A traditional Chinese medicine composition and application thereof in preparing a medicine for improving and / or treating male infertility

Abstract

The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition and application thereof in preparation of a medicine for improving and / or treating male infertility. The traditional Chinese medicine composition is prepared from Fructus Malli, Fructus Equiseti, Rhiocarpae, Radix Rehmanniae Preparatae, Rubus chingii Hu, Fructus Carpesii, Rhizoma Polygonati, Morus, Fructus Lycii, Semen Marsdeniae, Fructus Schisandrae Chinensis, Semen Plantaginis, Fructus Rosae Laevigatae, Radix Glycyrrhizae Preparatae. The traditional Chinese medicine composition is supplemented with multiple medicinal materials on the basis of a traditional prescription, forms a comprehensive conditioning scheme considering multiple viscera and multiple causes, a multi-dimensional syndrome differentiation and treatment strategy, and is beneficial to the treatment purpose of treating both the symptoms and the root cause, can more effectively cope with the multiple pathological changes of oligospermia and asthenospermia of male infertility, and improve the overall treatment effect. The traditional Chinese medicine composition is applied in cooperation with an assisted reproductive technology, and the effect is remarkable.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition and its application in the preparation of drugs for improving and / or treating male infertility. Background Technology

[0002] Male infertility is primarily caused by oligospermia and asthenospermia. The pathological mechanisms of oligospermia and asthenospermia are highly complex, with current research focusing mainly on oxidative stress, immune responses, apoptosis, signal transduction, and gene and protein expression. Current treatment methods include: drug therapy (such as antibiotics, hormones, and antioxidants), surgical treatment (for problems like vas deferens obstruction and severe varicocele), and assisted reproductive technologies (ART) including in vitro fertilization combined with embryo transfer (IVF) and intracytoplasmic sperm injection (ICSI). While drug therapy can improve semen quality, its effectiveness in treating oligospermia and asthenospermia with multiple etiologies remains limited. Clinical observations have revealed that oligospermia and asthenospermia often involve multiple organs, with kidney deficiency being the most common. Traditional Chinese medicine, with its holistic approach and syndrome differentiation, offers a feasible treatment option for oligospermia and asthenospermia.

[0003] Wuzi Yanzong Wan, a classic traditional Chinese medicine formula for treating oligospermia and asthenospermia, first appeared in the Tang Dynasty's *Xuanjie Lu* and is hailed as "the premier formula for fertility throughout history." This formula has been widely used clinically in treating male infertility, demonstrating good efficacy and safety, with no reported side effects to date. Its basic treatment principle is to tonify the kidneys, focusing on warming and tonifying kidney yang and nourishing kidney essence. However, the pathogenesis of oligospermia and asthenospermia is not simply limited to kidney deficiency, but involves a comprehensive manifestation of dysfunction of multiple organs and imbalance of qi, blood, yin, and yang. Therefore, the therapeutic effect of Wuzi Yanzong Wan has certain limitations, failing to comprehensively regulate overall function and thus unable to achieve the ideal therapeutic effect of holistic regulation. Furthermore, although some modern pharmacological studies have preliminarily indicated that Wuzi Yanzong Wan may improve sperm production and semen quality by regulating the hypothalamic-pituitary-gonadal axis (HPG axis), the specific molecular mechanism of action for oligospermia and asthenospermia has not yet been clearly elucidated.

[0004] In recent years, with the development and application of ART technology, traditional Chinese medicine has gradually permeated into all aspects of ART. However, the application of traditional Chinese medicine in in vitro fertilization-embryo transfer (IVF-ET) is relatively recent. In particular, the ambiguity of the mechanism of action of Wuzi Yanzong Wan has limited its synergistic application in assisted reproductive technology to some extent, and there is still a lack of high-quality clinical research support.

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

[0006] To address the shortcomings of existing technologies, this invention proposes a traditional Chinese medicine composition and its application in the preparation of drugs for improving and / or treating male infertility, thus solving the problems existing in the prior art.

[0007] To achieve the above-mentioned objectives, one of the technical solutions adopted by this invention is:

[0008] A traditional Chinese medicine composition is provided, which is made from the following raw materials in parts by weight: 10-20 parts of Broussonetia papyrifera fruit, 10-20 parts of Astragalus complanatus seed, 10-20 parts of processed Epimedium brevicornu, 10-20 parts of processed Rehmannia glutinosa, 10-20 parts of Rubus idaeus fruit, 5-15 parts of Allium tuberosum seed, 10-30 parts of Polygonatum sibiricum (processed with wine), 10-20 parts of Morus alba fruit, 10-30 parts of Lycium barbarum fruit, 10-30 parts of Cuscuta chinensis seed, 10-20 parts of processed Schisandra chinensis fruit, 10-20 parts of Plantago asiatica seed, 10-20 parts of Rosa laevigata fruit, and 5-10 parts of processed Glycyrrhiza uralensis root.

[0009] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15 parts of Broussonetia papyrifera fruit, 15 parts of Astragalus complanatus fruit, 12 parts of processed Epimedium brevicornu, 15 parts of Rehmannia glutinosa, 15 parts of Rubus idaeus, 9 parts of salted Allium tuberosum seed, 18 parts of Polygonatum sibiricum (processed with wine), 15 parts of Morus alba fruit, 30 parts of Lycium barbarum fruit, 30 parts of Cuscuta chinensis fruit, 12 parts of processed Schisandra chinensis fruit, 12 parts of Plantago asiatica seed, 15 parts of Rosa laevigata fruit, and 6 parts of processed Glycyrrhiza uralensis.

[0010] The second technical solution adopted in this invention is:

[0011] The use of the aforementioned traditional Chinese medicine composition in the preparation of drugs for improving and / or treating male infertility is provided.

[0012] Furthermore, the male infertility is oligospermia or asthenospermia; the dosage form of the traditional Chinese medicine composition is decoction, powder, capsule, tablet, granule, or pill. Decoction is preferred.

[0013] Furthermore, the traditional Chinese medicine composition is used in the preparation of a drug for treating male oligospermia and asthenospermia by directly targeting Wee1, PLK1, CCNA1 and CCNA2 to regulate the cell cycle pathway in the testes.

[0014] Furthermore, the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs used in conjunction with assisted reproductive technology.

[0015] Furthermore, the application of the aforementioned traditional Chinese medicine composition in the preparation of medicines for male infertility patients undergoing assisted reproductive technology intervention.

[0016] Furthermore, the assisted reproductive technology is intracytoplasmic sperm injection (ICSI) or in vitro fertilization combined with embryo transfer. The traditional Chinese medicine composition improves sperm concentration and motility in patients with oligospermia and asthenospermia, thereby increasing the rate of high-quality embryos in patients treated with ICSI.

[0017] The above-described solution of the present invention can bring the following beneficial effects:

[0018] 1. The herbal composition of this invention is a modified Wuzi Yanzong Wan (Five-Seed Progeny Pill), which, based on the traditional formula, is supplemented with multiple medicinal materials such as Rehmannia glutinosa, Astragalus complanatus, and Polygonatum sibiricum. It not only retains the original formula's basic effects of tonifying the kidneys and replenishing essence, consolidating essence and reducing urination, but also further enhances the effects of soothing the liver and relieving depression, replenishing qi and nourishing blood, and strengthening the spleen and stomach, forming a comprehensive conditioning plan that addresses multiple organs and causes. Its multidimensional syndrome differentiation and treatment strategy can holistically regulate the patient's organ functions, tonifying kidney essence while simultaneously addressing spleen and stomach function, liver qi circulation, and blood and qi sufficiency, achieving both symptomatic and root-cause treatment. This more effectively addresses the multifactorial pathological changes of oligospermia and asthenospermia, improving the overall treatment effect.

[0019] 2. This invention, the Modified Wuzi Yanzong Pill, takes a holistic approach, combining ingredients such as salted leek seeds, prepared rehmannia root, and polygonatum rhizome to achieve the dual effects of tonifying the kidneys and soothing the liver, nourishing yin and replenishing qi. Salted leek seeds warm and tonify kidney yang, warm the lower back and knees, and promote qi circulation and dissipate stagnation; prepared rehmannia root excels at nourishing yin and blood, replenishing kidney essence; and polygonatum rhizome tonifies the spleen and kidneys, nourishes yin and moistens the lungs. Together, they tonify the kidneys and replenish essence, soothe the liver and regulate qi, and nourish the liver and kidneys. This liver-kidney synergy makes the formula more in line with the TCM theory of treating both the liver and kidneys, effectively alleviating the negative impact of liver qi stagnation or insufficient liver blood on sperm production, achieving simultaneous tonification of the liver and kidneys and regulation of qi and blood. This reflects the advantages of TCM's "holistic view" and "differential treatment" in treating male infertility. Clinically, it demonstrates broader applicability and significant therapeutic advantages. Furthermore, the Modified Wuzi Yanzong Pill's treatment approach, based on regulating spleen and stomach function, overcomes the shortcomings of simple kidney-tonifying formulas and better meets the needs of patients with different constitutions in clinical practice.

[0020] 3. This invention, the modified Wuzi Yanzong Wan, follows the principle of Yin-Yang mutual support and generation in its drug formulation. Through the synergistic combination of different Chinese herbs, it achieves the effect of Yin-Yang harmony and water-fire balance. Rehmannia glutinosa, mulberry, and Polygonatum sibiricum primarily nourish Yin, effectively tonifying kidney essence and moistening kidney Yin. Other herbs in the formula, such as leek seed and Cuscuta chinensis, warm and tonify kidney Yang, thus achieving a balance between Yin-Yang nourishment and Yang tonification. This embodies the treatment principle of "seeking Yang within Yin and Yin within Yang," achieving the therapeutic goal of Yin-Yang complementarity and water-fire balance. Theoretically, leek seed and Rehmannia glutinosa belong to "water," Cuscuta chinensis and Rubus idaeus belong to "wood," and Polygonatum sibiricum also corresponds to "earth." The combination of herbs achieves mutual generation and organ harmony, not only tonifying kidney essence but also regulating the overall function of the organs and meridians, promoting the smooth generation and circulation of essence and qi.

[0021] 4. Treatment of oligospermia and asthenospermia requires gradual tonification rather than short-term excessive tonification. Compared to traditional Wuzi Yanzong Wan (Five-Seed Progeny Pill), the modified Wuzi Yanzong Wan of this invention emphasizes gradual and gentle tonification. The herbs in it, such as Rosa laevigata and Cuscuta chinensis, are mild and warm in nature, not only slowly nourishing kidney essence but also avoiding the drawbacks of excessive or simple tonification that could lead to qi stagnation or phlegm-dampness accumulation, thus achieving the therapeutic effect of "tonifying without stagnation" and "gradual treatment is best." Through this gentle and mild tonification method, the modified Wuzi Yanzong Wan ensures stable and lasting therapeutic effects, achieving long-term improvement in semen quality and reproductive function in patients with oligospermia and asthenospermia.

[0022] In summary, the modified Wuzi Yanzong Pill of this invention not only focuses on short-term improvement of semen quality or quantity, but also emphasizes the overall coordination of the functions of all organs in the body, ensuring the holistic and effective treatment outcome. This treatment approach is more in line with modern medicine's understanding of the multifactorial and multipathological mechanisms of oligospermia and asthenospermia, and better reflects its scientific nature, feasibility, and clinical applicability. Attached Figure Description

[0023] Figure 1 These are images of the testes of five groups of mice used in the animal experiments of this invention.

[0024] Figure 2 Figures showing the body weights of five groups of mice;

[0025] Figure 3 Figure 1 shows the testicular weight of five groups of mice; **** p <0.0001;

[0026] Figure 4 HE staining images of testes from five groups of mice;

[0027] Figure 5 Microscopic images of five groups of mouse sperm;

[0028] Figure 6 This section compares the sperm concentration and sperm motility of five groups of mice. p<0.01 , *** p<0.001 **** p<0.0001 ;

[0029] Figure 7 The expression levels of FSH and LH in the blood of five groups of mice are shown. **p<0.01, ***p<0.001, ****p<0.0001 ;

[0030] Figure 8 The results show the mRNA expression of blood-testis barrier markers in five groups of mice; **p<0.01, ***p<0.001, ****p< 0.0001 ;

[0031] Figure 9 Here are mRNA expression diagrams of inflammatory markers in five groups of mice; p <0.01, *** p<0.001, **** p <0.0001;

[0032] Figure 10 Here are mRNA expression diagrams of five mouse germ cell markers;* p <0.05, ** p <0.01, *** p <0.001,**** p <0.0001;

[0033] Figure 11 This invention relates to the effect of the modified Wuzi Yanzong Pill on the cell cycle pathway in mice with oligospermia and asthenospermia;* p <0.05,** p <0.01, *** p <0.001, **** p <0.0001;

[0034] Figure 12 This is a flowchart of the clinical trial process for this invention.

[0035] in, Figure 1 Image A shows mouse testes in the control group and the oligospermia group; Image B shows mouse testes in the L-carnitine group, the Jiawei Wuzi Yanzong Wan group, and the Wuzi Yanzong Wan group.

[0036] Figure 4 In the table, A, B, C, D, and E represent the HE staining results of mouse testes from the control group, oligospermia-asthenospermia group, L-carnitine group, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group, respectively.

[0037] Figure 5 Images A, B, C, D, and E in the table are, in order, microscopic images of testicular sperm from mice in the control group, oligospermia and asthenospermia group, L-carnitine group, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group.

[0038] Figure 6 In the table, A represents the sperm concentration statistics of the five groups of mice, and B represents the total sperm motility statistics of the five groups of mice.

[0039] Figure 7 In the table, A represents the serum follicle-stimulating hormone (FSH) level in the five groups of mice, and B represents the serum luteinizing hormone (LH) level in the five groups of mice.

[0040] Figure 8 In the table, A, B, and C represent the mRNA expression levels of the tight junction protein genes CLDN3, CLDN5, and Cldn11 in the testicular tissues of five groups of mice, respectively.

[0041] Figure 10In the table, A represents the mRNA expression level of the key gene SOX9 in the testicular tissue of five groups of mice, and B represents the mRNA expression level of Wnt4.

[0042] Figure 11 In the table, A, B, C, and D represent the mRNA expression levels of the genes Wee1, CCNA1, Plk1, and CCNA2 in the testicular tissues of five groups of mice, respectively. Detailed Implementation

[0043] To more clearly illustrate the overall concept of the present invention, a detailed description is provided below with reference to the accompanying drawings. The embodiments described below are merely preferred embodiments of the present invention. It should be noted that the following description is solely for explaining the present invention and does not constitute any limitation on the present invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention fall within the scope of the present invention.

[0044] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0045] Example 1

[0046] This embodiment is a decoction of the traditional Chinese medicine composition of the present invention, and the traditional Chinese medicine composition is named Jiawei Wuzi Yanzong Wan.

[0047] The prescription is as follows: 15g of Broussonetia papyrifera fruit, 15g of Astragalus complanatus fruit, 12g of processed Epimedium brevicornu, 15g of Rehmannia glutinosa, 15g of Rubus idaeus, 9g of salted Allium tuberosum seed, 18g of Polygonatum sibiricum (processed with wine), 15g of Morus alba fruit, 30g of Lycium barbarum fruit, 30g of Cuscuta chinensis seed, 12g of Schisandra chinensis (processed with wine), 12g of Plantago asiatica seed, 15g of Rosa laevigata fruit, and 6g of processed Glycyrrhiza uralensis.

[0048] The preparation method is as follows:

[0049] Preparation method: Soak the above-mentioned herbs in water in an earthenware pot for 1-2 hours. During the first decoction, the water level should be 2-3 cm above the herbs. During the second decoction, add water until the water level is 1-2 cm above the herbs. Decoction all herbs together, first bring to a boil over high heat, then simmer over low heat for 40-60 minutes to make a decoction.

[0050] Dosage and administration: Decocted in water, 400mL, one dose per day, taken in the morning and evening.

[0051] Example 2

[0052] This embodiment is a decoction of the traditional Chinese medicine composition of the present invention.

[0053] The prescription is as follows: 10g of Broussonetia papyrifera fruit, 10g of Astragalus complanatus fruit, 12g of processed Epimedium brevicornu, 10g of Rehmannia glutinosa, 10g of Rubus idaeus, 9g of salted Allium tuberosum seed, 18g of Polygonatum sibiricum (processed with wine), 15g of Morus alba fruit, 30g of Lycium barbarum fruit, 30g of Cuscuta chinensis fruit, 12g of Schisandra chinensis (processed with wine), 12g of Plantago asiatica seed, 15g of Rosa laevigata fruit, and 6g of processed Glycyrrhiza uralensis.

[0054] For preparation methods and usage, please refer to the preparation and usage of Example 1.

[0055] Example 3

[0056] This embodiment is a decoction of the traditional Chinese medicine composition of the present invention.

[0057] The prescription is as follows: 20g of Broussonetia papyrifera fruit, 20g of Astragalus complanatus fruit, 20g of processed Epimedium brevicornu, 20g of processed Rehmannia glutinosa, 20g of Rubus idaeus fruit, 9g of salted Allium tuberosum seed, 20g of Polygonatum sibiricum (processed with wine), 15g of Morus alba fruit, 30g of Lycium barbarum fruit, 30g of Cuscuta chinensis fruit, 20g of processed Schisandra chinensis fruit, 20g of Plantago asiatica seed, 15g of Rosa laevigata fruit, and 6g of processed Glycyrrhiza uralensis root.

[0058] For preparation methods and usage, please refer to the preparation and usage of Example 1.

[0059] The following experimental studies and verifications were conducted on the efficacy of the herbal composition of the present invention and the sample prepared from the reference drug in treating male infertility with oligospermia and asthenospermia, and in synergistic application with assisted reproductive technology, through animal experiments and clinical observations.

[0060] I. Animal Experiments

[0061] 1. Experimental materials

[0062] 1.1 Laboratory Animals

[0063] Forty male C57 mice, weighing 20±2g, aged 10 weeks, SPF grade, were obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd., China (License No.: SCXK (Lu) 20220099).

[0064] 1.2 Medicines and Reagents

[0065] 2. Reagent preparation

[0066] Busulfan solution: Dissolve 1 mL of 20.8 mg / mL busulfan in DMSO, then add it to 4 mL of PEG300 and mix well; then add 500 μL of Tween-80 to the above system and mix well; then add 4.5 mL of physiological saline to make up to 10 mL to obtain a clear solution of 2.08 mg / mL.

[0067] L-carnitine oral solution (1g: 10mL × 6 vials, approval number H19990372, manufacturer: Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.). This solution is adjusted to a concentration of 0.27g / Kg / d based on the body surface area index ratio of humans and mice, and is used for oral gavage in the L-carnitine group. It should be stored in a refrigerator and thoroughly mixed before use.

[0068] Wuzi Yanzong Wan (produced by Beijing Tongrentang Pharmaceutical Factory, 60g / bottle, approval number Z11020188. This medicine 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 ratio of human to mouse body surface area coefficient).

[0069] The modified Wuzi Yanzong Pill suspension was prepared by lyophilizing the decoction of modified Wuzi Yanzong Pill obtained in Example 1 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 to completely dissolve it. It was then stored in a refrigerator at 4°C and shaken well before use.

[0070] 3. Animal grouping and administration

[0071] This study used 40 10-week-old male C57 mice, which were randomly divided into five groups of 8 mice each: normal control group, oligospermia and asthenospermia group, L-carnitine group, Wuzi Yanzong Wan group, and Jiawei Wuzi Yanzong Wan group. Specific details can be found in Table 1, which outlines the medication schedule.

[0072] Establishment of the oligoasthenospermia model: Thirty-two mice in the oligoasthenospermia group, L-carnitine group, Wuzi Yanzong Wan group, and modified Wuzi Yanzong Wan treatment group were injected intraperitoneally with 20 mg / kg busulfan solution to induce the oligoasthenospermia model. Fourteen days after the model was established, the corresponding drug treatment was started.

[0073] Normal control group: Mice received intraperitoneal injections of 10% DMSO aqueous solution, the same dosage as busulfan, for 14 days. Starting the day after successful modeling, these mice were orally administered 1 mL / kg of purified water daily for 28 days.

[0074] Oligospermia and Asthenospermia 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 daily for 28 consecutive days.

[0075] L-carnitine group: Starting from the second day after the successful establishment of the model, this group of mice began to receive 0.27 g / kg / d of L-carnitine oral solution by gavage for 28 consecutive days.

[0076] The Wuzi Yanzong Pill group and the Jiawei Wuzi Yanzong Pill treatment group: Starting from the second day after the successful establishment of the model, the mice in these two groups received suspensions of Wuzi Yanzong Pill and Jiawei Wuzi Yanzong Pill, respectively, at a dose of 2g / kg, once a day for 28 days.

[0077]

[0078] 4. Experimental Procedure and Results

[0079] The data were statistically processed using SPSS 20.0 software. One-way ANOVA was used to compare the data of each group. If the variances were unequal, the rank-sum test was used. When the p-value was less than 0.05, the difference was considered to be statistically significant.

[0080] 4.1 Effects of Jiawei Wuzi Yanzong Wan on testes, body weight, and histopathology in mice with oligospermia and asthenospermia

[0081] Twenty-eight days after intervention with Jiawei Wuzi Yanzong Wan (a traditional Chinese medicine formula), mice were anesthetized. After disinfecting the abdomen with alcohol swabs, the abdominal cavity was quickly opened, and both testes were removed. The surrounding adipose tissue was gently removed using forceps and surgical scissors, the epididymis was separated, and fluids and blood from the testes and epididymis were gently absorbed using absorbent paper. The tissues were weighed on a balance, and the weight of each organ was recorded. After weighing, the testes were washed with PBS solution, and after absorbing the surface liquid with absorbent paper, they were quickly placed in testicular tissue fixation solution and liquid nitrogen for preservation.

[0082] like Figure 1 The image shows images of the testes of five groups of mice. Figure A represents the control group and the oligospermia / asthenospermia group; Figure B represents the L-carnitine group, the Jiawei Wuzi Yanzong Wan group, and the Wuzi Yanzong Wan group. It is evident that the testes of the other four groups of mice were significantly smaller than those of the control group.

[0083] Figure 2 Table 2 shows the changes in body weight of the five groups of mice from day 1, day 15, day 30, and day 44 after enrollment. As the experiment progressed, the body weight of the control group mice gradually increased. However, on day 30, the body weight of the mice in the oligospermia and asthenospermia group, as well as the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group, was significantly lower than that of the control group (P < 0.05). The most significant decrease was observed on day 44, before the end of the experiment. P <0.01), the weight of mice in the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group did not increase significantly after intervention. P >0.05).

[0084]

[0085] * Comparison between the oligospermia / asthenospermia group and the control group; # Comparison between the L-carnitine, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group and the oligospermia group; Δ Comparison between the Wuzi Yanzong Wan group and the 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.

[0086] Figure 3 Table 3 shows the statistical data of testicular weight in the five groups of mice. The results showed that the testicular weight of mice in the oligoasthenospermia group was significantly lower than that in the control group (P < 0.001). The testicular weight of mice in the L-carnitine group, the Jiawei Wuzi Yanzong Wan group, and the Wuzi Yanzong Wan group showed an increasing trend, with the Jiawei Wuzi Yanzong Wan group showing the most significant recovery (Table 4). Compared to the oligoasthenospermia group, the increase in testicular weight 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 detailed in Table 4, the statistical table of testicular weight in the five groups of mice.

[0087]

[0088] * Comparison between the oligospermia / asthenospermia group and the control group; # Comparison between the L-carnitine, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group and the oligospermia group; Δ Comparison between the Wuzi Yanzong Wan group and the 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.

[0089]

[0090] The above results indicate that there are no visually discernible differences between the Jiawei Wuzi Yanzong Wan group and the Wuzi Yanzong Wan group at the tissue morphology level.

[0091] 4.2 HE staining of mouse testicular tissue sections

[0092] Mouse testicular tissue was fixed in fixative for 24 hours, then removed and thoroughly rinsed with running water in a fume hood. The tissue was then dehydrated sequentially with 75%, 85%, and 95% alcohol, followed by two treatments with anhydrous ethanol and two treatments with xylene for further dehydration and clearing. The tissue was then embedded after three paraffin infiltrations. After cooling, 4 μm thick sections were cut from the paraffin-embedded tissue using a microtome, mounted on glass slides, and baked overnight in an oven. After dewaxing, hematoxylin-eosin (HE) staining was performed, followed by stepwise dehydration and mounting with neutral resin. The slides were then observed and imaged under 200x and 400x microscopes.

[0093] Figure 4 HE staining results of testes from five groups of mice are presented. In the control group, the seminiferous tubules of the mice were intact, the basement membrane was of uniform thickness, the Sertoli cells were of normal morphology, the interstitial cells were evenly distributed, and spermatogenic cells of different morphologies were visible, arranged closely from the base to the luminal surface with clear layers. Spermatogenesis was normal, and a large number of mature spermatocytes were visible in the middle of the tubules.

[0094] In mice with oligoasthenospermia, the seminiferous tubules had incomplete luminal structures, large spacing between seminiferous tubules, and shrunken, deformed, and disordered luminal shapes. A large number of interstitial cells connecting the seminiferous tubules disappeared, and interstitial cells in some areas were broken. The seminiferous epithelium was destroyed, the basement membrane was thinned, and apoptosis of germ cells was common in the lumen, forming a large number of cavities. A large number of spermatogenic cells and supporting cells at all levels atrophied and sloughed off, leaving only a small number of loosely arranged and irregularly arranged supporting cells and spermatogonia. Inflammatory cell infiltration was observed, and there were no mature sperm cells in the lumen.

[0095] In mice in the L-carnitine group and the Jiawei Wuzi Yanzong Wan group, the testicular tissue structure was restored, the lumens morphology was improved to some extent, the connections between seminiferous tubules were restored and tightly arranged, the number of interstitial cells increased, the interstitium thickened, the number of supporting cells increased, and some lumens structure gradually recovered. Cavities formed by germ cell apoptosis were still visible inside, and mature sperm cells were present in the lumen. There was slight inflammatory cell infiltration between interstitial cells, and the degree of damage was less. The asthenospermia group showed significant improvement, and the testicular morphology was improved.

[0096] The degree of testicular tissue damage in the Wuzi Yanzong Wan group was between that in the oligospermia and asthenospermia group and 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, Sertoli cells and spermatogonia was less than that in the asthenospermia group, but not as much as 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 sperm cells were scarce.

[0097] 4.3 Effects of Jiawei Wuzi Yanzong Wan on Sperm Count and Motility in Oligospermia and Asthenospermia Mice

[0098] Figure 5Microscopic images of sperm from five groups of mouse testes were presented, further confirming the significant changes in sperm morphology and quantity under different treatment conditions. In the control group, sperm morphology was regular and the number was relatively large, indicating normal spermatogenesis. The sperm heads in the control group exhibited a typical hook-shaped structure, with long, straight tails, uniform and smooth morphology, representing the typical phenotype of mature, functional sperm. In contrast, the oligoasthenospermia group showed a significant reduction in sperm count, with particularly prominent morphological abnormalities. The sperm heads in the oligoasthenospermia group were mostly pointed, split, or shrunken, and the tails frequently bent, folded, or even broken. The neck junction was deformed or shortened, indicating that the sperm differentiation process may have been interfered with, further demonstrating the severity of the spermatogenesis disorder.

[0099] In contrast, both the L-carnitine group and the Jiawei Wuzi Yanzong Wan treatment group showed some recovery in sperm count, although irregularly shaped sperm remained. Specifically, the L-carnitine group showed improvement in sperm head and tail morphology, but still partially retained characteristics of oligoasthenospermia, while the Jiawei Wuzi Yanzong Wan treatment group showed more significant improvement in sperm morphology. The sperm head structure in this group was more normalized, the tail morphology was more straight, and the neck junction 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. These results indicate that the Jiawei Wuzi Yanzong Wan group significantly improved sperm count in mouse testes compared to the Wuzi Yanzong Wan group, and the significant improvement in sperm count and morphology suggests that it plays a role in promoting spermatogenesis and improving sperm morphology.

[0100] Figure 6 Table 5 shows the comparison of sperm concentration and sperm motility in the five groups of mice, revealing the effects of different treatments 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 reduction in sperm count and successful model establishment. In contrast, the sperm concentration of mice in the Jiawei Wuzi Yanzong Wan group, L-carnitine group, and Wuzi Yanzong Wan group was significantly higher than that in the oligoasthenospermia group (P<0.001). Among them, the sperm concentration of the Jiawei Wuzi Yanzong Wan group was slightly higher than that of the L-carnitine group, both of which were better than the Wuzi Yanzong Wan group. Furthermore, the sperm concentration of the Jiawei Wuzi Yanzong Wan group was significantly higher than that of the Wuzi Yanzong Wan group, and the difference was statistically significant (P<0.001). This result proves that Jiawei Wuzi Yanzong Wan, L-carnitine, and Wuzi Yanzong Wan can restore sperm count, especially the Jiawei Wuzi Yanzong Wan group, which showed a more prominent effect.

[0101] Sperm motility: The sperm motility values ​​of mice in the oligoasthenospermia group were all 0, significantly lower than those in the control group (P < 0.0001), indicating that the model was successfully established and significantly reduced sperm motility. Overall, sperm motility in mice in the L-carnitine group, the Jiawei Wuzi Yanzong Wan group, and the Wuzi Yanzong Wan group did not show significant improvement; the sperm motility values ​​of most mice remained 0, and there was no significant difference compared to the oligoasthenospermia group. However, a few mice in the L-carnitine group and the Jiawei Wuzi Yanzong Wan group showed some sperm motility, suggesting that Jiawei Wuzi Yanzong Wan may have a certain improving effect on 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 was not yet significant.

[0102] Overall, the sperm count and motility in the oligoasthenospermia group were significantly lower than those in the control group, validating the successful establishment of the oligoasthenospermia model. Regarding sperm concentration, the Jiawei Wuzi Yanzong Wan group showed the best improvement, followed by the L-carnitine group; while the Wuzi Yanzong Wan group showed some improvement, the effect was relatively low. As for sperm motility, both Jiawei Wuzi Yanzong Wan and L-carnitine treatments showed some potential for improvement, but these did not reach statistical significance. Further extension of the intervention period or increase in dosage may be needed to clarify their effects.

[0103]

[0104] * Comparison between the oligospermia / asthenospermia group and the control group; # Comparison between the L-carnitine, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group and the oligospermia group; Δ Comparison between the Wuzi Yanzong Wan group and the 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.

[0105] 4.4 Effects of Jiawei Wuzi Yanzong Wan on the endocrine system of oligospermia and asthenospermia mice

[0106] Figure 7 Table 6 shows the results of ELISA detection of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in the serum of five groups of mice.

[0107] The FSH level in mice in the oligoasthenospermia group was significantly higher than that in the control group (P < 0.001), indicating that there may be dysfunction of the hypothalamus-pituitary-testis axis in the oligoasthenospermia model, leading to abnormally high FSH secretion. The FSH levels in the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group were all decreased, with the FSH levels in the Jiawei Wuzi Yanzong Wan group and the L-carnitine group being significantly lower than those in the oligoasthenospermia group (P < 0.01). Furthermore, the FSH levels in the Jiawei Wuzi Yanzong Wan group and the L-carnitine group were close to those in the control group, with no statistically significant difference compared to the control group (P > 0.05). In contrast, although the FSH level in the Wuzi Yanzong Wan group showed a decreasing trend, the difference compared to the oligoasthenospermia group was not statistically significant (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 hypothalamic-pituitary-testicular axis.

[0108] The LH level in mice with oligoasthenospermia was significantly higher than that in the control group. P <0.01), indicating that impaired testicular function not only affects spermatogenesis but also interferes with normal LH secretion. Compared with the oligospermia and asthenospermia group, the LH levels in the Wuzi Yanzong Wan group, the Jiawei Wuzi Yanzong Wan group, and the L-carnitine group were all significantly reduced, and the differences were statistically significant. P <0.05). The reduction was more significant in the Jiawei Wuzi Yanzong Wan group and the L-carnitine group. P The effect was <0.001), and it was superior to the Wuzi Yanzong Wan group. This further indicates that the modified Wuzi Yanzong Wan can more effectively improve the endocrine disorder in mice with oligospermia and asthenospermia.

[0109] Wuzi Yanzong Wan, Jiawei Wuzi Yanzong Wan, and L-carnitine can all regulate FSH and LH secretion levels in mice with oligoasthenospermia, with Jiawei Wuzi Yanzong Wan showing significantly better results than Wuzi Yanzong Wan. Jiawei Wuzi Yanzong Wan may significantly alleviate endocrine disorders and restore normal FSH and LH secretion by more efficiently regulating the hypothalamic-pituitary-testicular axis, thereby providing an effective endocrine environment for improving spermatogenesis.

[0110]

[0111] * Comparison between the oligospermia / asthenospermia group and the control group; # Comparison between the L-carnitine, Jiawei Wuzi Yanzong Wan group, and Wuzi Yanzong Wan group and the oligospermia group; Δ Comparison between the Wuzi Yanzong Wan group and the 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.

[0112] 4.5 Effects of Jiawei Wuzi Yanzong Wan on the expression of serum testis barrier marker mRNA, mouse inflammatory factor marker mRNA, germ cell marker mRNA, and cell cycle pathway in oligospermia and asthenospermia mice

[0113] 4.5.1 qPCR detection:

[0114] ① The TRIzol extraction kit used to extract total RNA from tissue samples was operated according to the instructions.

[0115] ② RNA concentration measurement: After centrifuging the obtained total RNA, take 1 μL to measure the concentration. (Wash three times first, then BLACK, then measure the concentration.) Divide the measured concentration by 1000. If the concentration is too low, divide by 500 ÷ the lowest concentration of the sample. Add ddH2O to make up to 7 μL.

[0116] ③ Removal of genomic DNA

[0117] Prepare the reaction mixture on ice according to the following ingredients. To ensure accurate preparation of the reaction mixture, prepare an additional 2 Master Mixes as needed for the required number of reactions, and then dispense them into the individual reaction tubes. Finally, add the RNA sample to each tube. See Tables 7, 8, and 9 for details.

[0118]

[0119] ④ 42℃ for 2 minutes (or room temperature for 5 minutes)

[0120] ⑤ The reverse transcription process should be carried out on ice to ensure the stability of the reaction solution. To improve the accuracy of the preparation, prepare two additional Master Mixes based on the required number of reactions, and distribute them evenly to each reaction tube. The reverse transcription process should be started immediately after mixing.

[0121]

[0122]

[0123] 4.5.2 Effects on the expression of blood-testis barrier marker mRNA

[0124] Figure 8This study demonstrates the mRNA expression levels of tight junction protein genes Cldn3, Cldn5, and Cldn11, which are associated with the blood-testis barrier (BTB), in testicular tissues of five groups of mice, detected by real-time quantitative PCR (qPCR). Using normal control mice as a reference, the results showed that the expression level of the Cldn3 gene was significantly decreased in the oligoasthenospermia group. P <0.05). Cldn3 (Claudin-3), as a key component of the tight junctions of the blood-testis barrier, participates in maintaining the structural stability and integrity of the testicular basement membrane, playing an important role in maintaining testicular microenvironment homeostasis and spermatogenesis. Therefore, the significant downregulation of Cldn3 expression indicates impaired testicular basement membrane structural integrity in oligoasthenospermia mice, thereby affecting the normal function of the blood-testis barrier. It was also found that Cldn5 and Cldn11, as important members of the tight junction protein family, also play crucial roles in maintaining blood-testis barrier function. Cldn5 is mainly expressed in testicular vascular endothelial cells, participating in the regulation of testicular tissue vascular permeability and microcirculatory homeostasis. Cldn11 is widely distributed in Sertoli cells and their junctions with spermatogonia, participating in the formation of the specific microenvironment required for spermatogenesis and maintaining the functional barrier between Sertoli cells and germ cells. In this study, the mRNA expression levels of Cldn5 and Cldn11 in the oligoasthenospermia group 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 decrease in Cldn3 expression, in an attempt to maintain the stability of the blood-testis barrier function.

[0125] In the testicular tissue of oligoasthenospermia mice treated with Jiawei Wuzi Yanzong Wan and L-carnitine, the expression level of the Cldn3 gene was significantly higher than that in the oligoasthenospermia model group. P The effect was <0.05%, and it was superior to the Wuzi Yanzong Wan group. This result suggests that Jiawei Wuzi Yanzong Wan has the effect of improving the structure of the testicular basement membrane and restoring the blood-testis barrier function.

[0126] The above results preliminarily reveal the expression changes of Cldn3, Cldn5, and Cldn11 in the oligoasthenospermia mouse model and their effects on the structure and function of the blood-testis barrier. They further clarify that Jiawei Wuzi Yanzong Wan 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 evidence and research ideas for the treatment of male oligoasthenospermia with traditional Chinese medicine.

[0127] 4.5.3 Effects on the expression of mouse inflammatory cytokine markers mRNA

[0128] Figure 9This study demonstrates the detection of CD45 mRNA expression levels in testicular tissues of five mouse groups using real-time quantitative PCR (qPCR). Compared with the normal control group, CD45 gene expression was significantly increased in the testicular tissues of mice in the oligoasthenospermia model group. P <0.05). CD45 is a transmembrane glycoprotein widely distributed on the surface of immune cells, often used to reflect the infiltration level and activity status of immune cells. Elevated CD45 expression is usually closely related to immune system activation and enhanced inflammatory responses. In this study, the significant upregulation of CD45 expression indicates that there may be significant immune cell infiltration and local inflammatory responses in the testicular tissue of oligoasthenospermia mice, suggesting a disruption of testicular microenvironment stability.

[0129] Damage to the blood-testis barrier can lead to the infiltration of immune cells into the testicular interstitium and seminiferous tubules, triggering a local inflammatory response that further aggravates testicular tissue damage and disrupts spermatogenesis and the normal physiological functions of Sertoli cells. Therefore, it is hypothesized that upregulation of CD45 expression may be closely related to damage to the blood-testis barrier function, suggesting that immune-mediated inflammatory responses play an important role in the pathogenesis of oligoasthenospermia. Long-term immune cell infiltration may lead to tissue fibrosis and apoptosis, ultimately severely affecting testicular development and spermatogenesis.

[0130] After intervention with Jiawei Wuzi Yanzong Wan, the expression level of CD45 was significantly reduced in the asthenospermia model group. P <0.05). In comparison, L-carnitine and L-carnitine treatment also significantly reduced CD45 expression, but the reduction was not as great as that in the Jiawei Wuzi Yanzong Wan group. This result indicates that Jiawei Wuzi Yanzong Wan has good therapeutic potential in improving the testicular immune microenvironment and inhibiting the inflammatory response, and is superior to Wuzi Yanzong Wan.

[0131] The above studies further confirmed the important role of immune inflammatory response in the pathogenesis of oligoasthenospermia mouse model through changes in CD45 expression. Furthermore, the modified Wuzi Yanzong Wan may promote sperm production by reducing immune cell infiltration and improving the testicular microenvironment, which provides experimental evidence for the treatment of oligoasthenospermia with traditional Chinese medicine.

[0132] 4.5.4 Effects on the expression of germ cell marker mRNA

[0133] Figure 10 This study demonstrates the detection of mRNA expression levels of SOX9 and Wnt4, key genes related to spermatogenesis, in testicular tissues of five groups of mice using real-time quantitative PCR (qPCR). The results showed that the expression of both SOX9 and Wnt4 genes was significantly increased in the testicular tissues of mice in the oligoasthenospermia group. P <0.05).

[0134] SOX9, an important transcription factor, plays a crucial role in testicular development and functional maintenance, particularly in the differentiation and functional regulation of Sertoli cells. SOX9 promotes Sertoli cell differentiation, maintains their morphology and function, and plays a vital regulatory role in the maintenance and further differentiation of spermatogonia. In this experiment, the upregulation of SOX9 expression may be a compensatory response of the body to damage to Sertoli cell function, attempting to maintain the number and function of Sertoli cells by activating SOX9 expression, thereby protecting and repairing the microenvironment of spermatogenesis.

[0135] Wnt4 is an important member of the Wnt signaling pathway and is widely involved in biological processes related to spermatogenesis, such as the development of seminiferous tubules, spermatogonial differentiation, and spermatocyte maturation. Abnormally elevated Wnt4 gene expression may disrupt the activity of the normal Wnt signaling pathway, leading to abnormalities in spermatogonial differentiation and spermatocyte maturation during spermatogenesis, thereby causing a significant decrease in sperm quality and quantity.

[0136] After intervention with Jiawei Wuzi Yanzong Wan, the mRNA expression levels of SOX9 and Wnt4 were significantly lower in the asthenospermia group. P The result was <0.05, suggesting that Jiawei Wuzi Yanzong Wan may help gradually improve spermatogenesis in oligoasthenospermia mice and gradually restore the stability of the testicular microenvironment by regulating the expression levels of SOX9 and Wnt4. Among them, the intervention effect of Jiawei Wuzi Yanzong Wan was better than that of Wuzi Yanzong Wan, showing the potential advantages of Jiawei Wuzi Yanzong Wan in improving the testicular microenvironment and promoting spermatogenesis.

[0137] In summary, this study further confirms the important roles of SOX9 and Wnt4 in the pathogenesis of oligoasthenospermia in mouse models and provides new experimental evidence for the use of Jiawei Wuzi Yanzong Wan in improving spermatogenesis in men with oligoasthenospermia.

[0138] 4.5.5 Impact on the Cell cycle pathway

[0139] Figure 11 This study demonstrates the mRNA expression levels of cell cycle pathway-related genes Wee1, Plk1, CCNA1, and CCNA2 in testicular tissues of five groups of mice, detected by real-time quantitative PCR (qPCR). The results showed that the expression levels of these genes in the testicular tissues of mice in the oligoasthenospermia model group were significantly decreased compared to the normal control group. P <0.05).

[0140] Cell cycle regulation is crucial for spermatogenesis, with Wee1 and Plk1 playing key roles in regulating cell cycle progression, spermatogonial proliferation, and spermatocyte differentiation. Wee1 is a cell cycle checkpoint kinase that negatively regulates the transition from G2 to M phase, ensuring genome stability and normal cell division. Plk1 (Polo-like kinase 1) plays an important role in multiple stages of the cell cycle, especially in regulating meiosis and spermatogenesis. CCNA1 and CCNA2, belonging to the cyclin A family, participate in regulating the transition from G1 to S phase and from G2 to M phase, and are of great significance for germ cell cycle regulation and normal spermatogenesis.

[0141] In this study, the significantly reduced expression of Wee1, Plk1, CCNA1, and CCNA2 in the oligoasthenospermia mouse model group suggests abnormal cell cycle regulation, which may lead to a significant inhibition of the proliferation and differentiation of spermatogonia and spermatocytes, thereby further interfering with the normal process of spermatogenesis.

[0142] After intervention with Jiawei Wuzi Yanzong Wan, the expression levels of the above-mentioned cell cycle-related genes were significantly higher in the asthenospermia group than in the asthenospermia group. P <0.05), and the expression level was higher than that of the Wuzi Yanzong Wan group, suggesting that the Jiawei Wuzi Yanzong Wan may improve the spermatogenic function of oligoasthenospermia mice by regulating the expression of genes related to the cell cycle pathway and promoting the normal division and differentiation of spermatogonia and spermatocytes.

[0143] The above research results reveal for the first time the mechanism by which Jiawei Wuzi Yanzong Wan may improve spermatogenesis through the cell cycle pathway, providing a theoretical basis and experimental support for further in-depth research on traditional Chinese medicine treatment of male oligospermia and asthenospermia.

[0144] II. Clinical Research Trials

[0145] (a) Retrospective cohort study

[0146] 1. Materials and Methods

[0147] Patient data for this study were obtained by searching the ART Management System (ARMS) of the reproductive department of a hospital in Shandong Province. The data included patients who registered for ICSI between January 2020 and December 2024. Inclusion criteria for the observation group: ① The male partner met the diagnostic criteria for oligospermia and asthenospermia; ② They had previously received treatment with Jiawei Wuzi Yanzong Wan (a traditional Chinese medicine formula). Inclusion criteria for the control group: ① The male partner met the diagnostic criteria for oligospermia and asthenospermia; ② They received only conventional Western medicine treatment. A total of 578 patients were ultimately included, with 367 in the observation group and 211 in the control group.

[0148] 2. Statistical methods

[0149] This study used R language (version 4.1.3, R Foundation for Statistical Computing, Vienna, Austria) for statistical analysis, and the test methods included Wilcox.test, etc., with significance levels... P <0.05 indicates that the difference is statistically significant.

[0150] 3 Results

[0151] 3.1 Comparison of basic data between the observation group and the control group

[0152] There were no statistically significant differences between the observation group and the control group in basic characteristics such as age, body mass index, abstinence time, sperm concentration, and progressively motile sperm. P >0.05), the two groups were comparable, and the specific data are shown in Table 10. There were also no significant differences between the two groups of female patients in terms of age, duration of infertility, and body mass index, etc. P (>0.05), which is also comparable; see Table 11 for details.

[0153]

[0154]

[0155] 3.2 Comparison of laboratory results between the observation group and the control group

[0156] The results showed that the observation group was superior to the control group in multiple embryonic development indicators. The observation group had significantly higher numbers of D3-I grade embryos, D3-II grade embryos, normal fertilized eggs, cleavage counts, 2PN cleavage counts, D1-2PN counts, total number of viable embryos, and total number of high-quality embryos than the control group, and the differences were statistically significant. P <0.001). Furthermore, although there was no statistically significant difference between the observation group and the control group in the total number of viable blastocysts formed on day 5 (…),… P >0.05), but it is on an upward trend. See Table 12 for details.

[0157]

[0158] 4. Summary

[0159] The results of this study showed that the observation group had significantly better results than the control group in multiple embryonic development indicators, including the number of D3-I grade embryos, the number of D3-II grade embryos, the number of normally fertilized eggs, the number of cleavages, the number of 2PN cleavages, the number of D1-2PN embryos, the total number of effective embryos, and the total number of high-quality embryos. P<0.001), sperm fertilization capacity, embryo quality, and early developmental potential were significantly improved. These findings demonstrate that Jiawei Wuzi Yanzong Wan (a traditional Chinese medicine formula) plays a positive role in improving embryonic development in patients with oligospermia and asthenospermia, indicating its significant clinical value in assisted reproductive technology for treating male factor infertility. This provides important theoretical basis and experimental support for the application of traditional Chinese medicine in clinical assisted reproductive technology.

[0160] (ii) Prospective randomized controlled trials

[0161] 1. Clinical Data

[0162] 1.1 Source of Cases

[0163] This study has been approved by the Medical Ethics Committee of a hospital in Shandong Province and registered with the Chinese Clinical Trial Registry. Ninety patients with infertility due to oligospermia or asthenospermia who visited the reproductive center of a hospital in Shandong Province between January 2023 and December 2024 were selected. This study employed a prospective, randomized, parallel-controlled trial design. The 90 patients receiving infertility treatment were randomly divided into two groups using R language version 3.5.1: an observation group and a control group, with 45 patients in each group. The control group received L-carnitine treatment, while the observation group received the same treatment plus the modified Wuzi Yanzong Wan (decoction) of this invention.

[0164] L-carnitine oral solution (1g: 10mL × 6 vials, approval number H19990372, manufacturer: Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.). Dosage adjusted according to weight: Individuals weighing 60-100 kg should take 2-3g daily; individuals weighing less than 60 kg should take 1g daily, with a maximum daily dose not exceeding 3g. Take twice daily (morning and evening) or three times daily (morning, noon, and evening) after meals.

[0165] 1.2 Sample Size Estimation

[0166] Previous studies have found that, after treatment, the concentration of motile sperm in the observation group was 10 × 10⁻⁶ under the null hypothesis. 6 / mL, which is 9 × 10⁹ under the alternative hypothesis. 6 / mL (the concentration of motile sperm in the control group was 9×10⁻⁶) 6 / mL). Sample size was estimated using the TwoSample T-Test in PASS 2021 software. The significance level α was set at 0.05, the power 1-β at 80%, and the ratio between groups at 1:1. Assuming a dropout rate of 15%, the study expected to include 90 participants, with 45 in each group.

[0167] 2. Research Methods

[0168] 2.1 See the test flowchart. Figure 12 .

[0169] 2.2 Study population

[0170] Inclusion criteria: (1) Males aged 22-35 years; (2) The diagnostic criteria for oligospermia and asthenospermia are based on the provisions of the "Laboratory Test Manual for Human Semen and Sperm-Cervical Mucus Interaction" (5th edition)

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

[0171] Exclusion criteria: (1) Males under 22 years of age or over 35 years of age; (2) Those who have received any drug treatment for oligospermia or asthenospermia within 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 alcohol and drug use.

[0172] Withdrawal criteria: (1) serious adverse reactions occur; (2) withdrawal is requested for personal reasons; (3) violation of the test protocol.

[0173] 2.3 Study Treatment Groups

[0174] Group A (control group): routine Western medicine treatment: L-carnitine; Group B (observation group): in addition to the treatment given to the control group, the group was given a modified Wuzi Yanzong Wan formula (decoction).

[0175] 2.4 Randomized grouping: Random number tables generated by computer software are used for random allocation to ensure fairness and randomness in the allocation.

[0176] 2.5 Research Procedure

[0177] 2.5.1 Study treatment period, as shown in Table 13 below.

[0178]

[0179] 2.5.2 Investigational Drugs

[0180] The formula for the modified Wuzi Yanzong Wan (Five-Seed Progeny Pill) is as follows: 15g mulberry, 15g raspberry, 15g astragalus seed, 15g plantain seed, 30g wolfberry, 18g processed polygonatum, 15g prepared rehmannia root, 15g paper mulberry fruit, 12g processed schisandra fruit, 6g prepared licorice root, 9g salted leek seed, 12g prepared epimedium, 15g rosehip kernel, and 30g dodder seed (all provided by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine). The decoction method and usage are the same as before.

[0181] 2.5.3 Administration method and dosage adjustment

[0182] In this study, the control group received levocarnitine oral solution (1g: 10mL × 6 vials, approval number H19990372, manufacturer: Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.). Dosage was adjusted according to weight: individuals weighing 60-100 kg took 2-3g daily; individuals weighing less than 60 kg took 1g daily, with a maximum daily dose not exceeding 3g. It was taken twice daily (morning and evening) or three times daily (morning, noon, and evening) after meals.

[0183] The observation group received the same levocarnitine treatment plus oral administration of Jiawei Wuzi Yanzong Wan decoction, twice daily (morning and evening). Both groups of patients received continuous treatment for three months, and efficacy was evaluated at the end of the treatment course.

[0184] 2.5.4 Accompanying treatment and follow-up visits

[0185] During this study, participants may receive additional treatments according to the following guidelines, but details should be recorded and reported to the researchers during follow-up visits.

[0186] 2.5.5 Patient compliance and withdrawal

[0187] Patient adherence is monitored and improved through regular follow-up visits and telephone calls. If a subject chooses to withdraw from the trial, their final data before withdrawal should be collected whenever possible.

[0188] 2.5.6 Deviation from the plan: Record all deviations from the plan and consider their potential impact during data analysis.

[0189] 2.6 Evaluation: Conduct a safety assessment.

[0190] 2.7 Adverse Event Reporting: Describes the interventions or protocols implemented in the process of collecting, assessing, reporting, and handling any adverse events and unexpected reactions. Adverse event assessment includes type and severity, timing, relevance, and outcome.

[0191] 2.8 Quality Management: This study will adhere to Good Clinical Practice (GCP) guidelines. All participants will receive training on clinical trial design, clinical observation and mechanism of action of Jiawei Wuzi Yanzong Wan in the treatment of oligospermia, implementation, monitoring, and reporting. Regular internal audits will be conducted to ensure the accuracy and completeness of the data.

[0192] 2.9 Statistical Analysis: This includes the analysis population (full analysis set, protocol-compliant analysis set, safety analysis set), efficacy analysis and statistical methods (T-test or Mann-Whitney U test to compare differences in efficacy indicators between the two groups), safety analysis and statistical methods (calculating the incidence of each adverse event and using chi-square test or Fisher's exact test to compare differences between the two groups), interim analysis, final analysis, and data monitoring committee.

[0193] 3. Clinical Results

[0194] 3.1 Statistical analysis of the two groups of cases

[0195] A total of 90 patients participated in this study, with 84 actually completing the trial. In the observation group, 2 patients withdrew from treatment due to personal reasons, and 2 patients were lost to follow-up. In the control group, both patients voluntarily withdrew from treatment for personal reasons. The dropout rate was 6.7%, and the dropout cases did not affect the observation results. See Table 14 for detailed information.

[0196]

[0197] 3.2 Comparison of basic data between the observation group and the control group

[0198] There were no significant differences between the observation group and the treatment group in terms of pre-treatment baseline data such as semen volume, sperm concentration, progressive motility, total sperm motility, and total sperm count. P >0.05), making them comparable. See Table 15 for details.

[0199]

[0200] 3.3 Comparison of treatment results between the observation group and the control group

[0201] After treatment, the observation group showed significant improvements compared to the control group in sperm concentration, total sperm count, progressive motility sperm concentration, non-progressive motility sperm concentration, motile sperm concentration, total progressive motility sperm count, total non-progressive motility sperm count, and total motile sperm count, with statistically significant differences. P <0.05). There were no significant differences in progressively motile sperm (PR) (%) and total motility (PR+NP) (%), indicating no statistical significance. P >0.05), but there is an increasing trend. See Table 16 for details.

[0202]

[0203] 3.4 Comparison of data before and after treatment in the observation group

[0204] In the comparison of data before and after treatment in the observation group, significant increases were observed in sperm concentration, total sperm count, progressive motility (PR) (%), total motility (PR+NP) (%), progressive motility sperm concentration, and total number of motile sperm after treatment, with statistically significant differences. P <0.05). See Table 17 for details.

[0205]

[0206] 3.5 Comparison of baseline data of the two groups of women before treatment

[0207] There were no statistically significant differences in baseline data between the observation group and the control group before treatment, including age, body mass index, duration of infertility, baseline FSH, baseline LH, baseline E2, parity, parity, and number of miscarriages. P >0.05), indicating comparability. Laboratory results for the female partners in both groups showed that the observation group had a significantly higher rate of high-quality embryos than the control group, with a statistically significant difference. P <0.05). Although there was no statistically significant difference in the number of high-quality embryos, there was still a clear increasing trend. P The value was close to less than 0.05. Although there was no statistically significant difference in the number of D5 blastocysts and the number of transferable embryos (…),… P (>0.05), but all showed a significant increase.

[0208] 4. Summary

[0209] The results of this study indicate that the observation group showed significant improvements compared to the control group in terms of sperm concentration, total sperm count, concentration of progressively motile sperm, concentration of non-progressively motile sperm, concentration of active sperm, total number of progressively motile sperm, total number of non-progressively motile sperm, and total number of active sperm. P <0.05). This indicates that Jiawei Wuzi Yanzong Wan has a significant advantage in improving sperm count and concentration. Furthermore, the observation group showed an upward trend in multiple embryo development indicators, especially in the rate of high-quality embryos, further confirming the potential role of Jiawei Wuzi Yanzong Wan in improving embryo quality and implantation potential. This is of great significance for promoting natural conception and improving the success rate of assisted reproductive technology.

[0210] The above description is merely an embodiment of the present invention and is not intended to limit the 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 principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, It is made from the following ingredients in parts by weight: 15 parts of Broussonetia papyrifera seeds, 15 parts of Astragalus complanatus seeds, 12 parts of processed Epimedium brevicornu, 15 parts of Rehmannia glutinosa, 15 parts of Rubus idaeus, 9 parts of salted Allium tuberosum seeds, 18 parts of Polygonatum sibiricum (processed with wine), 15 parts of Morus alba, 30 parts of Lycium barbarum, 30 parts of Cuscuta chinensis, 12 parts of processed Schisandra chinensis (processed with wine), 12 parts of Plantago asiatica seeds, 15 parts of Rosa laevigata fruit, and 6 parts of processed Glycyrrhiza uralensis.

2. The use of the traditional Chinese medicine composition as described in claim 1 in the preparation of a drug for improving and / or treating male infertility.

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

4. The use of the traditional Chinese medicine composition as described in claim 1 in the preparation of a drug for treating male oligospermia by targeting Wee1, PLK1, CCNA1 and CCNA2 to regulate the cell cycle pathway in the testes.

5. The use of the traditional Chinese medicine composition as described in claim 1 in the preparation of a medicament for treating male infertility in combination with assisted reproductive technology.

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

7. The use of the traditional Chinese medicine composition as described in claim 1 in combination with levocarnitine in the preparation of a drug for improving and / or treating male oligospermia and asthenospermia.

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