Zhuyang-Huazheng Formula for Preoperative Bleeding Control in Endometrial Polyp-Associated Abnormal Uterine Bleeding: A Retrospective Cohort Study

Summary

This retrospective cohort study evaluates the Zhuyang-Huazheng Formula for preoperative bleeding control in women with endometrial polyp-associated abnormal uterine bleeding and explores polyp Ki-67/p53 biomarker profiles associated with treatment outcomes.

Abstract

Endometrial polyps (EPs) are a common structural cause of abnormal uterine bleeding (AUB) in premenopausal women, and short-term bleeding control before hysteroscopic polypectomy is clinically important. This single-center retrospective cohort study evaluated preoperative bleeding outcomes associated with Zhuyang-Huazheng Formula (ZHF), a hospital-formulated traditional Chinese medicine regimen, compared with tranexamic acid (TXA), and explored Ki-67/p53 expression profiles in polyp tissue. A total of 181 premenopausal women with EP-associated AUB treated between January 2022 and December 2024 were included; 87 received ZHF and 94 received TXA. The primary endpoint was change in Pictorial Blood Loss Assessment Chart (PBAC) score. Secondary outcomes included PBAC responder rate, hemoglobin improvement, time to clinical stability (TtCS), adverse events, and Ki-67/p53 expression in polyp and adjacent endometrial tissues.

Baseline characteristics were comparable between groups. PBAC scores decreased significantly in both groups, from 210.70 ± 56.60 to 93.03 ± 27.56 with ZHF and from 221.04 ± 54.27 to 92.85 ± 29.56 with TXA. The absolute reduction in PBAC score was significantly greater with TXA than with ZHF (128.19 ± 34.11 vs. 117.67 ± 35.99; P = 0.045). No significant between-group differences were observed in PBAC responder rate, hemoglobin gain, TtCS, or adverse-event rate, and no serious adverse events occurred. Adjacent endometrial Ki-67 indices and p53 H-scores were similar between groups, whereas polyp tissue from the ZHF group showed lower Ki-67 indices and p53 H-scores. Higher polyp-to-adjacent biomarker ratios were associated with greater bleeding burden and longer TtCS. ZHF was associated with short-term improvement in preoperative bleeding among women with EP-associated AUB, although TXA produced a significantly greater reduction in PBAC score. Several secondary clinical outcomes did not differ significantly between groups. The lower polyp-tissue Ki-67 indices and p53 H-scores observed in the ZHF group are exploratory findings and should not be interpreted as evidence of treatment-induced biological modification.

Introduction

Endometrial polyps (EPs) are benign overgrowths of endometrial glands and stroma that project into the uterine cavity and commonly present with abnormal uterine bleeding (AUB). Contemporary reviews and guidelines indicate that AUB is the most frequent symptom of EPs, affecting roughly two-thirds of women with this condition¹. AUB impairs health-related quality of life and frequently leads to iron-deficiency anemia and repeated health-care use, underscoring the need for effective symptom control before definitive management².

For symptomatic EPs, hysteroscopic polypectomy is the standard of care because it removes the structural source of bleeding while permitting histopathology³. Because abnormal uterine bleeding caused by a structural intrauterine lesion may coexist with premalignant or malignant endometrial pathology, adequate diagnostic assessment remains essential before and during preoperative management. Clinical evaluation and transvaginal ultrasonography, together with hysteroscopic assessment when indicated, are important for characterizing the underlying lesion, while histopathological examination remains necessary for definitive diagnosis. Short-term pharmacological bleeding control should therefore be regarded as bridging management and should not delay or replace appropriate investigation of the underlying endometrial pathology. However, many patients continue to experience AUB while investigations are underway or while awaiting surgery. The NICE NG88 guideline therefore recommends short-term pharmacological control—specifically, offering tranexamic acid (TXA) and/or NSAIDs—during this interval⁴. TXA reduces menstrual blood loss by about 26%–60% and is generally well tolerated, although caution is advised in patients with thromboembolic risk.

Given the short duration of effect and potential risks of conventional agents, complementary approaches may add value in the pre-operative period. Traditional Chinese medicine (TCM) formulations that “activate blood and resolve stasis” have long been used clinically for bleeding and pain in gynecological disorders. Observational data from Taiwan suggest that women with dysfunctional uterine bleeding (a non-structural AUB category) frequently use TCM, and, in some analyses, complementary Chinese herbal medicine has been associated with lower subsequent surgery rates, although high-quality randomized evidence remains scarce⁵. A prior systematic review also suggested the potential benefit of Chinese herbal medicine for dysfunctional uterine bleeding, while emphasizing heterogeneity and methodological limitations^6,7. These signals support studying standardized, well-described formulas as adjunctive pre-operative options for symptom relief.

Beyond symptomatic control, exploratory assessment of tissue biomarker profiles may provide additional biological context for the clinical features observed during pre-operative management. Ki-67 is a canonical marker of cellular proliferation in endometrial tissues⁸, whereas p53 is a tumor-suppressor protein whose immunohistochemical expression is context-dependent and may reflect alterations in cell-cycle regulation⁹. Evaluation of Ki-67 and p53 in resected polyp tissue may therefore help characterize tissue biomarker patterns associated with the clinical phenotype, without implying that any between-group differences were caused by treatment¹⁰. Based on these considerations, this single-center retrospective cohort study aimed to evaluate short-term pre-operative bleeding outcomes associated with a standardized hospital-formulated TCM compound—the Zhuyang-Huazheng formula—in women with EPs awaiting hysteroscopic polypectomy, and to explore whether Ki-67 and p53 expression profiles in resected polyp tissue differed between the ZHF and TXA groups.

Protocol

The study protocol was reviewed and approved by the Ethics Committee of Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine, Hebei Province (Approval No. CZX2023-KY-164) and was conducted in accordance with the Declaration of Helsinki. The same committee waived the requirement for written informed consent due to the retrospective nature of the study and the use of fully anonymized data.

Study design and setting
This was a retrospective comparative cohort study conducted at Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine between January 2022 and December 2024. The study aimed to evaluate clinical hemostatic outcomes, exploratory tissue biomarker profiles, and safety of ZHF compared with TXA in premenopausal women presenting with AUB secondary to EPs.

Patient selection
Women aged 25–50 years who underwent hysteroscopic evaluation and subsequent hysteroscopic polypectomy, with histopathological confirmation of benign endometrial polyps, were screened for eligibility. Final inclusion required histopathological confirmation of benign EPs, thereby excluding lesions with hyperplasia, atypia, or malignancy from the analytic cohort.

Inclusion criteria were as follows: regular menstrual cycles prior to onset of AUB; ultrasound- or hysteroscopy-confirmed diagnosis of EPs; presentation with abnormal uterine bleeding attributable to endometrial polyps (AUB-P) persisting for at least two cycles; and complete clinical, laboratory, and follow-up data available for analysis. Exclusion criteria were the presence of uterine fibroids ≥3 cm, adenomyosis, or intrauterine adhesions; pregnancy, lactation, or perimenopausal status; use of hormonal therapy, intrauterine devices, or anticoagulants within the preceding 3 months; previous or current histopathological evidence of endometrial hyperplasia with atypia, atypical endometrial lesions, or malignancy; and incomplete medical records or follow-up shorter than one treatment cycle. After applying these criteria, a total of 181 patients were included: 87 received ZHF, and 94 received TXA.

Treatment protocols
Patients were classified into treatment groups according to the regimen prescribed as part of routine outpatient management before hysteroscopic polypectomy. Treatment selection was determined by routine clinical practice rather than by the study protocol and was not randomized.

In the ZHF group, patients received a standardized hospital-formulated Zhuyang-Huazheng Formula prepared and dispensed by the Traditional Chinese Medicine pharmacy of Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine. The formula had a fixed composition throughout the study period and was not individually modified according to patient symptoms. Each daily prescription consisted of Bupleuri Radix (Chaihu; Bupleurum chinense DC., root), 10 g; Curcumae Rhizoma (Ezhu; Curcuma phaeocaulis Valeton, rhizome), 10 g; Paeoniae Radix Alba (Baishao; Paeonia lactiflora Pall., root), 15 g; Sparganii Rhizoma (Sanleng; Sparganium stoloniferum Buch.-Ham., tuber), 10 g; Astragali Radix (Huangqi; Astragalus membranaceus [Fisch.] Bunge, root), 20 g; and Agrimoniae Herba (Xianhecao; Agrimonia pilosa Ledeb., aerial part), 30 g. The detailed composition, botanical sources, medicinal parts, and amounts of the formula are summarized in Supplementary Table 1.

All crude herbal materials were obtained through the hospital pharmacy and were subjected to routine identity and quality inspection in accordance with the applicable institutional standards and the Pharmacopoeia of the People’s Republic of China. For each daily prescription, the herbal materials were mixed, soaked in approximately 800 mL of purified water for 30 min, and subsequently decocted twice using the standardized hospital-pharmacy decoction procedure. The first extraction was performed for 30 min after boiling, followed by a second extraction with approximately 600 mL of water for 25 min. The two aqueous extracts were filtered, combined, and concentrated to a final volume of approximately 400 mL. The resulting decoction was divided into two sealed 200 mL single-dose packages and stored under refrigerated conditions (2–8 °C) until dispensing. Patients were instructed to warm the decoction before administration and to take 200 mL orally twice daily, in the morning and evening after meals, for 15 consecutive days during the preoperative treatment cycle.

The formulation, preparation procedure, dose, and treatment course were documented in sufficient detail to facilitate reproducibility in accordance with established recommendations for the precise reporting of Chinese herbal medicine interventions¹¹.

In the TXA group, patients received tranexamic acid tablets at 500 mg three times daily for 5 consecutive days during the corresponding preoperative treatment cycle. Both groups were advised to maintain their normal diet and exercise habits and to avoid using hormones or hemostatic drugs simultaneously. Subsequently, all patients underwent hysteroscopic polyp resection using standard electrosurgical techniques by experienced gynecologists. Hysteroscopic polypectomy was preferentially scheduled after cessation of menstrual bleeding and during the early proliferative phase when clinically feasible. However, because treatment and surgery were delivered as part of routine clinical practice, the timing of surgery was not strictly standardized to an identical menstrual-cycle phase in all patients.

Clinical and laboratory assessments
Baseline Characteristics:
Demographic and clinical parameters, including age, body mass index (BMI), menstrual cycle length, and parity, were extracted from medical records. Transvaginal ultrasonography was performed in the early proliferative phase to measure endometrial thickness and polyp size. The number of lesions was recorded as single or multiple.

Efficacy endpoints:
The primary efficacy endpoint was the change in menstrual blood loss assessed using the Pictorial Blood Loss Assessment Chart (PBAC). PBAC scores were compared between the pretreatment cycle (C–2) and the cycle immediately preceding hysteroscopic polypectomy (C–1).

Secondary endpoints:
Hemoglobin concentration (g/dL) before and after treatment;
Proportion of PBAC responders, defined as ≥50% reduction in PBAC from baseline;
Time to clinical stability (TtCS), defined as the number of days required for cessation or normalization of uterine bleeding during the treatment cycle.

Histopathological and biomarker evaluation:
Endometrial specimens were collected from both polyp tissue and adjacent normal endometrium at the time of hysteroscopic surgery. Adjacent endometrium was defined as non-polypoid endometrial tissue obtained from the same patient during the same hysteroscopic procedure and histologically distinct from the polyp body and the immediate polyp–endometrium transition zone. Only morphologically evaluable endometrial tissue with preserved glandular and stromal architecture was included for comparative assessment. Samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 µm thickness.

Immunohistochemistry (IHC) was performed using monoclonal antibodies against Ki-67 (clone MIB-1, 1:200) and p53 (clone DO-7, 1:100). Slides were incubated overnight at 4 °C, visualized using DAB chromogen, and counterstained with hematoxylin. Both antibody assays had been validated for routine diagnostic use in the institutional pathology laboratory according to established laboratory procedures. Appropriate positive-control sections were included in each staining run, and available internal positive staining was routinely reviewed as an additional quality-control measure to confirm adequate staining performance. Negative-control preparations processed without the primary antibody were also assessed according to the routine laboratory protocol. Only staining runs showing the expected control results were considered technically valid and included in the quantitative evaluation.

Before quantitative scoring, each slide was reviewed at low magnification to identify representative and well-preserved epithelial regions. Five non-overlapping high-power fields were then selected from these evaluable regions for quantitative assessment. Field selection was not based on areas showing the highest staining intensity. Areas with prominent inflammation, extensive hemorrhage, tissue breakdown or fragmentation, crush artifact, or otherwise inadequate tissue preservation were excluded from quantitative evaluation. All immunohistochemical slides were scored independently by two pathologists who were blinded to treatment-group information. Each pathologist independently evaluated the selected five high-power fields and recorded the Ki-67 index and p53 H-score without access to the other pathologist’s assessment. After completion of independent scoring, cases with discrepant assessments were jointly reviewed, and the final values used for analysis were established by consensus.

Ki-67 index (%) was calculated as the mean percentage of positively stained nuclei among ≥500 epithelial cells. p53 expression was quantified using the H-score method, ranging from 0 to 300 (intensity × percentage of positive nuclei). As specified above, the p53 H-score was treated as an exploratory continuous measure of immunohistochemical expression rather than as a formal pattern-based classification of p53 status. The polyp-to-adjacent expression ratios for Ki-67 and p53 were calculated as exploratory relative measures of immunohistochemical expression between paired tissue compartments.

Safety assessment:
Adverse events (AEs) were retrospectively collected from outpatient records and telephone follow-up logs. Assessed parameters included gastrointestinal reactions (nausea, vomiting, abdominal discomfort), hepatic enzyme elevations, renal function abnormalities (estimated glomerular filtration rate <60 mL/min/1.73 m²), and allergic manifestations. A composite non-gastrointestinal safety endpoint was predefined as the occurrence of hepatic function abnormality, renal function abnormality, allergic manifestation, or any serious adverse event (SAE).

Kaplan–Meier analysis of time to clinical hemostatic stability:
Kaplan–Meier–derived cumulative probability curves were constructed to visualize the time course of achieving clinical hemostatic stability after treatment initiation. Time to clinical stability was defined as the number of days from treatment initiation to the first documented cessation or normalization of uterine bleeding during the treatment cycle. The cumulative stabilization profiles of the ZHF and TXA groups were compared using the log-rank test.

Statistical analysis
Continuous variables were tested for normality using the Shapiro–Wilk test and expressed as mean ± standard deviation (SD). Intergroup comparisons were conducted using Student’s t-test or the Mann–Whitney U test, as appropriate. Categorical variables were analyzed using the χ² test or Fisher’s exact test. Paired t-tests were used for within-group pre/post comparisons. The Kaplan–Meier method with log-rank testing was applied to compare the cumulative probability of achieving clinical hemostatic stability between treatment groups. Spearman correlation analyses were performed to evaluate the associations between polyp-to-adjacent biomarker ratios and bleeding-related clinical parameters. Exploratory univariable and multivariable linear regression analyses were further conducted using baseline PBAC score and time to clinical stability (TtCS) as continuous dependent variables. For the baseline PBAC model, candidate covariates included age, body mass index, baseline hemoglobin, polyp size, multiple polyps, endometrial thickness, Ki-67 polyp-to-adjacent ratio, and p53 polyp-to-adjacent ratio. For the TtCS model, candidate covariates included treatment group, baseline PBAC score, baseline hemoglobin, polyp size, multiple polyps, Ki-67 polyp-to-adjacent ratio, and p53 polyp-to-adjacent ratio. All analyses involving Ki-67 and p53 expressions, including between-group comparisons, correlation analyses, and regression models, were considered exploratory and hypothesis-generating rather than confirmatory. Regression results are presented as standardized beta coefficients with 95% confidence intervals (CIs). A two-sided P < 0.05 was considered statistically significant. All statistical analyses were conducted using R software.

Results

Baseline characteristics of included patients
The patient selection process is summarized in Figure 1. A total of 181 premenopausal women with EP-associated AUB were included, among whom 87 received ZHF and 94 received TXA. Baseline demographic and clinical characteristics are summarized in Table 1. No statistically significant between-group differences were observed in age (38.08 ± 5.05 vs. 39.00 ± 5.42 years, P = 0.242), body mass index (23.97...

Discussion

This retrospective cohort study found substantial short-term improvement in bleeding-related outcomes in both the ZHF and TXA groups among women with EP-associated AUB. Importantly, the primary efficacy outcome differed between groups: TXA produced a significantly greater absolute reduction in PBAC score than ZHF (128.19 ± 34.11 vs. 117.67 ± 35.99, P = 0.045). In contrast, no statistically significant between-group differences were observed in PBAC responder rate, hemoglobin gain, or time to clinical s...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% neutral-buffered formalin	Servicebio Technology Co., Ltd.	G1101 or equivalent	Fixation of endometrial polyp and adjacent endometrial tissue
Adhesion microscope slides	Citotest Scientific Co., Ltd.	188105 or equivalent	Mounting of paraffin tissue sections for immunohistochemical staining
Automated immunohistochemistry stainer	Dako/Agilent Technologies	Autostainer Link 48	Standardized immunohistochemical staining workflow for Ki-67 and p53
DAB chromogen kit	Dako/Agilent Technologies	K3468	Chromogenic visualization of immunohistochemical staining
Diagnostic hysteroscopy system	KARL STORZ	26033AP / 26046BA or equivalent	Hysteroscopic evaluation and visualization of endometrial polyps
Digital microscope camera	Olympus Corporation	DP74	Acquisition of representative microscopic images of tissue staining
Electrosurgical generator	ERBE Elektromedizin GmbH	VIO 300D	Power source for hysteroscopic electrosurgical polypectomy
Hematoxylin staining solution	Servicebio Technology Co., Ltd.	G1004 or equivalent	Nuclear counterstaining after DAB visualization
Hysteroscopic electrosurgical resection system	Olympus / KARL STORZ	A22003A / 26055L or equivalent	Standard electrosurgical resection of endometrial polyps
Image analysis software	Image-Pro Plus / Media Cybernetics	Version 6.0	Assistance with microscopic image review and biomarker quantification when needed
Ki-67 primary antibody, clone MIB-1	Dako/Agilent Technologies	M7240	Immunohistochemical detection of Ki-67 expression; dilution 1:200
Light microscope	Olympus Corporation	BX53	Pathological review and high-power field assessment of IHC staining
Microsoft Excel	Microsoft Corporation	Microsoft 365 or equivalent	Data organization and preparation of tabular materials for submission
p53 primary antibody, clone DO-7	Dako/Agilent Technologies	M7001	Immunohistochemical detection of p53 expression; dilution 1:100
Paraffin embedding system	Leica Biosystems	EG1150H	Paraffin embedding of fixed endometrial tissue specimens
Pictorial Blood Loss Assessment Chart (PBAC)	Not applicable	N/A	Semi-quantitative assessment of menstrual blood loss before and after treatment
R software	R Foundation for Statistical Computing	version 4.2.2	Analysis of time to clinical hemostatic stability; Correlation analysis between biomarker ratios and bleeding-related parameters; Exploratory univariable and multivariable regression analysis
Rotary microtome	Leica Biosystems	RM2235	Preparation of 4 µm paraffin sections for histology and immunohistochemistry
Tranexamic acid tablets	Hunan Dongting Pharmaceutical Co., Ltd.	N/A	0.5 g/tablet; hospital formulary item;Antifibrinolytic comparator treatment; 500 mg three times daily for 5 days
Transvaginal ultrasound system	GE Healthcare	Voluson E8 Expert	Measurement of endometrial thickness and maximum polyp size
Zhuyang-Huazheng Formula oral liquid	Hospital pharmacy, Cangzhou Hospital of Integrated TCM-WM·Hebei	In-house preparation; N/A	Hospital-formulated traditional Chinese medicine regimen; 200 mL per dose twice daily for 15 days