Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization

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1 FERTILITY AND STERILITY Copyright ~ 1983 The American Fertility Society Vol. 39, No.5, May 1983 Printed in U.SA. Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization Lucinda L. Veeck, M.L.T. (A.S.C.P.)* J. W_ Edward Wortham, Jr., Ph.D.t Jeannine Witmyer, B.S.t Bruce A. Sandow, Ph.D.t Anibal A. Acosta, M.D.* Jairo E. Garcia, M.D. * Georgeanna S. Jones, M.D.* Howard W. Jones, Jr., M.D.* Eastern Virginia Medical School and Old Dominion University, Norfolk, Virginia Oocytes of varying stages of maturity were aspirated from follicles primed with either human menopausal gonadotropin (hmg) and human chorionic gonadotropin (hcg) or a combination offollicle-stimulating hormone (FSH), hmg and hcg. Of the aspirated oocytes from 44 cycles, 74 were considered to be immature by virtue of morphologic characteristics of the oocytes and the degree of intercellular expansion of the associated cumular and membrana granulosa cells. After incubation periods of 22 to 35 hours in a Ham's F-10-based culture medium, these immature oocytes were inseminated with sperm donated by the patient's husband. Ultimately, 44 conceptuses were transferred to the respective uteri. of 30 patients. Eight pregnancies were established as a result of these 30 transfers, two of which resulted from the transfer of only developed immature oocytes. Fertil Steril39:594, 1983 The ability of nonhuman mammalian nucleat-, ed oocytes to resume maturation when isolated from their follicles has been demonstrated by several investigators over the past 50 years.1-3 Studies done with human oocytes show the same results, and reports of efforts to fertilize these 00- Received November 29, 1982; revised and accepted January 25,1983. *Department of Obstetrics and Gynecology, Eastern Virginia Medical School. tdepartment of Biological Sciences, Old' Dominion University. :j:department of Anatomy, Eastern Virginia Medical School. Reprint requests: Howard W. Jones, Jr., M.D., Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 603 Medical Tower, Norfolk, Virginia Veeck et al. In vitro maturation of human oocytes cytes indicate that human oocytes matured in vitro are indeed capable of resuming meiosis, extruding a first polar body, and undergoing fertilization.4-6 There has been caution in handling immature human oocytes in clinical in vitro fertilization (IVF) programs, caution borne primarily out of fear of abnormal maturation, fertilization, or embryonic development. Fear of anaphase blockage at the first meiotic division, abnormal protein synthesis, abnormal movement of chromosomes along the spindle, fragmentation of the female pronucleus, and observations made of abnormal male pronucleus formation have caused clinicians and researchers to be wary of assuming normality in oocytes that have progressed to maturity in vitro and have subsequently undergone fertiliza-

2 Table 1. Classification of Oocyte Maturity Oocyte classification Preovulatory Immature Associated granulosa Cumulus Corona Ooplasm Mature, displaying cellular expansion and intercellular spacing-loosely aggregated Generally immature, displaying little intercell ular spacing-compact and tightly aggregated Present and expanded Expanded, radiant Round, healthy in appearance Generally absent, occasionally present and dense Compact, occasionally absent Generally obscured by corona cells, round and healthy, often germinal vesicle noted tion.4. 5, 7-14 Fortunately, many of these problems have not been noted in the limited work done with human oocytes. However, if it could be demonstrated that human oocytes matured in vitro were capable of normal fertilization and development, the number of patients receiving intrauterine transfer would be increased, as well as the number of conceptuses available for transfer. This paper deals with the attempts to mature and fertilize, in vitro, human oocytes during the course of a clinical IVF program in Norfolk. The study involves 74 immature oocytes collected from 44 cycles from September 1981 to July Special emphasis is placed on one pregnancy resulting from the transfer of three in vitro matured and fertilized oocytes. MATERIALS AND METHODS Oocytes were collected from women undergoing ovulation induction with human menopausal gonadotropin (hmg) (Pergonal, Serono Laboratories, Braintree, MA) and human chorionic gonadotropin (hcg) (Ayerst Laboratories, New York, NY). Eight of these 44 cycles were aaditionally stimulated with pure follicle-stimulating hormone (FSH) (National Pituitary Agency, Baltimore, MD), given concurrently with the hmg. Most patients exhibited secondary infertility due to blocked or absent fallopian tubes. Two patients, representing two cycles, were treated for idiopathic infertility. The details of hmg/hcg ovulation induction have been presented elsewhere.15 In brief, two ampules ofhmg were given daily from cycle day 3 until estradiol values reached 300 pg/ml as measured by radioimmunoassay (RIA) or until an estrogenic mediated shift in the vaginal maturation index and cervical mucus scores were noted for 3 consecutive days. Ultrasound measurements of follicular growth were also considered, and follicles averaging 12 to 15 Vol. 39, No.5, May 1983 mm in diameter were considered suitable at the time of hmg discontinuation. In cycles that were supplemented with FSH, equal numbers of ampules of FSH and hmg were administered on cycle days 3 to 5, and hmg alone was administered during the remainder of the stimulation. Ten thousand milli international units of hcg were given 50 hours after the last hmg, and laparoscopy for follicular aspiration was scheduled to be performed 36 to 38 hours later. The method of follicular aspiration was that of Jones et ai.,16 and involved the use of a 12-gauge needle with an inside diameter of 2.16 mm and overall length of 43 cm. Patients were prepared for laparoscopy in the routine manner, except that anesthesia was withheld until after the preparation was complete. The gas used for pneumoperitoneum was 100% CO 2, Follicular fluids were aspirated into 2 ml of Dulbecco's phosphate-buffered saline (PBS) (# , Gibco Laboratories, Grand Island, NY), and an additional 2 ml of Dulbecco's PBS was flushed through the aspiration system to clear away any remaining fluid or cells left within the needle or connecting tubing. Follicular fluids were examined microscopically in the adjoining culture laboratory, and characteristics of the fluid, including volume, color, and the morphologic characteristics of granulosa cells were noted. Rapid scanning of fluids was carried out with a dissecting stereomicroscope, and critical morphologic examinations were done with a Nikon MS inverted microscope (Nikon, Inc., Garden City, NY). Once located, oocytes were graded for maturity on the basis of the presence or absence of the extra coronal cumular mass, morphologic characteristics ofthe inner cumulus, i.e., corona cells, observations of the ooplasm, and morphologic characteristics of the associated detached membrana granulosa cells (Table 1). In contrast to a typical preovulatory oocyte Veeck et al. In vitro maturation of human oocytes 595

3 Figure 1 Examples of oocytes and their associated granulosa cells. (a), Preovulatory oocyte displaying expansion of the corona radiata (c) and expansion of the extra coronal cumulus (Cu). The cumulus cannot be seen well due to photographic enlargement. (b), Example of typical membrana granulosa cells found associated with preovulatory oocytes. Note loose aggregation of cells. (e), Typical immature oocyte with compact follicle cells, i.e., corona (c). (d), Immature oocyte displaying compact corona (c) and a germinal vesicle (gv). (e), Immature oocyte displaying a compact corona (c). A small amount of expanded cumulus (Cu) is also present. (fl, Immature membrana granulosa displaying little intercellular expansion, compact aggregation. (Fig. la), displaying expansion of both the corona radiata and the extra coronal cumulus and associated with membrana granulosa cells with mature characteristics (Fig. lb), a typical immature oocyte lacked cumular and coronal expansion, displayed a compact layer of cells adherent to the zona pellucida (Fig. lc), and was associated with immature membrana granulosa cells in the aspirated follicular fluid (Fig. If). Not quite as typical was the immature oocyte which displayed an absence or near absence of coronal cells (Fig. ld), or one that displayed a compact layer of coronal cells within a cumulus mass (Fig. Ie). Both of these types were generally associated with immature, compact membrana granulosa. When all follicle cells were absent, the presence of a germinal vesicle or first polar body was a guiding criterion for determining the maturation classification. 596 Veeek et al. In vitro maturation of human oocytes After assessment, oocytes were incubated before insemination for a period of time related to their maturation classification (Table 2). Preovulatory oocytes were routinely incubated for 8 hours prior to insemination, while immature 00- cytes were incubated for periods greater than 22 hours. Immature oocytes were examined for first polar body extrusion at approximately 22 to 24 hours after aspiration and again at 2-hour intervals if no polar body was noted on previous exam- Table 2. Length of Incubation Prior to Insemination Oocyte classification Preovulatory Immature Time 8 hours Inseminated as soon as possible after polar body extrusion If oocyte is obscured by cells and polar body status cannot be evaluated, inseminated by 35 hours after aspiration

4 Table 3. Results of Attempts to Mature and Fertilize Human Oocytes in Vitro as Compared with Attempts to Fertilize Preovulatory Oocytes During the Same Period (September 1,1981 to July 31,1982) Immature Preovulatory Total no. of oocytes Failed maturation 10 (13.5%) Inseminated prior to com- 2 (2.7%) pletion of maturation Failed fertilization after 5 (6.8%) 27 (12.5%) maturation Fertilized with more than 6 (8.1%) 19 (8.8%) two pronuclei Failed cleavage after 7 (9.5%) 6 (2.8%) apparently normal fertilization Total no. of concep- 44 (59.5%) 165 (76.4%) tuses undergoing fertilization and transfer Transferred with cleavage 35 (79.5%) 165 (100%) Transferred at pronuclear 9 (20.5%) 0(0%) stage or after pronuclear stage prior to cleavage inations. In most cases, collection of the semen sample was requested for immature oocytes as soon as first polar body extrusion was observed. Some oocytes were inseminated prior to polar body extrusion if other immature oocytes from the same cycle were being inseminated. In cases where polar body extrusion was not determinable because of an obscuring cover of cells, oocytes were inseminated by 35 hours after aspiration. Ham's F-10 medium (# , Gibco Laboratories) was used, supplemented with 7.5% (vol/vol) human fetal cord serum for insemination medium (1M) and 15% (vol/vol) human fetal cord serum for growth medium (GM). Insemination was carried out in a welled organ culture dish (#3037, Falcon Plastics, Oxnard, CA) with 1.5 x 10 6 motile sperm in the 3-ml volume of 1M. All phases of incubation involved the use of 5% CO2 in air and a relative humidity of 97% to 98%. Culture medium composition, granulosa cell and oocyte classifications, and routine laboratory and quality control techniques have been describep in greater detail previously. 6, 7 Intrauterine conceptus transfer was carried out with the patient in the knee-chest position. A Teflon transfer catheter with a lateral opening was 'introduced through the cervix a distance of 6 cm, and the conceptuses were expelled in a volume of GM by injection with an attached tuberculin syringe. In most cases, 250 mg oftetracycline was administered on luteal phase day + 2 through days + 3 or + 4 to cover the period of transfer. Additional supplements of 12.5 mg/day of progesterone 1M were given from the day of transfer until menses and continued if a pregnancy was confirmed. Results from serum j3-subunit hcg assays (RIA) were obtained approximately between day 11 and day 14 of the luteal phase to confirm or deny an early pregnancy. Table 4. Individual Pregnancies Associated with the Transfer of Aspirated Immature Oocytes Pregnancies #1 #2 #3 #4 #5 #6 #7 #8 Category of infer- Tubal Tubal Tubal Idiopathic Tubal Tubal + Tubal Tubal tility male Total no. concep tuses transferred No. conceptuses de veloping from preovulatory oocytes No. conceptuses de veloping from immature oocytes Hours of incubation prior to insemination for immature oocytes Preovulatory oocytes NA 4/404/40 8/50 4/68 3/45 3/454/45 5/456/45 NA (no. blastomeresl I no. hours after insemination) Immature oocytes 4/464/46 After 2 2/302/30 2/424/42 2 pronucleil 2/242 pro- (no. blastomeresl 5/46 pronuclei! 17 nuclei/24 After 2 pronucleil 4/45 no. hours after insemination) Outcome Normal First-tri- Normal Normal fe- Normal Normal In prog- Biochemical male in- mester male in- male in- male in- twins-- ress pregnancy fant spontane- fant fant fant male and ous abor- female tion Vol. 39, No.5, May 1983 Veeck et ai. In vitro maturation of human oocytes 597

5 PREGNANCIES ASSOCIATED WITH THE TRANSFER OF ASPIRATED IMMATURE OOCYTES (8) TOTAL NUMBER CONCEPTI TRANSFERRED -- TOTAL NUMBER PATIENTS RECEIVING TRANSFERS 30 (A) Number patients receiving transfers of multiple concepti resulting from development of aspirated preovulatory and immature ocx:ytes. 15 (all multiple I transfers) Number pregnancies 6 ("') (B) Number patients receiving transfer of concepti resulting from development of aspirated immature oocytes ~. 15 (5 multiple I transfers 10 single transfers) Number pregnancies 2 (13.ij\) Figure 2 Pregnancies associated with the transfer of aspirated immature oocytes (n = 8), RESULTS The results are summarized in Tables 3 and 4 and in Figure 2. Of the 74 immature oocytes where attempts were made to mature them in vitro, 10 failed to mature. Two of these exhibited no germinal vesicle breakdown (GVBD); eight succeeded in GVBD but failed to extrude a first polar body. Two oocytes were inseminated prior to extrusion of a first polar body and failed to fertilize. Five other oocytes matured through polar body extrusion but failed to fertilize for unknown reasons. Of the 57 oocytes displaying evidence of fertilization, 6 exhibited the presence of three or four pronuclei, and 7 failed to cleave after observations of two pronuclei. Thirty-five went on to divide and were transferred at the 2- to 8-cell stage. Nine were transferred early while in the pronuclear stage or just after breakdown of the pronuclear membranes prior to cleavage. These 44 conceptuses were transferred to 30 patients and may have been instrumental in the establishment of the ensuing eight pregnancies. The actu~l original classification of the conceptuses responsible for six of these pregnancies is not clear, however, because one or two other developing conceptuses of mature origin were transferred concurrent with the immature matured in vitro (lmiv) conceptuses. One twin implantation occurred. Of special interest are the two pregnancies that resulted from the transfer of IMIV conceptuses only. One of these patients, 37 years of age, was seen in December 1981 for treatment of secondary infertility due to tubal occlusion. Her regimen for ovarian stimulation commenced on cycle day 3, with two ampules hmg being given through day 598 Veeck et al. In vitro maturation of human oocytes 10. On day 12 at 6:00 P.M., heg was administered, and laparoscopy for oocyte aspiration was done 38 hours later. Her daily clinical monitoring through the follicular phase is shown in Figure 3. During laparoscopy, six small «1.5 ml follicular fluid) follicles were aspirated, this number being the total complement of follicles present, and four oocytes were recovered. One of these was considered to be atretic, and the other three appeared immature by virtue of the morphologic characteristics of the oocytes and associated follicle cells (Table 5). These three oocytes can be seen as observed at the time of aspiration in Figures 4 to 6. The oocyte from follicle 5 displayed a prominent germinal vesicle. The oocytes were incubated in 1M for a period of 23.5 hours, at which time the observation was made that the oocytes from follicles 2 and 5 had matured in vitro through first polar body extrusion. A semen sample from the patient's husband was called for and collected immediately, and insemination of all three 00- cytes took place after 26 hours of incubation (Figs. 7 to 9). At the time of insemination, oocyte 3 did not yet display an extruded polar body. Fourteen hours after insemination, all three 00- cytes exhibited the presence of two pronuclei Figure 3 S.K. Daily monitoring of vaginal smear maturation index, cervical mucus scores, estradiol, and ultrasound assessment during follicular phase of hmgihcg stimulated cycle.

6 Figure 4 S.K. Oocyte from follicle 2, aspiration. Figure 7 S.K. Oocyte 2, insemination. Figure 5 S.K. Oocyte from follicle 3, aspiration. Figure 8 S.K. Oocyte 3, insemination. Figure 6 S.K. Oocyte from follicle 5, aspiration. (Figs. 10 to 12), and by 46 hours after insemination, all had cleaved to the 4-cell stage. Intrauterine transfer was undertaken within the hour, and 'the three conceptuses were delivered to the uter- Vol. 39, No.5, May 1983 Figure 9 S.K. Oocyte 5, insemination. us without difficulty on cycle day 17. At the time of transfer, conceptuses from follicles 2 and 3 displayed 4 cells, and the conceptus from follicle 5 was at the 5-cell stage (Figs. 13 and 14). A tenacu- Veeck et al. In vitro maturation of human oocytes 599

7 Figure 10 S.K. Oocyte 2, 14 hours after insemination. Figure 13 S.K. Oocyte 2, 46 hours after insemination, at transfer. Figure 11 S.K. Oocyte 3, 14 hours after insemination. Figure 14 S.K. Oocyte 5, 46 hours after insemination, at transfer. S.K E I.i ~11i,OOO I'- II 11 ~'''''' 11- ".:: 1 I oil... a_ I.. :1 ~ i!... r Figure 12 S.K. Oocyte 5, 14 hours after insemination. lum was used to stabilize the cervix, and minor bleeding was noted at the external os. Pregnancy was confirmed by ~-subunit heg (RIA) values of 97.2 IU on cycle day 26 (luteal phase day + 12) 600 Veeck et ai. In vitro maturation of human oocytes rll1ml In Weeks Figure 15 S.K. Progesterone, estradiol, and J3-hCG values during first 21 weeks of gestational periqd. and 487 IU on cycle day 29 (luteal phase day + 15). The gestational period progressed without incident (Fig. 15). Due to advanced maternal age, the patient chose to have a second trimester amniocentesis for chromosomal analysis. The resulting karyotype showed a chromosome complement of 46,XY. In August 1982 a normal male infant was delivered by cesarean section.

8 Table 5. Characteristics of Follicles and the Three Associated Oocytes Which Were Matured and Fertilized in Vitro, One of Which Resulted in the Term Delivery of a Healthy Male Child Follicle 2 Follicle 3 Follicle 5 Follicle Small, Small, Small, description thick, thick, thin, opaque opaque transparent Volume fol- 1.0 ml 1.5 ml 1.0 ml licular fluid Membrana Immature Immature Immature granulosa cells Cumulus Absent Absent Absent Corona Compact Few cells, Compact compact Ooplasm Obscured Healthy, Prominent perivitelline germinal vesmembrane icle, perivipulled away telline memfrom zona pellucida brane pulled away from zona pellucida The second pregna:ncy. trnu; was established by the transfer of an IMIV conceptus resulted in early embryonic loss and was considered to be a biochemical pregnancy after three consecutive elevated f3-subunit hcg values and a delay in menses. DISCUSSION Although it has been observed in the past that human oocytes were capable of in vitro maturation, the ability of the fertilized conceptuses resulting from these oocytes to develop normally has been in question. McGaughey and Polge 17 had poor results in maturing pig oocytes in Ham's F-lO medium, and expressed caution in handling immature oocytes on the basis of work that they had done with analysis of chromosomes. Thibault et al. 13 concluded that maturation of rabbit 00- cytes outside an intact follicle resulted in abnormal development of the male pronucleus, and that in vitro maturation was only successful in cases where intact follicles were cultured. Various investigators have shown an increased ability of nonhuman oocytes to mature in vitro in the presence of luteinizing hormone added to the culture medium but had lower developmental rates in non-gonadotropin-supplemented media. 18-2o Other researchers found that fertilization rates were lower than expected in matured oocytes of mice and that development rarely proceeded past Vol. 39, No.5, May 1983 the blastocyst stage. 21 In work done with humans, Cohen et al. 22 found that less than one half of human oocytes undergoing maturation in vitro expelled a first polar body, and none fertilized. Our work indicates that human oocytes matured in vitro is indeed feasible, at least in those immature oocytes that have been exposed to a gonadotropin surge. The birth of at least one normal human offspring would substantiate this conclusion. Although a single normal birth certainly does not provide sufficient evidence to state that all such conceptuses are normal, it does indicate that the technique can be useful and valid for clinical procedures. Maturation, fertilization, and cleavage rates were not disappointing for immature oocytes, especially in light of the fact that they were cultured outside of their intact follicles, and without additional gonadotropin supplementation of the media. Of additional interest in this study is the fact that oocytes were exposed to a high O2 tension (20%) in the incubating atmosphere of 5% CO2 in air, and that all oocytes and conceptuses were photographed during the course of their development. The choice of utilizing these methods during the culturing of human specimens has been debated among clinicians and reproductive physiologists in the field of IVF; but with the current experience, neither of these methods seems necessarily detrimental. It is evident that during the course of any IVF program where smaller follicles are aspirated, immature oocytes will be recovered along with the anticipated preovulatory ones. Successful in vitro maturation of these oocytes ensures a wider range of fertilizable oocytes and should therefore increase the opportunity for securing a pregnancy. Besides increasing the chances that at least one conceptus will be transferred during any given cycle, preliminary evidence indicates that pregnancy rates may increase in direct relation to the number of conceptuses transferred. 15 Pregnancy rates may reach 40% to 50% when three conceptuses are returned to the uterus, and this probably accounts for the high rate of implantation demonstrated in Figure 2A, where all transfers were of multiple numbers. REFERENCES 1. Chang MC: The maturation of rabbit oocytes in culture and their maturation, activation, fertilization, and subsequent development in the fallopian tube. J Exp Zool 128:379, 1955 Veeck et al. In vitro maturation of human oocytes 601

9 2. Pincus G, Enzmann EV: The comparative behavior of mammalian eggs in vitro and in vivo. I. The activation of ovarian eggs. J Exp Med 62:665, Edwards RG: Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208:349, Edwards RG: Maturation in vitro of human ovarian 00- cytes. Lancet 2:926, Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221:632, Wortham JWE Jr, Veeck LL, Witmyer J, Jones HW Jr: Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction. Phase Fertil Steril. In press 7. WorthamJWE Jr, Veeck LL, WitmyerJ, Sandow B,Jones HW Jr: Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction. Phase Fertil Steril. In press 8. Thibault C: Are follicular maturation and oocyte maturation independent processes? Hammond Memorial Lecture. J Reprod Fertil 51:1, Thibault C, Gerard M: Facteur cytoplasmique necessaire a la formation du pronucleus male dans l'ovocyte de lapine. C R Acad Sci [D] (Paris) 270:2025, Thibault C, Gerard M: Cytoplasmic and nuclear maturation of rabbit oocytes in vitro. Ann BioI Anim Biochim Biophys 13:145, Soupart P: The need for capacitation of human sperm: functional and ultrastructural observations. In Biology of Spermatozoa: Transport, Survival, and Fertilizing Ability: Proceedings of the INSERM International Symposium, Nouzilly, November 4 to 7, 1973, Edited by ESE Hafez, CG Thibault. New York, S. Karger, Motlik J, Fulka J: Fertilization of pig follicular oocytes cultivated in vitro. J Reprod Fertil 36:235, Thibault C, Gerard M, Menezo Y: Preovulatory and ovulatory mechanisms in oocyte maturation. J Reprod Fertil 45:605, Warnes GM, Moor RM, Johnson MH: Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro. J Reprod Fertil 49:331, Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G: The program for in vitro fertilization at Norfolk. Fertil Steril 38:14, Jones HW Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, McGaughey RW, Polge C: Cytogenetic analysis of pig 00- cytes matured in vitro. J Exp Zool 176:383, Shalgi R: In Ovarian Follicular and Corpus Luteum Formation. New York, Plenum Press, 1979, p Moor RM, Trounson AO: Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 49: 101, Shalgi R, Dekel N, Kraicer P: The effect of LH on the fertilizability and developmental capacity of rat oocytes matured in vitro. J Reprod Fertil 55:429, Eppig J: Developmental potential of LT/Sv parthenotes derived from oocytes matured in vivo and iii vitro. Dev BioI 65:244, Cohen J, Mandelbaum J, Planchot M: Maturation et fecondation in vitro d'ovocytes humains. J Gynecol Obstet BioI Reprod (Paris) 9:523, Veeck et ai. In vitro maturation of human oocytes