*In vitro* Maturation, Oocyte Cryopreservation and Quality Management

#### **Chapter 4**

## *In vitro* Maturation (IVM) Perspectives

*Bassim Alsadi*

#### **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.

**Keywords:** In vitro maturation, oocyte maturation, infertility, antral follicles, folliculogenesis, nuclear and cytoplasmic maturation, polycystic ovary syndrome (PCOS)

#### **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.

#### In vitro *Maturation (IVM) Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109797*

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 mediumsized 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:


#### **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].

In vitro *Maturation (IVM) Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109797*

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].

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 [18–20].

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,

#### **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].*

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.

In vitro *Maturation (IVM) Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109797*

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 [27–29].

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

#### **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].*

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 [38–40].

#### **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.

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.

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

#### **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].*

#### **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].*

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, costeffectiveness, 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 [59–61] 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 [65–67].

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].

**Figure 5.** *Factors influencing the IVM of human oocytes [74].*

In vitro *Maturation (IVM) Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109797*

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.

#### **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 oocytecumulus 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.

### **Author details**

Bassim Alsadi Rome University, Rome, Italy

\*Address all correspondence to: balsadi@hotmail.com

© 2023 The Author(s). Licensee IntechOpen. 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.

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#### **Chapter 5**

## The Role of Oocyte Cryopreservation in Assisted Reproduction

*Timothy J. Gelety*

#### **Abstract**

Oocyte cryopreservation (OC) has progressed rapidly from an experimental procedure with limited success to a clinically accepted procedure, in large part due to significant improvements in the techniques and widespread laboratory adaptation of vitrification. With significant improvements in clinical outcome, elective oocyte cryopreservation has gained in popularity as a means of overcoming diminishing ovarian reserve associated with aging. With clinical pregnancy rates equal to utilizing retrieved oocytes, oocyte cryopreservation is being increasingly utilized as an adjunct to standard IVF and now plays a significant role in egg donation with the establishment of egg banks analogous to sperm banks. Continuing research and clinical experience will be instrumental in defining the role of OC going forward.

**Keywords:** oocyte cryopreservation, Vitrification, elective oocyte cryopreservation, decreased ovarian reserve, egg donation, egg banking

#### **1. Introduction**

The Nobel Prize winning work of RG Edwards lead to the first successful pregnancy resulting from in vitro fertilization (IVF) and the birth of Louise Brown in England in 1978 [1]. Numerous improvements in the technique of controlled ovarian hyperstimulation (COH), as well as laboratory fertilization and culture technique, subsequently resulted in rapid improvement in pregnancy success and worldwide clinical acceptance. Although the cryopreservation of mammalian embryos was originally described as early as 1947 [2], the first successful live births from cryopreserved/thawed embryos following IVF were reported in 1983 by Alan Trounson's group [3]. Cryopreservation of embryos following IVF quickly gained clinical acceptance by increasing overall pregnancy success and decreasing the need for additional procedures.

Cryopreservation of unfertilized human oocytes for the purpose of fertility preservation has always been an attractive alternative to cryopreservation and storage of human embryos, posing fewer ethical, moral, religious and legal problems. However, initial attempts at oocyte cryopreservation based on the laboratory success of cryopreservation of fertilized embryos was disappointing, resulting in only a handful of successful live births by 1987 [4].

Unfertilized oocyte cryopreservation had been vexed with the technical problems of potential meiotic spindle disruption, possibly resulting in aneuploidy when traditional slow freezing utilizing cryoprotectants was used [5]. In addition, although acceptable rates of freeze–thaw survival were observed, poor fertilization with IVF was common, as was polyspermic fertilization [6]. These problems were found to be a result of changes in the zona pellucida associated with cryopreservation [7]. As such, the procedure did not attain initial widespread clinical acceptance and was relegated to being considered an experimental procedure for many years.

The hurdle of cryopreservation of mature unfertilized human oocytes was overcome by the use of ultra-rapid freezing or "vitrification". Significant improvement in fertilization of cryopreserved-thawed oocytes was the result of the widespread clinical application of intracytoplamsic sperm injection (ICSI), where a single sperm is injected through the zona pelucida into the ooplasm, overcoming the problems of poor fertilization and potential polyspermic fertilization. As a result, many live-births have been achieved over the last 25 years, and the safety and efficacy of the procedure was confirmed [8, 9]. Of interest, following thousands of live-births world wide, the procedure was deemed no longer "experimental" in 2012 by the American Society for Reproductive Medicine (ASRM), leading to widespread clinical acceptance of the procedure [10].

Oocyte cryopreservation has long been suggested as a means of fertility preservation in young women wishing to delay childbearing into their fourth and fifth decades. Likewise, fertility preservation is paramount in young women faced with potentially sterilizing procedures such as chemotherapy or radiation treatment, as well as surgery associated with modern oncology treatments. Cryopreservation of mature unfertilized oocytes can also be a valuable adjunct in IVF, particularly when sperm for fertilization is not available on the day of oocyte retrieval. Finally, oocyte cryopreservation has the potential to play a significant role in oocyte donation: adding convenience as well as screening and expanded donor selection choices, analogous to sperm donation using commercial sperm banks.

#### **2. Vitrification**

Fertilized cleavage or blastocyst stage embryos contain a diploid chromosomal complement encased within the nucleus of each cell. Cryopreservation of embryos was done originally via slow freezing using cryoprotectants that were required to avoid damage associated with cooling to sub-zero temperatures. Cryoprotectants, either permeating such as propanediol (PROH), glycerol, or dimethysulfoxide (DMSO) or non-permeating such as sucrose, were utilized to displace intracellular water and avoid cellular damage from intracellular ice formation. Seeding, to induce ice formation outside the specimen, was followed by more rapid cooling to −196 degrees Celsius and storage in liquid nitrogen [11].

In 1985, Rall and Fahey introduced the process of vitrification for the cryopreservation of mammalian embryos [12]. Using higher concentrations of cryoprotectant and rapid cooling results in vitrification (glass formation), thereby avoiding intracellular ice formation. Trounson subsequently described ultrarapid freezing techniques in human embryos [5]. The potential toxicity of cryoprotectants at higher concentrations at room temperature requires their addition at lower temperatures, but the rapid exposure to low temperatures obviates the need for costly programmed biological freezing equipment. Significant advances in the techniques of vitrification have resulted in widespread clinical acceptance [13].

Unlike embryos, immature human oocytes, which are arrested in the diplotene stage of the first meiotic prophase, are recruited by gonadotropin stimulation at the

#### *The Role of Oocyte Cryopreservation in Assisted Reproduction DOI: http://dx.doi.org/10.5772/intechopen.107624*

beginning of the follicular phase at which time they experience significant antral growth or atresia. Developing antral follicles morphologically exhibit the germinal vesicle (GV) by light microscopy which contains the chromosomal complement. The oocyte resumes meiosis in response to the midcycle luetenizing hormone (LH) surge, as evidenced morphologically by extrusion of the first polar body which can be seen beneath the zona pellucida (ZP). The mature oocyte therefore demonstrates evidence of germinal vesicle breakdown (GVBD) which is associated with the appearance of the spindle apparatus and the presence of the first polar body, but remains arrested in the second meiotic metaphase (MII). With fertilization, meiosis is completed and the second polar body is extruded. The polar body contains the discarded haploid chromosomal complement of the oocyte, which is visible beneath the zona pelucida followed by formation of 2 pronuclei (2pn), visible morphologically on light microscopy.

Because MII oocytes contain chromosomes which are still attached to microtubular spindle, there were concerns regarding disassembly of the meiotic spindle and dispersal of the polar pericentriolar material upon freezing and thawing that could result in an increase in chromosomal aneuploidy. Early studies of murine oocyte ultrastructure using transmission electron microscopy (TEM) have shown little adverse effect on the meiotic spindle structure following freezing/thawing either with slow cooling or ultrarapid cooling [14]. Likewise, reports of cytogenetic evaluation following oocyte freezing/thawing followed by IVF have been reassuring in both mouse and in human [8, 9], suggesting no significant increase in meiotic nondisjunction resulting in aneuploidy.

Although early reports by Chen [15] suggested excellent survival following cryopreservation of mature oocytes, with the first successful pregnancy in humans reported in 1986, poor fertilization following freezing and thawing limited the clinical success of the procedure. Poor fertilization of previously cryopreserved mature oocytes as well as problems with polyspermic fertilization have suggested changes to the ZP, such as hardening of the zona, possibly resulting from premature cortical granule release associated with changes to the oolema as a consequence of freezing and thawing. TEM evaluations of cryopreserved/thawed oocytes have shown no evidence of premature cortical granule release [6], however using TEM studies in mammalian oocyte have revealed cracks in the ZP following freezing and thawing. This suggests that physical changes in the glycoprotein architecture of the ZP may be the cause of "hardening," preventing normal fertilization or conversely for polyspermic fertilization.

Intracytoplasmic Sperm Injection (ICSI), in which a single viable spermatozoa is injected through the ZP into the oolema, was developed as treatment for severe male factor infertility and resulted in the first live births in 1992 [16]. The development of the technique followed earlier attempts at zona drilling and sub-zonal insertion of sperm to enhance fertilization, which were also plagued by polyspermic fertilization. Extensive experience with ICSI has demonstrated excellent fertilization rates, often higher than seen with standard IVF [17] and obviates the problem of polyspermic fertilization through the injection of a single sperm. Based on these results, ICSI was subsequently found to provide high rates of normal fertilization in previously cryopreserved oocytes [18], resulting in the first live birth in 1997 [19].

#### **2.1 Fertility preservation prior to cancer treatment**

Over the past 50 years; significant advances in cancer therapies, and in particular chemotherapy, has resulted in major improvement in long-term patient survival [20]. Chemotherapy and radiation therapy as well as gonadectomy can decrease the

reserve of viable oocytes, resulting in immediate or premature ovarian failure (POF). The type of chemo/radiotherapy, duration, cumulative dose and patient age, have all been shown to predict POF [21]. As survival for cancer patients continues to improve, counseling prior to potential iatrogenic infertility due to planned oncology therapy in reproductive age patients has become a clinical necessity regarding the available options for fertility preservation prior to treatment [20].

For long term cancer survivors who have experienced POF due to gonadotoxic therapies or even gonadectomy, options for having children include adoption or oocyte donation. For reproductive age women in a stable committed relationship, standard IVF utilizing COH followed by fertilization using her partner's sperm and cryopreservation and storage of resulting embryos is a reasonable option for women wishing to preserve the chance of having their own biologic offspring. COH and egg retrieval can be accomplished in a relatively short time period (14–21 days), allowing time to schedule prior to starting chemotherapy, radiation therapy, or surgery, and can even be accomplished successfully after early potential gonadotoxic therapy has begun [22]. Cryopreservation of embryos offers a predictable likelihood of pregnancy success based on the age of the patient as well as the number and the quality of embryos stored. Although data on live birth rates from stored embryos prior to cancer therapy are limited, patients can be counseled based on live birth rates following use of cryopreserved embryos from the general infertility population [20].

Oocyte cryopreservation has the advantage of not requiring a partner for single women facing cancer therapy and has fewer ethical, moral, religious and legal problems than the current widespread cryopreservation of embryos. Mature oocyte cryopreservation also requires COH using gonadotropin therapy, followed by out-patient surgical oocyte retrieval and cryopreservation. Like cryopreservation of embryos, information on the pregnancy success rates following fertilization of mature oocytes from cancer patients is limited. It is clear that the age of the patient at vitrification and the number of oocytes stored are predictors of pregnancy success [23]. Live birth rates from donor and infertile patients can be a guide to counseling cancer patients however, with live birth rates as high as 46.8% reported for women less than 35 years of age [20].

For prepubertal and adolescent women facing cancer treatment, gonadotropin therapy and egg retrieval are not reasonable options for obtaining multiple mature oocytes for cryopreservation. However, ovarian biopsy, in which ovarian cortical tissue containing several hundreds or thousands of immature oocytes can be obtained for cryopreservation, is an option. Laparoscopic Ovarian Biopsy (LOB) has been shown to be a safe and effective method for obtaining ovarian tissue for cryopreservation [24]. It can be performed at the time of general anesthesia for lymph node biopsy or central line placement, immediately prior to planned cancer therapies. Histologic evaluation from ovarian biopsies performed in pre-pubertal or adolescent females have shown viable immature oocytes, even after initiation of conservative chemotherapy [24]. This allows for obtaining ovarian tissue for cryopreservation prior to more complete myeloablative therapies such as total body irradiation used in preparation for bone marrow transplantation (BMT) [24].

Due to the complexity and limited success of in vitro maturation, frozen–thawed immature human oocytes, like those found in cryopreserved ovarian tissue, require in vivo maturation [25]. This involves reimplantation of autologous cryo-thawed ovarian tissue back into the patient, survival of the autologous graft, and normal maturation of oocytes which can be harvested and used in conjunction with standard IVF to achieve pregnancy and live-births [26].

Two competing strategies have been championed. Oktay and others have pursued reimplantation of the previously cryopreserved-thawed ovarian tissue into the forearm [27].

This is analogous to the procedure used for preserving parathyroid gland function following total thyroidectomy. Technical problems related to monitoring the tissue, poor graft survival, and difficulty retrieving mature eggs, have limited the widespread acceptance of this procedure over the years.

An alternative option involves re-implantation of the cryo-thawed tissue into the remaining ovary or the ovarian bed as originally described by Gosden et al. in 1994 [25]. This approach has the advantage of superior graft survival, likely due to the excellent blood supply and high oxygen tension, ease of monitoring, and ease of oocyte retrieval which is unchanged from standard IVF and widely accessible to clinicians performing egg retrieval.

Concerns have been raised regarding the theoretic risk of reintroducing malignant cells or tissue, resulting in relapse of the original cancer or disease which prompted the cryopreservation of the ovarian tissue [28]. It is important to evaluate thawed ovarian tissue prior to autotransplantation and to involve pathology and oncology specialists in the consideration of its use. With proper screening, the risk appears small, with no reported recurrences [29]. With continued experience, overall the data on safety and efficacy, as well as reproductive outcomes, has by 2019 lead to ovarian tissue cryopreservation to be considered an established medical procedure [30, 31].

#### **3. Planned oocyte cryopreservation**

With improvements in vitrification of mature oocytes, as well as significantly improved fertilization using ICSI, the pregnancy rates using cryopreserved oocytes were found to be comparable to those found using fresh oocytes with IVF [32]. More importantly, studies of the health of babies born following the use of oocyte cryopreservation have shown no increase in congenital abnormalities [8, 9]. With these reassuring clinical results, OC was no longer considered experimental by the ASRM in 2012 [10].

The success of oocyte cryopreservation has led to increased interest in cryopreserving oocytes to extend the reproductive capacity in otherwise healthy women wishing to delay childbearing. Although the ASRM initially declined to recommend OC for the "sole purpose of circumventing reproductive aging in healthy women," intense interest and an undeniable increase in efficacy lead the organization to publish a risk/benefit "fact sheet" regarding OC by 2014, followed by a stronger supportive endorsement for the procedure by 2018 [33].

The rate of first birth to women age 35–39, as well as age 40–44, continues to increase in the U.S. [34]. Increasing emphasis on education, later age at marriage, access to effective contraception and opportunity for career advancement are among many of the reasons for this trend. However, with increasing maternal age, fertility decreases dramatically beginning at 35, due to decreasing oocyte quantity and quality, resulting in increasing chromosomal abnormalities seen in failure to conceive, miscarriage and birth defects [35].

Egg donation, using higher quality eggs from young healthy donors, has historically been the treatment of choice for women wishing to conceive in their fourth and fifth decades of life. OC performed at a younger age, prior to decreasing ovarian reserve, allows for having a child using a woman's own genetic material later in life. Because a woman's age, number and quality of oocytes strongly determine the chance of pregnancy, OC cryopreservation is likely to be most successful for younger women [23]. By the age of 38, research suggests that 25–30 oocytes may be required to provide a reasonable chance of pregnancy success [36]. The cost of the procedure must be considered, as well as the cost of long term storage, particularly in young women. In addition, the possibility of achieving pregnancy naturally or with standard fertility treatments, should be taken into consideration when considering planned OC [37]. Due to the fact that at age 20–30, the time of maximal career advancement also corresponds a woman's to optimal fertility, the available option for OC has prompted several large corporations to cover the costs associated with the procedure [38], providing additional incentive for career advancement and delayed childbirth.

#### **3.1 In vitro fertilization and oocyte cryopreservation**

The success and ready availability of OC has also resulted in increasing use of the technique as an adjunct to standard IVF procedures. On the day of egg retrieval, typically a fresh semen sample is required to prepare viable spermatozoa for either standard insemination or for ICSI to accomplish fertilization of multiple mature oocytes retrieved following COH. In cases of severe oligo-asthenospermia, several samples may be cryopreserved and "banked" to ensure adequate numbers of viable spermatozoa on the day of oocyte retrieval. In cases of obstructive azoospermia, surgical extraction of spermatozoa from the epididymis or testis is typically performed prior to the planned oocyte retrieval and cryopreserved, or the planned procedure for sperm retrieval can be scheduled on the same day to obtain a fresh specimen.

In clinical practice, there are cases when fresh sperm cannot be obtained on the day of egg retrieval, either because the male partner is unexpectedly unavailable or unable to provide a sample. Also, thawing of severely oligo-asthenic semen samples may yield insufficient viable spermatozoa to fertilize any or all of the mature oocytes retrieved, particularly in cases when several dozen oocytes are obtained following COH. Likewise, planned surgical extraction procedures can be unexpectedly delayed due to surgical scheduling requirements or fail to yield viable spermatozoa sufficient for fertilization. In these cases, cryopreservation of the unfertilized mature oocytes can be performed until such time adequate viable spermatozoa are available to accomplish IVF without compromising the success of the procedure.

Other common clinical situations can arise when insufficient viable mature oocytes are obtained at the time of egg retrieval, particularly in older patients or those with significantly decreased ovarian reserve. In these cases, additional cycles of COH and egg retrieval can be performed to increase the overall number of oocytes used in IVF and in particular when genetic screening using preimplantation genetic screening (PGS) is used [39], increasing the overall pregnancy success. In addition, for patients wishing to limit or avoid freezing embryos, supernumary mature oocytes retrieved following COH can be cryopreserved [40]. As seen for other indications for oocyte cryopreservation, pregnancy rates following transfer of fresh embryos in such cycles and embryos from previously cryopreserved "sister oocytes" from the same cycle are similar [41], suggesting no significant decrease in oocyte or embryo quality.

#### **4. Oocyte donation**

The clinical problem of infertility due to inadequate number or quality of oocytes available to produce a healthy pregnancy was overcome by the introduction of oocyte donation in 1984 [42]. Patients with diminished ovarian reserve due to advanced age, POF, or gonadectomy for cancer or benign disease could conceive a pregnancy with her partner, carry the pregnancy to term and deliver a healthy child using donated oocytes from a young, healthy woman, fertilized by the patient's husband's sperm, and the resulting embryos transferred to her uterus.

Options for obtaining oocytes include "known donors," such as family members including younger sisters, cousins or same-sex partners. Alternatively, anonymous donors, analogous to sperm donors, which are chosen by matching physical characteristics such as height, weight, hair color, eye color, ethnic background, etc., can be used. Anonymous donors must be carefully screened for the absence of infectious or heritable disease which could adversely affect the health of the offspring.

Sperm donors have the advantage of providing semen samples, which can be easily cryopreserved and banked, allowing for quarantine against potential infectious agents with long incubation periods and rapid availability of a wide selection of potential donors. Unlike sperm donors, oocyte donors initially require selection of a potential donor who would undergo COH using gonadotropin treatment and ultrasound monitoring followed by oocyte retrieval and insemination of the fresh mature oocytes using the patient's partner's fresh or previously banked semen sample.

Although thorough screening of a potential oocyte donor for health, infectious disease risk factors and family history of potential genetic disease is similar to semen donors, actual screening for infectious disease is required within 3 days of oocyte retrieval by the U.S. Food and Drug Administration (FDA), compared with a 6 week quarantine period required for banked semen samples. Cryopreservation of oocytes from healthy young donors allows for similar quarantining and "banking", with ready availability of healthy, screened oocytes which can be chosen by matching the donor's physical traits, as has been in use for sperm donors for many decades. However, concerns with respect to commercialization and marketing as well as cost effectiveness and accurate reporting of pregnancy outcomes remain.

Because commercial "egg banks" are not required to report clinical outcomes per cycle start, including pregnancy, miscarriage and live birth rates, as required by law under the auspices of the Society for Assisted Reproductive Technology (SART) to the centers of disease control (CDC), caution should be exercised when interpreting the pregnancy success rates advertised by such commercial enterprises as they compete for patients seeking donor eggs [43]. Known factors influencing pregnancy success using previously cryopreserved mature oocytes include younger age of the donor [44]. Also, donors who have had previous pregnancy success in fresh cycles are associated with a higher live birth rate using cryopreserved oocytes [45].

It is also clear that as the number of donor oocytes thawed increases, there is an associated increase in the cumulative live birth rate [45]. This raises the question of cost-effectiveness of utilizing commercial egg banks and their pricing structure in terms of the cost per oocyte. Considering the rate of fertilization, embryo cleavage, blastocyst formation, implantation and miscarriage, the chance of live birth has been estimated at 8% per thawed oocyte [46]. This must be compared with the multiple oocytes and embryos obtained through conventional egg donation, which may yield multiple embryos for transfer, as well as for cryopreservation of supernumary

embryos for additional attempts at pregnancy, which can increase the overall cumulative probability of successful live birth.

#### **5. Summary**

The clinical success of IVF has resulted in the rapid development and adoption of innovations including COH, embryo cryopreservation, ICSI and oocyte donation, which have been successful in overcoming infertility from multiple etiologies resulting in the birth of more than 1 million children as of 2012 [47]. By 2018, 40 years after the birth of Louise Brown, more than 8 million children have been born, worldwide [48]. Due to significant technical challenges, largely overcome by rapid improvements in vitrification and fertilization, the innovation of clinically successful oocyte cryopreservation has been much more recent, having been approved for widespread use only since 2012 [10].

The principal application of the technology remains preserving fertility potential, both for medically necessary and elective indications. Cryopreservation of unfertilized eggs has the advantage of significantly fewer ethical, moral, religious and potentially legal problems when compared with the cryopreservation and potential long-term storage of embryos. The clinical utility of OC is also clear as an adjunct to fertility treatment using IVF as well as having a significant potential role in oocyte donation. As with all important emerging innovations in the field of assisted reproductive technology, continuing research and clinical experience will be instrumental in defining the role of OC going forward.

#### **Author details**

Timothy J. Gelety The Arizona Center for Reproductive Endocrinology and Infertility, Tucson, Arizona, USA

\*Address all correspondence to: tgelety@infertility-azctr.tuccoxmail.com

© 2022 The Author(s). Licensee IntechOpen. 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.

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#### **Chapter 6**

## Quality Management System in Medical Assisted Reproductive Technology (MART)

*Delia Hutanu and Ioana Rugescu*

#### **Abstract**

A quality management system (QMS) refers to an organization's broader approach to minimize deficiencies and errors, to meet regulatory compliance standards, and to satisfy a specified set of inherent characteristics during the health care services provided to patients. According to the European directives and recommendations (European Commission, 2006a, c, 2012; Council of Europe, 2013), working in compliance with a QMS is mandatory. The requirements cover the organization, management, personnel, equipment and materials, facilities/premises, documentation, records, and quality review. The IVF clinics should consider total quality management (TQM) as an option, especially in these days when escalating regulatory scrutiny increases the pressure for professional accreditation. TQM is an integrative philosophy of management for continuously improving the quality of services and processes and includes quality assurance (QA), quality control (QC), quality improvement (QI), and risk assessment and risk management. QMS must become an essential topic for those who are working in MART.

**Keywords:** quality management system, total quality management

#### **1. Introduction**

Medical assisted reproductive technology (MART) represents the sum of medical procedures used by healthcare providers in order to help infertile patients to achieve a pregnancy. It is a complex process, and the success depends on various factors such as patient-related and some other not patient-related. The present chapter focuses on the quality management system involved in assisted reproductive facilities. Taking into account all European directives and recommendations, as well as the national laws/regulations, the present material wants to summarize the present status of the quality management system.

#### **2. Quality management system**

A quality management system (QMS) refers to an organization's broader approach to minimizing deficiencies and errors, meeting regulatory compliance standards, and satisfying a specified set of inherent characteristics during the health care services provided to patients.

According to the European directives and recommendations (European Commission, 2006a, c, 2012; Council of Europe, 2013), working in compliance with a QMS is mandatory. The requirements cover the organization, management, personnel, equipment and materials, facilities/premises, documentation, records, and quality review. Moreover, in vitro fertilization (IVF) clinics should consider total quality management (TQM) as an option, especially in these days when escalating regulatory scrutiny increases the pressure for professional accreditation. TQM is an integrative philosophy of management for continuously improving the quality of services and processes and includes quality assurance (QA), quality control (QC), quality improvement (QI), and even risk assessment and risk management. QMS must become the essential topic for those who are working in MART.

In the following pages, this chapter will bring together the most important information and tools that will allow the reader to start, implement, and maintain a QMS in his own unit, searched in literature, guidelines, and several consensuses. This chapter is intended to be a condensed pill of quality management information that will treat the reader's acute need for information on this topic.

The concept of "quality" has been defined over time in many ways and viewed from many angles and points of view, probably precisely because it has been and continues to be a topic of great importance and relevance. Any organization that respects itself and wants to be part of modern accreditation schemes has the entire activity based on the principles of a quality system. In 1979, Crosby defined five stages in the development of a quality management system and Garven in 1988 proposed a four-stage development model, so there it has been more than four decades since organizations are focused on good quality products or services. As a definition, although it seems simplistic, quality management; means coordinated activities to lead and control an organization in the field of quality.

#### **2.1 Quality management principles**

As an approach initially, eight principles were proposed as follows:


#### *Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*

In medically assisted human reproduction units, but especially in terms of testing, processing, cryopreservation, storage, distribution of reproductive cells and reproductive tissues in embryology and/or andrology laboratories it is imperative that the organization be based on ISO 9000 standards principles and other relational standards. In particular, the embryology part is especially important for the risk management and the minimization of errors.

As a result of the MART activity expansion and also taking into account the globalization that is taking place in the healthcare services sector, quality and risk management have become a necessity. In this context, in order to provide medical services that are intended to have the expected result in conditions of maximum safety, it was necessary that all licensed IVF units that offer such services operate in accordance with international standards, such as ISO 9001 [1], thus reflecting the current awareness of these services not only from a medical point of view but also from a commercial point of view.

The structure and organization of a licensed IVF unit may vary depending on a several number of factors, such as its size, and various types of organizational structures have been described in the literature by Dale, 1998; Heller and Hindle, 2003 [2, 3].

#### **2.2 Legislation, certification, accreditation/licensing**

Even if may vary from country to country, legislation, certification, and accreditation/licensing are often confused with each other or are seen as points of view in the management of an IVF unit, in fact, they are completely different concepts and all three work together for an integrated management system.

Legislation or the legislative requirements that an organization (IVF unit) must comply with in order to have permission to offer healthcare services. Compliance with legislative requirements is verified by an individual inspection of the unit and is confirmed by licensing/accreditation.

Generally, the legislative requirements are of the restrictive indicative type and provide precise indications regarding what an organization must comply with in accordance with the legislation in force.

The accreditation/licensing is the set of requirements and the process by which an organization must meet and is identified as complying, in order to be licensed / accredited and that they must maintain throughout the license/accreditation period. In EU member states, usually, this accreditation/licensing is issued by the Health Ministry and/or National Competent Authority. In other countries, the process may vary.

In addition, there is a certification that can be defined as a process by which an organization is identified as complying with and meeting certain criteria. In the case of IVF units, the ISO 9000 standards family applies (ISO 9001: 1994, ISO 9002: 1994, ISO 9003: 1994) with revised editions, for example, ISO 9001: 2002.

It is also possible to consider ISO 19011: 2002, as well as ISO 15189: 2003, and last but not least the ISO 17025: 2005 standard. The certification system implemented by ESHRE (European Society for Assisted Human Reproduction and Embryology-ARTCC) can also be considered. Regarding the legislative requirements for EU member states, the European Directives for Tissues and Cells and their implementation in the member state's national legislation must be taken into account. From this point of view, in the case of IVF clinics, we must consider the following:


In October 2020, the commission adopted its work program for 2021. The work program includes the revision of EU directives for tissues and cells. This revision comes after an evaluation of available translations of the preceding of the legislation, published in October 2019, confirming that the legislation had improved the safety and quality of blood, tissues, and cells used for transfusion, transplantation, or medically assisted reproduction. The evaluation also highlighted a number of gaps and shortcomings, which will be addressed to ensure the framework is up-to-date, fit for purpose, and future-proof. The initiative aims at updating the legislation in the direction of a more flexible alignment with scientific and technological developments tackling the re- emergence of communicable diseases, including lessons learned from the COVID-19 pandemic focusing on the increasing commercialization and globalization of the sector removing from legislation many technical provisions, which will allow a faster update of standards possibly merging the basic acts into a single instrument. The revision is planned to be adopted in the second quarter of 2022. The legal basis is provided by Article 168(4)(a) of the treaty on the functioning of the European Union [4].

Although the field of medically assisted human reproduction is very well known, quality management is much more used and implemented in embryology laboratories and it is based on monitoring cellular activity in terms of monitoring parameters in the workspace, equipment, etc.

It is well known that as a result of the action of some physic-chemical agents, the embryos are greatly affected, so that for correct implementation of a quality system all the parameters that can bring/produce changes regarding the embryonic development must be monitored.

Within an efficient quality management system, it is known that the performance indicators of the system play an important role, in our case the performance indicators of the activity.

In the quality management of an IFV unit, it is very important to be aware of the proactive tools in terms of risk management and to use these tools in a designed system in order to optimize the processes.

If we discuss the embryology or andrology laboratories, we must also standardize as much as possible the evaluation of oocytes, sperm, and embryos because they are considered to be essential components in the qualitative monitoring of the laboratory's processes.

Quality management is summarized as:


From the point of view of risk management, the most important aspects to consider are:

*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*


Regarding the management system:


At this point, risk management is considered to be an integral part of proactive quality management [5, 6].

#### **2.3 Indicators and benchmarks**

Indicators: (we cannot control something we cannot measure). That is why the indicators must be:


Routine data collection should not be difficult without a lot of extra work. In order to optimize the activity and the results, it must be taken into account that the whole process is governed by the biology of gametes and embryos; therefore, it must ensure optimal conditions for gametes and embryos; protect gametes and embryos from physiological stress; and protect gametes and embryos from adverse external factors.

Cellular stress is a high-energy consumer and can also lead to altered gene expression and/or, for example, imprinting. Suboptimal embryonic culture may lead to irreparable changes that may affect the future conception product.

Another important step in quality management is the recognition of all the factors of influence that affect the processes in the embryology/andrology laboratories.

Possible sources of influence can be derived from the patient but also derived from clinical processes: ovarian stimulation, ovarian puncture during oocyte pick-up, embryo transfer, and luteal support.

The environmental design and construction of space, design of the workflow, equipment, work circuit, and air can influence the procedures. Among other factors that can have an influence on the process are: temperature, Co2/pH, equipment calibration, and faulty operation.

The materials used in the IVF lab should be suitable for use, not to be embryo or cytotoxic, to be manufactured by a certified manufacturer in terms of quality (CE marking), or to be validated. All the methods used during the process have to be appropriate for the purpose, correctly chosen, SOP (documented), and lastly, the staff of the facility should be trained and skilled.

From the point of view of the sources of influence, the optimization of the system in the (embryology/andrology laboratories) for each component of the process must take into account the following:


#### **2.4 Key point indicators in IVF laboratory**

Another important step for an efficient quality management system is the selection of key indicators for the medically assisted human reproduction process. In this case, the Vienna consensus on performance indicators in medically assisted human reproduction laboratories must be taken into account.

Performance indicators (PIs) are objective measures for assessing critical areas (patient safety, efficacy, fairness, patient fairness, timeliness, and effectiveness of medical treatments). In the activity of medically assisted human reproduction, quality indicators are needed for the systematic monitoring and evaluation of its contribution to patient care (ISO15189-2012), and it is a vital element in the quality management system (QMS) [6].

Any performance indicator must be reliable and robust, and the collection of data for tracking the indicator should be straightforward. In addition, the biological or technical process that we want to monitor must be defined with certainty. Key performance indicators (KPIs) are indicators that are considered essential for evaluating the introduction of a technique or process; setting minimum standards of competence; monitoring ongoing performance in a quality management system (for quality control (IQC), external quality assurance (EQA)); and benchmarking and quality improvement.

In general, the results of a series of key performance indicators (KPIs) will provide you with an adequate overview of the most important steps in the medically assisted human reproduction process [7].

The requirement for defining a process within quality management are:

1.Defining the process to be monitored,


*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*


#### **Table 1.** *KPIs in IVF lab.*

In accordance with Vienna consensus [8], for a high-quality management system, three types of indicators have been identified that can be monitored:


Considering the cryopreservation, the Alpha consensus on cryopreservation key performance indicators and benchmarks divided these into the following categories:


#### *2.4.1 Oocytes*

#### *2.4.1.1 Morphological survival*

This KPI was defined as the proportion of morphologically intact oocytes, based on the intention to inject, at the time of ICSI. Oocytes with oolemma or abnormal ooplasm at the time of ICSI should not be excluded (**Table 2**).


**Table 2.**

*KPIS for morphological survival of cryopreserved oocytes.*

As this KPI may be affected by the number of cases and/or the experience of the practitioner, different values have been assigned to achieve competence for both slow freezing and vitrification. Competency values are those that should be achieved by any practitioner, while reference intervals are aspirational targets.

#### *2.4.1.2 Fertilization rate*

The fertilization rate indicator was defined as the proportion of oocytes with two pronuclei at the time of fertilization verification (17 ± 1 h after insemination). The fertilization rate should be no more than 10% lower than that for the fresh oocyte in the center.

#### *2.4.1.3 Embryonic development rate*

The embryonic development rate indicator is defined as the proportion of embryos in the developmental stage that reach the stage of development specifically for the time of observation (2-cell stage at 26 ± 1 h after ICSI, 4-cells stage at 44 ± 1 h after insemination, 8-cells stage at 68 ± 1 h after insemination, morula stage at 92 ± 2 h after insemination, and the blastocyst stage at 116 ± 2 h after insemination).

The rate of embryonic development for embryos from vitrified oocytes should be the same as for the comparable population of fresh embryos from the bank of reproductive cells and tissues. For embryos from cryopreserved by slow freezing oocytes, some developmental delays may occur, but no more than 10–30% lower than that for the fresh embryos at the center.

#### *2.4.1.4 Implantation rate*

The implantation rate indicator was defined as the proportion of ultrasoundconfirmed pregnancies with fetal heartbeat relative to the number of embryos transferred. The implantation rate for embryos from cryopreserved oocytes should be at most 10–30% lower than a comparable population of fresh embryos from the laboratory.

#### *2.4.2 Zygotes*

For zygotes produced by ICSI, the observations made during the ICSI procedure regarding the oocyte quality should always be recorded. This would allow a possible further analysis of the prevalence of oolemma/ooplasm abnormalities that could have been caused by the cryopreservation procedure.

#### *2.4.2.1 Morphological survival rate*

This KPI was defined as the proportion of morphologically intact zygotes immediately after thawing/devitrification compared to the number of morphologically

preserved zygotes. A morphologically intact zygote is one that is similar in appearance to a fresh zygote. The same survival rate should be achieved by slow freezing as well as vitrification.

#### *2.4.2.2 Cleavage rate*

This KPI was defined as the proportion of thawed/devitrified zygotes that divide to form a cleavage embryo. The rate of division should be the same as for the comparable population of fresh embryos in the bank of reproductive cells and tissues.

#### *2.4.2.3 Embryonic development rate*

The embryonic development rate indicator is defined as the proportion of embryos in the developmental stage that reach the stage of development specifically for the time of observation.

#### *2.4.2.4 Implantation rate*

The implantation rate indicator was defined as the proportion of fetal ultrasound-confirmed pregnancies with fetal heart rate relative to the number of embryos transferred. The implantation rate for embryos from cryopreserved zygotes should be no more than 10–30% lower than that for the comparable population of fresh embryos at the center (**Table 3**).

#### *2.4.3 Embryos*

For the KPI calculation, the embryos selected for cryopreservation should meet the criteria for an optimal embryo at the cleavage stage.

#### *2.4.3.1 Indicators - post-freezing survival rate from a morphological point of view*

The KPIs that assess the post-freezing survival rate for embryos are based on the proportion of thawed/devitrified embryos with 100% and ⩾50% of the total intact embryos. For the first category, embryos thawed with 100% of the total intact embryos, the competence value is 40% and the benchmark is 55%, and for the devitrified embryos, the competence value is 70% and the benchmark is 85%. For the category with embryos thawed with ⩾50% of the total intact cells, the competence value is 60% and the benchmark is 85% and for the devitrified embryos, the competence value is 85% and the benchmark is 95%. It should be noted that KPI values do


**Table 3.** *KPIs cryopreserved zygotes.* not prevent the transfer of embryos with suboptimal morphology, as this may be the only opportunity for patients.

#### *2.4.3.2 Development rate indicator after thawing/warming*

For the calculation of this key performance indicator, only the embryos with 100% intact morphological structure will be considered after thawing/warming. Post-cryopreservation development includes cleavage and further development at the blastocyst stage, as well as implantation rate, defined as the proportion of ultrasoundconfirmed pregnancies with fetal heartbeats relative to the number of embryos transferred.

The competence value for the rate of embryo development after thawing/warming should be at most 10% (relatively) lower than the comparable population of fresh embryos in the laboratory and the benchmark value should be the same as for the comparable population of fresh embryos at the center.

#### *2.4.4 Blastocysts*

As the in vitro growth rate is substantially affected by exogenous factors, no key differences were made between the performance indicators of post-freezing/ vitrification blastocysts and blastocyst stages (early, full, expanded blastocyst, hatched). Similarly, there is no recommendation on blastocyst collapse. The decision will be made by each lab accordingly. Regarding the reported results for cryopreserved embryos cryopreserved in the blastocyst stage, they are substantially better after vitrification than after slow freezing.

#### *2.4.4.1 Survival rate*

The blastocyst survival rate indicator after cryopreservation is defined as the proportion of surviving blastocysts relative to the total number of thawed/devitrified blastocysts and applies to blastocysts with at least 75% intact morphology.

#### *2.4.4.2 Transfer rate*

This KPI has been defined as the proportion of thawed/warmed blastocysts that are of sufficient quality to be transferred. This parameter assumes that the transfer decision is not subject to legislative limitations in terms of the number of embryos transferred per patient and does not take into account the transfer decisions of some suboptimal blastocysts. No matter the type of embryo transfer (single, double, or multiple).

#### *2.4.4.3 Implantation rate*

The implant rate indicator was defined as the proportion of ultrasoundconfirmed pregnancies with fetal heartbeats relative to the number of blastocysts transferred (**Table 4**).

*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*


#### **Table 4.**

*KPIs for cryopreserved blastocysts.*

#### *2.4.5 Sperm*

The performance indicators for sperm are related to:

1.Sperm recovery rate

#### 2.Sperm motility post-wash

The expected proportion of motile spermatozoa in the final washed preparation showed values of 90% for competency and 95% for the benchmark.

Sperm recovery rate, defined as the percentage recovery of progressively motile sperm after washing as compared to pre-washing, can be used as a laboratory KPI, providing useful information for inter-operator comparison and proficiency testing.

#### **2.5 Clinical KPI**

Performance indicators (PIs) are a valid method to be sure that the medical facility is of high quality and it operates within acceptable limits. In order to reach these goals in 2019 was published the Maribor consensus [9]. The paper recommends six PIs to be monitored in clinical work in ovarian stimulation for ART: cycle cancelation rate (before oocyte pick-up), rate of cycles with moderate/severe ovarian hyperstimulation syndrome, the proportion of mature oocytes at ICSI, complication rate after OPU, clinical pregnancy rate, and multiple pregnancy rate.


(polycystic ovary syndrome) [10]. The value of the rate of cycles with moderate/ severe ovarian hyperstimulation syndrome is 6.43% and 10.61% in regular and PCOS groups.


A well-developed system in terms of quality management in the ART clinic must be taken into account the moment when following the monitoring and evaluation of the processes we find that there are problems. That is why an evaluation and correction process must be identified and developed. For a good administration of a scheme for the evaluation and solution of a nonconformity, it is absolutely necessary the root cause analysis (RCA). Such an analysis is the basis of a process that must take place after a problem has arisen.


#### **Table 5.**

*Oocyte retrieval rate and proportion of mature oocytes at ICSI.*

*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*

In this case, it is necessary to identify the factors that led to the appearance of a nonconformity; therefore, we will classify the factors as follows:


In the latter case, the need arises to create or access the necessary data in order to run a new classification. If we identify a number of factors, it is necessary to prioritize them in order to build a risk mold. Following the identification and ranking of contributing factors, an action plan must be developed and implemented. In general, in an ART clinic, the appearance of nonconformities is due to an accumulation of contributing factors and less to a single cause.

Also, the literature recommends that the term "cause" not be used in the reports prepared due to the possible psychological and/or legal impact.

In order for a noncompliance process to be effective, it must be effective and:


Due to technological progress, it has been possible for four decades to get from the first cultures of animal embryos to the culture and transfer of genetically tested human embryos. All these advances require quality control and also quality assurance methods in assisted human reproduction laboratories precisely to ensure repeatable processes. If progress is constant all specialists recommend the introduction of a total quality management system (TQM).

Quality control (QC) in the activities of the bank of reproductive cells and tissues is essential for its smooth running. Quality control must work in parallel with the specific activities of the bank. Recording the temperature of the equipment is an example of a quality control activity. All these control activities have been specially planned to be able to demonstrate and verify if, for example, that equipment produces the same results every time.

In terms of quality assurance (QA), this consists of complex methods of monitoring and evaluating all the processes in a bank. While quality control is concomitant with banking activities, quality assurance is a retrospective process. Also, for good quality management, we have to take into account the qualitative improvements (QI) through which we raise the performance of the activities in the ART clinic.

QI is different from QA and QC and is specifically designed to identify and correct problems or errors in the processes and activities of the ART clinic.

An example of QI is to adapt the laboratory's procedures to new zygote evaluation technologies precisely to improve the criteria for selecting embryos for transfer.

Total quality management (TQM) is a combination of all three of these topics.

This orientation does not change the structure of authority in the organization, nor does it diminish the essential role of top management. The inverted hierarchy emphasizes "service delivery" relationships and the importance of the consumer to the organization, which is why it is the perfect model for healthcare organizations.

An important part of QMS is the environment in the processing space: Laboratory staff must inspect the equipment to ensure that it is in good working order. Instruments used to determine temperature, gas concentration, and relative humidity must be recalibrated at the latest within 1 year.

If the manufacturer recommends another interval, the manufacturer's recommendation will be followed. The maintenance of all equipment must also be considered (according to the manufacturer's instructions and in accordance with the organization's policy).

The recommended monitoring parameters documented measures of continuous evaluation, correction, and monitoring of the activity, as well as, but not limited to:


#### **2.6 Air quality**

The first references to this topic appeared in the 1990s when the first correlations between various toxic agents (bacteria, dust, and VOC = volatile organic components) and embryonic development were reported. Johnson published a study on the influence of VOCs on embryonic culture [14] and Boone published studies on this topic [15]. Other authors published similar findings [16, 17]. Kao published a study that showed improved results based on air quality in the processing area [18]. At

#### *Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*

present, it is mandatory in most countries to have purified air in the ART laboratory. Most laboratories use HEPA-type ventilation and purification systems (0.3 μm is the average of airborne particles found in measurements). Some of the ART laboratories that process reproductive cells have added ULPA filters, which bring improvements in air quality. It is recommended to have ISO class V regarding the number of airborne particles (grade A). It is also important to monitor the level of VOCs both in the air in the cell processing space and the level inside the incubators [19]. In any case, the HEPA/ULPA filtration system does not exclude the occurrence of VOCs neither in the air in the processing area nor in the incubator environment.

During the measurements carried out in various laboratories, aromatic hydrocarbons like (benzene, toluene, and xylene) were found, probably from the paints used as well as isoflurane due to the fact that the reproductive cell processing space is located in the immediate vicinity of the puncture room. Other VOCs found were propane and hexane, as well as aldehydes, probably from perfume and/or deodorants used by staff [20]. Incubation gas monitoring revealed benzene in CO2 cylinders, which leads to the recommendation to use special filters on the gas transport system [21].

Due to reports of ethylbenzene and benzaldehyde emissions from plastic consumables, special consumables that do not eliminate VOCs are currently being used [22]. Since VOCs are oil-soluble, even the closed culture system (under oil) does not protect embryos from these toxic substances.

Because over time, a number of VOCs from the outside air have been reported, taken over by the HEPA/ULPA ventilation system [23] or the air to be partially recirculated in case the outside air does not correspond to the norms. Fresh air supply of 30% is recommended to use, and also an active charcoal filtration system.

The quality system in an ART clinic is a component part of the total quality management system (TQM). The quality assurance system when performing the tests is formulated in the quality manual and can be developed according to the provisions of SR EN ISO/CEI 17025: 2002.

ISO 17025 is the standard that specifies the requirements for proficiency testing and/or calibration. This standard includes 15 quality management requirements and 10 technical requirements. These requirements show what an ART laboratory needs to do to be accredited.

#### **2.7 Risk analysis**

Risk analysis is a process that incorporates three components:


The person in charge of the reproductive cell and tissue bank has, among other responsibilities, the one related to the implementation of a risk management and prevention policy. According to European Directives and recommendations (European Commission, 2006a, c, 2012; Council of Europe, 2013), work in accordance with a quality management system (QMS) is mandatory. Requirements cover organization, management, personnel, equipment and materials, facilities/premises, documentation, records, and quality assessment. It includes, but is not limited to:


Risk assessment is used to describe the general process or method of identifying hazards and risk factors that have the potential to cause harm (identifying hazards), and analyzing and assessing the risk associated with that hazard (risk analysis and risk assessment).

A danger is anything that can cause harm; these can be physical health hazards, such as chemicals, electricity, working on stairs, an open drawer, or mental health.

The risk is high or low that someone will be injured by these or other hazards, along with an indication of how serious the injury may be.

There are five steps in risk assessment:

1.Hazard identification.


For ease of use for biological reproductive material, the Euro-GTP II Guide has been created, which provides structured guidance on how to use the tools and methodologies developed by the EuroGTP II project, namely, the use of a systematic mechanism based on risk analysis from the point of view of the degree of novelty as follows:


*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*


The general process requires to identify specific risks related to potential risk factors and the consequences of the risks. Each of these must be assessed individually to determine the residual risk of implementing the change. Assessment taking into account:


The instrument shall take into account the number of individual risks assessed to calculate the percentage value of the overall risk.

The result of this risk analysis will be a single global risk score (on a scale of 0 to 100)the final risk score, which can be used to define the extent and/or need for preclinical and clinical assessment needed to support the proposal to change or introduce a new type of product or service [24].

#### **3. Conclusion**

Due to the fact that infertility is a growing problem in our society, the natality being on a downward trend, medical assisted reproductive technologies have an important role. The success rate depends on numerous factors, patient and nonpatient-related. Among the latest, quality management systems with all the components of it play a crucial role in the whole process. The chapter summarizes this part of MART, emphasizing how and why the QMC can and does influence the final result.

#### **Author details**

Delia Hutanu1 \* and Ioana Rugescu<sup>2</sup>

1 West University of Timisoara, Biology-Chemistry Department, Timisoara, Romania

2 National Transplant Agency, Bucharest, Romania

\*Address all correspondence to: delia.hutanu@e-uvt.ro

© 2022 The Author(s). Licensee IntechOpen. 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.

*Quality Management System in Medical Assisted Reproductive Technology (MART) DOI: http://dx.doi.org/10.5772/intechopen.106172*

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Section 4
