321Ovary Transplantation for Fertility Preservation in Cancer Patients: Fresh and FrozenChapter 42

321

Ovary Transplantation for FertilityPreservation in Cancer Patients:Fresh and FrozenSJ Silber

AbstractA series of monozygotic (MZ) twin pairs discordant forpremature ovarian failure presented an unusual opportunityto study fresh ovarian transplantation. This unusual series thenhelped us to refine the techniques for both fresh and frozenovary transplantation in order to preserve fertility for cancerpatients who would otherwise suffer iatrogenic iatrogenicovarian failure from their chemotherapy and radiation.

Ten MZ twin pairs requested ovarian transplantation andnine of them have undergone fresh transplantation along withcryopreservation of spare donor ovarian tissue. Eight of themhad a fresh cortical tissue transplant, and two of those eightreceived a second frozen–thawed transplant after the firsttransplant ceased functioning years later. One of the 9 MZ twins

had a fresh whole ovary microvascular transplant rather than acortical graft. 62 cancer patients had had ovarian tissuecryopreserved from 1997 till the present, and one thus far hashad her frozen ovarian tissue transplanted back after she wascured and married, which was 12 years after her ovary hadbeen frozen and she had undergone menopause from her bonemarrow transplant.

All recipients reinitiated ovulatory menstrual cycles andnormal Day 3 serum FSH levels by 77–142 days. Seven of thenine fresh transplants have already conceived naturally (threeof them twice), producing 9 healthy babies. One required IVFbecause she had congenital absence of ovaries and fallopiantubes, and delivered healthy IVF twins. Currently, elevenhealthy babies (including two from frozen ovary transplantshave been delivered out of 14 pregnancies in 10 patients, two of

322 Clinical Infertility

whom were the cancer cases. There is only one patient out ofthe ten that has not had a healthy pregnancy, but she was 40years old when she first underwent the transplant. Onesuccessful fresh transplant ceased functioning by four years,but then this patient conceived again after a frozen–thawedsecondary transplant. Another ceased functioning after twoyears, but again conceived after a frozen–thawed secondarytransplant. There was no apparent difference in return of ovarianfunction between the nine fresh ovarian grafts and the threefrozen grafts. Ovarian transplantation appears to restoreovulatory function robustly. Fourteen pregnancies, leading to11 healthy babies, including two after cryopreservation, bodewell for application of ovarian tissue freezing and transplan-tation to fertility preservation. A comparison of slow freezetechnology to vitrification of ovarian tissue strongly favorsvitrification.

IntroductionSuccessful fresh human ovary transplantation was firstreported between monozygotic twins discordant forpremature ovarian failure (POF) using a cortical graftingtechnique.1,2 We have now reported a total of nineconsecutive successful fresh ovary transplants in meno-pausal women, with resumption of normal hormonalcycling and menstruation in all cases, and with12 pregnancies and nine healthy babies born from thesenine fresh ovary transplants.3-5 This experience helpedus refine the techniques necessary for successfulpreservation of fertility for cancer patients using ovariantissue freezing.

The great majority of women enter menopause in theirfifth or sixth decade of life, although ~ 1% undergomenopause prematurely, i.e. before 40 years of age.6-9

Premature ovarian failure (POF) frequently has a geneticaetiology and menopausal age normally is stronglyheritable judging by the greater concordance betweenmonozygotic (MZ) than dizygotic twins.10-12 However,another common cause for POF is treatment for cancerin young girls and women of reproductive age.13-21

It was remarkable to identify a MZ twin pair in whichone sister had undergone menopause for unexplainedreasons at age 14 years, whereas the other, aged 24, wasstill fertile with three naturally conceived children, aswell as normal ovulatory cycles and ovarian reserve.1,3-5

After the sterile twin received a graft of ovarian tissuefrom her sibling, she conceived naturally after her firstmenstrual cycle, and delivered a healthy baby at full-term.This case of discordant twins is not unique, however, andnine other twin pairs have subsequently consulted ourcenter for ovarian transplantation in preference toconventional oocyte donation. The present chapterprovides a clinical evaluation of the nine cases of freshovarian transplantation that have already undergonesurgery and its application then to 62 cases of ovarian

tissue freezing for cancer and 3 cases of frozen ovary tissuetransplantation.3,5,13,20-21 Furthermore, it delineates theexpected duration of function of these cortical ovariangrafts, both fresh and frozen.

Subject Recruitment and ConsentTen MZ twin pairs aged 24–40 years presented withdiscordant ovarian function, one sibling of each pairhaving undergone POF. Nine of the ten elected toundergo transplantation of an ovary from the normaltwin to the twin with POF. POF was diagnosed aftermore than 4 years of amenorrhea accompanied byelevated serum levels of gonadotrophins, over 50 miu/mL. Their sisters, in contrast, still had normal menstrualcyclicity, normal gonadotropin levels, and eight of thenine donors had successful pregnancy histories. Noneof the twin pairs was actively recruited. Each hadenquired about treatment to restore normal reproductiveendocrine function with fertile potential after hearingreports of the first successful ovarian transplant in a twinpair in 2005, as well as from researching an earlier testistransplant we had reported for anorchia.1,22,23 Thepatients volunteered many reasons for preferringtransplantation to conventional oocyte donation. Manyof them had previous failures with donor oocyte cycles,and did not want to go through more cycles. Some hadthe opportunity to donate an ovary at the same time ashaving surgery they required for other gynecologicproblems (such as fibroids or cysts). All of them foundthe possibility of natural conception more attractive thanIVF and egg donation. In most cases, the twins lived farapart (even in different countries) and the donorspreferred to make a single visit for a one-time donation,with the hope that frozen banked tissue could serve as abackup if the first transplant failed.

Clinical ProfileThese studies were carried out with informed consentunder a protocol approved by the Institutional ReviewBoard and the Ethics Committee of St. Luke’s Hospital,St. Louis, MO. The donors were informed that they mightreach menopause slightly earlier than normal based ontheoretical models and experimental studies24,25 andwere aware of the relative risks associated with unilateraloophorectomy.

The reproductive history of each twin pair wasreviewed and ovarian function was investigated bystandard gynecological procedures. Serum from peri-pheral blood was prepared for immunoassay of FSH,LH and estradiol (E2) three days after the start of menses

323Ovary Transplantation for Fertility Preservation in Cancer Patients: Fresh and Frozen

in women who were cycling naturally, but not on anyspecific day in those with POF. The antral follicle count(AFC) was recorded by transvaginal ultrasoundscanning. A tiny piece of spare tissue becoming availableduring oophorectomy of the donor, as well as resectedatrophied ovarian cortex of the recipient was preparedby fixation in Bouin’s fluid, embedding in paraffin,sectioning, and staining with haematoxylin and eosin(Figures 42.1A and B). Most of the spare donor tissuewas cryopreserved for future use.

Genetic StudiesDNA fingerprinting confirmed the genetic identity ofall ten twin pairs, who were also screened for common

genetic causes of POF. Peripheral lymphocytes wereprepared as DNA for testing genetic polymorphisms at15 loci (Paternity Testing Corporation, Columbia, MO),and cultures (and in some cases spare ovarian medullarytissue) were karyotyped by the G-banding technique andfluorescent in-situ hybridization. DNA was also screenedfor the number of CGG repeats in the FMR1 gene usingSouthern blot analysis or the polymerase chain reactionfor fragile X syndrome.1 In addition, genomic DNA andlymphoblastoid cell lines were prepared for futuregenetic studies of all the twin pairs in the hope of oneday identifying the loci responsible for prematureovarian failure (POF).

Fresh Cortical Ovarian TissueTransplantationThe patients were scheduled for surgery within twoweeks of confirming negativity for the human immuno-deficiency virus type 1 and hepatitis B and C viruses.Nine of the presenting identical twin pairs actuallyunderwent the surgery. Under general anesthesia, oneovary was removed from donor using laparoscopy orminilaparotomy. For the eight undergoing cortical tissueharvesting (excluding the intact whole ovary case), thewhole ovary was transferred to a Petri dish for dissectionby hand with a scalpel and toothed forceps. It was feltimportant to prepare a cortical tissue slice no thicker than~ 1.5 mm to facilitate rapid revascularization whilekeeping the tissue constantly irrigated with ice-coldLeibovitz L-15 medium (Figure 42.2A). We nowrecommend a special microtome tissue slicer rather thandissecting by hand, in order to get a thinner slice of only0.75 mm. But for these initial fresh cases, the hand paredcortex was divided into three or four pieces ofapproximately equal size for grafting, one piece to eachrecipient ovary. The remaining one-half to two-thirdsof the cortical tissue was cryopreserved in 1.5 M 1.2-propanediol and 0.2 M sucrose by slow freezing to liquidnitrogen temperatures.44,45 Remnants of the trimmedtissue were set aside for histology and genetic studies.

The recipients were prepared by minilaparotomy viaa 3.5 cm incision above the pubis. For cortical tissuetransplantation, recipient ovarian cortex was resectedto completely expose medullary tissue (Figure 42.2B);hemostasis was controlled with microbipolar forceps,and irrigation with heparinized saline was performedto avoid adhesion formation or a hematoma betweendonor and recipient tissues. The tissue graft was trimmedto the dimensions of the exposed surface of the recipientorgan and attached using 9-0 interrupted sutures underan operating microscope (Figure 42.2C). Very impor-tantly, the medullary bed was also sutured to the under

Figures 42.1A and B: (A) Showing the absence of primordialor preantral follicles in ovarian biopsies of this candidate forovarian transplantation compared with (B) that in her fertilesister

324 Clinical Infertility

surface of the cortical graft with 9-0 sutures to maintaintight tissue approximation again to avoid micro-hematoma formation under the cortical grafts. Irrigationand meticulous pinpoint hemostasis were rigorous toavoid adhesion formation. The same procedure was usedfor frozen–thawed tissue to replace the first graft thatmay have ceased to function (Figure 42.2D), and alsofor the cancer cases. After removing the first graft or theold cortex of the cancer patients, to accommodate thenew one, all discarded tissue was prepared by histologyand found to be completely devoid of follicles. Allpatients were released from the hospital the same dayor the following morning, and had a rapid and unevent-ful recovery. In the one case of bilateral absence of ovaryand ampulla, the graft was attached to the fallopian tubeisthmus.

Whole-Ovary TransplantationTo transplant an intact whole ovary, the donor ovarywas removed by clamping the infundibular pelvicligament at its base in order to obtain maximum length.The veins (3–5 mm) were easily identified, but theovarian artery (0.3 mm) was not grossly visible. Theentire specimen was placed in Leibovitz medium at 4°Cand two veins and one artery were dissected and isolatedunder the operating microscope. Germinal vesicleoocytes were aspirated from antral follicles for in vitromaturation and vitrification at the metaphase II stage.Meanwhile, the recipient’s infundibular pelvic ligamentwas clamped at the base and transected close to her non-functioning ovary. The donor’s ovarian veins were thenanastomosed to the recipient’s with 9-0 nylon interrupted

Figures 42.2A to D: Steps in the procedure of ovarian transplantation between MZ twin sisters: (A) preparation of donor ovariancortex by dissection in a Petri dish on ice; (B) Preparation of recipient ovarian medulla; (C) Attaching donor cortical tissue torecipient ovarian medulla; (D) Attaching thawed donor cortical tissue for re-transplant to the recipient

325Ovary Transplantation for Fertility Preservation in Cancer Patients: Fresh and Frozen

sutures, and the ovarian arteries were anastomosed with10-0 nylon interrupted sutures (Figures 42.3A to D).When the microvascular clamps were removed, bloodflow was observed by fresh bleeding from the surface ofthe ovary where a cortical slice had been taken forcryopreservation as a backup.

Details on the original chorionicity at birth of theseidentical twin sisters were available for six of the tentwin pairs consulting our center. Two were monochorio-nic-diamniotic, one was dichorionic-diamniotic andthree (50%) were monochorionic-monoamniotic, whichwas a surprisingly high incidence since the incidence ofmono/mono is normally only ~ 2% (P< 0.0005). It is clearthat late splitting, for whatever reason, predisposesotherwise identical twins to discordant germ celldeficiency.

Postoperative ResultsAll nine twin pairs underwent orthotopic ovarianisotransplantation between April 2004 and April 2008.

The recipients, for the most part, continued to cycle forover three years to six years or more, although two ofthem whose donor had low antral follicle counts (AFC)of less than 10, only functioned for 2 years. However,even these two cases had spare frozen cortical tissue thatwas available for further transplants. One has not yetdone this, but in the other, the frozen transplant wassuccessful, and so she also has now more than three yearsof ovarian function. Day 3 FSH levels returned to normalby 4.5 months of surgery in all cases (Figure 42.4A), soonafter ovulation had recommenced (judging by the resultsof BBT or home ovulation detection kits monitored bythe patients themselves). The refractory period forresuming menses after transplantation was 63–100 days,with the majority of subsequent cycles in the normalrange of duration.

The first case, a 25-year old recipient, becamepregnant the first time after her second menses withoutmedical assistance, and subsequently delivered a healthybaby girl in 2005 following an uneventful pregnancy.After nursing for several months, she resumed mensesand during the seventh cycle conceived naturally a

Figures 42.3A to D: Steps in the procedure of intact ovary microvascular transplantation: (A) Depiction of donor oophorectomy;(B) Microsurgical isolation of donor ovary blood supply; (C) End-to-end anastomosis of ovarian blood vessel; (D) Completedanastomosis of ovarian artery and veins

326 Clinical Infertility

second time, but this pregnancy miscarried. Over threeyears after her transplant, she ceased cycling andhormone levels, which were measured monthly, becamepost-menopausal again (82 mIU/mL FSH, 34 mIU/mLLH and 13 pg/mL E2). After transplanting cryopreservedspare tissue, her hormones again returned topremenopausal levels after four months, a delay identicalto her fresh transplant (Figure 42.4B). She conceivedagain without any intervening menses and without anymedical treatment approximately five months after herre-transplant and delivered another healthy baby.

The second case became pregnant at 39 years ofage without medical assistance after her fifth menses,eight months after transplantation. She too delivered ahealthy baby girl at full-term. She then conceived againat age 41, and delivered a healthy baby boy, again atfull-term. Her ovary is still functioning to date after sixyears.

Only one patient failed to conceive, but she had amarginal AFC and was 41 years old at the time of thetransplant. However, the ovary continued to functiondespite her age for over four years. One case had amicrovascular transplant and is continuing to cycleregularly. Her Day 3 FSH and LH fell to the lowest levelsof all the recipients (3.4 and 0.4 mIU/mL, respectively),and her post-operative ultrasound appeared normal. Shebecame pregnant and delivered a healthy baby two yearslater.

All patients remain pleased with their decision to haveundergone transplantation, and even the patientrequiring IVF preferred this option. Overall, there havebeen fourteen pregnancies and ten healthy live births inthis group of nine transplants.

Frozen Cortical OvarianTransplantationSince 1996, we have frozen ovary tissue for 62 youngwomen with solid organ cancer, or with great risk forPOF, of whom 16 had spare frozen tissue subjected todetailed viability testing before cryopreservation andafter thaw. All 62 had histologic review, and only onehad evidence of ovarian metastasis, a young woman withbreast cancer. With the classical slow freeze technique,only 41% of oocytes survived the thaw.20,21,26,27 However,with vitrification there was no difference between freshunfrozen controls and frozen tissue.21 It seemed likely,therefore, that vitrified ovarian tissue would give betterresults after transplantation than tissue cryopreservedby slow-freeze.

It is naturally to be expected that it could be a decadeor more before patients with frozen tissue come back tohave it thawed and re-implanted. Firstly, they need tosee if they are declared “cured” by their oncologist, andsecondly, in the modern era, it could be a while beforethey meet the right partner and decide to have children.Thus, far, only 3 of our patients have had their frozentissue transplanted back. In two cases, the tissue wascryopreserved with slow freeze and, in one case, withvitrification.

In addition to these 62 pathologic cases, 7 women havehad ovarian tissue frozen simply to allow them thepossibility of having children at an older age because theyhad to delay childbearing for strong personal andeconomic reasons. All of these women are more recentcases, and had their ovarian tissue frozen withvitrification.20,21

Figure 42.4A: The eight fresh transplant cases showed adramatic decline in day 3 serum FSH by 80–140 days post-operatively corresponding approximately to the resumption ofmenses. The results of the microvascular whole-ovarytransplant are not significantly different from cortical grafts

Figure 42.4B: After a frozen cortical re-transplant, serum FSHdeclined again to normal levels, similar to those of freshtransplants

327Ovary Transplantation for Fertility Preservation in Cancer Patients: Fresh and Frozen

The method we use for vitrification of ovarian tissuehas already been described in detail,21 but to summarize:Using a “Cryotissue” knife and silicone template, a3/4 mm deep slice is taken off the cortical surface of theovary, measuring centimeter wide by two centimeterslong. Then similar cortical slices are removed fromthe surface of the rest of the ovary, and usually10 to 16 slices can be obtained. The cortical slices areplaced in a large test tube containing 7.5% ethylene glycoland 7.5% DMSO in HEPES buffered media containing20% synthetic serum substitute for 25 minutes. This iscalled ES, or equilibrium solution. The thin tissue slicesare then transferred to a large test tube containing 20%ethylene glycol, 20% DMSO, and 0.5 molar sucrose, andkept there until the tissue slices sink to the bottom, whichis usually less than 15 minutes. They are then blotteddry and placed on a perforated copper grid (Kitazato,Japan), and plunged directly into liquid nitrogen. Aprotective cover is then screwed on and the vitrifiedtissue is transferred to a liquid nitrogen storage tank.The speed of cooling and warming is approximately20,000 degrees centigrade per minute, and successfulvitrification is easily observed by the absence of icecrystal formation on the tissue.

For thawing, the copper grid is plunged into a largetest tube containing thaw media at 37°C for 5 minutes.Thaw media contains 1.0 molar sucrose. The tissue thenis transferred to 0.5 molar sucrose at room temperaturefor 5 minutes following which it is transferred tostandard isotonic HEPES buffered transfer media. Thetechnique for transplantation of the thawed tissue is nodifferent from that which we described for fresh tissue.

Of the three cases of frozen transplanted tissue thusfar, all have had a robust return of ovulatory menstrualcycles with spontaneous pregnancy, but the case withvitrified tissue had the quickest return of function withher first period occurring at just 42 days post transplant.

DiscussionIncluding the first MZ twin pair presenting for ovariantransplantation in 2004, there have been eight morecomparable cases using fresh ovary so far. There havebeen three additional frozen cases, for a total of twelveauto- or homo-transplants. The MZ twin cases charac-terized by ovarian discordancy, a phenomenon that isnot as rare as first assumed.28 The ovaries with POF werediminutive, fibrous and completely lacking follicles atany stage, while serum gonadotrophins were corres-pondingly elevated and E2 was low. None of the medicalhistories provided an explanation for POF withafollicular ovaries in the recipients, except for one whohad received chemotherapy. The clinical histories of POF

in the other nine MZ cases were idiopathic and consistentwith congenital deficiency of germ cells. According to amathematical model,25 the follicle reserve at birth mustbe very small to account for POF as early as adolescenceor young adulthood.

With a single exception, the reproductive tracts of therecipients were structurally normal and both ovarieswere present, albeit as “streaks” in some cases. In theagonadal case, the ovaries and the tubal ampullae werebilaterally absent, indicating a concurrent Mulleriananomaly.29

There may be non-genetic explanations for discor-dancy in ovarian reserve since MZ twins, like animalclones, are not phenotypically identical and other, non-ovarian discordancies were observed in three twin pairs.One clue to ovarian discordancy might be the mono-amniotic, monochorionic twin pregnancies, which weremore frequent than expected.28,30,31 The MZ twinembryos are presumed to arise from late splitting post-implantation, perhaps as late as the primitive streak. Ifso, late splitting may be a risk factor for decreasedovarian reserve through misallocation of germ cellprecursors. Also perhaps, epigenetic factors may affectthe ovarian follicle reserve, since we know that there isan excess of imprinting defects in monozygotic twinpregnancies.31

Conventional oocyte donation is the first line oftreatment for patients with POF who want to becomepregnant. Nevertheless, the robust results obtained inevery case of this series of isogenetic twins givesconfidence in ovarian transplantation as an alternativestrategy for overcoming sterility. Although the surgerymight seem more burdensome than oocyte retrieval, itis a straightforward and uneventful outpatientprocedure, which has been effective in all nine cases inrestoring menstrual cycles and enabling establishmentof viable pregnancies. It has been equally uneventful inthe three frozen cases. After ovarian transplantation, thepatients were able to attempt natural conception everymonth without medical assistance. Accepting the risksof surgery, the procedure avoids the specific risksassociated with IVF, notably ovarian hyperstimulationsyndrome and multiple pregnancy, and moreover,allows spare tissue to be cryopreserved in the event ofgraft failure. The number of successful re-transplants willdepend on the age of the patient and the number offollicles surviving, but the results of fresh and frozencortical tissue from the first few patients suggest thatfertile potential may be restored cumulatively for overfifteen years by serial transplantation.

Ovarian cortical grafting was chosen for eight of theprocedures in preference to vascular anastomosis of theintact ovary because it is less invasive, carries minimaloperative risk and reduces recovery time. The peripheral

328 Clinical Infertility

location of primordial follicles is advantageous for rapidrevascularization, as well as for successful cryopreser-vation.1-4,38-40 The thinner slices used for vitrification mayalso allow for more rapid revascularization than thethicker slices used in earlier efforts. Heterotopic siteshave produced no successful pregnancies to date andour patients preferred the chance of natural concep-tion.42-45 The whole-ovary microvascular approach wasadopted at one patient’s request in the hope ofmaximizing the functional longevity of her transplant.41

We suspect follicle ischemia was minimal, as predictedfrom transplants of intact rat ovaries using vascularanastomosis.6 However, the long duration of functionof our ovarian cortical grafts suggest that ischemia maynot be problematic if very thin cortical slices are used.

There was remarkable consistency between subjectsin the return of menses after ovarian transplantation.The refractory period of about three months was similarto autotransplants in the sheep model,2 and consistentwith estimates of the time taken for small follicles to growto ovulatory size in humans.48 There was one exceptionof an extraordinarily rapid return of function in the onepatient who had vitrified tissue transplanted, and thiscould very well be due to the exceptionally thinness ofthe slice (0.75 mm) and the fact that vitrification in itselfcauses no tissue or cellular damage. In some case reportsof transplantation in cancer patients, a longer refractoryperiod of up to nine months has been reported,39,40,48

but it is not clear whether the difference is due totechnical aspects of surgery, the type of cryopreservation,or patient selection, either of which could influence thespeed of recovery and duration of graft function. Whilethis evidence suggests that follicle dynamics arerelatively normal once the transplant is fully active, thefollicle reserve might be compromised by ischemia. Graftlongevity might be affected by graft ischemia time or bycryopreservation damage. This is now the subject ofintense study.20,21,49

This is the most extensive clinical series of orthotopicovarian transplantation of which we are aware. Twinsare unlikely to be the main candidates for this procedurein the future, and the major application is likely to be forfertility preservation in cancer patients and possiblyother women who need to delay childbearing. Whileneither ovarian autografts nor isografts should presentany problem of histocompatability, allografts arepotentially at risk. However, allografts might occa-sionally be considered if ovarian tissue is available froma young woman who previously donated bone marrowto the same patient, or if mild immunosuppressionproves adequate to prevent rejection, which we arecurrently studying. Reassuringly, well-matched (HLA)kidney transplant recipients on immunosuppressionhave favourable obstetric outcomes.50

At the time of this writing, we are only aware of asmall number of other births or ongoing pregnanciesafter re-implanting frozen ovarian tissue, and these wereall for cancer patients.39,40,51-54 This is not surprising fora new procedure involving patients who must delaytransplantation until they are assured of long-termremission or await confirmation that their frozen tissueis free of malignant cells. We had no such concern in ourseries, which offers a better chance to rapidly evaluatethis technique in both fresh and frozen grafts from thesame individual. No such concerns applied to the healthytwins, and the evidence from this series of the effective-ness of both fresh and frozen transplants gives hope thatcryopreserved ovarian tissue for cancer patients shouldno longer be considered experimental.

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