Aytac Yuksel1, Gamze Bildik2, Filiz Senbabaoglu2, Nazli Akin2, Macit Arvas1, Fehmi Unal3, Yagmur Kilic2, Isil Karanfil2, Baldan Eryılmaz2, Pelin Yilmaz2, Can Ozkanbaş2, Cagatay Taskiran4, Senai Aksoy5, Yılmaz Guzel5, Basak Balaban5, Umit Ince6, Akira Iwase7, Bulent Urman8, Ozgur Oktem9. 1. Department of OB/GYN, Cerrahpasa Medical School of Istanbul University, Istanbul, Turkey. 2. Koc University School of Medicine and the Graduate School of Health Sciences, Istanbul, Turkey. 3. Department of OB/GYN, Istanbul Teaching and Training Hospital, Istanbul, Turkey. 4. Department of Obstetrics and Gynecology, Koc University School of Medicine, Istanbul, Turkey. 5. American Hospital Women's Health Center, Comprehensive Cancer Care and Fertility Preservation Programs, Assisted Reproduction Unit, Istanbul, Turkey. 6. Department of Pathology, Acıbadem University School of Medicine, Istanbul, Turkey. 7. Department of Obstetrics and Gynecology, The School of Medicine, Nagoya University, Nagoya, Japan. 8. Department of Obstetrics and Gynecology, Koc University School of Medicine, Istanbul, Turkey American Hospital Women's Health Center, Comprehensive Cancer Care and Fertility Preservation Programs, Assisted Reproduction Unit, Istanbul, Turkey. 9. Department of Obstetrics and Gynecology, Koc University School of Medicine, Istanbul, Turkey American Hospital Women's Health Center, Comprehensive Cancer Care and Fertility Preservation Programs, Assisted Reproduction Unit, Istanbul, Turkey ooktem@ku.edu.tr.
Abstract
STUDY QUESTION: Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER: Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY: It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION: This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS: For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION: This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.
STUDY QUESTION: Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER: Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY: It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION: This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS: For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION: This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.
Authors: Motohiro Kano; Amanda E Sosulski; LiHua Zhang; Hatice D Saatcioglu; Dan Wang; Nicholas Nagykery; Mary E Sabatini; Guangping Gao; Patricia K Donahoe; David Pépin Journal: Proc Natl Acad Sci U S A Date: 2017-01-30 Impact factor: 11.205