Ovarian Tissue Cryopreservation:
Current Experience
Ariel Revel MD.
Joseph G Schenker MD,FRCOG,FACOG (Hon)
Department of Obstetrics and Gynecology. Hebrew University Hadassah
Medical Center P.O.Box 12000, Jerusalem, 91120, Israel.
schenker@cc.huji.ac.il
1
Introduction
Progress in cancer treatment improves survival and cure. The extensive advance
in oncology, hematology and bone marrow transplantation (BMT) have resulted in a
90% remission rate in various diseases. Nevertheless, exposure of the ovaries to
chemotherapy and radiotherapy during the reproductive years predisposes patients to
ovarian failure.
The remarkable improvement in the survival rates due to progress in cancer
treatment has resulted in a large population of cancer survivors which require special
medical treatment. The National Cancer Institute estimated that about 1 per 1000
population will be survivors of childhood cancers. Due to the limited possibilities of
fertility preservation methods and the significant risk of ovarian failure, childhood
cancer patients request special attention. This is especially important when taking the
high 5 y survival rates in pediatric hemato-oncology patients (combined ~ 80 %, ALL
80-86%, Hodgkin’s disease>90%) (1-4) . A list of pediatric patients who face the risk
of ovarian failure due to cytotoxic treatment is detailed in table 1.
Whereas the cytotoxic-induced damage is reversible in other tissues of rapidly
dividing cells such as bone marrow, gastrointestinal tract and thymus, it appears to be
progressive and irreversible in the ovary, where the number of germ cells is limited,
fixed since fetal life, and cannot be regenerated (3) (5) . Cyclophosphamide is the
most commonly implicated agent in causing damage to oocytes and granulosa cells in
a dose dependent manner (6) . Relative risk of POF was reported between 4 and 9.3 in
patients receiving cyclophosphamide (7)
2
Radiotherapy is utilized to improve prognosis or to achieve local tumor control
in solid tumors presenting in the pelvis such as Ewing sarcoma, osteosarcoma,
retroperitoneal sarcomas, and in some benign bone tumors (8-11) .
A common concern of female patients and their families is the effect of
chemotherapy and radiotherapy on future fertility. An 8-fold increase in the risk of
premature ovarian failure (POF) has been observed in cancer survivors (12) . Women
who undergo bone marrow transplantation have a 99% risk for ovarian failure (13) .
Whereas menopausal symptoms and signs can be treated medically, no solution
is available to preserve female gametes. The resulting infertility and premature
menopause produces physical, psychological, and social consequences.
Options for Fertility Preservation
Embryo cryopreservation: Women may be offered in vitro fertilization (IVF) for
embryo cryopreservation prior to chemotherapy. This approach is the most successful
one as pregnancy rates following embryo cryopreservation. Results are reasonable,
although lower by one-third when compared with fresh IVF embryo transfer (14) and
are standard in IVF units. IVF should be offered prior to chemotherapy and not
afterwards, since diminished ovarian response was reported in women who underwent
IVF after systemic cancer treatment (15) . Although women with significant systemic
disease are reported to have poor ovarian response to ovulation induction (16) , most
cancer patients can expect an average of 11 embryos cryopreserved (15) . Since a
male partner is required for IVF, this option is inappropriate for single women who
reject the use of donor sperm. In addition, ovarian stimulation requires deferment of
chemotherapy and could be hazardous to estrogen-sensitive malignancies (e.g. breast
carcinoma). A sufficient number of viable embryos cannot be guaranteed, and a repeat
3
IVF cycle may be contraindicated. Finally, ethical issues such as the fate of stored
embryos, should the patient die, must be resolved at the outset of treatment.
Oocyte Cryopreservation
Oocyte cryopreservation would be an ideal technique for fertility preservation in
single patients. Its advantages include no need for a partner, which avoids legal,
regulatory and religious dilemmas. Nevertheless, this method has a very low success
rate. Although attempted for more than a decade (17) , only a few pregnancies have
been achieved from frozen–thawed human oocytes. While the introduction of new
cryoprotective agents (18) and intra-cytoplasmic sperm injection (ICSI), as a means
of bypassing the zona pellucida, has improved pregnancy rates of cryopreserved
oocytes, pregnancies and deliveries derived from cryopreserved human oocytes have
only resulted in a success rate averaging 2% per frozen oocyte (19) . The
disappointing success rates can be attributed, in part, to the human oocytes biological
properties (20) . Three major alternatives have been proposed in order to prevent
chilling damage to oocytes: cryopreservation of immature oocytes, oocyte vitrification
and ovarian cryo-preservation. Banking of germinal vesicle (GV) stage oocytes
involves cryo-preservation at prophase I before oocytes resume nuclear maturation
and progress to metaphase II. At this stage, a reduced risk of cytogenetic errors is
theoretically expected since the chromosomes are not aligned along the spindle.
Membranes of oocytes at the GV stage are, however, more sensitive to chilling injury
(21) . Vitrification refers to a form of cryopreservation where cooling rates are so
rapid (>20 000°/min) that ice does not have a chance to form, and the mixture of
cryoprotectant and oocyte forms a ‘glass-like’ gel. From a practical standpoint,
vitrification is simple and removes the need for expensive programmable controlledrate freezers. Following the first case report of pregnancy after oocyte vitrification
4
(22) , eight more pregnancies were reported (23, 24) . The need for high
concentrations of cryoprotectants and the paucity of human clinical data require
caution in the use of vitrification in clinical practice. A 2–4 month delay in cancer
treatment would be required to obtain enough oocytes to enable a single pregnancy.
This method cannot, at present, be routinely proposed to cancer patients.
Ovarian Tissue Cryopreservation
The emergence of ovarian tissue cryopreservation (OTCP) for fertility
conservation has led to a new worldwide trend of ovarian tissue banking for
reproductive cancer patients, scheduled to undergo chemotherapy or radiotherapy.
Since the survival of young women and children who undergo such curative anticancer treatment is increasing, it is imperative for the consulting clinician to have an
updated and accurate understanding of the proven benefits and the limitations of this
new and hitherto evolving technique. OTCP was shown to be successful in several
animal models during the last decades. In 1960, Parrot (25) reported successful
pregnancies in mice after implantation of frozen-thawed ovarian grafts. Other studies
reproduced the similar results in mice (26) and in sheep (27) . Despite these
promising preliminary animal outcome, the efficiency of ovarian tissue auto-grafting
whether orthotopic (the auto-transplantation to the ovarian pedicle) or heterotopic (the
auto-transplantation to a different site) has not yet been demonstrated in humans. The
initial hypothesis was that OTCP would be successful in humans, since the primordial
and primary follicles can survive the freezing-thawing procedures. It was hoped that
in vitro maturation (IVM) would be an important source for harvesting mature
oocytes (28) . However, the in vitro maturation of both animal and human follicles
was equally disappointing than auto-grafting. There still are no reports of harvesting
mature oocytes from in vitro culturing and maturation of primordial and primary
5
follicles. Hence, there is increasing interest in improving the cryopreservation, autografting and the in vitro culturing and maturation techniques, necessitating further
intensive research to increase their efficiency and to test their safety (29) .
Ovarian Tissue Cryopreservation Clinical Application
Human ovarian tissue banking is proposed as a method of preserving female
fertility and offers the potential of restoring normal ovarian function and natural
fertility. This procedure is applied in several medical centers worldwide. Indications
for ovarian tissue cryobanking include pre-menarche girls as well as young women
facing POF (30) . Cryopreservation of ovarian cortex rich in primordial and primary
follicles is a strategy for oocyte banking. The rationale is to cryopreserve immature
follicles within the ovarian tissue, before oocytes resume nuclear maturation. The
main advantage of this technique is that no ovarian stimulation is required and thus the
procedure can be performed on an urgent basis. Moreover, small immature follicles in
the ovarian cortex probably withstand cryopreservation better than mature ones (31) .
This has led to interest in the procedure as a potential strategy for preserving the
fecundity of patients at risk of POF (32) . In order to obtain ovarian cortex,
laparoscopic biopsies (33) or unilateral oophorectomy (34) can be carried out at any
stage of the menstrual cycle. Women electing ovarian preservation at the time of
abdominal surgery for other indications will incur no further risk from the operative
procedure. In most cancer patients, laparoscopic oophorectomy does not incur a
special surgical risk. Moreover, in many cases, this general anesthesia is used as an
opportunity to perform a bone marrow aspiration or to insert a porthacath for the
administration of chemotherapy. The technical method of ovarian cortex
cryopreservation has been well described (35) .
6
Contraindications for ovarian cryopreservation include patients at high surgical
risk and those >40 years old (36) . In order to prevent possible transmission of disease
by ovarian grafts, we routinely send samples for pathological and
immunohistochemical analysis. In patients with cancer involving the ovary,
transplantation should not be performed since this may result in transferring malignant
cells back to the patient (37, 38) . Xenotransplantation of human ovarian cortex
transmitted cancer in Hodgkin’s disease and leukemia but not in non-Hodgkin’s
lymphoma (39) . Frozen–thawed human ovarian tissue xenotransplanted into nude
mice enabled follicular maturation and oocyte retrieval (40) .
Human Ovarian Transplantation Techniques
Ideally, frozen–thawed ovarian cortex should be used for obtaining oocytes by
laboratory methods. In-vitro folliculogenesis entails harvesting mature oocytes in vitro
from frozen–thawed ovarian cortex by isolating small follicles (41) or oocytes (42)
from the surrounding stroma and growing them to maturity. Oocyte in-vitro
maturation (IVM) is currently feasible only in the latest stages of follicular
development and requires a lot of optimization before widespread clinical
implementation. Freshly aspirated GV stage (prophase I) oocytes can be matured
successfully in the laboratory to re-initiate and complete the first meiotic division to
metaphase II, including accompanying cytoplasmic maturation and fertilization. This
has resulted in the achievement of pregnancies and live births in polycystic ovary
syndrome (PCOS) patients (43) . It should be remembered, however, that the capacity
for oocyte maturation will be significantly lower when cryopreserved immature (GV
stage) oocytes are used. With the advancement in IVM of primordial follicles (44) ,
one could envisage the possibility of safely obtaining oocytes from cryopreserved
ovarian cortex. Oocytes competent for meiotic maturation, fertilization and
7
implantation could develop in vitro from primordial follicles. However, oocyte
development in vitro beginning with the primordial follicles is a complex and
prolonged process which includes follicular recruitment, tonic gonadotropin growth
and gonadotropin-dependent stages. The first live offspring produced proved that
development of oocytes in vitro from the primordial follicle stage is possible. A recent
revised protocol presents a significant advance in oocyte culture technology (45) .
Human Ovarian Transplantation Techniques
Although human data do not substantiate fertility resumption after ovarian
transplantation, animal data have demonstrated more success. Ovarian transplantation
has been tested with various animal models (25, 27, 46-48) . Xenotransplantation of
mouse ovarian cortex has enabled pregnancy (49) . The main obstacle to the wide
application of ovarian cortex cryopreservation is that the majority of transplanted
follicles are lost by ischemic damage by the time sufficient neoangiogenesis has
supplied the graft. A significant fraction of the follicles are lost, however, during the
ischemic phase present until neovascularization takes place (50, 51) . Ischemic
damage is more pronounced in frozen–thawed than in fresh grafts (52) . However, the
majority of ischemic damage observed in frozen–thawed avascular grafts occurs after
transplantation, confirming that grafting procedures are more deleterious for follicle
survival than cryopreservation (53) . Thus, optimal conditions have not yet been
established and the success of ovarian tissue transplantation depends on the ability of
the graft to support oocyte maturation and ovulation. It appears that ovarian slices can
convey only short-term function due to loss of most follicles by ischemia.
Ovarian transplantation preserving blood vessels could prevent ischemic
follicular loss. Lessons from nature demonstrate the feasibility of intact ovary
8
freezing. One such example is the Canadian wood-frog (Rana sylvatica) which
undergoes slow cooling and freezes for 6 months of winter (54) . Allografting
vascularized rabbit ovaries with their oviducts by microsurgical techniques resulted in
a vascular failure rate of only 5% of 40 grafts (55) . Intact ovarian freezing and
transplantation preserving blood vessels has been reported recently in rats (56) and
sheep (57, 58) . Until intact ovaries are successfully transplanted in humans, this
technique should also be considered an experimental one.
To our knowledge no pregnancy or embryo transfer was reported so far in
humans as a result of utilizing cryopreserved ovarian tissue. At present the clinical
experimental data showed the following results.
Oktay reported two cases of a forearm heterotopic ovarian transplantation
technique in two patients (59) . Both patients were menopausal immediately after
oophorectomy. One patient developed a dominant follicle 10 weeks after
transplantation, and her gonadotropin levels decreased to nonmenopausal levels.
Percutaneous aspiration of ovarian follicles yielded a metaphase I (M-I) oocyte that
was matured to metaphase II (M-II). The graft was functional for at least 21 months.
In the second patient, ovarian follicle development was detected 6 months after
transplantation, and periodic menstruation occurred thereafter. Spontaneous ovulation
was confirmed by a midluteal increase in her progesterone levels. Menstruation and
follicle development continued for more than 2 years after the transplant.
The same group recently reported a case where ovarian tissue was cryopreserved
for six year in a patient following chemotherapy and was transplanted beneath the
skin of her abdomen. Ovarian function returned in the patient 3 months after
transplantation, as shown by follicle development and estrogen production. The
9
patient underwent eight oocyte retrievals percutaneously and 20 oocytes were
retrieved. Of the eight oocytes suitable for in-vitro fertilization, one fertilized
normally and developed into a four-cell embryo (60) .
Ethical Aspects
The results of the attempts to achieve pregnancy by the use of ovarian tissue
banking as a method to preserve fertility in cancer treated female patients confirm that
this technique is still in its early stages. We believe that at this stage of experience and
outcome, OTCP should not be routinely proposed to patients as a treatment modality.
Cancer patients are a priori in a suboptimal health state and hence, the risk of invasive
surgical procedures for retrieving ovarian tissue may not be justified.
When applying this experimental approach women should be informed of the
current state of data concerning the success rates in order to prevent developing false
expectations and further disappointments.
Furthermore, it should be taken into consideration that ovarian transplantation
might be unsafe in some malignancies (e.g. acute leukemia) because of the risk of
ovarian involvement and the possible reseeding of malignant cells through the implant
On the other hand, other studies have shown that ovarian tissue harvested before high
dose chemotherapy for lymphomas may not carry a risk of disease transmission by
auto-transplantation (38, 39) . However, even in these low risk cases (e.g.
lymphomas), transmission of the malignant cells is difficult to exclude completely.
Thus, if auto-grafting is considered, testing for malignant cells in the tissue must be
performed using adequate techniques. The current techniques are still unequivocal
and inadequate and need further extensive research.
10
Studies on full organ cryopreservation (ovarian freezing and transplantation) are
in their first phase of research and are thus not applicable at present. This modality
may be a fertility conserving measure in the future.
Since OTCP is yet to be yielding and in the absence of other more promising
modalities, we believe that in order to preserve potential fertility in cancer patients,
they should be advised with one of the following:
Adult women with partners should be advised to undergo one cycle of ovarian
super-ovulation with subsequent IVF before chemotherapy or irradiation in order to
obtain embryos for cryopreservation. This strategy can be suggested to stable couples
in order to achieve embryos with the partner’s sperm. Frozen zygotes have an
acceptable rate of viability and are so far the only proven modality ensuring delayed
pregnancy. To our experience one cycle of ovarian super-ovulation, is adequately safe
and is equally acceptable by patients in most malignancies without significant
deleterious influence of the delay in chemotherapy or radiotherapy on patient’s
prognosis.
In single adolescent women donor sperm can be used for oocyte fertilization
and embryo cryopreservation. According to their desire and in the absence of any
clinical contraindication to get pregnant due to their malignancy, those single women
will have the option of embryo transfer. According to the present state of knowledge,
embryos can be cryopreserved and achieve pregnancy for several years (61) and in
some countries according to legislation or regulation even to ten years. Hence, women
can decide whether they are interested in the sustained cryopreservation and transfer
of these embryos.
11
In patients refusing to use donor sperm due to ethical, religious or traditional
attitudes, oocyte harvesting and cryopreservation might be attempted despite low
fertilization and pregnancy rates. This modality is still in its early experimental stages
and only few cases of pregnancies were achieved. At the present time, oocyte
cryopreservation is still more promising than OTCP in which no cases of pregnancies
were reported.
In pre-pubertal girls suffering from malignancies who are candidates for
chemotherapy or radiotherapy, a different approach for fertility preservation is
needed. In such girls, in whom ovulation induction can not be performed and mature
oocytes can not be achieved, the above mentioned recommendation for embryo
cryopreservation is impractical. The only option in this group is OTCP. If such a
recommendation is given, these patients and their parent’s ought to be informed that
this clinical approach is still in its early stages of experimentation and their chance to
preserve fertility can not be guaranteed according to present knowledge. A detailed
and up-to-date informed consent in such circumstances is crucial and mandatory.
In conclusion, we believe that OTCP should be used in experimental set up
and only when other modalities fail or cannot be implemented. Large scale human
transplantation studies should be investigated in order to improve their efficacy and
safety. Until then, OTCP should not be performed as routine therapeutic approach.
This is of utmost importance in order to avoid creating false expectations and
disappointments in these patients.
12
Table 1
Pediatric patients who face the risk of
ovarian failure due to cytotoxic treatment
•
•
•
•
•
•
•
•
13
Leukemias
Hodgkin’s lymphoma
Neuroblastoma
non- Hodgkin’s lymphoma
Wilm’s tumor
Ewing’s sarcoma
Osteosarcoma of the pelvis
genital Rhabdomyosarcoma.
References
1) Brenner H, Kaatsch P, Burkhardt-Hammer T, Harms DO, Schrappe M and
Michaelis J. Long-term survival of children with leukemia achieved by the end of the
second millennium. Cancer 2001;92:1977-83.
2) Jemal A, Murray T, Samuels A, Ghafoor A, Ward E and Thun MJ. Cancer
statistics, 2003. CA Cancer J Clin 2003;53:5-26.
3) Pui CH, Cheng C, Leung W, Rai SN, Rivera GK, Sandlund JT, et al. Extended
follow-up of long-term survivors of childhood acute lymphoblastic leukemia. N Engl
J Med 2003;349:640-9.
4) Robison LL and Bhatia S. Late-effects among survivors of leukaemia and
lymphoma during childhood and adolescence. Br J Haematol 2003;122:345-59.
5) Gougeon A. Dynamics of follicular growth in the human: a model from
preliminary results. Hum Reprod 1986;1:81-7.
6) Meirow D, Lewis H, Nugent D and Epstein M. Subclinical depletion of primordial
follicular reserve in mice treated with cyclophosphamide: clinical importance and
proposed accurate investigative tool. Hum Reprod 1999;14:1903-7.
7) Byrne J, Fears TR, Gail MH, Pee D, Connelly RR, Austin DF, et al. Early
menopause in long-term survivors of cancer during adolescence. Am J Obstet
Gynecol 1992;166:788-93.
8) Bacci G, Ferrari S, Mercuri M, Longhi A, Giacomini S, Forni C, et al. Multimodal
therapy for the treatment of nonmetastatic Ewing sarcoma of pelvis. J Pediatr
Hematol Oncol 2003;25:118-24.
9) Ozaki T, Flege S, Kevric M, Lindner N, Maas R, Delling G, et al. Osteosarcoma of
the pelvis: experience of the Cooperative Osteosarcoma Study Group. J Clin Oncol
2003;21:334-41.
10) Pisters PW, Ballo MT, Fenstermacher MJ, Feig BW, Hunt KK, Raymond KA, et
al. Phase I trial of preoperative concurrent doxorubicin and radiation therapy, surgical
resection, and intraoperative electron-beam radiation therapy for patients with
localized retroperitoneal sarcoma. J Clin Oncol 2003;21:3092-7.
11) Rodl RW, Hoffmann C, Gosheger G, Leidinger B, Jurgens H and Winkelmann W.
Ewing's sarcoma of the pelvis: combined surgery and radiotherapy treatment. J Surg
Oncol 2003;83:154-60.
12) Byrne J, Nicholson HS and Mulvihill JJ. Absence of birth defects in offspring of
women treated with dactinomycin [letter; comment]. N Engl J Med 1992;326:137.
13) Mertens AC, Ramsay NK, Kouris S and Neglia JP. Patterns of gonadal
dysfunction following bone marrow transplantation. Bone Marrow Transplant
1998;22:345-50.
14) ASRM/SART-registry. Assisted reproductive technology in the United States:
1999 results generated from the American Society for Reproductive Medicine/Society
for Assisted Reproductive Technology Registry. Fertil Steril 2002;78:918-31.
15) Ginsburg ES, Yanushpolsky EH and Jackson KV. In vitro fertilization for cancer
patients and survivors. Fertil Steril 2001;75:705-10.
14
16) Pal L, Leykin L, Schifren JL, Isaacson KB, Chang YC, Nikruil N, et al.
Malignancy may adversely influence the quality and behaviour of oocytes. Hum
Reprod 1998;13:1837-40.
17) Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;1:884-6.
18) Boldt J, Cline D and McLaughlin D. Human oocyte cryopreservation as an
adjunct to IVF-embryo transfer cycles. Hum Reprod 2003;18:1250-5.
19) Porcu E. Oocyte freezing. Semin Reprod Med 2001;19:221-30.
20) Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C. Human
oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod
2001;16:411-6.
21) Arav A, Zeron Y, Leslie SB, Behboodi E, Anderson GB and Crowe JH. Phase
transition temperature and chilling sensitivity of bovine oocytes. Cryobiology
1996;33:589-99.
22) Kuleshova L, Gianaroli L, Magli C, Ferraretti A and Trounson A. Birth following
vitrification of a small number of human oocytes: case report. Hum Reprod
1999;14:3077-9.
23) Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM, et al. Live births after
vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program.
Fertil Steril 2003;79:1323-6.
24) Katayama KP, Stehlik J, Kuwayama M, Kato O and Stehlik E. High survival rate
of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223-4.
25) Parrot DM. The fertility of mice with orthotopic ovarian grafts derived
from frozen tissue. J Reprod Fertil 1960;1:230 –41.
26) Carrol N and Gosden RG. Transplantation of frozen-thawed mouse primordial
follicles. Hum. Reprod. 1993;8:1163-67.
27) Gosden RG, Baird DT, Wade JC and Webb R. Restoration of fertility to
oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod
1994;9:597-603.
28) Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al.
Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediolsucrose as cryoprotectants. Hum Reprod 1996;11:1268-72.
29) Picton HM, Kim SS and Gosden RG. Cryopreservation of gonadal tissue and
cells. Br Med Bull 2000;56:603-15.
30) Donnez J, Godin PA, Qu J and Nisolle M. Gonadal cryopreservation in the young
patient with gynaecological malignancy. Curr Opin Obstet Gynecol 2000;12:1-9.
31) Hovatta O, Silye R, Abir R, Krausz T and Winston RM. Extracellular matrix
improves survival of both stored and fresh human primordial and primary ovarian
follicles in long-term culture. Hum Reprod 1997;12:1032-6.
32) Newton H. The cryopreservation of ovarian tissue as a strategy for preserving the
fertility of cancer patients. Hum Reprod Update 1998;4:237-47.
15
33) Meirow D, Fasouliotis SJ, Nugent D, Schenker JG, Gosden RG and Rutherford
AJ. A laparoscopic technique for obtaining ovarian cortical biopsy specimens for
fertility conservation in patients with cancer. Fertil Steril 1999;71:948-51.
34) Radford JA, Lieberman BA, Brison DR, Smith ARB, Russell SA, Watson AJ, et
al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose
chemotherapy for Hodgkin's lymphoma. Lancet 2001;357:1172-1175.
35) Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ and Gosden RG. Permeation
of human ovarian tissue with cryoprotective agents in preparation for
cryopreservation. Hum Reprod 1998;13:376-80.
36) Oktay K. Evidence for limiting ovarian tissue harvesting for the purpose of
transplantation to women younger than 40 years of age. J Clin Endocrinol Metab
2002;87:1907-8.
37) Shaw JM, Bowles J, Koopman P, Wood EC and Trounson AO. Fresh and
cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer
to graft recipients. Hum Reprod 1996;11:1668-73.
38) Meirow D, Ben Yehuda D, Prus D, Poliack A, Schenker JG, Rachmilewitz EA, et
al. Ovarian tissue banking in patients with Hodgkin's disease: is it safe? [editorial]
[see comments]. Fertil Steril 1998;69:996-8.
39) Kim SS, Radford J, Harris M, Varley J, Rutherford AJ, Lieberman B, et al.
Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for
autotransplantation. Hum Reprod 2001;16:2056-60.
40) Revel A and Laufer N. Protecting female fertility from cancer therapy. Mol Cell
Endocrinol 2002;187:83-91.
41) Smitz J and Cortvrindt R. Oocyte in-vitro maturation and follicle culture: current
clinical achievements and future directions. Hum Reprod 1999;14 Suppl 1:145-61.
42) Abir R, Franks S, Mobberley MA, Moore PA, Margara RA and Winston RM.
Mechanical isolation and in vitro growth of preantral and small antral human follicles.
Fertil Steril 1997;68:682-8.
43) Cha KY and Chian RC. Maturation in vitro of immature human oocytes for
clinical use. Hum Reprod Update 1998;4:103-20.
44) Vand Hur R, Abir R, Telfer EE and Bevers MM. Primate and bovine immature
oocytes and follicles as sources of fertilizable oocytes. Hum Reprod Update
2000;6:457-74.
45) O'Brien MJ, Pendola JK and Eppig JJ. A revised protocol for in vitro
development of mouse oocytes from primordial follicles dramatically improves their
developmental competence. Biol Reprod 2003;68:1682-6.
46) Harp R, Leibach J, Black J, Keldahl C and Karow A. Cryopreservation of murine
ovarian tissue. Cryobiology 1994;31:336-43.
47) Aubard Y, Newton H, Scheffer G and Gosden R. Conservation of the follicular
population in irradiated rats by the cryopreservation and orthotopic autografting of
ovarian tissue. Eur J Obstet Gynecol Reprod Biol 1998;79:83-7.
48) von Eye Corleta H, Corleta O, Capp E and Edelweiss MI. Subcutaneous
autologous ovarian transplantation in Wistar rats maintains hormone secretion. Fertil
Steril 1998;70:16-9.
16
49) Snow M, Cox SL, Jenkin G, Trounson A and Shaw J. Generation of live young
from xenografted mouse ovaries. Science 2002;297:2227.
50) Newton H, Aubard Y, Rutherford A, Sharma V and Gosden R. Low temperature
storage and grafting of human ovarian tissue. Hum Reprod 1996;11:1487-91.
51) Candy CJ, Wood MJ and Whittingham DG. Effect of cryoprotectants on the
survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997;110:11-9.
52) Nisolle M, Casanas-Roux F, Qu J, Motta P and Donnez J. Histologic and
ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in
nude mice. Fertil Steril 2000;74:122-9.
53) Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N and Driancourt MA.
Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum
Reprod 1999;14:2149-54.
54) Storey KB and Storey JM. Freeze tolerance and intolerance as strategies of winter
survival in terrestrially-hibernating amphibians. Comp Biochem Physiol A
1986;83:613-7.
55) Green CJ, Simpkin S, Grimaldi G and Johnson A. Pregnancy after autografting
and allografting vascularized ovaries and en bloc vascularized ovaries with adnexa in
rabbits. Br J Obstet Gynaecol 1982;89:645-51.
56) Wang X, Chen H, Yin H, Kim SS, Lin Tan S and Gosden RG. Fertility after intact
ovary transplantation. Nature 2002;415:385.
57) Revel A, Elami A, Bor A, Yavin S, Natan Y and Arav A. Whole ovary
cryopreservation and transplantation in sheep. Fertil Steril 2001;76:S42-43.
58) Jeremias E, Bedaiwy MA, Gurunluoglu R, Biscotti CV, Siemionow M and
Falcone T. Heterotopic autotransplantation of the ovary with microvascular
anastomosis: a novel surgical technique. Fertil Steril 2002;77:1278-82.
59) Oktay K, Buyuk E, Rosenwaks Z and Rucinski J. A technique for transplantation
of ovarian cortical strips to the forearm. Fertil Steril 2003;80:193-8.
60) Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, et al. Embryo
development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet
2004;363:837-40.
61) Revel A, Safran A, Laufer N, Lewin A, Reubinov BE and Simon A. Twin
delivery following 12 years of human embryo cryopreservation: case report. Hum
Reprod 2004;19:328-9.
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