**4. Factors affecting the in vitro maturation of oocytes**

### **4.1 Effect of culture time on in vitro maturation of oocytes**

Maturation of oocytes includes the nuclear and cytoplasmic maturation of oocytes. Nuclear maturation refers to the rupture of the germinal vesicle, separation of homologous chromosomes, appearance of the perivitelline space, and discharge of the first polar body. Cytoplasmic maturation refers to the completion of protein phosphorylation and dephosphorylation as well as the rearrangement of organelles in oocytes. Only the oocytes whose nucleus and cytoplasm are matured simultaneously can have adequate fertility and the potential for embryo development. Studies have found that most oocytes cultured in vitro can reach maturity within 24–48 h. The length of in vitro culture can affect the developmental potential of the embryo. The rate of nuclear maturation in oocytes cultured for 48 h in vitro is significantly higher than that for 24 h, but the rate of cytoplasmic maturation in oocytes cultured for 48 h is not significantly different from that for 24 h. Excessive culture time leads to the aging of oocytes and an increased level of associated genetic risks. When the culture time is too short, the maturation of cytoplasm and nucleus is not synchronized and will affect the subsequent development potential of the embryo. Wrenzycki et al. [13] found that oocytes only possess the ability to mature in the final stage of development. Therefore, adequate extension of IVM time can promote the necessary process of oocyte maturation, increase the rate of nuclear maturation in immature oocytes, and significantly improve in vitro developmental potential of oocytes and the rate of high-quality embryos.

#### **4.2 Effects of hormones on in vitro maturation of oocytes**

Gonadotropin can promote the expansion of cumulus cells and stimulate the maturation of the nucleus and cytoplasm of oocytes, thus facilitating the formation of embryos and blastocysts in the cleavage stage and playing an important role in follicular development [14]. The addition of follicle-stimulating hormone to the culture medium for oocyte maturation can promote the cytoplasmic maturation of oocytes. Some scholars believe that the effect of follicle-stimulating hormone is related to its concentration. When the concentration is 5 g/mL, a relatively high cleavage rate (79.1%) and blastocyst rate (16.1%) can be obtained [15]. The addition of LH or human chorionic gonadotropin to the IVM culture medium may promote protein synthesis, enhance oocyte metabolism, and facilitate oocyte maturation. The concentration of estradiol (E2) in the human body increases with an increasing volume of follicles. In addition, estradiol is involved in maintaining the meiotic arrest of oocytes and can promote the cytoplasmic maturation of oocytes. During in vitro culture of oocytes, nuclear maturation is faster than cytoplasmic maturation. Therefore, the addition of E2 to the culture medium helps synchronize the development of the nucleus and cytoplasm in oocytes.

#### **4.3 Effect of antioxidant addition on in vitro maturation of oocytes**

As a hydrophobic activator, forskolin (FSK) can increase the activity of adenylate cyclase in mammalian cells and the level of intracellular cAMP. By adding FSK to an IVM culture medium, Ezoe et al. [16] significantly improved the developmental capacity of oocytes in the GV stage. By adding FSK to the culture medium, Zeng et al. [17] promoted the synchronization of nuclear and cytoplasmic maturation and increased the rates of maturation, cleavage, and high-quality embryos. During IVM, the presence of oxidative stress may block oocyte maturation, lead to abnormal gene expression, and impair the cytoplasmic and nuclear development of oocytes, thereby resulting in the failure to obtain high-quality oocytes and decreasing the fertility and developmental capacity. The addition of antioxidants to the culture medium can reduce the damage caused by oxidative stress. By adding a-lipoic acid to the culture medium, Zavareh et al. [18] reduced the content of active oxygen, increased the total antioxidation capacity, and promoted the nuclear and cytoplasmic maturation of oocytes in vitro. The results of Mokhber et al. [19, 20] showed that an appropriate concentration of a natural antioxidant, crocin (100 g/mL), and aqueous extract of saffron (40 g/mL) can increase the concentration of glutathione (GSH), protect oocytes, and significantly increase IVM rate and fertility rate.

#### **4.4 Effect of co-culture with mural granulosa cells on in vitro maturation of oocytes**

Together with cumulus cells and follicular fluid, MGC form an in vivo environment for oocyte maturation. Co-culture with MGC can increase the rate of nuclear and cytoplasmic maturation of immature oocytes. Studies have shown that coculture with parietal MGC can improve the nuclear maturation of naked oocytes, slow down the nuclear maturation of naked oocytes, increase the content of glutathione in naked oocytes, reduce the activity of glucose-6-phosphate dehydrogenase in naked oocytes, increase the rate of cytoplasmic maturation, and facilitate the simultaneous development of the nucleus and cytoplasm of oocytes [21]. Although immature oocytes detached of MGC can still mature, they cannot undergo normal

**27**

preservation of fertility.

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

with naked oocytes at a 1:5 ratio.

nucleus and cytoplasm of murine oocytes.

ture oocytes obtained from cesarean section.

**5. Sources of immature oocytes**

**oocytes**

**oocytes**

fertilization and development because the cytoplasm is not synchronously matured [22]. The addition of a certain amount of MGC to the culture medium can delay the nuclear maturation of oocytes, so that the maturation of the nucleus and cytoplasm becomes more synchronized. However, there is currently no uniform standard for the amount of MGC addition. Choi et al. [23] significantly increased the development potential of embryos by co-culturing the oocyte-corona-cumulus complex

**4.5 Effect of co-culture with oviductal epithelial cells on in vitro maturation of** 

Some scholars have pointed out that the maturation of oocytes is completed in the fallopian tube, and hence some components of the fallopian tube may affect the maturation process of oocytes. Human tubal fluid (HTF) has been used to culture oocytes. Shirazi et al. [24] co-cultured ovine oocytes with oviductal epithelial cells (OECs) and conducted IVF, resulting in higher cleavage and blastocyst rates.

**4.6 Effect of co-culture with mesenchymal stem cells on in vitro maturation of** 

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

In addition to the potential of self-renewal and multidirectional differentiation, mesenchymal stem cells (MSCs) can also secrete a variety of cytokines and growth factors, and some biologically active factors can enhance the in vitro maturation of oocytes and subsequent developmental potential of embryos. By adding MSCs to a culture medium, Ling et al. [25] significantly increased the maturation rate and rate of blastocyst formation of immature murine oocytes. It can be seen that the co-culture system with MSCs can promote the simultaneous development of the

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 imma-

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

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

*Embryology - Theory and Practice*

**4.2 Effects of hormones on in vitro maturation of oocytes**

development of the nucleus and cytoplasm in oocytes.

**4.3 Effect of antioxidant addition on in vitro maturation of oocytes**

As a hydrophobic activator, forskolin (FSK) can increase the activity of adenylate cyclase in mammalian cells and the level of intracellular cAMP. By adding FSK to an IVM culture medium, Ezoe et al. [16] significantly improved the developmental capacity of oocytes in the GV stage. By adding FSK to the culture medium, Zeng et al. [17] promoted the synchronization of nuclear and cytoplasmic maturation and increased the rates of maturation, cleavage, and high-quality embryos. During IVM, the presence of oxidative stress may block oocyte maturation, lead to abnormal gene expression, and impair the cytoplasmic and nuclear development of oocytes, thereby resulting in the failure to obtain high-quality oocytes and decreasing the fertility and developmental capacity. The addition of antioxidants to the culture medium can reduce the damage caused by oxidative stress. By adding a-lipoic acid to the culture medium, Zavareh et al. [18] reduced the content of active oxygen, increased the total antioxidation capacity, and promoted the nuclear and cytoplasmic maturation of oocytes in vitro. The results of Mokhber et al. [19, 20] showed that an appropriate concentration of a natural antioxidant, crocin (100 g/mL), and aqueous extract of saffron (40 g/mL) can increase the concentration of glutathione (GSH), protect oocytes, and significantly increase

**4.4 Effect of co-culture with mural granulosa cells on in vitro maturation of** 

Together with cumulus cells and follicular fluid, MGC form an in vivo environment for oocyte maturation. Co-culture with MGC can increase the rate of nuclear and cytoplasmic maturation of immature oocytes. Studies have shown that coculture with parietal MGC can improve the nuclear maturation of naked oocytes, slow down the nuclear maturation of naked oocytes, increase the content of glutathione in naked oocytes, reduce the activity of glucose-6-phosphate dehydrogenase in naked oocytes, increase the rate of cytoplasmic maturation, and facilitate the simultaneous development of the nucleus and cytoplasm of oocytes [21]. Although immature oocytes detached of MGC can still mature, they cannot undergo normal

Gonadotropin can promote the expansion of cumulus cells and stimulate the maturation of the nucleus and cytoplasm of oocytes, thus facilitating the formation of embryos and blastocysts in the cleavage stage and playing an important role in follicular development [14]. The addition of follicle-stimulating hormone to the culture medium for oocyte maturation can promote the cytoplasmic maturation of oocytes. Some scholars believe that the effect of follicle-stimulating hormone is related to its concentration. When the concentration is 5 g/mL, a relatively high cleavage rate (79.1%) and blastocyst rate (16.1%) can be obtained [15]. The addition of LH or human chorionic gonadotropin to the IVM culture medium may promote protein synthesis, enhance oocyte metabolism, and facilitate oocyte maturation. The concentration of estradiol (E2) in the human body increases with an increasing volume of follicles. In addition, estradiol is involved in maintaining the meiotic arrest of oocytes and can promote the cytoplasmic maturation of oocytes. During in vitro culture of oocytes, nuclear maturation is faster than cytoplasmic maturation. Therefore, the addition of E2 to the culture medium helps synchronize the

**26**

IVM rate and fertility rate.

**oocytes**

fertilization and development because the cytoplasm is not synchronously matured [22]. The addition of a certain amount of MGC to the culture medium can delay the nuclear maturation of oocytes, so that the maturation of the nucleus and cytoplasm becomes more synchronized. However, there is currently no uniform standard for the amount of MGC addition. Choi et al. [23] significantly increased the development potential of embryos by co-culturing the oocyte-corona-cumulus complex with naked oocytes at a 1:5 ratio.

## **4.5 Effect of co-culture with oviductal epithelial cells on in vitro maturation of oocytes**

Some scholars have pointed out that the maturation of oocytes is completed in the fallopian tube, and hence some components of the fallopian tube may affect the maturation process of oocytes. Human tubal fluid (HTF) has been used to culture oocytes. Shirazi et al. [24] co-cultured ovine oocytes with oviductal epithelial cells (OECs) and conducted IVF, resulting in higher cleavage and blastocyst rates.

## **4.6 Effect of co-culture with mesenchymal stem cells on in vitro maturation of oocytes**

In addition to the potential of self-renewal and multidirectional differentiation, mesenchymal stem cells (MSCs) can also secrete a variety of cytokines and growth factors, and some biologically active factors can enhance the in vitro maturation of oocytes and subsequent developmental potential of embryos. By adding MSCs to a culture medium, Ling et al. [25] significantly increased the maturation rate and rate of blastocyst formation of immature murine oocytes. It can be seen that the co-culture system with MSCs can promote the simultaneous development of the nucleus and cytoplasm of murine oocytes.
