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

Retrieval of immature oocytes from non-stimulated ovaries, followed by in vitro maturation (IVM), was initially proposed in order to avoid side effects of gonadotropin administration. The goal is to eradicate or significantly decrease the risk of ovarian hyperstimulation syndrome (OHSS) in patients with polycystic ovary syndrome (PCOS) and to reduce drug cost and burden of patients. This technology was also proposed for treatment of normal ovulatory women, fertility preservation, or infrequent conditions as failure of oocyte to mature or repeated development of poor-quality embryos. There is no downregulation, and only a small amount of hormones are injected if at all. In vitro maturation of the oocyte procedure obtained up to 35% clinical pregnancy rate in young women, compared with in vitro fertilization (IVF) in many programs. The obstetric and perinatal outcomes of IVM cycles are comparable with IVF/ICSI cycles; therefore it may gradually substitute IVF in certain cases, as the technique continues to develop and pregnancy rates continue to increase. IVM holds great promises as an alternative to assisted reproductive technologies and may be the procedure of choice not only for infertile patients but also for obtaining oocytes for donation or fertility preservation.


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.

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).

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 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].

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][15][16][17].

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.

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 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).

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),   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.

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

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].

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. IVM treatments of 21.9-29.9% [28][29][30][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][34][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.

Fertilization failure
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.

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].

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

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 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].

Future concerns
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]: 1. Changing endometrial gene expression.
2. Causing immunologic changes to the endometrium.
3. Affecting endometrial-embryo interaction causing impairment on fetal development and growth.
4. Increasing the risk of abnormal placentation, leading to increased rates of low birth weight.

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].

Conclusions
The results of the process of IVM may be comparable and may have advantages over standard IVF. It is a simple procedure without pituitary downregulation. For stimulation of IVM cycle, a very small amount of hormones are administered if at all. Treatment time is short with low side effects (no OHSS), resulting in a reduced psychosocial impact.
The method of IVM holds great promise as an alternative to assisted reproductive technologies and may be the procedure of choice not only for infertile patients but also to obtain oocytes for donation or fertility preservation. Improving embryonic-endometrial synchrony through pharmaceutical or other manipulation of endometrial/uterine receptivity will hopefully improve IVM success rates. Appropriate counseling of the patients about the benefits and difficulties of the process should be done routinely [72].

Summary points
In vitro maturation of oocytes is a simple and less stressful process of treatment. Main indication: PCOS patients, selected cases with fertilization failure, and fertility preservation.
Pregnancy rates are comparable. [