Abstract. Introduction. RBMOnline - Vol 5. No Reproductive BioMedicine Online; on web 1 August

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1 RBMOnline - Vol 5. No Reproductive BioMedicine Online; on web 1 August Articles Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles Dr RC Chian Dr Chian is Assistant Professor at Division of Reproductive Biology, Department of Obstetrics and Gynecology, McGill University. After award of his PhD from Okayama University, he obtained a Ryobiteien International Award, also in Japan, for his outstanding research in Reproductive Biology. He produced the first pregnancies and live births from invitro matured (IVM) human oocytes derived from women with polycystic ovarian syndrome (PCOS) in Canada. He has published more than 60 research papers and presentations and acted as a referee for several scientific journals, including Human Reproduction, Fertility and Sterility, Reproduction, Fertility, Reproduction and Development. He is a member of the American Society for Reproductive Medicine (ASRM), Canadian Fertility and Andrology Society (CFAS), and Society for Study of Reproduction (SSR). In 2001, Dr Chian received First Prize from the Second Royan International Research Award of Iran for outstanding contribution in the field of IVM treatment, delivered the John Collins lecture at the CFAS meeting, and the President s lecture of the Japanese Society for Mammalian Ovum Research. His research interests include sperm DNA remodelling in mature and ageing oocytes, reprogramming of nuclear genomes in oocytes, and the mechanism of oocyte maturation. RC Chian 1, SL Tan Department of Obstetrics and Gynecology, McGill University, Montreal, Canada H3A 1A1 1 Correspondence: Women s Pavilion F3 46, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3E 1A1. Fax: ; ri-cheng.chian@muhc.mcgill.ca Abstract The present experiments compared the maturational and developmental competence of immature oocytes derived from stimulated cycles, following culture in a newly designed in-vitro maturation medium (IVM-medium) or in standard tissue culture medium (TCM-199; control). The results indicated that maturation and fertilization rates were comparable when the cumulus-free M-I stage oocytes were matured in the IVM-medium (78.6%) or the control medium (70.8%). However, there was a significant difference in blastocyst development (P < 0.05) when M-I oocytes were matured in these two media (19.6 versus 7.7%). Both maturation and early embryonic development rates of GV-stage oocytes were significantly higher (P < 0.01) in the IVM-medium (maturation: 75.7%; blastocyst: 12.9%) compared with control (maturation: 55.7%; blastocyst: 0.0%). Moreover, embryos developed to the blastocyst stage at a higher rate in both media if GV-stage oocytes had matured within 24 h compared with 48 h of culture. These results demonstrate that immature human oocytes derived from stimulated ovaries can achieve maturation and early embryonic development in vitro, especially in the new IVM-medium, which may allow additional embryos to be produced for clinical use at embryo transfer. Keywords: blastocyst, cumulus cells, germinal vesicle, immature oocytes, IVM, metaphase Introduction Since the achievement of the first successful human pregnancy following IVF (Steptoe and Edwards, 1978), assisted reproductive technology has helped hundreds of thousands of couples overcome infertility (Tan et al., 1992; Engmann et al., 1999). During the follicular phase of a woman s menstrual cycle, a single follicle usually grows to the preovulatory stage and then releases its oocyte for potential fertilization. In conventional IVF treatment, women are pretreated for approximately 2 or 3 weeks with gonadotrophin releasing hormone agonists (GnRHa). After pituitary suppression has been achieved, human menopausal gonadotrophin (HMG) or purified follicle-stimulating hormone (FSH) is administered to induce the development of multiple follicles (Tan et al., 1994). Once the mean diameter of the largest follicles exceeds 18 mm, human chorionic gonadotrophin (HCG) is administered to trigger final oocyte maturation 36 h before follicular aspiration with transvaginal ultrasound guidance. Although a large number of metaphase-ii (M-II) oocytes are generally collected, about 15% of recovered oocytes are immature at the germinal vesicle (GV) or metaphase-i (M-I) stage (Cha and 125

2 126 Chian, 1998). Attempts have been made to mature these oocytes in vitro but the maturation rates obtained have hitherto been very low (Nagy et al., 1996; Edirisinghe et al., 1997; Liu et al., 1997; Jaroudi et al., 1999). It would be clinically useful if the in-vitro maturation rates of these oocytes could be improved because some women produce only a few oocytes, while others may produce mostly immature oocytes (Tan and Child, 2002). Because of the costs of gonadotrophins and the side effects they may produce, including the risk of ovarian hyperstimulation syndrome (OHSS), it would be ideal if the use of FSH or HMG could be avoided altogether for clinical IVF treatment. In-vitro maturation (IVM) of immature oocytes could be developed and used as a new method for the treatment of infertile women (Tan and Child, 2002). The benefits of IVM include eliminating the need for hormonal stimulation of the ovaries, frequent blood sampling and ultrasound monitoring, reduced costs and the potential risk of OHSS. In human IVM, tissue culture medium 199 (TCM-199) has most often been used as the maturation medium (Trounson et al., 1994; Barnes et al., 1995; Chian et al., 1999a, 2000; Mikkelson and Lindenburg, 2001; Mikkelson et al., 2001) and even though different culture media have been used to mature human oocytes (Shea et al., 1975; Cha and Chian, 1998), no one medium has been shown to be clearly superior (Trounson et al., 1998, 2001). The ability to resume meiosis is independent of the presence of cumulus cells (Magnusson, 1980). Thus, the oocyte is able to undergo nuclear maturation without support from the cumulus and granulosa cells. However, it has been reported, in a wide variety of species, that such oocytes are not capable of undergoing normal fertilization and development (Staigmiller and Moor, 1984; Vanderhyden and Armstrong, 1989; Fukui, 1990; Chian et al., 1994; Gregrory and Leese, 1996). Animal study also indicated that the protein synthesis pattern is different between oocytes with and without cumulus cells and FSH modulates the protein synthesis pattern of cumulus cellintact oocytes (Chian and Sirard, 1995). Cumulus cells have different levels of steroid secretion and different responses to gonadotrophins (Dirnfeld et al., 1993; Goldman et al., 1993; Chian et al., 1999b), and they also differ in the expression of specific mrna compared with the mural granulosa cells (Braw-Tal, 1994). Nevertheless, it has been reported recently that a relatively high percentage of cumulus-denuded bovine oocytes can develop to the blastocyst stage in a chemically defined medium (Geshi et al., 2000). Intracytoplasmic sperm injection (ICSI) is often used to treat severe male factor infertility. ICSI requires enzymatic and mechanical removal of the cumulus cells from both mature (M-II) and immature oocytes (GV and M-I) because it is difficult to identify cumulus oocyte complex (COC) maturity under a microscope. Immature oocytes (GV and M-I) collected from stimulated cycles are discarded in most IVF clinics because those oocytes have been exposed to a bolus of HCG but were not mature and only a few live births have been obtained from IVM of cumulus-denuded oocytes derived from stimulated cycles (Nagy et al., 1996; Edirisinghe et al., 1997; Jaroudi et al., 1999). It would therefore be desirable to improve the maturational and developmental competence of cumulus-denuded immature oocytes. The present study was designed to determine if a new IVMmedium would stimulate increased rates of maturation and early embryonic development of immature human oocytes derived from stimulated and ICSI cycles. Materials and methods Source of immature oocytes A total of 521 immature (213 GV and 308 M-I stages) oocytes were collected with informed consent from 183 women aged years. This study was approved by the Ethical Committee of the Hospital. The women were undergoing ovarian stimulation and ICSI cycles using a standard long stimulation protocol. Thirty-six hours after HCG administration, oocyte retrieval was performed with ultrasound guidance. The collected COC were cultured in human tubal fluid medium (HTF; Irvine Scientific) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific) and placed in a 5% CO 2 incubator (37 C with high humidity) for at least 1 h before they were stripped from their cumulus cells. COC were stripped with 85 IU/ml hyaluronidase in modified HTF medium (mhtf; Irvine Scientific) and mechanical pipetting until all oocytes were completely denuded from their cumulus cells. Denuded oocytes were observed under an inverted microscope ( 200) to assess oocyte maturity. Mature (M-II) oocytes were determined by the presence of a first polar body (1PB) extrusion and were used for ICSI immediately. Immature oocytes were defined by the absence of 1PB and then classified as GV or M-I stages depending on whether a GV was visible. Immature oocytes without a visible GV were considered as M-I stage. The immature oocytes were cultured in HTF medium supplemented with 10% SSS (Irvine Scientific) in a 37 C and 5% CO 2 incubator for 3 4 h before transfer to either TCM-199 or the new IVM-medium. The time at which the immature oocytes were transferred into the maturation media was defined as the starting point for maturation in vitro. In-vitro maturation of immature oocytes Immature oocytes were classified two groups, (1) GV stage (n = 213) and (2) M-I stage (n = 308). Each group of oocytes was randomly allocated to be cultured in either one of two maturation media, namely, TCM-199 (GIBCO, Catalogue No ) or newly designed IVM-medium (Table 1). To make the new IVM-medium, the minimum effective concentrations of growth factors were chosen. Both media were supplemented with 10% SSS (Irvine Scientific), IU/ml FSH + LH (Humegon, Ontario, Canada) and 1.0 µg/ml oestradiol (Sigma). Additional supplements to TCM-199 included 0.25 mmol/l sodium pyruvate (Sigma), 0.05 IU/ml penicillin G and streptomycin (GIBCO). Before culture, the immature oocytes were washed at least twice in either IVM-medium or TCM One to 5 immature oocytes were cultured in each organ tissue culture dish (60 15 mm; Falcon) containing 1 ml of maturation medium (TCM-199 or IVM-medium) at 37 C in an atmosphere of 5% CO 2 and 95% air with high humidity. The maturity of the oocytes was determined as described above after 6 h for M-I stage oocytes and after 24 h and 48 h of culture for all oocytes.

3 Table 1. Composition of oocyte in-vitro maturation (IVM) medium. Concentrations given as mg/l unless otherwise specified. Inorganic salts CaCl KCl MgSO NaCl NaHCO NaH 2 PO 4.H 2 O Amino acids L-Alanine L-Arginine L-Asparagine L-Aspartic acid L-Cystine L-Glutamic acid L-Glutamine Glycine L-Histidine.HCI.H 2 O L-Isoleucine L-Leucine L-Lysine.HCI L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine Vitamins Biotin D-Ca pantothenate Choline chloride Folic acid i-inositol Nicotinamide Pyridoxal.HCI Riboflavin Thiamine.HCI Other components D-Glucose Sodium pyruvate Insulin Human transferrin Selenite Hydrocortisone FGF EGF Phenol red Penicillin G (IU) Streptomycin (µg) Intracytoplasmic sperm injection (ICSI) IVM oocytes (M-II) were inseminated by ICSI with spermatozoa from the patient s partner. All semen samples were obtained from the ejaculates provided for clinical use. The remaining samples were donated for this study under informed consent. Spermatozoa for ICSI were prepared by PureSperm (Nidacon, Gothenburg, Sweden) gradient separation (45%, 70% and 90%) by centrifugation at 560 g for 20 min. Following gradient separation, the sperm pellet was washed twice (200 g) with 2 ml of HTF medium (Irvine Scientific) supplemented with 10% SSS. A single spermatozoon was injected into each M-II oocyte. Because the oocytes did not mature synchronously during culture, sperm samples were kept in 5 ml Falcon tubes (tight tube cap) containing ml HTF medium supplemented with 10% SSS at room temperature for further insemination by ICSI at 24 h or 48 h respectively. Following ICSI, each oocyte was transferred into a 20 µl droplet of HTF medium supplemented with 10% SSS in a tissue culture dish (35 10 mm; Falcon) under mineral oil (Sigma). The appearance of two distinct pronuclei and two polar bodies was the criterion for fertilization h after ICSI. Culture of embryos Fertilized oocytes were cultured in HTF medium supplemented with 10% SSS until 72 h after ICSI, and then each embryo was transferred into a 20 µl droplet of G2.2 medium (VitroLife, Göteborg, Sweden) in a tissue culture dish (35 10 mm; Falcon) under mineral oil (Sigma) for an additional 48 h of culture. Embryo development was recorded at 24 h intervals following culture until day 5 (120 h). All embryos were destroyed at that stage. Statistical analysis Statistical significant differences in the percentages of oocyte maturation, fertilization and embryo cleavage as well as development between the groups were determined and compared by Student Newman Keuls test (Steel and Torrie, 1980). A P-value of <0.05 was considered statistically significant. Results As shown in Table 2, 52.0 and 46.1% of M-I stage oocytes were mature 6 h after culture in IVM-medium and TCM-199 respectively. Following further culture for 24 h and 48 h, there were no differences in oocyte maturation rates between IVMmedium (76.0 and 78.6%) and TCM-199 (67.5 and 70.8%). As shown in Table 3, the final fertilization rates were also not different between the two media (90.0 versus 84.4%). Furthermore, the final cleavage rates of the fertilized oocytes were not different between the oocytes matured in these two media (97.3 versus 98.9%). Although there were no significant differences in the total number of embryos that developed to the 8-cell stage between the two media (IVM-medium, 63.7%; TCM-199, 57.1%), total blastocyst formation rates were significantly different (P < 0.05) between the IVM-medium (19.6%) and TCM-199 (7.7%). As shown in Table 4, when GV-stage oocytes were matured in vitro, there were significant differences (P < 0.01) in the 127

4 Table 2. Maturation of human oocytes (metaphase-i stage) cultured 6 h, 24 h or 48 h in the IVMmedium or TCM-199. Culture medium No. of oocytes No. of oocytes matured at (%) examined 6 h 24 h 48 h IVM-medium (52.0) 117 (76.0) 121 (78.6) TCM (46.1) 104 (67.5) 109 (70.8) Table 3. Fertilization and embryonic development of human oocytes (metaphase-i stage) matured in the IVM-medium or TCM-199. Culture medium No. of oocytes No. of oocytes No. of oocytes No. of embryos inseminated fertilized (%) cleaved (%) developed to (%) 8-cell stage Blastocyst IVM-medium (90.9) 107 (97.3) 68 (63.6) 21 (19.6) a TCM (84.4) 91 (98.9) 52 (57.1) 7 (7.7) b a,b Different letters indicate significant differences within columns (P < 0.05). Table 4. Maturation of human oocytes (germinal vesicle stage) cultured 24 h or 48 h in the IVMmedium or TCM-199. Culture medium No. of oocytes No. of oocytes No. of oocytes examined matured at matured at 24 h (%) 48 h (%) IVM-medium (50.5) a 81 (75.7) a TCM (34.0) b 59 (55.7) b a,b Different letters indicate significant differences within columns (P < 0.05). Table 5. Fertilization and embryonic development of human oocytes (germinal vesicle stage) matured in the IVM-medium or TCM-199. Culture medium No. of oocytes No. of oocytes No. of oocytes No. of embryos inseminated fertilized (%) cleaved (%) developed to (%) 8-cell stage Blastocyst IVM-medium (86.4) 70 (100.0) 41 (58.6) a 9 (12.9) a TCM (78.0) 44 (95.7) 18 (40.9) b 0 (0.0) b a,b Different letters indicate significant differences within columns (P < 0.05). 128 maturation rates between the two media 24 h (50.1 versus 34.0%) and 48 h (75.7 versus 55.7%) after culture respectively. However, as shown in Table 5, the final fertilization rates were not different between the IVM-medium (86.4%) and TCM-199 (78.0%). Although the final cleavage rates were not different between the two media (100.0 versus 95.6%), there were significant differences (P < 0.05) in embryos that developed to the 8-cell stage (58.6 versus 40.9%) and blastocyst stage (12.9 versus 0.0%) between the IVM-medium and TCM-199. No pregnancy data are available from the present study. Two pregnancies have been achieved after pooling of embryos produced from cumulus-free immature oocytes with in-vivo matured ones but it was not clear whether immature or mature embryos gave rise to the pregnancies. Discussion The present study demonstrates that cumulus-free immature human oocytes derived from stimulated ovaries can undergo maturation and early embryonic development in vitro. In ovarian stimulation cycles, maturation of 85 90% oocytes may be triggered by the administration of HCG 36 h before oocyte collection. Thus, approximately 10 15% of the oocytes

5 could be still immature (Cha and Chian, 1998). In the present study, 19.0% (521/2745) of the collected oocytes from 183 women underwent for stimulation and ICSI cycles were at an immature stage (GV: 213; M-I: 308). It has been reported that the time course of germinal vesicle breakdown (GVBD) and oocyte maturation is different between GV-stage oocytes retrieved from ovulation induction (HMG/HCG) and unstimulated ovaries, but the final rates of oocyte maturation are comparable in these two groups (Cha and Chian, 1998). In the present study, it is unclear whether the immature oocytes were from the small antral follicles or from the large preovulatory follicles in which do not respond to a large bolus of HCG for oocyte maturation. It has previously been demonstrated that IVM, fertilization and pregnancy rates from immature human oocytes collected from unstimulated ovaries are potentially improved by priming with HCG 36 h before oocyte retrieval (Chian et al., 2000). Therefore, these authors suggest that immature oocytes collected from stimulated varies may have acquired initial maturational competence, because the oocytes had been exposed to HCG. Mammalian oocytes are surrounded by layers of compact cumulus cells during folliculogenesis. The cumulus cells respond to gonadotrophins and are known to secrete various substances. Beneficial effects of cumulus cells on oocyte maturation have been reported for rabbit (Van Blerkom and McGaughey, 1978a,b), mouse (Cross and Brinster, 1970), rat (Vanderhyden and Armstrong, 1989), ewe (Staigmiller and Moor, 1984), cow (Fukui, 1990; Chian and Sirard, 1995), and human (Kennedy and Donahue, 1969). Some important changes occur during oocyte maturation including transcription, synthesis and post-translational modification of proteins, energy metabolism, membrane transport and cellular co-operation. However, the results of the present study indicate that cumulus-free immature human oocytes can also support early embryonic development following IVM in the proper culture medium. Culture conditions employed for IVM of mammalian oocytes significantly influence fertilization rates and subsequent embryonic development (Brackett and Zuelke, 1993). The current rationale for choosing a specific medium for IVM appears to stem largely from methods developed for the culture of other cell types. More research is necessary to determine the specific metabolites needed and optimal culture conditions required by maturing oocytes. Complex culture media, such as TCM-199, contain many components and are designed for somatic cell culture in vitro. The major beneficial components for the oocytes seem already to be present in TCM-199 (Bevers et al., 1997). However, there is a paucity of information about the effects of culture medium on maturational and developmental competence of immature oocytes. TCM-199 buffered with bicarbonate or HEPES (N-2- hydroxyethyperazine N-2-ethane sulphonic acid), supplemented with various sera, gonadotrophins and steroids have been most widely used in clinical studies for IVM of human oocytes. It is uncertain whether each factor contained in the complex culture medium supports IVM. It has been suggested that there may be significantly different culture requirements for human oocyte maturation, fertilization and embryonic development (Trounson et al., 1998, 2001). Animal studies have shown that the culture medium used for oocyte maturation can markedly affect subsequent embryonic development at several stages, including the blastocyst stage (Rose and Bavister, 1992; Brackett and Zuelke, 1993). In the present study, it was found that oocyte maturation and early embryonic development was profoundly affected by the oocyte maturation medium. Both the designed IVM-medium and TCM-199 were supplemented with 10% SSS, gonadotrophins and oestradiol. Besides many components, the major differences between the IVM-medium and TCM-199 are that the IVM-medium contained insulin, human transferrin (TF), fibroblast growth factor (FGF) and epidermal growth factor (EGF). Normally, insulin is involved in energy metabolism and amino acid transportation into cells. TF is a 75 kda glycoprotein containing 679 amino acids and two glycan chains. For each, the last residue is a sialic (N-acetyl neuraminic) acid, which can be hydrolysed by neuraminidase. TF not only transports iron in all extracellular fluid but also exerts many additional effects, including cell growth stimulation (Gross-Weege et al., 1986). Furthermore, using nested reverse transcription-polymerase chain reaction (RT- PCR), TF mrna expression has been demonstrated in granulosa cells of the human and mouse ovary but not in the oocyte (Briggs et al., 1999). Growth factors may act on membranes of oocytes to positively stimulate oocyte maturation (Maruo et al., 1993; Goud et al., 1998). However, the mechanisms behind the effect of insulin, growth factors and human TF on oocyte maturation are largely unknown and must be studied further. In addition, different energy substrates and nutrients can greatly influence oocyte meiotic and cytoplasmic maturation (Downs and Mastropolo, 1994; Avery et al., 1998; Rose-Hellekant et al., 1998). Although it has been reported that culture medium without glucose seems to result in higher maturation rates of cumulus-free human oocytes when compared with medium containing glucose, there are no convincing data to support this contention (Cekleniak et al., 2001). The metabolism of glucose, pyruvate, lactate and glutamine in oocytes seems to be related to the presence of cumulus cells (Leese et al., 1993). However, the details of energy metabolism in oocytes are still unclear. There is a need to explore more extensively energy metabolism in oocytes. Culture conditions in vitro can affect gene expression and embryo metabolism in mouse model system (Ho et al., 1994; Lane and Gardner, 1998). Embryo culture conditions can also affect the expression of imprinted genes (Surani, 2001; Judson et al., 2002). However, it is uncertain at present how the factors contained in the oocyte maturation medium affect embryo development and gene expression later. Recently, concerns have been expressed regarding the uncertainty of whether assisted reproductive technology increases the incidence of congenital malformations, or has subtle, longterm, and adverse effects on behaviour and cognition (Schultz and Williams, 2002). Therefore, it seems that further animal model studies are required for gene expression and imprinting of IVM oocytes when using insulin, growth factors and human TF in the culture medium. Preliminary studies with IVMmedium have already been carried out in animals (Chian, unpublished) using established methods (Chian et al., 2002). The addition of FSH to maturation medium does not significantly increase nuclear maturation rates in rat (Vanderhyden and Armstrong, 1989), monkey (Morgan et al., 1991) and human (Durinzi et al., 1997) oocytes but it appears significantly to increase fertilization rates of mouse oocytes 129

6 Figure 1. A blastocyst derived from GV-stage oocytes following 24 h of maturation. There was no morphological difference between oocytes matured in vivo or in vitro. Scale bar indicates 5 µm length. 130 (Schroeder et al., 1988; Morgan et al., 1991). In women undergoing oocyte retrieval during stimulated cycles, significantly higher FSH concentrations have been found in the fluid of follicles containing M-I and M-II oocytes compared with those containing GV-stage oocytes (Laufer et al., 1984). FSH is necessary for the induction of LH receptors in pre-ovulatory follicles. Addition of LH to culture medium induces GVBD in cultured intact follicles (Eppig, 1993). Although it is conventionally believed that there are no LH receptors on the oocytes (Dekel, 1988), it has been recently reported that mrna for FSH and LH receptors are present in mouse oocytes, zygotes and preimplantation embryos, indicating a potential role for the gonadotrophins in the modulation of meiotic resumption and completion of oocyte maturation (Patsoula et al., 2001). Therefore, the mechanism of gonadotrophin action on oocyte maturation needs to be carefully re-evaluated. De Vos et al. (1999) reported that ~4% of retrieved oocytes were at the M-I stage, 11% at the GV stage, and 85% at the M- II stage, and that 26.7% of the M-I oocytes extruded the first PB within 4 h of culture. The results of the present study indicate that approximately half of the M-I oocytes were mature at 6 h of culture in both maturation media (52.0 versus 46.1%), most M-I oocytes were mature at 24 h of culture, and very few M-I oocytes matured after further culture to 48 h. Although there were no differences in rates of cleavage and embryos developing to the 8-cell stage between both media when the M-I oocytes were matured 6 h and 24 h, blastocyst formation rates were significantly different (P < 0.01) in IVM- medium when the M-I oocytes were matured 6 h (24.0%) compared with 24 h (9.7%). However, there were no such differences in the rates of blastocyst formation between 6 h (7.9%) and 24 h (7.1%) for TCM-199. This indicates that the ability of early embryonic development is different between M-I oocytes matured 6 h and 24 h and that IVM-medium can improve the developmental competence of the M-I stage oocytes when compared with TCM-199. It has been reported that there are differences in molecular and structural characteristics between GV-stage oocytes retrieved from ovulation induction (HMG/HCG) and unstimulated ovaries (Chian et al., 1997). Therefore, it appears that the initiation of the maturation process in vivo is important for the oocytes to acquire full developmental competence (Chian et al., 1999a, 2000). De Vos et al. (1999) have reported that the implantation rate of embryos resulting from IVM M-I stage oocytes is low (5.3%) compared with the overall implantation rates obtained with embryos resulting from M-II stage oocytes at retrieval (~20%). However, the present study indicates that similar embryo quality was obtained from M-I stage oocytes matured in vitro and in vivo in new IVM-medium. Therefore, it is possible to use these immature oocytes clinically, because it has been reported that healthy infants were born following transfer of those embryos derived from IVM oocytes (Edirisinghe et al., 1997; Jaroudi et al., 1999). Animal studies indicate that denuded GV-stage oocytes can be matured in vitro and undergo blastocyst formation (Geshi et al., 2000). Although it has been suggested that cumulus cells

7 have beneficial effects on oocyte cytoplasmic maturation (Mattioli et al., 1988; Chian et al., 1994), the results of the present study indicate that the role of cumulus cells on oocyte maturation seems to be less important if the oocyte was matured in the IVM-medium. Most oocytes were mature following 24 h of culture when the GV-stage oocytes cultured in vitro. Also, M-I oocyte maturation rates were significantly different between the IVM-medium and TCM-199 at 24 h and 48 h of culture. This suggests that the IVM-medium has apparent benefit for oocyte maturation compared with TCM-199. Although the overall rates of fertilization and cleavage were not different between GV-stage oocytes matured in the IVMmedium and TCM-199, the rates of embryo development to the 8-cell and blastocyst stage were higher (P < 0.01) in the IVM-medium compared with TCM-199. In addition, fertilization rates were not different in the IVM-medium when GV-stage oocytes were matured after 24 h (88.9%) and 48 h (81.5%) of culture. However, fertilization rates were significantly different (P < 0.05) in TCM-199 when these oocytes were matured at 24 h (83.3%) and 48 h (69.6%). It is important to mention that spermatozoa for ICSI in this study were obtained on the day of oocyte retrieval and kept at room temperature for a further 6 h, 24 h or 48 h for later use. Nevertheless, there appears to be no obvious detrimental effect of using spermatozoa to achieve fertilization until 48 h of culture at room temperature, because fertilization rates were not decreased following 48 h of culture in the IVM-medium group. Although the cleavage rates were not different between the IVM-medium and TCM-199 when the GV-stage oocytes were matured for 24 h (100.0 versus 96.7%) and 48 h (100.0 versus 93.8%), there were significant differences (P < 0.01) in rates of embryo development to 8-cell (75.0 versus 51.7%) and blastocyst stage (18.8 versus 0.0%) between these two media. Furthermore, this indicates that embryos developed to the 8- cell and blastocyst stages in both media at significantly higher rates (P < 0.01) when the GV-stage oocytes matured at 24 h (8- cell: 75.0 and 51.7%; blastocyst: 18.8 and 0.0% respectively) compared with oocytes matured at 48 h (8-cell: 22.7 and 20.0%; blastocyst: 0.0 and 0.0% respectively). These results suggest that there are differences in early embryonic development between oocytes that matured after 24 h and 48 h. Moreover, similar morphology of blastocysts was obtained from the GV-stage oocytes (Figure 1) and M-I stage oocytes matured in vitro compared with in-vivo matured oocytes. Taken together, this indicates that the IVM-medium is a more suitable medium for human oocyte maturation in vitro. Therefore, it is important to mature and inseminate these oocytes retrieved from stimulated cycles for embryo transfer, because relatively reasonable blastocyst formation rates are obtained following embryo culture in vitro. In conclusion, the present study indicates that the IVMmedium is a better medium for in-vitro maturation of human oocytes and that IVM cumulus-free oocytes can develop to the blastocyst stage in vitro. References Avery B, Bavister BD, Greve T 1998 Development of bovine oocytes, in vitro matured in a chemically defined protein-free medium, supplemented with different amino acid formulations. Theriogenology 49, 306. 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