Estrous cycle assessment and PCOS model confirmation
All female rats in the control group evidenced a normal estrus cycle (characterized by proestrus, estrus, metestrus and diestrus phases), by using vaginal cytology and Pap smear (Figure 1). Differently, the vaginal smear cytology of letrozole-treated rats displayed an irregular estrous cycle with a continuation of diestrus phase. Therefore, these data evidenced that the PCOS model was successfully induced.
H&E staining
Ovaries from Control and Sham groups showed a normal morphology, with primordial, primary, secondary, Graafian, some antral follicles and visible corpora lutea in the cortex (Fig. 2A), while the PCOS and PCOS-Sham groups showed many cysts (Fig. 2C). Differently, in the PCOS+Stem cells group the ovarian structure seemed to be ameliorated, with an increased number of follicles, and a decreased number of cysts (Fig. 2D).
Cell culture and MenSC characterization
MenSCs were isolated from menstrual blood and were cultured for 1-2 weeks until a confluency of 80%. Then, they were passaged until the dominant cell population was spindle shaped.
Confirmation of mesenchymal stem-cell markers
The profile of cell surface antigens in stem cells derived from menstrual blood in passage 3 was evaluated. Based on the results of table 1 and Fig. 4, all MenSCs significantly expressed CD90 and CD105 markers, while they were negative for CD34 and CD45.
Labeling of the MenSCs with PKH26
Homing of MenSCs cells after intraovarian injection was evaluated using fluorescent PKH26 staining (Sigma, USA) and Hoechst (Sigma, USA). then the cells were examined under a Fluorescence microscope (Olympus IX71, Japan) for injection into the ovaries of the animal
Angiogenesis assessment
The number of blood vessels in PCOS (17.10 P ≤ 0.0001) and PCOS-Sham (15.17, P ≤ 0.0001) groups significantly increased compared to the Control group (3.40). On the contrary, the number of blood vessels in the PCOS+Stem cells group (2.97) decreased significantly respect to PCOS (P ≤ 0.0001) and PCOS-Sham (P ≤ 0.0001) (Fig. 7).
Evaluation of folliculogenesis
The number of primordial follicles in the ovaries of PCOS (24.66) and PCOS-Sham groups (22.73) were significantly higher than those in the control (9.16, vs PCOS: P ≤ 0.001; vs PCOS-Sham; P ≤ 0.01) and Sham groups (8.16, vs PCOS and PCOS-Sham; P ≤ 0.001). However, the number of primordial follicles in the PCOS + Stem cells group (9.8) was significantly decreased related to PCOS (P ≤ 0.001) and PCOS-Sham (P ≤ 0.01) Groups. (Fig. 8A).
The number of primary follicles in the ovaries of PCOS (19.03) and PCOS-Sham groups (16.40) were significantly higher than those in the control (5.30, vs PCOS and PCOS-Sham; P ≤ 0.0001) and sham groups (3.70, vs PCOS and PCOS-Sham; P ≤ 0.0001). nonetheless, the number of primordial follicles in the PCOS + Stem cells group (8.16) was significantly decreased related to PCOS (P ≤ 0.0001) and PCOS-Sham (P ≤ 0.001) Groups, and higher than the sham group (P≤0.05). (Fig. 8B).
The number of secondary follicles in the ovaries of PCOS (2.6) and PCOS-Sham groups (2.66) were significantly higher than those in the Control (1.53, vs PCOS and PCOS-Sham; P ≤ 0.05) and sham groups (0.96, vs PCOS and PCOS-Sham; P ≤ 0.001). While, the number of secondary follicles in the PCOS + Stem cells group (1.46) was significantly decreased related to PCOS (P ≤ 0.05) and PCOS-Sham (P ≤ 0.01) groups. (Fig. 8 C)
As shown in Fig. 8D, the number of antral follicles in ovaries of PCOS (2.6) and PCOS Sham groups (2.66) were significantly higher than those in the control (1.53, vs PCOS: P ≤ 0.01 and PCOS-Sham; P ≤ 0.05) and sham groups (0.96, vs PCOS: P ≤ 0.01 and PCOS-Sham; P ≤ 0.05). While, the number of antral follicles in the PCOS + Stem cells group (1.46) was significantly decreased related to PCOS group (P ≤ 0.01) group.
The number of Graafian follicles in the PCOS (0) and PCOS-Sham (0) groups decreased significantly compared to the control (0.53, P ≤ 0.0001) and Sham (0.5, P ≤ 0.0001) groups. In addition, the number of Graafian follicles significantly increased in the PCOS + Stem cells group compared to PCOS (P ≤ 0.001) and PCOS-Sham (P ≤ 0.001) groups (Fig. 8 E).
The number of atretic follicles in the PCOS (2.26) and PCOS-Sham (2.03) groups significantly increased compared to the control (0.2, P ≤ 0.001) and Sham (0.36, vs PCOS: P ≤ 0.001 and vs PCOS-Sham: P ≤ 0.01) groups. Additionally, the reduction in the number of the atretic follicles in the PCOS + Stem Cell group (0.67, P ≤ 0.01) was significant compared to the PCOS and PCOS-Sham groups (Fig. 8f).
The number of corpora lutea in the PCOS (4) and PCOS-Sham (3.9) groups significantly increased compared to the control (1.2, P ≤ 0.01) and Sham (1.13, P ≤ 0.01) groups. Furthermore, the decrease in the number of corpora lutea in the PCOS + Stem cells group (1.16, P ≤ 0.01) was significant compared to the Control and Sham groups (Fig. 8G).
Finally, the number of cysts in the PCOS (3.5) and PCOS-Sham (3.7) groups was significantly increased compared to the Control (0, P ≤ 0.0001) and Sham (0, P ≤ 0.0001) groups, and its reduction in the PCOS + Stem cells group (0, P ≤ 0.0001) was significant compared to the PCOS and PCOS-Sham groups (Fig. H).
Measurement the volume and area of collagen and blood vessels
After Masson's Trichrome staining, collagen volume, blood vessels volume and blood vessel area were calculated with Cavalieri’s principle (Fig 6). After quantification, the area of blood vessels in the PCOS+Stem cells group (11.26 vs PCOS-Sham: P ≤ 0.02; vs PCOS: P ≤ 0.001) was significantly reduced compared to the PCOS (32.74) and PCOS-Sham (24.85) groups (Fig. 12).
Moreover, the Area of blood vessels in the PCOS (Vs Control and Sham: P ≤ 0.0001) and PCOS-Sham (Vs Control: P ≤ 0.003; Vs Sham ≤ 0.004) groups significantly increased compared to the control (6.68) and Sham (6.90) groups (Fig. 12).
The blood vessel volume in the PCOS (0.0168, P ≤ 0.02) and PCOS-Sham (0.015, P ≤ 0.01) groups significantly increased compared to control (0.0011) and Sham groups (0.0010) (Figs. 11A, B). On the contrary, in the PCOS+Stem cells group (0.0015) it appeared to be significantly reduced compared to the PCOS (P ≤ 0.03) and PCOS-Sham (P ≤ 0.01) group.
The collagen volume in the PCOS (0.072, P ≤ 0.02) and PCOS-Sham (0.06895, P ≤ 0.01) groups significantly increased compared to Control (0.0011 vs PCOS: P = 0.0008; vs PCOS-Sham: P = 0.0011) and Sham groups (0.0010 vs PCOS: P = 0.0008; vs PCOS-Sham: P = 0.0011) (Figs. 11 B). While, in the PCOS+Stem cells group (0.0029) it appeared to be significantly reduced compared to the PCOS (P = 0.0008) and PCOS-Sham (P = 0.0012) group.
Body and Ovary weight
The Ovarian weight in PCOS (0.047) and PCOS-Sham (0.044) groups increased significantly compared to Control (0.035, vs PCOS: P ≤ 0.0001; vs PCOS Sham: P ≤ 0.001) and Sham (0.033, vs PCOS and PCOS Sham: P ≤ 0.0001) groups. Ovary weight reduction in PCOS+Stem Cells group (0.043) was not significant compared to PCOS and PCOS Sham groups.
Ovary weight Ratio in PCOS (1.20 vs Control and Sham: P ≤ 0.0001) and PCOS Sham (1.16 vs Control: P ≤ 0.006; vs Sham: P ≤ 0.002) groups has increased significantly compared to Control (1.07) and Sham (1.06) groups. Ovary weight Ratio reduction in PCOS+Stem Cells group (0.89, vs PCOS and PCOS Sham: P ≤ 0.0001) was significant compared to PCOS and PCOS Sham groups.