In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles

FERTILITY AND STERILITY VOL. 74, NO. 6, DECEMBER 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles Byung-Ki Kim, D.V.M., Ph.D., a Sang-Chan Lee, M.D., Ph.D., b Keun-Joo Kim, M.Sc., a Chang-Hee Han, Ph.D., a and Jong-Heung Kim, Ph.D. b Dongeui University and Saewha Women s Clinic, Pusan, Korea Objective: To evaluate whether germinal vesicle (GV) oocytes from stimulated cycles can be a source of embryos. Design: In vitro model study. Setting: Specialized laboratory of women s clinic. Patient(s): Women in whom oocytes were retrieved at the GV stage. Intervention(s): After culture of GV oocytes in the modified TLP medium with human follicular fluid, oocytes that reached the metaphase II stage underwent ICSI. Potential of fertilization and subsequent cleavage of in vitro-matured oocytes was compared with that of an in vivo-matured control. Main Outcome Measure(s): Maturation rate, rate of pronuclei formation, and developmental activity. Result(s): The maturation rate of GV oocytes from follicles primed with gonadotropin was not affected by the presence or absence of cumulus. However, the maturation was more synchronous in oocytes with cumulus than in those without cumulus. Proportions of oocytes with two pronuclei and embryos cleaved to the 16-cell stage after ICSI were significantly lower in the oocytes matured in vitro than in the oocytes matured in vivo. Conclusion(s): Human GV oocytes from stimulated ovaries can be matured, fertilized, and developed in vitro. Production of embryos from GV oocytes will increase the opportunity for achieving pregnancy. (Fertil Steril 2000;74:1153 8. 2000 by American Society for Reproductive Medicine.) Key Words: Germinal vesicle, in vitro maturation, ICSI, pronuclei Received March 14, 2000; revised and accepted June 28, 2000. Reprint requests: Byung-Ki Kim, D.V.M., Ph.D., Department of Biology, Dongeui University, San 24 Kayadong, Pusan 614-714, Korea (FAX: 051-890-1521; E-mail: bkkim@hyomin.dongeui.ac.kr). a Department of Biology, Dongeui University. b Saewha Women s Clinic. 0015-0282/00/$20.00 PII S0015-0282(00)01617-4 In most mammals, including humans, the first meiosis of the oocytes is initiated during fetal life and arrested at the diplotene stage of the prophase before birth. Oocyte maturation, characterized by germinal vesicle (GV) breakdown, formation of the first meiotic spindle (metaphase I), expulsion of the first polar body, and arrest in metaphase of the second meiotic division (metaphase II), occurs in preovulatory follicles in response to the surge of gonadotropin and leads to an ovulated oocyte. Most mammalian oocytes released from the follicular environment are able to undergo the meiotic maturation to metaphase II spontaneously when they are placed in a suitable culture medium (1). In rodents, spontaneous oocyte maturation is achieved in 95% of oocytes removed from their follicular environment in gonadotropin-primed, as well as unprimed, animals (2). In humans, however, spontaneous maturation in vitro is achieved in only 30% 50% of oocytes (1, 3). The production of embryos by in vitro maturation, fertilization, and culture of oocytes collected from ovarian follicles during the stimulated cycle is performed in humans for therapeutic reasons. For production of embryos, ovarian stimulation is usually required and is carried out by using exogenous gonadotropin, such as hmg, FSH, and hcg. It is well known that hcg is essential for the final maturation of oocytes during ovarian stimulation. After ovarian stimulation with hmg or FSH and hcg, approximately 15% of oocytes are usually found in the GV or metaphase I stage (4). However, a conventional in vitro fertilization of the human oocytes matured in vitro often results in a poor outcome. Veek et al. (5) reported two pregnancies by the transfer of in 1153

vitro-matured oocytes from stimulated cycles in their IVF program. Because of the high fertilization and pregnancy rates, intracytoplasmic sperm injection (ICSI) may be useful in treating male factor infertility (6). On the basis of these results, the aim of the present study was to identify whether immature oocytes from stimulated cycles can be matured in vitro and whether the fertilization rate of in vitro-matured oocytes can be increased by the use of ICSI. MATERIALS AND METHODS Patients and Oocyte Recovery This study was approved by the Ethical Committee of the Korean Association of Obstetricians and Gynecologists and conducted in accordance with the Helsinki Declaration of 1975 on human experimentation. Between April 1998 and March 1999, all women who underwent embryo transfer at the Human Infertility and Genetics Institute of the Saewha Women s Clinic were included in the study. GV oocytes used in this study were collected from patients having an IVF rate of 60% of metaphase II oocytes in the current treatment cycle. The protocol for ovulation induction was identical in all patients. Pituitary desensitization was achieved by gonadotropin-releasing hormone agonist (Superfact; Hoechst, Germany) administration during the luteal phase. Serum estradiol levels below 50 pg/ml and negative vaginal ultrasound scans were used to define ovarian quiescence. FSH and hmg for ovarian stimulation were used when ovarian quiescence was achieved. On days 1 and 2 of ovarian stimulation, 2 ampules/day of hmg were administered together with two ampules of FSH. On days 3 and 4 of ovarian stimulation, 3 ampules/day of hmg were administrated to each patient. Beginning on day 4, hmg was administered on an individual basis according to the serum estradiol levels and transvaginal ovarian ultrasound scans. The criteria for hcg administration (10,000 IU) were the presence of three or more follicles with greatest diameter 16 mm and serum estradiol levels higher than 800 pg/ml. Oocytes were aspirated at 36 hours after hcg administration. Identification of GV oocytes was based on the method used by Farhi et al. (7) with some modification. The contents of the aspirated follicle were poured into a 60-mm dish. The dish was tilted under the stereoscope to allow better observation of the oocyte itself. GV oocytes under these conditions were identified by the typical appearance of a brown spot in the cytoplasm. In Vitro Maturation, Fertilization, and Cleavage GV oocytes were matured for 44 hours before insemination. Oocytes were scored for presence of a GV, absence of a GV or polar body (metaphase I), or extrusion of the first polar body (metaphase II) (8). The culture medium for oocyte maturation and development was modified from Tyrode s solution (9), which was composed of 110 mm of NaCl, 3.2 mm of KCl, 2.25 mm of CaCl 2, 0.5 mm of MgCl 2, 25.0 mm of NaHCO 3, 0.5 mm of sodium pyruvate, and 10.0 mm of sodium lactate and which was designated as modified (m) TLP supplemented with 0.2 mm of taurine, 1.0 mm of glutamine, 2.22 mm of MEM amino acid, 10% heat-inactivated human follicular fluid, and 31 g of sodium penicillin G/mL at ph 7.4 and 280 285 mosm. In all experiments, the oocytes were cultured at 37 C in an atmosphere of 5% CO 2 95% air with high humidity. Only oocytes that reached the metaphase II stage after in vitro maturation were used for the ICSI procedure. The sperm samples were prepared mainly by centrifugation through Percoll gradients (45%/70%/80%/90%). Spermatozoa were injected according to the method of Van Steirteghem et al. (4). In the ICSI cycles, the oocytes were denuded by using hyaluronidase (80 IU/mL); thereafter, one spermatozoon was deposited in the ooplasm of each oocyte. All oocytes were screened for pronuclei, and further cleavage was analyzed. Follicular Fluid Preparation Follicular fluid was obtained from women whose cycles were stimulated and who attended the IVF program of the Saewha Human Infertility and Genetics Institute. Oocyte retrieval and follicular fluid collection were carried out under ultrasound-guided aspiration. The total fluid content of each follicle was then aspirated and sent to the laboratory where the oocytes were collected. Follicular fluids were labeled and centrifuged at 300 g for 10 minutes to remove blood and granulosa cells. After the inactivation of supernatant at 56 C for 30 minutes, follicular fluid was filtered with a 0.22- m filter and stored at 20 C before use. Statistical Analysis Statistical analysis was performed with a standard computerized statistics program using 2. P.05 was considered statistically significant. RESULTS The number and the maturity of oocytes retrieved from stimulated cycles are shown in Table 1. From 135 stimulated cycles, the mean and total number of oocytes recovered were 10.3 4.5 and 1,367, respectively. When oocytes were collected, 78.5% (1,073 of 1,367) of the oocytes were matured and 12.3% (168 of 1,367) of the oocytes remained in a GV stage. Eighty immature oocyte-cumulus complexes (OCCs) were removed from cumulus through pipettes of suitable diameter to determine whether cumulus affects the in vitro maturation. The oocytes, together with the cumulus cells, including those removed by pipetting, were matured for 44 hours before insemination. Data for maturation based on 1154 Kim et al. Maturation of human GV oocytes and ICSI Vol. 74, No. 6, December 2000

TABLE 1 Number and maturity of oocytes retrieved from the stimulated cycles. Item Patients responses (total number) FIGURE 1 Time sequence of maturation of germinal vesicle oocytes with or without cumulus cells in the stimulated cycle. The numbers in parentheses are the number of metaphase II oocytes after maturation. No. of cycle 135 Mean ( SE) no. of oocytes retrieved/cycle 10.3 4.5 (1,367) Maturity of oocytes retrieved Mature 78.5% (1,073) Immature 12.3% (168) Degenerated 9.2% (126) cumulus cells is shown in Table 2. Of 88 oocytes with cumulus, 12 (13.6%) degenerated or fragmented, eight (9.1%) remained GVs, and eight (9.1%) matured to metaphase I. The proportions of OCCs and denuded oocytes that reached metaphase II stage were 68.2% and 65.0%, respectively. The maturational sequence of oocytes with or without cumulus cells is shown in Figure 1. Mature metaphase II stage of OCCs was first observed at 26 hours (12.5%), whereas 5.0% of denuded oocytes had already matured to the metaphase II stage at 22 hours. However, the maturation of OCCs and denuded oocytes was completed at 38 and 44 hours, respectively. Although there were no significant differences in the proportion of oocytes that reached metaphase II with the presence or absence of cumulus cells, it was noteworthy that the synchronization of maturation was observed in oocytes with cumulus cells. The pronuclei (PN) formation of metaphase II stage oocytes according to maturation condition after ICSI is shown in Table 3. Of 112 metaphase II oocytes matured in vitro, 59.8% (67 of 112) had PN (three with 1PN, 58 with 2PN, and six with 3PN). Of 328 metaphase II oocytes matured in vivo, 80.8% (265) had PN (four with 1PN, 245 with 2PN, and 16 with 3PN). The proportion of oocytes with 2PN after ICSI was significantly lower in the oocytes matured in vitro than in the oocytes matured in vivo. Table 4 depicts the in vitro developmental capacity of 2PN oocytes matured in vitro or in vivo through the examination of the proportions developed to 4 cells, 8 cells, and 16 cells at 40, 60, and 80 hours after ICSI, respectively. When 2PN oocytes matured in vitro were cultured, the proportion of embryos reaching the 4-cell, 8-cell and 16-cell stage at 60 and 80 hours after ICSI was 24.5%, 69.0% and 17.2%, respectively. In general, the proportion of embryos cleaved to the 16-cell stage after ICSI was significantly higher in the oocytes matured in vivo than in the oocytes matured in vitro. DISCUSSION In a stimulated cycle, an administered pharmacologic dose of gonadotropin creates a supraphysiologic hormonal environment and induces simultaneous growth of a cohort of TABLE 2 Effect of cumulus cells on the in vitro maturation of germinal vesicle oocytes from stimulated cycles. Presence or absence of cumulus cell No. of germinal vesicle oocytes Stage of oocytes after maturation (%) Degenerated GV Metaphase I Metaphase II Cumulus 88 12 (13.6) 8 (9.1) 8 (9.1) 60 (68.2) Cumulus 80 13 (16.3) 9 (11.3) 6 (7.4) 52 (65.0) Total 168 25 (14.9) 17 (10.1) 14 (8.3) 112 (66.7) FERTILITY & STERILITY 1155

TABLE 3 Effect of maturation condition on the pronuclei formation of oocytes after intracytoplasmic sperm injection in the stimulated cycles. Place of maturation No. of oocytes (metaphase II) injected No. of oocytes damaged PN status of oocytes injected (%) 0PN 1PN 2PN 3PN 1PN 2PN 3PN In vitro 112 15 (13.4) 30 (26.8) 3 (2.6) 58 (51.8) a 6 (5.4) 67 (59.8) a In vivo 328 41 (12.5) 22 (6.7) 4 (1.2) 245 (74.7) a 16 (4.9) 265 (80.8) a a P.05. large and small follicles. The heterogeneity of the oocyte population at the time of hcg administration leads to retrieval of oocytes at a different stage of maturation. The retrieved oocytes vary in maturity and in morphology from the OCCs. Hammitt et al. (10) found that at the time of oocyte retrieval, 15% of oocytes remained in prophase I of meiosis (GV stage), whereas 50% of them showed nuclear maturity (metaphase II stage). Approximately 70% 85% of the oocytes retrieved are at the metaphase II stage, and about half of the remaining oocytes (5% 15%) are classified as being at the GV stage (4). The growth speed of oocytes in stimulated ovaries may be variable and depends on the maturational stage of oocytes in the follicular phase at which gonadotropin stimulation was begun. Although oocytes that remain in the GV stage in spite of ovarian stimulation may be inherently lower in quality, 66.7% of those recovered matured after 22 44 hours of culture, 59.8% of the in vitro-matured oocytes activated, and 84.5% of the activated oocytes cleaved. Meanwhile, of the in vivo-matured oocytes recovered from stimulated ovaries, 80.8% activated and 90.6% of the activated oocytes cleaved. These data suggest that human GV oocytes obtained from stimulated ovaries can be matured, fertilized, and developed in vitro. Until now, there has been very little information available on the in vitro maturation of immature oocytes TABLE 4 Effect of maturation condition on the development of oocytes after intracytoplasmic sperm injection in the stimulated cycles. Place of maturation No. of oocytes (2PN) 4-cell stage, 40 hours No. of embryos cleaved and time of cleavage (%) 8-cell stage, 60 hours 16-cell stage, 80 hours In vitro 58 49 (84.5) 40 (69.0) 10 (17.2) a In vivo 245 222 (90.6) 181 (73.9) 72 (29.4) a a P.05. obtained from stimulated ovaries. According to Gomez et al. (11), approximately 16% of immature oocytes retrieved from patients whose ovaries were not stimulated reached the metaphase II stage after 48 hours of culture, but maturation rate was significantly increased with the addition of epidermal growth factor and insulin-like growth factor I to the culture medium. When immature oocytes retrieved from untreated patients with polycystic ovarian disease were cultured in a medium with gonadotropins, estrogen, and fetal calf serum, 65% of the oocytes cultured matured to metaphase II at 43 47 hours, and 81% were matured at 48 54 hours after culture (8). The maturation of human immature oocytes may be enhanced by the addition of gonadotropin, growth factors, and steroids. The mature follicular fluid collected after LH surge has an adequate level of gonadotropin and steroid hormone (12). We consider that the mature follicular fluid used in this study may contain various growth factors, gonadotropin, steroid hormones, and heat stable factors that are required for maturation. In agreement with a previous study (13), we found that 65.0% of oocytes without cumulus cells completed their first meiotic division. In this study, maturation of oocytes with cumulus cells was more synchronous than that of oocytes without cumulus cells. With regard to the maturation of oocytes, several investigators have reported evidence suggesting that camp controls the maintenance of meiotic arrest in mammalian oocytes and that purines, e.g., hypoxanthine, in follicular fluid increase the camp (14, 15). The gonadotropins act on the somatic cells, such as the follicle and cumulus cells, to produce substances that mediate resumption of meiosis in the oocyte (16). Richard and Sirard (17) have observed inhibitory substances in thecal cells capable of maintaining bovine oocytes in meiotic arrest, and Down (18) found evidence of both inhibitory and stimulatory signals from the cumulus cells via gap junction to the oocyte. From this study, we consider that the oocytes without cumulus cells also have the ability to mature in vitro because they still possess cumulus cell projection embedded in the 1156 Kim et al. Maturation of human GV oocytes and ICSI Vol. 74, No. 6, December 2000

zona pellucida (19), from which both inhibitory and stimulatory signals may be transferred to the oocytes. Cumulus cells and granulosa cells are known to secrete epidermal growth factor and other factors (20). Epidermal growth factor produced by cumulus cells may play a significant role in the final maturation of the oocytes. We also observed that protrusion of a first polar body occurred approximately 4 hours earlier in oocytes without cumulus cells than in those with cumulus cells. It can be interpreted as an indication of faster and easier removal of inhibitory and stimulatory maturation signals from denuded oocytes (13). Maturation in vitro of GV oocytes from an unstimulated cycle (1) or a stimulated cycle (21) requires 36 37 or 30 32 hours, respectively, after ovum pickup for full maturation to the metaphase II stage. When compared with GV oocytes from an unstimulated cycle, the maturation speed of GV oocytes from a stimulated cycle was accelerated in vitro. It is difficult to explain this phenomenon clearly. From this study, we found that the number of oocytes having 2PN and the subsequent developmental capacity were significantly reduced in in vitro-matured GV oocytes retrieved from a stimulated cycle. This apparently low proportion of oocyte activation and embryo development may represent abnormalities of cytoplasmic maturation of in vitro-matured oocytes, unsuitable culture conditions for these maturation and cleavage stages, and/or an intrinsic defect in developmental capacity. The increase in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities may be necessary for the onset of GV breakdown and metaphase progression during oocyte maturation and meiotic arrest (22, 23). MPF displays a cyclic activity that peaks at metaphase (24). A decrease in MPF and MAPK activity coincided with metaphase II exit and pronuclear formation, respectively (25). Therefore, the maturation and cleavage may depend on the establishment of these factors and their cyclic activity in the correct sequence of activation for syngamy, cleavage, and mitosis. The occurrence of aneuploidy is higher in mature oocytes collected from stimulated cycles (31%) than in mature oocytes from natural ovulatory cycles (20%) (26). Although human oocytes matured in vitro are morphologically normal, they showed gross meiotic aberrations (27). There was no difference in nuclear maturation rate after in vitro maturation among the two oocyte groups (with and without cumulus cells). However, cytogenetic data (28) derived from the IVF program indicate that the incidence of chromosomal aberration in zygotes fertilized in vitro is extremely high, which hinders the course of development. Furthermore, chromosomal abnormalities in morphologically normal bovine 2-cell embryo (29) and blastocysts (30) matured and fertilized in vitro were detected at rates of 34.2% and 25%, respectively, by use of the fluorescent in situ hybridization technique. We did not confirm whether cumulus cells during in vitro maturation can or cannot affect the chromosomal status of embryos. Further study will be required to identify the effect of cumulus cells on chromosomal constituents. Trounson et al. (8) found normal fertilization (i.e., oocytes showing 2PN) in 34% of all inseminated oocytes and 56% of the cultured pronuclear oocytes cleaved to the 8-cell stage or further. Barnes et al. (31) reported that 43% (23 of 54) of in vitro-matured oocytes from unstimulated regular cycling patients were fertilized. Unlike the results mentioned above, we observed the higher fertilization rate in this study, which was similar to the results in the previous work reviewed by Palermo et al. (6). The use of ICSI has produced consistent results in terms of fertilization, pregnancies, live births, and safety (6). ICSI seems to be the best option for fertilizing in vitro-matured oocytes from the GV stage even when sperm parameters are not impaired, although fertilization and pregnancy have been reported after standard insemination of in vitro-matured oocytes (5). Therefore, ICSI is recommended for fertilization of in vitro-matured oocytes from a stimulated cycle as a substitution for conventional IVF. Lee et al. (32) observed a much higher pregnancy rate after IVF-ET took place at the 16-cell stage than at the blastocyst stage. Therefore, subsequent in vitro development of in vitro-fertilized oocytes has been performed only up to the 16-cell stage. In conclusion, the present study shows that immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, and cleaved in vitro. 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