Review In-vitro maturation of oocytes: biological aspects

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1 RBMOnline - Vol 13 No Reproductive BioMedicine Online; on web 5 June 2006 Review In-vitro maturation of oocytes: biological aspects Dr Pierre Miron obtained his medical degree from the University of Sherbrooke (1980), completed an obstetrics & gynecology specialty at University of Montreal (1985) and became, the same year, fellow of the Royal College of Physicians and Surgeons of Canada. He pursued his academic training as a fellow in reproductive endocrinology and infertility at the Royal Women s Hospital, University of Melbourne, Australia (1986), under the guidance and mentorship of Professors Ian Johnston and John McBain. Dr Miron is currently professor at University of Montreal and founded more recently, FERTILYS Reproductive Center, Canada. Dr Pierre Miron A Ali 1, M Benkhalifa 1,2, P Miron 1,3,4 1 Centre de Fertilité et de Reproduction Fertilys, Laval, Québec, Canada; 2 ATL R and D Laboratory, Reproductive Biology and Genetics, La Verrière, France; 3 Department of Obstetrics and Gynecology, Hôpital Maisonneuve- Rosemont, Faculté de Médecine, Université de Montréal, Montréal, Canada 4 Correspondence: Department of Obstetrics and Gynecology, Hôpital Maisonneuve-Rosemont, 5415, Boulevard de l Assomption, Montréal (QC), Canada H1T 2M4. pierre.miron@fertilys.com Abstract Natural cycle and in-vitro maturation (IVM) of oocytes are becoming interesting alternatives to classical assisted reproduction technology approaches for patients, especially in those at high risk for ovarian hyperstimulation syndrome or with poor ovarian reserve. More than for their clinical and biological indications, natural cycle and IVM of oocytes can also be considered as good social and economic alternatives to the classical IVF treatment, based on their financial cost effectiveness with exclusion of expensive medications. To be successful, IVM must entail both nuclear and cytoplasmic maturation, and its maturation and success rates are affected by the number of collected cumulus layers, the degree of atresia and the maturation rate between 24 and 48 h. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. This complex process requires a better definition of each contributing factor affecting oocyte development and the resulting embryo quality. The clinical aspects of IVM have been documented earlier; the present paper will mainly focus on the biological aspect of oocyte maturation in vitro and the quality of derived embryos. Keywords: clinical application, cumulus cells, cytoplasmic maturation, developmental competence, nuclear maturation, oocyte Introduction The success of clinical IVF remains compromised by suboptimal culture conditions, resulting in impaired embryo development and subsequent loss of viability. Even though pregnancy rates have increased since the first human IVF attempts, this can be primarily attributed to the introduction of ovarian stimulation and the subsequent replacement of an increasing number of embryos. The initiation of oocyte in-vitro maturation (IVM) was first reported by Pincus and Enzman (1935) and Edwards et al. (1965, 1969). An interesting feature of the mammalian oocyte is that oocytes undergo spontaneous maturation upon removal from the follicle without stimulation factors (Edwards, 1965). Resumption of meiosis leads to germinal vesicle breakdown (GVBD), the first meiotic reduction division, and arrest at metaphase II (MII) until ovulation and fertilization. IVM of human oocytes is becoming an emerging application in IVF laboratories, and has great promise. Methods used in cattle for IVM of primary oocytes have been applied to women suffering from infertility (Cha et al., 1991). To be successful, the oocyte should achieve two levels of maturation, nuclear maturation through meiosis I and II progression, and cytoplasmic maturation. In the study of Sirard and colleagues (1989) on the timing of nuclear events during IVM, the germinal vesicle (GV) was observed from 0 to 6.6 h, GVBD occurred at h, chromatin condensation at h, metaphase I (MI) at h, anaphase I (AI) at h, telophase I (TI) at

2 h and metaphase II (MII) at h. In 1992, a direct relationship between premature chromosome condensation and IVM of oocytes was suggested (Santalo et al., 1992). More recently, Li and collaborators confirmed that IVM could have deleterious effects on the organization of the meiotic spindle and chromosome alignment of human oocytes (Li et al., 2006). Aneuploidy resulting from errors in meiotic chromosome segregation is the leading cause of pregnancy losses (Li et al., 2002). This could explain the reduction found in developmental competence of IVM human oocytes compared with those matured in vivo and supports the need for improving in-vitro culture conditions. Cytoplasmic maturation in itself describes both the ultrastructural changes that take place in the oocyte from the GV to the MII stage and the acquisition of developmental competence of the oocyte (Duranthon and Renard, 2001). Cytoplasmic maturation is indirectly and retroactively assessed as the ability of the mature oocyte to undergo normal fertilization, cleavage and blastocyst development. Other indirect morphological parameters taken into account to evaluate cytoplasmic maturation include cumulus cell expansion, expulsion of the polar body and an increased perivitelline space (Kruip et al., 1983). Oocyte maturation also involves complex interactions of different intracellular, paracrine and structural factors, including sterols, steroids, growth factors, cyclic adenosine monophosphate and gap junction (Jamnongjit and Hamnes, 2005). Many factors and regulatory molecules have been shown either to inhibit or to promote nuclear and cytoplasmic oocyte maturation (Chian et al., 2004), either directly or via the cumulus cells. Advances made in immature oocyte collection and culture conditions have increased the clinical feasibility of IVM (Son et al., 2005). However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and on how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence. In clinical application of IVM, nearly 50% of immature oocytes reach their maturation after h (Abdul-Jalil et al., 2001) with nearly 20 25% clinical pregnancy in patients with polycystic ovarian syndrome at risk for ovarian hyperstimulation syndrome or in normo-ovulatory patients (Le Du et al., 2005; Mikkelsen, 2005). This result is in contrast to studies in other species where MII is achieved much more easily. When immature oocytes were cultured in suitable conditions in vitro, a significant proportion of oocytes reached nuclear maturation (bovine, Ali and Sirard, 2002; sheep, Guler et al., 2000; rabbit, Lorenzo et al., 1996; pig, Ye et al., 2005). However, Barnes et al. (1996) reported in humans significantly higher rates of oocyte maturation and fertilization of immature oocyte from patients with regular cycles compared with irregular and anovulatory patients. This study also showed that the culture of immature oocytes for 48 h can increase the maturation rate (57 versus 82%) but did not affect the fertilization or the cleavage rate. The optimization of clinical and biological aspects of IVM cycles will enhance the take-home baby rate (Papanikolaou, 2005). Several factors determine the ultimate competence of the oocyte; these have been investigated and attempts made to mimic these conditions in vitro. The complexity of the orchestration of the events that control oocyte growth and final acquisition of developmental competence is under continuous investigation. The present review describes some of biological aspects of oocyte maturation in vitro to date. Oocyte follicle relationship Meiotic and developmental competence In each menstrual cycle, a number of follicles are activated to enter a growth phase characterized by granulosa cell proliferation and an increase in oocyte size. The capacity of oocyte maturation is closely related to follicular maturation. It has been found that in the human oocyte there is a decreased maturation rate in oocytes from small follicles (3 4 mm) when compared with those from larger follicles (9 15 mm) (Tsuji et al., 1985). The growing follicle must reach a minimum of 3 4 mm in size to acquire the capacity to respond to a developmental signal. This capacity will increase (percentage of oocytes capable of responding) as the follicle reaches a plateau or a reduction of its growth rate, either by dominance or early atresia. Moor and Trounson (1977) observed in sheep that as the follicle size increased, the frequency of oocytes progressing to the second meiotic metaphase increased as well. Their observation showed that follicle size could be used as an indictor for selecting oocytes with high meiotic competence. With respect to follicle size, oocytes recovered during the follicular phase show definitive differences in meiotic competence. However, oocytes recovered in the luteal phase, irrespective of follicle size, result in a meiotic competence similar to that of oocytes recovered from large follicles during the follicular phase (Machatkova et al., 2004). After fertilization, these luteal-phase oocytes also demonstrate a high potential to reach blastocyst stage. Also, in humans, early evidence suggested that immature oocytes collected in the luteal phase have significantly higher maturation rates (Cha et al., 1992). In addition, aspiration in the mid-follicular phase of antral follicles ( 8 mm) in women with regular menstrual cycles, prior to the emergence of a dominant follicle, offers the best oocytes for maturation and ultimately pregnancy rates (Mikkelsen et al., 1999). The relative influence of follicular and luteal phases on oocyte meiotic competence was not clearly defined, but may be related to follicular atresia. As expected, the follicle size from which an oocyte is derived is also related to its ability to progress from early cleavage to blastocyst stages. Oocyte developmental competence characterizes the ability of such oocytes to develop to the blastocyst stage in vitro. This observation is well demonstrated in mouse, ewe and cow, where oocytes from small growing follicles lack the ability to progress beyond the 2 8-cell stage. Conversely, oocytes derived from large follicles ( 8 mm) may achieve blastulation rates comparable to IVM oocytes (Moor and Trounson 1977; Eppig et al. 1992). It appears that oocytes require an additional prematuration to express this competence (Hendriksen et al., 2000). In vivo, this pre-maturation occurs during pre-ovulatory growth before the LH surge. This suggests that developmental competence is mainly acquired during oocyte growth within the follicle by an unknown mechanism (Sirard, 2001). Final steps in oocyte maturation are crucial to the acquisition of functional properties necessary for further development (Hyttel et al., 1997).

3 Current IVM protocols are generally inefficient at producing viable embryos, partly due to abnormal oocyte cytoplasmic maturation in vitro (Krisher and Bavister, 1998). Embryos produced after IVM of human oocytes are often arrested at pronuclear or 4 8-cell stage when attempting to culture them to blastocysts (Barnes et al. 1996). During IVM programmes, in most cases, embryos obtained after IVM have been replaced at 2 8-cell stage and their developmental potential to the blastocyst stage is relatively unknown. When considering the low pregnancy rate achieved after IVM of human oocytes, it appears that human oocytes acquire their ability to mature and develop to blastocyst relatively late during folliculogenesis. Allowing such development of IVM of human oocytes to blastocysts could be an important selection tool for embryo transfer (Barnes et al., 1995). Moreover, optimization of IVM protocols is vital not only for generating viable embryos, but also to support the development of subsequent offspring into normal adults (Eppig and O Brien, 1998). For single embryo selection prior to transfer, more than culture media development and improvement, there is a need for an indicator of viability. Other visual assessments used to evaluate developmental competence include morphological evaluations such as blastocoele expansion, number of blastomeres, and trophectoderm to inner-cell mass ratio (Ali et al., 2004). Moreover, functional evaluations such as the ability to resume development after freezing and induce a pregnancy should also be considered to provide a more complete idea of the developmental potential of the oocyte (Isachenko et al., 2004; Sirard et al., 2006). The ovarian follicular population is another parameter used to estimate the developmental competence of the oocyte. The number and size of the follicles present in the ovary at the time of aspiration may be used to select oocytes with higher developmental competence. Oocytes retrieved from ovaries that have at least one follicle larger than 10 mm or with more than 10 follicles of 2 5 mm have a high developmental potential. In contrast, oocytes retrieved from ovaries with fewer than 10 follicles of 2 5 mm or no follicle larger than 10 mm reach lower blastocyst rates with lower cell numbers (Gandolfi et al., 1997). Follicular quality It was observed that the development to blastocyst of an oocyte recovered from an atretic follicle (showing signs of cumulus expansion in the outer layers of the cumulus granulosa and slight granulations in the oocyte cytoplasm), irrespective of size, was equally good as that of an oocyte recovered from large non-atretic follicles. It is believed that some large follicles contain developmentally incompetent oocytes, while some medium-sized follicles contain competent oocytes (Blondin and Sirard, 1995). Follicular atresia may promote the acquisition of developmental competence (Hendriksen et al., 2000). Blondin et al. (1997) found that bovine oocytes derived from ovaries maintained at 35 C for 4 h yielded a higher frequency of blastocysts. Their data suggested that developmental competence was acquired shortly prior to IVM and depended on the handling conditions of post-mortem ovaries. Similar conclusions to those of Blondin and Sirard (1995) have been observed in human oocytes (Barnes et al., 1996). Human oocytes in which the outer layers of cumulus granulosa are partially expanded are classified as atretic. These oocytes results in high rates of maturation and fertilization when compared with oocytes with healthy, tightly compacted cumulus granulosa (Blondin and Sirard, 1995; Blondin et al., 1997). In the final steps of follicular growth, even in atretic follicles, oocytes could undergo a final maturational event at the cytoplasmic level that would render them competent to develop. Moreover, oocytes may retain their developmental competence acquired in the late follicular phase even when atresia is under way; in that regard, it was shown that the cumulus oocyte complex (COC) is the last part of the follicle to be affected by atresia (Kruip and Dieleman, 1982). It is well known that the follicle can profoundly influence the quality of the oocyte obtained at ovulation and, as a result, the quality of embryo obtained. Recently, bovine follicular fluid (bff) of competent follicles (>8 mm) from FSH-stimulated animals was compared with bff from small (2 5 mm) follicles during IVM. The objective was to evaluate if adding bff to the maturation medium level could influence the developmental competency of selected oocytes obtained from 2 5-mm-sized follicles (Ali et al., 2004). The conclusion was that follicular fluid originating from competent follicles increased the developmental competence of abattoir-derived oocytes and, as a result, the quality of embryo obtained. In humans, follicular vascularity and the level of intrafollicular oxygen appear to be important determinants of oocyte competence. Findings from several studies indicate that embryos with the highest implantation potential originate from follicles that are well vascularized and oxygenated (Van Blerkom, 1998, 2000). Metabolic coupling Within the follicle, there are several different somatic cell phenotypes that surround the oocyte. It is now widely recognized that bi-directional communication between the oocyte and follicular somatic cells is fundamentally important for folliculogenesis and oocyte growth and maturation (Eppig, 2001). The morphology of the cumulus surrounding an oocyte is commonly used as a selection criterion prior to IVM (Goud et al., 1998). The degree of expansion is also considered as a morphological indicator of oocyte quality and directly related to developmental capacity of the oocyte to reach maturation (Ali and Sirard, 2002a). Earlier studies have demonstrated that granulosa cells are metabolically coupled to oocyte via gap junctions during growth and initial stages of maturation (Brower and Schultz, 1982; de Loos et al., 1991). Cessation of metabolic coupling coincides with the breakdown of the gap junction, which generally occurs just prior to the first meiotic metaphase. Nutritional and regulatory elements responsible for growth, maintenance of meiotic arrest, and substrates for maturation and development are able to pass through these gap junctions (Tanghe et al., 2002). In cattle, it is speculated that granulosa cells, in response to LH, synthesize pyruvate, which is transferred to the maturing oocyte. These events do not occur in the absence of an LH surge or in 439

4 440 COC in which the oocyte has been removed (oocytectomized), indicating the interdependence of these cell types (Zuelke and Brackett, 1993). It is evident that cumulus cells provide many important functions not just for the oocyte but also for zygote, such as pronuclei formation, polar body extrusions, and organelle redistribution and cytoskeletal rearrangements. The cumulus provides not only a microenvironment that would consist in low concentrations of glucose and high lactate concentration, but also provides homeostasis regulation for the oocyte and the early embryo (Mori et al., 2000; Tanghe et al., 2002). The beneficial effect of cumulus oophorus during IVM can be attributed to the formation of a favourable microenvironment (biochemical or metabolic) around the oocyte (Tanghe et al., 2002). Cumulus cells participate in oocyte development during IVM, either by secreting soluble factors, which induce developmental competence or by removing inhibitory or toxic components from the maturation medium (Homa, 1995; Hashimoto et al., 1998). In addition, cumulus cells might have unknown promoting effects on subsequent oocyte development which might be attributable to intracellular changes such as ph or calcium ions (Mori et al., 2000). Another possible influence of cumulus cells during IVM of bovine oocytes might be that cumulus cells decrease oxygen tension in the immediate vicinity of the oocyte as a result of an active metabolism of the cumulus cells (Ali et al., 2003). By removing the cumulus cells artificially, it has been demonstrated that their presence during maturation is necessary for most oocytes to express competency (Ali et al., 2005). In this study, oocyte MII and development to the 2 8-cell stage 72 h after insemination were not affected by the absence of cumulus cells during maturation. However, development beyond the blastocyst stage was significantly lower in cumulus-free than in cumulus-intact oocytes, indicating the importance of cumulus cells during IVM of oocytes for cytoplasmic maturation and subsequent early development (Figure 1). Culture conditions It is known that treating the cause instead of the symptoms produces the most encouraging results. Early embryo development appears to be dependant on the maturation microenvironment of the oocyte (Table 1). Significant improvements in the overall rate of oocyte maturation and embryo development can be achieved by adding mature mammalian fluid from large follicles (containing oocytes more likely to develop into blastocysts) in the culture media (Blondin et al., 2002; Ali et al., 2004). Early embryo development is a complex mechanism, based on interactions between intracellular and extracellular cell biology of oocyte and spermatozoa development and maturation. It is therefore quite remarkable that oocytes matured and embryos produced in vitro still maintain a reasonable degree of functional and developmental competence. Studies have investigated a number of culture modifications, based on physiological conditions found in the female reproductive tract, to improve embryo developmental outcome. In-vitro conditions cannot truly replicate in-vivo conditions. Features of oocyte and embryo morphology, metabolic and biochemical properties can be altered by the in-vitro environment. These alterations can become evident as errors that may be compatible with early embryonic development but are deleterious for viability. In most reports, IVM of mammalian oocytes was performed in incompletely defined systems containing co-cultured somatic cells, blood serum, bovine serum albumin (BSA) or cell-conditioned medium. Besides being a potential source of infectious agents, these undefined components make it difficult to undertake proper quality control and to evaluate the basic requirements for metabolic substrates, nutritional, growth factors and hormones during IVM. Pituitary hormones and oocyte developmental competence In most mammalian oocytes, supplementation of in-vitro culture media by gonadotrophins, steroids and growth factors separately or in combination can stimulate or inhibit cumulus expansion and/or nuclear and cytoplasm maturation. It is now common practice in mammalian oocyte media to add these supplements during IVM. Supplementation with recombinant human FSH (rfsh) and 17β-oestradiol during IVM of bovine oocytes results in a positive effect by increasing the number of embryos after IVF (Ali and Sirard, 2002b). Media for IVM of human oocytes contain oestradiol to support cytoplasmic maturation, which is necessary for fertilization and early embryonic development (Tesarik and Mendoza, 1995). This is in agreement with the finding that oocytes matured in the presence of higher concentrations of oestradiol (1000 ng/ml) improved the developmental rate to blastocyst stage (Ali and Sirard, 2002b). Other observations in cattle suggest the possibility of including hyaluronic acid (HA) with oestradiol in culture media to increase the efficiency of in-vitro blastocyst production from IVM oocytes using completely defined conditions (Ali et al., 2002; Figure 2). Interestingly, when oestradiol and HA were added to the maturation medium, cumulus expansion was never observed. These results confirmed recent findings that the presence of cumulus cells during IVM might be necessary to express the competence of oocytes and that cumulus expansion is not required to improve this competence; at the least, there is no linear relationship between cumulus expansion and cytoplasmic maturation (Ali and Sirard, 2002a,b). Moreover, these results suggest that communication between the oocyte and surrounding cumulus cells is important in determining which factors are released by the cumulus cells (Ail and Sirard, 2005; Ali et al., 2005). In cattle, the concentration of oestradiol in the follicular fluid of a dominant follicle is higher than in others, indicating a possible role of oestradiol on the cytoplasmic maturation occurring before LH surge. Recently, studies in cattle have postulated that increased developmental competence of bovine oocytes in response to rfsh and oestradiol occurs by the production of factors that reach the oocyte through the cell cell coupling pathways or that the coupling per se results in physiological changes (Ali and Sirard, 2005; Ali et al., 2005). Moreover, these studies provide novel evidence of a direct role of rfsh in the regulation of gap junction communication between cumulus cells and oocytes and the consequences of such conditions on further competence.

5 Figure 1. Effect of the presence or absence of cumulus cells during different periods of in-vitro maturation on further development of bovine oocytes in vitro. Pooled data from three replicates (mean ± SEM). Values with different superscripts (a c) are significantly different within a column (P < 0.05). All treatments were replicated at the same time. Recombinant human FSH (rfsh) = synthetic oviductal fluid/bovine serum albumin (BSA + rfsh) (Ali et al., 2005). COC = cumulus oocyte complexes; DO = denuded oocyte; MII = metaphase II. Table 1. Some factors added to in-vitro maturation media to enhance oocyte maturation and subsequent embryo development. Factors Authors Year FSH Izadyar et al Ali and Sirard 2002b, 2005 Sp-cAMPS (PKA activator) Ali and Sirard 2005 PMA (PKC activator) Ali and Sirard 2005 Oestradiol Tesarik and Mendoza 1995 Ali and Sirard 2002b Growth hormone (GH) Iga et al Izadyar et al Hyaluronic acid (HA) Ali et al Follicular fluid (FF) Sirard et al Blondin et al Ali et al Serum Trounson et al Intracellular antioxidants (cysteine, cysteamine, Jeong and Yang 2001 glutamine, β-mercaptoethanol) Ali et al

6 Figure 2. Effect of hyaluronic acid (HA) added to the in-vitro maturation medium on the response to oestradiol on subsequent bovine embryo development. Pooled data from three replicates (mean ± SEM) (Ali et al., 2002). SOF = synthetic oviductal fluid. 442 The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in rfsh response), were investigated recently (Ali and Sirard, 2005) using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases respectively. The developmental competence of oocytes was measured by the rate of blastocyst formation after IVF. This study is one of the first to report such high development rates of bovine oocytes after IVM using defined conditions, especially after short-term treatment with rfsh. The data show that the PKC pathway is implicated in r-fsh, which improved oocyte competence, especially after short-term treatment. These results indicate that the PKA and PKC pathways can modulate the maturation of oocytes in vitro. A better understanding of the mechanism by which rfsh stimulates the oocytes can have important implications in the light of clinical interest in the use of rfsh in assisted reproduction protocols. During the period of folliculogenesis and oocyte maturation in vivo, growing evidence indicates an important role of both FSH and LH. In human IVF, Filcori (1999) reported that a minimal level of LH activity during exogenous stimulation protocols is required to optimize ovulation induction in patients. In fact, it has been postulated that profound LH suppression could affect optimal oocyte maturation and/or endometrial development (Lévy et al., 2000). Moreover, the final stages of oocyte maturation in vivo are induced by a rise in serum LH concentrations. Schoolcraft and co-workers (1999) reported that including LH in ovarian stimulation protocols using highly purified FSH preparations would improve blastocyst quality as reflected by increased embryo implantation and pregnancy rate. However, LH supplementation, particularly in ovarian stimulation protocols using GnRH agonists, and the concept of window for LH requirement, still remain today a matter of debate (Tesarik and Mendoza, 2002; Humaidan, 2006; Kolibianakis et al., 2006). The effect of LH during IVM on oocyte maturation and subsequent development has also been questioned. Initially, there have been many studies reporting the beneficial effects of LH on oocytes, as shown by an increase in embryo yield after IVF and in-vitro culture (Younis et al., 1989; Zuelke and Brackett, 1992; Gliedt et al. 1996; Choi et al., 2001). However, recent findings have clearly shown that LH had no effect on the developmental potential of bovine oocytes when added to an IVM defined medium (Ali and Sirard, 2002b). These results confirmed earlier findings from Izadyar and co-workers (1996), who reported that when bovine oocytes were cultured in the presence of FSH and human chorionic gonadotrophin (HCG), only FSH, and not LH, influenced IVM of oocytes. The absence of an in-vitro effect of LH during oocyte maturation can be observed by the fact that the effect of LH on oocyte is not direct. Oocyte donors with low serum LH also have low oestradiol concentrations (Tesarik and Mendoza, 2002). In fact, most COC used for IVM studies originated from small- and medium-sized follicles (2 6 mm), and it has been demonstrated that receptors for FSH but not LH are transcribed in the cumulus and granulosa cells of these follicles (Van Tol et al., 1996). Such contradictory results could partly be explained, in earlier studies demonstrating a beneficial effect of LH supplementation, by the possible use of LH preparations contaminated by FSH, thyroid stimulating hormone (TSH) or other contaminants. They could also be explained by the fact that LH receptor is not detected in COC from small antral follicles and that in-vitro FSH priming is initially required (Okazaki et al., 2003). Another pituitary hormone that has been proposed in assisted reproduction is growth hormone (GH). The role of GH in ovarian function, follicular growth, and steroidogenesis is well known and evidence shows a positive effect of GH on oocyte maturation. GH concentration in FF has been shown to be positively related to both normal fertilization and preimplantation embryo morphology and cleavage speed (Mendoza et al., 1999). Moreover, GH is known to enhance intrafollicular metabolic events required for oocyte maturation. A study by Mendoza and co-authors (2002) also reported that GH concentration in FF is significantly higher in conception IVF as compared with non-conception cycles. Other observations in women suggest the possibility of including GH during ovarian

7 stimulation in an ICSI programme to increase the efficiency of delivery and live birth rates in women of 40 years (Tesarik et al., 2005). The addition of GH during IVM has been shown to accelerate nuclear maturation and promote subsequent cleavage and embryonic development (Iga et al., 1998). As for FSH, GH exerts its effects during oocyte maturation through a cyclic adenosine monophosphate (camp) signal transduction pathway. The promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation (Izadyar et al., 1998). These results were confirmed later in humans by Menezo and co-workers (2003), who reported that GH receptor is present in oocytes and early preimplantation embryos. Protein sources Follicular fluid Follicular fluid composition changes during follicle growth. A study by Sirard and co-authors demonstrated that addition of selected follicular fluid influences the developmental potential of oocytes obtained from unselected ovaries (Sirard et al., 1995). In fact, the beneficial effect on development is present in dominant follicles but not in all growing or regressing dominant follicles, suggesting a fine tuning between growth and differentiation signals. Therefore, follicular supplementation, perhaps in the form of follicular fluid, influences oocyte competence and subsequent developmental capacity as well as embryonic quality In follicular fluid, oestradiol concentrations are higher in large than in small follicles (Gastal et al., 1999; Belin et al., 2000). In cattle, before LH surge, the oestradiol concentration in the follicular fluid is high (about 1 μg/ml) but sharply decreases subsequently (Fortune and Hansel, 1985). Although there is no evidence that oestradiol is involved in the resumption of meiosis, it might be that oestradiol is involved in the cytoplasmic changes that occur before LH surge. Follicular fluid supplementation of IVM medium has been proven to provide a beneficial microenvironment for further development of the immature oocyte. High developmental rates after IVM, especially after supplementation of bff were obtained (Ali et al., 2004). In this study, modified synthetic oviductal fluid (m- SOF) was used successfully for bovine oocyte maturation and gave good embryo development with respect to rate and quality, closer to that observed in vivo. Serum and IVM and fertilization Historically, to support sperm capacitation and/or fertilization ability, it was necessary to support basic media with proteins. Initially, follicular fluid was used because it contains factors that maintain sperm motility in vitro as well as components that support capacitation and stimulate acrosome reaction, which are prerequisites for sperm penetration of the zona pellucida (Yanagimachi, 1969). Later, it was discovered that follicular fluid could be replaced by serum albumin (Bavister, 1969). This protein source has been universally used ever since for sperm capacitation in IVF. Fetal calf serum (FCS) has long been recognized as an important constituent for in-vitro oocyte maturation. It is well known that FCS is necessary for FSH induced cumulus expansion in hamsters and cattle (Leibfried- Rutledge et al. 1986). Additionally, bovine oocytes matured in the presence of FCS had higher levels of sperm penetration. FCS contains a protein called fetuin that inhibits zona pellucida hardening initiated by cortical granule release (Schroeder et al., 1990). Low rates of fertilization have been documented following IVM of human oocytes (Barnes et al., 1995, 1996). While zona hardening has yet to be defined for IVM oocytes, there is no doubt that the zona pellucida of day 3 5 human embryos following IVM is extremely hard (Trounson et al., 1994) Serum and oocyte developmental competence Protein supplementation during IVM can affect the efficiency of the procedure. In-vitro-matured human and bovine oocytes have been shown to reduce protein content compared with in-vivomatured oocytes (Trounson et al., 2001), suggesting that proteins play a critical role in acquisition of developmental competence. FCS and maternal serum have been widely used and accepted as protein supplements in IVF. Although oocytes can be cultured without protein or hormone supplementation (Ali and Sirard, 2002a,b), embryo production seems to be improved when a protein supplementation is included in the culture medium. The role of FCS in oocyte developmental competence is still unclear. In the authors experience, bovine oocytes are often cultured in groups of 10 and, under these conditions, the effect of FCS does not appear to be beneficial (Ali and Sirard, 2002b). In this study, the authors suggested that the effect of FCS as a protein supplement during IVM of bovine oocytes depends on the kind of culture medium used. The same authors demonstrated that when FCS is replaced by BSA as the only protein source during IVM of oocytes, BSA not only delays nuclear maturation but also decreases the developmental capacity of oocytes. These oocytes yielded the lowest frequency of blastocyst development when compared with maturation medium without BSA supplementation. Influence of antioxidants on oocyte developmental competence Antioxidants may be beneficial additives to synthetic culture media. In well-defined media, there is a possible lack of serum factors or of other macromolecules that serve as reactive oxygen species scavengers (ROS). It is well known that oxygen concentration within the lumen of the female reproductive tract is about one-third (3 9%) that found under standard in-vitro conditions (Mastrioanni, 1965). Culture of embryos with a high oxygen tension in vitro (20%) can produce more free radicals (Fowler and Callingham, 1995) than embryo culture under 5% O 2 or 7% O 2 (Liu and Foote, 1995). Detrimental effects of oxygen-derived free radicals during in-vitro culture have been demonstrated in several species. ROS can induce mitochondrial dysfunction, DNA, RNA and protein damage (Comporti, 1989) as well as inhibiting sperm oocyte fusion (Aitken, 1993). To protect oocytes and embryos from oxidative stress during in-vitro culture, various antioxidants can be added to culture media (Ali et al. 2003). Correct conditions for oocyte and embryo culture are not well defined and any potential effect of a controlled O 2 environment 443

8 444 can be observed when optimum conditions are maintained. Cytoplasmic maturation of oocytes can be improved by reducing oxidative stress caused by COC production of reactive oxygen species due to the in-vitro culture environment (Ali et al., 2003). Metabolic pathways, mediated by enzymes, such as glutathione, can control ROS cellular concentrations and protect the oocyte against damaging effects of oxidative stress. Glutathione content of the oocyte can be increased by adding thiol compounds such as cysteine, cysteamine, glutamine, β-mercaptoethanol and/or follicular fluid to the maturation medium (Jeong and Yang, 2001; Ali et al., 2003). Apart from its protective action, glutathione also increases amino acid transport and stimulates DNA and protein synthesis (Lafleur et al., 1994). Conclusion Human oocyte maturation in vitro is becoming an effective alternative to classical IVF treatment. The procedure avoids ovarian stimulation, side effects of medical treatment and other risks such as ovarian hyperstimulation syndrome (Chian et al., 2004; Rao and Tan, 2005). Moreover, natural cycle and IVM of oocytes can be considered as an economic alternative to classical assisted reproduction based solely on its financial cost effectiveness in excluding the need to pharmacologically stimulate the ovaries. Following experience with the IVM procedure, the efficiency of producing homogene embryo cohort with good quality and viability is less than in classical IVF and intracytoplasmic sperm injection. When comparing oocytes matured in vivo versus in vitro, no apparent differences are seen at the level of nuclear maturation, in the rates of fertilization or cleavage, but rather in the developmental competence of the oocytes as exemplified by poor embryonic developmental competence and pregnancy rate. The clinical data have shown a biochemical pregnancy rate of approximately 18%, with a 40% early miscarriage rate (personal communication, Dr Benkhalifa). These observations indicate that the cytoplasmic competence must be different between in-vitro- and in-vivo-maturated oocytes. Advances made in immature oocyte isolation and maturation, zygote and embryo culture conditions have increased the clinical feasibility of IVM. However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and on how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence. Simple maturation systems now exist, using a fully defined medium in which the search for factors improving acquisition of both nuclear and cytoplasmic competences will be possible. 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