Effects of Single Transplantation and Multiple Transplantation of Human Umbilical Cord Mesenchymal Stem Cells on the Recovery of Ovarian Function in the Treatment of Premature Ovarian Failure in Mice

Background: Previous studies have reported that transplantation of mesenchymal stem cells (MSCs) from many human tissues can improve ovarian dysfunction. However, the therapeutic effects of single injection of MSCs and multiple injections of MSCs on premature ovarian failure (POF) have not been reported yet. In this study, we used long-term follow-up to study the effect of human umbilical cord mesenchymal stem cell (hUC-MSCs) on the functional recovery of mouse POF models. Methods: In this study, we used a mouse model of premature ovarian failure induced by the combination of 120mg/kg cyclophosphamide and 30mg/kg busulfan. Enzyme-linked immunosorbent assay (ELISA) was used to detect estradiol (E2) and follicle stimulating hormone (FSH) levels in mouse serum. Evaluate ovarian function by counting follicles, ovarian weight, number of proliferating cells, anti-Mullerian hormone (AMH) and oocytes. Results: Our study shows that hUC-MSCs have obvious therapeutic effect for the POF mice model, and treatment effect of multiple transplantation is better than single transplantation. Mesenchymal stem cells were detected in follicular granulosa cells with tracer of the ovarian tissue freezing slice, which show hUC-MSCs to selectively migration and stay in damaged tissues. Genome Array screened a number of differentially expressed genes, the hUC-MSCs can change the level of expression of certain genes in the ovarian tissue, thus affecting ovarian function, and laid the foundation for us to further explore the mechanism of hUC-MSCs treatment of premature ovarian failure. Conclusion: This study demonstrated that hUC-MSCs transplantation signi�cantly restored ovarian function after chemotherapy-induced damage.


Abstract
Background: Previous studies have reported that transplantation of mesenchymal stem cells (MSCs) from many human tissues can improve ovarian dysfunction.However, the therapeutic effects of single injection of MSCs and multiple injections of MSCs on premature ovarian failure (POF) have not been reported yet.In this study, we used long-term follow-up to study the effect of human umbilical cord mesenchymal stem cell (hUC-MSCs) on the functional recovery of mouse POF models.
Methods: In this study, we used a mouse model of premature ovarian failure induced by the combination of 120mg/kg cyclophosphamide and 30mg/kg busulfan.Enzyme-linked immunosorbent assay (ELISA) was used to detect estradiol (E2) and follicle stimulating hormone (FSH) levels in mouse serum.Evaluate ovarian function by counting follicles, ovarian weight, number of proliferating cells, anti-Mullerian hormone (AMH) and oocytes.
Results: Our study shows that hUC-MSCs have obvious therapeutic effect for the POF mice model, and treatment effect of multiple transplantation is better than single transplantation.Mesenchymal stem cells were detected in follicular granulosa cells with tracer of the ovarian tissue freezing slice, which show hUC-MSCs to selectively migration and stay in damaged tissues.Genome Array screened a number of differentially expressed genes, the hUC-MSCs can change the level of expression of certain genes in the ovarian tissue, thus affecting ovarian function, and laid the foundation for us to further explore the mechanism of hUC-MSCs treatment of premature ovarian failure.

Conclusion:
This study demonstrated that hUC-MSCs transplantation signi cantly restored ovarian function after chemotherapy-induced damage.

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Figures
Figure 1 The    (c) In the multiple group, the levels of E2 were signi cant differences at 28, 60 days post-induction.(d) In the multiple group, the levels of FSH were signi cant differences at 28, 60 days post-induction, *P<0.05.
Figure 5 The ovarian reserve capacity analysis.Immunohistochemistry for AMH in once intravenous group (a) and the multiple intravenous group (b).Strong expression of AMH were seen in Sham group at each time point, granulosa cells were negative in POF group, AMH expression reappeared in ovaries from MSCs group.Original magni cation: 100×.
Figure 6 The comparison of ovarian proliferation potential in each group.Immunohistochemistry for KI67 in once intravenous group (a) and the multiple intravenous group (b).Sham mice follicle highly expressed KI67, POF mice follicle lacked KI67 signal, the expression were observed in MSCs group.Original magni cation: 400×.

Figure 7
Figure 7 (d) Expression of INHIBINα in mouse ovary in the hUC-MSCs group injected multiple times.(e) Expression of INHIBINβ in mouse ovary in single injection of hUC-MSCs group.(f) Expression of INHIBINβ in mouse ovary in the hUC-MSCs group injected multiple times.*P<0.05;**P<0.01;***P<0.001.