2014 http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2014; 30(2): 135–140 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2013.860127 PUBERTY AND OVARIAN PROTECTION Protection from cyclophosphamide-induced ovarian damage with bone marrow-derived mesenchymal stem cells during puberty Sevtap Kilic 1 , Ferda Pinarli 1 , Candan Ozogul 2 , Nicel Tasdemir 3 , Gulce Naz Sarac 2 , and Tuncay Delibasi 1 1 Yildirim Beyazit Educational and Research Hospital, Laboratory Animal and Stem Cell Research Center, Ankara, Turkey, 2 Department of Histology and Embryology, School of Medicine, Gazi University, Ankara, Turkey, and 3 Department of Obstetric and Gynecology, Namik Kemal University School of Medicine, Degirmenalti, Tekirdag, Turkey Abstract Objective: In female cancer survivors, the accelerated loss of primordial follicles may lead to premature ovarian failure. We investigated the protective effects of bone marrow derived mesenchymal stem cells (BMMSC) and gonadotropin releasing hormone analogue (GnRHa) against chemotherapeutic-induced ovarian toxicity in a rat model. Material and methods: Forty-eight Wistar albino female rats were divided into four groups. Group 1 was composed of rats that were given 200 mg/kg cyclophosphamide injection for each cycle (two cycles for each rat). Both cyclophosphamide and 0.4 mg GnRHa were administered to Group 2. Cyclophosphamide and 4 million/kg BMMSC were administered to Group 3. Cyclophosphamide, GnRHa, and BMMSC were administered to Group 4. Germ cell apoptosis, DNA fragmentation and primordial follicular count were investigated with Cleave Caspase-9 and TUNEL analysis. The presence of the SRY gene on the Y chromosome in the ovary of the recipient female rats was checked with PCR. Results: Immunohistochemical staining (IHS) of Caspase-9 and TUNEL was higher in Group 1 than in Group 3 (p50.05). Similarly, Group 4 had higher values than Group 3 (p50.05). The presence of the SRY gene was detected in Groups 3 and 4 with the PCR analysis. The mean primordal follicle count was lowest in Group 1 and the mean primordial follicle counts were higher in Groups 2 and 3 than in Group 1. The difference between Group 1 and Group 4 was not significant. Conclusion: BMMSC therapy was found to be protective from germ cell apoptosis and DNA damage when it was used with chemotherapy regimens including alkylating agents. Keywords Apoptosis, chemotherapy, childhood cancer, mesenchymal stem cell, puberty History Received 19 September 2013 Revised 10 October 2013 Accepted 24 October 2013 Published online 6 December 2013 Introduction Cancer is one of the biggest public health problems all over the world. Cytotoxic drugs are used to treat cancer in children and adolescents. Cytotoxic agents are successful in prolonging the lives of patients with various malignant and nonmalignant diseases [1,2]. Approximately 78% of all patients with malignant diseases diagnosed younger than 15 years of age will survive for five years [3]. The majority of this population is expected to survive for many years after diagnosis. Infertility remains one of the most common and life-altering complications experienced by adults treated for cancer during childhood. The risk of infertility is generally related to the grade of disease, dose of application and combination of cytotoxic therapy. Moreover, age, sex and genetic factors influence the risk of permanent infertility [4]. Loss of ovarian function after chemotherapy leads to loss of hormone produc- tion due to decreased follicle count. As is known, the risk of early menopause is increased with the decreased ovarian function and follicular count [5–8]. The preservation of gonadal function or maintaining fertility is much more important in the cancer treatment in puberty. The main concern is side effects of the cytotoxic treatments on the primary follicles. Ways to protect gonads from the side effects of cytotoxic therapy are not yet clear. Recently, several clinical and experimental studies have supported stem cell therapy as a potential and alternative therapeutic modality, offering the possibility of repairing the damaged tissue and restoring its normal function. Research on regenerative medicine suggests that stem cells could be used to treat various human diseases, due to their self-renewal capacity and multiplex differentiation potential. Bone marrow-derived mesenchymal stem cells (BMMSCs) are promising for regenera- tive medicine applications such as renoprotection, cardioprotec- tion, and repair in acute kidney and myocardial injuries [9,10]. However, there is a lack of research about the effectiveness of fertility preservation of stem cells [11]. Chemotherapy treatments create acute tissue damage at the beginning of therapy. The potential benefit of stem cell therapy after acute tissue damage seems to be related to angiogenesis and decreased apoptosis. From this point of view, we can hypothesize that selected stem cells could be used to treat ovarian injury [12]. In this study, we designed an in vivo BMMSCs transplantation model, in order to evaluate the protective efficacy of BMMSCs and GnRh analogs by measuring apoptosis and DNA damage in ovarian tissue. Address for correspondence: Sevtap Kilic Assoc. Prof, MD, Yildirim Beyazit Educational and Research Hospital, Laboratory Animal and Stem Cell Research Center, Su ¨leymanbey Sok. 29/10 06570 Maltepe, Ankara, Turkey. Tel: þ90 312 230 65 85. Fax: þ90 312 430 07 18. E-mail: sevtapkilic@gmail.com Gynecol Endocrinol Downloaded from informahealthcare.com by Hacettepe Univ. on 02/03/14 For personal use only.