**4. Indications for IVM**

The indications for IVM include normal ovulatory women (mechanical factor or male infertility), PCOS patients or susceptible to develop ovarian hyperstimulation (OHSS), contraindication to hormonal administration, patients with estrogensensitive cancers, or those who require rapid fertility preservation before undergoing potentially gonadotoxic treatments. Other occasional indications may include fertilization failure; poor ovarian response [22]; rescue of oocytes, which have failed to mature in stimulated cycles [23]; or unexplained primarily poor-quality embryos [24].

### **4.1 IVM in women with normal ovulatory cycles**

In early studies, Mikkelsen et al. [7, 25, 26] reported a 17–18% pregnancy rates resulting from in vitro matured oocytes. These pregnancy rates were disappointingly low in comparison with regular IVF results obtained in that time. A constant improvement in pregnancy rate up to 30% was achieved during the last decade, mainly due to application of FSH/hCG priming in IVM protocol [6] and proper patient selection [27]. It seems that in normal ovulatory patients IVM may be an intriguing alternative to conventional IVF techniques resulting in comparable pregnancy rates. It removes the side effects of pituitary suppression and gonadotropin stimulation, especially OHSS; reduces the costs of the entire procedure, both in terms of time consumption and patient/society costs for drugs; and reduces psychological impact.

### **4.2 PCOS patients**

PCOS patients are likely to develop OHSS with conventional IVF treatments. Substituting IVM in PCOS patients eliminates the risk of OHSS and lowers the cost of the treatment. From the early 2000s until nowadays, studies have demonstrated a reassuring pregnancy and delivery rate in PCOS patients undergoing

**183**

delivered.

**4.3 Fertilization failure**

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice*

IVM treatments of 21.9–29.9% [28–31]. Recent studies reported up to 32–44% pregnancy and 22–29% delivery rates [32, 33], compared with IVF pregnancy rate results of 38–45% [33–35]. Junk and Yeap transferred a single blastocyst embryo obtained after IVM in patients with PCOS. A live birth rate of 42.4% per oocyte collection and 45.2% per embryo transfer were obtained [34]. Vitek et al. [35] have recently described implantation, pregnancy, and delivery rates of 17.5, 40, and 40%, respectively, in 20 estrogens suppressed in vitro maturation cycles. In a latest retrospective study comparing results of 61 IVM vs. 53 antagonist protocol cycles in young patients with PCOS, a comparable pregnancy and delivery rates of 30% vs. 40% and 21.3% vs. 28.8%, respectively, was obtained [36]. Those recent reports are encouraging, as in Europe the pregnancy and delivery rates in this group of patients undergoing ICSI was 35.5% and 24.3%, respectively [37]. de Ziegler et al. [38] opposed the need of IVM in the gonadotropin-releasing hormone (GnRH) antagonist era. However, his conclusion is based on outdated publication [28, 39, 40], with poor results in terms of pregnancy and delivery rates in IVM. However, to update, it is ascertained that such data has already improved. GnRH-agonist (GnRH-a) used as a trigger to control the risk of OHSS may cause higher pregnancy losses due to luteal phase defects [41]. In order to overcome this complication in antagonist protocol/agonist trigger, the dual-trigger approach (GnRH-a + low hCG) was proposed; 2.9% of OHSS complications developed [42]. GnRH trigger combined with intensive luteal support in OHSS high-risk patients can facilitate fresh embryo transfer; however, the occurrence of late OHSS was not totally eliminated [43]. Applying the policy of ovarian stimulation with a dual-trigger approach and freezing all of the oocytes or embryos for future use [44] do not, necessarily, eliminate totally OHSS. In a few patients after dual trigger and freeze all, severe OHSS was reported [45]. It seems that in PCOS patient, IVM is a simple, less stressful, and economical protocol of treatment. The puncture is simple and safe, and it may improve the disrupted endocrine environment and induce a spontaneous recovery of ovulation in women with PCOS [46]. It can also avoid short-term complications, such as OHSS, and elude massive hormonal stimulation and long-term complications, such

as hormone-dependent neoplasms including breast and ovarian cancer.

Repeated IVF failure is a highly upsetting condition for patients who have apparently normal ovarian stimulation and follicular development, which underwent numerous unsuccessful IVF cycles with no embryos for transfer. Often, these patients are referred to surrogacy or egg donation program, which is also a psychological and economic burden for the couples [47]. Failures following IVF treatment might occur due to many reasons, such as formation of low-quality embryos, maturation arrest of oocytes [48], uncertain diagnosis of oocyte factor, or empty follicle syndrome. Thus, IVM was also proposed for treating patients with poor ovarian response; moreover it might serve the last choice to achieve pregnancy in IVF [22]. Other indications can be applying IVM in rare conditions, such as to rescue oocytes which have failed to mature in stimulated cycles [49] or cases with unexplained primarily poor-quality embryos. Hourvitz et al. [23] examined efficacy of IVM in seven patients with three or more conventional IVF failures due to abnormal oocyte development due to empty follicle syndrome, oocyte maturation arrest, or failure of fertilization. Four women received minimal ovarian stimulation with FSH. Oocytes were obtained in all patients: mean maturation rate was 39.6%, and mean fertilization rate is 45.8%. Embryo transfer was performed in four women; two patients with previous empty follicle syndrome conceived and

*DOI: http://dx.doi.org/10.5772/intechopen.91802*

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice DOI: http://dx.doi.org/10.5772/intechopen.91802*

IVM treatments of 21.9–29.9% [28–31]. Recent studies reported up to 32–44% pregnancy and 22–29% delivery rates [32, 33], compared with IVF pregnancy rate results of 38–45% [33–35]. Junk and Yeap transferred a single blastocyst embryo obtained after IVM in patients with PCOS. A live birth rate of 42.4% per oocyte collection and 45.2% per embryo transfer were obtained [34]. Vitek et al. [35] have recently described implantation, pregnancy, and delivery rates of 17.5, 40, and 40%, respectively, in 20 estrogens suppressed in vitro maturation cycles. In a latest retrospective study comparing results of 61 IVM vs. 53 antagonist protocol cycles in young patients with PCOS, a comparable pregnancy and delivery rates of 30% vs. 40% and 21.3% vs. 28.8%, respectively, was obtained [36]. Those recent reports are encouraging, as in Europe the pregnancy and delivery rates in this group of patients undergoing ICSI was 35.5% and 24.3%, respectively [37]. de Ziegler et al. [38] opposed the need of IVM in the gonadotropin-releasing hormone (GnRH) antagonist era. However, his conclusion is based on outdated publication [28, 39, 40], with poor results in terms of pregnancy and delivery rates in IVM. However, to update, it is ascertained that such data has already improved. GnRH-agonist (GnRH-a) used as a trigger to control the risk of OHSS may cause higher pregnancy losses due to luteal phase defects [41]. In order to overcome this complication in antagonist protocol/agonist trigger, the dual-trigger approach (GnRH-a + low hCG) was proposed; 2.9% of OHSS complications developed [42]. GnRH trigger combined with intensive luteal support in OHSS high-risk patients can facilitate fresh embryo transfer; however, the occurrence of late OHSS was not totally eliminated [43]. Applying the policy of ovarian stimulation with a dual-trigger approach and freezing all of the oocytes or embryos for future use [44] do not, necessarily, eliminate totally OHSS. In a few patients after dual trigger and freeze all, severe OHSS was reported [45]. It seems that in PCOS patient, IVM is a simple, less stressful, and economical protocol of treatment. The puncture is simple and safe, and it may improve the disrupted endocrine environment and induce a spontaneous recovery of ovulation in women with PCOS [46]. It can also avoid short-term complications, such as OHSS, and elude massive hormonal stimulation and long-term complications, such as hormone-dependent neoplasms including breast and ovarian cancer.

### **4.3 Fertilization failure**

*Innovations in Assisted Reproduction Technology*

**3. Definitions of IVM in human**

**4. Indications for IVM**

psychological impact.

**4.2 PCOS patients**

according to oocyte cumulus complex classification. This was followed by oocyte intracytoplasmic sperm injection (ICSI) for the injection of a single sperm cell into the MII oocytes on the day of OPU or 1–2 days afterward, according to oocyte maturation (**Figure 4A** (a and b) and **B** (a and b)). Two to three embryos were

The biological definition of oocyte IVM is to aspirate GV oocytes from antral follicles and culture them for in vitro maturation to a MII stage. Apart from routine IVF, the clinical definition of an IVM cycle should include an understanding that it involves the retrieval of oocytes from small and intermediate-sized follicles in an ovary before the largest follicle has surpassed 13 mm in mean diameter [21]. However, since modifications of the IVM technique are commonly employed, it is suggested, though, to designating the cycle when an oocyte trigger is given. When oocyte triggering is not performed, the cycle should be designated as IVM without triggering. When the addition of gonadotropin stimulation is given for few days in the early follicular phase, the cycle should be designated as IVM with short gonadotropin stimulation or modified natural cycle IVF with early triggering combined

The indications for IVM include normal ovulatory women (mechanical factor or male infertility), PCOS patients or susceptible to develop ovarian hyperstimulation (OHSS), contraindication to hormonal administration, patients with estrogensensitive cancers, or those who require rapid fertility preservation before undergoing potentially gonadotoxic treatments. Other occasional indications may include fertilization failure; poor ovarian response [22]; rescue of oocytes, which have failed to mature in stimulated cycles [23]; or unexplained primarily poor-quality embryos [24].

In early studies, Mikkelsen et al. [7, 25, 26] reported a 17–18% pregnancy rates resulting from in vitro matured oocytes. These pregnancy rates were disappointingly low in comparison with regular IVF results obtained in that time. A constant improvement in pregnancy rate up to 30% was achieved during the last decade, mainly due to application of FSH/hCG priming in IVM protocol [6] and proper patient selection [27]. It seems that in normal ovulatory patients IVM may be an intriguing alternative to conventional IVF techniques resulting in comparable pregnancy rates. It removes the side effects of pituitary suppression and gonadotropin stimulation, especially OHSS; reduces the costs of the entire procedure, both in terms of time consumption and patient/society costs for drugs; and reduces

PCOS patients are likely to develop OHSS with conventional IVF treatments. Substituting IVM in PCOS patients eliminates the risk of OHSS and lowers the cost of the treatment. From the early 2000s until nowadays, studies have demonstrated a reassuring pregnancy and delivery rate in PCOS patients undergoing

transferred 48–78 h post ICSI. Supplementary embryos were vitrified.

with IVM (if hCG or GnRh-agonist triggering was delivered) [21].

**4.1 IVM in women with normal ovulatory cycles**

**182**

Repeated IVF failure is a highly upsetting condition for patients who have apparently normal ovarian stimulation and follicular development, which underwent numerous unsuccessful IVF cycles with no embryos for transfer. Often, these patients are referred to surrogacy or egg donation program, which is also a psychological and economic burden for the couples [47]. Failures following IVF treatment might occur due to many reasons, such as formation of low-quality embryos, maturation arrest of oocytes [48], uncertain diagnosis of oocyte factor, or empty follicle syndrome. Thus, IVM was also proposed for treating patients with poor ovarian response; moreover it might serve the last choice to achieve pregnancy in IVF [22]. Other indications can be applying IVM in rare conditions, such as to rescue oocytes which have failed to mature in stimulated cycles [49] or cases with unexplained primarily poor-quality embryos. Hourvitz et al. [23] examined efficacy of IVM in seven patients with three or more conventional IVF failures due to abnormal oocyte development due to empty follicle syndrome, oocyte maturation arrest, or failure of fertilization. Four women received minimal ovarian stimulation with FSH. Oocytes were obtained in all patients: mean maturation rate was 39.6%, and mean fertilization rate is 45.8%. Embryo transfer was performed in four women; two patients with previous empty follicle syndrome conceived and delivered.

### **4.4 Fertility preservation**

The emerging technology of IVM has recently become another option for fertility preservation. This process can be done without hormonal stimulation [50]. In the cases of cancer patients, who must be started on immediate chemotherapy, IVM might be the only option to preserve fertility by collecting oocytes during the follicular phase, within up to 13 days from cancer diagnosis, and cryopreservation [51, 52]. To shorten the period of time until cancer treatment, studies by Maman et al. [53] reported on luteal phase minimal ovarian stimulation with a reasonable number of harvested oocytes. Therefore, in the cases of cancer patients, especially in whom hormonal treatment is contraindicated and in those who must start chemotherapy without postponement, IVM might be the only choice to preserve fertility [53]. Recently, one successful pregnancy resulting from cryopreserved embryos obtained from IVM oocytes after oophorectomy in an ovarian cancer patient was reported [54]. Other studies have raised the possibility to preserve fertility even in pediatric patients.

Preserving in vitro matured oocytes from antral follicles found in harvested ovarian tissue is an experimental technique that offers a possible advantage over ovarian tissue cryopreservation. Using a mature, frozen, and later thawed oocyte for fertilization might serve as a safer option for fertility preservation than reimplantation of ovarian cortex tissue, due to the risk of malignant cell reseeding [55]. Caravani et al. followed a total of 84 chemotherapy-naïve patients ages < 1–18 years old, who were referred for fertility preservation. Thirty-three children were premenarche and 51 postmenarche. IVM was performed in the pre- and postmenarche groups and in subgroups of very young (up to age 5 years) and older (5–10 years) premenarche patients. The study concluded that IVM is feasible in the prepubertal age group. However, the success of in vitro maturation of those oocytes was correlated with the patient's age (more oocytes were obtained from the post pubertal vs. prepubertal); no mature oocytes are cryopreserved for girls under the age of five [55]. Additionally, it was found that fertilization potential of oocytes was negatively affected after vitrification of IVM oocytes [56]. This implies that vitrification/warming itself could also induce some detrimental effects on IVM oocytes. Actually, present vitrification methods have been adapted to use good-quality in vivo matured oocytes from young women. Therefore, studies to improve survival and further embryological developmental competence of the oocytes retrieved from IVM program are urgently required in order to successfully apply them to IVM fertility preservation program for cancer patients [57].

### **5. Pregnancy results**

Two thousand healthy infants have been born following immature oocyte retrieval and IVM [58].

### **5.1 Obstetric and fetal complications**

Soderstrom-Anttila et al. presented comparable complications and malformations for babies born after IVM and IVF [31]. Buckett et al. commented on a normal pregnancy course for IVM patients compared to routine IVF [59]. Fadini et al. performed a retrospective cohort study involving 196 babies born from IVM cycles compared with 194 children born from conventional ICSI cycles, which were performed during the same period of time. In single births, gestational age at delivery was comparable, but birth weight was significantly higher (P = 0.009) in children from IVM cycles (3091 ± 669 vs. 3269 ± 619 g). In a separate analysis of the IVM group, comparing

**185**

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice*

singleton births derived with certainty from oocytes matured in vitro (n = 71) or in vivo (n = 74), no statistically significant differences were observed in terms of birth weight (3311 ± 637 vs. 3194 ± 574 g, respectively) and gestational age (38.9 ± 2.4 vs. 38.4 ± 2.1 weeks, respectively). In twin births, gestational age was lower in IVM cycles, while weight at birth was comparable (ICSI, 2432 ± 540 g; IVM, 2311 ± 577 g). In single births, major and minor abnormalities were 2 (1.4%) and 6 (4.1%) in the ICSI group and 0 (0.0%) and 8 (5.2%) in the IVM category, respectively. In twin children, major and minor abnormalities were 1 (2.2%) and 2 (4.3%) in ICSI babies

Obstetric outcome and congenital anomalies of 1421 babies (960 singletons, 442 twins, 15 triplets, and 4 quadruplets) born from 1187 IVM pregnancies were recently summarized by Chian et al. Reassuring results were obtained. The incidence of congenital malformation was 2% in singletons and 1% in twins [61].

In vitro maturation as a part of assisted reproductive technologies, may not, yet, be free of possible unidentified future problems. Epigenetic modifications necessary for normal development are established during oocyte growth. In vitro maturation, therefore, may modify the normal maturation of the oocytes [62]. Moreover, the capability of reprogramming the male chromatin after fertilization is dependent upon the maturity of the oocyte. It is questionable, whether this process might be affected by IVM [63]. It was postulated that IVM oocytes were more likely to have abnormal chromosomal configurations and disorganized meiotic spindle microtubules [64]. This finding may be a probable explanation for the reduced developmental potential of oocytes matured in vitro compared to those matured in vivo. However, despite the great achievements obtained in treating infertile couples by standard IVF during the last 34 years, it has become apparent in recent years that ovarian stimulation may itself have disadvantageous effects on oogenesis, with production of aneuploidy [65], reduced embryo quality, and lower endometrial receptivity and might even contribute to perinatal effects [66]. Moreover, human and animal data have demonstrated the potential changes in the implanta-

3.Affecting endometrial-embryo interaction causing impairment on fetal devel-

4.Increasing the risk of abnormal placentation, leading to increased rates of low

There is no doubt that efforts should be made to improve IVM outcome. An adequate learning curve taking into consideration clinical decisions, retrieval

Improving culture condition to optimum must be determined, for instance, adding epidermal growth factor family molecules, such as amphiregulin and epiregulin

procedure, laboratory knowledge, and experience is required [33, 68].

and 0 (0.0%) and 2 (4.6%) in IVM cycles, respectively [60].

tion process that may occur following superovulation [67]:

2.Causing immunologic changes to the endometrium.

1.Changing endometrial gene expression.

opment and growth.

**7. Improving IVM outcome**

birth weight.

*DOI: http://dx.doi.org/10.5772/intechopen.91802*

**6. Future concerns**

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice DOI: http://dx.doi.org/10.5772/intechopen.91802*

singleton births derived with certainty from oocytes matured in vitro (n = 71) or in vivo (n = 74), no statistically significant differences were observed in terms of birth weight (3311 ± 637 vs. 3194 ± 574 g, respectively) and gestational age (38.9 ± 2.4 vs. 38.4 ± 2.1 weeks, respectively). In twin births, gestational age was lower in IVM cycles, while weight at birth was comparable (ICSI, 2432 ± 540 g; IVM, 2311 ± 577 g). In single births, major and minor abnormalities were 2 (1.4%) and 6 (4.1%) in the ICSI group and 0 (0.0%) and 8 (5.2%) in the IVM category, respectively. In twin children, major and minor abnormalities were 1 (2.2%) and 2 (4.3%) in ICSI babies and 0 (0.0%) and 2 (4.6%) in IVM cycles, respectively [60].

Obstetric outcome and congenital anomalies of 1421 babies (960 singletons, 442 twins, 15 triplets, and 4 quadruplets) born from 1187 IVM pregnancies were recently summarized by Chian et al. Reassuring results were obtained. The incidence of congenital malformation was 2% in singletons and 1% in twins [61].

### **6. Future concerns**

*Innovations in Assisted Reproduction Technology*

fertility preservation program for cancer patients [57].

Two thousand healthy infants have been born following immature oocyte

Soderstrom-Anttila et al. presented comparable complications and malformations for babies born after IVM and IVF [31]. Buckett et al. commented on a normal pregnancy course for IVM patients compared to routine IVF [59]. Fadini et al. performed a retrospective cohort study involving 196 babies born from IVM cycles compared with 194 children born from conventional ICSI cycles, which were performed during the same period of time. In single births, gestational age at delivery was comparable, but birth weight was significantly higher (P = 0.009) in children from IVM cycles (3091 ± 669 vs. 3269 ± 619 g). In a separate analysis of the IVM group, comparing

**5. Pregnancy results**

retrieval and IVM [58].

**5.1 Obstetric and fetal complications**

The emerging technology of IVM has recently become another option for fertility preservation. This process can be done without hormonal stimulation [50]. In the cases of cancer patients, who must be started on immediate chemotherapy, IVM might be the only option to preserve fertility by collecting oocytes during the follicular phase, within up to 13 days from cancer diagnosis, and cryopreservation

[51, 52]. To shorten the period of time until cancer treatment, studies by Maman et al. [53] reported on luteal phase minimal ovarian stimulation with a reasonable number of harvested oocytes. Therefore, in the cases of cancer patients, especially in whom hormonal treatment is contraindicated and in those who must start chemotherapy without postponement, IVM might be the only choice to preserve fertility [53]. Recently, one successful pregnancy resulting from cryopreserved embryos obtained from IVM oocytes after oophorectomy in an ovarian cancer patient was reported [54]. Other studies have raised the possibility to preserve fertility even in pediatric patients. Preserving in vitro matured oocytes from antral follicles found in harvested ovarian tissue is an experimental technique that offers a possible advantage over ovarian tissue cryopreservation. Using a mature, frozen, and later thawed oocyte for fertilization might serve as a safer option for fertility preservation than reimplantation of ovarian cortex tissue, due to the risk of malignant cell reseeding [55]. Caravani et al. followed a total of 84 chemotherapy-naïve patients ages < 1–18 years old, who were referred for fertility preservation. Thirty-three children were premenarche and 51 postmenarche. IVM was performed in the pre- and postmenarche groups and in subgroups of very young (up to age 5 years) and older (5–10 years) premenarche patients. The study concluded that IVM is feasible in the prepubertal age group. However, the success of in vitro maturation of those oocytes was correlated with the patient's age (more oocytes were obtained from the post pubertal vs. prepubertal); no mature oocytes are cryopreserved for girls under the age of five [55]. Additionally, it was found that fertilization potential of oocytes was negatively affected after vitrification of IVM oocytes [56]. This implies that vitrification/warming itself could also induce some detrimental effects on IVM oocytes. Actually, present vitrification methods have been adapted to use good-quality in vivo matured oocytes from young women. Therefore, studies to improve survival and further embryological developmental competence of the oocytes retrieved from IVM program are urgently required in order to successfully apply them to IVM

**4.4 Fertility preservation**

**184**

In vitro maturation as a part of assisted reproductive technologies, may not, yet, be free of possible unidentified future problems. Epigenetic modifications necessary for normal development are established during oocyte growth. In vitro maturation, therefore, may modify the normal maturation of the oocytes [62]. Moreover, the capability of reprogramming the male chromatin after fertilization is dependent upon the maturity of the oocyte. It is questionable, whether this process might be affected by IVM [63]. It was postulated that IVM oocytes were more likely to have abnormal chromosomal configurations and disorganized meiotic spindle microtubules [64]. This finding may be a probable explanation for the reduced developmental potential of oocytes matured in vitro compared to those matured in vivo. However, despite the great achievements obtained in treating infertile couples by standard IVF during the last 34 years, it has become apparent in recent years that ovarian stimulation may itself have disadvantageous effects on oogenesis, with production of aneuploidy [65], reduced embryo quality, and lower endometrial receptivity and might even contribute to perinatal effects [66]. Moreover, human and animal data have demonstrated the potential changes in the implantation process that may occur following superovulation [67]:


### **7. Improving IVM outcome**

There is no doubt that efforts should be made to improve IVM outcome. An adequate learning curve taking into consideration clinical decisions, retrieval procedure, laboratory knowledge, and experience is required [33, 68].

Improving culture condition to optimum must be determined, for instance, adding epidermal growth factor family molecules, such as amphiregulin and epiregulin to the culture media which augmented oocyte maturation [69], or brain-derived neurotrophic factor (BDNF) and glial-cell-derived neurotrophic factor (GDNF), which recently were reported to improve maturation rates in human oocytes [70], or addition of dibutyryl cyclic adenosine 3′,5′-monophosphate (cAMP) to mouse oocytes in vitro to arrest germinal vesicle break down, in order to combine it with the cytoplasmic maturation [71].
