**1. Introduction**

The concept of in vitro maturation of oocytes (IVM) was firstly mentioned in literature by Pincus and Enzmann, initially in 1935 [1]. They conducted experiments in which ova, taken from tubes at various intervals after fertile matting, were cultured in vitro*.* Thirty-four years later, Eppig and Schroeder [2] designed the possibility to use IVM rather than protocols of hormonal stimulation currently in use. These authors mentioned "it may be possible to recover immature oocytes from several antral follicles, excluding the dominant preovulatory follicle, and mature them in proper culture." At that time it was impossible to accomplish that assignment due to technical reasons. However, 2 years later, Cha et al. [3] mentioned, "this is an engineering problem with the ultrasound equipment that will be resolved in

the future." Recently, Cha et al. were the first to succeed with IVM in human using immature oocytes retrieved from antral follicles. Trounson et al. [4] were the first who put IVM firmly in the clinical realm, obtaining a live birth from oocytes, recovered from untreated polycystic ovarian patient who underwent in vitro maturation.

In vitro maturation of oocytes have potential advantages over conventional IVF: a simple protocol with decreased or no hormonal stimulation before oocyte retrieval, lower cost of the treatment cycle, and reduced psychological impact. Moreover, and not of less of importance, the risk of OHSS is entirely avoided. Despite these benefits, however, there are still many debatable problems surrounding this treatment. Until one can say that IVM could be an alternative to conventional IVF treatment, these advantages have to be weighed against the pregnancy and delivery rates, children outcome, and possible risks.

There are several basic differences between routine in vitro fertilization (IVF) and IVM. These differences might be related to the size of aspirated follicles, the nuclear and cytoplasmic maturity of the oocytes, and the laboratory procedures. In order to achieve proper fertilization and embryo development, Gougeon and Testart in [5] pointed out that the follicles at the time of the collection for IVM procedures should be in the early antral to antral stage (0.2–14 mm) vs. preovulatory stage (0.16–20 mm) for IVF. After retrieval, majority of the oocytes are immature (MI, GV). However, only few will proceed in vitro to metaphase II (MII) in culture media, some after 6 h, but most of them after 24–48 h and only then will be able to undergo successful fertilization after ICSI. This is in comparison with conventional IVF, where most of the retrieved oocytes are mature, and fertilization might occur immediately after ICSI.

There are many controversial areas of debate, especially regarding the process of oocyte maturation in vitro: (a) nuclear maturation—the process that reverses meiotic arrest at prophase I (GV), driving the progression to MII. This process is followed by the expansion of cumulus granulosa cells and loss of intercellular communications between the cumulus cells and also between cumulus and the surface of the oocyte (a visible laboratory course); (b) cytoplasmic maturation—metabolic and structural modifications within the oocyte that prepares the ovum for activation, fertilization, and embryonic development (an invisible laboratory process) [2, 4, 5]; it should, therefore, be kept in mind that IVM may not be free of possible future complications and counseling of the patients thoroughly before commencing the treatment is essential.

### **2. Guidelines for IVM treatment**

### **2.1 Ultrasound monitoring**

Follicle monitoring follow-up is mainly performed using ultrasound scans of the ovaries and endometrium. However, in contrast to IVF, in IVM cycles, there is no need for serum hormonal level (estradiol, progesterone) follow-up.

First ultrasound should be performed on days 2–3 of the menstrual (or induced) cycle in order to record the number and size of all follicles along with endometrial thickness and a second scan on days 6–8, to determine the presence and size of the largest follicle in each ovary. A third scan should be completed on day of hCG trigger, to measure the endometrial thickness (**Figure 1**).

### **2.2 FSH priming**

Fadini et al. [6] reported that 77.4% of retrieved immature oocytes underwent maturation in vitro followed by 29.8% pregnancy rate vs. 48.4 and 15.2% in primed and non-primed IVM cycles, respectively. Mikkelsen et al. [7] pointed that priming

**179**

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

with r-FSH for 2–3 days before the harvesting of immature oocytes from patients with polycystic ovarian syndrome (PCOS) may improve the maturational potential of the oocytes and the implantation rate. Similar results were described by other

**2.3 Human chorionic gonadotropin (hCG)/GnRh-agonist priming and retrieval** 

The beneficial effect of hCG priming in IVM cycles and extension of the period of time from 35 to 36 h (routinely administrated in IVF cycles) to 38 h from hCG administration was demonstrated by Son et al. [12]. This hCG priming promotes GV oocytes to reach MI stage and increases the maturation rate of immature oocytes in vitro. Gonadotropin-releasing hormone agonist (GnRH-a) has been used recently in triggering oocyte maturation. In this approach, small follicles were stimulated with gonadotropins for 3–5 days. GnRH-a administration was performed, to trigger ovulation, when the largest follicles were 10–12 mm in diameter. Many immature oocytes, which underwent maturation in vitro, were then harvested, fertilized, and subsequently developed into blastocysts that resulted in live births [13]. This interesting observation might have a great importance in follicular cytoplasmic maturation due to the FSH surge obtained after GnRH triggering. The FSH surge might promote formation of LH receptors on the granulosa cells enhancing LH activity, induce plasminogen activator activity causing dissociation of oocytes from somatic cells of the follicle (therefore more immature oocytes could be obtained), and maintain the opening of gap junction between cumulus cells and oocyte which

Son et al. [18] conducted a study in which patients were triggered when the leading follicle was <10 mm, 10–14 mm, or >14 mm. In the group with a leading follicle >14 mm, only one pregnancy was obtained. The authors of the current paper administer, therefore, hCG trigger once a leading follicle of 10–12 mm is developed.

In IVM, estradiol levels are physiological, and oocyte collection is done before the endometrium is fully estrogenized. After aspiration, there is an insufficient support from the corpus luteum for endometrial receptivity. It was demonstrated

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

investigators in PCOS patients [8–11].

*In vitro maturation oocytes: clinical protocol.*

contributes to oocyte maturation [14–17].

**2.4 Timing of collection: at what follicle size?**

**2.5 Endometrial preparation and luteal support**

**interval**

**Figure 1.**

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

**Figure 1.**

*Innovations in Assisted Reproduction Technology*

**2. Guidelines for IVM treatment**

**2.1 Ultrasound monitoring**

and delivery rates, children outcome, and possible risks.

the future." Recently, Cha et al. were the first to succeed with IVM in human using immature oocytes retrieved from antral follicles. Trounson et al. [4] were the first who put IVM firmly in the clinical realm, obtaining a live birth from oocytes, recovered from untreated polycystic ovarian patient who underwent in vitro maturation. In vitro maturation of oocytes have potential advantages over conventional IVF: a simple protocol with decreased or no hormonal stimulation before oocyte retrieval, lower cost of the treatment cycle, and reduced psychological impact. Moreover, and not of less of importance, the risk of OHSS is entirely avoided. Despite these benefits, however, there are still many debatable problems surrounding this treatment. Until one can say that IVM could be an alternative to conventional IVF treatment, these advantages have to be weighed against the pregnancy

There are several basic differences between routine in vitro fertilization (IVF) and IVM. These differences might be related to the size of aspirated follicles, the nuclear and cytoplasmic maturity of the oocytes, and the laboratory procedures. In order to achieve proper fertilization and embryo development, Gougeon and Testart in [5] pointed out that the follicles at the time of the collection for IVM procedures should be in the early antral to antral stage (0.2–14 mm) vs. preovulatory stage (0.16–20 mm) for IVF. After retrieval, majority of the oocytes are immature (MI, GV). However, only few will proceed in vitro to metaphase II (MII) in culture media, some after 6 h, but most of them after 24–48 h and only then will be able to undergo successful fertilization after ICSI. This is in comparison with conventional IVF, where most of the retrieved oocytes are mature, and fertilization might occur immediately after ICSI. There are many controversial areas of debate, especially regarding the process of oocyte maturation in vitro: (a) nuclear maturation—the process that reverses meiotic arrest at prophase I (GV), driving the progression to MII. This process is followed by the expansion of cumulus granulosa cells and loss of intercellular communications between the cumulus cells and also between cumulus and the surface of the oocyte (a visible laboratory course); (b) cytoplasmic maturation—metabolic and structural modifications within the oocyte that prepares the ovum for activation, fertilization, and embryonic development (an invisible laboratory process) [2, 4, 5]; it should, therefore, be kept in mind that IVM may not be free of possible future complications and counseling of the patients thoroughly before commencing the treatment is essential.

Follicle monitoring follow-up is mainly performed using ultrasound scans of the ovaries and endometrium. However, in contrast to IVF, in IVM cycles, there is no

First ultrasound should be performed on days 2–3 of the menstrual (or induced) cycle in order to record the number and size of all follicles along with endometrial thickness and a second scan on days 6–8, to determine the presence and size of the largest follicle in each ovary. A third scan should be completed on day of hCG trig-

Fadini et al. [6] reported that 77.4% of retrieved immature oocytes underwent maturation in vitro followed by 29.8% pregnancy rate vs. 48.4 and 15.2% in primed and non-primed IVM cycles, respectively. Mikkelsen et al. [7] pointed that priming

need for serum hormonal level (estradiol, progesterone) follow-up.

ger, to measure the endometrial thickness (**Figure 1**).

**178**

**2.2 FSH priming**

*In vitro maturation oocytes: clinical protocol.*

with r-FSH for 2–3 days before the harvesting of immature oocytes from patients with polycystic ovarian syndrome (PCOS) may improve the maturational potential of the oocytes and the implantation rate. Similar results were described by other investigators in PCOS patients [8–11].

### **2.3 Human chorionic gonadotropin (hCG)/GnRh-agonist priming and retrieval interval**

The beneficial effect of hCG priming in IVM cycles and extension of the period of time from 35 to 36 h (routinely administrated in IVF cycles) to 38 h from hCG administration was demonstrated by Son et al. [12]. This hCG priming promotes GV oocytes to reach MI stage and increases the maturation rate of immature oocytes in vitro. Gonadotropin-releasing hormone agonist (GnRH-a) has been used recently in triggering oocyte maturation. In this approach, small follicles were stimulated with gonadotropins for 3–5 days. GnRH-a administration was performed, to trigger ovulation, when the largest follicles were 10–12 mm in diameter. Many immature oocytes, which underwent maturation in vitro, were then harvested, fertilized, and subsequently developed into blastocysts that resulted in live births [13]. This interesting observation might have a great importance in follicular cytoplasmic maturation due to the FSH surge obtained after GnRH triggering. The FSH surge might promote formation of LH receptors on the granulosa cells enhancing LH activity, induce plasminogen activator activity causing dissociation of oocytes from somatic cells of the follicle (therefore more immature oocytes could be obtained), and maintain the opening of gap junction between cumulus cells and oocyte which contributes to oocyte maturation [14–17].

### **2.4 Timing of collection: at what follicle size?**

Son et al. [18] conducted a study in which patients were triggered when the leading follicle was <10 mm, 10–14 mm, or >14 mm. In the group with a leading follicle >14 mm, only one pregnancy was obtained. The authors of the current paper administer, therefore, hCG trigger once a leading follicle of 10–12 mm is developed.

### **2.5 Endometrial preparation and luteal support**

In IVM, estradiol levels are physiological, and oocyte collection is done before the endometrium is fully estrogenized. After aspiration, there is an insufficient support from the corpus luteum for endometrial receptivity. It was demonstrated

**Figure 2.** *Endometrial support.*

that endometrial thickness is an important predictor of IVM outcome [19]. Thus, endometrial preparation is an important factor for IVM success rates. The protocol for endometrial preparation, which the authors currently use, is similar to that originally described by Trounson et al. [19] and modified by Elizur et al. [20], depending upon the endometrial thickness at day of hCG administration: when endometrial thickness is less than 6 mm, 6–8 mm, or higher than 8 mm, supplementation with 10–12 mg/day, 8–10 mg/day, and 6 mg/day 17-beta estradiol is given, respectively, starting at the day of oocyte retrieval. This approach mimics the natural estrogen rise from the dominant follicle in a natural cycle. In terms of progesterone, supplementation usually begins on the day of oocyte aspiration (as this is the progesterone rise in a natural cycle) using vaginal micronized progesterone (Endometrin, Florish Ltd. Industrial Park Misgav, Israel, Ferring Pharmaceuticals Ltd.) 300 mg daily until pregnancy test is performed (**Figure 2**).

### **2.6 Oocyte retrieval by transvaginal ultrasound**

An high-resolution ultrasound device is obligatory. The oocyte retrieval is done with a single-channel needle 19 G (Swemed, Reduced Single Lumen, Vitrolife, Göteborg, Sweden AB), using a reduced aspiration pressure of 7.5 kPa. This is essential to minimize damage to the immature oocytes.

Usually general anesthesia/sedation is provided. However, local anesthesia with lidocaine 1%, 5 cc injected into the lateral fornixes, could be sufficient.

Oocytes from each ovary are aspirated in separate flask containing 15 ml of flushing medium (Origio, Denmark) and placed on a heated block. Collected cumulus oocyte complexes (OCCs) were classified immediately after OPU (Day 0).

Oocyte cumulus complexes could be classified in one of five groups: (1) expanded cumulus, slack and fluffy multilayer of granulosa cells; (2) full cumulus, multilayer of strictly compact and cubical granulosa cells; (3) full corona, thin layer of strictly compact and cubical granulosa cells; (4) partial cumulus, oocytes surrounded partially with cumulus cells; (5) nude, oocytes without cumulus cells (**Figure 3**). This classification may serve as a prognostic indication of the future maturation and fertilization rate of the immature collected oocytes.

Each complex was then separately cultured in IVM medium (Sage, Cooper Surgical Company, Trumbull, CT, USA) supplemented with FSH + LH (Menogon, Ferring GmbH, Kiel, Germany) with a final concentration of 75 mIU/ml (maturation medium). Oocyte maturation was assessed after 6 (a) and 24–30 h (b),

**181**

**Figure 4.**

*OPU (solid fill arrows); b*

*OPU (solid fill arrows); b*

*(A) In vitro maturation procedure. \*Day 0 = day of OPU; a*

*(B) in vitro maturation procedure. \*Day 0 = day of OPU; a*

*MII oocytes detected in maturation medium 6 h after* 

*MII oocytes detected in maturation medium 6 h after* 

*MII oocytes detected in maturation medium 24–30 h after OPU (open arrows) and* 

*MII oocytes detected in maturation medium 24–48 h after OPU (open arrows).*

**Figure 3.**

*Classification of collected cumulus complexes.*

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

*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*

**Figure 3.** *Classification of collected cumulus complexes.*

*Innovations in Assisted Reproduction Technology*

that endometrial thickness is an important predictor of IVM outcome [19]. Thus, endometrial preparation is an important factor for IVM success rates. The protocol for endometrial preparation, which the authors currently use, is similar to that originally described by Trounson et al. [19] and modified by Elizur et al. [20], depending upon the endometrial thickness at day of hCG administration: when endometrial thickness is less than 6 mm, 6–8 mm, or higher than 8 mm, supplementation with 10–12 mg/day, 8–10 mg/day, and 6 mg/day 17-beta estradiol is given, respectively, starting at the day of oocyte retrieval. This approach mimics the natural estrogen rise from the dominant follicle in a natural cycle. In terms of progesterone, supplementation usually begins on the day of oocyte aspiration (as this is the progesterone rise in a natural cycle) using vaginal micronized progesterone (Endometrin, Florish Ltd. Industrial Park Misgav, Israel, Ferring Pharmaceuticals

An high-resolution ultrasound device is obligatory. The oocyte retrieval is done

Usually general anesthesia/sedation is provided. However, local anesthesia with

Oocytes from each ovary are aspirated in separate flask containing 15 ml of flushing medium (Origio, Denmark) and placed on a heated block. Collected cumulus oocyte complexes (OCCs) were classified immediately after OPU (Day 0). Oocyte cumulus complexes could be classified in one of five groups: (1) expanded cumulus, slack and fluffy multilayer of granulosa cells; (2) full cumulus, multilayer of strictly compact and cubical granulosa cells; (3) full corona, thin layer of strictly compact and cubical granulosa cells; (4) partial cumulus, oocytes surrounded partially with cumulus cells; (5) nude, oocytes without cumulus cells (**Figure 3**). This classification may serve as a prognostic indication of the future

Each complex was then separately cultured in IVM medium (Sage, Cooper Surgical Company, Trumbull, CT, USA) supplemented with FSH + LH (Menogon, Ferring GmbH, Kiel, Germany) with a final concentration of 75 mIU/ml (maturation medium). Oocyte maturation was assessed after 6 (a) and 24–30 h (b),

with a single-channel needle 19 G (Swemed, Reduced Single Lumen, Vitrolife, Göteborg, Sweden AB), using a reduced aspiration pressure of 7.5 kPa. This is

lidocaine 1%, 5 cc injected into the lateral fornixes, could be sufficient.

maturation and fertilization rate of the immature collected oocytes.

Ltd.) 300 mg daily until pregnancy test is performed (**Figure 2**).

**2.6 Oocyte retrieval by transvaginal ultrasound**

essential to minimize damage to the immature oocytes.

**180**

**Figure 2.**

*Endometrial support.*

### **Figure 4.**

*(A) In vitro maturation procedure. \*Day 0 = day of OPU; a MII oocytes detected in maturation medium 6 h after OPU (solid fill arrows); b MII oocytes detected in maturation medium 24–30 h after OPU (open arrows) and (B) in vitro maturation procedure. \*Day 0 = day of OPU; a MII oocytes detected in maturation medium 6 h after OPU (solid fill arrows); b MII oocytes detected in maturation medium 24–48 h after OPU (open arrows).*

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