Macrophage-derived exosomes promote proliferation, migration, and invasion of endometrial stromal cells in endometriosis and are associated with exosomal lncRNA ZFAS1: A pilot translational study

Abstract

Endometriosis (EMs) is a prevalent gynecological disorder affecting reproductive-age women. Exosomes secreted by peripheral blood macrophages may participate in EMs progression. In this pilot translational study, exosomes from peripheral blood macrophages obtained from patients with EMs (n = 3) and control patients (n = 3) were isolated by ultracentrifugation, identified by transmission electron microscopy and exosomal markers, and cocultured with endometrial stromal cells. Quantitative reverse transcription polymerase chain reaction was used to detect long noncoding RNA zinc finger antisense 1 (ZFAS1) expression in macrophage-derived exosomes. Cell proliferation, migration, invasion, and apoptosis were evaluated using Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, wound healing, transwell, and flow cytometry assays. Gain- and loss-of-function experiments were performed in stromal cells to examine the biological role of ZFAS1. EMs-derived macrophage exosomes promoted endometrial stromal-cell proliferation, migration, and invasion and inhibited apoptosis compared with the blank and control-exosome groups. long noncoding RNA ZFAS1 expression was higher in EMs-derived exosomes than in control exosomes. In stromal cells, ZFAS1 overexpression enhanced proliferation, migration, and invasion and reduced apoptosis, whereas ZFAS1 knockdown produced the opposite effects. Macrophage-derived exosomes were associated with an aggressive stromal-cell phenotype, and exosomal ZFAS1 may contribute to this process. Because of the very small patient sample size and limited exosome characterization, these findings should be considered preliminary and hypothesis-generating.

1. Introduction

Endometriosis (EMs) is indeed a common gynecological disorder affecting women during their reproductive years.^[1] It is characterized by the presence of endometrial-like tissue outside the uterus, leading to pelvic pain, infertility, and discomfort.^[1,2] The incidence rate is significant, affecting approximately 7% to 15% of women of reproductive age.^[1] EMs is characterized by the presence of active endometrial glands and stromal cells outside the uterine cavity, which implant, grow, invade, and repeatedly bleed, forming nodules and masses.^[3] The lesions can affect all pelvic tissues and organs, most commonly the ovaries, rectovesical pouch, and uterosacral ligaments.^[2,4] The main clinical manifestations include dysmenorrhea, chronic pelvic pain, and infertility,^[5] which seriously affect the physical and mental health of women of reproductive age.

Although EMs is considered a benign disease, it exhibits malignant behavioral characteristics similar to tumors, such as invasion and recurrence.^[6] The disease is prone to recurrence with various manifestations and even malignant transformation. The recurrence rate of EMs patients is approximately 40% within 5 years after surgery, with an annual recurrence rate of about 20%.^[7] Therefore, adjuvant treatments are essential after surgery to reduce recurrence rates.

Recent research has increasingly focused on exosomes in tumor development and progression. Exosomes are a class of extracellular vesicles released by active cells, consisting of lipid bilayer membrane vesicles widely distributed in blood, urine, milk, cerebrospinal fluid, and other body fluids.^[8] These vesicles contain specialized proteins, lipids, and nucleic acids, including long noncoding RNA (lncRNA) and microRNA, which function as signaling molecules and can be transmitted to other cells through extracellular vesicles to regulate recipient cell functions, playing crucial roles in physiological and pathological processes.^[9,10]

lncRNA ZFAS1 (zinc finger antisense 1) is the antisense RNA of zinc finger, NFX1-type containing 1, located at 20q13, and serves as a carrier of 3 small nucleolar RNAs (SNORD12, SNORD12B, SNORD12C).^[11] Studies have demonstrated that lncRNA ZFAS1 is highly expressed in colorectal cancer, breast cancer, and lung cancer.^[12–14] Its expression positively correlates with cancer cell proliferation, lymph node metastasis, and advanced TNM staging, while negatively correlating with overall survival rates.^[15,16]

To investigate the role of macrophage-derived exosomal lncRNA ZFAS1 in endometriosis progression, we designed a pilot translational study integrating patient-derived exosomes with in vitro functional assays. We isolated and characterized exosomes secreted by peripheral blood macrophages from patients with endometriosis and controls, verified their identity through transmission electron microscopy and Western blotting for cluster of differentiation 9 (CD9) and CD81, and then assessed their effects on endometrial stromal-cell proliferation, migration, invasion, and apoptosis. We further manipulated ZFAS1 expression in stromal cells to examine whether this lncRNA could reproduce the observed phenotypes. Given the limited sample size and the absence of direct exosome-depletion or downstream pathway experiments, the present work was intended to provide preliminary biological evidence rather than definitive mechanistic proof.

2. Methods

2.1. Clinical samples and study design

This pilot translational study combined a small patient-sample comparison with in vitro functional experiments. The study was approved by the Ethics Committee of Haikou Municipal People’s Hospital (Approval No. 2021-017; March 11, 2021), and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Patients with ovarian endometriotic cysts diagnosed by laparoscopy and histopathology were included in the EMs group (n = 3). The control group (n = 3) included patients who underwent laparoscopic surgery for other benign gynecologic conditions. Peripheral venous blood samples were collected from all participants, and ectopic endometrial tissues were obtained from ovarian endometriotic cysts. Although the sample size was small, the observed direction of effect was consistent across the patient-derived comparisons. Because this exploratory pilot study was based on limited eligible cases and resource availability during the study period, no formal a priori power analysis was performed, and all patient-derived expression findings are interpreted cautiously.

2.2. Culture and characterization of endometrial stromal cells

Endometrial stromal cells (Procell, CP-H233) were purchased from Procell Life Science & Technology Co., Ltd. and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone, Cat. No. SH30087.01) and 1% penicillin-streptomycin solution (Hyclone, Cat. No. SH30010) at 37°C with 5% CO₂ in a humidified incubator (Thermo Fisher Scientific, HERACELL150i).

2.3. Isolation and identification of peripheral blood macrophages

Under sterile conditions, peripheral venous blood samples were collected from participants. Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). The isolated peripheral blood mononuclear cell fraction was resuspended in RPMI-1640 medium containing 10% exosome-free fetal bovine serum and cultured in a humidified atmosphere of 5% CO₂ at 37°C for 20 hours. Non-adherent cells were discarded, and adherent cells were used as peripheral blood macrophages for subsequent experiments.

2.4. Exosome isolation and identification

Culture supernatants of peripheral blood macrophages were collected for exosome isolation by sequential centrifugation. The supernatants were centrifuged at 1200 × g for 5 minutes using a low-speed centrifuge to remove cells and cellular debris, followed by ultracentrifugation at 100,000 × g for 4 hours at 4°C using an ultracentrifuge (Beckman Coulter; Optima L-100XP). The supernatant was discarded, and the pellet was gently resuspended in phosphate-buffered saline (PBS) to obtain purified exosomes. Exosome identity was assessed by transmission electron microscopy and Western blotting for the commonly used exosomal markers CD9 and CD81. Because of limited sample availability, negative-marker validation for cellular contaminants (e.g., calnexin or Golgi matrix protein of 130 kDa) and nanoparticle tracking analysis were not performed.

2.5. Western blotting detection of exosome marker proteins

Isolated exosomes were lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche) on ice for 30 minutes. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Thermo Fisher Scientific). Equal amounts of protein (20 μg) were mixed with 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (Bio-Rad), heated at 95°C for 5 minutes, and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel electrophoresis using a Mini-PROTEAN system (Bio-Rad). Proteins were transferred to polyvinylidene fluoride membranes (Millipore) using a semi transfer system (Bio-Rad). Membranes were blocked with 5% nonmilk (Bio-Rad) in tris-buffered saline with tween 20 for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against the exosomal markers CD81 (1:1000; Abcam, ab79559) and CD9 (1:1000; Abcam, ab236630). After washing 3 times with tris-buffered saline with tween 20, membranes were incubated with horseradishperoxidase-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 hour at room temperature. Protein bands were detected using enhanced chemiluminescence reagent (GE Healthcare) and visualized using a ChemiDoc imaging system (Bio-Rad).

2.6. Experimental design and grouping

This study was divided into 2 experimental phases. In the 1st phase, to evaluate the biological effects of macrophage-derived exosomes on endometrial stromal cells, 3 groups were established: blank group (cells cultured alone without exosome treatment), control-exosome group (cells cocultured with exosomes from control patients, 50 μg/mL), and EMs-derived exosome group (cells cocultured with exosomes from EMs patients, 50 μg/mL). Cells were treated for 48 hours before functional assays. In the 2nd phase, to examine whether ZFAS1 could phenocopy the observed exosomal effects, 5 transfection groups were established: untransfected cells (Ctrl) group, negative control (NC) group (empty vector), ZFAS1 overexpression (ZFAS1-OE) group, siRNA negative control (si-NC) group, and ZFAS1 knockdown (si-ZFAS1) group. Following stable transfection, both phases employed identical functional assays including Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) for proliferation, wound healing and transwell for migration, transwell with Matrigel for invasion, and flow cytometry for apoptosis detection. Direct validation using macrophage-derived exosomes with experimentally reduced ZFAS1 content remains a priority for future studies.

2.7. Cell transfection

Endometrial stromal cells were transfected with lncRNA ZFAS1 mimics, lncRNA ZFAS1 inhibitors, and their negative controls using a lentiviral vector system (GeneChem). The vector used was pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, carrying fluorescent markers. After virus packaging, the virus titer was adjusted to 10⁸ and added to cells for infection. Transfected cells were selected using puromycin (4 μg/mL) for 7 days, then maintained in medium containing 1 μg/mL puromycin to obtain stable cell lines.

2.8. CCK-8 assay for cell proliferation detection

Cell proliferation was assessed using the CCK-8 (Dojindo). Treated endometrial stromal cells were plated in 96-well plates at 1 × 10⁴ cells per well. At various time points (0, 24, 48, 72 hours), CCK-8 reagent was added at a 1:10 ratio. After 2-hour incubation at 37°C, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, multiscan MK3). Cell viability was calculated as: Cell viability (%) = (OD experimental group/OD control group) × 100%.

2.9. EdU assay for cell proliferation detection

Cell proliferation was evaluated using the EdU Cell Proliferation Kit (RiboBio). Cells were incubated with EdU for 2 hours, then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes. After permeabilization with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes, 100 μL of EdU detection cocktail was added to each well and incubated in darkness at room temperature for 30 minutes. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), and EdU-positive cells were counted under a fluorescence microscope (Olympus). Proliferation rate was calculated as: Proliferation rate (%) = (EdU-positive cells/DAPI-positive cells) × 100%.

2.10. Wound healing assay for cell migration detection

Horizontal reference lines were drawn on the back of 6-well plates (Corning). Fibronectin (10 μg/mL; Sigma-Aldrich) was added to each well and stored at 4°C overnight. Cells in the logarithmic growth phase (1 × 10⁶/mL) were uniformly seeded into 6-well culture plates. After cells formed a confluent monolayer, wounds were created using 10 μL sterile micropipette tips (Eppendorf), and cells were washed 2 to 3 times with PBS to remove detached cells. Migration was monitored at 0 and 48 hours using an inverted microscope (Olympus), and wound closure rates were calculated using ImageJ software (National Institutes of Health). Migration rate was calculated as: Migration rate (%) = [(Width at 0 hour − Width at 48 hours)/Width at 0 hour] × 100%.

2.11. Transwell assay for cell migration and invasion detection

For migration assays, treated cells (1 × 10⁵) were resuspended in 100 μL serum-free medium and added to the upper chamber of 24-well Transwell plates (8-μm pore size; Corning). Complete medium (600 μL) was added to the lower chamber. For invasion assays, Matrigel (BD Biosciences) was dissolved overnight at 4°C, diluted with precooled serum-free medium (1:3 ratio), and 40 μL was added to precooled transwell chambers. After 24-hour incubation, cells were fixed with 4% paraformaldehyde, stained with crystal violet (Sigma-Aldrich), and migrated/invaded cells were counted under a light microscope (Olympus) in 5 random fields.

2.12. Flow cytometry for apoptosis rate detection

Cell apoptosis was analyzed using the Annexin V-fluorescein isothiocyanate/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences). Cells were harvested and resuspended in binding buffer. Annexin V-fluorescein isothiocyanate (1.25 μL) was added and incubated in darkness at room temperature for 15 minutes. PI (10 μL) was added before analysis using a flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar). The total apoptosis rate was calculated as the sum of early apoptosis (Annexin V⁺/PI⁻) and late apoptosis (Annexin V⁺/PI⁺) rates.

2.13. Reverse transcription and real-time fluorescence quantitative PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Reverse transcription was performed using the ImProm-II Reverse Transcription System (Promega) following the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green qPCR SuperMix (Invitrogen) with specific primers for ZFAS1 and glyceraldehyde 3-phosphate dehydrogenase on an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems, Thermo Fisher Scientific). Relative expression levels were calculated using the 2^(−ΔΔCt) method with glyceraldehyde 3-phosphate dehydrogenase as the internal control.

2.14. Statistical analysis

Statistical analysis was performed using SPSS 25.0 software (IBM Corporation). Data are presented as mean ± standard deviation (x̄ ± s). Considering the very small sample size in the patient-derived experiments, formal assessment of data distribution had limited reliability; therefore, the inferential statistics are presented as exploratory only. Student t test was used for 2-group comparisons and 1-way analysis of variance followed by least significant difference post hoc testing was used for multi-group comparisons. Nonparametric approaches would be reasonable alternatives in future validation studies with larger sample sets. Two-sided P < .05 was considered statistically significant.

3. Results

3.1. Identification and characterization of exosomes

Exosomes from peripheral blood macrophages were obtained using differential centrifugation. Transmission electron microscopy revealed characteristic double-membrane vesicular structures with an approximate diameter of 100 nm (Fig. 1A). Western blotting demonstrated expression of the commonly used exosomal markers CD81 and CD9 in exosomes from both the EMs group and the control group, supporting successful exosome isolation (Fig. 1B). However, because negative markers for cellular contaminants and nanoparticle tracking analysis were not included, the characterization of exosome purity, size distribution, and particle concentration remains incomplete.

Figure 1.

Characterization of macrophage-derived exosomes. (A) Representative transmission electron microscopy images showing exosome morphology with characteristic double-membrane vesicular structures (scale bar = 200 nm). (B) Western blot analysis demonstrating expression of the exosomal markers CD81 and CD9 in the control and endometriosis groups. CD = cluster of differentiation, EMs = endometriosis.

3.2. Effects of different macrophage exosomes on endometrial stromal-cell proliferation and apoptosis

CCK-8 assays demonstrated significantly enhanced proliferation rates in the EMs-derived exosome group compared with both the blank group and the control-exosome group at 24, 48, and 72 hours (P < .05, Fig. 2A). There was no significant difference between the blank group and the control-exosome group. EdU incorporation assays also showed increased DNA synthesis in the EMs-derived exosome group, with proliferation rates significantly higher than those in both comparison groups (P < .05, Fig. 2B and C).

Figure 2.

Effects of different macrophage exosomes on endometrial stromal cell proliferation and apoptosis. (A) CCK-8 proliferation-rate curve for each group. (B) Representative EdU immunofluorescence images after treatment. Red, EdU; blue, DAPI. Scale bar, 50 µm. (C) EdU experiment proliferation rate bar chart for different treatment groups. (D) Representative flow cytometry apoptosis images for different treatment groups. (E) Apoptosis rate bar chart for different treatment groups. *P<.05, ***P < .001 versus blank; #P<.05, ###P < .001 versus control. CCK-8 = Cell Counting Kit-8, DAPI = 4′,6-diamidino-2-phenylindole, EdU = 5-ethynyl-2′-deoxyuridine, EMs = endometriosis, FITC = fluorescein isothiocyanate.

Flow cytometry analysis revealed that EMs-derived macrophage exosomes significantly inhibited endometrial stromal cell apoptosis compared with both the blank group and the control-exosome group (P < .05, Fig. 2D and E). The total apoptosis rate in the EMs-derived exosome group was significantly lower than that in the other 2 groups.

3.3. Effects of macrophage-derived exosomes on endometrial stromal cell migration and invasion

Wound healing assays demonstrated enhanced migratory capacity in the EMs-derived exosome group compared to the blank group and the control-exosome group (P < .05, Fig. 3A and B). No significant differences in migration rates were observed between the blank group and the control-exosome group. Transwell migration assays yielded consistent results, revealing increased cell migration in the EMs-derived exosome group with significantly higher numbers of migrated cells than in both comparison groups (P < .05, Fig. 3C and D).

Figure 3.

Effects of different macrophage exosomes on endometrial stromal cell migration and invasion. (A) Representative wound healing assay images at 0 and 48 hours for all groups. Scale bar, 100 μm. (B) Wound healing migration rate bar chart for different treatment groups. (C) Representative transwell migration images for all groups. Scale bar, 100 μm. (D) Transwell migration cell numbers bar chart for different treatment groups. (E) Representative transwell invasion images for all groups. Scale bar, 100 μm. (F) Transwell invasion cell numbers bar chart for different treatment groups. **P<.01, ***P < .001 versus blank; ###P < .001 versus control. EMs = endometriosis.

Transwell invasion assays demonstrated that EMs-derived macrophage exosomes significantly enhanced invasive capacity compared to the blank group and the control-exosome group (P < .05, Fig. 3E and F). The number of invaded cells in the EMs-derived exosome group was significantly higher than that in both comparison groups.

3.4. Expression of lncRNA ZFAS1 in macrophage-derived exosomes

Quantitative real-time PCR analysis revealed significantly elevated lncRNA ZFAS1 expression in exosomes derived from EMs patients compared to control patients (P < .05, Fig. 4A and B). Given the very small number of patient samples, this difference should be interpreted as preliminary and requires validation in a larger cohort.

Figure 4.

Expression of lncRNA ZFAS1 in macrophage-derived exosomes. (A) lncRNA ZFAS1 amplification and melting curves. (B) Relative expression levels of lncRNA ZFAS1 detected by qRT-PCR in the EMs and control groups. ***P < .001 versus control. EMs = endometriosis, lncRNA = long noncoding RNA, qRT-PCR = quantitative reverse transcription polymerase chain reaction, ZFAS1 = zinc finger antisense 1.

3.5. Effects of lncRNA ZFAS1 overexpression and knockdown on endometrial stromal-cell proliferation and apoptosis

Successful transfection was confirmed by fluorescence microscopy (Fig. 5A). In the CCK-8 assay, overall proliferation-rate curves varied among groups, but these differences were not statistically significant (Fig. 5B). By contrast, inhibition-rate analysis showed significant differences, with the si-ZFAS1 group displaying higher inhibition rates than the other groups (P < .05, Fig. 5C). These findings indicate that the proliferation-promoting effect of ZFAS1 was supported primarily by the EdU assay and the inhibition-rate analysis rather than by statistically significant differences in the overall CCK-8 proliferation curves.

Figure 5.

Effects of lncRNA ZFAS1 on endometrial stromal cell proliferation and apoptosis. (A) Representative transfection images showing stable cell lines for all groups. Scale bar, 100 μm. (B) CCK-8 proliferation rate bar chart for different treatment groups. (C) CCK-8 inhibition rate curve for different treatment groups. (D) Representative EdU experiment images for all transfected groups. (E) EdU proliferation rate bar chart for different treatment groups. (F) Representative flow cytometry apoptosis images for all transfected groups. (G) Apoptosis rate bar chart for different treatment groups. *P<.05, **P<.01, ***P<.001 compare with Ctrl; #P<.05, ##P<.01, ###P<.001 compare with NC; ΔΔP<.01, ΔΔΔP<.001 compare with ZFAS1-OE; &&P<.01, &&&P<.001 compare si-NC. CCK-8 = Cell Counting Kit-8, Ctrl = untransfected cells, EdU = 5-ethynyl-2′-deoxyuridine, EMs = endometriosis, FITC = fluorescein isothiocyanate, lncRNA = long noncoding RNA, NC = negative control, si-NC = siRNA negative control, si-ZFAS1 = zinc finger antisense 1 knockdown, ZFAS1-OE = zinc finger antisense 1 overexpression.

EdU incorporation assays demonstrated significant differences in DNA synthesis rates between groups (P < .05, Fig. 5D and E). The ZFAS1-OE group significantly enhanced proliferation rates compared to the Ctrl and NC groups, while the si-ZFAS1 group substantially reduced proliferation compared to all other groups.

Flow cytometry apoptosis analysis revealed significant differences in programmed cell death rates between groups (P < .05, Fig. 5F and G). The si-ZFAS1 group exhibited significantly higher apoptosis rates compared to all other groups, while the ZFAS1-OE group effectively reduced apoptosis rates compared to the Ctrl and NC groups.

3.6. Effects of lncRNA ZFAS1 overexpression and knockdown on endometrial stromal cell migration and invasion

Wound healing assays revealed significant differences in migratory capacity between treatment groups (P < .05, Fig. 6A and B). The ZFAS1-OE group markedly enhanced cell migration compared to the Ctrl and NC groups, while the si-ZFAS1 group substantially reduced migratory capacity compared to all other groups. No significant differences were observed among the Ctrl, NC, and si-NC groups.

Figure 6.

Effects of lncRNA ZFAS1 on endometrial stromal cell migration and invasion. (A) Representative wound healing assay images at 0 and 48 hours for all transfected groups. Scale bar, 100 μm. (B) Wound healing migration rate bar chart for different treatment groups. (C) Representative transwell migration images for all transfected groups. Scale bar, 100 μm. (D) Transwell migration cell numbers bar chart for different treatment groups. (E) Representative transwell invasion images for all transfected groups. Scale bar, 100 μm. (F) Transwell invasion cell numbers bar chart for different treatment groups. **P<.01, ***P<.001 compare with Ctrl; ##P<.01, ###P<.001 compare with NC; ΔΔP<.01, ΔΔΔP<.001 compare with ZFAS1-OE; &P<.05, &&&P<.001 compare si-NC. Ctrl = untransfected cells, lncRNA = long noncoding RNA, NC = negative control, si-NC = siRNA negative control, si-ZFAS1 = zinc finger antisense 1 knockdown, ZFAS1-OE = zinc finger antisense 1 overexpression.

Transwell migration assays confirmed these observations, demonstrating that the ZFAS1-OE group significantly promoted cell migration while the si-ZFAS1 group effectively inhibited migratory behavior (P < .05, Fig. 6C and D). The migratory capacity was highest in the ZFAS1-OE group, followed by the NC, Ctrl, and si-NC groups, with the lowest migration observed in the si-ZFAS1 group.

Transwell invasion assays showed consistent patterns, with the ZFAS1-OE group enhancing invasive capacity and the si-ZFAS1 group reducing the ability of cells to penetrate the extracellular matrix (P < .05, Fig. 6E and F). The number of invaded cells was significantly higher in the ZFAS1-OE group and significantly lower in the si-ZFAS1 group compared to control groups.

4. Discussion

Exosome isolation requires precise methodological approaches, with various techniques available, including ultracentrifugation, gradient ultracentrifugation, commercial kits, chromatographic methods, and novel enrichment techniques such as microfluidic filtration, contactless sorting, and immunoaffinity enrichment.^[17,18] In this study, differential centrifugation successfully isolated extracellular vesicles secreted by peripheral blood macrophages, and transmission electron microscopy showed vesicles with characteristic morphology of approximately 100 nm. Western blotting for the commonly used exosomal markers CD9 and CD81 supported successful isolation. Nevertheless, the absence of negative markers such as calnexin or Golgi matrix protein of 130 kDa and the lack of nanoparticle tracking analysis limited the rigor of exosome characterization and prevented a more complete assessment of purity, size distribution, and particle concentration. Even so, the consistent TEM morphology, positive CD9/CD81 signals, and reproducible biological activity across experiments support the interpretation that the analyzed vesicle fraction is relevant to the observed stromal-cell responses.

Our findings suggest that peripheral blood macrophages from patients with endometriosis secrete exosomes that can promote the proliferation, invasion, and migration of endometrial stromal cells while reducing apoptosis. However, because the patient-derived exosomes were obtained from only 3 cases per group, the observed differences should be regarded as preliminary and hypothesis-generating rather than definitive.

To identify the key cargo potentially mediating these effects, we examined lncRNA ZFAS1 expression in macrophage-derived exosomes. Quantitative reverse transcription polymerase chain reactionanalysis showed higher lncRNA ZFAS1 expression in EMs-derived exosomes than in control exosomes, suggesting that ZFAS1 may be enriched in exosomes from patients with endometriosis. Together with the stromal-cell gain- and loss-of-function experiments, these data support the biological plausibility that ZFAS1 contributes to the observed phenotype, although this patient-based expression result still requires confirmation in a larger dataset.

To examine whether lncRNA ZFAS1 could account for the observed phenotypes, we performed gain- and loss-of-function experiments in endometrial stromal cells. Elevated ZFAS1 expression enhanced invasion and migration and reduced apoptosis compared with control groups, whereas ZFAS1 knockdown produced the opposite pattern. These findings support the biological relevance and phenotypic sufficiency of ZFAS1. However, the necessity of exosomal transfer of ZFAS1 was not directly tested in the present study because exosomes derived from ZFAS1-silenced macrophages were not available. Therefore, our data support an association between exosomal ZFAS1 enrichment and the observed cellular phenotypes but do not establish direct exosome-transfer causality at this stage.

The downstream mechanism by which lncRNA ZFAS1 may promote endometriosis progression remains unclear. Previous cancer studies suggest that ZFAS1 can function as a competing endogenous RNA, modulate epithelial-mesenchymal transition-related signaling, and regulate RNA-binding proteins.^[19,20] Because no downstream targets or epithelial-mesenchymal transition markers were examined in this study, these mechanistic explanations remain speculative and should be interpreted cautiously. We therefore present these pathways as testable hypotheses rather than established conclusions.

Several additional limitations should also be noted. First, the small number of patient samples precluded robust assessment of between-patient heterogeneity and limited the reliability of formal statistical assumptions. Second, the experimental design combined patient-derived material with in vitro mechanistic assays and should therefore be considered a pilot translational study rather than a purely observational clinical study. Third, although the cell-based overexpression and knockdown experiments helped clarify the biological role of ZFAS1, they do not replace direct validation using macrophage-derived exosomes with altered ZFAS1 content. Future studies should prioritize: expanded patient cohorts with formal power analysis; comprehensive exosome characterization including negative markers and nanoparticle tracking analysis; ZFAS1-reduced macrophage-derived exosome experiments; and mechanistic validation of downstream pathways.

5. Conclusion

These findings suggest that peripheral blood macrophage-derived exosomes are associated with endometrial stromal-cell proliferation, migration, invasion, and apoptosis changes, and that exosomal lncRNA ZFAS1 may contribute to this phenotype. Given the pilot nature of the study, the limited patient sample size, the incomplete characterization of exosomes, and the lack of direct exosome-transfer mechanistic validation, the present conclusions should be regarded as preliminary.

Acknowledgments

The authors thank all participants who contributed samples for this study.

Author contributions

Conceptualization: Qiu Meng, Jing Zhang.

Data curation: Qiu Meng, Xingrun Xie, Huiting Chang.

Formal analysis: Qiu Meng, Shouguo Huang.

Funding acquisition: Qiu Meng.

Investigation: Qiu Meng, Haiyan Wang, Jie Wang, Huiting Chang.

Methodology: Shouguo Huang.

Project administration: Haiyan Wang, Jie Wang.

Resources: Haiyan Wang, Jie Wang.

Supervision: Jing Zhang.

Visualization: Xingrun Xie.

Writing – original draft: Qiu Meng.

Writing – review & editing: Qiu Meng, Jing Zhang.