Fig 2: Effects of naïve and primed AMSC-sEVs, alone or in combination with hMG, on AMH and FSHR mRNA expression in CTX-treated human granulosa cells. hGCs were treated with cyclophosphamide (CTX, 2 μM) in medium containing 0.25% FBS, with or without naïve or primed AMSC-derived small extracellular vesicles (sEVs; 104 and 108 particles/mL) and/or human menopausal gonadotropin (hMG, 1 IU/mL). (A) Relative mRNA expression of AMH. (B) Relative mRNA expression of FSHR. Gene expression levels were quantified by quantitative real-time PCR and normalized to 18S rRNA. Relative expression was calculated using the 2−ΔΔCt method with the untreated control group set as 1. Data are presented as mean ± SD (n = 6). (* p < 0.05, *** p < 0.001 compared with the control group; ### p < 0.001 compared with the CTX-treated group). Different bar patterns are used to distinguish the different experimental treatment groups.

Fig 3: Effects of CTX and hMG on AMH and FSHR mRNA expression in human granulosa cells. hGCs were treated with different concentrations of cyclophosphamide (CTX; 0–10 μM) or human menopausal gonadotropin (hMG; 0–10 IU/mL), either alone or in combination. (A,B) Relative mRNA expression of AMH and FSHR in hGCs treated with increasing concentrations of CTX (0, 0.1, 1, 2, 5, and 10 μM). (C,D) Relative mRNA expression of AMH and FSHR in hGCs treated with increasing concentrations of hMG (0, 0.1, 1, and 10 IU/mL). (E,F) Relative mRNA expression of AMH and FSHR in hGCs co-treated with CTX (2 μM) and increasing concentrations of hMG (0–10 IU/mL). Gene expression levels were quantified by quantitative real-time PCR and normalized to 18S rRNA. Relative expression was calculated using the 2−ΔΔCt method with the untreated control group set as 1. Data are presented as mean ± SD (n = 6). (*** p < 0.001 compared with the control group; ### p < 0.001 compared with the CTX-treated group).

Fig 4: Effects of naïve and primed AMSC-sEVs, alone or in combination with hMG, on AMH and estradiol (E2) secretion in CTX-treated human granulosa cells. hGCs were treated with cyclophosphamide (CTX, 2 μM) in medium containing 0.25% FBS, with or without naïve or primed AMSC-derived small extracellular vesicles (sEVs; 104 and 108 particles/mL) and/or human menopausal gonadotropin (hMG, 1 IU/mL). (A) Concentration of AMH in culture supernatants. (B) Concentration of E2 in culture supernatants. Hormone levels were quantified using ELISA according to the manufacturer’s instructions. Data are presented as mean ± SD (n = 6). (*** p < 0.001 compared with the control group; ### p < 0.001 compared with the CTX-treated group; $ p < 0.05, $$$ p < 0.001 compared with the CTX + hMG group). Actual AMH and E2 concentrations are presented as mean ± SD in Supplementary Table S1. Different bar patterns are used to distinguish the different experimental treatment groups.

Fig 5: Effects of naïve and primed AMSC- sEVs on AMH and FSHR mRNA expression in human granulosa cells. Human granulosa cells (hGCs) were treated with naïve or primed AMSC-derived small extracellular vesicles (sEVs; 104 and 108 particles/mL). (A) Relative mRNA expression of AMH. (B) Relative mRNA expression of FSHR. Gene expression levels were quantified by quantitative real-time PCR and normalized to 18S rRNA. Relative expression was calculated using the 2−ΔΔCt method with the untreated control group set as 1. Data are presented as mean ± SD (n = 6). (** p < 0.01, *** p < 0.001 vs. control).