{"paper_id":"74363cb0-a09a-460d-94a8-6d8e8a0540cb","body_text":"There is a concerning increase in cancer diagnoses\naccording to the Iranian Cancer Society. Today, due\nto medical advances, many cancers are treatable with\ntimely diagnosis and follow up. The patient can re-\nturn to a normal life after radiotherapy, chemothera-\npy, or surgical tumor excision. Therefore, many can-\ncers are no longer considered incurable. Although, in\nmany cases, chemotherapy and radiotherapy aim to\nsave lives, premature ovarian failure and reduction of\nfollicular reserve is undeniable. By taking into con-\nsideration the probable infertility of cancer patients,\npreservation of their reproductive ability prior to\nonset of cancer treatment is crucial ( 1 ,  2 ). Different\nmethods of assisted reproductive techniques that in-\nclude oocyte, embryo and ovarian tissue cryopreser-\nvation have helped these patients. The use of these\ntechniques in single or married, as well as young\nand older women differ. Hence, the most appropriate\ntechnique is selected according to the patients’ cir-\ncumstances ( 1 - 7 ). In cases where adequate time exists\nfor ovarian stimulation, embryo cryopreservation is\nconsidered the gold standard and an acceptable clini-\ncal technique. However, if embryo cryopreservation\nis not an option due to the absence of a sexual partner,\nunwillingness to use donor sperm, or for any other\nreason, the oocytes can be frozen ( 6 ). Ovarian tissue\ncryopreservation is another technique that has a long\nhistory of use, but with a new purpose. Limitations\nof oocyte cryopreservation exist, such as the impos-\nsibility of stimulating ovaries in patients with hyper-\nstimulation syndrome. Under these circumstances,\novarian tissue cryopreservation is more accepted and\napproved ( 2 ,  5 ). In this technique numerous follicles\nat different stages of maturity are preserved without\ndelays to cancer treatment. In addition, for single or\nyoung girls this is the best choice to preserve their\nreproductive ability ( 3 ,  4 ).\nThe Royan Human Ovarian Tissue Bank was es-\ntablished in 2010 with the intent to provide fertility\npreservation services to cancer patients eligible for\npreservation of reproductive ability. We have estab-\nlished the maximum age for inclusion in the Tissue\nBank as 35 years. Cases of malignancy where tu-\nmors have metastasized to the ovarian tissue are not\naccepted for cryopreservation. In other cases there is no exclusion for acceptance. Patients undergo an ini-\ntial consultation that determines individual factors of\nage, marital status, physical and mental conditions,\ncancer type, its progression stage and grade, level of\nprevious treatments, earlier infertility treatment and\nprognosis after treatment. After the initial consulta-\ntion, the best fertility preservation technique is se-\nlected. A contract is signed between the Ovarian Tis-\nsue Bank and the patient after the consultation. This\ncontract includes patients’ rights and sample mainte-\nnance insurance, as well as informing patients about\nthe use of her own sample after treatment, which is\napproved by the Royan Ethical Committee.\nThe procedure for ovarian tissue cryopreserva-\ntion at the Royan Human Ovarian Tissue Bank is as\nfollows. An ovarian tissue sample is removed from\nthe patient by laparoscopy, laparotomy, unilateral\nor bilateral oophorectomy according to the patient’s\ncondition. The sample is transferred to the Ovarian\nBank in the shortest possible time (approximately\n1 hour) in Medium 199+Heppes (HTCM, Gibco,\nPaisley, UK)+20% human serum albumin (HAS,\nBiotest, Germany) as transfer medium at 4°C and\non ice. In the laboratory, initially, the transferred\ntissue is washed in HTCM+20% HSA medium,\nafter which the medullary part is removed. Next,\nthe cortical part is thinned and 10×5×1 mm strips\nare obtained from the thin cortex. These steps are\nall performed on a cool pad. Finally, the stripes are\nvitrified in a two-step process, equilibration and\nvitrification. In the first step (equilibration), each\nstrip is washed in equilibrium medium composed\nof HTCM, ethylene glycol (EG, Sigma, St. Louis,\nMO, USA), Dimethyl sulphoxide (DMSO, Sigma,\nUSA, each 7.5%) and 20% HSA for 15 minutes at\n4°C. In the second step (vitrification), each strip\nis washed in 15% HTCM, 15% DSMO and 15%\nEG, 0.25 M sucrose, and 20% HSA for 10 minutes\nat 4°C. The extra medium is completely removed\nfrom the strips, after which they are directly trans-\nferred into liquid nitrogen. Of note, we randomly\nfix one strip before cryopreservation for histologi-\ncal evaluation (H&E staining and Semi thin). For\ntissue evaluation, one vitrified strip is warmed and\nassessed histologically. Warming is performed in\n4 steps in descending concentrations (1, 0.5, 0.25,\nand 0.125) of sucrose. The base medium is com-\nprised of HTCM+20% HSA. The histological as-\nsessment markers considered for tissue evaluation\ninclude total integrity, follicular population, oo-\ncyte degeneration, vacuolization and granulation\nof the nucleus, oolemma and ooplasm conditions,\nzona pellucida situation (in secondary or preantral\nfollicle), coherence and connectivity of granulosa\ncells ( Fig .1 ).\nPrimordial (short arrows), primary (long arrows) and preantral (black arrow) follicles in A., B., C. Control, D., E. and F. Vitrified hu-\nman ovarian tissues. Hematoxylin and eosin (H&E) staining (magnification: ×20, ×50 μm).\nFunctionality of the entire ovarian tissue is con-\nsidered by the presence or absence of the corpus\nluteum or corpus albicans in tissue. Finally, the in-\nformation is kept and filed in a histology descrip-\ntion form.","source_license":"CC-BY-4.0","license_restricted":false}