The keyword “Polycystic Ovary Syndrome” was used to search the Genecards database (9) (v5.15),¹ OMIM database (10) (Online Mendelian Inheritance in Man),² PharmGKB database (11) (Pharmacogenomics Knowledgebase),³ DisGeNet database (12) (v7.0),⁴ and DrugBank database (13) (v5.1.10).⁵ Similarly, the keyword “Female infertility” was queried in the Genecards, OMIM, PharmGKB, DisGeNet, and TTD databases (14) (Therapeutic Target Database).⁶ All database searches were conducted in October 2023. After removing duplicate entries from each search, the intersection of PCOS and infertility targets was extracted. A Venn diagram was generated to visualize the overlapping targets.

The list of common intersection targets was submitted to the Uniprot database (https://www.uniprot.org/, release 2023_04) to convert gene names into standardized protein names. The TCMSP database (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform)⁷ was then used to retrieve all compounds associated with these target proteins. The SMILES (Simplified Molecular Input Line Entry System) identifiers of the retrieved compounds were obtained from the PubChem database.⁸ Active compounds were screened using the SwissADME web tool⁹ based on two criteria: “GI absorption = High” and “Druglikeness ≥ 2 Yes.” These widely accepted criteria were chosen to enrich for compounds with favorable oral bioavailability and pharmacokinetic properties, thereby increasing their potential as therapeutic agents.

Herbs corresponding to the screened active compounds were identified using the TCMSP database. A comprehensive target-compound-herb network was constructed and visualized using Cytoscape software (v3.8.0). To identify the most influential components within this network, nodes (targets, compounds, and herbs) with a Degree value > 20 were defined as core components. The Degree > 20 threshold is a commonly used metric in network pharmacology to identify highly connected “hub” nodes that are considered functionally significant within the network. A subnetwork consisting of these core targets, compounds, and herbs was then extracted and visualized.

The frequency, nature (Four Natures), flavor (Five Flavors), meridian tropism, and therapeutic categories of the herbs associated with the active compounds were statistically analyzed using Microsoft Excel. Herbs with a usage frequency > 10 were classified as core candidates for further analysis. Association rule mining was performed on these core herbs using IBM SPSS Modeler 18.0 software, with thresholds set at support > 1 and confidence > 60% to identify significant co-occurrence patterns. Finally, hierarchical cluster analysis of the core herbs was conducted with IBM SPSS Statistics 26.0 to identify distinct medication patterns and herbal clusters.

A core herbal combination was formulated based on a synthesis of the results from the frequency statistics, association rule analysis, and cluster analysis. The targets associated with this core herbal combination were retrieved from the TCMSP database. These targets were then intersected with the previously identified 2,500 common PCOS-infertility targets. To elucidate the underlying biological mechanisms of the core combination, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the final set of overlapping targets. The analysis was conducted using the DAVID (Database for Annotation, Visualization and Integrated Discovery) platform, and terms with an adjusted p-value (FDR) < 0.05 were considered statistically significant.

From the five databases, we retrieved 5,476 PCOS-related targets and 5,210 infertility-related targets from Genecards; 1,310 PCOS targets and 8 infertility targets from OMIM; 165 PCOS targets and 226 infertility targets from PharmGKB; 988 PCOS targets and 37 infertility targets from DisGeNet; and 32 PCOS targets from DrugBank. The TTD database provided an additional 9 infertility-related targets. After removing all duplicate entries, a total of 6,488 unique PCOS-related targets and 5,291 unique infertility-related targets were retained. The intersection of these two sets identified 2,500 shared targets, which were considered the common therapeutic targets for PCOS-related infertility (Figure 1).

The 2,500 common targets were reverse-mapped through the TCMSP database, resulting in the identification of 25,540 associated compounds. These compounds were then subjected to pharmacokinetic screening using PubChem and SwissADME with the criteria of “GI absorption = High” and “Druglikeness ≥ 2 Yes.” This filtering process yielded 1,545 active compounds predicted to have therapeutic potential for PCOS-related infertility (Table 1).

The 1,545 active compounds were traced back to their herbal sources, corresponding to 488 unique herbs and a total of 180 targets. A preliminary target-compound-herb network was constructed using Cytoscape 3.8.0, which comprised 2,213 nodes and 15,465 edges. An analysis of the network topology revealed the top 10 herbs with the highest number of associated targets: Pueraria lobata (Gegen, 143 targets), Morus alba (Sangye, 136 targets), Oroxylum indicum (Muhudie, 135 targets), Zanthoxylum bungeanum (Huajiao, 126 targets), Carthamus tinctorius (Honghua, 121 targets), Eriobotrya japonica (Pipaye, 120 targets), Ginkgo biloba (Yinxingye, 120 targets), Syzygium aromaticum (Dingxiang, 119 targets), Astragalus membranaceus (Huangqi, 118 targets), and Ephedra sinica (Mahuang, 118 targets). To focus on the most significant interactions, nodes with a Degree > 20 (representing 40 targets, 79 compounds, and 89 herbs) were selected to reconstruct a refined, core target-compound-herb network (Figure 2).

Our intersection analysis identified 2,500 shared targets between PCOS and infertility. From these, the TCMSP database and Cytoscape analysis helped pinpoint 40 core targets related to active herbal compounds. Among these, PTGS1 and PTGS2 are known to exacerbate chronic inflammation by promoting PGE2 synthesis, which stimulates androgen secretion from theca cells, thus aggravating insulin resistance and metabolic dysregulation (19). Sex hormone receptors (ESR1, ESR2, PGR, and AR) are increasingly recognized for their dual roles in both reproductive function and the modulation of inflammation and oxidative stress (20–23). Clinical evidence shows that DPP-4 inhibitors can reverse polycystic ovarian morphology and reduce serum androgen levels in women with PCOS (24). Additionally, dysfunction of the ADRB2 gene contributes to energy metabolism disturbances by impairing fatty acid release, and its polymorphisms have been linked to PCOS susceptibility (25). These identified core targets predominantly mediate processes of inflammation, oxidative stress, immune dysregulation, and metabolic dysfunction, aligning with the known pathophysiology of PCOS.

Among the identified active compounds, quercetin, kaempferol, and palmitic acid ranked highest by Degree value. Quercetin, a flavonoid with well-documented antioxidant, anti-inflammatory, and immunomodulatory properties, is widely investigated in gynecology (26). In PCOS models, it has been shown to suppress the Ox-LDL/TLR-4/NF-κB pathway, downregulating ovarian IL-1β, IL-6, and TNF-α expression (27), while also improving levels of SOD, CAT, and MDA to restore ovarian function (26). It also inhibits androgen receptor (AR) expression, thereby lowering androgen levels (28). Recent studies highlight its dual action on both ovarian tissue and the pituitary-ovarian axis to promote folliculogenesis, marking it as a promising therapeutic candidate for PCOS (29). Kaempferol, another flavonol, exhibits potent free radical scavenging and antioxidant effects, showing therapeutic potential for oxidative stress-related conditions (30). It has been found to reduce body weight, fasting glucose, and insulin resistance in PCOS rats by modulating hypothalamic inflammation and energy balance (31). Conversely, palmitic acid, a prevalent saturated fatty acid, is often elevated in the serum of PCOS patients (32) and is known to induce inflammation (33), oxidative stress (34), mitochondrial dysfunction, and insulin resistance in vitro (35, 36). The identification of these compounds aligns with the pathophysiology of PCOS, as they primarily target oxidative stress, chronic inflammation, and metabolic dysregulation.

The 488 herbs identified in this study were predominantly Warm and Pungent, followed by Cold, Bitter, Neutral, and Sweet medicines. This distribution reflects TCM’s therapeutic principles: Pungent-Warm herbs are used to resolve phlegm-dampness, Bitter-Cold herbs to clear heat-toxicity, and Sweet-Neutral herbs to tonify visceral functions. A clinical study of 234 PCOS patients revealed that the most prevalent TCM syndrome was spleen deficiency with phlegm-dampness (44.4%), followed by kidney deficiency with liver stagnation (36.3%) and phlegm-stasis interaction (12.0%) (37), which is consistent with our findings on herbal flavor distribution. The meridian tropism analysis emphasized the Lung, Liver, Stomach, Spleen, and Kidney meridians, aligning with therapeutic strategies to regulate qi, resolve phlegm, clear heat, and tonify organs. The top five herbs screened by Degree value (>20), namely Ephedra sinica, Glycyrrhiza uralensis, Cinnamomum cassia, Bupleurum chinense, and Perilla frutescens, may be considered priority candidates for treating PCOS-related infertility.

Through a comprehensive analysis of frequency statistics, association rules, and cluster analysis, we identified a core herbal combination: “Ephedra sinica (Mahuang)–Magnolia officinalis (Houpo)–Bupleurum chinense (Chaihu)–Chrysanthemum morifolium (Juhua)–Angelica dahurica (Baizhi)–Morus alba (Sangye).”

The rationale for this combination can be understood through TCM theory. Classical texts suggest a strong link between lung function and menstruation. For example, a passage in The Yellow Emperor’s Inner Classic implies that when lung qi is obstructed, its descending function is impaired, which can disrupt the connection to the uterus and lead to amenorrhea (38). Modern lifestyle factors, such as excessive consumption of cold and sweet foods, can damage yang qi and lead to internal phlegm-dampness. Sedentary habits and lack of exercise can further impair the diffusion of lung qi, causing phlegm-dampness obstruction, which blocks uterine collaterals and leads to menstrual disorders and infertility. This aligns with the TCM axiom, “The spleen is the source of phlegm production, while the lungs store the phlegm.” Therefore, regulating lung-spleen function may improve polycystic ovarian morphology in PCOS patients.

The core formula employs Ephedra sinica, Bupleurum chinense, and Angelica dahurica to diffuse lung qi and release the exterior; Magnolia officinalis to dry dampness and strengthen the spleen; and Chrysanthemum morifolium and Morus alba to clear heat and drain fire. Interestingly, the classical formula Wuji San, used for accumulations of “qi, blood, phlegm, fluid, and food,” contains Ephedra sinica, Magnolia officinalis, and Angelica dahurica. Research suggests Wuji San may regulate inflammatory factors, thereby improving ovulation and pregnancy rates in PCOS patients (39).

The compatibility of this formula can be analyzed from a TCM perspective. Ephedra acts as the monarch herb, powerfully diffusing lung qi to open blockages. It is assisted by Bupleurum, which soothes liver qi, and Angelica dahurica, which dispels wind-dampness, together addressing both the upper (lung) and middle (liver/spleen) jiao. Magnolia officinalis serves as the minister herb, drying dampness and promoting qi circulation to resolve the phlegm-dampness pathology. Chrysanthemum and Morus alba act as assistant and envoy herbs, clearing heat that may arise from stagnation and guiding the formula’s actions downward. From a safety perspective, the inclusion of heat-clearing herbs like Chrysanthemum and Morus alba helps to balance the warming and drying nature of Ephedra and Magnolia officinalis, reducing the risk of depleting yin or generating excess heat. However, the use of Ephedra requires caution due to its cardiovascular and central nervous system stimulant effects; its dosage and the patient’s condition must be carefully monitored in any future clinical application. This predicted synergy and balancing mechanism provides a strong rationale for its validation in animal models.

Bupleurum chinense, with its exterior-releasing and liver-soothing properties, is widely used for gynecological disorders, especially those related to emotional stress and liver qi stagnation (40, 41). For instance, Xiaoyao San, which features Bupleurum, may enhance endometrial receptivity in PCOS by regulating VEGF signaling (42). Similarly, historical texts note that Morus alba (mulberry leaf) and Chrysanthemum morifolium (chrysanthemum) are key herbs for treating uterine bleeding caused by liver heat (43), a condition that can manifest in PCOS. Modern TCM practitioners like Professor Shen Shaogong have successfully used modified versions of formulas containing these herbs to regulate yin-yang balance in PCOS patients (44).

Modern pharmacological studies lend further support to this combination. Ephedrine from Ephedra sinica has anti-inflammatory effects and may improve metabolic rate and circulation, countering insulin resistance (45). Magnolol from Magnolia officinalis demonstrates potent antioxidant and anti-inflammatory activity and improves insulin resistance (46–48). Active compounds in Bupleurum chinense modulate pathways like PI3K-AKT to improve glucose metabolism (49). Components of Morus alba (50), Angelica dahurica (51), and Chrysanthemum morifolium (52) have also been shown to improve glucose tolerance, insulin secretion, and lipid metabolism while fighting oxidative stress.

Our study’s findings are consistent with prior network pharmacology research on PCOS, yet offer a unique perspective due to the reverse methodology. For example, a study on the Guizhi Fulingwan formula identified targets related to inflammation and apoptosis, such as AKT1, TNF, and IL6 (53), which overlap with our findings. Another study on the Zishen Yutai Pill highlighted pathways in cancer and PI3K-Akt signaling (54). While these studies confirm the importance of inflammation and metabolic regulation, our reverse approach started from a broader disease-target landscape, leading to the identification of a novel herbal combination that emphasizes the role of the lung system in PCOS, a connection less explored in conventional network pharmacology analyses.

The GO enrichment analysis highlighted oxidative stress-related processes, while KEGG analysis pointed to endocrine resistance, TNF signaling, and PI3K-Akt signaling pathways. These findings collectively suggest that the core herbs treat PCOS-related infertility by ameliorating oxidative stress and endocrine dysfunction, which aligns perfectly with the functions of the identified PCOS-infertility targets.

This study has several limitations that should be acknowledged. First, as a purely computational investigation, its findings are hypothesis-generating and lack experimental or clinical validation. Future studies involving cell culture experiments, animal models (e.g., letrozole-induced PCOS rats), and eventually clinical trials are necessary to verify the efficacy and safety of the predicted herbal combination. Second, the reliance on public databases for targets and compounds introduces potential biases, as some herbs or compounds may be underrepresented or incompletely annotated. The data does not account for tissue-specific gene expression or pharmacodynamics, which could influence therapeutic outcomes. Third, the statistical analysis of herbal properties, while informative, has inherent limitations. Our classification of herbs into therapeutic categories was based on their primary functions as documented in standardized texts, which does not account for their multiple effects or their specific roles (e.g., monarch, minister) within a formula. This could introduce bias, as suggested by the reviewer. For instance, high-frequency herbs with broad applications, such as licorice, might influence the distribution across categories. Future studies could employ weighted statistical methods and incorporate multi-center prescription data to refine these findings. The suggestion to use a Sankey diagram to visualize channel-efficacy flow is excellent and represents a valuable direction for future, more granular analyses. Fourth, the screening criteria for active compounds (e.g., high GI absorption, druglikeness) might have inadvertently excluded potentially effective natural metabolites that do not meet these stringent pharmaceutical filters but could still exert biological effects. Fifth, this study did not address crucial clinical considerations such as optimal dosage, potential herb-drug interactions, or toxicity of the proposed herbs. Finally, PCOS is a highly heterogeneous syndrome with multiple phenotypes (e.g., lean vs. obese PCOS) and diverse TCM syndromic patterns. Our analysis treated PCOS as a single entity and did not differentiate between these subtypes, which may require tailored therapeutic approaches.