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 e....

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 lev....

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 Fig.......

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