In Vitro Oocyte Maturation: Current Status

199 In Vitro Oocyte Maturation: Current Status Daniela Nogueira, Ph.D. 1 Jean Cl Sadeu, M.D., Ph.D. 2 Jacques Montagut, M.D. 1 1 LaboratoiredeBiologiedelaReproduction IFREARES, Clinique Saint Jean Languedoc, Toulouse, France 2 Department of Obstetrics and Gynecology-Reproductive Biology, McMaster University, Hamilton, Ontario, Canada Address for correspondence and reprint requests Daniela Nogueira, Ph.D.,LaboratoiredeBiologiedelaReproduction IFREARES, Clinique Saint Jean Languedoc, 2 route de Revel, 314 Toulouse, France (e-mail: daniela_nogueira@yahoo.com). Semin Reprod Med 212;3:199 213 Abstract Keywords In vitro maturation immature oocytes oocyte maturation assisted reproductive technologies Due to its numerous clinical applications, in vitro maturation () has emerged as a significant topic in the field of assisted reproduction. of germinal vesicle breakdown/metaphase I and germinal vesicle stage oocytes collected from in vitro fertilization (IVF) superovulation cycles are commonly applied with unsatisfactory results. The biological aspect of this so-called rescue in vitro oocyte maturation greatly differs from the actual practice. In the latter, immature oocytes are obtained from small antral follicles of unprimed or minimally stimulated cycles aiming to avoid ovarian hyperstimulation syndrome in high-risk patients or simply as an alternative to conventional IVF in normo-ovulatory patients. Over the past decade, cases reports regarding have been sporadically reported, with 25 peer-reviewed articles currently available. These studies present variable outcomes and deal with clinical approaches about selecting the most appropriate patient population that could benefit from technology. Although some of the studies are encouraging, the vast majority includes small sample sizes, thus making the data rather inconclusive. As such there is a certain reserve in the IVF community to embark on treatment cycles for in routine use. Laboratory parameters play an important role in the success of, and research for optimal culture conditions is warranted. Existing data from newborns assure us that may be a safe procedure provided in assisted reproductive technology. When optimized, it will serve, not only for infertile patients, but also as a more patient-friendly alternative than standard controlled ovarian stimulation to obtain oocytes for donation or preservation of fecundity. The first report showing that immature oocytes could spontaneously mature in vitro and be subsequently fertilized was reported by Pincus and Enzmann in 1935 using rabbit oocytes. 1 This pioneering work was the starting point of early investigations on in vitro oocyte maturation techniques; 3 years later, Edwards 2 showed similar features with human oocytes. This technology was thereafter disregarded in clinical use due to discouraging outcomes together with the emerging advances using in vivo matured oocytes. It was much later, in 1983, that the first human offspring from in vitro matured oocytes following in vitro fertilization (IVF) was reported using oocytes retrieved from superovulated cycles. 3 Subsequently, in the early 199s, the first reports on pregnancies and deliveries were published using clinical protocols developed intentionally for in vitro maturation (). 4,5 These successful cases were indeed encouraging and set the stage for further research and development with the aim of improving success rates. During the last decade, studies have shown growing interest not only in the development of laboratory conditions but also in the design of innovative clinical protocols. As a result, it is currently estimated that the number of healthy children born from surpasses 2. 6 Issue Theme Safety, Efficacy, and Complexities in the ART Laboratory; Guest Editors, Catherine Racowsky, Ph.D., H.C.L.D., and Douglas Carrell, Ph.D., H.C.L.D. Copyright 212 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 11, USA. Tel: +1(212) 584-4662. DOI http://dx.doi.org/ 1.155/s-32-1311522. ISSN 1526-84.

2 In Vitro Oocyte Maturation Nogueira As shown by the various studies and case reports published on, the quest for innovation has led to the establishment of different methodological approaches with the use of oocytes from different sources such as (1) oocytes retrieved from large antral follicles of superovulation treatment cycles: rescued oocyte maturation, and (2) immature oocytes retrieved from small antral follicles after mild gonadotropin priming (follicle-stimulating hormone [FSH] or human chorionic gonadotropin [hcg] or both) or from unprimed cycles (i.e., without FSH or hcg priming): intentional. Nowadays it is well established that the type of protocol selected for patients undergoing conventional IVF depends on several factors: purpose of therapy, the patient's age and history, and physician's experience. Most of the clinics use individually adjusted protocols to achieve the best possible response needed for a given patient. Likewise for because so far there is no standard hormonal stimulation protocol that perfectly suits all patients. Different protocols have been used in patients with distinct etiologies, including infertile patients with polycystic ovaries (PCOs), polycystic ovary syndrome (PCOS), or regular-cycling patients with normal ovaries. 7,8 This review focuses on outcomes from techniques reported in the past decade presenting clinical perspectives on the applicability of this practice in relation to laboratory outcomes. Clinical Application and Outcomes of in Patients Undergoing Superovulation Treatment Cycles At the time of oocyte retrieval, owing to the complexity of folliculogenesis, there are follicles with different diameters containing oocytes at different maturational stages. Most oocytes retrieved in IVF/intracytoplasmic sperm injection (ICSI) cycles originate from follicles that are 14 mm, depending on the policy of the fertility center. Following the removal of cumulus cells for ICSI, 15% of the retrieved oocytes in gonadotrophin-stimulated cycles are immature, either at the germinal vesicle breakdown/metaphase I (GVBD/ MI) or at the germinal vesicle (GV) stage. 9 These follicles containing immature oocytes at the time of retrieval may have been unable to respond synchronously to the exogenous hormonal stimulation due to local vascularization and/or metabolic deficiency. By maturing these oocytes in culture, the yield of metaphase II (MII) oocytes available for insemination may be increased, which is most important for poor responder patients to increase the number of embryos available. 1 It may also augment the cohort of supernumerary embryos available for cryopreservation and could open possibilities as a source of gametes/embryos for donation. The next sections describe the feasibility and reported outcomes of of cumulus-free immature oocytes currently used worldwide in IVF laboratories. In Vitro Maturation of Cumulus-Free GVBD/MI Oocytes From the first reports in the late 199s to date, suboptimal outcomes have been generated by in vitro maturing GVBD/MI oocytes. Maturation rates range greatly in relation to the exposure timing in culture ( Table 1). When oocytes are cultured for a maximum of 6 after retrieval, which fits the working for a routine IVF laboratory, maturation ranged broadly from 19% to 73%. Fertilization rates are 2% lower compared with their in vivo matured counterparts. Once oocytes fail to mature on the day of collection, prolonging culture to an overnight period assures a polar body (PB) extrusion rate >7%; however, fertilization rates are unchanged when compared with oocytes that take a short time to mature. These differences in maturation time are most likely related to the meiotic stage of the oocytes when placed in culture. In the absence of a germinal vesicle and of a PB, denuded oocytes with their nuclear apparatus in transition between GVBD and MI take longer to mature (12 of culture in humans), whereas PB extrusion for oocytes that have already reached the MI plate at retrieval occur in a shorter period of time (a maximum of 6 in humans). 19 However, without the help of a polarized microscope in an attempt to identify, noninvasively, the presence of an oocyte spindle, it is not feasible to determine the meiotic stage of these immature oocytes. Rescuing MI Oocyte Developmental Quality Few studies have been performed in humans in an attempt to evaluate the quality of MI oocytes in culture. For example, Cekleniak et al 2 showed that by supplementing two different basal media with the same additives (1% substitute supplement, hcg, FSH, estradiol [E 2 ], and epidermal growth factor [EGF]), maturation rates differed between media, with 48% more oocytes having normal spindle configurations following a 24-hour culture in P1 versus TC199 media. The differences in the composition of the two basal media regarding constituents such as energy substrates, glucose, proteins, and amino acids supplementation can alter oocyte metabolism in both cumulus-free and cumulus-enclosed oocytes (CEOs). 21 Based on earlier animal studies in mammalian species, Curnow et al 22 recently increased the maturation of MI to MII and fertilization rates of primate oocytes by adding glutathione (-ethyl ester) in basal media for 4 to 6 to compensate for the lack of surrounding cumulus cells. As for other additives to enhance maturation of GVBD/ MI oocytes, it is unlikely that hormones and growth factors would exert any influence on the maturation transition beyond GVBD. Yan et al 23 demonstrated that these additives failed to improve maturation or either fertilization or subsequent embryonic developmental quality even when these oocytes were cultured in a partially cumulus-enclosed fashion. However, when partially cumulus-enclosed GV-stage oocytes were cultured in the presence of gonadotropins, increased significantly. 23 These substances are mainly activators of meiosis resumption in pathways dependent on cyclic adenosine phosphate (camp) hydrolysis. Meiotic arrest at the GV stage is maintained by an inflow of camp within the granulosa cells and oocyte, driven by the gonadotropin environment. It is believed that, physiologically, reinitiation of meiosis is preceded by a transient increase of camp under the influence of gonadotropins followed by a fall Seminars in Reproductive Medicine Vol. 3 No. 3/212

In Vitro Oocyte Maturation Nogueira 21 Table 1 Retrospective Data Reported on Clinical Outcomes of Culture of GVDB/M I Oocytes Retrieved from Superovulated Cycles in Relation to Culture Time and Conditions Study No. of MI Oocytes Cultured Culture Conditions Hours of Culture % Maturation % Fertilization Pregnancy Newborns Pregnancies from Mixed Embryos Coetzee and Windt 11 42 2 74 68 1 N/A De Vos et al 9 4716 B2 in humidified 4 27 53 1/15 1 at 5% O 2,5%CO 2, 9% N 2 Chen et al 12 128 HTF plus 15% maternal 9 16 59 1/2 1 14 clinical pregnancies/ in humidified at 5% CO 2 4 transfers 2 miscarriages; 14 newborns/ongoing Strassburger et al 13 257 G1 series 2 (Vitrolife) in 3.5 6 45 44 2/4 1 humidified at 5% CO 2 Balakier et al 14 64 IVC-One in humidified 1 1.5 at 5% O 2,5%CO 2, 9% N 2 2 2.5 3 6 39 36 6 25 43 61 /3 5 clinical pregnancies/ 16 transfers Vanhoutte et al 15 3 Cook/Early cleavage gas Conditions not specified 2 4 41 43 2 pregnancies/ 21 transfers >4 7 42 54 >7 11 67 1 19 26 75 67 1 1 Jee et al 16 124 G2 supplemented with FSH, LH, EGF, plus hff or HSA in humidified at 5% CO 2 Reichman et al 17 96 78 SAGE culture in humidified at 5% CO 2 SAGE plus FSH, LH 4 73 56 2 newborns/ 12 transfers 3 6 24 19 78 67 5 pregnancies (33% pregnancy rate) Braga et al 1 227 G1 (Vitrolife) in 6% CO 2 24 76 65 2/17 N/A 15 pregnancies/ 86 transfers Li et al 18 252 G-IVF (Vitrolife) 3 5 overnight 4 87 69 58 1/3 1/5 1 miscarriage 1 miscarriage pregnancy/ 2 transfers MI, metaphase I; N/A, not assessed;, not described; HTF, human tubal fluid; FSH, follicle-stimulating hormone; LH, luteinizing hormone; EGF, epidermal growth factor; hff, human follicle fluid; HSA, human albumin;, in vitro maturation. Pregnancies involving patients where only embryos derived from retrieved MI were transferred in fresh cycles. From a single embryo derived from a MI-arrested oocyte in culture for 4. Pregnancies obtained when fresh embryos derived from in vitro matured MI oocytes were transferred with embryos derived from in vivo MII oocytes. In some reports information on the number of embryo transfers are missing. Seminars in Reproductive Medicine Vol. 3 No. 3/212

22 In Vitro Oocyte Maturation Nogueira in intra-oocyte camp concentrations by a cell-specific phosphodiesterase (PDE). Hydrolysis of camp to AMP by PDE causes a subsequent inactivation of camp-dependent protein kinase (PKA). Downstream of PKA deactivation, maturationpromoting factor (MPF), mitogen-activating protein kinase (MAPK), and other cyclin-dependent kinases function simultaneously and interact as key regulators in the oocyte meiotic cell cycle (see Nogueira et al for review 24 ). Activation of MPF in GV oocytes requires the formation of the p34 cdc2 -cyclin B complex followed by phosphorylation and dephosphorylation of p34 cdc2 itself. The rate of synthesis and control of activation of these factors are species dependent. For instance, in meiotically competent mice oocytes, pre-mpf (inactive MPF complex) is already present with the stockpile of cyclin B at comparable levels in both meiotically incompetent (growing) and competent (fully grown) GV-stage oocytes; it is assumed that activation of MPF (dephosphorylation) is sufficient for meiosis resumption. It seems that humanoocytes,onthecontrary,requireproteinsynthesis to undergo GVBD to form the pre-mpf complex before dephosphorylation can occur (see Nogueira et al for review 24 ).This need for neosynthesis of cyclin B explains the long time interval needed for the human oocyte to undergo spontaneous GVBD (12 to 18 in culture). 25 MPF reaches its peak at MI followed by a fall in activity as a result of cyclin degradation, to a basal level sufficient for maintenance of chromosome condensation. Due to de novo cyclin synthesis, MPF peaks again at MII. Therefore, the oocytes usually require less time for transition from MI to MII. 19 Oocytes need to be supplied with additives that will likely support this meiotic progression and ensure normal subsequent development. Studies along these lines will hopefully reveal factors that can have a positive impact on the quality of these oocytes to be applied more safely in clinical assisted reproductive technology (ART). Considering the importance of cumulus granulosa celloocyte exchanges during the final stages of oocyte maturation, 26 Zhu et al 27 demonstrated that supplying MI human oocytes with a flat monolayer of autologous cumulus cells allows PB extrusion to increase by 1% at 24 but not at 12-hour culture. Moreover, embryo development was low after insemination and few oocytes achieved blastocyst formation. Although physiologically there is a loss of gap junctional connections by the time oocytes reach MI, it seems that the cumulus cells surrounding the oocyte, in a natural threedimensional (3D) conformation, permit an adjacent communication that interferes with later development as shown in animal species. 28 Although there is no consensus regarding which culture system is the most appropriate to support progression of GVBD/MI oocytes to MII, the various systems described in the literature show that even when high rates of maturation are achieved, development after insemination remains lower than freshly collected MII oocytes ( Table 1). A study by Strassburger et al 29 in which fluorescent in situ hybridization (FISH) was performed for chromosomes X,Y and 18 clearly demonstrated that the longer MI oocytes take to mature, the higher the incidence of chromosomal abnormalities in the derived embryos. When oocytes extrude the PB 2 after retrieval, the incidence of abnormality was 4%, increasing to 67% at 4 to 8 of culture, reaching 1% at 24-hour culture. Suggestions of applying preimplantation diagnosis to select embryos for transfer have been put forward by the authors. However, they concede that the benefits of such an approach is unknown 29 and surely negative in terms of a costbenefit balance. Regardless, the timing of ICSI in relation to the extrusion of the PB should be considered, with the highest fertilization rate occurring when injection takes place 3 to 6 after PB extrusion in these rescued MI oocytes. 14 Clinical approaches have been used to increase the yield of in vivo matured oocytes in patients where half of the retrieved oocytes were immature in previous IVF cycles. By prolonging the time interval between hcg and oocyte retrieval by 3 to 4, a higher rate of MII oocytes was obtained with a lower rate of aspirated MI and GV-stage oocytes. 3 Moreover, ongoing pregnancy rates were significantly higher in those patients having the prolonged hcg-tooocyte retrieval interval compared with patients with the regular hcg interval of 35 to 36 (25% vs. 6%, respectively). By taking the approach of Raziel et al, 3 we performed oocyte retrieval 39 to 4 after hcg administration in 74 patients who had three or more previous cycles with 5% (5 to 1%) of oocytes at the GVBD/MI stage during a 5-year period. We obtained a 68% maturation rate and a 22% ongoing clinical pregnancy rate in these poor prognosis patients (unpublished data). This methodology, which allows oocyte maturation to occur in vivo under the influence of the follicular milieu, can serve as a better alternative to the 4- to 6-hour culture. Moreover, increasing the time of hcg exposure in vivo in patients with recurrent retrieval of high numbers of MI oocytes seems more effective in augmenting the yield of mature oocytes obtained than increasing the hcg doses. This might be due to a probable existing follicular threshold of response to hcg. 31,32 Prolonging the interval between hcg and retrieval might not be useful in patients with recurrent meiotic arrest at or beyond GVBD, where incompetence to progress to MII is likely inherent to cell signaling deficiencies in the oocyte itself. 33,34 Consistent with this theory, Combelles et al 33 presented a case report in which meiotically arrested MI oocytes showed clear defects in spindle and chromatin arrangements. In contrast, MI oocytes from patients with a recurrent history of high incidence of MI but where MIIs were also retrieved had a high incidence of normal meiotic apparatus structure. 33 Another report showed that calcium ionophore could induce first PB extrusion in MI oocytes from patients with a history of maturation arrest. However, these treated oocytes had abnormal spindles and failed embryonic development, 34 rendering this approach inadequate for those cases in clinical practice. In view of current data, transfer of embryos derived from in vitro matured MI oocytes should be discouraged. The derived embryos have a decreased capability for implantation that does not increase when transferred with embryos from fresh MII oocytes. However, if this approach is applied due to a lack of alternatives, a culture time of 5 after Seminars in Reproductive Medicine Vol. 3 No. 3/212

In Vitro Oocyte Maturation Nogueira 23 retrieval (2 to extrude the PB and 3 to culture before ICSI) seems to offer the most favorable results in terms of fertilization and embryonic developmental rates in vitro. In Vitro Maturation of Cumulus-Free GV-Stage Oocytes After the first pregnancies reported by Veeck et al 3 from in vitro matured GV-stage oocytes retrieved from IVF superovulated cycles, several case reports of deliveries using a similar approach were published sporadically. Despite a high fertilization rate, embryo developmental quality is poorer compared with in vitro matured MI oocytes, and implantation is considerably reduced. The cytogenetic constitution of the derived embryos is chaotic, associated with a high percentage of multinucleated blastomeres. 35 37 The origins of oocyte deficiency might be diverse and multifactorial. Defects can be inherent to the oocyte caused by a suboptimal intrafollicular environment prior to oocyte retrieval: These oocytes have been originally retrieved from a compromised population of follicles with a deficiency in their triggering mechanisms for responding to exogenous hcg. 24 Deficiency might be inherent to the GV-oocyte itself, presenting abnormalities in the chromatin configuration during the GV stage, an inability to maintain M-phase characteristics during meiotic progression, and a propensity to undergo spontaneous activation after metaphase arrest, 19 leading to a deficient coordination between nuclear and cytoplasmic maturation. Although fresh collected MII oocytes from healthy volunteers and donors have an aneuploidy rate of 8 38 to 13% 39 following hormone-induced superovulation, the rate of aneuploidy in these in vitro matured oocytes using FISH or M (multiple)- FISH techniques is fourfold higher (39% and >4%, respectively). 4,41 More recently, it was determined that these oocytes had dysregulation in either transcription or posttranscriptional modification of genes resulting in an incorrect temporal utilization of those that may culminate in developmental incompetence of any embryos derived from these oocytes. 42 Rescuing GV Oocyte Developmental Quality One of the adverse factors contributing to an unsuccessful rescue of GV oocytes likely involves the microenvironment in culture. These immature oocytes are often denuded from their surrounding cumulus cells and undergo maturation depleted from essential supplementation factors from their cellular counterparts. 43,44 Culture conditions affect the outcomes of oocyte maturation by altering gene expression, the kinetics of cell cycle progression, and spindle/chromatin organization, and thus ensuing embryonic development. Experimental studies have shown that the addition of EGF or the EGF family members amphiregulin and epiregulin to the culture improved maturation rates in cumulusfree oocytes, 43,45 whereas in oocytes surrounded by their cumulus cells, EGF had a positive effect on embryo development when combined with FSH and E 2. 43 Removal of cumulus cells limits the capability of the oocyte to synthesize GSH. Depletion of GSH content during culture leads to the generation of reactive oxygen species (ROS), the extent of which depends on the formulation. 22,46 In an experimental study on 13 cumulus-free GV oocytes, modification of culture media conditions in a -free environment improved the maturation rate by 2% and reduced the rate of multinucleated embryos obtained (61% mononucleated embryos versus 4%) compared to HTF supplemented with.5% HSA. 47 In other studies, the use of superficial systems to encapsulate denuded GV oocytes in a 3D co-culture system with cumulus cells resulted in increased MAPK activity patterns approaching those of in vivo matured oocytes. 48,49 Vanhoutte et al 49 developed a two-step culture system, first by exposing the 3D complex to an agent, a PDE type 3 inhibitor, to maintain intra-oocyte camp levels above the threshold required for maintenance of the oocytes arrested at the GV stage. As a second step, the 3D complexes were cultured for 24 in an arrester-free that encourages oocytes to undergo maturation. From these and previous experiments involving a similar two-step culture, we have observed improved spindle/chromosome organization in oocytes and an improved morphology and nuclear constitution of the derived human embryos. 49 52 These collective results suggest that attempts to enhance culture conditions may, at least during the preimplantation developmental steps, provide some support to enhance rescue of the oocyte cytoplasmic constituents and those mechanisms required for maturation activation. Considerable research is still needed, and no defined culture media have yet been identified as categorically safe for. So-called rescue is inefficient in generating competent embryos capable of implantation and should not be considered for transfer. However, these immature oocytes are a valuable source for research on cloning, stem cells, and the foremost assessment of culture systems in humans. *MEM plus supplements: MEM supplemented with 2 mm Glutamax-I,.4 mm Glycine, 1 ng/ml human insulin, 5 ng/ ml sodium selenite, 5 μg/ml human transferrin,.47 mm sodium pyruvate, 3.3 mm L-lactate, 1 ng/ml Long R3 insulin growth factor-i and 1 μm cysteamine, 1 miu/ml recombinant follicle-stimulating hormone,1 ng/ml recombinant epidermal growth factor, and.5% human albumin. Clinical Application and Outcomes of in Patients at Risk of Ovarian Hyperstimulation Syndrome Retrieving immature oocytes before administration of hcg could be an effective means of averting ovarian hyperstimulation syndrome (OHSS), which is caused by the combination of ovarian hyperstimulation by FSH and the administration of hcg. Although it has become apparent that oocytes from rescued result in a poor outcome, the quality of GV oocytes retrieved in a superovulation cycle prior to hcg administration might differ from the rescued oocytes. First, within the follicles with an advanced stage of growth, the enclosed oocytes would have had more time to undergo cytoplasmic maturation. Second, these GV oocytes are retrieved in a tight cumulus mass because hcg activation of the signaling cascades for cumulus expansion in vivo is omitted. Seminars in Reproductive Medicine Vol. 3 No. 3/212

24 In Vitro Oocyte Maturation Nogueira Thus these retrieved GV-stage oocytes have more favorable exchanges with their somatic cells in culture via the preserved gap junctional connections. However, there are no consistent studies to evaluate the quality of these immature oocytes. This is a relevant gap in our knowledge because patients at risk of OHSS during conventional ovarian stimulation commonly demonstrate elevated E 2 levels, combined with a significantly poor embryo quality and a lower pre- and postimplantation developmental capacity. 53 The few reports on attempting to recover the immature oocytes prior to hcg injection are usually described in patients presenting with multiple follicles >12 mm (2) and with high E 2 levels on the day of hcg cancellation (25 pg/ml). 54 56 The disadvantage of applying this methodology is the low recovery rate because without hcg priming it becomes more difficult to detach the oocytes from the follicle wall during aspiration. There is also the constraint of having to cryopreserve the oocytes/embryos obtained for transfer in a frozen embryo transfer cycle to avoid the added risk of OHSS caused by embryonic implantation. Another strategy to avoid OHSS is to administer hcg earlier in the cycle when multiple follicles of 12 to 14 mm are present. In this case, oocyte recovery rate is increased upon aspiration, with a possibility of retrieving 24% of in vivo matured oocytes next to the immature ones, thus increasing the chances of achieving pregnancy to 46%. 57 Currently technology involves the practice of minimizing or even avoiding exogenous gonadotropin administration to intentionally collect cumulus-enclosed immature oocytes from small antral follicles. There is a worldwide interest in applying for PCO or PCOS patients due to their high risk of developing OHSS in a conventional superovulation IVF cycle. This strategy involves the retrieval of immature oocytes around the time of the selection of a dominant follicle (1 to 12 mm), just after the process of atresia has been initiated in the pool of smaller antral follicles but before a prolonged exposure to the dominant follicle. in natural cycles in human and animal models suggest that the quality of the immature oocyte is not affected by the presence of a dominant follicle as previously thought. Immature oocytes may actually acquire maturational competence for using the early stages of this atresia-like process. 58 Strategies to Minimize Risk of OHSS Three main strategies have been attempted to minimize the risks of OHSS: (1) FSH priming but omission of hcg priming, (2) omission of FSH priming but the use of hcg, and (3) omission of priming with either FSH or hcg. In protocols applying FSH priming, FSH is administered for up to 6 days at minimal/low daily doses (37.5 to 75 IU) or is administered for 3 or alternate days at a higher dose (15 IU) until follicles reach a diameter between 1 and 12 mm. This may be followed or not by coasting for 24 to 72 before oocyte retrieval. In the hcg-priming approach established by Chian et al, 59 follicle growth occurs in a natural cycle and oocytes are retrieved after hcg priming at day 1 to 14, with best results obtained when the leading follicle is between 1 and 12 mm in diameter the day of hcg priming and has reached >1 to 14 mm at collection. 6 Others have shown that when hcg priming is performed even when the leading follicle is only 7 mm, pregnancy and implantation can achieve similar success rates compared with the other criteria for hcg priming. 61 In unprimed cycles, retrieval is performed the day after the leading follicle is 1 62,63 to 12 mm. 64 As for the efficacy of these protocols, most of the studies have been performed in patients <38 years of age with small sample sizes, and a few studies used control groups. In the largest studies (5 patients included), the average number of oocytes recovered in unprimed patients ranged from 13 to 17, 63,65,66 and in hcg-primed cycles the number ranged from 9 to 24 oocytes. 61,67 69,71,72 These results are generally derived from the most experienced teams because a learning curve must be mastered to improve the yield of oocytes retrieved. 7 Although pregnancy rates are quite similar to those of conventional IVF, rates of implantation are variably lower. In PCO patients, where gonadotropins were omitted, implantation rates ranged from 6 to 17%. Exceptionally, a study by Söderström-Anttila 62 presented pregnancy and implantation rates as high as 53% and 35%, respectively. However, 3% of these pregnancies miscarried. When only hcg was administered, implantation rates ranged from 6 to 12%, increasing up to 27% when embryos were transferred at the blastocyst stage. 7 Mikkelsen et al 73 showed a reasonably high implantation rate of 22% in a study group including 24 patients when FSH was the sole gonadotropin administered in PCO patients. However, a more recent report applying the same protocol with a few patients (n ¼ 12) showed an implantation rate of only 7%. 74 Two studies determined the efficacy of yet another strategy relative to the effect of hcg administration after 6 days of FSH priming starting on day 3 of the cycle in PCO patients. These studies involved 3 patients in each arm with an average oocyte recovery rate of 22% 7 and 9%, respectively. 75 Both studies presented <1% implantation rates. However, Wei et al 75 observed a positive effect when combining hcg pretreatment with metformin, significantly doubling implantation rates to 15%. Clinical Applications and Outcomes of in Patients With Normo-Ovulatory Response technology is being developed not only to apply to patients at risk of OHSS but also as a general alternative to conventional IVF protocols. For example, in patients in whom gonadotropin is contraindicated, such as cancer patients, performing can help avoid the supraphysiological increase in E 2 levels. Moreover, the use of in such patients before they undergo chemotherapy provides a strategy for shortening the time to retrieval to obtain oocytes for cryopreservation, or embryos, for later use. In these patients, can also be combined with the removal of ovarian tissue biopsies for cryopreservation. For patients with allergies to exogenous hormonal substances, an unprimed cycle could circumvent this issue. Finally, as a welcome protocol Seminars in Reproductive Medicine Vol. 3 No. 3/212

In Vitro Oocyte Maturation Nogueira 25 where male factor is the only cause of infertility, may reduce the patient's burden by shortening the duration, side effects, and cost of the treatment. Fadini et al 76 recently performed in 2 normo-ovulatory patients with nonobstructive azoospermia, and they reported 11% implantation and 16% abortion rates. As other proofs of concept, unprimed cycles have been applied successfully as an alternative to conventional IVF protocol for poor ovarian response 77 and for oocyte donation. 78 Similar clinical strategies used in PCO patients have been used in normo-ovulatory patients, with and without gonadotropin priming. About nine studies performed in normo-ovulatory women in unprimed cycles, and most of them reported large samples with >6 cycles included. 62,64,79 83 In normoovulatory women, achieving a lead follicle diameter of 1 to 13 mm may occur much faster than in women with ovulatory dysfunction, although the numbers of oocytes retrieved are generally much lower and vary according to the size of the lead follicle. In unprimed cycles, 3.8 to 6.1 oocytes were recovered when the leading follicle was <1 mm, 79,8 6.3 to 9. when retrieval was performed the day after the lead follicle was 1 mm, 62,81 4.7 when the lead follicles were between 1 and 12 mm, 64 and 4.1 to 5.3 when leading follicles reach 14 mm the day before retrieval. 82,83 Priming with FSH apparently does not increase the number of oocytes retrieved in normo-ovulatory women with an average of 5. retrieved oocytes when compared with unprimed, 82 84 hcg priming, 83 or FSH plus hcg priming 76 cycles. Throughout the past 1 years of experience, implantation rates have been variable with 11% in FSH-primed cycles, 83,84 1.5 67 to 19% in hcg primed cycles, 85 and 7 to 9% 81,82 to 23% in unprimed cycles. 62 Influence of the Endometrial Thickness on Cycles Overall, miscarriage rates are reported to range from 15% to 2% to a maximum of 3 to 39% in cycles. There is no consensus on the optimal endometrial thickness for embryo transfer following. Some authors use endometrial thickness as an embryo transfer cancellation parameter independent of the patient's etiology. For example, cycles were cancelled if endometrial thickness was <4 mm the day before retrieval. 82,83 Although some authors reported pregnancies with 3-mm endometrial thickness at retrieval, 62 others canceled embryo transfers if the endometrium was <7 mmat day 2 to 3 of transfer. 59,61 It is thought that endometrial development in an cycle is delayed at the time of embryo transfer because of its follicular growth phase that is truncated, which negatively contributes to implantation. Perhaps E 2 supplementation may be insufficient to optimize endometrial quality. With the advances of cryopreservation techniques (i.e., vitrification), implantation rates may be improved by replacing cryopreserved embryos in a subsequent cycle to achieve endometrium-embryo compatibility. In summary, the balance of evidence suggests a need for further investigation, with well-designed large studies to determine the association between and endometrial preparation in relation to outcomes. Cumulus-Enclosed Oocyte Morphology and Oocyte Maturation Capacity in Relation to Gonadotropin Priming Cumulus-Enclosed Oocyte Morphological Patterns The developmental pattern of CEOs retrieved from small antral follicles is influenced by the endocrine environment in which they are exposed in vivo. Although cumulus masses in unprimed and FSH-primed patients have similar compacted morphological patterns ( Fig. 1), CEOs retrieved from hcg-primed cycles have two distinct morphological patterns of cumulus cells: expanded and compacted. 24,6 Both patterns are found in hcg-primed PCO and non-pco patients. Oocytes at all three stages of maturation (MII, GVBD/ MI, and GV) can be found enclosed in expanded CEO. However, only GV-stage oocytes are enclosed in tightly compacted cumulus cells ( Fig. 1). In PCO patients, it has been demonstrated that cumulus cells with an expanded pattern have a higher expression of luteinizing hormone (LH) receptor mrna than those with a compacted pattern. 86 The LH/hCG effect is strongly associated with a rise in camp levels in the somatic cells of the follicle. It is thought that camp production might be elevated in the expanded CEOs in response to hcg action, thereby providing the oocyte with a rise in the level of this nucleotide. Moreover, CEOs with expanded cells have already been sensitized by paracrine factors originating from mural granulosa cells Figure 1 Pattern of cumulus-enclosed oocyte morphology in relation to in vitro maturation () patient treatment. (A) Immature oocytes are retrieved prior to the formation of a dominant follicle during a natural cycle or following a mild stimulation with follicle-stimulating hormone (FSH): Oocytes retrieved are enclosed in a compact mass of cumulus cells and most of the oocytes reach maturation between 28 and 36 of culture. (B, C) Immature oocytes when retrieved following the administration of human chorionic gonadotropin (hcg) with or without FSH-priming: maturation is more rapid, occurring within 24 to 3 of culture, and the oocytes retrieved from the most advanced follicles (C) have a more expanded cell morphology, maturing faster. Seminars in Reproductive Medicine Vol. 3 No. 3/212

26 In Vitro Oocyte Maturation Nogueira within the follicles in response to hcg. This is supported by the fact that LH/hCG indirectly causes cumulus expansion and oocyte maturation within preovulatory follicles, partly by inducing expression of EGF-like growth factors in mural granulosa cells. 87,88 In PCOS patients, it has been demonstrated that the distinct CEO patterns are related to the capability of the GV-stage oocytes to mature in vitro. For instance, 7% of expanded CEOs are already matured by 24 of culture as compared with 3% of compacted CEOs. Moreover, there is indication of a positive relationship between expanded CEOs and embryo developmental capacity, with 4% of fertilized oocytes derived from expanded CEOs forming blastocysts versus 23% derived from compacted cumulus cells. 86 Oocytes within complexes with a pattern of sparse cumulus cells can also be found in hcg-primed PCOS cycles. Such oocytes mature at lower rates than expanded CEOs 86 but at higher rates than compacted CEOs. 6 In normo-ovulatory patients, following superovulation for IVF, higher rates were demonstrated in expanded CEOs compared with compacted CEOs from small antral follicles (12 mm) (83% versus 46% MII, respectively). 5 In normoovulatory women, there are no comparative studies to determine any potential differences in rates between the different CEO morphological patterns in hcg-primed cycles. Oocyte Nuclear Maturation Data from the current literature ( Table 2) show a tendency toward differences in oocyte maturational stages between PCO/S and non-pco/s patients in relation to priming protocols. Although hcg priming may significantly affect the rate of maturation in vivo in PCO/S patients, 69,71,74,89,9 the effect of gonadotropin priming is less evident on the rate of maturation in vitro. Chian et al 59 showed higher maturation rate at 24 and 48 in patients primed with hcg compared with unprimed patients. Likewise, Mikkelsen et al 73 showed that maturation rates in vitro at 28 to 36 were significantly increased in PCOS patients primed with FSH compared with unprimed patients. In contradiction, Son et al 74 showed no differences in maturation rates at 24 to 3 or at 48 in vitro in patients primed with FSH, hcg, or both compared with unprimed patients. 74 Several reasons could explain the discrepancies between studies, including (1) the number of days of FSH priming: 2 days of 15 IU FSH was given in Son's study 74 versus 3 days in Mikkelsen's, 73 and (2) the differences in culture conditions and timing of evaluation between the studies (3 74 versus 36 73 ). Yet another possible explanation to clarify the higher maturation rates in hcg-primed patients observed by Chian 59 versus Son 74 is that all oocytes collected at the GVBD/MI stage were placed in culture for 24, 59 whereas in Son's study 74 only the GVBD/MI oocytes that did not become mature on the day of retrieval were cultured for an additional 24. Compared with the rates in PCO/S patients, 69,71,72,74,89 Fadini el al 83 showed that the effect of hcg priming alone on in vivo maturation rates in normo-ovulatory patients was less pronounced ( Table 2). In our experience, by using a combination of FSH and hcg priming, we retrieved more in vivo matured oocytes from PCOS patients (17%) than non- PCO/S patients (8%) in a comparative study. 9 However, in the study by Fadini et al, 83 a much higher proportion of in vivo matured oocytes was retrieved from non-pcos patients (2%) primed with both gonadotropins. It is likely that this high in vivo maturation rate might be related to follicle sizes at retrieval. In our study, hcg priming was performed the day a leading follicle reached 12 mm; 9 however, in the study by Fadini et al, 83 hcg was administered 24 to 48 after the leading follicle had reached 13 mm. This suggests that follicles were further developed under the influence of FSH with increased receptiveness to hcg. It seems that the timing of maturation of the GV-stage oocytes is a reflection of oocyte development and depends on the milieu of origin (i.e., whether retrieved from primed or unprimed patients). Rates of maturation in vitro seem to be significantly increased by 28 to 36 when normoovulatory patients are primed with FSH 84,91 or with hcg alone 83,85 and even higher when both FSH plus hcg priming is applied compared with unprimed patients. 83,9 Once CEOs from gonadotropin-primed patients are not matured by 24 to 32 of culture, insemination the day after (44 to 48 of culture) leads to embryos with a low capability to develop and implant in both PCOS or normo-ovulatory patients. 71,72,89 It seems that in FSH-primed cycles of normoovulatory patients, although a higher proportion of oocytes will be matured by 36 of culture compared with 3, 38,91 oocytes exhibit increased aneuploidy rates compared with in vitro matured oocytes analyzed after a 3-hour culture. 38 Therefore, oocyte aging in primed cycles may be prevented by the evaluation of oocyte maturity 24 to 28 (first evaluation) with subsequent evaluation 32 (second evaluation). While in unprimed cycles, the first evaluation of oocyte maturity can be later at 28 up to 36 ( Table 2). Although it is certain that mature oocytes cannot be found in cumulus cells in patients not exposed to hcg or endogenous LH, it is important to note that not all authors have disclosed the rate of in vivo matured oocytes collected in hcgprimed cycles. Their recovery will significantly increase pregnancy rates when transferred along with in vitro matured oocytes, independent of the patient's etiology. Recently, it was revealed that upon hcg priming, 9% of cycles in PCOS patients already had at least one MII oocyte at the time when the leading follicles were between 12 and 14 mm in diameter. 6 Interestingly 62% of these matured oocytes were retrieved from follicles whose diameters were 1 mm. 69 Furthermore, some mature oocytes can even be found in follicles with 6-mm diameters. 74 About 8% of all the oocytes were mature when retrieved from hcg-primed PCOS at the time when follicles had reached a 1-mm diameter. 86 The probability of retrieving both maturing (GVBD/MI) and matured oocytes increases with the increasing diameter of the leading follicles. 71 Extension of the time of hcg exposure to 38 increased the yield of in vivo matured oocytes and thus improved outcomes. 92 The same group showed that in vivo matured oocytes produce blastocysts of Seminars in Reproductive Medicine Vol. 3 No. 3/212

In Vitro Oocyte Maturation Nogueira 27 Table 2 Reported Conditions for Maturation of Oocytes Retrieved from Small Antral Follicles in Clinical in Vitro Maturation Programs and Outcomes of Culture Study Gonadotropin priming Basal Media Protein Supplementation Gonadotropin Supplementation Other Additives Hours of Culture Oxygen Condition %InVitro Maturation* Culture Conditions for % In Vivo Maturation %IR Treatment cycles in normo-ovulatory patients Mikkelsen None et al 84 FSH primed TCM199 1% patient.75 IU/mL FSH.5 IU/mL hcg Pyruvate 1 μg/ml estradiol 28 36 5% CO2 and 76 85 18.8 11.8 Mikkelsen None TCM199 1% patient et al 79.75 IU/mL FSH.5 IU/mL hcg Pyruvate 1 μg/ml estradiol 28 36 5% CO2 and 6 8.8 Mikkelsen and None TCM199 1% patient Lindenberg 73.75 IU/mL FSH.5 IU/mL hcg Pyruvate 1 μg/ml estradiol 28 36 5% CO 2 and 6 Yoon et al 81 None YS 7% hff 24/48/ 56 5% CO 2 and 41/71/74 6.5 Fadini et al 82 None Medicult 1% patients.75 IU/mL.1 IU/mL hcg 29 5% CO 2 and 56 8.5 Lim et al 85 hcg primed.75 IU/mL.75 IU/mL hcg 36 5% O 2 5% CO 2 and 9% N 2 65 Yes,?% 17. Fadini et al 83 None FSH primed hcg primed FSH plus hcg Medicult 1% patients.75 IU/mL.1 IU/mL hcg 3 5% CO 2 and 48 51 58 77 6% 2% 9.2 1.6 4. 16. Treatment cycles in PCO(S) patients Cha et al 65 None TCM199 2% FBS 1 IU/mL PMSG plus 1 IU/mL hcg Pyruvate 48 5% CO 2 and 62 6.9 Chian et al None 2 59 hcg primed TCM199 2% FBS 75 miu/ml hmg Pyruvate 24 /48 5% CO2 and 5/69 78/84 14.8 16.7 Mikkelsen None et al 8 FSH primed TCM199 1% patients.75 IU/mL FSH.5 IU/mL hcg Pyruvate 1 μg/ml estradiol 28 36 5% CO2 and 44 59 21.6 (Continued) Seminars in Reproductive Medicine Vol. 3 No. 3/212

28 In Vitro Oocyte Maturation Nogueira Table 2 (Continued) Study Gonadotropin priming Basal Media Protein Supplementation Gonadotropin Supplementation Other Additives Hours of Culture Oxygen Condition %InVitro Maturation* Culture Conditions for % In Vivo Maturation %IR Child et al 68 hcg primed Grouped in 1 ml of TCM199 2% maternal 75 miu/ml hmg Pyruvate 24/48 5% CO 2 and /76 9.5 Lin et al 7 FSH plus hcg primed hcg primed Grouped in TCM199 2% patients 75 miu/ml hmg Pyruvate 24/48 5% CO2 and 43/76 39/72 9.7 11.3 Le Du et al 61 hcg primed Grouped in 1 ml of TCM199 2% maternal.75 IU FSH plus.75 IU LH 24/48 5% CO2 and 54/63 1.9 Son et al 74 None FSH primed hcg primed YS Son et al 69 hcg primed YS Treatment cycles in normo-ovulatory patients 1 IU/mL FSH 1 IU/mL hcg 3% HFF 1 IU/mL FSH 1 IU/mL hcg 1 ng/ml EGF 1 ng/ml EGF 24 3/ 48 24 3/ 48 5% O 2 5% CO 2 and 9% N 2 5% O 2 5% CO 2 and 9% N 2 45/75 4/71 51/73 11% 48/61 Yes, <15% 6.5 6.8 12. 9.7 26.8 (blastocyst) Wei et al 75 FSH plus hcg primed FSH plus hcg plus Metform Medicult 2% patients.75 IU/mL.1 IU/mL hcg Pyruvate 24/48 5% CO2 and?/7%?/77% 6.2 15.3 Son et al 71 hcg primed (Cooper medical) None.75 IU/mL.75 IU/mL hcg 24/48 65% 7 15% 11.9 Ge et al 63 None TCM199 2% FBS A: FSH plus hcg B:1hFSH/FSH plus hcg C: FSH Pyruvate 36/48 6% CO2 and 69% (A) 65% (B) 67% (C) 15.6 (A) 16.1 (B) 17.9 (C) Benkhalifa hcg primed Medicult et al 72 15% patients.75 IU/mL.1 IU/mL hcg 1 miu rgh 26/34 5% CO 2 and 62/8% 6.4 Comparative studies on PCO and normo-ovulatory patients Child et al 67 RC PCO hcg primed hcg primed TCM199 2% maternal 75 miu/ml hmg Pyruvate 24 /48 5% CO 2 and 7/79% 6/76% 1.5 8.9 Seminars in Reproductive Medicine Vol. 3 No. 3/212

In Vitro Oocyte Maturation Nogueira 29 Table 2 (Continued) %IR Basal Media Study Gonadotropin priming Culture Conditions for % In Vivo Maturation Oxygen Condition Hours of Culture Other Additives Gonadotropin Supplementation Protein Supplementation 52/65 12 26.4 5% O 2 5% CO 2 and 9% N 2 24 3/ 48 52 1 ng/ml EGF 3% HFF 1 IU/mL FSH 1 IU/mL hcg Son et al 89 RC plus PCO hcg primed YS 17.9 14. 55/67 49/61 5% CO 2 and Pyruvate 24 36/ 48.75 IU/mL.1 IU/mL hcg 1% patient TCM199 or Medicult None None %InVitro Maturation* Söderström- RC Anttila et al 62 PCO 8.8 59% 49% 29 5% CO 2 and.75 IU/mL.1 IU/mL hcg 1% patients Medicult None FSH primed Dal Canto RC et al 64 PCO 8 17 76/86 75/83 5% O2 6% CO 2 and 9% N 2 24/32.75 IU/mL.1 IU/mL hcg 1% patients Medicult FSH plus hcg FSH plus hcg Nogueira RC et al 9 PCO, in vitro maturation; IR, implantation rate; FSH, follicle-stimulating hormone; hff, human follicle fluid;, not described; PCOS, polycystic ovary syndrome; EGF, epidermal growth factor. Cumulative maturation rate at each time point of observation. higher developmental quality than in vitro matured ones. 89 In non-pco patients, further follicle growth results in an increased oocyte maturation rate at the time of retrieval with up to 2% matured oocytes recovered when leading follicles are at 14 mm. 83 These data emphasize the importance of assessing the nuclear maturity of the oocyte within the cumulus on the day of retrieval because this improves the chance of pregnancy by preventing oocyte aging if the culture is extended to the next day. 24 Culture Condition Influences on Oocyte Maturation Besides the impact of oocyte morphology on outcomes, other laboratory parameters can strongly influence the quality of in vitro matured oocytes. Based on animal studies, the most frequent supplements to the basal clinical media include gonadotropins and, and less frequently growth factors (growth hormone, EGF) and E 2. In humans, gonadotropins are often added to in concentrations ranging from.75 IU/mL to 1 times higher FSH and.1 to 1 IU/mL hcg ( Table 2). There are few studies on the relationship between the choice and amount of gonadotropins selected for and the developmental competence of human oocytes. In CEOs removed from FSH-primed cycles, the same outcomes are observed regardless whether hcg or LH is added or not in culture. 93 A 1:1 ratio of FSH to LH following 24-hour culture with FSH shows some beneficial effects on the maturation of oocytes collected from unstimulated ovaries, with a higher embryonic developmental capacity than when FSH alone is added. 94 Ge et al showed that hcg is unnecessary in the maturation of oocytes from PCOS unprimed patients in culture containing 2% supplementation and FSH. 63 However, it should be noted that increasing FSH concentrations to supraphysiological levels speeds up the process of nuclear maturation with the onset of chromosomal abnormalities. 95 In the last decade, there have been no significant improvements of basal clinical media. One of the most commonly used basal media in clinical studies, TCM199, has been shown to be superior to HTF that is routinely used in IVF. 96 It is advisable not to supplement media with sources from other patients or animal origins, and therefore inactivated patient's has been used at concentrations raging from 2 to 1%, with 1% superior to 2% HSA. 97 The likely presence of additional compounds in sera and follicular fluids (growth factors, glycoproteins, steroid hormones, lipids, cytokines, etc.) might help promote maturation and therefore might explain the benefits of these biological fluids over a synthetic substitute or human albumin. It is relevant to note that inactivation will alter the integrity of some of its compounds, however. Nevertheless, from a scientific point of view, these additives are not standardized with different compositions among patients causing intervariability and challenging the evaluation of the quality of the culture. Besides, it is questionable whether using from PCOS patientsmightbedetrimentaltooocytequalityinculture, Seminars in Reproductive Medicine Vol. 3 No. 3/212