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REVIEW

Strategies for fertility preservation and gonadal protectionduring gonadotoxic chemotherapy and radiotherapy

Mohamed A. Bedaiwy, M.D.

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, and Mount Sinai Hospital, ON, Canada.

ABSTRACT

With the recent report of a pregnancy and delivery after autotransplantation of cryopreserved-thawed ovariancortical strips, preservation of the reproductive potential resurfaced. There is a growing academic and public interest in

exploring the available strategies for fertility preservation in patients at risk. This is due to the increasing incidence of

cancer during the reproductive age. The overall survival and cure rates of reproductive age cancers are improving due to

improvements in cancer therapy. Reproductive derangement is one of the major consequences of cytotoxic

chemotherapy and radiotherapy. GnRh analogues concomitant therapy, laparoscopic ovarian transposition, oocyte

cryopreservation, embryo cryopreservation and transplantation of cryopreserved-thawed ovarian tissue, are all strategies

for fertility preservation in patients at risk. However, no evidence-based strategy is available yet. This article discusses

the mechanisms of reproductive failure after gonadotoxic therapy and the currently available fertility preservation strategies.

Key words: chemotherapy/fertility preservation/ovarian failure/radiotherapy.

INTRODUCTION

About 15 % of treatments of childhood and

adolescent cancer carry a substantial risk to future

fertility. This risk varies according to the

presenting pathology and requires treatment. It has

been estimated that at the year of 2000, one in

1000 adults will be a survivor of childhood cancer

(1, 2). This was due to the current use of multi-

agent chemotherapy regimens and/or well-tailored

radiotherapy. However, this was on the expense of

reproductive functions and future fertility of those

patients (3, 4). Consequently, better attention has been

paid to prevent reproductive failure in those patients.

Address of Correspondence: Division of Reproductive

Sciences, Department of Obstetrics and Gynecology,

University of Toronto, and Mount Sinai Hospital, 600University Avenue, Room 876, Toronto, Ontario M5G 1X5,

Canada. Tel: 416-586 - 4800 ext. 2451 Fax: 416-586-8588

e-mail: [email protected]

Moreover, it was also reported that 650,000

new female cancer cases are estimated to bediagnosed in 2003 in the USA (2). Approximately

8% of them occur during the reproductive years

(5). In addition, there is a steady incremental

decline of cancer mortality rates for all

malignancies in women. It was estimated that the

decline rate is 0.6% per year during the period

from 1992 to 1999 despite an increase in the

incidence by 0.3% per year from 1987 to 1999 (2).

The National Cancer Institute (NCI) estimated the

number of survivors of all child hood cancers to be

1 per 1000 population in 1997 (6). It is estimated

that by the year 2010, 1 in 250 patients will have

survived malignancies (7). Summary of thedistribution of cancers among women in the

reproductive age is shown in table 1. Besides the

expected premature ovarian failure and its

consequences, multi-agent chemotherapy and high

dose radiotherapy may be associated with early

Middle East Fertility Society Journal Vol. 10, No. 1, 2005Copyright

Middle East Fertility Society

Vol. 10, No. 1, 2005 Bedaiwy Fertility preservation in cancer patients 1


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Table 1. Distribution of cancers among women in the reproductive age

Variable Number/percent/ratio Source

Female cancer cases in 2003 650,000 Jemal et al 2003

Percentage of cancers below the age of 40 ys 8% Oktay and Yih 2002Survivors of all childhood cancers 270,000 (1/1000 population) Simon 2003Survivors of all childhood cancers in 2010 1/250 patients Bleyer 1990

pregnancy loss, premature labor and low birth

weight (8, 9).

There is a growing concern regarding the future

reproductive functions in pediatric oncology

survivors. Moreover, the safety of offsprings of those

survivors may be subjects to future abnormalities.

Consequently, there is increasing demand for a

fertility preserving interventions. On the other side,there is an increase in the techniques to preserve

fertility via medical protection, surgical procedures,

assisted reproduction and cryopreservation (10). In

this article we will review the pathophysiology of

chemotherapy/radiotherapy induced gonadal toxicity

and current techniques of fertility preservation.

Possible future options will also be discussed.

CHEMOTHERAPY-INDUCED OVARIAN

FAILURE

Reproductive age malignancies treated withchemotherapy

Premature ovarian failure (POF) is a well-known

consequence of exposure of the female gonad to

chemotherapeutic drugs (11, 12). A wide variety of

malignant and non-malignant conditions during the

reproductive age are treated with gonadotoxic

chemotherapy (Table 2). Patients are exposed to

these agents for treatment of malignancies such as

acute lymphoblastic leukemia (ALL). ALL is the

commonest childhood malignancy and it is estimated

that more than 2000 patients are destined to be long-

term survivors of ALL each year (13). In addition toleukemias, patients with Hodgkin's lymphoma,

neuroblastoma, non-Hodgkin's lymphoma, Wilm's

tumor, Ewing's sarcoma and osteosarcoma of the

pelvis and genital rhabdomyosarcoma receiving

chemotherapy are at risk of developing POF (14-17).

In addition, breast cancer is the commonest

malignancy in women during the reproductive age

(18). There is steady rise in the incidence and decline

of the case fatality rate of female breast cancer (18).

It is estimated that 1 out of every 228 women will

develop breast cancer before the age of 40.

Moreover, 15% of all breast cancer cases are

estimated to occur at


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Table 2. Epidemiologic features of malignant and non-malignant conditions during reproductive age treated by chemotherapy:

Epidemiologic features

Prepubertal cancers:

Acute lymphoblastic leukaemia (ALL), Hodgkin's lymphoma,

Neuroblastoma, Non-Hodgkin's lymphoma, Wilm's tumour,Ewing's sarcoma Osteosarcoma of the pelvis and genital

rabdomyosarcoma.

-ALL is the commonest childhood maliganacy -5 y survival rate:

all cancers = 80%, ALL=80-86%, and >90% for Hodgkin'sdisease

-2000 patients are estimated to become long-term survivors of

ALL/year.

Breast cancer -Commonest malignant disease in reproductive age women (15%of cases


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Table 3. Effect of different chemotherapeutic agents on the ovarian functions

Variable Cell Cycle Phase-Specific Agents

Drug type G1 Phase S Phase G2 Phase M Phase

Individual drugs L-asparaginase,

Prednisone

Cytarabine, 5-

fluorouracil,

hydroxyurea,

methotrexate,thioguanine

Bleomyosin, etoposide Vinblastine, vincristine,

vindesine, paclitaxel

Extent of ovarian

damage

No /low risk No /low risk No /low risk No /low risk

Variable Cell Cycle Phase-NonSpecific Agents

Drug type Alkylators Antitumor Antibiotics Nitrosureas Miscellaneous

Individual drugs Busulfan, carboplatin,chlorambusil, cisplatin,

cyclophosphamide,

isofamide,

mechlorethamine,melphalan

Dactinomycin,daunorubicin,

doxorubicin,

mitomycin,

mitoxantrone

Carmustine, lomustine,streptozocin

Dacarbazine,procarbazine

Extent of ovarian

damage

High Intermediate Intermediate High

likelihood of POF. Alkylating agents such as

cyclophosphamide, L-Phenyalanine mustard, and

chlorambucil produce permanent damage to

gonadal tissue by interacting chemically with

DNA. They cause inaccurate base pairing and

produce single and double stranded breaks. Thus

DNA, RNA and protein synthesis is inhibited.

Agents that do not induce permanent ovarianfailure include: 5-Fluorouracil, methotrexate,

etoposide, and doxorubicin. In one study in which

seven women with osteosarcoma were treated with

methotrexate, there was no evidence of premature

ovarian failure or gonadal dysfunction (32). Also

gonadal dysfunction has not been demonstrated in

women treated with methotrexate for high risk

vesicular mole and choriocarcinoma.

Differential sensitivity of the different cellular

components of the ovary to chemotherapeutic agents

The close structural and functional relationship

between granulosa cells and the oocyte make it

difficult to establish the exact target of cytotoxic

drugs. Destruction of either one leads to demise of

the other. These drugs may impair follicular

maturation and/or deplete primordial follicles

(30,33). Temporary amenorrhea will result when

maturing follicles are destroyed by cytotoxic

drugs. Permanent amenorrhea or POF will result

when all primordial follicles are destroyed. There

is conflicting data regarding the sensitivity of the

primordial follicle to cytotoxic drugs. The

primordial follicles of the mouse are sensitive to

cyclophosphamide but those of the rat are not(34,35). Since alkylating agents are not cell cycle

specific, they may be able to act on both the oocyte

and pregranulosa cells of the primordial follicles

leading ultimately to their destruction (36,37).

The dose of the cytotoxic agents used

The cumulative dose of the cytotoxic drug being

administered is a key factor impacting permanent

ovarian failure. Goldhirsch and associates

demonstrated a steadily increasing rate of POF,

ranging from 10%- 61% as the cumulative dose of

cyclophosphamide increased(38). Younger women

require higher cumulative doses of cytotoxic drugs

than older women before amenorrhea is induced.

"The average dose of single agent

cyclophosphamide before the onset of amenorrhea

was 5200 mg, 9300 mg, and 20,400 mg among 40,

4 Bedaiwy Fertility preservation in cancer patients MEFSJ


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Table 4. Effect of radiation dose and age on ovarian function

Ovarian dose (cGy) Risk of ovarian failure

60 No deleterious effect150 No deleterious effect in young

women; some risk for sterilization inwomen older than 40

250-500 In women aged 15-40, 60%permanently sterilized; remainder

may suffer temporary amenorrhea. In

women older than 40, 100%permanently sterilized

500-800 In women aged 15-40, 60%-70%

permanently sterilized; remainder

may experience temporary

amenorrhea. No data available forwomen over 40.

>800 100% permanently sterilized

From Damewood and Grochow (175).

30, and 20 year olds respectively" (39). These

investigators also demonstrated that the

"cumulative dose appears to be more important

than the dose rate since administration of low daily

doses of cyclophosphamide given over several

months was only slightly less effective at inducing

permanent ovarian failure than a high dose in 4

days". Finally older women have a shorter duration to

onset of amenorrhea after exposure to cytotoxic

drugs. Bines's review showed that women younger

that 40 years experienced amenorrhea 6-16 months

after initiation of chemo versus women older than 40,

who experienced amenorrhea within 2-4 months (31).The effect of duration of treatment and route of

administration on gonadal toxicity is not yet clear.

The population of women that may potentially

require gonadal protection against alkylating

agents is quite large including breast cancer. In the

typical regimen of CMF ( cyclophosphamide 11-15

grams, methotrexate, 5-fluoro-uracil), two thirds of

breast cancer patients will become amenorrheic.

With the AC ( doxorubicin, cyclophosphamide)

protocol 34 % will be amenorrheic at 3 years. This

figure is worse if taxanes ( such as docetaxel) are

also used with this regimen.

Women age

Prepubertal girls seem less susceptible than

young women to cytotoxic drugs (40). Treatment

of girls with up to 50,000 mg of cyclophospahmide

failed to induce ovarian failure in this population

(41). Nicosia et al showed that while the number of

growing follicles was reduced in patients receiving

combination chemotherapy regimens, there was no

significant change in the number of primordial

follicles (42). This may explain the apparent

decreased susceptibility of the pre-pubertal ovary to

cytotoxic drugs. The true incidence of POF in this

population is unclear since long-term follow-up

studies are rare (40,43-46). Most of these studies

have documented subsequent menarche and

ovulatory cycles in these patients (46). When the

outcome of subsequent pregnancies in these patients

was reviewed, the frequency of congenital anomalies

in the children born to them was not increased

(47,48). However, others debated that young girls are

equally susciptable to chemotherapy-induced

gonadotoxicity (17,49). On the other hand, women

with advanced age are more susciptable to

chemotherapy induced gonadal toxicity as previously

discussed.

RADIOTHERAPY-INDUCED OVARIAN

FAILURE

Reproductive age malignancies treated with

radiotherapy

Several cancers afflicting young premenopausal

women can be cured with radiation therapy. Theseinclude cervical, vaginal and ano-rectal carcinomas,

some germ cell tumors, and CNS tumors. Due to

early detection, approximately half of the patients

with cervical cancer are premenopausal and about a

third under 40 years of age (50). An even higher

proportion of women are premenopausal with the

other aforementioned cancers (51). The radiation

doses to the ovaries with standard pelvic radiation

therapy will uniformly induce ovarian failure. Total

body irradiation has been also used in conditioning

regimens prior to HSCT to eradicate the host's pre-

existing bone marrow as in leukaemias. This is

commonly associated with ovarian failure (4).

Factors affecting the extent of radiotherapy-

induced gonadotoxicity

The ovarian follicles are remarkably vulnerable



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to DNA damage from ionizing radiation.

Irradiation results in ovarian atrophy and reduced

follicle stores (52). On the cellular level, oocytes

show rapid onset of pyknosis, chromosome

condensation, disruption of the nuclear envelope

and cytoplasmic vacuolization. Serum levels of

FSH and LH rise progressively within 4-8 weeks

following radiation exposure along with a decline

in serum E2 levels (53). The degree and

persistence of ovarian damage, and suppression of

ovarian function, is related to the patient's age and

the dose of radiation to the ovaries (1,51,53) (Table

4).

In addition, the extent and type of radiation

therapy whether abdominal, pelvic external beam

irradiation, or brachytherapy are contributory

factors. The fractionation of the total dose plays an

important role in determining the extent of ovarian

damage as irradiation is more toxic when given in

single dose compared to fractionated doses (1).

Two studies indicated that the break point for

radiation induced ovarian failure is around 300

cGy to the ovaries. Only 11 - 13% experienced

ovarian failure below 300 cGy versus 60 - 63%

above that threshold value (54). The addition of

chemotherapy increases the risk of premature

ovarian failure (55,56).

A dose-dependant reduction in the primordial

follicle pool was noted upon exposing the ovary to

radiotherapy (57). At ovarian dose >6 Gy

irreversible ovarian failure is encountered due tothe extremely sensitivity of the oocytes to

radiotherapy (58). It was also estimated that


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irradiation, and despite some reports showing a

protective effect, it seems unlikely that

radiotherapy-induced gonadal damage can be

prevented by gonadal suppression (66).

Multiple studies have failed to show a

protective effect of GnRHa on human male gonads

exposed to cytotoxic drugs (67,68). Waxman

performed one of the first clinical studies in which

GnRH-a was utilized to down regulate the ovaries

of eight women undergoing chemotherapy. At

follow up, 4 of 8 women were amenorrheic who

had received the agonist and 6 of nine controls

were amenorrheic. Thus it appeared that buserelin

was not effective in preserving fertility in this

population of humans (67). Blumenfeld and

colleagues have reported on the largest group of

females exposed to both cytotoxic drugs and

GnRH-a thus far. Sixty patients with lymphoma

were initiated on GnRH-a 7-10 days prior to their

chemotherapy regimen. The control group

consisted of 60 historical patients of similar age

range treated with chemotherapy but not GnRH-a.

The rate of POF was 5% versus 55% in the GnRH-

a/chemotherapy versus chemotherapy alone group

(69-71). However, this study was an observational

one, with historical control group and relatively

short follow up period.

Recently another preliminary report has been

released regarding the use of GnRH-a for

protection of the adolescent ovary during cancer

treatment. All GnRH-a treated adolescentsresumed cyclic ovarian function, where as all

chemo only patients experienced

hypergonadotropic hypogonadism (46).

However, others debated a role of gonadal

suppression in women in preserving ovarian

function against chemotherapy (17). They argued

that ovarian reserve is made up of primordial

follicles that constitute 90% of the total follicle

pool (72). Primordial follicles initiate follicle

growth through an unknown mechanism which is

FSH independent (73). FSH receptors are not

expressed in primordial and primary follicles, but

the expression is uniformly present in as early as 3-4 granulosa layer preantral follicles (17, 118). This

means that GnRH analogues preserve only follicles

that have initiated growth which constitute only


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sphingosine-1-phosphate therapy, resisted

apoptosis induced by doxorubicin (81). With

eventual identification of the molecular and genetic

framework of chemotherapy induced germ cell

death, apoptotic inhibitors may be one of the most

promising stratiegies for preventing oocytes loss.

Ovarian transposition

For patients subjected to gonadotoxic

radiotherapy, ovarian function could be maintained

by transposing the ovaries out of the field of

irradiation. The ovarian dose following

transposition is reduced to approximately 5 - 10%

of untransposed ovaries (58,82,83). The dose to

each transposed ovary is 126 cGy for intracavitary

radiation, 135 - 190 cGy for external radiation

therapy (4500 cGy) and 230-310 cGy with the

addition of para-aortic node irradiation (4500 cGy)

(82).

Medial transposition

Initially, ovarian transposition for Hodgkin's

disease patients receiving nodal irradiation was

performed by suturing the ovaries posterior to the

uterus and shielding them during treatment. Later,

the ovaries were mobilized laterally out of the

pelvic field. Gaetini reported that of the 3600 cGy

delivered to central axis with total nodal irradiation

for Hodgkin's disease, the ovaries in normalposition were exposed to 3,524 cGy whereas those

transposed medially received 534 cGy and those

transposed laterally 319 cGy (55). However, this

technique is currently abandoned.

Lateral transposition

On the other hand, lateral transposition entails

dissecting the ovarian pedicle and moving the

ovary up the lateral pelvic sidewall. Hadar et al

performed CT follow up of 7 cervical cancer

patients who underwent lateral ovarian

transposition and 9 Hodgkin's disease patients withmedial transposition prior to radiation therapy. Six

of the 7 patients with lateral transposition had the

ovaries outside field and all retained ovarian

function. Scattered doses to the ovaries were 100-

300 cGy. The 1 patient with ovaries within the

field received 450 cGy and developed ovarian

failure. Only 3 of the 13 identified ovaries

following medial transposition were outside the

field. Even the 3 ovaries outside the field received

approximately 300 cGy (84). A compilation of 10

case reports and small series comparing medial and

lateral transposition noted ovarian failure in 50%

and 14% respectively (85).

Lateral ovarian transposition was typically

performed by laparotomy at the time of radical

hysterectomy for cervical cancer or staging

laparotomy for Hodgkin's disease. The utero-

ovarian ligament is divided and the ovary is

mobilized with the ovarian vessels to the paracolic

gutters. Ideally, the vascular pedicles are kept

retroperitoneal to avoid tension, torsion or trauma

and bowel herniation while the ovaries remain

intraperitoneal to reduce cyst formation (50,86).

van Beurden reported that with a tumor dose of

4,000 cGy for cervical cancer, the ovaries receive

280 cGy at 3 cm and 200 cGy at 4 cm from the

radiation field edge due to scatter (87). Bidzinski

confirmed that ovarian function was preserved if

they were transposed at least 3 cm from the upper

border of the field (88). In another study, 100% of

the patients whose transposed ovaries were above

the iliac crest maintained ovarian function as

opposed to none of those whose ovaries were

below (89). Ovarian failure may result if the

ovaries are not moved far enough out of the

radiation field or if they migrate back to theiroriginal position. Ovarian failure following

transposition may also be due to compromising the

ovarian vessels from surgical technique or

radiation injury to the vascular pedicle (90).

Ovarian transposition is of limited value in

patients over 40 as they have an intrinsically

reduced fertilization potential as well as a much

higher risk for ovarian failure despite transposition

(91). Another concern with ovarian transposition is

the development of symptomatic ovarian cysts.

The mechanism causing the cysts is unknown (92).

Laparoscopic approach

Currently, it has been recommended that

ovarian transposition should be performed

laparoscopically just prior to initiating radiation

therapy. This will eliminate unnecessary



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laparotomy in the majority of cervical cancer cases

where radiation therapy is not required. An

important advantage of laparoscopic ovarian

transposition is that radiation therapy can be

initiated immediately postoperatively preventing

failure due to the ovaries migrating back (56,93-95).

In cases of vaginal or cervical cancers being treated

by brachytherapy, laparoscopic ovarian transposition

can be performed under the same anesthetic for

inserting the brachytherapy device (96).

Staging laparotomy and splenectomy is no

longer required for Stage I and II Hodgkin's

disease (56). This eliminates the need for

laparotomy as ovarian transposition can be

performed laparoscopically with the benefits of

being an outpatient procedure with more rapid

recovery, less discomfort, better cosmesis and

lower cost. Nearly all women with Stage I and II

Hodgkin's disease treated with radiation alone or

with minimal chemotherapy following

laparoscopic ovarian transposition retain their

ovarian function and fertility (56). Morice et al

reported that 79% preserved ovarian function after

laparoscopic ovarian transposition and radiation

therapy for various indications (97).

Reproductive functions of transposed ovaries

The transposed ovaries produce estradiol and

progesterone at pretreatment levels (98). The

endometrium has been shown to function afterbrachytherapy and external radiation, though the

effect of radiation on the endometrium was not

clearly defined (99,100). Critchley studied 10

patients with ovarian failure following whole

abdominal radiation. The uterine length was

significantly reduced; most had no uterine blood

flow detectable by Doppler ultrasonography and

30% showed no endometrial response to hormone

replacement therapy. The radiation effect on the

uterus was unpredictable but higher doses were

more likely associated with vascular and uterine

damage (101).

Term pregnancies have been reported afterintracavitary radiation for cervical cancer (82).

Morice et al noted a 15% pregnancy rate after

brachytherapy with or without external radiation

for vaginal clear cell carcinoma compared to 80%

for those treated with external radiation only for

dysgerminomas and pelvic sarcomas (102). The

much lower rates with vaginal clear cell carcinoma

were attributed to DES associated genital

malformations and possibly to the effects of

brachytherapy on the endometrium. Interestingly,

89% of the pregnancies were spontaneous with

75% occurring without repositioning the ovaries.

The ovaries were only repositioned in cases of

infertility and 11% of those patients conceived

with IVF (97). Tulandi and Al-Took (103) reported

a case of laparoscopic lateral ovarian transposition

in a patient with rectal adenocarcinoma whereby

the utero-ovarian ligaments were divided but the

ovaries remained attached to the distal fallopian

tubes potentially improving the chances for ovum

pickup. The patient achieved a spontaneous

pregnancy (95).

Several papers addressed the concerns

regarding pregnancy outcomes after pelvic

irradiation. There was no increase in fetal wastage

or birth defects in women treated for Hodgkin's

disease (82). Swerdlow et al confirmed that there

was no excess of stillbirths, low birth weight,

congenital malformations, abnormal karyotypes or

cancer in the offspring of women treated for

Hodgkin's disease (104). However, Fenig et al

cited an increase in low birth weight and

spontaneous abortions especially if conception

occurred less than a year after radiation exposure.

They advised delaying pregnancy for a year after

completing radiation therapy (105).There is only 1 case report of superovulation

and oocyte retrieval for IVF with transposed

ovaries that were not repositioned. A cervical

cancer patient underwent laparoscopic lymph node

dissection, unilateral ovarian transposition and

chemotherapy followed by brachytherapy and

external radiation before radical hysterectomy.

Transabdominal ultrasound-guided needle

aspiration after superovulation with gonadotropins

yielded 5 oocytes. The resulting embryos were

transferred to a gestational carrier who delivered

normal live born twins. It was noted that oocyte

retrieval was possible due to the superficiallocation and lack of mobility of the ovary (106).

To facilitate oocyte retrieval, as well as the

diagnosis and treatment of ovarian cysts, the

ovaries have been transposed subcuticularly. This

has the disadvantages of requiring a laparotomy



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and an additional abdominal incision and may also

be associated with a higher rate of cyst formation.

Transient ovulatory pain was reported by 81.5% of

the patients and 15.4% required needle aspiration

(107). IVF with donor oocytes has also been

reported in 6 women with ovarian failure following

treatment for Hodgkin's disease. All conceived

with pregnancy and implantation rates per cycle of

37 and 17% respectively (108).

Complications of oophropexy

Despite proper shielding, the transposed ovaries

are still subjected to a significant amount of

radiation, consequently ovarian failure could still

happen. Moreover, Fallopian tube infarction,

chronic ovarian pain, ovarian cyst formation, and

migration of ovaries back to their original position

before radiotherapy are all reported complications

of oophropexhy (1,56). Anderson et al. (109)

reported subsequent oophorectomy in nine of 51

patients (17.5%) for the management of painful

ovarian cysts after a mean duration of 46.8 months

from the procedure. Spontaneous pregnancy may

not be possible, unless a second procedure is

performed to relocate ovaries back to pelvis (102).

One study found that there was no increased

risk of ovarian metastasis in patients with Stage IB

cervical carcinoma without gross extracervical

disease (110). Another study confirmed no

significant difference in 5 year survival rates incervical cancer patients with and without retained

ovaries (111). However, those with tumors >3 cm

are at higher risk for metastasis to the ovaries and

are not candidates for ovarian transpostion (91).

The rate of ovarian metastases was not correlated

with histologic type (91,110). Ovarian metastases

occurred in 0.5% with Stage IB squamous cell

cervical carcinoma and 1.7% with adenosquamous

carcinoma (110). For comparison, the risk of

ovarian cancer after hysterectomy for benign

disease is 0.2% (112). In addition to the very low

rate of ovarian metastasis with early stage cervical

carcinoma, there is a concern of inducing ovariancancer due to radiation exposure. No excess cases

of ovarian cancer was observed in 2068 women

who received 500-1000 cGy to the ovaries for the

treatment of menorrhagia followed up for a mean

of 19 years (113).

Oocyte cryopreservation

In single women, freezing mature or immature

oocytes may be the only ethically accepted

practical option. The main factor that may

influence the outcome in oocyte cryopreservation

is its structural complexity. Oocyte subcellular

organelles are far more complex and perhaps more

sensitive to cryopreservation-thawing injury than

in preimplantation embryos (114,115). An ideal

oocyte cryopreservation protocol should appreciate

the essential role of cytoskeletal elements as well

as plasma membrane functions in subsequent

development (116).

Despite the resounding success of mouse

mature oocyte cryopreservation (117), these results

could not be duplicated in the human scenarios

with the same success profile. Among the

alternative strategies to nullify the damaging

effects of the freezing trauma on M II oocytes is to

cryopreserve earlier stages before resumption of

full maturation. The relatively inactive

metabolism, absence of zona pellucida and lack of

the meiotic spindle makes the cryopreservation

insult the lowest at the primordial follicle levels

(118). On the other hand, antral follicles contain

oocytes either in the prophase -1 or metaphase-II

stage, and their oocytes are much more liable to

cryodamage (119). Both prophase-1 and

metaphase-II stages have zona pellucida but MII

oocytes have large cell size and which makescryoinjury more pronounced in M II oocytes.

Moreover the meitotic spindle, a unique feature of

M-II oocytes, is of particular importance as

variations in the temperature may lead to its

permanent damage (120).

Fewer pregnancies have been reported out of

frozen-thawed germinal vesicles oocytes (121-

123). In a study to evaluate the cryopreservation of

immature human oocytes obtained from

unstimulated ovarian tissue, immature prophase I

oocytes were obtained from unstimulated follicles

and were either cryopreserved or cultured as

controls. Rates of cryosurvival and maturation tometaphase II were compared between control

oocytes and cryopreserved oocytes with 2 different

methods. Lower cryosurvival and maturation rates

were demonstrated in cryopreserved-thawed

immature oocytes compared to control ones (124).



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The fair success of cryopreservation is partially

attributed to the cryoinjury. A wide spectrum of

subcellular damages might happen as a

consequence of the freeze-thaw trauma. These

include meitotic spindle damage {Pickering, 1990

#142} that may lead to chromosomal instability.

Other injuries include destruction of the

microtubules, which are an essential component

for polar body extrusion, pronuclear migration and

cytokinesis (125,126). Furthermore, aneuploid

embryos have been obtained from oocytes exposed

to 1.5 M DMSO without cooling, which suggests

CPA induced DNA damage (127). Other reported

abnormalities from cryopreservation of oocytes

include hardening of zona pellucida by alteration

of its glycoproteins particularly ZP2, which might

lead to failure of hatching and implantation

(128,129) and abnormalities in cortical granules

causing a premature cortical reaction {Schalkoff,

1989 #146}. Better choice of CPA, efficient

instrumentation and refinement of the available

cryopreservation protocol could minimize or even

prevent cryoinjury.

Oocyte cryopreservation is currently used

electively in patients with ovarian hyperstimulation

syndrome. Given the improved cryosurvival,

fertilization rates, pregnancies and births, oocyte

cryopreservation can be used in cancer patients as a

fertility preservation procedure. Despite the early

disappointing results with low survival, fertilization,

and pregnancy rates after IVF of thawed oocytes(130), recent studies suggesting increased success

rates (131-133). Vitrification using ethylene glycol

and dimethylsulphoxide (DMSO) as cryprotectants

(131,133) are being used successfully by many

groups. The mean survival and fertilization rates are

68.4%, 48.5% respectively. However the pregnancy

rate is 1.7% per vitrified-thawed oocyte. Once the

oocyte cryopreservation technology becomes

established with post-thaw survival rates, fertilization

rates and pregnancy rates matching those of embryo

cryopreservation; it will represent an effective

ethically approved strategy for adolescents facing

POF.

IVF and cryopreservation of preimplantation

embryos

Results of cryopreservation of preimplantation

embryos

Embryo cryopreservation was introduced to

maximize the conception chances from a single

cycle. Delivery rates per embryo transfer utilizing

cryopreserved embryos are reported by SART

(134) to be 18.6 %. The post-thaw survival rate of

embryos range between 35 and 90%, implantation

rates between 8 and 30%, and cumulative

pregnancy rates can be >60% (135,136). However,

this option may not be acceptable to prepubertal,

adolescent and women without a partner. If

acceptable, long-term data is available about the

outcome of children born from these procedures.

Ovarian stimulation protocols in estrogen-sensitive

cancers

Typically a standard IVF protocol is used.

Because of time constraints, this is usually a short

flare protocol (137). However there is some

concern that in patients with breast cancer the high

estrogen levels obtained in a standard IVF cycle

may impact the long-term survival negatively. For

this reason, some centers have offered natural

cycle IVF that is oocyte aspiration in an

unstimulated cycle. However cancellation rates are

high and the pregnancy rates are very low (7.2%

per cycle and 15.8% per embryo transfer) (138).

The time window between surgery and

chemotherapy is main determinant before adoptingthis policy. This window is around 6 weeks in

breast cancer patients, which would be adequate to

perform ovarian stimulation and IVF. Given the

fact that, conventional COH is associated with

significant rise of serum estrogen, which might

affect the overall prognosis (139), other ovarian

stimulation protocols are more appropriate.

Consequently, tamoxifen, a non-steroidal anti-

estrogen, commonly used as a breast-cancer

chemopreventive agent, was recently investigated

for ovarian stimulation and IVF in breast cancer

survivors (140). Tamoxifen 40-60 mg was started

on day 2 or 3 of the cycle and given daily for 5-12days and the tamoxifen group was compared to a

natural cycle-IVF group. The tamoxifen group had

a significantly higher numbers of mature oocytes,

peak estradiol, and embryos (mean of 1.6 embryos

versus 0.6 embryos) than the natural cycle group



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(140).

In certain group of patients tamoxifen could not

be used due to well-known effects on cancer

growth as in endometrial carcinomas. Some

authors proposed the use of aromatase inhibitors

for in these patients (17). Aromatase P450

stimulates the conversion of androgenic substances

to estrogens. Letrozole is a potent and highly

selective third generation aromatase inhibitor that

was developed in the early 1990s. It competitively

inhibits the activity of aromatase enzyme, and has

a half-life of 48 h (141). Letrozole significantly

suppresses plasma estradiol, estrone and estrone

sulphate levels using doses as low as 0.1 and as

high as 5 mg/day. Letrozole was even more

effective than tamoxifen in the treatment of

advanced stage post-menopausal breast cancer

(142).

Moreover, letrozole was introduced as a

promising ovulation induction agent. Repeated

clinical studies documented its clinical feasibility

in ovulation induction. It could be used

independently or in combination with FSH. It was

also used for the treatment of poor responders

(143-145). Many groups are currently testing the

feasibility of ovarian stimulation with aromatase

inhibitors in breast and endometrial cancer

patients.

Recently, a successful pregnancy was reported

in a patient after conservative management of

endometrial adenocarcinoma. A 29-year-oldinfertile white woman with grade 1 endometrial

adenocarcinoma treated by high-dose progesterone

therapy underwent an IVF cycle with transfer of

three blastocysts after proven cure. The patient

delivered triplets by cesarean section.

Laparoscopic-assisted vaginal hysterectomy and

bilateral salpingo-oophorectomy was then done.

No residual endometrial cancer was evident in the

hysterectomy specimen, but a 1.1-cm cystic mixed

endometrioid and clear cell-type adenocarcinoma

was discovered in the left ovary (146).

Ovarian stimulation protocols in non-estrogensensitive cancers

In another study, 69 women underwent 113

IVF/gamete intrafallopian transfer (GIFT) cycles

after cancer treatment in one partner, and 13

women underwent 13 IVF cycles for embryo

cryopreservation before chemotherapy/radiation.

The women undergoing IVF after chemotherapy

had poorer responses to gonadotropins than did the

women with locally treated cancers even though

they were younger. The delivery rates after the

women had undergone chemotherapy tended to be

lower among the systemic treatment group than it

was for the local cancer treatment group. The

women who had cryopreserved all embryos before

chemotherapy produced more oocytes and

embryos than did the women who had had a

history of local cancer treatment (147).

Recently, a case report depicted the feasibility

of IVF for the sake of embryo cryopreservation in

a patient with colorectal cancer. Controlled ovarian

stimulation, IVF, and embryo freezing were

performed before fluorouracil-based

chemotherapy. The 28-year-old woman

subsequently underwent subtotal colectomy. Three

years later, when the clinical and hormonal

analysis confirmed ovarian failure, two thawed

embryos were transferred to the uterus. She gave

birth at term to a 3200g infant (148).

Cryopreservation and transplantation of

ovarian tissue

Ovarian cryopreservation and transplantation is

an experimental procedure introduced to preserve

fertility in women with threatened reproductivepotential (149). Studies investigating the effects of

cryopreservation insult on ovarian tissues have

been limited compared to those studying the same

effects on oocytes (150). Unlike a suspended single

cell, tissue cryopreservation presents serious

physical constraints related to heat and mass

transfer. Furthermore, because it is a multicellular

structure for which cell-to-cell interactions are

known to exist, the dynamics of cryoprotectant

permeation into and out of the tissue during

cryopreservation are of utmost importance for

subsequent tissue survival. Water will be

osmotically drawn to the extracellular spaces andblood vessels from the individual cells under the

effect of the hypertonic cryoprotectants. It is this

water that is responsible for the formation of ice

crystals, which is responsible for the freeze-thaw

injury (151). Consequently, better survival is



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expected from primordial follicles because of their

smaller size and lack of follicular fluid.

As in other reproductive technologies, animal

models have provided useful information in

transferring methods to treat human infertility.

With the sheep ovary providing a reliable tool,

Gosden and associates pioneered the sheep ovarian

transplantation model. Using cryopreserved-

thawed ovarian cortical strips, they showed

follicular survival and endocrine function, as well

as restoration of fertility after transplantation of

cryopreserved-thawed ovarian cortical strips

(152,153). Despite the initial success of orthotopic

transplantation of cryopreserved ovarian tissue the

longevity of the grafts were very short. This was

attributed to freezing small pieces of tissue and the

ischemic destruction of the ovarian germ cells.

Our efforts to improve on the graft longevity

and prevention of ischemic destruction of the

ovarian follicles went through a consequential

cascade of experimental techniques. First, we have

shown that increasing the ovarian cortical

specimen size and ischemia time up to 30 minutes

did not alter ovarian cortical tissue survival (154).

Then, we demonstrated that cryopreservation of an

entire ovary with its vascular pedicle is technically

possible (155). In order to develop a microvascular

anastomosis technique for the ovaries, we proved

that the anterior abdominal wall is a suitable

recipient site for the ovary. The ovarian vessels

were anastomosed effectively to the inferiorepigastric vessels using microvascualr anastomosis

(156). The same technique worked for

cryopreserved-thawed intact ovaries as well (157).

However, antiovarian antibodies were induced in

some cases by changing the site of the ovary (158).

We argued that -in theory- all subcutaneous vessels

with a matching caliber to those of the ovarian

vessels could be potential recipient sites (159).

Finally in our last experiment, two intact human

ovaries with their vascular pedicles were

cryopreserved, thawed and reassessed in vitro in

comparison to cryopreserved-thawed ovarian

cortical strips. Matching primordial folliclesviability and count were observed using the two

techniques (160).

There are several potential uses of

cryopreserved ovarian tissue. Firstly, the tissue can

be transplanted back into patient. The potential for

reintroduction of a cancer nidus may limit this use

in malignancies that are known to have a

predilection for the ovaries as leukaemias and

lymphomas. Using present techniques, ischemic

injury to the transplanted tissue results in the loss

of virtually the entire growing follicle population

and a significant number of primordial follicles.

This could limit its long- term viability.

Alternatively the primordial follicles in the ovarian

tissue can be matured in vitro or in an immune

deficient animal host. The former cannot be

accomplished with present technology and the

latter is unlikely to be acceptable to patients with a

high likelihood of trans-species viral infections.

Autografting of human ovarian tissue

Oktay and his associates developed 3 different

surgical techniques of ovarian cortical strips

transplantation. In their first experimental surgery,

they placed ovarian cortical strips beneath the

pelvic peritoneum of the ovarian fossa

laparoscopically. The patient was 17 when right

salpingo-oophrectomy was performed for dermoid

cyst and at the age of 28, left salpingo-

oophrectomy with subsequent cryopreservation of

the cortex as a treatment of intractable

menometrorrhagia was performed. Transplantation

was performed at the age of 29 where partial

viability of the cortical strips was proven by in

vitro studies. The cortical strips were threaded andanchored to an absorbable cellulose membrane

followed by transplantation to the selected site.

The graft establishes its blood supply as early as 3

weeks postoperatively as evidenced by doppler

ultrasound scanning. After 24 days, of

gonadotrophin stimulation, they were able to

document ovulation based on hormonal and

sonographic features followed by menstruation 16

days after the first hCG injection (161).

Another case of orthotopic transplantation of

ovarian cortical strips was reported in 32 years old

woman with stage IIIB Hodgkin's lymphoma after

high dose chemotherapy (162). Seven month aftertransplantation, she obtained menopausal

symptomatic relief and her E2 level rose up to

100pg\mL with a 2 cm follicle at the

transplantation site. Complete cessation of the graft

function occurred 9 month after transplantation.



14/21

The fact that the patient received many

chemotherapy cycles before cryopreservation may

in part be responsible for early failure of the graft.

In their second experimental surgery, Oktay and

his group used the forearm as the transplant

recipient site in two cases. The first patient was a

35-year-old woman with stage III B squamous cell

carcinoma. Fresh ovarian cortical strips were

grafted over the fascia of the brachiradialis muscle.

A fifteen mm follicle was detected

utrasonographically 10 weeks after the procedure.

Several oocytes were retrieved percutaneaously

after gonadotrophin stimulation. Intracytoplasmic

sperm injection was tried in a single oocyte but

fertilization did not occur. The FSH level was

normal at 18 month follow up. The second patient

was a 37-year old woman who underwent

oophrectomy for recurrent ovarian cysts.

Heterotopic transplantation was performed similar

to the first patient. The patient resumed her regular

menses and ovulated 3 months later. Her graft was

still functioning 10 months after the transplant

(163). In a final study by Callejo et al (164), these

authors evaluated the long-term function of both

fresh (3 patients) and cryopreserved (1 patient)

ovarian autografts in four premenopausal patients

aged 46-49 who underwent heterotopic ovarian

transplantation. Although ovarian function was

reestablished in three patients, a 2-7-fold increase

in FSH levels was reported. One did not have

recovery of ovarian function. The limitation in thestudy was the age of the study population.

However, in their third experimental trial Oktay

and his team transplanted frozen-banked ovarian

tissue underneath lower abdominal skin in a 36

years old breast cancer survivor. Hormonal

functions were restored. Percutaneous oocyte

aspiration resulted in the generation of a 4-cell

embryo (165). More recently, A 32-year-old

Belgian woman has given birth to a healthy baby 7

years after banking her ovarian tissue before

starting chemotherapy for Hodgkin's lymphoma.

Although she became infertile as a result of the

chemotherapy, re-implantation of her ovariantissue re-started ovulation 5 months later. She

became pregnant 11 months after re-

transplantation by natural fertilization. This is the

first case of a human live birth after success of

orthotopic autotransplantation of cryopreserved

ovarian tissue, in a patient from whom tissue was

collected and cryopreserved before chemotherapy

was initiated (166).

One of the potential limitations of ovarian

tissue cryopreservation and transplantation is loss

of a large fraction of follicles during the initial

ischaemia after transplantation. Previous work

indicated that whereas the loss due to freezing is

relatively small (153,167,168), up to two-thirds of

follicles are lost after transplantation. Given this

limitation, it has been recommended that ovarian

tissue freezing (17), should be restricted to patients


15/21

examination showed that significant numbers of

the follicles had initiated growth (171). In a similar

work by Weissman and associates (172), they

attempted to evaluate the development of follicles

in human ovarian cortex grafted under the skin of

non-obese diabetic SCID mice. Exogenous

gonadotropin administration 12 weeks after

transplantation resulted in follicle growth in 51%

of the grafts. Similarly, several groups showed

follicle development, ovulation and corpus luteum

formation after stimulation of xenografted human

ovarian tissue using gonadotrophins in

immunodeficient mive (173,174). Concerns

regarding retroviral infections may limit the use of

this option in clinical practice.

CONCLUSION

GnRh analogues are the only available medical

protection means for gonadotoxic chemotherapy.

However, to be recommended for routine use, their

benefits should be substantiated by prospective

randomized trials. Laparoscopic ovarian

transposition is viable option if radiotherapy is to

be used. Oocyte cryopreservation is gaining

popularity as a treatment option. The recent

improvement in the post-thaw survival,

fertilization, implantation and pregnancies rates

adds to the practical potential of this option. So far,

embryo cryopreservation is the most successfulfertility preservation modality. However, it is not

applicable in unmarried women due to ethical

considerations.

Despite the recent reports of embryo

development, pregnancy and delivery after

transplantation of cryopreserved-thawed ovarian

tissue, the technique remains experimental.

Because not enough clinical evidence is available

today and the demand usually requires urgent

answers, a first careful counseling is very

important for the best management of the patients.

Given the desire of many individuals to preserve

fertility after cancer, chronic illness, iatrogeniccomplications of treatment or simply with

advancing age, the ESHRE Task Force on Ethics

and Law considered ethical questions and specific

dilemmas surrounding the cryopreservation of

gametes and reproductive tissue. In view of the

transition time during which research becomes

therapy, the taskforce stops short of giving

recommendations in anticipation of the availability

of new evidence, specifically in the case of

cryopreservation of reproductive tissues, in-vitro

maturation and in-vitro follicle culture. Consent

needs to be obtained within a research context

rather than for therapy or preservation of fertility

per se (176). Finally, all oncologists,

gynecologists, and reproductive endoncrinologists

should be aware of the available fertility

preservation strategies. A multidisciplinary

approach is ideal for the management of

chemotherapy and /or radiotherapy-induced

gonadal failure.

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