**5. Sources of immature oocytes**

#### **5.1 Oocyte retrieval from cesarean section or gynecological surgery**

Immature oocytes retrieved from the ovarian cortex during cesarean section can be cultured in vitro to achieve maturation, fertilization, and healthy progeny. The mature oocytes cultured in this way are expected to be used as the source of oocytes to preserve female fertility [26]. At present, few studies have investigated the approach to obtain immature oocytes during cesarean section for in vitro culture, and hence more studies are needed to prove the safety and effectiveness of immature oocytes obtained from cesarean section.

In addition to cesarean section, immature oocytes can also be obtained via gynecological surgery in the follicular phase or luteal phase. The number of retrieved oocytes is mainly related to the age, pathological status, and stage of the menstrual cycle of the patient. Clinical studies have confirmed that oocyte retrieval carried out at different stages of the menstrual cycle does not affect the rate of in vitro maturation and the rate of fertilization of oocytes, suggesting that IVM technique can be used to preserve fertility in cancer patients during the follicular phase or luteal phase [27]. Therefore, for cancer patients who lack sufficient time for treatment and are unable to use hormone to induce ovulation, immature oocytes can be retrieved before chemotherapy to carry out IVM and vitrification to maximize the preservation of fertility.

#### **5.2 PCOS patients**

A large number of antral follicles are present in the ovary of infertile women with anovulatory PCOS. These antral follicles are more sensitive to gonadotropins, and hence the risk of OHSS is increased when hormones are used to induce ovulation. Therefore, for PCOS patients, immature oocytes can be retrieved from antral follicle for in vitro maturation [28]. The use of hCG at 36 h before oocyte retrieval in PCOS patients can promote the resumption of meiosis of immature oocytes and their in vitro maturation, improving the rate of pregnancy and clinical outcomes [29]. The use of small doses of gonadotropin before the retrieval of immature oocytes from PCOS patients is also beneficial to improve the maturation potential of oocytes, increasing the rate of embryo implantation and clinical pregnancy. In addition, IVM techniques can also be considered for some PCOS patients with no or low response to hormones [30].

#### **5.3 Women with normal ovaries and menstrual cycles**

Based on the advantages of low hormone dosage, low cost, and simple treatment process, IVM has been gradually applied to the treatment of infertile women with normal ovaries and regular menstrual cycles. However, it remains controversial as whether the use of hCG is required in the IVM treatment of this type of patient prior to oocyte retrieval. It should be noted that the hCG trigger exerts different effects on normal ovaries and PCOS patients. In the IVM treatment cycle of PCOS patients, dominant follicles are barely visible in the ovary, but MI-stage oocytes can be retrieved from small follicles after hCG-induced ovulation. However, after the hCG trigger is used in the normal ovary during the follicular phase, most oocytes retrieved from small follicles are oocytes in the GV stage. There is currently no evidence suggesting that the hCG trigger exerts a significant effect on the pregnancy rate, live birth rate, or abortion rate in the IVM of immature oocytes obtained from normal ovaries [31]. However, the accuracy of these findings is limited by the small number of samples. Therefore, a well-designed, randomized, and controlled clinical trial is needed to further confirm the optimal dosage and timing of hCG administration.

### **6. IVM culture system**

#### **6.1 Improvement of IVM media**

The in vitro maturation of oocytes is mainly affected by culture conditions. At present, the common media used for the IVM of immature human oocytes include TCM-199 medium, Ham's F10 medium, and Chang's medium. In addition, serum, gonadotropin [follicle-stimulating hormone and luteinizing hormone], growth factors, and steroids can be added in a basal medium to produce a complex medium. At present, commercial IVM media have been widely used. However, no breakthrough has been made in the research on improving the quality of oocytes by improving the IVM medium. In recent years, research and application of antioxidants and growth-promoting factors have promoted the advancement of this technology to some extent. In addition, using cell cycle regulators or inhibitors of mitotic spindle formation, the synchronization of nuclear and cytoplasmic maturation of immature oocytes can be achieved by inhibiting GVBD, thereby increasing the blastocyst rate and live birth rate in animal models [32]. However, the safety and efficacy of this method in human oocytes should be further verified.

**29**

IVM technology.

*Clinical Application of In Vitro Maturation of Oocytes DOI: http://dx.doi.org/10.5772/intechopen.87773*

**6.2 Optimization of IVM culture environment and process**

tions in areas such as IVM of immature human oocytes.

**7. Clinical application and safety of IVM**

The culture environment, equipment, and related operations in the IVM system may affect the in vitro maturation and embryo development of immature oocytes. Therefore, the optimization of the embryo culture environment and process of in vitro operation will help to maintain the potential of embryonic development [33, 34]. The three-dimensional culture system can support the development of follicles by using biological materials to maintain cell-to-cell information exchange. In addition, mature oocytes can be obtained by using a three-dimensional culture system in the in vitro culture of anterior follicles of nonhuman primates [35], although no reports are available regarding the use of a three-dimensional culture system in the in vitro culture of immature human oocytes. A past study has used microreactors to form three-dimensional bioreactors to support the growth of different types of cells [36]. Consisting of a drop of liquid encapsulated by hydrophobic powder particles, this system can provide a suitable microenvironment for in vitro maturation of oocytes. In addition, the development of microfluidic technology will exert an important impact in the field of human gametes and preimplantation embryo development and will have potential applications in the field of ART. This technology enables the creation of microfluidic models mimicking the "menstrual cycle of women" [37]. These models include interconnected 3D models of different tissues, such as 3D models of the ovaries, fallopian tubes, uterus, cervix, and vagina, in the female reproductive system and the endocrine cycle between various organ modules. The mechanical and biochemical properties of microfluidic systems still require intensive research before these systems can be applied to clinical applica-

At present, the in vitro maturation rate of immature human oocytes can reach

The results of current research showed that human oocytes matured in vivo or in vitro display no significant differences in terms of their spindle morphology, organelle distribution, cortical particle distribution, and mitochondrial morphology [38, 39]. By observing embryos dynamically using time-lapse videos, it was confirmed that oocytes matured in vivo or in vitro showed no significant differences in terms of the morphological dynamics observed during the early development of embryos derived from these oocytes [40]. Another study has also shown that the oocytes matured in vitro and in vivo are different in terms of their organelle function, distribution, and gene expression [41]. The different experimental conclusions mentioned above may be caused by different sources and quality of oocytes used in these studies. Therefore, attention should be paid to clarify the IVM efficiency of oocytes retrieved from different sources, so as to reasonably evaluate the safety of IVM. In terms of epigenetics, a study has reported that IVM exerts no significant effect on the methylation level of maternal imprinted genes, such as

70%, but the developmental potential of mature oocytes obtained in vitro is still lower than that of mature oocytes obtained in vivo. In addition, the rate of blastocyst development and the rate of implantation are relatively low after the fertilization of IVM oocytes. The main reason of such discrepancy may be related to non-synchronized nuclear and cytoplasmic maturation during IVM. With the further development in the basic and clinical research of IVM, the in-depth study on the mechanisms of oocyte maturation and mastery of key factors involved in oocyte maturation will contribute to the improvement and optimization of clinical

low response to hormones [30].

administration.

**6. IVM culture system**

**6.1 Improvement of IVM media**

method in human oocytes should be further verified.

**5.3 Women with normal ovaries and menstrual cycles**

A large number of antral follicles are present in the ovary of infertile women with anovulatory PCOS. These antral follicles are more sensitive to gonadotropins, and hence the risk of OHSS is increased when hormones are used to induce ovulation. Therefore, for PCOS patients, immature oocytes can be retrieved from antral follicle for in vitro maturation [28]. The use of hCG at 36 h before oocyte retrieval in PCOS patients can promote the resumption of meiosis of immature oocytes and their in vitro maturation, improving the rate of pregnancy and clinical outcomes [29]. The use of small doses of gonadotropin before the retrieval of immature oocytes from PCOS patients is also beneficial to improve the maturation potential of oocytes, increasing the rate of embryo implantation and clinical pregnancy. In addition, IVM techniques can also be considered for some PCOS patients with no or

Based on the advantages of low hormone dosage, low cost, and simple treatment process, IVM has been gradually applied to the treatment of infertile women with normal ovaries and regular menstrual cycles. However, it remains controversial as whether the use of hCG is required in the IVM treatment of this type of patient prior to oocyte retrieval. It should be noted that the hCG trigger exerts different effects on normal ovaries and PCOS patients. In the IVM treatment cycle of PCOS patients, dominant follicles are barely visible in the ovary, but MI-stage oocytes can be retrieved from small follicles after hCG-induced ovulation. However, after the hCG trigger is used in the normal ovary during the follicular phase, most oocytes retrieved from small follicles are oocytes in the GV stage. There is currently no evidence suggesting that the hCG trigger exerts a significant effect on the pregnancy rate, live birth rate, or abortion rate in the IVM of immature oocytes obtained from normal ovaries [31]. However, the accuracy of these findings is limited by the small number of samples. Therefore, a well-designed, randomized, and controlled clinical trial is needed to further confirm the optimal dosage and timing of hCG

The in vitro maturation of oocytes is mainly affected by culture conditions. At present, the common media used for the IVM of immature human oocytes include TCM-199 medium, Ham's F10 medium, and Chang's medium. In addition, serum, gonadotropin [follicle-stimulating hormone and luteinizing hormone], growth factors, and steroids can be added in a basal medium to produce a complex medium. At present, commercial IVM media have been widely used. However, no breakthrough has been made in the research on improving the quality of oocytes by improving the IVM medium. In recent years, research and application of antioxidants and growth-promoting factors have promoted the advancement of this technology to some extent. In addition, using cell cycle regulators or inhibitors of mitotic spindle formation, the synchronization of nuclear and cytoplasmic maturation of immature oocytes can be achieved by inhibiting GVBD, thereby increasing the blastocyst rate and live birth rate in animal models [32]. However, the safety and efficacy of this

**5.2 PCOS patients**

**28**

### **6.2 Optimization of IVM culture environment and process**

The culture environment, equipment, and related operations in the IVM system may affect the in vitro maturation and embryo development of immature oocytes. Therefore, the optimization of the embryo culture environment and process of in vitro operation will help to maintain the potential of embryonic development [33, 34]. The three-dimensional culture system can support the development of follicles by using biological materials to maintain cell-to-cell information exchange. In addition, mature oocytes can be obtained by using a three-dimensional culture system in the in vitro culture of anterior follicles of nonhuman primates [35], although no reports are available regarding the use of a three-dimensional culture system in the in vitro culture of immature human oocytes. A past study has used microreactors to form three-dimensional bioreactors to support the growth of different types of cells [36]. Consisting of a drop of liquid encapsulated by hydrophobic powder particles, this system can provide a suitable microenvironment for in vitro maturation of oocytes. In addition, the development of microfluidic technology will exert an important impact in the field of human gametes and preimplantation embryo development and will have potential applications in the field of ART. This technology enables the creation of microfluidic models mimicking the "menstrual cycle of women" [37]. These models include interconnected 3D models of different tissues, such as 3D models of the ovaries, fallopian tubes, uterus, cervix, and vagina, in the female reproductive system and the endocrine cycle between various organ modules. The mechanical and biochemical properties of microfluidic systems still require intensive research before these systems can be applied to clinical applications in areas such as IVM of immature human oocytes.
