**1. Introduction**

212 Topics in Cancer Survivorship

Schover, L. (2009). Patient attitudes toward fertility preservation. *Pediatr. Blood Cancer.*

Selick, C.; Hofmann, G.; Albao, C.; Horowitz, G.; Copperman, A.; Garrisi, G. & Navot, D.

Shaw, J.; Bowles, J.; Koopman, P.; Wood, E. & Trounson, A. (1996). Fresh and cryopreserved

Silber, S.; Lenahan, K.; Levine, D.; Pineda, J.; Gorman, K.; Friez, M.; Crawford, E. & Gosden,

Silber, S.; DeRosa, M.; Pineda, J.; Lenahan, K.; Grenia, D.; Gorman, K. & Gosden, R. (2008a).

Silber, S.; Grudzinskas, G. & Gosden, R. (2008b). Successful pregnancy after microsurgical transplantation of an intact ovary. *N. Engl. J. Med.* 11;359(24):2617-2618. Sobrinho, L.; Levine, R. & DeConti, R. (1971). Amenorrhea in patients with Hodgkin's disease treated with antineoplastic agents. *Am. J. Obstet. Gynecol*.109:135-139. Son, W.; Yoon, S.; Lee, S. & Lim, J. (2002). Pregnancy outcome following transfer of human

Sonmezer, M. & Oktay, K. (2006). Fertility preservation in young women undergoing breast

Tauchmanova, L.; Selleri, C.; De Rosa, G.; Pagano, L.; Orio, F.; Lombardi, G.; Rotoli, B. &

Wallace, W.; Thomson, A. & Kelsey, T. (2003). The radiosensitivity of the human oocyte.

Wallace, W.; Anderson, R. & Irvine, D. (2005). Fertility preservation for young patients with cancer: who is at risk and what can be offered? *Lancet Oncol.* ;6(4):209-218. Wandji, S.; Srsen, V.; Voss, A. & Fortune, J. (1996). Initiation in vitro of growth of bovine

Wang, J.; Yap, Y. & Mathews, C. (2001). Frozen-thawed embryo transfer: influence of clinical

Wang, X.; Chen, H.; Yin, H.; Kim, S.; Lin Tan, S. & Gosden, R. (2002). Fertility after intact

Warne, G.; Fairley, K.; Hobbs, J. & Martin, F. (1973). Cyclophosphamide- induced ovarian

factors on implantation rate and risk of multiple conception. *Hum. Reprod.* 16:2316-

premature ovarian failure. *N. Engl. J. Med.* 7;353(1):58-63.

(1995). Embryo quality and pregnancy potential of fresh compared with frozen embryos: is freezing detrimental to high quality embryos? *Hum. Reprod.*10:392-395. Senn, A.; Urner, F.; Chanson, A.; Primi, M.; Wirthner, D. & Germond, M. (2006).

Morphological scoring of human pronuclear zygotes for prediction of pregnancy

ovarian tissue samples from donors with lymphoma transmit the cancer to graft

R. (2005). Ovarian transplantation between monozygotic twins discordant for

A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation. *Hum. Reprod.* 23(7):1531-1537.

blastocysts vitrified on electron microscopy grids after induced collapse of the

Colao, A. (2002). High prevalence of endocrine dysfunction in long-term survivors after allogenic bone marrow transplantation for hematologic diseases. *Cancer* 

53(2):281-284.

outcome. *Hum. Reprod*. 234-239.

recipients. *Hum. Reprod.*11(8):1668-1673.

blastocoele. *Human. Reprod.*18:137-139.

cancer therapy. *Oncologist* 11(5):422-434.

primordial follicles. *Biol. Reprod.* 55(5):942-948.

ovary transplantation. Nature 24;415(6870):385.

failure. N. Engl. J. Med.289:1159-1162.

95:1076-1084.

2319.

*Hum. Reprod.*18:117-121.

Oocytes designed for reproductive function in women are the largest cell in the human body. They are surrounded by granulosa cells to form the follicles that in various stages of development are present in the ovarian cortex (Fig. 1).

Fig. 1. Primary follicle in frozen/thawed human ovarian cortex (20X magnification)

The ovary of a healthy woman contains a finite number of follicles that decreases over time. The peak number is reached in the fifth month of gestation with approximately 7 million follicles. This number reduces progressively due to atresia, so at birth there are only 1-2

Human Ovarian Tissue Cryopreservation as Fertility Reserve 215

However, cryopreservation of ovarian tissue may be the only acceptable method to preserve fertility for prepubertal girls, for women who cannot delay the start of cancer treatment for ovarian stimulation, and probably for women with hormone-sensitive malignancies. All these situations make ovarian tissue freezing the only option for female fertility

Therefore, an entire ovary or part of it, is removed laparoscopically, frozen and stored in liquid nitrogen at -196°C. After complete recovery of the patient, the tissue can be used to

Potential indications for ovarian tissue cryopreservation are patients diagnosed with malignant (extra-pelvic diseases: bone cancer, thyroid cancer, kidney cancer, breast cancer, melanoma, neuroblastoma; pelvic diseases: non-gynecological malignancy, gynaecological malignancy; systemic diseases: Hodkin's disease, non-Hodgkin's lymphoma, leukaemia, melanoblastoma) or benign diseases (recurrent ovarian cysts, etc), or with non malignant autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, autoimmune thrombocytopenia or other haematological diseases). These patients are being treated successfully with chemotherapy and/or radiotherapy or repeated surgery, greatly improved in recent years, but all these therapies can be toxic to the ovary. So, it is important to focus attention on the quality of life, including fertility preservation, as well as on survival of these

**2. Effect of radiotherapy, chemotherapy, or other toxic drugs on female** 

higher in young women (20 Gy) than older (6 Gy) (Lushbaugh & Casarett, 1976).

The risk of ovarian failure after anticancer treatments is assessed in relation to patient's age, treatment protocol and type of cancer as reported by Meirow (Meirow and Nugent, 2001). High-dose ionizing radiation is used to treat many types of cancer and hematologic malignancies. Ovarian transposition is not possible in case of total irradiation as is required for bone marrow transplantation (Meirow et al., 2010). The estimated dose at which half of the follicles are lost in humans (LD50) is 4 Gy (Wallace et al., 1989), but is age-dependent. Lashbaugh reported that the toxic ovarian dose, which leads to permanent loss of fertility is

Chemoterapic agents can be grouped into five classes of drugs based on their mode of action (alkylating agents, aneuploidy inducers, topoisomerase II inhibitors, antimetabolites and radiomimetics) as summarized by Meirow. It was found that alkylating agents imposed the highest risk in causing ovarian failure with an odd ratio (OR) of 3,98, followed by cisplatin

Schmidt, reported that the ovaries of healthy young girls contain a higher number of follicles than ovaries from older women, meaning young girls are more resistant to chemotherapy. Even if chemotherapeutic agents differ in their ovarian toxicity (Schmidt et al., 2010), they are often used in combination, so their adverse effects are increased and

In 2007, Oktem and Oktay, published an interesting paper on ovarian damage from chemotherapy. They present the first quantitative evidence in humans, by histological evaluation, that alkylating agents can significantly reduce ovarian reserve, and may affect stromal cell function (Oktem & Oktay, 2007). Meirow also reported that injury from chemotherapy is in stromal cells as well as in follicles and blood vessels (Meirow, 2010).

preservation.

young women.

**reproduction** 

restore gametogenic and/or steroidogenic function.

with an OR of 1.77 (Meirow and Nugent, 2001).

cannot always be easily evaluated.

million follicles that, at puberty, become 300.000. Approximately, 400 of these follicles become mature oocytes and ovulate during the fertile life of the female.

At the age of 37, there is usually an acceleration of follicular loss, which is correlated with an increase in follicle-stimulating hormone (FSH) level.

Last year Wallace reported with a mathematical model combined with histological evidence, the establishment and decline of non-growing follicles (NGFs) in the human ovary. Wallace shows for the first time that the rate of NGF recruitment increases from birth to age 14 years and then declines with age until menopause (Fig. 2) (Wallace & Kelsey, 2010).

Fig. 2. The best histological model for the establishment of the NGF population after conception and the subsequent decline until menopause (Kindly provided by Professor Wallace).

However, the central dogma of ovarian biology has been questioned by new discoveries. Johnson in 2004 reported that germline stem cells (GSCs) are present in the mouse ovary, and could become new follicles (Johnson et al., 2004). It seems that female germline stem cell reside in an extragonadal location, the bone marrow, and reach the ovary through the circulatory system (Johnson et al., 2005; Lee et al., 2007). De Felici reported that germline stem cells exist in adult mouse ovaries but are quiescent under physiological conditions, contributing to the oocyte reserve only in response to ovotoxic damage (De Felici, 2010). Recently, Bukovsky showed the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries (Bukovsky et al., 2009).

Despite these exciting new prospects to indicate that oocytes are continuously formed in the female adult, at the present it is known that the pool of follicles is limited, so in case of toxic ovarian events that may affect the ovary, opportune measures should be considered to preserve the fertility of young women. According to the law in different countries regulating assisted reproductive technologies, approaches to fertility preservation include embryo, oocyte cryopreservation, or ovarian tissue banking followed by ovarian tissue transplant or in in-vitro culture.

million follicles that, at puberty, become 300.000. Approximately, 400 of these follicles

At the age of 37, there is usually an acceleration of follicular loss, which is correlated with an

Last year Wallace reported with a mathematical model combined with histological evidence, the establishment and decline of non-growing follicles (NGFs) in the human ovary. Wallace shows for the first time that the rate of NGF recruitment increases from birth to age 14 years

become mature oocytes and ovulate during the fertile life of the female.

and then declines with age until menopause (Fig. 2) (Wallace & Kelsey, 2010).

Fig. 2. The best histological model for the establishment of the NGF population after conception and the subsequent decline until menopause (Kindly provided by Professor

premenopausal and postmenopausal human ovaries (Bukovsky et al., 2009).

However, the central dogma of ovarian biology has been questioned by new discoveries. Johnson in 2004 reported that germline stem cells (GSCs) are present in the mouse ovary, and could become new follicles (Johnson et al., 2004). It seems that female germline stem cell reside in an extragonadal location, the bone marrow, and reach the ovary through the circulatory system (Johnson et al., 2005; Lee et al., 2007). De Felici reported that germline stem cells exist in adult mouse ovaries but are quiescent under physiological conditions, contributing to the oocyte reserve only in response to ovotoxic damage (De Felici, 2010). Recently, Bukovsky showed the production of new eggs in cultures derived from

Despite these exciting new prospects to indicate that oocytes are continuously formed in the female adult, at the present it is known that the pool of follicles is limited, so in case of toxic ovarian events that may affect the ovary, opportune measures should be considered to preserve the fertility of young women. According to the law in different countries regulating assisted reproductive technologies, approaches to fertility preservation include embryo, oocyte cryopreservation, or ovarian tissue banking followed by ovarian tissue transplant or

increase in follicle-stimulating hormone (FSH) level.

Wallace).

in in-vitro culture.

However, cryopreservation of ovarian tissue may be the only acceptable method to preserve fertility for prepubertal girls, for women who cannot delay the start of cancer treatment for ovarian stimulation, and probably for women with hormone-sensitive malignancies. All these situations make ovarian tissue freezing the only option for female fertility preservation.

Therefore, an entire ovary or part of it, is removed laparoscopically, frozen and stored in liquid nitrogen at -196°C. After complete recovery of the patient, the tissue can be used to restore gametogenic and/or steroidogenic function.

Potential indications for ovarian tissue cryopreservation are patients diagnosed with malignant (extra-pelvic diseases: bone cancer, thyroid cancer, kidney cancer, breast cancer, melanoma, neuroblastoma; pelvic diseases: non-gynecological malignancy, gynaecological malignancy; systemic diseases: Hodkin's disease, non-Hodgkin's lymphoma, leukaemia, melanoblastoma) or benign diseases (recurrent ovarian cysts, etc), or with non malignant autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, autoimmune thrombocytopenia or other haematological diseases). These patients are being treated successfully with chemotherapy and/or radiotherapy or repeated surgery, greatly improved in recent years, but all these therapies can be toxic to the ovary. So, it is important to focus attention on the quality of life, including fertility preservation, as well as on survival of these young women.

## **2. Effect of radiotherapy, chemotherapy, or other toxic drugs on female reproduction**

The risk of ovarian failure after anticancer treatments is assessed in relation to patient's age, treatment protocol and type of cancer as reported by Meirow (Meirow and Nugent, 2001).

High-dose ionizing radiation is used to treat many types of cancer and hematologic malignancies. Ovarian transposition is not possible in case of total irradiation as is required for bone marrow transplantation (Meirow et al., 2010). The estimated dose at which half of the follicles are lost in humans (LD50) is 4 Gy (Wallace et al., 1989), but is age-dependent. Lashbaugh reported that the toxic ovarian dose, which leads to permanent loss of fertility is higher in young women (20 Gy) than older (6 Gy) (Lushbaugh & Casarett, 1976).

Chemoterapic agents can be grouped into five classes of drugs based on their mode of action (alkylating agents, aneuploidy inducers, topoisomerase II inhibitors, antimetabolites and radiomimetics) as summarized by Meirow. It was found that alkylating agents imposed the highest risk in causing ovarian failure with an odd ratio (OR) of 3,98, followed by cisplatin with an OR of 1.77 (Meirow and Nugent, 2001).

Schmidt, reported that the ovaries of healthy young girls contain a higher number of follicles than ovaries from older women, meaning young girls are more resistant to chemotherapy. Even if chemotherapeutic agents differ in their ovarian toxicity (Schmidt et al., 2010), they are often used in combination, so their adverse effects are increased and cannot always be easily evaluated.

In 2007, Oktem and Oktay, published an interesting paper on ovarian damage from chemotherapy. They present the first quantitative evidence in humans, by histological evaluation, that alkylating agents can significantly reduce ovarian reserve, and may affect stromal cell function (Oktem & Oktay, 2007). Meirow also reported that injury from chemotherapy is in stromal cells as well as in follicles and blood vessels (Meirow, 2010).

Human Ovarian Tissue Cryopreservation as Fertility Reserve 217

However, data on cryopreservation of human ovarian tissues by vitrification are still modest and controversial. This may be due to the fact that the same cryopreservation procedure is often used, such as slow freezing or vitrification, but different freezing protocol (types of cryoprotectant, concentration used and time of diffusion, etc.), this creates significant bias

In addition vitrification presupposes direct contact with liquid nitrogen, which is a potential

After 4 or 5 years when the patient is considered oncologically cured, the ovarian tissue stored in liquid nitrogen can be used. There are several ways to use tissue after thawing, such us reimplantation in various anatomic sites in the same patient or in a host animal or in

CULTURE

CONTAMINATION

IN VITRO

 RESULTS: STAGES OF FOLLICULAR DEVELOPMENT COMPLETED

source of microbial contamination, as reported by Isachenko (Isachenko et al., 2009). In conclusion, an ideal ovarian cryopreservation method has not yet been established.

vitro culture to grow primordial follicles present in large number (Fig. 3).

FROZEN/THAWED OVARIAN TISSUE

Fig. 3. Techniques using human cryopreserved ovarian tissue.

ORTHOTOPIC SITE

When dealing with tumours that can metastasize to the ovary, ovarian tissue reimplantation should be avoided due to the risk of retransmitting cancer cells. In these cases the only option is to develop other methods in which the primordial follicles are matured in vitro within ovarian tissue pieces. Only in mice was the full maturation of primordial follicles with livebirths achieved (Eppig et al., 1996), this is difficult to replicate in large animals and

**RESULTS:** 

IN VIVO IN ANIMAL HOST

 **LIVE BIRTHS** RISK OF CROSS

**4.1 Ovarian tissue culture** 

TRANSPLANTA TION

> RESULTS: EMBRYOS

humans.

that must be considered.

**4. How to use** 

HETEROTOPIC SITE
