Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells On Ameliorating Androgen-Induced Polycystic Ovary Syndrome

Background and objective Polycystic ovary syndrome(PCOS)is a prevalent female endocrine disorder worldwide,affecting 6%-20% of women of reproductive age.It is characterized by irregular ovulation,hyperandrogenemia,ovarian polycystic changes,and often accompanied by metabolic disorders such as obesity,glucose metabolism disorders,and cardiovascular diseases.The etiology of PCOS is multifactorial,involving genetics,diet,and environmental factors.Currently,individualized treatment for PCOS mainly focuses on clinical manifestations.This includes lifestyle improvements,reduction of androgen level using oral contraceptives,management of insulin resistance and type 2 diabetes with metformin,induction of ovulation with letrozole,laparoscopic ovarian drilling to address hormonal level imbalances,and Chinese medicine treatment.While most patients can achieve improved fertility through these interventions,a cure for the disease remains elusive and long-term complications prevention remains challenging.This has posed significant difficulties for both PCOS patients and healthcare providers.Therefore,scientists and clinicians have been actively seeking effective and comprehensive treatment options for PCOS.Given the close association between PCOS,chronic inflammatory,and abnormal autophagy,stem cells have emerged as a promising biological material with antiinflammatory and autophagy regulation effects for treating PCOS.Among various stem cells of biological origin,mesenchymal stromal cell(MSC)stand out due to their abundant sources,fewer ethical restrictions,and remarkable self-renewal and differentiation capabilities.Numerous studies have demonstrated that MSC,as a biological material,play a significant therapeutic role in regulate female reproductive system diseases,restoring damaged ovarian function,and repairing compromised uterus.However,the regulation of MSC on PCOS remains underexplored,requiring further investigation and research.Our study aimed to investigate the therapeutic effect of human umbilical cord mesenchymal stromal cells(hUC-MSCs)in treating dehydroepiandrosterone(DHEA)-induced PCOS mice.Additionally,we examined the impact of hUC-MSC treatment on ovulation,sex-related hormone levels,and estrous cycle.The results demonstrated that hUC-MSC treatment restored ovulation function and normalized the estrous cycle in PCOS mice.Furthermore,there was an improvement in sex hormone levels following hUC-MSC treatment.To further explore the underlying mechanisms,we assessed the autophagy level in granule cells collected from both PCOS and non-PCOS populations using transmission electron microscopy(TEM).Our findings revealed significantly elevated autophagy levels in granule cells of PCOS population,characterized by a high presence of autophagosomes and autophagolysosomes within the cytoplasm when compared with the non-PCOS population.These findings indicate a potential association between the development of PCOS and elevated autophagy levels in the ovarian tissue of PCOS patients.Finally,through animal and cell experiments,we elucidated that hUC-MSC improved PCOS by activating the mTOR/ autophagy pathway.Our research studied the effect of hUC-MSC in improving androgen-induced PCOS and its potential mechanism,which provided a reliable experimental basis for the application of hUC-MSC in the clinical treatment for PCOS patients,and further provided theoretical basis for the treatment of PCOS by hUC-MSC.Part 1 Therapeutic effect of hUC-MSC in DHEA-induced PCOSMethods hUC-MSC were extracted using the scraping method and identified by flow cytometry(FCM).The PCOS mouse model was constructed through subcutaneous injection of dehydroepiandrosterone(DHEA).Ovarian morphological changes and oocyte count in PCOS mice were observed using hematoxylin-eosin staining(HE).Serum samples from PCOS mice were collected for detection of sex hormone levels using enzyme linked immunosorbent assay(ELISA).The estrous cycle of PCOS mice was assessed through vaginal smear analysis.The estrous cycle of PCOS mice was assessed through vaginal smear analysis.Results 1.The majority of separated hUC-MSC displayed a fibrous morphology under optical microscope.Cytotoxicity assays confirmed successful induction of hUC-MSC into bone,cartilage,and lipid formations.The positive rates of MSC surface markers CD44,CD43,CD90 and CD105 were all above 90%,while the positive rate for HLA-DR was below 5%.These findings indicate successful isolation of hUC-MSC.2.Constructed the DHEA-induced PCOS mouse model.The DHEA group mice showed polycystic changes,enlarged ovarian section size,and a significant number of cystic follicles in the cortex,with majority of follicles being atretic.No luteal structures were found compared to the control group.After hUC-MSC treatment,there was a reduction in the number of cystic follicles in the ovarian cortex,thickening of the granulosa cell layer,and the presence of corpus luteum structures.These results indicated that hUC-MSC could restore ovulation function and facilitate oocyte maturation in PCOS mice.3.The T and LH/FSH levels significantly increased in DHEA group.However,after hUC-MSC treatment,there was a noticeable decrease in T and LH/FSH levels,almost returning to the same level as observed in the control mice.These results indicated that hUC-MSC could restore the hormone level in PCOS mice.4.Mice in the DHEA group did not exhibit the typical cytological features of the diestrus period and lacked the characteristic features of vaginal shedding cells in the four stages of the estrous cycle.However,following hUC-MSC treatment,the DHEA+MSC group displayed typical leukocytes of the metaoestrus to diestrus stage,and there was a significant recovery in the cytological features of vaginal shedding cells across all four periods.These results indicated that hUC-MSC could improve the disturbed estrous cycle in PCOS mice.Conclusion hUC-MSC can ameliorate the disturbed estrous cycle in PCOS mice and reduce the expression level of T and the LH/FSH ratio.Additionally,hUC-MSC can restore the ovulation function and promote oocyte maturation in PCOS mice.Part 2 hUC-MSCs improve DHEA-induced PCOS by activating mTOR/Autophagy pathwayMethods Collect the serum of the PCOS population(This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University,and informed consent was obtained).We detected the expression levels of autophagyrelated proteins ATG5 and Parkin,using ELISA.Western blot(WB)and real-time quantitative polymerase chain reaction(RT-q PCR)were employed to assess the expression of autophagy associated proteins in ovarian tissues,including LC3 B,Beclin1,mTOR,SIRT1.TEM was utilized to detect the changes in the number of autophagosomes in human granule cells,mouse granule cells and uterine epithelial cells.The mouse ovulation induction test was conducted to collect MI and MII stage oocytes and assess the oocyte maturation rate.KGN cells were used to establish co-culture cell model.Results1.The expression of autophagy related proteins ATG5 and Parkin in PCOS population was significantly increased.Additonally,the number of autophagosomes in granule cells was markedly increased,and mitochondrial ridge breakages were observed compared to the non-PCOS population.These findings suggested that excessive autophagy in granule cells may be associated with the onset and progression of PCOS.2.LC3 B,Beclin1 and SIRT1 expression were significantly increased in the DHEA group and the DHEA+ autophagy agonist(RAPA)group,while mTOR expression was decreased in both group compared to control group.In contrast,Beclin1 and SIRT1 expression were decreased in DHEA+ autophagy inhibitor(CQ)group and the DHEA+MSC group,while mTOR expression was significantly increased compared to the DHEA group.These results indicated that hUC-MSC could modulate the mTOR/ autophagy pathway,by activating mTOR expression and inhibiting the expression of LC3 B and Beclin1.3.The number of autophagosomes in mouse granule cells and endometrial epithelial cells significantly decreased in both DHEA+CQ and the DHEA+MSC group compared to the DHEA group.However,in the DHEA+RAPA+MSC group,the number of autophagosomes increased when compared to the DHEA+MSC group.These results indicated that hUC-MSC could reduce the number of autophagosomes in granule cells and endometrial epithelial cells of PCOS mice,and excessive activation of autophagy may hinder the therapeutic effect of hUCMSC on PCOS mice.4.The total number of oocytes decreased in both the DHEA+CQ group and the DHEA+MSC group,while the ratio of the MII oocyte ratio significantly increased compared to DHEA group.In addition,the DHEA+RAPA group exhibited a significantly higher number of dead cells compared to the other five groups.These results indicated that hUC-MSC and autophagy inhibitor could improve the oocyte maturation,while excessive activation of autophagy may impair ovarian function in mice.5.Serum level of T and LH/FSH significantly increased in the mice of the DHEA group and the DHEA+RAPA group.Conversely,T and LH/FSH levels decreased in both DHEA+MSC group and the DHEA+CQ group,with the LH/FSH levels in the DHEA+MSC group being restored to the level of the control group.These results indicated that activation of autophagy could cause abnormal hormone levels in PCOS mice,while inhibiting autophagy could ameliorate this condition.6.The estrous cycles of mice in the DHEA group were disturbed,mainly staying in the estrous phase.However,the DHEA+CQ group and DHEA+MSC group showed typical leukocytes from metaoestrus to diestrus phase,and continuous smears indicated that some mice had restored normal estrous cycles.Conversely,the DHEA+RAPA group remained in the estrus and proestrus phase.These results indicated that activating autophagy may contribute to the disruption of estrous cycle in the PCOS model mice,while inhibiting autophagy could improve this situation.Conclusions 1.hUC-MSC can improve the autophagy level in granule cells,facilitate oocytes maturation,regulate hormone levels,and restore the estrous cycle in PCOS mice.2.hUC-MSC can ameliorate ovarian dysfunction in PCOS mice by activating mTOR/Autophagy pathway.