Sun’s Bushen Huayu Formula Restores Fertility in Endometriosis by Modulating Th1 and Th2 Cytokine Balance in Clinical and Animal Models

Endometriosis (EMs) is a chronic, estrogen-dependent gynecological disorder characterized by the presence of endometrial glands and stroma outside the uterine cavity, primarily on the ovaries, pelvic peritoneum, and other pelvic organs^1,2. It affects approximately 10%-15% of women of reproductive age and is identified in up to 50% of infertile women³. The disease manifests as chronic pelvic pain, dysmenorrhea, and infertility, which severely affect women's quality of life and psychological well-being⁴. Epidemiological surveys indicate that the prevalence of endometriosis among infertile women ranges from 25% to 50%⁵, underscoring its strong association with reproductive failure.

The pathogenesis of EMs is multifactorial, involving retrograde menstruation, immune dysfunction, endocrine imbalance, and genetic and epigenetic factors⁶. Among these, immune dysregulation plays a pivotal role in both the establishment of ectopic lesions and infertility⁷. The endometrium of women with EMs exhibits elevated inflammatory cytokines and altered immune cell composition, which can impair gamete interaction, embryo implantation, and endometrial receptivity⁸.

A successful pregnancy requires a finely tuned maternal immune adaptation, characterized by a shift toward a Th2-dominant profile, which promotes tolerance at the maternal-fetal interface. In contrast, excessive Th1-type immune activation-marked by elevated interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)-can lead to cytotoxic damage, implantation failure, and early pregnancy loss⁹. An imbalance in the Th1/Th2 ratio, with predominant Th1 responses, is thus considered a key immunological mechanism linking EMs to infertility¹⁰.

Current therapeutic approaches, including hormonal suppression (such as gonadotropin-releasing hormone agonists, progestins, or oral contraceptives), surgical lesion removal, and assisted reproductive technologies, can alleviate pain and improve fertility to some extent¹¹. However, these methods are often associated with recurrence, drug-related side effects, or incomplete restoration of fertility¹². Consequently, there is a growing need for alternative or complementary interventions that target immune dysregulation and restore reproductive homeostasis.

From the perspective of Traditional Chinese Medicine (TCM), EMs belong to the category of dysmenorrhea or pelvic mass and are primarily attributed to Qi stagnation and blood stasis resulting from kidney deficiency and poor circulation¹³. The therapeutic principle of Bushen Huayu-tonifying the kidney and activating blood circulation-has long been employed to treat infertility and menstrual disorders associated with blood stasis¹⁴.

Sun's Bushen Huayu Formula (BSHYF) is a classical prescription developed based on this principle, composed of ten herbal ingredients including Cuscuta chinensis, Lycium barbarum, Rehmannia glutinosa, Angelica sinensis, Salvia miltiorrhiza, and Cyperus rotundus. These herbs have been shown to possess diverse pharmacological effects, such as anti-inflammatory, antioxidative, endocrine-regulating, and immunomodulatory activities^15,16. Clinically, BSHYF has demonstrated beneficial effects in improving ovulatory function, menstrual regularity, and conception rates among EM patients^17,18. Nevertheless, its immunological mechanism, particularly its impact on Th1/Th2 cytokine balance, remains incompletely understood.

Therefore, this study was designed to investigate whether Sun's Bushen Huayu Formula alleviates infertility associated with EMs by restoring Th1/Th2 immune balance or not. To this end, we analyzed IFN-γ and IL-4 levels in clinical serum samples and examined Th1/Th2 cell proportions in an EMs rat model. Our integrated clinical and experimental findings aim to provide mechanistic insight into how this traditional prescription exerts immunomodulatory and fertility-promoting effects.

This study was reviewed and approved by the Ethics Committee of Fuzhou Traditional Chinese Medicine Hospital (Approval No. LC-2022-131). All participants provided written informed consent in accordance with the Declaration of Helsinki. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Fujian University of Traditional Chinese Medicine (Approval No. IACUC FJABR2024030201) and conducted following institutional guidelines for the ethical care and use of laboratory animals.

Safety considerations

All experimental procedures were performed under biosafety level 2 (BSL-2) conditions with full personal protective equipment, including a lab coat, gloves, a mask, and protective eyewear. The 5% chloral hydrate anesthetic was handled inside a fume hood to avoid inhalation exposure. Surgical instruments and needles were disposed of in sharps containers. Estradiol valerate, progesterone, and gentamicin solutions were prepared and administered using chemical-resistant gloves. All biological and chemical waste, including blood samples and animal tissues, was collected separately in labeled containers and disposed of through institutional incineration.

Human sample collection

A total of 80 patients were recruited from the outpatient department of Fuzhou Traditional Chinese Medicine Hospital. The diagnostic criteria were based on the relevant content of the Guidelines for Clinical Research on New Chinese Medicines for the Treatment of Pelvic Endometriosis. Inclusion criteria for EMs are listed as follow: 1) progressive dysmenorrhea; 2) discomfort in the lower abdomen and lumbosacral region during menstruation, progressively worsening; 3) progressively worsening cyclical rectal stimulation symptoms; 4) palpable tender nodules in the posterior fornix, uterosacral ligaments, or uterine isthmus; 5) adnexal masses with adhesion and nodularity, with patent fallopian tubes; 6) significant changes in the size of adnexal masses before and after menstruation (without anti-inflammatory treatment). Inclusion criteria for infertility are listed as follows: 1) meeting the diagnostic criteria for infertility; 2) no use of hormonal medications in the 3 months prior to admission; 3) regular menstrual cycles and normal male semen parameters. Exclusion criteria for infertility are listed as follows: 1) presence of uterine fibroids, pelvic infections, or gynecological malignancies; 2) coexisting conditions such as cardiovascular, liver, kidney, hematological diseases, or rectal cancer; 3) use of Western medications within 3 months or use of other hormonal agents; 4) presence of psychiatric or neurological disorders. According to the design and objectives of this study, clinical data were collected from EM patients meeting the inclusion criteria and patients without EMs. These patients were classified into two groups based on infertility diagnostic criteria: the EMs fertile group and the EMs infertile group, as well as the fertile group and infertile group of patients without EMs. Blood collection was performed during the follicular phase (day 3-5 of the menstrual cycle) to minimize hormonal variation.

Sample processing

Approximately 3 mL of fasting venous blood was collected from each participant during the non-menstrual period using heparinized tubes. For cytokine stimulation, 1 mL of heparinized blood was transferred into sterile tubes and stimulated with 20 ng/mL phorbol-12-myristate-13-acetate (PMA), 1 µg/mL ionomycin, and 10 µg/mL brefeldin A (BFA), each prepared in phosphate-buffered saline (PBS). The samples were incubated in suspension for 4 h at 37 °C in a humidified 5% CO₂ incubator with gentle mixing. Following stimulation, cells were immediately processed for surface and intracellular staining by ELISA.

Animals

A total of 40 healthy adult female SPF rats, weighing 200-240 g, and 20 healthy adult male SD rats, weighing 250-280 g, were provided by the Animal Experiment Center of Fujian University of Traditional Chinese Medicine. Female and male rats were housed separately, with five rats per cage, under a 12 h light and 12 h dark cycle. After 1 week of acclimatization under standard housing conditions, vaginal smears were performed on the female rats according to the guidelines in the Animal Experiment Methodology to identify those in proestrus. The selected females were weighed, numbered, and randomly assigned to four groups: blank control, model, traditional Chinese medicine, and aspirin, using a random number table. The remaining females were retained for the next estrous cycle to repeat the procedure, resulting in 10 rats per group.

Preparation of Sun's Bushen Huayu Formula (BSHYF) and hormone solutions

The BSHYF decoction consisted of Cuscuta chinensis (15 g), Lycium barbarum (15 g), Rehmannia glutinosa (15 g), Angelica sinensis (12 g), Luffa cylindrica (10 g), Cyperus rotundus (10 g), Salvia miltiorrhiza (12 g), Achyranthes bidentata (10 g), Plantago asiatica (15 g), and Glycyrrhiza uralensis (6 g). All herbs were decocted in distilled water (10x volume), filtered, and concentrated to yield a final extract equivalent to 1 g of crude herb per mL. For hormonal synchronization, estradiol valerate (1 mg tablet) was dissolved in 20 mL of distilled water containing 0.1% ethanol to prepare a 0.05 mg/mL suspension. Progesterone (2.5 mg capsule) was dissolved in 62.5 mL of 1% polysorbate-80 solution to yield a 0.04 mg/mL suspension. All hormone solutions were freshly prepared before use and stored at 4 °C in amber bottles to avoid light exposure.

Establishment of the endometriosis model and administration

At 2 days prior to surgery, female rats were orally administered estradiol valerate (0.5 mg/kg) once daily to ensure synchronization of the estrous cycle, which was confirmed by vaginal smears showing predominantly anucleated keratinized epithelial cells. Rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (6 mL/kg). A ~2 cm midline incision was made approximately 3 cm above the urethral orifice. The left uterine horn was excised, trimmed of fat, and placed in sterile saline to prevent drying during transplantation. The endometrial surface was then sutured diagonally onto the right abdominal wall using 4-0 absorbable sutures. For the control group, only laparotomy and closure were performed without tissue transplantation. After surgery, 0.1 mL of gentamicin solution was injected intraperitoneally once daily for 3 consecutive days to prevent infection. Rats were allowed to recover on a heated pad and monitored until full mobility returned.

Drug administration methods

Starting on postoperative day 3, rats were treated for 4 weeks as follows: Control group: 2.5 mL/kg body weight saline by oral gavage 2x daily at 10:00 and 16:00. EMs model group: 2.5 mL/kg saline 2x daily at 10:00 and 16:00. BSHYF group: 2.5 mL/kg Sun's Bushen Huayu Formula 2x daily at 10:00 and 16:00. Aspirin group: 2 mg/kg aspirin once daily at 16:00. Doses were calculated based on individual body weight and adjusted weekly.

Animal cohousing and specimen collection and processing

On day 11 post-modeling, vaginal smears were performed daily each morning to identify females in estrus. Successful mating occurred each evening at 18:00, with males and females housed in a 2:1 ratio. The following morning at 08:00, the presence of vaginal plugs and sperm in the vaginal smears was assessed. Females exhibiting a vaginal plug or sperm were considered successfully mated, marking day 1 of gestation. Females that successfully mated were separated from those that did not. After mating, all rats ceased receiving treatment and were maintained under standard care conditions. On day 8 after confirmed mating (or at the corresponding time point for unmated rats), approximately 1 mL of blood was collected from the tail vein for cytokine detection. Pregnancy rate for each group was calculated as follows:
Pregnancy Rate (%) = (Number of Pregnant Rats / Total Number of Rats in the Group) x 100

Flow cytometry analysis

For each sample, approximately 1 x 10⁶ cells were transferred into 100 µL of staining buffer. Surface staining was performed by adding 5 µL of anti-CD3 and 5 µL of anti-CD8 antibodies, followed by incubation for 15 min at room temperature (20-25 °C) in the dark. After surface staining, 1 mL of red blood cell lysis buffer (ammonium chloride-based) was added, and the cells were incubated for 10 min at room temperature before centrifugation and washing 2x with PBS. Permeabilization was performed by adding 0.5 mL of saponin-based buffer, incubating for 10 min at 4 °C, and washing again. Intracellular staining was carried out by adding 5 µL of anti-IFN-γ-FITC and 5 µL of anti-IL-4-PE antibodies, followed by incubation for 20 min at 4 °C in the dark. Samples were washed 2x and resuspended in 500 µL of PBS before analysis.

Flow cytometry was conducted on a flow cytometer equipped with a 488 nm laser and 530/30 (FITC) and 585/42 (PE) bandpass filters. The voltages were set to 450 V for forward scatter (FSC), 500 V for side scatter (SSC), 480 V for FITC, and 520 V for PE. Compensation was adjusted using single-stained controls. Lymphocytes were first gated based on FSC/SSC parameters. CD³⁺CD^8- cells were identified as CD⁴⁺ helper T cells, and Th1/Th2 ratios were calculated based on IFN-γ⁺ (Th1) and IL-4⁺ (Th2) populations. A minimum of 10,000 lymphocyte events were collected per sample. Data were analyzed using FlowJo software, and Th1/Th2 ratios were calculated as IFN-γ⁺/IL-4⁺ cell percentages. Each sample was measured in duplicate to ensure analytical consistency.

Statistical analysis

Data were analyzed using SPSS 24.0. Continuous variables are expressed as mean ± standard deviation, with intergroup comparisons performed using t-tests. Categorical data are presented as percentages. A p-value of <0.05 was considered statistically significant.

Clinical cytokine analysis

A total of 80 serum samples were analyzed, including endometriosis (EMs) patients with infertility, EMs patients with successful pregnancy, infertile women without EMs, and healthy pregnant volunteers without EMs (n = 20 per group). The concentrations of IFN-γ and IL-4 in serum were determined by ELISA (Figure 1A). As shown in Figure 1B,C, the concentrations of IFN-γ and IL-4 in EM patients with infertility were higher than those in EM patients with pregnancy. Moreover, both cytokines showed higher mean levels in EMs groups compared with non-EMs controls. The IFN-γ/IL-4 ratio, representing the Th1/Th2 balance, was also elevated in EM patients with infertility compared with EM patients with pregnancy (Figure 1D). These findings indicate that Th1/Th2 imbalance is more pronounced in EM patients, especially those with infertility.

Establishment of the endometriosis animal model

The EM rat model was successfully established by autologous transplantation of uterine endometrial tissue onto the abdominal wall (Figure 2A). Representative surgical and postoperative images are shown in Figure 2B. All rats recovered normally after surgery without infection or complications, confirming successful modeling. The successful establishment of the EM rat model provided a reliable basis for evaluating the therapeutic effects of Sun's Bushen Huayu Formula (BSHYF).

Observation of pregnancy rate

Vaginal smears were used to monitor the estrus cycle, and representative cytological features at different stages are shown in Figure 3A. In the pre-estrus stage, vaginal smears predominantly contained nucleated epithelial cells; during estrus, keratinized anucleated epithelial cells were dominant; and during diestrus, leukocytes appeared in the background. These findings confirmed accurate estrus determination, providing a basis for subsequent pregnancy assessment. As shown in Figure 3B, the pregnancy rate in the EMs model group was the lowest (20%), while treatment with BSHYF increased the rate to 50%. The aspirin group, used as a positive control, exhibited a pregnancy rate of 70%. These results demonstrate that Sun's Bushen Huayu Formula significantly improved fertility outcomes in EM rats.

Regulation of Th1/Th2 balance by Sun's Bushen Huayu formula

Flow-cytometric analysis was performed to evaluate Th1/Th2 balance in peripheral blood lymphocytes (Figure 4). Representative plots of IFN-γ+CD4+ (Th1) and IL-4+CD4+ (Th2) cell populations are shown in Figure 4A-B. Quantitative analysis revealed that the proportion of Th1 cells was highest in the EMs group (9.5%) and decreased significantly following BSHYF treatment (4.9%), approaching the level of the aspirin group. Similarly, the percentage of Th2 cells was markedly elevated in EM rats and reduced after BSHYF administration (12.4% to 5.36%, p < 0.001) (Figure 4C-D). The Th1/Th2 ratio in EM rats was the lowest among all groups (79.06% ±3.89%), while treatment with BSHYF increased it to 110.21% ±13.05%, slightly exceeding the aspirin group (100.32% ± 12.53%; Figure 4E). These results suggest that Sun's Bushen Huayu Formula effectively restored Th1/Th2 immune balance, which may underlie its therapeutic effect on EMs-related infertility.

Collectively, both clinical and animal studies demonstrated that patients and rats with endometriosis exhibited a significant Th1/Th2 imbalance, characterized by elevated IFN-γ and IL-4 levels. Treatment with Sun's Bushen Huayu Formula restored immune balance and improved pregnancy outcomes, supporting its potential as a therapeutic approach for EM-associated infertility.

DATA AVAILABILITY

All data generated or analyzed during this study have been fully presented and discussed in the article. The raw data is provided in Supplementary File 1.

Figure 1: Detection of IL-4 and IFN-γ in clinical serum samples. (A) Schematic overview of sample grouping and cytokine detection design. 80 serum samples were divided into four groups: EMs with infertility, EMs with pregnancy, infertility without EMs, and pregnancy without EMs (n = 20 per group). (B-C) Serum concentrations of IFN-γ and IL-4 were measured by ELISA. (D) IFN-γ/IL-4 ratio representing Th1/Th2 balance. Data are presented as mean ± SD. n = 20 per group. Statistical analysis was performed using unpaired two-tailed t-tests; p < 0.05 was considered significant. Please click here to view a larger version of this figure.

Figure 2: Construction of the endometriosis (EMs) animal model. (A) Experimental timeline showing hormonal synchronization, uterine tissue autotransplantation, drug administration, and mating schedule. (B) Representative surgical and postoperative photographs illustrating uterine horn excision and implantation. Scale bars = 1 cm. Please click here to view a larger version of this figure.

Figure 3: Estrus cycle assessment and pregnancy outcomes in rats. (A) Representative vaginal smear cytology at different estrus stages stained with crystal violet. Pre-estrus: large nucleated epithelial cells; estrus: polygonal anucleated keratinized cells; diestrus: leukocytes and keratinized cells. Magnification 100x. (B) Pregnancy rate (%) in each group calculated as (Number of Pregnant Rats / Total Rats per Group) x 100. Data are shown as mean ± SD (n = 10 rats per group). Significance was determined using one-way ANOVA followed by Tukey's post hoc test. p < 0.05, ** p < 0.01 vs model group. Please click here to view a larger version of this figure.

Figure 4: Sun's Bushen Huayu Formula regulates Th1/Th2 balance in EMs rats. (A-B) Representative flow-cytometry plots showing IFN-γ+CD4+ (Th1) and IL-4+CD4+ (Th2) cell populations. (C-D) Quantitative analysis of Th1 and Th2 percentages. (E) Th1/Th2 ratio calculated as IFN-γ+/IL-4+ cell percentage. Data are expressed as mean ± SD (n = 10 rats per group). Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test. p < 0.05, ** p < 0.01 vs model group. Please click here to view a larger version of this figure.

Supplementary File 1: Raw data. Please click here to download this file.

This study demonstrates that immune imbalance plays a crucial role in EM-associated infertility and that Sun's Bushen Huayu Formula (BSHYF) may alleviate infertility by modulating Th1/Th2 cytokine balance. Clinically, we observed elevated IFN-γ and IL-4 levels and an increased IFN-γ/IL-4 ratio in EM patients, especially those with infertility, suggesting a disturbed Th1/Th2 equilibrium. In animal experiments, BSHYF treatment effectively reduced the Th1/Th2 ratio, restored cytokine homeostasis, and increased pregnancy rates from 20% to 50%, indicating that immune regulation may be a key mechanism underlying its therapeutic effect.

From an immunological standpoint, excessive Th1-type responses contribute to local inflammation, cytotoxicity, and embryo implantation failure, whereas Th2 dominance promotes tolerance and supports pregnancy maintenance^19,20. The ability of BSHYF to rebalance Th1/Th2 responses suggests that it helps create an immune environment conducive to implantation and early gestation. This aligns with emerging evidence that modulating cytokine profiles in the uterus and peripheral circulation can improve fertility outcomes in EMs²¹.

From a TCM perspective, EMs correspond to Qi stagnation and blood stasis with underlying kidney deficiency. The treatment principle of Bushen Huayu emphasizes nourishing the kidney, activating blood flow, and resolving stasis to restore reproductive harmony. The observed immunomodulatory effect of BSHYF provides a modern biological interpretation of this principle. Its herbal components have been individually reported to exert immunoregulatory actions: Salvia miltiorrhiza suppresses proinflammatory cytokine release and oxidative stress²²; Rehmannia glutinosa controls blood sugar²³; and Angelica sinensis modulates macrophage and T-cell responses²⁴. The synergy of these actions may contribute to the overall restoration of immune homeostasis and reproductive function observed in this study.

Despite these promising findings, certain limitations must be acknowledged. First, the clinical sample size was relatively modest, which may limit the generalizability of the results. Second, the rat autotransplantation model recapitulates major pathological features of EMs but cannot fully simulate the chronic and multifactorial nature of the human disease. Third, the treatment duration and follow-up period were relatively short, and long-term effects on recurrence and hormonal cycling remain to be investigated. Finally, this study focused primarily on Th1/Th2 cytokines; other immune axes, such as Treg/Th17 balance, NK cell cytotoxicity, and macrophage polarization, were not examined.

Alternative approaches could further strengthen future research. Broader immune profiling using multiplex cytokine assays, single-cell transcriptomics, or immunohistochemical analysis of ectopic lesions could provide deeper mechanistic insights. Longitudinal studies assessing cytokine dynamics throughout the menstrual cycle, combined with endometrial receptivity assays or uterine imaging, may clarify how BSHYF modulates the immune-endocrine interface during implantation.

The clinical implications of this work are twofold. First, by targeting immune imbalance rather than solely hormonal suppression, BSHYF offers a complementary therapeutic strategy for EM-associated infertility, particularly for patients seeking fertility preservation or those intolerant to hormonal therapy. Second, the integrated methodology combining clinical data, animal modeling, and flow cytometric immune assessment provides a reproducible framework for evaluating other TCM formulations with potential reproductive benefits.

In conclusion, our findings indicate that Sun's Bushen Huayu Formula may improve fertility in EMs by modulating Th1/Th2 immune balance, thereby restoring the immune microenvironment required for successful conception. Although additional studies are warranted to validate these findings and elucidate molecular pathways, this research provides important mechanistic evidence linking traditional therapeutic concepts with contemporary immunological mechanisms and highlights the potential of immunomodulatory herbal therapy as a novel adjunct in managing EMs-related infertility.