Oocyte diameter as a predictor of fertilization and embryo quality in assisted reproduction cycles Gustavo Salata Rom~ao, Ph.D., Maria Cristina Picinato Medeiros Araujo, B.S., Anderson Sanches de Melo, M.D., Paula Andrea de Albuquerque Salles Navarro, Ph.D., Rui Alberto Ferriani, Ph.D., and Rosana Maria dos Reis, Ph.D. Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil Objective: To assess the impact of the mean oocyte diameter (MOD) on occurrence of fertilization and embryo quality in assisted reproduction cycles. Design: Prospective observational study. Setting: Sector of Human Reproduction of the University Hospital, Faculty of Medicine of Ribeir~ao Preto, University of S~ao Paulo (HCFMRP-USP). Patient(s): Thirty-five women undergoing intracytoplasmic sperm injection (ICSI) at the University Hospital of Ribeir~ao Preto from May to October 2007. Intervention(s): MOD assessment. Main Outcome Measure(s): Occurrence of fertilization and qualitative embryo classification on 2nd and 3rd day after ICSI. Result(s): We divided 160 metaphase II oocytes according to MOD into groups A (MOD below the 25th percentile), B (MOD between 25th and 75th percentile), and C (MOD above the 75th percentile). There was no statistically significant association between MOD and the occurrence of fertilization or the qualitative embryo classification on days 2 and 3. There was no statistically significant difference between groups regarding number of cells or the qualitative embryo classification on days 2 and 3. Conclusion(s): The MOD of mature oocytes does not seem to be related to the occurrence of fertilization or to the developmental quality of human embryos on days 2 and 3 after ICSI. (Fertil Steril Ò 2010;93:621 5. Ó2010 by American Society for Reproductive Medicine.) Key Words: Mean oocyte diameter, ICSI, fertilization, embryo quality, assisted reproduction Received June 6, 2008; revised December 12, 2008; accepted December 19, 2008; published online May 6, 2009. G.S.R. has nothing to disclose. M.C.P.M.A. has nothing to disclose. A.S. de M. has nothing to disclose. P.A. de A.S.N. has nothing to disclose. R.A.F. has nothing to disclose. R.M. dos R. has nothing to disclose. Supported by the Laboratory of Human Reproduction, Hospital of Clinics of the Faculty of Medicine of Ribeir~ao Preto, University of S~ao Paulo. Reprint requests: Gustavo Salata Rom~ao, Ph.D., Department of Medicine, Federal University of S~ao Carlos, Rodovia Washington Luıs, km 235, SP-310, S~ao Carlos, S~ao Paulo, Brazil 13565-905 (FAX: 55-16-36106775; E-mail: gsalata@uol.com.br). During growth, mammalian oocytes stagnate in prophase I of the first meiotic division in the diplotene I stage, also known as the germinal vesical stage (GV). Fully grown oocytes can resume the division process and reach metaphase II both in vivo after the luteinizing hormone (LH) peak and in vitro after their release from the follicular units. In contrast, oocytes that have not completed their full growth cannot resume or complete meiosis (1). Competence for meiotic resumption is believed to be acquired during the phase of oocyte growth, when the processes of synthesis and storage of proteins and of ribosomal and heterogeneous RNA take place. These events lead not only to nuclear maturation but also to the cytoplasmic modifications necessary for the development of the future embryo. These modifications include [1] protein and RNA storage, [2] development of mechanisms that regulate intracellular calcium transport, [3] redistribution of cytoplasmic organelles, and [4] modulation of kinase protein activity (2). Some of the factors that determine the acquisition of meiotic competence are still controversial. Schultz and Wassarman (3) suggested that the acquisition of competence and the maturation of rat oocytes occur in two distinct stages. Initially, the oocytes acquire the ability to resume meiosis, and later they acquire the competence for meiotic maturation. These investigators observed that the growth of rat oocytes is accompanied by significant changes in the types of proteins synthesized in each phase. Additionally, oocyte metabolism seems to be modified during growth, in agreement with findings of an increase in the total activity of the enzymes lactate dehydrogenase and glucose-6-phosphate dehydrogenase. It has been demonstrated in many species that a critical oocyte size is necessary for the resumption of meiosis and maturation. According to Otoi et al. (4), canine oocytes acquire meiotic competence when they reach a diameter of more than 120 mm, although the ability of sperm penetration does not depend on oocyte diameter. In a study of oocytes from goats aged 1 to 2 months (prepubertal phase) with in vitro maturation (IVM) and fertilization (IVF), Anguita et al. (5) demonstrated that oocyte diameter was positively correlated with the percentage of 0015-0282/10/$36.00 Fertility and Sterility â Vol. 93, No. 2, January 15, 2010 621 doi:10.1016/j.fertnstert.2008.12.124 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
oocytes that reached metaphase II and with the percentage of fertilized oocytes that developed beyond the blastocyst stage. Oocyte diameter was also positively correlated with the intracellular amount of the p34 protein and with the degree of activity of maturation promoting factor. The highest percentage of blastocysts (12%) was obtained from oocytes with a diameter of more than 135 mm on the day of retrieval, which originated from follicles measuring 2 to 3 mm in diameter. The investigators concluded that the impaired development of embryos from oocytes of prepubertal goats may be related to oocyte diameter more than to the physiologic condition of the animals. Goat oocytes acquire meiotic competence when their diameter is more than 136 mm, both in adult (6) and prepubertal (7) females. Although several studies have evaluated the relation between oocyte diameter and the capacity for maturation and embryo development in different animal species, little information on this topic is available about human oocytes. Griffin et al. (8) carried out a comparative analysis of follicle and oocyte diameter in four different species rats, mice, pigs, and humans. The investigators described in detail for the first time the differences between experimental models of folliculogenesis and identified variations in the histologic pattern of the ovarian follicles among the four species studied. Their conclusion was that the relation between oocyte diameter and degree of maturation is species-specific and should not be generalized or extrapolated to other animal models. In a study using oocytes from women who were undergoing surgical procedures for nonovarian disorders, Durinzi et al. (9) demonstrated that the ability of human oocytes from nonstimulated cycles to resume meiosis and complete IVM was correlated with oocyte diameter at the time of retrieval. Thus, the maturation potential of immature oocytes must be related to the level of growth reached by the oocytes inside the follicles before the detachment provoked by ovulation. The specificity of and the mechanisms involved in the relationship of metabolism, protein synthesis, and cytoplasm maturation with meiotic competence and embryo development in human oocytes are still under investigation. In a recent study, Cavilla et al. (10) used IVM to evaluate oocytes obtained from women with polycystic ovary syndrome (POS) and demonstrated that larger oocytes had a better probability of maturation. In the same study, 20 mature oocytes retrieved from women undergoing intracytoplasmic sperm injection (ICSI) were evaluated, and no significant relation was observed between oocyte diameter and probability of fertilization or embryo cleavage, although a small number of oocytes was evaluated. The use of noninvasive measures such as mean oocyte diameter (MOD) for oocyte evaluation to predict the results of ICSI is of great clinical importance, permitting a better selection of the oocytes for insemination and better control of the quantity and quality of the embryos obtained, reducing to a minimum the ethical problems related to embryo freezing. Our main objective was to assess the impact of MOD on fertilization and embryo quality in assisted reproduction cycles. We also investigated the relations among MOD, maternal age, and basal follicle-stimulating hormone (FSH) levels. MATERIALS AND METHODS The study was conducted in the Sector of Human Reproduction of the University Hospital, Faculty of Medicine of Ribeir~ao Preto, University of S~ao Paulo (HCFMRP-USP), and the investigation project was submitted and approved by our institutional review board. Thirty-five women who underwent ovarian stimulation and oocyte retrieval for assisted fertilization at HCFMRP-USP were selected between May and October 2007 after giving written, informed consent to participate in the study. The age of the selected patients ranged from 24 to 42 years (mean þ standard deviation [SD]: 34.2 4.11), and their basal FSH levels ranged from 2.4 to 17.8 IU/L (mean þ SD: 7.16 3.82 IU/L). Patients with suspected endometriosis or confirmed disease by videolaparoscopy and patients whose sexual partners presented with severely compromised seminal parameters (sperm morphology of less than 8% in the spermiogram and/or sperm concentration of less than 2 million in the seminal fluid recovered) were excluded from the study. The investigation of conjugal infertility revealed the presence of male factor infertility in 25 couples (77.1%) (17 cases of asthenoteratozoospermia, four of asthenozoospermia, three of mild oligozoospermia, and one of teratozoospermia), of tubal factor infertility in nine couples (25.7%), ovulatory factor in eight (22.9%), and uterine factor in one (2.9%), with the presence of more than one infertility factor observed in nine couples. Four of the patients in the study (11.4%) were trying assisted reproduction for the first time, 16 (45.7%) for the second time, eight (22.9%) for the third time, five (14.3%) for the fourth time, and two (5.7%) for the fifth time. Protocol of Assisted Fertilization The menstrual period was programmed with the use of combined oral contraceptives started during the preceding cycle and discontinued 5 days before the date scheduled for the beginning of ovarian stimulation. For pituitary suppression, leuprolide acetate (Lupron, Abbott Pharmaceuticals, Abbott Park, IL; or Reliser, Serono, S~ao Paulo, Brazil; 10 IU/day by the subcutaneous route) was administered to nine patients (25.7%) and nafarelin (Synarel; Pharmacia, S~ao Paulo, Brazil; 400 mg/day by the nasal route) was administered to 26 patients (74.3%) starting 10 days before the programmed menstruation and maintained until the day of human chorionic gonadotropin (hcg) administration. Once ovarian suppression was confirmed by transvaginal ultrasound from the first to the third day of the menstrual cycle, controlled ovarian stimulation was started with recombinant FSH (Puregon, Organon, S~ao Paulo, Brazil; or Gonal-F, Serono) at the dose of 622 Rom~ao et al. Oocyte diameter and embryo quality Vol. 93, No. 2, January 15, 2010
200 to 450 IU/day on the first 5 days, adjusted according to the ovarian response starting on the six day of stimulation. Urinary human chorionic gonadotropin (Profasi; Serono), 10,000 IU, was administered intramuscularly, or recombinant hcg (Ovidrel, Serono), 250 mg, was administered subcutaneously when two or more follicles R18 mm in mean diameter were present. Oocytes were retrieved by puncture of the vaginal sac guided by transvaginal ultrasound 34 to 36 hours after hcg administration. The follicular content was aspirated under negative pressure of 100 mm Hg into Falcon tubes and kept at 37 C. The cumulus oocyte complexes (COC) were separated from the follicular fluid under a stereomicroscope, washed in human tubal fluid (HTF) culture medium modified with HEPES (Irvine Scientific, Santa Ana, CA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific), and transferred to culture plates previously incubated at 37 C in the presence of 5% CO 2 in air, containing HTF culture medium (Irvine Scientific) supplemented with 10% SSS. After 2 to 5 hours of incubation in this culture medium, cumulus cells were removed by exposing the COC to hyaluronidase (H-4272; Sigma-Aldrich, St. Louis, MO) at the concentration of 80 IU/mL, in HTF-HEPES supplemented with 10% SSS for 30 seconds, followed by mechanical removal with glass pipettes. Semen samples were obtained immediately after oocyte retrieval and were manipulated and analyzed for the ICSI procedure according to recommended procedures for male infertility. Before ICSI, the denuded oocytes were placed in 5-mL microdroplets of HTF-HEPES supplemented with 10% SSS on Falcon plates and were observed under an inverted microscope with Hoffman optic contrast at 400 magnification. The MOD was calculated immediately after oocyte denudation. We performed ICSI as described previously elsewhere, 36 to 41 hours after hcg administration. After ICSI, the oocytes were incubated separately in 25-mL microdroplets of HTF þ 10% SSS at 37 C in the presence of 5% CO 2 in air. Fertilization was determined 18 to 20 hours after ICSI on the basis of the presence of two pronuclei and of two polar corpuscles. Oocytes presenting one, three, or more pronuclei were classified as abnormal; they were considered inviable and later discarded. Cleavage was verified about 24 hours after fertilization by the observation of the first cell division. Embryo quality was assessed on the basis of variables such as percentage of cytoplasmic fragmentation, symmetry, and number of blastomeres (11). Embryos with four cells, symmetrical blastomeres, and no fragmentation on the second day after retrieval (D2) and embryos with eight or more cells and 20% fragmentation or less on the third day after retrieval (D3) were considered to be of good quality. Embryo transfer was performed on the third day after retrieval, and the luteal phase was supplemented with micronized progesterone administered orally or vaginally up to the 12th week of pregnancy or until the occurrence of menstruation. Pregnancy was confirmed by serum determination of b-hcg on the 14th day after transfer. Mean Oocyte Diameter (MOD) The MOD was determined on the day of retrieval immediately after the removal of cumulus cells using OCTAX Eye- Ware software (Universal Imaging Corp., Boston, MA). Two perpendicular measurements were made for each oocyte, excluding the zona pellucida. The maximum value obtained in each parallel measurement was used as the reference value. The MOD was obtained by the arithmetic mean of the two perpendicular measurements. Statistical Analysis The correlations among MOD values, maternal age, basal FSH levels, and number of cells on D2 and D3 were determined by the Pearson coefficient. The correlations among MOD values, occurrence of fertilization, number of cells, and qualitative embryo classification on D2 and D3 were determined by analysis of the receiver operating characteristic (ROC) curve. For further analysis of these parameters, oocytes were divided according to MOD into groups: group A, MOD below the 25th percentile; group B, MOD between 25th and 75th percentile; and group C, MOD above the 75th percentile. Comparisons among groups were performed by using the chi-square test. P<.05 was considered statistically significant. RESULTS A total of 160 metaphase II oocytes was obtained in 35 cycles from 35 women included in the study. The number of oocytes retrieved per patient ranged from one to 10 (mean SD: 4.61 2.24). The oocytes retrieved had a MOD of 104.03 to 121.78 mm, with a median of 111.89 mm and a mean SD of 112.21 3.31 mm. The MOD was not statistically significantly correlated with maternal age (r ¼ 0.1773; P¼.3082) or with basal FSH levels (r ¼ 0.3798; P¼.0611). Also, no correlation was observed between maternal age and basal FSH levels (r ¼ 0.3548; P¼.0818) in the patients studied. There was no statistically significant association between MOD and the occurrence of fertilization (P¼.5044), or qualitative embryo classification on D2 and D3 (P¼.2777 and 0.5450, respectively). There was no statistically significant association between MOD and the number of cells on D2 (r ¼ 0.05043; P¼.5975) or D3 (r ¼ 0.1574; P¼.1899). The comparisons of groups A (MOD <109.92 mm), B (MOD between 109.92 mm and 114.26 mm), and C (MOD > 114.26 mm) are shown in Table 1. The fertilization rates for groups A, B, and C were 82.5%, 68.75%, and 75%, respectively, with no statistically significant difference (P¼.2662). Fertility and Sterility â 623
TABLE 1 Comparison of the embryonic parameters of groups A, B, and C by the chi-square test. Day Parameter Group A Group B Group C P value: A versus B versus C 1 Number of oocytes 40 80 40 Mean oocyte diameter (mm) <109.92 a 109.92 a 14.26 b >114.26 b Fertilized 33 (82.5%) 55 (68.75%) 30 (75%).2662 2 R4 cells 12 (38.7%) 31 (56.4%) 15 (50%).2906 Good quality 16 (51.6%) 22 (42.3%) 10 (34.5%).4051 3 R8 cells 12 (57.1%) 16 (53.3%) 24 (77.4%).117 Good quality 10 (47.6%) 9 (31%) 11 (52.4%).2689 a 25th percentile. b 75th percentile. Rom~ao. Oocyte diameter and embryo quality. Fertil Steril 2010. There was no statistically significant difference between the groups regarding the number of cells (P¼.2906) or the qualitative embryo classification on D2 (P¼.4051) or D3 (P¼.117 and.2689, respectively). Among the 35 cycles analyzed, 32 embryo transfers were performed (91.4%), with a mean number of embryos per embryo transfer of two. DISCUSSION Studies on different species have elucidated the relation between oocyte diameter and competence for maturation and embryo development. Eppig and Schroeder (12) demonstrated in rats that the successive stages of meiotic maturation and embryogenesis were related to oocyte age and size. According to these findings, although the oocytes with a diameter of more than 60 mm from mice of younger than 13 days old were able to progress to germinal vesicle breakdown in culture, larger oocytes obtained from mice older than 15 days were more likely to complete metaphase II (MII) in culture. Hirao et al. (13) confirmed that the minimum oocyte diameter needed for meiotic maturation in mice is 60 mm, even for in vitro growth. Similar evidence was obtained with rats (14) and pigs (15), for which the minimum diameters needed for germinal vesicle breakdown were 55 and 90 mm, respectively. The continued transcription observed in bovine oocytes indicates that the maternal RNA complement, necessary for early embryo development, may still be incomplete, suggesting a possible mechanism for the association between oocyte growth and acquisition of the capacity for maturation (16). The capacity for cleavage after maturation and in vitro insemination is also related to the increase in oocyte age and diameter. In mouse oocytes, the competence for maturation was found to be acquired as the diameter increased from 65 to 75 mm, and the modifications occurring in the final stages of oocyte growth were critical to reach full maturation (17). However, the relation between oocyte diameter and degree of maturation may differ significantly between animal species. In the oocytes of Rhesus monkeys the oocyte diameter remains relatively stable as competence for germinal vesicle breakdown (GVBD) is acquired (18). Although several studies have evaluated the relation between oocyte diameter and the ability for maturation and embryo development in different animal species, little information is available about this topic for human oocytes despite the accessibility of this data during assisted fertilization procedures and IVM. In a study on humans, 49 oocytes were obtained from six women aged 25 to 39 years (a mean of 4 to 11 oocytes per patient) who were undergoing surgery for ovarian disease (9). The oocytes were evaluated for diameter (mm) and degree of maturation according to parameters established in the literature (GV, presence of the germinal vesicle; MI, rupture of the germinal vesicle; or MII, extrusion of the first polar corpuscle). Only one third of the oocytes with a diameter of less than 105 mm were able to resume meiosis or to mature, as opposed to more than two thirds of the oocytes with a diameter of more than 105 mm. Evaluation of oocyte diameter in the final stage of maturation revealed that, among oocytes with a diameter between 116 and 125 mm, 60% were in MII as opposed to only 44% of those with a diameter of less than 115 mm. It was also revealed that, among oocytes in the GV stage, those with a diameter between 116 and 125 mm progressed more rapidly to GV rupture and extrusion of the first polar corpuscle compared with oocytes with a diameter of less than 115 mm. These results support the idea that human oocytes, as is the case for other species, present a size-dependent latency to progress in the division process until complete maturation. Cavilla et al. (19), using computerized micrographic images of human oocytes in culture for IVM, confirmed the findings of Durinzi et al. (9), which had suggested the potential of noninvasive oocyte measurements as predictors of subsequent ability to develop. Cavilla et al. (10) also evaluated via IVM 86 oocytes obtained from women with polycystic 624 Rom~ao et al. Oocyte diameter and embryo quality Vol. 93, No. 2, January 15, 2010
ovary syndrome. The diameter of the 28 oocytes that survived and progressed in IVM ranged from 103 and 121 mm, with a median of 108 mm. Of these, 10 progressed to fertilization (presenting two pronuclei) after ICSI, with a diameter ranging from 105 to 121 mm (median: 112.5 mm). The investigators concluded that oocytes that were larger when collected for IVM had a greater probability to mature. In the same study, 20 mature oocytes retrieved for ICSI from 17 women aged 22 to 35 years (mean: 28.1 years) were evaluated. The MOD measured after the removal of cumulus cells and before ICSI ranged from 112 to 119 mm, with a mean of 116 mm. In contrast to the results for IVM, no statistically significant relation was observed between MOD and probability of fertilization or embryo cleavage, probably because of the small number of oocytes observed. The mean oocyte diameter observed in our study (112.21 mm) was similar to the value obtained by Cavilla et al. (10) for oocytes inseminated after IVM (112.5 mm) and was close to the value obtained for IVM oocytes submitted to ICSI (116 mm). As also reported in previous studies (10), our study found no statistically significant association between oocyte diameter and the occurrence of fertilization, number of cells, or embryo quality at D2 and D3. These results may have been due to the fact that among the 160 evaluated oocytes, only one had a MOD value below the cutoff level (105 mm) for which statistically significant differences were observed regarding the ability to resume meiosis or to obtain maturation (9). In comparison with the study by Cavilla et al. (10) in which the MOD of oocytes matured in vitro for ICSI ranged from 109 to 118 mm, we observed a greater oscillation of this variable (104.03 to 121.78 mm), which could be attributed in part to the different number of oocytes evaluated by Cavilla et al. (10) (n ¼ 20) and by us (n ¼ 160). In addition, Cavilla et al. (10) obtained median MOD values of 115 mm for oocytes that survived but were not matured in vitro, 114 mm for IVM oocytes, and 113 mm for fertilized oocytes after ICSI, as opposed to the median value of 111.89 mm obtained in our study. During the growth phase, the diameter of human oocytes increases from 30 to 110 mm over a period of at least 8 weeks (20). The diameter of IVM oocytes, excluding the zona pellucida, usually is 110 to 120 mm, while the thickness of the zona pellucida is 15 to 20 mm (11). However, as also concluded by Griffin et al. (8), it is possible that for mature oocytes the relations between oocyte diameter and degree of maturation are species specific and should not be generalized or extrapolated to other animal models. We conclude that the MOD of mature oocytes does not seem to be related to the occurrence of fertilization or to the developmental quality of human embryos on days 2 and 3 after ICSI. Acknowledgments: The authors thank Maria Albina Verceze Bortolieiro, Maria Auxiliadora Padua, Maria Aparecida Carneiro Vasconcelos, Marilda Hatsumi Yamada Dantas, Roberta Cristina Giorgenon, and Sandra Aparecida Cavichollo Vianna. REFERENCES 1. Edwards R. Maturation in vitro of human oocytes. Lancet 1965;6:926 9. 2. Crozet N, Motlik J, Sz oll osi D. Nucleolar fine structure and RNA synthesis in porcine oocytes during early stages of antrum formation. Biol Cell 1981;41:35 42. 3. Schultz RM, Wassarman PM. Biochemical studies of mammalian oogenesis: protein synthesis during oocyte growth and meiotic maturation in the mouse. J Cell Sci 1977;24:167 94. 4. Otoi T, Fujii M, Tanaka M, Ooka A, Suzuki T. Canine oocyte diameter in relation to meiotic competence and sperm penetration. Theriogenology 2000;54:535 42. 5. Anguita B, Jimenez-Macedo AR, Izquierdo D, Mogas T, Paramio MT. Effect of oocyte diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity in prepubertal goat oocytes. Theriogenology 2007;67:526 36. 6. Smedt De, Crozet N, Gall L. Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte. J Exp Zoo 1994;269:128 39. 7. Martino A, Mogas T, Palomo M, Paramio M. Meiotic competence of prepubertal goat oocytes. Theriogenology 1994;41:969 80. 8. Griffin J, Emery BR, Huang I, Peterson CM, Carrell DT. Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human). J Exp Clin Assist Reprod 2006;3:2 7. 9. Durinzi KL, Saniga EM, Lanzerdorf SE. The relationship between size and maturation in vitro in the unstimulated human oocyte. Fertil Steril 1995;63:404 6. 10. Cavilla JL, Kenedy CR, Byskov AG, Hartshorne GM. Human immature oocytes grow during culture for IVM. Hum Reprod 2008;23:37 45. 11. Veeck LL. Abnormal morphology of the human oocyte and conceptus. In: An atlas of human gametes and conceptuses. Carnforth, Lancashire, United Kindom: Parthenon, 1999:57 68. 12. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation and fertilization invitro. Biol Reprod 1989;41:268 76. 13. Hirao Y, Miyano T, Kato S. Acquisition of maturational competence in in vitro grown mouse oocytes. J Exp Zool 1993;267:543 7. 14. Daniel SAJ, Armstrong DT, Gore-Langton RE. Growth and development of rat oocytes in vitro. Gamete Res 1989;24:109 21. 15. Hirao Y, Nagai T, Kubo M, Miyano T, Miyake M, Kato S. In vitro growth and maturation of pig oocytes. J Reprod Fertil 1994;100:333 9. 16. Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995;42: 437 42. 17. Bao S, Obata Y, Carroll J, Domeki I, Kono T. Epigenetic modifications necessary for normal development are established during oocyte growth in mice. Biol Reprod 2000;62:616 21. 18. Schramm RD, Tennier MT, Boatman DE, Bavister BD. Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in non-stimulated rhesus monkeys. Biol Reprod 1993;48:349 56. 19. Cavilla JL, Kennedy CR, Baltsen M, Klentzeris LD, Byskov AG, Hartshorne GM. The effects of meiosis activating sterol on in vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries. Hum Reprod 2001;16:547 55. 20. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986;2:81 7. Fertility and Sterility â 625