Main strategies of female fertility preservation

Currently, patients who suffer from different kinds of hormone-dependent malignancies, such as breast cancer or autoimmune diseases like vasculitis, are offered an aggressive form of therapy, which negatively influences the health of both themselves and the gametes. For example, the treatment of breast cancer, which is a hormone-dependent kind of cancer, may involve a drastic method like oophorectomy. This procedure leads to a decrease in the production of sex hormones (Di Leva Gianpiero et al., Reference Di Leva, Piovan, Gasparini, Ngankeu, Taccioli, Briskin, Cheung, Bolon, Anderlucci, Alder, Nuovo, Li, Iorio, Galasso, Ramasamy, Marcucci, Perrotti, Powell, Bratasz, Garofalo, Nephew and Croce2013). Moreover, treatment of other cancer types includes radio- and chemotherapy, which negatively affects the ovarian status. It is well known that most of the alkylating chemotherapeutic agents are gonadotoxic (Bokemeyer et al., Reference Bokemeyer, Schmoll, van Rhee, Kuczyk, Schuppert and Poliwoda1994; Thomson et al., Reference Thomson, Critchley and Wallace2002). Use of these agents in a therapy protocol leads to the degradation of follicles and oocytes (Familiari et al., Reference Familiari, Caggiati, Nottola, Ermini, Di Benedetto and Motta1993; Oktem & Oktay, Reference Oktem and Oktay2007a). Moreover, such treatment may result in the formation of ovarian tissue fibrosis (Meirow, Reference Meirow, Fasouliotis, Nugent, Schenker, Gosden and Rutherford1999; Behringer et al., Reference Behringer, Breuer, Reineke, May, Nogova, Klimm, Schmitz, Wildt, Diehl and Engert2005). Chemotherapeutic agents induce apoptosis in ovarian stroma and in the follicular system (Verga Falzacappa et al., Reference Verga Falzacappa, Timperi, Bucci, Amendola, Piergrossi, D'Amico, Santaguida, Centanni and Misiti2012), which leads to a reduction in follicle numbers or, in other words, results in an increase in the risk of infertility (Oktem & Oktay, Reference Oktem and Oktay2007b).

The use of radiation therapy is dangerous for women over the age of 30, because the effective sterilizing dose of radiotherapy decreases with age (Wallace et al., Reference Wallace, Anderson and Irvine2005). At the same time, the risk of malignancy formation increases with age (Xu et al., Reference Xu, Lawson, Yeoman, Molskness, Ting, Stouffer and Zelinski2013). Given these facts, the use of aggressive antitumour therapy for women over 30 must be supplemented with procedures of fertility preservation.

In contemporary society, many women prefer to delay childbearing, but the number of follicles decrease and reproductive health becomes impaired with age, reducing the probability of normal fertilization and pregnancy (Wallace & Kelsey, Reference Wallace and Kelsey2010). Therefore, methods that allow us to preserve fertility could also help these women to bear healthy children.

In some cases, fertility preservation is assisted with a classical cycle of ovulation induction (Cardozo et al., Reference Cardozo, Thomson, Karmon, Dickinson, Wright and Sabatini2015; Sigismondi et al., Reference Sigismondi, Papaleo, Viganò, Vailati, Candiani, Ottolina, Di Mattei and Mangili2015). Obtained oocytes can be cryopreserved, or fertilized and the developed embryos can subsequently be cryopreserved. Use of such a protocol is possible only in cases in which the woman is absolutely healthy or when her disease was diagnosed as being at an early stage of development and does not depend on gonadotropic and steroid sex hormones. Otherwise, the use of this protocol may lead to growth of the tumour. Moreover, such a strategy of therapy is unsuitable for prepubertal patients (Mahajan, Reference Mahajan2015).

At the present time, a patient requiring fertility preservation is offered cryopreservation of ovarian tissue, followed by retransplantation of thawed fragments into the patient's body (Meirow et al., Reference Meirow, Baum, Yaron, Levron, Hardan, Schiff, Nagler, Yehuda, Raanani, Hourvitz and Dor2007). However, technologies of ovarian tissue in vitro culture are being actively developed (Roy & Treacy, Reference Roy and Treacy1993; Abir et al., Reference Abir, Franks, Mobberley, Moore, Margara and Winston1997, Reference Abir, Roizman, Fisch, Nitke, Okon, Orvieto and Ben Rafael1999, Reference Abir, Fisch, Nitke, Okon, Raz and Ben Rafael2001; Nayudu et al., Reference Nayudu, Wu and Michelmann2003; Xu et al., Reference Xu, Fazleabas, Shikanov, Jackson, Barrett, Hirshfeld-Cytron, Kiesewetter, Shea and Woodruff2011b). The main aim of these technologies is to produce mature oocytes capable of fertilization from pieces of in vitro cultured ovarian tissue (Filatov et al., Reference Filatov, Khramova and Semenova2015). Embryos obtained after the fertilization of oocytes may be cryopreserved and transferred into the patient's uterus after full recovery. Moreover, obtained embryos may be used in surrogacy programmes if the patient would like to have a child before the disease is cured.

Diseases whose treatment requires fertility preservation

Many diseases whose treatment requires aggressive methods of therapy and procedures of fertility preservation are known. These diseases can be divided into two groups: oncological and non-oncological diseases. Non-oncological diseases include autoimmune and haematological disorders that, like oncological ones, are treated with chemotherapy and radiotherapy (Donnez et al., Reference Donnez, Martinez-Madrid, Jadoul, Van Langendonckt, Demylle and Dolmans2006a,b, 2010a,b). The most common diseases the treatment of which requires procedures of fertility preservation are represented in Table 1.

In many cases, diagnosis of the disease occurs at a late stage, when treatment must begin immediately. In these situations, the usage of standard protocols of assisted reproductive technologies, followed by the cryopreservation of embryos obtained after in vitro fertilization of oocytes received via ovulation stimulation, is not possible (Sigismondi et al., Reference Sigismondi, Papaleo, Viganò, Vailati, Candiani, Ottolina, Di Mattei and Mangili2015, Cardozo et al., Reference Cardozo, Thomson, Karmon, Dickinson, Wright and Sabatini2015). Moreover, the use of classical programmes of assisted reproductive technologies for fertility preservation is not possible for prepubertal girls (Mahajan, Reference Mahajan2015). In these cases, the application of ovarian tissue cryopreservation technologies or the ovarian tissue in vitro culture technique seems to be the best strategy for female fertility preservation.

Ovarian tissue cryopreservation techniques

The two most commonly used methods of ovarian tissue cryopreservation are vitrification and slow freezing. The slow freezing technique was developed earlier than vitrification. The slow freezing protocol involves ovarian tissue impregnation with the cryoprotective agent, followed by a step-by-step temperature lowering. Usually, the slow freezing procedure involves the application of programmed freezers, which are special devices that allow the gradual lowering of temperature according to the program's settings. In most cases, tissue is cooled to −140°C and then placed into liquid nitrogen for storage (Newton et al., Reference Newton, Aubard, Rutherford, Sharma and Gosden1996, Reference Newton, Fisher, Arnold, Pegg, Faddy and Gosden1998; Fuller & Paynter, Reference Fuller and Paynter2004; Hovatta, Reference Hovatta2005).

Both slow freezing and vitrification require the exposure of ovarian tissue to a cryoprotective solution. The principal difference between vitrification and slow freezing is the very rapid cooling of the sample in the former. Rapid cooling allows the avoidance of ice crystal formation in the tissue. The appearance of ice crystals inside cells during cryopreservation has a detrimental effect on their viability: ice has a lower density than water, meaning that frozen water takes up more volume than the liquid form. Ice crystals can impair a cell's membrane and, after thawing, a cell with a destroyed membrane may die.

The vitrification technique has demonstrated good results in the cryopreservation of oocytes and early embryos (Kuwayama et al., Reference Kuwayama, Vajta, Kato and Leibo2005; Cobo et al., Reference Cobo, Domingo, Perez, Crespo, Remohi and Pellicer2009; Abedellhafez et al., Reference Abedellhafez, Desai, Abou-Setta, Falcone and Goldfarb2010; Cobo et al., Reference Cobo, Meseguer, Remohi and Pellicer2010; Edgar & Gook, Reference Edgar and Gook2012), but this method has grave disadvantages. Cryopreservation with a vitrification protocol requires the application of cryoprotective agents in high concentrations, which may negatively affect cryopreserved cells (Fahy et al., Reference Fahy, MacFarlane, Angell and Meryman1984; Isachenko et al., Reference Isachenko, Isachenko, Rahimi and Nawroth2003, Reference Isachenko, Isachenko, Reinsberg, Montag, van der Ven, Dorn, Roesing and van der Ven2007; Gandolfi et al., Reference Gandolfi, Paffoni, Papasso Brambilla, Bonetti, Brevini and Ragni2006; Keros et al., Reference Keros, Xella, Hultenby, Pettersson, Sheikhi, Volpe, Hreinsson and Hovatta2009; Kagawa et al., Reference Kagawa, Silber and Kuwayama2009; Amorim et al., Reference Amorim, David, Van Langendonckt, Dolmans and Donnez2011;). Moreover, the high concentration of cryoprotective agents can cause osmotic shock in cells (Fahy et al., Reference Fahy, Lilley, Linsdell, Douglas and Meryman1990). Only two clinical cases of a successful pregnancy followed by a live birth after the vitrification and retransplantation of ovarian tissue are known (Kawamura et al., Reference Kawamura, Cheng, Suzuki, Deguchi, Sato, Takae, Ho, Kawamura, Tamura, Hashimoto, Sugishita, Morimoto, Hosoi, Yoshioka, Ishizuka and Hsueh2013; Suzuki et al., Reference Suzuki, Yoshioka, Takae, Sugishita, Tamura, Hashimoto, Morimoto and Kawamura2015). Brief descriptions of cryopreservation protocols whose usage led to pregnancies and live births are listed in Table 2. Comparison of the effectiveness of vitrification vs. slow freezing requires further studies, which are to be carried out in the near future.

Effectiveness of ovarian tissue cryopreservation

The effectiveness of cryopreservation and thawing procedures can be estimated using different methods. Evaluation of a tissue's condition after cryopreservation and thawing can be performed via histological analysis. However, the only reliable way to prove that a cryopreservation and thawing technique is effective is to obtain mature oocytes capable of fertilization and further development, followed by the live birth of healthy baby.

At present, more than 25 cases of successful ovarian tissue cryopreservation that led to childbirth are known (Donnez et al., Reference Donnez, Dolmans, Demylle, Jadoul, Pirard, Squifflet, Martinez-Madrid and van Langendonckt2004, Reference Donnez, Squifflet, Dolmans, Martinez-Madrid, Jadoul and van Langendonckt2013; Meirow et al., Reference Meirow, Levron, Eldar-Geva, Hardan, Fridman, Zalel, Schiff and Dor2005; Chung et al., Reference Chung, Donnez, Ginsburg and Meirow2013). Most of these cases are listed in Table 3. However, although the data are very promising, it is not known exactly whether the oocytes ovulated from the retransplanted ovarian tissue or the remaining fragment of the ovary. In most cases, pieces of ovary remain in the patient's body after an ovarian tissue biopsy, which is required for better survival rates of retransplanted tissue. It is only in isolated instances that women need a total bilateral oophorectomy, so only in these cases is it possible to declare confidently that an embryo has developed successfully from an oocyte obtained from the thawed ovarian tissue.

Cryopreservation of ovarian tissue followed by retransplantation is not applicable in several cases. Many scientific groups (Meirow et al., Reference Meirow, Levron, Eldar-Geva, Hardan, Fridman, Zalel, Schiff and Dor2005; Silber et al., Reference Silber, Lenahan, Levine, Pineda, Gorman, Friez, Crawford and Gosden2005; Donnez et al., Reference Donnez, Martinez-Madrid, Jadoul, Van Langendonckt, Demylle and Dolmans2006a, Reference Donnez, Jadoul, Squifflet, Van Langendonckt, Donnez, Van Eyck, Marinescu and Dolmans2010a, Reference Donnez, Dolmans, Pellicer, Diaz-Garcia, Sanchez Serrano, Schmidt, Ernst, Luyckx and Andersen2013; Andersen et al., Reference Andersen, Rosendahl, Byskov, Loft, Ottosen, Dueholm, Schmidt, Andersen and Ernst2008; Sanchez et al., Reference Sanchez, Novella-Maestre, Teruel, Ortiz and Pellicer2008; Schmidt et al., Reference Schmidt, Rosendahl, Ernst, Loft, Andersen, Dueholm, Ottosen and Andersen2011; Silber, Reference Silber2011) agreed that, as a rule, 35 is the upper age limit for the cryopreservation of ovarian tissue. This situation is due to the fact that, after the procedure of cryopreservation of ovarian tissue, follicles are preserved mainly in the early stages of folliculogenesis (primary and primordial follicles), the number of which reduces significantly with age.

In the case of prepubertal girls, obtaining ovarian tissue, followed by its cryopreservation and retransplantation, will negatively influence the patient's health. Such procedures will provoke harmful hormonal stress. In these cases, it seems better to obtain mature oocytes from ovarian tissue cultured in vitro and use them in surrogate programmes.

In some instances, ovarian tissue cryopreservation cannot be used (for example, when the patient is a prepubertal girl) or is not very effective (when the patient is older than 35). In these cases, the ovarian tissue in vitro culture technique can be applied for female fertility preservation.

Ovarian tissue in vitro culture

There are two methods for an ovarian tissue in vitro culture technique: (i) two-dimensional (2D); and (ii) three-dimensional (3D) systems (Hirao, Reference Hirao2012). The principal drawback of 2D systems is the low correspondence between the microenvironment of the cultured fragment and the in vivo conditions. In the case of the 2D method, the ovarian tissue culture's stroma and follicular cells migrate along the plane, follicles lose their normal shape and oocytes lose their normal cellular environment. Regulation of the 2D system's conditions may be performed only by adding various chemical agents (growth factors, hormones, etc.) to the culture medium. Three-dimensional systems allow us to regulate the condition of cultivated ovarian tissue, not only by adding different chemical agents but also by variation of microenvironmental physical properties.

For in vitro culture, the whole ovary (Jin et al., Reference Jin, Lei, Shikanov, Shea and Woodruff2010), ovarian fragments or single follicles can be used (Xu et al., Reference Xu, Kreeger, Shea and Woodruff2006b). In vitro culture of a whole ovary or ovarian fragments has a significant disadvantage. This is due to the difficulties of nutrient delivery and oxygenation in the deep layers of cultivated tissue. Therefore, studies dedicated to isolated ovarian follicles in an in vitro culture seem to be the most effective. In the case of isolated ovarian follicles in vitro culture, all cultivated follicles receive required nutrients that penetrate into all of the follicular compartments via diffusion. Moreover, when single ovarian follicles are cultivated, it is possible to trace the development of each follicle, which is not possible in the cases of a whole ovary or ovarian tissue fragments in vitro culture.

Mammalian ovarian follicles in vitro culture can be performed using various substrates, e.g. agarose, collagen, Matrigel or alginates and their derivatives. The main parameters of different substrates used for ovarian follicles in vitro culture are listed in Table 4. Described substrates belong to the group of hydrogels, which are polymeric compounds that are powdery when dried. If water or another solvent is added to the dry hydrogel, its granules swell, resulting in the formation of a gel-like substance. This gel can be used further for the in vitro culture of various cell types, particularly for the culture of ovarian follicles.

The described substrates have various limitations in their applications. For instance, agarose gelation requires a high temperature but heating is very harmful to ovarian follicles. The most commonly methods used for hydrogel gelation (exposure to UV radiation, high temperatures and aggressive chemical treatment) are dangerous for the living objects.

Alginate hydrogel gelation does not require toxic agents or application of non-physiological conditions (high temperature, UV radiation, etc.). Only the supplementation of a solution containing cross-linking agents (Ca²⁺ or Mg²⁺ ions) is needed for alginate polymerization. The effect of the conditions of hydrogel preparation on living cells is less negative. This is because alginate hydrogel preparation does not require temperatures higher than 37°C (physiological temperature) and polymerization agents (divalent cations, added to most culture mediums) have no toxic effects on cells. Moreover, cultivated cells do not form contacts with an alginate hydrogel. The alginate hydrogel destruction procedure does not impair cultivated follicles, because substances that digest the alginate hydrogel are unable to destroy cell contacts.

One of the principal advantages of alginate substrates is their vegetative origins. Therefore, alginate hydrogels can be used in ‘animal-free’ projects. In some studies, e.g. human ovarian follicle culture, the application of substances of animal origin is forbidden because it may lead to the contamination of the culture medium by viruses, proteins and other molecules that are able to interact with the cultivated tissue. There is no consensus on horizontal gene transfer when tissues or substances from different species are cocultured.

Effectiveness of ovarian tissue in vitro culture

To date, the ovarian tissue in vitro culture technique is not a widely used technique in clinical practice because the developing methods are far from perfect. Furthermore, there are no standardized technique protocols for effectively obtaining oocytes capable of in vitro fertilization. There are two principally distinct strategies of ovarian tissue in vitro culture: in vitro culture of ovarian cortical tissue fragments (strips) and in vitro culture of isolated ovarian follicles.

The cortical compartment of an ovary contains a large number of primordial follicles. Most commonly, ovarian tissue fragments are obtained by dissection of the cortical tissue into strips, with their subsequent culturing in vitro. This approach allows to support the growth of primordial follicles (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008).

Another approach to culture of ovarian tissue in vitro is based on the culture of isolated ovarian follicles. Commonly, primary and secondary follicles are used for this technique. For the ovarian tissue in vitro culture technique, researchers use various substrates that allow the stimulation of follicle growth and oocyte maturation, e.g., alginate (Xu et al., Reference Xu, West, Shea and Woodruff2006a, Reference Xu, Barrett, West-Farrell, Kondapalli, Kiesewetter, Shea and Woodruff2009, Reference Xu, Fazleabas, Shikanov, Jackson, Barrett, Hirshfeld-Cytron, Kiesewetter, Shea and Woodruff2011b; Amorim et al., Reference Amorim, Van Langendonckt, David, Dolmans and Donnez2009; Hornick et al., Reference Hornick, Duncan, Shea and Woodruff2012; Choi et al., Reference Choi, Agarwal, Huang, Zhao and He2014; Skory et al., Reference Skory, Xu, Shea and Woodruff2015), collagen (Sharma et al., Reference Sharma, Dubey and Meur2009; Itami et al., Reference Itami, Yasuda, Yoshida, Matsui, Hashiura, Sakai and Tamotsu2011; Mochida et al., Reference Mochida, Akatani-Hasegawa, Saka, Ogino, Hosoda, Wada, Sawai and Shibahara2013), Matrigel (Oktem & Oktay, Reference Oktem and Oktay2007c; Oktem et al., Reference Oktem, Buyuk and Oktay2011; Guzel et al., Reference Guzel, Nur Şahin, Sekeroglu and Deniz2014) and agarose (Fujihara et al., Reference Fujihara, Comizzoli, Wildt and Songsasen2012). There are promising results showing that the ovarian tissue in vitro culture technique allows not only to stimulate follicular growth but to obtain mature oocytes that are competent for development after fertilization (Xu et al., Reference Xu, Kreeger, Shea and Woodruff2006b, Reference Xu, Lawson, Yeoman, Pau, Barrett, Zelinski and Stouffer2011a, Reference Xu, Lawson, Yeoman, Molskness, Ting, Stouffer and Zelinski2013; Shikanov et al., Reference Shikanov, Xu, Woodruff and Shea2009, Reference Shikanov, Xu, Woodruff and Shea2011; Jin et al., Reference Jin, Lei, Shikanov, Shea and Woodruff2010; Choi et al., Reference Choi, Agarwal, Huang, Zhao and He2014).

In comparison with the method of ovarian tissue cryopreservation followed by retransplantation, the method of in vitro culturing of ovarian tissue allows to obtain mature oocytes not only from primary and primordial follicles that can be cryopreserved but also from follicles at advanced stages of development, e.g., from the secondary follicles (Mochida et al., Reference Mochida, Akatani-Hasegawa, Saka, Ogino, Hosoda, Wada, Sawai and Shibahara2013).

It has been shown that the in vitro culturing of murine ovarian follicles can lead to the formation of mature oocytes that are able to undergo successful fertilization and further development. Furthermore, after the transfer of these embryos to a surrogate mother, the live birth of healthy pups has been obtained (Xu et al., Reference Xu, Kreeger, Shea and Woodruff2006b). However, live births of healthy pups after in vitro culturing of ovarian tissue have been shown in only a few studies (Yamamoto et al., Reference Yamamoto, Otoi, Koyama, Horikita, Tachikawa and Miyano1999; Hasegawa et al., Reference Hasegawa, Mochida, Ogasawara and Koyama2006; Xu et al., Reference Xu, Kreeger, Shea and Woodruff2006b; Mochida et al., Reference Mochida, Akatani-Hasegawa, Saka, Ogino, Hosoda, Wada, Sawai and Shibahara2013; Higuchi et al., Reference Higuchi, Maeda, Horiuchi and Yamazaki2015). Such significant results motivate researchers to develop more advanced systems that will allow us to obtain mature oocytes more effectively.

The latest studies of the ovarian tissue in vitro culture technique are dedicated to the reconstruction of the ovarian corticomedullary structure. During folliculogenesis, in vivo growing follicles migrate from the more rigid ovarian cortex to the soft ovarian medulla (Xu et al., Reference Xu, Lawson, Yeoman, Molskness, Ting, Stouffer and Zelinski2013). Therefore, in a natural environment, mechanical stress reduction occurs in follicular cells. Reconstruction of the corticomedullary ovarian structure in vitro can be performed using combined hydrogels; for example, a fibrin-alginate hydrogel can be used (Shikanov et al., Reference Shikanov, Xu, Woodruff and Shea2009, Reference Shikanov, Xu, Woodruff and Shea2011; Jin et al., Reference Jin, Lei, Shikanov, Shea and Woodruff2010). A growing follicle secretes proteases that destroy fibrin, resulting in mechanical stress decreasing around the follicle and thus promoting its development (Shikanov et al., Reference Shikanov, Xu, Woodruff and Shea2011).

Another approach to reconstruct the ovarian corticomedullary structure in vitro is using an alginate-collagen system. The drop in which a single follicle is cultivated contains two compartments. The centre of the drop consists of collagen, and its periphery is formed with alginate. This technique allows to create a system with heterogenic mechanical tensions. Collagen is softer than alginate, so collagen imitates the medulla and alginate imitates the cortex (Choi et al., Reference Choi, Agarwal, Huang, Zhao and He2014). It has been shown that follicular development up to the late antral stage occurs in 28% of cases when an alginate–collagen system is used (Choi et al., Reference Choi, Agarwal, Huang, Zhao and He2014). Preparation of such a complex culture system can be performed using microfluidic technologies (Streets & Huang, Reference Streets and Huang2014). These technologies allow to create drops consisting of various substances in the required proportions by using directed microflows of liquids. The type of microflow is determined by the microfluidic chip configuration.

Technologies of 3D ovary reconstruction in vitro are being actively developed (Vanacker et al., Reference Vanacker, Luyckx, Dolmans, Des Rieux, Jaeger, Van Langendonckt, Donnez and Amorim2012; Luyckx et al., Reference Luyckx, Dolmans, Vanacker, Scalercio, Donnez and Amorim2013). The main conception of this technology is the encapsulation of ovarian follicles with other ovarian cells into the matrix. At the present time, this technique is at the first stage of its development but it seems to be very promising in the future. These systems will allow better reconstitution of in vivo conditions, resulting in the better stimulation of follicles’ growth and maturation.

Rodent ovarian follicles are most commonly used to develop ovarian tissue in vitro culture techniques. However, it is impossible to create an effective technology for medical practice on the basis of rodent ovarian tissue. Firstly, rodents have shorter ovarian cycles than the human female's one. Moreover, the ovarian tissue of primates has a higher density than the rodent one. Therefore, for the development of medical technology, studies on primate ovarian tissue in vitro culture are needed.

There is little scientific research related to primate ovarian follicles in vitro culture. However, the obtained results seem to be very promising. It has been shown that baboon follicles can produce mature oocytes that are capable of fertilization and can develop until the morula stage after fertilization via the intracytoplasmic sperm injection (ICSI) procedure (Xu et al., Reference Xu, Fazleabas, Shikanov, Jackson, Barrett, Hirshfeld-Cytron, Kiesewetter, Shea and Woodruff2011b).

Other research that was performed on rhesus monkeys has demonstrated that more rigid alginate hydrogels allow for better development of follicles than softer ones do (Hornick et al., Reference Hornick, Duncan, Shea and Woodruff2012). Moreover, mature oocytes capable of fertilization and further development up to the morula stage after fertilization by using the ICSI procedure have been obtained during a study on rhesus monkey ovarian follicles, using a fibrin-alginate hydrogel culture system (Xu et al., Reference Xu, Lawson, Yeoman, Molskness, Ting, Stouffer and Zelinski2013).

Research studies dedicated to human ovarian follicles in vitro culture are few in number. However, there are very encouraging results among that research. In the early works on human ovarian follicles in vitro culture, follicles were encapsulated into the agar (Roy and Treacy, Reference Roy and Treacy1993) or collagen gel (Abir et al. Reference Abir, Franks, Mobberley, Moore, Margara and Winston1997, Reference Abir, Roizman, Fisch, Nitke, Okon, Orvieto and Ben Rafael1999, Reference Abir, Fisch, Nitke, Okon, Raz and Ben Rafael2001), and they maintained their morphology. Moreover, some of the follicles developed until the early antral stages. A recent study has shown that alginate hydrogel permits the growth of encapsulated human ovarian follicles, corresponding to the normal folliculogenesis in vivo. Furthermore, an increase of oestradiol, progesterone and inhibin A during the period of cultivation has been observed (Xu et al., Reference Xu, Barrett, West-Farrell, Kondapalli, Kiesewetter, Shea and Woodruff2009). These hormones are required for normal follicular development.

A scientific group from Belgium has shown that human ovarian follicles can grow in alginate hydrogel. Moreover, in 90% of cases, follicular cells and oocytes retained their viability (Amorim et al., Reference Amorim, Van Langendonckt, David, Dolmans and Donnez2009).

In more recent work, researchers from the USA developed an alginate culture system that imitates follicles’ microenvironment in vivo. Moreover, this scientific group (Skory et al., Reference Skory, Xu, Shea and Woodruff2015) has developed alginate hydrogel ovarian follicles in vitro culture system that reconstitutes natural conditions. Hormone levels (17β-oestradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone) of cultured follicles were observed by an enzyme-linked immunosorbent assay. When the level of 17β-oestradiol increased and stabilized on the highest value, ovulation was induced by the addition of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF) into the culture medium. Due to the monitoring of follicular hormone secretion, researchers imitated menstrual cycle conditions in vitro by adding hormones and growth factors to the culture medium.

The human ovary contains a large portion of connective tissue, the amount of which varies greatly from patient to patient. Therefore, it appears impossible to develop a standard protocol of a primordial follicle's isolation from human ovarian tissue. This is due to the fact that the period of enzymatic treatment of human ovarian tissue for the obtaining of primordial follicles varies noticeably in different patients. Moreover, the loss of the integrity of primordial follicles with surrounding somatic cells, which may occur during this procedure, negatively influences their condition (Laronda et al., Reference Laronda, Duncan, Hornick, Xu, Pahnke, Whelan, Shea and Woodruff2014). Therefore, for better female fertility preservation, both techniques can be used: ovarian tissue cryopreservation in the case of primordial follicles, and ovarian follicle in vitro culture for follicles of advanced stages.

A two-step culture system is another approach that can also be performed for primordial follicle preservation. In these systems, small fragments of ovarian cortical tissue that contain primordial follicles are first cultured in order to stimulate follicular development. After the cultivation, follicles of advanced stages of folliculogenesis (primary, secondary, etc.) are isolated from the ovarian tissue. Subsequent cultivation is carried out with the obtained follicles (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008; McLaughlin et al., Reference McLaughlin, Kinnell, Anderson and Telfer2014).

Akt-signalling pathway inactivation allows to stimulate growth of primordial follicles in vitro. The Akt-pathway suppressor is PTEN (phosphatase and tensin homologue). It has been shown that PTEN inactivation results in an increase in the number of growing ovarian follicles in the cases of humans (McLaughlin et al., Reference McLaughlin, Kinnell, Anderson and Telfer2014) and mice (Jagarlamudi et al., Reference Jagarlamudi, Liu, Adhikari, Reddy, Idahl, Ottander, Lundin and Liu2009).

Nowadays, mature oocytes that are capable of fertilization can be obtained not only from rodents but also from primates. Furthermore, these oocytes can develop after fertilization. Designing human ovarian tissue in vitro culture systems is a very complicated problem. Firstly, it is more difficult to obtain human ovarian tissue for the experiments than in the case of animal models. Secondly, human follicular development in vivo is longer than that of rodents, for example. Due to these facts, human ovarian tissue in vitro culture systems are at the very earliest stages of their development. However, the growth of human ovarian follicles in vitro has still been observed. It is likely that findings that will allow the obtaining of mature human oocytes from ovarian tissue using in vitro culture may appear in the near future.