**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-

Fertility Preservation for Pre-Pubertal Girls and Young Female Cancer Patients 199

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

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

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

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

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

vascular damage and reduced elasticity of the uterine musculature.

**4. Current options for fertility preservation** 

function in 80% of cases (Bisharah & Tulandi, 2003).

vitro (IVM) before they can be fertilised (Gosden, 2005).

cryoprotectant, resulting in the prevention of ice crystal formation.

**4.1 Ovarian transposition** 

**4.2 Vitrification of oocytes** 

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 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.
