Intravenous Infusion of Nucleated Peripheral Blood Cells Restores Fertility in Mice with Chemotherapy-Induced Premature Ovarian Failure

Abstract

Cancer treatment with specific chemotherapeutic agents has been well documented to have an adverse impact on female fertility leading to premature ovarian failure (POF). The objective of this study is to investigate if chemotherapeutic induced POF can be reversed by the infusion of autologous nucleated peripheral blood cells (PBMC). To reach our goal, mice were treated with a single intraperitoneal injections of busulfan and cyclophosphamide to induce POF. This was followed by transfusion of PBMC. The ovarian morphology and functional parameters were monitored by radioimmunoassay, real-time PCR, immunofluorescence and immunohistochemistry analysis. Our study showed that chemotherapy (CTX) protracted estrous cycle period and repressed E2 production. In addition, CTX decreased the expressions of steroidogenesis markers, CYP-17 synthesis, StAR (steroidogenic acute regulatory protein), and Connexin-43 protein expression in the ovarian follicles. We also observed reduced numbers and sizes of the primordial and primary follicles in CTX-treated mice compared to untreated controls (p < 0.05). When both CTX and untreated control groups were stimulated with gonadotrophin, the control group produced ten times more ova than the CTX group. Finally, the treatment of premature ovarian failure induced by CTX with autologous PBMC transfusion resulted in over-expression and a statistically significant increase in several stem cell markers and restoration of fertility. Infusion with PBMC in CTX further decreased the estrous cycle length by 2.5 times (p < 0.01). We found that transfusion of autologous PBMC to mice with chemotherapy induced POF was very effective at restoring fertility. These results are similar to other studies using bone marrow derived mesenchymal stem cells.

Keywords: PBMC (Peripheral blood mononucleated cells); cancer; chemotherapy; ovary; premature ovarian failure (POF).

Figure 1

Effect of chemotherapy treatment on estrus cycle and steroidogenesis. (A) Effect of CTX (Chemotherapy) on estrus cycle length; (B) comparison of plasma estradiol 17-β concentrations presented in pg/mL between CTX-treated mice group and control group at Proestrus stage (* p < 0.05); (C) plasma LH (Luteinizing hormones) levels presented in pg/mL and compared between CTX-treated mice group and control group at Proestrus stage (* p < 0.05).

Figure 2

Impact of chemotherapy on apoptosis in murine granulosa cells. (A) Tunel staining (green) on ovary cut section, positive staining showed by yellow arrows. Data supported by high magnification of the circle area; (B) graph bar of tunel positive granulosa cells number compared to the matched control (* p < 0.05).

Figure 3

Steroidogenesis markers expressions suppressed by chemotherapy. Cross-section of the ovaries in both the CTX-treated group on the left side and control group on the right side analyzed by immunohistochemistry staining for specific targets involved in the ovarian function supported by fold change (on the right side) for statistical analysis: (A) StAR (steroidogenic acute regulatory protein), (B) CYP-17, (C) aromatase and (D) Connexin-43. For the four markers * p < 0.05. Some slides contain yellow arrows indicating the positive staining in granulosa cells. A Total of 10 sections was analyzed for each markers.

Figure 4

Chemotherapy effects on ovarian stimulation and follicular development. Microscopic illustration of mouse oocytes from control (A) and CTX (B) exposed mice; (C) graph bar summarize the number of ovulated ova after gonadotropins injection in both the CTX-treated group of mice and the control group (* p < 0.05).

Figure 5

Effect of chemotherapy on antral follicle and corpora lutea numbers. (A) Graph bar of antral follicle number in CTX treated group versus untreated control group (* p < 0.05, ** p < 0.005); (B) numbers of corpora lutea formed following ovulated ova in the CTX treated group versus control group (* p < 0.05).

Figure 6

Impact on nucleated peripheral blood cells on estrus cycle and fertility. (A) Estrous cycle length presented in days comparing CTX to CTX treated with PBMC versus matched negative control (* p < 0.05); (B) length of estrus cycle stages presented in days of the three involved mice groups: Control, CTX and CTX treated with PBMC (Peripheral blood mononucleated cells) (* p < 0.05); (C) effect of PBMC on fertility presented in litter size compared between CTX to CTX treated with PBMC versus matched negative control (* p < 0.05).