{"paper_id":"ef59499f-adb7-4dc0-82c9-b164f1a6e590","body_text":"Because of timely diagnosis and new treatments, we\nencounter an increase in the survival rate for cancer.\nIncreased knowledge about the various cytotoxic\ntreatment impacts on the ovary has resulted in a\nsurge in the number of patients seeking to preserve\ntheir fertility before starting treatments. If there is\ntime for ovarian stimulation, embryo, and oocyte\ncryopreservation are standard techniques for fertility\npreservation. At present, ovarian cryopreservation\nis the only fertility preservation option that can\nbe offered to women who have ovary stimulation\nlimitations (such as inadequate time for stimulation or\nthe impossibility of stimulating) and prepubertal girls.\nA transplantation of cryopreserved ovarian tissue after\ncancer treatment is a promising fertility restoration\nstrategy that has already led to more than 200 live\nbirths worldwide ( 1 - 3 ).\nAfter a decade of investigations, the Royan Human\nOvarian Tissue Bank (OTB, ACECR, Tehran, Iran)\nwas established in 2010 and started patient reception\n( 4 ). In 2015, Royan Institute obtained international\ncertificate, ISO 9001:2015, for this bank. From that time,\nconsultations for approximately 1000 patients between\n7- 47 years have been directed and ovarian tissues of\nmore than 100 patients who had our criteria, have been\ncryopreserved.\nIn recent years, the Royan OTB has been requested\nfor only three cases of ovarian tissue transplantation\nfollowing cancer survival, that we will report two of\nthem here. Before transplantation, a general consultation\nwas conducted about the ovary transplantation and its\noutcomes in Iran and other countries. Consultation\nwith an oncologist was conducted to declare the\ncomplete remission of the underlying disease and a\nconsultation with the surgical team was carried out\nto manage possible adhesions during surgery for both\npatients. The informed consents were obtained from\nboth patients ( 4 ).\n\nFirst case: She was a 22 years old girl with a history of colon\ncancer. She was affected by rectal bleeding when she was 17.\nThe diagnosis of hyperplastic adenomatous polyps with focal\nhigh-grade dysplasia was established by colonoscopy biopsy.\nThe pathological examination of the colon identified an\ninvasive adenocarcinoma that its serosal surface and margin\nwas tumor free, and also four reactive hyperplasia pericolic\nlymph nodes were detected. Her right ovary was resected\nand vitrified in 28 strips in the Royan OTB after one course\nof chemotherapy. Thereafter, she received five courses of\nchemotherapy and 40 sessions of radiotherapy. Hormonereplacement therapy (HRT), including 0.625 mg of conjugated\nestrogens (Aburaihan Pharma.Co., Tehran, Iran) plus 10 mg\nof medroxyprogesterone acetate (Aburaihan Pharma.Co.,\nTehran, Iran), was administered for the relief of menopausal\nsymptoms monthly for 5 years. At the 5th year, she asked for\nthe ovary transplantation. Serum concentrations of folliclestimulating hormone (FSH), luteinizing hormone (LH),\nand Estradiol (E2) levels were measured before ovary\ntransplantation ( Table 1 ).\nSecond case: She was a 37-year old married woman who\nexperienced a radical abdominal hysterectomy and bilateral\nsalpingoophorectomy and pelvic lymph node dissection\nbecause of stage IA well-differentiated adenocarcinoma. She\ndid not receive any chemotherapy or radiotherapy before\novariectomy. Both ovaries were resected, and 40 strips were\nvitrified and stored in the Royan OTB. Four years later,\nshe asked for ovary transplantation. Serum concentrations\nof FSH, LH, and E 2  levels were measured before ovary\ntransplantation ( Table 2 ).\nIn each case, after ovarian tissue resection, ovaries were\nquickly transferred to the Royan OTB (during approximately\n1 hour) with a transfer medium at 4°C with ice packs. This\nmedium consisted of Medium 199+Heppes (HTCM, Gibco,\nPaisley, UK) that was supplemented with 20% human\nserum albumin (HSA, Biotest, Germany). The transferred\novary was washed in the HTCM+20% HSA medium. The\nmedullary part was removed and the thinnest cortical part\nwas cut into 10×5×1 mm strips. Finally, the strips were\nvitrified in a two-step procedure as following:\nFirst, each strip was transferred to an equilibration medium\ncomposed of HTCM, 7.5% ethylene glycol (EG, Sigma,\nSt. Louis, MO, USA), 7.5% dimethyl sulfoxide (DMSO,\nSigma, St. Louis, MO, USA), and 20% HSA for 15 minutes,\nand then the strips were washed in the vitrification medium\n[HTCM, 15% DMSO, 15% EG, 0.25 M sucrose, and 20%\nHSA] for 10 minutes. After the removal of the extra medium,\nthe strips were directly transferred into liquid nitrogen. All\nsteps were performed at 4°C ( 4 ).\nOf note, one strip was randomly selected and fixed for\nhistological and pathological evaluation via hematoxylin\nand eosin staining before cryopreservation ( 5 ).\nThe hormonal profile of the first patient\nFSH; Follicle-stimulating hormone and LH; Luteinizing hormone.\nThe hormonal profile of the second patient\nFSH; Follicle-stimulating hormone and LH; Luteinizing hormone.\nBoth patients underwent the laparoscopic autologous\northotopic transplantation. The surgery and postoperative\nperiod were uneventful.\nBefore transplantation, the patient’s ovarian\ncryopreserved strips were warmed and incubated at 37°C.\nWarming procedure was performed in four steps in a\ndescending sucrose concentration (1, 0.5, 0.25, and 0.125\nM). The base medium was composed of HTCM and 20%\nHSA. To make sure tumor cells free, a histopathological\nexamination of the storage tissue were performed before\ntransplantation.\nFor the first case, 15 strips were warmed and prepared\nin two forms, including separate (7 strips) and ribbonshaped (containing 8 strips). For preparing the ribbonshaped, the strips were sutured under the surgical\nmicroscope by 8/0 “Coated VICRYL® (polyglactin 910)\nSuture - Ethicon” and a 1.5×2 cm ribbon was made. For\ntransplantation, first, the pelvic area was evaluated well;\nno adhesion was observed, and the uterus was completely\nhealthy and free from any pathology. Because the patient’s\nright ovary had been removed and her left ovary was\nvery small, a peritoneal pouch in the left broad ligament,\nunder the fallopian tube was created. The ribbon-shaped\nstrip was transplanted into the pouch, the medullary side\nfacing the pelvic floor. Likewise, a peritoneal pouch was\nformed in the right broad ligament below the uterosacral\nligament and the separate strips were transplanted inside\nit. Peritoneal pouch was sutured using a Vicryl suture. The\npatient was discharged 24 hours after surgery without any\ncomplications. The transplantation procedure for the first\npatient was conducted in the gynecology and obstetrics\ndepartment of the Rasool_e_Akram University General\nHospital, Tehran, Iran.\nFor the second patient, 17 strips were warmed. Nine\nseparate strips were transplanted in the right ovarian\nfossa, and 8 separate strips in the left ovarian fossa. Under\ngeneral anesthesia, her pelvic area was evaluated, and then\nan incision (1 cm) was made in the parietal peritoneum\nbetween the infundibular pelvic and uterosacral ligament\non both sides. The sub-peritoneal pocket was bluntly\ndissected, and the ovarian strips were placed separately\nwith their medullary side facing the pelvic floor. Finally,\nthe peritoneal closure was performed with an interrupted\nsuture (Monocryl suture 3-0, W3326, ETHICON surgical\ntechnology, USA). The transplantation procedure for\nthe second patient was conducted in the gynecology and\nobstetrics department of the Royan Institute, Tehran, Iran.\nBoth patients’ follow-up was carried out until 9\nmonths after transplantation. Menstrual monitoring,\nultrasonography, and measurement of the hormonal\nprofile were conducted after the transplantation for the\nfirst patient. Her hormonal profile was reported in Table 1.\nMonthly sonography examinations revealed no follicular\ndevelopment and she didn’t report menstruation during a\nyear\nFor the second patient, according to her hysterectomy\nand bilateral salpingoophorectomy, only hormonal\nprofiles could be followed up which was reported in Table\n2. Same as our first patient, no decrease in FSH levels\nand no increase in estradiol was observed, and as a result\nthe transplanted ovarian strips are considered to be nonfunctional.\n\nIf a transplantation is successful and its related\ntransplanted ovarian tissue is functional, we must observe\na decrease in the FSH level and an increase in the E2\nlevel. Hormonal fluctuations are current after an ovarian\ntissue transplantation, in such a way that the FSH level\nwill be decreased after 4-5 months of transplantation, and\nthen will be returned to its premenopausal level ( 6 ). In the\nfirst patient, although 2-6 months after transplantation,\nthe FSH hormone decreased to 50-60 mIU/mL, suddenly,\nan increasing trend was observed in the following\nmonths. Unfortunately, in the second patient, no decrease\nin the FSH level was observed after 9 months following\ntransplantation.\nAs mentioned earlier, 200 live births have been reported\nworldwide but unfortunately we have not. It might be\ndue to various reasons such as cryopreservation method,\npre-cryopreservation ovarian tissue quality, follicle loss\nprior to the freezing process, size of the tissue, tissue\nrevascularisation, and re-transplantation performance.\nAlthough, ovarian tissue integrity was well preserved\nby the vitrification method, only 2 live births have been\nreported after transplantation of vitrified tissue ( 7 ).\nMost live births were reported after a slow-freezing\nprocedure ( 8 - 10 ). It seems that multiple transplantations,\ndouble or triple, may be successful in some patients\n( 9 ). Although, we suggested a re-transplantation plan\nto both of our patients, they did not accept because of\ntheir personal desire. As there are no standard protocols\nfor ovarian tissue cryopreservation and transplantation\nworldwide, differences in acquired results are predictable\n( 11 ).\n\nIt is very encouraging that ovarian tissue cryopreservation\nand transplantation have resulted in a live birth. We\nacquired ovary cryopreservation knowledge in 2010 and\ntried transplantation 6 years later, but unfortunately did\nnot result in a live birth. Continued research efforts are\nrequired to optimize our approach, and we hope to report\nsuccessful transplantation soon.","source_license":"public-domain-us","license_restricted":false}