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

Summary

Sun's Bushen Huayu formula alleviates endometriosis (EM)-related infertility by regulating the Th1/Th2 cytokine balance, as shown in clinical samples and rat experiments. It reduces the elevated IFN-γ/IL-4 ratio in EM patients, especially infertile ones, boosts pregnancy rates in EM rats from 20% to 50%, and outperforms aspirin in balancing Th1/Th2.

Abstract

Endometriosis (EMs) is a common gynecological disorder frequently associated with infertility, yet the underlying immune mechanisms remain poorly understood. Sun's Bushen Huayu Formula (BSHYF) has been clinically applied to improve fertility in patients with EMs, but its immunological mechanism remains unclear. This study investigated whether BSHYF alleviates EMs-related infertility by modulating the Th1/Th2 cytokine balance. A total of 80 serum samples from women with or without EMs were analyzed and further stratified into pregnancy and infertility groups (n = 20 each). Levels of IFN-γ and IL-4 were measured by ELISA, and the IFN-γ/IL-4 ratio was calculated. In parallel, an EMs rat model was established and divided into four groups: control, EMs, EMs + BSHYF, and EMs + aspirin. After treatment, fertility outcomes were evaluated, and flow cytometry was performed to assess Th1 (IFN-γ⁺) and Th2 (IL-4⁺) lymphocyte proportions. Clinically, EM patients-particularly those with infertility-showed a significantly elevated IFN-γ/IL-4 ratio compared with controls. In the animal model, BSHYF treatment improved the pregnancy rate from 20% to 50% and effectively corrected the Th1/Th2 imbalance. Notably, BSHYF demonstrated a stronger immunoregulatory effect on Th1/Th2 cytokine balance than aspirin, accompanied by improved reproductive performance. These findings indicate that BSHYF may alleviate infertility associated with EMs by restoring Th1/Th2 cytokine homeostasis, providing new mechanistic insights into immunomodulation and supporting its therapeutic potential as a complementary approach for EMs-related infertility.

Introduction

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.

Protocol

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.

Results

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 1...

Discussion

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 pre...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Rat CD4	MULTI SCIENCES	F3100403	Stream cytometry detects CD4
FITC anti-rat IFN-γ Antibody	Biolegend	507804	Stream cytometry detects IFN-γ
Flow cytometry analyzer	BD	FACSCalbur	Flow cytometry
Flow Cytometry Staining Buffer	MULTI SCIENCES	S1001	Flow cytometry detection buffer reagent
FoxP3/Transcription Factor Staining Buffer Kit	MULTI SCIENCES	IC001	Flow cytometry detection buffer reagent
PE anti-rat IL-4 Antibody	Biolegend	511906	Stream cytometry detects IL-4
PerCP anti-rat CD8a Antibody	Biolegend	201712	Stream cytometry detects CD8
SD rats	S(Beijing) Biotechnology Co., Ltd.	2019-0010	Model construction