smoking, drinking, and drug abuse.

[0114] Exit criteria: (1) occurrence of serious adverse reactions; (2) request for withdrawal due to personal reasons; (3) violation of the trial protocol.

[0115] 2.3 Study Treatment Groups Group A (control group): conventional western medicine treatment: L-carnitine; Group B (observation group): based on the control group, given modified Wuzi Yanzong Pills (decoction).

[0116] 2.4 Randomization: Random allocation is performed using a random number table generated by computer software to ensure fairness and randomness of the allocation.

[0117] 2.5 Research Procedure 2.5.1 Study treatment period, as shown in Table 13 below.

[0118]

[0119] 2.5.2 Investigational Drugs Modified Wuzi Yanzong Pills: 15g mulberry, 15g raspberry, 15g astragalus, 15g plantain, 30g wolfberry, 18g huangjing (sweet yellow stalk) wine, 15g rehmannia root, 15g chufa (paper mulberry), 12g schisandra (wine-processed schisandra), 6g roasted licorice root, 9g salt-processed leek seed, 12g roasted epimedium, 15g laevigata fruit, 30g dodder seed (all provided by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine). Decoction and usage are the same as above.

[0120] 2.5.3 Administration and Dosage Adjustment In this study, the control group received L-carnitine oral solution (1g: 10mL x 6 vials, Approval No. H19990372, Manufacturer: Shenyang First Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group). The dosage was adjusted based on body weight: patients weighing 60-100 kg should take 2-3g daily; those weighing less than 60 kg should take 1g daily, with a maximum daily dose of no more than 3g. The dosage should be taken twice a day, morning and evening, or three times a day, after meals.

[0121] The observation group received the same L-carnitine treatment, plus oral administration of Modified Wuzi Yanzong Pills, once daily in the morning and evening. Both groups received treatment for three consecutive months, with efficacy evaluations conducted after completion.

[0122] 2.5.4 Concomitant treatment and follow-up visits During this study, subjects may receive other treatments according to the following provisions, but the detailed information should be recorded and reported to the researchers during the follow-up visits.

[0123] 2.5.5 Patient compliance and withdrawal Monitor and improve patient compliance through regular follow-up visits and telephone calls. If a subject chooses to withdraw from the trial, the final data before withdrawal should be collected as much as possible.

[0124] 2.5.6 Protocol Deviations: All protocol deviations are recorded and their possible impact is considered during data analysis.

[0125] 2.6 Evaluation: Conduct safety assessment.

[0126] 2.7 Adverse Event Reporting: Describe the protocol for collecting, evaluating, reporting, and managing any adverse events and unexpected reactions to the intervention or implementation process. Adverse event assessment should include type and severity, timing, relevance, and outcome.

[0127] 2.8 Quality Management: This study will adhere to Good Clinical Practice (GCP). All participants will receive training on clinical trial design, clinical observation and mechanism of action of Jiawei Wuzi Yanzong Wan in the treatment of oligoasthenospermia, implementation, monitoring, and reporting. Regular internal audits will be conducted to ensure data accuracy and completeness.

[0128] 2.9 Statistical Analysis: Includes analysis populations (full analysis set, per-protocol analysis set, and safety analysis set), efficacy analysis and statistical methods (T-test or Mann-Whitney U test to compare the differences in efficacy indicators between the two groups), safety analysis and statistical methods (calculation of the incidence of each adverse event and comparison of the differences between the two groups using chi-square test or Fisher's exact test), interim analysis, final analysis, and data monitoring committee.

[0129] 3 Clinical Results 3.1 Statistics of the two groups of cases A total of 90 patients participated in this trial, of which 84 completed the study. Two patients in the observation group withdrew from treatment for personal reasons, and two dropped out due to loss to follow-up. Both patients in the control group voluntarily withdrew from treatment for personal reasons. The dropout rate was 6.7%, and these dropouts did not affect the observed results. See Table 14 for details.

[0130]

[0131] 3.2 Comparison of basic data between the observation group and the control group There was no significant difference between the observation group and the treatment group in terms of basic data such as semen volume, sperm concentration, forward motility, total motility and total sperm count before treatment ( P >0.05), and are comparable. Detailed information is shown in Table 15.

[0132]

[0133] 3.3 Comparison of post-treatment results between the observation group and the control group After treatment, the observation group had significantly higher sperm concentration, total sperm count, progressive motility sperm count, non-progressive motility sperm count, motile sperm count, total progressive motility sperm count, total non-progressive motility sperm count and total motile sperm count than the control group, and the differences were statistically significant ( P ﹤0.05). There was no significant difference in terms of forward motility of sperm (PR) (%) and total motility (PR+NP) (%), and there was no statistical significance ( P >0.05), but there is an increasing trend. Detailed information is shown in Table 16.

[0134]

[0135] 3.4 Comparison of data before and after treatment in the observation group In the comparison of the data before and after treatment in the observation group, there was a significant increase in sperm concentration, total sperm count, progressive motility (PR) (%), total motility (PR+NP) (%), progressive motility sperm concentration and total number of active sperm after treatment, and the differences were statistically significant ( P ﹤0.05). Detailed information is shown in Table 17.

[0136]

[0137] 3.5 Comparison of basic data of women in the two groups before treatment There was no statistically significant difference in age, body mass index, infertility years, basal FSH, basal LH, basal E2, pregnancy number, parity and abortion number between the observation group and the control group before treatment ( P﹥0.05), which is comparable. The laboratory results of the female patients in the two groups showed that the high-quality embryo rate in the observation group was significantly higher than that in the control group, and the difference was statistically significant ( P ﹤0.05). Although there is no statistical significance in the number of high-quality embryos, there is still a clear upward trend. P The value is close to less than 0.05. Although there is no statistical significance in the number of D5 blastocysts and the number of transferable embryos ( P ﹥0.05), but all of them increased significantly.

[0138] 4. Summary The results of this study showed that the observation group showed significant improvement in sperm concentration, total sperm count, progressive motility sperm concentration, non-progressive motility sperm concentration, motile sperm concentration, total progressive sperm count, total non-progressive sperm count and total motile sperm count compared with the control group ( P <0.05). This indicates that Jiawei Wuzi Yanzong Wan has a clear advantage in improving sperm count and concentration. Furthermore, the observation group showed an upward trend in multiple embryo development indicators, particularly in the rate of high-quality embryos. This further confirms the potential role of Jiawei Wuzi Yanzong Wan in improving embryo quality and implantation potential, which is of great significance for promoting natural conception and increasing the success rate of assisted reproductive technology.

[0139] The foregoing is merely an embodiment of the present invention and is not intended to limit the present invention. It will be apparent to those skilled in the art that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. A Chinese medicine composition, characterized in that The invention is prepared from the following raw materials in parts by weight: 15 parts of Cuscuta australis fruits, 15 parts of Astragalus complanatus fruits, 12 parts of roasted Epimedium, 15 parts of Rehmannia root, 15 parts of Rubus idaeus, 9 parts of salted Allium sativum seeds, 18 parts of Polygonatum odoratum, 15 parts of mulberries, 30 parts of Lycium barbarum fruits, 30 parts of Cuscuta australis seeds, 12 parts of Schisandra chinensis fruit with wine, 12 parts of Plantago seed, 15 parts of Rosa laevigata fruit pulp and 6 parts of roasted Licorice root.

2. Use of the Chinese medicine composition as claimed in claim 1 in preparing a drug for improving and / or treating male infertility.

3. The use according to claim 2, characterized in that The male infertility is oligospermia and asthenospermia; the dosage form of the Chinese medicine composition is decoction, powder, capsule, tablet, granule or pill.

4. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for treating male oligoasthenospermia by regulating the cell cycle pathway in the testis by targeting Wee1, PLK1, CCNA1 and CCNA2.

5. Use of the Chinese medicine composition according to claim 1 in the preparation of a medicament for treating male infertility in conjunction with assisted reproductive technology.

6. The use according to claim 5, characterized in that The assisted reproductive technology is intracytoplasmic sperm injection or in vitro fertilization combined with embryo transfer technology. The traditional Chinese medicine composition improves the sperm concentration and motility of patients with oligospermia and asthenospermia, thereby increasing the high-quality embryo rate of patients treated with intracytoplasmic sperm injection (ICSI).

7. Use of the Chinese medicine composition as claimed in claim 1 in combination with L-carnitine in the preparation of a medicament for improving and / or treating male oligoasthenospermia.

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