Fertility Preservation for Pre-Pubertal Girls and Young Female Cancer Patients

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Fertility Preservation for Pre-Pubertal
Girls and Young Female Cancer Patients
R. Gerritse1,2, L. Bastings1,3, C.C.M. Beerendonk1,
J.R. Westphal1, D.D.M. Braat1 and R. Peek1,4
1Radboud University Nijmegen Medical Centre,
Department Obstetrics and Gynaecology Nijmegen,
2Koningin Beatrix Ziekenhuis Winterswijk, The Netherlands,
3Jeroen Bosch Hospital ‘s Hertogenbosch,
The Netherlands
1. Introduction
New protocols in the early diagnosis and treatment of cancer have led to major
improvements in the long-term survival of patients. However, aggressive chemotherapy or
radiotherapy of the pelvic region, often lead to infertility, due to the damage of the follicles
and/or oocytes that are present in the ovaries. In women the probability of sterilization due
to cancer therapy varies with age, the type of treatment, and the follicular reserve in the
ovary. Safeguarding their reproductive potential is a very important issue for women that
have not yet started or completed their family, and even more so in pre-pubertal girls.
Several options, some of which are still in the experimental phase, can now be offered to
these women to (partially) preserve their fertility.
In this review, we will, after briefly describing the anatomy and physiology of an ovary,
discuss the detrimental effects of chemotherapy and radiation on ovarian function.
Subsequently, the various options that are currently available or are still in an experimental
phase, for preserving fertility in women and pre-pubertal girls, will be discussed. These
options (with the exception of option (i)), deal with cryopreserving either oocytes, embryos
or ovarian tissue until the patient has been cured.
i. Minimizing the effects of radiation of the inner pelvic region by transposing the ovaries
from the radiation area.
ii. Standard IVF procedures can be offered to women who are awaiting chemotherapy and
radiotherapy for neoplastic disease. This procedure results in the generation of embryos
that can be transferred after recovery of the disease. This option has its limitations, since
it not only requires the presence of a male partner, but also delays cancer treatment
during ovarian stimulation. In addition, the number of embryos that can be produced is
restricted, and the chance of achieving a pregnancy after transfer of a cryo-preserved
embryo is only 8-30%. Furthermore, the presence of estrogen-sensitive tumors is a
contra-indication for this type of treatment, as high estradiol levels are induced during
a normal IVF procedure, although alternative stimulation protocols with aromatase-
inhibitors are nowadays available for these specific patients. Most importantly, this

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treatment is not an option for pre-pubertal girls, or for post-pubertal girls who are not
yet involved in a stable relationship.
iii. Aspiration of oocytes, followed by cryopreservation and IVF (if necessary preceded by
in vitro maturation). This option has already been applied to a number of patients.
Although the same drawbacks that apply to standard IVF are applicable, this procedure
is mainly aimed at the treatment of post-pubertal girls/young women without a stable
relationship. Only limited scientific data are currently available to substantiate its
efficacy and long-term safety.
iv. As an alternative, cryopreservation of small ovarian cortex strips containing primordial
follicles can be offered. After the patient has been cured, these cortex strips can
subsequently be retransplanted either heterotopically or orthotopically. This procedure
has been successfully used to re-establish female fertility in humans in a limited
number of cases. A major problem with these avascular implants however is their
relative short life expectancy and follicular loss due to long term ischemic injury
directly after reimplantation.
v. Cryopreservation and subsequent reimplantation of intact ovaries may be a valuable
addition to the existing array of options, especially for pre-pubertal girls and post-
pubertal girls/women without a stable relationship. An important safety issue of this
procedure is obviously the chance of reintroduction of malignant cells that may be
present in the cryopreserved intact ovary. For this reason, patients with solid types of
tumor and diffuse types of cancer such as leukemia that have a high chance of
metastasizing to the ovaries, will have to be excluded from this kind of therapy. The
cryobiological and surgical aspects of the preservation and retransplantation of an
organ in toto, is technically clearly more challenging than the cryopreservation and
transfer of isolated cells or tissue strips. The advantages of this approach are obvious;
immediate revascularization of the transplanted ovary ensures that less ischemic
damage is inflicted to the ovarian tissue post-thawing, and that more follicles will
survive. In addition menses, normal long term reproductive functions, and normal
hormonal status will be restored.
Finally, we will go into the safety of the procedure. Inevitably the autotransplantation of
cortical strips or intact ovaries carries the risk of reintroducing malignant cells from the graft
into the recipient.
The increase in knowledge of the biology and treatment of cancer has been accompanied by
an increase in the efficacy of cancer therapies. Long term survival rates for many cancer
types have therefore increased accordingly (Gatta et al., 2009). Consequently, the quality of
life of cancer survivors is becoming an important issue.
The possibility to have genetically concordant progeny is for many people an event that is
essential for an unrestricted quality of life as an adult (Schover, 2009). The loss of fertility
that may result from cancer therapy, is therefore an additional complication on top of an
already difficult period spent on conquering a devastating disease.
With this in mind, it is of the utmost importance to explore the possibilities for fertility
preservation in patients that are to be treated with a gonado-toxic therapy. For post-pubertal
boys and men, this can be achieved relatively easy via the cryopreservation of their semen
prior to start of the therapy. For pre-pubertal boys this is not an option, as semen production
is initiated during puberty. Also for this group of patients options for fertility preservation
are being developed.

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In this paper we confine ourselves to fertility preservation for female patients. We discuss
the causes of anti-cancer therapy-related infertility, and review the current options for
fertility preservation. We illustrate this matter with two case reports from our own clinical
practice. In addition we discuss some as yet experimental procedures, that may in the future
be offered to patients requiring fertility preservation.
2. Ovaries, oocytes and female reproduction
The human ovary is spherical structure with a mean volume of 7 cm3 (range 2-15 cm3; Munn
et al., 1986). The inner ovarian mass, the medulla, consists mainly of stromal cells and
contains the larger blood vessels. The outer layer of the ovary consists of the cortical tissue,
spanning 2-3 mm. This tissue is rich in extra-cellular matrix proteins and poor in capillaries ,
and contains the vast majority of the follicles containing oocytes that comprise the ovarian
reserve. The most important role of the follicle is to protect the oocyte, and support its
development. Follicles are comprised of layer(s) of theca cells and granulosa cells. Different
stages of follicles can be distinguished, ranging from primordial follicles to primary follicles,
and via secondary finally to tertiary (antral) follicles.
In contrast to males in whom spermatogenesis is a continuous process resulting in the
uninterrupted generation of fresh spermatozoa, in women a fixed number of oocytes is formed
during embryogenesis from 1000-2000 germ cells. These germ cells are present in the human
embryo at 30 days after conception. After 9-10 weeks, these cells transform to oogonia (Baker,
1972), that degenerate for the greater part between 10 and 20 weeks of gestation. After 5
months of gestation, the first meiotic division is initiated in the remaining oogonia, resulting in
the differentiation to primary oocytes. At this stage the meiotic division process is arrested,
and the oocytes enter a stage of dormancy (Wandji, 1996). At birth only 300.000 to 400.000
oocytes remain in the ovaries. From birth, the number of oocytes gradually decreases, and at
the beginning of puberty around 200.000 oocytes remain. Under the influence of pituitary
gonadotropic hormones (Gougeon, 1996; Oktay, 1997), each month a cohort of primary oocytes
is recruited, and resumes development. Usually only one primary oocyt completes the first
meiotic division. This secondary oocyt again enters a stage of dormancy, and is ovulated. The
second dormant stage is only lifted after fertilization by a sperm cell. Around the age of 50
years, the total oocyte reserve is almost depleted and the woman enters menopause. In
addition to age, several factors may affect the follicular reserve, leading to an early exhaustion
and to premature ovarian insufficiency (POI). These factors include fertility-threatening
therapies that are discussed in more detail in the next section.
3. Effects of radio- and chemotherapy on female fertility
3.1 Chemotherapy
Cytotoxic therapy may affect all components of the follicle, including granulosa cells, theca
cells, and of course the oocyt itself (Sobrinho et al., 1971; Blumenfeld et al., 1999). In
addition, interactions between these cell types that are required for oocyt development may
be disturbed, resulting in the demise of the oocyt. Damage may become manifest by reduced
ovarian weight, stromal fibrosis and in a reduction in the number of oocytes and ovarian
follicles (Warne et al., 1973; Meirow et al., 1999; Oktem & Oktay, 2007).
The effect of chemotherapy on fertility is dependent on the type of the cytotoxic agent, the
dose, and the duration of the therapy. Alkylating agents such as cyclophosphamide, L-

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phenyalanine mustard, and chlorambucil permanently damage ovarian tissue by interacting
with DNA (Meirow et al., 1999; Manger et al., 2006; Oktem & Oktay, 2007). Analysis of a group
of 138 young females receiving the alkylating agent busulfan as a preparative regimen for
indicated that 83% of these women showed signs of fertility impairment, demonstrating the
potentially very severe effects of this type of compounds (Borgmann-Staudt et al., 2011).
Mertens et al. (1998) showed an even higher percentage of 99% in gonadal dysfunction for
women receiving allogeneic haematopoietic stem cell transplantation. The cumulative dose of
the cytotoxic drug being administered is an important factor in determining the level of
ovarian insufficiency (Goldhirsch et al., 1990). Permanent ovarian insufficiency was more often
induced when high dosages of drugs were administered during a short period of time,
compared to low doses given over a longer time (Koyama et al., 1977).
In addition the age of the patient is pivotal in determining the amount of damage that is
inflicted to the ovary. Older women, with an already decreased number of primordial
follicles, have a higher risk of developing acute complete POI, compared with young
women who still possess numerous primordial follicles (Schilsky et al., 1981; Sanders et al.,
1996; Tauchmanova et al., 2002). Prepubertal girls seem less vulnerable to cytotoxic drugs
than adults (Chiarelli et al., 1990). This may be explained by the fact that several
chemotherapeutical drugs affect DNA replication and/or RNA and protein synthesis, and
are therefore targeted at metabolically active cells. In prepubertal ovaries all follicles are in a
dormant, metabolically quiescent state, and therefore less prone to chemotherapy induced
damage. In contrast, in adult ovaries a number of follicles will be in an active state, and
therefore more prone to chemotherapy induced damage. Nicosia et al. (1985) actually
showed in ovarian autopsy material derived from patients having received chemotherapy,
that the number of growing follicles was reduced, whereas the number of primordial
follicles remained the same.
3.2 Radiotherapy
Similar to the effects of chemotherapeutical agents on DNA integrity, ionizing radiation,
amongst other effects, also interferes with DNA function. As a consequence, also
radiotherapy may negatively affect the ovarian reserve. Analogous to chemotherapy, the
(cumulative) dose and the fractionation schedule determine the degree of damage to the
ovary (Gosden et al., 1997). The human oocyte is exceptionally sensitive to radiation (Howell
& Shalet, 1998) and the estimate of the LD50 ( the lethal dose need to kill half the total
number of oocytes) seems to be less than 2 Gy (Wallace et al., 2003). Also for radiation
therapy, the age of the patient is an important factor in determining the level of damage. A
dose of 4 Gy leads to sterility in 30% of young women, and in 100% of women over 40.
Not surprisingly, the combination of radiotherapy with chemotherapy increases the risk of
POI (Williams et al., 1999; Wallace et al., 2005; Chemaitilly et al., 2006). Abdominal
radiotherapy in combination with alkylating agents increased the risk of POI 27-fold (Byrne
et al., 1992). By the age of 31, 42% of patients treated with this combination therapy, was
postmenopausal, compared with 5% of women in the normal population.
3.3 Effects on pregnancy and health of newborns
In addition to their effects on oocytes and follicles, chemotherapy and radiotherapy may
also influence uterine function. Radiation may lead to impaired uterine growth in
premenarchal girls and failure of uterine development during pregnancy, leading to

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miscarriages, premature births and intrauterine growth retardation (Ogilvy-Stuart et al.,
1997; Critchley, 1999; Critchley et al., 1992; 2002; Wallace et al., 2005). Comparable results
were described by Salooja et al. (2001), who showed that in women that had received total
body irradiation prior to autologous or allogeneic stem cell transplantation, are at high risk
for maternal and fetal complications. These problems are probably a consequence of uterine
vascular damage and reduced elasticity of the uterine musculature.
4. Current options for fertility preservation
4.1 Ovarian transposition
An way to prevent damage to the ovaries caused by ionizing radiation therapy applied to
the pelvic region, is to surgically move the ovary temporarily to a location outside the field
of radiation (Hadar et al., 1994; Howard, 1997). This procedure, referred to as oophoropexy,
can be performed laparoscopically. Potential ovarian insufficiency following transposition
may occur if the ovaries are not entirely moved outside the field of radiation, or when they
spontaneously migrate back to their original position. Ovarian failure can also occur when
the ovarian vascular pedicle has been compromised by the surgical procedure (Feeney et al.,
1995). Oophoropexy is a safe and effective procedure, allowing preservation of ovarian
function in 80% of cases (Bisharah & Tulandi, 2003).
4.2 Vitrification of oocytes
Cryopreservation of mature or immature oocytes is an obvious approach to preserve
fertility. As no fertilization of the oocytes is yet required, this is option is especially suitable
for women without a partner. The collection of mature oocytes requires stimulation with
follicle stimulating hormone (FSH). This procedure, that may have to be repeated to obtain a
sufficient number of oocytes, takes at least two weeks, and is therefore only suitable for
women for whom it is safe to postpone their cancer treatment. The use of high doses of FSH
makes this option unsuited for women with oestradiol-sensitive breast tumors, as high
levels of oestradiol are induced by the FSH treatment (Sonmezer & Oktay, 2006) This caveat
may be circumvented by the simultaneous use of aromatase inhibitors /anti oestrogens such
as letrozole or tamoxifen (Oktay et al., 2005b; Sonmezer & Oktay, 2006). Alternatively,
immature oocytes can be collected without prior stimulation. This procedure may also be
used for young (prepubertal girls). Evidently, these immature oocytes must be matured in
vitro (IVM) before they can be fertilised (Gosden, 2005).
After collection of the oocytes, they have to be cryopreserved in liquid nitrogen for long
term storage. The formation of ice crystals during the freezing process may severely damage
the oocyte, rendering it useless for further use. This is especially the case for mature oocytes,
as they possess a fragile and sensitive meiotic spindle. Immature oocytes are in this respect
less sensitive. Cryodamage can be prevented by freezing the oocytes in the presence of
cryoprotective agents via specific protocols, either by slow freezing, or via vitrification (Cao
et al., 2009; Chian et al., 2009; Kuwayama et al., 2005). During the latter procedure, that
appears to result in more oocytes surviving the process undamaged, the oocytes are frozen
extremely rapidly (> 12.000 °C/minute), in the presence of high concentrations of
cryoprotectant, resulting in the prevention of ice crystal formation.
A consequence of the cryopreservation procedure (either slow freezing or vitrification) is
hardening of the zona pellucida. Therefore, cryopreserved oocytes can only be fertilized via

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intracytoplasmatic sperm injection (ICSI). As the pregnancy rate per cryopreserved oocyte is
approximately 3% (Kuwayama et al., 2005; Cobo et al., 2007; Homburg et al., 2009), a large
number of oocytes, equivalent to several stimulation cycles and/or oocyte retrieval
procedures, are required to achieve a reasonable chance of progeny. The exact number of
children conceived with cryopreserved oocytes is unknown, but it is estimated to be over
500 worldwide. Postnatal parameters such as birth weight and incidence of congenital
anomalies, were comparable with the reference population, indicating the safety of this
procedure (Borini et al., 2007; Chian et al., 2008).
4.3 Cryopreservation of embryos
For women with a partner, the generation and cryopreservation of embryos is a suitable
option. Obviously, this option will generally require ovarian stimulation, and is therefore
subject to the same limitations as mentioned previously for the collection and
cryopreservation of mature oocytes – the cancer treatment has to be postponed to allow for
one or more ovarian stimulation(s), and extreme caution has to be taken when stimulating
women with hormone sensitive tumors. The factor time may be circumvented by skipping
the stimulation with FSH and collect immature oocytes instead. Evidently, in that case IVM
has to be performed prior to fertilisation of the oocytes. Employing tamoxifen or letrozole
based stimulation regimes may be used in the case of hormone sensitive tumors (see
previous paragraph) (Oktay et al., 2005a, 2005b; Sonmezer & Oktay, 2006). Oktay et al.
(2005b) has shown that in cancer patients who had been stimulated with this compound,
recurrence rates were not elevated compared to cancer patients who had not been receiving
any ovarian stimulation. Although we should keep in time that the follow up period was
confined to only a limited number of years.
Theoretically, in women with hormone sensitive tumors oocytes can also be collected in a
spontaneous (non-stimulated) cycle. However, the very limited number of oocytes that can
be collected this way (one or two per cycle) makes this a very inefficient option and is
therefore not advisable (Brown et al., 1996).
Embryo cryopreservation is an established and efficient technique, with reported implantation
rates per thawed embryo between 8 and 30% (Frederick et al., 1995; Selick et al., 1995; Wang et
al., 2001; Son et al., 2002; Senn et al., 2006), that has resulted in the birth of tens of thousands of
children worldwide. In the future, new cryopreservation techniques such as vitrification may
further improve the efficiency of this technique (Kuwayame et al., 2005).
4.3.1 Case report A: Emergency IVF in a patient with breast cancer
Mrs. X was diagnosed 3 years ago with breast cancer. She then underwent a lumpectomy of the right
breast, and received radiotherapy. Shortly thereafter a unilateral recurrence was found, and a
mastectomy with lymph node dissection was performed. Pathologic examination revealed an invasive
ductal carcinoma, positive for estrogen and progesterone receptors. No tumor cells were found in the
lymph nodes, and no other indications for metastatic disease were found. Additional chemotherapy
courses were planned.
At this stage the patient, now 35 years of age, and her partner visited the Centre for Reproductive
Medicine of our hospital, and expressed their interest in fertility preservation. After establishing that
the current reproductive status of herself as well as of her partner showed no abnormalities, the
possibilities for fertility preservation were discussed. Although oocyte vitrification and ovarian tissue
banking were in theory viable options, the couple was counseled to proceed with an emergency IVF-

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ICSI attempt, followed by cryopreservation of the embryos, as this would probably give the highest
chance of progeny within the time limit set by the oncologist.
The ovarian stimulation protocol was started one month after the mastectomy. Regarding the
hormone receptor positive status of the tumor, a regimen combining FSH and letrozol was selected in
order to avoid the high oestradiol levels associated with ovarian hyperstimulation. The treatment
eventually resulted in the retrieval of 13 oocytes, 12 of which could be inseminated via ICSI. Of the 7
resulting embryos, 3 were eligible for cryopreservation. The efficacy of the letrozol treatment was
demonstrated by the finding that during the stimulation with FSH, oestradiol levels did not rise
beyond 1000 pmol/L.
The patient than completed 5 cycles of chemotherapy. In addition, she received adjuvant hormonal
therapy. Menses had stopped and the patient suffered from hot flushes. Two years later at age 37, the
patient wanted to achieve pregnancy. After discontinuation of medication, the hot flushes diminished
and menses did resume. Five months later the patient conceived spontaneously, but unfortunately the
pregnancy ended in an abortion. As further spontaneous conceptions did not occur, two cycles of
fresh IVF were performed. Although the second cycle resulted in a pregnancy, this again ended in an
abortion. The patient is now being prepared to receive the embryos that were cryopreserved prior to
the start of her chemotherapy.
4.4 Cryopreservation of ovarian cortical tissue strips
As mentioned previously, each fertility preservation option is aimed at a specific group of
patients. When the patient is prepubertal, when there is no partner is available for the
generation of embryos, or when the cancer treatment cannot be postponed in order to
perform ovarian stimulation, cryopreservation of ovarian tissue strips may be an alternative
approach. Silber et al. (2005) showed previously that transplantation of fresh (non
cryopreserved) ovarian cortex strips between identical twin sisters was actually feasible. The
development of efficient freezing and thawing protocols for cortex strips has rendered this
technique applicable for fertility preservation purposes and has recently led to the thirteenth
live birth (Donnez et al., 2011).
Although still experimental, this option is nowadays being performed on an increasing
scale. Cortical fragments can be obtained laparoscopically, and slow frozen using DMSO as
a cryoprotectant. Care should be taken to minimize the thickness of the cortical strips to 1
mm, to facilitate diffusion of the cryoprotectant into the tissue. In addition, thin fragments
will suffer less from ischemic damage, which is a serious problem after retransplantation. A
significant proportion (60-95%) of (growing) follicles that survive the freezing and thawing
process, is actually lost due to warm posttransplantation ischemia (Baird et al., 1999; Nisolle
et al., 2000; Candy et al., 2000; Aubard et al., 1999, Aubard, 2003; Liu et al., 2002). Cortical
strips can be autotransplanted heterotopically (for instance subcutaneous in the forearm), or
orthotopically. Thus far, only orthotopic transplantation has led to the birth of a number of
healthy offspring (Donnez et al.,2004; Meirow et al.,2005, 2007; Demeestere et al., 2006; 2007;
2010; Andersen et al.,2008; Schmidt et al., 2011; Ernst et al., 2010).
Cryopreservation of ovarian cortical strips is applicable for a wide range of different
patients. Conception may require artificial reproductive techniques like IVF or ICSI, but may
occur spontaneously as well. An additional advantage of this technique is resumption of the
regular hormonal processes, leading to the reversal of the postmenopausal status that many
patients experience after their cancer therapy. Follicular development and restoration of
ovarian function usually occur 4-5 months after a transplantation procedure ( Donnez et al.,
2006a; 2008), as more than 120 days are required to initiate follicular growth and

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approximately 85 days to the reach final maturation stage from a pre-antral follicle
(Gougeon, 1985, Oktem & Oktay, 2008). Unfortunately, the survival time of a single
autotransplanted number of strips is usually limited to a few months, with exceptions of
survival up to approximately 3 years (Kim et al., 2009; Meirow et al., 2007; Silber et al.,
2008a), requiring another surgical intervention to transplant a new set of cortex strips.
4.4.1 Case report B: Ovarian tissue cryopreservation in a patient with Hodgkin’s
lymphoma
Mrs. Y was diagnosed with Hodgkin’s lymphoma at the age of twenty. As she was to start with six
cycles of chemotherapy the next month, she visited our fertility Centre to discuss the options for
fertility preservation.
Although the patient was at the time in a steady relationship, she regarded herself to young to start
emergency IVF, as this would confront both herself and her partner with the definitive choice of
having children together in the future. Ovarian hyper stimulation followed by cryopreservation of the
retrieved oocytes was not considered an optimal option, as the time to the start of her chemotherapy
was relatively short, allowing for only one cycle of hyperstimulation. As a consequence, only a limited
number of oocytes would be obtained.
Eventually the choice was made for cryopreservation of ovarian cortical strips. At that time we could
not offer her this procedure ourselves so we referred her to another centre. Biopsies of both ovaries
were taken via a laparoscopic procedure, and 13 strips were cryopreserved. She then started with the
chemotherapy. Since then, she has had two relapses, that were treated with chemotherapy,
radiotherapy, and stem cell transplantation.
At the age of 27, the patient and her partner visited our Centre as she wished to conceive. She had
now been in complete remission for 3 years. Hormonal examination showed that she was
postmenopausal, indicating that both spontaneous conception as well as IVF treatments were no
options to achieve pregnancy. The couple was referred back to the clinic where her ovarian tissue was
cryopreserved, and is now considering autotransplantation of the ovarian cortical strips.
5. Future options for fertility preservation
Several alternative procedures are being evaluated to expand the current array of fertility
preservation options. These include the isolation and cryopreservation of follicles from
ovarian tissue that is harvested laparoscopically (Bedaiwy & Falcone, 2007; Feigin et al.,
2007). However, isolation of follicles by either mechanical or enzymatic means is difficult,
especially from human ovaries (Dolmans et al., 2006). In addition, this approach requires
different cryopreservation techniques then for oocytes and embryos, and sophisticated in
vitro maturation protocols to obtain oocytes that can be fertilized in vitro by IVF or ICSI.
A more promising future option may comprise the cryopreservation of an intact ovary,
including its vascular pedicle. The vascular pedicle can be used to reconnect the thawed
ovary to the circulation, thereby preventing the devastating effects of warm ischemia that is
known to deplete the follicles in ovarian tissue transplanted without vascular anastomosis
(Newton et al., 1996; Nisolle et al., 2000; Candy et al., 1997; Aubard et al., 1999; Baird et al.,
1999; Aubard, 2003; Liu et al., 2008 ). However, the successful cryopreservation of an intact
organ represents an immense technical challenge. Pioneering work by Parrot (1960) on
murine ovaries provided proof of principle. Later reports showed that also in other
mammalian species this proved to be a viable approach. Freezing and autologous grafting of
whole ovaries has now been performed in rabbits (Chen et al., 2005), pigs (Imhof et al.,

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2004), and sheep (Bedaiwy et al., 2003; Arav et al., 2005; Imhof et al., 2006), yielding
promising results. In rats (Wang et al., 2002) and sheep (Imhof et al., 2006), this procedure
has actually resulted in live offspring. In humans, transplantation of fresh (non-
cryopreserved) intact ovaries has also been performed successfully. Ovarian
autotransplantation in the upper arm was performed before pelvic irradiation (Leporrier
1987, Hilders et al., 2004). Over a period of 16 years, the ovary remained functional
(Leporrier et al., 2002). A first full-term pregnancy was obtained using orthotopic fresh
whole ovary transplantation between identical twin sisters (Silber et al., 2008b).
Cryopreservation of an intact human ovary with its vascular pedicle has been described
previously (Martinez-Madrid et al., 2004, 2007; Bedaiwy et al., 2006). These authors showed
that perfusion of the ovary with cryoprotectants led to a certain degree of protection from
cryodamage. The subsequent autotransplantation of frozen and thawed human ovaries,
however, has thus far not been performed. Major obstacle in this respect is the much larger
volume of human ovaries compared to murine and ovine ovaries (Gerritse et al., 2008). This
larger volumes hampers the sufficient diffusion of cryoprotectant into the tissue (Donnez et
al., 2006b). In addition, the freezing kinetics in a bulky organ are bound to be completely
different from those in a small volume organ (Pegg, 2005). Finally, all components of the
organ, including the vascular pedicle, the inner vasculature, the stromal tissue and of course
the follicles, should be verifiably protected before retransplantation to human subjects can
be even considered. This requires the development of biologically relevant assays that are
able to quantify cryodamage in a reliable fashion. Understandably, efforts have focused
mainly on the survival of the follicles within the intact cryopreserved ovary. This has been
done by conventional histology (Bedaiwy et al., 2003, Arav et al., 2005 Courbiere et al., 2005,
2006; Martinez-Madrid et al., 2004; Imhof et al., 2006; Baudot 2007), immunohistochemistry
(Arav et al., 2005; Bedaiwy et al., 2006), determining the frequency of apoptosis (Bedaiwy et
al., 2003, 2006; Martinez-Madrid et al., 2007), using survival/viability/proliferation assays
(Bedaiwy et al., 2003, 2006; Martinez-Madrid et al., 2004, Arav et al., 2005, Courbiere et al.,
2005, 2006; Imhof et al., 2004; Baudot et al., 2007, Onions et al., 2008), transmission electron
microscopy (Martinez-Madrid et al., 2007) and estradiol assays (Huang et al., 2008;
Isachenko et al., 2007; Gerritse et al., 2010). These studies have produced relevant
information on the prevention of cryodamage in follicles, but have largely left out the main
component of the ovary, namely the stromal cell compartment that constitutes over 95% of
the ovarian mass. An additional reason to focus also on survival of stromal cells is the
observation that these cells are vital for optimal follicular development (McLaughlin and
McIver, 2009). Finally, the metabolically active stromal cells have been described to be more
sensitive to cryodamage than the quiescent primordial oocytes (Kim et al., 2004). These
observations emphasize the need for a cryopreservation protocol that not only efficiently
preserves the follicles/oocytes, but the stromal cell compartment as well.
We therefore decided to develop an assay that is capable of quantifying the basal
metabolism of the bulk of the tissue as a measure of cryodamage. For this purpose we
measured the uptake of glucose and the release of lacate by cultured ovarian tissue
fragments. We used bovine ovaries as a model system, as they are comparable to human
ovaries with respect to size, monthly cycle, and number of follicles that mature per cycle
(Gerritse et al., 2008). In this model system we were able to test different cryopreservation
protocols. Our results show that both immersion of the bovine ovary in cryoprotectant,
combined with perfusing it for a prolonged period of time, resulted in a nearly complete

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protection of the ovarian metabolism. This procedure did not affect the endothelium of the
vascular pedicle and the inner vasculature (Gerritse et al., submitted). We plan to
xenotransplant optimally cryopreserved bovine ovaries into immune deficient rats, in order
to test the ability of the follicles to develop in vivo and produce mature oocytes.
6. Safety aspects of ovarian tissue autotransplantation
A major point of concern when autotransplanting ovarian tissue to cured cancer patients, is
the possibility that (metastasized) tumor cells are present in the ovarian graft and are
reintroduced to the patient (Shaw et al., 1996). Thus far a limited number of patients has
received an autransplantation, and up to now no relapses have been reported. It should be
noted, however, that most patients receiving an autotransplantation suffered from early
stage cancer when their tissue was harvested. In addition, the follow up period after the
transplantation has been relatively short. As a consequence, the experience with this matter
is only limited, and retransplantation of the malignancy can never be ruled out completely.
Shaw et al. (1996) actually showed that lymphoma could be transmitted via cryopreserved
ovarian tissue in a mouse model. The physician therefore has the responsibility to counsel
the patient comprehensively on the risk of malignant cells being present in the ovarian
tissue, and the possible consequences after autotransplantation. Two different approaches
can be used to draft an advice.First, one can extrapolate on statistical data describing the
frequency with which a certain tumor in a certain stage will metastasize to the ovary. For a
number of solid tumor types, ovarian metastases have been described for advanced stages
but not for early stage tumors (Rosendahl et al., 2011). These include Hodgkin’s disease
(Khan et al., 1986), renal cell carcinoma (Insabato et al., 2003) and breast cancer (Horvath et
al., 1977). It should be noted, however, that systematically collected data are missing for
most tumor types, giving only a limited idea of the risk of tumor dissemination to the ovary.
In contrast to solid tumors, diffuse malignancies such as leukemia are likely to be present in
all blood-filled organs, including the ovary. Therefore, patients suffering from these kind of
diseases should probably be excluded from using (cryopreserved) ovarian tissue as a means
for fertility preservation. The second, and probably preferable, option, is to tailor a patient
specific approach, i.e. analyzing (part of) the tissue that is to be autotransplanted in the
future for the presence of (residual) disease. In an ideal situation, sensitive and specific tests
would be available for the detection of each tumor type in a tissue. Techniques that have
been used to asses the presence of malignant cells in ovarian tissue include conventional
histology (Azem et al., 2010; Donnez et al., 2011), immunohistochemistry (Rosendahl et al.,
2011), PCR amplification of tumor specific RNA/DNA (Rosendahl et al., 2010), and
xenotransplantation of ovarian tissue fragments to immune deficient mice (Dolmans et al.,
2010). In real life, however, these approaches encounter several obstacles. Histology is
relatively non-sensitive as individual tumor cells can be missed, and usually only a limited
number of sections is analyzed. Whereas immunohistochemistry is generally more sensitive
than histology, it requires specific tumor cell markers that are not available for most type of
cancers. While PCR in itself is a very sensitive technique, the ratio between the few
malignant cells that are potentially present in the graft and the large number of normal
ovarian cells impairs the reliability and sensitivity of this test. PCR results that indicate the
presence of residual tumor cells are therefore mostly qualitative and not quantitative.
Furthermore, PCR detects only the presence of relatively short stretches of specific
RNA/DNA sequences and not viable cells. Xenotransplantation experiments may provide

Fertility Preservation for Pre-Pubertal Girls and Young Female Cancer Patients
205
biologically relevant information, but are expensive and cumbersome and probablyl not
routinely applicable. Apart from these practical issues, positive tests results raise some more
questions. First, we do not know when a positive signal becomes biologically significant,. i.e.
predictive of relapse after transplantation. Examples of this notion are a positive PCR signal
that may be derived from a number of deceased cells and may therefore not be of clinical
relevance. We currently we do not know exactly how many malignant cells are required for
reintroduction of the tumor. In animal models as few as 200 lymphoblasts were sufficient to
introduce leukemia (Hou et al., 2007), but the same may not apply to the human situation.
Next, the ovarian tissue fragment that is being analyzed for residual disease, is evidently no
longer available for transplantation. The outcome of the analysis will therefore not
necessarily be applicable to the cortical fragments that are actually transplanted. The
importance of this notion was substantiated by the finding that malignant cell DNA was
found in an ovarian cortex fragment by PCR analysis, whereas the adjacent cortex fragment
from the same ovary was found to be PCR-negative (Rosendahl, 2010). Finally, the
autotransplantation of small volume cortex fragments is much less likely to reintroduce the
malignancy then the autotransplantation of an intact ovary.
7. Concluding remarks
The last decade has seen the development of a number of options for fertility preservation for
cancer patients. All the options that are currently available have their own specific indications
and contraindications. The choice for the appropriate option will be a shared decision of both
the patient and her physician, requiring a careful evaluation and counseling. Increasing the
awareness of physicians to address the issue of fertility preservation before starting
gonadotoxic therapy should be an integral part of medical education.
Current research, including intact ovary cryopreservation, may lead to several exciting new
options for fertility preservation. It should be noted that this option is not intended to
replace the current possibilities, but will rather have its own specific patient population that
may benefit most from this procedure. The obvious risk of intact ovary autotransplantation
is reintroduction of the malignancy. Evidently, more research into the development of valid
and biologically relevant tumor detection methods in ovarian tissue, as well as in the
prevalence of ovarian metastases in cancer patients with different types of primary tumors,
is urgently needed.
8. Acknowledgements
The authors would like to thank the Kika Foundation and Stichting Pink Ribbon for their
financial support. MSD/N.V. Organon is acknowledged for providing an unconditional grant.
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