IN VITRO FERTILIZATION Oocyte maturity and preimplantation development in relation to follicle diameter in gonadotropin-releasing hormone agonist or antagonist treatments Daniela Nogueira, Ph.D., a Shevach Friedler, M.D., b Morey Schachter, M.D., b Arieh Raziel, M.D., b Raphael Ron-El, M.D., b and Johan Smitz, Ph.D. a a Follicle Biology Laboratory, Free University of Brussels (Vrije Universiteit Brussel), Brussels, Belgium; and b IVF and Fertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, Tel Aviv, Israel Objective: To analyze the distribution of oocytes nuclear maturational stages and intracytoplasmic sperm injection (ICSI) outcome in relation to follicular size, after controlled ovarian hyperstimulation (COH), using gonadotropinreleasing hormone (GnRH) antagonist or agonist protocols. Design: Prospective comparative study. Setting: Fertility unit in an academic hospital. Patient(s): Normo-ovulatory patients with unexplained, male and/or tubal factor infertility undergoing ICSI treatment. Intervention(s): Oocytes were retrieved from small (7 12 mm) and large follicles ( 12 mm). Main Outcome Measure(s): Oocyte nuclear maturation stage, fertilization and pre-implantation embryonic development of the mature ova evaluated. Result(s): Distribution of oocyte maturation stages from small follicles did not differ between the two COH protocols. From large follicles, a statistically significantly higher percentage of immature oocytes was retrieved after antagonist compared with agonist treatment (16 vs 11%, respectively). No statistically significant differences existed regarding ICSI outcome of mature ova within the same follicle diameter range between COH protocols. Inseminated ova from small follicles presented a significantly higher number of embryos with delayed development on day 3 after ICSI. Conclusion(s): A greater heterogeneity of maturity was found in the population of oocyte stages that were present after the antagonist protocol. Matured oocytes retrieved from small follicles generated embryos of lower developmental potential than oocytes derived from larger follicles. (Fertil Steril 2006;85:578 83. 2006 by American Society for Reproductive Medicine.) Key Words: Follicles, oocyte maturation, oocyte development, GnRH analogues Received December 27, 2004; revised and accepted August 11, 2005. Presented at the European Society of Human Reproduction (ESHRE) 2004 20th Annual Meeting; Berlin, Germany; June 27 to 30, 2004. Reprint requests: Daniela Nogueira, Ph.D., Women and Infants Hospital, New England Medical Center, Tufts, 750 Washington Street, Box 036, Boston, Massachusetts (FAX: 1-617-636-5906; E-mail: daniela_nogueira@yahoo.com). Gonadotropin-releasing hormone (GnRH) antagonists recently joined the armamentarium of clinically available GnRH analogues to prevent premature luteinizing hormone (LH) surges during controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) (1 3). Although protocols using antagonists are of shorter duration compared with protocols using GnRH agonists, the preliminary reports of inferior rates of implantation and clinical pregnancies raised concerns (1 4). The potentially different effects of GnRH antagonists and agonists on ovarian folliculogenesis may lead to different responses and endocrine environments for maturing oocytes. Information about oocyte maturational stage in relation to follicular size after different superovulation protocols has been scanty. Early studies performed on IVF cycles using cumulus-enclosed oocytes indicated that oocytes retrieved from larger follicles, defined as 12 mm (5) or 16 mm (6 9), tended to achieve higher fertilization and cleavage rates. However, no difference in embryo quality was observed (5). Because oocyte denudation enables direct observation of developmental status, ICSI cycles facilitate precise assessment of oocyte nuclear maturation. Oocyte nuclear stage correlates with follicular diameter in some studies, but no studies have compared different COH protocols. In patients treated by short or long agonists COH protocols, a progressive and significant increase in the rates of polar body extruded oocytes was observed from small follicles, defined 578 Fertility and Sterility Vol. 85, No. 3, March 2006 0015-0282/06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2005.08.033
as 12 mm (10, 11) or 16 mm (8, 9), to larger diameter follicles. Variable outcomes were reported following ICSI, including a significant reduction in fertilization rates of oocytes from small follicles (8) and a trend toward lower fertilization rate compared with oocytes from larger follicles (11). Other studies showed similar fertilization rates (9, 10). Our study aimed to determine the distribution of oocyte maturation stages and outcome of ICSI in relation to follicular diameter after using antagonist or agonist protocols for COH. MATERIALS AND METHODS Study Population Our prospective study enrolled all women consecutively treated with ICSI in the IVF unit at Assaf Harofeh Medical Center from December 2003 to March 2004 who agreed to participate. Informed consent was obtained from all participants, and the investigation received the approval of the institutional review board of the Assaf Harofeh Medical Center. To minimize confounding factors, patients diagnosed with polycystic ovaries or with poor response (fewer than four mature follicles on ultrasound before oocyte retrieval) were excluded from the study. Only one treatment cycle per patient was analyzed. Because the proportion of agonist and antagonist cycles in our center is 3 to 1, an antagonist cycle was recruited for the study after three recruited agonist cycles. A total of 97 patients was included, 71 treated by agonists and 26 by antagonists. Mean ages ( standard deviation) were 30.7 4.7 years (range: 28 to 41 years) and 34.3 4.9 years (range: 22 to 31 years) for the agonist and antagonist groups, respectively. Major indications for IVF/embryo transfer (ET) included male factor infertility (76%), male and tubal factor infertility (15%), and unexplained infertility (9%). COH, Oocyte Retrieval, and ICSI Patients included in the study were treated by routine COH protocols for IVF using either agonists or antagonists. In the agonist group, COH consisted of midluteal pituitary downregulation, using a daily dose of GnRH-a (nafarelin acetate, 200 mg three times a day, via intranasal spray; or triptorelin, 0.1 mg/day, via subcutaneous injection), followed by an individually adjusted daily dose of urinary or recombinant gonadotrophin injections. In the antagonist group, COH using daily injections of gonadotrophins was administered, followed by a daily dose of GnRH antagonist (0.25 mg of Cetrotide, Serono Laboratories, Geneva, Switzerland; or Orgalutran, Organon, Oss, the Netherlands) once a lead follicle of 12 mm was observed or serum estradiol levels had reached 400 pg/ml. Oocytes were retrieved 35 to 38 hours after administration of 5000 IU hcg, which was given according to similar criteria in both groups (i.e., presence of at least two leading follicles of 18 mm). Routine oocyte pick-up was performed using a 17G oocyte aspiration needle. Once follicles larger than 12 mm had been aspirated ( large ), the needle attached to the test tube was washed with HEPES-containing media, and a new tube was attached to the needle for aspiration of smaller follicles ( small ) measuring 7 to 12 mm. Follicles were aspirated using 80 mm Hg pressure. Follicular aspirates from large and small follicles were collected in separate plastic Falcon tubes. Oocyte denudation was performed 2 to 5 hours after the oocyte retrieval procedure (40 hours after hcg administration). Following oocyte denudation, oocytes of each COH protocol were classified according to intactness (degenerated or not) and nuclear maturational stages (germinal vesicle [GV], germinal-vesicle breakdown [GVBD], or polar body extruded [PB]) under an inverted microscope (magnification 200; 400). For PBextruded oocytes, ICSI was performed. Oocytes were cultured singly in droplets of G1 media (Vitrolife, Kungsbacka, Sweden). Fertilization was assessed 16 to 18 hours after injection, and normal fertilizations (presence of two pronuclei) was recorded. Embryonic cleavage and morphologic appearance were assessed 40 to 44 hours and 64 to 68 hours after ICSI. A morphologic score was given for each embryo according to the degree of fragmentation: 1, no fragmentation; 2, 20% fragmentation; 3, 20% to 50% fragmentation; 4, 50% fragmentation. The outcome of ICSI was compared in PB oocytes retrieved from small and large follicles. For later identification and data analysis, the clinical and laboratory files of each patient were assigned a common number that was different from the patient s medical record number. This ensured that the biologists who handled the oocytes and recorded the outcomes would be blinded to the COH protocols. Statistical Analysis Differences in oocyte maturational stages, as well as fertilization and cleavage rates, were calculated by chi-square test and Fischer s exact test, as appropriate. Differences in means between two variables were calculated using Student s t-test or the Mann-Whitney U-test, as appropriate. P.05 was considered statistically significant. Comparative statistics were performed to include all ICSI cycles. RESULTS Clinical IVF outcomes are described in Table 1. The recovery rate of oocytes was recorded only for the small follicles, and it was comparable after both COH protocols (56% and 52% in antagonist and agonist groups, respectively; P.05) (Table 2). Among oocytes from small follicles, a larger proportion was degenerated at retrieval after the antagonist protocol compared with the agonist (P.05). The distribution of oocyte maturation stages of oocytes from small follicles did not differ after COH protocols using antagonist or agonist. In both treatment groups, about Fertility and Sterility 579
TABLE 1 Clinical outcome of patients treated with gonadotropin-releasing hormone antagonist or agonist protocols. Antagonist (n 26) Agonist (n 71) P value a Day of hcg administration (mean SD) 11.2 2.3 12.6 1.9.008 Mean dose of FSH/patient (IU per stimulation cycle) 2,627.9 1,057.4 2,448.8 1,092.4 NS E 2 at day of hcg (pg/ml) 2,046.2 1,744.3 2,582.5 1,239.5.03 Mean no. of embryos transferred SD 2.7 1.1 2.5 0.9 NS No. (%) of patients with cryopreservation 9 (34.6) 29 (40.8) NS No. (%) of cryopreserved embryos/cleaved embryos 29 (28.1) 157 (32.7) NS No. (%) of implanted embryos 10 (14.5) 38 (22.4) NS No. (%) of pregnancies 7 (26.9) 29 (40.1) NS Note: hcg human chorionic gonadotropin; FSH follicle-stimulating hormone; NS not statistically significant; SD standard deviation. a Mann-Whitney test, chi-square, or Fisher s exact test. 40% of oocytes retrieved from small follicles were at the PB stage (see Table 2). Among oocytes from large follicles, a higher proportion was at the GV stage (P.0001) and significantly fewer oocytes were at GVBD stage after the antagonist protocol. The proportion of PB oocytes was lower after the antagonist treatment, but this difference did not reach statistical significance (P.07). Comparing ICSI outcome between the antagonist and agonist treatments of PB oocytes retrieved from small or large follicles (Table 3), no statistically significant difference was observed regarding 2-PN fertilization rates or embryo developmental quality on day 2. Cleavage rates were comparable between the antagonist and agonist groups, with similar proportions of four-cell stage embryos on day 2 (approximately 60% from large follicle groups and approx- TABLE 2 Comparison of the distribution of oocyte nuclear maturation stages, between small and large follicles in patients treated by gonadotropin-releasing hormone antagonist or agonist controlled ovarian hyperstimulation protocols for intracytoplasmic sperm injection cycles. Follicular size Antagonist (n 26) Agonist (n 71) P value Small No. of oocytes 72 243 Maturation stages GV (%) 34 (47) 124 (51) NS GVBD (%) 2 (3) 21 (9) NS PB (%) 31 (43) 93 (38) NS Degenerated (%) 5 (7) 5 (2).05 Large No. of oocytes 179 821 Maturation stages GV (%) 25 (14) 37 (5).0001 GVBD (%) 3 (2) 47 (6).05 PB (%) 149 (83) 726 (88) NS Degenerated (%) 2 (1) 11 (1) NS Note: GV germinal vesicle; GVBD germinal-vesicle breakdown; NS not statistically significant; PB polar body extruded. 580 Nogueira et al. Oocyte development and follicle size Vol. 85, No. 3, March 2006
TABLE 3 Comparison of ICSI outcomes of oocytes with polar body extruded from small and large follicles, according to gonadotropin-releasing hormone antagonist or agonist protocols. Small follicles Large follicles Antagonist Agonist P value a Antagonist Agonist P value a No. of PB oocytes injected 31 93 149 726 % 2-PN 63 67 NS 71 68 NS % Cleaved/2-PN 98 97 NS 97 97 NS Day 2 embryo developmental quality Mean no. of blastomeres SD 3.9 1.1 3.7 0.9 NS 4.0 1.0 3.8 1.0 NS Mean fragmentation score SD 2.4 0.7 1.8 0.7 NS 1.8 0.8 2.0 0.6 NS Day 3 embryo developmental quality Mean no. of blastomeres SD 6.5 2.6 6.0 1.7 NS 6.5 0.9 6.5 1.7 NS Mean fragmentation score SD 2.6 0.7 2.0 0.7 NS 2.0 0.5 2.5 0.5 NS Note: NS not statistically significant; PB polar body extruded; PN pronuclear. a Chi-square test or Student s t-test. imately 55% from small groups) and eight-cell stage on day 3 (approximately 35% from large and approximately 20% from small follicle groups). The outcome of ICSI on oocytes from small or large sibling follicles derived from the same patient did not show statistically significant differences regarding 2-PN fertilization rates, mean blastomere number, or embryo quality on days2or3(table 4). No differences were found regarding the percentage of day-2 embryos with 4 cells (72% from small vs. 74% from large follicles; not statistically significant). However, by day 3 after ICSI, the oocytes from small follicles generated more embryos presenting only four cells (P.05). Although more embryos from large follicles were at the eight-cell stage on day 3 compared with the small follicles (P.05), the proportion of embryos attaining at least eight cells ( 8 cells) did not reveal a statistically significant difference between the embryos from large (42%) and small (29%) follicles (P.08). Embryos generated from TABLE 4 Outcome of intracytoplasmic sperm injection in oocytes with polar body extruded according to follicular size of sibling follicles derived from the same patient. Small follicles Large follicles P value a No. of PB oocytes injected 122 511 % 2-PN 66 73 NS % Cleaved/2-PN 96 96 NS Day 2 embryo developmental quality Mean no. of blastomeres SD 3.7 1.0 3.8 0.8 NS Mean fragmentation score SD 1.9 0.7 2.1 0.5 NS % 4-cell/total cleaved 28 26 NS % 4-cell/total cleaved 72 74 NS Day 3 embryo developmental quality Mean no. of blastomeres SD 6.1 1.9 6.4 1.1 NS Mean fragmentation score SD 2.1 0.7 2.1 0.5 NS % 4-cell/total cleaved 26 13.05 % 8-cell/total cleaved 29 42.08 Note: NS not statistically significant; PB polar body extruded; PN pronuclear. a Mann-Whitney test, chi-square, or Fisher s exact test. Fertility and Sterility 581
oocytes of small follicles had a lower capability for optimal rate of cleavage, with only 15% of embryos per total cleaved presenting four-cell stage on day 2 and subsequently presenting eight-cell stage on day 3. For large follicles, 31% of embryos per total cleaved had an optimal rate of cleavage from days 2 to 3 of culture. DISCUSSION Early studies performed after IVF (which does not require denudation of oocyte before insemination) indicated that oocytes retrieved from larger follicles tended to achieve higher fertilization and cleavage rates than the oocytes from follicles 12 mm (5) or 16 mm (6 9). However, no difference in embryo quality was observed (5). Scott et al. (12) suggested that, due to the lower recovery rates of mature oocytes from follicles 11 mm, the operating surgeon may choose not to aspirate these follicles. Follicles 15 mm provide the highest probability of retrieving mature oocytes (13). Exact assessment of oocytes maturational stage has been reported in a few studies that involved ICSI, where oocyte denudation is needed. In these studies, COH included agonists protocols only. The definition of small follicles varied from 16 mm to 10 mm in the studies located by our literature search. A progressive and significant increase in the rates of PB oocytes has been observed from small follicle groups of diameter 16 mm (8, 9) or 10 mm (10, 11) to the larger follicle groups. Moreover, a positive correlation has been observed between follicular diameter and fertilization rates and rates of embryos scored as good when oocytes were fertilized by ICSI (8, 10, 11). Similar rates of fertilization, cleavage, and implantation rates have been found by others, regardless of the size of the follicles from which oocytes were aspirated (small at 16 mm, or large at 16 mm) (9). Our review of the literature located no studies correlating oocyte maturational stage and follicular size according to COH protocol. Thus, our study was the first comparison of oocyte maturational rates after agonist or antagonist treatments. An important finding in our study was that a high percentage of PB extruded oocytes ( 40%) were retrievable from small follicles. This may be useful clinically; currently many IVF programs may routinely aspirate small follicles, regardless of the stimulation protocols applied. The reason for the high recovery of PB extruded oocytes from small follicles in our study is not clear. Although follicles are FSH-dependent when in antral stage, they can respond to FSH from the preantral stage onward. Recombinant FSH mrna is detected in follicles presenting only two or more layers of granulosa cells (14). Moreover, FSH upregulates expression of its own receptor and induces expression of LH receptor in granulosa cells. The prolonged exogenous FSH administration (up to when leading follicles are 18 mm) could have induced an increase in gene expression for LH receptors in granulosa cells even in the smaller follicles. Therefore, at the time of LH administration, the smaller follicles already would have achieved the ability to undergo meiotic maturation. The effects of GnRH-analogues on the ovary, the oocyte, follicle, embryo and endometrium are a matter of debate (15). At time of retrieval, about half the population of oocytes from small follicles were found to be at GV-stage, independent of the COH protocol. However, at the time of retrieval, we observed a trend toward increasing number of degenerated oocytes aspirated from small follicles after COH with antagonists. From large follicles, besides the trend to acquire a lower number of PB oocytes after antagonists treatment, aspirated follicles presented a significantly higher proportion of GV oocytes, and significantly lower proportion of GVBD compared to agonists groups. The capacity to resume meiosis is sequentially acquired in relation to follicular growth (16). Follicles recruited by both protocols vary in many aspects. In agonists desensitization cycles (long protocols), small antral follicles are recruited by the exogenous gonadotrophins after a variable period of endogenous gonadotrophin depletion. This population of follicles differs from the one recruited in antagonists cycles, which have been exposed for a few days to the endogenous inter-cycle FSH rise (1 3). The microenvironment of the oocyte might, therefore, be different in many aspects, and this hypothetically could explain the observed differences in nuclear maturation stages of oocytes. Whether antagonists treatment matures follicles to a less developed stage needs to be investigated. Research on the content and ratio of various proteins present in follicular fluid at retrieval after various COH protocols should prove helpful. Overall, the results of this study indicate the greater heterogeneity in follicle and/or oocyte development in patients receiving antagonists protocols. When we analyzed oocyte quality among the different follicle diameters of the same patient, we found that PB oocytes originating from small follicles had similar fertilization and developmental potential rates compared with PB originating from large follicles. Still, on day 3 after ICSI a statistically significant proportion of embryos derived from small follicles were retarded in their development (containing not more than four cells) compared with embryos derived from large follicles. Moreover, the rate of embryos presenting a higher number of cells, reaching the eight-cell stage, was less in small follicles group. Several studies indicate that a correlation exists between oocyte developmental competence and follicular size (16 18). The present findings indicate that there is a propensity for a lower developmental capacity when embryos are generated from in vivo matured oocytes of small antral follicles. Therefore, when aspirating these oocytes, one must take into account the differences in development when compared with oocytes from larger follicles. Although PB oocytes of small follicles can eventually be selected for transfer and/or freezing, more studies are needed to know to what extent they 582 Nogueira et al. Oocyte development and follicle size Vol. 85, No. 3, March 2006
form viable pregnancies, as their developmental rate is reduced on day 3 after ICSI. The pregnancy rate was not analyzed in our study because we could not relate the embryos that implanted to the follicle sizes. The collection of oocytes from small follicles could offer an extra source of matured oocytes for research. In conclusion, this study demonstrated the greater heterogeneity in the population of oocyte stages that are present in cycles using an antagonist protocol. Although the profile of nuclear maturation in oocytes retrieved after use of antagonist or agonist protocols differs, no statistically significant differences were found in the outcome of ICSI. These results suggest that in vivo matured oocytes derived from 12 mm follicles differ qualitatively from their counterparts derived from larger follicles. When oocytes from smaller follicles are used clinically, it must be taken into account that the resulting embryos will tend to have lower rates of embryonic development. Acknowledgments: The authors thank the embryologists Dr. D. Strassburger, Dr. O. Bern, M.Sc., E. Kasterstein, B.Sc., D. Komarovsky, and the medical staff at the Infertility Unit, Assaf Harofeh Medical Center-Tel Aviv University for their kind collaboration to this project. We also thank Dr. David Keefe for kindly reviewing the manuscript. REFERENCES 1. Elter K, Nelson LR. Use of third generation gonadotrophinfreleasing hormone antagonists in in vitro fertilization embryo transfer: a review. Obstet Gynecol Surv 2001;56:576 8. 2. Al-Inany H, Aboulghar M. Gonadotrophin-releasing hormone antagonists for assisted conception. Gonadotrophin-releasing hormone antagonists for assisted conception. Cochrane Database Syst Rev 2001;(4): CD001750. 3. Olivennes F, Cunha-Filho JS, Fanchin R, Bouchard P, Frydman R. The use of GnRH antagonists in ovarian stimulation. Hum Reprod Update 2002;8:279 90. 4. Hernandez ER. Embryo implantation and GnRH antagonists. Embryo implantation: the Rubicon for GnRH antagonists. Hum Reprod 2000; 15:1211 6. 5. Wittmaack FM, Tureck RW, Kreger DO, Mastroianni L Jr, Blasco L, Lessey BA. Effect of follicular size on oocyte retrieval, fertilization, cleavage and embryo quality in in vitro fertilization cycles: a 6-years data collection. Fertil Steril 1994;62:1205 10. 6. Dubey AK, Wang HA, Duffy P, Penzias AS. The correlation between follicular measurements, oocyte morphology and fertilization rates in an in vitro fertilization program. Fertil Steril 1995;64:787 90. 7. Miller KF, Goldberg JM, Falcone T. Follicle size and implantation of embryos from in vitro fertilization. Obstet Gynecol 1996;88:583 6. 8. Ectors FJ, Vanderzwalmen P, Hoeck JV, Nijs M, Verhaegen G, Delvigne A, Schoysman R, Leroy F. Relationship of human follicular diameter with oocyte fertilization and development after in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1997;12: 2002 5. 9. Bergh C, Broden H, Lundin K, Hamberger L. Comparison of fertilization, cleavage and pregnancy rates of oocytes from large and small follicles. Hum Reprod 1998;13:1912 15. 10. Sereepapong W, Ahnonkitpanit V, Chompurat D, Pansatha J, Suwajanakorn S, Pruksananonda K, et al. Correlation between human follicular diameter and oocyte recovery, metaphase II oocytes and fertilization rate in intracytoplasmic sperm injection programs. J Med Assoc Thai 2001;84(Suppl 1):367 70. 11. Triwitayakorn A, Suwajanakorn S, Pruksananonda K, Sereepapong W, Ahnonkitpanit V. Correlation between human follicular diameter and oocyte outcomes in an ICSI program. J Assist Reprod Genet 2003;20: 143 7. 12. Scott RT, Hofmann GE, Muasher SJ, Acosta AA, Kreiner DK, Rosenwaks Z. Correlation of follicular diameter with oocyte recovery and maturity at the time of transvaginal follicular aspiration. J In Vitro Fert Embryo Transf 1989;6:73 5. 13. Smitz J, Janssenswillen C, Liu J, et al. Retrospective analysis of superovulated cycles for ICSI yielding an increased number of immature oocytes. In: Proceedings of the 26th annual conference of the Australian Society for Reproductive Biology. Brisbane, Australia: Australian Society for Reproductive Biology, 1994: abstract 23. 14. Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 1997;82:3748 51. 15. Kol S, Lightman A, Hillensjo T, Devroey P, Fauser B, Tarlatzis B, et al. High doses of gonadotrophin-releasing hormone antagonist in invitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles. Hum Reprod 1999;14:2242 4. 16. Mermillod P, Oussaid B, Cognie Y. Aspects of follicular and oocyte maturation that affect the developmental potential of embryos. J Reprod Fertil Suppl 1999;54:449 60. 17. Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 1995;103: 293 8. 18. Marchal R, Vigneron C, Perreau C, Bali-Papp A, Mermillod P. Effect of follicular size on meiotic and developmental competence of porcine oocytes. Theriogenology 2002;57:1523 32. Fertility and Sterility 583