Cryopreservation and transplantation of ovarian tissue: results from one center in the USA

Over a period from 1997 to 2017 (20 years), 108 females between age 6 and 35 years were referred for possible ovary tissue freezing for fertility preservation. Ninety-two (85%) of these women underwent unilateral oophorectomy and cryopreservation either by slow freeze or vitrification. Sixty-six were for cancer, 5 for threatened premature ovarian failure (ovary tissue of discordant identical twin that was cryopreserved for her sister), 9 for social reasons, and 12 for a variety of conditions including Turner’s syndrome, multiple sclerosis, endometriosis, aplastic anemia, a daughter born with no ovary, or massive bilateral ovarian teratoma. Hodgkin’s disease accounted for 20 of the cancer cases (30%), breast cancer 13 (20%), leukemia 7 (11%), and non-Hodgkin’s lymphoma 5 (8%). The rest of the cancer cases (21) were a wide variety of less common cancers such as Ewing’s sarcoma, embryonal sarcoma of liver, colon cancer, calf sarcoma, spinal cord tumor, dysgerminoma, medulloblastoma, rhabdomyosarcoma, stomach cancer, carcinoid tumor, and brain cancer. Social reasons included not being ready to have children and wishing to have more children perhaps at a later date. Those who chose ovary freezing for social reasons did so before oocyte freezing was widely accepted, or because they were just too occupied in their hectic life to find time for three cycles of ovarian stimulation and oocyte retrieval, and did not want to go through hormonal stimulation. Nineteen of the cancer patients underwent slow freeze prior to September 2007, and 47 subsequent cases underwent vitrification of their ovarian tissue. Six of the 66 (10%) cancer patients have died, and 54 either underwent transplantation, or are prepared eventually to undergo transplantation. All patients were counseled in detail with the advice that the transplant might not ever be performed, or might not function. All underwent IRB consent.

Thirteen of the 92 ovary freeze cases (14%) have come back to have their ovary tissue thawed and transplanted back. Of those 13, the four most recent cases had been cryopreserved by vitrification, and the other 9 had been frozen by slow freeze. Ten were cancer survivors, and three were POF patients who had frozen ovary tissue from an identical twin sister. The 9 slow freeze cases had their ovary tissue frozen before 2007, and so naturally they composed the majority (9) of the cases of cryopreserved tissue thaw and that were transplanted. The other four cases of frozen ovary transplant had their tissue cryopreserved after 2007, and so these were vitrification cases. In addition to these cryopreserved ovary tissue transplant cases reported here, there have been 11 fresh transplants either between identical twins or allografts, that have already been reported, for a total of a large series of 24 ovary transplants that have been performed at one center with one technique [1]. All patients were menopausal for 3 to 20 years prior to the transplant. Three of the nine women undergoing transplant of their frozen tissue had leukemia, but their tissue was cryopreserved when they were in remission prior to their bone marrow transplant [38]. Assessment of multiple fragments by histology and immunochemistry by oncology and pathology departments revealed no tumor cells.

The technique for slow freeze and thaw has not changed since the original description by Gosden et al. in 1994 [18, 26]. Slow freezing is the earliest approach to ovarian cryopreservation. The cortex is removed from the medulla, divided into multiple strips of 1 cm by 2 cm by 0.1 cm and transferred to cryovials after incubation in 1.5 mol/L 1,2-propanedial ethylene glycol or DMSO, and 0.1 mol/L sucrose with 10% SSS at 37 °C for 30 min; then, 0.2 mol/L sucrose for 5 min, and after that cooled by computerized lowering of temperature [39]. Cooling was first at 2 °C/min to − 9 °C and then seeded. Thereafter, cooling rate was − 0.3 °C/min down to − 40 °C. Then, − 10 °C/min to − 140 °C and then plunged in liquid nitrogen. Thawing is performed rapidly (100 °C/min) in a warm bath and tissue is trimmed under an operating microscope before transplantation [6, 18, 26, 39]. This slow freeze method has worked quite well, and most of the many pregnancies and live births achieved so far have been with slow freeze. However, since 2007, we have used vitrification, because in vitro viability analysis studies shows no oocyte loss with vitrification, and a 50% oocyte loss with slow freeze [9, 10, 40]. Vitrification may show no difference in percent of morphological normal primordial follicles, but does result in less DNA damage and should cause and therefore probably less decrease in viability [41].

Nonetheless, slow freeze has had equally robust for ovarian tissue cryopreservation. One possible speculation is that if you lose half of 100,000 or 200,000 eggs, it might not affect the clinical result, because of a conceivably lower rate of primordial follicle recruitment. Whatever the reason, we cannot claim better clinical results with vitrification.

The method of both cooling and warming for vitrification are as follows [9, 10, 40]. For vitrification, the cortex tissue of each ovary is cut into slices 10 mm by 10 mm by 1 to 1.5 mm. The tissue slices are initially equilibrated in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in HEPES-buffered handling media supplemented with 20% synthetic serum substitute (SSM) for 25 min. Then, a second equilibration is performed in 20% EG and 20% DMSO with 0.5 M sucrose for 15 min or until the slices descend to the bottom of a 50-ml centrifuge tube indicating complete absorption of the cryoprotectants. The tissues are then placed on a thin metal strip (Kitayzato Bio Pharma, Japan) which is then plunged directly into sterile liquid nitrogen and inserted into a “closed” tube which contains liquid nitrogen for storage.

For thaw, the metal strip is immersed swiftly into 40 ml of 37 °C HEPES-buffered handling media supplemented with 1.0 mol sucrose for 1 min, and then 40 ml of 0.5 mol sucrose for 5 min at room temperature and washed twice for 10 min in standard handling media. Standard H+E histology and dye exclusion is performed to check for viability and presence of cancer cells.

The transplant technique has not changed since our first fresh transplant in 2004, which has been well described [18]. The ovary cortical strip after thawing is treated as though it were a full thickness skin graft (1–1.5 mm in thickness, and 1 cm by 1 cm size pieces). On average, only 25% of stored tissue was thawed and transplanted. The thawed strips or slices are then quilted together with a 9-0 nylon sutures and applied to the denuded ovarian medulla of the remaining intact ovary, (Fig. 1). Micro-hematoma formation under the graft as well as adhesions were avoided by meticulous micro-bipolar cautery with pulsatile heparinized saline irrigation, and multiple micropressure interrupted stitches of 9-0 nylon. Constant pulsatile irrigation is used to prevent adhesions, so as to allow spontaneous pregnancy with no need for IVF. All transplants were therefore orthotopic, with a healthy fallopian tube in good position for ovum pickup.

Before transplanting to the denuded ovarian medulla, the slices of thawed ovarian tissue were quilted together into one graft using 9-0 nylon interrupted sutures, before the patient was taken to the operating room and anesthetized (Figs. 1 and 2). All procedures were performed with minilaparotomy and an operating microscope. The reasons for using minilaparotomy over laparoscopy or robot are as follows: (1) we used an operating microscope with 9-0 nylon interrupted sutures and continuous irrigation with heparinized saline to avoid adhesions which could interfere with spontaneous pregnancy, and (2) we inject the operative site with a combination of ropivacaine toradol, and morphine, which provides 3 days of local anesthesia and minimal pain. So it is a quick outpatient procedure despite not being laparoscopic.

All patients underwent monthly hormone evaluation (FSH, estradiol, and AMH) and recording of the return of menstrual cycling. Once menstrual cycling returned, hormonal measurements were made on day 3 of each cycle. All patients were allowed to conceive spontaneously, and treatment intervention such as IVF or hormonal stimulation was not employed. All cases were approved by the IRB of St. Luke’s Hospital in St. Louis Missouri. This is a prospective series but not a randomized control (RCT) study.