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Summary

Background Cancer treatments, including chemotherapy,radiotherapy, and radical surgery, can induce prematuremenopause and infertility in hundreds of thousands ofwomen of reproductive age every year. One of the ways topossibly preserve fertility before these treatments is tocryopreserve ovarian tissue for later transplantation. Weaimed to restore fertility by cryopreservation andtransplantation of ovarian tissue.

Methods Ovarian tissue was cryopreserved from a 30-year-old woman with breast cancer before chemotherapy-inducedmenopause, and this tissue was transplanted beneath theskin of her abdomen 6 years later.

Findings Ovarian function returned in the patient 3 monthsafter transplantation, as shown by follicle development andoestrogen production. The patient underwent eight oocyteretrievals percutaneously and 20 oocytes were retrieved. Ofthe eight oocytes suitable for in-vitro fertilisation, onefertilised normally and developed into a four-cell embryo.

Interpretation Fertility and ovarian endocrine function can bepreserved in women by long-term ovarian tissue banking.

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IntroductionChemotherapy, radiotherapy, and radical surgery canresult in ovarian failure and infertility in hundreds ofthousands of women of reproductive age with cancer inthe USA alone. Thousands more patients receivechemotherapy and radiation for the treatment of disorderssuch as collagen vascular, haematological, and idiopathicdiseases.1 Women of reproductive age and their partnercan undergo a cycle of in-vitro fertilisation (IVF) tocryopreserve their embryos before chemotherapy.2

However, most cancer patients do not have sufficient timeto complete the necessary ovarian stimulation for IVF.This option is also not acceptable for single women whodo not want to use donor sperm and for children. Oocytecryopreservation can be considered for adult singlewomen who understand that pregnancy rates are low withthis experimental strategy, but this approach, similar toembryo freezing, also needs several weeks of ovarianstimulation. Against this background, an experimentalovarian cryopreservation and autotransplantationtechnique has been developed. After success by otherresearchers in animals,3,4 we reported ovarian functionafter orthotopic transplantation of frozen-thawed andheterotopic grafting of fresh human ovarian corticalpieces.5,6 In all women, ovarian function was restored, atleast temporarily. In one patient with fresh ovarian tissuethat was transplanted subcutaneously in the forearm, anintact oocyte was retrieved percutaneously but it did notbecome fertilised. In this study we aimed to restorefertility by subcutaneous transplantation of frozen bankedovarian tissue.

Embryo development after heterotopic transplantation ofcryopreserved ovarian tissue

Kutluk Oktay, Erkan Buyuk, Lucinda Veeck, Nikica Zaninovic, Kangpu Xu, Takumi Takeuchi, Michael Opsahl, Zev Rosenwaks

Center for Reproductive Medicine and Infertility, Joan and Sanford IWeill Medical College of Cornell University, 505 East 70th Street,HT-340, New York, NY 10021, USA (K Oktay MD, E Buyuk MD, L Veeck DSc, N Zaninovic MSc, K Xu PhD, T Takeuchi MD, Z Rosenwaks MD); and Genetics and IVF Institute, Fairfax, VA 22031, USA (M Opsahl MD)

Correspondence to: Dr Kutluk Oktay(e-mail: kuo9001@med.cornell.edu)

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Figure 1: Resumption of ovarian function after ovariantransplantationPeak oestradiol concentration accords with that of a typical cycle and isaccompanied by a luteinising hormone (LH) surge, but no FSH increase isseen.

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MethodsPatient’s information The patient was a woman who was diagnosed withstage IIB breast cancer at age 30 years. Before shereceived high-dose gonadotoxic chemotherapy, includingcyclophosphamide, we removed one ovary laparo-scopically. Cortical pieces, which invariably contain somestroma, were cryopreserved with a slow freezing protocol,with dimethylsulfoxide as a cryoprotectant.7 Aftercompleting her chemotherapy and bone-marrowtransplantation, the patient ceased to menstruate, and araised follicle-stimulating hormone (FSH) value of 117 IU/L confirmed menopause. The study was approved

by the institutional review board of Weill Medical Collegeof Cornell University and the patient gave writteninformed consent for experimental procedures.

Procedures6 years after ovarian cryopreservation, the patient’s tissueswere transported to our laboratory. We thawed one vial oftissue and tested the piece histologically to rule outovarian involvement with cancer and to establish thedensity of primordial follicles. On the basis of follicledensity and the number of pieces available, we estimatedthat the patient had about 11 000 follicles, which couldpotentially last for a year.8 Under local anaesthesia, weimplanted all 15 ovarian cortical pieces—with sizesranging from 5�5�1 mm to 15�5�2 mm—beneath theskin of the patient’s lower abdominal wall with a suturepull-through technique, as described.9,10 Before every cycleof ovarian stimulation, we achieved ovarian suppressionby administration of gonadotropin-releasing hormoneantagonist or agonist. We did ovarian stimulation with acombination of FSH and human menopausalgonadotropins. Oocyte maturity was triggered by 250 �gof recombinant human chorionic gonadotropin (Ovidrel,Serono, Norwell, MA, USA), and we undertook oocyteretrieval 36–40 h later. We gave progesteronesupplements in every cycle; thus, we could not fully assessspontaneous luteinisation. For IVF, the intracytoplasmicsperm injection technique was used for all but one oocyte.We used a complex, non-commercial sequential culturemedium, which we prepared every week onsite at theWeill Cornell facility. We matured and fertilised theoocyte and cultured the embryo in phase 1 of this mediumuntil about 48 h after injection. We did preimplantationgenetic diagnosis by fluorescence in-situ hybridisation, asdescribed.11

Role of the funding sourceThe sponsor had no role in study design, data collection,data analysis, data interpretation, or writing of the report.

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Figure 2: Subcutaneous ovarian follicle growth (A) Day 12 follicle. (B) Day 14 follicle. (C) Trilaminar endometrialdevelopment in response to oestrogen production.

Stimulation dose Peak oestradiol Follicle Oocyte(FSH/HMG) and (pmol/L)† size (mm)†duration*

Retrieval1 None 888 10·9 GV 3 3675/2175, 980 14·2 FZ

13 days 10·7 Degenerated8·9 GV7·3 FZ

4 None 1093 9·1 FZ5 3225/1725, 2257 14·5 FZ

11 days 13·1 M-I9·5 FZ6·4 GV

6 2025/2475, 1868 12·8 Mature10 days 10·8 FZ

8·4 FZ7 2100/2400, 921 11·8 Mature

10 days 10 GV6·7 Degenerated

8 2025/1500, 987 11 Degenerated10 days 9·9 Mature

9·8 GV6·4 GV

HMG=human menopausal gonadotropins. FZ=fractured zona pellucida.GV=germinal vesicle. M-I=metaphase-I. No oocytes were recovered during thesecond retrieval. Embryos were generated after in-vitro maturation during thefifth (M-I oocyte) and eighth (GV oocyte) IVF cycles. Immature oocytes arefurther categorised as GV or M-I stage. *Numbers represent total dose of FSHand HMG in IU. †On the day human chorionic gonadotropin was injected.

Stimulation type, peak oestradiol concentration, and folliclesizes on day of human chorionic gonadotropin administration,and oocyte retrieval outcome from ovarian transplant

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Results85 days after ovarian transplantation, the patient noticed apea-size lump at the transplant site. Resumption ofovarian endocrine function was confirmed by raisedoestradiol concentrations (477 pmolL), by lowered FSHconcentrations after 11 days (7·9 IU/L; figure 1), and bydemonstration of ovarian follicle development byultrasound (figure 2). Follicles could also be palpatedunderneath the skin. Simultaneous ultrasound monitoringof the menopausal ovary did not show ovarian follicledevelopment in any of the cycles.

We did eight consecutive percutaneous oocyteretrievals over the next 8 months, six after ovarianstimulation (table). Of the 20 oocytes obtained from thepatient’s transplanted tissue, eight were suitable for IVFwith her husband’s sperm. Three of these were mature atretrieval and five had to be matured in vitro. Althoughmature oocytes did not fertilise, two of the oocytes thatwere matured in vitro were fertilised via intracytoplasmicsperm injection. One embryo showed abnormalmorphology and its growth halted at the three-cell stage(figure 3, A). Fluorescence in-situ hybridisation analysiswas done on two cells,11 which showed an XX embryowith aneuploidy for several chromosomes. A secondoocyte that matured in vitro in 24 h showed signs ofnormal fertilisation (two pronuclei) 18 h afterintracytoplasmic sperm injection, which then progressedto a high-grade12,13 four-cell stage embryo 24 h later

(figure 3, B). The embryo was then transferred to thepatient’s uterus.

DiscussionDevelopment of a morphologically normal four-cellembryo indicates that ovarian follicles with viable andfunctional oocytes can develop in ovarian tissue thawedand transplanted to a heterotopic location. In addition tonormal morphological findings, the rate of in-vitromaturation14 and growth to four-cell stage accords withembryo viability.12,15–17 Our results also suggest that, eventhough oestrogen production is seemingly typical, oocytequality could be compromised at this heterotopic location.Alteration in quality could potentially be attributable tofreezing, thawing, and initial ischaemia aftertransplantation.18 Oocyte quality could also be alteredbecause of differences in temperature and blood flow inthe subcutaneous environment compared with pelvic(orthotopic) location. If this change is the case, the longerthe follicles are allowed to develop before retrieval thehigher the likelihood of compromise. This hypothesis islent support by the fact that the only viable embryo wegenerated was from a germinal vesicle stage oocyte thatwas retrieved from a small antral follicle. By contrast, anabnormal embryo was obtained from an oocyte of a largerfollicle matured from metaphase-I stage, and matureoocytes failed to fertilise. Because of this difference,aspiration of immature oocytes from smaller follicles

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Figure 3: Embryo development with in-vitro fertilised oocytes retrieved from transplanted ovarian tissue(A) Abnormal three-cell embryo with unequal blastomeres and extensive fragmentation (left). Multicolour fluorescence in-situ hybridisation shows that onecell was aneuploidic for chromosome 16 (middle) and the other for chromosomes 15, 16 (right), and 13, 18, 21 (not shown). X=yellow; 15=green;16=orange. Scale bar=50 �m. (B) Fertilisation and normal embryo formation after intracytoplasmic sperm injection. 18 h later, a two-pronuclear stageembryo was seen (left), which progressed to a high-grade four-cell stage embryo with minimal fragmentation (right). Scale bar=50 �m.

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followed by in-vitro maturation could be the preferredapproach to preserve competence to undergo fertilisation.Our findings also showed that ovarian follicles do notgrow to the same sizes in heterotopic locations comparedwith pelvic placement,5 which accords with findings ofprevious xenografting experiments.19 Oocyte maturityseems to be attained at 10–11 mm diameter, contrastingwith 16–17 mm in orthotopic ovaries.20 Our findings showthat fertility and ovarian endocrine function can bepreserved in women by long-term ovarian tissue banking.Because the probability of pregnancy with one embryooriginating from an in-vitro matured oocyte is 6–12%,13,21

many attempts or simultaneous transfer of multipleembryos might be needed to achieve a pregnancy. Eventhough the final proof of success of ovariancryopreservation and transplantation procedure will be aviable pregnancy in human beings, with the developmentof a human embryo, prospects for a pregnancy andliveborn are now more promising.

Contributors K Oktay was responsible for study design, invention and performance ofthe transplantation technique, ovarian stimulation, cycle monitoring,oocyte retrievals, embryo transfer, data analysis, and preparation of thereport. E Buyuk planned and did tissue thawing and preparation,undertook viability tests, collected and analysed data, and prepared thereport. Z Rosenwaks assisted with tissue preparation and gave technicaladvice in IVF-embryo transfer. K Xu undertook and interpreted theanalysis of fluorescence in-situ hybridisation. L Veeck and N Zaninovicprovided IVF laboratory support, did in-vitro maturation, and undertookembryo culture and assessment. T Takeuchi did intracytoplasmic sperminjection. M Opsahl removed and cryopreserved ovarian tissue.

Conflict of interest statementNone declared.

AcknowledgmentsWe thank Rodney Bosley of Cook OBGYN for providing aspirationneedles and technical advice. This study was sponsored by departmentalresearch funds.

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13 Veeck LL. The morphological assessment of human oocytes and earlyconcepti. In: Keel BA, Webster BW, eds. Handbook of the librarydiagnosis and treatment of infertility. Boca Raton: CRC Press, 1990:353–66.

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20 Scott RT, Hofmann GE, Muasher SJ, Acosta AA, Kreiner DK,Rosenwaks Z. Correlation of follicular diameter with oocyte recoveryand maturity at the time of transvaginal follicular aspiration. J In Vitro Fert Embryo Transf 1989; 6: 73–75.

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