Animals and Experimental Design

This study was conducted in accordance with international guidelines for the care and use of laboratory animals and was approved by the Pamukkale University Animal Experiments Ethics Committee (Approval No. PAUHDEK-2021/38). A total of 32 adult female Wistar albino rats (weighing 250–350 g) were housed under controlled standard laboratory conditions (temperature: 21 ± 1 °C, relative humidity: 65–70%, 12-hour light/dark cycle) with ad libitum access to standard rodent chow and water.

The animals were randomly allocated into four experimental groups (n = 8 per group):

1. Control group: received no interventions or injections throughout the study period.

2. Sham group: received daily intraperitoneal (i.p.) injections of physiological saline (0.9% NaCl) for 14 consecutive days.

3. POF group: premature ovarian failure was induced via an initial loading i.p. injection of CTX (200 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) on day 1, followed by daily maintenance i.p. injections of 8 mg/kg/day for 14 consecutive days.

4. DP-MSC group: following the 14-day CTX regimen as described above, animals received a local intraovarian injection of 2 × 10⁶ human DP-MSCs suspended in 0.2 mL phosphate-buffered saline (PBS) on day 15, administered bilaterally using a 1-mL insulin syringe equipped with a 26-gauge needle under aseptic conditions.

On day 25 (10 days post-transplantation), all animals were deeply anesthetized and euthanized. Blood samples were collected via cardiac puncture for biochemical analysis, and ovaries were harvested and processed for histological and immunohistochemical evaluations.

DP-MSC Isolation, Culture, and Characterization

Human DP-MSCs were isolated from healthy impacted third molars following approval from the Pamukkale University Non-Interventional Clinical Research Ethics Committee (Approval No. 17, dated July 8, 2020) and informed donor consent. Cells at passage 3 were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin at 37 °C in a humidified incubator containing 5% CO.

The immunophenotypic profile of DP-MSCs was verified by flow cytometry using monoclonal antibodies against human CD73, CD90, and CD34 (Beckman Coulter, Brea, CA, USA). Trilineage multipotency was validated by culturing cells in osteogenic, adipogenic, and chondrogenic differentiation media for 21 days, followed by specific staining with Alizarin Red S (calcium deposits), Oil Red O (neutral lipid vacuoles), and Alcian Blue (sulfated glycosaminoglycans), respectively.

Hormonal Analysis

Blood samples were collected via cardiac puncture under deep anesthesia, allowed to clot for 30 minutes at room temperature, and centrifuged at 3,000 × g for 15 minutes at 4 °C. Serum was separated and stored at −80 °C until analysis. Serum concentrations of AMH, FSH, and LH were measured using rat-specific enzyme-linked immunosorbent assay (ELISA) kits (Bioassay Technology Laboratory [BT LAB], Shanghai, China) according to the manufacturer’s instructions. All samples were analyzed in duplicate. Optical density was measured at 450 nm using a microplate reader (BioTek ELx800; BioTek Instruments Inc., Winooski, VT, USA), and absolute hormone concentrations were calculated using standard calibration curves.

Histological Evaluation and Differential Follicle Counting

Excised ovarian tissues were fixed in 10% neutral buffered formalin for 24 hours, dehydrated through an ascending ethanol series, cleared in xylene, and embedded in paraffin wax. Serial sections (5-µm thickness) were prepared and stained with hematoxylin and eosin (H&E) for structural evaluation.

For quantitative follicle counting, every 5th section (specifically the 1st, 5th, and 10th sections per ovary) was systematically selected to avoid double counting. Only follicles containing an oocyte with an identifiable nucleus were counted and classified according to standard morphological criteria11,12:

• Primordial follicles: an oocyte surrounded by a single layer of flattened, squamous pre-granulosa cells;

• Primary follicles: an oocyte surrounded by a single layer of cuboidal granulosa cells;

• Secondary follicles: an oocyte enclosed by two or more layers of cuboidal granulosa cells without an antral cavity;

• Tertiary (antral) follicles: a follicle exhibiting a well-defined, fluid-filled antral cavity;

• Atretic follicles: follicles displaying apoptotic/pyknotic granulosa cells, cellular detachment from the basement membrane, or degenerated oocytes;

• Cystic follicles and corpora lutea were also quantified across sections.

All microscopic examinations were performed using an Olympus BX53 light microscope by two independent histologists blinded to the study groups.

Immunohistochemical Analysis

Paraffin sections (5-µm thickness) were deparaffinized in xylene and rehydrated through a descending graded ethanol series. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) using a microwave oven, followed by cooling to room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. Non-specific binding was blocked using a protein blocking solution (ScyTek Laboratories, Logan, UT, USA).

Sections were incubated overnight at 4 °C with primary rabbit polyclonal antibodies against Wnt-1 (1:300; Bioss Antibodies, Woburn, MA, USA), Wnt-4 (1:300; Bioss Antibodies), and β-catenin (1:300; Bioss Antibodies). After PBS washes, sections were incubated with a biotinylated secondary antibody, followed by streptavidin–horseradish peroxidase conjugate (ScyTek Laboratories). Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as the chromogen substrate. Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. Negative controls were prepared by omitting the primary antibody. Immunoreactivity was evaluated semi-quantitatively based on staining intensity as negative (−), weak (+), moderate (++), or strong (+++) by two blinded observers.

Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviation (SD) for normally distributed data or as median with interquartile range (IQR; 25th–75th percentiles) for non-normally distributed data. Normality was evaluated using the Shapiro–Wilk test. Differences among multiple independent groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for normally distributed parameters, or the Kruskal–Wallis test followed by post hoc pairwise Mann–Whitney U tests with Bonferroni correction for non-normally distributed variables. All statistical tests were two-tailed, and a p-value < 0.05 was considered statistically significant.

Phenotypic and Functional Characterization of Human DP-MSCs

Flow cytometric immunophenotyping confirmed the characteristic mesenchymal surface profile of isolated human DP-MSCs. The cells demonstrated strong positive surface expression of characteristic mesenchymal markers CD73 (99.92%) and CD90 (96.76%), alongside negligible expression of the hematopoietic lineage marker CD34 (0.08%) (Figure 1, Panel I). Under phase-contrast microscopy, passage 3 DP-MSCs displayed typical spindle-shaped, fibroblast-like morphology with plastic-adherent growth (Figure 1, Panel IIA). Multilineage differentiation potential was verified by successful differentiation into adipocytes (Oil Red O-positive neutral lipid droplets; Figure 1, Panel IIB), osteocytes (Alizarin Red S-positive mineralized calcium nodules; Figure 1, Panel IIC), and chondrocytes (Alcian Blue-positive sulfated proteoglycans; Figure 1, Panel IID). Together, these phenotypic and functional assays confirmed the MSC identity of the isolated cell populations.

Body Weight Changes

At baseline, initial body weights did not differ significantly among the experimental groups (p = 0.583; Table 1). At the end of the experimental period, a statistically significant difference in final body weight was observed across groups (F = 3.673, p = 0.024), with the Control group exhibiting significantly higher final weights (288.60 ± 20.44 g) compared to the DP-MSC group (265.00 ± 14.93 g; p < 0.05). Furthermore, net body weight gain differed significantly among the groups (Kruskal–Wallis H = 9.046, p = 0.029), with the Sham group showing a significantly greater weight gain (19.00 ± 7.81 g) compared to the DP-MSC group (2.90 ± 15.41 g; p < 0.05; Table 1).

Serum Hormonal Profiles

Biochemical analysis of serum hormone concentrations revealed no statistically significant differences in AMH (p > 0.05) or FSH (p > 0.05) levels among the four experimental groups (Table 2). Serum FSH concentrations were lowest in the Control group (4.68 ± 1.16 mIU/mL) and highest in the POF group (4.99 ± 0.41 mIU/mL). Serum LH concentrations differed significantly among the groups (p = 0.017). Specifically, LH levels were significantly elevated in the untreated POF group (4.90 ± 0.51 mIU/mL) compared with the Sham group (4.04 ± 0.86 mIU/mL; p < 0.05; Table 2), reflecting gonadotropin hypersecretion secondary to chemotherapy-induced ovarian follicular damage.

Ovarian Follicle Dynamics and Morphometry

Histomorphometric follicle counts demonstrated profound alterations in follicular dynamics following CTX administration and substantial recovery following DP-MSC transplantation (Table 3).

The primordial follicle count was significantly decreased in the untreated POF group (10.67 ± 2.18) compared with the Control (19.80 ± 3.90), Sham (24.57 ± 4.72), and DP-MSC (20.00 ± 2.36) groups (p = 0.0001; Table 3).

Evaluation of developing follicles showed that primary follicle numbers were significantly reduced in the POF group (5.56 ± 2.24) relative to the Sham (9.43 ± 2.07) and DP-MSC (10.40 ± 3.75) groups (p = 0.006). Secondary follicle counts were significantly higher in the DP-MSC group (11.00 ± 2.79) than in the POF group (5.78 ± 3.27; p = 0.013). Similarly, tertiary (antral) follicle counts were significantly depleted in the POF group (1.44 ± 1.67) compared with both the Control (6.20 ± 1.92) and DP-MSC (6.60 ± 3.57) groups (p = 0.001). The number of functional corpora lutea was significantly increased in the DP-MSC group (28.60 ± 5.34) compared with the POF group (20.11 ± 7.41; p = 0.029).

Conversely, the POF group exhibited a marked increase in degenerate structures, demonstrating significantly higher numbers of atretic follicles (13.89 ± 4.01 vs. 3.80 ± 0.84 in Control, 2.71 ± 1.38 in Sham, and 5.50 ± 1.43 in DP-MSC; p = 0.0001) and cystic follicles (7.00 ± 2.55 vs. 2.20 ± 1.10 in Control, 2.29 ± 0.95 in Sham, and 2.50 ± 1.18 in DP-MSC; p = 0.001; Table 3).

Histopathological Findings

Histological evaluation of H&E-stained ovarian sections revealed distinct structural differences among the experimental groups (Figure 2).

In the Control and Sham groups, normal ovarian histoarchitecture was well preserved, featuring an intact single layer of cuboidal surface germinal epithelium, a distinct tunica albuginea, and organized cortical stroma. Follicles across various developmental stages—including primordial, primary, secondary, and tertiary follicles—along with healthy corpora lutea were regularly distributed throughout the cortex.

In contrast, the POF group displayed marked histopathological damage, characterized by severe cortical thinning, interstitial stromal fibrosis, and pronounced depletion of the primordial follicle pool. The developing follicular cohorts were drastically reduced, with a predominance of atretic follicles exhibiting apoptotic granulosa cells, cell detachment from the basement membrane, and distorted oocytes.

In the DP-MSC-treated group, ovarian architecture was notably preserved and restored. The surface epithelium remained intact, the cortical stroma was well organized, and an abundant population of primordial follicles was present. Primary, secondary, and tertiary follicles showed normal multilayered granulosa organization and clear antral spaces, alongside prominent corpora lutea and a substantial reduction in atretic and cystic degenerations (Figure 2).

Immunohistochemical Localization of Wnt-1, Wnt-4, and β-Catenin

Immunohistochemical staining was performed to evaluate the spatial expression and localization of Wnt-1, Wnt-4, and β-catenin in the ovary:

• Wnt-1: Strong Wnt-1 immunoreactivity was localized in primordial follicles of the Control, Sham, and DP-MSC groups, whereas staining intensity was noticeably reduced (weak-to-moderate) in primordial follicles of the POF group (Figure 3). Developing follicles (primary, secondary, and tertiary) and corpora lutea displayed positive Wnt-1 staining across all groups, with highest intensity in the corpora lutea of the Control group (Figure 3).

• Wnt-4: Wnt-4 immunoreactivity showed striking group- and stage-dependent differences (Figure 4). In primary follicles, strong Wnt-4 staining was detected in the Control, Sham, and DP-MSC groups, but was markedly diminished or absent in the POF group. In secondary follicles, positive staining was maintained across groups, although the POF group showed a distinct linear perioocytic pattern. In tertiary antral follicles, strong cytoplasmic staining was retained across all groups. Furthermore, corpora lutea exhibited strong Wnt-4 expression in the Control, Sham, and DP-MSC groups, compared to only moderate staining in the POF group (Figure 4), confirming that DP-MSC therapy restored Wnt-4 expression.

• β-Catenin: β-Catenin staining was consistently positive across all experimental groups (Figure 5). Strong immunoreactivity was observed in oocytes as well as in the cytoplasm and nuclei of granulosa cells within primary, secondary, and tertiary follicles. Robust β-catenin expression was also observed throughout the corpora lutea in all groups (Figure 5).