The field of female fertility preservation is based on the ability to successfully cryopreserve ovarian tissue , and this can now be offered to a wide range of patients worldwide [2, 3]. Fragments of cryopreserved ovarian cortex can be thawed and autografted to an orthotopic or heterotopic site to restore fertility [4, 5]. The potential of this tissue to restore fertility would be greatly enhanced if immature oocytes contained within the tissue could be grown to mature stages within the laboratory and this would be particularly beneficial for prepubertal girls who currently have fewer options to preserve and restore their fertility than adult women [2, 3].
Cryopreserved tissue contains the most immature stage of oocyte within primordial follicles and the main aim of culturing this tissue is to support in vitro gametogenesis/growth (IVG) to develop immature oocytes entirely in vitro . If this methodology could be demonstrated to be safe, it would maximize the potential of cryopreserved ovarian tissue and have many clinical applications [2, 3].
Success rates of assisted reproductive treatment (ART) have significantly improved over the past three decades. Nevertheless, controlled ovarian stimulation protocols are still associated with side effects. In a subset of patients, there is a substantial risk of ovarian hyperstimulation syndrome (OHSS) which, in its severe form, may result in significant morbidity and even mortality . The overall incidence of severe OHSS is < 2% , but in women with polycystic ovary syndrome (PCOS), who represent up to 30% of women eligible for ART , the risk of OHSS is higher. Strategies to prevent OHSS in patients with a high response to gonadotropins include the administration of a GnRH agonist instead of hCG to induce the final oocyte maturation  and in vitro maturation of oocytes (IVM). IVM avoids the risks and side effects of conventional ovarian hormonal stimulation because this approach involves retrieving immature oocytes from unstimulated or minimally stimulated ovaries. After immature oocyte collection, these oocytes are cultured, matured, and fertilized in vitro. Selected embryos are then transferred to an adequately primed endometrium, or, alternatively, they are cryopreserved and subsequently thawed or warmed in a natural or artificial cycle.
The first successful pregnancy and birth from IVM in an anovulatory patient with PCOS was reported in 1994 ; in spite of technical advances to the IVM protocol and improvements of the maturation method and culture media, pregnancy rates are generally lower compared to those after conventional ovarian stimulation, although expert IVM centers can achieve birth rates that are comparable to birth rates after IVF . Lower pregnancy rates in comparison with conventional ART, as well as concerns about the genetic health of IVM oocytes  and the long-term health of embryos, fetuses and children born after IVM, still preclude a more general acceptance of IVM in ART centers. The causes and mechanisms of the lower implantation rates of IVM derived embryos are still largely unknown, although they are almost certainly linked to a lack of complete cytoplasmic maturation  and to a suboptimal endometrial receptivity associated with IVM cycles. There is a need to optimize IVM culture media , to standardize the IVM treatment protocol with regard to the use of gonadotropins to prime the follicles , the aspiration technique and the in vitro maturation timings .
There is evidence that oocyte maturation, fertilization rates and blastocyst production are compromised when compared with in vivo matured oocytes , possibly because of a dyssynchronous nuclear and cytoplasmic maturation. Promising technologies have emerged in recent years to improve maturation rates and developmental competence of IVM derived embryos, based on methods that can compensate the limited in vitro maturation time (24–36 hours) of oocytes after their aspiration from antral follicles, compared to in vivo maturation within the ovary . Recent IVM culture system modifications include substances that increase the cyclic AMP (cAMP) levels in the oocyte environment and by doing so allow for a more physiological cascade of in vitro maturation triggering [13, 14].
This chapter will summarize the scope of IVM as a stand-alone assisted reproductive technology and will discuss the current status of IVM as an additional tool for fertility preservation.
The primordial follicle pool of the ovaries harbors oocytes that are arrested at prophase stage I of meiosis. The majority of these follicles become apoptotic; only a few follicles grow beyond the antral stage, at which time the oocyte continues to mature until ovulation. The molecular events that drive and regulate the process of oocyte maturation are not fully understood, although FSH is thought to play a major role in influencing the maturation process. After selection of the dominant follicle, this follicle becomes FSH dependent. The increasing estrogen levels from the pre-ovulatory follicle induce a luteinizing hormone (LH) rise, which in turn induces a cascade of secondary factors synthetized in the mural follicle cells that lead to breakdown of the germinal vesicle (GV) in the oocyte . The oocyte proceeds from meiotic metaphase I to telophase I, which is associated with extrusion of the first polar body. The full meiotic progress does not only occur after the LH surge, but also when the oocytes become detached from their follicular environment. Historical observations by Pincus and Enzmann in 1935 showed that immature oocytes have the ability to resume meiosis spontaneously when they are removed from the follicle . Edwards et al. confirmed this concept by showing that they reach metaphase I approximately 28 to 35 h after being released from the follicle, that they extrude the first polar body after being in culture between 36 and 43 h and that they can be fertilized .
IVM has a number of important advantages over IVF. Ovarian hormonal stimulation is either limited or even absent, which reduces the hormonal burden for the patient and eliminates ovarian swelling which would typically occur during stimulation with gonadotropins. Furthermore, the clinical IVM protocol requires less meticulous follow-up and reduces the number of visits during the treatment cycle. Finally, IVM is more affordable for the patients because the medication costs are significantly reduced. All of these contribute to a more “patient friendly” protocol compared to standard ART.
Cha et al. obtained the first in vitro maturation (IVM) pregnancy in 1991 . They had used the immature human oocytes retrieved during gynecologic operations in an oocyte donation program. The first IVM pregnancy with a patient’s own oocytes was achieved in 1994 and was obtained by Trounson et al. . However, pregnancy rates after IVM in those years were disappointingly low, until Chian et al. introduced human chorionic gonadotropin (hCG) priming for IVM in PCOS patients, 36 hours prior to the oocyte retrieval. They reported implantation rates of 32% and clinical pregnancy rates of 40%, which marked an important step forward . During the following years the same group achieved clinical pregnancy rates of up to 54% after IVM in unstimulated cycles . However, these high pregnancy rates were obtained after the transfer of 3 to 4 embryos irrespective of the age of the patient.
IVM pregnancy rates have consistently been lower than IVF pregnancy rates after conventional ovarian stimulation . The reduced success rates have been attributed to the asynchrony in the cytoplasmic and nuclear maturation of the oocyte as well as to an inferior endometrial thickness and receptivity . Nevertheless, live birth rates of 40% and higher in patients with polycystic ovaries have been reported from Western Australia . Key elements contributing to the success of the IVM protocol in expert centers include FSH priming and blastocyst culture. However, in comparison with conventional IVF, IVM has not been able to generate an equal number of blastocysts, although the implantation rate per embryo appears similar in both approaches.
Strictly speaking, IVM involves the aspiration of immature oocytes from antral follicles after minimal or no exogenous gonadotrophin administration. Follicle aspiration is typically performed when antral follicles are small (less than 10 mm), and in women with ovulatory cycles selection of a single dominant follicle is avoided to prevent any negative impact on development of subordinate follicles. After a baseline ultrasound scan to rule out the presence of cysts or other pathology, serial ultrasound scans are scheduled to assess the growth of the antral follicles and the thickness of the endometrium. Oocyte maturation rates in vitro are generally lower than maturation rates of oocytes retrieved in a conventional IVF program after administration of an ovulation trigger, suggesting that a considerable proportion of immature oocytes from small antral follicles are still meiotically incompetent and would have required more time within their follicular environment to accomplish physiological nuclear and cytoplasmic maturation.
Higher oocyte maturation rates can be obtained when a bolus of hCG is administered, typically 36–38 hours before oocyte retrieval . In these cases, meiotic resumption is initiated in vivo and a proportion of oocytes are found to have reached metaphase II at the time of oocyte retrieval – they are oocytes which have thus completed meiosis in vivo and can readily be inseminated. As such, the hCG triggered IVM system may represent a semantical contradiction, but it is applied more often than the ‘pure’ non-hCG triggered system, where all oocytes are at GV stage at the time of egg collection (Figure 31.1). Nevertheless, there is ongoing debate as to the most efficient clinical and laboratory protocol for patients undergoing IVM .
Figure 31.1 In the absence of an ovulatory hCG trigger, all oocytes retrieved are immature and surrounded by compact cumulus cells
Improving In Vitro Culture Techniques
The nuclear maturation through meiosis I and II is a prerequisite for successful oocyte maturation. Cytoplasmic maturation is equally important and includes relocation of organelles, synthesis and modification of proteins and mRNAs, and regulation of biochemical processes that support subsequent fertilization and embryonic development . Regulation of oocyte maturation in vivo involves complex signaling pathways that occur in the microenvironment of the maturing oocyte. The oocyte and cumulus cells communicate through gap junctions  that allow passage of regulatory molecules and growth factors. The oocyte is in meiotic arrest until meiotic progress is triggered. In vivo, maturation is triggered by the endogenous LH surge and mediated by growth factors, such as epidermal growth factor (EGF) family members amphiregulin, epiregulin and beta-cellulin . In vitro, oocyte maturation occurs spontaneously when the oocyte is removed from the follicular environment that inhibits meiotic progression . When immature oocytes are removed from small antral follicles, meiotic resumption will occur precociously, that is, before completion of cytoplasmic maturation. Therefore, the timing of resumption of meiosis is important in oocyte maturation. To solve this problem for in vitro maturation systems, some authors suggest delaying spontaneous nuclear maturation while promoting development of the cytoplasm at the same time . The intracellular messenger molecule cAMP plays a significant role in the regulation of mammalian oocyte maturation . High levels of cAMP and cAMP analogs prevent meiotic resumption . Spontaneous oocyte maturation in vitro can be inhibited or delayed by increasing the cAMP level within the cumulus-oocyte complex environment by adding any of the following substances to the media: (i) cAMP analogues such as dibutyryl cAMP, (ii) activators of adenylate cyclase, such as FSH, forskolin or invasive adenylate cyclase and (iii) phosphodieserase (PDE) inhibitors, such as the non-specific inhibitor IBMX, the PDE type 4-specific inhibitor rolipram or the PDE type 3-specific inhibitors milrinone, cilostamide or Org9935 . These agents delay germinal vesicle breakdown and simultaneously increase the extent and prolong the duration of oocyte-CC gap-junctional communication during the meiotic resumption phase [28, 29, 30], which in turn extends the exchange of regulatory factors and metabolites between the oocyte and the cumulus cells .
Although IVM research has not yet revolutionized IVM systems in the clinical setting, improved IVM systems are under way. Lessons have been learnt from experiments in animal models, where the addition of oocyte growth factors, such as Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) can result in substantially higher numbers of blastocysts . An alternative approach to enhance oocyte potential during maturation in vitro is based on modulation of cyclic adenosine 3′,5′-monophosphate (cAMP). Studies of systems involving cAMP modulators (IBMX and Forskolin) in the human model have revealed serious practical hurdles by the unexpected interaction with heparin (which is used routinely during egg collection to prevent the formation of blood clots in follicle aspirates) . A recent study has shown that the introduction of a prematuration culture (PMC) system that blocks meiosis using C-type Natriuretic Peptide (CNP) has the potential to narrow down the efficiency gap between IVM and conventional ART (Figure 31.2) . Oocyte maturation in vitro after PMC incubation appeared to result in increased blastocyst rates, which is likely to be related to maintenance of cumulus-oocyte transzonal projections (TZP) which can sustain the dynamic changes in oocyte chromatin remodeling.
Figure 31.2 Prematuration: an intriguing focus of IVM research. Spontaneous meiotic resumption occurs when the connections between cumulus and oocytes are not maintained after removal from the follicle. A prematuration phase should enable oocytes to remain under meiotic arrest while cumulus-oocyte connections are preserved. This phase of prematuration creates a capacitation window for the oocyte. During this phase, different factors may be supplemented to enhance oocyte potential, such as cumulus cell regulators and nutrients.
Although IVM is usually applied in the context of infertility and assisted reproduction, fertility preservation before cancer treatment represents another emerging indication for IVM, more specifically when there is no time for standard hormonal stimulation. Since antral follicles are present in the ovaries of women of reproductive age irrespective of the menstrual cycle, immature oocytes can be readily obtained from these antral follicles at any time during the cycle. This makes IVM a highly suitable technique to be applied in emergency situations when the start of gonadotoxic treatment is imminent.
In a prospective study in 248 breast cancer patients who were planned to have neoadjuvant chemotherapy, Grynberg et al. demonstrated that immature oocytes can be obtained from antral follicles in the follicular and luteal phase of the cycle . In this largest series of cancer patients undergoing IVM for FP so far, with a mean age of 31.5 ± 0.3 years, a mean number of 6.4 ± 0.3 mature oocytes were cryopreserved after IVM, which was comparable to the number of mature oocytes available for cryopreservation after standard ovarian stimulation with letrozole co-treatment in patients with breast cancer who were scheduled to have adjuvant chemotherapy. Nevertheless, based on a recent retrospective study in cancer patients, the developmental potential of in vitro matured oocytes is still inferior to that of oocytes that matured in vivo . Oocytes can also be retrieved from extracorporeal ovarian tissue from cancer patients , to mature in vitro, be fertilized, and result in live births [38, 39]. Cryopreservation of ovarian cortex, followed by ovarian tissue transplantation when the patient has become infertile after cancer treatment, is still considered experimental, although more than 130 live births have been reported following ovarian cortex transplantation procedures with a live birth rate per patient of 25% . Ovarian tissue cryopreservation is the only available method of fertility preservation in prepubertal girls and is also applied in post-pubertal girls and young women when there is no time available for ovarian stimulation to harvest mature oocytes. After ovariectomy, during tissue processing in the lab, small compacted oocyte–cumulus complexes can be retrieved from the laboratory dish or through direct aspiration from visible follicles (Figure 31.3). These oocytes are an additional source of gametes for cryopreservation. So far, at least 15 publications reporting this approach in a total number of 240 patients have documented this combined strategy of fertility preservation . Based on published series, on average 14.7 oocyte–cumulus complexes were retrieved from extracorporeal ovarian tissue and available for IVM. It appears that the retrieval of oocyte–cumulus complexes ex vivo is independent of the phase of the menstrual cycle or the use of oral contraceptives. These complexes can be incubated in specifically designed IVM media and result in mature oocytes, with an overall maturation rate of 39% according to the literature. Although this approach has only resulted in a low number of reported live births so far, it holds promise for the future of the large number of patients who undergo ovarian tissue cryopreservation but who have a high risk of malignant cell reintroduction after ovarian tissue grafting, for example, patients with leukemia . Nevertheless, although this combined approach is theoretically also feasible in prepubertal children, and although a proportion of immature oocytes from these children have reached full nuclear maturation in vitro, the true potential of this approach in prepubertal cancer patients is unknown. Although there is evidence from in vitro studies that ovaries from prepubertal girls harbor a large proportion of follicles with abnormal morphology, there is no information about the developmental capacity of immature oocytes derived from pre-antral and antral follicles in prepubertal ovaries . Nevertheless, the report of the first live birth in a patient who had her ovarian tissue harvested and cryopreserved before menarche illustrates that oocytes embedded in prepubertal ovaries can be cryopreserved and that fertility can be restored in these patients .