UCMSCs were isolated from fresh, full-term umbilical cords of women who gave birth at term in The Second Hospital of Hebei Medical University (Shijiazhuang, China) from January 2021 to December 2022 were collected, and informed consent was obtained from the six mothers who donated the umbilical cords for research purposes (n=6; mean age, 35.83±3.061 years), with each mother providing one umbilical cord. The inclusion criteria were: Healthy mothers with full-term deliveries. The exclusion criteria were: Mothers with pregnancy complications or pre-existing medical conditions, and those whose fetuses were diagnosed with developmental abnormalities during prenatal screening. The present study was approved by the Ethical Committee of the Second Hospital of Hebei Medical University (Shijiazhuang, China; approval no. 2020-R285). The cords were first washed with PBS supplemented with an antibiotic/antimycotic solution to mitigate microbial contamination. Subsequently, Wharton's jelly was dissected from the cords and cut into small explants of 1-2 cm³. These explants were then placed onto culture dishes pre-coated with 0.1% gelatin (HapCult™; Precision BioMedicals Co., Ltd.) to enhance cell adhesion, before the plates were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) enriched with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% L-glutamine (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in an atmosphere containing 95% air/5% CO₂. The medium was initially replaced after 72 h to eliminate non-adherent cells, and then replaced every 3 days. Once the cells had reached 80-90% confluence, they were detached using 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and subsequently passaged. Cells at passages 3-7 were utilized for further experiments to ensure consistency and vitality. The morphological features of UCMSCs were continuously monitored under an inverted light microscope (Carl Zeiss AG), where they displayed a typical fibroblast-like morphology.

The UCMSCs were identified as previously described (16). Cells were first observed under an inverted light microscope to confirm their morphology. Subsequently, phenotypic characterization was performed using flow cytometric (FCM) analysis with a BD FACSCanto™ II flow cytometer (BD Biosciences). Cells were stained with fluorescently labeled antibodies to confirm their mesenchymal lineage, whereas hematopoietic elements were excluded. Additionally, H&E staining was performed on cellular smears to further authenticate their morphology.

FCM analysis was performed to phenotypically identify the UCMSCs. Cells were detached using 0.25% trypsin-EDTA, after adding trypsin and incubating at room temperature for 1-3 min, PBS containing 2% FBS was added and stained with fluorescently labeled antibodies against MSC markers CD90-FITC (1:400; cat. no. 555595), CD44-PE (1:400; cat. no. 566803), CD105-PerCP (1:400; cat. no. 323215), CD73 PE (1:400; cat. no. 550257) and CD29-APC (1:400; cat. no. 559883) (all purchased from Biolegend, Inc.) and negative markers human leukocyte antigen (HLA)-DR-APC (1:400; cat. no. 307609) and CD45-FITC (1:400; cat. no. 555488) (both from BioLegend, Inc.). The number of cells per aliquot for antibody staining ranged from 5x10⁶ to 10x10⁶ cells. Following a 30-min incubation at 4˚C, the cells were washed, fixed in 1% paraformaldehyde at room temperature for 10-15 min. If prompt analysis on the machine was not feasible, fixation was required, followed by storage at 4˚C in the dark. Analysis was conducted within 24 h. If analysis was performed immediately, the fixation step was unnecessary (fixed with 1% paraformaldehyde at room temperature for 10-15 min). After antibody labeling, the cells were incubated at room temperature in the dark for 15 min. Subsequently, 1 ml of PBS was added to wash the cells. The cells were then centrifuged at 300 x g for 5 min at 4˚C, and the supernatant was discarded. Finally, the cells were resuspended in 500 to 1,000 µl of PBS and analyzed using flow cytometry (CellQuest; BD Biosciences). Data obtained from ≥10,000 events per sample confirmed a high expression level of mesenchymal markers and a low expression level of hematopoietic markers, verifying their MSC identity.

To track the cellular integration of UCMSC post-transplantation, CellTracker™ CM-DiI dye (cat. no. C7000; Life Technologies; Thermo Fisher Scientific, Inc.), a lipophilic fluorescent dye, was used for staining the cell membranes. The passage 3 UCMSCs were first incubated with the CM-Dil solution (the CM-Dil working solution was prepared in advance for fluorescence staining using a ratio of CM-Dil stock solution to medium of 1:1,000). Following an incubation at 37˚C in an atmosphere containing 5% CO₂ for a predetermined duration, the cells were washed with PBS to remove the excess dye. Successful dye incorporation was verified under a fluorescence microscope, with cell nuclei counterstained using DAPI at room temperature 5 min (cat. no. 0100-20; SouthernBiotech) to enhance visualization.

All animals were housed in a pathogen-free facility at the Animal Center of the Second Hospital of Hebei Medical University. The rats were kept in standard cages under controlled conditions (12-h light/dark cycle, temperature of 22-24˚C and 40-60% humidity) with ad libitum access to food and water. Humane endpoints were predefined to minimize animal suffering. Rats exhibiting signs of distress, severe illness or immobility were humanely euthanized using an overdose of pentobarbital sodium (100 mg/kg, intraperitoneal injection). The total duration of the experiment was 18 weeks, which included a 2-week post-modeling period for IUA induction, and 4 weeks of treatment and observation following UCMSC administration, followed by 12 weeks of the co-housing experiment. A total of 174 rats (149 female rats and 25 male rats) were used in the present study. All animals were humanely euthanized at the conclusion of the experiment. No animals reached the humane endpoint and no animals were found dead during the experiments.

The IUA model was established using female Sprague-Dawley (SD) rats, aged 6-8 weeks and weighing between 180-220 g, which were procured from the Hebei Medical University Animal Experiment Center. All animal experiments were conducted with approval from the Ethical Committee of the Second Hospital of Hebei Medical University (approval no. 2021-AE261). A total of 12 rats were randomly divided into two groups: A normal control group (n=6 rats) and a model group (n=6 rats). The use of 6 rats in the modeling group was based on previous studies and statistical power analysis that indicated this number to be sufficient for histological assessments (17). In similar studies, a sample size of 6 has been shown to provide reliable and reproducible data for evaluating tissue morphology and fibrosis (18).

The IUA model in the present study was based on a dual-injury approach: Mechanical injury (scratching) combined with lipopolysaccharide (LPS) application. The primary focus was on mechanical injury, with LPS used to represent secondary infection, which can occur following intrauterine procedures and may contribute to adhesion formation (17-19). This method was chosen to reflect clinical situations where infection may serve a role in adhesion development after surgical interventions.

The rat model of IUA was established following previously reported protocols (17-19). To quantify the migration of CM-Dil-labeled UCMSCs to the dual-injured uterus in SD rats, the following procedure was performed. Briefly, SD rats were anesthetized using an intraperitoneal injection of sodium pentobarbital at a dose of 40 mg/kg body weight and subjected to a midline abdominal incision (2-2.5 cm). The endometrial lining was then scraped using a 2.5-mm endometrial curette (RWD Life Science Co., Ltd.) until the uterine wall became visibly rough. LPS-coated sutures were subsequently placed into the cavity of one uterine horn, with one end fixed to the skin through the muscle layer. The sutures were removed after 48 h. Model stability was confirmed after three estrous cycles. Our previous research conducted observations at 1, 2, 4, 8 and 12 weeks after IUA modeling. When comparing the 2-week post-modeling period with 4, 8 and 12 weeks, there were no significant changes in endometrial thickness, glandular density, and extent of fibrotic tissue (17). Our research team (17-19) used the same IUA modeling method and confirmed that the 2-week IUA model reached a stable state. CM-Dil-labeled UCMSCs were injected into the abdominal cavity, tail vein, and uterine myometrium. The animals were euthanized on days 3, 7 and 14 post-transplantation, and the uteri were subjected to rapid frozen sectioning followed by immunofluorescence tracing. In the present study, the time points for euthanasia varied depending on the experimental purpose. During the fluorescent tracing experiment, rats were euthanized at D3, D7 and D14 to track the distribution and persistence of the transplanted cells over time. By contrast, for the stem cell treatment experiment, rats were euthanized only at D28 to perform histological analyses and evaluate the therapeutic effects of stem cell therapy on uterine tissues. Thus, D3, D7 and D14 were specifically used to investigate the localization of the transplanted stem cells, while D28 was used to assess the outcomes of the treatment.

Female SD rats were categorized into five groups, each comprising 6 animals for histological examination and 5 animals for fertility testing (totaling 11 rats in each group; Table I), to assess the impact of different UCMSC administration routes on IUA. The groups included the following: i) A normal group (Normal), with no modeling or treatment; ii) a model group (IUA group), in which the rats underwent IUA modeling without any treatment; iii) an intraperitoneal injection group (IP group), in which the rats received UCMSCs through intraperitoneal injection after IUA modeling; iv) an in-site injection group (IS group), where the rats were treated with UCMSCs directly into the intrauterine wall post-modeling; and v) an injection of caudal vein group (IOCV group), where the rats were administered UCMSCs through the tail vein following IUA induction.

To ascertain the therapeutic efficacy of UCMSCs on IUA, UCMSCs were administered to female SD rats through the three distinct routes (intraperitoneal injection, in-site injection and caudal vein injection), where each rat received a standardized dose of 1x106 cells. Over a 4-week post-transplantation period, the health of the rats was monitored, with particular attention given to any adverse reactions or complications. At the end of the observation period (D28), all animals were euthanized via intraperitoneal injection of an overdose of sodium pentobarbital (100 mg/kg). Death was confirmed by the cessation of heartbeat and pupil dilation. The uteri were subsequently collected for comprehensive histopathological evaluation. Histological assessments were performed using H&E and Masson's trichrome staining to evaluate endometrial regeneration and fibrosis. Quantitative analyses of endometrial thickness, gland density and fibrotic areas were performed using ImageJ software (Image J 1.53e. Java 1.8.0_172; National Institutes of Health), and immune responses were assessed by measuring serum IgG levels using ELISA.

To investigate the effect of different concentrations of intraperitoneally administered UCMSCs on the treatment efficacy for IUA, three dosage groups were established among the model rats: A low-dose group (with the administration of 0.5x10⁶ cells), a medium-dose group (receiving 1x10⁶ cells) and a high-dose group (treated with 5.0x10⁶ cells). The dosage selection was based on preliminary experiments that were conducted to identify the most appropriate and effective dosage range for treatment (17), ensuring the doses used were within the therapeutic window. The normal group underwent a sham operation (abdominal incision without injury, no IUA induction, and no stem cell treatment) and served as the baseline control. The model group received the IUA induction without stem cell treatment, providing a reference for evaluating the effects of UCMSC therapy. Each group consisted of 11 rats (6 for histological assessment and 5 for mating experiments; Table I), all of which had undergone IUA induction and received UCMSC administration through the route of intraperitoneal injection. After 4 weeks of monitoring the rats for adverse effects, they were euthanized and uterine tissue samples were collected. Histological assessment was subsequently performed as previously described in analysis of the effects of UCMSC administration routes on IUA treatment.

To assess the efficacy of UCMSCs on fertility after IUA, a cohabitation study was initiated after the initial 4-week treatment period. In total, 5 female Sprague-Dawley rats from each experimental group were randomly selected and paired with fertile male rats at a 1:1 ratio. The pairs were housed under controlled environmental conditions (temperature, 20-25˚C; humidity maintained at 50-60%; and the light/dark cycle of 12 h of light followed by 12 h of darkness) optimal for breeding for a duration of 12 weeks, with food and water provided ad libitum. The mating pairs were observed daily to monitor for signs of pregnancy, which were initially indicated by physical changes in the females and subsequently confirmed through palpation. Post-gestation, the number of offspring for each female was recorded. No offspring were sacrificed in this study. Instead, these offspring were donated to the Hebei Medical University Animal Experiment Center for use in other research projects. These data served as a direct indicator of the fertility potential of UCMSC treatment, providing insights into the success rate and possible enhancements in reproductive health, of the rats following the intervention for IUAs.

H&E staining was used to assess morphological changes in the uterine tissue post-treatment. Samples were fixed in 4% neutral buffered formalin at room temperature for 24 h, dehydrated in an ascending ethanol gradient, cleared in xylene and paraffin-embedded. Sections of 5 µm in thickness were deparaffinized, rehydrated and stained with hematoxylin for nuclei (at room temperature for 30 min) and counterstained with eosin (at room temperature for 30 sec) for cytoplasmic and extracellular components. The slides were subsequently dehydrated, cleared and mounted in a xylene-based medium. Microscopic examinations at magnifications of x100, x200 and x400 were undertaken to evaluate the endometrial thickness, cellular integrity and extent of fibrosis. Endometrial thickness was measured using H&E-stained sections imaged under a low-power light microscope. In total, four perpendicular points were selected within each section and the thickness was measured at these points. The average of these measurements was calculated to determine the final endometrial thickness. Finally, images were taken for comparative analysis among the experimental groups.

Masson's trichrome staining was used to assess the extent of fibrosis in the uterine tissues post-UCMSC treatment. Tissue sections, embedded in paraffin and sliced to a thickness of 5 µm, underwent deparaffinization in xylene and rehydration through a descending graded alcohol series. Staining involved the sequential application of Weigert's iron hematoxylin for 7 min, Biebrich scarlet-acid fuchsin for 15 min, and aniline blue for 2 min, and all procedures were completed at room temperature. Quantitative fibrosis analysis was performed using image processing software (Image J 1.53e. Java 1.8.0_172; National Institutes of Health) to measure fibrosis area and intensity in four randomly selected fields of view.

To assess the immunological response following transplantation of the UCMSCs, the serum IgG levels were measured using ELISA. Blood samples (1 ml per rat) were collected from the tail vein immediately prior to euthanasia after the 4-week treatment period and the serum was separated by centrifugation at 3,018.6 g for 15 min at room temperature, before being stored at -20˚C for analysis. Using a specific Rat IgG ELISA kit (cat. nos.866, Meimian Technology Co., Ltd.) for IgG according to the manufacturer’s instructions, 100 µl serum was added to anti-rat IgG-coated wells on a 96-well plate. After incubating and washing the plate, an HRP-conjugated secondary antibody was used to detect bound IgG. The reaction was developed with tetramethylbenzidine and stopped by the addition of sulfuric acid. The optical density was subsequently measured at 450 nm using a microplate reader and the IgG levels were quantified against a standard curve.

Each experiment was performed at least three times. Statistical analysis was performed using SPSS version 26.0 (IBM Corp.). Continuous variables were presented as mean ± standard deviation. One-way ANOVA was used for the homogeneous variance in ≥3 group comparisons followed by the Tukey's test for multiple comparisons between groups. Welch's ANOVA was applied to the uneven variance in ≥3 group comparisons followed by the Games-Howell method between groups. P<0.05 was considered to indicate a statistically significant difference. Each data analysis included at least three independent molecular experiments, with a minimum of three animal samples per group in each experiment.

After the UCMSCs had been successfully isolated and cultured within ~2 weeks, spindle-shaped cells began to migrate from the explanted tissue pieces and reached 80-90% confluence, displaying a characteristic ‘whirlpool-like’ pattern in their arrangement (Fig. 1A). Further phenotypic characterization through FCM analysis confirmed the mesenchymal identity of the cells. Specifically, the cells were found to exhibit high expression levels of MSC markers CD90, CD44, CD105, CD73 and CD29, with minimal expression of the hematopoietic markers HLA-DR and CD45, thereby validating their mesenchymal lineage from Wharton's jelly (Fig. 1B). Subsequently, the morphological characteristics of the UCMSCs were assessed using H&E staining, which revealed elongated, spindle-shaped cells with well-defined oval nuclei and light red cytoplasm, exhibiting a uniform fusiform shape, growing in a vortex-like or parallel pattern, and arranged in a neat and orderly fashion adherent to the substrate (Fig. 1C). These results confirmed the successful isolation and characterization of UCMSCs, where the specific marker profile supported their identity as MSCs.

Post-transplantation tracking of UCMSCs was achieved using CM-Dil. After completion of the labeling process, UCMSCs exhibited strong red fluorescence in the cytoplasm, confirming successful uptake of the dye. Their nuclei were counterstained with DAPI, producing a striking blue fluorescence (Fig. 1D). This dual-color imaging process enabled precise tracking of the UCMSCs in the rat IUA model, confirming that the CM-Dil-labeled cells both maintained their structural integrity and were easily identifiable within the target tissues.

The localization and persistence of UCMSCs within rat uterine tissues were tracked using fluorescence imaging following transplantation. Fig. 2 specifically shows the distribution of UCMSCs post-transplantation and does not include data from IUA-induced rats or a normal control group. The figure was designed solely to evaluate the in vivo localization of the transplanted cells. Cells were pre-labeled with a fluorescent dye for enhanced visualization in the tissue sections. By day 3, fluorescently labeled UCMSCs were detectable in all treatment groups, confirming successful engraftment (Fig. 2). By day 7, the IP group exhibited widespread fluorescence throughout the uterine muscular and interstitial tissues, whereas the IS group exhibited no detectable fluorescence in the muscular layer, indicating poor retention. The IOCV group displayed localized fluorescence at the endometrial-myometrial junction, indicating route-specific migration patterns. By day 14, all experimental groups exhibited fluorescent markers near the uterine cavity's inner lining, suggesting retention and potential integration of UCMSCs into the endometrial layer (Fig. 2).

Following the established modeling procedures, a rat model of IUA was developed (Fig. 3A-D). Histological analysis using H&E staining revealed a significant reduction in the endometrial thickness in the model group compared with that in the normal group (P<0.05; Fig. 3E and Table II). The number of endometrial glands also decreased significantly in the model group compared with that in the normal group (P<0.05), suggesting the occurrence of substantial glandular loss due to induced adhesions (Fig. 3E and Table II). Furthermore, the Masson's trichrome staining experiments demonstrated a significant increase in fibrotic areas within the endometrial interstitium of the model group compared with that in the normal group (P<0.05; Fig. 3E and Table II). Collectively, these data suggest the efficacy of the combined mechanical and infection-induced injury methods in replicating IUAs in rats.

The effectiveness of the different administration routes of UCMSCs that were investigated in the present study in terms of treating IUAs was evaluated through various histological and immunological assessments. Significant differences in endometrial thickness and gland numbers were observed comparing among the experimental groups (P<0.001 for thickness; P<0.001 for gland number; Fig. 4A, C and D). In particular, the IP group demonstrated an increase in endometrial thickness, which was around normal levels and was significantly higher compared with that in the IS and IOCV groups. Additionally, the gland density was also higher in the IP group (close to normal) compared with that in the IS group and the baseline IUA model.

The extent of fibrosis was also shown to be significantly different among the experimental groups (P<0.001), with the IP group showing the lowest percentage of fibrotic areas, a value that was significantly lower compared with that in the IS, IOCV and IUA groups (Fig. 4B and E). IgG levels, measured using ELISA, showed an initial rise within 3-7 days post-transplantation in the five groups, although this trend was followed by diverging trends afterwards. The IP group exhibited a rapid increase in IgG levels, which were later stabilized, whereas the IS and IOCV groups exhibited a more gradual increase (Fig. 4F). By day 28, the normal group had the lowest immune response, whereas the IOCV group showed markedly higher levels, reflecting the varying immunological impacts of the different treatments. Taken together, these findings underscored the distinct impact of different UCMSC administration routes on both structural restoration and the immunological environment in the IUA model of rats.

This series of experiments assessed the therapeutic efficacy of administering different concentrations of UCMSCs intraperitoneally in a rat model of IUA. Specifically, significant differences in endometrial thickness were observed among the five groups (P<0.001; Fig. 5A and C). The group treated with 1.0x10⁶ UCMSCs (the middle dose) exhibited a substantial increase in thickness, surpassing the low-dose (0.5x10⁶ cells) and high-dose (5.0x10⁶ cells) groups, where the thickness was comparable with that of the normal group, indicating effective endometrial regeneration. Gland numbers also varied significantly (P<0.001; Fig. 5A and D). The 5.0x10⁶ cells treatment group exhibited a significantly higher gland count compared with that in the 0.5x10⁶ cells treatment group and the baseline IUA model, which was comparable with the normal and 1.0x10⁶ cells groups, suggesting a dose-dependent effect on glandular restoration. The differences in fibrotic area were also found to be statistically significant (P<0.001; Fig. 5B and E). The 1.0x10⁶ cells (middle dose) group exhibited the lowest fibrotic percentage, which was significantly lower compared with that in the high dose, low dose and the baseline IUA model groups, highlighting the optimal dose for minimizing fibrosis. Taken together, these findings suggested that the concentration of UCMSCs that is administered intraperitoneally critically influences both endometrial tissue regeneration and fibrosis reduction.

Subsequently, the fertility restoration capabilities of various concentrations and administration routes of UCMSCs were next systematically evaluated. A 12-week co-housing experiment was performed to assess the reproductive outcomes by counting the offspring from different treatment and control groups (Fig. 6A). No pregnancies were observed in the IUA model group (Fig. 6B) or the low-dose UCMSC (0.5x10⁶ cells) group (Fig. 6C). Among the administration methods, intraperitoneal injection markedly outperformed the in-site injection and intravenous tail-vein injection groups in terms of offspring count (P<0.05). However, these numbers remained significantly lower compared with those of the normal group (Fig. 6B). The high-dose (5.0x10⁶ cells) and the middle-dose (1.0x10⁶ cells) groups produced similar reproductive outcomes, but were both significantly lower compared with the average number of pups in the normal group (P<0.05; Fig. 6C). Collectively, these results suggest that the treatment with UCMSCs could partly restore fertility in rats afflicted with IUA.