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 ± 3.16 vs. 23.70 ± 3.08 kg/m², P = 0.565), baseline PBAC score during the C–2 cycle (210.70 ± 56.60 vs. 221.04 ± 54.27, P = 0.212), hemoglobin concentration (11.61 ± 0.83 vs. 11.54 ± 0.96 g/dL, P = 0.601), endometrial thickness (9.39 ± 1.93 vs. 9.45 ± 1.98 mm, P = 0.836), or polyp size (1.31 ± 0.38 vs. 1.33 ± 0.43 cm, P = 0.713). The proportions of patients with single versus multiple polyps were also comparable between groups (72.4% vs. 63.8%, P = 0.731). These findings indicate that the measured baseline characteristics were broadly comparable between the two treatment groups.

Primary efficacy outcomes
To evaluate short-term preoperative bleeding control, we compared menstrual blood-loss reduction, hemoglobin improvement, PBAC responder rates, and time to clinical stability between the two treatment groups (Table 2, Figure 2). Both regimens were associated with marked reductions in menstrual blood loss during preoperative management. Mean PBAC scores decreased from 210.7 ± 56.6 to 93.0 ± 27.6 in the ZHF group and from 221.0 ± 54.3 to 92.9 ± 29.6 in the TXA group (both within-group P < 0.001; Figure 2A). The absolute reduction in PBAC score was significantly greater in the TXA group than in the ZHF group (128.19 ± 34.11 vs. 117.67 ± 35.99, P = 0.045; Figure 2B). However, PBAC responder rates, defined as a ≥50% reduction from baseline, were similar between groups (88.5% in ZHF vs. 90.4% in TXA, P = 0.858; Figure 2C). Hemoglobin concentrations also increased significantly after treatment in both groups, from 11.61 ± 0.83 to 12.23 ± 0.85 g/dL in the ZHF group and from 11.54 ± 0.96 to 12.13 ± 1.05 g/dL in the TXA group (both within-group P < 0.001; Figure 2D). The magnitude of hemoglobin gain did not differ significantly between groups (0.62 ± 0.30 vs. 0.59 ± 0.33 g/dL, P = 0.521; Figure 2E). No statistically significant between-group difference was observed by log-rank testing (P = 0.71), consistent with the absence of a significant between-group difference in TtCS shown in Figure 2F.

To further visualize the temporal dynamics of early bleeding stabilization, Kaplan–Meier–derived cumulative probability curves were constructed (Figure 3). The cumulative probability of achieving clinical hemostatic stability increased progressively over the treatment period in both groups, with closely overlapping stabilization profiles. No statistically significant between-group difference was observed by log-rank testing (P = 0.665), consistent with the comparable TtCS values shown in Figure 2F. Collectively, both regimens were associated with substantial short-term improvement in bleeding-related outcomes. However, the primary outcome favored TXA, which produced a significantly greater absolute reduction in PBAC score, whereas no statistically significant between-group differences were observed in PBAC responder rate, hemoglobin gain, or time to clinical stability.

Exploratory tissue biomarker analysis
To characterize tissue biomarker profiles, Ki-67 and p53 expressions were evaluated in both polyp tissue and adjacent endometrium (Figure 4, Table 3). Ki-67 index and p53 H-score in adjacent endometrium were comparable between the ZHF and TXA groups (Ki-67: 14.53 ± 5.20 vs. 15.28 ± 5.84, P = 0.355; p53: 93.77 ± 22.13 vs. 97.89 ± 24.08, P = 0.211). In contrast, polyp tissue from the ZHF group showed significantly lower Ki-67 index (18.97 ± 5.46 vs. 25.81 ± 5.67, P < 0.001) and p53 H-score (155.64 ± 40.51 vs. 210.45 ± 44.19, P < 0.001) than that from the TXA group. Consistently, the polyp-to-adjacent Ki-67 and p53 expression ratios were both significantly lower in the ZHF group (1.34 ± 0.32 vs. 1.92 ± 0.37 and 1.61 ± 0.38 vs. 2.15 ± 0.41, respectively; both P < 0.001). These findings describe between-group differences in polyp-associated Ki-67 and p53 expressions, whereas expression in adjacent endometrium remained similar between groups. Because the biomarkers were assessed in resected tissue at a single postoperative time point, these results were considered exploratory observations rather than evidence of treatment-mediated biological change.

Associations between tissue biomarker ratios and bleeding-related clinical parameters
To further explore the clinical relevance of the tissue biomarker findings, we assessed the associations between polyp-to-adjacent biomarker ratios and bleeding-related clinical parameters (Figure 5). The Ki-67 ratio was positively correlated with baseline PBAC score (Spearman ρ = 0.49, P < 0.001), and a similar positive correlation was observed for the p53 ratio (Spearman ρ = 0.55, P < 0.001). In addition, higher Ki-67 and p53 ratios were modestly associated with longer time to clinical stability (Ki-67 ratio: Spearman ρ = 0.27, P < 0.001; p53 ratio: Spearman ρ = 0.30, P < 0.001). These exploratory correlations suggest that higher polyp-associated biomarker ratios were associated with greater baseline bleeding burden and a longer time required to achieve clinical stability during preoperative management.

Exploratory regression analyses of baseline bleeding burden and time to clinical stability
To further evaluate whether tissue biomarker burden remained associated with clinical presentation after adjustment for other covariates, we performed exploratory univariable and multivariable linear regression analyses for baseline PBAC score and time to clinical stability (Figure 6). In the multivariable model for baseline PBAC score, lower hemoglobin concentration (adjusted β = -0.24, P < 0.001), larger polyp size (adjusted β = 0.14, P = 0.013), a higher polyp-to-adjacent Ki-67 ratio (adjusted β = 0.22, P < 0.001), and a higher polyp-to-adjacent p53 ratio (adjusted β = 0.29, P < 0.001) were independently associated with greater baseline bleeding burden. In contrast, age, body mass index, endometrial thickness, and multiple polyps were not independently associated with baseline bleeding severity.

In the multivariable model for time to clinical stability, higher baseline PBAC score (adjusted β = 0.24, P < 0.001), a higher Ki-67 ratio (adjusted β = 0.14, P = 0.023), and a higher p53 ratio (adjusted β = 0.17, P = 0.006) remained independently associated with longer TtCS. Treatment group was not an independent predictor of TtCS (TXA vs. ZHF: adjusted β = -0.04, P = 0.517), consistent with the comparable stabilization time observed in the primary efficacy analysis. Baseline hemoglobin showed a borderline inverse association with TtCS (adjusted β = -0.12, P = 0.051). Taken together, these exploratory analyses indicate that greater polyp-associated biomarker burden was independently associated with both heavier baseline bleeding and slower early clinical stabilization.

Safety outcomes
The safety profiles of ZHF and TXA were compared, revealing no significant differences in the incidence of adverse events between the two groups. As detailed in Table 4, in the ZHF group (n = 87), 7 patients (8.0%) experienced any adverse event, compared to 10 patients (10.6%) in the TXA group (n = 94), with a P-value of 0.57. Gastrointestinal reactions were the most common adverse events in both groups (5.7% in ZHF vs. 8.5% in TXA, P = 0.48), primarily manifesting as mild nausea (3.4% vs. 5.3%, P = 0.62) and abdominal discomfort (2.3% vs. 3.2%, P = 0.74).

Further analysis of specific adverse events, also presented in Table 4, indicated comparable rates of hepatic function abnormality (1.1% in ZHF vs. 2.1% in TXA, P = 0.62) and renal function abnormality (1.1% in ZHF vs. 3.2% in TXA, P = 0.38). Notably, no serious adverse events were reported in either treatment group. The composite non-gastrointestinal safety endpoint showed no statistically significant difference between the ZHF and TXA groups (2.3% vs. 5.3%, P = 0.29).

DATA AVAILABILITY:
The de-identified individual-level data supporting the findings of this study are provided in Supplementary File 1.

Figure 1: Patient selection, treatment-group allocation, and analytical framework of the retrospective cohort. Among 245 premenopausal women with endometrial polyps and abnormal uterine bleeding identified from hospital records, 64 were excluded according to prespecified criteria. The final retrospective cohort comprised 181 pathologically confirmed benign EP-AUB cases, including 87 patients treated with Zhuyang-Huazheng Formula and 94 treated with tranexamic acid. The included cohort was used for baseline comparison, clinical efficacy assessment, tissue biomarker analysis, and safety evaluation. Please click here to view a larger version of this figure.

Figure 2: Clinical hemostatic efficacy and hemoglobin improvement during preoperative management. (A) Pictorial Blood Loss Assessment Chart (PBAC) scores before and after treatment in the Zhuyang-Huazheng Formula (ZHF) and tranexamic acid (TXA) groups. Both groups showed significant within-group reductions in PBAC scores. (B) Absolute reduction in PBAC score from baseline to the preoperative assessment cycle, which was significantly greater in the TXA group. (C) Distribution of PBAC responder status, defined as a ≥50% reduction from baseline, showing similar responder rates between groups. (D) Hemoglobin concentrations before and after treatment showed significant within-group increases in both groups. (E) Hemoglobin gain from baseline to the preoperative assessment cycle, with no significant between-group difference. (F) Time to clinical stability, with no statistically significant difference between-group. Please click here to view a larger version of this figure.

Figure 3: Cumulative probability of achieving clinical hemostatic stability during preoperative management. Kaplan–Meier–derived cumulative probability curves depict the time course of achieving clinical stability after treatment initiation in the Zhuyang-Huazheng Formula (ZHF) and tranexamic acid (TXA) groups. The shaded areas represent 95% confidence intervals. No statistically significant difference in the cumulative stabilization profiles was observed between groups by log-rank testing. Numbers at risk are shown below the plot. Please click here to view a larger version of this figure.

Figure 4: Ki-67 and p53 expression profiles in polyp and adjacent endometrial tissues. (A–C) Ki-67 expression in adjacent endometrium, polyp tissue, and the polyp-to-adjacent expression ratio. (D–F) p53 expression in adjacent endometrium, polyp tissue, and the polyp-to-adjacent expression ratio. Ki-67 and p53 levels were comparable between groups in adjacent endometrium, whereas both markers and their corresponding polyp-to-adjacent ratios were significantly lower in the ZHF group than in the TXA group within polyp-related measures. Each point represents one patient; violins indicate data distributions, boxes indicate interquartile ranges with median lines, and diamonds indicate means. ZHF, Zhuyang-Huazheng Formula; TXA, tranexamic acid. Please click here to view a larger version of this figure.

Figure 5: Associations between tissue biomarker ratios and bleeding-related clinical parameters. (A) Correlation between the polyp-to-adjacent Ki-67 ratio and baseline PBAC score. (B) Correlation between the polyp-to-adjacent p53 ratio and baseline PBAC score. (C) Correlation between the polyp-to-adjacent Ki-67 ratio and time to clinical stability (TtCS). (D) Correlation between the polyp-to-adjacent p53 ratio and TtCS. Spearman correlation coefficients and corresponding P values are shown in each panel. The solid line indicates the fitted trend, and the shaded area indicates the 95% confidence interval. ZHF, Zhuyang-Huazheng Formula; TXA, tranexamic acid; PBAC, Pictorial Blood Loss Assessment Chart; TtCS, time to clinical stability. Please click here to view a larger version of this figure.

Figure 6: Univariable and multivariable predictors of baseline bleeding burden and time to clinical stability. (A) Forest plot showing univariable and multivariable linear regression analyses for predictors of greater baseline bleeding burden, with baseline PBAC score as the dependent variable. (B) Forest plot showing univariable and multivariable linear regression analyses for predictors of longer time to clinical stability (TtCS). Results are presented as standardized beta coefficients with 95% confidence intervals. Adjusted P values correspond to the multivariable models. PBAC, Pictorial Blood Loss Assessment Chart; TtCS, time to clinical stability; ZHF, Zhuyang-Huazheng Formula; TXA, tranexamic acid. Please click here to view a larger version of this figure.

Variable	ZHF group (n = 87)	TXA group (n = 94)	Statistical test	P-value
Age (years)	38.08 ± 5.05	39.00 ± 5.42	Student's t-test	0.242
BMI (kg/m²)	23.97 ± 3.16	23.70 ± 3.08	Student's t-test	0.565
PBAC (C-2 cycle)	210.70 ± 56.60	221.04 ± 54.27	Student's t-test	0.212
Hemoglobin (g/dL)	11.61 ± 0.83	11.54 ± 0.96	Student's t-test	0.601
Endometrial thickness (mm)	9.39 ± 1.93	9.45 ± 1.98	Student's t-test	0.836
Polyp size (cm)	1.31 ± 0.38	1.33 ± 0.43	Student's t-test	0.713
Polyp type (single/multiple)	63 (72.4%) / 24 (27.6%)	60 (63.8%) / 34 (36.2%)	χ²	0.731

Table 1: Baseline characteristics of premenopausal women with endometrial polyps and abnormal uterine bleeding. Data are presented as mean ± standard deviation (SD) for continuous variables and n (%) for categorical variables. Comparisons between the Zhuyang-Huazheng Formula (ZHF) group (n = 87) and the tranexamic acid (TXA) group (n = 94) were performed using Student’s t-test for continuous variables and the χ² test for categorical variables. BMI: Body Mass Index; PBAC: Pictorial Blood Loss Assessment Chart.

Variable	ZHF group (n = 87)	TXA group (n = 94)	P-value
PBAC (C-1 cycle)	93.03 ± 27.56	92.85 ± 29.56	0.31
ΔPBAC (C-2 to C-1)	117.67 ± 35.99	128.19 ± 34.11	0.045
PBAC responders (%)	88.5	90.4	0.858
Hemoglobin (C-1, g/dL)	12.23 ± 0.85	12.13 ± 1.05	0.48
ΔHemoglobin (g/dL)	0.62 ± 0.30	0.59 ± 0.33	0.521
TtCS (days)	4.73 ± 1.59	4.62 ± 1.43	0.6648

Table 2: Comparison of primary efficacy outcomes between the ZHF and TXA groups. Data are presented as mean ± standard deviation (SD) for continuous variables and as percentages for categorical variables. ΔPBAC: the absolute reduction in PBAC; ΔHemoglobin (g/dL): hemoglobin gain; TtCS: the time to clinical stability.

Variable	ZHF Mean ± SD	TXA Mean ± SD	P-value
Ki67_adjacent (%)	14.53 ± 5.20	15.28 ± 5.84	0.355
Ki67_polyp (%)	18.97 ± 5.46	25.81 ± 5.67	<0.001
Ki67_ratio	1.34 ± 0.32	1.92 ± 0.37	<0.001
p53_adjacent_hscore	93.77 ± 22.13	97.89 ± 24.08	0.211
p53_polyp_hscore	155.64 ± 40.51	210.45 ± 44.19	<0.001
p53_ratio	1.61 ± 0.38	2.15 ± 0.41	<0.001

Table 3: Comparison of Ki-67 and p53 expression between the ZHF and TXA groups. Data are presented as mean ± standard deviation (SD). The ratio variables (Ki-67_ratio and p53_ratio) indicate relative expression levels in polyp tissue compared to adjacent endometrium.

Variable	ZHF group (n = 87)	TXA group (n = 94)	P-value
Any adverse event	7 (8.0%)	10 (10.6%)	0.57
Gastrointestinal reactions	5 (5.7%)	8 (8.5%)	0.48
Mild nausea	3 (3.4%)	5 (5.3%)	0.62
Abdominal discomfort	2 (2.3%)	3 (3.2%)	0.74
Hepatic function abnormality	1 (1.1%)	2 (2.1%)	0.62
Renal function abnormality (eGFR < 60 mL/min/1.73 m²)	1 (1.1%)	3 (3.2%)	0.38
Allergic reaction	0 (0%)	1 (1.1%)	0.31
Serious adverse event	0 (0%)	0 (0%)	—
Composite safety endpoint	2 (2.3%)	5 (5.3%)	0.29

Table 4: Comparison of adverse events between the ZHF and TXA groups. Data are presented as n (%) for categorical variables.

Supplementary Table 1: Composition and botanical characteristics of the Zhuyang-Huazheng Formula. All amounts represent the crude-herb equivalents used for one daily prescription. The formula was prepared as a standardized aqueous decoction by the hospital pharmacy and dispensed as two 200 mL doses per day. Please click here to download this file.

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

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 stability. These findings therefore indicate overlapping but not identical clinical response patterns and should not be interpreted as demonstrating therapeutic equivalence between ZHF and TXA. The ZHF group also showed lower Ki-67 indices and p53 H-scores in polyp tissue, whereas corresponding values in adjacent endometrium did not differ significantly between groups. Exploratory correlation and regression analyses further showed that higher polyp-to-adjacent biomarker ratios were associated with greater baseline bleeding burden and longer clinical stability. No serious adverse events were reported in either treatment group.

An important clinical consideration is that short-term preoperative bleeding control should not be interpreted as a substitute for adequate evaluation of the underlying endometrial pathology. EP-associated AUB represents a structural cause of bleeding, and persistent or atypical bleeding patterns, suspicious intrauterine findings, or other concerning clinical features warrant timely diagnostic assessment and histopathological confirmation. In the present cohort, all included lesions were ultimately confirmed as benign endometrial polyps on histopathological examination. Accordingly, the present findings should be interpreted specifically in the context of short-term symptom control after appropriate diagnostic assessment and should not be extrapolated to patients in whom premalignant or malignant endometrial pathology has not been adequately excluded. Our clinical findings are consistent with existing literature on TXA, which remains an established nonhormonal option for managing heavy menstrual bleeding and typically reduces menstrual blood loss by 26–54% per cycle^12,13,14. The PBAC remains a widely used semiquantitative tool for assessing menstrual blood loss^15,16. The between-group difference in absolute PBAC reduction warrants specific consideration. TXA produced an additional mean reduction of 10.52 PBAC points compared with ZHF, and this difference reached statistical significance (P = 0.045). However, the present study did not prespecify a minimal clinically important difference for PBAC and was not designed as an equivalence or non-inferiority trial. Therefore, the clinical importance of the magnitude of this difference cannot be determined from the present data, and the absence of statistically significant differences in responder rate, hemoglobin gain, or TtCS should not be interpreted as evidence that the two regimens have equivalent efficacy. The findings are more appropriately interpreted on an outcome-specific basis, with the primary PBAC reduction favoring TXA while several secondary outcomes showed no statistically significant between-group differences. In the present study, a ≥50% reduction in PBAC score was prespecified as the responder definition to provide a clinically interpretable measure of short-term bleeding improvement. Preclinical and indirect evidence involving individual herbal components or related traditional formulations has suggested potential effects on oxidative, inflammatory, and cellular signaling pathways^17,18,19. However, these pathways were not evaluated in the present study, and their relevance to the bleeding improvement observed with ZHF remains uncertain. Accordingly, such mechanisms should be regarded as hypothesis-generating explanations for future investigation rather than established mechanisms of action of ZHF in EP-associated AUB.

Hormonal therapies, including combined hormonal contraceptives and progestin-based regimens, are established options for the medical management of AUB and may provide additional benefits such as cycle regulation, menstrual suppression, and contraception. ZHF represents a non-hormonal approach and may be of interest to patients who prefer to avoid hormonal treatment or for whom hormonal therapy is considered unsuitable. However, ZHF currently has a substantially more limited evidence base, requires standardized herbal sourcing and preparation, involved a 15-day treatment course in this study, and does not provide the contraceptive or cycle-regulating benefits of hormonal therapies. Because patients who had received hormonal therapy within the preceding 3 months were excluded, the present study does not provide a direct comparison of ZHF with hormonal treatment. Accordingly, ZHF should not be interpreted as superior or equivalent to hormonal therapy and requires prospective comparative evaluation.

The potential application of ZHF to other structural causes of AUB, including adenomyosis and uterine leiomyomas, warrants further investigation. However, these conditions differ from endometrial polyps in their anatomical substrates and pathophysiological mechanisms of bleeding, and patients with adenomyosis or uterine fibroids ≥3 cm were specifically excluded from the present study. Therefore, the current findings should not be extrapolated directly to these populations. Future prospective studies should evaluate ZHF separately in well-defined etiologic subgroups.

Beyond symptomatic control, the tissue analyses identified distinct Ki-67 and p53 expression profiles between the two treatment groups. Lower Ki-67 indices and p53 H-scores were observed in polyp tissue from the ZHF group, whereas corresponding values in adjacent endometrium did not differ significantly between groups. These findings should be interpreted as descriptive between-group differences rather than evidence that ZHF directly altered endometrial cellular activity, particularly because treatment exposure was not randomized and no pretreatment tissue measurements were available. Ki-67 is a well-established marker of cellular proliferation and is expressed during active phases of the cell cycle but is absent in resting cells²⁰. Higher Ki-67 expression in endometrial polyps has been associated with increased proliferative activity²¹. For p53, immunohistochemical interpretation in routine gynecologic pathology is generally based on characteristic staining patterns rather than quantitative staining intensity alone, and abnormal expression patterns have been described in endometrial hyperplasia and neoplastic lesions^22,23. Variable p53 immunoreactivity has also been reported in benign endometrial polyps²⁴. In the present study, p53 was summarized using the H-score as an exploratory continuous measure rather than a formal pattern-based classification. Accordingly, the lower p53 H-score in the ZHF group should not be interpreted as evidence of more favorable p53 biology, normalization of p53 function, therapeutic benefit, reduced neoplastic potential, or wild-type p53 status. Indirect preclinical literature has implicated oxidative, inflammatory, proliferative, and other molecular signaling pathways in the biological actions of some herbal components or related formulations^25,26; however, none of these pathways were measured in the present study. Their relevance to the observed Ki-67 and p53 profiles therefore remains hypothetical and should be regarded only as hypothesis-generating biological context.

The exploratory association analyses showed that higher polyp-to-adjacent Ki-67 and p53 ratios were associated with greater baseline PBAC scores and longer time to clinical stability. In multivariable analyses, both biomarker ratios remained associated with baseline bleeding burden and TtCS after adjustment for the included clinical covariates. These findings indicate covariation between tissue biomarker profiles and bleeding-related clinical features but do not establish either a causal role of Ki-67 or p53 expression in bleeding severity or a treatment-mediated biological effect of ZHF. Although preclinical studies involving related herbal constituents have described a variety of molecular regulatory pathways^27,28,29, such evidence cannot establish which, if any, mechanisms account for the present clinical or tissue findings. Prospective studies incorporating pretreatment and post-treatment tissue assessment together with direct measurement of relevant molecular pathways would be required to address these questions.

From a safety perspective, adverse events were uncommon in both treatment groups. No statistically significant between-group differences were observed in the overall adverse-event rate or the composite non-gastrointestinal safety endpoint, and no serious adverse events were reported. Given the retrospective design and sample size, these findings provide only a preliminary description of short-term tolerability and should not be interpreted as establishing comparative safety between treatment strategies^30,31.

The retrospective, non-randomized design represents an important limitation of the present study. Treatment selection arose from routine outpatient clinical practice rather than study-directed randomization, making the analysis susceptible to selection bias and confounding by indication. Although no statistically significant differences were observed in the measured baseline characteristics, residual confounding from unmeasured clinical, physician-related, or patient-level factors cannot be excluded. This limitation is particularly relevant to the biomarker analyses: the lower Ki-67 indices and p53 H-scores observed in the ZHF group cannot be confidently attributed to treatment and may reflect pre-existing biological differences, residual confounding, treatment-related effects, or a combination of these factors. The absence of pretreatment tissue measurements further prevents determination of within-patient biomarker changes. Another limitation is that the timing of hysteroscopic surgery and tissue collection was not strictly standardized to an identical menstrual-cycle phase in all patients. Although surgery was preferentially scheduled after cessation of menstrual bleeding and during the early proliferative phase when clinically feasible, residual cycle-related variability may have influenced the Ki-67 and p53 measurements and should be considered when interpreting these exploratory tissue findings.

Further limitation concerns the interpretation of p53 immunohistochemistry. p53 expression was quantified using an H-score rather than classified according to formal pattern-based criteria; therefore, the present analysis cannot determine wild-type versus abnormal p53 status, and differences in H-score should not be interpreted as indicating differences in neoplastic potential. The treatment strategies also differed in preparation and duration. ZHF required standardized hospital-pharmacy preparation and was administered for 15 consecutive days, whereas TXA was provided as a ready-to-use oral tablet for 5 consecutive days. These differences resulted in unequal treatment duration, cumulative exposure, and implementation burden and may have influenced adherence and practical feasibility. Accordingly, the present comparison should be interpreted as an observational comparison of two real-world preoperative management strategies rather than a duration-matched pharmacological equivalence assessment.

Future prospective multicenter studies using more rigorously controlled treatment allocation, standardized treatment and surgical timing, and longitudinal tissue assessment are needed to validate the present clinical findings and determine whether biomarker changes are temporally associated with treatment exposure. Direct measurement of relevant biological pathways would also be required before oxidative, inflammatory, proliferative, or other molecular mechanisms could be attributed to ZHF.

In summary, ZHF was associated with significant short-term improvement in preoperative bleeding among women with EP-associated AUB. TXA produced a significantly greater absolute reduction in PBAC score, whereas PBAC responder rate, hemoglobin gain, and time to clinical stability did not show statistically significant between-group differences. The ZHF group also showed lower polyp-tissue Ki-67 indices and p53 H-scores, although these biomarker findings remain exploratory and cannot be attributed confidently to treatment. Given the retrospective, non-randomized design and the absence of an equivalence or non-inferiority framework, the present findings support prospective comparative evaluation of ZHF as a potential non-hormonal strategy for short-term preoperative management rather than any conclusion of equivalence with TXA.

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