Royan Institute

Royan Human Ovarian Tissue Bank Background and Project According to published statistics by Iranian Cancer Society, cancer has had arising progress in recent years which brings many concerns. Today, by the advances in medical sciences, many cancers are considered treatable providing on time diagnosis and follow up. In this way, patients can return to their normal life after a period of radiotherapy or chemotherapy and tumor removal surgeries. But, many cancers are not considered incurable. Although in many cases the only and the best strategy for patient’s lifesaving is chemotherapy, the premature ovarian failure and reduction of follicular reserve is undeniable. Considering the probable infertility of the cancerous patients, preservation of their reproductive ability before mentioned treatments seems to be crucial. In this regard, different methods of Assisted Reproductive Techniques such as: oocyte, embryo and ovarian tissue cryopreservation have helped these patients. Applying these techniques in single, married, young and grown women are different and the most appropriate one is selected, due to all circumstances. In case for ovaries stimulation, embryo cryopreservation would be gold standard and acceptable technique in all clinics. However, if embryo cryopreservation could not be chosen due to any reason such as absence of sexual partner or unwilling to use donor sperm, oocytes can be frozen instead. Another technique is known as ovarian tissue cryopreservation with a long history of usage but with new purpose. Regarding to the limitations of oocyte cryopreservation such as impossibility of stimulating ovaries in patients with hyper stimulation syndrome, usage of ovarian tissue cryopreservation is more accepted and approved. In this technique, numerous follicles at different stages of maturity are preserved without any delay in cancer treatment. In addition, for single or young girls this is the best choice to keep their reproductive ability. About the Service In order to provide fertility preservation services to cancerous patients and those are candidates for reproductive ability preservation, the researchers in Royan Institute have begun a wide range of research on ovarian tissue cryopreservation from December 2000 till 2010. Eventually, after a decade, Royan Human Ovarian Tissue Bank was established in 2010 as one of the Embryology department subgroups. Since then, about 180 patients (ages between 7- 47) have been consulted and conclusively the ovarian samples of 54 patients (ages between 7- 35) have been reserved. Among cryopreserved ovaries, the most malignant cases are cervix adenocarcinoma (9 patients), breast cancer, Ewing’s Sarcoma and opposite side ovarian tumor (each case 7 patients), endometrial adenocarcinoma (4 patients), colon malignant tumor (3 patients) and Hodgkin’s lymphoma, major Thalassemia and acute lymphoblastic leukemia (each case 1-2 patients).It should be noted that two patients (one with colon cancer and the other with opposite side ovarian tumor) have requested for ovarian tissue transplantation after completion of their treatments. Acceptance Criteria The maximum age for admission in Royan Ovarian Tissue Bank is considered 35. If malignancy with tumors has metastasized the ovarian tissue, the sample is not accepted for cryopreservation. In other cases, there is no accepted exclusion. After the initial consultation, regarding to the individual factors including age, marital status, physical and mental condition, cancer type, its progression stage and grade, previous treatments and their level, earlier infertility treatment and prognosis after treatment, the best fertility preservation technique has been selected. Ethical Codes After consultation, a contract is signed between ovarian tissue bank and the patient in terms of “patients’ rights and the sample maintenance insurance”, as well as informing patients about the usage of her own sample after treatment” which is approved by Royan Ethical Committee. Procedure of human Ovarian Tissue Cryopreservation The ovarian tissue sample is taken from the mentioned patients by laparoscopy, laparotomy or unilateral or bilateral oophorectomy (depending on patient conditions). Sample is transferred to ovarian bank in the shortest possible time (approximately 1 hour) in HTCM (Gibco, Paisley, UK)+ 20% human serum albumin (HSA: Baxter) as transfer medium at 4^°c in ice. In laboratory, at first the transferred tissue is washed in HTCM + 20%HSA medium, then the medullary part is removed and cortical part is thinned and 10×5×1 mm strips are provided from thin cortex. All these steps are performed on cool pad. Finally, the stripes vitrified in two steps: first (equilibration): each strip is washed in equilibrium medium composed of HTCM, ethylene glycol (EG: Sigma, St Louis, MO, USA) and DMSO (Sigma, each 7.5%) and 20% HSA for 15 minutes at 4°C, Second (vitrification): each strip is washed in vitrification medium composed  of HTCM, DSMO and EG (each 15%), 0.25 M sucrose, 20% HSA for 10 minutes and at 4°C. After completing the removal of the extra medium from stripes, they are directly transferred into liquid nitrogen. It should be noted that one strip is randomly fixed before cryopreservation for histological evaluation (H&E staining and Semi thin). For tissue evaluation, one vitrified strip is warmed and assessed histologically. Warming is performed in 4 steps in descending concentrations (1, 0.5, 0.25, and 0.125) of sucrose. Base medium is HTCM + 20%HSA.The histological assessment markers considered for tissue condition evaluation are: total integrity, follicular population, Oocyte degeneration, vacuolization and granulation of nucleus, oolema and ooplasm condition, zona pelllucida situation (in secondary or pre antral follicle) and coherence and connectivity of granulosa cells. Moreover, the whole ovarian functionality is considered by presence or absence of corpus luteum or corpus albicant in tissue and finally the information is kept and filed in histology description form.