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Early cleavage does not predict treatment outcome following the use of GnRH antagonists in women older than 35
Early cleavage does not predict treatment outcome following the use of GnRH antagonists in women older than 35 Wen-Jui Yang, M.D.,a Yuh-Ming Hwu, M.D.,a,b,c Robert Kuo-kuang Lee, M.D.,a,c Sheng-Hsiang Li, Ph.D.,c Shyr-Yeu Lin, M.D.,a and Steven Fleming, Ph.D.d a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei; b Mackay Medicine, Nursing and Management College, Taipei; c Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan; and d Department of Obstetrics & Gynaecology, University of Sydney, Westmead Hospital, NSW 2145, Australia
Objective: To compare the early-cleavage rates and the implantation potential of embryos between the GnRH antagonist and agonist long stimulation protocols in women older than 35 years. Design: Retrospective analysis. Setting: Academic medical center. Patients: Two hundred twenty patients older than 35 years old underwent IVF. Intervention(s): Sixty-eight patients received GnRH antagonist protocol (GnRH antagonist group) and 152 patients received GnRH agonist long stimulation protocol (GnRH agonist group). Main Outcome Measure(s): Early-cleavage rate of zygotes, implantation rate, and pregnancy rate. Result(s): Early-cleavage rate of zygotes was significantly lower in the GnRH antagonist group than agonist group (21.8% vs. 32.6%, P<.0001). In the GnRH antagonist group, the pregnancy rate was not significantly different between the early-cleavage and late-cleavage subgroups (40.0% vs. 47.4%). In the GnRH agonist group, the pregnancy rate was significantly higher in the early-cleavage subgroup than in the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001). Conclusion(s): In women older than 35 years, the early-cleavage rate of zygotes is significantly lower in the GnRH antagonist group than the agonist group. Early-cleavage status of zygotes is not a reliable predictor for embryo implantation in patients receiving the GnRH antagonist protocol. (Fertil Steril 2007;88:1573–8. 2007 by American Society for Reproductive Medicine.) Key Words: Early cleavage, GnRH antagonist, GnRH agonist, long protocol
The ability to identify viable embryos is essential for improving the success of assisted reproductive therapy. In attempts to select the most viable embryos, numerous embryo scoring systems have been developed. Embryo selection is traditionally performed by using embryo morphology as the guideline. Other selection methods include oocyte and pronuclei polarity and morphology, blastomere symmetry, and blastocyst culture (1). Recently, the rate of zygote mitosis has been emphasized. Several studies have shown that early embryonic cleavage, which is the time period 25–27 hours postinsemination/intracytoplasmic sperm injection (ICSI), can be a strong indicator of viable embryos (2–8). In previous studies investigating early cleavage of embryos, most of the stimulation protocols for controlled ovarian hyperstimulation used GnRH agonists. However, in recent years, GnRH antagonists have also become available for assisted reproduction. GnRH antagonists competitively block pituitary GnRH receptors, inducing a rapid, reversible suppression of gonadotropin secretion (9). Previous studies have shown that GnRH antagonists are associated with a lower Reprint requests: Yuh-Ming Hwu, M.D., Department of Obstetrics and Gynecology, Mackay Memorial Hospital, No.92, Section 2, Chung-Shan North Road, Taipei, Taiwan (FAX: þ 886 2 25433642; E-mail: hu4416@ yahoo.com).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2007.01.052
usage of gonadotropins, a shorter duration of stimulation, and a lower cycle cancellation rate, especially in poor responders (10 –12). Nevertheless, no difference in pregnancy outcome was observed in these studies when using GnRH antagonists compared with GnRH agonists (10 –12). However, some animal studies have shown that GnRH receptor mRNA is present in developing embryos, and that preimplantation embryonic development is significantly decreased by GnRH antagonists in vitro (13). In our center, most of the patients stimulated with GnRH antagonist protocols were older than 35 years. Therefore, in this study we collected data from patients who were older than 35 years undergoing assisted reproduction treatment to investigate the clinical effect of GnRH antagonists. The primary purpose of this study was to compare rates of oocyte maturation and early embryonic development (especially the early cleavage of embryos) between patients administered GnRH antagonists and those administered GnRH agonists during a long protocol. Furthermore, implantation and pregnancy rates were also compared. MATERIALS AND METHODS Study Subjects This study was a retrospective study of IVF/ICSI outcome based on the medical records of patients in the fertility unit
Fertility and Sterility Vol. 88, No. 6, December 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
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of MacKay Memorial Hospital from the time period April 2004 to April 2006. The study protocol was approved by the institutional review board of the Mackay Memorial Hospital.
of a fetal heartbeat on ultrasonography after 12 weeks of gestation. Abortion was defined as fetal loss before 12 weeks of gestation.
To minimize confounding factors, patients diagnosed with polycystic ovaries, a severely poor response (fewer than two retrieved oocytes and an E2 level %300 pg/mL on the day of hCG administration), and cycles for oocyte donation were excluded from the study. Only patients undergoing their first stimulated cycle were included, and only one treatment cycle per patient was analyzed. A total of 220 embryo transfer cycles were included in this study; 68 patients were treated with antagonists and the other 152 patients with agonists.
Insemination and ICSI Patients with male factor infertility (which was defined by the presence of any of the following parameters: sperm concentration <10 106/mL, motility <50%, and normal morphology <5%) received ICSI as treatment while others, without male factor infertility, received conventional insemination.
Ovarian Stimulation Protocol Two stimulation protocols were used in this study; the GnRH antagonist protocol (GnRH antagonist group) and the long protocol for GnRH agonists (GnRH agonist group). In the GnRH antagonist group, recombinant FSH (Gonal-F; Serono Laboratories, Aubonne, Switzerland) and hMG (Pergonal; Serono Laboratories, Aubonne, Switzerland) were administrated daily from the second day of the menstrual cycle. The doses were adjusted according to the patient’s individual ovarian response. Cetrorelix (Cetrotide; Serono, Baxter Oncology GmbH, Halle, Germany) was administered subcutaneously at a dose of 0.25 mg daily when the dominant follicle reached 14 mm in mean diameter, until the day of hCG administration.
Conventional IVF or ICSI was performed 3–5 hours after oocyte aspiration. For conventional IVF, each oocyte was inseminated with 20 103 motile spermatozoa in a single drop of 20 mL medium (Quinn’s advantage fertilization HTF medium; SAGE In Vitro Fertilization, Inc., Trumbull, CT). For the ICSI procedure, 1–2 mL washed spermatozoa were placed in 7% polyvinylpyrrolidone (PVP; SAGE IVF Inc.) and injected using standard techniques. Each embryo was cultured in a single drop of 20 mL medium (Quinn’s Advantage protein Plus, Cleavage medium; SAGE IVF Inc.) supplemented with 10% synthetic serum substitute (Quinn’s Advantage SPS Serum Protein Substitute; SAGE IVF Inc.) covered by mineral oil in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 37 C.
In the long GnRH agonist group, leuprolide acetate (Takeda Pharma GmbH, Stolberg, Germany) was given at a daily dose of 1 mg, starting on day 21 of the previous cycle. Once serum levels of E2 <30 pg/mL were achieved, the daily dose of leuprolide acetate was reduced to 0.5 mg and recombinant FSH and hMG were given until the day of hCG administration. The doses were also adjusted according to the patient’s ovarian response. In both groups, 10,000 IU hCG (Profasi; Serono Laboratories, Aubonne, Switzerland) was administered when at least two follicles reached 18 mm in diameter, and oocyte retrieval was performed 34–36 hours later. Upon completion of oocyte collection and IVF/ICSI, embryos were graded morphologically, by the same embryologist. Embryo transfer was performed 72 hours after the oocyte retrieval. In each stimulation protocol, patients who had at least one early-cleavage embryo transferred were designated as the ‘‘early-cleavage subgroup.’’ Patients who had no early-cleavage embryos transferred were designated as the ‘‘late-cleavage subgroup.’’
Assessment of Fertilization, Early Cleavage, and Embryo Quality Normal fertilization was confirmed by the presence of two pronuclei and two polar bodies 16–20 hours (day 1) after insemination or ICSI. On the same day, early cleavage examination was performed at 25–27 hours after insemination or ICSI. Embryos displaying two cells at inspection were designated as ‘‘early cleavage,’’ whereas those embryos that had not yet cleaved to the two-cell stage were designated as ‘‘late cleavage.’’ Embryos were further examined for their quality at 44–46 hours (day 2) and 66–68 hours (day 3). The day 2 and day 3 embryo scoring system [modified from Veeck (14)] was as follows: for day 2 embryos, namely: score 1: preembryo with blastomeres of equal size and no fragmentation; score 2: preembryo with blastomeres of equal or unequal size and fragmentation %10% of the preembryo surface; score 3: preembryo with blastomeres of equal or unequal size and 11%–49% overall fragmentation; score 4: preembryo with blastomeres of equal or unequal size and fragmentation R50% of the preembryo surface. For the day 3 embryo scoring system, this was the same as described above except score 2 denotes %20% fragmentation and score 3 denotes 21%–49% fragmentation.
The luteal phase was supported by vaginal supplementation with 200 mg micronized natural progesterone (Progeffik; Effik, Paris, France) three times a day. To assess treatment outcome, serum hCG was measured 14 days after oocyte retrieval. An increase in serum hCG levels above 50 IU/L indicated pregnancy. Implantation was defined by the observation of a gestational sac in the uterus by transvaginal ultrasonography. An ongoing pregnancy was defined by the observation
Statistical Analysis Statistical analysis was performed using SPSS software version 10.1 for Windows (Statistical Package for Social Sciences, Inc., Chicago, IL). The differences of means between two variables were calculated using the Mann-Whitney U test. The differences in implantation rates, pregnancy rates, abortion rates, and early-cleavage rates were calculated using
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the chi-square test. A P value of < .05 was considered statistically significant. RESULTS A total of 220 treatment cycles were analyzed. Cycle characteristics between the antagonist group and agonist group are given in Table 1. There were no significant differences between the two groups in terms of age, percentage of patients requiring ICSI, mean number of ampoules of gonadotropins used, mean number of oocytes retrieved, mean number of mature oocytes, mean number of fertilized oocytes, mean number of embryos transferred, pregnancy rate, implantation rate, abortion rate, and ongoing pregnancy rate. The mean total number of ampoules of Cetrorelix (0.25 mg/vial) administered was 3.1 0.5 (mean SD). The E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group than in the agonist group (1,424.2 1,072 pg/mL vs. 2,155.3 1,082 pg/mL, P<.0001). The mean duration of stimulation in the GnRH antagonist group was significantly shorter (P¼.008) than in the agonist group. In this study, a total of 1,163 normally fertilized oocytes were derived, including 340 zygotes from the antagonist group and 823 zygotes from the agonist group (Table 2). In the antagonist group, 74 of 340 (21.8%) zygotes yielded early-cleavage embryos. In the agonist group, 268 of 823 (32.6%) zygotes yielded early-cleavage embryos. The early-cleavage rate of zygotes in the antagonist group was significantly lower than in the agonist group (21.8% vs. 32.6%, P<.0001; Table 2). There were no significant differences in day 2 or day 3 embryo scoring or in the mean number
of day 3 blastomeres (Table 2). Thirty of 68 (44.1%) patients receiving GnRH antagonists had at least one early-cleavage embryo following treatment, while 105 of 152 (69.1%) patients with GnRH agonists had at least one early-cleavage embryo. The percentage of patients with ‘‘early-cleavage’’ embryos was significantly higher in the GnRH agonist group compared with the GnRH antagonist group (69.1% vs. 44.1%, P<.001). Patients who had at least one early-cleavage embryo transferred were designated as the ‘‘early-cleavage subgroup.’’ Patients who had no early-cleavage embryos transferred were designated as the ‘‘late-cleavage subgroup.’’ In the antagonist group, 30 of 68 patients had at least one early-cleavage embryo transferred while 38 patients had only late-cleavage embryos transferred. In the antagonist group, a total of 30 patients conceived successfully (Table 3). Twelve of 30 (40%) women conceived in the early-cleavage subgroup. Eighteen of 38 (47.4%) women conceived in the late-cleavage subgroup. Although the pregnancy rate appeared higher in the late-cleavage subgroup (40.0% vs. 47.4%; Table 3), the difference was not statistically significant. In the GnRH agonist group, 105 patients had at least one early-cleavage embryo transferred (early-cleavage subgroup) while 47 patients had only late-cleavage embryos transferred (late-cleavage subgroup). In the GnRH agonist group, a total of 78 patients conceived. Sixty-four of 105 (61.0%) women conceived in the early-cleavage subgroup, while 14 of 47 (29.8%) women conceived in the late-cleavage subgroup. The pregnancy rate was significantly higher in the earlycleavage subgroup than in the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001; Table 3).
TABLE 1 Comparison of cycle characteristics between the antagonist and agonist groups of patients. Antagonist group (n [ 68)
Agonist group (n [ 152)
P
68 38.4 2.6 44.1 (30/68) 55.9 (38/68) 9.9 1.6 38.7 10.5 1,424.2 1,072.1 7.2 4.6 (488) 6.3 4.2 5.0 3.6 (340) 3.4 1.6 18.4 (41/223) 44.1 (30/68) 24.0 (6/30) 35.3 (24/68)
152 37.6 2.2 44.1 (67/152) 55.9 (85/152) 10.6 1.7 39.5 10.6 2,155.3 1,082.2 8.9 4.6 (1342) 7.8 4.3 5.4 3.7 (823) 3.9 1.7 21.2 (117/552) 51.3 (78/152) 23.1 (18/78) 39.5 (60/152)
NS NS NS .008 NS < .001 NS NS NS NS NS NS NS NS
No. of cycles Age of patients ICSI rate of cycles (%) IVF rate of cycles (%) Duration of stimulation (days) No. of ampoules used Peak E2 level No. of oocytes retrieved (total) No. of mature oocytes No. of normal fertilized oocytes (total) No. of embryos transferred Implantation rate (%) Pregnancy rate (%) Abortion rate (%) Ongoing pregnancy rate (%)
Note: Values are the mean SD. NS ¼ not significant; ICSI ¼ intracytoplasmic sperm injection. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
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TABLE 2 Comparison of early-cleavage of embryos and embryo scores between the antagonist and agonist groups of patients. Antagonist group (n [ 340)
Agonist group (n [ 823)
Pa
21.8 (74/340) 78.2 (266/340) 2.2 1.1 6.6 1.7 2.2 1.1
32.6 (268/823) 67.4 (555/823) 2.0 1.0 6.8 1.8 2.2 1.0
< .001 < .001 NS NS NS
Early-cleavage rate of embryos (%) Late-cleavage rate of embryos (%) Day 2 score No. day 3 blastomeres Day 3 score
Note: Values are the mean SD. NS ¼ not significant. a Chi-square test. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
DISCUSSION In the present study, we observed that the early-cleavage rate of the zygotes in the antagonist group was significantly lower than the agonist group (21.8% vs. 32.6%, P<.0001) in women over 35 years old. However, the mean number of fertilized oocytes, implantation rates, abortion rates, clinical pregnancy rates, and ongoing pregnancy rates were all similar between the antagonist and agonist groups. Furthermore, early-cleavage was not a reliable predictor of pregnancy in patients over 35 years old stimulated with GnRH antagonist protocols. To achieve the goals of maximal implantation rate and a singleton pregnancy, meticulous embryo selection is essential in assisted reproduction. Recently, Borini et al. (1) have reviewed several predictive factors for embryo implantation. Among these, embryo morphology is an important and easily accessible determining factor in embryo selection. Several studies have reported that early cleaving zygotes yield better embryo quality and higher implantation rates (2–8). The reason why early-cleavage zygotes have better embryo quality and higher implantation rates is still unknown. It has been suggested that early-cleavage zygotes arise from better quality gametes that require a shorter time for cell division (2–8). In those previous studies, all used long protocol GnRH agonists for down-regulation. In the present study, we analyzed and compared the early-cleavage rates of zygotes, and pregnancy rates between the GnRH antagonist and long GnRH agonist protocols. In the long GnRH agonist protocol group, the results were in accordance with previous studies in which the early-cleavage subgroup had a better pregnancy outcome than the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001; Table 3). However, in the antagonist group, there was no difference in the pregnancy rates between the early-cleavage subgroup and late-cleavage subgroup (40.0% vs. 47.4%; Table 3). Therefore, early cleavage is not a good predictor of pregnancy outcome in patients over 35 years old stimulated with GnRH antagonist protocols. A conflicting finding was reported by Rabinson et al. (15), who demonstrated that the use of GnRH agonists or antagonists had no significant effect on 1576
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the early-cleavage rate of human embryos. However, the age range of patients was not mentioned and the sample size was proportionally smaller (109 cycles) than in this study. In the present study, the early-cleavage rate of zygotes was significantly lower in the GnRH antagonist group compared with the agonist group (21.8% vs. 32.6%, P<.0001; Table 2). The exact mechanism underlying early cleavage in human embryogenesis is still unclear. Raga et al. (13) conducted a study to investigate the effect of GnRH antagonists on preimplantation embryos; they found that GnRH and GnRH receptor gene expression could be detected in preimplantation embryos. Furthermore, they showed that GnRH antagonist administration was able to block early embryonic development. Is it possible, therefore, that high doses of GnRH antagonists affect mitotic gene expression? GnRH antagonists act through competitive binding to GnRH receptors, a situation that can be reversed by GnRH. Previous studies have shown that continuous exposure to GnRH antagonists will induce down-regulation of the GnRH receptor and a decrease in GnRH receptor gene expression (16, 17). With the discovery of extrapituitary GnRH receptors in human ovaries and other sites (including uterus, endometrium, oocyte–cumulus complex and preimplantation embryo) (18–21), it is possible that the use of GnRH antagonists in reproductive medicine may lead to down-regulation of these receptors (21). To determine the mechanism of action of GnRH antagonists at the cellular level, several studies using cancer cell lines have been performed (18, 19, 22). In these in vitro studies, GnRH antagonists restricted cell growth by decreasing the synthesis of insulin-like growth factor (IGF) and inositol 1, 4, 5-trisphosphate (a second messenger of GnRH action). Furthermore, a decrease in epidermal growth factor (EGF) receptor mRNA and EGF peptide occurs in the presence of GnRH antagonists in vitro (23). IGF and EGF are growth factors that exert their actions through receptors with intrinsic tyrosine kinase activity (24). These receptors form the basis for the generation of an important mitogenic cascade, mediated by MAP kinase and activation of the c-fos and c-jun proto-oncogenes. MAP kinase controls the concentrations
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TABLE 3 Pregnancy rates of early-cleavage versus late-cleavage subgroups in antagonist and agonist groups of patients.
Antagonist group (n ¼ 68) Agonist group (n ¼ 152)
Pregnancy rate in early- cleavage subgroup (%)
Pregnancy rate in late-cleavage subgroup (%)
P
40.0 (12/30) 61.0 (64/105)
47.4 (18/38) 29.8 (14/47)
NS < .0001
Note: NS ¼ not significant. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
of cyclin D/CdK4 and cyclin E/CdK2. These cyclins control the overall proliferation process because entry into S-phase requires both cyclins to be activated (25). Theoretically, if IGF, EGF, and the MAP kinase pathway are inhibited by GnRH antagonists, first mitosis of the zygote (early cleavage) could be retarded. In recent studies, the presence of the IGF system in the human embryo has been demonstrated using the polymerase chain reaction (26). In the murine model, Raga et al. (13) showed that the presence of the GnRH receptor mRNA in the developing embryo and preimplantation embryonic development were significantly decreased by GnRH antagonists. This detrimental effect of the GnRH antagonist on preimplantation embryonic development was a specific receptor-mediated effect, rather than a toxic effect. In IVF cycles, the GnRH antagonist is suspended when hCG is applied, and the embryo is transferred between 5 and 7 days after the last dose of GnRH antagonist. The half-life of the GnRH antagonist reported by the manufacturer (Serono, Baxter Oncology GmbH, Halle, Germany) is 30 hours, and the time interval between the last dose of the GnRH antagonist to the early cleavage of zygotes is about 80 to 90 hours. Consequently, the GnRH antagonist may still have some effect on zygotes to delay the first mitosis. This could explain why lower rates of early cleavage were found in women stimulated with GnRH antagonist protocols. However, in the following 20 to 40 hours, the potential detrimental effect on the developing embryo may be significantly diminished. This could be the reason for a lack of effect of the GnRH antagonist on pregnancy outcome. In fact, several studies have shown that there are no differences in pregnancy outcome between GnRH antagonist and agonist protocols (10–12). The results of this study are compatible with these previous studies. Overall, we believe that by using a GnRH antagonist protocol for ovarian stimulation, there is a delay in the first mitosis of the zygote but later embryonic development is not affected because of the short half-life of the GnRH antagonist. The use of GnRH antagonist therapy in women undergoing assisted reproduction has a significant effect on ovarian follicular steroidogenesis, as demonstrated in several studies (27–29). In the present study, the E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group (P<.0001). Minaretzis et al. (27) have reFertility and Sterility
ported reduced aromatase activity in granulosa lutein cells from patients treated with GnRH antagonists. The reduced aromatase activity might explain the lower E2 concentrations. As mentioned above, GnRH receptor mRNA is found in the ovaries, especially in granulosa-lutein cells (20). If GnRH antagonists inhibit the synthesis of growth factors (IGF) involved in the cell cycle of granulosa cells, the rate of apoptosis within them will be accelerated (30), in turn affecting follicular steroidogenesis. Recently, Giampietro et al. (29) have reported that in human oocytes, therapy with GnRH agonists or antagonists was associated with comparable levels of apoptosis in the granulosa cells. On the other hand, the processes of apoptosis and steroidogenesis can coexist in the same cell population, as different mechanisms are presumably involved the regulation of the two processes. Interestingly, in the present study, although the E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group, the number of retrieved oocytes, and oocyte maturation and fertilization rates were similar in comparison with the GnRH agonist group. Furthermore, the implantation and pregnancy rates showed no statistically significant difference between the two groups. Previous studies have shown a lack of evidence supporting any association between the serum E2 level on the day of hCG administration and the clinical pregnancy rate (31–33). The finding of similar oocyte maturation and fertilization rates accompanied with lower serum E2 levels on the day of hCG administration when using GnRH antagonists was also observed in other studies (20, 28, 29). Schoot et al. (34) have suggested that the serum E2 level may act as a marker of oocyte maturation rather than as a factor controlling maturation. Therefore, this suggests a possible direct role of GnRH antagonists on E2 production within the ovary, without other effects upon follicular development. In conclusion, the results of this study indicate that in women older than 35 years, the early-cleavage rate of zygotes is significantly lower in the GnRH antagonist group compared with the long GnRH agonist group. The results also indicated that the early cleavage status of the embryo is not a reliable predictor for implantation in women over 35 years old received the GnRH antagonist protocol. Further studies are warranted to confirm the association between early cleavage status and pregnancy rate using GnRH antagonist 1577
stimulation protocols. We will extend this study in the future to recruit patients younger than 35 years old. REFERENCES 1. Borini A, Lagalla C, Cattoli M, Sereni E, Sciajno R, Flamigni C, et al. Predictive factors for embryo implantation potential. Repro Biomed Online 2005;10:653–68. 2. Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997;12:1531–6. 3. Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum Reprod 1998;13:182–7. 4. Lundin K, Bergh C, Hardarson T. Early embryo cleavage is a strong indicator of embryo quality in human IVF. Hum Reprod 2001;16:2652–7. 5. Bos-Mikich A, Mattos AL, Ferrari AN. Early cleavage of human embryos: an effective method for predicting successful IVF/ICSI outcome. Hum Reprod 2001;16:2658–61. 6. Fenwick J, Platteau P, Murdoch AP, Herbert M. Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. Hum Reprod 2002;17:407–12. 7. Salumets A, Hyden-Granskog C, Makinen S, Suikkari AM, Tiitinen A, Tuuri T. Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures. Hum Reprod 2003;18:821–5. 8. Windt ML, Kruger TF, Coetzee K, Lombard CJ. Comparative analysis of pregnancy rates after the transfer of early dividing embryos versus slower dividing embryos. Hum Reprod 2004;19:1155–62. 9. Diedrich K, Diedrich C, Santos E, Zoll C, al-Hasani S, Reissmann T, et al. Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation. Hum Reprod 1994;9:788–91. 10. Cheung LP, Lam PM, Lok IH, Chiu TTY, Yeung SY, Tjer CC, et al. GnRH antagonist versus long GnRH agonist protocol in poor responders undergoing IVF: a randomized controlled trial. Hum Reprod 2005;20:616–21. 11. D’Amato G, Caroppo E, Pasquadibisceglie A, Carone D, Vitti A, Vizziello GM. A novel protocol of ovulation induction with delayed gonadotropin-releasing hormone antagonist administration combined with high-dose recombinant follicle-stimulating hormone and clomiphene citrate for poor responders and women over 35 years. Fertil Steril 2004;81: 1572–7. 12. Takahashi K, Mukaida T, Tomiyama T, Goto T, Oka C. GnRH antagonist improved blastocyst quality and pregnancy outcome after multiple failures of IVF/ICSI–ET with a GnRH agonist protocol. J Assist Reprod Genet 2004;21:317–22. 13. Raga F, Casan EM, Kruessel J, Wen Y, Bonilia-Musoles F, Polan ML. The role of gonadotropin-releasing hormone in murine preimplantation embryonic development. Endocrinology 1999;140:3705–12. 14. Veeck L. Preembryo grading and degree of cytoplasmic fragmentation. In: Veeck L, ed. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Parthenon, 1999:46–51. 15. Rabinson J, Girsh E, Pinchasov M, Meltzer S, Bar-Hava I, Tur-Kaspa I. Early cleavage of GnRH agonist versus GnRH antagonist derived embryos. Fertil Steril 2003;80(Suppl. 3):66. 16. Schally AV, Halmos G, Pinski J. Terminology for luteinizing hormonereleasing hormone antagonists. Fertil Steril 1995;64:226. 17. Pinski J, Lamharzi N, Halmos G, Groot K, Jungwirth A, VadilloBuenfil M, et al. Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist Cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats. Endocrinology 1996;137:3430–6.
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18. Emons G, Ortmann O, Becker M, Irmer G, Springer B, Laun R, et al. High affinity binding and direct antiproliferative effects of LHRH analogues in human ovarian cancer cell lines. Cancer Res 1993;53: 5439–46. 19. Emons G, Schorder B, Ortmann O, Westphalen S, Schulz KD, Schally AV. High affinity binding and direct antiproliferative effects of luteinizing hormone-releasing hormone analogs in human endometrial cancer cell lines. J Clin Endocrinol Metab 1993;77:1458–64. 20. Minaretzis D, Jakubowski M, Mortola JF, Pavlou SN. Gonadotrophinreleasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J Clin Endocrinol Metab 1995;80:430–4. 21. Ortmann O, Diedrich K. Pituitary and extrapituitary actions of gonadotrophin-releasing hormone and its analogues. Hum Reprod 1999;14(Suppl. 1):194–206. 22. Sharoni Y, Bosin E, Miinster A, Levy J, Schally AV. Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 1989;86: 1648–51. 23. Moretti RM, Marelli MM, Dondi D, Poletti A, Martini L, Motta M, et al. Luteinizing hormone releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145. J Clin Endocrinol Metab 1996;81:3930–7. 24. LeRoith D, Werner H, Beitner-Johnson D, Roberts CT. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 1995;16:143–63. 25. Van Zoelen EJJ. Polypeptide growth factors and their role in regulating the cell cycle. In: Fauser BCJM, Urtherford AJ, Straus III JF, Van Steirteghem A, eds. Molecular biology in reproductive medicine. New York: Parthenon, 1999:65–77. 26. Liu HC, He ZY, Tang YX, Mele CA, Veeck LL, Davis O, et al. Simultaneous detection of multiple gene expression in mouse and human individual preimplantation embryos. Fertil Steril 1997;67:733–41. 27. Minaretzis D, Alper MM, Oskowitz SP, Lobel SM, Mortola JF, Pavlou SN. Gonadotropin-releasing hormone antagonist versus agonist administration in women undergoing controlled ovarian hyperstimulation: cycle performance and in vitro steroidogenesis of granulosa-lutein cells. Am J Obstet Gynecol 1995;17:1518–25. 28. Garcia-Velasco JA, Isaza V, Vidal C, Landazabal JR, Simon C, Pellicer A. Human ovarian steroid secretion in vivo: effects of GnRH agonist versus antagonist (cetrorelix). Hum Reprod 2001;16:2533–9. 29. Giampietro F, Sancilio S, Tiboni GM, Rana RA, Pietro RD. Levels of apoptosis in human granulosa cells seem to be comparable after therapy with a gonadotropin- releasing hormone agonist or antagonist. Fertil Steril 2006;85:412–9. 30. Parborell F, Irusta G, Vitale A, Gonzalez O, Pecci A, Tesone M. The gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary. Biol Reprod 2005;72:659–66. 31. Chen CH, Zhang X, Barnes R, Confino E, Milad M, Puscheck E, et al. Relationship between peak serum E2 levels and treatment outcome in in-vitro fertilization cycles after embryo transfer on day 3 or day 5. Fertil Steril 2003;80:75–9. 32. Papageorgiou T, Guibert J, Goffinet F, Patrat C, Fulla Y, Janssens Y, et al. Percentile curves of serum estradiol levels during controlled ovarian stimulation in 905 cycles stimulated with recombinant FSH show that high estradiol is not detrimental to IVF outcome. Hum Reprod 2002;17:2846–50. 33. Kosmas IP, Kolibianakis EM, Devroey P. Association of estradiol levels on the day of hCG administration and pregnancy achievement in IVF: a systematic review. Hum Reprod 2004;19:2446–53. 34. Schoot DC, Coelingh-Bennink HJ, Mannaerts BM, Lamberts SW, Bouchard P, Fauser BC. Human recombinant follicle-stimulating hormone induces growth of preovulatory follicles without concomitant increase in androgen and estrogen biosynthesis in a woman with isolated gonadotrophin deficiency. J Clin Endocrinol Metab 1992;74:1471–3.
GnRH antagonists and embryonic early cleavage
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Objective: To compare the early-cleavage rates and the implantation potential of embryos between the GnRH antagonist and agonist long stimulation protocols in women older than 35 years. Design: Retrospective analysis. Setting: Academic medical center. Patients: Two hundred twenty patients older than 35 years old underwent IVF. Intervention(s): Sixty-eight patients received GnRH antagonist protocol (GnRH antagonist group) and 152 patients received GnRH agonist long stimulation protocol (GnRH agonist group). Main Outcome Measure(s): Early-cleavage rate of zygotes, implantation rate, and pregnancy rate. Result(s): Early-cleavage rate of zygotes was significantly lower in the GnRH antagonist group than agonist group (21.8% vs. 32.6%, P<.0001). In the GnRH antagonist group, the pregnancy rate was not significantly different between the early-cleavage and late-cleavage subgroups (40.0% vs. 47.4%). In the GnRH agonist group, the pregnancy rate was significantly higher in the early-cleavage subgroup than in the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001). Conclusion(s): In women older than 35 years, the early-cleavage rate of zygotes is significantly lower in the GnRH antagonist group than the agonist group. Early-cleavage status of zygotes is not a reliable predictor for embryo implantation in patients receiving the GnRH antagonist protocol. (Fertil Steril 2007;88:1573–8. 2007 by American Society for Reproductive Medicine.) Key Words: Early cleavage, GnRH antagonist, GnRH agonist, long protocol
The ability to identify viable embryos is essential for improving the success of assisted reproductive therapy. In attempts to select the most viable embryos, numerous embryo scoring systems have been developed. Embryo selection is traditionally performed by using embryo morphology as the guideline. Other selection methods include oocyte and pronuclei polarity and morphology, blastomere symmetry, and blastocyst culture (1). Recently, the rate of zygote mitosis has been emphasized. Several studies have shown that early embryonic cleavage, which is the time period 25–27 hours postinsemination/intracytoplasmic sperm injection (ICSI), can be a strong indicator of viable embryos (2–8). In previous studies investigating early cleavage of embryos, most of the stimulation protocols for controlled ovarian hyperstimulation used GnRH agonists. However, in recent years, GnRH antagonists have also become available for assisted reproduction. GnRH antagonists competitively block pituitary GnRH receptors, inducing a rapid, reversible suppression of gonadotropin secretion (9). Previous studies have shown that GnRH antagonists are associated with a lower Reprint requests: Yuh-Ming Hwu, M.D., Department of Obstetrics and Gynecology, Mackay Memorial Hospital, No.92, Section 2, Chung-Shan North Road, Taipei, Taiwan (FAX: þ 886 2 25433642; E-mail: hu4416@ yahoo.com).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2007.01.052
usage of gonadotropins, a shorter duration of stimulation, and a lower cycle cancellation rate, especially in poor responders (10 –12). Nevertheless, no difference in pregnancy outcome was observed in these studies when using GnRH antagonists compared with GnRH agonists (10 –12). However, some animal studies have shown that GnRH receptor mRNA is present in developing embryos, and that preimplantation embryonic development is significantly decreased by GnRH antagonists in vitro (13). In our center, most of the patients stimulated with GnRH antagonist protocols were older than 35 years. Therefore, in this study we collected data from patients who were older than 35 years undergoing assisted reproduction treatment to investigate the clinical effect of GnRH antagonists. The primary purpose of this study was to compare rates of oocyte maturation and early embryonic development (especially the early cleavage of embryos) between patients administered GnRH antagonists and those administered GnRH agonists during a long protocol. Furthermore, implantation and pregnancy rates were also compared. MATERIALS AND METHODS Study Subjects This study was a retrospective study of IVF/ICSI outcome based on the medical records of patients in the fertility unit
Fertility and Sterility Vol. 88, No. 6, December 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
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of MacKay Memorial Hospital from the time period April 2004 to April 2006. The study protocol was approved by the institutional review board of the Mackay Memorial Hospital.
of a fetal heartbeat on ultrasonography after 12 weeks of gestation. Abortion was defined as fetal loss before 12 weeks of gestation.
To minimize confounding factors, patients diagnosed with polycystic ovaries, a severely poor response (fewer than two retrieved oocytes and an E2 level %300 pg/mL on the day of hCG administration), and cycles for oocyte donation were excluded from the study. Only patients undergoing their first stimulated cycle were included, and only one treatment cycle per patient was analyzed. A total of 220 embryo transfer cycles were included in this study; 68 patients were treated with antagonists and the other 152 patients with agonists.
Insemination and ICSI Patients with male factor infertility (which was defined by the presence of any of the following parameters: sperm concentration <10 106/mL, motility <50%, and normal morphology <5%) received ICSI as treatment while others, without male factor infertility, received conventional insemination.
Ovarian Stimulation Protocol Two stimulation protocols were used in this study; the GnRH antagonist protocol (GnRH antagonist group) and the long protocol for GnRH agonists (GnRH agonist group). In the GnRH antagonist group, recombinant FSH (Gonal-F; Serono Laboratories, Aubonne, Switzerland) and hMG (Pergonal; Serono Laboratories, Aubonne, Switzerland) were administrated daily from the second day of the menstrual cycle. The doses were adjusted according to the patient’s individual ovarian response. Cetrorelix (Cetrotide; Serono, Baxter Oncology GmbH, Halle, Germany) was administered subcutaneously at a dose of 0.25 mg daily when the dominant follicle reached 14 mm in mean diameter, until the day of hCG administration.
Conventional IVF or ICSI was performed 3–5 hours after oocyte aspiration. For conventional IVF, each oocyte was inseminated with 20 103 motile spermatozoa in a single drop of 20 mL medium (Quinn’s advantage fertilization HTF medium; SAGE In Vitro Fertilization, Inc., Trumbull, CT). For the ICSI procedure, 1–2 mL washed spermatozoa were placed in 7% polyvinylpyrrolidone (PVP; SAGE IVF Inc.) and injected using standard techniques. Each embryo was cultured in a single drop of 20 mL medium (Quinn’s Advantage protein Plus, Cleavage medium; SAGE IVF Inc.) supplemented with 10% synthetic serum substitute (Quinn’s Advantage SPS Serum Protein Substitute; SAGE IVF Inc.) covered by mineral oil in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 37 C.
In the long GnRH agonist group, leuprolide acetate (Takeda Pharma GmbH, Stolberg, Germany) was given at a daily dose of 1 mg, starting on day 21 of the previous cycle. Once serum levels of E2 <30 pg/mL were achieved, the daily dose of leuprolide acetate was reduced to 0.5 mg and recombinant FSH and hMG were given until the day of hCG administration. The doses were also adjusted according to the patient’s ovarian response. In both groups, 10,000 IU hCG (Profasi; Serono Laboratories, Aubonne, Switzerland) was administered when at least two follicles reached 18 mm in diameter, and oocyte retrieval was performed 34–36 hours later. Upon completion of oocyte collection and IVF/ICSI, embryos were graded morphologically, by the same embryologist. Embryo transfer was performed 72 hours after the oocyte retrieval. In each stimulation protocol, patients who had at least one early-cleavage embryo transferred were designated as the ‘‘early-cleavage subgroup.’’ Patients who had no early-cleavage embryos transferred were designated as the ‘‘late-cleavage subgroup.’’
Assessment of Fertilization, Early Cleavage, and Embryo Quality Normal fertilization was confirmed by the presence of two pronuclei and two polar bodies 16–20 hours (day 1) after insemination or ICSI. On the same day, early cleavage examination was performed at 25–27 hours after insemination or ICSI. Embryos displaying two cells at inspection were designated as ‘‘early cleavage,’’ whereas those embryos that had not yet cleaved to the two-cell stage were designated as ‘‘late cleavage.’’ Embryos were further examined for their quality at 44–46 hours (day 2) and 66–68 hours (day 3). The day 2 and day 3 embryo scoring system [modified from Veeck (14)] was as follows: for day 2 embryos, namely: score 1: preembryo with blastomeres of equal size and no fragmentation; score 2: preembryo with blastomeres of equal or unequal size and fragmentation %10% of the preembryo surface; score 3: preembryo with blastomeres of equal or unequal size and 11%–49% overall fragmentation; score 4: preembryo with blastomeres of equal or unequal size and fragmentation R50% of the preembryo surface. For the day 3 embryo scoring system, this was the same as described above except score 2 denotes %20% fragmentation and score 3 denotes 21%–49% fragmentation.
The luteal phase was supported by vaginal supplementation with 200 mg micronized natural progesterone (Progeffik; Effik, Paris, France) three times a day. To assess treatment outcome, serum hCG was measured 14 days after oocyte retrieval. An increase in serum hCG levels above 50 IU/L indicated pregnancy. Implantation was defined by the observation of a gestational sac in the uterus by transvaginal ultrasonography. An ongoing pregnancy was defined by the observation
Statistical Analysis Statistical analysis was performed using SPSS software version 10.1 for Windows (Statistical Package for Social Sciences, Inc., Chicago, IL). The differences of means between two variables were calculated using the Mann-Whitney U test. The differences in implantation rates, pregnancy rates, abortion rates, and early-cleavage rates were calculated using
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the chi-square test. A P value of < .05 was considered statistically significant. RESULTS A total of 220 treatment cycles were analyzed. Cycle characteristics between the antagonist group and agonist group are given in Table 1. There were no significant differences between the two groups in terms of age, percentage of patients requiring ICSI, mean number of ampoules of gonadotropins used, mean number of oocytes retrieved, mean number of mature oocytes, mean number of fertilized oocytes, mean number of embryos transferred, pregnancy rate, implantation rate, abortion rate, and ongoing pregnancy rate. The mean total number of ampoules of Cetrorelix (0.25 mg/vial) administered was 3.1 0.5 (mean SD). The E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group than in the agonist group (1,424.2 1,072 pg/mL vs. 2,155.3 1,082 pg/mL, P<.0001). The mean duration of stimulation in the GnRH antagonist group was significantly shorter (P¼.008) than in the agonist group. In this study, a total of 1,163 normally fertilized oocytes were derived, including 340 zygotes from the antagonist group and 823 zygotes from the agonist group (Table 2). In the antagonist group, 74 of 340 (21.8%) zygotes yielded early-cleavage embryos. In the agonist group, 268 of 823 (32.6%) zygotes yielded early-cleavage embryos. The early-cleavage rate of zygotes in the antagonist group was significantly lower than in the agonist group (21.8% vs. 32.6%, P<.0001; Table 2). There were no significant differences in day 2 or day 3 embryo scoring or in the mean number
of day 3 blastomeres (Table 2). Thirty of 68 (44.1%) patients receiving GnRH antagonists had at least one early-cleavage embryo following treatment, while 105 of 152 (69.1%) patients with GnRH agonists had at least one early-cleavage embryo. The percentage of patients with ‘‘early-cleavage’’ embryos was significantly higher in the GnRH agonist group compared with the GnRH antagonist group (69.1% vs. 44.1%, P<.001). Patients who had at least one early-cleavage embryo transferred were designated as the ‘‘early-cleavage subgroup.’’ Patients who had no early-cleavage embryos transferred were designated as the ‘‘late-cleavage subgroup.’’ In the antagonist group, 30 of 68 patients had at least one early-cleavage embryo transferred while 38 patients had only late-cleavage embryos transferred. In the antagonist group, a total of 30 patients conceived successfully (Table 3). Twelve of 30 (40%) women conceived in the early-cleavage subgroup. Eighteen of 38 (47.4%) women conceived in the late-cleavage subgroup. Although the pregnancy rate appeared higher in the late-cleavage subgroup (40.0% vs. 47.4%; Table 3), the difference was not statistically significant. In the GnRH agonist group, 105 patients had at least one early-cleavage embryo transferred (early-cleavage subgroup) while 47 patients had only late-cleavage embryos transferred (late-cleavage subgroup). In the GnRH agonist group, a total of 78 patients conceived. Sixty-four of 105 (61.0%) women conceived in the early-cleavage subgroup, while 14 of 47 (29.8%) women conceived in the late-cleavage subgroup. The pregnancy rate was significantly higher in the earlycleavage subgroup than in the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001; Table 3).
TABLE 1 Comparison of cycle characteristics between the antagonist and agonist groups of patients. Antagonist group (n [ 68)
Agonist group (n [ 152)
P
68 38.4 2.6 44.1 (30/68) 55.9 (38/68) 9.9 1.6 38.7 10.5 1,424.2 1,072.1 7.2 4.6 (488) 6.3 4.2 5.0 3.6 (340) 3.4 1.6 18.4 (41/223) 44.1 (30/68) 24.0 (6/30) 35.3 (24/68)
152 37.6 2.2 44.1 (67/152) 55.9 (85/152) 10.6 1.7 39.5 10.6 2,155.3 1,082.2 8.9 4.6 (1342) 7.8 4.3 5.4 3.7 (823) 3.9 1.7 21.2 (117/552) 51.3 (78/152) 23.1 (18/78) 39.5 (60/152)
NS NS NS .008 NS < .001 NS NS NS NS NS NS NS NS
No. of cycles Age of patients ICSI rate of cycles (%) IVF rate of cycles (%) Duration of stimulation (days) No. of ampoules used Peak E2 level No. of oocytes retrieved (total) No. of mature oocytes No. of normal fertilized oocytes (total) No. of embryos transferred Implantation rate (%) Pregnancy rate (%) Abortion rate (%) Ongoing pregnancy rate (%)
Note: Values are the mean SD. NS ¼ not significant; ICSI ¼ intracytoplasmic sperm injection. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
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TABLE 2 Comparison of early-cleavage of embryos and embryo scores between the antagonist and agonist groups of patients. Antagonist group (n [ 340)
Agonist group (n [ 823)
Pa
21.8 (74/340) 78.2 (266/340) 2.2 1.1 6.6 1.7 2.2 1.1
32.6 (268/823) 67.4 (555/823) 2.0 1.0 6.8 1.8 2.2 1.0
< .001 < .001 NS NS NS
Early-cleavage rate of embryos (%) Late-cleavage rate of embryos (%) Day 2 score No. day 3 blastomeres Day 3 score
Note: Values are the mean SD. NS ¼ not significant. a Chi-square test. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
DISCUSSION In the present study, we observed that the early-cleavage rate of the zygotes in the antagonist group was significantly lower than the agonist group (21.8% vs. 32.6%, P<.0001) in women over 35 years old. However, the mean number of fertilized oocytes, implantation rates, abortion rates, clinical pregnancy rates, and ongoing pregnancy rates were all similar between the antagonist and agonist groups. Furthermore, early-cleavage was not a reliable predictor of pregnancy in patients over 35 years old stimulated with GnRH antagonist protocols. To achieve the goals of maximal implantation rate and a singleton pregnancy, meticulous embryo selection is essential in assisted reproduction. Recently, Borini et al. (1) have reviewed several predictive factors for embryo implantation. Among these, embryo morphology is an important and easily accessible determining factor in embryo selection. Several studies have reported that early cleaving zygotes yield better embryo quality and higher implantation rates (2–8). The reason why early-cleavage zygotes have better embryo quality and higher implantation rates is still unknown. It has been suggested that early-cleavage zygotes arise from better quality gametes that require a shorter time for cell division (2–8). In those previous studies, all used long protocol GnRH agonists for down-regulation. In the present study, we analyzed and compared the early-cleavage rates of zygotes, and pregnancy rates between the GnRH antagonist and long GnRH agonist protocols. In the long GnRH agonist protocol group, the results were in accordance with previous studies in which the early-cleavage subgroup had a better pregnancy outcome than the late-cleavage subgroup (61.0% vs. 29.8%, P<.0001; Table 3). However, in the antagonist group, there was no difference in the pregnancy rates between the early-cleavage subgroup and late-cleavage subgroup (40.0% vs. 47.4%; Table 3). Therefore, early cleavage is not a good predictor of pregnancy outcome in patients over 35 years old stimulated with GnRH antagonist protocols. A conflicting finding was reported by Rabinson et al. (15), who demonstrated that the use of GnRH agonists or antagonists had no significant effect on 1576
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the early-cleavage rate of human embryos. However, the age range of patients was not mentioned and the sample size was proportionally smaller (109 cycles) than in this study. In the present study, the early-cleavage rate of zygotes was significantly lower in the GnRH antagonist group compared with the agonist group (21.8% vs. 32.6%, P<.0001; Table 2). The exact mechanism underlying early cleavage in human embryogenesis is still unclear. Raga et al. (13) conducted a study to investigate the effect of GnRH antagonists on preimplantation embryos; they found that GnRH and GnRH receptor gene expression could be detected in preimplantation embryos. Furthermore, they showed that GnRH antagonist administration was able to block early embryonic development. Is it possible, therefore, that high doses of GnRH antagonists affect mitotic gene expression? GnRH antagonists act through competitive binding to GnRH receptors, a situation that can be reversed by GnRH. Previous studies have shown that continuous exposure to GnRH antagonists will induce down-regulation of the GnRH receptor and a decrease in GnRH receptor gene expression (16, 17). With the discovery of extrapituitary GnRH receptors in human ovaries and other sites (including uterus, endometrium, oocyte–cumulus complex and preimplantation embryo) (18–21), it is possible that the use of GnRH antagonists in reproductive medicine may lead to down-regulation of these receptors (21). To determine the mechanism of action of GnRH antagonists at the cellular level, several studies using cancer cell lines have been performed (18, 19, 22). In these in vitro studies, GnRH antagonists restricted cell growth by decreasing the synthesis of insulin-like growth factor (IGF) and inositol 1, 4, 5-trisphosphate (a second messenger of GnRH action). Furthermore, a decrease in epidermal growth factor (EGF) receptor mRNA and EGF peptide occurs in the presence of GnRH antagonists in vitro (23). IGF and EGF are growth factors that exert their actions through receptors with intrinsic tyrosine kinase activity (24). These receptors form the basis for the generation of an important mitogenic cascade, mediated by MAP kinase and activation of the c-fos and c-jun proto-oncogenes. MAP kinase controls the concentrations
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TABLE 3 Pregnancy rates of early-cleavage versus late-cleavage subgroups in antagonist and agonist groups of patients.
Antagonist group (n ¼ 68) Agonist group (n ¼ 152)
Pregnancy rate in early- cleavage subgroup (%)
Pregnancy rate in late-cleavage subgroup (%)
P
40.0 (12/30) 61.0 (64/105)
47.4 (18/38) 29.8 (14/47)
NS < .0001
Note: NS ¼ not significant. Yang. GnRH antagonists and embryonic early cleavage. Fertil Steril 2007.
of cyclin D/CdK4 and cyclin E/CdK2. These cyclins control the overall proliferation process because entry into S-phase requires both cyclins to be activated (25). Theoretically, if IGF, EGF, and the MAP kinase pathway are inhibited by GnRH antagonists, first mitosis of the zygote (early cleavage) could be retarded. In recent studies, the presence of the IGF system in the human embryo has been demonstrated using the polymerase chain reaction (26). In the murine model, Raga et al. (13) showed that the presence of the GnRH receptor mRNA in the developing embryo and preimplantation embryonic development were significantly decreased by GnRH antagonists. This detrimental effect of the GnRH antagonist on preimplantation embryonic development was a specific receptor-mediated effect, rather than a toxic effect. In IVF cycles, the GnRH antagonist is suspended when hCG is applied, and the embryo is transferred between 5 and 7 days after the last dose of GnRH antagonist. The half-life of the GnRH antagonist reported by the manufacturer (Serono, Baxter Oncology GmbH, Halle, Germany) is 30 hours, and the time interval between the last dose of the GnRH antagonist to the early cleavage of zygotes is about 80 to 90 hours. Consequently, the GnRH antagonist may still have some effect on zygotes to delay the first mitosis. This could explain why lower rates of early cleavage were found in women stimulated with GnRH antagonist protocols. However, in the following 20 to 40 hours, the potential detrimental effect on the developing embryo may be significantly diminished. This could be the reason for a lack of effect of the GnRH antagonist on pregnancy outcome. In fact, several studies have shown that there are no differences in pregnancy outcome between GnRH antagonist and agonist protocols (10–12). The results of this study are compatible with these previous studies. Overall, we believe that by using a GnRH antagonist protocol for ovarian stimulation, there is a delay in the first mitosis of the zygote but later embryonic development is not affected because of the short half-life of the GnRH antagonist. The use of GnRH antagonist therapy in women undergoing assisted reproduction has a significant effect on ovarian follicular steroidogenesis, as demonstrated in several studies (27–29). In the present study, the E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group (P<.0001). Minaretzis et al. (27) have reFertility and Sterility
ported reduced aromatase activity in granulosa lutein cells from patients treated with GnRH antagonists. The reduced aromatase activity might explain the lower E2 concentrations. As mentioned above, GnRH receptor mRNA is found in the ovaries, especially in granulosa-lutein cells (20). If GnRH antagonists inhibit the synthesis of growth factors (IGF) involved in the cell cycle of granulosa cells, the rate of apoptosis within them will be accelerated (30), in turn affecting follicular steroidogenesis. Recently, Giampietro et al. (29) have reported that in human oocytes, therapy with GnRH agonists or antagonists was associated with comparable levels of apoptosis in the granulosa cells. On the other hand, the processes of apoptosis and steroidogenesis can coexist in the same cell population, as different mechanisms are presumably involved the regulation of the two processes. Interestingly, in the present study, although the E2 level on the day of hCG administration was significantly lower in the GnRH antagonist group, the number of retrieved oocytes, and oocyte maturation and fertilization rates were similar in comparison with the GnRH agonist group. Furthermore, the implantation and pregnancy rates showed no statistically significant difference between the two groups. Previous studies have shown a lack of evidence supporting any association between the serum E2 level on the day of hCG administration and the clinical pregnancy rate (31–33). The finding of similar oocyte maturation and fertilization rates accompanied with lower serum E2 levels on the day of hCG administration when using GnRH antagonists was also observed in other studies (20, 28, 29). Schoot et al. (34) have suggested that the serum E2 level may act as a marker of oocyte maturation rather than as a factor controlling maturation. Therefore, this suggests a possible direct role of GnRH antagonists on E2 production within the ovary, without other effects upon follicular development. In conclusion, the results of this study indicate that in women older than 35 years, the early-cleavage rate of zygotes is significantly lower in the GnRH antagonist group compared with the long GnRH agonist group. The results also indicated that the early cleavage status of the embryo is not a reliable predictor for implantation in women over 35 years old received the GnRH antagonist protocol. Further studies are warranted to confirm the association between early cleavage status and pregnancy rate using GnRH antagonist 1577
stimulation protocols. We will extend this study in the future to recruit patients younger than 35 years old. REFERENCES 1. Borini A, Lagalla C, Cattoli M, Sereni E, Sciajno R, Flamigni C, et al. Predictive factors for embryo implantation potential. Repro Biomed Online 2005;10:653–68. 2. Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997;12:1531–6. 3. Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum Reprod 1998;13:182–7. 4. Lundin K, Bergh C, Hardarson T. Early embryo cleavage is a strong indicator of embryo quality in human IVF. Hum Reprod 2001;16:2652–7. 5. Bos-Mikich A, Mattos AL, Ferrari AN. Early cleavage of human embryos: an effective method for predicting successful IVF/ICSI outcome. Hum Reprod 2001;16:2658–61. 6. Fenwick J, Platteau P, Murdoch AP, Herbert M. Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. Hum Reprod 2002;17:407–12. 7. Salumets A, Hyden-Granskog C, Makinen S, Suikkari AM, Tiitinen A, Tuuri T. Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures. Hum Reprod 2003;18:821–5. 8. Windt ML, Kruger TF, Coetzee K, Lombard CJ. Comparative analysis of pregnancy rates after the transfer of early dividing embryos versus slower dividing embryos. Hum Reprod 2004;19:1155–62. 9. Diedrich K, Diedrich C, Santos E, Zoll C, al-Hasani S, Reissmann T, et al. Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation. Hum Reprod 1994;9:788–91. 10. Cheung LP, Lam PM, Lok IH, Chiu TTY, Yeung SY, Tjer CC, et al. GnRH antagonist versus long GnRH agonist protocol in poor responders undergoing IVF: a randomized controlled trial. Hum Reprod 2005;20:616–21. 11. D’Amato G, Caroppo E, Pasquadibisceglie A, Carone D, Vitti A, Vizziello GM. A novel protocol of ovulation induction with delayed gonadotropin-releasing hormone antagonist administration combined with high-dose recombinant follicle-stimulating hormone and clomiphene citrate for poor responders and women over 35 years. Fertil Steril 2004;81: 1572–7. 12. Takahashi K, Mukaida T, Tomiyama T, Goto T, Oka C. GnRH antagonist improved blastocyst quality and pregnancy outcome after multiple failures of IVF/ICSI–ET with a GnRH agonist protocol. J Assist Reprod Genet 2004;21:317–22. 13. Raga F, Casan EM, Kruessel J, Wen Y, Bonilia-Musoles F, Polan ML. The role of gonadotropin-releasing hormone in murine preimplantation embryonic development. Endocrinology 1999;140:3705–12. 14. Veeck L. Preembryo grading and degree of cytoplasmic fragmentation. In: Veeck L, ed. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Parthenon, 1999:46–51. 15. Rabinson J, Girsh E, Pinchasov M, Meltzer S, Bar-Hava I, Tur-Kaspa I. Early cleavage of GnRH agonist versus GnRH antagonist derived embryos. Fertil Steril 2003;80(Suppl. 3):66. 16. Schally AV, Halmos G, Pinski J. Terminology for luteinizing hormonereleasing hormone antagonists. Fertil Steril 1995;64:226. 17. Pinski J, Lamharzi N, Halmos G, Groot K, Jungwirth A, VadilloBuenfil M, et al. Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist Cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats. Endocrinology 1996;137:3430–6.
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Vol. 88, No. 6, December 2007