טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTurgeman Orli
SubjectMinimizing Chemotherapy Associated Gonadotoxic Effect by
S1P Administration
DepartmentDepartment of Medicine
Supervisor Professor Zeev Blumenfeld
Full Thesis textFull thesis text - English Version


Abstract

Background: The increase in malignancy of young women in the recent decades, combined with a significant improvement in long term survival of young patients after gonadotoxic chemotherapy, have brought about a ubiquitous interest in preservation of fertility in these young patients. None of the currently used methods: IVF and cryopreservation of embrya, ova or ovarian tissue or administration of gonadotropin releasing hormone (GnRH) analog co-treatment is ideal and none guarantees future fertility.

Objective: Since Sphingosine-1-Phosphate (S1P) may minimize gonadotoxicity, we have examined its possible anti-gonadotoxic effect on human luteinized granulosa cells (GCs).

Methods: Human GC's were donated by women undergoing follicular aspiration for assisted reproductive technology, after informed consent and institutional approval of ethics committee (IRB, Helsinki). The GCs were separated from RBC's by centrifugation on ficoll and plated on 96 multiwell plates at a density of 20,000 - 25,000 cells/well for lactate dehydrogenase (LDH) assays, or 100,000 cells/well for XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide ) assays. Cells were also plated on 6 well plates at a concentration of 250,000 cells/well for flow cytometry. Each experiment, done between 2-7 days after plating, was conducted at least in triplicates and repeated at least three times.
Sprague-Dawley rats, 3 months old were weekly injected intraperitonally (IP) with Doxorubicin 10, 7.5 or 5 mg/kg  or Cyclophosphamide 50mg/kg and their follicular reservoir was evaluated.

Results: Doxorubicin, between concentration of 1 ?M and 2 μM was toxic to granulosa cells as evaluated by the XTT method and/or LDH concentration in the medium.

 Co-incubation with S1P at 1-5 μM for 3 days significantly decreased the gonadotoxic effect of Doxorubicin.
Cyclophosphamide, at 1 and 2 mg/mL, but not at 0.5 mg/mL, was toxic to GC's and S1P at concentration of 1 and 5 μM could inhibit the gonadotoxic effect.

Phospharamide mustard, active metabolite of cyclophosphamide, did not cause significantly more cell death than the cyclophosphamide in its inactive state.

Doxorubicin at the concentration of 10mg/kg was lethal to the rats, and depleted the ovaries from oocytes. Doxorubicin at concentration of 7.5mg/kg was almost as lethal as 10mg/kg for the rats, which were sacrificed after 3 weeks due to health concerns. Doxorubicin 5mg/kg allowed rats to survive up to 5 weeks. 
Rats exposed to weekly injection of Cyclophosphamide, 50mg/kg, survived up to 5 weeks, with no significant health problems.

Conclusion: Future development of a device that may deliver S1P directly to the gonads may possibly prevent chemotherapy induced gonadotoxicity and enable fertility preservation avoiding the risk of malignant cell reimplantation.