Objective:To investigate the reparative effects and underlying mechanisms of human umbilical cord mesenchymal stem cells (hUCMSCs) in conjunction with hyperbaric oxygen (HBO) on ovarian dysfunction in mice exposed to simulated high-altitude conditions.Methods:A total of 64 six-week-old female C57BL/6 mice were randomly allocated according to random number table, assigned to control group, model group, hUCMSCs group and hUCMSCs+HBO group, with 16 mice in each group. Mice in model group, hUCMSCs group and hUCMSCs+HBO group were placed in a hypobaric oxygen chamber to simulate a high-altitude environment at an elevation of 6 500 m for 15 d, thereby establishing a model of ovarian dysfunction. Beginning on the first day following model establishment, the control and model groups received intravenous injections of 0.2 mL saline via the tail vein. In contrast, the hUCMSCs group and the hUCMSCs+HBO group received 0.2 mL hUCMSCs (1×10 6 cells) per mouse through tail vein injection, administered once a week for three weeks, with continuous intervention lasting 15 d. Furthermore, the hUCMSCs+HBO group were subjected to daily hyperbaric oxygen treatment. The recorded variables included general health status, body weight, estrous cycle changes, serum hormone levels, bilateral ovarian wet weight, ovarian index, follicular development assessment, pathological alterations of ovarian tissue, ultrastructural changes, and the expression levels of transforming growth factor beta 1 (TGF-β1) and Smad family member 3 (Smad3) detected by Western blotting in ovarian tissue. Additionally, litter size and offspring body weight were measured across all groups to evaluate the reproductive capacity of the mice. Results:1) Compared with the hUCMSCs group, the hUCMSCs+HBO group exhibited no statistically significant differences in estrous cycle, body weight, bilateral ovarian wet weight, or ovarian index (all P>0.05). Furthermore, serum levels of anti-Müllerian hormone (AMH), estradiol, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), as well as counts of various stages of follicles in hUCMSCs+HBO group, did not demonstrate statistically significant differences compared with hUCMSCs group (all P>0.05). Notably, in the hUCMSCs+HBO group, the integrity of the nuclear membrane in granulosa cells of the ovarian tissue was preserved, with only mild swelling observed in individual mitochondria, while no expansion of the rough endoplasmic reticulum or swelling of the Golgi apparatus were noted. Additionally, expression levels of TGF-β1 and Smad3 proteins in the hUCMSCs+HBO group were significantly elevated compared with the hUCMSCs group ( P=0.010, P<0.001). The conditions of the offspring and litter size showed no statistically significant differences between the two groups (all P>0.05). 2)Compared with control group, the hUCMSCs+HBO group had slightly lower values for body weight, bilateral ovarian wet weight, ovarian index, serum levels of AMH, estradiol, and progesterone, while had slightly higher serum levels of FSH and LH without statistically significant differences (all P>0.05). Importantly, the ultrastructural characteristics of granulosa cells in the ovarian tissue of the hUCMSCs+HBO group closely resembled those of control group, displaying intact structures of the nuclear membrane, mitochondria, rough endoplasmic reticulum, and Golgi apparatus. There was no significant difference in TGF-β1 protein level between hUCMSCs+HBO group and control group ( P=0.253), but Smad3 protein level in hUCMSCs+HBO group was higher than that in control group ( P<0.001). No statistically significant differences were detected in offspring body weight, litter size, or behavioral tendencies (all P>0.05). Conclusion:Both hUCMSCs and the combination of hUCMSCs with HBO intervention demonstrated a safe and effective promotion of functional repair in damaged ovarian tissue under hypoxic conditions. Notably, the combination treatment of hUCMSCs with HBO exhibited a synergistic effect compared with hUCMSCs alone. The potential mechanism underlying this enhanced functional repair may involve the upregulation of the TGF-β1/Smad3 signaling pathway, which could ultimately improve fertility outcomes in mice subjected to hypoxic environments.

Objective:To investigate the reparative effects and underlying mechanisms of human umbilical cord mesenchymal stem cells (hUCMSCs) in conjunction with hyperbaric oxygen (HBO) on ovarian dysfunction in mice exposed to simulated high-altitude conditions.Methods:A total of 64 six-week-old female C57BL/6 mice were randomly allocated according to random number table, assigned to control group, model group, hUCMSCs group and hUCMSCs+HBO group, with 16 mice in each group. Mice in model group, hUCMSCs group and hUCMSCs+HBO group were placed in a hypobaric oxygen chamber to simulate a high-altitude environment at an elevation of 6 500 m for 15 d, thereby establishing a model of ovarian dysfunction. Beginning on the first day following model establishment, the control and model groups received intravenous injections of 0.2 mL saline via the tail vein. In contrast, the hUCMSCs group and the hUCMSCs+HBO group received 0.2 mL hUCMSCs (1×10 6 cells) per mouse through tail vein injection, administered once a week for three weeks, with continuous intervention lasting 15 d. Furthermore, the hUCMSCs+HBO group were subjected to daily hyperbaric oxygen treatment. The recorded variables included general health status, body weight, estrous cycle changes, serum hormone levels, bilateral ovarian wet weight, ovarian index, follicular development assessment, pathological alterations of ovarian tissue, ultrastructural changes, and the expression levels of transforming growth factor beta 1 (TGF-β1) and Smad family member 3 (Smad3) detected by Western blotting in ovarian tissue. Additionally, litter size and offspring body weight were measured across all groups to evaluate the reproductive capacity of the mice. Results:1) Compared with the hUCMSCs group, the hUCMSCs+HBO group exhibited no statistically significant differences in estrous cycle, body weight, bilateral ovarian wet weight, or ovarian index (all P>0.05). Furthermore, serum levels of anti-Müllerian hormone (AMH), estradiol, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), as well as counts of various stages of follicles in hUCMSCs+HBO group, did not demonstrate statistically significant differences compared with hUCMSCs group (all P>0.05). Notably, in the hUCMSCs+HBO group, the integrity of the nuclear membrane in granulosa cells of the ovarian tissue was preserved, with only mild swelling observed in individual mitochondria, while no expansion of the rough endoplasmic reticulum or swelling of the Golgi apparatus were noted. Additionally, expression levels of TGF-β1 and Smad3 proteins in the hUCMSCs+HBO group were significantly elevated compared with the hUCMSCs group ( P=0.010, P<0.001). The conditions of the offspring and litter size showed no statistically significant differences between the two groups (all P>0.05). 2)Compared with control group, the hUCMSCs+HBO group had slightly lower values for body weight, bilateral ovarian wet weight, ovarian index, serum levels of AMH, estradiol, and progesterone, while had slightly higher serum levels of FSH and LH without statistically significant differences (all P>0.05). Importantly, the ultrastructural characteristics of granulosa cells in the ovarian tissue of the hUCMSCs+HBO group closely resembled those of control group, displaying intact structures of the nuclear membrane, mitochondria, rough endoplasmic reticulum, and Golgi apparatus. There was no significant difference in TGF-β1 protein level between hUCMSCs+HBO group and control group ( P=0.253), but Smad3 protein level in hUCMSCs+HBO group was higher than that in control group ( P<0.001). No statistically significant differences were detected in offspring body weight, litter size, or behavioral tendencies (all P>0.05). Conclusion:Both hUCMSCs and the combination of hUCMSCs with HBO intervention demonstrated a safe and effective promotion of functional repair in damaged ovarian tissue under hypoxic conditions. Notably, the combination treatment of hUCMSCs with HBO exhibited a synergistic effect compared with hUCMSCs alone. The potential mechanism underlying this enhanced functional repair may involve the upregulation of the TGF-β1/Smad3 signaling pathway, which could ultimately improve fertility outcomes in mice subjected to hypoxic environments.