**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).

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.


*Diversity of Extracellular Vesicles (EV) in Plasma of Cancer Patients DOI: http://dx.doi.org/10.5772/intechopen.101760*

for overcoming some, but probably not all, of the barriers we might face in the future while attempting to study the diversity EVs in body fluids. As this diversity is of key importance in understanding the role EV subsets such as, e.g., TEX, play in health and disease, immunoaffinity EV capture is likely to remain the method of choice for selective TEX pulldown. However, only when performed correctly, immunoaffinity capture of TEX yields valuable insights into their potential as cancer biomarkers and as markers of immune competence.


**1. Introduction**

The relationship between food and health has been investigated for many years, and therefore, the development of foods that promote health and well-being is a key research priority of the food industry [1].  Unfortu‐ nately, the daily intake of fruits and vegetables is estimated to be lower than the recommen‐ dation of the World Health Organization (WHO) [2], who suggest a dietary intake of 450 and 500 g of fruits and vegetables, respectively. Vegetables are strongly recommended in the human diet because they are rich in antioxidants, vitamins, dietary fibres and minerals. The majority of vegetables consumed in the human diet are fresh, minimally processed, pasteurised or cooked by boiling in water or microwaving, and vegetables can be canned, dried, or juiced or made into pastes, salads, sauces, or soups. Fresh vegetables or those that have been minimally processed have a particularly short shelf-life because they are subjected to rapid microbial spoilage. In addition, the above cooking processes can cause a number of potentially undesirable changes in physical characteristics and chemical composition [3,4].

Therefore, these drawbacks could be reduced by novel technologies, such as new packaging systems, high-hydrostatic pressure processing, ionisation radiation and pulsed electric fields [5-7]. The use of natural antimicrobial preservatives is considered to be the simplest and most valuable biological technique to keep and/or enhance the safety, nutrition, palatability and shelf-life of fruits and vegetables [5]. Lactic acid fermentation of vegetables, currently used as the bio-preservation method for the manufacture of finished and half-finished foods, is an important biotechnology for maintaining and/or improving safety, nutritional, sensory and

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

shelf-life properties of vegetables. Three technology options are usually considered for lactic acid fermentation of vegetable: spontaneous fermentation by autochthonous lactic acid bacteria, fermentation by starter cultures that are added into raw vegetables, and fermentation of mild heat-treated vegetables by starter cultures [18]. For thousands of years, microorganisms have been used to produce and preserve foods through the process of fermentation. Fermented foods have been adopted in various ways depending on the properties of the available raw materials and the desired features of the final products [8-10]. Food produced by traditional methods has become popular among consumers who know that their food is manufactured from high quality raw materials, without preservatives and other synthetic additives that are characterised by unique flavour values [11].


**1. Introduction**

Non-coding RNAs (ncRNAs) are a broad spectrum of functional RNA molecules that are transcribed from DNA but not translated into proteins [1]. The discovery of microRNAs (miRNAs) in 1993 followed by developments and discoveries in small RNA biology have hinted the importance of RNA in the post-transcriptional regulation of genes, especially in eukaryotes [2, 3]. With the development of highthroughput RNA sequencing technologies and bioinformatics methods, thousands of new ncRNAs have been discovered. Circular RNAs (circular RNAs, circRNAs) are a novel class of highly conservative endogenous ncRNA generated by premRNA back splicing, which is characterized by a covalently closed-loop structure (**Figure 1**) [4]. It was originally discovered that circRNAs are universally present in human and mouse, but then they were found to be common across essentially all eukaryotes [5]. Although most circRNAs are generally expressed at low levels, some of them are more abundant than their linear counterparts and often exhibit

**Figure 1.** *Back-splicing and canonical splicing of a single pre-mRNA.*

cell-type-specific and tissue-specific patterns [6, 7]. CircRNA levels are highly enriched in the brain, both embryonic and adult stage, and many of them play important roles in synaptic plasticity and neuronal function [8, 9]. Accumulating data suggest key roles for circRNAs in Alzheimer's disease (AD) by affecting mechanisms such as neuronal plasticity, autophagy, apoptosis, and inflammation [10]. This review covers the expression and function of circRNAs in the brain, as well as their role involvement in initiation and progression of AD.


**2. IC10 and IC50 in HEp-2 and Ca9-22 cells**

We used the same IC10 and IC50 concentrations (Table 1) (Kogashiwa et al. 2010) as our previous study. At IC10 concentrations, anti-proliferative effect was not observed.

Table 1 IC10 or IC50 values in two cells

IC10 or IC50 values after 1 hour drug exposure followed by a 96 hours incubation in two head and neck cell lines.

Microtubule and Cdc42 are the Main Targets of

repeated 4 times.

were repeated 4 times.

Docetaxel's Suppression of Invasiveness of Head and Neck Cancer Cells 153

Fig. 2. 3D gel culture of HEp-2 cell spheroids at IC50 concentration.

**5. Tubulin bundle was formed by docetaxel treatment**

HEp-2 cell spheroids were treated with cisplatin or docetaxel at IC50 followed by 96-hour incubation. bars= 250 µm. 4 replicate were used in each experiments and experiments were

Taxanes, including docetaxel, function as a mitotic spindle toxin by inhibiting microtubule turnover. They bind to microtubules and enhance tubulin polymerization. We hypothesized that docetaxel may exert similar effect on cytosolic, non-centrisome associated microtubules resulting in decreased cell motility. We therefore examined the structure of microtubules as well as actin filaments. Consistent with previous observation, filopodia formation was less in docetaxel treatment compared to cisplatin treatment. But no gross abnormality was found in actin filament structure between the treatments. On the other hand, tubulin bundle formation was noted in docetaxel treatment but not in cisplatin treatment (Fig.3.). Then, we attempted to find the mechanism that connect deformed microtubule and decreased filopodia formation.

Fig. 3. Staining of α-tubulin (by antibody) in HEp-2 cells treated by cisplatin, docetaxel or no treatment at IC50. Bars=50 µm. 3 replicate were used in each experiments and experiments

#### **3. Docetaxel inhibits the migration of head and neck cancer cells**

To assess cell migration a wound healing assay was employed. These results have been reported (Kogashiwa et al. 2010). Briefly, both in HEp-2 cell and CA9-22 cell, wound closure relative to no treatment condition is significantly reduced in docetaxel treatment while cisplatin treatment does not affect the cell migration (Fig.1.) (Kogashiwa et al. 2010).

Fig. 1. Migration assay at IC10 in two head and neck cancer cell lines. Migration rate is compared to control cell migration rate. Each data point represents mean ± SE. \*\*\*p < 0.001. 15 replicate were used in each experiments and experiments were repeated 4 times.

#### **4. Docetaxel inhibits the invasiveness of multicellular tumor spheroids.**

The similar results are obtained in three-dimensional multicellular tumor spheroid culture (Kogashiwa et al. 2010). At IC10 determined in monolayer culture, either cisplatin or docetaxel does not affect filopodia formation. However, at IC50 determined in monolayer culture, docetaxel, but not cisplatin, significantly decreases filopodia formation in HEp-2 cells in spheroid culture (Fig.2.) (Kogashiwa et al. 2010). Taken together, In the previous study, we have shown that docetaxel, but not cisplatin inhibits cell migration both in 2D and 3D culture.

To assess cell migration a wound healing assay was employed. These results have been reported (Kogashiwa et al. 2010). Briefly, both in HEp-2 cell and CA9-22 cell, wound closure relative to no treatment condition is significantly reduced in docetaxel treatment while

**3. Docetaxel inhibits the migration of head and neck cancer cells**

Fig. 1. Migration assay at IC10 in two head and neck cancer cell lines.

repeated 4 times.

3D culture.

Migration rate is compared to control cell migration rate. Each data point represents mean ± SE. \*\*\*p < 0.001. 15 replicate were used in each experiments and experiments were

The similar results are obtained in three-dimensional multicellular tumor spheroid culture (Kogashiwa et al. 2010). At IC10 determined in monolayer culture, either cisplatin or docetaxel does not affect filopodia formation. However, at IC50 determined in monolayer culture, docetaxel, but not cisplatin, significantly decreases filopodia formation in HEp-2 cells in spheroid culture (Fig.2.) (Kogashiwa et al. 2010). Taken together, In the previous study, we have shown that docetaxel, but not cisplatin inhibits cell migration both in 2D and

**4. Docetaxel inhibits the invasiveness of multicellular tumor spheroids.**

cisplatin treatment does not affect the cell migration (Fig.1.) (Kogashiwa et al. 2010).

Fig. 2. 3D gel culture of HEp-2 cell spheroids at IC50 concentration. HEp-2 cell spheroids were treated with cisplatin or docetaxel at IC50 followed by 96-hour incubation. bars= 250 µm. 4 replicate were used in each experiments and experiments were repeated 4 times.