**4.1 Ovarian tissue culture**

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 humans.

Human Ovarian Tissue Cryopreservation as Fertility Reserve 219

(Donnez et al., 2004), followed recently by other live births (Donnez et al., 2011-a; Donnez et al., 2011-b; Meirow & Levron, 2005; Meirow et al., 2007; Demeestere et al., 2007; Demeestere et al., 2009; Anderson et al., 2008; Ernst et al., 2010; Silber et al., 2010; Piver et al., 2009;

The major disadvantage of transplanting ovarian cortical strips is that revascularization of the graft needs several days, according to the species. So, revascularization occurrs within 48h after transplantation in rats and within 1 week of grafting in sheep. This leads to great loss of follicles from the grafts in transplantation due to ischemic injury, which can however be avoided by performing a whole ovarian transplantation thus providing immediate revascularization of the transplant (Onions et al., 2009; Bedaiwy & Falcone, 2004; Bedaiwy et al., 2006; Bromer & Patrizio, 2009). However, whole ovarian transplantations have a high rate of vascular complication, such as risk of thrombosis. Therefore, further work is needed before whole ovarian tissue transplantation can be considered a viable option for fertility

Exogenous factors such as antioxidants, growth factors or hormones have been tested to improve follicular survival in ovarian grafts by reducing ischaemia-reperfusion injury

Until now, slow programmed ovarian tissue freezing is the only procedure that has resulted live births following orthotopic transplantation of frozen/thawed human ovarian tissue slices, but it is unknown or difficult to quantify how many women have attempted ovarian

The risk of ovarian tissue reimplantation includes transplantation of the primary tumour. However, most malignant diseases encountered during the reproductive years do not metastasize to the ovaries, except blood-borne malignancies such as leukemias, neuroblastoma and Burkitt's lymphoma. In the 1996 Shaw reported the transmission of lymphoma from a donor to a graft recipient (Shaw et al., 1996). Recently, Dolmans demonstrated, by quantitative RT-PCR, ovarian contamination by malignant cells by reimplant cryopreserved ovarian tissue

So, it is highly recommended to use the best assessment to detect micro-metastases on a small portion of the harvested tissue before cryopreservation, it is also useful to freeze a less

As suggested by Von Wolff, sophisticated techniques are required to exclude first macroscopic ovarian pathology, such as ovarian metastasis, using imaging (sonography, CT scan, etc.). Then immunohistochemistry and polymerase chain reaction (PCR) to exclude single malignant cells, and identified minimal residual disease (MRD) by highly sensitive RT-PCR. Another effective method is xenotransplant small sample of ovarian tissue in a

Xenotransplantation falls between reimplantation and in vitro culture, so the follicles within the tissue are growing in vivo in an animal host (T-and B-cell-deficient SCID mice). It has been applied to assess the risk of reimplanting malignant cells after human ovarian tissue

Last year Dath compared four grafting (intraperitoneal, ovarian bursa, sub-cutaneous, and intramuscular) sites for xenotransplantation of human ovarian tissue to nude mice, concluding that all four sites equally supported early follicular growth and preserved some

(Nugent et al., 1998; Torrents et al., 2003; Demeestere et al., 2009).

in acute and chronic leukaemia patients (Dolmans et al., 2011).

valuable part of the cortex, such as ovarian medulla.

immunodeficient animal host (Von Wolff et al., 2009).

reimplantation, and to observe follicle development.

Sanchez-Serrano et al., 2010; Revel et al., 2011; Roux et al., 2010), as shown in table 1.

preservation.

tissue reimplant.

**4.3 Xenotransplantation** 

In 2008 Picton published an interesting review on in vitro growth and maturation of follicles, which showed a number of different culture systems. It showed that culture of thin cortical strips has many advantages such as avoiding the damage caused by mechanical or enzymatic recovery of follicle present in the cortex, and provides a complex support system that resembles the ovary in vivo as the follicles also remain in contact with the surrounding stromal cells. This contact is very important at least until development of secondary follicles, when the follicles from the surrounding stroma should be left to facilitate further development. Thus, a multi-step strategy for the complete in vitro growth and maturation of follicles is promoted (Picton et al., 2008).

Recently another interesting review was published by Fabbri which summarizes the most relevant literature of the last ten years on in vitro cultures of ovarian cortical pieces. The conclusion was that the current optimal method for growing and maturing human follicles remains whole tissue culture. The authors underline the difficult of graduating nutrient concentration and especially what is best to add to the culture media and when (Fabbri et al., 2009).

In conclusion the culture of ovarian cortex strips is under development and currently not applicable to the human species, owing to the long period necessary for the follicle to complete development, almost six months compared to the couple of weeks in mice.

However, as suggested by Picton before these strategies can be utilised therapeutically, extensive testing is required to confirm the epigenetic health and genetic normality of in vitro growth derived oocytes (Picton et al., 2008).

We must not forget that oocytes are matured in vitro, after full development of follicles in vitro for a period exceeding 90 days, with the addition of nutrients that are not exactly what you get in their natural anatomic site, both as concentration and as timing of administration. So, even if complete follicle development in vitro is far from being clinically applicable, this does not mean that patients with malignancies that metastasize to the ovary should be excluded from the eventuality of cryopreserved tissue. We cannot know whether the study of in vitro cultures will make progress in a few years time.

#### **4.2 Ovarian tissue reimplantation**

The main option of this strategy is to transplant cortical ovarian tissue into the pelvic cavity, into its original location (orthotopic site) or in a different site, such as forearm, arm, abdominal wall, etc. (heterotopic site) when anticancer treatment is completed and the patient is disease-free.

Human transplantation history begins in the heterotopic site, such as the arm or the forearm. First studies on heterotopic reimplantation of an organ date back to 1975 and were applied to parathyroid gland. Several authors demonstrated that parathyroid autografts secret hormone and maintain normal serum calcium in the host (Wells et al., 1975; Hickey & Samaan, 1975). Oktay was the first to begin reimplanting of heterotopic tissue in the forearm, paving the way to human reimplantation history. In 2004 Oktay published a paper on the first embryo development after heterotopic transplantation of cryopreserved ovarian tissue (Oktay et el., 2004).

Successful orthotopic transplantation of ewe cryopreserved ovarian tissue was first performed by Gosden in 1994 (Gosden et al., 1994). The orthotopic transplantation is the site most suitable compared to the heterotopic site, because it is the natural environment, with comfortable temperatures and pressures for proper follicular development.

After several animal studies, ten years later, the first live human birth after frozen-thawed ovarian cortex orthotopic reimplantation was achieved by the Belgian group led by Donnez

In 2008 Picton published an interesting review on in vitro growth and maturation of follicles, which showed a number of different culture systems. It showed that culture of thin cortical strips has many advantages such as avoiding the damage caused by mechanical or enzymatic recovery of follicle present in the cortex, and provides a complex support system that resembles the ovary in vivo as the follicles also remain in contact with the surrounding stromal cells. This contact is very important at least until development of secondary follicles, when the follicles from the surrounding stroma should be left to facilitate further development. Thus, a multi-step strategy for the complete in vitro growth and maturation of

Recently another interesting review was published by Fabbri which summarizes the most relevant literature of the last ten years on in vitro cultures of ovarian cortical pieces. The conclusion was that the current optimal method for growing and maturing human follicles remains whole tissue culture. The authors underline the difficult of graduating nutrient concentration and especially what is best to add to the culture media and when (Fabbri et

In conclusion the culture of ovarian cortex strips is under development and currently not applicable to the human species, owing to the long period necessary for the follicle to

However, as suggested by Picton before these strategies can be utilised therapeutically, extensive testing is required to confirm the epigenetic health and genetic normality of in

We must not forget that oocytes are matured in vitro, after full development of follicles in vitro for a period exceeding 90 days, with the addition of nutrients that are not exactly what you get in their natural anatomic site, both as concentration and as timing of administration. So, even if complete follicle development in vitro is far from being clinically applicable, this does not mean that patients with malignancies that metastasize to the ovary should be excluded from the eventuality of cryopreserved tissue. We cannot know whether the study

The main option of this strategy is to transplant cortical ovarian tissue into the pelvic cavity, into its original location (orthotopic site) or in a different site, such as forearm, arm, abdominal wall, etc. (heterotopic site) when anticancer treatment is completed and the

Human transplantation history begins in the heterotopic site, such as the arm or the forearm. First studies on heterotopic reimplantation of an organ date back to 1975 and were applied to parathyroid gland. Several authors demonstrated that parathyroid autografts secret hormone and maintain normal serum calcium in the host (Wells et al., 1975; Hickey & Samaan, 1975). Oktay was the first to begin reimplanting of heterotopic tissue in the forearm, paving the way to human reimplantation history. In 2004 Oktay published a paper on the first embryo development after heterotopic transplantation of cryopreserved ovarian

Successful orthotopic transplantation of ewe cryopreserved ovarian tissue was first performed by Gosden in 1994 (Gosden et al., 1994). The orthotopic transplantation is the site most suitable compared to the heterotopic site, because it is the natural environment, with

After several animal studies, ten years later, the first live human birth after frozen-thawed ovarian cortex orthotopic reimplantation was achieved by the Belgian group led by Donnez

comfortable temperatures and pressures for proper follicular development.

complete development, almost six months compared to the couple of weeks in mice.

follicles is promoted (Picton et al., 2008).

vitro growth derived oocytes (Picton et al., 2008).

**4.2 Ovarian tissue reimplantation** 

patient is disease-free.

tissue (Oktay et el., 2004).

of in vitro cultures will make progress in a few years time.

al., 2009).

(Donnez et al., 2004), followed recently by other live births (Donnez et al., 2011-a; Donnez et al., 2011-b; Meirow & Levron, 2005; Meirow et al., 2007; Demeestere et al., 2007; Demeestere et al., 2009; Anderson et al., 2008; Ernst et al., 2010; Silber et al., 2010; Piver et al., 2009; Sanchez-Serrano et al., 2010; Revel et al., 2011; Roux et al., 2010), as shown in table 1.

The major disadvantage of transplanting ovarian cortical strips is that revascularization of the graft needs several days, according to the species. So, revascularization occurrs within 48h after transplantation in rats and within 1 week of grafting in sheep. This leads to great loss of follicles from the grafts in transplantation due to ischemic injury, which can however be avoided by performing a whole ovarian transplantation thus providing immediate revascularization of the transplant (Onions et al., 2009; Bedaiwy & Falcone, 2004; Bedaiwy et al., 2006; Bromer & Patrizio, 2009). However, whole ovarian transplantations have a high rate of vascular complication, such as risk of thrombosis. Therefore, further work is needed before whole ovarian tissue transplantation can be considered a viable option for fertility preservation.

Exogenous factors such as antioxidants, growth factors or hormones have been tested to improve follicular survival in ovarian grafts by reducing ischaemia-reperfusion injury (Nugent et al., 1998; Torrents et al., 2003; Demeestere et al., 2009).

Until now, slow programmed ovarian tissue freezing is the only procedure that has resulted live births following orthotopic transplantation of frozen/thawed human ovarian tissue slices, but it is unknown or difficult to quantify how many women have attempted ovarian tissue reimplant.

The risk of ovarian tissue reimplantation includes transplantation of the primary tumour. However, most malignant diseases encountered during the reproductive years do not metastasize to the ovaries, except blood-borne malignancies such as leukemias, neuroblastoma and Burkitt's lymphoma. In the 1996 Shaw reported the transmission of lymphoma from a donor to a graft recipient (Shaw et al., 1996). Recently, Dolmans demonstrated, by quantitative RT-PCR, ovarian contamination by malignant cells by reimplant cryopreserved ovarian tissue in acute and chronic leukaemia patients (Dolmans et al., 2011).

So, it is highly recommended to use the best assessment to detect micro-metastases on a small portion of the harvested tissue before cryopreservation, it is also useful to freeze a less valuable part of the cortex, such as ovarian medulla.

As suggested by Von Wolff, sophisticated techniques are required to exclude first macroscopic ovarian pathology, such as ovarian metastasis, using imaging (sonography, CT scan, etc.). Then immunohistochemistry and polymerase chain reaction (PCR) to exclude single malignant cells, and identified minimal residual disease (MRD) by highly sensitive RT-PCR. Another effective method is xenotransplant small sample of ovarian tissue in a immunodeficient animal host (Von Wolff et al., 2009).

#### **4.3 Xenotransplantation**

Xenotransplantation falls between reimplantation and in vitro culture, so the follicles within the tissue are growing in vivo in an animal host (T-and B-cell-deficient SCID mice). It has been applied to assess the risk of reimplanting malignant cells after human ovarian tissue reimplantation, and to observe follicle development.

Last year Dath compared four grafting (intraperitoneal, ovarian bursa, sub-cutaneous, and intramuscular) sites for xenotransplantation of human ovarian tissue to nude mice, concluding that all four sites equally supported early follicular growth and preserved some

Human Ovarian Tissue Cryopreservation as Fertility Reserve 221

As reported by Maltaris it is important to assess the 'ovarian reserve' that is the available pool of primordial follicles in the ovary and is a major determinant of female fertility potential. This information is important for a correct strategy of fertility preservation before cancer treatment. In general, ovarian reserve tests are either biochemical or biophysical

In conclusion, a common goal of life-saving methods must be to protect the fertility of young women to this aim it is important to form a closely collaborative team comprising gynaecologists, surgeons, oncologists, haematologists, biologists and psychologists. Specifically, it is important that the oncologist gives an assessment of the degree of ovarian

It is known that for each malignant disease only few protocols are commonly used, so it is possible to analyse the risk of ovarian failure. Meirow reported that the ovarian failure rate was 50% for breast cancer, 44% for non-Hodgkin's lymphoma, and 32% for Hodgkin's

Before proceeding to cryopreserve ovarian tissue, it is important to identify a series of tests for morphological analyses (such as staining with hematoxylin and eosin for observation by an optical microscope, or a more thorough evaluation of cell components by electron microscopy), for functional analyses (such as immunohistochemistry), or for viability analyses (such as trypan blue); in order to assess the degree of overall tissue preservation

In addition, it is also important to put an age limit to the preservation of ovarian tissue. The patient can use it only after cancer remission, therefore not before few years, so the cryopreservation of ovarian tissue of a patient older than 38 years old may be a useless procedure but could give psychological support to women. In any case, patients should be properly informed about the real possibility of recovering fertility and be able to get

In conclusion, we agree with Donnez that ovarian tissue cryopreservation should be offered before anticancer treatments in all cases where there is a high risk of premature ovarian failure and where emergency in vitro fertilization is not possible (Donnez et al., 2011-c). If the dogma of reproductive biology is that of a finite number of follicles in the ovary which, moreover, undergo atresia during reproductive life, all procedures to preserve fertility should be carried out before acting on the ovary with drugs, therapies, surgery or potentially toxic treatments. Therefore, live births after cryoprserved ovarian tissue

The authors thank Rosalind Roberts for reviewing the English language, and Elena Gismano

Andersen CY, Rosendahl M, Byskov AG, Loft A, Ottosen C, et al. (2008) Two successful

pregnancies following autotransplantation of frozen/thawed ovarian tissue. Hum

trasnplantation opened up new chapter in the field of infertility preservation.

damage in order to decide how much ovarian tissue to retrieve and store.

**5. Conclusion** 

pregnant.

**6. Acknowledgment** 

**7. References** 

(Maltaris et al., 2006), but new methods are required.

disease (Meirow & Nugent, 2001).

with the procedure adopted in the laboratory.

(biologist) for assistance with chapter preparation.

Reprod.;23(10):2266-72.

quiescent follicles (Dath et al., 2010). However, Sonmezer reported an asynchrony between oocytes and granulosa cell development (Sonmezer & Oktay, 2010).


Table 1. Live births after frozen/thawed orthotopic transplantation of human ovarian tissue.

In conclusion, xenotransplantation should be carefully considered in its human clinical application because of the risk of cross-species retroviral infection.
