Preclinical Effect of Shaofu Zhuyu Decoction on Decidualization and the Expression of PR-β and HNRNPL in a Mouse Model of Endometriosis-Associated Infertility

To investigate the effects of Shaofu Zhuyu Decoction on decidualization and the expression of progesterone receptor-β (PR-β) and heterogeneous nuclear ribonucleoprotein L (HNRNPL) in a mouse model of endometriosis.

An endometriosis mouse model was established by allogeneic uterine transplantation. Sixty recipient mice were randomized into model (Model), medium-dose (SF-M), and high-dose (SF-H) Shaofu Zhuyu Decoction groups (n=20 each), with 20 normal mice as controls (NC). Doses (9.403 and 18.806 g·kg− 1·day− 1) were derived from clinical equivalence via body surface area conversion. After 4 weeks of gavage, females were mated. Mice were sacrificed on gestational days (GD) 4.5, 5.5, and 7.5 (n=6-7/group/time point). Pregnancy rates, implantation sites, and histology were assessed. IGFBP1, PRL, PR-β, and HNRNPL protein expression were detected by Western blotting.

Compared with NC, the Model group showed a lower pregnancy rate (66.67% vs 100%) although this difference was not statistically significant (p > 0.05), however, it exhibited significantly reduced implantation numbers (median 7 vs 9, p < 0.05). IGFBP1 was decreased (p < 0.01), while PRL was increased (p < 0.01). PR-β was lower at GD4.5 but higher at GD5.5 and GD7.5 versus NC (p < 0.05), whereas HNRNPL was consistently lower in the Model group (p < 0.01). Both SF-M and SF-H significantly increased implantation numbers versus Model (p < 0.05). SF-H also upregulated IGFBP1 (p < 0.01), reduced PRL (p < 0.05), normalized the abnormal PR-β temporal pattern, and increased HNRNPL (p < 0.05).

Shaofu Zhuyu Decoction, particularly at the high dose, improved impaired decidualization in endometriosis model mice, potentially via regulation of PR-β and HNRNPL. These preclinical findings support further investigation of this formula for endometriosis-associated infertility. However, as an observational protein-level study without mechanistic validation, the results should be considered exploratory.

Shaofu Zhuyu Decoction, endometriosis, decidualization, progesterone receptor-β, heterogeneous nuclear ribonucleoprotein L

Endometriosis (EMs) is a disease characterized by the presence of endometrial glands and stroma outside the uterine cavity and is closely associated with estrogen dependence and chronic inflammation.1 The detection rate of endometriosis in infertile patients ranges from 25% to 40%, and women with endometriosis have approximately a twofold higher risk of infertility compared with women without a history of endometriosis.2 Furthermore, infertility affects 30–50% of women diagnosed with endometriosis.3 Several studies suggest that impaired decidualization of the eutopic endometrium in patients with endometriosis may be an important cause of infertility.4,5 Decidualization refers to the process by which endometrial stromal cells differentiate into secretory decidual stromal cells under the stimulation of increased local progesterone levels and cyclic adenosine monophosphate (cAMP), through complex signaling cascades and extensive gene reprogramming. This process is essential for the establishment and maintenance of pregnancy.5 A growing body of evidence suggests that impaired decidualization in the eutopic endometrium of patients with endometriosis may contribute to infertility or reduced fertility, and this impairment is closely associated with progesterone resistance.6–8 Insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL) are classical markers produced by decidual stromal cells during decidualization.9 These factors promote trophoblast growth and invasion, reduce immune rejection, regulate the survival of uterine natural killer cells, and facilitate angiogenesis.10 In menstrual blood from patients with endometriosis, the number of IGFBP1-positive decidual stromal cells is significantly reduced.11 Cytological studies have also shown that during decidualization induction, PRL expression in eutopic endometrial stromal cells from patients with endometriosis is significantly lower than that in normal stromal cells, indicating impaired decidualization.5 However, how the expression of IGFBP1 and PRL changes during the progression of decidualization in normal endometrium and eutopic endometrium in endometriosis has not yet been fully described. During decidualization, progesterone signals through its receptors (PGRs). Upon ligand binding, PGRs translocate to the nucleus and regulate progesterone-responsive genes by interacting with promoter/enhancer elements or transcriptional co-regulators. In mammals, PGRs exist as two isoforms, PR-α and PR-β, which exert distinct and species-specific functions in decidualization. In humans, PR-β is the more potent isoform for mediating decidualization in endometrial stromal cells, whereas in mice, PR-α appears to play the more dominant role, with PRα-null mice exhibiting endometrial epithelial hyperplasia and an inflammatory phenotype.12 In the endometrium of patients with endometriosis, the PR-β isoform, which is critical for decidualization, is significantly downregulated.13 During the late proliferative-to-early secretory phase transition, attenuated PR expression in ectopic endometrial tissues shifts the PR-α:PR-β ratio toward PR-α dominance, thereby diminishing stromal cell responsiveness to progesterone.8,14 Given these species-specific differences, findings from mouse models regarding PR isoform function should be interpreted with caution when considering translational implications for human endometriosis. Heterogeneous nuclear ribonucleoprotein L (HNRNPL) is an RNA-binding protein closely associated with mRNA function. It specifically binds to CA-rich conserved sequences in target mRNAs through its RNA recognition motifs (RRMs), thereby regulating mRNA metabolism. Mathew et al reported that HNRNPL has great potential roles in driving embryo and placental formation and differentiation.15 Given the potential link between RNA metabolism and endometrial function, the regulatory role of HNRNPL-mediated RNA metabolism in the decidualization of the eutopic endometrium in endometriosis warrants investigation; however, no relevant studies have been reported to date. In TCM, endometriosis is classified as a blood stasis pattern. Shaofu Zhuyu Decoction, a representative formula for activating blood circulation and resolving stasis that originates from the Yilin Gaicuo by the Qing Dynasty physician Wang Qingren, is widely used in clinical practice to treat dysmenorrhea and hypogastric pain. Clinical studies have suggested that this decoction can reduce serum CA-125 levels and alleviate pain (as measured by VAS) in patients with endometriosis.16,17 Our previous study indicated that Shaofu Zhuyu Decoction does not affect the estrous cycle in a rat model of endometriosis.18 However, whether Shaofu Zhuyu Decoction can improve decidualization defects associated with endometriosis remains unclear. Based on the above evidence, we hypothesized that Shaofu Zhuyu Decoction would restore normal decidualization markers (IGFBP1 and PRL) and normalize PR-β and HNRNPL expression in endometriosis model mice. Therefore, in this study, we treated an endometriosis mouse model with Shaofu Zhuyu Decoction to explore its potential mechanism.

Experimental Animals A total of 110 specific pathogen-free (SPF) female C57BL/6J mice aged 6–8 weeks (body weight 18–20 g) and 40 male C57BL/6J mice aged 12–14 weeks (body weight 30–34 g) were used. All animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production license No. SYXK (Beijing) 2023-0011; Animal certificate No. 110011241101484885). Sample size was determined based on previous studies19,20 that reported significant differences in fertility outcomes with group sizes of 8–10 mice, with an estimated 30% attrition due to modeling failure (the observed failure rate in this study was 35%, consistent with this estimate). The experiments were conducted at the Animal Experiment Center of Beijing University of Chinese Medicine from March to June 2024. All mice were allowed to acclimate to the housing conditions for one week prior to any experimental procedures. Mice were housed under controlled conditions with a temperature of 22 ± 2°C, humidity of 40%–70%, and a 12 h light/dark cycle, with free access to food and water. All experimental procedures were approved by the Experimental Animal Ethics Committee of North China University of Science and Technology (LAEC-NCST-2020083, valid from 2020 to 2024 with annual renewal) and the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (BUCM-2024031807-1159). All animal work was carried out in compliance with the internationally recognized “3R” principles—Replacement, Reduction, and Refinement—and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978) and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Main Instruments and Reagents The main instruments included a vertical electrophoresis system (Bio-Rad Mini Protean Tetra), transfer apparatus (Bio-Rad Mini Trans-Blot), digital shaker (DLAB MX-S), and refrigerated centrifuge (Thermo Fisher Fresco17), and a ChemiDoc MP Imaging System (Bio-Rad). The Shaofu Zhuyu Decoction consisted of the following herbal components: Angelica sinensis (9 g), Pollen Typhae (9 g), Trogopterorum Faeces (6 g), Paeoniae Radix Rubra (6 g), Foeniculum vulgare (3 g), Corydalis yanhusuo (vinegar-processed, 3 g), Myrrha (3 g), Ligusticum chuanxiong (3 g), Cinnamomum cassia (3 g), and Zingiber officinale (processed ginger, 0.6 g). All herbal slices were purchased from Beijing Qiancao Traditional Chinese Medicine Co., Ltd. (Batch No. 230100005). Herbal materials were authenticated by pharmacognosist according to the Chinese Pharmacopoeia standards. Progynova (estradiol valerate tablets, 1 mg/tablet) was obtained from Bayer Healthcare Co., Ltd. (Batch No. 242A). The following antibodies were used: HNRNPL antibody (Abcam, ab6106), insulin-like growth factor binding protein-1 (IGFBP1) antibody (Abways, CY8219), prolactin (PRL) antibody (Abclonal, A1618), PR-β antibody (CST, 3157S), and GAPDH antibody (Abcam, ab181602). Secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG (Chengdu Zhengneng Biotechnology Co., Ltd.), and molecular weights were verified using a pre-stained protein ladder (Biosun Biotechnology Co., Ltd., PM1180). Hematoxylin staining solution (G1080) and eosin staining solution (G1100) were purchased from Solarbio (Beijing). Sodium pentobarbital was obtained from Beijing Chemical Reagent Company. Experimental Grouping and Treatment Drug Preparation Shaofu Zhuyu Decoction was prepared by conventional decoction of the herbal ingredients and concentrated to a final concentration of 0.5 g/mL, and stored at 4°C. Fresh decoction was prepared weekly to minimize batch-to-batch variability. High-performance liquid chromatography (HPLC) fingerprinting confirmed that the stability of the active constituents exceeded 90% under this preparation protocol, and the detailed chromatographic data are provided in Figure S1 and Table S1. Progynova suspension was prepared by grinding one estradiol valerate tablet and dissolving it in 10 mL of normal saline to obtain a concentration of 0.1 mg/mL. Endometriosis Model Establishment and Experimental Grouping A homologous uterine transplantation method was used to establish the endometriosis model.19,20 After one week of acclimatization, the 60 recipient female mice were randomly allocated using a random number table into three experimental groups: Model group (n = 20), Shaofu Zhuyu Decoction medium-dose group (SF-M, n = 20), and Shaofu Zhuyu Decoction high-dose group (SF-H, n = 20). All recipient mice underwent the same transplantation procedure, and the group allocation was fixed prior to surgery. An additional 30 female mice served as uterine tissue donors, and 20 normal female mice were used as the normal control group (NC). Donor mice treatment: Donor mice were administered estradiol valerate (0.5 mg/kg) by gavage for two consecutive days to synchronize the estrous cycle. Food and water were withheld for 12 hours before surgery. Mice were anesthetized with 3% sodium pentobarbital at a dose of 30 mg/kg. After shaving and disinfection with iodophor, a midline abdominal incision was made to expose the uterus. The bilateral uterine horns were excised at the bifurcation, and surrounding adipose and omental tissues were removed. The tissues were rinsed with normal saline, longitudinally opened to expose the endometrium, and cut into approximately 5 mm × 5 mm fragments using ophthalmic scissors. The fragments were stored in phosphate-buffered saline (PBS) until transplantation. Each donor uterus was equally divided for transplantation into two recipient mice. The number of donor mice (n = 30) was calculated based on the need for two fragments per recipient (n = 60), with each donor providing sufficient tissue for two recipients. Recipient mice treatment: Recipient mice were administered estradiol valerate (0.5 mg/kg) for two consecutive days prior to surgery. After anesthesia and abdominal preparation, the abdominal cavity was opened. Endometrial fragments were sutured onto vascular-rich areas of the bilateral abdominal wall using 5–0 sterile sutures, with one fragment on each side. Penicillin (8 IU, 0.06 mL) was administered intraperitoneally. The abdominal wall was closed layer by layer, followed by iodophor disinfection. Model confirmation and inclusion criteria: Four weeks after surgery, three recipient mice (one randomly selected from each of the three groups) were randomly selected for laparotomy to confirm successful model establishment. Endometriotic lesions were observed in the abdominal cavity as irregular cystic nodules, raised translucent nodules, or cyst-like vesicles filled with yellow or transparent serous fluid. The lesions were covered by connective tissue with neovascularization and showed varying degrees of adhesion to surrounding tissues, confirming successful establishment of the endometriosis model. The abdomen was then closed and disinfected. These three mice were excluded from subsequent experimental analyses. Subsequently, all remaining recipient mice underwent laparotomy to assess lesion formation. Only mice with visible ectopic lesions were included in the subsequent analyses; this exclusion criterion was pre-specified. Among the 60 recipient mice, 39 developed visible ectopic lesions and were included, whereas 21 mice without visible lesions were excluded (Model: 7/20 excluded, yielding 13 successful; SF-M: 6/20 excluded, yielding 14 successful; SF-H: 8/20 excluded, yielding 12 successful). Drug Administration Drug doses were calculated based on body surface area conversion from the clinical dosage.18 Assuming a human height of 165.5 cm and body weight of 60 kg and a mouse body weight of 20 g, the equivalent dose was calculated. The SF-M group received Shaofu Zhuyu Decoction at 9.403 g·kg−1·day−1 (equivalent to the clinical human dose scaled to mouse body surface area), while the SF-H group received 18.806 g·kg−1·day−1 (twice the equivalent adult dose) by gavage. The NC and Model groups received 0.2 mL normal saline by gavage. Drug administration commenced immediately after the 4-week post-surgery confirmation and continued for 4 weeks. Mating and Sample Collection After four weeks of intervention, female mice from all groups were housed with proven fertile male C57BL/6J mice aged 12–14 weeks at a ratio of 2:1 at 16:00 during estrus. Pregnancy was monitored daily. The presence of a vaginal plug the following morning was considered day 0.5 of pregnancy (Day 0.5). Mice were sacrificed on gestational Day 4.5, Day 5.5, and Day 7.5 for tissue collection. The numbers of mice available at each gestational time point from the 39 successfully modeled animals were as follows: Model group, n = 4 at GD4.5, n = 5 at GD5.5, and n = 4 at GD7.5; SF-M group, n = 5, 5, and 4; SF-H group, n = 3, 4, and 5. For the NC group, n = 6, 7, and 7, respectively. The overall experimental design and workflow are summarized in Figure 1. | Figure 1 Mouse Study Experimental Flowchart. | Sample Processing Under sterile conditions, the entire uterus was rapidly excised. Uterine morphology, color, and pregnancy status were observed, and the number of embryo implantation sites was counted. Afterward, embryos at the implantation sites were separated, and residual adipose tissue and blood vessels were removed. One uterine horn was cut and immediately placed in a cryogenic tube, snap-frozen in liquid nitrogen, and stored at −80 °C for Western blot analysis. The other uterine horn and endometriotic lesion tissues were fixed in 4% paraformaldehyde for hematoxylin and eosin (HE) staining. The numbers of mice used for analysis at each gestational time point were as described in Mating and Sample Collection. Observation Indicators and Detection Methods Pregnancy Rate and Number of Embryo Implantation Sites The number of pregnant mice and embryo implantation sites on gestational Day 5.5 and Day 7.5 were recorded. Pregnancy outcomes were assessed only in mice that met the predefined inclusion criteria. Data from GD5.5 and GD7.5 were pooled for the pregnancy analysis to achieve sufficient sample size for the chi-square test, resulting in nine mice available for analysis in each group (NC: 5 + 4; Model: 5 + 4; SF-M: 5 + 4; SF-H: 4 + 5). The number of implantation sites was expressed as the median (interquartile range) of the count of implantation sites per pregnant mouse. Hematoxylin and Eosin (HE) Staining Tissues were fixed, dehydrated, embedded in paraffin, and sectioned into 5 μm-thick slices. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin. Histopathological changes in endometriotic lesions and uterine endometrial tissues were observed under a light microscope. Qualitative morphological assessment was performed. Detection of Decidualization Markers and PR-β and HNRNPL Proteins in Mouse Endometrial Tissue For Western blotting, total protein was extracted from uterine tissues using RIPA lysis buffer and quantified by BCA assay. A total of 20 µg of protein per sample was resolved by 10% SDS-PAGE (for all target proteins) and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with primary antibodies against IGFBP1 (1:1000), PRL (1:500), PR-β (1:1000), and HNRNPL (1:2000) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Membranes were stripped using stripping buffer (Thermo Scientific) and re-probed with GAPDH antibody as the loading control to ensure equal loading. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and imaged with a ChemiDoc MP Imaging System (Bio-Rad). A pre-stained protein ladder (Biosun, PM1180) was run in parallel to verify molecular weights. Band intensities were quantified using ImageJ software (NIH, version 1.53) and normalized to GAPDH. Statistical Analysis Statistical analyses were performed using SPSS version 26.0. Continuous data were expressed as mean ± standard deviation (x ± SD). For data that conformed to a normal distribution (tested by Shapiro–Wilk test), comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by pairwise comparisons using the LSD test. Data that did not follow a normal distribution were expressed as the median (interquartile range) [Md (P25, P75)] and analyzed using non-parametric tests (Kruskal–Wallis test for multiple groups). Categorical data (pregnancy rate) were expressed as rates or proportions and analyzed using Fisher’s exact test. The number of implantation sites was analyzed using the Kruskal–Wallis test, followed by Mann–Whitney U-test for pairwise comparisons. A value of P < 0.05 was considered statistically significant. Post hoc power analyses were performed using G*Power software (version 3.1.9.7; Universität Düsseldorf, Germany). For the pregnancy rate, the effect size (w) was derived from the observed proportions using a chi-square test for contingency tables. With a total sample size of 36 animals (n = 9 per group) and a significance level of α = 0.05, the achieved power (1–β) was calculated to be 0.75 for the observed effect size (w = 0.522).

Gross and Histological Observations of Ectopic Lesions and Endometrium In successfully modeled mice, vesicle-like ectopic lesions were observed on the abdominal wall. These lesions appeared translucent and firm with abundant surrounding blood vessels, or presented as raised cystic vesicles filled with yellowish or clear serous fluid (Figure 2A). Hematoxylin and eosin (HE) staining revealed the presence of both endometrial glands and stromal components within the lesions (Figure 2B). The endometrial morphology of mice in each group is shown in Figure 3. In the NC group, uterine glands were moderate in number, lined by orderly single-layered columnar epithelium with regular, uniform nuclei and no pyknosis or atypia. Mild secretory changes were noted, with abundant cytoplasm and no atrophy or necrosis. Stromal microvessels and spiral arterioles were evenly distributed, with intact walls and continuous endothelium; lumina were mildly engorged, without hyperemia, wall thickening, or obliteration. Decidual cells were abundant, large and polygonal, with ample pale cytoplasm and central round nuclei; cells were loosely arranged with mild stromal edema. In the model group, uterine glands were markedly atrophic and reduced in number, with collapsed and obliterated ducts. Epithelial cells showed disorganized layering, flattening, and cytoplasmic loss. Stromal vessels were prominently dilated and congested; spiral arterioles exhibited thickened, edematous walls, with occasional stromal hemorrhagic foci and focal small-vessel obliteration. The normal loose decidual architecture was replaced by extensive inflammatory infiltration and fibrous tissue proliferation. In the SF-M and SF-H groups, uterine gland numbers were significantly restored, with well-formed morphology and reestablished orderly single-layered columnar epithelium showing normal nuclei. Spiral arterioles normalized in wall thickness with intact endothelium; no hemorrhage or obliteration was observed, and microvessels were evenly distributed. Decidual cells were abundant and plump, with pale cytoplasm and normal nuclei; only mild stromal edema remained. Pregnancy Rate and Average Number of Embryo Implantation Sites The gross uterine morphology of mice in each group is shown in Figure 4, and the pregnancy rate and average number of embryo implantation sites are presented in Table 1. | Table 1 Pregnancy Rate and Average Number of Embryo Implantation Sites in Each Group | Compared with the NC group (100%, 9/9), the pregnancy rate in the Model group was reduced (66.67%, 6/9), but this reduction was not statistically significant (p > 0.05). In contrast, the median number of implantation sites in the Model group was significantly lower than that in the NC group [7 (P25–P75: 5–8.25) vs 9 (7.5–9.5); p < 0.05]. Furthermore, both the SF-M and SF-H groups had significantly more implantation sites than the Model group [8 (7.5–9.5) and 8 (7–9.5) vs 7 (5–8.25), respectively; both p < 0.05]. Expression of Decidualization Markers and PR-β and HNRNPL Proteins in Mouse Endometrial Tissue As shown in Figure 5, the expression levels of IGFBP1, PRL, PR-β, and HNRNPL proteins in endometrial tissues were analyzed at gestational Days 4.5, 5.5, and 7.5. Error bars in all bar graphs represent standard deviation (SD). IGFBP1 protein expression in the Model group was significantly lower than that in the NC group at all three time points: Day 4.5 (0.53 vs 1.23, p < 0.01), Day 5.5 (0.62 vs 1.03, p < 0.01), and Day 7.5 (0.25 vs 0.72, p < 0.01). Compared with the Model group, IGFBP1 expression was significantly increased in both the SF-M and SF-H groups at Day 4.5 (0.53 vs 0.85 and 0.53 vs 1.08, respectively; both p < 0.01), Day 5.5 (0.62 vs 0.77 and 0.62 vs 0.91, respectively; both p < 0.01), and Day 7.5 (0.25 vs 0.51 and 0.25 vs 0.69, respectively; both p < 0.01). The SF-H group exhibited higher IGFBP1 expression than the SF-M group at all three time points (Day 4.5: 1.08 vs 0.85, p < 0.05; Day 5.5: 0.91 vs 0.77, p < 0.01; Day 7.5: 0.69 vs 0.51, p 0.05). For PRL protein expression, the Model group showed significantly higher levels than the NC group on Day 4.5 (0.72 vs 0.27, p < 0.01), Day 5.5 (0.68 vs 0.32, p < 0.01), and Day 7.5 (0.84 vs 0.36, p < 0.01). On Day 4.5, PRL expression in the SF-M and SF-H groups was significantly lower than that in the Model group (0.57 vs 0.72 and 0.42 vs 0.72, respectively; both p < 0.01), but remained higher than that in the NC group (0.57 vs 0.27 and 0.42 vs 0.27, respectively; both p < 0.01). On Days 5.5 and 7.5, PRL expression in the SF-H group was significantly lower than that in the Model group (Day 5.5: 0.44 vs 0.68, p < 0.05; Day 7.5: 0.53 vs 0.84, p < 0.01). On gestational Day 4.5, PR-β protein expression in the Model group was significantly lower than that in the NC group (0.49 vs 0.75, p < 0.05). PR-β expression in the SF-H group was significantly higher than that in the Model group (0.75 vs 0.49, p 0.05). On Days 5.5 and 7.5, PR-β expression was significantly higher in the Model group than in the NC group (Day 5.5: 0.75 vs 0.34; Day 7.5: 0.56 vs 0.09; both p < 0.01). Treatment with SF-H significantly reduced PR-β expression compared with the Model group at both time points (Day 5.5: 0.47 vs 0.75; Day 7.5: 0.20 vs 0.56; both p 0.05), indicating full recovery. In contrast, the SF-M group showed only partial reduction, with PR-β levels remaining significantly higher than those in both the SF-H group (Day 5.5: 0.65 vs 0.47; Day 7.5: 0.37 vs 0.20; both p < 0.05) and the NC group (Day 5.5: 0.65 vs 0.34; Day 7.5: 0.37 vs 0.09; both p < 0.01), suggesting a dose-dependent effect of Shaofu Zhuyu Decoction. For HNRNPL protein expression, levels in the Model group were significantly lower than those in the NC group at all three time points (Day 4.5: 0.43 vs 0.94; Day 5.5: 0.42 vs 0.72; Day 7.5: 0.14 vs 0.79; all p < 0.01). Treatment with SF-M and SF-H both significantly upregulated HNRNPL expression compared with the Model group at all time points [SF-M: Day 4.5 (0.66 vs 0.43, p < 0.01), Day 5.5 (0.59 vs 0.42, p < 0.05), Day 7.5 (0.49 vs 0.14, p < 0.01); SF-H: Day 4.5 (0.82 vs 0.43), Day 5.5 (0.74 vs 0.42), Day 7.5 (0.64 vs 0.14); all p < 0.01]. Notably, this upregulation occurred in a dose-dependent manner, as SF-H consistently yielded higher HNRNPL levels than SF-M at each time point (Day 4.5: 0.82 vs 0.66, p < 0.01; Day 5.5: 0.74 vs 0.59, p < 0.05; Day 7.5: 0.64 vs 0.49, p 0.05). However, at Days 4.5 and 7.5, SF-H levels remained significantly different from NC levels (Day 4.5: 0.82 vs 0.94; Day 7.5: 0.64 vs 0.79; both p < 0.05).

The uterine tissue transplantation model is one of the most commonly used mouse models for studying endometriosis. Previous studies have suggested a dose-dependent relationship between the number of transplanted uterine tissues and reproductive outcomes. When two or more uterine tissue fragments are transplanted, pregnancy rates are significantly reduced compared with control animals.19,20 Here, an endometriosis model was established by transplantation of two uterine tissue fragments. The Model group exhibited a lower pregnancy rate (66.67% vs 100%), but this difference was not statistically significant (p > 0.05), likely attributable to the limited sample size. Nevertheless, the significant reduction in implantation site numbers (median 7 vs 9, p < 0.05) supports the notion that the model impairs reproductive outcomes, consistent with previous reports.19,20 In contrast, pregnancy rate and implantation sites in the SF-H group were comparable with those in the NC group. These findings suggest that transplantation of two uterine tissue fragments impairs reproductive outcomes in mice with endometriosis, whereas Shaofu Zhuyu Decoction may partially improve fertility-related outcomes. The mechanisms underlying infertility in endometriosis are complex and may involve inflammatory alterations in the pelvic microenvironment and impaired function of the eutopic endometrium.20 Among these mechanisms, defective decidualization has been increasingly recognized as an important contributor to implantation failure. We observed that IGFBP1 protein expression in the Model group was lower than that in the NC group at gestational Days 4.5, 5.5, and 7.5, whereas PRL expression was increased at these time points. The increased PRL expression observed during decidualization in the endometriosis model requires cautious interpretation. Clinical studies have reported that hyperprolactinemia occurs more frequently in infertile patients with endometriosis and is associated with disease severity.21–23 However, endometrial PRL is produced by decidual stromal cells under progesterone stimulation and is regulated independently of circulating PRL levels.24,25 Under physiological conditions, progesterone induces PRL synthesis during the mid-luteal phase, with peak expression occurring during the late luteal phase, coinciding with decidualization. In endometriosis, increased immune cell infiltration and alterations in the pelvic microenvironment may contribute to abnormal local PRL regulation in lesions or eutopic endometrium.26 PRL has been reported to possess pro-inflammatory, pro-angiogenic, and immunomodulatory properties; however, its precise role in endometriosis-associated decidualization remains incompletely understood.24,27 Previous studies using decidual monocytes and trophoblast cell models have suggested that excessively high PRL levels may activate the JAK2/STAT5 signaling pathway and increase inflammatory cytokine production, including IL-6 and IL-1β.27 However, whether this pathway contributes to impaired decidualization in endometriosis requires further experimental validation. In this study, elevated PRL expression in the uterus of endometriosis model mice may reflect an altered decidualization-related state. Furthermore, the SF-H group showed increased IGFBP1 expression and reduced PRL expression compared with the Model group, with expression patterns closer to those of the NC group. These findings suggest that Shaofu Zhuyu Decoction may partially ameliorate decidualization-associated abnormalities in endometriosis. PR-β is considered a key regulator of decidualization in human endometrial stromal cells (HESCs). Previous studies have demonstrated that PR-β regulates multiple decidualization-related genes, and knockdown of PR-β significantly reduces the expression of decidualization markers, including IGFBP1 and PRL.28,29 In addition, mouse studies have suggested that altered progesterone receptor isoform expression may influence estrogen receptor signaling and downstream pathways involved in endometrial receptivity.30 Our findings showed that PR-β expression in the NC group decreased from gestational Day 4.5 to Days 5.5 and 7.5. In contrast, the Model group exhibited lower PR-β expression at Day 4.5 but higher expression at later time points compared with the NC group. The SF-H group displayed an expression pattern more similar to that of the NC group. These results suggest that PR-β expression dynamics may be disrupted during decidualization in endometriosis model mice, and Shaofu Zhuyu Decoction may partially normalize this altered expression pattern. Using immunofluorescence analysis, Mathew demonstrated the localization and expression patterns of HNRNPL in embryos and decidual (or placental) tissues from gestational Day 4.5 to Day 18.5. HNRNPL expression remained relatively stable in decidual tissues, except for a transient absence near implantation sites at Day 5.5.15 These observations suggest that HNRNPL may participate in early pregnancy regulation, although its specific function during endometrial decidualization remains unclear. Previous work by Lei et al showed that HNRNPL regulates PRL synthesis in pituitary cells. Knockdown of HNRNPL in pituitary cells significantly reduced PRL synthesis, and HNRNPL was demonstrated to bind to a non-conserved CA-rich intronic region within the Prl precursor mRNA transcript, thereby preventing the generation of cryptic terminal exons and truncated mRNAs.31 Although these findings provide important insights into HNRNPL-mediated regulation of PRL in pituitary cells, whether a similar regulatory mechanism exists in endometrial stromal cells during decidualization remains unknown. Differences in cellular context, transcriptional regulation, and physiological function between pituitary cells and endometrial stromal cells should be considered. In this study, HNRNPL protein expression was significantly reduced in the endometrium of endometriosis model mice compared with controls, whereas treatment with Shaofu Zhuyu Decoction partially increased HNRNPL expression. These findings indicate an association between altered HNRNPL expression and impaired decidualization in this model. However, the current data do not establish a direct causal relationship between HNRNPL and decidualization, nor do they clarify whether HNRNPL directly regulates PRL or other decidualization-related genes in endometrial cells. Further studies using cell-type-specific gain- and loss-of-function approaches, together with transcriptomic analyses, are required to determine the biological role of HNRNPL in decidualization. To our knowledge, this is the first study investigating the potential effects of Shaofu Zhuyu Decoction on decidualization in an endometriosis mouse model by evaluating IGFBP1 and PRL expression at multiple stages of early pregnancy. These findings extend the potential biological effects of this traditional formula beyond symptom relief and suggest a possible role in fertility-associated regulation. Impaired decidualization contributes to infertility in patients with endometriosis, whereas effective therapeutic strategies targeting this defect remain limited.32 Previous studies have mainly focused on in vitro models using eutopic endometrial stromal cells from patients with endometriosis, including investigations of compounds such as quercetin and aspirin.33,34 In an endometriosis mouse model, oleuropein was reported to suppress lesion progression without apparent reproductive toxicity and partially improve decidualization capacity in human endometriotic stromal cells.35 Shaofu Zhuyu Decoction was originally formulated by Wang Qingren and has traditionally been considered to have potential reproductive benefits. Previous studies have demonstrated that this formula may improve endometrial receptivity in hyperinsulinemic mice and rats with impaired implantation conditions.36,37 Consistent with these findings, our results provide additional preclinical evidence suggesting that Shaofu Zhuyu Decoction may influence decidualization-related abnormalities associated with endometriosis. Despite these findings, several limitations should be acknowledged. First, the experimental design did not include a sham surgery control group, which limits the ability to distinguish the effects of endometriosis lesions from potential influences of surgical procedures. Second, the mechanistic investigation was limited to protein-level analyses. The absence of functional experiments, including siRNA knockdown, overexpression, and co-immunoprecipitation assays, prevents definitive conclusions regarding whether the HNRNPL–PR-β axis directly regulates decidualization. Third, mRNA validation by quantitative PCR was not performed; therefore, protein-level alterations could not be confirmed at the transcriptional level. Fourth, compared with the NC group (100%, 9/9), the pregnancy rate in the Model group was reduced (66.67%, 6/9), but this reduction was not statistically significant (p > 0.05). Although the observed effect size (w = 0.522) was large and the post hoc power (0.76) approached the acceptable level, the limited sample size (n = 9 per group) and the extremely low number of non-pregnant events (n = 3) still warrant cautious interpretation of the pregnancy rate comparisons. Fifth, because PR-α/PR-β biology differs between mice and humans, the translational relevance of these findings should be interpreted cautiously. Finally, although no obvious adverse effects were observed during the intervention period, systematic toxicological evaluation was not conducted, and the safety profile of Shaofu Zhuyu Decoction during pregnancy requires further investigation. The present findings suggest that Shaofu Zhuyu Decoction may represent a potential strategy for improving decidualization-associated abnormalities in endometriosis. However, these conclusions remain preliminary and require validation through well-designed clinical studies. Future investigations should further clarify the molecular mechanisms involving HNRNPL and progesterone receptor signaling using cellular and multi-omics approaches, and evaluate the clinical efficacy and safety of this formula.

In this study, endometriosis model mice exhibited reduced fertility and altered expression of decidualization-related proteins. Shaofu Zhuyu Decoction partially improved reproductive outcomes and normalized abnormal IGFBP1 and PRL expression patterns in the endometrium. These effects may be associated with changes in PR-β and HNRNPL expression, although the underlying molecular mechanisms remain to be elucidated. This study provides preliminary preclinical evidence suggesting that Shaofu Zhuyu Decoction may have potential value in improving endometriosis-associated decidualization impairment and warrants further investigation in clinical settings. Ethical Approval This study was approved by the Experimental Animal Ethics Committee of North China University of Science and Technology (LAEC-NCST-2020083) and the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (BUCM-2024031807-1159). Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Funding Hebei Natural Science Foundation (No. H2020209123); The Fifth Batch of National Traditional Chinese Medicine Excellent Clinical Talents Training Project (Announcement from the Personnel and Education Department of the National Administration of Traditional Chinese Medicine, No. 2022-239). Disclosure The authors declare that there are no conflicts of interest.

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