In vitro Maturation (IVM) Perspectives

Abstract

The basic concept of in vitro maturation (IVM) of oocytes in practice clinic consists of the collection of immature oocytes from small antral follicles before spontaneous ovulation and then left to mature in vitro. IVM is based on the observations of Pincus and Enzmann in 1935 and Edwards in 1965, which highlighted the spontaneous nuclear maturation of the follicles when they were removed from their ovarian context and matured in vitro, and these first discoveries of in vitro folliculogenesis laid the foundations for the present research on the technique of in vitro maturation. In vitro folliculogenesis represents not only the possibility of extending the availability of female gametes in terms of the number of fertilizable oocytes but also a model within which to understand the complex mechanisms that regulate the synergistic development between the follicle and the female gamete. Deeper understanding of the complex orchestration of maturation, nuclear and cytoplasmic, of the oocyte based on research of bases on animal oocytes allowed the clinical application of the IVM technique to begin in reproductive medicine.

1. Introduction

The in vitro maturation method (IVM) is a new approach to the treatment of infertility. The aim is to reduce the cost of drugs in IVF procedures and the risk of severe forms of OHSS. In order to establish the optimal protocol, historically, the method had undergone a different transformation, starting with the rejection of the use of gonadotropins for stimulation to a method with minimal application of gonadotropins. In recent years, the application of this method has decreased, but IVM will find its place and remain one of the main methods of infertility treatment.

The benefits of IVM in clinical practice have been widely recognized in, first of all, the lack of the use of gonadotropins for controlled ovarian stimulation and consequently the absence of the risk of ovarian hyperstimulation syndrome especially in women with polycystic ovary syndrome (PCOS).

In vitro folliculogenesis represents not only the possibility of extending the availability of female gametes in terms of the number of fertilizable oocytes but also a model within which to understand the complex mechanisms that regulate the synergistic development between the follicle and the female gamete.

Deeper understanding of the complex orchestration of the nuclear and cytoplasmic maturation of the oocyte based on the basic research in animal oocytes allowed us to begin the clinical application of the IVM technique in human reproductive medicine.

The most promising technique to avoid ovarian hyperstimulation syndrome (in women with PCOS and non-PCOS) is to bypass the entire controlled ovarian stimulation phase by culturing immature oocytes to produce fertilizable eggs in a process called in vitro maturation.

However, in vitro maturation is not yet fully optimized universally in various medically assisted reproduction centers.

The mature human oocyte is the key ingredient for fertility, highly specialized in the process of oogenesis, which includes growth, differentiation, and maturation of the female gamete.

There are multiple differences between in vivo maturation and in vitro maturation of oocytes. Unlike oocytes matured in vivo, the oocyte cumulus complex (CCO) is typically recovered from medium antral follicle dimensions that have not reached complete “oocyte capacity,” the mechanism that describes the changes that occur in the oocyte of the dominant follicles before the LH hormone peak that allows the oocyte to achieve full development competence [1, 2].

In the IVM technique, the oocytes are inevitably retrieved from the follicles in various stages of development, ranging from antral to atresic follicles, and grown in optimal conditions. By understanding the recovery meiotic division as well as through increased optimization of culture media with specific formulation for the oocytes maturation in vitro, we will be able to develop IVM techniques in optimal conditions, not only to enhance the competence for the development of oocytes but also to improve the results in terms of an increase in the fertilization and implanting rates.

The growth and quality of the oocytes depend on the normal development and its differentiation process. However, the egg itself also carries out a direct function in the follicular environment, for example, by preventing early luteinization, by regulating both the secretion of the cumulus oophorus cells and the cumulus matrix. Another direct function of the oocyte includes the expression of LH hormone receptors on the cells of the cumulus and granulosa cells. The oocyte-cumulus complex is aspirated from the small antral follicles for the IVM technique with the aim of replacing intrafollicular conditions.

For the success of IVM, there are various factors that influence the competence for the development of the oocyte in vitro, which include the choice of culture medium, additives such as serum, various growth factors, as well as the somatic cells that make up and surround the oocyte. The various culture media were originally proposed for somatic cells, and for this, there is a clinical need for formulation of specific culture media for the in vitro maturation of human oocytes.

2. Oocytes maturation

The maturation of oocytes is a complex process involving the nuclear maturation (the progression of the meiotic cycle) and cytoplasmic maturation.

In vitro studies have provided information about the importance of substances that affect the maturation of oocytes and its inhibition such as cAMP, growth factors, gonadotropins, purines, and steroids.

The development of the oocytes is gradually acquired during their prolonged period of growth in which the oocytes remain arrested in the same phase of first meiosis but undergo a noticeable increase in volume and alterations in cellular behavior; this is indicated by an intense metabolic activity, which, in turn, is reflected in marked biosynthetic changes and ultrastructural variations of oocytes. It is around this time that many of the macromolecules essential for further development, both before and after ovulation, get produced and accumulate within the oocyte; in addition, the rate of protein synthesis and total protein increase in parallel with the expansion of cell volume.

IVM differs from in vivo oocyte maturation in three fundamental ways.

Firstly, the cumulus-oocyte complex is usually collected from small or medium-sized antral follicles (6–12 mm of diameter), which have not completed their final maturation or capacitation of oocytes and, therefore, do not have the necessary organization for cytoplasmic maturation to support early embryogenesis [1, 2].

Secondly, the mechanical removal of the oocyte-cumulus complex from the follicle would lead to the loss of the natural environment of meiotic inhibition with the resulting spontaneous or “premature” meiotic maturation in vitro. In this way, nuclear maturation occurs before the cytoplasm has reached full maturity.

Thirdly, the population of small antral follicles collected from the oocyte-cumulus complex for IVM is very heterogeneous regarding their stages of developments and atresia.

The oocyte acquires an increasingly greater competence in a gradual and progressive manner as it passes through the various stages of folliculogenesis, from the moment in which the oocyte begins to grow through the differentiation of the somatic cells that surround the oocytes.

For this reason, it is important that the egg cell is adequately protected from the action of all chemical or physical agents able to damage it and compromise its development [3, 4]. This primary role is played by the follicular cells surrounding the female gamete, creating around it a microenvironment suitable for correct maturation [5, 6].

The oocytes are arrested in the prophase of the first meiosis during the prenatal life for several years until sexual maturity is reached, and the follicular growth is completed by the actions of gonadotropins [7]. However, the oocytes will be able to resume meiosis spontaneously when they are removed from the follicles [8]. This shows the presence of inhibitory molecules in the follicle that keep the oocytes in arrested meiotic.

In addition, several possible inhibitory molecules such as intracellular 3′,5′- cyclic adenosine monophosphate [cAMP], Hypoxanthine, steroid hormones, and several other factors derived from the granulosa cells have a role in keeping the oocytes meiotic arrest [9, 10].

A sophisticated complex of numerous intercellular interactions, including growth factors, cAMP, and gap junction between oocytes and granulosa cells, are involved in the arrest process in the first phase of meiosis in vivo.

3. Nuclear maturation

Nuclear maturation typically includes the period between recovery of the meiosis of an oocyte, which was arrested in prophase I, and the passage to metaphase II (MII) stage when the oocyte undergoes a new arrest pending fertilization.

Morphologically, these events are represented by the rupture of the germinal vesicles (GV) and asymmetric cytoplasmic division of the oocyte subsequent to the extrusion of the first polar body (PB). The germinal vesicle (GV) is the nucleus of the oocyte.

4. Cytoplasmic maturation

Cytoplasmic maturation can be defined as the process in which the female gamete passes from an incompetent evolutionary cell to a state of functional capacity of addressing and supporting the events of fertilization and early embryonic development, and it was first described by Delage in 1901 to clarify that the concept of cytoplasmic maturation was not necessarily synchronous with nuclear maturation [4].

Assisting at the microscopic level, the process of cytoplasmic maturation is more difficult compared to nuclear maturation, which is a relatively evaluable process at the microscopic level.

Cytoplasmic maturation involves the accumulation of mRNA and proteins and post-translational modifications that are necessary to achieve a competence in the development of the oocyte. In addition, a number of cytoplasmic organs (Golgi complexes, mitochondria, endoplasmic reticulum) proliferate in ooplasm as a result of their peripheral dislocation, which is regulated from a system of microtubules [4].

5. Role of gonadotropins in meiotic recovery

The signals involved in the resumption of meiosis in the oocyte are little known, but two experimental in vitro models provided the framework for a large part of our understanding of this process.

In 1935, Pincus and Enzmann observed that mammalian oocytes spontaneously resumed meiosis when removed from the follicular environment.

This observation led to the hypothesis that follicle cells sent inhibitory signals to the oocyte to maintain meiotic arrest.

Other studies have shown that LH promotes the maturation of the oocytes indirectly through the activation of granulosa cells [11, 12].

In vivo, meiosis resumes in response to a pre-ovulatory luteinizing LH hormone increase; in this way, the primary oocyte completes the first meiotic division and stops at the level of II° metaphase with the formation of two haploid cells:

• a cell that preserves almost all of the oocyte’s cytoplasm, called secondary oocyte

• a cell with very little cytoplasm, called the 1st polar globe.

6. Cellular communication and gap junction

The oocyte and follicular cells are intimately associated and are in communication across a vast network of gap junctions and are composed of membrane protein structures called connexins. Several types of different connexins have been located in the ovarian follicle [13].

Gap junctions play an important role in maintaining the oocyte in meiotic arrest by transferring inhibitory molecules such as cAMP, which are generated from somatic follicular cells [14].

Recently, it has been supposed that heterologous gap junctions play a role in the regulation of oocyte chromatin configuration [15].

The communication between the oocyte and the granulosa cells is possible through the presence of the connexins (Cx 43). Experimental evidence has shown that in mice with deficiency of the connexin 37 gene, there is no formation of Graafian follicles, the ovulatory process fails, they develop many inappropriate corpora lutea, and also the growth of the oocyte is arrested earlier before it reaches maturation competences. This shows that the intercellular communication via gap junctions regulates and coordinates critically the complex mechanism of cellular interactions for the maturation of oocytes [16, 17].

The second mechanism of communication between oocyte and granulosa cells is mediated by paracrine factors (Figure 1), which play a fundamental role in directing the growth and differentiation of ovarian follicles. Paracrine factors secreted by the oocyte are essential for the expansion of cumulus cells and to keep their own phenotype [19].

Figure 1.

Communication between oocyte and somatic cells is essential for the growth and development of both the female gamete and the follicle. This bi-directional communication axis is mediated by paracrine factors (solid arrows) and by the exchange of small molecules via gap junctions (dashed arrow) [18].

Most of the studies concerning paracrine factors have focused on some members of the transforming growth factor β (TGFβ) superfamily, such as Growth Differentiation Factor 9 (GDF9), Bone Morphogenetic Protein 15 (BMP15), and others. The great interest in these paracrine factors is mainly due to the fact that an alteration of the expression of their respective genes greatly impairs ovarian function and fertility [18, 19, 20].

Studies carried out on knockout mice have shown that deletions at the level of such factors involved in the proliferation of granulosa cells, in particular of GDF9, induce the production of a sterile phenotype in which the development of the follicles is arrested, obtaining a single layer of cells that delimit a large oocyte [21]. This demonstrates how granulosa cells need GDF9 for their own proliferation.

The feedback communication between oocyte and somatic cells is also needed to coordinate the resumption of meiosis by the female gamete and the ovulation process. Immature or non-competent oocytes have been shown not to interact in appropriate manner in this communication system and do not progress to ovulation [22, 23].

Although several studies have highlighted important aspects of the relationship between oocyte and somatic cells, much remains to be investigated regarding the cellular communication pathways that are selectively activated. Since this bidirectional communication arrangement seems to be a prerequisite to ensuring proper oocyte development, it is important to know and study the molecular basis of such events.

7. The role of the cyclic AMP (cAMP)

The cyclic adenosine monophosphate (cAMP) is the second messenger for the transduction of the signal of gonadotropins. The FSH and LH hormones exert their biological function, activating membrane receptors of target cells and, consequently, activating adenyl cyclase, which leads to the production of cyclic AMP, which is one of most important intracellular signaling molecules that is responsible for the maintenance of meiotic arrest in the oocytes.

Resumption of meiosis occurs after a drop in cyclic AMP levels; the cAMP acts as a regulator of gap junction communication [24].

Phosphodiesterases (PDEs) are important regulators and play a critical role in the maturation of oocytes and their meiotic recovery. In mammals, the Phosphodiesterases (PDE) constitute a large family of various isoenzymes and are classified into 11 subtypes, PDE1–PDE11. Their regulation is cell-tissue specific.

The mechanisms by which cAMP maintains meiotic arrest are related to the diffusion of cAMP from somatic cells (granulosa and cumulus cells) to the oocyte and the increase in the intra-oocyte level of cAMP, preventing the maturation of oocytes [25].

The precise mechanism by which the intracellular concentrations of cAMP may produce a stimulation or an inhibitory response in the oocyte during the meiosis is not entirely clear.

As previously discussed, high levels of cAMP keep the oocyte in the meiotic arrest, and this is supported by in vitro studies.

Adding substances to the culture media able to maintain a high level of intracellular cAMP or agents that prevent the degradation of cAMP will maintain meiotic arrest of oocytes. The stimulatory or inhibitory effect of cAMP is dependent on the levels of cAMP in the different compartments of the follicle.

The mammalian oocyte acquires a series of competencies during the follicular development, involving chromatin remodeling occurring in the germinal vesicles (GV). The chromatin configuration in the germinal vesicles is correlated with increased competence in the development of oocytes in different mammalian species in which diffuse chromatin condenses into a perinuclear ring.

Ovarian folliculogenesis is regulated by a delicate balance between several intraovarian factors. An imbalance or any dysfunction between these various factors causes abnormal folliculogenesis and, consequently, directly compromises the competence of oocyte development.

It appears that the interactions between hormones and growth factors produced locally in the follicular microenvironment are highly organized, and the timing and extent of these interactions are pivotal to establishing the intrafollicular cascade of the ovarian follicle development.

Albuz et al. [26] evaluated the role of cyclic AMP modulators added to pre-IVM of bovine or mouse cumulus-oocyte complexes (COCs) and observed an almost 100-fold increase in COCs’ cyclic AMP levels. With this technique, they simulated the physiological maturation of the oocytes, giving the definition of “simulated physiological maturation of the oocytes” (SPOM). SPOM imitates oocyte maturation in vivo and has benefits for IVM, which can be used in IVM protocols to optimize clinical outcomes [26].

8. Epidermal growth factor (EGF)

Epidermal growth factor (EGF) is a growth factor that plays an important role in the regulation of cell proliferation and differentiation [27]. In the human oocyte, EGF is found in the follicular microenvironment (Figure 2) regulating the development and maturation of oocytes [27, 28, 29].

Figure 2.

Epidermal growth factor (EGF)-like growth factors in the human follicular fluid. Luteinizing hormone (LH) induces the expression of AREG in the preovulatory follicle, and AREG (amphiregulin) acts in an autocrine and paracrine manner to mediate LH effects throughout the follicle, including the promotion of oocyte meiotic resumption and cumulus expansion [27].

In vitro studies show that exposure of the cumulus-oocyte complex (CCO) to the growth factor EGF stimulates the expansion of cumulus cells (CC) and improves nuclear and cytoplasmic maturation of oocytes from metaphase I (MI) to metaphase II (MII) both in human oocytes and in other mammals, facilitating the fertilization and embryonic development [30].

Other studies suggest that EGF levels in the follicular microenvironment have an inverse correlation with oocyte maturation [27, 31, 32].

In women with polycystic ovary syndrome (PCOS), EGF levels in the follicular microenvironment are higher compared to non-PCOS women, and this may suggest the role of the EGF factor in the maintenance of the PCOS phenotype [33, 34].

The EGF inhibits estrogen synthesis in granulosa cells and is involved in the maintenance of the PCOS phenotype with the arrest of follicle growth in PCOS women [34].

Therefore, it is assumed that an alteration of the regulation of EGF synthesis and/or its action mediated through its specific receptor (EGF-R) can cause anovulatory infertility in women with polycystic ovary syndrome [28].

The correlation between high levels of EGF growth factor in the follicular microenvironment and the quality and competence of the oocytes is yet to be clarified.

In addition, other various factors called EGF-like factors have been identified such as amphiregulin, epiregulin, and betacellulin in the follicular microenvironment [35, 36]; however, the specific physiological function of the various EGF-like factors in PCOS patients remains unknown.

9. Fibroblast growth factor (FGF)

Fibroblast growth factors (FGFs) are a group of polypeptides that play a fundamental role in cell growth, development, tissue repair, and cell transformation. They are expressed in the granulosa (GC) and theca cells of growing follicles and are considered physiological regulators of FSH action [34, 37].

Recent studies have revealed high levels of fibroblast growth factor (FGF) in the follicular microenvironment and serum of polycystic ovary syndrome patients (PCOS) versus non-PCOS patients, leading to an inverse correlation of oocyte maturity; this contributes to alterations in the intrafollicular environment with consequent arrest of follicular development in patients with polycystic ovary syndrome [34, 37].

Therefore, the alterations of FGF in the follicular microenvironment and in the serum remain controversial, and the impact of FGF on the maturation of oocytes and the embryonic development requires further elucidation in PCOS patients.

10. Transforming growth factor-β family (TGF-β family)

Among the many growth factors in the intrafollicular microenvironment, the various members of the TGF-β family play an important biological role in the growth of follicle and oocyte development. These members of the TGF-β family include: anti-Mullerian hormone, activin, follistatin, inhibin, and growth differentiation factor-9 (GDF-9).

Under different physiological conditions, the various TGF-β family factors can promote or block the growth of the ovarian follicle and/or the differentiation of the granulosa-oocyte complex that is related to the pathogenesis of PCOS [38, 39, 40].

11. FF meiosis-activating sterol (follicle fluid meiosis-activating sterol)

FF meiosis-activating sterol (FF-MAS) is an endogenous signaling molecule present in the follicular microenvironment of the oocyte and is an intermediate metabolite in cholesterol biosynthesis [41].

Many in vitro studies show that FF-MAS exposure can promote nuclear and cytoplasmic maturation of the oocyte [42] and improve the fertilization rate [41, 43, 44].

Recent in vitro studies have showed that FF-MAS improves the quality of oocytes retrieved from women with PCOS to undergo the IVM technique [41, 45].

12. Growth differentiation factor-9 and bone morphogenetic protein-15 (GDF-9/BMP-15)

Growth differentiation factor-9 and bone morphogenetic protein-15 (GDF-9/BMP-15) are members of the transforming growth factor beta (TGF-β) superfamily and are highly expressed in oocytes during their development and growth [46, 47].

BMP-15 and GDF-9 have a fundamental role in regulating the functions of cumulus cells (CC) through the process of mitosis, proliferation, apoptosis, and the signal transduction mechanism [38, 46, 47].

Data from in vitro experiments on animal models show that coincubation of cumulus cells(CC) with either BMP-15 or GDF-9 greatly promotes the maturation of oocytes and improves the production of blastocytes [6].

An altered expression of BMP-15 or GDF-9 during folliculogenesis can be related to female infertility [46, 48, 49] and an increase in correlations with the pathogenesis of polycystic ovary syndrome (PCOS) [46, 48, 50, 51].

A correlation was observed between a high BMP-15 level in the follicular fluid and an improvement in oocyte quality, higher rates of fertilization, and embryonic development in women who underwent to IVF, suggesting that BMP-15 can be a good indicator of the maturity of the oocyte and its potential for fertilization [52].

GDF-9 expression in cumulus cells is lower in patients with PCOS and can lead to premature luteinization and decreased oocyte development [34, 53].

The decreased expression of GDF-9 in cumulus cells (CC) can also be related to the high rate of miscarriage in women with polycystic ovary syndrome syndrome [51].

The expression of BMP-15 and GDF-9 in oocytes and cumulus cells (CC) may provide valuable support for the regulation of the follicular microenvironment during the maturation process of the oocytes.

A recent study has demonstrated that the expression of GDF-9 and BMP-15 tends to be higher in PCOS patients when compared with a control group and therefore may be involved in follicular dysplasia in PCOS [51].

Further studies on the role of BMP-15 or GDF-9 in folliculogenesis will be essential in understanding those factors involved in the regulation of the pathogenesis of PCOS and help to improve in vitro oocyte maturation (IVM) in women with PCOS.

13. Optimizing in vitro maturation (IVM) in clinical practice and outcomes

It is actually not surprising that current in vitro culture systems fail to support the differentiation of oocytes with their maximum potential development. In fact, only some of the follicular characteristics are maintained in vitro by culture of the intact cumulus-oocyte complex (CCO) with the supplementation of certain growth factors in the culture media (Figure 3). In addition, the supports by the granulosa cells, the follicular fluid, the basal lamina, and theca cells are missing altogether in the phase of oocytes in vitro maturation. A strategy aimed to maintain meiotic arrest in vitro can allow events that take place in the cumulus-oocyte complex (COC) to progress further. Even with this strategy, however, the oocyte cannot remain under the influence of cumulus cells for as long as it would be in vivo, in addition to the interaction from other follicular compartments that are still missing.

Figure 3.

The absence of the antral follicle environment remains an obstacle to in vitro maturation (IVM) success and other clinical assisted reproductive technology (ART) protocols [54].

New approaches that more closely mirror the follicular conditions are essential.

Recently, a two-stage approach, the first phase of which includes the in vitro prematuration stage (pre-IVM) and the second phase includes prolongation of the in vitro maturation (extended-IVM) combined with the use of FSH and drugs that arrest meiosis, has shown better egg development in vitro [26]. In this way, a phase of prematuration of immature oocytes could be beneficial to enable the biochemical processes that accompany the cytoplasmic rearrangements to develop in a more physiological way (Figure 4). The possible strategy could be to adopt the in vitro follicle coculture system using specific pharmacological agents that act on the metabolism of the cAMP, on the synthesis protein, and the inhibition of phosphodiesterase.

Figure 4.

Conventional and biphasic IVM culture systems. (A) Conventional IVM system, only includes IVM culture phase. (B) Biphasic IVM system, includes a pre-IVM culture phase before IVM culture. The pre-IVM culture inhibits resumption of meiosis in immature oocytes and provides time for acquiring developmental potential. MII, metaphase II [55].

Egg retrieval during IVM has similar aspects to the conventional IVF technique, but smaller diameter needle and lower suction pressures are used generally to recover intact cells of the cumulus complex from small follicles. The diameter of the aspiration needle involved varies from 17 to 20 gauge, and the aspiration pressure is around 80 mmHg to avoid traumatizing the cumulus complex cells and denuding the oocyte.

During the oocyte retrieval in the IVF, the follicle curetting technique is frequently practiced, which consists of the rapid and delicate rotation of the needle clockwise and counterclockwise inside the follicle after the complete aspiration of the follicular liquid, with the advantage of an increased likelihood of aspirating all oocytes and a reduced risk of ovarian hyperstimulation syndrome (OHSS) secondary to the removal of more granulosa cells. This technique may enhance the oocyte yield by 22% [56].

IVM has attracted attention in clinical practice for its safety, repeatability, cost-effectiveness, and almost no risk of OHSS along with acceptable clinical pregnancy rates and live-birth rates [57].

Siristatidis et al. in a systematic review and meta-analysis reviewed IVM in patients with and without PCOS and concluded that IVM was an effective treatment option when offered to infertile women with PCOS [58].

Edwards conducted several studies [59, 60, 61] on the in vitro maturation of the human oocyte (IVM), and the first techniques of human IVF were based on the use of IVM. IVM is considered the progenitor of the current in vitro fertilization treatment [57, 62].

The collection of mature oocytes from preovulatory follicles in women with normal cycles became possible after the introduction of laparoscopy into gynecological practice in the 1970s [63] and with the advent of in vitro fertilization and the successful birth of Louise Brown; IVF with controlled ovarian stimulation has become a common practice.

Although the primary indication of IVM was in patients with polycystic ovarian syndrome (PCOS), IVM has much broader indications including poor ovarian reserve and repeated IVF failures [64]. IVM can also be used in cases of resistant ovary syndrome and fertility preservation [65, 66, 67].

Other potential indications of IVM may be in normo–ovulatory patients, patients with previous failed IVF attempts and a history of OHSS (ovarian hyperstimation syndrome), emergency oocyte retrieval due to malignant tumors in patients who are candidates for ovarian chemotoxic therapy, poor responders, and IVM for rescue IVF cycles.

In vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in an oocyte donation program after IVF in which Cha et al. reported the first IVM birth from immature oocytes egg donors [68].

In vitro maturation and developmental proficiency of oocytes retrieved from patients with untreated polycystic ovaries resulted in the first IVM from the mother’s own immature oocytes in 1994 [69]. Over 5000 babies have been born since then with IVM technique [70].

Seok et al. studied the predictive role of the anti-Müllerian hormone (AMH) on IVM selection in PCOS patients and concluded that AMH was a valuable factor in predicting clinical outcomes in such patients who preferred IVM as the treatment of choice [71].

Other predictive factors in IVM have been investigated and evidenced that Estradiol, FSH concentration, and AFC (antral follicular counts) were found to be predictive factors in the decision on whether to initiate IVM, and endometrial thickness and leading follicle size were predictive factors for the timing of the retrieval of immature oocytes [72].

IVM offers a possibility in cancer patients with a desire to preserve their fertility by retrieving immature oocytes in the luteal phase, which can be successfully matured in vitro; therefore, if there is insufficient time for a conventional retrieval of the follicular phase oocytes in a stimulated/unstimulated cycle prior to chemotherapy, a luteal phase retrieval could be considered as an option [73].

Factors influencing IVM of human oocyte are shown in Figure 5: the application of a biphasic IVM culture system, culture medium conditions, different protein sources, womens’ age, criopreservation, and different follicular priming methods. Prospective RCT studies in the future may put in place their specific roles and implications in IVM and the possibility for increasing the successful outcome rate of IVM.

Figure 5.

Factors influencing the IVM of human oocytes [74].

14. Conclusion

In the reproductive system of mammals, the development of oocytes takes place within the highly specialized microenvironment of an ovarian follicle. The follicle has the task of facilitating the complex and the delicate process of oogenesis.

Egg differentiation ultimately depends on the cooperation and coordination of the function of the antral follicle as a whole. By understanding the differentiation of the oocyte, we must clarify the functions of the compartments of the antral follicles as well as their relationship to each other.

Further efforts must continue to reveal how the components of the follicular microenvironment drive egg differentiation.

The ability of the oocyte to modulate its development is codependent between interaction between the oocytes and their respective follicles which presents a further obstacle to in vitro culture of oocytes.

The state of the art of the IVM technique attempts to replicate the essential components of the follicular microenvironment for the benefit of oocytes in vitro but must also try to understand how, when, and why the oocyte induces changes to this follicular microenvironment.

Although nuclear and cytoplasmic maturation of oocytes can proceed independently of each other, both processes must be coordinated in order to ensure the competence of oocyte development. Therefore, maintaining the transzonal connections between the granulosa cells and oocyte for a continuous exchange of substances and regulatory factors between the two cell compartments.

Maintaining the oocyte in a state of meiotic arrest using both coculture with pharmacological agents and providing growth factors and hormone supplements to support the completion of cytoplasmic maturation of the oocyte could theoretically lead to an improvement in development of the oocyte.

Knowledge of the molecular mechanisms of oocyte maturation are still Insufficient, and the culture media currently in use are not capable of supporting the complex paracrine events of in vitro maturation.

One possible strategy to improve oocyte-development competence in the IVM technique is to align meiotic and cytoplasmic maturation by delaying spontaneous meiotic recovery. It is speculated that this may provide the time for cytoplasmic changes (e.g., storage of mRNA and proteins, morphological changes, ultrastructural remodeling) and could improve the synchronization of immature oocytes.

The ovaries host various local growth factors involved in folliculogenesis, and the physiological significance of autocrine/paracrine regulation, the integrated effects of their action, and their implication in reproduction medicine remain to be established.

Oocyte quality is a key factor in female fertility, yet we have a poor understanding of what constitutes oocyte quality and the mechanisms that govern it. The ovarian follicular microenvironment through the granulosa cells (GC) and the cumulus cells (CC) is responsible for the growth and gradual acquisition of competence in the development of the oocyte; however, the communications between the oocyte, granulosa cells (GC), and cumulus cells (CC) are bidirectional, with the oocyte secreting growth factors acting locally to direct the differentiation and function of the cumulus cells (CC).

The ability of oocytes to regulate their own microenvironment constitutes one important component of oocyte quality, and improving our knowledge of the oocyte-cumulus cell (CC) interactions will improve IVM efficiency and thus provide new options for infertility treatment.

Establishing a global registry for all births with the IVM technique would be desirable for the long-term and follow-up of perinatal and postnatal outcomes.

By evaluating the cellular structures of the oocytes (such as the reticulum endoplasmic, the mitochondrion, and the Golgi apparatus), the sensitivities of the reserves of calcium, chromosome dynamics, and apoptosis during embryogenesis are essential topics for the structural study of the oocyte and its optimization of the IVM technique in clinical practice.

Overall, current efforts are focused on understanding the complex interaction between the oocyte and cumulus cells in an attempt to overcome the artifacts and to develop a system of in vitro maturation that is capable of supporting oocyte developmental competence.